MINISTRY OF

EDUCATION AND TRAINING

VIETNAM ACADEMY OF

SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY FOR SCIENCE AND

TECHNOLOGY

-----------------------------

PHAM THI NINH

STUDY ON CHEMICAL CONSTITUENTS AND

BIOLOGICAL ACTIVITIES OF LITSEA GLUTINOSA

(LOUR). C. B. ROB. (LAURACEAE) AND LEPISANTHES

RUBIGINOSA (ROXB.) LEENH. (SAPINDACEAE) IN

VIETNAM

Research field : Organic chemistry

Code : 62.44.01.14

SUMMARY OF THE DOCTORAL THESIS

HA NOI - 2018

2

This thesis is carried out in the Graduate University for

Science and Technology, VAST

Scientific supervisers:

1. Prof. Dr. Sc. TRAN VAN SUNG

2. Dr. TRAN THI PHUONG THAO

Thesis Reviewer 1:

Thesis Reviewer 2:

Thesis Reviewer 3:

The thesis will be defended in the Graduate University for

Science and Technology (GUST) council at Vietnam Academy of

Science and Technology (VAST) at ... on .... 2018

The thesis may be found in the library of the GUST and

National Library of VietNam

1

I. INTRODUCTION

1. Reason of the study

The genus Litsea Lam. (Lauraceae) and Lepisanthes Blume

(Sapindaceae) are wildly distributed in Vietnam, especially in the

mountainous areas. The reports in the literatures indicated that these two

genus contain many constituents with interesting structures and potential

biological activities. In order to find interesting substances for

development of new pharmaceutical ingredients or functional food

products we would like to conduct the resarch thesis ‘‘Study on

Chemical constituents and biological activities of Litsea glutinosa

(Lour.) Rob. (Lauraceae) and Lepisanthes rubiginosa (Roxb.)

(Sapindaceae) in Vienam:

2. Objective of the study in the thesis

The samples of Litsea glutinosa (Lour.) Roxb. [Lauracea] and

Lepisanthes rubiginosa (Roxb.) (Sapindaceae) collected in Vietnam

3. The new contributions of the thesis

- For the first time in Vietnam chemical constituents and

biological activities of two species Litsea glutinosa (Lour.) Roxb.

[Lauracea] and Lepisanthes rubiginosa (Roxb.)Leenh are systematically

investigated. From Litsea glutinosa, 21 compounds were isolated and

structural elucidated, 15 among them are isolated from this species for

the first time. The ethanol-water extract of Litsea glutinosa exhibits

inhibition activity on all 4 cancer cell lines terterd: HepG2, KB, Lu-1 and

MCF-7. Some of the isolated pure compounds from Litsea glutinosa

leaves showed antioxidant activity on DPPH method.

- For the first time in Vietnam, chemical constituents and

biological activities of species Lepisanthes rubiginosa (Roxb.) Leenh are

investigated. From a buthanol extract of this species, 11 compounds have

been isolated and nine ones of them were isolated from L. Rubiginosa

for the first time. The new compounds are glycoside of oleanolic acid

and farnesol.

2

II. The content of the thesis

● Introduction: Interpretation of the reason for the study as well as the

importance in the science and practice of the thesis.

Part 1: Review on the published results of the research objectives

- Review on the reported chemical constituents, biological activities,

distribution in the nature and utilization in the folk medicine related to

two species in the thesis, L. glutinosa and L. rubiginosa

1.1. Botanical characteristics, chemical constituents, and biological

activities of some selected species in the genus Litsea Lam.

1.2. Botanical characteristics, chemical constituents and biological

activities of some selected species in the genus Lepisanthes Blume

Part 2: Experimental

2.1. Chemicals and Equipments for the study

2.2.Plant samples for the study

Twigs and barks of L. glutinosa were collected from Thai Nguyen

province in October 2014 and identified by Ngo Van Hai, Institute of

Ecology and Natural Resources, VAST. An authentic sample is

deposited in Department for Org. Syn., Institute of Chemistry, VAST

(PTN01).

● Leaves and barks of L. glutinosa collected from Thua Thien Hue

province in October 2015 were identified by Ngo Van Hai, Institute of

Ecology and Natural Resources, VAST. An authentic sample is kept in

Department for Org. Syn., Institute of Chemistry, VAST (PTN02).

● Leaves and twigs of L. rubiginosa were collected from the beach of

Phu Loc district, Thua Thien - Hue province in October 2014 and

identified by Do Xuan Cam, university for Agricubture and Forest, Hue

City ( ND01-2014). An authentic sample is deposited in Department for

Org. Syn., Institute of Chemistry, VAST (PTN0).

2.3. Methods in study

2.3.1. Extraction and separation

2.3.2. Determination of the structures

3

2.3.3. Bioassays

Used of: DPPH methol; cytotoxic activity in vitro, diminution of blood

sugar levels in animal test, inhibition of the enzym α –glucosidase,

determination of the acute toxicity in animals.

2.4. Separation, purification of compounds from two studied species

2.4.1. From L. glutinosa (Lour.) Roxb.

2.4.1.1. Compounds from L. glutinosa collected from Thua Thien - Hue-

province.

The dried, powdered leaves were extracted and separated according Fig

2.1.

Fig. 2.1: Extraction and saparation the compounds from L. glutinosa

collected from Thua Thien - Hue province.

4

* Compounds isolated from the water extract

● Compound BL01 ( Nicotiflorin) yellow powder .IR (Kbr): νmax (cm-1

):

3339; 2929; 1655; 1554; 1361; 1058; UV (MeOH), λmax (nm): 212,1;

265,8; 349,0 nm; (+) ESI-MS m/z: 617.1 [M+Na]+; (-) ESI-MS m/z:

593,1 [M-H]-;

1H,

13C-NMR, 1D, 2D, spectral data are in agreement with

those in [152].

● Compound BL02 (Rutin) yellow powder; IR (KBr) νmax (cm-1

): 3392;

1614; 1518; 1455; 1244; 1007; UV (MeOH) λmax (nm): 206,9; 257,3;

358,0; (+) ESI-MS m/z: 611,0 [M+H]+; (-) ESI-MS m/z: 609,1 [M-H]

-;

1H,

13C-NMR, 1D, 2D spectral data are in agreement with those in [153].

● Compound BL03 (Afzelin) yellow powder; IR (KBr) νmax (cm-1

): 3379;

3100; 2920; 1646; 1562; 1459; 1074; UV (MeOH) λmax (nm): 206,9;

265,4; 316,6; (+) ESI-MS m/z: 432,8 [M+H]+; (-) ESI-MS m/z: 431,0

[M-H]-;

1H,

13C-NMR, 1D, 2D spectral data are in agreement with those

in [154].

* Compounds from ethyl acetatee extract

● Compound BL04 (Quercitrin) yellow powder ; IR (KBr) νmax (cm-1

):

3415, 3253, 2970, 1655, 1557, 1471, 1052; UV (MeOH), λmax (nm):

206,9 nm); (+) ESI-MS m/z: 471,0 [M+Na]+; (-) ESI-MS m/z: 447,0 [M-

H]-;

1H,

13C-NMR, 1D, 2D spectral data are in agreement with those in

[155].

The dried, powdered were extracted and separated according to Fig 2.2.

5

Fig. 2.1: Extraction and saparation from compounds the barks and

twigs of L. glutinosa collected from Thua Thien Hue- province.

* Compounds isolated from the water extract

● Compound BL05 (Magnocurarine chloride) yellow powder. HR-

ESIMS m/z: 314,1751 [M-Cl]+ (Molecular formula is C19H24NO3

+

314.1756). 1H,

13C-NMR, 1D, 2D spectral data are in agreement with

those in [156].

* Compounds from n-butanol extract

Compound BL06 (Oblongine chloride) yellow powder ; IR (KBr) νmax

(cm-1

): 3482, 3239, 2989, 1656, 1556, 1061; UV (MeOH), λmax (nm):

204,8; 245,3; 295,2; Phổ ESI-MS m/z: 314,0 [M-Cl]+; positive HR-

ESIMS m/z: 314,1741 [M-Cl]+; Molecular formula is C19H24NO3

+

6

314,1756); 1H,

13C-NMR, 1D, 2D spectral data are in agreement with

those in [157].

● Compound BL07 (Boldine methochloride) yellow powder: ESI-MS

m/z: 342,0 [M-Cl]+;

1H,

13C-NMR, 1D, 2D spectral data are in

agreement with those in [158, 159].

● Compound BL08 (Pallidine) yellow powder: ESI-MS m/z: 328.0

[M+H]+; negative ESI-MS m/z: 326,0 [M-H]

-;

1H,

13C-NMR, 1D, 2D

spectral data are in agreement with those in [160].

● Compound BL09 (Predicentrine) yellow powder: ESI-MS m/z: 342,0

[M+H]+.

1H,

13C-NMR, 1D, 2D spectral data are in agreement with those

in [158, 159].

● Compound BL10 (Criptorodine) yellow powder. ESI-MS m/z: 309,9

[M+H]+;

1H,

13C-NMR, 1D, 2D spectral data are in agreement with those

in [161].

Compound BL11 (Reticuline) yellow powder. ESI-MS m/z: 330,0

[M+H]+;

1H,

13C-NMR, 1D, 2D spectral data are in agreement with those

in [162].

● Compound BL12 (Aripuanin) yellow powder; (+)-ESI-MS m/z: 267,0

[M+Na]+;

1H,

13C-NMR, 1D, 2D spectral data are in agreement with

those in [163].

● Compound BL13 (Blumenol A) oil yellow powder ; (+)-ESI-MS m/z:

247 [M+Na]+ data;

1H,

13C-NMR, 1D, 2D spectral data are in agreement

with those in [164].

* Compounds from n-hexane extract

● Compound BL14 (2-phyten-1-ol) white powder: 1H,

13C-NMR, 1D,

2D spectral data are in agreement with those in [165].

2.4.1.2. Extraction and separation the compounds from L. glutinosa

collected in Thai Nguyen

* Compounds from ethyl acetate extract

● Compound BL15 (cis-5,8,11,14,17-eicosapentaenoic acid methyl

erterr) yellow powder. IR (KBr, cm-1): 2923,11 (CH); 1740,14 (COO).

7

ESI-MS m/z: 317,0 (25 %, [M+H]+).

1H,

13C-NMR, 1D, 2D spectral data

are in agreement with those in [166].

● Compound BL16: Spatozoate yellow powder. IR (KBr, cm-1): 2964

(C-H), 1724 (COOR- erter), 1283 (C-O). ESI-MS (m/z, %): 313,0 (98

%) [M+H]+ data:

1H,

13C-NMR, 1D, 2D spectral data are in agreement

with those in [167].

● Compound BL17: β-sitosterol; white powder C 1H,

13C-NMR, 1D, 2D

spectral data are in agreement with those in [168].

● Compound BL18: Daucosterol; white powder 1

H, 13

C-NMR, 1D, 2D

spectral data are in agreement with those in [169].

* Compounds from n-hexane extract

● Compound BL19 (1-heptadecanol) white powder. ESI-MS m/z: 256,1

(98 %), [M]+, Molecular formula is C17H36O.

1H,

13C-NMR, 1D, 2D

spectral data are in agreement with those in [170, 171].

● Compound BL20 (1-eicosanol) white powder. ESI-MS m/z: 298,2 (15

%) [M]+, 338,2 (80 %) [M+K+H]

2+, Molecular formula is C20H42O;

1H,

13C-NMR, 1D, 2D spectral data are in agreement with those in [170,

171].

● Compound BL21 (Glycerol 1,3-di-(9Z, 12Z-octadecadienoate) 2-

hexadecanoate) yellow powder. ESI-MS m/z: 875,7 (98 %)

[M+H2O+3H]+; 595,4 (10 %) [M+3H-CO(CH2)14CH3)]

+; Molecular

formula is C55H98O6. 1H,

13C-NMR, 1D, 2D spectral data are in

agreement with those in [172].

8

Hình 2.3: Extraction and saparation compounds from

the barks and twigs of L. glutinosa collected in Thai Nguyen

2.4.2. Extraction and separation the compounds from L. rubiginosa

* Compounds from ethyl acetate extract

● Compound ND1 (lupeol) white powder; ESI-MS: m/z 409 (100,

[M+1-H2O]+; M = 426 ; Molecular formula is C30H50O.

1H,

13C-NMR,

1D, 2D spectral data are in agreement with those in [173, 174].

● Compound ND2 (Diosmetin); yellow powder, Molecular formula is

C16H12O6. ESI-MS: m/z 300,9 [M+H]+ và m/z 298,9

[M-H]

- data.

1H,

13C-NMR, 1D, 2D spectral data are in agreement with those in [175].

● Compound ND3: Heptadecanoic acid (Margaric acid, Daturinic acid).

ESI-MS: m/z 271 [M+H]+ data;

1H,

13C-NMR, 1D, 2D spectral data are

in agreement with those in [176].

● Compound ND4: β-sitosterol

● Compound ND5: Daucosterol

9

* Compounds from n-butanol extract

● Compound ND6 white powder . ESI-MS m/z 803,4 [M+Na]+; 815,4

[M+Cl-]

- data.

1H,

13C-NMR, 1D, 2D spectral data are in agreement with

those in [178].

● Compound ND7 white powder . ESI-MS data m/z 803,4 [M+Na]+;

815,4 [M+Cl-]

-;

1H,

13C-NMR, 1D, 2D spectral data are in agreement

with those in [178].

● Compound ND8 white powder. ESI-MS data tại m/z 977,3 [M+Na]+;

989,4 [M+Cl-]

-. HR-ESIMS data m/z 977,5065.

1H và

13C NMR data in

(Fig 3.18).

● Compound ND9 white powder. ESI-MS data, compound of ND9 m/z

935,4 [M+Na]+. HR- ESIMS data pic ion: m/z 935,4929 [M+Na]

+.

1H-

NMR và 13

C-NMR spectral data are in agreement with those in (Table

3.19).

● Compound ND10 white powder. ESI-MS data pic tại m/z 963,4

[M+Na]+ và 939,4 [M-H]

-; HR- ESI-MS ; m/z 963,4314 [M+Na]

+.

1H-

NMR và 13

C-NMR spectral data are in agreement with those in (Table

3.19).

● Compound ND11 white powder. ESI-MS data m/z 979; HR-ESI-MS

m/z 979,41760. 1H-NMR và

13C-NMR spectral data are in agreement

with those in (Table 3.19).

[173, 174].

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Hình 2.5: Extraction and separation the compounds from L.

rubiginosa

Part 3: Results and discussions

3.1. About L.glutinosa

3.1.1. Biological activity of the extracts of L.glutinosa from Thua

Thien Hue province

3.1.1.1. Inhibition activity of α- glucosidase of the extracts in vitro

The activity in decreasing of the blood sugar levels of the ethanol/ water

extracts of the barks and leaves of L.glutinosa from Thua Thien Hue

province was terterd on the inhibition of α- glucosidase activity. The

result showed that the EtOH/H2O (80:20) extracts possessed the

inhibition of α- glucosidase activity with the IC50 value of 194,9 (barks)

and 197.3 μg/ml (leaves). Acarbose was the reference (IC50 165 μg/ml).

3.1.1.2. The activity in decreasing the blood sugar level on the diabet-

induced mice of EtOH/H2O (80:20) extracts.

11

● Influence of the extract on the body weights of the mice: The body

weights of mice before and after use of EtOH/H2O extracts are given in

table 7. The result: In the doses of 250mg extract and 500mg extract/kg

body weight, the weights of mice do not changed.

● The effect of EtOH/H2O extract on the serum glucose concentration of

the diabet- induced mice (Table 3.3): The extract showed significant

activity in the decreasing the serum glucose level of the terterd mice.

3.1.2. Chemical constituents of L. glutinosa

3.1.2.1. Compounds from L. glutinosa collected in Thua Thien Hue

From the leaves, twigs and barks of this sample, 15 compounds have

been isolated and elucidated. They included: 4 flavonol glycosides

(compounds BL01-BL04), 7 aporphin alkaloids (compounds BL 05-

BL11), 2 megastigmanes (BL12 , BL13) and 1 phytol (BL14).

3.1.2.2. Compounds from L. glutinosa collected in Thai Nguyen

From n-hexane , ethyl acetate of the barks and twigs 10 compounds were

isolated and structural determined. The compounds from two samples

collected in Thai Nguyen and Thua Thien Hue province are presented in

Table 3.20

Table 3.20: Compounds from L. glutinosa

Bảng 3.20: Compounds from L. glutinosa isolated from in Thai nguyên va

Thua Thien Hue

L. glutinosa Thai Nguyen L. glutinosa Thua Thien -

Hue

BL15 (Cis-5,8,11,14,17-eicosapentaenoic

acid methyl erter)

BL01 (Nicotiflorin)

12

BL16 (Spatozoate)

BL02 (Rutin)

BL17 (β-sitosterol)

BL03 (Afzelin)

BL18 (Daucosterol)

BL04 (Quercitrin)

BL19 (1-heptadecanol)

BL05 (Magnocurarine chloride)

BL20 (1-eicosanol)

BL06 (Oblongine chloride)

BL21 (Glycerol 1,3-di(9Z,12Z-

octadecadienoat)-2-hexadecanoate)

BL07 (Boldine methochloride)

13

BL08 (Pallidine)

BL09 (Predicentrine)

BL09 (Predicentrine)

BL10 (Criptorodine)

BL10 (Criptorodine)

BL11 (Reticuline)

BL11 (Reticuline)

BL12 (Aripuanin)

BL13 (Blumenol A)

BL14 (2-phyten-1-ol)

* From a literature search, it resulted that: Among 21 isolated

compounds there are 15 compounds, which were first time isolated from

the species L. glutinosa (Lour.) Roxb. (BL01, BL05- BL10, BL12-

BL16, BL19-BL21)

14

3.1.3. Biological activity of pure isolated compounds

3.1.3.1. Cytotoxic activity

The compounds BL01- BL11 were terterd on four human cancer cell

lines as KB, HepG2, Lu and MCF-7, Ellipticine was used as positive

control. The results are given in Table 3.10

Table 3.10: Cytotoxic activity of the compounds BL01- BL11, isolated

from Thua Thien Hue sample

Compound

IC50 (g/ml)

KB Hep

G-2 Lu MCF-7

BL01 >128,0 >128,0 >128,0 >128,0

BL02 >128,0 >128,0 >128,0 >128,0

BL03 >128,0 >128,0 >128,0 >128,0

BL04 >128,0 >128,0 >128,0 >128,0

BL05 67,66 117,08 >128,0 >128,0

BL06 68,49 82,75 >128,0 >128,0

BL07 >128,0 >128,0 >128,0 >128,0

BL08 21,29 13,56 >128,0 29,24

BL09 17,72 25,6 56,32 54,21

BL10 68,39 >128,0 >128,0 >128,0

BL11 >128,0 >128,0 >128,0 >128,0

Ellipti

cine

0,31 0,38 0,41 0,60

As the results: Some aporphin alkaloids (BL05, BL08, BL09) showed

cytotoxic activity on four terterd cell lines. This is the fist report on the

cytotoxic activity of the above alkaloids.

3.1.3.2. The antioxidant of the pure compounds

The flavonoids BL01-BL04 were terterd on the antioxidant activity.

Quercetrin was used as a reference with the IC50 =9.45µg/ml (Table 3.5).

As the result: Compound BL02 (rutin) and BL04 (quercitrin) exhibited a

good antioxidant activity with the IC50=23.73 and 38.20µg/ml,

respectively. This result confirmed that the flavonoid compounds are

responsible for the antioxidant activity of L. glutinosa.

15

3.1.3.3. The inhibition activity of α - glucosidase of the pure isolated

compounds

The compounds BL01-BL11 were evaluated on the inhibition of α -

glucosidase activity. As the results: From these compounds only

compound BL04 (quercitrin) showed a good inhibition of α –glucosidase

with IC50 149.5µg/ml in comparison to acarbose (IC50 165.7µg/ml) The

remained compounds showed no inhibition of α -glucosidase (Table

3.10). This result is in agreement with the result published before.

According to [142] quercitrin isolated from the ethyl acetate of Amomum

xanthioides, was determined as the inhibitor of α - glucosidase, with

55.7% inhibition in a concentration of 5mg/ml, higher than acarbose

(32.4%) in the same concentration.

3.1.4. The acute toxicity of the EtOH/H2O extract

Based on a bigger number of constituents in Thua Thien Hue sample we

carried out the determination of the acute toxicity of the EtOH/H2O

(80/20) extracts from this sample. This sample also will be the starting

material for the farther research. The obtained result showed: This

extract had a low toxicity. The lethal dose (LD50) of it on the animal is

>20g/kg body weight (LD50 > 20g/kgP) According to the classification of

GHS, this extract is non-toxic.

3.2 The Lepisanthes rubiginosa (Roxb.) Leenh.

3.2.1. Biological activity

3.2.1.1. Cytotoxic activity

The n-BuOH extract of leaves and twigs of Lepisanthes rubiginosa

(NDL- Bu) was evaluated for cytotoxic activity in vitro. The result is

given in Table 3.14

Table 3.14: Cytotoxic activity of the n-BuOH extract of the L.

rubiginosa

STT extract IC50 (µg/ml)

KB HepG2 MCF7 Lu

1. NDL-Bu 21,33 23,42 18,37 20,01

Ellipticine 0,40 0,41 0,48 0,45

16

The result in Table 3.14 showed that the n-BuOH extract of the leaves

and twigs of L. rubiginosa (NDL- Bu) is active against all four terterd

cancer cell lines (KB, HepG2, Lu, MCF-7). Among them, the activity

against the cell MCF-7 is good. (IC50 =18.37µg/ml), followed by the Lu

(IC50 =20.01µg/ml), KB (IC50 =21.33µg/ml) and HepG2 (IC50

=23.42µg/ml).

3.2.2. Chemical constituents of L. rubiginosa

● Compound ND8 (New compound)

Compound ND8 was isolated as a white amorphous powder.

The 1H and

13C NMR compound ND8 indicated this compound is

a glycoside with oleanolic acid as aglycone, connected to sugar

moiety at C-3 (Matsuda et al. 1998). The ESI-MS spectra of ND8

contained the pseudomolecular ion peaks at m/z 977.3 [M+Na]+,

989.4 [M+Cl]- and 977.5065 [M+Na]

+ (calcl. for C49H48O18Na:

977.5086. Its NMR spectra indicated the presence of an acetyl

group (δH 2.08 (3H, s)/ δC 20.63, 172.57). That means, the sugar

moiety should consist of 17 carbon atoms, equal to two hexoses

and one pentose, which are proved by three anomeric atoms [(δH

4.49 (d, J = 8.0 Hz), 4.73 (d, J = 8.0 Hz), 5.24 (d, J = 2.0 Hz)/ δC

103.74, 105.38, 110.93], three oxymethylene groups [δC 65.01, δH

4.35 (dd, J = 11.5, 3.0 Hz); 4.16 (d, J = 11.5); δC 63.67, δH 3.83-

3.87 overlapped, 3.56-3.59 overlapped; δC 62.76, δH 3.88-3.89,

3.72-3.73 overlapped], together with eleven oxymethine groups. In

comparison with the data in (Andesegun et al. 2008; Chaturvedula

et al. 2003) the sugar residue is a trisaccharide consisted of one β-

D-glucopyranose, one α-D-galactopyranose and one α-L-

arabinofuranose which is binded to the C-3 of oleanolic acid by β-

D-glucopyranose. This suggestion was supported by HMBC

correlations between: H-1’ (δH 4.49) and C-3 (δC 92.31) and H-3

17

(δH 3.23, dd, J = 4.0, 10.5 Hz) and C-1’ (δC 103.74). The α-L-

arabinofuranose is connected to β-D-glucopyranose at C-3’ (δC

78.57), which is evidenced by the HMBC correlations between H-

1’’ (δH 4.73) and C-3’ (δC 78.57); H-3’ (δH 3.64, t, J = 9.5 Hz) and

C-1’’ (δC 110.93). The α-D-galactopyranose is connected to β-D-

glucopyranose at C-4’, indicating by the HMBC correlations from

H-1’’’ (δH 5.24) to C-4’ (δC 82.13) and C-1’’’ (δC 105.38) to H-

4’(δH 3.75, t, J = 9.5 Hz). Finally, the acetyl group is proved to

bind to C-6’ (δC 65.01, δH 4.35; 4.15) by the correlations between

H-6’a (δH 4.35), H-6’b (δH 4.15) to C-5’ (δC 77.99), C-4’ (δC 82.13)

and COCH3 (δC 172.57) in the HMBC spectrum (Fig. 3.34). From

the above data and by comparison with the similar compounds in

(Andesegun et al. 2008) compound ND8 was determined as 3-O-

{α-L-arabinofuranosyl-(1→3)-[α-D-galactopyranosyl-(1→4)]-6-

O-acetyl-β-D-glucopyranosyl}-oleanolic acid. This is a new

compound and is named as Lepisantheside A. The NMR spectral

data of compound ND8 were given in Table 3.18.

● Compound ND9 (New compound)

Compound ND9 was isolated as a white amorphous

powder. It showed the pseudomolecular ion peaks at m/z 935.4

[M+Na]+ in its ESI-MS and 935.4929. [M+Na] in HR- MS spectra

(Calc. for C17H76O17Na : 935,4980. This data suggest 2 is also a

trisaccharide of a triterpenoid. The 1H and 13C NMR spectra in the

aglycon region are quite similar to those of compound 1. So that

the aglycone of ND9 should be also oleanolic ac id, which is

binded to the sugar moiety at C-3 position. (δC-3: 92.31, δH-3: 3.23

(dd, J = 12.0, 4.5). The trisaccharide residue also composed from

two hexose and one pentose, which were identified as two β-D-

glucopyranose and one β-D-xylopyranose by comparison with the

similar sugar moiety in (Nagao T. et.al.1991; Ito A. et.al. 2004).

The β-D-glucopyranose is binded to C-3 of oleanolic acid, which

18

is evidenced by the HMBC correlations between H-1’ (δH 4.49) and

C-3 (δC 92.31; H-3 (δH 3.23) and C-1’ (δC 105.55); H-1’ (δH 4.49) and

C-2’ (δC 78.54). The second β-D-glucopyranose is connected to C-

2’ which is evidenced by the HMBC correlation between H-1’’ (δH

4.99) and C-2’ (δC 78.54), while the β-D-xylopyranose was bound

to C-3’ through the cross peak between H-1’’’ (δH 4.61) and C-3’

(δC 87.95). Besides, the following correlations were observed in the

HMBC spectrum of compound ND9: H-5’’’ axial (δH 3.94, dd, J =

5.5, 11.5) to C-4’’’ (δC 69.88) and C-1’’’ (δC 105.03); H-2’ (δH 3.74)

to C-3’ (δC 87.95) and C-1’ (δC 105.55); H-2’’ (δH 3.15, t, J = 9.5 Hz)

to C-5’ (δC 78.32) and C-6’ (δC 63.66 (Fig.1). The H,H-COSY and

ROESY spectra of compound ND9 also supported the results of

the HMBC spectrum. The correlations between H-1’ (δH 4.49) and

H-3 (δH 3.23); H-1’’ (δH 4.99) and H-2’ (δH 3.74); H-1’’’ (δH 4.61)

and H-3’ (δH 3.70, t, J = 9.0 Hz) in the ROESY spectrum as well as

the correlations between the protons in one sugar molecule moiety

to each other in the H,H-COSY spectrum were observed. From the

above data the structure of compound ND9 was elucidated as 3-O-

{β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-

Dglucopyranosyl}-oleanolic acid. This is a new compound and is

named as Lepisantheside B. The NMR data of ND9 were given in

Table 3.18

Table 3.18: NMR spectral data of compound ND8 and ND9

Vị trí ND8 ND9

δC δH (J = Hz) δC δH (J = Hz)

1 39,80 1,62-1,59 m; 1,03-1,00

m

39,81 1,63-1,61 m; 1,03-1,00

m

2 27,08 1,98-1,97 m; 1,78-1,76

m

27,08 1,99-1,97 m; 1,78-1,73

m

3 92,31 3,23 (dd; J = 10,5; 4,0) 92,31 3,23 dd (J = 12,0; 4,5)

4 40,58 - 40,57 -

5 57,01 0,81-0,79 m 57,01 0,81 -0,79 m

6 19,33 1,62-1,59 m; 1,43-1,42

m

19,32 1,53-1,50 m; 1,43-1,41

m

7 34,02 1,56-1,52 m; 1,23-1,20

m

34,00 1,53-1,50 m; 1,34-1,31

m

8 40,50 - 40,50 -

19

9 49,51 1,61-1,59 m 49,51 1,59-1,56 m

10 37,89 - 37,88 -

11 24,54 1,92-1,91 m 24,54 1,93-1,91 m

12 123,55 5,26 (brs) 123,55 5,26 (brs)

13 145,32 - 145,33 -

14 42,91 - 42,90 -

15 28,88 1,78-1,76 (m); 1,17-1,13

(m)

28,87 1,78-1,76 m; 1,17-1,13

m

16 24,14 2,00-1,98 m; 1,56-1,52

m

24,21 1,98-1,97 m; 1,59-1,56

m

17 48,49 - 48,49 -

18 42,75 2,87 (d; J = 11,5) 42,88 2, 89 (d; J = 12,0)

19 47,34 1,71-1,68 m; 1,17-1,13

m

47,34 1,71-1,68 m; 1,17-1,13

m

20 31,62 - 31,63 -

21 34,96 1,43-1,41 m; 1,23-1,20

m

34,96 1,43-1,41 m; 1,23-1,208

m

22 33,90 1,52-1,50 m; 1,34-1,32

m

34,01 1,53-1,50 m; 1,34-1,31

m

23 28,32 1,09 s 28,28 1,09 s

24 16,83 0,88 s 16,84 0,89 s

25 15,90 0,97 s 15,91 0,97 s

26 17,78 0,84 s 17,78 0,84 s

27 26,39 1,17 s 26,39 1,17 s

28 181,90 - 181,90 -

29 33,59 0,92 s 33,60 0,92 s

30 24,00 0,96 s 24,00 0,96 s

-OCOCH3 172,57 -

-OCOCH3 20,63 2,08 s

Đường

Glc-1’ 103,74 4,49 (d; J = 8,0) 105,55 4,49 (d; J = 8,0)

2’ 75,96 3,75-3,73 m 78,54 3,74 (m)

3’ 78,57 3,64 (t, J = 9,5) 87,95 3,70 (t, J = 9,0)

4’ 82,13 3,75 (t, J = 9,5) 72,55 3,09 (t, J = 9,5)

5’ 77,99 4,21 (dt; J = 6,5; 3,0) 78,32 3,31-3,29 m

6’ 65,01 4,35 (dd; J = 11,5; 3,0)

4,16 (d, 11,5)

63,66 3,88-3,83 m

3,67 (dd; J = 12,0; 5,0)

Xyl-1’’’ 105,03 4,61 (d; J = 7,5)

2’’’ 75,27 3,26-3,28 m

3’’’ 78,27 3,29-3,28 m

4’’’ 77,65 3,52-3,54 m

5’’’ 67,17 3,94 (dd; J = 11,5; 5,5)

3,28-3,31 m

20

Glu-1’’ 103,18 4,99 (d; J = 7,5)

2’’ 76,19 3,15 (t; J = 9,5)

3’’ 78,43 3,29-3,28 m

4’’ 70,99 3,08 (t; J = 9,5)

5’’ 78,53 3,33-3,31 m

6’’ 62,74 3,84-3,83 m; 3,58-3,55

m

Gal-1’’’ 105,38 5,24 (d; J = 2,0)

2’’’ 72,46 3,23-3,18 m

3’’’ 78,26 3,41-3,39 m

4’’’ 70,19 3,09 (t; J = 9,5)

5’’’ 77,58 3,66-3,65 m

6’’’ 63,67 3,87-3,83 m; 3,59-3,56

m

Ara-1’’ 110,93 4,73 (d; J = 8,0)

2’’ 83,32 3,67-3,66 m

3’’ 78,31 3,89-3,88 m

4’’ 87,86 3,62-3,54 m

5’’ 62,76 3,89-3,88 m; 3,73-3,72

m

* Eleven compounds have been isolated and identified from L.

rubiginosa , among them 4 compounds were identified as new

compounds. They are glycosides of oleanolic acid (ND8, ND9) and

pentaglycosides of farnesol (ND10, ND11). Seven other compounds

were isolated from this plant for the first time. The isolated compounds

are presented in Table 3.20

Bảng 3.20: compounds isolated from (Lepisanthes rubiginosa (Roxb.)

Leenh.)

ND1 (Lupeol)

ND2 (Diosmetin;

5,7,3'-Tryhydroxy-4'-

methoxyflavone)

21

ND3 (Heptadecanoic acid;

Magaric acid; Daturinic acid) ND4 (R=H: β-sitostosterol)

ND5 (R= β-D-glucose; sitosterol-

3-O-β-D-glucopyranoside)

ND6 (3-O-β-sophoroside)

ND7 (3-O-[β-D-xylopyranosyl

(1→3)-

β-D-glucopyranosyl-]-oleanoic

acid)

ND8 (Lepisantheside A) mới

ND9 (Lepisantheside B) mới

22

Conclusion and suggestion

Conclusion

● Chemical constituents of L. glutinosa (Lour.) Rob.

- From the Thua Thien Hue sample: 14 compounds were isolated and

identified, among them 7 aporphine alkaloid , 4 flavonoid glycosides,

two megastigmanes and one sterol.

- From the Thai Nguyen sample 10 compounds were isolated and

elucidated including 3 aporphine alkaloid (two of them are also found in

Thua Thien Hue sample) and derivatives of fatty acid and fatty alcohols.

- 15 isolated compounds were found in L. glutinosa for the first time.

● Chemical constituents and Biological activity of L. rubiginosa (Roxb.)

Leeh.

- 11 compounds were isolated and identified from L. rubiginosa,

including 4 new compounds, which are the glycosides of oleanolic acid

(ND8, ND9), and farnesol ND10, ND11. They are named as

Lepisantheside A, B, C, D, respectively). Seven compounds were

isolated from this species for the first time.

● Biological activity

The blood sugar level lowering effect on diabet- induced mice of the

EtOH/H2O (8/2) extract of Thua Thien Hue sample was evaluated . The

result: With the doses 250 and 500mg /kg body weight, the blood sugar

23

level of animal were decreased significantly in comparison to the

control. The 500mg/kg body weight showed an effect which

comparatible with acarbose 5mg/kg body weight.

The LD50 of the EtOH/H2O (8/2 vv) extract of Thua Thien Hue was

determined. The LD50 is > 20g/kg P. So that this extract is considered as

none toxic. The cytotoxic, antioxidant and α-glucosidase inhibition

activity of 11 pure isolated compounds were evaluated. Some of them

showed moderate activity.The n- BuOH extract of L. rubiginosa

exhibited the inhibition on all four terterd cancer cell lines: KB, Hep G2,

MCF-7 Lu-1

Suggestion

- Further studies on the mechanism of the blood sugar lowering effect of

the plant studied

- Further investigation on the cytotoxic acticity of – BuOH extracts of L.

rubiginosa

New contribution of the thesis

- Systematical investigation on the chemical constituents of L.

glutinosa

- Isolation of 21 com compounds from L. glutinosa,among them

15 compounds were isolated for the first time from this species.

- The cytotoxic, antioxidant and α-glucosidase inhibition

,antidiabetic of pure compounds from L. glutinosa were reported

for the first time.

- 4 new compounds (glycosides of oleanolic acid and farnesol)

were isolated from L. rubiginosa together with 7 ones isolated

for the first time from this species.

24

LIST OF PUBLICATIONS RELATED TO THE THESIS

1. Pham Thi Ninh, Nguyen Thi Luu, Nguyen The Anh, Tran Van Loc,

Nguyen Thi Ha Mi, Tran Thi Phuong Thao, Tran Van Sung, Chemical

constituents of the barks of Litsea glutinosa collected in Thai Nguyen

province, Vietnam, Vietnam Journal of Chemistry 2015, 53(5) 652-656

2. Pham Thi Ninh1,2

, Tran Thi Phuong Thao1*, Tran Van Loc

1, Nguyen

Thi Dung1, Đo Xuan Cam

3, Tran Van Sung

1, Chemical from from etyl

axetat extract of Lepisanthes rubiginosa collected in Thua Thien Hue

province, Vietnam, , Journal of Chemistry, 2017, 53 (1): 1-5.

3. Tran Thi Phuong Thao,1 Pham Thi Ninh,

1,2 Tran Van Loc,

1Nguyen

Tuan Thanh,1 and Tran Van Sung,

1* Chemical constituents, cytotoxic

and alpha-glucosidase inhibitory activity of the isolated compound from

Litsea glutinosa collected in Thua Thien Hue province, Vietnam

Accepted by Chemistry of Natural Compounds.

4. Pham Thi Ninh1,2

, Tran Van Loc1, Tran Thi PhuongThao

1*, “Activity

and Chemical of (Litsea glutinosa) collected in Thua Thien Hue province,

Vietnam, Journal of Chemistry, 56 (1), . 60-64.

5. Tran Van Loc, Pham Thi Ninh1,2

, Tran Thi Phuong Thao, Tran Van

Sung. Two new oleanolic acid glycoside from Lepisanthes rubiginosa, a

mangrove plant collected from the beach of Thua Thien - Hue province,

Vietnam, Nat. Prod. Res. 2018, submitted.