Nghiên cứu điều chế cao từ cỏ sữa lá lớn (euphobia hirta l ) và ứng dụng chế biến thực phẩm dinh dưỡng kiểm soát glucose máu tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH NATIONAL INSTITUTE OF NUTRITION NGUYEN MANH THANG A RESEARCH ON MAKING EUPHORBIA HIRTA L EXTRACT IN APPLICATION FOR PRODUCING NUTRITIOUS FOOD TO CONTROL THE GLUCOSE IN BLOOD DOCTORAL THESIS ON NUTRITION Code: 9720401 SUMMARY OF DOCTORAL THESIS Hanoi – 2020 THE WORK WAS COMPLETED AT NATIONAL INSTITUTE OF NUTRITION Science instructors: Prof Dr Nguyen Cong Khan Assoc Prof Dr Truong Tuyet Mai Reviewer 1: Reviewer 2: Reviewer 3: The thesis is presented and approved from Thesis Council at Institute level at the National Institute of Nutrition Time: ……….o’clock, day……… month…… year…… The thesis can be found at: - The National library - The Library of the National Institute of Nutrition INTRODUCTION Relevance of the study In recent decades, studies of plants that can help preventing and treating diabetes and diabetes complications are increasingly drawing the attention of scientists around the world The asthma plant with the scientific name Euphorbia hirta L., belonging to Euphorbiaceae family, a wild plant that grows in humid lands in tropical countries, including the southern provinces of Vietnam The field survey shows that Binh Duong is a province with satisfactory weather and soil conditions for the development of plants in the castor family Many studies around the world such as India, Malaysia, and Japan have shown the effects of Euphorbia hirta L plants on diabetes through the mechanism of controlling blood glucose, inhibiting enzymes α-amylase and α-glucosidase by conjugation Flavonoids are found in this herb Up to now, in our country, there are limited systematic studies on Euphorbia hirta L applying this medicinal source to the production of nutritious food to meet the needs of public health care With this regard, the thesis titled "A research on making Euphorbia hirta L extract in application for producing nutritious food to control the glucose in blood” was carried out The objectives of the study To describe the botanical characteristics and identify some of the chemical components of the Euphorbia hirta L To make and evaluate the safety and effectiveness of this plant’s extract in controlling blood glucose To design a technological process to produce nutritious beverages from Euphorbia hirta L.used in blood glucose control Research content Studying the botanical characteristics and analyzing some chemical components of the Euphorbia hirta L Working on the process of extracting Euphorbia hirta L., evaluating the safety and efficiency of the extract in controlling blood glucose Building a technological process to produce nutritious beverages from Euphorbia hirta L extract used in blood glucose control Research hypothesis At present, there is a limitation of well-ground and systematic scientific studies on the botanical characteristics and chemical components of Euphorbia hirta L in different regions of Vietnam The extract from Euphorbia hirta L that has been studied to undergo optimal conditions to achieve appropriate flavonoid content will be able to inhibit the enzymes α-amylase and α-glucosidase in vitro, control blood glucose in diabetic rats Euphorbia hirta L extract products can be used as raw materials to create various products, including nutritious drinks with high-quality and food safety, allowing the community to access and use them with ease New contribution of the study - Provides scientific evidence on the botanical characteristics and some chemical components of naturally grown Euphorbia hirta L in Binh Duong, proving the blood sugar-lowering mechanism of Euphorbia hirta L is due to its ability to inhibit carbohydrate hydrolytic enzymes such as α-amylase and α-glucosidase This is the scientific basis to demonstrate the effect of Euphorbia hirta L (Euphorbia hirta L.) in reducing the glycemic index in mice with diabetes on an experimental scale - The research has built a technological process for extracting Euphorbia hirta L on the basis of determining the appropriate technological conditions Many diabete products (capsules, powder or liquid) can be produced from this extract with ease products in diabetes prevention (in capsule, powder or liquid) The author has successfully tested and trial-produced a flavonoid-rich nutritional beverage from naturally sourced milk grass This is an area in Vietnam that has not yet been developed much, and should receive more attention in the coming time to contribute to improve community health Structure of the thesis The thesis consists of 148 pages (excluding references and appendixes), in which there are pages of the introduction; page of research objectives and hypothesis; 48 pages of literature review; 26 pages of the subjects and method of the study; 41 pages of the results; 25 pages of the discussion; pages of the conclusion and page of the recommendations The thesis consists of 26 tables, 33 figures and 167 references, which include 58 references in Vietnamese, 107 references in English and 02 websites Chapter 1: REVIEW 1.1 Overview of disease pattern: Current situation and intervention solutions to facilitate the prevention and treatment of diabetes and blood glucose disorders 1.1.1 Current state of diabetes Currently, the disease pattern in Vietnam has many changes compared to this a decade ago It is a rapid rise in untransmittable chronic diseases in terms of prevalence and mortality rates This is the result of a shift in demographic patterns, with the proportion of the elderly approaching 11%, which is considered to reflect an aging population and result from socio-economic, diet and life style changes Thus, we are facing the enormous challenge of the double burden of this disease One of the most common untransmittable chronic diseases is diabetes In 2019, the American Diabetes Association introduced diagnostic criteria for detecting diabetes based on blood glucose (capillary or venous) and HbA1c levels Result Dignostic criteria FPG (no caloric intake for at least h) �7.0 (mmol/L) 2h-PG (a glucose load containing the equivalent of 75-g anhydrous Diabetes glucose dissolved in water) �11.1 (mmol/L) Or HbA1c �6.5% FPG (no caloric intake for at least h) from 5.6 to 6.9 (mmol/L) Pre2h-PG (a glucose load containing the equivalent of 75-g anhydrous diabetes glucose dissolved in water) from 7,8 to 11,0 (mmol/L) Or HbA1c from 5.7% to 6.4% Accoding to the latest IDF report in 2017, about 425 million people aged 20-79 have diabetes, equivalent to in 11 people with diabetes This number will be 629 million people (up 48%), equivalent to in 10 people with diabetes by 2045 In Vietnam, the Ministry of Health reported that in 2017 there were 3.5 million adults with diabetes, equivalent to 6% of the population, and it is expected that by 2040 there will be 6.1 million adults who can have diabetes This seriously affects the public health and socioeconomic development of the country Therefore, it is very important to find solutions to help prevent and treat diabetes through blood glucose control in the community, including a combination of many solutions, from nutritional solutions, enhancing physical activity, reasonable diet, lifestyle changes to the use of plant-based products, especially polyphenols-rich plants are trending in current research application 1.1.2 Intervention solutions to support the prevention and treatment of diabetes mellitus and blood glucose disorders The main measures in the prevention and treatment of diabetes and blood glucose disorders include changing lifestyles, having a reasonable diet and increasing physical activities, in addition to using effective products and drugs for controlling blood glucose In recent decades, researches on plant species capable of helping to prevent diabetes are of greater concerns Many plants have been recommended for use as a supplement or as an alternative to diabetic medicines, mainly based on the polyphenol and flavonoid compounds available in plants 1.2 Overview of Plant Polyphenol Compound Group There exist about 1200 plants worldwide that have been acknowledged to have positive effects on diabetic patients Each plant may contain one or many different active ingredients which can limit hyperglycemia such as polyphenols, flavonoids, alkanoids, terpenoids, anthocyanins and other active ingredients isolated from plants Polyphenol is a compound found in natural plants with color and smell Polyphenols are composed of benzene rings with different OH radicals in different groups: non-flavonoids and flavonoids In plants, flavonoids exist in two forms: the free form (aglycon) and the sugarlinked form (glycoside) The research results have shown that flavonoids act as insulin stimulants or mimic the function of insulin, affecting the activity of enzymes in sugar metabolism, which helps slow the breakdown and absorption of carbohydrates in the diet by limiting the hydrolysis of straight or branched oligosaccharides such as dextrin, maltose and maltotriose to glucose production, thereby blocking glucose absorption Currently, Vietnamese scientists continue to study plants that have been used according to traditional experience in preventing diabetes such as bitter melon, cyanosis… In the southern provinces, Euphorbia hirta L has been widely used in the treatment of some diseases such as dysentery, thirst, diabetes, and high polyphenol content Therefore, the research to promote the potential of using naturally grown Euphorbia hirta L plants in diabetes prevention and treatment is essential This is the reason why the author chose Euphorbia hirta L as the main raw material to conduct the research and takes into account the industrial production of nutrient-rich drinking water from Euphorbia hirta L plants as a health supplement in the future, contributing to the diversification of beverage products as well as supplementary products for the prevention and treatment of diabetes 1.3 Euphorbia hirta L Euphorbia hirta L belongs to the castor family (Euphorbiaceae) Herbaceous plant, growing all year around, is 30-60 cm high with tap-root of 3-5 mm diameter The stem is usually less branched, branching from the center or above, light red, with white latex and with many long, yellow-brown protective hairs Leaves are simple, in opposite pairs with two small triangular leaves or hard hairs, size 0.8-1.7 mm, and deciduous The petiole is 13.5 mm long, the leaf blade is oblong, elongated, or lanceolate, 10-50 x 3-6 mm in size, sometimes with purplish spots along the midrib Both sides are hairy thick The leaf blades are round, slightly deviated Leaf margin on the lower part is fully or partially serrated, the upper part is half serrated, pointed leaf tip, or conical The inflorescence grows in leaf axils, with stalks 25 mm long, all parts hairy, and single flowers A flower cluster consists of 4-5 male flowers, female flowers have short stalks, high gourds, protruding from the total bell-shaped, 3-compartment pots, sparse hairs, 2-lobed stigma In the Medicinal Plants and Animals document, Euphorbia hirta L is mentioned to help control blood glucose in the animals In fact, this plant has been used by the Southern people according to the Oriental medicine method to treat diabetic patients This plant can be considered as a potential source of natural raw materials to support diabetes prevention and food production in general and nutrient-rich beverages in particular Euphorbia hirta L 1.4 Overview of extraction and preparation of plant extracts Extraction is a technological process that involves separating the pharmacologically active parts from inactive ingredients in the plant tissue using solvents The finished products are usually impure, in fluid or semi-solid (thickened), or powdered (highly dried) forms This is the first step to separating the desired product from natural ingredients Extraction methods include: solvent extraction, distillation, pressing and sublimation according to the respective principles In which, solvent extraction is a widely used method In industry, commonly used methods include: extraction, soak extraction or reflux extraction The rest of the methods, such as microwave assisted, ultrasonic, supercritical enzymes or fluids, apply for specific purposes or groups of active ingredients that are susceptible to thermal degradation Flavonoids are considered to be quite resistant to heat and have a good solubility in water Its applicable extraction methods include immersion, percolation, soxhlet or reflux extraction In Euphorbia hirta L., flavonoid compounds, polyphenolic and tannin are quite polar and easily soluble in water Therefore, it is possible to use reflux extraction with water or a liquid alcohol solvent system However, reflux extraction with water is more economical and produces higher extraction efficiency, with the collected extract can be easily used in beverage preparation 1.5 The production technology of herbal beverage for blood glucose prevention and control The trend of using beverages with natural ingredients, especially those extracted from herbs and fruits, is growing This type of beverage is a refreshing drink, providing the body with nutrients and has become popular in the market, will gradually replace carbonated drinks The production line for beverages prepared from natural or synthetic sources is shown in the figure below Chapter 2: SUBJECTS AND METHODS OF THE STUDY 2.1 Research materials 2.1.1 Main material Euphorbia hirta L plants,collected in Binh Duong province, dried to the humidity ≤ 11%, cut into segments 4-6 cm long and stored in a plastic bag, kept in a dry and ventilated place The minimum time from harvesting to putting into experiments is 30 days From this raw material, the author has conducted preparing Euphorbia hirta L extract In the study, the author uses a number of other materials such as: Honey, Ginseng, sweeteners, Cinnamon, Ginger, Water and aluminum package cans 2.1.2 Chemicals and tools - In this study, the main chemicals used include: absolute ethanol, methanol, quercetin, L-aspartate and L-alanin substrate, Blocked p-nitrophenyl maltoheptaoside (BPNPG7) substrate, p-nitrophenyl substrate - α-D-glucopyranoside (PNPG), quercitrin; reagents used in qualitative reactions and chromatography reagents (Mayer, Dragendorff, Bouchardat, dd FeCl3 5%, dd gelatin 1%, dd lead acetate 10%, Lugol, Fehling A, Fehling B, vanillin- sulfuric acid) - Tools: Glucose dosing kit, HbA1c (Wako Pure Chemicals, Japan), syringes, needles and basic laboratory tools 2.1.3 Experimental animals In the study, the experimental animals were the white mice of Swiss albino used in the assessment of acute toxicity and blood glucose decrease and Wistar white rats used in semi-chronic toxicity 2.1.4 Device Basic devices used in the laboratory such as analytical balance, optical meter, oven, moisture meter, microscope, automatic blood analyzer, In test production, the devices includes: ordinary oven; Herbal cutting machine; Pharmaceutical grinder; Ultrasound with heating; Rotary distillation machine; Pumping equipment; Automatic mixer; Canned machine system; Pasteurization system 2.1.5 Research location After directly field surveying the naturally grown Euphorbia hirta L in Binh Duong, the researcher has collected the fresh (original) Euphorbia hirta L., washed it and transported it to Hanoi to conduct experiments The author has carried out the following studies: Plant characteristics and chemical components of Euphorbia hirta L in the Department of Medicinal Materials, Hanoi University of Pharmacy; Euphorbia hirta L preparation at the Department of Plant Chemistry under the Institute of Medicinal Materials; Evaluation of acute and semi-chronic toxicity in the Department of Pharmacology and Biochemistry of the Institute of Medicinal Materials; Determination of inhibitory activity of α-amylase and α-glucosidase enzymes at the National Institute of Food Safety and Hygiene under the Ministry of Health; Efficiency evaluation was conducted at the Center for standard laboratory animal research and production under the National Institute of Hygiene and Epidemiology; Formulating the formula and process of nutritional beverage products from Euphorbia hirta L.extract at the Aroma Alcohol and Beverage Joint Stock Company; Test production and canning at the Institute for Fruit and Vegetable Research and Community Acceptance Assessment in Hanoi and Hung Yen 2.2 Research Methods 2.2.1 Surveying the botanical characteristics and chemical components of the Euphorbia hirta L 2.2.1.1 Plant characteristics study Morphological characterization and scientific name determination: Euphorbia hirta Lsamples were described according to the analytical description method, compared with the genus Euphorbia key, the description of Euphorbia hirta L species in Plants Chinese Journal and the Course of Identifying and the Vietnamese Oil Contractor surname of Prof Dr Nguyen Nghia Thin to identify scientific names Description of stem and leaf micro-anatomy: The stems and leaves are cut microsurgery by hand cutting machine, bleached with Cloramin B, and dyed by double dyeing method Look under a microscope, take pictures and describe microscopic features Description of stem powder, leaves, flowers, fruit: Dried medicinal herbs (stem, leaf, flower, fruit) in an oven at 60oC, then crushed by boat and mortar Sieve the fine powder, use a lance needle to take the medicinal powder onto the glass slide, already drip a drop of distilled water, and put the lamina on Observe under a microscope, take pictures and describe powder characteristics Take photos of micro-anatomical features and powder with Canon camera Image processing by software PHOTOSHOP CS6 2.2.1.2 Chemical component research Qualitative groups of compounds by chemical reaction: Conducted according to the method stated in the Document Research Methodology of Medicinal Materials, Department of Medicinal Materials - University of Medicine and Pharmacy, Ho Chi Minh City, Ho Chi Minh City Qualitative n-hexane, chloroform, and ethyl acetate extracts by thin layer chromatography - Extraction of the large foliar above ground milky fraction: - Medicinal herbs (1.0320 kg, H = 9.4%) boiled with distilled water for hour, sometimes adding distilled water Then hot filter by vacuum pump Add more distilled water and perform the second and third extraction as above Combine the extracts and concentrate the extracts to the ratio of kg of medicinal herbs: L of extract, allow to cool The obtained extract is extracted with n-hexane, chloroform, and ethyl acetate solvents, resulting in n-hexane fraction, chloroform fraction, ethyl acetate fraction, respectively Store and recover the extract fractions under reduced pressure and evaporate the corresponding fractional bites, n-hexane bites (0,1001 g), chloroform bites (0.5912 g), ethyl acetate bites (14.4499 g) - Prepare solutions for chromatograph: Test solution: Place mg of bite each fraction into test tubes and then add ml of methanol to each tube; Standard solution: Quercitrin solution of 0.1 mg / ml in methanol and rutin solution of 0.1 mg / ml in methanol - Reagent: vanilin-sulfuric acid - Implementation process: The F254 silica gel thin sheet was activated at 110oC for hour Spot the extracts on the plate and develop with the corresponding solvent system Observe the stain in normal light, then look under ultraviolet light (UV254 and UV366) Show traces with vanillin-sulfuric acid reagent The ethyl acetate fraction was compared with quercitrin and standard rutin Quantification of flavonoids present in Euphorbia hirta L dairy plants by photometric method - Prepare stock standard solutions Accurately weigh 0.98 mg of standard quercitrin, place into a 10 ml volumetric flask, add ml of methanol, and shake to dissolve completely Make up to the mark with methanol to obtain a stock standard solution S of concentration 98 μg / ml - Survey the maximum absorption Accurately take ml of the stock standard solution to a 10 ml volumetric flask, add 0.3 ml of 10% aluminum chloride solution R, 0.3 ml of 1M sodium acetate solution R, make up to volume with distilled water, Shake well, then let stand for 30 minutes Conduct spectral scan from 200-600 nm Spectral scan results show that the solution has absorption peaks at the wavelengths of 207.5 nm, 270.5 nm and 412.0 nm Therefore, choose the wavelength of 412.0 nm as the photometric wavelength - Construction of standard roads  Prepare standard series: Get exactly 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1,2; 1,3 ml of stock standard solution S, in turn, into 10-ml volumetric flasks, evaporate the solvent, allow to cool, add ml of methanol shake to dissolve, add 0.3 ml 10% aluminum chloride solution R, 0,3 ml sodium acetate solution M R, add distilled water to the mark, shake well, obtain a series of standard symbols respectively S1, S2, S3, S4, S5 , S6, S7, S8 have respective concentrations of 5.88; 6.86; 7.84; 8,82; 9.80; 10.78; 11.76; 12.74 μg / ml  Building a calibration curve: Measure the absorbance of the S1 - S8 standard solution series at a wavelength of 412.0 nm with a blank sample prepared by taking exactly ml of methanol, 0.3 ml of aluminum chloride solution 10% Make up to the mark with 0.3 ml of 1M sodium acetate solution R into a 10 ml volumetric flask The results of the absorbance measurement of the standard series and the graph showing the linear correlation between the absorbance and quercitrin concentration are shown in the following table and figure The dependence of the absorbance on the standard quercitrin concentration Độ hấp thụ Nồng độ quercitrin (μg/ml) Lần Lần Lần Trung bình 5,88 0,233 0,233 0,233 0,233 6,86 0,280 0,280 0,280 0,280 7,84 0,321 0,321 0,321 0,321 8,82 0,366 0,365 0,366 0,366 9,80 0,394 0,392 0,397 0,394 10,78 0,432 0,433 0,434 0,433 11,76 0,480 0,480 0,480 0,480 12,74 0,516 0,516 0,516 0,516 The graph showing the linear correlation between the absorbance and quercitrin concentration shows that in the survey concentration range, the absorbance and quercitrin concentration have a very close linear correlation with the correlation coefficient R = 0.9989 (coefficient of determination R2 = 0.9978) Linear regression equation: D = 0.0406.C - 0.0004 (*) In which: D: Absorption of the solution 13 temperature After months, determine the chemical, physical, and microbiological parameters of the product 2.2.3.2 Testing methods Physical and microbiological criteria: use reliable methods to determine Preparation efficiency: The extraction efficiency is determined by the ratio of the extracted high mass to the extracted volume of medicinal powder - The formula is as follows: - Inside: - M: Extraction efficiency(%) - M2: High volumes are obtained(mg) - M1: The amount of medicinal powder (Euphorbia hirta L powder) is put into extraction(mg) 2.2.3.3 Evaluation of food quality and safety Assessing food quality and safety according to the QCVN 6-2: 2010 / BYT National Technical Regulation for non-alcoholic beverages and the Vietnamese standard TCVN 7041: 2009 on non-alcoholic beverages 2.2.3.4 Evaluate community acceptance The Euphorbia hirta L nutritional drink product, when it was tested, was assessed for community's acceptance by a taste score test conducted on a sample size of 98 mature consumers in Hanoi and Hung Yen average 24-65 years old, both men and women, currently not smoking, not suffering from nose and throat diseases and voluntarily participating in the study Chapter 3: RESULTS 3.1 Botanical characteristics and chemical components of Euphorbia hirta L 3.1.1 Botanical characteristics 3.1.1.1 Morphological characterization Herbaceous plant, annual life, 20-50 cm high, has white latex Stems erect sometimes folded, cylindrical, 1-4 mm diameter, light green (when young) to pink, redpurple (when mature), divided into several segments, the nodes near the roots are often short more than 2-3 cm long, the upper segments to cm long, the nodules between the trunk segments slightly bulging Leaves are simple, opposite, in a row, with leaves with thin, hard hair, 1-2 mm long on either side of the petiole Leaf stalks short, 1-3 mm long, hairy Leaf blade oval or long elliptical, young leaves 1-2 cm long, 0.4-0.8 cm wide; The leaf base is asymmetric, one side is rounded, one side is shield-shaped, the leaf edge is serrated, the leaf tip is pointed 3.1.1.2 Determine the scientific name of the research sample Based on the morphological characteristics of the study sample, comparing with the classification key Euphorbia, the description of species Euphorbia hirta L of the Chinese plant lice, and the GS Dr Nguyen Nghia Thin with the help of TS Nguyen 14 Quoc Huy, a sample of Euphorbia hirta L.studied, has been identified as Euphorbia hirta L., and belongs to the Euphorbiaceae family 3.1.1.3 Microsurgery characteristics Characteristics of leaf microsurgery Characteristics of body microsurgery Note: Leaf vein Dear 1.Feather cover Libe Gandular hair 2.Lower epidermis wood Epidermis Soft tissue Hedgehog Soft shell tissue Thick tissue 10 Upper epidermis Libe Soft tissue cells surround the phloem-wood bundle wood Blade 11 Feather cover 14 Hedgehog 12 Lower epidermis 15 Upper epidermis 13 Soft tissue 3.1.1.4 Characteristics of medicinal powder: Leaf powder is green, fragrant, tasteless Stem powder has a golden brown color, aroma, and a slightly bitter taste The flower powder is brown, fragrant, has a slightly bitter taste and the fruit powder is brown, fragrant, tasteless 3.1.2 Chemical components determination 3.1.2.1 Qualitative groups of compounds by chemical reaction Conducting a qualitative reaction of the groups of compounds present in the upper part of the Euphorbia hirta L milkgrass grass shows that in the upper part of the ground, the large-leaved milkgrass has groups of compounds: flavonoid, tannin, saponin, alcaloid, sterol, reducing sugar , polysaccharide 3.1.2.2 Qualify the extract fractions by thin layer chromatography Qualitative extracts of n-hexane, chloroform and ethyl acetate by thin layer chromatography with the corresponding solvent systems are: toluene-ethyl acetate-acid formic (7: 1,5: 0,1); toluen-chloroform-methanol (5: 4: 0.5) and toluen-ethyl acetatemethanol-acid formic (3: 6: 1: 1) Comparing with the standard, it was found that in the ethyl acetate fraction with quercitrin had high density, which showed that quercetrin was a major ingredient in the ethyl acetate fraction 3.1.2.3 Quantification of total flavonoids in Euphorbia hirta L Quantification of total flavonoid in the upper part of the Euphorbia hirta L field shows that, the total flavonoid content in the upper part of the Euphorbia hirta L 15 ground, calculated as quercitrin, is 0.375 ± 0.014% in absolute dry medicinal herbs The quantitative results have acceptable accuracy with RSD = 3,676% 3.2 Euphorbia hirta L preparation, evaluating the safety and effectiveness of the product in blood glucose control 3.2.1 Modulation of Euphorbia hirta L 3.2.1.1 Investigation of factors influencing on the preparation of Euphorbia hirta L Effect of temperature Effect of ratio of solvents and medicinal herbs Euphorbia hirta L medicine, cut into 1-3 cm pieces, humidity