Salicylic Acid and Methyl Gallate from the Roots of Conyza Canedensis

Salicylic acid and Methyl gallate from the roots of Conyza canedensis

Javid A Banday*1, FA Mir2, Saleem Farooq3, Mushtaq A Qurishi1, Surrinder Koul3 and TK Razdan4 1Department of Chemistry, University of Kashmir, Hazratbal, Srinagar-190006, J&K, India 2University Science Instrumentation Centre (USIC), University of Kashmir, Srinagar-190006, J&K, India 3Bio-organic Division, Indian Institute of Integrative Medicine(CSIR), Canal Road, Jammu, J&K, India 4Department of Chemistry, University of Jammu, Ambedkar Road, Jammu, J&K, India

From the root part of Conyza canedensis (Astereacea), Salicylic acid (2-Hydroxybenzoic acid) (1) and Methyl gallate (Methyl- 3,4,5-trihydroxybenzoate) (2) were isolated for the first time, along with five known compounds (3-7). The structures of all these compounds were elucidated on the basis of their spectral data.

Keywords: Conyza canedensis, Astereacea, Salicylic acid, Methyl gallate.

INTRODUCTION

Genus Conyza (Family Astereaceae), consists of more than 70 Plant Material: The root part of Conyza canedensis (5.4 Kg) species and is an annual or perennial plant. The plants are were collected from Hazratbal, Srinagar (J&K, India) ) in June mainly distributed in tropical and sub-tropical regions. The 2007. The specimen was identified by Akhtar H. Malik, species Conyza canedensis Linn. is native to North America Curator, Centre for Biodiversity & Taxanomy, University of and is distributed in almost all parts of the world. In India, Kashmir (Specimen deposited under accession No. 33214 and Conyza canedensis is widespread in northern areas. The whole Collection No. 1202- Javid, Kash). plant is locally used for the treatment of edema, hematuria, hepatitis and cholecystitis[1]. Extraction and isolation: The air dried, finely powdered root material (1Kg) was extracted for 72 hours sequentially with Earlier, aerial parts of Conyza canedensis have been shown to petroleum ether (60-80oC), ethyl acetate and methanol in a possess sphingolipids[2,3], phenolic acids[3], steroids and soxhlet apparatus to afford the respective extracts, which triterpenoids[3,4], acetylenes, 16β,20β-dihydroxytaraxastane-3- were concentrated under reduced pressure. The methanol β-O-palmitate, 8R,9R-dihydroxymatricarine methyl ester [4], extract (06 g) was charged on silica gel (60-120 mesh) column phenyl esters[5], β-sitosterol, α-spinasterol[6], stigmasterol[7], and eluted with a gradient of ethyl acetate and methanol matricarine methyl ester, matricane lactone[8], friedelin[9] (19:1, 9:1, 4:1, 7:3). Four major fractions were collected. and friedelinol[10], etc. Since, the chemistry of the roots of the plant seemed not to have been worked out, the present The ethyl acetate-methanol fraction (19:1, 0.1g) showed one investigation was undertaken to determine the components of major spot on TLC with some impurities in traces. It was the roots of the plant. In this paper we report the isolation and purified with column chromatography using silica gel (60- structural elucidation, by extensive spectral methods, of two 120 mesh) as adsorbent and eluted with chloroform-methanol aromatic compounds, Salicylic acid (1) and Methyl gallate (2) (11.5:1) to give compound (3) (13 mg). The ethyl acetate- (figure 1), (already reported in other species of the genus) [11], methanol fraction (9:1, 0.3g) on silica gel column for the first time, in addition to five known compounds: 3β- chromatography (60-120 mesh), with chloroform-methanol Erythrodiol (3), 3,5-Dimethoxybenzoic acid (4), 4- (4:1) as eluent, gave compound (4) (12 mg) and a mixture (71 hydroxybenzoic acid (5), 3,5-Dihydroxybenzoic acid (6), mg). The mixture was subjected to silica gel column 3β-Hydroxyolean-12-en-28-oic acid (7) from the methanol chromatography (100-200 mesh) and eluted with chloroform- extract of the root part of the plant. methanol (17:3) to yield the compounds (1) (11 mg) and (5) (10 mg). The ethyl acetate-methanol fraction (4:1, 0.2g) was MATERIALS AND METHODS subjected to silica gel column chromatography (60-120 mesh), with chloroform-methanol (3:1) as eluent, to give more of the General: Melting points were determined in centigrade scale compound (5) (4 mg) and (6) (07 mg). The ethyl acetate- in one end open capillary on Buchii 570 melting point methanol fraction (7:3, 0.09g) gave three major spots on TLC. apparatus and are uncorrected. IR spectrum was recorded It was separated by silica gel column chromatography (60-120 using KBr discs on Perkin-Elmer Paragon-1000 mesh), using chloroform-methanol (3:1) as eluent to give spectrophotometer, Esquire 3000 spectrometer. 1H and 13C- more of the compound (6) (5 mg) and a mixture (64 mg). The NMR spectra were recorded by a Bruker 500 and 125 MHz mixture was subjected to silica gel column chromatography instruements, respectively, using TMS as internal standard (100-200 mesh) and eluted with chloroform-methanol (7:3) to yield the compounds (2) (14 mg) and (7) (09 mg). and CDCl3 and CD3OD as solvent. The chemical shift values are reported in ppm (δ) units and the coupling constants (J) are in Hz. Column chromatography was carried out with Salicylic acid (2-Hydroxybenzoic acid) (1): o Merk silica gel (60-120 mesh and 100-200 mesh). Aluminium Colourless crystals, m.p.160.5 C, UV (MeOH) λmax. nm 302, -1 sheets, precoated with silica gel 60 F254 (20x20 cm, 0.2 mm 234, 208. IR (KBr) νmax : 3237(OH), 1662(C=O), 1609, 2856, 1 thick; E-Merck) were used for TLC to check the purity of the 2991, 1440, 1292, 1246, 1154, 892. H-NMR (CD3OD, 500 MHz) compounds and were visualized under UV light (254 and 366 δ: 7.89 (1H, d, J = 7.3 Hz, H-6), 7.48 (1H, t, J = 7.4 Hz, H-4)), 7.1 nm) followed by cerric sulfate as spraying reagent. (1H, d, J = 8.1 Hz, H-3) , 6.96 (1H, t, J = 7.3 Hz, H-5)), 12.1 (1H, 13 s, COOH). C-NMR (CD3OD, 125 MHz) δ: 174.2(C=O), 162.0 Corresponding Author: Javid A Banday, Department of Chemistry, University of Kashmir, Hazratbal, Srinagar-190006, J&K, India

(C-2), 136.4(C-4), 130.5(C-6), 119.4(C-5), 117.3(C-3), 111.5(C-1). 3,5-Dihydroxybenzoic acid (6): o MS: m/z at (rel.int.) 138.0314(calculated for C7H6O3, 138.0316) Colourless crystals, m.p.236-238 C, UV (MeOH) λmax. nm 218, -1 (M+) (54), 120(100), 107(86), 92(99), 77(33). 250, 299. IR (KBr) νmax : 3515(O-H), 1710(C=O), 1 1630(Aromatic). H-NMR (CD3OD, 500 MHz) δ: 7.11 (2H, d, From these results, the structure of the compound (1) was J= 1.8Hz, H-2/H-6), 6.65(1H, d, J= 1.8Hz, H-4). 13C- NMR identified as Salicylic acid, which was confirmed by (CD3OD, 125 MHz) δ: 169.4(C=O), 159.0(C-3/C-5), 139.8(C-1), comparison of its spectral data with the data values reported 109.0(C-2/C-6), 108.3(C-4). MS: m/z at (rel. int.) 155.0259 + in literature [11,12]. (calculated for C7H7O4, 155.0266) (M+), 136(M-H2O) , 121(90), 72(14), 71(46). Methyl gallate (Methyl-3,4,5-trihydroxybenzoate) (2): o Colourless amorphous solid, m.p.203 C, UV (MeOH) λmax. The spectral data were in complete agreement with that -1 [17,18] nm 330, 260 , 236. IR (KBr) νmax : 3597, 2963, 1742, 1697, 1237, reported in literature. 1 1016. H-NMR (CD3OD, 500 MHz) δ : 3.79 (3H s,-OCH3), 7.1 13 (2H, s, H-2/H-6). C- NMR (CD3OD, 125 MHz) δ: 170(C=O), 3β-Hydroxyolean-12-en-28-oic acid (7): o -1 145.3(C-3/C-5), 139.8(C-4), 121(C-1), 110.4(C-2/C-6), Colourless crystals, m.p.305-306 C. IR (KBr) νmax : 3400-2640, 1 51.8(OCH3). MS: m/z at (rel. int.) 184.0971(calculated for 1700, 1660, 820. H-NMR (CD3OD, 500 MHz) δ : 5.24 (2H,t, J= C8H8O5, 184.0968) (M+) (38), 154(100), 126(32), 107(10), 79(23), 3.45Hz, H-12), 3.60(1H,dd,J= 4.1, 9.9Hz, H-3), 1.12, 1.03, 0.98, 13 69(07), 53(21), 51(22). 0.97, 0.91, 0.90, 0.89 (3H each, s, CH3) ; C- NMR (CD3OD, 125 MHz) δ :183.4(C-28), 143.6(C-13),122.7(C-12),79.0(C-3),55.2(C- From these results, the structure of the compound (2) was 5),47.6(C-9), 46.5(C-17), 45.9(C-19), 41.6(C-14), 41.0(C-18), identified as Methyl gallate, which was confirmed by 39.1(C-8), 38.7(C-4), 38.4(C-1), 37.1(C-10), 33.8(C-21), 33.0(C- comparison of its spectral data with the data values reported 29), 32.6(C-7), 32.4(C-22), 30.6(C-20), 28.1(C-23), 27.7(C-15), in literature.[11,13,14] 27.2(C-2), 25.9(C-27), 23.5(C-30), 23.4(C-11), 23.4(C-16), 18.3(C- 6), 17.1(C-26), 15.6(C-24), 15.3(C-25). MS: m/z at (rel. int.) 3β-Erythrodiol (3): 456.3610 (calculated for C8H8O5, 456.3603) 456(M+) (4), o -1 Colourless crystals, m.p.229 C, IR (KBr) νmax : 3580, 3430(O- 248(98), 208(12), 203(60), 133(53). 1 H), 1610(C=C). H-NMR (CD3Cl3, 500 MHz) δ : 5.70 (1H, t, J =3.6Hz, H-12), 3.12 (1H, d, Jaa=11.2 Hz, Jae=4.5 Hz,H-12), The spectral data were in complete agreement with that 1.15(3H,s, H-27), 0.98(3H,s,H-23), 0.93(3H,s,H-26), reported in literature. [15, 19-21] 0.92(3H,s,H-25), 0.87(3H,s,H-30), 0.86(3H,s,H-29), 0.78(3H,s,H- 13 24). C- NMR (CD3Cl3, 125 MHz) δ : 144.2(C-13), 122.4(C-12), Fig. 1: Structures of the Compounds (1 - 7) 79.0(C-3), 69.5(C-28), 55.1(C-5), 47.6(C-9), 46.5(C-19), 42.3(C- 18), 41.8(C-14), 39.8(C-8), 38.8(C-4), 38.6(C-1), 36.9(C-10/17), 34.1(C-21), 33.2(C-29), 32.6(C-7), 31.0(C-22), 30.9(C-20), 28.1(C- OH O 23), 27.2(C-2), 25.9(C-27), 25.6(C-15), 23.6(C-11/30), 28.1(C-23), 22.0(C-16), 18.4(C-6), 16.6(C-26), 15,6(C-24), 15.5(C-25). MS: OH m/z at (rel. int.) 442.3798(calculated for C30H50O2, 442.3812) (M+) (8), 409(9), 234(32), 216(14), 207(15), 204(28), 203(100),

189(11), 107(10), 95(14), 81(12), 69(17). (1)

The spectral data were in complete agreement with that reported in literature. [15, 16] O

HO 3,5-Dimethoxybenzoic acid (4): O o Colourless crystals, m.p.182 C, UV (MeOH) λmax. nm 220, -1 251, 289. IR (KBr) νmax : 3500(O-H), 1703(C=O), HO 1 1629(Aromatic). H-NMR (CD3OD, 500 MHz) δ: 7.09(2H, d, J= OH 2.0Hz, H-2/6)), 6.72 (1H, d, J= 2.1Hz, H-4), 3.82(6H, s, 2 x (2) 13 OCH3). C- NMR (CD3OD, 125 MHz) δ : 167.4(C=O),161.0(C- 3/5),129.7(C-1),105.2(C-2/C-6), 101.3(C-4). MS: m/z at (rel. int.) 182.1739(calculated for C9H10O4, 182.1740) 182 (M+).

The spectral data were in complete agreement with that reported in literature. [17] H CH2OH

H 4-hydroxybenzoic acid (5): o HO Colourless crystals, m.p.184 C, UV (MeOH) λmax. nm 216, 255, H -1 296. IR (KBr) νmax : 3510, (Aromatic C-H), 1750(C-O), (3) 1 1626(Aromatic). H-NMR (CD3OD, 500 MHz) δ: 11.92 (1H s, COOH), 7.92 (2H, d, J= 8.5Hz, H-3/H-5), 6.72(2H, d, J= 8.5Hz, 13 H-2/6). C- NMR (CD3OD, 125 MHz) δ: 180.0(C=O), 160.3(C- 4), 131.5(C-2/6), 122.4(C-1), 116.5(C-3/C-5). MS: m/z at (rel. int.) 138.0310(calculated for C7H6O3, 138.0316) 138 (M+).

The spectral data were in complete agreement with that reported in literature. [17] 130.5 and 136.3. The mass spectrum of compound (1) revealed COOH the successive loss of 18 amu and 28 amu, corresponding to loss of a water molecule and carbon dioxide to give abundant ion peaks at m/z 120 and m/z 92.

Compound (2), m.p. 203oC, in its mass spectra, showed the H3CO OCH3 molecular ion peak at m/z 184.0971(calculated for C8H8O5, (4) 184.0968). The compound exhibited IR spectra displaying -1 - absorption at νmax.cm 3597 due to hydroxyl group, νmax.cm 1 -1 COOH 1742 for carbonyl group and νmax.cm 1697 for conjugated C=C, besides other absorption peaks at 2963, 1237 and 1016. The UV-spectrum of compound (2) displayed absorption peaks at λmax. nm 330, 260 and 236.

The 1H-NMR spectrum of compound (2) displayed just two resonance signals; a characteristic resonance signal at δ 3.79 (s, 3H) for methoxy protons and a signal at δ 7.1 assigned to two OH aromatic protons (H-2 and H-6). In its 13C-NMR spectrum, (5) compound (2) displayed prominent resonance signals at δ C 51.8 and δ 170, which were assigned to methoxy group C carbon and carbonyl carbon, respectively. Besides, the COOH compound displayed resonance signals for quaternary carbons at δC 139.8 & 145.3 and a signal at δC 110.4. The mass spectrum of compound (2) revealed the successive loss of 30 amu and 28 amu, corresponding to loss of methoxy (-OCH3) group and carbon dioxide molecule to give abundant ion HO OH peaks at m/z 154 and m/z 126.

(6) CONCLUSION

In this paper, we report the isolation of salicylic acid and methyl gallate from the roots of Conyza canedensis for the first time.

H COOH Salicylic acid (2-Hydroxybenzoic acid) (1) colourless crystals, o - H m.p.160.5 C, UV (MeOH) λmax nm 302, 234, 208. IR (KBr) νmax 1 HO : 3237, 1662, 1609, 2856, 2991, 1440, 1292, 1246, 1154, 892. H (7) 1H-NMR(δ) : 7.89 (1H, d, J = 7.3 Hz, H-6), 7.48 (1H, t, J = 7.4 Hz, H-4)), 7.1 (1H, d, J = 8.1 Hz, H-3) , 6.96 (1H, t, J = 7.3 Hz, RESULTS AND DISCUSSION H-5)), 12.1 (1H, s, COOH).13C- NMR(δ) : 174.2(C=O),162.0 (C- 2), 136.4(C-4), 130.5(C-6), 119.4(C-5), 117.3(C-3), 111.5(C-1). Extensive chromatography of the methanol extract of the MS: m/z at (rel. int.) 138.0314(calculated for C7H6O3, roots of Conyza canedensis over silica gel, using graded solvent 138.0316) (M+)(54), 120(100), 107(86), 92(99), 77(33). systems, afforded seven known compounds, including salicylic acid (1) and methyl gallate (2), for the first time from Methyl gallate (Methyl-3, 4, 5-trihydroxybenzoate) (2) colourless o this plant. crystals, m.p.203 C, UV (MeOH) λmax. nm 330, 260 , 236. IR -1 1 (KBr) νmax : 3597, 2963, 1742, 1697, 1237, 1016. H-NMR (δ): 13 Compound (1), m.p.160.5oC in its mass spectra, showed the 3.79 (3H s,-OCH3), 7.1 (2H, s, H-2/H-6). C- NMR (δ): molecular ion peak at m/z 138.0314(calculated for C7H6O3, 170(C=O), 145.3(C-3/C-5), 139.8(C-4), 121(C-1), 110.4(C-2/C- 138.0316). The compound exhibited IR spectra displaying 6), 51.8(OCH3). MS: m/z at (rel. int.) 184.0971(calculated for -1 -1 absorptions at νmax.cm 3237 due to hydroxyl group, νmax.cm C8H8O5, 184.0968) (M+) (38), 154(100), 126(32), 107(10), 79(23), -1 1662 for carbonyl group and νmax.cm 1609 for conjugated 69(07), 53(21), 51(22). C=C, besides other absorption peaks at 2856, 2991, 1440, 1292, 1246, 1154 and 892. The UV-spectrum of compound (1) ACKNOWLEDGEMENT displayed absorption peaks at λmax. nm 302, 234 and 208. 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