Anti-hyperglycemic and Anti-oxidant activities of Ethanolic extract of Lantana camara Leaves

The present study has been undertaken to evaluate In-vivo Anti–hyperglycemic and anti–oxidant activities of ethanolic Lantana camara leaves extract (LCE). The extract was prepared by cold percolation process. Phytochemical screening indicates that the presence of flavanoids, phenols, carbohydrates etc. The Anti-hyperglycemic activity of the extract was studied by In-vivo using Streptozotocin induced diabetes rat models. Glibenclamide was used as a standard drug to compare the blood glucose level. The groups treated with Lantana camara leaves extract at dose 200 and 400 mg/kg prevented the diabetes condition in a dose related manner. LCE and Glibenclamide treated rats showed decreased LPO that is associated with increased activity of SOD and catalase. The increase in the level of lipid peroxides in plasma generally is thought to be the consequence of increased production of and liberation in to the circulation of tissue lipid peroxides due to pathological changes. This action shows the Anti-peroxidative effect of LCE. The results obtained thus suggest that 50% ethanolic Lantana camara leaves extract possesses potent Anti-hyperglycemic and Anti-oxidant activity.


Introduction
Diabetes is defined as a state in which homeostasis of carbohydrate and lipid metabolism is improperly regulated by insulin. It leads to hyperglycemia that in due course turns into a syndrome called Diabetes mellitus [1]. Diabetes mellitus (predominantly type 2 diabetes) is a major and growing health problem in almost all the countries. Globally, the number of people with diabetes will be more than double over the next 25 years, to reach a total of 366 million by 2030 [2]. Medicinal plants and their bioactive constituents are used for the treatment of Diabetes throughout the world. Many indigenous medicinal plants have found to be useful to manage diabetes. The synthetic drugs are either too expensive or have undesirable effects or contraindications. Moreover, as the heterogeneity of this disorder increases, there is need to look for more efficacious agents with lesser side effects [3]. In the present study, attempt was made to prove the antihyperglycemic effect of Lantana camara. The stem, root and leaves contain many of the bioactive compounds responsible for various therapeutic applications such as cancers, swellings, chicken pox, asthma and curing infections [4].

Chemicals and Instruments
 Drug for induction of Diabetes: Steptozotocin.  Standard drug used: Glibenclamide  All other reagents used were of analytical grade.  Instruments used: Percolator, UV-Visible Spectrophotometer.

Plant Material and Extraction Procedure
The leaves of Lantana camara (Family -Verbenaceae) were collected from Local area, Attur, Salem, Tamilnadu, India (Fig 1). The plant specimen (No: BSI/SRC/5/23/2016/Tech/1211) was authenticated by Dr. M. Palanisamy, Scientist D, In-charge, Botanical Survey of India, Southern Regional Centre, Coimbatore, TamilNadu, India. The dried leaves were ground to coarse powder by grinder. Ethanolic extract of Lantana camara was produced by cold percolation. The percentage yield of extract was found to be 9.5 % w/w.

Preliminary Phytochemical Analysis
50% ethanolic extract of Lantana camara was subjected to qualitative tests for the identification of various active constituents' viz. carbohydrate, glycosides, alkaloids, amino acids, flavanoids, fixed oil, tannins, gum and mucilage, phytosterols etc. according to Khandelwal [5].

Animals
Sprague-Dawley rats (150-185 g) and Swiss albino mice (20-25 g) of either sex and of approximately the same age were procured from the animal house of J. K. K. Nattraja College of Pharmacy, Kumarapalayam, Namakkal, Tamilnadu. All animals were maintained under standard laboratory conditions (Temperature: 22±2°C and humidity: 45±5%) with 12 hours day: 12 hours night cycle. Animals were provided with standard rodent pellet diet (Dayal, India) and the food was withdrawn 18-24 h before the experiment though water was allowed ad labitum. All experiments were performed in the morning according to current guidelines for investigation of experimental pain in conscious animals [6].
 The overnight fasted mice were weighed and selected.  Ethanolic extract was dosed in a stepwise procedure, with the initial dose being selected as the dose expected to produce some signs of toxicity and were observed for a period of two weeks (Fig 2).
 The toxic doses were selected based on the Guideline 423.
 The result of the LD50 study was done by mice using guideline 423 method.

Streptozocin induced Diabetes Mellitus in models
All animals were allowed to adapt to metabolic cages for 3 days, after which they were fasted overnight and 150 mg/kg Streptozotocin freshly dissolved in normal saline was injected intraperitoneally. Blood glucose levels were measured 3 days after Streptozotocin injection. The animals having a blood glucose level higher than 200 mg/dL was considered diabetic and was used for the experiments [7].

Experimental design
Group I -Control rats received vehicle solution (1% CMC). Group II -Diabetic control rats received vehicle solution (1% CMC). Group III -Diabetic rats treated with extract 200 mg/kg body weight in 1% CMC Group IV -Diabetic rats treated with extract 400 mg/kg body weight in 1% CMC Group V -Diabetic rats treated with Glibenclamide 5 mg/kg in aqueous solution.

Total cholesterol estimation
The serum cholesterol level was estimated by Wybenga and Pileggi method using cholesterol diagnostic reagent kit (span). Absorbance was measured at 560 nm.

Triglycerides estimation
The triglycerides level was estimated by Glycerol phosphate oxidase (GPO) method.

Reaction
Triglycerides Lipases → glycerol + free fatty acid

Reagents Test Standard Blank
Working Reagent 1.0 ml 1.0 ml

Determination of Serum glutamic oxaloacetic transaminase (SGOT)
The SGOT activity was determined according to the method of IFCC modified method using SGOT (Liquizone diagnostic reagent kit).

Calculation
Calculate the average change in absorbance per minute (Δ Abs / min).

Determination of Serum glutamate pyruvate transaminase (SGPT) or ALAT
The SGPT activity was determined according to the method of IFCC modified method using SGPT (Liquizone diagnostic reagent kit).

Calculation
Calculate the average change in absorbance per minute (Δ Abs/min).

Determination of serum alkaline phosphatase (SALP)
The alkaline phosphates level was estimated by p-Nitro phenyl phosphate (PNPP) method (Qualigens diagnostic reagent kit).

Test (T) Blank (B)
Working reagent 1.0 ml Distilled water Sample 20 µl Distilled water

IU /L of Alkaline phosphatase = Δ A /min × 2713
Where F = 2713 is calculated on the basis of molar extinction coefficient for p-nitro phenol and total assay volume to sample volume.

Assay of lipid per oxidation
The concentration of thiobarbituric acid reactive substances (TBARS) was measured (lipid per oxidation product maondialdehyde (MDA) was estimated) in liver using the method of Okhawa et al. The concentration was expressed as n moles of MDA per mg of protein using 1, 1, 3, 3,-tetra-ethoxypropane as the standard [8].

Catalase activity
CAT activity was determined by monitoring the enzyme-catalyzed decomposition if hydrogen peroxide by potassium permanganate according to Cohen et al. [9]. The measured activities were normalized to the protein content of each sample.

Superoxide dismutase activity
A system devoid of enzymes served as control. One unit of the enzyme activity is defined as the enzyme concentration required to inhibit the optical density at 560 nm of chromogen production by 50 % in one min under the assay conditions and expressed as specific activity in mill units/ mg protein [10].

Glutathione peroxidase
The 50% ethanolic extracts of leaves ofLantana camara and Glibenclamide treated rats showed decreased LPO that is associated with increased activity of SOD, catalase and GPx.

Statistical Analysis
The data expressed as mean ± SEM was statistically analyzed by using Prism software package (version 3.0). Statistical significance of differences between the control and experimental groups was assessed by One-way ANOVA followed by Newman-Keuls Multiple Comparison Test. The value of probability less than 5% (P < 0.05) was considered statistically significant.

Plant Material and Extraction
The dried leaves of ethanolic extract of Lantana camara was produced by cold percolation. The percentage yield of extract was found to be 9.5 % w/w (Table 1).

General behavior and acute toxicity studies
50% ethanolic extract of selected plant Lantana camara Leaves upto 2000 mg/kg did not cause any mortality in mice. None of the doses tested produced any gross apparent effect on general motor activity, muscular weakness, fecal output, feeding behavior etc. during the period of observation.

Streptozotocin induced diabetic rats after 0 day and 21 days
Streptozotocin induced the significant increase in the blood glucose level at 0 day (72.33-292.66, p<0.0001). The 50% ethanolic extract of the Lantana camara showed the significant effect compared with the respective diabetic control group, decrease the blood glucose level at a dose of 200 mg/kg and 400 mg/kg (274.75-205.05, 274.75-199.83, p<0.0001), the standard drug Glibenclamide 5 mg/kg also showed the significant decrease the blood glucose level after 21 days (274.75-162.16, p<0.0001). Finally the 400 mg/kg and the standard drug showed the significant decrease in the blood glucose level after 21 days treatment (p<0.0001) as given in (Table 3).

Effects of 50% ethanolic extract of Lantana camara on the blood glucose Levels of Streptozotocin induced diabetic rats
The blood glucose levels of diabetic rats treated with LCE at doses of 200 and 400 mg/kg showed significant differences at 2, 3 and 4 h from initial levels (P < 0.0001). Only diabetic rats with blood glucose level of 252.0± 1.59 mg/dl showed a significant and pronounced reduction of glycemia (P <0.0001) 2, 3 and 4 hr after oral Glibenclamide (5 mg/kg) administration. The LCE was found to be slow and less effective than Glibenclamide in diabetic rats as given in (Table  4).

Effect of 50% ethanolic extract of Lantana camara on Cholesterol and Triglyceride, in Streptozotocin induced diabetic rat serum
It is clearly evident that Streptozotocin caused significant elevation of serum markers. The STZ treated group, the level of cholesterol (72.82-103.61, p<0.0001), triglyceride (81.25-105.79, p<0.0001). In contrast, the groups treated with Lantana camara Leaves extract at dose 200 and 400 mg/kg once daily for 21 days prevented the diabetes condition in a dose related manner. The range of protection were found to be, Cholesterol (103.61-79.54, p<0.05, p<0.0001), Triglyceride (105.79-84.01, p<0.05, p<0.0001) and Glibenclamide (5 mg/kg) also showed the significantly decrease with respect to diabetic control group (103.61-76.71, p<0.0001, p<0.01, 105.79-84.01, p<0.0001, p<0.05) ( Table 5).  (Table 6). The differences in mean SGOT, SGPT, SALP, in extract treated groups are not significantly different from control group at the end of study (21 days).

Discussion
Diabetes mellitus is a serious complex chronic condition that is a major source of ill health worldwide. Plant drugs are frequently considered to be less toxic and freer from side effects than synthetic ones [11]. Streptozotocin induced diabetes cause an increase in blood glucose level in rats [12]. Our studies show that oral administration of 50 % ethanolic Lantana camara Leaves extractdecreases blood glucose in diabetic rats. The increase in the level of lipid peroxides in plasma generally is thought to be the consequence of increased production of and liberation in to the circulation of tissue lipid peroxides due to pathological changes [13]. This action shows the Anti-per oxidative effect of LCE. Changes in the levels of antioxidants are observed in diabetic conditions [14].
The present study demonstrated that the 50% ethanolic Lantana camara Leaves extracthad an anti-hyperglycemic effect in the Streptozotocin induced diabetic rats when administered orally.Administration of LCE and Glibenclamide increased the activities of GPx and GST in diabetic conditions. SOD and catalase are two major scavenging enzymes that remove the toxic-free radical in vivo. LCE and Glibenclamide treated rats showed decreased LPO that is associated with increased activity of SOD and catalase [15]. The results obtained thus suggest that 50% ethanolic Lantana camara Leaves extract possesses potent anti-hyperglycemic and anti-oxidant activity.

Conclusion
It is hoped that the activity-guided isolation of the extract of this plant may yield valuable therapeutic compound(s) useful for developing powerful hypoglycemic or anti-oxidant drugs. The study also demonstrates that pharmacological screening based on the ethno medical leads can yield faster hits in search of therapeutic agents from plants.