Evidence-Based Complementary and Alternative Medicine

Volume 2012 (2012), Article ID 878365, 8 pages

doi:10.1155/2012/878365

Research Article

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

Young Sook Kim,¹ Youngseop Lee,¹ Junghyun Kim,¹ Eunjin Sohn,¹ Chan Sik Kim,¹ Yun Mi Lee,¹ Kyuhyung Jo,¹ Sodam Shin,¹ Yoojin Song,¹ Joo Hwan Kim,² and Jin Sook Kim¹

¹Korean Medicine-Based Herbal Drug Research Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM), Daejeon 305-811, Republic of Korea

²Department of Life Science, Gachon University, Seongnam, Kyonggi-do 461-701, Republic of Korea

Received 14 August 2012; Revised 18 October 2012; Accepted 11 November 2012

Academic Editor: R. Govindarajan

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.

Abstract.

To identify effective herb to treat obesity, we screened 115 herbal extracts for inhibition of porcine pancreatic lipase (triacylg-ycerol acylhydrolase, EC 3.1.1.3) 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 [16–19]. 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).

Table 1: Lipase inhibitory activities of extracts from herbs.

Scientific name
Family
Part used
Conc. (μg/mL)
Inhibition (%)a
IC₅₀ (μg/mL)

2.5

Solidago  serotina
Compositae
Whole plant
5

5.16

7.5

5

Acer  mono
Aceraceae
Branch, leaf
7.5

7.7

10

5

Cudrania  tricuspidata
Moraceae
Leaf
7.5

9.91

10

10

Kalopanax  pictus
Araliaceae
Bark
50

10.51

100

5

Cudrania  tricuspidata
Moraceae
Branch, stem
10

13.8

50

10

Oenothera  odorata
Onagraceae
Whole plant
50

23.34

100

10

Platycarya  strobilacea
Juglandaceae
Branch, stem
50

25.51

100

10

Actinidia  arguta
Actinidiaceae
Fruit
50

26.7

100

10

Tilia  amurensis
Tiliaceae
Branch, leaf
50

28.5

100

10

Actinidia  arguta
Actinidiaceae
Stem
50

28.51

100

20

Euscaphis  japonica
Staphyleaceae
Branch
30

28.62

40

10

Actinidia  arguta
Actinidiaceae
Root
50

31.34

100

10

Carpinus  cordata
Betulaceae
Branch, stem
50

31.39

100

10

Rhus  sylvestris
Anacardiaceae
Branch, leaf
50

32.14

100

10

Celtis  sinensis
Ulmaceae
Branch, stem
50

35.89

100

10

Prunus  serrulata
Rosaceae
Branch, leaf
50

42.55

100

10

Potentilla  fragarioides
Rosaceae
Whole plant
50

42.58

100

10

Tilia  mandshurica
Tiliaceae
Flower, leaf
50

48.21

100

10

Actinidia  arguta
Actinidiaceae
Stem, leaf, fruit
50

54.09

100

10

Hypericum  ascyron
Hypericaceae
Whole plant
50

56.12

100

10

Rhus  chinensis
Anacardiaceae
Branch, leaf
50

56.9

100

10

Picrasma  quassioides
Simaroubaceae
Branch, stem
50

60.47

100

10

Prunus  persica
Rosaceae
Branch, leaf
50

62.12

100

10

Actinidia  arguta
Actinidiaceae
Root
50

69.17

100

10

Spiraea  pubescens
Rosaceae
Branch, leaf, flower
50

74.62

100

10

Tilia  mandshurica
Tiliaceae
Branch, stem
50

79.67

100

10

Acer  ginnala
Aceraceae
Branch, leaf
50

82.29

100

10

Elsholtzia  splendens
Labiatae
Root
50

83.98

100

10

Staphylea  bumalda
Staphyleaceae
Branch, leaf
50

84.28

100

80

Pinus  densiflora
Pinaceae
Stem
90

87.58

100

10

Machilus  thunbergii
Lauraceae
Leaf, branch
50

90.9

100

10

Deutzia  glabrata
Saxifragaceae
Branch, leaf, flower
50

91.09

100

10

Indigofera  kirilowii
Leguminosae
Branch, leaf, flower
50

94.98

100

Opuntia  ficus-indica
Opuntiacae
Stem
100

>100

Hibiscus  syriacus
Malvaceae
Root
100

>100

Actinidia  arguta
Actinidiaceae
Bark
100

>100

Euonymus  oxyphyllus
Celastraceae
Branch
100

>100

Eucommia  ulmoides
Eucommiaceae
Branch, leaf
100

>100

Asarum  sieboldii
Aristolochiac
Root
100

>100

Bupleurum  longeradiatum
Umbelliferae
Whole plant
100

>100

Plantago  asiatica
Plantaginacea
Root
100

>100

Alisma  plantago-aquatica
Alismataceae
Root
100

>100

Duchesnea  chrysantha
Rosaceae
Whole plant
100

>100

Cuscuta  japonica
Convolvulaceae
Whole plant
100

>100

Clematis  apiifolia
Ranunculaceae
Stem, leaf, flower
100

>100

Prunus  serrulata
Rosaceae
Branch
100

>100

Colocasia  antiquorum
Araceae
Aerial part
100

>100

Lespedeza  cuneata
Leguminosae
Aerial part
100

>100

Lespedeza  cuneata
Leguminosae
Root
100

>100

Mallotus  japonicas
Euphorbiaceae
Aerial part
100

>100

Alisma  canaliculatum
Alismataceae
Aerial part
100

>100

Alisma  canaliculatum
Alismataceae
Root
100

>100

Magnolia  denudata
Magnoliaceae
Flowers
100

>100

Scopolia  japonica
Solanaceae
Stem, leaf
100

>100

Scopolia  japonica
Solanaceae
Root
100

>100

Chloranthus  japonicus
Chloranthaceae
Whole plant
100

>100

Barbarea  orthoceras
Cruciferae
Whole plant
100

>100

Caulophyllum  robustum
Berberidaceae
Stem, leaf
100

>100

Caulophyllum  robustum
Berberidaceae
Root
100

>100

Carduus  crispus
Compositae
Stem, leaf
100

>100

Carduus  crispus
Compositae
Flower
100

>100

Styrax  japonica
Styracaceae
Flower
100

>100

Cornus  controversa
Cornaceae
Branch, leaf
100

>100

Cornus  controversa
Cornaceae
Flower
100

>100

Magnolia  sieboldii
Magnoliaceae
Branch, leaf
100

>100

Magnolia  sieboldii
Magnoliaceae
Flower
100

>100

Prunus  persica
Rosaceae
Fruit
100

>100

Rhamnus  yoshinoi
Rhamnaceae
Branch, leaf
100

>100

Erigeron  annuus
Compositae
Whole plant
100

>100

Styrax  japonica
Styracaceae
Branch, leaf
100

>100

Quercus  aliena
Fagaceae
Branch, leaf
100

>100

Callicarpa  japonica
Verbenaceae
Branch, leaf
100

>100

Ligustrum  obtusifolium
Oleaceae
Branch, leaf
100

>100

Lindera  obtusiloba
Lauraceae
Branch, leaf
100

>100

Lespedeza  bicolor
Leguminosae
Branch, leaf
100

>100

Carpinus  laxiflora
Betulaceae
Branch, leaf
100

>100

Machilus  thunbergii
Lauraceae
Bark
100

>100

Hedera  rhombea
Araliaceae
Whole plant
100

>100

Arenaria  serpyllifolia
Caryophyllaceae
Whole plant
100

>100

Paulownia  coreana
Paulowniaceae
Flower
100

>100

Thlaspi  arvense
Brassicaceae
Whole plant
100

>100

Vicia  villosa
Leguminosae
Whole plant
100

>100

Descurainia  pinnata
Brassicaceae
Whole plant
100

>100

Ribes  fasciculatum
Saxifragaceae
Branch, leaf, fruit
100

>100

Corydalis  speciosa
Fumariaceae
Whole plant
100

>100

Clematis  fusca
Ranunculaceae
Whole plant
100

>100

Deutzia  parviflora
Saxifragaceae
Branch, leaf, stem, flower
100

>100

Rosa  multiflora
Rosaceae
Branch, leaf, stem, flower
100

>100

Parthenocissus  tricuspidata
Vitaceae
Leaf, stem
100

>100

Chelidonium  majus
Papaveraceae
Whole plant
100

>100

Platycarya  stobilacea
Juglandaceae
Leaf
100

>100

Platycarya  stobilacea
Juglandaceae
Flower
100

>100

Carpinus  cordata
Betulaceae
Leaf
100

>100

Celtis  sinensis
Ulmaceae
Leaf
100

>100

Orixa  japonica
Rutaceae
Leaf
100

>100

Orixa  japonica
Rutaceae
Branch, stem
100

>100

Orixa  japonica
Rutaceae
Fruit
100

>100

Picrasma  quassioides
Simaroubaceae
Leaf
100

>100

Picrasma  quassioides
Simaroubaceae
Fruit
100

>100

Tilia  mandshurica
Tiliaceae
Leaf
100

>100

Aralia  cordata
Araliaceae
Whole plant
100

>100

Viburnum  sargentii
Caprifoliaceae
Branch, leaf
100

>100

Polygonatum  odoratum
Liliaceae
Root　 　
100

>100

Astragalus  membranaceus
Leguminosae
Root　
100

>100

Pleuropterus  multiflorus
Polygonaceae
Root　
100

>100

Torilis  japonica
Umbelliferae
Fruit　
100

>100

Phaseolus  angularis
Leguminosae
Fruit　
100

>100

Phaseolus  radiates
Leguminosae
Fruit　
100

>100

Artemisia  scoparia
Compositae
Aerial part　
100

>100

Solanum  tuberosum
Solanaceae
Tuber
100

>100

Brassica  juncea
Cruciferae
Leaf　
100

>100

Arctium  lappa
Compositae
Root
100

>100

Cucumis  sativus
Cucurbitaceae
Fruit　
100

>100

Diospyros  kaki
Ebenaceae
Fruit　 　
100

>100

Artemisia  princeps
Compositae
Aerial part　
100

>100

0.0005

Orlistat (positive control)

0.005

0.036 (0.073 μM)

0.05

aResults are the mean ± SD ().

(a)

(b)

Figure 1: Inhibitory effect of Cudrania tricuspidata leaf extract on porcine pancreatic lipase. (a) Porcine pancreatic lipase activity at different concentrations of C. tricuspidata leaves. (b) Orlistat was used as a positive control. Data are the mean ± S.D. ().

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 [20–22]. 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.

Figure 2: Inhibitory effect of Cudrania tricuspidata leaves on rat plasma triacylglycerol levels. Plasma triacylglycerol levels, at the time marked by an asterisk, significantly differ between the control and C. tricuspidata (250 mg/kg) groups (). Orlistat (a lipase inhibitor) was used as a positive control ( versus control).

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.

Acknowledgments

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

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