INTRODUCTION:

Obesity is the accumulation of abnormal or excessive fat that can harm health. Obesity is a metabolic disorder caused by lifestyle disorders related to lack of physical activity. Obesity is a cause of concern throughout the world, not only because of the dangers of being overweight¹, but obesity and lipid metabolism disease are global problems because they are risk factors for people with cancer, insulin resistance, diabetes mellitus, endocrine disorders, and cardiovascular disease².

The development of obesity is very rapid and is a world problem to this day; therefore, preventing obesity is a big challenge in both developed and developing countries^3,4

There are two types of therapy to prevent obesity: first, inhibiting the absorption of fat and carbohydrates to reduce the energy absorption of food, second, reducing appetite and increasing the feeling of fullness. Inhibition of fat absorption through the intestinal wall by inhibiting lipase enzyme activity is a widespread target. The pancreatic lipase enzyme is the main fat-digesting enzyme, hydrolyzing triglycerides into fatty acids and liquid products⁵. Currently, lipase inhibitors are receiving more attention⁶. Orlistat is a powerful antiobesity agent with a mechanism that inhibits the lipase enzyme in the gastrointestinal tract, which does not work on the central nervous system or blood flow7^,8. However, Orlistat has side effects on the digestive tract, such as oily stools and flatulence. Therefore, it is necessary to carry out investigations to find new antiobesity agent compounds without adverse side effects^6,8,⁹.

Pancreatic lipase inhibition is one of the most widely studied mechanisms for determining the antiobesity activity of natural products. Compounds from natural ingredients are considered suitable for this purpose because they offer less risk when compared to synthetic drugs^10-12. Lipase inhibitors derived from natural ingredients are currently a potential research center. Screening for lipase enzyme inhibition of several medicinal plant extracts has been conducted. However, the mechanism of action and active compounds that contribute are still being explored. Therefore, research is necessary to determine active compounds with a mechanism of action that inhibits the lipase enzyme as a candidate antiobesity agent from natural ingredients8^,9,10,13-14.

Extracts from Kaempferia rotunda rhizomes have been reported to have various pharmacological activities, including antioxidant, antinociceptive, antimicrobial, anticancer, antihyperglycemic, wound healing, antityrosinase, anti-allergic, antiandrogenic, anthelmintic and lipase inhibitor^15-22. Previous research reported that Kaempferia rotunda extract inhibits the lipase enzyme (water extract at 65.1% and ethanol extract at 36.5%)²³. Still, the active compound that contributes to this activity has never been reported. Meanwhile, the compounds that have been isolated from Kaempferia rotunda rhizomes include a group of polyoxygenated cyclohexane derivatives, such as crotepoxide, pinostrobin compounds, and two flavone compounds, namely 7-hydroxy-5-methoxy flavanone, 5,7-dihydroxy flavanone^24-26. Crotepoxide is a cyclohexane derivative, such as diepoxide, the major component in Kaempferia rotunda rhizomes^27-29. Crotepoxide was also found in Croton macrotachyus, Kaempferia angustifolia, and Piper attenuatum plants^30-33 and has been reported to have antibacterial, antifeedant, antioxidant, insecticidal, and anti-HIV activities3^1-39.

One of the abilities of chemical compounds in plants is to bind enzymes and inhibit the action of certain enzymes40. Research on inhibiting lipase enzyme activity by natural ingredients has been widely reported^2,3,5,34–3⁶. However, the studies are limited to the activity of the extracts, therefore, it is necessary to carry out research that focuses on isolating lipase enzyme inhibitor compounds. This study is the first to report on the activity of Crotepoxide from KRE as an inhibitor of the lipase enzyme in vitro using p-nitro phenylbutyrate as an artificial substrate.

MATERIALS AND METHODS:

Materials:

Rhizome of K. rotunda (L.) was collected and identified (identification number: YK.01.03/2/677/2021) at the Research and Development Center for Medicinal Plants and Traditional Medicine (B2P2TOOT), Tawangmangu, Karanganyar, Central Java, in November 2019. The specimen (number: 12/kdr/301-01) is stored in the pharmaceutical biology laboratory of the Faculty of Pharmacy, Bhakti Wiyata Kediri Institute of Health Sciences.

From Sigma-Aldrich (Schnelldorf, Germany), the following were purchased: Orlistat, dimethyl sulfoxide, p-nitrophenyl butyrate, crude porcine pancreatic lipase powder type II (Sigma, EC 3.1.1.3), and Phosphate Buffer Saline. Orlistat (control) from Sigma-Aldrich (Schnelldorf, Germany). Silica Gel For Thin Layer Chromatography PF₂₅₄ from Merck. Dichloromethane, chloroform, ethyl acetate, n-hexane, methanol, ethanol, and all other chemicals and solvents were of analytical grade (Smartlab, Indonesia).

Solvent Extraction, Fractionation, and Isolation Methods:

Ten kilograms of Kaempferia rotunda simplicia powder was extracted by maceration with 70% ethanol 1:10 w/v (3x24 h) at room temperature and repeated twice with the same solvent system. The filtrate was concentrated under reduced pressure using a rotary evaporator to give a dark brown gummy Kaempferia rotunda Extract (KRE) residue.

In this research, the solid-liquid partition of the KRE was carried out in stages. KRE is stirred with a solvent with increasing solvent polarity [n-hexane, ethyl acetate, and ethanol] to produce n-hexane (HF), ethyl acetate (EaF), ethanol (EF), and an insoluble residue fraction (RF). The activity of the four fractions was measured against the pancreatic lipase enzyme in vitro. ELISA reader was used to calculate the inhibitory activity (%) of the fraction based on the hydrolysis power of the pancreatic lipase enzyme on the substrate.

The most active fraction was then further fractionated using Vacuum Liquid Chromatography (VLC) with a column 8 cm diameter filled with Silica Gel For TLC 60 (Merck)]. The mobile phase used with the gradient system uses dichloromethane 100%, dichloromethane: chloroform, [8:2], [6:4], [4:6], [2:8] v/v, chloroform 100%, ethyl acetate 100% and 100% methanol v/v. Eight fractions were analyzed using TLC with mobile phase n-hexane: ethyl acetate (7:3 v/v). Through TLC monitoring, four combined sub-fractions were obtained based on the similarity of compound profiles. The four combined fractions (Ea1, Ea2, Ea3, and Ea4) were again tested for their inhibitory activity against the lipase enzyme. The most potential sub-fraction, namely Ea3, becomes the target which will be further separated using the Preparative Thin Layer Chromatography (P-TLC). The stationary phase was silica gel for P-TLC F₂₅₄ 60 (Merck), eluted with n-hexane: ethyl acetate (7:3 v/v). The separation and purification process produced the major compound [Crotepoxide (82.9mg)], whose purity was then identified using TLC using different mobile phases and analyzed under UV₂₅₄ and UV₃₆₆ lights as well as spot visualization using Ce(SO₄)₂, which is a universal spotting agent for organic compounds. Spectroscopic characterization of Crotepoxide has been reported in our previous study31.

Pancreatic Lipase Inhibition In-vitro Assay:

This study's lipase enzyme inhibitory activity test was conducted using an ELISA reader instrument (BIO-TEK, Synergy HT, USA). The instrument was used to measure the hydrolysis of the pNPB substrate by the lipase enzyme to p-nitrophenol at a wavelength of 415 nm in a 96-well plate. Test samples for triturated fractions, combined sub-fractions resulting from VLC, and isolated compounds were prepared at 0.1mg/mL concentration in 1% DMSO. Meanwhile, KRE was prepared at 0.5mg/mL and Orlistat (control) at 0.1 mg/mL concentration in 1% DMSO. An enzyme solution with 1 mg/mL concentration was made by dissolving Porcine Pancreatic Lipase (PPL) enzyme powder (Sigma-Aldrich) in phosphate buffer pH 7 and given a concentration of 50mM. The enzyme solution was centrifuged for 5minutes to remove insoluble parts. The concentration of the buffer solution was adjusted to 0.1mg/ml for every 1mg of solid PPL powder dissolved in the volume of the buffer solution. The PNPB substrate was prepared at a concentration of 2.5mM in 1% DMSO. To a 96-well plate containing 50μL of sample solution, 50μL of lipase enzyme solution was added. The mixture was then incubated at 37⁰C for 10 minutes.

After incubation, 50μL of substrate solution was added to each sample, including Orlistat as a positive control, and then set again at 37⁰C for 10 minutes. A negative control, DMSO, was used in measurements with and without inhibitors. At 415nm, measurements of positive and negative control samples and blank measurements were carried out without the addition of enzymes and substrates⁹. The test was carried out three times in replication, and the calculation of percent inhibition was the average of the three tests. At 37⁰C, the reaction rate that produces one mole of p-nitrophenyl butyrate is used as the definition of one unit of activity. When PPL is incubated with a test sample, the amount of lipase activity inhibited is determined by calculating the percentage reduction in that activity. Lipase inhibition/lipase inhibition (%) is calculated based on the following formula

Inhibition of lipase (1%) = 100- [(B-b)/(A-a)x100]

A is an activity that occurs without an inhibitor, and a represents a negative control that occurs without an inhibitor. At the same time, B is an activity with an inhibitor, and b is a negative control with an inhibitor.

Data analysis:

All determinations of pancreatic lipase inhibition were conducted in triplicate. Experimental data were expressed as mean±SD. The data were analyzed using a one-way analysis of variance (ANOVA) using IBM ver. 25. A Post hoc test was used to determine differences between the means. It was accepted at a level of 5% (significance level p<0.05).

RESULT:

Bioassay Guided Isolation of Pancreatic Lipase Inhibitor Compound:

The results obtained from the maceration process were a thick extract with a yield of 9.84%w/w. Using the trituration method, solid-liquid partition has four types of fractions (HF, EAF, EF, and RF). The trituration procedure separates extracts based on increasing solvent polarity because it is easy to apply while simultaneously employing large samples.

The results of the activity test of the trituration fractions on the pancreatic lipase enzyme showed that EAF had the highest percent inhibition, namely 30.54 ±0.95%, while HF, EF, and RF had percent inhibition respectively: 9.75±0.50%, 27.18±1.01%, and 13.18±0.74%. Meanwhile, KRE obtained a percent inhibition of 20.81±0.29%, and Orlistat inhibited 61.48 ±1.87% as a positive control.

In the VLC process, the EAF fraction produces eight fractions from the gradient solvent which are then combined into four sub-fractions (Ea1, Ea2, Ea3, and Ea4) based on the similarity of metabolite profiles and Rf. These four subfractions were monitored using a pancreatic lipase-inhibitory activity bioassay to determine the active fraction, further separated using P-TLC. The results, it can be seen that Ea3 (48.29 ±1.26%) is the subfraction that most strongly inhibits lipase enzyme activity compared to Ea1 (12.38± 0.36%); Ea2 (20.18±0.95%) and Ea4 (15.95±4.89%) while Orlistat as a positive control inhibited 64.46±0.32%. These results showed that the Ea3 is the target for separating active compounds that inhibit the pancreatic lipase enzyme using the P-TLC method.

Fig 1. Pancreatic Lipase Inhibition (%) of KRE, n-hexane (H), ethyl acetate (Ea), ethanol (E) and insoluble residue fraction (R) from trituration (A), Pancreatic Lipase Inhibition (%) of Ea1, Ea2, Ea3 fractions from VLC, and Orlistat in percent inhibition (B). Values are mean±SD (n = 3). **** are significant (p<0.05), ns = not significant (p>0.05)

Pancreatic Lipase Inhibition Activity of Crotepoxide:

Compound 1 (colorless needle) was isolated using the P-TLC method from fraction Ea.3, was identified as Crotepoxide. Isolation procedure and spectroscopic data of Crotepoxide have been reported in our previous study³¹. Crotepoxide activity against the lipase enzyme in this study was carried out using the same method as extracts and fractions using an Elisa reader at a wavelength of 415nm with incubation conditions at 37 ⁰C for 10minutes. Crotepoxide gave a percentage value of 42.80±0.49%, while Orlistat was 74.04±0.13%.

Fig 2. Structure of Crotepoxide

DISCUSSION:

The lipase enzyme is an enzyme that plays an essential role in breaking down triglycerides into free fatty acids, which are secreted in the pancreas. Inhibition of lipase enzyme activity is the most widely studied mechanism of action in finding antiobesity agents derived from natural ingredients8. To guide the fractionation stages using the VLC method, the triturated fractions were tested for their activity inhibiting the pancreatic lipase enzyme in vitro. In this study, the method used to test the inhibitory activity of the pancreatic lipase enzyme was the Alias, 2017 method41. The inhibitory activity of the pancreatic lipase enzyme was tested using a colorimetric assay that measures the release of p-nitrophenol ions (yellow), which is monitored during the enzymatic hydrolysis process of the PNPB substrate in l 415nm⁴¹.

From the figure 1.A., it can be concluded that each group has significant (p<0.05) differences. This indicates that the compounds in each fraction provide different activities inhibiting the lipase enzyme^42,43. The finding of percent inhibition showed that KRE provides very low inhibition to the lipase enzyme. It could be due to the varying composition of compounds in KRE, which had synergistic or antagonistic against PPL. Meanwhile, in EAF, groups of compounds have various mechanisms of action and potential synergistic effects5^,42.

Ea.3 contains several compounds that synergize to provide the strongest inhibition of lipase, the major compound in this fraction was the target for isolation (compound 1). Although EA3 has significant differences to orlistat, this fraction is worthy of further development. Pancreatic lipase inhibitory activity by fractions and isolated compound from KRE have never been reported. The reported studies presented the findings at the extract level. This study differs in the type of extraction solvent and the pancreatic lipase-inhibitory activity test method used²³. A study conducted by Pradono (2012) reported the activities of the water extract (65.1%) and ethanol extract (36.5%), which was different from our study (20.81±0.29%). Apart from the effect of the type of solvent and test method used, the type of synthetic substrate used also affects the in vitro test results.

Pancreatic Lipase Inhibition Activity of Crotepoxide:

In this study, however, Crotepoxide was less potent than Orlistat inhibiting the Lipase enzyme These findings showed that Crotepoxide inhibits the lipase enzyme in the medium (42.80±0.49%) inhibition. Based on previous research⁴¹, it was reported that inhibition of the lipase enzyme provided high inhibition (>70%), while medium inhibition (30-70%) showed low inhibition (<30%) or no inhibition when incubated with PPL at the final concentration of extract at 500µg/ml for 10 minutes at 37°C. At this point, the percent inhibition of Ea.3 (48.29±1.26%) is stronger than the isolated compound due to the presence of active and non-active compounds that provide inhibition of the lipase enzyme³⁷. Besides, Ea.3 may contain active compounds that synergis to inhibit PPL.

In many cases, the pharmacological activities of these plants are attributable to the presence of secondary metabolites such as polyphenols, saponins, tannins, terpenes, flavonoids, and alkaloids, which are active inhibitors of pancreatic lipase^29,30,38.

CONCLUSION:

This study shows that extract, fraction, and compound isolated from KRE (Crotepoxide) inhibited the lipase enzyme activity. The finding proves that the isolated compound has lower pharmacological activity than the fraction. The study also indicates that other compounds in the fraction may provide stronger inhibition than Crotepoxide. There is still no report found about inhibitory Porcine Pancreatic Lipase by Crotepoxide from K. rotunda rhizome. However, the medium inhibition indicates that the fraction level and the isolated compound are not potent inhibitors, suggesting that these compounds may contribute to obesity via other mechanisms. Further research is needed to evaluate other compounds in the Ea.3 fraction and explore compounds that provide stronger inhibition than Crotepoxide.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

AUTHORS' DECLARATION:

The author declares that the work in this article is original and that they will bear all responsibility related to the content of this article.

ACKNOWLEDGMENTS:

The research was funded by the Indonesia Endowment Funds for Education (LPDP) Center for Higher Education Funding (BPPT) through Indonesian Education Scholarship Program (BPI), the Indonesian Ministry of Education and Culture (Kemendikbud RI).

REFERENCES:

1. Mewada PS. Patel CK. Rami CS. et al. New Emerging Targets for Obesity. Asian J. Research Chem. 2010; 3(2): 278-287.

2. Jaradat N. Zaid AN. Hussein F. et al. Anti-Lipase Potential of the Organic and Aqueous Extracts of Ten Traditional Edible and Medicinal Plants in Palestine; a Comparison Study with Orlistat. Medicines. 2017; 4(89): 1-13. doi.org/10.3390/medicines4040089

3. Ong SL. Mah S. H. Lai HY. Porcine Pancreatic Lipase Inhibitory Agent Isolated from Medicinal Herb and Inhibition Kinetics of Extracts from Eleusine indica (L.) Gaertner. J Pharm. 2016; 2016: 1–9. doi.org/10.1155/2016/8764274.

4. Bustanji Y. Mohammad M. Hudaib M. et al. Screening of Some Medicinal Plants for Their Pancreatic Lipase Inhibitory Potential. Jordan J Pharm Sci. 2011; 4(2): 81–88. doi.org/10.5897/JMPR10.399.

5. Gandhi SP. Gawahne AR. Nanda RK. et al. In-vitro and In-silico Approach to Study Phyllanthus amarus Extract Against Obesity. Research J. Pharm. and Tech. 2019; 12(12): 6091-6096. doi: 10.5958/0974-360X.2019.01058.8

6. Fernando WIT. Attanayake AMKC. Perera HKI. et al. Isolation, identification and characterization of pancreatic lipase inhibitors from Trigonella foenum-graecum seeds. South African J Bot. 2019; 121: 418–421.

7. Rashida JN. Gowri VR. Aruna A. A Study on Ethanolic Extract of Dalbergia sissoo roxb. Leaves for Pancreatic Lipase Inhibition. Research J. Pharm. and Tech. 2012; 5(4): 497-500.

8. Liu TT. Liu XT. Chen QX. Shi Y. Lipase Inhibitors for Obesity: A Review. Biomed Pharmacother. 2020; 128: 1–9. doi.org/10.1016/j.biopha.2020.110314.

9. Huang R. Zhang Y. Shen S. et al. Antioxidant and pancreatic lipase inhibitory effects of flavonoids from different citrus peel extracts: An in vitro study. Food Chem. 2020; 326: 1-10. doi.org/10.1016/j.foodchem.2020.126785.

10. Atigari RGD. Sheeba DSH. Ramesh C. Evaluation of Anti-Obesity Activity of Lantana camara var Linn on butter induced Hyperlipidemia in Rats. Research J. Pharmacology and Pharmacodynamics. 2012; 4(5): 315-318.

11. Shukla A. Bioassay: An uncomplicated methodologies for ensure safety of Traditional Formulations. Research J. Pharmacognosy and Phytochemistry. 2009; 1(1): 01-04.

12. Sankpal S. Pathade KS. Kondawar M. Coffea arabica: Herbal Drug on Global Health Issue-Obesity. Res. J. Pharmacognosy and Phytochem. 2020; 12(4): 201-206. doi: 10.5958/0975-4385.2020.00034.5

13. Arun A. Pavitrra RS. Kanimozhi S.. Screening of Phytochemical and Quantitative Lipase Inhibition of Honey and Cinnamon Paste: A Synergistic Effect. Res J Pharm Technol. 2023; 16(8): 3709–3713. doi:10.52711/0974-360X.2023.00611.

14. Bajes HR. Almasri I. Bustanji Y. Plant Products and Their Inhibitory Activity Against Pancreatic Lipase. Rev Bras Farmacogn. 2020; 30: 321–330. doi.org/10.1007/s43450-020-00055-z.

15. Aryantini D. Astuti P. Yuniarti N. Wahyuono S. Extraction and Isolation of Phytochemicals from Kaempferia rotunda Linn. (white turmeric) for Pharmacological Application: A Review. Trop. J. Nat. Prod. Res. 2022; 6: 1359–1366. doi.org/10.13057/BIODIV/D240665.

16. Madaka F. Tewtrakul S. Anti-allergic Activity of Some Selected Plants in the Genus Boesenbergia and Kaempferia. Songklanakarin J. Sci. Technol. 2011; 33(3): 301–304.

17. Imam SA. Rout KS. Sutar N. Shankar SU. Sutar R. Wound Healing Activity of Kaempferia rotunda Linn Leaf Extract. Int J Curr Microbiol App Sci. 2013; 2(12): 74–78.

18. Agrawal S. Bhawsar A. Choudary P. et al. In-Vitro Anthelmintic Activity of Kaempferia rotunda. Int J Pharm Life Sci. 2011; 2(9): 1062–1064.

19. Malahayati N. Widowati TW. Febrianti A. Total Phenolic, Antioxidant and Antibacterial Activities of Curcumin Extract of Kunci Pepet (Kaempferia rotunda L). Res J Pharm Biol Chem Sci. 2018; 9(3): 129–135. doi.org/10.33887/rjpbcs/2019.9.3.

20. Desmiaty Y. Winarti W. Nursih AM. Nisrina H. Finotory G. Antioxidant and Antielastase Activity of Kaempferia Rotunda and Curcuma Zedoaria. Res J Chem Environ. 2018; 22(Special Issue): 95–98.

21. Panyakaew J. Chalom S. Sookkhee S. et al. Kaempferia Sp. Extracts as UV Protecting and Antioxidant Agents in Sunscreen. J Herbs Spices Med Plants. 2020; 27(1): 1-20. doi.org/10.1080/10496475.2020.1777614.

22. Rachkeeree A. Kantadoung K. Puangpradub R. Suksathan R. Phytochemicals, Antioxidants and Anti-tyrosinase Analyses of Selected Ginger Plants. Pharmacogn J.2020; 12(4): 872–883. doi.org/10.5530/pj.2020.12.125.

23. Pradono DI. Darusman LK. Susanti A. Inhibisi Lipase Pankreas secara In Vitro oleh Ekstrak Air dan Etanol Daun Asam Jawa (Tamarindus indica) dan Rimpang Kunci Pepet (Kaempferia rotunda). J Natur Indones. 2012; 13(2): 146-154. doi.org/10.31258/jnat.13.2.146-154.

24. Lotulung PDN. Minarti Kardono LBS. Kawanishi K. Antioxidant Compound from the Rhizomes of Kaempferia rotunda L. Pakistan J Biol Sci. 2008; 11(20): 2447–2450. doi.org/10.3923/pjbs.2019.518.526.

25. Stevenson PC. Veitch NC. Simmonds MSJ. Polyoxygenated Cyclohexane Derivatives and Other Constituents from Kaempferia rotunda L. Phytochemistry. 2007 68(11): 1579–1586. doi.org/10.1016/j.phytochem.2007.03.015.

26. Pancharoen O. Tuntiwachwuttikul P. Taylor WC. Cyclohexane Diepoxides from Kaempferia rotunda. Phytochemistry. 1996; 43(1): 305–308. doi.org/10.1016/0031-9422(96)00218-X.

27. Diastuti H. Chasani M. Suwandri. Antibacterial Activity of Benzyl Benzoate and Crotepoxide from Kaempferia rotunda L. Rhizome. Indones J Chem. 2020; 20(1): 9–15. doi: 10.22146/ijc.37526

28. Gelaw H. Adane L. Tariku Y. Hailu A. Isolation of Crotepoxide from Berries of Croton Macrostachyus and Evaluation of Its Anti-Leishmanial Activity. J Pharmacogn Phytochem. 2012; 1(4): 15–24.

29. Yeap YSY. Kassim N. Ng RC. et al. Antioxidant Properties of Ginger (Kaempferia angustifolia Rosc.) and its chemical markers. Int J Food Prop. 2017; 2014: 1158–1172. doi.org/10.1155/2014/417674

30. Reddy SD. Siva B. Poornima D. et al. New Free Radical Scavenging Neolignans from Fruits of Piper attenuatum. Pharmacogn Mag. 2015; 11(42): 235–241. doi.org/ 10.4103/0973-1296.153063.

31. Aryantini D. Astuti P. Yuniarti N. Wahyuono S. Bioassay-guided Isolation of the Antioxidant Constituent from Kaempferia rotunda L. Biodiversitas J Biol Divers. 2023; 24(6): 3641-3647. doi.org/ 10.13057/BIODIV/D240665

32. Manivasagan V , Saranya K , Poojashree S, Ankitha K, Niyathi V. In vitro Antioxidant, Antidiabetic and Antibacterial Activities of Orthosiphon diffusus. Research Journal of Pharmacognosy and Phytochemistry. 2022; 14(3): 163-0. doi: 10.52711/0975-4385.2022.00030

33. Nugroho BW. Schwarz B. Wray V. Proksch P. Insecticidal constituents from rhizomes of Zingiber cassumunar and Kaempferia rotunda. Phytochemistry. 1996; 41(1): 129–132. doi.org/ 10.1016/0031-9422(95)00454-8

34. Rotich W. et al. HIV-1 integrase inhibitory effects of major compounds present in CareVidTM: An anti-hiv multi-herbal remedy. Life. 2022; 12(3): 417-427. doi.org/ 10.3390/life12030417.

35. Jamous RM. Abu-Zaitoun SY. Akkawi RJ. Ali-Shtayeh MS. Antiobesity and Antioxidant Potentials of Selected Palestinian Medicinal Plants. Evidence-based Complement Altern Med. 2018; 2018: 1-21. doi.org/10.1155/2018/8426752.

36. Zheng CD., Duan YQ. Gao JM. Ruan Z. G. Screening for Anti-lipase Properties of 37 Traditional Chinese Medicinal Herbs. J Chinese Med Assoc. 2010; 73(6): 319–324. (2010). doi.org/10.1016/S1726-4901(10)70068-X.

37. Kendre N. Wakte P. LC-MS/MS-QTOF Screening and Identification of Phenolic compounds from Ethyl Acetate Fraction of Madhuca longifolia Leaves. Research Journal of Pharmacy and Technology. 2024; 17(4): 1435-0. doi: 10.52711/0974-360X.2024.00227

38. Sagar TV. Byndoor Y. Das A. Prescribing Pattern of Antihypertensive, Anti-Diabetic, Hypolipidaemic and Anti-Obesity Drugs in Diabetic Nephropathy. Research Journal of Pharmacy and Technology. 2022; 15(5): 2240-3. doi: 10.52711/0974-360X.2022.00372

39. Bustanji Y. et al. Inhibition of hormone sensitive lipase and pancreatic lipase by Rosmarinus officinalis extract and selected phenolic constituents. J Med Plants Res. 2010; 4(21): 2235–2242. doi.org/10.5897/JMPR10.399.

40. Chatsumpun N. Sritularak B. Likhitwitayawuid K. New biflavonoids with α-glucosidase and pancreatic lipase inhibitory activities from Boesenbergia rotunda. Molecules. 2017; 22(11): 1862-1875. doi.org/10.3390/molecules22111862.

41. Alias N. et al. Anti-obesity Potential of Selected Tropical Plants via Pancreatic Lipase Inhibition. Adv Obesity, Weight Manag Control. 2017; 6(4): 122–131. doi.org/10.15406/aowmc.2017.06.00163.

42. Pliego J. et al. Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate. Sensors (Switzerland). 2015; 15: 2798–2811. doi.org/10.3390/s150202798.

43. Shivsharan U. Kothari Y. Antimicrobial Activity and Isolation of Lawsone from Lawsonia inermis using Column Chromatography. Res. J. Pharmacognosy and Phytochem. 2020; 12(4): 219-223. doi: 10.5958/0975-4385.2020.00036.9

Received on 18.04.2024 Revised on 12.07.2024

Accepted on 16.09.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):857-862.

DOI: 10.52711/0974-360X.2025.00126

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