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
1.
2.
3.

4.

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 trong rễ Thổ phục linh (Similax
glabra) bằng 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 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.
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 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


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/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


5

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: H O (97:3)
2
and EtOAc:H O:EtOH (95:3:2)
2

F3-F4

F1-F2

F5

silica gel: EtOAc: MeOH
(96:4)

Sephadex LH-20, EtOH

Crystallized CH Cl
2

D1.1
(12.8mg)

D5.3
(251.2mg)

F1.
2

F1.
1

2

F6

F7-F10

silica gel: EtOAc: MeOH
(95:5)

D6.4

(198.7mg)

Kết tinh CH Cl
2

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


6

3.3.4 Isolation and purification of glycosides from flower of
Styphnolobium japonicum (L.) Schott
Characteristic of the compound:
compound melting point:
point 242oC
1
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


7
`

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]: 1
2 100
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

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 LiyanaPathirama 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:
-OD sample
x100
DPPH scavenging activity (%) = OD blank
ODblank
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-Dglucopyranoside (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 αglucosidase 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:


9

% inhibitor of ACE = (Acontrol – Asample)/(Acontrol– Ablank)
Where
Where, A sample and A blank were the optical density of the extract at
different concentrations and the blank sample.
Captopril was used as positive control
CHAPTER 4. RESULTS AND DISCUSSION
The aim of the research is to study the hydrolysis of glycoside
compounds from plants. Therefore, we firstly isolated the fungal βglucosidase.
4.1 Isolation and properties of fungal beta
beta-glucosidases
glucosidases
4.1.1 Isolation of fungal beta
beta-glucosidases:
glucosidases:

Fig 4.1:
4.1 Colonies of fungal were isolated from root of Clerodendron

cyrtophyllum Turcz
We isolated 5 fungi (C1, C2, C3, C4, C5) from roots of
Clerodendron cyrtophyllum Turcz and screened them for prodution of
beta-glucosidase
glucosidase enzyme. The fungal isolate C5 gave maximum enzyme
when tested with β-pNG
β pNG method. Analysis of the culture filtrate of C5
showed the presence of β-glucosidase
β glucosidase with the activity was 33,628U/ml
after six days culture. The fulgal isolate C5 was identified in next
experiment.
4.1.2. Identification of fungal beta
beta-glucosidases:
glucosidases:
Colonies of C5 are fast growing in shades of green mostly
consisting of a dense felt of conidiophores. Microscopically, phialides
like a brush
brush-like
like appearance (a penicillus).
DNA sequence analysis methods are objective, reproducible and
rapid means of identification, and thus gaining importance and have
commonly
ommonly been used to identify the fungal. We used 5.8S gene and
flanking ITS1/ITS4
ITS1/
4 regions
regions for fungal identification.
identification. Constructing

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
4 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
5 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 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. BGLP 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 4nitrophenyl 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 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 5 plant species:
No Symbol
Structure
Name of compound
D1.1
1
Genistein


12

2

3

D1.2

D5.3

Daidzein

Genistein 7-O-beta-Dglucoside

4

D6.4

Daidzein7-O-beta-Dglucoside

5

S3.1
MB5

catechin

6

S5.2

quercetin-3-O-βgalactoside

7

S7.3

quercetin

8

S8.4

kaemferol

9

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

7

O

3'
2'

1''

1'

O

9

8

2
6

OH

5'
6'

3

10
5

OH
4'

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 3 –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--7 (δC 162,8)/C
162,8)/C-8
8 ((δC 104,7)/C-9(δ
104,7
δC 156,1)) comfirmed the
position of glucose at C--8
8 of A ring. The HMBC correlations between H2’, 6’(δδH8,01)) and C-2
C 2 (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

7

2'

1'

O

9

8

5'
2

6

3

10
5

OH

4

2''

OH

HO

4'

O

8

HO

5'
2

6

6'

3

10

OH

OH
4'

1'

O

9

7

6'

5

O

3'
2'

1''

4

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 5 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, 6

OH
O

HiÖu

s

OH
COOH

O

OH
O

0oC

OH

uÊt: 9
0%

HO

O

OH

su

O

OH

O

OH
OH

OH

HO
O

O

HiÖu suÊt: 2 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 2
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 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-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 5
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 min
100% P.citrinium was killed in direct experiment and after 60 min in
indirect experiment.
0

15

30

45

60

thời gian (phút)

0

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)
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 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 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
IC50
No
Name of compound
(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
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
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, 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 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, quercetin3-O-β-D-glucuronide, vitexin, isovitexin, luteolin, taxifolin, rutin,
resveratrol, picied, three anthraquinone glycoside and aglycone