Varadarassou Mouttaya Mounnissamy et al. / Journal of Pharmacy Research 2011,4(1),237-240 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Phytochemical investigation of Cansjera rheedii J.Gmelin () Varadarassou Mouttaya Mounnissamy1*, Subramanian Kavimani1, Sabarimuthu Darlin Quine2, Kuppuswamy Subramani3 1College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences (MTPG & RIHS), (A Govt. of Puducherry Institution), Indira Nagar, Gorimedu, Puducherry-605 006, . 2School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudhram, Thanjavour-613 412, Tamil Nadu, India. 3Department of Chemistry, Kanchi Mamunivar Centre for Post-Graduate Studies (KMCPGS), Lawspet, Puducherry-605 008, India. Received on: 15-09-2010; Revised on: 15-10-2010; Accepted on:13-12-2010

ABSTRACT A Phytochemical study of an ethanol extract from the aerial parts of Cansjera rheedii J.Gmelin(Opiliaceae), a traditional Indian medicinal , led to the isolation of 5 compounds, including a mixture of 2 phenyl propenoic acid derivatives and 3 flavonols glycosides: 3, 4-dihydroxy cinnamic acid (1), 4-hydroxy 3-methoxy cinnamic acid (2), 3, 5, 7, 3’, 4’-pentahydroxy flavone (quercetin) (3), 5, 7, 3’, 4’-tetrahydroxy -3-O-ß-D-glucopyranosyl flavones (quercetin-3-O-ß-glucoside) (4) and 5, 7, 3’, 4’-tetrahydroxy-3-O-(6-O-a-L-rhamnopyanosyl)-ß-D-glucopyranosyl flavone (quercetin-3-O-ß-rutinoside) (5). Structures were established by spectroscopic and chemical method. This is the first time that 5 compounds have been isolated and identified from this plant.

Key words: Cansjera rheedii, Opiliaceae, Flavonoids, quercetin, quercetin-3-O-ß-D- glucoside, quercetin-3-O-ß-D-rutinoside.

INTRODUCTION

Cansjera rheedii J Gmelin (Opiliaceae) is a climbing shrub, sometimes armed, Extraction and isolation generally found in India through Malaya to Hong Kong and North [1-2]. The air dried and coarsely powdered aerial parts (1.0Kg) were extracted with The tribes of Nilgiris in Tamil Nadu, India using the plant extract for the treat- boiling 95% ethanol (3 X 5l) and the extract was concentrated to about 250 ml. ment of post-natal pain [3], intermittent fever [4] and poisonous bites and skin The insoluble green residue was removed by filtration and the soluble in the filtrate diseases [5]. In our earlier studies, the ethanol extract of aerial parts of C.rheedii (150 ml) were fractioned into C6H6, Et2O, EtOAc and EtCOMe. The C6H6 frac- has been reported to have hepatoprotective [6], cytotoxic [7], anthelmintic [8], tion after concentration yielded a pale yellow needle, recrystallized from MeOH anti-inflammatory and membrane stabilizing property [9], antipyretic [10], anti- and designated as compound 1(910mg). The Et2O concentrate was column nociceptive [11] and diuretic [12] activities. The preliminary phytochemical chromatographed over sephadex LH-20 using MeOH. 35 fractions of 50ml each screening reveals the presence of flavonoids, Phenolic and aromatic acids [13]. In were collected. Fraction 4-29 gave colourless needles, recrystallized from MeOH the absence of any report on a systematic examination of this plant and in and designated as compound 2(800mg). The EtOAc concentrate was column continuation of our studies on the flavonoids of Indian medicinal [14-15], chromatographed over Sephadex LH-20 using MeOH. 44 fractions of 50ml each the aerial parts of C.rheedii were investigated for polyphenolics and the results where collected, fractions 7-32 gave yellow needles, recrystallized with MeOH and leading to the isolation and characterization of 3, 4-dihydroxy cinnamic acid (1), designated as Compound-3(1.1g). The EtCOMe concentrate was chromatographed 4-hydroxy 3-methoxy cinnamic acid (2), 3, 5, 7, 3’, 4’-pentahydroxy flavone on a column of Sephadex LH-20 using MeOH as eluent. 107 fractions of 50ml (quercetin) (3) 5, 7, 3’, 4’-tetrahydroxy -3-O-ß-D-glucopyranosyl flavones (quer- each were collected, fractions 6-35 deposited a homogenous yellow solid recrys- cetin-3-O-ß-D-glucoside) (4) and 5, 7, 3’, 4’-tetrahydroxy-3-O-(6-O-a-L- tallized from MeOH and designated as compound-4 (89mg). Fractions 36-98 gave rhamnopyanosyl)-ß-D-glucopyranosyl flavone (quercetin-3-O-ß-D-rutinoside) (5) a pale yellow homogenous solid, recrystallized from MeOH and were designated as are here presented. This is the first time that these compounds are reported from compound-5(530mg). the plant. 3, 4-dihydroxy cinnamic acid (1) EXPERIMENTAL Pale yellow needles; mp. 210.5°C gave brisk effervescence with saturated NaHCO3 General experimental procedures 3+ solution [16], light blue with Fe and decolourised Br2 water. Blue under UV and 1D and 2D NMR spectra were recorded on a JEOL 600 MHZ spectrometer, che- deep blue under UV/NH . UV l (MeOH): 240, 324 nm; (+ NaOMe) 250, 302sh, mical shifts (ppm) are related to (CH ) Si as TMS as internal standard. Optical 3 max 3 4 343 nm; (+ CH3COONa) 244, 295sh, 327 nm; (+CH3COONa /H3BO4) 244, 298sh, rotations were determined on a JASCO P-1020 Polarimeter in MeOH. Elemental 326 nm; (+AlCl ) 246, 305sh, 341nm; (+AlCl /HCl) 244, 300sh, 328 nm; 1H analysis by CHNSO Analyser (Thermofinnigan -Flash EA 1112 series). IR spectra 3 3 NMR (500MHz), DMSO-d6: d 7.38 (d, J=16.0 Hz, 1H, H-a); d 6.99 (d, J=2.3 Hz, were recorded on a Perkin-Elmer FTIR spectrometer. UV-Visible spectropho- 1H, H-2); d 6.92 (dd, J=8.4 & 2.3 Hz, 1H, H-6); d 6.72 (d, J=8.4 Hz, 1H, H-5); d tometer (Shimadzu-UV-2500PC series) of each compound was determined in MeOH 13 6.15 (d, J=16.05 Hz, 1H, H-ß). C NMR (500MHz), DMSO-d6: d 168.51(s, and after addition of different shift reagents such as AlCl3, AlCl3/HCl, CH3COONa, =C=O); d148.64 (s, C-ß); d146.05 (C-3); d145.16 (C-4); d126.19 (C-1); d 121.79 CH3COONa/H3 BO4 and NaOMe at 190-500nm. Mass spectra were recorded on (C-6); d 116.25 (C-5); d115.59 (C-2); dd 115.0 (C-a). MS (-ve) observed m/z: 179 GCMS-Celuras-500 (Perkin-Elmer). Melting point determinations by Differen- + (M , 100) calculated for C9H8O4; 180 (M+H); 178 (M-H); IR (gmax, KBr): 3427, tial Scanning Calorimeter (DSC-60) (Shimadzu Co., Japan). Open column chro- 2558, 1654, 1605, 1534, 1449, 1283, 1217, 1176, 1110, 969, 901, 851, 809 matography was carried out on Sephadex LH-20 (Amersham Pharmacia Biotech cm-1. Elemental analysis (%): C (60.69): H (4.24): O (0.33). Co., UK) as packing material and Whatmann No.1 filter paper and TLC-Silica gel 60 F sheets( Merck Co., Germany) 254 4-hydroxy 3-methoxy cinnamic acid (2)

Plant material Colourless needles, mp. 210.8°C gave effervescence with NaHCO3 solution, 3+ The aerial parts of the plant C.rheedii (Opiliaceae) were collected from Auroville, decolourized Br2 water and green colour with Fe [16]. It was blue under UV Puducherry in June 2006 and it was identified and authenticated by Auro Her- changing to bright blue under UV/NH3; UV lmax(MeOH): 233, 296, 319nm; barium Sakthi Botanical Survey Department, Auroville, India . A Voucher speci- (+NaOMe): 234, 304sh, 346; (+ CH3COONa): 227, 284sh, 323nm; (+CH3COONa / H BO ): 224, 296sh, 322nm; (+AlCl ):237, 302sh, 331nm; (+AlCl /HCl): 234, men has been kept in our laboratory for future reference (VS-12). 3 4 1 3 3 297sh, 323nm; H NMR (500MHz), DMSO-d6; d 7.46 (d, J=16.0 Hz, 1H, H-a); *Corresponding author. d 7.24 (d, J=1.55 Hz, 1H, H-2); d 7.05 (dd, J=1.55 & 1.50 Hz, 1H, H-6); d 6.76 (d, 13 Research Schloar, J=8.4 Hz, 1H, H-5); d 6.34 (d, J=16.05 Hz, 1H, H-ß). C NMR (500MHz), SASTRA University, Thanjavour, T.N. DMSO-d6; d 168.55 (C-9); d 148.42 (C-7); d 149.59 (C-3); d145.05 (C-4); d Tel.: +91-9442288084 126.28 (C-1); d 123.37 (C-6); d 116.12 (C-5); d111.60 (C-2); d116.01 (C-8); d E-mail: [email protected] Journal of Pharmacy Research Vol.4.Issue 1. January 2010 237-240 Varadarassou Mouttaya Mounnissamy et al. / Journal of Pharmacy Research 2011,4(1),237-240

56.18 (C-10). MS(-ve): (m/z, rel. int. %) 193(M+, 100%) calculated for C10H10O4; Figure 1. 3, 4-dihydroxy cinnamic acid (Compound: 1) 194(M+H); 192 (M-H); IR (gmax, cm-1, KBr): 3436, 2903, 2841, 1686, 1609, 1514, 1424, 1277, 1173, 1116, 1033, 941, 852, 803, 749. Elemental analysis O (%): C (61.53): H (5.20): O (33.27) OH 3, 5, 7, 3’, 4’-pentahydroxy flavone : quercetin (3) HO

Yellow needles, mp.305.83°C, gave yellow colour with NH3, Na2CO3 and NaOH, pink with Mg-HCl and olive green with Fe3+[16]. It was yellow under UV and UV/ OH NH3: UV lmax (MeOH): 256, 271sh, 305sh, 373nm; (+NaoMe) 243sh, 330, 432 nm(decompose); (+CH3COONa) 266, 305sh, 358sh, 427nm; (+CH3COONa / H3BO4) 265, 299sh, 362sh, 426nm; (+AlCl3 ) 270, 302sh, 332sh, 447nm; (+AlCl3/ Figure 2 . 4-hydroxy-3-methoxy cinnamic acid (Compound: 2) 1 HCl) 265, 302sh, 359sh, 426nm; H NMR (500MHz), DMSO-d6; d 12.45 (s, 1H, 5-OH); d10.75 (s, 1H, OH-7); d 9.37 (s, 1H, OH-3); d 9.29 (s, 1H, OH-3’); d 9.57 HO (s, 1H, OH-4’); d 7.65 (d, J= 2.3 Hz, 1H, H-2’); d 7.50 (dd, J=8.3 & 2.3Hz, 1H, H- 6’); d 6.87 (d, J=3.8Hz, 1H, H-5’); d 6.38 (d, J=5..3Hz, 1H, H-8); d 6.16 (d, J=1.55 13 OH Hz, 1H, H-6). C NMR (500MHz), DMSO-d6; d 176. 35 (C-4); d 164.39 (C-7); d 161.24 (C-5); d 156.64 (C-9); d 148.21 (C-4’); d 147.30 (C-2); d 145.57(C-3’); H3CO d 136.26 (C-3); d 122.48 (C-1’); d 120. 49 (C-6’); d 116.12 (C-5’); d 115.58 (C- 2’); d 103.53 (C-10); d 98.70 (C-6); d 93.87 (C-8). MS(+ve) (m/z,rel.int %); O 303(M+, 100%), 304(M+H), 302 (M-H), 286 (M+-17), 259 (M+ -44); IR (KBr): -1 cm 3387, 2604, 1662, 1608, 1516, 1432, 1371, 1313, 1251, 1198, 1162, 1092, Figure: - 3. 3, 5, 7, 3’, 4’-pentahydroxy flavone (Compound-3) 1003, 931, 820 and 709. Acetylation and Methylation of Compound (3) gave colourless needles of quercetin penta acetate mp. 194.27°C and quercetin penta OH methyl ether, mp 151.27°C respectively. Elemental analysis(%): C (58.52): H (3.29): O (38.19). OH

5, 7, 3’, 4’-tetrahydroxy -3-O-ß-D-glucopyranosyl flavones (4) HO O Yellow powder, mp. 219.47° C gave yellow colour with alkalis, pink with Mg-HCl, Olive green with Fe3+ and positive Molisch’s test [16]. It was purple under UV and yellow under UV/NH3. UV lmax (MeOH): 256, 297sh, 356nm; (+NaOMe): 272, OH 324, 412nm ;(+CH3COONa):268,299sh, 358,402nm; (+CH3COONa /H3BO4): OH O 268,299sh, 358,402nm; (+AlCl3): 274,300sh, 425nm ;( + AlCl3/HCl): 268,298sh, 1 360,403nm. H NMR (500MHz) DMSO-d6 ; d 12.57 (s, 1H, 5-OH); d 10.56 (s, 1H, OH-7); d 9.26(s, 1H, OH-4’); d 9.12 (s, 1H, OH-3’); d 7.53 (d, J= 2.2 Hz, 1H, Figure: - 45, 7, 3’, 4’-tetrahydroxy-3-O-â-D-glucopyranosyl flavone (Com- H-2’); d 7.52 (dd, J=8.8 & 2.2Hz, 1H, H-6’); d 6.81 (d, J=8.8Hz, 1H, H-5’); d 6.37 pound: 4) (d, J=2.2Hz, 1H, H-8); d 6.16 (d, J=1.5 Hz, 1H, H-6) of aglycone; d 5.40 (d, J=8.80 Hz, 1H, H-1’’); d 3.30 (d, J=8.8 Hz, 1H, H-2’’); d 3.19 (m,4H, H-3’’, H-4’’, H-6’’ OH a); d 3.05 (m, 1H, H-5’’); d 3.71 (dd, J= 10.3 Hz & 8.8 Hz, 1H, H-6’’b) of glucose. 13 OH C NMR (500MHz) DMSO-d6; assignment based on HSQC and HMBC, d 156. 20 (s, C-2); d 133.30 (s, C-3); d 174.61 (s, C-4); d 161.70 (s, C-5); d 94.10 (d, C-6); d 164.4 (s, C-7); d 93.40 (d, C-8);d 156.60 (s, C-9); d 103.9 (s, C-10); d121.60 (s, HO O C-1’); d 115.71 (d, C-2’); d 144.70 (s, C-3’); d 149.40(s, C-4’); d 116.48 (d, C-5’); d 121.92 (d, C-6’) of aglycone. d 101.82 (d, C-1’’); d 76.58 (d, C-2’’); d 77.98 (d, C-3’’); 76.99 (d, C-4’’); 70.37 (d, C-5’’); 61.20 (t, C-6’’) of glucose. MS (+ve d d d O C H2O H + + - - and -ve), m/z 487 [M+Na] , 325 [M+Na-162] , 463 [M-H] , 301 [M-H-162] . OH O IR (KBr) cm-1: 3365 br, 2921, 1657, 1608, 1502, 1450, 1365, 1303, 1198, 1058, O H 1010, 937, 809, 722, 655 and 592. Elemental analysis (%): C (54.30): H (4.26): H H OH O (41.44). H OH O H H 5,7,3’,4’-tetrahydroxy-3-O-(6-O-a-L-rhamnopyanosyl)-ß-D glucopyrano syl flavones:Quercetin-3-O-ß-D-rutinoside (5) Figure:-5 .Quercetin -3-O-ß-D-rutinoside (Compound: 5) Yellow needles, mp.188.48°C, [a]D -2.8° (c=0.1, MeOH), gave yellow colour with alkalis, pink with Mg-HCl and greenish brown with Fe3+ and positive Molisch’s test O H [16]. It was under UV and yellow under UV/NH . UV l (MeOH): 257, 300sh, 3 max O H 360nm; (+NaoMe): 273, 320sh, 412nm; (+CH 3COONa): 268, 300sh, 364sh, 402nm;(+ CH 3COONa / H3BO4): 268, 300sh, 364sh, 401 nm; (+AlCl3) :275,303sh, 1 430 nm ;(AlCl3/HCl): 269, 303sh, 361, 402nm. H NMR (500MHz), DMSO-d6; HO O d12.53 (s, 1H, 5-OH), d 9.40(s, 1H, OH-4’); d 9.17 (s, 1H, OH-3’); d 7.51 (d, J= 9.5 Hz, 1H, H-2’); d 7.49 (dd, J=9.5 & 2.2Hz, 1H, H-6’); d 6.81 (d, J=8.5Hz, 1H, H-5’); d 6.38 (d, J=1.5Hz, 1H, H-8); d 6.16 (d, J=1.5 Hz, 1H, H-6) of aglycone; d 5.35 (d, J=3.05 Hz, 1H, H-1’’); d3.22 (d, J=12.9 Hz, 1H, H-2’’); d3.20 (m,4H, H- 3’’, H-4’’, H-6’’ a); d 3.04 (m, 1H, H-5’’); d 3.68 (dd, J= 9.9 Hz & 8.1 Hz, 1H, H- O H O 6’’b) of glucose; d 4.35 (d, J=12.9 Hz, 1H, H-1’’’); d 3.36 (d, J= 8.8 Hz, 1H, H- CH 3 2’’’); d 3.24 (m, 2H, H-3’’’, H-5’’’), d 3.04 (m, 1H, H-4’’’); d0.95 (d, J=6.12, 3H, C H O H 13 2 O H-6’’’) of rhamnose. C NMR (500MHz), DMSO-d6; assignment based on HSQC H and HMBC, d177. 89 (s, C-4); d164.69 (s, C-7); d161.73 (s, C-5); d157.14 (s, C- O O H O H 2); d156. 94 (s, C-9); d148.92 (s, C-4’); d 145.25 (s, C-3’); d 133.3(s, C-3); d H O H 121.50 (s, C-1’); d121.69 (d, C-6’); d 116. 74 (d, C-5’); d 115.74 (d, C-2’); d H O H 104.48 (s, C-10); d 99.20 (d, C-6); d 94.12 (d, C-8) of aglycone. d 101.25 (d, C- 1’’); d 76.41 (d, C-3’’); d 76.30 (d, C-4’’); d74.20 (d, C-2’’); d71.10 (d, C-5’’); d H O H O H H

67.52 (t, C-6’’) of glucose. d101.20 (d, C-1’’’); d71.80 (d, C-4’’’), d71.07 (d, C- OH H 3’’’); d69.90 (d, C-2’’’); d68.76 (d, C-5’’’); d18.10 (q, C-6’’’) of rhamnose. MS + (positive and negative) m/z; 633[M+Na] , 30), 465 (MH-rhamnose,10), 303 + + - - (aglycone +H , 25), 610 (M , 30), 609 (M-H , 100), 302 (agly, 7), 301 (agly-H , Journal of Pharmacy Research Vol.4.Issue 1. January 2010 237-240 Varadarassou Mouttaya Mounnissamy et al. / Journal of Pharmacy Research 2011,4(1),237-240

Table-1 Rf values of the isolated compounds from Cansjera rheedii Table-6 2D NMR values for compound 5 Compound Solvent system Protons (d) Correlated with carbons,

H 2O 5%OHAc 15%OHAc30%OHAc 50%OHAcBAW Seikal Phenol Forestal t-BAW 2 & / or 3 bond correlations

A 70 53 58 63 75 87 87 42 88 87 7.51(H-2’) C-2 (157.14), C-3’ (145.25) B 75 65 70 78 84 88 93 85 90 95 7.49 (H-6’) C-2’ (115.74) C 0 01 10 17 33 85 87 46 48 72 6.81 (H-5’) C-3’ (145.25) D 13 40 53 68 72 71 74 71 83 84 6.38 (H-8) C-10 (104.48), C-6 (99.20) E 32 43 51 60 64 53 63 51 76 65 6.16 (H-6) C-10 (104.48); C-8 (94.12) 5.35 (H-1’’ glu) C-3 quercetin (133.30) (Rf X 100, Whatman No.1, ascending, 28± 2°C) 4.35 (H-1’’’ rham) C-6 glu (67.52) 3.24 (H-3’’’) C-5’’’(68.76); C-1’’’(101.20) BAW : nBuOH : HOAc : H2O (4:1:5) top layer Seikal : 27% HOAc: nBuOH (1:1) 3.2 4(H-5’’’) C-3’’’(71.07) PhOH : Phenol saturated with water 3.36(H-2’’’) C-4’’’(71.80) Forestal : HOAc : H O : Conc HCl (30:10:3) 3.22 (H-2’’) C-4’’(76.30) 2 3.20 (H-3’’) C-5’’ (70.10); C-1’’ (101.20) t-BAW : tBuOH: HOAc: H2O (3:1:3) 3.20 (H-4’’) C-2’’(74.20) 3.04 (H-4’’’) C-2’’’(69.90), C-6’’’(18.10) Table-2 R Values of Sugars f HMBC (for confirmation of assignment) Sugar BAW PhOH t-BAW BEW BBPW 32). IR (KBr), cm-1; 3428, 1656, 1595, 1502, 1360, 1290, 1205, 1063, 1003, D-rutinose 12 33 36 12 18 879, 804, 725 and 528. Elemental analysis (%): C (53.11): H (4.96): O (41.93). D-glucose 18 42 41 15 20 L-rhamnose 42 60 62 42 44 RESULTS AND DISCUSSION From the ethanolic extract of the aerial parts of Cansjera rheedii (Opiliaceae) a (R X100, Whatman No.1, ascending, 28±2°C) f mixture of phenyl propenoic acids and flavonoids containing the compounds 1-5

BAW : nBuOH : HOAc : H2O (4:1:5) top layer were isolated. t-BAW : tBuOH: HOAc: H2O (3:1:3) PhOH : Phenol saturated with water Compound 1(fig-1), C9H8O4 had UV spectrum showed lmax (MeOH) 240, 324 nm. BEW : nBuOH: EtOH: H2O (4:1:1) A hypsochromic shift of 17nm in band I of AlCl3 spectrum, shows clearly that BBPW : C6H6: nBuOH: Pyridine: H2O (1:5:3:3) there is an orthodihydroxy substitution and had Rf (Table-1) typical of a phenyl Table-3 2D NMR values for compound 4 propeonic acid. Acetylation of this compound yielded a diacetate mp. 201.56°C. 1H NMR [17] spectrum gave signals for 1, 3, 4-tri substituted benzene derivative Protons Correlated Assignment at d 6.99 (d, J=2.3 Hz, 1H, H-2); d 6.92 (dd, J=8.4Hz & 2.3 Hz, 1H, H-6); d 6.72 chemical shift (d) Carbon (d, J=8.4 Hz, 1H, H-5). The trans stereochemistry was deduced from the peaks at 7.53(H-2’) 116.48 C-5’ d 7.38 and d 6.15 (d, J=16.0 Hz). Further the 13C NMR spectrum showed one 7.52 (H-6’) 121.92 C-6’ signal for C=O at d 168.51, 2 signals for 2 carbons with OH at d 146.0(C-3) & d 6.81 (H-5’) 115.71 C-2’ 145.1(C-4) in addition to the other characteristic chemical shift for carbon at d 6.37 (H-8) 93.40 C-8 6.16 (H-6) 94.10 C-6 148.64 (C-b), d 126.19 (C-1), d121.79 (C-6), d116.25 (C-5), d115.59 (C-2) and 5.40 (H-1’’ Glu) 101.82 C-1’’ d115.0 (C-a). The Mass spectrum having molecular ion peaks at m/z 180 along 3.61 (H-6’’ ß) 61.20 C-6’’ with other characteristic peaks. It had IR bands at 3427, 1176 (Phenolic OH and 3.19 (H-3’’) 77.98 C-3’’ OH of COOH), 1654 (C=O), 1449 (C=C), 1217, 1110, 969 and 809 cm-1 (Substi- 3.19 (H-4’’) 76.99 C-4’’ 3.19 (H-2’’) 76.58 C-2’’ tuted benzene). Based on these discussions compound 1 was characterized as 3, 4- 3.05 (H-5’’) 70.37 C-5’’ dihydroxy cinnamic acid, whose identity was further confirmed by co-PC, melting 1H- 13C (HSQC) (for confirmation of assignment) Point, Mass, NMR and IR spectrum datas obtained from literature [16-18].

Table-4 2D NMR values for compound 4 Compound 2(fig-2), C10H10O4, had lmax (MeOH) 233, 296 & 319 nm and a hypso- chromic shift of 12nm in band I of AlCl spectrum, shows clearly that there is an Protons (d) Correlated with carbons, 3 2 & / or 3 bond correlations ortho disubstitution in benzene ring had Rf similar to hydroxy cinnamic acid (Table-1). 1H NMR spectrum [17] gave signals for 1, 3, 4-tri substituted benzene 7.53(H-2’) C-2 (156.20), C-3’ (144.7) derivative at d 7.24 (d, J=1.55 Hz, 1H, H-2); d7.05 (dd, J=1.55 & 8.4 Hz, 1H, H- 7.52 (H-6’) C-2’ (115.71) 6); d 6.76 (d, J=8.4 Hz, 1H, H-5). The Trans stereochemistry was deduced from 6.81 (H-5’) C-3’ (144.7) the peaks at d 7.46 and d 6.34 (d, J=16.0 Hz). Further the 13 C NMR spectrum of 6.37 (H-8) C-10 (103.9), C-6 (94.1) 6.16 (H-6) C-10 (103.9); C-8 (93.4) compound-B showed one signal at d 168.55 (C=O), one signal for OH at d 145.05(C- 5.40 (H-1’’ glu) C-3 quercetin (133.3) 4), one signal at d149.59 (-OCH3), in addition to the other characteristic chemical 3.30 (H-2’’) C-4’’(76.99) shift for carbon at d 148.42 (C-b), d126.28 (C-1), d123.37 (C-6), d111.60 (C-2), 3.19 (H-3’’) C-5’’ (70.37); C-1’’ (101.8) d116.12 (C-5) and d116.01 (C-a). The Mass spectrum having molecular ion 3.19(H-4’’) C-2’’(76.8) peaks at m/z 194 along with other characteristic peaks. Compound (2) had IR Table-5 2D NMR values for compound 5 absorption frequencies at 3436, 1173 (Phenolic OH and OH of COOH), 1686 (C=O), 1609 (C=C), 1277 and 1116 (-OCH ) cm-1.Thus, compound (2) was Protons Correlated Assignment 3 chemical shift (d) Carbon identified as 4-hydroxy-3-methoxy cinnamic acid. The identity was further confirmed by co-PC, melting point, Mass, NMR and IR Spectrum datas obtained 7.51(H-2’) 116.74 C-5’ 7.49 (H-6’) 121.69 C-6’ from literature[16-18]. 6.81 (H-5’) 115.74 C-2’ 6.38 (H-8) 94.12 C-8 Compound 3(fig-3), C H O had R characteristic of flavonoid aglycone (Table- 6.16 (H-6) 99.20 C-6 15 10 7, f 5.35 (H-1’’ Glu) 101.2 C-1’’ 1)[16] and ?max (MeOH) 256, 271sh, 305sh, and 373 nm. A Characteristic 4.35 (H-1’’’ rham) 101.2 C-1’’’ bathochromic shift in band II of NaOAC spectrum with decomposition of band I 3.68 (H-6’’ ß) 67.52 C-6’’ 3.36 (H-2’’’) 69.90 C-2’’’ suggested free 3’, 7 and 4’ –OH groups. A bathochromic shift in band II of NaOAC 3.24 (H-3’’’) 71.07 C-3’’’ spectrum (10nm) suggested free –OH at C-7. A bathochromic shift in NaOH 3.24 (H-5’’’) 68.76 C-5’’’ spectrum with fast decomposition showed the presence of 3’, 4’-OH. A 3.20 (H-6’’ a) 67.52 C-6’’ 3.20 (H-3’’) 76.41 C-3’’ bathochromic shift of 53nm in band I of AlCl3 / HCl spectrum was indicative of the 3.20 (H-4’’) 76.30 C-4’’ presence of 3 and / or 5-OH groups. A hypsochromic shift of 21nm in band I of 3.22 (H-2’’) 74.20 C-2’’ AlCl /HCl spectrum compared to AlCl spectrum indicated orthodihydroxy in ring 3.04 (H-4’’’) 71.80 C-4’’’ 3 3 3.04(H-5’’) 70.10 C-5’’ B, which was further supported by 53 nm bathochromic shift of band I in 0.95 (H-6’’’) 18.10 C-6’’’ 1 CH3COONA/ H3BO4 spectrum. Further the H NMR spectrum showed signal for five (5, 7, 4’, 3 and 3’) OH protons at d12.45, d 10.75, d 9.57, d 9.37 and d 9.29 1H- 13C (HSQC) (for confirmation of assignment) Journal of Pharmacy Research Vol.4.Issue 1. January 2010 237-240 Varadarassou Mouttaya Mounnissamy et al. / Journal of Pharmacy Research 2011,4(1),237-240 ppm besides giving the characteristic -chemical shift and splitting pattern ex- vone. The position of inter linkage can be made by 1H and mainly 13 C NMR have pected for the five (2’, 6’, 5’,8 and 6) aromatic protons at d7.65, d 7.50 d 6.87, been employed to characterize the sugars. Thus the 1H NMR spectrum exhibited d 6.38 and d 6.16ppm . Further the 13C NMR spectrum showed signals for five (C- a doublet at d 5.35 (H-1’’, J=3.0 Hz) and a broad multiplicate at d 0.95 (H-6’’’, 7, C-5, C-4’, C-3’and C-3) carbons with OH at d 164.39, d 161.24, d 147.30, d J=6.1Hz) clearly indicated the presence of rhamnose and its attachment to C-6 of 145.57 & d136.26 ppm and C=O carbon at d 176.35 ppm in addition to the other glucose. The site at which a second sugar attached to the sugar of a flavonoid characteristic chemical shift for carbon at d156.60 (C-9), d121.92 (C-6’), d mono-O-glucoside is readily determined by 13C NMR spectroscopy and this is 121.60 (C-1’), d 116.48 (C- 5’), d 115.71 (C-2’), d 103.9 (C-10), d 94.10 (C-6) perhaps the most significant information contained in the spectrum, as it is and d 93.40 (C-8). The compound had characteristic IR absorption frequencies at difficult to obtain it by other methods. A sizable downfield shift of d 5.9 ppm in 3387 (Phenolic-OH), 1662 (C=O), 1608 (C=C). The compound yielded a penta the resonance of C-6 of glucose carbon and up field shift of d 0.5 ppm in the acetyl derivative with mp.194.27°C and penta methyl ether mp.151.27 °C agree- resonance of C-5 of glucose carbon and without affecting the rest of the spectrum ing with the values reported. These properties led to the identification of the compared to quercetin-3-O-b-D-glucoside clearly indicated that terminal sugar compound (3) as 3, 5, 7, 3’, 4’- pentahydroxy flavone (quercetin). The identity rhamnose is attached to C-6 of glucose. This fact was further proved beyond was further confirmed by co-PC, Mass, NMR and IR Spectrum data obtained from doubt on the basis of HMBC & HSQC data (Table-5&6) connecting rhamnose H- literature [17-21]. 1 (d 4.35) to C-6 of glucose (67.52 ppm). Similarly, connectivity between H-1 of glucose (d 5.35 with C-3 of quercetin (133.81 ppm) was also established. Thus the Compound 4(fig-4), C21H20O12had the characteristic Rf (Table-1) and UV lmax structure of compound (5) was established as 5, 7, 3’, 4’-tetrahydroxy 3-O-(6-O- (MeOH):256, 297sh, 360nm. Acidhydrolysis [22] yielded an aglycone identified a-L-rhamnopyranosyl ) –b –D- glucopyranosyl flavone ( or) Quercetin-3-O-b- as quercetin and a sugar identified as D-glucose.The above two products are iden- D-rutinoside. The identity was further confirmed by co-PC, Mass, NMR and IR tified by co-PC with authentic sample of glucose (Table-2).This suggested com- Spectrum data obtained from literature [24-27] pound (4) to be a glucoside of quercetin. Purple fluorescence of the glycoside under UV compared to yellow of its aglycone indicated the involvement of 3-OH in ACKNOWLEDGEMENTS glycosylation. A bathochromic shift of 12 nm in band II of CH3COONa spectrum The authors are greatful to Dr.K.V.Raman, Dean, MTPG & RIHS, Puducherry for compared to MeOH spectrum revealed the presence of free 7-OH and a provide facilities to carry out the experiment and IIT-Madras for NMR spectra, bathochromic shift of 56nm shift in band I in NaOH spectrum indicated the Mr. C. Iyyappan for LCMS and Mr. T. Vasudevan of M/s. Shashan drugs(Formulation presence of free 4’-OH. A bathochromic shift of 47 nm in band I of AlCl3/HCl division), Puducherry. spectrum compared to MeOH spectrum indicated free 5-OH. A hypsochromic shift of 23nm in band I of AlCl3/HCl spectrum compared with AlCl3 spectrum REFERENCES indicated the presence of ortho dihydroxyring in ring B. The presence of free 1. Gamble JS, Flora of the Presidency of Madras. In: Botanical Survey of India, Calcutta, Vol.I, 1981; 5,7, 3’ and 4’ were further confirmed by 1H and 13C NMR data. Further 1H NMR pp. 137-38. 2. Matthew KM, An Excursion flora of Central Tamil Nadu, India, Oxford and IBH Publications, New spectrum showed a characteristic signal for anomeric proton at d 5.40 ppm (d, Delhi, 1991; 647. J=7.6 Hz) showing b-glycosidic link and the site of attachment at 3-OH. This was 3. Ravikumar K, Vijayashankar R, Ethanobotanyof Malayali Tribes in Melpattu village, Javvadhu further confirmed by 13C NMR signals for anomeric carbon appeared at d 101.82 Hills of Eastern Ghats, Thiruvannamalai District, Tamil Nadu. J Econ Taxo Bot 2003; 27:715-26. 1 4. Hosagoudar VB, Henry AN, Ethanobotany of tribes irular, Kurumban and pariyan of Nilgiris in (glu-C-1) ppm and for C-3 at d 133.30 ppm respectively. The H NMR peaks at Tamil Nadu, Southern India, J. Econ. Taxon. Bot, 1996, 12, 272-283. d 12.57, d 10.56, d 9.12 and d 9.26 suggested the presence of free 5, 7, 3’ and 4’- 5. Ayyanar M, Ignacimuthu S, Medicinal plants used by the tribals of Tirunelveli hills, Tamil Nadu 13 to treat poisonous bites and skin diseases. Indian Journal of Traditional Knowledgev2005; 4(3): 229- OH. This was further confirmed by the appearance of C NMR peaks at d 164.4 236. ppm for C-7, d 161.7 ppm for C-5, d 144.7ppm for C-3’ and d 149.4ppm for C- 6. Mounnissamy VM, Kavimani S, Balu V, Darline Quine S, Effect of ethanol extract of Cansjera 4’. The presence of H-6, H-8, H-2’, 5’ and 6’ was also confirmed unamguiously rheedii J.Gmelin (Opiliaceae) on Hepatotoxicity, J. Pharmacol Toxicol., 2008, 3(2), 158-162. 1 7. Mounnissamy VM, Kavimani S, Balu V, Darlin Quine s, Cytotoxic effect of various extracts of from H NMR by characteristic peaks at d 6.16, d 6.37, d 7.53, d 6.81 & d Cansjera rheedii J. Gmelin (Opiliaceae) on human cancer cell lines, Amala Res. Bulletin, 2007, 7.52ppm. The anomeric proton of the sugar appeared at d 5.40 in 1H NMR and 27, 252-253. the anomeric carbon appeared at d 101.82 ppm in 13C NMR was in close agree- 8. Mounnissamy VM, Kavimani S, Balu V, Darlin Quine S, Anthelminthic activity of Cansjera rheedii J. Gmelin (Opiliaceae), J. Biol. Sci, 2008, 8(4), 831-833. ment with expected values for Quercetin-3-O-glucoside. Further the molecular 9. Mounnissamy VM, Kavimani S, Balu V, Darlin Quine S, Evaluation of anti-inflammatory and ion peak at m/z 464(M+Na+, 100) was in agreement with the molecular formula membrane stabilizing properties of ethanol extract of Cansjera rheedii J. Gmelin (Opiliaceae), Iranian J. Pharmacol. Therapeutics, 2007, 6, 235-237. C21H20O12. The other characteristic peaks appeared are at m/z 301 supports the 10. Mounnissamy VM, Kavimani S, Balu V, Darlin Quine S, Antipyretic activity of ethanol extract of above structure. This fact was further proved beyond dought on the basis of Cansjera rheedii J. Gmelin (Opiliaceae), J. Pharmacol. Toxicol, 2008, 3(5), 378-381. HMBC and HSQC datas (Table-3&4) connecting glucose H-1 (d 5.40ppm) to C- 11. Varadarassou M. Mounnissamy, Subramanian Kavimani, Vaithialingam Balu, Gnanapragasam Sankari, Sabarimuthu D. Quine, Anti-nociceptive activity of Cansjera rheedii J. Gmelin (Opiliaceae), 3 of quercetin (d 133.30 ppm) was also established. Thus compound D was Maejo Int. J. Sci. Technol., 2009, 3(02),306-312. identified as 5, 7, 3’, 4’-tetrahydroxy-3-O-b-D-glucopyranosyl flavone. [21-24]. 12. Varadarassou Mouttaya Mounnissamy, Subramanian Kavimani, Gnanapragasam Sankari, S. Dhayalamurthi, Sabarimuthu Darlin Quine, K. Subramani, Effect of Cansjera rheedii J. Gmelin Compound 5(fig-5), C27H30O16 had UV lmax (MeOH) 257, 300sh, 359 nm and Rf (Opiliaceae) on diuretic activity in rats, J. Pharm. Res., 2009, 2(10),1627-1628. (Table-1) typical of flavonol glycoside. The compound on acid hydrolysis [22] 13. Mounnissamy VM, Kavimani S, Balu V, Darlin Quine S, Preliminary Phytochemical screening with 2N HCl yielded an aglycone and two different monosaccharides in of Cansjera rheedii J. Gmelin (Opiliaceae), Int. J. Pharmacol. Biol. Sci., 2008, 2(3), 157-160. 14. Mounnissamy VM, Kavimani S, Gunasegaran R, Saraswathi A, Anti-inflammatory activity of equimolecular ratio. The aglycone was found to be identical with compound (3) Gossypetin isolated from Hibiscus sabdariffa, Ind. J. Heterocyclic Chem., 2002, 12, 85-86. 15. Mounnissamy VM, Kavimani S, Gunasegaran R, Saraswathi A, Diuretic activity of Gossypetin and sugars as D-glucose and L-rhamnose by Rf values and co-PC with authentic samples(Table-2). Glycosylation of 3-OH was inferred by the different UV fluo- Isolated from Hibiscus sabdariffa in rats, Hamdard Medicus, 2002, XLV(2),68-70. 16. Harborne JB, Phytochemical methods, London Chapman and Hall, 1973. rescence of the glycoside (Purple) from the aglycone (Yellow). The presence of 17. Martin GE, Zektzer AS, Two-dimentional NMR methods for establishing molecular connectivity. free 7-OH was indicated by the bathochromic shift of 11nm in band II of New York: VCH, 1988. 18. Durust N, Ozden S, Umur E, Durust Y, Kucukislamoglu M, The isolation of Carboxylic acids from CH3COONa spectrum compared to MeOH spectrum. A bathochromic shift of 70 the flowers of Delphinium formosum, Turk.J. Chem.,2001, 25, 93-97. nm in band I of AlCl3 spectrum relative to the MeOH spectrum indicated the 19. Jia LY, Sun QS, Huang SW, Isolation and identification of flavonoids from Chrysanthemum presence of free 5-OH. A bathochromic shift of 41 nm in band I of CH COONa moriflolium ramat, Chin. J. Med. Chem., 2003, 13, 159-161. 3 20. Li X, Shi RB, Liu B, Chen YP, Study on chemical components from effective fraction of /H 3BO4 revealed the presence of ortho dihydroxyl groups in ring B. Further Qingnaoxuanqiao Formula (II), J.Beijing Univ. Trad. Chin. Med., 2006, 29, 545-550. evidence of free 3’ and 4’-OH was obtained from a hypsochromic shift of 28nm 21. Yu-Lan Li, Jun Li, Nai-Li Wang, Xin-Sheng Yao, Flavonoids and a new polyacetylene from Bidens parviflora Willd, Molecules, 2008, 13, 1931-1941. in band I of AlCl3/HCl relative to AlCl3 spectrum. On H2O2 oxidation of compound 22. Lawrence O. Arot Manguro, Ivar Ugi, Peter Lemmen, Rudolf Hermann, Flavonol glycosides of (5) [20] yielded a disaccharide, identified by Rf (Table-2). It was not affected by Warburgia Ugandensis leaves, Phytochemistry, 2003, 64, 891-896. enzyme ß-glucosidase indicating that glucose was not the terminal sugar. Mass 23. Wang JH, Wang YL, Luo FC, Study on the chemical constituents from the seeds of Sophora + Japonica, Chin. Trad. Herb. Drugs, 2001, 32, 471-473. spectrum (positive and negative) showed peaks at 634 (MH + Na , 30), 633 (M + 24. Harborne, JB, The Flavonoids: Advances in Research since 1986; Chapman & Hall: London, UK, + + + + 1994. Na , 100), 611 (MH , 45), 465 (MH -rhamnose, 10), 303 (MH -rutinose, 25) corresponding to a molecular formula C H O and other fragment ions charac- 25. Agarwal PK, In Agarwal PK (Ed), Studies in Organic Chemistry, Carbon-13 of Flavonoids, 27 30 16 Elsevier, Amsterdam, 1989. teristic of the attachment of rhamnosyl-glucose residue to the aglycone quercetin. 26. Fattemeh Fathiazad, Abbas Delazar, Roya Amiri, Satyajit D. Sarker, Extraction of flavonoids and The 1H NMR spectrum exhibited signals corresponding to aromatic protons at 6, Quantification of Rutin from waste tobacco leaves, Iran. J. Pharma. Res., 2006, 3, 222-227. 27. Gunasegaran R, Subramani K, Azantha Parimala P, Ramachandran Nair AG, Rodriguez B, 8, 2’, 5’ and 6’ and OH groups at 5, 7, 3’ and 4’. The characteristic signals in the Madhusudanan KP, 7-O-(6-O-Benzoyl –ß-D-glucopyranosyl)-rutin from Canthium dicoccum, aliphatic region were assigned to the anomeric proton and other sugar protons Fitoterapia, 2001, 72, 201-205. showing the compound 5 as a diglycoside of 3,5,7,3’, 4’-penta oxygenated fla-

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Journal of Pharmacy Research Vol.4.Issue 1. January 2010 237-240