Turk J Biochem 2018; 43(4): 375–384 Research Article Min-Jin Kim, Sang Suk Kim, Ji Hye Ko, Young Il Moon, Kyung-Jin Park, Hyun Joo An, Young Hun Choi, Nam Ho Lee and Chang-Gu Hyun* Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 cells https://doi.org/10.1515/tjb-2016-0209 Keywords: Cyclooxygenase-2; Inducible Nitric Oxide Syn- Received January 12, 2017; accepted February 9, 2017 thase; Inflammation; MAPK; Shiranuhi Flower. Abstract Özet Objective: In this study, we evaluated the anti-inflamma- tory effect of Shiranuhi flower in RAW 264.7 cells. Amaç: Bu çalışmada, RAW 264.7 hücrelerinde Shiranuhi Methods: The effects of the extracts and solvent fractions çiçeğinin anti-inflamatuar etkisini değerlendirdik. on cell viability and LPS-induced inflammatory responses Yöntem: Ekstraktların ve çözücü fraksiyonlarının hücre were investigated in RAW 264.7 cells. sağlığı ve LPS ile uyarılan inflamatuvar tepkileri üzerin- Results: The results showed that the ethyl acetate fraction deki etkileri, RAW 264.7 hücrelerinde araştırılmıştır. (HEF) significantly decreased NO production in RAW 264.7 Bulgular: Sonuçlar etil asetat fraksiyonunun (HEF) RAW cells; however, cell viability was not affected. In addition, 264.7 hücrelerinde NO üretimini önemli ölçüde azalttığını ELISA assay revealed that HEF significantly inhibited gösterdi; ancak hücre canlılığı etkilenmemiştir. Buna ek the productions of PGE , TNF-α, and IL-6. As well, using 2 olarak, ELISA tahlili, HEF’in PGE2, TNF-α ve IL-6 üretim- Western blot analysis, it was observed that HEF signifi- lerini önemli ölçüde inhibe ettiğini ortaya koymuştur. cantly reduced the expression levels of iNOS and COX-2 Sonuçlar etil asetat fraksiyonunun (HEF) RAW 264.7 hüc- in a dose dependent manner. Furthermore, we detected relerinde NO üretimini önemli ölçüde azalttığını gösterdi; a reduced phosphorylation of mitogen-activated protein Ancak hücre canlılığı etkilenmemiştir. Buna ek olarak, kinases such as p38, JNK, and ERK1/2. This indicates that ELISA tahlili, HEF’in PGE2, TNF-α ve IL-6 üretimlerini HEF regulates LPS-induced inflammatory responses, at önemli ölçüde inhibe ettiğini ortaya koymuştur. Bunun least in part, via suppressing the MAPK signaling pathway. yanı sıra, Western blot analizi kullanılarak, Hef’in doza Correlation analysis also showed that anti-inflammatory bağımlı olarak iNOS ve COX-2 ekspresyonu anlamlı bir activities were highly correlated to antioxidant activities şekilde azalttığı gözlenmiştir. Dahası, p38, JNK ve ERK1/2 in this study. Characterization of the Shiranuhi flowers for gibi mitojenle aktive olan protein kinazların fosforilasyo- flavonoid contents using HPLC showed varied quantity of nunun azaldığını tespit edilmiştir. Bu bulgular, HEF’in, en narirutin and hesperidin. azından kısmen MAPK sinyal yolunu baskılayarak, LPS Conclusion: Overall, the results demonstrate that HEF ile uyarılan inflamatuvar tepkileri regüle ettiğini gösterir. may be a potential anti-inflammatory agent. In addition, Ayrıca bu çalışmada yapılan korelasyon analizi, anti-inf- our findings contribute to understanding the molecular lamatuvar aktivitelerin antioksidan aktivitelerle yüksek mechanism underlying the anti-inflammatory effect of oranda korele olduğunu göstermiştir. Shiranuhi çiçeğinin Shiranuhi flower. flavonoid içeriği HPLC kullanarak karakterize edildiğinde çeşitli miktarlarda narirütin ve hesperidin bulunmuştur. Sonuç: Genel olarak, sonuçlar HEF’in potansiyel bir *Corresponding author: Chang-Gu Hyun, Department of Chemistry anti-inflamatuar ajan olabileceğini göstermektedir. Buna and Cosmetics, Jeju, Korea (the Republic of), e-mail: [email protected] ek olarak, bulgularımız Shiranuhi çiçeğinin anti-infla- Min-Jin Kim, Ji Hye Ko and Nam Ho Lee: Department of Chemistry matuar etkisinin altında yatan moleküler mekanizmanın and Cosmetics, Jeju, Korea (the Republic of) anlaşılmasına katkıda bulunmaktadır. Sang Suk Kim, Young Il Moon, Kyung-Jin Park, Hyun Joo An and Young Hun Choi: Citrus Research Institute, Seogwipo, Anahtar Sözcükler: Siklooksijenaz-2; Indüklenebilir nitrik Korea (the Republic of) oksit sentaz; Inflamasyonu; MAPK; Shiranuhi çiçeği. 376 Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide Introduction anti-inflammatory [21], antioxidant [22], anti-obesity [23], and anti-angiogenic effects [24]. In addition, the Inflammation is a highly regulated defensive process that peel has been noted to inhibit breast cancer cell migra- results from tissue injury or external stimuli. It leads to tion. However, potential biological activities of Shiranuhi the release of numerous inflammatory mediators and flower have not been explored. Therefore, in this study, ultimately triggers the restoration of tissue structure the Shiranuhi flower was investigated for its potential and function [1–3]. However, uncontrolled and excessive anti-inflammatory activity. To the best of our knowledge, inflammatory responses can cause severe tissue damage this is the first study to demonstrate that Shiranuhi flower and secondary inflammatory injuries, such as sepsis, shows an anti-inflammatory activity. asthma, rheumatoid arthritis, vascular diseases, and cancer [4, 5]. Macrophages play a crucial role in provid- ing an immediate defense by directly counteracting the aforementioned stimuli by releasing cellular signaling Materials and methods molecules such as inflammatory mediators and cytokines [6, 7]. In a normal state, these inflammatory mediators Preparation of Shiranuhi flower extracts and cytokines released from macrophages are essential for host survival and tissue repair [8]. Lipopolysaccha- Shiranuhi flowers were collected from Citrus Research ride (LPS) is a molecule obtained from Gram-negative Institute (RDA, Jeju Island) and were identified by Dr. bacteria. It induces the release of many pro-inflamma- Young Hun Choi. A voucher specimen was prepared and tory mediators and cytokines such as nitric oxide (NO), deposited at the Cosmetic Science Center at Jeju National prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, University (Korea). interleukin (IL)-6, and IL-1β from macrophages [9, 10]. The dried flowers (1.39 kg) were soaked in 5 L of Therefore, a decrease in LPS-induced expression of pro- 80% ethanol at room temperature for 24 h, after which inflammatory mediators and cytokines in macrophages the solvent was evaporated under vacuum. The residue may be considered a significant therapeutic property obtained was freeze-dried and stored in a desiccator at 4°C during the development of anti-inflammatory agents before use. The weight of the dried sample was 52.48 g; [11–13]. Accumulating evidence indicates that mitogen- therefore, the yield of the extraction was 3.78%. The dried activated protein kinases (MAPKs) also induce the pro- extract (20 g) was then sequentially suspended in water duction of inflammatory molecules and immune-related (1 L) and extracted with ethyl acetate (1 L). The recoveries cytotoxic factors via a sequence of phosphorylation of the extract from the ethyl acetate and water fractions events. The MAPK family is composed of subfamilies, were 12.5% (2.49 g) and 70.5% (14.1 g), respectively. which include members such as extracellular signal- For the water extract, the dried flowers (700 g) were regulated kinase (ERK), c-Jun N-terminal kinase (JNK), ground into powder and extracted with 3 L of water in and p38. Furthermore, many studies have demonstrated a water bath at 80°C for 5 h. Next, the mixture was fil- that MAPKs are important in modulating the expressions tered and the filtrate was concentrated to about 250 mL. of cyclooxygenase (COX)-2 and inducible NO synthase The water extract of the Shiranuhi flower was thereafter (iNOS) and the productions of NO, PGE2, and pro-inflam- obtained by lyophilizing the concentrate. The yield was matory cytokines, such as IL-1β, TNF-α, and IL-6 [14–16]. 25.4 g (3.6%). In this study, each fraction and extracts were Thus, regulating the activation of MAPK pathways may be dissolved in DMSO and filtered through syringe filters a useful strategy in modulating inflammatory responses (0.45 μm pore size). [17–19]. Shiranuhi [(Citrus unshiu Marcov × C. sinensis Osbeck) × C. reticulata Blanco] is a seedless and sweet Reagents and cell culture variety of mandarin orange that has been cultivated on Jeju Island (Korea) since 1988, after its development in Murine RAW 264.7 macrophages were purchased from Japan in 1972. Its trade name is ‘Hallabong’, named after American Type Culture Collection (Manassas, VA, USA). the Hallasan Mountain located on Jeju Island. Shiranuhi Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal is also the name of a genus of flowering plants in the bovine serum (FBS) were purchased from Gibco (Grand Rutaceae family. The Shiranuhi fruit is primarily used Island, NY, USA). LPS, antibiotics (penicillin and strepto- for its juice; however, its peel has diverse pharmacologi- mycin), and bovine serum albumin (BSA) were obtained cal properties including anti-tumor, anti-metastatic [20], from Sigma-Aldrich (St. Louis, MO, USA). Lactate Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide 377 dehydrogenase (LDH) cytotoxicity detection kit and Determination of cytokine levels enzyme-linked immunosorbent assay (ELISA) antibody sets were purchased from Promega (Madison, WI, USA) The concentrations of PGE2, TNF-α, and IL-6 in the and R&D Systems Inc. (Minneapolis, MN, USA),