EVALUATION OF ANTIDIABETIC ACTIVITY OF LEAF EXTRACT OF PLUMBAGO ZEYLANICA IN ALLOXAN - INDUCED DIABETIC RATS

About Author:
Vijay Kumar Dadi*
Department Of Pharmacology,
Vinayaka Missions College Of Pharmacy, Salem (Tamilnadu)

Abstract
The Present investigation was carried out to study the antidiabetic effect of the methanolic extract of plumbago zeylanicain alloxan induced diabetic rats.TheLD50 determination was done in mice by OECD guidelines 423. The LD50 of the plumbago zeylanica was found to be 1000mg/kg. Metformin ( 500 mg/kg p.o) was given as a standard drug. The methanolic extract ( 200 mg/kg p.o) have shown significant antidiabetic activity than ( 100 mg/kg p.o) in alloxan induced diabetic rats by reducing serum Cholesterol, Triglycerides,LDL and increased HDL levels. Histopathological studies also confirmed  the antidiabetic nature of the extract. Our results indicate that extract of plumbago zeylanica have prominent antidiabetic effect in experimental diabetes and can therefore be used as an alternative remedy for the treatment of diabetes mellitus and its complications.

REFERENCE ID: PHARMATUTOR-ART-1137

Introduction
Diabetes mellitus (DM) is common endocrine disorder affecting more than 150 million people worldwide and this number is likely to increase to 300million by the year 2025[1]out of which more than one?fifth are Indians. According to the International Diabetes Federation, India has been declared as the diabetes capital of the world[2]. Plants have been used as sources of drugs for treatment of diabetes in developing countries where the cost of conventional medicines is a burden to the population [3].Despite the introduction of anti hyperglycemic agents from natural and synthetic sources, diabetes and its secondary complications continue to be a major medical problem. Many indigenous Indian medicinal plants have been found to be useful to successfully manage diabetes. One of the great advantages of medicinal plants is that these are readily available and have no side effects.[4] World Health Organization (WHO)[5] has suggested the evaluation of the potential of plants as effective therapeutic agents, especially in areas in which we lack safe modern drugs. Earlier chemical examination of the plant revealed that root contains plumbagin, 3- chloro plumbagin ,zeylinone, chitranone, droserone. The leaves contain little or no plumbagin. Literature review indicated that the antidiabetic activity of leaves of plumbagozeylanica have not been clinically evaluated so far. The objective of present study was to investigate the effect of methanolic extract of plumbago zeylanica leaves in normal and alloxan induced diabetic rats.

Materials and Methods
Plant material
The medicinal properties of plants have been investigated in the light of recent scientific developments throughout the world, due to their potent pharmacological activities and economic viability. As per the informations collected from local vaidhyas and other traditional medicine practitioners, the plant Plumbago zeylanica was selected for our study. The leaves of Plumbago zeylanica (plumbaginaceae) were collected  from the village of  Rajavommangi, East Godavari district, Andhra Pradesh in the month of November 2010. The plant material was authenticated by the Dr. B.Badriah, Head, Department of Botany, OsmaniaUniversity, Hyderabad-500007, (Voucher No: 0524).

Extraction procedure
The freshly collected leaves (2 kg) of Plumbago zeylanica were first air dried and then dried in tray drier under control conditions and powdered. The air dried leaves ofPlumbago zeylanicawere extracted successively with petroleum ether for 15 hrs, methanol for 48 hrs using soxhlet apparatus. The extracts were concentrated using rotary vaccum evaporator(Buchi USA) and then dried in lyophizier (Labconco USA) under reduced pressure. The dried extracts were stored in airtight container and placed in refrigerator.

Phytochemical analysis
Preliminary phytochemical screening of methanol extract revealed the presence of triterpenes, steroids, tannins, flavonoids and alkaloids.[6]

Animals
Albino-Wistar rats (200-250g) and Swiss albino mice (20-25g) of either sex and of approximately the same age were procured from the  animal house of  nishka labs, hyderabad. They were kept in the departmental animal house at 26 ±2 °C and relative humidity 44 – 56 % in Polypropylenecages. The animals were exposed to alternate 12 hrs of darkness and light each..Animals were provided with standard rodent pellet diet and the food was withdrawn 18-24 h before the experiment though water was allowed ad libitum. All experiments were performed in the morning according to current guidelines for investigation of experimental pain in conscious animals. The standard orogastric cannula was used for oral drug administration in experimental animals. The experiment protocols were approved by Instituonal Animal Ethical Committee (IAEC  No-CPCSEA/282/10-11/05)

Chemicals
Alloxan Monohydrate  was purchased Sigma Aldrich Chemicals Pvt, Ltd, Bangalore. All other chemicals and reagents used were of analytical grade.

Evaluation of  Toxicological studies of plumbago zeylanica

Oral acute toxicity studies
Acute toxicity was generally carried out for the determination of LD₅₀ value in experimental animals. The LD₅₀ determination was done in mice by OECD guidelines 423. The aim of performing acute toxicity study is for establishing therapeutic index of particular drug and to ensure safety invivo. LD₅₀ of methanolic extractplumbago zeylanica was found to be 1000 mg/kg.

Table 1: LD₅₀ of methanolic extract ofplumbago zeylanica

Animals used: Swiss albino mice                   Weight of animals: 20-25 g

No of animals:  3                                            Route: oral

LD 50 =1000mg/kg                   ED 50 =100mg/kg

Antihyperglycemic Studies
Experimental induction of diabetes
All animals were allowed to adapt to cages for 3 days, after which they were fasted overnight and 150 mg/kg of alloxan monohydrate freshly dissolved in normal saline was injected intra-peritoneally. After alloxan treatment, all animals were given free access to food and water. Blood glucose levels were measured 2 days after alloxan injection and used as parameters to obtain matching pairs of rats with diabetes of similar level of severity. Only rats with fasting blood glucose levels greater than 200 mg/dl were considered to be diabetic and were used in the experiment. The mean blood concentration of glucose in normoglycemic rats was 95 mg/dl. The animals were randomly assigned to four different groups i.e. group II to V. Group I served as normal control containing 6 normal rats. All treatments started 2 days after alloxan injection.[7]

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Experimental Design
Experimental rats were divided into five groups of six animals each and treatedfor 21 days as follows.

Group I: Normal control  received  1% CMC (10 ml/kg)

Group II: Diabetic controls  received  1% CMC(10 ml/kg)

Group III: Animals Received Methanolic extract of Plumbago zeylanica (100 mg/kg, p.o)

Group IV: Animals Received Methanolic extract of Plumbago zeylanica (200 mg/kg, p.o)

Group V: Animals Received  Metformin (500 mg/kg  p.o).

The vehicles and the drugs were administered orally using oral gavage tube daily for three weeks. Blood samples were collected for the measurement of blood glucose level from the tail vein on 0 day , 7th , 14th and 21st day. The blood glucose level was determined by glucometer (Accucheck). The values of sample treated were compared with that of the standard group which was treated with Metformin.

Estimation Of Biochemical Parameters
On 21 day, blood was collected from retro-orbital plexus of overnight fasted rats under light anesthesia and kept aside for ½ hr for clotting.Serum was separated by centrifuging the sample at 6000 rpmfor 20 min. The serum was analyzed for cholesterol, triglycerides, HDL, LDL.

Histopathological studies
Pancreas tissues from all groups were subjected to histopathological studies. The whole pancreas from each animal was removed after sacrificing the animal under  anesthesia and was collected in 10% formalin solution and immedietly processed by paraffin technique. Sections of 5μm thickness were cut and stained by hematoxyllin and eosin (H and E).

Statistical analysis
All the values were expressed as mean ± SEM (standard error mean) for six rats. Statistical analysis was carried out by using PRISM software package (version 5.0). Statistical significance of differences between the control and experimental groups was assessed by One-way ANOVA followed by Dunnets Multiple Comparision Test. The value of probability less than 5% (P < 0.05) was considered statistically significant.

Table No :2. EFFECT OF METHANOLIC EXTRACT OF PLUMBAGO ZEYLANICA ON BLOOD GLUCOSE LEVEL

IN ALLOXAN INDUCED DIABETIC RATS (24 Hrs Study).

All Values are expressed as Mean ±SEM (n=6) , *p≤0.05, **p≤0.01, ***p≤0.001

*Group I compared with Group II,  # Group II compared with Group III, IV, V.

Table No :3.EFFECT OF METHANOLIC EXTRACT OF PLUMBAGO ZEYLANICA ON BODY WEIGHT IN ALLOXAN INDUCED DIABETIC RATS­ (21Day Study).

All Values are expressed as Mean ±SEM (n=6) , *p≤0.05, **p≤0.01, ***p≤0.001

*Group I compared with Group II,  # Group II compared with Group III, IV, V.

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Table No: 4. EFFECT OF METHANOLIC EXTRACT OF PLUMBAGO ZEYLANICA ON BIOCHEMICAL PARAMETERS MEASURED IN ALLOXAN  INDUCED DIABETIC RATS (21Day Study).

All Values are expressed as Mean ±SEM (n=6) , *p≤0.05, **p≤0.01, ***p≤0.001

*Group I compared with Group II,  # Group II compared with Group III, IV, V

Fig No1:EFFECT OF METHANOLIC EXTRACT OF PLUMBAGO ZEYLANICA ON BLOOD GLUCOSE LEVEL IN ALLOXAN INDUCED DIABETIC RATS (21Day study).

Discussion:
Diabetes mellitus arises from the irreversible destruction of the pancreatic beta cells causing degranulation and reduction of insulin secretion[10]. The present study demonstrated that the methanolic extract of plumbago zeylanica had an antihyperglycemic effect in the alloxan induced diabetic rats when administered orally. It has been demonstrated that insulin deficiency in diabetes mellitus leads to a variety of derangements in metabolic and regulatory process, which in turns leads to accumulation of lipids such as cholesterol and triglyceride in diabetic patients. The abnormal high concentration of serum lipids in the diabetic subject is mainly due to increase in the mobilization of free fatty acids from the peripheral fat depots.  In present study, methanolic extract of the leaves  of plumbago zeylanica at the dose of 200 mg/kg body weight possesses significant antidiabetic activity in rats. It was also found effective in normalizing blood lipids like total cholesterol, total triglyceride,HDL&LDL.