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-Dlucopyranosyl]-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.
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sis 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.
5
- 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-
6
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.
Code
species Sample
OD % cell viability
1
Blank
0,049
2
DMSO
0,805 100
3
PT
P. trimera
MeOH extract
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
PR3
P. trimera PR3 fraction
0,895 111,13
8
PR4
P. trimera PR 4 fraction
0,805 100,02
7
9
PR5
P. trimera PR 5 fraction
0,192 23,84
10 ML M. longissima EtOH extract 0,86 102,25
11
W
M. longissima aqueous
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
8
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
2 Paracetamol 400mg/kg
715,75 ± 253,94
p<0,05 so với (1)
558,25 ± 296,54
p<0,05 so với (1)
3 metanol extract10g/kg
116,50 ± 34,65
p<0,05 so với (2)
p>0,05 so với (5)
81,25 ± 8,59
p<0,05 so với (2)
p>0,05 so với (5)
4 aqueous extract 10g/kg
266,00 ± 170,92
p<0,05 so với (2)
p<0,05 so với (5)
113,25 ± 27,41
p<0,05 so với (2)
p<0,05 so với (5)
5 Silymarin 50mg/kg
101,33 ± 23,03
p<0,05 so với (1)
p<0,05 so với (2)
46,67 ± 11,47
p<0,05 so với (1)
p<0,05 so với (2)
Frigue 4.1. Histopathological changes of liver tissue (A): Control,
(B): Paracetamol 400mg/kg, (C): metanol extract 10g/kg, (D)
aqueous extract 10g/kg, (E): Silymarin 50mg/kg
4.2. Chemical compositions of Paramignya trimera
Compound PT-1 to PT-10 were isolated from the roots of
Paramignya trimera. The structures of all isolated compounds (PT-1–10)
were elucidated on the basis of spectroscopic data (high resolution (HR)-
MS, and one and two dimensional (1/2D)-NMR).
9
PT-1 (ostruthin), C19H22O3, 298
PT-2 (ninhvanin) mới, C20H24O4, 328
O
OH
1
2
3
4 5
6
7
8
9
10
11
12
13
14
PT-3 (6-(2-Hydroxyethyl)-2,2-
dimethyl-2H-1-benzopyran),
C13H16O2, 203
PT-4 (citrusinine-I), C16H15NO5, 301
PT-5 (paramitrimerol) mới, C14H16O3, 232
PT-6 6-(6-hydroxy-3,7-
dimethylocta-2,7-dienyl)-7-
hydroxycoumarin, C19H22O4, 314
PT-7 (ninhvanin B) mới, C19H22O4, 314
PT-9 (paratrimerin B) mới, C40H44O16, 780
PT-8 (paratrimerin A) mới, C45H52O20, 912
PT-10 (axit parabacunoic) mới, C32H44O13, 636
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]+,
10
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
11
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-6as 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 9S,11S or 9R,11R.
Figure 4.11. 1H-NMR of compound PT-8
12
2 vòng
coumarin thế
2 lần
2 vòng đường
H-9, H-10
H-11’
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)
C 13C-NMR
(MeOD,
125 MHz)
1H-NMR ( MeOD,
500 MHz)
HMBC
13C1H
HMBC
1H13C
COSY
1H ->
1H
NOESY
1H ->
1H
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,
8a
- 1''
8a 155,22 (s) - 4, 5, 8 - -
9 121,23 (d) 6,41, d, 16,5 Hz 5 5, 10 10 5, 11-
CH3
13
10 140,92 (d) 6,37, d, 16,5 Hz 9, 11-CH3 9, 11, 11-
CH3
9 11-CH3
11 40,22 (s) - 9, 12, 11-
CH3
- 9, 10, 9'
12 31,95 (t) 1,65-1,88, 2H, m 11-CH3 10, 11,
12'
12'
11-
CH3
25,82 (q) 1,31, s 12 10, 11, 9',
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',
8'a
- 1'''
8'a 155,03 (s) - 4', 5', 8' -
9' 44,34 (d) 4,13, s 12, 5', 11'-
CH3
12, 11-
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
2,16, m
12, 11'-CH3 12 8
11'-
CH3
23,66 (q) 1,85, s - -
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
3,35-3,39, m
4''
1''' 102,49 (d) 4,93, d, 7,5 Hz 7' 2''' 8'
2''' 75,01 (d)
74,77 (d)
3,58, dd, 8,0, 8,0 Hz 1'''
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).
14
Damnacanthal (ML-1)
C16H10O5, 282
Lucidin-ω-methyl ether (ML-2)
C16H12O5, 284
Sorandidiol (ML-3)
C15H10O4, 254
Morindone-5-methyl ether (ML-
4), C16H12O5, 284
Rubiadin (ML-5)
C15H10O4, 254
Rubiadin-3-methyl ether(ML-
6), C16H12O4,268
Damnacanthol (ML-7)
C16H12O5, 284
Morindone (ML-8)
C15H10O5, 270
1-hydroxy-2-methyl-6-
methoxy anthraquinone
(ML-9) C16H12O4, 268
Morindone-6-methyl ether (ML-
10)
C16H12O5, 284
Morindone-6-O-β-
Gentiobioside(ML-11)
C27H30O15, 594
Lucidin-3-O-β-
primeveroside (ML-12)
C26H28O14, 564,5
morinlongoside A (ML-
13)
C29H38O15, 626
morinlongoside B (ML-
14)
C27H36O13, 568
morinlongoside C (ML-
15)
C22H32O15, 536
15
Geniposidic acid (ML-
16)
C16H22O10, 374
3-O-[β-D-Xylopyranosyl-(1-6)-β-
D-glucopyranosyl]
(3R)-l-octen-3-ol (ML-17)
C19H33O10, 421
Cistanoside-E (ML-18)
C21H32O12, 476,47
Ethyl-β-D-
galatopyranoside (ML-
19) C8H16O6, 208,09
Isoacteoside (ML-20)
C29H36O15, 624
Acteoside (ML-21)
C29H36O15, 624
Quercetin (ML-22)
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
16
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-
2H-1-benzopyran (PT-3)
10 µM
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
Conc. OD (450nm) SE
HBsAg
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
(PT-3)
20 µM 1,276 0,044 104,61
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
Conc. Abs 450 SE
HBsAg
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
17
morindone
(ML-8)
2,5 µM 1,829 0,026 95,53
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
(ML-1)
5 µM 1,711 0,067 89,11 32,54±3,05
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)
5 µM 1,809 0,019 94,44 45,82±10,79
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)
1
Control 84,20 ± 3,90
29,20 ± 2,59
2
paracetamol 400mg/kg 324,00 ± 50,54
p< so với (1)
495,67 ± 61,50
p< so với (1)
3
ostruthin 50 mg/kg
97,00 ± 15,72
p> so với (1)
p< so với (2)
p>so với (5)
62,00 ± 23,30
p< so với (1)
p<0,01 so với(2)
p> so với (5)
18
4
ninhvanin 50 mg/kg
283,00 ± 11,13
p>so với (2)
p< so với (5)
162,67 ± 72,51
p< so với (2)
p< so với (5)
5
silymarin 50 mg/kg
90,32 ± 19,06
p> so với (1)
p<so với (2)
56,00 ± 12,53
p< so với (1)
p< so với (2)
Figure 4. 1 Histopathological changes of liver tissue (A): Control
group, (B): Paracetamol 400mg/kg, (C): Silymarin 50mg/kg, (D)
ostruthin 50 mg/kg P, (E): ninhvanin 50 mg/kg
CONCLUSION
Two medicinal species, Paramignya trimera (Xao tam
phan) and Morinda longissima (Nho dong) were studied
systematically on chemical composition and biological activities.
Biological activities of two plants extracts:
Both ethanol and water extracts of Morinda longissima
stems and roots exhibited in vitro anti-HBV activity through
inhibition of HBsAg secretion of HepG2.2.15 cells with IC50 values
of 146.90 ± 30.71 μg/ml and 297.76 ± 58.14 μg/ml, respectively.
The aqueous extracts of Paramignya trimera roots at the
oral dose of 10 g/kg/day weight has reduced serum AST and ALT
concentrations, decreased liver histopathological injury by
paracetamol (single dose 400 mg/kg, oral). The methanol extract of
Paramignya trimera roots exhibited potential hepatoprotective
effect which was similar to those of the positive control (silymarin
at the dose of 50 mg/kg/day).
Chemical composition:
From the stems and roots of Paramignya trimera, 10
compounds, including four coumarins: ostruthin (PT-1); ninhvanin
19
(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.
20
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
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