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In Vitro Glucose Entrapment and Alpha-Glucosidase Inhibition of Mucilaginous Substances from Selected Thai Medicinal Plants

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  • Siam Quality Starch Co., Ltd

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As a continuous searching for anti-diabetic(type II) substances, seven mucilage polysaccharides from selected plants were studied as follow: aerial parts of Basella alba Linn., fruits of Hibiscus esculentus Linn., leaves of Litsea glutinosa (Lour.) C.B. Robinson, seeds of Ocimum canum Sims., seeds of Plantago ovata Forssk., fruits of Scaphium scaphigerum G. Don. and seeds of Trigonella foenum-graecum Linn. The bioactive properties for entrapping glucose, inhibiting enzyme alpha-glucosidase and free radical scavenger were in vitro studied compared to glucomannan. The physical characteristics for water holding capacity and viscosity were determined. The chemical characteristics were assayed for monosaccharide composition using methanolysis, TMS-derivatization and gas chromatography. O. canum mucilage superiorly entrapped glucose compared to glucomannan. This activity was relevant to its highly viscous gelation. S. scaphigerum showed another property of alpha-glucosidase inhibition. S. scaphigerum mucilage (0.5%) inhibited the enzyme activity by 82.6%, compared to 1-Deoxynorjirimycin (by 47.6%). Most mucilages, except O. canum and P. ovata, showed DPPH scavenging activity higher than glucomannan. Galacturonic acid was found in 3 from 7 mucilages namely B. alba, P. ovata and S. scaphigerum. Whereas rhamnose was common sugar found in all seven mucilages. Monosaccharide components of these mucilages were compared to the results from the previous reports.
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Research article Open Access
In Vitro
Glucose Entrapment and Alpha-
Glucosidase Inhibition of Mucilaginous
Substances from Selected Thai Medicinal Plants
Chanida PALANUVEJ 1, Sanya HOKPUTSA 2,
Tanasorn TUNSARINGKARN 1, Nijsiri RUANGRUNGSI * 1,3
1 College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2 Research and Development Institute, Government Pharmaceutical Organization, Bangkok 10400, Thailand
3 Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
* Corresponding author. E-mail: nijsiri.r@chula.ac.th (N. Ruangrungsi)
Sci Pharm. 2009; 77: 837–849 doi:10.3797/scipharm.0907-17
Published: October 10th 2009 Received: July 25th 2009
Accepted: October 9th 2009
This article is available from: http://dx.doi.org/10.3797/scipharm.0907-17
© Palanuvej et al.; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Abstract
As a continuous searching for anti-diabetic(type II) substances, seven mucilage
polysaccharides from selected plants were studied as follow: aerial parts of
Basella alba Linn., fruits of Hibiscus esculentus Linn., leaves of Litsea glutinosa
(Lour.) C.B. Robinson, seeds of Ocimum canum Sims., seeds of Plantago ovata
Forssk., fruits of Scaphium scaphigerum G. Don. and seeds of Trigonella
foenum-graecum Linn. The bioactive properties for entrapping glucose,
inhibiting enzyme alpha-glucosidase and free radical scavenger were in vitro
studied compared to glucomannan. The physical characteristics for water
holding capacity and viscosity were determined. The chemical characteristics
were assayed for monosaccharide composition using methanolysis, TMS-
derivatization and gas chromatography. O. canum mucilage superiorly
entrapped glucose compared to glucomannan. This activity was relevant to its
highly viscous gelation. S. scaphigerum showed another property of alpha-
glucosidase inhibition. S. scaphigerum mucilage (0.5%) inhibited the enzyme
activity by 82.6%, compared to 1-Deoxynorjirimycin (by 47.6%). Most mucilages,
except O. canum and P. ovata, showed DPPH scavenging activity higher than
glucomannan. Galacturonic acid was found in 3 from 7 mucilages namely B.
alba, P. ovata and S. scaphigerum. Whereas rhamnose was common sugar
found in all seven mucilages. Monosaccharide components of these mucilages
were compared to the results from the previous reports.
838 C. Palanuvej et al.:
Sci Pharm. 2009; 77; 837–849.
Keywords
Mucilage • Polysaccharide • Anti-diabetic potential • Free radical scavenger •
Physicochemical property
Introduction
Type 2 diabetes comprises 90% of people with diabetes around the world and is one of the
major public health challenges of the 21st century. The number of cases worldwide in
2000 is estimated to be about 171 million and is projected to rise to 366 million in 2030.
The World Health Organization (WHO) projects that without urgent action, diabetes-related
deaths will increase by more than 50% in the next 10 years. Especially in upper-middle
income countries, diabetes deaths are projected to increase by over 80% between 2006
and 2015 [1]. This circumstance results that the demand for medical care in type 2
diabetes will continue to increase. The substantial care and cost are due to the
management of complications of the disease at both the starting point and the degree of
deterioration over time. Macrovascular complications (ischemic heart disease, peripheral
vascular disease, and cerebrovascular disease) has been estimated to be the largest cost
component followed by microvascular complications (nephropathy, neuropathy and
retinopathy) [2]. Endothelial dysfunction is considered to be an integral component of
vascular diseases. Impaired endothelial function induces vasoconstriction, inflammatory
and proliferative changes in the arterial wall and promotes atherosclerotic lesion growth.
Prevention or normalization of endothelial function, contributes to the prevention of
vascular lesion progression or destabilization [3]. Hyperglycemia has been proposed to be
a crucial factor inducing endothelial dysfunction. High concentration of blood glucose as
well as high glucose fluctuation during postprandial period correlates with the increase in
reactive oxygen species or oxidative stress. Reactive oxygen species mediates the
activation of the imbalance in vasoregulating factors (vasodilators and vasoconstrictors)
then affects endothelial homeostasis and triggers atherogenic changes, including
increases in low-density lipoprotein oxidation, sympathetic tone, vasoconstriction, and
thrombogenicity [4]. Accordingly, glucose control is an important goal to diminish the risk of
long term health complications of type 2 diabetes. In addition to glycated haemoglobin and
fasting plasma glucose, postprandial glucose is recently recommended as essential target
for diabetes management [5]. Alpha-glucosidase inhibition is one of the powerful
interventions. Alpha-glucosidase is intestinal enzyme which catalyzes the degradation of
diet polysaccharides to absorbable monosaccharide. Natural or synthetic glucosidase
inhibitors are of therapeutic interest to delay postprandial hyperglycemia in type 2
diabetes. Amongst these, saccharide derivatives, for example Acarbose and Miglitol, have
been approvable for anti-diabetic drugs [6]. Non-starch polysaccharides designated as
soluble dietary fibers are also useful functional foods according to their association with
the reduced risks of diabetes and cardiovascular diseases [7]. The viscous characteristics
due to excellent water-holding and gel-forming capabilities have been proposed as an
important mechanistic factor to delay gastric emptying and delay absorption of glucose in
gastrointestinal tract [8]. Plant mucilage has been credited as one of plant chemical
constituents showing hypoglycemic activity [9, 10]. This study investigated in vitro
properties for anti type 2 diabetic potential among selected mucilaginous plants compared
to a well known soluble dietary fiber, glucomannan. Some physico-chemical characteristics
of these mucilages were also characterized.
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Sci Pharm. 2009; 77; 837–849.
Results and Discussion
Mucilage extraction
Seven mucilaginous plants were studied as follow: aerial parts of Basella alba Linn.
(Basellaceae), fruits of Hibiscus esculentus Linn. (Malvaceae), leaves of Litsea glutinosa
(Lour.) C.B. Robinson (Lauraceae), seeds of Ocimum canum Sims. (Labiatae), seeds of
Plantago ovata Forssk. (Plantaginaceae), fruits of Scaphium scaphigerum G. Don.
(Sterculiaceae) and seeds of Trigonella foenum-graecum Linn. (Papilionaceae). The
mucilages from selected plants yielded range from 3.5% to 23.0% as shown in table 1.
Tab. 1. Mucilage polysaccharides from selected plants
Plants Used parts % Yield
Basella alba Linn. aerial parts 3.5
Hibiscus esculentus L. fruits 5.6
Litsea glutinosa Lour. leaves 12.0
Ocimum canum Sims. seeds 17.6
Plantago ovata Forssk. seeds 19.0
Scaphium scaphigerum G. Don. fruits 23.0
Trigonella foenum-graecum L. seeds 15.0
In vitro property of entrapping glucose
Dialysis tubing technique is a simple model to evaluate the potential of soluble dietary
fibers to additionally retard the diffusion and movement of glucose in the intestinal tract
[11]. Movement in this system is not by the true diffusion but is assisted by the convective
activity of intestinal contractions [12]. The entrapment ability of mucilage gel resulted in
decreasing of glucose diffusion into the external solution. The retardation of the nutrient
flow into the external medium is an indication of the modulating effect of that fiber on
glucose absorption in the jejunum [11]. From the studied model, all mucilages showed
concentration response (0.5, 1.0 and 2.0%w/v) on glucose entrapment activity. The
percentage of glucose releasing from 2% mucilage suspension were 61.6, 70.8, 71.7,
80.6, 83.4, 85.8 and 92.8 % for O. canum, P. ovata, T. foenum-graecum, L. glutinosa, H.
esculentus, B. alba and S. scaphigerum respectively. Glucomannan showed 65.4% of
glucose releasing at the same concentration (Figure 1). Glucomannan seems to be the
most impressive natural fiber with increasing importance in the biomedical and
pharmaceutical fields. It has been found to decrease the serum glucose levels and also
the postprandial insulin flow which aiding diabetic control [13]. Psyllium seeds from P.
ovata and fenugreek seeds from T. foenum-graecum are also reported as an adjunct to
dietary therapy in patients with type II diabetes, to reduce glucose and glycosylated
hemoglobin [14, 15]. Figure 1 showed that the retardation effect on glucose movement of
the mucilages from P. ovata and T. foenum-graecum were lower degree than
glucomannan. Whilst O. canum mucilage of all concentration superiorly entrapped glucose
compared to glucomannan. There have been previous studies of hypoglycemic effect of O.
canum but from leaves extract [16].
840 C. Palanuvej et al.:
Sci Pharm. 2009; 77; 837–849.
In vitro property of alpha-glucosidase inhibition
The effect of selected mucilages against α-glucosidase was evaluated. At the
concentration of 0.5% mucilage, S. scaphigerum, L. glutinosa, H. esculentus, O. canum, T.
foenum-graecum, P. ovata, B. alba and glucomannan showed the inhibitory percentage of
82.6, 41.0, 37.6, 32.8, 30.6, 27.0, 25.0 and 19.7 % respectively. Whereas, 1-Deoxy-
norjirimycin at the same concentration showed the inhibition of 47.6% (Figure 2). S.
scaphigerum mucilage was further investigated and found that the concentration for 50%
inhibition of α-glucosidase activity (IC50) was 0.17% (Figure 3). S. scaphigerum or Malva
nut tree is mostly found in the East of Thailand. The gel made from malva nuts is used as
ingredients in dishes and beverages. Malva nut drink is traditionally used to relief coughing
and sore throats. The previous study in type 2 diabetic patients reported that after the
intake of malva nut drink, fasting plasma glucose and glycosylated hemoglobin decreased
significantly [17]. The present study showed a possible mechanism in alpha-glucosidase
inhibition.
DPPH radical-scavenging activity
Tab. 2. DPPH scavenging activity as IC50 among mucilage polysaccharides from
selected plants
Mucilages IC50 (mg/ml) Mucilages IC50 (mg/ml)
B. alba 2.00 S. scaphigerum 0.61
H. esculentus 0.70 T. foenum-graecum 1.52
L. glutinosa 0.49
O. canum >10 Glucomannan 4.15
P. ovata >10 Ascorbic acid 0.02
a mean of 3 runs
glucose releasing
(
%
)
*
**
*
*
*
***
*
*
*
**
*
*
*
Fig. 1. Percentages of glucose releasing from 0.5, 1, 2% mucilage containing 2% glucose
after 2 hr dialysis (n=4). The significance of differences from the control (0%
mucilage, 99.8±0.9%) was determined by ANOVA followed by Dunnett’s test
(*p<0.01, **p<0.05).
*
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Sci Pharm. 2009; 77; 837–849.
Fig. 2. Percentage of alpha-glucosidase inhibition by 0.5% mucilage (n = 3).
# %inhibition was not significantly different from control (Deoxynorjirimycin)
(p>0.05); * %inhibition was significantly higher from control (p<0.05);
* %inhibition was significantly lower than control (p<0.05) (determined by
ANOVA followed by Dunnett’s test).
Concentration (%)
0.0.2.4.6.81.01.2
0
20
40
60
80
100
Fig. 3. Percentage of alpha-glucosidase inhibition from various concentration of S.
scaphigerum mucilage
Endothelial dysfunction is a key factor in all stages of atherosclerosis development. This
term refers to an imbalance in the production of vasodilators at the circumstance of high
concentrations of reactive oxygen species or oxidative stress [4]. Scavenging of the stable
free radical diphenylpicrylhydrazyl (DPPH) is the basis of a common antioxidant assay.
There have been widely different protocols which differed in the conditions (i.e. pH,
*
*
#
#
*
*
*
*
*
alpha-glucosidase
inhibition (%)
842 C. Palanuvej et al.:
Sci Pharm. 2009; 77; 837–849.
solvents, wavelength of measurement), yielding different results. In the present study, the
polysaccharide samples were incubated with 100 µM DPPH in methanol for 30 min using
ascorbic acid as standard antioxidant. The number of DPPH reduced by one molecule of
ascorbic acid (antioxidant stoichiometry) was 2.5 which was higher than the theoretic value
of 2.0 but close to the previous experiments [18, 19]. Table 2 demonstrated the
scavenging activity of the polysaccharide samples on the DPPH radical. Except for O.
canum and P. ovata, other mucilages showed scavenging activities higher than
glucomannan. Especially the mucilages of L. glutinosa, S. scaphigerum and H. esculentus
had IC50 less than 1 mg/ml. Although the abilities were lower than that of ascorbic acid,
these mucilages counteracted with DPPH as strongly as Ganoderma polysaccharides
(IC50 between 3–13 mg/ml) [20].
Swelling measurements and viscosity
There are a series of physical interactions in the gastrointestinal tract which affect
absorption, as follow: diffusion of nutrients from the bulk solution to the intestinal epithelia,
the rate of removal of waters of hydration from a complex nutrient solvent system, counter
diffusion of nutrients away from the intestinal surface and diffusion of nutrients along the
epithelial surface to an appropriate absorptive site [21]. Water can be held within the
polysaccharide matrix causing considerable swelling and viscous solution or gelation.
Viscous polysaccharide gels may slow absorption by trapping nutrients, digestive enzymes
or bile acids in the matrix and by slowing mixing and diffusion in the intestine [22]. These
physical properties of selected mucilages were studied (Table 3). O. canum and S.
scaphigerum mucilages showed highest values of swelling volume (SV) and water
absorption index (WAI) followed by P. ovata and T. foenum-graecum respectively. Other
mucilages showed the same values of SV and WAI as glucomannan. Viscosity was
determined using falling ball viscosity method. O. canum mucilage had highest viscosity
value even at low concentration (0.5%). Viscous character seemed to be a prominent
factor affected the hypoglycemic potential of O. canum but not S. scaphigerum mucilage.
Tab. 3. Swelling volume (SV), water absorption index (WAI) and viscosity among
mucilage polysaccharides from selected plants
Viscosity (mPa s)
Mucilages SV
(ml/g)
WAI
(g/g) 0.5% 1.0% 2.0%
glucomannan 23.9 ± 1.9 22.1 ± 1.9 21.1 ± 0.3 143.2 ± 8.9 4582.8 ± 60.3
B. alba 25.7 ± 6.3 15.8 ± 3.0 2.2 ± 0.3 3.5 ± 0.4 7.0 ± 0.4
H. esculentus 22.2 ± 4.6 20.7 ± 4.3 7.7 ± 0.7 17.1 ± 0.9 45.1 ± 2.0
L. glutinosa 27.5 ± 8.5 20.6 ± 4.6 1.8 ± 0.2 5.9 ± 0.6 19.1 ± 2.6
O. canum 115.9 ± 17.3* 111.1 ± 17.1* 581.3 ± 59.1 >5000 >5000
P. ovata 60.4 ± 7.7* 48.3 ± 3.2* 6.2 ± 0.3 18.5 ± 1.1 1575.3 ± 57.5
S. scaphigerum 210.5 ± 4.3* 102.8 ± 1.6* 0 0 0
T. foenum-graecum 38.9 ± 1.0 19.1 ± 1.4 6.7 ± 0.4 29.6 ± 1.9 213.0 ± 17.9
a mean of 3 runs; * the significance of differences of SV and WAI from the control (glucomannan) was
determined by ANOVA followed by Dunnett’s test (p<0.01).
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Sci Pharm. 2009; 77; 837–849.
Monosaccharide analysis and protein content
The selected mucilages as well as glucomannan were analyzed for the primary structures
of their monosaccharide compositions as shown in Table 4 and 5. Konjac glucomannan in
this study showed the mannose : glucose molar ratio of around 1.6 : 1 which was in the
range reported elsewhere. Galacturonic acid was found in 3 from 7 mucilages namely
B. alba, P. ovata and S. scaphigerum. Whereas rhamnose was common sugar found in all
studied mucilages. Chemical analyses of the mucilages in this study showed some
differing results from the previous reports. This was due to the differences in either plant
origin or methodology of extraction and analysis. Literature reviews of monosaccharide
composition among these mucilages were summarized in Table 6. H. esculentus mucilage
from this study had highest glucose (79%) composition compared to 44% and 5% from the
literatures. L. glutinosa mucilage from the leaves in this study had xylose/ arabinose ratio
differed from the reported barks. P. ovata and T. foenum-graecum mucilages displayed the
typical characters of arabinoxylan and galactomannan respectively. The water extracts of
S. scaphigerum mucilages in this report had similar ratio of monosaccharide with the
alkaline extracts reported elsewhere. B. alba mucilage was firstly revealed the composition
of arabinose, rhamnose, galactose, galacturonic acid and glucose (24:5:41:13:16). Total
protein contents in these crude polysaccharides ranged from 2% in P. ovata to 38% in L.
glutinosa (Table 4).
Tab. 4. Monosaccharide composition and total protein content (µg/mg) among mucilage
polysaccharides from selected plants
Arab Rham Xy Man
glucomannan 493.8 ± 11.6
B. alba 43.5 ± 0.8 10.7 ± 0.1
H. esculentus 27.8 ± 0.6
L. glutinosa 84.1 ± 1.2 6.0 ± 0.2 56.4 ± 0.5 9.7 ± 0.1
O. canum 47.8 ± 1.1 28.0 ± 0.7 98.43 ± 2.5 37.5 ± 1.5
P. ovata 165.2 ± 13.5 52.0 ± 3.4 697.5 ± 1.3
S. scaphigerum 121.7 ± 3.4 155.2 ± 4.8 20.8 ± 0.7
T. foenum-graecum 18.8 ± 0.4 20.4 ± 0.2 310.4 ± 1.2
Gal Galn Glu TPc
glucomannan 309.6 ± 3.6 107.8 ± 0.04
B. alba 88.7 ± 1.0 33.7 ± 1.3 34.6 ± 0.8 235.5 ± 0.04
H. esculentus 46.5 ± 1.4 285.7 ± 1.4 152.7 ± 0.04
L. glutinosa 29.7 ± 0.6 88.9 ± 0.5 375.1 ± 0.01
O. canum 156.2 ± 0.8 66.30 ± 0.2 81.8 ± 0.02
P. ovata 51.7 ± 3.0 20.3 ± 0.02
S. scaphigerum 144.6 ± 0.3 173.4 ± 2.9 21.1 ± 0.2 195.3 ± 0.03
T. foenum-graecum 302.2 ± 1.2 82.2 ± 0.5 209.5 ± 0.01
a mean of 3 runs; b monosaccharides found in these mucilages included arabinose (ara),
rhamnose (rham), xylose (xy), mannose (man), galactose (gal), galacturonic acid (galn) and
glucose (glu). Fucose (fu) and glucuronic acid (glun) were absent; c Total protein.
844 C. Palanuvej et al.:
Sci Pharm. 2009; 77; 837–849.
Tab. 5. Monosaccharide composition (% mole ratio) among mucilage polysaccharides
from selected plants
Ara Rham Xy Man Gal Galn Glu
glucomannan 61 39
B. alba 24 5 41 13 16
H. esculentus 8 13 79
L. glutinosa 33 2 22 3 10 29
O. canum 12 6 25 8 34 14
P. ovata 18 5 74 4
S. scaphigerum 23 24 4 23 23 3
T. foenum-graecum 3 3 42 41 11
Tab. 6. Monosaccharide composition among mucilage polysaccharides from literatures
glucomannan man : gluc 1.6 : 1 [23]
rham : gal : galn: glu : glun 1 : 2.5 : 1.8 : 0.3 : 0.2 [24]
H. esculentus ara : rham : xyl : man : gal : galn : glu : glun 5 : 3 : 5 : 3 : 17 :
16 : 44 : 7
[25]
L. glutinosa ara : xy 3.4 : 1.0 (barks) [26]
O. canum ara : rham : xy : man : gal : glu 1 : 2 : 1 : 2 : 5 : 8 (uronic acids
8.15%)
[27]
P. ovata ara : rham : xy : man : gal : glu 20.7 : 1.1 : 50.3 : 1.1 : 4.8 : 2.0 [28]
S. scaphigerum ara :rham : gal 1.1 : 1.0 : 1.0 (%w/w) [29]
ara: rham : man : gal : glu 0.5 : 0.2 : 31.4 : 26.2 : 0.6 [30]
T. foenum-
graecum gal : man 1.00 : 1.02 to 1.00 : 1.14 [31]
Conclusion
The plants bearing mucilage in this study have been well known in Thailand as both edible
and medicinal plants. Mucilagenous typed polysaccharides from these plants were
investigated for the biological activities especially antidiabetic potential. Despite the
limitations of this in vitro study, there seemed to be various mechanisms possibly involved
by mucilages due to their physico-chemical characteristics. The in vitro models could be
beneficial tools for the survey of other potential plant mucilages. Moreover they could
refine the possible and capable research designs prior to the expensive further studies of
either the animal models or the clinical trials.
Experimental
Seven mucilaginous plants were studied as follow: aerial parts of Basella alba Linn.
(Basellaceae), fruits of Hibiscus esculentus Linn. (Malvaceae), leaves of Litsea glutinosa
(Lour.) C.B. Robinson (Lauraceae), seeds of Ocimum canum Sims. (Labiatae), seeds of
Plantago ovata Forssk. (Plantaginaceae), fruits of Scaphium scaphigerum G. Don.
(Sterculiaceae) and seeds of Trigonella foenum-graecum Linn. (Papilionaceae).
Glucomannan flour (the Siam Konjac Co., Ltd.) was used for comparison. Chemicals and
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Sci Pharm. 2009; 77; 837–849.
reagents included p-nitrophenyl-α-D-glucopyranoside and α-glucosidase (EC 3.2.1.20) from
Saccharomyces cerevisiae, 1-Deoxynojirimycin, 1,1-Diphenyl-2-picrylhydrazyl (DPPH),
standard monosaccharides (Sigma Chemical Co. Ltd, St. Louis, MO.); methanolic HCl,
trimethylchlorosilane, hexamethyldisilazane (Supelco, Bellefont, PA); Glucose Liquicolor
kit (Human Gesellschaft für Biochemica und Diagnostica mbH, Germany); Lactated
Ringers Buffer pH 7 (General Hospital Product Public Co., LTD., Thailand), All other
chemicals were analytical grade. Dialysis tubing cellulose membrane (molecular weight cut
off = 12,000 Da) was from Sigma Chemical Co. Ltd, St. Louis, MO. Dialysis tubing cellulose
membrane (molecular weight cut off = 3,500 Da) was from Spectrum Medical Industries,
Inc., Los Angeles, CA.
Mucilage extraction
The mucilage were extracted from the specified plant parts with warm water and
concentrated by lyophilization. The lyophilized samples were re-dissolved in water,
precipitated twice with 2 volume of 80% ethanol and dialyzed against distilled water in a
dialysis tubing cellulose membrane (molecular weight cut off = 3,500 Da). The samples
were lyophilized, ground and kept in refrigerator for further studies.
In vitro property of entrapping glucose
The mucilage and glucomannan were dissolved in Ringers buffer. Glucose was added to
make the final concentration of 2% glucose and 0, 0.5, 1 and 2 % w/v polysaccharide gel.
Four milliliter of each concentration was dialysed against 60 ml of Ringers buffer in a
dialysis tubing cellulose membrane (molecular weight cut off = 12,000 Da) for 2 hours
under rotationally shaking at 150 rpm. The released glucose was determined by glucose
oxidase - phenyl ampyrone (GOD-PAP) colorimetric method using Glucose Liquicolor kit
according to manufacturer's instructions.
In vitro property of alpha-glucosidase inhibition
Alpha-glucosidase activity was assayed using 0.1M sodium phosphate buffer at pH 6.9,
and 1 mM p-nitrophenyl-α-D-glucopyranoside was used as a substrate [32]. The
concentration of α-glucosidase was 1 U/mL in each experiment. The enzyme (4 μl) was
incubated in the absence or presence of various concentrations of tested polysaccharides
at 37 °C. The preincubation time was specified at 10 min and the substrate (95 μL) was
added to the mixture. The reaction was carried out at 37 °C for 20 min, and then 100 μL of
1M Na2CO3 was added to terminate the reaction. Enzymatic activity was quantified by
measuring the absorbance of p-nitrophenol at 405 nm. One unit of α-glucosidase is
defined as the amount of enzyme liberating 1.0 μmol of p-nitrophenol per minute under the
conditions specified. 1-Deoxynojirimycin was used as the positive control.
DPPH radical-scavenging activity
The potential antioxidant activity of polysaccharide samples was determined on the basis
of the scavenging activity of the stable DPPH free radical [33]. Various concentrations
of polysaccharides samples (0.5 ml) were added to 1.5 mL of a 0.004% methanolic
solution of DPPH. Absorbance at 517 nm was determined after 30 min, and the percent
scavenging activity was calculated by the following formula:
Scavenging effect (%) = (1 Asample/Acontrol) x 100
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Where Acontrol is the absorbance of control (DPPH solution plus water), Asample is the test
sample (DPPH solution plus test sample or positive control) and the percent inhibition
activity was calculated. IC50 values denote the concentration of sample required to
scavenge 50% DPPH free radicals.
Swelling measurements and viscosity
Swelling volume (SV) and water absorption index (WAI) were determined from the ratio of
the volume and weight of swollen gel to the dry weight of sample respectively [34,35]. A
0.050 g ground sample (<60 mesh) was suspended in 25 ml of water in a 25-ml graduated
cylinder for 2 hours. The volume of swollen gel was measured. The supernatant liquid was
removed, the swollen gel was weighed and SV and WAI were calculated. Viscosities at
various concentrations were measured with a falling ball viscometer (HAAKE Mess-Technik
GmbH u. Co, Germany) at 20 °C.
Monosaccharide analysis and protein content
The polysaccharide samples (1 mg) were subjected to methanolysis with 4 M methanolic
HCl at 80°C for 24 h. Mannitol was added as an internal standard. The samples were dried
with nitrogen, methanol was added and the samples were dried again. This washing was
repeated twice [36]. Prior to gas chromatographic analysis, the samples were
trimethylsilylated using trimethylchlorosilane : hexamethyldisilazane : Pyridine 1:2:5 (0.4
ml) at room temperature for 30 min. Instrumentation was performed on a Finnigan Trace
GC Ultra with DSQ MS detector and a split–splitless injector. The column was a ZB-5
fused silica capillary column (30 m × 0.25 mm i.d.) with film thickness 0.25 μm. Helium
was used as carrier gas at a flow rate of 1.0 ml/min. The injector and detector temperature
were 260 and 300°C respectively. The column temperature was initially 140°C, then an
increase of 1°C/min to 170°C and followed by 6°C/min to 250°C. The protein contents of
the samples were determined by Lowry method using bovine serum albumin as protein
standard.
Statistical analysis
The significance of differences between the mean values was determined by analysis of
variance (ANOVA), followed by Dunnett’s test, and a p value of less than 0.05 was
considered statistically significant.
Acknowledgements
C.P. wishes to thank the Thailand Research Fund through the Royal Golden Jubilee Ph.D.
Program and Chulalongkorn University Graduate School Thesis Grant for financial
supports. The authors also express their gratitude to the Siam Konjac Co., Ltd. for
standard glucomannan.
Authors’ Statement
Competing Interests
The authors declare no conflict of interest.
In Vitro Glucose Entrapment and Alpha-Glucosidase Inhibition of Mucilaginous Substances from … 847
Sci Pharm. 2009; 77; 837–849.
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