Nghiên cứu phản ứng thủy phân các glycozit tự nhiên bằng enzym b glucosidase và đánh giá hoạt tính sinh học của các sản phẩm nhận được tóm tắt LA tiếng anh

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY -LE THI TU ANH STUDY OF HYDROLYSIS OF NATURAL GLYCOSIDES BY β-GLUCOSIDASE ENZYME AND BIOACTIVITIES OF THEIR PRODUCTS Major: Organic chemistry Code: 62.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi – 2018 The thesis was completed in Graduate University Science and Technology, Vietnam Academy of Science and Technology Supervisor 1: Assoc.Prof Dr Le Truong Giang Institute of Chemistry, Vietnam Academy of Science and Technology Supervisor 2: Dr Doan Duy Tien Institute of Chemistry, Vietnam Academy of Science and Technology 1st Reviewer: 2nd Reviewer: 3rd Reviewer: The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at date month 2018 Thesis can be found in - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology - The National Library of Vietnam PUBLICATIONS WITHIN THE SCOPE OF THESIS Lê Thị Tú Anh, Đoàn Duy Tiên, Bá Thị Châm, Nguyễn Văn Tuyến, Nghiên cứu phân lập chủng vi sinh vật thủy phân glycosit thành aglycon có hoạt tính sinh học cao Tạp chí Hóa học, 2016 , 54 (6e2): 84-89 Lê Thị Tú Anh, Bá Thị Châm, Nguyễn Thu Hà, Nguyễn Thanh Trà, Nguyên Văn Tuyến, Nghiên cứu thủy phân astilbin rễ Thổ phục linh (Similax glabra) vi sinh vật, Tạp chí Hóa học, 2016, 54 (6e2): 223-227 Nguyễn Thị Thu Hà, Phạm Thị Thu Hằng, Nguyễn Thanh Trà, Bá Thị Châm, Lê Thị Tú Anh, Đặng Thị Tuyết Anh, Nguyễn Hà Thanh, Thành phần hóa học hoạt tính ức chế enzym khử HMG-Coenzym A vỏ đậu xanh (Vigna radiata), Tạp chí hóa học 2017, 55 (4e23), 21-26 Nguyễn Thị Thu Hà, Nguyễn Thanh Trà, Bá Thị Châm, Lê Thị Tú Anh, Đặng Thị Tuyết Anh, Nguyễn Hà Thanh, Thành phần hóa học hoạt tính ức chế enzym khử HMG-Coenzym A Sen hồng (Nelumbo nucifera), Tạp chí hóa học 2017, 55 (4e23), 261-266 INTRODUCTION The urgency of the thesis Nowadays, environmental protection has become a necessity in every aspect of life In the field of chemistry, looking for catalytic enzymes, supporting the conversion process, organic synthesis is considered to be environmentally friendly green development Thanks to its superior advantages over other catalysts: they produce very little byproduct, operate at amazing speeds, are usually harmless and not require expensive and rare elements to produce them… enzyme catalysis not only improves reaction efficiency but also contributes to reducing environmental pollution β-glucosidases (BGL) are member of cellulase enzyme complex, they catalyze the hydrolysis of the β-glycosidic linkages in carbohydrate structures Hydrolysis of glycoconjugates such as aminoglycosides, alkyl glucosides, and fragments of phytoalexin-elicitor oligosaccharides is an important application of β-glucosidases Flavonoids, a group of natural substances with variable phenolic structures, are considered as an indispensable component in a variety of nutraceutical, pharmaceutical, medicinal and cosmetic applications The natural flavonoids almost all exist as their O-glycoside or C-glycoside forms in plants However, their aglycone usually has more activity in comparison with their glycoside forms Therefore, the development of bio-catalyzed hydrolysis of flavonoids glycoside and the study of the activity of these substances are very important to predict potential applications and manufacturing by industry In the proceeding of research and development of enzyme, the amount of microorganism must to be cultured Negative effects of these microorganisms on the environment are the reason of the necessary of a disinfection process before disposal so to ensure an environmentally friendly process, For research purposes: looking for potential biologically active glycosides, aglycones from plants and developing new research methods – bio-catalysis applied, we select thesis topic: "Study on hydrolysis of natural glycosides by β-Glucoside enzyme and bioactivities of their products" In this study, P.citrinum were isolated from Clerodendron cyrtophyllum Turcz roots, identified and biosynthesized as β-glucosidase The extracted glycosides from Vietnamese plants are hydrolyzed by this β-glucosidase The flavonoids and their corresponding metabolites are evaluated for bioavailability The fungus after fermentation was studied sterilization by advanced oxidation process The aim of the thesis Study on applied of enzyme on hydrolysis of natural glycosides to produce new potential biologically active compound Develop a new methods supporting the conversion process, organic synthesis is considered to be environmentally friendly green development The main contents of the thesis: - Identification of microorganisms capable of producing β-glucosidase - Fermentation, evaluation of kinetic parameters of free and fixed βglucosidase from P citrinum - Research on sterilization after fermentation by advanced oxidation - Study on the extraction of flavonoids glycoside compounds from Vietnamese plants - Study the hydrolysis of glycoside compounds from plants with βglucosidase enzyme - biological activity of glycoside and aglycone compounds CHAPTER 1: OVERVIEW Overview of national and international researches related to my study 1.1 β-D-glucosidase enzyme Presentation of contents related to β-glucosidase: basic contents related to the definition, classification, reaction mechanism, purification and evaluation of enzyme activity Next, the content of diversity and the ability of biosynthesis of β-glucosidase in microorganisms, on the improvement of seed sources for the purpose of increasing BGL production and related to commercial BGL production Finally, on the multidisciplinary application of β-glucosidase 1.2 Flavonoid compounds Presentation of flavonoid-related content: baseline, group classification, biosynthesis, reagent identification and bioactivity of the substance group 1.3 Flavonoid glycosides and their aglycon Presentation of the content related to the uptake, metabolism of flavonoid glycose from which the potential of the aglycon compared with their glycoside This is followed by an overview of the globally published flavonoid glycozite metabolites 1.4 Biosafety in research Strict adherence to biosafety procedures is absolutely essential for researchers working with pathogens because the exact transmission pathways of these pathogens are unclear, and specific preventives and therapeutics are generally unavailable It would only take a single mistake in handling infectious materials to cause a full-on disaster One painful example of this occurred at Beijing's Institute of Virology where a lab researcher was infected by severe acute respiratory syndrome-coronavirus in a sample that was improperly handled, resulting in the death of the researcher's mother and the infection of several others.Thus, researchers should be particularly careful in handling laboratory-generated organism CHAPTER 2: EXPERIMENTAL AND RESULTS 2.1 Materials Residue seeds of Glycine max from Quang Minh vegetable oil joint stock company, Kim Dong, Hung Yen Dry leafs of Nelumbo nucifera and seed coat of Vigna radiate from Hanoi, Bac Giang Flower of Styphnolobium japonicum (L.) Schott from Nam Dinh The rhizomes of Rhizoma Polygoni cuspidati from Nghia Trai, Hung Yen 2.2 Chemical and equipments: 2.3 Methods 2.3.1 Methods for isolation, identification of microorganism 2.3.1.1 Method of isolation 2.3.1.2 Method of identification: phenotypic identification, genotypic identification 2.3.2 Enzymatic activities and kinetic properties of β-glucosidase: p-nitrophenyl-β-glucopyranosid (pNPG) method 2.3.3 Methods for isolation and structural elucidation glycosides: Chromatographic methods such as thin layer chromatography (TLC), column chromatography (CC) Physical parameters and modern spectroscopic methods such as electrospray ionization mass spectrometry (ESI-MS) and high-resolution ESI-MS (HR-ESI-MS), one/twodimension nuclear magnetic resonance (NMR) spectra 2.3.4 Method for hydrolysis of glycosides by β-glucosidase: free enzyme and immobilized enzyme 2.3.5 Sterilization of microorganisms 2.3.6 Biological assays - DPPH method of antioxidant assay - Inhibitor enzyme activity of α-glucosidase - Inhibitor enzyme activity of Angiotensin I CHAPTER 3: RESEARCH METHODOLOGY 3.1 Isolation and identification of a fungal β-glucosidase 3.1.1 Isolation of a fungal β-glucosidase We isolated fungus from roots of Clerodendron cyrtophyllum Turcz The most active β-glucosidase fungus will be used in the next study 3.1.2 Identification of a fungal β-glucosidase Phenotypic and rDNA internal transcribed spacer sequence analyses indicated that the isolate belongs to Penicillium citrinum 3.2 Purification and Characterization of a β-Glucosidase Fermentation condition (pH,carbon source) was optimized for producing the enzyme in shake flask cultures Kinetic parameters for hydrolysis β-pNG, ability to catalyzes the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature were investigated Study on the immobilized BGL-P, performance of immobilized enzyme is calculated by equation: Performance of immobilized enzyme (%) = (Et- Es)/Et x100 Et is the enzymatic activity before the immobilization Es is the enzymatic activity after the immobilization 3.3 Isolation and purification of glycosides from Vietnamese plants 3.3.1 Isolation and purification of glycosides from residue seeds of Glycine max EtOH extract extracted by acetone times solvent removal by vacuum evaporation Acetone extract Dissolve by EtOAc Extracted by H2O EtOAc extract H2O extract silica gel: EtOAc: H O (97:3) and EtOAc:H O:EtOH (95:3:2) F3-F4 F1-F2 F5 silica gel: EtOAc: MeOH (96:4) Sephadex LH-20, EtOH Crystallized CH Cl D1.1 (12.8mg) D5.3 (251.2mg) F1 F1 F6 F7-F10 silica gel: EtOAc: MeOH (95:5) D6.4 (198.7mg) Kết tinh CH Cl 2 D1.2 (3.4 mg) 3.3.2 Isolation and purification of glycosides from leave of Nelumbo nucifera 3.3.3 Isolation and purification of glycosides from coat of green bean seeds Vigna radiate 3.3.4 Isolation and purification of glycosides from flower of Styphnolobium japonicum (L.) Schott Characteristic of the compound: compound melting point: point 242oC H NMR (500 MHz, DMSO-d DMSO 6): =0,99 =0,99 ppm (3H, d, J= J= 6,5Hz, HRha6’’’); 3,09 3,09- 5,00 (proton (protons CH-OH OH ); 5,2 (1H, brs, HRha-1’’’); 1’’’); 5,34 (1H, d, J= = 7,0 Hz, HGlc-1’’); -1’’); 6,19 (1H, d, J= = 2,0 Hz, H-6); H 6); 6,38 (1H, d, J= = 2,0 Hz, H-8); 8); 6,84 (1H, d, J= = 8,0 Hz, H H 5’); 5’); 7,52 (1H, d, J = 2,0 Hz, H-2’); 2’); 7,55 (1H, dd, J=2,0; =2,0; 8,0 Hz, H-6’); H 6’); 12,58 (1H, s, OH OH-5) 5) 13 C NMR (125 MHz, DMSO-d C-NMR DMSO 6):  156,5 (C (C-2); 2); 133,3 (C-3); (C 3); 177,4 (C-4); 4); 161,2 (C (C-5); 5); 100,1 (C-6); (C 6); 164,1 (C (C-7); 7); 93,6 (C-8); (C 8); 156,4 (C-9); (C 9); 103,9 (C (C-10); 10); 121,1 (C (C-1’); 1’); 115,2 (C-2’); (C 2’); 144,7 (C (C-3’); 3’); 148,4 (C (C-4’); 4’); 116,2 (C (C-5’); 5’); 121,5(C-6’); 121,5(C 6’); 101,2 (CGlc-1’’); 1’’); 74,1 (CGlc-2’’); 2’’); 76,4 (CGlc3’’); 70, 70,3 (CGlc-4’’); -4’’); 75,8 (CGlc-5’’); 5’’); 66,9 (CGlc-6’’); 6’’); 98,7 (CRha-1’’’); 1’’’); 70,5 (CRha-2’’’); 2’’’); 71,3(CRha-3’’’); 3’’’); 71,8 (CRha-4’’’); 4’’’); 68,2 (CRha-5’’’); 5’’’); 18,6 (CRha6’’’) 3.3.5 Isolation and purification of glycosides from Rhizoma Polygoni Polygoni cuspidati ` CC extract (15 g) Silicagel 0,063 ÷ 0,2 - CH2Cl2 : CH3OH F1 F2 Crystallized F3 F4 F8 F7 (2,0g) F6 F5 (2,4g) - Silicagel CH2Cl2 /CH3OH - Silicagel CH2Cl2 : CH3OH C2.1 (290mg) 7-1 7-2 7-3 7-4 7-5 Crystallized 5-1 5-2 5-3 5-4 C7.3 (155mg) Crystallized C5.2 (97mg) C8.4 (20mg) C8.5 (15mg) 3.4 Hydrolysis glycoside compounds: Percentage of hydrolysis [140]: 100 Percentage of hydrolysis (%) = Qc: the amount of hydrolyzed product Qo: the amount of glycoside initially put into the reaction M1: molecular weight of glycoside M2: molecular weight of hydrolysis product 3.5 Disinfection of study microorganisms using Advanced oxidation processes 3.5.1 Prepaire of Advanced oxidation processes: electro-disinfection 3.5.2 Studies on the Electrochemical Disinfection of B cereus as an indicator 3.5.2.1 Studies on the effect of electric current on the disinfection 3.5.2.2 Studies on the effect of pH of electrolysis water on the disinfection 10 phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statistical statistical errors Bootstrap consensus is applied to the constructed tree so as to read maximum sequence replications replications Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate C5 It is because more than 100 BLAST hits belon belonged ged to Penicillium citrinum, citrinum, thus strongly recommending our isolate as a member of this group Fig 4.4: Colonies, Colonies phialides of C5 4.2 Purification and properties of β glucosidase glucosidase from culture Partial purification of β β-BGL BGL was carried out by ammonium sulphate precipitation, followed by sephadex sephadex,, lyophilized Activity of the BGL from Penicillium citrinum partially purified enzyme (BGL (BGL P) P) was determined using 4-Nitrophenyl Nitrophenyl β-D β D-glucopyranoside glucopyranoside (5 mM) as substrate BGL P P was used at free enzyme and an d immobilized enzyme 4.2.1 Properties of BGL BGL-P: Optimum pH and temperature for enzyme assay β-glucosidase glucosidase activity was observed at 40, 50, 60, 70 and 80°C The results showed that the BGL activity increased from 50 to 70°C 0°C after which decrease in activity was observed The best temperature for BGL-P BGL P o activity was 60 C Temperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also prevent event contamination to allow the reaction to proceed at higher range of temperature As far as pH is concerned, the plot obtained by the expected bell curve and maximum activity was observed in the pH range of 5.0 to 6.5 and the BGL-P P was optimized at pH 6.0 Kinetic parameters for BGL-P BGL 11 Different concentrations of pNPG (0-25 mM) were used to estimate the kinetic parameters, Km and Vmax using double reciprocal LineweaverBurk plot The results were Km = 0,01µmol Vmax = 13,91 µmol/min 4.2.2 Properties of BGL-P immobilized: Immobilization of BGL-P in calcium alginate: Sodium alginate of 4% concentration and 4% CaCl2 solution were found to be best with respect to immobilization efficiency and calcium alginate beads so obtained were not much susceptible to breakage BGLP entrapped in large calcium alginate beads was used successfully for cycles for the conversion of pNPG into product without much damage to the beads under stirring conditions Immobilization of BGL-P onto spent coffee grounds: Spent coffee grounds, discarded as environmental pollutants, were adopted as enzyme immobilisation solid carriers instead of commercialised solid supports to establish an economical catalytic system β-Glucosidase was covalently immobilised onto spent coffee grounds Conditions were determined to be 40 °C and pH using 4nitrophenyl β-D-glucuronide as an indicator Operational reusability was confirmed for batch reactions Table 4.3 Kinetic parameters for free BGL-P and immobilized Forms Temperature pH Km R2 * Vmax (oC) (µmol/min) (µmol) Free forms 60 6.0 13,91 0,011 0,9994 Immobilized in 50 6.0 13,09 0,034 0,9978 alginat Immobilized onto 40 6.0 14,45 0,022 0,9992 spent coffee grounds * is R2 of Lineaweaver and Burk plot 4.2 Chemical structure of isolated compounds This section presents the detailed results of spectral analysis and structure determination of 20 isolated compounds from plant species: No Symbol Structure Name of compound D1.1 Genistein 12 D1.2 D5.3 Daidzein Genistein 7-O-beta-Dglucoside D6.4 Daidzein7-O-beta-Dglucoside S3.1 MB5 catechin S5.2 quercetin-3-O-βgalactoside S7.3 quercetin S8.4 kaemferol S8.5 isorhamnetin-3-O-β-Dglucuronide 13 10 S8.6 quercetin-3-O-β-Dglucuronide HO 11 MB3 apigenin-6-C-glucoside (vitexin) OH OH 4'' 6'' 3'' 5'' 2'' HO HO O 3' 2' 1'' 1' O OH 5' 6' 10 OH 4' O 12 MB4 apigenin-6-C-glucoside (isovitexin) 13 MB1 luteolin 14 MB2 taxifolin 15 HH1 quercetin-3-Orutinoside (rutin) 16 C2.1 resveratrol 17 C5.2 Resveratrol –betamono-D-glucoside (picied) 14 18 C7.3 emodin 19 C8.4 emodin-8-O-β-Dglucopyranoside 20 C8.5 physcion-8-O-β-Dglucopyranoside Example: Compound MB1: vitexin (MB1) Compound was obtained as a yellow amorphous powder and its molecular formula MB1was determined as C21H20O10 on the basic of ESI-MS at m/z 433 [M+H]+ and the melting point at 247-249ºC The 1H-NMR spectrum of MB1 showed a doublet proton at δ 8.01 corresponding to H-2’ and H-6’ proton Another doublet proton occurs at δ 6.89 corresponding to H-3’ and H-5’ Two protons appeared at δ 6.75 and δ 6.24 as singlets corresponding to H-3 and H-6 protons respectively, one proton anomeric at  4,71 corresponding to H-1’’, which suggested the structure of flavone with one sugar moiety The 13CNMR and DEPT spectrum of the compound showed 21 signals for the vitexin Carbon bonded to the carbonyl group C-4 appeared at δ 182.1 The carbonyl carbon, C-4 resonates around δ 175178, when the carbonyl is not hydrogen bonded But in the presence of Hbonding to 5-hydroxy group, it moves downfield to about δ 182 When 3hydroxy group is alone it resonates at δ 171- 173 When both 3- hydroxyl and 5-hydroxyl groups are present, it resonates at δ176.Carbon bonded to the hydroxyl group C-5, C-7 and C-4’ appeared at δ 160,4, δ 162,8, δ 161.1 respectively Signals of C-6 from C-8 and signals of C-5 from C-9 are distinguished with the help of 13C-1H coupling data The degree of coupling identifies each carbon and demonstrates that C-9 resonates at higher field from C-6 while C-8 resonates at higher field from C-6.The degree of coupling identifies each carbon and demonstrates that C-9 resonates upfield from C-5 while C-8 resonates up field in comparison to C-6 15 The HMBC correlations HMBC between OH-5 OH (δH13,15) and C C-55 (δC160,4)/C 160,4)/C-66 ((δC98,4)/C-10 98,4)/C (δC104,6), between H-6 (δδH6,24) and C C-55 (δC 160,4)/C 160,4)/C-77 ((δC 162,8)/C-8 162,8 (δδC 104,7)/C )/C-10 (δδC 104,7), ), between H-1’’ 1’’ (δH4,71) and C C (δC 162,8)/C 162,8)/C-8 ((δC 104,7)/C-9(δ 104,7 δC 156,1)) comfirmed the position of glucose at C 8 of A ring The HMBC correlations between H2’, 6’(δδH8,01)) and C-2 C (δ ( C164,2), between H-3’, 3’, 5’ (δ ( H 6,89) and C-1’ C ’ (δC121,1)/C 121,1)/C-4’ (δC161,1) suggested the the structure of B ring and the link between C C-1’ 1’ and C-2 C The structure of C ring was confirmed by HMBC correlation from H-3 (δH6,75) to C-2 (δC164,2)/C-4 164,2)/C (δC182,1)/ CC 10 (δC104,7) Thus, the structure of MB1 was determined and named vitexin HO HO OH OH OH OH 4'' 4'' 6'' 6'' 3'' 3'' 5'' 2'' 5'' O 3' HO 1'' HO 2' 1' O 5' 10 OH 2'' OH HO 4' O HO 5' 6' 10 OH OH 4' 1' O 6' O 3' 2' 1'' O Figure 4.44 Chemical structure and the important HMBC correlations of MB1 4.3 Hydroly Hydrolyzation zation of glycosides from Vietnamess plants by BGL BGL-P 4.4.1 Hydrol Hydrolyzation zation by free BGL BGL-P : 4.4.1.1 1.1 Hydrolyzation of quercetin glycosides: glycoside Quercetin-3-O-beta Quercetinbeta-galactoside galactoside was hydrolysed by different BGL BGL-P P enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected Experimental xperimental design esign was processed and analyzed using Modde 5.0 software Figure 4.74: The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time 16 Figue 4.75: The effect of enzyme concentration and reaction time on the rate of the hydrolysis Hydrolyzation of quercetinquercetin-3-O-β-D D-glucuronide glucuronide After h of enzymatic reaction catalyzed by BGL BGL-P, P, heated at o 60 C,, significant amounts of quercetin (approximately 90%) were obtained OH O OH HO O HO O OH 5h, OH O HiÖu s OH COOH O OH O 0oC OH uÊt: 0% HO O OH su O OH O OH OH OH HO O O HiÖu suÊt: 5% Öu Hi OH O 8h, 60oC C % 60o 5h , Êt: 98 OH HO H O OH O OH OH HO O HO O OH OH O OH O O O H3C HO O HO OH Figure 4.77 Hydrolyzation of quercetin glycosides by BGL-P BGL Hydrolyzation quercetin-3-O-rutinoside quercetin rutinoside (rutin) Normally, the transformation of rutin catalyzed by mixture of enzymes: α-L-rhamnosidase rhamnosidase and β-D glucosidase glucosidase α-L L-Rhamnosidase Rhamnosidase catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and the same time, β-D-glucosidase β glucosidase catalyzes the cleavage of terminal rutinoside groups from rutin to quercetin and rutinose rutinose In this study, β-D D-glucosidase glucosidase (BGL-P) (BGL was used to hydrolysis and the obtained maximal yield of quercetin was 25% % when the enzymatic reaction time was 6h 17 4.4.1.2 Hydrolyzation of other glycosides: Genistin and daidzin were hydrolysed in the condition that the BGL-P enzyme concentration was 0.4U / ml with an incubation time of hours at 60oC The reaction was stopped by adding 10 ml of MeOH Hydrolysis results showed high yield to both genistin and daidzin 98% Hình 4.78 Hydrolyzation of genistin and daidzin Maximum yield of picied hydrolisis was 99% when the enzymatic reaction time was 5h at 60oC Hình 4.79 Thủy phân hợp chất picied Under the same conditions, isorhamnetin-3-O-β-D-glucuronide was hydrolysed with the yield 85% after hour Figue 4.80 Hydrolyzation of isorhamnetin-3-O-β-D-glucuronide BGL-P hydrolysed isoapigenin-6-C-glucoside and apigenin-8-Cglucoside, with similar yield Figue 4.81 Hydrolyzation of isoapigenin-6-C-glucoside and apigenin-8C-glucoside 18 4.4.1.3 Hydrolyzation of anthraquinone glycosides: glycosides The results of hydrolysis of eemodin modin-8-O-β D-glucopyranoside glucopyranoside (19) and physcion-8 physcion 8-O-β-D D-glucopyranoside glucopyranoside (20) showed that that: BGL-P BGL P can hydrolysed anthraquinone glycosides with the same yeild of flavonoid glycosides OH HO O H HO O O O OH OH HO O OH 4h h, 60oC (96% %) OH HO CH3 CH3 O O ( (18) (19) OH H HO HO O O O OH OH OH O OH 4h, 60oC 95% % H3 CO C CH H3 O (20) H3 CO CH3 O (23) Figue 4.82: Hydrolyzation of anthraquinone glycosides 4.4.2 Hydrolyzation by BGL-P BGL immobilization: immobilization Quercetin -3-O-beta beta-galactoside galactoside was hydrolized by BGL-P P immobilization at the appropriate conditions Figue 4.84: Reusable of BGL-P BGL P immobilized onto Ca-alginate Ca alginate and spent coffee grounds Enzyme BGL BGL-P P immobilized onto alginate can be reused in times before lost 50% activity This is a potential results for applied to 19 hydrolysis of glycoside compounds with the higher performance and the lower cost 4.5 Disinfection of microorganisms by electro-chemical method: In microbiological studies, keeping the environment safe, controlling the spread of microorganisms during and after the study is of utmost importance Therefore, after each microbial experiment, it should be carefully disinfected before disposal Bacillus spores are resistant to disinfection methods and they represent a potential threat that requires improved methods to ensure water safety In this study, Bacillus cereus spores were used to investigate the effectiveness of the electrochemical (EC) disinfection process The results of study show that the optimum conditions of the electro-chemical disinfection method is: electric potential 2A, water contain 50mg/L Cl-, pH 6,8 with 0,01M phosphate buffer Applied this results on the disinfection of P citrinum, after 30 100% P.citrinium was killed in direct experiment and after 60 in indirect experiment 15 30 45 60 thời gian (phút) Log N/No -1 -2 trực tiếp -3 Gián tiếp -4 -5 Figure 4.88: Effect of storage time to the disinfection ability of spore of P citrinum by electro-disinfection 4.6 Biological activities of isolated compounds and their aglycone: 4.6.1 Antioxidant activity by DPPH assay 22 compounds were evaluated for their antioxidant activity by DPPH assay: Table 4.14 : Antioxidant activity by DPPH assay of aglycone and glycosides No Name of compound EC50 20 (mM) (µg/ml) Quercetin 0,027 8,2 quercetin-3-O-β-galactoside 0,055 25,6 quercetin-3-O-rutinoside 0,143 87,4 quercetin-3-O-β-D-glucuronide 0,047 22,5 Luteolin 0,015 4,3 Kaemferol 0,060 17,2 Isorhamnetin 0,032 10,1 isorhamnetin-3-O-β-D-glucuronide 0,133 65,5 Apigenin 0,047 12, 10 apigenin-6-C-glucoside 0,055 23,8 11 apigenin-8-C-glucoside 0,055 23,8 12 Genistein >0,948 >256 13 Genistein 7-O-beta-D-glucoside 0,256 110,7 14 Daidzein 0,532 135,3 15 Daidzein7-O-beta-D-glucoside >0,615 >256 16 Resveratrol 0,036 8,2 17 Resveratrol –beta-mono-D-glucoside 0,043 16,8 18 Emodin 0,205 55,4 19 emodin-8-O-β-D-glucopyranoside >0,592 >256 20 Physcion 0, 731 207,8 21 physcion-8-O-β-D-glucopyranoside >0,573 >256 22 Catechin 0,038 11,0 As results, almost compounds had antioxidant activity and the aglycone usually had higher activity than their glycosides such as EC 50 of quercetin and quercetin-3-O-rutinoside were 0,027 μM and 0,143 μM, respectively or isorhamnetin and isorhamnetin-3-O-β-D-glucuronide were 0,032 μM and 0,133 μM, respectively DPPH scavenging activity of compounds is given in descending order as follows: quercetin > quercetin-3-O-β-D-glucuronide > quercetin-3-O-β-galactoside > rutin; hay resveratrol > resveratrol –beta-mono-D-glucoside; isorhamnetin > isorhamnetin-3-O-β-D-glucuronide So the hydrolysis of glycosides onto aglycone help to create 21 compounds with higher antioxidant capacity to enhance application 4.6.2 α-Glucosidase inhibition: To assess the applicability of the treatment of diabetes, compounds were evaluated for their α-Glucosidase inhibition activity Table 4.15 : Results of α-Glucosidase inhibition activity IC50 No Name of compound (µg/ml) (mM) quercetin 6,3 0,021 quercetin-3-O-β-galactoside 44,1 0,095 quercetin-3-O-rutinoside 131,9 0,216 quercetin-3-O-β-D-glucuronide 68,9 0,144 apigenin 53,46 0,198 apigenin-6-C-glucoside 117,2 0,271 apigenin-8-C-glucoside 107,2 0,248 genistein 13,5 0,050 genistein 7-O-beta-D-glucoside >256 >0,592 10 daidzein 26,9 0,106 11 daidzein7-O-beta-D-glucoside >256 >0,615 12 acarbose 192,1 0,297 Flavonoids group isolated from vietnamess plants had great potential for use in treatment of diabetes, many compounds are able to inhibit enzyme α- glucosidase higher than acarbose In this test, quercetin, apigenin, genistein and daidzein had an IC50 value at lower concentrations than their glycosides 4.6.3 An angiotensin converting enzyme inhibitor Angiotensin-converting enzyme (ACE) inhibitors is widely used in the treatment of hypertension, chronic kidney disease, and heart failure In addition to efficacy, these agents have the additional advantage of being particularly well tolerated since they produce few idiosyncratic side effects and not have the adverse effects on lipid and glucose metabolism seen with higher doses of diuretics or beta blockers To compare Angiotensin-converting enzyme (ACE) inhibitors of aglycone and their glycosides, we evaluted the bioactivity of them 22 Table 4.16: Results of an angiotensin converting enzyme inhibitor IC50 Name of compound (µg/ml) (mM) Quercetin 23,6 0,078 quercetin-3-O-rutinoside 70,34 0,115 quercetin-3-O-β-D-glucuronide >256 >0,535 0,000326 0,000015 Captopril Therefore, ACE inhibitors of compounds sample were much lower than the control, however the results showed that: effects of quercetin was higher than their glycosides CONCLUSION Isolated and identified of Penicillium citrinum which hight produced β-glucosidase from roots of Clerodendron cyrtophyllum Turcz: - Fermentation and evaluation of kinetic parameters of free and immobilized β-glucosidase from P citrinum - P citrinum cultured on Pd medium at days, 27oC, 200 rpm Free BGL-P showed Michaelis–Menten kinetics for pNPG substrates tested with Km values 0,011 µmol, Vmax 13,91 µmol/min, t o 60o - BGP-L immobilized on Ca-alginate: 50oC, pH: 5,5-6,2, Km= 0,034 µmol, Vmax = 13,09 µmol/min Reused from to times dependent on substances - BGP-L immobilized on spent coffee grounds: 40oC, pH: 6,0, Km= 0,022 µmol, Vmax= 14,45 µmol/min Seventeen flavonoide glycoside and aglycone compounds were isolated: Genistein, daidzein, genistin, daidzin, catechin, hyperoside, quercetin, kaempferol, isorhamnetin-3-O-β-D-glucuronide, quercetin3-O-β-D-glucuronide, vitexin, isovitexin, luteolin, taxifolin, rutin, resveratrol, picied, three anthraquinone glycoside and aglycone 23 compounds were isolated: emodin, emodin-8-O-β-D-glucopyranoside, physcion-8-O-β-D-glucopyranoside The optimum condition of hydrolysis by BGL-P process: reaction at 60oC in 5h, 0,4U/ml BGL-P in citrate buffer pH 6,0 Hydrolyzed ten glycoside compounds in the optimum condition: the yield of reactions from 85 to 98%, except rutin (25%) The optimum conditions of the electro-chemical disinfection method is: electric potential 2A, water contain 50mg/L Cl-, pH 6,8 with 0,01M phosphate buffer The bioactivity of most aglycone were higher than their glycosides - DPPH scavenging activity of compounds is given in descending order as follows: quercetin > quercetin-3-O-β-D-glucuronide > quercetin-3-O-β-galactoside > rutin; hay resveratrol > resveratrol –beta-mono-D-glucoside; isorhamnetin > isorhamnetin-3-O-βD-glucuronide - α-Glucosidase inhibition: quercetin, apigenin, genistein and daidzein had an IC50 value at lower concentrations than their glycosides - ACE inhibitors of compounds sample were much lower than the control, however the results showed that: effects of quercetin was higher than their glycosides RECOMMENDATIONS BGL-P isolated from P citrinum had high activity and it can hydrolysis many kind of substances Therefore, there should be more research is needed to expand the scope of the application Further research is needed on the applicability of aglycone compounds in practice 24 NEW FINDINGS OF THE THESIS Isolated and identified of Penicillium citrinum which hight produced β-glucosidase Purificated enzyme beta-glucosidase from Penicilium citrinum to hydrolysis glycosides with high yield This is an interdisciplinary study with the combination of chemistry, biology, electrochemistry solving the whole problem from isolation to application and finally treatment without affecting the environment ... Hydrolyzation by BGL-P BGL immobilization: immobilization Quercetin -3-O-beta beta-galactoside galactoside was hydrolized by BGL-P P immobilization at the appropriate conditions Figue 4.84: Reusable of BGL-P... aglycon có hoạt tính sinh học cao Tạp chí Hóa học, 2016 , 54 (6e2): 84-89 Lê Thị Tú Anh, B? ? Thị Châm, Nguyễn Thu Hà, Nguyễn Thanh Trà, Nguyên Văn Tuyến, Nghiên cứu thủy phân astilbin rễ Thổ phục... Therefore, we firstly isolated the fungal ? ?glucosidase 4.1 Isolation and properties of fungal beta beta-glucosidases glucosidases 4.1.1 Isolation of fungal beta beta-glucosidases: glucosidases: Fig