Protective Effect of Luffa cylindrica Fruit Extracts on Alloxan Intoxicated Diabetic Rats

INTRODUCTION

Diabetes is a complex and multifarious group of disorders characterized by hyperglycemia that has reached epidemic proportions in the present century. Several drugs such as sulfonylureas and biguanides are currently available to control hyperglycemia in diabetes mellitus. However, these drugs are not free from side effects and thus searching for the new class of drugs which are essential to overcome these problems.1

Management of diabetes without any side effects is still a challenge to the medical community. There is continuous search for alternative drugs. Therefore, it is prudent to look for options in herbal medicine for diabetes as well. Medicinal plants used in the management of diabetes in the traditional system of medicine might provide new oral hypoglycemic compounds, which can counter the high cost and poor availability of the allopathic medicines for many rural populations in developing countries. India is well known for its herbal wealth. Medicinal plants like Syzigium cumini foenum graecum, Allium sativum, Gymnema slyvestre and Syzigium cumini have been studied for treatment of diabetes mellitus.2

Luffa cylindrica (family: Cucurbitaceae) is one such plant cultivated for different purposes such as vegetable and spice. The leaves and fruits of this plant are used as a highly nutritive vegetable in many countries, particularly in India and China. The leaves of the plant are used in traditional medicine to overcome liver diseases, iron deficiency, stoppage of abnormal menses, shortness of breath, amenorrhea, testicular inflammation, burns, ulcers, ringworm and snake bite.3,4 The major chemical components of Luffa cylindrica are triterpenoid saponins, as well as flavonoids.5,6 Pharmacological studies indicate that Luffa cylindrica has the effects of oxytocic7 , abortifacient,8 antioxidant,9 bronchodilator,10 antifungal11 and hypolipidemic12 properties. Leaves and seeds of the title plant are known to exhibit significant antidiabetic activity.13,14 Extensive literature survey afforded no scientific data on antidiabetic potential of fruits of the title plant. In view of this the present investigation was undertaken to evaluate antidiabetic activity of the fruit extracts of the title plant.

MATERIALS AND METHODS

Plant materials

The fruits of Luffa cylindria were collected in the month of July from the surrounding garden of the Bijapur (Karnataka) after the sample was authenticated by Dr. M. B. Mulimani, Professor of Botany, S.B. Arts and K.C.P. Science College, Bijapur.

Preparation of extracts

The fruits were dried in shade at room temperature. The dried fruits were powdered by using grinder to coarse powder, packed into Soxhlet’s column and subjected to successive extraction using petroleum ether, 70% ethanol and distilled water. Excess of solvents from the extracts were recovered using rotary flash evaporator resulted to obtain semisolid crude extracts. The obtained crude extracts were stored in airtight container in refrigerator below 100C for further studies.

The stock solution of aqueous and ethanolic extracts were prepared using distilled water whereas petroleum ether extract by suspending in 2% aqueous gum tragacanth and subjected to the following studies.

1. Preliminary phytochemical screening of crude extracts.

2. Determination of acute toxicity.

3. Evaluation of antidiabetic activity.

1. Preliminary phytochemical screening

Preliminary phytochemical investigation was carried out on all three extracts such as petroleum ether extract of fruits of Luffa cylindrical (PEEFLC), hydro alcoholic extract of fruits of Luffa cylindrica (HAEFLC) and aqueous extract of fruits of Luffa cylindrica (AEFLC) for detection of various phytochemicals by following standard methods described in practical Pharmacognosy by C.K. Kokate15 and R.K. Khandelwal.16

Experimental animals

Male albino rats (150-180 g) and albino mice (20-25 g) of either sex were procured from Sri. Venkateshwara Enterprises, Bangalore. The animals were acclimatized for a period of 10 days under standard environmental conditions such as temperature (26 + 20 C), relative humidity (45- 55%) and 12 hrs dark/light cycle were maintained in the quarantine. All the animals were fed with rodent pellet diet and water allowed ad-libitum under strict hygienic conditions. Ethical clearance for performing the experiments on animals was obtained from Institutional Animal Ethics Committee (IAEC) (Reg. No. 1076/c/07 CPCSEA).

2. Determination of acute toxicity (LD50)

The acute toxicity for all three extracts of Luffa cylindrica was determined in albino mice. The animals were fasted overnight prior to the experiment, fixed dose method was adopted as per OECD Guideline No. 423; (Annexure-2d). Group of three mice were taken for each test dose.17

3.Evaluation of anti-diabetic activity18,19

Fasting blood glucose of animal was determined after depriving food for 16 hrs with free access of drinking water. Hyperglycemia was induced by a single i.p. injection of 160 mg/kg of alloxan monohydrate in sterile saline. After 3 days of alloxan injection, the hyperglycemic rats (glucose level between 250-300 mg/dl) were separated and divided into different groups comprising of 6 rats each for the antidiabetic study. The treatment (p.o.) was started from the same day except normal control and diabetic control groups for a period of 7 days. During this period, animals in all groups had free access to standard diet and water. Blood glu-cose level was estimated on 1st and 7th day of the treatment. On the 7th day, blood samples were collected from overnight fasted rats by carotid bleeding under mild ether anesthesia for biochemical estimations.

The various groups used in experiment

Group A - Served as normal control and did not receive any treatment.

Group B - Served as diabetic control and received alloxan monohydrate and ve-hicle

(0.5 ml / 100 g, 2% aqueous aqueous gum tragacanth)

Group C - Alloxan monohydrate + Glibenclamide (10 mg/kg, p.o.) and served as Standard.

Group D - Alloxan monohydrate + PEEFLC (500 mg/kg, p.o.)

Group E - Alloxan monohydrate + EEFLC (500 mg/kg, p.o.)

Group F - Alloxan monohydrate + AEFLC (500 mg/kg, p.o.)

The biochemical parameters studied were as follows

1. Fasting blood glucose.

2. Serum urea.

3. Serum total cholesterol

4. Serum triglycerides.

Statistical analysis

The data obtained from the above findings subjected to statistical analysis using one way ANOVA followed by Turkey Kramer Multiple Comparison Test to assess the statistical significance of the results.

RESULTS

Percentage yield of extracts

The percentage yield of Luffa cylindrica fruit extracts are as follows

PEEFLC - 1.37 %

HAEFLC - 14.85 %

AEFLC - 8.18%

Preliminary phytochemical screening

Results of the preliminary phytochemical investigation of petroleum ether, 70% ethanolic and aqueous fruit extracts of Luffa cylindrica are shown in the Table 1.

Acute toxicity study LD50

The Petroleum ether, ethanolic and aqueous extracts were studied for acute toxicity at dose of 2000 mg/kg i.p. in albino mice. The extracts were found devoid of mortality of the animals. Hence 2500 mg/kg was considered as LD50 cut off value for all the three extracts.

The screening doses chosen for the evaluation of antidiabetic activity as per OECD guidelines No. 423 (Annexure - 2D) fixed dose method are stated below.

1. 500 mg/kg PEEFLC (1/5th of 2500 mg/kg).

2. 500 mg/kg HAEFLC (1/5th of 2500 mg/kg).

3. 500 mg/kg AEFLC (1/5th of 2500 mg/kg).

Antidiabetic study

Effect of Luffa cylindrica fruit extracts on fasting blood glucose level in alloxan intoxicated diabetic rats

Petroleum ether, ethanolic and aqueous fruit extracts of Luffa cylindrica were subjected to anti-diabetic activity in rats where alloxan monohydrate (160 mg/kg b.w., i.p.) used as the diabetogenic agent. A marked rise in fasting blood glucose level was observed in diabetic control compare to normal control rats. All the three tested extracts of the title plant significantly lowered the elevated serum blood glucose level on 7th day post treatment. Glibencla-mide produced a significant reduction in blood glucose as compare to diabetic control. The antihyperglycemic potency of the tested extracts was found to be in the following order EEFLC > PEEFLC > AEFLC. However, the effect of test extracts on FBS level was found to be less potent than the reference standard drug, glibenclamide. The results are shown in the Table 2.

Effect of Luffa cylindrica fruit extracts on biochemical parameters

Serum cholesterol, triglycerides and urea levels were decreased significantly by ethanolic fruit extract of Luffa cylindrica followed bypetroleum ether and aqueous extracts on 7th day of post treatment, when compared to diabetic control group. However, the effect of these ex-tracts on above said biochemical parameters was found to be less potent than the standard drug glibenclamide. Results are shown in Table- 3.

DISSCUSSION

Diabetes mellitus is a chronic disease characterized by high blood glucose levels due to absolute or relative deficiency of circulating insulin levels. Though different types of oral hypoglycemic agents are available along with insulin for the treatment of diabetes mellitus, there is a growing interest in herbal remedies, due to the side effects associated with these therapeutic agents. Because of their perceived effectiveness, minimal side effects in clinical experience and relatively low costs, herbal drugs are prescribed widely even when their biologically active compounds are unknown.20

Alloxan causes massive reduction in insulin release by the destruction of β cells of islets of Langerhans there by inducing hyperglycemia. Insulin deficiency leads to various metabolic alteration in animal viz increase blood glucose, triglycerides, cholesterol and urea.21,22

In the present study, treatment with Luffa cylindrica fruit extracts in alloxan intoxicated diabetic rats exhibited significant antihyperglycemic effect. The observed hypoglycemic effect of the test extracts may be due to due to regeneration of pancreatic β cells that were partially destroyed by alloxan.

Diabetes associated with kidney malfunction. Urea, uric acid and creatinine are markers of renal function.23 In our study also increase in serum urea level was observed in diabetic rats and this level was significantly reduced in animals treated with test extracts of the title plant suggests that the tested extracts also possess nephro protective property.

DM also been associated with an increased risk of arteriosclerosis and coronary heart disease due to hypertriglyceridemia and hypercholesteremia which are secondary complications of diabetes.24 An ideal treatment for diabetes would be a drug that not only controls blood glucose level but also prevent arteriosclerosis and other cardiovascular complications associated with diabetes.25

It was observed in the current study, triglycerides and cholesterol concentrations were significantly lowered in alloxan mediated diabetic rats treated with all the three tested extracts. Thus, we postulate that the test extracts of the title plant could modulate blood lipid abnormalities.

Oxidative stress has been shown to have a significant effect in the causation of diabetes as well as diabetes related complications in human beings. Oxidative stress in diabetes has been shown to coexist with a reduction in the antioxidant status.26

Natural antioxidants strengthen the endogenous antioxidant defense mechanism against reactive oxygen species (ROS) and restore the optimal balance by neutralizing the reactive species.27 In the present investigation, antioxidant property of the title plant reported in the literature could be the one of the reason for the observed antidiabetic activity of the fruit extracts of the Luffa cylindrica. 9

There are several literature reports revealed that the flavonoids present in the plant extracts exhibiting potential antidiabetic activity.28 In the present study also presence of flavonoids in the ethanolic and aqueous extracts indicated by preliminary phytochemical investigation may be attributed for the observed antidiabetic activity.

Previous researchers reported that medicinal plants containing sterols possess antidiabetic activity.29 Based on this evidence it can be concluded that the presence of sterols in the petro-leum ether extract may be responsible for exhibiting antidiabetic activity of the title plant.

CONCLUSION

In conclusion, the results of the present study demonstrated the blood glucose lowering effect of Luffa cylindrica fruit extracts in alloxan induced hyperglycemic rats and justifies the utility of fruits of the title plant in the management of diabetes mellitus.