This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
Author's personal copy Fitoterapia 80 (2009) 223–225
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Humarain: A new dimeric gallic acid glycoside from Punica granatum L. bark Mudasir Ahmad Tantray a,⁎, Seema Akbar b, Reehana Khan a, Khurshid Ahmad Tariq c, Abdul Sami Shawl a a b c
Natural Product Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Sanatnagar Srinagar 190 005, India Phytochemistry Division, Regional Research Institute of Unani Medicine (CCRUM), University of Kashmir, Srinagar 190 006, India Parasitology Division, Department of Zoology, Faculty of Science, University of Kashmir, Srinagar 190 006, India
a r t i c l e
i n f o
Article history: Received 22 October 2008 Received in revised form 22 January 2009 Accepted 22 January 2009 Available online 2 February 2009
a b s t r a c t A new dimeric gallic acid glycoside named Humarain (1) was isolated from stem bark of Punica granatum. The structure of the compound was determined by spectroscopic data including 1D and 2D NMR spectral analysis. © 2009 Elsevier B.V. All rights reserved.
Keywords: Punica granatum Humarain Spectral techniques
1. Introduction Punica granatum L. (Pomegranate) is a shrub distributed originally in Afghanistan, introduced into China in second century BC [1,2]. It is uniformly distributed in the hilly areas of Kashmir. In family Punicaceae this is the only species which has prominent biological activity [3]. The aerial parts are valued as astringents in diarrhea and dysentery [4–6]. In folk medicine pomegranate preparations of the dried pericarp and the juice of the fruits are employed as an oral medication in the treatment of colic, colitis, leucorrhea, menorrhagia, oxyuriasis, paralysis and rectocele and external application to caked breast [7] and to the nape of the neck in mumps [8] and headache [9]. A number of therapeutic actions of these materials have been described including vermifugal, taenicidal, astringent, antispasmodic, antihysteric, diuretic, carminative, sudorific, galactogogue and emmenagogue [10]. Pomegranate peel is used for treating the infection of male and female sexual organs, mastitis, acne, folliculitis, piles,
⁎ Corresponding author. Tel.: +91 194 2431255; fax: +91 194 2430779. E-mail address:
[email protected] (M.A. Tantray). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.01.013
allergic dermatitis, tympanitis [11] and for the treatment of oral diseases [12]. Biological screening of Punica granatum extracts and compounds have shown antioxidant [13], antiperoxidative [14], antidiarrhoeal [15] and antibacterial [16], inflammation [17], hepatoprotective [18] effects. Punica granatum is a good source of tannins, alkaloids, glycosides, triglycerides, flavonoids, polyphenols [19–24]. 2. Experimental 2.1. General Melting point is uncorrected and was determined on BUCHI melting point apparatus. UV spectra was recorded in methanol in nm on Specard S 100. IR was recorded on a Bruker Vector 22 spectrometer as KBr pellets with absorption given in cm− 1. 1H NMR and 13C NMR run on 500 MHz Bruker Daltonics instrument using TMS as internal standard. Mass spectra was recorded by using Bruker Daltonics electrospray ionization. Column was run using silica gel (60–120 mesh), TLC was run on silica gel G and fluorescent aluminium TLC using solvents CHCl3–MeOH. Spots were visualized on TLC under UV light, ferric chloride, cerric ammonium suplhate, exposure to iodine vapour in an iodine chamber and also by heating the chromatoplates at 100 °C in an oven.
Author's personal copy 224
M.A. Tantray et al. / Fitoterapia 80 (2009) 223–225
Fig. 1. Significant 1H–13C correlations observed in HMBC spectra of 1.
2.2. Plant material Punica granatum bark was collected in September 2005 from F/S Pulwama (Kashmir), India. A voucher specimen (No. 206/05) was deposited in the hebarium of the institute and Centre of Plant Taxonomy (COPT), University of Kashmir. 2.3. Extraction and isolation The air dried and coarsely powdered (stem bark) plant material (9.3 kg) was extracted exhaustively with methanol at room temperature and the solvent was evaporated in vacuo to give a dark brown residue (325 g). The residue was chromatographed on a silica gel (60–120 mesh) column packed in hexane, eluting with a step gradient of hexane– CHCl3 (0–75% CHCl3 in hexane) followed by CHCl3 and finally with CHCl3–MeOH (0–100% MeOH in CHCl3), all fractions being monitored by TLC. The relevant fractions were combined and further chromatographed. Elution of one of the fraction with CHCl3–MeOH (4:6) gradient afford (1) as a white amorphous powder, mp 205.6 °C; UVmax (MeOH): 268.0 nm; IR bands (KBr): 3450.1, 2993.6, 1725.7, 1636.3, 1510.5,1478.5,1332.4,1182.7,1081.2, 967.5, 834.6, 756.3 cm− 1; ESI-MS: 647 [M + H]; 1H-NMR (500 MHz, CD3OD-d4) δ: 7.09 (1H, bs, H-3), 7.09 (1H, bs, H-7), 7.10 (1H, bs, H-14), 4.35 (1H, d, J 7.8 Hz, H-1′), 3.26 (1H, dd, J 8.3, 7.8 Hz, H-2′), 3.31 (1H, dd, J 9.3, 8.3 Hz, H-3′), 3.33 (1H, dd, J 9.3, 9.3 Hz, H-4'), 3.39 (1H, m, H-5′), 3.70 (1H, dd, J 11.3, 5.6 Hz, H-6′α), 4.02 (1H, bd, J 11.3 Hz, H-6′β ), 4.84 (1H, bs, H-1′′), 3.28 (1H, dd, J 8.4, 7.7 Hz, H-2′′), 3.32 (1H, dd, J 9.1, 7.8 Hz, H-3′′), 3.33 (1H, dd, J 9.3, 9.1 Hz, H-4′′), 3.49 (1H, m, H-5′′), 3.71 (1H, dd, J 11.5, 5.7 Hz, H-6′′α), 4.03 (1H, bd, J 11.5 Hz, H-6′′β); 13C-NMR (500 MHz, CD3OD-d4) δ: 168.2 (C-1), 121.6 (C-2), 110.5 (C-3), 146.5 (C-4), 139.9 (C-5), 146.5 (C-6), 110.5 (C-7), 168.2 (C-8), 121.5 (C-9), 110.6 (C-10), 146.5 (C-11), 139.9 (C-12), 146.5 (C-13), 110.5 (C-14), 103.2 (C-1′), 75.2 (C-2′), 78.1 (C-3′), 71.6 (C-4′), 76.9 (C-5′), 67.9 (C-6′), 102.2 (C-1′′), 74.5 (C-2′′), 77.2 (C-3′′), 72.2 (C-4′′), 75.8 (C-5′′), 68.5 (C-6′′). 3. Results and discussion The dried plant material was extracted with methanol, and the extract, after sequential fractionation on silica gel with CHCl3–MeOH gradient led to the isolation of (1).
The compound (1) was isolated as a white amorphous powder. The mass spectrum showed m/z at 647 [M+H], corresponding to the molecular formula C26H30O19. The λmax at 268 nm in UV spectrum revealed substituted benzene moiety. The IR showed band at 3450 cm−1 of hydroxyl functionality, 2993 cm− 1 of aromatic –C-H stretching, 1725 cm− 1 of carboxyl group as ester and 967 cm−1 of substituted benzene. 1H NMR swept at 500 MHz showed two broad singlets of two aromatic protons H-3 and H-7 each at δ 7.09 and another two broad singlets of another two aromatic protons H-10 and H-14 each at δ 7.10. The series of signals of absorbing frequencies at δ 4.35 (1H, d, J 7.8 Hz), 3.26 (1H, dd, J 8.3, 7.8 Hz), 3.31 (1H, dd, J 9.3, 7.8 Hz), 3.33 (1H, dd, J 9.3, 9.3 Hz), 3.39 (1H, m) and 3.70 (1H, dd, J 11.3, 5.6 Hz), 4.02 (1H, bd, J 11.3 Hz) are diagnostic signals of protons H-1′ to H6′ of glucose moiety attached through glycosidic linkage to C1. The another series of such type of absorption frequencies at δ 4.84 (1H, bs), 3.28 (1H, dd, J 8.4, 7.7 Hz), 3.32 (1H, dd, J 9.1, 7.8 Hz), 3.33 (1H, dd, J 9.3, 9.1 Hz), 3.41 (1H, m) and 3.71 (1H, dd, J 11.5, 5.7 Hz), 4.03 (1H, bd, J 11.5 Hz) are diagnostic signals of protons H-1′′ to H-6′′ of another glucose moiety attached through glycosidic linkage to C-8. In short disaccharide is attaching two galloyl moieties as shown in structure 1. 13C NMR (125 MHz) agrees with the C26 carbon skeleton. DEPT showed fourteen methines and two methylenes. The most downfield signals were assigned to carbonyl carbons each at δ 168.2 (C-1 and C-8). The other downfield signals were assigned to aromatic carbons which are attached to oxygen function at δ 146.5, 139.9, 146.5, 146.5, 139.9 and 146.5 of carbons C-4, C-5, C-6, C-11, C-12 and C-13 respectively. The frequencies at values δ 103.2, 75.2, 78.1, 71.6, 76.9 and 67.9 were assigned carbons C-1′, C-2′, C-3′, C-4′, C-5′ and C-6′ respectively of glucose moiety attached to carbonyl carbon (C-1) via ester linkage. Remaining frequencies absorbing at δ 102.2, 74.5, 77.2, 72.2, 75.8 and 68.5 were assigned to carbons C-1′′, C-2′′, C-3′′, C-4′′, C-5′′ and C-6′′ respectively of another glucose moiety attached to carbonyl carbon (C-8) via another ester linkage [25]. The chemical shift assignments were confirmed by HMBC, COSY and NOESY techniques. The COSY and HMBC showed connectivity of H-7 (δ 7.09) with C-1 (δ 168.2) Fig. 1. Significant COSY and HMBC connectivity is efficient of C-6 hydroxyl proton with C-5 (δ 139.9) and C-4 hydroxyl with C-3 (δ 110.5). Same type of connectivity was observed of C-13 hydroxyl proton with C-12 (δ 139.9) and C-14 (δ 110.5). The
Author's personal copy M.A. Tantray et al. / Fitoterapia 80 (2009) 223–225
HMBC correlation is being efficiently observed C-2′ hydroxyl proton with C-1′ (δ 103.2) and C-4′ hydroxyl proton with C-5′ (δ 76.9). C-2′′ and C-6′′ hydroxyl protons HMBC correlation is efficiently observed from another sugar moiety with C-1′′ (δ 102.2) and C-4′′ (δ 72.2) respectively. Based on the above data supported by 2D experiments the structure 1 was assigned to the compound and was named Humarain Fig. 1. References [1] Flora Republicae Popularis Sinicae, Tomus 52. Beijing: Science Press; 1983. p. 120. [2] The flora of Chinese drugs, vol. 3. Beijing: Peoples Health Press; 1961. p. 288. [3] Dar GH, Khroo AA, Khan ZS, Dar AR. J Himalayan Ecol Sustain Dev 2007;2:13–9. [4] Sathyavati GV, Gupta AK, Neeraj T. Medicinal plants of India: New Delhi Indian Council of Medicinal Research; 1987. p. 540. [5] Prashanth D, Asha MK, Amit A. Fitoterapia 2001;72:171. [6] Bruni A, Nicoletti M. Fitoterapia 2003;74:717. [7] Duke AJ, Ayensu SE. Medicinal plants of China. Algonac, MI: Reference Publications; 1985. [8] Boulos L. Medicinal plants of North Africa. Algonac, MI: Reference Publications; 1983.
225
[9] Ayensu SE. Medicinal plants of West Indies. Algonac, MI: Reference Publications; 1981. [10] Bianchini F, Corbeta F. Health plants of the world. New York: Newsweek; 1979. [11] Hu W. Chinese patent, 1997: 1156617A. [12] Fengehun H, Liu X. Chinese patent, 1997:1145793A. [13] Ricci D, Giamperi L, Bucchini A, Fraternale D. Fitoterapia 2006;77:310. [14] Sudheesh S, Vijayalakshmi NR. Fitoterapia 2005;76:181. [15] Das AK, Mandal SC, Banerjee SK, Sinha S, Das J, Saha BP, Pal M. J Ethnopharmacol 1999;68(1–3):1205. [16] Prashant D, Asha MK, Amit A. Fitoterapia 2001;72:171. [17] Lansky EP, Newman RA. J Ethnopharmacol 2007;109(2):177. [18] Celik I, Temur A, Ismail I, Ali M, Aeri V, Bhowmik M, Sultana S. Food Chem Toxicol 2009;47(1):145. [19] Ziyyad A, Legssyer A, Mekhfilt A, Dassouli A, Serhrouchni M, Benjelloun WJ. J Ethnopharmacol 1997;58:45. [20] Sahar AM, Hussein H, Barakat H, Merfort I, Mahmoud A, Nawwar M. Phytochemistry 1997;45(4):819. [21] Yusuph M, Mann J. Phytochemistry 1997;45(7):1391. [22] Mahmoud A, Nawwar M, Sahar AM, Hussein H, Merfort I. Phytochemistry 1994;37(4):1175. [23] Wang R, Wang W, Wang L, Liu R, Ding Y, Du L. Fitoterapia 2006;77:534. [24] Tanaka T, Nonaka G, Nishioka I. Phytochemistry 1985;24(9):2075. [25] Breitmair E, Voelter W. 13C NMR spectroscopy: methods and application in organic chemistry. New York: Verlag Chemie Weinheim; 1978. p. 250.
