Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 40, Num. 6, 2008, pp. 256-260

Indian Journal of Pharmacology, Vol. 40, No. 6, November-December, 2008, pp. 256-260

Research Article

Antidiabetic activity of flower buds of Michelia champaca Linn.

Jarald EEdwin, Joshi SB, Jain DC

Department of Pharmacognosy, B. R. Nahata College of Pharmacy and Research Centre, Mandsaur, Madhya Pradesh
Correspondence Address:Department of Pharmacognosy, B. R. Nahata College of Pharmacy and Research Centre, Mandsaur, Madhya Pradesh
jaraldpharm@rediffmail.com

Date of Submission: 14-Dec-2007
Date of Decision: 30-Jul-2008
Date of Acceptance: 22-Nov-2008

Code Number: ph08073

Abstract

Keywords: Alloxan, antidiabetic activity, hypoglycemic activity, lipid profile, Michelia champaca

Introduction

Diabetes mellitus, a chronic metabolic disorder, has now become an epidemic, with a worldwide incidence of 5% in the general population. The number of people suffering from diabetes has soared to 246 million and the disease now kills more people than AIDS. ^[1] Decreased physical activity, increasing obesity, stress and changes in food consumption have been implicated in this increasing prevalence in the past two decades.^[2] In conventional therapy, Type I diabetes is treated with exogenous insulin and Type 2 with oral hypoglycemic agents (sulphonylureas, biguanides etc). ^[3] Though different types of oral hypoglycemic agents are available along with insulin for the treatment of diabetes, there is an increased demand by patients to use natural products with antidiabetic activity. ^[4] Since time immemorial, patients with non-insulin dependent diabetes have been treated orally in folk medicine, with a variety of plant extracts. In India, a number of plants are mentioned in ancient literature (Ayurveda) for the treatment of diabetic conditions.

Flower buds of Michelia champaca Linn. belonging to the family Magnoliaceae is commonly used by many traditional healers (e.g., Dr. G. Elias, Reg. No. 01323, Leela Vilasam Siddha Varma Vaidyasalai, Karungal, Tamil Nadu) in most of the herbal preparations for diabetes ^[5] and kidney diseases. ^[6] Traditionally, it is being used in fever, colic, leprosy, post partum protection ^[7] and in eye disorders. ^[8] It has been reported to possess antipyretic, anti-inflammatory, ^[9] insecticidal, ^[10] antimicrobial ^[7] and leishmanicidal activities. ^[11] The active constituents reported in this plant are alkaloids, saponins, tannins, sterols, flavonoids and triterpenoids. ^[7]

In Ayurveda, traditional usages of plants are generally and most commonly in the form of their aqueous extracts only. Concurrently, some of the papers searched focus testing plants in their ethanolic or aqueous extracts and some have also reported activity in petroleum ether, benzene and chloroform extracts. ^{[12],[13],[14]} Keeping these facts in view, the present study was undertaken to create a scientific base for the traditional use of the extract of M. champaca as an antidiabetic in diabetes associated complications, and to identify the active antidiabetic.

Materials and Methods

Plant material
Flower buds of M. champaca were collected in March 2006 from Tamil Nadu, India. The taxonomical identification of the plant was done by Dr. H. S. Chatree, Botanist, Government Arts and Science College, Mandsaur, India. The voucher specimen (BRNCP/M/003/2006) was deposited in the Herbarium of the Department of Pharmacognosy, B. R. Nahata College of Pharmacy, Mandsaur.

Preparation of extracts
Dried and powdered plant material (500 g) was successively Soxhlet extracted with petroleum ether (60-80 ^o ), chloroform, acetone, ethanol and water for 72 h each. Crude aqueous extract of this plant was prepared separately by boiling the plant material (25 g) with 200 ml of water for 15 min. The obtained extracts were evaporated in vacuum to give residues and their percentage yields were determined.

Phytochemical screening
In order to determine the presence of alkaloids, glycosides, flavones, tannins, terpenes, sterols, saponins, fats and sugars, a preliminary phytochemical study (color reactions) with various plant extracts was performed. ^[15]

Animals and treatment
Healthy Wistar rats of either sex (150-180 g), with no prior drug treatment, were used for the present studies. The animals were fed with commercial pellet diet (Kamadenu Agencies, Bangalore, India) and water ad libitum. The animals were acclimatized to laboratory hygienic conditions for 10 days before starting the experiment. Animal study was performed in the Division of Pharmacology, B R Nahata College of Pharmacy, Mandsaur, after approval from the Institutional Animal Ethics Committee (registration number 918/ac/05/CPCSEA).

Acute toxicity studies
The acute toxicity test of the extracts was determined according to the OECD (Organization for Economic Co-operation and development) guidelines No. 420. Female Wistar rats (150-180 g) were used for this study. After the sighting study, starting dose of 2000 mg/kg (p.o.) of the test samples was given to various groups containing five animals in each group. The treated animals were monitored for 14 days, for mortality and general behavior. No death was observed till the end of the study. The test samples were found to be safe up to the dose of 2000 mg/kg, and, from the results, 400 mg/kg dose was chosen as the maximum dose for further experimentation.

Antihyperglycemic activity
Antihyperglycemic activity was studied in glucose overloaded hyperglycemic rats. ^[16] The animals were divided into eight groups (n = 5). Group 1 was treated with 5 mg/kg of glibenclamide and the negative control group animals; group 2, received only vehicle; the remaining six groups were treated with 400 mg/kg of various extracts of plant suspended in 1% Tween 80. Zero hour blood sugar level was determined from overnight fasted animals. After 30 min of the drug treatment (p.o.), the animals were fed with glucose (4 g/kg) and blood glucose was determined after 0.5, 1, 2, and 3 hours of the glucose load. Blood glucose concentration was estimated by the glucose oxidase enzymatic method, using a commercial glucometer and test-strips (Accu-chek Active ^TM Test meter).

Hypoglycemic activity
The animals were classified in to four groups (n = 5). Group 1 was kept as control, and was given a single dose of 0.5 ml/100 g of the vehicle; group 2 was treated with glibenclamide (5 mg/kg) as the hypoglycemic reference drug. Groups 3 and 4 were treated with ethanolic extract at two dose levels (200 and 400 mg/kg) (p.o.), as mentioned in [Table - 2]. Blood samples were collected from the tail tip at 0 (before oral administration), 0.5, 1, 2, and 3 h after administration. ^[17] The blood sugar level was measured using Accu-chek Active ^TM Test strips in Accu-chek Active ^TM Test meter.

Antidiabetic activity in alloxan induced diabetes model
Alloxan induced diabetic model was selected to confirm the effectiveness of active antihyperglycemic extract in experimental diabetic conditions. Diabetes was induced in rats by injecting 120 mg/kg of Alloxan monohydrate intraperitoneally in 0.9% w/v NaCl to overnight-fasted rats. The rats were then kept for the next 24 h on 10% glucose solution bottles, in their cages, to prevent hypoglycemia. After 72 h of injection, fasting blood glucose level was measured. The animals that did not develop more than 300 mg/dl glucose levels, were rejected. ^[18],[19] The selected diabetic animals were divided into four groups (n = 5) and one more group of normal non-alloxanised animals was also added to the study. Group 1 was kept as normal control (non-alloxanised rats), which received a single dose of 0.5 ml/100 g of the vehicle; group 2 was kept as negative control, alloxan induced and received a single dose of 0.5 ml/100 g of the vehicle; group 3, diabetic induced, was treated with glibenclamide (5 mg/kg) as the reference drug. Groups 4 and 5, diabetic induced were treated with ethanolic extract that exhibited antihyperglycemic at two dose levels (200 and 400 mg/kg), as mentioned in [Table - 3]. The treatment was continued for seven consecutive days (p.o.). At the end of the 7 ^th day, the rats were fasted for 16 h and blood parameters were determined.

Collection of blood and estimation of biochemical parameters
The blood sugar level was measured using Accu-chek Active ^TM Test strips in Accu-chek Active ^TM Test meter by collecting the blood from rat tail vein. For other plasma profiles, blood was collected from retro-orbital plexus of the rats, under light ether anesthesia, using capillary tubes into eppendorf tubes containing heparin. The plasma was separated by centrifugation (5 min, 5000 rpm) and was analyzed for lipid profiles (serum cholesterol, serum triglyceride, HDL cholesterol, LDL cholesterol), serum creatinine, serum urea, hemoglobin and glylosylated hemoglobin. The plasma profiles were measured by standard enzymatic methods, with an automatic analyzer (Tulip, Goa, Model No. Evaluation 300). ^[14] and glycosylated hemoglobin by colorimetric method.

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. P < 0.05 was considered significant.

Results

Preliminary phytochemical screening
The percentage yields of petroleum ether, chloroform, acetone, ethanol, aqueous and crude aqueous extracts were found to be 0.40, 6.40, 2.00, 4.10, 10.20 and 12.00% w/w respectively. The petroleum ether contained fats and terpenoids. The chloroform extract contained steroids and alkaloids. Acetone extract contained only tannins. Ethanol extract contained carbohydrates, flavonoids, alkaloids and tannins. Aqueous extract contained carbohydrates, alkaloids, flavonoids and saponins. Crude aqueous extract contained carbohydrates, alkaloids, flavonoids, tannins and saponins.

The effect of extracts in glucose loaded hyperglycemic animals
[Table - 1] shows the antihyperglycemic effect in glucose loaded hyperglycemic rats, after administration of the plant extracts at a dose of 400 mg/kg. After 0.5 h of the glucose load, there was a significant rise in the blood glucose levels of the control animals, and at the end of two hours, the glucose level declined. Ethanolic extract exhibited significant antihyperglycemic activity at 1, 2 and 3 h after glucose load, compared to control. Crude aqueous extract and petroleum ether extract exhibited an effect only at 1 h. The remaining extracts did not show significant activity.

The effect of ethanolic extract in fasted normal rats
Based on the antihyperglycemic activity, the active ethanolic extract was subjected to hypoglycemic studies at two dose levels (200 and 400 mg/kg) and the results are given in [Table - 2]. The ethanolic extract of M. champaca did not show any hypoglycemic activity.

The effect of ethanolic extract in alloxan induced diabetic rats
The basal blood glucose levels of all the groups were statistically not different from each other. Three days after alloxan administration, blood glucose values were five-folds higher in all the groups and were not statistically different from each other. After seven days, the values of blood glucose were decreased in all the treatment groups and the diabetic rats showed a slight increase in blood glucose level. The administration of plant extract and glibenclamide to diabetic rats restored the level of blood glucose significantly ( P < 0.01).

The level of total hemoglobin, glycosylated hemoglobin, serum urea, serum creatinine and lipid profiles of different experimental groups are represented in [Table - 3]. The diabetic rats showed a significant decrease in the level of total hemoglobin and a significant increase in the level of glycosylated hemoglobin. The administration of plant extract and glibenclamide to diabetic rats restored the changes in the level of total hemoglobin and glycosylated hemoglobin to near normal levels ( P < 0.05) ( P < 0.01). But 200 mg/kg of ethanolic extract did not show significant effect in increasing the total hemoglobin content.

Alloxan induced diabetic rats showed significant hypercholesterolemia, as compared with control. Treatment with plant extract (400 mg/kg) showed significant decrease in cholesterol levels ( P < 0.01); at the same time, it showed an increase in HDL-c. Hypercholesterolemia was associated with hypertriglyceridemia, as compared with the control animals. Hypertriglyceridemia was also significantly prevented by treatment with the plant extract ( P < 0.05). Diabetic control rats showed a significant increase in creatinine and urea levels, as compared with the control animals. Treatment with ethanolic extract of M. champaca significantly decreased these values ( P < 0.01) ( P < 0.05). The extract was found effective in alleviating experimental diabetes and diabetes related complications [Table - 3].

Discussion

The present study was undertaken to examine the antidiabetic activity of M. champaca and to find out the active antihyperglycemic extract prepared using various solvents of this plant. Also, the antihyperglycemic activities of extracts obtained by successive solvent extraction method and crude aqueous extract prepared in a traditional manner were compared. The effect of this plant in diabetes changes in associated complications biochemical parameters was also assessed.

Effective blood glucose control is the key for preventing or reversing diabetic complications and improving the 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. ^[20] On the basis of this statement, we have selected the glucose induced hyperglycemic model to screen the antihyperglycemic activity of the plant extracts.

In the glucose loaded hyperglycemic model, the plant tested for antihyperglycemic activity exhibited significant antihyperglycemic activity at a dose level of 400 mg/kg. Excessive amount of glucose in the blood induces the insulin secretion. This secreted insulin will stimulate peripheral glucose consumption and control the production of glucose through different mechanisms. ^[21] However, from the study (glucose control), it was clear that the secreted insulin requires 2-3 h to bring back the glucose level to normal. In case of the ethanolic extract, petroleum ether extract, crude aqueous extract and drug treated groups, the glucose levels did not exceed the control group, giving an indication regarding the supportive action of the extracts and drug in the glucose utilization. The most active antihyperglycemic ethanolic extract, when tested for hypoglycemic activity, did not exhibit hypoglycemic activity, suggesting its mechanism being similar to biguanides. Biguanides do not increase insulin secretion; they promote tissue glucose uptake and reduce hepatic glucose output, thereby producing antihyperglycemic effect and not hypoglycemic effect. ^[21]

Alloxan produces hyperglycemia by selective cytotoxic effect on pancreatic beta cells. Our investigations indicate the efficiency of the plant extract in the maintenance of blood glucose levels in alloxan induced diabetic rats, may be possibly by the above mentioned mechanism. In uncontrolled or poorly controlled diabetes, the excess glucose present in the blood reacts with hemoglobin. Therefore, the total hemoglobin level is decreased and glycosylated hemoglobin is increased in alloxan diabetic rats. ^[22] Administration of ethanolic extract for seven days prevented a significant elevation in glycosylated hemoglobin, thereby increasing the level of total hemoglobin ( P < 0.05) ( P < 0.01) in diabetic rats. This could be due to the result of improved glycemic control produced by the plant extract.

The levels of serum lipids are usually elevated in diabetes mellitus and such an elevation represents a risk factor for coronary heart disease. This abnormally high level of serum lipids is mainly due to the uninhibited actions of lipolytic hormones on the fat depots, mainly due to the action of insulin. Under normal circumstances, insulin activates the enzyme lipoprotein lipase, which hydrolyses triglycerides. However, in diabetic state, lipoprotein lipase is not activated due to insulin deficiency, resulting in hypertriglyceridemia ^[23] and insulin deficiency is also associated with hypercholesterolemia due to metabolic abnormalities. ^[24] In our study also, the diabetic rats showed hypercholesterolemia and hypertriglyceridemia and the treatment with plant extract significantly decreased both cholesterol and triglyceride levels. This implies that the ethanolic extract of M. champaca flower buds may prevent or be helpful in reducing the complications of lipid profile seen in some diabetics in whom hyperglycemia and hypercholesterolemia coexist quite often. ^[25]

The diabetic hyperglycemia induced by alloxan produces elevation of plasma levels of urea and creatinine, which are considered significant markers of renal dysfunction. ^[26] After treatment of alloxan-diabetic rats with ethanolic extract, the level of urea and creatinine were significantly decreased, compared to the mean value of diabetic group. This further suggests the potential utility of these plants in diabetes associated complications. ^[27]

Mostly, it was believed that the formation of the artefacts during the preparation of crude aqueous extracts would be responsible for the biological activities. ^[28] In our study, the crude aqueous extract of M. champaca was found active only at 1 h after glucose administration. Though the main classes of active constituents are present in the crude aqueous extract of this plant, no significant activity was observed. This may be due to the lower concentrations of active constituents present at 400 mg/kg of crude aqueous extracts, when compared to 400 mg/kg of successively fractionated extracts. Babayi et al.^[29] suggested the same for the better activity of fractions, compared to extracts. However, the reason behind the less activity of crude aqueous extract was unknown. Hence, the antidiabetic activity of the extracts was caused by substances that naturally exist in the plant, and not due to transformations induced by heating.

We conclude that the plant has shown potential activity in decreasing the serum glucose level and other complications associated with experimental diabetes. This research supports the inclusion of this plant in traditional antidiabetic preparations.

Acknowledgment

References

1.	Anonymous. Diabetes now a global threat gets own day. Sunday Times of India 2006;24:11.  Back to cited text no. 1
2.	Shastri K. Comments on Charaka Samhita. Varanasi: Chanukah Bharati; 1980.  Back to cited text no. 2
3.	Pepato MT, Mori DM, Baviera AM, Harami JB, Vendramini RC, Brunetti IL. Fruit of the Jambolan tree (Eugenia jambolana Lam.) and experimental diabetes. J Ethnopharmacol 2005;96:43-8.   Back to cited text no. 3
4.	Venkatesh S, Reddy GD, Reddy BM, Ramesh M, Apparao AV. Antihyperglycemic activity of Carulluma attenuate. Fitoterapia 2003;74:274-9.  Back to cited text no. 4
5.	Rajagopalan PM, Siddha medicine. Madurai: Siddha Maruthuva Gurukulam; 2000.  Back to cited text no. 5
6.	Nadkarni M. Indian Materia Medica. 1st ed. Mumbai: Popular Book Depot; 1954.  Back to cited text no. 6
7.	Khan MR, Kihara M, Omoloso AD. Antimicrobial activity of Michelia champaca. Fitoterapia 2002;73:744-8.  Back to cited text no. 7
8.	Sobhagini N, Soumit KB, Malaya KM. Ethno-medico-botanical survey of Kalahandi district of Orissa. Indian J Trad Knowledge 2004;3:72-9.  Back to cited text no. 8
9.	Vimala R, Nagarajan S, Alam M, Susan T, Joy S. Antiinflammatory and antipyretic activity of Michelia champaca Linn., (white variety), Ixora brachiata Roxb. and Rhynchosia cana (Willd.) D.C. flower extract. Indian J Exp Biol 1997;35:1310-4.  Back to cited text no. 9
10.	Ulla J, Vijaya K, Shantini S. Sesquiterpene lactones from Michelia champaca. Phytochemistry 1995;39:839-43.  Back to cited text no. 10
11.	Takahashi M, Fuchino H, Satake M, Agatsuma Y, Sekita S. In vitro screening of leishmanicidal activity in myanmar timber extracts. Biol Pharm Bull 2004;27:921-5.  Back to cited text no. 11
12.	Nagarajan NS, Murugesh N, Thirupathy KP, Radha N, Murali A. Antidiabetic and antihyperlipidemic effects of Cleome feline. Fitoterapia 2005;76:310-5.   Back to cited text no. 12
13.	Nalamolu KR, Srinivasu N. Antidiabetic and Renoprotecticve effects of chloroform extract of Terminalia chebula seeds in streptozotocin induced diabetic rats. BMC Complement Altern Med online publication 13 November 2006; doi: 10.1186/1472-6882-6-17.   Back to cited text no. 13
14.	Phuong ML, Ali BA, Aziz E, Abdellatif S, Yahia C, Pierre SH. The petroleum ether extract of Nigella sativa exerts lipid lowering and insulin-sensitizing action in the rats. J Ethnopharmacol 2004;94:251-9.  Back to cited text no. 14
15.	Kokate CK, Purohit AP, Gokhale SB. Text book of pharmacognosy. 33rd ed. Pune: Nirali Prakashan; 2001.  Back to cited text no. 15
16.	Babu V, Ganga DT, Subramonium A. Antihyperglycemic activity of Cassia kleinii leaf extract in glucose fed normal rats and alloxan induced diabetic rats. Indian J Pharmacol 2002;34:409-13.  Back to cited text no. 16
17.	Sezik E, Aslan M, Yesilada E, Ito S. Hypoglycaemic activity of Gentiana olivieri and isolation of the active constituent through bioassay-directed fractionation techniques. Life Sci 2005;76:1223-38.   Back to cited text no. 17  [PUBMED]  [FULLTEXT]
18.	Jamal AA, Issa AA, Mohammad HH. Hypoglycaemic and antihyperglycaemic effects of Trigonella foenum-graecum leaf in normal and alloxan induced diabetic rats. J Ethnopharmacol 1997;58:149-55.  Back to cited text no. 18
19.	Sabu MC, Subburaju T. Effect of Cassia auriculata Linn. on serum glucose level, glucose utilization by isolated rat hemidiaphragm. J Ethnopharmacol 2002;80:203-6.  Back to cited text no. 19
20.	Latha M, Pari L, Sitasawad S, Bhonde R. Insulin-secretagogue activity and cytoprotective role of the traditional antidiabetic plant Scoparia dulcis (Sweet Broomweed). Life Sci 2004;75:2003-14.   Back to cited text no. 20  [PUBMED]  [FULLTEXT]
21.	Andrew JK. Diabetes. New York: Churchill Living Stone; 2000.  Back to cited text no. 21
22.	Pari L, Amarnath SM. Antidiabetic activity of Boerhaavia diffusa L.: Effect on hepatic key enzymes in experimental diabetes. J Ethnopharmacol 2004;91:109-13.  Back to cited text no. 22
23.	Pushparaj PN, Low HK, Manikandan J, Tan BK, Tan CH. Anti-diabetic effects of Cichorium intybus in streptozotocin-induced diabetic rats. J Ethnopharmacol 2007;111:430-4.  Back to cited text no. 23  [PUBMED]  [FULLTEXT]
24.	Murali B, Upadhyaya UM, Goyal RK. Effect of chronic treatment with Enicostemma littorale in non-insulin dependent diabetic (NIDDM) rats. J Ethnopharmacol 2002;81:199-204.  Back to cited text no. 24  [PUBMED]  [FULLTEXT]
25.	Sharma SB, Nasir A, Prabhu KM, Murthy PS, Dev G. Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of Eugenia jambolana in alloxan-induced diabetic rabbits. J Ethnopharmacol 2003;85:201-6.  Back to cited text no. 25  [PUBMED]  [FULLTEXT]
26.	Alarcon-Aguilar FJ, Calzada-Bermejo F, Hernandez-Galicia E, Ruiz-Angeles C, Roman-Ramos R. Acute and chronic hypoglycaemic effect of Ibervillea sonorae root extracts-II. J Ethnopharmacol 2005;97:447-52.  Back to cited text no. 26
27.	El-Demerdash FM, Yousef MI, Abou El-Naga NI. Biochemical study on the hypoglycemic effects of onion and garlic in alloxan-induced diabetic rats. Food Chem Toxicol 2005;43:57-63.  Back to cited text no. 27
28.	Mukherjee KP. Quality control of herbal drugs. New Business Horrizons. New Delhi: Pharmaceutical Publishers; 2002.  Back to cited text no. 28
29.	Babayi H, Kolo I, Okogun JI, Ijah UJ. The antimicrobial activities of methanolic extracts of Eucalyptus camaldulensis and Terminalia catappa against some pathogenic microorganisms. Biokemistri 2004;16:106-11.   Back to cited text no. 29

Copyright 2008 - Indian Journal of Pharmacology

The following images related to this document are available:

Photo images