Lipase inhibitory activity of Carica papaya , Chrysophyllum cainito , Corcorus olitorius , Cympogon citrates and Syzygium cumini extracts

The lipase inhibitory action of Carica papaya, Chrysophyllum cainito, Corcorus olitorius, Cymbopogon citrates and Syzygium cumini were evaluated to explore for the presence of anti-obesity compounds and their potential weight-lowering activity. Enzyme inhibition results of the alcoholic extracts of the five plants showed that C. cainito has the highest percent inhibition at 74.91% while S. cumini, C. citratus, C. olitorius and C. papaya obtained less than 50% average inhibition. C. cainito was partitioned using hexane and ethyl acetate to further concentrate the bioactive compounds. The lipase inhibition assay of hexane and ethyl acetate extracts showed 92.11% inhibition and 21.9% inhibition, respectively. The greater activity in the former may imply that majority of potential antilipase constituents are found in the hexane portion.


Introduction
According to the 2014 data of the World Health Organization, more than 1.9 billion adults are overweight and 600 M of which are obese (WHO, 2016).Obesity has been considered as a global epidemic that needs immediate prevention and control (Brug and Crawford, 2009).It results from an imbalance between energy intake and energy expenditure (Abete et al., 2010).The imbalance leads to an abnormal weight gain and increases the risk for chronic illnesses including cardiovascular diseases (Lavie et al., 2016), cancer (Noureddin and Rinella, 2015) and diabetes (Genser et al., 2016).
Despite the growing awareness of the importance of active lifestyle and healthy diet, obesity is still widespread and is now a common health problem (Lim et al., 2012).Although physical activity and lifestyle change are crucial for reducing body weight and elevating average daily metabolic rate, these approaches do not offer long-term effects.It is ideal to combine these approaches with inhibitors of fat absorption (Garza et al., 2011).The most commonly marketed weight loss agent is Orlistat, an irreversible inhibitor of gastric and pancreatic lipase, which is the key enzyme for dietary triacylglycerol digestion (Kaila and Raman, 2008).By inhibiting these lipases, systemic fat absorption can be significantly reduced.
Pharmaceutical drugs such as Orlistat may moderately reduce fat absorption, but side effects like steatorrhea, oily stools, diarrhea, cholelithiasis, and incontinence render them as undesirable means of weight management (Flippatos et al., 2008).Several researchers have focused on screening plant extracts for potential lipase inhibition activities since medicinal plants have been used as dietary supplements for weight control (Lee et al., 2015;Seo et al., 2015;Belfeki et al., 2016;Chien et al., 2016;Glisan et al., 2017).
Since food is the major source of unwanted calories, this study focuses on plants used for human consumption.
Syzygium cumini (black plum), Chrysophyllum cainito (star apple), Cymbopogon citratus (lemon grass), Corchorus olitorius (bush okra) and Carica papaya (papaya) were evaluated for their potential to inhibit lipase activity.These plants were chosen due to their abundance, availability, and ease of access in the local setting.
There is limited literature on lipase inhibition activities of the aforementioned plants.However, plants from the same family and genus have been previously studied.Ado et al. (2013)  namely Chrysophyllum roxburghii showed significant dose-dependent activity against lipase (Prashith et al., 2013).Some members of Poaceae family, where C. citratus belongs, were also found to have lipase inhibitors (Sharma et al., 2005).Using 80% methanol solvent, Setaria italica extracts exhibited 80.3% lipase inhibition at 0.2 mg/mL solution concentration.Extracts of Eriochloa villosa showed 83% inhibition of lipase activity.Hibiscus sabdariffa (Malvaceae) also showed moderate anti-lipase activity at 60.9% (Ado et al., 2013).
Abroma augusta from Malvaceae family gave 88.6% inhibition at 100 mg/mL.The activity of plant was also concentration-dependent (Gupta et al., 2012)

Crude extraction and solvent partitioning
The leaves were washed and air-dried.Grinding and homogenization of samples were done using a kitchen blender.Methanol, hexane, and ethyl acetate solvents used were singly distilled prior to use.Ground samples were soaked in methanol.The methanol solvent was later filtered out and concentrated in vacuo using a rotary evaporator at 40°C.
The concentrate with the highest inhibition activity was partitioned using hexane and ethyl acetate.The methanol extract was re-dissolved in water for partitioning with hexane followed by ethyl acetate.Approximately 10 g of the methanol extract was dissolved in 100 mL distilled water and 600 mL hexane.The mixture was allowed to settle until a distinct separation between the organic and aqueous layer was obtained.
The organic layer was collected and concentrated in vacuo using a rotary evaporator at 40 o C. The process was repeated two more times.A similar process was done for the partitioning with ethyl acetate.

Lipase inhibition assay
Lipase inhibition assay was adapted from Kim et al. (2007).3-morpholinopropane-1-sulfonic acid (MOPS)ethylenediaminetetraacetic acid (EDTA), porcine pancreatic lipase solution, Tris-HCl buffer, p-nitrophenyl butyrate (p-NPB) substrate solution were freshly prepared.Lipase from porcine pancreas (Sigma L3126) was used.Orlistat served as the positive control.Samples were placed on a 96 well-quartz microplate.The total reaction volume inside one well is 200 mL.Fifteen mL each of lipase and p-NPB were placed in each well.For the uninhibited reaction, 170 mL of buffer was added.For the positive control, 150 mL of the buffer was added with 20 mL of Orlistat prepared from 1 mg of Orlistat in 1 mL of DMSO.For the sample reaction, 150 mL of buffer was used with 20 mL of plant extract prepared from 1 mg of extract dissolved in 3% DMSO solution.
The reaction was monitored using a Multiskan TM GO UV/Vis Microplate Spectrophotometer.The samples were then incubated at 37°C for 10 min to allow interaction of samples with the enzyme.After incubation, the p-NPB substrate was added to the samples.Solutions were shaken for 15 seconds and scanned at 405 nm for 40 times at 30-second intervals.For each extract, 2 trials were performed with 2 replicates per trial.

Percent inhibition was calculated using the equation % Inhibition =
where A sample is the activity of the solution with inhibitor and A uninhibited is the activity of the solution without the inhibitor.

Results and discussion
The methanol extracts of the samples were obtained and the percent yields are shown in Figure 1 with the percent inhibitory activities of the samples against pancreatic lipase.C. cainito had the highest activity at 74.91% while C. olitorius had the lowest anti-lipase activity 37.33%.The plants that were frequently studied for their possible anti-obesity effects were C. olitorius, C. papaya and C. cainito; however, the first two plants exhibited moderate activity (<50% inhibition).It is possible that the anti-obesity mechanism of C. olitorius and C. papaya is different from the inhibition of the pancreatic lipase enzyme.C. olitorius contains the flavonol glycoside Q3MG which is responsible for the antioxidant activities that subdue increase in body FULL PAPER 100 - adiposity, reduces plasma and hepatic lipid levels and triggers lipid catabolism in the liver (Wang et al., 2011).These effects are attained via alteration of the gene expression of specific enzymes that are linked to lipid and glucose metabolism (Honda et al., 2009).Suppression of NADPH oxidase expression and enhancement of b-oxidation are claimed to be one of the targets of polyphenolic compounds with regards to obesity control (Dávalos et al., 2009).
C. papaya has constituents that follow the same antiobesity mechanism as C. olitorius extracts.Studies showed regulation in total cholesterol, triglycerides, phospholipids and free fatty acid in manifested in highdiet fed mice (Manjula et al. 2014).Athesh et al. (2012) suggested that the substantial reduction of total cholesterol is by inhibition of 3-hydroxy-3methylglutaryl coenzyme A reductase which limits cholesterol biosynthesis.In addition, increase in endothelium-bound lipoprotein lipase may have caused the decrease in total triglyceride count.Heightened activity of lipoprotein lipase can then lead to activation of b-oxidation in the liver, along with the decline in gene expression for fatty acid synthesis (Athesh et al., 2012).
C. cainito methanol extract was further partitioned using hexane and ethyl acetate and the inhibitory activity are shown in Figure 2. The hexane extract showed high inhibitory activity at 92.11% while the ethyl acetate extract is lower (21.93%)than the methanol extract (74.91%).Results suggest that the pancreatic lipase inhibitory agents were concentrated in the hexane extract.Phytochemical screening of C. cainito leaves showed the presence of alkaloids, flavonoids and sterols (N'guessan et al., 2009).The triterpenoids ursolic acid, b-sitosterol and lupeol were isolated from the leaves, along with the phenolic compound gallic acid (Shailajan and Gurjar, 2014).Flavonoids and alkaloids have been previously shown to inhibit lipase activity (Ruiz et al., 2006).Cyclic di-terpenoids from Calotropis procera were found to competitively inhibit pancreatic lipase (Patil et al., 2015).It is possible that the C. cainito extracts contain similar secondary metabolites that affect the pancreatic lipase activity.
Future in vivo studies will be carried out to further explore the potential of C. cainito as dietary supplements or consumed for weight management.Current efforts include the isolation and identification of compounds responsible for the lipase inhibitory activity.Athesh, K., Karthiga, D. and Brindha, P. (2012)

FULLPAPERFigure 1 .
Figure 1.Percent yield of methanol extracts and their corresponding lipase inhibitory activity Figure 2. Lipase inhibitory activity of methanol, hexane and ethyl acetate extracts of C. cainito pointed out that the methanol leaf extract of Syzygium malaccense can inhibit up to 99% of pancreatic lipase activity, while Psidium guajava from the same family Myrtaceae can inhibit lipase up to 85.6%.Another species of the Chrysophyllum genus eISSN: 2550-2166 © 2017 The Authors.Published by Rynnye Lyan Resources papaya leaves were collected from the University of the Philippines Diliman campus and submitted to the Jose Vera Memorial Herbarium, Institute of Biology, University of the Philippines-Diliman for authentication and verification.C. olitorius leaves were purchased at Regalado Wet Market and also submitted to the herbarium for authentication and verification.The voucher specimen number for S. cumini is 9469, for C. cainito is 019337 , for C. citratus 14618, for C. papaya 21403 and for C. olitorius 9398.
. Antiobesity Effect of Aqueous Fruit Extract of Carica papaya L. In Rats Fed on High-Fat Cafeteria Diet.