Inhibitory effect of chemical constituents from Artemisia scoparia Waldst. et Kit. on triglyceride accumulation in 3T3-L1 cells and nitric oxide production in RAW 264.7 cells

We investigated the anti-obesity effect of the aerial part of Artemisia scoparia Waldst. et Kit. (Compositae). An 80 % aqueous EtOH extract of the aerial part inhibited triglyceride (TG) accumulation and the nitric oxide (NO) production activity. A new chromane derivative was isolated from the aerial part of A. scoparia Waldst. et Kit. along with 18 known compounds. The structure of the new chromane, scopariachromane (1), was elucidated by spectroscopic analyses. The inhibitory effects of the compounds on TG accumulation activity were examined. Among these, cirsiliol (11) inhibited TG accumulation in 3T3-L1 preadipocytes. Jaceosidin (12) inhibited NO production in a murine macrophage-like cell line (RAW 264.7). These results indicate that the 80 % aqueous EtOH extract and compounds isolated from the aerial part of A. scoparia Waldst. et Kit. may improve obesity-related insulin resistance.


Introduction
Obesity is one of the leading metabolic diseases worldwide [1] and is closely associated with coronary heart disease, hypertension, type 2 diabetes mellitus, cancer, respiratory complications, and osteoarthritis [2]. Obesity is a condition in which adipocytes accumulate a large amount of fat and become enlarged. At the cellular level, this condition is characterized by an increase in the number and size of adipocytes that differentiate from 3T3-L1 preadipocytes in the adipose tissue [3]. Recent studies have demonstrated that the obese adipose tissue is characterized by enhanced macrophage infiltration [4]. Macrophages produce various inflammatory proteins such as tumor necrosis factor alpha (TNF-a), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO), which are implicated in insulin resistance and metabolic disorders [5]. NO is a diffusible, liposoluble, free radical gas produced from L-arginine by a family of enzymes known as inducible NO synthases (iNOS) [6]. In a co-culture system of RAW 264.7 macrophages and 3T3-L1 adipocytes, marked increases in the secretion of inflammatory mediators such as TNF-a, MCP-1, and NO were observed [7]. Triglyceride (TG) is synthesized from glucose and fatty acid that is incorporated by glucose transporter 4 and fatty acid transporter (CD36) into 3T3-L1 preadipocytes [8,9]. Cultured 3T3-L1 adipocytes have many properties similar to those of normal adipocytes. Thus, this cell line is a suitable model system for obesity-related research [10][11][12]. Herbal extracts from plants such as Blumea balsamifera [13], Ginkgo biloba [14], Wasabia japonica [15], Zizyphus jujuba [16], Morus alba var. multicaulis [17], and Albizia julibrissin Durazz [18] have been shown to possess anti-obesity effects. Thus, we aimed to screen for crude drugs and natural products with inhibitory effects on TG accumulation in adipocytes and NO production in activated macrophages.
Artemisia scoparia Waldst. et Kit. belongs to the family Compositae and is native to Japan, Korea, and Mongolia. The aerial part of this plant is used in traditional medicine as an antiphlogistic, as a diuretic, for the treatment of hepatitis and urticaria, and as an antimold agent. Phytochemical investigations of the aerial part of A. scoparia Waldst. et Kit. resulted in the isolation of flavonoids, coumarins, and essential oils [19].  (Fig. 1).
Scopariachromane (1) was isolated as an amorphous colorless powder, and its molecular formula was determined as C 15 H 18 O 5 by HR-EI-MS, with seven degrees of unsaturation. The UV spectrum of 1 showed absorption at 295 and 232 nm, and its IR spectrum showed the characteristic absorption bands of hydroxyl groups (3420 cm -1 ), a conjugated carbonyl group (1694 cm -1 ), and conjugated double bonds (1633 and 1609 cm -1 ). The 1 H NMR chemical shifts of 1 (Table 1) (Table 1) and DEPT spectra revealed the presence of one carbonyl group, one methoxy group, two methyls, seven methines, and five quaternary carbons. All protonated carbons were assigned by HMQC analysis. Furthermore, the 1 H-1 H COSY ( 126.1 (C-10) and d C 69.1 (C-4); H-4 and d C 129.2 (C-5) and C-10; and H-3 and C-10 indicated that the prenyl-type unit was located on C-4. The degree of unsaturation at seven also indicated that the prenyl-type unit participated in a cyclic structure. The NOESY spectrum of 1 ( Fig. 3
The isolated compounds were evaluated for their inhibitory effects against intracellular TG accumulation in 3T3-L1 cells. To determine their nontoxic concentrations, 3T3-L1 cells were treated with various concentrations (3-100 lM) of 1-19, and the cell viability was measured by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. None of the compounds were cytotoxic at concentrations up to 30 lM. Thus, we used a concentration of at most 30 lM. TG measurements were corrected for the amount of DNA. Quercetin, which has been reported to have inhibitory effects on TG accumulation, was used as a positive control [37]. Treatment with 7, 10, 11, and 16 (30 lM) inhibited TG accumulation ( Table 2). In particular, 11 potently inhibited intracellular TG accumulation. Compared with 10, 11 showed a stronger inhibitory effect on TG accumulation. These results suggest that the presence of a hydroxy group at C-3 0 could be important. We also examined the effects of 7, 10, 11, and 16 on the uptake of [1-3 H]-2deoxy-D-glucose (Table 3). TG is synthesized from glucose and fatty acid that are incorporated by glucose transporter 4 and fatty acid transporter CD36 [8,38]. These compounds inhibited glucose uptake for TG synthesis in 3T3-L1 adipocytes. In addition, we have examined the inhibitory effects of 1-19 on NO production in RAW 264.7 cells. As shown in Table 4, compounds 1-14, 16, 17, and 19 showed inhibitory activity. These compounds did not have cytotoxic effects (10-30 lM). In the assay, aminoguanidine (IC 50 17.5 lM), which has been reported to have inhibitory effects on NO production in LPS-activated RAW 264.7 macrophages via the downregulation of iNOS, was used as a positive control [39]. Compounds 4,5,[7][8][9][10][11][12][13][14]16, and 17 showed strong or moderate inhibitory effects on NO production compared with aminoguanidine (Table 4). Of the 19  Chemical shifts are given in ppm; coupling constants J (in parentheses) are given in Hz compounds tested, compound 12 exhibited the highest inhibitory activity against NO production (IC 50 5.9 lM).
In brief, the results presented here showed that an 80 % aqueous EtOH extract of the aerial part of A. scoparia Waldst. et Kit. and the isolated compounds may be used to reduce obesity, which is a serious health problem in industrialized countries, by inhibiting NO production by activated macrophages, and intracellular TG accumulation and glucose uptake in mature adipocytes.

Experimental
General experimental procedure Optical rotation (OR) was measured in MeOH using a JASCO P-1020 polarimeter. The UV spectra were obtained in MeOH using a JASCO V-550 spectrophotometer, and the IR spectra were recorded using a JASCO IR A-2 spectrophotometer. The NMR spectra were recorded using a JEOL ECX-500 spectrometer ( 1 H NMR, 500 MHz; 13 C NMR, 125 MHz), with TMS as an internal standard. Mass spectra were obtained using a JEOL GCmate spectrometer.

Nitrite assay
The cells were seeded at 1.2 9 10 6 cells/mL onto 96-well flat-bottom plates (Sumitomo Bakelite) and incubated at 37°C for 2 h. The test sample was then added to the culture simultaneously with Escherichia coli LPS (100 ng/ mL) and recombinant mouse IFN-c (0.33 ng/mL), and the cells were incubated at 37°C, usually for 16 h. After incubation, the cells were chilled on ice. The culture supernatant (100 lL) was placed in wells in duplicate 96-well flat-bottom plates. A standard solution of NaNO 2 was also placed in other wells on the same plates. To quantify nitrite, 50 lL Griess reagent, 1 % sulfanilamide in 5 % H 3 PO 4 , and 0.1 % N-(1-naphthyl)ethylenediamide dihydrochloride were added to each well. After 10 min, the reaction products were colorimetrically quantified at 550 nm with subtraction of the background absorbance at 630 nm, using a model 3550 microplate reader (BIO-RAD).
TG assay 3T3-L1 preadipocytes (American Type Culture Collection) were plated in 24-well plates and maintained in DMEM supplemented with 10 % (v/v) fetal calf serum (FCS) and 1 % (v/v) penicillin-streptomycin at 37°C in a humidified 5 % CO 2 incubator. To induce differentiation, 3-day postconfluent 3T3-L1 preadipocytes (day 0) were stimulated by adipogenic agents (500 lM 3-isobutyl-1-methylxanthine, 1 lM dexamethasone, and 10 lg/mL insulin) that were added to DMEM with 10 % (v/v) fetal bovine serum (FBS) culture medium. After 3 days, the medium was replaced with DMEM containing 10 % (v/v) FBS and 5 lg/mL insulin, and it was subsequently replaced every 3 days. The cells were harvested 8 days after the initiation of differentiation. The cells were washed with PBS (-), scraped on ice in 500 lL of sonication buffer (25 mM Tris buffer and 1 mM EDTA; pH 7.5), and sonicated to homogenize the cell suspension. The total TG content of the cells was determined using the LabAssay TM triglyceride kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The DNA concentration was determined using the DNA quantity kit (Primary Cell Co., Ltd., Sapporo, Japan). The TG concentration per microgram of DNA in 3T3-L1 cells was expressed as the ratio (%) relative to the control value. The test sample dissolved in DMSO was added.
Glucose uptake assay 3T3-L1 adipocytes were harvested 8 days after the initiation of differentiation. After the differentiation, the medium was replaced with serum-free DMEM for 2 h, and the cells were then washed and incubated in KRH buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM KH 2 PO 4 , 1 mM CaCl 2 , 1 mM MgSO 4 ; pH 7.4) containing insulin (200 nM) for 10 min at 37°C. After exposure to insulin, the cells were washed and treated with samples in KRH buffer for 15 min. After incubation, [1-3 H]-2-deoxy-D-glucose (0.25 lCi/mL) was added, and incubation was continued for 10 min. The cells were washed twice with ice-cold KRH buffer and then solubilized with 0.1 % SDS. The incorporated radioactivity was measured by liquid scintillation counting. Nonspecific uptake was determined in the presence of 20 lM cytochalasin B and was subtracted from the total value. The test sample dissolved in DMSO was added.

Cell viability assay
Cell viability was assessed using MTT. The 3T3-L1 cells (1.0 9 10 5 cells/mL) were seeded in 96-well plates and incubated for 24 h at 5 % CO 2 and 37°C and then treated with samples. After 24 h of incubation, 20 lL of MTT solution (1 mg/mL) was added to the cell culture, and the cells were further incubated at 37°C and 5 % CO 2 for 4 h. After removing the medium, the MTT formazan crystals were dissolved in DMSO, following which the absorbance in individual wells was determined at 570 nm using a microplate reader and the background absorbance (655 nm) was subtracted. The test sample dissolved in DMSO was added.