Summary of Chemistry doctoral thesis: Study on chemical constituents and biological activities of Vitex limonifolia Wall. Ex C.B.Clarke and Vitex trifolia L.

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Study on chemical constituents of leaves of two Vitex species including Vitex limonifolia Wall. ex C.B.Clarke and Vitex trifolia L. growing in Vietnam; evaluate anti-inflammatory and antiviral activities of isolates to find out bioactive compounds.

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Nội dung Text: Summary of Chemistry doctoral thesis: Study on chemical constituents and biological activities of Vitex limonifolia Wall. Ex C.B.Clarke and Vitex trifolia L.

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ----------------------------- NGUYEN THI KIM THOA STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF Vitex limonifolia WALL. EX C.B.CLARKE AND Vitex trifolia L. Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi - 2019
This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Advisor 1: Dr. Nguyen Xuan Nhiem Institute of Marine Biochemistry - Vietnam Academy of Science and Technology Advisor 2: Prof. Dr. Ninh Khac Ban Institute of Marine Biochemistry - Vietnam Academy of Science and Technology 1st Reviewer: 2nd Reviewer: 3rd Reviewer: The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at hour date month 2019 The thesis can be found in - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology. - The National Library of Vietnam.
1 INTRODUCTION 1. The urgency of the thesis In the recent decades, the chemical and biological investigation of medicinal plants and animals to find out natural bioactive compounds supplying for pharmaceutical industry is getting more and more attention from scientists worldwide. According to Dictionary of Vietnamese medicinal plants, the species of the Vitex genus were used for treating many diseases, such as: cough, colds, dizziness, headache, asthma, malaria, skin diseases,… [1]. Phytochemical investigation of Vitex genus led to the isolation of numerous terpenoids, flavonoids, ecdysteroids, lignans, and other compounds. Pharmacological studies had shown that the extractions and isolated compounds possess antitumor, anti-inflammatory, antibacterial, antioxidant activities, and so on. However, there are few researches on the chemical components and biological activities of Vitex species growing in Vietnam [2-6]. Therefore, the mission of studying the chemical and bioactive components of Vitex species growing in Vietnam, developing health care production is very important. From above reasons, thesis title was chosen to be “Study on chemical constituents and biological activities of Vitex limonifolia Wall. Ex C.B.Clarke and Vitex trifolia L.” 2. The aim of the thesis Study on chemical constituents of leaves of two Vitex species including Vitex limonifolia Wall. ex C.B.Clarke and Vitex trifolia L. growing in Vietnam. Evaluate anti-inflammatory and antiviral activities of isolates to find out bioactive compounds. 3. The main contents of the thesis 1. Isolate compounds from the leaves of V. limonifolia and V. trifolia;
2 2. Elucidate chemical structures of the isolated compounds; 3. Evaluate the anti-inflammatory activity of the isolated compounds from V. limonifolia; 4. Evaluate the antiviral activity of the isolated compounds from V. limonifolia and V. trifolia. CHAPTER 1: OVERVIEW Overview of national and international researches related to my study of the chemical constituents and biological activities of Vitex genus and about inflammation and antivirus. 1.1. Introduction to Vitex genus The genus Vitex includes approximately 250 known species of trees and shrubs in tropical and sub-tropical regions. In Vietnam, Vitex genus includes 20 species [7]. Since ancient times, civilization used Vitex plants for treating many health problems, such as malaria, herpes, itches, dermatitis or controlling menstruation. The chemical constituents of Vitex include terpenoids, flavonoids, ecdysteroids, lignans, iridoids,…. In Vietnam, there are only 5 published reports about phytoconstituents of some Vitex species, there is no report about biological activities. 1.2. Introduction to inflammation Introduction of inflammation, anti-inflammatory drugs and the role of nitric oxide in inflammatory disease. 1.3. Introduction to antivirus Introduction to antivirus, some types of antiviral drugs are naturally derived.
3 CHAPTER 2: EXPERIMENTAL AND RESULTS 2.1. Plant materials The leaves of Vitex limonifolia Wall. ex C.B.Clarke and Vitex trifolia L. were collected in Bachma National park, Thua Thien Hue, Vietnam in September, 2015. 2.2. Methods 2.2.1. Methods for isolation of secondary metabolites Chromatographic methods such as thin layer chromatography (TLC), column chromatography (CC). 2.2.2. Methods for determination of chemical structure of compounds Physical parameters and modern spectroscopic methods such as optical rotation ([]D), electrospray ionization mass spectrometry (ESI-MS) and high-resolution ESI-MS (HR-ESI-MS), one/two-dimension nuclear magnetic resonance (NMR) spectra, circular dichroism spectrum (CD). 2.2.3. Biological assays - Anti-inflammatory activity of the compounds was assessed on the basis of inhibiting NO production in lipopolysaccharide (LPS) activated BV2 cells. - Antivirus activity was determined by the SRB assay. 2.3. Isolation of compounds This section presents outlines of the general methods to isolate pure substances from the plants samples. 2.3.1. Isolation of compounds from Vitex limonifolia
4 This section presents the process of isolating the compounds from Vitex limonifolia. Figure 2.1. Isolation of compounds from V. limonifolia 2.3.2. Isolation of compounds from Vitex trifolia This section presents the process of isolating the compounds from Vitex trifolia. Figure 2.2. Isolation of compounds from V. trifolia
5 2.4. Physical properties and spectroscopic data of the isolated compounds 2.4.1. Physical properties and spectroscopic data of the isolated compounds from V. limonifolia This section presents physical properties and spectroscopic data of 12 compounds from V. limonifolia. 2.4.2. Physical properties and spectroscopic data of the isolated compounds from V. trifolia This section presents physical properties and spectroscopic data of 16 compounds from V. trifolia. 2.5. Results on biological activities of isolated compounds 2.5.1. Results on anti-inflammatory activity of compounds from Vitex limonifolia - 12 compounds (VL1-VL12) were evaluated for their anti- inflammatory activities on the basis of inhibiting nitric oxide production in LPS-activated BV2 cells. Table 2.1. Inhibition effects of VL1-VL12 on NO production in the LPS-activated BV2 cells at concentration of 20 μM Comp. Cell viability (%) IC50 (µM) VL1 87.062.43 >50 VL2 120.757.80 2.500.34 VL3 147.828.55 7.130.87 VL4 87.1211.28 24.701.52 VL5 104.519.50 39.673.14 VL6 86.633.23 19.161.09 VL7 96.935.10 45.315.31 VL8 104.519.50 >50 VL9 141.198.73 44.232.48 VL10 119.536.65 15.881.17
6 Comp. Cell viability (%) IC50 (µM) VL11 59.045.83 - VL12 80.503.25 >50 L-NMMA 22.101.20 (-) Do not evaluate anti-inflammatory activity because cell viability was small (< 80%) 2.5.2. Results on antiviral activity of the isolated compounds From V. limonifolia - 12 compounds (VL1-VL12) were evaluated for antiviral activities against enterovirus including coxsackievirus B3 (CVB3), human rhinovirus 1B (HRV1B), and enterovirus 71 (EV71). Table 2.2. Antiviral activities against CVB3, HRV1B, and EV71 viruses of some compounds from V. limonifolia Kí hiệu CC50 (M) IC50 (M) Coxsackievirus B3 (CVB3) VL4 >50 0.21±0.06 VL6 >50 1.86±0.18 Rupuntrivir >50 0.12±0.06 Human rhinovirus 1B (HRV1B) VL4 >50 0.61±0.21 Ribavirin >50 48.07±1.46 Enterovirus 71 (EV71) VL4 >50 32.05±0.94 Rupuntrivir >50 0.11±0.05 From V. trifolia - 12 compounds (VT1-VL16) were evaluated for antiviral activities against coxsackievirus B3, human rhinovirus 1B, and enterovirus 71 at concentration of 10 M.
7 Table 2.3. Screening of antiviral activities against CVB3, HRV1B, and EV71 viruses of the isolated compounds from V. trifolia Cell viability (%) Comp. Coxsackievirus Human rhinovirus Enterovirus B3 1B 71 VT1 2.38 5.41 1.30 VT2 3.52 4.23 5.97 VT3 4.99 -1.51 -4.34 VT4 13.27 0.71 3.37 VT5 1.94 -0.12 2.29 VT6 -2.31 -1.87 -5.16 VT7 4.20 -3.10 6.44 VT8 1.85 -0.68 1.42 VT9 77.14 80.20 43.35 VT10 -5.98 -1.75 -4.99 VT11 3.44 1.03 -4.94 VT12 1.89 -3.34 -1.17 VT13 6.23 -0.95 -1.87 VT14 -0.63 -0.20 1.53 VT15 -0.19 -2.30 -0.19 VT16 1.32 -1.79 -8.83 CHAPTER 3: DISCUSSIONS 3.1. Chemical structure of isolated compounds This section presents the detailed results of spectral analysis and structure determination of 28 isolated compounds from V. limonifolia and V. trifolia.
8 * 12 compounds from V. limonifolia (Figure 3.3.1), including: 3 new compounds, namely vitexlimolides A-C (VL1-VL3); and 9 known, 5,4′- dihydroxy-3,7-dimethoxyflavone (VL4), vitecetin (VL5), 5,4′-dihydroxy- 7,3′-dimethoxyflavone (VL6), verrucosin (VL7), 2α,3α-dihydroxyurs-12- en-28-oic acid (VL8), euscaphic acid (VL9), 2α,3α-dihydroxy-19-oxo- 18,19-seco-urs-11,13(18)-dien-28-oic acid (VL10), maslinic acid (VL11), and maltol O-β-D-glucopyranoside (VL12). 5 compounds VL4, VL6, VL7, VL10 and VL12 were reported from Vitex genus for the first time. Figure 3.1. Chemical structure of compounds from V. limonifolia
9 * 16 compounds from V. trifolia (Figure 3.2): Figure 3.2. Chemical structure of compounds from V. trifolia 2 new compounds, 3α-hydroxylanosta-8,24E-dien-26-oic acid (VT1), and matairesinol 4′-O-β-D-glucopyranoside (VT2); 14 known,
10 ecdysone (VT3), 20- hydroxyecdysone (VT4), 20-hydroxyecdysone 2,3- monoacetonide (VT5), turkesterone (VT6), polypodine B (VT7), rubrosterone (VT8), luteolin (VT9), (2S)-7,4'-dihydroxy-5- methoxyflavanone (VT10), vitexin (VT11), orientin (VT12), homoorientin (VT13), 2-O-rhamnosylvitexin (VT14), euscaphic acid (VT15) and tormentic acid (VT16). Compound VT5 was reported from Vitex genus for the first time. 3 compounds VT3, VT4, and VT6 were reported from V. trifolia for the first time. 3.1.1. Chemical structure of isolated compounds from V. limonifolia This section presents the detailed results of spectral analysis and structure determination of 12 isolated compounds from V. limonifolia. 3.1.1.1. Compound VL1: Vitexlimolide A (new compound) Figure 3.3. Chemical structure of VL1 and the reference compound Compound VL1 was obtained as a white amorphous powder. The HR-ESI-MS of VL1 showed a pseudo-ion peak at m/z 369.1830 [M+Cl]‒ (Calcd. for [C20H30O4Cl]‒, 369.1838), revealed the molecular formula to be C20H30O5. The 1H-NMR spectrum of VL1 (in CD3OD) showed the following signals: three tertiary methyl groups at δH 0.74, 0.86, and 0.92 (each 3H, s), two hydroxymethine protons at δH 4.38 (t, J = 3.0 Hz) and 4.58 (dd, J = 4.0, 8.0 Hz), and three olefinic protons at δH 4.65 (s), 5.13 (s), and 6.01 (d, J = 2.0 Hz). The 13C-NMR and DEPT spectra of VL1
11 revealed the signals of 20 carbons including three methyl carbons at δC 14.3, 22.0, and 33.8, seven methylenes at δC 20.4, 32.0, 32.5, 39.8, 43.3, 67.2, and 110.0, five methines 47.6, 48.8, 67.2, 74.7, and 114.4, four quaternary carbons at δC 34.1, 40.5, 150.6, 177.5, and one carbonyl carbon at δC 176.5. The 1H- and 13 C-NMR data analysis indicated the structure of VL1 as a labdane-type diterpene [42]. In addition, the NMR data of VL1 were similar to those of vitexolide E (VL1a) [42] except for an additional hydroxyl group at C-7. Figure 3.4. The important HMBC, COSY and NOESY correlations of VL1 The position of hydroxyl group at C-7 and double bond at C-8/C-17 were confirmed by the HMBC correlations from H-17 (δH 4.65 and 5.13) to C-7 (δC 74.7)/C-8 (δC 150.6)/C-9 (δC 47.6) and from H-7 (δH 4.38) to C-5 (δC 48.8)/C-6 (δC 32.5)/C9 (δC 47.6). The configuration of hydroxyl group at C-7 (the multiplicity of H-7: δH 4.38 t, J = 3.0 Hz) was proved as axial (α-oriental), based on comparing the coupling constants of H-6 and H-7 of 7β-hydroxyl compound [7β-hydroxyisocupressic acid: δH 3.83 (1H, dd, J = 5.0, 11.5 Hz, H-7), recorded in CD3OD)] [120] and 7α- hydroxyl compound [7α-hydroxylabd-8(17)-en-15,18-dioic acid-15- methyl ester: δH 4.38 (br s, H-7), recorded in CDCl3] [121]. The HMBC correlations from H-14 (δH 6.01) to C-12 (δC 67.2)/C-13 (δC 177.5)/C-15 (δC 176.5)/C-16 (δC 73.0); from H-16 (δH 5.01) to C-14 (δC 114.4)/C-15
12 (δC 176.5); from H-12 (δH 4.58) to C-9 (δC 47.6)/C-11 (δC 32.0)/C-13 (δC 177.5)/C-14 (δC 114.4)/C-16 (δC 73.0) confirmed the presence of the α,β- unsaturated γ-lactone at β-carbon (C-13) in C ring and hydroxyl group at C-12. The multiplicity of H-12 (δH 4.58, dd, J = 4.0, 8.0 Hz) confirmed the α-configuration of hydroxyl group at C-12 by comparing with the corresponding data of the vitexolide A [multiplicity of H-12 (4.56 br d, J = 10.6 Hz), recorded in acetone-d6] and 12-epivitexolide A [multiplicity of H-12 (4.61 br s), recorded in acetone-d6] [42]. Furthermore, the absolute configuration of hydroxyl group at C-12 of VL1 was also elucidated by the comparison of its experimental CD spectrum with those calculated spectra. The TDDFT calculated CD spectra of two epimers (1a-1b) [122] are shown in Figure 3.5. The CD spectrum of VL1 was recorded at the concentrations of 10-4 M (for the wavelength of 200-245 nm) and 10-2 M (for the wavelength of 200-245 nm). The CD spectra of VL1 were found similar to that of 1a indicating the configuration of the hydroxyl group at C-12 as α (R). Consequently, the structure of VL1 was defined as 7α,12α-dihydroxylabda-8(17),13-dien-15,16-olide and named vitexlimolide A. 1 .0 -4 1 (c = 1 0 M) -2 1 (c = 1 0 M) 1a 0 .5   , r e la tiv e u n its 1b 0 .0 -0 .5 -1 .0 210 220 230 240 250 260 270 280  , nm Figure 3.1. Experimental CD spectra of compound 1 and calculated CD spectra their epimers 1a and 1b.
13 Table 3.1. NMR spectral data of VL1 and the reference compound C δ C δCa,b DEPT δHa,c (J = Hz) 1 39.7 39.8 CH2 1.19 (ddd, 3.0, 12.5, 13.0, α)/1.74 (m, β) 2 20.1 20.4 CH2 1.54 (m, α)/1.64 (m, β) 3 43.0 43.3 CH2 1.30 (m, α)/1.45 (brd, 13.0, β) 4 34.3 34.1 C - 5 56.4 48.8 CH 1.75 (m) 6 25.3 32.5 CH2 1.60 (m, α)/1.91 (ddd, 2.5, 3.0, 14.0, β) 7 39.0 74.7 CH 4.38 (t, 3.0) 8 149.5 150.6 C - 9 52.8 47.6 CH 2.60 (dd, 4.0, 9.0) 10 40.1 40.5 C - 11 32.1 32.0 CH2 1.76 (m) 12 67.3 67.2 CH 4.58 (dd, 4.0, 8.0) 13 176.3 177.5 C - 14 114.2 114.4 CH 6.01 (d, 2.0) 15 174.1 176.5 C - 16 71.8 73.0 CH2 5.01 (m) 17 107.1 110.0 CH2 4.65 (s)/5.13 (s) 18 22.1 33.8 CH3 0.92 (s) 19 34.0 22.0 CH3 0.86 (s) 20 15.2 14.3 CH3 0.74 (s) C of vitexolide E (VL1a, recorded in acetone-d6) [42], arecorded in CD3OD, b125MHz, c500MHz. # 3.1.1.2. Compound VL2: Vitexlimolide B (new compound) Compound VL2 was isolated as a white amorphous powder. The molecular formula of VL2 was established as C20H30O8 from the pseudo-
14 molecular ion at m/z 385.1772 [M+Cl]‒ in HR-ESI-MS spectrum (Calcd. for [C20H30O5Cl]‒, 385.1787). Figure 3.14. Chemical structure of VL2 and the reference compound (VL1) The 1H- and 13C-NMR spectrum of VL2 (in CD3OD) showed a pattern similar to those of compound VL1. However, the signals of oxymethylene (δH 5.01 and δC 73.0) in VL1 were replaced by hemiacetal (δH 6.23 and δC 100.2) in VL2. The broad and short signals in γ-hydroxy- γ-latone group was hard to observe in 1H- and 13C-NMR, suggesting the formation of conformational equilibriums of two C-16 epimers. The position of hydroxyl group at C-7 and double bond at C-8/C-17 were confirmed by the HMBC correlations from H-17 (δH 4.78 and 5.13) to C- 7 (δC 74.7)/C-8 (δC 150.0)/C-9 (δC 47.6) and from H-7 (δH 4.38) to C-5 (δC 49.0)/C-6 (δC 32.4)/C9 (δC 47.6). Figure 0.2. The important HMBC, COSY and NOESY correlations of VL2
15 Table 3.2. NMR spectral data of VL2 and the reference compound C δ C δCa,b DEPT δHa,c (J = Hz) 1 39.8 39.8 CH2 1.19 (ddd, 3.0, 13.0, 13.0)/1.71 (m) 2 20.4 20.3 CH2 1.53 (m)/1.63 (m) 3 43.3 43.3 CH2 1.26 (m)/1.45 (brd, 13.0) 4 34.1 34.1 C - 5 48.8 49.0 CH 1.75 (m) 6 32.5 32.4 CH2 1.61 (dd, 3.0, 13.0)/1.90 (m) 7 74.7 74.7 CH 4.38 (t, 3.0) 8 150.6 150.0 C - 9 47.6 47.6 CH 2.62 (br d, 12.0) 10 40.5 40.5 C - 11 32.0 31.2 CH2 1.65 (m)/ 1.73 (m) 12 67.2 66.9 CH 4.58 (br s) 13 177.5 173.1 C - 14 114.4 117.1 CH 6.04 (s) 15 176.5 173.1 C - 16 73.0 100.2 CH 6.23 (s) 17 110.0 110.5 CH2 4.78 (s)/5.13 (s) 18 33.8 33.8 CH3 0.92 (s) 19 22.0 22.0 CH3 0.86 (s) 20 14.3 14.2 CH3 0.74 (s) C of vitexlimolide A (VL1, recorded in CD3OD), arecorded in CD3OD, b125MHz, c500MHz. # The position of hydroxyl group at C-12 was confirmed by HMBC correlation between H-11 (δH 1.65 and 1.73) and C-12 (δC 66.9) and COSY correlations of H-9 (δH 2.62)/H-11 (δH 1.65 and 1.73)/H-12 (δH 4.58). The configuration of this hydroxyl group was determined as β by comparing the multiplicity of H-12 (δH 4.58, br s) of 7α-hydroxyl compound [vitexolide A: multiplicity of H-12 (δH 4.56 br d, J = 10.6 Hz), recorded in acetone-d6] and 7β-hydroxyl compound (12-epivitexolide A) [multiplicity of H-12 (δH 4.61 br s), recorded in acetone-d6] [42]. Based on these, the structure of VL2 was determined as 7α,12β,16- trihydroxylabda-8(17),13-dien-15,16-olide and named vitexlimolide B.
16 3.1.2. Chemical structure of isolated compounds from V. trifolia This section presents the detailed results of spectral analysis and structure determination of 16 isolated compounds from V. trifolia. 3.1.2.2. Compound VT2: Matairesinol 4’-O-β-D-glucopyranoside (new compound) Figure 3.51. Chemical structure of VT2 and the reference compound Compound VT2 was obtained as a white amorphous powder. Its molecular formula was determined as C26H32O11 on the basic of HR-ESI- MS ion at m/z 521.2009 [M+H]+ (Calcd. for [C26H33O11]+, 521.2017). The 1 H-NMR spectrum of VT2 (in CD3OD) showed the signals for two pairs of ABX aromatic protons at δH 6.49 (dd, J = 1.6, 8.0 Hz), 6.55 (d, J = 1.6 Hz), and 6.66 (d, J = 8.0 Hz); 6.64 (dd, J = 1.6, 8.0 Hz), 6.73 (d, J = 1.6 Hz), and 7.04 (d, J = 8.0 Hz), two methoxy groups at δH 3.75 (s) and 3.79 (s), one anomeric proton at δH 4.84 (d, J = 8.0 Hz). The 13C-NMR and DEPT spectra revealed 26 carbon signals, of which, 18 were assigned to a lignan moiety, 2 carbons belonged to two methoxy groups, and 6 carbons contributed to a sugar moiety. The 1H- and 13C-NMR data of VT2 were very similar to those of VT2a (matairesinol 4-O-β-D-glucopyranoside) [134] except for the change position of glucopyranosyl moiety from C-4 to C-4′. Butanolide ring was confirmed by HMBC correlation between H- 9 (δH 3.91 and 4.16) and C-9′ (δC 181.5) as well as COSY correlations of H-8′ (δH 2.66)/H-8 (δH 2.47)/H-9 (δH 3.91 and 4.16).
17 Figure 3.52. The important HMBC, COSY and NOESY correlations of VT2 The HMBC correlations between H-7 (δH 2.52) and C-1 (δC 131.3)/C-2 (δC 113.3)/C-6 (δC 122.2)/C-8 (δC 42.6)/C-9 (δC 72.9)/C-8′ (δC 47.6); between methoxy protons (δH 3.75) and C-3 (δC 149.0); between H-7′ (δH 2.85) and C-1′ (δC 134.2)/C-2′ (δC 114.8)/C-6′ (δC 123.0)/C-8′ (δC 47.6)/C-9′ (δC 181.5)/C-8 (δC 42.6); and between methoxy protons (δH 3.79) and C-3′ (δC 150.6) suggested the positions of two 3-methoxy-4- hydroxyphenyl groups at C-7 and C-7′. The coupling constant, JH-1″/H-2″ = 8.0 Hz of sugar moiety and its 13C-NMR chemical shifts: C-1′′ (δC 102.9), C-2′′ (δC 74.9), C-3′′ (δC 77.8), C-4′′ (δC 71.3), C-5′′ (δC 78.1), and C-6′′ (δC 62.5) suggested the presence of β-D-glucopyranosyl moiety. This was further confirmed by acidhydrolysis of VT2 (identified as TMS derivative). In addition, the HMBC correlation between glc H-1″ (δH 4.84) and C-4′ (δC 146.8) determined the glucose moiety at C-4′ of aglycone. The absolute configuration of aglycone was determined by the NOESY, CD spectra. The NOE correlations between H-8′ (δH 2.66) and Hα-9 (δH 3.91); H-8 (δH 2.48) and Hβ-9 (δH 4.16)/H-7 (δH 2.52); H-7 (δH 2.52) and Hα-9 (δH 3.91), suggested the configurations of H-8 and H-8′ to be trans. The two negative Cotton effects at 226 and 275 nm in the CD spectrum indicated a (8R,8′R)-configurations in matairesinol [134]. Consequently, the new compound VT2 was determined to be matairesinol 4′-O-β-D-glucopyranoside.
18 Table 3.12. NMR spectral data of VT2 and the reference compound C δ C δCa,b δHa,c (J = Hz) 1 132.6 131.3 - 2 112.9 113.3 6.55 (d, 1.6) 3 148.8 149.0 - 4 145.1 146.2 - 5 115.3 116.2 6.66 (d, 8.0) 6 120.5 122.2 6.49 (dd, 1.6, 8.0) 7 36.9 38.9 2.52 (m) 8 40.8 42.6 2.47 (m) 9 70.8 72.9 3.91 (dd, 8.0, 8.8, α) 4.16 (dd, 7.2, 8.8, β) 3-OMe 55.6 56.4 3.75 (s) 1 129.0 134.2 - 2 113.5 114.8 6.73 (d, 1.6) 3 147.5 150.6 - 4 145.1 146.8 - 5 115.4 117.8 7.04 (d, 8.0) 6 121.6 123.0 6.64 (dd, 1.6, 8.0) 7 33.8 35.3 2.85 (dd, 6.8, 12.8) 8 45.7 47.6 2.66 (m) 9 178.6 181.5 - 3-OMe 55.6 56.7 3.79 (s) 4-O-glc 1 100.2 102.9 4.84 (d, 8.0) 2 73.3 74.9 3.46 (dd, 8.0, 9.2) 3 77.1 77.8 3.45 (m) 4 69.7 71.3 3.38 (m) 5 76.9 78.1 3.38 (m) 6 60.7 62.5 3.67 (dd, 4.0, 10.8) 3.85 (br d, 10.8) C of matairesinol 4-O-β-D-glucopyranoside (recorded in DMSO-d6) [128], arecored in CD3OD, # b 100MHz, c400MHz.