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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 6  |  Page : 1350-1354

In vitro anticancer, anti-inflammatory, and antioxidant potentials of Ephedra aphylla


1 Department of Biology and Biotechnology, American University of Madaba, Madaba, Jordan
2 Department of Basic Sciences and Humanities, American University of Madaba, Madaba, Jordan
3 Department of Horticulture and Crop Science, Faculty of Agriculture, The University of Jordan, Amman, Jordan
4 Department of Clinical Pharmacy and Therapeutics, Applied Science University, Amman, Jordan
5 Department of Medical Laboratories, American University of Madaba, Madaba, Jordan

Date of Web Publication28-Nov-2018

Correspondence Address:
Wajdy Al-Awaida
Department of Biology and Biotechnology, American University of Madaba, Madaba
Jordan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.196760

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 > Abstract 


Purpose: The goal of our study is to test whether a naturally occurring plant, Ephedra aphylla, will show antiproliferative ability against tested cell lines and to test its anti-inflammatory and antioxidative potentials.
Materials and Methods: In our study, we used four solvents with different polarities – aqueous, chloroform, methanol, and n-hexane – to extract different compounds from the aerial parts of E. aphylla. Antioxidant activity of E. aphylla was determined by measuring nitric oxide (NO) and hydrogen peroxide (H2O2) scavenging activities. The anti-inflammatory activity was studied using the inhibition of albumin denaturation assay. Finally, the antiproliferative activity of breast cancer cell lines (T47D, MCF-7) and Vero cell line (African green monkey kidney) was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
Results: Phytochemical screening for various extracts of E. aphylla showed the presence of medicinally important compounds including cardiac glycosides, alkaloids, triterpenes, tannins, and flavonoids. The scavenging activity for H2O2 of various solvent extracts was in the order of methanol > aqueous > chloroform > ethyl acetate > n-hexane. In addition, E. aphylla solvent extracts also exhibited a scavenging activity for NO in the order of methanol > ethyl acetate > aqueous > chloroform > n-hexane. All of the solvent extracts showed IC50 inhibition of albumin denaturation at a concentration between 209.5 ± 8.1 and 225 ± 11 μg/ml. Moreover, all extracts displayed strong antiproliferative potential against MFC7, T47D tested cell lines and very weak cytotoxic activity against Vero normal cell line.
Conclusions: E. aphylla has a promising potential to be used as a drug source for breast cancer treatment based on its strong antiproliferative activity.

Keywords: Anti-inflammatory, antioxidant, antiproliferative, Ephedra aphylla, phenolics


How to cite this article:
Al-Awaida W, Al-Hourani BJ, Akash M, Talib WH, Zein S, Falah RR, Aburubaiha Z. In vitro anticancer, anti-inflammatory, and antioxidant potentials of Ephedra aphylla. J Can Res Ther 2018;14:1350-4

How to cite this URL:
Al-Awaida W, Al-Hourani BJ, Akash M, Talib WH, Zein S, Falah RR, Aburubaiha Z. In vitro anticancer, anti-inflammatory, and antioxidant potentials of Ephedra aphylla. J Can Res Ther [serial online] 2018 [cited 2020 Jun 1];14:1350-4. Available from: http://www.cancerjournal.net/text.asp?2018/14/6/1350/196760




 > Introduction Top


Plants are rich in different types of natural compounds. About 25% of the prescription products in the world originate from wild or cultivated plants.[1] Only a small percentage of the 250,000–500,000 plant species worldwide have been studied for pharmacological properties.[2] In the Mediterranean region, more than 200 herbs are used in treating different human diseases.[3] In Jordan, of the 2500 plant species recorded,[4] more than 500 plant species are used in traditional medicine.[5] Recent research in synthetic chemistry reported a good potential of natural compounds to provide better aspects of treatment and prevention of many diseases.[6],[7],[8],[9] Plant-derived anticancer drugs, such as vincristine, vinblastine, camptothecin, and taxol are a part of the battle against tumor.[1] Continuing search for new antitumor natural compounds is a promising avenue for its prevention or treatment.[10] Plant compounds such as alkaloids, phenylpropanoids, and terpenoids were also reported to have anticancer activity.[11],[12]

Ephedra is a genus of Ephedraceae family, which includes forty species worldwide. Many of which have been used in traditional medicine.[13] They are mostly used to treat bronchial disorders. In addition, Ephedra has been used by the Chinese to treat asthma for more than 5000 years ago.[13] Ephedrine-type alkaloids include ephedrine, pseudoephedrine, and norephedrine.[14] There are very few reports on ephedrine-type alkaloids although there are many on tannins[15] and flavonoids.[16] Aqueous extracts of the Ephedra aphylla plant revealed strong growth inhibitory activity against the malaria-causing parasite. On the other hand, it did not show any inhibitory activity against cultured melanoma cell lines.[17] In our study, we evaluated the antioxidant, anti-inflammatory, and antiproliferative activity of E. aphylla against breast cancer cell lines; MFC7 and T47D.


 > Materials and Methods Top


Ephedra aphylla extract preparation

The aerial part of the plant was collected from around the aquatic ponds from the Wadi systems – banks from an area called Wadi al Wala, located south of Jordan. The plant samples were dried at room temperature and then powdered and extracted in a Soxhlet extractor using different solvents; methanol, chloroform, ethyl acetate, or distilled water. The extracts were collected under reduced pressure and stored at −20°C. The taxonomic identity was authenticated by Hatem Taifour, the Head Botanist at the Royal Botanic Garden in Jordan. Plant materials used in this study have been deposited in the Royal Botanic Garden with accession number NHJ.2016.0024. All chemicals were purchased from Sigma-Aldrich

Cell lines and culture conditions

Breast cancer cell lines (T47D, MCF-7) and Vero cell line (African green monkey kidney) (ATCC, USA) were studied. All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat inactivated fetal bovine serum (Gibco, UK), 40 μg/ml gentamicin, and 29 μg/ml L-glutamine. Cells were incubated at 37°C at 95% humidity and 5% CO2.

Phytochemical evaluation

Different extracts of E. aphylla were studied for their phytoconstituents such as cardiac glycosides, alkaloids, tannins, flavonoids, triterpenes, saponins, and steroids, by performing different phytochemical tests.[18]

Quantitative analysis of antioxidative components

Determination of total phenolic content

Total phenols (TPs) were measured according to Kim et al.,[19] where Folin–Denis reagent with gallic acid was used as the standard. In brief, 1 ml of dissolved extract was added to solution containing 10 ml of deionized water and 1 ml of Folin–Denis reagent. After 5 min, 2 ml of sodium carbonate was added to the mixture. The solution was gently mixed and allowed to stand at 25°C for 1 h, and then the absorbance of the solution was measured at 724 nm. The total phenols (TPs) contents were expressed as gallic acid equivalents g/g of dry plant extract by reference to a standard curve (y = 0.0214x + 0.031, R2 = 0.988)..

Determination of total flavonoids content

TFs were measured according to Karadeniz et al.,[20] using aluminum chloride colorimetric method. Briefly, 5 ml of 2% aluminum trichloride in methanol was mixed with the same volume of plant extract for 10 min at 25°C. Then, the absorbance was measured at 415 nm in triplicate. The results were expressed as milligram of rutin equivalents per gram of dry plant extract. The total flavonoid (TF) contents are reported as milligram rutin equivalent per gram of extract powder by reference to a standard curve (y = 0.0013x − 0.0088, R2 = 0.994).

In vitro antioxidant evaluation

Hydrogen peroxide scavenging activity assay

Radical scavenging activity of the E. aphylla extracts against hydrogen peroxide (H2O2) was measured according to Ruch et al.[21] Extracts with different concentrations were added to 3.4 ml of 0.1 M phosphate buffer solution (pH 7.4), followed by gentle mixing with 600 μl of 43 mM H2O2. After 10 min, H2O2 concentration was measured at 230 nm. Ascorbic acid was used as a reference compound.

Nitric oxide scavenging activity assay

Nitric oxide (NO) released from sodium nitroprusside was measured using Griess reaction.[21],[22] Sodium nitroprusside (5 mM) in phosphate-buffered saline was gently mixed with different concentrations of the extract (dissolved in dimethyl sulfoxide) and incubated at room temperature for 2.5 h. At the end of the incubation, 500 μl samples of each sample were diluted with 500 μl of Griess reagent. The reaction mixture absorbance was determined at 546 nm and referenced to the absorbance of potassium nitrite treated in the same way with Griess reagent.

In vitro anti-inflammatory evaluation

Inhibition of albumin denaturation

The anti-inflammatory activity of the different extracts was measured according to Mizushima and Kobayashi,[23] using the inhibition of albumin denaturation technique. The reaction mixture consisted of 1% aqueous solution of bovine albumin and test extracts fraction. The solution was incubated at 37°C for 20 min, and then temperature was increased to 51°C for 20 min; after cooling, the absorbance was measured at 660 nm in triplicate. In this assay, diclofenac sodium, a powerful non-steroidal anti-inflammatory drug used as a positive control. The Percentage inhibition of protein denaturation was calculated as follows: Percentage inhibition = (Abs Control –Abs Sample) × 100/Abs control.

In vitro antiproliferative evaluation

The antiproliferative activity of various solvent extracts prepared from E. aphylla was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay; Sigma, USA). A modification of the method used by Chao et al.[24] was used that actively dividing cells were washed and seeded at 15,000 cells/well in 96-well microplates. After 24 h incubation, a partial monolayer was created, the media were removed, and then 200 μl of medium containing the extract was added and re-incubated for 48 h. Then, 100 μl of the medium was removed and 15 μl of the MTT solution was added to the remaining medium in each well. After 4 h contact with the MTT solution, blue crystals were made. One hundred microliters of the stop solution was added and incubated for one more hour. Reduced MTT was assayed at 550 nm using enzyme-linked immunosorbent assay Plate Reader (VWR, USA). Vincristine sulfate was used as a positive control. IC50 values were calculated as the average of three replicates.

Statistical analysis

Collected data were subjected to statistical analysis using analysis of variance, followed by Fisher protected least significant difference mean separation. SAS software (SAS Institute, North Carolina State University, version 9.2) was used for data analysis.


 > Results Top


Phytochemical screening

Phytochemical screening of various extracts of E. aphylla showed the presence of cardiac glycosides, alkaloids, triterpenes, tannins, and flavonoids [Table 1].
Table 1: Qualitative analysis of the phytochemicals of various extracts of Ephedra aphylla

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Total phenol and flavonoid contents

Our results show that the extraction with methanol and aqueous resulted in the highest amount of TPs and flavonoid compounds [Table 2].
Table 2: Total phenolics and flavonoid of different extracts of Ephedra aphylla

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In vitro antioxidant activity

The scavenging effect of various extracts of E. aphylla on H2O2 and NO is shown in [Table 3]. Methanol and aqueous extracts displayed a strong scavenging activity compared to that of the standard, ascorbic acid, which exhibited 44 ± 2.1 for H2O2 scavenging activity and 58 ± 2.4 for NO scavenging activity [Table 3]. The scavenging activity for H2O2 of various solvent extracts was in the order of methanol > aqueous > chloroform > ethyl acetate > n-hexane. While the scavenging activity for NO of various solvent extracts from E. aphylla was in the order of methanol > ethyl acetate > aqueous > chloroform > n-hexane.
Table 3: Antioxidant potential of Ephedra aphylla

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In vitro anti-inflammatory activity

All solvent extracts showed the IC50 inhibition of albumin denaturation at concentration between 209.5 ± 8.1 and 225 ± 11.5 in the following order ethyl acetate > methanol > n-hexane > chloroform > aqueous. Diclofenac sodium, a standard anti-inflammatory drug, showed IC50 inhibition at a concentration of 65.5 ± 3.1 μg/ml [Table 4].
Table 4: Anti-inflammatory potential of various extracts of Ephedra aphylla

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In vitro antiproliferative activity

All extracts displayed strong antiproliferative potential against the tested cell lines. The determined IC50 values for extracts on all cell lines are shown in [Table 5]. Moreover, E. aphylla extracts showed a little cytotoxicity activity against the Vero normal cell line. The antiproliferative activity of various solvent extracts against MFC7 cell line was in the order of aqueous > methanol > chloroform > ethyl acetate > n-hexane.
Table 5: The half maximal inhibitory concentration values of different solvent extracts prepared from Ephedra aphylla tested against MFC7, T47D, and Vero cell lines

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 > Discussion Top


The phytochemical analysis of E. aphylla showed the presence of alkaloids, flavonoids, condensed tannins, triterpenes, and cardiac glycosides; compounds that were reported to have medicinal activity against various diseases.[25] Our study demonstrated that E. aphylla is rich in phenolic compounds. Phenolic compounds are considered very important plant components for their antioxidant activity due to their ability to eradicate free radicals.[26] Flavonoids and other phenolic compounds of plants play important preventive role in cancer development.[27] In several studies, flavonoids showed a wide range of antibacterial, anticancer, antiviral, and anti-inflammatory activities.[28],[29] Flavonoids are also known to be very active scavengers of many free radicals.[30] Flavonoids and polyphenols are natural antioxidants but have also been shown to significantly increase antioxidant enzyme activities.[31] It is known that free radicals show a vital role in the progress of many diseases.[32] Therefore, the antioxidant potential of various Ephedra aphylla extracts is most likely due to the presence of phenolic components.

Naturally, H2O2 occurs at low concentration in the blood.[33] Initiation of lipid peroxidation that causes DNA damage is due to the creation of hydroxyl radicals (OH) by H2O2 decomposition.[34] Aqueous and methanol extracts of E. aphylla displayed a strong scavenging activity of H2O2 which may be attributed to the presence of phenolic groups.

NO plays a significant role in several inflammatory processes. High concentration of NO in the tissue is very toxic and may contribute to various carcinomas. When NO reacts with superoxide radicals, its toxicity increases by forming a highly reactive peroxy nitrate anion.[35] Aqueous, methanol, and ethyl acetate extracts of E. aphylla showed a strong scavenging activity of NO. This may be due to the antioxidant compounds in this extract, which competes with oxygen to react with NO, thereby inhibiting generation of nitrite. Plant extracts may have the ability to reduce the effect of NO formation, which is important in preventing the effects of excessive NO generation in vivo.[36]

Inflammation is the response of tissues to injury. It involves a cascade of enzyme activation, mediator release, tissue breakdown, and repair.[37] Most biological proteins are not functional when denatured; denaturation of proteins plays an important role in developing inflammation response.[38] Many anti-inflammatory drugs show a dose-dependent ability to inhibit protein denaturation caused by heat.[39] In our study, all of the E. aphylla solvent extracts inhibited the albumin denaturation. The anti-inflammatory characteristics of E. aphylla extract may be due to the presence of medicinally active flavonoids.[40],[41]

Moreover, all extracts displayed strong antiproliferative potential in the tested cell lines; however, the inhibition of cancer cell proliferation by the extracts may not be totally due to their polyphenolic content. The presence of flavonoids, saponins, triterpenes, and tannins is believed to contribute partially by their antiproliferative activity through their antioxidant activity.[42],[43] Tannins and polyphenolic compounds have significant effects on cancer prevention and anticancer activity.[44],[45] Thus, the antiproliferative activity may be due to the presence of phytochemicals such as alkaloids, phenols, and flavonoids.


 > Conclusions Top


E. aphylla showed strong antiproliferative potential against the breast cancer cell lines MFC7and T47D. The potential antiproliferative activities may be due to the presence of phytochemicals such as alkaloids, phenols, and flavonoids.

Acknowledgments

We would like to thank the American University of Madaba for the financial support. We also would like to thank Hatem Taifour, the Head Botanist at the Royal Botanic Garden in Jordan, for the identification of the E. aphylla.

Financial support and sponsorship

Funding for this study was kindly provided by the American University of Madaba.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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