Phytochemical characterization and anti-cancer properties of extract of Ephedra foeminea (Ephedraceae) aerial parts

Purpose: To evaluate the phytochemical profile of methanol extract of Ephedra foeminea and assess its anti-carcer effect on a large set of normal and cancerous cell lines Methods: Extraction of air-dried powder of aerial parts of E. foeminea was carried out with methanol. The bioactive compounds in the extract were determined using gas chromatography/mass spectrometry (GC-MS). The anti-cancer effect of the extract was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay against various types of normal and cancer cell lines. Serial concentrations of plant extract were used, ranging from 7.812 to1000 μg/mL. Doxorubicin (DOX) served as standard drug. The half-maximal concentration (IC50) values of the extract and DOX for each cell line were determined, and the selectivity index (SI) was computed. Results: Phytochemical analysis showed that the extract contained several bioactive compounds, including alkaloids, flavonoids, sterols and fatty acids. The hazardous ephedra alkaloids (ephedrine and pseudoephedrine) were absent in the plant extract. The extract showed significant anti-proliferative activity against cancer cell lines, when compared with the positive control, doxorubicin (p < 0.05). Selective and concentration-dependent cytotoxicity was exhibited in cancer cell lines of breast (MCF-7), lung (A549), colon (Caco-2), liver (HepG-2) and prostate (PC-3). Weak selectivity was produced in other cancer cell lines, i.e., human epithelioma (Hep-2) and cervical carcinoma (Hela). Interestingly, noncancerous cells showed no or weak cytotoxicity. Conclusion: Ephedra foeminea exerts potential selectivity in anti-proliferative effect against some cancer cell lines. Thus, it is a promising drug source for the production of new and selective anti-cancer medicines.


INTRODUCTION
The genus Ephedra is a member of the Ephedraceae family containing approximately 69 species of non-flowering seed plants. It can be found in different arid and semi-arid areas of the world, especially North Africa, Asia, America and Europe.
These Ephedra species are characterized by ecological, economic, and recently, medicinal values [1]. The Ephedra species are associated with many traditional medicinal uses, including treatment of allergies, cough, oedema, bronchial asthma, headaches, fever, flu, nasal congestion, chills and colds [2]. Moreover, they are the main natural sources of bioactive compounds, including alkaloids such as ephedrine and pseudoephedrine; flavonoids and phenolic compounds such as proanthocyanidins, amino acid derivatives, and volatile organic compounds in essential oils such as aromatic and terpenoid compounds [3].
Recently, the idea of using Ephedra as an alternative to cancer therapy has become popular. This is particularly true of Ephedra foeminea, which many cancer patients in the Middle East region utilise because of the belief that it has cancer-curative properties [4]. Based on these observations, limited studies have investigated the effect of crude extracts of various types of Ephedra foeminea on different cancer cell lines [4][5][6][7]. In a study using an aqueous decoction of Ephedra foeminea, there were no significant effects on the viability of MDA-MB231 and SKBR3 breast cancer cell lines [5]. In contrast, another study showed that extracts and fruit juice of Ephedra foeminea significantly decreased the viability of colon cancer cells (HTC116) and breast cancer cells (MDA-MB-213), but no toxic effect was exerted on lung carcinomatous cells (A549) [7]. Likewise, Ephedra foeminea extracts exhibited a dosedependent decrease in viability of human osteosarcoma cells (U2OS) [7]. These contradictory results may be attributed mainly to differences in constituents of various types of Ephedra foeminea extracts. There are limited data on the phytochemical constituents of crude extracts of Ephedra foeminea. A recent study reported the absence of ephedrine and pseudoephedrine alkaloids from Ephedra foeminea [5]. The results obtained from the above studies are contradictory, and further investigations about anti-cancer properties and phytochemical analysis of Ephedra foeminea are required.
Since there are limited studies and contradictory results on the anti-cancer properties of Ephedra foeminea, this study was aimed at phytochemically analysing a methanol extract of Ephedra foeminea, and evaluating its cancerkilling effects on different cancer cell lines.

Plant material
The aerial parts of Ephedra foeminea were collected from South Jordan, Mutah, Al-Karak, Jordan during the fruiting stage from May to July 2020. They were washed, dried in the shade, and ground to a fine powder. The plant was identified by Professor Al-Gohary, a taxonomist in Department of Plant Ecology and Range, Desert Research Centre, Cairo, Egypt. A voucher specimen (CAIH-0439S) was deposited at the Herbarium of the Centre.

Plant extraction
One hundred grams of air-dried powder of the plant was extracted with methanol (500 ml × 3 times) for 72 h using the cold percolation method. The methanolic extract was filtered using a Buchner funnel. Then, the filtrate was evaporated in a rotary evaporator at a temperature below 40 °C, after which the residue was dried in a dissector to give 25 g/100 g dry weight (DW) of Ephedra foeminea. The crude methanol extract was subjected to GC/MS analysis for the determination of bioactive compounds.

Phytochemical screening
The samples of Ephedra foeminea were analyzed using GC-MS (Shimadzu GC/MS-QP 5050A) with a DBI column and He carrier gas maintained at a flow rate of 1 ml/min. Injector and ion source temperature was 280 °C, and oven temperature was raised from 40 °C to 280 °C with a ramp of 2 °C/min and a withholding period of 7.5 min. The amount of sample injected was 1 μL. Sample ionization took place in electron impact mode at an ionization voltage of 70 eV and mass span of 50 -650 m/z. The data interpretation was carried out with Wiley & Nist library databases.

In vitro cytotoxic effect of Ephedra foeminea
The cytotoxic effect of Ephedra foeminea was evaluated using the MTT assay. The assay relies on the mitochondrial-dependent reduction of yellow MTT which is converted to purple crystals of formazan through a mitochondrial reduction reaction [9]. Once 70 % confluence was reached, the cells were plated in 96-well plates at a density of 1x10 4 cells/mL (180 µL/well), followed by overnight incubation at 37 C in a 5-% CO₂ incubator. After forming a confluent layer of cells, diluted plant extract (0.1 mL) or medium was put in appropriate wells. Serial dilutions of plant extract were obtained by dissolving the extract in growth medium to make final concentrations of 7.812; 15.625; 31.25; 62.5; 125; 250; 500 and 1000 μg/mL, followed by incubation as before, for 96 h in the dark. Thereafter, each well medium was decanted and replaced with 20 μL of 5 mg/mL MTT solution. To allow the development of formazan crystals, the plates were incubated in the dark for 4 h at 37 o C. The supernatant was removed and formazan crystals formed were re-suspended in 150 µL of DMSO. The absorbance of each of the formazan solutions was read at 560 nm in Mindray-96A microplate reader. Doxorubicin was used standard drug, while the medium served as negative control. The assay was carried out in triplicate for each group.

Determination of IC50
The IC50 values of the extract and DOX for the cell lines were determined with GraphPad Prism ver. 7 software California, U.S.A. Cell growth inhibition (GI) was calculated using Eq 1.

Selectivity index
The degree of plant extract selectivity towards cancer cells was calculated with selectivity index (SI). This is the ratio of IC50 value of plant extract in normal cells (WI-38) to IC50 value of plant extract in each cancer cell line. Values >3 suggest that the extract has in vitro selective cancer activity, relative to non-cancer cells [10].

Statistical analysis
Statistical analysis was performed using Statistical Packages for Social Sciences (SPSS) software version 19. The results are presented as mean ± standard deviation (SD). Statistical significance of differences was evaluated with ttest. Values of p < 0.05 were considered statistically significant.

Phytochemical profile of extract
The GC/MS analysis of the methanol extract revealed that the aerial parts of the Ephedra foeminea contained different classes of bioactive metabolites, including alkaloids, flavonoids, sterols and fatty acids (Table 1 and Figure 1)

In vitro cytotoxicity
The cytotoxicity assay (MTT) of the methanol extract was done at different concentrations i.e. 7.812, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL with respect to seven human cancer cell lines. For each cell line, complete concentration response curve and IC50 value were developed. The principle employed to classify the cytotoxic effect of the extract was adopted from the U.S. National Cancer Institute (NCI) and Geran protocol [11,12]. The classification is as follows: IC50 ≤ 20 μg/mL = highly cytotoxic, IC50 of 21 -200 μg/mL = moderately cytotoxic, IC50 of 201 -500 μg/mL = weakly cytotoxic, and IC50 > 501 μg/mL = not cytotoxic.  Each value represents mean ± SD from three independent experiments.

Figure 1: GS/MS spectra of E. foeminea
Results showed marked dose-dependent reduction in the viability of all cancer cell lines, relative to DOX (  Microscopic analysis of the various cell lines treated for 72 h with Ephedra foeminea extract at a dose of 125 μg/mL was conducted. The examination showed that the plant extract treatment caused the cells of PC-3, MCF-7, Caco-2 and A549 to shrink and become rounded and detached, when compared to untreated control cells. On the other hand, no changes in cell morphology were displayed in Hela, HepG-2 and Hep-2 cells (Figure 2).

Selective cytotoxic activity
Methanol extract of Ephedra foeminea produced highly selective cytotoxicity in MCF-7, A549, Caco-2 cell lines, while weak cytotoxic selectivity was observed against HepG-2 and PC-3 (Table  4). In contrast, no cytotoxic selectivity was exhibited for the plant extract in Hep-2 and Hela cell lines (values < 3).

DISCUSSION
Medicinal herbs are currently playing a leading role in modern drug development. Indeed, many traditional medicines are basically copies or synthetic alterations of natural chemical compounds present in plants. Nowadays, key research investments are dedicated to identifying and characterising new plants with the potential to prevent or hinder development of cancer [13]. Many current techniques may be useful in successful discovery of cancer-suppressing principles from plant sources, as well as elucidation of their modes of action [14]. Although Ephedra foeminea has neither a recognised traditional nor an ethno-botanic rationale for curing cancer, the popularity of the herb is on the increase, particularly in the Middle East region [5]. Therefore, this research, explored the phytochemical constituents of methanol crude extract of Ephedra foeminea. In addition, we display for the first time, strong evidence of cancer-suppressing effect of Ephedra foeminea on a large array of cancer cell lines.
Various plants have been examined for their content of different bioactive compounds and how they affect cell physiological functions, as well as their anti-cancer potential and capacity to prevent cancer cell growth [14]. The phytochemical investigations showed that, unlike other ephedra plants, ephedra alkaloids were absent in Ephedra foeminea. This finding is consistent with a previous study which confirmed the absence of potentially hazardous ephedra alkaloids from Ephedra foeminea [5].
Interestingly, the analysis found that the major component of the plant methanol extract was 4Hpyran-4 one,2,3-dihydro-3,5-dihydroxy-6-methyl. This compound has been shown to reduce the growth of colon cancer cells by causing apoptotic cell death through inhibition of NF-κB [15]. Furthermore, 9-Octadecenoic acid was one of the main free fatty acids found in the plant methanolic extract. This fatty acid produced potent cytotoxicity against a range of cancer cell lines, particularly colon cancer. This is consistent with the cytotoxicity data which displayed its strong anti-cancer activity against colon cancer cell lines [16,17]. The second major free fatty acid found in the plant extract was hexadecanoic . acid. It has been reported that hexadecanoic acid extracted from Aquilaria malaccensis leaves exerted antioxidant and anti-cancer effects in various cancer cells [18]. Overall, the major bioactive compounds found in the methanolic extract of Ephedra foeminea seem to exert synergetic anti-cancer activity against several cancer cell lines. Further studies are required to fully characterise, isolate and assess the anticancer activity of all the bioactive compounds in Ephedra foeminea.
Nowadays, naturally-derived compounds are of research interest because they are known to be less toxic than conventional cancer therapy such as chemotherapy. This is particularly true because these compounds may have the potential to target tumour cells without affecting normal cells [14]. The present study reveals, for the first time, a clear selective cytotoxicity in cancer cell lines with no, or minimal effect on normal cells. This selectivity in cytotoxic activity was exhibited only against MCF-7, A549, Caco-2, HepG-2 and PC-3 cell lines. The highest potential cytotoxic activity was reached at certain optimal extract concentrations. The extract showed less selectivity on other cell lines (Hela and Hep-2). These interesting results are consistent with previous studies which demonstrated strong cytotoxic activity of different types of Ephedra foeminea crude extracts against certain types of cancer cell lines [4,7]. The ethanolic extract and fruit juice of Ephedra foeminea exhibited significant growth-inhibitory activity against colon cancer cells (HTC116) and breast cancer cells (MDA-MB-213) [4]. Moreover, potent cytotoxic activity was displayed in human osteosarcoma cells (U2OS) by treatment with ethyl acetate, ethanol, and water extracts of Ephedra foeminea [7]. Importantly, all the IC50 values of Ephedra foeminea reported here are relatively lower than those obtained in previous studies [4,7]. However, these results contrast with a previous study where water decoction of Ephedra foeminea showed no significant cytotoxic effect on MDA-MB231 and SKBR3 breast cancer cell lines [5]. This discrepancy may be attributed to the potency of Ephedra foeminea, which probably differs from one cell line to another. In addition, the extraction method used in each study has been shown to influence the final physical, chemical and biological properties of the extract [19]. Alcoholic extracts of Ephedra foeminea seem to exert robust cytotoxic activity due to prominent bioactive contents. So far, there is still no mechanistic explanation of the anti-proliferative potential of Ephedra foeminea. However, the antiproliferative potential should be investigated further in future studies to better understand the mechanism involved in the anti-cancer activity of Ephedra foeminea.

CONCLUSION
The present study has revealed the presence of several bioactive medicinal components in aerial parts of the Ephedra foeminea plant. Importantly, the methanol crude extract of the plant did not contain the hazardous toxic ephedra alkaloids. The extract exerts pronounced cytotoxic activity against most of the various cancer cell lines studied, but not in normal cell line treated with the crude extract. The selectivity in the antiproliferative activity of Ephedra foeminea against certain cancer cell lines holds considerable promise for the isolation of bioactive compounds, thereby providing a basis for developing novel anti-cancer therapies.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
This study was done by the authors named in this manuscript, and the authors accept all liabilities resulting from claims which relate to this article and its contents. The study was conceived and designed by Yousef M Al-saraireh, Ahmed M.M. Youssef and Sameeh A Al-Sarayreh. Data were collected and analyzed by Jehad M Al-Shuneigat, Hamzeh M Alrawashdeh and Samir S Mahgoub. Yousef M Al-saraireh wrote the manuscript. All authors read and approved the manuscript for publication.

Open Access
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