110654 (048) Biosci. Biotechnol. Biochem., 76 (2), 110654-1–5, 2012 Inhibitory Effects of Actinidiamide from Actinidia polygama on Allergy and Inflammation Myun-Ho BANG,1 In-Gyeong CHAE,2 Eun-Ju LEE,2 Nam-In BAEK,1 Yoon-Su BAEK,1 y Dae-Young LEE,1 In-Seon LEE,2 Sam-Pin LEE,2;3 and Seun-Ah YANG3; 1Department of Oriental Medicine and Processing, College of Life Sciences, Kyung Hee University, Yongin, Gyeonggi 446-701, Korea 2Department of Food Science and Technology, Keimyung University, Daegu 704-701, Korea 3The Center for Traditional Microorganism Resources (TMR), Keimyung University, Daegu 704-701, Korea Received September 2, 2011; Accepted November 5, 2011; Online Publication, February 7, 2012 [doi:10.1271/bbb.110654] Actinidia polygama Max. was subjected to super- cells.3) Moreover, it inhibited the vascular permeability critical fluid extraction (SFE), and the resulting ethanol induced by acetic acid and the edema induced by extract of marc (SFEM) was subjected to sequential carrageenan in mouse and rat.3) It is not clear, however, fractionation with various solvents. Each extract and whether -linoleic acid is the main component respon- fraction was assayed for anti-inflammatory effect. The sible for reducing inflammation and allergic reactions. ethyl acetate fraction (EtOAc) contained the highest iNOS can be induced to produce NO by bacterial LPS levelAdvance (70.8% inhibition) of anti-inflammatory activity. View In or immunological stimuli such as interferon (IFN)- , order to identify the active constituents, the EtOAc and sustained NO synthesis mediates inflammatory fraction was further fractionated by silica gel and ODS reactions in macrophages and other cells. Macrophages column chromatography. By activity-guided fractiona- play a major role in innate immunity, initiating and tion, an active ceramide was identified as the anti- propagating defensive reactions against pathogens. Ex- inflammatory component, and its structure was deter- cess NO production by macrophages and other cells mined by NMR and MS analysis. The novel ceramide exposed to endotoxins can contribute to cerebral was named actinidiamide, and was found significantly to injury,4) myocardial ischemia,5) inflammatory disorders, inhibit nitric oxide (NO) production (30.6% inhibition diabetes and other diseases.6–8) Although iNOS activa- at 1 g/mL) in lipopolysaccaride (LPS)-stimulated tion is not a major event in all of these diseases, the RAW 264.7 cells and -hexosaminidase release (91.8% proinflammatoryProofs effects of NO generated by iNOS inhibition at 1 g/mL) in IgE-sensitized RBL-2H3 cells. contribute to pathophysiology in each case. Therefore, Thus the presence of actinidiamide conveys allergy and inhibiting NO synthesis is potentially beneficial in inflammation treatment ability to A. polygama. preventing inflammatory reactions. The murine macro- phage-like cell line RAW 264.7 has previously been Key words: Actinidia polygama Max.; actinidiamide; used to characterize the actions of various immunomo- anti-allergy; anti-inflammation; nitric oxide dulatory compounds at the molecular level.9–11) (NO) production Mast cells are distributed throughout the body and play a primary role in acute inflammation and allergic Actinidia polygama Max. (silver vine) is used as a responses.12,13) They are also important in mediating the folk medicine in Korea for treating pain, gout, and inflammatory response in allergic reactions by producing inflammation. Only a few studies have provided detailed cytokines, including IL-4, IL-5, IL-6, and tumor necrosis information on the medicinal effects of A. polygama factor (TNF)- .14) The rat basophilic leukemia cell line fructus extracts. A water extract of A. polygama sup- RBL-2H3 is a mucosal mast cell line, and is the major pressed rat paw edema induced by carrageenan, inhib- model system for antigen-induced degranulation.15) ited the production of nitric oxide (NO) induced by Several studies have shown that allergic inflammation lipopolysaccharide (LPS), and inhibited the expression mediated by mast cells is suppressed by phenolics of inducible NO synthase (iNOS) protein in RAW 264.7 or flavonoids, such as rutin and chlorogenic acid,16) macrophages.1) Additionally, it demonstrated anti-asth- EGCG,17) paeonol,18) fisetin, kaempferol, myricetin, matic effects in a murine model of asthma.2) Recently, quercetin, rutin,19) and morin.20) -linoleic acid was identified as an active component of Supercritical fluid extraction (SFE) is used to improve A. polygama fruits. It strongly inhibits NO production, the flavor of A. polygama. We investigated the anti- iNOS, cyclooxygenase (COX)-2, and tumor necrosis allergic effects of the ethanol extract of marc (SFEM), factor (TNF)- expression by blocking the activation of which results from SFE.21) Our aim was to isolate the nuclear factor kappa (NF-k) B and mitogen-activated constituent in the ethanol extract with the greatest anti- protein kinases (MAPKs) in LPS-activated RAW 246.7 inflammatory activity and to determine its structure. y To whom correspondence should be addressed. Tel: +82-53-580-6449; Fax: +82-53-580-6447; E-mail: [email protected] Abbreviations: SFE, supercritical fluid extraction; SFEM, marc obtained after supercritical fluid extraction of Actinidia polygama; LPS, lipopolysaccharide; NO, nitric oxide; iNOS, inducible NO synthase; COX-2, cyclooxygenase-2; TNF- , tumor necrosis factor; NF-kB, nuclear factor kappa B; IFN- , interferon- 110654-2 M.-H. BANG et al. Materials and Methods microplates. The next day, the cells were treated with various concentrations (0, 100, 200, and 400 mg/mL) of A. polygama extract Plant sample. Dried fruit of A. polygama was purchased from a at 37 C. After 48 h, 100 mL of MTT (5 mg/mL) was added to each Chinese drug store in Yeongcheon City, Gyeongbuk, Korea. A voucher well, and the plates were incubated at 37 C for 4 h. One hundred mL specimen (KHU-090723) was deposited at the Laboratory of Natural of DMSO was added to each well to dissolve the cells. The plates Products Chemistry, Kyung Hee University, Yongin, Korea. were kept at room temperature for 5 min, and the absorbance was measured at 550 nm using a multiwell spectrophotometer (Molecular Instruments and reagents. Melting points were determined using a Devices, Sunnyvale, CA). The cytotoxicity of the extract in the IgE- sensitized RBL-2H3 cells was also assessed by MTT assay. The Fisher-John apparatus (Fisher Scientific, Pittsburgh, PA) and were not cells (3 Â 104 cells/well) were seeded and incubated with 450 ng/mL corrected. Optical rotation was measured on a Jasco P-1010 digital polarimeter (Jasco, Tokyo). The IR spectrum was collected on a of DNP-specific IgE at 37 C overnight before treatment with the Perkin-Elmer Spectrum One FT-IR Spectrometer (Perkin-Elmer, extract. Norwalk, CT). EIMS was recorded on a JEOL JMS 700 (JEOL, Tokyo). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were Determination of LPS-induced NO production from RAW 264.7 5 assessed using a Varian Unity Inova AS 400 FT-NMR spectrometer cells. RAW 264.7 cells were seeded in 96-well plates (1 Â 10 cells/well) overnight and treated with various concentrations of test (Palo Alto, CA). CHCl3-d1 with TMS as internal standard was purchased from Sigma (St. Louis, MO). Dinitrophenyl (DNP)-specific samples (in 0.1% DMSO) in the presence of LPS (final concentration, IgE, Earle’s basal salt solution (EBSS), trypsin solution, 3-(4,5- 100 ng/mL) at 37 C for 24 h. After LPS stimulation for 24 h, NO dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and production in the cell culture medium was measured by the Griess 23) LPS were from Sigma (Sigma, St. Louis, MO). Fetal bovine serum Reagent System. Briefly, the culture supernatant (100 mL) was mixed (FBS), minimal essential medium (MEM), and other cell culture with same volume of Griess reagent (1% sulfanilamide and 0.1% reagents were from Gibco BRL (Grand Island, NY). DNP-bovine naphtylethylendiamine in 2.5% phosphoric acid) for 10 min, and the serum albumin was from Alpha Diagnostic International (San Antonio, absorbance was measured at 540 nm. The NO concentration was TX). All other reagents were from Sigma Aldrich (St. Louis, MO). calculated from a standard curve of NaNO2. Preparation of crude extract, marc of the supercritical fluid extract, Determination of antigen-induced -hexosaminidase release from and ethanol extract of it from A. polygama. One kg of dried RBL-2H3 cells. The inhibitory effect of the extract on the release of A.Advance polygama was ground and extracted 3 times with a 10-fold View excess -hexosaminidase in the RBL-2H3 cells was evaluated as reported by w/v of 70% ethanol at room temperature over 24 h. After filtering of Matsuda et al.,24) with modifications. In brief, RBL-2H3 cells seeded in the extract solutions, the combined filtrate was concentrated by rotary 24-well plates (2 Â 105 cells/well) were sensitized with anti-DNP IgE vacuum evaporator (Eyela Uni Trap UT-1000, Tokyo, Japan) at 55 C (450 ng/mL) at 37 C overnight. The cells were washed with and freeze-dried to use as the ethanol extract of A. polygama (EtOH, Siraganian buffer and then incubated in 160 mL of the incubation 306.9 g). Essential oils from A. polygama were extracted using the buffer at 37 C for 20 min. Then the cells were treated with 20 mLof Supercritical Fluid Extraction (SFE) apparatus (SFT-100XW, Super- extract for 10 min, followed by the addition of 20 mL of antigen (DNP- critical Fluid Technologies, Newark, DE). Briefly, 40 g of A. polygama BSA, 10 mg/mL) at 37 C for 10 min to stimulate the cells to granulate. were placed in an extraction vessel (100 mL), and SFE was statically The reaction was stopped by cooling in an ice bath for 10 min.