Pancreatic Lipase Inhibition by C-Glycosidic Flavones Isolated from Eremochloa ophiuroides

Activity-guided fractionation of a methanolic extract of the leaves of Eremochloa ophiuroides (centipede grass) using a pancreatic lipase inhibitory assay led to the isolation and identification of a new C-glycosidic flavone, luteolin 6-C-β-D-boivinopyranoside (1), as well as eight known compounds. The structures of these compounds were established on the basis of interpretation of their spectroscopic data. Among these isolates, the C-glycosidic flavones 1–5 showed potent inhibitory effects on pancreatic lipase, with IC50 values ranging from 18.5 ± 2.6 to 50.5 ± 3.9 μM.


Introduction
Plants belonging to the genus Eremochloa (Poaceae) consist of eight species, mainly distributed throughout China, Southeast Asia, and South America. One of these, centipede grass (Eremochloa ophiuroides), is native to China and Southeast Asia and has become one of the most popular lawn grasses in South America [1]. Several C-glycosidic flavones and phenolic constituents have been isolated from this plant [2]. However, information on the bioactivity of this biomass is very limited.
Obesity is caused by an imbalance between energy intake and expenditure and is widely recognized as a major public health problem. Obesity can lead to a variety serious diseases, including hypertension, hyperlipidemia, arteriosclerosis, and type II diabetes [3]. Pancreatic lipase plays a key role for triglyceride absorption in the small intestine. This enzyme is secreted from the pancreas and hydrolyzes triglycerides into glycerol and fatty acids [4]. Therefore, pancreatic lipase inhibitors are considered to be a valuable therapeutic reagent for treating diet-induced obesity in humans. The success of orlistat [5], which is a specific pancreatic lipase inhibitor, has prompted research to identify new pancreatic lipase inhibitors derived from natural sources, such as the constituents of Platycodin grandiflorum [6], Salvia officinalis [7], Salacia reticulate [8], and oolong tea [9].
As part of our continuing search for bioactive natural products, the ethyl acetate (EtOAc)-soluble partition of a methanolic extract from the leaves of E. ophiuroides was found to exhibit an inhibitory effect against porcine pancreatic lipase. In the present work, activity-guided fractionation of the EtOAc-soluble portion using a pancreatic lipase inhibition assay led to the isolation of a new Cglycosidic flavone (1), along with several known compounds. Herein, we describe the isolation and structure elucidation of compound 1 and a biological evaluation of all metabolites obtained in this investigation.
Pancreatic lipase plays an important role in lipid metabolism and is one of the most widely studied models for the evaluation of the potential efficacy of natural products as antiobesity agents [32]. The isolation and characterization of constituents 1-9 from E. ophiuroides was guided by their pancreatic lipase inhibitory activity, using orlistat as a positive control. The flavonoids 1-5 isolated in the highest quantities all had a C-glycosyl group in the A-ring and potently inhibited pancreatic lipase with IC 50 values ranging from 18.5 ± 2.6 to 50.5 ± 3.9 M. The known compound methyl chlorogenate (8) was found to be active (IC 50 = 33.6 ± 2.0 M), but the structurally related compounds chlorogenic acid (7) and caffeic acid (9) were inactive (IC 50 > 200 M). The inhibitory activity of luteolin (6), which does not have a C-glycosyl group on the A-ring of its flavonoid moiety, against pancreatic lipase was much weaker than those of C-glycosylated derivatives on their A-ring. Furthermore, the C-glycosylated flavone 2 at C-8 was more potent than a C-6 glycosylated flavone 3. The presence of at least one C-glycosylated sugar moiety at the 6 or 8-position of the A-ring is essential for the enzyme inhibitory activity of the luteolin skeleton. The C-glycosylated sugar number and attached position in the A-ring appear to be particularly important for strong inhibitory activity.

General
Optical rotations were measured with a JASCO DIP-4 digital polarimeter. UV spectra were acquired on a JASCO V-530 UV-vis spectrometer. The 1 H-and 13 C-NMR spectra were measured on a Varian VNS600 instrument operating at 600 and 150 MHz, respectively. The chemical shifts are given in  (ppm) values relative to that of the solvent CD 3 OD ( H 3.35;  C 49.0) on a tetramethylsilane (TMS) scale. The standard pulse sequences programmed into the instruments were used for each 2D measurement. The J CH value was set at 8 Hz in the HMBC spectra. FAB-MS using 3-nitrobenzyl alcohol as the matrix agent, including HRFAB-MS, was performed on a Micro Mass Auto Spec OA-TOF spectrometer. HPLC analysis was carried out on a YMC-Pack ODS A-302 column (4.6 mm i.d. × 150 mm; YMC Co., Ltd.) and the solvent system consisted of a linear gradient that started with 5 % (v/v) MeCN in 0.1% HCOOH/H 2 O (detection: UV 280 nm; flow rate: 1.0 mL/min; at 40 o C), increased to 95 % MeCN over 30 min, and then increased to 100% MeCN over 5 min. At the end of the run, 100 % MeCN was allowed to flush the column for 5 min, and an additional 10 min of post-run time was set to allow for equilibration of the column. Column chromatography was carried out on Toyopearl HW-40 (coarse grade; Tosoh Co.), YMC GEL ODS AQ 120-50S (YMC Co., Ltd), and Sephadex LH-20 (Pharmacia Fine Chemicals Co., Ltd). Thin-layer chromatography (TLC) was performed on Kieselgel 60 F254 plates (0.2 mm layer thickness, Merck), and the spots were detected by ultraviolet irradiation (254, 365 nm) and by spraying with 10% H 2 SO 4 reagent.

Plant Material
In September, 2007, the leaves of Eremochloa ophiuroides were collected at the Advanced Radiation Technology Institute (ARTI), Jeongeup, Korea, and identified by Dr. Seung Sik Lee. A voucher specimen (KAJ-0053) was deposited at the Natural Product Chemistry Laboratory, College of Herbal Bio-industry, Daegu Hanny University.

Extraction and Isolation
Fresh milled E. ophiuroides plant material (5.0 kg) was extracted with MeOH (18 L × 3) at room temperature, and the solvent was evaporated in vacuo. The combined crude MeOH extract (102.1 g) was suspended in 20% MeOH (6 L), and then partitioned in turn with n-hexane (6 L × 3) and EtOAc

Assay for Pancreatic Lipase Activity
The ability of the compounds to inhibit porcine pancreatic lipase was evaluated using previously reported methods with a minor modification [10,11]. Briefly, an enzyme buffer was prepared by the addition of 30 L (10 units) of a solution of porcine pancreatic lipase (Sigma, St. Louis, MO) in 10 mM MOPS (morpholinepropanesulphonic acid), and 1 mM EDTA, pH 6.8 to 850 L of Tris buffer (100 mM Tris-HC1 and 5 mM CaCl 2 , pH 7.0). Then, 100 L of compound at the test concentration or Orlistat (Roche, Switzerland) was mixed with 880 L of enzyme buffer, and incubated for 15 min at 37 o C, then, 20 L of the substrate solution [10 mM p-NPB (p-nitrophenylbutyrate) in dimethyl formamide] was added and the enzymatic reaction was allowed to proceed for 15 min at 37 o C. Pancreatic lipase activity was determined by measuring the hydrolysis of p-NPB to p-nitrophenol at 405 nm using an ELISA reader (Tecan, Infinite F200, Austria). Inhibition of lipase activity was expressed as the percentage decrease in the OD when porcine pancreatic lipase was incubated with the test compounds.

Conclusions
In summary, we have investigated the pancreatic lipase inhibitory activity of a new flavonoid (1) and eight known compounds (2-9) isolated from the leaves of E. ophiuroides. Among the isolates, the C-glycosyl flavonoids were compared to estimate the optimal position and number of glycosyl groups on the flavone skeleton for significant and specific pancreatic lipase inhibitory activity. Compound 5, a C-glycosyl flavone with two sugar moieties at C-6 of the A-ring, showed especially potent activity (IC 50 = 18.5 ± 2.6 M) when compared to the other tested compounds.