STUDY OF HYDROLYSIS OF NATURAL GLYCOSIDES β- …gust.edu.vn/media/26/uftai-ve-tai-day26666.pdf ·...

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

1. 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

2. Lê Thị Tú Anh, Bá Thị Châm, Nguyễn Thu Hà, Nguyễn Thanh Trà, Nguyên Văn Tuyến, Nghiên cứu thủy phân astilbin trong rễ Thổ phục linh (Similax glabra) bằng vi sinh vật, Tạp chí Hóa học, 2016, 54 (6e2): 223-227

3. 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 và hoạt tính ức chế enzym khử HMG-Coenzym A của vỏ đậu xanh (Vigna radiata), Tạp chí hóa học 2017, 55 (4e23), 21-26.

4. 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 và hoạt tính ức chế enzym khử HMG-Coenzym A của lá Sen hồng (Nelumbo nucifera), Tạp chí hóa học 2017, 55 (4e23), 261-266.

1

INTRODUCTION 1. 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 do 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.

2 The flavonoids and their corresponding metabolites are evaluated for bioavailability. The fungus after fermentation was studied sterilization by advanced oxidation process.

2. 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.

3. The main contents of the thesis: - Identification of microorganisms capable of producing β-glucosidase. - Fermentation, evaluation of kinetic parameters of free and fixed β-glucosidase 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 β-glucosidase 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

3

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

4 (ESI-MS) and high-resolution ESI-MS (HR-ESI-MS), one/two-dimension 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

5

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

EtOH extract extracted by acetone 3 times solvent removal by vacuum evaporation

Acetone extract - Dissolve by EtOAc

Extracted by H2O

EtOAc extract H2O extract

silica gel: EtOAc: H2O (97:3)

and EtOAc:H2O:EtOH (95:3:2)

F1-F2 F3-F4 F7-F10 F5 F6

Sephadex LH-20, EtOH silica gel: EtOAc: MeOH (96:4)

silica gel: EtOAc: MeOH (95:5)

D5.3 (251.2mg)

D6.4 (198.7mg) F1.

1 F1.2

Crystallized CH2Cl

2

D1.1 (12.8mg)

D1.2 (3.4 mg)

Kết tinh CH2Cl

2

3.3.4 Isolation andStyphnolobium japonicum

Characteristic of the compound1H NMR (500 MHz, DMSO6’’’); 3,09J= 7,0 Hz, HH-8); 6,84 (1H, d, (1H, dd,

13C(C-4); 161,2 (C103,9 (C116,2 (C3’’); 70,(CRha-2’’’); 71,3(C6’’’). 3.3.5 Isolation cuspidati

Isolation andStyphnolobium japonicum

Characteristic of the compoundH NMR (500 MHz, DMSO

6’’’); 3,09- 5,00 (proton= 7,0 Hz, HGlc-

8); 6,84 (1H, d, (1H, dd, J=2,0; 8,0 Hz, H

C-NMR (125 MHz, DMSO4); 161,2 (C

103,9 (C-10); 121,1 (C116,2 (C-5’); 121,5(C3’’); 70,3 (CGlc-

2’’’); 71,3(C

Isolation cuspidati

Isolation and purificationStyphnolobium japonicum

Characteristic of the compoundH NMR (500 MHz, DMSO

5,00 (proton-1’’); 6,19 (1H, d,

8); 6,84 (1H, d, J= 8,0 Hz, H=2,0; 8,0 Hz, HNMR (125 MHz, DMSO

4); 161,2 (C-5); 100,1 (C10); 121,1 (C5’); 121,5(C-6’); 101,2 (C

-4’’); 75,8 (C2’’’); 71,3(CRha-3’’’); 71,8 (C

Isolation and purification

purification Styphnolobium japonicum (L.) Schott

Characteristic of the compoundH NMR (500 MHz, DMSO-d6

5,00 (protons CH-OH ); 5,2 (1H, brs, H1’’); 6,19 (1H, d,

= 8,0 Hz, H-=2,0; 8,0 Hz, H-6’); 12,58 (1H, s, OHNMR (125 MHz, DMSO

5); 100,1 (C-6); 164,1 (C10); 121,1 (C-1’); 115,2 (C

6’); 101,2 (C4’’); 75,8 (CGlc-5’’); 66,9 (C

3’’’); 71,8 (C

purification

6

of glycosides fromSchott

Characteristic of the compound: melting point6): =0,99 ppm (3H, d,

OH ); 5,2 (1H, brs, H1’’); 6,19 (1H, d, J= 2,0 Hz, H

-5’); 7,52 (1H, 6’); 12,58 (1H, s, OH

NMR (125 MHz, DMSO-d6): 6); 164,1 (C

1’); 115,2 (C-2’); 144,7 (C6’); 101,2 (CGlc-1’’); 74,1 (C

5’’); 66,9 (C3’’’); 71,8 (CRha-4’’’); 68,2 (C

purification of glycosides from

glycosides from

melting point=0,99 ppm (3H, d,

OH ); 5,2 (1H, brs, H= 2,0 Hz, H-6); 6,38 (1H, d,

5’); 7,52 (1H, d, 6’); 12,58 (1H, s, OH-5).

156,5 (C6); 164,1 (C-7); 93,6 (C

2’); 144,7 (C1’’); 74,1 (C

5’’); 66,9 (CGlc-6’’); 98,7 (C4’’’); 68,2 (C

glycosides from

glycosides from flower of

melting point: 242oC=0,99 ppm (3H, d, J

OH ); 5,2 (1H, brs, HRha-1’’’); 5,34 (1H, d, 6); 6,38 (1H, d, d, J = 2,0 Hz, 5).

156,5 (C-2); 133,3 (C7); 93,6 (C-8); 156,4 (C

2’); 144,7 (C-3’); 148,4 (C1’’); 74,1 (CGlc-2’’); 76,4 (C

6’’); 98,7 (C4’’’); 68,2 (CRha-5’’’); 18,6 (C

glycosides from Rhizoma

flower of

C J= 6,5Hz, H

1’’’); 5,34 (1H, d, 6); 6,38 (1H, d, J= 2,0 Hz,

= 2,0 Hz, H-2’); 7,55

2); 133,3 (C-3); 177,4 8); 156,4 (C

3’); 148,4 (C2’’); 76,4 (C

6’’); 98,7 (CRha-1’’’); 70,5 5’’’); 18,6 (C

Rhizoma P

= 6,5Hz, HRha-1’’’); 5,34 (1H, d,

= 2,0 Hz, 2’); 7,55

3); 177,4 8); 156,4 (C-9);

3’); 148,4 (C-4’); 2’’); 76,4 (CGlc-

1’’’); 70,5 5’’’); 18,6 (CRha-

Polygoni

1’’’); 5,34 (1H, d, = 2,0 Hz, 2’); 7,55

3); 177,4 9);

4’);

1’’’); 70,5

olygoni

7

`

C8.4 (20mg) C8.5 (15mg)

CC extract (15 g)

F1 F2 F7 (2,0g) F6 F5 (2,4g) F4 F3

Silicagel 0,063 ÷ 0,2

- CH2Cl2 : CH3OH

Crystallized

C2.1

(290mg)

- Silicagel CH2Cl2

/CH3OH

7-2 7-3 7-4 7-5 7-1

Crystallized

C7.3 (155mg)

- Silicagel CH2Cl2

: CH3OH

5-1 5-2 5-3

C5.2 (97mg)

5-4

F8

Crystallized

3.4. Hydrolysis glycoside compounds:

Percentage of hydrolysis [140]: Percentage of hydrolysis (%) =

12 100

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

8 3.5.3 Applied the Electrochemical Disinfection on P. citrinum 3.6 Bioactivity of glycosides and the products of hydrolysis 3.6.1 Antioxidant activity by DPPH assay [117-119]

Compound was determined by modified methods of Liyana-Pathirama et al. (2005) and Thirugnanasampandan et al. (2008). Two milliliter of different concentrations (0.5 to 128 µg/ml) of each compound in methanol was added to 0.2 ml of DPPH radical solution in methanol (final concentration of DPPH was 1.0 mM). The mixture was shaken vigorously and allowed standing for 60 min in the dark. The absorbance of the resulting solutions, the blank and the control were measured at 517 nm using Bioteck spectrophotometer. Standard antioxidant compound resveratrol was used as positive control. DPPH scavenging activity of the compound was calculated using the following formula:

DPPH scavenging activity (%) = OD blank-OD sampleODblank

x100 Where OD sample and OD blank were the optical density of the extract

at different concentrations and the blank sample. The effective concentration providing 50% inhibition (EC50) was calculated from the graph of percentage inhibition against each extract concentrations. 3.6.2 α-Glucosidase inhibition assay:

The enzyme solution contained 20 μl α-glucosidase (0.5 unit/ml) and 120 μl 0.1 M phosphate buffer (pH 6.9). p-Nitrophenylα-D-glucopyranoside (5 mM) in the same buffer (pH 6.9) was used as a substrate solution.

10 μl of test samples, dissolved in DMSO at various concentrations, were mixed with enzyme solution in microplate wells and incubated for 5 min at 37°C. 10 μl of substrate solution were added and incubated for an additional 30 min. The reaction was terminated by adding 100 μl of 0.2 M sodium carbonate solution. Absorbance of the wells was measured with a Bioteck spectrophotometer at 405 nm, while the reaction system without compound was used as control. The system without α-glucosidase was used as blank, and acarbose was used as positive control

3.6.3 An angiotensin converting enzyme inhibitor [124-126]: Reaction at 37o C, pH 7,0, in 30 min. Absorbance of the wells was

measured with a Bioteck spectrophotometer at 410 nm (A). Percentage inhibitor of ACE was calculated using the following

formula:

Wheredifferent concentrations and the blank sample.

Captopril was used as positive

The aim of the research is to study the hydrolysis of glycoside compounds from plants. Therefore, we firstly isolated the fungalglucosidase.4.1 Isolation and properties of fungal beta

4.1.1

Fig 4.1

We isolated 5 fungi (C1, C2, C3, C4, C5) from Clerodendron cyrtophyllum beta-glucosidase enzyme. The fungal isolatewhen tested with βshowed the presence of βafter six days culture. The fulgal isolate C5 experiment.4.1.2. Identification of

Colonies of C5 are fast growing in shades of greenconsisting of a dense felt of conidiophores. Microscopically, phialides like a brush

DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have commonly been used to identifyflanking ITS1/

% inhibitor of ACE Where, A

different concentrations and the blank sample. Captopril was used as positive

CHAPTERThe aim of the research is to study the hydrolysis of glycoside

compounds from plants. Therefore, we firstly isolated the fungalglucosidase.

Isolation and properties of fungal beta4.1.1 Isolation of fungal beta

Fig 4.1: Colonies of fungal were isolated from root of

We isolated 5 fungi (C1, C2, C3, C4, C5) from Clerodendron cyrtophyllum

glucosidase enzyme. The fungal isolatewhen tested with βshowed the presence of βafter six days culture. The fulgal isolate C5 experiment.

Identification ofColonies of C5 are fast growing in shades of green

consisting of a dense felt of conidiophores. Microscopically, phialides like a brush-like appearance (a penicillus).

DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have ommonly been used to identify

flanking ITS1/

% inhibitor of ACE A sample and A


CHAPTERThe aim of the research is to study the hydrolysis of glycoside

compounds from plants. Therefore, we firstly isolated the fungal

Isolation and properties of fungal betaIsolation of fungal beta

Colonies of fungal were isolated from root of

We isolated 5 fungi (C1, C2, C3, C4, C5) from Clerodendron cyrtophyllum

glucosidase enzyme. The fungal isolatewhen tested with β-pNG method. Analysis of the culture filtrate of C5 showed the presence of βafter six days culture. The fulgal isolate C5

Identification of fungal betaColonies of C5 are fast growing in shades of green

consisting of a dense felt of conidiophores. Microscopically, phialides like appearance (a penicillus).


flanking ITS1/ITS4 re

% inhibitor of ACE and A blank


CHAPTER 4. RESULTS AND DISCUSSIONThe aim of the research is to study the hydrolysis of glycoside


Isolation and properties of fungal betaIsolation of fungal beta

Colonies of fungal were isolated from root of cyrtophyllum

We isolated 5 fungi (C1, C2, C3, C4, C5) from Clerodendron cyrtophyllum Turcz

glucosidase enzyme. The fungal isolatepNG method. Analysis of the culture filtrate of C5

showed the presence of β-glucosidaseafter six days culture. The fulgal isolate C5

fungal betaColonies of C5 are fast growing in shades of green



4 regions for fungal identification

9

= (Acontrol blank were the optical density of the extract at

different concentrations and the blank sample. Captopril was used as positive control

RESULTS AND DISCUSSIONThe aim of the research is to study the hydrolysis of glycoside


Isolation and properties of fungal betaIsolation of fungal beta-glucosidases:

Colonies of fungal were isolated from root of cyrtophyllum

We isolated 5 fungi (C1, C2, C3, C4, C5) from Turcz and screened them for prodution

glucosidase enzyme. The fungal isolatepNG method. Analysis of the culture filtrate of C5

glucosidase with theafter six days culture. The fulgal isolate C5

fungal beta-glucosidases:Colonies of C5 are fast growing in shades of green


DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have ommonly been used to identify the fungal.

gions for fungal identification

control – Asamplewere the optical density of the extract at

different concentrations and the blank sample. control



Isolation and properties of fungal beta-glucosidasesglucosidases:

Colonies of fungal were isolated from root of cyrtophyllum Turcz

We isolated 5 fungi (C1, C2, C3, C4, C5) from and screened them for prodution

glucosidase enzyme. The fungal isolate C5 gave maximum enzyme pNG method. Analysis of the culture filtrate of C5

with the activity was after six days culture. The fulgal isolate C5

glucosidases:Colonies of C5 are fast growing in shades of green


DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have

the fungal. We used 5.8S gene and gions for fungal identification

sample)/(Acontrolwere the optical density of the extract at



glucosidasesglucosidases:

Colonies of fungal were isolated from root of Clerodendron

We isolated 5 fungi (C1, C2, C3, C4, C5) from and screened them for prodution

C5 gave maximum enzyme pNG method. Analysis of the culture filtrate of C5

activity was after six days culture. The fulgal isolate C5 was identified

glucosidases: Colonies of C5 are fast growing in shades of green

consisting of a dense felt of conidiophores. Microscopically, phialides


We used 5.8S gene and gions for fungal identification

control– Ablankwere the optical density of the extract at

RESULTS AND DISCUSSION The aim of the research is to study the hydrolysis of glycoside


glucosidases

Clerodendron

We isolated 5 fungi (C1, C2, C3, C4, C5) from roots of and screened them for prodution


activity was 33,628U/ml was identified

Colonies of C5 are fast growing in shades of green consisting of a dense felt of conidiophores. Microscopically, phialides


We used 5.8S gene and gions for fungal identification. Constructing

blank) were the optical density of the extract at

The aim of the research is to study the hydrolysis of glycoside compounds from plants. Therefore, we firstly isolated the fungal β-

Clerodendron

roots of and screened them for prodution of


33,628U/ml in next

mostly consisting of a dense felt of conidiophores. Microscopically, phialides


We used 5.8S gene and . Constructing

The aim of the research is to study the hydrolysis of glycoside

roots of of


33,628U/ml in next

mostly consisting of a dense felt of conidiophores. Microscopically, phialides


We used 5.8S gene and . Constructing

phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statisticconsensus is applied to the constructed tree so as to read maximum sequence replicationsa clear picture for identifying fungal isolate 100 BLAST hits belonrecommending our isolate as a member of this group.

4.2 Purification and properties ofPartial purification of β

precipitation, followed by sephadexfrom Penicillium citrinumdetermined using 4substrate.4.2.1 Properties of BGL

Optimum pH and temperature for enzyme assayβ-glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The

results showed that the BGL activity increased from which decrease in activity was 60activity. Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also prevent 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 was optimized at pH

Kinetic parameters for

phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statisticconsensus is applied to the constructed tree so as to read maximum sequence replicationsa clear picture for identifying fungal isolate 100 BLAST hits belonrecommending our isolate as a member of this group.

Purification and properties ofPartial purification of β

precipitation, followed by sephadexPenicillium citrinum

determined using 4substrate. BGL-

Properties of BGLOptimum pH and temperature for enzyme assay

glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The results showed that the BGL activity increased from which decrease in activity was 60activity. Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also

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

P was optimized at pH Kinetic parameters for

phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statisticconsensus is applied to the constructed tree so as to read maximum sequence replications. Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate 100 BLAST hits belonrecommending our isolate as a member of this group.

Fig.Purification and properties of

Partial purification of βprecipitation, followed by sephadex

Penicillium citrinumdetermined using 4-Nitrophenyl β

-P was used at free enzyme anProperties of BGL

Optimum pH and temperature for enzyme assayglucosidase activity was observed at 40, 50, 60, 70 and 80°C. The

results showed that the BGL activity increased from which decrease in activity was observed.activity was 60oC. 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

event contamination to allow the reaction to proceed at higher range of


P was optimized at pH Kinetic parameters for

phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statisticconsensus is applied to the constructed tree so as to read maximum

Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate 100 BLAST hits belonged to recommending our isolate as a member of this group.

Fig. 4.4: ColoniesPurification and properties of

Partial purification of β-BGL was carried out by precipitation, followed by sephadex

Penicillium citrinum partially purified enzyme (BGLNitrophenyl β

P was used at free enzyme anProperties of BGL-P:


results showed that the BGL activity increased from activity was observed.

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



P was optimized at pH 6.0.Kinetic parameters for BGL

10


Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate

ged to Penicillium citrinumrecommending our isolate as a member of this group.

Colonies, phialidesPurification and properties of β-

BGL was carried out by precipitation, followed by sephadex, lyophilized.

partially purified enzyme (BGLNitrophenyl β-D

P was used at free enzyme an


results showed that the BGL activity increased from activity was observed.




6.0. BGL-P


Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate C5.

Penicillium citrinumrecommending our isolate as a member of this group.

phialides-glucosidase

BGL was carried out by , lyophilized.

partially purified enzyme (BGLD-glucopyranoside (5 mM) as

P was used at free enzyme and immobilized enzyme.


results showed that the BGL activity increased from The best temperature for BGL




phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statistical errors. Bootstrap consensus is applied to the constructed tree so as to read maximum

Neighbour joining tree with bootstrapping gave us It is because more than

Penicillium citrinum, thus strongly recommending our isolate as a member of this group.

phialides of C5 glucosidase from culture

BGL was carried out by ammonium sulphate , lyophilized. Activity of the BGL

partially purified enzyme (BGLglucopyranoside (5 mM) as

d immobilized enzyme.

Optimum pH and temperature for enzyme assay glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The

results showed that the BGL activity increased from 5The best temperature for BGL




phylogenetic tree is crucial in molecular identification, since BLAST al errors. Bootstrap

consensus is applied to the constructed tree so as to read maximum Neighbour joining tree with bootstrapping gave us

It is because more than , thus strongly

from culture ammonium sulphate Activity of the BGL

partially purified enzyme (BGL-glucopyranoside (5 mM) as


glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The 50 to 70°C after

The best temperature for BGLTemperature 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






from culture ammonium sulphate Activity of the BGL

-P) was glucopyranoside (5 mM) as


glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The 0°C after

The best temperature for BGL-P Temperature is an important factor for enzymatic







ammonium sulphate Activity of the BGL

P) was glucopyranoside (5 mM) as

glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The 0°C after

P Temperature is an important factor for enzymatic




11

Different concentrations of pNPG (0-25 mM) were used to estimate the kinetic parameters, Km and Vmax using double reciprocal Lineweaver-Burk plot. The results were Km = 0,01µmol và 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. BGL-P entrapped in large calcium alginate beads was used successfully for 7 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 6 using 4-nitrophenyl β-D-glucuronide as an indicator. Operational reusability was confirmed for 2 batch reactions.

Table 4.3 Kinetic parameters for free BGL-P and immobilized Forms Temperature

(oC) pH Vmax

(µmol/min) Km

(µmol) R2 *

Free forms 60 6.0 13,91 0,011 0,9994 Immobilized in alginat

50 6.0 13,09 0,034 0,9978

Immobilized onto spent coffee grounds

40 6.0 14,45 0,022 0,9992

* 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 5 plant species: No Symbol Structure Name of compound 1 D1.1

Genistein

12

2 D1.2

Daidzein

3 D5.3 Genistein 7-O-beta-D-glucoside

4 D6.4

Daidzein7-O-beta-D-glucoside

5 S3.1 MB5

catechin

6 S5.2

quercetin-3-O-β-galactoside

7 S7.3

quercetin

8 S8.4

kaemferol

9 S8.5

isorhamnetin-3-O-β-D-glucuronide

13 10 S8.6

quercetin-3-O-β-D-glucuronide

11 MB3

O

OH

HO

OH O

OHO

OH

HOOH

2

3

45

106

7 89

1'

2'

3'

4'

5'

6'

1''

2''

3''

4''

5''

6''

apigenin-6-C-glucoside (vitexin)

12 MB4

apigenin-6-C-glucoside (isovitexin)

13 MB1

luteolin

14 MB2

taxifolin

15 HH1

quercetin-3-O-rutinoside

(rutin)

16 C2.1

resveratrol

17 C5.2

Resveratrol 3 –beta-mono-D-glucoside

(picied)

14 18 C7.3

emodin

19 C8.4

emodin-8-O-β-D-glucopyranoside

20 C8.5

physcion-8-O-β-D-glucopyranoside

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 175-178, when the carbonyl is not hydrogen bonded. But in the presence of H-bonding to 5-hydroxy group, it moves downfield to about δ 182. When 3-hydroxy 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.

The HMBC correlations HMBC between

(δC160,4)/C(δC 160,4)/C(δH4,71) and Cposition of glucose at 2’, 6’(δ(δC121,1)/Clink between CHMBC correlation from10 (δC104,7)

Thus, the structure of

4.3 Hydroly4.4.1 Hydrol4.4.1.1

Quercetinenzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected. design was processed and analyzed using

Figure

The HMBC correlations HMBC between 160,4)/C-6 (160,4)/C-7 (4,71) and C-

position of glucose at δH8,01) and C

121,1)/C-4’ link between CHMBC correlation from

104,7) . Thus, the structure of

and the important HMBC correlations of Hydrolyzation of glycosides from Vietnamess plants by BGLHydrolyzation by free BGL

1.1 Hydrolyzation of Quercetin-

enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected.

esign was processed and analyzed using

Figure 4.74: The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.

The HMBC correlations HMBC between 6 (δC98,4)/C7 (δC 162,8

-7 (δC 162,8)/Cposition of glucose at C-

) and C-2 ( (δC161,1)

link between C-1’ and CHMBC correlation from

Thus, the structure of

HO

HO

OH

HO

6

7

2''

3''

Figureand the important HMBC correlations of zation of glycosides from Vietnamess plants by BGLzation by free BGL

Hydrolyzation of -3-O-beta



The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.

The HMBC correlations HMBC between 98,4)/C-10 (δC162,8)/C-8 (δ

162,8)/C-8 (-8 of A ring.

2 (δC164,2), 161,1) suggested the

1’ and C-2. The structure of C ring was confirmed by HMBC correlation from H-3 (δH

Thus, the structure of MB1

O

OH O

O

OH

HOOH

2

3

45

10

89

1'

1''

2''

4''

5''

6''

Figure 4.44. and the important HMBC correlations of zation of glycosides from Vietnamess plants by BGLzation by free BGL

Hydrolyzation of quercetinbeta-galactoside was hydrolysed by different BGL




15

The HMBC correlations HMBC between C104,6), between δC 104,7)/C8 (δC 104,7

8 of A ring. The HMBC correlations between 164,2), between

suggested the The structure of C ring was confirmed by

H6,75) to

MB1 was determined and named

OH2'

3'

4'

5'

6'

HO

4.44. Chemical structure and the important HMBC correlations of zation of glycosides from Vietnamess plants by BGLzation by free BGL-P :

quercetin glycosidegalactoside was hydrolysed by different BGL




The HMBC correlations HMBC between OH104,6), between

)/C-10 (δ104,7)/C-9(δThe HMBC correlations between

between H-3’, 5’ (suggested the the structure of B ring and the

The structure of C ring was confirmed by C-2 (δC164,2)/C

was determined and named

OHO

OH O

OHO

OH

HOOH

45

106

7

8

9

1''

2''

3''

4''

5''

6''

Chemical structure and the important HMBC correlations of zation of glycosides from Vietnamess plants by BGL

glycosides: galactoside was hydrolysed by different BGL


esign was processed and analyzed using Modde 5.0 software.


OH-5 (δH104,6), between H-6 (δ

δC 104,7), betweenδC 156,1) comfirmed the

The HMBC correlations between 3’, 5’ (δH

the structure of B ring and the The structure of C ring was confirmed by

164,2)/C

was determined and named

O

O

2

3

1'

2'

3'

4'

5'

6'

Chemical structure and the important HMBC correlations of MB1zation of glycosides from Vietnamess plants by BGL

galactoside was hydrolysed by different BGL


Modde 5.0 software.


H13,15) and CδH6,24) and C), between) comfirmed the

The HMBC correlations between H 6,89) and C


164,2)/C-4 (δC182,1)/ C

was determined and named vitexin.

OH4'

MB1 zation of glycosides from Vietnamess plants by BGL

galactoside was hydrolysed by different BGLenzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected. Experimental

Modde 5.0 software.


13,15) and C-5 6,24) and C-5

), between H-1’’ ) comfirmed the

The HMBC correlations between H-6,89) and C-1’


182,1)/ C-

vitexin.

zation of glycosides from Vietnamess plants by BGL-P

galactoside was hydrolysed by different BGL-P enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h,

xperimental

The regression plot represents the optimum region of the

5 5

1’’ ) comfirmed the

’ the structure of B ring and the

The structure of C ring was confirmed by

P enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h,

xperimental

Figue 4.75:

Hydrolyzation of After 5 h of enzymatic reaction catalyzed by BGL

60oC, significant amounts of quercetin (approximately 90%) were obtained.

HydrolyzationNormally, the transformation of rutin catalyzed by mixture of 2

enzymes: catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and catalyzes the cleavage of terminal rutinoside groups from rutin to quercetin and rutinoseto hydrolysis the enzymatic reaction time was 6h.

4.75: The

Hydrolyzation of After 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were

obtained.

H

Figure 4.77 Hydrolyzation quercetin

Normally, the transformation of rutin catalyzed by mixture of 2 enzymes: α-L-rhamnosidase and catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and catalyzes the cleavage of terminal rutinoside groups from rutin to quercetin and rutinoseto hydrolysis and the enzymatic reaction time was 6h.

The effect of

Hydrolyzation of quercetinAfter 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were

OHO

OH O

OHO

OH O

4.77 Hydrolyzation of quercetin

Normally, the transformation of rutin catalyzed by mixture of 2 rhamnosidase and

catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and catalyzes the cleavage of terminal rutinoside groups from rutin to quercetin and rutinose. In this study,

and the obtained maximal yield of quercetin was the enzymatic reaction time was 6h.

of enzyme concentrationrate of the

quercetin-After 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were

O

O

O

OH

O

O

O

O

OH

O

HO

HO

Hydrolyzation of quercetin-3-O-rutinoside

Normally, the transformation of rutin catalyzed by mixture of 2 rhamnosidase and

catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and catalyzes the cleavage of terminal rutinoside groups from rutin to

. In this study, the obtained maximal yield of quercetin was

the enzymatic reaction time was 6h.

16

enzyme concentrationrate of the hydrolysis

-3-O-β-DAfter 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were

OH

OHCOOH

OH

OH

OHOH

OH

5

5h, 60

HiÖu su

HiÖu s

Hydrolyzation of quercetinrutinoside (rutin)

Normally, the transformation of rutin catalyzed by mixture of 2 rhamnosidase and β-D-

catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and the same time, catalyzes the cleavage of terminal rutinoside groups from rutin to

. In this study, β-Dthe obtained maximal yield of quercetin was

the enzymatic reaction time was 6h.

enzyme concentration and reaction hydrolysis

D-glucuronideAfter 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were

HO

OH

HO

O

5h, 60oC

60oC

suÊt: 90%

u suÊt: 98%

quercetin glycosides by (rutin)

Normally, the transformation of rutin catalyzed by mixture of 2 -glucosidase

catalyzes the cleavage of terminal rhamnoside groups from rutin to the same time,

catalyzes the cleavage of terminal rutinoside groups from rutin to D-glucosidase

the obtained maximal yield of quercetin was

and reaction

glucuronide After 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were

O

O

OH

O

O

O

OH

OH

O

O

HO

H3HO

8h,6

0oC

HiÖ

usu

Êt:2

5%

glycosides by

Normally, the transformation of rutin catalyzed by mixture of 2 glucosidase. α-L

catalyzes the cleavage of terminal rhamnoside groups from rutin to the same time, β

catalyzes the cleavage of terminal rutinoside groups from rutin to glucosidase (BGL

the obtained maximal yield of quercetin was

and reaction time on the

After 5 h of enzymatic reaction catalyzed by BGL-P, heated at , significant amounts of quercetin (approximately 90%) were

OH

O OH

OH

OH

OH

O

O3CO

HO OH glycosides by BGL

Normally, the transformation of rutin catalyzed by mixture of 2 L-Rhamnosidase

catalyzes the cleavage of terminal rhamnoside groups from rutin to β-D-glucosidase

catalyzes the cleavage of terminal rutinoside groups from rutin to (BGL-P) was used

the obtained maximal yield of quercetin was 25% when

time on the

P, heated at , significant amounts of quercetin (approximately 90%) were

BGL-P

Normally, the transformation of rutin catalyzed by mixture of 2 Rhamnosidase

catalyzes the cleavage of terminal rhamnoside groups from rutin to glucosidase

catalyzes the cleavage of terminal rutinoside groups from rutin to was used % when

P, heated at , significant amounts of quercetin (approximately 90%) were

Normally, the transformation of rutin catalyzed by mixture of 2 Rhamnosidase

catalyzes the cleavage of terminal rhamnoside groups from rutin to glucosidase

catalyzes the cleavage of terminal rutinoside groups from rutin to was used % when

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 5 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 5 hour.

Figue 4.80 Hydrolyzation of isorhamnetin-3-O-β-D-glucuronide BGL-P hydrolysed isoapigenin-6-C-glucoside and apigenin-8-C-

glucoside, with similar yield.

Figue 4.81 Hydrolyzation of isoapigenin-6-C-glucoside and apigenin-8-

C-glucoside

4.4.1.3

The results of hydrolysis of eand physcionhydrolysed glycosides.

4.4.2 Hydrolyzation by Quercetin

immobilization at the appropriate conditions.

Figue

Enzyme BGLtimes before lost 50% activity. This is a potential

4.4.1.3 Hydrolyzation ofThe results of hydrolysis of ephyscion-8

hydrolysed anthraquinone glycosides with the same yeild of flavonoid glycosides.

HHO

H3C

HOHO

OH

Figue Hydrolyzation by Quercetin


Figue 4.84:

Enzyme BGLtimes before lost 50% activity. This is a potential

Hydrolyzation ofThe results of hydrolysis of e

8-O-β-Danthraquinone glycosides with the same yeild of flavonoid

HO

OHOO

OH

OH

O

CO

O

O

O

H

OH

(20)

Figue 4.82: Hydrolyzation by BGLQuercetin -3-O-beta


4.84: Reusable of

Enzyme BGL-P immobilized onto alginate can be reused in 5 times before lost 50% activity. This is a potential

Hydrolyzation of anthraquinone glycosidesThe results of hydrolysis of e

D-glucopyranoside (20) anthraquinone glycosides with the same yeild of flavonoid

O OHO

O

(19)

OH

CH

O

O

Hydrolyzation of BGL-P immobilizationbeta-galactoside was hydrolized by


Reusable of BGLspent

P immobilized onto alginate can be reused in 5 times before lost 50% activity. This is a potential

18

anthraquinone glycosidesThe results of hydrolysis of emodin

glucopyranoside (20) anthraquinone glycosides with the same yeild of flavonoid

OH

CH3

4h(96%

H3

95%

Hydrolyzation of immobilization

galactoside was hydrolized by immobilization at the appropriate conditions.

BGL-P immobilized spent coffee grounds


anthraquinone glycosidesmodin-8-O-β-

glucopyranoside (20) showed thatanthraquinone glycosides with the same yeild of flavonoid

HO

h, 60oC%)

4h, 60oC%

Hydrolyzation of anthraquinone glycosidesimmobilization:

galactoside was hydrolized by immobilization at the appropriate conditions.

P immobilized coffee grounds


anthraquinone glycosides: -D-glucopyranoside (19)

showed thatanthraquinone glycosides with the same yeild of flavonoid

OH O

O(

OH

H3CO

anthraquinone glycosides

galactoside was hydrolized by

P immobilized onto Ca

P immobilized onto alginate can be reused in 5 times before lost 50% activity. This is a potential results for applied to

glucopyranoside (19) showed that: BGL

anthraquinone glycosides with the same yeild of flavonoid

OH

CH3

(18)

OHO

O(23)


galactoside was hydrolized by

Ca-alginate and

P immobilized onto alginate can be reused in 5 results for applied to

glucopyranoside (19) BGL-P can


CH3


galactoside was hydrolized by BGL-P

alginate and


glucopyranoside (19) P can


P


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 min 100% P.citrinium was killed in direct experiment and after 60 min in indirect experiment.

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

-5

-4

-3

-2

-1

00 15 30 45 60

Log

N/N

o

thời gian (phút)

trực tiếp

Gián tiếp

20

(mM) (µg/ml) 1 Quercetin 0,027 8,2 2 quercetin-3-O-β-galactoside 0,055 25,6 3 quercetin-3-O-rutinoside 0,143 87,4 4 quercetin-3-O-β-D-glucuronide 0,047 22,5 5 Luteolin 0,015 4,3 6 Kaemferol 0,060 17,2 7 Isorhamnetin 0,032 10,1 8 isorhamnetin-3-O-β-D-glucuronide 0,133 65,5 9 Apigenin 0,047 12, 6 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 3 –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 EC50 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 3 –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

No Name of compound IC50

(µg/ml) (mM) 1 quercetin 6,3 0,021 2 quercetin-3-O-β-galactoside 44,1 0,095 3 quercetin-3-O-rutinoside 131,9 0,216 4 quercetin-3-O-β-D-glucuronide 68,9 0,144 5 apigenin 53,46 0,198 6 apigenin-6-C-glucoside 117,2 0,271 7 apigenin-8-C-glucoside 107,2 0,248 8 genistein 13,5 0,050 9 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 do 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

Name of compound IC50

(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

Captopril 0,000326 0,000015

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

1. 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 6 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, to 60o

- BGP-L immobilized on Ca-alginate: 50oC, pH: 5,5-6,2, Km= 0,034 µmol, Vmax = 13,09 µmol/min. Reused from 5 to 7 times dependent on substances

- BGP-L immobilized on spent coffee grounds: 40oC, pH: 6,0, Km= 0,022 µmol, Vmax= 14,45 µmol/min.

2. Seventeen flavonoide glycoside and aglycone compounds were isolated: Genistein, daidzein, genistin, daidzin, catechin, hyperoside, quercetin, kaempferol, isorhamnetin-3-O-β-D-glucuronide, quercetin-3-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

3. 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.

4. Hydrolyzed ten glycoside compounds in the optimum condition: the yield of reactions from 85 to 98%, except rutin (25%).

5. 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.

6. 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 3 –beta-mono-D-glucoside; isorhamnetin > isorhamnetin-3-O-β-D-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

1. 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 2. Further research is needed on the applicability of aglycone compounds in practice.

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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.

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