Chemical constituents of Albizia myriophylla wood and the HPLC determination of some high yield compounds as markers

Cite this paper: Vietnam J. Chem., 2020, 58(5), 597-601 Article DOI: 10.1002/vjch.202000018 597 Wiley Online Library © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Chemical constituents of Albizia myriophylla wood and the HPLC determination of some high yield compounds as markers Pham Thi Tam1, Phung Van Trung2, Vo Thi Nga3, Nguyen Thi Anh Tuyet4, Nguyen Kim Phi Phung5, Ngo Thi Thuy Duong5, Nguyen Thi Hoai Thu6* 1Sai Gon University, 273, An Duong Vuong, Dist. 5, Ho Chi Minh City 70000, Viet Nam 2Center for Research and Technology Transfer, Vietnam Academy of Science and Technology, 18, Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 3University of Technology and Education Ho Chi Minh City, 1, Vo Van Ngan, Thu Duc, Ho Chi Minh City 70000, Viet Nam 4University of Education Ho Chi Minh City, 280, An Duong Vuong, Dist. 5, Ho Chi Minh City 70000, Viet Nam 5University of Science, Vietnam National University - Ho Chi Minh City, 227, Nguyen Van Cu, Dist.5, Ho Chi Minh City 70000, Viet Nam 6University of Medicine and Pharmacy at Ho Chi Minh City, 217, Hong Bang, Dist. 5, Ho Chi Minh City 70000, Viet Nam Submitted February 21, 2020; Accepted March 19, 2020 Abstract Spinasterol (1) and four flavonoids (2-5) were isolated from Albizia myriophylla wood collected at Phu Yen province, Vietnam. The chemical structures of these compounds were elucidated by NMR spectroscopy as well as compared with data in the literature. The contents of some high yield isolated compounds (2-4) in the ethanol extract of A. myriophylla were quantified by high-performance liquid chromatography. These HPLC chromatograms could be used to reveal the presence of isolated compounds as markers of the sample. Keywords. Albizia myriophylla, flavonoid, content determination, HPLC. 1. INTRODUCTION Albizia myriophylla is widely distributed in Asia such as India, Laos, Cambodia, Myanmar, Thailand and Vietnam etc.[1] In Vietnamese traditional medicine, it was used to cure cough, bronchitis, toothache, obesity, dyspepsia, diabetes, cancer etc.[1- 3] There were many researches about chemical constituents and bioactivities of Albizia myriophylla.[2-5] However, in Vietnam, there has been no chemical study on this plant although it is now cultivated at Phu Yen province for the internal commercial use thanks to its above-mentioned medicinal value. In this paper, we report the isolation of five compounds, spinasterol (1), 7,3',4'-trihydroxy-3- methoxyflavone (2), fisetin (3), (2S)-7,3',4'- trihydroxyflavanone (4) and (2S,3S,4S)-7,3,4,3',4'- pentahydroxyflavane (5). The contents of some high yield compounds (2, 3, 4) were determined by HPLC. 2. MATERIALS AND METHODS 2.1. General experimental procedures The 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer. LR-MS spectra were recorded by UPLC-MS (Ultimate RS 3000 MSQ Pluc. Thermo, USA. Optical rotations were measured on a Kruss (Germany) polarimeter with the length of the tube of 2 decimetres. HPLC analyses were carried out on a HPLC Hewlett Packard 1050 series system include a Quartenary pump, an Autosampler, a Thermostatted column and a DAD detector. All moduls and the data processing were controlled by Chemstation software (Ver. A.10). The column was Phenomenex Vietnam Journal of Chemistry Nguyen Thi Hoai Thu et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 598 LUNA 5u C18 (250×4.6 mm, 5 m). The mobile phase was composed of methanol (A) and aqueous formic acid 0.1 % (B): from 0-18 min, 35 % A, 65 % B; from 19-23 min, 70 % A, 30 % B; from 24-30 min, 100 % A, 0 % B; at a flow rate 1.0 mL/min. Injection volume was of 10 L. All sample and pure compounds were filtered by 0.45 m membrane before injection. 2.2. Plant material Albizia myriophylla wood sample was collected at Hoa Hiep Nam industrial zone, Dong Hoa district, Phu Yen province, Vietnam in June, 2018. This was the donation of Mr. Hoang Xuan Lam, Middle Vietnam Research and Manufacturing Organic Medicinal Herb Centre in Phu Yen province. 2.3. Extraction and isolation The dried powder wood (3.1 kg) of A. myriophylla was exhaustedly extracted with ethanol by maceration at room temperature. After evaporating, the ethanolic filtrated solution gave crude extract (210 g). This crude was chromatographed on silica gel and successively eluted with n-hexane, ethyl acetate and methanol to afford three fractions, respectively. The ethyl acetate extract was chromatographed, eluted with n-hexane:ethyl acetate (20:80, 0:100), ethyl acetate:methanol (90:10, 80:20, 70:30, 0:100) to give 5 sub-fractions, EA1EA5. The sub-fraction EA2 (7.0 g) was applied to a silica gel column, and eluted with n-hexane:ethyl acetate (96:4) to afford 1 (15 mg). The same procedure for the sub-fraction EA3 (5.0 g) was carried out, eluted with n-hexane:ethyl acetate (60:40) to obtain 2 (110 mg), 3 (24 mg), 4 (15 mg) and 5 (8.5 mg). Spinasterol (1): white powder. ESI-LRMS: m/z 473.6 [M+CH3COOH+H]+. 1H-NMR (CDCl3,  ppm, J in Hertz): 3.59 (1H, m, H3), 5.15 (1H, brs, H7), 0.55 (3H, s, H18), 0.80 (3H, s, H19), 1.02 (3H, d, 6.5, H21), 5.16 (1H, dd, 14.5, 9.0, H22), 5.03 (1H, dd, 15.0, 8.5, H23), 0.85 (3H, d, 6.0, H26), 0.80 (3H, d, 6.0, H27), 0.81 (3H, t, 7.0, H29). 13C-NMR (CDCl3,  ppm): 37.1 (C1), 31.4 (C2), 71.0 (C3), 37.9 (C4), 40.2 (C5), 29.5 (C6), 117.4 (C7), 139.5 (C8), 49.4 (C9), 34.1 (C10), 21.5 (C11), 39.4 (C12), 43.2 (C13), 55.0 (C14), 22.9 (C15), 28.4 (C16), 55.8 (C17), 11.9 (C18), 12.9 (C19), 40.7 (C20), 21.3 (C21), 138.1 (C22), 129.4 (C23), 51.2 (C24), 31.8 (C25), 21.0 (C26), 18.9 (C27), 25.3 (C28), and 12.1 (C29). 7,3',4'-Trihydroxy-3-methoxyflavone (2): yellow crystal. ESI-LRMS m/z 301.8 [M+H]+. 1H- NMR (CDCl3,  ppm, J in Hertz): 7.95 (1H, d, 8.5, H5), 6.81 (1H, dd, 8.5, 2.0, H6), 6.80 (1H, d, 2.0, H8), 7.60 (1H, d, 1.5, H2), 6.86 (1H, d, 8.5, H5), 7.49 (1H, dd, 8.5, 1.5, H6), 3.70 (3H, s, OCH3). 13C- NMR (CDCl3) data, see table 2. Fisetin (3): yellow crystal. ESI-LRMS m/z 287.3 [M+H]+. 1H-NMR (CD3OD, 500 MHz,  ppm, J in Hertz): 7.99 (1H, d, 9.0, H5), 6.92 (1H, brd, 9.0, H6), 6.91 (1H, brs, H8), 7.78 (1H, brs, H2), 6.89 (1H, d, 8.0, H5), 7.67 (1H, brd, 8.0, H6). 13C-NMR (CD3OD) data, see table 2. (2S)-7,3',4'-Trihydroxyflavanone (4): yellow powder. ESI-LRMS m/z 271.1 [M-H]-. [α]D 25 -22.7 (c 0.75, EtOAc). 1H-NMR (CDCl3,  ppm, J in Hertz): 5.23 (1H, dd, 13.0, 2.0, H2), 2.93 (1H, dd, 16.5, 13.0, H3a), 2.66 (1H, dd, 16.5, 2.5, H3b), 7.70 (1H, d, 8.5, H5), 6.44 (1H, dd, 8.5, 1.5, H6), 6.31 (1H, d, 1.5, H8), 6.87 (1H, d, 2.0, H2), 6.77 (1H, d, 8.5, H5), 6.74 (1H, dd, 8.5, 2.0, H6). 13C-NMR (CDCl3) data, see table 2. Table 1: The contents of some isolated compounds in the ethanol extract of A. myriophylla Compound Rt (min) Intergration C (ppm) m (mg) a (%) Content (mg/g) 2 14.299 299.312 11.62 121.9 15 1.12 3 14.105 49.500 4.354 121.9 15 0.42 4 12.083 581.433 12.60 121.9 15 1.22 C: Concentration of the compound; m: Mass of the ethanol extract; a: Humidity of the ethanol extract. (2S,3S,4S)-7,3,4,3',4'-Pentahydroxyflavane (5): brown powder. ESI-LRMS m/z 289.1 [M-H]-. [α]D 25 +38.9 (c 4.25, MeOH). 1H-NMR (CD3OD, 500 MHz,  ppm, J in Hertz): 5.02 (1H, brs, H2), 3.99 (1H, d, 4.0, H3), 4.95 (1H, d, 4.0, H4), 7.32 (1H, d, 8.5, H5), 6.45 (1H, dd, 8.5, 2.5, H6), 6.32 (1H, d, 2.0, H8), 7.05 (1H, d, 1.5, H2), 6.80 (1H, d, 8.5, H5), 6.86 (1H, dd, 8.0, 1.5, H6). 13C-NMR (CD3OD, 125 MHz) data, see table 2. 2.4. HPLC determination of major compounds in the ethanol extract of A. myriophylla Compounds 2, 3, 4 were studied because of their good isolated amount and bioactivities.[5] They were used as standards with the purity of about 98 %. Vietnam Journal of Chemistry Chemical constituents of Albizia myriophylla © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 599 Establish of standard curve of pure compounds To establish of standard curve of pure compounds, 10 mg of each compound was weighted and dissolved perfectly in 10 mL MeOH as stock solution. A certain amount of this solution of each compound was then taken and diluted to 10 mL in a volumetric flask to get standard solutions. The standard curve for each compound was performed as shown in figures 1 and 2. Figure 1: Standard curves of (2), (3), (4) Preparation of sample 100 mg of the ethanol extract was weighted and dissolved perfectly in 10 mL MeOH then filtered by using a 0.45-m membrane before injection. Content calculation of 2-4 in the ethanol extract The content of each compound in the ethanol extract was determined by the following formula and the result was presented in table 1. C: Concentration of compound (ppm) calculated from standard curve equation; V: Volumetric volume (mL); m: Mass of ethanol extract (g); a: Humidity of ethanol extract (%). Figure 2: HPLC chromatograms of 2, 3, 4 (the lower lines) and ethanol extract (the upper lines) at  = 350 nm, 350 nm, 280 nm, respectively 3. RESULTS AND DISCUSSION Compound 1 was isolated as a white powder. The 1H-NMR spectrum showed signals of three olefin protons at H 5.15 (H7), 5.16 (H22), 5.03 (H23), and one oxymethine group at H 3.59 (H3). In the higher magnetic field, there were six signals of methyl groups including two singlets (H 0.55, H18), 0.80 (H19)], three doublets (H 1.02, H21), 0.85 (H26), 0.80 (H27)], and one triplet (H 0.81, H29)]. The 13C-NMR spectrum indicated the presence of 29 carbons with two C=C bonds, one oxymethine group and six methyl groups. 1 was elucidated as spinasterol by the good compatibility of its MS and NMR data with those in the literature.[6] Compound 2 was isolated as a yellow crystal. The 1H-NMR spectrum displayed three signals of min2.5 5 7.5 10 12.5 15 17.5 20 22.5 mAU 0 20 40 60 80 100 DAD1 E, Sig=350,16 Ref=550,100 (KQ_HPLC1\CT_AM414.D) 1 4. 25 9 DAD1 E, Sig=350,16 Ref=550,100 (KQ_HPLC1\CT_EA-40.D) 5 .0 51 5 .7 36 6 .1 84 6 .6 19 6 .9 46 8 .8 59 9 .9 12 1 0. 38 9 1 0. 84 2 1 1. 28 3 1 2. 02 6 1 3. 37 2 1 4. 29 9 1 4. 65 5 1 5. 02 1 1 5. 50 8 1 6. 60 2 1 7. 88 1 1 8. 18 5 1 8. 47 2 1 8. 65 9 1 8. 92 6 1 9. 10 0 2 0. 68 7 Ethanol extract Compound 2 min2.5 5 7.5 10 12.5 15 17.5 20 22.5 mAU 0 20 40 60 80 DAD1 E, Sig=350,16 Ref=550,100 (D:\KETQUA~1\CHI_TAM\CT_EA000.D) 1 .8 47 2 .1 06 2 .5 65 1 0. 79 3 1 3. 77 8 1 4. 10 5 1 4. 51 6 2 3. 02 6 DAD1 E, Sig=350,16 Ref=550,100 (D:\KETQUA~1\CHI_TAM\CT_AM802.D) 1 4. 14 1 Ethanol extract Compound 3 min2 4 6 8 10 12 14 16 18 mAU -20 0 20 40 60 80 100 120 DAD1 D, Sig=280,16 Ref=550,100 (D:\KETQUA~1\CHI_TAM\CT_EA000.D) 4 .5 02 5 .7 51 6 .8 54 7 .6 29 8 .4 05 9 .6 52 1 1. 47 9 1 2. 08 3 1 3. 78 0 1 4. 51 8 DAD1 D, Sig=280,16 Ref=550,100 (D:\KETQUA~1\CHI_TAM\CT_AM500.D) 1 2. 07 0 DAD1 D, Sig=280,16 Ref=550,100 (D:\KETQUA~1\CHI_TAM\CT_AM500.D) 1 2. 07 0 Compound 4 Ethanol extract Vietnam Journal of Chemistry Nguyen Thi Hoai Thu et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 600 aromatic protons at H 7.95 (1H, d, 8.5 Hz, H5), 6.81 (1H, dd, 8.5, 2.0 Hz, H6), 6.80 (1H, d, 2.0 Hz, H8), confirming the presence of a 1,2,4-trisubstituted A benzene ring system of flavonoids. The 1,3,4- trisubstituted B benzene ring was determined by the signals of three aromatic protons at H 7.60 (1H, d, 1.5 Hz, H2), 6.86 (1H, d, 8.5 Hz, H5), 7.49 (1H, dd, 8.5, 1.5 Hz, H6). Additionally, the 1H-NMR spectrum showed one methoxy group at H 3.70. The 13C-NMR spectrum corresponded to 15 carbons, including one carbonyl carbon at C 175.3 (C4), six oxygenated aromatic carbons and eight aromatic carbons (table 2). The HMBC correlation between protons of -OCH3 with C3 confirmed this methoxy group attached to C3. Based on all the aforementioned analysis and the comparison of MS and NMR data (table 2) with those reported in the literature,[7] 2 was determined as 7,3',4'-trihydroxy- 3-methoxyflavone. Table 2: 13C-NMR data of isolated compounds No C (ppm) 2a 3b 4a 5b 2 156.1 148.5 80.4 78.5 3 140.1 138.4 44.6 69.4 4 175.3 174.3 192.8 67.2 5 126.9 127.3 129.8 128.3 6 115.1 115.8 111.6 108.3 7 162.5 164.1 165.8 157.4 8 102.3 102.8 103.7 101.9 9 157.2 158.3 164.6 155.1 10 116.9 115.3 114.7 115.0 1 122.5 124.2 131.3 130.4 2 115.3 116.1 114.1 114.0 3 144.5 147.4 145.9 144.6 4 147.6 146.1 145.5 144.6 5 114.9 115.8 115.9 114.6 6 121.4 121.7 119.2 118.0 3-OCH3 59.5 - - - a) In CDCl3; b) In CD3OD. Compound 3 was isolated as a yellow crystal. The NMR data of 3 were similar to those of 2, except that the methoxy group at C3 was replaced by a hydroxyl group. Based on the MS and NMR data (table 2) compared to the ones in published data,[8] 3 was identified as fisetin. Compound 4 was isolated as a yellow powder. The NMR data demonstrated that 4 was a flavanone with the lack of double bond at C2. The comparison of the measured specific optical rotation value of 4 {[α]D 25 –22.7 (c 0.75, EtOAc)} with the ones of (2S)- 7,3',4'-trihydroxyflavanone isomer {[α]D 22 -36.1 (c 0.8, MeOH) or [α]D 30 -18.7 (c 0.5, MeOH)},[9] as well as the good compatibility of its MS and NMR data (table 2) with those published in the literature,[9,10] 4 was thus (2S)-7,3',4'- trihydroxyflavanone. Figure 3: Structures of isolated compounds from Albizia myriophylla wood Compound 5 was obtained as a brown powder. The NMR data suggested that 5 was a flavanane derived from 3 with the disappearance of the double bond at C2 and the carbonyl group at C4 and the appearance of two hydroxyl groups at C3 and C4. According to Drewes et al.,[11] the coupling constants J2,3 and J3,4 (in Hertz) of flavan-3,4-diol derivatives were around 1.0 and 4.4 in (+)-2,3-cis- 3,4-cis isomer; 1.0 and 2.7 in (+)-2,3-cis-3,4-trans isomer; 10.0 and 3.4 in (+)-2,3-trans-3,4-cis isomer; and 9.0 and 7.1 in (+)-2,3-trans-3,4-trans isomer, respectively. Compound 5 possessed a set of proton signals including 5.02 (brs, H-2), 3.99 (dd, 1.0, 4.0 Hz, H-3) and 4.95 (d, 4.0 Hz) corresponding to the coupling constants of (+)-2,3-cis-3,4-cis isomer. MS and NMR data (table 2) of 5 were compatible with those of published ones.[2,12] The dextrorotatory value of 5 {[α]D 25 +38.9 (c 4.25, MeOH)} matched with the one of [α]D 20 +48.5 (c 0.8, acetone-H2O) reported for (2S,3S,4S)-7,3,4-trihydroxy-2,3-cis- flavan-3,4-cis-diol.[13] Therefore 5 was assigned as (2S,3S,4S)-7,3,4,3',4'-pentahydroxyflavane. The contents of some isolated compounds in the ethanol extract of A. myriophylla determined by HPLC were displayed in table 1 and figures 1-2. From the results, these flavonoids 2-4 were found as major compounds in the ethanol extract of A. myriophylla wood. These HPLC chromatograms could be used to reveal the presence of isolated compounds as markers of the sample. Fisetin (3) had Vietnam Journal of Chemistry Chemical constituents of Albizia myriophylla © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 601 a small content in the ethanol extract, however it possessed potential interest in the prevention or treatment of cancer in vivo, antioxidant and anti- inflammatory[5] The bioactivities of the two rest flavonoids (2, 4) have not much been studied. Compound 2 showed no active in assays of the inhibition of xanthine oxidase[14] as well as antimalarial activity against Plasmodium falciparum.[15] Compound 4 was tested for free radical scavenging activity and its percentage of reduced DPPH was 14.5 % at a dose of 410-8 mol.[15] Therefore, it needs necessary research for the biological properties of these compounds in order to support the traditional use of A. myriophylla wood. 4. CONCLUSION From the Albizia myriophylla wood, collected at Phu Yen province, spinasterol (1) and four flavonoids (2- 5) were isolated and elucidated the structures. This is the first time these compounds are known in A. myriophylla. The contents of some high yield isolated compounds (2-4) in the ethanol extract were determined by HPLC and these chromatograms could be used as markers to verify the presence of major flavonoids of the sample. Acknowledgements. We are grateful to Mr. Hoang Xuan Lam (Co-Founder of HerbEco Company) for Albizia myriophylla sample and M.Sc. Bui Ngoc Duy for the good image of the plant. REFERENCES 1. T. A. T. Nong, Q. T. Nguyen, T. M. T. Nguyen, T. H. Dao. Study on the phytomorphology of the Albizia myriophylla Benth. plants collected in Thai Nguyen, Vietnam J. Sci. Tech., 2017, 17(6), 10-12. 2. N. Joycharat, C. Boonma, S. Thammavong, B. Yingyongnarongkul, S. Limsuwan, S. P. Voravuthikunchai. Chemical constituents and biological activities of Albizia myriophylla wood, Pharm. Biol., 2016, 54, 62-73. 3. M. Yoshikawa, T. Morikawa, K. Nakano, Y. Pongpiriyadacha, T. Murakami, H. Matsuda. Characterization of new sweet triterpene saponins from Albizia myriophylla, J. Nat. Prod., 2002, 65, 1638-1642. 4. N. Joycharat, S. Thammavong, S. Limsuwan, S. Homlaead, S. P. Voravuthikunchai, B. Yingyongnarongkul, S. Dejadisai, S. Subhadhirasakul. Antibacterial substances from Albizia myriophylla wood against cariogenic Streptococcus mutans, Arch. Pharm. Res., 2013, 36, 723-730. 5. Y. S. Touil, N. Auzeil, F. Boulinguez, H. Saighi, A. Regazzetti, D. Scherman, G. G. Chabot. Fisetin disposition and metabolism in mice: Identification of geraldol as an active metabolite, Biochem. Phar., 2011, 82, 1731-1739. 6. L. J. Goad, Toshihiro Akihisa. Analysis of sterols, Blackie Academic and Professional, London, UK, 1997, 388. 7. J. H. Wu, Y. T. Tung, S. C. Chien, S. Y. Wang, Y. H. Kuo, L. F. Shyur, S. T. Chang. Effect of phytocompounds from the heartwood of Acacia confusa on inflammatory mediator production, J. Agri. Food Chem., 2008, 56, 1567-1573. 8. M. P. Costa, M. C. V. Bozinis, W. M. Andrade, C. R. Costa, A. L. Silva, C. M. Oliveira, L. Kato, O. F. Fernandes, L. K. H. Souza, M. R. R. Silva. Antifungal and cytotoxicity activities of the fresh xylem sap of Hymenaea courbaril L. and its major constituent fisetin, BMC Comple. Alte. Med. 2014, 14, 245-252. 9. A. R. Jassbi, P. Singh, V. Krishna, P. K. Gupta, S. Tahara. Antioxidant study and assignments of NMR spectral data for 3’,4’,7-trihydroxyflavanone 3’,7-di- O-β-D-glucopyranoside (butrin) and its hydrolyzed product, Chem. Nat. Com., 2004, 40(3), 250-253. 10. W. L. Kuo, C. C. Chen, P. H. Chang, L. Y. Cheng, B. J. Shien, Y. L. Huang. Flavonoids from Guizotia abyssinica, J. Chin. Med., 2007, 18, 121-128. 11. S. E. Drewes, D. G. Roux. Optically active diastereoisomers of (+)-mollisacacidin by epimerization, Biochem. J., 1965, 94, 482-487. 12. P. J. Steynberg, J. P. Steynberg, E. V. Brandt, D. Ferreira, R. W. Hemingway. Oligomeric flavonoids. Part 26. Structure and synthesis of the first profisetinidins with epifisetinidol constituent units, J. Chem. Soc. Perkin Trans., 1997, 28(45), 1943-1950. 13. S. E. Drewes, A. H. Ilsley. Isomeric leucofisetinidins from Acacia mearnsii, Phytochemistry, 1969, 8(6), 1039-1042. 14. Y. T. Tung, S. T. Chang