Volume 3. Issue 2. Pages 113-302. 2008 ISSN 1934-578X (printed); ISSN 1555-9475 (online) www.naturalproduct.us
Natural Product Communications
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2008 Vol. 3 No. 2 199 - 203
Natural Product Communications
Phenolic Constituents of Platanus orientalis L. Leaves Taha S. El-Alfya, Hamida M.A. El-Goharya, Nadia M. Sokkara, Amani A. Sleemb and Dalia A. Al-Mahdya a
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Egypt
Department of Pharmacology, National Research Center, Giza, Egypt
Received: September 10th, 2007; Accepted: November 24th, 2007
A new flavonoid glycoside, 5,7,4´-trihydroxy-3,6-dimethoxyflavone-3´-O-β-D-xylopyranoside (axillarin-3´-O-β-Dxylopyranoside), was isolated from the ethyl acetate fraction of the ethanolic extract of Platanus orientalis L. leaves, along with nine known flavonoid aglycones and glycosides, a phenolic acid and a phenolic acid derivative. Their structures were established on the basis of detailed spectral analysis. The total ethanolic, aqueous and ethyl acetate extracts of the leaves were tested for antihepatotoxic, antioxidant and cytotoxic activities. The extracts were found to be effective in protection against CCl4 liver injury by significantly lowering alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels. The extracts also significantly restored depleted levels of glutathione, indicating a strong antioxidant activity, while they showed variable cytotoxic activity. Keywords: Platanus orientalis L., antihepatotoxic, antioxidant, antitumor, flavonoids.
Many phytochemicals, especially phenolic constituents, have shown promise for the protection and/or treatment of hepatotoxicity cases in which oxidative stress plays a key role [1-3]. Platanus orientalis L., known as oriental plane (family Platanaceae), is a deciduous tree native to south-west Asia, south-east Europe and the Mediterranean regions [4,5]. The leaves were used in folk medicine in opthalmia, the bark was boiled in vinegar and given for diarrhea, dysentery and toothache [6,7], while the buds were used as a urinary tract antiseptic and antimicrobial . Only one report has been found dealing with the flavonoidal content of the buds and their cytotoxic activity . Nothing could be traced concerning either the phytochemical content or biological activities of the leaves. This work reports the isolation of a new flavonoid glycoside, 5,7,4´-trihydroxy-3,6dimethoxyflavone-3´-O-β-xylopyranoside (axillarin3´-O-β-xylopyranoside) (1), in addition to nine known flavonoid aglycones and glycosides (2-10), a phenolic acid (11), and a phenolic acid
derivative (12). Also, the antihepatotoxic, antioxidant and cytotoxic activities of the total ethanolic extract 5` 6` HO
(TEE), total aqueous extract (TAE), and the flavonoid-rich ethyl acetate fraction (EAF) were evaluated and reported. Compound 1 was obtained as a yellow powder. The molecular formula was calculated as C22H22O12 from the ESI-MS [M-H]- molecular ion peak appearing at m/z 477. A peak observed at m/z 345 after ionization indicated the loss of a pentose sugar. The NMR spectra suggested a flavonoid skeleton for the compound. The 1H NMR spectrum showed signals of ring B (2 doublets at δ 6.99, d, J = 8.4 Hz and δ 7.83, d, J = 2.1 Hz) indicating ortho and meta couplings of protons 5´ and 2´ with H-6´,
200 Natural Product Communications Vol. 3 (2) 2008
respectively. This was confirmed by a doublet of doublets assigned to H-6´ (δ 7.65, dd, J = 8.4, 2.1 Hz). The downfield shift of H-2´ (0.7 ppm) indicates the presence of a substituent at the 3´position [10,11]. In addition, ring A protons were represented by a singlet at δ 6.52, assigned to H-8. The absence of other signals characteristic of ring A suggested substitution at the 3 and 6 positions, with the two methoxyl groups displayed at δ 3.80 and 3.74, respectively. The anomeric proton of the sugar unit appeared as a doublet at δ 4.79. This upfield shift, together with its coupling constant (J = 6.9 Hz), confirmed the sugar attachment to be at the 3´ position having a β- configuration [10,11]. All the 13 C NMR signals were assigned and the sugar moiety was identified as β-xylopyranoside by comparison with reported data . The HMBC experiment showed correlations between δ 4.79 (H-1´´) and 144.9 (C-3´), between δ 3.74 (CH3-6) and 131.2 (C-6) and between δ 3.80 (CH3-3) and 137.3 (C-3). Thus, compound 1 was identified as 5,7,4´-trihydroxy-3,6-dimethoxyflavone-3´-O-βD-xylopyranoside. Compounds 2-12 were identified as kaempferol (2), quercetin (3), kaempferol-3-O-α-L-(2´´,3´´-di-E-pcoumaroylrhamnopyranoside) (platanoside) (4), kaempferol-3-O–β-D-(6´´-E-p-coumaroylglucopyranoside) (tiliroside) (5), kaempferol-3-O-α-Lrhamnopyranoside (afzelin) (6), quercetin-3-O-α-Lrhamnopyranoside (quercitrin) (7), quercetin-3-O-βD-galactopyranoside (hyperoside) (8), kampferol -3O-β- rutinoside (nicotiflorin) (9), quercitin-3-Orutinoside ( rutin) (10), p-coumaric acid (11), and 4´caffeoylquinic acid (cryptochlorogenic acid) (12) by comparing their UV,1H and 13C NMR spectroscopic data with those published [10,12-18].
El-Alfy et al.
Table 1: Effect of TEE, TAE extracts and EAF of P. orientalis leaves on blood glutathione level of alloxan-induced diabetic rats.(n=6). GSH
36.8 ± 1.2b
23.4 ± 0.3a
Diab. treated with TEE
33.1 ± 0.7b
Diab. treated with TAE
33.1 ± 0.9b
Diab. treated with EAF
33.7 ± 1.2b
Diab. treated with Vitamin E
35.9 ± 0.7b
a Significantly different from normal value at P=0.01. b Significantly different from diabetic control value at P=0.05.
Toxicological studies revealed that the LD50 of both the TEE and TAE of P. orientalis leaves were 7.3 and 7.6 g/kg, respectively and can be considered safe . The antioxidant activity was determined in alloxaninduced diabetic rats . All the extracts restored the depleted levels of glutathione (GSH) (Table 1) with the EAF being the most potent extract (93.8%). The tested extracts reversed the elevated levels of the serum liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) (Table 2). These effects were comparable to and in some cases higher than that of the reference drug, silymarin. TEE was the most potent extract as it decreased the levels of ALT, AST and ALP by 52.7%, 43% and 39%, respectively. All the tested extracts showed dose dependent cytotoxicity against the three human cancer cell lines, MCF7, HEPG2 and H460 (Table 3). It was observed that EAF showed the highest activity against HEPG2 cell line.
Table 2: Effect of TEE, TAE and EAF of P. orientalis on ALT, AST and ALP using silymarin as the reference standard in liver damaged rats. (n=10).
- ve control TEE (100 mg/kg) TAE (100 mg/kg) EAF (100 mg/kg) Silymarin (25 mg/kg) a 1
Liver enzymes ALT AST ALP
Zero 25.9±0.7 28.2±1.3 7.2±0.2
30 days1 25.2±0.3 27.9±0.8 7.1±0.1
72 hours2 131.6±4.3 124.3±4.1 28.2±1.2
30 days3 142.8±5.1 136.7±5.1 31.2±1.3
ALT AST ALP
28.6±0.8 26.5±0.9 7.6±0.1
27.9±1.1 26.1±0.3 7.1±0.1
58.4±1.7a 54.8±1.6a 13.1±0.9a
27.6±0.9ab 31.2±1.1ab 8±0.2 ab
52.7 43 39
ALT AST ALP ALT AST ALP
27.2±0.4 29.1±0.8 7.5±0.1 28.2±1.4 29.2±1.2 7.4±0.2
27.5±0.8 27.6±0.5 7.0±0.2 26.8±1.3 28.8±0.8 7.1±0.1
52.6±1.7a 51.4±1.2a 11.5±0.3a 53.5±2.4a 50.4±2.1a 15.2±0.8a
29.9±1.1ab 30.6±1.4ab 8.3±0.5 ab 34.7±1.6ab 34.0±0.7ab 9.3±0.4 ab
43 40.4 27.8 35 32.5 38.8
ALT AST ALP
28.4±0.9 28.7±0.6 7.3±0.2
27.6±1.2 28.2±0.4 7.2±0.1
41.6±0.9a 43.7±1.3a 9.1±0.3a
24.7±0.4ab 25.8±0.7 ab 6.5±0.1 ab
40.6 41 28.5
% change -
Significantly different from control group at P=0.01, b Significantly different from 72 hours after injection of CCl4 at P=0.01. 30 days after treatment with the extracts, 2 72 hours after injection with CCl4, 3 30 days after administration of the extracts to liver damaged rats.
Constituents from Platanus orientalis
Natural Product Communications Vol. 3 (2) 2008 201
Table 3: Effect of TEE, TAE and EAF of the leaves of P. orientalis on MCF7, HEPG2 and H460 human carcinoma cell lines. Conc μg/mL 0.000
MCF7 = breast carcinoma cell line, HEPG2 = hepatocellular carcinoma cell line, H460= lung carcinoma cell line.
This result, in addition to its potent antioxidant activity and its ability to lower the elevated levels of the liver enzymes could be attributed to its phenolic content. Experimental General: Melting points were determined on Electrothermal 9100 equipment. Optical rotation [α]D was determined using a Perkin-Elmer 241 polarimeter (MeOH, c in g/100 mL). IR spectra were measured using a Shimadzu-IR-435 Infrared spectrophotometer. UV spectra were measured using a Shimadzu UV 1650 PC spectrophotometer. EI-MS were measured on a Finnigan SS Q 7000. ESI-MS (negative mode) of compound 1 was measured on Thermo Finnigan (ion trap) equipment. NMR spectra were measured in DMSO-d6 with a Varian Mercury instrument (NMR, 300 MHz, 13C, 75 MHz). TLC was performed on sheets pre-coated with either silica gel F254 (Fluka) or with RP-18 F254 (Merck, Germany). Compounds were visualized under UV light (254 and 366 nm) and by spraying with aluminium chloride and NP-PEG reagents. Column chromatography was performed over Silica gel H 60 (Sigma) for VLC, Sephadex LH-20 (Pharmacia), silica gel 60 (Fluka) and silica gel C18–reversed phase (70-230 mesh, Fluka). Plant material: The leaves of Platanus orientalis L., were collected from El-Orman Botanical Garden, Giza, Egypt. A voucher specimen is kept in the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. Material for pharmacology: Chemicals used in this study were: Alloxan (Sigma Co., USA); Vitamin E (dl α-tocopheryl acetate) Pharco Pharmaceutical Co,
Egypt; and Silymarin ®, Sedico Co., Egypt. Kits for biochemical assessment of AST, ALT and ALP were purchased from Biomerieux, France, and the glutathione kit from Wak Company, Germany. Extraction and isolation: The air dried, powdered and defatted leaves of P. orientalis (2 kg) were macerated with 70% EtOH till exhaustion. The ethanolic extract was evaporated under reduced pressure to give 197 g of dark green residue, of which 180 g was suspended in water (600 mL) and partitioned successively with CHCl3, EtOAc and n-BuOH to afford 22 g, 25 g and 13 g of dried residues, respectively. Another 100 g of the air dried, powdered leaves was boiled with 500 mL water, followed by evaporation under reduced pressure. The residue was dissolved in sterile water before administration to rats. EAF (20 g) was chromatographed on Silica gel H 60, VLC (Ø7 x 12.5 cm, 200 g) and eluted with CHCl3, gradients of CHCl3-EtOAc, EtOAc and gradients of EtOAc-MeOH up to pure methanol. Fractions, each of 200 mL, were collected and monitored by TLC. Similar fractions were pooled together to give 9 collective fractions. Fractions 1 (270 mg), 2 (410 mg), 3 (850 mg) and 4 (600 mg) were subjected to repeated CC on Sephadex LH-20 to give compounds 11 (22 mg, 0.11%), 2 (38 mg, 0.19%), 3 (35 mg, 0.17%), 4 (15 mg, 0.07%) and 5 (20 mg, 0.1%). Fractions 5 (950 mg), 6 (3.1 g), 7 (3.2 g), 8 (2 g) and 9 (1.5 g) were purified on Sephadex LH-20 followed by CC on silica gel 60 and Silica gel C18– reversed phase to give compounds 6 (22 mg, 0.11%), 1 (30 mg, 0.15%), 7 (23 mg, 0.11%), 8 (32 mg, 0.16%), 9 (20 mg, 0.1%), 10 (35 mg, 0.17%) and 12 (32 mg, 0.16%). 5,7,4´-Trihydroxy-3,6-dimethoxyflavone-3´-O-βD-xylopyranoside (axillarin-3´-O-β-D-xylopyranoside) (1) Yellow powder. MP: 240-242oC. [α]D: -36.2º (c 0.1, MeOH). Rf: 0.62 (CHCl3-MeOH, 8:2 and 2 drops formic acid). IR (KBr): 3399, 2950, 2922, 1659, 1597, 1499, 1150, 1075, 834, 800 cm-1.
202 Natural Product Communications Vol. 3 (2) 2008
UV/Vis λmax (MeOH) nm: 254 (IIb), 272 (IIa), 344, (NaOMe): 277, 328, 401, (AlCl3): 261, 280, 308sh, 364, (AlCl3/HCl): 260, 282, 309sh, 363, (NaOAc): 274, 334 sh, 401, (NaOAc./H3BO3): 272, 350. 1 H NMR (300 MHz, DMSO-d6) δ: 7.83 (1H, d, J = 2.1 Hz, H-2´), 6.99 (1H, d, J = 8.4 Hz, H-5´), 7.65 (1H, dd, J = 8.4, 2.1 Hz, H-6´), 6.52 (1H, s, H-8), 3.80 (3H, s, OMe-3), 3.74 (3H, s, OMe-6). Xylose: 4.79 (1H, d, J = 6.9 Hz, H-1''). 13 C NMR (75 MHz, DMSO-d6): Aglycone: 177.9 (C-4), 161.7 (C-7), 154.7 (C-2), 152.2 (C-5), 151.5 (C-9), 150.3 (C-4´), 144.9 (C-3´), 137.3 (C-3), 131.2 (C-6), 123.6 (C-1´), 120.7 (C-6´), 116.8 (C-5´), 116.3 (C-2´), 104.2 (C-10), 94.0 (C-8), 59.8 (OMe-3), 59.5 (OMe-6). Xylose: 102.8 (C-1´´), 73.03 (C-2´´), 75.7 (C-3´´), 69.3 (C-4´´), 65.7 (C-5´´). MS (EI, 70 eV) m/z (%): 478 (M+, 1.02%), 345 (M+ - C5H9O4, 9.01%), 463 (M+- CH3, 1.22%), 158 (100%). Negative ESI-MS: m/z [M-H]- calcd for C22H22O12: 477.00, MS/MS fragment at m/z 345 [M-xylose]Yield: 0.15%. Animals and cell lines: Adult male albino rats (130-140 g) were obtained from the animal-breeding unit of the National Research Center, Dokki, Giza, Egypt. Rats were fed on standard normal laboratory diet and water ad libidum. Doses of the drugs were calculated according to the method of Paget and Barnes  and were administrated orally by gastric tube. MCF7, HEPG2 and H460 cell lines were obtained from the National Cancer Institute, Kasr ElAiny Street, Cairo, Egypt. Determination of LD50: LD50 values of both TEE and TAE of the leaves of P. orientalis were determined following Karber procedures . Antioxidant activity: Thirty six adult male albino rats were used in the experiment and divided into 6 groups. One group was kept as control. All the remaining groups were injected intraperitoneally with a single dose of alloxan (150 mg / kg b. wt.) to
El-Alfy et al.
induce hyperglycaemia . The second group was kept as diabetic control and the third group was given Vitamin E orally in a daily dose of 100 mg/kg. The other three groups received 100 mg/kg orally of the TEE, TAE and the EAF of P. orientalis leaves, respectively. Blood GSH levels were determined and recorded . Anti-hepatotoxic activity: The tested extracts were evaluated and compared with that of silymarin (reference standard), according to the method of Klassen and Plaa . Fifty adult male albino rats were divided into 5 groups as following: the first group received 1 mL saline and kept as the reference control group; the second group received silymarin (25 mg/kg b. wt. daily) and groups three, four and five received a daily oral dose (100 mg/kg) of TEE, TAE and EAF, respectively. The extracts and silymarin were administered for a period of 30 days, followed by induction of hepatotoxicity through intraperitoneal injection of 5 mL/kg of 25% CCl4 in liquid paraffin, then further administration of the extracts for 30 days. Liver damage and/or recovery were assessed by measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Cytotoxic activity: The antitumor activity of the extracts against MCF7, HEPG2 and H460 cell lines was determined using the method of Skehan et al. . The relation between the surviving tumor fraction and drug concentration was determined to obtain the survival curve of each tumor cell line after the specified tested extract. All data are expressed as mean±S.E., and the statistical significance was evaluated by One-Way Analysis of Variance ANOVA . Acknowledgments - The authors wish to thank Prof. Dr Wafaa M. Amer, Prof. of Taxonomy and Flora, Botany Department, Faculty of Science, Cairo University, for identification of the plant material.
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Seasonal Variation of Hypericin and Pseudohypericin Contents in Hypericum scabrum L. Growing Wild in Turkey Ali Kemal Ayan, Cüneyt Çırak and Kerim Güney
Molluscicidal Polyphenols from Species of Fucaceae Asmita V. Patel, David C. Wright, Maricela Adrian Romero, Gerald Blunden and Michael D. Guiry
Anti-diabetic Activity of Triphala Fruit Extracts, Individually and in Combination, in a Rat Model of Insulin Resistance Venkateshan S. Prativadibhayankaram, Samir Malhotra, Promila Pandhi and Amritpal Singh
Biotransformation of Mefenamic Acid by Cell Suspension Cultures of Solanum mammosum Suzana Surodjo, Angela A. Salim, Suciati, Achmad Syahrani, Gunawan Indrayanto and Mary J. Garson
Natural Variability in Enantiomeric Composition of Bioactive Chiral Terpenoids in the Essential Oil of Solidago canadensis L. from Uttarakhand, India Chandan S. Chanotiya and Anju Yadav
Germacrone Dominates the Leaf Oil of Siparuna grandiflora from Monteverde, Costa Rica William N. Setzer, Brittany R. Agius, Tameka M. Walker, Debra M. Moriarity and William A. Haber
Leaf Oil Composition of Piper aduncum subsp. ossanum (C. CD.) Saralegui from Cuba Orlando Abreu and Jorge A. Pino
Volatile Constituents from the Leaves of Phyllanthus salviaefolius H. B. K. from the Venezuelan Andes Silvana Villarreal, Luis B. Rojas, Alfredo Usubillaga, Irama Ramírez and Mariana Solórzano
Synergistic Antifungal Activities of Thymol Analogues with Propolis Chi-Pien Chen and Ai-Yu Shen
Review /Account Argan oil, Functional Food, and the Sustainable Development of the Argan Forest Zoubida Charrouf and Dominique Guillaume
Chemical Constituents of Selected Japanese and New Zealand Liverworts Yoshinori Asakawa, Masao Toyota, Fumihiro Nagashima and Toshihiro Hashimoto
Natural Product Communications 2008 Volume 3, Number 2 Contents Original paper
New cis-Chrysanthenyl Esters from Eryngium planum L. Emilia Korbel, Ange Bighelli, Anna Kurowska, Danuta Kalemba and Joseph Casanova
Secondary Metabolites from Eremostachys laciniata İhsan Çalış, Ayşegül Güvenç, Metin Armağan, Mehmet Koyuncu, Charlotte H. Gotfredsen and Søren R. Jensen
A Novel Iridoid from Plumeria obtusa Firdous Imran Ali, Imran Ali Hashmi and Bina Shaheen Siddiqui
Terpenoids from Neolitsea dealbata Xiujun Wu, Bernhard Vogler, Betsy R. Jackes and William N. Setzer
Volatile Components from Selected Mexican, Ecuadorian, Greek, German and Japanese Liverworts Agnieszka Ludwiczuk, Fumihiro Nagashima, Rob S. Gradstein and Yoshinori Asakawa
New ent–Kaurane type Diterpene Glycoside, Pulicaroside-B, from Pulicaria undulata L. Nasir Rasool, Viqar Uddin Ahmad, Naseem Shahzad, Muhammad A. Rashid, Aman Ullah, Zahid Hassan, Muhammad Zubair and Rasool Bakhsh Tareen
Anti-babesial Quassinoids from the Fruits of Brucea javanica Ahmed Elkhateeb, Masahiro Yamasaki, Yoshimitsu Maede, Ken Katakura, Kensuke Nabeta and Hideyuki Matsuura
Triterpenoids and Alkaloids from the Roots of Peganum nigellastrum Zhongze Ma, Yoshio Hano, Feng Qiu, Gang Shao, Yingjie Chen and Taro Nomura
Saikosaponins from Bupleurum chinense and Inhibition of HBV DNA Replication Activity Feng Yin, Ruixiang Pan, Rongmin Chen and Lihong Hu
Brauhenoside A and B: Saponins from Stocksia brauhica Benth. Viqar Uddin Ahmad, Sadia Bader, Saima Arshad, Faryal Vali Mohammad, Amir Ahmed, Shazia Iqbal, Saleha Suleman Khan and Rasool Bakhsh Tareen
Saponins from Fresh Fruits of Randia siamensis (Lour) Roem. & Schult. (Rubiaceae) Rapheeporn Khwanchuea, Emerson Ferreira Queiroz, Andrew Marston, Chaweewan Jansakul and Kurt Hostettmann
New Alkaloid from Aspidosperma polyneuron Roots Tatiane Alves dos Santos, Dalva Trevisan Ferreira, Jurandir Pereira Pinto, Milton Faccione and Raimundo Braz-Filho
Acanthomine A, a new Pyrimidine-β-Carboline Alkaloid from the Sponge Acanthostrongylophora ingens Sabrin R. M. Ibrahim, RuAngelie Ebel, Rainer Ebel and Peter Proksch
Phytochemical and Microscopic Characterization of the Caribbean Aphrodisiac Bois Bandé: Two New Norneolignans Ingrid Werner, Pavel Mucaji, Armin Presser, Christa Kletter and Sabine Glasl
3-Acetoxy-7-methoxyflavone, a Novel Flavonoid from the Anxiolytic Extract of Salvia elegans (Lamiaceae) Silvia Marquina, Yolanda García, Laura Alvarez and Jaime Tortoriello
Struthiolanone: A Flavanone-Resveratrol Adduct from Struthiola argentea Sloan Ayers, Deborah L. Zink, Robert Brand, Seef Pretorius, Dennis Stevenson and Sheo B. Singh
Continued inside back cover