Inhibitory Effects of Constituents from Morus alba var. multicaulis on Differentiation of 3T3-L1 Cells and Nitric Oxide Production in RAW264.7 Cells

A new arylbenzofuran, 3′,5′-dihydroxy-6-methoxy-7-prenyl-2-arylbenzofuran (1), and 25 known compounds, including moracin R (2), moracin C (3), moracin O (4), moracin P (5), artoindonesianin O (6), moracin D (7), alabafuran A (8), mulberrofuran L (9), mulberrofuran Y (10), kuwanon A (11), kuwanon C (12), kuwanon T (13), morusin (14), kuwanon E (15), sanggenon F (16), betulinic acid (17), uvaol (18), ursolic acid (19), β-sitosterol (20), oxyresveratrol 2-O-β-d-glucopyranoside (21), mulberroside A (22), mulberroside B (23), 5,7-dihydroxycoumarin 7-O-β-d-glucopyranoside (24), 5,7-dihydroxycoumarin 7-O-β-d-apiofuranosyl-(1→6)-O-β-d-glucopyranoside (25) and adenosine (26), were isolated from Morus alba var. multicaulis Perro. (Moraceae). Their structures were determined by spectroscopic methods. The prenyl-flavonoids 11–14, 16, triterpenoids 17,18 and 20 showed significant inhibitory activity towards the differentiation of 3T3-L1 adipocytes. The arylbenzofurans 1–10 and prenyl-flavonoids 11–16 also showed significant nitric oxide (NO) production inhibitory effects in RAW264.7 cells.


Introduction
Obesity has become a global epidemic in both developed and developing countries, and it is often accompanied by hyperglycemia, hypertension, and hyperlipidemia, which together are called metabolic syndrome [1]. Adipose tissue is composed of various cell types; mature adipocytes, preadipocytes, fibroblasts, endothelial cells, vascular cells, and macrophages [2]. Obesity is a condition in which adipocytes accumulate a large amount of lipids and become enlarged. It is characterized at the cellular level by an increase in the number and size of adipocytes differentiated from fibroblastic preadipocytes in adipose tissue [3]. 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 [4]. Recent studies indicate that obesity is associated with low-grade chronic inflammation of adipose tissues, and that such inflammation is one of the potential mechanisms leading to insulin resistance [5]. It has been demonstrated that 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 NO were observed [6,7]. NO is a short-lived free radical produced from L-arginine by nitric oxide synthase (NOS). 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 [8]. Therefore, we attempted to identify natural anti-inflammatory compounds that not only inhibit triglyceride accumulation by 3T3-L1 adipocytes, but also inhibit the secretion of NO from RAW 264.7 cells.
Compounds 1-26 were evaluated for their inhibitory effects on triglyceride accumulation and GPDH activity in 3T3-L1 cells at the concentration of 20 μM (Table 2). Quercetin, which has been reported to have inhibitory effects on triglyceride accumulation, was used as a positive control [40]. No compound, except for 19, exhibited cytotoxic effects in 3T3-L1 cells based on microscopic observation, furthermore, the percentage of survival cells examined by DNA quantity assays, and the cell viability of 19 was near 10%. As shown in Table 1, the prenyl-flavonoids 11-14,16, triterpenoids 17,18 and β-sitosterol showed significant inhibition of adipogenesis in 3T3-L1 adipocytes, with TG inhibition values of 47.1%, 40.0%, 39.9%, 51.6%, 36.0%, 56.0%, 43.2% and 34.5%, respectively, which were stronger than the positive control, and quercetin whose TG inhibition was 34%. The flavones, 12 and 14 showed particularly strong inhibition of GPDH activity (60.1% and 70.0% inhibition, respectively), the flavanones, 15 and 16 showed weaker inhibitory effects of GPDH activity (12.0% and 18.1% inhibition, respectively) than other flavones. These results indicate that the prenyl moiety at C-3 and A-ring may have enhanced the anti-differentiation activities of 3T3-L1 cells. The 2-arylbenzofurans showed moderate or weak inhibitory effects on TG and GPDH activity. Comparing 1 with 2, 3 with 6 and 9 with 10, compounds 1, 6 and 10 showed stronger TG and GPDH activity inhibition than 2, 3 and 9, respectively, these results suggest that the methoxy group may enhance their activities. The major component, mulberroside A (22, 0.23% yield), showed 16.7% TG inhibition and 22.8% GPDH activity inhibition.  It has been demonstrated that macrophages treated with LPS and/or IFN-γ, increase secretion of several cytokines. LPS and IFN-γ responsive pathways in macrophages are distinct and independent, and these two inducers can cooperate with each other in achieving full macrophage activation [41,42]. Macrophages, treated with LPS and IFN-γ, produce 5-6 fold higher amounts of NO than with LPS alone [43]. Therefore, compounds 1-26 were also examined with respect to their inhibition of NO production stimulated by LPS and IFN-γ in RAW 264.7 cells (Table 3). In the assay, aminoguanidine (IC 50 17.5 μM), which has been reported to have inhibitory effects on NO production in LPS activated RAW 264.7 macrophages via the down-regulation of inducible nitric oxide synthase (iNOS), was used as a positive control [44]. As shown in Table 3, most of the arylbenzofurans (compounds 1-10) and prenyl-flavonoids (compounds 11-16) showed strong or moderate inhibitory effects on NO production compared with aminoguanidine. The triterpenoids 17 and 19 also exhibited weak or strong NO production inhibitory activity; however, 18 did not show such activity may because of a lack of the carbonyl group at C-28. Furthermore, effects of isolated compounds on cell viability of RAW264.7 cells were measured by MTT assay. As shown in Figure 3, the isolated compounds had no effects on viability of RAW264.7 at concentration of IC 50 values.

Plant Material
The root barks of M. alba var. multicaulis Perro. were collected in Chiba, Japan, in March 2007 and were identified by Dr. Heran Li of the College of Pharmaceutical Science, Soochow University, People's Republic of China. Voucher specimens have been deposited at the Laboratory of Pharmacognosy, School of Pharmacy, Nihon University.

Extraction and Isolation
The root barks of M. alba var. multicaulis Perro. (9.0 kg) were extracted three times with 80% methanol and concentrated to give the methanol extract. The extract (1311 g) was dissolved and suspended in water (6 L) and partitioned to form a chloroform layer (8.7 g), ethyl acetate layer (356.9 g), n-butanol layer (225.0 g) and water layer (758.3 g).

Triglyceride (TG) content and Glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 cells
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.

NO Production in Activated
Macrophage-Like Cell Line, RAW 264.73 [46,47] 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% CO 2 atmosphere. The RAW264.7 cells were seeded at 1.2 × 10 6 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. Cell viability was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay method.

Conclusions
Obesity is associated with low-grade chronic inflammation of adipose tissues, and obese adipose tissue is characterized by increased infiltration of macrophages. The root bark extract of Morus alba var. multicaulis Perro. showed inhibitory effects on the differentiation of 3T3-L1 cells and NO production in RAW264.7 cells. Bioguided assay of bioactive constituents led to the identification of a new compound, 3',5'-dihydroxy-6-methoxy-7-prenyl-2-arylbenzofuran (1) and 25 known constituents. The isolated compounds, especially the prenyl-flavonoids and 2-arylbenzofurans, suppressed adipogenesis in 3T3-L1 adipocytes and NO production in RAW264.7 cells. In conclusion, the findings of the present study may account for the use of M. alba var. multicaulis Perro. in traditional medicine to treat inflammation, and the isolated compounds might be a source of antiobesity and anti-inflammatory agents to improve the symptoms of metabolic syndrome.