Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837

Research Article ISSN: 0974-6943

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Phytochemical and antimicrobial studies of a herbal Medicinal plant Aeschynomene aspera L. Leaf extracts Aruna Chittamuri*, Suvarnalatha Anchapakula, Alekhya Cheruku, Chaithra Dandu, Yasodamma Nimmanapalli. Meerasaheb Chittoor, Department of Botany, S.V.University, Tirupati-517502 Andhra Pradesh, India.

Received on:01-01-2012; Revised on: 17-02-2012; Accepted on:20-03-2012 ABSTRACT Aeschynomene aspera is administered against tuberculosis, skin infections, antidote to snake venom, menstrual disorders and small pox. Hence the phytochemical and antibacterial studies were carried out. Preliminary phytochemical studies revealed the presence of Alkaloids, Flavonoids, Phenols, Terpinoids, Anthocynadins, Indoles, Glycosides, Saponins and Tannins in Hot water, Cold Water, Alcohol and Methanol extracts. The qualitative analysis reveals the presence of 9 identified and 2 unidentified phenolic compounds along with 3 flavonoid constituents through paper chromatography. Antibacterial activity on four selected pathogens like Bacillus subtilis, Staphylococcus aureus (Gram +ve) Escherichia coli, Pseudomonas aeruginosa (Gram- ve) bacterial strains were observed with all extracts. Alcoholic extracts are more effective on all the bacteria at 10 mg concentration with an average inhibitory zone between 40 to 45 mm nearly double to that of the controls i.e., 20-25mm (Ampicillin-10 mg and Gentamycin-30mg).But Staphylococcus aureus is more resistant showing the equal inhibition to that of the controls with all the extracts. Minimum Inhibitory Concentration values in all the extracts on all the selected bacteria ranges between 0.42mg to 0.78 mg. Key words: Phytochemical screening, Antimicrobial activity, Qualitative analysis, Aeschynomene aspera L.

INTRODUCTION Aeschynomene aspera (Fabaceae) is a tall erect sub shrub in swampy areas, with stout glabrous nodular stems, full of white pith. It is commonly known as Niru-jilugu (Telugu) Sola pith plant (English) Sola (Hindi) Aatrunetti (Tamil). It is used as a substitute for cork material and as a sun hats fide [1] .Pith is used as helmets and floats [2]. Magico religious belief of the tribe Oraon root is used for jaundice [3]. This species in North Bihar region is used as ornamental purpose and used for making berths (fish boats) also for several handicraft items like garlands, maur (head crown), Jhamp and paag which were variously used in auspicious occasions. These people called it as pith plant. In Siddha it is called as Aatrunetti where the leaves are used to cure joint pains and swellings [4-5]. In Ayurveda this species is known as Pashenabhed recommended for painful micturition and for breaking uric acid calculi [6].It is a weed of rice paddies [7]. The roots are boiled in Plant of A.aspera(Terrestrial)

less quantity of water and made in to paste applied on mumps [8]. Aerial part juice is given to cure cold, cough, and fever. Dried young shoot powder with half tea spoon powdered candy is given to increase the consistency of semen; local herbalists used it for urinary troubles [9]. A.aspera is also recognized as leafy vegetable [10].

Plant of A.aspera(Sub-merged)

*Corresponding author. C.Aruna Dept .of Botany, S.V.University, Tirupati-517502, A.P,India.

Phenolic compounds like Protocatechuic acid prevents spore germination and growth of smudge fungi and other fungi. It acts as anti amoebic [11]; modulates the cellular enzymes, acts as anti- oxidant and anti mutagenic [12]. Chemo preventive against tumor production [13]. Polymers of protocatechuic acid acts against influenza virus [14]. Induces apoptosis in human leukemia cells and also malignant HSG cells of oral cavities [15]. Chlorogenic acid acts as anti-inflamatory and antifungal [16] anti bacterial [17]; acts as anticoagulants [18]. Chlorogenic acid, neo-chlorogenic acid, caffeic acid along with the flavonoids shows synergic action against bronchial diseases [19]. It also prevents cardio vascular diseases [20]; Chlorogenic acid has been known for antioxidant due to its scavenging activity of hydroxyl ions [21] and anti viral [22], Neo chlorogenic acid may act as anti oxidant and released in to the blood stream after a meal [23] Iso chlorogenic acid improves immunity [24], along with Caffeic acid it prevents type 2 diabetes mellites [25]. Caffeic acid and Ferrulic acid acts as anti-inflammatory and antifungal [26-27].Caffeic acid and its derivative CAPE acts as anti-inflammatory and suppresses the intestinal carcinogenesis’ and the estrogenic activity [28]; It also acts as anti

Journal of Pharmacy Research Vol.5 Issue 3.March 2012

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Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 immuno-modulatory, anti inflammatory, protects from U.V radiation effects on skin cells [29-30]. Ferrulic acid and Cinnamic acid of onion epidermal cells shows resistance in preventing fungal cell wall degradation [31]. Polyphenols are noticed important natural Anti-Oxidant sources from plants by adsorbing and neutralizing free radicals [32]. Trans-p- coumaric acid acts as an anti bacterial [33]. Hydroxy Cinnamic acid amide acts against influenza and Herpes virus [34]. Hydroxy benzoic acid, Hydroxy Cinnamic acid, and Flavonoids like Quercetin, Catchin, Rutin acts as natural Anti-oxidants [35].

Qualitative estimation of phenolic compounds:

Coumarins are having narcotic and sedative action also oxytocic [36] anti inflamatory [37] antithrombotic [38] . Spasmolytic and oxytocic [39] , vasodialatory [40]; Coumarins of Coriander decrease the content of P450 in induced hepatic microsomes without a major effect on heme content [41]. Oral administration of Citrus Coumarin and Iso pimpinellin blocks DNA adduct formation and skin tumor initiation in mice [42]. Coumarins suppresses the expression of T3SS genes of the plant pathogen Dickeya dadanti, proves that plants can also defend against bacterial pathogens by manipulating the expression of the T3SS PCA (P-Coumaric Acid) which regulates genes through the Hrpx/y two component system [43].

Extraction of phenolic compounds: Fresh leaves were collected and phenolic extract was collected through the standard method [62] .About 30 gm of the healthy and fresh, leaves were macerated in approximate 100ml of 2N HCl. The homogenate was digested on a boiling water bath for about 30min. The contents were cooled and filtered through Whatmann no: 1 filter paper. The filtrate was extracted with Peroxide free diethyl ether (solvent ether) repeatedly. The cooled extract was concentrated to a small volume and was treated thrice with 25ml of 5% anhydrous Na2Co3 solution. The Cooled Na2Co3 solution was adjusted to 2 P H with concentrated HCl. The acidified fraction at 2 P H was then extracted with equal volumes of fresh diethyl ether for three times and the combined ether extracts were washed with 2ml of distilled water for 3times to remove the traces of HCl. The ether soluble water was removed by freezing the extract and then the ether was evaporated to dryness on water bath at 98°C. The resulting residue was dissolved in 1ml of 95% ethanol and was preserved at low temperature in a dark container until used.

Flavonoid compounds may exhibit vast biological activities including antiallergic, antiviral, anti-inflammatory and vasodialatory in human beings44. These are used in the treatment of cold [45-46] against allergy, x rays and other radiation injuries, hemorrhage, dermatitis and albuminuric diseases [47]. Flavonoid acts as natural food antimicrobial systems [48]. Quercitin used against Urethritis and cystitis. It exerts antiseptic action on the urinary tract and other allergic/inflammatory mediators. In vitro studies of Quercetin shows anti tumour activities [49-50] .It is known for the treatment of capillary fragility and phlebosclerosis [51]; anti hemorrhagic [52]. It shows anti-tumor activity against chronic prostitis [53];Quercitin shows positive effects in preventing prostate cancer, heart disease, cataracts, allergies, inflammations and respiratory diseases such as bronchitis and asthma [54]; It also acts as antiinflammatory and antioxidant properties [55].Myricetin plays an important role in inhibiting the tumor growth and enhancing the diuretic and diaphoretic activities. Higher doses of Myricetin lowers the rates of prostate cancer [56].The three important Flavones (Kaempferol, Quercetin and Myricetin) reduce the risk of pancreatic cancer by 23 percent [57]. It is also used against Jaundice and hepatitis [58]. Apigenin shows its anti inflammatory activity [59]. MATERIALS AND METHODS: Plant material: Plant material A.aspera was collected at Mallemadugu dam along the water hedges near dodlamitta area of Renigunta Mandal. The botanical identity of the plant was determined and authenticated from the literature available in the Department of Botany and the voucher specimens (CA.27) were deposited in the Department Herbarium. The present work was carried out in the Department of Botany, S.V.University, Tirupati. Leaf material was thoroughly washed and dried under shade at 28 ± 2°C for about 10 days. The dried leaves were ground well into a fine powder in a mixer grinder and sieved to give particle size of 50-150mm. The powder was stored in air sealed polythene bags at room temperature. Phytochemical analysis: Solubility: The solubility was studied in eight solvents (Hot water, Cold water, Benzene, Hexane, Ethyl acetate, Chloroform, Methanol and Ethanol) based on polarity gradience. Preliminary Phytochemical analysis of secondary metabolites: All the extracts were subjected to preliminary Phytochemical qualitative screening for the Presence or absence of various secondary metabolites such as alkaloids, flavonoids, phenols, Terpinoids, steroids, Anthocynadins, anthroquinones, saponins, tannins, lignins, Indoles and glycosides were carried out by the standard methods [60-61].

Chemicals, Glassware & Equipment: Peroxide free Diethyl ether, Anhydrous Na2Co3 , HCl, Ethanol, Benzene, Acetic Acid Sodium formate, Formic Acid, Sulphanilic Acid, Sodium Nitrite, Sodium Carbonate, Para Nitriline, Ferric Chloride, Separatory funnel, Beakers, Conical flasks, Measuring Jars, Capillary tube, Test tubes, Glass tanks, Petri plates and Automizer.

Separation of phenolic compounds:1gm of the extract was spotted on 23 × 29cm Whatmann No: 1 chromatographic paper with the help of a micropipette. The origin of the spot was dried immediately with the help of hair drier. The dried sheets were run in bi-dimensional ascending chromatography by using rectangular chromatographic glass tank. The Chambers are saturated with the chromatographic solvents one day before. The development of Chromatograms has to be carried out at 22°-24°C. Solvent I Benzene: Acetic acid: water (60: 70: 30) v/v/v (upper layer of this mixture used at first direction) Solvent II Sodium formate: Formic acid: water (10: 1: 200) v/v/v (used for second direction). The paper after development was removed from the tank and dried at room temperature. The dried sheet was examined under UV light and the fluorescent regions were marked. Then the paper was exposed to ammonia vapors were also observed under UV light and the new fluorescent spots were also marked. Identification of phenolic compounds using chromogenic spraying reagents: To identify the phenolic compounds separated on the chromatograms were sprayed with Diazotized Sulphanilic acid, Paranitranilins and ferric chloride reagent with the help of an automizer. The phenolic compounds were identified by calculating their Rf values of individual spot colors with chromogenic sprays and finally confirmed with authentic samples by cochromatography. Preparation of Chromogenic spray reagents: Diazotized Sulphanilic acid: Sulphanilic acid Solution, 5% Sodium Nitrite Solution: 20% Anhydrous Sodium Carbonate solution, Diazotized Para Nitraniline Reagent, 5%Anhydrous Sodium Nitrite solution, 10% Sodium Carbonate solution, Ferric Chloride reagent. Qualitative estimation of flavonoids compounds Chemicals &Glassware: - Methanol, Chloroform, Alcohol 95%, Isopropyl alcohol, Ammonia, Sulphanilic acid, Concentrated HCl, Sodium Ni-

Journal of Pharmacy Research Vol.5 Issue 4.April 2012

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Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 trate, Sodium Carbonate, Separatory funnel, Beakers, Conical flasks, Measuring jar, Capillary tube, Test tubes, Glass tanks, Petri plates, Atomizer. . Extraction of flavonoids compounds The flavonoid compounds were extracted according to the standard methods [63-64]. About 2g of dried leaf powder was taken in a boiling test tube heated up to 40°C and then 18ml of methanol and 2ml of water (9:1) was added. Shaken well and was kept for about 24 hrs at room temperature. After that the upper clear solution of the extract was transferred to another test tube. To the remaining residue in the test tube, again 5ml of methanol and 5ml of water (1:1) was added. Stirred well and the contents were kept for 24 hours and the clear extract thus obtained was cooled up with the earlier sample. The combined extract was mixed well and filtered through the cotton. The filtrate was evaporated to about 1/3 of the original volume and the resultant aqueous extract was taken into a separatory funnel and then extracted with 10ml of chloroform. This process was repeated 2or 3 times. All the chloroform extracts were combined and evaporated to dryness under vaccume in a rotatory evaporator and the dried residue was dissolved in 1ml of 95% alcohol which was stored at low temperature in the dark until used. Separations of flavonoid compounds 1 gm of the extract was spotted on 23x29cm Whatmann No: 1 chromatographic filter paper with help of a micropipette. The origin of the spot areas was dried immediately with the shelp of hair drier. The dried sheets were run in unidimensional ascending chromatography by using rectangular chromatographic glass tanks which can accommodate 4 sheets at a time. The chromatographic chambers were saturated with any of the following solvents one day before the development of the chromatograms at 22-24°C. 1. Isopropyl alcohol: Ammonia (25%): Water (8: 1: 1) v/v/v. 2. n-Butanol: Acetic acd: Water (4: 1: 5) v/v/v (top layer was used) 3. Conc.HCl: Acetic acid: water (3: 30: 10) v/v/v. 4. Phenol: Water (3: 1) v/v. The chromatograms after unidimensional development were removed from the tanks and dried at room temperature. The dried sheet was observed under UV light and the fluorescent regions were marked. The paper while exposed to ammonia was also observed under UV light and the new fluorescent spots also marked. Identification of flavonoids using chromogenic spray reagents: To identify the flavonoid compounds separated on the chromatograms were sprayed with chromogenic spray reagents with the help of an atomizer. The flavonoid compounds were identified by calculating their Rf values of individual compound along with authentic samples by co-chromatographic studies. Preparation of Chromogenic sprays reagents: Sulphanilic acid solution. Sodium Nitrate solution, anhydrous sodium carbonate solution. Antibacterial activity: Extracts preparation: Dried leaf powder (25g) was packed in a Whatmann no.1 filter paper and was extracted in a Soxlet apparatus using 100ml of solvent. Methanol, Alcohol, Hot water and Cold water extracts were dried and stored in a refrigerator at 4°C. Bacterial cultures: The bacterial cultures Bacillus subtilis (MTCC- 441) causes Pneumonia, diarrhoea. ,Staphylococcus aureus (MTCC- 737) causes bone and joint pains, skin infections, boils; Escherichia coli (MTCC- 443) causes urinary tract infections, Pneumonia, Pseudomonas aeruginosa (MTCC- 741) causes urinary infections and Pneumonia. The above strains were procured from the Department of Microbiology S.V.University Tirupati and also from the SVIMS Tirupati.

Preparation of inoculums: Stock cultures were maintained at 4°C of nutrient agar slants. Active cultures for experiments were prepared by transferring a loop full of cells from the stock cultures to test tubes of nutrient agar medium and were incubated without agitation for 24hrs at 37°C. Preparation of the medium: To prepare 1lit of nutrient agar medium 3g of beef extract, 3g of peptone, 15g of agar was used. The ingredients were accurately weighed using digital electronic balance and dissolved in liter of distilled water before the addition of agar; the P H of the medium was adjusted to 7.0 by adding few drops of 0.1N NaoH/HCl using digital PH meter. Later this medium was transferred to conical flasks and plugged with nonabsorbent cotton. Medium was then sterilized by autoclaving at 15lbs for 20min cooled to 4 °C and used for the study. Agar well diffusion assay: The antibacterial activity of the leaf extracts was determined using agar well diffusion method [65] with slight modifications. Nutrient agar was inoculated with the selected microorganisms by spreading the bacterial inoculums on the media. Wells (8 mm diameter) were punched in the agar and filled with plant extracts. Control wells containing pure solvents (negative control) or standard antibiotic (positive control) viz., Gentamycin (30mg) and Ampicillin (10 mg). The plates were incubated at 37°C for 18hrs. The antibacterial activity was assessed by measuring the diameter of the zone of inhibition for the respective drug. The relative antibacterial potency was calculated by comparing its zone of inhibition with that of the standard drug. The data of crude drugs activity is given the mean of quadruplicates along with the standard error. Minimum inhibitory concentration: MIC is defined as the lowest concentration where no visible turbidity is observed in the test tube (bacterial concentration).The method [66] modified by Usman [67] was employed. In this method the broth dilution technique was used, where the leaf extract was prepared to the highest concentration of 10mg/ml (stock concentration). By adding sterile distilled water and serially diluted (two fold dilution) using the nutritive broth and later inoculated with 0.2ml standardized suspension of the test organisms. After 18hrs of incubation at 37°C., the test tubes were observed for turbidity .The lowest concentration of the tube that did not show any visible growth can be considered as the MIC. RESULTS: Solubility: Solubility with eight solvents of leaf powder revealed in increased gradience from hot water to alcohol (Hot water> Cold water> Benzene> Hexane> Ethyl acetate> Chloroform > Methanol >Ethanol). It is highest in Alcohol and less in Hot water respectively. Preliminary phytochemical screening- secondary metabolites :-( Table-1) Out of eight extracts in Methanol (8) and Alcohol (7) secondary metabolites were observed. In cold water (5) and Hot water (5), further Ethyl acetate (3) Chloroform (1) Benzene (1) and Hexane (1). The important secondary metabolites are Alkaloids, Terpinoids, and Tannins along with few Steroids, Anthocyanidins, and Glycosides. But flavonoids are present only in cold water and hot water extracts, where as Alkaloids, Phenols and glycosides present both in Alcohol and Methanol, and Anthocyanidins are present only in Methanolic extract. Phenols :- (Table-2) Nearly 11 phenolic compounds were observed in the leaves of A.aspera out of which 2 are un identified. Mainly Neo-chlorogenic acid is more in quality with bright brown colour, and Homo-protocatechuic acid in more quantity spreading in larger area with buff colour. Other compounds are

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Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 Table.1. Preliminary phytochemical screening L. leaf extracts S.no

Name of the test

1

Alkaloids Mayers test Wagners test Flavonoids Shinodons test Fecl3 test Phenols Fecl3 test Ellagicacid test Terpinoides Liebermann Burchards test SteroidsSalkowski test Liebermann’s Burchard test Anthocyanidins Saponins Tannins Gelatin test Fecl3 test Lignins Indols Enrilich test Glycosides Keller-kilani test

2

3

4

5

6 7 8

9 10 11

of Aeschynomene aspera

Cold water

Hot water Alcohol

Methanol

-

-

+++ +++

++++ ++++

+++ +++

+++ +++

-

-

-

-

+++ +++

+++ +++

+++

+++

+++

+++ +

+++ -

+++ -

++

+++

+++

+++

-

+++ -

+++ +++ -

+++ +++ -

+++ +++ +++

+++ +++ +++

-

-

-

-

-

-

+++

+++

is most susceptible with Alcoholic extracts 10mg-48 mm activity when compared to control as Ampicillin10 mg- 25mm, Gentamycin 30mg-19 mm and S. aureus is more resistant as 10 mg -29 mm. Other pathogens as B.subtilis and E. coli also shows more susceptible with alcohol extracts when compared to other extracts Effective inhibition of the leaf extracts is observed highest with Alcohol < Methanol