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alpha amylase study

alpha amylase activity<br>

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alpha amylase study

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  1. Journal of Ethnopharmacology 147 (2013) 622–630 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep In vitro and in vivo anti-diabetic activity of Swertia kouitchensis extract Luo-sheng Wan, Cui-ping Chen, Zuo-qi Xiao, Yong-long Wang, Qiu-xia Min, Yuedong Yue, Jiachun Chenn Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of Pharmaceutical Sciences, Huazhong University of Science and Technology, People's Republic of China a r t i c l e i n f o a b s t r a c t Ethnopharmacological relevance: Swertia kouitchensis has long been used as a folk medicine to treat hepatitis and diabetes in central-western China. Therefore, this study was aimed to evaluate the anti- diabetic activity of the plant ethanol extract. Materials and methods: Firstly, the extract was tested for its inhibitory activity on α-amylase and α- glucosidase in vitro. Following that, insulin secretion test in NIT-1cell was performed. Then, oral sucrose or starch tolerance test of the extract were carried out in normal mice. After that, acute effect of the extract was executed in normal and streptozotocin-induced (60 mg/kg) diabetic mice. Eventually, long term effect of the extract was performed in diabetic mice for 4 weeks. Oral glucose tolerance test and biochemical parameters were estimated at the end of the study. Results: Swertia kouitchensis extract could remarkably inhibit the activity of α-amylase and α-glucosidase and stimulate insulin secretion in vitro. And also the extract displayed anti-hyperglycemic activity, improved antioxidant capacity, ameliorated the hyperlipidemia and carbohydrate metabolism in diabetic mice. Conclusions: Swertia kouitchensis exhibits considerable anti-diabetic activity and metabolic alterations in diabetic mice. These results provide a rationale for the use of Swertia kouitchensis to treat diabetes mellitus. Article history: Received 31 October 2012 Received in revised form 15 March 2013 Accepted 18 March 2013 Available online 6 April 2013 Keywords: Swertia kouitchensis Ethanol extract Xanthone α-Amylase and α-glucosidase Diabetic mice & 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction xanthones and triterpenoids, such as gentiacaulein, methylswer- tianin, bellidifolin, swertianin and oleanolic acid, from the whole plant ethanol extract were reported. And He et al. (2011) found three new secoiridoid glycosides with their anti-hepatitis B virus activity from Swertia kouitchensis. Some plants in the Swertia genus, like Swertia japonica (Basnet et al., 1994) and Swertia chirayita (Chandrasekar et al., 1990; Kar et al., 2003), have been reported for their anti-diabetic activity; other researchers have found the anti-diabetic activity of some xanthones, such as mangiferin, bellidifolin and methylswertianin (Muruganandan et al., 2005; Tian et al., 2010), which also have been found in Swertia kouitchensis. However, there is still no direct scientific report of Swertia kouitchensis for its anti-diabetic activity up to now. So the present investigation was designed to evaluate the hypoglycemic and anti-diabetic effect of the Swertia kouitchensis ethanol extract by using in vitro and in vivo models, with a view to establish the pharmacological basis for its anti-diabetic use in folk medicine. Diabetes mellitus (DM), characterized by hyperglycemia and carbohydrate, protein and fat metabolism disturbances, is a wide- spread metabolic disease (American Diabetes Association, 2011). And because of the complex mechanism involved in DM, many of the current anti-diabetic chemical agents have some limitations and even some severe adverse-effects (Bhatnagar,1998; May et al., 2002). Therefore, these situations have encouraged the searches for alternative therapeutic agents from not only synthetic chemi- cals but also natural plants. As a plant belonging to Swertia genus, Swertia kouitchensis Franch. (Gentianaceae), locally named as “Guizhou Zhangyacai”, “Shuihuanglian” or “Silengcao”, has long been used in the folk medicine to treat jaundice hepatitis, diabetes, dysentery, pneumo- nia, tonsillitis and gynecological inflammation in central-western China (State Administration of Traditional Chinese Medicine, 1999). Especially, in the area of Tujia ethnic minority of Hunan and Hubei provinces, Swertia kouitchensis extract was commonly used to manage diabetes and hepatitis (Cai et al., 2004). In previous phytochemical investigations of Swertia kouitchensis (Qing et al., 2004), the isolations and structure elucidations of 2. Materials and methods 2.1. Plant materials and extract preparations The whole plant of Swertia kouitchensis (S.k.) was collected from natural habitat in Hubei province, China, and identified by nCorresponding author. Tel./fax: +86 27 83692793. E-mail address: homespringchen@126.com (J. Chen). 0378-8741/$-see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.03.052

  2. 623 L.-s. Wan et al. / Journal of Ethnopharmacology 147 (2013) 622–630 Prof. Jiachun Chen (Tongji medical school of HUST, Wuhan, China). A voucher specimen with the number S.k.2010-1010 was deposited in the University herbarium for future reference. The whole plant was dried at room temperature and ground into powder. And then the dried ground powder 15 kg was extracted with 95% ethanol under reflux successively (each 2 h,120 L?2 times). All the extract were combined and concentrated under vacuum to give a residue 3 kg (yielding 20%). The obtained extract was stored in a refrig- erator at 4 1C until time of use. included in the experiments. Diabetic mice continued to be fed with the diets high in fat and fructose to the end of the experi- ments (Srinivasan et al., 2005). 2.6. In vitro α-amylase and α-glucosidase activity test α-Amylase (A3176, Sigma) activity was determined according to the method of Bernfeld (1955), with minor modifications. Briefly, the sample (S.k. or acarbose) 500 μl and the α-amylase 500 μl (2 unit/ml) were mixed and pre-incubated in 20 mM sodium phosphate buffer (pH 6.7) for 5 min at 37 1C. Then, 1 ml of 0.2% (w/v) starch dissolved in the buffer was added to the reaction mixture to make a total volume of 2 ml, and the whole was incubated for 5 min at 37 1C. After the incubation, 1 ml of dini- trosalicylic acid (DNS) color reagent was added and placed in a boiled bath for exactly 5 min. And then, this mixture was cooled on ice to room temperature and added another 6 ml of deionized water. α-Amylase activity was determined by measuring the absorbance of the mixture at 540 nm. α-Glucosidase (G5003, sigma) activity was determined accord- ing to the method of Feng et al. (2011), with minor modifications. Inhibition of α-glucosidase activity was determined spectrophoto- metrically in a 96-well microtiter plate. Briefly, the sample (S.k. or acarbose) 120 μl and 20 μl α-glucosidase (1 unit/ml) were pre- incubated in 0.1 M potassium phosphate buffer (pH 6.8) for 15 min at 37 1C. 20 μl of 5 mM para-nitrophenyl-α-D-glucopyranoside (PNPG) in 0.1 M potassium phosphate buffer was added to initiate the reaction, and the mixture was further incubated for 15 min. The reaction was terminated by the addition of 80 μl of 0.2 M Na2CO3in 0.1 M potassium phosphate buffer, and the absorbance of the mixture was recorded at 405 nm. The results were expressed as % inhibition of enzyme activity and calculated according to the following equation: 2.2. HPLC analysis of the extract The quality of the extract was measured by HPLC, with the conditions: stationary phase: Alltima C18 (250?4.6 mm,5 μm), Agilent-1100 HPLC system, Agilent, USA; mobile phase: methanol (A) and 0.05%(V/V) phosphoric acid aqueous solution (B) in gradient (0 min, A:B¼20:80, 50 min, A:B¼80:20, 55 min, A: B¼100:0); flow rate: 1 ml/min; detective wave length: 254 nm; temperature: 30 1C. We used mangiferin (1), swertianolin (2) and bellidifolin (3), which were prepared from our previous work and identified by comparing the (Tan et al., 2000; Qing et al., 2004; Ge et al., 2011), as the standard markers to indicate the main components of the extract. 13C-NMR data with the literatures 2.3. Animals Male BALB/c mice (18–22 g each) were obtained from the Center for Disease Prevention and Control in Hubei province, China (Reg. no. SCXK (Hubei) 2008-0005). The study had been carried out in accordance with the “Principles of Laboratory Animal Care” (World Health Organization, 1985). Animals were maintained in an animal laboratory with a controlled environment of 21–23 1C, 45–65% humidity, and a 12 h (07:30–19:30) light/dark cycle. A standard pellet diet (Center for Disease Prevention and Control in Hubei province, China) and water were given ad libitum. After a week of pellet diets, the normal mice were randomly divided into two groups. One group was continually fed with pellet diets as normal mice; the other group was fed with diets high in fat and fructose (consisting of 10% fat, 20% fructose,10% egg and 60% basic diet (w/w)), and used to induce diabetic mice (Zheng et al., 2011). %Inhibition ¼ 100 ? ðabsorbance of control−absorbance of sampleÞ=absorbance of control 2.7. In vitro NIT-1 cell insulin secretion test Insulin secretion test was used the method based on Verspohl (2002) and Verspohl et al. (2005), with some modifications. Mouse insulinoma cell line, NIT-1 cells (CRL-2055, ATCC), were grown in RPMI 1640 containing 11.1 mM glucose and supplemented with 10% fetal calf serum, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin in an atmosphere of 95% air/5% CO2. Cells were grown in micro-well for 48–96 h to reach confluence (cell density in half-confluence about 1–2?106cells/ml). Prior to the experi- ment, cells were washed three times and incubated in Krebs– Ringer buffer containing 10 mM HEPES and 5% bovine serum albumin (KRBH). When measuring confluent cells were incubated for 90 min at 37 1C, in the presence or absence of the S.k. extract (0.1, 1, 10, 100, 1000 μg/ml) or gliclazide (10 μg/ml) diluted in the KBRH buffer containing 5.6 or 16.7 mM glucose, which would represent normal and diabetic conditions respectively. After incubation, cells were centrifuged at 1500g for 10 min at 4 1C and the supernatants were transferred to fresh tubes for insulin assay by ELISA (Yihan, Shanghai, China). 2.4. Acute toxicity test According to Horn's method (Ministry of Health, P.R. China, 2003), 50 normal mice fed with pellet diets of either sex were randomly divided into five groups with ten mice per group (each group with five male mice and five female mice) and admini- strated with 0.464, 1.00, 2.15, 4.64 and 10.0 g/kg of the S.k extract orally by gastric gavage. The animals were observed for general behavioral changes, signs of toxicity and mortality continuously for 1 h after treatment, then intermittently for 4 h, and thereafter over a period of 24 h (Twaij et al., 1983). Further, the mice were observed for up to 14 days following the treatment for any lethality and death (Jain et al., 2010). insulin secretion, half- 2.5. Induction of diabetes in mice According to Sharma et al. (2010), mice fed with diets high in fat and fructose for 4 weeks were 12 h fasted but free access to water. Then, diabetes was induced by intraperitoneal (i.p.) injec- tion of streptozotocin (STZ) dissolved in 0.1 M cold citrate buffer (pH¼4.4) at a dose of 60 mg/kg body weight. On the third day after STZ injection, fasted blood glucose levels were measured by hand-held glucose meter (BAYER ContourTM). Only diabetic mice with a fasting blood glucose level of at least 11.1 mmol/l were 2.8. Oral starch and sucrose tolerance test in normal mice According to Wu et al. (2011), normal mice were classified into 4 groups (1–4), each of them with 10 mice. Group 1 served as the normal control group and received 0.3% CMC-Na solution (0.1 ml/ 10 g, as vehicle), while Group 2 were treated with standard oral hypoglycemic drug, acarbose, with a dose of 15 mg/kg in vehicle. The mice of Groups 3 and 4 were treated with the S.k. extract at

  3. 624 L.-s. Wan et al. / Journal of Ethnopharmacology 147 (2013) 622–630 with vehicle; two groups received the S.k. extract at the doses of 250 and 500 mg/kg, respectively; one group received gliclazide (15 mg/kg) as standard drug group. The normal control group was also treated with vehicle. Blood glucose levels were determined in 12 h fasted mice before the first administration of drugs (1st day) and at weekly interval until the end of the study. two doses (250 mg/kg and 500 mg/kg per os, dissolved in vehicle). Test samples, standard drug and vehicle were given orally to 12 h fasted mice, 30 min before the administration of starch 6 g/kg. Blood glucose levels were measured before and after starch loading at 0, 0.5, 1, 2 h. The oral sucrose tolerance test was designed similar to the starch tolerance test, only replacing the loading of 6 g/kg starch with the loading of 4 g/kg sucrose, and measured the blood glucose levels at 0, 0.5, 1, 2 h. Both glucose responses during the experiments were calculated from the area under the curve (AUC) by using the trapezoidal rule (Le Floch et al., 1990). 2.11. Oral glucose tolerance test in diabetic mice The day before mice sacrificed, oral glucose tolerance test (OGTT) was performed in 12 h fasted diabetic mice by feeding glucose (2.5 g/kg) per os. S.k. extract, gliclazide and vehicle were orally administered 0.5 h before glucose administration. Blood glucose levels were determined at 0, 0.5, 1, 2 h after glucose administration subsequently. Glucose responses during the OGTT were also calculated from the area under the curve (AUC) by using the trapezoidal method. 2.9. Acute effect of S.k. extract on Blood glucose in normal and diabetic mice The 12 h fasted mice were divided into 8 groups (10 animals in each group): groups 1/5 were normal (NC)/diabetic (DC) control groups, respectively, which received 0.3% CMC-Na solution. Groups 2/6 were normal mice/diabetic mice both treated with standard drug gliclazide (G.D) (15 mg/kg per os, dissolved in vehicle). Groups 3/4 were normal mice respectively treated with S.k. extract at two doses (250 mg/kg and 500 mg/kg per os, dissolved in vehicle); groups 7/8 were diabetic mice respectively treated with S.k. extract at the same doses as groups 3/4. The hypoglycemic effect of the S.k. extract was assessed by measuring blood glucose levels at 0 h, 0.5,1, 2, 4, 6 h after drug administration. 2.12. Estimation of biochemical parameters At the end of the experimental period, the 12 h fasted animals were sacrificed under anesthesia (thiopental 50 mg/kg). Blood samples were collected and the serum samples obtained by centrifugation (2000g for 20 min) were stored at −20 1C for further assay. Serum insulin levels were measured by ELISA. The contents of Malondialdehyde (MDA), the activities of Superoxidase dismu- tase (SOD) and lipids levels (TC, TG, HDL, LDL) in the serum were analyzed by using commercial diagnostic kits (Jiancheng, Nanjing, China). The liver tissues were extracted from the sacrificed mice, washed in ice-cold saline to remove the blood and stored at −80 1C for further assay. Glucokinase activities (GK), glucose 2.10. Long term effect of S.k. extract in diabetic mice Diabetic mice, divided into 4 groups, were daily treated for 4 weeks as the following: the diabetic control group was treated Fig. 1. The chromatographic profile of S.k ethanol extract: 1. Mangiferin; 2. Swertianolin; and 3. Bellidifolin. HPLC conditions: stationary phase: Alltima C18 (250?4.6 mm, 5 μm), Agilent-1100 HPLC system, Agilent, USA; mobile phase: methanol (A) and 0.05% (V/V) phosphoric acid aqueous solution (B) in gradient (0 min, A:B¼20:80, 50 min, A: B¼80:20, 55 min, A:B¼100:0); flow rate: 1 ml/min; detective wavelength: 254 nm; temperature: 30 1C.

  4. 625 L.-s. Wan et al. / Journal of Ethnopharmacology 147 (2013) 622–630 6-phosphatase activities (G6Pase) and glycogen level were assayed in the liver tissues by the methods of Newgard et al. (1983), Alegre et al. (1988) and Morales et al. (1975), respectively. 2.13. Statistical analysis All data were expressed as mean7S.D. Results were analyzed by one-way analysis of variance (ANOVA), and significant differ- ences were determined by post-hoc Tukey test using SPSS 11.0 software. Differences were statistically significant at Po0.05. 3. Results 3.1. HPLC analysis The chromatographic profile of the extract (Fig.1) indicated the presence of mangiferin, swertianolin and bellidifolin, which are the main components of S.k. The content of mangiferin, swertianolin and bellidifolin in the extract was determined to be 0.61%, 0.88%, 4.93% (w/w). 3.2. Acute toxicity test The Maximum Tolerated Dose (MTD) of the S.k. extract was found to be 410g/kg (per os) in mice. Tested mice of each group did not show any overt signs of toxicity during 24 h and 14 days observation. No mortality was recorded throughout 14 days monitoring. Fig. 2. Influence of different concentrations of S.k extract on α-amylase (A) and α- glucosidase (B) activity in vitro. Acarbose 0.03 mg/ml (A) and 0.8 mg/ml (B) were positive control. Each value were represented as mean7S.D., n¼4 independent experiments. 3.3. The inhibitory potency of S.k. extract on α-amylase and α-glucosidase The extract exhibited α-amylase inhibitory activity in a dose- dependent manner as 4.4%, 34.8%, 63.7%, 68.1% at the concentra- tions of 0.06, 0.13, 0.25, 0.50 mg/ml, respectively (Fig. 2A). The inhibitory effect of S.k. extract against α-glucosidase was also in a dose-dependent manner, as shown in Fig. 2B, and the increments were 19.3%, 43.2%, 65.4%, 80.6%, 85.2% at the concentrations of 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml. IC50values of S.k. extract against α-amylase and α-glucosidase were 0.17 and 0.94 mg/ml while acarbose were 0.04 and 0.78 mg/ml, respectively. Though the results of IC50were obviously (Po0.05) higher than that of acarbose, they still revealed the potential hypoglycemic activity of the S.k. extract. 3.6. Acute effect of S.k. extract on blood glucose levels in normal and diabetic mice The effects of S.k. extract on fasting blood glucose levels of normal and diabetic mice were presented in Fig. 5A and B, respectively. In the normal mice, both doses of S.k. extract displayed slow but significant hypoglycemic effects from 1st to 6th hour after extract administrations, while gliclazide took a more immediate and intensive hypoglycemic effect at 0.5th hour after the drug administration, as shown in Fig. 5A. The maximum hypoglycemic rates of the treated mice were 12.3% (Po0.01) at 2nd hour for S.k. 250 mg/kg, 15.8% (Po0.01) at 6th hour for S.k. 500 mg/kg, 48.7% (Po0.01) at 6th hour for gliclazide 15 mg/kg, compared with the normal control group at each time point. In diabetic mice, as shown in Fig. 5B, both doses of S.k. extract and gliclazide had a time dependent hypoglycemic activity, and maximumly brought blood glucose levels down by 11.3% (Po0.05) at 6th hour for S.k. 250 mg/kg, 11.9% (Po0.05) at 4th hour for S.k. 500 mg/kg, and 18.5% (Po0.01) at 6th hour for gliclazide 15 mg/kg, compared with the diabetic control group at each time point. 3.4. Effects of S.k. extract on NIT-1 cell insulin secretion ability Changes of insulin secretions in NIT-1 cells treated with S.k. extract or gliclazide were given in Fig. 3. After 90 min treatment, there were obvious increase in groups S.k. (1, 10, 100, 1000 mg/ml) and gliclazide (10 mg/ml) at both 5.6 mM and 16.7 mM glucose, compared with respectively negative control, suggesting that S.k. extract may have similar effects as gliclazide. 3.5. Oral sucrose and starch test in normal mice As shown in Fig. 4A and C, oral administration of 250 mg/kg or 500 mg/kg S.k. extract both significantly slowed down the increase of postprandial blood glucose. And the areas under the curve (AUC) for glucose were reduced by 13.5% (Po0.01) for S.k. 250 mg/kg, 16.7% (Po0.01) for S.k. 500 mg/kg and 23.4% (Po0.01) for acarbose 15 mg/kg in oral starch tolerance test (Fig. 4B); by 9.8% (no significant) for S.k. 250 mg/kg, 11.6% (Po0.05) for S.k. 500 mg/kg and 20.3% (Po0.01) for acarbose 15 mg/kg in oral sucrose tolerance test (Fig. 4D). 3.7. Long term effect of S.k. extract on blood glucose levels in diabetic mice As shown in Fig. 6, after 4 weeks' treatment, the STZ-induced hyperglycemia was significantly (Po0.01) ameliorated by S.k. extract. Whereas the gliclazide 15 mg/kg had a maximal reduction of 60.3% (Po0.01), two doses of S.k. extract reduced the glucose level by 41.3% (Po0.01) for S.k. 250 mg/kg and 40.7% (Po0.01) for

  5. 626 L.-s. Wan et al. / Journal of Ethnopharmacology 147 (2013) 622–630 Fig. 3. Effects of S.k. extract on NIT-1 cell insulin secretion ability. Each column represented the mean7S.D., n¼4 independent experiments; *Po0.05, **Po0.01, vs. control group at glucose 5.6 mM;+Po0.05,++Po0.01, vs. control group at glucose 16.7 mM. S.k. 500 mg/kg, respectively, compared with the diabetic control group at each week. Table 1 The effects of S.k. extract on serum MDA and SOD levels. Groups MDA (nmol/ml) SOD (U/ml) 3.8. Long term effect of S.k. extract on OGTT Normal control Diabetic control Diabetic+G.D. (15 mg/kg) Diabetic+S.k. (250 mg/kg) Diabetic+S.k. (500 mg/kg) 5.2170.87nn 9.1170.79 7.2371.05nn 6.9870.92nn 6.9170.84nn 139.4727.3nn 107.2719.3 124.2724.5 130.6721.9n 133.2722.3n In the OGTT study, as shown in Fig. 7A, the oral glucose tolerance of the diabetic group were severely impaired and the blood glucose levels were at a sustained high level until 1st hour. However, the S.k. extract treated groups showed significant (Po0.01) and steady declines of blood glucose levels from 0.5th hour. And the areas under the curve (AUC) for glucose were reduced by 25.6% (Po0.01) for S.k. 250 mg/kg, 29.7% (Po0.01) for S.k. 500 mg/kg, and 49.2% (Po0.01) for gliclazide 15 mg/kg (Fig. 7B). Each value was expressed as mean7S.D. n¼10. nPo0.05. nnPo0.01 vs. diabetic control group. 3.11. Long term effect of S.k. extract on serum lipid profile As shown in Table 2, the increased serum levels of TG, TC, LDL were significantly suppressed decreased serum HDL levels (Po0.01, o0.05) by S.k. extract in diabetic mice. In the diabetic mice treated with S.k 250 mg/kg, S.k. 500 mg/kg and gliclazide 15 mg/kg, the TC levels were decreased by 29.2%, 34.5%, 24.3%, respectively; the TG levels were decreased by 24.1%, 29.3%, 14.5%; the LDL levels were decreased by 50.5%, 58.6%, 38.4%; while the HDL levels were elevated by 23.7%, 17.9%, 16.0%, compared with the diabetic control mice. 3.9. Long term effect of S.k. extract on serum insulin level in diabetic mice (Po0.01, were o0.05) significantly while improved the As shown in Fig. 8, the serum insulin levels of each treated group were significantly (Po0.01) ameliorated after 4-weeks’ treatment. The insulin levels of gliclazide 15 mg/kg, S.k. 250 mg/kg, S.k. 500 mg/kg groups were significantly (Po0.01) increased by 83.0%, 35.9%, 40.4%, respectively, compared with the diabetic control group. 3.10. Long term effect of S.k. extract on serum MDA and SOD levels 3.12. Long term effect of S.k. extract on key markers of carbohydrate metabolism As shown in Table 1, Serum MDA levels were significantly (Po0.01) increased while the activities of SOD were significantly (Po0.01) decreased in diabetic control group. However, after 4 weeks’ treatment with S.k. extract (250 mg/kg or 500 mg/kg), the MDA levels were remarkably (Po0.01) decreased while the activities of SOD were significantly (Po0.05) increased. Gliclazide 15 mg/kg significantly (Po0.01) decreased the MDA levels but had no significant effect on the activities of SOD. As shown in Table 3, the hepatic GK activities were significantly (Po0.01) lower in the diabetic control group, compared to the normal control group, while the G6Pase activities in diabetic control group were remarkably (Po0.01) higher. However, after 4 weeks administration, S.k. 250 mg/kg, S.k. 500 mg/g and glicla- zide 15 mg/kg supplements significantly elevated hepatic GK activities compared to the diabetic control group by 44.1%, 41.2%

  6. 627 L.-s. Wan et al. / Journal of Ethnopharmacology 147 (2013) 622–630 Fig. 4. Effects of S.k. extract on blood glucose levels after 6 g/kg starch (A and B) and 4 g/kg sucrose (C and D) administration in normal mice. Each value was expressed as mean7S.D., n¼10 independent experiments. Normal control groups were treated with equal volume of vehicle. c Po0.05, cc Po0.01, stand for S.k. extract (250 mg/kg) vs. control in A–D; d Po0.05, dd Po0.01, stand for S.k. extract (500 mg/kg); e Po0.05, ee Po0.01, stand for acarbose (15 mg/kg). Table 2 The effects of S.k. extract on serum lipid profiles. Groups TC (mmol/l) TG (mmol/l) LDL (mmol/l) HDL (mmol/l) Normal control Diabetic control Diabetic+G.D. (15 mg/kg) Diabetic+S.k. (250 mg/kg) Diabetic+S.k. (500 mg/kg) 4.1270.63** 8.3570.99 6.3270.87** 5.9170.94** 5.4770.87** 1.3270.17** 2.4970.31 2.1370.37* 1.8970.41** 1.7670.39** 0.3270.08** 0.9970.14 0.6170.12** 0.4970.07** 0.4170.06** 2.4970.17** 1.5670.24 1.8170.15* 1.9370.32** 1.8470.27* Each value was expressed as mean7S.D. n¼10. nPo0.05. nnPo0.01 vs. diabetic control group. and 76.5%, respectively, while the G6Pase activities were signifi- cantly reduced by 22.7%, 25.3% and 36.6%, respectively. The hepatic glycogen level was significantly higher in both S.k. treated groups and gliclazide group, as compared to the diabetic control group. with that of the standard drug acarbose. And in the following oral sucrose and starch tolerance test in normal mice, both doses of S.k. extract showed a remarkable inhibitory activity, compared with the normal control group. These findings suggested that S.k. extract could decrease the postprandial glucose level by inhibiting the activity of α-amylase and α-glucosidase, which are important enzymes in the digestion of the complex carbohydrates into adsorbable monosaccharides in the food. And recently, some reports found that xanthones (Fouotsa et al., 2012; Ryu et al., 2011), xanthone glycosides (Feng et al., 2011) and triterpenoids (Ali et al., 2006) could effectively inhibit the activity of α-amylase and/or α-glucosidase to decrease the absorption of carbohydrates from food. So the xanthones and triterpenoids in S.k. might take the responsibility for the postprandial antihyperglycemic effect of 4. Discussion The present study was designed to testify the potential anti- diabetic activity of S.k. ethanol extract (S.k. extract) by using in vitro enzyme assay and insulin secretion test, along with in vivo normal/STZ-induced diabetic mice model. The results of the in vitro α-amylase and α-glucosidase inhibi- tion test displayed that S.k. extract had a similar inhibitory effect

  7. 628 L.-s. Wan et al. / Journal of Ethnopharmacology 147 (2013) 622–630 S.k. extract. It was very interesting to point out that S.k. extract was more efficient for complex carbohydrate disaccharide sucrose. This result was consistent with the finding that S.k. extract could inhibit both α-amylase and α-glucosidase, since the digestion of starch involved both of these enzymes. In vitro insulin secretion test were carried out on NIT-1 cells, which is a mouse insulinoma cell line and a suitable model for evaluating the stimulating activity of insulin secretion (Xia et al., 2011). The test revealed that S.k. extract could significantly increase NIT-1 insulin secretion under 5.6 mM or 16.7 mM glucose level in medium, which could represent normal and diabetic conditions respectively, just as Brogden, 1993), suggesting that S.k. extract made this effect probably by enhancing the sensitivity of NIT-1 cell to glucose. In an early report, researchers found that a xanthone swerchirin isolated from the hexane fraction of Swertia chirayita could stimulate insulin release from islets (Saxena et al., 1993), so xanthones in the S.k. extract may be responsible for its stimulating insulin secretory ability. Then in vivo test, single administration of the S.k. extract significantly decreased the blood glucose levels in normal mice and diabetic mice. However, the hypoglycemic effect of S.k. extract was less intensive and immediate than that of gliclazide in normal mice, suggesting that S.k. extract might reduce the side effect of hypoglycemia during the treatment of diabetes. In further, the hypoglycemic effect of no matter S.k. extract or gliclazide in diabetic mice were lower than that in normal mice, starch than the gliclazide did (Palmer and Table 3 The effects of S.k. extract on key markers of carbohydrate metabolism. GK (nmol/ (min mg protein)) G6Pase (nmol/(min mg protein)) Hepatic glycogen (mg/g) Normal control Diabetic control Diabetic+G.D. (15 mg/kg) Diabetic+S.k. (250 mg/kg) Diabetic+S.k. (500 mg/kg) 15.6271.47** 7.3371.29 12.9470.87** 498.34778.41** 437.74735.74** 786.52756.23 20.2471.96** 13.4672.24 18.4873.26** 10.5671.68** 607.91769.58** 17.1072.98** 10.3571.23** 587.55770.52** 16.9272.06** Fig. 5. Acute effect of S.k. extract on blood glucose levels of normal (A) and diabetic (B) mice. Values were expressed as mean7S.D., n¼10. c Po0.05, cc Po0.01, stand for S.k extract (250 mg/kg) vs. normal control (A)/diabetic control (B); d Po0.05, dd Po0.01, stand for S.k. extract (500 mg/kg); e Po0.05, ee Po0.01, stand for gliclazide (15 mg/kg). Each value was expressed as mean7S.D. n¼10. nnPo0.01 vs. diabetic control group. Fig. 6. Long term effect of daily administration of S.k. extract on blood glucose levels in diabetic mice during a 4-weeks’ treatment. Values were expressed as mean7S.D, n¼10; c Po0.05, cc Po0.01, stand for S.k. extract (250 mg/kg) vs. diabetic control group; d Po0.05, dd Po0.01, stand for S.k. extract (500 mg/kg); e Po0.05, ee Po0.01, stand for gliclazide (15 mg/kg).

  8. 629 L.-s. Wan et al. / Journal of Ethnopharmacology 147 (2013) 622–630 Fig. 7. Effects of S.k. extract on glucose tolerance in diabetic mice. OGTT was performed on week 4 of treatment (A), and the AUC of OGTT was calculated (B). All results above were presented as mean7S.D., n¼10; c Po0.05, cc Po0.01, stand for S.k. extract (250 mg/kg) vs. diabetic control group; d Po0.05, dd Po0.01, stand for S.k. extract (500 mg/kg); e Po0.05, ee Po0.01, stand for gliclazide (15 mg/kg); *Po0.05, **Po0.01, stand for normal control group. Fig. 8. Effect of S.k. extract on serum insulin levels in diabetic mice. Each column represented the mean7S.D., n¼10; c Po0.05, cc Po0.01, stand for S.k. extract (250 mg/kg) vs. diabetic control group; d Po0.05, dd Po0.01, stand for S.k. extract (500 mg/kg); e Po0.05, ee Po0.01, stand for gliclazide (15 mg/kg); *Po0.05, **Po0.01, stand for normal control group. which may be due to the failure of β-cells to release sufficient insulin after stimulated by the extract or gliclazide, since the pancreatic β-cells were partially destroyed after injection of STZ (Strandell et al., 1988). And further, after fed with S.k. extract for 4 weeks, there were significant decrease in blood glucose levels and increase in serum insulin levels when compared with the diabetic control group, and the impaired oral glucose tolerance in diabetic mice were also greatly improved in the S.k. extract treated groups, indicating the beneficial effect of S.k. extract on ameliorat- ing function of islets in diabetic mice. In another report, researchers found that the antioxidant treatment was beneficial for treating diabetes and could provide protection to β-cells against glucose toxicity in diabetic mice (Kaneto et al., 1999). Recently a report revealed that the ethanolic extract of Swertia chirayita possesses in vitro and in vivo antiox- idant effects (Chen et al., 2011). In our present study, the antioxidant enzyme SOD activities in the serum, which can stand against the chronic oxidative stress on the β-cells (Wu et al., 2012), were remarkably improved in the S.k. extract treated groups with the decreasing MDA levels. So as the main components in S.k., xanthones with their antioxidant activity (Scartezzini and Speroni, 2000) may directly or indirectly preserve and regenerate β-cells function in the duration of hyperglycemia. The dysfunction of lipid metabolism associated with diabetes mellitus may be attributed to insulin deficiency (Morel and Chisolm, 1989). Insulin deficiency results in failure to activate lipoprotein lipase, which hydrolyzes triglycerides in normal mice, thereby causing hypertriglyceridemia (Shirwaikar et al., 2004). In the present long term study, the increased TG, TC and LDL levels and decreased HDL levels were observed in the diabetic mice. After treated with S.k. extract or gliclazide for 4 weeks, levels of TG, TC and LDL were decreased while HDL levels were increased, all of which maybe contributed to the relatively sufficient insulin secretion, and these amelioration of lipids metabolisms suggested that the S.k. extract had some beneficial effects on hyperlipidemia. The liver is an important organ that plays a pivotal role in glycolysis and gluconeogenesis. GK in liver is not only an insulin- dependent but also an insulin-sensitive enzyme and is almost completely inactivated in the diabetic liver in the absence of insulin (Gupta et al., 1999). GK insufficiency in diabetes can cause decreased utilization of glucose for energy production (Vats et al., 2003). G6Pase, which normally is suppressed by the action of insulin, catalyzes the last enzymatic reaction that is common to gluconeo- genesis and makes the liver release glucose into the blood, resulting in the rising of blood glucose level (Argaud et al., 1996; Mevorach et al., 1998). In the present study, GK activity was significantly increased while G6Pase activity was significantly decreased in the liver of diabetic mice after 4 weeks treatment with S.k extracts, which may be due to the enhanced insulin secretion. The significant increase in glycogen levels of the S.k. extract and gliclazide treated groups toward normal level further supported the beneficial effect on carbohydrate metabolism of S.k. (Golden et al., 1979).

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