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:

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

- 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



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

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

(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);

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


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



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

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


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


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


OH

OH

4''
6''

3''
5''
2''

HO
HO

7

O

3'
2'

1''

1'

O

9

8

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


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

3''

5''
2''

5''

O

3'

HO 1''
HO

7

2'

1'

O

9

8

5'
2

6


3

10

OH

OH
4'

1'

O

9

7

6'

5

O

3'
2'

1''

4


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


OH
OH

OH

HO
O

O

HiÖu suÊt: 2 5%

Öu
Hi

OH
O

8h, 60oC

C
%
60o
5h , Êt: 98

OH

HO
H

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


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

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

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


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

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

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)

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