Design and Development of Anti-obesity Herbal Formulation and Its Pre-clinical Evaluations

Abstract

Obesity (also known as Sthoulya in Ayurveda) is one of the greatest health threats of modern times. It is a state of excess adipose (lipid storing) tissue mass and having a body mass index (BMI) > 25 kg/m2. It is a non-communicable lifestyle disorder having many implications (Hu et al., 2003). Etiology of obesity includes lack of physical activity with increased intake of food, industrialization, stress, dietary habits especially fast food, frozen fruits, soft drinks and canned foods. WHO has projected that by 2015, approximately 2.3 billion adults will be overweight and more than 700 million will be obese. Various approaches like diet therapy, physical activity and behavior therapy have no extraordinary result and all synthetic drugs approved till date are withdrawn except orlistat. Moreover bariatric surgery has many preoperative, operative and post-operative complications. Hence, because of dissatisfaction with high costs and potentially hazardous side effects, the plant derived natural products for treating obesity is under exploration, and this may be an excellent alternative strategy for developing future effective, safe anti-obesity drugs (Mayer et al., 2009; Nakayama et al., 2007; Park et al., 2005). A variety of natural products, including crude extracts and isolated compounds from plants, can induce body weight reduction and prevent diet-induced obesity. (Han et al., 2005; Moro and Basile, 2000; Rayalam et al., 2008). About 70% of the Indian population uses plants or plant products for their healthcare (Vaidya and Devasagayam, 2007). It was estimated that approximately 5– 15% of the total 250,000 species of higher plants have been systematically investigated, and yet there is potential of many plantsto be a good source of novel bioactive compounds (Cragg and Newman, 2005). Pterocarpus marsupium has been recommended as early as 1000 BC by Sushruta for different pharmacological activity. It is exploited for its timber and its medicinal bark (Sambath et. al., 2006). The bark exudes a red gummy substance called ‘Gum Kino’ when injured. It is found to contain many compounds like epicatechin, pterosupin, marsupinol, stilbene , catechin (koul et al., 2006). It also contains various phenolics which is responsible for antihyperglycemic activity while flavonoids like liguritigenin and pterostilbene have hypolipidemic activity (Prathap B. et al., 2012). It also possesses anti-inflammatory, cardiotonic activity, anti-bacterial, anti-cataract and hepatoprotective activity. Achyranthes aspera also known as Aghedo (Gujarati) is an important medicinal herb found as weed throughout India. Though all parts of the plant are used in traditional system of medicine, seeds, roots and shoots are the most important medicinally. (Srivastava S et al., 2011). It is found to contain betaine, oleanolic acid and other long chain fatty acids, β asarone, ecdysterone and various saponin C and D (Misra et al., 1996, Banerji et al., 1970). It posses various pharmacological activities like hypoglycemic, anti-oxidant, antiparasitic, wound healing, anti-depressant, immunomodulatory and abortifacient (Shibeshi et al., 2006, Akhtar et al., 1991, Zahir et al., 2009, Chakraborty et al., 2002). The previous study done in our laboratory and reports available with us has suggested that both Achyranthes aspera and Pterocarpus marsupium possess ability to reduce weight. In the previous studies done in the laboratory, methanolic and aqueous extracts of Pterocarpus marsupium were used to assess their anti-obesity potential in which the aqueous extract was found to have better potential than methanolic. On the other hand previous finding and research suggest that methanolic extract of Achyranthes aspera has potential as an antiappetite agent rather than the aqueous extract. Hence aqueous extract of Pterocarpus marsupium and methanolic extract of Achyranthes aspera were taken and a step further was taken by making a formulation and evaluating its anti-obesity potential. The extract of Ptercarpus marsupium was given the code AEPM while that of Achyranthes aspera was given the code MEAA. To get better yield, three different extraction techniques were tried and compared for % yield w/w. Normally extraction is done by hot or cold extraction, decoction, maceration or by using soxhlet apparatus. Conventionally Pterocarpus marsupium is extracted by hot extraction technique while Achyranthes aspera by soxhlet method. Both of them involve heating which might not be suitable for thermolabile compounds. Hence two different techniques were tried: sonication probe (involves vibrational movement) and microwave assisted extraction (uses microwaves) to get better yield. Comparison was done between all the three methods of extraction i.e. conventional (1.73%w/w AEPM, 0.29%w/w MEAA), sonication probe (9.03 %w/w AEPM, 0.29%w/w MEAA) and microwave assisted extraction (6.25%w/w AEPM, 0.88%w/w MEAA) for both the plants on basis of their % yield and time to get exhaustive/maximum yield. Maximum % yield was obtained with sonication probe method. The optimum extraction efficiency was found for each plant and it’s reproducibility was also checked. Attempt was made to formulate herbal granules which can be dissolved in water and consumed as a drink. Wet granulation technique was used for making the formulation. The excipients were selected based on their cost, ease of use and availability. The concentration of sweeteners; aspartame and mannitol was optimized to 6:20% while that of flavors; peppermint oil and chocolate to (0.1:0.2ml) in 5 gm batch. Optimisation of other parameters like sieve size, drying temperature and time required for drying was also done. PEG 4000 was used as solubility enhancer. Various evaluations mentioned for granules were done and also evaluation of taste masking was done with the help of human volunteers (discreet). The formulation was given the code: PMAA13001 The in vivo study was done on Sprague Dawley rats to evaluate the anti-obesity activity of PMAA13001, its acute toxicity study according to OECD guidelines 420 and anti-appetite activity of MEAA at the dose of 10 mg/kg. For anti-obesity activity, we gave HFD (High Fat Diet) for 30 days alongwith the treatment. The formulation (50mg/kg, p.o., OD) was compared with the standard marketed herbal formulation Ayurslim (144 mg/kg, p.o. OD). Various parameters like food intake and body weight were monitored daily for 30 days. BMI was also calculated from the body weight at the start and end of treatment. While parameters like TG, cholesterol, HDL, LDL and total protein were estimated from the serum on the 0 day and 30th day of treatment. There was significant reduction in food intake, body weight, BMI (Body Mass Index), LDL (Low Density Lipoprotein) (p<0.0001), TG (Triglycerides), and cholesterol (p<0.05) while increase (p<0.05) in HDL (High Density Lipoprotein) in treated animals. Hence it was concluded that PMAA13001 is as effective as standard herbal formulation. For acute toxicity studies, sighting and main study were performed for 14 days at the dose of 2000 mg/kg. Observations were made for any sign of toxicity and at the end of the study, all surviving animals were sacrificed and histopathology was done for five major organs: heart, brain, liver, kidney and spleen. Anti-appetite activity was also done for MEAA (10 mg/kg, p.o., OD) for 30 days with normal pellet diet and water ad libitum. Parameters like the body weight and food intake were estimated daily while TG, Cholesterol, HDL, LDL and total protein were estimated at 0 day and 30th day of study. There was significant reduction (p<0.0001) in food intake and body weight while less significant reduction (p<0.05) for TG (Triglycerides), LDL (Low Density Lipoprotein) and cholesterol was observed with increase in HDL (High Density Lipoprotein) (p<0.05) in treated rats. In vitro (Roh C. et al.; 2012) pancreatic lipase inhibition study was done for PMAA13001, AEPM and MEAA at an increasing concentration of 10,100 and 1000 ppm. It was found as the concentration increases % inhibition increases. Moreover the % inhibition of MEAA was found to be same as PMAA13001. From the entire study it can be concluded that: 1) Sonication probe gives the highest % yield compared to the other two methods for both Pterocarpus marsupium and Achyranthes aspera. 2) The granules have good flow property along with good taste, proven by the data of human volunteers. 3) The formulation has anti-obesity potential with no toxicity at dose of 2000 mg/kg. Hence it was classified as GHS (Globally Harmonised System) category V. 4) The MEAA has anti-appetite potential even at the potent dose of 10 mg/kg. 5) From in vitro activity, possible mechanism of action for both the extracts and formulation was found to be pancreatic lipase inhibition but the % inhibition of MEAA alone was same as the formulation PMAA1300.