Chlorogenic acid from the Japanese herbal medicine Kinginka (Flos Lonicerae japonicae) suppresses the expression of inducible nitric oxide synthase in rat hepatocytes

Background: Flos Lonicerae japonicae (FLJ; Kinginka) is the dried flowers and buds of the Japanese honeysuckle Lonicera japonica Thunberg. FLJ has been used as a Japanese Kampo medicine to treat infectious and inflammatory diseases. However, it is not clear which constituent of FLJ is responsible for its pharmacological effects. Methods: FLJ was extracted with methanol and fractionated by hydrophobicity. We measured the effects of each fraction on the induction of the inflammatory mediator nitric oxide (NO), which was induced by interleukin 1β in primary cultured rat hepatocytes. To estimate cytotoxicity, the activity of lactate dehydrogenase released from the hepatocytes was measured. The expression of inducible nitric oxide synthase (iNOS) was analyzed by Western blot analysis and reverse transcription-polymerase chain reaction. Results: The methanol extract was fractionated into hydrophobic (11.1%), butanol-soluble (16.4%), and water-soluble fractions (72.5%). These three fractions dose-dependently suppressed the induction of NO and reduced the level of iNOS protein in interleukin 1β-stimulated hepatocytes. Chlorogenic acid, a major constituent of the water-soluble fraction, significantly reduced the levels of NO production, iNOS protein, and iNOS mRNA. Chlorogenic acid also decreased the levels of mRNAs encoding cytokines and chemokines that are involved in inflammatory disease. Caffeic acid, which is formed by the hydrolysis of chlorogenic acid, markedly reduced the induction of NO, although it did not exist at a detectable level in the water-soluble fraction. In contrast, other constituents of the water-soluble fraction, such as inositol fructose, glucose, and sucrose, did not affect the induction of NO. Conclusions: The anti-inflammatory effects of the FLJ extract and its constituents were analyzed by measuring the induction of NO and iNOS in hepatocytes. We demonstrated that chlorogenic acid, one of the main constituents of FLJ, is involved in the anti-inflammatory effect of the FLJ extract, suggesting its therapeutic potential. © 2012 Nishizawa et al; licensee Herbert Publications Ltd. This is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). This permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: [email protected] 1 Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan. Full list of Author’s information is available at the end of the article Background The Japanese honeysuckle Lonicera japonica Thunberg is a vine shrub that commonly grows in Japan, Korea, and China, and flowers of Lonicera japonica are sweetly scented and produce nectar [1]. The flowers are white at first and then become yellow. Flos Lonicerae japonicae (FLJ; Kinginka in Japanese), the dried flowers and buds of Lonicera japonica, is included in various prescriptions of Japanese Kampo medicine to treat infectious and inflammatory diseases due to its antiviral, antibacterial, anti-inflammatory, and antipyretic effects [2]. FLJ is popularly used also in China as a traditional medicine, such as Yin Hua tea and Jin Yin wine [2]. However, a mechanism of the pharmacological effects of FLJ is not well understood. More than 100 constituents have been isolated from the flowers, leaves, and stems of Lonicera japonica [2]. Some of the most prominent constituents of Lonicera japonica are chlorogenic acids, which is a family of esters formed between trans-cinnamic acids and quinic acid that is also abundant in coffee beans [2,3]. The most common chlorogenic acid is an ester of caffeic acid and quinic acid, also known as 5-O-caffeoylquinic acid. The contents of chlorogenic acid in the FLJ samples collected in different locations are determined to be 19.8–29.5 mg/g 50% methanol extract by high-performance liquid chromatography (HPLC) coupled with diode-array and evaporative light scattering detectors [4]. A few constituents involved in the pharmacological activity of Lonicera japonica have been reported. Luteolin, a flavonoid isolated from the flowers of Lonicera japonica, suppresses the expression of tumor necrosis factor α (TNF-α) and cyclo-oxygenase 2 (COX-2) in human mast cells [5]. In addition, the purified extract from the stems of Lonicera japonica, which are rich in loganin and sweroside, has anti-inflammatory and analgesic activity in mice [5]. However, luteolin, loganin, and sweroside are not major constituents of the Lonicera japonica extract [4]. It remains unclear which constituent in FLJ is primarily responsible for its anti-inflammatory activity and how the constituent suppresses inflammation. Not only TNF-α and COX-2 but also other molecules that are involved in inflammation remain to be investigated to understand the anti-inflammatory activity of FLJ. Herbal medicines generally consist of many compounds, and it is difficult to isolate and identify their pharmacologically active constituents. According to our data to purify more than 20 herbal medicines, cytotoxic compounds were often constituents of the total extract of herbal medicine, for example, the rhubarb Rhei Rhizoma [7]. Such cytotoxic compounds, including saponins and tannins, may mask the pharmacological activities of an herbal extract in HOAJ Biology ISSN 2050-0874 Nishizawa et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-2.pdf 2 doi: 10.7243/2050-0874-1-2 in vitro assays. Therefore, we established a standardized protocol to fractionate the herbal extract of FLJ by hydrophobicity and estimate the pharmacological activity of each fraction. We analyzed the anti-inflammatory effect of the FLJ extract on the induction of the inflammatory mediator nitric oxide (NO), which was induced by the pro-inflammatory cytokine interleukin 1β (IL-1β) in rat hepatocytes. The induction of NO and inducible nitric oxide synthase (iNOS) in the IL-1β-stimulated hepatocytes mimics liver injury [8,9]. We simultaneously estimated the cytotoxicity of the FLJ fractions by measuring the activity of lactate dehydrogenase (LDH) released from the injured hepatocytes. Furthermore, the FLJ extract may affect on expression of the cytokines and chemokines that are induced by IL-1β. In this study, we indicated that chlorogenic acid, the main constituent of the FLJ extract, suppresses induction of NO production and expression of the cytokines and chemokines that are involved in inflammation. We demonstrated that chlorogenic acid is the main constituent responsible for the anti-inflammatory activity of FLJ in hepatocytes and discuss the structure-activity relationship of chlorogenic acid and its metabolites with respect to their therapeutic potential. Materials & Methods Plant material, extraction, and fractionation (ABC fractionation): The flowers and buds of Lonicera japonica Thunberg, which were collected in Henan Province, China, and identified and authenticated by Dr. Yutaka Yamamoto (Tochimoto Tenkaido Co. Ltd., Osaka, Japan) and Professor Yukinobu Ikeya, a co-author of this paper, were purchased from Tochimoto Tenkaido Co. Ltd. The voucher specimen was deposited in the Ritsumeikan Herbarium of Pharmacognosy, Ritsumeikan University (Kusatsu, Shiga, Japan) under code number RIN-LJ-010. The dried flowers and buds (length, 3.0–11.0 mm; diameter, 0.9–1.4 mm) of Lonicera japonica (100.1 g) were extracted twice with absolute methanol under reflux for 1 h. The solvent was evaporated under reduced pressure, yielding the methanol extract. The resultant methanol extract was resuspended in water. After filtration of the suspension, it was successively partitioned with ethyl acetate and n-butanol. These layers were concentrated to give an ethyl acetate-soluble fraction (fraction A; hydrophobic), an n-butanol-soluble fraction (fraction B), and a water-soluble fraction (fraction C; hydrophilic) (Figure 1). Fraction C was further fractionated by ultrafiltration with a U-Tube Concentrator 2H-2 (Novagen, Madison, WI, USA) at a molecular weight (MW) cutoff of 2,000 Da, and the filtrate was collected by centrifugation at 1,600 × g for 9 h. The analysis of chlorogenic acid by HPLC: Quantitative analyses of chlorogenic acid in fraction C were performed using a Shimadzu LC-20A series HPLC instrument equipped with an SPD-20S detector at 254 nm (Shimadzu Corporation, Kyoto, Japan). Samples were separated by a Cosmosil Cholester column (4.6 mm internal diameter × 150 mm; Nacalai Tesque Inc., Kyoto, Japan) at 0.8 mL/min with a mobile phase of absolute acetonitrile:5% (v/v) acetic acid (10:90 to 50:50 over 35 min). Chlorogenic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used as an analytical standard. The analysis of sugar composition by HPLC: The fraction C sample was dissolved in water and injected into a Cosmosil Sugar D column (4.6 mm internal diameter × 250 mm; Nacalai Tesque Inc.) equilibrated with 80% (v/v) acetonitrile at 1 mL/ min delivered by a Shimadzu LC-20 AT (Shimadzu Corporation). Elution was monitored by refractive index using a Shimadzu RID-10A. Data were collected and processed by a Shimadzu CR-7A. As analytical standards, fructose, glucose, sucrose, and myo-inositol (Wako Pure Chemical Industries, Ltd.) were used. Preparation of primary cultured rat hepatocytes: Hepatocytes were isolated from the livers of male Wistar rats (Charles River Laboratories Japan Inc., Yokohama, Japan) by collagenase perfusion as previously described [10]. Briefly, dispersed cells were centrifuged at 50 × g for 70 sec and further purified by centrifugation three times to remove non-parenchymal cells. The pellet was resuspended in Williams’ E medium (Sigma-Aldrich Corp., St. Louis, MO), seeded at 1.2 × 106 cells/dish, incubated at 37°C for 2 h, and the medium was replaced twice with fresh medium containing 10% newborn bovine serum (SAFC Biosciences, Inc., Lenexa, KS, USA). The purity of the resultant hepatocytes was greater than 99% by microscopic observation (data not shown). The hepatocytes were incubated at 37°C overnight and treated with 1 nM rat IL-1β (PeproTech, Rocky Hill, NJ, USA) and a FLJ fraction or a compound. The animal experiments were approved by the Animal Care Committee of Ritsumeikan University, Biwako-Kusatsu Campus. Figure 1. A flowchart of the procedures to fractionate the FLJ extract. The fractionation procedures are schematically shown. The weights of the fractions and the constituents included in each fraction are indicated. The dried flowers and buds of Lonicera japonica (FLJ) were extracted with methanol and dried. This FLJ extract was sequentially fractionated by ethyl acetate (fraction A), n-butanol (fraction B), and water (fraction C). See also Table 2. Nishizawa et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-2.pdf 3 doi: 10.7243/2050-0874-1-2 Determination of NO production and LDH activity: To indirectly measure the production of NO, triplicate measurements of nitrite (a stable metabolite of NO) by the Griess method [11] in the culture medium were performed, and the half-maximal inhibitory concentration (IC50) was determined in triplicate (n = 3 dishes per point) with at least three different concentrations. When a fraction or compound are not cytotoxic to hepatocytes, the NO levels at the concentrations are inversely proportional to log10[concentration] (i.e., dose-dependent) and thus used to determine the IC50 values. As an indicator of cell viability and cytotoxicity, the LDH activity in the culture medium was measured, in triplicate (3 dishes per point), using LDH Cytotoxicity Detection Kits (Takara Bio Inc., Otsu, Japan). Caffeic, ferulic, isoferulic, and p-coumaric acids were purchased from Wako Pure Chemical Industries, Ltd. or Nacalai Tesque Inc. Western blot analysis: Total cell lysates were prepared essentially as described previously [12]. Briefly, cells (1 × 106 cells/35-mm dish) were lysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (final concentrations of 125 mM Tris-HCl, pH 6.8, 5% glycerol, 2% SDS, and 2% 2-mercaptoethanol), subjected to SDSPAGE and electroblotted onto a Sequi-Blot membrane (Bio-Rad, Hercules, CA, USA). Immunostaining was performed using primary antibodies against rat iNOS (Thermo Fisher Scientific, Waltham, MA, USA) and rat β-tubulin (internal control; Cell Signaling Technology, Inc., Danvers, MA, USA), followed by visualization with an Enhanced Chemiluminescence Blotting Detection Reagent (GE Healthcare Biosciences Corp., Piscataway, NJ, USA). Reverse transcription-polymerase chain reaction (RT-PCR): Total RNA was prepared from hepatocytes (2 dishes/point) using Sepasol I Super (Nacalai Tesque Inc.) and TURBO DNA-free kits (Applied Biosystems, Austin, TX, USA). The cDNA was reverse-transcribed in a strand-specific manner with an oligo(dT) primer and a sense (forward) primer for mRNA and as RNA, respectively [13]. Step-down PCR [14,15] was performed with paired primers (Table 1), and mRNA levels were estimated in triplicate by real-time PCR with SYBR Green I and the Thermal Cycler Dice Real Time System (Takara Bio Inc.), as described previously [13]. The values obtained were normalized to elongation factor 1α (EF) mRNA. Statistical analysis: The results in the figures and Table 2 are representative of at least three independent experiments yielding similar findings. Values are represented as the mean ± standard deviations (SD). Differences were analyzed using the Student’s t-test. Statistical significance was set at P < 0.05.