Screening for anti-pancreatic lipase properties of 28 traditional Thai medicinal herbs

Ananya Dechakhamphu，Nattapong Wongchum1Thai Traditional Medicine Program，Faculty of Thai Traditional and Alternative Medicine，Ubon Ratchathani Rajabhat University，Ubonratchathani 34000，Thailand

2Biology Program，Faculty of Science，Ubon Ratchathani Rajabhat University，Ubonratchathani 34000，Thailand

Screening for anti-pancreatic lipase properties of 28 traditional Thai medicinal herbs

Ananya Dechakhamphu1*，Nattapong Wongchum2
1Thai Traditional Medicine Program，Faculty of Thai Traditional and Alternative Medicine，Ubon Ratchathani Rajabhat University，Ubonratchathani 34000，Thailand

2Biology Program，Faculty of Science，Ubon Ratchathani Rajabhat University，Ubonratchathani 34000，Thailand

ARTICLE INFO

Article history:

in revised form 20 Jul 2015

Accepted 15 Aug 2015

Available online 3 Oct 2015

Obesity

Medicinal plant

Herb

Anti-pancreatic lipase

Phenolic

Alkaloid

Flavonoid

Objective:To screen the effect of 28 medicinal plants on inhibition of pancreatic lipase and evaluate the phytochemical contents of extracts.

Methods:The ethanolic extracts of 28 traditional Thai herbal medicines were assayed for their in vitro activities against porcine pancreatic lipase using p-nitrophenyl butyrate as a substrate.Quantitative estimation of flavonoids，phenolics，and alkaloids was done. Results:Extracts from four herbs，Memecylon edule Roxb.，Garcinia vilersiana Pierre，Cryptolepis elegans Wall.and Phyllanthus chamaepeuce Ridl.，at a concentration of 100μg/mL，strongly inhibited porcine pancreatic lipase by 90.97%，92.04%，94.64%and 95.38%，respectively.There was a significant positive correlation between phenolic content and inhibition activity.Inhibition activity was significantly correlated with flavonoid and with alkaloid contents.

Conclusions:From this result，it could be concluded that herbs represent a rich of antipancreatic lipase compounds，in particular，Cryptolepis elegans Wall.and Phyllanthus chamaepeuce Ridl.It is suggested that the phytochemical compounds from these plants may be applied for the prevention and treatment of obesity or hyperlipidemia.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2015.09.012

1.Introduction

Obesity is becoming a worldwide epidemic，resulting in a major risk factor for coronary heart diseases including diabetes mellitus，metabolic syndrome，stroke，and some cancers［1］. Globally，around 39%of adults aged 18 and over were overweight in 2014 and 13%of them were clinically obese［2］. Therefore，prevention and treatment of obesity become an important factor for a healthy condition.The reduction of nutrient digestion and absorption by developing of enzyme inhibitors without altering major mechanism in gastrointestinal system became the most important strategies in the treatment of obesity［3，4］.The major source of unwanted calories is dietary lipids，therefore，lipid metabolism play a major role in maintaining energy homeostasis［5］.The identification andcharacterizationofseveralenzymesinvolvedinlipid metabolism have yielded a rich pool of potential targets for drugs to treat obesity and other metabolic disorders［6］. Pancreatic lipase is the key enzyme for dietary fat digestion and absorption.Therefore，inhibition of this enzyme would be in effect to reduce lipid absorption from intestine and lead to a consequence suppress of weight gain.Orlistat，a specific drug forinhibitingpancreaticlipasethatreducesdietaryfat absorption by 30%，has been approved for clinical use［4，7，8］. However，Orlistat can result in adverse side effects，such as fecal incontinence，flatulence，and steatorrhea［9，10］.Therefore，the investigation to find new safety medication for anti-obesity is still needed.The significant progress of the development of anti-obesity from medicinal plants has provided potential therapeutic targets for obesity［11，12］.It has been recently reported that natural compounds from plants and other organisms have been approved as anti-pancreatic lipase activities.For example，ethanolicextractfromTerminaliapaniculatabark［13］，polyphenols from Oolong tea［14］，Abroma augusta extract［15］，pomegranateleavesethanolextract［16］，andother components from other kinds of herbs.However，it remains necessary to search for more effective lipase inhibitors from traditional herbs.In this study，we investigated the ethanolicextracts of 28 traditional Thai herbal medicines for their in vitro activities against porcine pancreatic lipase using p-nitrophenyl butyrate（p-NPB）as a substrate.

2.Materials and methods

2.1.Materials

Porcine pancreatic lipase，p-NPB，morpholinepropanesulphonic acid，quercetin，colchicine and gallic acid were purchased from Sigma Aldrich.The fresh leaves of 28 plants were collectedfromPlantGeneticConservationForest，Ubon Ratchathani Rajabhat University，Ubonratchathani Province，Thailand.All plant species were identified and authenticated by Mr.Prakorb Boonma，Senior Plant Taxonomist，Ubon Ratchathani Rajabhat University，Thailand.

2.2.Preparation of plant extracts

The leaves were dried in hot air oven at 50°C for 48 h and grounded into fine powder.A total of 50 g powder was extracted in 95%ethanol and concentrated at 55°C in a rotary vacuum evaporator（Heidorf，Germany）.The obtained extracts were stored at-20°C until use.

2.3.Porcine pancreatic lipase inhibition assay

Lipase activity was measured using p-NPB as a substrate. The method was modified from the previously described by Kim et al.［17］.Briefly，an enzyme buffer was prepared by the addition 30μL of solution of porcine pancreatic lipase（2.5 mg/mL in 10 mmol/L morpholinepropanesulphonic acid and 1 mmol/L ethylenediamine tetraacetic acid，pH 6.8）to 850μL of Tris buffer（100 mmol/L Tris-HCl and 5 mmol/L CaCl2，pH 7.0）.Then，either 100μL of the plant extracts（100μg/mL）or Orlistat was added and incubated for 15 min at 37°C.Ten microliter of substrate（10 mmol/L p-NPB in dimethyl formamide）was then added and incubated for 30 min at 37°C.Lipase activity was determined by measuring the hydrolysis of p-NPB to p-nitrophenol at 405 nm using an ELISA reader（Biochrome，England）.The inhibitory activity（I）was calculated according to the following formula:

whereAistheactivityoftheenzymewithoutinhibitor，andaisthe negative control without inhibitor；B is the activity of the enzyme with inhibitor，and b is the negative control with inhibitor.

2.4.The half maximal inhibitory concentration（IC50）determination

The IC50value of extracts was determined at a concentration of 500.0，250.0，125.0，100.0，25.0，12.5 and 5.0μg/mL.Orlistat was used as a positive control.IC50value was calculated by the following formula: formula:where LowInh%/HighInh%signify%inhibition directly below/above 50%inhibition，and LowConc/HighConcare the corresponding concentrations of extract.

2.5.Determination of total phenolic content

According to a previously described protocol［18］，Folin-Ciocalteu reagent was used to determine the total phenolic content of extracts.Absorbance was measured at 725 nm.All tests were performed 6 times.The phenolic content was calculated based on a gallic acid standard curve.

2.6.Determination of total flavonoid content

Total flavonoid content was determined according to a previously discussed method［18］using quercetin as a standard.The absorbance was measured at 510 nm.The flavonoid content was calculated based on a quercetin standard curve.

2.7.Quantification of alkaloid content

Quantification of alkaloid content for extracts was carried out using a method described earlier［19］.The absorbance was taken at 500 nm and all tests were performed 6 times.The alkaloid content was evaluated based on the colchicine standard curve.

2.8.Statistical analysis

Statistical analysis of the data was performed using the SPSS 16.0 program.The comparison between Orlistat control and extract group was conducted using the Mann-Whitney U test，and the correlations between parameters were determined using the Spearman's rank test.

3.Results

A total of 28 extracts were prepared from leaf part of the traditional Thai medicinal herbal medicines and were tested at a concentration of 100μg/mL for porcine pancreatic lipase inhibition（Table 1）.

Table 1 Lipase inhibitory effects of 28 selected traditional Thai medicinal herbs.%.

Table 1（continued）

Among28 herbs，9 plantswere found to have stronginhibitory activity of＞50%against porcine pancreatic lipase:59.15%with Diospyros filipendula，67.47%with Cratoxylum formosum，70.00%with Congea siamensis，80.11%with Prismatomeris sessiliflora，87.55%with Xantolis cambodiana，90.97%with Memecylon edule，92.04%with G.vilersiana，94.64%with C. elegans，and 95.38%with P.chamaepeuce.The extracts of GomphiaserrataandAzadirachtaindicashowed＞30%inhibition of pancreatic lipase.There were several herbs showed slightly effect on inhibition against pancreatic lipase.Besides，some of herbs increased in the activity of pancreatic lipase，such as Syzygium gratum，which promoted the activity of enzyme by 34.93%.

The different concentration of crude extracts of G.vilersiana，C.elegans，and P.chamaepeuce were measured for IC50at a concentrationof500.0，250.0，125.0，100.0，25.0，12.5and5.0μg/ mL.Theextractsof G.vilersiana，C.elegans andP.chamaepeuce had IC50values of 0.11，0.30 and 0.45μg/mL，respectively. Whereas，Orlistat had IC50value of 0.08μg/mL（Figure 1）.

The correlations between phytochemical content measured in extracts and inhibition activity were shown in Figure 2.There was a significant positive correlation between phenolic content and inhibition activity.Inhibition activity was significantly correlated with flavonoid and alkaloid contents.

4.Discussion

Pancreatic lipase is a key enzyme for lipid absorption by hydrolysis of total dietary fats.Therefore，inhibition of pancreatic lipase is suggested to be an effective therapy in the regulation of obesity.Although Orlistat has anti-obesity effects by inhibiting pancreatic lipase activity，however，it can cause adverse side effects，such as fecal incontinence，flatulence，and steatorrhea［9，10］.Therefore，the investigation of new agent for pancreatic lipase inhibitor is still needed.Our finding is the first time to show that crude extracts of G.vilersiana，C.elegans and P.chamaepeuce exhibited strong anti-lipase activity.This suggests that these herbs seem to be the potential candidates as the inhibitor of pancreatic lipase.However，further in vivo studies on animal model must be conducted in order to confirm this hypothesis.

Our results showing a significant positive correlation between phenolic，flavonoid，alkaloid contents and inhibition activity，which provide strong support that these phytochemical compounds are key agents for pancreatic lipase inhibition.This assumption is further supported by the previous results showing that phenolic compounds exhibit the ability to inhibit pancreaticlipase activity［14，20-23］.Published research also reported that flavonoids and alkaloid be able to inhibit pancreatic lipase［24，25］.The study in vivo model indicated that polyphenols and flavonoid glycoside derived from Salix matsudana leaf showed decreased in body weight gain in Wistar rats［21］.According to these reports，we hypothesized that these three compounds may be the main contributors to the inhibition of pancreatic lipase.Experimental proof for this assumption is now required.

Although high activities on pancreatic lipase inhibition were detected in crude extracts of G.vilersiana，C.elegans and P.chamaepeuce，the further investigation for both in vitro and in vivo should be performed to elucidate the bioactive compounds，to clarify the molecular mechanism and to verify the main effective phytochemicals in these three candidates which are responsible for the inhibition of pancreatic lipase activity. For example，caffeine，chlorogenic acid，feruloylquinic acids derived from Coffea canephora showed to inhibit pancreatic lipase in vitro and decrease body weight gain of mice by 157%［26］.It has been reported that crocetin derived from Gardenia jasminoides showed inhibitory activity of pancreatic lipase in vitro and 25%decreased in body weight gain of Triton WR-1339-induced hyperlipidemia mice［27，28］.

We concluded that phenolic，flavonoid and alkaloid compounds in G.vilersiana，C.elegans and P.chamaepeuce are key agents for pancreatic lipase inhibition in vitro.These three plants should be explored as dietary supplements or nutraceutical foods with anti-obesity properties.To the best of our knowledge，the purification of active compounds of certain pancreatic lipase inhibitor is under investigation.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

This research was supported by grants funded by Ubon Ratchathani Rajabhat University through Plant Genetic Conservation Project Under The Royal Initiative of Princess Maha Chakri Sirindhorn Program （Grant No.UBRU_RSPG_2556）. The authors thank the Faculty of Thai Traditional and Alternative Medicine，Ubon Ratchathani Rajabhat University for providing instruments.We thank Dr.Chayada Danuwong，English Program，Ubon Ratchathani Rajabhat University，for proofreading the manuscript.

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7 Jul 2015

Ananya Dechakhamphu，Thai Traditional Medicine Program，Faculty of Thai Traditional and Alternative Medicine，Ubon Ratchathani Rajabhat University，Ubonratchathani 34000，Thailand.

Tel:+66 942818585

E-mail:ananya.d@ubru.ac.th

Peer review under responsibility of Hainan Medical University.

Foundation Project:Supported by Ubon Ratchathani Rajabhat University through Plant Genetic Conservation Project Under The Royal Initiative of Princess Maha Chakri Sirindhorn Program（Grant No.UBRU_RSPG_2556）.