Background
Diabetes mellitus is a metabolic disorder characterized by abnormal carbohydrate, protein and fat metabolism due to deficient insulin action on target tissues which results in hyperglycemia [
1]. Diabetes mainly is of two types Type-1 and Type-2 diabetes i.e. insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM) respectively [
2,
3]. According to International Federation of Diabetes report it is estimated that 387 million people have been suffering from diabetes till 2014, in which 90% cases of type-2 diabetes. The numbers of diabetic people are expected to rise to 592 million by 2035 [
4]. Increase incidence of visceral obesity is closely associated with the risk of diabetes mainly Type-2 diabetes [
5]. There is a pronounced correlation between an increased quantity of visceral fat, metabolic disorder and heart related diseases [
6].
Bauhinia racemosa Lam. (BR) belongs to the family
Caesalpiniaceae, popularly known as ‘Sonpatti’ in India. Its leaves are simple, bilobed, and alternate; stipules small, caducous, petiole 10–33 mm long, slender, pubescent, swollen at the base and at the tip. Poor and harsh climate is favorable for its growth of rare medicinal tree. The tree is widely distributed throughout India, Srilanka, Ceylon, China and Timor [
7]. The deciduous tree is propagated easily from seed. Almost each & every part of this tree has some medicinal values. The tree has small, creamy white or yellow colored flowers in axillary or terminal racemes. The flowers of BR are laxative & seeds are anti-bacterial [
8] and [
9]. BR commonly used in the treatment of diabetes, however, its other species like
Bauhinia vahlii, Bauhinia variegata, Bauhinia purpurea, Bauhinia tomentosa has been used as antidiarrhoeal, antispasmodic, anti-inflammatory, carminative, vermicidal and hepatoprotective activity [
10,
11]. The tree yields a useful gum & fibers, bark is used for dyeing. Juice of BR which is extracted from the tender shoots is mixed with mother’s milk and used to clean and cool eyes. The bark fiber is used to make ropes to make ladders and to tie cows [
12].
The phytochemistry of
Bahuinia revealed the presence of a new tetracyclic lupeol, betulin, β-sitosterol, and tetracyclic 2, 2-dimethyl chroman isolated from the roots [
13]. Stem bark revealed the presence of alkaloids, glycosides, carbohydrates, saponins, flavonoids, triterpenoids (ß-amyrin), anthroqunonine, steroid (ß-sitosterol) stilbene (resveratrol) [
14] and [
15]. It has been reported that different plant isolates possess insulin releasing, pancreatic beta cells re-generating and fighting the insulin resistance problems.
Methods
Collection, authentication and extraction
Leaves of BR were collected from forest of Ambikapur (23.1200° N, 83.2000° E) district of Chhattisgarh in the month of December 2014 authenticated by Dr. N. K. Satti, Department of Natural Products, Institute of Integrative Medicine, Jammu, and leaves were deposited in the herbarium of the Institute. 1 kg leaves of BR were extracted with petroleum ether by cold percolation at b.p. 40–60 °C and percentage yield was found to be 6.42%. The extract was dried with the help of evaporator (Buchi, USA) under reduced pressure, temperature (37 – 40 °C). Dried extract was placed in an airtight container and further used for studies as antidiabetic, antiadipogenic, and hypolipidemic studies [
16].
Phytochemical screening
The petroleum ether extract of BR leaves was subjected to different chemical tests separately for identification of various active constituents employing standard protocols recommended by WHO [
17].
Experimental animals
Wistar rats were selected for the study weighing 170–190 g. Experimental protocol was prepared to take the minimum number of animals for in-vivo and acute toxicity study and selected animals were approved by the Institutional Animal Ethical Committee (IAEC reg no- 68/99/CPCSEA/reg) of Indian Institute of Integrative Medicine (IIIM), CSIR, Jammu. The animals kept in the arrangement of 12 h light and 12 h dark conditions with temperature maintained at 22 ± 2 °C and humidity 47 ± 65%. Pelleted diet was used for animals feeding throughout the study and water ad libitum. Animals were divided into five different groups as normal control (NC), diabetic control (DC), reference group (RG), test group-1 (BR-1) (250 mg/kg), test group-2 (BR-2) (500 mg/kg) each group containing six animals.
Cell line
3T3-L1 cell line was used for evaluating in vitro antiadipogenic activity which was procured from National Centre for Cell Science (NCCS), Pune, India.
Chemicals used
Dexamethasone, Dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), Gentamycin, Insulin, Penicillin, Phosphate Buffer Saline (PBS), Roswell Park Memorial Institute medium (RPMI), Streptomycin, Trypsin, Sodium Pyruvate were purchased from Sigma Chem. Co., USA. Other chemicals are Cholesterol (Qualigens fine chem., glaxo India Ltd.), Ethylenediamine tetraacetic acid, disodium salt (EDTA) (HiMedia Laboratories Pvt. Ltd., Mumbai), Formaldehyde (Qualigens fine chem., glaxo India Ltd.), 3-isobutyl-1-methylxanthine (IBMX), Isopropyl alcohol (Sisco Research Laboratories. Pvt. Ltd., Mumbai), Sodium bicarbonate (NaHCO3) (HiMedia Laboratories Pvt. Ltd., Mumbai), Oil Red O Dye, Blood glucose, Triglyceride, Total Cholesterol, FFA estimation kits (Siemens Medical Solutions Diagnostics Ltd., Baroda, Gujarat, India), Rat Insulin ELISA kit (Mercodia, Sweden), Simavastatin, Glibenclamide (Nicholas Piramal Research Limited Mumbai).
Pharmacological screening methods
In-vivo experimental methods
Discussion
This study suggested that BR possess definite antidiabetic, hypolipidemic and antiadipogenic activity in STZ-induced diabetic rats. BR extract showed a dose dependent fall in FSG with 250 and 500 mg/kg dose in experimental diabetic animals. Moreover, the daily administration of the BR extract to STZ-diabetic rats for four weeks caused a significant reduction in food and water intakes, and an increase in the body weight. STZ is a cytotoxic compound which induces diabetes by damaging β pancreatic cells that causes a reduction in insulin release. [
25], reported that extracts of medicinal plants causes activation of β cells as an antidiabetic effect and hence showing insulinogenic action. STZ causes persistent hyperglycemia through destruction of pancreatic β-cells (type-I diabetes mellitus). The possible mechanism through which BR extracts showed an antidiabetic effect might have been due to increased utilization of glucose by peripheral tissues, improved sensitivity of target tissues for insulin or it may be due to β-cell stimulation. Extract of BR exhibited hypocholesterolemic and hypotriglyceridemic effects, while increasing the levels of HDL in diabetic rats. These findings have been well justified by various reports in the literature stating that some medicinal plants show antidiabetic as well as hypolipidemic effects [
26].
The elevation of serum insulin in BR extract treated STZ-induced diabetic rats could either be due to the insulinotropic substances present in the extract, which induce the intact functional β-cells to produce insulin, or the protection of the functional β-cells from further deterioration so that they remain active and produce insulin. Similarly the extracts of
Medicago sativa [
27],
Eucalyptus globulus [
25,
28] and
Sambucus nigra [
29] have been reported to possess antidiabetic property by insulin-releasing action. Since insulin inhibits the activity of Glc-6-Pase in the liver of diabetic rats and controls hepatic glucose production (HGP), the insulinotropic effect of BR might play an immense role in the control of diabetes in STZ-induced diabetic rats. The suppression of Glc-6-P hydrolysis could also be one of the reasons for the hypoglycemic effect of the BR extract in diabetic rats. The improvement in lipid profile by these extracts also supports their antidiabetic activity along with antihyperlipidemic activity [
30].
BR extract showed more than 75% of viability of cells during in-vitro study. The extract of BR revealed significant inhibition in differentiated cell as compared to non-differentiated cell in 3T3-L1 cell line [
31]. Improvement in lipid profile reduces the possibilities of diabetes induced obesity and cardiovascular diseases [
32]. BR extract normalized the amount of triglycerides (TG) and total cholesterol (TC) in STZ-induced diabetic rats and may be effective for the treatment of cardiovascular disease like coronary artery disease, atherosclerosis etc. Triglycerides (TG) are prepared from carbohydrates as a major energy source or derived from fats, by taking food [
33]. Calories present in food is not absorbed immediately by tissues; it is converted into TG and transported to fat cells for storage. Release of triglycerides from fat tissues is regulated by hormones to meet the body’s needs for energy between meals. Excess triglycerides in plasma are called hypertriglyceridemia. Elevation in triglyceride level may be a consequence of another disease like untreated diabetes mellitus. STZ-induced diabetes and adipocyte differentiation assay have been used as a model to evaluate the effect of test products on lipid/fat metabolism [
34,
35]. To develop the antidiabetic drugs and antiadipogenic drugs the lipid centric approach has been widely used.
Conclusion
It is revealed from the present study that BR extract possess antidiabetic activity due to decreasing blood glucose level of STZ-induced diabetic rats. Furthemore, BR extract also exhibits lowering effect on lipid level and adipose tissue. Due to this property the extract showed significant antiadipogenic and hypolipidemic effect. Pharmacologically, the extract improved the lipid profile by decreasing the levels of serum triglycerides, total cholesterol, LDL and increasing HDL cholesterol. Conclusively, these effects of BR may be useful for the development of potent herbal medicine for diabetes. The relevance of anti-obesity mechanism of BR extractmay be more appropriate. Further studies to isolate, identify and characterize the active principle(s) are in progress.
Acknowledgement
Authors are thankful to Dr. Zabeer Ahmed, Principal Scientist, Department of Pharmacology, Indian Institute of Integrative Medicine (IIIM), Jammu for their guidance and providing space in laboratory to carry out work and also showing gratitude towards Indian Institute of Integrative Medicine (IIIM), Jammu for their assistance to provide all the facilities required during the study.
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