Journal of Ethnopharmacology 150 (2013) 1119–1127
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Antihyperglycemic, antihyperlipidemic and antioxidant effects of ethanol and aqueous extracts of Cyclocarya paliurus leaves in type 2 diabetic rats Qingqing Wang a,b, Cuihua Jiang b, Shengzuo Fang c, Junhu Wang a,b, Yun Ji a,b, Xulan Shang c, Yicheng Ni b,d, Zhiqi Yin a,n, Jian Zhang b,nn a Department of Natural Medicinal Chemistry, China Pharmaceutical University, No.24, Tongjiaxiang, Gulou District, Nanjing 210009, Jiangsu Province, PR China b Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, No. 100, Shizi Street, Hongshan Road, Nanjing 210028, Jiangsu Province, PR China c College of Forest Resources and Environment, Nanjing Forestry University, Nanjing 210042, Jiangsu Province, PR China d KU Leuven, Faculty of Medicine, Herestraat 49, 3000 Leuven, Belgium
art ic l e i nf o
a b s t r a c t
Article history: Received 23 May 2013 Received in revised form 21 October 2013 Accepted 23 October 2013 Available online 30 October 2013
Ethnopharmacological relevance: Cyclocarya paliurus (CP) Batal., the sole species in its genus and native to China, is a herbal tea, which has been traditionally used in the folk medicine for the treatment of diabetes and hyperlipidemia in China. To evaluate the antihyperglycemic, antihyperlipidemic and antioxidant effects of ethanol and aqueous extracts from CP in high fat diet (HFD) and streptozotocin (STZ) induced diabetic rats. Materials and methods: Type 2 diabetes was induced in 140 rats by feeding with HFD and high sugar water for 6 weeks and single injection of STZ (30 mg/kg, intraperitoneally). CP ethanol extract (CPEE) and aqueous extract (CPAE) at three doses at 2, 4 and 8 g/kg/day were orally administered once daily for four weeks. Blood glucose, serum insulin, oral glucose tolerance test (OGTT), insulin tolerance test (ITT), free fatty acid (FFA), total cholesterol (TC), triglyceride (TG), low density lipoprotein–cholesterol (LDL–C), high density lipoprotein–cholesterol (HDL–C), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), blood urea nitrogen (BUN), creatinine (CREA) and glycated serum protein (GSP) were examined. The content of total flavonoids and polysaccharides in CPEE and CPAE were assayed by ultraviolet spectrophotometry. Results: Both CPEE and CPAE increased OGTT, ITT, HDL–C, SOD and GSH-Px, while they decreased FFA, TC, TG, LDL–C, MDA, BUN, CREA and GSP. The amount of total flavonoids was found in CPEE (30.41 mg/g extract), followed by CPAE (6.75 mg/g extract). Similarly, the polysaccharides content (4.13 mg/g extract) was observed in CPAE, while absent in CPEE. Conclusions: The results suggested that CPEE and CPAE exhibited the similar antihyperglycemic, antihyperlipidemic and antioxidant effects in type 2 diabetic rats, and there were no significant differences between these two extracts. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cyclocarya paliurus Type 2 diabetes Antihyperglycemic Antihyperlipidemic Antioxidant
1. Introduction Diabetes mellitus is a worldwide prevalent disease. It is a chronic endocrine metabolic disease with high incidence of complications (Stamler et al., 1984). About 97% of the diabetic patients suffer from type 2 diabetes mellitus (International Diabetes Federation, 2009), which is characterized by hyperglycemia and associated with impaired glucose metabolism that leads to increase in lipid and lipoprotein levels as well as free radical production (Snehal et al., 2009). n
Corresponding author. Tel.: þ 86 25 86185371. Corresponding author. Tel.: þ 86 25 52362107; fax: þ 86 25 85637817. E-mail addresses: [email protected] (Z. Yin), [email protected] (J. Zhang). nn
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.10.040
The disorders in lipid and protein metabolism result in progressive complications due to free radicals generated by lipoprotein oxidation (Kuyvenhoven and Meinders, 1999). Meanwhile, free radicals can initiate peroxidation of lipids, which in turn stimulates glycation of protein, antioxidant enzymes and oxidative stress, implicated in diabetes associated complications (Baynes, 1991b; Mehta et al., 2006). Most available clinical anti-diabetic agents such as insulin and synthetic drugs do not have synergetic effects on lipid profile and antioxidant stress (Aissaoui et al., 2011), whereas traditional herbal medicines may play an important role of multiple effects. Cyclocarya paliurus (CP) (Batal.) Iljinsk (family Cyclocaryaceae), native to China, is the sole species in its genus that only grows in the highland of southern China (Shu et al., 1995). According to the ancient Chinese pharmacopoeia Zhong Hua Ben Cao, CP is
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a medicinal herb with Qingrejiedu (to clear heat and toxin) and Shengjinzhike (to help produce saliva and slake thirst) efficiency. Based on the traditional Chinese medicine theory, “damp” and “heat” are main pathological factors associated with obesity and diabetes. Over the years, CP leaves have been widely used as drug formulation in the folk medicine for the treatment of diabetes and hyperlipidemia in China. Recent epidemiological research showed that diabetes and hyperlipidemia are very rare in the Xiushui County, Jiangxi Provence where CP leaves are daily consumed as a beverage (Xie and Li, 2001). Furthermore, CP is reported to be of significant effects for the treatment of diabetes, hypertension, hyperlipidemia and obesity (Ren, 1994; Ye, 2002). In addition, CP antihyperglycemic herbal tea has been approved by the United States Food and Drug Administration (FDA), which was the first health tea from China certificated by FDA (Wang and Cao, 2007). Previous studies found that CP water extract could inhibit the activity of α-glucosidase, which was attributed to the antihyperglycemic effect of CP (Kurihara et al., 2003b; Li et al., 2011). Meanwhile, lipid-lowering effect shown with CP water extract was suggestively related to the suppression of digestive lipase activity in the hyperlipidemic mice (Kurihara et al., 2003a), and water-soluble polysaccharide of CP exerted significant scavenging effects on DPPH radicals in antioxidant assays (Xie et al., 2010). However, so far the effects of ethanol extract versus aqueous extract have not been compared. Hence, in the present study, we investigated antihyperglycemic, antihyperlipidemic and antioxidant activities of both CP ethanol extract (CPEE) and CP aqueous extract (CPAE) in high fat diet (HFD) and streptozotocin (STZ) induced type 2 diabetic rats. Comparison of activities between CPEE and CPAE has been undertaken to provide the scientific evidence for its traditional use. In addition, CP total flavonoids (CPF) and CP polysaccharides (CPP) were supposed to be the active antidiabetic components (Shangguan et al., 2010; Xie et al., 2012; Xie et al., 2006; Yang et al., 2007), we thus tested the CPF and CPP contents from the both extracts.
Bioengineering Institute (Nanjing, Jiangsu, China). All other chemicals were of analytical grade. 2.3. Preparation of the extracts Leaves of CP were collected from Nanjing Forestry University in March and authenticated by Prof. Min-Jian Qin from China Pharmaceutical University (Nanjing, Jiangsu, China), and a voucher specimen (No. L20100033) was deposited in the herbarium of the university. The air-dried and powdered leaves (2.5 kg) were extracted with 80% ethanol and distilled water (3 20 L, each 2 h), respectively. The combined ethanol extracts were concentrated to yield crude extract (466 g) under reduced pressure, while 530 g aqueous crude extract was obtained. 2.4. Determination of CP total flavonoids (CPF) and CP polysaccharides (CPP) CPEE and CPAE were weighed exactly 2.0 g for each. Extraction of CPF was performed as described previously and the CPF content was determined by aluminum chloride method with rutin as standard at 410 nm (Li et al., 2006; Feng et al., 2009). The yield of CPF was calculated by the following equation:
CPF yield ðmg=gÞ ¼
total f lavonoids weight extract weight
The same amounts of extracts were weighted. CPP was precipitated by 95% (v/v) ethanol, and then separated by centrifugation (8000 g for 5 min). The precipitate was dissolved in distilled water and the CPP content was determined by phenol-sulfuric acid method with D-glucose as a standard at 490 nm (Shangguan et al., 2006; Xie et al., 2012). The yield of CPP was calculated by the following equation: CPP yield ðmg=gÞ ¼
2. Materials and methods
polysaccharides weight extract weight
2.1. Animals
2.5. Induction of diabetes in rats
SD male rats (180–220 g) were purchased from Shanghai Super - B&K laboratory animal Corp. Ltd. [Certificate No. SCXK (HU) 2008-0016] and bred in our animal facility. The animals were kept in controlled conditions of temperature (24 72 1C), relative humidity (60 710%) and 12/12 h light/dark cycle (light from 08:00am to 08:00pm) and water ad libitum. The care and treatment of these rats were maintained in accordance with the Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation.
The rats were fed with standard laboratory diet for 2 weeks and those weighing 260–300 g were selected for study. For the development of diabetes, Rats were fed with HFD diet, consisting of 18% fat, 20% carbohydrate, 3% egg and 59% basic diet (w/w) (Srinivasan et al., 2005). HFD rats were placed in cages allowing limited physical activity (just enough space to move around to eat and drink, but not enough to run) for a period of up to 6 weeks; they had free access to 5% sucrose-containing water. After six weeks, rats were injected intraperitoneally with a single dose of 30 mg/kg STZ in citrate buffer (PH¼ 4.5). After 5 days, the blood samples were collected from the orbital venous plexus under mild anesthesia and centrifuged (Beckman Colter Allegra 64R Centrifuge) at 3000 r/min for 15 min to separate serum. The rats with blood glucose411.1 mmol/L were used for study.
2.2. Drugs and reagents Streptozotocin (STZ) was purchased from Sigma Chemical Co. (St. Louis, MO, USA).; Metformin Hydrochloride Tablets (MHT) was a product from Sino-American Shanghai Squibb Pharma (Shanghai, China) and Xiaoke Pill (XKP) from Guangzhou Zhongyi Pharmaceutical Enterprise (Guangdong, China). The ELISA kit for insulin detection was purchased from R&D Systems (Minneapolis, MN, USA). The kits for the assay of blood glucose (GLU), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL–C), low density lipoprotein cholesterol (LDL–C), free fatty acid (FFA), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), blood urea nitrogen (BUN), creatinine (CREA) and glycated serum protein (GSP) were purchased from Nanjing Jiancheng
2.6. Study design Animals were divided in 10 groups of 10 rats each as follow: Group 1: non-diabetic control (NC), fed 10 mL/kg/day of distilled water. Group 2: diabetic control (DC), fed 10 mL/kg/day of distilled water. Group 3, 4, 5: diabetic rats treated with CPEE (IL, IM, IH; 2, 4, 8 g/kg), respectively.
Q. Wang et al. / Journal of Ethnopharmacology 150 (2013) 1119–1127
Group 6, 7, 8: diabetic rats treated with CPAE (IIL, IIM, IIH; 2, 4, 8 g/kg), respectively. Group 9: diabetic rats treated with MHT (250 mg/kg). Group 10: diabetic rats treated with XKP (1.73 g/kg) (Wang et al., 2003; Xu et al., 2004; Wang et al., 2010). Each extract was dissolved or suspended in distilled water. Each group was administered by gavage once a day for 4 weeks. MHT and XKP, used as positive controls, and distilled water, as a negative one, were given to rats by the same administration route. The following experimental procedure was showed in Fig. 1.
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after insulin injection. Serum glucose concentrations were determined with glucose kits.
2.10. Determination of fasting serum insulin (FINS) and insulin sensitivity index (ISI) The fasting serum insulin was measured with rat insulin (R&D Corporation, USA) Elisa kits. Insulin sensitivity index was calculated according to the following formula: ISI¼ Ln[1/(FINS FBG)]. Where FINS referred to fasting serum insulin; FBG referred to fasting blood glucose; ISI referred to insulin sensitivity index.
2.7. Observe the general condition of the rats Mental activity, fur condition, water intake, food intake, urine output and survival of the rats were observed every day. Body weight and food intake of the rats were determined every week. 2.8. Oral glucose tolerance test (OGTT) The oral glucose tolerance test was performed on overnightfasted rats on 28th day of the treatment. Different doses of CPEE, CPAE and controls were administered 60 min prior to glucose administration (2 g/kg, i.g.). The blood samples were collected from the orbital venous plexus under mild anesthesia just before glucose load (0 min) and at 30, 60 and 120 min after glucose administration. Serum glucose concentrations were determined with glucose kit based on glucose oxidase method.
2.11. Blood sampling and biochemical analysis On 3rd day after ITT, overnight-fasted rats were anesthetized by 10% chloral hydrate (0.3 mL/100 g). The blood samples from abdominal aortic were collected and centrifuged at 3000 r/min for 15 min and serum was separated and stored at 80 1C until analysis was done. FFA, TC, TG, HDL–C and LDL–C were measured using commercial assay kits according to the manufacturer's directions. The contents of MDA and the activity of SOD and GSH-Px were determined with commercially available kits according to the manufacturer's directions.
2.12. Histological evaluation of kidney sample by hematoxylin eosin (HE) staining and renal function
2.9. Insulin tolerance test (ITT) The insulin tolerance test was performed on overnight-fasted rats on 3rd day after OGTT. Different doses of CP extracts and controls were administered 60 min before insulin administration (0.5 U/kg, subcutaneous injection). The blood samples were collected from the orbital venous plexus under mild anesthesia just before insulin administration (0 min) and at 30, 60 and 90 min
After the blood samples were gathered, the renal tissue samples were collected. Renal tissue was fixed with neutral formalin solution for 48 h, dehydrated through ascending grades of alcohol, cleared in benzene and embedded in low melting point paraffin wax. 3 μm thick sections were cut and stained with hematoxylin and eosin (HE staining) for light microscopic examinations.
Fig. 1. Experimental procedure. CPEE and CPAE were given to HFD-STZ induced diabetic rats for 4 weeks, following with the experiments of glucose metabolism, lipid metabolism, antioxidant activity and renal function. Groups: NC¼ non-diabetic control; DC¼ diabetic control; IL,IM,IH ¼groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT ¼ metformin hydrochloride tablets; XKP ¼xiaoke pill. FFA ¼free fatty acid; TG ¼ triglyceride; TC¼ total cholesterol; HDL–C ¼ high density lipoprotein cholesterol; LDL–C ¼low density lipoprotein cholesterol; MDA ¼malondialdehyde; SOD ¼superoxide dismutase; GSH-Px ¼ glutathione peroxidase; BUN ¼blood urea nitrogen; CREA ¼ creatinine; GSP ¼glycated serum protein.
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The level of BUN, CREA and GSP in serum was measured by using standard diagnostic kits according to the manufacturer's instructions.
Table 1 Effect of CP treatment on food intake in STZ induced diabetic rats (g, n¼ 10). Group Before administration (week) 1
2
3
4
5
After administration (week) 6
1
2
3
4
1750 1353 1327 1354 1383 1231 1299 1286 1219 1232
1750 1388 1283 1327 1311 1209 1258 1262 1197 1225
1750 1404 1177 1281 1297 1187 1203 1240 1182 1190
2.13. Statistical analysis Statistical analysis was performed using SPSS software package, Version 19.0. The numerical data were expressed by the means 7standard deviation. Within group comparisons were performed using independent-samples T test. A value of p o0.05 or p o0.01 was considered statistically significant.
3. Results 3.1. General features of experimental rats
NC DC IL IM IH IIL IIM IIH MHT XKP
1750 1750 1750 1750 1750 1750 1750 866 1090 1189 1219 1259 1295 1321 853 1132 1177 1239 1314 1361 1385 913 1168 1197 1331 1310 1329 1379 972 1204 1213 1296 1285 1358 1406 919 1187 1156 1220 1277 1288 1280 891 1123 1216 1224 1297 1347 1316 838 1218 1176 1276 1262 1288 1326 901 1189 1113 1198 1241 1247 1255 861 1135 1169 1187 1236 1256 1286
Groups: NC¼non-diabetic control; DC ¼diabetic control; IL, IM, IH ¼ groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT¼ metformin hydrochloride tablets; XKP ¼ xiaoke pill.
Compared to the non-diabetic control, diabetic rats had tarnished fur and significantly decreased body weight with symptoms i.e. polydipsia, polyuria and thickened urine smell (Fig. 2). Treatment of CPEE and CPAE improved these general features. Food intake of diabetic rats was less than that of non-diabetic control probably in the first week, then return to stability of intake. Chronic treatment with CP or MHT and XKP prevented the body weight loss, polydipsia, and polyphagia, also showed a certain downward trend in food intake (Table 1). As showed in Fig. 3, CP treatment groups had lower mortality rates than diabetic control group and survival rate reduced gradually from low dose to high dose whether CPEE or CPAE group.
3.2. Oral glucose tolerance test (OGTT) Blood glucose reached the highest level at 60 min and then showed a certain downward trend. Both CPEE and CPAE inhibited the increase of blood glucose level at 60 and 120 min after glucose loading when compared to the diabetic control group. The effect of HMT and XKP on serum glucose levels appeared a similar pattern (Table 2).
Fig. 3. Survival during the experiment in STZ induced diabetic rats (n¼ 6). Groups: NC¼ non-diabetic control; DC¼ diabetic control; IL,IM,IH ¼ groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT¼ metformin hydrochloride tablets; XKP¼ xiaoke pill.
3.3. Insulin tolerance test (ITT) Both CPEE and CPAE increased the blood glucose level after 60 and 90 min of insulin administration in comparison to diabetic control group. However, the blood glucose level of high dose CPEE and CPAE closed to pretreatment level after 90 min. The effect of HMT and XKP administration on serum glucose levels followed a similar pattern (Table 3).
3.4. Fasting insulin (FINS) and insulin sensitivity index (ISI) in serum As shown in Fig. 4A, diabetic rats exhibited significant hyperglycemia with a corresponding hypoinsulinaemia compared with non-diabetic control rats. The insulin levels appeared to be a slight upward trend in both CPEE and CPAE from low dose to high dose. Moreover, ISI was tested to evaluate the insulin metabolism in diabetic rats. The serum insulin level in diabetic control rats abnormally raised and subsequently triggered insulin resistance. The high given dose of CPEE and CPAEs and positive controls groups were notably lower than the diabetic control group (Fig. 4B).
3.5. Effect of CP on lipid profile and FFA level in diabetic rats
Fig. 2. Effect of CP on body weight in STZ induced diabetic rats (n¼ 10). Groups: NC¼non-diabetic control; DC ¼diabetic control; IL,IM,IH ¼groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT ¼metformin hydrochloride tablets; XKP¼ xiaoke pill.
As shown in Table 4, the increased serum levels of TC, TG, LDL–C and FFA were found to be significant (po0.05, po0.01), whereas HDL–C was found to be significantly (po0.05, po0.01) decreased when treated with CPEE and CPAEs' high dose in diabetic rats versus diabetic control group.
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Table 2 Effect of CP treatment on OGTT in STZ induced diabetic rats (n¼ 6, mean 7 SD). Blood glucose levels (mmol/L) Group
NC DC IL IM IH IIL IIM IIH MHT XKP
Dose
– – 2 g/kg 4 g/kg 8 g/kg 2 g/kg 4 g/kg 8 g/kg 250 mg/kg 1.73 g/kg
0 min
30 min
60 min
120 min
3.46 7 0.22 21.687 2.58 18.60 7 1.78n 18.147 4.57 17.317 1.41nn 18.05 7 3.10 18.577 3.33 17.517 1.42nn 17.117 4.52 16.90 7 6.49
5.55 7 0.58 24.087 1.10 19.647 4.82 19.09 7 4.95n 21.247 4.19 23.157 2.63 21.117 3.59 19.38 7 2.91nn 20.717 5.24 21.81 7 3.97
5.647 0.71 25.277 2.99 20.40 7 2.55n 20.62 7 2.70n 21.617 1.74n 24.007 1.68 23.32 7 0.91 19.917 2.17nn 20.25 7 3.24 21.92 7 3.19
5.37 70.59 24.6070.88 19.15 71.88nn 18.81 73.98nn 18.72 73.55nn 22.94 72.07 22.64 7 3.75 18.68 74.71nn 18.86 72.74nn 19.17 73.88n
Groups: NC¼ non- diabetic control; DC ¼ diabetic control; IL, IM, IH ¼ groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT ¼metformin hydrochloride tablets; XKP ¼xiaoke pill. OGTT ¼oral glucose tolerance test. n
po 0.05 compared with the diabetic control using independent-samples T test. p o0.01 compared with the diabetic control using independent-samples T test.
nn
Table 3 Effect of CP treatment on ITT in STZ induced diabetic rats (n¼ 6, mean7 SD). Blood glucose levels (mmol/L) Group
NC DC IL IM IH IIL IM IH MHT XKP
Dose
– – 2 g/kg 4 g/kg 8 g/kg 2 g/kg 4 g/kg 8 g/kg 250 mg/kg 1.73 g/kg
0 min
30 min
60 min
90 min
4.27 70.48 27.78 70.96 21.0776.48n 25.73 72.98 21.34 71.91nn 22.42 74.54n 24.6275.10 21.88 75.67nn 23.79 73.17n 21.92 74.31nn
2.617 0.80 20.067 2.51 17.22 7 7.05 18.81 7 2.62 18.81 7 5.25n 17.117 4.95 18.03 7 4.06 16.067 3.74n 16.60 7 4.73n 16.917 5.30n
2.917 0.60 21.26 7 5.88 18.87 7 4.22 19.50 7 3.74 19.277 3.53nn 19.357 5.00 18.82 7 3.43 18.58 7 3.10nn 18.08 7 4.64n 20.617 4.58n
3.117 0.25 23.727 3.83 19.137 1.16n 20.85 7 4.27 20.46 7 3.32n 20.29 7 4.49 19.39 7 2.07n 20.677 2.70nn 20.40 7 3.95n 20.69 7 1.27nn
Groups: NC¼ non-diabetic control; DC ¼diabetic control; IL,IM,IH ¼ groups treated by CPEE; IIL, IIM, IIH ¼groups treated by CPAE; MHT¼ metformin hydrochloride tablets; XKP ¼xiaoke pill. ITT ¼ insulin tolerance test. n
p o0.05 compared with the diabetic control using independent-samples T test. p o 0.01 compared with the diabetic control using independent-samples T test.
nn
3.6. Effect of CP on MDA level, antioxidant enzymes activity in diabetic rats The MDA level shown in Table 5 has significantly (p o0.05, po 0.01) decreased, whereas SOD and GSH-Px activity has significantly (p o0.05, p o0.01) increased in CP treatment groups compared to diabetic control group. The effect of CP groups even better than both two positive controls. Additionally, treatment of CPEE and CPAE in all three doses caused a conspicuous increase in the activity of SOD. 3.7. Effect of HE staining and renal function
Fig. 4. The level of FINS and ISI in serum of each group (n¼ 6, mean7 SD). nnp o 0.01, np o 0.05 compared with the diabetic control and ##p o 0.01, # p o 0.05 compared with the non-diabetic control using independent-samples T test. Groups: NC¼ non-diabetic control; DC ¼diabetic control; IL,IM,IH ¼ groups treated by CPEE; IIL, IIM, IIH ¼ groups treated by CPAE; MHT ¼ metformin hydrochloride tablets; XKP¼ xiaoke pill. FINS¼ fast serum insulin; ISI ¼ insulin sensitivities index.
Histological changes of the renal tissue are presented in Fig. 5. Light microscopy of HE stained sections showed the absence of damage of renal tissue in non-diabetic group which thickened a little in glomerular basement membrane. CP treatment groups attenuated the above pathologic changes, but there was no obvious difference in each group. It is evident from Table 6 that BUN, CREA and GSP levels increased significantly in diabetic rats as compared to non-diabetic rats, indicating the presence of renal injury. To some extent, treatment with CPEE and CPAE could reduce the levels of those
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Table 4 Effect of CP on lipids and lipoprotein in diabetic rats (n ¼6, mean 7 SD). Group
TC (mmol/L)
TG (mmol/L)
HDL–C (mmol/L)
LDL–C (mmol/L)
FFA (μmol/L)
NC DC IL IM IH IIL IIM IIH MHT XKP
1.46 7 0.23 1.99 7 0.19 1.52 7 0.31n 1.577 0.20nn 1.517 0.22nn 1.46 7 0.32nn 1.54 7 0.30n 1.477 0.19nn 1.34 7 0.27nn 1.447 0.40n
0.82 7 0.26 1.217 0.25 0.96 7 0.22 0.81 7 0.35n 0.747 0.14nn 1.25 7 0.15## 1.067 0.24 0.717 0.25nn 0.63 7 0.16nn 0.667 0.11nn
1.65 7 0.21 1.047 0.20 1.26 7 0.22# 1.317 0.26# 1.59 7 0.18nn 1.23 7 0.30# 1.337 0.30 1.50 7 0.26nn 1.65 7 0.20nn 1.62 7 0.26nn
0.84 7 0.21 1.147 0.20 0.977 0.23 0.99 7 0.18 0.84 7 0.20n 0.977 0.16 1.02 7 0.16 0.79 7 0.20n 0.707 0.11nn 0.777 0.09
145.117 36.25 508.167 74.73 295.747 28.06nn, ## 238.58 7 29.41nn, ## 195.92 7 30.87nn, # 276.81 7 64.75nn, # 256.067 72.13nn, ## 218.187 53.73nn, # 504.087 58.27## 468.777 77.22##
DC¼ diabetic control; IL, IM, IH ¼ groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT ¼metformin hydrochloride tablets; XKP¼ xiaoke pill. TC ¼total cholesterol; TG ¼ triglyceride; HDL–C ¼high density lipoprotein cholesterol; LDL–C ¼low density lipoprotein cholesterol; FFA ¼free fatty acid. n
po 0.05 compared with the diabetic control p o0.01 compared with the diabetic control p o0.05 compared with the non-diabetic control using independent-samples T test. ## p o 0.01 compared with the non-diabetic control using independent-samples T test. Groups: NC¼non-diabetic control. nn
#
Table 5 Effect of CP on MDA level, SOD and GSH-Px activity in diabetic rats (n¼6, mean 7SD). Group
MDA (nmol/mL)
SOD (U/mL)
GSH-Px (μmol/L)
NC DC IL IM IH IIL IIM IIH MHT XKP
13.65 7 1.72 19.88 7 1.50 16.50 7 1.29nn, ## 15.93 7 0.76nn, # 15.52 7 0.89nn, # 17.54 7 2.02n, ## 16.247 1.97nn, # 15.84 7 0.75nn, # 18.247 0.57n, ## 18.81 7 1.50##
10.03 7 2.25 4.53 7 1.91 8.107 1.55nn 10.117 2.44nn 10.417 2.30nn 12.177 2.25nn 14.23 7 1.52nn, ## 15.39 7 2.06nn, ## 7.687 1.20nn, # 9.137 1.49nn
1313.317 144.30 837.94 7 169.54 1036.017 130.65n, # 1192.45 7 93.44nn 1216.447 102.59nn 1076.80 7 161.81n, # 1171.747 79.90nn 1224.407 55.96nn 1064.83 7 149.39n, # 1060.087 164.64n, #
Groups: NC¼non-diabetic control; DC¼ diabetic control; IL, IM, IH ¼groups treated by CPEE; IIL, IIM, IIH ¼groups treated by CPAE; MHT ¼metformin hydrochloride tablets; XKP¼ xiaoke pill. MDA ¼ malondialdehyde; SOD¼ superoxide dismutase; GSH-Px ¼ glutathione peroxidase. n
po 0.05 compared with the diabetic control p o0.01 compared with the diabetic control p o0.05 compared with the non-diabetic control using independent-samples T test. ## p o 0.01 compared with the non-diabetic control using independent-samples T test. nn
#
indicators in a dose-dependent manner. Thus, the results demonstrated that the treatment groups improved diabetic rats' renal function. 3.8. Content of CPF and CPP Content of CPF and CPP in both CPEE and CPAE were showed in Table 7.
4. Discussion Type 2 diabetes mellitus is a complex and heterogeneous disorder characterized by a progressive decline in insulin action. Rat diabetic model induced by HFD and low-dose STZ reveals similar metabolic characteristics of the type 2 diabetes in humans (Srinivasan et al., 2005). In our study, such models were applied to evaluate the pharmacological effects of CP extracts. Meanwhile, living condition and physical activity of model rats were constrained in order to cause significant changes such as lipid metabolism disorder, oxidative damage and even early diabetic nephropathy.
To assess the effect of CP on glucose homeostasis in rats, we first performed OGTT, which is an important index for evaluation of islet function (Thomas et al., 2002). HFD-STZ treatment attenuated insulin glucose-lowering action and delayed glucose disposal by direct influence on insulin sensitivity. CP extracts may reverse these changes and decrease blood glucose as well as insulin level, suggesting their insulin sensitivity-enhancing properties. In uncontrolled type 2 diabetes mellitus, the disorders of lipid metabolism is associated with decreased HDL–C and increased TG, TC and LDL–C (Arvind et al., 2002). In addition, FFA plays a major role in the pathogenesis of insulin resistance in type 2 diabetes (Kovacs and Stumvoll, 2005). In the present study, a marked increase in serum lipids and lipoprotein was observed in diabetic rats and regular administrations of CPEE and CPAE restored the disorders of lipid metabolism. The elevation of serum lipids are usually elevated in diabetes mellitus, which is a risk factor for coronary heart disease (Sakatani et al., 2005). The risk of the development of atherosclerosis in diabetes mellitus can be reduced by elevating HDL–C level (Vasan et al., 2003). Thus, our results suggest that CP treatment might be useful to prevent diabetic complications through improving lipid metabolism. Hyperglycemia facilitates the production of free radicals and depletes natural antioxidants, which are associated with diabetes complications (Friel et al., 2004; Mehta et al., 2006). SOD and GSHPx as two important enzymes for scavenging oxygen free radicals could protect the pancreatic tissue against oxidative stress injury (Kim et al., 2009). In addition, the increase of MDA content reflected the degree of the lipid peroxides (Adewole et al., 2006). The present investigation showed that CP could lower the MDA content and improve SOD and GSH-Px activities in diabetic rats, suggesting that these extracts may have effective antioxidative properties. Furthermore, the enhancement of lipid peroxidation and reduction in activities of antioxidative enzymes in diabetic rats support the idea of a significant contribution of free radicals to the pathogenesis of diabetes (Baynes, 1991a). CP might also mediate glucolipid metabolism and boost the antioxidant ability to fight against hyperglycemia and hyperlipidemia in diabetic rats. Diabetic nephropathy (DN) is the leading cause of end stage renal disease and the STZ-induced diabetic rat model has been widely used to study early diabetic renal changes (Neuhofer and Pittrow, 2006). Prolonged hyperglycemia is a risk factor for the development and progression of DN (Ghule et al., 2012). An increase in the renal function indicators such as BUN, serum CREA and GSP accompanying DN were observed in diabetic patients (Ritz and Orth, 1999). Elevated levels of CREA and BUN may be due
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Fig. 5. Renal tissue slice from non-diabetic control rats (NC; A) showed the normal tissue structure. The tissue slice from HFD feeding and STZ-induced diabetic control rats (DC; B) showed a mild thickening of the glomerular basement membranes. The tissue sections from type 2 diabetic rats that received CPEE (IL of 2 g/kg, IM of 4 g/kg, IH of 8 g/kg; C, D and E, respectively), CPA (IIL of 2 g/kg, IIM of 4 g/kg, IIH of 8 g/kg; F, G and H, respectively), positive controls (MHT of 250 mg/kg, XKP of 1.73 g/kg; I and J, respectively) demonstrated ameliorated the above pathological changes. One representative microphotograph from each of the 10 experimental groups was shown. Original magnification 400 and H&E staining.
to hyperglycemia, which causes osmotic diuresis and depletion of extracellular fluid volume (Mogenson and Christensen, 1985). Our study showed that CP administration restored changes in the levels of BUN, CREA and GSP in HFD-STZ induced diabetic rats, which was parallel with the decrease in glucose levels by CP treatment. Moreover, HE staining biopsy revealed that glomerular capillary basement membranes were diffusely thickened in HFDSTZ induced diabetic rats and CP treatment could ameliorate the above pathological changes. In brief, CP might be beneficial to the management of early diabetic nephropathy. Phytochemicals constituents of CP such as carbohydrates, flavones, phenols acids, triterpenoid, sterols and carotenoids were reported (Fu and Fang, 2009). Flavonoids and polysaccharides were proved to be effective antihyperglycemic agents (Li et al., 2008; Li et al., 2011; Shangguan et al., 2010). It is reported that polysaccharide in CP can decrease the contents of glucose and insulin (Shi et al., 2009), and a purified polysaccharide from CP water extract revealed significant antioxidative activity (Xie et al., 2010). Polysaccharide was extracted by water medium and might
be the antidiabetic and antioxidative ingredient in CP (Jiang et al., 2013; Wang and Zhu, 2007). In the present study, our data showed that CPEE and CPAE had similar bioactivities. We found that polysaccharide was observed in CPAE, while absent in CPEE. Polysaccharide did not appear to be the active antidiabetic constituent in CP. However, CPAE elevated the SOD activity much better than CPEE, which indicates polysaccharide might be the potential antioxidative composition in CP. On the other hand, total flavonoids content was higher in CPEE than that in CPAE. This suggested that there were other substances playing a more important role in bioactivity of CP and it would be necessary to further investigate the effective chemical materials in the future.
5. Conclusion CPEE and CPAE had the similar antihyperglycemic, antihyperlipidemic and antioxidant activities in HFD-STZ induced diabetic
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Table 6 Effect of CP treatment on BUN, CERA and GSP levels in diabetic rats (n ¼6, mean 7SD). Group
Bun (mmol/mL)
Crea (μmol/mL)
GSP (μmol/L)
NC DC IL IM IH IIL IIM IIH MHT XKP
4.417 0.37 6.26 7 1.01 4.667 0.41nn 4.247 0.89nn 4.197 0.52nn 4.93 7 0.72n 4.59 7 1.20n 4.017 0.25nn 4.87 7 0.98n 5.28 7 0.37n, ##
27.50 7 2.43 33.507 3.39 31.50 7 3.73 29.177 3.87 29.007 4.10 30.50 7 4.09 28.177 2.32n 27.007 2.00nn 31.83 7 5.95 31.83 7 5.95
148.007 14.48 305.677 40.90 259.83 7 28.48n, ## 251.83 7 24.75n, ## 242.337 32.69n, ## 281.83 7 15.3## 240.177 34.48n, ## 202.83 7 14.51nn, ## 291.007 63.72## 299.007 59.81##
#
p o 0.05 compared with the non-diabetic control using independent-samples T test. Groups: NC¼non-diabetic control; DC¼ diabetic control; IL, IM, IH ¼ groups treated by CPEE; IIL, IIM, IIH ¼groups treated by CPAE; MHT ¼ metformin hydrochloride tablets; XKP ¼xiaoke pill. BUN ¼blood urea nitrogen; CREA ¼creatinine; GSP¼ glycated serum protein. n
po 0.05 compared with the diabetic control p o0.01 compared with the diabetic control ## p o 0.01 compared with the non-diabetic control using independent-samples T test. nn
Table 7 Content of CPF and CPP in both CPEE and CPAE (mg/g). Extracts
Product
Abs
Yield (mg/g)
Mean yield (mg/g)
RSD/%
CPAE
CPF
0.51
CPF
30.41
1.02
CPAE
CPP
4.13
1.75
CPEE
CPP
6.76 6.71 6.77 30.07 30.68 30.48 4.08 4.21 4.08 –
6.75
CPEE
0.4362 0.4324 0.4370 0.7901 0.8070 0.8013 0.9191 0.9367 0.9192 –
–
–
CPAE ¼CP aqueous extract; CPEE ¼ CP ethanol extract; CPF¼ CP total flavonoids; CPP¼ CP polysaccharides.
rats, and there were no significant differences between these two extracts. Comparison of activities between CPEE and CPAE provided the scientific evidence for its traditional use. Polysaccharide did not appear to be the active antidiabetic constituent, but it might be the potential antioxidative composition. In addition to total flavonoids, there might be other active ingredients for the multiple pharmaceutical effects.
Acknowledgments This work was partially supported by the grants awarded by the National Natural Science Foundation of China (No.81071828, 81001379), Jiangsu Province Natural Science Foundation (BK2010594), The Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. ZJ11175) and sponsored by Qing Lan Project. References Adewole, S.O., Caxton-Martins, E.A., Ojewole, J.A., 2006. Protective effect of quercetin on the morphology of pancreatic beta-cells of streptozotocintreated diabetic rats. Afr. J. Tradit. Complement. Altern. Med. 4, 64–74. Aissaoui, A., Zizi, S., Israili, Z.H., Lyoussi, B., 2011. Hypoglycemic and hypolipidemic effects of Coriandrum sativum L. in Meriones shawi rats. J. Ethnopharmacol. 137, 652–661. Arvind, K., Pradeepa, R., Deepa, R., Mohan, V., 2002. Diabetes and coronary artery diseases. Indian J. Med. Res 116, 163–176.
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Antihyperglycemic, antihyperlipidemic and antioxidant effects of ethanol and aqueous extracts of Cyclocarya paliurus leaves in type 2 diabetic rats Qingqing Wang a,b, Cuihua Jiang b, Shengzuo Fang c, Junhu Wang a,b, Yun Ji a,b, Xulan Shang c, Yicheng Ni b,d, Zhiqi Yin a,n, Jian Zhang b,nn a Department of Natural Medicinal Chemistry, China Pharmaceutical University, No.24, Tongjiaxiang, Gulou District, Nanjing 210009, Jiangsu Province, PR China b Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, No. 100, Shizi Street, Hongshan Road, Nanjing 210028, Jiangsu Province, PR China c College of Forest Resources and Environment, Nanjing Forestry University, Nanjing 210042, Jiangsu Province, PR China d KU Leuven, Faculty of Medicine, Herestraat 49, 3000 Leuven, Belgium
art ic l e i nf o
a b s t r a c t
Article history: Received 23 May 2013 Received in revised form 21 October 2013 Accepted 23 October 2013 Available online 30 October 2013
Ethnopharmacological relevance: Cyclocarya paliurus (CP) Batal., the sole species in its genus and native to China, is a herbal tea, which has been traditionally used in the folk medicine for the treatment of diabetes and hyperlipidemia in China. To evaluate the antihyperglycemic, antihyperlipidemic and antioxidant effects of ethanol and aqueous extracts from CP in high fat diet (HFD) and streptozotocin (STZ) induced diabetic rats. Materials and methods: Type 2 diabetes was induced in 140 rats by feeding with HFD and high sugar water for 6 weeks and single injection of STZ (30 mg/kg, intraperitoneally). CP ethanol extract (CPEE) and aqueous extract (CPAE) at three doses at 2, 4 and 8 g/kg/day were orally administered once daily for four weeks. Blood glucose, serum insulin, oral glucose tolerance test (OGTT), insulin tolerance test (ITT), free fatty acid (FFA), total cholesterol (TC), triglyceride (TG), low density lipoprotein–cholesterol (LDL–C), high density lipoprotein–cholesterol (HDL–C), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), blood urea nitrogen (BUN), creatinine (CREA) and glycated serum protein (GSP) were examined. The content of total flavonoids and polysaccharides in CPEE and CPAE were assayed by ultraviolet spectrophotometry. Results: Both CPEE and CPAE increased OGTT, ITT, HDL–C, SOD and GSH-Px, while they decreased FFA, TC, TG, LDL–C, MDA, BUN, CREA and GSP. The amount of total flavonoids was found in CPEE (30.41 mg/g extract), followed by CPAE (6.75 mg/g extract). Similarly, the polysaccharides content (4.13 mg/g extract) was observed in CPAE, while absent in CPEE. Conclusions: The results suggested that CPEE and CPAE exhibited the similar antihyperglycemic, antihyperlipidemic and antioxidant effects in type 2 diabetic rats, and there were no significant differences between these two extracts. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cyclocarya paliurus Type 2 diabetes Antihyperglycemic Antihyperlipidemic Antioxidant
1. Introduction Diabetes mellitus is a worldwide prevalent disease. It is a chronic endocrine metabolic disease with high incidence of complications (Stamler et al., 1984). About 97% of the diabetic patients suffer from type 2 diabetes mellitus (International Diabetes Federation, 2009), which is characterized by hyperglycemia and associated with impaired glucose metabolism that leads to increase in lipid and lipoprotein levels as well as free radical production (Snehal et al., 2009). n
Corresponding author. Tel.: þ 86 25 86185371. Corresponding author. Tel.: þ 86 25 52362107; fax: þ 86 25 85637817. E-mail addresses: [email protected] (Z. Yin), [email protected] (J. Zhang). nn
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.10.040
The disorders in lipid and protein metabolism result in progressive complications due to free radicals generated by lipoprotein oxidation (Kuyvenhoven and Meinders, 1999). Meanwhile, free radicals can initiate peroxidation of lipids, which in turn stimulates glycation of protein, antioxidant enzymes and oxidative stress, implicated in diabetes associated complications (Baynes, 1991b; Mehta et al., 2006). Most available clinical anti-diabetic agents such as insulin and synthetic drugs do not have synergetic effects on lipid profile and antioxidant stress (Aissaoui et al., 2011), whereas traditional herbal medicines may play an important role of multiple effects. Cyclocarya paliurus (CP) (Batal.) Iljinsk (family Cyclocaryaceae), native to China, is the sole species in its genus that only grows in the highland of southern China (Shu et al., 1995). According to the ancient Chinese pharmacopoeia Zhong Hua Ben Cao, CP is
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a medicinal herb with Qingrejiedu (to clear heat and toxin) and Shengjinzhike (to help produce saliva and slake thirst) efficiency. Based on the traditional Chinese medicine theory, “damp” and “heat” are main pathological factors associated with obesity and diabetes. Over the years, CP leaves have been widely used as drug formulation in the folk medicine for the treatment of diabetes and hyperlipidemia in China. Recent epidemiological research showed that diabetes and hyperlipidemia are very rare in the Xiushui County, Jiangxi Provence where CP leaves are daily consumed as a beverage (Xie and Li, 2001). Furthermore, CP is reported to be of significant effects for the treatment of diabetes, hypertension, hyperlipidemia and obesity (Ren, 1994; Ye, 2002). In addition, CP antihyperglycemic herbal tea has been approved by the United States Food and Drug Administration (FDA), which was the first health tea from China certificated by FDA (Wang and Cao, 2007). Previous studies found that CP water extract could inhibit the activity of α-glucosidase, which was attributed to the antihyperglycemic effect of CP (Kurihara et al., 2003b; Li et al., 2011). Meanwhile, lipid-lowering effect shown with CP water extract was suggestively related to the suppression of digestive lipase activity in the hyperlipidemic mice (Kurihara et al., 2003a), and water-soluble polysaccharide of CP exerted significant scavenging effects on DPPH radicals in antioxidant assays (Xie et al., 2010). However, so far the effects of ethanol extract versus aqueous extract have not been compared. Hence, in the present study, we investigated antihyperglycemic, antihyperlipidemic and antioxidant activities of both CP ethanol extract (CPEE) and CP aqueous extract (CPAE) in high fat diet (HFD) and streptozotocin (STZ) induced type 2 diabetic rats. Comparison of activities between CPEE and CPAE has been undertaken to provide the scientific evidence for its traditional use. In addition, CP total flavonoids (CPF) and CP polysaccharides (CPP) were supposed to be the active antidiabetic components (Shangguan et al., 2010; Xie et al., 2012; Xie et al., 2006; Yang et al., 2007), we thus tested the CPF and CPP contents from the both extracts.
Bioengineering Institute (Nanjing, Jiangsu, China). All other chemicals were of analytical grade. 2.3. Preparation of the extracts Leaves of CP were collected from Nanjing Forestry University in March and authenticated by Prof. Min-Jian Qin from China Pharmaceutical University (Nanjing, Jiangsu, China), and a voucher specimen (No. L20100033) was deposited in the herbarium of the university. The air-dried and powdered leaves (2.5 kg) were extracted with 80% ethanol and distilled water (3 20 L, each 2 h), respectively. The combined ethanol extracts were concentrated to yield crude extract (466 g) under reduced pressure, while 530 g aqueous crude extract was obtained. 2.4. Determination of CP total flavonoids (CPF) and CP polysaccharides (CPP) CPEE and CPAE were weighed exactly 2.0 g for each. Extraction of CPF was performed as described previously and the CPF content was determined by aluminum chloride method with rutin as standard at 410 nm (Li et al., 2006; Feng et al., 2009). The yield of CPF was calculated by the following equation:
CPF yield ðmg=gÞ ¼
total f lavonoids weight extract weight
The same amounts of extracts were weighted. CPP was precipitated by 95% (v/v) ethanol, and then separated by centrifugation (8000 g for 5 min). The precipitate was dissolved in distilled water and the CPP content was determined by phenol-sulfuric acid method with D-glucose as a standard at 490 nm (Shangguan et al., 2006; Xie et al., 2012). The yield of CPP was calculated by the following equation: CPP yield ðmg=gÞ ¼
2. Materials and methods
polysaccharides weight extract weight
2.1. Animals
2.5. Induction of diabetes in rats
SD male rats (180–220 g) were purchased from Shanghai Super - B&K laboratory animal Corp. Ltd. [Certificate No. SCXK (HU) 2008-0016] and bred in our animal facility. The animals were kept in controlled conditions of temperature (24 72 1C), relative humidity (60 710%) and 12/12 h light/dark cycle (light from 08:00am to 08:00pm) and water ad libitum. The care and treatment of these rats were maintained in accordance with the Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation.
The rats were fed with standard laboratory diet for 2 weeks and those weighing 260–300 g were selected for study. For the development of diabetes, Rats were fed with HFD diet, consisting of 18% fat, 20% carbohydrate, 3% egg and 59% basic diet (w/w) (Srinivasan et al., 2005). HFD rats were placed in cages allowing limited physical activity (just enough space to move around to eat and drink, but not enough to run) for a period of up to 6 weeks; they had free access to 5% sucrose-containing water. After six weeks, rats were injected intraperitoneally with a single dose of 30 mg/kg STZ in citrate buffer (PH¼ 4.5). After 5 days, the blood samples were collected from the orbital venous plexus under mild anesthesia and centrifuged (Beckman Colter Allegra 64R Centrifuge) at 3000 r/min for 15 min to separate serum. The rats with blood glucose411.1 mmol/L were used for study.
2.2. Drugs and reagents Streptozotocin (STZ) was purchased from Sigma Chemical Co. (St. Louis, MO, USA).; Metformin Hydrochloride Tablets (MHT) was a product from Sino-American Shanghai Squibb Pharma (Shanghai, China) and Xiaoke Pill (XKP) from Guangzhou Zhongyi Pharmaceutical Enterprise (Guangdong, China). The ELISA kit for insulin detection was purchased from R&D Systems (Minneapolis, MN, USA). The kits for the assay of blood glucose (GLU), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL–C), low density lipoprotein cholesterol (LDL–C), free fatty acid (FFA), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), blood urea nitrogen (BUN), creatinine (CREA) and glycated serum protein (GSP) were purchased from Nanjing Jiancheng
2.6. Study design Animals were divided in 10 groups of 10 rats each as follow: Group 1: non-diabetic control (NC), fed 10 mL/kg/day of distilled water. Group 2: diabetic control (DC), fed 10 mL/kg/day of distilled water. Group 3, 4, 5: diabetic rats treated with CPEE (IL, IM, IH; 2, 4, 8 g/kg), respectively.
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Group 6, 7, 8: diabetic rats treated with CPAE (IIL, IIM, IIH; 2, 4, 8 g/kg), respectively. Group 9: diabetic rats treated with MHT (250 mg/kg). Group 10: diabetic rats treated with XKP (1.73 g/kg) (Wang et al., 2003; Xu et al., 2004; Wang et al., 2010). Each extract was dissolved or suspended in distilled water. Each group was administered by gavage once a day for 4 weeks. MHT and XKP, used as positive controls, and distilled water, as a negative one, were given to rats by the same administration route. The following experimental procedure was showed in Fig. 1.
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after insulin injection. Serum glucose concentrations were determined with glucose kits.
2.10. Determination of fasting serum insulin (FINS) and insulin sensitivity index (ISI) The fasting serum insulin was measured with rat insulin (R&D Corporation, USA) Elisa kits. Insulin sensitivity index was calculated according to the following formula: ISI¼ Ln[1/(FINS FBG)]. Where FINS referred to fasting serum insulin; FBG referred to fasting blood glucose; ISI referred to insulin sensitivity index.
2.7. Observe the general condition of the rats Mental activity, fur condition, water intake, food intake, urine output and survival of the rats were observed every day. Body weight and food intake of the rats were determined every week. 2.8. Oral glucose tolerance test (OGTT) The oral glucose tolerance test was performed on overnightfasted rats on 28th day of the treatment. Different doses of CPEE, CPAE and controls were administered 60 min prior to glucose administration (2 g/kg, i.g.). The blood samples were collected from the orbital venous plexus under mild anesthesia just before glucose load (0 min) and at 30, 60 and 120 min after glucose administration. Serum glucose concentrations were determined with glucose kit based on glucose oxidase method.
2.11. Blood sampling and biochemical analysis On 3rd day after ITT, overnight-fasted rats were anesthetized by 10% chloral hydrate (0.3 mL/100 g). The blood samples from abdominal aortic were collected and centrifuged at 3000 r/min for 15 min and serum was separated and stored at 80 1C until analysis was done. FFA, TC, TG, HDL–C and LDL–C were measured using commercial assay kits according to the manufacturer's directions. The contents of MDA and the activity of SOD and GSH-Px were determined with commercially available kits according to the manufacturer's directions.
2.12. Histological evaluation of kidney sample by hematoxylin eosin (HE) staining and renal function
2.9. Insulin tolerance test (ITT) The insulin tolerance test was performed on overnight-fasted rats on 3rd day after OGTT. Different doses of CP extracts and controls were administered 60 min before insulin administration (0.5 U/kg, subcutaneous injection). The blood samples were collected from the orbital venous plexus under mild anesthesia just before insulin administration (0 min) and at 30, 60 and 90 min
After the blood samples were gathered, the renal tissue samples were collected. Renal tissue was fixed with neutral formalin solution for 48 h, dehydrated through ascending grades of alcohol, cleared in benzene and embedded in low melting point paraffin wax. 3 μm thick sections were cut and stained with hematoxylin and eosin (HE staining) for light microscopic examinations.
Fig. 1. Experimental procedure. CPEE and CPAE were given to HFD-STZ induced diabetic rats for 4 weeks, following with the experiments of glucose metabolism, lipid metabolism, antioxidant activity and renal function. Groups: NC¼ non-diabetic control; DC¼ diabetic control; IL,IM,IH ¼groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT ¼ metformin hydrochloride tablets; XKP ¼xiaoke pill. FFA ¼free fatty acid; TG ¼ triglyceride; TC¼ total cholesterol; HDL–C ¼ high density lipoprotein cholesterol; LDL–C ¼low density lipoprotein cholesterol; MDA ¼malondialdehyde; SOD ¼superoxide dismutase; GSH-Px ¼ glutathione peroxidase; BUN ¼blood urea nitrogen; CREA ¼ creatinine; GSP ¼glycated serum protein.
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The level of BUN, CREA and GSP in serum was measured by using standard diagnostic kits according to the manufacturer's instructions.
Table 1 Effect of CP treatment on food intake in STZ induced diabetic rats (g, n¼ 10). Group Before administration (week) 1
2
3
4
5
After administration (week) 6
1
2
3
4
1750 1353 1327 1354 1383 1231 1299 1286 1219 1232
1750 1388 1283 1327 1311 1209 1258 1262 1197 1225
1750 1404 1177 1281 1297 1187 1203 1240 1182 1190
2.13. Statistical analysis Statistical analysis was performed using SPSS software package, Version 19.0. The numerical data were expressed by the means 7standard deviation. Within group comparisons were performed using independent-samples T test. A value of p o0.05 or p o0.01 was considered statistically significant.
3. Results 3.1. General features of experimental rats
NC DC IL IM IH IIL IIM IIH MHT XKP
1750 1750 1750 1750 1750 1750 1750 866 1090 1189 1219 1259 1295 1321 853 1132 1177 1239 1314 1361 1385 913 1168 1197 1331 1310 1329 1379 972 1204 1213 1296 1285 1358 1406 919 1187 1156 1220 1277 1288 1280 891 1123 1216 1224 1297 1347 1316 838 1218 1176 1276 1262 1288 1326 901 1189 1113 1198 1241 1247 1255 861 1135 1169 1187 1236 1256 1286
Groups: NC¼non-diabetic control; DC ¼diabetic control; IL, IM, IH ¼ groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT¼ metformin hydrochloride tablets; XKP ¼ xiaoke pill.
Compared to the non-diabetic control, diabetic rats had tarnished fur and significantly decreased body weight with symptoms i.e. polydipsia, polyuria and thickened urine smell (Fig. 2). Treatment of CPEE and CPAE improved these general features. Food intake of diabetic rats was less than that of non-diabetic control probably in the first week, then return to stability of intake. Chronic treatment with CP or MHT and XKP prevented the body weight loss, polydipsia, and polyphagia, also showed a certain downward trend in food intake (Table 1). As showed in Fig. 3, CP treatment groups had lower mortality rates than diabetic control group and survival rate reduced gradually from low dose to high dose whether CPEE or CPAE group.
3.2. Oral glucose tolerance test (OGTT) Blood glucose reached the highest level at 60 min and then showed a certain downward trend. Both CPEE and CPAE inhibited the increase of blood glucose level at 60 and 120 min after glucose loading when compared to the diabetic control group. The effect of HMT and XKP on serum glucose levels appeared a similar pattern (Table 2).
Fig. 3. Survival during the experiment in STZ induced diabetic rats (n¼ 6). Groups: NC¼ non-diabetic control; DC¼ diabetic control; IL,IM,IH ¼ groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT¼ metformin hydrochloride tablets; XKP¼ xiaoke pill.
3.3. Insulin tolerance test (ITT) Both CPEE and CPAE increased the blood glucose level after 60 and 90 min of insulin administration in comparison to diabetic control group. However, the blood glucose level of high dose CPEE and CPAE closed to pretreatment level after 90 min. The effect of HMT and XKP administration on serum glucose levels followed a similar pattern (Table 3).
3.4. Fasting insulin (FINS) and insulin sensitivity index (ISI) in serum As shown in Fig. 4A, diabetic rats exhibited significant hyperglycemia with a corresponding hypoinsulinaemia compared with non-diabetic control rats. The insulin levels appeared to be a slight upward trend in both CPEE and CPAE from low dose to high dose. Moreover, ISI was tested to evaluate the insulin metabolism in diabetic rats. The serum insulin level in diabetic control rats abnormally raised and subsequently triggered insulin resistance. The high given dose of CPEE and CPAEs and positive controls groups were notably lower than the diabetic control group (Fig. 4B).
3.5. Effect of CP on lipid profile and FFA level in diabetic rats
Fig. 2. Effect of CP on body weight in STZ induced diabetic rats (n¼ 10). Groups: NC¼non-diabetic control; DC ¼diabetic control; IL,IM,IH ¼groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT ¼metformin hydrochloride tablets; XKP¼ xiaoke pill.
As shown in Table 4, the increased serum levels of TC, TG, LDL–C and FFA were found to be significant (po0.05, po0.01), whereas HDL–C was found to be significantly (po0.05, po0.01) decreased when treated with CPEE and CPAEs' high dose in diabetic rats versus diabetic control group.
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Table 2 Effect of CP treatment on OGTT in STZ induced diabetic rats (n¼ 6, mean 7 SD). Blood glucose levels (mmol/L) Group
NC DC IL IM IH IIL IIM IIH MHT XKP
Dose
– – 2 g/kg 4 g/kg 8 g/kg 2 g/kg 4 g/kg 8 g/kg 250 mg/kg 1.73 g/kg
0 min
30 min
60 min
120 min
3.46 7 0.22 21.687 2.58 18.60 7 1.78n 18.147 4.57 17.317 1.41nn 18.05 7 3.10 18.577 3.33 17.517 1.42nn 17.117 4.52 16.90 7 6.49
5.55 7 0.58 24.087 1.10 19.647 4.82 19.09 7 4.95n 21.247 4.19 23.157 2.63 21.117 3.59 19.38 7 2.91nn 20.717 5.24 21.81 7 3.97
5.647 0.71 25.277 2.99 20.40 7 2.55n 20.62 7 2.70n 21.617 1.74n 24.007 1.68 23.32 7 0.91 19.917 2.17nn 20.25 7 3.24 21.92 7 3.19
5.37 70.59 24.6070.88 19.15 71.88nn 18.81 73.98nn 18.72 73.55nn 22.94 72.07 22.64 7 3.75 18.68 74.71nn 18.86 72.74nn 19.17 73.88n
Groups: NC¼ non- diabetic control; DC ¼ diabetic control; IL, IM, IH ¼ groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT ¼metformin hydrochloride tablets; XKP ¼xiaoke pill. OGTT ¼oral glucose tolerance test. n
po 0.05 compared with the diabetic control using independent-samples T test. p o0.01 compared with the diabetic control using independent-samples T test.
nn
Table 3 Effect of CP treatment on ITT in STZ induced diabetic rats (n¼ 6, mean7 SD). Blood glucose levels (mmol/L) Group
NC DC IL IM IH IIL IM IH MHT XKP
Dose
– – 2 g/kg 4 g/kg 8 g/kg 2 g/kg 4 g/kg 8 g/kg 250 mg/kg 1.73 g/kg
0 min
30 min
60 min
90 min
4.27 70.48 27.78 70.96 21.0776.48n 25.73 72.98 21.34 71.91nn 22.42 74.54n 24.6275.10 21.88 75.67nn 23.79 73.17n 21.92 74.31nn
2.617 0.80 20.067 2.51 17.22 7 7.05 18.81 7 2.62 18.81 7 5.25n 17.117 4.95 18.03 7 4.06 16.067 3.74n 16.60 7 4.73n 16.917 5.30n
2.917 0.60 21.26 7 5.88 18.87 7 4.22 19.50 7 3.74 19.277 3.53nn 19.357 5.00 18.82 7 3.43 18.58 7 3.10nn 18.08 7 4.64n 20.617 4.58n
3.117 0.25 23.727 3.83 19.137 1.16n 20.85 7 4.27 20.46 7 3.32n 20.29 7 4.49 19.39 7 2.07n 20.677 2.70nn 20.40 7 3.95n 20.69 7 1.27nn
Groups: NC¼ non-diabetic control; DC ¼diabetic control; IL,IM,IH ¼ groups treated by CPEE; IIL, IIM, IIH ¼groups treated by CPAE; MHT¼ metformin hydrochloride tablets; XKP ¼xiaoke pill. ITT ¼ insulin tolerance test. n
p o0.05 compared with the diabetic control using independent-samples T test. p o 0.01 compared with the diabetic control using independent-samples T test.
nn
3.6. Effect of CP on MDA level, antioxidant enzymes activity in diabetic rats The MDA level shown in Table 5 has significantly (p o0.05, po 0.01) decreased, whereas SOD and GSH-Px activity has significantly (p o0.05, p o0.01) increased in CP treatment groups compared to diabetic control group. The effect of CP groups even better than both two positive controls. Additionally, treatment of CPEE and CPAE in all three doses caused a conspicuous increase in the activity of SOD. 3.7. Effect of HE staining and renal function
Fig. 4. The level of FINS and ISI in serum of each group (n¼ 6, mean7 SD). nnp o 0.01, np o 0.05 compared with the diabetic control and ##p o 0.01, # p o 0.05 compared with the non-diabetic control using independent-samples T test. Groups: NC¼ non-diabetic control; DC ¼diabetic control; IL,IM,IH ¼ groups treated by CPEE; IIL, IIM, IIH ¼ groups treated by CPAE; MHT ¼ metformin hydrochloride tablets; XKP¼ xiaoke pill. FINS¼ fast serum insulin; ISI ¼ insulin sensitivities index.
Histological changes of the renal tissue are presented in Fig. 5. Light microscopy of HE stained sections showed the absence of damage of renal tissue in non-diabetic group which thickened a little in glomerular basement membrane. CP treatment groups attenuated the above pathologic changes, but there was no obvious difference in each group. It is evident from Table 6 that BUN, CREA and GSP levels increased significantly in diabetic rats as compared to non-diabetic rats, indicating the presence of renal injury. To some extent, treatment with CPEE and CPAE could reduce the levels of those
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Table 4 Effect of CP on lipids and lipoprotein in diabetic rats (n ¼6, mean 7 SD). Group
TC (mmol/L)
TG (mmol/L)
HDL–C (mmol/L)
LDL–C (mmol/L)
FFA (μmol/L)
NC DC IL IM IH IIL IIM IIH MHT XKP
1.46 7 0.23 1.99 7 0.19 1.52 7 0.31n 1.577 0.20nn 1.517 0.22nn 1.46 7 0.32nn 1.54 7 0.30n 1.477 0.19nn 1.34 7 0.27nn 1.447 0.40n
0.82 7 0.26 1.217 0.25 0.96 7 0.22 0.81 7 0.35n 0.747 0.14nn 1.25 7 0.15## 1.067 0.24 0.717 0.25nn 0.63 7 0.16nn 0.667 0.11nn
1.65 7 0.21 1.047 0.20 1.26 7 0.22# 1.317 0.26# 1.59 7 0.18nn 1.23 7 0.30# 1.337 0.30 1.50 7 0.26nn 1.65 7 0.20nn 1.62 7 0.26nn
0.84 7 0.21 1.147 0.20 0.977 0.23 0.99 7 0.18 0.84 7 0.20n 0.977 0.16 1.02 7 0.16 0.79 7 0.20n 0.707 0.11nn 0.777 0.09
145.117 36.25 508.167 74.73 295.747 28.06nn, ## 238.58 7 29.41nn, ## 195.92 7 30.87nn, # 276.81 7 64.75nn, # 256.067 72.13nn, ## 218.187 53.73nn, # 504.087 58.27## 468.777 77.22##
DC¼ diabetic control; IL, IM, IH ¼ groups treated by CPEE; IIL, IIM, IIH¼ groups treated by CPAE; MHT ¼metformin hydrochloride tablets; XKP¼ xiaoke pill. TC ¼total cholesterol; TG ¼ triglyceride; HDL–C ¼high density lipoprotein cholesterol; LDL–C ¼low density lipoprotein cholesterol; FFA ¼free fatty acid. n
po 0.05 compared with the diabetic control p o0.01 compared with the diabetic control p o0.05 compared with the non-diabetic control using independent-samples T test. ## p o 0.01 compared with the non-diabetic control using independent-samples T test. Groups: NC¼non-diabetic control. nn
#
Table 5 Effect of CP on MDA level, SOD and GSH-Px activity in diabetic rats (n¼6, mean 7SD). Group
MDA (nmol/mL)
SOD (U/mL)
GSH-Px (μmol/L)
NC DC IL IM IH IIL IIM IIH MHT XKP
13.65 7 1.72 19.88 7 1.50 16.50 7 1.29nn, ## 15.93 7 0.76nn, # 15.52 7 0.89nn, # 17.54 7 2.02n, ## 16.247 1.97nn, # 15.84 7 0.75nn, # 18.247 0.57n, ## 18.81 7 1.50##
10.03 7 2.25 4.53 7 1.91 8.107 1.55nn 10.117 2.44nn 10.417 2.30nn 12.177 2.25nn 14.23 7 1.52nn, ## 15.39 7 2.06nn, ## 7.687 1.20nn, # 9.137 1.49nn
1313.317 144.30 837.94 7 169.54 1036.017 130.65n, # 1192.45 7 93.44nn 1216.447 102.59nn 1076.80 7 161.81n, # 1171.747 79.90nn 1224.407 55.96nn 1064.83 7 149.39n, # 1060.087 164.64n, #
Groups: NC¼non-diabetic control; DC¼ diabetic control; IL, IM, IH ¼groups treated by CPEE; IIL, IIM, IIH ¼groups treated by CPAE; MHT ¼metformin hydrochloride tablets; XKP¼ xiaoke pill. MDA ¼ malondialdehyde; SOD¼ superoxide dismutase; GSH-Px ¼ glutathione peroxidase. n
po 0.05 compared with the diabetic control p o0.01 compared with the diabetic control p o0.05 compared with the non-diabetic control using independent-samples T test. ## p o 0.01 compared with the non-diabetic control using independent-samples T test. nn
#
indicators in a dose-dependent manner. Thus, the results demonstrated that the treatment groups improved diabetic rats' renal function. 3.8. Content of CPF and CPP Content of CPF and CPP in both CPEE and CPAE were showed in Table 7.
4. Discussion Type 2 diabetes mellitus is a complex and heterogeneous disorder characterized by a progressive decline in insulin action. Rat diabetic model induced by HFD and low-dose STZ reveals similar metabolic characteristics of the type 2 diabetes in humans (Srinivasan et al., 2005). In our study, such models were applied to evaluate the pharmacological effects of CP extracts. Meanwhile, living condition and physical activity of model rats were constrained in order to cause significant changes such as lipid metabolism disorder, oxidative damage and even early diabetic nephropathy.
To assess the effect of CP on glucose homeostasis in rats, we first performed OGTT, which is an important index for evaluation of islet function (Thomas et al., 2002). HFD-STZ treatment attenuated insulin glucose-lowering action and delayed glucose disposal by direct influence on insulin sensitivity. CP extracts may reverse these changes and decrease blood glucose as well as insulin level, suggesting their insulin sensitivity-enhancing properties. In uncontrolled type 2 diabetes mellitus, the disorders of lipid metabolism is associated with decreased HDL–C and increased TG, TC and LDL–C (Arvind et al., 2002). In addition, FFA plays a major role in the pathogenesis of insulin resistance in type 2 diabetes (Kovacs and Stumvoll, 2005). In the present study, a marked increase in serum lipids and lipoprotein was observed in diabetic rats and regular administrations of CPEE and CPAE restored the disorders of lipid metabolism. The elevation of serum lipids are usually elevated in diabetes mellitus, which is a risk factor for coronary heart disease (Sakatani et al., 2005). The risk of the development of atherosclerosis in diabetes mellitus can be reduced by elevating HDL–C level (Vasan et al., 2003). Thus, our results suggest that CP treatment might be useful to prevent diabetic complications through improving lipid metabolism. Hyperglycemia facilitates the production of free radicals and depletes natural antioxidants, which are associated with diabetes complications (Friel et al., 2004; Mehta et al., 2006). SOD and GSHPx as two important enzymes for scavenging oxygen free radicals could protect the pancreatic tissue against oxidative stress injury (Kim et al., 2009). In addition, the increase of MDA content reflected the degree of the lipid peroxides (Adewole et al., 2006). The present investigation showed that CP could lower the MDA content and improve SOD and GSH-Px activities in diabetic rats, suggesting that these extracts may have effective antioxidative properties. Furthermore, the enhancement of lipid peroxidation and reduction in activities of antioxidative enzymes in diabetic rats support the idea of a significant contribution of free radicals to the pathogenesis of diabetes (Baynes, 1991a). CP might also mediate glucolipid metabolism and boost the antioxidant ability to fight against hyperglycemia and hyperlipidemia in diabetic rats. Diabetic nephropathy (DN) is the leading cause of end stage renal disease and the STZ-induced diabetic rat model has been widely used to study early diabetic renal changes (Neuhofer and Pittrow, 2006). Prolonged hyperglycemia is a risk factor for the development and progression of DN (Ghule et al., 2012). An increase in the renal function indicators such as BUN, serum CREA and GSP accompanying DN were observed in diabetic patients (Ritz and Orth, 1999). Elevated levels of CREA and BUN may be due
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Fig. 5. Renal tissue slice from non-diabetic control rats (NC; A) showed the normal tissue structure. The tissue slice from HFD feeding and STZ-induced diabetic control rats (DC; B) showed a mild thickening of the glomerular basement membranes. The tissue sections from type 2 diabetic rats that received CPEE (IL of 2 g/kg, IM of 4 g/kg, IH of 8 g/kg; C, D and E, respectively), CPA (IIL of 2 g/kg, IIM of 4 g/kg, IIH of 8 g/kg; F, G and H, respectively), positive controls (MHT of 250 mg/kg, XKP of 1.73 g/kg; I and J, respectively) demonstrated ameliorated the above pathological changes. One representative microphotograph from each of the 10 experimental groups was shown. Original magnification 400 and H&E staining.
to hyperglycemia, which causes osmotic diuresis and depletion of extracellular fluid volume (Mogenson and Christensen, 1985). Our study showed that CP administration restored changes in the levels of BUN, CREA and GSP in HFD-STZ induced diabetic rats, which was parallel with the decrease in glucose levels by CP treatment. Moreover, HE staining biopsy revealed that glomerular capillary basement membranes were diffusely thickened in HFDSTZ induced diabetic rats and CP treatment could ameliorate the above pathological changes. In brief, CP might be beneficial to the management of early diabetic nephropathy. Phytochemicals constituents of CP such as carbohydrates, flavones, phenols acids, triterpenoid, sterols and carotenoids were reported (Fu and Fang, 2009). Flavonoids and polysaccharides were proved to be effective antihyperglycemic agents (Li et al., 2008; Li et al., 2011; Shangguan et al., 2010). It is reported that polysaccharide in CP can decrease the contents of glucose and insulin (Shi et al., 2009), and a purified polysaccharide from CP water extract revealed significant antioxidative activity (Xie et al., 2010). Polysaccharide was extracted by water medium and might
be the antidiabetic and antioxidative ingredient in CP (Jiang et al., 2013; Wang and Zhu, 2007). In the present study, our data showed that CPEE and CPAE had similar bioactivities. We found that polysaccharide was observed in CPAE, while absent in CPEE. Polysaccharide did not appear to be the active antidiabetic constituent in CP. However, CPAE elevated the SOD activity much better than CPEE, which indicates polysaccharide might be the potential antioxidative composition in CP. On the other hand, total flavonoids content was higher in CPEE than that in CPAE. This suggested that there were other substances playing a more important role in bioactivity of CP and it would be necessary to further investigate the effective chemical materials in the future.
5. Conclusion CPEE and CPAE had the similar antihyperglycemic, antihyperlipidemic and antioxidant activities in HFD-STZ induced diabetic
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Table 6 Effect of CP treatment on BUN, CERA and GSP levels in diabetic rats (n ¼6, mean 7SD). Group
Bun (mmol/mL)
Crea (μmol/mL)
GSP (μmol/L)
NC DC IL IM IH IIL IIM IIH MHT XKP
4.417 0.37 6.26 7 1.01 4.667 0.41nn 4.247 0.89nn 4.197 0.52nn 4.93 7 0.72n 4.59 7 1.20n 4.017 0.25nn 4.87 7 0.98n 5.28 7 0.37n, ##
27.50 7 2.43 33.507 3.39 31.50 7 3.73 29.177 3.87 29.007 4.10 30.50 7 4.09 28.177 2.32n 27.007 2.00nn 31.83 7 5.95 31.83 7 5.95
148.007 14.48 305.677 40.90 259.83 7 28.48n, ## 251.83 7 24.75n, ## 242.337 32.69n, ## 281.83 7 15.3## 240.177 34.48n, ## 202.83 7 14.51nn, ## 291.007 63.72## 299.007 59.81##
#
p o 0.05 compared with the non-diabetic control using independent-samples T test. Groups: NC¼non-diabetic control; DC¼ diabetic control; IL, IM, IH ¼ groups treated by CPEE; IIL, IIM, IIH ¼groups treated by CPAE; MHT ¼ metformin hydrochloride tablets; XKP ¼xiaoke pill. BUN ¼blood urea nitrogen; CREA ¼creatinine; GSP¼ glycated serum protein. n
po 0.05 compared with the diabetic control p o0.01 compared with the diabetic control ## p o 0.01 compared with the non-diabetic control using independent-samples T test. nn
Table 7 Content of CPF and CPP in both CPEE and CPAE (mg/g). Extracts
Product
Abs
Yield (mg/g)
Mean yield (mg/g)
RSD/%
CPAE
CPF
0.51
CPF
30.41
1.02
CPAE
CPP
4.13
1.75
CPEE
CPP
6.76 6.71 6.77 30.07 30.68 30.48 4.08 4.21 4.08 –
6.75
CPEE
0.4362 0.4324 0.4370 0.7901 0.8070 0.8013 0.9191 0.9367 0.9192 –
–
–
CPAE ¼CP aqueous extract; CPEE ¼ CP ethanol extract; CPF¼ CP total flavonoids; CPP¼ CP polysaccharides.
rats, and there were no significant differences between these two extracts. Comparison of activities between CPEE and CPAE provided the scientific evidence for its traditional use. Polysaccharide did not appear to be the active antidiabetic constituent, but it might be the potential antioxidative composition. In addition to total flavonoids, there might be other active ingredients for the multiple pharmaceutical effects.
Acknowledgments This work was partially supported by the grants awarded by the National Natural Science Foundation of China (No.81071828, 81001379), Jiangsu Province Natural Science Foundation (BK2010594), The Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. ZJ11175) and sponsored by Qing Lan Project. References Adewole, S.O., Caxton-Martins, E.A., Ojewole, J.A., 2006. Protective effect of quercetin on the morphology of pancreatic beta-cells of streptozotocintreated diabetic rats. Afr. J. Tradit. Complement. Altern. Med. 4, 64–74. Aissaoui, A., Zizi, S., Israili, Z.H., Lyoussi, B., 2011. Hypoglycemic and hypolipidemic effects of Coriandrum sativum L. in Meriones shawi rats. J. Ethnopharmacol. 137, 652–661. Arvind, K., Pradeepa, R., Deepa, R., Mohan, V., 2002. Diabetes and coronary artery diseases. Indian J. Med. Res 116, 163–176.
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