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Myricetin from Rhodomyrtus tomentosa (Aiton) Hassk fruits attenuates inflammatory responses in histamine-exposed endothelial cells
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Myricetin from Rhodomyrtus tomentosa (Aiton) Hassk fruits attenuates inflammatory responses in histamine-exposed endothelial cells Thanh Sang Voa,*, Young-Sang Kimb, Dai-Nghiep Ngoc, Dai-Hung Ngod,* a
NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City, Viet nam Department of Chemistry, Pukyong National University, Busan, 608-737, Republic of Korea c Department of Biochemistry, Faculty of Biology and Biotechnology, University of Science, Vietnam National University, Ho Chi Minh City, Viet nam d Faculty of Natural Sciences, Thu Dau Mot University, Thu Dau Mot City, Binh Duong Province, Viet nam b
A R T I C LE I N FO
A B S T R A C T
Keywords: Adhesion molecules Endothelial cells MCP-1 Myricetin Rhodomyrtus tomentosa
Histamine plays a key role in inflammatory responses via increasing chemokine and adhesion molecule productions and augmenting vascular permeability in endothelial cells. Rhodomyrtus tomentosa has been found as a rich source of structurally diverse and biologically active metabolites. In this study, the role of phenolic compound from Rhodomyrtus tomentosa (Aiton) Hassk fruits in down-regulation of histamine-induced EA.hy926 endothelial cell activation was investigated. Herein, myricetin was successfully isolated from R. tomentosa fruits by HPLC and its characterization was identified by mass spectrometer and nuclear magnetic resonance spectroscopy. It was revealed that myricetin was effective in suppression of IL-8 and MCP-1 productions and adhesion molecule generation. Moreover, NF-κB activation was inhibited by myricetin via reducing IκB-α phosphorylation and p50/p65 subunit level. Notably, myricetin potentially attenuated vascular permeability of endothelial cells through decrease in eNOS phosphorylation and intracellular calcium elevation. These results indicate that myricetin from R. tomentosa possesses a promissing pharmaceutical property for the amelioration of endothelial inflammatory responses.
1. Introduction Mast cell activation plays an important role in allergic inflammatory reaction by release of various preformed and synthesized mediators [1]. Notably, histamine mediating its receptors causes inflammatory responses due to increase in vasodilation, edema, vascular permeability, and blood flow [2]. Several studies have determined that histamine induced vascular permeability by activating endothelial cells in vitro and in vivo [3–5]. This action of histamine facilitates the transition of leukocytes such as neutrophil, eosinophil, and T cells to the late-phase reaction that leads to the development of inflammatory responses [6]. Thus, the inhibition of histamine-induced endothelial cell activation has been considered as a key therapeutic strategy for amelioration of allergic inflammatory development. Flavonoids and other phenolic compounds are the main class of secondary metabolites and widely distributed in fruits, vegetables, and beverages [7]. They are known to contain an aromatic ring bearing at least one hydroxyl groups and are biosynthesized by the shikimate or acetate/malonate pathway to produce a wide range of phenolic compounds with different structures [8]. So far, almost 8000 phenolic
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compounds as naturally occurring substances in different parts of the plants have been identified in numerous studies [9]. Interestingly, the potential pharmaceutical and medical applications of phenolic compounds have been revealed due to antioxidant, anti-inflammation, antiallergy, anticancer, antimicrobial, anti-diabetes, cardio-protection, UVprotection, and immune-promotion activities [10,11]. Therefore, phenolic compounds have got much attention by researchers regarding their versatile benefits for human health. In this sense, Rhodomyrtus tomentosa, a flowering plant in family Myrtaceae, has been found as a rich source of structurally diverse and biologically active metabolites of phenolic compounds [12]. In the previous study, the inhibitory effect of R. tomentosa fruit extract on mast cell activation has been determined via suppression of degranulation [13]. This activity of R. tomentosa fruit extract was suggested due to phenolic compounds. In the present study, myricetin was purified from R. tomentosa fruit extract, and its suppressive effect on histamine-induced endothelial cell activation was examined.
Corresponding authors. E-mail addresses: [email protected] (T.S. Vo), [email protected] (D.-H. Ngo).
https://doi.org/10.1016/j.procbio.2020.02.004 Received 5 September 2019; Received in revised form 3 February 2020; Accepted 3 February 2020 1359-5113/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Thanh Sang Vo, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2020.02.004
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fluorescence probe, Fura 3-AM. EA.hy926 cells (1 × 104 cells/ml) were treated with myricetin for 24 h before incubated with Fura-3/AM (2 μM, final concentration) for 60 min at 37 °C. The cells were washed with PBS buffer and exposed to histamine (10 μM, final concentration) at 37 °C for 10 min. The fluorescence intensity was monitored under a fluorescence microscope (CTR 6000, Leica, Wetzlar, Germany).
2. Materials and methods 2.1. Materials R. tomentosa fruits were collected from Duong Dong Town, Phu Quoc district, Kien Giang province. Enzyme immunoassay kits for IL-8 and MCP-1 were purchased from R&D Systems (Minneapolis, MN, USA). Specific antibodies including p-IκB-α, p50, p65, p-eNOS, and βactin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). All other reagents including calcein-AM, Fura-3/AM, histamine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide were purchased from Sigma–Aldrich (St. Louis, MO, USA). EA.hy926 cells (ATCC® CRL2922™) and U937 cells (ATCC® CRL1593.2™) were purchased from ATCC (PO Box 1549 Manassas, VA 20108 USA).
2.7. Western blot analysis EA.hy926 cells were treated with myricetin (10, 20, or 40 μM) for 24 h before stimulated with histamine (10 μM, final concentration). The cells were lysed in RIPA lysis buffer and the detection was performed as described by Vo et al. [15]. Briefly, cell lysates (25 μg of protein/ sample) were subjected to 10 % SDS-PAGE, transferred onto a nitrocellulose membrane, and blocked by TBS-T buffer containing 5 % (w/v) bovine serum albumin (BSA). The membrane was washed and probed with primary antibodies (diluted 1:1000) for at least 1 h. Subsequently, horseradish peroxidase (HRP)-conjugated IgG secondary antibody (diluted 1:5000) was applied for 1 h. Protein bands were visualized by an enhanced chemiluminescent ECL assay kit (Amersham Pharmacia Biosciences, UK) and LAS3000® Luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan).
2.2. Extraction and isolation The powdered fruits of R. tomentosa (1 kg) were extracted with ethyl acetate (EtOAc) at 40 °C for 24 h. Crude extract was evaporated under vacuum to achieved 11.2 g EtOAc extract. This extract (1 g) was suspended in ethyl acetate and separated by silica gel column. It was respectively eluted with hexan-chloroform (10:0 to 2:8), chloroform-ethyl acetate (10:0 to 2:8), ethyl acetate-ethanol (10:0 to 2:8), and ethanol to achieved 16 fractions. The fraction of ethyl acetate-ethanol (8:2) was subjected to Supelco column (250 × 21.2 mm, 10 μm) of HPLC (Shimadzu LC 8A, Japan), eluted with H2O-acetonitril (90:10 to 5:95), and detected at 360 nm to obtain compound A (15.1 mg). The compound A was characterized by high-resolution MicrOTOF QII Mass Spectrometer (Bruker Daltonics, Bremen, Germany) and NMR (Bruker, 20251 Hamburg, Germany).
2.8. Statistical analysis Statistical analysis was performed by using the analysis of variance (ANOVA) test of statistical package for the social sciences (SPSS). The statistical significance of differences among groups was analyzed using Duncan’s multiple range tests wherein p < 0.05 was considered significant. 3. Results and discussion
2.3. Cell viability 3.1. Structure evaluation of myricetin from R. Tomentosa The cell viability levels were determined by MTT assay. EA.hy926 cells (2 × 105 cells/ml) were treated with myricetin (10, 20, or 40 μM) for 24 h. The cell viability was measured as described by Ngo et al. [14]. Briefly, the cells were incubated with MTT solution (1 mg/ml) for 4 h, and DMSO (100 μl) was subsequently added. The absorbance was measured at 540 nm using a microplate reader (GENios® Tecan Austria GmbH, Austria). The cell viability was quantified as a percentage compared to blank.
Compound A was obtained as yellow needles. The 1H-NMR and 13CNMR spectrum were showed in Fig. 1. The 1H-NMR spectrum showed a down field signal at δ 12.15 of a chelated hydroxyl group (1H, s, 5−OH). It also exhibited a characteristic meta-coupling proton signal at δ 6.26 (1H, d, J =2.0 Hz, H-6) and 6.50 (1H, d, J =2.0 Hz, H-8) for the A-ring of flavonoid. The AX coupling system at δ 7.42 (2H, s) was assigned to H-2′ and H-6′ of B ring. The 13C-NMR spectrum showed 13 signals for 15 carbons of a flavone skeleton including a conjugated ketone group at δC 176.6 (C-4) (Table 1). By comparing the NMR spectral data with those reported in literature, the structure of compound A was determined as myricetin (Fig. 2) [16].
2.4. Measurement of IL-8 and MCP-1 production EA.hy926 cells (4 × 104 cells/ml) were treated with myricetin (10, 20, or 40 μM) for 24 h before stimulated with histamine (10 μM, final concentration) for 24 h. The supernatants were collected, and the productions of IL-8 and MCP-1 were quantified by sandwich immunoassays following the protocol of R&D systems.
3.2. The inhibitory effect of myricetin on IL-8 and MCP-1 productions Endothelial cells are known to involve in the initiation of inflammatory process [17]. The activation of endothelial cells causes increase in vascular permeability and up-regulation of pro-inflammatory cytokines, chemokines, and adhesion molecules [18,19]. Notably, NF-κB pathway-mediated productions of monocyte chemoattractant protein (MCP)-1 and IL-8 contribute to the inflammatory endothelial cell response [20]. Therefore, the inhibition of these chemokine productions could suppress inflammatory responses. Herein, the inhibitory effect of myricetin on IL-8 and MCP-1 productions was examined in culture supernatants by enzyme-linked immunosorbent assay. It was shown that IL-8 and MCP-1 productions were increased from histamine-exposed EA.hy926 cells. The levels of IL-8 and MCP-1 were produced up to 132 ± 9 and 615 ± 15 pg/ml, respectively. Meanwhile, these increases were significantly lowered in a dose-dependent manner by myricetin treatment. Myricetin reduced IL-8 and MCP-1 productions up to 51 ± 6 and 207 ± 11 pg/ml at concentration of 40 μM, respectively (Fig. 3A and B). It indicated that myricetin
2.5. In vitro cell adhesion assay U937 cells (2 × 104 cells/ml) were incubated with calcein-AM (5 μM, final concentration) in the dark at room temperature for 60 min. Meanwhile, EA.hy926 cells (1 × 104 cells/ml) were treated with myricetin for 24 h before stimulated with histamine (10 μM, final concentration) for 24 h. Thereafter, EA.hy926 cells were overlaid with calcein-AM-labelled U937 cells for 1 h. The unbound cells were removed by washing the plates. Fluorescence images were visualized and photographed under fluorescence microscope (CTR 6000, Leica, Wetzlar, Germany). 2.6. The intracellular Ca2+ elevation assay Intracellular calcium level was measured using the calcium reactive 2
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Fig. 1. Spectra 1H-NMR (A) and
13
C-NMR (B) of myricetin from R. tomentosa fruits.
myricetin on IL-8 and MCP-1 productions was not due to cytotoxicity.
treatment suppressed endothelial cell activation that caused the reduction of IL-8 and MCP-1 productions in histamine-exposed EA.hy926 cells. According to Jang and colleagues, scoparone, a major component of Artemisia capillaris shoot, has been reported to inhibit IL-8 and MCP-1 productions in PMA-stimulated U937 cells [21]. Moreover, Hao and colleagues have determined that cafestol, a diterpenoid in coffee beans, significantly inhibited IL-8 and MCP-1 productions from cyclic-straininduced vascular endothelial cells (HUVEC cells) [22]. It was observed that the inhibitory effect of myricetin on IL-8 and MCP-1 productions was similar with that of scoparone. However, cafestol was shown to be more effective than myricetin on the inhibition of IL-8 and MCP-1 productions. In order to exclude the inhibition of myricetin on IL-8 and MCP-1 productions due to cytotoxic effect, MTT test was further examined on EA.hy926 cells. The result showed that myricetin did not cause significantly cytotoxic effect on EA.hy926 cells at concentration of 10, 20, or 40 μM (Fig. 3C). It indicated that the inhibitory activity of
3.3. The inhibitory effect of myricetin on adhesion molecule production So far, numerous adhesion molecules have been identified due to their structure and function, including the selectin, integrin and immunoglobulin families of adhesion proteins [23]. Adhesion molecules are known to participate in the pathogenesis of inflammatory diseases including atherosclerosis, rheumatoid arthritis, psoriasis, diabetes, multiple sclerosis, and inflammatory bowel diseases [24]. The production of adhesion molecules on surface of the activated endothelial cells plays a vital role in inflammatory responses, such as leukocyte emigration, lymphocyte recirculation, platelet adhesion, and phagocytosis [25]. Therefore, suppression of adhesion molecule production from the activated endothelial cells can block leukocyte emigration and lymphocyte recirculation that leads to alleviation of inflammatory 3
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Table 1 1 H (500 MHz) and C
that the density of calcein-AM-labelled U937 cells were significantly increased on the monolayer of EA.hy926 cells in the control group, indicating increase in adhesion molecule production from EA.hy926 cells. Meanwhile, the density of calcein-AM-labelled U937 cells was reduced in the presence of myricetin at concentration of 40 μM (Fig. 4). Likewise, it was reported that fisetin and quercetin inhibited the expression of adhesion molecules, causing to decrease in THP-1 monocyte adhesion to inflammatory cells (A549 cells or ARPE-19 cells) [26,27]. Especially, anti-inflammatory drugs such as dexamethasone, 5-aminosalicylic acid, methotrexate, and 6-mercaptopurine inhibited adhesion molecule expression, thus blocking leucocyte adhesion and transmigration in the microvasculature [28]. Accordingly, the inhibitory activity of myricetin on adhesion molecule production may contribute to the alleviation of leucocyte adhesion and transmigration into the latephase of inflammatory reaction.
C (125 MHz) NMR data for compound A in acetone-d6. Myricetina
Compound A
2 3 4 5 6 7 8 9 10 1' 2′, 6′ 3', 5′ 4' 5-OH a
13
δH (mult., J = Hz)
δC
δH (mult., J = Hz)
δC
— — — — 6.26 (1H, d, 2.0) — 6.50 (1H, d, 2.0) — — — 7.42 (2H, s) — — 12.15 (1H, s)
147.1 136.5 176.6 162.4 99.2 165.1 94.5 157.9 104.2 122.9 108.4 146.5 137.0 —
— — — — 6.13 (brs) — 6.41 (brs) — — — 7.17 (s) — — —
147.1 136.1 176.0 160.9 98.6 164.4 93.7 156.5 103.2 121.1 107.5 146.0 136.2 —
3.4. The inhibitory effect of myricetin on NF-κB activation
δH and δC of myricetin recorded in DMSO-d6.
Nuclear factor-κB (NF-κB), a family of inducible transcription factors, is composed of five subunits including p50, p52, p65, RelB, and cRel, and is normally regulated by inhibitory protein IκBα in the cytoplasm. NF-κB serves as a key mediator of inflammatory responses through regulating the expression of various pro-inflammatory genes [29]. Notably, the role of NF-κB on regulation of the expression of chemokines and adhesion molecules in endothelial cells has been
responses. In this study, we also examined whether myricetin can reduce the production of adhesion molecules in histamine-stimulated EA.hy926 cells. In this regard, histamine-stimulated EA.hy926 cells were overlaid with calcein-AM-labelled U937 cells and fluorescence intensity was visualized under fluorescence microscope. It was shown
Fig. 2. HPLC-UV–vis chromatogram (A), mass chromatogram (B), and chemical structure (C) of myricetin from R. tomentosa fruits. 4
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Fig. 3. The inhibitory effect of myricetin on IL8 and MCP-1 productions and the cytotoxic effect of myricetin on EA.hy926 cells. (A and B) The cells were treated with myricetin before stimulation of histamine. The production levels of IL-8 and MCP-1 were quantified in culture media using commercial ELISA kits. (C) EA.hy926 cells were treated with myricetin for 24 h. Cell viability was assessed by MTT method, and the results were expressed as percentage of surviving cells over blank cells. Each determination was made in three independent experiments, and the data were shown as means ± SD. Different letters a–e indicate significant difference among groups (p < 0.05).
Fig. 4. The suppressive effect of myricetin on the production of adhesion molecules. EA.hy926 cells were treated with myricetin before stimulation of histamine. The monolayer of EA.hy926 cells was then overlaid with calcein-AM-labelled U937 cells for 1 h. The level of monocyte adhesion onto EA.hy926 cells was monitored using light microscope (10× magnification). Each determination was made in three independent experiments.
pathway [31]. Moreover, the inhibition of NF-κB activation by triptolide from Tripterygium wilfordii resulted in attenuation of cytokine and chemokine productions, adhesion molecule expression, and monocyte adhesion [32]. Similarly, Cynandione A from Cynanchum wilfordii roof exhibited inhibitory effect on the expression of adhesion molecule (VCAM‑1), pro‑inflammatory (IL‑1β, IL‑6, and TNF-α), and chemoattractant cytokines (IL-8 and MCP‑1) via suppressing transcriptional activity of NF‑κB [33]. Especially, the inhibition of adhesion molecules (VCAM-1, ICAM-1, and E-selectin) by flavones (luteolin and apigenin) was also found due to interfering NF-κB-dependent transcription pathway [34]. Accordingly, the suppressive effect of myricetin on NF-
determined [20,30]. In order to examine whether myricetin can suppress NF-κB activation, EA.hy926 endothelial cells were pre-treated with myricetin before stimulated with histamine, and NF-κB activation level was measured by western blot analysis. The results showed that NF-κB was significantly activated in histamine-exposed EA.hy926 cells via augmenting phosphorylation of IκB-α and the level of p50 and p65 subunits. However, these increases were markedly reduced by myricetin treatment at concentration of 40 μM (Fig. 5). Recently, Davallia bilabiata has been determined to suppress the expression of adhesion molecules (ICAM, VCAM, and E-selectin) and chemokines (CX3CL1, MCP-1, and RANTES) through inhibition of NF-κB/IκBα/IKK signaling 5
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activation from endothelial cells may attenuate the microvascular permeability, contributing to amelioration of inflammatory responses. As shown in Fig. 6A, histamine increased phosphorylation of eNOS in the activated EA.hy926 cells. However, this increase was significantly suppressed by myricetin treatment in a dose-dependent manner. According to Breslin and colleagues, the inhibition of eNOS activation by NG-nitro-L-arginine methyl ester abolished VEGF-induced hyperpermeability in HUVECs [38]. Likewise, Lal and colleagues have reported that blockade of eNOS activation by NG-monomethyl-L-arginine completely inhibited VEGF-induced enhancement of permeability in HUVECs [39]. Furthermore, Hatakeyama and colleagues have determined the eNOS as an important regulator of microvascular permeability in inflammation [37]. As the result, the inhibitory activity of myricetin on eNOS activation may diminish the vascular permeability in endothelial cells, leading to alleviation of inflammatory responses. Importantly, the activation of the eNOS is dependent to intracellular free calcium elevation [40], which is induced by an influx of extracellular calcium or the release from endoplasmic reticulum [41]. Interestingly, histamine-induced intracellular calcium elevation was also blocked by myricetin treatment in a dose-dependent manner. This effect was observed via the reduction of fluorescent intensity by myricetin as compared with control group (Fig. 6B). According to Wang and colleagues, the blockade of IP3 receptor by heparin or the inhibition of PKC molecules by PKC 412 reduced intracellular calcium elevation and subsequently abolished eNOS activation in tentacle extract-exposed HUVECs [42]. On the other hand, Hamabata and colleagues have evidenced that 5,6-DiHETE inhibited endothelial calcium elevation, leading to decrease in vascular hyperpermeability during inflammation [43]. Consequently, the mitigation of intracellular calcium level by myricetin may result in the inhibition of eNOS activation and subsequent attenuation of vascular
Fig. 5. The down-regulative effect of myricetin on NF-κB activation. EA.hy926 cells were treated with myricetin before stimulation of histamine. The NF-κB activation level was assessed by Western blotting. β-actin was used as internal controls. Each determination was made in three independent experiments.
κB activation may mitigate the production of adhesion molecules and chemoattractant cytokines in histamine-activated endothelial cells. 3.5. The inhibitory effect of myricetin on eNOS activation and calcium elevation Endothelial nitric oxide synthase (eNOS) has been reported to play a predominant role in vascular endothelial growth factor-induced vascular permeability under inflammatory conditions [35]. The activation of eNOS has been shown to increase microvascular permeability [36]. Numerous studies have reported that microvascular hyperpermeability in response to an inflammatory challenge is mainly regulated by endothelial cells through eNOS [35,37]. Hence, the suppression of eNOS
Fig. 6. The alleviative effect of myricetin on eNOS activation and intracellular calcium elevation. (A) EA.hy926 cells were treated with myricetin before stimulation of histamine. The eNOS phosphorylation level was assessed by Western blotting. β-actin was used as internal controls. Each determination was made in three independent experiments. (B) EA.hy926 cells were treated with myricetin and incubated with Fura-3/AM before stimulation of histamine. The level of calcium release was monitored by a light microscope with 10× magnification. Each determination was made in three independent experiments.
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4. Conclusion In this study, myricetin from R. tomentosa fruits have been determined for its inhibitory activity on inflammatory responses in histamine-activated endothelial cells. The inhibitory activity was evidenced via suppression of chemokine and adhesion molecule productions, blockade of NF-κB activation, and attenuation of endothelial vascular permeability. These results indicated that myricetin could be applied as a functional ingredient for down-regulation of histamine-induced inflammatory responses. However, the further studies should be conducted to clarify the mechanism of action of myricetin on the alleviation of intracellular calcium. Moreover, an in vivo experimental model is also necessary for confirmation of bioactivity as well as cytotoxicity of myricetin. Declaration of Competing Interest There are no conflicts to declare. Acknowledgments This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106NN.02-2016.68. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2020.02.004. References [1] I. Puxeddu, A. Piliponsky, I. Bachelet, F. Levi-Schaffer, c. biology, Mast cells in allergy and beyond, Int. J. Biochem. Cell Biol. 35 (12) (2003) 1601–1607. [2] P. Benly, Research, Role of histamine in acute inflammation, J. Pharm. Sci. Res. 7 (6) (2015) 373. [3] K. Ashina, Y. Tsubosaka, T. Nakamura, K. Omori, K. Kobayashi, M. Hori, H. Ozaki, T. Murata, Histamine induces vascular hyperpermeability by increasing blood flow and endothelial barrier disruption in vivo, PLoS One 10 (7) (2015) e0132367. [4] L. Leach, B.M. Eaton, E.D. Westcott, J.A. Firth, Effect of histamine on endothelial permeability and structure and adhesion molecules of the paracellular junctions of perfused human placental microvessels, Microvasc. Res. 50 (3) (1995) 323–337. [5] D. Kugelmann, L.T. Rotkopf, M.Y. Radeva, A. Garcia-Ponce, E. Walter, J. Waschke, Histamine causes endothelial barrier disruption via Ca 2+-mediated RhoA activation and tension at adherens junctions, Sci. Rep. 8 (1) (2018) 13229. [6] S.J. Galli, M. Tsai, A.M. Piliponsky, The development of allergic inflammation, Nature 454 (7203) (2008) 445. [7] A. Panche, A. Diwan, S. Chandra, Flavonoids: an overview, J. Nutr. Sci. 5 (e47) (2016) 1–15. [8] G. Ferrazzano, I. Amato, A. Ingenito, A. Zarrelli, G. Pinto, A. Pollio, Plant polyphenols and their anti-cariogenic properties: a review, Molecules 16 (2) (2011) 1486–1507. [9] D. Tungmunnithum, A. Thongboonyou, A. Pholboon, A. Yangsabai, Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview, Medicines 5 (3) (2018) 93. [10] H. Cory, S. Passarelli, J. Szeto, M. Tamez, J. Mattei, The role of polyphenols in human health and food systems: a mini-review, Front. Nutr. 5 (87) (2018) 1–9. [11] T.S. Vo, D.H. Ngo, S.-K. Kim, Pharmaceutical properties of marine polyphenols: an overview, Acta Pharm. Sci. 57 (2) (2019) 217–242. [12] T.S. Vo, D.H. Ngo, The health beneficial properties of Rhodomyrtus tomentosa as potential functional food, Biomolecules 9 (2) (2019) 76. [13] T.S. Vo, Y.-S. Kim, D.-N. Ngo, D.-H. Ngo, The Role of Rhodomyrtus tomentosa(Aiton) Hassk. Fruits in Downregulation of Mast Cells-Mediated Allergic Responses, Biomed Res. Int. 2019 (3505034) (2019) 1–7. [14] D.-H. Ngo, D.-N. Ngo, T.T.N. Vo, T.S. Vo, Mechanism of action of Mangifera indica leaves for anti-diabetic activity, Sci. Pharm. 87 (2) (2019) 13. [15] T.-S. Vo, C.-S. Kong, S.-K. Kim, Inhibitory effects of chitooligosaccharides on degranulation and cytokine generation in rat basophilic leukemia RBL-2H3 cells, Carbohyd. Polym. 84 (1) (2011) 649–655. [16] A.E.-S.I. Mohammed, Phytoconstituents and the study of antioxidant, antimalarial and antimicrobial activities of Rhus tripartita growing in Egypt, J. Pharmacogn. Phytochem. 4 (2) (2015) 276–281.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Myricetin from Rhodomyrtus tomentosa (Aiton) Hassk fruits attenuates inflammatory responses in histamine-exposed endothelial cells Thanh Sang Voa,*, Young-Sang Kimb, Dai-Nghiep Ngoc, Dai-Hung Ngod,* a
NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City, Viet nam Department of Chemistry, Pukyong National University, Busan, 608-737, Republic of Korea c Department of Biochemistry, Faculty of Biology and Biotechnology, University of Science, Vietnam National University, Ho Chi Minh City, Viet nam d Faculty of Natural Sciences, Thu Dau Mot University, Thu Dau Mot City, Binh Duong Province, Viet nam b
A R T I C LE I N FO
A B S T R A C T
Keywords: Adhesion molecules Endothelial cells MCP-1 Myricetin Rhodomyrtus tomentosa
Histamine plays a key role in inflammatory responses via increasing chemokine and adhesion molecule productions and augmenting vascular permeability in endothelial cells. Rhodomyrtus tomentosa has been found as a rich source of structurally diverse and biologically active metabolites. In this study, the role of phenolic compound from Rhodomyrtus tomentosa (Aiton) Hassk fruits in down-regulation of histamine-induced EA.hy926 endothelial cell activation was investigated. Herein, myricetin was successfully isolated from R. tomentosa fruits by HPLC and its characterization was identified by mass spectrometer and nuclear magnetic resonance spectroscopy. It was revealed that myricetin was effective in suppression of IL-8 and MCP-1 productions and adhesion molecule generation. Moreover, NF-κB activation was inhibited by myricetin via reducing IκB-α phosphorylation and p50/p65 subunit level. Notably, myricetin potentially attenuated vascular permeability of endothelial cells through decrease in eNOS phosphorylation and intracellular calcium elevation. These results indicate that myricetin from R. tomentosa possesses a promissing pharmaceutical property for the amelioration of endothelial inflammatory responses.
1. Introduction Mast cell activation plays an important role in allergic inflammatory reaction by release of various preformed and synthesized mediators [1]. Notably, histamine mediating its receptors causes inflammatory responses due to increase in vasodilation, edema, vascular permeability, and blood flow [2]. Several studies have determined that histamine induced vascular permeability by activating endothelial cells in vitro and in vivo [3–5]. This action of histamine facilitates the transition of leukocytes such as neutrophil, eosinophil, and T cells to the late-phase reaction that leads to the development of inflammatory responses [6]. Thus, the inhibition of histamine-induced endothelial cell activation has been considered as a key therapeutic strategy for amelioration of allergic inflammatory development. Flavonoids and other phenolic compounds are the main class of secondary metabolites and widely distributed in fruits, vegetables, and beverages [7]. They are known to contain an aromatic ring bearing at least one hydroxyl groups and are biosynthesized by the shikimate or acetate/malonate pathway to produce a wide range of phenolic compounds with different structures [8]. So far, almost 8000 phenolic
⁎
compounds as naturally occurring substances in different parts of the plants have been identified in numerous studies [9]. Interestingly, the potential pharmaceutical and medical applications of phenolic compounds have been revealed due to antioxidant, anti-inflammation, antiallergy, anticancer, antimicrobial, anti-diabetes, cardio-protection, UVprotection, and immune-promotion activities [10,11]. Therefore, phenolic compounds have got much attention by researchers regarding their versatile benefits for human health. In this sense, Rhodomyrtus tomentosa, a flowering plant in family Myrtaceae, has been found as a rich source of structurally diverse and biologically active metabolites of phenolic compounds [12]. In the previous study, the inhibitory effect of R. tomentosa fruit extract on mast cell activation has been determined via suppression of degranulation [13]. This activity of R. tomentosa fruit extract was suggested due to phenolic compounds. In the present study, myricetin was purified from R. tomentosa fruit extract, and its suppressive effect on histamine-induced endothelial cell activation was examined.
Corresponding authors. E-mail addresses: [email protected] (T.S. Vo), [email protected] (D.-H. Ngo).
https://doi.org/10.1016/j.procbio.2020.02.004 Received 5 September 2019; Received in revised form 3 February 2020; Accepted 3 February 2020 1359-5113/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Thanh Sang Vo, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2020.02.004
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fluorescence probe, Fura 3-AM. EA.hy926 cells (1 × 104 cells/ml) were treated with myricetin for 24 h before incubated with Fura-3/AM (2 μM, final concentration) for 60 min at 37 °C. The cells were washed with PBS buffer and exposed to histamine (10 μM, final concentration) at 37 °C for 10 min. The fluorescence intensity was monitored under a fluorescence microscope (CTR 6000, Leica, Wetzlar, Germany).
2. Materials and methods 2.1. Materials R. tomentosa fruits were collected from Duong Dong Town, Phu Quoc district, Kien Giang province. Enzyme immunoassay kits for IL-8 and MCP-1 were purchased from R&D Systems (Minneapolis, MN, USA). Specific antibodies including p-IκB-α, p50, p65, p-eNOS, and βactin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). All other reagents including calcein-AM, Fura-3/AM, histamine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide were purchased from Sigma–Aldrich (St. Louis, MO, USA). EA.hy926 cells (ATCC® CRL2922™) and U937 cells (ATCC® CRL1593.2™) were purchased from ATCC (PO Box 1549 Manassas, VA 20108 USA).
2.7. Western blot analysis EA.hy926 cells were treated with myricetin (10, 20, or 40 μM) for 24 h before stimulated with histamine (10 μM, final concentration). The cells were lysed in RIPA lysis buffer and the detection was performed as described by Vo et al. [15]. Briefly, cell lysates (25 μg of protein/ sample) were subjected to 10 % SDS-PAGE, transferred onto a nitrocellulose membrane, and blocked by TBS-T buffer containing 5 % (w/v) bovine serum albumin (BSA). The membrane was washed and probed with primary antibodies (diluted 1:1000) for at least 1 h. Subsequently, horseradish peroxidase (HRP)-conjugated IgG secondary antibody (diluted 1:5000) was applied for 1 h. Protein bands were visualized by an enhanced chemiluminescent ECL assay kit (Amersham Pharmacia Biosciences, UK) and LAS3000® Luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan).
2.2. Extraction and isolation The powdered fruits of R. tomentosa (1 kg) were extracted with ethyl acetate (EtOAc) at 40 °C for 24 h. Crude extract was evaporated under vacuum to achieved 11.2 g EtOAc extract. This extract (1 g) was suspended in ethyl acetate and separated by silica gel column. It was respectively eluted with hexan-chloroform (10:0 to 2:8), chloroform-ethyl acetate (10:0 to 2:8), ethyl acetate-ethanol (10:0 to 2:8), and ethanol to achieved 16 fractions. The fraction of ethyl acetate-ethanol (8:2) was subjected to Supelco column (250 × 21.2 mm, 10 μm) of HPLC (Shimadzu LC 8A, Japan), eluted with H2O-acetonitril (90:10 to 5:95), and detected at 360 nm to obtain compound A (15.1 mg). The compound A was characterized by high-resolution MicrOTOF QII Mass Spectrometer (Bruker Daltonics, Bremen, Germany) and NMR (Bruker, 20251 Hamburg, Germany).
2.8. Statistical analysis Statistical analysis was performed by using the analysis of variance (ANOVA) test of statistical package for the social sciences (SPSS). The statistical significance of differences among groups was analyzed using Duncan’s multiple range tests wherein p < 0.05 was considered significant. 3. Results and discussion
2.3. Cell viability 3.1. Structure evaluation of myricetin from R. Tomentosa The cell viability levels were determined by MTT assay. EA.hy926 cells (2 × 105 cells/ml) were treated with myricetin (10, 20, or 40 μM) for 24 h. The cell viability was measured as described by Ngo et al. [14]. Briefly, the cells were incubated with MTT solution (1 mg/ml) for 4 h, and DMSO (100 μl) was subsequently added. The absorbance was measured at 540 nm using a microplate reader (GENios® Tecan Austria GmbH, Austria). The cell viability was quantified as a percentage compared to blank.
Compound A was obtained as yellow needles. The 1H-NMR and 13CNMR spectrum were showed in Fig. 1. The 1H-NMR spectrum showed a down field signal at δ 12.15 of a chelated hydroxyl group (1H, s, 5−OH). It also exhibited a characteristic meta-coupling proton signal at δ 6.26 (1H, d, J =2.0 Hz, H-6) and 6.50 (1H, d, J =2.0 Hz, H-8) for the A-ring of flavonoid. The AX coupling system at δ 7.42 (2H, s) was assigned to H-2′ and H-6′ of B ring. The 13C-NMR spectrum showed 13 signals for 15 carbons of a flavone skeleton including a conjugated ketone group at δC 176.6 (C-4) (Table 1). By comparing the NMR spectral data with those reported in literature, the structure of compound A was determined as myricetin (Fig. 2) [16].
2.4. Measurement of IL-8 and MCP-1 production EA.hy926 cells (4 × 104 cells/ml) were treated with myricetin (10, 20, or 40 μM) for 24 h before stimulated with histamine (10 μM, final concentration) for 24 h. The supernatants were collected, and the productions of IL-8 and MCP-1 were quantified by sandwich immunoassays following the protocol of R&D systems.
3.2. The inhibitory effect of myricetin on IL-8 and MCP-1 productions Endothelial cells are known to involve in the initiation of inflammatory process [17]. The activation of endothelial cells causes increase in vascular permeability and up-regulation of pro-inflammatory cytokines, chemokines, and adhesion molecules [18,19]. Notably, NF-κB pathway-mediated productions of monocyte chemoattractant protein (MCP)-1 and IL-8 contribute to the inflammatory endothelial cell response [20]. Therefore, the inhibition of these chemokine productions could suppress inflammatory responses. Herein, the inhibitory effect of myricetin on IL-8 and MCP-1 productions was examined in culture supernatants by enzyme-linked immunosorbent assay. It was shown that IL-8 and MCP-1 productions were increased from histamine-exposed EA.hy926 cells. The levels of IL-8 and MCP-1 were produced up to 132 ± 9 and 615 ± 15 pg/ml, respectively. Meanwhile, these increases were significantly lowered in a dose-dependent manner by myricetin treatment. Myricetin reduced IL-8 and MCP-1 productions up to 51 ± 6 and 207 ± 11 pg/ml at concentration of 40 μM, respectively (Fig. 3A and B). It indicated that myricetin
2.5. In vitro cell adhesion assay U937 cells (2 × 104 cells/ml) were incubated with calcein-AM (5 μM, final concentration) in the dark at room temperature for 60 min. Meanwhile, EA.hy926 cells (1 × 104 cells/ml) were treated with myricetin for 24 h before stimulated with histamine (10 μM, final concentration) for 24 h. Thereafter, EA.hy926 cells were overlaid with calcein-AM-labelled U937 cells for 1 h. The unbound cells were removed by washing the plates. Fluorescence images were visualized and photographed under fluorescence microscope (CTR 6000, Leica, Wetzlar, Germany). 2.6. The intracellular Ca2+ elevation assay Intracellular calcium level was measured using the calcium reactive 2
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Fig. 1. Spectra 1H-NMR (A) and
13
C-NMR (B) of myricetin from R. tomentosa fruits.
myricetin on IL-8 and MCP-1 productions was not due to cytotoxicity.
treatment suppressed endothelial cell activation that caused the reduction of IL-8 and MCP-1 productions in histamine-exposed EA.hy926 cells. According to Jang and colleagues, scoparone, a major component of Artemisia capillaris shoot, has been reported to inhibit IL-8 and MCP-1 productions in PMA-stimulated U937 cells [21]. Moreover, Hao and colleagues have determined that cafestol, a diterpenoid in coffee beans, significantly inhibited IL-8 and MCP-1 productions from cyclic-straininduced vascular endothelial cells (HUVEC cells) [22]. It was observed that the inhibitory effect of myricetin on IL-8 and MCP-1 productions was similar with that of scoparone. However, cafestol was shown to be more effective than myricetin on the inhibition of IL-8 and MCP-1 productions. In order to exclude the inhibition of myricetin on IL-8 and MCP-1 productions due to cytotoxic effect, MTT test was further examined on EA.hy926 cells. The result showed that myricetin did not cause significantly cytotoxic effect on EA.hy926 cells at concentration of 10, 20, or 40 μM (Fig. 3C). It indicated that the inhibitory activity of
3.3. The inhibitory effect of myricetin on adhesion molecule production So far, numerous adhesion molecules have been identified due to their structure and function, including the selectin, integrin and immunoglobulin families of adhesion proteins [23]. Adhesion molecules are known to participate in the pathogenesis of inflammatory diseases including atherosclerosis, rheumatoid arthritis, psoriasis, diabetes, multiple sclerosis, and inflammatory bowel diseases [24]. The production of adhesion molecules on surface of the activated endothelial cells plays a vital role in inflammatory responses, such as leukocyte emigration, lymphocyte recirculation, platelet adhesion, and phagocytosis [25]. Therefore, suppression of adhesion molecule production from the activated endothelial cells can block leukocyte emigration and lymphocyte recirculation that leads to alleviation of inflammatory 3
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Table 1 1 H (500 MHz) and C
that the density of calcein-AM-labelled U937 cells were significantly increased on the monolayer of EA.hy926 cells in the control group, indicating increase in adhesion molecule production from EA.hy926 cells. Meanwhile, the density of calcein-AM-labelled U937 cells was reduced in the presence of myricetin at concentration of 40 μM (Fig. 4). Likewise, it was reported that fisetin and quercetin inhibited the expression of adhesion molecules, causing to decrease in THP-1 monocyte adhesion to inflammatory cells (A549 cells or ARPE-19 cells) [26,27]. Especially, anti-inflammatory drugs such as dexamethasone, 5-aminosalicylic acid, methotrexate, and 6-mercaptopurine inhibited adhesion molecule expression, thus blocking leucocyte adhesion and transmigration in the microvasculature [28]. Accordingly, the inhibitory activity of myricetin on adhesion molecule production may contribute to the alleviation of leucocyte adhesion and transmigration into the latephase of inflammatory reaction.
C (125 MHz) NMR data for compound A in acetone-d6. Myricetina
Compound A
2 3 4 5 6 7 8 9 10 1' 2′, 6′ 3', 5′ 4' 5-OH a
13
δH (mult., J = Hz)
δC
δH (mult., J = Hz)
δC
— — — — 6.26 (1H, d, 2.0) — 6.50 (1H, d, 2.0) — — — 7.42 (2H, s) — — 12.15 (1H, s)
147.1 136.5 176.6 162.4 99.2 165.1 94.5 157.9 104.2 122.9 108.4 146.5 137.0 —
— — — — 6.13 (brs) — 6.41 (brs) — — — 7.17 (s) — — —
147.1 136.1 176.0 160.9 98.6 164.4 93.7 156.5 103.2 121.1 107.5 146.0 136.2 —
3.4. The inhibitory effect of myricetin on NF-κB activation
δH and δC of myricetin recorded in DMSO-d6.
Nuclear factor-κB (NF-κB), a family of inducible transcription factors, is composed of five subunits including p50, p52, p65, RelB, and cRel, and is normally regulated by inhibitory protein IκBα in the cytoplasm. NF-κB serves as a key mediator of inflammatory responses through regulating the expression of various pro-inflammatory genes [29]. Notably, the role of NF-κB on regulation of the expression of chemokines and adhesion molecules in endothelial cells has been
responses. In this study, we also examined whether myricetin can reduce the production of adhesion molecules in histamine-stimulated EA.hy926 cells. In this regard, histamine-stimulated EA.hy926 cells were overlaid with calcein-AM-labelled U937 cells and fluorescence intensity was visualized under fluorescence microscope. It was shown
Fig. 2. HPLC-UV–vis chromatogram (A), mass chromatogram (B), and chemical structure (C) of myricetin from R. tomentosa fruits. 4
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Fig. 3. The inhibitory effect of myricetin on IL8 and MCP-1 productions and the cytotoxic effect of myricetin on EA.hy926 cells. (A and B) The cells were treated with myricetin before stimulation of histamine. The production levels of IL-8 and MCP-1 were quantified in culture media using commercial ELISA kits. (C) EA.hy926 cells were treated with myricetin for 24 h. Cell viability was assessed by MTT method, and the results were expressed as percentage of surviving cells over blank cells. Each determination was made in three independent experiments, and the data were shown as means ± SD. Different letters a–e indicate significant difference among groups (p < 0.05).
Fig. 4. The suppressive effect of myricetin on the production of adhesion molecules. EA.hy926 cells were treated with myricetin before stimulation of histamine. The monolayer of EA.hy926 cells was then overlaid with calcein-AM-labelled U937 cells for 1 h. The level of monocyte adhesion onto EA.hy926 cells was monitored using light microscope (10× magnification). Each determination was made in three independent experiments.
pathway [31]. Moreover, the inhibition of NF-κB activation by triptolide from Tripterygium wilfordii resulted in attenuation of cytokine and chemokine productions, adhesion molecule expression, and monocyte adhesion [32]. Similarly, Cynandione A from Cynanchum wilfordii roof exhibited inhibitory effect on the expression of adhesion molecule (VCAM‑1), pro‑inflammatory (IL‑1β, IL‑6, and TNF-α), and chemoattractant cytokines (IL-8 and MCP‑1) via suppressing transcriptional activity of NF‑κB [33]. Especially, the inhibition of adhesion molecules (VCAM-1, ICAM-1, and E-selectin) by flavones (luteolin and apigenin) was also found due to interfering NF-κB-dependent transcription pathway [34]. Accordingly, the suppressive effect of myricetin on NF-
determined [20,30]. In order to examine whether myricetin can suppress NF-κB activation, EA.hy926 endothelial cells were pre-treated with myricetin before stimulated with histamine, and NF-κB activation level was measured by western blot analysis. The results showed that NF-κB was significantly activated in histamine-exposed EA.hy926 cells via augmenting phosphorylation of IκB-α and the level of p50 and p65 subunits. However, these increases were markedly reduced by myricetin treatment at concentration of 40 μM (Fig. 5). Recently, Davallia bilabiata has been determined to suppress the expression of adhesion molecules (ICAM, VCAM, and E-selectin) and chemokines (CX3CL1, MCP-1, and RANTES) through inhibition of NF-κB/IκBα/IKK signaling 5
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activation from endothelial cells may attenuate the microvascular permeability, contributing to amelioration of inflammatory responses. As shown in Fig. 6A, histamine increased phosphorylation of eNOS in the activated EA.hy926 cells. However, this increase was significantly suppressed by myricetin treatment in a dose-dependent manner. According to Breslin and colleagues, the inhibition of eNOS activation by NG-nitro-L-arginine methyl ester abolished VEGF-induced hyperpermeability in HUVECs [38]. Likewise, Lal and colleagues have reported that blockade of eNOS activation by NG-monomethyl-L-arginine completely inhibited VEGF-induced enhancement of permeability in HUVECs [39]. Furthermore, Hatakeyama and colleagues have determined the eNOS as an important regulator of microvascular permeability in inflammation [37]. As the result, the inhibitory activity of myricetin on eNOS activation may diminish the vascular permeability in endothelial cells, leading to alleviation of inflammatory responses. Importantly, the activation of the eNOS is dependent to intracellular free calcium elevation [40], which is induced by an influx of extracellular calcium or the release from endoplasmic reticulum [41]. Interestingly, histamine-induced intracellular calcium elevation was also blocked by myricetin treatment in a dose-dependent manner. This effect was observed via the reduction of fluorescent intensity by myricetin as compared with control group (Fig. 6B). According to Wang and colleagues, the blockade of IP3 receptor by heparin or the inhibition of PKC molecules by PKC 412 reduced intracellular calcium elevation and subsequently abolished eNOS activation in tentacle extract-exposed HUVECs [42]. On the other hand, Hamabata and colleagues have evidenced that 5,6-DiHETE inhibited endothelial calcium elevation, leading to decrease in vascular hyperpermeability during inflammation [43]. Consequently, the mitigation of intracellular calcium level by myricetin may result in the inhibition of eNOS activation and subsequent attenuation of vascular
Fig. 5. The down-regulative effect of myricetin on NF-κB activation. EA.hy926 cells were treated with myricetin before stimulation of histamine. The NF-κB activation level was assessed by Western blotting. β-actin was used as internal controls. Each determination was made in three independent experiments.
κB activation may mitigate the production of adhesion molecules and chemoattractant cytokines in histamine-activated endothelial cells. 3.5. The inhibitory effect of myricetin on eNOS activation and calcium elevation Endothelial nitric oxide synthase (eNOS) has been reported to play a predominant role in vascular endothelial growth factor-induced vascular permeability under inflammatory conditions [35]. The activation of eNOS has been shown to increase microvascular permeability [36]. Numerous studies have reported that microvascular hyperpermeability in response to an inflammatory challenge is mainly regulated by endothelial cells through eNOS [35,37]. Hence, the suppression of eNOS
Fig. 6. The alleviative effect of myricetin on eNOS activation and intracellular calcium elevation. (A) EA.hy926 cells were treated with myricetin before stimulation of histamine. The eNOS phosphorylation level was assessed by Western blotting. β-actin was used as internal controls. Each determination was made in three independent experiments. (B) EA.hy926 cells were treated with myricetin and incubated with Fura-3/AM before stimulation of histamine. The level of calcium release was monitored by a light microscope with 10× magnification. Each determination was made in three independent experiments.
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4. Conclusion In this study, myricetin from R. tomentosa fruits have been determined for its inhibitory activity on inflammatory responses in histamine-activated endothelial cells. The inhibitory activity was evidenced via suppression of chemokine and adhesion molecule productions, blockade of NF-κB activation, and attenuation of endothelial vascular permeability. These results indicated that myricetin could be applied as a functional ingredient for down-regulation of histamine-induced inflammatory responses. However, the further studies should be conducted to clarify the mechanism of action of myricetin on the alleviation of intracellular calcium. Moreover, an in vivo experimental model is also necessary for confirmation of bioactivity as well as cytotoxicity of myricetin. Declaration of Competing Interest There are no conflicts to declare. Acknowledgments This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106NN.02-2016.68. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2020.02.004. References [1] I. Puxeddu, A. Piliponsky, I. Bachelet, F. Levi-Schaffer, c. biology, Mast cells in allergy and beyond, Int. J. Biochem. Cell Biol. 35 (12) (2003) 1601–1607. [2] P. Benly, Research, Role of histamine in acute inflammation, J. Pharm. Sci. Res. 7 (6) (2015) 373. [3] K. Ashina, Y. Tsubosaka, T. Nakamura, K. Omori, K. Kobayashi, M. Hori, H. Ozaki, T. Murata, Histamine induces vascular hyperpermeability by increasing blood flow and endothelial barrier disruption in vivo, PLoS One 10 (7) (2015) e0132367. [4] L. Leach, B.M. Eaton, E.D. Westcott, J.A. Firth, Effect of histamine on endothelial permeability and structure and adhesion molecules of the paracellular junctions of perfused human placental microvessels, Microvasc. Res. 50 (3) (1995) 323–337. [5] D. Kugelmann, L.T. Rotkopf, M.Y. Radeva, A. Garcia-Ponce, E. Walter, J. Waschke, Histamine causes endothelial barrier disruption via Ca 2+-mediated RhoA activation and tension at adherens junctions, Sci. Rep. 8 (1) (2018) 13229. [6] S.J. Galli, M. Tsai, A.M. Piliponsky, The development of allergic inflammation, Nature 454 (7203) (2008) 445. [7] A. Panche, A. Diwan, S. Chandra, Flavonoids: an overview, J. Nutr. Sci. 5 (e47) (2016) 1–15. [8] G. Ferrazzano, I. Amato, A. Ingenito, A. Zarrelli, G. Pinto, A. Pollio, Plant polyphenols and their anti-cariogenic properties: a review, Molecules 16 (2) (2011) 1486–1507. [9] D. Tungmunnithum, A. Thongboonyou, A. Pholboon, A. Yangsabai, Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview, Medicines 5 (3) (2018) 93. [10] H. Cory, S. Passarelli, J. Szeto, M. Tamez, J. Mattei, The role of polyphenols in human health and food systems: a mini-review, Front. Nutr. 5 (87) (2018) 1–9. [11] T.S. Vo, D.H. Ngo, S.-K. Kim, Pharmaceutical properties of marine polyphenols: an overview, Acta Pharm. Sci. 57 (2) (2019) 217–242. [12] T.S. Vo, D.H. Ngo, The health beneficial properties of Rhodomyrtus tomentosa as potential functional food, Biomolecules 9 (2) (2019) 76. [13] T.S. Vo, Y.-S. Kim, D.-N. Ngo, D.-H. Ngo, The Role of Rhodomyrtus tomentosa(Aiton) Hassk. Fruits in Downregulation of Mast Cells-Mediated Allergic Responses, Biomed Res. Int. 2019 (3505034) (2019) 1–7. [14] D.-H. Ngo, D.-N. Ngo, T.T.N. Vo, T.S. Vo, Mechanism of action of Mangifera indica leaves for anti-diabetic activity, Sci. Pharm. 87 (2) (2019) 13. [15] T.-S. Vo, C.-S. Kong, S.-K. Kim, Inhibitory effects of chitooligosaccharides on degranulation and cytokine generation in rat basophilic leukemia RBL-2H3 cells, Carbohyd. Polym. 84 (1) (2011) 649–655. [16] A.E.-S.I. Mohammed, Phytoconstituents and the study of antioxidant, antimalarial and antimicrobial activities of Rhus tripartita growing in Egypt, J. Pharmacogn. Phytochem. 4 (2) (2015) 276–281.
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