Evaluation of anti-diabetic activity of Trianthema portulacastrum Linn in Dithizone induced diabetes in Wistar rats

Trianthema portulacastrum Linn. Whole plant is an extensively used plant for medication in India for the treatment of various health issues. This study was focused to pre igure the antidiabetic potency of Chloroform extract of the Trianthema portulacastrum (CETP) whole plant administered at two doses (100mg/kg and 200mg/kg) for the duration of 21days in Dithizone induced diabetic rats. The rats were separated into ive groups, each group containing six animals. All Groups except Group I were made diabetic by intraperitonial administration of Dithizone(50mg/kg). Group I served as control group, Group II served as diabetic control received Dithizone(50mg/kg), Group III rats were administered with glibenclamide (10mg/kg), a standard oral hypoglycemic agent while Group IV and Group V diabetic rats were served with 100 mg/kg and 200 mg/kg of CETP whole plant respectively. Potency of the plant extract as an antidiabetic was assessed in dithizone induced diabetic models by comparing biochemical parameters like blood glucose level, Glucose tolerance, lipid pro iles along with the liver antioxidant enzymes, glycogen content and Glycogenic enzymes which were quanti ied using standard experimental procedures. There exists a signi icant reduction in levels of blood glucose, raise in glucose tolerance, and improved imbalance in lipid metabolism in diabetic rats after administration of the extract at 100mg/kg and 200mg/kg. T.portulacastrum with 200mg/kg of the extract showed the best hypoglycemic action by comparing favorably well with Glibenclamide. This investigation clearly showed that, the extract is endowed with hypoglycemic activity, T.portulacastrum may also defend the deterioration of liver due to diabetes.

Trianthema portulacastrum, Dithizone-induced diabetes, oral hypoglycemic agent, anti-diabetic activity A Trianthema portulacastrum Linn. Whole plant is an extensively used plant for medication in India for the treatment of various health issues. This study was focused to pre igure the antidiabetic potency of Chloroform extract of the Trianthema portulacastrum (CETP) whole plant administered at two doses (100mg/kg and 200 mg/kg) for the duration of 21days in Dithizone -induced diabetic rats. The rats were separated into ive groups, each group containing six animals. All Groups except Group I were made diabetic by intraperitonial administration of Dithizone(50mg/kg). Group I served as control group, Group II served as diabetic control received Dithizone(50mg/kg), Group III rats were administered with glibenclamide (10mg/kg), a standard oral hypoglycemic agent while Group IV and Group V diabetic rats were served with 100 mg/kg and 200 mg/kg of CETP whole plant respectively. Potency of the plant extract as an antidiabetic was assessed in dithizone induced diabetic models by comparing biochemical parameters like blood glucose level, Glucose tolerance, lipid pro iles along with the liver antioxidant enzymes, glycogen content and Glycogenic enzymes which were quanti ied using standard experimental procedures. There exists a signi icant reduction in levels of blood glucose, raise in glucose tolerance, and improved imbalance in lipid metabolism in diabetic rats after administration of the extract at 100mg/kg and 200mg/kg. T.portulacastrum with 200mg/kg of the extract showed the best hypoglycemic action by comparing favorably well with Glibenclamide. This investigation clearly showed that, the extract is endowed with hypoglycemic activity, T.portulacastrum may also defend the deterioration of liver due to diabetes.

INTRODUCTION
WHO reports Diabetes is a major endocrinal disorder which will affect a near population of 10% worldwide in the year 2020 (King et al., 1998). Diabetes mellitus is a group of metabolic disorders with hypoglycemia where there is a rise in blood sugar level in an individual due to either reduced insulin production or no utilization the insulin by cells that are produced in. This raised blood sugar associated with the classical signs like polyphagia, polydipsia and polyuria. According to the traditional Indian system of medicine (Ayurveda), a men-tion was made on a good number of plants for the cure of diabetes or 'madhumeha' and some of them have been experimentally screened and the active principles have been isolated and identi ied (Grover et al., 2002). It is crucial to induce diabetes in experimental animals which open window for the researcher to develop a new therapy in order to terminate the side effects including patient factors and environmental parameters that nulli ies a clinical investigation (Potenza et al., 2011). However, search for the new anti-diabetic drugs continues. The current study was developed to evaluate the anti-diabetic potency of chloroform extract of Trianthema portulacastrum in dithizone induced diabetic rats. The potency was comparable with Glibenclamide (a standard hypoglycemic drug).

Plant material and Extraction
The whole plant of Trianthema portulacastrum was collected from the forests of Maisammaguda, Secunderabad situated in the state of Telangana (India) and shade dried. The plant specimen was authenticated by a botanist of Osmania University and authenticated voucher specimen Number 145 of the plant has been potted in the department for future reference. The plant material was shade dried, then milled to coarse powder mechanically and successively extracted with Petroleum ether, Chloroform, Ethyl acetate and Methanol using Soxhlet-extractor. Method of maceration was followed for water for 72 hours. The rotary evaporator was used for concentrating the extracts, dried in vacuum desiccators, properly labelled and weighed, stored thereafter in the refrigerator until further use. Preliminary Phytochemical screening for the above plant extracts was conducted (Divya, 2020). Based on the presence of phytochemical constituents, chloroform extract was considered for the evaluation of antidiabetic potential.

Animals
Ethical approval for this experimental study was obtained from the Institutional Animal Ethical Committee with an Approval no: CPC-SEA/IAEC/JLS/11/11/19/14. Wistar albino rats with average body weight from 150g to 250 g were utilized in this study. They were procured from Sanzyme Bio-analytical lab, Plot no. 8 Sys.No.542, Kothur(V), Shameerpet, R.R. Dist. The rats were caged in polypropylene cages and maintained under standard conditions (12 h light and dark cycles at 25 ± three • C and35-60 % humidity). Standard pellet feed and tap water were provided ad-libitum.

Experimental Design
The rats were split into two sets, each comprising ive groups (n = 6 in each group): Former one for the evaluation of anti-diabetic/toxicity studies and the latter for the evaluation of glucose tolerance. Diabetes was induced in all groups other than Group-I by injecting Dithizone at 50mg/kg b/w intraperitoneally. Development of diabetes was allowed for 3 days. Group-I regarded as control received normal saline (1ml/kg p.o.) as a vehicle for a period of 21 days. Group-II regarded as diabetic control, were administered with Dithizone (50mg/kg). Group-III received Glibenclamide (10mg/kg). Groups IV and V were administered with 100mg/kg and 200 mg/kg body weight/day of Chloroform Extract of Trianthema portulacastrum (CETP).

Estimation of Blood Glucose level
At the end of the study, the experimental rats were subjected to overnight fasting. On the 22 nd day, Blood samples (3ml) were withdrawn from the tail vein and subjected for the estimation of blood glucose level using a glucometer (Accu-Chek, Roche Products (Pty) Ltd., South Africa) at 0, 1/2, 1, 2, 4, 6, 8hr.

Estimation of Lipid parameters and Total Protein
Parameters like triglycerides, total cholesterol, HDL cholesterol, and LDL-cholesterol concentrations in serum were determined by automatic analyser technique (Beckman Coulter Inc., Ireland). Total protein in the serum was estimated using bovine serum albumin as a standard (Henry et al., 1974).

Biochemical estimation of markers of oxidative stress
At the end of the study, on 22 nd day, animals were sacri iced and the liver tissue from all the experimental groups of animals was removed carefully followed by washing thoroughly with ice-cold saline, Wet tissue of 0.5 gms was taken exactly and allowed for homogenization in 0.1M Tris-Hydrochloric acid buffer at pH 7.4, the temperature of 4 0 C in a Remi homogenizer with a Te lon pestle rotated at 600 rpm for 30 min. The homogenate was allowed for centrifugation at 2500 rpm for 10 min at 4 o C using a refrigerated centrifuge. The resultant supernatant was utilised for performing the assay of lipid peroxidation products and antioxidant enzymes such as malondialdehyde (MDA) (Uchiyama and Mihara, 1978), reduced glutathione (GSH) (Sedlak and Lindsay, 1968), superoxide dismutase (SOD) (Richard et al., 1976), Catalase (CAT) (Aebi, 1984).

Oral Glucose Tolerance Test
On day 22, the rats from groups I to V of the latter set were received glucose orally (2 g/kg body weight) after 30 min from the administration of the extract/drug (Joy and Kuttan, 1999). Blood samples were withdrawn from the tail vein prior to glucose administration and at 30, 60, and 90min after glucose loading for immediate measurement of blood glucose levels.

Estimation of Glycogen content and Gluconeogenic enzymes
Hepatic glycogen content was estimated by the method of Carroll et al., (Carroll et al., 1956), hepatic gluconeogenic enzymes like glucose-6-phosphatase was estimated by the method described by Koide and Oda (Hikaru and Toshitsugu, 1959), succinate dehygrogenase was estimated by the method proposed by Slater EC et al., (Slater and Bonner, 1952).

Statistical Analysis
Data were expressed as mean ± SEM of six replicates and subjected to one-way analysis of variance (ANOVA) followed by Duncan's multiple range test to determine signi icant differences in all the parameters. Values were considered statistically signi icant at P < 0.05.

Estimation of Blood Glucose Level
On the last day of experiment, it was proven that there existed a signi icant fall in blood glucose levels when the rats were administered continuously with chloroform extract of T. portulacastrum in diabetic rats (Table 1 and Figure 1). The effect was more noticeable in the rats treated with 200 mg/kg body weight of the plant extract and compared fairly well with rats received glibenclamide. The results from the current study revealed that the Chloroform extract (200 mg/kg) of Trianthema portulacastrum whole plant exhibited signi icant antihyperglycemic effect in Dithizone -induced diabetic rats by fall in the fasting blood glucose level. The marked reduction in the levels of fasting blood glucose in diabetic rats treated with the Chloroform Extract Of Trianthema portulacastrum (CETP) may be either due to the increased secretion of insulin from beta cells of pancreas or stimulation of the residual pancreatic mechanism, probably by increasing peripheral utilization of glucose.

Serum Lipid Pro ile and Total Protein
From the experimental study, it was observed that there was an elevation in the levels of serum cColesterol, triglycerides, and LDL and reduced HDL and protein concentrations in diabetic rats when compared with the control group ( Table 2)

Measurement of Liver Function Parameters
The Group-II diabetic rats exhibited a marked increase in levels of ALT, ALP, AST and bilirubin concentrations in serum when compared with the control (group I) (Table 3). Continuous administration of chloroform extract of T.portulacastrum to diabetic rats for 21 days was able to restore all the liver function indices back to normalcy.

Biochemical estimation of markers of oxidative stress
From the results obtained on the last of the experimental study (on 22 nd day of the experimental study) were listed in Table 4. Declined levels of malondialdehyde (MDA) in the standard drugtreated group shown statistical signi icance in comparison with the control group. In a similar way, with 100mg/kg dose and 200 mg/kg of chloroform extracts of the plant were shown a reduction with statistical signi icance compared with the control group. The enzymatic levels of CAT, GSH and SOD, were lowered signi icantly in diabetic rats when compared with normal control rats. The standard drug, the test extracts (100 mg/kg and 200 mg/kg ) showed elevated levels of these enzymes with statistical signi icance. Diabetes and experimental animal models exhibit high oxidative stress due to unremitting and chronic hyperglycemia, which thereby lessen the activity of the antioxidative defence system and thus pop in the free radicals generation. The present investigation reports a signi icant increase in hepatic MDA of diabetic control rats which suggest that peroxidative injury may be involved in the development of diabetes. The extract treated diabetic animals showed a signi icant fall in MDA, which indicate that the Chloroform Extract Of Trianthema portulacastrum (CETP) is having. Potential to inhibit the oxidative damage of hepatic tissues. In the current study, Chloroform Extract of this plant shown a signi icant increase in hepatic GSH levels, which may be the contributing property of the Tanacetum parthenium chloroform extract to exerts its anti-diabetic potential. An enzymatic antioxidant such as SOD and CAT were considered as priliminary enzymes since they are involved in the direct elimination of reactive oxygen species (ROS) (Arulselvan and Subramanian, 2007). ROS causes non-enzymatic glycosylation and oxidation resulting in the inactivation and inhibition of antioxidant enzymes such as CAT and SOD (Al-Azzawie and Alhamdani, 2006). In the current study, it was observed that long term treatment with the extract reverses the activities of these enzymatic antioxidants (SOD, CAT), by signi icantly increasing the activity of such enzymes.

Oral Glucose Tolerance Test
Table 5 and Figure 2 represents the blood glucose levels of the experimental rats after glucose intake (oral). The blood glucose level in the control rats raised to the high level after 30 min of glucose intake and decreased to near normalcy at 90min. In the diabetic control group (Group II), the blood glucose concentration was high after 30 min of glucose intake and remained high for the next 60 min. Trianthema portulacastrum (Group IV and V) and drug ( glibenclamide) treated diabetic rats (Group III) were shown a signi icant decrease in blood glucose level in comparison to diabetic control rats (Group II). Table 6 represents the effect of Trianthema portulacastrum on Glycogen content and Gluconeogenic enzymes. Hepatic glycogen content in diabetic rats (group II) was found to be signi icantly reduced when compared with the control (group I). Treatment with the standard drug (group III), Trianthema portulacastrum (group IV and V) enhanced the glycogen storage ef iciency of the liver of diabetic rats (treated) in comparison with diabetic control animals. In the fasting stage, the Glycogen content of normal animals was slightly higher than diabetic animals and this may be due to breakdown of glycogen to retain the normal blood glucose levels, whereas glycogen levels in diabetics were found to be very low against high blood glucose levels, is maybe possibly due to lower levels of glycogen synthase activity.

Analysis of Glycogen content and Gluconeogenic enzymes
The activity of glucose-6-phosphatase, the regulatory enzyme of hexose monophosphate shunt (HMP shunt pathway) was found to be lowered in diabetic animals and increased in drug (Glibenclamide), Trianthema portulacastrum chloroform extract-treated animals. The activity of this enzyme was higher in comparison to untreated diabetic animals, indicating an improvement in glucose utilization by this (HMP shunt) pathway. Succinate dehygrogenase activity was decreased in diabetic control animals (Group II), was found to be increased in standard (Glibenclamide), Trianthema portulacastrum treated animals (Group IV and V). Increase in succinate dehygrogenase activities in treated animals (Groups III, IV and V) indicates better utilization of energy-yielding intermediates by Krebs cycle.

CONCLUSIONS
It is evidenced that oral administration of chloroform extract of Trianthema portulacastrum exerted marked hypoglycemic activity in Dithizone-induced diabetes using Glibenclamide as standard in experimental Wistar rats. The rise lipid parameters like total cholesterol, total bilirubin and hypoproteinemia were also critical parameters taken into consideration in case of liver damage. The results also revealed the bene icial effects of this medicinal plant in improving the imbalance in lipid metabolism experienced during diabetes, as evi-denced by antioxidant defence properties. It can, therefore, be concluded from this study that the chloroform extract of Trianthema portulacastrum, besides its hypoglycemic action, could protect the liver against impairment due to diabetes.