MINISTRY OF EDUCATION
VIETNAM ACADEMY OF
AND TRAINING
SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY
Tran Thi Nu
STUDIES ON EXTRACTION, PURIFICATION AND HYDROLYSIS
OF GLUCOMANNAN FROM MORPHOPHALLUS KONJAC K.KOCH IN
LAM DONG, VIETNAM AND ITS ANTI-DIABETIC ACTIVITIES
Major: Organic chemistry
Code: 9.44.01.14
SUMMARY OF CHEMISTRY DOTORAL THESIS
Hanoi – 2020
This thesis was completed at: Graduate University of Science
and Technology, Vietnam Academy of Science and Technology.
Advisor 1:Prof. Dr. Do Truong Thien
Advisor 2: Dr. Tran Thi Y Nhi
1st Reviewer: …
2st Reviewer: …
3st Reviewer: ….
This thesis will be defended at Graduate University of Science
and Technology, Vietnam Academy of Science and Technology
at .......hour ........date........ month ......... 2020.
The thesis can be found in:
- The Library of Graduate University of Science and
Technology, Vietnam Academy of Science and Technology.
- Vietnam National Library.
INTRODUCTION
1. The urgency of the thesis
Glucomannan, a water soluble polysaccharide, is
composed of a linear chain of β-1,4-linked D-glucose and D-
mannose residues in a molar ratio of 1:1.6, with side branches
through β-1,6-glucosyl units. The acetyl groups along the
glucomannan backbone are located, on average, every 9–19
sugar units at the C-6 position. Glucomannan is a low-calorie
dietary fiber that has been used as diatary food for dieters to
lose weight, reduce blood cholesterol and postprandial glucose
response. In addition, glucomannan is one of the most viscous
dietary fibres known which has been used in various fields such
as food thickener, elastic gels, films ...
Glucomannan is extracted from the tubers of some
Amorphophallus species. In some subtropical Asia countries
such as China and Japan Thai Lan, A. konjac is regarded as an
agronomically important crop which has great potential in both
domestic and international markets.
Glucomannan is found in many different
Amorphophallus species which has different structure and
properties in each species. Amorphophallus konjac K. Koch
(Amorphophalus konjac K. Koch) is a species with high content
of glucomannan that become a industrial key crop in some East
Asian and Southeast Asian countries such as China, Japan and
Thailand. There are more than 25 Amorphophallus species in
1
Vietnam which distributed in different regions of the country.
Amorphophallus konjac K. Koch was recently discovered in
2012 in some northern mountainous provinces.
Despite its hydrophilicity, glucomannan is poorly
soluble in water (solubility of around 30%) due to its high
molecular weight 1.9 106÷2 106Da, which limits its
application range in certain areas [3]. In order to increase its
solubility, glucomannan is hydrolyzed to lower molecular
weigh and the process attracts the attention of many scientists.
In addition to the general properties of glucomannan
(KGM), hydrolyzed glucomannan (LMWG) also has many
biological activities such as probiotics, antioxidants, immune
regulators, etc. Hydrolyzed glucomannan is also used as drug
delivery carriers.
With the potential application in food and
pharmaceuticals, studies on the methods of preparing low
molecular weight glucomannan have been of interest to many
authors worldwide, including enzymatic hydrolysis [13]–[23],
combination of -irradiation and β-mannanase [24] hydrochloric
acid [14][25], hydrochloric acid combined with ultrasound [26]
treatment with -irradiation combined with ethanol [8], alkaline
hydrolysis combined with heat [28]... However, the above-
mentioned studies in the world have almost focused on methods
of low molecular weight glucomannan preparation. Studies on
properties, chemical structure, and the relationship between
their structure and biological activity have not paid enough
attention. Especially, the ability to reduce blood sugar
2
absorption when using low molecular weight and mechanism
has not been studied. Such studies are hardly ever been
investigated in Vietnam
In order to contribute a new fundamental research on
glucomannan originating in Vietnam and to improve the value
of glucomannan for pharmaceutical and functional food
products, we have chosen the Doctor thesis entitled “Studies on
extraction, purification and hydrolysis of glucomannan from
Amorphophallus konjac K.Koch in Lam Dong, Vietnam and its
anti-diabetic activities”.
1. The objectives of the thesis
- Extraction, chemical charaterization of glucomannan
from the tubers of Amorphophallus Konjac K.Koch in Lam
Dong, Vietnam
- Parameter optimization for glucomannan hydrolysis
reaction to make different types of low molecular weight
glucomannan by different methods
- Evaluate the hypoglycemic activity and hypoglycemic
mechanism of hydrolyzate products.
2. The main content of the dotoral thesis
* Study on main chemical constituents of tuber from
A.Konjac K.Koch. Physico-chemical charaterization of
glucomannan: chemical constituents, manose/glucose ratio,
molecuar weight by IR, NMR, TGA, …
* Hydolysis parameter optimization, physico-chemical
3
characterization of low molecular weight gluocomannan
* AMPK activation by low molecular weihgt
glucomannan in vitro and Oral Glucose Tolerance Test .
3. New finding of the thesis
Fully investigation on the main composition of tuber of
A.konjac planted in Lam Dong province, glucomannan
extraction and purification process, physico-chemical
characterization of glucomannan.
The hydrolysis parameters were optimized by response
surface methodology, using β-1,4-mannanase from Bacillus sp.
as catalysis. A three level, four variable Box-Behnken factorial
designs (BBD) was applied to determine the best combination
for viscosity. The optimal conditions were pH at 7.24, temperature at 42.4oC, and incubation time at 5.7 h, substrate
concentration at 0.54%. Under optimized conditions, predicted
Y was 57.5 mpa.s and experimentally value Y was 60.85 mpa.s. The hydrolysis product (LMWG-E) consisting of beta-(1 4)-
linked D-glucose (G) and D-mannose (M) in a proportion of
1:1.2; the degree of acetylation was determined to be
approximately 7.56%, molecular weight was calculated to be
2051.77 g/mol, solubility of 92.5%.
LMWG-E significantly increased AMPK
phosphorylations in a dose dependent manner. Treatment with
KGM 100 μg/ml and 50μg/ml caused 1.47-fold and 1.81-fold
phosphorylation of AMPK, respectively (p<0.05). LMWG-E at
the dose of 6 g/kg significantly attenuated the elevated blood
glucose levels seen following glucose loading at this time point
4
compared to the control (p<0.05).
4. Outline of the thesis
The thesis consists of 124 pages with 29 tables, 33 figures, 9
schemes and 137 references. The thesis consists of 4 chapters:
Introduction (2 pages), Chapter 1: Liturature overview (40
pages); Chapter 2: Materials and Methods (18 pages); Chapter 3:
Results and Discussion (53 pages); Conclusion (2 pages);
Publications related to the thesis (1 page); References (8 pages).
CHAPTER 1: OVERVIEW
This chapter provides an overview on national and
international researches related to the thesis: general
introduction about glucomannan; A.konjac K. Koch and
glucomannan extraction and purification process; hydrolysis of
glucomannan; AMPK enzyme and its role in hypoglycemia;
researchs on glucomannan extracted from A.konjac in Vietnam.
1.1. General introduction to glucomannan
1.1.1. Sources and chemical structure of glucomannan
1.1.2. The physical properties of glucomannan.
1.1.3. The chemical properties of glucomannan
1.1.4. Biological activity and pharmacological effects of
glucomannan
1.2. The review of A.konjac K. Koch and glucomannan
extraction and purification process
1.2.1. Introduction to Amorphophallus Konjac K.Koch
1.2.2. Extraction and purification of glucomannan from tuber of
A.konjac
1.3. The review of hydrolysis of glucomannan
5
1.3.1. Depolymerization by physico-chemical methods
1.3.2. Introduction to Enzyme hydrolysis
1.4. The review of AMPK enzyme and its role in
hypoglycemia
1.4.1. Glucose metabolism in the body
1.4.2. Overview of Adenoidin 5'-monophosphat kích hoạt
protein kinase (AMPK ).
1.4.3. Method of activation of AMPK
1.5. The review of glucomannan extracted from A.konjac in
Vietnam
CHAPTER 2. METERIAL AND METHODS
2.1. Plant materials
- Three-year-old tuber of Amorphophallus Konjac
K.Koch planted in Lam Dong province, Vietnam was collected
in November, 2016 and identified by Dr.Nguyen Van Du,
Institute of Ecology and Biological Resources, Vietnam
Academy of Science and Technology, VAST. The specimens
were kept in a sample storage house in Dak Nong province of
the Center for High Technology Development - Vietnam
Academy of Science and Technology deposited in the Institute
of Chemistry, VAST.
- Bacillus substilis và Bacillus lichenifomis were
supplied by An Thai Production & Service Co., Ltd. Both
strains are beneficial bacteria, bio safety and clear origin and
have a genetic sequence of the original strain attached to the
6
appendix of this thesis.
- endo-1,4 β-Mannanase (Bacillus sp.) EC
3.2.1.78 CAZy Family: GH26 CAS: 37288-54-3 was from Enzyme Megazyme Company.
- The C2C12 myoblasts (CRL-1772) were purchased
from the American Type Culture Collection (Manassas, VA,
U.S.A.).
- White mice (of Swiss strains), both male and female,
weighing 18÷22 grams, having healthy physiology. Dulbecco’s
modified Eagle’s medium (DMEM), fetal bovine serum (FBS),
horse serum (HS), and penicillin−streptomycin (PS) were
obtained from WelGENE (Daegu, Korea).
- All other chemicals were of analytical grade
2.2. Method
2.2.1. Determination of glucomannan content.
DNS method: Acid hydrolysis of glucomannan will
produce two kinds of reducing sugar: D-mannan and D-glucose.
Reducing sugars will be reduced to a brownish red amino-
compound when co-boiled with 3,5-dinitro salicylic acid in an
alkali medium. To some extent, the amount of the reducing
sugars is in positive correlation with the color strength and,
therefore, glucomannan can be determined with
spectrophotometry.
2.2.2. Extraction of glucomannan from the tubers of A.Konjac.
Two-stage technique for extraction of glucomannan
from A.Konjac K.Koch was chosen as follow:
Step 1: Tubers of A.konjac were washed, peeled,
7
sliced, and immersed into NaHSO3 0.25 ‰.
Step 2: adding in to the mixture a volume of
ethanol/water (1.5:1 v/v) with tubers/solution ½ (w/v). Then
the crushing process was operated in 20 minutes.
Step 3: Centrifugation to get precipitate (paste form).
Step 4: drying to get KGM powder.
Step 5: purification of the product by dissolving KGM
powder into hot ethanol 40%, stirring, centrifuging to collect
the precipitate, and removing the filtrate. Repeat 3 times to
obtain refined KGM.
2.2.2. Methods for determination of chemical structure of
compounds
Physicochemical characterization was investigated by
modern spectroscopic methods such as IR, one/two-dimension
nuclear magnetic resonance (NMR) spectra, thermal analysis,
Brookfield DV2T viscometer, OSOMAT 090...
Degree of acetylation (DA) of glucomanno-
oligosaccharides was determined by using the 1H NMR
0
spectroscopy. The DA value was estimated from the formula:
I
3/
0
CH
DA
100 3 1 HI
Where: ICH3 was the integral of the hydrogen atom in –
COCH3 group and IH1was total integral of the hydrogen atom
of C1 in both glucose and mannose units.
The mannose/glucose ratio in GO molecule was
H1
Man
calculated using the integrals of H1 in the 1H NMR spectrum
R
Man/Glu
I I
H1
Glu
8
(2.3) :
In which: RGlu/Man ratio of glucose/mannose
IH1-Glu is the integral of H1 of glucose.
IH1 -Man is the integral of H1 of mannose.
2.2.3. Hydrolysis of glucomannan
2.2.3.1. Hydrolysis with hydrochloric acid
- Glucomannan (10g) was dispersed in a mixture of HCl
and CH3COOH solution. The mixture was stirred to get
homogeneous solution for both acid and ultrasound combined
acid hydrolysis.
- For ultrasound combined acid hydrolysis, the solution
was subjected to sonication for 30 min at 20 kHz. Then both
were carried out at specified concentrations at a given time or
temperature. Viscosity measurements of the reaction mixture
were carried out at the specific time of the studies.
- After treatment, the mixture was rinsed with ethanol to
neutral, left to evaporate off the ethanol before being dried in a vacuum oven at 60oC. The product obtained by acid hydrolysis
method named as and the other was named as LKGM-1.
- Parameter investigation in the range as follow:
[H+] 0,05M, 0,1M, 0,15M, 0,2M, 0,25M; temperature: 50 oC, 60 oC, 70 oC, 80 oC, time duration: 1 hours, 2 hours, 3 hours, 4 hours, 5
hours, glucomanan/solution: 1/5; 1/10; 1/15; 1/20 (g/ml)
Hydrolysis efficiency was assessed by viscosity.
2.2.3.2. Enzymatic hydrolysis
* Qualitative determination: two microorganism strains
9
that can produce enzyme β-mannanase were selected to
hydrolysis glucomannan: bacillus subtilis and bacillus
licheniformis.
* Enzymatic hydrolysis
After qualitative determination, we used commercial β-
mannanase from Megazyme Company for further experiments.
A three level, four variable Box-Behnken factorial
design (BBD) was applied to determine the best combination
for viscosity. Temperature, pH, time and E/S ratio were chosen
as independent variables. The range and central point values of
four independent variables presented in Table 2.1 were based on
the results of our preliminary single-factor experiments. All the
experiments were done in triplicate and viscosity was selected
as the response (Y)
Table 2.1: Independent variables and their levels
Code level -1 0 +1 Independent variables
X1: Temperature (oC) 30 40 50
X2: Time (h) 4 6 8
pH (X3) 5 7 9
X4: E/S (w/w) 0.1 0.4 0.7
A 27-run BBD with four factors and three levels was
used to fit a second-order response surface in order to
optimize the extraction conditions. Glucomannan powder
(10g) was dissolved in 300 ml of desired pH solution, then
mixed with endo-1,4 β-Mannanase 0.01÷0.7 (w/w) to start
the reaction. The mixture was incubated at pH 5÷9 for
10
reaction time ranging from 4÷8 hours while the temperature
of the water bath was kept steadily at given temperature ranged from 40 60oC. The reaction was stopped by boiling the samples for 10 min. The hydrolysate obtained was
concentrated with a rotary evaporator, mixed with ethanol
and then had been collected as a precipitate by centrifugation
at 4000 rpm for 20 min, was resuspended in ethanol three
times for further investigated (named as LKGM-E)
2.2.3. Biological assays
2.2.3.1. AMPK activation in vitro
The anti-diabetic effects in C2C12 myotube occur via
activation of AMPK were investigated using Western Blot
Analysis. The experiment was done at Department of
Pharmacology, Hanoi University of Pharmacy.
2.2.3.1. Oral Glucose Tolerance Test (OGTT)
Oral glucose tolerance test of different doses of
LMWG-E was conducted in white, non-diabetic mice (of Swiss
strains), both male and female, weighing 18-22 grams. The
mice were fed daily with synthetic feed supplied by the Institute
of Vaccines and Biologicals. The experiment was done at
Department of Pharmacology, Hanoi University of Pharmacy.
Chapter 3. RESULTS AND DISCUSSION
3.1. Extration and Purification process, physic-chemical
properties of glucomanan from A.konjac
3.1.1. Determination of glucomannan content in tubers of
A.Konjac
This section presents the results of glucomannan
11
content in tuber of A.konjac and some physical characteristics
of glucomannan. The glucomannan content was 12.26% (wet
weight). The extracted glucomannan powder is white, solubility
in water of 32%, ash content of 4.17%, water absorbency of 9%,
Asen content was 0.208 ppm, Pb content was 0,184 ppm.
Glucomannan content in tuber of A.konjac was much higher
than that of in other Amorphophallus species such as A.
Paeonnifolius (glucomannan content was 1.67%), A.
Corrugatus (glucomannan content of 1.67%). This finding
confirmed the role of Amorphophallus Konjac K.koch in the
development orientation of Amorphophallus species in Vietnam.
3.1.2. Chemical structure of glucomannan.
This section presents the detailed results of spectral
analysis and structure determination of glucomannan extracted
from tuber of A.Konjac. Structure determination of the KGM
was investigated by IR, NMR 1H, 13C, HSQC and TGA.
Table 3.5: 1H NMR chemical shift data of (δ ppm) LKGM-1 Signals Mannose (δ ppm) Glucose (δ ppm)
H1 5,65;5,04 5,30; 5,60
H2 3,94÷3,99 4,24;4,29
H3 4,20÷4,23 4,32÷4,69
H4 3,79 3,80÷3,89
H5 4,06÷4,08 3,65÷3,65
H6 4,18÷4,19 4,29;4,27
12
H of CH3CO- 2,52
Glucomannan obtained as a white, amorphophallus,
glucose/manose ratio of 1.6/1, degree of acetylation 8%,
branched at C6, molecular weight was 1.598 kDa. The high DA
value makes glucomannan soluble in water so that glucomannan
has been used for food and pharmaceutical application.
From the 1H, 13C and HSQC spectra, the cross peaks of both substituted and nonsubstituted mannosyl and glucosyl
residues were assigned as follows: Cross-peaks of mannose
residues: C1/H1 (94.71;94.33/5.30; 5.60), C2/H2
(71.04/4,24;4.29), C3/H3 (71.45/4.32÷4.69),
C4/H4(76.76/3.80÷3.89), C5/H5(74.976/3.651÷3.658),
C6/H6(61.97/4.29;4.27). The cross peaks of glucosyl residues:
C1/H1 (96.68;92.78/5.65;5.04), C2/H2 (72,27;72,18/3,94÷3,99),
C3/H3(73,12/4,20÷4,23), C4/H4(76,60; 76,56/3,79),
C5/H5(73.82/4.06÷4.08), C6/H6(61.63; 61.54/4.18÷4.19).
3.2. Hydrolysis with hydrochloric acid
This section presents the detailed results of parameter
optimization for hydrolysis reaction and physico-chemical
characteristic of hydrolysis products. Based on the experimental
results, suitable hydrolysis parameters for ultrasound mediated
acid hydrolysis were: CH3COOH 10%, [HCl] 0.15M, KGM/ acid solution ratio of 1/10(g/ml), temperature of 50oC in 4 hours.
For acid hydrolysis only, the chosen/optimal parameter were:
CH3COOH 10%, [HCl] 0.15M, KGM/ acid solution ratio of 1/10(g/ml), temperature of 50oC, in 6 hours. The molecular
weight of the hydrolysis product reduced from 1598 kDa to
13
88.561kDa. Solubility in water was 82.6%. Structure
determination of the hydrolysis product was investigated by IR,
NMR 1H, 13C and TGA.
Table 3.13: 1H NMR chemical shift data of (δ ppm) LKGM-1
Signals Mannose (δ ppm) Glucose (δ ppm)
H1 5.17 5.54; 5.34
H2 4.88; 4.87 3.96
H3 4.69 4.87
H4 4.49 4,58
H5 4.14 4.43; 4.41
H6 4.28; 4.26 4.35
H of CH3CO- 2.49
able 3.14: 13C NMR chemical shift data of (δ ppm) LKGM-1
Signals Mannose (δ ppm) Glucose (δ ppm)
C1 101.45 100.86
C2 71.03 72.49
C3 71.18 73.93
C4 76.86 79.34; 79.16
C5 76.09 75.87
C6 64.26; 63.68 61.53
C of CH3CO- 69.80; 66.67
The results showed that the main chain of LKGM-1 consisting of beta-(14)-linked D-glucose (G) and D-mannose
(M) in a proportion of 1:1.2, DA of 7.03, molecular weight of
88.561 kDa and less heat-stable in comparion with its parent
14
glucomannan.
3.3. Enzymatic hydrolysis of glucomannan
3.3.1. Qualitative determination to select β-mannanase
hydrolyses
Experimental results showed that both enzymes
produced from Bacillus subtilis and bacillus licheniformis can
hydrolyzes of the glycosidic bond (p<0.05). However, enzyme
from bacillus subtillis hydrolyzes glucomannan better than that
of mẫu bacillus licheniformis (p<0.05). So that we chose
endo-1,4 β-Mannanase (Bacillus sp.) EC 3.2.1.78
CAZy Family: GH26 CAS: 37288-54-3 came from Megazyme Enzyme Company.
3.3.2. Response surface methodology for parameter
optimization
A 27-run BBD with four factors and three levels was
used to fit a second-order response surface in order to optimize
the extraction conditions. The following quadratic model
explains the experimental data.
Y = 62.21 – 12.81X1 – 6.85X2 – 25.03X3–14.95 X4 +
2 + 94.30 X3
2 + 27.20 X2
2
20.02 X1X2 + 8.01X1X3 – 10.02 X1X4 + 3.75 X2X3 + 3.72 2+ 38.45 X2X4 – 17,34 X3X4 + 46.94 X1
X4
The fit of the model was checked by determination of the coefficient R2, which was calculated to be 0.9988, indicating
that 99.9 % of the variability in the response of Y can be explained by the model equation (2). This high R2 value
indicated that the models are well adapted to the responses. The
15
predicted R² of 0.993 is in reasonable agreement with the
adjusted R² of 0.997. The ANOVA results were shown in the
table 3.20.
Table 3.20: ANOVA for quadratic model
Sum of Mean Source df F-value p-value Squares Square
65344,95 14 4667,50 695,95 < 0,0001 Model
A-Temp. 1915,47 1 1915,47 285,61 < 0,0001
B-Time 563,07 1 563,07 83,96 < 0,0001
C-pH 7412,76 1 7412,76 1105,29 < 0,0001
D-E/S 2681,43 1 2681,43 399,82 < 0,0001
AB 1604,00 1 1604,00 239,17 < 0,0001
AC 223,35 1 223,35 33,30 < 0,0001
AD 402,00 1 402,00 59,94 < 0,0001
BC 57,00 1 57,00 8,50 0,0130
BD 55,35 1 55,35 8,25 0,0140
CD 1200,62 1 1200,62 179,02 < 0,0001
A² 11662,98 1 11662,98 1739,02 < 0,0001
B² 3973,30 1 3973,30 592,44 < 0,0001
C² 47246,57 1 47246,57 7044,76 < 0,0001
D² 7920,57 1 7920,57 1181,01 < 0,0001
Residual 80,48 12 6,71
Lack of fit 78,77 10 7,88 9,19 0,1021
16
R2 0,9988
Design-Expert® Software
Design-Expert® Software
Factor Coding: Actual
Factor Coding: Actual
Y (mpa.s)
Y (mpa.s)
Design points above predicted value
Design points above predicted value
Design points below predicted value
Design points below predicted value
61.15
250.15
61.15
248
250
300
X1 = A: Temperature
X1 = A: Temperature
X2 = D: E/S
X2 = C: pH
250
200
Actual Factors
Actual Factors
B: Time = 6
200
B: Time = 6
150
C: pH = 7
D: E/S = 0.4
150
) s . a p m
100
(
) s . a p m
Y
(
100
Y
50
50
0.7
50
0.6
45
9
50
0.5
0.4
40
8
45
0.3
35
D: E/S
A: Temperature (oC)
0.2
7
40
0.1
30
6
35
C: pH
A: Temperature (oC)
5
30
b) E/S and temperature
Design-Expert® Software
Factor Coding: Actual
Design-Expert® Software
Factor Coding: Actual
a) pH and temperature
Y (mpa.s)
Design points above predicted value
Y (mpa.s)
Design points below predicted value
Design points above predicted value
61.15
250.15
Design points below predicted value
61.15
250.15
300
300
X1 = A: Temperature
X2 = B: Time
250
X1 = B: Time
X2 = C: pH
250
Actual Factors
200
C: pH = 7
Actual Factors
200
D: E/S = 0.4
A: Temperature = 40
D: E/S = 0.4
150
150
) s . a p m
(
100
Y
) s . a p m
(
100
Y
50
50
8
50
9
8
7
45
8
7
6
40
7
6
5
35
B: Time (h)
A: Temperature (oC)
6
5
C: pH
B: Time (h)
4
30
5
4
c) time and temperature d) Time and pH
Fig.3.23. Response surface (3-D) showing the effect of time,
temperature, pH and E/S on the response Y
Optimization of hydrolysis conditions: The optimal
conditions were extracted by Design Expert Software for the
minimum value of the response (Y) were pH at 7.24,
temperature at 42.3oC, and incubation time at 5.68 h and
substrate concentration at 0.54%. Under these conditions, value
17
Y of 57.5 mpa.s was obtained.
13C, HSQC. The 1H NMR chemical shifts of LKGM-E signals
Chemical characterization was investigated by IR, 1H,
were assigned as in table 3.23.
Table 3.23:1H NMR chemical shift data of LKGM-E
Signals Mannose ( ppm) Glucose ( ppm)
5.257 4.405 4.298 4.324 4.014 4.200 5.031;5.021 3.873 4.219; 4.157 4.076 4.491 H1 H2 H3 H4 H5 H6 H of CH3CO- 2.702
The 1H NMR chemical shifts of LKGM-E signals were
assigned as in table 3.24.
Tín hiệu Mannose (δ ppm) Glucose (δ ppm)
C1 102,15;101,99 104,45
C2 72,52;72,18 75,35;75,11
C3 73,63; 73,15 76,19
C4 77,79÷78,90 80,86
C5 77,26 76,97;76,83
C6 62,92;62,78 62,57;62,45
22,17; 176,80
71,77
C của CH3CO- β-Man(14)-β-Glc β-Man(14)-β-Man 71,56
The cross-peaks of mannose residues were assigned as
follows: C1/H1 (102.15;101.99/5.257), C2/H2
(72.52;72.18/4.405), C3/H3 (73.63; 73.15/4.298), C4/H4
18
(77.79÷78.90/4.324), C5/H5 (77.26/4.014),
C6/H6(62.92;62.78/4.277; 4.200). The cross peaks of glucosyl
residues: C1/H1 (104.45/5.031;5.021), C2/H2
(75.35;75.11/3.873), C3/H3 (76.19/4.219), C4/H4 (80.86/4.157),
C5/H5 (76.97;76.83/4.076), C6/H6(62.57;62.45/4.491).
The main chain of LKGM-E consisting of beta-(1 4)-
linked D-glucose (G) and D-mannose (M) in a proportion of
1:1.2, DA of 7.03, molecular weight of 2051 kDa and less
heat-stable in comparion with its parent glucomannan, solubility
of 92,5%.
3.4. AMPK assay and Western Blot Analysis
This section presents the detailed results of anti-diabetic
effects of LKGM-E via AMPK acitivation in C2C12 myotube
control AICAR m100 m50 m25 m12,5 m6,25
in vitro
actin
p-AMPK
Figure 3.31: The relative phosphorylation levels of AMPK- Thr172 (p-AMPK) in C2C12 myotubes The experiments were performed in differentiated
C2C12 myotubes by Western lot method. This is a modern
method with high sensitivity that detecting protein by specific
antigen - antibody reaction. The relative phosphorylation levels
of AMPK-Thr172 (p-AMPK) in C2C12 myotubes was shown
19
in fig.3.31.
LKGM-E significantly increased AMPK
phosphorylations in a dose dependent manner. Treatment with
KGM 100 μg/ml and 50μg/ml for 1 hour caused 1.47-fold and
1.81-fold phosphorylation of AMPK, respectively (p<0.05).
The results indicated that glucomanan at concentration of
100μg/ml and 50μg/ml activated AMPK.
3.5. Oral Glucose Tolerance Test (OGTT)
This section presents the detailed results of anti-diabetic
effects of LKGM-E in vivo.
The test was conducted in white, non-diabetic mice (of
Swiss strains). The results showed that blood glucose levels had
rapidly increased 30 min after administration of the glucose
load. The dose at 3g/kg produced no significant hypoglycemic
effect in normal mice (p>0.05). However, LKGM-E at the dose
of 6 g/kg significantly attenuated the elevated blood glucose
levels seen following glucose loading at this time point (p <
0.05), indicating enhanced insulin sensitivity, compared to the
control. It demonstrated noteworthy anti-hyperglycemic effect
from 90 min onward (p<0.05). In the OGTT, LKGM-E
treatment increased utilization of peripheral glucose in mice,
resulting in improved glucose tolerance. LKGM-E showed
concentration-dependant reduction in the blood glucose level.
The results were compared with glyclazid that has been used for
20
many years to treat diabetes and stimulated insulin secretion.
This study provides good evidence that LKGM-E has excellent
potential to be used as a functional food for glycemic control.
Table3.29. The end of oral glucose tolerance test (OGTT) result
Treament
t = 0
t = 30 min t = 60 min t = 120 min
Control
4,51 ± 0,51 3,98 ± 0,44 5,32 ± 0,14 5,02 ±0.25 4,12 ± 0,25
Group 2
3,53 ± 0,34
5,58 ± 0,24
5,17 ± 0,31
4,04 ± 0,41
LMGM-E
4,42 ± 0,51
P>0,05
P>0,05
p>0,05
p>0,05
(3g/kg)
Group 3
3,22 ± 0,43
5,35 ± 0,55
5,05 ± 0,43
4,18 ± 0,55
LMGM-E
4,28 ± 0,59
P>0,05
P<0,05
P<0,05
P>0,05
(6g/kg)
Group 4
3,58 ± 1,14
5,55 ± 0,32
5,12 ± 0,23
4,10 ± 0,23
Glucomannan
4,38 ± 0,28
P>0,05
P>0,05
P<0,05
p>0,05
(6g/kg)
Group 5
3,17 ± 0,51
4,06 ± 0,11
3,74 ± 0,29
3,17 ± 0,66
4,4 ± 0,62
Glyclazid (10
P<0,01
P<0,01
P<0,01
P<0,01
mg/kg)
Blood glucose levels (mg/dl)
The results were in agrement with experimental results
on activation of AMPK in vitro. Because LKGM-E is capable
of activating enzyme AMPK, that stimulates energy generation
processes [84–88]. Therefore, when glucose is absorbed into the
bloodstream, it is transported into cells and converted into
Glucose-6-phosphate which was burned to create energy for
21
cellular activities.
CONCLUSION
1. Etraction and purification of glucomannan from tuber
of A.konjac ( Amorphophallus Konjac K.Koch):
- The glucomannan content in tuber of A.konjac was
12.26% (wet weight). The extracted glucomannan powder is
white, with 90% purity, ash content of 4.17%, water
absorbency of 9%, Asen content was 0.208 ppm, Pb content
was 0.184 ppm.
- The extracted glucomannan powder is white powder,
amorphous, solubility in water of 32%, consisting of beta-
(1 4)-linked D-glucose (G) and D-mannose (M) in a
proportion of 1.6/1, DA ≈8%, Mw 1.598 kDa, branched at C6.
2. Hydrolysis with hydrochloric acid
- Optimal parameters for acid hydrolysis were:
CH3COOH 10%, [HCl] 0,15M, KGM/ acid solution ratio of
1/10(g/ml), temperature of 50oC, in 6 hour for acid hydrolysis
only and in 4 hours for ultrasound mediated acid hydrolysis
- The molecular weight of the hydrolysis product
(LKGM-1) reduced from 1598 kDa to 88,561kDa. Solubility in
water was 82.6%. The main chain structure of LKG-1 was
almost unchanged in comparison with the parent glucomannan,
the M / G ratio was 1.51, and the DA of LKGM-1 was 7.03%.
22
3. Enzymatic hydrolyis of glucomannan
- Parameter conditions for glucomannan hydrolysis
were successfully optimized by response surface methodology
(RSM) using Box-Behnken design. The optimal conditions
were pH at 7.24, temperature at 42.3oC, and incubation time at
5.68 h and substrate concentration at 0.54%. Under these
conditions, value Y of 57.5 mpa.s was obtained and
experimentally value Y was 60.85 mpa.s.
- The main chain of LKGM-E consisting of beta-
(1 4)-linked D-glucose (G) and D-mannose (M) in a
proportion of 1:1.2, DA of 7.03, molecular weight of 2051
kDa and less heat-stable in comparion with its parent
glucomannan, solubility of 92,5%.
4. AMPK assay and Western Blot Analysis
- LKGM-E significantly increased AMPK
phosphorylations in a dose dependent manner. Treatment with
KGM 100 μg/ml and 50μg/ml for 1 hour caused 1.47-fold and
1.81-fold phosphorylation of AMPK, respectively (p<0,05).
The results indicated that glucomanan at concentration of
100μg/ml and 50μg/ml activated AMPK
5. Oral glucose tolerance test (OGTT) in non-diabetic rats.
LKGM-E at the dose of 6 g/kg significantly attenuated
the elevated blood glucose levels. LKGM-E can reduce
intestinal absorption of glucose and had better ability to
stimulate glucose tolerance than glucomannan. This difference
23
is statistically significant (p < 0.05)
Based the obtained results, glucomannan from
Amorphophallus konjac K. Koch and its hydrolyzed products
24
have many potential applications, especially in health care.
“Response surface methodology
glucomannan producing flour for
PUBLICATIONS WITHIN THE SCOPE OF THESIS 1. Do Truong Thien, Tran Thi Nu, Nguyen Hong Vinh, “Preparation of Low Molecular Weight Glucomannan from A. Konjac K. Koch in Vietnam by Enzyme Catalyzed Hydrolysis Reaction and its Prospective use to Lower Blood Sugar Levels”, Academic journal of polymer science, Volume 2 - issue 2- January 2019. 2. Tran Thi Nu, Tran Thi Y Nhi, Do Truong Thien, “Chemical structure of Konjac glucomannano oligosaccharides prepared by endo-1-4 β mannanase” Tạp chí Hóa học, Vol 57 (4e3,4), p.291-295, 2019. 3. Tran Thi Y Nhi, Tran Thi Nu, Do Truong Thien, Lai Thi Thuy, Le Thi Thanh Ha, Pham Thi Bich Hanh, Le Quang Tuan, Trinh Duc Cong, for hydorolysis parameter optimization of Konjac glucomannan using endo-1,4 β mannanase” Tạp chí Hóa học, Vol 57 (4e3,4), p.296-300, 2019. 4. Trần Thị Nữ, Trần Thị Ý Nhi, Đỗ Trường Thiện, Phạm Thị Bích Hạnh, “Nghiên cứu phản ứng thủy phân glucomannan bằng axit kết hợp sử dụng sóng siêu âm và khảo sát cấu trúc của sản phẩm”, Tạp chí Hóa học, 54 (6e1), trang 61-66, 2016 5. Nguyen Van Minh Khoi, Do Truong Thien, Le Minh Ha, Tran Van Thanh, Le Ngoc Hung, Tran Thi Nu, “New lab-scale process from Amorphophallus plant in Viet Nam and their characterization, part 2”, proceedings of scientific workshop on progress and trends in science and technology, p.225-232, 2016. 6. Trần Thị Ý Nhi, Trần Thị Nữ, Lại Thị Thúy, Đỗ Trường Thiện, Nguyễn Văn Dư, Phạm Thị Bích Hạnh, “Nghiên cứu cấu trúc hóa học của glucomannan từ củ cây Nưa Amorphophallus Konjac K. Koch thu tại Hà Giang Việt Nam”, Tạp chí Hóa học 52(6A), p.228-232, 2014
