INTRODUCTION:

Diabetes is a severe chronic metabolic disease. Diabetes mellitus (DM) is generally a heterogeneous syndrome which symptoms are characterized by high levels of blood glucose level¹. Diabetes is caused by acquired or inherited deficiency in production of insulin by the beta cell of pancreas, or by the ineffectiveness of the insulin produced in the body².In the long duration, this disease can affect most of the organ systems in the body³. The patients of diabetes belong to all age groups. It is now considered as a major global health problem. Globally an estimated number of about 387 million people are currently diagnosed to have diabetes⁴.

The use of insulin and different oral hypoglycaemic drugs are associated with various side effects. Apart from the currently available different synthetic drugs, many herbal drugs have been recommended for the treatment of the diabetic patients.

There are many plants present in nature which possess marked antidiabetic activity³. Medicinal plants have high amount and variety of phytochemicals like flavonoids, alkaloids, polyphenol etc. and these served as rich source of therapeutic agents⁵.

Musa paradisiaca is widely distributed throughout the tropical regions. It is commonly known as banana tree or kela tree. It is found all over the India. It is a tall herbaceous plant. Fruits are found to be fleshy, oblong, 6-8cm long in wild form and more longer in the cultivated varieties. Cylindric fruits are berries in several clusters and golden yellow or yellowish green in colour when ripe. Its unripe fruits are used as vegetable. This plant has a robust tree like pseudo-stem. Green leaves are 8 ft long and 2 ft broad. It is generally cultivated in different parts of India due to its wide consumption. Almost all parts of this plant are traditionally used for several medicinal purposes. Fruit of the plant is employed for traditional folk medicine in the management of different diseases like diabetes, wound healing and ulcer. In different literature it has been reported that various parts of Musa paradisiacal are traditionally used in folk medicine for the treatment of dysentery, diarrhea, hypertension, intestinal lesions in ulcerative colitis, cardiac disease, diabetes, nephritis and gout. It is reported that mature green fruit of Musa paradisiaca are used to treat diabetes in traditional herbal medicine^6,7,8. This study was performed to evaluate the hypoglycemic effect of ethanol extract of Musa paradisiacal unripe fruit pulp in normal and diabetic mice. Effect of Musa paradisiacal extract on body weight in normal and allox an-induced diabetic mice was also investigated.

MATERIALS AND METHOD:

Collection of plant material:

The fresh unripe fruit of Musa paradisiacal were collected from local village area of Nalbari district, Assam, located in between 26°N and 27°N latitude and 91°E and 91°47′ E longitude during the month of April to September. The collected plant parts were identified and authenticated in the Botany Department of Gauhati University.

Preparation of Ethanol extracts of Musa paradisiacal:

The matured green unripe fruits of Musa paradisiacal were collected. The fruits were peeled off. The fruits were cut into small pieces. Then the pieces of the fruits were dried under shade. The pieces were mechanically crushed and ground into powder. A portion (150g) of the powdered fruits of Musa paradisiacal was weighed into a 1000 ml round bottom flask and 300 ml of ethanol was added to it. The resultant mixture was magnetically stirred in the round bottom flask for 72 hours and at room temperature and then allowed to stand for another 24 hours. The ethanol extract was prepared by decanting, followed by filtration using cotton and Whatman filter paper to obtain a clear filtrate. The filtrate was evaporated to dryness at 50-60 °C under reduced pressure in a vacuum rotary evaporator. Thus crude ethanol extract was obtained.

Animal used:

The white laboratory mice of both sexes with average weight of 24-30g were used as the experimental animal for the study. After an acclimatization period of 30 days, they were used for experimentation. Animal were housed under uniform environmental conditions in plastic cages of 18cm×12 cm size. The animals were fed on commercial pellet diet and were given drinking water. The protocol for the study was approved as well as all animals were carefully monitored and maintained in accordance with ethical recommendations of the Ethical Committee of Animal Research of Gauhati University.

Induction of diabetes mellitus with Alloxan monohydrate:

Alloxan is an oxygenated pyrimidine derivative. Alloxan selectively destroys the insulin producing beta-cells in the pancreas, hence it is used to induce diabetes in laboratory animals. Alloxan monohydrate freshly dissolving in distilled water was injected intraperitoneally at a dose of 180 mg/kg body weight to mice. After 7 days, mice with diabetes mellitus indicated by hyperglycemia were used for present study. Alloxan monohydrate was obtained from Sigma-Aldrich and all the other chemicals used were of analytical grade and were acquired from commercial sources.

Experimental design of various groups of animals:

The mice were randomly divided into four groups, each containing six mice. Standard diet and water was provided to the animals.

Group I (Untreated normal control mice):

Normal control mice received only normal diet and water during the experimental period but without any therapy.

Group ll (Plant extract treated normal mice):

Normal mice treated with a single dose of ethanol extract of Musa paradisiaca orally at a dose of 500 mg/ kgbwt daily one time, for 14 consecutive days.

Group III (Diabetic control mice):

Mice of this group were alloxan-induced diabetic model and were served as diabetic controls throughout the experimental period but without any therapy.

Group IV (Plant extract treated diabetic mice):

Diabetic models of mice treated with a single dose of ethanol extract of Musa paradisiaca orally at a dose of 500 mg/kgb wt daily one time, for 14 consecutive days.

Treatment continued for 14 consecutive days. Before the treatment and at the end of 7^th and 14^th day fasting blood glucose level were estimated by using a blood glucose test strip and a one touch commercial glucometer. Blood samples were collected from the tail tip. All blood samples were collected by cutting the tail-tip of the mice. The blood glucose level was measured in milligram per deciliter.

Statistical analysis:

Results were expressed as mean values ± standard error of mean (Mean±SEM). The significances of the differences between the means of the tests and control studies were established by t-test and ANOVA. Values of p<0.05 were considered statistically significant.

RESULT:

Preliminary phytochemical screening:

The preliminary phytochemical screening of the crude extracts of the unripe fruit of Musa paradisiacal was carried out in order to ascertain the presence of its constituents by utilizing standard conventional protocols. The phytochemical study indicated the presence of flavonoids, carbohydrates, tannins and phenolic compounds.

Toxicity studies for Musa paradisiacal:

The crude ethanol extract of green unripe fruit of Musa paradisiacal was tested for their toxicity in mice. Single oral administration of the ethanol extract of Musa paradisiacal in different doses (500, 1000, 2000, 3000 and 4000 mg/kg body weight) were administered to different groups of mice. The general gross behavior was observed for a period of 72 hours. Upto the highest dose (i.e. 4000mg/kg body wt.) did not reveal any behavioral changes or mortality.

Effect on body weight:

One of the characteristics of diabetes is loss of body weight, which was also seen in this study. Normal control animals were found to be almost stable in their body weight during the study period. In group II, ethanol extract of Musa paradisiacal treated normal mice, there is a slight increase of body weight of the mice during the study period i.e. 21 days. But in the groups of diabetic mice a reduction in body weight was found. The alloxan-induced diabetic mice exhibited loss of body weight. Before starting the experiment, all the groups had no significant difference in initial body weight. A significant decrease in body weight was detected in the all groups of diabetic mice, compared to the normal control group from 7 days after alloxan injection. All the animals treated with alloxan in diabetic control group (Gr III) showed a loss in body weight (from 27.83 g to 19.72 g) which was persistently observed till the end of the study period i.e. 21 days (Table 1). In group IV, the initial body weight was reduced (from 28.78 g to 21.73 g) after alloxan treatment. But it has been found that the body weights in the group IV mice were gradually increased (from 23.67 g to 24.12 g) as compared to those of the diabetic control by the treatment with ethanol extract of unripe fruit of Musa paradisiaca. Thus initially there was a decrease in body weight of diabetic mice and after treatment of ethanol extract of unripe fruit of Musa paradisiaca (500 mg/kgbwt), it prohibited the reduction in body weight in the alloxan-treated diabetic mice and showed gain in weight as in normal control animals. Results were analyzed statistically by One-way Analysis of Variance (ANOVA). Values of p<0.05 were considered statistically significant.

Table-1.Effect of ethanol extract of unripe fruit of Musa paradisiacal on body weight (g) in normal and alloxan-induced diabetic mice.

Groups	Treatment	Mean body weight in gram
Groups	Treatment	Initial	0^th day	7^th day	14^th day
Group I (Untreated normal control mice)	Without any therapy	27.93 ± 0.53	28.21± 0.07	28.43± 0.14	28.57± 0.23
Group ll (Plant extract treated normal mice)	plant extract	28.31± 0.21	28.55± 0.17	29.53± 0.42*	29.73± 0.48*
Group III. (Diabetic control mice)	Alloxan only	27.83± 0.23	22.92± 0.56	20.34± 0.21	19.72 ± 0.14
Group IV. (Plant extract treated diabetic mice)	Alloxan + plant extract	28.78 ± 0.55	21.73 ± 0.17*	23.67± 0.42	24.12±0.31*

Results were expressed as mean values ± standard error of mean (Mean ± SEM) for 6 mice in each group (n=6); *p ˂ 0.05 Vs Control.

Fig-1.Effect of ethanol extract of unripe fruit of Musa paradisiacal on body weight (g) in normal and alloxan-induced diabetic mice

Table-2.Effect of Ethanol extract of unripe fruit of Musa paradisiaca on blood glucose level of normal and alloxan-induced diabetic mice.

Groups	Treatment	Blood glucose level (mg/dl )
Groups	Treatment	0^thday	7^th day	14^th day
Group I.(Untreated normal control mice)	Without any therapy	95 ± 3.53	88 ± 2.16	96 ± 5.35
Group ll.(Plant extract treated normal mice)	plant extract	103 ± 4.10	94 ± 1.58*	90 ± 2.12
Group III. (Diabetic control mice)	Alloxan only	292 ±7.31	312 ± 5.40	308 ± 4.53
Group IV. (Plant extract treated diabetic mice)	Alloxan + plant extract	305 ± 4.38*	264 ±5.40*	240 ± 6.59*

Results were expressed as mean values ± standard error of mean (Mean ± SEM) for 6 mice in each group (n=6); *p ˂ 0.05 Vs Control.

Fig-2.Effect of ethanol extract of unripe fruit of Musa paradisiaca on blood glucose level of normal and alloxan-induced diabetic mice

DISCUSSION:

Diabetes is one of the major and main culprits responsible in degrading the health of human being in this stressful life. Different medicinal plants are used to treat illness and diseases like diabetes for thousands of years^9,10. Many plant species in traditional folk medicine were used for their hypoglycemic activities and therefore used to treat and manage diabetes. Apart from the various literatures available in the form of Ethics, Journals, Books etc., a wide range of electronic information systems are also now available which cover different aspects of Ethnobotany. Previously it was reported that various parts of Musa paradisiacal plant have antidiabetic efficacy. The present study was undertaken to assess the hypoglycemic activity of ethanol extract of Musa paradisiacal unripe fruit pulp.

Alloxanmonohydrate produced significant increase in blood glucose level of experimental mice by damaging pancreatic beta-cells, resulting decrease in the insulin secretion, which decreases the proper utilization of the glucose by the various tissues of the body^11,12. In the present study, ethanol extract of unripe fruit of Musa paradisiacal at a dose of 500 mg/kgbwt could produce a significant fall in blood glucose levels by about 21.31% in diabeticmice, after 14 days of treatment.

Reduction in the body weight in the case of diabetic animals as well as in humans is very common and well known. In case of diabetes, the body weight will increase when normal blood glucose levels are achieved which is generally observed in the case of oral hypoglycemic drugs or insulin. Ethanol extract of Musa paradisiacas lightly increases the body weight in diabetic mice, which might be due to the increased insulin secretion in the pancreas and better glyceamic control. The potency of Musa paradisiacal ethanol extract may be increased by the purification of the crude extractor isolation of the proper active constituents from the ethanol extract.

The main mechanism by which the extract brings hypoglycemic effects is not clearly known. But the probable cause of reduction of blood glucose level might be due to increased sensitivity of insulin receptor, increased uptake of glucose peripherally or increased insulin secretion by pancreatic beta cell¹³. Phytochemicals present in the medicinal plants are known to possess potent hypoglycemic activity¹⁴. The phytochemical screening test results showed that ethanol extract of Musa paradisiacal fruit contains flavonoids, carbohydrates, tannins and phenolic compounds. The hypoglycemic activity of Musa paradisiaca may probably be due to the presence of these phytochemicals. Thus, it has been observed from the study that Musa paradisiacal has therapeutic usefulness in diabetes Mellitus as adjunct drug.

From the present investigation we may conclude that Musa paradisiaca extract, was effective in reducing the blood glucose level under our experiment conditions and the crude extract was found to be safe for further biological studies. Additional studies are needed for purification, separation, isolation and identification of bioactive compounds or antidiabetic components from the unripe fruit of this important medicinal plant. Further studies are also required to elucidate its mode of action.

ACKNOWLEDGEMENTS:

The authors are thankful to the Department of Chemistry, Gauhati University for providing facilities and encouragement.

REFERENCES:

1. Panal S. Characterization Simplisia and Ethanolic Extract of Pirdot(Saurauia Vulcani, Korth) Leaves and Study of Antidiabetic Effect in Alloxan Induced Diabetic Mice. International Journal of ChemTech Research. 2015; 8(6): 789-794.

2. Sajeeth I, Manna PK, Manavalan R. Der Pharmacia Sinica.2011; 2 (2): 220-226.

3. Devi S, Kumar D, Kumar M. Ethnobotanical values of antidiabetic plants of M.P. region, India. Journal of Medicinal Plants Studies. 2016; 4(3): 26-28.

4. Kanungo S, Mahapatra T, Bhowmik K, MahapatraS, Saha J, Pal D, Sarkar K.Diabetes scenario in a backward rural district population of India and need for restructuring of health care delivery services. Epidemiol.2016; 6(2): 1-10.

5. Ayman YA, Tobgy El, HematS. Phytochemical detection and Therapeutical properties of Moringaoleifera leaves. International Journal of ChemTech Research. 2016; 9(9):156-168.

6. Imam MZ and Akter S. Musa paradisiaca L. and Musa sapintum L.:A phytochemical and pharmacological review. Journal of Applied Pharmaceutical Science. 2011; 1(5): 14-20.

7. Kumar S, Kumar C. Phytoconstituents and pharmacological activities of Musa paradisiaca Linn. Asian Journal of Biochemical and Pharmaceutical Research. 2012;4(2): 199-206.

8. Banik G, Bawari M, Choudhury MD. Choudhury S.Some anti-diabetic plants of Southern Assam. Assam University, Journal of Science & Technology, Biological and Environmental Sciences, 2010; 5(1): 114-119.

9. Garg P and Deep A. Antidiabetic profile of herbal drugs: A review. Hygeia. J. D. Med. 2015; 7(2): 42-50.

10. Piero MN, Nzaro GM, Njagi JM. Diabetes mellitus – a devastating metabolic disorder. Asian Journal of Biomedical and Pharmaceutical Sciences. 2014; 4(40): 1-7.

11. Viana GS, Medeiros AC, Lacerda AM, Leal LK, Vale TG, Matos FJ. Hypoglycemic and anti-lipemic effects of the aqueous extract from Cissussicyoides. BMC Pharmacol.2004; 8: 4-9.

12. Battell ML, Yuen VG, Verma S. Other models of type-1 diabetes, In: Experimental models of diabetes, Florida, USA: CRC Press LLC.1999; 219-229.

13. Mohamed B, Abderrahim Z, Hassane M, Abdelhafid T, Abdelkhaleq L. Medicinal plants with potential antidiabetic activity – A review of ten years of herbal medicine research (1990-2000). International Journal of Diabetes Metabol. 2006; 14(6): 1-25.

14. Kadali VN, Pola SR, Ramesh T and Sandeep BV. Anti diabetic plants present in West Godavari district of Andhra Pradesh India- A short review. International Journal of Pharma Sciences and Research (IJPSR).2016; 7(2): 72-76.

Received on 06.10.2017 Modified on 17.11.2017

Research J. Pharm. and Tech. 2018; 11(3): 1048-1052.

DOI: 10.5958/0974-360X.2018.00196.8

Evaluation of Hypoglycemic effect of Ethanol extract of Musa paradisiaca unripe fruit pulp on normal and alloxan induced diabetic mice.