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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.
MINISTRY OF VIETNAM ACADEMY OF EDUCATION AND TRAINING 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 This thesis is carried out in the Graduate University for Science and Technology, VAST Scientific supervisers: Prof Dr Sc TRAN VAN SUNG 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 I INTRODUCTION 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: Objective of the study in the thesis The samples of Litsea glutinosa (Lour.) Roxb [Lauracea] and Lepisanthes rubiginosa (Roxb.) (Sapindaceae) collected in Vietnam 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 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 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 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 - Hueprovince 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 * Compounds isolated from the water extract ● Compound BL01 ( Nicotiflorin) yellow powder IR (Kbr): νmax (cm-1): 3339; 2929; 1655; 1554; 1361; 1058; UV (MeOH), λ max (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]-; 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) λ max (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 [MH]-; 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 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 HRESIMS 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 HRESIMS m/z: 314,1741 [M-Cl]+; Molecular formula is C19H24NO3+ 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) 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 1H, 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) 2hexadecanoate) 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] 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]+ 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 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 diabetinduced mice of EtOH/H2O (80:20) extracts 10 ● 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 The result: In the doses of 250mg extract and 500mg extract/kg body weight, the weights of mice 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: flavonol glycosides (compounds BL01-BL04), aporphin alkaloids (compounds BL 05BL11), megastigmanes (BL12 , BL13) and 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) 11 BL01 (Nicotiflorin) 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,12Zoctadecadienoat)-2-hexadecanoate) BL07 (Boldine methochloride) 12 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, BL12BL16, BL19-BL21) 13 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 IC50 (g/ml) Compound BL01 BL02 BL03 BL04 BL05 BL06 BL07 >128,0 >128,0 >128,0 >128,0 67,66 68,49 >128,0 Hep G-2 >128,0 >128,0 >128,0 >128,0 117,08 82,75 >128,0 BL08 BL09 21,29 17,72 13,56 25,6 >128,0 56,32 29,24 54,21 BL10 BL11 68,39 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 Ellipti 0,31 0,38 0,41 0,60 KB Lu MCF-7 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 >128,0 cine 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 14 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 NDL-Bu Ellipticine IC50 (µg/ml) HepG2 MCF7 23,42 18,37 0,41 0,48 KB 21,33 0,40 15 Lu 20,01 0,45 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 H 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 3.833.87 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 βD-glucopyranose, one α-D-galactopyranose and one α-Larabinofuranose which is binded to the C-3 of oleanolic acid by βD-glucopyranose This suggestion was supported by HMBC correlations between: H-1’ (δH 4.49) and C-3 (δC 92.31) and H-3 16 (δH 3.23, dd, J = 4.0, 10.5 Hz) and C-1’ (δC 103.74) The α-Larabinofuranose is connected to β-D-glucopyranose at C-3’ (δC 78.57), which is evidenced by the HMBC correlations between H1’’ (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-Dglucopyranose 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 H4’(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)]-6O-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 is also a trisaccharide of a triterpenoid The H and C NMR spectra in the aglycon region are quite similar to those of compound So that the aglycone of ND9 should be also oleanolic ac id, which is binded to the sugar moiety at C-3 position (δ : 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-Dglucopyranose 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 + 13 C-3 17 is evidenced by the HMBC correlations between H-1’ (δ 4.49) and C-3 (δ 92.31; H-3 (δ 3.23) and C-1’ (δ 105.55); H-1’ (δ 4.49) and C-2’ (δ 78.54) The second β-D-glucopyranose is connected to C2’ which is evidenced by the HMBC correlation between H-1’’ (δ 4.99) and C-2’ (δ 78.54), while the β-D-xylopyranose was bound to C-3’ through the cross peak between H-1’’’ (δ 4.61) and C-3’ (δ 87.95) Besides, the following correlations were observed in the HMBC spectrum of compound ND9: H-5’’’ axial (δ 3.94, dd, J = 5.5, 11.5) to C-4’’’ (δ 69.88) and C-1’’’ (δ 105.03); H-2’ (δ 3.74) to C-3’ (δ 87.95) and C-1’ (δ 105.55); H-2’’ (δ 3.15, t, J = 9.5 Hz) to C-5’ (δ 78.32) and C-6’ (δ 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’ (δ 4.49) and H-3 (δ 3.23); H-1’’ (δ 4.99) and H-2’ (δ 3.74); H-1’’’ (δ 4.61) and H-3’ (δ 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)]-βDglucopyranosyl}-oleanolic acid This is a new compound and is named as Lepisantheside B The NMR data of ND9 were given in Table 3.18 H C H C H C H C H C H C C C C C H H C H H H H H H Table 3.18: NMR spectral data of compound ND8 and ND9 Vị trí δC 39,80 27,08 92,31 40,58 57,01 19,33 34,02 40,50 ND8 δH (J = Hz) 1,62-1,59 m; 1,03-1,00 m 1,98-1,97 m; 1,78-1,76 m 3,23 (dd; J = 10,5; 4,0) 0,81-0,79 m 1,62-1,59 m; 1,43-1,42 m 1,56-1,52 m; 1,23-1,20 m - 18 δC 39,81 27,08 92,31 40,57 57,01 19,32 34,00 40,50 ND9 δH (J = Hz) 1,63-1,61 m; 1,03-1,00 m 1,99-1,97 m; 1,78-1,73 m 3,23 dd (J = 12,0; 4,5) 0,81 -0,79 m 1,53-1,50 m; 1,43-1,41 m 1,53-1,50 m; 1,34-1,31 m - 10 11 12 13 14 15 49,51 37,89 24,54 123,55 145,32 42,91 28,88 16 24,14 17 18 19 48,49 42,75 47,34 20 21 31,62 34,96 22 33,90 23 24 25 26 27 28 29 30 -OCOCH3 -OCOCH3 Đường Glc-1’ 2’ 3’ 4’ 5’ 6’ 28,32 16,83 15,90 17,78 26,39 181,90 33,59 24,00 172,57 20,63 103,74 75,96 78,57 82,13 77,99 65,01 1,61-1,59 m 1,92-1,91 m 5,26 (brs) 1,78-1,76 (m); 1,17-1,13 (m) 2,00-1,98 m; 1,56-1,52 m 2,87 (d; J = 11,5) 1,71-1,68 m; 1,17-1,13 m 1,43-1,41 m; 1,23-1,20 m 1,52-1,50 m; 1,34-1,32 m 1,09 s 0,88 s 0,97 s 0,84 s 1,17 s 0,92 s 0,96 s 2,08 s 49,51 37,88 24,54 123,55 145,33 42,90 28,87 4,49 (d; J = 8,0) 3,75-3,73 m 3,64 (t, J = 9,5) 3,75 (t, J = 9,5) 4,21 (dt; J = 6,5; 3,0) 4,35 (dd; J = 11,5; 3,0) 4,16 (d, 11,5) 105,55 78,54 87,95 72,55 78,32 63,66 Xyl-1’’’ 2’’’ 3’’’ 4’’’ 5’’’ 24,21 48,49 42,88 47,34 31,63 34,96 34,01 28,28 16,84 15,91 17,78 26,39 181,90 33,60 24,00 105,03 75,27 78,27 77,65 67,17 19 1,59-1,56 m 1,93-1,91 m 5,26 (brs) 1,78-1,76 m; 1,17-1,13 m 1,98-1,97 m; 1,59-1,56 m 2, 89 (d; J = 12,0) 1,71-1,68 m; 1,17-1,13 m 1,43-1,41 m; 1,23-1,208 m 1,53-1,50 m; 1,34-1,31 m 1,09 s 0,89 s 0,97 s 0,84 s 1,17 s 0,92 s 0,96 s 4,49 (d; J = 8,0) 3,74 (m) 3,70 (t, J = 9,0) 3,09 (t, J = 9,5) 3,31-3,29 m 3,88-3,83 m 3,67 (dd; J = 12,0; 5,0) 4,61 (d; J = 7,5) 3,26-3,28 m 3,29-3,28 m 3,52-3,54 m 3,94 (dd; J = 11,5; 5,5) 3,28-3,31 m Glu-1’’ 2’’ 3’’ 4’’ 5’’ 6’’ 103,18 76,19 78,43 70,99 78,53 62,74 Gal-1’’’ 2’’’ 3’’’ 4’’’ 5’’’ 6’’’ 105,38 72,46 78,26 70,19 77,58 63,67 Ara-1’’ 110,93 5,24 (d; J = 2,0) 3,23-3,18 m 3,41-3,39 m 3,09 (t; J = 9,5) 3,66-3,65 m 3,87-3,83 m; 3,59-3,56 m 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 4,99 (d; J = 7,5) 3,15 (t; J = 9,5) 3,29-3,28 m 3,08 (t; J = 9,5) 3,33-3,31 m 3,84-3,83 m; 3,58-3,55 m * Eleven compounds have been isolated and identified from L rubiginosa , among them 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.) ND2 (Diosmetin; 5,7,3'-Tryhydroxy-4'methoxyflavone) ND1 (Lupeol) 20 ND3 (Heptadecanoic acid; Magaric acid; Daturinic acid) ND4 (R=H: β-sitostosterol) ND5 (R= β-D-glucose; sitosterol3-O-β-D-glucopyranoside) ND7 (3-O-[β-D-xylopyranosyl (1→3)β-D-glucopyranosyl-]-oleanoic acid) ND6 (3-O-β-sophoroside) ND8 (Lepisantheside A) ND9 (Lepisantheside B) 21 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 aporphine alkaloid , flavonoid glycosides, two megastigmanes and one sterol - From the Thai Nguyen sample 10 compounds were isolated and elucidated including 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 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 22 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 - new compounds (glycosides of oleanolic acid and farnesol) were isolated from L rubiginosa together with ones isolated for the first time from this species 23 LIST OF PUBLICATIONS RELATED TO THE THESIS 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 Pham Thi Ninh1,2, Tran Thi Phuong Thao1*, Tran Van Loc1, Nguyen Thi Dung1, Đo Xuan Cam3, Tran Van Sung1, Chemical from from etyl axetat extract of Lepisanthes rubiginosa collected in Thua Thien Hue province, Vietnam, , Journal of Chemistry, 2017, 53 (1): 1-5 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 Pham Thi Ninh1,2, Tran Van Loc1, Tran Thi PhuongThao1*, “Activity and Chemical of (Litsea glutinosa) collected in Thua Thien Hue province, Vietnam, Journal of Chemistry, 56 (1), 60-64 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 24 ... (Lour.) Rob (Lauraceae) and Lepisanthes rubiginosa (Roxb.) (Sapindaceae) in Vienam: Objective of the study in the thesis The samples of Litsea glutinosa (Lour.) Roxb [Lauracea] and Lepisanthes rubiginosa. .. 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. .. 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