http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.935864
ORIGINAL ARTICLE
Antidiabetic, antilipidemic, and antioxidant activities of Gouania longipetala methanol leaf extract in alloxan-induced diabetic rats Maxwell Ikechukwu Ezeja1, Aruh Ottah Anaga2, and Isaac U. Asuzu2 1
Department of Veterinary Physiology, Pharmacology and Biochemistry, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria and Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, Enugu State, Nigeria
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Abstract
Keywords
Context: Gouania longipetala Hemsl. (Rhamnaceae) is used in folkloric medicine for treating diabetes mellitus and its associated symptoms. Objective: This study evaluated the antidiabetic antilipidemic and antioxidant activities of the plant methanol leaf extract. Materials and methods: Diabetes was induced in rats by intraperitoneal injection of alloxan monohydrate (160 mg/kg). Three test doses (50, 100, and 150 mg/kg) of G. longipetala extract (GLE) were administered orally and the effects were compared with glibenclamide (2 mg/kg). The effect of GLE on hyperglycemia and sub-acute study for 21 d were carried out using its effect on fasting blood sugar (FBS) level. Serum biochemistry and antioxidant activity were evaluated. Histopathological evaluation of the pancreas was also done. Results: The LD50 of G. longipetala was found to be 44000 mg/kg. The extract significantly (p50.0001) decreased the FBS levels of treated rats from 16.2 ± 2.03 to 6.5 ± 1.52 mM/L at 150 mg/kg within 24 h. The extract decreased FBS levels of rats by 62.0, 74.8, and 75.0% on day 21 at 50, 100, and 150 mg/kg, respectively. GLE reduced the level of malondiadehyde from 23.0 ± 1.34 to 10.3 ± 0.43 mg/dL, increased superoxide dismutase activities from 2.97 ± 0.34 to 5.80 ± 0.53 IU/L at 150 mg/kg, and improved the serum lipid profile of treated rats. GLE also caused restoration of the altered histopathological changes of the pancreas. Discussion and conclusion: Gouania longipetala demonstrated significant antidiabetic, antilipidemic, and antioxidant activities that may be due to its multiple effects involving both pancreatic and extra-pancreatic mechanisms.
Fasting blood sugar, glibenclamide, histopathology, hyperglycemia
Introduction Diabetes mellitus is a group of metabolic conditions characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both (Edwin et al., 2007). In the disease condition, blood glucose remains high either because the body does not produce enough insulin or because cells do not respond to insulin produced (Rakesh et al., 2010). Diabetes mellitus is a disease condition of both humans and animals. According to the World Health Organization (WHO) (2011), it is estimated that about 350 million people have diabetes, the prevalence being similar in both high and low income countries and it is predicted that global diabetes prevalence will increase by 50% in 2030 (Wild et al., 2004). Marked increases in levels of serum lipids have been observed in diabetes mellitus and are considered to be a prime cause of coronary heart disease in diabetics (Murugan et al.,
Correspondence: Maxwell I. Ezeja, Department of Veterinary Physiology, Pharmacology and Biochemistry, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. Tel. +2348033238975. E-mail: [email protected]
History Received 28 October 2013 Revised 30 April 2014 Accepted 13 June 2014 Published online 21 October 2014
2009). Oxidative stress is increased in diabetes mellitus owing to an increase in the production of oxygen-free radicals such as superoxide (O 2 ), hydrogen peroxide (H2O2), and hydroxide (OH) radicals and deficiency in antioxidant defense mechanisms (Moussa, 2008). Ultimately, the goal of all treatment strategies for diabetes mellitus is to lower blood glucose concentrations and to lessen the risk for development and progression of the disease complications, emphasizing cardiovascular risk reductions and focusing particularly on hypertension control and correction of dyslipidemia (Holman et al., 2008). Current therapies in the management of diabetes include life style intervention through diet modification and exercise and the use of oral hypoglycemic therapy and insulin treatment (Holman et al., 2008). Other management strategies in diabetes mellitus include transplantation of pancreas (Larsen, 2011), islet cell transplantation (Lakey et al., 2003), and bariatric surgery (Robinson, 2009). The agents used in clinical practice have serious adverse effects such as hematological effects, hypoglycemic coma, and disturbances of liver and kidney (Distefano & Watanabe, 2010). Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment
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of diabetes due to their effectiveness, less side effects, and relatively low cost (Nanu et al., 2008). Gouania longipetala Hemsl. (Rhamnaceae) is a scandent shrub or liana mainly present in closed forests, forest margins, and in jungle regrowths (Burkill, 1985). Its salient characteristics are the watch-spring tendrils, spike-like thyrsus, a more or less lobed disc, inferior ovary, and longitudinally 3-winged septicidal fruits (Buerki et al., 2011). The plant has been used in ethnomedicine for the treatment of different ailments. The leaves are used for the treatment of swelling, edema venomous stings, gout, febrifuges, etc. The leaf sap is used for eye treatments, as pain killers and for treating heart diseases (Abbiw, 1990; Burkill, 1985). The stem of the plant has been shown to possess antibacterial and anti-inflammatory activities (Ekuadzi et al., 2012). In Orba, Udenu local Government Area of Enugu State, South Eastern Nigeria (where the plant is known as ‘‘Asha’’), the leaves of Gouania longipetala are used to treat diabetes mellitus and malaria (Focho et al., 2009). No scientific study has been carried out to authenticate the use of G. longipetala for the treatment of diabetes mellitus and to establish the efficacy of the plant in the modulation of oxidative stress and hyperlipidemia associated with diabetes mellitus. Hence, the present study was undertaken to evaluate the methanol leaf extract of G. longipetala for its antidiabetic effect with a view to establishing the pharmacological basis for its folkloric use as an antidiabetic plant, and to determine the effect of sub-acute administration of G. longipetala on lipid profile and in vivo and in vitro antioxidant parameters of alloxan-induced diabetic rats.
Materials and methods Plant collection and identification Fresh leaves of Gouania longipetala were collected from its natural habitat at Orba-Nsukka, Enugu state, Nigeria in July 2011 and identified by a taxonomist, Mr. A. O. Ozioko of the Bioresources Development and Conservation Programme, Aku Road, Nsukka, Enugu State where the voucher specimen number VPP/2011/68 was deposited in the herbarium. Extraction of plant material The plant was extracted by the cold maceration method. The leaves were washed, cut into small pieces, dried at room temperature, and pulverized into coarse powder of about 1 mm in diameter. The plant material (500 g) was macerated in 80% methanol for 48 h with intermittent shaking at 2 h interval. The extract was then filtered using Whatman No. 1 filter papers and concentrated in vacuo using rotary evaporator at 40 C and 210 milibar and a vacuum lyophilizer. The yield of the extract was calculated and it was stored in a refrigerator at 4 C as G. longipetala extract (GLE) until the time of use (Mansi & Lahham, 2008). Experimental animals Mature Wistar albino rats, bred in the laboratory animal unit of the Faculty of Veterinary Medicine, University of Nigeria Nsukka, were used for the experiments. They were housed in
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an environment of normal ambient temperature and the lighting period was about 12 h daily. The relative humidity was between 40 and 60%. The weight of the rats varied between 107 and 185 g. The rats were kept in stainless steel cages, supplied with clean drinking water, and fed ad libitum with standard commercial pelleted feed (Vital feedÕ , UAC, Lagos, Nigeria). Ethical conditions governing the conduct of experiments with life animals were strictly observed as stipulated by Ward and Elsea (1997) and all animal experiments were conducted in compliance with NIH Guide for Care and Use of Laboratory Animals (Pub. no. 85-23, revised 1985). The experimental protocol was approved by the institution’s ethical committee for the use of laboratory animals (Number: VPP/EC/012/36). Acute toxicity effect of the methanol leaf extract of G. longipetala in rats The method of Lorke (1983) was adopted for this study. Mature albino rats (35) of both sexes were randomly grouped into seven groups (1–7) of five rats per group. Groups 1–6 were given GLE at the doses of 100, 500, 1000, 2000, 3000, and 4000 mg/kg, using oral gavage. Group 7 rats received distilled water (10 ml/kg). The rats were allowed free access to feed and drinking water and were observed for 48 h for signs of toxicity and death. Induction of experimental diabetes Diabetes was induced in male albino Wistar rats using the method described by Venugopal et al. (1998). Thirty male rats were used for this experiment. The rats were fasted for 18 h. The fasting blood sugar (FBS) levels of the rats were determined with blood from the rats’ tail vein using an auto analyzer (AccuCheck Advantage IIÕ , Roche, Branford, CT) glucose kit. Diabetes was then induced in the rats by a single intraperitoneal injection of alloxan monohydrate at 160 mg/ kg. The FBS levels of the rats were checked every other day. On the sixth day, diabetes was established in the rats and rats with FBS of 8 mMol/L were considered diabetic (Vinuthan et al., 2007).
Effect of graded doses of G. longipetala on blood glucose level of alloxan-induced hyperglycemia in rats (dose–response study) Thirty alloxan-induced diabetic male rats were used for the study. The rats were randomly grouped into five groups of six rats per group and treated as follows after measuring their FBS at 0 h (on establishment of diabetes). Group 1 rats received 10% Tween 20 (10 ml/kg) and served as a negative control. Group 2 rats were given glibenclamide (2 mg/kg) and served as a positive control, while groups 3, 4, and 5 served as treatment groups and were given 50, 100, and 150 mg/kg of GLE, respectively, by gastric gavage. The FBS of all the rats was measured at 0, 1, 3, 6, and 24 h post drug or extract treatment using an auto analyzer (AccuCheck Advantage IIÕ , Roche, Branford, CT) glucose kit. The blood samples were collected from the tail vein after a snip (Vinkatesh et al., 2008).
Antidiabetic activity of G. longipetala
DOI: 10.3109/13880209.2014.935864
Effect of GLE on oral glucose tolerance test (OGTT) in rats
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The method described by Aslan et al. (2007) was adopted for this study. Thirty mature male albino rats were fasted for 18 h and were randomly divided into five groups of six rats each. The FBS at 0 h was measured before treatment. Group 1 rats (negative control) were given 10 ml/kg of 10% Tween 20 solutions. Group 2 rats were given 2 mg/kg glibenclamide (positive control) while groups 3, 4, and 5 rats were given 50, 100, and 150 mg/kg of GLE, respectively. They were dosed orally with the aid of gastric gavage. The rats in all the groups were loaded with glucose at the dose of 2 g/kg per os 30 min after drug or extract administration. Blood samples were then collected from the tail vein after a snip for measurement of the glucose levels at 30, 60, 120, and 180 min. Evaluation of in vitro antioxidant activity of GLE 1-1-Diphenyl-2-picril hydrazyl photometric assay The method of Mensor et al. (2001) was adopted for this study. Test extract (2 ml) at concentrations ranging from 10 to 400 mg/ml was each mixed with 1 ml of 0.5 mM diphenyl-2picril hydrazyl (DPPH) (in methanol). Absorbance at 517 nm was taken after 30 min incubation in the dark at room temperature. The concentrations were prepared in triplicates. The percentage antioxidant activity was calculated as follows: % Antioxidant activity ½AA absorbance of sample 100 absorbance of blank ¼ 100 Absorbance of control Methanol (1 ml) plus 2 ml of the extract was used as a blank while 1 ml of 0.5 mM DPPH solution plus 2 ml of methanol was used as a control. Ascorbic acid was used as a reference standard. Ferric-reducing antioxidant power assay Total antioxidant activity was measured by the ferric reducing antioxidant power (FRAP) assay (Benzie & Strain, 1999). Sample (100 ml) of different concentrations of the extract (10, 50, 100, 200, and 400 mg/ml) was mixed with 3 ml of the working FRAP reagent. The absorbance was immediately taken at 593 nm at 0 min. Thereafter samples were placed in water bath at 37 C and the absorbance was measured after 4 min with a spectrophotometer. FRAP value of the samples was calculated using the following formula: Change in absorbance from 0 to 4 min FRAP value of standard FRAP value : Change in absorbance of standard from 0 to 4 min Effect of 21-day oral administration of GLE on FBS, serum biochemistry, lipid peroxidation/in vivo antioxidant activities in alloxan-induced diabetic rats (sub-acute antidiabetic study) Fifty male albino rats were randomly grouped into five groups of 10 rats per group. Diabetes was induced in the rats by
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single intraperitoneal injection of 160 mg/kg of alloxanmonohydrate. On establishment of diabetes using the method earlier described, the rats were re-grouped into five groups of eight rats per group and were treated as follows: group 1 rats which served as a negative control received 10% Tween 20 solution. Group 2 rats received 2 mg/kg glibenclamide as a reference drug while groups 3, 4, and 5 rats were administered 50, 100, and 150 mg/kg GLE, respectively, through gastric gavage. The drug or extract and the vehicle were administered to the rats for 21 d. The FBS levels and the body weights of the rats were measured on days 0, 1, 7, 14, and 21 (Verma et al., 2010). On day 21, being the last day of the treatment, blood samples were collected from the retro-orbital plexus of the eye under light ether anesthesia for serum biochemistry, lipid peroxidation/in vivo antioxidant activities. The rats were later sacrificed by chloroform and the pancreas was collected in sample (bijou) bottles for histopathology. Lipid profile assay Total cholesterol was evaluated using the enzymatic colorimetric chod-pap test method described by Allain et al. (1974) as described in Quimica Clinica Applicada test kit (Quimica Clinica Aplicada S.A, Amposta, Spain); triglycerides were also determined spectrophotometrically using the method of Tietz (1990); high density lipoprotein (HDL) was evaluated by the method of Grove (1979) as described in Quimica Clinica Applicada test kit; lowdensity lipoprotein (LDL) was determined as the difference between total cholesterol and cholesterol content of the supernatant after precipitation of the LDL fraction by polyvinyl sulfate (PVS) in the presence of polyethylene– glycol monomethyl ether (Bergmenyer, 1985) and very LDL (VLDL) was calculated according to the method of Wilson et al. (1981) as VLDL ¼ 0.2 TG (where TG is triglycerides). Liver marker enzymes Aspartate aminotransferase (AST) was evaluated using the method of Reitman and Frankel (1957) as described by Randox Laboratories, Belfast, United Kingdom, using Randox kits; alanine aminotransferase (ALT) was measured by monitoring the concentration of pyruvate hydrazone formed with 2,4,-dinitrophenylhydrazine using the method of Reitman and Frankel (1957) as described in Randox kits; alkaline phosphatase (ALP) was assayed based on the methods of Kind and King (1972); total protein in serum was assayed using the direct biuret method (Gornall et al., 1949) while total bilirubin was determined according to the method of Jendrassik and Grof (1938). Lipid peroxidation/in vivo antioxidant activities of G. longipetala Malondialdehyde Lipid peroxidation was determined spectrophotometrically by measuring the level of lipid peroxidation product, malondialdehyde (MDA) as described by Draper and Hadley (1990).
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Table 1. Effect of graded doses of Gouania longipetala extract on the fasting blood glucose levels of alloxan-induced diabetic rats. Mean fasting blood glucose levels ± SEM (mmol/L) Treatment
0h
1h
3h
6h
24 h
% Reduction at sixth hour
% Reduction at 24th hour
Tween 20 (10 ml/kg) Glibenclamide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
19.9 ± 4.02 21.8 ± 4.32 12.8 ± 1.22 15.3 ± 1.48 16.2 ± 2.03
20.2 ± 3.80 17.3 ± 3.28* 8.9 ± 2.27* 8.3 ± 2.31** 8.5 ± 2.62***
19.3 ± 4.05 13.0 ± 3.12** 7.8 ± 1.72** 7.9 ± 1.76*** 8.3 ± 2.41***
20.1 ± 3.50 7.7 ± 1.42** 6.6 ± 1.82*** 6.0 ± 0.65*** 5.2 ± 1.52***
22.8 ± 2.45 8.6 ± 1.40*** 7.2 ± 1.55** 6.3 ± 0.42*** 6.5 ± 1.52***
– 64.7 48.4 60.8 70.1
– 60.6 43.8 58.8 60.0
Group 1. 2. 3. 4. 5.
MDA reacts with thiobarbituric acid (TBA) to form a red or pink colored complex which absorbs maximally in acid solution at 532 nm.
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Estimation of superoxide dismutase
5
Adrenaline (10 mg) was dissolved in 17 ml of distilled water to make adrenaline solution. Serum (0.1 ml) was added to 0.9 ml of phosphate buffer (pH 7.8). The extract (0.2 ml) was taken in triplicate and 2.5 ml of buffer added inside a cuvette and 0.3 ml of adrenaline solution added, mixed well, and absorbance was read at 450 nm at 30 s interval for five times (Xin et al., 1991). Histopathology
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Blood glucose level
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*p50.01, **p50.001, ***p50.0001 when compared with the negative control.
4 3 vehicle 2
Glibenclamide 2 mg/kg GLE 50 mg/kg
1
GLE 100 mg/kg GLE 150 mg/kg
Tissue samples (pancreas) collected after sacrifice of the rats, on the completion of 21 d treatment with drug or extract, were fixed in 10% formal saline for 24 h. They were washed in ascending grades of ethanol, cleared with xylene, embedded in paraffin wax, sectioned with a microtome, and stained with hematoxylin and eosin (H and E) and mounted on Canada balsam (Sigma-Aldrich, St. Louis, MO) (Bancroft & Stevens, 1977). All the sections were examined under a light microscope under different (100 and 400) magnifications. Photomicrographs of lesions were taken with an Olympus photo microscope (Olympus Scientific Equipment, Ashburn, VA) for observations and documentation of histopathological lesions.
Acute toxicity test
Phytochemical analysis of G. longipetala leaf extract
Effect of graded doses of G. longipetala on the blood glucose levels of alloxan-induced diabetic rats (dose–response study)
Phytochemical analysis of G. longipetala leaf extract was carried out to identify the chemical families present in it using the methods of Trease and Evans (1996). Data analysis The results were presented as mean ± SEM and analyzed using one-way analysis of variance (ANOVA). The differences between the means were tested using post hoc LSD and values of p50.05 were considered statistically significant.
Results
0 0 min
30 min
60 min Time
90 min
180 min
Figure 1. Effect of GLE on oral glucose tolerance test (OGTT) in rats.
After 48 h, no death or signs of toxicity were observed in the rats treated with different doses (100, 500, 1000, 2000, 3000, and 4000 mg/kg) of GLE. The LD50 of G. longipetala is, therefore, 44000 mg/kg.
The result of the effect of G. longipetala on the FBS of alloxan-induced hyperglycemic rats is presented in Table 1. Gouania longipetala (50, 100, and 150 mg/kg) and glibenclamide caused significant (p50.01–0.0001) dose- and time-dependent decrease in the fasting blood glucose levels of treated rats when compared with the negative control rats. The extract caused a decrease in FBS levels of the rat by 48.4, 60.8, and 70.1% at the sixth hour and 43.8, 58.8, and 60.0% at the 24th hour, respectively as against 64.7% and 60.6% reduction by the reference drug at 6th and 24th hour, respectively. The extract at all doses and the reference drug had their best activities at the 6th hour.
Extraction of G. longipetala The methanol leaf extract of G. longipetala was dark green in color with a pasty consistency and the percent yield was 16.3% w/w dry matter.
Effect of GLE on OGTT in alloxan-induced diabetic rats Figure 1 shows the effect of GLE on the blood glucose levels of rats after glucose induced hyperglycemia. After the glucose
Antidiabetic activity of G. longipetala
DOI: 10.3109/13880209.2014.935864
load, the mean blood glucose level of rats in group 1 (negative control) increased from 4.14 ± 0.07 mmol/L to 6.42 ± 0.19 mmol/L within 30 min representing 35.5% increase and continued to gradually decrease up to 180th minute. Also, there were various levels of increase in the blood glucose
GLE
Evaluation of in vitro antioxidant activity of GLE
80
ASCORBIC acid
DPPH photometric assay
30
The percent antioxidant activity of GLE and ascorbic acid standard is shown in Figure 2. The result showed that the extract exhibited a concentration-dependent antioxidant activity by increasing the percent antioxidant from 16.6% at the concentration of 10 mg/ml to 65.5% at the concentration of 400 mg/ml. The ascorbic acid standard had an antioxidant activity of 31.6% at the concentration of 10 mg/ml and 79.7% at the concentration of 400 mg/ml.
20
FRAP assay
10
The total antioxidant activity of GLE using FRAP is presented in Figure 3. The result showed that the extract had a concentration-dependent total antioxidant activity increasing the FRAP value from 0.40 mM at the concentration of 10 mg/ml to 1.64 mM at the concentration of 400 mg/ml.
% anoxidant acvity
60 50 40
0 10 µg/ml
50 µg/ml 100 µg/ml 200 µg/ml 400 µg/ml Treatment
Figure 2. Percent antioxidant activity of G. longipetala extract in DPPH photometric assay.
2.5
Effect of sub-acute administration of GLE on alloxan-induced diabetic rats Fasting blood sugar
GLE
The result of the effect of G. longipetala leaf extract on the mean FBS levels of control and experimental groups of rats over a period of 21 d is presented in Table 2. The reference drug and the extract (50, 100, and 150 mg/kg) caused various levels of significant (p50.001–0.0001) dose- and timedependent reduction of the FBS of the rats on days 1, 7, 14, and 21 when compared with a negative control with the extract at the doses of 50, 100, and 150 mg/kg reducing the FBS, by 62.0, 74.8, and 75.0%, respectively, as against 73.4% by the reference drug glibenclamide (2 mg/kg) on day 21. The activity of the extract at the doses of 150 mg/kg was comparable with glibenclamide (2 mg/kg) reference drug on day 21.
Ascorbic Acid 2
Frap value (µm)
levels of the treated rats at the 30th minute. GLE and glibenclamide caused a dose- and time-dependent reduction of the blood glucose levels from the 60th to 180th minute with the extract (50, 100, and 150 mg/kg) reducing the blood glucose levels by 15.0, 15.5, and 28.0%, respectively, at 180th minute as against 36.8% by glibenclamide (2 mg/kg).
90
70
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5
1.5
1
0.5
0 10 µg/ml GLE
50 µg/ml GLE
100 µg/ml 200 µg/ml 400 µg/ml GLE GLE GLE
Effect of GLE on the body weights of alloxan-induced diabetic rats
Treatment
Figure 4 shows the weight changes of control and alloxaninduced diabetic rats treated with G. longipetala leaf extract
Figure 3. Total antioxidant activity of GLE using FRAP assay.
Table 2. Effect of GLE on the FBS of alloxan-induced diabetic rats treated for 21 d. Mean fasting blood glucose levels ± (mmol/L) Group 1. 2. 3. 4. 5.
Treatment
Pre-diabetic FBS
Tween 20 (10 ml/kg) Glibenclamide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
4.1 ± 0.37 4.2 ± 0.31 4.0 ± 0.24 4.0 ± 0.15 3.9 ± 0.02
Day 0
Day 1
Day 7
Day 14
Day 21
15.7 ± 0.59 14.7 ± 0.77 15.7 ± 1.48 15.8 ± 1.19 16.5 ± 0.32 15.4 ± 1.22 8.2 ± 1.05* 6.8 ± 0.59** 6.0 ± 0.40** 4.1 ± 0.43** 13.7 ± 1.30 9.0 ± 1.09* 8.4 ± 0.89** 5.8 ± 0.50** 5.2 ± 0.42** 16.7 ± 1.25 10.9 ± 1.25* 7.4 ± 0.75** 5.5 ± 0.37** 4.2 ± 0.26** 14.2 ± 1.04 8.0 ± 0.56* 6.4 ± 0.36** 4.4 ± 0.19** 3.6 ± 0.15**
*p50.001, **p50.0001 when compared with the negative control.
% reduction (day 21) – 73.2 62.0 74.8 75.0
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reduction in the serum levels of total cholesterol, triglycerides, LDL, and VLDL of the rats when compared with the negative control rats. The activity of the extract at the dose of 150 mg/kg was comparable with glibenclamide (2 mg/kg). The extract at the dose of 50 mg/kg caused some level of reduction of the total cholesterol, triglycerides, LDL, and VLDL of the rats but the reduction was not statistically significant (p40.05). Also GLE at all the doses used caused dose-dependent and significant (p50.05) increase in the HDL of the rats when compared with the negative control with the effect of extract at the doses of 100 and 150 mg/kg being comparable with the reference drug.
for 21 d. In the negative control, there was a gradual decrease in the mean body weights of the rats from the pre-induction mean weight of 147.3 ± 1.33–145.5 ± 0.92 g at day 0 to 142.2 ± 1.22 g on day 21. In the glibenclamide (2 mg/kg) and GLE-treated groups of rats, there was a sharp fall in the mean body weight on day 0 of the diabetes from the pre-diabetes induction weights and then followed by gradual increase in the weight on days 7, 14, and 21 with the best result being observed with the highest dose of the GLE (150 mg/kg).
Table 3 shows the mean serum lipid profile of the controland GLE-treated groups of alloxan-induced diabetic rats. Glibenclamide (2 mg/kg) and GLE (100 and 150 mg/kg) caused various levels of significant (p50.05–0.0001) 160 155
Effect of G. longipetala on liver marker enzymes of alloxan-induced diabetic rats The results of the effect of GLE on liver marker enzyme activities of alloxan-induced diabetic rats are presented in Table 4. There was a significant (p50.05) decrease in mean AST, ALT, and ALP activities in the glibenclamide and GLE (100 and 150 mg/kg) treated rats when compared with the negative control group. Total protein and bilirubin levels were not significantly (p40.05) changed in all the test groups when compared with the negative control group.
GLE 100 mg/kg GLE 150 mg/kg
vehicle Glibenclamide 2 mg/kg GLE 50 mg/kg
150 Body weight (g)
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Effect of GLE on lipid profile of alloxan-induced diabetic rats
145
Effect of GLE on lipid peroxidation (in vivo antioxidant activity) in alloxan-induced diabetic rats
140 135
The results of the effect of GLE on MDA and SOD of alloxaninduced diabetic rats are presented in Table 5. Glibenclamide (2 mg/kg) and GLE (50, 100, and 150 mg/kg) caused a dosedependent and significant (p50.01–0.001) reduction in the level of MDA of rats when compared with the negative control group. The extract at the doses of 100 and 150 mg/kg also caused a significant (p50.001) increase in the SOD activities from 2.97 ± 0.51 mg/dl in the negative control group to 4.55 ± 0.39 mg/dl at the dose of 100 mg/kg and 5.80 ± 0.53 at 150 mg/kg as against 3.62 ± 0.56 mg/dl by glibenclamide (2 mg/kg).
130 125 120 before inducon
Day 0
Day 7
Day 14
Day 21
Duraon of treatment
Figure 4. Effect of G. longipetala on mean body weights (g) of alloxaninduced diabetic rats. Table 3. Effect of GLE on the lipid profile of alloxan-induced diabetic rats. Group 1. 2. 3. 4. 5.
Treatment
Total cholesterol (mg/dl)
Triglycerides (mg/dl)
LDL (mg/dl)
HDL (mg/dl)
VLDL (mg/dl)
Tween 20 (10 ml/kg) Glibenclanide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
74.5 ± 2.50 63.7 ± 4.51* 68.5 ± 4.17 64.6 ± 4.34* 54.2 ± 6.30**
76.5 ± 5.01 58.2 ± 4.74** 73.7 ± 3.67 63.7 ± 3.37* 61.5 ± 2.30*
64.0 ± 2.16 40.8 ± 3.56** 51.7 ± 2.70 41.6 ± 3.67** 40.5 ± 2.67**
15.5 ± 1.57 33.3 ± 4.62*** 21.5 ± 2.03* 38.3 ± 2.76*** 47.8 ± 4.97***
15.3 ± 1.80 11.8 ± 4.67* 14.7 ± 1.53 12.7 ± 1.21* 12.3 ± 0.46*
*p50.05, **p50.001, ***p50.0001 when compared with the negative control group. Table 4. Effect of GLE on the liver enzyme markers of alloxan-induced diabetic rats. Group 1. 2. 3. 4. 5.
Treatment
AST (IU/L)
ALT (IU/L)
ALP (IU/L)
Bilirubin (mg/dl)
Total protein (g/dl)
Tween 20 (10 ml/kg) Glibencdanide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
127.8 ± 2.1 86 ± 1.89* 124.0 ± 2.32 93. 0 ± 2.01* 92.3 ± 1.56*
123.6 ± 3.22 90.0 ± 1.18* 121.0 ± 2.44 91.2 ± 2.52* 90.0 ± 1.43*
220.5 ± 1.20 180.0 ± 2.44* 214.1 ± 3.44 193.1 ± 1.11* 182.4 ± 0.82*
0.51 ± 0.51 0.50 ± 0.22 0.52 ± 1.13 0.50 ± 0.26 0.52 ± 0.06
6.01 ± 1.23 6.12 ± 1.69 6.28 ± 1.50 6.18 ± 0.37 6.34 ± 0.16
*p50.05 when compared with the negative control group.
Antidiabetic activity of G. longipetala
DOI: 10.3109/13880209.2014.935864
Histopathology
and showed evidence of cell regeneration and also contained cells in the process of maturation which was more prominent in the 150 mg/kg GLE-treated rats (group 5).
Plate 1 shows the photomicrograph of sections of the pancreas with diabetic-untreated group (group 1) showing shrunken pancreatic islets (PI) having fewer but degenerate cells. The glibenclamide-treated group indicated that there were more pancreatic islet cells when compared with the negative control group. Although there were few cells in 50 mg/kg GLEtreated group (3), the cells were more when compared with the negative control group of rats. In the 100 and 150 mg/kg GLE-treated rats (groups 4 and 5), the islet cells were many
Phytochemical analysis of G. longipetala Phytochemical analysis showed that GLE contains saponins, starch, flavonoids, alkaloids, glycosides, sterols/terpenes, and tannins.
Discussion Acute toxicity tests indicated that the extract was well tolerated and safe at the doses used in this study. The antidiabetic activity of Gouania longipetala was evaluated, among others, by testing the ability of the extract to reduce the FBS level of rats raised by a single intraperitoneal administration of 160 mg/kg of alloxan monohydrate. The FBS test is a carbohydrate metabolic test which measures blood glucose after a fast (usually 12–18 h), during which the body stimulates the release of the hormone glucagon which in turn releases glucose into the blood stream through catabolic processes (Kelly, 2008).
Table 5. Effect of GLE on the MDA levels and SOD activities of alloxan-induced diabetic rats.
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Group 1. 2. 3. 4. 5.
Treatment
MDA (mg/dl)
SOD (IU/l)
Tween 20 (10 ml/kg) Glibenclamide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
23.0 ± 1.34 15.3 ± 1.75* 14.7 ± 0.56* 10.7 ± 2.11** 10.3 ± 0.43**
2.97 ± 0.34 3.62 ± 0.56 3.57 ± 0.79 4.55 ± 0.39** 5.80 ± 0.53**
*p50.01, **p50.001 when compared with the negative control.
Plate 1. Photomicrograph of sections of the pancreas from experimental rats after 21 d of treatment with GLE. See Table 5 for respective treatment groups. PI, pancreatic islets; AC, Acinar cell.
AC
AC
PI PI
1
2 AC
AC
PI
PI
3
4 AC
PI
5
7
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Alloxan monohydrate is one of the usual substances used for the induction of diabetes mellitus apart from streptozotocin (Etuk, 2010) and it induces diabetes by destruction of the beta cells of the islets of Langerhans of the pancreas (Lenzen, 2008). This results in a decrease of endogenous insulin secretion which paves way for the decreased utilization of glucose by body tissues and consequently elevation of blood glucose level, decreased protein content, and increased levels of cholesterol and triglycerides (Dhanabal et al., 2007). This study showed that the methanol leaf extract of G. longipetala reduced the FBS levels of treated rats (Tables 1 and 2). It is well documented that antidiabetic drugs treat diabetes by lowering glucose levels in the blood. Results obtained from this study clearly show that GLE caused marked hypoglycemic activity in the alloxan-induced diabetic rat model which is an indication of its antidiabetic potential. Hypoglycemic activities of G. longipetala may be attributed to its ability to restore the functions of pancreatic tissues thereby causing increase in insulin output or insulin utilization just like other antidiabetic medicinal plants (Neelish et al., 2010). GLE may also have suppressed hepatic gluconeogenesis, stimulated glycolysis, or inhibited or decreased intestinal absorption of glucose (Tanko et al., 2009). Sulfonylureas such as glibenclamide, which was used as the reference drug in this study, are potent secretagogues and they act by stimulating insulin secretion (Distefano & Watanabe, 2010). It is also possible that GLE acted like the sulfonylureas. To further demonstrate the efficacy of GLE in the reduction of blood glucose level, OGTT was conducted in normolgycemic rats. OGTT measures the body’s ability to metabolize glucose or glucose homeostasis (Olson et al., 2010). The significant time- and dose-dependent reduction of the blood glucose levels by GLE seen in OGTT (Figure 1) further suggests that the antidiabetic activity of GLE may involve extra-pancreatic mechanism. Some hypotheses have been proposed to explain the decrease in blood glucose of rats treated with medicinal plants in OGTT. These include lowered intestinal glucose absorption, inhibition of renal glucose re-absorption, improved tissue glucose uptake or neoglycogenesis inhibition which is highly activated by 16 h fasting before glucose load (Oliveira et al., 2008). There are convincing experimental and clinical evidence that the generation of ROS is increased in both Type 1 and Type 2 diabetes and that the onset of diabetes is closely associated with oxidative stress (Johansan et al., 2005). In this study, the antioxidant activity of GLE was evaluated using both in vitro (DPPH and FRAP) and in vivo (SOD and MDA) models. DPPH and to a lesser extent FRAP are a fast, reliable, and reproducible method widely used to measure the in vitro general antioxidant activity of pure compounds as well as plant extracts and needs no sample separation (Badarinath et al., 2010). Substances that increase FRAP value and percentage antioxidant activity in DPPH photometric assay as seen in the concentration-dependent increase in DPPH and FRAP by GLE in this study (Figures 2 and 3) are assumed to have antioxidant activity (Ramdas & Seema, 2010). This antioxidant activity may have slowed or terminated the production of ROS thereby reversing
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the diabetic condition as seen in the reduction of FBS with GLE-treated rats. Antioxidant enzymes such as SOD are known to be inhibited in diabetes mellitus as a result of non-enzymatic glycosylation and oxidation (Al-Azzawie & Allamdani, 2006). The increase in the activities of SOD of GLE-treated rats may have contributed to the antidiabetic activity of the plant extract since the increase in the activity of the enzymatic antioxidant (SOD) may have helped in reversing the action of the alloxan monohydrate through the above mechanism. Lipid peroxidation has been shown to be increased in both Type 1 and Type 2 diabetes and it could cause initial b-cell damage in Type 1 diabetes or impaired insulin production, release, or function in Type 2 diabetes (West, 2000). It is measured by determining the concentration of MDA in the blood (Ohkawa et al., 1979). The decrease in MDA levels of extract-treated rats (Table 5) may have increased the activity of glutathione peroxidase in the rats and hence caused inactivation of lipid peroxidation as suggested by Afshari et al. (2007) or may have halted b-cell damage or reversed impaired insulin production, release, or function as suggested by West (2000). Tissue wasting or weight loss is characteristic of poor glycemic control in diabetes mellitus and weight measurement is a valuable tool used in diabetes study to monitor severity and/or response to treatment plan (Mayfield, 1998). The observed weight decrease in the untreated rats in this study agrees with earlier reports (Atangwho et al., 2007). Gouania longipetala-treated rats for 3 weeks showed signs of recovery in body weight gain which was more prominent in the 150 mg/kg GLE-treated rats (Figure 4). Marked increases in levels of serum lipids have been observed in diabetes mellitus and are considered to be a prime cause of coronary heart disease in diabetics (Murugan et al., 2009). Typical findings are elevation of total cholesterol, triglycerides, LDL, very LDLs (VLDL), and decreases in HDL (Anonymous, 2007) as observed in the negative control group in this study and which were reversed in the GLE-treated rats (Table 3). These reductions could be beneficial in preventing progression of the disease and also diabetic complications and the reduction may have been presumably mediated by a control of lipid metabolism (Cho et al., 2002). This finding suggests that GLE can ameliorate the cardiac risks associated with diabetes mellitus; hence it may possess a cardio-protective property. Damage to the liver in diabetes, which can be induced by alloxan, leads to leakage and increased levels of liver enzymes into the blood (Edet et al., 2011) as seen in the negative control group of rats in this study. The significant dose-dependent reduction in the elevated serum levels of these enzymes after administration of GLE suggests a clear manifestation of the anti-hepatotoxic effect of GLE in diabetes and may have been brought about by plasma membrane stabilization as well as repair of tissue damage as suggested by Argawal et al. (2012). The slightly higher protein level of the treated rats when compared with untreated diabetic rats agrees with the work of Duncan and Bond (1981), where they found that the protein levels of diabetic rodents were only slightly reduced after 3 weeks of induction of diabetes. Also there was no
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DOI: 10.3109/13880209.2014.935864
significant difference in the serum bilirubin of both treated and control rats in this study and this may be due to the duration of the experimental diabetes. In the histopathology, the degeneration observed in the pancreas of alloxan-induced diabetic rats may be due to necrotic action of alloxan monohydrate on the b-cells (Bansal, 2002). The degenerative changes induced by alloxan monohydrate in rats pancreas were ameliorated by GLE and glibenclamide which was followed by regeneration of the islet cells (Plate 1), with attendant improved insulin production and reduction of FBS in rats (Tables 1 and 2). This effect was more pronounced at 150 mg/kg GLE. It has been demonstrated that b-cells can regenerate from stem cells located in pancreatic ducts or from progenitor cells residing inside murine islets (Liu et al., 2010). Regeneration of the islet cells by GLE may involve promotion of the above mechanisms. Metformin, a standard antidiabetic drug, has also been reported to regenerate b-cells and this directly improves insulin action (Distefano & Watanabe, 2010). Histopathology of the pancreas reinforces the regeneration of b-cells by GLE which may be responsible for its antidiabetic action. Several phytochemical constituents including flavonoids, alkaloids, glycosides, saponins, tannins, and others obtained from various plant sources have been reported to be potent hypoglycemic agents (Al-Quattan et al., 2008). From the phytochemical analysis of G. longipetala, it was found to contain most of these constituents. These phytochemcal constituents are known natural antioxidants that possess free radical scavenging effects and rejuvenating potentials (Biswas et al., 2011). Therefore, the hypoglycemic activity of GLE may have been contributed to by the presence of these active phytoconstituents. In conclusion, G. longipetala leaf methanol extract has demonstrated significant antidiabetic activity in this study and thus establishes the pharmacological basis for its use as an antidiabetic plant in the folk medicine. The antidiabetic activity may be due to its multiple effects involving both pancreatic and extra pancreatic mechanisms. It has also been shown that G. longipetala possesses potent antioxidant activity and can inhibit lipid peroxidation. GLE has also demonstrated possible cardioprotective and hepatoprotective activities. Regeneration of the pancreatic islet cells as seen in the histopathology by GLE may be responsible for its antidiabetic activity. These effects and its phytochemical constituents may also be responsible for its antidiabetogenic activity. However, further studies are on-going in our laboratory to isolate and elucidate the bioactive compound(s) responsible for its antidiabetic activity and its molecular mechanism of action.
Declaration of interest The authors report no declarations of conflict of interest.
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ORIGINAL ARTICLE
Antidiabetic, antilipidemic, and antioxidant activities of Gouania longipetala methanol leaf extract in alloxan-induced diabetic rats Maxwell Ikechukwu Ezeja1, Aruh Ottah Anaga2, and Isaac U. Asuzu2 1
Department of Veterinary Physiology, Pharmacology and Biochemistry, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria and Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, Enugu State, Nigeria
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Abstract
Keywords
Context: Gouania longipetala Hemsl. (Rhamnaceae) is used in folkloric medicine for treating diabetes mellitus and its associated symptoms. Objective: This study evaluated the antidiabetic antilipidemic and antioxidant activities of the plant methanol leaf extract. Materials and methods: Diabetes was induced in rats by intraperitoneal injection of alloxan monohydrate (160 mg/kg). Three test doses (50, 100, and 150 mg/kg) of G. longipetala extract (GLE) were administered orally and the effects were compared with glibenclamide (2 mg/kg). The effect of GLE on hyperglycemia and sub-acute study for 21 d were carried out using its effect on fasting blood sugar (FBS) level. Serum biochemistry and antioxidant activity were evaluated. Histopathological evaluation of the pancreas was also done. Results: The LD50 of G. longipetala was found to be 44000 mg/kg. The extract significantly (p50.0001) decreased the FBS levels of treated rats from 16.2 ± 2.03 to 6.5 ± 1.52 mM/L at 150 mg/kg within 24 h. The extract decreased FBS levels of rats by 62.0, 74.8, and 75.0% on day 21 at 50, 100, and 150 mg/kg, respectively. GLE reduced the level of malondiadehyde from 23.0 ± 1.34 to 10.3 ± 0.43 mg/dL, increased superoxide dismutase activities from 2.97 ± 0.34 to 5.80 ± 0.53 IU/L at 150 mg/kg, and improved the serum lipid profile of treated rats. GLE also caused restoration of the altered histopathological changes of the pancreas. Discussion and conclusion: Gouania longipetala demonstrated significant antidiabetic, antilipidemic, and antioxidant activities that may be due to its multiple effects involving both pancreatic and extra-pancreatic mechanisms.
Fasting blood sugar, glibenclamide, histopathology, hyperglycemia
Introduction Diabetes mellitus is a group of metabolic conditions characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both (Edwin et al., 2007). In the disease condition, blood glucose remains high either because the body does not produce enough insulin or because cells do not respond to insulin produced (Rakesh et al., 2010). Diabetes mellitus is a disease condition of both humans and animals. According to the World Health Organization (WHO) (2011), it is estimated that about 350 million people have diabetes, the prevalence being similar in both high and low income countries and it is predicted that global diabetes prevalence will increase by 50% in 2030 (Wild et al., 2004). Marked increases in levels of serum lipids have been observed in diabetes mellitus and are considered to be a prime cause of coronary heart disease in diabetics (Murugan et al.,
Correspondence: Maxwell I. Ezeja, Department of Veterinary Physiology, Pharmacology and Biochemistry, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. Tel. +2348033238975. E-mail: [email protected]
History Received 28 October 2013 Revised 30 April 2014 Accepted 13 June 2014 Published online 21 October 2014
2009). Oxidative stress is increased in diabetes mellitus owing to an increase in the production of oxygen-free radicals such as superoxide (O 2 ), hydrogen peroxide (H2O2), and hydroxide (OH) radicals and deficiency in antioxidant defense mechanisms (Moussa, 2008). Ultimately, the goal of all treatment strategies for diabetes mellitus is to lower blood glucose concentrations and to lessen the risk for development and progression of the disease complications, emphasizing cardiovascular risk reductions and focusing particularly on hypertension control and correction of dyslipidemia (Holman et al., 2008). Current therapies in the management of diabetes include life style intervention through diet modification and exercise and the use of oral hypoglycemic therapy and insulin treatment (Holman et al., 2008). Other management strategies in diabetes mellitus include transplantation of pancreas (Larsen, 2011), islet cell transplantation (Lakey et al., 2003), and bariatric surgery (Robinson, 2009). The agents used in clinical practice have serious adverse effects such as hematological effects, hypoglycemic coma, and disturbances of liver and kidney (Distefano & Watanabe, 2010). Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment
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of diabetes due to their effectiveness, less side effects, and relatively low cost (Nanu et al., 2008). Gouania longipetala Hemsl. (Rhamnaceae) is a scandent shrub or liana mainly present in closed forests, forest margins, and in jungle regrowths (Burkill, 1985). Its salient characteristics are the watch-spring tendrils, spike-like thyrsus, a more or less lobed disc, inferior ovary, and longitudinally 3-winged septicidal fruits (Buerki et al., 2011). The plant has been used in ethnomedicine for the treatment of different ailments. The leaves are used for the treatment of swelling, edema venomous stings, gout, febrifuges, etc. The leaf sap is used for eye treatments, as pain killers and for treating heart diseases (Abbiw, 1990; Burkill, 1985). The stem of the plant has been shown to possess antibacterial and anti-inflammatory activities (Ekuadzi et al., 2012). In Orba, Udenu local Government Area of Enugu State, South Eastern Nigeria (where the plant is known as ‘‘Asha’’), the leaves of Gouania longipetala are used to treat diabetes mellitus and malaria (Focho et al., 2009). No scientific study has been carried out to authenticate the use of G. longipetala for the treatment of diabetes mellitus and to establish the efficacy of the plant in the modulation of oxidative stress and hyperlipidemia associated with diabetes mellitus. Hence, the present study was undertaken to evaluate the methanol leaf extract of G. longipetala for its antidiabetic effect with a view to establishing the pharmacological basis for its folkloric use as an antidiabetic plant, and to determine the effect of sub-acute administration of G. longipetala on lipid profile and in vivo and in vitro antioxidant parameters of alloxan-induced diabetic rats.
Materials and methods Plant collection and identification Fresh leaves of Gouania longipetala were collected from its natural habitat at Orba-Nsukka, Enugu state, Nigeria in July 2011 and identified by a taxonomist, Mr. A. O. Ozioko of the Bioresources Development and Conservation Programme, Aku Road, Nsukka, Enugu State where the voucher specimen number VPP/2011/68 was deposited in the herbarium. Extraction of plant material The plant was extracted by the cold maceration method. The leaves were washed, cut into small pieces, dried at room temperature, and pulverized into coarse powder of about 1 mm in diameter. The plant material (500 g) was macerated in 80% methanol for 48 h with intermittent shaking at 2 h interval. The extract was then filtered using Whatman No. 1 filter papers and concentrated in vacuo using rotary evaporator at 40 C and 210 milibar and a vacuum lyophilizer. The yield of the extract was calculated and it was stored in a refrigerator at 4 C as G. longipetala extract (GLE) until the time of use (Mansi & Lahham, 2008). Experimental animals Mature Wistar albino rats, bred in the laboratory animal unit of the Faculty of Veterinary Medicine, University of Nigeria Nsukka, were used for the experiments. They were housed in
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an environment of normal ambient temperature and the lighting period was about 12 h daily. The relative humidity was between 40 and 60%. The weight of the rats varied between 107 and 185 g. The rats were kept in stainless steel cages, supplied with clean drinking water, and fed ad libitum with standard commercial pelleted feed (Vital feedÕ , UAC, Lagos, Nigeria). Ethical conditions governing the conduct of experiments with life animals were strictly observed as stipulated by Ward and Elsea (1997) and all animal experiments were conducted in compliance with NIH Guide for Care and Use of Laboratory Animals (Pub. no. 85-23, revised 1985). The experimental protocol was approved by the institution’s ethical committee for the use of laboratory animals (Number: VPP/EC/012/36). Acute toxicity effect of the methanol leaf extract of G. longipetala in rats The method of Lorke (1983) was adopted for this study. Mature albino rats (35) of both sexes were randomly grouped into seven groups (1–7) of five rats per group. Groups 1–6 were given GLE at the doses of 100, 500, 1000, 2000, 3000, and 4000 mg/kg, using oral gavage. Group 7 rats received distilled water (10 ml/kg). The rats were allowed free access to feed and drinking water and were observed for 48 h for signs of toxicity and death. Induction of experimental diabetes Diabetes was induced in male albino Wistar rats using the method described by Venugopal et al. (1998). Thirty male rats were used for this experiment. The rats were fasted for 18 h. The fasting blood sugar (FBS) levels of the rats were determined with blood from the rats’ tail vein using an auto analyzer (AccuCheck Advantage IIÕ , Roche, Branford, CT) glucose kit. Diabetes was then induced in the rats by a single intraperitoneal injection of alloxan monohydrate at 160 mg/ kg. The FBS levels of the rats were checked every other day. On the sixth day, diabetes was established in the rats and rats with FBS of 8 mMol/L were considered diabetic (Vinuthan et al., 2007).
Effect of graded doses of G. longipetala on blood glucose level of alloxan-induced hyperglycemia in rats (dose–response study) Thirty alloxan-induced diabetic male rats were used for the study. The rats were randomly grouped into five groups of six rats per group and treated as follows after measuring their FBS at 0 h (on establishment of diabetes). Group 1 rats received 10% Tween 20 (10 ml/kg) and served as a negative control. Group 2 rats were given glibenclamide (2 mg/kg) and served as a positive control, while groups 3, 4, and 5 served as treatment groups and were given 50, 100, and 150 mg/kg of GLE, respectively, by gastric gavage. The FBS of all the rats was measured at 0, 1, 3, 6, and 24 h post drug or extract treatment using an auto analyzer (AccuCheck Advantage IIÕ , Roche, Branford, CT) glucose kit. The blood samples were collected from the tail vein after a snip (Vinkatesh et al., 2008).
Antidiabetic activity of G. longipetala
DOI: 10.3109/13880209.2014.935864
Effect of GLE on oral glucose tolerance test (OGTT) in rats
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The method described by Aslan et al. (2007) was adopted for this study. Thirty mature male albino rats were fasted for 18 h and were randomly divided into five groups of six rats each. The FBS at 0 h was measured before treatment. Group 1 rats (negative control) were given 10 ml/kg of 10% Tween 20 solutions. Group 2 rats were given 2 mg/kg glibenclamide (positive control) while groups 3, 4, and 5 rats were given 50, 100, and 150 mg/kg of GLE, respectively. They were dosed orally with the aid of gastric gavage. The rats in all the groups were loaded with glucose at the dose of 2 g/kg per os 30 min after drug or extract administration. Blood samples were then collected from the tail vein after a snip for measurement of the glucose levels at 30, 60, 120, and 180 min. Evaluation of in vitro antioxidant activity of GLE 1-1-Diphenyl-2-picril hydrazyl photometric assay The method of Mensor et al. (2001) was adopted for this study. Test extract (2 ml) at concentrations ranging from 10 to 400 mg/ml was each mixed with 1 ml of 0.5 mM diphenyl-2picril hydrazyl (DPPH) (in methanol). Absorbance at 517 nm was taken after 30 min incubation in the dark at room temperature. The concentrations were prepared in triplicates. The percentage antioxidant activity was calculated as follows: % Antioxidant activity ½AA absorbance of sample 100 absorbance of blank ¼ 100 Absorbance of control Methanol (1 ml) plus 2 ml of the extract was used as a blank while 1 ml of 0.5 mM DPPH solution plus 2 ml of methanol was used as a control. Ascorbic acid was used as a reference standard. Ferric-reducing antioxidant power assay Total antioxidant activity was measured by the ferric reducing antioxidant power (FRAP) assay (Benzie & Strain, 1999). Sample (100 ml) of different concentrations of the extract (10, 50, 100, 200, and 400 mg/ml) was mixed with 3 ml of the working FRAP reagent. The absorbance was immediately taken at 593 nm at 0 min. Thereafter samples were placed in water bath at 37 C and the absorbance was measured after 4 min with a spectrophotometer. FRAP value of the samples was calculated using the following formula: Change in absorbance from 0 to 4 min FRAP value of standard FRAP value : Change in absorbance of standard from 0 to 4 min Effect of 21-day oral administration of GLE on FBS, serum biochemistry, lipid peroxidation/in vivo antioxidant activities in alloxan-induced diabetic rats (sub-acute antidiabetic study) Fifty male albino rats were randomly grouped into five groups of 10 rats per group. Diabetes was induced in the rats by
3
single intraperitoneal injection of 160 mg/kg of alloxanmonohydrate. On establishment of diabetes using the method earlier described, the rats were re-grouped into five groups of eight rats per group and were treated as follows: group 1 rats which served as a negative control received 10% Tween 20 solution. Group 2 rats received 2 mg/kg glibenclamide as a reference drug while groups 3, 4, and 5 rats were administered 50, 100, and 150 mg/kg GLE, respectively, through gastric gavage. The drug or extract and the vehicle were administered to the rats for 21 d. The FBS levels and the body weights of the rats were measured on days 0, 1, 7, 14, and 21 (Verma et al., 2010). On day 21, being the last day of the treatment, blood samples were collected from the retro-orbital plexus of the eye under light ether anesthesia for serum biochemistry, lipid peroxidation/in vivo antioxidant activities. The rats were later sacrificed by chloroform and the pancreas was collected in sample (bijou) bottles for histopathology. Lipid profile assay Total cholesterol was evaluated using the enzymatic colorimetric chod-pap test method described by Allain et al. (1974) as described in Quimica Clinica Applicada test kit (Quimica Clinica Aplicada S.A, Amposta, Spain); triglycerides were also determined spectrophotometrically using the method of Tietz (1990); high density lipoprotein (HDL) was evaluated by the method of Grove (1979) as described in Quimica Clinica Applicada test kit; lowdensity lipoprotein (LDL) was determined as the difference between total cholesterol and cholesterol content of the supernatant after precipitation of the LDL fraction by polyvinyl sulfate (PVS) in the presence of polyethylene– glycol monomethyl ether (Bergmenyer, 1985) and very LDL (VLDL) was calculated according to the method of Wilson et al. (1981) as VLDL ¼ 0.2 TG (where TG is triglycerides). Liver marker enzymes Aspartate aminotransferase (AST) was evaluated using the method of Reitman and Frankel (1957) as described by Randox Laboratories, Belfast, United Kingdom, using Randox kits; alanine aminotransferase (ALT) was measured by monitoring the concentration of pyruvate hydrazone formed with 2,4,-dinitrophenylhydrazine using the method of Reitman and Frankel (1957) as described in Randox kits; alkaline phosphatase (ALP) was assayed based on the methods of Kind and King (1972); total protein in serum was assayed using the direct biuret method (Gornall et al., 1949) while total bilirubin was determined according to the method of Jendrassik and Grof (1938). Lipid peroxidation/in vivo antioxidant activities of G. longipetala Malondialdehyde Lipid peroxidation was determined spectrophotometrically by measuring the level of lipid peroxidation product, malondialdehyde (MDA) as described by Draper and Hadley (1990).
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Table 1. Effect of graded doses of Gouania longipetala extract on the fasting blood glucose levels of alloxan-induced diabetic rats. Mean fasting blood glucose levels ± SEM (mmol/L) Treatment
0h
1h
3h
6h
24 h
% Reduction at sixth hour
% Reduction at 24th hour
Tween 20 (10 ml/kg) Glibenclamide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
19.9 ± 4.02 21.8 ± 4.32 12.8 ± 1.22 15.3 ± 1.48 16.2 ± 2.03
20.2 ± 3.80 17.3 ± 3.28* 8.9 ± 2.27* 8.3 ± 2.31** 8.5 ± 2.62***
19.3 ± 4.05 13.0 ± 3.12** 7.8 ± 1.72** 7.9 ± 1.76*** 8.3 ± 2.41***
20.1 ± 3.50 7.7 ± 1.42** 6.6 ± 1.82*** 6.0 ± 0.65*** 5.2 ± 1.52***
22.8 ± 2.45 8.6 ± 1.40*** 7.2 ± 1.55** 6.3 ± 0.42*** 6.5 ± 1.52***
– 64.7 48.4 60.8 70.1
– 60.6 43.8 58.8 60.0
Group 1. 2. 3. 4. 5.
MDA reacts with thiobarbituric acid (TBA) to form a red or pink colored complex which absorbs maximally in acid solution at 532 nm.
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Estimation of superoxide dismutase
5
Adrenaline (10 mg) was dissolved in 17 ml of distilled water to make adrenaline solution. Serum (0.1 ml) was added to 0.9 ml of phosphate buffer (pH 7.8). The extract (0.2 ml) was taken in triplicate and 2.5 ml of buffer added inside a cuvette and 0.3 ml of adrenaline solution added, mixed well, and absorbance was read at 450 nm at 30 s interval for five times (Xin et al., 1991). Histopathology
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Blood glucose level
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*p50.01, **p50.001, ***p50.0001 when compared with the negative control.
4 3 vehicle 2
Glibenclamide 2 mg/kg GLE 50 mg/kg
1
GLE 100 mg/kg GLE 150 mg/kg
Tissue samples (pancreas) collected after sacrifice of the rats, on the completion of 21 d treatment with drug or extract, were fixed in 10% formal saline for 24 h. They were washed in ascending grades of ethanol, cleared with xylene, embedded in paraffin wax, sectioned with a microtome, and stained with hematoxylin and eosin (H and E) and mounted on Canada balsam (Sigma-Aldrich, St. Louis, MO) (Bancroft & Stevens, 1977). All the sections were examined under a light microscope under different (100 and 400) magnifications. Photomicrographs of lesions were taken with an Olympus photo microscope (Olympus Scientific Equipment, Ashburn, VA) for observations and documentation of histopathological lesions.
Acute toxicity test
Phytochemical analysis of G. longipetala leaf extract
Effect of graded doses of G. longipetala on the blood glucose levels of alloxan-induced diabetic rats (dose–response study)
Phytochemical analysis of G. longipetala leaf extract was carried out to identify the chemical families present in it using the methods of Trease and Evans (1996). Data analysis The results were presented as mean ± SEM and analyzed using one-way analysis of variance (ANOVA). The differences between the means were tested using post hoc LSD and values of p50.05 were considered statistically significant.
Results
0 0 min
30 min
60 min Time
90 min
180 min
Figure 1. Effect of GLE on oral glucose tolerance test (OGTT) in rats.
After 48 h, no death or signs of toxicity were observed in the rats treated with different doses (100, 500, 1000, 2000, 3000, and 4000 mg/kg) of GLE. The LD50 of G. longipetala is, therefore, 44000 mg/kg.
The result of the effect of G. longipetala on the FBS of alloxan-induced hyperglycemic rats is presented in Table 1. Gouania longipetala (50, 100, and 150 mg/kg) and glibenclamide caused significant (p50.01–0.0001) dose- and time-dependent decrease in the fasting blood glucose levels of treated rats when compared with the negative control rats. The extract caused a decrease in FBS levels of the rat by 48.4, 60.8, and 70.1% at the sixth hour and 43.8, 58.8, and 60.0% at the 24th hour, respectively as against 64.7% and 60.6% reduction by the reference drug at 6th and 24th hour, respectively. The extract at all doses and the reference drug had their best activities at the 6th hour.
Extraction of G. longipetala The methanol leaf extract of G. longipetala was dark green in color with a pasty consistency and the percent yield was 16.3% w/w dry matter.
Effect of GLE on OGTT in alloxan-induced diabetic rats Figure 1 shows the effect of GLE on the blood glucose levels of rats after glucose induced hyperglycemia. After the glucose
Antidiabetic activity of G. longipetala
DOI: 10.3109/13880209.2014.935864
load, the mean blood glucose level of rats in group 1 (negative control) increased from 4.14 ± 0.07 mmol/L to 6.42 ± 0.19 mmol/L within 30 min representing 35.5% increase and continued to gradually decrease up to 180th minute. Also, there were various levels of increase in the blood glucose
GLE
Evaluation of in vitro antioxidant activity of GLE
80
ASCORBIC acid
DPPH photometric assay
30
The percent antioxidant activity of GLE and ascorbic acid standard is shown in Figure 2. The result showed that the extract exhibited a concentration-dependent antioxidant activity by increasing the percent antioxidant from 16.6% at the concentration of 10 mg/ml to 65.5% at the concentration of 400 mg/ml. The ascorbic acid standard had an antioxidant activity of 31.6% at the concentration of 10 mg/ml and 79.7% at the concentration of 400 mg/ml.
20
FRAP assay
10
The total antioxidant activity of GLE using FRAP is presented in Figure 3. The result showed that the extract had a concentration-dependent total antioxidant activity increasing the FRAP value from 0.40 mM at the concentration of 10 mg/ml to 1.64 mM at the concentration of 400 mg/ml.
% anoxidant acvity
60 50 40
0 10 µg/ml
50 µg/ml 100 µg/ml 200 µg/ml 400 µg/ml Treatment
Figure 2. Percent antioxidant activity of G. longipetala extract in DPPH photometric assay.
2.5
Effect of sub-acute administration of GLE on alloxan-induced diabetic rats Fasting blood sugar
GLE
The result of the effect of G. longipetala leaf extract on the mean FBS levels of control and experimental groups of rats over a period of 21 d is presented in Table 2. The reference drug and the extract (50, 100, and 150 mg/kg) caused various levels of significant (p50.001–0.0001) dose- and timedependent reduction of the FBS of the rats on days 1, 7, 14, and 21 when compared with a negative control with the extract at the doses of 50, 100, and 150 mg/kg reducing the FBS, by 62.0, 74.8, and 75.0%, respectively, as against 73.4% by the reference drug glibenclamide (2 mg/kg) on day 21. The activity of the extract at the doses of 150 mg/kg was comparable with glibenclamide (2 mg/kg) reference drug on day 21.
Ascorbic Acid 2
Frap value (µm)
levels of the treated rats at the 30th minute. GLE and glibenclamide caused a dose- and time-dependent reduction of the blood glucose levels from the 60th to 180th minute with the extract (50, 100, and 150 mg/kg) reducing the blood glucose levels by 15.0, 15.5, and 28.0%, respectively, at 180th minute as against 36.8% by glibenclamide (2 mg/kg).
90
70
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5
1.5
1
0.5
0 10 µg/ml GLE
50 µg/ml GLE
100 µg/ml 200 µg/ml 400 µg/ml GLE GLE GLE
Effect of GLE on the body weights of alloxan-induced diabetic rats
Treatment
Figure 4 shows the weight changes of control and alloxaninduced diabetic rats treated with G. longipetala leaf extract
Figure 3. Total antioxidant activity of GLE using FRAP assay.
Table 2. Effect of GLE on the FBS of alloxan-induced diabetic rats treated for 21 d. Mean fasting blood glucose levels ± (mmol/L) Group 1. 2. 3. 4. 5.
Treatment
Pre-diabetic FBS
Tween 20 (10 ml/kg) Glibenclamide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
4.1 ± 0.37 4.2 ± 0.31 4.0 ± 0.24 4.0 ± 0.15 3.9 ± 0.02
Day 0
Day 1
Day 7
Day 14
Day 21
15.7 ± 0.59 14.7 ± 0.77 15.7 ± 1.48 15.8 ± 1.19 16.5 ± 0.32 15.4 ± 1.22 8.2 ± 1.05* 6.8 ± 0.59** 6.0 ± 0.40** 4.1 ± 0.43** 13.7 ± 1.30 9.0 ± 1.09* 8.4 ± 0.89** 5.8 ± 0.50** 5.2 ± 0.42** 16.7 ± 1.25 10.9 ± 1.25* 7.4 ± 0.75** 5.5 ± 0.37** 4.2 ± 0.26** 14.2 ± 1.04 8.0 ± 0.56* 6.4 ± 0.36** 4.4 ± 0.19** 3.6 ± 0.15**
*p50.001, **p50.0001 when compared with the negative control.
% reduction (day 21) – 73.2 62.0 74.8 75.0
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reduction in the serum levels of total cholesterol, triglycerides, LDL, and VLDL of the rats when compared with the negative control rats. The activity of the extract at the dose of 150 mg/kg was comparable with glibenclamide (2 mg/kg). The extract at the dose of 50 mg/kg caused some level of reduction of the total cholesterol, triglycerides, LDL, and VLDL of the rats but the reduction was not statistically significant (p40.05). Also GLE at all the doses used caused dose-dependent and significant (p50.05) increase in the HDL of the rats when compared with the negative control with the effect of extract at the doses of 100 and 150 mg/kg being comparable with the reference drug.
for 21 d. In the negative control, there was a gradual decrease in the mean body weights of the rats from the pre-induction mean weight of 147.3 ± 1.33–145.5 ± 0.92 g at day 0 to 142.2 ± 1.22 g on day 21. In the glibenclamide (2 mg/kg) and GLE-treated groups of rats, there was a sharp fall in the mean body weight on day 0 of the diabetes from the pre-diabetes induction weights and then followed by gradual increase in the weight on days 7, 14, and 21 with the best result being observed with the highest dose of the GLE (150 mg/kg).
Table 3 shows the mean serum lipid profile of the controland GLE-treated groups of alloxan-induced diabetic rats. Glibenclamide (2 mg/kg) and GLE (100 and 150 mg/kg) caused various levels of significant (p50.05–0.0001) 160 155
Effect of G. longipetala on liver marker enzymes of alloxan-induced diabetic rats The results of the effect of GLE on liver marker enzyme activities of alloxan-induced diabetic rats are presented in Table 4. There was a significant (p50.05) decrease in mean AST, ALT, and ALP activities in the glibenclamide and GLE (100 and 150 mg/kg) treated rats when compared with the negative control group. Total protein and bilirubin levels were not significantly (p40.05) changed in all the test groups when compared with the negative control group.
GLE 100 mg/kg GLE 150 mg/kg
vehicle Glibenclamide 2 mg/kg GLE 50 mg/kg
150 Body weight (g)
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Effect of GLE on lipid profile of alloxan-induced diabetic rats
145
Effect of GLE on lipid peroxidation (in vivo antioxidant activity) in alloxan-induced diabetic rats
140 135
The results of the effect of GLE on MDA and SOD of alloxaninduced diabetic rats are presented in Table 5. Glibenclamide (2 mg/kg) and GLE (50, 100, and 150 mg/kg) caused a dosedependent and significant (p50.01–0.001) reduction in the level of MDA of rats when compared with the negative control group. The extract at the doses of 100 and 150 mg/kg also caused a significant (p50.001) increase in the SOD activities from 2.97 ± 0.51 mg/dl in the negative control group to 4.55 ± 0.39 mg/dl at the dose of 100 mg/kg and 5.80 ± 0.53 at 150 mg/kg as against 3.62 ± 0.56 mg/dl by glibenclamide (2 mg/kg).
130 125 120 before inducon
Day 0
Day 7
Day 14
Day 21
Duraon of treatment
Figure 4. Effect of G. longipetala on mean body weights (g) of alloxaninduced diabetic rats. Table 3. Effect of GLE on the lipid profile of alloxan-induced diabetic rats. Group 1. 2. 3. 4. 5.
Treatment
Total cholesterol (mg/dl)
Triglycerides (mg/dl)
LDL (mg/dl)
HDL (mg/dl)
VLDL (mg/dl)
Tween 20 (10 ml/kg) Glibenclanide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
74.5 ± 2.50 63.7 ± 4.51* 68.5 ± 4.17 64.6 ± 4.34* 54.2 ± 6.30**
76.5 ± 5.01 58.2 ± 4.74** 73.7 ± 3.67 63.7 ± 3.37* 61.5 ± 2.30*
64.0 ± 2.16 40.8 ± 3.56** 51.7 ± 2.70 41.6 ± 3.67** 40.5 ± 2.67**
15.5 ± 1.57 33.3 ± 4.62*** 21.5 ± 2.03* 38.3 ± 2.76*** 47.8 ± 4.97***
15.3 ± 1.80 11.8 ± 4.67* 14.7 ± 1.53 12.7 ± 1.21* 12.3 ± 0.46*
*p50.05, **p50.001, ***p50.0001 when compared with the negative control group. Table 4. Effect of GLE on the liver enzyme markers of alloxan-induced diabetic rats. Group 1. 2. 3. 4. 5.
Treatment
AST (IU/L)
ALT (IU/L)
ALP (IU/L)
Bilirubin (mg/dl)
Total protein (g/dl)
Tween 20 (10 ml/kg) Glibencdanide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
127.8 ± 2.1 86 ± 1.89* 124.0 ± 2.32 93. 0 ± 2.01* 92.3 ± 1.56*
123.6 ± 3.22 90.0 ± 1.18* 121.0 ± 2.44 91.2 ± 2.52* 90.0 ± 1.43*
220.5 ± 1.20 180.0 ± 2.44* 214.1 ± 3.44 193.1 ± 1.11* 182.4 ± 0.82*
0.51 ± 0.51 0.50 ± 0.22 0.52 ± 1.13 0.50 ± 0.26 0.52 ± 0.06
6.01 ± 1.23 6.12 ± 1.69 6.28 ± 1.50 6.18 ± 0.37 6.34 ± 0.16
*p50.05 when compared with the negative control group.
Antidiabetic activity of G. longipetala
DOI: 10.3109/13880209.2014.935864
Histopathology
and showed evidence of cell regeneration and also contained cells in the process of maturation which was more prominent in the 150 mg/kg GLE-treated rats (group 5).
Plate 1 shows the photomicrograph of sections of the pancreas with diabetic-untreated group (group 1) showing shrunken pancreatic islets (PI) having fewer but degenerate cells. The glibenclamide-treated group indicated that there were more pancreatic islet cells when compared with the negative control group. Although there were few cells in 50 mg/kg GLEtreated group (3), the cells were more when compared with the negative control group of rats. In the 100 and 150 mg/kg GLE-treated rats (groups 4 and 5), the islet cells were many
Phytochemical analysis of G. longipetala Phytochemical analysis showed that GLE contains saponins, starch, flavonoids, alkaloids, glycosides, sterols/terpenes, and tannins.
Discussion Acute toxicity tests indicated that the extract was well tolerated and safe at the doses used in this study. The antidiabetic activity of Gouania longipetala was evaluated, among others, by testing the ability of the extract to reduce the FBS level of rats raised by a single intraperitoneal administration of 160 mg/kg of alloxan monohydrate. The FBS test is a carbohydrate metabolic test which measures blood glucose after a fast (usually 12–18 h), during which the body stimulates the release of the hormone glucagon which in turn releases glucose into the blood stream through catabolic processes (Kelly, 2008).
Table 5. Effect of GLE on the MDA levels and SOD activities of alloxan-induced diabetic rats.
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Group 1. 2. 3. 4. 5.
Treatment
MDA (mg/dl)
SOD (IU/l)
Tween 20 (10 ml/kg) Glibenclamide (2 mg/kg) GLE (50 mg/kg) GLE (100 mg/kg) GLE (150 mg/kg)
23.0 ± 1.34 15.3 ± 1.75* 14.7 ± 0.56* 10.7 ± 2.11** 10.3 ± 0.43**
2.97 ± 0.34 3.62 ± 0.56 3.57 ± 0.79 4.55 ± 0.39** 5.80 ± 0.53**
*p50.01, **p50.001 when compared with the negative control.
Plate 1. Photomicrograph of sections of the pancreas from experimental rats after 21 d of treatment with GLE. See Table 5 for respective treatment groups. PI, pancreatic islets; AC, Acinar cell.
AC
AC
PI PI
1
2 AC
AC
PI
PI
3
4 AC
PI
5
7
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Alloxan monohydrate is one of the usual substances used for the induction of diabetes mellitus apart from streptozotocin (Etuk, 2010) and it induces diabetes by destruction of the beta cells of the islets of Langerhans of the pancreas (Lenzen, 2008). This results in a decrease of endogenous insulin secretion which paves way for the decreased utilization of glucose by body tissues and consequently elevation of blood glucose level, decreased protein content, and increased levels of cholesterol and triglycerides (Dhanabal et al., 2007). This study showed that the methanol leaf extract of G. longipetala reduced the FBS levels of treated rats (Tables 1 and 2). It is well documented that antidiabetic drugs treat diabetes by lowering glucose levels in the blood. Results obtained from this study clearly show that GLE caused marked hypoglycemic activity in the alloxan-induced diabetic rat model which is an indication of its antidiabetic potential. Hypoglycemic activities of G. longipetala may be attributed to its ability to restore the functions of pancreatic tissues thereby causing increase in insulin output or insulin utilization just like other antidiabetic medicinal plants (Neelish et al., 2010). GLE may also have suppressed hepatic gluconeogenesis, stimulated glycolysis, or inhibited or decreased intestinal absorption of glucose (Tanko et al., 2009). Sulfonylureas such as glibenclamide, which was used as the reference drug in this study, are potent secretagogues and they act by stimulating insulin secretion (Distefano & Watanabe, 2010). It is also possible that GLE acted like the sulfonylureas. To further demonstrate the efficacy of GLE in the reduction of blood glucose level, OGTT was conducted in normolgycemic rats. OGTT measures the body’s ability to metabolize glucose or glucose homeostasis (Olson et al., 2010). The significant time- and dose-dependent reduction of the blood glucose levels by GLE seen in OGTT (Figure 1) further suggests that the antidiabetic activity of GLE may involve extra-pancreatic mechanism. Some hypotheses have been proposed to explain the decrease in blood glucose of rats treated with medicinal plants in OGTT. These include lowered intestinal glucose absorption, inhibition of renal glucose re-absorption, improved tissue glucose uptake or neoglycogenesis inhibition which is highly activated by 16 h fasting before glucose load (Oliveira et al., 2008). There are convincing experimental and clinical evidence that the generation of ROS is increased in both Type 1 and Type 2 diabetes and that the onset of diabetes is closely associated with oxidative stress (Johansan et al., 2005). In this study, the antioxidant activity of GLE was evaluated using both in vitro (DPPH and FRAP) and in vivo (SOD and MDA) models. DPPH and to a lesser extent FRAP are a fast, reliable, and reproducible method widely used to measure the in vitro general antioxidant activity of pure compounds as well as plant extracts and needs no sample separation (Badarinath et al., 2010). Substances that increase FRAP value and percentage antioxidant activity in DPPH photometric assay as seen in the concentration-dependent increase in DPPH and FRAP by GLE in this study (Figures 2 and 3) are assumed to have antioxidant activity (Ramdas & Seema, 2010). This antioxidant activity may have slowed or terminated the production of ROS thereby reversing
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the diabetic condition as seen in the reduction of FBS with GLE-treated rats. Antioxidant enzymes such as SOD are known to be inhibited in diabetes mellitus as a result of non-enzymatic glycosylation and oxidation (Al-Azzawie & Allamdani, 2006). The increase in the activities of SOD of GLE-treated rats may have contributed to the antidiabetic activity of the plant extract since the increase in the activity of the enzymatic antioxidant (SOD) may have helped in reversing the action of the alloxan monohydrate through the above mechanism. Lipid peroxidation has been shown to be increased in both Type 1 and Type 2 diabetes and it could cause initial b-cell damage in Type 1 diabetes or impaired insulin production, release, or function in Type 2 diabetes (West, 2000). It is measured by determining the concentration of MDA in the blood (Ohkawa et al., 1979). The decrease in MDA levels of extract-treated rats (Table 5) may have increased the activity of glutathione peroxidase in the rats and hence caused inactivation of lipid peroxidation as suggested by Afshari et al. (2007) or may have halted b-cell damage or reversed impaired insulin production, release, or function as suggested by West (2000). Tissue wasting or weight loss is characteristic of poor glycemic control in diabetes mellitus and weight measurement is a valuable tool used in diabetes study to monitor severity and/or response to treatment plan (Mayfield, 1998). The observed weight decrease in the untreated rats in this study agrees with earlier reports (Atangwho et al., 2007). Gouania longipetala-treated rats for 3 weeks showed signs of recovery in body weight gain which was more prominent in the 150 mg/kg GLE-treated rats (Figure 4). Marked increases in levels of serum lipids have been observed in diabetes mellitus and are considered to be a prime cause of coronary heart disease in diabetics (Murugan et al., 2009). Typical findings are elevation of total cholesterol, triglycerides, LDL, very LDLs (VLDL), and decreases in HDL (Anonymous, 2007) as observed in the negative control group in this study and which were reversed in the GLE-treated rats (Table 3). These reductions could be beneficial in preventing progression of the disease and also diabetic complications and the reduction may have been presumably mediated by a control of lipid metabolism (Cho et al., 2002). This finding suggests that GLE can ameliorate the cardiac risks associated with diabetes mellitus; hence it may possess a cardio-protective property. Damage to the liver in diabetes, which can be induced by alloxan, leads to leakage and increased levels of liver enzymes into the blood (Edet et al., 2011) as seen in the negative control group of rats in this study. The significant dose-dependent reduction in the elevated serum levels of these enzymes after administration of GLE suggests a clear manifestation of the anti-hepatotoxic effect of GLE in diabetes and may have been brought about by plasma membrane stabilization as well as repair of tissue damage as suggested by Argawal et al. (2012). The slightly higher protein level of the treated rats when compared with untreated diabetic rats agrees with the work of Duncan and Bond (1981), where they found that the protein levels of diabetic rodents were only slightly reduced after 3 weeks of induction of diabetes. Also there was no
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DOI: 10.3109/13880209.2014.935864
significant difference in the serum bilirubin of both treated and control rats in this study and this may be due to the duration of the experimental diabetes. In the histopathology, the degeneration observed in the pancreas of alloxan-induced diabetic rats may be due to necrotic action of alloxan monohydrate on the b-cells (Bansal, 2002). The degenerative changes induced by alloxan monohydrate in rats pancreas were ameliorated by GLE and glibenclamide which was followed by regeneration of the islet cells (Plate 1), with attendant improved insulin production and reduction of FBS in rats (Tables 1 and 2). This effect was more pronounced at 150 mg/kg GLE. It has been demonstrated that b-cells can regenerate from stem cells located in pancreatic ducts or from progenitor cells residing inside murine islets (Liu et al., 2010). Regeneration of the islet cells by GLE may involve promotion of the above mechanisms. Metformin, a standard antidiabetic drug, has also been reported to regenerate b-cells and this directly improves insulin action (Distefano & Watanabe, 2010). Histopathology of the pancreas reinforces the regeneration of b-cells by GLE which may be responsible for its antidiabetic action. Several phytochemical constituents including flavonoids, alkaloids, glycosides, saponins, tannins, and others obtained from various plant sources have been reported to be potent hypoglycemic agents (Al-Quattan et al., 2008). From the phytochemical analysis of G. longipetala, it was found to contain most of these constituents. These phytochemcal constituents are known natural antioxidants that possess free radical scavenging effects and rejuvenating potentials (Biswas et al., 2011). Therefore, the hypoglycemic activity of GLE may have been contributed to by the presence of these active phytoconstituents. In conclusion, G. longipetala leaf methanol extract has demonstrated significant antidiabetic activity in this study and thus establishes the pharmacological basis for its use as an antidiabetic plant in the folk medicine. The antidiabetic activity may be due to its multiple effects involving both pancreatic and extra pancreatic mechanisms. It has also been shown that G. longipetala possesses potent antioxidant activity and can inhibit lipid peroxidation. GLE has also demonstrated possible cardioprotective and hepatoprotective activities. Regeneration of the pancreatic islet cells as seen in the histopathology by GLE may be responsible for its antidiabetic activity. These effects and its phytochemical constituents may also be responsible for its antidiabetogenic activity. However, further studies are on-going in our laboratory to isolate and elucidate the bioactive compound(s) responsible for its antidiabetic activity and its molecular mechanism of action.
Declaration of interest The authors report no declarations of conflict of interest.
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