Establishment of a Cell Suspension Culture of Ageratina pichinchensis for the Improved Production of Anti-inflammatory Compounds

Many species of the Asteraceae family are used in traditional Mexican medicine for possessing healing properties. Ageratina pich inchensis (Asteraceae) is a plant used for the treatment of gastric ulcers, deep wounds and for its antifungal effects. The aim of this study was to establish a cell suspension culture of A. pichinchensis, quantify the anti-inflammatory constituents 2,3-dihydrobenzofuran and 3-epilupeol, to evaluate the anti-inflammatory potential of its extracts and perform a phytochemical analysis. Cell suspension cultures were established in MS culture medium supplemented with 30 g L-1 sucrose and 1.0 g L-1 α-naphthaleneacetic acid (NAA) plus 0.1 mg L-1 6-furfurylaminopurine (KIN). The ethyl acetate extracts of cell suspension cultures analyzed by GC revealed that the maximum production of compounds The anti-inflammatory activity of these extracts exhibited significant inhibition of NO production. Furthermore, the phytochemical study of EtOAc and MeOH extracts of the biomass on day 20 led to the identification of 17 known compounds. The structures of compounds were assigned by analysis of 1D and 2D NMR data and the remainder by GC-MS. This is the first report of the production of the (-)-Artemesinol, (-)-Artemesinol glucoside, Encecalin and 3,5-diprenyl-acetophenone compounds by a cell suspension cultures of A. pichinchensis.


Cell Suspension Culture and Growth Kinetics
It has been shown that friable callus is most suitable for establishing cell suspension cultures of any plant species. A. pich inchensis cells transferred to MS liquid culture medium with the same growth regulators as in callus cultures (1.0 mg L -1 NAA and 0.1 mg L -1 KIN) grew easily, which showed abundant biomass and slightly yellowish appearance (Fig. 1a, b, c). Similar results were observed in suspension cell cultures of Stevia rebaudiana which were disintegrated in a period of 7 days and cells also acquired a yellowish appearance [28]. In this study, cell growth of A. pich inchensis was faster (22 days) in liquid culture medium MS than in callus cultures, in contrast, callus reached their maximum growth between and 30 -40 days [27]. This can be caused by the facilitated absorption of nutrients in liquid medium [29].  The growth kinetic of A. pich inchensis cell suspension cultures was maintained until 22 days, over which it showed a typical growth curve (Fig. 2 ). The growth kinetic was characterized by a lag phase of 4 days, during which the biomass reached 2.37 g L -1 DW; subsequently, the cells entered the exponential phase and lasted until day 16. During this time, the maximum biomass accumulation was observed (13.28 g L -1 ) which was about 5.6-fold over 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 (Fig. 2 ). In terms of the consumption of total sugars, an abrupt decrease of the sugar content was observed until day 6, but the biomass increased; the remainder sugar was stable after day 14 (Fig. 2), which may be due to consumption of the nutrients and the lack of oxygen in the medium [30,31]. Growth kinetics is similar to that for Spilanthes acmella, in which, the cell suspension culture reached a specific growth rate of 0.279 days -1 , during the exponential phase the doubling time was 2.50 days and the maximum biomass was 8.5 g L -1 at day 15 [32]. On the other hand, of 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 doubling time was 7.6 days -1 [33]. Similarly, cell suspension cultures of Helianthus annuus, produced 12.7 g L -1 dry biomass at 9 days of culture showing a specific growth rate of 0.21 days -1 and doubling time of 3.31 days -1 [34]. These results suggest that A.
pichinchensis cell cultures have similar tendency to other cell suspension cultures and although biomass yield differs for each species, it can also be produced in a short time.
Compounds 1-5 were isolated by column chromatography as described in the experimental part, these were identified by analysis of their 1 H, 13 C NMR data and by comparison with the literature data [27]. While compounds 10-14 were identified by GC-MS analysis (NIST 1.7a). Compounds 4-5 and 10-11 were previously reported as constituents of the aerial parts of A. pich inchensis wild plant [15,17], compounds 1-3 and 12-13 were previously reported in a callus culture of A. pich inchensis [27]. and 80.86 (C-9, C-6, C-10 and C-2) and two methyl groups in δ 26.32 and 23.49 (C-12 and C-13).
According to the data obtained in 1 H, and 13 C NMR, compound 6 was characterized as (-)-Artemesinol (6) [40]. Compound 7 was obtained as a semi-solid mass from MSR-M-4C fractions group. The 1 H NMR spectrum of (7) displayed signals in δ 1.30 (3H, s, CH3-13), δ 2.44 (3H, s, CH3-12),   The production of compound 2 started from the lag phase and the maximum production (510.75 ± 29.10 µg g -1 of dry biomass) occurred on day 8 of the exponential phase; then it gradually decreased until day 22. Similar results have been observed in Capsicum chinense Jacq suspension cell cultures, in which, the capsaicin compound reaches a maximum production of 567.4 µ g g -1 of dry biomass at 25 days [44]. On the other hand, in Celosia cristata cell suspension cultures, the betalaine production was observed at the beginning of the exponential phase and then decreased, remaining stable during the exponential and stationary pha ses [45].
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 consequently cellular death [46]. In another species such as Eurycoma long ifolia, was reported that cell suspension cultures produce the quassinoid eurycomanone and this also occurs from the beginning of the growth kinetics reaching its maximum (1.7 mg g -1 of dry biomass) [47].
These results are important given that they improve those reached by callus cultures whose maximum production was identified on day 30, producing 650 µg g-1 of dry biomass for compound 2 and 201.10 µg g -1 of dry biomass of compound 3 [27], reducing the time of production of the Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 August 2020 doi:10.20944/preprints202008.0443.v1 compounds is a desirable characteristic in suspension cell cultures, in addition to being more homogeneous cultures compared to callus cultures, it is additionally possible to increase the production of bioactive compounds by adding elicitor s, additionally it is it can then be scaled up to reactors to mass produce the compounds [48][49][50].
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 concentration during the exponential phase (day 8) (Fig. 6). Antifungal, gastroprotective and antinociceptive effects have been reported for compounds 10 and 11 [43,17]. The highlight of both compounds is that their production on in v itro cultures is reported for the first time. Based on the importance of its biological effects, the cell suspension cultures of A. pichinchensis is a useful alternative for production of compound 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 A. pich inchensis suspension cell culture was assessed at different times of the growth kinetics (8, 12 and 16 days).
Firstly, the extracts were evaluated for their effect on via bility of RAW 264.7 cells at different concentrations (5, 10, 20, 30 and 40 µg mL -1 ). All extracts did not exhibit 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 (Fig. 7). To assess the effect of the extracts from D8, D12 and D16 on nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells, cells were treated with the extracts at the same concentrations used in the viability assay. The experimental results showed that NO level was increased in LPS-stimulated RAW cells, and this effect was decreased significantly by treatment with extracts at the concentrations tested (Fig. 8). The results showed that D12 and D16 extracts were the most active to inhibit 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 day 8 (D8) showed 24.69 ± 6.17% inhibition in NO production, at proportionally also increases. However, in a pure way, both compounds have an important anti-inflammatory effect. The indomethacin (positive control) showed an inhibition of 47.45 ± 7.41% at 30 µg mL -1 (Fig. 8). The results obtained in this work are of great interest because the (2S,3R)-5-acetyl-7,3α-dihydroxy-2β-(1-isopropenyl)-2,3-dihydrobenzofuran (2) and 3-epilupeol (3) compounds have important pharmacological properties. Compound 2 inhibits the secretion of NO, IL-6 and TNF-α in RAW 264.7 macrophages, as well as the activation of NF-κB in RAW-blue macrophages [27], while compound 3 has shown antiviral [51], anti -inflammatory [52], antitubercular [53] and cytotoxic activity [54]. About the anti-inflammatory activity, the 3-epilupeol (3) compound has shown marked inhibition of the edema induced by TPA in mice and exhibits the nitric oxide (NO) production inhibitory activity in LPS-activated macrophages [52,55]. Nitric oxide (NO) is a short-lived bioactive molecule which plays an important role in host defense response against various pathogens such as bacteria, viruses, fungi and parasites, and is thought to be various pathophysiological processes such as neuronal communicat ion, vasodilatation, and neurotoxicity [56].
The importance of the anti-inflammatory effects of 3-epilupeol (3 ), lies in that the excessive production of NO causes tissue damage, extensive systemic vasodilatation and hypotension [57]. In addition, NO is involved in inflammatory disorders including bowel diseases, rheumatoid arthritis, chronic hepatitis, pulmonary fibrosis and colon cancer [58 -60].

Establishment of Cell Suspension Cultures
Friable callus cultures of A. pichinchensis was previously established by our working group [27].
Callus were subcultured in the same MS semisolid culture medium containing 3% sucrose, 1. To increase the biomass, the cells were subcultured every 15 days for 6 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 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 grow was allowed until 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 (td) was computed from the µ exponential data.

Cell Viability
The cell viability of 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 3 times with three replicates each.

Sugar Quantification and pH Measurement
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 August 2020 doi:10.20944/preprints202008.0443.v1 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 S_-25CW) and total sugar content by 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 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 H2SO4 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 w as dried in an oven at 40 °C (12. Compounds 1-5 were identified using 1 H-and 13 C-NMR and comparing with reported data and by direct comparison with authentic samples available in our laboratory [27].

(±)-Artemesinol Glucoside (7)
Compound (7) was isolated as a semi-solid mass. 1   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 accep table linearity with correlation coefficients r2= 0.9926 and r2= 0.9997 respectively ( Fig. S11 and S2). The quantification of compound 2 and 3 in the extracts was expressed as µg g -1 dry biomass (µg g -1 DW).

Determination of NO Concentration
Nitrite, the stable end-product of NO, was used as an indicator of NO production in the cell-free