Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine / 2012 / Article
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Integrating Complementary and Alternative Medicine with Primary Health Care

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Research Article | Open Access

Volume 2012 |Article ID 878365 | 8 pages | https://doi.org/10.1155/2012/878365

Inhibitory Activities of Cudrania tricuspidata Leaves on Pancreatic Lipase In Vitro and Lipolysis In Vivo

Academic Editor: R. Govindarajan
Received14 Aug 2012
Revised18 Oct 2012
Accepted11 Nov 2012
Published10 Dec 2012


To identify effective herb to treat obesity, we screened 115 herbal extracts for inhibition of porcine pancreatic lipase (triacylg-ycerol acylhydrolase, EC activity in vitro. Of the extracts tested, Cudrania tricuspidata leaves exhibited the most pronounced inhibitory effect on lipase activity with an value of 9.91 μg/mL. Antilipid absorption effects of C. tricuspidata leaves were examined in rats after oral administration of lipid emulsions containing 50 or 250 mg  C. tricuspidata/kg body weight. Plasma triacylglycerol levels 2 h after the oral administration of emulsions containing C. tricuspidata were significantly reduced compared to the untreated group (). These results suggest that C. tricuspidata leaves may be useful for the treatment of obesity.

1. Introduction

Obesity is a significant risk factor for increased morbidity and mortality from cardiovascular disease and diabetes; however, it is also associated with many other medical conditions including cancer, liver and kidney diseases, sleep apnea, and depression [1]. The recent National Health and Nutrition Examination Survey showed that 68.0% of those studied were considered overweight (basal metabolic rate (BMI) ≥ 25) and 33.8% were obese (BMI ≥ 30) [2]. The inhibition of dietary fat absorption is a logical target for managing obesity, and pancreatic lipase is a key enzyme involved in triglyceride absorption in the small intestine. It is secreted from the pancreas and hydrolyzes triglycerides into glycerol and free fatty acids. Thus, inhibitors of digestive lipases are suggested to function as antiobesity agents [3]. Orlistat, which can be found in global markets, inhibits the action of gastrointestinal lipase and thus reduces absorption of dietary fat. However, it has serious side effects, such as steatorrhea, stomach pain, irregular menstrual periods, and headaches [4]. Recently, studies have searched for new lipase inhibitors in natural resources with minimal adverse effects. In a series of investigations to evaluate potential lipase inhibitors derived from plants, researchers showed that certain plant extracts significantly inhibited porcine pancreatic lipase in vitro [5, 6]. In this study, as a preliminary evaluation of natural antiobesity products, we tested 115 herbal extracts for inhibition of pancreatic lipase activity in vitro and verified the suppression of lipid absorption by C. tricuspidata leaves in vivo. The fruits of C. tricuspidata suppress development of atopic dermatitis in animal model and the roots of it exhibit immunomodulatory and anti-oxidant activities in vitro [7, 8]. These results show that C. tricuspidata leaves extracts have on lipase and dietary fat absorptionactivities and may be useful in the treatment of obesity and metabolic disease.

2. Material and Methods

2.1. Plant Materials and Chemicals

Herbs were collected from Republic of Korea from September 2005 to July 2009 and identified by Professor Kim, Division of Life Science, Gachon University, Republic of Korea. Samples were deposited at the Herbarium of Diabetic Complication Research Team, Korea Institute of Oriental Medicine. Porcine pancreatic lipase (type II), orlistat, and p-nitrophenyl butyrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents were of biochemical grade.

2.2. Animals

Male Wistar rats (6 weeks of age) were purchased from Koatech (Kyungkido, Korea) and housed for 1 week in a 12-h/12-h light/dark cycle in a temperature- and humidity-controlled room. The animals were given free access to food and water. After adaptation to these conditions for 1 week, healthy animals were used in the present study. The Animal Studies Committee of Korea Institute of Orient Medicine approved the experimental protocol.

2.3. Preparation of Herbal Extracts

Dried and ground herbs (200 g) were extracted with 1 L of 80% EtOH 3 times by maceration. The extracts were concentrated and dried in vacuo at 40°C. Concentrated extracts were stored at −20°C for further studies. Extracts were dissolved in dimethyl sulfoxide at concentrations that in the total volume (3%) did not affect enzyme activity.

2.4. Measurement of Porcine Pancreatic Lipase Inhibitory Activity

The ability of the herbs to inhibit pancreatic lipase was measured using the method previously reported by Kim et al. [9, 10]. Briefly, an enzyme buffer was prepared by the addition of 6 μL porcine pancreatic lipase solution (Sigma-Aldrich) in buffer containing 10 mM MOPS (morpholinepropanesulphonic acid) and 1 mM EDTA, pH 6.8, to 169 μL Tris buffer (100 mM Tris-HC1 and 5 mM CaCl2, pH 7.0). Then, 20 μL of either the herbal extracts at the test concentration (0, 0.313, 0.625, 1.25, 2.5, 5, 7.5, 10, 50, and 100 μg/mL) or orlistat (Roche, Basel, Switzerland) were mixed with 175 μL enzyme buffer and incubated for 15 min at 37°C with 5 μL substrate solution (10 mM p-NPB (p-nitrophenylbutyrate) in dimethyl formamide); the enzymatic reactions were allowed to proceed for 15 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 (BIO-TEK, Synergy HT, Winooski, VT, USA). Inhibition of lipase activity was expressed as the percentage decrease in OD when porcine pancreatic lipase was incubated with the test materials. Lipase inhibition (%) was calculated according the following formula: where is the activity without inhibitor, is the negative control without inhibitor, is the activity with inhibitor, and is the negative control with inhibitor. The results were expressed as an average .

2.5. Estimation of Plasma Triacylglycerol after Oral Administration of Lipid Emulsion in Rats

Plasma triacylglycerol levels were estimated using the method previously reported by Kim et al. [11]. Rats (7 weeks of age, body weight  g) that had fasted overnight were orally administered 3 mL lipid emulsion consisting of corn oil (6 mL), cholic acid (80 mg), cholesteryloleate (2 g), and saline (6 mL) with or without C. tricuspidata leaves (at doses of 50 or 250 mg C. tricuspidata leaves/kg body weight). Blood was taken from the tail vein at 0, 1, 2, 3, and 4 h after oral administration of the lipid emulsion and centrifuged at 5500 ×g for 5 min to obtain the plasma. Triacylglycerol levels were determined using the Cleantech TS-s kit (ASANPHARM, Seoul, Korea).

2.6. Statistical Analysis

All experiments were repeated three times, and representative data are shown. Data are expressed as the mean ± S.D. Differences between groups were analyzed using a one-way ANOVA followed by the Tukey multiple comparison test (PRISM software, Graph Pad, CA, USA). Values of were considered statistically significant.

3. Results and Discussion

3.1. Pancreatic Lipase Activity of Herbal Extracts

Currently, obesity is considered a global epidemic, and many medications have been studied and developed to treat this condition. However, there is presently only one drug—orlistat—globally approved for long-term treatment of overweight patients after sibutramine was withdrawn in January 2010 from the European market [12, 13]. Although this compound strongly inhibits the activity of pancreatic lipase, which is an important enzyme associated with fat digestion, orlistat may cause serious adverse effects on the gastrointestinal, nervous, endocrine, and renal systems and interferes with the absorption and effectiveness of many drugs and vitamins [4, 14]. Therefore, researching a safe and effective natural inhibitor of pancreatic lipase has been a major target for the development of new drugs to treat obesity [15]. Among them, extracts isolated from natural sources such as Sorbus commixta, Morus bombycis, Panax ginseng, and Ginkgo biloba have been reported as potential agents in pancreatic lipase inhibition action [1619]. Our previous studies have also identified some natural products as new pancreatic lipase inhibitors [11, 18, 19]. In this study, 115 herbal extracts were prepared from selected parts of plants and tested at various concentrations as inhibitors of pancreatic lipase. The lipase inhibitory effects of the extracts are indicated by percentage (%) and IC50 values (Table 1). Eighteen extracts had IC50 values less than 50 μg/mL, and of these extracts, three samples (i.e., the whole Solidago serotina plant, the branches and leaves of Acer mono, and the leaves of C. tricuspidata) had IC50 values less than 10 μg/mL. Notably, C. tricuspidata leaves exhibited an IC50 value of 9.91 μg/mL (Figure 1).

Scientific nameFamilyPart usedConc. (μg/mL)Inhibition (%)aIC50 (μg/mL)

Solidago  serotina CompositaeWhole plant5 5.16
Acer  mono AceraceaeBranch, leaf7.5 7.7
Cudrania  tricuspidata MoraceaeLeaf7.5 9.91
Kalopanax  pictus AraliaceaeBark50 10.51
Cudrania  tricuspidata MoraceaeBranch, stem10 13.8
Oenothera  odorata OnagraceaeWhole plant50 23.34
Platycarya  strobilacea JuglandaceaeBranch, stem50 25.51
Actinidia  arguta ActinidiaceaeFruit50 26.7
Tilia  amurensis TiliaceaeBranch, leaf50 28.5
Actinidia  arguta ActinidiaceaeStem50 28.51
Euscaphis  japonica StaphyleaceaeBranch30 28.62
Actinidia  arguta ActinidiaceaeRoot50 31.34
Carpinus  cordata BetulaceaeBranch, stem50 31.39
Rhus  sylvestris AnacardiaceaeBranch, leaf50 32.14
Celtis  sinensis UlmaceaeBranch, stem50 35.89
Prunus  serrulata RosaceaeBranch, leaf50 42.55
Potentilla  fragarioides RosaceaeWhole plant50 42.58
Tilia  mandshurica TiliaceaeFlower, leaf50 48.21
Actinidia  arguta ActinidiaceaeStem, leaf, fruit50 54.09
Hypericum  ascyron HypericaceaeWhole plant50 56.12
Rhus  chinensis AnacardiaceaeBranch, leaf50 56.9
Picrasma  quassioides SimaroubaceaeBranch, stem50 60.47
Prunus  persica RosaceaeBranch, leaf50 62.12
Actinidia  arguta ActinidiaceaeRoot50 69.17
Spiraea  pubescens RosaceaeBranch, leaf, flower50 74.62
Tilia  mandshurica TiliaceaeBranch, stem50 79.67
Acer  ginnala AceraceaeBranch, leaf50 82.29
Elsholtzia  splendens LabiataeRoot50 83.98
Staphylea  bumalda StaphyleaceaeBranch, leaf50 84.28
Pinus  densiflora PinaceaeStem90 87.58
Machilus  thunbergii LauraceaeLeaf, branch50 90.9
Deutzia  glabrata SaxifragaceaeBranch, leaf, flower50 91.09
Indigofera  kirilowii LeguminosaeBranch, leaf, flower50 94.98
Opuntia  ficus-indica OpuntiacaeStem100 >100
Hibiscus  syriacus MalvaceaeRoot100 >100
Actinidia  arguta ActinidiaceaeBark100 >100
Euonymus  oxyphyllus CelastraceaeBranch100 >100
Eucommia  ulmoides EucommiaceaeBranch, leaf100 >100
Asarum  sieboldii AristolochiacRoot100 >100
Bupleurum  longeradiatum UmbelliferaeWhole plant100 >100
Plantago  asiatica PlantaginaceaRoot100 >100
Alisma  plantago-aquatica AlismataceaeRoot100 >100
Duchesnea  chrysantha RosaceaeWhole plant100 >100
Cuscuta  japonica ConvolvulaceaeWhole plant100 >100
Clematis  apiifolia RanunculaceaeStem, leaf, flower100 >100
Prunus  serrulata RosaceaeBranch100 >100
Colocasia  antiquorum AraceaeAerial part100 >100
Lespedeza  cuneata LeguminosaeAerial part100 >100
Lespedeza  cuneata LeguminosaeRoot100 >100
Mallotus  japonicas EuphorbiaceaeAerial part100 >100
Alisma  canaliculatum AlismataceaeAerial part100 >100
Alisma  canaliculatum AlismataceaeRoot100 >100
Magnolia  denudata MagnoliaceaeFlowers100 >100
Scopolia  japonica SolanaceaeStem, leaf100 >100
Scopolia  japonica SolanaceaeRoot100 >100
Chloranthus  japonicus ChloranthaceaeWhole plant100 >100
Barbarea  orthoceras CruciferaeWhole plant100 >100
Caulophyllum  robustum BerberidaceaeStem, leaf100 >100
Caulophyllum  robustum BerberidaceaeRoot100 >100
Carduus  crispus CompositaeStem, leaf100 >100
Carduus  crispus CompositaeFlower100 >100
Styrax  japonica StyracaceaeFlower100 >100
Cornus  controversa CornaceaeBranch, leaf100 >100
Cornus  controversa CornaceaeFlower100 >100
Magnolia  sieboldii MagnoliaceaeBranch, leaf100 >100
Magnolia  sieboldii MagnoliaceaeFlower100 >100
Prunus  persica RosaceaeFruit100 >100
Rhamnus  yoshinoi RhamnaceaeBranch, leaf100 >100
Erigeron  annuus CompositaeWhole plant100 >100
Styrax  japonica StyracaceaeBranch, leaf100 >100
Quercus  aliena FagaceaeBranch, leaf100 >100
Callicarpa  japonica VerbenaceaeBranch, leaf100 >100
Ligustrum  obtusifolium OleaceaeBranch, leaf100 >100
Lindera  obtusiloba LauraceaeBranch, leaf100 >100
Lespedeza  bicolor LeguminosaeBranch, leaf100 >100
Carpinus  laxiflora BetulaceaeBranch, leaf100 >100
Machilus  thunbergii LauraceaeBark100 >100
Hedera  rhombea AraliaceaeWhole plant100 >100
Arenaria  serpyllifolia CaryophyllaceaeWhole plant100 >100
Paulownia  coreana PaulowniaceaeFlower100 >100
Thlaspi  arvense BrassicaceaeWhole plant100 >100
Vicia  villosa LeguminosaeWhole plant100 >100
Descurainia  pinnata BrassicaceaeWhole plant100 >100
Ribes  fasciculatum SaxifragaceaeBranch, leaf, fruit100 >100
Corydalis  speciosa FumariaceaeWhole plant100 >100
Clematis  fusca RanunculaceaeWhole plant100 >100
Deutzia  parviflora SaxifragaceaeBranch, leaf, stem, flower100 >100
Rosa  multiflora RosaceaeBranch, leaf, stem, flower100 >100
Parthenocissus  tricuspidata VitaceaeLeaf, stem100 >100
Chelidonium  majus PapaveraceaeWhole plant100 >100
Platycarya  stobilacea JuglandaceaeLeaf100 >100
Platycarya  stobilacea JuglandaceaeFlower100 >100
Carpinus  cordata BetulaceaeLeaf100 >100
Celtis  sinensis UlmaceaeLeaf100 >100
Orixa  japonica RutaceaeLeaf100 >100
Orixa  japonica RutaceaeBranch, stem100 >100
Orixa  japonica RutaceaeFruit100 >100
Picrasma  quassioides SimaroubaceaeLeaf100 >100
Picrasma  quassioides SimaroubaceaeFruit100 >100
Tilia  mandshurica TiliaceaeLeaf100 >100
Aralia  cordata AraliaceaeWhole plant100 >100
Viburnum  sargentii CaprifoliaceaeBranch, leaf100 >100
Polygonatum  odoratum LiliaceaeRoot   100 >100
Astragalus  membranaceus LeguminosaeRoot 100 >100
Pleuropterus  multiflorus PolygonaceaeRoot 100 >100
Torilis  japonica UmbelliferaeFruit 100 >100
Phaseolus  angularis LeguminosaeFruit 100 >100
Phaseolus  radiates LeguminosaeFruit 100 >100
Artemisia  scoparia CompositaeAerial part 100 >100
Solanum  tuberosum SolanaceaeTuber100 >100
Brassica  juncea CruciferaeLeaf 100 >100
Arctium  lappa CompositaeRoot100 >100
Cucumis  sativus CucurbitaceaeFruit 100 >100
Diospyros  kaki EbenaceaeFruit   100 >100
Artemisia  princeps CompositaeAerial part 100 >100
Orlistat (positive control)0.005 0.036 (0.073 μM)

aResults are the mean ± SD ( ).
3.2. Inhibitory Effect of C. tricuspidata on Lipolysis In Vivo

Next, we focused on C. tricuspidata on lipolysis in vivo. C. tricuspidata has been used as an important folk medicine for the treatment of cancer in Korea and has also been used as a traditional medicine for the treatment of hypertension, neuritis, and inflammation in Asia [2022]. To evaluate the antilipolytic effects of C. tricuspidata leaves in vivo, we analyzed plasma triacylglycerol levels after oral administration of lipid emulsions with or without the C. tricuspidata leaves to rats. Figure 2 shows plasma triacylglycerol levels after oral administration of lipid emulsion with or without C. tricuspidata as a function of time. After oral administration, low concentrations of C. tricuspidata (50 mg/kg body weight) reduced plasma triacylglycerol levels and high concentrations of C. tricuspidata (250 mg/kg body weight) delayed lipid absorption significantly; however, these effects were weaker than that of the positive control, orlistat.

C. tricuspidata is a rich source of xanthones and flavonoids, including cudraflavone C [23]. A recent study reported that cudraflavone C from Artocarpus nitidus inhibited pancreatic lipase activity (IC50 = μM) [24]. Thus, cudraflavone C may be a potential as one of active compounds for preventing and treating obesity.

4. Conclusion

In this paper, we screened 115 herbal extracts for inhibition of porcine pancreatic lipase to identify effective herb to treat obesity. C. tricuspidata leaves show the most pronounced effect on pancreatic lipase activity and are able to suppress dietary fat absorption in vivo. Up until now, C. tricuspidata leaves extracts have not been reported on lipase and dietary fat absorptionactivities. Thus, it is worthwhile to further investigate these extracts for their potential pharmacological effect in antiobesity and attempt should be made to characterize phytoactive compounds to be used as safer therapeutic agents in future.

Authors’ Contribution

Y. S. Kim and Y. Lee contributed equally to this work.

Conflict of Interests

The authors declare no conflict of interests.


This research was supported by Grants (K11040 and K12040) from the Korea Institute of Oriental Medicine (KIOM).


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Copyright © 2012 Young Sook Kim et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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