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THESIS ON
PHYTOCHEMICAL INVESTIGATION AND PHARMACOLOGICAL SCREENING OF SOME INDIAN MEDICINAL PLANTS SUBMITTED FOR THE AWARD OF DOCTOR OF PHILOSOPHY DEGREE IN
PHARMACEUTICAL SCIENCES BY DHARMENDRA KUMAR Reg. No.: SU/SPS/Ph.D/FT/08/27
UNDER THE SUPERVISION OF PROF. (DR.) RANJIT SINGH DIRECTOR SCHOOL OF PHARMACEUTICAL SCIENCES
SCHOOL OF PHARMACEUTICAL SCIENCES
SHOBHIT INSTITUTE OF ENGINEERING & TECHNOLOGY A DEEMED-TO-BE-UNIVERSITY MODIPURAM, MEERUT – 250110 (INDIA)
2012
Certificate This is to certify that the thesis entitled “Phytochemical Investigation and Pharmacological Screening of Some Indian Medicinal Plants” submitted by Mr. Dharmendra Kumar for the award of the Degree of Doctor of Philosophy in Pharmaceutical Sciences to School of Pharmaceutical Sciences, Shobhit Institute of Engineering & Technology, a Deemed-to-be-University, Modipuram, Meerut is a record of authentic work carried out under my supervision. The matter embodied in this thesis is original work of the candidate and has not been submitted for the award of any other degree or diploma. It is further certified that he worked with me for the required period in the School of Pharmaceutical Sciences, Shobhit Institute of Engineering & Technology, a Deemedto-be-University, Modipuram, Meerut.
Date
:
Place :
Prof. (Dr.) Ranjit Singh (Supervisor)
Declaration I, hereby, declare that the work presented in this thesis entitled “Phytochemical Investigation and Pharmacological Screening of Some Indian Medicinal Plants” in fulfilment of the requirements for the award of Degree of Doctor of Philosophy in Pharmaceutical Sciences, submitted to School of Pharmaceutical Sciences, Shobhit Institute of Engineering & Technology, a Deemed-to-be-University, Modipuram, Meerut is an authentic record of my own research work under the supervision of Prof. (Dr.) Ranjit Singh. I also declare that the work embodied in the present thesis; (i) is my original work and has not been copied from any Journal/Thesis/Book, and (ii) has not been submitted by me for any other degree or diploma.
Date
:
Place :
Dharmendra Kumar
ACKNOWLEDGEMENT Those who love hard work and love it because it is hard, Those who try and fail and keep on trying, Those who suffer the loss of months of hard labou labour and start all over again, Those who cannot be discouraged no matter whatever happens, Are invited to undertake “RESEARCH” To work under the esteemed guidance of a genius scientist is a matter of pride. Destiny has bestowed upon me this golden opportunity to work under the supervision of Prof. Ranjit Singh, Director, School of Pharmaceutical Sciences, the pillar and backbone of my research work. His plausible, valuable guidance, motivation urge instilled in me immense confidence to continue my search right from the stage of identifying problem to the accomplishment of goals. I wish to express my gratitude and respect to Dr. Shobhit Kumar, Chancellor, Kunwar Shekhar Vijendra, Pro-chancellor and Prof. R.P. Agarwal, Vice Chancellor, Shobhit Institute of Engineering & Technology for their blessings and constant unconditional support. I record my profound sense of gratitude to Prof. Shailendra K. Saraf, Director, Faculty of Pharmacy, Northern India Engineering College, Lucknow and Prof. Shubhini A. Saraf, Professor and Head, Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar Central University, Lucknow for their constant guidance, supervision, unflagging interest, critical comments and valuable suggestion during the course of this work. I am extremely thankful to Prof. S. K. Jain, Professor of Pharmaceutics, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Central University, Sagar, whose stimulating criticism, constructive suggestion, keen interest and inspiration have contributed immensely towards the successful completion of the work. I wish to express my warm and sincere thanks to Brig. (Retd.) S. K. Sareen, Registrar, Shobhit Institute of Engineering & Technology, a Deemed-to-be-University, Meerut, whose nimble attitude, moral support and intellectual suggestions helped me a lot in the accomplishment of the project. I specially thank Divya Prakash Maurya for providing me the logistic support, affection, co-operation, loving companionship, unflagging interest during my project work.
During this work I have collaborated with many colleagues for whom I have great regard. I wish to extend my warmest thanks to Mr. Mukesh Maithani, Mr. Anand Gaurav, Mr. Mayank Yadav, Mr. Santosh K. Mishra, Dr. Vikas Jain, Mr. V. D. Ashwlayan. and Mr. Amrendra K. Chaudhary. I also wish to extend my warmest thanks to Mrs. Vertika Gautam, and Mrs. Deepika Jain. It is due to their keen interest, encouragement and fruitful suggestions that I could complete my work. I wish to express my thanks to Dr. Vijay Jyoti Kumar and Mr. Rajinder Guleria for the help and support. My humble regards are due to Mr. V. D. Sharma, Mr. Pawan Kumar and Mr. U. P. Tomar for their support. I would like to express my thanks to Mr. Rajpal, Mr. Manoj and Mr. Suraj for continuous support during my project work. I warmly thank Mr. Subodh Arora, Mrs. Indu Arora, Shruti and Mehul for their affection, co-operation and loving companionship during my project work. I wish to thank Kushal Lata Mam, Shivani, Akshit and Arushi for their blessings, kind co-operation, loving companionship & moral support. My parents deserve special mention for their inseparable support and prayers. My Father, in the first place is the person who showed me the joy of intellectual pursuit ever since I was a child. My Mother is the one who sincerely raised me with her caring and gentle love. I would like to express my heartfelt thanks and great debts of gratitude to my Father in law, Mother in law, Brothers in law, Sisters, Brother, Bhabhi and dearest nephews Apoorv Raj, Abhinav Akarsh and Niharika Raj for being supportive and caring. Words elude me to express my appreciation to my son Adit Advay (Yash) and my wife Jayshree whose dedication, love and persistent confidence in me, has taken the load off my shoulder. She helped me in different ways & with whom I shared my joys & sorrows. Besides this, there are several other people who have knowingly or unknowingly helped me in the successful completion of this project. I thank all those people for every ounce of efforts they contributed, with my sincere apology, if I could not mention anyone. Above all, I bow my head before the Almighty God with whose grace and beatitude, I moved through this venture. I finish with a final silence of gratitude for my life. Dharmendra Kumar
CONTENTS Contents
Page No.
List of Abbreviations
i-iii
List of Tables
iv-v
List of Figures
vi-viii
Chapter 1: Introduction
1-32
1.1. Introduction
1
1.2. Traditional and orthodox medicine
2
1.3. Herbal medicine used in ocular activity
2
1.4. Diseases and disorder of eyes
2
1.5. Anatomy of eye lens
3
1.5.1. Position, size, and shape
4
1.5.2. Variations among vertebrates
4
1.5.3. Lens structure and function
4
1.5.3.1. Lens capsule
5
1.5.3.2. Lens epithelium
5
1.5.3.3. Lens fibers
5
1.5.4. Accommodation: changing the power of the lens
6
1.5.5. Crystallins and transparency
6
1.5.6. Development and growth of human lens
7
1.5.7. Nourishment of the lens
8
1.6. Cataract; general overview
8
1.7. Factors implicated in cataractogenesis
9
1.7.1. Smoking
9
1.7.2. Diabetes
9
1.7.3. Gender
9
1.7.4. Steroids
9
1.7.5. Nitric oxide
10
1.8. Etiology of cataract
10
1.9. Hypothesis for cataract
10
1.10. Models proposed to study of cataract
11
1.11. Classification of cataract
13
1.11.1. Congenital and developmental cataract
13
1.11.2. Blue dot cataract
13
1.11.3. Coronary cataract
13
1.11.4. Capsular or polar cataract
13
1.11.5. Sutural cataract
14
1.11.6. Coralliform cataract
14
1.11.7. Floriform cataract
14
1.11.8. Central cataract
14
1.11.9. Lamellar or zonular cataract
14
1.11.10. Total cataract
14
1.11.11. Acquired cataract
14
1.12. Mechanisms associated with cataract
15
1.12.1. Non enzymatic glycation
16
1.12.2. Oxidative stress
17
1.12.3. Polyol pathway
17
1.13. Pharmacological strategies for prevention of cataract
18
1.13.1. Aldose reductase inhibitors
18
1.13.2. Non steroidal anti-inflammatory drugs
19
1.13.3. Agents which act on glutathione
19
1.13.4. Vitamins, minerals, antioxidants and herbal drugs
20
References
24-32
Chapter 2: Literature Review
33-56
2. Literature Review
33-56
2.1. Acorus calamus
33
2.2. Vitex negundo
37
2.3. Butea frondosa
41
2.4. Research Envisaged
48
2.5. Plan of Work
49
References
Chapter 3: Plant(s) Profile 3. Plant(s) Profile
50-56
57-74 57-74
3.1. Acorus calamus
57
3.1.1. Classification
57
3.1.2. Morphology
58
3.1.3. Cultivation
58
3.1.4. Chemical constituents
59
3.1.5. Medicinal uses
60
3.2. Vitex Negundo
64
3.2.1. Classification
65
3.2.2. Morphology
65
3.2.3. Cultivation
66
3.2.4. Chemical constituents
66
3.2.5. Medicinal uses
68
3.3. Butea frondosa
69
3.3.1. Classification
69
3.3.2. Morphology
70
3.3.3. Cultivation
70
3.3.4. Chemical Constituents
70
3.3.5. Medicinal Uses
71
References
73-74
Chapter 4: Experimental Work
75-111
4. Experimental Work
75-111
4.1. Materials and Methods
75
4.1.1. Collection and authentication of roots Acorus calamus
75
4.1.2. Drying of roots
76
4.1.3. Size reduction of roots
76
4.1.4. Extraction of Acorus calamus
76
4.1.5. Phytochemical investigations
78
4.1.6. Chromatographic studies
81
4.1.7. Thin layer chromatographic studies
81
4.1.8. Column chromatographic studies
83
4.1.9. Methodology
83
4.1.10. Isolation of compound from Acorus calamus (roots)
85
4.1.11. Characterization of isolated compound by spectral analysis 4.2. Materials and Methods
85 89
4.2.1. Collection and authentication of leaves of Vitex negundo
89
4.2.2. Drying of leaves
89
4.2.3. Size reduction of leaves
89
4.2.4. Extraction of Vitex negundo
89
4.2.5. Phytochemical investigations
90
4.2.6. Chromatographic studies
92
4.2.7. Thin layer chromatographic studies
92
4.2.8. Column chromatographic studies
93
4.2.9. Methodology
93
4.2.10. Isolation of compound from Vitex negundo (leaves)
94
4.2.11. Characterization of the isolated compound by spectral analysis
95
4.3. Material and Methods
99
4.3.1. Collection and authentication of leaves of Butea frondosa
99
4.3.2. Drying of leaves
99
4.3.3. Size reduction of leaves
99
4.3.4. Extraction of Butea frondosa
99
4.3.5. Phytochemical investigations
100
4.3.6. Chromatographic studies
101
4.3.7. Thin layer chromatographic studies
102
4.3.8. Column chromatographic studies
102
4.3.9. Methodology
103
4.3.10. Isolation of compound from Butea frondosa (leaves)
104
4.3.11. Characterization of isolated compound by spectral analysis
105
References
Chapter 5: Pharmacological Activity
111
112-129
5.1. Anticataract activity
112
5.2. Experimental design
112
5.3. Dulbecco's modified eagle's medium (DMEM)
112
5.4. Standard drugs
112
5.5. Effect of extract on Hydrogen peroxide (oxidative stress) 0.5 mM induced cataract in goat lens.
112
5.6. Biochemical studies
113
5.6.1. Reduced glutathione (GSH)
113
5.6.2. Estimation of malondialdehyde (MDA)
113
5.6.3. Estimation of total protein
113
5.7. Result of Acorus calamus (roots)
114
5.7.1. Effect on GSH, MDA and Protein estimation in hydrogen peroxide induced oxidative stress
114
5.8. Result of Vitex negundo (leaves)
116
5.8.1. Effect on GSH, MDA and Protein estimation in hydrogen peroxide induced oxidative stress
116
5.9. Result of Butea frondosa (leaves)
118
5.9.1. Effect on GSH, MDA and Protein estimation in hydrogen peroxide induced oxidative stress
118
References
129
Chapter 6: Summary and Conclusion
130
6.1. Extraction of the roots of Acorus calamus
130
6.2. Phytochemical study
130
6.3. Structure elucidation of isolated compound AC-1 by spectral analysis
131
6.4. Pharmacological activity
131
6.5. Effect on GSH, MDA and Protein estimation in Hydrogen peroxide induced oxidative stress
132
6.6. Extraction of the leaves of Vitex negundo
132
6.7. Phytochemical study
132
6.8. Structural elucidation of isolated compound VN-1by spectral analysis
133
6.9. Pharmacological activity
133
6.10. Effect on GSH, MDA and Protein estimation in Hydrogen peroxide induced oxidative stress
134
6.11. Extraction of the leaves of Butea frondosa
134
6.12. Phytochemical study
134
6.13. Structure elucidation of isolated compound BF-1 by spectral analysis
135
6.14. Pharmacological activity
136
6.15. Effect on GSH, MDA and Protein estimation in Hydrogen peroxide induced oxidative stress 6.16. Conclusion
136 137
LIST OF ABBREVIATIONS Abbreviation
Full form
α
Alfa
AC
Acorus calamus
AED
Antiepileptic drug
b.w
Body weight
BMA
Butea monosperma agglutinin
β
Beta
BF
Butea frondosa
C
Carbon
CP
Crude powder
CDCl3
Deuterated chloroform
Contd.
Continued
CHCl3
Chloroform
CBH
Cambo ball head
CCl4
Carbon tetra Chloride
cm 0
-1
Centimeter inverse
C
Degree Celsius
δ
Delta
DNA
Deoxyribonucleic acid
DMEM
Dulbacco modified eagle medium
etc.
et cetera
EPG
Egg per gram
ED
Effective dose
FAB
Fast Ion Bombardment
FDA
Food and Drug Administration
Fig.
Figure
GOT
Glutamate Oxaloacetate Transaminase
GPT
Glutamate Pyruvate Transaminase
GGT
Gamma Glutamyl Transpeptidase i
G
Gypsum
γ
Gamma
g
Gram
GABA
Gamma amino butyric acid
GM-CSF
Granulocyte macrophage colony stimulating factor
H2O2
Hydrogen peroxide
HIV
Human Immunodeficiency virus
HT
Hydroxy tryptamine
Hz
Hertz
ICAM
Intracellular adhesion molecule
IL
Interleukin
IR
Infrared
IP
Indian Pharmacopoeia
Ig
Immunoglobulin
Kg
Kilogram
K
Potassium
LOCH
Lens Organ Culture Hydrogen Peroxide
LDH
Lactate dehydrogenase
LD
Lethal dose
LTs
Leukotrienes
mM
Millimole
MES
Maximum electroshock
MDA
Malondialdehyde
MCF
Michigan cancer foundation
MEAC
Methanolic extract of Acorus calamus
MEVN
Methanolic extract of Vitex negundo
MEBF
Methanolic extract of Butea frondosa
µl
Microlitre
ml
Millilitre
mg
Milligram ii
MHz
Mega Hertz
NMR
Nuclear Magnetic Resonance
NFkB
Nuclear factor kappa B
ODC
Ornithine decarboxylase
PDG
Platelet derived growth
PAF
Platelet aggregation factor
p.e
Paw edema
ppm
Parts per million
PTZ
Pentylenetetrazol
PCA
Principal Component Analysis
PGs
Prostaglandins
PD
Protection dose
%
Percentage
P
Phosphorous
Rf
Retention factor
SALP
Serum alkaline phosphatase
SGPT
Serum glutamate pyruvate transaminase
TNF
Tumor Necrosis Factor
TAA
Thioacetamide
TLC
Thin Layer Chromatography
TSM
Traditional System of Medicine
TBM
Triterpene Butea monosperma
USP
United State of Pharmacopoeia
UV
Ultraviolet
VCAM
Vascular cell Adhesion Molecule
VN
Vitex negundo
w/v
Weight upon volume
iii
iv
List of Tables Table No.
Title
Page No.
1.1
Vitamins, antioxidants and herbal drugs for the prevention and 22 treatment of cataract
3.1
Marketed ayurvedic formulations containing Acorus calamus
64
4.1
Chemical and solvents
75
4.2
Instruments/Apparatus
76
4.3
Physical nature and percentage yield of various root extracts of
77
Acorus calamus 4.4
Phytochemical test(s) of Acorus calamus (roots)
4.5
Column chromatography of methanolic extracts of Acorus calamus 84 roots
4.6
Interpretation of FTIR spectra of the isolated compound of Acorus 86 calamus
81
Interpretation of NMR spectra of the isolated compound of Acorus 4.7
87
calamus Interpretation of Mass spectra of the isolated compound of Acorus
4.8
88
calamus Physical nature and percentage yield of various extract of Vitex
4.9
4.10
90
negundo Phytochemical test(s) of Vitex negundo leaves
91
Column chromatography of methanolic extracts of Vitex negundo 4.11
4.12
94
leaves FTIR spectra of the isolated compound of Vitex negundo
96
Interpretation of NMR spectra of the isolated compound of Vitex 4.13
4.14
97
negundo Interpretation of Mass spectra of the isolated compound of Vitex
iv
98
Table No.
Title
Page No.
4.15
Physical nature and percentage yield of various leaves extracts of 100 Butea frondosa
4.16
Phytochemical test(s) of Butea frondosa (leaves)
101
Column chromatography of methanolic extracts of Butea frodosa 4.17
4.18
104
leaves FTIR spectra of the isolated compound of Butea frondosa
106
Interpretation of NMR spectra of the isolated compound of Butea 4.19
108
frondosa Interpretation of Mass spectra of the isolated compound of Butea
4.20
109
frondosa
Anticataract activity of the extract of Acorus calamus and 5.1
isolated compound against hydrogen peroxide induced 114 cataract.
5.2
Effect of various treatments on GSH, MDA and protein levels
115
against hydrogen peroxide induces cataract. Anticataract activity of the extract of Vitex negundo and 5.3
isolated compound against hydrogen peroxide induced 116 cataract
5.4
Effect of various treatments on GSH, MDA and protein levels
117
against hydrogen peroxide induced cataract Anticataract activity of the extract of Butea frondosa and 5.5
isolated compound against hydrogen peroxide induced 118 cataract
5.6
Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induced cataract
v
119
List of Figures Figure No.
Title
Page No.
1.1
Major risk factors implicated in cataractogenesis
10
1.2
Mechanisms associated with cataractogenesis
15
1.3
Formation of advanced glycation end products
16
1.4
Polyol pathway
18
3.1
Sweet flags specimen showing whole parts
57
3.2
Cultivation of Acorus calamus
58
3.3
Phytoconstituents of Acorus calamus
60
3.4
Vitex negundo plant
65
3.5
Phytoconstituents of Vitex negundo
67
3.6
Leaves of Butea frondosa
69
3.7
Phytoconstituents of Butea frondosa
71
4.1
TLC of isolated compound in visible region
82
4.2
TLC of isolated compound in ultraviolet region
82
4.3
FTIR spectra of the isolated compound of Acorus calamus
85
4.4
NMR spectra of the isolated compound
86
4.5
Mass spectra of the isolated compound
87
4.6
Chemical structure of β asarone
88
4.7
FTIR spectra of isolated compound of Vitex negundo
95
4.8
NMR spectra of isolated compound
96
4.9
Mass spectra of isolated compound
97
vi
Figure No.
Title
Page No.
4.10
Chemical structure of negundin A
98
4.11
FTIR spectra of isolated compound of Butea frondosa
105
4.12
NMR spectra of isolated compound
107
4.13
NMR spectra of isolated compound
107
4.14
Mass spectra of the isolated compound
109
4.15
Chemical structure of tridecanoic acid
110
5.1
Goat eyes
120
5.2
Goat lens being withdrawn from goat eye
120
5.3
Goat lenses (control and standard group) before treatment
121
Goat lenses (isolated compound and methanolic extract of 5.4
(Acorus calamus) before treatment
122
Goat lenses (isolated compound and methanolic extract of 5.5
(Vitex negundo) before treatment
123
Goat lenses (isolated compound and methanolic extract of 5.6
(Butea frondosa) before treatment
124
Goat lenses after incubation in control group complete 5.7
cataractogenesis after 17 hrs. and standard group after 46 hrs. 125 appears slightly hazy Goat lenses after incubation in isolated group after 42 hrs.
5.8
and methanolic extract group after 45 hrs. appears slightly 126 hazy
5.9
Goat lenses after incubation in isolated group after 40 hrs. and methanolic extract group after 42 hrs. appears slightly
vii
127
hazy Goat lenses after incubation in isolated group after 41 hrs. 5.10
and methanolic extract group after 44 hrs. appears slightly 128 hazy
viii
Chapter I
Introduction
1.1 Introduction Ayurveda is believed to be prevalent since last 5000 years in India. It is one of the most noted systems of medicine in the world. Ayurveda is based on the hypothesis that everything in the universe is composed of five elements viz. space, air, energy, liquid and solid. These elements exist in the human body in combined forms like Vata (space and air), Pitta (energy and liquid) and Kapha (liquid and solid). Vata, Pitta and Kapha together are called Tridosha (three pillars of life). Some important herbs from ayurveda include Rauwolfia serpentina, Asparagus racemosus, Cassia angustifolia, Sesamum indicum, Holarrhena antidysenterica, Withania somnifera, Aconitum napellus and Piper longum etc.1 Natural products, including plants, animals and minerals have been the basis of treatment of diseases from time immemorial. History of medicine dates back practically to the existence human civilization. The current accepted modern medicine or allopathy has gradually developed over the years by scientific and observational efforts of scientists. However, the basis of its development remains rooted in traditional medicine and therapies. The history of medicine includes many ludicrous therapies. Nevertheless, ancient wisdom has been the basis of modern medicine and will remain as one important source of future medicine and therapeutics. The future of natural products drug discovery will be more holistic, personalized and involve wise use of ancient and modern therapeutic skills in a complementary manner so that maximum benefits can be accrued to the patients and the community.2 Plants have played a crucial role in maintaining human health and improving the quality of human life for thousands of years. The World Health Organization has estimated that 80% of the earth’s inhabitants rely on traditional medicine for their health care needs, and most of this therapy involves the use of plants extracts or their active components. Therefore, therapeutic approach of several traditional medicines is rather more holistic. Majority of fundamental concepts of their medicinal systems still cannot be explained using modern tools.3 Medicinal plants sector has traditionally occupied an important position in the sociocultural, spiritual and medicinal area of rural and tribal lives of India. The global thrust areas for drugs from medicinal plants include disease conditions, whose incidence is unavailable or unsatisfactory. International market of medicinal plants is over US $ 60 billion per year, which is growing at the rate of 7% annually.4
School of Pharmaceutical Sciences, Shobhit University, Meerut
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Chapter I
Introduction
1.2 Traditional and orthodox medicine Traditional remedies invariably involve crude plant extracts containing multiple chemical constituents, which vary in potency from highly active (e.g. Digitalis leaf) to very weak (e.g. Cinnamon bark). In contrast, orthodox medicine relies heavily on single (or a very small member of) chemically well-characterized active ingredients exhibiting selective activities at, in many cases, well-established biological targets. These medicines are generally very potent and many exhibit fairly narrow windows between an effective and a toxic dose. Orthodox medicines are formulated into doses that are carefully standardized for bioavailability. Amongst our most invaluable orthodox medicines derived from compounds in higher plants are analgesic agents (e.g. morphine and codeine), antimalarial treatments (e.g. quinine), antitumour drugs (e.g. vincristine and taxol) and asthma therapies (e.g. cromoglycate).5 1.3 Herbal medicine used in ocular activity Traditional medicines such as Emblica officinalis (Euphorbiaceae), Atropa belladonna (Solanaceae), Azadirachta indica (Meliaceae), Berberis aristata (Berberidaceae), Acorus calamus (Araceae), Butea monosperma (Fabaceae), Cadaba indica (Capparaceae),Rosa indica (Rosaceae), Terminalia belerica (Combretaceae) Vitex negundo (Verbenaceae), Solanum nigram (Solanaceae), Tinospora cordifolia (Menispermaceae) are used in eye disorders.6-10 1.4 Diseases and disorders of the lens •
Cataracts are opacities of the lens. While some are small and do not require any treatment, others may be large enough to block light and obstruct vision. Cataracts usually develop as the aging lens becomes more and more opaque, but cataracts can also form congenitally or after injury to the lens. Diabetes is also a risk factor for cataract.
•
Presbyopia is the age-related loss of accommodation, which is marked by the inability of the eye to focus on nearby objects. The exact mechanism is still unknown, but age-related changes in the hardness, shape, and size of the lens have all been linked to the condition.
•
Ectopia lentis is the displacement of the lens from its normal position.
•
Aphakia is the absence of the lens from the eye. Aphakia can be the result of surgery or injury, or it can be congenital.
•
Nuclear sclerosis is an age-related change in the density of the lens nucleus that occurs in all older animals.
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Chapter I •
Introduction
Age-related macular degeneration (AMD) is a disease that blurs the sharp, central vision you need for "straight-ahead" activities such as reading, sewing, and driving. AMD affects the macula, the part of the eye that allows you to see fine detail. AMD causes no pain.
•
Amblyopia is the medical term used when the vision in one of the eyes is reduced because the eye and the brain are not working together properly. The eye itself looks normal, but it is not being used normally because the brain is favoring the other eye. This condition is also sometimes called lazy eye.
•
Microphthalmia is a disorder in which one or both eyes are abnormally small.
•
Anophthalmia is the absence of one or both eyes. These rare disorders develop during pregnancy and can be associated with other birth defects.
•
Refractive errors include nearsightedness and farsightedness, eye conditions that are very common. Most people have one or more of them. Refractive errors can usually be corrected with eyeglasses or contact lens.
•
Blepharitis is a common condition that causes inflammation of the eyelids. It can affect the inside or outside of the eyelids. The condition can be difficult to manage because it tends to recur.
•
Dry eye occurs when the eye does not produce tears properly, or when they evaporate too quickly. Dry eye can make it difficult to do some activities, such as using a computer or reading for an extended period of time, and it can decrease tolerance for dry environments, such as the air inside an airplane.
•
Glaucoma is a group of diseases that can damage the eye's optic nerve and result in vision loss and blindness. Open-angle glaucoma is the most common form of the disease.11
1.5 Anatomy of lens The crystalline lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. The lens, by changing shape, functions to change the focal distance of the eye so that it can focus on objects at various distances, thus allowing a sharp real image of the object of interest to be formed on the retina. This adjustment
School of Pharmaceutical Sciences, Shobhit University, Meerut
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Chapter I
Introduction
of the lens is known as accommodation. It is similar to the focusing of a photographic camera via movement of its lenses. The lens is flatter on its anterior side. The lens is also known as the aquula (Latin, a little stream, dim. of aqua, water) or crystalline lens. In humans, the refractive power of the lens in its natural environment is approximately 18 dioptres, roughly one-third of the eye's total power. 1.5.1 Position, size, and shape The lens is part of the anterior segment of the eye. Anterior to the lens is the iris, which regulates the amount of light entering into the eye. The lens is suspended in place by the suspensory ligament of the lens, a ring of fibrous tissue that attaches to the lens at its equator12, 13 and connects it to the ciliary body. Posterior to the lens is the vitreous body, which, along with the aqueous humor on the anterior surface, bathes the lens. The lens has an ellipsoid, biconvex shape. The anterior surface is less curved than the posterior. In the adult, the lens is typically circa 10 mm in diameter and has an axial length of about 4 mm, though it is important to note that the size and shape can change due to accommodation and because the lens continues to grow throughout a person’s lifetime.14 1.5.2 Variations among vertebrates In many aquatic vertebrates, the lens is considerably thicker, almost spherical, to increase the refraction of light. This difference compensates for the smaller angle of refraction between the eye's cornea and the watery medium, as they have similar refractive indices.15 Even among terrestrial animals; however, the lens of primates such as humans is unusually flat. In reptiles and birds, the ciliary body touches the lens with a number of pads on its inner surface, in addition to the zonular fibres. These pads compress and release the lens to modify its shape while focusing on things at different distances; the zonular fibres perform this function in mammals. In fish and amphibians, the lens is fixed in shape, and focusing is instead achieved by moving the lens forwards or backwards within the eye. 16 1.5.3 Lens structure and function The lens has three main parts: the lens capsule, the lens epithelium, and the lens fibers. The lens capsule forms the outermost layer of the lens and the lens fibers form the bulk of the interior of the lens. The cells of the lens epithelium, located between the lens capsule and the outermost layer of lens fibers, are found only on the anterior side of the lens.
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1.5.3.1 Lens capsule The lens capsule is a smooth, transparent basement membrane that completely surrounds the lens. The capsule is elastic and is composed of collagen. It is synthesized by the lens epithelium and its main components are Type IV collagen and sulfated glycosaminoglycans (GAGs). The capsule is very elastic and so causes the lens to assume a more globular shape when not under the tension of the zonular fibers, which connect the lens capsule to the ciliary body. The capsule varies from 2-28 micrometres in thickness, being thickest near the equator and thinnest near the posterior pole. The lens capsule may be involved with the higher anterior curvature than posterior of the lens.13 1.5.3.2 Lens epithelium The lens epithelium, located in the anterior portion of the lens between the lens capsule and the lens fibers, is a simple cuboidal epithelium. The cells of the lens epithelium regulate most of the homeostatic functions of the lens. As ions, nutrients, and liquid enter the lens from the aqueous humor, Na+/K+ ATPase pumps in the lens epithelial cells pump ions out of the lens to maintain appropriate lens osmolarity and volume, with equatorially positioned lens epithelium cells contributing most to this current. The activity of the Na+/K+ ATPases keeps water and current flowing through the lens from the poles and exiting through the equatorial regions.17 The cells of the lens epithelium also serve as the progenitors for new lens fibers. It constantly lays down fibers in the embryo, fetus, infant, and adult, and continues to lay down fibers for lifelong growth. 18 1.5.3.3 Lens fibers The lens fibers form the bulk of the lens. They are long, thin, transparent cells, firmly packed, with diameters typically between 4-7 micrometres and lengths of up to 12 mm long. The lens fibers stretch lengthwise from the posterior to the anterior poles and, when cut horizontally, are arranged in concentric layers rather like the layers of an onion. If cut along the equator, it appears as a honeycomb. The middle of each fiber lies on the equator. These tightly packed layers of lens fibers are referred to as laminae. The lens fibers are linked together via gap junctions and interdigitations of the cells that resemble "ball and socket" forms. The lens is split into regions depending on the age of the lens fibers of a particular layer. Moving outwards from the central, oldest layer, the lens is split into an embryonic nucleus, School of Pharmaceutical Sciences, Shobhit University, Meerut
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the fetal nucleus, the adult nucleus, and the outer cortex. New lens fibers, generated from the lens epithelium, are added to the outer cortex. Mature lens fibers have no organelles or nuclei. 18 1.5.4 Accommodation: changing the power of the lens The lens is flexible and its curvature is controlled by ciliary muscles through the zonules. By changing the curvature of the lens, one can focus the eye on objects at different distances from it. This process is called accommodation. At short focal distance the ciliary muscle contracts, zonule fibers loosen, and the lens thickens, resulting in a rounder shape and thus high refractive power. Changing focus to an object at a greater distance requires the relaxation of the ciliary muscle, which in turn increases the tension on the zonules, flattening the lens and thus increasing the focal distance. The refractive index of the lens varies from approximately 1.406 in the central layers down to 1.386 in less dense layers of the lens. This index gradient enhances the optical power of the lens. Aquatic animals must rely entirely on their lens both for focusing and to provide almost the entire refractive power of the eye as the water-cornea interface does not have a large enough difference in indices of refraction to provide significant refractive power. As such, lenses in aquatic eyes tend to be much rounder and harder.18 1.5.5 Crystallins and transparency Crystallins are water-soluble proteins that compose over 90% of the protein within the lens. The three main crystallin types found in the human eye are α-, β-, and γ-crystallins. Crystallins tend to form soluble, high-molecular weight aggregates that pack tightly in lens fibers, thus increasing the index of refraction of the lens while maintaining its transparency. β and γ crystallins are found primarily in the lens, while subunits of α -crystallin have been isolated from other parts of the eye and the body. α-crystallin proteins belong to a larger superfamily of molecular chaperone proteins, and so it is believed that the crystallin proteins were evolutionarily recruited from chaperone proteins for optical purposes. The chaperone functions of α -crystallin may also help maintain the lens proteins, which must last a human for his/her entire lifetime.19 Another important factor in maintaining the transparency of the lens is the absence of light-scattering organelles such as the nucleus, endoplasmic reticulum, and mitochondria within the mature lens fibers. Lens fibers also have a very extensive cytoskeleton that maintains the
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precise shape and packing of the lens fibers; disruptions/mutations in certain cytoskeletal elements can lead to the loss of transparency.20 1.5.6 Development and growth of human lens Development of the human lens begins at the 4 mm embryonic stage. Unlike the rest of the eye, which is derived mostly from the neural ectoderm, the lens is derived from the surface ectoderm. The first stage of lens differentiation takes place when the optic vesicle, which is formed from out pocketing in the neural ectoderm, comes in proximity to the surface ectoderm. The optic vesicle induces nearby surface ectoderm to form the lens placode. At the 4 mm stage, the lens placed is a single monolayer of columnar cells. As development progresses, the lens placode begins to deepen and invaginate. As the placode continues to deepen, the opening to the surface ectoderm constricts and the lens cells forms a structure known as the lens vesicle. By the 10 mm stage, the lens vesicle has completely separated from the surface ectoderm. After the 10 mm stage, signals from the developing neural retina induces the cells closest to the posterior end of the lens vesicle begin to elongate toward the anterior end of the vesicle.[12] These signals also induce the synthesis of crystallins. These elongating cells eventually fill in the lumen of the vesicle to form the primary fibers, which become the embryonic nucleus in the mature lens. The cells of the anterior portion of the lens vesicle give rise to the lens epithelium. Additional secondary fibers are derived from lens epithelial cells located toward the equatorial region of the lens. These cells lengthen anteriorly and posteriorly to encircle the primary fibers. The new fibers grow longer than those of the primary layer, but as the lens gets larger, the ends of the newer fibers cannot reach the posterior or anterior poles of the lens. The lens fibers that do not reach the poles form tight, interdigitating seams with neighboring fibers. These seams are readily visible and are termed sutures. The suture patterns become more complex as more layers of lens fibers are added to the outer portion of the lens. The lens continues to grow after birth, with the new secondary fibers being added as outer layers. New lens fibers are generated from the equatorial cells of the lens epithelium, in a region referred to as the germinative zone. The lens epithelial cells elongate, lose contact with the capsule and epithelium, synthesize crystallin, and then finally lose their nuclei (enucleate) as they become mature lens fibers. From development through early adulthood, the addition of secondary School of Pharmaceutical Sciences, Shobhit University, Meerut
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lens fibers results in the lens growing more ellipsoid in shape; after about age 20, however, the lens grows rounder with time.21 1.5.7 Nourishment of the lens The lens is metabolically active and requires nourishment in order to maintain its growth and transparency. Compared to other tissues in the eye, however, the lens has considerably lower energy demands. By nine weeks into human development, the lens is surrounded and nourished by a net of vessels, the tunica vasculosa lentis, which is derived from the hyaloid artery. Beginning in the fourth month of development, the hyaloid artery and its related vasculature begin to atrophy and completely disappear by birth.21 In the postnatal eye, Cloquet’s canal marks the former location of the hyaloid artery. After regression of the hyaloid artery, the lens receives all its nourishment from the aqueous humor. Nutrients diffuse in and waste diffuses out through a constant flow of fluid from the anterior/posterior poles of the lens and out of the equatorial regions, a dynamic that is maintained by the Na+/K+ ATPase pumps located in the equatorially positioned cells of the lens epithelium.22 Glucose is the primary energy source for the lens. As mature lens fibers do not have mitochondria, approximately 80% of the glucose is metabolized via anaerobic respiration. The remaining fraction of glucose is shunted primarily down the pentose phosphate pathway. The lack of aerobic respiration means that the lens consumes very little oxygen as well.23 1.6 Cataract: General overview Cataracts are described as an opacification (cloudiness) of the lens that leads to the scattering of light entering the eye and a loss of vision. Cataracts, which affect more than 50 million people
24
are the most common cause of blindness in the world. In first world countries,
old age is the single largest cause of cataracts: only about 5% of Caucasian Americans aged 5264 years have cataracts, where as 18% of those aged 65-75 and 46% of those aged 75-85 are affected by cataracts.25 As the average life span increases, the prevalence of cataract also increases. Cataract formation cannot be prevented or reversed,26 however it can be cured by surgical replacement of the lens. There have been significant advances in surgical techniques and refinement of intraocular lens implants which have benefited cataract patients. In India alone around 30 million people suffer from cataract. Thus, the expense and unavailability of surgery mean that non surgical medical therapy or nutritional treatment to inhibit the formation School of Pharmaceutical Sciences, Shobhit University, Meerut
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or slow the progression of cataracts is an important goal in experimental eye research to benefit patients and reduce the huge economic burden. 27 1.7 Factors implicated in cataractogenesis Several risk factors have been identified in the pathogenesis of cataract. Apart from aging, smoking, diabetes, gender, steroids and nitric oxide are responsible for the development of cataract. These risk factors have been associated with different morphological type of cataract. 1.7.1 Smoking: Smoking is thought to increase the risk of cataract, at least in part by increasing the oxidative stress in the lens caused by the generated free radicals. In the presence of tobacco smoke, these free radicals may directly damage lens proteins and the fiber cell membrane in the lens.28, 29 Tobacco leaves contain a significant amount of cadmium (Cd), which is absorbed into the body when a person smokes or chews tobacco and this Cd replace the bivalent metals like Zinc (Zn), copper (Cu) and manganese from super oxide dismutase (SOD), a powerful antioxidant.30 1.7.2 Diabetes: There are several ways that diabetes can affect the eyes but the most common cause of loss of vision is cataract. Cataractogenesis is one of earliest secondary complications of diabetes mellitus, a severe metabolic disorders characterized by hyperglycemia. Some mechanisms have been proposed for cataract formation in diabetes mellitus such as excessive tissue sorbitol concentrations, abnormal glycosylation of lens proteins and increased free radical production. 31 1.7.3 Gender: A number of epidemiological studies using cross sectional data have shown an increased prevalence of cataract in women compared with men.32 The cause of the gender differences in cataract occurrences is not clear but could be related to the hormonal differences between women and men. Postmenopausal estrogen deficiency may be a factor. Recent epidemiologic data provided some evidence that estrogen and hormone replacement therapy may play a protective role in reducing the incidence of age related cataract. 33 1.7.4 Steroids: The association between steroid use and development of cataract is well established. There seems to be a consensus that higher the dose of steroid and longer the duration of use, the higher will be the risk for posterior sub capsular cataract.34 Steroids cause an inhibition of the cation pump in the lens capsule and the resulting electrolyte/water imbalance is responsible for cataract formation.35
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1.7.5 Nitric oxide: O2- in itself is not highly toxic but it may react with other molecules yielding more reactive compounds. For example, the reaction with nitric oxide (NO) generates peroxynitrite (ONOO-), which causes extensive cell damage and can also have an important role in diabetic cataract formation. 36, 37 Apart from the above mentioned risk factors, genetic factors, socioeconomic status, illiteracy, malnutrition, diarrhea, myopia, renal failure, hypertension, sunlight, ultraviolet exposure, obesity, chemical burn, glaucoma and alcohol.38,39 have also been implicated in cataracogenesis. Major risk factors implicated in cataractogenesis are shown in figure 1.1.
Fig. 1.1 Major risk factors implicated in cataractogenesis 1.8 Etiology of cataract Developing anti-cataract agents has been difficult because cataract is not a single disease with a single aetiology. There are three major categories of cataract (nuclear, cortical, and posterior sub-capsule), each of which is multifactorial in aetiology and highly variable in severity and rate of progression. Further complicating the situation, the factors contributing to age-related cataractogenesis are a combination of pathological and normal aging processes, which have no obvious borders to distinguish them. Cataracts may be prevented if the mechanisms of formation are known. Based on the available knowledge of the biology of the normal lens and the cataractogenic process, three hypotheses have been proposed for the aetiology of cataract and three approaches have accordingly been adopted in the design of anti-cataract agents. 27
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1.9 Hypothesis for cataract The first hypothesis is that chronic oxidative stress is a major factor in the aetiology of age related cataract. Experimental evidence suggests that oxidative stress due to the generation of free radicals plays a role in the pathogenesis of cataracts and that the process can be prevented or ameliorated by antioxidants. Therefore, agents with anti-oxidative properties have received the most attention. Such compounds comprise of three categories including antioxidant vitamins (e.g., E, C, β-carotene); functional mimics of antioxidant enzymes; and a wide variety of low molecular weight compounds with antioxidant activity. 40 The second hypothesis is that phase separation phenomena are integral to cataract development. Phase separation results from non-covalent attractive interactions between proteins in concentrated solutions, creating protein-rich and protein poor regions. In the lens, formation of such domains creates light scattering, leading to cataract. Two putative phase separation inhibitors, pantethine and the radio protective phosphorothioate WR-77913, were tested in several acute animal models of cataract and displayed the delay of the onset of cataract. 41 The third hypothesis is the “protease hypothesis”. Calcium activated neutral enzymes, calpains, can induce proteolysis and truncate crystalline to precipitate and scatter light to form cataract.42 Therefore, research on calpain inhibitors is another approach to prevent or inhibit cataract formation. It has been reported that, when lambs with an inherited cataracts were treated with eye drops containing the calpain inhibitor SJA6017 for 4 months, progression of cataracts were slowed down in treated eyes compared with non-treated eyes.43 1.10 Models proposed to study cataract In order to study cataracts and possible treatments for cataracts, a number of in vivo animal models have been developed. For example, administration of L-buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis, to preweanling mice (aged ≤ 12-days) provides a model system for the induction of cataracts by depletion of lens glutathione.44 The strong sulfhydryl oxidant, selenite, has been used to produce cataract in rats.45 This selenite-induced cataract model has been extensively utilized to demonstrate that calpain-induced proteolysis causes truncated crystallins to precipitate and scatter light. Other in vivo experimental animal models such as hyperbaric oxygen, and UVA light, have also been utilized to investigate the mechanism of formation of human senile nuclear cataract.46 School of Pharmaceutical Sciences, Shobhit University, Meerut
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for example, H2O2-induced cataract in cultured lenses from rabbit.47 and rat
48
diamide (a thiol-
specific oxidant)-induced cataract in Sprague-Dawley rat cultured lens model.49 Ionomycin cataract in rat cultured lens model,
50
4-bromo-calcium ionophore A23187 (Br-A23187)-induced
cataract in guinea pig and rabbit cultured lens model 27
rhesus monkey lens model.
51
and sugar xylose-induced cataract in
These studies have established the underlying premise that a lens
organ culture model system can be used to screen potential anti-cataract agents. The lens, which is a vascular and non-innervated in vivo, can be maintained in a fully viable state in organ culture. Opacity can be induced in cultured lenses by various chemical or environmental perturbations, and prevention or inhibition of opacification can be observed after addition of appropriate agents to counteract the cataractogenic stresses.27 Sheep lenses are considered to be more appropriate models of the humans lens than the rodent lenses commonly used for lens research. Rats and mice have lenses which are smaller than human and are spherical in shape compared to the flattened disc shape of the human lens. Also in sheep and human, the biconvex lens shape is altered by the ciliary muscle whereas the lens is moved backwards and forwards to focus light on the retina in the rodent eye.52 Oxidative stress, an excess of pro-oxidants relative to antioxidants and a key factor in the gradual loss of lens transparency, is implicated in the initiation of maturity onset cataract which appears late in life and is probably not associated with congenital conditions or other diseases, such as diabetes.53 Evidence from epidemiological studies, model systems and human lenses obtained after cataract surgery, has indicated a role for oxidation in this opacification process. This has fuelled interest in the role of diet and dietary supplements in slowing down the progression of cataract 54 concluded that dietary antioxidants have a significant impact on cataract development based on the epidemiological evidence. Experimental studies have shown that, pretreatment of the plant antioxidant, quercetin, at concentrations of 30 µM for 24 h, inhibited hydrogen peroxide-induced oxidation of the rat lens.55, 56 α-lipoic acid, which plays an essential role in mitochondrial dehydrogenase reactions, has recently gained considerable attention as an antioxidant. Lipoate, or its reduced form dihydrolipoate, reacts with reactive oxygen species such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also protects membranes by interacting with vitamin C, which may in turn recycle vitamin E. In addition to its antioxidant School of Pharmaceutical Sciences, Shobhit University, Meerut
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activities, dihydrolipoate may exert proxidant actions through reduction of iron. α-lipoic acid administration has been shown to be beneficial in a number of oxidative stress models such as ischemia-reperfusion injury, diabetes (both α-lipoic acid and dihydrolipoic acid exhibit hydrophobic binding to proteins such as albumin, which can prevent glycation reactions), cataract formation, HIV activation, neurodegeneration, and radiation injury. Furthermore, lipoate can function as redox regulator of proteins such as myoglobin, prolactin, thioredoxin and NF-κB transcription factor.57 1.11 Classification of cataract
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1.11.1 Congenital and developmental cataract Congenital cataract is present at birth, and developmental cataract is that cataract which develops during the development of the lens. These type of cataracts are developed due to some disturbance, at a certain phase of growth of the lens, therefore, these types of opacities of the lens are usually stationary and they may be of various types as noted below. Underlying cause is not known but it may be due to•
Maternal malnutrition
•
Maternal infection, particularly by virus of German measles or rubella.
•
Deficient oxygenation duo to placental hemorrhage.
1.11.2 Blue-dot cataract •
Tiny bluish white opaque spot scattered all over the lens.
•
No visual disturbance.
1.11.3 Coronary cataract •
Club shaped opacities in the peripheral part of the cortex
•
Arranged like a corona or crown.
•
Develops at puberty.
•
No visual disturbance.
•
Axial area of the lens remains clear.
1.11.4 Capsular or polar cataract Anterior capsule or polar cataract It forms as a result of delayed formation of the anterior chamber during the development of the lens. A white plaque is formed in the anterior lens capsule in the pupillary
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area. Sometimes, this opacity may project into the anterior chamber in the form of a pyramid, then it is called anterior pyramidal cataract. Posterior Capsular Cataract Due to persistence of the posterior part of the vascular sheath of the lens, the opacity is usually tiny and there is very little visual disturbance. 1.11.5 Sutural cataract Tiny opaque dots situated in the Y sutures of the lens. No visual disturbance. 1.11.6 Coralliform cataract Minute opacities situated in the central area of the lens, in the form of a coral. No visual disturbance. 1.11.7 Floriform cataract The opacities are annular in shape arranged like petals of flowers and situated in the axial part of the lens. No visuals disturbance. 1.11.8 Central cataract The nucleus of the lens shows opacity. The opacity may be granular when there is no visual disturbance or the whole of the nucleus may be opaque associated with visual disturbance. This type of cataract may be unilateral or bilateral. 1.11.9 Lamellar or zonular cataract It is the most common variety of cataract in children and is bilateral. It may develop at the later part of intra-uterine life or early infancy. Sometimes it is hereditary. 1.11.10 Total cataract It may be unilateral or bilateral and is usually congenital. The entire lens is opaque. The lens matter may remain soft or may liquefy to form milky fluids contained in the capsule. 1.11.11 Acquired cataract Acquired cataracts develop in the intrauterine period, and the opacity generally does not enlarge or change with age. In acquired cataracts, parts of the lens almost invariably remain transparent, and visual acuity is not completely impaired. Depending on the site of the opacities, cataracts may be anterior or posterior polar (limited opacities of the capsule of the lens), lamellar, and so forth.59
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1.12 Mechanisms associated with cataract Loss of transparency during human cataract formation results from a variety of complex metabolic and physiological mechanisms, which act in combination to change the refractive index.60 Studies on lens proteins indicate that post translational modifications occurs in the lens proteins during cataractogenesis as a result of chemical actions that include oxidation, glycation, Schiff base formation, proteolysis, transmidation, carbamylation, phosphorylation, and elevated calcium levels.61 The post translational modifications alter attractive forces between lens proteins to favour aggregation, disruption of normal lens cell structure and opacification.62 Mechanisms associated with cataractogenesis are shown in figure 1.2.
Fig. 1.2 Mechanisms associated with cataractogenesis
The multiple mechanisms proposed for cataractogenesis, the role of the following pathways in cataract development. •
Non enzymatic glycation
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•
Oxidative stress
•
Polyol pathway
1.12.1 Non enzymatic glycation Under hyperglycemic conditions, part of the excess glucose reacts non enzymatically with proteins or other tissue or blood constituents, thus increasing the physiological rate of non enzymatic glycation.63 Chronic, irreversible abnormalities unaffected by normalization of blood glucose levels primarily involve long lived molecules, extra cellular matrix, eye lens crystallins, and chromosomal DNA. Due to their characteristic chemical properties, advanced products of non enzymatic glycation play a critical role in the evolution of sugar cataract. The formation of advanced glycation end products (AGEs) begins with the attachment of a glucose carbonyl group to a free amino group of proteins or amino acids to form a labile Schiff base adduct as the first step of the complex Millard process. Levels of the unstable Schiff base increase rapidly, and equilibrium is reached after several hours. Once formed, Schiff base adducts undergo slow chemical rearrangement over a period of weeks to form more stable, but still chemically reversible, Amadori products. 64 Specific chemical characterization of AGE proteins has been difficult, as Amadori products can theoretically undergo a large number of potential rearrangements. Immunological and chemical evidence indicates that progressive accumulation of AGEs in the diabetic eye lens contributes to accelerate cataractogenesis in hypoglycemic experimental animals and diabetic humans. 65, 66 Formation of advanced glycation end products are presented in figure 1.3.
Fig.1.3 Formation of advanced glycation end products School of Pharmaceutical Sciences, Shobhit University, Meerut
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1.12.2 Oxidative stress The osmotic and exogenous or endogenous oxidative stresses play an important role in the pathogenesis of cataract.67 Oxidative stress may result from an imbalance between the production of reactive oxygen species (ROS) and the cellular antioxidant defense mechanisms. In the cells of the eyes, ROS may initiate a surge of toxic biochemical reactions such as peroxidation of membrane lipids and extensive damage to proteins causing intracellular protein aggregation and precipitation and eventually leading to lens opacification.68, 69 On exposure of the eye to oxidative stress, the redox set point of the single layer of the lens epithelial cells quickly changes, going from a strongly reducing to an oxidizing environment. Almost concurrent with this change is extensive damage to the DNA and membrane pump systems, followed by loss of epithelial cell viability and death by necrotic and apoptotic mechanisms leading to cataract.70, 71
1.12.3 Polyol pathway The mechanism involved in the progression of diabetic cataracts is different from senile cataracts. The accumulation of polyols within the lens is the primary contributing factor. Certain tissues of the body, including the eye lens, do not require insulin for glucose and other simple sugars to enter. In diabetes, sugar is in high concentration in the aqueous humor and can diffuse passively into the lens. The enzyme aldose reductase within the lens converts glucose to sorbitol or galactose to galctitol. These polyols cannot diffuse passively out of the lens and accumulate or converts to fructose. The accumulation of polyols results in an osmotic gradient, which encourages diffusion of fluid from the aqueous humor. The water drags sodium with it and the swelling and electrolyte imbalances result in cataract formation. Polyol pathway is shown in figure 1.4.
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Fig.1. 4 Polyol pathway 1.13 Pharmacological strategies for prevention of cataract Drugs have been developed which are aimed to interact at the level of altered lens metabolism and lens pathophysiology. The anticataract agents claimed to be effective in vitro, in vivo and in epidemiological studies may be broadly classified in the following categories: •
Aldose reductase inhibitors
•
Non steroidal antiinflammatory drugs
•
Agents acting on glutathione
•
Vitamins, minerals, antioxidants and herbal drugs
•
Miscellaneous agents
1.13.1 Aldose reductase inhibitors ARIs are aimed to block the metabolic pathways of glucose responsible for diabetic vascular dysfunction. Their role in the prevention of diabetic cataract in animals is now well established.72,73 Numerous natural and synthetic compounds have been found to inhibit aldose reductase. These so called ARI bind to aldose reductase, inhibiting polyol production. The rationale of using sorbitol -lowering agents has eroded over the years because the aldose reductase is remarkably sluggish with glucose. Furthermore, adult human lenses incubated in high glucose media do not accumulate sorbitol. There are a number of ARI known to possess anticataract potential and delay the galactose induced cataract in different experimental models.74 Some of these include alrestatin, sorbinil, sulindac, naproxen, aspirin, tolrestat, statil, and bioflavonoid. Flavonoids are among the most potent naturally occurring ARI. Several evaluations
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of in vitro animal lenses incubated in high sugar mediums have found flavonoids to inhibit aldose reuctase.75, 76 1.13.2 Non steroidal anti-inflammatory drugs Non steroidal anti-inflammatory drugs (NSAIDs) have emerged as another group of drugs with anticataract potential. The first indication regarding the probable use of NSAIDs as prophylactic anticataract agents came from studies on aspirin use in patients with rheumatoid arthritis and diabetes.77 subsequently; a number of NSAIDs with diverse chemical structures were reported to delay the phenomenon in experimental animals. The NSAIDs extensively studied are aspirin, paracetamol, ibuprofen, naproxen, sulindac and bendazec.78-80 The anticataract activity of these drugs is explained by virtue of their effect on different biochemical pathways. The mechanism associated with the protective effect of NSAIDs includes acetylation, inhibition of glycosylation and carbamylation of lens proteins.81 Anticataract activity of aspirin, sulindac and naproxen eye drops was also studied and they were found to delay both onset and progression of cataract in different models of cataractogenesis, moreover, there were no adverse side effects even after long term application.82 Subsequent studies further confirmed that aspirin is a potential anticataract agent.83 Bendazac, a compound resembling indomethacin in its structure, emerged as a potential radical scavenger and anticataract agent. Bendazac protects lens and serum proteins denaturation in vitro and in vivo.84,85 5- hydroxybendazac, a derivative, was found to be more effective than the parent compound in protecting lens protein against cyanate, glucose-6-phosphate and galactose.86 1.13.3 Agents which act on glutathione The most important function of glutathione (GSH) is to deactivate and render excess free radicals and keep them harmless. GSH is composed of the amino acids cysteine, glutamic acid and glycine and its synthesis within the lens takes place in two ATP dependent steps. There are several ways in which GSH or its depletion can affect the opacity of the lens.87 The mechanisms of cataract prevention including; •
maintaining sulphydryl (SH) groups on proteins in their reduced form preventing disulfide cross linkage.
•
protecting SH groups on proteins important for active transport and membrane permeability and
•
preventing oxidative damage from H2O2.
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88
cataract.
GSH has been reported to control calcium influx and protect lens protein against
damaging effects of osmotic and oxidative stress .89,90 Large amount of research has been done on antioxidants and vitamins, and the role of GSH in the prevention of cataract has been reported. A recent study indicates that vitamin E protects the antioxidative defense mechanisms directly or indirectly through increased levels of GSH.91 The anticataract effect of melatonin (a scavenger of free radical), was demonstrated which was reported to be due to its stimulatory effect on GSH production.92
1.13.4 Vitamins, minerals, antioxidants and herbal drugs Vitamins: The potential role of vitamins in preventing cataract is well documented, especially vitamin C or ascorbic acid which plays an important part in lens biology, both as an antioxidant and as a UV filter.93 Dietary deficiency of vitamin C leads to reduction in lens concentrations of ascorbate.94 A research study on guinea pigs shows that ascorbate inhibits galactose cataract.95 Similarly another study reveals that intake of ascorbate increases the level of vitamin C in rat lens.96 Vitamin E also has an important part to play in lenticular antioxidant status. A number of studies have evaluated the anticataract potential of vitamin E and found to be effective against galactose, steroids and UV radiation induced cataract.97-100
Minerals: The excessive free radical attack implicated in the development of cataract can be prevented by dietary intake of micronutrients such as zinc, copper and manganese. Copper and zinc are required for the catalytic activity of metal protein and SOD.101 Plasma levels of zinc and copper were found to be significantly low in cataract patients.102 Selenium is an integral part of the enzyme, glutathione peroxidase. A decrease in glutathione peroxidase activity has been found in the lenses of selenium deficient rats. 103 Antioxidants: It is widely accepted that oxidative stress is a significant factor in the progression of cataractogenesis.104,105 Oxidative stress is associated with increased reactive oxygen species and is known to accelerate cataract formation since superoxide is converted to a toxic substance, namely hydrogen peroxide. This reaction is prevented by antioxidant enzymes, namely catalase, superoxide dismutase and glutathione peroxidase. Antioxidants are key prophylactic agents in preventing oxidation related cataractogenesis. A large number of epidemiological and School of Pharmaceutical Sciences, Shobhit University, Meerut
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interventional studies have been investigated for the role of dietary antioxidant supplement in the incidence of cataract. Carotenoids are natural lipid –soluble antioxidants. It is reported that persons with a high intake of carotene reduce the incidence of risk of cataract,106 and the relationship between nuclear cataract and intake of alpha carotene, beta carotene, lutein, lycopene and cryptoxanthin stratifying by gender and by regular multivitamin use.107 Among all carotenoids lycopene has a high antioxidative activity and experts a protective effect in varios diseases.108 Curcumin, the active principle of turmeric, has been shown to have antioxidant activity in vitro and in vivo.109 The effect of curcumin on cataract has also been established. Curcumin delays the onset and maturation of galactose- induced110 and streptozotocin induced diabetic cataracts.111 Curcumin also prevents oxidative stress induced cataract.112
Herbal drugs: In recent years, great emphasis has been laid on exploring the possibility of using our natural resources to delay the onset and progression of cataract. A great number of medicinal plants and their formulations are reported to possess antioxidant properties and offer protection against cataract. The aqueous extract of Ocimum sanctum possesses potential anticataract activity against oxidative stress induced experimental cataractogenesis. The protective effect was supported by restoration of the antioxidant defense system.113 The aqueous extracts of well known herbal antidiabetic drugs namely Pterocarpus marsupium and Trigonella foenum graceum exerted a favorable anticataract effect.114 A recent research study found that grape seed proanhocyanidin extract effectively suppressed cataract formation in rats.115 Flavonoids from Emilia sonchifolia modulate the lens opacification and oxidative stress in selenite induced cataract.116 Dregea volubilis is a traditionally used medicinal plant for the treatment of various eye ailments, now its potential anticataract effect has been reported which is attributed to drevogenin D, a triperpenoid aglycone.117 Certain herbal drugs, especially Ginkgo biloba extract have been found to possess potential therapeutic effect in radiation induced cataract.118 The anticataract activity of green tea (Camellia sinensis) has been studied extensively and antioxidative potential is noted to be major mechanism in the prevention of cataractogenesis.
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Miscellaneous agents: Various substances with diverse chemical structures and properties are reported to have protective effect against cataract in different experimental models. ACE inhibitors have found to afford protection from free radical damage in many experimental conditions.119 Recently, the anticataract activity of lisinopril and enalapril was evaluated in glucose induced cataract in vitro and found to offer significant protection. It was concluded that the effect might be due to the antioxidant and free radical scavenging activity, as evidenced by a decrease in malondialdehyde in treated lens.120 Various supplements and mechanism of action of vitamins, antioxidants and herbal drugs for the prevention and treatment of cataract are presented in table 1.1.
Table 1.1 Vitamins, antioxidants and herbal drugs for the prevention and treatment of cataract Supplements
Mechanism of action
Vitamin C
Preserves glutathione levels; protects the Na+/K+ pump.
Riboflavin
Precursor to FAD, coenzyme for glutathione reductase which recycles glutathione.
Vitamin E
Antioxidant; increases glutathione; supplementation associated with prevention.
Glutathione
Deficiency noted in cataractous lenses; important component of the innate antioxidant system in the lens.
Carotenes
Antioxidant; higher levels associated with decreased risk of cataract.
Lycopene
Major carotenoids; possesses potential antioxidative property; reduces the risk associated with osmotic stress.
Curcumin
Antioxidant; reduces apoptosis in sugar cataract; inhibits the enzyme AR in polyol pathways
Stobadine
A novel synthetic pyridoindole, an antioxidant, effective against diabetic cataract.
Ocimum sanctum
Restores the antioxidant defense system; inhibits lens protein degradation.
Emillia sonchifolia
Acts as an antioxidant and inhibits the lipid peroxidation
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Chapter I
Introduction reaction.
Emblica officinalis
Potent inhibitors of aldose reductase; reduce the osmotic stress.
Dregea volubilis
Preserves the antioxidant mechanisms and lower the level of lipid peroxidation.
Vaccinium myritillus
Potent antioxidant.
Ginkgo biloba
Antioxidant that protects the lens from various oxidative stress.
Camellia sinensis
Inhibits oxidative stress by balancing the antioxidant defense mechanism.
Pterocarpus marsupium
Prevents diabetic cataract by reduces the risk associated with osmotic stress.
Trigonella foenum-graceum
Prevents diabetic cataract by reducing the risk associated with osmotic stress.
Grape seed
Increases glutathione level; reduces the lipid peroxidation.
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References 1. Kokate, C. K.; Purohit, A. P. Pharmacognosy, Nirali Prakashan. 1999, 12, 4-5. 2. Patwardhan, B.; Hooper, M., Int. J. Alternative complements Med. 1992, 10, 9-11. 3. Bruneton, J.; Hatton, C. K. Pharmacognosy, translator Paris Lavousier publisher. 1995, 30-35. 4. Tewari, D. N. Report of the taskforce on conservation and sustainable use of medicinal plants. Planning commission. 2000, 1-18. 5. Evans, W. C., Trease and Evans. Pharmacognosy, Saunders publication. 2002, 15, 109. 6. Sukhdev. A selection of prime Ayurvedic plant drugs, Anamaya publisher. 2006, 118. 7. The Wealth of India. National Institute of Science Communication, CSIR, Vol. III. 1952, 168. 8. Pillay, P. P.; Iyer, K. M. Current science. 1958, 27, 256. 9. Karrer. Supplement-1, 181,234, supplement-2,(Part-1). 205, 219,814. 10. Kokate, C. K.; Purohit, A. P., Pharmacognosy, Nirali Prakashan. 1999,12, 217. 11. http://www.nei.nih.gov/health/cataract/ 12. http://www.biology-online.org/dictionary/Equator_of_lens. 13. http://medical-.thefreedictionary.com/equator+of+the+crystalline+lens. 14. Forrester, J.; Dick, A.; McMenamin, P.; William, L. The Eye: Basic Sciences in Practice. London: W.B. Saunders Company Ltd. 1996, 28, 1790-6. 15. Kardong, K. Vertebrates: Comparative anatomy, function, evolution, 5th ed. Boston: McGraw-Hill. 2008, 676-677. 16. Alfred, R.; Sherwood, P.; Thomas, S. The Vertebrate Body. Philadelphia, PA: HoltSaunders International. 1977, 463–464. 17. Candia, O. Electrolyte and fluid transport across corneal, conjunctival and lens epithelia. Experimental Eye Research. 2004, 78 (3): 527-535. 18. Eye, "Encyclopædia Britannica from Encyclopædia Britannica 2006 Ultimate Reference Suite DVD. 2009. 19. Eugene, H. Optics, 2nd ed.Addison Wesley. 1987, 178. 20. Andley, U. Crystalline in the eye: function and pathology. Progress in Retinal and Eye Research. 2006. 26 (1): 78-98.
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21. Bloemendal, H.; De Jong,W.; Jaenicke, R.; Lubsen, N.H.; Slingsby, C.;Tardieu, A. Aging and vision: structure, stability, and function of lens crystallins. Progress in Biophysics and Molecular Biology. 2004, 86 (3), 407-485. 22. The Eye: Basic Sciences in Practice.102, 1790-6. 23. Whikehart, R. D. Biochemistry of the Eye, 2nd ed..Philadelphia: Butterworth Heinemann. 2003, 107-8. 24. Taylor, A.; Davies, K. J. Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens. Free Radic Biol Med. 1987, 3(6), 371-377. 25. Kahn, H. A.; Leibowitz, H. M.; Ganley, J. P.; Kini, M. M.; Colton, T.; Nickerson, R. S.; Dawber, T. R. The Framingham Eye Study. I. Outline and major prevalence findings. Am J Epidemiol. 1977, 106(1), 17-32. 26. Bhatt, K. S. Nutritional status of thiamine, riboflavin and pyridoxine in cataract patients. Nutritional Reports International. 1987, 36(3), 685-692. 27. Zigler, J. S.; Qin, C.; Kamiya, T.; Krishna, M. C.; Cheng, Q. F.; Tumminia, S.; Russell, P. Tempol- H inhibits opacification of lenses in organ culture. Free Radical Biology & Medicine. 2003, 35(10), 1194-1202. 28. Sulochana, K, N.; Punitham, R.; Ramakrishnan, S.; Effect of cigarette smoking on cataract, antioxidant enzymes and constituent minerals in the lens and blood of humans. Indian J Pharmacology. 2002, 34, 428-431. 29. Lindblad, B.E.; Hakansson N.; Svensson, H.; Philipson, B.; Wolk, A. Intensity of smoking and smoking cessation in relation to risk of cataract extraction: A prospective study of women. Am J epidemiol. 2005, 167, 73-79. 30. Ramakrishnan, S.; Sulochana, K.N.; Selvaraj, T.; Abdulrahim, A.; Lakshmi, M.; Arunagiri, K.; Smoking of beedies and cataract: cadmium and vitamin C in the lens and blood. Br J Ophthalmol. 1995, 79, 202-206. 31. Aksoy, H.; Keles, S.; Kocer, F. Diabetic cataract and the total antioxidant status in aqueous humor. Clin Chem Lab Med. 2001, 39,143-5. 32. Mc Carty, C.A.; Mukesh, B.N.; Fu, C.L.; Taylor, H.R. The epidemiology of cataract in Australia. Am J Ophthalmology. 1999, 128, 446-65. 33. Christine, Y.; Paul, M.; Robert, G.C.; Panchapakesan, J.; Elena, R.; Angela, M.H. Hormone replacement therapy, reproductive factors, and the incidence of cataract and School of Pharmaceutical Sciences, Shobhit University, Meerut
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cataract surgery: The blue mountains eye study. Am J Epidemiology. 2002, 155,9971006. 34. Jaime, E.D.; Eric, D.; Abbot, F.L. Steroid induced cataract: New perspectives from in vitro and lens culture studies. Exp Eye Res. 1997, 65,507-16. 35. Mayman, C.L.; Miller, D.; Tijerina, M.L. In vitro production of steroid cataract in bovine lens. Acta Ophthalmol. 1979,57,1107-16. 36. Eva, M.O.; Stefan, L.M.; Anders, B. Glucose-induced cataract in CuZn-SOD null lenses: An effect of nitric oxide? Free Radical Biol Med. 2007, 42, 1098-105. 37. Varma, S.D.; Hegde, K.R. Susceptibility of the ocular lens to nitric oxide: Implications in cataractogenesis. J Ocul Pharmacol Ther. 2007,23,188-95. 38. Harding, J. Cataract: Biochemistry, epidemiology and pharmacology. London: Chapman-Hall. 1991,83-124. 39. Ito, Y.; Nabekura, T.; Takeda, M.; Nakao, M.; Terao, M.; Hori, R. Nitric oxide participates in cataract development in selenite treated rats. Current Eye Research. 2001, 22, 215-220. 40. Robertson, J. M.; Donner, A. P.; Trevithick, J. R. Vitamin E intake and risk of cataracts in humans. Ann N Y Acad Sci. 1989, 570, 372-382. 41. Benedek, G. B. Theory of the transparency of the eye. Applied Optics. 1971, 10, 459473. 42. Andersson, M.; Sjostrand, J.; Karlsson, J. O. Calpains in the Human Lens: Relations to Membranes and Possible Role in Cataract Formation. Ophthalmic Research. 1996, 28 (Suppl 1): 51-54. 43. Robertson, L. J.; Morton, J. D.; Yamaguchi, M.; Bickerstaffe, R.; Shearer, T. R.; Azuma, M. Calpain may contribute to hereditary cataract formation in sheep. Invest Ophthalmol Vis Sci. 2005, 46(12), 4634-4640. 44. Calvin, H. I.; Medvedovsky, C.; Worgul, B. V. Near-total glutathione depletion and age-specific cataracts induced by buthionine sulfoximine in mice. Science. 1986, 233 (4763), 553-555. 45. Shearer, T. R.; David, L. L.; Anderson, R. S.; Azuma, M. Review of selenite cataract. Current Eye Research. 1992, 11(4), 357-369.
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46. Giblin, F. J. Glutathione: a vital lens antioxidant. J Ocul Pharmacol Ther. 2000, 16(2), 121-135. 47. Giblin, F. J.; McCready, J. P.; Schrimscher, L.; Reddy, V. N. Peroxide inducedn effect on lens cation transport following inhibition of glutathione reductase activity in vitro. Experimental Eye Research. 1987, 45, 77-91. 48. Lou, M. F.; Dickerson, J. E.; Jr. Garadi, R. The role of protein-thiol mixed disulfides in cataractogenesis. Exp Eye Res. 1990, 50(6), 819-826. 49. Azuma, M.; Shearer, T. R. Involvement of Calpain in Diamide-Induced Cataract in Cultured Lenses. FEBS. 1992, 307(3), 313-317. 50. Sanderson, J.; Marcantonio, J. M.; Duncan, G. Calcium ionophore induced proteolysis and cataract: inhibition by cell permeable calpain antagonists.Biochemical and Biophysical Research Communications. 1996, 218, 893-901. 51. Fukiage, C.; Azuma, M.; Nakamura, Y.; Tamada, Y.; Shearer, T. R. Nuclear cataract and light scattering in cultured lenses from guinea pig and rabbit. Current Eye Research. 1998, 17, 623-635. 52. Augusteyn, R. C.; Stevens, A. Macromolecular Structure of the Eye Lens. Progress in Polymer Science. 1998, 23(3): 375-413. 53. Spector, A. Oxidative Stress-Induced Cataract: Mechanism of Action. FASEB. 1995, 9:1173-1182. 54. Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine. Oxford University Press Inc, New York. 1999, 617-783. 55. Sanderson, J.; Lauchlar, W. R. M.; Williamson, G. Free Radical Biology and Medicine. 1999, 26(5), 639-645. 56. Cornish, K. M.; Williamson, G.; Sanderson, J. Quercetin metabolism in the lens: role in inhibition of hydrogen peroxide induced cataract. Free Radic Biol Med. 2002, 33(1), 63-70. 57. Packer, L.; Witt, E.H.; Tritschler, H.J. alpha-Lipoic acid as a biological antioxidant. Free Radic Biol Med. 1995, 19(2), 227-50. 58. Roy, I. S. Handbook of ophthalmology, CBS Publishers and Distributers. 1988, 4, 154157. 59. http://encyclopedia2.thefreedictionary.com/acquired+cataract. School of Pharmaceutical Sciences, Shobhit University, Meerut
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Chapter I 60. Clark, J. I.
Introduction Development and maintenance of lense transpetency. In: Albers EM,
jakobiec FA, editors. Principles and practices of opthalmology. Philadelpia: .B. Saunders. 1994, 4-23. 61. Ito, Y.; Nabekura, T.; Takeda, M.; Nakao, M.; Terao, M.; Hori, R. Nitric oxide participates in cataract development in selenite treated rats. Current Eye Research. 2001, 22, 215-220. 62. Kyselova, Z.; Stefek, M.; Bauer, V. Pharmacological prevention of diabetic cataract. J Diabetes Compl. 2004, 18,129-140. 63. Brownlee, M. Advanced glycation end products in diabetes and ageing. Ann Rev Med. 1996, 46,223-34. 64. Monnier, V.M.; Sell, D.R.; Nagaraj, R.H.; Miyata, S.; Grandee, S.; Odetti, P. Maillard reaction mediated molecular damage to extra cellular matrix and other those tissue protein in diabetes, ageing and uremia. Diabetes. 1992, 41, 36-41. 65. Araki, N.; Ueno, N.; Chakrabati,
B.; Morino, Y.; Horicuchi, S. Immunological
evidence for the presence of advanced glycation end product in human lens proteins and its positive correlation with ageing. J Biol Chem. 1992, 267,102, 11-4. 66. Duhaiman, A. S. Glycation of human lens protiens from diabetic and (nondiabetic) senile cataract patients. Glycoconjugate Jr. 1995, 12, 618-21. 67. Halliwell B. Antioxidant: The basic what they are and how to evaluate them. Adv Pharmacol.1997; 38:3-19. 68. Micelli-Ferrari, T.; Vendemiale, G.; Grattogilango, I. Role of lipid peroxidation in the pathogenesis of myopic and sensile cataract. Br J Opthalmol. 1996, 80, 840-3. 69. De Haan, J.B.; Cristiano, F.; Jannello, R.C.; Kola, I. Cu/Zn- superoxide dismutase and glutathione peroxidase during ageing. Biochem Mol Biol Int. 1995, 35, 1281-97. 70. Spector, A. Oxidative stress – induced cataract: mechanism of action. FASEB. 1995, 9, 1173-82. 71. Spector, A. Review: Oxidative stress and disease.J Ocul Pharmacol Ther. 2000, 16,193201. 72. Steinberg, E. P.; Javitt, J. C.; Sharkey, P. D.; Zuckerman, A.; Legro, M. W.; Anderson, G. F.; Bass, E. B.;
O'Day, D. The content and cost of cataract surgery. Arch
Ophthalmol. 1993, 111(8), 1041-1049. School of Pharmaceutical Sciences, Shobhit University, Meerut
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73. Zenon, G.J.; Abobo, C.V.; Carter, B.L.; Ball, D.W. Potential use of aldose reductase inhibitors to prevent diabetic complications. Clin Pharm. 1990, 9, 446-57. 74. Harding, J. J.; Pharmacological treatment strategies in age related cataracts. Drugs ageing.1992, 2, 287-300. 75. Nakai, N.; Fujji, Y.; Kobashi, K. Aldose reductase inhibitors: Flavanoids, alkaloids, acetophenins, benzophenones, and spirohydantoins of chroman. Arch Biochem Biophysics. 1985, 239, 491-6. 76. Varma, S. D.; Mikuni, I.; Kinosita, J.H. Flavonoids as inhibitor of lens aldose reductase. Science. 1975, 188, 1215-6. 77. Cotlier, E.; Sharma, Y.R. Aspirin and senile cataracts in rheumatoid arthritis. Lancet. 1981, 1, 338-9. 78. Harding, J.J.; Egerlon, M.; Harding, R.S. Protection against cataract by aspirin, paracetamol and ibuprofen. Acta Opthalmol. 1989, 67, 518-24. 79. Gupta, S.K.; Joshi, S. Relationship between aldose reductase inhibiting activity and anti-cataract action of various NSAIDs. Dev Opthalmol. 1991, 21, 151-6. 80. Bono, A.; Milletelo, A.; Bongiomo, A. Effects of benzadac L-lysine salts on some metabolic enzymes of glutathione in the rabbit lens after x-ray irradiation. Indian J Biochem. 1987, 36, 153-65. 81. Harding, J.J.; Egerlon, M.; Harding, R.S. Protection against cataract by aspirin, paracetamol and ibuprofen. Acta Opthalmol.1989, 67, 518-24. 82. Gupta, S.K.; Joshi, S.; Tandon, R.; Mathur, P. Topical aspirin provides protection against galactosemic cataract. Indian J Opthalmol. 1997, 45, 221-5. 83. Eckerskorn, V.; Hockwin, O.; Muller Breienkamp, R.; Chen,T.T.; Knowles, W. Evaluation of cataract related risk factors using detailed classification systems and multiiarate statistical methods. Dev Opthalmol. 1987, 15, 82-91. 84. Silvestrini, B. Rationale for benzadac. In: Ermo et al, editors. Recent development in the pharmacological treatment of cataract. Amsterdam: Kugler.1987, 1-9. 85. Guglielmotti, A.; Capezzone, De JonnanonA, Cazolla N, Marchetti M, Sollo L, Cavallo G. Radical scavenger activity of benzadac: An anti cataract non steroidal antiinflammatory agents. Pharmacol Res. 1995, 32, 369-73.
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86. Levis, B.S.; Harding, J.J. The major metabolite of benzadac inhibitors the glycosylation of soluble lens protiens: A possible mechanism for a delay in cataractogenesis. Exp Eye Res. 1988, 47, 217-25. 87. Reddy, V.N.; Giblin, F.J. Metabolism and function of glutathione in the lens. Human cataract foundation Pitman, London (Ciba Foundation Symposium). 1984, 106, 65-87. 88. Kathleen, N.D. Natural therapies for ocular disorders part two: Cataracts and glaucoma. Altern Med Rev. 2001,6, 141-66. 89. Gupta, S.K.; Mohantly, I.; Joshi, S.; Trivedi, O.; Srivastava, S. Lycopene prevents sugar-induced morphological changes and modulates antioxidant status of human lens epithelial cells. Br J Nutr. 2002, 88, 347-54. 90. Varma, S.D.; Hedge, K.R. Effect of alpha ketoglutarate against selenite cataract formation. Exp. Eye Res. 2004, 79, 913-8. 91. Ayala, M.N.; Soderberg, P.G.; Vitamin C can protect against ultraviolet radiation induced cataract in albino rats. Ophthalmic Res. 2004, 36, 264-9. 92. Abe, M.; Reiter, R.J.; Orhii, P.B.; Hara, M.; Poeggeler, B. Inhibitory effect of melatonin on cataract formation in new born rats: Evidence for an antioxidative role for melatonin. J Pineal Res. 1994, 17, 94-100. 93. David, L.W. Oxidation, antioxidants and cataract formation. A Literature Review. Vet Opyhalmol. 2006, 9, 292-8. 94. Ohta, Y.; Niwa, T.; Yamasaki, T. Effect of prolonged marginal ascorbic acid deficiency lenticular level of antioxidants and lipid peroxidase in guienia pig. Int J Vitamin Nutr Res. 2001, 71,103-9. 95. Yokoyama, T.; Sasaki, H.; Giblin, F.J.; Reddy, V.N. A physiological level of ascorbate inhibits galactose cataract in guinea pigs by decreasing plyol accumlation in the lens epithelium: A dehydroascorbate-linked mechanism. Exp Eye Res. 1994, 58, 207-18. 96. Mody, V.C.; Kakar, M.; Elfving, A.; Soderberg, P.G.; Lofgren, S. Ascorbate in the rat lens: Dependence on dietary intake. Ophthalmic Res. 2005, 37, 142-9. 97. Labgele, U.W.; Wolf, A.; Cordier, A. Enhancement of SDZ ICT 32-induced cataracts and skin changes in rats following vitamin E and selenium deficient diet. Arch Toxicol. 1997, 71, 283-9.
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98. Kojima, M.; Shui, Y.B.; Murano, M.; Nagata, O.; Hockwin, K.; Sasaki, K. Low vitamin E level as a subliminal risk factor in a rat model of predisolone-induced cataract. Investig Ophthalmol Visual Sci. 2002, 43, 1116-20. 99. Reddy, G.B.; Nayak, S.; Reddy, P.Y.; Seetharam Bhatt, K. Reduced levels of rats lens antioxidant vitamins upon in vitro UVB irradiation. J Nutr Biochem. 2001, 12, 121-4. 100. Nath, B.; Srivastava, S.K. Accumulation of copper and inhibition of lactate dehyrogenase activity in human senile cataractous lens. Indian J Biol. 1969, 7, 25-6. 101. Bhatt, K.S. Nutritional status of thiamine, riboflavin and pyridoxine in cataract patients. Nutr Rep Inter. 1987, 36, 385-92. 102. Karakucuk, S.; Ertugrul Mirza, G.; Faruk Ekinciler, O.; Saraymen, R.; Karakucuk, I.; Ustdal, M. Selenium concentration in serum, lens and aqueous humour of patients with senile cataract. Acta Opthalmol Scand. 1995, 73, 329-32. 103. Truscott, R.J. Age related nuclear cataract-oxidation is the key. Exp Eye Res. 2005, 80, 709-25. 104. Spector, A.; Wang, G.M.; Wang, R.R.; Li, W.C.; Kuszak, J.R. A brief phytochemically induced oxidative insult caused irreversible lens damage and cataract: Mechanism of action. Exp Eye Res. 1995, 60, 483-93. 105. Gupta, S.K.; Trivedi, D.; Srivastava, S.; Joshi, S.; Halder, N.; Varma, S.D. Lycopene atteruates oxidative stress induced experimental cataract development. An in vitro and invivo study. Nutrition. 2003, 19, 794-9. 106. Cumming, R.C.; Mitchell, P.; Smith, W. Diet and cataract: The blue mountain eye studies. Opthalmology. 2000, 107, 450-6. 107. Mares-Perlman, J.A.; Brady, W.E.; Klein, B.E.; Klein, R.; Haus, G.J.; Palta, M. Diet and nuclear lens opacities. Am J Epidemiol. 1995, 41, 322-34. 108. Clinton, S.K. Lycopene: Chemistory, biology and implications for human health and disease. Nutr Rev. 1998, 56, 35-51. 109. Rao, K.E. Oxygen radical scavenging activity of curcumin. Int J Pharmacol. 1990, 58, 237-40. 110. Suryanaraya, P.; Krishnaswamy, K.; Reddy, G.B. Effect of curcumin on galactose induced cataractogenesis in rats. Mol Vis. 2003, 9, 223-30.
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111. Suryanarayan, P.; Saraswat, M.; Murdula, T.; Krishna, T.P.; Krishnaswamy, K.; Reddy, G.B. Curcumin and turmeric delay streptozocin-induced diabetic cataract in rats. Invest Ophthalmol Vis Sci. 2005, 46, 2092-9. 112. Padmaja, S.; Raju, T.N. Antioxidant effect of curcumin in selenium induced cataract of wistar rats. Indian J Exp Biol. 2004, 42, 601-3. 113. Gupta, S.K.; Srivastava, S.; Trivedi, D.; Joshi, S.; Nabanita, H. Osmium sanctum modulates selenite-induced cataractogenic changes and prevents rat lens opacification. Curr Eye Res. 2005,30,583-91. 114. Vats, V.; Yadav, S.P.; Biswas, N.R.; Grover, J.K. Anticataract activity of Pterocarpus marsupium bark and Trigonella foenum graceum seeds extract in alloxan diabetic rats. J Ethnopharmacol. 2004, 93, 289-94. 115. Durukan, A.H.; Evereklioglu, C.; Hurmeric, V.; Kerimoglu, H.; Erdurman, C.; Bayraktar, M.Z. Investigation of IH636 grape seed proanthocyanidin extract to prevent selenite-induced oxidative stress in experimental cataract. J Cataract Refract Surg. 2006, 32, 1041-5. 116. Lija, Y.; Biju, P.G.; Reeni, A.; Cibin, T.R.; Sahasranamam, V.; Abraham, A. Modulation of selenite cataract by the flavonoid fraction of Emilia sonchifolia in experimental animal models. Phytother Res. 2006, 20, 1092-5. 117. Biju, P.G.; Devi, V.G.; Lija, Y.; Abraham, A. Protective against selenite cataract in rat lens by drevogenin D: Atriterpenoid aglycone from Dregea volubilis. J Med Food. 2007, 10, 308-15 118. Ertekin, M.V.; Koccer, I.; Taysi, S.; Gepdiremen, A.; Sezen, O. Effects of oral Ginkgo biloba supplementation on cataract formation and oxidative stress occurring in lenses of rats exposed to total cranium radiotherapy. Jpn. J Ophthalmol.2004, 48,499-502. 119. Elena, M.V.; De Cavanagh, E.M.; Inserra, F.; Ferder, L.; Fraga, C.G.; Enalapril and captopril enhance glutathione dependent antioxidant defense in mouse tissues. Am J Physiol Regular Inter Comp Physiol. 2000, 278, 572-7. 120. Langade, D.G.; Rao, G.; Girme, R.C.; Patki, P.S.; Bulakh, P.M. In vitro prevention by ACE inhibitors of cataract induced by glucose. Indian J Pharmacol. 2006, 38, 53, 12539.
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Chapter 2
Literature Review
2. Literature Review 2.1 Acorus calamus Reddy et al., (2010) discussed the effect of natural memory enhancing drugs on dementia. They concluded that natural memory enhancing drugs controlled the activity of acetylcholinesterase (AChE).Which modulates acetylcholine (ACh) to proper levels to maintain constant ACh deficiency leading to memory and cognitive impairements. Glycyrrhiza glabra (Fabaceae), Commiphora whighitti (Burseraceae), Panax ginseng (Araliaceae), Acorus calamus (Araceae) were reported as natural memory enhancing agents.1 Si et al., (2010) reported the insulin releasing and α glucosidase inhibitory activities of the ethyl acetate fraction of Acorus calamus (ACE) in vitro and in vivo. In vivo, ACE (400 and 800 mg/kg) significantly decreased fasting serum glucose, and suppressed the increase of blood glucose levels in normal mice. They also reported that ACE may have hypoglycemic effects via mechanisms of insulin releasing and α glucosidase inhibition, and thus used in postprandial hyperglycemia and cardiovascular complications.2
Agarwal et al., (2009) evaluated the effect of supplemental UV- ß radiation (SUV-ß) induced changes in essential oil composition and total phenolic compound of A. calamus. They concluded that the level of phenolic compound, p-cymene and carvacrol content of essential oil increased with exposure to UV- β, whereas β asarone content of essential oil decreased.3
Ganjewala et al., (2009) evaluated antimicrobial activity of Acorus calamus rhizome and leaf extracts obtained with different solvents viz., petroleum ether, chloroform, hexane and ethyl acetate. They reported that minimum inhibitory concentration (MIC) of the rhizome and leaf extracts for antifungal activity was except against Penicillium chrysogenum. They also indicated that A. calamus rhizomes and leaves might possess active principle α and ß asarones which is believed to be responsible for their antimicrobial activities.4
Nalamwar et al., (2009) performed in vitro licicidal activity of different extracts of A.calamus (rhizomes). Chloroform, n-hexane, methanol and distilled water were used for exhaustive sequential extraction. Goat-lice damalinia caprae (trichodectidae) used as experimental organism. It was concluded that only n-hexane and chloroform fractions exhibited licicidal activity.5 School of Pharmaceutical Sciences, Shobhit University, Meerut
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Literature Review
Wu et al., (2009) performed in vitro and in vivo insulin sensitizing activity of ethyl acetate fraction of Acorus calamus extract (ACE). They reported that ACE (12.5 and 25g/ml) increased glucose consumption mediated by insulin in L6 (Langerhansch’s cells). It was concluded that ACE decreased the intake of food and water, and did not increase body weight whereas rosiglitazone (standard antidiabetic drug) did.6
Lee et al., (2009) reported anti-inflammatory activity of aqueous extract of Acorus calamus leaves (ACL) on human keratinocyte cells. They reported that ACL inhibits the production of pro-inflammatory cytokines through multiple mechanisms and may be a novel and effective anti-inflammatory agent for the treatment of skin diseases.7
Yende et al., (2009) performed anticonvulsant activity of Acorus calamus. They reported that administration of Acorus calamus (ED50 at 185 mg/kg) significantly potentiates the anticonvulsant action of phenytoin by reducing its ED50 value (13.5 mg/kg to 9.25 mg/kg) and phenobarbital by reducing its ED50 value (8 mg/kg to 5 mg/kg) .8 Ahmad et al., (2009) studied the in vitro antioxidant activity and total phenolic content of the four Indian medicinal plants (methanolic extract) i.e. Plumbago zeylanica (root), A.calamus (rhizome), Hemidesmus indicus (stem) and Holarrhena antidysenterica (bark).
Ferric
thiocyanate (FTC) assay method was used for evaluating the antioxidant activity and the same was then compared with thiobarbituric acid (TBA) method. The order of antioxidant potential according to FTC assay method was reported. Plumbago zeylanica showed highest activity followed by Holarrhena antidysenterica, A.calamus and Hemidesmus indicus. 9
Gyawali et al., (2009) reported various volatile organic compounds of Nepalese A.calamus rhizomes. They identified and detected several bioactive compounds such as β asarone( 46.78%), linalool (0.41%), farnesol (11.09%), methyleugenol (6.10%), α and β pinene ( both 0.06%), [E]-caryophyllene (0.11%),
β elemene (0.39%), ocimene(0.7%), aromadendrene
(0.26%), camphor (0.03%).10
Chattopadhayay et al., (2009) reported the effect of bioactive fraction of A.calamus rhizomes in experimentally induced hyperlipidemic rats. They revealed that bioactive fraction
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caused significant decreased in the total cholesterol level, which was related to decrease in LDL (low density lipoprotein) level in experimentally induced hyperlipidemic rats.11
Sharma et al., (2008) studied the effect of Acorus calamus rhizomes oil on hemogram and ultrastructure of hemocytes of the Tobacco armyworm, Sodoptera litura. Scanning electron microscopic (SEM) and transmission electron microscopic (TEM) methods demonstrated calamus oil treatment on plasmatocytes (PLs) and granular hemocytes (GRs) .12
Lee et al., (2007) studied the fungicidal property of A. gramineus rhizome derived materials against Botrytis cineria, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani in vivo. They reported that the hexane fraction of A.gramineus rhizomes at dose of 2000 mg/l exhibit strong fungicidal activities against R. solani and P. infestans.13
Tkachev et al., (2006) isolated acorafuran, from essential oil of A.calamus. They elucidated structure of acorafuran (6-isopropyl-4-methyl-7, 8-dihydro-6H-naphtho [1, 8-b-c] furan), which was a new cadalin-type sesquiterpenoid of A.calamus essential oil by using spectral data (IR, NMR, Mass spectrometry).14
Ghosh et al., (2006) discussed antifungal properties of haem peroxidase from A.calamus leaf (ACL). They fractionated A.calamus protein by using cation exchange chromatography and gel filtration which inhibited the hyphen extension of phytopathogens .15
Bains et al., (2005) studied novel lectins from Acorus species rhizomes. They used affinity chromatography on mannose linked epoxy-activated sepharose 6B. Acorus calamus lectin (ACL) and Acorus gramineus lectin (AGL) both were reported to have mitogenic activity and inhibition of murine cancer cell lines. 16 Lee et al., (2005) reported antioxidative activity of volatile extract isolated from Angelica tenuissimae (root), peppermint leave, pine needles and Acorus calamus (leave).17
Devi et al., (2005) discussed antioxidant property of α asarone, active principal component of A.calamus against noise stress induced changes in different regions of rat brain.
They
reported that α asarone treatment restored the activities and levels of antioxidant machinery School of Pharmaceutical Sciences, Shobhit University, Meerut
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i.e superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), vitamin C, vitamin E, protein thiols and lipid peroxidation (LPO) in noise stress. It may be due to its antioxidant property.18
Houghton et al., (2004) reported the screening of Korean herbal medicine used to enhance memory and cognitive function in old age for anticholinesterase properties using the Ellman colorimetric method. They worked on methanolic extracts of seven herbs (A.calamus, A.gramineus, Bupleurm facaltum, Dioscorea batatas, Epimedium koreanum, Poria cocos and Zizyphi jujuba) for cholinesterase inhibitory activity.19
Houghton et al., (2003) discussed the plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function including neurodegenerative diseases such as Alzheimer’s disease (AD).20
Singh et al., (2003) evaluated the anticellular and immunomodulatory properties of ethanolic extract of A.calamus rhizomes in vitro. They reported that this extract inhibit proliferation of mitogen (phytohaemagglutinin; PHA) and antigen (purified protein derivative; PPD), stimulated human peripheral blood mononuclear cells (PBMCs).21
Mengi et al., (2002) performed hypolipidemic activity of Acorus calamus in rats. They reported that 50% ethanolic extract (100 and 200 mg/kg) and saponins (10 mg/kg) isolated from the Acorus calamus demonstrated significant hypolipidemic activity.22
Shobha et al., (2001) evaluated the effect of aqueous and methanolic plant extracts of Acorus calamus rhizome), Pongamia glabra (leaves), Aegle marmelos (unripe fruit) and Strychnos nux-vomica (root bark) as antidiarrhoeal agent against castor oil induced diarrhoea in mice. They noted that the methanolic plant extract significantly reduced induction time of diarrhoea and total weight of feces whereas aqueous extract could not.23
Chernenko et al., (2001) studied the lipids of Acorus calamus (leaves, stems, rhizomes and roots). They evaluated neutral-lipid classes and fatty-acid composition of neutral, glyco, and phospholipids, by using steam distillation methods.24
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Chapter 2 Almeida et al., (2001) discussed analgesic activity of different
Literature Review plants such as Acorus
calamus, Cannabis sativa, Morinda citrifolia, Panax ginseng, Piper umbellatum, Ricinus communis, Strychnos nux-vomica etc. with special focus on those plants which act on the central nervous system.25
Orpean et al., (1998) used GC-MS and thin layer chromatographic (TLC) techniques for determination of α and ß asarone isomers of A.calamus and Asarum europaeum. They reported that GC-MS is more sensitive and mathematical computations for spots optimization & interference elimination could improve the TLC quality results.26
Kuroyangi et al., (1996) isolated sesquiterpene from methanol extract of A.calamus rhizomes as germination inhibitors. They elucidated sesquiterpenes as cadinane, acorane and eudesmane skeletons.27
Vohora et al., (1990) evaluated CNS activity of ethanol extract of A.calamus rhizomes. They reported large number of actions similar to α asarone, but different from the responses to electroshock, apomorphine and isolation-induced aggressive behaviour, amphetamine toxicity in aggregated mice, behavioural despair syndrome in forced swimming due to the presence of chemical substances other than either α asarone or ß-asarone. 28
Koul et al., (1990) isolated asarones from Acorus calamus and reported effect on feeding behavior and dietary utilization in Peridroma saucia. They also concluded that cis-asarone is toxic in addition to having strong antifeedant activity, whereas the trans isomer acts only as an anti feedant with no appreciable toxicity. 29
2.2 Vitex negundo Panda et al., (2009) studied antibacterial activity along with phytochemical screening of leaves and bark of Vitex negundo. They reported that the crude extracts of ethanol and methanol of leaves and petroleum ether, chloroform extracts of bark exhibited significant antibacterial activity and properties that support folkloric use in the treatment of some diseases as broad spectrum antibacterial agents.30
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Zheng et al., (2009) reported analgesic activity and chemical constituents of Vitex negundo seeds. They demonstrated the analgesic activity of the acetoacetate fraction of Vitex negundo seeds in the test models of nociception induced by chemical stimuli. Bioassay guided isolation from the acetoacetate fraction led to a productive compound, 6-hydroxy-4-(4-hydroxy-3methoxy-phenyl)-3-hydroxy- methyl-7-methoxy-3, 4-dihydro-2-naphthaldehyde, with potent analgesic properties that could partially explain the analgesic effect of Vitex negundo seeds extract.31
Ahirrao et al., (2009) reported anthelmintic activity of leaves of Jatropha circus and Vitex negundo. Both the leaves of Jatropha curcas and Vitex negundo show good anthelmintic activity. After comparative study the leaves of Jatropha curcas gives more potent anthelmintic activity than that of leaves of Vitex negundo.32
Bansod et al., (2009) reviewed phytochemical constituents, traditional uses and pharmacological properties of Vitex negundo. The extensive survey of literature revealed that Vitex negundo is important medicinal plant with diverse pharmacological spectrum. The plant shows presence of many chemical constituents, which are responsible of the various activities of the plant such as anticonvulsant effect, CNS depressant activity ,antiarthritic effect, antiallergic activity were reported in literature.33
Adnaik et al., (2008) reported laxative activity of Vitex negundo (leaves). The aqueous extract of Vitex negundo (100 and 200 mg/kg, oral) showed significant and dose dependant increase in faecal output of rats at selected dose levels. The effect was comparable with that of standard (agar-agar). During the phytochemical analysis of the extract, test for anthracene derivatives was found to be positive. The laxative activity may be attributed to the presence of anthracene derivatives in the leaves.34
Tasduq et al., (2008) extracted negundoside and an irridiod glycoside from leaves of Vitex negundo. They concluded that negundoside (NG) protects against carbon tetra chloride induced toxicity and oxidative stress.35
Tandon et al., (2008) reported hepatoprotective activity of Vitex negundo leaves extract against anti tubercular drugs induced hepatotoxicity. They concluded that the ethanolic extract School of Pharmaceutical Sciences, Shobhit University, Meerut
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(leaves) of Vitex negundo possesses hepatoprotective activity against anti-tubercular drugs induced hepatotoxicity (HT) at dose of 250 mg/kg and high 500 mg/kg doses.36
Sahare et al., (2008) reported antimicrofilarial activity of methanolic extract of Vitex negundo and Aegle marmelos and carried out their phytochemical analysis. They concluded the initial phytochemical analysis provided important mechanistic clue for antifilarial effect of Vitex negundo and Aegle marmelos.37
Khokra et al., (2008) studied essential oil composition and antibacterial potential of Vitex negundo extracts. They concluded that each of essential oils and extracts gives promising results against B. subtilis and E.coli. Ethyl acetate and ethanol extracts showed prominent antibacterial activity against all the tested strains. Fruit and leaf oils were found to be most active against E. coli and S. aureus, respectively. Only flower oil was found to be active against P. aeruginosa.38
Karunamoorthi et al., (2008) evaluated leaf extracts of Vitex negundo (family Verbenaceae) against larvae of Culex tritaeniorhynchus and for repellent activity on adult vector mosquitoes. They concluded that the Vitex negundo leaf extracts served as a potential larvicidal agent against Japanese encephalitis vector C. tritaeniorhynchus and additionally acted as a promising repellent against various adult vector mosquitoes.39
Devi et al., (2007) studied the effect of Vitex negundo leaf extract as free radicals scavengers in complete Freund’s adjuvant induced arthritic rats. They concluded the altered levels of free radicals, antioxidant enzymes were reverted to a considerable extent with the oral administration of the leaf extract in the arthritic rats, proving its antioxidant property.40
Sathiamoorthy et al., (2007) reported new antifungal flavonoid glycoside from Vitex negundo.
They
isolated
five
known
compounds
(1)
5’-hydroxy–3’,4’,3,6,7-
pentamethoxyflavone (2) luteolin (3) agnuside (4) negundoside (5) iso-orientin and a new flavonoid glycoside from Vitex negundo through activity guided fractionation and discovered the potent antifungal activity of isolated compounds.41
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Tiwari et al., (2007) reported antioxidant properties of different fractions of Vitex negundo. They concluded that leaves of Vitex negundo contain a number of antioxidant compounds, which can effectively scavenge various reactive oxygen species/ free radicals under in vitro conditions. They also have mild metal chelation properties. This property may be attributed more to the polar fraction, because the alcoholic fraction showed better response on all the tested parameters.42
Banerjee et al., (2007) reported the endometrial membrane response of Vitex negundo in Mus musculus during implantation. They observed a decrease in superoxide dismutase (SOD) activity at the time of implantation in both control and treated groups which suggests that the Vitex negundo extract does not have a significant anti estrogenic activity.43
Haq et al., (2006) reported tyrosinase inhibitory lignans from the methanol extract of the roots of Vitex negundo and studied their structure activity relationship. They concluded that the lignans compounds isolated from the roots of Vitex negundo, can be effective inhibitors of tyrosinase enzyme and exhibit potential to be used for the treatment of hyperpigmentation associated with the high production of melanocytes.44
Tandon et al., (2006) studied anti-inflammatory activity of Vitex negundo and its mechanism of action. These findings suggested that Vitex negundo probably produce anti-inflammatory action against acute inflammation by inhibiting prostaglandin synthesis, although the result is not conclusive, as other mediators in addition to PG’s like 5HT and calcium have a role to play in contractions of rat uterus. They suggested that anti-inflammatory and pain suppressing activities of Vitex negundo are possibly mediated via PG synthesis.45
Gupta et al., (2005) reported antinociceptive activity of Vitex-negundo leaves extract. They observed that Vitex negundo possesses both central and peripheral analgesic activities. It may be useful in relieving both the visceral and integumental pain.46 Tandon et al., (2005) carried out an experimental evaluation of anticonvulsant activity of Vitex negundo. They concluded that Vitex negundo possesses anticonvulsant activity particularly against pentylenetetarazole (PTZ) induced convulsion. Moreover, the potentiation of diphenylhydantoin valporic acid by Vitex negundo indicates that it may be useful as an
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adjuvant therapy along with standard anticonvulsant and lower the requirement of diphenylhydantoin and valporic acid.47
Dharmasiri et al., (2003) reported anti-inflammatory and analgesic activities of mature fresh leaves of Vitex negundo. Their observations provided evidence for the anti-inflammatory and analgesic properties of mature fresh leaves of Vitex negundo as claimed in Ayurveda medicine.48
Alam et al., (2003) studied snake venom neutralization by Indian medicinal plants (Vitex negundo and Emblica officinalis) root extracts.49
2.3 Butea frondosa Ngamrojanavaich et al., (2007) reported
carpin (3-hydroxy-9-methoxypterocarpan)
medicarpin and four isoflavones, viz. 7-hydroxy-4’-methoxy-isoflavone (formononetin), 7,4’dimethoxyisoflavone,
5,4’-dihydroxy-7-methoxy-isoflavone,
and
7-hydroxy-6,4’-
dimethoxyisoflavones, from the tuber roots of Butea superb Roxb. compounds, formononetin and prunetin showed moderate cytotoxic activity on KB cell lines.50 HO
O
O
OCH3 7-Hydroxy-3-p-tolyl-chromen-4-one; compound with methanol
Manosroi et al., (2006) reported the effects of Butea superba on the reproductive system in male wistar rats. The animals were fed daily with the powdered crude drug suspended in distilled water by a gastric tube at the dose of 2, 25, 250 and 1250 mg/kg body weight for 8 weeks. Rats fed with 1 ml distilled water were used as a negative control. The weights of all vital organs in all treated groups were noted to be same as that of control, except that the testis of the group fed with 1250 mg/kg was significantly different from the control and the other treated groups. In addition, the sperm counts in this group was about 16% more than the control group.51
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Iqbal et al., (2006) reported that seeds of Butea monosperma when administered as crude powder (CP) at doses of 1, 2 and 3 g/kg to sheep, naturally infected with mixed species of gastrointestinal nematodes, exhibited a dose and a time-dependent anthelmintic effect. The maximum reduction of 78.4% in eggs per gram of faeces (EPG) was recorded on day 10 after treatment with 3 g/kg. Levamisole (7.5 mg/kg), a standard anthelmintic agent, exhibited 99.1% reduction in EPG.52
Somani et al., (2006) studied the antihyperglycemic activity of the ethanolic extract of Butea monosperma (BMEE) in glucose loaded and alloxan induced diabetic rats. 53
Sumitra et al., (2005) reported that wound healing occurs as a fundamental response to tissue injury. Several natural products had been shown to accelerate the healing process. The investigation was undertaken to determine the efficacy of topical administration of an alcoholic bark extract of Butea monosperma on cutaneous wound healing in rats.54
Gunakkunru et al., (2005) studied the anti-diarrhoeal potential of the ethanolic extract of stem bark of Butea monosperm, in wistar albino rats. The extract inhibited castor oil induced diarrhoea and PGE2 induced enter pooling in rats. It also reduced gastrointestinal motility after charcoal meal administration. The results obtained establish the efficacy and substantiate the use of this herbal remedy as a non-specific treatment for diarrhoea in folk medicine. 55
Cherdshewasrt et al., (2004) studied the differential anti-proliferation effect of white (Pueraria mirifica), red (Butea superba) and black (Mucuna collettii) Kwao krua plant extracts on the growth of MCF-7 cells and evaluated after 4 days of incubation. The percent cell growth comparison was based on protein determination of the harvested cells in parallel with the control group and Pueraria lobata treatment group. Pueraria lobata led to no proliferation and a mild anti-proliferation effect on the growth of MCF-7 cells. Pueraria mirifica caused proliferation at 1 g/ml and an anti-proliferative effect on the growth of MCF-7 cells at 100 and 1000 g/ml with an ED50 value of 642.83 g/ml. Butea superba led to no proliferation and an anti-proliferation effect on the growth of MCF-7 cells at 10, 100 and 1000 g/ml with an ED50 value of 370.91 g/ml. Mucuna collettii led to no proliferation and an anti-proliferation effect on the growth of MCF-7 cells at 100 and 1000 g/ml with an ED50 value of 85.36 g/ml. The results demonstrated that only Pueraria mirifica showed an School of Pharmaceutical Sciences, Shobhit University, Meerut
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estrogenic effect on MCF-7 cell growth and a clear antagonistic effect with E2 at high concentration. Butea superba and Mucuna collettii exhibited only anti-proliferation effects on the growth of MCF-7 cells in relation with a possible anti-estrogen mechanism or a potent cytotoxic effect. 56
Ramachandaran et al., (2004) reported the aphrodisiac activity of Butea frondosa, (Papillionaceae) bark extract. The extract (400 mg/kg. body wt./day) was administered orally by gavage for 28 days. Mount latency (ML) intromission latency (IL) ejaculation latency (EL) mounting frequency (MF) intromission frequency (IF) ejaculation frequency (EF) and post ejaculatory interval (PEI) were the parameters observed before and during the sexual behavior study at day 0, 7, 10, 14, 21 and 28. The extract reduced ML, IL, EL, and PEI significantly (P< 0.05). The extract also increased MF, IF, and EF significantly (P< 0.05). These effects were observed in sexual active and inactive male rats. 57
Ingkaninan et al., (2003) collected 32 plants used in Thai traditional rejuvenating and neurotonic remedies. The plant methanolic extracts were tested for AChE inhibitory activity using Ellman’s colorimetric method in 96-welled microplates. The results showed that the methanolic extracts from roots of Stephania suberosa and Tabernaemontana divaricata, at concentration of 0.1 mg/ml inhibited more than 90% of AChE activity. At the same concentration, four extracts, i.e. stems of Piper interruptum, seeds of Piper nigrum, and root barks of Butea superba and roots of Cassia fistula, extracts showed 50–65% inhibitory activity on AChE. The rest of the extracts showed the AChE inhibitory activity below 50%.58
Kasture et al., (2002) carried out bioassay guided fractionation of dried flowers of Butea monosperma (BM) to isolate the active principle responsible for its anticonvulsant activity. The petroleum ether extract was fractionated by column chromatography using solvents of varying polarity such as n-hexane, n-hexane: ethyl acetate, ethyl acetate, and methanol. The anticonvulsive principal of B. monosperma was found to be a triterpene (TBM) present in the n-hexane: ethyl acetate (1:1) fraction of the petroleum ether extract. TBM exhibited anticonvulsant activity against seizures induced by maximum electroshock (MES) and its PD50 was found to be 34.2 ± 18.1 mg/kg. TBM also inhibited seizures induced by pentylenetetrazol (PTZ), electrical kindling, and the combination of lithium sulfate and pilocarpine nitrate (Li-Pilo). However, TBM was not effective against seizures induced by School of Pharmaceutical Sciences, Shobhit University, Meerut
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strychnine and picrotoxin. TBM exhibited depressant effect on the central nervous system. After repeated use for 7 days, the PD50 (MES) of TBM increased to 51.5 ± 12.1 mg/kg. Similarly, after repeated use of TBM, the duration of sleep induced by pentobarbital was not reduced significantly. 59
Gawale et al., (2001) reported that acetone-soluble part of petroleum ether and ethanolic extract exhibit nootropic activity in laboratory animals. 60
Prashanth et al., (2001) showed that methanol extract of Butea monosperma seeds, tested in vitro, possesses significant anthelmintic activity. 61
Kasture et al., (2000) reported that the ethanolic extracts of leaves of Albizzia lebbeck, flowers of Hibiscus rosa sinesis and the petroleum ether extract of flowers of Butea monosperma exhibited anticonvulsant activity. The bioassay guided fractionation indicated that the anticonvulsant activity lies in the methanolic fraction of chloroform soluble part of ethanolic extract of the leaves of A. lebbeck, acetone soluble part of ethanolic extract of H. rosa sinesis flowers and acetone soluble part of petroleum ether extract of B. monosperma flowers. The fractions protected animals from maximum electro shock, electrical kindling and pentylenetetrazole-induced convulsions in mice. The fractions also inhibited convulsions induced by lithium, pilocarpine and electrical kindling. However, they failed to protect animals from strychnine-induced convulsions. The fractions antagonized the behavioural effects of D-amphetamine and potentiated the pentobarbitone- induced sleep. The fractions raised brain contents of γ-amino butyric acid (GABA) and serotonin. These fractions were found to be anxiogenic and general depressant of central nervous system. 62
Shukla et al., (2000) studied sigmasterol-3-α-arabinopyranoside and four new compounds isolated from the stem of Butea monosperma and
characterized them as 3-methoxy-8,9-
methylenedioxypterocarp-6-ene, 21-oxooctacosanoic acid methyl ester, 4-pentacosanylphenol and pentacosanyl –β-D-glucopyranoside by spectral data and chemical studies. 63 Mengi et al., (1999) reported that in the carrageenan-induced rat paw edema, the aqueous extract of Butea frondosa leaves showed dose-dependent (25-100 mg/kg p.e.) antiinflammatory activity, which at the highest dose was almost comparable to ibuprofen (25 mg/kg p.e.).The LD50 was higher than 5g/kg p.e. in rats. 64 School of Pharmaceutical Sciences, Shobhit University, Meerut
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Rane et al., (1998) tested methanolic and water extracts of the wood of Pterocarpus marsupium Roxb. and leaves of Butea frondosa koen. ex Roxb. for antihepatotoxic activity in albino rats intoxicated with CCl4, liver weight, phenobarbitone sleep time and biochemical parameters were studied. Both the plants lowered the elevated levels of SGOT, SGPT and SALP indicating promising antihepatotoxic activity. 65
Yadav et al., (1998) isolated a new flavonoid 3, 7-dihydroxy-8-methoxyflavone, 7-o-αrhamnopyranoside, from Butea superba stems and identified the same by spectral analysis and chemical degradation. 66
Edwards et al., (1998) indicated that crude powder of B. monosperma seeds (CP) showed a dose-dependent (1–3 g/kg) and a time-dependent anthelmintic activity in sheep. CP showed a maximum reduction of 78.4% in eggs per gram of faeces (EPG) on day 10 post-treatment which was maintained till day 14 with the dose of 3 g/kg. In comparison to this, the standard anthelmintic agent was levamisole. 67
Mengi et al., (1995) evaluated roots and leaves of Butea frondosa for ocular antiinflammatory activity in rabbits. Experimentally, induced ocular inflammation manifested as rise in intraocular pressure, leucocytosis and meiosis following the breakdown of blood aqueous humor barrier. The study involved the assessment of anti-inflammatory activity of Butea frondosa preparation by determination of percentage reduction in total leucocyte count at various time intervals. The arkas were prepared using the two parts of the plant. Commercially available eye drops of flurbiprofen and a marketed Butea frondosa arks were used to compare these arks. Statistical evaluation revealed significant difference between the aqueous preparations. However gel formulations of Butea frondosa leaves, arks and flurbiprofen prepared by using a commercially available polymer revealed no significant difference statistically. Moreover the gel revealed prolonged effect until 24 hours. 68
Cheng et al., (1995) isolated butein (2’, 4’, 3, 4-tetrahydroxychalcone), from Dalbergia odorifera. 69
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O
CH2
OH
HO
OH OH CO CH CH
OH
O
Wongkham et al., (1994) reported a new lectin, which was purified from the seeds of Butea monosperma by affinity chromatography on N-acetylgalactosamine-agarose. The purified lectin had an apparent molecular mass of 67,000 dalton by gel filteration on a separose 6 HR 10/30 column. The lectin appeared to be comprised of two non-covalently bound subunits with molecular masses of 32,000 and 34,000 dalton and contained 8% neutral sugar. The lectin agglutinated human erythrocytes but not those of rats, mouse, hamster, goose and pigeon. The agglutinating activity was inhibited by N-acetylgalactosamine and did not require a divalent ion it was also stable at up to 80oC for 60 minutes. 70
Chopra et al., (1993) reported that seeds of Butea frondosa were very effective in round worm infection and ineffective against hookworms in ayurvedic system of medicine. 71
Shah et al., (1992) studied the isolation and identification of free sugar and free amino acids from the petals of Butea frondosa, commonly known as Palash, which is an indigenous medicinal plant distributed through India. Flavonoids are constituents of Butea frondosa flowers. Dried powder of flower petals was continuously extracted with petroleum ether (60800C) for 8 hours to remove fatty materials. The defatted residue was soaked in hot boiling 70% ethanol and kept overnight and then filtered. 72
Mishra et al., (1991) reported pterocarpans, phenols and lipids from the stem of Butea monosperma. 73
Guha et al., (1990) reported a new imide, palasimide that was isolated from the pods of Butea monosperma and identified as palasonin-N-phenyl imide. 74
Zaffar et al., (1989) reported antimicrobial properties of the methanolic extract of the leaves of the Butea monosperma. The interaction studies of gentamycin and dried methanolic extract
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showed that the effect of gentamycin was neither enhanced nor inhibited by the dried methanolic extract containing antimicrobial component. The phytochemical investigation revealed the presence of steroidal compound, which was responsible for the antimicrobial properties. 75
Bandara et al., (1989) studied the petroleum and ethyl acetate extracts of stem bark from Butea monosperma and antifungal activity against Cladosporium cladosporioides. The active constituent of low polarity was isolated by bioassay monitored chromatographic fractionation and identified as (-) medicarpin, by comparison of physical data. The antifungal activity of (-) medicarpin was found to be greater than that of benlate, a standard fungicide, while (-) medicarpin acetate also exhibited significant activity against Cladosporium cladosporioides. 76
Bhargava et al., (1986) reported butin isolated from the seeds of Butea monosperma and administered orally to adult female rats at the doses of 5,10,and 20 mg/rats from day 1 to day 5 of pregnancy showed anti-implantation activity in 40%,70%, and 90% of the treated animals respectively. At lower dose, there was a dose dependent termination of pregnancy and reduction in the number of implantation sites. In ovariectomised young female rats, the butin exhibited estrogenic activity at comparable anticonceptive doses, but was devoid of antiestrogenic activity. Butin is a weak estrogen, in that a significant uterotrophic effect was discerned even at 1/20 th the anticonceptive dose. 77
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2.4 Research Envisaged Traditional medicine is very important and well recognized aspect of health care. Most of the population in the developing countries still relies mainly on indigenous traditional medicine to address their primary health care needs. Herbal medicine plays a vital role in restoring health of individuals and communities, but there is a need to develop quality assurance methods for their standardization. Literature survey reveals that the plants Acorus calamus, Vitex negundo and Butea frondosa have immense potential in the treatment of ocular diseases. It has been well documented that Acorus calamus, Vitex negundo and Butea frondosa possess ocular anti-inflammatory, anthelmintic as well as aphrodisiac properties. Ethnopharmacology studies reveal that different parts of these plants (leaves, bark, flower and seed) are useful in various physiological processes and hence form an integral part of various medicinal preparations. It has been noted that anticataract potential of leaves, roots and bark of these plants is not studied till date, therefore the present study was aimed at evaluating the effect of leaves, roots and bark against cataract and ocular inflammation and generating scientific data to support various ethnopharmacological claims. A well defined ocular activity has been reported in Acorus calamus, Vitex negundo and Butea frondosa plants but there are no report regarding the effect of leaves, bark on one of the major ocular problem i.e. cataract and ocular inflammation. Various constituents of these plants could also be tested for other pharmacological activities. Still majority of their activities are without any scientific backing. The present work involved ethnopharmacological studies of Acorus calamus, Vitex negundo and Butea frondosa to generate scientific data to support various claims.
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2.5 Plan of Work The work was carried out on the following lines
Exhaustive literature survey Collection of plant(s) Authentication of plant material Air drying of plant material i.e. Leaves, Bark and Flower Air drying of plant material i.e. Leaves, Bark and Flower Size reduction of plant material Extraction with the help of polar and non polar solvent system Phytochemical Investigation(s) Chromatographic studies Fractionation Characterization of isolated compound (UV, IR, Mass, NMR spectroscopy) Pharmacological studies. Compilation and submission of the thesis
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References 1. Reddy, K.Y.; Lakshmi, M..; Kumar, A.S. Review on effect of natural memory enhancing drugs on dementia. International Journal of Phytopharmacology. 2010. 1, 1.
2. Si,Mm..; Loua, J.S. ; Zhou,C.X. ; Shena,J-N. ; Wua, H.H.; Yanga, B.; Hea,Q. J.; Wua, H-S. Journal of Ethnopharmacology. 2010,128, 154. 3. Agrawal, S.B.; Kumari, R.; Singh, S.; Dubey, N.K. Ecotoxicology and Environmental Safety, 2009, 72, 2013. 4. Ganjewala, D.; Devi, A. Acta Biologica Szegediensis. 2009, 53 (1), 45-49. 5. Nalamwar, V.P.; Khadbadi, S.S.; Aswar, P.B.; Kosalge, B.B.; Rajurkar, R. M.. In vitro Licicidal Activity of Different Extracts of Acorus calamus Linn. (Araceae) Rhizome. International Journal of Pharm tech Reasearch.. 2009, 1, 96. 6. Wu, H-S.; Zhu, D-F.; Zhou,C-X.; Feng, C-R.; Lou, Y. J.; Yang, B.; He, Q. J. Insulin sensitizing activity of ethyl acetate fraction of Acorus calamus L. in vitro and in vivo. Journal of Ethnopharmacology. 2009, 123, 288. 7. Lee, S.G.; Kim, H.; Han,T. H. Anti-infammatory activity of a water extract of Acorus calamus L. leaves on keratinocyte HaCaT cells. Journal of Ethnopharmacology. 2009,122, 149. 8. Yende, S.R.; Harle, U.N.; Bore, V.V.; Bajaj, A.O.; Shroff, K.K.; Vetal, Y.D. Reversal of neurotoxicity induced cognitive impairment associated with phenytoin and phenobarbital by acorus calamus in mice. Journal of Herbal Medicine & Toxicology. 2009, 111. 9. Ahmad, I.; Zahin, M.; Aqil, F. in vitro antioxidant activity and total phenolic content of four Indian medicinal plants. International Journal of Pharmacy and Pharamceutical Sciences. 2009, 1, 88. 10. Gyawali, R.; Kim. K. S. Volatile organic compounds of medicinal values from nepalese Acorus calamus. Katmandu university journal of science, engineering and technology. 2009, 5, 51. 11. Chattopadhayay, S.; Souja, T.D.; Mengi, S.A.; Hassarajani, S. Efficacy study of the bioactive fraction (F-3) of Acorus calamus in hyperlipidemia. Indian Journal of Pharmacology. 2007, 39, 196. 12. Sharma, P.R.; Sharma O.P.; Saxena, B.P. Effect of sweet flag rhizome oil (Acorus calamus) on hemogram and ultrastructure of hemocytes of the tobacco armyworm, spodoptera litura (lepidoptera: noctuidae) Micron. 2008, 39, 544. School of Pharmaceutical Sciences, Shobhit University, Meerut
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13. Lee, H.S. Fungicidal property of active component derived from Acorus gramineus rhizome against phytopathogenic fungi. Bioresource Technology. 2007, 98, 1324. 14. Tkachev, A.V.; Gaurav, A.M.; Yusubov, M.S. Acorafuran, a new sesquiterpenoid from Acorus calamus essential oil.
Chemistry of natural compound. 2006, 42 (6),
696. 15. Ghosh, M. Antifungal properties of haem peroxidase from Acorus calamus. Annals of botany. 2006, 98, 1145. 16. Bains, J.S.; Dhuna, V.; Singh, J.; Kamboj, S.S.; Nijjara, K.K.; Agrewal, J.N. Novel lectins from rhizomes of two Acorus species with mitogenic activity and inhibitory potential towards murine cancer cell lines.International Immunopharmacology. 2005, 5, 1470. 17. Lee, K.G.; Ka, M.H.; Choi, E.H.; Chun, H.S. Antioxidative activity of volatile extract isolated from Angelica tenuissimae roots, Peppermint leaves, Pine needles and Sweet flag leaves J. Agric Chem. 2005, 53, 4124. 18. Devi, R.S.; Manikandan, S. Antioxidant property of α-asarone against noise-stressinducedchanges in different regions of rat brain. Pharmacological Research.. 2005, 52, 467. 19. Houghtona, P.J.; Oh, M.H.; Whang, W.K.; Cho, J.H. Screening of Korean herbal medicines used to improve cognitive function for anti-cholinesterase activity. Phytomedicine. 2004, 11, 544. 20. Houghton, P. J.; Howes, M. J. R. Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function. Biochemistry and Behavior. 2003, 75, 513. 21. Singh, V.K.; Mahrotra, S.; Mishra, K.P.; Maurya, R.; Srimal, R.C.; Yadav, V.S.; Pandey, R. Anticellular and immunosuppressive properties of ethanolic extract of Acorus calamus rhizome. International Immunopharmacolog. 2003, 3, 53. 22. Mengi, S.A.; Parab, R.S. Hypolipidemic activity of Acorus calamus L. in rats. Fitoterapia. 2002, 73, 451. 23. Shobha, F.G.; Thomas, M. Study of antidiarrhoeal activity of four mrdicinal plants in castor-oil induced diarrhoea. Journal of Ethnopharmacology. 2001, 76 (1), 73-76. 24. Chernenko, T.V.; Glushenkova, A.L. Lipids of Acorus calamus. Chemistry of natural compound. 2001, 37(4), 304.
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25. Almeida, R.N.; Navarro, D.S.; Barbosa-Filho, M. Plants with central analgesic activity.Phytomedicine. 2001, 8(4), 310. 26. Oprean, R.; Tamas,M.; Sandules, R.; Roman, L. Essential oils analysis. Journal of Pharmaceutical and Biomedical Analysis. 1998, 18, 651. 27. Kuroyanagi, M.; Nawamak, K. Sesquiterpenoids from Acorus calamus as germination Inhibitors. Phytochemistry. 1996, 43, 1175.9 28. .Vohora, S.B.; Shah, S.A.; Dandiya, P.C. Central nervous system studies on an ethanolextract of Acorus calamus rhizomes. Journal of Ethnopharmacology. 1990, 28, 53. 29. Koul, O.; Smire, M.J.; Isman, B.M. Asarones from Acorus calamus L. oil their effect on feeding behavior and dietary utilization Peridroma saucia in Journal of Chemical Ecology. 1990, 16, 1991. 30. Panda, S.K.; Thatoi, H.N.; Dutta, S.K. Antibacterial activity and phytochemical screening of leaf and bark extracts of Vitex negundo l. from similipal biosphere reserve, Orissa. Journal of medicinal plants research. 2009, 3 (4), 294-300. 31. Zheng, C.J.; Tang, W.Z.; Huang, B.K.; Zhang, Q.Y.; Zhang, H.; Qin, L.P. Bioactivity guided fractionation for analgesic properties and constituents of Vitex negundo seeds. Phytomedicine. 2009, 16, 560-567. 32. Ahirrao, R.A.; Pawar, S.P.; Borse, S.L.; Desai, S.G.; Muthu A.K. Anthelmintic activity of leaves of Jatropha circus and Vitex negundo. Pharmacologyonline. 2009, 1, 276-279. 33. Bansod, M.S.; Harle, U.N. Vitex negundo phytochemical constituents, traditional uses and Pharmacological properties: comprehensive review. Pharmacologyonlin.2009, 1, 286-302. 34. Adnaik, R.S.; Pai, P.T.; Mule, S.N.; Naikwade, N.S.; Magdum, C.S. Laxative activity of Vitex negundo leaves. Asian journal exp. Science. 2008, 22(1), 159-160. 35. Tasduq, S.A.; Kaiser, P.J.; Gupta, B.D.; Gupta, V.K.; Johri, R.K. Negundoside, an irridiod glycoside from leaves of Vitex negundo, protects human liver cells against calcium mediated toxicity induced by carbon tetrachloride. World journal of gastroenterology. 2008, 14(23), 3693-3709. 36. Tandon, V.R.; Khajuria, V.; Gupta, R.K. Hepatoprotective activity of Vitex negundo leaf extract against anti-tubercular drugs induced hepatotoxicity. Fitoterapia. 2008, 52, 220-227. School of Pharmaceutical Sciences, Shobhit University, Meerut
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37. Sahare, K.N.; Anandhraman, V.; Meshram, V.G.; Meshram, S.U.; Reddy, M.V.R.; Tumane, P.M.; Goswami, K. Antimicrofilarial activity of methanolic extracts of Vitex negundo and Aegle marmelos and their phytochemical analysis. Indian journal of experimental biology. 2008, 46, 128-131. 38. Khokra, S.L.; Prakash, O.; Jain, S.; Aneja, K.R.; Dhingra, Y. essential oil composition and antibacterial studies of Vitex negundo extracts. Indian Journal of Pharmaceutical Sciences. 2008, 70, (4), 522-526. 39. Karunamoorthi, K.; Ramanujam, S.; Rathinasamy, R. evaluation of leaf extracts of Vitex negundo (family Verbenaceae) against larvae of Culex tritaeniorhynchus and repellent activity on adult vector mosquitoes. Springer-Verlag. 2008, 103, 545-550. 40. Devi, P.R.; Kumari, K.K.; and Kokilavani, C. Effect of Vitex negundo leaf extract on the free radicals scavengers in complete freund’s adjuvant induced arthritic rats. Indian Journal of Clinical Biochemistry. 2007, 22 (1), 143-147. 41. Sathiamoorthy, B.; Gupta, P.; Kumar, M.; Chaturvedi, A.K.; Shukla, P.K.; and Maurya, R. New antifungal flavonoid glycoside from Vitex negundo. Bioorganic and Medicinal Chemistry Letters. 2007, 17, 239-242. 42. Tiwari, O.P.; and Tripathi, Y.B. Antioxidant properties of different fractions of Vitex negundo. Food Chemistry. 2007, 100, 1170-1176. 43. Banerjee, A.; Vaghasiya, R.; Shrivastava, N.; Padh, H.; and Nivsarkar, M. Endometrial membrane response in Mus musculus during implantation by Vitex negundo. Anim. Reprod. 2007, 4(1/2), 46-50. 44. Haq, A.U.; Malik, A.; Khan, M.T.Z.; Haq, A.U.; Khan, S.B.; Ahmad, A.; Choudhary, M.I. Tyrosinase inhibitory lignans from the methanol extract of the roots of Vitex negundo and their structure–activity relationship. Phytomedicine. 2006, 13, (4), 255260. 45. Tandon, V.R.; Gupta, R.K. Vitex negundo leaf extract as an adjuvant therapy to standard anti-inflammatory drugs. Indian Journal Med. Research. 2006, 124, 447-450. 46. Gupta, R.K.; and Tandon, V.R. Antinociceptive activity of Vitex-negundo leaf extract. Indian Journal Physiol Pharmocol. 2005, 49(2), 163-170. 47. Tandon, V.R.; Gupta, R.K. An experimental evaluation of anticonvulsant activity of Vitex negundo. Indian Journal Physiol Pharmocol. 2005, 49(2), 199-205.
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48. Dharmasiri, M.G.; Jayakody, J.R.A.C.; Galhena, G.; Liyanage, S.S.P.; and Ratnasooriya, W.D. Anti-inflammatory and analgesic activities of mature fresh leaves of Vitex negundo. Journal of Ethnopharmacology. 2003, 87, 199-206. 49. Alam, M.I.; and Gomes, A. Snake venom neutralization by Indian medicinal plants (Vitex negundo and Emblica officinalis) root extracts. Journal of Ethnopharmacology. 2003, 86, 75-80. 50. Ngamrojanavanich, N.; Loontaisong, A.; Pengpreecha, S.; Cherdshewasart, W.; Pornpakakul, K.; Roengsumaran, S.; Petsom, A. Cytotoxic constituents from Butea superba Roxb., Journal of Ethnopharmacology. 2007, 109, 354-358. 51. Manosroi, A.; Sanphet, K.; Saowakon, S.; Aritajat, S.; Manosroi, J. Effects of Butea suberba on reproductive system of rats, Fitoterapia. 2006, 77(6), 435-438. 52. Iqbal, Z.; Lateef, M.; Jabbar, A.; Ghayur, M. N.; Gilani, A. H. In vivo anthelmintic activity of Butea monosperma against Trichostrongylid nematodes in sheep Fitoterapia. 2006, 77, 137- 140. 53. Somani, R.; Kasture, S.; Singhai, A.K., Antidiabetic potential of Butea monosperma in rats. Fitoterapia. 2006, 77(2), 86-90. 54. Sumitraa, M.; Manikandan, P.; Sugunab, L. Efficacy of Butea monosperma on dermal wound healing in rats. The International Journal of Biochemistry & Cell Biology. 2005, 37, 566–573. 55. Gunakkunru, A.; Padmanaban, K.; Vengatesan,
Thirumal, P.;
Pritila, J.; Parimala, G.;
N.; Gnanasekar, N.; James, B; Perianayagam, K.; Sharma, S. K.;
Pillai, K. K. Antidiarrhoeal activity of Butea monosperma in experimental animals, Journal of Ethnopharmacolog., 2005, 98, 241–244. 56. Cherdshewasart, W.; Cheewasopit, W.; Picha, P. The differential anti-proliferation effect of white (Pueraria mirifica), red (Butea superba) and black (Mucuna collettii) Kwao krua plant extracts on the growth of MCF-7 cells, Journal of Ethnopharmacology. 2004,
93, 255–260.
57. Ramachandran, S.; Sridhar, Y.; Sam, S.K G.; Saravanan, M.; Leonard, T.H.; Anbalagan, N.; Sridhar, S. K. Aphrodisiac activity of Butea frondosa Koen. Ex Roxb. extract in male rats, Phytomedicine. 2004, 11, 165-168. 58. Ingkanina, K.; Temkitthawon, P.; Chuenchom, K,; Yuyaem, T.; Thongnoi, W. Screening for acetylcholinesterase inhibitory activity in plants used in Thai traditional
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Literature Review Journal
of Ethnopharmacology. 2003, 89,
261–264. 59. Kasture,V. S.; Kasture, S. B.; Chopde, C. T. Anticonvulsive activity of Butea monosperma flowers in laboratory animals,
Pharmacology, Biochemistry and
Behavior. 2002, 72, 965-972. 60. Gawale, N. S.; Pal, S. C.; Kasture, V. S.; Kasture, S. B. acetone-soluble part of petroleum ether and ethanolic extract exhibit nootropic activity in laboratory animals. Journal of Natural Remedies. 2001,1,33-41. 61. Prashanth, D.; Asha, M. K.; Amit, A.; Padmaja, R. Anthelmintic activity of Butea monosperma, Fitoterapia. 2001, 72, 421-422. 62. Kasture, V. S.; Chopde, C.T.; Deshmukh, V. K. Anticonvulsive activity of Albizzia lebbeck, Hibiscus rosasinesis and
Butea monosperma in experimental animals,
Journal of Ethnopharmacology. 2000, 71, 65 – 75. 63. Shukla, Y. N.; Mishra, M.; Kumar, S. Euphane triterpenoid and lipid constituents from Butea monosperma, Phytochemistry. 2000, 54, 835-838. 64. Mengi, S. A.; Deshpande, S. G. Antiinflammatory activity of Butea frondosa leaves, Fitoterapia. 1999, 70, 521-522. 65. Rane, A.; Gramurohit, N. D. Hepatoprotective activity Pterocarpus marsupium Roxb. and leaves of Butea frondosa koen. ex Roxb.
Indian Journal of Pharmaceutical
Sciences. 1998, 182-184. 66. Yadav, R. N.; Reddy, K. I. A novel glycoside from the stem of Butea superba, Fitoterapia. 1998, 69 (3), 269-270. 67. Edward, G.; Breckenridge, A. M. Clinical pharmacokinetics of anthelmintic drugs, Clinical Pharmacokinetic. 1998, 15, 67-93. 68. Mengi, S. A.; Deshpande, S. G. Evaluation of ocular anti-inflammatory activity of Butea frondosa, Indian Journal of Pharmacology. 1995, 27, 116-119. 69. Cheng, Z. J.; Kuo, S. C.; Yu, S. M. Isolation of butein from Dalbergia odorifera ,Europian Journal of Pharmacology. 1995, 280, 69-77. 70. Wongkham, S.; Wongkham, C.; Trisonthi, C.; Boonsiri, P.; Simasathiansophon, S.; Atisook, K. Isolation and properties of a lectin from the seeds of Butea monosperma, Plant science. 1994, 103, 121-126. 71. Chopra, R. N.; Ghosh, S.; Chatterjee, N. R. Anthelmintic and their uses in medical and veterinary practice, Indigenous Drugs of India. 1933, 303-304. School of Pharmaceutical Sciences, Shobhit University, Meerut
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72. Shah, K. C.; Baxi, A. J.; Dave, K. K. Isolation and identification of free sugar and free amino acids from the petals of Butea frondosa, Indian Drugs. 1992, 29 (9), 422-423. 73. Mishra, D. N.; Dixit, V.; Mishra, A. K. Isolation of pterocarpans, phenols and lipids from the stem of Butea monosperma, Indian Drugs. 1991, 28(7), 300-303. 74. Guha, P. K.; Poi, R.; Bhattacharyya, A. A new imide isolated from the pods Butea monosperma, Phytochemistry. 1990, 29(6), 2017. 75. Zafar, R.; Singh, P.; Siddiqui, A. A. Antimicrobial and preliminary studies on leaves of Butea monosperma Linn., Indian Journal of Forestry. 1989, 12(4), 328-329. 76. Bandara, B. M.; Kumar, N. S.; Samaranayake, K. M. S. An antifungal constituent from the stem bark of Butea monosperma, Journal Ethnopharmacology. 1989, 25, 7375. 77. Bhargava, S. K. Estrogenic and postcoital anticonceptive activity in rats of butin isolated from Butea monosperma seed, Journal of Ethnopharmacology. 1986, 18, 95101.
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3. Plant(s) Profile 3.1 Acorus calamus It is a small genus of herbs of the monocotyledonous family Araceae. It is comprises two species i.e. Acorus calamus and Acorus gramineus Soland. The plant is found in the northern temperate and subtropical regions of Asia, North America, and Europe. The plant prefers swampy or marshy habitats. The plant exhibits polyploidy and three karyotypes in India, the plant is found growing wild as well as cultivated up to altitude of 2200 m in the Himalayas. It is plentiful in the marshy tracts of Kashmir, Himachal Pradesh, Manipur, and Naga hills, and is regularly cultivated in Karnataka. Various therapeutic potentials have been attributed to Acorus calamus in the traditional system of medicine as well as in folk fore practices. Acorus calamus has been used as traditional Chinese and Indian prescription for its beneficial effects.1 Sweet flags specimens showing whole parts are presented in figure 3.1.
Fig. 3.1 Sweet flags specimen showing whole parts 3.1.1Classification 2,3 Kingdom – Plantae Division – Angiospermae. Class – Monocotyledoneae Order – Spathiflorae School of Pharmaceutical Sciences, Shobhit University, Meerut
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Family – Araceae Genus – Acorus Species – calamus
3.1.2 Morphology Acorus calamus possesses grass-like or sword shaped long slender leaves that fan out from a pinkish base and grows up to 1.5m in length. The flower stem, or scape curved spadix is curved with small yellowish-green to brown flowers. The plant bears green, angular, 1-3 seeded berries. The seeds are oblong in shape. The rhizomes of the plant are occasionally orange-brown in color, cylindrical to flat with distinct node and internodes. The rhizomes possess strong, characteristic aromatic odor and bitter in taste. The transversely cut surface of the rhizome in color with pinkish tinge and differentiated into narrow cortical and large stellar regions. Microscopically, epidermis is radically elongated cells with heavily thickened walls. The cortical region consists of thin walled parenchymatous cells in chains, having large intercellular spaces, sheathed collateral vascular bundles, and patches of fibers.1 3.1.3 Cultivation The plant is propagated through rhizomes. The sprouted rhizomes are taken from old vigorous plants. Cultivation of Acorus calamus is shown in figure 3.2.
Fig. 3.2 Cultivation of Acorus calamus School of Pharmaceutical Sciences, Shobhit University, Meerut
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Plant Profile Before planting propagates should be washed and treated with 0.3% solution of
brassical for 5 min. The planting is done in furrows at depth of 6cm with the spacing of 30cm between the rows and 35cm between the plants. Light irrigation after planting is essential to get uniform good growth of rhizomes. The ideal time of planting is between the second week of June and second week of July.4
3.1.4 Chemical constituents A wide variety of chemical constituents have been reported from the rhizomes of Acorus calamus. The oil of Acorus rhizomes has been analyzed by various researchers for their chemical constituents. The oil contains varying concentration of α-asarone(a), β-asarone, γasarone, calamine, calamemenol, calamenone(b), α –pinene(c), β-pinene(d), camphene, eugenyl acetate, eugenol(e), isoeugenol(f), methyl eugenol(g), calamol, azulene(h), eugenol methyl ether, dipentene(i), α- caryophyllene(j) and hydrocarbons. The oil also contains fatty acids such as palmitic acid and its ester, heptylic acid, an ester of butyric acid, acoramone (k). The 50% ethanolic extract of the rhizome contains glycosides, flavonoids, sterols and terpenoids. Two sesquiterpenoids ketones of the guaiane-type calamusenone (l) and its isomer were isolated from sweet flag oil.
Sesquiterpenoids, namely shybunone(m), isoshybunone(n), isocalamendiol,
dehydroxycalamendiol, and epishyobunone were isolated from Acorus, in addition to calamenone. The thermal isomerization of shybunone an elememe(o) type sesquiterpene, resulted in the formation of preisocalamendiol a germacrone-type compound and acorone(p).1 Phytoconstituents of Acorus calamus are shown in figure 3.3.
OH
H3CO
CHCHCH3
H3CO
OCH3
1,2,4-trimethoxy-5-(2-propenyl)benzene
O OH
α--pinene
(c)
cis-asarone (a) Calamenone (b)
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H3CO
OCH3
H3CO
CH2COCH3
Acoramone(k) α-caryophyllene
O
Shybunone(m) Calamusenone(l)
O O
Elemene(o)
Epishybunone(n)
O
Acoromone(p)
Fig. 3.3 Phytoconstituents of Acorus calamus
3.1.5 Medicinal uses Uses described in traditional medicine In the ayurvedic system, the rhizomes of Acorus calamus are considered to possess aromatic, stimulant, bitter tonic, emetic, expectorant, aphrodisiac, laxative, diuretic, antispasmodic, carminative and anthelmintic properties. They are used for the treatment of a host of diseases such as mental ailments like epilepsy, schizophrenia, memory disorders, chronic diarrhea, dysentery, cough, and asthma, glandular and abdominal tumors. They are also
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employed for kidney and liver troubles, rheumatism and eczema. The skin of the rhizomes are said to be haemostatic. The rhizomes are used in the form of powder, balms, enemas, and pills. 1 Uses described in folklore medicine The rhizomes are used as an emetic and to kill lice. The leaves are used on wounds to kill worms. The stem is used in cough, cold and toothache. The rhizome is useful in diseases of nervous system, throat, and in diarrheal diseases. It is used for protection against smallpox in respiratory, gastrointestinal tract diseases, snakebite and in dysmenorrheal.1
Sedative and hypnotic effect The volatile oils from Acorus showed potentiation of the sedative activity of phenobarbitone in mice. The steam volatile fractions responsible for the activity resided in the hydrocarbon fraction of the oil or in an oxygenated sleeping time in mice with pentobarbital, hexobarbital and ethanol. The sedative enhancement activity is highest in the volatile fraction of the petroleum ether extract.
CNS depressant activity The effect of the ethanol extract of Acorus was studied on spontaneous electrical activity and monoamine levels of the brain. The serotonin level was increased in the cerebral cortex but decreased in the midbrain. Similarly, the dopamine level was increased in the caudate nucleus and midbrain but decreased in cerebellum. Thus, Acorus showed its action by changing electrical activity and by differentially altering brain monoamine levels in different brain regions.1 Acorus calamus leaf extract inhibits the production of pro-inflammatory cytokines through multiple mechanisms and act as a novel and effective anti-inflammatory agent for the treatment of skin diseases.5
Anticancer activity Lectins purified from the rhizomes of Acorus (sweet flag) species, by using affinity chromatography exhibited potent antimitogenic activity toward mouse splenocytes and human lymphocytes.
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Behavioral changes The steam volatile fractions of the rhizome of Acorus calamus decreased blood pressure of anesthetized cats, caused hypothermia in mice, and potentiated the action of acetylcholine, histamine, and barium chloride on the isolated gut of guinea pigs. The in vitro studies of the oil on the rat brain revealed that it inhibited the oxygen uptake of brain tissues.1
Memory-enhancing effect In ayurveda, herbal medicines with rasayana effects are believed to be restorative, to attain longevity, intelligence and freedom from age related disorder. Acorus calamus possesses beneficial memory enhancing property on memory impairment, learning performance and behavior modifying. 1 The rhizomes of Acorus calamus are used in loss of memory given in combination with other drugs like Centella asiatica, Bacopa monniera and Reserpine serpentine.6
Antioxidant activity The ethyl acetate extract of Acorus was found to be a potent antioxidant by inhibition of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical. In vitro antioxidant activity by DPPH scavenging at three different concentration (0.2, 0.3, and 0.01 g/ml) showed a maximum activity of 86.43% at 0.2g/ml. The ethyl acetate and methanol extracts of Acorus protected most of the changes of oxidative stress status in the rat brain induced by noise stress.1
Action on cardiovascular system The essential oil was reported to have negative inotropic and antiarrythmic properties.α -asarone and β -asarone showed cardiac depressant activity on frog and rabbit heart perfusion experiments. Ethyl acetate fraction of Acorus calamus extract, in vitro and in vivo has the potential to be useful for the treatment of diabetes and cardiovascular complications without body weight gain.8
Anticonvulsant activity Acorus calamus is a traditionally used as neuroprotective and anticonvulsant. Acorus calamus showed mild to moderate anticonvulsant activity at dose of 250 mg/kg, while it was School of Pharmaceutical Sciences, Shobhit University, Meerut
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found to effective at dose of 150 mg/kg in maximal electroshock and pentelenetetrazole induced convulsion. It also reduces the dose of phenytoin and phenobarbital showed synergistic anticonvulsant activity in adult albino mice.9
Insecticidal activity Acorus calamus has also attracted attention for its insecticidal activity. It is a cheap, effective and simple to handle insecticide with no adverse effects on sored graing, domenstic animal and human being.1
Bactericidal activity Dried rhizome revealed antibacterial activity for Bacillus subtilis, Escherichiaa coli, and Staphylococcus aureus. Acorus calamus rhizomes and leaves must possess active principle α asarone and β asarone which is believed to be responsible for their antimicrobial activity. Methanolic extract of the rhizome exhibited high activity against filamentous fungi, Trichophyton rubrum, Microsporum gupseum and Penicillium marneffei. 10
Antidiarrheal activity A study was performed to evaluate the effect of aqueous and methanol extracts of Acorus calamus rhizome for its antidiarrheal potential against castor oil induced diarrhea in mice. The methanolic plant extracts significantly reduced induction time of diarrhea and total weight of the feces.11
Immunosuppressive activity The in vitro immunomodulatory properties of the ethanol extract of Acorus calamus rhizomes were evaluated. This extract inhibited proliferation of mitogen (phytohaemagglutinin; PHA) and antigen (purified protein derivative; PPD)-timulated human peripheral blood mononuclear cells (PBMCs). It also inhibited production of nitric acid (NO), interleukin-2 (IL-2) and tumor necrosis factor-α (TNF- α).12 Some of the ayurvedic drugs in which Acorus calamus is one of the ingredients shown in table 3.1.13
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Table 3.1 Marketed ayurvedic formulations containing Acorus calamus S. No.
Ayurvedic Drugs
Uses
1.
Krumina
For intestinal diseases
2.
Automer powder
To check ear infection
3.
Cybil Tab
As tranquilizer
4.
Libobel drops
In liver disorder
5.
Galachol
To enhance lactation
6.
Sepra tab
For mental disorder
7.
Traquinil
For tranquillizer
8.
Ciladin
To cure Pschychosometic disorder
9.
Phortage
For sex tonic
10.
Suktin
For acidity and peptic ulcer
3.2 Vitex negundo Plants and plant derived agents have long history as source of potential chemotherapeutic agents in Ayurvedic and Unani system of medicine. Vitex negundo, belonging to family Verbenaceae (which comprises 75 genera and nearly 2500 species), commonly known as five leaved chaste tree (Eng), Nirgundi (Hindi), is a deciduous shrub, occur in tropical to temperate regions (up to 2200 m from east to west) grows gregariously in wastelands and is also widely used as a hedge plant. Plants used in traditional medicine represent a priceless tank of new bioactive molecules. Vitex negundo is one of the important plants from traditional system of medicine found all over the world. Vitex negundo is large aromatic shrub distributed throughout India. Plant is easily grown in light well drained loamy soil in a warm sunny position. The various chemical constituents like flavonoids, flavone glycosides, volatile oil, triterpenes, tannins and lignin many others were identified in this plant. This review gives a bird’s eye view mainly on the pharmacognostic characteristics, traditional uses, phytochemistry and pharmacological actions of Vitex negundo.14 School of Pharmaceutical Sciences, Shobhit University, Meerut
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3.2.1 Classification of Vitex negundo (Verbanaceae) Kingdom: Plantae Subkingdom: Tracheobionta – Vascular plants, Super division: Spermatophytes – Seed plants, Division: Magnoliophyta – Flowering plants, Class: Magnoliopsida – Dicotyledons, Subclass: Asteridae, Order: Lamiales, Family: Verbenaceae, Genus: Vitex, Species-negundo Vitex negundo plant is shown in figure 3.4.
Fig. 3.4 Vitex negundo plant
3.2.2 Morphology Vitex negundo is large and erect aromatic shrubs grow to height 2–5 m and slender tree with quadrangular branchlets distributed throughout India. The leaves have five leaf lets in a School of Pharmaceutical Sciences, Shobhit University, Meerut
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palmately arrangement, which are lanceolate, 4–10 cm long, hairy beneath and pointed at both ends, quadrangular whites fine tomentum, the leaves are 3-5 foliate, leaflets are lanceolate (5-10 cm), acute terminal leaflet (16-32 mm) with petiolate having 1-1.3 cm long, with a very short petiolate. The bluish purple flowers are numerous. The fruit is succulent, black when ripe, rounded and about 4 mm in diameter.
3.2.3 Cultivation Vitex negundo is easily grown plant; it prefers a light well-drained loamy soil in a warm sunny position sheltered from cold drying winds. The plants require abundant summer sunshine in order to ripen their wood fully; the well-ripened wood is more frost resistant. The leaves and stems are strongly aromatic. The flowers have a most pronounced musk-like perfume.
3.2.4 Chemical constituents
Phytochemical investigation of this plant indicated the presence of monoterpenes agnuside, The flavonoids are casticin, chryso-splenol and vitexin, vitexicarpin, 5,3’-dihydroxy3,6,7,4’-tetramethoxyllavone and hydroxy-3,6,7,3’,4’-penta methoxy flavone from the leaves. Seeds of Vitex negundo agorded a new lignan characterized as 6-hydroxy-4-(4-hydroxy-3methoxyphenyl), 3-hydroxymethyl-7- methoxy-3,4-dihydro-2-naphthaldehyde by spectroscopic methods and triterpenoids (betulinic acid and ursolic acid), lignans (negundins, vitedonin), alkaloid (vitrafalal) and diterpene (vitedoin) investigated. Isolation of the acetoacetate fraction yielded two major lignans: 6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7methoxy-3,4-dihydro-2 napthededyde and vitedoamine A.
Chromatography of an ethanolic extract of Vitex negundo resulted in the isolation of another new iridoid glucoside which was characterized as 6’-p-hydroxybenzoyl mussaenosidic acid
with
derivatives
2-p
hydroxyl
benzyl
mussaenosidic
acid
(1b),
6-
p-
hydroxybenzoylmussaenosidic acid (la), p-hydroxybenzoyl ester of mussaenosidic acid, phydroxybenzoyl methyl ester of mussaenosidic acid. Sequiterpenes flavone glycosides, iridoid glycosides, eurotoside aucubin, stilbenes have been isolated from roots of Vitex negundo. School of Pharmaceutical Sciences, Shobhit University, Meerut
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Chasteberry is contains a progesterone like compound. The leaves of Vitex negundo are known to possess various antioxidant chemical constituents like flavonoids, vitamin C and carotene which may have a modulatory effect on oxidative stress or endogenous antioxidants. Tannins were found in the aerial parts of Vitex negundo. Triterpenoids present in Vitex negundo and Emblica officinalis may involve in venom inactivation processes. Flavonoids are known to inhibit the enzyme prostaglandin synthetase, more specifically the endoperoxidase and reported to produce significant anti-inflammatory effect. Bioassay-guided fractionation of the chloroform-soluble extract of the leaves of Vitex negundo following isolation gives the known flavone vitexicarpin, which exhibited broad cytotoxicity in a human cancer cell line. Phytoconstituents of Vitex negundo is shown in figure 3.5.
H3CO
O
H3CO
CH2OH
HO
CH2OR
O
HO
OCH3
OCH3
OCH3
5 R=H 6 R=B-D- Glucose
OH
OH
(+)-lyoniresinol 5 (+)-lyoniresinol-3a-O-b-D-Glucoside 6
Negundin A
O H3CO
R
H 3 CO
NH CH 2 OH
HO
HO
OCH 3 OH
2 R=CH2OH 3 R=CHO
Negundin - 2 6-hydroxy-4-(4-hydroxy-3-methoxyethyl)7-methoxy-3,4-dihydro-2-nepthphaldehyde
OCH3
OH Vitedomine A
Fig. 3.5 Phytoconstituents of Vitex negundo School of Pharmaceutical Sciences, Shobhit University, Meerut
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3.2.8 Medicinal Uses Leaves The leaves Vitex negundo are antibacterial, antitumor, astringent, febrifuge, sedative, tonic and vermifuge. They are useful in dispersing swellings of the joints from acute rheumatism, and of the testes from suppressed gonorrhoea. The juice of the leaves is used for removing foetid discharges. They are harvested in early summer and used fresh or dried. Extracts of the leaves have shown bactericidal and antitumor activity.
Stem The stems of Vitex negundo is used in the treatment of burns and scalds. Fruit The dried fruits of Vitex negundo are vermifuge. The fruit is also used in the treatment of angina, colds, coughs, rheumatic difficulties etc. The fresh berries are pounded to a pulp and used in the form of a tincture for the relief of paralysis, pains in the limbs, weakness etc. Root The roots of Vitex negundo are expectorant, febrifuge and tonic. It is used in the treatment of colds and rheumatic ailments. It is harvested in late summer and autumn, and dried for later use. Edible Uses Seeds of Vitex negundo are occasionally used as a condiment, it has pepper substitute. When washed to remove the bitterness it can be ground into a powder and used as a flour, though it is very much a famine food used only when all else fails .A tea is made from the roots and leaves. Therapeutic uses mentioned in Ayurvedic Pharmacopoeia Sul, Vatavyadhi, Amavata, Kustha, Kandu, Kasa, Pradara, Adhmana, Piniroga, Gulma,Aruci,Karma,Vrana Nadi Vrana, Karnasula, Sutika, Jwara.15
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3.3 Butea frondosa 3.3.1 Classification Kingdom: Plantae (unranked) Angiosperms (unranked): Eudicots (unranked): Rosids Order: Fabales Family: Fabaceae, Subfamily: Faboideae, Tribe: Phaseoleae, Genus: Butea Common name – Palash, Dhak, Tesu.
The botanical name of Palash is Butea frondosa (Leguminosae) synonym; Butea monosperma. The plant is moderate sized deciduous tree, with bright yellowish red to orangish red flowers. Commonly called “flame of the forest” (English) and Tesu (Hindi) found throughout India up to a height 1230 meter, except in arid zones.16 Leaves of Butea frondosa are presented in figure 3.6.
Fig. 3.6 Leaves of Butea frondosa
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Species Butea acuminate, Butea affinis, Butea africana, Butea monosperma (Butea frondosa).17
3.3.2 Morphology The trunk is somewhat crooked, has light brown or bluish grey bark and exudes a ruby red gum (Bengal kino). When incised, leaves are alternate long petiolated, 3-folioate and large, 25-45 cm long. Flowers are large, orange red or scarlet on racemes, 15-20 cm long on bare branches; pods are silvery white, 10-15 cm long, pendulous contains large, flat kidney shaped seeds. Mature seeds are glossy, wrinkled and deep reddish brown in colour. The plants flower from January to April depending on the locality.
3.3.3 Cultivation Butea frondosa is easily grown plant; it prefers a light well-drained loamy soil in a warm sunny position sheltered from cold drying winds. The plants require abundant summer sunshine in order to ripen their wood fully; the well-ripened wood is more frost resistant. The leaves and stems are strongly aromatic.
3.3.4 Chemical constituents Butea frondosa contains several γ- pyrone and furan derivatives, and related compounds (e.g., flavones, chalkones, aurones and their glycosides) had been isolated mostly from its flowers. Butin, Isobutin from flowers, Palasonin from seeds, and also proteolytic enzymes, steroid stigmasterol, triterpenoid, gum, leucocyanidin, gallic acid. Phytoconstituents of Butea frondosa are shown in figure 3.7.
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OH OR
HO
O
CH 2
OH
RO
O
OR OR' OR OR
O
RO
Butein (2’, 4’, 3, 4-tetrahydroxychalcone)
O
OR' OR
O
OR
R'O
H 3CO
O O
OR OR
RO R'O OR RO O
O
OR
O OR
(1) R=R'=H (2) R=R'=COCH3
O
(3) R=OCH3,R'=H (4) R=OCH3,R'=COCH3
HO
O
O
OCH3 7-Hydroxy-3-p-tolyl-chromen-4-one; compound with methanol
Fig. 3.7 Phytoconstituents of Butea frondosa
3.3.5 Medicinal Uses Flower of Butea monosperma is traditionally used as anticonvulsant, antioxidant, antistress, memory and behaviour stimulant, antigout, diuretic, antileprotic, antiinflammatory, antiulcer, astringent and antihepatotoxic. Flower is also used to treat enlarged spleen, menstrual disturbances, burning sensation and eye diseases. Leaf of B. monosperma is traditionally used as antiinflammatory, antitumor, diuretic, antidiabetic, antimicrobial, anthelmintic, appetizer, carminative, astringent and aphrodisiac. These are also used to treat stomach disorders, diabetic 71 School of Pharmaceutical Sciences, Shobhit University, Meerut
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soar throat, irregular bleeding during menstruation, flatulent colic, cough and cold. Stem bark is traditionally used as aphrodisiac, antidysentery, antiulcer, antitumor, antimicrobial, antifungal, antipyretic, blood purifier and anti-asthmatic. It is also used in bleeding hemorrhoid disorder, dysmenorrheal, hydrocele, liver disorders, gonorrhoea, wound, worm infections, scorpion sting, cough and cold. Root is used in night blindness, elephantiasis, impotency and in snake bite. It also causes temporary sterility in women and is applied in sprue, piles, ulcers, tumors and dropsy. Seed of B. monosperma is used in inflammation, skin and eye diseases, bleeding piles, urinary stones, abdominal troubles, intestinal worms and tumour. When seeds are pounded with lemon juice and applied to the skin, they act as a rubefacient.18
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References 1. Mukherjee, P.K.; Kumar, V.; Mal, M.; Houghton, P.J. Acetylcholinesterase inhibitors from plants. Pharm. Biology Pharm. Biology. 2007, 45(8), 651. 2. Mukharji, K.P. Quality Control of Herbal Drugs. Business Horizons, 1st ed.; 2002; p. 6. 3. Motley, T.J. The ethnobotany of sweet flag, Acorus calamus (araceae).Eco.Bot.1994, 48, 397. 4. Kulkarni, V.M. In vitro propagation of sweet flag (Acorus calamus).J. Med. Aromatic Plant Science. 1999,21,325. 5. Kim, H.; Han, T.H.; Lee, S.G. Anti-infammatory activity of a water extract of Acorus calamus L. leaves on keratinocyte HaCaT cells. Journal of Ethnopharmacology. 2009,122, 149. 6. Wu, H-S.; Zhu, D-F.; Zhou,C-X.; Feng, C-R.; Lou, Y-J.; Yang, B.; He, Q. J. Insulin sensitizing activity of ethyl acetate fraction of Acorus calamus L. in vitro and in vivo Journal of Ethnopharmacology. 2009. 123, 288–292 7. Yende, S.R.; Harle, U.N.; Bore, V.V.; Bajaj, A.O.; Shroff, K.K.; Vetal, Y.D. Reversal of neurotoxicity induced cognitive impairment associated with phenytoin and phenobarbital by Acorus calamus in mice Journal of Herbal Medicine & Toxicology. 2009, 111. 8. Devi, A.; Ganjewala, D. Antimicrobial activity of Acorus calamus L. rhizome and leaf extract Acta Biologica Szegediensis. 2009, 53(1), 45-49. 9. Shobha, F.G.; Thomas, M. Study of antidiarrhoeal activity of four mrdicinal plants in castor-oil induced diarrhoea. Journal of Herbal Ethnopharmacology. 2001, 73. 10. Rathee, P.; Chaudhary, H..; Rathee, S.; Rathee, D. Natural memory boosters pharmacognosy rev. 2008, 2(4), 249.
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11. Singh, V.K.; Mahrotra, S.; Mishra, K.P.; Maurya, R.; Srimal, R.C.; Yadav, V.S.; Pandey, R. Anticellular and immunosuppressive properties of ethanolic extract of Acorus calamus rhizome.International Immunopharmacolog. 2003, 3, 53. 12. Kumar, V.S.; Srivastva, R.K.; Tomar, V.K.S.; Singh, A.K.; Kumar, S. Cultivation, chemistry, biology and utilization of bach (Acorus calamus ). J.Med. Aromatic Plant Science. 2000, 22, 338. 13. Kumar, V.S; Srivastva,R.K; Tomar, V.K.S; Singh, A.K; Kumar, S. Cultivation, chemistry, biology and utilization of bach (Acorus calamus ). J.Med. Aromatic Plant Science. 2000, 22, 338. 14. Gautam, L.N.; Shrestha, S.L.; Wagle, P.; Tamrakar, B.M. Chemical constituents from Vitex negundo of Nepalese origin. Scientific World. 2008, 6, 6. 15. Bansod, M.S.; Harle, U.N. Vitex negundo phytochemical constituents, traditional uses and Pharmacological properties: comprehesive review. Pharmacologyonline. 2009, 1, 286302. 16. Khare, C. P., Encyclopedia of Indian Medicinal Plants, Springer publisher. 2004, 113114. 17. www. Enwikipedia.org. 18. Sukhdev. A selection of prime Ayurvedic Plant Drugs, Anamaya publisher. 2006, 118.
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4. Experimental Work 4.1 Materials and methods 4.1.1 Collection and authentication of roots of Acorus calamus The roots of Acorus calamus was collected from Haridwar, Uttarakhand, India. The plant was identified and authenticated by Dr. K. Pradheep, Senior Scientist at National Bureau of Plant Genetic Resources (ICAR) New Delhi. A voucher specimen no. NHCP/NBPGR/2010-36/ is deposited in the herbarium. The chemical and solvents used during the study are tabulated in table 4.1 and the instruments and apparatus are mentioned in table 4.2
Table 4.1 Chemical and solvents S. No
Name
Specification
Manufacturer/Supplier
Petroleum ether o
1.
(60-80 C)
LR grade
Rankem, RFCL Ltd. New Delhi
2.
Methanol
LR grade
Rankem, RFCL Ltd. New Delhi
3.
Ethyl acetate
LR grade
Rankem, RFCL Ltd. New Delhi
4.
Chloroform
LR grade
Rankem, RFCL Ltd. New Delhi
5.
Acetone
LR grade
Rankem, RFCL Ltd. New Delhi
6.
Acetic acid glacial
LR grade
Rankem, RFCL Ltd. New Delhi
7.
Toluene
LR grade
Rankem, RFCL Ltd. New Delhi
8.
Benzene
LR grade
Rankem, RFCL Ltd. New Delhi
9.
n-hexane
LR grade
Rankem, RFCL Ltd. New Delhi
10.
Ammonia
LR grade
Rankem, RFCL Ltd. New Delhi
11.
LR grade
Rankem, RFCL Ltd. New Delhi
12.
Sulphuric acid Silica-gel (60-120) mesh size
LR grade
Rankem, RFCL Ltd. New Delhi
13. 14.
Silica gel G α- Naphthol
LR grade LR grade
Rankem, RFCL Ltd. New Delhi E- merk, Mumbai, India
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15.
Fehling Solution A and B LR grade
16.
Ferric chloride
LR grade
17.
Picric acid Potassium iodide
LR grade
18. 19.
Lead acetate Mercuric chloride
20.
LR grade
E- merk, Mumbai, India Oualigens fine chemicals, Glaxo India Oualigens fine chemicals, Glaxo India Oualigens fine chemicals, Glaxo India
LR grade
Central Drug House New Delhi
LR grade
Central Drug House New Delhi
Table 4.2 Instruments/Apparatus S.No.
Instruments/Apparatus
Manufacturer
1.
U V Cabinet
Perfit India
2.
Magnetic stirrer
Remi Equipments Pvt. Ltd.
3.
Water bath
Narang scientific works Pvt. Ltd.
4.
Heating mantle size (1000 ml)
Perfit India
5.
Oven Universal (max. temp.250 oC)
Narang scientific works Pvt. Ltd.
6.
Rotary Vacuum Bath
Gupta scientific industries
7.
FTIR spectrometer
8.
1
Perkin Elmer Bruker advanced spectrometer
9.
MASS spectrometer
H NMR spectrometer
II
400
ESI-ToF Mass spectrometer
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4.1.2 Drying of Acorus calamus (roots) The roots of Acorus calamus were dried at room temperature.
4.1.3 Size reduction of Acorus calamus (roots) Size reduction of plant material was done with the help of grinder and stored in desiccator.
4.1.4 Extraction of Acorus calamus (roots) 500g of the dried roots powder was placed in soxhlet apparatus (Perfit, India) and subjected to successive extraction using petroleum ether (60°-80°C), methanol, and macerated to form an aqueous extract. Subsequently, various extracts were filtered and the filtrate was evaporated using vacuum evaporator (Perfit, India) under reduced pressure at ≤ 50° C temperature1. The crude extract obtained after evaporation was stored in desiccator. After extraction with various solvent remaining residue of root was discarded and extract was weighed. Physical nature and percentage yield of various extract are recorded in Table 4.3. 4 The % yield of various extracts was evaluated using the formula:
% Yield= Weight of extract (g)/ Weight of dry powder(g) ×100
Table 4.3 Physical nature and percentage yield of various root extracts of Acorus calamus
Extract(s)
Physical nature and color(s)
% Yield
Pale yellow
6.58
Methanol
Deep red
25.58
Aqueous
Dark brownish
21.10
Petroleum ether
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4.1.5 Phytochemical investigations1 Chemical test for carbohydrates Mono-saccharides are the building blocks of carbohydrates. Di, oligo and polysaccharides on hydrolysis in presence of mineral acid yield monosaccharide units. These are optically active compounds and respond to various color reaction and identification tests. 1
Molish test Aqueous solution of the extract mixed with few drops of Molish reagent (α naphthol) and conc. H2SO4 (sulphuric acid) was added along the wall of the tube. Formation of purple colored ring at junction indicated presence of carbohydrates.
Fehling solution test It is generally used for reducing sugar and composed of two solutions, which are mixed in situ. Fehling solution A composed of 0.5% of copper sulphate whereas Fehling solution B composed of sodium potassium tartarate. Equal volume of Fehling A and Fehling B solution were mixed (1ml each) and 2ml of aqueous solution of extract was added followed by boiling for 5-10.Minutes on water bath. Formation of reddish brown colored precipitate due to formation of cuprous oxide indicated presence of reducing sugar. 1
Benedict’s test It is used for reducing sugars and composed of mainly copper sulphate and sodium hydroxide. To the aqueous solution of extract, 1 ml of Benedict solution was added and heated almost to boiling. Formation of green precipitate in order of increasing concentration of simple sugar in the test solution, due to formation of cuprous oxide. 1 Chemical tests for lipids Biuret test The aqueous solution of protein in hot water, few drops of Biuret reagent (potassium hydroxide, Copper sulphate and sodium potassium tartarate) were added, which turned blue reagent to violet.
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Chemical tests for alkaloids Dragendroff’s test The test was carried out by taking sodium iodide with 5.2g of basic bismuth carbonate in 50 ml of glacial acetic acid and boiling for few minutes. It was allowed to stand overnight. The precipitates were filtered off. To the red brown filtrate, ethyl acetate and water were added. Then 20 ml of acetic acid was added and volume was made up to 100 ml with water. Extract was added to this solution. Reddish brown precipitate showed the presence of alkaloid.
Mayer’s test The test was performed by dissolving mercuric chloride in distilled water (A)and 5g of potassium iodide in of distilled water (B). Solution A and B were mixed together and the volume adjusted to 100 ml with water followed by addition of extract. Cream color precipitate showed the presence of alkaloid. 1
Hager test This was performed by dissolving 10 mg of picric acid in 100 ml distilled water and adding the extract to it. Yellow color precipitate showed the presence of alkaloid.
Wagner’s Test This test was performed by taking 1.27 g of iodine and 2 g of potassium iodide in 5 ml of water and the volume was made up to with water and 2 ml of extract was added. It produced reddish brown precipitate with the alkaloid. Chemical tests for glycosides Borntragor’s test In this test 5-10 ml of dilute hydrochloric acid (HCl) was added in 0.5 gm of extract and boiled on water bath for 10 minutes. Solution was filtered and filtrate was extracted with benzene and mixed with ammonia solution. Red color was obtained in ammonia layer that indicated the presence of anthraquinone glycosides. 1
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Keller Killani test In this test alcoholic extract of the drug was mixed with equal volume of water and 0.5 ml of strong lead acetate solution was added followed by stirring. Filtrate was extracted with equal volume of chloroform. Chloroform extract was evaporated to dryness and residue was dissolved in 3 ml of glacial acetic acid with addition of few drops of ferric chloride solution. The resultant solution was transferred to a test tube containing 2 ml of conc. sulphuric acid. Reddish brown layer was formed, which turned bluish green after standing due to presence of digitoxose .1
Chemical tests for saponin Foam test In this test 0.5 gm of extract was added in 10-20 ml of water, shaken for few minutes. Formations of frothing which persisted for 60-120 seconds, showed presence of saponins. 2
Chemical test for steroids Libermann Bruchard test In this test alcoholic extract of crude drug was extracted with chloroform. Few drops of conc. sulphuric acid were added from the side of the wall of test tube. Formation of violet to blue colored ring at the junction of two liquid, indicated the presence of steroid moiety.
2
Chemical tests for flavonoids Ammonia test In this test filter paper dipped in alcoholic solution of extract was exposed to ammonia vapor. Formation of yellow spot on filter paper showed the presence of flavonoids.
Chemical tests for phenolic compounds In this test extract solution was treated with 10 % ethanolic ferric chloride. Formation of bluish green or dark blue color indicated the presence of phenolic compounds. 3
Different phytochemical test of Acorus calamus (roots) are shown in table 4.4. School of Pharmaceutical Sciences, Shobhit University, Meerut
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Table 4.4 Phytochemical test(s) of Acorus calamus (roots) Chemical test(s) Extracts Carbohydrate
Petroleum ether
Methanol
Aqueous
Fehling’s test
-
-
-
Benedict’s test
-
+
-
Dragendroff’s test
-
+
+
Picric acid test
+
+
+
Wagner’s test
+
+
+
Borntragor’s test
+
+
+
Keller Killani test
+
-
-
-
+
+
Alkaloids
Glycosides
Saponins Foam test Phenolic compounds and tannin Ferric chloride test
-
+
+
(+) = Present, (-) = Absent. 4.1.6 Chromatographic studies4 Chromatography is a separation process which depends on the differential distribution of the components of a mixture between a mobile bulk phase and an essentially thin film stationary phase. This technique is used as analytical tool to establish the complexity of mixtures and the purity of samples, and as preparative tools for the separation of mixtures into individual components.
4.1.7 Thin layer chromatographic studies (TLC) TLC
is based on adsorption chromatography in which separation depends on the
selective adsorption of the components of a mixture on the surface of solid. The stationary phase School of Pharmaceutical Sciences, Shobhit University, Meerut
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was in the form of a thin layer adhering to a suitable form of backing material over which the mobile phase was allowed to ascend by capillary action. Traditionally analytical TLC has found application in the detection and monitoring of compound through a separation process. Precoated plates are also available for TLC technique in which backing material may be either of aluminium foil or a solvent resistant polyester sheet. These sheets can be cut to the desired size and activated if necessary. Thin layer chromatographic studies are shown in figure 4.1 and 4.2.
Fig.4.1 TLC of isolated compound in Visible region
Fig.4.2 TLC of isolated compound in Ultraviolet region
Thin layer Chromatographic studied was performed by application of solute as a spot. TLC plate was then placed in a tank containing sufficient solvent system for proper development School of Pharmaceutical Sciences, Shobhit University, Meerut
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via capillary action. The
developed chromatogram provided information about number of
compound in a mixture. The developed chromatogram was observed under short-wavelength UV light (254 nm). The Rf value is the constant and characteristic of the substance which indicates its movement relative to the solvent front in a given chromatographic system. 4
Rf= Distance traveled by compound/Distance traveled by solvent Rf value depends on number of variables such as the particle size of different batches of adsorbent. the solvent composition and the degree of saturation of the tank atmospheres with solvent vapors. prior activation and storage condition of the plates. the thickness of adsorbent layer. 4.1.8 Column chromatographic studies The stationary phase is in form of a packed column through which a mobile phase is allowed to flow under gravity, or under pressure applied to the top of the solvent reservoir. The choice solvent for transferring the mixture to be chromatographed to the column depends upon the solubility characteristics of the mixture. Frequently the most non-polar solvent for introducing the mixture on to the column and the initial solvent for chromatogram development. 4 Eluting solvent with increasing the polarity applied for solvent selection, all these solvents have sufficient low boiling points to permit ready recovery of eluted material. The solvents include hexane, cyclohexane, carbon tetrachloride, trichloroethylene, toluene, dichloromethane,
chloroform, diethylether, ethylacetate, acetone, propanol, ethanol, and
methanol. 4.1.9 Methodology The column assembly was set for the separation of compounds from methanolic extract. The silica gel (100-200 mesh size) was used for column packing. The slurry method was School of Pharmaceutical Sciences, Shobhit University, Meerut
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adopted for filling of the column. Slurry was made with chloroform. The adsorbent was allowed to settle evenly and free of air bubbles, by with gentle tapping of the column with a wooden rod. The top of the column was frequently covered with a circle of filter paper or a layer of clean sand to prevent disturbances of the surface during subsequent loading. Semisolid methanolic extract was poured through a funnel into the packed column. The solvent level was maintained 2.5 cm above the extract. For continuous supply of solvent at the constant rate the reservoir was set and filled with the solvent. The elutes were collected drop by drop in 10 ml volumetric flasks at the bottom of column. TLC was carried out for the identification of constituents which were eluted by different eluent solvent systems. Eluted fractions which have same Rf on the TLC were pooled. Afterwards other fractions were collected and subjected to thin layer chromatographic studies. At last the column was eluted with methanol. 4
Column chromatography of methanolic extracts of Acorus calamus roots are shown in table 4.5
Table 4.5 Column chromatography of methanolic extracts of Acorus calamus roots
S.No.
Eluent solvent (200ml )
Fractions
TLC Solvent System (chloroform: methanol) : Rf 80:20 90:10
1.
Chloroform
1-11
-
-
2.
Chloroform
12-18
3.
Chloroform
19-25
4.
Chloroform
26-32
5.
Chloroform:Methanol (90:10)
24-26
0.80,0.86
0.82, 0.87,0.73
6.
Chloroform:Methanol (80:20)
27-34
0.75,0.80
-
7.
Chloroform:Methanol (70:30)
35-41
0.65,0.71
0.84, 0.84
0.72, 0.74 0.82, 0.830.84,0.86,0. 87, 0.95, 0.78, 0.90, 0.83, 0.87
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0.85, 0.85, 0.92
0.81
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4.1.10 Isolation of compound from Acorus calamus roots The green residue obtained from methanolic extract was dissolved in minimum amount of chloroform in a round bottom flask and absorbed over silica gel mesh size 60-120 to make slurry. The slurry was dried under reduced pressure on rotatory evaporator (Perfit India). The completely dried silica gel slurry containing the methanolic extract was poured in the column (30×2 cm) packed earlier with slurry of silica gel (60-12- mesh size) in chloroform. Successively, elution was carried out with different solvents in increasing order of polarity. The fractions collected in conical flask were marked. The fractions were subjected to thin layer chromatography in order to check the homogeneity of various fractions. The fractions having same Rf value were pooled and concentrated. Finally the obtained fractions from 12 to 18 having same Rf (0.85) were isolated to give a single compound (AC-1).
4.1.11 Characterization of the isolated compound (AC-1) by spectral analysis Isolated compound (AC 1) was subjected to FTIR, 1H NMR and Mass spectroscopy. FTIR spectra of the isolated compound (AC 1) of Acorus calamus (roots) presented in figure 4.3.
Fig. 4.3 FTIR spectra of the isolated compound of Acorus calamus The FTIR spectrum featured bands at 2929.7.2 cm-1 indicated C-H stretching of alkanes. Medium and weak peaks were observed at 1645.4 cm-1 and 1510.5 cm-1 indicated C=C stretching of aromatic ring. Strong peak of ether (C-O stretching) was observed at 1053.5 cm-1.
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Sharp peak of tetra substituted benzene ring was observed at 802 cm-1. 5 Interpretation of FTIR spectra of the isolated compound of Acorus calamus is presented in table 4.6.
Table 4.6 Interpretation of FTIR spectra of the isolated compound of Acorus calamus Reported Peak(cm-1)
Observed Peak
Inference
3000-2850
2929.7(s)
Alkanes(C-H stretching)
1600, 1475(m-w)
1645.4(m), 1510.5(w)
Aromatic (C=C stretching)
1300-1000(s)
1053.5(s)
800-850(s)
866.3
Ether (C-O stretching) Tetra substituted benzene ring
1
H NMR spectra confirmed two singlet aromatic protons (δ 6.8 and7.4), two plefinic
protons (δ1.4and1.6), three methoxyl protons (δ 3.71,3.82,3.90), and methyl protons (δ 0.4) indicating sixteen protons signals. NMR spectra of the isolated compound is presented in figure 4.4.
Fig. 4.4 NMR spectra of the isolated compound Interpretation of NMR spectra of the isolated compound of Acorus calamus is presented in table 4.7.
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Table 4.7 Interpretation of NMR spectra of the isolated compound of Acorus calamus Reported Peak
Observed Peak
Inference
6.86,6.65
7.4. 6.8
Two singlet aromatic protons
6,42, 5.68
1.4,1.6
Two olefinics protons
3.85, 3.78, 3.77
3.71, 3.82, 3.90
Three methoxy protons
1.84
0.4
Methyl proton
Mass spectra confirmed a molecular peak at m/z 209 (M+). Fragment peaks were also observed at 60.4 M+ -C6H5+CO. Mass spectra of the isolated compound is shown in figure 4.5. 104.5 21665
%
2.17e4
360.6 7448
60.4 2395
338.6 2113
118.5 1612
0 50
100
150
200
250
300
350
365.3 2142
400
450
500
550
600
650
700
750
800
850
900
950
m /z 1000
Fig .4.5 Mass spectra of compound the isolated compound
Interpretation of Mass spectra of the isolated compound of Acorus calamus is presented in table 4.8.
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Table 4.8 Interpretation of Mass spectra of the isolated compound of Acorus calamus
Reported peak
Observed Peak
Inference
208
209
M+
105
104.5
M+ -C6H5+CO
From the spectral data the structure of the compound AC-1 was characterized as β asarone with molecular formula C12H16O3. Chemical structure of β asarone is presented in figure 4.6.
H3CO
OCH3
H3CO
asarone
Fig. 4.6 Chemical structure of β asarone
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4.2 Materials and methods 4.2.1 Collection and authentication of leaves of Vitex negundo The leaves of Vitex negundo was collected from Haridwar, Uttarakhand, India. The plant was identified and authenticated by Dr. K.C Bhatt, Senior Scientist at National Bureau of Plant Genetic Resources (ICAR) New Delhi. A voucher specimen no. NHCP/NBPGR/2010-39/ is deposited in the herbarium. The leaves were air dried at School of Pharmaceutical Sciences, Shobhit University, Meerut.
4.2.2 Drying of Vitex negundo (leaves) The leaves of Vitex negundo were dried at room temperature.
4.2.3 Size reduction of Vitex negundo (leaves) Size reduction of plant material was done with the help of grinder and stored in desiccator.
4.2.4 Extraction of Vitex negundo (leaves) 500g of the dried leaves powder was placed in soxhlet apparatus (Perfit, India) and subjected to successive extraction using petroleum ether (60°-80°C), methanol, and macerated to form an aqueous extract. Subsequently, various extracts were filtered and the filtrate was evaporated using vacuum evaporator (Perfit, India) under reduced pressure at ≤ 50° C temperature1. The crude extract obtained after evaporation was stored in desiccator. After extraction with various solvent remaining residue of leaves was discarded and extract was weighed. Physical nature and percentage yield of various extract are recorded in table 4.9. 4
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Table 4.9 Physical nature and percentage yield of various extract of Vitex negundo Extract(s)
Physical nature and color(s)
% Yield
Petroleum ether
Light Brownish
3.07
Methanol
Dark Greenish
12.90
Aqueous
Dark Brownish
13.51
4.2.5 Phytochemical investigations1 Preliminary phytochemical screening was carried out to find the presence of the active chemical constituents in methanolic extract such as alkaloids, flavonoids, tannins, phenolic compounds, saponins, steroids, fixed oils and fats. In general, tests for the presence of phytochemical compounds involved the addition of appropriate chemical reagent(s) to the extract in test tubes. The mixture was then shaken and/or heated as the case may be. The alkaloid was tested by using Dragendorff’s, Mayer, Wagner’s and Hager’s test. The flavonoids were tested by alkaline reagent and Shinoda test. The tannins were tested by ferric chloride and gelatin test. The total phenolic content in extract was determined by Bromine water and Liebermann tests. Saponin content of Vitex negundo was determined by froth formation test. The steroids and triterpenoids were tested by Salkowski test. The presence of fats and fixed oils was determined by using 2, 4dinitrophenylhydrazine test. The different extracts were subjected to qualitative analysis for identifying various constituents. The observations are recorded in table 4.10.
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Table 4.10 Phytochemical test(s) of Vitex negundo leaves Chemical test(s) Extracts
Petroleum ether
Methanol
Aqueous
Fehling’s test
-
-
-
Benedict’s test
-
+
-
-
+
+
+
+
+
+
+
+
Borntragor’s test
+
+
+
Keller Killani test
+
-
-
-
+
+
Carbohydrate
Alkaloids Dragendroff’s test Picric acid test
Wagner’s test Glycosides
Saponins Foam test Phenolic compounds and tannin Ferric chloride test + Present
-
+
+
- Absent
The tests were done to check the presence or absence of the active chemical constituents such as alkaloids, flavonoids, tannins, phenolic compounds, saponins, fixed oils and fats
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4.2.6 Chromatographic studies4 Chromatography is a separation process which depends on the differential distribution of the components of a mixture between a mobile bulk phase and an essentially thin film stationary phase. This technique is used as analytical tool to establish the complexity of mixtures and the purity of samples, and as preparative tools for the separation of mixtures into individual components.
4.2.7 Thin layer chromatographic studies (TLC) TLC
is based on adsorption chromatography in which separation depends on the
selective adsorption of the components of a mixture on the surface of solid. The stationary phase was in the form of a thin layer adhering to a suitable form of backing material over which the mobile phase was allowed to ascend by capillary action. Traditionally analytical TLC has found application in the detection and monitoring of compound through a separation process. Precoated plates are also available for TLC technique in which backing material may be either of aluminium foil or a solvent resistant polyester sheet. These sheets can be cut to the desired size and activated if necessary.
Thin layer Chromatographic studied was performed by application of solute as a spot. TLC plate was then placed in a tank containing sufficient solvent system for proper development via capillary action. The developed chromatogram provided information about number of compound in a mixture. The developed chromatogram was observed under short-wavelength UV light (254 nm). The Rf value is the constant and characteristic of the substance which indicates its movement relative to the solvent front in a given chromatographic system. 4
Rf= Distance traveled by compound/Distance traveled by solvent Rf value depends on number of variables such as the particle size of different batches of adsorbent. School of Pharmaceutical Sciences, Shobhit University, Meerut
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the solvent composition and the degree of saturation of the tank atmospheres with solvent vapors. prior activation and storage condition of the plates. the thickness of adsorbent layer. 4.2.8 Column chromatographic studies The stationary phase is in form of a packed column through which a mobile phase is allowed to flow under gravity, or under pressure applied to the top of the solvent reservoir. The choice solvent for transferring the mixture to be chromatographed to the column depends upon the solubility characteristics of the mixture. Frequently the most non-polar solvent for introducing the mixture on to the column and the initial solvent for chromatogram development. 4 Eluting solvent with increasing the polarity applied for solvent selection, all these solvents have sufficient low boiling points to permit ready recovery of eluted material. The solvents include hexane, cyclohexane, carbon tetrachloride, trichloroethylene, toluene, dichloromethane,
chloroform, diethylether, ethylacetate, acetone, propanol, ethanol, and
methanol.
4.2.9 Methodology The column assembly was set for the separation of compounds from methanolic extract. The silica gel (100-200 mesh size) was used for column packing. The slurry method was adopted for filling of the column. Slurry was made with chloroform. The adsorbent was allowed to settle evenly and free of air bubbles, by with gentle tapping of the column with a wooden rod. The top of the column was frequently covered with a circle of filter paper or a layer of clean sand to prevent disturbances of the surface during subsequent loading. Semisolid methanolic extract was poured through a funnel into the packed column. The solvent level was maintained 2.5 cm above the extract. For continuous supply of solvent at the constant rate the reservoir was set and filled with the solvent. The elutes were collected drop by drop in 10 ml volumetric flasks at the bottom of column. TLC was carried out for the identification of constituents which were eluted by different eluent solvent systems. Eluted fractions which have same Rf on the TLC were
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pooled. Afterwards other fractions were collected and subjected to thin layer chromatographic studies. At last the column was eluted with methanol. 4 Column chromatography of methanolic extracts of Vitex negundo leaves are shown in table 4.11.
Table 4.11 Column chromatography of methanolic extracts of Vitex negundo leaves S.No.
Eluent solvent (200ml )
Fractions
1.
Chloroform
1-5
2.
Chloroform
6-10
TLC Solvent System (chloroform: methanol) : Rf 80:20 90:10 -
0.75, 0.60
-
0.70
3.
Chloroform
11-15
0.45, 0.54, 0.71, 0.61
4.
Chloroform
16-20
0.61, 0.52, 0.90, 0.73
0.53,0.65, 0.32 0.31,0.74, 0.56
5.
Chloroform: Methanol (90:10)
21-25
0.55, 0.79
0.45,0.62, 0.35, 0.25
6.
Chloroform: Methanol (80:20)
26-30
0.95, 0.95
0.58,0.35, 0.72, 0.80
7.
Chloroform: Methanol (70:30)
31-35
0.54, 0.90,
0.25, 0.45
4.2.10 Isolation of compound from Vitex negundo leaves The greenish residue obtained from methanolic extract was dissolved in minimum amount of chloroform in a round bottom flask and absorbed over silica gel mesh size 60-120 to make slurry. The slurry was dried under reduced pressure on rotatory evaporator (Perfit India). The completely dried silica gel slurry containing the methanolic extract was poured in the column (30×2 cm) packed earlier with slurry of silica gel (60-12- mesh size) in chloroform. Successively, elution was carried out with different solvents in increasing order of polarity. The fractions collected in conical flask were marked. The fractions were subjected to thin layer chromatography in order to check the homogeneity of various fractions. The fractions having same Rf value were pooled and concentrated. Finally the obtained fractions from 26 to 30 having same Rf (0.95) were isolated to give a single compound (VN-1). School of Pharmaceutical Sciences, Shobhit University, Meerut
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4.2.11 Characterization of the isolated compound (VN 1) by spectral analysis Isolated compound (VN 1) was subjected to FTIR, 1H NMR and Mass spectroscopy. FTIR spectra of the isolated compound (VN 1) of Vitex negundo (leaves) presented in figure 4.7.
Fig. 4.7 FTIR spectra of isolated compounds of methanolic extract of Vitex negundo The FTIR spectrum featured bands at 3435.77 cm-1 indicated O-H stretching of phenols. Medium and weak peaks were observed at 2921.26 cm-1 and 2850.56 cm-1 indicated C=H stretching of alkenes and aldehyde. Strong peak of ether (C=O stretching) was observed at 1719.01 cm-1. Sharp peak of ester was observed at 1267.13 cm-1. 5Interpretation of FTIR spectra of the isolated compound of Vitex negundo is presented in table 4.12.
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Table 4.12 FTIR spectra of the isolated compound of Vitex negundo Reported Peaks (cm-1)
Observed Peaks
3529-3266 2925
3435.77 2921.26
Inference
O-H (s) Phenols C=H (s) Alkenes
2850.56 2850.56
C-H (s) Aldehyde
1719.01
1719.01
C=O (s) Carbonyl group
1461
1458.67
CH (b) Aldehyde
1266
1267.13
C=O (s) Esters
1
H NMR CDCl3 at 400 MHZ, triplet at δ 6.20 ppm was assigned to 8 protons of
fused aromatic compound. 1H NMR displayed 6 protons of terminal methyl at δ 3.66 ppm. Chemical shift of the 2 protons adjacent phenol group appeared at δ 5.17 ppm. NMR spectra of the isolated compound (VN 1) of Vitex negundo (leaves) presented in figure 4.8.
Fig. 4.8 NMR spectra of the isolated compound Interpretation of NMR spectra of the isolated compound of Vitex negundo is presented in table 4.13. School of Pharmaceutical Sciences, Shobhit University, Meerut
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Table 4.13 Interpretation of NMR spectra of the isolated compound of Vitex negundo
Reported Peak
Observed Peak
Inference
6.18- 6.75
6.20 – 6.70
Eight singlet protons
fused aromatic
3.60
3.66-4.26
Six protons of terminal methyl
3.85, 3.78, 3.77
5.17, 5.19
Two protons adjacent phenol group
Mass spectroscopy showed the molecular ion at m/z 353 (M+1) corresponding to molecular formula (C20H16O6) 352, as supported by 1H NMR, on the basis of their analysis. Mass spectra of isolated compound is shown in figure 4.9.
Fig. 4.9 Mass spectra of the isolated compounds
Interpretation of Mass spectra of the isolated compound of Vitex negundo is presented in table 4.14.
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Table 4.14 Interpretation of Mass spectra of the isolated compound of Vitex negundo
Reported peak
Observed Peak
352
Inference M+
353
From the spectral data the structure of the compound VN-1 was characterized as negundin A with molecular formula C20H16O6. Chemical structure of negundin A is presented in figure 4.10.
H3CO
O O
HO
OCH3 OH
Negundin A
Fig. 4.10. Chemical structure of lignans (Negundin A)
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4.3 Materials and methods 4.3.1 Collection and Authentication of leaves of Butea frondosa The leaves of Butea frondosa was collected from Meerut, Uttar Pradesh, India. The plant was identified and authenticated by Dr. E. Roshini Nayar, Principal Scientist at National Bureau of Plant Genetic Resources (ICAR) New Delhi. A voucher specimen no. NHCP/NBPGR/2010-35/ is deposited in the herbarium.
4.3.2 Drying of Butea frondosa (leaves)
The leaves of Butea frodosa were dried at room temperature.
4.3.3 Size reduction of Butea frondosa (leaves) Size reduction of plant material was done with the help of grinder and stored in desiccator.
4.3.4 Extraction of Butea frondosa (leaves) 500g of the dried leaves powder was placed in soxhlet apparatus (Perfit, India) and subjected to successive extraction using petroleum ether (60°-80°C), methanol, and macerated to form an aqueous extract. Subsequently, various extracts were filtered and the filtrate was evaporated using vacuum evaporator (Perfit, India) under reduced pressure at ≤ 50° C temperature1. The crude extract obtained after evaporation was stored in desiccator. After extraction with various solvent remaining residue of leaves was discarded and extract was weighed. Physical nature and percentage yield of various extract are recorded in table 4.15. 4
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Table 4.15 Physical nature and percentage yield of various leaves extracts of Butea frondosa Extract(s)
Physical color(s)
nature
and % Yield
Petroleum ether
Light Brownish
5.18
Methanol
Dark Greenish
9.00
Aqueous
Dark Brownish
8.10
4.3.5 Phytochemical investigation1 Preliminary phytochemical screening was carried out to find the presence of the active chemical constituents in methanolic extract such as alkaloids, flavonoids, tannins, phenolic compounds, saponins, steroids, fixed oils and fats. In general, tests for the presence of phytochemical compounds involved the addition of appropriate chemical reagent(s) to the extract in test tubes. The mixture was then shaken and/or heated as the case may be. The alkaloid was tested by using Dragendorff’s, Mayer, Wagner’s and Hager’s test. The flavonoids were tested by alkaline reagent and Shinoda test. The tannins were tested by ferric chloride and gelatin test. The total phenolic content in extract was determined by Bromine water and Liebermann tests. Saponin content of Butea frondosa was determined by froth formation test. The steroids and triterpenoids were tested by Salkowski test. The presence of fats and fixed oils was determined by using 2, 4-dinitrophenylhydrazine test. The different extracts were subjected to qualitative analysis for identifying various constituents. The observations are recorded in table 4.16.
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Table 4.16 Phytochemical test(s) of Butea frondosa (leaves) Chemical test
Petroleum ether
Methanol
Aqueous
extract
extract
extract Alkaloids Mayer reagent
-
+
+
Hager reagent
-
+
-
Wagner reagent
-
+
-
Molisch’s reagent
-
+
+
Fehling test
-
+
+
-
+
+
-
+
+
-
+
+
-
+
+
+
+
-
Carbohydrate
Glycosides Legal test Saponins Foaming test Phenolic compounds and tannin Ferric chloride test Protein Ninhydrin Test Oils and fats Solubility test
(+) = Present
(-) = Absent
4.3.6 Chromatography studies4 Chromatography is a separation process which depends on the differential distribution of the components of a mixture between a mobile bulk phase and an essentially thin film stationary phase. This technique is used as analytical tool to establish the complexity of mixtures and the purity of samples, and as preparative tools for the separation of mixtures into individual components. School of Pharmaceutical Sciences, Shobhit University, Meerut
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4.3.7 Thin layer chromatographic studies (TLC) TLC
is based on adsorption chromatography in which separation depends on the
selective adsorption of the components of a mixture on the surface of solid. The stationary phase was in the form of a thin layer adhering to a suitable form of backing material over which the mobile phase was allowed to ascend by capillary action. Traditionally analytical TLC has found application in the detection and monitoring of compound through a separation process. Precoated plates are also available for TLC technique in which backing material may be either of aluminium foil or a solvent resistant polyester sheet. These sheets can be cut to the desired size and activated if necessary. Thin layer Chromatographic studied was performed by application of solute as a spot. TLC plate was then placed in a tank containing sufficient solvent system for proper development via capillary action. The
developed chromatogram provided information
about number of compound in a mixture. The developed chromatogram was observed under short-wavelength UV light (254 nm). The Rf value is the constant and characteristic of the substance which indicates its movement relative to the solvent front in a given chromatographic system. 4
Rf= Distance traveled by compound/Distance traveled by solvent Rf value depends on number of variables such as the particle size of different batches of adsorbent. the solvent composition and the degree of saturation of the tank atmospheres with solvent vapors. prior activation and storage condition of the plates. the thickness of adsorbent layer. 4.3.8 Column chromatographic studies The stationary phase is in form of a packed column through which a mobile phase is allowed to flow under gravity, or under pressure applied to the top of the solvent reservoir. The choice solvent for transferring the mixture to be chromatographed to the column depends upon School of Pharmaceutical Sciences, Shobhit University, Meerut
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the solubility characteristics of the mixture. Frequently the most non-polar solvent for introducing the mixture on to the column and the initial solvent for chromatogram development. 4 Eluting solvent with increasing the polarity applied for solvent selection, all these solvents have sufficient low boiling points to permit ready recovery of eluted material. The solvents include hexane, cyclohexane, carbon tetrachloride, trichloroethylene, toluene, dichloromethane,
chloroform, diethylether, ethylacetate, acetone, propanol, ethanol, and
methanol.
4.3.9 Methodology The column assembly was set for the separation of compounds from methanolic extract. The silica gel (100-200 mesh size) was used for column packing. The slurry method was adopted for filling of the column. Slurry was made with chloroform. The adsorbent was allowed to settle evenly and free of air bubbles, by with gentle tapping of the column with a wooden rod. The top of the column was frequently covered with a circle of filter paper or a layer of clean sand to prevent disturbances of the surface during subsequent loading. Semisolid methanolic extract was poured through a funnel into the packed column. The solvent level was maintained 2.5 cm above the extract. For continuous supply of solvent at the constant rate the reservoir was set and filled with the solvent. The elutes were collected drop by drop in 10 ml volumetric flasks at the bottom of column. TLC was carried out for the identification of constituents which were eluted by different eluent solvent systems. Eluted fractions which have same Rf on the TLC were pooled. Afterwards other fractions were collected and subjected to thin layer chromatographic studies. At last the column was eluted with methanol. 4
Column chromatography of methanolic extracts of Butea frondosa leaves are shown in table 4.17
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Table 4.17 Column chromatography of methanolic extracts of Butea frodosa leaves
S.No.
Eluent solvent (200ml )
Fractions
TLC Solvent System (chloroform: methanol) : Rf 80:20 90:10
1.
Chloroform
1-11
-
-
2.
Chloroform
12-18
0.26, 0.24
0.27, 0.27
Chloroform: Acetic acid
19-25
0.30,0.32,0.35 ,0.36, 0.37
0.29
Chloroform:Methanol (95:05)
26-32
0.42, 0.43, 0.44, 0.45, 0.44
0.41
4.
Chloroform:Methanol (90:10)
0.54,0.53
0.54,0.55,0.56
5.
24-26
Chloroform:Methanol (80:20)
0.58,0.60
0.61
6.
27-34
Chloroform:Methanol (70:30)
0.66,0.62
0.69, 0.70
7.
35-41
3.
4.3.10 Isolation of compound from Butea frondosa leaves The brown residue obtained from methanolic extract was dissolved in minimum amount of chloroform in a round bottom flask and absorbed over silica gel mesh size 60-120 to make slurry. The slurry was dried under reduced pressure on rotatory evaporator (Perfit India). The completely dried silica gel slurry containing the methanolic extract was poured in the column (30×2 cm) packed earlier with slurry of silica gel (60-12- mesh size) in chloroform. Successively, elution was carried out with different solvents in increasing order of polarity. The fractions collected in conical flask were marked. The fractions were subjected to thin layer chromatography in order to check the homogeneity of various fractions. The fractions having same Rf value were pooled and concentrated. Finally the obtained fractions from 12 to 18 having same Rf (0.27) were isolated to give a single compound (BF-1).5
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4.3.11 Characterization of the isolated compound (BF-1) by spectral analysis Isolated compound (BF 1) was subjected to FTIR, 1H NMR and Mass spectroscopy. FTIR spectra of the isolated compound (BF 1) of Butea frondosa (leaves) presented in figure 4.11.
Fig. 4.11 FTIR spectra of isolated compound of Butea frondosa The FTIR (cm-1) spectrum displayed the absorption band at 2925 and 2856 cm-1, for C-H stretching of methylene group, C=O stretching vibration of aliphatic acid absorb near the 1721 cm-1. Two band arising from C=O stretching vibration and OH bending of COOH at about 1218 cm-1, and at 1375 cm-1.6,7 5 Interpretation of FTIR spectra of the isolated compound of Butea frondosa is presented in table 4.18.
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Table 4.18 FTIR spectra of the isolated compound of Butea frondosa Reported Peaks (cm-1) 2925 2850.56
Observed Peaks 2925 2856
Inference
C=H (s) Alkenes C-H (s) Aldehyde
1
1719.01
1721
C=O (s) Carbonyl group
1461
1218
O-H (b)
1370
1375
COOH
H NMR CDCl3 at 300 MHz, One broad singlet at δ 1.25 ppm was assigned to 18
protons of methylene group. 1
H NMR displayed 3 protons of terminal methyl at δ 0.88 and 0.85 ppm. Chemical
shift of the 2 proton on the methylene carbon adjacent to COOH in aliphatic compound appeared at δ 2.34 and at δ 2.32 ppm. Chemical shift of the 3 protons adjacent methyl group appeared at δ 1.46 and at δ 1.42.8, 9 NMR spectra of the isolated compound (BF1) of Butea frondosa (leaves) presented in figure 4.12, 4.13.
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Fig. 4.12 NMR spectra of the isolated compound
Figure 4.13 NMR spectra of the isolated compound School of Pharmaceutical Sciences, Shobhit University, Meerut
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Interpretation of NMR spectra of the isolated compound of Butea frondosa is presented in table 4.19.
Table 4.19. Interpretation of NMR spectra of the isolated compound of Butea frondosa
Reported Peak
Observed Peak
1.22-1.30
1.25
1.40-1.50
1.46, 1.42
Inference 18 protons of methylene group. 3 protons adjacent methyl group appeared 3
0.83-0.92
0.88, 0.85
methyl
proton
proton
on
methylene 2 Carbon adjacent to COOH (or) at
2.28-2.40
2.32, 2.30
α position to COOH
FAB Mass spectroscopy showed the molecular ion at m/z 215(M+) corresponding to molecular formula (C13H26O2) 214. Mass spectra of isolated compound is shown in figure 4.14.
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Figure 4.14 Mass spectra of the isolated compound
Interpretation of Mass spectra of the isolated compound of Butea frondosa is presented in table 4.20.
Table 4.20.Interpretation of Mass spectra of the isolated compound of Butea frondosa
Reported peak 214
Observed Peak 215
Inference M+
From the spectral data the structure of the compound BF 1 was characterized as tridecanoic acid with molecular formula C13H26O2. Chemical structure of tridecanoic acid is presented in figure 4.15.
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OH O Tridecanoic acid
Fig. 4.15 Chemical structure of tridecanoic acid
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References 1.
Evans, W.C.; Trease, G.E. A text book of Pharmacognosy. Bailliare Tindall and Cassel. 14th ed.; 2008,13.
2.
Mojab. F.; Kamalinejad. M.; Ghaderi. N., Vahidipour, H. R. Phytochemical Screening of Some Species of Iranian Plants, Iranian Journal of Pharmaceutical Research, 2003, 2, 77-82.
3.
Kapoor, L. D.; Singh, A.; Kapoor, S L.; and Srivastava, S N. Survey of Indian plants for alkaloids and flavonoids. I. Lloydia, 1969, 32, 297-304.
4.
Vogel’s “Textbook of Practical Organic Chemistry” Furniss, B.S, Hamaford, A.J, Smiths, P.G, Tatchell.,A.R. 1at., Dorling Kindersley Pvt Ltd., 2007,197.
5.
Kemp, W. Organic spectroscopy, Palgrave publishers Ltd, New York, 2007, 3, 19-56.
6.
Pavia, D. L.; Lampman, G. M..; Kriz, G. S. Introduction to Organic Laboratory Techniques, Harcourt Brace college publishers, 2008, 3, 593-629.
7.
Silverstein, R. M..; Webster, F. X. Spectrometric Identification of Organic Compound, John Wiley and Sons publishers, 2004, 6, 168-189.
8.
Silverstein, R. M.; Webster, F. X.. Spectrometric Identification of Organic Compound, John Wiley and Sons publishers, 2004, 6, 81-109.
9.
Silverstein, R. M..; Webster, F. X. Spectrometric Identification of Organic Compound, John Wiley and Sons publishers, 2004, 6, 12-23.
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5. Ocular activity 5.1. Anticataract activity Acorus calamus, Vitex negundo and Butea frondosa, have been used traditionally as a topical formulation in the treatment of eye diseases and disorders.1 Cataract has been the major eye disorder afflicting mankind since ancient times. Oxidative stress is one of the factor implicated in pathogenesis of cataract.2 Acorus calamus, Vitex negundo and Butea frondosa is mentioned in Ayurveda to be beneficial in treatment of eye diseases. The present study was done to screen Acorus calamus, Vitex negundo and Butea frondosa for its anticataract potential.
5.2. Experimental design Goat lenses were obtained from the freshly frozen eyeballs transported immediately from the slaughter house. They were dissected out carefully and placed in a sterile tissue culture dish having the Dulbecco's modified eagle's medium (DMEM).
5.3. Dulbecco's modified eagle's medium (DMEM). Liquid, with 1000 mg/L glucose and sodium bicarbonate, L-glutamine, pyridoxine (substitutes pyridoxine HCl for pyridoxal HCl), Cell culture tested Endotoxin tested, Sterilefiltered, Sodium Chloride (NaCl = 8.0 g), Potassium Chloride (KCl= 0.2 g), Calcium Chloride (CaCl2= 1.8 g), Magnesium Chloride (MgCl2= 0.1-1.0 g), Sodium bicarbonate (NaHCO3= 1.0 g), Sodium dihydrogenphosphate (NaH2PO4= 0.05 g), Glucose (C6H12O6= 2.0 g). 3 5.4. Standard drugs Catlon eye drop, manufactured by Ophtho remedies pvt. Limited, contains Potassium Iodide IP 3.3% w/v, Sodium Chloride IP 0.83% w/v, Calcium Chloride IP 1% w/v, Chlorbutol IP 0.5% w/v) and Tobramycin Ophthalmic Solution USP. 5.5. Effect of extract on Hydrogen peroxide (oxidative stress) 0.5 mM induced cataract in goat lens. The in vitro protective effects of Acorus calamus, Vitex negundo and Butea frondosa, against hydrogen peroxide induced damage was assessed by culturing freshly excised goat lenses
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in DMEM containing 0.5 mM hydrogen peroxide with or without Acorus calamus and by measuring the time taken by lenses to become opaque. 5.6. Biochemical studies 5.6.1. Reduced glutathione (GSH) The GSH content was estimated by the method of Moron et al. Half of the lenses from each group were weighed and homogenized in 1 ml of 5% trichloroacetic acid (TCA), and a clear supernatant was obtained by centrifugation at 5000 rpm for 15 min. To 0.5 ml of this supernatant, 4.0 ml of 0.3 M disodium phosphate and 0.5 ml of 0.6 mM 5, 5’- dithiobis-2nitrobenzoic acid in 1% tri sodium citrate were added in succession. The intensity of the resulting yellow color was read spectrophotometrically at 410 nm. Reduced GSH was used as a standard. 5.6.2. Estimation of malondialdehyde (MDA) The extent of lipid peroxidation was determined by the method of Ohkawa et al. Briefly, 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.81% thiobarbituric acid aqueous solution were mixed. To this reaction mixture, 0.2 ml of the tissue sample (lens homogenate prepared in 0.15 M Potassium chloride) was added. The mixture was then heated in boiling water for 60 min. After cooling to room temperature, 5 ml of butanol: pyridine (15:1 v/v) solution was added. The mixture was then centrifuged at 5000 rpm for 15 min. The upper organic layer was separated, and the intensity of the resulting pink colour was read at 532 nm. Tetramethoxypropane was used as an external standard. The level of lipid peroxide was expressed as nmoles of MDA formed in µmol/g wet weight for lenses. 5.6.3. Estimation of total protein For total protein estimation the lens homogenate was prepared in 5% trichloroacetic acid. The precipitated protein was dissolved in sodium hydroxide and used as aliquots for the estimation of total proteins. Soluble and insoluble fractions of the protein were estimated by preparing homogenate in double distilled water. The water soluble supernatant was used for estimation of soluble protein while the residue dissolved in sodium hydroxide was used for the estimation of insoluble protein. The protein content of the samples was determined by the method of Lowry et al using bovine serum albumin as the standard. School of Pharmaceutical Sciences, Shobhit University, Meerut
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5.7. Result of Acorus calamus (roots) All the lenses in DMEM alone were transparent. However, lenses after 52 h of incubation in the presence of hydrogen peroxide developed dense opacity. Acorus calamus (methanolic extract) was found to afford significant, concentration dependent protection against hydrogen peroxide (H2O2) damage to goat lenses. Acorus calamus extract at a dose of 0.25 mg/ml prevented opacity up to 21 to 44 hrs as compared to the control, which became opaque in 17 hrs, where as β asarone prevented opacity for 42 hrs and standard drug (Catlon + Tobramycin) prevented opacity for 46 hrs. Anticataract activity of the extract of Acorus calamus and isolated compound against hydrogen peroxide induced cataract shown in table 5.1. Table 5.1 Anticataract activity of the extract of Acorus calamus and isolated compound against hydrogen peroxide induced cataract. S.No.
Treatment
Treatment H2O2 (0.5mM)+ DMEM Time of Opacity (Top) in Hrs.
Time to Opacity (Top) in Hrs.
1
Control
----
17±2
2
Methanolic extract (0.25mg/ml)
----
45±3**
3
Isolated Compound(0.25mg/ml)(AC1)
----
42±2
4
Standard drug (Catlon+ Tobramycin)
----
46±4**
Each values represents mean ± S.E. n=6; **Represents statistical significance vs. control (p < 0.01) 5.7.1. Effect on GSH, MDA and Protein contents in hydrogen peroxide induced oxidative stress GSH level of 0.12±0.05µmol/g of lenses was observed in control group and the Acorus calamus extract and β asarone at the concentration of 0.25 mg/ml significantly restored the GSH level at 0.49±0.01 and 0.32±0.01 of the lens, as also the standard drug could restore the GSH level at 0.55±0.002. School of Pharmaceutical Sciences, Shobhit University, Meerut
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Administration of the Acorus calamus extract and β asarone at the concentration of 0.25 mg/ml significantly prevented the rise in MDA level i.e 1.69±0.01 and 1.50±0.02nmol/mg. Hydrogen peroxide 0.5mM treated lenses also showed significantly low concentrations of protein in the lens homogenate as compared to control group. Acorus calamus, β asarone and standard drug treatment exhibited significantly higher concentrations of total lens protein as compared to hydrogen peroxide group. Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induces cataract shown in table 5.2.
Table 5. 2 Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induces cataract.
Control (Group I)
MEAC (Group II)
MDA (nmol/mg)
4.75±2.15
1.69±0.01*
Isolated compound (AC1) (Group III) 1.50±0.02
Standard (Group IV)
GSH (µmol/g) Protein
0.12±0.05
0.49±0.01*
0.32±0.01
0.55±0.002*
0.59±0.005
2.159±0.001*
1.571±0.004
2.298±0.001*
2.89±0.01*
All values are expressed as mean ±SD, Group I: Control, lenses exposed to H2O2 only. Group II: lenses exposed to H2O2 and MEAC, Group III: lenses exposed to H2O2 and β asarone and Group IV: lenses exposed to H2O2 and standard drug. Values are mean ± SD, n=6 statistically significant difference (*P < 0.01) when compared with group II values. GSH: glutathione; MDA: malondialdehyde.
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5.8. Result of Vitex negundo (leaves) All the lenses in DMEM alone were transparent. However, lenses after 52 h of incubation in the presence of hydrogen peroxide developed dense opacity. Vitex negundo (methanolic extract) was found to afford significant, concentration dependent protection against hydrogen peroxide (H2O2) damage to goat lenses. Vitex negundo extract at a dose of 0.25 mg/ml prevented opacity up to 21 to 42 hrs as compared to the control, which became opaque in 17 hrs, where as negundin A prevented opacity for 40 hrs and standard drug (Catlon + Tobramycin) prevented opacity for 46 hrs. Anticataract activity of the extract of Vitex negundo and isolated compound against hydrogen peroxide induced cataract shown in table 5.3.
Table 5.3 Anticataract activity of the extract of Vitex negundo and isolated compound against hydrogen peroxide induced cataract. S.No.
1
Control
------
Time to Opacity (Top) in Hrs. 17±2
2
Methanolic extract (0.25 mg/ml) (VN 1)
------
42±3**
3
Isolated Compound(0.25 mg/ml) (VN 2)
------
40±2
Standard drug
-------
46±4**
4
Treatment
Treatment H2O2 (0.5mM)+ DMEM Time of Opacity (Top) in Hrs.
Each values represents mean ± S.E. n=6; **Represents statistical significance vs. control (p < 0.01)
5.8.1. Effect on GSH, MDA and Protein estimation in hydrogen peroxide induced oxidative stress GSH level of 0.12±0.05µmol/g of lenses was observed in control group, and the Vitex negundo extract and negundin A at the concentration of 0.25 mg/ml significantly restored the School of Pharmaceutical Sciences, Shobhit University, Meerut
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GSH level at 0.40±0.01 and 0.30±0.01 of the lens as also the standard drug could restore the GSH level at 0.55±0.002. MDA level in control group was found to be 4.75±2.15 nmol/mg of lens weight. Administration of the Vitex negundo extract and negundin A at the concentration of 0.25 mg/ml significantly prevented the rise in MDA level i.e. 1.51±0.01 and 1.42±0.02 nmol/mg. Hydrogen peroxide 0.5mM treated lenses also showed significantly low concentrations of protein in the lens homogenate as compared to control group. Vitex negundo, negundin A and standard drug treatment exhibited significantly higher concentrations of total lens protein as compared to hydrogen peroxide group. Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induced cataract shown in table 5.4. Table 5.4 Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induced cataract.
Control (Group I)
MEVN (Group II)
VN1 (Group III)
Standard (Group IV)
MDA (nmol/mg)
4.75±2.15
1.51±0.01*
1.42±0.02
2.89±0.01*
GSH (µmol/g)
0.12±0.05
0.40±0.01*
0.30±0.01
0.55±0.002*
Protein
0.59±0.005
2.100±0.001*
1.451±0.004
2.298±0.001*
All values are expressed as mean ±SD, Group I: Control, lenses exposed to H2O2 only. Group II: lenses exposed to H2O2 and MEVN, Group III: lenses exposed to H2O2 and Negundin A and Group IV: lenses exposed to H2O2 and standard drug. Values are mean ± SD, n=6 statistically significant difference (*P < 0.01) when compared with group II values. GSH: glutathione; MDA: malondialdehyde.
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5.9. Result of Butea frondosa (leaves) All the lenses in DMEM alone were transparent. However, lenses after 52 h of incubation in the presence of hydrogen peroxide developed dense opacity. Butea frondosa (methanolic extract) was found to afford significant, concentration dependent protection against hydrogen peroxide (H2O2) damage to goat lenses. Butea frondosa extract at a dose of 0.25 mg/ml prevented opacity up to 21 to 42 hrs as compared to the control, which became opaque in 17 hrs, where as Tridecanoic acid prevented opacity for 41 hrs and standard drug (Catlon + Tobramycin) prevented opacity for 46 hrs. Anticataract activity of the extract of Butea frondosa and isolated compound against hydrogen peroxide induced cataract presented in table 5.5. Table 5.5 Anticataract activity of the extract of Butea frondosa and isolated compound against hydrogen peroxide induced cataract. S.No.
Treatment
Treatment H2O2 (0.5mM)+ DMEM Time of Opacity (Top) in Hrs.
1
Control
-----
Time of Opacity (Top) in Hrs. 17±2
2
Methanolic extract (0.25 mg/ml) Isolated Compound(0.2 5 mg/ml) (BF 1) Standard drug
-----
44±3**
-----
41±2
-----
46±4**
3
4
Each values represents mean ± S.E. n=6; **Represents statistical significance vs. control (p < 0.01) 5.9.1. Effect on GSH, MDA and Protein estimation in hydrogen peroxide induced oxidative stress
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Butea frondosa extract and Tridecanoic acid at the concentration of 0.25 mg/ml significantly restored the GSH level at 0.45 ±0.01 and 0.35 ±0.01 of the lens as also the standard drug could restore the GSH level at 0.55±0.002. MDA level in control group was found to be 4.75±2.15 nmol/mg of lens weight. Administration of the Butea frondosa extract and Tridecanoic acid at the concentration of 0.25 mg/ml significantly prevented the rise in MDA level i.e. 1.59±0.01 and 1.45±0.02nmol/mg. Hydrogen peroxide 0.5mM treated lenses also showed significantly low concentrations of protein in the lens homogenate as compared to control group. Butea frondosa, Tridecanoic acid and standard drug treatment exhibited significantly higher concentrations of total lens protein as compared to hydrogen peroxide group. Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induced cataract shown in table 5.6. Table 5.6 Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induced cataract.
MDA (nmol/mg) GSH (µmol/g) Protein
Control (Group I)
MEBF (Group II)
BF1 (Group III)
Standard (Group IV)
4.75±2.15
1.59±0.01*
1.45±0.02
2.89±0.01*
0.12±0.05
0.45±0.01*
0.35±0.01
0.55±0.002*
0.59±0.005
2.132±0.001*
1.475±0.004
2.298±0.001*
All values are expressed as mean ±SD, Group I: Control, lenses exposed to H2O2 only. Group II: lenses exposed to H2O2 and MEBF, Group III: lenses exposed to H2O2 and Tridecanoic acid and Group IV: lenses exposed to H2O2 and standard drug. Values are mean ± SD, n=6 statistically significant difference (*P < 0.01) when compared with group II values. GSH: glutathione; MDA: malondialdehyde. School of Pharmaceutical Sciences, Shobhit University, Meerut
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Anticataract activity
Fig. 5.1 Goat eyes
Fig. 5.2 Goat lens being withdrawn from goat eye School of Pharmaceutical Sciences, Shobhit University, Meerut
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Control
Standard Drug
Fig. 5.3 Goat lenses (control and standard group) before treatment
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Isolated Compound (Acorus calamus) (roots)
Methanolic Extract (Acorus calamus) (roots)
Fig. 5.4 Goat lenses (Isolated compound and methanolic extract group of Acorus calamus) before treatment
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Isolated Compound (Vitex negundo) (leaf)
Methanolic Extract (Vitex negundo) (leaf)
Fig. 5.5 Goat lenses (isolated compound and mehanolic extract of Vitex negundo) before treatment
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Isolated Compound (Butea frondosa) (leaf)
Methanolic extract (Butea frondosa) (leaf)
Fig. 5.6 Goat lenses (Isolated compound and methanolic extract of Butea frondosa) before treatment
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Control
Time to Opacity = 17 hrs.
Standard Drug
Time to Opacity = 46 hrs. Fig. 5.7 Goat lenses after incubation in control group complete cataractogenesis after 17 hrs. and standard group after 46 hrs. appears slightly hazy.
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Isolated Compound (Acorus calamus) (roots)
Time to Opacity = 42 hrs.
Methanolic Extract (Acorus calamus) (roots)
Time to Opacity = 45 hrs. Fig. 5.8 Goat lenses after incubation in isolated group after 42 hrs. and methanolic extract group 45 hrs. appears slightly hazy.
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Isolated Compound (Vitex negundo) (leaf)
Time to Opacity = 40 hrs.
Methanolic Extract (Vitex negundo) (leaf)
Time to Opacity = 42 hrs. Fig. 5.9 Goat lenses after incubation in isolated group after 40 hrs. and methanolic extract group 42 hrs. appears slightly hazy. School of Pharmaceutical Sciences, Shobhit University, Meerut
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Isolated Compound (Butea frondosa) (leaf)
Time to Opacity = 41 hrs.
Methanolic extract (Butea frondosa) (leaf)
Time to Opacity = 44 hrs.
Fig. 5.10 Goat lenses after incubation in isolated group after 41 hrs. and methanolic extract group 44 hrs. appears slightly hazy School of Pharmaceutical Sciences, Shobhit University, Meerut
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References 1. Mengi, S. A.; Deshpande, S. G. Indian Journal of Pharmacology, 1995, 27, 116-119. 2. Nobuyuki, O.; Masakatsu, F.; Tetsuo, S.; Yasuo, T. British J. of Ophthalomol, 1999, 83, 1064-1068. 3. Kulkarni, S. K. Hand Book of Experimental Pharmacology, Vallabh Prakashan Publisher, 2003, 3, 11. 4. Zar, Jerrold H. Biostatistical Analysis, Pentice Hall Publishers, 1974, 205-208. 5. Vogel Gerhard, H. Drug Discovery and Evaluation, Library of Congress Publisher, 2002, 2, 751-762. 6. Kokate, C. K.; Purohit, A. P. Pharmacognosy, Nirali Prakashan Publisher, 1999, 12, 111. 7. Kulkarni, S. K. Hand Book of Experimental Pharmacology, Vallabh Prakashan Publisher, 2003, 3, 128-131. 8. Vogel, G. H. Drug Discovery and Evaluation, Library of Congress Publisher, 2002, 2, 751762.
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Summary and Conclusion
6.0 Summary and Conclusion The present study was undertaken with the objective to explore the anticataract potential of extract of Acorus calamus (roots), Vitex negundo (leaves) and Butea frondosa (leaves) against cataract developed by oxidative stress (Hydrogen peroxide) in goat eyes (lens). Acorus calamus is commonly known as Vach and the plants belong to family Araceae. Vitex negundo is commonly known as nirgundi and belongs to family Verbenaceae. Butea frondosa is commonly known as palash and the plant belongs to family Leguminosae. Exhaustive literature survey revealed that sufficient studies have been done on flowers and seeds of these plants. While quite a less attention have been paid towards leaves and roots. Therefore, the present work was undertaken to explore leaves and roots for their ocular activities as these are considered as rich source of phytoconstituents. The seeds, flowers, bark and gum of these plants have been used for inflammation, intestinal worm infection, urinary disorders, piles, natural colorants, persistent dysentery, backache, aphrodisiac, and skin diseases. In ayurveda, it is mentioned to possess ocular activity. As there is no scientific record of this activity, so, the present work was undertaken to evaluate anticataract potential and to support of the traditional claims. 6.1 Extraction of the roots of Acorus calamus Extraction of the roots of Acorus calamus was carried out in petroleum ether and methanol and aqueous. The yield of petroleum ether extracts was found to 6.58 % w/w. The extract was pale yellow semisolid. The yield of methanolic extracts was found to 25.58 % w/w in root. The extract was a deep red semisolid. The yield of aqueous extracts was found to 20.10 % w/w in root. The extract was a dark brownish semisolid. It was noted that all extracts were semisolid in nature in roots of Acorus calamus. 6.2 Phytochemical study Evaluation from phytochemical screening of three extracts (petroleum ether, methanol and aqueous) of roots confirmed the presence of various constituents such as alkaloid, flavonoids, tannins, carbohydrates, steroidal compounds, phenolic compounds, fats and fixed oil. However, flavonoids and phenolic compounds were found in almost all the extracts. School of Pharmaceutical Sciences, Shobhit University, Meerut
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6.3 Structure elucidation of isolated compound (AC-1) by spectral analysis The FTIR spectra showed sharp peaks at 2929.7.2 cm-1indicating C-H stretching of alkanes. Medium and weak peaks were observed at 1645.4 cm-1cm and 1510.5 cm-1cm indicated C=C stretching of aromatic ring. Strong peak of ether(C-O stretching) was observed at 1053.5 cm-1. Sharp peak of tetra substituted benzene ring was observed at 802 cm-1. 1
H NMR spectra confirmed two singlet aromatic protons (δ 6.8 and7.4), two olefinic
protons (δ1.4and1.6), three methoxyl protons (δ 3.71,3.82,3.90), and methyl protons (δ 0.4) indicating sixteen protons signals. Mass spectra confirmed a molecular peak at m/z 209 (M+). Fragment peaks were also observed at 60.4 M+ -C6H5+CO From the spectral data the structure of the compound AC-1 was elucidated as β asarone with molecular formula C12H16O3.
H3CO
OCH3
H3CO
asarone
β asarone 6.4 Pharmacological activity All the lenses in DMEM alone were transparent. However, lenses after 52 h of incubation in the presence of hydrogen peroxide developed dense opacity. Acorus calamus (methanolic extract) was found to afford significant, concentration dependent protection against hydrogen peroxide (H2O2) damage to goat lenses. Acorus calamus extract at a dose of 0.25 mg/ml prevented opacity up to 21 to 44 hrs as compared to the control, which became opaque in 17 hrs, where as β asarone prevented opacity for 42 hrs and standard drug (Catlon + Tobramycin) prevented opacity for 46 hrs. Anticataract activity of the extract of Acorus calamus and isolated compound against hydrogen peroxide induced cataract shown in table 5.1. School of Pharmaceutical Sciences, Shobhit University, Meerut
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6.5 Effect on GSH, MDA and Protein estimation in hydrogen peroxide induced oxidative stress GSH level of 0.12±0.05µmol/g of lenses was observed in control group and the Acorus calamus extract and β asarone at the concentration of 0.25 mg/ml significantly restored the GSH level at 0.49±0.01 and 0.32±0.01 of the lens, as also the standard drug could restore the GSH level at 0.55±0.002. MDA level in control group was found to be 4.75±2.15 nmol/mg of lens weight. Administration of the Acorus calamus extract and β asarone at the concentration of 0.25 mg/ml significantly prevented the rise in MDA level i.e 1.69±0.01 and 1.50±0.02nmol/mg. Hydrogen peroxide 0.5mM treated lenses also showed significantly low concentrations of protein in the lens homogenate as compared to control group. Acorus calamus, β asarone and standard drug treatment exhibited significantly higher concentrations of total lens protein as compared to hydrogen peroxide group. Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induces cataract shown in table 5.2.
6.6 Extraction of the leaves of Vitex negundo Extraction of the leaves of Vitex negundo was carried out in petroleum ether and methanol and aqueous. The yield of petroleum ether extracts was found to 3.07 % w/w. The extract was Light brownish semisolid. The yield of methanolic extracts was found to 12.90 % w/w in leaves. The extract was a dark greenish semisolid. The yield of aqueous extracts was found to 13.51 % w/w in leaves. The extract was a dark brownish semisolid. It was noted that all extracts were semisolid in nature in leaves of Vitex negundo. 6.7 Phytochemical study Phytochemical screening of three extracts (petroleum ether, methanol and aqueous) of leaves confirmed the presence of various constituents such as alkaloid, flavonoids, tannins, carbohydrates, steroidal compounds, phenolic compounds,
fats and fixed oil. However,
flavonoids and phenolic compounds were found in almost all the extracts.
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6.8 Structural elucidation of isolated compound (VN -1) by spectral analysis FTIR, NMR and Mass spectroscopy were carried out for the detection of isolated compound. The isolated compound was also confirmed through FTIR, NMR and Mass spectra. IR (cm-1) spectrum displayed 3435.77 O-H (s), 2921.26 C=H (s), 2850 C-H (s), 1719.01 C=O (s), 1458.67 CH (b), 1267.13 C=O (s). 1
H NMR CDCl3 at 400 MHZ, triplet at δ 6.20 ppm was assigned to 8 protons of fused
aromatic compound. NMR displayed 6 protons of terminal methyl at δ 3.66 ppm. Chemical shift of the 2 protons adjacent phenol group appeared at δ 5.17 ppm. Mass spectroscopy showed the molecular ion at m/z 353 (M+1) corresponding to molecular formula (C20H16O6) 352. The isolated compound was Negundin A. H3CO
O O
HO
OCH3 OH
Negundin A
6.9 Pharmacological activity All the lenses in DMEM alone were transparent. However, lenses after 52 h of incubation in the presence of hydrogen peroxide developed dense opacity. Vitex negundo (methanolic extract) was found to afford significant, concentration dependent protection against hydrogen peroxide (H2O2) damage to goat lenses. Vitex negundo extract at a dose of 0.25 mg/ml prevented opacity up to 21 to 42 hrs as compared to the control, which became opaque in 17 hrs, where as negundin A prevented opacity for 40 hrs and standard drug (Catlon + Tobramycin) prevented opacity for 46 hrs. Anticataract activity of the extract of Vitex negundo and isolated compound against hydrogen peroxide induced cataract shown in table 5.3.
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6.10 Effect on GSH, MDA and Protein estimation in hydrogen peroxide induced oxidative stress GSH level of 0.12±0.05µmol/g of lenses was observed in control group, and the Vitex negundo extract and negundin A at the concentration of 0.25 mg/ml significantly restored the GSH level at 0.40±0.01 and 0.30±0.01 of the lens as also the standard drug could restore the GSH level at 0.55±0.002. MDA level in control group was found to be 4.75±2.15 nmol/mg of lens weight. Administration of the Vitex negundo extract and negundin A at the concentration of 0.25 mg/ml significantly prevented the rise in MDA level i.e. 1.51±0.01 and 1.42±0.02 nmol/mg. Hydrogen peroxide 0.5mM treated lenses also showed significantly low concentrations of protein in the lens homogenate as compared to control group. Vitex negundo, negundin A and standard drug treatment exhibited significantly higher concentrations of total lens protein as compared to hydrogen peroxide group. Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induced cataract shown in table 5.4. 6.11 Extraction of the leaves of Butea frondosa Extraction of the leaves of Butea frondosa was carried out in petroleum ether and methanol and aqueous. The yield of petroleum ether extracts was found to 5.18 % w/w. The extract was Light brownish semisolid. The yield of methanolic extracts was found to 9.00 % w/w in leaves. The extract was a dark greenish semisolid. The yield of aqueous extracts was found to 8.10 % w/w in leaves. The extract was a dark brownish semisolid. It was noted that all extracts were semisolid in nature in leaves of Butea frondosa. 6.12 Phytochemical study Phytochemical screening of three extracts (petroleum ether, methanol and aqueous) of roots confirmed the presence of various constituents such as alkaloid, flavonoids, tannins, carbohydrates, steroidal compounds, phenolic compounds,
fats and fixed oil. However,
flavonoids and phenolic compounds were found in almost all the extracts. School of Pharmaceutical Sciences, Shobhit University, Meerut
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6.13 Structural elucidation of isolated compound (BF -1) by spectral analysis After isolation of desired compound, it was subjected to characterization. For characterization studies; Melting point/ Range and spectroscopic technique (IR, NMR, and Mass) were employed. TLC of isolated compound was performed using solvent system of chloroform and methanol (9:1), detected under short wave UV spectrophotometer. The Rf value of isolated compound was found to be 0.26. IR spectrum of isolated compound gave characteristic signals of groups present in the isolated compound like, IR (cm-1) spectrum displayed the absorption band at 2925 and 2856 cm-1, for C-H stretching of methylene group, C=O stretching vibration of aliphatic acid absorb near the 1721 cm-1. Two band arising from C=O stretching vibration and OH bending of COOH at about 1218 cm-1, and at 1375 cm-1. Mass spectroscopy of isolated compound was performed showing the molecular ion peak at M+1 i.e. at 215, while calculated molecular weight of Tridecanoic acid (molecular formula: C13H26O2) is 214. Proton NMR spectroscopy was used for the confirmation of structure of isolated compound. NMR gave characteristic signals of protons present in the isolated compound, according to which: 1H NMR CDCl3 at 300 MHz, One broad singlet at δ 1.25 ppm was assigned to 18 protons of methylene group. 1 H NMR displayed 3 protons of terminal methyl at δ 0.88 and 0.85 ppm. Chemical shift of the 2 proton on the methylene carbon adjacent to COOH in aliphatic compound appeared at δ 2.34 and at δ 2.32 ppm. Chemical shift of the 3 protons adjacent methyl group appeared at δ 1.46 and at δ 1.42. On the basis of characterization studies of isolated compound, it was found to be Tridecanoic acid.
OH O Tridecanoic acid School of Pharmaceutical Sciences, Shobhit University, Meerut
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6.14 Pharmacological activity All the lenses in DMEM alone were transparent. However, lenses after 52 h of incubation in the presence of hydrogen peroxide developed dense opacity. Butea frondosa (methanolic extract) was found to afford significant, concentration dependent protection against hydrogen peroxide (H2O2) damage to goat lenses. Butea frondosa extract at a dose of 0.25 mg/ml prevented opacity up to 21 to 42 hrs as compared to the control, which became opaque in 17 hrs, where as Tridecanoic acid prevented opacity for 41 hrs and standard drug (Catlon + Tobramycin) prevented opacity for 46 hrs. Anticataract activity of the extract of Butea frondosa and isolated compound against hydrogen peroxide induced cataract presented in table 5.5. 6.15 Effect on GSH, MDA and Protein estimation in hydrogen peroxide induced oxidative stress GSH level of 0.12±0.05µmol/g of lenses was observed in control group, and the Butea frondosa extract and Tridecanoic acid at the concentration of 0.25 mg/ml significantly restored the GSH level at 0.45 ±0.01 and 0.35 ±0.01 of the lens as also the standard drug could restore the GSH level at 0.55±0.002. MDA level in control group was found to be 4.75±2.15 nmol/mg of lens weight. Administration of the Butea frondosa extract and Tridecanoic acid at the concentration of 0.25 mg/ml significantly prevented the rise in MDA level i.e. 1.59±0.01 and 1.45±0.02nmol/mg. Hydrogen peroxide 0.5mM treated lenses also showed significantly low concentrations of protein in the lens homogenate as compared to control group. Butea frondosa, Tridecanoic acid and standard drug treatment exhibited significantly higher concentrations of total lens protein as compared to hydrogen peroxide group. Effect of various treatments on GSH, MDA and protein levels against hydrogen peroxide induced cataract shown in table 5.6.
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6.16 Conclusions The conclusion derived from the results obtained reveal a defined role of methanolic extract of Acorus clamus (roots), Vitex negundo (leaves) and Butea frondosa (leaves) in delaying the onset of cataract. It is also clear from the study that the opacity of lens in methanolic extract took 45, 42 and 44 hours, while for standard drug it was 46 hours. The plants Acorus clamus (roots), Vitex negundo (leaves) and Butea frondosa (leaves) thus seems to be a promising candidate with respect to its anticataract activity and may be used as adjuvant to dietary therapy and drug treatment for controlling cataract. Active constituents, i.e. β asarone, negundin A and tridecanoic acid, has been isolated from Acorus clamus (roots), Vitex negundo (leaves) and Butea frondosa (leaves). β asarone, negundin A and tridecanoic acid is used as antioxidant; further studies may establish the exact mechanism of its protective action. Cataract is multifactorial diseases associated with several risk factors and it is responsible for 50% of blindness worldwide. In the present study, Acorus calamus extract, β asarone, Vitex negundo extract, negundin, Butea frondosa extract and tridecanoic acid, delayed the progression of cataract in goat eyes and also extract prevented the cataract development in goat lenses. Although, multiple mechanisms may contribute to these effects, the antioxidant effect of Acorus calamus extract, β asarone, Vitex negundo extract, negundin, Butea frondosa extract and tridecanoic acid appears to be the predominant mechanism of action. In conclusion, Acorus calamus and β asarone, negundo extract, negundin, Butea frondosa extract and tridecanoic acid showed anticataract activity against hydrogen peroxide induced cataract in goat eyes. This effect is attributed to the protection of antioxidant defense system.
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Volume 11, Issue 2, November – December 2011; Article-022
ISSN 0976 – 044X
Research Article ANTICATARACT ACTIVITY OF ACORUS CALAMUS LINN. AGAINST HYDROGEN PEROXIDE INDUCED CATARACTOGENESIS IN GOAT EYES Dharmendra Kumar*, Ranjit Singh School of Pharmaceutical Sciences, Shobhit University, NH-58, Modipuram, Meerut-250110, UP, India.
Accepted on: 30-08-2011; Finalized on: 30-11-2011. ABSTRACT The anticataract activity of Acorus calamus roots extract against induced hydrogen peroxide cataractogenesis was assessed in Goat lenses. The in vitro phase of study was performed on lenses from Goat eyes incubated for 52 h at 37ᵒC in Dulbecco Modified Eagle Medium (DMEM) containing H2O2 (0.5 mM) (Group I), in DMEM containing H2O2 (0.5 mM) and methanolic extract of Acorus calamus (MEAC), (Group II), in DMEM containing H2O2 (0.5 mM) and isolated compound (β asarone), (Group III), in DMEM containing H2O2 (0.5 mM) and standard drug (Catlon and Tobramycin) (Group IV). Gross morphological examination of these lenses revealed dense opacification (cataract formation) in Group I. At the end of the experiment, the biochemical study was performed for estimation of MDA, GSH and proteins. The data suggest that methanolic extract of Acorus calamus (MEAC), isolated compound (β asarone) and standard drug (Catlon and Tobramycin) are able to significantly retard experimental hydrogen peroxide induced cataractogenesis. Keywords: Cataractogenesis, Hydrogen peroxide, Methanolic extract Acorus calamus, MDA, GSH and protein estimation.
INTRODUCTION Cataract is the leading cause of blindness worldwide. Several risk factors have been identified for the development of human cataract which includes: aging, diabetes, diarrhea, malnutrition, poverty, sunlight, smoking, hypertension and renal failure.1 Although cataract is a multifactorial disease, oxidative stress has been identified as an initiating factor for the development of cataract. 2 The oxidizing agent, hydrogen peroxide (H2O2) is present in normal aqueous humor at concentrations of approximately 20-30 µM and is reported to be raised (up to 660 µM) in patient with cataract.3 In in vitro lens culture experiments, Hydrogen peroxide at these higher concentrations causes lens opacification and produces a pattern of oxidative damage similar to that found in human cataract.4 Evidence suggests that Hydrogen peroxide is likely to be a major oxidant involved in cataract formation in man. Physiological antioxidants such as pyruvate5 and nutritional antioxidants such as ascorbate, flavonoids, vitamin E and carotenoids are reported to delay the development of experimental cataract.6 Acorus calamus belonging to the family Araceae is a small genus of herbs of the monocotyledonous; the plant is found in the northern temperate and subtropical region of Asia, North America and Europe. Various therapeutic potentials have been attributed to Acorus calamus in the traditional system of medicine as well as in folk fore practices.7 The plant is known to be effective as stimulant, emetic, expectorant, aphrodisiac, anthelmintic, epilepsy, memory disorders, homeostatic7, antioxidants 8 and skin diseases9 In this study the effect of methanolic extract
and isolated compound of Acorus calamus was assessed against H2O2 (0.5 mM) induced opacity in goat lenses. MATERIALS AND METHODS Animals Goat lenses were obtained from the freshly frozen eye balls transported immediately from the slaughter house. They were dissected out carefully and placed in a sterile tissue culture dish having the DMEM. Collection and authentication of plant material The plant material was collected from Haridwar, Uttarakhand, India. The plant was identified and authenticated by Dr. K. Pradheep, Senior Scientist at National Bureau of Plant Genetic Resources (ICAR) New Delhi. A voucher specimen no. NHCP/NBPGR/2010-36/ is deposited in the herbarium. Extraction The powdered Acorus calamus roots material was extracted with petroleum ether and methanol by soxhlet extraction method. The filtrate was evaporated using rotary vacuum evaporator under reduced pressure
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