Establishment of a Cell Suspension Culture of Ageratina pichinchensis (Kunth) for the Improved Production of Anti-Inflammatory Compounds

Ageratina pichinchensis (Kunth) is a plant used in traditional Mexican medicine to treat multiple ailments. However, there have not been biotechnological studies on producing compounds in in vitro cultures. The aim of this study was to establish a cell suspension culture of A. pichinchensis, quantify the anti-inflammatory constituents 2,3-dihydrobenzofuran (2) and 3-epilupeol (3), evaluate the anti-inflammatory potential of its extracts, and perform a phytochemical analysis. Cell suspension cultures were established in a MS culture medium of 30-g L−1 sucrose, 1.0-mg L−1 α-naphthaleneacetic acid, and 0.1-mg L−1 6-furfurylaminopurine. The ethyl acetate extract of the cell culture analyzed by gas chromatography (GC) revealed that the maximum production of anti-inflammatory compounds 2 and 3 occurs on days eight and 16, respectively, improving the time and previously reported yields in callus cultures. The anti-inflammatory activity of these extracts exhibited a significant inhibition of nitric oxide (NO) production. Furthermore, a phytochemical study of the ethyl acetate (EtOAc) and methanol (MeOH) extracts from day 20 led to the identification of 17 known compounds. The structures of the compounds were assigned by an analysis of 1D and 2D NMR data and the remainder by GC–MS. This is the first report of the production of (-)-Artemesinol, (-)-Artemesinol glucoside, encecalin, and 3,5-diprenyl-acetophenone by a cell suspension culture of A. pichinchensis.


Introduction
The genus Ageratina (Asteraceae) consists of about 1200 species and is distributed in temperate and subtropical regions of the America, Europe, Africa, and Asia [1][2][3][4]; in Mexico, about 164 species of Ageratina have been reported [5,6]. Several species of this genus have been studied, and these studies have demonstrated bactericidal, antifungal, antiviral, analgesic, cytotoxic, and anti-inflammatory effects, as well as the ability to treat gastric ulcers [7][8][9][10][11][12]. In the state of Morelos, Mexico, Ageratina pichinchensis is traditionally used to treat gastric ulcers and heal deep wounds. Phytochemically, the aerial parts of A. pichinchensis are characterized by containing sterols, triterpenes, benzochromenes, and benzofurans [13,14]. The benzochromenes isolated from A. pichinchensis showed insecticidal [12], antifungal [15,16], and gastroprotective activities [17]. However, the production of these compounds can be unsustainable through the large-scale planting of A. pichinchensis, because many secondary Similar results were observed in cell suspension cultures of Stevia rebaudiana that were disintegrated within seven days, and the cells also acquired a yellowish appearance [28]. In this study, the cell growth of A. pichinchensis was faster (22 days) in the MS liquid culture medium than in callus cultures; in contrast, callus reached their maximum growth after 30-40 days [27]. This might be caused by the facilitated absorption of nutrients in the liquid medium [29].
The growth kinetic of A. pichinchensis cell suspension cultures was maintained for 22 days, during which it showed a typical growth curve ( Figure 2). The growth kinetic was characterized by a lag phase of four days, reaching 2.37-g L −1 dry weight (DW); subsequently, the cells entered the exponential phase and lasted until day 16. During this time, a maximum biomass accumulation was observed (13.28-g L −1 ) of about 5.6-fold the initial dry weight. The specific growth rate (µ) was 0.20 days −1 , and doubling time (Dt) was 3.01 days −1 until reaching a stationary phase, in which the cell culture showed a brown appearance and decreased growth ( Figure 2). Regarding the consumption of total sugars, an abrupt decrease in the sugar content was observed until day six, but the biomass increased; the remainder of the sugar was stable after day 14 ( Figure 2), perhaps due to the consumption of nutrients and lack of oxygen in the medium [30,31].
The growth kinetics is similar to that for Spilanthes acmella, in which the cell suspension culture reached a specific growth rate of 0.28 days −1 ; during the exponential phase, the doubling time was 2.50 days −1 , and the maximum biomass was 8.5-g L −1 at day 15 [32]. On the other hand, Satureja khuzistanica cell suspension cultures reached a maximum dry biomass of 19.7-g L −1 at 21 days, with a specific growth rate of 1.5 days −1 and a doubling time of 7.6 days −1 [33]. Similarly, cell suspension cultures of Helianthus annuus produced 12.7-g L −1 dry biomass after nine days of culture, showing a specific growth rate of 0.21 days −1 and a doubling time of 3.31 days −1 [34]. These results suggest that A. pichinchensis cell cultures have a similar tendency as other cell suspension cultures, and, although the biomass yield differs for each species, it can also be produced in a short time.

Cell Viability and pH in the Culture Medium
Cell viability was suitable in the cell suspension culture, decreasing only slightly during the 22 days of culture. On the first day of culture, 89.57% of the viability was present, and by 22 days, it only decreased to 77.19% ( Figure 3).
These results confirm that the establishment of the A. pichinchensis cell suspension culture was successful since the viability was greater than 50%. In other species, such as Taxus globose, the viability decreases to 45-50% at the end of the growth culture [35], which was lower than that of the A. pichinchensis culture. The microscopic image shows that the A. pichinchensis cell suspension culture produced single cells and some small aggregates (Figure 1d). In a study conducted in Taxus cuspidate cell cultures [35], it was found that the size of cell aggregates is important, since at average sizes greater than 800 µm, there is a decrease in the production of paclitaxel; however, in our study, the aggregates were smaller (Figure 1d). On the other hand, there was a gradual increase in the pH values (5.0-5.3) during the exponential phase (from day four to 18); later, between days 18 and 22, the pH values remained stable at 5.3, but these changes did not affect the biomass or the production of compounds 2 and 3 during the growth kinetics. Likewise, the cell viability experienced a similar behavior; at the beginning of the kinetics, the cell viability was 89%, remaining stable until day 10 (exponential phase), and on subsequent days, a gradual decrease was observed up to 77%, possibly as a consequence of the depletion of nutrients and alkalinization of the culture medium ( Figure 3).
In cell suspension cultures of Dioscorea deltoidea, there was an acidification of the culture medium during the lag phase, an alkalization during the exponential phase, and a steady state of pH in the stationary phase. At the same time, the exponential growth of the cell culture medium may be associated with the development of alkaline reactions that occur during metabolism [36]. The observed changes in pH values may be related to the use of ammonium and nitrate or the absorption of sugar by the transport mechanism with H + [37]. Moreover, plant cells can also modify the external pH; they can increase or decrease the values according to the pH range in which they are grown, until a balance is produced [38].
The quantification of compounds 2 and 3 was performed by analyzing the peaks at the retention time (RT) = 20.67 min (compound 2) and 38.70 min (compound 3); the molecular ion peaks were observed at m/z = 234 for 2 and 426 for 3 in GC-MS ( Figures S19 and S20). The production of compound 2 started from the lag phase, and the maximum production (510.75 ± 29.10-µg g −1 DW) occurred on day eight of the exponential phase; then, it gradually decreased until day 22. Similar results were observed in Capsicum chinense Jacq. cell suspension cultures, in which the capsaicin compound reached a maximum production of 567.4-µg g −1 DW at 25 days [31]. On the other hand, in Celosia cristata cell suspension cultures, betalaine production was observed at the beginning of the exponential phase and then decreased, remaining stable during the exponential and stationary phases [30]. Regarding the 3-epilupeol compound (3), an association with the culture growth was observed, obtaining its maximum yield (410.59 ± 36.91-µg g −1 DW) at day 16 ( Figure 5). Likewise, the production of the fatty acid amide spilanthol by cell suspension cultures of Spilanthes acmella Murr. presented a trend associated with its growth, reaching a maximum yield during the exponential phase and, subsequently, decreasing rapidly due to the lack of nutrients and consequent cellular death [32]. In another species, Eurycoma longifolia, it was reported that cell suspension cultures produce the quassinoid eurycomanone; this also occurs from the beginning of the growth kinetics, reaching a maximum amount of 1.7-mg g −1 DW [44].
These results surpass those reached by callus cultures, whose maximum production was identified on day 30, producing 650-µg g −1 DW for compound 2 and 201.10-µg g −1 DW of compound 3 [27]. Reducing the time of production of the compounds is a desirable characteristic in cell suspension cultures; in addition to being more homogeneous compared to callus cultures, it is possible to increase the production of bioactive compounds by adding elicitors and by scaling up reactors [45][46][47].
On the other hand, encecalin (10) (m/z 232, RT = 18.5 min) and 3,5-diprenyl-1,4-hydroxyacetophenone (11) (m/z 272, RT = 21.87 min) were detected in very low concentrations during the exponential phase (day eight) ( Figure 6). Antifungal, gastroprotective, and antinociceptive effects were reported for compounds 10 and 11 [17,43]. The highlight of both compounds is that their production on in vitro cultures was reported for the first time. Based on the importance of its biological effects, cell suspension cultures of A. pichinchensis are a useful alternative for the production of compounds 10 and 11, which could be increased by inductors.

In Vitro Anti-Inflammatory Activity
The anti-inflammatory activity of the ethyl acetate extracts of the biomass of the A. pichinchensis suspension cell culture was assessed at different times of the growth kinetics: day 8 (D8), day 12 (D12), and day 16 (D16). Firstly, the extracts were evaluated for their effect on the viability of RAW 264.7 cells at different concentrations (5,10,20,30, and 40-µg mL −1 ). No extracts exhibited a significant reduction in the viability of macrophages compared with the control group, while the positive control (etoposide) showed a significant reduction in the cellular viability at 40-µg mL −1 (Figure 7).  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 dimethyl sulfoxide (DMSO), ETOP (etoposide), and extracts compared with the control group).
To assess the effect of the extracts from D8, D12, and D16 on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, cells were treated with the extracts at the same concentrations used in the viability assay. It was reported that LPS, an essential component of the outer membrane of Gram-negative bacteria, can induce inflammatory responses in RAW 264.7 macrophages to produce proinflammatory molecules such as NO [48,49]. The experimental results showed that the NO level was increased in LPS-stimulated RAW cells, and this effect was decreased significantly by treatment with extracts at the concentrations tested ( Figure 8). The results showed that D12 and D16 were the most active extracts that inhibited NO production at 40-µg mL −1 , with 35.14% ± 7.55% and 34.42% ± 7.15% inhibition, respectively. On the other hand, the extract from D showed 24.69% ± 6.17% inhibition in NO production; at this point of the kinetics, compound 2 is produced in greater quantity compared to compound 3 (510.75 ± 29.10-µg g −1 DW and 127.85 ± 14.86-µg g −1 DW, respectively). In the extract from D12, the concentrations of the compounds were 434.01 ± 32.25-µg g −1 DW (2) and 244.89 ± 13.34-µg g −1 DW (3); finally, it was observed that the extract from D16 contains more of compound 3 (410.60 ± 36.91-µg g −1 DW), which showed a greater effect with respect to compound 2, which was identified at 335.45 ± 21.72-µg g −1 DW. This result corroborates the outstanding effect of compound 3: as it increases in content in the extracts, the anti-inflammatory effect also increases proportionally. However, in a pure way, both compounds have an important anti-inflammatory effect. Indomethacin (the positive control) showed an inhibition of 47.45% ± 7.41% at 30-µg mL −1 (Figure 8).
The importance of the anti-inflammatory effects of 3-epilupeol (3) lies in the fact that the excessive production of NO causes tissue damage, extensive systemic vasodilatation, and hypotension [54]. In addition, NO is involved in inflammatory disorders, including bowel diseases, rheumatoid arthritis, chronic hepatitis, pulmonary fibrosis, and colon cancer [56,[58][59][60].

Establishment of Cell Suspension Cultures
Friable callus cultures of A. pichinchensis were previously established by our working group [27]. Calluses were subcultured in the same Murashige and Skoog (MS) semisolid culture medium containing 3% sucrose, 1.0-mg L −1 α-naphthaleneacetic acid (NAA), and 0.1-mg L −1 6-furfurylaminopurine (KIN). The 20-day-old calluses were used to establish the cell suspension cultures. Fresh calluses (5 g) were transferred to 250-mL Erlenmeyer flasks containing 50 mL of MS liquid culture medium using the same conditions and plant growth regulator as in the callus cultures. Cell suspension cultures were placed on an orbital shaker at 110 rpm and incubated at 25 ± 2 • C under a photoperiod of 16-h with white fluorescent light (50-µmol m -2 s −1 ). When an increase of biomass was shown in the flasks, cells were harvested and screened with 200-µm nylon mesh filters (Whatman No. 1) to obtain a homogeneous cell culture. To increase the biomass, the cells were subcultured every 15 days for six months using an inoculum size of 10% (v/v) in 500-mL Erlenmeyer flasks with 100 mL of liquid culture medium.

Growth Kinetics
The growth kinetics of the cell suspension culture was carried out in 250-mL flasks without modifying the composition of the culture medium. Each flask containing 50 mL of medium MS was inoculated with 2 g of fresh cells and incubated in the same conditions mentioned above. Three flasks were harvested every two days, and the culture was allowed to grow for 22 days. Harvested cells were washed with distilled water, filtered with a cellulose filter (Whatman No. 1), and dried in an oven at 55 • C for 24 h. Then, dry-weight biomass (DW) data were recorded to perform the culture growth curve. The specific cell growth rate (µ) was calculated by plotting the natural logarithm of the cell growth data versus time. The doubling time (Dt) was computed from the µ exponential data.

Cell Viability
The cell viability of the cell suspension culture was measured by Evan's blue day exclusion method [61]. A sample of 1 mL of cell suspension was taken from each flask and incubated into 0.25% Evan's blue stain for 5 min, and at least 700 cells were counted. Viable cells were considered those that were not stained. All the experiments were repeated three times, with three replicates each.

Sugar Quantification and pH Measurement
During each sampling of growth kinetics, 5-mL aliquots from the residual culture medium of each biomass sample were taken; their pH was measured with a potentiometer (Science Med SM-25CW) and total sugar content by the phenol-sulfuric method [62]. A calibration curve was performed using sucrose as a standard at concentrations of 0.1 to 1.0-µg mL −1 . A sample aliquot (2 mL) of the sample was mixed with 2 mL of phenol reagent at 5% in digester tubes and placed in a rack submerged in a cold-water bath. Then, 5 mL of H 2 SO 4 concentrated was added to the mixture and allowed to stand for 15 min and analyzed in a spectrophotometer at 490 nm against a blank.

Extraction and Isolation of Compounds from Cell Suspension Cultures
Biomass harvested on day 20 was dried in an oven at 40 • C (12.30 g) and extracted with 100 mL of ethyl acetate by sonication (30 min); the extraction process was performed in triplicate. The excess of solvent was eliminated in a rotatory evaporator under reduced pressure, and a brown residue (1.3 g) was obtained. A second extraction was carried out with methanol (3 × 100 mL); the solvent was removed by distillation, giving rise to a resinous residue (2.4 g).

3-Epilupeol (3)
Compound (3) -27). These data match those in the literature [27]. Spectra of 1 H and 13 C NMR are in Figures S5 and S6. 3.4.6. (±)-Artemesinol Glucoside (7) Compound (7)  Compounds 8 and 9 and 12-17 were identified by comparing the GC relative retention times and MS fragmentation pattern of a single compound with those from the NIST 1.7a mass spectral library. GC-MS chromatograms of compounds 8 and 9 and 12-17 are in Figures S15-S17. Compounds 10 and 11 were identified in the suspension cells by comparing their GC relative retention times and MS fragmentation patterns. The standards used in the analysis were as follows: encecalin (10) and 3,5-diprenyl-4-hydroxyacetophenone (11), previously isolated from A. pichinchensis. GC-MS chromatograms are in Figure S18.

Quantification of Compounds 2 and 3 by GC-MS
Biomass collected at 0, 2,4,6,8,10,12,14,16,18,20, and 22 days were dried in an oven at 55 • C for 24 h. Subsequently, each sample was extracted by sonication with EtOAc (25 mL × 3) and MeOH (25 mL × 3) and concentrated in a rotatory evaporator. Maximum production of compounds 2 and 3 was identified in ethyl acetate extracts on days 8 (D8) and 16 (D16), respectively. For the quantitative analysis, a standard curve of compounds 2 and 3 was prepared in triplicate and analyzed by GC-MS. Concentrations of 2.2, 1.1, 0.55, 0.275, 0.1375, and 0.06875-mg mL −1 were used for compound 2 and of 0.350, 0.175, 0.0875, 0.04375, and 0.02187-mg mL −1 for compound 3. Each standard solution was analyzed in triplicate to calculate the peak area ratio (y) and relative concentration (x); these data were used to construct a linear calibration curve, which showed acceptable linearity with correlation coefficients r 2 = 0.9926 and 0.9997, respectively ( Figures S9 and S10). The quantification of compounds 2 and 3 in the extracts was expressed as µg g −1 biomass dry weight (µg g −1 DW).
3.6. In Vitro Anti-Inflammatory Activity 3.6.1. Cell Culture RAW 264.7 cells were maintained in DMEM/F12 medium supplemented with 10% heat-inactivated FBS, without antibiotics. Cells were cultured at 37 • C in a humidified atmosphere containing 5% CO 2 and subcultured by scraping and seeding them in 25-cm 2 flasks of 96-well plates [52,53].

Assay for Cell Viability
Cells (1 × 10 4 cells/well in 100 µL of medium) were seeded in a 96-well plate and incubated for 24 h. Then, the cells were incubated for 22 h in the presence of extracts at various concentrations (5-40 µg mL −1 ) or vehicle (DMSO, 0.21%, v/v) or etoposide (40-µg mL −1 ), which served as a positive control, and cells without treatment were considered a negative control. Cell viability was determined by adding MTS solution to each well and incubating the cells for another 2 h. The optical density was measured at 490 nm on a microplate reader.

Treatment with LPS
Cells (2 × 10 4 cells/well in 200 µL of medium) were plated and incubated for 24 h in 96-well plates. After that, the cells were incubated for 1 h in the presence of extracts at noncytotoxic concentrations (5 to 40-µg mL −1 ), a vehicle (DMSO, 0.21%, v/v), or indomethacin (30-µg mL −1 ), which served as a positive control; cells without treatment were considered a negative control. Then, the cells were incubated at 37 • C for 20 h with LPS at 4-µg mL −1 (for wells with extracts, vehicle, indomethacin, and 100% stimulus control) as a proinflammatory stimulus and without LPS (negative control). Finally, cell-free supernatants were collected and used for NO quantification.

Determination of NO Concentration
Nitrite, the stable end product of NO, was used as an indicator of NO production in the cell-free supernatants and was measured according to the Griess reaction. Briefly, 50 µL of each supernatant were mixed with 100 µL of Griess reagent (50 µL of 1.0% sulfanilamide and 50 µL of 0.1% N-(1-naphtyl)ethylenediamine dihydrochloride in 2.5% phosphoric acid solution) in a new 96-well plate and incubated for 10 min at room temperature. The optical density at 540 nm (OD 540 ) was measured with a microplate reader, and the nitrite concentrations in the samples were calculated by comparison with the OD 540 of a standard curve of NaNO 2 in a fresh culture medium [27,57,60].

Statistical Analysis
The results shown were obtained from at least three independent experiments and are presented as the means ± standard deviation. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparisons test. For suspension cell cultures, the values of each experiment are means, and the bars represent the standard error of triplicate determinations. All statistical analyses were performed using GraphPad Prism®version 8.0 software; p-values < 0.5 were considered to indicate statistical significance. Microsoft®Excel®for Office 364 MSO (16.0.11425.20242) 32-bit software was used for analysis.

Conclusions
The establishment of a cell suspension culture of A. pichinchensis is reported for the first time. This cell suspension culture retained the ability to produce the anti-inflammatory compounds 2,3-dihydrobenzofuran (2) and 3-epilupeol (3) identified previously in a callus culture from this species. Moreover, the yield of both compounds was improved, and the production time was reduced by almost half compared with callus cultures. Phytochemical analysis of the cell cultures led to the identification of 17 bioactive components, of which compounds 2-5 and 8-11 were previously described to have anti-inflammatory, antimicrobial, antifungal, and gastroprotective properties. Furthermore, the in vitro anti-inflammatory efficacy of the cell culture extracts and the identification for the first time of (-)-Artemesinol (6), (-)-Artemesinol glucoside (7), encecalin (10), and 3,5-diprenyl-acetophenone (11) in cell cultures of A. pichinchensis offer a biotechnological tool for bioreactor scale-up to produce anti-inflammatory compounds in a sustainable way.