Cytotoxic Activity of Wild Plant and Callus Extracts of Ageratina pichinchensis and 2,3-Dihydrobenzofuran Isolated from a Callus Culture

Ageratina pichinchensis (Kunth) R.M. King & H. Rob. belongs to the Asteraceae family and is a plant native to Mexico to which several biological properties are attributed. In this study, the cytotoxic effect of four extracts from the wild plants and two extracts from A. pichinchensis callus culture were evaluated against carcinogenic cell lines including prostate carcinoma, cervical cancer, hepatocellular carcinoma, hepatoma human, lung cancer, and cellular keratinocytes. The extracts were obtained with ethyl acetate and methanol using both leaves and stems or the callus. Only the ethyl acetate extract of the callus culture influenced the cervical cancer cell line (HeLa) with an IC50 of 94.79 ± 2.0 µg/mL. From the ethyl acetate callus extract, 2,3-dihydrobenzofuran was isolated and purified and also evaluated against cancer cells. The cytotoxic evaluation of this compound showed a significant effect against the HeLa cell line with an IC50 of 23.86 ± 2.5 µg/mL. Our results contribute to the development of biotechnological alternatives and extraction processes to produce compounds with possible potential against certain types of human cancer.


Introduction
Ageratina pichinchensis (Kunth) R.M. King & H. Rob.belongs to the family Asteraceae and is an endemic plant species of America.In the state of Morelos, Mexico, this species grows mainly in the municipalities of Amatlán and Tepoztlán.A. pichinchensis is popularly known as "water leaf" and "axihuitl".Traditionality, this plant is used by communities to treat gastric ulcers, healing, and diseases related to inflammatory events [1]. A. pichinchensis is described as a shrub up to 1.5 m tall, stem erect, highly branched, or slightly puberulent [2].In this regard, scientific evidence dealing with the phytochemical and pharmacological studies has validated its ethnomedical uses [3][4][5][6][7][8].Moreover, biotechnological studies have shown that A. pichinchensis can biosynthesize anti-inflammatory

Cytotoxic Evaluation of Extracts
We evaluated the cytotoxic activity of the ethyl acetate and methanol extracts obtained from different parts of the wild plant A. pichinchensis, including its leaves, stems, and callus cultures.The extracts were evaluated using concentrations of 200, 100, 50, 25, and 12.5 µg/mL against various cell lines (Table 1), including human prostate carcinoma (PC-3), cervical cancer (HeLa), hepatocellular carcinoma (Huh-7), human hepatoma (HepG2) and breast tumor cells (MCF-7).These cell lines represent cell types that, unfortunately, have high incidence and mortality rates globally.In addition, cytotoxicity was evaluated in keratinocytes (HaCat), a human epithelial cell line that exhibits normal differentiation, and was used as a selectivity control [30].
The methanolic and ethyl acetate extracts of the wild plants did not demonstrate a significant cytotoxic effect in any of the cell lines analyzed, even with the maximum concentration evaluated (200 µg/mL).
On the other hand, it was detected that the ethyl acetate extract from wild plant leaves exhibited a mild cytotoxic effect on HeLa and PC-3 cell lines, with mean inhibitory concentration (CI50) values of 161.49µg/mL and 188.6 µg/mL, respectively.By contrast, the methanolic extract from wild plant leaves showed no significant cytotoxic effect on these same cell lines.
Interestingly, although the methanolic extract of stems and leaves did not show a significant cytotoxic effect on any of the cell lines, the methanolic extract of callus cultures did have a cytotoxic effect on the HeLa and PC-3 lines, with IC50 values of 150.9 µg/mL and 168.6 µg/mL, respectively.However, the most prominent cytotoxic effect was observed with ethyl acetate extract of callus cultures, especially against the HeLa cell line (IC50 = 94.79 µg/mL), followed by PC-3, HepG2, Huh-7, and MCF-7 (IC50).
To corroborate the efficacy of our cytotoxic study, we used paclitaxel (PTX) as the reference drug; it is a chemotherapeutic drug widely used in the treatment of various types of cancer, including breast, cervical, prostate, and liver cancers [31,32].The results reveal a remarkable sensitivity of PXT-treated cell lines with IC50 values in the nanomolar (nM) range, underscoring the potential efficacy of this reference drug as an anti-tumor agent in our research context.

Cytotoxic Evaluation of Extracts
We evaluated the cytotoxic activity of the ethyl acetate and methanol extracts obtained from different parts of the wild plant A. pichinchensis, including its leaves, stems, and callus cultures.The extracts were evaluated using concentrations of 200, 100, 50, 25, and 12.5 µg/mL against various cell lines (Table 1), including human prostate carcinoma (PC-3), cervical cancer (HeLa), hepatocellular carcinoma (Huh-7), human hepatoma (HepG2) and breast tumor cells (MCF-7).These cell lines represent cell types that, unfortunately, have high incidence and mortality rates globally.In addition, cytotoxicity was evaluated in keratinocytes (HaCat), a human epithelial cell line that exhibits normal differentiation, and was used as a selectivity control [30].The methanolic and ethyl acetate extracts of the wild plants did not demonstrate a significant cytotoxic effect in any of the cell lines analyzed, even with the maximum concentration evaluated (200 µg/mL).
On the other hand, it was detected that the ethyl acetate extract from wild plant leaves exhibited a mild cytotoxic effect on HeLa and PC-3 cell lines, with mean inhibitory concentration (CI 50 ) values of 161.49µg/mL and 188.6 µg/mL, respectively.By contrast, the methanolic extract from wild plant leaves showed no significant cytotoxic effect on these same cell lines.
Interestingly, although the methanolic extract of stems and leaves did not show a significant cytotoxic effect on any of the cell lines, the methanolic extract of callus cultures did have a cytotoxic effect on the HeLa and PC-3 lines, with IC 50 values of 150.9 µg/mL and 168.6 µg/mL, respectively.However, the most prominent cytotoxic effect was ob-served with ethyl acetate extract of callus cultures, especially against the HeLa cell line (IC 50 = 94.79 µg/mL), followed by PC-3, HepG2, Huh-7, and MCF-7 (IC 50 ).
To corroborate the efficacy of our cytotoxic study, we used paclitaxel (PTX) as the reference drug; it is a chemotherapeutic drug widely used in the treatment of various types of cancer, including breast, cervical, prostate, and liver cancers [31,32].The results reveal a remarkable sensitivity of PXT-treated cell lines with IC 50 values in the nanomolar (nM) range, underscoring the potential efficacy of this reference drug as an anti-tumor agent in our research context.
These results suggest that ethyl acetate extract of A. pichinchensis callus cultures could be a potential interest in future applications relating to the treatment of certain types of cancer, especially in the case of the HeLa cell line.It was therefore important to delve into the composition of the extract and identify if there is any compound to which the activity can be attributed.

Chemical Profile of Ethyl Acetate Extract of Callus Cultures
Since the ethyl acetate extract prepared with callus cultures had an important cytotoxic effect, the chemical content was analyzed by mass gas chromatography coupled with mass spectrometry (Figure 2).The chromatographic and mass spectrometric analysis allowed us to identify 11 main compounds [9].These results suggest that ethyl acetate extract of A. pichinchensis callus cultures could be a potential interest in future applications relating to the treatment of certain types of cancer, especially in the case of the HeLa cell line.It was therefore important to delve into the composition of the extract and identify if there is any compound to which the activity can be attributed.

Chemical Profile of Ethyl Acetate Extract of Callus Cultures
Since the ethyl acetate extract prepared with callus cultures had an important cytotoxic effect, the chemical content was analyzed by mass gas chromatography coupled with mass spectrometry (Figure 2).The chromatographic and mass spectrometric analysis allowed us to identify 11 main compounds [9].In addition, according to an NIST (National Institute of Standards and Technology) database search, the identities of the 11 compounds of the structures in Figure 3 are known, and their biological effects have been reported.
Fatty acids (1) and ( 2) have been reported as constituents of many plant species such as Commiphora, Orchis chusua, Salvia verbenaca, and Cleidion javanicum Bl., and these compounds have shown antioxidant and antimicrobial effects on gram-positive and gram- Fatty acids (1) and ( 2) have been reported as constituents of many plant species such as Commiphora, Orchis chusua, Salvia verbenaca, and Cleidion javanicum Bl., and these compounds have shown antioxidant and antimicrobial effects on gram-positive and gram-negative microorganisms, and their antifungal effect has also been reported [33][34][35][36][37]. Particularly, the hexadecenoic acid (1) compound has been shown to be an important inhibitor of the PLA2 enzyme that plays a role in inflammation of blood vessels and favors the development of atherosclerosis [38].However, compound (2) has only been associated with antimicrobial effects [39,40].These compounds are common in the essential oils of many medicinal plants such as Chasmanthe aethiopica, Zostera japonica, and Jatropha curcas [41][42][43], and their effect has been reflected in antimicrobial and anti-inflammatory evaluations.
In addition, according to an NIST (National Institute of Standards and Technology) database search, the identities of the 11 compounds of the structures in Figure 3 are known, and their biological effects have been reported.microbial and antifungal effect [47][48][49].
The compound campesterol (10) has been reported as an important phagocytosis suppressor and inhibitor of lipopolysaccharide in RAW 264.7 macrophage cells [82]; in addition, extracts of different plant species with anti-inflammatory effects have revealed that compound (10) participates in the biological effects attributed to the extracts, such as in Cajanus cajan L. seeds, Ananas comosus leaves, Allium schoenoprasum L. leaves, and in Opuntia ficus-indica seed oil [83][84][85][86].
These scientific reports demonstrate that the cytotoxic effect observed in the ethyl acetate extract of A. pichinchensis callus in our study is due to biologically active compounds that act synergistically, significantly enhancing its cytotoxic effect on PC-3 and HeLa cell lines.This chemical content is constant, because the callus are cultured under controlled conditions and their nutrients are the same.This advantage has allowed numerous callus cultures to be a source of bioactive compounds, opening up the applications of the cultures in the design of alternative treatments for health problems.
Due the compound 2,3-dihydrobenzofuran having been found to be related to an anti-inflammatory effect with pro-inflammatory potential, it was selected to evaluate its cytotoxic effect; the rest of the compounds have been deeply studied at a pharmacological level, and according to the literature, 2,3-dihydrobenzofurans commonly exhibits cytotoxic, antiviral, antioxidant, antimicrobial, and anti-tumor activities [86,87].

Cytotoxic Effect of the Compound 2,3-Dihydrobenzofuran
Considering the pharmacological properties of 2,3-dihydrobenzofurans, we analyzed the cytotoxic effect of compound (7) on the study cell lines.The results show a remarkable effect on the inhibition of HeLa cells, while the rest of the cell lines revealed no inhibitory effect (Figure 4).
It is notable that HeLa cervical cancer cells were the most sensitive to the treatment using 2,3-dihydrobenzofuran (7) (Table 2) with an IC 50 of 23.86 µg/mL, which is 3.97 times lower than the IC 50 of ethyl acetate extract of callus cultures of A. pichinchensis.This may suggest that the cytotoxic activity observed in the extract is associated with the effect of this compound; selectivity against cancer cells was also observed.The selectivity index is commonly reported in the literature as a ratio of IC 50 values calculated for healthy cells and cancer cells [30,88], with values greater than 1 indicating desirable selectivity against cancer cells.The selectivity index of 2,3-dihydrobenzofuran (7) was 5.17, 1.53, 1.54, 1.65, and 1.15 for HeLa, PC-3, Huh-7, HepG2, and MCF-7 cells, respectively.This finding is outstanding because compound ( 7) is only biosynthesized by callus cultures, but not in the wild plants of A. pichinchensis, which provides a notable contribution Pharmaceuticals 2023, 16, 1400 8 of 14 from callus cultures, as a controlled and stable source of compounds of therapeutic interest.Moreover, compound (7) exhibits chirality, which complicates the development of synthesis routes to obtain it.On the other hand, our research group has reported the production of the compound through suspension cell cultures in flasks as well as in airlift reactors, significantly improving its production and possible scaling [10,11], which provides a viable and sustainable alternative to produce the compound.
Methanolic and ethanolic extracts of other wild plant species have reported important cytotoxic effects against HeLa cells, suggesting that there are still plants that can be a source of important cytotoxic compounds; for example, Artemisia ludoviciana, Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L., Moringa oleifera, and Ficus carica L. [89][90][91][92][93].Likewise, pure compounds have also exhibited a significant inhibitory effect on cell proliferation of the HeLa line, such as the compound benzobijuglone, which was isolated from Juglans mandshurica, and the gymnemagenol compound from Gymnesa sylvestre [94,95].
Therefore, it is necessary to continue establishing callus cultures of medicinal plants that are a potential source of cytotoxic compounds, and it is even possible to biosynthesize them in species that have not reported a cytotoxic effect, as occurs with A. pichinchensis, which is widely used in traditional Mexican medicine.It is used to treat diseases caused by fungal and skin infections and wounds, as well as to relieve pain and treat gastric ulcers, and anti-inflammatory effects have been reported.Extracts from this plant have shown antifungal activity against Trichophyton mentagrophytes, T. rubrum, and Candida albicans, and have shown therapeutic effectiveness in patients with vulvovaginal candidiasis [7,96].

Plant Material from the Wild Plants
The plants were collected in their natural habitat in the San Juan Tlacotenco neighborhood of the municipality of Tepoztlán in the state of Morelos, Mexico.Plants were identified by our research group in previously reported works and assigned the voucher number 39913.The specimen is under protection in the HUMO herbarium of the Autonomous University of the State of Morelos (UAEM), whose taxonomic identification was carried out by Biol.Gabriel Flores Franco [9].

Plant Material from Callus Cultures
Calluses were previously established by our research group using leaf explants in Murashige and Skoog culture medium supplemented with 30 g/L sucrose, 1 mg/L naphthaleneacetic acid, 0.1 mg/L kinetin, and 3 g/L phytagel [8].The culture medium was sterilized at 121 • C, 15 psi, for 15 min using an autoclave.Calluses were subcultured every 20 days and incubated at 25 ± 2 • C under a photoperiod of 16 h with white fluorescent light (50 µmol/m 2 s).

Obtaining Organic Extracts
The wild plants were dried at room temperature and the leaves were separated from the stems and ground to a fine powder.For the extraction process, 145.18 g of dried leaves and 78.13 g of dried stems were used.On the other hand, 13.20 g of ground dried calluses were used.The dry plant material (leaves and stems) and dry biomass (callus culture) were extracted by maceration (72 h, at room temperature) with ethyl acetate.The same biomass was then extracted with methanol with three extraction cycles each.The solvent was removed by distillation under reduced pressure, using a rotary evaporator, finally obtaining three ethyl acetate extracts, i.e., leaves (17.20 g), stems (7.31 g), and callus culture (0.924 g), and three methanol extracts, i.e., leaves (59.09 g), stems (28.47 g), and callus culture (2.346 g).This methodology is summarized in Figure 5.
were used.The dry plant material (leaves and stems) and dry biomass (callus culture) were extracted by maceration (72 h, at room temperature) with ethyl acetate.The same biomass was then extracted with methanol with three extraction cycles each.The solvent was removed by distillation under reduced pressure, using a rotary evaporator, finally obtaining three ethyl acetate extracts, i.e., leaves (17.20 g), stems (7.31 g), and callus culture (0.924 g), and three methanol extracts, i.e., leaves (59.09 g), stems (28.47 g), and callus culture (2.346 g).This methodology is summarized in Figure 5.
Cells (4 × 10 3 cells/well) were seeded in 96-well plates.The cells were treated with the investigated samples (at 200, 100, 50, 25, and 12.5 µg/mL) and incubated at 37 • C in 5% CO 2 for 48 h.Paclitaxel was used as a positive control.For determining the number of viable cells in proliferation we used a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA), following the manufacturer's instructions.Cell viability was determined by absorbance at 450 nm using an automated ELISA reader (Promega, Madison, WI, USA).Stock solutions of all compounds were prepared in DMSO at a maximum concentration of 0.5%.The experiments were conducted in triplicate in three independent experiments.Data were analyzed using the Prism 8.0 statistical program (Graphpad Software Inc., La Jolla, CA, USA) and the half-maximal inhibitory concentrations (IC 50 ) were determined by regression analysis.

Conclusions
For the first time, it is shown that the chemical content of the ethyl acetate extract of A. pichinchensis callus cultures exhibits a significant cytotoxic effect on the HeLa cell line, whose activity is suggested to be attributed to the 2,3-dihydrobenzofuran compound.These results contribute to the development of alternatives for the treatment of cervical cancer, which has become a health problem causing many deaths worldwide; moreover, this finding provides the possibility of new applications of callus culture extracts, since wild plant extracts did not show the cytotoxic effect.This is because in vitro cultures produce the compounds of interest in a constant and controlled manner, while wild plants are dependent on environmental, geographical, seasonal, and other factors.

Figure 1 .
Figure 1.Typical plant of Ageratina pichinchensis growing naturally in the state of Morelos, Mexico.Adult plant (A); callus induced from leaf explants (B).

Figure 1 .
Figure 1.Typical plant of Ageratina pichinchensis growing naturally in the state of Morelos, Mexico.Adult plant (A); callus induced from leaf explants (B).

Figure 2 .
Figure 2. Chromatogram of GC-MS analysis of ethyl acetate callus extract.The red numbers correspond to the compounds identified in the extract.

Figure 2 .
Figure 2. Chromatogram of GC-MS analysis of ethyl acetate callus extract.The red numbers correspond to the compounds identified in the extract.

Figure 3 .
Figure 3. Compounds identified by GS-MS analysis in the ethyl acetate callus extract of A. pichinchensis.

Figure 5 .
Figure 5. Extraction process and cytotoxic evaluation of A. pichinchensis.

Figure 5 .
Figure 5. Extraction process and cytotoxic evaluation of A. pichinchensis.

Table 1 .
Cytotoxic activity (IC 50 ) of ethyl acetate and methanol extracts of callus cultures and wild plants of A. pichinchensis.

Table 1 .
Cytotoxic activity (IC50) of ethyl acetate and methanol extracts of callus cultures and wild plants of A. pichinchensis.