Abstract: Hydroalcoholic extracts of flowers, leaves, stems and roots of Catharanthus roseus Linn. (Apocynaceae) were tested for antihyperglycaemic and hypoglycaemic activities. Antihyperglycaemic activity was tested in glucose over loaded hyperglycemic rats and hypoglycaemic activity in fasted normal rats at two dose levels, 100 and 200 mg kgG-1, respectively. Glimepride 0.1 mg kgG-1 was used as the reference drug for both the activities. Results showed that the hydroalcoholic extracts of every part tested, exhibited significant antihyperglycaemic and hypoglycaemic activity. Comparatively the hydroalcoholic extract of leaves exhibited better activity, next to this stems and flowers were equally effective followed by roots. This study gives an indication to traditional healers those who use different parts of this plant to use the active part that has the ability to manage the complications of diabetes.
INTRODUCTION
Diabetes mellitus, a chronic metabolic disorder, has now become an epidemic with a worldwide incidence of 5% in the general population. More than 100 million of the world’s population has already reached the diabetic mark. The number of people suffering from diabetes has soared to 246 million and the disease now kills more people than AIDS (Anonymous, 2006). Decreased physical activity, increasing obesity, stress and changes in food consumption have been implicated in this increasing prevalence in the past two decades (Shastri, 1980). Overt diabetes affects 2-3% of the total world population. In conventional therapy, Type 1 diabetes is treated with exogenous insulin and Type 2 with oral hypoglycaemic agents (sulphonylureas, biguanides etc.) (Pepato et al., 2005). Though different types of oral hypoglycemic agents are available along with insulin for the treatment of diabetes, there is an increase demand by patients to use the natural products with antidiabetic activity (Venkatesh et al., 2003). Since time immemorial, patients with non-insulin requiring diabetes have been treated orally in folk medicine with a variety of plant extracts. In India a number of plants are mentioned in ancient literatures (Ayurveda) for the management of diabetic complications. Catharanthus roseus Linn. (Rosy Periwinkle or Madagascar Periwinkle) is a species of the genus Catharanthus in the family Apocynaceae that is commonly used in most of the herbal preparations for diabetes. It has been reported to possess anticancer activity (Jean et al., 1999), antidiabetic activity (flowers and leaves) (Sumana and Suryawanshi, 2001), hypolipidemic activity (Antia and Okokon, 2005), antioxidant activity (Jaleel et al., 2007) wound healing activity, has vasodilating, blood thinning, memory-enhancing actions and also used in alzheimer’s disease (Nayak et al., 2007). The main active constituents present in this plant are alkaloids (Jean et al., 1999), flavonoids (Gilles et al., 1999) and steroids (Akirai, 1999). As mentioned earlier it has been used effectively in various traditional systems of medicines for the treatment of diabetes. Few practitioners may use the leaves alone and others may use whole plant. In this direction our efforts were directed to identify the most effective antidiabetic part of C. roseus.
MATERIALS AND METHODS
Plant Material
Leaves, flowers, stems and roots of C. roseus were collected from
Mandsaur in the month of July and authenticated by Dr. H.S. Chattree, Department
of Botany, Government PG College Mandsaur (MP). A voucher specimen (BRNCP/V/001/2006)
was deposited in the herbarium of Department of Pharmacognosy, BRNCP, Mandsaur
(MP) for future reference.
Extraction
The collected plant parts (Flower, Leaves, Stems and Roots) were dried and
powdered. The powdered material (500 g) was firstly defatted with petroleum
ether and extracted with hydroalcohol (50:50) for 72 h in soxhlet apparatus.
The extract was evaporated under reduced pressure to obtain solid mass. Percent
yield and phytoconstituents (Brain and Turner, 1975; Khandelwal, 2005) present
in the extracts are given in Table 1.
Animals
After getting approval from the Institutional Animal Ethical committee (registration
number 918/ac/05/CPCSEA), Wistar strain rats (weighing between 100-150 g) procured
from the animal house of B.R. Nahata College of Pharmacy, Mandsaur were used
for investigation. The animals were housed in standard environmental conditions
of temperature (21±2°C), humidity (55±10%) and a 12 h light-dark
cycle. Rats were supplied with standard pellet diet and water ad libitum.
Acute Toxicity Studies
The acute toxicity test (LD50) of the extract was determined
according to the OECD guidelines No. 420 (Organization for Economic Co-operation
and development). Female albino rats (100-150 g) were used in this study. After
the sighting study, starting dose of 2000 mg kg-1 (P.O.) of the extracts
were given to 5 animals in each groups. The treated animals were monitored for
14 days for mortality and general behavior. No death was observed till the end
of the study. The extract was found to be safe up to the dose of 2000 mg kg-1
and from the results two doses of 100 and 200 mg kg-1 were chosen
for further experimentation.
Antihyperglycemic Activity
Antihyperglycaemic activity was studied in glucose overloaded hyperglycemic
rats (Gupta et al., 2005; Babu et al., 2002). Animals were divided
in to 10 groups (n = 5), group 1 was kept as vehicle control, treated only with
vehicle, 1% Tween 80. The positive control group animals, group 2, received
0.1 mg kg-1 of Glimepride, remaining 8 groups were treated with extracts
of different parts of plants suspended in 1% Tween 80 at two dose levels, 100
and 200 mg kg-1 respectively. In zero hour blood sugar level was
determined from overnight fasted animals.
| Table 1: | Percentage yield and phytochemical screening of hydroalcoholic extracts of Catharanthus roseus |
After 30 min of the drug treatment animals were fed with glucose (1.5 g kg-1) and blood glucose was determined after 1/2, 1, 2 and 3 h of the glucose load. Blood glucose concentration was estimated by the glucose oxidase enzymatic method using a commercial glucometer and test-strips (Accu-chek ActiveTM Test meter).
Hypoglycaemic Activity
Animals were classified in to 10 groups (n = 5). Group 1 was kept as control,
received a single dose of 0.5 mL/100 g of the vehicle, group 2 was treated with
0.1 mg kg-1 of Glimepride as hypoglycemic reference drug. Groups
3 to 10 were treated with various plant extracts at two dose levels (100 and
200 mg kg-1) as mentioned in Table 3. Blood samples
were collected from the tail tip at 0 (before oral administration), 1/2, 1,
2, 3, 4 and 5 h after vehicle, extract and drug administration (Ekrem et
al., 2005). The blood sugar level was measured using Accu-chek Active TM
Test strips in Accu-chek ActiveTM Test meter.
Statistical Analysis
The values are expressed as mean±SEM. The results were analyzed for
statistical significance using one-way ANOVA followed by Dunnet’s test.
RESULTS
Results of the phytochemical investigations carried out for various parts are given in Table 1. Alkaloids, carbohydrates, flavonoids, tannins and steroids are found in all the extracts. Saponins are present in the flower, leaves and roots and triterpenoids were identified only in leaves and roots (Table 1). The acute toxicity studies of hydroalcoholic extracts were determined using OECD guidelines No. 420. At a starting dose of 2000 mg kg-1 no mortality was observed for any extracts. So 1/10th of this dose was selected for all the in vivo studies as a maximal dose and the activity was studied using two dose levels 100 and 200 mg kg-1, respectively.
In order to identify the active antihyperglycaemic part, the hydroalcoholic extracts of various parts of C. roseus were studied for their antihyperglycaemic activity using glucose induced hyperglycaemic model. The different hydroalcoholic extracts of the plant parts of Cathranthus were studied at two different doses (100 and 200 mg kg-1 b.wt.) on glucose fed hyperglycaemic animals. The results of the experiment indicated that all the extract of different parts prevented the rise of blood glucose level significantly compared to negative control group (Table 2). Also the extracts of flowers, stems and leaves reduced blood glucose levels below the normal values, indicating their hypoglycaemic effects.
| Table 2: | Effect of hydroalcoholic extracts of various parts of C. roseus Linn. on glucose loaded hyperglycemic rats |
| Values are expressed in Mean±SEM, (n = 5); a: p<0.001, b: p<0.01 vs control (Dunnet’s test) | |
| Table 3: | Effect of hydroalcoholic extracts of various parts of C. roseus on fasted normal rats |
| Values are expressed in Mean±SEM, (n = 5); a: p<0.001, b: p<0.01 vs control (Dunnet’s test) | |
Comparatively the hydroalcoholic extract of leaves showed the maximum activity at a dose of 200 mg kg-1 and the activity was found dose dependant.
The hydroalcoholic extracts of various parts of C. roseus were subjected to hypoglycaemic studies at two dose levels (100 and 200 mg kg-1) and the results are given in Table 3. The extracts of all the parts exhibited hypoglycaemic activity at 1st, 2nd and 3rd h, but only the leaves extract was found to exhibit the hypoglycaemic activity up to 5th h. Comparatively the extract of leaves exhibited better antihyperglycaemic and hypoglycaemic activity than any other part. Next to this stems and flowers exhibited the mentioned activities and root part was found to contain less activity compared to remaining three parts.
DISCUSSION
This study was performed to identify the most effective antihyperglycaemic and hypoglycaemic part of C. roseus. Various traditional practitioners are using this plant in the treatment of diabetes. Diabetes is a major health problem affecting major populations worldwide. Epidemiological studies and clinical trials strongly support the notion that hyperglycemia is the principal cause of complications. Effective blood glucose control is the key for preventing or reversing diabetic complications and improving quality of life in patients with diabetes. Thus sustained reduction in hyperglycemia will decrease the risk of developing microvascular complications and most likely reduce the risk of macrovascular complications (Muniappan et al., 2004). On the basis of this statement we have selected the glucose induced hyperglycaemic model to identify the active antihyperglycaemic part of this plant. Any drug that is effective in diabetes will have the ability to control the rise in glucose level by different mechanisms and the ability of the extracts to prevent hyperglycaemia could be determined by glucose loaded hyperglycaemic model.
In the glucose loaded hyperglycaemic model, all the plant parts tested for antihyperglycaemic activity exhibited significant antihyperglycaemic activity at both the dose levels 100 and 200 mg kg-1. This supports the claim of Sumana and Suryawanshi (2001), who reported the antidiabetic effects of flowers and leaves. The activity of leaves and flowers was found to be more or less same in their studies. Contradictory to this, in present study the hydroalcoholic extract of leaves was found to exhibit better activity and, stems and flowers were found to be equally effective. Excessive amount of glucose in the blood induces the secretion of insulin. This secreted insulin will stimulate peripheral glucose consumption and controls the production of glucose through different mechanisms (Andrew, 2000). However from the study (negative control) it was clear that the secreted insulin requires 2-3 h to bring back the glucose level to normal. In case of the extracts and standard drug treated groups, the glucose levels have not exceeded than that of the negative control, giving an indication regarding the supportive action of the extracts and drug in the glucose utilization. The effect of glimepride, standard drug used in this study, on glucose tolerance has been attributed to enhanced activity of beta cells of the pancreas resulting in secretion of larger amounts of insulin. So the mechanism behind this antihyperglycaemic activity of plant extracts involves an insulin-like effect, probably, through peripheral glucose consumption or enhancing the sensitivity of beta cells to glucose, resulting in increased insulin release (Muniappan et al., 2004). Further the observed hypoglycaemic activity of the plant extracts supports the suggested mechanisms. In these contexts a number of other plants have also been reported to have hypoglycemic effects (Leila et al., 2007).
In conclusion, the findings described in the present study provide further evidence in support of the folkloric claim of traditional practitioners on C. roseus herb for the regulation of blood sugar and using the leaves alone in herbal preparations may provide better therapeutic effects to minimize the complications of diabetes. Works are in progress to isolate the active principle from the leaves responsible for antihyperglycaemic and hypoglycaemic activity.
ACKNOWLEDGMENT
The authors are thankful to the Director, BRNCOP for providing all the necessary facilities to carry out this research work.