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Extraction and purification of pumpkin polysaccharides and their hypoglycemic effect
International Journal of Biological Macromolecules 98 (2017) 182–187
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Extraction and purification of pumpkin polysaccharides and their hypoglycemic effect Shuang Wang, Aoxue Lu, Lu Zhang, Meng Shen, Tian Xu, Wangyang Zhan, Hui Jin, Yongjun Zhang ∗ , Weimin Wang College of Life Sciences, China JiLiang University, Hangzhou, Zhejiang, 310018, China
a r t i c l e
i n f o
Article history: Received 15 December 2016 Received in revised form 13 January 2017 Accepted 25 January 2017 Available online 30 January 2017 Keywords: Pumpkin polysaccharide Anti-diabetic effects Proliferation activity
a b s t r a c t The anti-diabetic activity of the polysaccharides (PPs) obtained from the dried pumpkin pulp was studied in this paper. The PPs were administered by intraperitoneal injection to the alloxan-induced diabetic male ICR mice. The PPs hypoglycemic effect was evaluated by testing the fast blood glucose level, fasting serum insulin and hepatic glycogen. After 7 h administration, the PPs showed a significantly hypoglycemic effect (p < 0.01) and could significantly increase hepatic glycogen and insulin level (p < 0.05). PPs-e with potential to enhance the islet cells proliferation activity by MTT assays in vitro was obtained from the PPs through alcohol fractional precipitation and gel chromatography. PPs-e composed of rhamnose, arabinose, glucose, galactose, and little amount of inositol could maintain the blood glucose at a low level in diabetic mice and could even last for more than 24 h. These results suggest the potential hypoglycemic effect of the pumpkin polysaccharides in alloxan-induced diabetic mice and for the treatment of diabetic mellitus. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a group of endocrine and metabolic disorder diseases characterized by high blood glucose, which might lead to disturbances of carbohydrate, fat, and protein metabolism [1,2]. At present, the common treatments for DM are insulin and oral hypoglycemic agents, such as biguanides, sulfonylureas, and thiazolidinediones, which can control hyperglycemia effectively. However, those drugs might also induce some significant side effects, such as, weight gain and gastrointestinal disturbance. As a result, it is very urgent to search for a new kind of natural product with less side effects. The pumpkin (Cucurbita moschata) pulp belonging to an annual herbaceous plant of the family Cucurbitaceae has been frequently used as functional food or medicine [3] and is one of the most popular vegetables in the world [4]. Pumpkin powder is rich in nutrition [5]. For hundreds of years, pumpkin has been regarded as a folk medicine used for prevention various human diseases, such as hypolipidemic, hypoglycemia and antioxidant activities [6–8]. It has been reported that the polysaccharides from pumpkin
∗ Corresponding author at: College of Life Sciences, China JiLiang University, Xueyuan Street, Xiasha, Hangzhou, Zhejiang, 310018, PR China. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.ijbiomac.2017.01.114 0141-8130/© 2017 Elsevier B.V. All rights reserved.
fruit had hypoglycemic activity by increasing plasma insulin in the normal and diabetic mice, and protected islets cells from streptozotocin (STZ) injury in vitro via increasing the levels of super-oxide dismutase (SOD) and malondialdehyde (MDA) and reducing the production of NO [9,10]. Our previous studies have investigated the hypoglycemic effects of the pumpkin crude polysaccharides [11,12], However, the hypoglycemic effects of more pure pumpkin polysaccharides and the probable hypoglycemic mechanisms were still remain elusive. The present study was performed to evaluate the effects of polysaccharides isolated from pumpkin step by step on the alloxan-induced diabetic mice by testing the fast blood glucose level, fasting serum insulin and hepatic glycogen. Furthermore, the effects of pumpkin polysaccharides on the activity of injured islet cells in vitro were also investigated as well.
2. Materials and methods 2.1. Plant materials and chemicals Pumpkins (Cucurbita moschata) were purchased from the local market (Hangzhou, China), and selected for their uniformity in shape, weight and color. This specie of pumpkin is unique in southern China. The fresh pumpkin was peeled, seeded and sliced into pieces (0.3 cm*0.5 cm*3.0 cm). The slices were dried under sun light
S. Wang et al. / International Journal of Biological Macromolecules 98 (2017) 182–187
for two days until completely dry. Dried slices were ground into powder. The islet cells (NIT-1) were purchased from Bioleaf Biotech Co., Ltd (Shanghai, China). Alloxan and Methyl thiazolyl tetrazolium (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal calf serum (FCS) was purchased from Thermo Fisher Scientific. GLP-1 was purchased from Shanghai Baiyu Co. (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), dimethyl sulfoxide (DMSO) and trypsin 0.25% solution were purchased from Hefei Bomei Biotechnology Co. (Hefei, China). 2.2. Composition analysis of pumpkin powder
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rhamnose. The relative molar ratios of monosaccharides were analyzed using the area normalization method. Inositol and amino sugar composition were determined using chemical titration and Morgan-Elson method, respectively. 2.6. Animals and their care Male ICR mice (22 ± 2 g) were obtained from Hangzhou Normal University. All mice were acclimated for 7 days. They were free access to tap water and standard laboratory diet under conditions of temperature (25 ± 2 ◦ C) and a 12 h light-dark cycle. (the animal center of Hangzhou Normal University), The experiment was approved by animal ethics guidelines of the Institutional Animal Ethics Committee.
The basic ingredients of pumpkin powder were measured accordingly. Ash was quantified gravimetrically after 12 h at 550 ◦ Cand then 4 h at 900 ◦ C for the further processing. Total content of lipids was determined gravimetrically by extraction with diethyl ether using a Soxhlet apparatus. Total nitrogen content was determined by the Kjeldahl method using Tecator equipment (digester model 2020 and Distillation and Tritation Kjeltec 1035/38 system). Then removed the nitrogen from glucosamine, and protein content was estimated by the factor 6.25, converting from nitrogen. Reducing sugar was determined by 3, 5-dinitrosalicylic acid colorimetry. Total carbohydrate content was estimated by [100(moisture + crude protein + ash + crude fat)]%.
The diabetic mice were induced by an intraperitoneal injection with alloxan (200 mg/kg body weight, freshly prepared in sodium chloride injection, 5%). Alloxan-induced diabetes mice were allowed free access to food and water until the start of the experiment. Five days later, all alloxan-injected mice were assessed by measuring glucose levels in tail vein. Glucose levels over 15.6 mmol/L were considered as diabetic.
2.3. Preparation of pumpkin crude polysaccharides
2.8. Experimental design
The pumpkin powders were extracted by distilled water and ethanol, and purified by aether and n-butyl alcohol step by step. Deproteinization was performed with sevage’ reagent. After concentration and filtration, absolute ethanol was added to the filtrate to make a final 80% ethanol. The filtrates were precipitated at 4 ◦ C over night, pumpkin crude polysaccharides (PPs) were obtained after centrifugation, and freeze-dried after being dialysed. Protein in PPs were not detected with the Brabender colorimetric method. The pumpkin powders were extracted with hot distilled water and ethanol. The aqueous extract was purified by aether and n-butyl alcohol step by step. Deproteinization was performed with sevag reagent. After concentration and filtration, 4 folds of cold anhydrous ethanol was added to the filtrate which kept overnight at 4 ◦ C. The crude polysaccharides (PPs) were obtained after centrifugation and lyophilization. Protein in PPs were not detected using Bradford colorimetric method.
The mice used for the experiment were randomly divided into 10 groups (5 mice per group). The groups were Group I: normal mice as control group and injected with saline (0.86% NaCl). Group II: alloxan-induced diabetic mice, the model group and injected with 0.86% NaCl only. Group III: alloxan-induced diabetic mice were administered Xiaoke pill (the Chinese medicine pill is widely used in the clinical treatment of diabetes in China) at 750 mg/kg of BW in 0.86% NaCl, as positive control group. Group IV: alloxan-induced diabetic mice administered 150 mg/kg of BW PPs by intraperitoneal injection. The dose of PPs was selected based on the results of our pilot study (data not shown). Group V ∼ X: alloxan-induced diabetic mice administered 150 mg/kg of BW PP-a, -b, -c, -d, -e, -f, respectively.
2.4. Isolation and purification of PPs PPs (approximate 200 mg) were re-dissolved in distilled water. After centrifugation, the polysaccharides in the supernatant were purified according to the method in Fig. 1. 2.5. Analysis of polysaccharides The monosaccharide compositions of PP-e was determined by gas chromatography that using the previous method [11] with some modification. The dried PP-e (20 mg) was hydrolyzed with 2.0 mL HCl (2 mol/L) at 105 ◦ C for 4 h. Then, the hydrolyzed product was cooled to room temperature and 2 mol/L NaOH was added to neutralise it. The distilled water was used to dialyze for 24 h and then taken away by vacuum drying. The sample, including 0.6 mL pyridine and 10 mg hydroxylamine hydrochloride, was incubated at 90 ◦ C for 30 min, then cooled down and added 0.6 mL acetic oxide, then was incubated at 90 ◦ C for another 30 min. The 0.22 M membrane was used for the supernatant filtration. Monosaccharide compositions were analyzed by comparing with six standard sugars including fucose, arabinose, mannose, glucose, galactose and
2.7. Induction of the diabetic mice model
2.9. Biochemical analysis Blood glucose (BG) levels in tail vein before and after 7 h intraperitoneal injection were determined using a glucose meter. Insulin concentration in serum was determined using commercial mouse insulin ELISA detection kits. Liver was homogenized, then centrifuged for 10 min at 3000 rpm. The supernatant was collected for determination of hepatic glycogen by anthrone spectrophotometric method [13]. 2.10. Cell viability assay Subcultured NIT-1 was cultured in DMEM supplemented with 10% FCS, 100 units/mL penicillin, 100 g/ml streptomycin and grown in a humidified incubator containing 5% CO2 and 95% air at 37 ◦ C. The cells were plated in 96-wells plate and incubated for 24 h, and then changed to dosing medium containing PPe from 0.1 to 5.0 mg/ml for 4 h exposure according to different experiments described in Table 1. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were incubated with 5 mg/ml MTT at 37 ◦ C for 4 h. After this, the medium was removed and replaced with 200 L of DMSO. After
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Fig. 1. The isolation and purification of the pumpkin polysaccharides.
Table 1 The handing methods on different cell groups. Groups
Handing Methods
Control DM GLP-1 PPe-1 PPe-2 PPe-3
Do not add any processing factors Only Alloxan(final concentration was 32 mmo1/L) Add Alloxan and GLP-1(final concentration of GLP-1 was 50 nmol/L) Add Alloxan and PPe (final concentration of PPe was 0.1 mg/ml) Add Alloxan and PPe (final concentration of PPe was 1.0 mg/ml) Add Alloxan and PPe (final concentration of PPe was 5.0 mg/ml)
general pipetting, absorbance was measured at 490 nm on the microplate reader (BIO-RAD, JAPAN). 2.11. Statistical analysis Data were expressed as means ± standard deviations of three replicated determinations. One way of variance analysis was applied for determining significant difference at P < 0.05 between the results. 3. Results and discussion 3.1. The chemical compositions of pumpkin powder The main nutritional components of the pumpkin powders are shown in Table 2. As shown in Table 2, pumpkin powders were rich in carbohydrates, protein, minerals. The total carbohydrate was the highest components in pumpkin powders. In carbohydrate part, the concentration of reducing sugar is high. Therefore, the reducing sugar should be removed as far in order to avoid the influence of the blood glucose. 3.2. Effects of PPs on the levels of BG, hepatic glycogen and insulin in alloxan- induced diabetic mice 3.2.1. Dynamic changes of BG levels in alloxan-induced diabetic mice During the alloxan intraperitoneal injection for 5 d, the dynamic changes of BG levels within 1 d are shown in Table 3.
Alloxan(2,4,5,6,tetraoxohexahydropyr- imidine) is a known compound which induced an insulin deficiency in most animal experimental models with some remarkable similarities to the human type 1 diabetes mellitus [14]. This diabetogenic agent is a hydrophilic, and is toxic to pancreatic -cells [15]. There is a possibility for the survival of a few -cells and this has been proved by several research groups who observed antihyperglycemic activity with oral hypoglycemic agents in alloxan induced diabetic rats [16,17]. As shown in Table 3, a stable high blood glucose model formed after 5 d alloxan injection, and there was no significant difference in blood glucose in mice within 1 d (P > 0.05).
3.2.2. Effects of PPs on BG, glycogen and insulin level in diabetic mice The effects of PPs on BG, glycogen and insulin level of diabetic mice induced by alloxan were shown in Table 4. As shown in Table 4, BG levels in the positive group (III) showed that Xiaoke pill had significantly hypoglycemic effect (p < 0.05) compared to the negative group. Meanwhile PPs treatment also caused significantly hypoglycemic effect compared to the negative group (p < 0.01). However, Xiaoke pill had no effect on liver glycogen and insulin level of mice (P > 0.05). While PPs had significantly effect on liver glycogen and insulin level of mice (P < 0.05) compared to the negative group. The liver was the only organ that regulates the metabolism of sugar in the body. When glucoses ingested into body, they entered Krebs cycle by glycolysis, and then released energy by oxidation decomposition completely. Meanwhile, the glycogens of synthesis by glucose-1-phosphoric acid were stored. The increase of hepatic glucose output (HGO) was a main pathological features for type 2 diabetes, and it could be used for maintaining the blood glucose levels at the starvation condition. The core of glycogen metabolism was the cycle of “glucose and glucose 6-phosphate”, which were associated with glycogen metabolism and gluconeogenesis through 6-phosphate glucose (Fig. 2). Glycogen output for type 2 diabetes was positively correlated with fasting glucose, which is the main reason for the fasting hyperglycemia of type 2 diabetes. From the experimental data in Table 4, pumpkin polysaccharides could restore liver glycogen and insulin level to the normal value, and decrease the blood sugar levels significantly (P < 0.01), suggesting pumpkin polysaccharides played an
S. Wang et al. / International Journal of Biological Macromolecules 98 (2017) 182–187
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Table 2 The main nutritional components of the pumpkin powders.
pumpkin powder(%)
Moisture
Crude protein
Ash
Crude fat
Reducing sugar
Total carbohydrate
10.16 ± 0.19
13.61 ± 0.27
5.11 ± 0.08
3.24 ± 0.05
32.28 ± 0.31
67.88
Table 3 The dynamic changes of BG levels within 1 d in alloxan-induced diabetic mice. Time (h)
0
1
2
4
8
24
BG level (mmol/L)
22.54 ± 4.28
23.34 ± 4.19
23.91 ± 4.37
24.13 ± 4.81
23.71 ± 4.58
22.71 ± 4.35
Table 4 The effects of PPs on BG, glycogen and insulin level in alloxan-diabetic mice. Group
Liver glycogen (mg/g)
Pancreatic insulin (mIU/L)
Group I (Control) Group II (Negative) Group III (Positive) Group IV (PPs)
31.16 ± 4.24* 20.14 ± 5.22 22.54 ± 4.55 32.91 ± 5.11*
26.51 ± 5.22* 17.42 ± 7.42 21.02 ± 8.32 29.11 ± 8.76*
* **
BG levels (mmol/L) 0h
7h
5.12 ± 0.25 22.27 ± 5.02 22.67 ± 4.94 23.33 ± 4.14
5.37 ± 0.25 22.88 ± 4.73 15.81 ± 3.16* 10.41 ± 7.18**
p < 0.05, compared to the negative group, n = 5, means ± SD. p < 0.01, compared to the negative group, n = 5, means ± SD. Table 6 Effects of PPs-e and PPs-f on BG level in alloxan-induced diabetic mice x¯ ± SD(mmol/l). Group
PPs-e PPs-f * **
Time (h) 0
4
8
12
24
24.3 ± 1.94 24.7 ± 2.80
13.7 ± 7.64* 12.7 ± 4.47*
5.43 ± 6.57** 5.48 ± 1.41**
1.65 ± 0.93** 13.6 ± 9.98*
1.90 ± 1.05** 22.0 ± 7.08*
p < 0.05, compared to the negative group, n = 5, means ± SD. p < 0.01, compared to the negative group, n = 5, means ± SD.
3.4. Effects of two components (PPs-e, -f) on the levels of BG in alloxan- induced diabetic mice Fig. 2. The circulation and transformation of glycogen.
Table 5 Effects of PPs-a, PPs-b, PPs-c and PPs-d on BG level in alloxan-induced diabetic mice¯x ± SD(mmol/l). Group
PPs-a PPs-b PPs-c PPs-d * **
Time(h) 0
7
13.18 ± 4.65 12.94 ± 3.63 12.88 ± 3.34 12.88 ± 2.61
8.48 ± 1.59* 8.00 ± 2.81* 5.40 ± 1.23** 4.74 ± 0.73**
p < 0.05, compared to the negative group, n = 5, means ± SD. p < 0.01, compared to the negative group, n = 5, means ± SD.
positive role to improve glucose metabolism and inhibit glycogen output.
The effects of two components (PPs-e, PPs-f) on the levels of BG in diabetic mice are shown in Table 6. The two components (PPs-e, Ps-f) could reduce hyperglycemia to normal levels within 8 h in alloxan-induced diabetic mice, and the effect of PPs-e on diabetic mice was more persistent. PPs-e could remain blood sugar in diabetic mice at low levels for more than 24 h. PPs-f could gradually reduce blood sugar in diabetic mice within 8 h, and BG levels in mice start to increase after 12 h. After 24 h, they returned to high values. Compared with Sephadex G-200 gel column elution curves, it showed that the peak volume of PPs-f was almost equal to the dead volume of the column, which could be a rough estimate that the molecular weight of PPs-f was not less than 105 Da. The retention time of PPs-e was later than PPs-f, speculating that the molecular weight of PPs-e should be far less than 105 Da. Consequently, the significant hypoglycemic effect of PPs-e mainly related to the small molecules of pumpkin polysaccharide. So, the next step was to analyze the molecular composition of PPs-e.
3.3. Effects of four components (PPs – a, b, c, d) on the levels of BG in alloxan-induced diabetic mice
3.5. Effect of PPs-e on islet cells proliferation activity in vitro
The effects of four components (PPs -a, -b, -c, -d) on the levels of BG in diabetic mice are shown in Table 5. All four components had the effect of lowering blood glucose. The effects of PPs-c and PPs-d were significant at 0.01 level, while the effects of PPs-a and PPs-b were significant at 0.05 level. During alcohol fractionated precipitation, the yield of PPs-c was higher than that in PPs-d, so PPs-c would be used and purified in further experiment.
Effect of PPs-e on the cell proliferation in islet cells were shown in Table 7. According to Table 7, alloxan significantly reduced islet cell proliferation activity compared with the control group. GLP-1 is a kind of peptide hormone secreted by the intestinal L cells, which has good therapeutic effect on diabetes. GLP-1 obviously enhanced the proliferation in alloxan-injured islet cell. Someone showed that GLP-1 promoted islet  cells to secrete insulin and reduced islet
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Table 7 Effect of PPs-e on the proliferation activity of islet cells. Group
Control
DM
GLP-1
PPe-I
PPe-II
PPeIII
A490
0.86 ± 0.01**
0.50 ± 0.07
0.58 ± 0.03
0.56 ± 0.04
0.55 ± 0.09
0.60 ± 0.04*
* **
p < 0.05, compared to the DM group, n = 5, means ± SD. p < 0.01, compared to the DM group, n = 5, means ± SD.
Table 8 Mixed monosaccharide standard equation of regression. Monosaccharides
Retension time/min
Equation of regression
R2
Linear range
Rhamnose Fucose Arabinose Mannose Glucose Galactose
10.132 10.643 10.803 12.935 13.313 13.520
Y = 56137x + 3566.3 Y = 90828x-8265.5 Y = 46246x + 2011.1 Y = 89954x-6919.3 Y = 85083x-8765.1 Y = 79545x-8697.5
0.9984 0.9954 0.9983 0.9986 0.9985 0.9976
0.5–109.8 mol/mL 0.3–133.2 mol/mL 0.3–52.2 mol/mL 0.2–47.6 mol/mL 0.2–43.3 mol/mL 0.2–47.6 mol/mL
Y refers to peak area. X is the concentration of monosaccharide mmol/mL.
levels compared to the negative group (P < 0.01). It can be speculated from the present study that the pumpkin polysaccharides would potentially be beneficial to improve the therapy of diabetes mellitus, and might be used as a functional and nutraceutical food ingredient. Conflict of interest The authors have declared that no conflict of interest exist. Fig. 3. GC chromatogram of PP-e.
␣ cells to secrete glucagon with different concentration of glucose [18]. The low concentration was not significant compared with DM group (P > 0.05), while high concentration was significant (P < 0.05) and showed dependence with PPs-e dose on islet cel proliferation. The results suggested that pumpkin polysaccharide could partly restore injury in pancreatic islet cells induced by alloxan. 3.6. Analysis of molecular composition of PPs-e The retention time of mixed monosaccharides standard were shown in Table 8, the gas chromatograms of the sample as shown in Fig. 3. Based on the mixed monosaccharide retention time, the main components of PPs-e were rhamnose, arabinose, glucose and galactose. The approximate ratio of rhamnose, arabinose, glucose, galactose was 1.12:5.19: 1.00: 3.91. PPs-e contained little amounts of inositol according to the result of chemical titration, while no amino sugar was found, based on Morgan-Elson method. 4. Conclusions In this experiment, solvent extraction (water and alcohol) and organic solvent fractional extraction were used to extract crude polysaccharides (PPs) from the dried pumpkin pulp. PPs-e was obtained by using alcohol fractional precipitation and Sephadex G-200 gel chromatography. PPs-e was a type of heteropolysaccharide with rhamnose, arabinose, glucose and galactose as its main compositions and little amount inositol. In the in vitro test, the pumpkin polysaccharides had the potential to enhance the islet cells proliferation activity. By using the alloxan-induced diabetic male ICR mice as the model, administration of the pumpkin polysaccharides could make liver glycogen and insulin back to the normal level and significantly lowered the BG
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Extraction and purification of pumpkin polysaccharides and their hypoglycemic effect Shuang Wang, Aoxue Lu, Lu Zhang, Meng Shen, Tian Xu, Wangyang Zhan, Hui Jin, Yongjun Zhang ∗ , Weimin Wang College of Life Sciences, China JiLiang University, Hangzhou, Zhejiang, 310018, China
a r t i c l e
i n f o
Article history: Received 15 December 2016 Received in revised form 13 January 2017 Accepted 25 January 2017 Available online 30 January 2017 Keywords: Pumpkin polysaccharide Anti-diabetic effects Proliferation activity
a b s t r a c t The anti-diabetic activity of the polysaccharides (PPs) obtained from the dried pumpkin pulp was studied in this paper. The PPs were administered by intraperitoneal injection to the alloxan-induced diabetic male ICR mice. The PPs hypoglycemic effect was evaluated by testing the fast blood glucose level, fasting serum insulin and hepatic glycogen. After 7 h administration, the PPs showed a significantly hypoglycemic effect (p < 0.01) and could significantly increase hepatic glycogen and insulin level (p < 0.05). PPs-e with potential to enhance the islet cells proliferation activity by MTT assays in vitro was obtained from the PPs through alcohol fractional precipitation and gel chromatography. PPs-e composed of rhamnose, arabinose, glucose, galactose, and little amount of inositol could maintain the blood glucose at a low level in diabetic mice and could even last for more than 24 h. These results suggest the potential hypoglycemic effect of the pumpkin polysaccharides in alloxan-induced diabetic mice and for the treatment of diabetic mellitus. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a group of endocrine and metabolic disorder diseases characterized by high blood glucose, which might lead to disturbances of carbohydrate, fat, and protein metabolism [1,2]. At present, the common treatments for DM are insulin and oral hypoglycemic agents, such as biguanides, sulfonylureas, and thiazolidinediones, which can control hyperglycemia effectively. However, those drugs might also induce some significant side effects, such as, weight gain and gastrointestinal disturbance. As a result, it is very urgent to search for a new kind of natural product with less side effects. The pumpkin (Cucurbita moschata) pulp belonging to an annual herbaceous plant of the family Cucurbitaceae has been frequently used as functional food or medicine [3] and is one of the most popular vegetables in the world [4]. Pumpkin powder is rich in nutrition [5]. For hundreds of years, pumpkin has been regarded as a folk medicine used for prevention various human diseases, such as hypolipidemic, hypoglycemia and antioxidant activities [6–8]. It has been reported that the polysaccharides from pumpkin
∗ Corresponding author at: College of Life Sciences, China JiLiang University, Xueyuan Street, Xiasha, Hangzhou, Zhejiang, 310018, PR China. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.ijbiomac.2017.01.114 0141-8130/© 2017 Elsevier B.V. All rights reserved.
fruit had hypoglycemic activity by increasing plasma insulin in the normal and diabetic mice, and protected islets cells from streptozotocin (STZ) injury in vitro via increasing the levels of super-oxide dismutase (SOD) and malondialdehyde (MDA) and reducing the production of NO [9,10]. Our previous studies have investigated the hypoglycemic effects of the pumpkin crude polysaccharides [11,12], However, the hypoglycemic effects of more pure pumpkin polysaccharides and the probable hypoglycemic mechanisms were still remain elusive. The present study was performed to evaluate the effects of polysaccharides isolated from pumpkin step by step on the alloxan-induced diabetic mice by testing the fast blood glucose level, fasting serum insulin and hepatic glycogen. Furthermore, the effects of pumpkin polysaccharides on the activity of injured islet cells in vitro were also investigated as well.
2. Materials and methods 2.1. Plant materials and chemicals Pumpkins (Cucurbita moschata) were purchased from the local market (Hangzhou, China), and selected for their uniformity in shape, weight and color. This specie of pumpkin is unique in southern China. The fresh pumpkin was peeled, seeded and sliced into pieces (0.3 cm*0.5 cm*3.0 cm). The slices were dried under sun light
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for two days until completely dry. Dried slices were ground into powder. The islet cells (NIT-1) were purchased from Bioleaf Biotech Co., Ltd (Shanghai, China). Alloxan and Methyl thiazolyl tetrazolium (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal calf serum (FCS) was purchased from Thermo Fisher Scientific. GLP-1 was purchased from Shanghai Baiyu Co. (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), dimethyl sulfoxide (DMSO) and trypsin 0.25% solution were purchased from Hefei Bomei Biotechnology Co. (Hefei, China). 2.2. Composition analysis of pumpkin powder
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rhamnose. The relative molar ratios of monosaccharides were analyzed using the area normalization method. Inositol and amino sugar composition were determined using chemical titration and Morgan-Elson method, respectively. 2.6. Animals and their care Male ICR mice (22 ± 2 g) were obtained from Hangzhou Normal University. All mice were acclimated for 7 days. They were free access to tap water and standard laboratory diet under conditions of temperature (25 ± 2 ◦ C) and a 12 h light-dark cycle. (the animal center of Hangzhou Normal University), The experiment was approved by animal ethics guidelines of the Institutional Animal Ethics Committee.
The basic ingredients of pumpkin powder were measured accordingly. Ash was quantified gravimetrically after 12 h at 550 ◦ Cand then 4 h at 900 ◦ C for the further processing. Total content of lipids was determined gravimetrically by extraction with diethyl ether using a Soxhlet apparatus. Total nitrogen content was determined by the Kjeldahl method using Tecator equipment (digester model 2020 and Distillation and Tritation Kjeltec 1035/38 system). Then removed the nitrogen from glucosamine, and protein content was estimated by the factor 6.25, converting from nitrogen. Reducing sugar was determined by 3, 5-dinitrosalicylic acid colorimetry. Total carbohydrate content was estimated by [100(moisture + crude protein + ash + crude fat)]%.
The diabetic mice were induced by an intraperitoneal injection with alloxan (200 mg/kg body weight, freshly prepared in sodium chloride injection, 5%). Alloxan-induced diabetes mice were allowed free access to food and water until the start of the experiment. Five days later, all alloxan-injected mice were assessed by measuring glucose levels in tail vein. Glucose levels over 15.6 mmol/L were considered as diabetic.
2.3. Preparation of pumpkin crude polysaccharides
2.8. Experimental design
The pumpkin powders were extracted by distilled water and ethanol, and purified by aether and n-butyl alcohol step by step. Deproteinization was performed with sevage’ reagent. After concentration and filtration, absolute ethanol was added to the filtrate to make a final 80% ethanol. The filtrates were precipitated at 4 ◦ C over night, pumpkin crude polysaccharides (PPs) were obtained after centrifugation, and freeze-dried after being dialysed. Protein in PPs were not detected with the Brabender colorimetric method. The pumpkin powders were extracted with hot distilled water and ethanol. The aqueous extract was purified by aether and n-butyl alcohol step by step. Deproteinization was performed with sevag reagent. After concentration and filtration, 4 folds of cold anhydrous ethanol was added to the filtrate which kept overnight at 4 ◦ C. The crude polysaccharides (PPs) were obtained after centrifugation and lyophilization. Protein in PPs were not detected using Bradford colorimetric method.
The mice used for the experiment were randomly divided into 10 groups (5 mice per group). The groups were Group I: normal mice as control group and injected with saline (0.86% NaCl). Group II: alloxan-induced diabetic mice, the model group and injected with 0.86% NaCl only. Group III: alloxan-induced diabetic mice were administered Xiaoke pill (the Chinese medicine pill is widely used in the clinical treatment of diabetes in China) at 750 mg/kg of BW in 0.86% NaCl, as positive control group. Group IV: alloxan-induced diabetic mice administered 150 mg/kg of BW PPs by intraperitoneal injection. The dose of PPs was selected based on the results of our pilot study (data not shown). Group V ∼ X: alloxan-induced diabetic mice administered 150 mg/kg of BW PP-a, -b, -c, -d, -e, -f, respectively.
2.4. Isolation and purification of PPs PPs (approximate 200 mg) were re-dissolved in distilled water. After centrifugation, the polysaccharides in the supernatant were purified according to the method in Fig. 1. 2.5. Analysis of polysaccharides The monosaccharide compositions of PP-e was determined by gas chromatography that using the previous method [11] with some modification. The dried PP-e (20 mg) was hydrolyzed with 2.0 mL HCl (2 mol/L) at 105 ◦ C for 4 h. Then, the hydrolyzed product was cooled to room temperature and 2 mol/L NaOH was added to neutralise it. The distilled water was used to dialyze for 24 h and then taken away by vacuum drying. The sample, including 0.6 mL pyridine and 10 mg hydroxylamine hydrochloride, was incubated at 90 ◦ C for 30 min, then cooled down and added 0.6 mL acetic oxide, then was incubated at 90 ◦ C for another 30 min. The 0.22 M membrane was used for the supernatant filtration. Monosaccharide compositions were analyzed by comparing with six standard sugars including fucose, arabinose, mannose, glucose, galactose and
2.7. Induction of the diabetic mice model
2.9. Biochemical analysis Blood glucose (BG) levels in tail vein before and after 7 h intraperitoneal injection were determined using a glucose meter. Insulin concentration in serum was determined using commercial mouse insulin ELISA detection kits. Liver was homogenized, then centrifuged for 10 min at 3000 rpm. The supernatant was collected for determination of hepatic glycogen by anthrone spectrophotometric method [13]. 2.10. Cell viability assay Subcultured NIT-1 was cultured in DMEM supplemented with 10% FCS, 100 units/mL penicillin, 100 g/ml streptomycin and grown in a humidified incubator containing 5% CO2 and 95% air at 37 ◦ C. The cells were plated in 96-wells plate and incubated for 24 h, and then changed to dosing medium containing PPe from 0.1 to 5.0 mg/ml for 4 h exposure according to different experiments described in Table 1. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were incubated with 5 mg/ml MTT at 37 ◦ C for 4 h. After this, the medium was removed and replaced with 200 L of DMSO. After
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Fig. 1. The isolation and purification of the pumpkin polysaccharides.
Table 1 The handing methods on different cell groups. Groups
Handing Methods
Control DM GLP-1 PPe-1 PPe-2 PPe-3
Do not add any processing factors Only Alloxan(final concentration was 32 mmo1/L) Add Alloxan and GLP-1(final concentration of GLP-1 was 50 nmol/L) Add Alloxan and PPe (final concentration of PPe was 0.1 mg/ml) Add Alloxan and PPe (final concentration of PPe was 1.0 mg/ml) Add Alloxan and PPe (final concentration of PPe was 5.0 mg/ml)
general pipetting, absorbance was measured at 490 nm on the microplate reader (BIO-RAD, JAPAN). 2.11. Statistical analysis Data were expressed as means ± standard deviations of three replicated determinations. One way of variance analysis was applied for determining significant difference at P < 0.05 between the results. 3. Results and discussion 3.1. The chemical compositions of pumpkin powder The main nutritional components of the pumpkin powders are shown in Table 2. As shown in Table 2, pumpkin powders were rich in carbohydrates, protein, minerals. The total carbohydrate was the highest components in pumpkin powders. In carbohydrate part, the concentration of reducing sugar is high. Therefore, the reducing sugar should be removed as far in order to avoid the influence of the blood glucose. 3.2. Effects of PPs on the levels of BG, hepatic glycogen and insulin in alloxan- induced diabetic mice 3.2.1. Dynamic changes of BG levels in alloxan-induced diabetic mice During the alloxan intraperitoneal injection for 5 d, the dynamic changes of BG levels within 1 d are shown in Table 3.
Alloxan(2,4,5,6,tetraoxohexahydropyr- imidine) is a known compound which induced an insulin deficiency in most animal experimental models with some remarkable similarities to the human type 1 diabetes mellitus [14]. This diabetogenic agent is a hydrophilic, and is toxic to pancreatic -cells [15]. There is a possibility for the survival of a few -cells and this has been proved by several research groups who observed antihyperglycemic activity with oral hypoglycemic agents in alloxan induced diabetic rats [16,17]. As shown in Table 3, a stable high blood glucose model formed after 5 d alloxan injection, and there was no significant difference in blood glucose in mice within 1 d (P > 0.05).
3.2.2. Effects of PPs on BG, glycogen and insulin level in diabetic mice The effects of PPs on BG, glycogen and insulin level of diabetic mice induced by alloxan were shown in Table 4. As shown in Table 4, BG levels in the positive group (III) showed that Xiaoke pill had significantly hypoglycemic effect (p < 0.05) compared to the negative group. Meanwhile PPs treatment also caused significantly hypoglycemic effect compared to the negative group (p < 0.01). However, Xiaoke pill had no effect on liver glycogen and insulin level of mice (P > 0.05). While PPs had significantly effect on liver glycogen and insulin level of mice (P < 0.05) compared to the negative group. The liver was the only organ that regulates the metabolism of sugar in the body. When glucoses ingested into body, they entered Krebs cycle by glycolysis, and then released energy by oxidation decomposition completely. Meanwhile, the glycogens of synthesis by glucose-1-phosphoric acid were stored. The increase of hepatic glucose output (HGO) was a main pathological features for type 2 diabetes, and it could be used for maintaining the blood glucose levels at the starvation condition. The core of glycogen metabolism was the cycle of “glucose and glucose 6-phosphate”, which were associated with glycogen metabolism and gluconeogenesis through 6-phosphate glucose (Fig. 2). Glycogen output for type 2 diabetes was positively correlated with fasting glucose, which is the main reason for the fasting hyperglycemia of type 2 diabetes. From the experimental data in Table 4, pumpkin polysaccharides could restore liver glycogen and insulin level to the normal value, and decrease the blood sugar levels significantly (P < 0.01), suggesting pumpkin polysaccharides played an
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Table 2 The main nutritional components of the pumpkin powders.
pumpkin powder(%)
Moisture
Crude protein
Ash
Crude fat
Reducing sugar
Total carbohydrate
10.16 ± 0.19
13.61 ± 0.27
5.11 ± 0.08
3.24 ± 0.05
32.28 ± 0.31
67.88
Table 3 The dynamic changes of BG levels within 1 d in alloxan-induced diabetic mice. Time (h)
0
1
2
4
8
24
BG level (mmol/L)
22.54 ± 4.28
23.34 ± 4.19
23.91 ± 4.37
24.13 ± 4.81
23.71 ± 4.58
22.71 ± 4.35
Table 4 The effects of PPs on BG, glycogen and insulin level in alloxan-diabetic mice. Group
Liver glycogen (mg/g)
Pancreatic insulin (mIU/L)
Group I (Control) Group II (Negative) Group III (Positive) Group IV (PPs)
31.16 ± 4.24* 20.14 ± 5.22 22.54 ± 4.55 32.91 ± 5.11*
26.51 ± 5.22* 17.42 ± 7.42 21.02 ± 8.32 29.11 ± 8.76*
* **
BG levels (mmol/L) 0h
7h
5.12 ± 0.25 22.27 ± 5.02 22.67 ± 4.94 23.33 ± 4.14
5.37 ± 0.25 22.88 ± 4.73 15.81 ± 3.16* 10.41 ± 7.18**
p < 0.05, compared to the negative group, n = 5, means ± SD. p < 0.01, compared to the negative group, n = 5, means ± SD. Table 6 Effects of PPs-e and PPs-f on BG level in alloxan-induced diabetic mice x¯ ± SD(mmol/l). Group
PPs-e PPs-f * **
Time (h) 0
4
8
12
24
24.3 ± 1.94 24.7 ± 2.80
13.7 ± 7.64* 12.7 ± 4.47*
5.43 ± 6.57** 5.48 ± 1.41**
1.65 ± 0.93** 13.6 ± 9.98*
1.90 ± 1.05** 22.0 ± 7.08*
p < 0.05, compared to the negative group, n = 5, means ± SD. p < 0.01, compared to the negative group, n = 5, means ± SD.
3.4. Effects of two components (PPs-e, -f) on the levels of BG in alloxan- induced diabetic mice Fig. 2. The circulation and transformation of glycogen.
Table 5 Effects of PPs-a, PPs-b, PPs-c and PPs-d on BG level in alloxan-induced diabetic mice¯x ± SD(mmol/l). Group
PPs-a PPs-b PPs-c PPs-d * **
Time(h) 0
7
13.18 ± 4.65 12.94 ± 3.63 12.88 ± 3.34 12.88 ± 2.61
8.48 ± 1.59* 8.00 ± 2.81* 5.40 ± 1.23** 4.74 ± 0.73**
p < 0.05, compared to the negative group, n = 5, means ± SD. p < 0.01, compared to the negative group, n = 5, means ± SD.
positive role to improve glucose metabolism and inhibit glycogen output.
The effects of two components (PPs-e, PPs-f) on the levels of BG in diabetic mice are shown in Table 6. The two components (PPs-e, Ps-f) could reduce hyperglycemia to normal levels within 8 h in alloxan-induced diabetic mice, and the effect of PPs-e on diabetic mice was more persistent. PPs-e could remain blood sugar in diabetic mice at low levels for more than 24 h. PPs-f could gradually reduce blood sugar in diabetic mice within 8 h, and BG levels in mice start to increase after 12 h. After 24 h, they returned to high values. Compared with Sephadex G-200 gel column elution curves, it showed that the peak volume of PPs-f was almost equal to the dead volume of the column, which could be a rough estimate that the molecular weight of PPs-f was not less than 105 Da. The retention time of PPs-e was later than PPs-f, speculating that the molecular weight of PPs-e should be far less than 105 Da. Consequently, the significant hypoglycemic effect of PPs-e mainly related to the small molecules of pumpkin polysaccharide. So, the next step was to analyze the molecular composition of PPs-e.
3.3. Effects of four components (PPs – a, b, c, d) on the levels of BG in alloxan-induced diabetic mice
3.5. Effect of PPs-e on islet cells proliferation activity in vitro
The effects of four components (PPs -a, -b, -c, -d) on the levels of BG in diabetic mice are shown in Table 5. All four components had the effect of lowering blood glucose. The effects of PPs-c and PPs-d were significant at 0.01 level, while the effects of PPs-a and PPs-b were significant at 0.05 level. During alcohol fractionated precipitation, the yield of PPs-c was higher than that in PPs-d, so PPs-c would be used and purified in further experiment.
Effect of PPs-e on the cell proliferation in islet cells were shown in Table 7. According to Table 7, alloxan significantly reduced islet cell proliferation activity compared with the control group. GLP-1 is a kind of peptide hormone secreted by the intestinal L cells, which has good therapeutic effect on diabetes. GLP-1 obviously enhanced the proliferation in alloxan-injured islet cell. Someone showed that GLP-1 promoted islet  cells to secrete insulin and reduced islet
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Table 7 Effect of PPs-e on the proliferation activity of islet cells. Group
Control
DM
GLP-1
PPe-I
PPe-II
PPeIII
A490
0.86 ± 0.01**
0.50 ± 0.07
0.58 ± 0.03
0.56 ± 0.04
0.55 ± 0.09
0.60 ± 0.04*
* **
p < 0.05, compared to the DM group, n = 5, means ± SD. p < 0.01, compared to the DM group, n = 5, means ± SD.
Table 8 Mixed monosaccharide standard equation of regression. Monosaccharides
Retension time/min
Equation of regression
R2
Linear range
Rhamnose Fucose Arabinose Mannose Glucose Galactose
10.132 10.643 10.803 12.935 13.313 13.520
Y = 56137x + 3566.3 Y = 90828x-8265.5 Y = 46246x + 2011.1 Y = 89954x-6919.3 Y = 85083x-8765.1 Y = 79545x-8697.5
0.9984 0.9954 0.9983 0.9986 0.9985 0.9976
0.5–109.8 mol/mL 0.3–133.2 mol/mL 0.3–52.2 mol/mL 0.2–47.6 mol/mL 0.2–43.3 mol/mL 0.2–47.6 mol/mL
Y refers to peak area. X is the concentration of monosaccharide mmol/mL.
levels compared to the negative group (P < 0.01). It can be speculated from the present study that the pumpkin polysaccharides would potentially be beneficial to improve the therapy of diabetes mellitus, and might be used as a functional and nutraceutical food ingredient. Conflict of interest The authors have declared that no conflict of interest exist. Fig. 3. GC chromatogram of PP-e.
␣ cells to secrete glucagon with different concentration of glucose [18]. The low concentration was not significant compared with DM group (P > 0.05), while high concentration was significant (P < 0.05) and showed dependence with PPs-e dose on islet cel proliferation. The results suggested that pumpkin polysaccharide could partly restore injury in pancreatic islet cells induced by alloxan. 3.6. Analysis of molecular composition of PPs-e The retention time of mixed monosaccharides standard were shown in Table 8, the gas chromatograms of the sample as shown in Fig. 3. Based on the mixed monosaccharide retention time, the main components of PPs-e were rhamnose, arabinose, glucose and galactose. The approximate ratio of rhamnose, arabinose, glucose, galactose was 1.12:5.19: 1.00: 3.91. PPs-e contained little amounts of inositol according to the result of chemical titration, while no amino sugar was found, based on Morgan-Elson method. 4. Conclusions In this experiment, solvent extraction (water and alcohol) and organic solvent fractional extraction were used to extract crude polysaccharides (PPs) from the dried pumpkin pulp. PPs-e was obtained by using alcohol fractional precipitation and Sephadex G-200 gel chromatography. PPs-e was a type of heteropolysaccharide with rhamnose, arabinose, glucose and galactose as its main compositions and little amount inositol. In the in vitro test, the pumpkin polysaccharides had the potential to enhance the islet cells proliferation activity. By using the alloxan-induced diabetic male ICR mice as the model, administration of the pumpkin polysaccharides could make liver glycogen and insulin back to the normal level and significantly lowered the BG
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