Antidiabetic Effect of Luffa acutangula Fruits and Histology of Organs in Streptozotocin Induced Diabetic in Rats

 

S. Mohan Raj*, Shahid Mohammed1, Vinoth Kumar S.2, Santhosh Kumar C3. and Subal Debnath3,

1Jogaiah Institute of Technology and Sciences College of Pharmacy, Kalagampudi, W.G, Andhra Pradesh

2Gogulakrishna College of Pharmacy, Sulurpet, Andhra Pradesh.

3Srikrupa Institute of Pharmaceutical Sciences, Vil. Velkatta, Kondapak (Mdl), Dist. Medak, Siddipet, Andhra Pradesh – 502 277.

 

ABSTRACT:

The Antidiabetic activity of fruits and seeds ethanolic extract of Luffa acutangula  (Cucurbitaceae) was studied in a Streptozotocin {STZ) induced diabetic in rats. The acute toxicity and lethality (LD50) and the Phytochemicals analysis of the extract were also evaluated. The results showed that the extract (200 and 400 mg/kg) significantly (P<0.05) reduced fasting blood sugar of Streptozotocin diabetic rats in a dose-related manner, with maximum hypoglycemic effect at after 21 days. Acute toxicity and lethality test of the extract in rats gave an oral LD50 greater than 5 g/kg. It is clearly evident from the study that the streptozotocin administration caused the significant increase in the blood glucose level at 0 day (p<0.001). The 50% ethanolic extract of the fruits of Luffa acutangula 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 (p<0.001), the standard drug glibenclamide also showed the significant decrease the blood glucose level after 21 days (241.33-105.33, p<0.001). Finally the 400 mg/kg and the standard drug showed the significant decrease in the blood glucose level after 21 days treatment (p<0.001). The findings indicate that the leaves of Luffa Acutangula may be beneficial as an Antidiabetic therapy.

 

KEYWORDS: Streptozotocin, Luffa Acutangula, Antidiabetic

 

INTRODUCTION:

Diabetes Mellitus is considered as one of the five leading causes of death in the world. An epidemic by all standards, nearly 250 million people suffer from diabetes across the world. Synthetic anti-diabetic drugs increase the insulin secretion or decrease the blood glucose level but they are also producing many harmful effects. Since, increase in the use of these drugs in diabetes therapy leads to many side effects and undesirable hazards, there is worldwide trend to go back to natural resources, i.e., traditional plant1. In industrialized countries the people are seeking for safer alternative to allopathic medicine because of the increasing realization on the adverse side effects of many modern remedies. The current interest in and demand for herbs is a worldwide phenomenon, WHO currently encourages, recommends and promotes traditional/ herbal remedies in national healthcare programmes because such drugs are easily available at low cost, are comparatively safe and people have faith in such remedies2. Luffa Acutangula wild (Cucurbitaceae), commonly known as “Turai” (Hindi), “Peerkku” (Tamil), and Kritavedhana” (Sanskrit), is an pale yellowish – brown and cylindrical or club – shaped. The seeds are pale green and bitter in taste and cultivated in wastelands especially along the coastal areas in throughout India.


The  plant is commonly used all over the world for the treatment of diabetes, anti- inflammatory , bronchodialator , antimicrobial activity, nutritional values, antiproliferative and anti- antiangiogenic activity and potent CNS depressant (1-4). The ethno medicinal use of the plant in the treatment of diabetes in the all over world, we evaluated the Antidiabetic potentials of L.acutangula fruits extract in diabetic rats.

 

MATERIALS AND METHODS:

Animals:

Sprague-Dawley rats (150-185g) and Swiss albino mice (20-25 gm) of either sex and of approximately the same age were selected for the study. The study was carried in accordance with the rules and regulations laid by the Institutional Animal Ethics Committee. They were kept in the departmental animal house at 26 ± 2°C and relative humidity 44 – 56% in polypropylene cages. The animals were exposed to alternate 12 hours of darkness and light each. 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 libitum. All experiments were performed in the morning according to current guidelines for investigation of experimental pain in conscious animals64. The standard orogastric cannula was used for oral drug administration in experimental animals.

 

Plant material:

The fruits of Luffa Acutangula (Family-Cucurbitaceae) were collected from the local market in month of January 2011. The plant material was authenticated by Dr. A.B.S Murthy, Botanist. A voucher specimen has been deposited at the museum of our college.

 

Preparation of plant extract:

The powdered plant material (1000g) was macerated with petroleum ether to remove fatty substances and filtered by using Whatman filter paper no. 1. The filtrate is dried under reduced pressure by using rotavapour (Buchi, USA) and then dried in lyophilizer (Labconco, USA). The residue was then stored in a desiccator. The marc left was dried and then further exhaustively extracted with of 50% ethanol for 3 days (3 X 5L). The extract was separated by filtration and concentrated on rotavapour (Buchi, USA) and then dried in lyophilizer (Labconco, USA) under reduced pressure and low temperature to obtain 87.52 g of solid residue (yield 8.75 % w/w). The extract obtained was further subjected to toxicological and pharmacological investigations, which was subsequently subjected to phytochemical analysis using standard procedures3.

 

Acute toxicity tests (OECD 423):

This test involves the administration of a simple bolus dose of test substances to faster healthy young adult rodents by oral gavage, observation for up to 15days after dosing and recording of body weight and the necropsy of all the animals. In this method pre-specified fixed doses of the test substances were used i.e, 5mg/Kg, 50mg/Kg, 300mg/Kg, 2000mg/Kg and the mortality due to these doses were observed. Generally female animals were used for this study and each dose group should consist of 3 animals (Table no-3).

 

IN VIVO ANTIDIABETIC ACTIVITY:

Experimental induction of diabetes:

All animals were allowed to adapt to metabolic cages for 3 days, after which they were fasted overnight and 65mg/kg of Streptozotocin freshly dissolved in normal saline was injected intraperitoneally. After Streptozotocin treatment, all animals were returned to their cages and given free access to food and water. Blood glucose levels were measured 3 days after Streptozotocin injection and used as parameter 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 diabetic and then included in this study. The mean blood concentration of glucose in normoglycemic rats was 95 mg/dl. Diabetic rats were randomly assigned to four different groups (n = 6 animal/groups). All treatments started 3 days after Streptozotoicn  injection.

 

Experimental Design:

Group I- Control rats received vehicle solution (1% carboxy methyl cellulose)

Group II-Diabetic control rats received 30 mg/kg body weight in (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 10 mg/kg body Weight in aqueous solution

 

The vehicles and the drugs were administered orally using intra gastric tube daily for three weeks. After three weeks of treatment the rats were fasted overnight, the blood samples were analyzed for blood glucose content. Then the animal was sacrificed by cervical decapitation. The liver, kidney and pancreas was exposed and per fused with cold phosphate buffer saline of pH 7.4. Blood free liver and kidney were taken out and homogenized in a glass Teflon homogenizer separately (10% w/v). Incubations were done at 37ºC under controlled conditions for biochemical estimations. Fresh blood drawn was centrifuged for 10 min at 2000 rpm. Erythrocyte sediment is resuspended twice in physiological NaCl solution (1:10) and centrifuged again in same solution. 250 ml of washed erythrocytes are then resuspended in 1000 ml physiological NaCl solution and stored at 4°C in the dark until SOD measurement4.

 

Glucose estimation:

The collected serum samples of different study group were subjected to the serum glucose level estimation by enzymatic GOD-POD method by using glucose diagnostic kit (Auto-span).We can also measure the change of optical density directly from Bio Chemical analyzer at 505 nm5.

Total cholesterol estimation

The serum cholesterol level was estimated by wybenga and pileggi method using cholesterol diagnostic reagent kit (span). Reagents were Mixed well and kept in the boiling water bath exactly for 90 seconds. Mixer was allowed to cool to room temperature under running tap water. Absorbance was measured at 560 nm6.

 

Triglycerides estimation:

The triglycerides level was estimated by Glycerol phosphate oxidase (GPO) method. Mix and Incubate at 37oC for 10 minutes. Read the absorbance against reagent blank on a spectrophotometer at 520 nm against reagent blank. The final colour stable for 30 minutes7.

 

Bio-chemical parameters:

Serum was analyzed for the following parameters Aspartate aminotransferase/Serum glutamic oxaloacetic transaminase (ASAT)/ (SGOT), Alanine amino transferase/Serum glutamate pyruvate transaminase (ALAT)/(SGPT), Alkaline phosphates (ALP) and cholesterol9,8,10.

 

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).

 

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).

 

Determination of serum alkaline phosphatase (SALP):

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

 

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 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.

 

RESULTS AND DISCUSSION

The main property in this regard is their Antidiabetic activity which enables them to attenuate the problem of hypoglycemia. Antidiabetic plants an important role in inhibiting the glucose level and, thus providing protection to human against hyperglycemia11. Realizing the fact, this research was carried out to evaluate the Antidiabetic activity of 50% ethanolic extract of fruits of Luffa Acutangula in streptozotoicn induced diabetic rats. The phytoconstituents were extracted by using different solvent of increasing polarity like Petroleum ether, Ethanol (50%).The extractive values were presented in (Table no.1)

 

Phytochemical evaluation

The phytoconstituents were identified by chemical tests which showed the various phytoconstituents in the both extracts, the phytochemical evaluation shows the presence of flavonoids, phenolic compounds, tannins, glycosides, saponins, phytosterols and carbohydrate in the ethanolic extract.

 

LD50 value of 50 % Ethanolic extract of Luffa Acutangula.

There were no mortality is observed by using 2000mg/kg b.w to the mice after 48 hours observation. So 1/10 and 1/5 of the dose i.e., 200mg/kg and 400mg/kg dose were selected for the study

 

In vivo Anti diabetic studies

Effect of 50% ethanolic extract of fruits of Luffa Acutangula on streptozotocin induced diabetic rats after 0 day and 21 days.

It is clearly evident from (Table no.2) that the streptozotocin administration caused the significant increase in the blood glucose level at 0 day (71.83-250.66, p<0.001). The 50% ethanolic extract of the fruits of Luffa Acutangula  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 (241.33-185.26, 241.33-118.16,p<0.001), the standard drug glibenclamide also showed the significant decrease the blood glucose level after 21 days (241.33-105.33, p<0.001). Finally the 400 mg/kg and the standard drug showed the significant decrease in the blood glucose level after 21 days treatment (p<0.001). Streptozotocin diabetes causes an increase in blood glucose level in rats. Streptozotocin exerts its diabetogenic action when it is administered parenterally. The action of Streptozotocin in the pancreas is preceded by its rapid uptake by the β cells. Rapid uptake by insulin-secreting cells has been proposed to be one of the important features determining Streptozotocin diabetogenicity. Our studies show that oral administration of 50 % ethanolic extract of the fruits of Luffa Acutangula decreases blood glucose level in diabetic rats.

 


 

 

Table No. 1. Percentage yield of various extracts of Luffa Acutangula

Plant used

Part used

Method

Percentage yield

Luffa Acutangula

 

Fruits

Cold percolation

Pet. ether

50%ethanol

2.35% w/w

8.75%w/w

 

Table No. 2. Anti-diabetic effect (Glucose level) of 50% ethanolic extract of Luffa Acutangula (ELA) on streptozotocin induced diabetic rats after 0 and 21 days.

Groups

Treatment

Dose

0 day (mg/dl)

After  7 day (mg/dl)

After 14 days

(mg/dl)

After 21 days

(mg/dl)

I

Control

V.1% CMC

71.83 ±7.12

72.33±6.26

73.16±5.72

72.16±6.57

II

Dia.Control

30 mg/kg

250.66±23.59***

243.12±23.23***

246.32±24.72***

241.33±23.33***

III

STZ +ELA 200

200mg/kg

246.33±22.43*

222.16±21.34*

205.12±19.76*

185.26±18.12*

IV

STZ +ELA 400

400mg/kg

240.32±21.26*

198.16±18.12**

182.16±17.7**

118.16±10.67***

V

Gliben-Clamide

10mg/kg

235.84±20.12***

152.84±14.67***

132.26±12.27***

105.33±10.52***

Values are expressed as Mean ± SEM of 6 rats in each group and 4 rats in Diabetic control group. *P < 0.05, ** P < 0.01, ***P < 0.001 compared with diabetic control group, and normal control group.

 

Table No. 3. Effect of 50% ethanolic extract of Luffa Acutangula (ELA) on the glucose level, cholesterol, and triglyceride in blood serum

Groups

Treatment

Dose

Glucose level

(mg/dl) after 21 days

Cholesterol

(mg/dl)

Triglycerides

(mg/dl)

I

Control

V.1%CMC

72.16±6.57

73.52±6.27

80.16±7.57

II

Diabetic

30 mg/kg

241.33.±23.33***

118.38±7.12***

130.23±12.27***

III

STZ +ELA 200

200mg/kg

185.26±18.12*

98.27±7.62*

98.27±9.16*

IV

STZ + ELA 400

400mg/kg

118.16±10.67**

88.16±6.67**

88.16±6.67**

V

Glibenclamide

10 mg/kg

105.33±10.52***

83.12±7.31***

83.12±7.72***

Values are expressed as Mean ± SEM of 6 rats in each group and 4 rats in Diabetic control group. * P < 0.05, ** P < 0.01, ***P < 0.001 compared with diabetic control group, compared and normal control group.

 

Table No. 4. Effect of 50% ethanolic extract of Luffa Acutangula (ELA) SGOT, SGPT,

SALP.

Groups

Treatment

Dose

SGOT (U/l)

SGPT (U/l)

SALP (U/l)

I

Control

V.1%CMC

64.73±6.23

22.16±2.62

231.62±22.12

II

Diabetic

30 mg/kg

105.45±8.79***

48.42±5.12***

283.23±27.16***

III

STZ+ELA 200

200 mg/kg

71.84±7.12*

32.17±2.82*

239.57±21.96*

IV

STZ + ELA 400

400mg/kg

65.38±6.25***

25.17±22.2***

235.91±22.79***

V

Glibenclamide

10 mg/kg

66.34±6.56***

23.29±2.16***

233.23±22.77***

Values are expressed as Mean ± SEM of 6 rats in each group and 4 rats in Diabetic control group. * P < 0.05, **P < 0.01, ***P < 0.001 compared with diabetic control group and normal control group

 

 

Table No. 5. Effect 50% ethanolic extract of fruits of Luffa Acutangula (ELA) on body weight of rats during anti-diabetic study

Group

Treatment

Dose

0 week (gm)

1st week (gm)

2ndweek (gm)

3rd week (gm)

I

Control

V.1%CMC

178.83±1.03

180.67±.86

182.17±.86

183.50±1.21

II

Diabetic

30 mg/kg

180.83±1.33x

180.66±1.11x

178.66±1.09x

164.50±1.06x

III

STZ +ELA 200

200 mg/kg

154.83±1.35c

155.33±1.66c

157.16±1.23c

161.16±1.06c

IV

STZ + ELA 400

400mg/kg

153.83±1.35c

156.33±1.28c

158.67±0.95c

162.67±0.86c

V

Glibenclamide

10 mg/kg

170.17±1.13c

173.17±1.09c

175.5±1.06c

178.83±0.97c

a P < 0.05, b P < 0.01, cP < 0.001  compared with diabetic control group. x P < 0.05, y P < 0.01, zP < 0.001 compared with normal control group.

 

 


Effect of 50% ethanolic extract of Luffa Acutangula on streptozotocin on cholesterol and triglyceride, in streptozotocin induced diabetic rat’s serum. 

It is clearly evident that Streptozotocin caused significant elevation of serum markers. The Streptozotocin treated group, the level of cholesterol (73.52-118.38, p<0.001), triglyceride (80.16-130.23, p<0.001). In contrast, the groups treated with 50 % ethanolic extract of the fruits of Luffa Acutangula at dose 200 and 400mg/kg once daily for 21 days prevented the diabetes condition in a dose related manner. The range of protection were found to be, cholesterol (118.38-98.27, 118.38-88.16 p<0.05, p<0.001), triglyceride (130.23 - 98.27, 130.23- 88.16 p<0.05, p<0.001) and glibenclamide (10mg/kg) also showed the significantly decrease with respect to diabetic control group (118.38-83.12, p<0.001, p<0.01, 130.23-83.12,p<0.001,p<0.05) in( Table no.3). It has been demonstrated that insulin deficiency in diabetes mellitus leads to a variety of derangements in metabolic and regulatory process, which in turn 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 due mainly to increase in the mobilization of free fatty acids from the peripheral fat depots. In present study the 50 % ethanolic extract of the fruits of Luffa Acutangula decreases the cholesterol level in a significant manner. The 50 % ethanolic extract of the fruits of Luffa Acutangula also decreases the triglycerides and lipid levels in diabetic rats.

 

 

Effect of the 50% ethanolic extract of fruits of Luffa Acutangula on SGOT, SGPT and SALP levels in blood serum.

It is clearly evident that Streptozotocin caused significant increase in SGOT (64.73 105.45, p<0.05, p<0.001), SGPT(22.16-48.42, p<0.05, p<0.001) and SALP (231.62-283.23, p<0.0001).The 50% ethanolic extracts of 200, and 400 mg/kg of fruits of Luffa Acutangula  decreases the SGOT (105.45-71.84, 105.45 65.38, p<0.001, p<0.01, p<0.05), SGPT (48.42-32.17, 48.42-25.17, p<0.001, p<0.05) and SALP (283.23-239.57, 283.23-235.91, p<0.05, p<0.001) levels with respect to diabetic control group and glibenclamide (10mg/kg) also showed the significantly decrease with respect to diabetic control group(105.45-66.34, 48.42-23.29, 283.23-233.23, p<0.0001, p<0.05) as shown in table no. 5 and in fig. no. 4. A marked hike observed in the activities of SGOT, SGPT and ALP in serum of diabetic rats indicates injury caused to tissue due to STZ. When cell membrane gets damaged, the enzyme which is normally located in the cytosol leaked into the blood stream thus manifesting damage affected the liver and other tissues. Thus the 50% ethanolic extract of fruits of Luffa acutangula  200, and 400 mg/kg of  decreases the SGOT, SGPT and SALP levels with respect to diabetic control group and glibenclamide (10mg/kg) also showed the significantly decrease up to normal level. The results were showed in (Table no 4)

 

Effect of 50% ethanolic extract of Luffa Acutangula on body weight and food intake of rats   

The 50% ethanolic extract of fruits of Luffa Acutangula   at a dose of 200 and 400 mg per kg of body weight, there is no significant difference on body weight (183.5-178.83) and food intake (16.16-16.17) in the end of the study (Table.no.5).

 

Histopathological studies on Pancreas:

In the pancreas of normal control group rats, many round and elongated islets were evenly distributed throughout the cytoplasm, with their nucleus lightly stained than the surrounding cells. In Diabetic Control group rats, the islets were damaged, shrunken in size and infiltration of lymphocytes was observed. The treatment with 400 mg of Luffa Acutangulat extract   significantly reduced pancreatic β-cells damage compare to 200mg plant extract.

The histological sections of the pancreas, kidney, and tissues were observed to know the effect of Luffa Acutangula fed in non-diabetic and diabetic rats. This was done to observe any protective or harmful effect of Luffa Acutangula extract on non-diabetic and diabetic rats.

.         

CONCLUSION:

In conclusion, the present study clearly demonstrates that the ethanol fruits and seeds extract of Luffa acutangula is an effective antidiabetic agent with multiple therapeutic effects mediated by a combination of preventing the β-cell destruction, histological architecture of the pancreatic islets, improving glucose disposa12. As a result indicates that Luffa acutangula extracts were more useful effective in comparison with glibenclamide in attenuating the increased serum parameters resulting from damage of STZ-induced diabetic rats and that the Luffa acutangula extract treatment may be of use as an antidiabetic supplements. Overall, our report from the present analysis should be ground data for undertaking further study, and it is useful information for investigating new sorghum materials for food additives and human health.

 

REFERENCE:

1.     S Punit Bansala, Piya Paula, Jayesh Mudgala, Pawan G. Nayaka, Steve Thomas Pannakal B, K.I. Priyadarsinic, M.K. Unnikrishnana, Antidiabetic, antihyperlipidemic andantioxidant effects of the flavonoids rich fraction of Pilea microphylla(L.) inhighfatdiet/streptozotocin-induced diabetes in mice. Experimental and Toxicologic Pathology, 2011, 1-8.

2.     Min Chung, Eun-Hye Kim, Min-A Yeo, Sun-Jin Kim, Myong–Cheol Seo, Hyung-In Moon, Antidiabetic effects of three Korean sorghum phenolic extracts in normal and streptozotocin-induced diabetic rats, Food Research International, 2011,44:127–132.

3.     Rucha Pandita, Ashish Phadke, Aarti Jagtap, Antidiabetic effect of Ficus religiosa extract in streptozotocin-induced diabetic rats, Journal of Ethnopharmacology, 2010, 128: 462–466.

4.     S Kumar, V Kumar, Om Prakash, Antidiabetic and anti-lipemic effects of Cassia siamea leaves extract in streptozotocin induced diabetic rats, Asian Pacific Journal of Tropical Medicine, 2010, 871-873.

5.     Ivorra M.D., Pay´AA M., Villarb A, A review of natural products and plants as potential hypoglycemic drugs. Journal of Ethnopharmacology, 1989, 27: 243–275.

6.     Katsumata K, Katsumata K, Jr., Katsumata Y: Protective effect of diltiazem hydrochloride on the occurrence of alloxan- or streptozotocin-induced diabetes in rats. Horm Metab Res 1992, 24: 508-510.

7.     Kim Hr, Rho Hw, Park Jw, Kim Js, Kim Uh, Chung my, Role of ca2+ in alloxan-induced pancreatic beta-cell damage. Biochim Biophys Acta, 1994, 1227: 87-91.

8.     Badole SL, BodhankarSL, Antidiabeticactivityofcycloart-23-ene-25-diol (B2) isolated from Pongamia pinnata (L. Pierre) in streptozotocin-nicotinamide induced diabeticmice. Eur J Pharmacol, 2010, 632:103–9.

9.     Anwar M, Meki A. M. R. (2003). Oxidative stress in streptozotoicn induced diabetic rats: Effects of garlic oil and melatonin. Comparative Biochemistry and Physiology, 2003, 135: 539−547.

10.   Eidi A., Eidi, M., Darzi, R. Antidiabetic effect of Olea europaea L. in normal and diabetic rats. Phytotherapy Research, 2009, 23(3): 347−350.

11.   Kamalak kannan N., Prince P., Anti-hyperglycaemic and antioxidant effect of rutin, a polyphenolic flavonoid, in streptozotocin-induced diabetic wistar rats. Basic & Clinical Pharmacology & Toxicology, 2006, 98:97–103.

12.   Prakasam A., Sethupathy S., Pugalendi K., Effect of Casearia esculenta root extract on blood glucose and plasma antioxidant status in streptozotocin diabetic rats. Polish Journal of Pharmacology, 2003, 55, 43–49.

 

 

Received on 06.12.2011

Modified on 24.12.2011

Accepted on 12.02.2012        

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 4(2): March-April  2012, 64-69