- About this Journal ·
- Abstracting and Indexing ·
- Advance Access ·
- Aims and Scope ·
- Annual Issues ·
- Article Processing Charges ·
- Articles in Press ·
- Author Guidelines ·
- Bibliographic Information ·
- Citations to this Journal ·
- Contact Information ·
- Editorial Board ·
- Editorial Workflow ·
- Free eTOC Alerts ·
- Publication Ethics ·
- Reviewers Acknowledgment ·
- Submit a Manuscript ·
- Subscription Information ·
- Table of Contents
Evidence-Based Complementary and Alternative Medicine
Volume 2012 (2012), Article ID 878365, 8 pages
Inhibitory Activities of Cudrania tricuspidata Leaves on Pancreatic Lipase In Vitro and Lipolysis In Vivo
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.
To identify effective herb to treat obesity, we screened 115 herbal extracts for inhibition of porcine pancreatic lipase (triacylg-ycerol acylhydrolase, EC 22.214.171.124) 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.
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 . 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) . 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 . 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 . 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.
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. . 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 . 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).
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.
C. tricuspidata is a rich source of xanthones and flavonoids, including cudraflavone C . A recent study reported that cudraflavone C from Artocarpus nitidus inhibited pancreatic lipase activity (IC50 = μM) . Thus, cudraflavone C may be a potential as one of active compounds for preventing and treating obesity.
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.
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).
- X. Pi-Sunyer, “The medical risks of obesity,” Postgraduate Medicine, vol. 121, no. 6, pp. 21–33, 2009.
- K. M. Flegal, M. D. Carroll, C. L. Ogden, and L. R. Curtin, “Prevalence and trends in obesity among US adults, 1999–2008,” Journal of the American Medical Association, vol. 303, no. 3, pp. 235–241, 2010.
- Y. Gargouri, S. Ransac, and R. Verger, “Covalent inhibition of digestive lipases: an in vitro study,” Biochimica et Biophysica Acta, vol. 1344, no. 1, pp. 6–37, 1997.
- T. D. Filippatos, C. S. Derdemezis, I. F. Gazi, E. S. Nakou, D. P. Mikhailidis, and M. S. Elisaf, “Orlistat-associated adverse effects and drug interactions: a critical review,” Drug Safety, vol. 31, no. 1, pp. 53–65, 2008.
- D. S. Jang, G. Y. Lee, J. Kim et al., “A new pancreatic lipase inhibitor isolated from the roots of Actinidia arguta,” Archives of Pharmacal Research, vol. 31, no. 5, pp. 666–670, 2008.
- P. Slanc, B. Doljak, S. Kreft, M. Lunder, D. Janeš, and B. Štrukelj, “Screening of selected food and medicinal plant extracts for pancreatic lipase inhibition,” Phytotherapy Research, vol. 23, no. 6, pp. 874–877, 2009.
- H. Lee, H. Ha, J. K. Lee et al., “The fruits of Cudrania tricuspidata suppress development of atopic dermatitis in NC/Nga mice,” Phytotherapy Research, vol. 26, pp. 594–599, 2012.
- L. Shi and Y. Fu, “Isolation, purification, and immunomodulatory activity in vitro of three polysaccharides from roots of Cudrania tricuspidata,” Acta Biochimica et Biophysica Sinica, vol. 43, no. 5, pp. 418–424, 2011.
- J. H. Kim, H. J. Kim, H. W. Park, S. H. Youn, D. Y. Choi, and C. S. Shin, “Development of inhibitors against lipase and α-glucosidase from derivatives of monascus pigment,” FEMS Microbiology Letters, vol. 276, no. 1, pp. 93–98, 2007.
- C. Roh and U. Jung, “Screening of crude plant extracts with anti-obesity,” International Journal of Molecular Sciences, vol. 13, pp. 1710–1719, 2012.
- J. Kim, D. S. Jang, H. Kim, and J. S. Kim, “Anti-lipase and lipolytic activities of ursolic acid isolated from the roots of Actinidia arguta,” Archives of Pharmacal Research, vol. 32, no. 7, pp. 983–987, 2009.
- J. Galhardo, N. Davis, S. Matthai, and J. P. Shield, “European Medicines Agency withdrawal for sibutramine,” Archives of Disease in Childhood, vol. 95, no. 10, p. 856, 2010.
- M. L. Vetter, L. F. Faulconbridge, V. L. Webb, and T. A. Wadden, “Behavioral and pharmacologic therapies for obesity,” Nature Reviews Endocrinology, vol. 6, no. 10, pp. 578–588, 2010.
- M. Li and B. M. Y. Cheung, “Pharmacotherapy for obesity,” British Journal of Clinical Pharmacology, vol. 68, no. 6, pp. 804–810, 2009.
- A. L. de La Garza, F. I. Milagro, N. Boque, J. Campión, and J. A. Martínez, “Natural inhibitors of pancreatic lipase as new players in obesity treatment,” Planta Medica, vol. 77, no. 8, pp. 773–785, 2011.
- Y. Bustanji, I. M. Al-Masri, M. Mohammad et al., “Pancreatic lipase inhibition activity of trilactone terpenes of Ginkgo biloba,” Journal of Enzyme Inhibition and Medicinal Chemistry, vol. 26, no. 4, pp. 453–459, 2011.
- N. Karu, R. Reifen, and Z. Kerem, “Weight gain reduction in mice fed Panax ginseng saponin, a pancreatic lipase inhibitor,” Journal of Agricultural and Food Chemistry, vol. 55, no. 8, pp. 2824–2828, 2007.
- Y. S. Kim, Y. M. Lee, H. Kim et al., “Anti-obesity effect of Morus bombycis root extract: anti-lipase activity and lipolytic effect,” Journal of Ethnopharmacology, vol. 130, no. 3, pp. 621–624, 2010.
- Y. M. Lee, Y. S. Kim, Y. Lee et al., “Inhibitory activities of pancreatic lipase and phosphodiesterase from Korean medicinal plant extracts,” Phytotherapy Research, vol. 26, pp. 778–782, 2012.
- D. G. Kang, T. Y. Hur, G. M. Lee et al., “Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension,” Life Sciences, vol. 70, no. 22, pp. 2599–2609, 2002.
- B. W. Lee, J. H. Lee, S. W. Gal, Y. H. Moon, and K. H. Park, “Selective ABTS radical-scavenging activity of prenylated flavonoids from Cudrania tricuspidata,” Bioscience, Biotechnology and Biochemistry, vol. 70, no. 2, pp. 427–432, 2006.
- Y. H. Rho, B. W. Lee, K. H. Park, and Y. S. Bae, “Cudraflavanone A purified from Cudrania tricuspidata induces apoptotic cell death of human leukemia U937 cells, at least in part, through the inhibition of DNA topoisomerase I and protein kinase C activity,” Anti-Cancer Drugs, vol. 18, no. 9, pp. 1023–1028, 2007.
- Y. S. Zou, A. J. Hou, and G. F. Zhu, “Isoprenylated xanthones and flavonoids from Cudrania tricuspidata,” Chemistry and Biodiversity, vol. 2, no. 1, pp. 131–138, 2005.
- T. Zhao, G. R. Yan, S. L. Pan, H. Y. Wang, and A. J. Hou, “New isoprenylated 2-arylbenzofurans and pancreatic lipase inhibitory constituents from Artocarpus nitidus,” Chemistry and Biodiversity, vol. 6, no. 12, pp. 2209–2216, 2009.