Anti-diabetic activity of aqueous extract of Trichilia prieureana A. Juss leaves in fructose-fed streptozotocin-induced diabetic male Wistar rats

Abstract

Background and aim

Ethanolic extract of Trichilia prieureana leaves have been reported to exhibit anti-hyperglycemic activities without detailed information on the anti-diabetic activities. This study investigated the anti-diabetic activity of aqueous extract of T. prieureana leaves (AETPL) in type 2 diabetic (T2DM) male rats.

Experimental procedure

T2DM rats (induced with 10 % fructose solution ad libitum for 2 weeks and streptozotocin [STZ]; 40 mg/kg body weight {BW}) in groups B, C, D, E, and F were also administered distilled water (DW), metformin (100 mg/kg BW), 11.2, 22.3, and 44.6 mg/kg BW of AETPL, respectively, whilst non-diabetic rats in Group A received DW only (Sham Control, SC) for 14 days. The T2DM-related parameters were then evaluated.

Results

The fructose-fed streptozotocin-(FSTZ) treatment related significant (p < 0.05) increases in FBS, HbA1c, fructosamine, HOMA-IR, G6P, GP, TC, TG, LDL-C, urea, bilirubin, hepatic and pancreatic MDA levels; decreases in BW, serum insulin, creatinine, albumin, HOMA-β, glycogen, G6PD, HK, HDL-C, hepatic and pancreatic SOD, GPX, RG, catalase, Hb, PCV, MCH, MCHC, MCV, RBC, WBC, differentials and the destruction of the pancreatic β-cells, hepatocyte degeneration, and central hepatic vein congestion were reversed by AETPL, to values that compared well with SC in most cases. In the IpGGT model, the intraperitoneally administered AETPL reduced the blood glucose and elevated the plasma insulin levels. The AETPL at 44.6 mg/kg BW exhibited the most pronounced effects.

Conclusion

AETPL (44.6 mg/kg BW) restored T2DM-glycemic control and associated biochemical changes via up-regulation of insulin, carbohydrate metabolizing enzymes and restoration of pancreatic and hepatic histoarchitecture.

Graphical abstract

List of abbreviations

ADP

Adenosine Diphosphate

AETPL

Aqueous Extract of Trichilia prieureana Leaves

AI

Atherogenic Index

ALP

Alkaline Phosphatase

ALT

Alanine Aminotransferase

AST

Aspartate Aminotransferase

ATP

Adenosine Triphosphate

BW

Body Weight

CAI

Coronary Artery Index

cAMP

Cyclic Adenine Mononucleotide Phosphate

DM

Diabetes Mellitus

DNA

Deoxyribonucleic Acid

FBS

Fasting Blood Sugar

FRIN

Forestry Research Institute of Nigeria

FSTZ

Fructose-fed Streptozotocin

ELISA

Enzyme-linked Immunosorbent Assay

G6P

Glucose-6-Phosphatase

G6PD

Glucose-6-Phosphate Dehydrogenase

GP

Glycogen Phosphorylase

GPX

Glutathione Peroxidase

Hb

Haemoglobin

HbA1c

Glycated Haemoglobin

HDL-C

High Density Lipoprotein-Cholesterol

HK

Hexokinase

HOMA-β

Homeostasis Model Assessment of β-cell Score

HOMA-IR

Homeostasis Model Assessment of Insulin Resistance

IpGTT

Intraperitoneal Glucose Tolerance Test

LDL-C

Low Density Lipoprotein-Cholesterol

MCH

Mean Corpuscular Haemoglobin

MCHC

Mean Corpuscular Haemoglobin Concentration

MCV

Mean Corpuscular Volume

MDA

Malondialdehyde

NAD⁺

Nicotinamide Adenine Dinucleotide (Oxidized)

NADH

Nicotinamide Adenine Dinucleotide (Reduced)

NADPH

Nicotinamide Adenine Dinucleotide Phosphate (Reduced)

NC

Negative Control

NO

Nitric Oxide

PC

Positive Control

PCV

Packed Cell Volume

RBC

Red Blood Cell

RG

Reduced Glutathione

RNA

Ribonucleic Acid

SC

Sham Control

SOD

Superoxide Dismutase

STZ

Streptozotocin

T2DM

Type 2 Diabetes Mellitus

TC

Total Cholesterol

TG

Triglycerides

UERC

University of Ilorin Ethical Review Committee

VLDL-C

Very Low Density Lipoprotein-Cholesterol

WBC

White Blood Cells

1. Introduction

Diabetes Mellitus (DM) is a major public health problem caused by alterations in carbohydrate, protein, and lipid metabolism, resulting in elevated blood glucose levels due to the inability of the pancreatic cells to secrete insulin and/or the inability of the body to utilize the secreted insulin.¹ In 2021, a total of 537 million (representing 10.5 % of the global adult population) people worldwide were estimated to be living with diabetes, whereas more than 24 million and 7.7 million people in Africa and Nigeria, respectively, are living with diabetes.² According to the International Diabetes Federation² (IDF), 783 million people worldwide, 55 million people in Africa, and 6.6 million people in Nigeria will have diabetes, by the year 2045. Diabetes mellitus present as two distinct primary types: Type 1 Diabetes Mellitus (Insulin-dependent diabetes with 5–10 % prevalence) and Type 2 Diabetes Mellitus (T2DM, Non-insulin Dependent Diabetes Mellitus with a prevalence of 90–95 %).

Type 2 Diabetes Mellitus (T2DM), characterised by chronic hyperglycemia and dyslipidemia which has been linked to insulin resistance and partial pancreatic β-cell dysfunction, is the most common type of DM, accounting for more than 90 % of diabetic morbidity and mortality.² The production of free radicals via diverse pathways, triggered by auto-oxidation of the excess glucose in the bloodstream, may account for the chronic hyperglycemia in T2DM and T2DM-associated macro-and microvascular complications.³

A number of oral hypoglycemic drugs (metformin, glibenclamide, and thiazolidinedione) currently in use for the management of T2DM have not been entirely efficient and are associated with side effects that include nausea, hypoglycemia, cardiovascular diseases, and body weight gain.⁴ All of these have thus, led to the need to screen for alternative and complementary therapies in the genus of Trichilia species, Trichilia prieureana, in this instance.

Trichilia prieureana A. Juss. (family: Meliaceae), also known as monkey apple (English), and in Nigeria as Urere (Yoruba) and Atantan (Igbos) is widespread from Senegal to South-western Ethiopia, Nigeria, Uganda, Tanzania, Angola (Cabinda), and Zambia.⁵ It is a forest tree, usually in the understorey, that grows between 10 and 46 feet high, but sometimes extends up to 70 feet. The bole is conspicuously fluted, with a grey-brown flaking bark. The flowers, fruits and seeds with red arils are greenish white, pink and black in colour, respectively. Traditionally, the bark is claimed to be used to treat venomous stings and bites, wounds, pain, rheumatism, fever, poisoning, ascites, sexual inadequacies, cough and constipation.⁶ In addition, the bark and roots have been purportedly used for treating piles, stomach troubles, venereal diseases and haemorrhoids.⁶ Furthermore, the fruits have been used as foods whereas the leaves have been used in folk medicine for the treatment of ascariasis, bronchitis, edema, leprosy and gonorrhea.⁶ It is claimed that the leaves of T. prieureana are being used to treat diabetes.⁷

Generally, information in the open scientific literature on the studies of T. prieureana leaves is scanty. For instance, Kuglerova et al.⁸ and Arotupin et al.⁹ have only reported on the antimicrobial activity of the T. prieureana. Although, Kangbeto et al.,⁷ reported the antioxidant and anti-diabetic effects of ethanolic extract of T. prieureana leaves at 200 and 400 mg/kg BW, per os, in short (3 h, oral glucose tolerance test) and long (8 weeks) term studies in high-fat diet-induced type 2 diabetic rats, focusing specifically on very few parameters like blood glucose, insulin, circulating lipids (TG, TC, LDL-C), antioxidant enzymes (SOD, glutathione reductase), liver and kidney function indices (ALP, ALT, AST, creatinine), the absence of a comprehensive profile on the anti-diabetic activity of aqueous extract of T. prieureana leaves in T2DM rat model necessitated the current study. In addition, this study also focused on T2DM because DM prevalence rates are driven almost entirely by T2DM, which accounted for more than 96 % of DM cases worldwide in 2021.¹⁰ It was against this backdrop that the authors considered it worthwhile to provide more comprehensive information on the anti-diabetic activity of T. prieureana leaves in a T2DM rat model. Hence, the current study evaluated the anti-diabetic activity and mechanism of anti-T2DM action of aqueous extract of T. prieureana leaves in fructose-fed streptozotocin-induced T2DM male Wistar rats.

2. Materials and methods

2.1. Chemicals

Streptozotocin (cat. No.: CAS: 18883-66-4), fructose, 5,5-dithio-bis-2-nitrobenzoic acid, trichloroacetic acid, thiobarbituric acid, acarbose, α-amylase and α-glucosidase assay kits were products of Sigma-Aldrich, Steinheim, Germany. Glycogen phosphorylase (GP), hexokinase (HK), glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PD), insulin (cat. No.: RAB0237-1 KT), rat glycosylated haemoglobin Enzyme Linked Immunosorbent Assay (ELISA) (HbA1C, cat. No.: ELISA: LOT 09652), total cholesterol (TC, cat. No.: GTIN05055273207324), triglycerides (TG, cat. No.: GTIN05055273206593), high density lipoprotein-cholesterol (HDL-C, cat. No.: GTIN05055273206584), low density lipoprotein-cholesterol (LDL-C), catalase, superoxide dismutase (SOD), glutathione peroxidase (GPX), reduced glutathione (RG), alkaline phosphatase (ALP, cat. No.: GTIN050552732007543), aspartate aminotransferase (AST, cat. No.: GTIN050552732007555), alanine aminotransferase (ALT, cat. No.: GTIN050552732007564), albumin (cat. No.: GTIN05055273200850), bilirubin (total and direct, (cat. No.: GTIN05055273200865), urea (cat. No.: GTIN05055273200633) and creatinine (cat. No.: GTIN05055273200542) assay kits were products of Randox Laboratories Ltd., Antrim, UK. The Folin-Ciocalteu reagent was a product of Merck (Darmstadt, Germany), while methanol was factory-made by British Drug House Chemicals Ltd. (Poole, England). All other chemicals used were of analytical grade obtained from Sigma-Aldrich, Steinheim, Germany.

2.2. Plant material

Fresh leaves of T. prieureana were obtained from a farm settlement at Ikire (GPS: Latitude: 7° 21′ 36.00″N; Longitude: 4° 11′ 6.00″E), Osun State, Nigeria. The plant was authenticated by Mr. K. Odewo of the Forestry Research Institute of Nigeria (FRIN). A prepared herbarium sample of the leaves was deposited at FRIN under FHI 110985 for future reference. Trichilia prieureana was checked at http://www.theplantlist.org/tpl1.1/on 23-03-2012 and found to be accepted.

2.3. Experimental animals

A total of 90 male Wistar rats (135.54 ± 10.52 g, 6 weeks 4 days old) made up of 48 male Wistar rats for the diabetic study and another 42 male Wistar rats for the IpGTT study were obtained from the Animal Holding Unit of the Department of Biochemistry, Federal University of Technology, Akure (GPS: Latitude: 7° 15′ 9.22″N; Longitude: 5° 11′ 35.23″E), Ondo State. The animals were housed in well-ventilated cages that were placed in the Animal House of Landmark University, Omu Aran (GPS: Latitude: 8° 08′ 18.85″N; Longitude: 5° 06′ 9.36″E), Nigeria, under the regulated housing conditions of temperature (22 ± 3 °C), light/dark cycle (12 h/12 h), relative humidity (40–45 %). The animals were maintained on rat feed (Top Feeds, a product of Premier Feed Mills of Nigeria PLC, Ogorode, Sapele, Delta State, Nigeria) and tap water ad libitum.

2.4. Preparation of aqueous extract of Trichilia prieureana leaves (AETPL)

The fresh leaves of T. prieureana (2218 g) were air-dried at 25 °C for three weeks to give 1600 g of dried leaves. This was pulverized using an electric blender (Kenwood, Model BL490, Huizhou, China) to yield 1001 g of the powder. The powder was then extracted for 48 h in 16,000 L of DW and freeze-dried (Modulyo Freeze Dryer, Edward, England) to yield 184.99 g of the lyophilized powder. Calculated amount of the lyophilized powder corresponding to the desired doses (11.2, 22.3 and 44.6 mg/kg BW) was reconstituted in DW and administered to the T2DM rats in the present study.

2.5. Secondary metabolite screening of AETPL

Standard procedures were adopted in screening for the presence of secondary metabolites in AETPL.^11,12 Furthermore, the detected secondary metabolites were quantified as described for saponins,¹³ tannins, alkaloids, flavonoids and total phenolics.¹⁴

2.6. Determination of in vitro α-amylase and α-glucosidase inhibitory activities of AETPL

The in vitro inhibitory effects of AETPL at 20, 40, 60, 80 and 100 μg/mL on α-amylase and α-glucosidase activities using acarbose as positive control were determined as described by Ademiluyi and Oboh.¹⁵

2.7. Induction of T2DM

Type 2 diabetes mellitus was induced in the male rats by adopting the procedure described by Wilson and Islam.¹⁶ Male Wistar rats (48 in total; average weight, 135.54 ± 10.52 g) were subjected to 12 h fasting (without food, but water) prior to the commencement of administration. The FBS level of the rats was determined from the tails of the animals before exposure to FSTZ using a portable glucometer (Glucoplus Inc., Saint-Laurent, Quebec, Canada) according to the procedure described by Adams and Yakubu.¹⁷ Thereafter, 40 male rats were selected and ad libitum maintained on 10 % fructose solution for two weeks to induce insulin resistance. At the end of the two weeks of exposure to the fructose solution, this same set of rats were intraperitoneally administered single dose of 40 mg/kg BW of STZ (dissolved in citrate buffer, pH 4.5).¹⁷ The FBS levels were determined again after 72 h of STZ administration. Male rats with FBS levels greater than 250 mg/dl were declared to have been induced with T2DM.¹⁷

2.8. Animal grouping, administration of AETPL and determination of body weight of the rats

The forty-eight male rats were completely randomized into six groups (A, B, C, D, E, and F) of eight animals each as follows:

Group A: Non-diabetic rats + DW (Sham Control, SC).

Group B: Diabetic rats + DW (Negative Control, NC).

Group C: Diabetic rats + 100 mg/kg BW of metformin (Positive Control, PC).

Group D: Diabetic rats + 11.2 mg/kg BW of AETPL.

Group E: Diabetic rats + 22.3 mg/kg BW of AETPL.

Group F: Diabetic rats + 44.6 mg/kg BW of AETPL.

The animals were orally administered 1 mL each of the metformin and AETPL that corresponded to their respective doses, once daily for 14 days, using metallic oropharyngeal cannula. The doses of 11.2, 22.3, and 44.6 mg/kg BW used in this study for AETPL were derived from the information obtained during the ethnobotanical survey carried out on the plant by the authors within the locality of Omu-Aran. The 11.2 and 44.6 mg/kg BW were equivalent of “a pinch” and “a table spoonful” respectively and consumed as a remedy for diabetes by a 70 kg adult man, while the 22.3 was half of the highest dose. Also, the 100 mg/kg BW of metformin was adopted from a previous study on hyperglycemia-mediated oxidative stress by Lekshmi et al.¹⁸ The BW of each of the animals (computed as average body weight) was determined before the administration of fructose and STZ (Day 0; initial body weight), and 14 days (final body weight) after the exposure to FSTZ. Metformin was used in this study because it is frequently prescribed as the first line therapy for the management of T2DM.^19,20

2.9. Animal grouping for the IpGTT of AETPL

Intraperitoneal glucose tolerance test (IpGTT) was performed in fasted (6 h, without food, but water) male rats according to the procedure of de Oliveira et al.²¹ The 42 male rats (n = 7), grouped into 6 with those in group A receiving physiological saline while the FSTZ-exposed rats in groups B, C, D, E and F received intraperitoneal dose of 2 g/kg of glucose, and after 2 h, were intraperitoneally administered 2.0 mL/200 g of physiological saline (NaCl, 0.9 %), metformin (100 mg/kg body weight), AETPL at 11.2, 22.3 and 44.6 mg/kg body weight, respectively. Blood samples from the IpGTT model were collected from the tail vein on day 14 at 0, 15, 30, 60 and 120 min after glucose administration for the determination of blood glucose and plasma insulin (determined at the end of the day 14) as previously described.^17,22

Ethical approval

Ethical approval (UERC Approval number: UERC/ASN/2017/900) was obtained on the 8th of June 2017, from the University of Ilorin Ethical Review Committee before the commencement of the study. In addition, all the animals in this study were handled in strict accordance with the Guidelines on the Care and Use of Laboratory Animals of the National Research Council, Washington DC, USA.²³

2.10. Preparation of serum and tissue supernatants

The method described by Adams and Yakubu¹⁷ was adopted for the preparation of serum and tissue supernatants. After an overnight fast and under diethyl ether anesthesia,²⁴ the jugular veins after being slightly displaced (to avoid contamination with interstitial fluid) were cut with sterile scalpel blade; 5 mL of the blood was collected into a plain bottle and left for 30 min to clot. The serum was thereafter aspirated with Pasteur pipette after centrifugation at 894×g for 15 min. Furthermore, the liver and pancreas of the rats were removed, blotted, weighed and homogenized in a 0.25 M sucrose solution (1:5 w/v) as described by Akanji and Yakubu.²⁵ The homogenates of the liver and pancreas were separately centrifuged at 1398×g for 20 min. The resulting supernatants were frozen for 24 h before being used for the analyses.

2.11. Determination of biochemical parameters

Standard procedures adopted were as described for FBS,¹⁷ insulin,²² HbA1c,²⁶ HOMA-IR and HOMA-β,²⁷ fructosamine,²⁸ glycogen,²⁹ GP,³⁰ HK,³¹ G6P,³² G6PD,³³ catalase,³⁴ SOD and GPX,³⁵ RG,³⁶ and MDA.³⁷

Other procedures adopted were as described for TC and triglycerides,³⁸ LDL-C, HDL-C and VLDL-C,³⁹ AI,⁴⁰ CAI,⁴¹ albumin,⁴² bilirubin (total and direct),⁴³ urea,⁴⁴ creatinine,⁴⁵ ALP,⁴⁶ AST and ALT.⁴⁷

The Hb, RBC, PCV, MCH, MCV, MCHC, WBC, platelets, lymphocytes, eosinophils, neutrophils, and monocytes were determined using the Albacus Junior Haematology Analyzer (Model 111478, Diatron GmbH, Vienna, Austria).

2.12. Histopathological examination of the liver and pancreatic tissues

A standard laboratory protocol for paraffin embedding was used to treat the formalin-preserved liver and pancreatic tissues of the male rats.⁴⁸ Tissue sections (4 mm) were fixed to the slides, de-paraffinized in p-xylene, rehydrated in ethanol (100, 80, 70, and 50 %) and rinsed with water. Slides were stained in hematoxylin for 5 min, rinsed with water, counter stained in eosin, mounted in dibutylphthalate polystyrene xylene, cover-slipped, and viewed at x 100 with a Leica slide scanner (SCN 4000, Leica Biosystems, Wetzlar, Germany).⁴⁸ The histoarchitectural changes in the pancreas and liver of AETPL-treated were compared with those of the SC and PC.

2.13. Data analysis

Results were expressed as the mean ± SEM; n = 3 for in vitro α-amylase and α-glucosidase inhibition study, n = 8 for in vivo anti-diabetic study and n = 7 for the IpGGT study. The means were analyzed using a one-way analysis of variance followed by a Duncan Multiple Range post hoc Test at p < 0.05 using Graphpad version 8.0 (GraphPad Software, Inc., San Diego, California, USA).

3. Results

3.1. Secondary metabolites in AETPL

The detected secondary metabolites in AETPL included saponins (3681.70 ± 179.87 mg/100 g), flavonoids (120.64 ± 4.67 mg/100 g), total phenolics (603.56 ± 12.86 mg/100 g), tannins (76.93 ± 9.31 mg/100 g), and alkaloids (51.15 ± 1.26 mg/100 g). In contrast, cardiac glycosides and terpenoids were not detected in AETPL. In all, the saponins (3681.70 ± 179.87 mg/100 g) were the relatively most abundant, whilst the least abundant were the alkaloids (51.15 ± 1.26 mg/100 g).

3.2. In vitro inhibition of α‐amylase and α‐glucosidase activities

The AETPL inhibited the activities of α‐amylase (IC₅₀ of 10.19 ± 1.12 μg/mL vs IC₅₀ of 1.54 ± 0.02 μg/mL for acarbose, Fig. 1) and α‐glucosidase (IC₅₀ of 12.20 ± 1.02 μg/mL vs IC₅₀ of 0.13 ± 0.01 μg/mL for acarbose, Fig. 2).

Fig. 1.

In vitro α-amylase inhibitory activities of aqueous extract of Trichilia prieureana leaves.

Fig. 2.

In vitro α-glucosidase inhibitory activities of aqueous extract of Trichilia prieureana leaves.

3.3. Effects of AETPL on fasting blood sugar

Animals treated with FSTZ produced FBS that was greater than 250 mg/dl after 48 h of treatment from the basal FBS level of 85.83 mg/dl and this was sustained throughout the 14 days of administration, climaxing at 366.33 mg/dl (Table 1). Administration of AETPL at 11.2, 22.3, and 44.6 mg/kg BW significantly (p < 0.05) and progressively reduced the FBS levels and by the end of the experimental period had reduced by 69 %, 74 %, and 74 % respectively, as against 57 % by metformin. However, the reduction in the FBS of the animals administered 22.3 and 44.6 mg/kg BW compared well (p > 0.05) with those of the SC animals by the end of the treatment period (Table 1).

Table 1.

Blood glucose levels of streptozotocin-induced diabetic male rats after oral administration of aqueous extract of Trichilia prieureana leaves.

Fasting blood glucose (mg/dl)
Treatment groups	Before administration of fructose + STZ	After 48 h of fructose + STZ administration	After 7 days of administration of fructose + STZ	After 14 days of administration of fructose + STZ
Distilled water	92.50 ± 1.71a	92.50 ± 1.09a	91.67 ± 1.67a	92.67 ± 1.67a
Fructose + STZ + distilled water	85.83 ± 2.90a	315.17 ± 17.74b	343.00 ± 17.36d	366.33 ± 12.78c
Fructose + STZ + 100 mg/kg body weight of metformin	96.33 ± 4.92a	321.00 ± 14.55b	256.17 ± 15.27c	156.33 ± 14.88b
Fructose + STZ + 11.2 mg/kg body weight of AETPL	90.33 ± 2.36a	335.33 ± 15.28b	214.67 ± 15.05bc	110.50 ± 6.55ab
Fructose + STZ + 22.3 mg/kg body weight of AETPL	90.67 ± 3.08a	328.50 ± 14.98b	170.67 ± 9.81b	91.50 ± 2.09a
Fructose + STZ + 44.6 mg/kg body weight of AETPL	88.83 ± 1.74a	349.33 ± 13.88b	168.17 ± 8.89b	85.67 ± 1.69a

Data are presented as the mean ± SEM of 8 determinations. Values with superscripts b, c, and d different from their respective control, a, along the row are significantly different (p < 0.05).

AETPL = Aqueous extract of Trichilia prieureana leaves.

STZ = Streptozotocin.

3.4. Effect of AETPL on body weight of male rats

Administration of FSTZ significantly (p < 0.05) lowered the BW of the male rats by 25.86 % (Table 2). In contrast, the BW of the diabetic rats increased after the treatment with 11.2, 22.3, and 44.6 mg/kg BW of AETPL in a manner similar to that of PC rats (Table 2). By the end of the experimental period, the 11.2, 22.3, and 44.6 mg/kg BW of AETPL had increased the BW of the animals by 3.76, 5.11, and 8.45 %, respectively, as against the 4.17 % produced by the PC, with the 44.6 mg/kg BW producing the most profound increase (8.45 %) (Table 2).

Table 2.

Body weight of diabetic male rats after treatment with aqueous extract of Trichilia prieureana leaves.

Groups	Initial body weight (g) (Day 0)	Final body weight (g) (Day 14)	Weight Change (%)
Distilled water	138.66 ± 3.81a	171.89 ± 3.45d	23.97↑
Fructose + STZ + distilled water	136.60 ± 3.97a	101.27 ± 3.26e	−25.86↓
Fructose + STZ + 100 mg/kg body weight of metformin	135.21 ± 3.55a	140.85 ± 2.22b	4.17↑
Fructose + STZ + 11.2 mg/kg body weight of AETPL	141.44 ± 4.01a	146.76 ± 2.04b	3.76↑
Fructose + STZ + 22.3 mg/kg body weight of AETPL	140.38 ± 3.01a	147.55 ± 2.20b	5.11↑
Fructose + STZ + 44.6 mg/kg body weight of AETPL	140.02 ± 3.99a	151.85 ± 2.13c	8.45↑

Data are presented as the mean ± SEM of 8 determinations. Values with superscripts b, c, d and e different with their respective control, a, along a column are significantly (p < 0.05) different.

AETPL = Aqueous extract of Trichilia prieureana leaves.

STZ = Streptozotocin.

3.5. Effects of AETPL on glycated haemoglobin

Administration of FSTZ significantly elevated the levels of glycated haemoglobin by 63 % in diabetic male rats (Table 3). In contrast, AETPL at 11.2, 22.3, and 44.6 mg/kg BW significantly (p < 0.05) reduced the levels of glycated haemoglobin by 33 %, 43 %, and 62 %, respectively, as against the 19 % reduction by the PC. Finally, the reduction in glycated haemoglobin by AETPL at 44.6 mg/kg body weight compared favourably (p > 0.05) with that of the SC (Table 3).

Table 3.

Effects of administration of aqueous extract of Trichilia prieureana leaves on biochemical parameters of fructose-fed streptozotocin-induced diabetic male Wistar rats.

Parameters	Animal Grouping
	Distilled water	Fructose + STZ + distilled water	Fructose + STZ +100 mg/kg body weight of metformin	Fructose + STZ +11.2 mg/kg body weight of AETPL	Fructose + STZ +22.3 mg/kg body weight of AETPL	Fructose + STZ +44.6 mg/kg body weight of AETPL
Glycated haemoglobin (HbA1c) %	16.36 ± 0.43a	44.21 ± 1.53b	35.92 ± 0.38c	29.81 ± 0.51d	24.98 ± 0.69e	16.61 ± 0.31a
Insulin (U/l)	86.54 ± 0.76a	24.38 ± 1.01b	54.22 ± 1.33c	66.86 ± 0.83d	73.78 ± 1.66e	84.66 ± 0.96a
HOMA-IR	2.72 ± 0.02a	3.07 ± 0.13b	2.92 ± 0.07c	2.54 ± 0.03d	2.39 ± 0.08e	2.75 ± 0.03a
HOMA-β	151.54 ± 1.22a	4.04 ± 0.17b	29.15 ± 0.71c	70.63 ± 0.87d	110.59 ± 2.49e	154.17 ± 1.50a
Fructosamine (μmol/l)	30.20 ± 1.85a	144.68 ± 4.39b	79.13 ± 1.70c	61.62 ± 0.55d	42.41 ± 1.17e	33.72 ± 0.53a
Liver glycogen (mg/g)	173.46 ± 7.64d	37.43 ± 4.58a	134.35 ± 3.60b	140.87 ± 4.81bc	143.67 ± 2.09bc	151.69 ± 3.47c
Liver glycogen phosphorylase activity (μmol of phosphate liberated/mg protein)	15.98 ± 0.41a	52.12 ± 3.06b	40.93 ± 2.40c	36.96 ± 1.63bc	32.25 ± 0.63bc	14.36 ± 2.23a
Hexokinase (U/mg protein)	53.24 ± 1.21a	19.34 ± 1.67b	28.93 ± 0.97c	35.56 ± 2.18d	38.91 ± 1.07d	51.52 ± 0.77a
Glucose-6-phosphatase (U/mg protein)	13.73 ± 1.29a	27.57 ± 1.87b	18.80 ± 0.39c	17.06 ± 0.64c	15.91 ± 0.22a	15.22 ± 0.28a
Glucose-6-phosphate dehydrogenase (U/mg protein)	168.85 ± 4.81a	21.99 ± 0.95b	56.77 ± 5.56c	90.92 ± 5.99d	116.60 ± 7.09e	157.64 ± 11.55a
Total cholesterol (mg/dl)	44.62 ± 0.98a	115.22 ± 18.09b	90.72 ± 2.36c	72.65 ± 1.59d	63.13 ± 1.06d	42.72 ± 2.41a
Triglycerides (mg/dl)	27.57 ± 0.27a	84.23 ± 4.17b	66.07 ± 1.16c	56.52 ± 1.20d	44.35 ± 1.03e	27.10 ± 1.83a
LDL- C (mg/dl)	2.46 ± 0.67a	85.08 ± 8.41b	57.88 ± 1.78c	38.42 ± 1.69d	28.03 ± 2.09e	3.26 ± 0.41a
VLDL- C (mg/dl)	5.51 ± 0.05a	16.85 ± 0.83b	13.01 ± 0.32bc	11.30 ± 0.24c	8.87 ± 0.21d	6.42 ± 0.36a
HDL- C (mg/dl)	36.65 ± 1.45a	13.29 ± 1.55b	19.63 ± 0.96c	22.92 ± 1.21c	26.34 ± 1.26cd	34.13 ± 0.70a
Atherogenic index	0.22 ± 0.03a	8.30 ± 2.27b	3.65 ± 0.16c	2.20 ± 0.15cd	1.43 ± 0.15e	0.25 ± 0.08a
Coronary artery index	1.22 ± 0.03a	9.30 ± 2.26b	4.65 ± 0.15c	3.23 ± 0.13cd	2.43 ± 0.15e	1.15 ± 0.08a
Liver superoxide dismutase (U/mg protein)	4.25 ± 0.99a	0.07 ± 0.02b	1.29 ± 0.07c	2.13 ± 0.04d	2.99 ± 0.06de	4.41 ± 0.09a
Liver glutathione peroxidase (U/mg/protein)	27.82 ± 0.47a	4.46 ± 0.24b	15.03 ± 1.13c	17.46 ± 1.98cd	20.60 ± 1.51e	25.07 ± 1.02a
Liver glutathione reduced (U/mg/protein)	2.90 ± 0.02a	0.88 ± 0.02b	4.19 ± 0.19c	3.84 ± 0.07d	3.40 ± 0.09d	3.02 ± 0.06a
Liver catalase (U/mg/protein)	3.68 ± 0.40a	1.21 ± 0.08b	2.33 ± 0.04bc	2.41 ± 0.02bc	2.48 ± 0.03bc	3.94 ± 0.12a
Liver malondialdehyde (x10−8nmd/ml)	47.33 ± 4.37a	98.26 ± 0.85b	75.07 ± 1.24c	60.76 ± 2.29d	60.56 ± 1.83d	42.52 ± 0.77a
Pancreatic superoxide dismutase (U/mg protein)	9.72 ± 2.01f	3.11 ± 1.10e	5.72 ± 1.41bc	6.82 ± 1.49cd	7.81 ± 1.69d	8.28 ± 1.84e
Pancreatic glutathione peroxidase (U/mg/protein)	76.21 ± 3.68a	22.50 ± 0.32b	47.25 ± 1.66c	53.94 ± 1.75c	58.45 ± 1.36d	75.18 ± 2.08a
Glutathione reduced (U/mg/protein)	17.50 ± 1.22a	7.13 ± 0.65b	11.50 ± 1.29c	12.89 ± 1.62c	14.11 ± 1.88c	18.43 ± 1.85a
Pancreatic catalase (U/mg/protein)	12.42 ± 1.52a	4.92 ± 1.05b	7.11 ± 1.61c	7.68 ± 1.98c	9.48 ± 1.12d	11.98 ± 1.47a
Malondialdehyde (x10−8nmd/ml)	2.70 ± 0.31a	7.54 ± 1.62b	4.64 ± 1.17c	3.88 ± 1.16c	3.54 ± 1.12c	2.61 ± 0.78a

Data are presented as the mean ± SEM of 8 determinations. Values with different superscript letters (a-f) down the column are significantly different. HOMA-IR, Homeostasis model assessment-insulin resistance; HOMA-β, Homeostasis model assessment- β; LDL-C, Low density lipoprotein-cholesterol; HDL-C, High density lipoprotein-cholesterol; and VLDL-C, Very low density lipoprotein-cholesterol; AETPL = Aqueous extract of Trichilia prieureana leaves; STZ = Streptozotocin.

3.6. Effects of AETPL on serum insulin levels, HOMA-IR and HOMA-β scores

The FSTZ exposure significantly (p < 0.05) decreased the serum insulin levels and HOMA-β scores, whereas serum fructosamine and HOMA-IR scores were significantly (p < 0.05) increased (Table 3). In contrast, AETPL administration at 11.2, 22.3, and 44.6 mg/kg BW significantly (p < 0.05) and dose dependently increased the serum insulin content and HOMA-β scores whereas the serum fructosamine level and HOMA-IR were dose dependently decreased (Table 3). However, oral administration of AETPL at 44.6 mg/kg BW produced levels of serum insulin, fructosamine, HOMA-β and HOMA-IR scores in T2DM animals that compared favourably (p > 0.05) with those of SC (Table 3). In addition, metformin produced values of serum insulin, fructosamine, HOMA-β and HOMA-IR scores that did not compare well (p > 0.05) with those of SC (Table 3).

3.7. Effects of AETPL on liver glycogen content, carbohydrate metabolizing enzymes, serum lipids and antioxidant enzymes

In the male rats, administration of FSTZ significantly (p < 0.05) reduced liver glycogen and increased GP activities when compared with those of SC rats (Table 3). In contrast, all the doses of AETPL significantly and dose relatedly increased the liver glycogen levels and GP activities when compared with the NC rats (Table 3).

Compared with the SC rats, administration of FSTZ significantly (p < 0.05) decreased both the activities of HK and G6PD and increased G6P activity (Table 3). Contrarily, all the doses of AETPL increased (p < 0.05) the FSTZ-treatment related decreases in HK and G6PD activities with those of 44.6 mg/kg BW treated diabetic rats comparing favourably with that of SC. Furthermore, the elevated G6P activity by FSTZ was reduced (p < 0.05) by all the doses of the AETPL. The AETPL-induced changes in the hepatic enzyme activities by the 44.6 mg/kg BW were similar to those produced by the SC.

The FSTZ significantly (p < 0.05) increased the levels of TC, triglycerides, LDL-C, VLDL-C, AI and CAI, and reduced the levels of HDL-C when compared with those of SC rats (Table 3). Furthermore, the oral administration of AETPL at 11.2, 22.3, and 44.6 mg/kg BW significantly (p < 0.05) and dose dependently reduced the FSTZ-treatment related increases in the levels of serum TC, triglycerides, LDL-C, VLDL-C, AI and CAI. In addition, the reduction in the levels of HDL-C by the FSTZ were dose dependently increased (p < 0.05) by the AETPL. Again, the oral administration of 44.6 mg/kg BW of AETPL produced levels of serum TC, TG, LDL-C, VLDL-C, AI, CAI and HDL-C that compared favourably (p > 0.05) with those of SC rats (Table 3). The metformin also produced trends of lipid profile that were similar to those of AETPL-treated diabetic rats when compared with the NC, but were not comparable with any of the lipid profile of the SC (Table 3).

Administration of FSTZ significantly (p < 0.05) depleted the levels of SOD, GPX, RG and catalase in the liver and pancreas of the animals, whereas the MDA contents increased significantly in both tissues (Table 3). However, oral administration of AETPL at 11.2, 22.3, and 44.6 mg/kg BW significantly (p < 0.05) increased the FSTZ-treatment related decreases in the activities of SOD, GPX, RG and catalase in both the liver and pancreas. This trend was similar to those of diabetic animals treated with metformin. Furthermore, the elevated MDA levels induced by the FSTZ were reduced (p < 0.05) by the AETPL and metformin in the liver and pancreas of the animals (Table 3). In addition, the 44.6 mg/kg BW displayed the most pronounced effects on the activities of SOD, GPX, RG, catalase and MDA contents in both the liver and pancreas and also compared well (p > 0.05) with those of SC rats.

3.8. Effects of AETPL on function indices of liver and kidney and haematological parameters

Fructose and STZ significantly (p < 0.05) increased the levels of serum ALP, AST, ALT, urea, total and direct bilirubin and also decreased the creatinine and albumin contents in the male rats (Table 4). In contrast, the AETPL reduced the FSTZ-related increases in the levels of ALP, AST, ALT, urea, total and direct bilirubin as well as the FSTZ-treatment related decreases in the concentrations of creatinine and albumin in the serum of the animals in a manner similar to those of the PC rats. In all, the treatment of the animals with 44.6 mg/kg BW of AETPL produced serum ALP, AST, ALT, urea, creatinine, albumin, total and direct bilirubin levels that compared favourably (p > 0.05) with those of SC rats (Table 4).

Table 4.

Organ function indices and haematological parameters of fructose and streptozotocin-induced diabetic male rats after oral administration of aqueous extract of Trichilia prieureana leaves.

Parameters	Animal Grouping
	Distilled water	Fructose + STZ + distilled water	Fructose + STZ +100 mg/kg body weight of metformin	Fructose + STZ +11.2 mg/kg body weight of AETPL	Fructose + STZ +22.3 mg/kg body weight of AETPL	Fructose + STZ +44.6 mg/kg body weight of AETPL
Alkaline phosphatase (U/L)	98.40 ± 6.20a	241.20 ± 15.04b	156.12 ± 10.46c	151.04 ± 1.12c	140.11 ± 3.17cd	100.42 ± 3.19a
Aspartate aminotansferase (U/L)	32.50 ± 0.84a	72.85 ± 1.21b	62.55 ± 0.92c	49.94 ± 2.37d	43.54 ± 3.09d	30.97 ± 1.27a
Alanine aminotransferase (U/L)	28.71 ± 1.68a	58.84 ± 3.37b	41.59 ± 1.86c	40.32 ± 0.89c	39.45 ± 1.81bc	30.87 ± 0.81a
Urea (mg/dl)	5.18 ± 0.08a	10.56 ± 0.25b	7.13 ± 0.04c	6.32 ± 0.11c	5.69 ± 0.02c	5.33 ± 0.08a
Creatinine (mg/dl)	33.39 ± 1.72a	17.69 ± 3.57b	45.21 ± 2.57c	42.99 ± 1.24c	37.05 ± 0.08a	36.01 ± 0.37a
Albumin (mg/dl)	2.22 ± 0.02a	7.71 ± 0.07b	5.44 ± 0.11c	4.29 ± 0.14c	3.85 ± 0.11c	2.11 ± 0.15a
Total Bilirubin (mg/dl)	8.15 ± 0.11a	14.30 ± 0.62b	11.65 ± 0.24bc	9.65 ± 0.26c	9.14 ± 0.23c	8.67 ± 0.14a
Direct Bilirubin (mg/dl)	7.52 ± 0.25a	12.21 ± 0.37b	9.74 ± 0.08ac	8.88 ± 0.78c	8.23 ± 0.13c	7.66 ± 0.19a
Packed Cell Volume (%)	43.20 ± 9.80a	25.20 ± 1.88b	33.00 ± 0.71c	36.00 ± 1.64bc	38.00 ± 0.77bc	49.00 ± 0.71a
Haemoglobin (g/dl)	12.52 ± 0.36a	4.22 ± 1.04b	6.66 ± 0.57c	7.82 ± 1.43bc	9.62 ± 0.58cd	11.78 ± 0.48a
White Blood Cell ( × 109/L)	7.56 ± 0.42a	2.46 ± 0.24b	4.14 ± 0.05c	4.46 ± 0.29c	4.70 ± 0.53c	7.26 ± 0.21a
Neutrophil (%)	68.40 ± 0.68a	40.00 ± 1.64b	65.40 ± 1.03a	66.40 ± 0.75a	66.80 ± 0.49a	67.80 ± 0.92a
Lymphocytes (%)	30.00 ± 0.01a	18.40 ± 0.93b	27.00 ± 0.71a	26.80 ± 1.32b	26.80 ± 1.32b	29.20 ± 0.58a
Monocytes (%)	3.80 ± 0.73a	1.40 ± 0.24b	3.00 ± 0.32a	3.40 ± 0.24a	3.80 ± 0.37a	3.80 ± 0.49a
Red Blood Cell (x12/l)	7.89 ± 0.15a	3.60 ± 0.66b	5.47 ± 0.99c	6.12 ± 1.02ab	6.43 ± 0.13ab	8.08 ± 0.38a
Mean Corpuscular Haemoglobin Concentration (g/dl)	27.84 ± 0.92a	19.60 ± 0.60b	24.52 ± 1.68cb	25.20 ± 1.38a	25.54 ± 1.10a	26.46 ± 0.25a
Mean Corpuscular Volume (fl)	55.54 ± 0.69a	37.72 ± 1.77b	43.60 ± 0.93c	44.80 ± 0.49c	46.00 ± 0.95c	56.80 ± 2.48a
Mean Corpuscular Haemoglobin (pg)	15.86 ± 0.34a	7.14 ± 0.27b	10.30 ± 1.35c	12.02 ± 1.43c	13.74 ± 1.25cd	16.36 ± 0.64a
Platelets ( × 109/L)	293.00 ± 8.30a	173.40 ± 9.66b	200.20 ± 3.47c	225.86 ± 8.03d	232.80 ± 5.51d	289.40 ± 7.87a

Data are presented as mean ± SEM of 8 determinations. Values with superscripts a, b, c, d and e along a row are significantly different at p < 0.05.

AETPL = Aqueous extract of Trichilia prieureana leaves.

STZ = Streptozotocin.

Administration of FSTZ significantly (p < 0.05) reduced the levels of PCV, Hb, WBC, neutrophils, lymphocytes, monocytes, RBC, MCHC, MCV, MCH, and platelets in the male rats, compared with the SC rats (Table 4). However, oral administration of AETPL at 11.2, 22.3, and 44.6 mg/kg WB as well as metformin significantly (p < 0.05) increased the levels of PCV, Hb, WBC, neutrophils, lymphocytes, monocytes, RBC, MCHC, MCV, MCH, and platelets in the FSTZ-treated rats when compared with those of the NC (Table 4). The levels of Hb, WBC, neutrophils, lymphocytes, RBC and MCV after administration of 44.6 mg/kg BW of AETPL compared favourably with SC. Furthermore, all the doses of AETPL produced levels of neutrophils and monocytes that compared well with those of SC whereas the 22.3 and 44.6 mg/kg BW of AETPL produced MCH and MCHC levels that were not significantly different from those of SC (Table 4).

3.9. Effects of AETPL on liver and pancreas histoarchitecture

The β-cells of the Islet of Langerhans in SC rats were intact and of normal histoarchitecture (Plate 1A). The FSTZ-treatment related partial destruction of the β-cells in the pancreatic islets (Plate 1B) became milder and/or returned to normal histoarchitecture after treatment with AETPL (Plates 1C, D, E and F). The liver histoarchitecture displayed normal appearance of radiating hepatocytes, portal triad, central vein and hepatic sinusoids lined with endothelial cells (Plate 2A). The exposure of the male rats to FSTZ disrupted the normal arrangement of the hepatocytes, induced congestion in the central hepatic vein (HV), caused degeneration in the hepatocytes of the peripheral area of the central vein, and induced massive vacuolization in the lobules (Plate 2B). In contrast, the liver of the male rats treated with AETPL showed mild to moderate degree of hepatocyte degeneration, vascular congestion, and edema (Plates 2D, E and F) in a manner similar to those treated with metformin (Plate 2C) and the SC (Plate 2A).

Plate 1.

Cross section of the pancreas of male rats after administration of aqueous extract of Trichilia prieureana leaves (H&E; x100). (A) SC, Sham control; (B) NC, Negative Control; (C) Positive Control; (D) DTp11.2, Diabetic + aqueous extract of T. prieureana leaves (11.2 mg/kg body weight); (E) DTp22.3, Diabetic + aqueous extract of T. prieureana leaves (22.3 mg/kg body weight); (F) DTp44.6, Diabetic + aqueous extract of T. prieureana leaves (44.6 mg/kg body weight).

Plate 2.

Cross section of the liver of male rats after administration of aqueous extract of Trichilia prieureana leaves (H&E; x100).

(A) SC, Sham control; (B) NC, Negative Control; (C) Positive Control; (D) DTp11.2, Diabetic + aqueous extract of T. prieureana leaves (11.2 mg/kg body weight); (E) DTp22.3, Diabetic + aqueous extract of T. prieureana leaves (22.3 mg/kg body weight); (F) DTp44.6, Diabetic + aqueous extract of T. prieureana leaves (44.6 mg/kg body weight).

3.10. Fasting blood glucose and plasma insulin levels of FSTZ animals in IpGTT

The blood glucose levels were significantly different between the AETPL, metformin and physiological saline-intraperitoneally administered animals during the IpGTT study (Table 5). The AETPL dose dependently reduced the glycemia of the animals as the time increased from 0 min to 120 min with the 44.6 mg/kg body weight producing the most profound glycemic reduction when compared with their respective physiological saline-intraperitoneally administered control. The area under the curve for the AETPL was similarly reduced from 19530.13 for the FSTZ to 8723.88 for the highest dose of the extract (44.6 mg/kg body weight) as against 9621.58 for the metformin-treated animals. The reduction in the blood glucose levels of the animals by the AETPL after the intraperitoneal glucose overload (IpGTT) was accompanied by increase in the plasma insulin concentrations with the 44.6 mg/kg body weight of AETPL-treated animals producing insulin levels that compared favourably (p > 0.05) with that of the physiological saline-intraperitoneally administered control by the end of the experimental period (Table 5).

Table 5.

Fasting blood glucose and plasma insulin of FSTZ-induced type 2 diabetic rats after intraperitoneal administration of glucose (IpGTT).

Glycemia (mg/dl)
Time/Groups	Control (PS)	FSTZ	Metformin	AETPL (11.2 mg/kg bw)	AETPL (22.3 mg/kg bw)	AETPL (44.6 mg/kg bw)
0 min	64.06 ± 6.66a	65.10 ± 5.11a	65.87 ± 3.98a	63.46 ± 6.10a	64.32 ± 5.77a	61.95 ± 7.06a
15 min	66.00 ± 2.18a	132.63 ± 8.13b	96.47 ± 8.32b	100.46 ± 7.44b	104.36 ± 9.01b	94.62 ± 6.39b
30 min	62.96 ± 7.88a	152.44 ± 10.49c	82.74 ± 5.90b	100.22 ± 8.46b	90.30 ± 8.80c	84.62 ± 2.07c
60 min	65.15 ± 4.22a	172.38 ± 9.21d	80.21 ± 4.51b	95.63 ± 4.76b	83.25 ± 8.40d	70.32 ± 2.28e
120 min	63.71 ± 4.65a	184.63 ± 7.14e	68.22 ± 5.18a	83.46 ± 7.35c	72.04 ± 6.37e	60.09 ± 4.18a
Area Under the Curve of the IpGTT
	7786.20 ±	19530.13	9621.58	11160.48	10100.57	8723.88 ±
	121.37	±1782.04	±214.06	±367.91	±150.27	104.88
Plasma Insulin Concentration (μg/l)
	24.57 ± 0.32a	0.42 ± 0.03b	20.69 ± 3.46a	12.53 ± 1.44c	16.22 ± 2.082d	26.81 ± 0.11a

Note: PS = Physiological saline; FSTZ = Fructose-fed Streptozotocin; AETPL = Aqueous extract of Trichilia prieureana leaves; STZ = Streptozotocin.

4. Discussion

Type 2 diabetes mellitus, the most prevalent of the type of diabetes mellitus, has constituted a global health challenge. Therefore, there is the need for continued research on the use of complementary and alternative medicine in the management of the disease. The anti-T2DM properties of several medicinal plants have been attributed to bioactive principles like phenolics, flavonoids, alkaloids, cardiac glycosides, saponins, tannins, terpenoids, chalcones, and phlobatannins.¹⁷ For instance, saponins have been putatively reported to have a glucagon reducing effect, improve glucose utilization, and stimulate the release of insulin from the pancreas in T2DM models. Conversely, flavonoids in medicinal plants may act as a secretagogue, facilitating insulin secretion and regeneration of damaged pancreatic β-cells in T2DM rats whereas tannins and phenolics may enhance the cellular uptake of glucose from the blood and prevent and/or mop up free radicals generated in T2DM rat models, respectively.¹⁷ Although, the bioactive agent have not been clearly elucidated in the AETPL, it is evident that the AETPL contained anti-diabetic agents that ameliorated T2DM in the male rats. The exact anti-T2DM bioactive agent(s) in AETPL may however, have to await further study. Alkaloids, flavonoids and tannins detected by Kagbento et al.⁷ in the ethanolic extract of T. prieureana leaves were also found in AETPL in the present study. Saponins detected in AETPL in the current study was not detected in the ethanolic extract of T. prieureana leaves, probably due to the different extractant used and the different locations where the plants were obtained from.

The inhibition of the activities of key enzymes like α-amylase and α-glucosidase has been considered to be an effective strategy for glycemic control in T2DM patients.⁴⁹ Therefore, the inhibitory activity of AETPL on α-amylase and α-glucosidase in the present study could suggest possible delay in carbohydrate digestion and absorption and consequently a reduction in postprandial hyperglycemia in the T2DM rats. It is possible that the constituents of the AETPL might have acted directly by blocking the active sites of the enzymes and slowing down the rate at which starches are converted to glucose, thus limiting the bioavailability of blood glucose in T2DM rats. Although, no correlation study was done between the inhibitory activities of α-amylase and α-glucosidase and the secondary metabolites, the reduction in the activities of these carbohydrate metabolizing enzymes by AETPL might be attributed to the natural inhibitors of α-amylase and α-glucosidase activities such as saponins, tannins, phenolics, flavonoids, alkaloids, among others, which have been reported as the constituents of some plant extracts with anti-T2DM activities.¹⁷ The precise secondary metabolites responsible for the inhibitory effects on these enzymes may have to await further study. It is noteworthy that the α-amylase inhibitory activity of AETPL was essentially similar to that of acarbose, the most widely used agent in experimental studies to regulate the inhibition of digestive enzyme for the treatment of T2DM.

Fructose and STZ have been widely accepted as a model for the induction of T2DM in experimental animals.¹⁶ Fructose, a sweetener and a known inducer of oxidative stress causes T2DM by up-regulation of PKR (double-stranded RNA-dependent protein kinase) resulting in impairment in insulin signaling pathway and metabolic dysfunction in T2DM.⁵⁰ On the other hand, the STZ, after being transported into the β-cells of the pancreas by Glucose transporter 2 interrupts a number of important cellular processes, culminating in DNA damage. This results in reduction in β-cell mass that manifests as insulin deficiency. The insufficiency of insulin and the contribution of glucose are required to account for the insulin resistance and glucose intolerance characteristic of T2DM. The combination of FSTZ results in the development of insulin resistance by the pancreas and decreased insulin production from the pancreatic β-cells and sensitivity for glucose uptake by tissues that are characteristic of T2DM.¹⁶ Therefore, the resultant effect of the selective cytotoxicity of the β-cells of the pancreas by the FSTZ was hyperglycemia as it is in the present study.³⁰ However, the reduction in blood glucose levels in both the AETPL- and metformin-treated animals suggests the ability of AETPL and metformin to promote the regeneration of aberrant β-cells and overcome insulin resistance,⁵¹ and this consistently emphasizes the anti-hyperglycemic activity of AETPL. Such dose-dependent reduction in FBS level by AETPL, could mean that it contained some bioactive principles that increased the uptake of glucose in skeletal muscles^52,53 probably as a result of increased liver HK activity, by de novo means, with its attendant stimulatory effect on glycolysis that led to clearance of glucose from the blood of the T2DM male rats. This mechanism of increased glucose uptake is similar to that of metformin, the referenced anti-diabetic agent. The pattern of reduction in FBS by AETPL is similar to a previous report by Djoupo et al.,⁵⁴ after administering aqueous and ethanolic stem bark extracts of Trichilia emetic (a specie of Trichilia) at 100 and 200 mg/kg BW each, to alloxanized diabetic rats as well as after the administration of flavonoid rich fraction of T. emetica leaves in an animal model of Type 2 diabetes mellitus.⁵⁵ Furthermore, the reduction in FBS in this study was corroborated by the report of Gomes et al.⁵⁶ following the administration of ethylacetate fraction of T. catigua bark to streptozotocin-induced type 1 diabetic rats.

Type 2 DM is a diabetic condition in which insulin is ineffective and is initially countered by an increase in insulin production to maintain glucose homeostasis, but over time, insulin production decreases. The FSTZ induced T2DM via three-and a half-fold decrease in insulin in which the body cells do not respond to this inadequate insulin (insulin resistance) to produce the T2DM model currently adopted in this study. The administration of AETPL restored the production and secretion of insulin which the body cells might have become sensitive to. These restorative effects might have been the result of alleviation of oxidative stress and regeneration of the pancreatic β-cells as evident in this study. However, the highest dose, 44.6 mg/kg BW produced the most unique amelioration that was even higher than that of metformin. The amelioration of insulin by AETPL is similar to a previous report by Djoupo et al.,⁵⁴ after the administration of aqueous and ethanolic stem bark extracts of Trichilia emetic at 100 and 200 mg/kg BW each, to alloxanized diabetic rats.

Another important feature of T2DM as induced by the FSTZ is severe loss in body weight, attributable to a reduction in glucose metabolism, elevation in metabolism of fats, and structural breakdown of proteins to serve as an alternative source of energy.^16,17 However, the AETPL reversed the T2DM-induced weight loss due to the enhanced glycemic control through improved insulin secretion from the remaining β-cells or regenerated β-cells which could have activated the hormones needed for fat storage.⁵⁷ The AETPL might have favoured the balance between energy intake and expenditure that resulted in the change in body mass of the T2DM rats.¹⁷ The findings with respect to the attenuation of FSTZ-induced body mass loss in the current study by AETPL is similar to the report of Gomes et al.⁵⁶ after the administration of ethylacetate fraction of T. catigua bark in streptozotocin-induced type 1 diabetic rat model.

Glycated haemoglobin, often measured as HbA1c, has been widely used as an index of glycemic control in T2DM rats.⁵⁸ Increased non-enzymatic glycosylation is one of the possible mechanisms linking hyperglycemia and vascular complications of diabetes.⁵⁸ The increase in HbA1c content in the T2DM is the consequence of persistent hyperglycemia that stimulated glycation of haemoglobin as a result of exposure to FSTZ.⁵⁸ However, the suppression of HbA1c by AETPL could be attributed to its ability, notably at the highest dose (44.6 mg/kg BW), to reduce the hyperglycemia as earlier alluded to in this study, via increased glucose uptake arising from enhanced glycolysis.

Fructosamine is a marker of glucose control reflecting the average glycemic level over a certain period of time.^59,60 The 4.7-fold increase in the serum fructosamine after exposure of the male rats to FSTZ is an indication of prolonged hyperglycemia and a poor glycemic control. The dose-dependent reduction in the fructosamine content of the T2DM rats after oral administration of AETPL further reflects a positive glycemic control with the highest dose displaying the best activity.

Type 2 DM is characterised by pancreatic β-cell dysfunction and insulin resistance, which prevents insulin from being secreted in response to hyperglycemia.¹⁷ The HOMA-IR and HOMA-β are indices of insulin resistance and insulin production/β-cell function, respectively.^16,17 The FSTZ induced T2DM characterised by impaired glucose tolerance and insulin resistance as reflected by the higher HOMA-IR and reduced HOMA-β scores in the current study. In another vein, the reduction in HOMA-IR and increase in HOMA-β after AETPL administration are consequences of improved insulin secretion and insulin sensitivity, which suggests that AETPL modulated β-cell function and ameliorated T2DM-related biochemical parameters. This effect may be attributed to the flavonoid content of AETPL, which has been implicated in promoting both pancreatic β-cell islet regeneration and insulin release in T2DM animal models.¹⁷ It is noteworthy that the highest dose (44.6 mg/kg BW) of AETPL produced the most outstanding effect, even with the metformin in the present study.

Hexokinase, G6P, and G6PD are the key enzymes of glucose metabolism involved in the pathways of glycolysis, gluconeogenesis and glycogenolysis and pentose phosphate, respectively. The reduction in the activity of HK in FSTZ-induced T2DM animals could be due to impaired glycolytic pathway resulting in underutilization of glucose for energy production in the T2DM animals. Therefore, the 2-fold increase in the G6P activity further corroborated the hyperglycemia induced by the FSTZ since such increase in activity will lead to higher glucose production by gluconeogenesis and glycogenolysis. The G6PD activity is a useful biomarker of oxidative stress and poor glycemic control in T2DM.⁶¹ Therefore, the FSTZ-induced reduction in the activity of G6PD was hyperglycemia-related and negatively correlated to oxidative stress markers in the present study. The fall in the activity of G6PD is an indication of poor glycemic control in the FSTZ exposed animals. However, the resulting increase in the activities of HK by AETPL, most profoundly at the 44.6 mg/kg BW, might have enhanced the utilization of glucose for the purpose of energy production; this is suggestive of an enhanced glucose uptake from the blood by the hepatocytes. In addition, the reduction in the activity of G6P by AETPL might have impacted on gluconeogenesis through the enhanced secretion of insulin, modulation of the enzyme activity via regulation of cAMP, or inhibition of glycolysis and gluconeogenesis. Although, no correlation study was done, it is still possible that the modulatory effect of AETPL on G6P activity could be attributed to the presence of flavonoids and phenolics, which have been implicated in stimulatory effects on this carbohydrate metabolizing enzyme via enhanced insulin secretion and antioxidant means.⁶² Again, the increase in the activity of G6PD by AETPL, most profoundly at 44.6 mg/kg BW, might be due to increased insulin secretion, which could have enhanced NADPH formation, lipogenesis, and influx of glucose into the pentose monophosphate shunt to dispose of the excess glucose.¹⁷ All these continuously emphasizes the ability of the components of AETPL to enhance glucose utilization by the hepatocytes and confers anti-T2DM activity on the plant extract, AETPL in this instance.

Dyslipidemia, which is a well-recognized complication in T2DM patients, characterized by high levels of TC, triglycerides, LDL-C, VLDL-C and AI as well as lowered levels of HDL-C in the present study after exposure of the male Wistar rats to FSTZ, may be ascribed to overproduction of VLDL-C by the liver, decrease in insulin activity (as evident herein), increased free fatty acid flux secondary to insulin resistance, decrease in insulin resistance which consequently blunt the inhibition of TG lipolysis, and inactivation of lipoprotein lipase since insulin inhibits hormone sensitive lipase.^63,64 The restorative effects of FSTZ-dependent derangement in lipid profile of the T2DM animals by the AETPL could be due to the enhanced insulin dependent inhibitory activity on lipase and the subsequent reduction in the rate of lipolysis, conversion of free fatty acids to phospholipids, and cholesterol in the liver. The ameliorative effect of AETPL on the computed AI is an indication that the T2DM rats are not likely to be predisposed to cardiovascular risk. The restoration of dyslipidemia in the T2DM rats by the most effective dose of the AETPL, the 44.6 mg/kg BW, produced a better effect than the referenced drug, metformin, in this study. The findings with respect to lipid profile after AETPL administration in this study are similar to those earlier reported by Kangbeto et al.⁷ after the administration of ethanolic extract of Trichilia prieureana leaves in high-fat diet-induced type 2 diabetic rat model.

Oxidative stress contributes to the pathogenesis of T2DM and aggravates the pathology and complications by interfering in the regulatory pathways involved in insulin resistance and β-cell dysfunction.⁶⁵ The manifold reduction in the activities of SOD, GPX, RG and catalase in both the pancreatic and hepatic tissues by the FSTZ which correlated with increase in the levels of MDA are pointers to the fact that FSTZ has induced oxidative stress in the T2DM animals.^18,66 The FSTZ by inducing hyperglycemia might have produced free radicals which affected the imbalance of the radical/antioxidant defense system that eventually led to the oxidative stress in this study. The oxidative stress is one of the factors that play a role in the pathogenesis of insulin resistance, impaired insulin secretion, glucose utilization and impaired hepatic glucose metabolism, which have all been demonstrated in the FSTZ-induced T2DM earlier in the present study. The restoration/amelioration of the FSTZ-depleted/enhanced levels of SOD, GPX, RG, catalase and MDA in the T2DM rats by the AETPL suggests that the AETPL contained chemical compounds that have improved the T2DM status by regulating glucose metabolism, promoting insulin secretion and sensitivity, decreasing insulin resistance, enhancing vascular functions, and regulating the levels of HbA1c and biomarkers of T2DM. The ability of AETPL to attenuate the FSTZ-treatment related reduction in the activities of the antioxidant enzymes could be a pointer to the free radical scavenging potential of the plant extract, made possible probably by the presence of phenolics and flavonoids in AETPL, which have been reported to exhibit free radical scavenging activity.⁶⁷ The findings here are similar to the report by Lartey et al.⁶⁸ after the administration of aqueous stem bark extract of Annickia polycarpa to T2DM rats. It is however, interesting to note that the AETPL at 44.6 mg/kg BW produced the best restorative/ameliorative effects on the oxidative stress markers than the referenced drug, metformin.

Glycogen levels in tissues, particularly the liver, correlate well with insulin activity since insulin enhances intracellular glycogen deposition via inhibiting glycogen phosphorylase.¹⁷ The depletion of glycogen levels after the exposure of animals to FSTZ could be a consequence of decrease in the activity of HK. In contrast, the restoration of the hepatic glycogen after the administration of AETPL, mainly at 44.6 mg/kg BW, may be due to its ability to stimulate/improve the secretion of insulin from the pancreas,^17,19 made possible by the presence of saponins and polyphenols which have earlier on been conferred with insulinomimetic activity, responsible for the direct peripheral glucose uptake.^17,19

Hyporcreatininemia, hypoalbuminemia, hyperuremia and hyperbilirubinemia have been reported to occur in T2DM rats.¹⁷ High levels of urea are associated with increase in insulin⁶⁹; suppressing insulin secretion and sensitivity and suggesting reduction in kidney- and liver-related diabetic parameters in T2DM, as is the case with FSTZ-exposed animals in the current study. Reduced creatinine and albumin as well as high levels of bilirubin after exposure of the male rats to FSTZ is associated with increased glucose and an increased risk for T2DM.⁷⁰ Therefore, the restoration of creatinine, albumin bilirubin (direct and total) and urea after exposure to AETPL are indications that the AETPL enhanced insulin secretion and sensitivity and consequently, reduction in glycemia or enhanced glycemic control. This consistently emphasizes recovery of the male rats from these metabolic disorders. This further supports the potential of AETPL in restoring some of the complications like liver and kidney dysfunction that are associated with T2DM.

Type-2 DM complications have been reported to include increase in the activities of ALP, AST and ALT.⁷¹ In the FSTZ diabetic rats, elevated levels of these serum enzymes are directly associated with poor glycemic control and fatty liver which are makers of insulin resistance.⁷¹ It has also been reported that increased aminotransferase activities during insulin deficiency are responsible for the enhanced gluconeogenesis and ketogenesis during T2DM.⁷² Such increase in the activities of these enzymes may be associated with T2DM-related liver dysfunction. In contrast, the treatment of the T2DM rats with AETPL, most profoundly at 44.6 mg/kg BW, reduced the activities of these enzymes which suggest enhanced insulin sensitivity and good glycemic control. Earlier report on increases in alloxan-treatment related reduction in creatinine, urea, ALT and AST after the administration of aqueous and ethanolic stem bark extracts of Trichilia emetic at 100 and 200 mg/kg BW each by Djoupo et al.⁵⁴ is also similar to what was obtained in the current study. Furthermore, the reduction in the activities of ALP, ALT and AST in this study is corroborated by the report of Gomes et al.⁵⁶ following the administration of ethylacetate fraction of T. catigua bark in streptozotocin-induced type 1 diabetic rat model as well as those of Kangbeto et al.⁷ after the administration of ethanolic extract of Trichilia prieureana leaves in high-fat diet-induced type 2 diabetic rat model.

In T2DM, haematological changes are directly associated with endothelial dysfunction and inflammation. Hyperglycemia and its metabolic syndrome can be related to the alteration of different haematological parameters such as morphology, size and physiological functions of RBC, WBC and platelets.⁷³ Therefore, the reduction in the haematological parameters of male rats exposed to FSTZ may be an indication of T2DM complications related to hyperglycemia. The restoration of the FSTZ-induced alterations in the haematological parameters after the administration of AETPL may be a consequence of the enhanced insulin secretion and sensitivity arising from the improved glycemic control by the plant extract.

Histological examination, the gold standard for tissue examination is normally carried out to corroborate structural changes with biochemical alterations in animals, since the latter manifests much earlier than the former. The destruction of the β-cells of pancreatic islet and the congestion in the central hepatic vein, degeneration in the hepatocytes of the peripheral areas of central vein and massive vacuolization in the lobules of the liver after exposure of the male rats to FSTZ is quite understandable since glucose fluctuations (hyperglycemia) influences the magnitude of oxidative stress in T2DM models. Therefore, the oxidative stress might be a possible mechanism for the T2DM-associated destruction of β-cells of the pancreatic islet and hepatocyte degeneration. Furthermore, the enhanced antioxidant status in the T2DM rats by the constituents of AETPL might have assisted in protecting the β-cell mass of the pancreas by inhibiting β-cell apoptosis and dedifferentiation and the hepatocytes from oxidative stress, and consequently improved the insulin secretion and sensitivity and glycemic control in the T2DM rats. The present finding is similar to the previous report by Arora et al.,⁷⁴ who attributed the improved diabetes mellitus control of Tephrosa purpurea to the pancreatic β cell regeneration potential of its rutin content and that of Gomes et al.,⁵⁶ following the administration of ethylacetate fraction of T. catigua bark in streptozotocin-induced type 1 diabetic rat model.

The IpGTT model measures the clearance of an intraperitoneally injected glucose load from the body. It is used to detect disturbances in the metabolism of glucose in relation to diabetes mellitus or other metabolic syndromes. In the present study, the comparison of physiological saline-intraperitoneally administered control rats, FSTZ- and the AETPL-treated rats in terms of their glycemia, the area under the curve and the insulin levels suggest a relative improvement of insulin sensitivity and a reduction of insulin resistance in the AETP-treated animals. Furthermore, the change in the pattern of insulin in intraperitoneally administered AETPL (60 min vs 120 min; 44.6 mg/kg body weight) when compared with the FSTZ suggests a quicker response of the β cells to postprandial hyperglycemia. The hypoinsulinemia in FSTZ-treated animals implies that the peripheral tissue insulin resistance occurred after the partial destruction of the β cells. In contrast, the improvement of insulin after the intraperitoneal administration of the AETPL, notably at 44.6 mg/kg body weight, is suggestive of amelioration of insulin resistance. All of these are indications of the ability of AETPL to stimulate insulin secretion from the rejuvenated β cells of the pancreas. It is also possible that AETPL plays the important role of insulin sensitizer, enhancing insulin secretion and sensitivity from the rejuvenated pancreatic β cells and reducing or abolishing insulin resistance.

5. Conclusion

The findings in the present study indicated that the aqueous extract of Trichilia prieureana leaves exhibited anti-diabetic activity against type 2 diabetes mellitus via up-regulation of the secretion and sensitivity to insulin, improvement of glucose homeostasis, antioxidant mechanism, and restoration of pancreatic histoarchitecture. The presence of tannins, saponins, alkaloids, flavonoids, and phenolics might have conferred the desired anti-diabetic activity on Trichilia prieureana leaves with the most pronounced anti-T2DM activity at 44.6 mg/kg BW. The future perspective of the study will be to investigate the bioactive principle(s) in Trichilia prieureana leaves that is responsible for the anti-T2DM activity in the FSTZ rat model, the mechanism of anti-diabetic action and safety profile of the bioactive principle(s) in male rats.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors’ contributions

OOA and YMT designed the study. OOA collected and analyzed the data. OOA and YMT interpreted the data. OOA drafted the manuscript. YMT reviewed the draft for correctness and scientific content. OOA and YMT read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.