Biological activity of Bauhinia racemosa against Diabetes and Interlinked Disorders like Obesity and Hyperlipidemia
© The Author(s). 2017
Received: 10 August 2016
Accepted: 10 February 2017
Published: 8 March 2017
Bauhinia racemosa Lam. (BR) has been used widely for the treatment of diabetes since ancient times. Along with its medicinal importance; the tree having great traditional value in India. The protocol was performed using Wistar rats to determine the effect of petroleum ether extract of BR leaves on blood glucose level, adipose tissue and lipid profile. The study was further evaluted to determine insulin level, glucose tolerance, food and water intake of the animals.
Wistar rats of both sexes were selected for the study. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) 45 mg/kg. Animals were divided into five groups namely control group, diabetic group, reference group (glibenclamide) and two test groups BR-1 (250 mg/kg extract) and BR-2 (500 mg/kg) each group containing 6 animals. Daily dosing was performed for 28 days and feed and water consumption was observed regularly at the end of the study.
The main finding of the study for the test extract was that the petroleum ether extract of BR leaves prevented a rise in blood glucose level in STZ induced diabetic animal. Furthermore, extract showed significant antiadipogenic and antihyperlipidemic effect. It improved lipid profile by decreasing the levels of serum triglycerides, total cholesterol, low-density lipoprotein (LDL) and increasing high-density lipoprotein (HDL) cholesterol. Extract exhibited the concentration-dependent inhibitory effect with an IC50 value of 660.26 μg/ml.
It can be concluded from the study that BR extract posses signigicant antidiabetic activity by reducing blood glucose level of diabetic rats. It also normalized adipose tissues and lipid level. Due to its potential, it may be an effective drug for treatment of diabetes and related complications like obesity and dyslipidemia.
KeywordsAdipocyte differentiation assay Oral glucose tolerance test Diabetes Hyperlipidemia Obesity
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 . 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 . Increase incidence of visceral obesity is closely associated with the risk of diabetes mainly Type-2 diabetes . There is a pronounced correlation between an increased quantity of visceral fat, metabolic disorder and heart related diseases .
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 . 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  and . 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 .
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 . Stem bark revealed the presence of alkaloids, glycosides, carbohydrates, saponins, flavonoids, triterpenoids (ß-amyrin), anthroqunonine, steroid (ß-sitosterol) stilbene (resveratrol)  and . It has been reported that different plant isolates possess insulin releasing, pancreatic beta cells re-generating and fighting the insulin resistance problems.
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 .
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 .
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.
3T3-L1 cell line was used for evaluating in vitro antiadipogenic activity which was procured from National Centre for Cell Science (NCCS), Pune, India.
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-vitro cytotoxicity assay
Here, OD (Optical Density/Absorbance), OD sample = absorbance of cell at different dose, OD negative control = absorbance of 100% viable cell
Dose response curve for the toxicity studies
Adipocyte Differentiation assay
In-vivo experimental methods
Acute toxicity study
The acute toxicity study was carried out on male Wistar rats weighing 150–200 g by administering extract orally at one of the levels (150, 500, 1000, 2000 mg/kg) once only. The dose that shows toxicity signs/morality is the toxic dose and 1/10th of this toxic dose is considered for therapeutic exploration . Toxicity study of extracts was performed as per OECD guideline No.423.
Eighteen hours fasted Rat model
Male Wistar rats, six animals in each group were fasted overnight. Blood glucose determination was done initially at 0 h prior to drug treatment and finally at 3 h after drug treatment has over. Animals were divided into four groups, namely normal control 1% v/v Tween 80 (10 ml/kg), reference group (glibenclamide; 5.0 mg/kg), test group; BR-1 (250 mg/kg extract) and BR-2 (500 mg/kg extract).
Oral Glucose Tolerance Test (OGTT)
Oral glucose tolerance test was performed on overnight fasted male Wistar rats. Animals were divided into different groups as normal control (untreated), glucose primed control, reference group (glucose primed + glibenclamide), test groups (glucose primed + test extract). Glucose was given in the dose of 1.5 g/kg of 10% solution to all groups except normal control. Estimation of blood was done at 0 min (prior to any treatment), 30 min and 90 min .
Induction of diabetes in rats
Diabetes was induced by single intraperitoneal injection of freshly prepared STZ (65 mg/kg body weight) in 0.1 M citrate buffer (pH 4.5) in a volume of 1 ml/kg body weight. Nicotinamide was given in the dose of 110 mg/kg prior to induction of diabetes to reduce the beta cytotoxic effect of streptozotocin. Diabetes was established in these STZ treated rats over a period of 4 days. The control animals were treated with citrate buffer (pH 4.5). After 4 days blood was collected from the retro-orbital route and the plasma glucose level of each rat was determined. Animals were evaluate by using glucometer and considered diabetic when fasting blood glucose (FSG) level higher than 250 mg/dL .
Treatment Protocol for STZ induced diabetic rats model
The diabetic rats were divided randomly into five different groups, each containing six animals. Normal and Diabetic control animal groups received 1% v/v Tween 80 (1 ml/kg p.o.), Reference group received glibenclamide 5.0 mg/kg p.o. and extract of BR was given in the doses of 250 and 500 mg/kg in 1% v/v of Tween 80 (1 ml/kg p.o.) for a period of 28 days to different treatment groups. The blood sample was collected on 0, 7, 14, 21 and 28 days.
The principle of the Rat Insulin ELISA is based on a solid phase two-site enzyme immune assay. It is based on the direct Sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. During incubation insulin in the sample reacted with peroxidase-conjugated anti-insulin antibodies and bound to microtitration well. A simple washing step removes the unbound enzyme labeled antibody. The bound conjugate was detected by reaction with 3, 3β, 5, 5β-tetramethylbenzidine. The reaction was stopped by adding acid to give a colorimetric endpoint that was read spectrophotometrically .
Different lipid parameters in blood like Triglycerides (TGs), Total Cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL) were estimated using suitable assay procedures. Triglycerides are the chemical form in which most fat exists in food as well as in the body. Triglycerides (TGs) were determined by enzymatic colorimetric method (Adipogenesis assay kit). Triglyceride concentration in the sample was measured at 546 nm. The determination of serum cholesterol was considered to be significant in Diabetes Mellitus and various other diseases. Total cholesterol (TC) was estimated by the enzymatic method as described by . HDL and LDL were estimated using commercial kits procured from Bayer Diagnostics & Siemens Healthcare, India with the aid of a clinical chemistry analyser Chem-7 (Erba, Manheim, Germany).
All values were expressed as Mean ± SEM. The results were analyzed statistically using two way analysis of variance (ANOVA) followed by Bonferroni post-tests to calculate the level of significance. Values are expressed as mean ± SEM (Number of animals, n = 6); significantly different at *p < 0.05, **p < 0.01, ***p < 0.001, when compared with diabetic control group.
The preliminary phytochemical screening of BR petroleum ether extract revealed the presence of saponins, sterols, carbohydrates, flavonoids, triterpenes and proteins.
In vitro toxicity of BR extract on 3T3-L1 cell lines
In vitro toxicity of BR extract on 3T3-L1 cell lines
93.33 ± 0.84
88.00 ± 0.73
83.33 ± 0.71
79.00 ± 0.25
91.33 ± 1.11
85.00 ± 1.06
80.16 ± 0.60
76.66 ± 0.66
Antiadipogenic activity of BR extract on 3T3-L1 cell line
Antiadipogenic activity of BR extract on 3T3-L1 cell Lines
20.66 ± 1.28
31.16 ± 1.47
39.33 ± 0.88
23.16 ± 0.65
33.83 ± 1.32
41.66 ± 1.05
32.50 ± 1.36
41.33 ± 0.49
51.83 ± 1.85
In-vivo experimental methods
Acute toxicity study
An acute toxicity study revealed that BR extract did not produce any toxic sign and symptoms even no any mortality when administered per orally to rats at a dose up to 2000 mg/kg. According to the dose safety level upto 2000 mg/kg two doses of BR extract, i.e. 250 and 500 mg/kg were selected for in-vivo antidiabetic study.
Effect of BR extract on eighteen hours fasted Rat model
Effect of BR extract on Oral Glucose Tolerance Test (OGTT)
Effect of BR extract on blood glucose level of glucose primed rats
Blood Glucose Level (mg/dl)
77.71 ± 0.87
78.02 ± 0.79
78.68 ± 1.13
81.32 ± 1.08
172.91 ± 1.85***
178.65 ± 1.13***
RG + glucose
79.34 ± 0.80
152.87 ± 2.12***
90.44 ± 2.42***
82.30 ± 1.15
157.78 ± 2.45***
123.57 ± 3.49***
81.97 ± 1.04
152.92 ± 1.02***
115.65 ± 1.45***
STZ Induced diabetes model
Effect of BR extract on fasting blood glucose level
Effect of BR extract on STZ induced diabetes in rats
Blood Glucose Level (mg/dL)
79.12 ± 0.78
89.83 ± 1.10
86.36 ± 1.03
87.60 ± 1.43
87.46 ± 2.37
80.20 ± 0.56
370.04 ± 6.28
338.30 ± 13.18
297.97 ± 2.66
297.76 ± 2.97
81.35 ± 0.49
353.33 ± 3.49**
261.48 ± 2.69***
182.82 ± 3.18***
122.70 ± 3.26***
81.37 ± 0.47
361.00 ± 2.80
281.10 ± 2.55***
204.49 ± 3.79***
143.88 ± 2.06***
79.72 ± 0.74
355.41 ± 3.17*
264.23 ± 2.56***
191.24 ± 2.69***
134.70 ± 1.58***
Effect of BR extract on insulin level in diabetic rats
Effect of BR extract on lipid profile in diabetic rats
Effect of BR extract on Lipid profile of STZ induced diabetes in rats
Lipid profile (mg/dL)
88.90 ± 0.92
97.08 ± 1.003
57.37 ± 0.57
47.52 ± 0.57
106.47 ± 2.12
148.91 ± 0.80
33.39 ± 1.27
105.16 ± 3.61
85.88 ± 0.34***
106.00 ± 0.40***
57.30 ± 0.65***
53.75 ± 0.78***
89.84 ± 0.78***
126.29 ± 1.37***
45.14 ± 1.32***
60.14 ± 1.31***
87.34 ± 0.77***
117.38 ± 0.75***
52.74 ± 0.44***
57.66 ± 1.21***
Effect of BR extract on body weight of diabetic rats
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. , 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 .
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 , Eucalyptus globulus [25, 28] and Sambucus nigra  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 .
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 . Improvement in lipid profile reduces the possibilities of diabetes induced obesity and cardiovascular diseases . 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 . 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.
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.
Dulbecco’s modified Eagle’s medium
Dexamethasone, Dimethyl sulfoxide
Ethylenediamine tetraacetic acid
Fetal bovine serum
High density lipoprotein
Insulin dependent diabetes mellitus
Low density lipoprotein
Non-insulin dependent diabetes mellitus
Roswell Park Memorial Institute medium
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.
VK carried out the pharmacology evaluation, participated in the dosing, animal care, feeding etc. KR carried out the in-vitro antiadipogenic studies. PJ participated in the design of the study, performed the statistical analysis and drafted manuscript. ZA conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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