Isolation and structural determination of some chemical constituents from andrographis paniculata nees (acanthaceae) growing in Hung Yen province

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From the ethanol extract of dried powders of Andrographis paniculata Nees leaves growing in Hung Yen Province, five compounds were isolated and identified: Andrographolide, dodecahexanoic acid, B-sitosterol, a mixture of 14-deoxy-11,12-didehydroandrographolide and 14-deoxyandrographolide (in ratio of 11:5).

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Nội dung Text: Isolation and structural determination of some chemical constituents from andrographis paniculata nees (acanthaceae) growing in Hung Yen province

JOURNAL OF SCIENCE OF HNUE 2011, Vol. 56, N◦ . 1, pp. 27-33 ISOLATION AND STRUCTURAL DETERMINATION OF SOME CHEMICAL CONSTITUENTS FROM Andrographis paniculata NEES (ACANTHACEAE) GROWING IN HUNG YEN PROVINCE Pham Huu Dien(∗) and Hoang Thi Hoa Hanoi National University of Education (∗) E-mail: dienhp@gmail.com Abstract. From the ethanol extract of dried powders of Andrographis pan- iculata Nees leaves growing in Hung Yen Province, five compounds were isolated and identified: andrographolide (1), dodecahexanoic acid (2), β- sitosterol (3), a mixture of 14-deoxy-11,12-didehydroandrographolide (4a) and 14-deoxyandrographolide (4b) (in ratio of 11:5). Their structures were confirmed by various spectral methods (IR, NMR, EIMS, 1 H-NMR, 13 C- NMR, etc.) and by comparison with those of references. Keywords: Andrographis paniculata Nees, andrographolide, dodeca- hexanoic acid, β-sitosterol, 14-deoxy-11,12-didehydroandrographolide, 14- deoxyandrographolide, isolation. 1. Introduction Xuyen Tam Lien is the Vietnamese name of the Andrographis paniculata Nees (Acanthaceae) plant, which is wildely cultivated in Southern Asia, including Viet- nam. Mostly the leaves and the roots have been traditionally used over the centuries for different medicinal purposes as a folklore remedy for a wide spectrum of ailments such as fever, common colds, hepatitis, diabetes, etc. [1]. Recent research on An- drographis paniculata Nees revealed that the plants have very large pharmacological effects such as anti-inflammation [2], anti-diarrhoea [3], antivirus [4], anti-malaria [5], anti-cancer [6], etc. Numerous studies have shown that bioactive constituents from Andrographis paniculata Nees are diterpenes, diterpene lactones and its glycosides, such as andro- grapholide, panniculitis A-C [2, 3]. In this paper we report our investigation on some chemical constituents of Andrographis paniculata Nees, widely growing in Hung Yen Province. 27
Pham Huu Dien and Hoang Thi Hoa 2. Content 2.1. Experiments ∗ General experimental procedures: IR spectra were recorded on SHIMADZU-FTIR 8101M spectrophotometer using KBr disks. NMR spectra (1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC) were recorded on Brucker Avance 500MHz. The chemical shift values (δ) are given in ppm with TMS as internal standard, coupling constant J (by Hz). EIMS spectra were recorded on HP 5989B mass spectrometer. Silica gel (Merck Co., Germany) was used for flash chromatography. TLC was carried out on precoated Si gel GF254 (Merck Co., Germany) and TLC spots were viewed at 254 nm and visualized by spraying with vanillin-10% H2 SO4 solution. ∗ Plant material: Xuyen tam lien plants collected in Hung Yen Province (in May, 2008) were identified as Andrographis paniculata Nees by Dr. Ninh Khac Ban (Institute of Ecol- ogy & Biological Resources, Vietnam Academy of Science and Technology, VAST). A voucher specimen (No.DHP20070521) is deposited in the Herbarium of Dept. of Organic Chemistry, Hanoi National University of Education. ∗ Extraction and Isolation: The air-dried aerial parts of Andrographis paniculata Nees (3.0 kg) were ground into powder and extracted with 80% methanol (5L x 7 days x 3 times). After evaporation of collected percolation, the crude extract was extracted in CH2 Cl2 :H2 O (1:1) biphasic solvent system. From the aqueous phase were precipitated some white amorphous powders, that recrystallized in acetone to give compound (1) (25 mg). Evaporating the organic phase gave 100 g of condensate. The crude condensate was subjected to column chromatography over silica gel and fractions eluted with n-hexane:ethyl acetate (100:0, 50:1, 10:1, 1:1, 1:2), ethyl acetate and ethyl acetate : methanol (4:1, 1:1, 1:2, 1:4), giving fraction (Fr.) A (1.48 g), Fr. B (1.57 g), Fr. C (6.8 g), Fr. D (1.50 g), Fr. E (1.65 g), Fr. F (1.43 g), Fr. G (0.85 g), Fr. H (1.45 g) and Fr. I (0.98 g). By repeating column chromatography, 60 mg of compound (2) from Fr. D and 22 mg of compound (3) from Fr. A and 16 mg of compound (4) from Fr.F were isolated. Andrographolide (1): white layer crystals, m.p.: 244 - 245◦ C; Rf = 0.35 (n- hexane:EtOAC = 1:10), well dissolved in methanol, ethanol; IR (KBr, cm−1 ): 3402 (wide), 3330, 2932, 1727, 1672, 1460, 1368, 1224, 1038, 983, 914, 716, 572; EIMS (m/z, %): 350 [M]+ (55), 332 (92), 314 (100), 296 (990), 282 (37), 254 (20), 240 (18), 226 (7); 1 H-NMR and 13 C-NMR (see Table 1). 28
Isolation and structural determination of some chemical constituents... Dodecahexanoic acid (2): white needles, m.p.: 153◦ C; Rf = 0.6 (n-hexane: EtOAC = 1:4), well dissolved in methanol, ethanol; IR (KBr, cm−1 ): 2921, 2865, 2681, 1707, 1626, 1467, 1296 and 1117; 1 H-NMR (δ, ppm): 2.34 (2H, t, 7.5Hz), 1.63 (2H, m), 1.20 - 1.40 (44H, m), 0.89 (3H, t, 7.0 Hz); EIMS (m/z, %): 396 [M]+ (25), 353 (6), 297 (9), 241 (8), 199 (6), 185 (12), 171 (7), 129 (30), 111 (22), 83 (50), 60 (62), 57 (100). β-Sitosterol (3): white needles, m.p.: 139.5◦C - 140.5◦ C; Rf = 0.33 (n-hexane: EtOAC = 1:4), well dissolved in n-hexane, ethyl acetate, chloroform; IR (KBr, cm−1 ): 3450 (wide), 2936, 2856, 1647, 1461, 1376, 1059, 963, 803, 588; 1 H-NMR (δ, ppm): 5.35 (1H, m), 3.49 (1H, m), 2.34 (2H, t, 7.5 Hz), 1.63 (2H, t, 7.5 Hz), 1.15-1.38 (45H, m), 0.683 (3H, s), 1.00 (3H, s), 0.918 (3H, d, J = 5.5Hz), 0.824 (3H, d), 0.811 (3H, d), 0.849 (3H, d); EIMS (m/z, %): 414 [M]+ (17), 396 (18), 367 (5), 329 (4), 303 (8), 273 (7), 255 (25), 187 (10), 159 (33), 145 (60), 95 (92), 81 (100). 14-Deoxy-11,12-didehydroandrographolide and 14-deoxyandrographolide (4a & 4b, in ratio of 11:5): white cylindes; Rf = 0.5 (n-hexane:EtOAC = 1:10), m.p: 210 - 214◦ C; IR (KBr, cm−1 ): 3282, 3069, 2941, 2870, 1750, 1641, 1444, 1350, 1207, 1086, 1037, 904, 717; 1 H-NMR and 13 C-NMR (see Table 2); EIMS (m/z, %): 332 [M]+ (5), 316 (8), 286 (20), 259 (18), 120 (100) for 4a; 334 [M]+ (5), 317 (8), 297 (20), 121 (100) for 4b; 1 H-NMR and 13 C-NMR (see Tables 2, 3). 2.2. Results and discussion ∗ Compound 1: The MS of (1) afforded m/z [M]+ 350. The strong, wide IR absorption at 3401 cm−1 (with small shoulder at 3330 cm−1 ) assigned for three OH band absorptions, in good corresponding with 1 H-NMR (1H, 3.42 ppm, t, 13.0 Hz; 1H, 4.96 ppm, d, 6.0 Hz; 2H, 3.30 and 4.17 ppm, d, 11.0 Hz) and 13 C-NMR signals (79.8 65.4 and 63.9 ppm). The IR (strong peak at 1727 cm−1 , assigned for C=O group, 1648 cm−1 , for C=C group), 1 H-NMR (2H, 4.22 ppm and 4.44 ppm, assigned for cyclic CH2 O group, 6.93 ppm, for olefinic protion) and 13 C-NMR signals (one at 170.9 ppm, assigned for carbonyl carbon, two at 127.9 ppm and 148.6 ppm, for C=C group, another at 74.6 ppm, for cyclic CH2 O) reveal that (1) must be a typical α, β- unsaturated γ-lactone. Besides, 1 H-NMR (1H, 4.63 ppm, s; 1H, 4.89 ppm, s), 13 C-NMR signals (108.6 and 146.6 ppm) showed the existing of uncyclic C=CH2 group in (1) (its medium IR band absorption, at 3095 - 3020 cm−1 , was overlaped by OH one, so it couldnt be observed clearly). From above analysis of IR, NMR, MS spectra and melting point of (1) in comparison with those in [7], we made a conclusion that (1) is andrographolide, C20 H30 O5 , a main diterpene from Andrographis paniculata Nees. 29
Pham Huu Dien and Hoang Thi Hoa Table 1. 1 H-NMR and 13 C-NMR spectral data of (1) in comparison with those of andrographolide in [7] 13 C-NMR 1 H-NMR No. (1) [7] (1) [7] 1 36.9 38.13 2.56 2H t 13.0 2.45 2H m 2 27.7 29.03 18 2H m 1.82 2H m 3 79.8 80.92 3.42 1H t 8.5 3.44 1H m 4 42.3 43.68 - - 5 55.0 56.32 1.23 1H m 1.23 1H d 13.0 Ha 1.89 m, 6 24.5 25.21 1.8 2H m Hb 1.33 d 13.0 7 37.6 38.97 Ha 1.98 dd 13.0 5.0; Hb 2.41 t 13.0 2.06 2H m 8 146.6 148.77 - - 9 55.9 57.39 1.93 1H m 1.95 1H m 10 38.6 39.96 - - 11 22.5 25.71 2.23 2H m 2.63 2H m 12 148.6 149.35 6.93 1H t 1.5 6.87 td 6.1 1.7 13 127.9 129.80 - - 14 65.4 66.65 4.96 1H d 6.5 5.03 1H 6.1 Ha 4.49 6.1; 15 74.6 76.14 Ha 4.22 dd 2.0; Hb 4.45 dd 6.0 Hb 4.47 1H 6.1 16 170.9 172.64 - - Ha 4.91 s; 17 108.6 109.22 Ha 4.89 1H s; Hb 4.63 1H s; Hb 4.69 s 18 23.6 15.54 0.71 3H s 0.77 3H s Ha 4.14 d 10.0; 19 63.9 64.98 Ha 4.17 d 11.0; Hb 3.30 d 11.0; Hb 3.41 d 10.0 20 14.9 23.38 1.23 3H s 1.24 3H s ∗ Compound 2: The MS of (2) afforded m/z [M]+ 396. The strong, wide IR peak at 3372 cm−1 assigned for OH band absorptions, another strong peak at 1706 cm−1 , assigned for C=O carboxylic band absorption, the rest, at 2921 cm−1 and 2865 cm−1 , assigned for saturated CH band absorptions. The 1 H-NMR spectral data of (2) showed three methyl protons at 0.89 ppm, 48 olefinelic protons at 1.20 ppm - 2.34 ppm. From IR, 1 H-NMR, MS spectra and melting point of (2), we made a suggestion that (2) be dodecahexanoic acid (CH3 [CH2 ]24 COOH). ∗ Compound 3: The EI-MS data of (3) afforded m/z [M]+ 414, corresponding to molecular formula of C29 H50 O. The strong, wide IR absorption at 3450 cm−1 (assigned for 30
Isolation and structural determination of some chemical constituents... OH band absorption), at 1646 cm−1 (assigned for olefinic C=C band absorption) together with 1 H-NMR signals at 3.49 ppm (1H, m, H-3), 5.35 ppm (1H, brd m, H-6) suggested the presence of OH (at C-3) and C=C groups (at C5-C6). Especially six methyl signals at 0.683 (3H, s, H-18), 1.00 (3H, s, H-19), 0.918 (3H, d, J = 5.5Hz, H-21), 0.824 (3H, d, J = 6.5 Hz, H-26), 0.811 (3H, d, J = 6.5 Hz, H-27), 0.849 (3H, d, J = 7.5, H-29) are typical for sterols. From above analysis of IR, 1 H-NMR, MS spectra and melting point of (3) and in comparison with those of stigmast-5-en-3β-ol (or β-sitosterol) [8], it shows that (3) must be the β-sitosterol, one of the most popular sterols in plants. ∗ Compound 4: The MS of (4) afforded m/z [M]+ 332 and 334 (in ratio of 11: 5). The strong, wide IR absorption at 3285 cm−1 , a small shoulder at 3069 cm−1 , assigned for two OH band absorptions of (4a) and (4b). A strong peak at 1750 cm−1 (assigned for C=O group), together with two medium at 1641 cm−1 and 1444 cm−1 (assigned for C=C absorption) revealed the existance of unsaturated γ-lactones. The 1 H-NMR data of (4) showed four singles at 4.88, 478 (2H) and 4.60 ppm, 5.52 ppm (2H) (in ratio of 11:5), assigned for four (4a) and (4b) exocyclic C=CH2 protons; two other cyclic olefinic protons (in ratio of 11:5) at 7.09 ppm and 7.17 ppm are in conjugation with C=O groups. Besides, two cyclic oxymethine protons at 3.46 ppm and 3.48 ppm are typical for H-3 labdans with OH-β orientation. The 13 C-NMR and DEPT spectra indicated that (4) has 40 carbons including 7 overlap couples at 28.3 (C6), 43.0 (C4), 64.2 (C19), 70.0 (C15), 143.8 (C14), 147 (C8) and 174 (C16), confirming the mixture of (4a, 4b):(4a) has 2 methyl, 7 methylen, 6 methine, 5 quaternary carbons; (4b) has 2 methyl, 7 methylen, 4 methine, 5 quaternary carbons. Table 2. Some specific 1 H-NMR signals of (4a), (4b) in comparison with those of 14-DHA and 14-DOA in [7, 9] No. (4a) 14-DHA [7] (4b) 14-DOA [9] 3 1H 3.48 m 1H 3.48 m 1H 3.46 m 1H 3.46 m 11 1H 6.88 1H 6.87 brs - - 12 1H 6.10 1H 6.12 d 15.8 - - 14 1H 7.18 1H 7.17 br 1H 7.10 1H 7.09 brs 15 1H 4.80 1H 4.81 d 1.5 1H 4.78 1H 4.77 d 2.1 1Ha 4.78 1Ha 4.78 brs 1H 4.88 1H 4.88 brs 17 1Hb 4.50 1Hb 4.52 brs 1H 4.68 1H 4.65 brs 18 3H 0.82 s 3H 0.82 s 3H 0.64 3H 0.63 s 1Ha 4.20 1Ha 4.21 d 11.0 1Ha 4.18 1Ha 4.17 d 11.0 19 1Hb 3.36 1Hb 3.35 d 11.0 1Hb 3.30 1Hb 3.31 d 11.0 20 3H 1.24 s 3H 1.24 s 3H 1.26 s 3H 1.26 s 31
Pham Huu Dien and Hoang Thi Hoa From IR, MS, NMR data analysis of (4) in comparison with those of 14-deoxy- 11,12-didehydroandrographolide (14-DHA) and 14-deoxyandrographolide (14-DOA) [7, 9], we made a conclusion that (4) must be a mixture of 14-DHA and 14-DOA in ratio of 11:5. Table 3. The 13 C-NMR spectral signals of (4a), (4b) in comparison with those of 14-DHA and 14-DOA in [7, 9] No. (4a) 14-DHA [7] (4b) 14-DOA [9] 1 38.3 38.6 39.1 39.0 2 22.1 21.9 22.7 22.7 3 80.7 80.5 80.9 80.7 4 43.07 42.8 43.1 42.9 5 55.5 55.3 54.9 54.7 6 28.3 28.0 28.3 28.2 7 36.7 36.6 38.3 38.3 8 147.0 148.1 146.9 146.9 9 61.8 61.7 56.2 56.1 10 38.3 38.6 39.1 39.1 11 134.9 136.0 22.0 21.9 12 121.1 121.1 24.6 24.5 13 129.0 129.3 134.8 134.7 14 143.8 143.1 143.8 144.2 15 70.0 69.7 70.0 70.2 16 174.0 172.4 174.0 174.4 17 109.0 109.1 107.4 107.3 18 15.9 15.9 15.2 15.2 19 64.2 64.2 64.2 64.2 20 24.1 22.8 22.7 22.7 Structure of compounds from leaves of Andrographis paniculata Nees (Acan- thaceae) is below: Andrographolide (1) β-Sitosterol (3) 14-DHA (4a) 14-DOA (4b) 32
Isolation and structural determination of some chemical constituents... 3. Conclusion We isolated and structurally identified five compounds from leaves of An- drographis paniculata Nees (Acanthaceae) by various spectral methods: IR, NMR, EIMS, 1 H-NMR, 13 C-NMR. Five compounds are andrographolide (1), dodecahex- anoic acid (2), β-sitosterol (3), a mixture of 14-deoxy-11,12-didehydroandrographolide (4a) and 14-deoxyandrographolide (4b) (in ratio of 11:5). Acknowledgement This work is supported by the Ministry of Education and Training (project number: B2010-17-275TD). REFERENCES [1] Vo Van Chi, 1999. Dictionary of Vietnamese Trad. Medicinal Plants. Medicine Pub. House. [2] Chiou W. F., Chen C. F., Lin J. J., 1998. Andrographolide suppresses the ex- pression of inducible nitric oxide synthase in macrophage and restores the vaso- construction in rat ocorta treated with lipopolysaccharide. Br. J. Pharmcol., 125, pp. 327-334. [3] Gupta S., Choudhry M. A., Yadava J. N. S., Srivastava V., Tando J. S., 1990. Antidiarrhoeal activity of diterpenes of Andrographis paniculata Nees agaist E.coli enterotoxin in vivo models. Tnt. J. Crude Drug Res., 28, pp. 273-283. [4] Wiart C., Kumar K., Yusof M. Y., Hamimah H., Fanzi Z. M. and Sulaiman M., 2005. Antiviral properties of ent-labdenes diterpenes of Andrographis paniculata Nees, inhibitors of Herpes simplex virus type 1. Phytother.Res. 19, pp. 1069-1070. [5] Misra P., Pal N. L., Guru P. U., Katiyar J. C., Srivastava V., Tandon J. S., 1992. Antimalarial activity of Andrographis paniculata Nees against Plasmodium bergei NK65 in Mastomys natalensis. Int. J. of Pharm., 30, pp. 263-274. [6] Kumar R. A., Sridevi K., Kumar N. V. Nanduri S., Rajagopal S., 2004. Anti- cancer and immonostimulatory compounds from Andrographis paniculata Nees. J. Ethnopharmacol., 92, pp. 291-295. [7] Cava M. R., Chan W. R., Haynes J., Johson I. F., Weinstein B., 1962. The structure of Andrographolide. Tetrahedron, No. 18, pp. 397-403. [8] John G. L., Toshihiro A., 1997. Analysis of sterols. Blackie Acad. & Professional, Chapman & Hall, p. 378. [9] Balmain A. and Connolly J. D., 1973. Minor diterpenoid constituents glucoside from Andrographis paniculata Nees. J.C.S. Perkin, No. 1, pp. 1247-1251. 33