Aneela Wahab et al.

/ Journal of Pharmacy Research 2012,5(4),1984-1987

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

Chemical investigation of Xanthium strumarium Linn and biological activity of its different fractions
Aneela Wahab, 1 Amina Sultana, 2* Khalid M. Khan, 1 Ayesha Irshad, 1,2 Nida Ambreen, 2 Muhammad Ali, and 2 Muhammad Bilal 1 Federal Urdu University of Arts, Science and Technology, Gulshan-e-Iqbal, Karachi, Pakistan 2 H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
1

Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012 ABSTRACT

Chemical investigation of the ethyl acetate extract of the aerial parts of Xanthium strumarium Linn has resulted in the isolation of a pentacyclic triterpene, 3- acetoxy lup-20(29)-ene (1), two sterols, stigmasterol (2) and -sitosterol (3) and a fatty acid, palmitic acid (4). Their structures were elucidated with the help of spectroscopic methods. Compound 1, however, a known compound but there is no report of its isolation from this plant. This is the first report of isolation of compound 1 from this plant with detailed spectral and chemical studies along with its important HMBC (Hg C) interactions that has not been reported earlier. The methanol extract of X. strumarium was fractionated on the basis of polarity and investigated for their antibacterial and antioxidant properties. All fractions exhibited weak to moderate antibacterial and weak antioxidant activity. Key words: Xanthium strumarium Linn, pentacyclic triterpene, antibacterial and antioxidant activities. INTRODUCTION: Xanthium strumarium Linn, commonly known as Chotagokhru or Chotadhatura in Hindi, the trade name is Cocklebur or Bur-weed, belongs to the family compositeae [1]. The genus Xanthium is distributed in nearly all parts of the world [2]. It is an annual herb found in tropical and temperate regions of Eurasia, North America and South America [3]. In Pakistan the noxious weed is found abundantly in plain to 8000 feet throughout the country and in Kashmir [4]. It is also found in fallow paddy fields and hotter parts of India, Srilanka and the Western Himalayas up to the height of 5000 feet [5]. The genus Xanthium is represented by its twenty-five species in the world [6]. Out of this X. strumarium, X. spinosum, X. macrocarpum and X. sibiricum are found in Pakistan and India [7]. The species of Xanthium have been used as traditional herbal medicines for the treatment of nasal sinusitis, headache, urticaria, arthritis [8,9], fever, scrofula, herpes and cancer [10,11]. The whole plant X. strumarium is of great medicinal importance. The compounds isolated from the aerial parts showed anti-malarial activity. Carboxyatractyloside possessed hypoglycemic activity [3,14], however, a sesquiterpene xanthanol is an antimicrobial agent [12]. Aerial parts also contain mixture of alkaloids, sesquiterpenoids and sesquiterpene lactones (xanthinin, xanthatin, xanthumin). Xanthumin is CNS depressant and also shows antibacterial activity. The seeds are potential source of fatty oil comprises of saturated and unsaturated fatty acids. Hydroquinone and choline are the basic toxic principles of seeds [7]. The seeds contain (-) - xanthienopyran which is a new inhibitor of superoxide anion generation by human neutrophils [13]. The fruits are rich in vitamin C and are effective in treating small-pox. The fruits are comprises of carbohydrates, organic acids, phosphatides, phytosterol, tetrahydroxyl flavones

and an antiinflammatory agent, -sitosterol--D-glucoside [7]. Caffeic acids found in fruits possess antihyperglycemic effect [15]. The n-butanol fraction of fruits showed analgesic and antiinflammatory activity [16]. The fruits are used as tonic, diuretic, sedative and diaphoretic. The leaves are astringent, antisyphilitic, diuretic and alterative. The leaves contain an essential oil that shows potent antifungal activity [7]. The sesquiterpene lactones found in leaves exhibit cytotoxic activity against human tumor cell lines [17,18]. The roots are bitter tonic, useful in cancer, scrofula and herpes [11]. All parts of this plant contain iodine [19]. Besides these, the extracts of this plant also exhibit antinociceptive [13], antidiabetic, antitrypanosomal [20], antimitotic [10], anticancer [21] and antitumor properties [22]. Reports about the pharmacological importance about the genus Xanthium fascinated us to investigate the aerial parts of X. strumarium. Herein we report the isolation and characterization of lupenyl acetate (1) reported for the first time from this plant along with other known compounds stigmasterol (2), -Sitosterol (3) and hexadecanoic acid (4). Differ ent extracts of this plant were tested for their antibacterial activity and antioxidant activity. All the extracts showed weak to moderate antibacterial activity (Table -1) and weak antioxidant activity (Table-2).

*Corresponding author.
Prof. Dr. Khalid M. Khan Ph.D. (Kar.), P.D.R. (Germany), M.C.S.P H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan

O H3C O

Figure-1: Significant HMBC (H

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C) interactions of compound (1)

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Aneela Wahab et al. / Journal of Pharmacy Research 2012,5(4),1984-1987 Table-1: In vitro Antibacterial activity of different extracts of X. strumarium Zone of inhibition diameter (mm)
Microorganism Extracts XS-HX XS-DC XS-BU XS-ME Standard Ampicilin

Bacteria Gram-positive Bacillus cereus Bacillus thuringiensis Staphylococcus aureus Streptococcus saprophytious Streptococcus faecalis Gram-negative Proteus mirabilis Pseudomonas aeruginosa ATCC 9027 Salmonetta typhi 10 08 08 > 35 >30 > 30 08 09 09 09 09 13 > 25 > 45 > 30 > 35 > 30

Values are inhibition zones (mm) and an average of triplicate. Extracts: XS = Xanthium strumarium, HX = Hexane, DC = Dichloromethane, BU = n-Butanol, ME =Methanol Table- 2: Antioxidant Activity of extracts of Xanthium strumarium S. No. 1 2 3 4 5 Sample code XS-DC XS-BU XS-HX XS-ME Ascorbic Acid % inhibition 41.66 30 -4.16 25 80 %

A voucher specimen (G.H. No.86398, No. 01) has been deposited in the herbarium at Department of Botany, Faculty of Science, University of Karachi, Karachi, Sindh, Pakistan. Isolation The air dried aerial parts of this plant (10 kg) were ground to fine powder and extracted repeatedly with methanol at room temperature. The solvent was evaporated under vacuum to obtain 250 g crude extract. The resultant dark greenish brown gummy mass was partitioned with n-hexane, dichloromethane, ethyl acetate and n-butanol fractions. Each fraction was concentrated in vacuum to have 13 g n-hexane, 14 g dichloromethane, 97 g ethyl acetate and 21 g n-butanol soluble fractions. The ethyl acetate soluble fraction (97 g) was subjected to column chromatography (CC) on a silica gel gravity column eluting successively with n-hexane and n-hexane: EtOAc in increasing order of polarity which furnished 17 fractions. The FR-2 eluted with n-hexane: EtOAc (9.5:0.5) gave a white crystalline solid which showed single spot on TLC using n-hexane: EtOAc (9.5:0.5) as solvent system was identified as lupenyl acetate (1) (40 mg) while the mother liquor (2 g) of FR2 was further subjected to pencil CC over silica gel eluting with n-hexane, nhexane: EtOAc as mobile phase in increasing order of polarity furnished 38 fractions. The FR-5 eluted by n-hexane: EtOAc (9.5:0.5) yielded a colorless solid which after purification over TLC using n-hexane: EtOAc (9:1) as solvent system was identified as stigmasterol (2) (20 mg), Fr-11eluted with n-hexane: EtOAc (9:1) to afford colorless solid which showed a single spot on TLC using n-hexane: EtOAc (8.5:1.5) as mobile phase was identified as -sitosterol (3) (25 mg). The FR-3 of the main EtOAc fraction on elution with n-hexane: EtOAc (9:1) gave two major spots on TLC using same solvent system. These were purified by thick layer chromatography using n-hexane: EtOAc (9:1) as mobile phase provided palmitic acid (4) (22 mg) and an unidentified compound. Lupenyl acetate (1) Colourless needles (40 mg) ; M.P: 214-215 C; IR (KBr) max cm-1 : 3050, 1735, 1630, 1200 and 800; EIMS: m/z 468[M]+, 453, 408, 249, 218 and 204; HR-EIMS: m/z 468.3970 (Calcd for C32 H52 O2 , 468.3969) 1 H-NMR (CDCl3 , 300 MHz): 4.67 and 4.55 (each 1H, d, J = 2.3 Hz, H-29), 4.46 (1H, dd, J = 10.0, 5.7 Hz, H-3), 2.36 (1H, m, H-19), 2.02 (3H, s, COCH3 ), 1.68 (3H, s, H-30), 1.05 (3H, s, H-26), 0.98 (3H, s, H-27), 0.85 (9H, s, H25, 24, 23), 0.81 (3H, s, H-28); 13 C-NMR (CDCl3 , 75 MHz): 38.3 (C-1), 23.8 (C-2), 81.0 (C-3), 37.8 (C-4), 55.3 (C-5), 18.2 (C-6), 34.2 (C-7), 40.8

RESULTS AND DISCUSSION: The methanolic extract of X. strumarium was fractionated into n-hexane, chloroform, ethyl acetate and n-butanol fractions. The ethyl acetate fraction was subjected to a series of column chromatographic techniques (see experimental for details) to provide four known compounds. The compound lupenyl acetate (1) is reported for the first time from this species, other compounds are stigmasterol (2), -sitosterol (3) and palmitic acid (4), respectively. Theirstructures have been elucidated through spectroscopic techniques. The structure of lupenyl acetate (1) is further confirmed by its significant heteronuclear multiple bond coherence (HMBC) interactions (Fig.1) reporting first time. In addition different extracts of this plant were tested for their antibacterial and anti-oxidant activities. All the extracts showed weak to moderate antibacterial activity (Table-1) while weak antioxidant activity (Table-2). EXPERIMENTAL: General Melting points were determined with a Gallenkamp melting point apparatus and are uncorrected. Column chromatography was carried out on silica gel (70-230 mesh, Merck). Thin layer chromatography (TLC) was performed on pre-coated silica gel GF-254. IR spectra were recorded on a Jasco-302-A spectrophotometer. The mass spectra were scanned on a Jeol-JMS HX-110 mass spectrometer. The 1 H and 13 C-NMR spectra were recorded on a Bruker spectrometer operating at 500, 300 and 75 MHz. The chemical shift values are reported in (ppm) relative to SiMe4 (TMS) as an internal standard. The coupling constant (J) are given in Hz. Plant Material The Xanthium strumarium was collected by Mohammad Farman Ali from Lakimarwat (Khyber Pakhtun Khawa) Pakistan and identified by Dr. Sahar.

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Aneela Wahab et al. / Journal of Pharmacy Research 2012,5(4),1984-1987

(C-8), 50.3 (C-9), 37.1 (C-10), 20.9 (C-11), 25.1 (C-12), 38.0 (C-13), 42.8 (C-14), 27.4 (C-15), 35.5 (C-16), 43.0 (C-17), 48.0 (C-18), 48.3 (C-19), 150.9 (C-20), 29.8 (C-21), 40.0 (C-22), 27.9 (C-23), 16.5 (C-24), 16.1 (C25), 15.9 (C-26), 14.5 (C-27), 18.0 (C-28), 109.3 (C-29), 19.2 (C-30), 171.0 (C=O), and 21.3 (COCH3 ). The spectral data showed complete agreement with the literature values [23,24]. Stigmasterol (2) Colourless needles (20 mg); M.P: 169-170 C; IR (KBr) max cm-1 : 3380, 1660; EIMS: m/z 412 [M]+, 397, 394, 379, 367, 273, 255, 229, 211, 199, 173, 145, 119; HR-EIMS: m/z 412.3742 (Calcd for C29 H48 O, 412.3707); 1 H-NMR (CDCl3 , 300 MHz): 5.34 (1H, br. s, H-6), 5.17 (1H, m, H-22), 5.03 (1H, m, H-23), 3.50 (1H, m, H-3), 0.99 (3H, s, Me-19), 0.83 (3H, d, J = 6.7Hz, Me-27), 0.81 (3H, t, J = 7.0Hz, Me-29), 0.78 (3H, d, J = 6.7Hz, Me-26), 0.67 (3H, s, Me-18).The spectral data coincided with the literature values [25,26,27] -Sitosterol (3) Colourless solid (25 mg); M.P: 134-135 C; IR (KBr) max cm-1 : 3400, 1640; EIMS: m/z 414 [M]+, 396, 381, 273, 255, 213, 133 and 55; HR-EIMS: m/ z 414.3862 (Calcd for C29 H50 O, 414.3864); 1 H-NMR (CDCl3, 500 MHz): 5.33 (1H, br. s, H-6), 3.40 (1H, m, H-3), 1.00 (3H, s, Me-19), 0.90 (3H, d, J = 6.0Hz, Me-21), 0.82 (3H, t, J = 7.0Hz, Me-29), 0.80 (3H, d, J = 6.5Hz, Me-26), 0.78 (3H, d, J = 6.5Hz, Me-27), 0.67(3H, s, Me-18). The spectral data showed complete agreement with the reported values [25,28,29]. Palmitic acid (4) White crystals (22 mg); M.P: 61-62 C; IR (KBr) max cm-1 : 3450-2610, 2925, 2850, 1700 and 1120; EIMS: m/z 256 [M]+, 227, 213, 199, 171, 129, 73 and 45; HR-EIMS: m/z 256.2389 (Calcd for C16 H32 O2, 256.2403); 1 HNMR (MeOD, 300 MHz): 2.25 (2H, t , J = 7.5Hz, H-2), 1.26 (26H, br. s, 13 x CH2 chain), 0.85(3H, t, J =6.6Hz, Me-16). The spectral data agreed with the reported values [30]. BIOLOGICAL ASSAY: Preparation of Culture media and inoculum Mueller Hinton agar and Mueller Hinton broth was used as the media for the culturing of bacterial strains and SDA and distilled water was used as the media for fungal strains. Antibacterial Assay Antibacterial activities of extracts of Xanthium strumarium were tested by using disc diffusion method (Kirby Bauer et al., 1966). The suspensions of the bacterial strains were prepared according to 0.5 McFarland scale and swabbed on to the surface of sterile Mueller Hinton agar plates. Sterile filter paper discs (7 mm in diameter) were impregnated with each extract, allow to air dry to eliminate remaining solvent and were placed on the surface of the inoculated plates. The discs impregnated with DMSO served as the negative control. The plates were incubated at 37 C for 24 hours. The results of antibacterial activity were noted based on the measurement of the diameter of inhibition zone appeared around the disc. Antioxidant Assay Antioxidant activity of the extracts of Xanthium strumarium was determined by using the method described by Lee et al. (1998). 1,1-Diphenyl-2picrylhydrazyl (DPPH) was prepared in ethanol (300 uM). Briefly, 10 L of test sample and 90 L solution of stable radical, 1,1-diphenyl-2picrylhydrazyl (DPPH) was added in 96-well micro titer plates and incubated at 37 C for 30 minutes. Absorbance was measured at 515 nm by using a spectrophotometer. Percent inhibition of radicals by treatment of test sample was determined by comparison with a DMSO treated control group.
% Inhibition = (absorbance of the control-absorbance of the test sample) x 100 Absorbance of the control

Ascorbic acid was used as standard control. The EC50 value calculated denotes the concentration (in ug/ml) of sample required to scavenge 50% of DPPH. REFERENCES: 1. G.I Chopra, Angiosperum, Rehber Publishers, Karachi (Pakistan), p.256 (1984). 2. Lauras S. Favier, Alejandra O.M Maria, G.H. Wendel, E.J. Borkowski and O.S Giordano, J.of Ethano pharmacology, 100, 260 (2005). 3. T. Pullaiah, Encyclopaedia of World Medicinal Plants, Regency Publication, New Delhi (India), Vol. 4, p.2076 (2006). 4. E. Nasir, Flora of West Pakistan, Fakhri Printing Press, Karachi (Pakistan), p. 801 (1972). 5. Dr K.M Nadkarni, Indian Materia Medica, Popular Prakashan Pvt. Ltd. Bombay (India), p. 1298 (1976). 6. T. Han, H.L. Li, Q.Y. Zhang and L.P. Qin, Phytomedicine, 14, 825 (2007). 7. B.N. Sastri, The Wealth of India, CSIR, New Delhi (India), Vol-10, p. 1 (1976). 8. Ting Han, Hui Liang, Q Zhang, H. Zhang and Luping Qin, Chemistry of Natural Compounds, 42, 5, 567 (2006). 9. Y.T.Ma, Mu-Chi Huang, F.L.Hsu and Hsiu Foug chang, Phytochemistry, 48, 6, 1083 (1998). 10. Ahmed A. Mahmoud, Ahmed A. Ahmed, Shar S. Al-Shihry and Otmar spring, Natural Product Research, 19, 6, 585 (2005). 11. Dr. Ravindra Sharma, Medicinal Plants of India- An Encyclopaedia, Daya Publishing House, Delhi (India), p.263 (2003). 12. A.I.M Jawad, M.I Mahmoud and Al-Naib, Fitoterapia, LIX, 3, 220 (1988). 13. C.L. Lee, P.C Huang, P.W. Hseih, T.L. Hwang, Yu-Yihau, F.R. Chang, Y.C.Wu, Planta Med, 74, 1276 (2008). 14. Horace G. Gutler and Richard J. Cole, Journal of Natural Products, 46, 5, 609 (1983). 15. F.L.Hsu, Y.C. Chen and J.T Cheng, Planta Medica, 66, 228 (2000). 16. T. Han, H.L. Li, Q.Y. Zhang, P.Han, H.C. Zheng, K. Rehman, and L.P. Qin, Phytomedicine, 14, 825 (2007). 17. J.W. Ahn, Z. NO, S.Y. Ryu, O.P.Zee and S.K. Kim, Natural Product Sciences, 1, 1, 1 (1995). 18. Y.S. Kim, J.S. Kim, S.H. Park, S.U Choi, C.O.Lee, S.K. Kim, Y.K. Kim, S.H. Kim and S.Y.Ryu, Letter. . . Planta Med, 69, 375 (2003). 19. N.T.UI chen ko and A.I Glushen Kova, Chemistry of Natural Compounds, 36, 2, 128 (2000). 20. Sankar Kumar Maitra, B.N. Chatterjee, Debi Chakravarti and B.C Maiti, J. Indian Chem. Soc., 83, 513 (2006). 21. Ahmed A. Mahmoud, Planta Medica, 64, 724 (1998). 22. V.K. Saxena and Manju Mishra, Fitoterapia, LXVI, 2, 159 (1995). 23. Viqar ud din Ahmed, Atta-ur-Rehman, Hand Book of Natural Product Data, Pentecyclic Triterpeniods, Elsevier Science B.V., The Netherland, Vol. 2, p. 1134 (1994). 24. Sunil K.T., K.M. Shrestha, M.K. Pal, A. Basak and B. Tala patra,

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Phytochemistry, 28, 12, 3437 (1989). 25. Pateh etal, Nig Journ. Pharm. Sci., 8, 01, 9 (2009). 26. Sankar Kumar, B.N. Chatterjee and B.C. Maiti, J. Indian Chem. Soc., 83, 513 (2006). 27. S.G. Wyllie and B.A. Amos, J.Org. Chem., 42, 4, 725 (1977). 28. S. Ferheen, E. Ahmed, N. Afza and A. Malik, J. Chem. Soc. Pak., 27, 2, 219 (2005). 29. S. Gupta, Mohd. Ali, M.S. Alam, M. Niwa and and T. Sakai, Phytochemistry, 31, 7, 2558 (1992). 30. Blaise, Fairness, M and Soulier, J. Am. Oil Chem. Soc., 74 727, (1997).

Source of support: Nil, Conflict of interest: None Declared

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