Summary of Doctoral thesis in Chemistry: Research on chemical compositions and biological activities of (Paramignya trimea (Oliv.) Guill. (Xao tam phan) and (Morinda longissima Y. Z. Ruan

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The task of studying their chemical composition and biological activities of Morinda longissima and Paramignya trimera species in our country is important and urgent. From that point, the topic “Research on chemical compositions and biological activities of (Paramignya trimea (Oliv.) Guill. (Xao tam phan) and (Morinda longissima Y. Z. Ruan (Nho dong) '' was chosen.

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Nội dung Text: Summary of Doctoral thesis in Chemistry: Research on chemical compositions and biological activities of (Paramignya trimea (Oliv.) Guill. (Xao tam phan) and (Morinda longissima Y. Z. Ruan

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY -------------------------------- TRAN THU HUONG RESEARCH ON CHEMICAL COMPOSITIONS AND BIOLOGICAL ACTIVITIES OF PARAMIGNYA TRIMERA (OILV.) GUILL.) AND MORINDA LONGISSIMA Y. Z. RUAN Speciality: Chemistry of Natural Products Code: 9.44.01.17 SUMMARY OF DOCTORAL THESIS IN CHEMISTRY Ha Noi, 2020 1
The thesis was completed at: Graduate University of Science and Technology – Vietnam Academy of Science and Technology Scientific Supervisors 1: Assoc. Prof. Nguyen Manh Cuong Scientific Supervisors 2: Assoc. Prof. Đo Thi Thao Reviewer 1: … Reviewer 2: … Reviewer 3: …. This thesis is going to be defended at the council of doctoral thesis examiners of Graduate University of Science and Technology, Vietnam Academy of Science and Technology 18 Hoang Quoc Viet, Cau Giay, Hanoi, at ..., ...., ...2020. The thesis can be found at: - Library of Graduate University of Science and Technology - National Library 2
INTRODUCTION 1. The necessity of the dissertation subject In Vietnam, seven Paramignya species have been identified, distributed mostly in the south of the country. Paramignya trimera (Oliv.) Guill., local name “Than duoc” or “Xao tam phan”, is an endemic plant in South Vietnam. The stems and roots of the plant have been used in folk medicine to treat liver disease, especially cirrhotic ascites. Morinda longissima Y.Z. Ruan. (Rubiaceae) is a small-to- medium tree, contributed in mountainous area in the North provinces of Vietnam. The stems and roots of medicinal plant Morinda longissima has been used by Thai's ethnic people in Son La and Lai Chau provinces for treatment of liver disorders, collitis and oedema. Therefore, the task of studying their chemical composition and biological activities of Morinda longissima and Paramignya trimera species in our country is important and urgent. From that point, the topic “Research on chemical compositions and biological activities of (Paramignya trimea (Oliv.) Guill. (Xao tam phan) and (Morinda longissima Y. Z. Ruan (Nho dong) '' was chosen. 2. The objectives of the thesis - Study on chemical composition of Paramignya trimera and Morinda longissima species. - Evaluation of hepatoprotective and anti-HBV activities of extracts and pure compounds from stems and roots of Xao tam phan (Paramignya trimera (Oliv.) Guill.) and Nho dong (Morinda longissima Y.Z. Ruan). 3. The main contents of the thesis - Evaluation of hepatoprotective and anti-HBV activity of herbal extracts. - Isolation of compounds from stems and roots of Paramignya trimera (Oliv.) Guill. and Morinda longissima Y.Z. Ruan. - Determination of chemical structures of isolated compounds. 3
- Evaluation of hepatoprotective and anti-HBV activity of isolated compounds. Outline of the thesis: The thesis consists of 139 pages with 45 tables, 21 figures, 4 schemes of isolation and 137 references. The thesis consists of 4 chapters: Introduction (2 pages), Chapter 1: Liturature overview (28 pages); Chapter 2: Materials and Methods (02 pages); Chapter 3: Results (10 pages), Chapter 4: Discussion (83 pages); Conclusion (2 pages); Suggestion (1 page); Publications related to the thesis (1 page); References (15 pages); Appendix (31 pages). CHAPTER 1: LITERATURE OVERVIEW This part reviews the following points: - The chemical composition and biological activities of compounds isolated from Paramignya plants were presented thoroughly. - The chemical compositions and biological activities of compounds isolated from Morinda plants were presented thoroughly. - An overview of biological techniques for screening hepatoprotective and anti-HBV activity was presented in detail. CHAPTER 2: MATERIALS AND METHODS 2.1.Plant Materials The stems and roots of Paramingya trimera and Morinda longissma were collected in Khanh Hoa province and in Son La province, Vietnam, respectively. The plants were identified by the botanist Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature, VAST. Voucher specimens (C-499 and C-547) are deposited in the herbarium of the Institute of Natural Products Chemistry, VAST, Hanoi, Vietnam. 2.2. Method - Phytochemical techniques - Chromatographic TLC analysis on precoated plates of silica gel 60 F254, separation methods including column chromatography on silica gel, C-18 silica gel, Diaion HP-20, Sephadex LH-20 were used to isolate natural compounds from plant extracts. 4
- Structures of isolated compounds were identified by physical, chemical and spectroscopic methods including ESI-MS, HR-ESI- MS, IR, UV, 1D và 2D-NMR. - Evaluation of biological activity The biological activities of the extracts and pure natural compounds were evaluated including: -In vitro anti-HBV activity on HepG2.2.15 cell line based on HBsAg expression levels with 50% inhibitory concentration values (IC50). - In vivo hepatoprotective activities against paracetamol-induced hepatotoxicity in BALB/c mice. CHAPTER 3: EXPERIMENTAL AND RESULTS 3.1. Evaluation of hepatoprotective and anti-HBV activities of herbal extracts 3.2. Isolation of natural compounds from Paramignya trimera From the stems and roots of Paramignya trimera, 10 compounds, including four coumarins: ostruthin (PT-1); ninhvanin (PT-2, new compound); 6-(6-hydroxy-3,7-dimethylocta-2,7- dienyl)-7-hydroxycoumarin (PT-6); ninhvanin B (PT-7, new compound); two dimeric monoterpene-linked coumarin glycosides, paratrimerin A (PT-8, new compound); paratrimerin B (PT-9, new compound); a new alcohol, paramitrimerol (PT-5, new compound), a chromene: 6-(2-hydroxyethyl)-2,2-dimethyl- 2H-1-benzopyran (PT-3), an alkaloid: citrusinine-I (PT-4) and a new limonoid, parabacunoic acid (PT-10, new compound) were isolated. - Spectroscopic data of isolated compounds. 3.2. Isolation of natural compounds from Morinda longissima From the stems and roots of Morinda longissima, 22 compounds, including twelve anthranoids: damnacanthal (ML-1); lucidin-ω- methyl ether (ML-2); soranjidiol (ML-3); morindone -5-methyl ether (ML-4); rubiadin (ML-5); rubiadin-3-methyl ether (ML-6); damnacanthol (ML-7); morindone (ML-8); 1-hydroxy-2-methyl-6- methoxy anthraquinone (ML-9); morindone-6-methyl ether (ML- 5
10); morindone-6-O-β-gentiobioside (ML-11); lucidin-3-O-β- primeveroside (ML-12), two new naphthalene glycosides: morinlongoside A (ML-13, new compound); morinlongoside B (ML-14, new compound), two iridoid glycosides: morinlongoside C (ML-15, new compound); geniposidic acid (ML-16), five glycosides: (3R)-3-O-[β-D-xylopyranosyl-(1→6)-β-D- lucopyranosyl]-l-octen-3-ol (ML-17); acteoside (ML-18); cistanoside E (ML-19); ethyl-β-D-galatopyranoside (ML-20); isoacteoside (ML-21) and a flavonoid: quercetin (ML-22) were isolated. - Spectroscopic data of isolated compounds. 3.4. Evaluation of hepatoprotective and anti-HBV activities of several pure compounds CHAPTER 4: DISCUSSION This chapter presented a discussion of the results of the structural identification and biological evaluation of extract and pure compounds from stems and roots of Morinda longissima and Paramignya trimera 4.1. Evaluation of hepatoprotective and anti-HBV activities of herbal extracts 4.1.1. Evaluation of anti-HBV activities Table 4.1. Effect on viability of HepG2.2.15 cells of extracts No. species Sample Code OD % cell viability 1 Blank 0,049 2 DMSO 0,805 100 3 P. trimera MeOH extract PT 0,920 114,23 4 PR P. trimera aqueous extracts 0,799 99,27 5 PR1 P. trimera PR1 fraction 0,811 100,70 6 PR2 P. trimera PR2 fraction 0,832 103,31 7 P. trimera PR3 fraction PR3 0,895 111,13 8 PR4 P. trimera PR 4 fraction 0,805 100,02 6
9 PR5 P. trimera PR 5 fraction 0,192 23,84 10 ML M. longissima EtOH extract 0,86 102,25 11 M. longissima aqueous W extracts 0,82 107,28 08 samples with cell viability ≥ 80 will be further studied for inhibitory activity of secretion of HBV surface antigen (HBsAg). Table 4. 1. Inhibitory activity of HBsAg secretion in HepG2.2.15 cells of extracts No. Code Sample Conc. OD (450nm) SE Inhibition ( %) 1 Blank 0,190 0,140 0 2 DMSO 0 µM 1,228 0,016 100 3 Lamivudine 50 µM 0,635 0,022 42,86 4 PT MeOH extract 60 µg/ml 1,351 0,026 111,88 5 PR aqueous extracts 60 µg/ml 1,647 0,066 140,37 6 PR1 PR1 fraction 60 µg/ml 1,393 0,011 115,89 7 PR2 PR2 fraction 60 µg/ml 1,406 0,016 117,19 8 PR3 PR3 fraction 60 µg/ml 1,310 0,035 107,93 9 PR4 PR 4 fraction 60 µg/ml 1,269 0,014 103,98 10 ML EtOH extract 60 µg/ml 0,91 0,028 69,37 11 W aqueous extracts 60 µg/ml 1,051 0,06 85,04 Ethanol and aqueous extracts of Morinda longissima showed inhibitory activity of HBsAg secretion in HepG2.2.15 cells. - Table 4. 3. The IC50 values of two active extracts Conc, Abs 450 SE Inhibition (%) IC50 (μg/ml) Blank 0,071 0,007 0 DMSO 0 µM 1,911 0,018 100 Lamivudine 50 µM 0,905 0,040 45,30 19,92±0,18 ML 15 µg/ml 1,856 0,001 97,02 30 µg/ml 1,754 0,081 91,48 146,90±30,17 60 µg/ml 1,567 0,129 81,31 7,5 µg/ml 1,966 0,000 102,98 W 7,5 µg/ml 1,917 0,008 100,34 15 µg/ml 1,894 0,038 99,09 297,76±58,14 30 µg/ml 1,843 0,028 96,29 60 µg/ml 1,841 0,107 90,77 7
The ethanol (ML) and aqueous extracts (W) of stems and roots of Morinda longissima showed significant inhibitory anti- HBV activities in vitro. 4.1.2. The hepatoprotective activities Both ethanol and aqueous extracts of Morinda longissima stems and roots exhibited hepatoprotective activities against paracetamol-induced hepatotoxicity in BALB/c mice. Table 4. 2. Effect of methanol extract and aqueous extract on the serum levels of ALT, AST No. Sample AST (UI/L) ALT (UI/L) 1 Control 87,80 ± 1,48 28,70 ± 2,59 715,75 ± 253,94 558,25 ± 296,54 2 Paracetamol 400mg/kg p
4 5 11 OH 10 3 6 12 2 13 7 9 O 1 8 14 PT-3 (6-(2-Hydroxyethyl)-2,2- dimethyl-2H-1-benzopyran), PT-1 (ostruthin), C19H22O3, 298 C13H16O2, 203 PT-2 (ninhvanin) mới, C20H24O4, 328 PT-4 (citrusinine-I), C16H15NO5, 301 PT-5 (paramitrimerol) mới, C14H16O3, 232 PT-6 6-(6-hydroxy-3,7- dimethylocta-2,7-dienyl)-7- PT-7 (ninhvanin B) mới, C19H22O4, 314 hydroxycoumarin, C19H22O4, 314 PT-9 (paratrimerin B) mới, C40H44O16, 780 PT-10 (axit parabacunoic) mới, C32H44O13, 636 PT-8 (paratrimerin A) mới, C45H52O20, 912 Compound PT-8 was obtained as a white amorphous powder with a negative optical rotation ([α]D25–25.0o, c = 0.02, MeOH). Its molecular formula was determined to be C40H44O16 based on the quasi- molecular ion peak observed at m/z 781.2691 [M+H]+ (calculated for C40H45O16, 781.2702) and m/z 803.2548 [M+Na]+ (calculated for C40H44O16Na, 803.2522) in the positive HR-ESI-MS spectrum. A further fragment ion was identified at m/z 457.1637 ([M–2xC6H10O5+H]+, 9
calculated for C28H25O6, 457.1651), indicating the presence of two glycosyl moieties in PT-8. The IR spectrum had bands at max 3379, 1646 cm-1, ascribable to hydroxyls and lactone carbonyl groups, respectively. The UV spectrum showed absorbtion maxima, max at 204, 256, 292, and 330 nm, characteristic of a 7-oxygenated coumarinchromophore. The 13C- NMR /DEPT spectra of PT-8 (CD3OD) contained 40 carbon signals (12 x C, 22 x CH, 4 x CH2, 2 x CH3), assignable to two glucosyl moieties, two coumarin nuclei, and a monoterpene ring. Its 1H-NMR spectrum of PT-8, displayed patterns similar to those of ostruthin, with characteristic signals of two pairs of cis-located olefinic protons H-3/H-4 and H-3/H-4(d, J=9.5 Hz, each), as well as two pairs of para-singlet aromatic protons H- 5/H-8, and H-5/H-8 (s, each), indicative of two 6,7-disubstituted coumarin nuclei. The structures of two coumarin nuclei in PT-8 were confirmed based on analysis of HSQC, HMBC, COSY and NOESY spectra. Two anomeric proton signals in PT-8 were observed at δH 4.99 (d, H-1) and δH 4.93 (d, H-1) (each J = 8.0 Hz), together with six pair- wise carbon signals at δC 101.6 (C-1)/102.5 (C-1), 74.8 (C-2)/75.0 (C-2), 78.2 (C-3)/78.3 (C-3), 71.7 (C-4)/71.2 (C-4), 78.4 (C- 5)/78.4 (C-5) and 62.7 (C-6)/62.5 (C-6) (Table 1). This indicated the presence of two glucosyl moieties in the β-configuration. The positions of the β-glucopyranosyl moieties at oxygenated carbons C-7/C- 7 of the two coumarin rings was determined based on the HMBC correlations (Figure 4.12) between the anomeric protons H-1(δH 4.99, d, J = 8.0 Hz) and H-1 (δH 4.93, d, J = 8.0 Hz) to carbons C-7 (δC 158.3) and C-7 (δC 160.0), respectively (Table 4.11, Figure 4.12). The NOESY spectra of PT-8 also confirmed the location of two sugar units based on the cross-peaks from the anomeric protons H-1 and H-1 to the singlet aromatic protons H-8 and H-8, respectively. The structure of the monoterpene (C10) bridge, determined as an 1,4-dimethyl-4- vinylcyclohexene, was similar to that of some monoterpene–linked biscoumarins isolated from other rutaceous species, such as bisparasin from Citrus paradise and thamnosin from Thamnosma montana. The coupling constant 3J9-10 = 16.5 Hz was indicative of two vicinal protons H-9 (δH 6.41) and H-10 (δH 6.37) in the trans position (Figure 4.12). Furthermore, the monoterpene bridge attached to two glucopyranosyl coumarin units at the non-protonated carbons C-6 (δC 127.7) and C-6(δC 10
131.6) could be directly determined based on the HMBC cross-peaks (3J– correlations) from the coumarinic protons H-5 (δH 7.47) and H-5 (δH 7.39) to the tertiary carbons of the vinylcyclohexene moiety C-9 (δC 121.2) and C-9 (δC 44.3), respectively (Table 4.11, Figure 4.12). Furthermore, two coumarinic protons H-5 and H-5 have NOESY interactions with the vinylic proton H-9 (δH 6.41) and proton H-9 (δH 4.13), respectively. These above spectroscopic evidences confirmed the linkage at C-6/C-9 and C-6/C-9 between the two glucosidic coumarins and the 1,4-dimethyl-4-vinylcyclohexene moiety. The bis coumarins comprising a cyclohexene ring acting as a bridge between two coumarins are quite common in nature. The cyclohexene ring can be substituted with between 3 to 5 substituents. The most common type is 1,3,4,4-tetrasubstituted cyclohexene (as in phebalin), whereas 1,3,4,5,5-pentasubstituted cyclohexene (as in toddalosin) is less common. Two coumarin nuclei can link through a 1,4- dimethyl-4-vinylcyclohexene chain at positions C-6/C-8 as in isothamnosin A, at C-8/C-6as in bisparasin, C-8/C-8 as in phebalin, and at C-6/C-6 as in thamnosin and our new compound PT-8. The NOESY spectrum of PT-8 showed a correlation of the methyl signal 11-CH3 (δH 1.31) to proton H-9 (δH 4.13), indicative of a cis-configuration of the 11- CH3 group and proton H-9, as well as a cis-arrangement of two coumarin units on the 1,4-dimethyl-4-vinylcyclohexene ring, similar to the case of bisparasin, a biscoumarin isolated from Citrus plants. Furthermore, the 11-CH3 group has NOESY correlation to the methylene protons H-12 (δH 1.87, m) and H-12 (δH 2.16, m) (Table 4.14), indicating that the cyclohexene ring adopts a half-chair conformation and the 11-CH3 group and proton H-9 are oriented axial- and pseudo-axial on the ring, respectively. Thus, the relative stereochemistry of paratrimerin A (PT-8) was determined to be 9S,11S or 9R,11R. Figure 4.11. 1H-NMR of compound PT-8 11
2 vòng đường H-9, H-10 2 vòng H-11’ coumarin thế 2 lần Figure 4.12. Selected (COSY, NOESY and HMBC) correlations of compound PT-8 Table 4. 13. 1H and 13C-NMR data of (-)-paratrimerin A (PT-8) 13 C C-NMR 1 HMBC HMBC COSY NOESY H-NMR ( MeOD, 13 (MeOD, C1H 1 H13C 1 H -> 1 H -> 500 MHz) 1 1 125 MHz) H H 2 163,28 (s) - 3, 4 - 3 114,31 (d) 6,28, d, 9,5 Hz - 2, 4a 4 4 4 146,00 (d) 7,95, d, 9,5 Hz 5 2, 8a, 5 3 3 4a 115,19 (s) - 3, 8 - - 5 126,60 (d) 7,47, s 4 4, 7, 8a - 9 6 127,66 (s) - 8, 10 - - 7 158,35 (s) - 5, 8, 1'' 6, 7 - 8 103,99 (d) 7,03, s - 4a, 6, 7, - 1'' 8a 8a 155,22 (s) - 4, 5, 8 - - 9 121,23 (d) 6,41, d, 16,5 Hz 5 5, 10 10 5, 11- CH3 12
10 140,92 (d) 6,37, d, 16,5 Hz 9, 11-CH3 9, 11, 11- 9 11-CH3 CH3 11 40,22 (s) - 9, 12, 11- - 9, 10, 9' CH3 12 31,95 (t) 1,65-1,88, 2H, m 11-CH3 10, 11, 12' 12' 11- 25,82 (q) 1,31, s 12 10, 11, 9', - CH3 12' 2' 163,49 (s) - 2', 3' - - 3' 114,08 (d) 6,22, d, 9,5 Hz - 2', 4'a 4' 3' 4' 145,89 (d) 7,88, d, 9,5 Hz 5' 2', 5', 8'a 3' 4' 4'a 114,40 (s) - 3', 8' - - 5' 130,95 (d) 7,39, s 4' 4', 7', 8'a - 6' 131,62 (s) - 8' - - 7' 160,03 (s) - 5', 8', 1''' - - 8' 103,63 (d) 7,09, s - 4'a, 6', 7', - 1''' 8'a 8'a 155,03 (s) - 4', 5', 8' - 9' 44,34 (d) 4,13, s 12, 5', 11'- 12, 11- - CH3 CH3, 5' 10' 124,60 (d) 5,35, s 11'-CH3 11' 135,60 (s) - 12, 11'-CH3 12' 28,56 (t) 2,29, m 12, 11'-CH3 12 8 2,16, m 11'- 23,66 (q) 1,85, s - - CH3 1'' 101,62 (d) 4,99, d, 8 Hz 7 2'' 8 2'' 74,77 (d) 3,35-3,4, m 1'' 3'' 78,19 (d) 3,42 -3,53, m 4'' 71,71 (d) 3,23, 1H, t, 9 Hz 5'' 78,42 (d) 3,42 -3,53, m 6'' 62,72 (t) 3,85, dd, 12, 2 Hz 4'' 3,35-3,39, m 1''' 102,49 (d) 4,93, d, 7,5 Hz 7' 2''' 8' 2''' 75,01 (d) 3,58, dd, 8,0, 8,0 Hz 1''' 74,77 (d) 3''' 78,34 (d) 3,42 -3,53, m 4''' 71,21 (d) 3,33-3,39, m 5''' 78,38 (d) 3,42 -3,53, m 6''' 62,48 (t) 3,88, dd, 12, 2 Hz 3,70, dd, 12, 6,5 Hz 4.3. Chemical compositions of Morinda longissima Compound ML-1 to ML-22 were isolated from the stems and roots of Morinda longissima. The structures of all isolated compounds (ML-1 - ML-22) were elucidated on the basis of spectroscopic data (high resolution (HR)-MS, one and two dimensional (1/2D)-NMR). 13
Damnacanthal (ML-1) Lucidin-ω-methyl ether (ML-2) Sorandidiol (ML-3) C16H10O5, 282 C16H12O5, 284 C15H10O4, 254 Morindone-5-methyl ether (ML- Rubiadin (ML-5) Rubiadin-3-methyl ether(ML- 4), C16H12O5, 284 C15H10O4, 254 6), C16H12O4,268 Morindone (ML-8) 1-hydroxy-2-methyl-6- Damnacanthol (ML-7) C15H10O5, 270 methoxy anthraquinone C16H12O5, 284 (ML-9) C16H12O4, 268 Morindone-6-methyl ether (ML- Morindone-6-O-β- Lucidin-3-O-β- 10) Gentiobioside(ML-11) primeveroside (ML-12) C16H12O5, 284 C27H30O15, 594 C26H28O14, 564,5 morinlongoside A (ML- morinlongoside B (ML- morinlongoside C (ML- 13) 14) 15) C29H38O15, 626 C27H36O13, 568 C22H32O15, 536 14
3-O-[β-D-Xylopyranosyl-(1-6)-β- Geniposidic acid (ML- D-glucopyranosyl] Cistanoside-E (ML-18) 16) (3R)-l-octen-3-ol (ML-17) C21H32O12, 476,47 C16H22O10, 374 C19H33O10, 421 Ethyl-β-D- Isoacteoside (ML-20) galatopyranoside (ML- C29H36O15, 624 19) C8H16O6, 208,09 Acteoside (ML-21) Quercetin (ML-22) C29H36O15, 624 C15H10O7, 302 4.4. Evaluation of hepatoprotective and anti-HBV activities of of several compounds 4.4.1. Evaluation of anti-HBV activities Viability of HepG2.2.15 cell was tested using MTS assay after 48 hr incubation. Table 4.4. Effect on viability of HepG2.2.15 cells of pure compounds No. Sample Conc. OD % cell viability 1 Blank 0,049 2 DMSO - D6 0,805 100 15
3 ostruthin (PT-1) 10 µM 0,165 20,55 4 ninhvanin (PT-2) 10 µM 0,625 77,60 5 6-(2-Hydroxyethyl)-2,2-dimethyl- 10 µM 2H-1-benzopyran (PT-3) 0,905 112,37 6 paratrimerin A (PT-8) 10 µM 0,900 111,81 7 paratrimerin B (PT-9) 10 µM 0,713 88,59 8 parabacunoidc acid (PT-10) 10 µM 0,53 65,81 9 morindone (ML-8) 10 µM 1,011 125,60 10 damnacanthal (ML-1) 10 µM 0,800 99,39 11 rubiadin (ML-5) 10 µM 1,014 125,97 05 compounds with cell viability ≥ 80 will be further studied for inhibitory activity of secretion of HBV surface antigen (HBsAg). Table 4. 5. Inhibitory activity of HBsAg secretion in HepG2.2.15 cells of pure compounds No. Sample HBsAg Conc. OD (450nm) SE Inhibition (%) 1 Blank 0,190 0,140 0 2 DMSO 0 µM 1,228 0,016 100 3 Lamivudine 50 µM 0,635 0,022 42,86 4 6-(2-Hydroxyethyl)-2,2- dimethyl-2H-1-benzopyran 20 µM 1,276 0,044 104,61 (PT-3) 5 paratrimerin A (PT-8) 20 µM 1,300 0,061 106,92 6 morindone (ML-8) 20 µM 0,992 0,067 77,28 7 damnacanthal (ML-1) 20 µM 1,077 0,094 85,42 8 rubiadin (ML-5) 20 µM 1,058 0,077 83,64 Morindone (ML-8), rubiadin (ML-5) and damnacanthal (ML-1) showed significant inhibitory activity of HBsAg secretion. Table 4. 6. The IC50 values of anthraquinone ML-8, ML-1 and ML-5 HBsAg Conc. Abs 450 SE Inhibition (%) IC50 (µM) Blank 0,071 0,007 0 DMSO 0 µM 1,911 0,018 100 Lamivudine 50 µM 0,905 0,040 45,30 19,92±0,18 16
morindone 2,5 µM 1,829 0,026 95,53 (ML-8) 5 µM 1,577 0,015 81,85 32,06±3,21 10 µM 1,465 0,056 75,74 20 µM 1,315 0,089 67,61 2,5 µM 1,813 0,009 94,66 damnacanthal 5 µM 1,711 0,067 89,11 32,54±3,05 (ML-1) 10 µM 1,471 0,107 76,06 20 µM 1,275 0,003 65,41 2,5 µM 1,858 0,081 97,13 rubiadin (ML- 5 µM 1,809 0,019 94,44 45,82±10,79 5) 10 µM 1,657 0,042 86,20 20 µM 1,409 0,053 72,72 Morindone (ML-8), rubiadin (ML-5) and damnacanthal (ML-1) exbihited anti-HBV in vitro effect on HepG2.2.15 cell line based on HBsAg expression levels. .4.4. The hepatoprotective activities of isolated conpound Ninhvanin (PT-2, new compound), at the 50 mg/kg/day reduced the serum ALT concentration as well as partly limited the liver injury induced by paracetamol (400 mg/kg). Ostruthin (PT-1) at the 50 mg/kg/day reduced the serum ALT and AST concentrations and limited the liver injury induced by paracetamol. Ostruthin exhibited the strong hepatoprotective activities equal to those of silymarin at the same dose of 50 mg/kg/day. Table 4. 7. Effect of ostruthin and ninhvanin on the serum levels of ALT, AST Lô Sample AST (UI/L) ALT (UI/L) Control 84,20 ± 3,90 29,20 ± 2,59 1 paracetamol 400mg/kg 324,00 ± 50,54 495,67 ± 61,50 2 p< so với (1) p< so với (1) ostruthin 50 mg/kg 97,00 ± 15,72 62,00 ± 23,30 p> so với (1) p< so với (1) 3 p< so với (2) pso với (5) p> so với (5) 17
ninhvanin 50 mg/kg 162,67 ± 72,51 283,00 ± 11,13 p< so với (2) 4 p>so với (2) p< so với (5) p< so với (5) silymarin 50 mg/kg 56,00 ± 12,53 90,32 ± 19,06 p< so với (1) 5 p> so với (1) p< so với (2) p
(PT-2, new compound); 6-(6-hydroxy-3,7-dimethylocta-2,7- dienyl)-7-hydroxycoumarin (PT-6); ninhvanin B (PT-7, new compound); two dimeric monoterpene-linked coumarin glycosides, paratrimerin A (PT-8, new compound); paratrimerin B (PT-9, new compound); a new alcohol, paramitrimerol (PT-5, new compound), a chromene: 6-(2-hydroxyethyl)-2,2-dimethyl-2H-1-benzopyran (PT-3), an alkaloid: citrusinine-I (PT-4) and a new limonoid, parabacunoic acid (PT-10, new compound) were isolated. From the stems and roots of Morinda longissima, 22 compounds, including twelve anthranoids: damnacanthal (ML-1); lucidin-ω-methyl ether (ML-2); soranjidiol (ML-3); morindone -5- methyl ether (ML-4); rubiadin (ML-5); rubiadin-3-methyl ether (ML-6); damnacanthol (ML-7); morindone (ML-8); 1-hydroxy-2- methyl-6-methoxy anthraquinone (ML-9); morindone-6-methyl ether (ML-10); morindon-6-O-β-gentiobioside (ML-11); lucidin-3- O-β-primeveroside (ML-12), two new naphthalene glycosides: morinlongoside A (ML-13, new compound); morinlongoside B (ML-14, new compound), two iridoid glycosides: morinlongoside C (ML-15, new compound); geniposidic acid (ML-16), five glycosides: (3R)-3-O-[β-D-xylopyranosyl-(1→6)-β-D- lucopyranosyl]-l-octen-3-ol (ML-17); acteoside (ML-18); cistanoside E (ML-19); ethyl-β-D-galatopyranoside (ML-20); isoacteoside (ML-21) and a flavonoid: quercetin (ML-22) were isolated. Biological effects of several pure compounds Three anthranoids, morindone (ML-8), damnacanthal (ML- 1), rubiadin (ML-5) exhibited an inhibitory activity on HBsAg secretion of HepG2.2.15 cells with IC50 values of 32,06 ± 3,21 μM, 32,54 ± 3,05 μM and 45,82 ± 10,79 μM, respectively. Ninhvanin (PT-2, new compound), at the 50 mg/kg/day reduced the serum ALT concentration as well as partly limited the liver injury induced by paracetamol (400 mg/kg). Ostruthin (PT-1) at the 50 mg/kg/day reduced the serum ALT and AST concentrations and limited the liver injury induced by paracetamol. 19
Ostruthin exhibited the strong hepatoprotective activities equal to those of silymarin at the same dose of 50 mg/kg/day. RECOMMENDATIONS The study results of chemical composition and biological activity of Paramignya trimera and Morinda longissima demonstrate that: - Further study on ostruthin (PT-1) is necessary to establish the efficacy, safety, and exact mechanism of action as a moral alternative in the treatment of liver disorders. - Three anthranoids, morindone (ML-8), damnacanthal (ML-1) and rubiadin (ML-5) are potential compounds for research and development of drugs to treat HBV. Therefore, further anti-HBV evaluation of these compounds need to be studied at in vivo level. NEW CONTRIBUTIONS OF THE THESIS - This is the first study on the chemical constituents and biological activities of Paramignya trimera and Morinda longissima in the world. - From the the stems and roots of Paramignya trimera, 06 new compounds (ninhvanin (PT-2), ninhvanin B (PT-7), paratrimerin A (PT-8), paratrimerin B (PT-9), paramitrimerol (PT-5), parabacunoic acid (PT-10) were isolated and elucidated the chemical structure. Especially, paratrimerin A, and paratrimerin B are the first monoterpen-linked biscourmarin glycosides isolated from nature. - Two new naphthalene glycosides, morinlongoside A (ML-13), morinlongoside B (ML-14), an iridoid glycosides, morinlongoside C (ML-15) were isolated from the stems and roots of Morinda longissima. - The metanol extract and ostruthin from stems and roots of Paramignya trimera exhibited the strong hepatoprotective activities against paracetamol-induced hepatotoxicity in BALB/c mice. - This is the first time, the ethanol and aqueous extracts and morindone (ML-8), damnacanthal (ML-1), rubiadin (ML-5) from 20