Iridoids and anthraquinones from the roots of Morinda officinalis

Cite this paper: Vietnam J. Chem., 2021, 59(1), 27-31 Article DOI: 10.1002/vjch.202060082 27 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Iridoids and anthraquinones from the roots of Morinda officinalis Vu Thi Phuong Anh 1 , Vo Van Minh 2 , Vo Chau Tuan 2 , Nguyen Van Khanh 2 , Duong Thi Dung 3 , Nguyen Xuan Nhiem 3,4 , Phan Van Kiem 3,4* 1 Quangnam University, 102 Hung Vuong, Tam Ky, Quang Nam 51000, Viet Nam 2 University of Science and Education, The University of Danang, 459 Ton Duc Thang, Lien Chieu, Da Nang 50000, Viet Nam 3 Graduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 4 Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam Submitted May 15, 2020; Accepted July 9, 2020 Abstract Three iridoids, asperulosidic acid (1), geniposidic acid (2), and galioside (3) and three anthraquinones, 2- hydroxymethyl-3-hydroxyanthraquinone (4), rubiadin-1-methyl ether (5), and anthragallol-2-methyl ether (6) were isolated from the methanol extract of the Morinda officinalis leaves. Their chemical structures were elucidated by ESI- MS, 1D- and 2D-NMR spectra and comparison with the data reported in the literature. Galioside (3) was reported from Morinda genus for the first time. Keywords. Morinda officinalis, iridoid, anthraquinone. 1. INTRODUCTION Morinda officinalis How (Rubiaceae), a lianoid shrub, is commonly cultivated in subtropical and tropical areas. The roots of this plant have been used as a tonic or nutrient supplements, including impotence, rheumatoid arthritis, osteoporosis, dermatitis, and depression. [1] Chemical constituents of M. officinalis indicates the presence of iridoid glycosides, anthraquinones, and saccharides. [1] The biological studies of M. officinalis showed various biological effects such as anti-rheumatoid arthritis, [2] anti- fatigue, [3] antiosteoporotic, [4] anti-oxidant, [5] and anti- inflammatory activities. [6,7] This paper reported the isolation and structure elucidation of three iridoid glycosides and three anthraquinones from M. officinalis. 2. MATERIALS AND METHODS 2.1. Plant materials The roots of M. officinalis were collected in Danang, Vietnam in December, 2019, and identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources. A voucher specimen (MO2012) was deposited at Institute of Marine Biochemistry. 2.2. General experimental procedures The used characterization equipments and detailed experimental procedures are the same as described in our published work. [15] 2.3. Extraction and isolation The dried roots of M. officinalis (3.5 kg) were sonicated in MeOH three times at 50 o C to yield a MeOH extract (600 g). The MeOH extract was suspended in water and successively partitioned with n-hexane and EtOAc to obtain n-hexane (MO1, 20.0 g) and EtOAc (MO2, 5.2 g) extracts after removal solvent in vacuo and water layer (MO3). MO1 and MO2 were combined, then loaded on a silica gel column eluting with a solvent system of CH2Cl2- MeOH (40:1, 20:1, 10:1, 5:1, 1:1, v/v) to give five smaller fractions, MO1A-MO1E. MO1A fraction was chromatographed on an RP-18 CC eluting with acetone-water (3:1, v/v) to give three sub-fractions, MO1A-MO1C. MO1B fraction was chromatographed on a Sephadex LH-20 CC, using Vietnam Journal of Chemistry Phan Van Kiem et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 28 MeOH-water (2:1, v/v) as the eluent solvent to yield compound 6 (11.0 mg). MO1C fraction was chromatographed on a Sephadex LH-20 CC, using eluent solvent of MeOH:water (1:1, v/v) to give five smaller fractions, MO1C1:MO1C5. MO1C3 fraction was chromatographed on an HPLC column (J’sphere, ODS H-80, 4 µm, 250×20 mm) with a flow rate of 3 mL/min eluting 40 % acetonitrile in water to yield 4 (6.0 mg). MO1C5 was crystallized in methanol to give compound 5 (40.0 mg). MO3 fraction was chromatographed on a Diaion HP-20P column eluting with water to remove sugars and polar components then increasing concentration of MeOH in water (25, 75, and 100 %) to obtain three sub-fractions, MO3A (8.0 g), MO3B (8.0 g), and MO3C (4.0 g). MO3B fraction (8.0 g) was chromatographed on a silica gel column eluting with CH2Cl2-MeOH-water (8:1:0.02, v/v/v) to give five smaller fractions, MO3B1-MO3B5. MO3B2 fraction was chromatographed on an HPLC column (J’sphere, ODS H-80, 4 µm, 250×20 mm) with a flow rate of 3 mL/min eluting 6 % acetonitrile in water to yield compound 3 (29.0 mg). MO3B3 fraction was chromatographed on an HPLC column (J’sphere, ODS H-80, 4 µm, 250×20 mm) with a flow rate of 3 mL/min eluting 20 % acetonitrile in water to yield compound 1 (6.0 mg). Finally, compound 2 was obtained from the MO3B5 fraction using HPLC column (J’sphere, ODS H-80, 4 µm, 250×20 mm) with a flow rate of 3 mL/min eluting 6 % acetonitrile in water. Figure 1: Chemical structures of compounds 1-6 Asperulosidic acid (1): White amorphous powder, C18H24O12, : +30.7 (c = 0.1, MeOH), HR-ESI-MS m/z: 431.1181 [M-H] - (Calcd. for [C18H23O12] - , 431.1195), 1 H- and 13 C-NMR (CD3OD), see table 1. Geniposidic acid (2): White amorphous powder, C16H22O10, : -40.0 (c = 0.1, MeOH), HR-ESI-MS m/z: 373.1130 [M-H] - (Calcd. for [C16H21O10] - , 373.1140), 1 H- and 13 C-NMR (CD3OD), see table 1. Galioside (3): White amorphous powder, C17H24O11, : -56.0 (c = 0.1, MeOH), HR-ESI- MS m/z: 439.0995 [M+Cl] - (Calcd. for [C17H24O11Cl] - , 439.1013), 1 H- and 13 C-NMR (CD3OD), see table 1. 2-Hydroxymethyl-3-hydroxyanthraquinone (4): Yellow amorphous powder, C15H10O4, HR-ESI- MS m/z: 253.0506 [M-H] - (Calcd. for [C15H9O4] - , 253.0506), 1 H- and 13 C-NMR (CD3OD), see table 2. Rubiadin-1-methyl ether (5): Yellow amorphous powder, C16H12O4, HR-ESI-MS m/z: 267.0661 [M-H] - (Calcd. for [C16H12O4] - , 267.0663), 1 H- and 13 C-NMR (CD3OD), see table 2. Anthragallol-2-methyl ether (6): Yellow amorphous powder, C15H10O5, HR-ESI-MS m/z: 269.0461 [M-H] - (Calcd. for [C15H9O5] - , 269.0455), 1 H- and 13 C-NMR (CD3OD), see table 2. 3. RESULTS AND DISCUSSION Compound 1 was obtained as a white amorphous powder. The molecular formula of 1 was determined to be C18H24O12 from HR-ESI-MS ion peak at m/z 431.1181 [M-H] - (Calcd. for [C18H23O12] - , 431.1195). The 1 H-NMR spectrum of 1 (CD3OD) 25][ D 25][ D 25][ D Vietnam Journal of Chemistry Iridoids and anthraquinones from the © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 29 Table 1: 1 H- and 13 C-NMR data for compounds 1-3 and reference compounds C 1 2 3 C $ δC a,b δH a,c (mult., J = Hz) C # δC a,b δH a,c (mult., J = Hz) C % δC a,b δH a,c (mult., J = Hz) 1 101.26 101.3 5.08 (d, 9.0) 98.0 98.3 5.18 (d, 7.5) 95.0 95.3 5.61 (d, 3.0) 3 155.41 155.2 7.67 (d, 1.0) 153.3 155.2 7.53 (d, 1.0) 151.9 152.5 7.41 (d, 1.0) 4 108.27 108.3 - 113.8 112.9 - 111.0 110.9 - 5 42.45 42.6 3.04 (dd, 1.0, 6.0, 7.5) 35.4 36.7 2.79 (dd, 7.5, 8.5) 37.7 39.0 3.57 (m) 6 75.35 75.4 4.83 (m) 40.9 39.7 2.14 (m)/2.85 (m) 132.7 133.9 5.63 (dd, 2.0, 6.0) 7 131.92 132.0 6.03 (d, 1.5) 130.4 128.5 5.82 (br s) 137.9 137.7 6.19 (dd, 2.5, 6.0) 8 145.92 145.9 - 142.5 144.8 - 85.5 86.0 - 9 46.23 46.3 2.65 (dd, 7.5, 9.0) 47.2 47.0 3.20 (m) 44.7 45.7 2.70 (dd, 3.0, 8.5) 10 63.78 63.8 4.82 (d, 15.0) 4.98 (dd, 1.0, 15.0) 61.9 62.6 4.20 (d, 14.5) 4.33 (d, 14.5) 67.1 68.4 3.57 (d, 5.0) 11 171.00 171.0 - 173.2 171.1 - 170.2 169.1 - 10-MeCO 172.53 172.6 - 10-MeCO 20.74 20.7 2.11 (s) 11-OMe 52.6 51.7 3.71 (s) 1' 100.58 100.6 4.74 (d, 7.5) 100.1 100.3 4.74 (d, 8.0) 99.0 100.0 4.66 (d, 8.0) 2' 74.91 75.0 3.25 (dd, 7.5, 8.5) 74.0 74.9 3.17 (t, 8.0) 73.3 74.6 3.20 (dd, 8.0, 9.0) 3' 78.54 77.9 3.40 (dd, 8.5, 9.0) 76.9 77.8 3.42 (m) 76.3 77.9 3.37 (t, 9.0) 4' 71.57 71.6 3.28 (m) 70.8 71.5 3.28 (m) 70.3 71.4 3.30 (m) 5' 77.87 78.6 3.30 (m) 77.5 78.3 3.30 (m) 76.9 78.2 3.30 (m) 6' 62.98 63.0 3.64 dd (6.0, 12.0) 3.87 dd (2.0, 12.0) 61.0 61.4 3.66 (dd, 4.0, 12.0) 3.87 (br d, 12.0) 61.4 62.6 3.67 (dd, 4.5, 12.0) 3.87 (dd, 1.5, 12.0) a) Measured in CD3OD, b) 125 MHz, c) 500 MHz, $C of asperulosidic acid in CD3OD, [8] #C geniposidic acid in D2O, [9] %C of galioside in CD3OD. [10] exhibited signals for one acetyl group at δH 2.11 (3H, s), two olefinic protons at δH 6.03 (1H, d, J = 1.5 Hz) and 7.67 (1H, d, J = 1.5 Hz), and one anomeric proton at δH 4.74 (1H, d, J = 7.5 Hz). The 13 C-NMR and HSQC spectra revealed signal of 18 carbons, including ten carbons at δC 171.0, 155.2, 145.9, 132.0, 108.3, 101.3, 75.4, 63.8, 46.3, and 42.6 assigned to a iridoid aglycone, six carbons at δC 100.6, 78.6, 77.9, 75.0, 71.6, and 63.0 assigned to a monosaccharide, one carbonyl and one methyl at δC 172.6 and 20.7 assigned to the presence of a acetyloxy group. Analysis of 1 H- and 13 C-NMR data indicated the structure of 1 was identical to asperulosidic acid. [8] The HMBC correlations between H-3 (δH 7.67) and C-1 (δC 101.3)/C-4 (δC 108.3)/C-5 (δC 42.6)/C-11 (δC 171.0) suggested the position of a double bond at C-3/C-4 and a carboxylic group at C-4. The HMBC correlations between H-10 (δH 4.82 and 4.98) and C-7 (δC 132.0)/C-8 (δC 155.2)/C-9 (δC 46.3) demonstrated the location of a double bond at C-7/C-8. The position of the acetyloxy group at C-10 was confirmed by HMBC correlations from H-10 (δH 4.82 and 4.98) to Ac (δC 172.6). The large coupling constant between glc H-1′ and H-2′ (J = 7.5 Hz) and 13 C-NMR chemical shifts of monosaccharide moiety at δC 100.6, 75.0, 77.9, 71.6, 78.6, and 63.0 confirmed the presence of β-D-glucopyranosyl moiety. The position of this sugar at C-1 of the iridoid was confirmed by the HMBC correlation from glc H-1' (δH 4.74) and C-1 (δC 101.26) as well as between H-1 (δH 5.09) and glc C-1' (δC 100.58). Thus, the structure of 1 was elucidated as asperulosidic acid. [8] Vietnam Journal of Chemistry Phan Van Kiem et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 30 The molecular formula of compound 2 was determined as C16H22O10 based on the HR-ESI-MS ion at m/z: 373.1130 [M-H] - (Calcd. for [C16H21O10] - , 373.1140). The 1 H-NMR of 2 (CD3OD) revealed signals of two olefinic protons at δH 7.53 (1H, d, J = 1.0 Hz) and 5.82 (1H, br s), and one anomeric proton at δH 4.74 (1H, d, J = 8.0 Hz). The 13 C-NMR and HSQC showed the signals of one iridoid moiety (three non-protonated carbons at δC 171.1, 144.8, and 112.9, five methines at δC 155.2, 128.5, 98.3, 47.0, and 36.7, and two methylenes at 62.6 and 39.7), one glucopyranosyl moiety (five methines at δC 100.3, 78.3, 77.8, 74.9, and 71.5 and one methylene at δC 61.4). The structure of 2 was elucidated as geniposidic acid by comparing their 1 H- and 13 C-NMR data with reported in the literature. [9] The molecular formula of compound 4 was identified as C15H10O4 by HRESIMS ion at m/z 253.0506 [M-H] - (Calcd. for [C15H9O4] - , 253.0506). The 1 H-NMR spectrum of 4 showed signals of four aromatic protons of ortho disubstituted benzene at δH 7.85 (1H, ddd, J = 2.0, 7.5, 7.5 Hz), 7.88 (1H, ddd, J = 2.0, 7.5, 7.5 Hz), 8.13 (1H, dd, J = 2.0, 7.5 Hz), and 8.16 (1H, dd, J = 2.0, 7.5 Hz), two para aromatic protons at δH 7.52 (1H, s) and 8.23 (1H, s). The 13 C-NMR and HSQC of 4 revealed signals of 15 carbons, including two carbonyls at δC 181.4 and 182.6 featured for the anthraquinone skeleton, six non-protonated carbons at δC 124.9, 133.1, 133.2, 133.4, 136.3, and 159.8, six methines at δC 111.2, 126.2, 126.4, 126.5, 134.3, and 133.8. Comparison with those in the literature data, [4] compound 4 was identified as 2-hydroxymethyl-3- hydroxyanthraquinone. Table 2: NMR data for compounds 4-6 and reference compounds C 4 5 6 C $ δC a,b δH a,c (mult., J = Hz) δC a,b δH a,c (mult., J = Hz) C # δC a,b δH a,c (mult., J = Hz) 1 126.2 126.2 8.23 (s) 160.6 - 156.9 155.8 - 2 125.0 124.9 - 126.1 - 140.3 138.6 - 3 159.6 159.8 - 162.2 - 157.1 155.0 - 4 111.2 111.2 7.52 (s) 109.2 7.46 (s) 109.5 108.0 7.22 (s) 5 126.5 126.4 8.13 (dd, 2.0, 7.5) 125.9 8.05 (dd, 1.0, 7.5) 113.5 127.3 8.10 (dd, 2.0, 7.5) 6 133.8 133.8 7.85 (ddd, 2.0, 7.5, 7.5) 133.1 7.78 (ddd, 1.0, 7.5, 7.5) 163.6 133.4 7.87 (m) 7 134.3 134.3 7.88 (ddd, 2.0, 7.5, 7.5) 134.3 7.84 (ddd, 1.0, 7.5, 7.5) 121.5 133.5 7.88 (m) 8 126.5 126.5 8.16 (dd, 2.0, 7.5) 126.5 8.11 (dd, 1.0, 7.5) 139.8 126.7 8.16 (dd, 2.0, 7.5) 9 181.4 181.4 - 182.6 - 187.1 187.7 - 10 182.5 182.6 - 180.0 - 183.2 181.1 - 11 133.2 133.2 - 133.7 - 136.0 133.9 - 12 133.0 133.1 - 134.5 - 126.0 134.3 - 13 133.3 133.4 117.5 - 111.1 111.7 - 14 136.3 136.3 132.0 - 130.0 129.6 - 15 57.7 57.8 4.59 (s) 9.0 2.13 (s) OMe 60.5 3.77 (s) 60.9 61.1 3.86 (s) a) Measured in CD3OD, b) 125 MHz, c) 500 MHz, $C of 2-hydroxymethyl-3-hydroxyanthraquinone in DMSO-d6, [4] #C of anthragallol-2-methyl ether in CDCl3. [11] The HRESIMS of 5 showed an ion peak at m/z 267.0661 [M-H] - (Calcd. for [C16H12O4] - , 267.0663), resulting in the molecular formula of C16H12O4. The 1 H- and 13 C-NMR spectra of compound 5 exhibited the feature of an anthraquinone with two carbonyl carbons at C-9 (δC 182.6) and C-10 (δC 180.0), two oxygenated carbons at C-1 (δC 160.6) and C-8 (δC 162.2), one ortho-disubstituted benzene at δH 8.05 (dd, J = 1.0, 7.5 Hz, H-5) and δC 125.9 (C-5), δH 7.78 (1H, ddd, J = 1.0, 7.5, 7.5 Hz, H-6) and δC 133.1 (C-6), δH 7.84 (1H, ddd, J = 1.0, 7.5, 7.5 Hz, H-7) and δC 134.3 (C-7), and δH 8.11 (1H, dd, J = 1.0, 7.5 Hz, H-8) and δC 126.5 (C-8). On the basis of the above evidence and comparison with respective literature data, [4] compound 5 was identified as rubiadin-1-methyl ether. The remaining compounds were characterized as galioside (3) [10] and anthragallol-2-methyl ether (6) [11] by comparing their observed and reported physical data (figure 1). Galioside (3) were reported for the first time from Morinda genus. Compounds, asperulosidic acid (1), [12] geniposidic acid (2), [13] 2- Vietnam Journal of Chemistry Iridoids and anthraquinones from the © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 31 hydroxymethyl-3-hydroxyanthraquinone (4), [4] rubiadin-1-methyl ether (5), [12] and anthragallol-2- methyl ether (6) [14] were already reported from M. officinalis. Acknowledgment. This research has inherited the research findings from the province-level study on titled Completing the process of producing Morinda officinalis How in Tay Giang by plant tissue culture and experientially planting it in Quang Nam. REFERENCES 1. J. H. Zhang, H. L. Xin, Y. M. Xu, Y. Shen, Y. Q. He, Y. Hsien, B. Lin, H. T. Song, L. Juan, H. Y. Yang, L. P. Qin, Q. Y. Zhang, J. Du. Morinda officinalis How. - A comprehensive review of traditional uses, phytochemistry and pharmacology, J. Ethnopharmacol., 2018, 213, 230-255. 2. F. Wang, L. Wu, L. Li, S. Chen. Monotropein exerts protective effects against IL-1β-induced apoptosis and catabolic responses on osteoarthritis chondrocytes, Int. Immunopharmacol., 2014, 23, 575-580. 3. H. L. Zhang, J. Li, G. Li, D. M. Wang, L. P. Zhu, D. P. Yang. Structural characterization and anti-fatigue activity of polysaccharides from the roots of Morinda officinalis, Int. J. Biol. Macromol., 2009, 44, 257- 261. 4. Y. B. Wu, C. J. Zheng, L. P. Qin, L. N. Sun, T. Han, L. Jiao, Q. Y. Zhang, J. Z. Wu. Antiosteoporotic activity of anthraquinones from Morinda officinalis on osteoblasts and osteoclasts, Molecules, 2009, 14, 573-583. 5. Z. Q. Wu, D. L. Chen, F. H. Lin, L. Lin, O. Shuai, J. Y. Wang, L. K. Qi, P. Zhang. Effect of bajijiasu isolated from Morinda officinalis F. C. How on sexual function in male mice and its antioxidant protection of human sperm, J. Ethnopharmacol., 2015, 164, 283-292. 6. J. Choi, K. T. Lee, M. Y. Choi, J. H. Nam, H. J. Jung, S. K. Park, H. J. Park. Antinociceptive anti- inflammatory effect of monotropein isolated from the root of Morinda officinalis, Biol. Pharm. Bull., 2005, 28, 1915-1918. 7. I. T. Kim, H. J. Park, J. H. Nam, Y. M. Park, J. H. Won, J. Choi, B. K. Choe, K. T. Lee. In-vitro and in- vivo anti-inflammatory and antinociceptive effects of the methanol extract of the roots of Morinda officinalis, J. Pharm. Pharmacol., 2005, 57, 607-615. 8. V. H. Giang, N. K. Ban, T. M. Linh, L. Q. Lien, N. X. Nhiem, D. T. Dung, B. H. Tai, H. L. T. Anh, P. H. Yen, C. V. Minh, P. V. Kiem. Iridoid glycosides from Morinda tomentosa and their endoplasmic reticulum stress modulation activity, Vietnam J. Chem., 2015, 53, 112-115. 9. C. S. Yuan, Q. Zhang, W. D. Xie, X. P. Yang, Z. J. Jia. Iridoids from Pedicularis kansuensis forma albiflora, Pharmazie, 2003, 58, 428-430. 10. S. R. Jensen, B. J. Nielsen. Iridoid glucosides in Fouquieriaceae, Phytochemistry, 1982, 21, 1623- 1629. 11. A. D. Pawlus, B.-N. Su, W. J. Keller, A. D. Kinghorn. An anthraquinone with potent quinone reductase-inducing activity and other constituents of the fruits of Morinda citrifolia (Noni), J. Nat. Prod., 2005, 68, 1720-1722. 12. X. Zhao, W. Kong, Y. Zhou, J. Wei, M. Yang. Evaluation and quantitative analysis of 11 compounds in Morinda officinalis using ultra-high performance liquid chromatography and photodiode array detection coupled with chemometrics, J. Sep. Sci., 2017, 40, 3996-4003. 13. J. Shi, X. Ren, J. Wang, X. Wei, B. Liu, T. Jia. Effects of the salt-processing method on the pharmacokinetics and tissue distribution of orally administered Morinda officinalis How. Extract, J. Anal. Methods Chem., 2020, 5754183. 14. M. Wang, Q. Wang, Q. Yang, X. Yan, S. Feng, Z. Wang. Comparison of anthraquinones, iridoid glycosides and triterpenoids in Morinda officinalis and Morinda citrifolia using UPLC/Q-TOF-MS and multivariate statistical analysis, Molecules, 2020, 25(1), 160. doi: 10.3390/molecules25010160. 15. P. V. Kiem, D. T. H. Yen, N. V. Hung, N. X. Nhiem, B. H. Tai, D. T. Trang, P. H. Yen, T. M. Ngoc, C. V. Minh, S. J. Park, J. H. Lee, S. Y. Kim, S. H. Kim. Five new pregnane glycosides from Gymnema sylvestre and their α-glucosidase and α-amylase inhibitory activities, Molecules, 2020, 25, 2525. doi: 10.3390/molecules25112525. Corresponding author: Phan Van Kiem Institute of Marine Biochemistry Vietnam Academy of Science and Technology 18, Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam E-mail: [email protected].