Nghiên cứu phân lập và tác dụng điều trị bệnh đái tháo đường type 2 của các hoạt chất sinh học từ một số loại thực vật thu hái tại miền trung TT TIENG ANH

1 INTRODUCTION The justification of research Diabetes mellitus (DM, or diabetes for short) is a metabolic disorder characterized by chronic hyperglycemia- the condition where blood sugar (glucose) levels are abnormally high, as a result of insulin deficiency or decreased activity or a combination of both The consequences of hyperglycemia are serious complications that can be life-threatening Globally, the proportion of people with diabetes in 2016 was 8.5% of the adult population (422 million people) and the number is expected to rise to 9.9% by 2045 The prevalence of diabetes is more common in low- and middle-income countries, where more than 80% of deaths occur The World Health Organization believes that DM will be one of the seven leading causes of death by 2030 Plant species are always a valuable source of medicinal materials, with many of the drugs currently available on the market derived directly or indirectly from them There are at least 1,200 plant species used in traditional medicine to combat diabetes, but only about 450 plants have been studied with scientific publications “Chè dây” plant (Ampelosis cantoniensis (H & A.) PL.), belonging to Grape family (Vitacae), although has been investigated on the chemical composition and antioxidant activity, but so far there has been no scientific research on anti-diabetic activity In addition, “lá đắng” (Vernonia amygdalina Del.), belonging to Aster family (Asteraceae) have had investigated on the world against diabetes, but the studies are not comprehensive Therefore, the search for new diabetes medicines from natural plants is still highly interesting because the plants can contain new and safer compounds in the treatment of diabetes On that basis, to contribute to the research and development of antidiabetic drugs from plants in the Central region of Vietnam, I have conducted the thesis: “Studies on isolation and effective treatment for type diabetes mellitus of bioactive compounds from some plant species collected in Central region” The objectives of the thesis - Screening various local plants with hypoglycemic effect on type diabetic mouse model and identify plants with the best hypoglycemic effect, thereby purifying active compounds - Determining their chemical structures, and investigating the mechanism of action of their hypoglycemia - Combining plants that have been studied effectively in the treatment of diabetes mellitus to form an extract mixture that is more effective in treating diabetes, thereby studying the mechanism of hypoglycemia and determining the acute toxicity of the extract mixture Content of the thesis - Collecting specimens of 20 species of plants in the Central region, most of them are considered to be effective in treating diabetes Extracting of plant samples with ethanol 70% (v/v) and screening the hypoglycemic effect of the extracts on type diabetic mouse model - Extracting “chè dây” and “lá đắng” with solvents of different polarities and testing the hypoglycemic abilities of these extract fractions on type diabetic mice - Isolate and determine the structure of the compounds from the fractions of “chè dây” and “lá đắng” extract with the best hypoglycemic activity - Investigating inhibiting effects of purified compounds on α-glucosidase and α-amylase enzymes; testing their effects on proinflammatory cytokine production of TNF-α, IL -6, IL-8 and IL-10, which have been associated with the development of insulin resistance, and restoring effect on the expression of cellular pIRS-1 and pY20 proteins, which are involved in the insulin signaling pathway - Studying the hypoglycemic effect of an extract mixture, and the effect of this mixture on blood cholesterol, triglyceride levels, liver’s glycogen content as well as inhibiting activities of α-glucosidase and α-amylase enzymes in the type diabetic mice The acute toxicity of this extract mixture has also been assessed New contributions of the thesis After a literature review of related researches in the country and the world, the results of the thesis have made new scientific and practical contributions as follows: In this research, herbal sources “chè dây” (A cantoniensis) , papaya seeds (C papaya), papaya leaves (C papaya), leaves and stems of sweet grass (S rebaudiana), and “lá đắng” (V amygdalina Delile) have been studied the hypoglycemic activity for the first time in Vietnam, particularly “chè dây” has not been studied for hypoglycemic activity in the world nor has it been mentioned in folklore about this use From the CEtOAc fraction of the ethanol extract of A cantoniensis, five purified compounds have been isolated, of which quercetin is not a new substance but is isolated for the first time in this tree For the first time, an investigation of α-amylase and α-glucosidase inhibitory activity of compounds isolated from A cantoniensis has been carried out Phloretin compound (isolated from A cantoniensis) was studied for the effect of reducing insulin resistance in 3T3-L1 cells caused by TNF-α as well as restoring the expression of pIRS-1 and pY20 proteins of 3T3-L1 cells From the CBuOH fraction of the ethanol extract of V amygdalina, a new compound, called vernonioside VN (LDB), has been isolated and determined the structure This is a novel compound isolated from V amygdalina and also isolated from nature for the first time Vernonioside VN was investigated on the ability to inhibit the expression of proinflammatory cytokines- TNF-α, IL-6, IL-8, and noninhibiting effect on anti-inflammatory cytokines IL-10 4 Providing the first proof that the ethanol extract of A cantoniensis (at the dose of 500mg/kg) enabling the recovery of damaged pancreas and liver in the type diabetic mouse model For the first time, developing an extract mixture, which not only has a good effect on hypoglycemia in type diabetic mice (64.40% reduction compared with h) but also reduces cholesterol and triglyceride concentrations, prevents glycogen depletion in liver tissue as well as inhibits α-glucosidase and α-amylase The structure of the thesis The thesis consists of 138 pages The opening of 04 pages, conclusion and recommendations of 04 pages, published works of 01 page, 18 pages for references and appendices The main content of the thesis is divided into 03 chapters: Chapter 1: Literature Review consists of 40 pages; Chapter 2: Materials and Research Methods is 21 pages; and Chapter 3: Results and discussion 50 pages CHAPTER LITERATURE REVIEW The literature review brings together the latest national and international studies on issues related to diabetes (concept and impact), type diabetes, and herbal resources in the treatment of diabetes CHAPTER MATERIALS AND RESEARCH METHODS 2.1 Materials and subjects of study - Materials: 20 different plants including: “chè dây” (A cantoniensis), “lá vằng” (J subtriplinerve), “lá nếp” (P amaryllifolius), “quả chuối hột” (M acuminate), “bùm bụp” (P angulate), papaya seeds (C papaya), papaya leaves, figs (F racemosa), “quả ké đầu ngựa” (X strumarium), stems and leaves of “nở ngày đất” (G globosa), corn silk (Z mays), “lá đắng” (V amygdalina), Breadfruit’s leaves (A altilis), “giảo cổ lam” (G pentaphyllum), “dây thìa canh” (G sylvestre), lotus leaves (N nucifera), “lược vàng” (Callisia fragrans), cinnamon bark (C loureirii), basil leaves (O 5 basilicum), stems and leaves of sweet grass (S rebaudiana) Plant samples were collected mainly in July-September in 2016-2017, and some samples were purchased locally - Research subjects: + Swiss white male mouse, weight from 18-22 g, provided by Suoi Dau breeding facility - Institute of Vaccines and Biological Products in Nha Trang + Raw macrophage cells 264.7 and 3T3-L1 fat cells were provided by The National Laboratory of Enzyme Technology of the National University of Hanoi 2.2 Research Methods 2.2.1 Extraction method: plant sample processing; solid phase extraction with ethanol 70% v/v; fractional extraction with solvents of increasing polarity: n-hexane, ethylacetate, n-buthanol 2.2.2 Preparation of type diabetic mouse model: raising fat mice through a high-fat diet (35% fat, 34% carbohydrate, 20% protein and 11% other ingredients - HFD-high fat diet); determining biochemical indicators (cholesterol, triglyceride); inducing type diabetic mice by STZ injection at a dose of 120 mg/kg; quantification of blood glucose, glucose tolerance test, as well as quantification of blood insulin concentration by ELISA technique 2.2.3 Screening for hypoglycemic effects of 20 plant samples on the type diabetic mice 2.2.4 Biological slice samples of investigated mice’s liver and pancreas 2.2.5 Investigating the hypoglycemic effect of fractional extracts of “chè dây” and “lá đắng” in the type diabetic mice 2.2.6 Investigation of the ability to inhibit in-vitro enzymes α-glucosidase and α-amylase of compounds isolated from “chè dây” and “lá đắng” 2.2.7 Evaluation of the effect on the expression of proinflammatory cytokines and on the improvement of insulin resistance based on essays in Raw 264.7 and 3T3-L1 cell lines: The process of cell activation; Toxicity Assessment of purified compounds to the viability of Raw 264.7 and 3T3-L1 cells; Evaluation of the cytokine production by ELISA kit; Method for assessing the ability to inhibit insulin resistance in cells 2.2.8 Preparing an extract mixture of herbs with the ability to lower blood sugar: combining herbal plants to increase the effectiveness of diabetes treatment; determining glycogen content 2.2.9 Method of isolation of compounds: thin layer chromatography; liquid column chromatography 2.2.10 Methods of determining chemical structure 2.2.11 Data processing: The experiments were repeated with appropriate numbers of times Data processing using SPSS software (Version 22) with appropriate statistical tests CHAPTER RESULTS AND DISCUSSION 3.1 Results of sample extraction The results of extracting 20 plant samples with ethanol 70% v/v showed that different plant samples had different extraction rate into alcohol extract Leaves of “chè dây”, and “dây thìa canh”, gave the highest extract yields, 26.00% and 22.62% of total dry matter in the material, respectively “Lược vàng”, and papaya seeds had the lowest extract yields, 11.25% and 9.25% of the total dry matter in the material, respectively 3.2 Establishment of experimental type diabetic mouse model Mice were divided into two groups: the “common food” group (ND) and the “high-fat food” group (which contains 35% fat) called the fat-fed group (HFD) After weeks of the fat-fed diet, the mice were measured their blood fat index, then were injected with STZ to cause diabetes and after 10 days of STZ injection, their insulin concentrations were measured to confirm type diabetes by comparison with the ND group 7 Figure 3.1: Establishment of experimental type diabetic mouse model Notes: * indicates p-value < 0,05 (p-value of t-test compared HFD group to the control one at the same time the test); A Body weights of mice between HFD and ND groups; B Differences in lipid concentrations in HFD and ND mouse groups; C Blood glucose levels as the result of HFD and single STZ injection as compared to the control group; D Insulin level of different mouse groups The results of Figure 3.1 showed that the change in body weight of mice after weeks of feeding, the group of mice raised on a high-fat diet (HFD) had an average weight of 60.42 ± 1.03 g, which was tripled from the baseline (20.69 ± 0.36 g) and nearly doubled compared to the group that ate standard food (ND) at the same time (38.95 ± 0.68 g) (panel A) The fat index measured by cholesterol and triglyceride levels of HFD mice statistically significantly increased when compared to the ND mouse lot (t-test had p