INTRODUCTION:

Obesity is energy equilibrium disorder and basically measured as a disarray of lipid metabolism¹. It is considered by extreme fat deposition in adipose tissue and other inner organs like liver, heart, skeletal muscle and pancreatic islet. In the present circumstances obesity remnants as a main global public health problem because of its prevalence, cutting across all age groups, sex and race^2,3. Worldwide about 1.9million adults are overweight and 600million of them are clinically obese⁴. Only drug named Orlistat is currently approved by USFDA for long term obesity treatment. But it has unwanted side effects like, dyspepsia, nephrotoxicity, flatulence, respiratory infection oily stools, abdominal pain, menstrual and psychiatric disorders⁵.

Natural products recognized from conventional medicinal plants encompass constantly existing a thrilling chance for the expansion of newer remedial agents. A big amount of Indigenous plants have been claimed to have anti-obesity effect in the Indian system of medicine. Many medicinal plants like Camellia sinensis, Allium sativum, Nelumbo nucifera, Argyreia nervosa and Sida rhomboidea, were reported to have anti-obesity effect in different animal models⁶.

Pithecellobium dulce belongs to family Leguminosae and subfamily Mimosoideae is an evergreen tree widely distributed in the greater part of India and is also found in Southeast Asia. P. dulce is one of the familiar species, commonly referred as manila tamarind^7,8.

The active compound of the plant includes flavonoids, sterols, tannins, and triterpenoids and the health promoting properties like proteins, carbohydrates, steroids and diseases preventing properties such as antioxidant, antifungal, antiviral, antibacterial, antidiabetic, diastolic, diuretic, anthlmintic, antipyretic, anti-inflammatory, hypoglycemic and sedative activities^9-12.

But the effectiveness of the peel of Pithecellobium dulce for treatment of obesity has not been scientifically reported. For this reason, the seek of the current study was to assess the anti-obesity effectiveness of petroleum ether (PE), ethyl acetate (EA) and methanolic (MA) extract of peel of Pithecellobium dulce in high fat diet (HFD) induced obese rats.

MATERIAL AND METHODS:

Animals:

Wistar rats (150-200gm) of both sexes were selected after obtaining IAEC approval (CPCSEA/IAEC/JIPS/19/1/1). The selected Wistar rats were maintained under standard laboratory conditions temperature at 25±1⁰C, relative humidity 55±10% and with 12h light/dark cycle^13,14. The animals were fed with standard pellet diet and water ad libitum. Animals were acclimatized to the laboratory conditions for one week.

Collection and Preparation of Plant Extract:

Peel of Pithecellobium dulce were collected and air dried for a period of two weeks, crushed and later pulverized into fine powder using electric blender. The powder was sieved through a mesh (2mm) and used for preparation of PE, EA and MA extracts by Soxhlet method. The excess solvent in each extract was removed entirely under vacuum till a dark semisolid residue was obtained. The phytochemical studies (Table 1) conducted as per standard procedures which match with the past reports^15,16,17,18. The extracts were stored in desiccators and a weighed (1gm) quantity of extracts was suspended in distilled water using 2% carboxyl methyl cellulose as suspending agent prior to administration.

Dose Selection:

In the present study, two doses of the Pithecellobium dulce peel extract were selected as 100 and 200mg/kg p.o. These doses were selected on the basis of prior reports of the acute toxicity studies achieved by the single oral administration of PE, EA and MA extract of Pithecellobium dulce at the concentrations of 100, 500, 1000 and 2000mg/kg, which shows no signs of toxicity in tested rats¹⁹.

High-Fat Diet Formula:

The animal feed was prepared with following ingredients to prepare high fat diet (HFD): Casein (20%), corn starch (15%), sucrose (27.5%), cellulose powder (5%), L-methionine (0.3%), mineral mixture (3.5%), vitamin mixture (1%), choline bitartrate (0.2%), lard oil (17.6%) and corn oil (9.9%). The HFD was prepared every day freshly, dried, powdered and administered throughout the treatment period^20,21.

Experimental Design:

Rats were divided into nine groups of six in each group²²:

Group-I: Normal control rats fed with standard chow diet and received vehicle for 40 days.

Group-II: Obesity control rats fed with HFD and received vehicle for 40 days.

Group-III: Rats fed with HFD and Orlistat (50mg/kg) orally for 40 days.

Group-IV: Rats fed with HFD and treated with PEPD 100 mg/kg for 40 days.

Group-V: Rats fed with HFD and treated with PEPD 200 mg/kg for 40 days.

Group-VI: Rats fed with HFD and treated with EAPD 100 mg/kg for 40 days.

Group-VII: Rats fed with HFD and treated with EAPD 200 mg/kg for 40 days.

Group-VIII: Rats fed with HFD and treated with MAPD 100 mg/kg for 40 days.

Group-IX: Rats fed with HFD and treated with MAPD 200 mg/kg for 40 days.

Assessment of anti-obesity activity:

Body weight gain and food intake:

The body weight in grams was documented on day one and 40 using a digital weighing balance. In addition to this, intake of the daily food for each group was also measured²³.

Body Temperature:

The body temperature was noted using a rectal thermometer before and after drug administration at 120 minutes with a contact time of 1 minute²³.

Organ and fat pad weights:

The liver, kidney and fat pads (retroperitoneal, epididymal and mesenteric fat pads) were dissected out, washed in ice cold saline and weighed^22,24.

Statistical analysis:

The data was expressed as mean ±SD. Mean values between the groups was considered statistically significant p<0.05 after analyzing by one-way ANOVA and was compared using Tukey’s post Hoc test.

RESULTS:

Table 1: Phytochemical constituents

Phytochemical constituents	Petroleum ether extract	Ethyl acetate extract	Methanolic extract
Alkaloids	-	+	+
Glycosides	-	-	+
Phenols	+	+	+
Flavonoids	-	+	+
Steroids	+	-	-
Tannins	-	+	+
Terpenoids	+	+	+
Saponins	-	-	+

+ = present, - = absent

Effect on body weight gain and food intake:

Table 2 showed body weight and intake of food. The normal rats body weight increases gradually as the rats grow during the test. By comparison, during the trial the bodyweight of animals on the HFD increased rapidly^25,26. Weight gains were 33.23±1.42 and 109.02 ±3.50gm respectively in regular diet groups and for highly fat diet control groups. Bodyweight gain reduces dramatically when contrasted to HFD control group when PEPD, EAPD, and MAPD (P<0.001) are administered orally which is dose-dependently.

Effect on Rectal Body Temperature:

It was found that the rectal body temperature, estimated between 0 and 120 minutes on the 40th day, of rats with an HFD. Based on a diagnosis for EAPD and MAPD, the body temperature (Table 2) has improved (P<0.05), but PEPD has not modified the body's temperature.

Table 2: Effect of PEPD, EAPD and MAPD on body weight gain, food intake and temperature changes in obese rats

Groups	Body weight gain (gm)	Food intake (gm)	Temperature
Groups	Body weight gain (gm)	Food intake (gm)	0 min	120 min
Normal Control	33.23±1.42	14.24±0.15	38.29±1.12	38.46±1.29
HFD	109.02±3.50^¥¥¥	19.43±0.62^¥¥¥	37.92±2.30^ns	22.39±1.79^¥¥¥
Standard	50.48±2.46^***	16.60±0.09^***	36.20±1.39^ns	33.59±2.32^*
PEPD (100mg/kg)	72.28±1.90^***	19.32±0.28^ns	37.60±0.88^ns	37.14±1.34^ns
PEPD (200mg/kg	67.39±1.82^***	19.08±0.26^ns	37.01±1.50^ns	37.10±0.94^ns
EAPD (100mg/kg)	68.42±1.68^***	19.01±0.42^ns	36.80±0.90^ns	38.17±1.42^*
EAPD (200mg/kg)	62.50±1.89^***	18.76±0.38^ns	36.05±1.60^ns	38.10±0.98^*
MAPD (100mg/kg)	60.29±1.72^***	18.92±0.29^ns	37.90±0.92^ns	38.20±1.62^*
MAPD (200mg/kg)	55.62±1.99^***	17.58±0.31^***	37.10±1.49^ns	39.90±0.92^*

^¥¥¥𝑃 < 0.001 when compared with the normal control group, * 𝑃 < 0.05, **𝑃 < 0.01, *** 𝑃 < 0.001; when compared with the normal HFD group

Fig. 1: Body weight changes in rats

Fig. 2: Food intake in rats

Fig. 3: Body temperature changes in rats

Fig. 4: Liver weight changes in rats

Fig. 5: Kidneys weight changes in rats

Effect on Organ Weight:

When contrast with the normal group, the overweight community displayed considerably higher organ weight (liver) (P<0.001). When the PEPD, EAPD and MAPD extracts were administrated, the organ became significantly lower (liver). Nonetheless, with HFD on kidneys, no significant increase was found. (Table-3).

Effect on fat pad weights:

The weight of the fat pad (Epididymal, Mesenteric and Retroperitoneal) in HFD control rats was significantly higher than that of standard monitoring rats. The once-daily oral treatment for fat pad weights, obesity index and adiposity index in comparison to the HFD control group was considerably reduced for 40 days for standard group animals (Orlistat), PEPD, EAPD, and MAPD (P<0.001) Table 4.

Fig. 6: Effect of PEPD, EAPD and MAPD on pad weight in obese rats

Table 4: Effect of PEPD, EAPD and MAPD on pad weight in obese rats

Groups	Retroperitoneal Fat (mg)	Epididymal Fat (mg)	Mesenteric Fat (mg)
Normal Control	2.79±0.50	1.25±0.52	2.82±0.23
HFD	7.39±0.59^¥¥¥	4.25±0.63^¥¥¥	7.02±0.53^¥¥¥
Standard	2.74±0.68^***	1.20±0.60^***	2.65±0.49^***
PEPD (100mg/kg)	5.42±0.73^***	3.62±0.49^***	6.19±0.68^***
PEPD (200mg/kg	3.59±0.68^***	2.56±0.58^***	4.89±0.54^***
EAPD (100mg/kg)	5.36±0.92^***	3.50±0.46^***	6.02±0.78^***
EAPD (200mg/kg)	3.56±0.89^***	2.42±0.42^***	4.75±0.55^***
MAPD (100mg/kg)	5.98±0.96^***	3.36±0.40^***	5.60±0.65^***
MAPD (200mg/kg)	3.07±0.50^***	2.03±0.44^***	3.06±0.61^***

^¥¥¥𝑃 < 0.001 when compared with the normal control group, * 𝑃 < 0.05, **𝑃 < 0.01, *** 𝑃 < 0.001; when compared with the normal HFD group

DISCUSSION:

The lethal dose of P. dulce provided a healthy and non-toxic extract of up to 5 gm / kg. In earlier studies includes, anti-inflammatory, antibacterial, antioxidant, antidiabetic, antimicrobial, cardiac, antidiarrheal, antiulcer, and antifungal activities have been observed in all sections²⁷. A variety of herbal extracts were used for their anti-obesity practices in traditional medicine. There is still no proof of anti-obesity ability in various P. dulce extracts. The research was therefore designed to prove that P. dulce has an anti-obesity impact in extremely fatty obesity induced by diet in rats.

Obesity is the most common medical disorder in the developed world and is synonymous with dietary deficiency, which is a major risk factor in the development of major human disorders, including heart, diabetes and cancer. Daily fat-rich diets tend to encourage obesity²⁸. A high level of caloric (energy and fat) intake in humans and animals always encourages storage of the fat, increased weight of the body as well as adiposity^29,30. There is a significant rise in the prevalence of obesity and there is therefore an immense need for safe and efficient long-term drugs in this epidemic³¹. HFD, due to its high similarities in the usual path of obesity episodes in humans and therefore its role as a credible tool to study obesity, has been considered a most common template in scientists, provided that they will quickly gain weight while fed on HFD^32,33. Human studies have shown that elevated consumption of fat is associated with increased body weight, which can lead to obesity and other metabolism. This experiment therefore showed a major increase in animal body weight and thus confirmed obesity when rats were exposed to HFD for 2 weeks³⁴.

In previous studies, HFD mediated obesity was stated to be a good model for rats because they have a close resemblance to human obesity. Like previous studies, HFD was also fed to rats to cause obesity in the present study also. The results of this study revealed that the progression of obesity in Wistar rats was representative of dietary fat lipid, which is consistent with previous reports of HFD mediated high body fat deposition and increased body weight. The elevation in body weight could mainly result from an increase in the energy consumption of fat and the Lee-Index of HFD animals, which could be classified as obese, was higher than 300.³⁵ Although the body weight of the HFD was significantly different from normal diet groups, no significant difference was found in animal daily intakes except for MAPD with 200mg/kg. There was also no significant difference. This observation leads to the fact that the weight of the body increases regardless of how many animals are eaten.

As observed in previous reports of different plant extracts like Argyreia nervosa, Nelumbo nucifera, Sida rhomboidea, animals which received P. dulce extracts exhibited reduced body weight in dose dependent manner³⁶. Further, decrease in food intake was also observed in P. dulce treated animals which may be due to hypophagic effect³⁷.

Body temperature variations are related to significant metabolic rate changes³⁸. The hypothesis has been confirmed by various overweight animal models and a reported hyperphagia, a decreased metabolic rate and a decreased core body temperature was found in the leptin-deficient ob/ob mouse and the polyphonic obese mouse³⁹. This argument is supported by our results; HFD rats feed show lower body temperature than normal rats. P. dulce treatment showed sharply an increase in the temperature of the rectal body. Rectal body temperature increase can be caused by the overall stimulant and thermogenic properties of the extracts phytoconstituents.

The results of this study have also supported previous reports, showing significant changes in weights of organs and fat pads for HFD test animals⁴⁰. P. dulce dosage based on organ and fat pad weights decreased which could result in adiposis mobilization and lipid catabolism.

Plants such as P. dulce are not shocking to see that they include a high amount of quercetin, hormones, saponins, lipids, phospholipids, glycosides, polysaccharides, kaempferol, dulcitol, and afezilin^41,42. The P. dulce in this experiment has worked on fatty liver, and anti-obesity interventions have been shown.

CONCLUSION:

The present study states that, the methanolic extract of peel of Pithecellobium dulce (MAPD) exhibit a promising role in management of high fat induced obesity than other two extracts EAPD and PEPD. The present study provides scientific evidence and support for the use of peel of Pithecellobium dulce in traditional medicine to treat obesity.

REFERENCES:

1. Birari RB, Gupta S, Mohan CG and Bhutani KK. Anti-obesity and lipid lowering effects of Glycyrrhiza chalcones: Experimental and computational studies. Phytomedicine. 2011; 18: 795-801.

2. World Health Organization, 1998. Obesity: Preventing and managing the global epidemic. Report of a WHO Consultation on obesity, WHO/NUT/NCD/98, Geneva, Switzerland. 1-275.

3. M. Selvakumar, Vijayalakshmi Ch, Venkata Rathina Kumar Th. Anti-obesity activity of Ficus religiosa on high fat diet induced model. Research J. Pharm. And Tech. 2015; 8(6): 679-682. - 3

4. Centre WM (2015) Obesity and overweigh. World Health Organization.

5. Jong Won Yun. Possible anti-obesity therapeutics from nature – A review. Phytochemistry. 2010; 71 (14-15): 1625-1641.

6. Satyajit Patra, S Nithya, B Srinithya and Meenakshi SM. Review of medicinal plants for anti-obesity activity. Translational Biomedicine. 2015; 6(3:21): 1-22.

7. Duke JA, Wain KK. Medicinal plants of the world. Computer index with more than 85,000 entries. 1981; 3: Longman group UK Limited.

8. M Sugumaran, T Vetrichelvan, S Darlin Quine. Anti-inflammatory activity of Folklore: Pithecellobium dulce Benth. Research J. Pharm. and Tech. 2009; 2(4): 868-869.

9. Megala J and Geetha A. Free radical-scavenging and H⁺/K⁺ ATPase inhibition activities of Pithecellobium dulce. Food Chem. 2010; 121: 1120-1128.

10. Sukantha TA, Shubashini K. Sripathi, Ravindran NT and Balashanmugam P. Evaluation of in-vitro anti-oxidant and anti-bacterial activity of Pithecellobium dulce benth fruit peel. Inter. J. of current Research. 2011; 3(1): 378-382.

11. Prasenjit Manna, Sudip Bhattacharyya, Joydeep Das, Jyotirmoy Ghosh and Parames C Sil. Phytomecicinal role of Pithecellobium dulce against CCl₄-medicated hepatic oxidative impairment and necrotic cell death. Evidence Based Complementary and Alternative Medicine. 2011; Article ID 832805: 1-17.

12. Mule VS, Naikwade NS, Magdum CS and Jagtap VA. Effect of Pithecellobium dulce benth leaves in dexamethasone induced diabetic rats. Inter. J. of Pharm. Pharmc. Seci. 2016; 8(9): 317-320.

13. Shetty Sudeep Dinesh, Karunakar Hegde. Anti-obesity activity of ethanolic extract of Citrus maxima leaves on cafeteria diet induced and drug induced obese rats. Research J. Pharm. And Tech. 2016; 9(7): 907-912.

14. Shetty Sudeep Dinesh, Karunakar Hegde. Anti-obesity activity of ethanolic extract of Citrus maxima leaves on cafeteria diet induced and drug induced obese rats. Research J. Pharm. And Tech. 2016; 9(7): 907-912.

15. R Kalavani, R Sabitha Banu, KA Jeyanthi, T Umasankar and A Vinoth Kanna. Evaluation of anti-inflammatory and anti-bacterial activity of Pithecellobium dulce (benth) extract. Biotechnol Res. 2016; 2(4): 148-154.

16. Kasarla Raju and Jagadeeshwar K. Phytochemical investigation and hepatoprotective activity of ripe fruits of Pithecellobium dulce in albino rats. Sch. Acal. J. Pharm. 2014; 3(6): 449-454.

17. SP Anand and S Deboran. Preliminary phytochemical screening of wild edible fruits from Boda and Kolli hills. Journal of Medicinal Herbs and Ethnomedicine. 2017; 3: 8-12.

18. Sunil HG, Archana MR. TLC profiling of Pithecellobium dulce seed. Research J. Pharmacognosy and Phytochemistry. 2012; 4(4): 220-222.

19. Jayaram Megala and Arumugam Geetha. Acute and Sub-acute toxicity study of hydroalcoholic fruit extract of Pithecellobium dulce. Natural Product Research. 2012; 26(12): 1167-1171.

20. Vasselli JR, Weindruch R, Heymsfield SB, Pi-Sunyer FX, Boozer CN, Yi N, Wang C, pietrobelli A and Allison DB. Intentional weight loss reduces mortality rate in a rodent model of dietary obesity. Obes Res. 2005; 13(4): 693-702.

21. Rashmi Saxena Pal, Nikita Saraswat, Yogendra Pal, Pranay Wal, Ankita Wal, AK Rai. Alcoholic extract of poly herbal powder mixture for anti-obesity effect on wistar rats. Research J. Pharm. And Tech. 2019; 12(4): 1857-1864.

22. Sai Sruthi Kaveripakam, Adikay Sreedevi and Gandhi Mathi. Anti-obesity efficacy of roots of Stereospermum suaveolens in high fat-induced obese rats. Journal of Young Pharmacists. 2017; 9(2): 234-238.

23. Manjula B, Rayappa Hunasagi and Shivalinge Gowda KP. Anti-obesity activity of ethanalic extract of Moringa oleifera seeds in experimental animal. Research J. Pharmacology and Pharmacodynamics. 2011; 3(6): 3018-328.

24. Souravh Bais, Guru Sewak Singh and Ramica Sharma. Anti-obesity and Hyperlipidemic activity of Moringa oleifera leaves against high fat diet-induced obesity in rats. Advances in Biology. 2014; Article ID 162914: 1-9.

25. A Sowmya, T Ananthi. Hypolipidemic activity of Mimosa pudica Linn on butter induced hyperlipidemia in rats. Asian J. Res. Pharm. Sci. 2011; 1(4): 123-126.

26. Preeti Tiwari. Antihyperlipidemic potential of Balarishta prepared by traditional and modern methods in high fat diet induced hyperlipidemic rats. Asian J. Res. Pharm. Sci. 2014; 4(1): 07-11.

27. Srinivas G, Geeta HP, Shashikumar JN and Champawat. A review of Pithecellobium dulce: A potential medicinal tree. International Journal of Chemical Studies. 2018; 6(2): 540-544.

28. Yogesh Saxena, Vartika Saxena, Jyoti Dvivedi and RK Sharma. Evaluation of dynamic function tests in normal obese individuals. Indian J. Physiol. Pharmacol. 2008; 52(4): 375-382.

29. Bray GA, Loveloy JC, Smith SR, DeLany JP, Lefevre M, Hwang D, Ryan DH and York DA. The influence of different fats and fatty acids on obesity, insulin resistance and inflammation. J. Nutr. 2002; 132(9): 2488-2491.

30. Estadella D, Oyama LM, Damaso AR, Ribeiro EB and Oller do Nascimento CM. Effect of palatable hyperlipidic diet in lipid metabolism of sedentary and exercised rats. Nutrition. 2004; 20(2): 2018-224.

31. Yilmaz A, Suleyman H, Umudum Z and Sahim YN. The effect of adrenalectomy on leptin levels and some metabolic parameters in rats with diet-induced obesity. Biol Pharm Bull. 2002; 25(5): 580-583.

32. Buettner R, Scholmerich J and Bollheimer LC. High-fat diets: Modeling the metabolic disorders of human obesity in rodents. Obesity. 2007; 15: 798-808.

33. AM Gajda. High fat diets for diet-induced obesity models. Open diet purified formula for rats. Obesity. 2009; 1-9.

34. Neyrinck AM, Bindels LB, De Backer F, Pachikian BD, Cani PD and Delzenne NM. Dietary supplementation with chitosan derived from mushrooms changes adipocytokine profile in diet-induced obese mice, a phenomenon linked to its lipid-lowering action. International Immunopharmacology. 2009; 9(6): 767-773.

35. Bernardis LL and Patterson BD. Correlation between ‘Lee index’ and carcass fat content in weanling and adult female rats with hypothalamic lesions. J. Endocrinol. 1968; 40(4): 527-528.

36. Arch JR. The discovery of drugs for obesity, the metabolic effects of leptin and variable receptor pharmacology: perspectives from beta3-adrenoceptor agonists. Naunyn Schmiedebergs Arch Pharmacol. 2008; 378(2): 225-240.

37. Shivaprasad HN, Gopalakrishna S, Mariyanna B, Thekkoot M, Reddy R and Tippeswamy BS. Effect of Coleus forskohlii extract on cafeteria diet- induced obesity in rats. Pharmacog. Res. 2014; 69: 41-45.

38. L. Landsberg, JB Young, WB Leonard, RA Linsenmeier and FW Turek. Isobesity associated with lower body temperatures? Core temperature: a forgotten variable in energy balance. Metabolism: Clinical and Experimental. 2009; 58(6): 871-876.

39. MJ Heikens, AM Gorbach, HS Eden et al., Core body temperature in obesity. American Journal of Clinical Nutrition. 2011; 93(5)963-967.

40. Yu Wen Huang, Yue Liu, Slavik Dushenkov, Chi-Tang Ho and Mou Taun Huang. Anti-obesity effects of epigallocatechin-3-gallate, orange peel extract, black tea extract, caffeine and their combinations in a mouse model. Journal of Functional Foods. 2009; 1(3): 304-310.

41. Mukesh Kumar, Kiran Nehara, Duhan JS. Phytochemical analysis & antimicrobial efficacy of leaf extract of Pithecellobium dulce. Asian Journal of Pharmaceutical & Clinical Research. 2013: 6(1):70-76.

42. M Sugumaran, T Vetrichelvan, S Darlin Quine. Antidiabetic potential of aqueous and alcoholic leaf extracts of Pithecellobium dulce. Asian J. Research Chem. 2009; 2(1): 83-85.

Received on 08.04.2020 Modified on 13.05.2020

Research J. Pharm. and Tech 2021; 14(3):1447-1452.

DOI: 10.5958/0974-360X.2021.00258.4

Groups	Liver (gm)	Right Kidney (gm)	Left Kidney (gm)
Normal Control	5.62±0.06	0.75±0.01	0.74±0.01
HFD	8.79±0.82^¥¥¥	1.74±0.09^¥¥¥	0.93±0.06^¥¥¥
Standard	6.20±0.78^***	0.79±0.04^ns	0.74±0.04^ns
PEPD (100mg/kg)	8.09±0.69^*	0.90±0.03 ^ns	0.89±0.02^ns
PEPD (200mg/kg	7.50±0.72^**	0.88±0.01^ns	0.82±0.03^ns
EAPD (100mg/kg)	7.92±0.85^*	0.89±0.01^ns	0.85±0.04^ns
EAPD (200mg/kg)	7.04±0.78^**	0.87±0.02^ns	0.80±0.05^ns
MAPD (100mg/kg)	7.83±0.82^*	0.87±0.02^ns	0.81±0.01^ns
MAPD (200mg/kg)	6.83±0.75^***	0.85±0.03^ns	0.79±0.02^ns