International Journal of Drug Development and Research

Keywords

Mirabilis jalapa, Streptozotocin, Metformin, Antidiabetic, Hyperglycemia

Introduction

Mirabilis jalapa Linn (Nyctaginaceae) is a perennial herb and is known as “Gulambasa” in ayurveda and commonly known as four o’clocks plant [1]. Infusion prepared from aerial part of the plant is used for treatment of diabetes by local healers of Mexico [2]. The present study has been designed to evaluate the effectiveness of Hydro-Alcoholic extract of aerial parts of Mirabilis jalapa L. (HAMJ) for potential antihyperglycemic activity, against normoglycemic and streptozotocin induced hyperglycemic rats.

Materials and Methods

Collection of Plant Material & Preparation of extract

The aerial parts of M. jalapa L. were collected in the month of July – August 2010, from the rural area of the dist. Cuttack, Odisha, India and authenticated by Dr. Kshetra Mohan Das, Senior Scientist of the Central Rice Research Institute, Cuttack, Odisha, India and a specimen voucher (Specimen No. 11- 11/SPS/SOAU) has been kept in the University for future reference. The aerial parts were shade dried and made into coarse powder. The powdered material was initially defatted with petroleum ether followed by 72-hours extraction with 1: 1 mixture of methanol and water using cold maceration process for 72-hours. The extract was filtered and concentrated by rotary evaporator and kept in vacuum desiccators until use. The yield of the extract was 18.5 % w/w with respect to dried powder.

Preparation of Interventions

The Hydro-Alcoholic Extract of Mirabilis jalapa (HAMJ) (at dose levels of 200 mg/kg and 400 mg/kg) was suspended in distilled water using 25% Tween 20 as suspending agent. The standard drug Metformin (300mg/kg) was also prepared in a similar manner. Suspension of distilled water and 25% Tween 20 was used as solvent treatment throughout the study at a dose of 2 ml/kg. The solvent, test samples and standard drugs were administered by oral route based on dose and corresponding weight of the animals.

Animals

Healthy Wistar albino rats (150 – 250 gm body weight) supplied by Central Animal House of School Of Pharmaceutical Sciences, SOA University, Bhubaneswar, Odisha, India were used in the experiments. The animals were acclimatised to laboratory conditions for one week before commencement of experiment. The study was approved by University Animals Ethics Committee (Regd. No. 1171/C/08/CPCSEA).

Induction of Diabetes

Diabetes was induced by administration of multiple low doses (40 mg/kg) of Streptozotocin (STZ) to the overnight fasted rats for five consecutive days intraperitoneally. STZ solution was prepared freshly in ice- cold citrate buffer (0.01 M, pH 4.5) [3]. After 12days of STZ administration, the fasting blood glucose levels (FBG) were measured and the rats with FBG level >250 mg/dL were considered to be diabetic and were used in the study.

Blood Glucose Level and Biochemical Parameters Measurement

Glucose level was estimated by using Glucometer (One Touch Horizon, Lifescan, Johnson and Johnson Company). On terminating the dosing, the rats were fasted for 12 h, sacrificed by decapitation, blood samples were collected by standard method for estimation of serum triglycerides and cholesterol by using commercially available diagnostic kit.

Acute Toxicity Study

Healthy Wistar albino rats of either sex starved overnight, were divided into five groups (n=4). Group I-IV animals were orally fed with HAMJ in increasing dose levels of 0.5, 1.0, 1.5 and 2.0 g/kg. b.w., while group V (untreated) served as control. The animals were observed continuously for the first 2 h for any gross change in behavioral, neurologic and autonomic profiles or any other symptoms of toxicity and mortality if any, and intermittently for the next 6 h and then again at 24 h , 48 h and 72 h for any lethality or death. One-tenth and one-fifth of the maximum safe dose of the extract tested for acute toxicity were selected for the experiment [4].

Single Oral Dose Hypoglycemic Study

The effect of single dose administration of HAMJ on blood glucose level was studied by following previously published methods [5-7]. The normal and diabetic rats were divided into different groups and treatments were given as follows; Group I (Normal rats treated with solvent 2 ml/kg), Group II (Normal rats treated with HAMJ 200 mg/kg), Group III (Normal rats treated with HAMJ 400 mg/kg), Group IV (Normal rats treated with Metformin 300 mg/kg), Group V (Diabetic control rats treated with solvent 2 ml/kg), Group VI (Diabetic rats treated with HAMJ 200 mg/kg), Group VII (Diabetic rats treated with HAMJ 400 mg/kg), Group VIII (Diabetic rats treated with Metformin 300 mg/kg). After 12hrs of fasting, each group of animals received single dose of either solvent or HAMJ (200 mg/kg and 400 mg/ kg) or Metformin 300 mg/kg and the experiment was carried out under fasting condition. Blood sample was collected from tail vein at 0, 1, 2, 4, 6 and 12 hrs after administration of intervention to estimate blood sugar.

Oral Glucose Tolerance Test

Oral glucose tolerance test has been conducted as per established methods [7, 8]. The apportionment of animals into different groups and administration of different interventions has been done as explained in single oral dose hypoglycemic study. The normal rat groups were loaded with glucose (2 g/kg/p.o.) 30 minutes after administration of interventions; whereas diabetic rats were loaded with glucose (5 g/kg) 1hr after administration of interventions. Blood samples were collected from the tail vein at 0, 30, 60, 120 and 180 minutes after glucose loading and glucose levels were measured immediately.

Multiple Oral Dose Hypoglycemic Study

Multiple dose study has been conducted by following methods established previously [5, 7]. The apportionment of animals into different groups similarly as it has been done in previous experiments. Each group either received solvent (2ml), or HAMJ 200 mg/kg, or HAMJ 400 mg/ kg, or Metformin 300 mg/kg everyday 30 minutes before food throughout the experiment duration.

In case of normal rats the treatment was given continuously for 9 days and fasting blood sample was collected on day 0, 1, 3, 6 and 9 for estimating blood sugar. On 10th day blood sample was collected for estimation of serum lipid profile. During the 10 days of observation of the rats were observed for any changes in the body weight relative to day 0, i.e. before the start of the treatment. In case of diabetic rats the treatment was given continuously for 14 days and fasting blood sample was collected on day 0, 1, 3, 6, 9 and 14 for estimating blood sugar. Blood sample was collected for estimation of serum lipid profile on day 15. In case of diabetic rats; food and water intake habits of the rats has been observed.

Statistical Analysis

Results are expressed as Mean ± SE. The data are analyzed by one way ANOVA followed by Turkey – Kramer Multiple Comparison Test. Confidence Interval has been considered as 95% and p < 0.05 are considered significant.

Results

Effect of single dose administration of HAMJ on blood glucose level

Single dose administration of HAMJ at both the dose levels (200 mg/kg and 400 mg/kg) showed significant decrease in blood glucose level after two hours of administration compared to the control group. In normal rats the percentage decrease in blood glucose level at end of study is 16.52%, 22.13% on treatment with HAMJ 200 mg/kg and HAMJ 400 mg/kg respectively (Table 1). In hyperglycemic rats, at 12th hour of observation, the percentage reduction in blood glucose level caused by HAMJ is 27.34% and 37.38% with respect to dose level of HAMJ 200 mg/kg and HAMJ 400 mg/kg (Table 2).

Effect of HAMJ on blood glucose levels during oral glucose tolerance test

During oral glucose tolerance test, it has been observed that blood sugar level reaches maximum after 30minutes of glucose loading. In case of normal rats; the blood glucose level of the groups treated with HAMJ are significantly (p < 0.001) less as observed at 30, 60, 120 and 180 minutes when compared with control rats, with percentage reduction in blood glucose is recorded as 33.64 % and 36.48 % with respect to HAMJ 200 mg/kg and HAMJ 400mg/kg treatment (Table 3). In diabetic rats; the fall in blood glucose level from maximum can be observed in all treatment groups after 60 minutes of glucose administration (p < 0.001 for HAMJ 400 mg/kg, p < 0.05 for HAMJ 200 mg/kg) with percentage reduction in blood glucose from maximum level being 23.63 % and 30.16 % with HAMJ 200 mg/kg and HAMJ 400 mg/kg treatment respectively (Table 4).

results of all the single dose administration experiment indicate that, HAMJ significantly reduces the blood sugar level in both STZ-induced and glucose loaded hyperglycemic rats as well as in normoglycemic rats.

Effect of multiple dose administration of HAMJ on blood glucose levels

In normal rats (Table 5), HAMJ was capable of reducing the sugar level significantly (p < 0.001) on 9th day by 20.65 % & 31.36 % with dose levels of 200 mg/kg and 400 mg/kg respectively. In diabetic rats irrespective of the sampling day (Table 6), HAMJ at both the dose levels of 200 mg/kg (7.78 % - 18.28 %) and 400 mg/kg (10.17 % - 21.29 %) showed antihyperglycemic action. After two weeks of administration of HAMJ, the percentage decrease in blood glucose level is 46.45 % & 55.11 % with respect to HAMJ 200 mg/kg and HAMJ 400 mg/kg.

The results of repeated dose study reveal that with repeated administration, the efficacy of the extract increases proportionately with increase in dose level and in higher dose level (400 mg/kg) the effect resembles with that of the Metformin. Considering these results it may be hypothesized that; the hypoglycemic effect of the extract may involve its Metformin like actions i.e., decreasing intestinal absorption of glucose or improving insulin sensitivity by increasing peripheral glucose uptake and utilization or decreasing hepatic glucose production.

Effect of multiple dose administration of HAMJ on serum lipid profile

Effect of HAMJ on serum lipid profile (Total Cholesterol and Triglyceride) has been depicted in Table 7 & Table 8. Both in normal rats (9 days observation) (Table 7) and diabetic rats (14 days observation) (Table 8) it has been observed that; on treatment with HAMJ serum lipid levels (cholesterol and triglycerides) were appreciably reduced at both the dose levels when compared with control group.

Effect of multiple dose administration of HAMJ on Body Weight

In normal rats (Table 9, 9 days observation), it has been observed that the body weight of rats has increased at both the dose levels of HAMJ 200 mg/kg (8.77 %) and HAMJ 400 mg/kg (7.84 %) relative to day 0, and can be compared with control group (6.22 %).

The percentage decrease in body weight of different groups of hyperglycemic rats (Table 10, 14 days observation) are 34.24 %, 30.26 %, 26.75 % and 30.69% with respect to treatment with solvent, HAMJ 200 mg/kg, HAMJ 400 mg/kg and Metformin 300 mg/kg.

Effect of multiple dose administration of HAMJ on Food and Water Intake of hyperglycemic rats

HAMJ at both the dose levels considerably (p < 0.001) reduced average weekly food intake (Table 11). The percentage decrease in food intake with HAMJ 200 mg/kg and 400mg/kg is found to be 32.34 % and 46.57 % respectively relative to Week- 0 (i.e. before Experiment).

The similar trend also has been observed in average water intake (Table 12) of hyperglycemic rats in comparison to control group.The percentage decrease in water intake for HAMJ 200 mg/kg and HAMJ 400mg/kg is found to be 27.80 % & 40.05 % respectively relative to Week-0 (i.e. before Experiment).

Values are expressed as Mean ± SEM.; (n = 6); One Way ANOVA followed by Turkey – Kramer Multiple Comparison test; *p<0.05, **p<0.01, ***p<0.001 vs. Control Group; “-” sign indicates decrease in the value from the initial value

Discussion

STZ produces oxygen radicals in the body, which cause pancreatic injury and could be responsible for increased blood sugar seen in animals [9]. Diabetes is associated with profound alteration in the plasma lipid and lipoprotein profile. Under normal circumstances, insulin activates enzyme lipoprotein lipase and hydrolyses triglycerides. Insulin deficiency results in failure to activate the enzymes, thereby causing hypertriglyceridemia and hypercholesterolemia [10]. It is well known that streptozotocin-provoked hyperglycemia accompanied by symptoms like loss of weight, polydipsia and polyphagia [11]. Induction of diabetes with streptozotocin associated with a characteristic loss of body weight, which is probably due to muscle wasting [12].

From the results of present investigation, it has been found that HAMJ supplementation is quite beneficial in controlling the blood glucose level of STZ-induced rats; additionally, it improves lipid metabolism. The extract did not significantly prevent loss in body weight in hyperglycemia condition but diminished food intake and the water consumption when compared with diabetic control rats. The similar pattern has been observed in the rats treated with standard drug Metformin. The improvements of these parameters could be attributed to the hypoglycemic properties of the plant. The results of all the repeated dose study reveals that the effect of HAMJ on blood glucose, serum lipid profile, change in physical parameters confirms the hypoglycemic effect of the plant. The result of this study is also in agreement with the previous studies done to evaluate the hypoglycemic activity of roots of Mirabilis jalapa [13]. The control of blood glucose level may be attributed to pancreatic or expancreatic effect of the extract or increased utilization of glucose by the tissues. Studies also suggest that Mirabilis jalapa possess a potent antioxidant activity [14]. The antioxidant potential of the plant may have a major role in reducing oxidative stress associated with streptozotocin induced diabetes. Hence, the aerial parts of M. jalapa L. extract can be considered as a potent source of hypoglycemic agents, which may be attributed to the one or more chemical constituent(s) of the extract.

Conclusion

The hydro-alcoholic extract of the aerial parts of M. jalapa L. has antidiabetic potential. Further studies are necessary to substantiate the above observation and to isolate the bioactive principle(s) from it & to work out the exact mechanism of action involved in the antidiabetic activity of this plant.

Conflict of Interest

NIL

Source of Support

NONE

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12

References

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