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Original Article
66 (
4
); 241-250
doi:
10.25259/IJPP_333_2022

Influence of concurrent administration of the methanol leaf extract of Leptadenia hastata (Pers) decne (Apocynaceae) with metformin on blood glucose in diabetic rats

, , ,
1Department of Pharmacology and Therapeutics, Usmanu Danfodiyo University, Sokoto, Nigeria.
2Department of Medical Biochemistry, College of Health Sciences, Usmanu Danfodiyo University,Sokoto, Nigeria.
3Department of Pharmacology and Toxicology, Usmanu Danfodiyo University, Sokoto, Nigeria.

*Corresponding author: Omobhude Fidelis Aluefua, Department of Pharmacology and Therapeutics, Usmanu Danfodiyo University, Sokoto, Nigeria. aluefuafo@gmail.com

Received: , Accepted: ,
© 2022 Published by Scientific Scholar on behalf of Indian Journal of Physiology and Pharmacology
Licence
This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

How to cite this article: Aluefua OF, Chika A, Ishaka A, Abubakar K. Influence of concurrent administration of the methanol leaf extract of Leptadenia hastata (Pers) decne (Apocynaceae) with metformin on blood glucose in diabetic rats. Indian J Physiol Pharmacol 2022;66:241-50.

Abstract

Objectives:

Leptadenia hastata (Pers) Decne (Apocynaceae) is a common medicinal plant used in northern Nigeria either singly or together with conventional drugs to treat diabetes. This study investigated the influence of concurrent administration of the methanol leaf extract of L. hastata with metformin in streptozotocin/ nicotinamide-induced diabetic rats.

Materials and Methods:

Possible synergistic activity between the extract and metformin was assessed using 3 models of synergy analysis (Loewe additivity, Bliss independent and highest single agent [HSA] models). Eleven groups of Wistar rats (eight animals per group) consisting of ten groups of diabetic rats and one normal control group were used in this study. Six groups were administered with either the extract or metformin at three different doses each (50, 150 and 500 mg/kg for the extract and 30, 100 and 300 mg/kg for metformin), while another three groups were co-administered with the extract and metformin at three different ratios each (50 mg/kg: 30 mg/ kg, 150 mg/kg: 100 mg/kg and 500 mg/kg: 300 mg/kg). An oral glucose tolerance test (OGTT) was conducted at baseline and on day 14.

Result:

The results revealed that the extract-metformin combination brought about a synergistic reduction in the total area under the OGTT curve (based on Loewe and HSA models) as well as a synergistic reduction in blood glucose (based on Loewe, Bliss and HSA models).

Conclusion:

The methanol leaf extract of L. hastata produced a synergistic antidiabetic activity in streptozotocin/ nicotinamide-induced diabetic rats when combined with metformin.

Keywords

Diabetes
Blood glucose
Leptadenia hastate
Metformin
Synergy

INTRODUCTION

The term diabetes mellitus describes a metabolic disorder of multiple aetiology characterised by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The effects of diabetes mellitus include long-term damage, dysfunction and failure of various organs.[1] The worldwide prevalence of diabetes mellitus and impaired glucose tolerance in adults has been on the rise in recent years. The rate at which the prevalence of diabetes mellitus changes in many countries and regions has been enhanced by rapid urbanisation and the increasing tendencies for a sedentary lifestyle. According to the world health organisation, about 108 million people were estimated to be diabetic in 1980. This estimate rose to 422 million individuals in 2014.[2] There has been increasing interest in drugs derived from higher plants especially those with medicinal value in recent times.[3] Awodele and Osuolale, reported that 48.7% of patients occasionally or always use herbal medication alongside their antidiabetic medications. Herbal medications have been discovered to cause interactions with conventional medications; these interactions can either be beneficial or harmful depending on the agent implicated. Some herbal medications were found to have either synergistic effects or antagonistic effects while others had no evidence of efficacy.[4]

About one-third of outpatients attending diabetic clinics in Nigeria use herbal products concurrently with conventional antidiabetic drugs.[4] Concurrent use of herbal products with conventional antidiabetic drugs, which is common in Nigeria, may influence the antidiabetic effect of the drugs.[5-7] Metformin is the most frequently prescribed antidiabetic drug in Nigeria.[8] Leptadenia hastata is a common medicinal plant used in Northern Nigeria either singly or together with conventional drugs to treat diabetes mellitus.[9,10] It is not known whether L. hastata will have an influence on the antidiabetic effect of metformin when the two are used concurrently. The study aimed to evaluate the influence of concurrent administration of the methanol leaf extract of L. hastata (pers) decne (Apocynaceae) with metformin on blood glucose in streptozotocin/nicotinamide-induced diabetic rats.

MATERIALS AND METHODS

Materials

Drugs and reagents

Metformin, streptozotocin and nicotinamide were ordered from Chem Cruz, USA. Glucose (kermel) and methanol were ordered from Sigma Aldrich USA. All reagents were of analytical grade.

Animals

Ethical clearance with clearance number PTAC/Lh/(Me) OT/35-21 was gotten from Usmanu Danfodiyo University Sokoto (UDUS) ethical clearance committee on Jan 18, 2021 before the commencement of the study. Male Wistar rats weighing 120–150 g (4–6 weeks) were obtained from IMRAT, UCH, Ibadan, Nigeria. The rats were maintained according to the minimum requirements of the International guidelines for the use of animals.[11] During the 2 weeks of acclimatisation, the animals were fed a commercial diet (Vital Feed Nig. Ltd) and allowed access to water ad libitum.[11] They fasted for 8 h before the commencement of the experiment.

Methods

Collection of plant material

Fresh leaves of L. hastata were collected from the Biological garden on the main Campus of Usmanu Danfodiyo University, Sokoto (UDUS). The plant sample was authenticated at the Herbarium of the Botany unit of the same Institution. A voucher number PCG/UDUS/ASCL/0002 was collected for the plant material specimen, which was deposited at the herbarium of the same unit.

Preparation of leaf extract

The leaves were allowed to air dry (to constant weight) at room temperature and pulverised into a fine powder. One hundred grams of the sample was separately extracted with 500 mL of 95% methanol for 24 h.[9] The extract was filtered and concentrated to semisolid form under reduced pressure using a rotary evaporator at 35°C. This was then finally dried in an aerated oven at 35°C. The percentage yield was calculated for the extract. The dried extract obtained was preserved at 4°C until needed for use.

Phytochemical analysis

Qualitative phytochemical screening

The extract was subjected to various chemical tests using standard procedures.[12-14]

Gas chromatography-mass spectroscopy (GCMS) analysis of plant extract

GCMS analysis was performed using Agilent Intuvo 9000 GC system coupled with detector system 5977B MSD with split/ splitless injector. A DB - 5 MS (5% phenyldimethylsiloxane) fused silica capillary column 30 m, 320 mm i. d., 0.25 mm of film thickness was used with Helium gas (99.999% purity) as carrier gas at flow rates of 1.2 mL/min. Inlet temperature was set at 300°C, MS Source at 230°C and MS Quad at 150°C. The oven temperature was programmed as follows: 50°C for 5 min, increased to 300°C at 10°C min−1 and hold for 20 min. The analysis was run for a total of 50 min. Data were acquired by GCMSD/Enhanced Mass Hunter Software and processed GCMSD Data analysis software incorporated with the 2017 Version of NIST Library. 1 mL of the sample extract was injected in split less mode into the GC system using Agilent Automated Liquid Sampler G4513A.

Experimental design of the in vivo study

Induction of diabetes

Out of 88 male Wistar rats, eight were randomly selected to serve as normal control. Diabetes mellitus was induced in the remaining animals using streptozotocin reconstituted in 0.1M citrate buffer (pH 4.5) and nicotinamide 230 mg/kg dissolved in normal saline. Using an insulin syringe, streptozotocin (65 mg/kg) was administered intraperitoneally 15 min after the intraperitoneal injection of nicotinamide.[15,16] Fasting blood glucose was determined after 72 h using a standardised glucometer (AccuChek® Active). Rats with blood glucose levels of 198–252 mg/dL (moderate diabetes) were selected for the subsequent study.[17]

Determination of the effect of co-administration of the extract and metformin (at selected ratios) on blood glucose of streptozotocin/nicotinamide-induced diabetic rats

Eighty-eight male Wistar rats (150–180 g each) comprising 80 diabetic rats and eight normal rats were employed. The 80 diabetic rats were randomised using decision analyst into ten groups of eight rats each. Two groups served as vehicle-administered normal and diabetic control, respectively, and received distilled water. Six groups were administered with either the extract or the metformin at three different doses each (50, 150 and 500 mg/kg for the extract and 30, 100 and 300 mg/kg for metformin), while the remaining three groups were co-administered with the extract and metformin at three different ratios each (50 mg/kg: 30 mg/kg, 150 mg/kg: 100 mg/kg and 500 mg/kg: 300 mg/kg). After an 8-h fast, an oral glucose tolerance test (OGTT) was conducted (as described below) at baseline and on day 14, a day before the sacrifice of the animals at the end of the experiment. The duration of this stage of the study was 15 days.

OGTT

Glucose (2 g/kg) was given orally 30 min after the administration of the respective treatments. Blood samples were collected (by tail tipping) at 0 min (just before glucose loading), 30 min, 60 min, 90 min and 120 min after glucose administration for glucose determination using a standardised glucometer (Accu Check®). Gentle digital pressure was applied to the tail tip for 30–45 s with a clean gauze pad to stop any bleeding before the animals were placed back in their cages.

Statistical analysis

Data were analysed using GraphPad Prism Version 7.0. The data were summarised as mean ± Standard Deviation. Analysis of Variance (ANOVA) with post hoc tests (Tukey Kramer) was used to compare the difference between different groups. P < 0.05 was considered to be significant. Synergy was analysed employing Combenefit software (Version 2.02), using Loewe additivity, Bliss independent and highest single agent (HSA) models. The sum of synergy and antagonism was calculated by the use of Combenefit software and a single value was given. Value >0 was synergism, if <0, antagonism. 0 indicated additivity.

RESULTS

Percentage yield of the methanol leaf extract of L. hastata

The percentage yield of the methanol leaf extract of L. hastata was 9.3% [Table 1].

Table 1: Weight and percentage yield of methanol leaf extract of L. hastata.
Plant Weight of extract (g) % yield
L. hastata 186 9.3

L. hastata: Leptadenia hastata

Phytochemical analysis of the methanol leaf extract of L. hastata

Phytochemical analysis of the methanol leaf extract of L. hastata revealed the presence of cardiac glycosides, steroids/triterpenoids, phenol, flavonoids, carbohydrates, tannins, saponins and alkaloids [Table 2].

Table 2: Phytochemical analysis of the methanol leaf extract of Leptadenia hastata.
S. No. Phytochemical constituents Results
1. Cardiac Glycosides +
2. Anthraquinones ND
3. Steroids/Triterpenoids +
4. Phenols +
5. Flavonoids +
6. Carbohydrates +
7. Tannins +
8. Saponins +
9. Alkaloids +

+: Present, ND: Not detected

GCMS analysis of the methanol leaf extract of L. hastata

GCMS analysis of the methanol leaf extract of L. hastata revealed the presence of active principles with their retention time, molecular formula, molecular weight and peak area (%) as shown in [Table 3 and Figure 1].

Table 3: Gas chromatography-mass spectroscopy analysis of the methanol leaf extract of Leptadenia hastata,
S. No. Retention time (min) Name of the compound Molecular formula Molecular weight Peak Area %
1. 5.911 Docosanoic acid C22H44O2 340.6 0.24
2. 7.891 Phosphoric acid H3PO4 97.994 0.19
3. 8.023 2 (1H)-Pyrimidinone C4H4N2O 96.09 0.36
4 8.635 2-butenedioic acid C4H4O4 174.15 0.24
5 13.745 Silane SiH4 32.12 0.28
6. 20.205 Naphthalene C10H4 128.17 3.32
7, 20.663 2-Dodecene C12H24 168.32 1.31
8. 22.476 Cyclohexane, octyl- C14H26 196.37 3.00
9. 28 0.336 2-Tetradecene, (E)- C14H28 196.37 7.61
10. 28.622 Tridecane C13H28 184.37 1.93
11. 35.185 Cetene C16H33 225.43 14.93
12. 35.431 Hexadecane C16H34 226.41 2.74
13. 37.634 Pentacos-1-ene C25H50 350.7 10.57
14. 41.353 1-Octadecene C18H36 252.49 14.85
15. 43.700 Thiazole C3H3NS 85.13 3.45
16. 43.917 Benzenepropanoic acid C9H1202 152.19 9.28
17. 46.973 1-Hexacosene C26H52 364.7 5.40
Mass spectra of identified compound from methanol leaf extract of L. hastata.
Figure 1:
Mass spectra of identified compound from methanol leaf extract of L. hastata.

Induction of diabetes

Following intraperitoneal administration of streptozotocin and nicotinamide, the majority (75.2%) of the rats induced exhibited an elevation in the level of their fasting blood glucose (compared with normal controls), within the acceptable range of 198–252 mg/dl.

Effect of co-administration of the methanol leaf extract of L. hastata and metformin on OGTT curve in streptozotocin/ nicotinamide-induced diabetic rats at baseline

At baseline, all the diabetic groups exhibited a significant increase in the total area under the OGTT curve (P < 0.05), compared with the vehicle-treated normal control group. Administration of the extract or metformin (at three increasing doses each), either singly or in combination, brought about a significant reduction in the total area under the OGTT curve (P < 0.05) when compared with the vehicle-treated diabetic rats. Other inter-group comparisons were not statistically significant [P > 0.05; Figure 2].

Effects of co-administration of the methanol leaf extract of Leptadenia hastata and metformin on the total area under the glucose tolerance curve in streptozotocin/nicotinamide-induced diabetic rats at baseline. Data are presented as mean ± SEM; bsignifies P < 0.05 when compared with athe vehicle-treated diabetic group, n= 8. One-way ANOVA with Tukey Kramer multiple comparison post hoc tests were used to arrive at the P value.
Figure 2:
Effects of co-administration of the methanol leaf extract of Leptadenia hastata and metformin on the total area under the glucose tolerance curve in streptozotocin/nicotinamide-induced diabetic rats at baseline. Data are presented as mean ± SEM; bsignifies P < 0.05 when compared with athe vehicle-treated diabetic group, n= 8. One-way ANOVA with Tukey Kramer multiple comparison post hoc tests were used to arrive at the P value.

Effect of co-administration of the methanol leaf extract of L. hastata and metformin on OGTT curve in streptozotocin/nicotinamide-induced diabetic rats at day 14 of treatment

Administration of the extract or metformin (at three increasing doses each) either singly or in combination, brought about a significant reduction in the total area under the OGTT curve (P < 0.05) compared with the vehicle-treated diabetic rats [Figure 3]. Other inter-group comparisons were not statistically significant [P > 0.05; Figure 3].

Effects of co-administration of the methanol leaf extract of Leptadenia hastata and metformin on the total area under the glucose tolerance curve in streptozotocin/nicotinamide-induced diabetic rats on day 14 of treatment. Data are presented as mean ± SEM; bSignifies P < 0.05 when compared with athe vehicle-treated diabetic group, n = 8. One-way ANOVA with Tukey Kramer multiple comparison post hoc tests were used to arrive at the P value. OGTT: Oral Glucose Tolerance Test.
Figure 3:
Effects of co-administration of the methanol leaf extract of Leptadenia hastata and metformin on the total area under the glucose tolerance curve in streptozotocin/nicotinamide-induced diabetic rats on day 14 of treatment. Data are presented as mean ± SEM; bSignifies P < 0.05 when compared with athe vehicle-treated diabetic group, n = 8. One-way ANOVA with Tukey Kramer multiple comparison post hoc tests were used to arrive at the P value. OGTT: Oral Glucose Tolerance Test.

Dose-response curves of the change in total area under the glucose tolerance test curve following 14 days of treatment with either the methanol leaf extract of L. hastata or metformin in streptozotocin/nicotinamide-induced diabetic rats

The dose-response curves of the methanol leaf extract of L. hastata and metformin showed median effective doses (ED50s) of 8.078 mg/kg and 29.56 mg/kg, respectively [Figure 4].

Dose-response curves of the change in total area under the glucose tolerance test curve following 14 days of treatment with either the methanol leaf extract of Leptadenia hastata or Metformin in streptozotocin/nicotinamide-induced diabetic rats. ED50 = Effective dose50.
Figure 4:
Dose-response curves of the change in total area under the glucose tolerance test curve following 14 days of treatment with either the methanol leaf extract of Leptadenia hastata or Metformin in streptozotocin/nicotinamide-induced diabetic rats. ED50 = Effective dose50.

HSA synergy and antagonism metrics of the change in total area under the glucose tolerance test curve following 14 days of co-administration of the methanol leaf extract of L. hastata and metformin in streptozotocin/ nicotinamide -induced diabetic rats

With the HSA model, the value of the sum of synergy and antagonism of the change in total area under the glucose tolerance test curve for increasing doses of methanol leaf extract of L. hastata co-administered with metformin to streptozotocin/nicotinamide-induced diabetic rats for 14 days was 1.189958. The maximum synergy and antagonism values were 9.366548 and −3.31782, respectively [Figure 5].

HSA synergy and antagonism Matrix of the change in total area under the glucose tolerance test curve following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced diabetic rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.
Figure 5:
HSA synergy and antagonism Matrix of the change in total area under the glucose tolerance test curve following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced diabetic rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.

Loewe’s synergy and antagonism metrics of the change in total area under the glucose tolerance test curve following 14 days of co-administration of the methanol leaf extract of L. hastata and metformin in streptozotocin/ nicotinamide-induced diabetic rats

With the Loewe’s model, the value of the sum of synergy and antagonism of the change in total area under the glucose tolerance test curve for increasing doses of methanol leaf extract of L. hastata co-administered with metformin to streptozotocin/nicotinamide-induced diabetic rats for 14 days was 0.659006. The maximum synergy and antagonism values were 3.3329 and −3.31782, respectively [Figure 6].

Loewe’s synergy and antagonism Matrix of the change in total area under the glucose tolerance test curve following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced diabetic rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.
Figure 6:
Loewe’s synergy and antagonism Matrix of the change in total area under the glucose tolerance test curve following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced diabetic rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.

Bliss synergy and antagonism metrics of the change in total area under the glucose tolerance test curve following 14 days of co-administration of the methanol leaf extract of L. hastata and metformin in streptozotocin/ nicotinamide-induced diabetic rats

With the bliss model, the value of the sum of synergy and antagonism of the change in total area under the glucose tolerance test curve for increasing doses of the methanol leaf extract of L. hastata co-administered with metformin to streptozotocin/nicotinamide-induced diabetic rats for 14 days was −0.54078. The maximum synergy and antagonism values were 2.505578 and −3.31782, respectively [Figure 7].

Bliss synergy and antagonism metrics of the change in total area under the glucose tolerance test curve following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced Diabetic Rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.
Figure 7:
Bliss synergy and antagonism metrics of the change in total area under the glucose tolerance test curve following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced Diabetic Rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.

Loewe synergy and antagonism metrics of the mean fasting blood glucose following 14 days of co-administration of the methanol leaf extract of L. hastata and metformin in streptozotocin/nicotinamide-induced diabetic rats

With Loewe’s model, the value of the sum of synergy and antagonism of the mean fasting blood glucose for increasing doses of the methanol leaf extract of L. hastata co-administered with metformin to streptozotocin/ nicotinamide-induced diabetic rats for 14 days was 3.309851. The maximum synergy and antagonism values were 12.49756 and −12.4975, respectively [Figure 8].

Loewe’s synergy and antagonism metrics of the mean fasting blood glucose following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced Diabetic Rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.
Figure 8:
Loewe’s synergy and antagonism metrics of the mean fasting blood glucose following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced Diabetic Rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.

HSA synergy and antagonism metrics of the mean fasting blood glucose following 14 days of co-administration of the methanol leaf extract of L. hastata and metformin in streptozotocin/nicotinamide-induced diabetic rats

With the HSA model, the value of the sum of synergy and antagonism of the mean fasting blood glucose for increasing doses of the methanol leaf extract of L. hastata co-administered with metformin to streptozotocin/nicotinamide-induced diabetic rats for 14 days was 3.660699. The maximum synergy and antagonism values were 12.49763 and −12.4975, respectively [Figure 9].

HSA synergy and antagonism metrics of the mean fasting blood glucose following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced diabetic rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.
Figure 9:
HSA synergy and antagonism metrics of the mean fasting blood glucose following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced diabetic rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.

Bliss synergy and antagonism metrics of the mean fasting blood glucose following 14 days of co-administration of the methanol leaf extract of L. hastata and metformin in streptozotocin/nicotinamide-induced diabetic rats

With the Bliss model, the value of the sum of synergy and antagonism of the mean fasting blood glucose for increasing doses of the methanol leaf extract of L. hastata co-administered with metformin to streptozotocin/ nicotinamide-induced Diabetic rats for 14 days was 0.06061. The maximum synergy and antagonism values were 12.49763 and −12.4975, respectively [Figure 10].

Bliss synergy and antagonism metrics of the mean fasting blood glucose following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced diabetic rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.
Figure 10:
Bliss synergy and antagonism metrics of the mean fasting blood glucose following 14 days of concurrent treatment with the methanol leaf extract of Leptadenia hastata (LH) and metformin in streptozotocin/nicotinamide-induced diabetic rats. Value of the sum of synergy and antagonism (as shown in the square brackets) > or < 0 indicated synergism or antagonism, respectively. NaN: No data inserted.

DISCUSSION

Plant chemical constituents constitute an indispensable part of medicinal plants and are responsible for their numerous bioactivities. The preliminary phytochemical screening of the methanol leaf extract of L. hastata revealed the presence of cardiac glycosides, steroids/triterpenoids, phenols, flavonoids, carbohydrates, tannins, saponins and alkaloids. The presence of some of these phytoconstituents in the methanol leaf extract of L. hastata has been previously documented.[18-20]

In this study, type 2 diabetes mellitus was induced in Wistar rats using streptozotocin in combination with nicotinamide. Streptozotocin induces diabetes mellitus in experimental animals by the process of methylation, generation of free radicals and production of nitric oxide which brings about the destruction of pancreatic islet cells.[21] It is preferable to alloxan in the induction of diabetes mellitus because of its greater selectivity towards b-cells, reduced mortality rates as well as more prolonged or irreversible induction.[22] Nicotinamide has been used together with streptozotocin to induce diabetes mellitus within 3–7 days after a single intraperitoneal injection, especially in the Wistar rat model. Several studies have shown that nicotinamide can enhance the energy status in ischaemic tissues, and exhibits antioxidant properties as well as slow apoptosis.[23-25]

Streptozotocin/nicotinamide-induced diabetic murine model is one of the commonest models of type 2 diabetes mellitus.[22] The antihyperglycaemic effect of the plant as observed in this study agrees with the findings of several studies using different leaf extracts of the plant in both alloxan- and streptozotocin-induced murine models.[9,19,26,27] Since streptozotocin-induced diabetes mellitus is associated with resistance to insulin action, the mechanism of L. hastata methanol leaf extract may be through the improvement of insulin sensitivity.[22] Some of the phytochemicals found in the extract, namely, cardiac glycosides, triterpenoids, flavonoids, tannins, saponins and alkaloids have been reported to possess antidiabetic activity.[28-31] Therefore, the antidiabetic activity of the extract may be due to the presence of any of these phytochemicals either singly or in combination.

The mass spectra of identified compounds from L. Hastata are presented in [Table 3 and Figure 1]. Some of the identified phytocompounds with possible antidiabetic potentials include Docosanoic acid,[32] 2(1H)-Pyrimidinone,[33] 2-Tetradecene,[34] Hexadecane,[35] 1-Octadecene,[36] Thiazole,[37] Benzenepropanoic acid,[38] and 1-Hexacosene.[32]

At baseline, single-dose treatment with either the extract or metformin (at three escalating doses each), alone or in combination resulted in a significant reduction in the total area under the OGTT curve compared to the vehicle-treated diabetic rats. This suggests that the methanol leaf extract of L. hastata possesses some antihyperglycaemic properties in diabetic rats even after a single dose treatment. Metformin is relevant in this study as a standard drug for type 2 diabetic models because it is the recommended first-line agent in the management of type 2 diabetes mellitus with proven clinical efficacy and lower incidence of adverse effect.[39] Furthermore, it is the most frequently prescribed oral antidiabetic agent globally. Metformin is ineffective in pancreatectomised animals and type 1 diabetes.[39] The dose-response curves of the methanol leaf extract of L. hastata and metformin showed median effective doses (ED50s) of 8.078 mg/kg and 29.56 mg/kg, respectively. Doses that give around 50% of the maximum possible drug effect (the effective dose 50 [ED50]) often prove to be sufficient. The ED50 helps clinicians decide the initial drug dose in patients as it is an essential clinical starting point for practitioners when prescribing medications, as adjustments are made to balance efficacy and toxicity.[40,41]

We believe that this study is the first of its kind to find the synergistic activity in reducing the level of fasting blood glucose (using all the three models of synergism assessment) of L. hastata methanol leaf extract when co-administered with metformin, as evidenced by the observed values of the sum of synergy and antagonism being greater than 0 as well as a synergistic activity between the extract and metformin in improving OGTT (using Loewe and HSA models, but not Bliss model).

The bliss-independent model assumes independence of the mechanism of action of the individual drugs in the combination. It also does not take into consideration the dose-response curve of the effect of the combination. It is only accurate if the mechanism of action is independent which has not been established in this study. The findings from the Loewe model are standard and accurate and it takes into consideration the dose-response curve of the combination.[42]

We also believe that this is the first study to evaluate the potential synergistic activity between herbal extracts and antidiabetic agents, using the internationally accepted models: Loewe additivity and Bliss independent models. However, various studies have reported synergistic activity between metformin and extracts of different plants using other models such as the HSA model.[43-45] Synergism was assessed with three increasing doses of the extract-metformin combination. Experiments in which any drug is tested at less than three dose levels are therefore not likely to be sufficient to demonstrate synergy.[42]

The antihyperglycaemic effects observed by L. hastata extracts could be attributed to one or more possible mechanisms which include the activation of b-cells and subsequent release of insulin and stimulation of the insulin receptors. It may also be due to the facilitation of blood glucose transport to the peripheral tissue.[9,26] Other possible mechanisms include the inhibition of the activity of alpha-glucosidase, inhibition of glycogenolysis or stimulation of glycogenesis in the liver and skeletal muscle of diabetic rats.[19] However, the exact mechanism of the antihyperglycaemic activity of the extract administered singly as well as the mechanism of the observed synergistic antihyperglycaemic activity between the extract and metformin has not been investigated in this study.

One of the limitations of this study is that the possibility of pharmacokinetic interaction between the extract and metformin has not been evaluated by assaying the concentration of metformin in the blood.

CONCLUSION

It could be concluded that the methanol leaf extract of L. hastata when coadministered with metformin act synergistically in producing antidiabetic activity in streptozotocin/nicotinamide-induced diabetic rats.

Declaration of patient consent

Patient’s consent not required as there are no patients in this study.

Conflicts of interest

There are no conflicts of interest.

Financial support and sponsorship

Nil.

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