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 Viet Nam.
Trang 1MINISTRY 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
Trang 2This 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
The thesis may be found in the library of the GUST and National Library of VietNam
Trang 3I 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
Trang 42
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
Trang 52.3.3 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-
Trang 64
* 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]-; 1
H, 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
Trang 7Fig 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+
Trang 86
314,1756); H, C-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: 1
H, 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)
Trang 9ESI-MS m/z: 317,0 (25 %, [M+H] ) H, C-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 1
H, 13C-NMR, 1D, 2D spectral data are in agreement with those in [168]
● Compound BL18: Daucosterol; white powder 1
H, 13C-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, 13
C-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]
Trang 108
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, 13
C-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
Trang 11* 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]+ 1NMR và 13C-NMR spectral data are in agreement with those in (Table 3.19)
H-● 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]+ 1NMR và 13C-NMR spectral data are in agreement with those in (Table 3.19)
H-● 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]
Trang 123.1.1 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/H 2 O (80:20) extracts
Trang 13● 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
Trang 15* 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)
Trang 16Table 3.10: Cytotoxic activity of the compounds BL01- BL11, isolated from Thua Thien Hue sample
Compound
IC 50 (g/ml)
KB Hep
G-2 Lu MCF-7 BL01 >128,0 >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