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.
27 trang |
Chia sẻ: honganh20 | Ngày: 04/03/2022 | Lượt xem: 291 | Lượt tải: 0
Bạn đang xem trước 20 trang tài liệu Studies on extraction, purification and hydrolysis of glucomannan from morphophalluskonjack.koch in Lamdong,vietnamanditsanti - Diabeticactivities, để xem tài liệu hoàn chỉnh bạn click vào nút DOWNLOAD ở trên
d 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
absorption when using low molecular weight and mechanism
3has 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
characterization of low molecular weight gluocomannan
4* 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-(14)-
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
compared to the control (p<0.05).
54. 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
1.3.1. Depolymerization by physico-chemical methods
61.3.2. 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
appendix of this thesis.
7- Enzyme endo-1,4 β-Mannanase (Bacillus sp.) EC
3.2.1.78 CAZy Family: GH26 CAS: 37288-54-3 was from
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,
sliced, and immersed into NaHSO3 0.25 ‰.
8Step 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
spectroscopy. The DA value was estimated from the formula:
1 0
0
3 3/100
HI
I
DA CH
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
calculated using the integrals of H1 in the 1H NMR spectrum:
Glu
H1
ManH1
Man/Glu I
I
R (2.3)
9In 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
that can produce enzyme β-mannanase were selected to
10
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
Independent variables
-1 0 +1
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
reaction time ranging from 4÷8 hours while the temperature
11
of the water bath was kept steadily at given temperature
ranged from 4060oC. 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
content in tuber of A.konjac and some physical characteristics
12
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 5,30; 5,60 5,65;5,04
H2 4,24;4,29 3,94÷3,99
H3 4,32÷4,69 4,20÷4,23
H4 3,80÷3,89 3,79
H5 3,65÷3,65 4,06÷4,08
H6 4,29;4,27 4,18÷4,19
H of CH3CO- 2,52
13
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
88.561kDa. Solubility in water was 82.6%. Structure
14
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
glucomannan.
15
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
Enzyme endo-1,4 β-Mannanase (Bacillus sp.) EC 3.2.1.78
CAZy Family: GH26 CAS: 37288-54-3 came from Megazyme
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 +
20.02 X1X2 + 8.01X1X3 – 10.02 X1X4 + 3.75 X2X3 + 3.72
X2X4 – 17,34 X3X4 + 46.94 X12 + 27.20 X22 + 94.30 X32+ 38.45
X42
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
predicted R² of 0.993 is in reasonable agreement with the
16
adjusted R² of 0.997. The ANOVA results were shown in the
table 3.20.
Table 3.20: ANOVA for quadratic model
Source
Sum of
Squares
df
Mean
Square
F-value p-value
Model 65344,95 14 4667,50 695,95 < 0,0001
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
R2 0,9988
17
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
Y of 57.5 mpa.s was obtained.
a) pH and temperature
Design-Expert® Software
Factor Coding: Actual
Y (mpa.s)
Design points above predicted value
Design points below predicted value
61.15 250.15
X1 = A: Temperature
X2 = C: pH
Actual Factors
B: Time = 6
D: E/S = 0.4
5
6
7
8
9
30
35
40
45
50
50
100
150
200
250
300
Y
(
m
p
a.
s)
A: Temperature (oC)C: pH
b) E/S and temperature
Design-Expert® Software
Factor Coding: Actual
Y (mpa.s)
Design points above predicted value
Design points below predicted value
61.15 248
X1 = A: Temperature
X2 = D: E/S
Actual Factors
B: Time = 6
C: pH = 7
0.1
0.2
0.3
0.4
0.5
0.6
0.7
30
35
40
45
50
50
100
150
200
250
Y
(
m
p
a.
s)
A: Temperature (oC)D: E/S
Design-Expert® Software
Factor Coding: Actual
Y (mpa.s)
Design points above predicted value
Design points below predicted value
61.15 250.15
X1 = A: Temperature
X2 = B: Time
Actual Factors
C: pH = 7
D: E/S = 0.4
4
5
6
7
8
30
35
40
45
50
50
100
150
200
250
300
Y
(m
p
a.
s)
A: Temperature (oC)B: Time (h)
c) time and temperature
Design-Expert® Software
Factor Coding: Actual
Y (mpa.s)
Design points above predicted value
Design points below predicted value
61.15 250.15
X1 = B: Time
X2 = C: pH
Actual Factors
A: Temperature = 40
D: E/S = 0.4
5
6
7
8
9
4
5
6
7
8
50
100
150
200
250
300
Y
(
m
p
a.
s)
B: Time (h)C: pH
d) Time and pH
18
Chemical characterization was investigated by IR, 1H,
13C, HSQC. The 1H NMR chemical shifts of LKGM-E signals
were assigned as in table 3.23.
Table 3.23:1H NMR chemical shift data of LKGM-E
Signals Mannose ( ppm) Glucose ( ppm)
H1 5.257 5.031;5.021
H2 4.405 3.873
H3 4.298 4.219;
H4 4.324 4.157
H5 4.014 4.076
H6 4.200 4.491
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
C của CH3CO- 22,17; 176,80
β-Man(14)-β-Glc 71,77
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
(77.79÷78.90/4.324), C5/H5 (77.26/4.014),
19
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
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
in fig.3.31.
control AICAR m100 m50 m25 m12,5 m6,25
20
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
many years to treat diabetes and stimulated insulin secretion.
21
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
Blood glucose levels (mg/dl)
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
LMGM-E
(3g/kg)
4,42 ± 0,51
3,53 ± 0,34
P>0,05
5,58 ± 0,24
P>0,05
5,17 ± 0,31
p>0,05
4,04 ± 0,41
p>0,05
Group 3
LMGM-E
(6g/kg)
4,28 ± 0,59
3,22 ± 0,43
P>0,05
5,35 ± 0,55
P<0,05
5,05 ± 0,43
P<0,05
4,18 ± 0,55
P>0,05
Group 4
Glucomannan
(6g/kg)
4,38 ± 0,28
3,58 ± 1,14
P>0,05
5,55 ± 0,32
P>0,05
5,12 ± 0,23
P<0,05
4,10 ± 0,23
p>0,05
Group 5
Glyclazid (10
mg/kg)
4,4 ± 0,62
3,17 ± 0,51
P<0,01
4,06 ± 0,11
P<0,01
3,74 ± 0,29
P<0,01
3,17 ± 0,66
P<0,01
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
cellular activities.
22
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/
Các file đính kèm theo tài liệu này:
- studies_on_extraction_purification_and_hydrolysis_of_glucoma.pdf