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ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 1  |  Page : 49-54  

Lack of antidiabetic effect of ethanolic extract of leaves of Solanum trilobatum Linn (Solanaceae)


Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Kedah, Malaysia

Date of Web Publication21-Aug-2018

Correspondence Address:
Subramani Parasuraman
Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong 08100, Kedah
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpnr.JPNR_9_18

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   Abstract 


Objective: The objective was to study the antidiabetic properties of ethanolic extract of the leaves of Solanum trilobatum (EEST) in streptozocin (STZ)-induced diabetics in Sprague-Dawley (SD) rats. Materials and Methods: EEST was prepared by using hot percolation method and the extract was used for antidiabetic screening. The SD rats were divided into six groups each of six animals, namely normal control, diabetic control, glibenclamide and EEST-treated groups at 125, 250, and 500 mg/kg. Except normal control animals, all the other animals were induced diabetes with intraperitoneal injection of STZ (55 mg/kg). The control and diabetic animals were treated with respective assigned treatment once daily for 21 consecutive days. The blood glucose was monitored at regular intervals and biochemical parameters such as aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea, and creatinine were measured with terminal sample. At the end of the study, the animals were sacrificed; lung, heart, stomach, liver, and kidney were harvested and absolute organ weight was measured. Results: The rats administrated with the extracts at dose of 500 mg/kg body weight (BW)/day showed significant antidiabetic activity and this effect was comparable with that of glibenclamide. The diabetic control animals showed significant increased levels of total cholesterol, high-density lipoprotein (HDL), HDL ratio, very-low-density lipoprotein, and glibenclamide, and EEST prevented STZ-induced cholesterol impairments. EEST did not show any significant antidiabetic effect at 125 and 250 mg/kg BW/day treated rats. Conclusion: EEST showed significant antidiabetic activity at 500 mg/kg and prevented STZ-induced metabolic changes in experimental animals. EEST did not show any antidiabetic activity in diabetic animals treated with 125 and 250 mg/kg of EEST.

Keywords: Antidiabetic activity, ethanolic extract of Solanum trilobatum, streptozocin


How to cite this article:
Parasuraman S, Hoong SS, Zou LN, De Wei DL, Loshini S. Lack of antidiabetic effect of ethanolic extract of leaves of Solanum trilobatum Linn (Solanaceae). J Pharm Negative Results 2018;9:49-54

How to cite this URL:
Parasuraman S, Hoong SS, Zou LN, De Wei DL, Loshini S. Lack of antidiabetic effect of ethanolic extract of leaves of Solanum trilobatum Linn (Solanaceae). J Pharm Negative Results [serial online] 2018 [cited 2018 Dec 18];9:49-54. Available from: http://www.pnrjournal.com/text.asp?2018/9/1/49/239515




   Introduction Top


Diabetes is a chronic metabolic disorder in which the amount of insulin produced by the body falls below the normal range. High blood glucose levels are symptoms of diabetes mellitus as a consequence of inadequate pancreatic insulin secretion or poor insulin-directed mobilization of glucose by target cells.[1] Diabetes develops when sufficient quantity of insulin is failed to be produced by the pancreas. Chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, heart, kidneys, nerves, and blood vessels.[2]

Diabetes is classified as type 1, type 2, gestational, or other specific types. Type 1 diabetes mellitus is caused by cell-mediated autoimmune destruction of the pancreatic cells and type 2 diabetes mellitus is associated with insulin resistance and elevations in plasma glucose and free fatty acids that stimulate reactive oxygen species levels which in turn activate inflammatory signaling pathways such as mitogen-activated protein kinases and nuclear factor-κB.[3] During the past years, this complication has attracted the attention and interest of a number of researchers, resulting in expanding knowledge of the pathogenesis of this disorder and drug discovery. Natural products including medicinal plants are having significant contribution on drug development, which includes the plants from the family of Solanaceae.

Solanum L. (Solanaceae) is a flowering plant genus containing some 1400 species distributed globally in tropical and temperate zones.[4]Solanum trilobatum Linn. (Solanaceae) plant parts such as leaves, berries, and flowers are commonly used for the treatment of respiratory illness such as common cold, cough, and asthma in the regions of South India.[5] The leaves of S. trilobatum are used as food supplement in Tamil Nadu, India, and consumed as fries by mildly frying it in oil or ghee and then it is grinded. S. trilobatum is full of thorns including the leaf part; the thorns must be removed before cooking because the thorns are known to have mild toxicity.[6] Other than treating respiratory disorders, it also possesses many pharmacological properties including reducing blood glucose level and antibacterial, antifungal, antioxidant, and antitumor properties (Sundari et al., 2013). In preclinical experiment, aqueous and methanolic extracts of S. trilobatum showed antidiabetic and α-amylase inhibitory activities, respectively.[7],[8] The antidiabetic potential of organic extracts of S. trilobatum remains unclear. Hence, the present study was planned to study the antidiabetic potential of ethanolic extract of S. trilobatum using streptozocin (STZ)-induced diabetic rats.


   Materials and Methods Top


Identification and collection of plant

Plant morphology

S. trilobatum Linn. is a purple-fruited pea eggplant. It is a climbing shrub with short compressed spines with sharp recurved leaves containing high amount of iron, carbohydrates, calcium, proteins, fats, phosphorus, crude fibre, and minerals.[9] The leaf structure of S. trilobatum is as follows: leaves are obtusely 3–5 lobed, rarely hastate and alternate, lamina ovate; petiole with prickles; deeply lobed, glabrous, apex obtuse, margin entire, prickles on the veins, glabrous 4–6 cm × 2–4 cm; round-shaped petioles in cross section with numerous curved, broad-base, yellowish prickles and longer than lamina; vascular bundle covered with bundle sheath in the middle, which contains 8–10 xylem strands each containing about 5–6 xylem vessels. There are two small round vascular traces at the adaxial side near to the epidermis. The stomata are anomocytic, the guard cells are covered with 4–5 subsidiaries, and palisade is build up by single vertical layer of cells.[10]

Collection of plant

Taxonomically identified S. trilobatum (Solanaceae) plants were collected from Vellore (12.92°N, 79.13°E, 220 m above the mean sea level) India, after confirmation of its identification and authentication by a botanist. The plant leaves were dried under shade for a few days and powdered using an electrical grinder.

Extraction of leaves

The powdered leaves of S. trilobatum were weighed and extracted with ethanol (absolute alcohol) using Soxhlet apparatus at 75°C ± 5°C. When the solvent became clear (approximately 6–8 cycles), extraction was completed. The extract was concentrated to a dry mass by evaporation under reduced pressure by rotary evaporator at 60°C ± 2°C. The ethanolic extract of S. trilobatum (EEST) was stored at room temperature until use. The yield of EEST was 6.2 g w/w (dry weight basis).

Animals

Healthy, adult, male Sprague-Dawley (SD) rats, weighing 200 ± 10 g, were obtained from Central Animal house, AIMST University, Malaysia. The animals were housed in large, spacious polyacrylic cages at an ambient room temperature with 12-h-light/12-h-dark cycle. The animals were fed with water and normal rats pellet diet ad libitum. The study was approved by AIMST University Human and Animal Ethics Committee (AUHAEC/FOP/2017/13), and the study was conducted according to the Animal Research Review Panel guidelines.

Antidiabetic screening of ethanolic extract of the leaves of Solanum trilobatum

The rats were fasted overnight and diabetes was induced by administering single intraperitoneal injection of freshly prepared STZ 55 mg/kg body weight (BW) in 0.1 M citrate buffer (pH 4.5) in a volume of 0.5 ml/kg BW. Fasting blood glucose levels were measured after 48 h of induction to confirm diabetes in the STZ-treated rats. The rats were given 5% w/v of glucose solution (2 mL/kg BW) after 24 h of STZ injection to prevent hypoglycemic mortality.[11] Rats with fasting blood glucose of more than 200 mg/dL or 11.1 mmol/L were considered as diabetics and they were divided randomly into five different groups (Groups II–VI) with six animals in each group as follows.

  • Group I: Normal control
  • Group II: Diabetic control
  • Group III: Diabetic rats treated with glibenclamide (10 mg/kg)
  • Group IV: Diabetic rats treated with EEST (125 mg/kg)
  • Group V: Diabetic rats treated with EEST (250 mg/kg)
  • Group VI: Diabetic rats treated with EEST (500 mg/kg).


Glibenclamide and EEST were suspended in 1% w/v carboxymethyl cellulose and administered once daily through oral gavage for 21 consecutive days. Few drops of venous blood were collected through tail vein on the 7th and 14th days of treatment to estimate blood glucose using glucometer (Accu-Chek ®, Roche Diagnostics (M) Sdn Bhd, Malaysia).[12] On day 21, oral glucose tolerance test (OGTT) was performed.[13],[14] At the end of the experiment, the blood samples were withdrawn from all the experimental animals through retro-orbital plexus puncture in plain glass tubes for biochemical analysis under diethyl ether anesthesia. The blood sample was centrifuged at 3000 rpm, and serum was separated and stored at −20°C until further biochemical analysis.

Body weight analysis

The BWs of each rat of each group were measured and recorded at weekly intervals.

Oral glucose tolerance test

The OGTT was performed on 21st day of the experiment. The rats were fasted overnight and blood was collected from the tail vein to estimate blood glucose using glucometer. OGTT was performed by oral administration of glucose (2 g/kg BW). Blood samples were collected at 0.5 h prior to drug administration and 1, 2, and 4 h after glucose challenge through tail vein for glucose estimation.[14]

Biochemical analysis

At the end of the experiment, the blood samples were withdrawn from all the experimental animals through retro-orbital plexus puncture in plain glass tubes for biochemical analysis under diethyl ether anesthesia. The blood sample was centrifuged at 3000 rpm, and serum was separated and stored at −20°C until further biochemical analysis. The serum sample was used for estimation of biochemical markers such as glucose, total serum cholesterol, serum triglyceride, high-density lipoprotein (HDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, creatinine, and urea using a biochemical analyzer (Reflotron Plus System, Hoffmann-La Roche, USA).

Low-density lipoprotein (LDL), HDL ratio, and very LDL (VLDL) levels were calculated mathematically. The LDL was calculated using Friedewald formula: LDL = total cholesterol − HDL − triglycerides (TGs)/5 mg/dL;[15] HDL ratio was calculated using the following formula: HDL − cholesterol/TC – HDL − cholesterol ×100%; and VLDL was calculated using the following formula: LDL/5.[12]

Organ weight analysis

At the end of the experiment, all the experimental animals were sacrificed and organs such as lung, heart, stomach, liver, and kidney were harvested and absolute organ weight was measured. Later, relative organ weights of lung, heart, stomach, liver, and kidney were calculated.

Statistical analysis

The results were expressed as mean ± standard error of mean. The repeated measures ANOVA, followed by Tukey's post hoc test, was used for the statistical test. P < 0.05 was considered statistically significant.


   Results Top


In the BW analysis, no significant change in the BW was observed in diabetic control, glibenclamide, and EEST 250- and 500 mg/kg-treated groups. The significant reduction (P < 0.01) in BW was observed with EEST 125 mg/kg-treated group on the 21st day [Figure 1]. Throughout the study, the animals in diabetic control group showed a significant increase of blood glucose levels (P < 0.001) when compared to the normal control group. The standard and EEST 500 mg/kg-treated groups of rats showed a significant reduction in blood glucose level when compared to the diabetic control group. However, the group of animals treated with EEST at 125 mg/kg showed increased glucose levels when compared to the normal control group, and EEST 250 mg/kg-treated group showed significant reduction in the levels of blood glucose from the 14th day onward when compared to the diabetic control group. S. trilobatum (500 mg/kg)- and glibenclamide (10 mg/kg)-treated group resulted with significant reduction in the blood glucose levels on the 7th, 14th, and 21st days when compared to the diabetic control group [Table 1].
Figure 1: Effect of ethanolic extract of Solanum trilobatum on body body weight of diabetic rats. All the values are mean ± SEM (n = 6). **P < 0.01 compared with control (Repeated measures ANOVA, followed by Tukey's post hoc test). SEM: Standard error of mean, EEST: Ethanolic extract of Solanum trilobatum

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Table 1: Effect of ethanolic extract of the leaves of Solanum trilobatum on blood glucose levels (mmol/L) in diabetic rats

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In OGTT, the significant increase in the levels of fasting blood glucose levels was observed in diabetic control and EEST-treated groups [Figure 2]. Throughout the test, glibenclamide and EEST 250- and 500 mg/kg-treated animals showed significant reduction in the glucose levels when compared to the control and diabetic control animals. Whereas EEST 125 mg/kg-treated animals did not show any significant reduction in the glucose levels when compared to the control and diabetic control animals.
Figure 2: Effect of ethanolic extract of Solanum trilobatum on oral glucose tolerance test body in diabetic rats. All the values are mean ± SEM (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control; cP < 0.001 compared with diabetic control (Tukey's post hoc test). EEST: Ethanolic extract of Solanum trilobatum, SEM: Standard error of mean

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On biochemical parameter analysis, diabetic control group showed significant increased levels of AST (P < 0.001) and creatinine (P < 0.05) when compared to the normal control group. The extract and glibenclamide had ameliorative effect on liver and renal enzymes and inhibited the STZ-induced metabolic changes [Table 2]. On lipid profile, diabetic control group showed significant elevated levels of total cholesterol (P < 0.001), LDL (P < 0.001), and VLDL (P < 0.001) levels and reduced levels of HDL (P < 0.001) and HDL radio (P < 0.001), when compared to the normal control group. Glibenclamide and EEST-treated groups did not showed any changes in the levels of total cholesterol, TG, HDL, LDL, VLDL, and HDL ratio when compared to the normal control animals and inhibited the STZ-induced metabolic changes [Table 3].
Table 2: Effect of ethanolic extract of the leaves of Solanum trilobatum on biochemical parameters of diabetic rats

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Table 3: Effect of ethanolic extract of the leaves of Solanum trilobatum on lipid profile of diabetic rats

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In organ weight analysis, no significant changes in absolute and relative organ weights were observed in glibenclamide and EEST-treated groups when compared to the normal and diabetic control groups [Table 4] and [Table 5].
Table 4: Effect of ethanolic extract of the leaves of Solanum trilobatum on absolute organ weight (g) of diabetic rats

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Table 5: Effect of ethanolic extract of the leaves of Solanum trilobatum on relative organ weight (g) of diabetic rats

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   Discussion Top


EEST at 500 mg/kg showed a significant antidiabetic activity and prevents STZ-induced hepatic and renal enzyme abnormalities in rats. Doss et al. also studied the antidiabetic activity of water extract of S. trilobatum in alloxan-induced diabetes in rats which showed significant antidiabetic activity and the effect was comparable with that of glibenclamide.[8]

Elevated levels of liver transaminases such as ALT and AST are measured biomarkers of hepatocellular damage, associated with fatty liver disease and hyperglycemia in diabetes. EEST-treated animals significantly reduced the levels of ALT and AST and the results suggest that EEST may improve STZ-induced hepatic damage in diabetic rats.[16]

STZ is used as the most prominent diabetogenic agent for diabetes induction in animals by inhibiting insulin secretion and causes insulin-dependent diabetes mellitus.[17] It is a glucosamine-nitrosourea derived from Streptomyces achromogenes (Gram-positive bacterium) and it is used for the treatment of pancreatic beta-cell carcinoma and to induce diabetes mellitus in rodents. STZ causes hyperglycemia after 2 h of injection, hypoglycemia in 6 h, and finally hyperglycemia by decreasing the insulin levels through the inhibition/destruction of pancreatic beta-cell function.[3],[17] In our research, diabetes is characterized by low glycemic intensity, which was induced to the rats by an STZ injection (dose of 55 mg/kg BW) intraperitoneally. STZ completely destroys beta-cells by accumulating in pancreatic beta-cells via the low-affinity glucose transporter GLUT2 in the plasma membrane and by generation of reactive oxygen species.[17]

In our study, diabetic control rats showed decrease in their BW and this may be due to increased muscle wasting and tissue proteins. Weight loss is one of the significant clinical manifestations of diabetes and this is due to frequent urination and overconversion of glycogen to glucose.[18] Increased levels of renal markers and liver enzymes are predictors of renal damage and liver disease associated with insulin resistance and the same was observed with diabetic control animals.[19],[20] The rats treated with glibenclamide and EEST 500 mg/kg prevented the diabetes-induced weight loss and abnormalities in biochemical parameter.

The diabetic control animals also showed abnormalities in lipid profile. The increased levels of total cholesterol, LDL, VLDL and decreased levels of HDL were found in diabetic animals. This may be due to exogenous fat loading, enhancement of intestinal CoA-dependent esterification, and an abnormal increase in small intestinal acyl coenzyme A: cholesterol acyltransferase activity.[12],[21] The EEST inhibited the hyperglycemia induced by STZ in SD rats, which may be due to its free radical scavenging properties. S. trilobatum is known for its antioxidant activity. The chloroform extract of S. trilobatum exhibited free radical scavenging properties against α,α-diphenyl-β-picryl hydrazyl radicals, and the aqueous extract of S. trilobatum attenuated thioacetamide-induced oxidative stress.[22],[23]

S. trilobatum contains chemical compounds such as soladunalinidine, tomatidine, solanine, sobatum, solasodine, diosgenin, and β-solamarine, in that diosgenin exhibited antidiabetic activity.[23],[24] Ghosh et al. isolated diosgenin from Dioscorea bulbifera and studied its antidiabetic activity and the activity is due to inhibition of pancreatic α-amylase and α-glucosidase.[25] Sobatum, a phytoconstituent of S. trilobatum, exhibited chemoprotective effect in lithium-induced oxidative damage and inhibited peritoneal tumors induced by Dalton's lymphoma ascites and Ehrlich ascites tumor cell in rodents.[26],[27]S. trilobatum is one of the important tropical plants of potential therapeutic importance. Further exploration may give the lead for the development of newer therapeutic agents.


   Conclusion Top


EEST exhibited significant antidiabetic activity at a dose of 500 mg/kg BW. EEST also prevented STZ-induced hyperlipidemia at the dose levels of 125, 250, and 500 mg/kg BW. EEST did not show any antidiabetic activity in diabetic animals treated with 125 and 250 mg/kg of EEST.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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