Inhibitory Effects of Constituents from Euphorbia lunulata on Differentiation of 3T3-L1 Cells and Nitric Oxide Production in RAW264.7 Cells

A new flavonol galactopyranoside, myricetin 3-O-(2'',3''-digalloyl)-β-D-galactopyranoide (1), and 23 known constituents, including myricetin 3-O-(2''-galloyl)-β-D-galactopyranoide (2), myricitrin (3), myricetin (4), quercetin 3-O-(2'', 3''-digalloyl)-β-D-galactopyranoide (5), quercetin 3-O-(2''-galloyl)-β-D-galactopyranoide (6), hyperin (7), isoquercetrin (8), quercetin (9), kaempferol (10), apigenin (11), luteolin (12), 3-O-methylquercetin (13), 5,7,2',5'-tetrahydroxyflavone (14), 1,3,4,6-tetra-O-galloyl-β-D-glucose (15), 1,2,6-tri-O-galloyl-β-D-glucose (16), 1,3,6-tri-O-galloyl-β-D-glucose (17), gallic acid (18), protocatechuic acid (19), 3,4,5-trimethoxybenzoic acid (20), 2,6-dihydroxyacetophenone (21), 3,3'-di-O-methylellagic acid (22), ellagic acid (23) and esculetin (24) were isolated from Euphorbia lunulata Bge. Their structures were determined by spectroscopic analysis. Isolated hydrolysable tannins, flavonoids, and flavonol galactopyranoside gallates showed significant inhibition of the differentiation of 3T3-L1 preadipocytes and triglyceride accumulation in maturing adipocytes, and nitric oxide production in RAW 264.7 cells.


Introduction
Euphorbia lunulata Bge. (Euphorbiaceae) is a perennial herbaceous plant that has long been used in China as a traditional crude drug for the treatment of chronic bronchitis. Previous investigations of E. lunulata Bge. have yielded some flavonoids and coumarins that have been reported to have anticancer, antioxidant, insulin and interleukin-10 (IL-10) mimicking activities [1][2][3][4][5].
It is well known that obesity is often accompanied by hyperglycemia, hypertension, and hyperlipidemia, which are together known as metabolic syndrome. Obesity is the result of an increase in the number and size of adipocytes, in which adipocytes accumulate a large amount of lipids and become enlarged. Cultured 3T3-L1 adipocytes show many properties similar to those of normal adipocytes, lipids are accumulated during the differentiation and maturing process, the cytosolic enzyme glycerol-3-phosphate dehydrogenase (GPDH) appears to have an important role in the conversion of glycerol into triglyceride (TG), and the level of activity of GPDH increases during the conversion of 3T3 cells. Recent studies indicate that obesity is associated with low-grade chronic inflammation of adipose tissues, and the obese adipose tissue is characterized by increased infiltration of macrophages. In a coculture system of 3T3-L1 adipocytes and RAW264 macrophages, marked increases in secretion levels of inflammatory mediators such as TNF-α, MCP-1, and nitric oxide (NO) were observed [6][7][8]. NO mediates diverse functions by acting on various cells through interactions with different molecular targets, and excessive NO production is involved in various types of inflammation [9]. Therefore, as a part of our continuing investigation of anti-adipogenesis and anti-inflammatory agents from natural sources [10], the chemical composition of the whole plant of E. lunulata Bge. has been examined.
Compound 1 was obtained as a yellow amorphous powder and gave positive Mg-HCl and FeCl 3 tests. The molecular formula of 1 was established as C 35 H 28 O 21 by HR-FAB-MS. The UV spectrum exhibited absorption maxima at 268 and 355 nm, suggesting the presence of aromatic rings in the molecule. The IR spectrum contained absorption bands for a hydroxyl group (3,396 cm −1 ), ester carbonyl (1,703 cm −1 ) and conjugated carbonyl (1,656 cm −1 ), and aromatic ring (1,608, 1,497 cm −1 ). The 1 H-NMR spectrum of 1 indicated meta-coupled proton signals at δ 6.31 (1H, d, J = 2.1 Hz) and 6.14 (1H, d, J = 2.1 Hz), and an A 2 -type aromatic proton signal at δ 7.27 (2H, s). These data for the aromatic region matched those of myricetin 3-O-(2''-galloyl)-β-D-galactopyranoide (2) and myricetin (4), indicating the aglycone was most likely a myricetin (Table 1). Except for the aglycone signals, the 1 Hand 13    Compounds 2-24 were identified as known compounds by detailed comparisons of their spectroscopic data with that in the literature.
Compounds 1-24 were next evaluated for their inhibitory effects on triglyceride accumulation and GPDH activity in 3T3-L1 cells at the concentration of 30 μM (Figure 3). The major component, quercetin (9, 0.038% yield), which has been reported to have inhibitory effects on triglyceride accumulation, was used as a positive control [10,33]. As shown in Figure 3, the hydrolysable tannins, 15-17, 22 and 23 showed significant inhibition of adipogenesis in 3T3-L1 adipocytes, with TG inhibition values of 75.7%, 72.2%, 56.6%, 64.2% and 72.8%, respectively. The flavonol galactopyranoside gallates, 1, 2, 5 and 6 showed moderate TG inhibitory activities (43.7%, 30.5%, 55.5% and 32.5% inhibition, respectively). Comparing 1 with 2 and 5 with 6, compounds 1, 5 showed stronger TG and GPDH activity inhibition than 2 and 6, respectively; these results suggest that the galloyl group may enhance their activities. The flavonols, 7, 8, 9, 10 and 13 showed moderate TG inhibitory activities (35.8%, 27.7%, 42.4%, 40.8% and 25.9% inhibition, respectively), which were stronger than the flavones, 11, 12 and 14 with TG inhibitions 20.3%, 13.7%, and 15.9%, respectively. The phenylpropanoids 18-21 and coumarin 24 showed weak inhibitory effects on TG and GPDH activity. Furthermore, effects of isolated compounds on cell viability of 3T3-L1 cells were measured by MTT assay. As shown in Figure 3, the isolated compounds of 15-17, 22 and 23 had effects on viability of 3T3-L1 at concentration of 30 μM. However, these compounds at low concentrations (10, 3, 1 μM, respectively) suppressed the differentiation of 3T3-L1 preadipocytes and TG accumulation in maturing adipocytes without exerting cytotoxicity (Figure 4)   Compounds 1-24 also examined with respect to their inhibition of NO production stimulated by LPS and IFN-γ in RAW 264.7 cells (Table 2). In the assay, aminoguanidine, which has been reported to have inhibitory effects on NO production in LPS activated RAW 264.7 macrophages by downregulation of inducible nitric oxide synthase (iNOS), was used as a positive control (IC 50 17.5 μM) [34]. As shown in In contrast, the descending orders of the NO inhibitory activities were: 4 > 2 > 1 > 3 (myricetin derivatives), and 9 > 6 > 5 > 7, 8 (quercetin derivatives). These results showed that the free flavonoids exerted stronger activities than the corresponding glycosides, and the conjugation of gallic acid may have enhanced the activities of the flavonoid glycosides, even though gallic acid (18) itself showed no inhibition. Furthermore, effects of isolated compounds on cell viability of RAW264.7 cells were measured by MTT assay. As shown in Figure 5, the isolated compounds had no effects on viability of RAW264.7 at concentration of IC 50 values.

General
The UV spectrum was obtained in MeOH on a Shimadzu UV-160 spectrophotometer, and the IR spectrum was recorded on a JASCO FT/IR 300E spectrophotometer. Optical rotations were measured in MeOH on a JASCO P-1020 polarimeter. The NMR spectra were recorded on a Varian Mercury 300 spectrometer, with TMS as an internal standard. The MS were obtained on a JEOL GCmate mass spectrometer. Column chromatography was carried out with Diaion HP-20 (Mitsubishi Chemical Co

Plant Material
Whole plants of E. lunulata Bge. were collected in Hebei Province, China, in October 2006 and were identified by Professor Naili Wang (Shenyang Pharmaceutical University, China). Voucher specimens have been deposited at the Laboratory of Pharmacognosy, School of Pharmacy, Nihon University.

Acid Hydrolysis of 1 and Identification of Sugar
Compound 1 (5 mg) was dissolved in 10% H 2 SO 4 and heated at 85 °C for 3 h. After cooling, the reaction mixture was neutralized by passage though an Amberlite IRA-93ZU (Organo) column and then partitioned between ethyl acetate and water. The ethyl acetate layer was concentrated and the concentrate was passed through an ODS column, and then successively eluted with 20% MeOH and 50% MeOH. Gallic acid was recovered from the 20% MeOH fraction, and myricetin from the 50% fraction by direct comparison with authentic samples. Silica gel TLC of the water layer in BuOH:
The 3T3-L1 preadipocytes were seeded at 1.0 × 10 5 cells/mL onto 24-well plates (Sumitomo Bakelite, MS-80240, Tokyo) and incubated at 37 °C. A test sample was added to the medium on day 0, and added at the time of every medium change during the 8 days of incubation. After removing the medium, the cells were washed twice with 500 μL of PBS. The cells were collected in 500 μL of cold sonication buffer (pH 7.5 25 mM Tris buffer containing 1 mM EDTA) and sonicated in ice-cold water. After centrifugation, the cell lysate was used to measure the TG content with LabAssay TM Triglyceride (WAKO Pure Chemical Industries Ltd.), GPDH activity with a GPDH Assay Kit (TaKaRa Bio Inc.), and DNA quantity with a DNA Quantity Kit (Primary Cell Co., Ltd.), according to the manufacturer's protocol. Inhibition of TG and GPDH activity was calculated using the following formula: Inhibition (%) = [(Cn − S)/Cn] × 100, where S is TG amount or GPDH activity when cells incubated with sample and divided by the amount of DNA for each well; Cn is TG amount or GPDH activity when cells incubated with DMSO (control) were divided by the amount of DNA for each well). Cell viability was confirmed by microscopic observation, and was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay method.

NO Production in Activated
Macrophage-Like Cell Line, RAW 264.7 [10,36] The macrophage-like cell line, RAW 264.7, was obtained from American Type Culture Collection. The cells were cultured in Ham's F12 medium with 10% FBS (SAFC Biosciences) at 37 °C under a humidified 5% CO2 atmosphere. The RAW264.7 cells were seeded at 1.2 × 106 cells/mL onto 96-well plates (Sumitomo Bakelite, MS-8096R, Tokyo) and then incubated at 37 °C for 2 h. A test sample was then added to the culture simultaneously with both Escherichia coli LPS (100 ng/mL) and recombinant mouse IFN-γ (0.33 ng/mL), and the cells were incubated at 37 °C, usually for 16 h. The amount of nitrite in culture supernatants was measured using the Griess assay. Cytotoxicity was measured using the MTT assay method.

Statistical Analysis
The values represent mean ± S.D. of at least three experiments. Statistical significant differences were evaluated by the one-way ANOVA followed by the Student's t-test for paired populations, and a P value of 0.05 or less among the means were considered significant.

Conclusions
Obesity is associated with low-grade chronic inflammation of adipose tissues, and obese adipose tissue is characterized by increased infiltration of macrophages. The whole plant extract of E. lunulata Bge. showed inhibitory effects on the differentiation of 3T3-L1 cells and NO production in RAW264.7 cells. Bioassay-directed investigation of active constituents, led to the identification of a new flavonol galactopyranoside gallate, myricetin 3-O-(2'',3''-digalloyl)-β-D-galactopyranoide (1) and 23 known phenolic constituents. The isolated compounds, especially hydrolysable tannins, flavonoids, and flavonol galactopyranoside gallates, showed significant inhibition towards the differentiation of 3T3-L1 adipocytes and nitric oxide production in RAW 264.7 cells. These studies suggest that the extract of E. lunulata Bge. and the isolated compounds might be a source of anti-obesity and antiinflammatory agents to improve the symptoms of metabolic syndrome.