Establishment and Phytochemical Analysis of a Callus Culture from Ageratina pichinchensis (Asteraceae) and Its Anti-Inflammatory Activity

A protocol was established to produce bioactive compounds in a callus culture of Ageratina pichinchensis by using 1 mg L−1 NAA with 0.1 mg L−1 KIN. The phytochemical study of the EtOAc extract obtained from the callus biomass, allowed the isolation and characterization of eleven secondary metabolites, of which dihydrobenzofuran (5) and 3-epilupeol (7), showed important anti-inflammatory activity. Compound 5 inhibits in vitro the secretion of NO (IC50 = 36.96 ± 1.06 μM), IL-6 (IC50 = 73.71 ± 3.21 μM), and TNF-α (IC50 = 73.20 ± 5.99 μM) in RAW (Murine macrophage cells) 264.7 macrophages, as well as the activation of NF-κB (40% at 150 μM) in RAW-blue macrophages, while compound 7 has been described that inhibit the in vivo TPA-induced ear edema, and the in vitro production of NO, and the PLA2 enzyme activity. In addition, quantitative GC-MS analysis showed that the anti-inflammatory metabolites 5 and 7 were not detected in the wild plant. Overall, our results indicated that A. pichinchensis can be used as an alternative biotechnological resource for obtaining anti-inflammatory compounds. This is the first report of the anti-inflammatory activity of compound 5 and its production in a callus culture of A. pichinchensis.

Phytochemical investigations established the presence of chromenes and acetophenones [13,14], benzofurans [1], and flavonoids [8] in different extracts obtained from the aerial parts of A. pichinchensis. These groups of compounds, mainly chromenes and bezofurans, are of great importance since they possess a wide variety of biological activities such as anticancer, antimicrobial, antiviral, anti-inflammatory, and antioxidant, between others [15,16].
To date, all phytochemical studies carried out in A. pichinchensis have been conducted directly from the wild plant, contributing to the imbalance in the ecosystem. Whereby, plant cell culture offers an alternative to produce bioactive secondary metabolites under a controlled environment, independent of seasonal and geographical conditions. One of the possibilities for producing valuable metabolites under in vitro conditions is based on callus culture. To our knowledge, chromene and benzofuran production by in vitro cultures has not been reported. Furthermore, there is no information related to the production of these metabolites in callus cultures of A. pichinchensis.
In the current research, a callus culture protocol from leaf explants of A. pichinchensis was established for the first time.

Callus Production
Callus induction was obtained from leaf explants of A. pichinchensis seedlings (aged 30-days old) grown under in vitro conditions. Leaf explants were submitted to different concentrations of plant growth regulators (PGR, auxins and cytokinin), such as 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram) in combination with 6-furfurylaminopurine (KIN). It was identified that the best combination of PGR for callus induction and secondary metabolites was NAA and KIN, so a second experimental design was established to identify the appropriate concentration of NAA and KIN (Table 1). Morphogenetic response occurred at 15 days of culture, and greater effect was shown, mainly by combining α-naphthaleneacetic acid (NAA) and kinetin (KIN). The callus produced was friable in appearance and beige in color. The maximum percentage of callus formation (81.7%) was obtained when 1.0 mg L −1 NAA with 0.1 mg L −1 KIN were added to culture medium ( Figure 1a). Simultaneously, callus with roots (20 to 50%, Figure 1b) or roots (15 to 51.7%, Figure 1c) were formed in the treatments containing KIN (1 or 2 mg L −1 ). This result is like to that obtained for A. adenophora, where the highest callus induction frequency (87.2%) was obtained from leaf segments, using MS medium supplemented with 0.5 mg L −1 2,4dichlorophenoxyacetic acid (2,4-D) and 2.0 mg L −1 6-benzylaminopurine (BA) [17]. These letters mean that the data is statistically significant

TPA-Induced Auricular Edema in Mice
The ethyl acetate extract obtained from callus biomass of A. pichinchensis was evaluated at different doses (0.005 to 0.10 mg/ear) in TPA-induced auricular edema in mice following the protocol described by Gutiérrez-Rebolledo et al. (2016) [18]. This study showed that the extract has an important anti-inflammatory effect. Indeed, the extract reduced the edema in mouse by 35.11 ± 3.79% at the dose of 0.1 mg/ear, which was similar to that obtained for indomethacin (27.66 ± 1.16% inhibition at the dose of 0.1 mg/ear). This result confirms the anti-inflammatory activity of extracts from the wild populations of A. pichinchensis previously reported by Romero-Cerecero et al. (2013) [8], who also suggested the presence of secondary metabolites capable to diminishing TPA-induced This result is like to that obtained for A. adenophora, where the highest callus induction frequency (87.2%) was obtained from leaf segments, using MS medium supplemented with 0.5 mg L −1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mg L −1 6-benzylaminopurine (BA) [17].

TPA-Induced Auricular Edema in Mice
The ethyl acetate extract obtained from callus biomass of A. pichinchensis was evaluated at different doses (0.005 to 0.10 mg/ear) in TPA-induced auricular edema in mice following the protocol described by Gutiérrez-Rebolledo et al. (2016) [18]. This study showed that the extract has an important anti-inflammatory effect. Indeed, the extract reduced the edema in mouse by 35.11 ± 3.79% at the dose of 0.1 mg/ear, which was similar to that obtained for indomethacin (27.66 ± 1.16% inhibition at the dose of 0.1 mg/ear). This result confirms the anti-inflammatory activity of extracts from the wild populations of A. pichinchensis previously reported by Romero-Cerecero et al. (2013) [8], who also suggested the presence of secondary metabolites capable to diminishing TPA-induced inflammation. Thus, we continued with the fractionation to identify the active anti-inflammatory compounds.

In Vitro Anti-Inflammatory Activity
In order to assess the anti-inflammatory activity of the isolated compounds 2 and 5, these were first evaluated for their cytotoxicity on RAW 264.7 cells. The ethyl acetate extract does not affect the viability of the macrophages up to 30 µg mL −1 . On the other hand, compounds 2 and 5 at concentrations from 9.4 to 150 µM do not exhibit a significant reduction in viability of macrophages compared with the control group ( Figure 3). Therefore, these concentrations were used to test in vitro anti-inflammatory effect of the extract and compounds.

In Vitro Anti-Inflammatory Activity
In order to assess the anti-inflammatory activity of the isolated compounds 2 and 5, these were first evaluated for their cytotoxicity on RAW 264.7 cells. The ethyl acetate extract does not affect the viability of the macrophages up to 30 μg mL −1 . On the other hand, compounds 2 and 5 at concentrations from 9.4 to 150 μM do not exhibit a significant reduction in viability of macrophages compared with the control group ( Figure 3). Therefore, these concentrations were used to test in vitro anti-inflammatory effect of the extract and compounds. The activity against production of NO and cytokines in macrophages stimulated by LPS was evaluated using RAW 264.7 cells. The results indicated that the ethyl acetate extract showed a limited but significant NO inhibitory activity at 30 μg mL −1 (17.52 ± 0.72%). Compound 2 had no significant effect against NO production, even at 150 μM (Figure 4a), nevertheless compound 5 showed an inhibitory effect at all concentrations tested, reaching 100% inhibition at 150 μM, with a dosedependent relationship (Figure 4b), with a CI50 = 36.96 ± 1.06 μM.  The activity against production of NO and cytokines in macrophages stimulated by LPS was evaluated using RAW 264.7 cells. The results indicated that the ethyl acetate extract showed a limited but significant NO inhibitory activity at 30 µg mL −1 (17.52 ± 0.72%). Compound 2 had no significant effect against NO production, even at 150 µM (Figure 4a

In Vitro Anti-Inflammatory Activity
In order to assess the anti-inflammatory activity of the isolated compounds 2 and 5, these were first evaluated for their cytotoxicity on RAW 264.7 cells. The ethyl acetate extract does not affect the viability of the macrophages up to 30 μg mL −1 . On the other hand, compounds 2 and 5 at concentrations from 9.4 to 150 μM do not exhibit a significant reduction in viability of macrophages compared with the control group ( Figure 3). Therefore, these concentrations were used to test in vitro anti-inflammatory effect of the extract and compounds. The activity against production of NO and cytokines in macrophages stimulated by LPS was evaluated using RAW 264.7 cells. The results indicated that the ethyl acetate extract showed a limited but significant NO inhibitory activity at 30 μg mL −1 (17.52 ± 0.72%). Compound 2 had no significant effect against NO production, even at 150 μM (Figure 4a   Because only compound 5 showed activity in the NO assay, it was the only one that was tested for its ability to inhibit Interleukin 6 (IL-6) and TNF-α secretion, two pro-inflammatory cytokines, using the LPS-stimulated RAW cell model. The secretion of IL-6 and TNF-α was totally inhibited at 150 µM and the effect showed a concentration dependence, with a CI 50 = 73.71 ± 3.21 and 73.20 ± 5.99 µM, respectively (Figure 5a,b). Because only compound 5 showed activity in the NO assay, it was the only one that was tested for its ability to inhibit Interleukin 6 (IL-6) and TNF-α secretion, two pro-inflammatory cytokines, using the LPS-stimulated RAW cell model. The secretion of IL-6 and TNF-α was totally inhibited at 150 μM and the effect showed a concentration dependence, with a CI50 = 73.71 ± 3.21 and 73.20 ± 5.99 μM, respectively (Figure 5a,b).
Activation of factor NF-κB is important for the pro-inflammatory response of macrophages, including IL-6 and TNF-α secretion, whereby, the ability of compound 5 to avoid this process was evaluated. We observed that this compound inhibits the activation of NF-κB, but 100% inhibition was not achieved even at 150 μM. Nonetheless, an effect associated with concentration is observed, reaching a 40% of inhibition at 150 μM (Figure 5c). . The values are expressed as the mean ± SD of three independent experiments (n = 3). Significance was determined using ANOVA followed by Dunnett's multiple comparisons test ( #### p < 0.0001). LPS compared with control group; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 INDO (indomethacin) or UA (ursolic acid) or compound 5, all compared with LPS group. . The values are expressed as the mean ± SD of three independent experiments (n = 3). Significance was determined using ANOVA followed by Dunnett's multiple comparisons test ( #### p < 0.0001). LPS compared with control group; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 INDO (indomethacin) or UA (ursolic acid) or compound 5, all compared with LPS group.
Activation of factor NF-κB is important for the pro-inflammatory response of macrophages, including IL-6 and TNF-α secretion, whereby, the ability of compound 5 to avoid this process was evaluated. We observed that this compound inhibits the activation of NF-κB, but 100% inhibition was not achieved even at 150 µM. Nonetheless, an effect associated with concentration is observed, reaching a 40% of inhibition at 150 µM (Figure 5c).
The NO is an important pro-inflammatory mediator and is involved in some inflammatory disorders including rheumatoid arthritis, chronic hepatitis, and pulmonary fibrosis [25,26]. The results suggested that compound 5 play an important role in the anti-inflammatory activity of A. pichinchensis. On the other hand, results indicate that compound 5 could be considered as an anti-inflammation agent because this compound has potential to inhibit NF-κB activation, NO, IL-6 and TNF-α secretion. Overall, our results could suggest that compound 5 suppressed the expression of NF-κB target genes such as Inducible nitric oxide synthase (iNOS) and pro-inflammatory pathway.

Quantification of 2,3-Dihydrobenzofuran (5) in Wild Plant and Callus Extracts of A. pichinchensis
These results indicated that compound 5 has an important anti-inflammatory activity, and taken in consideration that 3-epilupeol (7) has been described as an anti-inflammatory agent, we decide to quantify both in the callus biomass. For the quantitative GC-MS analyses of 5 and 7 in the EtOAc extracts of wild plant and callus biomass, identification of compounds was obtained by analysis of the peaks at R T = 20.67 min (for 5) and 38.70 min (for 7), and the molecular ion peaks at m/z = 234 for 5 and 426 for 7 observed in the GC-MS analysis ( Figures S1 and S2, supplementary material). According to the values of dry biomass (13.92 g), callus from leaf explants produced 0.65 ± 0.011 mg/g dry biomass of compound 5, and 0.2011 ± 0.015 mg/g dry biomass of 3-epilupeol (7) (Figures S3 and  S4, supplementary material); by contrast, these compounds were not detected in wild plant extract ( Figures S5 and S6, supplementary material). This result indicates that the addition of NAA and KIN (1.0/0.1 mg L −1 ) to the culture medium was essential for production of the anti-inflammatory metabolites 5 and 7 through callus culture derived from segments leaf of in vitro plantlets.

General
Compounds were isolated by means of open column chromatography (CC). Analytical TLC was carried out on precoated silica gel 60F254 plates (Merck, Darmstadt, Germany). The isolation procedures and purity of compounds were checked by thin layer chromatography (TLC), visualized by means of UV light, and sprayed with Ce(SO 4 ) 2 2(NH 4 ) 2 SO 4 2H 2 O. All 1 H-and 13  flow). The inlet temperature was set at 250 • C while oven temperature was initially at 40 • C (held for 1 min) and increased to 280 at 10 • C/min. The mass spectrometer was operated in positive electron impact mode with ionization energy of 70 eV. Detection was performed in selective ion-monitoring (SIM) mode and peaks were identified and quantitated using target ions.

Plant Material
Plants and seeds of Ageratina pichinchensis were collected in Tepoztlán Morelos, Mexico. Both plant and seed were identified by Biol. Gabriel Flores Franco and deposited at the HUMO Herbarium of the Universidad Autónoma del Estado de Morelos (UAEM), with the voucher number 33913.

Callus Induction and Maintenance
The seeds of A. pichinchensis were surface disinfected and germinated in sterile semisolid MS culture medium with 3% sucrose, plant growth regulators (PGRs)-free, pH adjusted to 5.8 and 0.2% phytagel. Culture medium was previously sterilized at 121 • C, 15 psi, for 15 min using an autoclave. After four weeks, the leaves of the seedlings were cut into pieces and four explants were transferred into jars containing 25 mL the same MS medium and PGRs (auxins and cytokinin). First, a screening of tree auxins (α-naphthaleneacetic acid, NAA; 2,4-dichlorophenoxyacetic acid, 2,4-D; picloram, PIC) at 0.1 or 1.0 mg L −1 , each combined with a cytokinin (kinetin, KIN) at 0.1 mg L −1 , were evaluated to determine which auxin induced better callus formation at 20 days of culture. At 20 days, we observed that NAA showed better response on leaves explant. Therefore, to determine the best treatment of callus induction, different concentrations of NAA (0.0, 0.1, 1.0 or 2.0 mg L −1 ) were combined with KIN (0.0, 0.1, 1.0 or 2.0 mg L −1 ). Five jars containing four leaf explants for each treatment were used to determine the percentage of callus induction at 20 days of culture and the experiment was repeated three times. Callus subcultures were carried out every 30 days for 12 months using culture medium with the same conditions. All cultures were incubated at 25 ± 2 • C under photoperiod of 16 h with white fluorescent light (50 µmol m −2 s −1 ).
Compounds 1, 2, 5, and 7 were identified using 1 H-and 13 C-NMR, and comparison with reported values. Compounds 3, 4 and 8 were characterized by direct comparison with authentic samples available in our laboratory [27,28].

GC/MS Quantification of the Anti-Inflammatory Metabolites 5 and 7
For the quantitative analysis, a series of solutions containing the standard molecule (2S,3R)-5-acetyl-7,3α-dihydroxy-2β-(1-isopropenyl)-2,3-dihydrobenzofuran (5) in known concentrations (2.2, 1.1, 0.55, 0.275, 0.1375, 0.06875 mg mL −1 ) and (0.350, 0.175, 0.0875, 0.04375, 0.02187 mg mL −1 ) for the standard molecule 3-epilupeol (7) were prepared in triplicate and analyzed by GC-MS. The linear calibration curves were plotted to quantify the compounds 5 and 7 in the ethyl acetate extract obtained from biomass of callus culture. Each standard solution was analyzed in triplicate to calculate the peak area ratio (y) and relative concentration (x), these data were used to construct the linear calibration curve, which showed acceptable linearity with correlation coefficients r 2 = 0.9926 and r 2 = 0.9997, respectively ( Figures S1 and S2 of supplementary material). The results were obtained by analysis in Microsoft Excel 2010.

Preparation of Extracts for GC-MS Quantification
Dried leaves of A. pichinchensis (15.2 g) were exhaustively extracted with EtOAc (3 × 100 mL) by maceration. After extraction and evaporation of the solvent 1.76 g of residue was obtained. An aliquot (12.5 mg mL −1 ) was diluted with chloroform (1 mL) and analyzed by GC-MS.
After 30 days of culture, the calli were harvested, dried and ground into fine powder. The finely ground samples (13.92 g) were extracted with EtOAc (3 × 100 mL) in a sonication bath for 30 min. The EtOAc extracts were filtered under vacuum through Whatman No. 1 filter paper and dried in vacuum to yield 550 mg of EtOAc extract. An aliquot (11.3 mg) was diluted with chloroform (1 mL) and immediately injected in a GC-MS for the quantification of compounds 5 and 7. The identification was based on the comparison of retention time and MS data with those of the authentic sample. Quantification was performed by using the calibration curve previously constructed in the same conditions and expressed in terms of mg g −1 dry mass. The analysis was done using three replicates per treatment.

TPA-Induced Mouse Ear Edema
Mouse ear edema was evaluated according to the method described previously [22]. All experiments were carried out using five animals per treatment. Adult male CD-1 mice with a body weight of 30 ± 5 g, were used. Experiments were performed according to the Official Mexican Rule: NOM-062-ZOO-1999 Guidelines (Technical Specifications for the Production, Care, and Use of Laboratory Animals) and international ethical guidelines for the care and use of experimental animals. The experimental protocol used was approved by Comité para el Cuidado y Uso de los Animales de Laboratorio (CCUAL) de la Facultad de Medicina (No. 06-2015). Mice were maintained under standard laboratory conditions (Bioterio de la Facultad de Medicina, Universidad Autónoma del Estado de Morelos) at 22 • C ± 3 • C, 70% ± 5% of humidity, 12 h light/dark cycle and food/water ad libitum. A negative control group received acetone as vehicle and indomethacin was used as anti-inflammatory drug as positive control group. Animal ear inflammation was induced with 2.5 µg/ear of TPA dissolved in 20 µL of acetone applied to the internal and external surface of the right ear to cause edema. Sample doses of 0.005, 0.01, 0.05, and 0.1 mg/ear of the EtOAc extract, as well as the anti-inflammatory drug of reference indomethacin (0.005, 0.01 and 0.1 mg/ear) were applied. All the samples of the different treatments were dissolved in (10 µL of acetone) and applied topically on the right ear immediately after TPA application; on the left ear, acetone was applied as vehicle. Four hours after application of the samples of interest as possible anti-inflammatory agents, the animals of each treatment were sacrificed by cervical dislocation. Circular sections of 6 mm in diameter were taken from both: the treated (t) and the non-treated (nt) ears, which were weighed to determine the inflammation. Percentage of inhibition was determined by the formula expressed below: where ∆ w = w t − w nt ; being w t the weight of the section of the treated ear and w nt the weight of the section of the non-treated ear. RAW 264.7 cells (1 × 10 4 cell/well in 100 µL of medium) were seeded in a 96-well plate and incubated for 24 h, the cells were treated with various concentrations (9.4-150 µM) of compounds 2 and 5 or vehicle (DMSO, 0.5%, v/v) or etoposide (68 µM) and incubated for 20 h. Cell viability was determined by MTS assay, using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation assay (Promega), according to the manufacturer's instructions. Briefly, 20 µL of MTS was added to each well, and the cells were incubated for another 4 h. The optical density was measured at 490 nm on a microplate reader.
3.6.4. Treatment of Macrophages RAW 264.7 with LPS RAW 264.7 cells (3 × 10 4 cells/well in 200 µL of medium) were plated and incubated for 24 h at 37 • C into 96-well plates. After that, the cells were incubated for two hours with the compounds 2 and 5 at various concentrations (9.4-150 µM), vehicle (DMSO, 0.5%, v/v) or indomethacin (84 µM) and then were incubated with LPS (1 µg mL −1 ) at 37 • C for 20 h as a pro-inflammatory stimulus. Finally, cell-free supernatants were collected and a part was used to NO quantification in fresh and another one was kept at −20 • C until IL-6 and TNF-α quantification.

Determination of NO Concentration
Nitrite, the stable end-product of NO, was measured according to Griess reaction. Briefly, 50 µL of each supernatant were mixed with 100 µL of Griess reagent [50 µL of 1% sulfanilamide and 50 µL of 0.1% N-(1-naphtyl) ethylenediamine dihydrochloride in 2.5% phosphoric acid], for 10 min at room temperature. The optical density at 540 nm (OD 540 ) was measured with a microplate reader and nitrite concentration in the samples were calculated by comparison with the OD 540 of a standard curve of NaNO 2 prepared in fresh culture medium [34].
3.6.6. Measurement of IL-6 and TNF-α The quantitative measurement of murine IL-6 and TNF-α secretion in the supernatants was performed using the PEPROTECH Murine IL-6 and TNF mini ABTS ELISA kit, according to the instructions provided by manufacturer.
3.6.8. Determination of NF-κB Activation RAW-Blue cells (3 × 10 4 cells/well in 100 µL of medium) were plated and incubated into 96-well plates for 24 h at 37 • C. After that, the cells were incubated for two hours with the compound 5 at 75 and 150 µM or DMSO (0.5%, v/v) or indomethacin (84 µM) or ursolic acid (17.5 µM, positive control) and then were incubated with LPS (1 µg mL −1 ) at 37 • C for 20 h to stimulate NF-κB activation [35,36]. Finally, cell-free supernatants were collected and were used in fresh to determine NF-κB activation, related to alkaline phosphatase secretion using QUANTI-Blue™ (InvivoGen), according to the instructions provided by manufacturer.

Statistical Analysis
The results shown were obtained at least by three independent experiments and are presented as means ± SDs. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. The mean comparation test for callus percentages was performed according to Tukey's method. All statistical analyses were performed using the GraphPad Prism ® , Version 6.0 software. p values <0.05 were considered to indicate statistical significance.

Conclusions
A protocol has been developed to produce anti -inflammatory metabolites in callus culture of A. pichinchensis. NAA and KIN found to be essential for the production of friable callus. Callus from in vitro leaf explant in presence of 1.0 mg L −1 NAA with 0.1 mg L −1 KIN in MS medium after 30 days of culture produce eleven secondary metabolites (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), of which, metabolites 3, 4, 6, 7, 9 and 10 were previously described with anti-inflammatory properties. In this work, we demonstrated that the dihydrobenzofuran 5, was the major constituent of the callus biomass, and effectively inhibited the LPS-induced production of proinflammatory factors, such as NO, IL-6, and TNF-α in RAW 264.7 macrophages, and the activation of NF-kB in RAW-blue macrophages without causing cytotoxicity. A possible mechanism for this effect involves the ability of 5 to activate a signaling cascade, which results in the repression of NF-κB, in LPS-challenged macrophages. Although further investigation is needed to clarify the precise mechanisms by which 5 inhibits NF-κB activation, this natural dihydrobenzofuran, produced by a callus culture of A. pichinchensis, may be considered a potential therapeutic agent for the treatment of inflammation-related disease.
Quantitative analysis demonstrated that the most active compounds 5 and 7 were not produced in wild plants. Therefore, callus culture of A. pichinchensis can serve as a good source for the obtaining of anti-inflammatory metabolites. This is the first report of the production of metabolites 1, 2, 4-7 in a callus culture of A. pichinchensis, and the first report of the anti-inflammatory activity of compound.