Study on chemical constituents and biological activities of litsea glutinosa (lour). c. b. rob. (lauraceae) and lepisanthes rubiginosa leenh in Vietnam

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] - ; 1 H, 13 C-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, 13 C-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] - ; 1 H, 13 C-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] - ; 1 H, 13 C-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). 1 H, 13 C-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); 1 H, 13 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] + ; 1 H, 13 C-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] - ; 1 H, 13 C-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] + . 1 H, 13 C-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] + ; 1 H, 13 C-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] + ; 1 H, 13 C-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] + ; 1 H, 13 C-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; 1 H, 13 C-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] + ). 1 H, 13 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: 1 H, 13 C-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. 1 H, 13 C-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; 1 H, 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. 1 H, 13 C-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. 1 H, 13 C-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. 1 H, 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; 1 H, 13 C-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. 1 H, 13 C-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 - ] - ; 1 H, 13 C-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. 1 H và 13 C 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] + . 1 H- 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] + . 1 H- 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. 1 H-NMR và 13 C-NMR spectral data are in agreement with those in (Table 3.19). [173, 174]. 10 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 1 H and 13 C 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