Four flavonols from the seed of Hibiscus sabdariffa Linn growing in Binh Thuan province

Science & Technology Development Journal, 22(4):348- 351 Open Access Full Text Article Scientific Report 1Faculty of Environmental Science, Sai Gon University, Ho Chi Minh City 2High School Education Office, Department of Education and Training of Binh Thuan Province 3Department of Nature, Dong Nai University, Dong Nai Province 4Faculty of Basic Science, University of Medicine and Pharmacy, Ho Chi Minh City 5Faculty of Chemistry, Ho Chi Minh City University of Education, Ho Chi Minh City, Vietnam 6Faculty of Chemistry, University of Science, Ho Chi Minh City Correspondence Duong Thuc Huy, Faculty of Chemistry, Ho Chi Minh City University of Education, Ho Chi Minh City, Vietnam Email: huydt@hcmue.edu.vn History  Received: 2019-06-25  Accepted: 2019-11-01  Published: 2019-11-30 DOI : 10.32508/stdj.v22i4.1690 Four flavonols from the seed of Hibiscus sabdariffa Linn. growing in Binh Thuan Province PhamNguyen Kim Tuyen1, Truong Van Tien2, Tran The Anh Huy1, Nguyen Thi Hoa1, Huynh Bui Linh Chi3, Nguyen Thi Hoai Thu4, Duong Thuc Huy5,*, Nguyen Kim Phi Phung6 Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: Hibiscussabdariffa Linn. (Malvaceae) is a medicinal plant popularly distributed in Asian countries. As of 2019, no chemical investigations from the Vietnamese plant were found. This paper reported the isolation and elucidation of compounds isolated from the seeds ofHibiscus sab- dariffa Linn. (Malvaceae) growing in Binh Thuan province. Method: Themacerationwas applied to the seeds of theplant to afford the crudeextractwhichwas then fractionatedby liquid-liquid extrac- tion to obtain the ethyl acetate extract. The extract was carried out by using normal phase silica gel column chromatography and thin-layer chromatography. Analysis of spectroscopic data including HR-ESI-MS, 1D and 2D-NMR and a comparison of the NMR data with that in the literature led to the structural elucidation of isolated compounds. Results: Four flavonols comprising quercetin 3- O-a-L-rhamnopyranosyl-(1!6)-b -D-glucopyranoside (1), quercetin 3-O-b -D-glucopyranoside (2), quercetin (3) and kaempferol (4) were isolated from the ethyl acetate extract. Conclusions: This is the first time the isolated compounds were found from the seeds of this plant. Key words: Hibiscus sabdariffa Linn, Malvaceae, flavonols, quercetin INTRODUCTION Hibiscus is one of the largest Malvaceae genera and comprises about 300 species widely distributed in Central and West Africa, and South East Asia 1,2. Hibiscus sabdariff a Linn. (Malvaceae) can be used as herbal drinks, beverages and flavouring agents. Leaves and powdered seeds of H. sabdariffa are used as foods in West Africa 2. The plant has traditionally been used in Mexico for the treatment of hyperten- sion, diaphoretic, diuretic and hyperlipidemia 3. In Vietnam, this plant is an important medicinal plant1 with folk treatments. Various chemical investiga- tions on flowers, calyces, and leaves of H. sabdariffa were reported so far, providing a number of organic compounds such as phenolic acid, pectin, polysac- charides, anthocyanin and flavonoids. Nevertheless, the phytochemical data on Vietnamese H. sabdariffa plants have not been studied while the chemical con- stituents of the seeds of H. sabdariffa are scarce with the reports of fatty acids, proteins, lipids4. More re- cently, Habid and coworkers (2015) 5 only reported the presence of ellagic acid in the seeds of H. sabdar- iffa via LCMS without isolation5 . The current manuscript reports on the isolation of several metabolites from the seeds of H. sabdariffa growing in Vietnam using various chromatographic methods consisting of normal phase silica gel column chromatography and thin-layer chromatography on the ethyl acetate extract. The chemical structures of isolated compounds were elucidated based on 1D, 2D NMR, and MS along with comparison with the data in the literature. As a result, four compounds have been afforded, including quercetin 3-O-a-L- rhamnopyranosyl-(1!6)-b -D-glucopyranoside (1), quercetin 3-O-b -D-glucopyranoside (2), quercetin (3) and kaempferol (4). This is the first time the iso- lated compounds found from the seed of this plant. MATERIALS ANDMETHODS General experimental procedures The HR-ESI-MS and ESI-MS spectra were carried on a Bruker microTOF Q-II and a MSQ plus Mass, re- spectively. TLC was carried out on pre-coated silica gel 60 F254 (MerckMillipore, Billerica,Massachusetts, USA) and spots were visualized by spraying with 10% H2SO4 solution followed by heating. Column chro- matography was conducted with silica gel 60 (0.040 – 0.063 mm) (HiMedia, Mumbai, India). Plant material The seeds of H. sabdariffa were collected in Lien Huong Town, Tuy Phong District, Binh Thuan Province, Vietnam in October 2016. The scientific name was identified by Dr. Dang Van Son, Institute Cite this article : Kim Tuyen P N, Van Tien T, Anh Huy T T, Thi Hoa N, Linh Chi H B, Thi Hoai Thu N, Huy D T, Phi Phung N K. Four flavonols from the seed of Hibiscus sabdariffa Linn. growing in Binh Thuan Province. Sci. Tech. Dev. J.; 22(4):348-351. 348 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Science & Technology Development Journal, 22(4):348-351 Figure 1: Structure of four isolated flavonols 1 – 4. of Tropical Biology, Vietnam. A voucher specimen (No.HSA/SGU-0008) was deposited in laboratory of Faculty of Environmental Science, Saigon University. Extraction and isolation The cleaned, air-dried and ground material (14.0 kg) wasmacerated in ethanol (7 x 5 L) at the ambient tem- perature then the filtrated solution was concentrated under reduced pressure to afford the c rude ethanol extract (1.0 kg). This crude extract was dissolved in ethanol: water (1:9, v/v), then was partitioned with n-hexane, chloroform, and ethyl acetate to give n- hexane (140.0 g), chloroform (70.0 g) and ethyl ac- etate (80.0 g) extracts, respectively. The ethyl acetate extract (80.0 g) was subjected to sil- ica gel CC and eluted consecutively with a mixture of n-hexane and ethyl acetate (stepwise 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, v/v) to yield 6 fractions EA1-6. Frac- tion EA2 (11.0 g) was applied to normal phase sil- ica gel column chromatography, eluted with a gradi- ent of n-hexane and ethyl acetate (7:3 to 6:4) to ob- tain 7 fractions EA2.1-7. Fraction EA2.2 (50.0 mg) was then rechromatographed using a gradient system of n-hexane-ethyl acetate (8:2 to 7:3, v/v) to afford 3 (32.0 mg). Fraction EA2.7 (317.0 mg) was purified by CC, eluted with chloroform-methanol (9:1, v/v) to afford 4 (12.0 mg). Fraction EA3 (7.5 g) was chro- matographed by CC, eluted with the solvent system of n-hexane-ethyl acetate (stepwise 7:3, 6:4, 4:6, 2:8) and then methanol to yield 9 fractions EA3.1- 3.9. Frac- tionEA3.7 (2.3 g)was subjected to silica gel CC, eluted with n-hexane-ethyl acetate (stepwise 4:6, 3:7, 2:8, 1:9, 0:1) then themixture of ethyl acetate-methanol (a gra- dient from 9:1- 0:1) to afford 15 subfractions EA3.7.1- 15. Further purification of the fraction EA3.7.8 (150.0 mg) using the same manner as described previously for the fraction EA3 to afford two compounds 1 (40.0 mg) and 2 (27.0 mg). • Quercetin 3-O-a-L-rhamnopyranosyl-(1!6)- b -D-glucopyranose or rutin (1). Yellow amor- phous powder. HR-ESI-MS, positive mode: m/z 611.1611 [M+H]+ (calcd. for C27H31O16, 611.1612). The 1H-NMRdata (DMSO-d6): 6.19 (1H, d, 2.0, H-6), 6.38 (1H, d, 2.0, H-8), 7.53 (1H, d, 2.0, H-2’), 6.83 (1H, d, 8.5, H-5’), 7.54 (1H, dd, 8.5, 2.0, H-6’), 5.33 (1H, d, 8.5, H- 1”), 4.39 (1H, s, H-1”’), 0.99 (2H, d, 6.5, H-6”’) and 12.58 (1H, s, OH-5). The 13C-NMR data (DMSO-d6): 156.4 (C-2), 133.3 (C-3), 177.4 (C- 4), 161.2 (C-5), 98.6 (C-6), 164.1 (C-7), 93.6 (C- 8), 156.6 (C-9), 104.0 (C-10), 121.2 (C-1’), 116.2 (C-2’), 144.7 (C-3’), 148.4 (C-4’), 115.2 (C-5’), 121.6 (C-6’), 101.2 (C-1”), 74.1 (C-2”), 76.4 (C- 3”), 70.5 (C-4”), 75.9 (C-5”), 68.2 (C-6”), 100.7 (C-1”’), 70.3 (C-2”’), 70.0 (C-3”’), 71.8 (C-4”’), 67.0 (C-5”’) and 17.7 (C-6”’). • Quercetin 3-O-b -D-glucopyranoside or iso- quercitrin (2). Yellow amorphous powder. HR- ESI-MS, positive mode: m/z 465.1028 [M+H]+ (calcd. for C21H21O12 465.1033). The 1H- NMR data (DMSO-d6): 6.20 (1H, d, 2.0, H-6), 6.40 (1H, d, 2.0, H-8), 7.58 (1H, d, 2.0, H-2’), 6.84 (1H, d, 8.5, H-5’), 7.58 (1H, dd, 8.5, 2.0, H-6’), 5.45 (1H, d, 8.5, H-1”) and 12.63 (1H, s, OH-5). The 13C-NMR data (DMSO-d6): 156.2 (C-2), 133.3 (C-3), 177.4 (C-4), 161.2 (C-5), 98.6 (C-6), 164.2 (C-7), 93.5 (C-8), 156.3 (C-9), 103.9 (C-10), 121.6 (C-1’), 115.2 (C-2’), 144.8 (C-3’), 148.4 (C-4’), 116.2 (C-5’), 121.1 (C-6’), 100.8 (C-1”), 74.1 (C-2”), 76.5 (C-3”), 69.9 (C-4”), 77.5 (C-5”) and 61.0 (C-6”). 349 Science & Technology Development Journal, 22(4):348-351 • Quercetin (3). Yellow amorphous powder. The 1H-NMR data (DMSO-d6): 6.21 (1H, d, 2.0, H- 6), 6.42 (1H, d, 2.0, H-8), 7.77 (1H, d, 2.0, H-2’), 6.91 (1H, d, 8.5, H-5’), 7.67 (1H, dd, 8.5, 2.0, H- 6’) and 12.57 (1H, s, OH-5). The 13C-NMR data (DMSO-d6): 148.8 (C-2), 137.2 (C-3), 177.5 (C- 4), 162.5 (C-5), 99.3 (C-6), 165.6 (C-7), 94.4 (C- 8), 158.3 (C-9), 104.5 (C-10), 124.2 (C-1’), 116.0 (C-2’), 146.2 (C-3’), 148.0 (C-4’), 116.2 (C-5’) and 121.7 (C-6’). • Kaempferol (4). Yellow amorphous powder. The 1H-NMR data (methanol-d4): 6.26 (1H, d, 2.0, H-6), 6.53 (1H, d, 2.0, H-8), 8.15 (2H, d, 8.0, H-2’, H-6’), 7.01 (2H, d, 8.0, H-3’, H-5’) and 12.31 (1H, s, OH-5). The 13C-NMR data (methanol-d4): 160.4 (C-2), 137.2 (C-3), 177.4 (C-4), 164.5 (C-5), 99.3 (C-6), 165.6 (C-7), 94.4 (C-8), 158.1 (C-9), 104.5 (C-10), 123.7 (C-1’), 130.5 (C-2’, C-6’) and 116.2 (C-3’, C-5’). RESULTS ANDDISCUSSION Compound 1 was obtained as a yellow amorphous powder. The NMR spectra of 1 revealed character- istic signals of a flavonol skeleton. Indeed, the 1 H- NMR spectrum of 1 showed a hydroxyl group at d 12.58 (1H, s) which was assigned to 5-OH of a 5,7- dihydroxy A ring system in flavonoid while twometa –coupled protons at dH 6.19 (1H, d, 2.0, H-6) and dH 6.38 (1H, d, 2.0, H-8) assignable to H–6 and H– 8, respectively. Moreover, a 3’,4’–dihydroxy benzene ring of 1 was defined due to the ABX system of three aromatic proton s at dH 7.53 (1H, d, 2.0, H-2’), 6.83 (1H, d, 8.5, H-5’), and 7.54 (1H, dd, 8.5, 2.0, H-6’). In addition, the 1H-NMR spectrum also displayed sig- nals of two sugar units including two anomeric pro- ton s at dH 5.33 (1H, d, 8.5, H-1”) and 4.39 (1H, s, H- 1”’) and oxygenated methine and methylene groups in the zone of dH 3.34–3.78. Combined, 1 was de- fined as a flavonol glycoside6. The 13C-NMR spec- trumwas further supported this findingwith the pres- ence of 27 carbon signals, including a carbonyl car- bon at dC 177.4 (C-4) and 14 carbons from 93.6 to 164.1 ppm of a flavonol unit together with 12 car- bons of two sugar units. The attachment of a b -D- glucopyranosyl unit was defined as C-3 due to the key HMBC cross peak of the anomeric proton at dH 5.33 (1H, d, 8.5 Hz, H–1”) to C-3 (dC 133.3). Likewise, the a-L -rhamnopyranosylmoiety connected to the b -D- glucopyranosyl unit via the linkage C-1”’-C-6”, thanks to the HMBC correlation of H-1’ at dH 4.39 (1H, s, H–1”’) to C-6” (dC 68.2). Additionally, in the HMBC spectra, the cross peak between the anomeric proton dH 4.39 (1H, s, H-1”’) and oxygenated carbon of the b -D-glucopyranosyl moiety (C-6”) was observed that demonstrated thea-L-rhamnopyranoside attached to the b -D-glucopyranosyl moiety at C-6”. The molec- ular formula of 1 was determined as C27H30O16 through the protonated molecular ion peak at m/z 611.16110 [M+H]+ inHRESIMS, strongly supported the structure of 1. TheNMRdata of 1were consistent with those of rutin 7, thus 1 was determined as rutin. Figure 2: The key HMBC correlations of compounds 1 and 2. Compound 2 was isolated as a yellow amorphous powder. NMR data of 2 was reminiscent to those of 1, except for the absence of thea-L-rhamnopyranosyl unit. The molecular formula of 2 was established as C21H20O12 based on a protonated molecular ion peak atm/z 465.1028 ([M+H]+) of HR-ESI-MS spec- trum. NMR data of 2 was identical with those of iso- quercitrin8, thus, 2 was elucidated as isoquercitrin. Compound 3 was obtained as a yellow amorphous powder. Analysis of 1D NMR data of 3 indicated that 3 had the same structure as the aglycone moiety of 1 and 2. The comparison of NMR data of 3 with those reported in the literature8 led to the chemical struc- ture of 3 to be quercetin. Compound 4 was isolated as a yellow powder. Com- parison of NMR data of 4 and 3 resulted in their sim- ilar structure, except for the absence of the hydroxyl group at C-4’ in 4when comparing to that of 3. NMR data of 4 was consistent with those of kaempferol re- ported in the literature6, accordingly, 4 was deter- mined as kaempferol. Rutin (1) and quercetin (3) have been described as cell-protecting agents on oxaliplatin-induced painful peripheral neuropathy based on their antioxidant properties9. Isoquercitrin (2) and quercetin (3) re- vealed the strong antimicrobial and antioxidant activ- ities6. Particularly, quercetin (3) showed the potent antimicrobial activity against Staphylococcus aureus, withMICvalue of 6.25 mg/mL9. These bioactive com- pounds occurred asmajor phenolic compounds in the extracts of leaves, flowers, and calyces ofH. sabdariffa growing in various regions in theworld. Nevertheless, 350 Science & Technology Development Journal, 22(4):348-351 it is worthy noting that isolated compounds 1 - 4were found in the seeds ofH. sabdariffa for the first time2 . CONCLUSION From the ethyl acetate extract of the seeds of H. sab- dariffa (Malvaceae) collected in BinhThuan province, four compounds rutin (1), isoquercitrin (2), quercetin (3), and kaempferol (4) were isolated and elucidated using modern chromatographic and spectroscopic methods. All these compounds were known to be present for the first time from the seed of H. sabdar- iffa. Further studies on the chemical constituent and biological activity of this plant are under progress. ABBREVIATIONS 1 HNMR: Proton nuclear magnetic resonance 13 C NMR: Carbon-13 nuclear magnetic resonance CC: column chromatography TLC: Thin layer chromatography HSQC: Heteronuclear single quantum coherence HMBC: Heteronuclear multiple bond correlation s: singlet d: doublet m: multiplet CONFLICTS OF INTEREST The authors declare no competing financial interest. AUTHOR CONTRIBUTION Pham N.K.T has contributed in conducting exper- iments, acquisition of data, and interpretation of data. Truong V.T., Tran T.A.H., Nguyen T.H., Huynh B.L.C., Nguyen T.H.T. interpreted NMR and MS data as well as searched the bibliography. Duong T.H. and NguyenK.P.P. gave final approval of themanuscript to be submitted. ACKNOWLEDGMENTS We would like to thank Dr. Dr. Dang Van Son for the identification of the scientific name. REFERENCES 1. Pham HH. An Illustrated Flora of Vietnam. Young Publishing House. 2003;1:523–528. 2. Da-Costa-Rocha I, Bonnlaender B, Sievers H, Pischel I, Heinrich M. 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