Anti-diabetic effects of the acetone fraction of Senna singueana stem bark in a type 2 diabetes rat model

Anti-diabetic effects of the acetone fraction of Senna singueana stem bark in a type 2 diabetes rat model Mohammed Auwal Ibrahim a,b, M. Shahidul Islam a,n a Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban 4000, South Africa b Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria a r t i c l e i n f o Article history: Received 3 September 2013 Received in revised form 28 January 2014 Accepted 18 February 2014 Available online 26 February 2014 Keywords: Senna singueana Type 2 diabetes α-Glucosidase α-Amylase Rats Chemical compounds studied in this article: Streptozotocin (CID 29327) α-Glucosidase (E.C. 3.2.1.20) α-Amylase (E.C. 3.2.1.1) p-Nitrophenyl-α-d-glucopyranoside (pNPG) (CID 11197369) p-Nitrophenol (CID 980) Starch (CID 439341) Dinitrosalicylic acid (DNS) (CID 11873) Maltose (CID 6255) Absolute ethanol (CID 702) Ethyl acetate (CID 8857) Hydrogen peroxide (CID 784) a b s t r a c t Ethnopharmacological relevance: Senna singueana is currently used in the traditional treatment of diabetes mellitus in Nigeria. The present study examined the anti-diabetic activity of the Senna singueana acetone fraction (SSAF) of stem bark in a type 2 diabetes (T2D) rat model. Materials and methods: Crude ethyl acetate extract of the Senna singueana stem bark was fractionated with various solvents and the acetone fraction was selected for in vivo studies based on the high α- glucosidase and α-amylase inhibitory activities. In the in vivo study, male Sprague-Dawley rats were induced with T2D and treated with the SSAF at 150 and 300 mg/kg body weight. Several T2D-related parameters were measured in the study. Results: After 4 weeks of intervention, non-fasting blood glucose concentrations were significantly decreased and the glucose tolerance ability was significantly improved in the SSAF treated groups compared to the diabetic control group. Serum insulin concentrations, pancreatic β-cell function (HOMA- β) and liver glycogen were significantly (Po0.05) increased while serum alanine transaminase, alkaline phosphatase and urea were significantly decreased in the SSAF treated diabetic rats compared to the diabetic control group. Though insignificantly (P40.05), other T2D-induced abnormalities such as food and fluid intake, body weight, serum lipids, serum fructosamine level and peripheral insulin resistance (HOMA-IR) were also partially ameliorated by the SSAF treatment. Conclusion: Data of this study suggest that orally administered SSAF could ameliorate most of the T2D- induced abnormalities in a T2D model of rats. & 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose level resulting from defects in insulin secretion, insulin action or both (ADA, 2005). According to a projec- tion of the International Diabetes Federation (IDF), approximately 366 million people are living with diabetes and this figure is projected to increase to 552 million by the year 2030 (IDF, 2011). Among two major types of diabetes, type 2 is more prevalent than type 1, with more than 90% of the total diabetic patients are suffering from it. Type 2 diabetes (T2D) is a heterogeneous disorder character- ized by a progressive decline in insulin action (insulin resistance), followed by the inability of pancreatic β-cells to compensate for insulin resistance (β-cell dysfunction) which leads to hyperglycemia (DeFronzo, 2004). The control of hyperglycemia is therefore of prime impor- tance to retard the progression of the disease. At present, the use of insulin secretagogues and sensitizers constitute the predominant line of therapy, however, the use of inhibitors of carbohydrate digesting enzymes in order to reduce the intest- inal absorption of glucose is also vital as they do not interfere Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology http://dx.doi.org/10.1016/j.jep.2014.02.042 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved. Abbreviations: ADA, American diabetes association; ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; BRU, biomedical resource unit; DBC, diabetic control; DCH, diabetic high dose; DCL, diabetic low dose; DMF, diabetic metformin; DNS, dinitrosalicylic acid; ELISA, enzyme-linked immunosor- bent assay; FBG, fasting blood glucose; GC–MS, gas chromatography–mass spec- troscopy; HOMA-β, homeostatic model assessment – β-cell function; HOMA-IR, homeostatic model assessment – insulin resistance; IDF, international diabetes federation; NC, normal control; NCT, normal toxicological dose; NFBG, non-fasting blood glucose; OGTT, oral glucose tolerance test; pNPG, p-nitrophenyl-α-d-gluco- pyranoside; T2D, type 2 diabetes; SSAF, Senna singueana acetone fraction; STZ, streptozotocin n Corresponding author. Tel.: þ27 31 260 8717; fax: þ27 31 260 7942. E-mail addresses: [email protected], [email protected] (M.S. Islam). Journal of Ethnopharmacology 153 (2014) 392–399 with the carbohydrate metabolism and help to control hyper- glycemia in a noninvasive manner (Ghadyale et al., 2012). The α-glucosidase inhibitors are the current class of inhibitors of intestinal carbohydrate absorption which are shown to control postprandial hyperglycemia. However, the leading glucosidase inhibitors, acarbose and miglitol, are often reported to produce diarrhea and other intestinal disturbances, with corresponding bloating, flatulence, cramping and abdominal pain (Fujisawa et al., 2005). Hence, research on diabetes therapy is focused on the search for alternative agents which could decrease post- prandial hyperglycemia and other diabetic complications with fewer or no side effects. Senna singueana Delile, also known as golden shower, is a member of the family Caesalpiniaceae and commonly used by traditional medical practitioners to treat diabetes mellitus (Etuk et al., 2010) as well as bath for nursing mothers. Preliminary reagent-based phyto- chemical screening revealed that the methanol extract of the root of the plant contains phenols, saponins, tannins and anthraquinones (Adzu et al., 2003) while the methanol extract of the leaves contains alkaloids, tannins, sterols and terpenes (Ode and Onakpa, 2010). In a recent study, the in vitro anti-oxidative potential of the methanolic extract from the leaves was reported (Madubunyi and Ode, 2012). However, in a most recent study, we subjected different solvent crude extracts of the stem bark, root and leaves of the plant to anti-oxidative activity assays using several models and reported that the ethyl acetate extract of the stem bark showed the best anti-oxidative activity among all other extracts (Ibrahim et al., 2013). Subsequently, the phytochemical compounds present in the ethyl acetate extract were identified using GC–MS analysis where it was found to contain phenolic compounds such as 4-propylphenol and resorcinol, sterols such as 6-dehydroestradiol, aromatic esters such as methyl benzoates including some others such as dehydroxylevodopa and 2,3-dihydro- benzofuran (Ibrahim et al., 2013). Along with others, the major compound found in this extract was resorcinol (relative abundance 54.03%) which has been reported to have anti-diabetic activities via enhancing the glucose lowering efficacy of an engineered insulin called acylated insulin degludec (Steensgaard et al., 2013) and by stimulating hepatic glycogen synthesis and storage via leptin and insulin mediated pathways (Aiston and Agius, 1999). Additionally, in another recent study, a plant derived dibenzofuran has been reported to have potent anti-hyperglycemic activity at an oral dose of 20 mg/ kg bw/day in a db/db mouse model (Carney et al., 2002). Hence, some of the above-mentioned compounds may have direct or indirect anti- diabetic effects. Hence, in the present study, the ethyl acetate extract was further fractionated across solvents of different polarity and the acetone fraction derived from it was found to have the highest α- glucosidase and α-amylase inhibitory activities (among other solvent fractions). Subsequently, the acetone fraction was sub- jected to a detailed anti-diabetic study in an experimentally- induced T2D rat model. 2. Materials and methods 2.1. Chemicals and reagents Streptozotocin (STZ), powdered Saccharomyces cerevisiae α- glucosidase (E.C. 3.2.1.20), powdered porcine pancreatic α-amylase (E.C. 3.2.1.1), p-nitrophenyl-α-d-glucopyranoside (pNPG), and p- nitrophenol were obtained from Sigma-Aldrich through Capital Lab Supplies, New Germany, South Africa. Starch, dinitrosalicylic acid (DNS), maltose, absolute ethanol, ethyl acetate, and hydrogen per- oxide reagent were obtained from Merck Chemical Company, Durban, South Africa. 2.2. Plant material The stem bark of Senna singueana was collected in the month of January, 2011 from Zaria, Kaduna state, Nigeria. The plant samples were identified and authenticated by the herbarium unit of the Department of Biological Science, Ahmadu Bello University, Zaria and a voucher specimen number 6863 was deposited. The stem bark was immediately washed and shade-dried for two weeks to constant weights. The dried samples were ground to fine powder using a kitchen blender, and stored individually in air-tight zip-loc polythene bags to transport to the University of KwaZulu-Natal, Westville Campus, Durban, South Africa for subsequent analysis. 2.3. Extraction and solvent-solvent fractionation Three kilograms (3 kg) of the fine powdered stem bark of the plant was defatted with hexane. The defatted material was extracted with 10 l of ethyl acetate by soaking for 48 h and filtered through Whatmann filter paper (No. 1). The resultant extract was evaporated in vacuum using a rotary evaporator (Buchi Rotavapor II, Buchi, Germany) at 40 1C under reduced pressure to obtain the crude ethyl acetate extract with a yield of 4.04%. Forty grams of the crude ethyl acetate extract of the stem bark was dissolved in 500 ml of distilled water:methanol (9:1) mixture and successively fractionated with hexane (2�500 ml), dichloro methane (2�500 ml) and acetone (2�500 ml). The fractions were evaporated to dryness in vacuum at 40 1C under reduced pressure whereas the remaining aqueous fraction was dried in water bath at 50 1C. The dried fractions were transferred to micro tubes and stored at 4 1C until further analysis. 2.4. α-Glucosidase (E.C. 3.2.1.20) inhibitory activity of the solvent fractions The α-glucosidase (E.C. 3.2.1.20) inhibitory activity was deter- mined according to the method described by Ademiluyi and Oboh (2013) with slight modifications. Briefly, 250 ml of each fraction or acarbose at different concentrations (30–240 mg/ml) was incu- bated with 500 ml of 1.0 U/ml α-glucosidase solution in 100 mM phosphate buffer (pH 6.8) at 37 1C for 15 min. Thereafter, 250 ml of pNPG solution (5 mM) in 100 mM phosphate buffer (pH 6.8) was added and the mixture was further incubated at 37 1C for 20 min. The absorbance of the released p-nitrophenol was measured at 405 nm and the inhibitory activity was expressed as percentage of control without inhibitors. The α-glucosidase inhibitory activity was calculated according to the following formula: Inhibitory activity ð%Þ ¼ 1� As Ac � � � 100 where As is the absorbance in the presence of sample and Ac is the absorbance of control. 2.5. α-Amylase (E.C. 3.2.1.1) inhibitory activity of the solvent fractions The α-amylase (E.C. 3.2.1.1) inhibitory activity was determined according to the method described by Shai et al. (2010) with slight modifications. A volume of 250 ml of each fraction or acarbose at different concentrations (30–240 mg/ml) was incubated with 500 ml of porcine pancreatic amylase (2 U/ml) in 100 mM phosphate buffer (pH 6.8) at 37 1C for 20 min. A 250 ml of 1% starch was dissolved in 100 mM phosphate buffer (pH 6.8) then added to the reaction mixture and incubated at 37 1C for 1 h. One ml of DNS color reagent was then added and boiled for 10 min. The absorbance of the resul- ting mixture was measured at 540 nm and the inhibitory activity was expressed as percentage of a control without inhibitors. The α- amylase inhibitory activity was calculated using the following M.A. Ibrahim, M.S. Islam / Journal of Ethnopharmacology 153 (2014) 392–399 393 formula: Inhibitory activity ð%Þ ¼ 1� As Ac � � � 100 where As is the absorbance in the presence of sample and Ac is the absorbance of control. Based on the above assays, the Senna singueana acetone fraction (SSAF) was selected for the in vivo anti-diabetic study. 2.6. Experimental animals Six-week-old male Sprague-Dawley rats were procured from the Biomedical Resource Unit (BRU) located at the University of KwaZulu-Natal (Westville Campus), Durban, South Africa with initial mean body weight (bw) 207.6074.27 g. Animals were housed as 2 in one medium size poly-carbonated cage in a temperature and humidity controlled room with a 12 h light–dark cycle. A standard rat pellet diet was supplied ad libitum during the entire experimental period and the animals were maintained according to the rules and regulations of the Experimental Animal Ethics Committee of the University of KwaZulu-Natal, South Africa (Ethical approval number: 022/12/Animal). 2.7. Animal grouping and induction of type 2 diabetes Animals were randomly divided into six groups of eight animals each namely; NC: Normal Control, DBC: Diabetic Control, DCL: Diabeticþ low dose (150 mg/kg bw) of SSAF, DCH: Diabeticþhigh dose (300 mg/kg bw) of SSAF, DMF: Diabeticþmetformin (300 mg/ kg bw), NCT: Non-diabeticþhigh dose (300 mg/kg bw) of SSAF. After one week adaptation period, the animals in DBC, DCL, DCH and DMF groups were supplied with a 10% fructose solution ad libitum for two weeks to induce insulin resistance followed by a single injection (i.p.) of STZ (40 mg/kg bw) dissolved in citrate buffer (pH 4.5) to overnight fasted animals to induce partial pancreatic β-cell dysfunctionwhen the animals in NC and NCT groups were supplied with normal drinking water and injected with citrate buffer instead of 10% fructose and STZ injection, respectively (Wilson and Islam, 2012). One week after the STZ injection, the non-fasting blood glucose (NFBG) levels of all animals were measured in the blood collected from tail vein by using a portable glucometer (Glucoplus Inc., Saint-Laurent, Quebec, Canada). Animals with a NFBG level 418mmol/l were considered to be diabetic (Islam, 2011) while animals with a NFBG level o18 mmol/l were excluded from the study. 2.8. Intervention trial After the confirmation of diabetes, a respective dose of the fraction was orally administered 5 days per week by using a gastric gavage needle to the animals in DCL and DCH and NCT groups while the animals in controls (NC and DBC) and DMF groups were treated with a similar volume of the vehicle and metformin respectively for a 4-week experimental period. The SSAF was given 5 days in a week with 2 days interval between two consecutive weeks in order to reduce esophageal as well as overall stress to the animals. During this period, daily food and fluid intake as well as weekly body weight and NFBG was measured in all animal groups. 2.9. Oral glucose tolerance test To measure the glucose tolerance ability of each animal, the oral glucose tolerance test (OGTT) was performed in the last week of the 4-week intervention period. To perform this test, a single dose of glucose solution (2 g/kg bw) was orally ingested to each animal and the levels of blood glucose were measured at 0 (just before glucose ingestion), 30, 60, 90 and 120 min after the ingestion of glucose. 2.10. Collection of blood and organs At the end of the experimental period, animals were euthanized by halothane anesthesia and blood and organ samples were col- lected. The whole blood of each animal was collected via cardiac puncture and immediately preserved in a refrigerator until further processing. The blood samples were centrifuged at 3000 rpm for 15 min and serum was separated and preserved at �30 1C for further analysis. The liver was collected from each animal, washed with normal saline, wiped with filter paper, weighed and preserved at �30 1C until subsequent analysis. A small piece of pancreatic tissue from each animal was cut and placed in a 10% neutral buffered formalin solution and preserved at room temperature for histopatho- logical study. The neutral buffered formalin of each pancreatic tissue sample was replaced weekly during the entire preservation period. 2.11. Analytical methods The serum insulin concentrations were measured by an enzyme- linked immunosorbent assay (ELISA) method using an ultrasensitive rat insulin ELISA kit (Mercodia, Uppsala, Sweden) in a multi plate ELISA reader (Biorad-680, BIORAD Ltd., Japan). The serum lipid profile, fructosamine, urea and creatinine concentrations as well as liver function enzymes; aspartate and alanine transaminases (AST and ALT) and alkaline phosphatase (ALP) were measured using an Automated Chemistry Analyzer (Labmax Plenno, Labtest Co. Ltd., Lagoa Santa, Brazil) with commercial assay kits from the same company. Homeostatic model assessment (HOMA-IR and HOMA-β) scores were calculated using fasting serum insulin and FBG concen- trations measured at the end of the experimental period according to the following formula: HOMA� IR¼ ½ðFasting serum insulin in U=L �Fasting blood glucose in mmol=LÞ=22:5� HOMA�β¼ ðFasting serum insulin in U=L �20=Fasting blood glucose in mmol=L–3:5Þ Conversion factor: insulin (1U/L¼7.174 pmol/l) Liver glycogen concentrations were measured by a phenol- sulfuric acid method as described by Lo et al. (1970). 2.12. Histopathological examination of pancreatic tissue The formalin preserved pancreatic tissues were treated accord- ing to a standard laboratory protocol for paraffin embedding. Sections were cut at a size of 4 mm. Then, slides were deparaffi- nized in p-xylene and rehydrated in changes of ethanol concen- trations (100%, 80%, 70%, 50%) and rinsed with water. Slides were stained in hematoxylin for 5 min and rinsed with water and counterstained in eosin, mounted in DPX, cover-slipped and viewed with Leica slide scanner (SCN 4000, Leica Biosystems Germany). 2.13. Statistical analysis All data are presented as the mean7SD. Data were analyzed by using a statistical software package (SPSS for Windows, version 18, IBM Corporation, NY, USA) using Tukey's-HSD multiple range post- hoc test. Values were considered significantly different at Po0.05. 3. Results Analysis of the α-glucosidase and α-amylase inhibitory activ- ities of the fractions revealed that the more polar fractions demonstrated significantly higher (Po0.05) α-glucosidase and α- amylase inhibitory activities than the less polar fractions. However, M.A. Ibrahim, M.S. Islam / Journal of Ethnopharmacology 153 (2014) 392–399394 within the more polar fractions, the α-glucosidase and α-amylase inhibitory activities demonstrated by the acetone fraction were significantly higher (Po0.05) than all other solvent fractions (Table 1). Based on the high activity of the acetone fraction in most of the assays described above, it was selected for further in vivo studies. The mean food and fluid intake per animal per day over the entire experimental period is presented in Fig. 1. The food and fluid intake of the DBC group was significantly higher (Po0.05) compared to NC and NCT groups; however the SSAF treated groups consumed an insignif- icantly (P40.05) lower food and fluid than the DBC group. The fluid intake of DCH group was markedly lower compared to the DBC, DCL and DMF groups. Although there was no significant difference in the body weight between the different animal groups during the first 2 weeks of the experiment but the body weight gains of the diabetic groups were significantly reduced compared to NC and NCT groups during the remaining experimental period (Fig. 2). The blood glucose concentrations of the diabetic groups were significantly higher than the NC and NCT groups at week 1, however, as soon as the intervention started, the DCL and DCH groups maintained a significantly lower (Po0.05) blood glucose levels than the DBC group throughout the experimental period (Fig. 3). The blood glucose lowering effect of the fraction seems to be dose dependent because the DCH group had a relatively lower blood glucose levels than DCL group throughout the intervention period. These results also suggest the significant effects of SSAF on both non-fasting (week 0–3) and fasting blood glucose (week 4) levels compared to the DBC group (Fig. 3). The data for OGTT are shown in Fig. 4. The glucose tolerance abilities of DCL and DCH groups were significantly (Po0.05) better than the DBC group during the entire experimental period. Even better glucose tolerance was observed for DCH group compared to the metformin consuming DMF group at 120 min point of the glucose tolerance test. The glucose tolerance ability of DCH group was also Table 1 IC50 values for the inhibition of α-glucosidase and α-amylase by various solvent fractions of the ethyl acetate extract of Senna singueana stem bark. Fractions/standard IC50 (mg/ml) α-Glucosidase α-Amylase Aqueous 80.9970.97c 157.2474.96b Acetone 67.5873.59b 108.3672.64a Dichloromethane 175.5971.68d 769.547165.71d Hexane 278.66748.64e NIL Acarbose 55.5975.22a 256.66720.52c Data are presented as mean7SD of triplicate determinations. a–eValues with different letters along a column are significantly different from each other (Tukey's-HSD multiple range post hoc test, Po0.05). 0 20 40 60 80 100 120 140 160 Food intake (g/rat/day) Fluid intake (ml/rat/day) Fo od a nd fl ui d in ta ke (u ni ts ) NC DBC DCL DCH DMF NCT a a ab b b b a a b b b b Fig. 1. Food and fluid intake of the different groups during the experimental period. Data are presented as mean7SD of eight animals. a,bValues with different letters over the bars for a given parameter are significantly different from each other (Tukey's-HSD multiple range post hoc test, Po0.05). NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose. 150 200 250 300 350 400 WK 0 WK 1 WK 2 WK 3 WK 4 WK 5 WK 6 WK 7 M ea n bo dy w ei gh t ( g) Experimental weeks NC DBC DCL DCH DMF NCT a a a a a ab b ab ab ab a b b b b ab b b ab b ab b ab b STZ injection T2D induction Treatment Fig. 2. Mean body weight gain for all groups of experimental animals over the seven weeks experimental period. Data are presented as the mean7SD of eight animals. a,bValues with different letters for a given week are significantly different from each other (Tukey's-HSD multiple range post hoc test, Po0.05). NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose. 0 5 10 15 20 25 30 35 WK 0 WK 1 WK 2 WK 3 WK 4 B lo od g lu co se le ve l ( m m ol /L ) Weeks after intervention NC DBC DCL DCH DMF NCT a a a a a b c c c bc c c b b b b b b c b b NFBG FBG Fig. 3. Weekly blood glucose concentrations of different animal groups. Data are presented as the mean7SD of eight animals. a–cValues with different letters for a given week are significantly different from each other (Tukey’s-HSD multiple range post hoc test, Po0.05). NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose; NFBG, Non- fasting Blood Glucose; FBG, Fasting Blood Glucose. M.A. Ibrahim, M.S. Islam / Journal of Ethnopharmacology 153 (2014) 392–399 395 significantly better than the DCL group at almost the entire period of the test. Table 2 presents the data for serum insulin and fructosamine concentrations as well as the calculated HOMA-IR and HOMA-β scores. A significantly (Po0.05) higher serum insulin concentra- tion and better β-cell function (HOMA-β) was observed in the DCL and DCH groups compared to the DBC group. The serum fructo- samine levels and insulin resistance (HOMA-IR) were also markedly decreased in the DCL and DCH groups compared to the DBC group (Table 2). The data for liver weights and liver glycogen levels are presented in Table 3 and there were no significant differences in the liver wei- ghts of all groups of experimental rats but the relative liver weights of the diabetic groups were significantly higher than NC and NCTgroups. The DCL, DCH and DMF groups had insignificantly (P40.05) lower relative liver weights compared to the DBC group. A significantly higher liver glycogen contents were detected in the NC, DCL, DCH and DMF groups compared to DBC and NCT groups. The concentrations of the serum lipids are presented in Fig. 5. Although there were no significant differences in the concentrations of total cholesterol and LDL cholesterol among the experimental groups, the DBC had relatively higher values which were reduced in the DCL and DCH groups. The DBC group also recorded a significantly lower level of HDL cholesterol compared to the DCL and DCH groups. Furthermore, T2D caused a significant increase in the serum triglycer- ides which was ameliorated in the DCL, DCH and DMF groups. The data for serum AST, ALT, ALP, urea and creatinine concen- trations are presented in Table 4. The serum levels of ALT, ALP and urea were significantly elevated in the DBC group compared to the NC group but the DCL, DCH and DMF groups recorded significantly lower values of these biochemical parameters when compared to the DBC group (Table 4). Conversely, relatively higher serum levels of creatinine was observed in the DMF group compared to the DBC group whereas no significant difference was observed for serum AST levels among all experimental groups. In the histopathological examination of the pancreas, the DBC group had a reduced number of β-cells which were highly dispersed compared to the NC group. Higher number of β-cells was observed in the DCL and DMF groups compared to the DBC group although the size of the islets was morphologically smaller compared to the NC group (Fig. 6). 4. Discussion The development of anti-diabetic agents that are devoid of adverse effects is still a challenge to the health care systems 0 5 10 15 20 25 30 35 0 30 60 90 120 B lo od g lu co se le ve l ( m m ol /L ) Time after glucose ingestion (min) NC DBC DCL DCH DMF a a a a a b bc c d b b b c c c bc c bc c b b c d b Fig. 4. Oral glucose tolerance test (OGTT) for all groups of animals in the last week of the experimental period. Data are presented as the mean7SD of eight animals. a–dValues with different letters for a given time are significantly different from each other (Tukey's-HSD multiple range post hoc test, Po0.05). NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose. Table 2 Serum insulin and fructosamine concentrations as well as HOMA-IR and HOMA-β scores at the end of the experimental period. NC DBC DCL DCH DMF NCT Serum insulin (pmol/L) 153.1279.91b 64.2729.93a 119.71717.80b 138.33712.58b 119.19713.00b 140.44717.82b Serum fructosamine (mmol/L) 196.3375.57a 258.40710.92b 268.40714.11b 214.40728.97ab 253.83730.36b 185.0078.41a HOMA-IR 4.9671.05a 16.2475.65b 13.0375.69b 13.4074.99b 9.8473.78b 4.5670.69a HOMA-β 304.07753.72d 9.6875.24a 20.2974.69b 32.76716.65bc 54.85727.51c 232.42755.48d Data are presented as the mean7SD of eight animals. a–dValues with different letters along a row are significantly different from each other (Tukey's-HSD multiple range post hoc test, Po0.05). NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose. HOMA� IR¼ ½ðFasting serum insulin in U=L � Fasting blood glucose in mmol=LÞ=22:5� HOMA�β¼ ðFasting seruminsulin in U=L � 20=Fasting blood glucose in mmol=L–3:5Þ Conversion factor: insulin (1 U/L¼7.174 pmol/l). Table 3 Liver weights and liver glycogen concentrations in different animal groups at the end of the experimental period. NC DBC DCL DCH DMF NCT Liver weight (g) 11.8072.35 10.9871.96 10.5971.18 9.8770.83 10.3971.73 10.8271.36 Relative liver weight (%) 3.2870.33a 3.9670.15b 3.8070.13b 3.7770.18b 3.6770.12b 3.1270.13a Liver glycogen (mg/g tissue) 3.5370.83c 1.5470.13a 3.1870.23bc 2.7270.15b 2.6570.38b 1.4670.64a Data are presented as the mean7SD of eight animals. a–cValues with different letters along a row are significantly different from each other (Tukey’s-HSD multiple range post hoc test, Po0.05). NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose. M.A. Ibrahim, M.S. Islam / Journal of Ethnopharmacology 153 (2014) 392–399396 globally. Thus, medicinal plants are constantly being explored with the hope of developing a relatively safe antidiabetic plant-based product alone or in combination with other agents (Tamiru et al., 2012). In the present study, we reported the anti-diabetic activity of an acetone fraction of the stem bark of Senna singueana in a newly developed model of T2D. Senna singueana is used for the traditional remedy of diabetes mellitus and the acetone fraction was selected for the study based on the higher α-glucosidase and α-amylase inhibitory activity showed by the fraction as compared to other solvent fractions. Polyphagia and polydipsia with concomitant reduction of body weight are major symptoms of diabetes mellitus (ADA, 2007) which were also evidently observed in the diabetic groups of our experiment. These parameters are usually dependent on the energy expenditure, urinary excretion and catabolic processes (Kamalakkannan and Prince, 2006) among others. In our study, although treatment with the either dosages of SSAF did not affect the food intake, the dose-dependently lower fluid intake and relatively better body weight gain in the SSAF treated groups compared to the DBC group suggest its possible anti- diabetic effects at least for T2D. However the period of our interven- tion was relatively short (4 weeks) and these effects of SSAF could be improved in a long-term study. Fasting or postprandial hyperglycemia is a common pathogen- esis of T2D which is induced by insulin resistance and partial pancreatic β-cell destruction (Wilson and Islam, 2012). Effective control of the blood glucose level is a key step in preventing or reversing diabetic complications and improving the quality of life in type 2 diabetic patients (Ross, 2004). In the present study, oral treatment of diabetic animals with the SSAF resulted in a significant, consistent and dose dependent decrease in blood glucose levels throughout the experimental period, indicating its potent anti-diabetic activity. Furthermore, in the OGTT analysis, better glucose tolerance abilities were observed in the SSAF treated groups than the DBC group. These results could be linked to the potent α-glucosidase and α-amylase inhibitory activity exhibited by the fraction which could cause a decrease in the digestion of carbohydrates and intestinal absorption of glucose (Ademiluyi and Oboh, 2013). On the other hand, the SSAF stimulated insulin secretions and improved pancreatic β-cell function whereas insulin resistance was not affected. Based on the foregoing observations, it can be suggested that the mechanism of anti-T2D action of the SSAF is elicited through delaying glucose absorption, improving β-cell function and stimulating insulin secretions rather than by increas- ing insulin sensitivity when resorcinol, the major bioactive com- pound of the extract (Ibrahim et al., 2013), might play a crucial role by enhancing the glucose lowering efficacy of insulin (Steensgaard et al., 2013). Interestingly, the above hypothesis was further supported by the histopathological examinations of the pancreatic islets where SSAF groups had more as well as healthier pancreatic islets than the DBC group. The SSAF was found to have high in vitro anti-oxidative activity (data not shown) and thus, we speculated that the protection of the pancreatic islets could be mediated through an anti-oxidative dependent mechanism because oxida- tive stress is an important contributor to the pancreatic β-cell damage in T2D (Huang et al., 2011). Previous studies reported contradictory findings on the effect of diabetes on liver weights. Some reports have shown an increase in hepatic weight in animals as well as humans while others have reported no change (Habibuddin et al., 2008). Our findings revealed that relative but not absolute liver weights were elevated by the disease and was not significantly reversed by the treatments. Hepatic glycogenesis is associated with extracellular glucose concentration and insulin availability. In vivo glycogen metabolism is regulated by some multifunctional enzymes such as glycogen synthase and glycogen phosphorylase (Verma et al., 2013). Reduced hepatic glycogen reserve in diabetic animals has been attributed to reduced activity of glycogen synthase and increased activity of glycogen phosphorylase. Thus, the ability of the SSAF to restore the depleted glycogen reserves in diabetic animals could indicate that the fraction also decreases the activity of glycogen phosphorylase and/or increases glycogen synthase activity finally increases the liver glycogen synthesis as well as storage via leptin and insulin mediated pathways when resorcinol (Ibrahim et al., 2013) might play a crucial role in this regard (Aiston and Agius, 1999). This could further suggest that the fraction not only acts as insulin secretagogue but also inhibits hepatic glycogenolysis thereby reducing an upsurge in blood glucose concentrations. Type 2 diabetes is associated with profound changes in the serum lipid and lipoprotein profile with an increased risk in cardiovascular diseases. Hyperlipidemia is a recognized complication in diabetic 0 20 40 60 80 100 120 140 160 180 200 Total cholesterol HDL-chol LDL-chol Triglycerides Se ru m li pi ds (m g/ dl ) NC DBC DCL DCH DMF NCT a b ab b b ab a a a a a ab Fig. 5. The serum lipid profile in different animal groups at the end of the experimental period. Data are presented as the mean7SD of eight animals. a,bValues with different letters for a given parameter are significantly different from each other (Tukey’s-HSD multiple range post hoc test, Po0.05). NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose. Table 4 Serum biochemical parameters for all groups of animals at the end of experimental period. NC DBC DCL DCH DMF NCT AST (U/L) 88.00714.88 73.6077.63 78.3374.88 76.1673.62 88.66717.87 86.00713.65 ALT (U/L) 56.25712.55a 77.7574.57b 64.6079.15ab 62.3379.26a 63.33718.18ab 48.50715.38a ALP (U/L) 188.80719.86b 898.607174.49e 320.40725.84c 344.60745.36c 472.00774.99d 117.50712.45a Urea (mg/dl) 48.0076.16b 93.00716.40c 69.20710.66bc 78.00710.94bc 42.00711.78a 30.2074.54a Creatinine (mg/dl) 582.00770.49a 485.00740.41a 570.007104.64a 545.00784.08a 675.00795.23b 470.00767.82a Data are presented as the mean7SD of eight animals. a–eValues with different letters along a row are significantly different from each other (Tukey's-HSD multiple range post hoc test, Po0.05). NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose. M.A. Ibrahim, M.S. Islam / Journal of Ethnopharmacology 153 (2014) 392–399 397 patients characterized by elevated levels of cholesterol, triglycerides and changes in lipoprotein composition (Kumar et al., 2011). This abnormally high level of serum lipids is mainly due to the uninhibited actions of lipolytic hormones on the fat depots, mainly due to the impairment of insulin secretions in the diabetic state. In our study, serum total cholesterol and LDL-cholesterol were relatively elevated but triglycerides were significantly increased in the DBC group compared to the NC group. Interestingly, the SSAF treated groups had relatively lower serum lipid concentrations than DBC group. Although the reductions of lipid profile in SSAF treated groups were nearly similar to the NC group, the datawere not significantly different due to high standard deviations. This observation is possibly linked to the stimulation of insulin secretion by the fraction which could modulate the actions of the lipolytic hormones in fat reserves. The serum levels of ALT and ALP but not AST were significantly increased in the untreated diabetic animals indicating impaired liver function, which is obviously due to hepatocellular necrosis. Diabetic complications such as increased gluconeogenesis and ketogenesis may be due to elevated transaminase activities (Ghosh and Suryawansi, 2001). Therefore, restoration of these biomarker enzymes towards normal level indicates decreased diabetic complications in the SSAF treated groups. Furthermore, the fraction reversed the T2D-induced increase in serum urea level which could suggest decreased renal impairments associated with diabetic complications. Apart from assessing renal damage, crea- tinine was reported to be a predictor for T2D and insulin resistance (Harita et al., 2009). Creatinine is a metabolite derived from creatine, which is predominantly found in skeletal muscle, the major site for insulin action and subsequent glucose disposal. It was proposed that inversely proportional relationship of muscle mass to insulin resistance and directly proportional to serum creatinine level could provide a conclusive measure to assess insulin resistance (Harita et al., 2009). Thus, previous studies have demonstrated lower serum creatinine level in type 2 diabetic human subjects compared to normal individuals (Harita et al., 2009; Hjelmesæth et al., 2010) which corroborates with our findings. From the data of our study, it is thus possible to suggest that the insigni- ficant decrease of insulin resistance (HOMA-IR) in DCL and DCH groups compared to the DBC group led to an insignificant elevation in the serum creatinine levels in these groups. In conclusion, data of our study supports the traditional use of Senna singueana in the treatment of diabetes mellitus because NC DBC DCL DCH DMF NCT Fig. 6. Histopathological examinations of the pancreas of different experimental groups at the end of the experiment. The NC had high number of β-cells while the DBC had highly dispersed and morphologically deformed β-cells. The DCL, DCH and DMF groups had higher number of β-cells (compared to DBC) but morphologically smaller whereas relatively reduced number of β-cells (compared to NC) but not morphologically deformed. NC, Normal Control; DBC, Diabetic Control; DCL, Diabetic Senna singueana Low dose; DCH, Diabetic Senna singueana High dose; DMF, Diabetic Metformin; NCT, Normal Control Toxicological (high) dose. M.A. Ibrahim, M.S. Islam / Journal of Ethnopharmacology 153 (2014) 392–399398 the acetone fraction of the stem bark of the plant has a strong anti- T2D activity and could also ameliorate other diabetes-related complications. The mechanism of action is mediated through inhibition of carbohydrate-hydrolyzing enzymes, modulation of β- cell function and stimulation of insulin secretions when some of the major compounds such as resorcinol and dibenzofuran might be actively involved in this process. Acknowledgments This study was supported by a Competitive Research Grant from the Research office, University of KwaZulu-Natal (UKZN), Durban; an Incentive Grant for Rated Researchers and a Grant Support for Women and Young Researchers from the National Research Foundation (NRF), Pretoria, South Africa. The first author was awarded a Ph.D. study scholarship by the Education Trust Fund desk office, Ahmadu Bello University, Zaria, Nigeria and also received a doctoral research Grant from the Research office, University of KwaZulu-Natal (Westville Campus), Durban, South Africa. The authors would like to thank Linda Bester, Dr. Sunday Oyedemi and Mr. Talent Chipiti for their technical assistance during the study. 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Islam / Journal of Ethnopharmacology 153 (2014) 392–399 399 Anti-diabetic effects of the acetone fraction of Senna singueana stem bark in a type 2 diabetes rat model Introduction Materials and methods Chemicals and reagents Plant material Extraction and solvent-solvent fractionation α-Glucosidase (E.C. 3.2.1.20) inhibitory activity of the solvent fractions α-Amylase (E.C. 3.2.1.1) inhibitory activity of the solvent fractions Experimental animals Animal grouping and induction of type 2 diabetes Intervention trial Oral glucose tolerance test Collection of blood and organs Analytical methods Histopathological examination of pancreatic tissue Statistical analysis Results Discussion Acknowledgments References