Invitro Evaluation of Anti-Inflammatory Activity of “Habanero” Chili Pepper (Capsicum chinense) Seeds Extracts Pretreated with Cellulase

The aim of this study was to explore the effect of capsaicin and particular phenolic compounds profile from cellulase assisted extracts of Habanero (Capsicum chinense) chili pepper seeds (CPS) on the concentration of cytokines (IL-2, IL-6, TNF-α, IL-1β) in murine macrophages (RAW 264.7) stimulated with lipopolysaccharides (LPS). Capsaicin was quantified by HPLC–DAD, and the phenolic profile was determined by UPLC-MS-QqQ. Anti-inflammatory activity was evaluated by Mouse Cytokine/Chemokine Magnetic Bead Panel 96-well plate assay. Among the 15 different phenolics found in CPS extracts obtained at 120 or 150 min of maceration with 2,500 UI/L at 30 ºC or 45 ºC in a 1:15 (w:v) proportion, the most abundant was vanillic acid (7.97–12.66 µg/g). The extract obtained at 30 ºC and 120 min, showed similar effects than the observed for synthetic anti-inflammatory drugs indomethacin and dexamethasone, and capsaicin standard. Beyond capsaicin, salicylic, protocatechuic and trans-cinnamic acids as well as vanillin in CPS extracts were correlated with the anti-inflammatory effect. On the other hand, capsaicin and chlorogenic acid contents were potential immunostimulants whose concentration varied depending on the cellulase treatment time.


Introduction
Capsaicin has been described as an efficient anti-inflammatory compound [1]. It links with transient receptor potential vanilloid (TRPV-1) and constrains the discharge of inflammatory mediators, including nitric oxide (NO) and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interleukin (IL)-6, in lipopolysaccharide (LPS)-stimulated human cells [2]. In general, chili pepper waste is an important source of compounds with interesting biological activities for pharmaceutical purposes [3]. In addition to capsaicin, fatty acids, and a variety of phenolic compounds and their glycosides are present in chili pepper seeds (CPS) extracts [4]. Glycosides of luteolin and quercetin obtained from chili pepper showed antioxidant and anti-inflammatory activities by inhibiting the release of TNF-α and IL-6 [5]. Acquavia et al. [6], demonstrated the presence of quercetin and several of its derivates in "Habanero" extracts and their bioactivity. Additionally, Pappalardo et al. [7] proved liposomes as delivery system for white "Habanero" chili extracts to reduce cell viability of leukemia and other tumoral cells, and decrease the inflammation response in in vitro models.
Capsaicin and its analogues inhibited mRNA expression of IL-6, macrophage-inflammatory protein-3α (CCL20) and monocyte chemoattractant protein-1 (MCP-1) [8]. It also overturned T-cell activation and the advancement of cell division to S phase due to the attenuation of IL-2, interferon (IFN)γ, IL-4 and IL-5 production. Additionally, in in vivo 1 3 models, capsaicin decreased the plasma level of TNF-α and IL-6 and augmented the concentration of the anti-inflammatory IL-10 [9].
Cortés-Ferré et al. [10] demonstrated that enzyme assisted extraction (EAE) allowed the recovery of antiinflammatory compounds from CPS using water as solvent. Although significant differences were observed in the CPS extracts obtained at different times, temperature and cellulase concentration, no statistically significant correlation was found between capsaicinoids or total phenolic content (TPC) with the NO synthesis [10]. For this purpose, the aim of this study was to investigate the effect of capsaicin and particular phenolic compounds from cellulase assisted extracts of Habanero (Capsicum chinense) CPS on the concentration of inflammatory cytokines (IL-2, IL-6, TNF-α, IL-1β) in murine macrophages (RAW 264.7) stimulated with lipopolysaccharides (LPS).

Enzymatic Pre-Treatment
Habanero seeds were mixed with the enzyme solution (2,500 UI/L) in a 1:15 (w:v) proportion and incubated at a controlled temperature water bath. Enzymatic pre-treatment was performed at 30 ºC (T1) or 45 ºC (T2) and samples were obtained at 120 or 150 min to compare the effect of time and temperature. To study the effect of enzymatic pre-treatment time, a kinetic was performed at 30 ºC for 150 min (taking a sample every 30 min). The supernatant was recovered by filtration using a cloth and stored at -20 ºC until use.

Capsaicin Quantification
The identification and quantification of capsaicin were performed according to the methodology of Othman et al. [12]. The chromatographic analysis of capsaicin (CAP) and dihydrocapsaicin (DHC) was performed using high-performance liquid chromatography (HPLC) with DAD (Agilent 1100 Series Santa Clara, CA, USA) with a Zorbax Eclipse Plus C 18 (particle size 4.6 × 100 mm 3.5 μm) column at 40 °C, a flow of 0.8 mL/min, and injection volume of 10 µL. The mobile phase consisted of (A) water with 0.1% of formic acid and (B) methanol. Chromatograms were obtained at λ = 280 nm. The CAP and DHC concentrations in samples were expressed as μg of capsaicin equivalents/mL of extract.

Phenolic Compounds Composition And Quantification
The identification and quantification of phenolic compounds was performed as previously reported in Juárez-Trujillo et al. [11]. Briefly, filtered samples were placed in 2 mL UPLC vials and the chromatography was performed in an ultra high performance liquid chromatograph (UPLC, Agilent 1290 series) coupled to a triple quadrupole mass spectrometer (MS-MS, Agilent 6460). The mobile phases were water with 0.1% of formic acid (A) and acetonitrile with 0.1% formic acid (B), at a flow of 0.3 mL/min and 2 μL of each sample were injected in the system. The column was an Eclipse Plus C18, 2.1 × 50 mm, 1.8 μm (Agilent Technologies, Santa Clara, CA, USA) used at a temperature of 40 ºC. Mass spectrometer operated in positive and negative electrospray ionization modes with the desolvation temperature of 300 °C, the cone gas (N 2 ) flow of 5 L/min, the nebulizer pressure of 45 psi, the sheath gas temperature of 250 °C, the sheath gas flow of 11 L/min, the capillary voltage (positive and negative) was 3,500 V and the nozzle voltage (positive and negative) of 500 V. The data was processed with the MassHunter Workstation Software version B.06.00 (Agilent Technologies) and the results are expressed as the average ± standard deviation of mg/g of extract and mg/mL for correlation analysis.

Evaluation of the Anti-Inflammatory Activity of CPS Extracts
Murine macrophage cell line (RAW 264.7) was maintained in DMEM with 5% FBS and 1% penicillin/ streptomycin at 37 ºC in a humidified atmosphere containing 5% CO 2 . For the bioassay, 1 × 10 4 cells/well were inoculated into 96-well plates using 100 µL of cell suspension. After 24 h incubation at 37 °C, the cells were pretreated with 50 µL of CPS extracts (previously diluted in cell culture media at 1%) or capsaicin standard (CAPstd) or indomethacin or dexamethasone (1 µg/mL) for 48 h before adding 50 µL of growth media (for control wells) or 50 µL LPS (10 µg/mL). After 24 h, 100 µL the supernatant of LPS treated cells was used to evaluate the expression of IL-1β, IL-2, IL-6, and TNF-α using a custom Mouse Cytokine/ Chemokine Magnetic Bead Panel 96-well plate assay (MCYTOMAG-70 k, Merck Millipore, Boston, MA, USA). The antibody beads were prepared following the instructions specified in the kit. The cytokine concentrations were expressed as pg/mL.

Statistical Analyses
Data were expressed as means ± standard deviation of at least three independent experiments unless otherwise indicated. All variables were analyzed using ANOVA followed by a post hoc Tukey multiple comparison test (p < 0.05). A multivariate principal components analysis of (p < 0.05) and Spearman ρ non-parametric correlation analysis were also performed using JMP 16.0 (SAS Institute Inc., Cary, NC, USA).

Phenolic Compounds Profile and Quantification
As can be observed in Table 1, the most abundant phenolic compound found in the extracts was vanillic acid (7.97-12.66 mg/g). Kim et al. [13] analyzed 11 chili pepper varieties and found out that the main phenolic compounds in the methanolic extracts from the fruits were p-hydroxybenzoic, vanillic, p-coumaric, ferulic and sinapic acids. Similarly, dos Anjos et al. [14] found that on Brazilian chili pepper (Capsicum frutescens) pericarp one of the main phenolic compounds was vanillic acid (1.9 mg/g).
Phenylalanine (phenolics' precursor) was the second most abundant compound, ranging from 4.90 to 9.31 mg/g followed by protocatechuic, ferulic and 4-hydroxybenzoic acids (1.07 to 2.11 mg/g). The concentration of salicylic acid was higher after 150 min of cellulase treatment at 45 ºC (0.31 mg/g) than the observed at 30 ºC (0.18 mg/g). Surendran et al. [15] demonstrated that the presence of salicylic acid and its derivates inhibited cellulose activity and therefore it affected its release from the cellular matrix.
Chlorogenic acid content was affected by the extraction time with the highest yield at 30 ºC and 120 min of enzyme exposure (0.22 mg/g) that contrasted to the obtained at 150 min (0.06 mg/g). Temperature affected vanillin concentration, reaching higher contents at 45 ºC (0.98 -1.06 mg/g) than the observed at 30 ºC (0.67-0.82 mg/g). Finally, caffeic acid content was higher in the extracts obtained at 120 min (0.05-0.10 mg/g). As with chlorogenic acid, caffeic acid concentration was lower or not detected in the CPS treated with cellulase for 150 min. Pettinato et al. [16] showed that after 60 min of extraction, the concentration of caffeic acid decreased while increasing that of its derivates.

Anti-Inflammatory Activity of CPS Extracts
The CPS extracts obtained at 30 ºC and 150 min or at 45 ºC and 120 min decreased (p < 0.05) the expression of IL-1β (Fig. 1a), a pro-inflammatory cytokine [17], to levels like those obtained with dexamethasone (4.76 pg/mL) or indomethacin (4.93 pg/mL). Only the CPS extracts obtained at 30 ºC for 120 min, containing 0.30 µg/mL of capsaicin, had a moderately higher expression of this interleukin than capsaicin at 1 µg/mL. The CPS extract obtained at 30 ºC and 150 min had a lower concentration of capsaicin (0.2 µg/mL) and had a better inhibitory effect on the expression of IL-1β. The capsaicin concentration in the CPS extracts tested in vitro was almost ten times lower than the reported IC 50 (7.2 μM or 2.2 μg/mL) for this compound in LPS-induced NO production in RAW264.7 [18]. Therefore, other phytochemicals extracted after 120 min of cellulase treatment should be evaluated to correlate them with the reduction on the expression of this cytokine. Although no significant differences were observed in the CPS extracts' concentration of phenylalanine, it has been correlated with the NO inhibitory effect of Cleome gynandra ethanolic extract [19]. All the treatments down-regulated the expression of IL-2 without significant differences among the CPS extracts or standards used as control (Fig. 1b). This is not just an immune and inflammatory cytokine that also activates the itchy response in nervous system [20]. For IL-6, the CPS extracts obtained at 30 ºC for 120 min did not show significantly different levels than the obtained for LPS control (Fig. 1c). In contrast, the concentration of IL-6 in cells treated with CPS extracts obtained at 30 ºC for 150 min or 45 ºC and 120 or 150 min (6946.60 -8,512.35 pg/mL) was similar to the observed with synthetic anti-inflammatory drugs (4,828.43 -5,961.49 pg/mL) or capsaicin standard (5,246.46 pg/mL). Previously, it has been demonstrated the synergistic effect of other phytochemicals, like curcumine, with the capacity of capsaicin to reduce the expression and synthesis of this cytokine [21]. TNF-α expression was completely inhibited by dexamethasone (Fig. 1d) since it is an effective anti-inflammatory treatment. No significant differences were observed in the concentrations obtained with capsaicin, indomethacin and treatments performed at 30 ºC (Fig. 1d). In contrast to aqueous extracts of Capsicum baccatum, that did not reduce the TNF-α production when tested at 300 μg/mL, cellulase treated CPS have a similar potential to C. baccatum ethanolic, buthanolic or dichloromethane extracts [22]. Only the CPS extract obtained at 45 ºC, and 120 min did not show significant difference in the concentration of this cytokine compared to LPS control. Based on these cytokines analysis, the CPS extract obtained at 30 ºC for 150 min showed the best results, leading us to analyze the cytokine expression over time of extraction (kinetics).
Similarly with our results, Hudita et al. [23] showed a significant decrease in IL-1β, IL-6 and TNF-α using alginate-encapsulated capsaicin (1.77 mg/mL). Bok et al. [24] demonstrated that the administration of peritoneal capsaicin reduced the expression of cytokines IL-1β and IL-6 after surgery injury in animal models. Cho et al. [5] found that the extracts rich in phenolics and flavonoids from chili pepper fruits decreased the expression of IL-6 and TNF-α in dose-dependent manner. Malagarie-Cazenave et al. [25] found that capsaicin standard (10 µM) reduce the expression of IL-6 and TNF-α in PC-3 cells of human prostate cancer. We demonstrated that CPS extracts with reduced capsaicin content (CAP = 0.98 µM) decreased the over-expression of these cytokines and therefore their bioactivity was also related with phenolic compounds.
The concentration of most phenolic compounds explained 46.8% of the variation, while cytokine expression mainly affected the principal component 2 (Fig. 2a). TNF-α was positively correlated with capsaicin and caffeic acid concentration in the CPS extracts ( Fig. 2b and 2c). IL-2 expression was positively related with chlorogenic acid concentration (Fig. 2d). These positive correlations indicate that capsaicin, caffeic acid and chlorogenic acid at the concentrations tested in these experiments may act as immunostimulants. The administration of 1.8 mg/kg of intraperitoneal capsaicin attenuated cellular immunosuppression [26]. Caffeic acid (10 mg/kg) improved immune response in animal models targeting cytokines as TNFα and IL-6 [27]. Chlorogenic acid loaded microcapsules (35 mg/kg) enhanced T-cell labor and by consequence inhibited the immunosuppressive factor [28].

Changes in the Anti-Inflammatory Activity of CPS Extracts Obtained at Different Times of Enzymatic Treatment
Among the four cytokines evaluated with CPS extracts obtained at 30 ºC and different times of enzymatic treatment, only IL-6 did not show significant differences (Fig. 3). For the IL-1β, the extracts obtained at 90 and 120 min were less anti-inflammatory than the obtained at 150 min (Fig. 3a). The extract at 120 min contained a higher amount of chlorogenic acid (0.22 µg/g) than the obtained at 150 min (0.06 µg/g) and the immunostimulant effect of this compound was clearly observed in the changes of concentration of IL-2 (Fig. 3b). The results of Spearman ρ test (p < 0.05) exhibited a positive correlation between the overexpression of this interleukin and chlorogenic acid concentration. TNF-α showed a positive correlation with capsaicin concentration (Fig. 3d). Based on the kinetics results, the CPS extracts obtained in less than 90 min of cellulase treatment reached their maximum inhibitory effect over the cytokines related with the inf lammatory process. After 90 min, CPS extracts obtained from cellulase treatment may be used as immunostimulant.

Conclusions
We demonstrated that the CPS extracts obtained after cellulase treatment produced an anti-inflammatory effect like indomethacin, dexamethasone, and capsaicin standard. These aqueous CPS extracts have a lot of potential to be used as a food ingredient or supplement due to their anti-inflammatory or immunostimulants activities. For anti-inflammatory purposes, the CPS extract obtained at 30 ºC for 150 min reduced the concentration of IL-1β, IL-2, IL-6, and TNF-α in RAW264.7 cells stimulated with LPS. Among the most anti-inflammatory compounds found in CPS extracts were salicylic, protocatechuic and trans-cinnamic acids along with vanillin that synergistically interacted with capsaicinoids. Based on the kinetic of enzyme assisted extraction, potential immunostimulants were released after 90 min. Further experimentation is required to evaluate the immunostimulant effect observed in CPS extracts obtained at 120 and 150 min of cellulase treatment and confirm the role of capsaicin, chlorogenic and caffeic acids on this bioactivity.

Acknowledgements
The authors acknowledge to Italmesa Company to provide the Habanero chili pepper seeds and to ENMEX S.A. de C.V. to contribute with Celluzyme used in this research. Funding This work was financially supported by the "Consejo Nacional de Ciencia y Tecnologia (CONACyT)" for the scholarship 2018-000068-02NACF-07952 and by the "Instituto Tecnológico de Estudios Superiores de Monterrey". J.A.G.A. and J.L.M.V. acknowledge funding from CONACyT (grant number 316998) for the annual maintenance of the UPLC-MS platform used in this work for phenolics profiling.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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