An initial study on chemical constituents of bischofia javanica - Nguyen Thi Mai

Journal of Science and Technology 55 (2) (2017) 188-194 DOI: 10.15625/0866-708X/55/2/8608 AN INITIAL STUDY ON CHEMICAL CONSTITUENTS OF BISCHOFIA JAVANICA Nguyen Thi Mai University of Transport and Communications, 3 Cau Giay, Lang Thuong, Dong Da, Ha Noi *Email: maidhgt@yahoo.com.vn Received: 25 July 2016; Accepted for publication: 22 February 2017 ABSTRACT From the methanol extract of Bischofia javanica Blume leaves, five compounds including 5'-β-D-glucopyranosyloxyjasmonic acid methyl ester (1), 2-(4-hydroxy-3-methoxyphenyl)ethyl- O-β-D-glucopyranoside (2), hexyl-O-β-D-glucopyranoside (3), friedelan-3-one (4), and gallic acid (5) were isolated. Their structures were elucidated by NMR spectra as well as in comparison with previously reported data. This is the first report on the isolation of 1, 2, and 3 from Bischofia javanica. Keyword: Bischofia javanica, jasmonic acid derivative, phenylethanoid. 1. INTRODUCTION Bischofia javanica Blume species, belonging to the family Euphorbiaceae, is found widely in Vietnam, India, China, Indonesia, and Philippine [1]. In folk medicinal remedies, B. javanica was used for treatment of various diseases such as cancer, inflammation, tuberculosis, diarrhea, sore throat, burns and different allergic conditions. The barks, leaves, roots, and fruits of this plant are used to treat diphtheria, pharyngitis, tonsillitis, different skin diseases, and nervous disorders [2]. Antileukemic activity of the leaves extract of B. javanica was evaluated on human leukemic cell lines by Lingadurai et al. The methanol extract of this plant showed significant cytotoxicity against HL-60 cell line with an IC50 value as low as 3.5 µg/ml. In addition, methanol extract B. javanica (10 µg/ml) was demonstrated to induce apoptosis of HL-60 cancer cells which was strongly supported for the ethno-medicinal use of B. javanica leaves in the treatment of cancer [3]. Betulinic acid and its derivatives from chloroform extract of the bark of B. javanica were found to be catalytic inhibitors of Topo II activities with IC50 values ranging from 0.38 to 58 µM [4]. Besides, methanol extract of B. javanica leaves was reported to have antioxidant, antiinflammatory and antinociceptive activities [5, 6]. However, chemical compositions from B. javanica have not been extensively investigated to date. Several triterpenoids and phenolics such as betulinic acid, ursonic acid, β-amyrine, chrysoeriol, quercetin have been isolated from the leaves of B. javanica [4, 7, 8]. To clarify the active components, the methanol extract of leaves of B. javanica was subjected to chemical study. Herein, we report the isolation and structural elucidation of five Chemical constituents of Bischofia javanica 189 compounds, 5'-β-D-glucopyranosyloxyjasmonic acid methyl ester (1), 2-(4-hydroxy-3- methoxyphenyl)ethyl-O-β-D-glucopyranoside (2), hexyl-O-β-D-glucopyranoside (3), friedelan-3- one (4), and gallic acid (5) from the methanol extract of B. javanica. 2. EXPERIMENTAL 2.1. Plant material The leaves of Bischofia javanica Blume were collected at Melinh, Vinhphuc province, Vietnam in June, 2012. Its scientific name was identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (BJ1-2012) is deposited at the Faculty of Basic Science, University of Transport and Communications. Figure 1. Chemical structures of 1 – 5. 2.2. General experimental procedures The 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on Agilent 400-MR-NMR spectrometer and TMS was used as an internal standard. Column chromatography was performed using silica gel (Kieselgel 60, 70 - 230 mesh and 230 – 400 mesh, Merck, Whitehouse Station, NJ) or RP-18 resins (30 – 50 µm, Fuji silysia Chemical Ltd.). Thin layer chromatography (TLC) was carried out using pre-coated silica-gel 60 F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm, Merck). 2.3. Extraction and isolation The dried and powdered of B. javanica leaves (2.0 kg) were extracted with methanol at 40oC for three times (10.0 L each). The organic layer was filtered and removed under vacuo to obtain 100.0 g of crude extract. This extract was suspended in distilled water (2.0 L) and successively partitioned with dichloromethane, ethyl acetate to give dichloromethane (BJC, 30.0 g), ethyl acetate (BJE, 12.0 g) extracts, and water soluble part. The BJC extract (30.0 g) was chromatographed on a silica gel column, eluting with a gradient elution of dichloromethane – methanol (50/1, 20/1, 5/1, 1/1, 0/1; v/v) to yield 5 fractions (BJC1 - BJC5). Fraction BJC2 (4.0 g) was chromatographed on a silica gel column, eluting with dichloromethane – acetone (10/1; v/v) to obtain 2 smaller fractions, named BJC2A and BJC2B. Compound 4 (10.0 mg) was obtained from fraction BJC2A using a silica gel column and eluted with dichloromethane – methanol (12/1; v/v). Compound 5 (7.0 mg) was obtained from fraction BJC2B using a RP-18 column and eluted with acetone/water (2/1; v/v). Nguyễn Thị Mai 190 Fraction BJC4 (3.0 g) was chromatographed on a RP-18 column, eluting with methanol/water (2/1; v/v) to obtain 3 sub-fractions, named BJC4A – BJC4C. Fraction BCJ4A was chromatographed on a RP-18 column, eluting with methanol/water/formic acid (2.5/1/0.01, v/v/v) to yield compound 3 (10.0 mg). Fraction BJC4C was chromatographed on a silica gel column eluting with dichloromethane/methanol (6/1; v/v) and further chromatographed on a RP- 18 column eluting with acetone/water/formic acid (1/1/0.01; v/v/v) to obtain compounds 1 (6.0 mg) and 2 (8.0 mg). 5’-β-D-Glucopyranosyloxyjasmonic acid methyl ester (1): white powder. MF: C19H30O9 (M = 402). ESI-MS: m/z 425 [M+Na]+. 1H-NMR (400 MHz, CD3OD) and 13C-NMR (100 MHz, CD3OD); see Table 1. 2-(4-Hydroxy-3-methoxyphenyl)ethyl-O-β-D-glucopyranoside (2): amorphous powder. MF: C15H22O8 (M = 330). ESI-MS: m/z 331 [M+H]+. 1H-NMR (400 MHz, CD3OD) and 13C-NMR (100 MHz, CD3OD); see Table 1. Hexyl-O-β-D-glucopyranoside (3): white amorphous powder. MF: C12H24O6 (M = 264). 1H- NMR (400 MHz, CD3OD) δH (ppm): 4.22 (1H, d, J = 7.6 Hz, H-1′), 3,88 (1H, dd, J = 6.8, 15.2 Hz, Ha-1), 3.84 (1H, d, J = 12.0 Hz, Ha-6′), 3.64 (1H, dd, J = 4.8; 12.0 Hz, Hb-6′), 3.51 (1H, dd, J = 7.2; 15.2 Hz, Hb-1), 3.32* (1H, H-3′), 3.30* (1H, H-5′), 3.24* (1H, H-4′), 3.14 (1H, t, J = 7.6 Hz, H-2′), 1.59 (2H, m, J = 6.8 Hz, H-2), 1.35* (2H, H-3), 1.29* (2H, H-5), 1.28* (2H, H-4) and 0.89 (3H, t, J = 6.8 Hz, H-6). 13C-NMR (100 MHz, CD3OD) δC: 104.4 (C-1′), 78.1 (C-3′), 77.9 (C-5′), 75.1 (C-2′), 71.6 (C-4′), 70.9 (C-1), 62.7 (C-6′), 32.9 (C-4), 30.8 (C-2), 26.8 (C-3), 23.7 (C-5) and 14.4 (C-6). *: overlapped signals. Friedelan-3-one (4): white amorphous powder. MF: C30H50O (M = 426). 1H-NMR (400 MHz, CDCl3) δH (ppm): 1.16 (s, 3H), 1.02 (s, 1H), 0.98 (s, 3H), 0.97 (s, 3H), 0.93 (s, 3H), 0.83 (d, J = 6.5 Hz, 3H), 0.84 (s, 3H) and 0.70 (s, 3H). 13C-NMR (100 MHz, CDCl3) δC (ppm): 213.4 (C-3), 59.4 (C-10), 58.2 (C-4), 53.1 (C-8), 42.7 (C-18), 42.1 (C-5), 41.5 (C-2), 41.2 (C-6), 39.7 (C-13), 39.2 (C-22), 38.2 (C-14), 37.4 (C-9), 35.9 (C-11), 35.6 (C-16), 35.3 (C-19), 35.0 (C-29), 32.7 (C-15), 32.4 (C-21), 32.0 (C-30), 31.7 (C-28), 30.5 (C-12), 29.9 (C-17), 28.1 (C-20), 22.3 (C-1), 20.2 (C-27), 18.6 (C-26), 18.2 (C-7), 17.9 (C-25), 14.6 (C-24), and 6.83 (C-23). Gallic acid (5): yellow amorphous powder. MF: C7H6O5 (M = 170). 1H-NMR (400 MHz, CD3OD) δH: 7.03 (2H, s, H-2, H-6). 13C-NMR (100 MHz, CD3OD) δC: 171.08 (COOH), 146.28 (C-3, C-5), 139.21 (C-4), 122.86 (C-1) and 110.33 (C-2, C-6). 3. RESULTS AND DISCUSSION Compound 1 was obtained as white powder. The 1H-NMR showed the presence of two olefin protons at δH 5.38 (1H, m) and 5.50 (1H, m); an anomeric proton at δH 4.25 (1H, d, J = 8.4 Hz) assigned for a sugar moiety; and a methoxy group at δH 3.66 (3H, s). The 13C-NMR and DEPT of 1 showed 19 carbon signals, including two carbonyl groups (δC 221.6 and 174.5); two olefin carbons (δC 129.0 and 128.9); an oxymethylene carbon (δC 70.0); two methine carbons and three methylene carbons from δC 26.3 to 55.0; and five oxymethine and an oxymethylene of a β-glucopyranoside (δC 104.3, 75.1, 78.1, 71.6, 77.9 and 62.7). The one bond proton-carbon signals were assigned on the basis of HSQC correlations (Table 1). The COSY correlations between H-1 (δH 2.38) and H-2 (δH 1.97); between H-1′ (δH 2.40) and H-2′ (δH 5.38); between H- 2′ (δH 5.38) and H-3′ (δH 5.50); between H-3′ (δH 5.50) and H-4′ (δH 2.42); between H-4′ (δH 2.42) and H-5′ (δH 3.54 and 3.88) confirmed the constitutional fragments (Figure 2). The HMBC correlations from H-5′ (δH 3.54 and 3.88) to carbons C-3′ (δC 129.0), C-4′ (δC 29.0), C-1′′′ (δC Chemical constituents of Bischofia javanica 191 104.3); from H-2′ (δH 5.38) to carbons C-4′ (δC 29.0), C-2 (δC 55.0); from H-3′ (δH 5.50) to carbon C-1′ (δC 39.5) confirmed the position of double bond at C-2′/C-3′ and the position of glucose moiety at C-5′. Besides, the HMBC correlations from methoxy group (δH 3.66) and H-2′ (δH 2.38) to carbonyl carbon C-1′ (δC 174.5) were observed confirming the linkage of methyl ester at C-2′. Based on the above evidence, chemical structure of 1 was established as 5'-β-D- glucopyranosyloxyjasmonic acid methyl ester [9] and shown in Figure 1. This compound was previously isolated from leaves of Thyus vulgaris [10], Phyllanthus urinaria [11] and possessed a weak cytotoxic activity against CHO (Chinese hamster ovary) and J774 (Murine macrophage) cells [11]. Table 1. 1H- and 13C-NMR data for 1-2 and reference compounds. 1 2 C dδC# [9] a,bδC a,cδH (J, Hz) C aδC$ [12] a,bδC a,cδH (J, Hz) 1 39.2 39.2 2.38 (m) 1 131.8 131.5 - 2 55.0 55.0 1.97 (dt, 5.0; 10.1) 2 114.0 113.6 6.80 (s) 3 221.6 221.6 - 3 148.9 148.8 - 4 38.6 38.6 2.39* 2.12 (m) 4 146.0 145.8 - 5 28.1 28.1 2.20 (m) 1.50 (m) 5 116.2 116.0 6.63* 1′ 26.4 26.3 2.40* 6 122.5 122.4 6.63* 2′ 128.9 128.9 5.38 (m) 7 36.8 36.7 2.79 (t, 6.8) 3′ 129.0 129.0 5.50 (m) 8 72.0 72.0 4.00 (m) 3.66 (m) 4′ 29.0 29.0 2.42* 1′ 104.4 104.3 4.24 (d, 7.5) 5′ 70.2 70.2 3.88* 3.54 (dd, 7.2, 10.0) 2′ 75.2 75.1 3.13 (dd, 7.5, 9.0) 1″ 174.5 174.5 - 3′ 78.2 78.1 3.31 (t, 9.0) 2″ 39.5 39.5 2.71 (dd, 3.6, 14.6) 2.40* 4′ 71.8 71.6 3.22 (t, 9.0) COOMe 52.1 52.1 3.66 (s) 5′ 78.0 77.9 3.21 (m) 1′′′ 104.4 104.3 4.25 (d, 7.5) 6′ 62.9 62.7 3.81 (dd, 3.0, 12.0) 3.62 (dd, 5.0, 12.0) 2′′′ 75.1 75.1 3.15 (dd, 7.5, 9.0) OMe 56.6 56.3 3.78 (s) 3′′′ 78.1 78.1 3.32 (t, 9.0) 4′′′ 71.6 71.6 3.30 (t, 9.0) 5′′′ 77.9 77.9 3.29 (m) 6′′′ 62.8 62.7 3.84 (dd, 3.0, 12.0) 3.64 (dd, 5.0, 12.0) Measured in a) CD3OD b)100 MHz, c)400 MHz. *overlapped signals. δC# 5'-β-D-glucopyranosyloxyjasmonic acid methyl ester [9], δC$ 2-(4-hydroxy-3-methoxyphenyl)ethyl-O-β-D-glucopyranoside [12]. Compound 2 was obtained as amorphous powder. The 1H-NMR showed three protons of a 1,3,4-trisubstituted aromatic ring at δH 6.80 (s, H-2), 6.63 (overlapped, H-5 and H-6); an anomeric proton at δH 4.24 (1H, d, J = 7.6 Hz); and a methoxy group at δH 3.78 (3H, s). The 13C- NMR and DEPT showed six carbon signals of a 1,3,4-trisubstituted aromatic ring at δC 148.8 (C-3), 145.8 (C-3), 131.5 (C-1), 122.4 (C-6), 116.0 (C-5) and 113.6 (C-2); six carbons of a Nguyễn Thị Mai 192 β-glucose at δC 104.3 (C-1′), 75.1 (C-2′), 78.1 (C-3′), 71.6 (C-4′), 77.9 (C-5′) and 62.7 (C-6′). Besides, the signals of a oxymethylen carbon at δC 72.0 (C-8); a methylene carbon at δC 36.7 (C- 7); and a methoxy group at δC 56,3 were observed. The NMR data of 2 were similar to those of 2-(4-hydroxy-3-methoxyphenyl)ethyl-O-β-D-glucopyranoside [12]. The chemical structure of 2 was assigned with the aid of HSQC (Table 1) and HMBC (Figure 2) spectra. The HMBC correlation from anomeric proton H-1′ (δH 4.24) to carbon C-8 (δC 72.0) confirmed the position of β-glucose moiety at C-8. The position of methoxy group at C-3 was confirmed by the HMBC correlations from methylene proton H-7 (δH 2.79) to carbons C-2 (δC 113.6)/C-6 (δC 122.4), from H-6 (δH 6.62) to carbon C-4 (δC 145.8) and from methoxy signal (δH 3.78) to carbon C-3 (δC 148.8). Based on the above evidence, chemical structure of 2 was established as 2-(4-hydroxy-3- methoxyphenyl)ethyl-O-β-D-glucopyranoside and shown in Figure 1. Compound 2 were previously isolated from various plant such as Tetrastigma hemsleyanum [13], Nanophyton erinaceum [14] and Laurus nobilis [12]. It was reported to have antioxidant and hepatoprotective effects [15]. O O COOMe O HO OH OH OH MeO HO O O HO OH OH OH 1 2 HMBC COSY Figure 2. The key COSY and HMBC correlations of 1 and 2. Compounds 3, 4, and 5 were identified as hexyl-O-β-D-glucopyranoside [16], friedelan-3- one [17], and gallic acid [18] based on their spectral evidence, which were in agreement with those of the reported data in the literature, respectively. Among isolated compounds, the compounds 1, 2 and 3 were firstly isolated from B. javanica leaves. Meanwhile, compounds 4 and 5 have been previously isolated from this plant, showing broad range of biological activities [7, 8]. Gallic acid (5) is known as the active principle responsible for the regeneration of β-cells and normalizing all the biochemical parameters related to the patho-biochemistry of diabetes mellitus and hence it could be used as a potent antidiabetic agent [18]. 4. CONCLUSIONS Five compounds 5'-β-D-glucopyranosyloxyjasmonic acid methyl ester (1), 2-(4-hydroxy-3- methoxyphenyl)ethyl-O-β-D-glucopyranoside (2), hexyl-O-β-D-glucopyranoside (3), friedelan-3- one (4), and gallic acid (5) were isolated from dichloromethane soluble fraction of B. javanica leaves. Compounds 1, 2 and 3 were isolated from B. javanica for the first time. Chemical constituents of Bischofia javanica 193 REFERENCES 1. Chi V. V. - Dictionary of Vietnamese Medicinal Plants, Medical Publishing House, 2 (2012) 364. 2. Rajbongshi P., Zaman K., Boruah S., Das S. - A review on traditional use and phytopharmacological potential of Bischofia javanica Blume, Int. J. Pharm. Sci. Rev. Res. 24 (2) (2014) 24-29. 3. Lingadurai S., Roy S., Joseph R. V., Nath L. K. - Antileukemic activity of the leaf extract of Bischofia javanica blume on human leukemic cell lines. Indian J. Pharmacol. 43(2) (2011) 143-149. 4. Wada S., Tanaka R. - Betulinic acid and its derivatives, potent DNA topoisomerase II inhibitors, from the bark of Bischofia javanica. Chem. Biodivers. 2(5) (2005) 689-694. 5. Lingadurai S., Kar P. K., Nath L. K., Besra S. E., Joseph R. V. S. - Free radical scavenging activity of leaves of Bischofia javanica Blume and Fraxinus floribunda Wallich. Pharmacologyonline 1 (2009) 1324-1332. 6. Lingadurai S., Nath L. K., Kar P. K., Besra S. E., Joseph R. V. - Antiinflammatory and antinociceptive activities of methanolic extract of leaves of Bischofia javanica Blume on experimental animals. Asian J. Chem. 19(7) (2007) 5150-5156. 7. Gupta D. R., Dhiman R. P., Naithani S., Ahmed B. - Chemical investigation of Bischofia javanica Blume. Pharmazie 43(3) (1988) 222-223. 8. An N. T. - Isolation and identification of Epi-friede lanol acetate, β-sitosterol and gallic acid from the leaves of Bischofia javanica (Blume). Tap Chi Duoc Hoc 49 (4) (2009) 41-43. 9. Fujita T., Terato K., Nakayama M. - Two jasmonoid glucosides and a phenylvaleric acid glucoside from Perilla frutescens. Biosci. Biotechnol. Biochem. 60 (4) (1996) 732-735. 10. Kitajima J., Ishikawa T., Urabe A. - A new hydroxyjasmone glucoside and its related compounds from the leaf of thyme. Chem. Pharm. Bull. 52 (8) (2004) 1013-1014. 11. Thanh N. V., Huong P. T. T., Nam N. H., Cuong N. X., Thao N. P., Dejaegher B., Gordien A., Vander Heyden Y., Quetin-Leclercq J., Minh C. V. - A new flavone sulfonic acid from Phyllanthus urinaria. Phytochem. Lett. 7 (2014) 182-185. 12. De Marino S., Borbone N., Zollo F., Ianaro A., Di Meglio P., Iorizzi M. - Megastigmane and phenolic components from Laurus nobilis L. leaves and their inhibitory effects on nitric oxide production, J. Agric. Food Chem. 52 (25) (2004) 7525-7531. 13. Liu H. X., He L., Huang R. M., Qiu S. X. - Chemical constituents of the rhizomes of Tetrastigma hemsleyanum, Chem. Nat. Compd. 51 (6) (2015) 1077-1079. 14. Han J., Li, L., Han L., Huang X., Yuan T. - Phenylpropanoid amides and phenylethanols from Nanophyton erinaceum, Biochem. Syst. Ecol. 61 (2015) 399-401. 15. Guo Y., Zheng C., Xu W., Si Y., Dou S., Yang Y. - Free radical scavenging and hepatoprotective effects of salidroside analogs on CCl4-induced cytotoxicity in LO2 cells, Med. Chem. Res. 22 (5) (2013) 2524-2530. 16. Kishida M., Nishiuchi M., Kato K., Akita H. - Chemoenzymatic synthesis of n-hexyl and O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranosides, Chem. Pharm. Bull. 52(9) (2004) 1105-1108. Nguyễn Thị Mai 194 17. Hisham A., Kumar G. J., Fujimoto Y., Hara N. - Salacianone and salacianol, two triterpenes from Salacia beddomei, Phytochemistry 40 (4) (1995) 1227-1231. 18. Latha R. C. R., Daisy P. - Insulin-secretagogue, antihyperlipidemic and other protective effects of gallic acid isolated from Terminalia bellerica Roxb. in streptozotocin-induced diabetic rats, Chemico-Biological Interactions 189 (1–2) (2011) 112-118.