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# Biochemical and tissue physiopathological evaluation of the preclinical efficacy of Solanum torvum Swartz leaves for treating oxidative impairment in rats administered a β-cell-toxicant (STZ)

Saidu Sani, Bashir Lawal, Jerius N Ejeje, Tawakalitu B Aliu, Amos S Onikanni, Onwe O Uchewa, Joy C Ovoh, Faith U Ekpa, Chikezie D Ozoagu, Tochukwu S Akuma, et al.

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Saidu Sani, Bashir Lawal, Jerius N Ejeje, Tawakalitu B Aliu, Amos S Onikanni, et al. Biochemical and tissue physiopathological evaluation of the preclinical efficacy of Solanum torvum Swartz leaves for treating oxidative impairment in rats administered a β-cell-toxicant (STZ). Biomedicine and Pharmacotherapy, 2022, 154, pp.113605. 10.1016/j.biopha.2022.113605. hal-04589376

HAL Id: hal-04589376

https://nantes-universite.hal.science/hal-04589376v1

Submitted on 27 May 2024

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Biochemical and tissue physiopathological evaluation of the preclinical efficacy of *Solanum torvum* Swartz leaves for treating oxidative impairment in rats administered a β-cell-toxicant (STZ)

Saidu Sani<sup>a,1</sup>, Bashir Lawal<sup>b,c,1</sup>, Jerius N. Ejeje<sup>a,d</sup>, Tawakalitu B. Aliu<sup>e</sup>, Amos S. Onikanni<sup>d,f</sup>, Onwe O. Uchewa<sup>g</sup>, Joy C. Ovoh<sup>a</sup>, Faith U. Ekpa<sup>a</sup>, Chikezie D. Ozoagu<sup>a</sup>, Tochukwu S. Akuma<sup>a</sup>, Success C. Onyeji<sup>a</sup>, Amara Obialor<sup>a</sup>, Saqer S. Alotaibi<sup>h</sup>, Sarah M. Albogami<sup>h</sup>, Michel De Waard<sup>i,j,k</sup>, Gaber El-Saber Batiha<sup>l</sup>, Tse Hung Huang<sup>m,n,o,p,q,r</sup>, Alexander T.H. Wu<sup>s,t,u,v,*</sup>

ARTICLE INFO

Keywords:

Solanum torvum Swartz

Oxidative stress

Diabetes mellitus

Organ histopathology

Antioxidant

A B S T R A C T

The current study evaluated the protective role of *Solanum torvum* Swartz against diabetes-induced oxidative stress and tissue impairment in streptozotocin (STZ)-intoxicated rats. Rats with STZ (40 mg/kg intraperitoneally (i.p.))-induced diabetes were divided into five groups (n = 5) and treated with (i) normal saline, (ii) 150 mg/kg body weight (BW) of the ethanol extract of *S. torvum* leaf (EESTL), (iii) 300 mg/kg BW EESTL, (iv) 100 mg/kg BW metformin, and (v) 50 m/kg BW metformin + 100 mg/kg BW EESTL orally for 21 days. Our results revealed that the EESTL displayed dose-dependent ferric-reducing antioxidant power (FRAP) activity, scavenged DPPH radicals (IC₅₀) = 13.52 ± 0.45 µg/mL), and inhibited lipid peroxidation in an *in vitro* models. In addition, the EESTL demonstrated dose-dependent inhibitory activity against α-amylase (IC₅₀ = 138.46 ± 3.97 µg/mL) and promoted glucose uptake across plasma membranes of yeast cells in a manner comparable to that of metformin. Interestingly, the extract demonstrated *in vivo* blood glucose normalization effects with concomitant increased

<footnote><sup>a</sup> Department of Biochemistry, Faculty of Biological Science, Alex Ekuweme Federal University Ndufu Alike Ikwo, PMB 1010 Abakalike, Ebonyi State, Nigeria</footnote>

<footnote><sup>b</sup> PhD Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 11031, Taiwan</footnote>

<footnote><sup>c</sup> Graduate Institute of Cancer Biology & Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan</footnote>

<footnote><sup>d</sup> Biochemistry Unit, Department of Chemical Sciences, Afe Babalola University, Ado-Ekiti, Ekiti State, Nigeria</footnote>

<footnote><sup>e</sup> Department of Medical Biochemistry, Faculty of Basic Medical Sciences, Alex-Ekuweme Federal University Ndufu Alike Ikwo, Ebonyi State, Nigeria</footnote>

<footnote><sup>f</sup> Graduate Institute of Biomedical Science, College of Medicine, China Medical University, Taipei, Taiwan</footnote>

<footnote><sup>g</sup> Department of Human Anatomy, College of Basic Medical Science, Alex Ekuweme Federal University Ndufu Alike Ikwo, PMB 1010 Abakalike, Ebonyi State, Nigeria</footnote>

<footnote><sup>h</sup> Department of Biotechnology, College of Science, Taif University, Taif 21944, Saudi Arabia</footnote>

<footnote><sup>i</sup> Smartox Biotechnology, 6 rue des Platanes, 38120 Saint-Egrève, France</footnote>

<footnote><sup>j</sup> L'institut du Thorax, INSERM, CNRS, UNIV NANTES, F-44007 Nantes, France</footnote>

<footnote><sup>k</sup> LabEx Ion Channels, Science & Therapeutics, Université de Nice Sophia-Antipolis, F-06560 Valbonne, France</footnote>

<footnote><sup>l</sup> Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University, Damanhour 22511, AlBeheira, Egypt</footnote>

<footnote><sup>m</sup> Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung 204, Taiwan</footnote>

<footnote><sup>n</sup> School of Traditional Chinese Medicine, Chang Gung University, Kweishan, Taoyuan 333, Taiwan</footnote>

<footnote><sup>o</sup> School of Nursing, National Taipei University of Nursing and Health Sciences, Taipei 112, Taiwan</footnote>

<footnote><sup>p</sup> Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan</footnote>

<footnote><sup>q</sup> Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan</footnote>

<footnote><sup>r</sup> Department & Graduate Institute of Chemical Engineering & Graduate Institute of Biochemical Engineering, Ming Chi University of Technology, New Taipei City 243, Taiwan</footnote>

<footnote><sup>s</sup> The PhD Program of Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan</footnote>

<footnote><sup>t</sup> TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei 11031, Taiwan</footnote>

<footnote><sup>u</sup> Clinical Research Center, Taipei Medical University Hospital, Taipei Medical University, Taipei 11031, Taiwan</footnote>

<footnote><sup>v</sup> Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei 114, Taiwan</footnote>

<footnote>* Corresponding author at: The PhD Program of Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan.</footnote>

<footnote>E-mail address: chaw1211@tmu.edu.tw (A.T.H. Wu).</footnote>

<footnote n="1">¹ These authors contributed equally to this work</footnote>
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activities of antioxidant parameters (superoxide dismutase (SOD), catalase, and reduced glutathione (GSH)) while decreasing malondialdehyde (MDA) levels when compared to untreated rats. Similarly, serum biochemical alterations, and tissues (liver, kidney, and pancreas) histopathological aberrations in untreated rats with STZ-induced diabetes were attenuated by treatment with the EESTL. Biometabolite characterization of the extract identified gallic acid (45.81 ppm), catechin (1.18 ppm), p-coumaric acid (1.43e⁻¹ ppm), DL-proline 5-oxomethyl ester (9.16 %, retention time (RT): 8.57 min), salicylic acid (3.26% and 7.61 min), and butylated hydroxytoluene (4.75%, RT: 10.18 min) as the major polyphenolic compounds in the plant extract. In conclusion, our study provides preclinical evidence of the antioxidant properties and oxidative stress-preventing role of *S. torvum* in STZ-dosed diabetic rats. Taken together, the EESTL represents a reserve of bioactive metabolites for managing diabetes and associated complications.

# 1. Introduction

Diabetes is a chronic, metabolic disease characterized by hyperglycemia and a variety of complications resulting from defects in insulin's action and/or secretion [1]. The rapid increase in the incidence of diabetes has imposed a severe burden on healthcare systems worldwide [2, 3]. A considerable amount of literature has shown that the generation of free radicals is associated with the DM pathogenesis [4,5]. Several studies showed that depleted levels of endogenous antioxidants in addition to increases in the generation of free radicals cause destruction of pancreatic β-cells [6], oxidative stress, excessive lipid peroxidation, and impairment of cellular organelles, which result in the development of secondary complications, including diabetic retinopathies, nephropathies, neuropathies, and cardiovascular diseases [7,8]. The early and proper management of secondary complications of diabetes is of significance in preventing diabetes-induced pathological impairments of vital organs [7,9].

Current strategies for managing diabetes include the use of pharmacological agents, physical exercise, and changes in lifestyle and diet [10,11]. However, questions have been raised about the effectiveness and safety of prolonged use of current antidiabetic agents. Conventional antidiabetic drugs are characterized by ineffectiveness and toxic side effects on vital organs including the heart, liver, kidneys, and brain [12, 13]. These global concerns have therefore prompted an urgent search for new antidiabetic agents. In recent years, there has been increasing interest in medicinal plants which are considered potential sources of affordable, safe, and more-effective antidiabetic agents [14,15]. This idea was derived from the historical use of medicinal plants for treating diabetes in traditional medicine. As an example, several antidiabetic herbal drugs have been approved for commercial use by the health regulatory agency in China [16], and other parts of the world [17]. The use of these naturally derived agents of plant origin as antidiabetics, suggests other medicinal plants may be potential reservoirs of lead antidiabetic agents. Several plants are reportedly used in folkloric medicine for managing diabetes and have received sufficient scientific validation in both *in vitro* and *in vivo* diabetic models [18–20]. One of such plant is *Solanum torvum*.

*Solanum torvum* Swartz, commonly known as Turkey berry, belongs to the family Solanaceae [21]. It is a small shrub that is widely distributed in Nigeria, Pakistan, India, Malaya, China, the Philippines, and tropical America [22]. In traditional medicine, *S. torvum* is used to treat colds and coughs, cure cracked feet, reduce body heat, control bacterial and fungal diseases, treat asthma, diabetes, hypertension, vermifuge, liver diseases, and tuberculosis, and as an anti-anemic [23]. Pharmacological studies showed that *S. torvum* possesses antiviral [24], anti-microbial [25], analgesic, anti-inflammatory [26], anti-ulcerogenic [27], nephroprotective [28], and antioxidant [29] activities. Gandhi et al. [30] reported the antidiabetic and antioxidant effects of *S. torvum* fruit extract against streptozotocin-induced diabetic rats. Several other studies have also reported the antidiabetic properties of the fruit extract of this plant [31,32]. However, far too little information is available on the antidiabetic and antioxidants effect of the leaves. In this study, we showed that the ethanol extract of *Solanum torvum* leaf (EESTL) possesses antioxidant activities in *in vitro* models, improved the uptake of

glucose by yeast cells, and inhibited activity against α-amylase in dose-dependent manners. Furthermore, the EESTL exhibited blood glucose normalization effects in rats with STZ-induced diabetes and lessened the events of oxidative stress and tissue pathophysiological aberrations in our experimental animal model.

# 2. Materials and methods

## 2.1. Plant collection and extraction

Mature leaves of *S. torvum* were obtained from the vicinity of Alex-Ekwueme Federal University Ndufu Alike Ikwo (FUNAI) (Abakaliki, Nigeria). Leaves were authenticated through the kind assistance of a botanist at Ebenyoni State University (Abakaliki, Nigeria). Leaves were prepared by washing, air-drying, and milling to a powdery form, after which a measured amount (200 g) of the powered sample was subjected to extraction by percolating in 2.5 L of 80% ethanol under continual agitation for 48 h. The extract was then filtered using filter paper (Whatman), concentrated at 40 °C using a rotary evaporator, after which it was lyophilized in a freeze-dryer to obtain the dried ethanol extract of the *S. torvum* leaf (EESTL) which was stored in an air-tight container until use.

## 2.2. Phytochemical quantification

Folin-Ciocalteau's protocols of the total phenol content assay of EESTL were based on the method of Singleton et al. [33], while the total flavonoid content of the sample was determined according to a method of Chang et al. [34]. Gallic acid and quercetin standards were used to respectively prepare the calibration curves for total phenols and total flavonoids. The total terpenoid content was quantified using the protocol of Ghorai et al. [35]. Steroids and tannins were quantified using a method of Sofowora [19], while glycoside was quantified by a method of Chassagne et al. [36].

## 2.3. Determination of in vitro antioxidant activities

**2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay:** The radical (DPPH)-scavenging effects of the EESTL was assessed according to a method of Oyaizu [37] as described by Tsado et al. [38]. Briefly, 0.1 mL of the EESTL (at a concentration range of 15.63–250 µg/mL) was added to 0.5 mL of a DPPH solution (4.5 mL of methanol, 50 mg/mL) and incubated in the dark for 45 min at room temperature. The decrease in absorbance was determined at 517 nm.

**Ferric-reducing antioxidant power (FRAP) assay:** FRAP effects of the EESTL were assessed on the basis of its ability to reduce ferric tripyridyl triazine (TPTZ) to a colored ferrous form, following the method described by Oyaizu [37]. Briefly, the EESTL (100 µL) was incubated at 37 °C with a FRAP solution (3 mL) for 30 min. The production of ferrous (Fe²⁺) through the reduction of the ferric (Fe³⁺) complex was determined by monitoring changes in the absorbance at 593 nm.

**In vitro lipid peroxidation (LPO) assay:** The inhibition of lipid peroxidation (LPO) by the EESTL was assessed by spectrophotometric measurements of thiobarbituric acid (TBA)-reactive substances
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(TBARSs) as outlined by Moore and Roberts [39]. Briefly, the EESTL at various concentrations was incubated with a TBA-trichloroacetic acid (TCA) solution at a 1:4 (v/v) ratio. The reaction mixture was incubated for 20 min in a water bath, allowed to cool, and then centrifuged for 10 min. The change in absorbance at a wavelength of 535 nm was used to monitor reaction changes.

## 2.4. Determination of in vitro hypoglycemic activities

**α-Amylase inhibition assay:** The *in vitro* α-amylase inhibitory activity of the EESTL was evaluated by following the description of Worthington [40]. Briefly, graded concentrations of the EESTL were incubated with α-amylase (0.5 mg/mL) in a 0.02 M sodium phosphate buffer solution at 25 °C for 10 min. The resulting solution was further re-incubated with 1% of a starch solution (pH 6.9) at 25 °C for 10 min, after which the reaction process was terminated following color development by the addition of the dinitrosalicylic acid reagent and incubated in a water bath at 100 °C for 5 min. The change in absorbance of the solution was observed at 540 nm.

**Glucose uptake by yeast cells:** The assay for the glucose-uptake capacity of yeast cells was carried out following a method by Cirillo [41]. The EESTL was mixed with a glucose solution (5–25 mM) at 37 °C for 10 min, followed by incubation with a prepared baker's yeast suspension (100 µL) at 37 °C for 1 h. The reaction solution was centrifuged at 2500 × g for 5 min. The level of glucose in the supernatant was assessed, and the percentage increase in glucose uptake by the yeast was computed.

## 2.5. *In vivo* antidiabetic study

Male albino Wistar rats (125.76 ± 4.72 g of BW) were obtained from the animal facility of AE-FUNAI. Rats were housed in a controlled environment with a 12-h dark/light cycle and fed a pelleted diet and water ad libitum. The *in vivo* experiments adhered to the regulations by the Ethics Committee on Animal Use of AE-FUNAI. Diabetes was inducted by injecting streptozotocin (STZ; 40 mg/kg BW, intraperitoneally (i.p.)) followed by administration of a 5% glucose solution 24 h after STZ dosing. Diabetic rats (as determined by a fasting blood glucose (FBG) level of > 250 mg/dL) [42], were subsequently separated into five groups (n = 5) and treated with (i) normal saline, (ii) 150 mg/kg body weight (BW) of the ethanol extract of *S. torvum* leaf (EESTL), (iii) 300 mg/kg BW EESTL, (iv) 100 mg/kg BW metformin, and (v) 50 m/kg BW metformin + 100 mg/kg BW EESTL. The extract and drug were reconstituted in normal saline, and treatments were administered daily for 21 days through the oral route with the aid of an oesophagal cannula. The extract doses were selected based on the literature and our pilot studies. The FBG, rat BW, and water and food consumption were monitored throughout the study period.

### 2.5.1. Blood and tissue sample collection and processing

After the final administration of the extract, rats were deprived of feed for 12 h and were euthanized under diethyl ether vapor, and blood was collected into clean tubes via cardiac puncture. Blood was allowed to coagulate and then was centrifuged at 3000 × g for 15 min to separate the serum [43,44]. Serum was aspirated and kept frozen at -20 °C [45]. The liver, kidneys, and pancreas were gently excised, rinsed in ice-cold KCl, and blotted with filter paper. Part of the liver was homogenized in 0.1 M phosphate buffer (pH 7.4), and the homogenate was centrifuged at 5000 rpm for 10 min at 4 °C. The kidney, pancreas, and remaining liver tissues were fixed with 10% formalin for a histopathological analysis.

### 2.5.2. Assays for enzymatic and non-enzymatic antioxidant parameters in tissue homogenates

Reduced glutathione (GSH) levels were determined by a modified colorimetric protocol [46]. An aliquot of the liver homogenate was precipitated with 20% TCA (200 µL) and centrifuged for 5 min at 2000 rpm and 4 °C. Thereafter, 50 µL of the supernatant was mixed with 100 and 50 µL of 1 M phosphate buffer and 5 mM dinitrothiocyanobenzene (DNTB). The LPO assay was carried out by measuring TBARSs as outlined by Shagirtha et al. [47]. Superoxide dismutase (SOD) activity in the tissue (liver) homogenate was assessed following a protocol described by Kakkar et al. [48], while a method outlined by Sinha [49] was used to assess catalase (CAT) activity in the liver homogenate.

### 2.5.3. Analysis of serum lipid profiles and biochemical parameters

Serum levels of lipid profile parameters, including total cholesterol (TC), high density lipoprotein (HDL)-cholesterol, and triglycerides (TGs), were measured by a colorimetric enzymatic approach using a Randox biochemical kit (Randox Laboratories, UK), following the description in the user's manual. Very-low-density lipoprotein (VLDL)-cholesterol was assessed as TG/5, and low-density lipoprotein (LDL)-cholesterol (mg/dl) was computed as [TC - (HDL + VLDL)] [50]. Serum levels of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALK) were analyzed by enzymatic methods using commercial biochemical kits (Olympus, Hamburg, Germany) on an automated analyzer (Olympus AU800). The serum creatinine concentration was assayed based on the formation of a colored complex when creatinine reacted with picric acid [51], while urea levels were assayed using a spectrophotometric protocol of Berthelot's reaction in the presence of urease [52].

### 2.5.4. Histopathological analysis of the liver, kidneys, and pancreas

Liver, kidney, and pancreas tissues were prepared for microscopic evaluation of organ histology by a conventional paraffin-embedded protocol [53]. Liver, kidney, and pancreas sections were embedded in paraffin and stained with hematoxylin and eosin (H&E). Pathophysiological changes were observed under a light microscope (Leica DM2500, Wetzlar, Germany) with a 40x objective and total magnification of 400x.

## 2.6. Characterization by gas chromatography-mass spectroscopy (GC-MS)

The EESTL was subjected to GC-MS characterization using a fused silica column and capillary column (250 µm in diameter, 30 m long, and 0.25 µm thick) in a Clarus 500 PerkinElmer system (PerkinElmer, city, USA) (with an AOC-20i auto-sampler). Relative amounts of the phytoconstituents are presented as the percentage with area peak normalization [54]. The compounds were identified based on comparisons of peak areas/heights, retention times, and mass spectral patterns with a database of authentic compounds in the library of National Institute of Standards and Technology (NIST).

## 2.7. Characterization by high-performance liquid chromatography (HPLC)

The EESTL was characterised by an HPLC analysis according to a description in our earlier study [55]. One hundred milligrams (100 mg) of the extract was dissolved in 5 mL of HPLC-grade methanol. The extract solution was filtered and run on an HPLC system (1200; Agilent Technologies) under standard chromatographic conditions of a mobile phase of acetonitrile (ACN) and 0.1% formic acid, a stationary phase of Hypersil C18, column dimensions of 250 × 4.0 mm, a flow rate of 0.6 mL/min, an injection volume of 10 µL, a detector wavelength of 280 nm, and gradient elution. Molecular docking studies was docking was conducted using Schrödinger tool. The crystal structures of glucose metabolizing enzyme (alpha amylase) was obtained from the PDB database, while the ligand structures were prepared via the Avogadro molecular builder and visualization tool [56]. The receptor and ligand preparations were conducted as described previous studies [57–60], The docked complexes were analyzed for interaction affinities as expressed in kcal/mol.
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<caption>**Table 1**</caption>

Phytochemical composition of the ethanol extract of *Solanum torvum* leaf (EESTL).

<table><thead><tr><th>Phytochemicals in the EESTL</th><th>Inference</th><th>Conc. (mg/100 g)</th></tr></thead><tbody><tr><td>Steroids</td><td>+</td><td>0.61 ± 0.10</td></tr><tr><td>Saponins</td><td>-</td><td>ND</td></tr><tr><td>Terpenoids</td><td>+</td><td>5.58 ± 0.72</td></tr><tr><td>Glycosides</td><td>++</td><td>7.81 ± 0.01</td></tr><tr><td>Tannins</td><td>++</td><td>10.50 ± 0.20</td></tr><tr><td>Flavonoids</td><td>++</td><td>22.09 ± 0.11</td></tr><tr><td>Alkaloids</td><td>+</td><td>92.78 ± 2.78</td></tr><tr><td>Reducing sugar</td><td>++</td><td>116.10 ± 1.10</td></tr><tr><td>Total phenols</td><td>++</td><td>687.50 ± 42.76</td></tr></tbody></table>

+, positive; ++, strong positive; -, negative. Value are mean ± standard error of the mean (SEM) of three replicate. ND; not detected

## 2.8. Statistical analysis

All analyses were conducted on several replicates, and data were analyzed using GraphPad prism software vers. 7.0. Results were statistically analyzed via a one-way analysis of variance (ANOVA) and Tukey's multiple-range post-hoc test. In addition, Student's t-test was used to evaluate the statistical significance between groups. All experimental data are expressed as the mean of at least three replicates ± standard error of the mean (SEM). Significant differences were accepted at *p* < 0.05, **p** < 0.01, ***p** < 0.001, and ****p** < 0.0001.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** : *In vitro* antioxidant effects of the ethanol extract of *Solanum torvum* leaf (EESTL). Effect of the EESTL and a standard drug on (A) DPPH, (B) thiobarbituric acid-reactive substances (TBARS), and (C) ferric-reducing activity power (FRAP). Values are the mean±SEM. of triplicate data.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** : *In vitro* hypoglycemic effects of the ethanol extract of *Solanum torvum* leaf (EESTL). Effect of EESTL on (A) α-amylase and (B) glucose uptake by yeast cells.</figcaption></figure>
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<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Effects of the ethanol extract of *Solanum torvum* leaf (EESTL) on (A, B) fasting blood glucose (FBG) levels, (C) average water intake, and (D) average food intake of rats with streptozotocin-induced diabetes. Histograms represent the mean ± SEM (n = 5). For all panels: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.</figcaption></figure>

### 3. Results

#### 3.1. Phytochemicals, in vitro antioxidants, and anti-hyperglycemic effects of the EESTL

We evaluated the phytochemical constituents in the EESTL and used different *in vitro* approaches to evaluate its antioxidant and anti-hyperglycemic properties. Our results revealed that phenols are the most abundant phytochemicals composition of EESTL. The extract also contains good amounts of alkaloids, reducing sugar and flavonoids while saponins are not detected (Table 1). We demonstrated that the EESTL displayed dose-dependent DPPH free radical-scavenging activities with an IC₅₀ of 13.52 ± 0.45 µg/mL (Fig. 1A). In addition, the EESTL also

demonstrated *in vitro* antioxidant activities in TBARS (Fig. 1B) and FRAP (Fig. 1C) assays. However, activity of the extract against TBARS was found to be dose-independent; It increase from 15.63 to 31.25 µg/mL after which a lower activity was observed at higher doses. We next examined the anti-hyperglycemic effect of the EESTL using *in vitro* models. Results showed that the EESTL demonstrated concentration-dependent α-amylase inhibitory activity with an IC₅₀ value of 138.46 ± 3.97 µg/mL (Fig. 2A). The EESTL also promoted glucose uptake across plasma membranes of yeast cells in a manner comparable to that of metformin (Fig. 2B). Taken together, our data strongly suggested promising antioxidant and hypoglycemic properties of the EESTL, thus forming the basis for the *in vivo* evaluation of its efficacy.

<caption>**Table 2**</caption>

Effect of the ethanol extract of *Solanum torvum* leaf (EESTL) on body weight (BW) changes in rats with streptozotocin-induced diabetes.

<table><thead><tr><th>Group</th><th>DAY 1</th><th>Day 7</th><th>Day 14</th><th>Day 21</th><th>Weight gain/loss</th></tr></thead><tbody><tr><td>Normal control</td><td>132.81 ± 2.42</td><td>137.73 ± 6.93</td><td>145.62 ± 7.41</td><td>140.31 ± 4.096</td><td>7.51 ± 1.66<sup>***</sup></td></tr><tr><td>Diabetic control</td><td>141.33 ± 8.63</td><td>125.51 ± 9.51</td><td>137.82 ± 1.60</td><td>134.80 ± 7.71</td><td>-6.20 ± 0.91<sup>**</sup></td></tr><tr><td>Diabetic+ 150 mg/kg BW EESTL</td><td>148.41 ± 2.92</td><td>122.54 ± 2.50</td><td>153.50 ± 13.55</td><td>150.72 ± 10.74</td><td>2.30 ± 7.81<sup>**</sup></td></tr><tr><td>Diabetic+ 300 mg/kg BW EESTL</td><td>136.70 ± 4.41</td><td>128.53 ± 8.50</td><td>138.80 ± 4.71</td><td>142.83 ± 4.69</td><td>6.11 ± 0.28<sup>**</sup></td></tr><tr><td>Diabetic+ 100 mg/kg BW met.</td><td>137.41 ± 4.33</td><td>122.50 ± 12.50</td><td>155.43 ± 4.60</td><td>140.31 ± 4.09</td><td>2.92 ± 0.23<sup>***</sup></td></tr><tr><td>Diabetic+EESTL+met.</td><td>132.82 ± 2.42</td><td>133.52 ± 5.51</td><td>135.14 ± 4.24</td><td>137.33 ± 6.36</td><td>6.50 ± 3.93<sup>***</sup></td></tr></tbody></table>

Data are presented as the mean ± SEM (n = 5).

<footnote>\*\*\* *p* < 0.001,</footnote>

<footnote>\*\*\*\* *p* < 0.0001. met., metformin.</footnote>
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<figure><img src="image_6.png" /><figcaption>**Fig. 4.** : Oxidative stress biomarkers in the liver of diabetic rats treated with the ethanol extract of *Solanum torvum* leaf (EESTL). Representative histograms showing the effect of the EESTL on (A) catalase (CAT), (B) superoxide dismutase (SOD), (C) reduced glutathione (GSH), and (D) malondialdehyde (MDA) in the liver of rats with streptozotocin-induced diabetes. Histograms represent the mean±SEM (n = 5). For all panels: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001; ns, non-significant.</figcaption></figure>

## 3.2. Effects of the EESTL on fasting blood glucose levels, BW, and food and water intake by rats with streptozotocin-induced diabetes

The effect of the EESTL on fasting blood glucose levels of rats with STZ-induced diabetes is shown in Fig. 3A. We demonstrated that i.p. administration of 40 mg/kg BW of STZ caused a significant elevation in the FBG (Fig. 3A, B) and loss of BW (Table 2) in experimental rats compared to the normal glycemic rat controls. Moreover, treatment with the EESTL and the standard drug alone or in combination caused a significant decrease in FBG compared to untreated diabetic rats (Fig. 3A). In addition, a significant improvement in BW was recorded in rats receiving the extract compared to untreated diabetic rats (Table 2). We next evaluated the effect of the EESTL on food and water intake of rats with STZ-induced diabetes. Results demonstrated significant decreases in the levels of water (Fig. 3C), and food intake (Fig. 3D) of diabetic rats administered different doses of the EESTL and metformin alone or in combination compared to untreated diabetic control rats.

## 3.3. Effect of the EESTL on oxidative stress in the liver

We evaluated the protective role of the EESTL in hepatic oxidative stress induced by STZ administration. We found that the EESTL exhibited dose-dependent antioxidant activity by significantly increasing the activities of the liver enzymatic antioxidants, including

CAT (Fig. 4A) and SOD (Fig. 4A), and the level of the non-enzymatic antioxidant GSH (Fig. 4C) compared to untreated diabetic rats. The antioxidant activities displayed by the EESTL at 300 mg/kg BW were higher compared to the antioxidant activities displayed by the standard drug (metformin). On the other hand, levels of the stable end-product of lipid peroxidation, MDA, had significantly decreased in rats treated with the EESTL and metformin alone or in combination compared to untreated diabetic rats (Fig. 4D). Overall, these results indicated that the EESTL exhibited significant antioxidant activities *in vitro* and *in vivo* in the liver of rats with STZ-induced diabetes, thus suggesting that the EESTL could have a beneficial effect on the liver if used as a hypoglycemic agent to treat diabetes.

## 3.4. The EESTL attenuated diabetes-induced dyslipidemia

We next examined the effects of the EESTL on lipid profiles of experimental animals. Data indicated a significant decrease in serum levels of HDL-cholesterol (Fig. 5A), and significant increases in serum triglycerides (TGs) (Fig. 5B), LDL-cholesterol (Fig. 5C) and total cholesterol (Fig. 5D) in untreated diabetic rats compared to normal control rats. Oral administration of the EESTL (at 150 and 300 mg/kg BW) and metformin each alone or in combination significantly decreased TC, TGs, and LDL-cholesterol and increased HDL-cholesterol compared to untreated diabetic control rats, thus indicating the
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** The ethanol extract of *Solanum torvum* leaf (EESTL) attenuated diabetes-induced dyslipidemia in rats with streptozotocin-induced diabetes. Levels of (A) high-density lipoprotein-cholesterol (HDL-C) (B) triglycerides (TGs), (C) low-density lipoprotein-cholesterol (LDL-C), and (D) total cholesterol (TC) in serum of rats with streptozotocin-induced diabetes. Histograms represent the mean±SEM (n = 5). For all panels: \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; ns, non-significant.</figcaption></figure>

hypolipidemic property of *S. torvum*.

**3.5. Effects of the EESTL on biochemical parameters of hepatorenal integrity**

We evaluated the protective effects of the EESTL on STZ-induced serum biochemical alterations. Our results revealed that the activities of serum ALT (Fig. 6A), ALP (Fig. 6B), AST (Fig. 6C), creatinine (Fig. 6D), and urea (Fig. 6E) significantly increased in untreated diabetic rats compared to normal control rats. Treatment with the EESTL, however, significantly reduced the activities of ALT, AST, and ALP, and concentrations of creatinine and urea compared to untreated diabetic rats. Taken together, our data suggest the protective effect of the EESTL against hepatotoxicity produced by STZ administration, and it can ameliorate liver injury in rats with T2DM.

**3.6. Effect of the EESTL on pancreatic tissue histology**

A photomicrograph of pancreatic tissue sections of normal control rats revealed normal well-perfused pancreatic tissues with active islets of Langerhans, pancreatic acini and well outlined acini cell (Fig. 7A). STZ-administration results in severe pancreatic degeneration with ground glass (GG) pancreatic acni, appearance of necrosis in islets of Langerhans, focal area of hemorrhage, loss of pancreatic tissue, and a non-distinct appearance of the acini cells (Fig. 7B). Oral administration

of the EESTL (150 mg/kg BW) improved the histological appearance of the pancreases, with the appearance of moderate ground glass pancreatic acini, pyknotic islet of Langerhan and focal area of hemorrhage (Fig. 7C). While treatment with the 300 mg/kg BW produced more pronounced amelioration of the pancreatic histopathology (Fig. 7D). Treatment with metformin results in appearance of an active pancreatic acini cells and islet of Langerhans. However, mild aggregate of inflammatory cells and congestion of the blood vessel were detected (Fig. 7E). Treatment with metformin combination with the extract result in better histological architecture with the appearance of mild fibrosis, and aggregate of inflammatory cell around the fibrotic area when compared with rats treated with metformin alone (Fig. 7F).

**3.7. Effects of the EESTL on liver histology**

The beneficial role of the EESTL on liver injury was demonstrated using H&E staining of rat liver sections. Photomicrograph of liver tissue sections of normal control (non-diabetic) rats revealed a normal hepatic architecture with portal triad, central vein, and hepatocytes (Fig. 8A). Intraperitoneal administration of STZ to experimental rats resulted in severe degeneration, moderate portal inflammation, and severe intrahepatic inflammation (Fig. 8B). As expected, only minor histopathological changes, including mild portal inflammation and mild intrahepatic inflammation were observed in experimental animals administered the EESTL at 150 and 300 mg/kg BW (Fig. 8C, and D). Rats
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Effects of the ethanol extract of *Solanum torvum* leaf (EESTL) on biochemical parameters associated with the hepatorenal integrity of rats with streptozotocin-induced diabetes. Levels of (A) alanine transaminase (ALT), (B) alkaline phosphatase (ALP), (C) aspartate transaminas (AST), (D) creatinine, and (E) urea in serum of rats with streptozotocin-induced diabetes. Histograms represent the mean±SEM (n = 5). For all panels: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.</figcaption></figure>

treated with metformin shows normal liver histology with mild intra hepatic inflammation and mild congestion of the central vein (Fig. 8E). This result was similar to that of diabetic rats treated with the combination of the extract and metformin (Fig. 8F). Together these results provide important insights into the protective role of the EESTL against liver injury.

### 3.8. Effects of the EESTL on kidney histology

A photomicrograph of kidney tissue sections of normal control rats revealed a normal renal architecture with glomeruli, Bowman space, renal tubules, and tubular cells (Fig. 9A). STZ administration results in appearance of severe coagulative necrosis of the renal tubules, severe intra renal inflammation, tubular and glomerular atrophy, and severe

intrarenal hemorrhage (Fig. 9B). Interestingly, oral administration of the EESTL (150 mg/kg BW) showed mild regeneration with moderate fatty change (steatosis) and intra renal inflammation when compared with the STZ-induced diabetic non treated rats (Fig. 9C). Similarly, a high dose of the EESTL (300 mg/kg BW) showed partially preserved kidney histology with only minor extravasations of the blood within the glomeruli and a pyknotic appearance of the glomeruli (Fig. 9D). Rats treated with metformin showed remarkable histological differences compared with the STZ-induced diabetic non treated rats. They had regular kidney architecture, mild intra hepatic inflammation, and very minor fatty changes (Fig. 9E). Similar effects were found in rats treated with the combination of metformin and extract (Fig. 9F).
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Histopathological changes in the pancreas of diabetic rats administered the ethanol extract of *Solanum torvum* leaf (EESTL). A) Normal control, B) untreated diabetes, C) 150 mg/kg body weight (BW) EESTL, D) 300 mg/kg BW EESTL, 4) 100 mg/kg BW metformin, F) 50 mg/kg BW metformin + 100 mg/kg BW EESTL. ILs: islets of Langerhans, PA: pancreatic acini, H: hemorrhaging, PPA: pyknotic pancreatic acini, N: necrotic, AICs: aggregates of inflammatory cells, CBV: congestion of the blood vessel, P: pyknotic islet of Langerhans, and F: fibrosis.</figcaption></figure>

### 3.9. Characterization of the *S. torvum* leaf extract

The current study found that the EESTL demonstrated promising *in vitro* and *in vivo* antioxidant and antidiabetic properties. Therefore, we next characterized the extract using GCMS and HPLC analyses. Our results revealed the presence of several bioactive compounds in the leaf extract of *S. torvum* (Fig. 10, Table 3). The three major polyphenolic compounds identified were DL-proline 5-oxo-methyl ester (9.16 %, retention time (RT): 8.57 min), salicylic acid (3.26%, 7.61 min), and butylated hydroxytoluene (4.75 %, RT: 10.18 min). Other biologically active compounds identified included azelaic acid and several fatty acids.

The HPLC analysis revealed the presence of five polyphenolic compounds including gallic acid, catechin, p-coumaric acid, fisetin, and myricetin with retention times of 4.66, 12.82, 19.80, 26.90, and 27.86 min, respectively (Fig. 12). Gallic acid (45.81 ppm), catechin (1.18 ppm), and p-coumaric acid (1.43e-1 ppm) were the most abundant bioactive compound present in the EESTL as characterized by the HPLC. Molecular docking is a simulation of binding interactions between protein and drug candidate for targeted inhibition of the protein [90,91] Interesting, our molecular docking profile revealed that the identified compounds particularly Myricetin (–9.11 kcal/mol) demonstrated high and strong binding efficacy with glucose metabolizing

enzyme (Fig. 12). Fisetin, Catechin, gallic acid, and Cis-P coumaric also demonstrated target binding affinities of – 6.99, – 6.41, – 4.88 and – 4.39 kcal/mol respectively against the glucose metabolizing enzymes. The ligand-receptor complexes are stabilized by several hydrogen bonding, hydrophobic, and halogen interactions, and several pi-interactions including pi-cation, and pi-pi stacking (Fig. 12). These compounds were implicated in various biological and therapeutic efficiencies of several medicinal plants. Overall, it is possible to hypothesize that the presence of these compounds in the EESTL was responsible for the observed reductions in the deleterious effects of ROS in the liver, pancreas, and kidneys, and they thus protected the experimental animals against diabetes-induced oxidative stress.

### 4. Discussion

The ineffectiveness of conventional antidiabetics and the increased global burden of this disease are reflected in increasing calls for a search for more-effective therapeutic options with few or no side effects [61]. Medicinal plants are considered an important therapeutic option that could provide a cheap and safe source of more-effective antidiabetic agents [18]. In this study, we documented the *in vitro/in vivo* antidiabetic and antioxidant potential of the EESTL in rats with STZ-induced diabetes.
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Light photomicrographs of Masson-stained sections of the liver. A) Normal control, B) untreated diabetic rats, C) 150 mg/kg body weight (BW) of the ethanol extract of *Solanum torvum* leaf (EESTL), D) 300 mg/kg BW of the EESTL, E) 100 mg/kg BW of metformin, F) 50 mg/kg BW of metformin + 100 mg/kg BW of the EESTL. PT: portal triad, CV: central vein, PI: portal inflammation, IHI: intrahepatic inflammation (IHI).</figcaption></figure>

Suppression of carbohydrate-hydrolyzing enzymes such as the pancreatic α-amylase is considered an important therapeutic approach for regulating postprandial blood glucose levels [7,62,63]; however, synthetic inhibitors of this enzymes are besieged with side effects. These led to a heightened search for pharmacologically active phytoconstituents as inhibitors of these enzymes [40,64,65]. It was interesting to note that the EESTL effectively inhibited the activity of pancreatic α-amylase. This would block the digestion of polysaccharides into absorbable monosaccharides, thus leading to retardation of an elevation of blood sugar and suppression of postprandial hyperglycemia [20,66]. The transport of glucose across membranes of yeast cells is considered an interesting therapeutic approach for *in vitro* screening of hypoglycemic agents. Studies suggested facilitated diffusion of glucose in yeast cells down the concentration gradient, which effectively functions based on the removal of intracellular glucose [67,68]. Interestingly, the EESTL promoted glucose uptake across plasma membranes of yeast cells in a manner comparable to that of metformin. The EESTL also demonstrated dose-related antioxidant activities. Generally, *in vitro* antioxidant and hypoglycemic activities demonstrated by the EESTL were lower than those for the standard drug. However, the EESTL was more effective in scavenging DPPH free radicals compared to ascorbic acid. Taken together, our data strongly suggested promising antioxidant and hypoglycemic properties of the EESTL, thus forming the rationale for our *in vivo* evaluation of its efficacy.

The increased fasting blood glucose concentration coupled with the loss of BW recorded in STZ-intoxicated rats could have resulted from oxidative stress induced by treatment with STZ, which agrees with findings from previous studies [9,55]. The decreased BW might have resulted from increased catabolism of fats and proteins, which consequently leads to a reduction in muscle mass and loss of weight. Importantly, our results demonstrated blood glucose normalization effect and improved BW in diabetic rats treated with the EESTL, thus suggesting an antidiabetic effect of *S. torvum*.

The pathogenesis of diabetes is associated with oxidative stress. Considering the implied role of oxidative stress in the etiology of diabetes [6,69], the use of antioxidants is an indispensable therapeutic approach for its treatment. The prevention of free radical-induced oxidative stress by antioxidants of plant origins has been attributed to the reducing power of the active principles to donate hydrogen. Our *in vitro* study revealed that the EESTL effectively scavenged free radicals and inhibited the generation of lipid peroxidation. These findings hinted at the potential of the EESTL to prevent lipid peroxidation of tissues and manage oxidative stress in diabetes mellitus.

Several antioxidant enzymes present in the human system, including SOD, CAT, and GSH, are important parts of the defense mechanism that protects cells from oxidative damage [44,70]. For example, GSH was reported to play important roles in detoxification and metabolism either as a cofactor or as a substrate for other enzymes, thus serving as an
<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Histopathological changes in the kidneys of diabetic rats administered the ethanol extract of *Solanum torvum* leaf (EESTL). A) Normal control, B) untreated diabetes, C) 150 mg/kg body weight (BW) EESTL, D) 300 mg/kg BW EESTL, E) 100 mg/kg BW metformin, F) 50 mg/kg BW metformin + 100 mg/kg BW EESTL. G: glomeruli, BS: bowman space, RTS: renal tubules, TCs: tubular cells, CN: coagulative necrosis, IRI: intrarenal inflammation, GA: tubular and glomerular atrophy, IRH: severe intrarenal hemorrhaging, EB: extravasations of the blood, P: pyknotic, FC: fatty changes.</figcaption></figure>

antioxidant agent that protects tissues from oxidative stress [71]. SOD and CAT are two major free radical-scavenging enzymes. While SOD detoxifies superoxide anions by converting them to H₂O₂ and water, on the other hand, CAT catalyses the reduction of H₂O₂, thus protecting tissues from damage caused by hydroxyl radicals [72]. In the current study, the increased lipid peroxidation and reduction of the antioxidant enzyme activities in rats with STZ-induced diabetes indicated the increased generation of free radicals in the liver of untreated diabetic rats, leading to oxidative damage to membranes, proteins, and lipids with consequential decreases in endogenous levels of antioxidants (SOD, CAT, and GSH) [9]. Interestingly, our data revealed that the EESTL decreased hepatic MDA levels and increased hepatic GSH and levels of the antioxidants enzymes in STZ-dosed rats.

STZ is a cytotoxic agent that induced the generation of free radicals which led to the destruction of β cells of pancreatic islets, leading to decrease insulin secretion, insulin resistance, high blood glucose levels, and decrease glucose utilization by various tissues [73]. Therefore, the blood glucose normalization effects and the histological preservation of the pancreas in extract-treated rats could partially be attributed to the antioxidants activities of the extract (as revealed by *in vitro* and *in vivo* data) which causes improvement of pancreatic function, insulin secretion from β-cell of islets, augmenting insulin sensitivity [74], and thus restored the glycemic status of the rats. The hypoglycemic effect

observed could also be attributed to the induction of effective glucose uptake and utilization [75] by the extract as observed in our *in vitro* studies. Our study therefore provides preclinical evidence of the hypoglycemic, antioxidant properties and oxidative stress-preventing role of the EESTL in STZ-dosed rats. The findings observed in this study mirror those of previous studies, where natural antioxidants were used to treat oxidative stressed rats [76-78]. These findings are also supported by histomorphological improvements in EESTL-treated rats compared to the untreated controls.

Collectively, results of the present study corroborate with the findings of Gandhi et al. [30] who worked on the antidiabetic and antioxidant effects of the fruit extract of *Solanum torvum* against streptozotocin-induced diabetic rats. However, Iyer et al. [79] found that the dietary supplementation with dried fiber of *Solanum torvum* fruit produced no significant effects on the levels of glucose, glycated proteins, and lipid profile in patients with T2DM. This inefficacy could be attributed to the use of the sun-dried fibre content of *Solanum torvum* as opposed to the phytochemical-rich extract used in the preclinical studies [30-32]. Nevertheless, our findings have contributed to the existing knowledge by providing the preclinical evidence of the therapeutic efficacy of *Solanum torvum* leave extract in modulating the glycemic levels, lipid profile and oxidative stress in DM Taken together, our data suggest that the EESTL could be effective in managing oxidative impairment in
<figure><img src="image_12.png" /><figcaption>Fig. 10. Gas chromatographic-mass spectrometric (GC-MS) chromatogram of the leaf extract of *Solanum torvum*.</figcaption></figure>

<table><caption>**Table 3**
Phytoconstituents identified in the leaf extract of *Solanum torvum* by GC-MS.</caption><thead><tr><th>Compound name</th><th>RT</th><th>Area (%)</th><th>Molecular formula</th><th>MW (g/mol)</th><th>Compound nature</th></tr></thead><tbody><tr><td>Ethyl 2-cyanopropionate</td><td>4.75</td><td>1.40</td><td>C6H9NO2</td><td>82.10</td><td></td></tr><tr><td>Salicylic acid</td><td>7.61</td><td>3.26</td><td>C7H6O3</td><td>138.12</td><td>Phenol</td></tr><tr><td>DL-Proline, 5-oxo-, methyl ester</td><td>8.57</td><td>9.16</td><td>C6H9NO3</td><td>143.14</td><td>Flavonoid</td></tr><tr><td>Ethene, 1-(anthracen-9-yl)- 2-(5-nitro-1-naphthyl)-, (E)-</td><td>9.30</td><td>1.18</td><td>C26H17NO2</td><td>375.43</td><td>Anthracene</td></tr><tr><td>Butylated Hydroxytoluene</td><td>10.18</td><td>4.75</td><td>C15H24O</td><td>220.35</td><td>Phenol</td></tr><tr><td>Azelaic acid</td><td>11.07</td><td>1.24</td><td>C9H16O4</td><td>188.22</td><td>Organic compound</td></tr><tr><td>Ethanone, 1-[2-methyl-5-(1-methylethenyl)cyclopentyl (1.alpha.,2.alpha.,5 beta.)</td><td>13.86</td><td>0.71</td><td>C11H18O</td><td>166.26</td><td></td></tr><tr><td>n-Hexadecanoic acid</td><td>15.05</td><td>9.81</td><td>C16H32O2</td><td>256.40</td><td>Fatty acid</td></tr><tr><td>9,12-Octadecadienoic acid, methylester</td><td>16.27</td><td>2.22</td><td>C19H34O2</td><td>294.50</td><td>Fatty acid</td></tr><tr><td>Methyl stearate</td><td>16.55</td><td>11.55</td><td>C19H38O2</td><td>298.50</td><td>Fatty acid</td></tr><tr><td>Linoelaidic acid</td><td>16.64</td><td>1.99</td><td>C18H32O2</td><td>280.40</td><td>Fatty acid</td></tr><tr><td>Octadecanoic acid</td><td>16.90</td><td>6.59</td><td>C18H36O2</td><td>284.48</td><td>Fatty acid</td></tr><tr><td>Eicosanoic acid, methyl ester</td><td>18.31</td><td>1.82</td><td>C21H42O2</td><td>326.55</td><td>Fatty acid ester</td></tr><tr><td>9-Octadecenamide</td><td>18.65</td><td>1.07</td><td>C18H35NO</td><td>281.50</td><td></td></tr><tr><td>Docosanoic acid, methyl ester</td><td>20.56</td><td>2.22</td><td>C23H46O2</td><td>354.61</td><td>Fatty acid</td></tr><tr><td>1,1':4',1'':4'',1'':4'',1'''':4''',1'''':Quinquephenyl</td><td>21.10</td><td>2.13</td><td>C30H22</td><td>382.50</td><td></td></tr></tbody></table>

RT, retention time; MW, molecular weight.

<figure><img src="image_13.png" /><figcaption>Fig. 11. HPLC chromatogram of compounds identified from the leaf extract of *Solanum torvum*.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Fig. 12.** Molecular docking profile of the identified compounds identified from the leaf extract of *Solanum torvum* with glucose metabolizing enzymes</figcaption></figure>

diabetes.

Studies showed that levels of common biochemical markers of liver damage, including ALP and transaminases are known to increase following liver damage [80,81]. Therefore, the increased activities of these enzymes following i.p. administration of STZ in experimental rats is an indicator of the hepatotoxic effect of STZ. However, administration of the EESTL resulted in decreased activities of ALT, ALP, and AST, suggesting a hepatoprotective role of the EESTL in liver cells against free radical damage.

Several metabolic disorders including hyperlipidemia were reported to result from insulin resistance [82]. Reduced action of insulin leading to increased lipase activity with consequential release of free fatty acids from adipose tissues is the main cause of hyperlipidemia [83]. An accumulation of fat in the liver due to disorders of lipid metabolism, leading to a reduced detoxification capacity of the liver, is among the major problems of T2DM [84]. The current study demonstrated the efficacy of the EESTL in modulating hyperlipidemia induced by STZ administration. We found that the EESTL effectively reduced levels of TGs, TC, and LDL-cholesterol, while concurrently elevating the HDL-cholesterol level compared to untreated rats. Findings of the current study are consistent with those of Eze et al. [85] who demonstrated the potency of plant extracts in alleviating hyperlipidemia.

Our histological study of the livers of STZ-induced diabetic untreated rats showed severe degeneration, moderate portal inflammation (PI) and severe intrahepatic inflammation (IHI). Interestingly, the degenerative changes and severe PI and IHI were ameliorated upon treatment with the EESTL, thus suggesting the hepatoprotective effect of the EESTL as a potent antioxidant. Similarly, histological impairments of the

kidneys and pancreas were attenuated by extract treatment when compared with the diabetic non treated rats. Conclusively, our serum and tissue biochemical analyses correlated with the histological observations, and strongly suggested the therapeutic role of the EESTL in managing DM and its associated complications.

The liver is a central organ that plays a significant function in regulating glucose homeostasis [86]. As a group of insulin-sensitive tissues, the liver is among the primary organs highly susceptible to the effects of oxidative stress and hyperglycemia, [87]. The generation of free radicals during hyperglycemia results in oxidative modifications of cellular macromolecules (lipids, proteins, and carbohydrates) [73], and triggered a cascade of inflammatory events that damages the hepatocytes [88,89]. Therefore, the beneficial effect of the extract on biochemical and tissue histology could be attributed to the concerted effects of both the glucose normalization and antioxidants effects of the plant extract.

## 5. Conclusions

In conclusion, our study provides preclinical evidence of the antioxidant properties and oxidative stress-preventing role of *Solanum torvum* in STZ-dosed diabetic rats. Characterization of the extract revealed the presence and abundance of several bioactive polyphenols with implied roles in the biological activities of natural products. Taken together, the EESTL therefore represents a reserve of bioactive metabolites for managing diabetes and its associated complications.
## Funding

Alexander TH Wu is funded by the Ministry of Science and Technology with grant numbers: 111-2314-B-038-098 and 111-2314-B-038-142. Tse Hung Huang was supported by grant nos. CMRPG2H0362 and CORPG2J0042.

## CRediT authorship contribution statement

**Sani Saidu:** Conceptualization, Methodology, Data analysis, Software, Investigation, Writing – original draft preparation. **Bashir Lawal:** Conceptualization, Methodology, Data analysis, Software, Investigation, Visualization, Writing – original draft preparation. **Jerius N. Ejeje:** Conceptualization, Methodology, Data analysis, Software, Investigation, Writing – original draft preparation. **Tawakalitu B. Ali:** Conceptualization, Methodology, Data analysis, Software, Investigation, Writing – original draft preparation. **Amos Sunday Onikanni:** Conceptualization, Methodology, Data analysis, Software, Investigation, Writing – original draft preparation. **Onwe O. Uchewa:** Conceptualization, Data curation, Data analysis, Software, Writing – review & editing. **Joy C. Ovoh:** Conceptualization, Data curation, Data analysis, Software, Writing – review & editing. **Faith U. Ekpa:** Conceptualization, Methodology, Data analysis, Software, Investigation, Writing – original draft preparation. **Chikezie D. Ozoagu:** Data curation, Investigation, Software, Validation, Writing – review & editing. **Tochukwu S. Akuma:** Data curation, Investigation, Software, Validation, Writing – review & editing. **Success C. Onyeji:** Data curation, Investigation, Software, Validation, Writing – review & editing. **Amara Obialor:** Data curation, Investigation, Software, Validation, Writing – review & editing. **Saqer S. Alotaibi:** Data curation, Investigation, Software, Validation, Writing – review & editing. **Sarah M. Albogami:** Data curation, Investigation, Software, Validation, Funding acquisition, Writing – review & editing. **Michel Dewaard:** Conceptualization, Investigation, Writing – review & editing, Software, Funding acquisition. **Gaber El-Saber Batih:** Conceptualization, Supervision, Writing – review & editing, Software, Funding acquisition. **Alexander T.H Wu:** Conceptualization, Methodology, Data analysis, Software, Funding, Supervision, Writing – review & editing. **Tse Hung Huang:** Methodology, Data analysis, Software, Investigation, Funding acquisition, Writing – review & editing.

## Author contributions

All authors participated in the study design, data accumulation, analysis, and manuscript preparation. All authors read and approved the final version of the manuscript.

## Conflict of interest statement

The authors declare no conflict of interest.

## Acknowledgements

Acknowledgment: M.D.W. thanks the French Agence Nationale de la Recherche and the Région Pays de la Loire for financial support (CoV2-E-TAR[1]GET-grant number: 2020 07132). Author would also like to acknowledge the Taif University Researchers Supporting Project [number TURSP-2020/202], Taif University, Taif, Saudi Arabia.

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