Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 12, Number 1, April 2023 | Pages: 33-44 | DOI: 10.14421/biomedich.2023.121.33-44 ISSN 2540-9328 (online) Solanum anomalum Leaf Extract and Fractions Attenuate Oxidative Stress and Liver Injuries in Alloxan-Induced Diabetic Rats Idongesit Charles Etuk1, John Akpan Udobang2, Nwakaego Omonigho Ebong3, Jude Efiom Okokon1,* 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria. 2Department of Clinical Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, University of Uyo, Uyo, Nigeria. 3Department of Pharmacology and Toxicology, Faculty of Pharmacy, Madonna University, Elele Campus. Rivers State, Nigeria. Corresponding author* judeefiom@yahoo.com Manuscript received: 03 September, 2022. Revision accepted: 20 September, 2022. Published: 04 October, 2022. Abstract The leaf of Solanum anomalum used in ethnomedicine for the treatment of various ailments such as diabetes was evaluated for antioxidative stress and hepatoprotective potentials against hepatic injuries in alloxan-induced diabetic rats. Antioxidative stress and hepatoprotective activities of leaf extract and fractions (70-210 mg/kg) were assessed by determining oxidative stress markers levels, liver function indices and histopathological study of livers of treated rats. The leaf extract and fractions caused significant (p<0.05 – 0.001) increases in the levels of oxidative stress markers (SOD, CAT, GPx, GSH) in the livers of the treated diabetic rats. The extract/fractions treatment caused reduction in liver enzymes (ALT, AST and ALP), total and direct bilirubin. Histology of the livers revealed absence or significant reductions in pathological features in the treated diabetic rats compared to untreated diabetic rats. The results show that the leaf extract and fractions of S. anomalum has antioxidative stress and hepatoprotective potentials which may be due to the antioxidant activities of their phytochemical constituents. Keywords: Solanum anomalum; Medicinal plant; liver protective; antioxidant; antioxidative stress. Abbreviations: Gas chromatography mass spectrometry (GCMS) INTRODUCTION Diabetes mellitus is a chronic metabolic disorder which is associated with increased generation of free radicals especially Reactive Oxygen Species (ROS) (Okutana et al., 2005). Alloxan biotransformation to dialuric acid in the body is accompanied by generation of H2O2, •OH and superoxide radicals via iron catalyst which attack organs like kidney, liver and pancreas etc (Mathews and Leiter, 1999) and cause oxidative stress which is implicated in the pathogenesis of diabetes complications in animals and humans (Baynes and Thorpe,1999). ROS produce cellular and tissue injury through covalent binding, DNA strand breaking, lipid peroxidation and augment fibrosis which is also implicated in other disease conditions (Paradies et al., 2011). Medicinal plants which are used traditionally in the management of diabetes, therefore serve as a great repository for anti- diabetic remedies having the advantage of being safer and providing many therapeutic effects. Solanum anomalum Thonn. ex Schumach, a plant whose fruits and leaves are used medicinally and nutritionally, is commonly found growing in West and East Africa sub-regions. Its parts are utilised locally to treat diabetes, gastrointestinal disorders, infections, inflammation and pains (Burkill, 2000; Bukenya and Hall, 1988; Offor and Ubengama, 2015). Hypoglycemic and antidiabetic activities of the fruits and leaves have been reported (Offor and Ubengama ,2015; Okokon et al., 2022). More so, in vivo and in vitro antiplasmodial (Okokon et al., 2016; Okokon et al., 2017a), anti- oedema (Okokon et al., 2017b), antioxidant and antiulcer (Okokon et al., 2019a), anticonvulsant and depressant (Okokon et al., 2019b), analgesic (Okokon et al., 2020) and antidiarrhoeal (Udobang et al., 2022) properties of the leaf extract have also been reported. Phytochemical constituents such as alkaloids, flavonoids, saponins, tanins, diosgenin, a diosgenin glycoside (25(R)-diosgenin-3-O-α-L-rhamnopyranosyl- (1→4)-β-D-glucopyranoside, uracil, 5-methyluracil, 1- octacosanol, and octacosane have been reported on the leaves of the plant (Okokon et al., 2016; Okokon et al., 2022). We report in this study the effect of the leaf extract and fractions S. anomalum on oxidative stress markers, liver function indices and histology of alloxan- induced diabetic rats. https://doi.org/10.14421/biomedich.2023.121.33-44 34 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 33-44 MATERIALS AND METHODS Plants collection Fresh leaves of Solanum anomalum were collected in compounds in Uruan area, Akwa Ibom State, Nigeria in August, 2020. The plant was identified and authenticated by a taxonomist in the Department of Botany and Ecological Studies, University of Uyo, Uyo, Nigeria. Hebarium specimen was deposited at Department of Pharmacognosy and Natural Medicine Herbarium, University of Uyo (UUH.75a). Extraction Fresh leaves of S. anomalum were washed, cut into smaller pieces and dried under shade for two weeks. The leaves were further pulverized to powder using electric grinder. The powdered leaves material was divided into two parts; one part (1.5 kg) was macerated in 50% ethanol (7.5 L) for 72 hours at room temperature (28 ±2 °C). While the other part, (1.5 kg) was successively and gradiently macerated for 72 hours in each of these solvents (2 x 5L), n-hexane, dichloromethane, ethyl- acetate and methanol to give corresponding fractions of these solvents. These were thereafter filtered and the liquid filtrates were concentrated and evaporated to dryness in vacuo 40°C using a rotary evaporator (BuchiLab, Switzerland). The extract and fractions were stored in a refrigerator at -4°C, until used for the proposed experiments. Animals Wistar rats (138-150 g) of either sex were obtained from the University of Uyo animal house. They were maintained on standard animal pellets and water ad libitum. Permission and approval for animal studies were obtained from the Faculty of Pharmacy Animal Ethics committee, University of Uyo. Induction of Experimental Diabetes using Alloxan Monohydrate Sixty (60) healthy albino Wistar rats (male and female) of known weights were fasted for 24 hours, they were reweighed before the induction by a single intra peritoneal injection of freshly prepared solution of alloxan monohydrate (150 mg/kg) in ice cold 0.9% saline (NaCl solution). According to the method of Pari and Saravanan, (2002), the animals were given 2 mL of 5% dextrose solution using orogastric tube immediately after induction to overcome the drug induced hypoglycaemia. A rest period of 72 hours was allowed during which the rats were allowed access to food and water and the diabetes to be fully developed during these 72 hours. After the rest period, rats with moderate diabetes, having persistent glycosuria, and hyperglycaemia (i.e with blood glucose levels 200 mg/dL and above), (Lenzen, 2008) were considered diabetic and selected for the experiments. The diabetic animals were randomised and divided into 9 (nine) treatment groups of 6 rats each. Based on the value of previously determined median lethal dose (LD50) (Okokon et al., 2022), suitable dose regimens were selected and the rats were treated as follows. Group 1 was given 10 mL/kg/day of normal saline orally for 14 days, Group 2 was administered with 5 mg/kg/day of Glibenclamide orally for 14 days, Group 3 was given 70 mg/kg/day of S. anomalum leaf extract orally for 14 days and group 4 rats were given 140 mg/kg/day of S. anomalum leaf extract orally for 14 days, Group 5 was administered with 210 mg/kg/day of S. anomalum leaf extract orally for 14 days. Groups 6 -9 were respectively administered with 140 mg/kg/day of n-hexane, dichloromethane, ethyl acetate and methanol fractions of S. anomalum leaves orally for 14 days. Effect of Administration of Leaf Extract and Fractions of S. anomlum on Fasting Blood Glucose of Alloxan-induced Diabetic Rats. The fasting blood glucose (FBG) of all the rats was measured after 14 days of administration of the leaf extract and fractions. The method that was employed was “the tail-tipping method”. The blood obtained from the tail vein of the rats was dropped on the dextrostix reagent pad and the pad inserted into a microprocessor digital blood glucometer and the readings were recorded (WHO, 1980). All the treatments were administered between 7.00- 8.00 am daily throughout the experimental period and food was withdrawn from the experimental animals 12 hours before measurement of FBG to create the necessary fasting period for measurement of the fasting blood glucose concentrations. Determination of the Body Weights Changes of the Treated Diabetic Rats Throughout the experimental period the body weights of the experimental animals were monitored and recorded at the following points; just before the fasting in preparation for induction of the diabetes, after induction, on stabilization of diabetes and after the prolonged study Collection of Blood Samples and Organs After 14 days of treatment (24 hours after the last administration) the rats were weighed again and sacrificed under light diethyl ether vapour. Blood samples were collected by cardiac puncture and used immediately. Blood were collected into plain centrifuge tubes and EDTA bottles. The blood in the centrifuge tubes were centrifuged immediately at 1500 rpm for 15 min to separate of serum at room temperature to avoid haemolysis and used for biochemical assays. Blood that was collected into EDTA bottles were taken for haematological analysis. The livers and kidneys of the diabetic rats were surgically removed, weighed and fixed in 10% formaldehyde for histological process. Etuk et al. – Biological activities of Solanum anomalum 35 Hematological Study After the animals were sacrificed under diethyl ether anesthesia, blood samples were collected from each rat by cardiac puncture using 21 gauge (21 G) needles mounted on a 5 ml syringe into ethylene diamine tetra- acetic acid (EDTA) – coated sample bottles for analyzed. Hematological parameters such as Red blood cell count (RBC), hemoglobin, (Hb), packed cell volume (PCV), platelet concentration (PLC) and total and differential white blood cell count (WBC). These parameters were analyzed using automatic hematological system (Sysmex Hematology – Coagulation system, Model MO-1000 I, Trans Asia, Japan). Liver Function Test The following parameters were determined; Aspartate transaminase (AST), alanine aminotransferase (ALT), total cholesterol, alkaline phosphatase (ALP), total and direct bilirubin. The determinations were done spectrophotometrically using Randox analytical kits according to standard procedures of manufacturer’s protocols (Tietz, 1976) at the Chemical Pathology Department of University of Uyo Teaching Hospital. Evaluation of the Protective Effect of the Leaf Extract and Fractions on Biochemical Parameters and Histology of Livers of Alloxan-induced Diabetic Rats Serum was separated from the blood of each animal sacrificed and the sera were stored at -20oC until used for biochemical determinations such as total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphotase (ALP), direct and total bilirubin. The determinations were done spectrophotometrically using Randox analytical kits according to standard procedures of manufacturer’s protocols (Reitman and Frankel, 1957; Tietz, 1976)). The livers of the animals were surgically removed, weighed and a part of each fixed in 10% formaldehyde for histological processes, while the other part was washed with ice cold 0.9% NaCl and homogenates were made in a ratio of 1 g of wet tissue to 9 ml of 1.25% KCl by using motor driven Teflon-pestle. The homogenates were centrifuged at 7000 rpm for 10 min at 4˚C and the supernatants were used for the assays of superoxide dismutase (SOD) (Marklund and Marklund, 1974), catalase (CAT) (Sinha,1972), glutathione peroxidase (GPx) (Lawrence and Burk,1976), and reduced glutathione (GSH) (Ellman,1959). Malondialdehyde (MDA) and gutathione-S-transferase (GST) were measured using commercial diagnostic kits (Sigma- Aldrich, USA) according to standard manufacturer's protocols. Gas Chromatography-Mass spectrometry (GCMS) Analysis GC-MS analyses of hexane and dichloromethane fractions were performed using an Agilent 7890A gas chromatograph connected with an 5975C MSD detector (Agilent Technologies, USA). 1-2 µL of each fraction was injected to a HP5-MS column (5 % phenyl- methylpolysiloxane, 30 m × 0.25 mm × 0.25 µm) and eluted with helium gas under a pressure of 10 psi. The oven temperature gradient was: keeping at 150 °C for 3 min, increasing to 300 °C at 10 °C/min, and keeping at 300 °C for 5 min. Electron ionization mode of GC-MS at 70 eV was applied (Okokon et al., 2022). The compounds in the hexane and Dichloromethane fractions were determined by comparison of spectral data in the NIST 2011 database. Statistical Analysis Data obtained from this work were analysed statistically using one –way ANOVA followed by Tukey-Kramer multiple comparison test using Instat Graphpad software, (San Diego, USA). Differences between means were considered significant at 5% level of significance i.e. p≤ 0.05. RESULTS AND DISCUSSION Effect of Leaf Extract and Fractions on Body Weights of Rats The body weights of the treated and untreated alloxan- induced diabetic rats were observed to have changed considerably within the period of study (Table 1). Treatment of the diabetic rats with the leaf extract and fractions produced a non – dose dependent increases in the body weight of the diabetic rats with the middle dose (140 mg/kg) having the highest weight increase. These increases which were significant (p<0.05-0.001) when compared to control were highest in the middle dose treated group (10.02%) followed by methanol fraction- treated group (8.77 %). (Table 1). Effect of Extract and Fractions on Weights of Organs Treatment of alloxan–induced diabetic rats with leaf extract and fractions of S. anomalum caused prominent decreases in the liver weights of the treated rats though non dose-dependently (Table 1). These decreases which were pronounced in groups treated with higher doses of the extract (140 and 210 mg/kg) and n-hexane fraction were only significant (p<0.001) in the group treated with methanol fraction (Table 1). Antidiabetic Activity of the Leaf Extract and Fractions during Prolonged Treatment The leaf extract produced significant (p<0.05-0.001) reductions in FBG levels of the diabetic rats following repeated treatment. These reductions which were non- 36 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 33-44 dose dependent were sustained throughout the duration of the study. These effects were comparable to that the standard drug, glibenclamide. On day 14, the effects were 64.08%, 63.43%, 65.43% and 64.54% for 70, 140, 210 mg/kg and glibenclamide respectively (Table 1). The various fractions produced sustained significant (p<0.05-0.001) reductions in FBG of the diabetic rats. The effects of the fractions on day 14 were 67.99, 54.66, 66.08 and 66.77% respectively for n-hexane, dichloromethane, ethyl acetate and methanol fractions. The effects exerted by n-hexane, ethyl acetate and methanol were better than that of standard drug, glibenclamide (Table 1). Effect of Leaf extract and Fractions on Liver Function Test Parameters of Diabetic Rats. Treatment of the diabetic rats with leaf extract and fractions of S. anomalum caused significant (p<0.05- 0.001) reductions in the levels of total bilirubin, ALT, ALP and AST when compared to control. Direct bilirubin level was not affected by treatment with the extract and fractions. Although reductions were observed in AST levels of the treated diabetic rats, it was only significant (p< 0.01-0.001) at the highest dose (210 mg/kg) and methanol fraction treated group (Table 2). Effect of Leaf Extract and Fraction on Liver Antioxidant Enzymes. Treatment of alloxan-induced diabetic rats with leaf extract and fractions of S. anomalum caused significant (p<0.05-0.001) dose- dependent elevation in the levels of the antioxidant enzymes (SOD, CAT, GPX) when compared to control. Similarly, GSH level was significantly (p<0.001) elevated following treatment with the extract and fractions when compared to control. n-hexane fraction followed by methanol fraction exerted the highest activity. Similarly, there were significant (p<0.05-0.01) reductions in the level of MDA of the treated rats with ethyl acetate and DCM fractions having the most significant effects. Also, significant (p<0.05- 0.01) increases in enzymes and GSH levels as well as significant (p<0.01) reductions in MDA level were observed with the standard drug, glibenclamide (Table 3). Effect of Extract and Fractions on the Histology of Livers of Diabetic Rats Histologic sections of livers of untreated diabetic rats revealed numerous hepatocytes with pyknotic nucleus, cellular and vascular degeneration, vascular congestion, hepatocytes hyperplasia and periportal inflammation. Livers of diabetic rats treated with glibenclamide (10 mg/kg) revealed reduced pathological signs with normochromic pyknotic nucleus. (Figure 1). Livers of rats treated with leaf extract (70 – 210 mg/ kg) revealed hyperchromic cellular profile with vascular congestion and numerous pyknotic nucleus. Livers of diabetic rats treated with the middle dose of the extract (140 mg/kg) showed no visible cellular abnormality (Figure 1). Livers of diabetic rats treated with n-hexane, dichloromethane, ethyl acetate and n-butanol fractions (140 mg/kg) revealed normochromic cellular profile containing periportal inflammation and pyknotic nuclei. There was no obvious cellular abnormality in the livers of diabetic rats treated with ethyl acetate fraction of the leaf. It could be considered slightly affected. (Figure 1). Effect of Leaf extract and Fractions on Haematological Indices of Diabetic Rats Administration of the leaf extract and fractions of S. anomalum caused significant (p< 0.05-0.001) increases in the levels of white blood cells, lymphocytes, and eosinophils, which was significant at the middle dose (140 mg/kg) when compared to control. Neutrophils, monocytes, basophils and platelets were reduced, while RBC, Hb concentration, platelets and pack cell volume were not affected significantly (p>0.05) by the administration of extract and fraction (Table 4). Gas Chromatography-Mass Spectroscopy (GCMS) analysis The phytochemical analysis of the most active fractions (n-hexane and dichloromethane) of S. anomalum revealed the presence of major compounds. The compounds in n-hexane fraction were bicyclo[3.1.1]heptanes-2,5,6-trimethyl-(1.alpha 2-beta, 5alpha-, squalene and beta.-sitosterol trimethylsilyl ether (Table 5). While 3,7,11,15-tetramethyl-2-hexadecen-1- ol, squalene and heptacosane were revealed to be present in DCM fraction (Table 6). Etuk et al. – Biological activities of Solanum anomalum 37 Table 1. Effect of leaf extract and fractions of Solanum anomalum on body and Liver weights of alloxan- induced diabetic rats. Treatment Dose mg/kg Body Weight (g) Weights of Liver (g) Fasting Blood Glucose (mg/dl) Day 0 Day 15 % Increase 0 HR 14th Day Control normal saline - 132.6 ± 18.34 129.3± 20.43 -2.48 6.72±0.98 266.0±17.16 249.0±14.01 Glibenclamide 10 132.0 ± 9.45 141.3± 12.33 7.04 6.10±0.15 233.0±12.00 82.6±8.19b Extract 70 152.8 ± 15.56 163.0± 9.29 6.67 6.28±0.23 279.3±14.74 100.3±16.69b 140 143.6 ± 12.55 158.0 ± 3.15 10.02 5.80±0.32 260.6±8.32 95.3±11.29 a 210 140.8 ± 13.29 152.6 ± 7.22 8.38 5.82±0.67 257.0±3.71 88.3±8.83b n- hexane fraction 140 142.9 ± 8.54 153.6 ± 6.48 7.48 5.82±0.65 264.3±14.50 84.6±40.05c Dichloromethane fraction 140 138.4 ± 6.26 144.6± 13.71 4.68 6.14±0.35 236.0±37.60 107.0±14.52b Ethyl acetate fraction 140 144.31 ± 8.50 156.6± 9.88 8.51 7.07±0.22 271.3±13.04 92.0±14.29a Methanol fraction 140 145.8 ± 7.45 158.6± 10.55 8.77 0.98±0.07c 245.6±12.33 81.6±18.49b Data are expressed as MEAN ± SEM, Significant at ap<0.05, bp< 0.01, cp< 0.001, when compared to control. (n=6). Table 2. Effect of S. anomalum leaf extract and fractions on the liver function parameters of alloxan-induced diabetic rats. Treatment Dose (mg/ kg) ALT (IU/L) ALP (IU/L) AST (IU/L) Total Bilirubin (µmol/L ) Direct Bilirubin (µmol/L) Control 10 mg/ml 97.00± 4.35 109.53±8.95 172.63±10.68 2.16±0.14 1.60±0.10 Crude extract 70 77.90±9.49 92.11± 2.67 162.42± 1.45 2.06± 0.29 1.16±0.03 140 75.10±12.61 70.73±8.67 149.69±15.94 1.80± 0.05 1.10±0.15 210 53.23±2.25c 61.92±8.67b 128.07±18.57a 1.50± 0.05 1.10±0.00 n- hexane Fraction 140 63.73±3.91b 100.23±9.68 130.48±8.54 1.60±0.00 1.10±0.10 Dichloromethane fraction 140 68.86±3.33a 62.92±6.76b 155.42±18.27 1.43±0.20a 1.10±0.05 Ethyl acetate fraction 140 70.40±4.19 67.43±5.44b 145.13±15.21 1.86 ± 0.31 1.10±0.11 Methanol fraction 140 58.2±5.51c 53.33±6.09c 106.98±0.75c 1.36± 0.08a 1.10±0.10 Glibenclamide 10 61.12±10.94b 60.40±6.54b 122.00±4.00b 1.66± 0.12 1.30±0.10 Data is expressed as MEAN ± SEM, Significant at ap<0.05, bp< 0.01, cp< 0.001, when compared to control. (n=6). Table 3. Effect of S. anomalum leaf extract on liver antioxidative stress markers in alloxan-induced diabetic in rats. Parameters/ Treatment Dose mg/kg SOD (µg/mL) CAT (IU/L) GST (µg/mL) GSH (µg/mL) GPx (µg/mL) MDA (µMol/mL) Diabetic Control 10 ml/kg 0.18±0.03 3.68± 0.28 0.16±0.01 0.71±0.02 0.039±0.001 0.54±0.02 Crude extract 70 0.24±0.04a 4.11±0.23 0.21±0.02 0.72±0.03 0.055±0.002 0.52±0.02 140 0.20±0.02 2.95±0.28 0.23±0.01a 0.81±0.02 a 0.056±0.002c 0.48±0.03 210 0.30± 0.02c 3.53±0.17 0.31±0.01c 1.01±0.03c 0.059±0.002c 0.41±0.02c n-hexane fraction 140 0.30±0.02c 5.86± 0.48c 0.23±0.01a 0.98±0.02c 0.057±0.002c 0.44±0.0b Dichloromethane fraction 140 0.20 ±0.04 2.87±0.18 0.31±0.01c 0.82±0.01a 0.058±0.002c 0.38±0.01c Ethyl acetate fraction 140 0.33 0.02c 4.640.26 0.26±0.01 c 0.92± 0.02c 0.048± 0.002a 0.31± 0.02c Methanol fraction 140 0.37 0.03c 4.330.56 0.32±0.01 c 0.87± 0.02c 0.049± 0.002a 0.43± 0.02b Glibenclamide 10 0.29±0.02c 5.69±0.18 b 0.25±0.02 b 0.85±0.01c 0.051±0.003b 0.40±0.02c Data were expressed as mean  SEM. Significant at ap< 0.05, bp< 0.01, cp< 0.001 when compared to diabetic control. (n = 6). Table 4. Effect of Solanum anomalum leaf extract and fractions on hematological parameters of alloxan-induced diabetic rats. Treatment Dose mg/kg WBC (L) NEUT. (%) LYM (%) MONO (%) EOSINO (%) BASO (%) RBC (L) HGB (g/dL) PCV (%) PLATELETS. (L) Control normal saline 10 9.27± 1.95 59.00±1.60 35.66±1.65 4.66± 0.28 0.26± 0.21 1.00± 0.10 6.00± 0.28 12.13± 0.78 43.76±3.22 940.0± 35.76 Crude extract 70 11.60±1.56 51.86± 3.16 45.86±2.06 a 2.96± 0.24 a 0.16 ±0.06c 0.90± 0.05 6.72 ± 0.53 12.63± 0.56 43.20±2.36 709.3± 54.14a 140 15.15±3.34 a 50.40±0.64 45.30±0.88 a 2.63± 0.29 a 0.76 ± 0.12c 1.10± 0.20 7.83± 0.78 14.10± 1.25a 53.76±7.61a 684.6± 92.13a 210 9.86±1.66 57.93 ± 0.60 36.53±4.27 3.76± 0.38 0.66± 0.12c 0.50± 0.00 7.17± 0.18 11.10± 0.68 40.80±1.55 925.6± 61.50 n- hexane Fraction 140 9.90±1.51 59.50 ± 0.60 34.90±1.44 4.96± 0.03 0.43 ± 0.17b 0.60± 0.26 6.08± 0.69 12.46± 0.08 44.50±0.40 768.0± 82.92 Dichloromethane fraction 140 9.32±2.31 48.10±14.13 65.30±5.71c 3.40± 0.58 0.36 ± 0.51 0.90 ± 0.05 6.65 ± 0.90 12.56± 1.07 45.90±7.50 723.0± 35.80 Ethyl acetate Fraction 140 10.31±1.64 50.66±0.82 43.46±0.73 3.60 ± 1.01 0.30± 0.51 0.50± 0.17 7.28± 0.17 12.13± 1.21 43.90±4.43 775.0±59.53 MethanolFraction 140 14.55 ± 0.98a 47.60±6.45a 46.93±6.59 a 4.16± 0.72 0.40± 0.15c 0.53 ± 0.03 6.70 ± 0.63 13.33 ± 0.74 48.90±2.82 867.3±74.71 Glibenclamide 10 12.03±3.76 33.16±0.79b 61.56±1.66 c 2.23± 0.28 a 0.06± 0.03c 0.55± 0.03 7.56 ± 0.31 12.66±1.18 47.90±2.82 888.0±47.05 Data is expressed as MEAN ± SEM, Significant at ap<0.05, bp<0.01, cp<0.001, when compared to control. (n=6). 3 8 B io lo g y , M ed ic in e, & N a tu r a l P r o d u c t C h em istr y 1 2 (1 ), 2 0 2 3 : 3 3 -4 4 Etuk et al. – Biological activities of Solanum anomalum 39 Table 5. GCMS analysis of column hexane fraction of S. anomalum. PEAK RT COMPOUND NAME FORMULA MOL. MASS 1. 10.044 Bicyclo[3.1.1]heptanes-2,5,6-trimethyl-(1.alpha 2-beta, 5alpha- C19H38O5Si4 458.18 2. 18.315 Squalene C30H50 410.39 5. 18.326 .beta.-Sitosterol trimethylsilyl ether C32H58OSi 486.43 Table 6. GCMS analysis of column DCM fraction of S. anomalum. PEAK RT (min) COMPOUND NAME FORMULA MOL. MASS 1. 10.044 cis-Pinane C10H18 138.25 2. 10.524 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C20H40O 296.1 3. 10.547 1,4-Eicosadiene C20H38 278.5 4 18.315 Squalene C30H50 410.39 5. 22.836 Heptacosane C27H56 380.44 Keys: Vascular congestion (VC), Vascular degeneration (VD), Cellular degeneration (CD), Periportal inflammation (PI), Pyknotic nucleus (Pn), Red blood cell (RBC), Hepatocytes (H), Hepatocytes hyperplasia(H). Hepatic vein (HV), Bile duct (BD), and Pyknotic nucleus (Pn) Figure 1: Histological sections of Livers of alloxan-induced diabetic rats treated with Normal saline 10 mL/kg (1), Glibenclamide 10 mg/kg bw (2), leaf extract 70 mg/kg bw (3), leaf extract 140 mg/kg bw (4), leaf extract 210 mg/kg bw (5), n-hexane fraction 140 mg/kg (6) dichloromethane fraction 140 mg/kg (7), ethyl acetate 140 mg/kg (8), Methanol fraction 140 m/kg (9), at Magnification A(x400), stained with H&E Method. Discussion The leaf of S. anomalum is use in ethnomedicine for the treatment of various ailments such as malaria, gastrointestinal disorders and diabetes (Okokon et al., 2016). This work was designed to evaluate antioxidative and hepatoprotective potentials of leaf extract and fractions of S. anomalum in alloxan-induced diabetic rats. The body weights of diabetic rats were found to increase significantly following treatment with the leaf extract and fractions. Diabetes is associated with a severe loss in body weight due to loss or degradation of structural proteins (Rajkumar and Govindarajulu, 1991). Treatment with the leaf extract and fractions remedied this situation perhaps due to the alleviation of hyperglycemic state and stimulation of protein synthesis. 987 6 321 54 40 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 33-44 The leaf extract and fractions treatments were observed in this study to cause significant increases in WBC, lymphocytes and eosinophils, while neutrophils, monocytes and basophils were significantly reduced. Other parameters were not affected. The increases maybe due to immunological responses by the body defence mechanism to heal or repair the injuries caused by alloxan (Soetan et al., 2013) which can be attributed to the immunostimulatory effect of diosgenin (Nimbalkar et al., 2018) and squalene. Metabolism of alloxan to dialuric acid causes the production of H2O2, •OH and superoxide radical (Mathews and Leiter, 1999)., which destroys β-cell necrosis and induces diabetes by partial destruction of pancreatic β-cells of islet of Langerhans (Cakici et al., 1994; Abdel-Barry et al., 1997). This reduces insulin levels, raise the blood glucose level thereby leading to type 2 diabetes mellitus, but with residual pancreatic β- cells with potentials to secrete insulin (Sheela and Augusti, 1992; Subramoniam et al., 1996). In this study, S. anomalum leaf extract/fractions were observed to demonstrate sustained significant antidiabetic activities during prolonged study with the methanol, n-hexane and ethyl acetate fractions exerting prominent activities. The fasting blood glucose (FBG) levels of the treated diabetic rats were significantly reduced when compared to those of untreated diabetic rats (control). The antidiabetic results observed in this study corroborate that of other species of Solanum such as S. nigrum (Sengottaiyan et al., 2012; Umamageswari et al.,2017), S. trilobatum (Doss et al., 2009), S. xanthocarpum (Selvi and Yogananth, 2016) and Solanum villosum (Nyaga et al., 2019) Thus, confirming strongly the antidiabetic potentials of this plant in ethnomedicine. The leaf extract and fractions were observed in this study to cause significant decrease in weight of liver. Generally, internal organs weights are considered as important indicator to injury and toxicities (Farah et al., 2013). Hypertrophy of organs often indicates toxicity and damaged to organ (Ping et al., 2013). This often results from oedema due to inflammation of the organs. Free radicals generated during alloxan metabolism cause destruction of hepatic, pancreatic and kidney cells and tissues (Mathews and Leiter, 1999). The decrease in weights of liver by the extract/fractions especially DCM and methanol fractions-treated group, is as a result of protective effect of the fractions against the effect of free radicals generated by alloxan and diabetic condition perhaps due to its hypoglycemic and antioxidant activities of the phytoconstituents (Okokon et al., 2019a) such as diosgenin (Kanchan et al., 2016), 1-octacosanol and octacosane (Leng et al., 2020; Sengupta et al., 2018; Rhetso et al., 2018 ), squalene (Gunes, 2013; Micera et al., 2020), β-sitosterol (Gupta et al., 2011; Baskar et al., 2012) and phenolic compounds in the leaf extract. Metabolism of alloxan leads to the generation of free radicals which attack organs and cause oxidative stress which contributes to diabetes complications in animals or humans (Baynes and Thorpe, 1999). Reactive oxygen species (ROS) produce cellular and tissue injury through covalent binding, DNA strand breaking, lipid peroxidation (LPO) and augment fibrosis which is also implicated in other disease conditions (Sirnivasan et al., 2007; Paradies et al., 2011). The results of this study show that oxidative stress was duly induced by alloxan in the diabetic rats as reflected in the marked reductions in the levels of SOD, CAT, GSH and GPx in hepatic tissues as well as significant increase in MDA levels in the liver of the diabetic rats. This finding agrees with earlier findings that the activities of these antioxidants are known to reduce during diabetes (Parmar and Kar,2008; Kostolanska et al., 2009; Dixit and Kar, 2010). The oxidative stress status of the diabetic rats is further supported by marked elevations in the serum levels of AST, ALT, ALP, and total bilirubin levels of the diabetic rats. Reduction in liver levels of these oxidative markers following treatments with the leaf extract and fractions strongly suggest the great potentials of leaf extract and fractions in attenuating oxidative stress associated with type II diabetes mellitus which was probably mediated via free radical scavenging activities and improving glutathione status in the tissues by its phytochemical constituents such as diosgenin, 1- octacosanol, octacosane and β-sitosterol (Gupta et al., 2011; Baskar et al., 2012;Kanchan et al., 2016;Leng et al., 2020; Sengupta et al., 2018; Rhetso et al., 2018). Disease conditions such as diabetes affect the liver and changes the hepatic function as shown by increases in the levels of AST, ALT, ALP, total and direct bilirubin indicates liver damage which are also detected in human diabetes (Takaike et al., 2004). Damage to liver cells often results in leakage of enzymes such as aspartate aminotransferase (AST), and alanine aminotransferase (ALT) into the blood, while blood levels of alkaline phosphatase (ALP) rise when the bile flow is slow or blocked (Prat and Kaplan, 2000). In this study, treatment of the diabetic rats with the leaf extract and fractions was found to cause significant reductions in the levels of ALT, ALP and total bilirubin. The integrity of hepatocytes is assessed by levels of the serum liver enzyme markers such as AST, ALT and ALP (Moss and Henderson, 1999). Serum aminotransferases levels (ALT and AST) are two of the most useful measures of liver parenchymal cell injury. AST is raised in acute liver damage, but is also present in RBCs, kidney, testis, cardiac and skeletal muscles, so it is not specific to the liver, while ALT is almost exclusively found in the liver (Nyeblom et al., 2006). Elevated levels of AST and ALT indicate liver damage but are not good measures of liver function since they do not reliably reflect the synthetic ability of the liver and may come from tissues other than the liver such as muscles. The leaf extract and fractions produced Etuk et al. – Biological activities of Solanum anomalum 41 significant reduced the elevated levels of these enzymes. This further corroborates histologic findings. The increased activities of transaminases, which are active in the absence of insulin due to the availability of amino acids in the blood of diabetics and are also responsible for the increased gluconeogenesis and ketogenesis (Udayakumar et al., 2009). Diabetes and hyperlipidaemia also cause cell damage by altering the cell membrane architecture, which results in enhanced activities of ALP in diabetic rats (Udayakumar et al., 2009). This suggests that alloxan-induced diabetes caused lipid peroxide mediated tissue damage in the liver. The increase in the levels of these enzymes in diabetes may be as a result of the leaking out from the tissues and then migrating into the blood stream. The decrease in AST, ALT and ALP levels in leaf extract and fractions treated groups indicates the protective effect on the liver. Total bilirubin level which was increased in the untreated diabetic rats was observed in this study to be significantly reduced in groups treated with the husk extract and fractions. Bilirubin, is a metabolic product of hemoglobin which undergoes conjucation with glucuronic acid in hepatocytes to increase its water solubility. Determination of bilirubin represent an index for the assessment of hepatic function, severity of necrosis, conjugation and excretory capacity of hepatocytes and any abnormal increase in the level of bilirubin in the serum indicate hepatobililiary disease and severe disturbance of hepatocellular function (Martin and Friedman, 1992). Decrease in serum bilirubin level of the diabetic rats after treatment with the leaf extract and fractions indicated extract/fractions effectiveness to restore normal functional status of the liver. The above activities of the extract/fraction show that the leaves extract and fractions posses hepatoprotective potential against alloxan-induced hepatic injury. These results corroborate histologic findings which show significant hepatoprotective potentials of the extract/fractions. This effect could be due to the antioxidant /free radical scavenging activities of the extract and fractions (Okokon et al., 2019a), squalene, β-sitosterol (Ivorra et al., 1988; Gupta et al., 2011; Baskar et al., 2012; Micera et al., 2020) as well as the isolated compounds; diosgenin, 1-octacosanol and octacosane (Kanchan et al., 2016; Leng et al., 2020; Sengupta et al., 2018; Rhetso et al., 2018). Reports have been indicated that persistent hyperglycemia causes increased production of oxidative stress in alloxan-induced diabetes (Bonnefont et al., 2000). Hence, excessive ROS produced leads to oxidative damage and increased Lipid Peroxidation (LPO). The results of this study showed a significant increase in LPO levels resulting in high level of MDA in alloxan-induced diabetic rats liver with accompanying reduction in the levels of oxidative stress markers (SOD, CAT, GPx and GSH) in the liver. This reduction in the levels of antioxidant enzymes in this study corroborates those of earlier reports (Parmar and Kar, 2008; Kostolanska et al., 2009; Dixit and Kar, 2010). In in vivo experimental models, tissue oxidative stress markers such as SOD, CAT and GSH are useful and reliable markers of antioxidant status while MDA is a sensitive and reliable marker for lipid peroxidation (Kumar et al., 2010; Feillet-Coudray et al., 1999). SOD plays an important role in oxygen defense metabolism by intercepting and reducing superoxide to hydrogen peroxide, which in mammals is readily reduced to water principally by CAT and GPx. GPx plays a pivotal role in minimizing the oxidative stress. GPx and GST work together with GSH and decompose H2O2 and other organic hydroperoxides to non-toxic products. Glutathione, reduced form GSH is ubiquitous tripeptide thiol, is a vital intra/extra-cellular protective antioxidant against oxidative/nitrosative stress. Glutathione reductase is an enzyme responsible for its conversion back to the reduced state (Kayali and Tarhan, 2006; Okutana et al., 2005). The antioxidant enzymes levels were found to be reduced in untreated alloxan-induced diabetic rats. Hence, the changes of these biomarkers is in accordance with the decrease in antioxidant state in the body as other reports (Ei-Missiry and Ei-Gindy, 2000; Meral et al., 2001). In the present study, administration of the extract and fractions significantly counteract the changes of oxidative stress biomarkers in alloxan-induced rats thus preventing the accumulation of excessive oxidative stress with corresponding increases in the levels of antioxidant enzymes/oxidative stress markers. This activity is due to the antioxidant activities of the phytochemicals such as squalene, β-sitosterol, diosgenin, 1-octacosanol, octacosane and other phenolic compounds found to be present in the leaf extract. Similarly, β-sitosterol and diosgenin present in the extract/fractions have been reported to cause reduction in hepatic lipid peroxidation and elevation in the activities of catalase, superoxide dismutase and glutathione (Gupta et al., 2011b; Baskar et al., 2012; Kanchan et al., 2016). The liver of the extract/fractions treated diabetic rats were found to be significantly protected from the effects of free radicals generated by alloxan. This protection was visible as the livers of the treated rats lacked or had reduced pathological signs such as vascular degeneration and congestion as well as periportal inflammation observed in the untreated diabetic rats. These findings corroborated that of chemical pathology and therefore suggest hepatoprotective activity against oxidative stress induced by alloxan. The protection is due to the free radical scavenging potentials of the phytoconstituents of the extract and fractions such as squalene, β-sitosterol, diosgenin, 1-octacosanol, octacosane as well as the antioxidant activity of other phenolic compounds present in the extract (Okokon et al., 2019a). 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