Five compounds as (–)catechin (1), gallocatechin (2), phloretin 4OβDglucopyranoside (3), chlorogenic acid (4), and protocatechuic acid (5) were isolated from the roots of Kadsura coccinea. Their chemical structures were elucidated on the basis of detailed spectroscopic analyses (NMR and MS) and comparing to those in the literature. Among them, compounds 2 and 3 are reported for the first time from this plant.
Flavanol, dihydrochalcone, and phenolic constituents from the roots of Kadsura coccinea Pham Quoc Tuan1, Ha Quang Loi1, Ha Thanh Hoa1, Nguyen Mai Nam1, Nguyen Quoc Tuan1, Phuong Thien Thuong2, Nguyen Duc Hung1,* Center for Drug Research and Technology Transfer, Phu Tho College of Medicine and Pharmacy, Phu Tho Province, Vietnam National Institute of Medicinal Materials, Ha Noi, Vietnam *Corresponding author: duchungnguyen1310@gmail.com Summary Five compounds as (–)-catechin (1), gallocatechin (2), phloretin 4'-O-β-D- glucopyranoside (3), chlorogenic acid (4), and protocatechuic acid (5) were isolated from the roots of Kadsura coccinea Their chemical structures were elucidated on the basis of detailed spectroscopic analyses (NMR and MS) and comparing to those in the literature Among them, compounds and are reported for the first time from this plant Keywords: Kadsura coccinea, flavanol, dihydrochalcone, phenolic Introduction Kadsura coccinea (Lem.) A.C Smith a climbing plant with woody stems, belongs to Schisandraceae family It is widely distributed in Asia [1] In Vietnam, K coccinea is vernacularly known as na rừng, dây xưn xe, nắm cơm, and ngũ vị nam, [2] Its dried roots and stems are used for the treatment of rheumatoid arthritis, gastric, and duodenal ulcers in the folk medicine [3] Previous studies revealed that the major constituents were reported as dibenzocyclooctadiene lignans, triterpenoids, and sesquiterpenoids [3, 4] They exhibited on anti-proliferative, anti-hepatitis, and nitrogen oxide inhibitory activities [5, 6] To investigate the chemical constituents, the EtOAc fraction of the EtOH extract from K coccinea was conducted for researching The results led to the isolation and identification of five substances, including two flavanols, one dihydrochalcone glycoside, and two phenolic compounds In this paper, we reported the extraction, isolation, purification, and structural elucidation of the isolated compounds from K coccinea Materials and methods 2.1 General procedures NMR spectra were measured in methanol-d4 on an Oxford AS 400 MHz instrument (Varian, Palo Alto, CA, USA) Mass spectra were recorded using a LCMS-8045 (Shimadzu) mass spectrometer The optical rotations were determined using a P3001/RS polarimeter (Kruss, Germany) The column chromatography was performed using silica gel (Merck, Darmstadt, Germany), RP-18 (Merck, Darmstadt, Germany) Thin layer chromatography (TLC) tests experimented on silica gel 60 F254 plates (Merck, Darmstadt, Germany) Fractions were monitored by TLC and spots were visualized by spraying with the EtOH solution containing 10% H2SO4, followed by heating All other agents and solvents were analytical grade 2.2 Plant material The roots of K coccinea were collected on December 2016, in Xuan Son National Park, Phu Tho province The plant was identified by Associate Professor Phuong Thien Thuong (National Institute of Medicinal Materials) A voucher specimen (KC-CDDPT.2016-009) has been deposited at the Herbarium of Department of Pharmacognosy, Phu Tho College of Medicine and Pharmacy 2.3 Extraction and isolation The dried powered root of K coccinea (4.2 kg) was extracted with 80% EtOH (3 × 12 L) at room temperature for 20 days The EtOH extract was concentrated under reduced pressure, giving 0.8 kg crude extract The crude extract was suspended in H 2O (1.5 L), and then partitioned successively with n-hexane (2L × 5); to yield 89 g, EtOAc (2 L × 5) to yield 273 g, n-BuOH (2 L × 5) to yield 309 g, and H2O fractions The EtOAc fraction was separated on silica gel column chromatography (size: 120 x 15 cm), using a gradient solvent system of CH2Cl2/MeOH/H2O (100:0:0 to 1:2:0.1, v/v/v) to give 12 fractions (A‒M) The fraction D (2.1 g) was subjected by RP-18 column chromatography (size: 40 x cm), eluting with acetone/H2O (1:10 to 1:1, v/v) to afford six sub-fractions (D1‒D6) The sub-fraction D3 (412 mg) was continuously chromatographed on RP-18 column chromatography, using the elution solution of acetone/H2O (2:6, 2:5, 2:4, v/v) to give (39.5 mg) and (21.3 mg) The subfraction D4 (395 mg) was purified on a RP-18 column chromatography, using the elution solution of acetone/H2O (2:6, 2:5, 2:4, and 2:3, v/v), led to provide compound (18.1 mg) The sub-fraction C (2.0 g) was separated on a RP-18 column chromatography, and then eluted with a gradient system of MeOH/H2O (1:6, 1:4, 1:2, 1:1, and 2:1, v/v) to obtain eight subfractions (C1‒C8) The sub-fraction C3 (328 mg) was chosen for continuing purification on a RP-18 column with mobile phase MeOH/H2O (1:3, 1:2, and 1:1, v/v) to yield compounds (15.6 mg) and (46.1 mg) (–)-Catechin (1): a colorless powder; ESI-MS m/z 289.1 [M-H]- ; 1H NMR (CD3OD, 400 MHz) δH: 4.56 (1H, d, J = 7.5 Hz, H-2), 3.97 (1H, m, H-3), 2.85 (1H, dd, J = 5.3, 16.1 Hz, H4a), 2.50 (1H, dd, J = 8.2, 16.1 Hz, H-4b), 5.85 (1H, s, H-6), 5.92 (1H, s, H-8), 6.83 (1H, s, H2'), 6.71 (1H, d, J = 8.2 Hz, H-5'), 6.76 (1H, d, J = 8.2 Hz, H-6'); 13C NMR (CD3OD, 100 MHz) δC: 82.9 (C-2), 68.8 (C-3), 28.5 (C-4), 157.6 (C-5), 96.3 (C-6), 157.8 (C-7), 95.5 (C-8), 156.9 (C-9), 100.8 (C-10), 132.2 (C-1'), 115.2 (C-2'), 146.2 (C-3'), 146.2 (C-4'), 116.1 (C-5'), 120.0 (C-6') Gallocatechin (2): a white amorphous powder; ESI-MS m/z 305.1 [M-H]-; 1H NMR (CD3OD, 400 MHz) δH: 4.53 (1H, d, J = 7.1 Hz, H-2), 3.97 (1H, m, H-3), 2.81 (1H, dd, J = 5.1, 16.2 Hz, H-4a), 2.50 (1H, dd, J = 7.8, 16.2 Hz, H-4b), 5.86 (1H, s, H-6), 5.92 (1H, s, H- 8), 6.40 (2H, s, H-2', 6'); 13C NMR (CD3OD, 100 MHz) δC: 82.8 (C-2), 68.7 (C-3), 28.1 (C-4), 157.8 (C-5), 96.2 (C-6), 157.6 (C-7), 95.5 (C-8), 156.8 (C-9), 100.7 (C-10), 131.5 (C-1'), 107.2 (C-2', 6'), 146.8 (C-3', 5'), 134.0 (C-4') Phloretin 4'-O-β-D-glucopyranoside (3): a colorless powder; ESI-MS m/z 435.2 [M-H]-; H NMR (CD3OD, 400 MHz) δH: 7.06 (2H, d, J = 8.1 Hz, H-2,6), 6.68 (2H, d, J = 8.1 Hz, H- 3, 5), 6.18 (1H, s, H-3'), 5.96 (1H, s, H-5'), 3.46 (2H, m, H-α), 2.88 (2H, t, J = 7.6 Hz, H-β), 5.04 (1H, d, J = 6.2 Hz, H-1''), 3.91 (1H, d, J = 12.0 Hz, H-2''), 3.71 (1H, dd, J = 4.6, 11.8 Hz, H-3''), 3.46 (3H, m, H-4'', 5'', 6''); 13C NMR (CD3OD, 100 MHz) δC: 133.8 (C-1), 130.3 (C-2, 6), 116.1 (C-3, 5), 156.4 (C-4), 106.9 (C-1'), 165.3 (C-2'), 96.4 (C-3'), 165.0 (C-4'), 96.4 (C5'), 165.3 (C-6'), 47.5 (C-α), 31.8 (C-β), 101.5 (C-1''), 74.6 (C-2''), 78.2 (C-3''), 71.1 (C-4''), 77.9 (C-5''), 62.4 (C-6''), 206.5 (CO) Chlorogenic acid (4): a white power; ESI-MS m/z 353.2 [M-H]-; 1H NMR (CD3OD, 400 MHz) δH: 2.20 (2H, m, H-2), 5.32 (1H, m, H-3), 3.72 (1H, dd, J = 3.1, 8.5 Hz, H-4), 4.16 (1H, dd, J = 3.1, 5.2 Hz, H-5), 2.04 (2H, m, H-6), 7.04 (1H, d, J = 1.8 Hz, H-2'), 6.77 (1H, d, J = 8.2 Hz, H-5'), 6.95 (1H, dd, J = 1.8, 8.2 Hz, H-6'), 7.55 (1H, d, J = 15.9 Hz, H-7'), 6.25 (1H, d, J = 15.9 Hz, H-8'); 13C NMR (CD3OD, 100 MHz) δC: 76.1 (C-1), 38.2 (C-2), 71.3 (C-3), 73.4 (C-4), 72.0 (C-5), 38.7 (C-6), 177.0 (COOH), 127.8 (C-1'), 115.2 (C-2'), 144.5 (C-3'), 149.5 (C-4'), 123.0 (C-5'), 116.5 (C-6'), , 147.1 (C-7'), 115.2 (C-8'), 168.6 (C-9') Protocatechuic acid (5): a pale amorphous powder; ESI-MS m/z 153.2 [M-H]-; 1H NMR (CD3OD, 400 MHz) δH: 7.39 (1H, s, H-2), 7.38 (1H, d, J = 7.9 Hz, H-5), 6.76 (1H, d, J = 7.9 Hz, H-6); 13C NMR (CD3OD, 100 MHz) δC: 169.0 (COOH), 114.3 (C-1), 116.3 (C-2), 144.6 (C-3), 150.0 (C-4), 121.8 (C-5), 122.4 (C-6) Results and Discussions The chromatographic separation and purification of the EtOAc fraction from roots of K coccinea, led to the isolation of five compounds (1–5) The structures of isolated compounds were elucidation based on the analyses of their NMR, MS data and comparing to the valuable publication in the previous reports Figure The structures of isolated compounds from the roots of K coccinea Compound was obtained as a colorless powder The molecular formula of was identified as C15H14O6 by the negative mode ESI-MS data at m/z 289.1 [M-H]- (calculated for C15H14O6, m/z 289.1) The 1H NMR data of exhibited two aromatic protons at δH 5.92 (1H, s, H-8) and 5.85 (1H, s, H-6) An ABX proton system at δH 6.83 (1H, s, H-2'), 6.76 (1H, d, J = 8.2 Hz, H-6'), 6.71 (1H, d, J = 8.2 Hz, H-5') was also observed In addition, 1H NMR spectrum of also displayed two oxygenated methine group at δH 3.97 (1H, m, H-3), 4.56 (1H, d, J = 7.5 Hz, H-2), and a methylene group at δH 2.85 (1H, dd, J = 5.3, 16.1 Hz, H-4a) and 2.50 (1H, dd, J = 8.2, 16.1 Hz, H-4b) From the analyses of 1H NMR, compound is considering as flavan-3-ol In 13C NMR data of showed 15 carbon signals at δC: 82.9 (C-2), 68.8 (C-3), 28.5 (C-4), 157.6 (C-5), 96.3 (C-6), 157.8 (C-7), 95.5 (C-8), 156.9 (C-9), 100.8 (C-10), 132.2 (C-1'), 115.2 (C-2'), 146.2 (C-3'), 146.2 (C-4'), 116.1 (C-5'), and 120.0 (C-6') suggesting that was catechin The valuable coupling constant between H-2 and H-3 are 7.5 Hz in agreement with β-form of 3-OH To identify was (+)-catechin or (-)-catechin In addition to comparison experimental NMR, the compound was continuously measured the optical rotation ([α]= -20,3 (c 0.2, Me2CO), suggesting that the compound such as (-)catechin [7] Base on the above evidence and comparison with the literature data [7, 8], compound was identified as (-)-catechin Compound was obtained as a white amorphous powder The molecular formula of was identified as C15H14O7 by the negative mode ESI-MS data at m/z 305.1 [M-H]- The NMR spectroscopic data of exhibited a flavan-3-ol backbone Its spectral data were closely similar to those of 1, except for the presence of a hydroxyl group at C-5' of at C-ring, which leading to observe a singlet peak δH 6.40 (2H, s) in 1H NMR of 2, thus, leading to conclusion that the compound was gallocatechin by comparing to those in the literature [9] Compound was isolated as a colorless powder, which is suggesting for a molecular formula of C21H24O10 by observation an ion peak at m/z 435.2 [M-H]-; in ESI-MS spectrum, together with experimental signals of 13C NMR spectrum The 1H NMR spectrum of showed characteristic signals for a phloretin moiety by observation AABB-type aromatic ring protons at δH 7.06 (2H, d, J = 8.1 Hz, H-2, 6) and 6.68 (2H, d, J = 8.1 Hz, H-3, 5), two meta-coupled aromatic protons at δH 6.18 (1H, s, H-3') and 5.96 (1H, s, H-5'), together with two methylene groups at δH 3.46 (2H, m, H-α), 2.88 (2H, t, J = 7.6 Hz, H-β) The 13C NMR spectrum of displayed 15 carbon signals, including a β-D-glucopyranosyl moiety, two methylene groups, six methines, six quaternary carbons, and a carbonyl group All detailed analyses of NMR experiments, its spectroscopic data led to the conclusion that the structure of was phloretin 4'-O-β-D-glucopyranoside, which was confirmed by comparison to valuable publication in the reference [10] Compound obtained as a white powder The [M-H]- molecular ion observed at m/z 353.2 in negative ESI-MS data, the combination with the 13 C-NMR spectroscopic data indicated a molecular formula of C16H18O97 The 1H NMR spectrum of exhibited an ABX spin system at δH 7.04 (1H, d, J = 1.8 Hz, H-2'), 6.77 (1H, d, J = 8.2 Hz, H-5'), 6.95 (1H, dd, J = 1.8, 8.2 Hz, H-6') and a pair of trans-olefinic proton at δH 7.55 (1H, d, J = 15.9 Hz, H-7') and 6.25 (1H, d, J = 15.9 Hz, H-8') This data suggested that chemical structure of contain a caffeoyl moiety Two methylene groups [δH 2.20 (2H, m, H-2) and 2.04 (2H, m, H-6),], three oxygenated protons [δH 5.32 (1H, m, H-3), 3.72 (1H, dd, J = 3.1, 8.5 Hz, H-4), and 4.16 (1H, dd, J = 3.1, 5.2 Hz, H-5)] were observed With above evidence, the compound was caffeoyl quinic acid In the 13 C NMR spectrum of showed a carboxylic group at δC 177.0 and a caffeoyl substitute at δC 127.8 (C-1'), 115.2 (C-2'), 144.5 (C-3'), 149.5 (C-4'), 116.5 (C-5'), 123.0 (C-6'), 147.1 (C-7'), 115.2 (C-8') and 168.6 (C-9') Base on above data and comparison NMR data of to those reported in the literature [11], the compound was elucidated as chlorogenic acid Compound was isolated as a pale amorphous powder The molecular formula of was identified as C7H6O4 by the negative mode ESI-MS data at m/z 153.2 [M-H]- (calculated for C7H6O4, m/z 153.0) The 1H NMR data of showed an ABX system [δH 7.39 (1H, s, H-2), 7.38 (1H, d, J = 7.9 Hz, H-5), 6.76 (1H, d, J = 7.9, H-6)] The 13C NMR data of displayed a carbonyl group at δC 169.0, along with six carbon signals at δC 114.3 (C-1), 116.3 (C-2), 144.6 (C-3), 150.0 (C-4), 121.8 (C-5), and 122.4 (C-6) Thus, the chemical structure of was determined as protocatechuic acid by comparing its NMR data to those previous reports in the literature [12] Catechin (1) and gallocatechin (2) belong to flavanol groups, the part of the chemical family of flavonoids, they are popular in fruits and in tea, cocoa, and berries Their chemical structures are contained the hydroxyl group, which are reported for radical scavenging, and antioxidant activities Previous studies showed that both have the capacity to scavenging hydroxyl, peroxyl, and 2,2-diphenyl-1-picrylhydrazyl radicals and chelate the iron ion [13] Phloretin 4'-O-β-D-glucopyranoside (3), a dihydrochalcone were reported as insectantifeedant from nature [14] Chlorogenic acid (4) is a phenolic acid with vicinal hydroxyl groups on aromatic residues that is derived from the esterification of a caffeic acid It found widely in beverages from herbs, fruits (e.g., apples, pears, many berries), and vegetables [15] The previous studies reported that chlorogenic acid possesses many biological properties, including antibacterial, antioxidant, and anti-inflammatory activities [15] Protocatechuic acid (5) is a phenolic compound appeared in many food plants Several investigations have shown that the compound is a major metabolite of complex polyphenols, and has great benefit to health such as antioxidant, anti-inflammatory, anti-hyperglycemic, anti-apoptosis versus proapoptotic, and anti-microbial activities [16] Conclusion Using various chromatography techniques, five compounds were isolated from the EtOH extract of the roots of K coccinea These isolated compounds were elucidated by NMR, MS spectra, and comparison to those the literature, namely, such as (-)-catechin (1), gallocatechin (2), phloretin 4'-O-β-D-glucopyranoside (3), chlorogenic acid (4), and protocatechuic acid (5) Compounds and are the first time for publication from K coccinea Acknonowledgements: This study is supported in the project financially funded by the Phu Tho Department of Science and Technology References Liu J., Qi Y., Lai H., Zhang J., Jia X., Liu, H., Zhang B., Xiao P (2014), Genus Kadsura, a good source with considerable characteristic chemical constituents and potential bioactivities Phytomedicine, 21 (8), 1092-1097 Vo V.C (2012), Dictionary of Vietnamese medicinal Plant., vol 2, 192 Medical Publishing House Fang L., Xie C., Wang H., Jin D.-Q., Xu J., Guo Y., Ma Y (2014), Lignans from the roots of Kadsura coccinea and their inhibitory activities on LPS-induced NO production Phytochemistry Letters, 9, 158-162 Hu,Z.-X., Shi Y.-M., 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H.-D (2012), Lignans from Kadsura angustifolia and Kadsura coccinea Journal of Asian Natural Products Research, 14 (2), 129134 Li H., Wang L., Yang Z., Kitanaka S (2007), Kadsuralignans H−K from... considerable characteristic chemical constituents and potential bioactivities Phytomedicine, 21 (8), 1092-1097 Vo V.C (2012), Dictionary of Vietnamese medicinal Plant., vol 2, 192 Medical Publishing... inhibitory effects Journal of Natural Products, 70 (12), 1999-2002 El-Razek M H Abd (2007), NMR Assignments of four Catechin epimers Asian Journal of Chemistry, 19, 4867-4872 Watanabe M (1998), Catechins