Background
There has been an ongoing increase in the incidence of chronic non-communicable diseases (CNCDs) worldwide, with cancer as one example of such diseases [
1]. In fact, the World Health Organization estimated that in the year 2030, 11 million people will die due to cancer [
2]. The traditional chemotherapy treatment against cancer causes undesirable side effects, and complementary and alternative medicine (CAM) has thus emerged as a possible solution. Among CAM treatments, phytotherapy is currently the most commonly used [
3]. Several studies have focused on the natural antioxidant intake because oxidation is closely related to cancer development [
4].
The genus
Ephedra is the only genus in the Ephedraceae family (which contains 35 to 45 species in total, commonly found worldwide) [
5]. This genus has been studied due its high contents of ephedrine alkaloids [
6]. However, several secondary metabolites as alkaloids (amphetamine-type, imidazole, quinoline, pyrrolidine, and others), flavonoids (flavonols, dihydroflavonol, flavanone, flavanols, flavones, anthocyanin), tannins (dimmer, trimmer and tetramer of proanthocyanidins), lignans, naphthalenes, esters, terpenoids, phenolic acids, and quinones have been reported in the Ephedra genus plants [
7]. In addition, some
Ephedra species have anti-inflammatory, antiviral, hepatoprotective, antibacterial and antifungal activities, as well as anticancer activities [
7]. In fact,
E. foeminea and
E. alata are used in CAM for cancer treatment in south-eastern Europe [
8]. By contrast, Chile has one such species, namely
Ephedra chilensis K Presl, commonly known as pingo-pingo [
9]. It is particularly abundant in the central zone and has a pink fruit that is fleshy and edible [
10]. The ethnopharmacological information showed that
E. chilensis is used for treating ulcers, abscesses, and clearing pus, as an astringent, anti-inflammatory, diuretic, and tonic, and for treatment of colds, and stomach and bladder pain, and is beneficial in the treatment of asthma, gonorrhoea, and syphilis [
10]. Additionally, potential bioactive applications of
E. chilensis have been explored. Examples of such applications are sun protection properties and growth-inhibitory activity against some bacterial cultures [
11,
12].
Despite the previous ethnopharmacological applications of E. chilensis, no studies have been reported on its effect on cancer or on the antioxidant capacity of this species. Therefore, the goals of this work are to measure the phytochemical content (anthraquinones, phenols, and flavonoids), to evaluate the antioxidant activity and the cytotoxicity against cancer cells (MCF-7, HT-29, PC-3, and DU-145) and non-tumour (CoN) lines for different E. chilensis extracts, and to identify the chemical composition (GC-MS analysis) of the extracts that present the greatest activity against the cancer cell lines.
Discussion
The anthraquinone and flavonoid contents are concentrated in the CH
2Cl
2 extracts (
p < 0.05, see Table
1), and comparing these results with other
Ephedra species, we found that there are no reports of anthraquinones in any
Ephedra species. However, flavonoids and related compounds have been reported in
E. aphylla,
E. sinica,
E. campylopoda, and
E. alata [
7]. Furthermore, the obtained flavonoid content showed a three-fold decrease compared to that of
E. major [
25]. In addition, phenolic compounds were mainly found in the CH
2Cl
2 and EtOH extracts (see Table
1), and our results for both of these extracts have similar values to those of
E. major, [
25] and a higher phenolic content than
E. sinica [
26]. These compound types have not been reported in
E. chilensis (except ephedrine). However, other
Ephedra species have been isolated and some of these compounds have been identified [
25,
27].
The antioxidant activity of
E. chilensis extracts was evaluated in a series of in vitro tests (see Table
2). The DPPH assay showed that EtOH extracts of
E. chilensis have the highest antioxidant activity, and present better activity than
E. laristanica and
E. Sarcocarpa (IC
50 = 4.6 and IC
50 = 5.3 mg / mL, respectively) [
28,
29]. Despite these results, all extracts have lower antioxidant activity than the positive controls. The FRAP and TRAP assays showed that the Hex extract has less antioxidant activity than the CH
2Cl
2 and the EtOH extracts. However, FRAP assay showed that all extracts are more active than the positive control (between 2.1 and 17.3 times more active than Gallic acid). TRAP assay showed that the extracts have similar activity to the positive controls. In addition, phytochemical content has been associated with the antioxidant activity [
30,
31]. The DPPH scavenging activity found in the present work is consistent with the total phenolic content (r = − 0.942, see Table
3), which is similar to the previous reports on
E. sinica [
32]. For the FRAP and TRAP assays, we found correlations between the total phenolic content (r > 0.9,
p < 0.05, see Table
3). Based on the results obtained in the FRAP assay, the
E. chilensis extracts manifest their antioxidant effect as reductive substances acting as single electron transferrers [
33]. The TRAP assay on
E. chilensis extracts showed great affinity of the extract with the peroxyl radical [
34].
The cytotoxic effect of
E. chilensis extracts was evaluated in vitro against several cell lines (see Table
4). The
E. chilensis extracts present higher activity for the MCF-7 and PC-3 cell lines (IC
50 < 1.0 μg / mL), while the HT-29 and DU-145 cell lines are resistant to the same treatment (IC
50 > Doxo in each cell line, see Table
4). The cytotoxic effect is related to several factors among which we can mention the solvent used for the extraction and the species; e.g.
, decoctions of
E. foeminea and
E. alata are used for breast cancer treatment in south-eastern Europe [
8].
E. alata extract has no cytotoxic effect against human liver cancer or against the leukaemia cell line, [
35]
E. sinica alcoholic extracts has a low cytotoxic effect against melanoma and non-cancer, [
36] and the CHCl
3 extract of
E. viridis does show cytotoxic action against leukaemia cells [
37]. The values obtained for MCF-7 and PC-3 cancer cell lines show promising anticancer properties for possible drug discovery and development according to the American National Center Institute [
21]. In fact, the non-polar extracts possess more activity than the antineoplastic drug for the MCF-7 and PC-3 cancer cell lines (as much as 3.9 times higher activity for MCF-7 and up to 7.2 times higher for PC-3, see Table
4). Exogenous antioxidant intake could be associated with chemoprevention of chronic diseases such as cancer, together with the presented correlation between phytochemical content and the antioxidant activity [
31,
38]. The correlation between the phytochemical content of
E. chilensis extracts and the cytotoxic effect was evaluated, but it was found that there was no relationship between the antiproliferative effect and the phytochemical content or antioxidant activity (r < 0.5, see Table
5). In addition to the cytotoxic effect, the selectivity for the cancer cell lines as a possible drug development pathway is a very relevant feature that must be evaluated considering the undesirable side effects of traditional chemotherapy [
39]. In this regard, the Hex and CH
2Cl
2 extracts of
E. chilensis have better selectivity index values than doxorubicin (up to 1.8 times more selective for MCF-7 and 3.3 times more selective for PC-3, see Table
6).
Based on the above discussion, the Hex and CH
2Cl
2 extracts were analysed by GC-MS (see Table
7 and Table
8). The analysis did not identify ephedrine, which is a typical compound of this species. The ephedrine concentration in
Ephedra species is variable; e.g.
, E. major,
E. fragilis,
E. distachya, and
E. monosperma have different ephedrine concentrations, while this alkaloid was not identified in
E. tweediana and
E. foeminica [
40,
41]. Nevertheless, the effect of ephedrine as a cytotoxic compound is not important because comparing both extracts (those free of ephedrine and those that do have ephedrine), similar activity is observed for non-small cell lung cancer [
42]. For breast cancer cells, ephedrine has a poor cytotoxic activity [
43]. Moreover, the GC-MS of both
E. chilensis non-polar extracts (Hex and CH
2Cl
2) showed high concentrations of long-chain fatty acids, and some of these have been identified in other
Ephedra species, e.g.
, n-tetradecanoic,
n-pentadecanoic,
n-hexadecanoic, and
n-octadecanoic acids were found in seeds of
E. nevadensis,
E. viridis,
E. przewalskii,
E. geradiana,
E. campylopoda, and
E. sinica, and in the leaves of
E. equizetina [
44‐
46]. Regarding the cytotoxic effect, these fatty acids can inhibit abnormal breast cancer cells [
47]. In fact,
n-tetradecanoic acid,
n-dodecanoic acid, and
n-octadecanoic acid have differentiation and/or cytotoxic and/or apoptotic effects on breast cancer cells [
48‐
50].
n-octadecanoic acid has cytotoxic effects for prostate carcinoma [
51,
52]. For
n-hexadecanoic acid, there have been no reports of cytotoxic activity on breast or prostate cancer cells. However,
n-hexadecanoic acid affects colon cancer cell growth, while its ethyl ester derivative can inhibit the DNA topoisomerase I and is an apoptosis inductor in leukaemia and neuroblastoma cells [
53‐
55]. Furthermore, alcohol derivatives of fatty acids such as
trans-9-hexadecen-1-olare only present in the CH
2Cl
2 E. chilensis extract. This compound has a growth inhibition effect on breast cancer [
56]. Other fatty alcohol derivatives such as 1-Eicosanol and lignoceric alcohol have not been reported to show cytotoxic effects on breast or prostate cancer. Nevertheless, they show antiproliferative activity for other cancer cell lines [
57,
58]. Regarding the fatty acids and their derivative content in non-polar
Ephedra extract, they could be related with activity and selectivity due to the
n-tetra,
n-penta,
n-hexa, and
n-octadecanoic acids present in the hexane extract corresponding to 41.37% of the total extract (see Table
7). The dichloromethane extract has fewer
n-tetra and
n-octadecanoic acids (2.40% of total extract, see Table
8). However, the synergistic effect between the fatty acids and other secondary metabolites cannot be ruled out.
Terpenoid derivatives were also identified, mainly in the dichloromethane extract of
E. chilensis (see Table
8). Among these, we found phytol which was also found in
E. campylopoda [
59]. This compound showed cytotoxic activity for a wide range of cancer cell lines, and particularly for the MCF-7 and PC-3 cancer cell lines [
60]. Others terpenic derivatives such as 4-(hydroxy-ethyl)-γ-butanolactone and loliolide were identified in the same extract (CH
2Cl
2, see Table
8). These compounds have highly similar structures. The extracts that present loliolide have high activity for the breast cancer MCF-7 cell line [
61]. Moreover, in the same extract, phenolic compounds such as isovanillin and (
E)-coniferyl alcohol were identified (see Table
8). Similar compounds (vanillin, ferulic acid, and lignin) have been found in
E. breana and
E. alata [
62,
63]. These compounds have a common core (benzene-3,4-OR) which is a fragment present in several molecules with antiproliferative activity such as lignins and benzaldehydes [
64‐
66].
On the other hand, in both non-polar extracts, we identified long-chain alkanes such as
n-heptadecane,
n-triacontane, and
n-hexatriacontane (see Table
7 and Table
8), of which
n-heptadecane and
n-triacontane had cytotoxic effects on several cancer cell lines, with a particularly pronounced effect on breast cancer observed for
n-triacontane [
67,
68].
Other compounds present in the Hex extract such as 6,10-dimethyl-2-undecanone and 3-(4-hydroxyphenyl)propionitrile were identified (see Table
7). The first compound has a cytotoxic effect on the breast cancer cell lines [
69]. A compound similar to 3-(4-hydroxyphenyl)propionitrile presents β-oestrogen receptor-selective inhibition which is important for breast cancer cells [
70,
71].
Finally, despite the above discussion of the cytotoxic effect of the principal components identified in the non-polar extracts, the synergic effect between them is not ruled out.