Food and Chemical Toxicology 66 (2014) 358–365

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Food and Chemical Toxicology

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Polyphenols-rich Cyamopsis tetragonoloba (L.) Taub. beansshow hypoglycemic and b-cells protective effects in type 2 diabetic rats

http://dx.doi.org/10.1016/j.fct.2014.02.0010278-6915/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: b wt., body weight; FBG, fasting blood glucose; HFD, high-fatdiet; ITT, insulin tolerance test; OGTT, oral glucose tolerance test; T2DM, type 2diabetes mellitus; TC, total cholesterol; TG, triglycerides; STZ, streptozotocin.⇑ Corresponding author. Address: Entomology Research Institute, Loyola College,

Nungambakkam, Chennai 600034, India. Tel.: +91 044 28178348; fax: +91 04428175566.

E-mail address: entolc@hotmail.com (S. Ignacimuthu).

Gopalsamy Rajiv Gandhi, Pautu Vanlalhruaia, Antony Stalin, Santiagu Stephen Irudayaraj,Savarimuthu Ignacimuthu ⇑, Michael Gabriel PaulrajDivision of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai 600034, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 October 2013Accepted 3 February 2014Available online 10 February 2014

Keywords:Cyamopsis tetragonolobaType 2 diabetesPolyphenolsBiochemical parametersHistologyImmunohistochemical

The aim of this study was to evaluate the antidiabetic activity of Cyamopsis tetragonoloba (L.) Taub.(Fabaceae) beans in high-fat diet (HFD) fed-streptozotocin (STZ)-induced type 2 diabetic rats. Dosedependent response of oral treatment of C. tetragonoloba beans’ methanol extract (CTme) (200 and400 mg/kg b wt.) was assessed by measuring fasting blood glucose, changes in body weight, plasma insu-lin, homeostasis model assessment of insulin resistance (HOMA-IR), total cholesterol, triglycerides, oralglucose tolerance, intraperitoneal insulin tolerance, hepatic glycogen, marker enzymes of carbohydratemetabolism in HFD fed-STZ-induced type 2 diabetic rats. Histology and immunohistochemical analysisof pancreatic islets were also performed. High-performance liquid chromatography (HPLC) analysis ofCTme showed the presence of polyphenols such as gallic acid and caffeic acid in the concentrations of2.46% (W/W) and 0.32% (W/W). CTme significantly reverted the altered biochemical parameters to nearnormal levels in diabetic rats. Furthermore CTme showed the protective effect on the b-cells of pancreatictissues in diabetic rats. These findings indicate that C. tetragonoloba beans have therapeutic potential inHFD fed-STZ-induced hyperglycemia; therefore this can be used in the management of type 2 diabetes.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Type 2 diabetes mellitus (T2DM) (non-insulin dependent dia-betes mellitus) has become an important threat to people’s healthby accounting for more than 90–95% of all diabetes (Naik et al.,2013). Defects in insulin action, depressed b-cell function and im-paired insulin secretion together with destruction in the b-cellmass are the decisive features of T2DM (Koning et al., 2008).The major diabetic complications are the micro- and macro-vas-cular problems such as neuropathy, nephropathy, retinopathy,cardiovascular and peripheral vascular disease (Dewanjee et al.,2009). Synthetic antidiabetic agents cause some side effects.Therefore, herbal medications with less side effects are encour-aged (Lim et al., 2012).

Cyamopsis tetragonoloba (L.) Taub. (Fabaceae) is also known ascluster bean. It is a drought resistant annual herb ranging from 2to 4 feet in height cultivated in semi-arid regions throughout India.C. tetragonoloba beans are consumed as food; the gum acquiredfrom endosperm of the seeds is beneficial for health. Pharmacolog-ical studies in rats have demonstrated the antiulcer, cytoprotective,anticholinergic, anticoagulant, antimicrobial, anti-asthmatic andanti-inflammatory activities of C. tetragonoloba (Sharma et al.,2011). Polyphenol composition of the plant includes gallic acid,caffeic acid, gallotannins, genistein, catechol, luteolin, myricetin-7-glucoside-3-glycoside, chlorogenic acid, ellagic acid, quercetin,daidzein, rutin, catechin, naringenin, kaemferol, 2,4,3,-trihydroxybenzoic acid, texasin-7-O-glucoside, hydroxycinnamic acid andp-coumaroylquinic acid (Daniel, 1989; Kobeasy et al., 2011).

C. tetragonoloba beans have been reported to possess hypogly-cemic and hypolipidemic effects in alloxan-induced insulin defi-cient animal model (Shrivastava et al., 1987). Mukhtar et al.(2006) have also reported antihyperglycemic property of C. tetrag-onoloba beans in alloxan-induced hyperglycemic rats. However, nostudies are available on the antidiabetic property of C. tetragono-loba beans in high-fat diet (HFD) fed-streptozotocin (STZ)-inducedtype 2 diabetic rats. Therefore the present investigation aims to

G.R. Gandhi et al. / Food and Chemical Toxicology 66 (2014) 358–365 359

study the hypoglycemic and b-cells protective effects of C. tetrag-onoloba beans along with associated biochemical parameters inHFD fed-STZ-induced type 2 diabetic insulin resistant rat model.

2. Materials and methods

2.1. Chemicals and reagents

STZ and all fine chemicals were purchased from Sigma–Aldrich (St. Louis, MO,USA). Ultrasensitive rat insulin ELISA kit was obtained from Crystal Chem, Inc.(USA). Organic solvents were obtained from Merck (Germany). Pioglitazone andall other laboratory chemicals of the highest grade were obtained from local orga-nizations (India).

2.2. Plant material

Fresh matured C. tetragonoloba (L.) Taub. (Fabaceae) beans were collected fromthe medicinal farm at Koyambedu, Chennai, India during April 2012 and were dulyauthenticated by Dr. M. Ayyanar, plant taxonomist, Department of Botany, Pachaiy-appa’s College, Chennai, India. A voucher specimen (No. CT/ERI/189) was preservedat the herbarium of the institute for future reference.

2.3. Extraction of crude extracts of C. tetragonoloba beans

The matured C. tetragonoloba beans (38 kg) were cut into pieces and were shadedried, pulverized in an electric grinder; 4 kg of the powdered material were succes-sively extracted with 12 L of hexane, chloroform, ethyl acetate and methanolrespectively by cold maceration for 48 h with random mechanical shaking. Totalaqueous extract was prepared separately. The filtrates obtained from each solventwere concentrated under reduced pressure using rotary evaporator at 55–65 �C toproduce green soluble residues of hexane (64 g), chloroform (172 g), ethyl acetate(78 g), methanol (489 g) and aqueous (198 g) respectively. The residues were fur-ther dried in an oven at 30 �C to remove traces of solvents and stored in an airtightglass container at 4–5 �C until use. The crude extracts dissolved in vehicle (0.2%polysorbate-80, 0.5% sodium carboxy methyl cellulose, 0.9% sodium chloride, 0.9%benzyl alcohol and 97.5% distilled water) (Lee, 2001) served as the material forexperimentation. In our initial studies, among hexane, chloroform, ethyl acetate,methanol and aqueous extracts of the beans, only the methanol extract exhibiteda significant blood glucose lowering effect. Hence the present study assesses theefficacy of methanol extract of C. tetragonoloba beans (CTme) on HFD fed-STZ-in-duced type 2 diabetic rats.

Table 1Composition of HFD (%, W/W).

Ingredients HFD (g/kg)

Lard 310

2.4. HPLC analysis

Qualitative and quantitative analyses of major polyphenol constituents in CTmewere carried out on Shimadzu HPLC system equipped with LC-10ATVP pump, SPD-M10AVP Photodiode Array detector (PDA) in combination with CLASS-VP 6.12 SP5integration software. The reversed phase C18-ODS (Octadecylsilane), LiChrospherRP-18e (5 lm) (Merck) with a Phenomenex guard column (4 mm � 2 mm i.d:5 lm) was eluted with the mobile phase system consisting of methanol:water50:50 (A) and Acetonitrile (B) in the ratio of 70:30 (A:B, v/v), with a flow rate of1 ml/min. The samples were injected using a 20 ll loop (Rheodyne Rohnet Park,CA, USA) and the separations were monitored with PDA signal at 280 nm. Peak pur-ity was checked and the quantification was done by the calibration curve appearingin the standard chromatogram using the following equation:

CðcÞ ¼ AðcÞAðstÞ � CðstÞ

where C(c) is the concentration of the constituent in the sample, A(c) is the peak areaof the constituent in the sample chromatogram, C(st) is the concentration of thestandard in the reference solution and A(st) is the area of the peak for the standardin the reference chromatogram (Vinholes et al., 2011).

Dalda (saturated fat) 110Casein 250Cholesterol 10Cornstarch 120Sucrose 85Cellulose 50Vitamin mixture 30Mineral mixture 30

DL-Methionine 03

L-Cystine 01

Sodium chloride 01Total metabolizable energy (kcal/kg) 5941

2.5. Experimental animals

Five-weeks-old healthy male Wistar rats weighing 180–200 g were securedfrom central animal house of Entomology Research Institute. They were housedin sterile polypropylene cages containing disinfected paddy husk as bedding withinthe facility at temperature of 22 ± 2 �C, a relative humidity of 60 ± 5%, and 12/12 h day/night cycle (lights on 6.00 a.m.) for 7 days. The animals were given free ac-cess to water and commercial standard pelleted diet purchased from Sai DurgaFeeds and Foods, Bangalore before commencement of the experiment. The animalfacilities and all experimental procedures were approved by the Institutional Ani-mal Ethics Committee and by the regulatory bodies of the government (IAEC-ERI-LC-03/13).

2.6. Acute oral toxicity study and fixation of doses

Normal healthy male rats fasted for 16 h were equally divided into three groupsof six rats each. CTme was given oral doses of 2 and 4 g/kg b wt. concentrations sus-pended in vehicle (3 ml/100 g b wt.) for treatment groups and the control group re-ceived the vehicle alone. The rats were then allowed free access to food and water4 h after the drug administration. All animals were closely observed for the initial4 h and then every day over a period of 14 days for signs of mortality, behavior,body weight changes, food and water consumption, urinalysis and feces. On the15th day the animals were sacrificed and all the organs were observed for grosspathological lesions. CTme (2 and 4 g/kg b wt.) did not show any abnormal behaviorexcept for mild physiological aberrations at 4 g/kg b wt. concentration for initial 4 hafter drug administration. No treatment associated gross pathological lesions wereobserved. Hence, doses of 200 and 400 mg/kg b wt. were preferred for subsequentexperiments based on Oliveira et al. (2008).

2.7. Development of T2DM

After acclimatization experimental rats excluding the normal control were fedfree access of standardized HFD (Srinivasan et al., 2005) (Table 1) for two weeksprior to intraperitoneal injection with STZ (40 mg/kg b wt.). Citrate buffer (carrier)alone was injected to normal control rats. Blood samples were withdrawn 5 daysafter STZ induction from the tail vein under mild diethyl ether inhalation anesthesiafrom overnight (9.00 p.m. to 9.00 a.m.) fasted rats with only free access to water;hyperglycaemia was assessed by measuring fasting blood glucose (FBG) level as de-scribed by Trinder (1969). The rats with a FBG level above 250 mg/dl were classifiedas diabetic and included in the study. The diabetic rats were appeased for 3 daysand on the subsequent day (day 0) the experiment was started. HFD was continuedthroughout the study.

2.8. Experimental design

In the experiment 30 rats (24 diabetic persisting rats, 6 normal rats) were used.Animals were randomly divided into five groups of six animals each according

to the following experimental design

Group I: Normal control rats received vehicle (1 ml/100 g b wt.).Group II: Diabetic control rats received vehicle (1 ml/100 g b wt.).Group III: Diabetic treated rats received CTme 200 mg/kg b wt. suspended invehicle (1 ml/100 g b wt.).Group IV: Diabetic treated rats received CTme 400 mg/kg b wt. suspended invehicle (1 ml/100 g b wt.).Group V: Diabetic treated rats received pioglitazone 10 mg/kg b wt. suspendedin vehicle (1 ml/100 g b wt.).

The vehicle or test drugs were administered orally once daily between 12.00p.m. and 2.00 p.m., using intragastric tube for 30 days.

2.9. Biochemical analysis

FBG and body weights were measured on days 0, 14, 21 and 28 of the experi-ment. Plasma insulin (Ultrasensitive rat insulin ELISA kit), total cholesterol (TC)(Henley, 1957) and triglycerides (TG) (Foster and Dunn, 1973) were determinedon days 0 and 28. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by standard equation as described by Matthews et al. (1985).At day 15 oral glucose tolerance test (OGTT) was performed. Briefly, the animalswere fasted for 6 h; a glucose solution (2 g/kg b wt.) was given to each rat orally

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with feeding syringe exactly after 30 min post administration of extracts, standarddrug and vehicle. Blood glucose of each rat was assayed at time 0 (prior to the glu-cose infusion) and 30, 60 and 120 min after glucose administration. At day 25, insu-lin tolerance test (ITT) was carried out. Briefly, after fasting for 6 h, plasma sampleswere collected. Then the animals were treated with 1.2 U/kg b wt. of insulin(Huminsulin R; Eli Lilly) in normal saline intraperitoneally. Plasma samples werecollected at 30 and 60 min after insulin injection for the estimation of glucose.On the 30th day, the animals were anaesthetized and sacrificed by cervical decap-itation following animal ethical guidelines. The liver was removed and washed inice cold saline. The homogenate prepared in ice chilled 10% potassium chloride in0.1 M phosphate buffer (pH 6.5) solution was used to measure the activities ofhexokinase (Brandstrup et al., 1957), glucose-6-phosphatase (Koide and Oda,1959), fructose-1,6-bisphosphatase (Gancedo and Gancedo, 1971) and hepatic gly-cogen (Van Handel, 1965), respectively.

2.10. Histology

Histology of pancreas was done according to the method described previously(Gandhi et al., 2012).

2.11. Immunohistochemical analysis

Immunohistochemical experiment was performed as described by Gandhi et al.(2011). Results were scored by multiplying the percentage of positive cells (P) bythe intensity of staining (I). Formula: Score = P � I (McDonald and Pilgram, 1999).

Fig. 1. HPLC fingerprint chromatogram of CTme with gallic acid and caffeic acid (A) andgallic acid, CF – caffeic acid.

2.12. Statistical analysis

All results were presented as means ± SEM. Significance of differences of meanswas calculated using one-way analysis of variance (ANOVA) followed by Dunnett’sC post hoc test. The differences were considered statistically significant at P < 0.05.

3. Results

3.1. HPLC analysis

HPLC fingerprint chromatogram of CTme is shown in Fig. 1A.The major polyphenols contents of CTme were recognized by com-parison to the retention times and absorption spectra of authenticstandard markers in overlaid chromatograms (Fig. 1B). Quantifica-tion of gallic acid (Fig. 2A) and caffeic acid (Fig. 2B) disclosed theircontents to be 2.46% (w/w) and 0.32% (w/w). Retention times forgallic acid and caffeic acid were 3.7 and 4.6 min.

3.2. Effect of CTme on OGTT and ITT

As shown in Table 2, oral administration with glucose progres-sively increased blood glucose levels in all the groups at 30 minand it remained unaffected over the next 120 min in diabetic

the overlaid chromatograms of standards gallic acid and caffeic acid (B), Peak; GA –

Gallic acid (A)

Caffeic acid (B)

Fig. 2. Structures of polyphenols.

Table 3Effect of CTme on ITT.

Groups Blood glucose level (mg/dl)

0 min 30 min 60 min

Normal control 134.3 ± 2.0 108.3 ± 2.1 104.6 ± 2.4Diabetic control 326.3 ± 12.3a 332.1 ± 7.4a 338.1 ± 7.1a

Diabetic + CTme (200 mg/kg b wt.) 144.2 ± 7.1b 113.2 ± 2.2b 124.0 ± 6.1b

Diabetic + CTme (400 mg/kg b wt.) 128.0 ± 2.1b 103.4 ± 5.1b 111.9 ± 7.3b

Diabetic + Pioglitazone (10 mg/kg b wt.) 133.7 ± 4.0b 117.2 ± 6.9b 121.3 ± 7.3b

Values indicate mean ± standard error of the mean (SEM) of six rats per group.a P < 0.05, compared with normal control values.b P < 0.05, compared with diabetic control values.

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control rats. CTme (200 and 400 mg/kg b wt.) significantly(P < 0.05) suppressed the increase in blood glucose levels at 30,60 and 120 min after glucose administration. Moreover CTme(200 and 400 mg/kg b wt.) supplemented diabetic rats exhibiteda significant (P < 0.05) clearance of glucose over the complete per-iod during the ITT compared to diabetic control rats (Table 3).

3.3. Effect of CTme on FBG and body weight

Tables 4 and 5 present the levels of FBG and body weight ofexperimental rats on 0, 14, 21 and 28th days after drug treatment.CTme (200 and 400 mg/kg b wt.) supplemented diabetic ratsexhibited significant (P < 0.05) reduction in blood glucose after14, 21 and 28 days of treatment compared to diabetic control rats.The body weight gain of diabetic control group was significantly(P < 0.05) higher than normal control group over the entire periodduring the experimentation. The supplementation of CTme (200and 400 mg/kg b wt.) to diabetic rats showed control in bodyweight gain compared to diabetic control group.

3.4. Effect of CTme on plasma insulin, TC and TG

The levels of plasma insulin and HOMA-IR was increased signif-icantly (P < 0.05) in diabetic control rats when compared withnormal control rats. Oral administration of CTme (200 and400 mg/kg b wt.) to diabetic rats significantly (P < 0.05) restoredthe changes in the level of insulin resistance to near normal duringthe study period. There was a significant (P < 0.05) increase in TCand TG of diabetic control rats compared with the normal control.However CTme treatment (200 and 400 mg/kg b wt.) produced asignificant (P < 0.05) decrease in TC and TG in diabetic rats(Table 6).

Table 2Effect of CTme on OGTT.

Groups Blood glucose level (mg/dl)

0 min

Normal control 131.7 ± 4.6Diabetic control 331.7 ± 12.1a

Diabetic + CTme (200 mg/kg b wt.) 229.4 ± 8.7a,b

Diabetic + CTme (400 mg/kg b wt.) 230.5 ± 7.7a,b

Diabetic + Pioglitazone (10 mg/kg b wt.) 233.1 ± 8.0a,b

Values indicate mean ± standard error of the mean (SEM) of six rats per group.a P < 0.05, compared with normal control values.b P < 0.05, compared with diabetic control values.

3.5. Effect of CTme on hexokinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and hepatic glycogen

As shown in Table 7, the activities of glucose-6-phosphataseand fructose-1,6-bisphosphatase were found to be significantly in-creased (P < 0.05) while hexokinase and glycogen content were sig-nificantly (P < 0.05) decreased in diabetic rats when compared tonormal control rats. After oral administration of CTme (200 and400 mg/kg b wt.) to the diabetic rats for 30 days the activities ofglucose-6-phosphatase and fructose-1,6-bisphosphatase and levelof glycogen content were significantly reversed (P < 0.05) to nearnormal levels. Besides, the hexokinase in the treated diabetic ratsshowed a gradual slight increase compared to the diabetic controlrats.

3.6. Histology

Histological examinations in the pancreatic islet tissues ofexperimental rats are represented in Fig. 3. As shown in Fig 3A,b-cells in the normal control rat displayed granulated cytoplasmand uniform nuclei. In contrast, islet of diabetic control presentedsevere pancreatic disruption along with degranulated b-cells(Fig. 3B). The pathological examination in the tissues of CTme(200 and 400 mg/kg b wt.) and pioglitozone (10 mg/kg b wt.) trea-ted diabetic rats exposed distinct granulated and protective effecton b-cells (Fig. 3C–E).

3.7. Immunohistochemical study

The outcomes of immunohistochemical observations in the islettissues of experimental rats are showed in Fig. 4. Diabetic controlrats showed a significant (P < 0.05) reduction in the level of insu-lin-immunostaining expression as compared to normal controlrats. Conversely, CTme (200 and 400 mg/kg b wt.) treated diabeticgroups exhibited significant (P < 0.05) increase in the insulin-immunostaining expression compared to the vehicle receiveddiabetic control group. Fig. 5A depicts islets with positive insu-lin-staining of b-cells in the islet of normal rat. Diabetic rat showedlow amount of insulin-staining (Fig. 5B). There was rise in the

30 min 60 min 120 min

184.4 ± 6.2 178.8 ± 3.7 132.4 ± 2.4389.4 ± 11.3a 390.6 ± 9.1a 343.6 ± 8.7a

281.2 ± 8.9a,b 240.4 ± 11.2a,b 232.7 ± 9.8a,b

279.2 ± 6.9a,b 236.4 ± 9.6a,b 231.4 ± 6.7a,b

277.3 ± 7.1a,b 244.0 ± 7.3a,b 236.8 ± 5.2a,b

Table 4Effect of CTme on FBG.

Groups FBG level (mg/dl)

0 day 14th day 21st day 28th day

Normal control 98.9 ± 3.2 128.1 ± 2.9 131.4 ± 4.1 130.2 ± 4.2Diabetic control 272.1 ± 6.1a 318.2 ± 5.0a 324.2 ± 7.4a 339.8 ± 8.9a

Diabetic + CTme (200 mg/kg b wt.) 283.3 ± 7.1a 233.0 ± 5.0a,b 152.3 ± 4.0a,b 128.6 ± 5.3b

Diabetic + CTme (400 mg/kg b wt.) 298.2 ± 5.2a 229.4 ± 5.2a,b 146.4 ± 2.1b 124.6 ± 6.0b

Diabetic + Pioglitazone (10 mg/kg b wt.) 294.7 ± 6.9a 231.6 ± 7.8a,b 144.7 ± 8.1b 129.8 ± 4.4b

Values indicate mean ± standard error of the mean (SEM) of six rats per group.a P < 0.05, compared with normal control values.b P < 0.05, compared with diabetic control values.

Table 5Effect of CTme on body weight.

Groups Body weight (g)

0 day 14th day 21st day 28th day

Normal control 184.0 ± 10.3 188.0 ± 13.7 191.5 ± 13.3 195.7 ± 10.1Diabetic control 194.6 ± 12.0 216.3 ± 11.4a 229.2 ± 10.6a 236.8 ± 15.2a

Diabetic + CTme (200 mg/kg b wt.) 193.4 ± 13.5 210.5 ± 8.7a 216.7 ± 12.0a 219.2 ± 12.0a

Diabetic + CTme (400 mg/kg b wt.) 184.6 ± 11.9 205.4 ± 9.4a 210.4 ± 12.2a 216.9 ± 8.1a

Diabetic + Pioglitazone (10 mg/kg b wt.) 203.4 ± 10.6a 217.6 ± 13.4a 221.7 ± 12.1a 216.0 ± 11.6a

Values indicate mean ± standard error of the mean (SEM) of six rats per group.a P < 0.05, compared with normal control values.

Table 6Effect of CTme on plasma insulin, HOMA-IR, TC and TG.

Groups Plasma insulin (lU/ml) HOMA-IR TC (mg/dl) TG (mg/dl)

0 day 28th day 0 day 28th day 0 day 28th day 0 day 28th day

Normal control 16.0 ± 0.6 15.9 ± 1.3 4.1 ± 0.7 5.1 ± 0.9 75.5 ± 4.0 74.8 ± 2.1 78.3 ± 4.5 72.2 ± 2.2Diabetic control 21.3 ± 0.4a 26.1 ± 1.4a 14.4 ± 1.2a 22.9 ± 2.6a 97.9 ± 3.5a 128.9 ± 5.1a 152.3 ± 6.9a 172.9 ± 7.9a

Diabetic + CTme (200 mg/kg b wt.) 22.6 ± 0.8a 16.6 ± 0.8b 15.6 ± 1.1a 5.8 ± 0.9b 93.4 ± 5.9a 78.7 ± 2.2b 144.1 ± 3.2a 98.7 ± 2.3a,b

Diabetic + CTme (400 mg/kg b wt.) 21.3 ± 1.2a 16.2 ± 0.7b 15.7 ± 1.8a 5.0 ± 0.6b 99.9 ± 2.4a 67.7 ± 1.4b 158.6 ± 3.9a 85.5 ± 1.9b

Diabetic + Pioglitazone (10 mg/kg b wt.) 20.6 ± 1.0a 15.9 ± 0.7b 14.9 ± 2.3a 5.1 ± 1.3b 94.4 ± 2.0a 72.6 ± 1.2b 144.9 ± 3.2a 88.6 ± 4.2b

Values indicate mean ± standard error of the mean (SEM) of six rats per group.a P < 0.05, compared with normal control values.b P < 0.05, compared with diabetic control values.

Table 7Effect of CTme on hexokinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and hepatic glycogen.

Groups Hexokinase(U/mg protein/min)

Glucose-6-phosphatase(U/mg protein/min)

Fructose-1,6-bisphosphatase(U/mg protein/min)

Hepatic glycogen(mg/g tissue)

Normal control 7.5 ± 0.6 0.2 ± 0.1 0.4 ± 0.3 22.8 ± 1.7Diabetic control 4.4 ± 0.4a 0.6 ± 0.1a 0.7 ± 0.3a 7.01 ± 1.6a

Diabetic + CTme (200 mg/kg b wt.) 5.3 ± 0.4a 0.4 ± 0.1b 0.4 ± 0.1b 19.4 ± 2.1b

Diabetic + CTme (400 mg/kg b wt.) 4.9 ± 0.9a 0.3 ± 0.1b 0.4 ± 0.1b 20.2 ± 1.1b

Diabetic + Pioglitazone (10 mg/kg b wt.) 4.8 ± 0.7a 0.3 ± 0.1b 0.3 ± 0.1b 19.1 ± 0.9b

Values indicate mean ± standard error of the mean (SEM) of six rats per group.a P < 0.05, compared with normal control values.b P < 0.05, compared with diabetic control values.

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expression of insulin-immunoreactivity for the presence of insulin-containing b-cells with consistent dark brown insulin granules inthe CTme (200 and 400 mg/kg b wt.) and pioglitozone (10 mg/kgb wt.) treated diabetic rats compared with the diabetic controlrat (Fig. 5C–E).

4. Discussion

T2DM has been recognized as the most common health syn-drome across the world and is becoming one of the leading causesof morbidity and mortality in human populations (Steppan et al.,

2001). T2DM induced by combination of standardized HFD andlow-dose STZ (40 mg/kg b wt.) to rats, mimicks the condition sim-ilar to insulin-resistant state in humans (Srinivasan et al., 2005).The feeding of HFD induces insulin resistance through reducedtyrosine phosphorylation of insulin receptor substrate (IRS), acrucial receptor which facilitates the signals that stimulate insulinaction (Petersen and Shulman, 2006). Furthermore, low dose STZprompts mild impairment to pancreatic b-cells which is parallelto the metabolic characteristics of the later stage of T2DM.Formerly other investigators have also used this animal model toattain T2DM (Zhang et al., 2010; Krol and Krejpcio, 2011; Veerapuret al., 2012).

Fig. 3. Histology of experimental rats’ pancreas (H and E, 100�). (A) Normal control rat showing b-cells with displayed granulated cytoplasm and uniform nuclei. (B) Diabeticcontrol rat showing severe pancreatic disruption along with degranulated b-cells. (C–E) Pancreas of CTme (200 and 400 mg/kg b wt.) and Pioglitozone (10 mg/kg b wt.)treated diabetic rats showing distinct granulated and protective effect on b-cells.

0

10

20

30

40

50

60

70

80

90

Inte

nsity

of

insu

lin-i

mm

unos

tain

ing

A B C D E

a

b b b

Fig. 4. Histogram showing intensity of insulin-immunostaining expression of b-cells in islet of Langerhans. (A) Normal control. (B) Diabetic control. (C and D)Diabetic + CTme (200 and 400 mg/kg b wt.). (E) Diabetic + Pioglitozone (10 mg/kg bwt.). Bars represent the mean ± standard error of the mean (SEM) of six rats pergroup aP < 0.05, compared with normal control values; bP < 0.05, compared withdiabetic control values.

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In this study CTme supplementation significantly controlled theglucose and insulin tolerance levels compared to the diabetic con-trol. These results indicate that CTme administration directed toinsulin facilitated glucose uptake into peripheral tissues. In addi-tion these data also show that CTme performed as a real insulinsensitizer likely due to enhanced glucose uptake in the main targetorgans. There was a significant restoration of raised insulin leveland HOMA-IR in CTme treated diabetic rats compared to the dia-betic control rats suggesting that CTme exhibited significant insu-lin sensitization activity as well as improvement in the glucosehemostasis probably due to normalized b-cell function. In an ear-lier study ursolic acid noticeably showed similar insulin sensitizingeffect in HFD fed STZ-induced diabetic mice (Jang et al., 2009).

In our study HFD-STZ treatment caused a significant increase inFBG level of diabetic rats indicating that insulin resistance has beenestablished in these rats. Hyperglycemia is the imperative featureof diabetes mellitus which ultimately leads to the formation ofreactive oxygen species and tissue degradation thereby contribut-ing to diabetic complications (Zhang et al., 2010). CTme signifi-cantly reduced the FBG level in the diabetic rats. Thehypoglycemic prospective exerted by CTme might be due to itsabundant polyphenol components of the extract. Polyphenols suchas gallic acid and caffeic acid constitute 2.78% W/W of the CTme,

Fig. 5. Light photo micrographs of rat pancreas showing insulin-immunostaining of b-cells in islet of Langerhans. (A) Normal control rat. (B) Islet of diabetic control rat. (C andD) Islet of CTme treated (200 and 400 mg/kg b wt.) diabetic rats. (E) Islet of Pioglitozone (10 mg/kg b wt.) treated diabetic rats.

364 G.R. Gandhi et al. / Food and Chemical Toxicology 66 (2014) 358–365

together with other bioactive polyphenolic compounds like geni-stein, daidzein, quercetin, gallotannins, ellagic acid, kaemferol, ru-tin, catechin, hydroxycinnamic acid, naringenin and chlorogenicacid could potentiate the antidiabetic activity of the CTme. Evi-dence showed that these bioactive polyphenols exhibited glucoselowering effects in diabetic animal models (Vedavanam et al.,1999; Vessal et al., 2003; Klein et al., 2007; Malini et al., 2011;Zhang and Liu, 2011; Gandhi et al., 2011; Singh et al., 2012; Annad-urai et al., 2012; Ong et al., 2013). Similarly, polyphenols in Aristot-elia chilensis also acted synergistically to produce hypoglycemicand insuin sensitizing effects in type 2 diabetic mice (Rojo et al.,2012).

HFD fed STZ-induced diabetic rats exhibited higher levels of TCand TG. Hypercholesterolaemia and hypertriglyceridaemia presentin these diabetic rats were due to the increased dietary cholesterolintake and severe accumulation of epididymal adipose tissue masscharacterized by abnormal lipid metabolism that has contraryreaction of body weight gain (Veerapur et al., 2012). CTme de-creased significantly the levels of TC and TG in diabetic rats and ar-rested the body weight gain caused by HFD confirming its remedialpotential on metabolic defect. In a previous related study Tamarin-dus indica pulp aqueous extract also markedly lowered TC and TGcontents and controlled body weight gain in HFD induced obeserats (Azman et al., 2012).

Hexokinase is the key glycolytic enzyme that is decreased dur-ing diabetic condition (Vestergard, 1999). Gluconeogenic enzymessuch as glucose-6-phosphatase and fructose-1,6-bisphosphatasewere elevated in the diabetic state that contributes to hyperglyce-mia (Saxena et al., 1984). CTme expressively restored the activitiesof these enzymes. The normalized insulin action and regulatedblood glucose level in diabetic rats treated with CTme might bethe outcome of restoration of these carbohydrate metabolism en-zymes. Nonexistence of insulin action can lead to glycogenolysiswith reduced hepatic glycogen content in diabetic rats. Insulin reg-ulates glycogen metabolism via activation or inhibition of variousenzymes and proteins (Ferrer et al., 2003). Our study results indi-cated that CTme significantly improved the hepatic glycogen con-tent in treated diabetic rats. This could be the reflection ofinsulin-sensitizing effect in the hepatic tissue.

Yuan et al. (2001) have suggested that insulin resistance in theperipheral tissues resulting in hyperinsulinaemia eventually dam-ages the structural integrity of b-cells. Impaired and degranulatedb-cells with decrease of insulin-positive staining b-cells werefound in the diabetic control group. Oral supplementation of CTmeto diabetic rats preserved the b-cell mass. Meanwhile the extensiveincrease in insulin-immunoreactive expression in CTme treateddiabetic rats substantiated the protective role of CTme in the rever-sion of pancreatic b-cell damage caused by HFD-STZ.

G.R. Gandhi et al. / Food and Chemical Toxicology 66 (2014) 358–365 365

5. Conclusion

In the present study C. tetragonoloba beans exerted hypoglyce-mic action due to insulin sensitizing effect in HFD fed-STZ-induceddiabetic rats. C. tetragonoloba beans also protected b-cell mass andlessened hyperlipidemia in insulin resistant animal model. Hencethis can be considered for use in the management of T2DM.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgement

This work was supported by the Council of Scientific and Indus-trial Research (CSIR), Government of India (a Grant-In-Aid to GRG,Sanction No. 08/293(0035)/2012/EMR-I).

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