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

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 IC50 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 (P < 0.05). These results suggest that C. tricuspidata leaves may be useful for the treatment of obesity.


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 antioxidant 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.

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 temperatureand 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.

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.

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 CaCl 2 , 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 A is the activity without inhibitor, a is the negative control without inhibitor, B is the activity with inhibitor, and b is the negative control with inhibitor. The results were expressed as an average (n = 3).

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 190 ∼ 230 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).

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 P < 0.05 were considered statistically significant.

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 drugorlistat-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][17][18][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 IC 50 values (Table 1). Eighteen extracts had IC 50 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 IC 50 values less than 10 μg/mL. Notably, C. tricuspidata leaves exhibited an IC 50 value of 9.91 μg/mL ( Figure 1).

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][21][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 [23]. A recent study reported that cudraflavone C from Artocarpus nitidus inhibited pancreatic lipase activity (IC 50 = 17.0 ± 0.7 μM) [24]. Thus, cudraflavone C may be a potential as one of active compounds for preventing and treating obesity.

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.