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Antidiabetic effect of a newly identified component of Opuntia dillenii polysaccharides.

ARTICLE INFO

Keywords;

Diabetes mellitus Opuntia

Polysaccharides Strepiozotocin

ABSTRACT

The aim of this study was to determine the most effective hypoglycemic component of polysaccharides from Opuntia dillenii Haw. By preliminary screening and to specifically study the antidiabetic effects of O. dillenii polysaccharide (ODP)-Ia in mice with streptozotocin (STZ)-induced diabetes. Three kinds of ODPs - ODP-Ia, ODP-Ib, and ODP-II' - were isolated by using an ultrasonic extraction method and diethylaminoethyl (DEAE)-Sepharose fast-flow column chromatography. The mice were administered ODPs for 3 weeks. Gavage administration of ODP-Ia significantly decreased (P < 0.05) their intake of food and water; the fasting levels of blood glucose (BG), total cholesterol (TC), triglycerides (TGs), plasma urea nitrogen (PUN), and malondialdehyde (MBA); and the activity of glucose-6-phosphatase (G-6-Pase). In contrast, it significantly increased (P < 0.05) the body weights, hepatic glycogen (HG) levels, high-density lipoprotein cholesterol (HDL-C) levels, and the hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in diabetic mice. However, ODP-Ia did not significantly increase insulin levels in the mice with STZ-induced diabetes. We propose that ODP-Ia exerts its antihyperglycemic effect by protecting the liver from peroxidation damage and by maintaining tissue function, thereby improving the sensitivity and response of target cells in diabetic mice to insulin.

[c] 2011 Elsevier GmbH. All rights reserved.

Introduction

Diabetes mellitus is the most common serious metabolic disorder and is 1 of the 3 leading causes of death worldwide (Islam and Choi, 2009). It can be treated by diet, exercise, and pharmaceutical therapy. However, the drugs used to treat this disorder are either expensive or have adverse effects or contraindications. Many natural plant-derived drugs have been used worldwide for treating diabetes (Balakrishnan et al., 2009). These plant-derived agents may be less toxic and have fewer side effects than synthetic agents (Gurib-Fakim et al., 2005). Thus, natural plant-derived drugs provide several potential options for the control of diabetes (Buyukbalci and El, 2008; Jaiswal et al., 2009).

Opuntia cacti are wild xerophytes that are used in many regions of the world, primarily for food, fodder, and medicine; they generally grow in dry sunny areas, where there are few other deep-rooted plants. These plants contain high levels of important nutrients, such as polysaccharides; betalains; phenolic compounds; organic acids; lipids; minerals; vitamins; and amino acids, including taurine. Therefore, they can be used in the manufacture of health foods and medicines (Feugang et al., 2006; Salim et al., 2009).

Recent studies on polysaccharides derived from Opuntia spp. have shown that these polysaccharides contain arabinose, xylose, fructose, glucose, galacturonic acid, and rhamnose units (Zhao et al., 2007a; Cai et al., 2008), and possess versatile functional properties, such as protective effects against [H.sub.2 O.sub.2]-induced damage, immunostimulatory effects, free radical-scavenging and antiinflammatory activity, antitumor activity, blood lipid-lowering effects, and wound-healing activity (Huang et al., 2008; Panico et al., 2007; Schepetkin et al., 2008; Cho et al., 2006; Trombetta et al, 2006). Some studies have reported the antidiabetic effects of Opuntia extracts, and a clinical study has also been conducted on the effectiveness of tablets derived from Opuntia dillenii Haw. In the treatment of type 2 diabetes mellitus (Alarcon-Aguilar et al., 2003; Yang et al., 2008).

However, few studies have been performed to determine which component of O. dillenii polysaccharides (ODPs) is most effective against diabetes mellitus or to determine the mechanism underlying the antihyperglycemic action of ODPs. In this study, we isolated and purified ODP-Ia - one of the active components of O. dillenii - and studied its antidiabetic effects in mice with streptozotocin (STZ)-induced diabetes.

Materials and methods

Plant materials and chemicals

Fresh O. dillenii cladodes of uniform shape and maturity were collected from Donghai Island, Zhanjiang City, Guangdong Province, China. ODP-Ia (chromatographically pure, molecular weight [Mr]: 60kDa) was obtained from O. dillenii aqueous extracts by low-pressure chromatography. STZ and gliquidone were purchased from Sigma Chemical Co., USA and Beijing Wanhui Double-Crane Pharmaceutical Co., China, respectively. Blood glucose (BG) and total cholesterol (TC) levels were measured using kits from Shanghai Rongsheng Biotechnology Co., China. Triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) levels were estimated using kits from Zhejiang Dongou Bioengineering Co., China. Superoxide dismutase (SOD), malondialdehyde (MDA), plasma urea nitrogen (PUN), glutathione peroxidase (GSH-Px), and serum calcium were detected using kits from the Nanjingjiancheng Bio-Engineering Research Institute, China. Glucose-6-phosphatase (G-6-Pase) activity was assayed according to the method described by Koide and Oda (1959). Insulin levels were determined using a radioimmunoassay kit from Beijing Biosino Biotechnology Co., China. All the other chemicals used in our study were of analytical grade.

Animals

Male Chinese Kunming mice (age, 5 weeks; weight, 18-22g) were purchased from the Centre of Experimental Animals, Guangdong Medical University, China. The mice were housed in a room with a 12/12-h light/dark cycle at an ambient temperature of 22-25[degrees]C and fed a commercial stock diet containing (w/w) 9% fiber, 23% protein, and 65% carbohydrate, together with adequate amounts of vitamins and mineral nutrients, in accordance with the institutional guidelines. The animals were used for the experiment after an acclimatization period of 1 week.

Extraction and purification of ODPs

Liposoluble substances were extracted from fresh 0. dillenii plants by soaking the plants 5 times in 80% ethanol; this was done after removing the thorns and peeling the plants, rinsing them with clean water, followed by chopping and homogenization. The ethanol was evaporated to dryness, and the residue sieved through a 200-mesh sieve to obtain O. dillenii powder. Some of this powder was added to phosphate buffer (pH 7.12) and subjected twice to ultrasonic extraction for 46 min, with a material-to-water ratio of 38ml/g - these conditions have been previously optimized in our laboratory (Lan et al., 2006). The filtrates were collected and concentrated to a ropy consistency by using a rotary evaporator at 50[degrees]C, at a low pressure of 80Mbar. When this solution was cooled and added to 95% ethanol (triple the volume of the solution) at 4[degrees]C, a white precipitate was formed. The precipitate was collected by centrifugation (1810 x g, 20min) and redissolved in distilled water. The resulting solution was dialyzed against distilled water for 3 d, with a molecular weight cut-off of 3500 Da; the solution in the dialysis bag was then centrifuged again (17,300 x g, 15 min) to remove any precipitate. After concentration, the proteins in the solution were removed by repeated freezing and thawing at temperatures ranging from - 20[degrees]C to room temperature. ODPs powder was obtained from the supernatant by centrifugation (17,300 x g, 15 min), performed to remove any precipitate, and freeze-drying.

Deproteinized ODP powder (0.15 g) was dissolved in 10 ml of 25 mM phosphate buffer (pH 7.6). After intensive dissolution, 6 ml of the solution was injected into a diethylaminoethyl (DEAE)-Sepharose fast-flow column (2.5 cm x 30 cm). After elution with 0.01 M NaCl solution, the components were separated by gradient elution using 0.01-0.2 M NaCl solution. The elution volume, elution flow rate, and volume per tube on the fraction collector were 480 ml, 1 ml/min, and 6 ml, respectively. The polysaccharide content of the solution per tube was measured using the phenolsulfuric acid method, with arabinose as the standard (Yang et al., 2005). The eluents corresponding to the single polysaccharide peak were combined and loaded on a Sephadex G-100 or Sephadex C-150 column (1.5 cm x 75 cm) and eluted with 0.01 M NaCl solution. The elution flow rate and volume per tube on the fraction collector were 18 ml/h and 3 ml, respectively. The polysaccharide content of the eluent was detected as described above. The purified polysaccharide components - ODP-Ia, ODP-Ib, and ODP-II' - were obtained after the eluents corresponding to the main peak were combined, dialyzed, concentrated, precipitated with alcohol, washed, and lyophilized.

Determination of the molecular weight, content, composition and optical rotation of ODPs

The molecular weights of the ODPs were determined by a gel filtration method. A Sephadex G-200 column (1.0 cm x 100 cm) was equilibrated for 60 h with 0.1 M NaCl solution, at a constant flow rate (20ml/h). We injected 3 mg each of blue dextran (2000 kDa) and standard dextran samples (T500, T70, T40, and T10) into the column. The void volume [V.sub.0] was obtained first by eluting the blue dextran, and the elution volume [V.sub.e] for the standard dextran samples was obtained by eluting them, collecting the effluent (volume, 1 ml per tube) on the fraction collector, and measuring them by the phenol-sulfuric acid method. The regression equation was obtained using [V.sub.e]/[V.sub.0] as the abscissa and log([M.sub.r]) as the ordinate. Three kinds of polysaccharides - ODP-Ia, ODP-Ib, and ODP-II' - were injected into the column by the same method, and the [V.sub.e] was determined for each polysaccharide. The molecular weights of these polysaccharides was determined from the regression equation, using their [V.sub.e]/[V.sub.0] value.

The ODPs were measured by the phenol-sulfuric acid method as mentioned above. The polysaccharide content was calculated using the following formula: Polysaccharide content = measured weight/sample weight. The chemical compositions of ODP-Ia, ODP-Ib and ODP-II' were determined by high performance lipid chromatography (Tao et al., 2006). Rhamnose, arabinose, galactose, glucose, mannose, xylose, and fructose were as the standard monose sample. The glycuronic acids of the three kinds of polysaccharides were measured using the carbazole method, with arabinuronic acid as the standard (Bitter and Muir, 1962), and their optical rotations were measured by polarimeter.

Determination of protein content and nitrogen content of ODPs

Protein content of ODPs was determined using the Bradford method with bovine serum albumin as the standard. Nitrogen contents of ODPs were measured using the micro-kjeldahl method (Miller and Houghton, 1945). Nonprotein nitrogen contents of ODPs were measured using the micro-kjeldahl method after removing the protein by 5% trichloroacetic acid.

Cellulose acetate film electrophoresis

Cellulose acetate films (2 cm x 8 cm) were immersed in 0.05 mol/1 borate buffer solution (pH 10) for 30min. Thin stripy polysaccharide samples (approximately 50[micro]g each) were added to the films with a sample applicator and inserted in the electrophoresis cell. The voltage and electrophoresis times were 250V and 20min, respectively. The films were dyed using 1% toluidine blue solution for 10 min and then rinsed in 90% ethanol until no background color remained.

Establishment of the diabetes mice model

Diabetes was induced in the mice by administering a single intraperitoneal injection of STZ (120 mg kg body [weight.sub.-1]), dissolved in citrate buffer (pH 4.5), after 12 h of fasting (Abeeleh et al., 2009). After receiving the injection, the animals were placed in metabolic cages with ad libitum access to water and the same food they had received before administration of the drug. Diabetic mice with BG concentrations greater than 14.5 mmol/1 were used for the experiment; the eligibility of the mice was confirmed by measuring the fasting BG concentration at 96 h after injecting STZ.

Preliminary screening for the ODPs component with hypoglycemic effects

The mice were segregated into the following groups: the normal control group; hyperglycemic model group; and ODPs, ODP-I, and ODP-II treatment groups. The ODPs group was further divided into 3 dose subgroups - 100, 200, and 400mg kg body [weight.sub.-1] [d.sub.-1]-while the ODP-I and ODP-II groups were each divided into the 50, 100, and 200 mg kg body [weight.sub.-1] [d.sub.-1] dose subgroups. Every group and subgroup comprised 10 animals. The mice in the normal control group and hyperglycemic model group were intragastrically administered the same volume of normal saline. During the experimental period, i.e., for 22 d, the mice had ad libitum access to food and water. The water and food intake of the mice were recorded every day, and the body weights of the mice were recorded in the first and third weeks of the study period. After 22 d of intragastric administration, BG was measured by drawing blood from the tail veins of the mice.

[FIGURE 1 OMITTED]

Experimental design

The mice were randomly segregated into 6 groups of 10 animals each, as described below. Group I mice, i.e., the normal control group mice, were only administered 0.86% NaCl. The other groups comprised mice with STZ-induced diabetes (groups II-VI). Group II mice, i.e., the model group mice, were also administered only 0.86% NaCI. Group III mice, i.e., the positive control mice, were administered 100 mg kg body [weight.sub.-1] of gliquidone in 0.86% NaCl. The mice in groups IV, V, and VI were administered 50, 100, and 200 mg kg body [weight.sub.-1] of ODP-Ia, respectively. ODP-Ia was intragastrically administered once daily for 3 weeks. After 21 d of treatment, the animals were sacrificed by cervical decapitation after 12 h of fasting. Their blood was collected in tubes containing heparin sodium, for the estimation of BG and lipid levels. Mice livers were collected and stored at - 70[degrees]C for future assays involving measurement of enzyme activities.

Statistical analyses

All values are given as means [+ or -] standard error (S.E.) for each group. Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's multiple-range test (DMRT) using the SAS system (version 8.2). P values of less than 0.05 were considered to indicate statistically significant differences.

Results

Extraction and purification of ODPs

ODP-I and ODP-II were obtained from ODPs by DEAE-Sepharose fast-flow column chromatography, with yields of 52.95% and 18.06%, respectively (Fig. 1A). ODP-Ia and ODP-Ib were obtained from ODP-I by Sephadex G-100 column chromatography, with yields of 54.48% and 27.18%, respectively (Fig. IB), and ODP-II' was obtained from ODP-II by Sephadex G-150 column chromatography, with a yield of 82.95% (Fig. 1C). The protein content, the total nitrogen content and the nonprotein nitrogen content in ODPs were 1.93%, 0.512%, 0.182% respectively. The other 5 plysaccharides in this study were all obtained in the form of white powders and readily dissolved in water but not in organic solvents such as alcohol, acetone, ether, chloroform, or butanol. The reaction with the phenol-sulfuric acid reagent was positive and that with ninhydrin was negative, indicating that these 5 polysaccharides did not contain amino acids or proteins. Further, the reaction with the iodide-potassium iodide reagent was negative, indicating that they were non-starch polysaccharides. The total nitrogen content and the nonprotein nitrogen content in ODP-I were 0.152% and 0.145%, respectively, and the total nitrogen content and the nonprotein nitrogen content in ODP-Ia were 0.147% and 0.143%, respectively, indicating that ODP-I and ODP-Ia contained nitrogenous compound. The total nitrogen content in ODP-II was 0.014%, indicating that it did not contain nitrogenous compound nearly.

The average polysaccharide content of ODPs, ODP-I, ODP-II, ODP-Ia, ODP-Ib and ODP-II' were 78.69%, 92.44%, 88.78%, 94.60%, 87.43%, and 90.36%, respectively. The purified ODP-Ia, ODP-Ib and ODP-II' fractions each presented as a single blue band on the cellulose acetate films, and the Sephadex G-200 column chromatograms for each polysaccharide showed single symmetric peaks (Fig. 1D-F), indicating that ODP-Ia, ODP-Ib, and ODP-II' were all homogeneous polysaccharides and chromatographically pure. The regression equation was y = - 1.6004, x + 7.2763 and [R.sub.2] = 0.9403 (y represents log[[M.sub.r]] and x represents [V.sub.e]/[V.sub.0]).The [V.sub.0] values of the 3 polysaccharides - ODP-Ia, ODP-Ib, and ODP-II' - were 22.3, 25.0, and 22.4mL, respectively, and the [V.sub.e] values were 34.8, 50.0, and 35.0 mL, respectively. Thus, the molecular weights of the 3 polysaccharides -ODP-Ia, ODP-Ib, and ODP-II' - obtained using this equation were approximately 60, 12, and 167 kDa, respectively. ODP-Ia was comprised mainly of rhamnose, arabinose, galactose and glucose, with 15.13% (w/w) of arabinuronic acid. ODP-Ib was composed of rhamnose, arabinose, and galactose, with 52.66% (w/w) of arabinuronic acid. ODP-II' was composed of rhamnose, glucose, with 26.38% (w/w) of arabinuronic acid. These polysaccharides were viscous. The specific rotations [[[[alpha]].sup.25].sub.D] (c 0.1, water) of ODP-Ia, ODP-Ib, and ODP-II' were +55 [degrees], +125 [degrees] and +21 [degrees] respectively.

Preliminary screening of the ODPs component with hypoglycemic effects

After 22 d of intragastric administration of ODPs, ODP-I, and ODP-II, the BG concentrations of the diabetic mice decreased to different degrees (Table 1). On comparison with the BG concentrations of the mice in the hyperglycemic model group, it was found that the BG concentrations of the diabetic mice in the 3 ODPs dose groups (100, 200, and 400 mg kg body [weight.sup.-1] [d.sup.-1]) had decreased by 2.9%, 16.54% (P < 0.05), and 17.26% (P < 0.01), respectively; those in the 3 ODP-I dose groups (50, 100, and 200 mg kg body [weight.sup.-1] [d.sup.-1]) had decreased by 20.62% (P < 0.01), 23.29% (P < 0.01), and 31.08% (P < 0.01), respectively; and those in the 3 ODP-II dose groups (50, 100, and 200 mg kg body [weight.sup.-1] [d.sup.-1]) had decreased by 4.58%, 14.99% (P < 0.01), and 20.25% (P < 0.01), respectively. All 3 polysaccharides could improve the symptoms of polydipsia and polyphagia in the diabetic mice and restore their body weight; the effects of ODP-I were most evident. These results showed that the hypoglycemic effect of ODP-I was stronger than that of ODPs or ODP-II.
Table 1

Effects of the different ODPs on the BC concentrations, body weights,
water intake, and food intake of mice with STZ-induced diabetes.

Croup Dose BG Body Water Food
 weight intake intake

 mg mmol/l g ml/d g/d
 [kg.sub.-1]
 [d.sub.-1]

Control - 4.60 [+ 3 9.21 7.2 [+ 6.2 [+
group or -] [+ or or -] or -]
 0.35 -] 2.71 1.3 0.5

Model - 22.07 [+ 29.83 [+ 28.2 [+ 14.9 [+
group or -] or -] or -] or -]
 3.01(a) 4.59(a) 2.2 1.8
 (a) (a)

ODPs 100 21.41 [+ 30.80 [+ 28.2 [+ 11.8 [+
group or or -] or -] or -]
 -]3.84 5.80 1.8 1.2
 (c)
 200 13.42 [+ 36.79 [+ 26.0 [+ 10.7 [+
 or or -] or -] or -]
 -]4.43 3.69 2.4 1.3
 (b) (b) (c)
 400 18.26 [+ 38.02 [+ 22.7 [+ 9.9 [+
 or -] or -] or or -]
 3.18 3.22 -]2.5 1.6
 (c) (c) (c) (c)

ODP-I 50 17.52 [+ 37.52 [+ 25.8 [+ 13.1 [+
group or -] or -] or -] or -]
 3.83 3.96 1.8 1.4
 (c) (c) (b) (c)
 100 16.93 [+ 38.04 [+ 24.3 [+ 11.3 [+
 or or -] or -] or -]
 -]3.35 4.42 1.9 1.5
 (c) (c) (c) (c)
 200 15.21 [+ 39.05 [+ 22.4 [+ 10.3 [+
 or or -] or -] or -]
 -]4.18 3.41 1.3 1.2
 (c) (c) (c) (c)

ODP-II 50 21.06 [+ 32.56 [+ 26.6 [+ 12.4 [+
group or -] or -] or -] or -]
 1.70 3.81 1.5 1.6
 (c) (c)
 100 18.76 [+ 34.04 [+ 25.2 [+ 12.5 [+
 or -] or -] or -] or -]
 2.01 2.60 2.2 0.9
 (c) (c) (c)
 200 17.60 [+ 33.46 [+ 25.3 [+ 12.5 [+
 or -] or -] or -] or -]
 1.33 3.83 2.0 1.4
 (c) (c) (c)

(a) P < 0.01, compared with the control group, n = 10, mean
[+ or -] S.E.
(b) P < 0.05. n = 10, mean [+ or -] S.H.
(c) P < 0.01 compared with the model group, n = 10, mean
[+ or -] S.E.


Effects of ODP-Ia on the body weight, water intake, and food intake of the mice

ODP-Ia had dose-dependent effects on body weight, water intake, and food intake in the experimental mice (Table 2). The mean body weight of the mice in the model group was 28.58 g and was lower than that of the mice in the normal control group, i.e., 39.92 g. No significant difference (P > 0.05) was found between the body weights of the mice in the positive and normal control groups. The body weights of the mice treated with ODP-Ia at 50, 100, and 200 mg kg body [weight.sup.-1]] doses were significantly higher (P < 0.05) than those of the mice in the model group, but no significant difference (P > 0.05) was found between the body weights of the mice in the 3 ODP-Ia dose subgroups. The body weights of the mice treated with 200 mg kg body [weight.sup.-1] ODP-Ia were similar to (P > 0.05) those of the normal control mice. The food and water intake of the mice in the positive control and ODP-Ia treatment groups were significantly (P < 0.05) lower than those of the mice in the model group.
Table 2

Effects of ODP-Ia on the body weights, water intake, food intake, and
fasting BC concentrations of the diabetic mice (n = 10, and mean
[+ or -] S.E).

Group Dose Body Water Food
 weight intake intake

 mg g ml/d g/d
 [kg.sup.-1]
 [d.sup.-1]
 Day 0 Day
 21

Normal - 20.10 39.92 7.93 [+ 6.71 [+
control [+ or [+ or or -] or
group -] -] 2.6 1.64 -]0.60
 1.51 l (a) (e) (d)

Model - 19.72 28.58 30.25 15.67[+
group [+ or [+ or [+ or or -]
 -] -] -] 2.50 1.31
 1.18 4.66 (a) (a)
 (c)

Positive 100 19.39 38.79 19.83 9.70 [+
control [+ or [+ or [+ or or -]
group -] -] -] 2.23 1.19
 1.25 3.02 (d) (c)
 (ab)

ODP-Ia 50 19.59 35.93 27.12 12.58 [+
treatment [+ or [+ or [+ or or -]
group -] -] -] 1.58 1.12
 0.71 4.39 (b) (b)
 (b)
 100 20.22 38.46 26.17 11.60 [+
 [+ or [+ or [+ or or -]
 -] -] -] 2.23 1.97
 1.12 3.45 (b) (b)
 (ab)
 200 20.00 39.12 22.42 9.50 [+
 [+ or [+ or [+ or or -]
 -] -] -] 1.50 1.33
 1.06 2.71 (c) (c)
 (ab)

Group BG
 concentration

 mmol/l
 Day 0 Day Day 21
 11

Normal 6.50 [+ or -] 5.68 6.29 [+
control 0.45 (b) [+ or or -]
group -] 0.56
 0.33 (e)
 (e)

Model 19.95[+ or -] 23.15 30.69 [+
group .57 (a) [+ or or -]
 -] 1.88
 4.21 (d)
 (a)

Positive 20.34 [+ or -] 11.94 10.28 [+
control 2.90 (a) [+ or or -]
group -] l.31
 1.32 (d)
 (d)

ODP-Ia 19.28 [+ or -] 18.37 17.36 [+
treatment 2.34 (a) [+ or or -]
group -] 3.62
 1.54 (b)
 (b)
 19.43 [+ or -] 17.33 16.67[+
 2.01 (a) [+ or or -]
 -] 2.28
 1.99 (b)
 (bc)
 20.28 [+ or -] 16.24 14.30 [+
 2.32 (a) [+ or or -]
 -] 2.44
 1.76 (c)
 (c)

In each column, values that do not share a common superscript differ
significantly at P < 0.05 (ANOVA followed by DMRT).


Dose-dependent effects of ODP-Ia on fasting BG concentrations in the diabetic mice

During the 21-d experimental period, the fasting BG concentrations of the mice in the normal control group were mostly constant and were significantly (P < 0.05) lower than those of the diabetic mice in the other 5 groups (Table 2). The fasting BG concentration significantly increased (P < 0.05) from 19.95 [+ or -] 2.57 mmol/1 to 30.69 [+ or -] 1.88 mmol/l in the mice from the model group. The BG concentrations in the 3 ODP-Ia dose groups (50, 100, and 200 mg kg body [weight.sup.-1]) were 43.43%, 45.68%, and 53,4%, respectively, lower than those in the model group, while those in the positive control group were 66.5% lower than those in the model group.

Effects of ODP-la on the plasma lipid levels of the mice

After 21 d of treatment, the TC and TG levels of the mice in the normal control group were significantly lower (P < 0.05) than those of the mice in the other groups, and the HDL-C levels of the mice in the normal control group were significantly higher (P < 0.05) than those of the mice in the other groups (Table 3). The TC levels in the mice in the positive control group and ODP-Ia treatment groups, except the 50 mg [kg.sup.-1] [d.sup.-1] dose group, were significantly (P < 0.05) lower than those of the mice in the model group. The TG levels of the mice in the positive control group and ODP-Ia treatment groups were significantly lower (P < 0.05) than those of the mice in the model group, hut no significant (P > 0.05) difference was observed between the TG levels of the mice in the ODP-Ia treatment groups. The HDL-C levels of the mice in the positive control group and ODP-Ia treatment groups were significantly higher (P < 0.05) than those of the mice in the model group, but no significant difference was found between the HDL-C levels of the mice in the positive control group and ODP-Ia treatment groups (P > 0.05).
Table 3

Effects of ODP-Ia on plasma lipid, serum calcium, and PUN concentrations
in diabetic mice (n = 10, and mean [+ or -] S.E.).

Group Dose TC TG
 concentration concentration

 mg mmol/l mmol/l
 [kg.sup.-1]
 [d.sup.-1]

Normal - 1.98 [+ or -] 0.92 [+ or -]
control 0.15 (d) 0.1 l (d)
group

Model - 2.44 [+ or -] 2.00 [+ or -]
group 0.20 (a) 0.36 (a)

Positive 100 2.09 [+ or -] 1.73 [+ or -]
control 0.24 (d) 0.37 (b)
group

ODP-Ia 50 2.40 [+ or -] 1.32 [+ or -]
treatment 0.14 (ab) 0.28 (c)
group
 100 2.25 [+ or -] 1.27 [+ or -]
 0.11 (bc) 0.20 (c)

 200 2.20 [+ or -] 1.23 [+ or -]
 0.18 (c) 0.23 (c)

Group HDL-C Serum calcium PUN
 concentration concentration concentration

 mmol/l mmol/l mmol/l

Normal 1.87 [+ or -] 1.50 [+ or -] 6.56 [+ or -]
control 0.07 (a) 0.18 (a) 0.51 (c)
group

Model 1-52 [+ or -] 1.30 [+ or -] 13.82 [+ or -]
group 0.13 (d) 0.l2 (b) 1.56 (a)

Positive 1.73 [+ or -] 1.47 [+ or -] 10.05 [+ or -]
control 0.13 (bc) 0.12 (a) 1.42 (b)
group

ODP-Ia 1.63 [+ or -] 1.46 [+ or -] 10.81 [+ or -]
treatment 0,14 (c) 0.13 (a) 1.03 (b)
group
 1.66 [+ or -] 1.59 [+ or -] 10.62 [+ or -]
 0.09 (c) 0.14 (a) 1.42 (b)
 1.77 [+ or -] 1.60 [+ or -] 10.37 [+ or -]
 0.11 (b) 0.19 (a) 2.03 (b)

In each column, values that do not share a common superscript differ
significantly at p < 0.05 (ANOVA followed by DMRT).


Effects of ODP-Ia on the serum calcium and PUN concentrations of the mice

The serum calcium concentrations of the mice in the normal control, positive control, and ODP-Ia treatment groups were significantly higher (P < 0.05) than those of the mice in the model group; however, no significant difference was found between the values for the control, positive control, and ODP-Ia treatment groups (P > 0.05; Table 3). No significant differences were observed between the PUN concentrations of the mice in the positive control and ODP-Ia treatment groups (P > 0.05), but the PUN concentrations of the mice in these groups were significantly higher (P < 0.05) than those of the mice in the normal control group and significantly lower (P < 0.05) than those of the mice in the model group.

Effects of ODP-Ia on the antioxidative enzyme activity and MDA levels in the livers of diabetic mice

After 21 d of oral administration, the SOD activity of the mice in the positive control group and ODP-la treatment groups, except the 50 mg [kg.sup.-1] [d.sup.-1] dose group, was significantly higher (P < 0.05) than that of the mice in the model group (Table 4). The SOD activity of the mice in the 200 mg [kg.sup.-1] [d.sup.-1] dose group was similar to (P > 0.05) that of the mice in the normal control group, but no significant difference (P > 0.05) was found between the SOD activity for the 100 and 200 mg [kg.sup.-1] [d.sup.-1] ODP-Ia treatment groups. The GSH-Px activity of the mice in the positive control and ODP-Ia treatment groups was significantly higher (P < 0.05) than that of the mice in the model control group. The GSH-Px activity in the ODP-Ia treatment groups was similar to (P > 0.05) or higher than (P < 0.05) that in the normal control group. After 21 d of treatment, the MDA levels of the mice in the positive control and ODP-Ia treatment groups were significantly lower (P < 0.05) than those of the mice in the model group. The MDA levels of the mice in the 100 and 200 mg [kg.sup.-1] [d.sup.-1] dose groups were similar (P > 0.05) to those of the mice in the normal control group, but no significant difference (P > 0.05) was found between the MDA levels of the mice in the ODP-Ia treatment groups.
Table 4

Effects of ODP-Ia on antioxidative enzyme and G-6-Pase activity and on
MDA, HG, and insulin levels in the livers of diabetic mice (n = 10,
and mean [+ or -] S.E.).

Group Dose SOD GSH-Px MDA
 activity activity content

 mg U/mg U/mg nmol/mg
 [kg.sup.-1] protein protein protein
 [d.sup.-1]

Normal - 379.42 [+ 114.09 [+ 2.34 [+
control or -] or -] or -]
group 21.02 4.31 (b) 0.77
 (a) (d)

Model - 294.96 [+ 70.92 [+ 4.18 [+
group or -] or -] or -]
 25.60 5.66 (d) 0.44
 (c) (a)

Positive 100 333.93 [+ 88.66 [+ 3.51 [+
control or -] or -] or -]
group 22.19 4.18 (c) 0.38
 (b) (b)

ODP-Ia 50 304.32 [+ 109.15 [+ 3.04 [+
treatment or -] or -] or -]
group 30.82 6.65 (b) 0.60
 (c) (bc)
 100 347.47 [+ 112.05 [+ 2.56 [+
 or -] or -] or -]
 25.85 5.63 (b) 0.58
 (b) (cd)
 200 359.14 [+ 128.94 [+ 2.50 [+
 or -] or -] or -]
 26.10 5.87 (a) 0,26
 (ab) (cd)

Group G-6-Pase activity HG Insulin
 content level

 nmol [min.sup.-1] mg/g mU/l
 mg tissue
 [protein.sup.-1]

Normal 6.74 [+ or -] 0.49 6.69 [+ 34.50 [+
control (b) or -] or -]
group 0.77 1.51
 (ab) (a)

Model 8.49 [+ or -] 1.34 2.63 [+ 12.11 [+
group (a) or -] or -]
 0.61 0.51
 (c) (c)

Positive 5.99 [+ or -] 1.01 6.04 [+ 24.63 [+
control (bc) or -] or -]
group 0.65 1.45
 (b) (b)

ODP-Ia 4.83 [+ or -] 1.20 6.45 [+ 12.43 [+
treatment (c) or -] or -]
group 1.59 0.61
 (ab) (c)

 3.36 [+ or -] 1.76 6.65 [+ 12.72 [+
 (d) or -] or -]
 1.20 0.77
 (ab) (c)

 3.19 [+ or -] 1.83 7.67 [+ 12.88 [+
 (d) or -] or -]
 1.52 0.55
 (a) (c)

A unit of SOD activity was defined as the amount of enzyme that
inhibited the photoreduction of nitro blue tetrazolium chloride (NBT)
by 50%. SOD activity values are given in units per milligram of
protein. The unit of GSH-Px activity was defined as[mu]mol/l of
glutathione consumed per minute. GSH-Px activity values are given
in units per milligram of protein; MDA levels, nmol/mg protein;
G-6-Pase activity, in nmol inorganic phosphate liberated per minute
per milligram protein; HG levels, mg/g tissue; and insulin levels,
mU/1. In each column, values that do not share a common superscript
differ significantly at P < 0.05 (ANOVA followed by DMRT).


Effects of ODP-Ia on C-6-Pase activity and hepatic glycogen levels in the livers of diabetic mice

The G-6-Pase activity in the mice of the model group was higher than that in the mice of the normal control group (Table 4). After 21 d of oral administration, the G-6-Pase activity in the mice of the positive control group was not significantly different (P < 0.05) from that in the mice of the normal control group and ODP-Ia 50 mg [kg.sup.-1] [d.sup.-1] dose group. The G-6-Pase activity in the mice of the 100 and 200 mg [kg.sup.-1], [d.sup.-1] dose groups was lower than (P > 0.05) that in the mice of the other groups, but no significant difference (P > 0.05) was found between the G-6-Pase activity in the mice of the 100 and 200 mg [kg.sup.-1] [d.sup.-1] dose groups. The hepatic glycogen (HG) levels of the mice in the model group were lower (P > 0.05) than those of the mice in the other groups. However, no significant difference (P > 0.05) was observed in the HG levels of the mice in the other groups. The insulin levels of the mice in the normal and positive control groups were higher (P > 0.05) than those of the mice in the model group, but no significant difference was found between the insulin levels of the mice in the model group and ODP-Ia treatment groups (P > 0.05).

Discussion

STZ-induced diabetes is characterized by apoptosis of pancreatic ([beta]-cells and by attenuation of insulin gene expression and reduction of insulin synthesis, which is induced by oxyradicals. Pancreatic ([beta]-cells normally maintain BG concentrations within a narrow range by modulating their insulin secretion rate in response to the glucose concentration in blood (Patel et al., 2006; Yin et al., 2006; Priel et al., 2007; Jiang et al., 2007; George et al., 2002; Zhang et al., 2004). Apoptosis of pancreatic [beta]-cells is traditionally considered the primary factor that ultimately causes hyperglycemia. The symptoms of STZ-induced diabetes include polydipsia; polyphagia; and a severe loss in body weight, which is caused by loss or degradation of structural proteins (Rajkumar et al., 1991). In this study, we investigated the effects of ODP-Ia on a series of biochemical indicators in diabetic mice. We found that ODP-Ia could significantly (P < 0.05) increase the body weight and improve the food and water intake of diabetic mice in a dose-dependent manner.

Decrease in SOD and GSH-Px activity and increase in MDA levels may also accelerate the pathogenesis progress of diabetes (Pari and Satheesh, 2006; Armagan et al., 2006). The hypoglycemic mechanisms of many polysaccharides in Chinese drugs such as Anyctylodes macrocephala Koidz polysaccharides, Cordyceps sinensis polysaccharides and Dentrobium chrysotoxum Lindl polysaccharides are closely related to their antioxidant activities (Shan et al., 2009; Li et al., 2006; Zhao et al., 2007b). A previous study from our laboratory showed that ODPs also have a strong antioxidant property and can scavenge reactive oxygen species and improve the activity of antioxidant enzymes of hyperlipidemic rats in vivo (Yang et al., 2009). In the current study, diabetic mice treated with ODP-Ia showed significantly increased antioxidative ability in the liver. The SOD activity and MDA levels of the mice in the 200 mg [kg.sup.-1] [d.sup.-1] ODP-Ia treatment group were similar (P > 0.05) to those of the mice in the normal control group, but the GSH-Px activity of the mice in 200 mg [kg.sup.-1] [d.sup.-1] ODP-Ia treatment group was higher than that of the mice in the normal control group. The antioxidant activity of ODP-Ia might have played the primary role in the development of diabetes.

The fundamental mechanism underlying hyperglycemia in diabetes mellitus involves overproduction (excessive hepatic glycogenosis and gluconeogenesis) and decreased utilization of glucose by the tissues, and the pathogenesis of diabetes always involves disturbances in carbohydrate, fat, and protein metabolism. These complex multifactorial metabolic changes often lead to damage in functional impairment of many organs, most importantly that of the cardiovascular system, in both types of diabetes (Momose et al., 2002), and the associated disturbances are typically characterized by low HDL-C concentrations, combined with hyperglycemia and hypertriglyceridemia (Valcheva-Kuzmanova et al., 2007; Tunali and Yanardag, 2006; Yeh et al., 2006; Pari and Latha, 2006; Ravi et al., 2005). However, our results indicate that ODP-Ia significantly reduced the BC, TG, and TC concentrations in diabetic mice and significantly increased plasma HDL-C concentrations (P < 0.05); however, these effects developed slowly. In addition, the BG-lowering effect in the ODP-Ia 200 mg kg body [weight.sup.-1] dose group was stronger than that in the 50 and 100 mg kg body [weight.sup.-1] dose groups. ODP-Ia may also play a important role in the recovery of liver function and utilization of glucose as do other polysaccharides in Chinese drugs such as Phellinus linteus polysaccharides and Hedysarum polybotrys polysaccharides (Kim et al., 2001; Hu et al., 2010).

Diabetes is often accompanied by abnormally high BG concentrations and osmotic diuresis. In diabetic patients, the reabsorption capacity of the renal tubules, which plays an important role in calcium and phosphate metabolism, is inhibited. Decrease in serum calcium and phosphate concentrations can cause osteoporosis (Botolin and McCabe, 2007; Yamaguchi et al., 2007). We found that ODP-Ia prevented loss of blood calcium and increase in PUN concentrations in diabetic mice; this may be attributable to the recovery of renal function by ODP-Ia.

G-6-Pase is a key gluconeogenic enzyme (Doi et al., 2007; Lee et al., 2007). In this study, we found that its activity significantly increased in the livers of the diabetic mice, possibly because of insulin deficiency. However, the G-6-Pase activity in the livers of diabetic mice treated with ODP-Ia was significantly lower (P < 0.05) than that in the livers of mice of the model and normal control groups, and the insulin levels in the mice with STZ-induced diabetes were not significantly affected by ODP-Ia. ODP-Ia can decrease glycogenesis as do other polysaccharides in Chinese drugs such as Portulaca oleracea L. polysaccharides and Panax ginseng CA Meyer polysaccharides. However, it did not promote insulin secretion or enhance insulin levels as these polysaccharides do (Li et al., 2009; Yang et al., 1990; Xie et al., 2004). Diabetes mellitus is known to impair the normal liver function of HG synthesis (Golden et al., 1979). The HG levels in diabetic mice treated with ODP-Ia were similar to those in the normal control mice.

Endotoxin (LPS) as a known immunomodulator is often a contaminant in biological preparations. In this study, ODPs were extracted from intact, fresh O. dillenii plants after soaking the plants 5 times for 7-10 d in 80% ethanol. Isolation of ODPs was conducted under conditions that minimized the possibility of bacterial contamination, and ODPs were filtered through 0.22[micro]m filters before injecting into columns every time and before intragastrically administering. Therefore, contamination of LPS was unlikely. We are confident that the results in our study are not a contaminating artifact.

On the basis of this analysis, we propose that ODP-Ia, an effective hypoglycemic ODP, can scavenge reactive oxygen species, thereby protecting liver tissue from peroxidation damage. Furthermore, it aids the recovery of tissue function and improves the sensitivity of target cells to insulin. Thus, ODP-Ia controls the BG and serum lipid levels and regulates the metabolic balance, for example, by regulating carbohydrate, fat, protein, calcium, and phosphate metabolism. On the basis of the results of this study, ODP-Ia merits further evaluation for its clinical potential in diabetes treatment.

Acknowledgements

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0944-7113/$ - see front matter[C] 2011 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2011.01.001

* Corresponding author at: College of Life Science and Technology, Zhanjiang Normal University, Cunjin Road No. 29, Zhanjiang 524048, Guangdong Province, China. Tel.: +86 13828288702; fax: +86 07593183271.

E-mail address: zengfuhua@gmail.com (F.H, Zeng).

(1) The first and second authors have contributed equally to this work.

L.Y. Zhao (a), (b), (1), Q.J. Lan (a), (b), (1), Z.C. Huang (a), L.J. Ouyang (a), F.H. Zeng (a), *

(a) College of Life Science and Technology, Zhanjiang Normal University, Zhanjiang 524048, Guangdong, China

(b) College of Biological Science and Technology, Hunan Agricultural University, Changsha, Hunan, China
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Author:Zhao, L.Y.; Lan, Q.J.; Huang, Z.C.; Ouyang, L.J.; Zeng, F.H.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Jun 15, 2011
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