Five metabolites, scopoletin 1, ursolic acid 2, 5-(hydroxymethyl)furan-2-carbaldehyde 3, B-sitosterol 4 and daucosterol 5, were isolated from the fruit of the Morinda citrifolia Linn. plant, collected in Ho Chi Minh City. Their structures were elucidated by spectroscopic analysis.
JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol 58, No 9, pp 9-14 This paper is available online at http://stdb.hnue.edu.vn THE CHEMICAL CONSTITUENTS OF Morinda citrifolia LINN FRUIT Ma Thi Cam Van, Lam Thi Hai Yen and Pham Huu Dien Faculty of Chemistry, Hanoi National University of Education Abstract Five metabolites, scopoletin 1, ursolic acid 2, 5-(hydroxymethyl)furan-2-carbaldehyde 3, β-sitosterol and daucosterol 5, were isolated from the fruit of the Morinda citrifolia Linn plant, collected in Ho Chi Minh City Their structures were elucidated by spectroscopic analysis Keywords: Morinda citrifolia Linn., fruit, scopoletin, ursonic acid Introduction Morinda citrifolia Linn (family Rubiaceae) is usually a small tree or bush occurring in South Pacific tropical islands but it is also widely distributed in southern Vietnam All parts of the plants can be used to treat boils, abscess, inflammations of various origins and fungal infection [6] Pharmacological research has revealed that Morinda citrifolia plant material has anticancer [2], anti-inflammation [8], antioxidant [4], liver protection [11] and anti-AIDS properties [10] From the fruit of Morinda citrifolia Linn many metabolites such as glucosides, anthraquinones, damnacanthal, nordamnacanthal, coumarins, seronin and sterols have been isolated [1, 5, 9] Herein we report the results of our recent study on the chemical constituents of Morinda citrifolia Linn fruit collected in Ho Chi Minh City 2.1 Content Material and methods * General experimental procedure: IR spectra were recorded using a Shimadzu-FTIR 8101M spectrophotometer with a KBr disk and NMR (1 H, 13 C-NMR, DEPT, HSQC, HMBC) spectra were recorded on a Bruker Avance 500 MHz The chemical shift (δ) values are given in ppm with TMS as an internal standard, coupling constant J by Hz EIMS spectra were recorded making use of a HP 5989B mass spectrometer LC-MS spectra were recorded using an Agilent LC mass spectrometer Received August 28, 2013 Accepted October 16, 2013 Contact Pham Huu Dien, e-mail address: dienph@hnue.edu.vn Ma Thi Cam Van, Lam Thi Hai Yen and Pham Huu Dien 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, 302 and 366 nm and visualized by spraying the samples with a vanillin/10% H2 SO4 solution * Plant material: Fruit of Morinda citrifolia plants was collected in Ho Chi Minh City in October 2009 The plant material was identified by Nguyen The Anh, MSc., Institute of Chemistry, Vietnam Academy of Sciences and Technologies * Extraction and isolation: Air-dried fruits of Morinda citrifolia Linn (3.2 kg) were ground into a powder and an crude extract (159.6 g) was obtained using 95% MeOH (5L × 3) at room temperature which was evaporated under vacuum The extract was then exposed to n-hexane, ethyl acetate and methanol, giving corresponding fractional extracts The ethyl acetate extract was subjected to column chromatography over silica gel and eluted with produce a gradient with chloroform-methanol solvents, from 10:1 to 1:4 Eight fractions were obtained in succession Fraction (210 mg) was separated by column chromatography (SiO2 ; CH2 Cl2 − MeOH, 10 : 1) to afford (28 mg); fraction (120 mg, SiO2 ; CH2 Cl2 - MeOH, 4:1) to afford (13 mg); and fraction (78 mg, SiO2 ; CH2 Cl2 - MeOH, 1:4) to afford (15 mg) The methanol extract was separated by CC over reversed phase Si (RP18), eluted by methanol-water solvents to afford (8 mg) The n-hexane fraction was subjected to column chromatography over silica gel and eluted with n-hexane-ethyl acetate solvents, from 100 : to : 4, giving ten fractions From fraction (205 mg) were precipitated white crystals (125 mg) Compound (Scopoletin): Yellowish crystals, well dissolved in methanol, chloroform, H and 13 C-NMR (CDCl3 ) See Table ESI-MS (m/z): 193,1 [M+H]+ Compound (Ursolic acid): White powders, m.p.: 261 - 262 C; Rf = 0.7 (n-hexane: EtOAc = : 2), well dissolved in methanol, ethanol IR (KBr, cm−1 ): 3451 (OH), 2931, 2867, 1693, 1462, 1382, 1279, 1185, 1036, 999, 734 H and 13 C-NMR (δ, ppm) See Table LC-MSD (m/z): 455[M − H]+ Compound [5-(hydroxymethyl)furan-2-carbaldehyde]: Greenish viscous oils H 13 and CNMR(CDCl3 ) See Table LC-MSD (m/z): 125[M − H]+ Compound (β-sitosterol): White needle crystals, m.p.: 139.5 - 140.5 C; Rf = 0.33 (n-hexane : EtOAc = : 4), well dissolved in n-hexane, ethyl acetate, chloroform IR (KBr, cm−1 ) : 3450 (wide), 2936, 2856, 1647, 1461, 1376, 1059, 963, 803, 588 H-NMR (CDCl3 , 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) Compound (Daucosterol): White amorphous powders, m.p.: 280 C; dissolved in DMSO, methanol, CHCl3 , Rf (in n-hexane: EtOAc = : 1): 0,28 IR (KBr, cm−1 ): 3440 (OH), 2948, 1638, 1463, 1377, 1020, 600 LC-MSD (m/z): 577[M + H]+ HNMR (δ, ppm): 5.37 (1H, brd, H-6), 3.35-4.29 (7H, m, H-1’-6’), 3.24 (1H, t 7.5, H-3), 1.03 (3H, s, H-19), 0.92 (3H, d 5.5, H-21), 0.85 (3H, t 6.5, H-29), 0.84 (1H, d 7.5, H-25), 0.83 (3H, d 6.5, H-27), 0.68 (3H, s, H-18) 10 The chemical constituents of Morinda citrifolia Linn fruit 2.2 Results and discussion Compound The IR spectrum of showed one strong absorption of 3348 cm−1 that was assigned to the OH group, and another strong absorption at 1700 cm−1 , assigned to a carbonyl group conjugating with a double bond C=C (1608 cm−1 ) Its molecular formula was deduced to be C10 H8 O4 on the basic of a pseudo-molecular ion [M + H]+ peak at m/z 193.1 in ESI-MS (positive) spectrum, in combination with its H ans 13 C NMR spectra The H-NMR spectrum showed seven protons, including two aromatic (δH 6.78 and 7.12 ppm), two olefinic conjugating with a carbonyl group (δH 6.22, d, J = 9.0 Hz and 7.86, d, J = 9.0 Hz) and three methoxy (δH 3.93 ppm) The 13 C-NMR spectrum showed ten carbon peaks, including one methoxy (δC 56.8 ppm), six aromatic (δC 103.9 109.9 112.5 147.1 151.4 and 152.9 ppm), two olefinic (δC 112.6 and 146.1 ppm), that conjugated with one carbonyl (δC 164.0) Based on the spectroscopic data, the HMQC, HMBC correlation and reference comparison [1], was identified as scopoletin No 10 CH3 O-6 HO-7 Table The H and 13 C-NMR (CDCl3 ) data of 1 13 H-NMR, δ(ppm), J (Hz) C-NMR, δ(ppm) 164.0 1H, 6.22 d, J = 9.0 Hz 112.6 1H, 7.86, d, J = 9.0 Hz 146.1 1H, 6.78, s 109.9 147.1 151.4 1H, 7.12, s 103.9 112.5 152.9 3H, 3.93, s 56.8 1H, 4.91, s - Compound The LC-MSD data of afforded m/z[M]+ 456 The strong, wide IR absorption at 3451 cm−1 (assigned for free O-H band absorption), the strong one at 1693 cm−1 (assigned for a carboxylic C=O band absorption) and the 13 C-NMR signal at 180.4 ppm suggests the presence of carboxylic group COOH One middle peak at 1444 cm−1 is assigned for a cyclic C=C absorption band The H NMR of showed triterpene-type proton signals in a high field with seven methyl (mainly single) groups, with (δH from 0.77 to 1.04 ppm One CH signal at 3.2 ppm is assigned for a proton linked to C3 − OH group; another with δH 5.23 ppm for a cyclic C=C group The 13 C-NMR and DEPT spectra indicated that has thirty carbons, including seven CH3 (δC from 16.6 to 30.2 ppm), nine CH2 (δC from 18.2 to 39.3 ppm), seven cyclic CH (δC from 39.4 to 77.3 ppm) and six cyclic C (δC from 36.8 to 47.7 ppm, not including COOH) From the above analysis of the IR, NMR and MS spectra, and the melting point of and in comparison with those of ursolic acid, C30 H48 O3 [7], was identified as ursolic acid 11 Ma Thi Cam Van, Lam Thi Hai Yen and Pham Huu Dien Table The H and 13 C-NMR (CDCl3 ) data of No 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OH H-NMR 1H 3.20 m 1H 5.23 m 1H 2.18 m 3H 0.67 s 3H 0.89 s 3H 0.87 s 3H 0.75 s 3H 1.04 s 3H 0.81 d 6.25 3H 0.91 m 1H 4.27 q 13 C-NMR 39.3 27.8 767.3 39.3 55.2 18.2 32.9 39.7 47.7 36.8 24.0 125.4 138.0 41.9 27.9 24.3 47.7 52.7 39.4 39.3 30.6 37.0 30.2 16.6 16.8 18.1 24.1 180.4 18.1 20.9 - Compound The H-NMR spectrum of has protons which resonated in the low and middle fields, including two protons from a hydroxymethylene group at δH = 4.63 ppm, other ortho aromatic protons with δH = 6.60, 7.40 ppm (J = 3.5 Hz), and one aldehyde proton with δH = 9.54 ppm 13 C-NMR of showed carbon peaks, including one carbinol (CH2 OH) with δC = 57.59 ppm, aromatic with δC = 110.87, 124.83, 153.81 and 163.15 ppm, and one aldehyde carbon with δC = 179.43 ppm Considering both DEPT and HMBC spectra, we suggest that should be 5-(hydroxymethyl)furan-2-carbaldehyde, C6 H6 O3 12 The chemical constituents of Morinda citrifolia Linn fruit No CHO CH2 OH Table The H and 13 C-NMR (CDCl3 ) data of H-NMR, δ(ppm), J 13 C-NMR, δ(ppm) HMBC, C → H (Hz) 153.81 3, 4, 7.40 d J = 3.5Hz 1H 124.83 6.60 d J = 3.5 Hz 1H 110.83 3, 163.15 3, 4, 9.54 s 1H 179.43 4.63 s 2H 57.93 - Compound The IR spectrum of showed a strong absorption band at 3450 cm−1 that was assigned to the OH group, other middle absorptions at 1647, 1461 cm−1 , assigned to a double bond C=C The molecular formula of was deduced to be C29 H50 O on the basic of the molecular ion [M]+ peak at m/z 414 in EIMS spectrum The H-NMR spectrum of showed 50 protons, which mainly resonated in the strong field (δH 0.68 - 2.34 ppm), besides two other protons: one with δH 5.35 ppm (1H, m), assigned to the olefinic proton H-6, and another with δH 3.49 ppm (1H, m), assigned to the carbinol proton H-3 From the above IR, EIMS and H-NMR analysis, and a comparison with those of β-sitosterol [3], was confirmed to be β-sitosterol, one of the most common sterols in plants Compound The IR spectrum of showed a strong wide absorption band at 3440 cm−1 , assigned to the OH group, and other two absorption bands at 1638, 1463 cm-1, assigned to the double bond C=C The molecular formula of was deduced to be C35 H60 O6 on the basic of the pseudo-molecular ion [M + H]+ peak at m/z 577 in the LC-MS spectrum The H-NMR spectrum of had 56 protons, including 49 protons 13 Ma Thi Cam Van, Lam Thi Hai Yen and Pham Huu Dien which resonated in the fields, liked of 4; protons, assigned to the β-D-glucopyranoside protons (δH 3.35-4.29 ppm) Four left OH protons were not shown in the H-MNR From the above IR, LC-MS and H-NMR analysis and a comparison with those of daucosterol [3], was confirmed to be daucosterol, one of the common glucoside sterols in plants Conclusion From the fruit of Morinda citrifolia Linn plants, collected in Ho Chi Minh City, five compounds were isolated and structurally elucidated: scopoletin 1, ursolic acid 2, 5-(hydroxymethyl)furan-2-carbaldehyde 3, β-sitosterol and daucosterol REFERENCES [1] Bina S.S., Fouzia A.S Fayaz A., Sabira B., 2007 Isolation and structural elucidation of chemical constituents from the fruits of Morinda citrifolia Linn Arch Pharm Res., 30, No 8, pp 919-923 [2] Hirazumi A., Furusawa E., Chou S.C , Hokama Y., 1996 Immuno-modulation contributes to the anticancer activity of Morinda citrifolia fruit juice Proc West Pharmacol.Soc., 39, pp.7-9 [3] John G.L., Toshihiro A., 1997 Analysis of sterols, Blackie Acad & Professional, Chapman & Hall, p 378 [4] Liu C.H., Xue Y.R., Ye Y.H., Yuan F.F., Liu J.Y., Shuang J.L., 2007 Extraction and characterization of antioxidant compositions from fermented fruit juice of Morinda citrifolia (Noni) Agric Sci Chin., 6, pp 1494-1501 [5] Levand O., Larson H., 2009 Some Chemical Constituents of Morinda citrifolia Planta Medica, 36, No 6, pp 186-187 [6] Do Tat Loi, 1977 Medicinal plants and Herb medicines of Vietnam Publ House of Sci and Tech., pp 323-324 [7] Maria Paula P.Ramos et al., 2003 Computer-aided identification of chemical constituents isolated from Cybistax antisyphilitica J of Molecular Design, No 2, pp 1232-1237 [8] Nitteranona V., Zhanga G.D., Darienb B.J., Parkin K., 2011 Isolation and synergism of in vitro anti-inflammatory and quinine redutase (QR) inducing agents from the fruits of Morinda citrifolia (noni) Food Res Int., 44, pp 2271-2277 [9] Rusia K., Srivastava S.K., 1989 A new anthraquinone from the roots of Morinda citrifolia Linn Curr.Sci., 58, p 249 [10] Selvam P., Maddali K., Marchand C., 2010 Studies of HIV intergrase inhibitory activity of Morinda citrifolia L fruit extracts Antrivir Res., 86, pp 45-46 [11] Wang M.Y., Nowiski D., Anderson G., Jensen J., West B., 2008 Liver protective effects of Morinda citrifolia (Noni) Plant Foods Hum.Nutr., 63, pp 59-63 14 ... H-18) 10 The chemical constituents of Morinda citrifolia Linn fruit 2.2 Results and discussion Compound The IR spectrum of showed one strong absorption of 3348 cm−1 that was assigned to the OH... characterization of antioxidant compositions from fermented fruit juice of Morinda citrifolia (Noni) Agric Sci Chin., 6, pp 1494-1501 [5] Levand O., Larson H., 2009 Some Chemical Constituents of Morinda citrifolia. .. 5-(hydroxymethyl)furan-2-carbaldehyde, C6 H6 O3 12 The chemical constituents of Morinda citrifolia Linn fruit No CHO CH2 OH Table The H and 13 C-NMR (CDCl3 ) data of H-NMR, δ(ppm), J 13 C-NMR, δ(ppm) HMBC,