|Year : 2017 | Volume
| Issue : 3 | Page : 74-85
Validation of the antidiabetic effects of Vernonia amygdalina delile leaf fractions in fortified diet-fed streptozotocin-treated rat model of type-2 diabetes
Stanley Irobekhian Reuben Okoduwa1, Ismaila Alhaji Umar2, Dorcas Bolanle James2, Hajiya Mairo Inuwa2
1 Department of Biochemistry, Ahmadu Bello University, Samaru-Zaria; Directorate of Research and Development, n Institute of Leather and Science Technology, Zaria, Nigeria
2 Department of Biochemistry, Ahmadu Bello University, Samaru-Zaria, Zaria, Nigeria
|Date of Web Publication||29-Dec-2017|
Stanley Irobekhian Reuben Okoduwa
Directorate of Research and Development, Nigerian Institute of Leather and Science Technology, PMB 1034, Zaria
Source of Support: None, Conflict of Interest: None
Background: Vernonia amygdalina (VA) is used in the traditional management of diabetes in Nigeria. Previous scientific verification of VA is on Type-1 diabetes model, in spite of the continuous increase in Type-2 diabetes (T2D) among adults. This study aimed to validate the antidiabetic effects of VA leaf fraction (VALF) in a unique T2D rat model. Materials and Methods: Methanol crude extract of VA leaf was fractionated with solvents of increasing order of polarity (n-hexane, chloroform, ethyl-acetate, n- butanol and water). The antidiabetic activities of the fractions were evaluated in vivo in T2D model rats. Albino Wistar rats were induced with T2D and treated with the VALF. Several T2D-related parameters were measured. Results: T2D rats showed significant increase in serum levels of fasting blood glucose (FBG), liver and kidney biomarkers. At 28-day post-oral treatment with the VALF, FBG levels were significantly (P < 0.05) reduced (n- hexane [29.3%], chloroform [66.7%], ethyl acetate [36.2%], n- butanol [45.59%] and aqueous [39.3%]). The glucose tolerance ability was significantly improved in the chloroform fraction (Vernonia amygdalina chloroform fraction [VAc])-treated groups compared to the other fractions-treated group and diabetic control group. Furthermore, the VAc was found to be most effective as it ameliorates most of the alterations caused in the studied parameters in diabetic rats when compared with n- hexane, ethyl acetate, n- butanol and aqueous fractions. Conclusion: The study validates the anti-diabetic effects of VALF in fortified diet-fed streptozotocin-treated rat model of T2D, and suggests that the VAc is a potential candidate for development of a more effective drug for the management of T2D.
Keywords: Anti-hyperglycaemia, hyperlipidaemia, rats, type-2 diabetes, Vernonia amygdalina
|How to cite this article:|
Reuben Okoduwa SI, Umar IA, James DB, Inuwa HM. Validation of the antidiabetic effects of Vernonia amygdalina delile leaf fractions in fortified diet-fed streptozotocin-treated rat model of type-2 diabetes. J Diabetol 2017;8:74-85
|How to cite this URL:|
Reuben Okoduwa SI, Umar IA, James DB, Inuwa HM. Validation of the antidiabetic effects of Vernonia amygdalina delile leaf fractions in fortified diet-fed streptozotocin-treated rat model of type-2 diabetes. J Diabetol [serial online] 2017 [cited 2018 Jul 20];8:74-85. Available from: http://www.journalofdiabetology.org/text.asp?2017/8/3/74/222085
| Introduction|| |
Diabetes mellitus is a group of chronic metabolic disorders characterised by hyperglycaemia as a result of defects in insulin secretion, action or both. The ever-increasing incidence of diabetes mellitus worldwide among all age groups,, irrespective of gender, race, socioeconomic status  or ethnicity, constitutes a global public health burden., The global report on diabetes by the World Health Organization affirmed that, with a prevalence rate of 8.5%, the number of adults living with diabetes has increased by almost four times since 1980 – 422 million adults in 2015., Moreover, the International Diabetes Federation reported that, on an average, a person dies from diabetes every 6 s. In Nigeria, about 5 million people are still living with diabetes, while 1.56 million cases were recorded in 2015 with 105,091 deaths documented as of 2014. This dramatic rise is largely due to increase in Type-2 diabetes (T2D), which accounts for 95% of all reported cases.,
Notwithstanding the high incidence of T2D which has become a serious threat all over the world and the advancement made thus far on basic and clinical investigations into diabetes, (particularly in this region where healthcare resources are scarce), an ultimate therapy does not exist. The existing therapeutic strategies are limited and involve insulin and four main classes of oral antihyperglycaemic agents such as glucosidase inhibitors, biguanide, sulfonylurea and troglitazone. Each of these agents suffers from generally inadequate efficacy and a number of serious adverse effects such as liver problems, diarrhoea and lactic acidosis., This has necessitated the scientific search for relatively less toxic natural resources from medicinal plants with therapeutic values as substitute.
Medicinal plants have been shown with substantial scientific documentation to attest for their effectiveness, relatively non-toxic and efficacy in diabetes management.,, In Nigeria, numerous plants have been screened and found to lower glycaemia in chemical-induced diabetes. Out of 185 plant species from 75 families that have been investigated for antidiabetic potential in Africa, Vernonia amygdalina Delile (VA) is one of the most studied plants.,
VA is commonly known as bitter leaf in English due to its bitter taste. African familiar names for VA include chusar-doki (Hausa), onugbu (Igbo), etidot (Efik, Ijaw and Ibibio), ewuro (Yoruba), oriwo (Edo) and ndoleh (Cameroon).,,,, It belongs to the family Asteraceae (Compositae) and genus Vernonia. VA grows as a small shrub of above 3 to 5 m high and found abundantly in African countries such as Nigeria, Cameroon and Zimbabwe., Traditional medical practitioners use VA leaf extract to treat diabetes mellitus ,,, and as anti-malarial, anti-helminth, digestive tonic, appetiser and for the treatment of wounds. The stems of VA are used in some parts of Nigeria, as chew sticks, for the management of dental problems and oral hygiene.
Recently, it was reported that 96 out of the 115 plants reviewed to have been investigated in various in vivo animal models of diabetes within the last few decades, alloxan and/or streptozotocin (STZ), were the repeatedly used animal models of diabetes worldwide., These chemical agents at high doses are cytotoxic to the pancreatic β-cells, producing a form of insulin deficiency, akin to type-1 diabetes (T1D), with resultant hyperglycaemia., Regrettably, there is a paucity of information documented on the antidiabetic effects of VA leaf on T2D.
Although a series of researches by various investigators have affirmed to the efficacy of VA leaf in the management of diabetes, it is, however, unfortunate that greater parts of those studies were conducted using either normoglycaemia or animal models of T1D., For instance, induction of diabetes using either >100 mg/kg bodyweight (b.w.) of alloxan or >40 mg/kg of STZ results in insulin deficiency typical for T1D. These methods were used by previous investigators like Ojieh et al., in Delta State, Abdulazeez et al., in Zaria, Atangwho et al.,, in Calabar and Osinubi  in Lagos. In another study conducted in Owerri by Iwuji et al., normoglycaemic rabbits were used. Asante et al. used multiple i.p. daily doses of 40 mg/kg b.w. STZ, a condition that also results in T1D. All these observations are in conformity with the recent review reported by Chikezie et al. and Mohammed et al. that none of the studies on VA were conducted on T2D model. However, Okolie et al. used non-diabetic human subjects in Nsukka. In the study, they administered the leaves of VA by 'squeeze-wash-drink' and 'chew-raw' options to normoglycaemic humans and found that VA had significant antihyperglycaemic effects (post-prandial) at 30-min intervals for 2 h. The question of if the same outcome could be replicated or mimic in T2D is yet to be studied.
T2D is the major problem of diabetes mellitus in the world today and it deteriorates the quality of human life, yet greater part of antidiabetic research is on T1D., A classical procedure towards the development of reliable therapeutic strategy for the likely cure of a disease is the study of the pathophysiology of the disease in an apt animal model.,, This study, therefore, seeks to validate the antidiabetic (antihyperglycaemic, antihyperlipidaemic, pancreatic, nephroprotective and hepatoprotective) effects of VA leaf fractions (VALFs) in a unique rat model of T2D.
| Materials and Methods|| |
Fresh leaves of VA plant were harvested in the month of May, 2015, from a local farm in Samaru, Zaria, Kaduna state, Nigeria. Samples of the leaves were identified and authenticated at the herbarium unit of the Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria, and a voucher specimen number 1166 was deposited.
Wistar Albino rats weighing 150–200 g were used for the research. They were acquired from the animal house of the Department of Pharmacology, Ahmadu Bello University, Zaria, Nigeria. The rats were kept in well-aerated cages where bedding was replaced each day, at a room temperature of about 27°C and 12 h light/dark cycle. They were allowed to acclimatise for 2 weeks prior to experimentation. During this period, they were all provided with the same commercially available rat pellets and tap water ad libitum. The Institutional Animal Research Ethics Committee reviewed and certified the experimental protocol in conformity with guidelines that are in compliance with the National and International Laws and Guidelines for Care and Use of Laboratory Animals in Biomedical Research. Strict adherence to the Ethical Committee's directive was observed. Efforts were made to reduce suffering by the animals. The criterion of anaesthesia was the lack of body or limb movement in reaction to a standardised tail clamping stimulus.
Chemical and reagents
Strepozotocin (STZ) was procured from Adooq Bioscience, LLC, USA., and fructose (Kem Light Laboratories PVT Ltd, India), Mission Cholesterol Meter (ACON Lab. Inc., USA), Simas Margarine (PT Salim Ivomas Pratama Tbk, Indonesia), On-call plus glucometer, normal diet feed (NDF) (Grand Cereals Limited, Nigeria), liver enzyme kits (Biovision, Milpitas USA), rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Mercodia AB, Uppsala, Sweden), rat C-peptide ELISA kit (WKEA Med supplies, China) and all other reagents were of existing analytical grade and procured from appropriate manufacturing companies.
Extraction of the plant crude extracts
The leaves of VA were sorted out to obtain only fresh samples and washed with distilled water without squeezing to remove debris. Samples of the VA leaves were dried for 7 days in the shade at room temperature to invariable weight. Using a laboratory milling machine (Thomas-Wiley Laboratory mill Model 4, USA), the dried samples were crushed into fine particles. The powdered samples were collected together and extracted using cold maceration method described by Okoduwa et al. In brief, 1250 g of the powdered leaves of VA was soaked in 80% methanol solvent (3 × 4.5 L) for 72 h, and then filtered. After extraction, the methanol solvent was evaporated completely to concentrate it in vacuum using rotary evaporator (Senco Rotary Evaporator, Model RE 801) at 40°C in reduced pressure. The solvent-free methanol crude extract was kept at 4°C in a refrigerator until required.
Fractionation of the plant crude extracts
The solvent-free methanol crude extract (100 g) was suspended in 500 ml distilled water and then fractionated using organic solvents in an increasing order of polarity (n-hexane, chloroform, ethyl acetate, n- butanol and water). In brief, the crude extract was first soaked in n-hexane in a separating funnel, shaken and allowed to stand for phase separation into two fractions. The n-hexane portion was cautiously decanted after separation and extra n- hexane solvent was added and the procedure was repeated until no more colour change was observed in the extra n- hexane. The n- hexane-soluble portion was obtained and allowed to dry at room temperature to obtain the n-hexane fraction. The same process was repeated for chloroform, ethyl-acetate and n- butanol to obtain the appropriate solvent fractions. The resultant insoluble residue was dissolved in water to get an aqueous fraction. Each fraction obtained was concentrated using a rotary evaporator, the residual solvent in the extract was allowed to evaporate at room temperature to an invariable weight. Each of the fractions obtained was screened in vivo for antidiabetic activities using T2D rat model.
Phytochemical analysis of the fractions
The VA leaf extract was screened for the presence or absence of various secondary metabolites using standard phytochemical screening protocols described by Trease and Evans. The extract was tested for flavonoids, saponins, glycosides, carbohydrates and steroids.
Oral acute toxicity studies
The lethal dose (LD50) of the extracts in Wistar Albino rats was determined using method described by Lorke. The procedure for determining the lethal dose involves the increment of the concentration of each extract administered to the rats (per kg b.w.) in each group consisting of six rats per group. The concentrations used ranged from 10 mg/kg up to 5000 mg/kg. The animals were monitored constantly for changes in respiration, palpitations, behaviour, toxicity and other signs of morbidity and/or mortality starting at 24 h for up to 7 days.
Determination of minimum therapeutic dose
The hypoglycaemic activity of VA leaf extract was evaluated to determine the minimum dosage of extract to be administered. Diabetic rats were divided into several groups (V100, V150, V200., diabetic control [DC]) of three rats each. The animals of group DC served as untreated control whereas the other groups were administered with the methanol crude extract of VA at a single dose of 100, 150, 200 mg/kg, etc. Blood glucose was estimated at 0, 1, 2, 3 and 4 h calculated for each group using the following formula:
Where Go and Gt were the values of initial blood glucose (0 h) and blood glucose at time 1, 2, 3 and 4 h, respectively. The blood glucose levels at different time intervals of different groups were compared. The extract dose that lowered the glucose level by 25% at 4 h was considered the minimum hypoglycaemic dosage.
Induction of type-2 diabetes
Our previously described method was adopted from the study of Okoduwa et al. In brief, commercially available NDF was fortified with margarine at a ratio of 10 g NDF per gram of margarine. This was administered along with 20% fructose solution as drinking water to the rats ad libitum for 6 weeks to induce insulin resistance, after which they were fasted overnight and injected (i.p.) with STZ (dissolved in a citrate buffer pH 4.5) at a single low dose of 35 mg/kg b.w. In the first 24 h after STZ induction, the rats were provided with 5% glucose solution as drinking water.
Confirmation and verification of diabetes
Confirmation was done 3 days after STZ induction, with On-call plus glucometer using blood samples which were obtained via tail puncture of the rats. Verification of diabetes was done 10 days after STZ induction. Blood sample was obtained by single prick on the tail tip of each rat (allowing only one drop to come off) using a glucometer to ensure stable hyperglycaemia. Animals with fasting blood glucose (FBG) ≥300 mg/dl were verified as diabetic and incorporated in the study as diabetic animals.
Grouping of experimental animals
The rats were divided into eight groups of six rats each.
- Group NC: Normal control (NC): Normoglycaemic rats given distilled water
- Group DC: Diabetic rats treated with vehicle alone
- Group VAh: Diabetic rats treated with VA hexane fraction
- Group VAc: Diabetic rats treated with VA chloroform fraction
- Group VAe: Diabetic rats treated with VA ethyl acetate fraction
- Group VAb: Diabetic rats treated with VA n-butanol fraction
- Group VAa: Diabetic rats treated with VA aqueous fraction
- Group positive control (PC): Diabetic rats treated with metformin (500 mg/kg b.w.).
Treatment of animals with plant fractions
The plant fractions were administered at a dose of 250 mg/kg b.w. by oral intubation to the diabetic rats daily for 4 weeks. This dose used in the treatment was derived from the minimum oral therapeutic study described above.
Observation of animals after treatment
Daily feed/fluid intake and weekly body weight changes were measured during the entire experimental period.
Oral glucose tolerance test
Three hours subsequent to the last dose of treatment, oral glucose tolerance test (OGTT) was carried out to assess the treatment. Rats were orally dosed with a D-glucose solution (2.0 g/kg b.w.) and blood glucose levels were afterwards measured at 0 (just prior to oral glucose dosing), 30, 60, 90 and 120 min after the oral dosing of glucose using blood samples obtained from tail puncture of the rats.
At the end of the experimental phase, all animals were fasted overnight and thereafter euthanised by halothane anaesthesia. Blood was collected in plain bottles through cardiac puncture and placed immediately on ice for 3 h, and then centrifuged at 3000 rpm for 15 min to obtain serum which was separated and stored at −30°C until further investigations. The pancreatic tissues from each rat were collected, washed with normal saline, wiped, weighed and preserved in buffered formalin (10% formaldehyde in phosphate-buffered saline) for histopathological study.
The serum insulin levels were measured by an ELISA method using an ultrasensitive rat insulin ELISA kit (Mercodia AB, Uppsala, Sweden) and Rat C-peptide ELISA kit (WKEA Med supplies, China) in a multiplate ELISA reader (Biorad-680, BIORAD Ltd., Japan). The serum lipid profiles were determined using Mission Cholesterol Meter (ACON Lab. Inc., USA). Serum urea, uric acid and creatinine levels, as well as liver function enzymes, aspartate and alanine transaminases (AST and ALT) and alkaline phosphatase (ALP), were measured using an automated chemistry analyser (Labmax Plenno, Labtest Co. Ltd., Lagoa Santa, Brazil) with commercial assay kits. Homeostatic model assessment-insulin resistance (HOMA-IR and HOMA-β) scores were calculated using fasting serum insulin and FBG concentrations measured at the end of the experimental period according to the formula of Matthew et al. as given below 
Histopathological examination of pancreatic tissue
The formalin-preserved pancreatic tissues were treated according to a standard laboratory protocol for paraffin embedding. Sections were cut at a size of 4 mm. Then, slides were deparaffinised in p-xylene and rehydrated in changes of ethanol concentrations (100%, 80%, 70% and 50%) and rinsed with water. Slides were stained in haematoxylin for 5 min and rinsed with water and counterstained in eosin, mounted on slides with cover-slips and viewed under a Leica scanner at ×100 magnification.
All statistical analyses were conducted using the computer software, Statistical Package for the Social Sciences (SPSS Cary, NC, USA) version 20.0. The results were expressed as mean ± standard deviation. The data were analysed by one-way analysis of variance and post hoc test. Differences between fractions and animal groups were compared using Duncan Multiple Range Test. P < 0.05 was considered statistically significant.
| Results|| |
Percentage yield and phytochemical screening of the plant fractions
The results of the percentage yield and the colour characteristics of the various fractions of the plant leaf extracts are presented in [Table 1]. It was observed that chloroform and n- hexane fractions of the methanol crude extract had the highest percentage recovery yield of 28.09% and 18.06%, respectively, when compared to ethyl acetate, n- butanol and aqueous fractions. The VAe leaf had the least percentage recovery yield of 1.73%.
|Table 1: Percentage yield of Vernonia amygdalina leaf extract and fractions|
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The presence of tannin and saponin was detected in VAc and VAb but they were absent in VAh, VAe and VAa. Flavonoids were not detected in VAh. Anthraquinone was not detected in all the fractions. VAc and VAb had higher concentrations of the majority of phytochemicals tested [Table 2].
|Table 2: Qualitative phytochemicals of Vernonia amygdalina leaf fractions|
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Determination of minimum oral therapeutic dose of Vernonia amygdalina leaf extracts
The minimum therapeutic dose as defined by the amount of extract capable of reducing the glucose level of diabetic rats by at least 25% within 4 h of oral administration was examined. It was observed that a VA leaf extract dose of 200 mg/kg b.w. decreased the blood glucose of diabetic rats by 18.42%, after 4 h. However, a 250 mg/kg b.w. was able to decrease the blood glucose of diabetic rats by 21.08 and 33.23% after 2 and 4 h, respectively. The observed decrease in blood glucose of the treated diabetic rats was highly significant (P < 0.05) when compared with the untreated DC group. Hence, the dose of 250 mg/kg b.w. was considered as the minimum therapeutic dose for this study [Table 3].
|Table 3: Determination of minimum oral therapeutic dose of Vernonia amygdalina leaf extract in rat model of type 2 diabetes|
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Mean feed and fluid intake
The mean feed and fluid intake of each experimental animal on a daily basis throughout the experimental duration is depicted in [Figure 1] and [Figure 2], respectively. Few days after diabetic induction with STZ, a significant decrease in feed intake was observed among the STZ-induced groups when compared with the NC group which were not induced. A few days after the confirmation of diabetes, however, it was observed that the feed intake had significantly increased among the diabetic groups when compared with the NC group. From 2 weeks of treatment till the end of the experiment, it was observed that the feed intake among the diabetic rats treated with VALF was significantly (P < 0.05) lower when compared with the untreated diabetic group (DC), but higher than the NC group. There were no significant (P > 0.05) differences in the feed intake among the various fractions-treated groups [Figure 1]. The fluid intake was observed to be significantly (P < 0.05) increased among the STZ-induced groups when compared with the NC group. All through the duration of the experiment, it was observed that the fluid intake among the diabetic rats treated with VALF was significantly (P < 0.05) higher than that of the NC but lower than the DC group. The fluid intake was almost comparable between the PC group and the VAc fraction-treated group at 4 weeks of treatment when compared with the NC group [Figure 2].
|Figure 1: Effect of treatment with Vernonia amygdalina leaf fractions on feed intake throughout the experimental duration. Data are presented as mean of six animals per group. NC: Normal control; DC: Diabetic control; PC: Positive control; VAh: Vernonia amygdalina n-hexane fraction; VAc: Vernonia amygdalina chloroform fraction; VAe: Vernonia amygdalina ethyl-acetate fraction; VAb: Vernonia amygdalina n-butanol fraction; VAa: Vernonia amygdalina aqueous fraction|
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|Figure 2: Effect of treatment with Vernonia amygdalina leaf fractions on fluid intake throughout the experimental duration. Data are presented as mean of six animals per group. NC: Normal control; DC: Diabetic control; PC: Positive control; VAh: Vernonia amygdalina hexane fraction; VAc: Vernonia amygdalina chloroform fraction; VAe: Vernonia amygdalina ethyl-acetate fraction; VAb: Vernonia amygdalina n-butanol fraction; VAa: Vernonia amygdalina aqueous fraction|
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Effect of treatment on total body weight
Two weeks after STZ induction, a slight decrease in b.w. was observed among the diabetic group when compared with the NC group and their initial b.w. just before commencement of the experiment. A significant difference (P < 0.05) in growth rate and weight increase was observed among the diabetic rats treated with VALF and PC groups when compared with the NC group. A decrease in b.w. was observed in the DC group. With respect to all the diabetic-treated groups, the VAc fraction-treated group and the PC group had increased b.w. significantly (P < 0.05) in a comparable manner to the NC group [Figure 3].
|Figure 3: Effect of treatment with Vernonia amygdalina leaf fractions on body weight. Data are presented as mean of six animals per group. NC: Normal control; DC: Diabetic control; PC: Positive control; VAh: Vernonia amygdalina n-hexane fraction; VAc: Vernonia amygdalina chloroform fraction; VAe: Vernonia amygdalina ethyl-acetate fraction; VAb: Vernonia amygdalina n-butanol fraction; VAa: Vernonia amygdalina aqueous fraction|
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Effect of post-treatment on blood glucose levels
The blood glucose levels (mg/dl) of rats after 28 days of administration of leaf fractions of VA are presented in [Figure 4]. There was a significant (P < 0.05) increase in blood glucose after diabetic induction before the commencement of treatment. After commencement of treatment, gradual decrease in blood glucose was observed among the VALF-treated group and the PC group when compared with the NC and DC groups. The blood glucose level of the DC group was significantly (P < 0.05) high than those of all the treated groups. The percentage change in blood glucose was compared weekly in all the groups with respect to their initial blood glucose level recorded after confirmation of diabetes, before commencement of treatment. The results showed a significant difference (P < 0.05) in the percentage change in blood glucose level among and within the treated groups [Table 4]. From the results, between and 1 and 2 weeks of treatment, there were no significant (P < 0.05) percentage change in blood glucose among the VAc, VAe, VAb and the PC group, except for the VAh-treated group with a negative decrease in blood glucose at 1-week post-treatment. At 3- and 4-week post-treatment, it was observed that the VAc fraction and the PC groups recorded a comparable percentage change in blood glucose without significant (P > 0.05) difference [Table 4].
|Figure 4: Effect of weekly post-treatment with Vernonia amygdalina leaf fractions on blood glucose. Data are presented as mean of six animals per group. NC: Normal control; DC: Diabetic control; PC: Positive control; VAh: Vernonia amygdalina n-hexane fraction; VAc: Vernonia amygdalina chloroform fraction; VAe: Vernonia amygdalina ethyl-acetate fraction; VAb: Vernonia amygdalina n-butanol fraction; VAa: Vernonia amygdalina aqueous fraction|
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|Table 4: Percentage decrease in fasting blood glucose of diabetic rats, after treatment with Vernonia amygdalina leaf fractions|
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Effect of Vernonia amygdalina leaf fractions on oral glucose tolerance test and area under the curve
The blood glucose levels of rats in the NC, DC and diabetic groups treated for 28 days with VALF demonstrated a significant change after oral loading, with 2 g/kg b.w. glucose solution [Figure 5]. The rats in the diabetic groups (DC) had significant elevation in blood glucose level throughout the total measurement period (120 min) with respect to the NC group and VALF-treated groups. Moreover, the glucose level did not return to the initial values (at 0 min), even at the end of the 2-h period. Treatment of diabetic rats with metformin or VALF resulted in significant reduction in area under the curve (AUC) for glucose when compared to the DC group. Although the various VALFs had antihyperglycaemic effects, the VAc was observed to have the highest antihyperglycaemic effect with 49.01% while the VAh had the least antihyperglycaemic activity of 31.69% [Table 5].
|Figure 5: Changes in blood glucose concentration during oral glucose tolerance test in diabetic rats treated with Vernonia amygdalina leaf fractions. Data are presented as mean of six animals per group. NC: Normal control; DC: Diabetic control; PC: Positive control; VAh: Vernonia amygdalina n-hexane fraction; VAc: Vernonia amygdalina chloroform fraction; VAe: Vernonia amygdalina ethyl-acetate fraction; VAb: Vernonia amygdalina n-butanol fraction; VAa: Vernonia amygdalina aqueous fraction|
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|Table 5: Total area under the curve induced after glucose loading (2 g/kg b.w.) in diabetic rats pre-treated with Vernonia amygdalina leaf fractions (250 mg/kg/day) for 28 days|
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Effect of treatment on biochemical parameters
The levels of serum liver marker enzymes (AST, ALT and ALP) and biomarkers of kidney function (serum urea, uric acid and creatinine) were significantly elevated in the diabetic DC group as compared to the NC, PC and VALF-treated groups [Table 6]. When VALF was administered to diabetic rats for 28 days, the serum urea, uric acid and creatinine contents decreased and the VAc shows better significant (P < 0.05) modification. Similarly, oral administration of VALF for 28 days restored the enzyme levels significantly (P < 0.05) to near normal.
|Table 6: Liver and kidney function biomarkers in diabetic rats treated with Vernonia amygdalina leaf fractions (250 mg/kg/day) for 28 days|
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[Table 7] summarizes the effect of the various VALFs on serum lipid profile levels in diabetic rats. Our results indicated that serum total cholesterol (Tc), serum triacylglycerol (TG), serum low-density lipoprotein cholesterol (LDLc) and Tc/high-density lipoprotein cholesterol ratio (Tc/HDLc) were significantly increased (P < 0.05) in the diabetic DC group as compared to the NC group and the VALF-treated groups. However, HDLc levels were significantly lower in the DC group when compared to the NC group and the VALF-treated group [Table 7].
|Table 7: Effects of treatment with Vernonia amygdalina leaf fractions for 28 days on serum lipid profile of diabetic rats|
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The HOMA-IR, β-cell function (HOMA-β-cell), serum C-peptide, serum insulin and insulin sensitivity for the diabetic-treated and untreated rats are shown in [Table 8]. The results of our findings indicate that HOMA-IR index was significantly (P < 0.05) higher in the DC group when compared to the NC, PC and VALF-treated groups. There was no significant (P > 0.05) difference in the HOMA-IR index between the various VALFs. The insulin sensitivity and HOMA-β cell functioning index were significantly lower in the DC group when compared to the NC, PC and the VALF-treated groups [Table 8].
|Table 8: Quantification of insulin sensitivity, resistance and β-cell function in diabetic rats treated with Vernonia amygdalina leaf fractions (250 mg/kg b.w.) for 28 days|
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Histopathological examination of the pancreatic tissues
[Figure 6] shows the photomicrographs of pancreas of the rats in all the experimental groups. The photomicrographs revealed pancreatic islets with partial destruction of the β-cells in the DC when compared with the NC and PC groups with apparently healthy pancreatic β-cells. Treatment with 250 mg/kg b.w./day for 28 days improves the damaged pancreatic islet cells. A considerable number of healthy pancreatic β-cells were observed in the group treated with VAc. However, the islet cells were significantly smaller compared to the NC and PC groups [Figure 6].
|Figure 6: Photomicrographs of liver tissues of diabetic/non-diabetic rats, pre-treated with Vernonia amygdalina leaf fractions (250 mg/kg b.w.) for 28 days (H and E, stain, ×100). (a) NC: Normal control (non-diabetic group): Section shows normal histology of pancreas with high number of β-cells. (b) DC: Diabetic control: Islets show highly dispersed and morphologically deformed β-cells. (c) PC: Positive control: It has normal arrangement of islet cells and their β-cells are likened to that of the NC group. (d) VAh: Vernonia amygdalina n-hexane fraction; (e) VAc: Vernonia amygdalina chloroform fraction; (f) VAe: Vernonia amygdalina ethyl acetate fraction; (g) VAb: Vernonia amygdalina n-butanol fraction; (h) VAa: Vernonia amygdalina aqueous fraction. The VAh, VAc, VAe, VAb and VAa groups had better structural architecture compared to the DC group but morphologically distorted β-cells. The VAe and VAb show sign of regeneration but cellularity is less pronounced as compared to VAc|
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| Discussion|| |
Due to the high prevalence of type 2 diabetes (T2D) worldwide, extensive research is still being performed to develop new antidiabetic agents. This work evaluated the antidiabetic effect of VALF in fortified diet-fed (FDF) STZ-treated rat (FDF-STZ) model of T2D. Recently, we demonstrated that the FDF-STZ Albino Wistar rat model is a suitable model of T2D for screening antidiabetic plants. This FDF-STZ model exhibited frank hyperglycaemia without severe insulin deficiency. The normoglycaemia state presented by the normal diet-fed STZ-treated rat group explained the characteristic feature of the FDF-STZ experimental model of T2D used in this present study to evaluate the antidiabetic activities of VALF.
Water is used generally by most traditional healers to make herbal decoctions of VA, but sometimes alcohol is also used. In this study, a sequential method of extraction using different solvents in the order of increasing polarity (n-hexane, chloroform, ethyl-acetate, n- butanol and water) was employed to obtain VALF from the crude extract. This was done to extract both polar and non-polar components from the leaf extract. The method was chosen primarily since the nature, polarity and solubility of the antidiabetic bioactive constituents in the leaf of VA were unknown. In general, n- hexane is used to extract compounds of low polarity such as fatty acids, waxes, some alkaloids and terpenoids. Chloroform and ethyl-acetate are known to extract both medium polarities and some polar compounds such as flavonoids, tannins and some terpenoids. On the other hand, n- butanol and water are known to extract hydropolar compounds such as carbohydrates, amino acids and their derivatives. The presence of medicinal active constituents was revealed in various fractions of the leaf extract. This observation is in agreement with the reports presented by previous investigators.,
Although plants used in ethno medicine are considered to be relatively safe, there is still the need to formally examine the safety level of such plants when used in scientific investigation. This is especially imperative if the preparation procedure differs from that of the traditional method, such as using organic versus aqueous solvent. Second, the age of plant, geographical location and season and time of harvest affect the phytochemical compositions of the plant  and ultimately affect its toxicity. For instance, Asante et al. reported that flavonoids were present in the old and not the young leaves of VA ethanol extract. In this study, single oral acute toxicity study of VA leaf extracts at 5 g/kg b.w. did not show any apparent toxic symptom or mortality even 7 days after oral administration. This indicated that LD50 of VA was >5 g/kg b.w. Our observations were in agreement with that of Nwanjo  and Adiukwu et al., who reported that VA leaf extract had no clinical symptoms of toxicity effect or adverse toxicological potentials at doses of 500–2000 mg/kg/day for 14 successive days. Several other investigators have also reported the non-toxic nature of the leaf extract either to the kidney or the liver.,,
In this study, the effective minimum oral therapeutic dose of VA leaf extract was found to be 250 mg/kg b.w. The dose was observed to have reduced the hyperglycaemia of diabetic rats by 33.5% at 4 h post-oral administration. This finding was consistent with the report of the 25% reduction criteria suggested by Oguanobi et al. Different doses of VA leaf extracts ranging from 100 to 500 mg/kg b.w. have been reported by several investigators without clear-cut criteria on how the doses were derived.,,, The antihyperglycaemic efficacy of VA leaf extract at different dosages could be due to the different amounts of phytoconstituents present in the plant. This hypoglycaemic potency of VA leaf extract has been attributed to the basic phytochemical constituents of the plant which include saponins, tannins, flavonoids, phenol, glycosides and steroid glucosides.
One week after confirmation of diabetes, before the commencement of treatment, a significant (P < 0.05) increase in fluid intake and decrease in feed intake were noted. This may probably be due to the obligatory renal water loss combined with hyperosmolarity in diabetes which tends to deplete intracellular water, triggering the osmoreceptor of the thirst centre of the brain and polydipsia occurrence, which leads to water intake. At this stage, there is decreased appetite and hence catabolic effect prevails resulting in weight loss which was evident in the DC group in this study. This observation corroborates with what was reported by Ekam et al. and Goje et al. in their studies 3 days post-induction of diabetes with alloxan. They observed a decrease in b.w. in all the groups administered with alloxan though not significant (P > 0.05), but that was not the case with their NC group which did not show any reduction in b.w. This was also consistent with that of Russell et al. that there is decreased appetite in diabetes. The maintenance of near b.w. in diabetic rats administered VALF for 28 days may be related to the preservation of feed intake or the protection against catabolism through increased insulin availability to promote the anabolic processes. Increased muscle degradation could be the reason for loss of b.w. in diabetes. Insulin reverses b.w. loss through stimulating protein and lipid synthesis together with glycogen storage. The relatively improved b.w. gain in the VALF-treated groups as compared to the DC group suggests its antidiabetic efficacy for T2D. In particular, the observed improved ameliorating effect of VAc leaf fraction may be due to the presence of saponins. Saponins possess insulin-like properties, which stimulate glucose uptake enhancing Glut4 expression and contributing to storage of glucose as glycogen in adipocytes.
The data from this study show that VALFs produced a significant (P < 0.05) reduction in blood glucose level in T2D rat model; hexane (29.3%), chloroform (66.7%), ethyl acetate (36.2%), butanol (42.9%) and aqueous (39.3%). This observation agrees with those of Ojieh et al., who reported that different VALFs produced a fall in the FBG level in T1D rats; chloroform (65.85%), ethyl acetate (69.65%), benzene (45.59%) and butanol (37.31%). The highest reduction in blood glucose (71.7%) was observed in the PC group (treated with metformin), followed by treated rats that received 250 mg/kg VAc (66.7%). The least reduction rate (29.3%) was observed in the group treated with the VAh. However, the DC group manifested continuous increase in glucose levels from day 0 to day 28. This study confirms an earlier report by other investigators on the antihyperglycaemic effect of the leaf extract of VA in rats. STZ is a diabetogenic agent which induces diabetes by damaging the pancreatic β-cells of the islets leading to hyperglycaemia. These observations correlate with previous research reports,,, where antidiabetic medicinal plants significantly reduced the high blood glucose level in STZ-induced diabetic rats.
The DC rats in this study demonstrated significant increase in AUC of the glucose concentration after oral glucose loading. This observation from this study corroborates with a similar previous investigation by Elberry et al. This could be due to the reduction of glucose tissue utilisation and increased hepatic glucose production as a result of decreased insulin production. Treatment with VALF for 28 days resulted in a significant reduction in the AUC of diabetic rats. The implication is that VALF stimulates increased glucose utilisation and glucose tolerance through body tissues of the diabetic rats. Among all the VALFs administered, VAc was noticed to have the most significant effect in most of the parameters measured.
Hyperglycaemia in diabetic condition is linked with changes in the metabolism of glucose and lipid as well as modification in liver enzymic activities. One of the most common severe complications of diabetes is renal disease. Serum creatinine, uric acid and urea are used as biomarkers for prediction of renal dysfunction, because they increase significantly in diabetic conditions., In this study, high levels of these biomarkers were observed in the DC group as compared to the NC group. This finding is in agreement with that of previous investigators.,, The significant high levels of serum urea and creatinine is an indication of diminished ability of the kidney to filter these waste products from the blood and excrete them in urine. However, treatment with VALF for 28 days was able to significantly (P < 0.05) reduce the serum concentration of urea and creatinine in diabetic rats. Among the various fractions administered, it was observed that the VAc fraction was able to significantly reduce the serum levels of urea and creatinine in a similar manner more comparable with the normal drug metformin. Data from this study suggest that the VAc may have enhanced the ability of the kidneys to get rid of these waste products from the blood. It could also be explained that the VAc has the ability to confer protective effect on the kidney or might be able to directly improve the structural and functional integrities of cells of the blood, kidney and liver.
Hepatocyte injury caused impairment in the liver cell membrane permeability. Consequently, cytoplasmic enzymic activities such as transaminase enzymes (AST and ALT) escape into the circulatory system and their activities in serum increase. Alteration of membrane-bound ALP affects membrane permeability and produces derangement in the transport of metabolites. In this study, the serum levels of liver biomarkers (AST, ALT and ALP) were considerably increased in the DC group when compared to the NC group, indicating impaired liver function, which is evidently due to hepatocellular necrosis. Treatment of diabetic rat model with VALF caused a reduction in the activity of the enzymes (AST, ALT and ALP). This observation is consistent with an earlier report that elevated activities of serum transferases are common signs of liver diseases which are observed more often in diabetic condition., The increase in serum levels of these enzymes indicated that STZ-induced diabetes produced alteration in hepatic function, which consequently results in leakage of these hepatic enzymes from the liver cytosol into the blood stream. Ghosh and Suryawansi  explained that diabetic complications such as elevated gluconeogenesis and ketogenesis may be the result of increased transaminase activities. Hence, restoration of these biomarker enzymes towards normal level signifies decreased diabetic complications as observed in the VALF-treated groups. Among the various fractions tested, the VAc was the most active in reducing the activities of the liver enzymes.
Dyslipidaemia is a known complication associated with diabetes. The abnormally high concentration of serum TG, TC, LDLc and low HDLc observed in diabetic rats compared to control rats is in consonant with reports from previous studies,,,, indicating that an increase in glucose level on induction of diabetes results in an equivalent rise in blood lipids. The live is an insulin-dependent tissue that plays a significant role in glucose and lipid homeostasis and is severely affected in diabetes. Hyperlipidaemia is a complication of diabetes which is characterised by raised levels of cholesterol, phospholipids, triglycerides and other lipoproteins. High serum lipids in diabetes have been reported on one hand to be the effect of increased mobilisation of free fatty acids from peripheral fat depots due to inhibition of the hormone-sensitive lipase by insulin, and conversely, could be due to catecholamines, glucagon and other hormones which enhance lipolysis. The excess fatty acids produced are converted into cholesterol and phospholipids which, together with excess triacylglycerols produced at the same time in the liver, are released into the blood stream in the form of lipoproteins. Hence, the significant hyperlipidaemia observed in diabetic rats may be regarded as a consequence of uninhibited actions of lipolytic hormones in fat depots. Elevated levels of TC, prominently LDLc, is one of the most important coronary risk factors, which is the major cause of morbidity and deaths in diabetic patients. Treatment of diabetic rats with VALF caused alleviations of all the lipid profile parameters, indicating its hypolipidaemic potency. This effect may be ascribed to the presence of phytoconstituents present in the fractions which inhibited cholesterol and/or bile acid absorption.
The hypertriglyceridaemia observed among the T2D rats may be because of increased absorption and production of triglycerides in the form of chylomicrons subsequent to exogenous consumption of FDF or through increased endogenous production of TG-enriched hepatic very low density lipoprotein and decreased TG uptake in peripheral tissues. Hypercholesterolaemia may be attributed to the increased dietary cholesterol absorption from the small intestine following the intake of FDF in a diabetic condition. However, the levels of serum TG and TC were significantly reduced in the VAc-treated T2D rats.
Histopathological results showed normal pancreatic islet β-cells in the NC group, with the NC group showing evidence of deformation and cytoplasmic changes in the cells of the islets, specifically within the middle portion of the islets where there was an architectural deformation of β-cells. The NC and PC had large islet cells with high number of β-cells whereas the DC had morphologically deformed islet cells with disperse β-cells. The VA fraction treated groups had higher (but morphologically smaller) number of β-cells compared to DC, but fewer numbers compared to NC. The VALF-treated groups showed rejuvenation of the β-cells of the pancreas, and this observation was evidently supported by the significant reduction in hyperglycaemia. These changes were observed consistently in all the animals within the groups. However, VAc manifested the greatest pancreas-regenerative ability compared to the other groups. This study affirms the work done by previous investigators.,,,,
| Conclusion|| |
This study showed that the daily administration of hexane, chloroform, ethyl acetate, butanol and aqueous fractions of VA resulted in reduction in blood glucose levels in FDF-STZ-induced T2D rats. The results suggest that the FDF-STZ-treated rat model is a suitable model of T2D for pharmacological screening. The findings and data from this study give credence to the existing reports that VA is useful in the ethno-therapeutic management of diabetes mellitus.,,, Regardless of the fact that all the five fractions proved to be effective, the VAc was most effective, in that it had the satisfactory capacity to restore diabetic alterations to near normal. Although the intervention duration in our study was relatively short for just 28 days, it is promising that the effects of VAc could be enhanced in a long-term study. Work is in progress to isolate the bioactive component(s) present in the VAc for structural elucidation and clinical investigation since it is a promising drug candidate for the management of T2D.
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Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]