A Study on Ethanolic Extract of Dalbergia sissoo roxb. Leaves for Pancreatic Lipase Inhibition.
Josephin Nerling Rashida G.*, T. Venkatarathnakumar, R. Gowri, Ajithadhas Aruna
Department of Pharmacognosy, Madurai Medical College, Madurai-625020, Tamilnadu, India.
*Corresponding Author E-mail: rashipharm@gmail.com.
ABSTRACT:
A high fat diet increased the body weight and produce adiposity in humans. Pancreatic lipase plays a major role in digestion and absorption of dietary fat because the dietary fat is not directly absorbed from intestine. So it is possible to reduce the absorption of dietary fat by the inhibition of pancreatic lipase, thereby obesity can be managed. Orlistat is one of the clinically approved drugs for treatment of obesity in the mechanism of pancreatic lipase inhibition. The aqueous ethanolic extract (70%) of Dalbergia sissoo Roxb. leaves (DSEE) and orlistat as reference has been studied for potent pancreatic lipase inhibition using chicken pancreas (Gallus domesticus) in dose dependent manner. The preliminary phytochemical analysis of extract showed the presence of flavonoids, glycosides, tannins, and terpenoids. The pancreatic lipase inhibition (IC50) value for the orlistat and extract was found to be 2.049±0.98 and 3.89±0.97 which might be attributed to the presence of phytoconstituents. Hence DSEE can be used as an anti-obesity agent in suitable form.
KEYWORDS: Pancreatic lipase, Dalbergia sissoo, Gallus domesticus, orlistat
INTRODUCTION:
Introduction
The cause of obesity is characterized by a chronic imbalance between energy intake and energy expenditure and it is also due to changing lifestyles and inadequate dietary habits1-3. Excess energy is stored in fat cells, to reduce body weight and adipose tissues, an alteration in lifestyle and habits are still the crucial cornerstone4. Physical activity might be helpful in the prevention of obesity by elevating average daily metabolic rate and increased energy expenditure1. Moreover, obesity is a strong risk factor for various diseases, such as hyperlipidemia, atherosclerosis, hypertension, diabetes, chronic kidney diseases, cardiovascular diseases, and cancer7, 8. The therapeutic approach for the treatment and prevention of obesity is to decrease the absorption of fatty acids by the inhibition of lipase in the digestive organs5. Pancreatic lipase is the enzyme which catalyses the hydrolysis of triacylglycerols in the GIT, it is generally thought that a potent and specific inhibitor of pancreatic lipase could be useful in the treatment of obesity6. Orlistat is one of the potent pancreatic lipase inhibitor and best-selling drug, but it has some side effects such as oily stools and fecal incontinence.17, 18
So, the development of natural products for the treatment of obesity is a challenging task, which can be launched faster and cheaper than conventional single-entity pharmaceuticals9. Many medicinal plants may provide safe, natural, and cost-effective alternatives to synthetic drugs10-12. Currently, one of the most important strategies in the treatment of obesity includes development of inhibitors of lipid digestion and absorption.
Dalbergia sissoo Roxb, (Fabaceae) used for stimulant, appetiser, anthelmintic, used in dyspepsia, diarrhoea, obesity, cutaneous affections, leprosy, spasmolytic, CNS active and hypothermic. The leaves are alternately arranged, compound and oddly pinnate with 3-5 glabrous, leathery leaflets, elliptical to ovate, tapering to a point and native to Pakistan, Oman, Bhutan, India, Nepal has a long history of human consumption13. In the development of a search for natural pancreatic lipase inhibitors from herbal medicine, the leaves of Dalbergia sissoo were chosen for more detailed investigation. Our finding is to indicate that an ethanolic extract of Dalbergia sissoo (DSEE) showed a significant in vitro inhibitory effect on pancreatic lipase.
MATERIALS AND METHODS:
Materials
The leaves of Dalbergia sissoo were collected in an around Madurai and it was authenticated by Dr. Stephan, the Botanist, American college, Madurai, Tamil Nadu, India. Fresh chicken (Gallus domesticus) pancreas from slaughter houses, Sucrose, Sodium hydroxide and Ammonium sulphate were purchased from Himedia Labs. Standard drug orlistat was obtained from Lupin pharma. Phosphate buffer (pH 7) 0.02M was used. All chemicals were of analytical reagent grade and were used without further purification.
Extraction and Phytochemical Analysis
The Dalbergia sissoo (DS) leaves were washed thoroughly with running tape water which was shade dried and powdered using mechanical blender. The powdered plant material was sieved (Sieve No 10/40) and defatted using petroleum ether and subjected to triple maceration in 70% of ethanol. The extract was filtered, concentrated using rotary flash evaporator and dried in the desiccators. Then DSEE was subjected to preliminary phytochemical screening to detect secondary metabolites.
Extraction of Lipase from Chicken Pancreas
Pancreas of freshly slaughtered chicken was collected with the guidance of veterinary surgeon. It was washed and placed in ice cold sucrose solution (0.01M). It was homogenized in 0.01M sucrose and centrifuged. The supernatant was separated and treated with ammonium sulphate precipitation (50% saturation). The pellet obtained from precipitate after centrifugation was dissolved in sucrose solution and again saturated with 50% ammonium sulphate and centrifuged. Finally pellets were used as enzyme source by dissolving in Phosphate buffer (pH 7).
Determination of Chicken Pancreatic Lipase Activity
The chicken pancreatic lipase activity was determined by incubating an emulsion containing 8ml of Olive oil (Dietary fat), 0.4ml of phosphate buffer and 1ml of freshly prepared chicken pancreatic lipase for an hour, the reaction was stopped by addition of 1.5 ml of a mixture solution containing acetone and 95% ethanol (1:1). Appearance of pink color from yellow color shows the liberated fatty acids, which was determined by titrating the solution against 0.02M sodium hydroxide (NaOH) (standardized by potassium hydrogen phthalate) using phenolphthalein as an indicator.
Pancreatic Lipase Inhibitory Activity
Pancreatic lipase inhibitory activity was studied using Anil kumar et al., 14 methods with slight modification. Orlistat (standard drug) and DSEE (sample) were prepared in different concentrations such as 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL, and 3.0 mg/mL. A 100µl of each concentration of sample and standard, pancreatic lipase was mixed with 8ml of olive oil, 0.4ml of phosphate buffer and 1ml of freshly prepared chicken pancreatic lipase and it was incubated for 60 minutes. The reaction was stopped by adding 1.5 ml of a mixture solution containing acetone and 95% ethanol (1:1). The liberated fatty acid was determined by titrating the solution against 0.02M NaOH using phenolphthalein as indicator and the percentage inhibition of lipase activity was calculated using the formula:
Lipase inhibition = A - B / A X 100
Where A is lipase activity,
B is activity of lipase when incubated with the standard and test compounds.
RESULTS AND DISCUSSION:
The phytochemical screening of Dalbergia sissoo ethanolic extract (Table 1) showed the presence of flavonoid, glycosides, tannins, terpenes, phenols and saponins. Flavonoid and their antioxidative metabolites 27 have the potential to be obesity-preventive agents in the following Alpinia officinarum 28, 29 Taraxacum officinale 30, Actinidia arguta 31, Aesculus turbinate, Arachis hypogaea reduce the TG plasma level and body weight gain. 26 Camellia sinensis reduce body weight gain and visceral fat 32. Eleuthero coccussenticosus decrease abdominal fat, TG in liver and serum and LDL in serum. [33] Humuluslupulus decreases body weight gain and blood glucose levels34.
Table 1: Preliminary Phytoconstituents analysis of DSEE
Phytoconstituents |
Ethanolic Extract |
Flavonoid |
+ |
Glycoside |
+ |
Alkaloid |
- |
Terpenes |
+ |
PhytoSterols |
- |
Terpenes |
+ |
Tannins |
+ |
‘+’ indicates Presence; ‘-‘indicates Absence
One of the important strategies in the treatment of obesity includes the development of inhibitors of lipid digestion and absorption, in an attempt to reduce the energy intake through gastrointestinal mechanisms, without altering any central mechanisms 15, 16. Pancreatic lipase inhibition is one of the most widely studied mechanisms used to determine the potential efficacy of natural products as antiobesity agents.
Our finding is to indicate that Dalbergia sissoo inhibits pancreatic lipase. The inhibitory activities of different concentrations of standard and tests compounds were tested against chicken pancreatic lipase using olive oil as the substrate. The activity of lipase was affected when incubated with the standard and test compounds. From figure 1 the IC50 values of the orlistat, DSEE were calculated from the least regression fit to be 2.049±0.98, 3.89±0.97 respectively. The dose dependent pancreatic lipase inhibition was observed i.e., inhibition of enzyme was increased on increasing the concentration of extract.
A number of plants and natural products have been screened for their pancreatic lipase inhibitory activity but just some of them have gone up to clinical studies20, 21. Orlistat is currently in clinical use, although others are under investigation. Some of them are Panax ginseng22, Camellia sinensis 23, Eleuthero coccussenticosus24, Malusdo mestica 25 and Arachis hypogaea 26. Some plant extracts show 40 % invitro anti lipase activity such as Levisticum officinale 35, Quercus infectoria 11, Rosmarinus officinalis 35, Vaccinium myrtillus 35, and Glycyrrhiz auralensis 22. Dalbergia sissoo inhibited pancreatic lipase, it may affect fat absorption and the uptake of fatty acids in the periphery, if enough of the active components can be absorbed and enter the circulation.
Figure 1: Pancreatic lipase inhibition of ethanolic extract of Dalbergia
sissoo (DSEE)
The clinical effect of orlistat showed that, the reducing the absorption of fat may be an extra effect to dieting in obese patients. Orlistat is a prescription medication available as a pancreatic lipase inhibitor that is not absorbed and, hence, works only within the intestine. Orlistat reduces the absorption of dietary fat by 30% in adults. Long-term clinical trials have shown that orlistat results in about 5% greater weight loss 18. However, pancreatic lipase hydrolyzes the low-density lipoproteins and chylomicrons, and release free fatty acids for uptake into adipocytes 19, 20. Thus, the inhibition of pancreatic lipase may slow the deposition of fat into adipose tissue.
CONCLUSION:
Obesity is the major risk factors for the development of chronic diseases such as type II diabetes, CVS risk and cancer. Although it is one of the best-selling drugs worldwide, it has some side effects such as flatulence, oily stools and oily spotting etc. At present, the potential of natural products for the treatment of obesity is still largely unexplored and pancreatic lipase might be an excellent alternative strategy for the development of safe and effective antiobesity drugs. The phytoconstituents present in the extract inhibit the pancreatic lipase, thus it reduce the risks of obesity. Further studies on isolation of active constituents and their lipase inhibitory activity are under investigation. Naturally occurring DSEE present an exciting opportunity for the discovery of newer anti-obesity agents.
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Received on 13.02.2012 Modified on 05.04.2012
Accepted on 06.04.2012 © RJPT All right reserved
Research J. Pharm. and Tech. 5(4): April 2012; Page 497-500