Novel compounds in fruits of coriander (Coşkuner & Karababa) with anti-inflammatory activity

Coriander, Coriandrum Sativum L., is one of the commonest food and medicinal plants in many countries, but its chemical ingredients and pivotal role in anti-inflammatory activity have not been fully explored. The present study aimed to identify new compounds in the fruits of coriander and explore their anti-inflammatory activity. The compounds were isolated by chromatographic seperations and identified using spectroscopic and spectrometric methods. RAW264.7 macrophage cells were used to detect the anti-inflammatory activity of the compounds via Griess assay, western blotting, ELISA, and flow cytometry methods. The study resulted in the discovery of four new compounds, which were identified as: 4α-(furo[2,3-d]pyrimidin-6′-ylmethyl)-9αpropylnonolactone (1), 4-(formyloxy)-4-(6′-methylcyclohex-1-en-1-yl)butanoate(2), (7α,8α)-3α-hydroxyl12,13α-dimethyl-5(6)-en-bicyclo[5,3,0]caprolactone (3), 7-methoxy-4-methyl-5,6-dihydro-7H-(2-hydroxypropan-2-yl)furo[2,3-f] coumarin (4). Compound 3 showed the highest anti-inflammatory activity with IC50 of 6.25 μM for an inhibitory effect on nitrite oxide (NO) level. In addition, compound 3 decreased the lipopolysaccharides-stimulated generations of ROS and the inflammatory cytokines (IL-6 and TNF-α). Mechanism exploration indicated that compound 3 suppressed inflammatory mediators’ expression, like iNOS and COX-2. Furthermore, the NF-κB and MAPK pathways were involved in the anti-inflammatory process of compound 3.


Introduction
Inflammation plays a crucial role in the progression of many diseases, excessive inflammation augments the activation of immune cells, which can destroy the tissues and body heath (Cárdeno et al., 2014;Franceschi & Campisi, 2014;Lee et al., 2020). Multiple pro-inflammatory mediators are over-produced when inflammation occurs and leads to a series of diseases, such as rheumatism, diabetes, and cardiovascular ailments (Golia et al., 2014;Karam, Chavez-Moreno, Koh, Akar, & Akar, 2017).
Accumulated studies have confirmed that reactive oxygen species (ROS) are involved in the response of inflammation (Chelombitko, 2018). The enhanced generation of ROS will exacerbate inflammation and lead to tissue injury (Mittal, Siddiqui, Tran, Reddy, & Malik, 2014). Therefore, restraining the NF-κB signaling pathway, MAPK pathways, and ROS generation might be a potential strategy to attenuate inflammatory diseases.
Coriandrum Sativum L. (coriander) is an annual or biennial plant affiliated with the Coriandrum genus of Umbelliferae family (Laribi, Kouki, M'Hamdi, & Bettaieb, 2015;Lee et al., 2020). It is one of the oldest aromatic vegetables used as food or/and medicine for over 2000 years. The fresh green leaves called cilantro are commonly used as a vegetable, and the dried fruits are traditionally used as spice in cooking (Abascal & Yarnell, 2012). Coriander has been cultivated largely in North Africa, central Europe, and Asia. The green leaf is a rich source of vitamins, minerals, iron, but is low in cholesterol (Slavin & Lloyd, 2012). The dried ripe fruit of coriander is not only used as food and spice, it is also used as traditional medicine for the treatment of different ailments in many countries. Coriander alone or in combination with other traditional herbs has been used for treating many diseases like rheumatism, diabetes, cough, bronchitis, insomnia, gastrointestinal, and flatulence (Emamghoreishi, Khasaki, & Aazam, 2005;Gray & Flatt, 1999;Hosseinzadeh, Alaw Qotbi, Seidavi, Norris, & Brown, 2014). However, coriander's anti-inflammatory activity and underlying mechanisms are not clear.
In this study, LPS-stimulated RAW264.7 macrophage cells were used to unravel the anti-inflammatory activity of the ingredients in coriander fruits, four new compounds (1-4) were isolated and identified, along with four known ones. Compound 3 exhibited the highest anti-inflammatory activity for the inhibitory effect on NO level in Griess assay. The mechanism of anti-inflammatory activity for compound 3 was further investigated by using various cellular assays targeting IL-6, TNF-α, iNOS, COX-2, ROS, NF-κB, and MAPK signaling pathways. The results showed that compound 3 exerted an anti-inflammatory effect via the NF-κB and MAPK pathways and has an inhibitory effect on oxidative stress.

Plant materials
The Coriandrum Sativum L. plants were grown in Qionggacun, Qusum, Shannan, Tibet, China and were verified by Prof. Xiaoran Li. When matured, the plants were harvested and dried in a ventilated area, then the fruits were collected. A voucher specimen (No. YS2018-10-11) was stored in the herbarium of the college of pharmacy at Guangxi University of Chinese Medicine.

Cell Culture
RAW264.7 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM medium with 10% FBS and antibiotics at 37°C in a humidified environment with 5% CO 2 .

Cell viability assay
Cell viability studies were performed on the RAW264.7 cells at a density of 5 × 10 4 cell/ well (100 μL) in 96-well plates, which were subsequently treated with indicated compounds for 24 h. Cell viability was evaluated by MTT according to the manufacturer's protocols, the absorbance at 570 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA) is recoded.
2.6. Griess reagent assay RAW264.7 macrophages (2 × 10 5 cells/well) were seeded in 24wells (500 μL) and pretreated with compounds at indicated concentrations for 1 h, and then treated with LPS for another 24 h. The collected medium was used to measure the nitrite levels by Griess reagent, the absorbance at 540 nm was detected by a microplate reader.

Flow cytometry assay
The cells were seeded in 24-well plates at a starting density of 2 × 10 5 cells per well (500 μL) and cultured in an incubator overnight. DCFH 2 -DA was employed to analyze the ROS generation. At the end of compound 3 incubation for the indicated time point, DCFH 2 -DA (1 μM, 30 min) was added to the medium, followed by signal collection by a FACScan Flow cytometer ((Becton-Dickinson, Franklin Lakes, NJ, USA).

Cytokines release assessment
Enzyme-linked immunosorbent assay (ELISA) was employed to detect the release of TNF-α and IL-6 according to the manufacture's protocols. Cells were seeded in 24-well plates at a starting density of 2 × 10 5 cells per well (500 μL) and pretreated with compound 3 for 1 h, then co-cultured with LPS for another16 h. The medium was collected to determine the release of the cytokines.
2.9. Western blotting analysis RAW264.7 macrophages (1 × 10 6 cells/well) were seeded in dishes (3 mL), and which were subsequently pretreated with compound 3 for 1 h, then stimulated with LPS for another 4 h. Preparation of cell lysates, proteins' quantification, electrophoresis, and immunoblotting were performed as described previously (Kang et al., 2019;Yuan et al., 2019). The primary antibodies and secondary antibodies were diluted into 1:1000 and 1:10000, respectively. Chemiluminescence signals were determined by using a ChemiDoc™ MP Imaging System with Image Lab version 5.1 software (Bio-Rad, Hercules, CA, USA).

Immunofluorescence assay
Immunofluorescence assay was used to evaluate the translocation of NF-κB/p65. RAW264.7 cells were cultured in the confocal dish (SPL, Pocheon, Korea) overnight. At the end of the indicated time point for compound 3 incubation, the primary antibodies and fluorescence secondary antibodies were diluted into 1:100 and 1: 500, respectively. Cells were imaged using a Leica TCS SP8 laser confocal microscope (Leica, Germany) and the processing of images was performed by ImageJ software.  2.11. Data analysis Two-sided Student's t-test was used to compare the differences between two groups. One-way-ANOVA and Dunńs multiple comparisons were used to compare the differences in more than two groups using GraphPad Prism 6.0 software. A p-value of < 0.05 was considered statistically significant.
Compound 2 was calculated with a molecular formula of C 13 H 20 O 4 in terms of its HR-ESI-MS [M−H]data. In the 1 H NMR spectrum, an olefinic proton signal at δ H 6.75 (1H, dd, J = 7.0, 17.5 Hz, H-2′) suggests the existence of a carbon-carbon double bond, which was supported by the carbon signals at δ C 130.8 (C-1′) and 135.6 (C-2′) in its 13 C NMR spectrum. The proton signals of one methyl at δ H 1.76 (3H, d, J = 7.0 Hz, H-7′), five methylene at between δ H 1.19-2.41, and one oxymethine at δ H 4.04 (1H, m, H-4), as well as one methoxy at δ H 3.66 (3H, s, H-6) were observed in its 1 H NMR and SHQC spectra. In the 13 C NMR spectrum, 13 carbon resonances were observed, which were attributed to two CH 3 , five CH 2 , four CH, and two quaternary carbons by the analysis of its HSQC spectrum. The HSQC correlation between H-5 at δ H 9.72 and C-5 at δ C 170.9 suggests that the existence of a formyl group. Accompanying with the HMBC correlations between H-6 at δ H 3.66, H-2 at δ H 2.41, H-3 at δ H 1.80 and C-1 at δ H 174.0, the molecular skeleton of compound 2 was established as butanoate by its H-H COSY spectrum. Similarly, the existence of a 6′-methylcyclohex-1-en-1 group was inferred.
Compound 3 was calculated with a molecular formula of C 12 H 18 O 3 in terms of the HR-ESI-MS [M−H 2 O + H] + data. In the 1 H NMR spectrum, the signals of an olefinic proton at δ H 6.54 (1H, d, J = 3.1 Hz, 0.8 Hz, H-6), two sets of methyl protons at δ H 1.40 (3H, s, H-12), 0.94 (3H, d, J = 5.5 Hz, H-13), and two sets of oxymethine protons at δ H 4.40 (1H, m, H-3), 4.02 (1H, m, H-8) were observed. In the 13 C NMR spectrum, 12 carbon resonances were observed including a signal for carbonyl carbon at δ C 170.3 (C-2). Besides, two methyl carbons, three methylene carbons, five methine carbons, and two quaternary carbons were assigned by the analysis of its HSQC and 1 H NMR spectra. The downfield shifting of C-8 at δ C 85.6 (+42.4) suggests the presence of a lactone group, which was supported the HMBC correlation between H-8 at δ H 4.02 and C-2 at δ C 170.3. The two olefinic carbon signals at δ C 133.3, 135.7 indicated the existence of a carbon-carbon double bond in the structure of compound 3, which should be attributed to C-5 and C-6 by the HMBC correlations between H-8 at δ H 4.02, H-4 at δ H 2.19 and C-6 at δ C 135.7, and between H-3 at δ H 4.40, H-7 at δ H 2.62 and C-5 at δ C 133.3. Accordingly, the basic skeleton of compound 3 was deduced to be a bicyclo [5,3,0]caprolactone by the analysis of its H-H COSY spectrum. In the HMBC spectra, Me-13 at δ H 0.94 showed correlations to C-10 at δ C 27.3 and C-8 at δ C 85.6, and Me-12 at δ H 1.40 showed correlations to C-4 at δ C 31.0 and C-6 at δ C 135.7, which indicated that the two methyl groups were located at C-9 and C-5 respectively. The NOESY spectrum indicated that there is a correlation between H-7 at δ H 2.62 and H-9 at δ H 1.76, H-3 at δ H 4.40 and H-11 at δ H 2.10, suggesting that H-7 and H-9 have the same spatial orientation, while H-7 and H-3 have an opposite orientation. Finally, compound 3 was (7α,8α)-3αhydroxyl-12,13α-dimethyl-5(6)-en-bicyclo[5,3,0]caprolactone named coriander lactone C.

Effect of isolates on the nitrite level in RAW264.7 cells
The dried ripe fruit of coriander is recorded as a traditional medicine against various symptoms. However, the pharmacological research about the coriander's anti-inflammatory activity and the underlying mechanisms are not clear. Therefore, the suppressive effects of inflammation of coriander's bioactive products and their anti-inflammatory mechanisms using macrophages have been investigated for the first time in this study. NO, IL-6 and TNF-α are the mediators of the inflammatory response by inhibiting or promoting inflammation through numerous pathways. In this study, apart from four new compounds, four known compounds (5-8) were identified as aurantiamide, 2-O-E-feruloyl-1-(4-hydroxyphenyl) ethane-1, 2-diol, methyl gallate, and methyl p-hydroxybenzoate, respectively. The anti-inflammatory activity of all isolated compounds was investigated in terms of nitrite level in RAW264.7 cells. Nitrite is the mediator of an inflammatory response by inhibiting or promoting inflammation through numerous pathways. Connelly, et al. found that nitrite can activate NF-κB, and the activated NF-κB promotes the release of proinflammatory cytokines, including TNF-α and IL-6 to accelerate the progress of inflammation (Brasier, 2010). Therefore, inhibition of nitrite may suppress the inflammatory response. In this study, the anti-inflammatory effects of four new compounds (1-4) and four known ones (5-8) were investigated in terms of nitrite level in LPS-stimulated RAW264.7 macrophage cells using the Griess assay. Several compounds had anti-inflammatory activity, pretreatment with compounds suppressed LPS treatment stimulated production of nitrite on RAW264.7 macrophage cells, while compound 3 had a better inhibitory effect on nitrite level with IC 50 of 6.25 μM than the other seven compounds, and treatment of compound 3 showed a dose-dependent decrease in the production of nitrite level (Fig. 4A). The cytotoxicity of 8 compounds was evaluated by MTT assay, and the results showed that all compounds almost have no cytotoxicity (data were not shown).

Suppression of inflammatory responses by compound 3
The pro-inflammatory mediators like NO, iNOS, COX-2, etc.play a pivotal role in many inflammatory models due to their changes (Katarzyna Popko, 2010;Murakami & Ohigashi, 2007;Qingjie Xue, 2018). It has been demonstrated that excess NO production, most of which promotes the iNOS expression, subsequently regulates COX-2 expression in inflammatory models. Therefore, iNOS and COX-2 are potential targets for the prevention of inflammation (Murakami & Ohigashi, 2007). ROS is central to the progress of inflammatory, of which contributes to LPS-induced production of proinflammatory cytokines IL-6, TNF-α, and IL-1L, and the activation of the MAPK pathway (Gabriele, Pucci, Árvay, & Longo, 2018;Naik & Dixit, 2011;Patruno et al., 2015). The main factors of TNF-α and IL-6 are responsible for the induction of inflammatory response and also act as the markers of inflammation in LPS-induced macrophage cells. In the present study, pretreatment with compound 3 decreased iNOS and COX-2 proteins' expression in a dose-dependent manner in LPS-induced RAW264.7 cells (Fig. 4B). ROS is an important inflammatory response signal (Chelombitko, 2018). As shown in Fig. 4C and D, flow cytometry results indicated that treatment of LPS significantly increased ROS production in RAW264.7 cells, and compound 3 suppressed the generation of ROS in a dose-dependent manner. In addition to nitrite and ROS levels stimulated by LPS, the generation of TNF-α and IL-6, etc. will be also increased in RAW264.7 cells (Hochdörfer et al., 2013;Mittal et al., 2014). The results of this study indicates that compound 3 significantly decreased TNF-α and IL-6 level in LPS-stimulated RAW264.7 cells ( Fig. 4E and F). Collectively, these data indicate that compound 3 exhibited both anti-inflammatory and anti-oxidant properties through suppressing the expression of iNOS and COX-2, the generation of ROS, and the releasing of TNF-α and IL-6 in LPS-stimulated RAW264.7 macrophage cells.  R. Yuan, et al. Journal of Functional Foods 73 (2020) 104145 3.4. Exploration of the role of compound 3 in the NF-κB and MAPKs pathways The transcriptional factor NF-κB is a member of Rel family and involves in the LPS-stimulated inflammatory progress (Lu et al., 2007), which has been validated as a regulator, and participates in the regulation of inflammatory factors, including iNOS, COX-2, IL-6, IL-1β, and TNF-α (Lawrence, 2009;Shang & Wu, 2020). The NF-κB signaling pathway also has a vital role in the chronic inflammatory disease. It has been found that NF-κB is inactive in the cytoplasm, IKKα and IKKβ are essential proteins in the activation of NF-κB, which promote the phosphorylation of IκB and subsequent degradation in LPS-induced RAW 264.7 cells (Castejon et al., 2019). NF-κB/p65 may be phosphorylated by the degraded IκB and translocated into the nucleus, which can further promote the release of proinflammatory cytokines and accelerate the inflammatory injury (Tak & Firestein, 2001). Therefore, we hypothesized that the NF-κB pathway may be involved in the anti-inflammatory activity of compound 3. In this study, the expression of p- Fig. 4. Compound 3 decreased LPS-induced pro-inflammatory responses in RAW264.7 cells. (A) RAW264.7 cells were pretreated with the indicated concentration of the 8 compounds for 1 h, which were co-cultured with LPS (1 μg/mL) for another 24 h. The medium was collected to detect the nitrite level using the Griess assay. (B) RAW264.7 cells pretreated with compound 3 (5, 10, 20 μM) for 1 h, then co-cultured with LPS (1 μg/mL) for another 16 h. The proteins' expression of iNOS and COX-2 were determined by Western blotting assay. (C) RAW264.7 cells were pretreated with compound 3 (5, 10, 20 μM) for 1 h, then co-cultured with LPS (1 μg/mL) for another 6 h. Cells labeled with DCFH 2 -DA (1 μM) for 30 min and determined by flow cytometry. (D) The statistical analysis of ROS fluorescence intensity. (E and F) RAW264.7 cells pretreated with compound 3 (5, 10, 20 μM) for 1 h, and co-cultured with LPS for another 24 h. The supernatants were collected and the proinflammatory cytokines TNF-α and IL-6 were determined by using the ELISA kit. *P < 0.05, ** P < 0.01, *** P < 0.001 compared to the LPS alone group. Fig. 5. NF-κB and MAPKs pathways were involved in compound 3′s anti-inflammatory process. (A and C) RAW264.7 cells were pretreated with compound 3 (5, 10, 20 μM) for 1 h, and stimulated with LPS for another 4 h. The proteins' expression of p-p65, p65, p-IκB-α, IκB-α, p-IKK-α/β, IKK-α, IKK-β, p-JNK1/2, JNK1/2, p-ERK1/ 2, ERK1/2, p-p38, and p38 were determined by western blotting assay. (B) RAW264.7 cells were pretreated with compound 3 for 1 h, then co-treated with LPS for another 2 h. The translocation of p65 was determined by using the immunofluorescence assay described in the Methods sections. The primary antibody anti-NF-κB/ p65 (1:100) and secondary antibody goat anti-Rabbit Alexa Fluor 568 (1:200). *P < 0.05, ** P < 0.01, and *** P < 0.001, compared to LPS alone group. R. Yuan, et al. Journal of Functional Foods 73 (2020) 104145 p65, p-IKKα/β, p-IκBα increased, which were markedly suppressed by compound 3 in LPS-induced RAW264.7 cells (Fig. 5A). Furthermore, the immunofluorescence staining analysis indicates that compound 3 suppressed the translocation of NF-κB/p65 into the nucleus (Fig. 5B). These results suggest that the NF-κB pathway was involved in the antiinflammatory activity of compound 3. The MAPK signaling pathway, including JNK1/2, ERK1/2, and p38MAPK, are involved in cellular signaling cascades wherein intracellular or extracellular stimuli induce inflammation, and has a response to inflammation (Chen et al., 2015). Previous studies validated that the MAPK pathway has a mediated effect on the activation of iNOS and COX-2 in LPS-induced macrophage cells (Zhang, Luna-Vital, & Gonzalez de Mejia, 2019). Therefore, the effects of compound 3 on the activation of JNK1/2, ERK1/2, and p38MAPK in LPS-induced RAW264.7 cells were evaluated by western blotting assay. As shown in Fig. 2B, pretreatment with compound 3 sharply decreased p-JNK1/2, p-ERK1/2, and p-p38MAPK expression in RAW264.7 cells induced by LPS, but did not affect total JNK1/2, ERK1/2, and p38MAPK expression (Fig. 5C). Those results demonstrated that compound 3 could prevent extracellular stimuli in LPS-stimulated macrophage cells, by inhibiting the phosphorylation of MAPKs. The findings indicate that NF-κB and MAPK pathways might account for compound 3′s anti-inflammatory activity.

Conclusions
In this study, we investigated the bioactive products of coriander and their anti-inflammatory mechanisms in chemical and pharmacological perspective, results demonstrated that eight compounds were identified from the dried ripe fruit of coriander, including four new compounds (1-4) and four known compounds (5-8), and compound 3 showed the most significant anti-inflammatory effect through inhibiting nitrite level, ROS production, and the generation of IL-6 and TNF-α. The mechanism exploration indicates that NF-κB and MAPKs pathways participated in compound 3′s anti-inflammatory effect. Collectively, our findings indicate that compound 3 exhibits a prominent inhibitory effect on inflammation. Ethics Statements Our research "Novel Compounds in Fruits of Coriander (Coriandrum Sativum L.) with Anti-inflammatory Activity" did not include any human subjects and animal experiments.

Declaration of Competing Interest
None.