Summary of Chemistry dotoral thesis: Studies on extraction, purification and hydrolysis of glucomannan from Amorphophallus konjac K.Koch in Lam Dong, Vietnam and its anti-diabetic activities

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The objectives of the thesis: Extraction, chemical charaterization of glucomannan from the tubers of Amorphophallus Konjac K.Koch in Lam Dong, Vietnam; parameter optimization for glucomannan hydrolysis reaction to make different types of low molecular weight glucomannan by different methods.

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Nội dung Text: Summary of Chemistry dotoral thesis: Studies on extraction, purification and hydrolysis of glucomannan from Amorphophallus konjac K.Koch in Lam Dong, Vietnam and its anti-diabetic activities

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY Tran Thi Nu STUDIES ON EXTRACTION, PURIFICATION AND HYDROLYSIS OF GLUCOMANNAN FROM MORPHOPHALLUS KONJAC K.KOCH IN LAM DONG, VIETNAM AND ITS ANTI-DIABETIC ACTIVITIES Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOTORAL THESIS Hanoi – 2020
This thesis was completed at: Graduate University of Science and Technology, Vietnam Academy of Science and Technology. Advisor 1:Prof. Dr. Do Truong Thien Advisor 2: Dr. Tran Thi Y Nhi 1st Reviewer: … 2st Reviewer: … 3st Reviewer: …. This thesis will be defended at Graduate University of Science and Technology, Vietnam Academy of Science and Technology at .......hour ........date........ month ......... 2020. The thesis can be found in: - The Library of Graduate University of Science and Technology, Vietnam Academy of Science and Technology. - Vietnam National Library.
INTRODUCTION 1. The urgency of the thesis Glucomannan, a water soluble polysaccharide, is composed of a linear chain of β-1,4-linked D-glucose and D- mannose residues in a molar ratio of 1:1.6, with side branches through β-1,6-glucosyl units. The acetyl groups along the glucomannan backbone are located, on average, every 9–19 sugar units at the C-6 position. Glucomannan is a low-calorie dietary fiber that has been used as diatary food for dieters to lose weight, reduce blood cholesterol and postprandial glucose response. In addition, glucomannan is one of the most viscous dietary fibres known which has been used in various fields such as food thickener, elastic gels, films ... Glucomannan is extracted from the tubers of some Amorphophallus species. In some subtropical Asia countries such as China and Japan Thai Lan, A. konjac is regarded as an agronomically important crop which has great potential in both domestic and international markets. Glucomannan is found in many different Amorphophallus species which has different structure and properties in each species. Amorphophallus konjac K. Koch (Amorphophalus konjac K. Koch) is a species with high content of glucomannan that become a industrial key crop in some East Asian and Southeast Asian countries such as China, Japan and Thailand. There are more than 25 Amorphophallus species in Vietnam which distributed in different regions of the country. 1
Amorphophallus konjac K. Koch was recently discovered in 2012 in some northern mountainous provinces. Despite its hydrophilicity, glucomannan is poorly soluble in water (solubility of around 30%) due to its high molecular weight 1.9  106÷2  106Da, which limits its application range in certain areas [3]. In order to increase its solubility, glucomannan is hydrolyzed to lower molecular weigh and the process attracts the attention of many scientists. In addition to the general properties of glucomannan (KGM), hydrolyzed glucomannan (LMWG) also has many biological activities such as probiotics, antioxidants, immune regulators, etc. Hydrolyzed glucomannan is also used as drug delivery carriers. With the potential application in food and pharmaceuticals, studies on the methods of preparing low molecular weight glucomannan have been of interest to many authors worldwide, including enzymatic hydrolysis [13]–[23], combination of -irradiation and β-mannanase [24] hydrochloric acid [14][25], hydrochloric acid combined with ultrasound [26] treatment with -irradiation combined with ethanol [8], alkaline hydrolysis combined with heat [28]... However, the above- mentioned studies in the world have almost focused on methods of low molecular weight glucomannan preparation. Studies on properties, chemical structure, and the relationship between their structure and biological activity have not paid enough attention. Especially, the ability to reduce blood sugar absorption when using low molecular weight and mechanism 2
has not been studied. Such studies are hardly ever been investigated in Vietnam In order to contribute a new fundamental research on glucomannan originating in Vietnam and to improve the value of glucomannan for pharmaceutical and functional food products, we have chosen the Doctor thesis entitled “Studies on extraction, purification and hydrolysis of glucomannan from Amorphophallus konjac K.Koch in Lam Dong, Vietnam and its anti-diabetic activities”. 1. The objectives of the thesis - Extraction, chemical charaterization of glucomannan from the tubers of Amorphophallus Konjac K.Koch in Lam Dong, Vietnam - Parameter optimization for glucomannan hydrolysis reaction to make different types of low molecular weight glucomannan by different methods - Evaluate the hypoglycemic activity and hypoglycemic mechanism of hydrolyzate products. 2. The main content of the dotoral thesis * Study on main chemical constituents of tuber from A.Konjac K.Koch. Physico-chemical charaterization of glucomannan: chemical constituents, manose/glucose ratio, molecuar weight by IR, NMR, TGA, … * Hydolysis parameter optimization, physico-chemical characterization of low molecular weight gluocomannan 3
* AMPK activation by low molecular weihgt glucomannan in vitro and Oral Glucose Tolerance Test . 3. New finding of the thesis Fully investigation on the main composition of tuber of A.konjac planted in Lam Dong province, glucomannan extraction and purification process, physico-chemical characterization of glucomannan. The hydrolysis parameters were optimized by response surface methodology, using β-1,4-mannanase from Bacillus sp. as catalysis. A three level, four variable Box-Behnken factorial designs (BBD) was applied to determine the best combination for viscosity. The optimal conditions were pH at 7.24, temperature at 42.4oC, and incubation time at 5.7 h, substrate concentration at 0.54%. Under optimized conditions, predicted Y was 57.5 mpa.s and experimentally value Y was 60.85 mpa.s. The hydrolysis product (LMWG-E) consisting of beta-(1  4)- linked D-glucose (G) and D-mannose (M) in a proportion of 1:1.2; the degree of acetylation was determined to be approximately 7.56%, molecular weight was calculated to be 2051.77 g/mol, solubility of 92.5%. LMWG-E significantly increased AMPK phosphorylations in a dose dependent manner. Treatment with KGM 100 μg/ml and 50μg/ml caused 1.47-fold and 1.81-fold phosphorylation of AMPK, respectively (p
4. Outline of the thesis The thesis consists of 124 pages with 29 tables, 33 figures, 9 schemes and 137 references. The thesis consists of 4 chapters: Introduction (2 pages), Chapter 1: Liturature overview (40 pages); Chapter 2: Materials and Methods (18 pages); Chapter 3: Results and Discussion (53 pages); Conclusion (2 pages); Publications related to the thesis (1 page); References (8 pages). CHAPTER 1: OVERVIEW This chapter provides an overview on national and international researches related to the thesis: general introduction about glucomannan; A.konjac K. Koch and glucomannan extraction and purification process; hydrolysis of glucomannan; AMPK enzyme and its role in hypoglycemia; researchs on glucomannan extracted from A.konjac in Vietnam. 1.1. General introduction to glucomannan 1.1.1. Sources and chemical structure of glucomannan 1.1.2. The physical properties of glucomannan. 1.1.3. The chemical properties of glucomannan 1.1.4. Biological activity and pharmacological effects of glucomannan 1.2. The review of A.konjac K. Koch and glucomannan extraction and purification process 1.2.1. Introduction to Amorphophallus Konjac K.Koch 1.2.2. Extraction and purification of glucomannan from tuber of A.konjac 1.3. The review of hydrolysis of glucomannan 1.3.1. Depolymerization by physico-chemical methods 5
1.3.2. Introduction to Enzyme hydrolysis 1.4. The review of AMPK enzyme and its role in hypoglycemia 1.4.1. Glucose metabolism in the body 1.4.2. Overview of Adenoidin 5'-monophosphat kích hoạt protein kinase (AMPK ). 1.4.3. Method of activation of AMPK 1.5. The review of glucomannan extracted from A.konjac in Vietnam CHAPTER 2. METERIAL AND METHODS 2.1. Plant materials - Three-year-old tuber of Amorphophallus Konjac K.Koch planted in Lam Dong province, Vietnam was collected in November, 2016 and identified by Dr.Nguyen Van Du, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, VAST. The specimens were kept in a sample storage house in Dak Nong province of the Center for High Technology Development - Vietnam Academy of Science and Technology deposited in the Institute of Chemistry, VAST. - Bacillus substilis và Bacillus lichenifomis were supplied by An Thai Production & Service Co., Ltd. Both strains are beneficial bacteria, bio safety and clear origin and have a genetic sequence of the original strain attached to the appendix of this thesis. 6
- Enzyme endo-1,4 β-Mannanase (Bacillus sp.) EC 3.2.1.78 CAZy Family: GH26 CAS: 37288-54-3 was from Megazyme Company. - The C2C12 myoblasts (CRL-1772) were purchased from the American Type Culture Collection (Manassas, VA, U.S.A.). - White mice (of Swiss strains), both male and female, weighing 18÷22 grams, having healthy physiology. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum (HS), and penicillin−streptomycin (PS) were obtained from WelGENE (Daegu, Korea). - All other chemicals were of analytical grade 2.2. Method 2.2.1. Determination of glucomannan content. DNS method: Acid hydrolysis of glucomannan will produce two kinds of reducing sugar: D-mannan and D-glucose. Reducing sugars will be reduced to a brownish red amino- compound when co-boiled with 3,5-dinitro salicylic acid in an alkali medium. To some extent, the amount of the reducing sugars is in positive correlation with the color strength and, therefore, glucomannan can be determined with spectrophotometry. 2.2.2. Extraction of glucomannan from the tubers of A.Konjac. Two-stage technique for extraction of glucomannan from A.Konjac K.Koch was chosen as follow: Step 1: Tubers of A.konjac were washed, peeled, sliced, and immersed into NaHSO3 0.25 ‰. 7
Step 2: adding in to the mixture a volume of ethanol/water (1.5:1 v/v) with tubers/solution ½ (w/v). Then the crushing process was operated in 20 minutes. Step 3: Centrifugation to get precipitate (paste form). Step 4: drying to get KGM powder. Step 5: purification of the product by dissolving KGM powder into hot ethanol 40%, stirring, centrifuging to collect the precipitate, and removing the filtrate. Repeat 3 times to obtain refined KGM. 2.2.2. Methods for determination of chemical structure of compounds Physicochemical characterization was investigated by modern spectroscopic methods such as IR, one/two-dimension nuclear magnetic resonance (NMR) spectra, thermal analysis, Brookfield DV2T viscometer, OSOMAT 090... Degree of acetylation (DA) of glucomanno- oligosaccharides was determined by using the 1H NMR spectroscopy. The DA value was estimated from the formula:  I  100 0 0  / 3  DA   CH 3   I  H1  Where: ICH3 was the integral of the hydrogen atom in – COCH3 group and IH1was total integral of the hydrogen atom of C1 in both glucose and mannose units. The mannose/glucose ratio in GO molecule was calculated using the integrals of H1 in the 1H NMR spectrum: I H1  Man (2.3) R Man/Glu  I H1  Glu 8
In which: RGlu/Man ratio of glucose/mannose IH1-Glu is the integral of H1 of glucose. IH1 -Man is the integral of H1 of mannose. 2.2.3. Hydrolysis of glucomannan 2.2.3.1. Hydrolysis with hydrochloric acid - Glucomannan (10g) was dispersed in a mixture of HCl and CH3COOH solution. The mixture was stirred to get homogeneous solution for both acid and ultrasound combined acid hydrolysis. - For ultrasound combined acid hydrolysis, the solution was subjected to sonication for 30 min at 20 kHz. Then both were carried out at specified concentrations at a given time or temperature. Viscosity measurements of the reaction mixture were carried out at the specific time of the studies. - After treatment, the mixture was rinsed with ethanol to neutral, left to evaporate off the ethanol before being dried in a vacuum oven at 60oC. The product obtained by acid hydrolysis method named as and the other was named as LKGM-1. - Parameter investigation in the range as follow: [H+] 0,05M, 0,1M, 0,15M, 0,2M, 0,25M; temperature: 50 oC, 60 oC, 70 oC, 80 oC, time duration: 1 hours, 2 hours, 3 hours, 4 hours, 5 hours, glucomanan/solution: 1/5; 1/10; 1/15; 1/20 (g/ml) Hydrolysis efficiency was assessed by viscosity. 2.2.3.2. Enzymatic hydrolysis * Qualitative determination: two microorganism strains that can produce enzyme β-mannanase were selected to 9
hydrolysis glucomannan: bacillus subtilis and bacillus licheniformis. * Enzymatic hydrolysis After qualitative determination, we used commercial β- mannanase from Megazyme Company for further experiments. A three level, four variable Box-Behnken factorial design (BBD) was applied to determine the best combination for viscosity. Temperature, pH, time and E/S ratio were chosen as independent variables. The range and central point values of four independent variables presented in Table 2.1 were based on the results of our preliminary single-factor experiments. All the experiments were done in triplicate and viscosity was selected as the response (Y) Table 2.1: Independent variables and their levels Code level -1 0 +1 Independent variables X1: Temperature (oC) 30 40 50 X2: Time (h) 4 6 8 pH (X3) 5 7 9 X4: E/S (w/w) 0.1 0.4 0.7 A 27-run BBD with four factors and three levels was used to fit a second-order response surface in order to optimize the extraction conditions. Glucomannan powder (10g) was dissolved in 300 ml of desired pH solution, then mixed with endo-1,4 β-Mannanase 0.01÷0.7 (w/w) to start the reaction. The mixture was incubated at pH 5÷9 for reaction time ranging from 4÷8 hours while the temperature 10
of the water bath was kept steadily at given temperature ranged from 40  60oC. The reaction was stopped by boiling the samples for 10 min. The hydrolysate obtained was concentrated with a rotary evaporator, mixed with ethanol and then had been collected as a precipitate by centrifugation at 4000 rpm for 20 min, was resuspended in ethanol three times for further investigated (named as LKGM-E) 2.2.3. Biological assays 2.2.3.1. AMPK activation in vitro The anti-diabetic effects in C2C12 myotube occur via activation of AMPK were investigated using Western Blot Analysis. The experiment was done at Department of Pharmacology, Hanoi University of Pharmacy. 2.2.3.1. Oral Glucose Tolerance Test (OGTT) Oral glucose tolerance test of different doses of LMWG-E was conducted in white, non-diabetic mice (of Swiss strains), both male and female, weighing 18-22 grams. The mice were fed daily with synthetic feed supplied by the Institute of Vaccines and Biologicals. The experiment was done at Department of Pharmacology, Hanoi University of Pharmacy. Chapter 3. RESULTS AND DISCUSSION 3.1. Extration and Purification process, physic-chemical properties of glucomanan from A.konjac 3.1.1. Determination of glucomannan content in tubers of A.Konjac This section presents the results of glucomannan content in tuber of A.konjac and some physical characteristics 11
of glucomannan. The glucomannan content was 12.26% (wet weight). The extracted glucomannan powder is white, solubility in water of 32%, ash content of 4.17%, water absorbency of 9%, Asen content was 0.208 ppm, Pb content was 0,184 ppm. Glucomannan content in tuber of A.konjac was much higher than that of in other Amorphophallus species such as A. Paeonnifolius (glucomannan content was 1.67%), A. Corrugatus (glucomannan content of 1.67%). This finding confirmed the role of Amorphophallus Konjac K.koch in the development orientation of Amorphophallus species in Vietnam. 3.1.2. Chemical structure of glucomannan. This section presents the detailed results of spectral analysis and structure determination of glucomannan extracted from tuber of A.Konjac. Structure determination of the KGM was investigated by IR, NMR 1H, 13C, HSQC and TGA. Table 3.5: 1H NMR chemical shift data of (δ ppm) LKGM-1 Signals Mannose (δ ppm) Glucose (δ ppm) H1 5,30; 5,60 5,65;5,04 H2 4,24;4,29 3,94÷3,99 H3 4,32÷4,69 4,20÷4,23 H4 3,80÷3,89 3,79 H5 3,65÷3,65 4,06÷4,08 H6 4,29;4,27 4,18÷4,19 H of CH3CO- 2,52 12
Glucomannan obtained as a white, amorphophallus, glucose/manose ratio of 1.6/1, degree of acetylation 8%, branched at C6, molecular weight was 1.598 kDa. The high DA value makes glucomannan soluble in water so that glucomannan has been used for food and pharmaceutical application. From the 1H, 13C and HSQC spectra, the cross peaks of both substituted and nonsubstituted mannosyl and glucosyl residues were assigned as follows: Cross-peaks of mannose residues: C1/H1 (94.71;94.33/5.30; 5.60), C2/H2 (71.04/4,24;4.29), C3/H3 (71.45/4.32÷4.69), C4/H4(76.76/3.80÷3.89), C5/H5(74.976/3.651÷3.658), C6/H6(61.97/4.29;4.27). The cross peaks of glucosyl residues: C1/H1 (96.68;92.78/5.65;5.04), C2/H2 (72,27;72,18/3,94÷3,99), C3/H3(73,12/4,20÷4,23), C4/H4(76,60; 76,56/3,79), C5/H5(73.82/4.06÷4.08), C6/H6(61.63; 61.54/4.18÷4.19). 3.2. Hydrolysis with hydrochloric acid This section presents the detailed results of parameter optimization for hydrolysis reaction and physico-chemical characteristic of hydrolysis products. Based on the experimental results, suitable hydrolysis parameters for ultrasound mediated acid hydrolysis were: CH3COOH 10%, [HCl] 0.15M, KGM/ acid solution ratio of 1/10(g/ml), temperature of 50oC in 4 hours. For acid hydrolysis only, the chosen/optimal parameter were: CH3COOH 10%, [HCl] 0.15M, KGM/ acid solution ratio of 1/10(g/ml), temperature of 50oC, in 6 hours. The molecular weight of the hydrolysis product reduced from 1598 kDa to 88.561kDa. Solubility in water was 82.6%. Structure 13
determination of the hydrolysis product was investigated by IR, NMR 1H, 13C and TGA. Table 3.13: 1H NMR chemical shift data of (δ ppm) LKGM-1 Signals Mannose (δ ppm) Glucose (δ ppm) H1 5.17 5.54; 5.34 H2 4.88; 4.87 3.96 H3 4.69 4.87 H4 4.49 4,58 H5 4.14 4.43; 4.41 H6 4.28; 4.26 4.35 H of CH3CO- 2.49 able 3.14: 13C NMR chemical shift data of (δ ppm) LKGM-1 Signals Mannose (δ ppm) Glucose (δ ppm) C1 101.45 100.86 C2 71.03 72.49 C3 71.18 73.93 C4 76.86 79.34; 79.16 C5 76.09 75.87 C6 64.26; 63.68 61.53 C of CH3CO- 69.80; 66.67 The results showed that the main chain of LKGM-1 consisting of beta-(14)-linked D-glucose (G) and D-mannose (M) in a proportion of 1:1.2, DA of 7.03, molecular weight of 88.561 kDa and less heat-stable in comparion with its parent glucomannan. 14
3.3. Enzymatic hydrolysis of glucomannan 3.3.1. Qualitative determination to select β-mannanase hydrolyses Experimental results showed that both enzymes produced from Bacillus subtilis and bacillus licheniformis can hydrolyzes of the glycosidic bond (p
adjusted R² of 0.997. The ANOVA results were shown in the table 3.20. Table 3.20: ANOVA for quadratic model Sum of Mean Source df F-value p-value Squares Square Model 65344,95 14 4667,50 695,95 < 0,0001 A-Temp. 1915,47 1 1915,47 285,61 < 0,0001 B-Time 563,07 1 563,07 83,96 < 0,0001 C-pH 7412,76 1 7412,76 1105,29 < 0,0001 D-E/S 2681,43 1 2681,43 399,82 < 0,0001 AB 1604,00 1 1604,00 239,17 < 0,0001 AC 223,35 1 223,35 33,30 < 0,0001 AD 402,00 1 402,00 59,94 < 0,0001 BC 57,00 1 57,00 8,50 0,0130 BD 55,35 1 55,35 8,25 0,0140 CD 1200,62 1 1200,62 179,02 < 0,0001 A² 11662,98 1 11662,98 1739,02 < 0,0001 B² 3973,30 1 3973,30 592,44 < 0,0001 C² 47246,57 1 47246,57 7044,76 < 0,0001 D² 7920,57 1 7920,57 1181,01 < 0,0001 Residual 80,48 12 6,71 Lack of fit 78,77 10 7,88 9,19 0,1021 R2 0,9988 16
Software Design-Expert® Software ctual Factor Coding: Actual Y (mpa.s) s above predicted value Design points above predicted value s below predicted value Design points below predicted value 50.15 61.15 248 300 250 ture X1 = A: Temperature 250 X2 = D: E/S 200 Actual Factors 200 B: Time = 6 C: pH = 7 150 Y (mpa.s) 150 Y (mpa.s) 100 100 50 50 0.7 50 0.6 9 50 45 0.5 0.4 40 8 45 0.3 D: E/S 35 A: Temperature (oC) 7 40 0.2 0.1 30 C: pH 6 35 A: Temperature (oC) 5 30 b) E/S and temperature Design-Expert® Software actor Coding: Actual a) pH and temperature Design-Expert® Software Factor Coding: Actual Y (mpa.s) Design points above predicted value Y (mpa.s) Design points below predicted value Design points above predicted value 1.15 250.15 Design points below predicted value 300 61.15 250.15 1 = A: Temperature 300 2 = B: Time 250 X1 = B: Time X2 = C: pH 250 Actual Factors 200 C: pH = 7 Actual Factors D: E/S = 0.4 200 A: Temperature = 40 150 D: E/S = 0.4 Y (mpa.s) 150 Y (mpa.s) 100 100 50 50 8 50 9 8 7 45 8 7 6 40 7 6 B: Time (h) 5 35 A: Temperature (oC) C: pH 6 5 B: Time (h) 4 30 5 4 c) time and temperature d) Time and pH Fig.3.23. Response surface (3-D) showing the effect of time, temperature, pH and E/S on the response Y Optimization of hydrolysis conditions: The optimal conditions were extracted by Design Expert Software for the minimum value of the response (Y) were pH at 7.24, temperature at 42.3oC, and incubation time at 5.68 h and substrate concentration at 0.54%. Under these conditions, value Y of 57.5 mpa.s was obtained. 17
Chemical characterization was investigated by IR, 1H, 13 C, HSQC. The 1H NMR chemical shifts of LKGM-E signals were assigned as in table 3.23. Table 3.23:1H NMR chemical shift data of LKGM-E Signals Mannose ( ppm) Glucose ( ppm) H1 5.257 5.031;5.021 H2 4.405 3.873 H3 4.298 4.219; H4 4.324 4.157 H5 4.014 4.076 H6 4.200 4.491 H of CH3CO- 2.702 The H NMR chemical shifts of LKGM-E signals were 1 assigned as in table 3.24. Tín hiệu Mannose (δ ppm) Glucose (δ ppm) C1 102,15;101,99 104,45 C2 72,52;72,18 75,35;75,11 C3 73,63; 73,15 76,19 C4 77,79÷78,90 80,86 C5 77,26 76,97;76,83 C6 62,92;62,78 62,57;62,45 C của CH3CO- 22,17; 176,80 β-Man(14)-β-Glc 71,77 β-Man(14)-β-Man 71,56 The cross-peaks of mannose residues were assigned as follows: C1/H1 (102.15;101.99/5.257), C2/H2 (72.52;72.18/4.405), C3/H3 (73.63; 73.15/4.298), C4/H4 (77.79÷78.90/4.324), C5/H5 (77.26/4.014), 18