Journal Information
Journal ID (publisher-id): chemical
Title: Journal of the Korean Chemical Society
Translated Title (ko): 대한화학회지
ISSN (print): 1017-2548
ISSN (electronic): 2234-8530
Publisher: Korean Chemical Society대한화학회
INTRODUCTION▼
Shiranuhi is the general name of a fruit harvested from a hybrid Citrus species [(Citrus unshiu Marc.×C. sinensis Osbeck)×(C. Reticulata Blanco)]. It was originally introduced from Japan and then named Hallabong in Korea, as its round shape with a folded neck looks like Halla mountain. This fruit is characterized by its bulky size and sweet taste, and is one of the major crops grown in Citrus orchards in Jeju Island, Korea.1
We have investigated the peel of Shiranuhi fruit as a source of bioactive ingredients available for cosmetic formulations. This fruit is rich in polyphenols, vitamins, limonoids, carotenoids, terpene and flavonoids that are functional and valuable dietary ingredients for human health.2 A polymethoxyflavone (PMF) component was isolated from its immature fruit and the anti-inflammatory effect was elucidated in our study.3 The other biological effects reported in Shiranuhi peels include anti-oxidative,4 anti-obesity5 and anti-microbial6 activities. PMFs are natural products, mainly found in Citrus species in plant kingdom, with a chemical structure bearing two or more methoxy groups in their basic benzo-γ-pyrone flavone skeleton.7 PMFs have shown broad biological spectrum such as anti-inflammatory, anti-cancer,8 anti-mutagenic, anti-microbial9 and anti-aging10 activities.
As a continuing effort, we conducted the phytochemical study using the branch parts of the Shiranuhi tree. Most of the citrus research is focused on the fruit parts with interest in the food efficacy. In terms of finding bioactive constituents from plants in Jeju Island, we became interested in not only citrus fruits but also tree stems. Here, in this study, the ethanol extract of Shiranuhi branches was screened to exhibit the inhibition activities on NO production in LPS-induced murine macrophage RAW264.7 cells. Then, five PMFs were isolated, and their anti-inflammatory effects were determined along with the analysis of structure-activity relationships.
EXPERIMENTAL▼
General
The medium-pressure liquid chromatography (MPLC, Biotage Co., Sweden) was performed with ODS gel column (KP-C18-HS, Biotage Co., Sweden). Column chromatography was carried out using Sephadex LH-20 (10-25 μm, GE healthcare) or silica gel (40-630 μm, Merck, Germany) as stationary phases. Nuclear magnetic resonance (NMR) spectra were recorded with a JNM-ECX 400 instrument (FT-NMR system, JEOL, Japan).
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco Co. (USA). Lipopolysaccharide (LPS), Griess reagent and RIPA lysis buffer were purchased from Sigma-Aldrich (USA). ELISA kits were provided from R&D systems (USA) for PGE2 and IL-1β as well as Invitrogen (USA) for TNF-α and IL-6.
Plant Material
The branches of Shiranuhi tree were collected from the Citrus Research Institute (Jeju Island, Korea) in March 2015. A voucher specimen (No. 465) was prepared and deposited at the laboratory of Natural Product Chemistry, Department of Chemistry and Cosmetics, Jeju National University.
Extraction and Isolation
The Dried branches of Shiranuhi tree (1.3 kg) were extracted three times with 70% ethanol (EtOH, 26 L) at room temperature for 24 h under stirring. The combined solutions were concentrated under reduced pressure and dried to powder (187.4 g). The extract (180.0 g) was suspended in water (6 L) and then partitioned into n-hexane (Hex, 18 L), ethyl acetate (EtOAc, 18 L) and n-butanol (BuOH, 18 L) soluble fractions. Since the EtOAc fraction exhibited potent anti-inflammatory activities in LPS-induced RAW264.7 cells, the EtOAc fraction (5.0 g) was divided into 48 fractions (Fr. MP1-Fr. MP48) by MPLC with elution of H2O:MeOH gradient (10% to 100%). Out of these subfractions, fraction MP25 and 26 (146.8 mg) was subjected to Sephadex LH-20 column chromatography eluting with CHCl3:MeOH (15:1) to afford compound 4 (33.6 mg). Fraction MP29 to 32 (285.6 mg) was further purified using a silica gel column with Hex:EtOAc (1:2) providing compounds 1 (15.4 mg), 2 (47.2 mg), 3 (11.9 mg) and 5 (12.5 mg).
Cell Culture and Viability Assay
The murine macrophage cell line RAW264.7 was purchased from American Type Cell Culture (ATCC). The cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin, and incubated in a humidified atmosphere of 5% CO2 at 37 ℃.
The cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. RAW264.7 cells (2.0×105 cells/well) for 18 h, were induced with LPS (1 μg/mL) and treated with various concentrations of samples. After 24 h incubation, MTT reagent (500 μg/mL) was added to the medium, and which was allowed to stand for 4 h. After removing the supernatant, the formazan crystals were dissolved in DMSO. The optical density of each well was measured at 570 nm using a microplate reader (TECAN, Austria).
Measurement of NO, PGE2 and Cytokines Concentration
The NO production was assayed by measuring the nitrite in the supernatants of cultured cells. After RAW264.7 cells (2.0×105 cells/well) were incubated for 18 h, the cells were treated with LPS and samples for 24 h. Then, the equal volumes of supernatant and Griess reagent were mixed at room temperature for 10 min. Absorbance was measured at 540 nm using a microplate reader. The nitrite amounts in the test samples were calculated from NaNO2 standard curve.
The production of PGE2 and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) was determined in the culture supernatants. The amounts of PGE2, TNF-α, IL-1β and IL-6 produced by the cells were determined by ELISA kits.
Western Blotting
RAW264.7 cells (3.0×105 cells/well) were cultured in 60 mm culture dishes for 18 h and induced with LPS (1 μg/mL) in the presence or absence of samples. After 24 h incubation, the cells were washed with ice-cold phosphatebuffered saline (PBS) and lysed with RIPA lysis buffer. The cells were centrifuged for 20 min (15,000 rpm, 4 ℃) and the protein concentration in the cell lysate was quantified using Bradford assay. Protein samples (20 μg) were loaded on SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After transfer, the membranes were blocked with 5% skim milk in TTBS (Tris-buffered saline and 0.1% Tween 20) overnight at 4 ℃ and then probed with primary antibodies (iNOS; Santa Cruz, COX-2; BD, β-actin; Sigma). After 2 h incubation followed by five washes, the membranes were incubated with appropriate secondary antibodies (iNOS; Cell signaling, COX-2, β-actin; Santa Cruz) for 1 h. After final five washes with TTBS, the protein bands were visualized by ECL reagent and the densities of the bands were detected using Chemidoc (Fusion solo, VILBER LOURMAT, Germany).
Statistical Analysis
All experiments were performed in triplicate. The experimental data were expressed as the mean ± standard deviation (S.D.). Statistical analysis of the results was performed using the student’s t-test for independent samples. Values of *p<0.05 and **p<0.01 were considered significant.
RESULTS AND DISCUSSION▼
EtOH extract and solvent fractions were prepared using Shiranuhi tree branch parts, and their inhibitory activities on NO production was screened using LPS-stimulated RAW264.7 cells. The cells were incubated in the presence or absence of the samples (100 μg/mL), and NO generation was analyzed by Griess reagent. In order to determine the toxicity of the fractions to the cells, MTT assay was simultaneously conducted. As shown in Fig. 1A, among the tested samples, the EtOAc fraction exhibited most significant NO inhibition with little loss of cell viability. Within a concentration range from 20 to 60 μg/mL, the EtOAc fraction decreased NO generation in a dose dependent manner (IC50 30.3 μg/mL) without causing cell toxicity (Fig. 1B).
Figure1.
Effects of EtOH extract, solvent fractions (100 μg/mL) (A) and EtOAc fraction (B) on NO production and cell viability in LPS-induced RAW264.7 cells.
Based on the observed activity data, the EtOAc fraction was selected for further purification to identify the active chemical components. Use of column chromatography with silica gel or Sephadex LH-20 as well as MPLC with a reversed phase ODS gel led to the isolation of five constituents. All of the isolates were chemical species of PMF, whose structure were identified by comparative analysis of the NMR data to the literature values;11−14 nobiletin (1), sinensetin (2), tetramethylscutellarein (3), 6-hydroxy-5,7,3’,4’-tetramethoxyflavone (4) and 5-desmethylsinensetin (5). The PMFs 1-3 have earlier been identified in Shiranuhi2, 15 while the compounds 4 and 5 are reported here for the first time from this plant.
Compounds 4 and 5 are isomers in which hydroxy (-OH) and methoxy (-OCH3) groups are replaced each other at C-5 and C-6 positions. The compound 5 has a hydroxy at C-5 position inducing intramolecular hydrogen bond with carbonyl group at C-4. Therefore, the compound 5 shows a characteristic down field chemical shift of 182.7 ppm for C-4 in 13C NMR spectrum. In comparison, δC for carbonyl carbon (C-4) is within the range of 175.6-177.5 ppm for the compounds 1-4 all bearing methoxy groups at C-5.
The effects of isolated PMFs (1-5) on NO levels were measured using LPS-induced macrophage cells. As shown in Table 1, all of the compounds showed inhibitory activities under the reaction conditions. Among the isolates, the compound 4 showed the most potent inhibition on NO generation with IC50 value of 18.2 μM. With the isolated PMFs 1-5 at hand, it seems meaningful to analyze the relationship between the structure and activity in NO production. Compared with the nobiletin (1, IC50 98 μM), the sinensetin (2, IC50 116 μM) and tetramethylscutellarein (3, IC50 96 μM) exhibit similar activity. This suggests that even without methoxy groups in position R4 or R5, there is no significant difference on the activity. In terms of chemical structure, the most active compound 4 could be compared to the compound 2. The only difference between 2 and 4 lies at R2 position, where 2 and 4 have methoxy and hydroxy group respectively. Since the activity of 4 was observed more than six times higher than that of 2, this implies that the effect of hydroxy group at R2 position is important in the NO inhibition. Compared to 2, the compound 5 has the structure bearing hydroxy instead of methoxy at R1. Given 5’s activity is no higher than 2’s, the hydroxy group at R1 is considered to have little effect on the activity. At this point, analysis of given compounds 1-5 suggests that hydroxy group at R2 is important to exhibit higher activity, while changes of functional groups in other position have no significant impact.
Table1.
IC50 values of the isolated compounds1-5
Previously, nobiletin (1) and its metabolites have been investigated for the anti-inflammatory activity. A research group has identified that the demethylated nobiletin metabolites (e.g., 3’- or 4’-demethylnobiletin) isolated in mouse urine possess more potent activity than the parent compound (nobiletin, 1) in the study using RAW264.7 cells.16 This observation is in agreement of our result that the presence of hydroxy group in PMFs can affect the anti-inflammatory activity.
The compound 4 was investigated in more detail searching for the mechanism related to the anti-inflammation. Therefore, production of prostagladin E2 and pro-inflammatory cytokines were monitored by treating the PMF 4 on macrophage cells (Fig. 3). As shown in Fig. 3, while the compound 4 did not affect PGE2 production, it effectively decreases the levels of TNF-α (IC50 10.1 μM), IL-1β (IC50 22.3 μM) and IL-6 (IC50 20.4 μM) in dose dependent manner.
Since iNOS and COX-2 are major enzymes inducing inflammatory mediators such as NO and PGE2,17 the effect of their protein expression by PMF 4 was investigated (Fig. 4). The compound 4 was observed to significantly decrease iNOS level in a dose dependent mode with 71.1% inhibition at 30 μM concentration. As presented in Figure, on the while, COX-2 level was slightly decreased (11.3%) at 30 μM.
PMFs exist almost exclusively in Citrus genus18 and more than 20 PMFs have been identified from different tissues of the citrus plants.19 One of the most significant biological properties of the PMFs is their anti-inflammatory activity.20 Flavonoids with methoxy groups have been identified to inhibit the enzymes like iNOS and NADPH oxidase that generate free radicals like NO and superoxide anion.21 The anti-inflammatory effect of the 6-hydroxy-5,7,3’,4’-tetramethoxyflavone (4) has not been released yet. The results obtained in this study are well in line with the anti-inflammatory effects of the reported PMFs.
CONCLUSION▼
This study revealed that the EtOAc fraction of Shiranuhi tree branches significantly inhibited NO production in RAW264.7 cells. Further purification process identified five polymethoxyflavons (1-5) as active constituents. Among the isolates from Shiranuhi tree branches, 6-hydroxy-5,7,3’,4’-tetramethoxyflavone (4) exhibited the most potent inhibitory effect on NO production. From the careful analysis of the structure, it is suggested that hydroxy group at R2 (Fig. 2) exerts important effect on the NO inhibition activity. In addition, PMF 4 suppressed the levels of iNOS protein and pro-inflammatory cytokines. Isolation of the compounds 4 and 5 from Shiranuhi as well as anti-inflammatory activities of 4 were reported for the first time in this study. Based on these results, it was suggested that the extracts possessing PMFs from Shiranuhi tree branches could be potentially applicable as a functional ingredient against inflammation.