INTRODUCTION:

Obesity is characterized by increased adipocytes in the body¹. It is estimated that there are over 600 million obese adults globally, comprising around 205 million males and 297 million women. According to research conducted by the World Health Organisation (WHO), being overweight or obese causes at least 2.8 million deaths annually². There are numerous strategies to stop or manage obesity, such as food plans, physical activity, and medicine. However, it has been noted that using anti-obesity medications like sibutramine and orlistat can have negative side effects^3,4.

As a consequence, additional research has been done on the application of herbal remedies that have been shown to have both in-vitro as well as in-vivo anti-obesity potential. Because of their natural origin, affordability, and few side effects, these herbal remedies attracted attention⁵.

Bauhinia purpurea L. (BP), a member of Fabaceae family has become a potential source of therapeutic agents in the recent research. Traditionally, B. purpurea has been used to treat rheumatism, pain, fever, ulcers, and gastric cancers^6,7. This study hypothesizes that BP bark ethanolic extract possesses anti-lipase and antioxidant activities.

Triacylglycerol acylhydrolase, or pancreatic lipase, an enzyme from pancreatic secretion is essential for the absorption of dietary lipids as well as their digestion⁸. Pancreatic lipase is one of the lipases that hydrolyzes 50–70% of the total lipids in food⁹. It is well established that pancreatic lipase inhibition, which lowers fat absorption, helps control obesity¹⁰. Ser152 residue of pancreatic lipase is substrate for hydrolysis and lipolysis activity¹¹.

Because of greater synthetic and secretory actions, the pancreas is a prime target for free radicals, which may contribute to oxidative damage and adverse effects on exocrine and endocrine function. According to experimental data, there are a variety of distinct sources of oxidative stress in obesity, including altered mitochondrial function, endothelial dysfunction, hyperglycemia, hyperleptinemia, insufficient antioxidant defenses, elevated muscle lipids, elevated muscle activity, and increased free radical formation rates. Numerous studies have indicated that naturally occurring substances like phytochemicals may play a significant role in regulating the risks related to obesity^12,13.

Hence this study aimed to evaluate phytochemicals in different extracts of Bauhinia purpurea and screening of selected extract for anti-lipase and antioxidant properties using in-vitro techniques.

MATERIALS AND METHODS:

Plant Material:

Identification of plant and Collection was done by Dr. V. Chelladurai, Plant Taxonomy and Medico botany and Authentication was done for the Plant Bauhinia purpurea from the Centre for Biodiversity and Biotechnology, Xavier Research Foundation, St Xavier's by Dr. S. Mutheeswaran. M.Sc, M.Phil., Ph.D. Scientist and Ethnobotanist. The herbarium number of the Bauhinia purpurea is XCH-40486 /2023.

Extraction:

The Bauhinia purpurea bark were collected, washed with water and dried in sunlight for 1 hour and then dried in shade. Then the extract is pulverized by mechanical blender and coarse powder obtained was subjected to extraction. Initially the extract was processed for Maceration. The entire or roughly ground-up crude substance was placed into a stoppered maceration flask along with 96% ethanol as the solvent, and it is let to remain at ambient temperature for three days while being constantly stirred until the soluble material dissolves. After that, the combination is strained, the marc—a moist solid material—is pressed, and both solvents are separated by filtration. Finally, the solvent is evaporated to obtain the dry extract. Then the dry extract was dissolved in distilled water and further processed with ether, chloroform and ethanol as described in previous studies¹⁴.

To determine the presence of phytoconstituents (alkaloids, phenol, tannins, carbohydrates, glycosides, saponins, steroids, flavonoids, terpenoids, resins, and proteins) in three distinct extracts, a preliminary qualitative phytochemical analysis was carried out¹⁵. All the chemicals used in this study were of analytical grade.

Antioxidant Assay:

Different Bauhinia purpurea extracts were tested for their ability to scavenge free radicals employing DPPH assay using the established methodology described by Olaleye et al. One millilitre of a 0.1 millilitre DPPH solution in 95% ethanol was made, and it was combined with three millilitres of different plant extract concentrations and one millilitre of a reference component (ascorbic acid, 1–5mg). Using a UV visible spectrophotometer, absorbance was measured at 517nm after 30 minutes. The test and control samples' absorbance levels were noted. The absorbance readings of test and control samples have been compared to determine the percentage of inhibition. The half maximum inhibitory concentration (IC₅₀) was determined using.

% of Inhibition = Absorbance of Blank – Absorbance of Test Absorbance of Blank × 100

Pancreatic Lipase Inhibition Assay:

Porcine pancreatic lipase (PPL) was tested for inhibitory activity utilising p-nitrophenyl butyrate (p-NPB) as a substrate. A distinct method was used to quantify the pancreatic lipase action from the one that Lai et al. had previously described. Method: The extracts at final concentrations of 25, 50, and 100μg/mL and Orlistat at the same concentrations were initially incubated using PPL for an hour at thirty degrees Celsius in a potassium phosphate buffer (0.1mM, pH 7.2, 0.1% Tween 80) before to assessing the PPL activity. Following five minutes of incubation at thirty degrees Celsius, the amount of p-NPB generated in the reaction was measured at 405nm using a UV-visible spectrophotometer. The PL inhibition capacity was computed using the standard formula.

Inhibition = 100 – [(B – b) ÷ (A - a) × 100]

Where A = activity of the enzyme without inhibitor, a = negative control without inhibitor, B = activity of the enzyme with inhibitor, b = negative control with inhibitor.

The IC50 value of extracts was determined at a concentration of 25, 50, 100µg/ml. Orlistat was used as a positive control. IC50 value was calculated by the following formula:

IC50 = 50% - Low inhibition % (High Inhibition % - Low Inhibition %) × (High Concentration – Low Concentration) + Low Concentration

Where, Low inhibition % /High inhibition % signify % inhibition directly below/above 50% inhibition, and Low Concentration/High Concentration are the corresponding concentrations of extract.

RESULTS:

Effect of extraction technique on percentage yield:

The crude extract was processed for preparing ethanolic, ether and chloroform extracts. The final extract yield obtained was higher in ethanolic extract and least in chloroform extract comparatively (Table 1). The phytochemical analysis revealed presence of carbohydrates, polyphenols, flavonoids, amino acids, diterpenes in ethanol, petroleum ether and chloroform extracts of BP. Apart from this, ethanolic extract contained saponins, terpenoids, proteins, and steroids also. In comparsion, ethanolic extract constituted higher composition of polyphenols and flavonoids (Table 2 and Figure 1-3).

Table 1: Extraction methods and final yield.

Solvent	BP Powder weight	Extract obtained	% Of Yield (w/w)
Ethanol	25g	4.5 g	18 %
Petroleum ether	25g	4 g	16 %
Chloroform	25g	3.5 g	14 %

Table 2: Phytochemical analysis

S. No.	Constituent	Ethanol	Petroleum ether	Chloroform
1	Alkaloids	-	-	-
2	Carbohydrates	+	+	+
3	Saponins	++	-	-
4	Polyphenols	++	+	+
5	Flavonoids	++	+	+
6	Amino acids	+	+	+
7	Diterpenes	+	+	+
8	Tannins	-	-	-
9	Terpenoids	+	-	-
10	Proteins	+	-	-
11	Steroids	+	-	-

Figure 1: Phytochemical analysis of Petroleum ether extract of Bauhinia purpurea

Figure 2: Phytochemical analysis of Chloroform extract of Bauhinia purpurea

Figure 3: Phytochemical analysis of Ethanolic extract of Bauhinia purpurea

Antioxidant effects of ethanolic extract of Bauhinia purpurea:

In Table 3, it was shown that the methanolic extracts of BP exhibited notable dose-dependent in vitro DPPH radical scavenging capabilities. The DPPH radical scavenging capabilities of the examined methanolic plant extract were significantly lower than those of the standard, L-ascorbic acid (Table 3).

Table 3: Antioxidant effects of ethanolic extract of Bauhinia purpurea

S. No	Concentration	% inhibition of extract
1	20	39.71
2	40	64.15
3	60	64.90
4	80	96.43
5	100	96.46

Effects of ethanolic extract of Bauhinia purpurea on pancreatic lipase:

The ethanolic extract of BP showed dose dependent inhibitory action on pancreatic lipase. At 100mg/ml, the percentage inhibition was 78.22±0.76 percent. Orlistat, a lipase inhibitor used as the positive control, strongly inhibited lipase activity with an IC50 of 88mg/ml. With an IC₅₀ of 97mg/ml, BPE analogously reduced lipase activity in a concentration-dependent manner (Figure 4).

Figure 4: Comparison of pancreatic lipase inhibition between ethanolic extract of Bauhinia purpurea and Orlistat.

DISCUSSION:

The present study evaluated phytochemicals in ethanolic, petroleum ether and chloroform extracts of Bauhinia purpurea, and screened ethanolic extract for anti-lipase and antioxidant properties using in-vitro techniques. High yield of the extract and maximum phytoconstituents in the extract was seen with ethanolic extraction method. The pancreatic lipase inhibitory activity of the BP ethanolic extract was dose-dependent and the mean values were near to comparability to orlistat. The antioxidant effect of the BP ethanolic extract was also in dose-dependent manner.

Antioxidant effects and pancreatic lipase inhibition are very crucial in the management of obesity. The emergence of dyslipidemia is influenced by increased ROS production. In cellular signalling, free radicals serve as physiological signal transducers to preserve homeostasis¹⁶. However, when these pathways are disturbed, they can lead to dyslipidemia^17,18. In comparison to petroleum ether and chloroform extracts, BP ethanolic extracts expressed higher concentrations of flavonoids and phenols. By chelating redox-active metal ions, deactivating lipid-free radical chains, and preventing hydroperoxide transformations into reactive oxyradicals, the phenolic molecule, as well as a flavonoid, functions as an additional antioxidant agent^19,20.

New strategies for treating obesity have recently attempted to lower energy intake via gastrointestinal processes without affecting any essential functions. One such approach is pancreatic lipase inhibition, and a lot of study has been done on the possible effectiveness of natural compounds as medicines against obesity⁴. Physiologically, pancreatic lipase helps in growth of adipocytes. As such, 90% of dietary fats are made up of mixed triglycerides, which must be digested by different lipases in order to be absorbed. The three primary human lipases that break down dietary lipids are pancreatic, gastric, and lingual lipases²¹. Of these lipases, 50–70% of dietary fats are hydrolyzed to produce monoglycerides and fatty acids by pancreatic lipase. Lipid hydrolysis releases these, which combine with cholesterol, lysophosphatidic acid, and bile salts to produce mixed micelles. Following that, mixed micelles are taken up by enterocytes, which is where triglyceride is resynthesized. Lastly, the main energy source that adipocytes store is triglyceride⁹. The BP ethanolic extract exhibited dose dependent inhibition of pancreatic lipase in our study and its IC50 was comparable to orlistat which is positive control.

However, in this study we have demonstrated the antioxidant and pancreatic lipase inhibitory effects of the ethanolic extract of BP alone based upon phytoconstituent profile, while we did not perform these in-vitro experiment with petroleum ether and chloroform extract which could be taken as limitation of this study.

CONCLUSION:

The BP ethanolic extracts expressed high constitution of phenols, saponins and flavonoids in comparison to chloroform and petroleum ether extracts. The BP ethanolic extract exhibited antioxidant and pancreatic lipase inhibitory properties demonstrated through in-vitro methods. This extract may taken forward for further evaluations and preparation of formulations which can benefit obesity population.

CONFLICT OF INTEREST:

None declared.

REFERENCES:

1. Qureshi K, Abrams GA. Metabolic liver disease of obesity and role of adipose tissue in the pathogenesis of nonalcoholic fatty liver disease. World J. Gastroenterol. 2007; 13(26): 3540-53. doi: 10.3748/wjg.v13.i26.3540.

2. Rahman HA, Sahib NG, Saari N, Abas F, Ismail A, Mumtaz MW, Hamid AA. Anti-obesity effect of ethanolic extract from Cosmos caudatus Kunth leaf in lean rats fed a high fat diet. BMC Complementary and Alternative Medicine. 2017; 17: 1-7. doi: https://doi.org/10.1186/s12906-017-1640-4.

3. Thurairajah PH, Syn WK, Neil DA, Stell D, Haydon G. Orlistat (Xenical)-induced subacute liver failure. Eur J Gastroenterol Hepatol. 2005; 17(12): 1437-8. doi: 10.1097/01.meg.0000187680.53389.88.

4. Yun JW. Possible anti-obesity therapeutics from nature--a review. Phytochemistry. 2010; 71(14-15): 1625-41. doi: 10.1016/j.phytochem.2010.07.011.

5. Slanc P, Doljak B, Kreft S, Lunder M, Janes D, Strukelj B. Screening of selected food and medicinal plant extracts for pancreatic lipase inhibition. Phytother Res. 2009; 23(6): 874-7. doi: 10.1002/ptr.2718.

6. Janardhanan K, Vadivel V, Pugalenthi M. Biodiversity in Indian underexploited/tribal pulses. InImprovement Strategies of Leguminosae Biotechnology 2003 (pp. 353-405). Dordrecht: Springer Netherlands. doi: https://doi.org/10.1007/978-94-017-0109-9_17

7. Zakaria ZA, Wen LY, Abdul Rahman NI, Abdul Ayub AH, Sulaiman MR, Gopalan HK. Antinociceptive, anti-inflammatory and antipyretic properties of the aqueous extract of Bauhinia purpurea leaves in experimental animals. Med Princ Pract. 2007; 16(6): 443-9. doi: 10.1159/000107749.

8. Veeramachaneni GK, Raj KK, Chalasani LM, Annamraju SK, Js B, Talluri VR. Shape based virtual screening and molecular docking towards designing novel pancreatic lipase inhibitors. Bioinformation. 2015; 11(12): 535-42. doi: 10.6026/97320630011535.

9. Birari RB, Bhutani KK. Pancreatic lipase inhibitors from natural sources: unexplored potential. Drug Discov Today. 2007; 12(19-20): 879-89. doi: 10.1016/j.drudis.2007.07.024.

10. Ahn JH, Liu Q, Lee C, Ahn MJ, Yoo HS, Hwang BY, Lee MK. A new pancreatic lipase inhibitor from Broussonetia kanzinoki. Bioorg Med Chem Lett. 2012; 22(8): 2760-3. doi: 10.1016/j.bmcl.2012.02.088.

11. Bourne Y, Martinez C, Kerfelec B, Lombardo D, Chapus C, Cambillau C. Horse pancreatic lipase. The crystal structure refined at 2.3 A resolution. J Mol Biol. 1994; 238(5): 709-32. doi: 10.1006/jmbi.1994.1331.

12. Vincent HK, Taylor AG. Biomarkers and potential mechanisms of obesity-induced oxidant stress in humans. Int J Obes (Lond). 2006; 30(3): 400-18. doi: 10.1038/sj.ijo.0803177.

13. Vanacore D, Messina G, Lama S, Bitti G, Ambrosio P, Tenore G, Messina A, Monda V, Zappavigna S, Boccellino M, Novellino E, Monda M, Stiuso P. Effect of restriction vegan diet's on muscle mass, oxidative status, and myocytes differentiation: A pilot study. J Cell Physiol. 2018; 233(12): 9345-9353. doi: 10.1002/jcp.26427.

14. Chaabane F, Pinon A, Simon A, Ghedira K, Chekir-Ghedira L. Chloroform leaf extract of Daphne gnidium inhibits growth of melanoma cells and enhances melanogenesis of B16-F0 melanoma. South African Journal of Botany. 2014; 90: 80-6. doi: https://doi.org/10.1016/j.sajb.2013.10.009

15. Dubale S, Kebebe D, Zeynudin A, Abdissa N, Suleman S. Phytochemical Screening and Antimicrobial Activity Evaluation of Selected Medicinal Plants in Ethiopia. J Exp Pharmacol. 2023; 15: 51-62. doi: 10.2147/JEP.S379805.

16. Ghanta M, Panchanathan E, Lakkakula BV, Narayanaswamy A, Murkunde Y, Tamrakar S. 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one attenuates oxidative trauma and recuperate beam walk and adhesive removal behavior in MPTP Parkinsonian mice model. Biomedical and Pharmacology Journal. 2018; 11(4): 2011-7. doi : https://dx.doi.org/10.13005/bpj/1576

17. Pignatelli P, Menichelli D, Pastori D, Violi F. Oxidative stress and cardiovascular disease: new insights. Kardiol Pol. 2018; 76(4): 713-722. doi: 10.5603/KP.a2018.0071.

18. Czaja AJ. Autoimmune hepatitis: focusing on treatments other than steroids. Can J Gastroenterol. 2012; 26(9): 615-20. doi: 10.1155/2012/512132. Erratum in: Can J Gastroenterol. 2012; 26(10): 758.

19. Patro G, Bhattamisra SK, Mohanty BK, Sahoo HB. In vitro and In vivo Antioxidant Evaluation and Estimation of Total Phenolic, Flavonoidal Content of Mimosa pudica L. Pharmacognosy Res. 2016; 8(1): 22-8. doi: 10.4103/0974-8490.171099.

20. Yang X, Kang SM, Jeon BT, Kim YD, Ha JH, Kim YT, Jeon YJ. Isolation and identification of an antioxidant flavonoid compound from citrus-processing by-product. J Sci Food Agric. 2011; 91(10): 1925-7. doi: 10.1002/jsfa.4402.

21. Mukherjee M. Human digestive and metabolic lipases—a brief review. Journal of Molecular Catalysis B: Enzymatic. 2003; 22(5-6): 369-76. doi: https://doi.org/10.1016/S1381-1177(03)00052-3.

Received on 04.04.2024 Revised on 25.07.2024

Accepted on 29.09.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1047-1051.

DOI: 10.52711/0974-360X.2025.00150

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.