Nghiên cứu thành phấn hóa học và hoạt tính sinh học loài vitex limonifolia wall ex c b clark và vitex trifolia l tt tiếng anh

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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 INTRODUCTION 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.” 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 The main contents of the thesis Isolate compounds from the leaves of V limonifolia and V trifolia; 2 Elucidate chemical structures of the isolated compounds; Evaluate the anti-inflammatory activity of the isolated compounds from V limonifolia; 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 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 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 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 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 antiinflammatory 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 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 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 2.38 5.41 1.30 VT1 3.52 4.23 5.97 VT2 4.99 -1.51 -4.34 VT3 13.27 0.71 3.37 VT4 1.94 -0.12 2.29 VT5 -2.31 -1.87 -5.16 VT6 4.20 -3.10 6.44 VT7 1.85 -0.68 1.42 VT8 VT9 77.14 80.20 43.35 -5.98 -1.75 -4.99 VT10 3.44 1.03 -4.94 VT11 1.89 -3.34 -1.17 VT12 6.23 -0.95 -1.87 VT13 -0.63 -0.20 1.53 VT14 -0.19 -2.30 -0.19 VT15 1.32 -1.79 -8.83 VT16 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 * 12 compounds from V limonifolia (Figure 3.3.1), including: new compounds, namely vitexlimolides A-C (VL1-VL3); and known, 5,4′dihydroxy-3,7-dimethoxyflavone (VL4), vitecetin (VL5), 5,4′-dihydroxy7,3′-dimethoxyflavone (VL6), verrucosin (VL7), 2α,3α-dihydroxyurs-12en-28-oic acid (VL8), euscaphic acid (VL9), 2α,3α-dihydroxy-19-oxo18,19-seco-urs-11,13(18)-dien-28-oic acid (VL10), maslinic acid (VL11), and maltol O-β-D-glucopyranoside (VL12) 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 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 7αhydroxyl 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,βunsaturated 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 (c =   , r e la tiv e u n its (c = -4 M) -2 M) 1a 1b 0 -0 -1 210 220 230 240 250 260 270 280  , nm Figure 3.1 Experimental CD spectra of compound 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 39.7 39.8 CH2 1.19 (ddd, 3.0, 12.5, 13.0, α)/1.74 (m, β) 20.1 20.4 CH2 1.54 (m, α)/1.64 (m, β) 43.0 43.3 CH2 1.30 (m, α)/1.45 (brd, 13.0, β) 34.3 34.1 C - 56.4 48.8 CH 1.75 (m) 25.3 32.5 CH2 1.60 (m, α)/1.91 (ddd, 2.5, 3.0, 14.0, β) 39.0 74.7 CH 4.38 (t, 3.0) 149.5 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 17 107.1 110.0 CH2 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) δCa,b DEPT δHa,c (J = Hz) 150.6 C - 5.01 (m) 4.65 (s)/5.13 (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-hydroxyγ-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 C7 (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 δCa,b DEPT δHa,c (J = Hz) δ C 39.8 39.8 CH2 1.19 (ddd, 3.0, 13.0, 13.0)/1.71 (m) 20.4 20.3 CH2 1.53 (m)/1.63 (m) 43.3 43.3 CH2 1.26 (m)/1.45 (brd, 13.0) 34.1 34.1 C 48.8 49.0 CH 1.75 (m) 32.5 32.4 CH2 1.61 (dd, 3.0, 13.0)/1.90 (m) 74.7 74.7 CH 4.38 (t, 3.0) 150.6 150.0 C 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,16trihydroxylabda-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-ESIMS ion at m/z 521.2009 [M+H]+ (Calcd for [C26H33O11]+, 521.2017) The 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, carbons belonged to two methoxy groups, and 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 H9 (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-4hydroxyphenyl 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) 132.6 131.3 112.9 113.3 6.55 (d, 1.6) 148.8 149.0 145.1 146.2 115.3 116.2 6.66 (d, 8.0) 120.5 122.2 6.49 (dd, 1.6, 8.0) 36.9 38.9 2.52 (m) 40.8 42.6 2.47 (m) 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, 100MHz, c400MHz # b 19 3.2 Chemical structure of isolated compounds 3.2.1 Anti-inflammatory activities of isolated compounds from V limonifolia Firstly, 12 compounds from V limonifolia were tested for their cytotoxicity on BV2 cells at concentration 20 M The screening results showed that VL11 has a strong toxicity (cell viability of 59.04% < 80%), so this compound was not evaluated anti-inflammatory activity The remaining compounds not have toxicity on BV2 cell (cell viability > 80%) Then, compounds VL1-VL10, VL12 were evaluated for their inhibitory activity on NO production in LPS-activated BV2 cells at concentration of 50 µM As the screening result, all of the compounds showed the significant inhibitory effects (% inhibition > 50%), therefore they were evaluated for their inhibitory activities at smaller concentrations : 1.0, 5.0, 10, 20 µM to find IC50 Among these, VL2 and VL3 showed potent inhibitory activity on NO production in LPSactivated BV2 cells with IC50 values of 2.500.34 and 7.130.87 M, respectively VL6 and VL10 showed significiant inhibitory activity on NO production, with IC50 values of 19.161.09, 15.881.17 M, respectively, as compared with positive control L-NMMA (IC50 22.101.20 M) Compounds VL4, VL5, VL7, and VL9 inhibited NO production with IC50 values ranging from 24.70 đến 45.31 M The results of inhibition NO production in LPS-activated BV2 cells of compounds from V limonifolia might suggest some comments on the relationship between chemical structure and anti-inflammatory activity of compounds: - Labdane compounds VL2, and VL3 with a hydroxy group at C-16 exhibited higher inhibitory effects than VL1 Furthermore, VL2 with 20 more hydroxy group at C-12 exhibited higher inhibitory effects than VL3 - The isolated flavonoid compounds (VL4, VL5, and VL6) exhibied moderate inhibitory activity of NO production with IC50 values ranging from 19.16 to 39.67 M 3.2.2 Antiviral activity of isolated compounds 3.2.2.1 Antiviral activity of isolated compounds from V limonifolia 12 compounds VL1-VL12 from V limonifolia were evaluated for antiviral activities against some enterovirus, including: coxsackievirus B3 (CVB3), human rhinovirus 1B (HRV1B), and enterovirus 71 (EV71) As the results, flavonoids VL4 and VL6 showed potent antiviral activities against CVB3 virus with IC50 of 0.21 ± 0.06, 1.86 ± 0.18, respectively, as compared with the positive control rupintrivir at IC50 0.12 ± 0.06 μM Furthermore, VL4 showed inhibitory effect on HVR1B virus with IC50 of 0.61±0.21 μM, higher than the positive control ribavirin (IC50 of 48.07±1.46 μM) This compound also elicited antiviral activity against EV71 virus with IC50 of 32.05±0.94 μM The results suggested broadspectrum antiviral activity of VL4 against EVs including CVB3, HRV1B, and EV71 From the results of antiviral activity isolated compounds from V limonifolia, two flavonoids (VL4 and VL6) exhibited potent antiviral effects In addition, many published researches also reported the antiviral activities against enterovirus of flavonoid compounds Therefore, this results of the thesis suggested the antiviral effects against enterovirus of flavonoid structure 3.2.2.2 Antiviral activity of isolated compounds from V trifolia 16 compounds VT1-VT16 from V trifolia were evaluated for antiviral activities against CVB3, HRV1B, and EV71 viruses at concentration of 10 M 21 As the screening results of antiviral activities against some enterovirus, compound VT9 showed the positive effects on CVB3/ HRV1B/ EV71 virus at concentration 10 M with the percentage of cell viability values 77.14%, 80.20%, 43.35%, respectively CONCLUSIONS From the leaves of two species of Vitex limonifolia and Vitex trifolia growing in Vietnam, we isolated and determined 28 compounds and evaluated the bioactivity of these compounds 12 compounds were isolated and identified from Vitex limonifolia, including: three new compounds, vitexlimolide A (VL1), vitexlimolide B (VL2), and vitexlimolide C (VL3); five compounds were isolated from Vitex genus for the first time, 5,4′-dihydroxy-3,7dimethoxyflavone (VL4), 5,4′-dihydroxy-7,3′-dimethoxyflavone (VL6), verrucosin (VL7), 2α,3α-dihydroxy-19-oxo-18,19-seco-urs-11,13(18)dien-28-oic acid (VL10), and maltol O-β-D-glucopyranoside (VL12); four known compounds, vitecetin (VL5), 2α,3α-dihydroxyurs-12-en-28oic acid (VL8), euscaphic acid (VL9), and maslinic acid (VL11) 16 compounds were isolated and identified from Vitex trifolia, including: two new compounds, 3α-hydroxylanosta-8,24E-dien-26-oic acid (VT1), and matairesinol 4′-O-β-D-glucopyranoside (VT2); one compounds were isolated from Vitex genus for the first time, 20hydroxyecdysone 2,3-monoacetonide (VT5); three compounds were isolated from Vitex trifolia for the first time, ecdysone (VT3), 20hydroxyecdysone (VT4), and turkesterone (VT6); ten known compounds, polypodine B (VT7), rubrosterone (VT8), luteolin (VT9), (2S)-7,4'dihydroxy-5-methoxyflavanone (VT12), homoorientin (VT10), (VT13), vitexin (VT11), 2-O-rhamnosylvitexin euscaphic acid (VT15), and tormentic acid (VT16) orientin (VT14), 22 Twelve compounds (VL1-VL12) from Vitex limonifolia were tested for their inhibitory activities on nitric oxide production in activated BV2 cells As the results, compounds VL2, VL3 exhibited potent inhibitory activity on NO production in LPS-activated BV2 cells with IC50 values of 2.500.34, 7.130.87 M, respectively; compounds VL6, VL10 showed significant inhibitory activity on NO production with IC50 values of 19.161.09, 15.881.17M, respectively, as compared with L-NMMA (positive control, IC50 of 22.101.20 µM) Compounds VL4, VL5, VL7, and VL9 inhibited NO prodution with IC50 values ranging from 15.88 to 72.50 M Twelve isolated compounds (VL1-VL12) from Vitex limonifolia and sixteen isolated compounds (VT1-VT16) from Vitex trifolia were evaluated for antiviral activities against virus coxsackievirus B3, human rhinovirus 1B and enterovirus 71 Among them, VL4 and VL6 showed potent antiviral activity against coxsackievirus B3 infection cells with the IC50 values of 0.21±0.06 and 1.86±0.18 (µM), respectively Compound VL4 also elicited antiviral activity against human rhinovirus 1B (with IC50 values 0.61±0.21 μM) and enterovirus 71 (with IC50 values 32.05±0.94 μM) In screening of antiviral activity of compounds (VT1VT16) against coxsackievirus B3, human rhinovirus 1B, and enterovirus 71, only VT9 showed positive results, with percentage of cell viability values 77.14%, 80.20%, 43.35%, respectively 23 RECOMMENDATIONS From the research results: Labdane VL2, VL3 showed potent inhibitory activity on NO production in LPS-actived BV2 cells Therefore, it is necessary to study more about the anti-inflammatory activity of these compounds by animal in vivo studies for orienting their applicabilities Compound VL4 elicited broad-spectrum antiviral activity of against EVs including CVB3, HRV1B, and EV71 These results indicate that VL4 might be a potential anti-rhinovirus agent A detailed pharmacological investigation, including animal in vivo studies, should follow to confirm this promising in vitro data Compound VT9 showed positive results in screening of antiviral activity against CVB3/HRV1B/EV71 Therefore, further research is needed on antiviral activities of this compound 24 NEW FINDINGS OF THE THESIS 28 compounds were isolated and identified from Vitex limonifolia and Vitex trifolia, including: - new compounds: new compounds from Vitex limonifolia, namely vitexlimolides A-C (VL1-VL3); and new compounds from Vitex trifolia, 3α-hydroxylanosta-8,24E-dien-26-oic acid (VT1), and matairesinol 4′-O-β-D-glucopyranoside (VT2) - compounds were isolated for the first time from Vitex genus: 5,4′-dihydroxy-3,7-dimethoxyflavone (VL4), 5,4′-dihydroxy-7,3′- dimethoxyflavone (VL6), verrucosin (VL7), 2α,3α-dihydroxy-19-oxo18,19-seco-urs-11,13(18)-dien-28-oic acid (VL10), maltol O-β-D- glucopyranoside (VL12), and 20-hydroxyecdysone 2,3-monoacetonide (VT5) - compounds were isolated for the first time from Vitex trifolia: ecdysone (VT3), 20-hydroxyecdysone (VT4), and turkesterone (VT6) Compounds from Vitex limonifolia were evaluated for their NO production inhibitory activity in LPS-activated BV2 cells for the first time As the results, vitexlimolides B and C (VL2 and VL3), 5,4′dihydroxy-7,3′-dimethoxyflavone (VL6), 2α,3α-dihydroxy-19-oxo- 18,19-seco-urs-11,13(18)-dien-28-oic acid (VL10) showed potent antiinflamatory activity with IC50 values ranging from 2.50 µM to 19.16 µM 28 compounds from Vitex limonifolia and Vitex trifolia were evaluated for their antiviral activity against coxsackievirus B3 (CVB3), human rhinovirus 1B (HRV1B), and enterovirus 71 (EV71) for the first time As the results, 5,4′-dihydroxy-3,7-dimethoxyflavone (VL4) showed potent antiviral activities against CVB3/HRV1B/EV71 virus with IC50 values 0.21, 0.61, 32.05 μM, respectively Compound 5,4′-dihydroxy7,3′-dimethoxyflavone (VL6) showed significiant antiviral activities against CVB3 virus with IC50 values 1.86 μM 25 PUBLICATIONS WITHIN THE SCOPE OF THESIS Ninh Khac Ban, Nguyen Thi Kim Thoa, Tran My Linh, Do Thi Trang, Phan Van Kiem, Nguyen Xuan Nhiem, Bui Huu Tai, Chau Van Minh, Jae-Hyoung Song, Hyun-Jeong Ko, Seung Hyun Kim Labdanetype diterpenoids from Vitex limonifolia and their antivirus activities Journal Natural Medicine, 2018, 72, 290-297 Ninh Khac Ban, Nguyen Thi Kim Thoa, Tran My Linh, Vu Huong Giang, Do Thi Trang, Nguyen Xuan Nhiem, Bui Huu Tai, Tran Hong Quang, Pham Hai Yen, Chau Van Minh, Phan Van Kiem Chemical constituents of Vitex trifolia leaves Natural Product Communications 2018, 13 (2), 129-130 Nguyen Thi Kim Thoa, Ninh Khac Ban, Do Thi Trang, Tran My Linh, Vu Huong Giang, Nguyen Xuan Nhiem, Phan Van Kiem Triterpenes from Vitex limonifolia Vietnam Journal of Chemistry, 2017, 55(6), 715 -719 Nguyen Thi Kim Thoa, Ninh Khac Ban, Do Thi Trang, Tran My Linh, Vu Huong Giang, Nguyen Xuan Nhiem, Phan Van Kiem Flavonoid and other compounds from Vitex limonifolia Vietnam Journal of Chemistry, 2018, 56(6), 679-683 Nguyen Thi Kim Thoa, Ninh Khac Ban, Do Thi Trang, Tran My Linh, Vu Huong Giang, Nguyen Xuan Nhiem, Phan Van Kiem Ecdysteroids from leaves of Vitex trifolia Vietnam Journal of Chemistry, 2018, 56(2), 127-261 Nguyen Thi Kim Thoa, Vu Kim Thu, Ninh Khac Ban, Do Thi Trang, Phan Van Kiem Triterpenoids and ecdysteroids from leaves of Vitex trifolia Journal of Military Science and Technology Research, 2018, Special number CBSE2, 83-87 (in Vietnamese) Nguyen Thi Kim Thoa, Nguyen Thi Thu Hien, Ninh Khac Ban, Nguyen Xuan Nhiem, Do Thi Trang Triterpenes and lignan from leaves of Vitex limonifolia Proceedings of the Conference Basic Science and Earth Science 2nd (CBES2), 2018, 17-22 (in Vietnamese) ... chemical constituents and biological activities of Vitex limonifolia Wall Ex C. B. Clarke and Vitex trifolia L. ” The aim of the thesis Study on chemical constituents of leaves of two Vitex species including... 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... isolated and identified from Vitex limonifolia and Vitex trifolia, including: - new compounds: new compounds from Vitex limonifolia, namely vitexlimolides A -C (VL1-VL3); and new compounds from Vitex
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