Potential Antiproliferative Activity and Evaluation of Essential Oil Composition of the Aerial Parts of Tamarix aphylla (L.) H.Karst.: A Wild Grown Medicinal Plant in Jordan

Essential (volatile) oil from aerial parts of Tamarix aphylla (L.) H.Karst. (Tamaricaceae) grown wild in Jordan was hydrodistilled by Clevenger apparatus and analyzed by means of GC and GC-MS techniques. In vitro screening of potential cytotoxicity of the aqueous (AE) and ethanol (EE) extracts was also evaluated against human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (Caco-2), and pancreatic carcinoma (Panc-1) cancer cell lines as well as normal human fibroblasts. GC-MS analysis of T. aphylla EO revealed its richness in nonterpenoid nonaromatic hydrocarbons (52.39%), with predominance of 6,10,14-trimethyl-2-pentadecanone as the principal component. Biologically, the plant extracts exhibited cytotoxicity effects in dose-dependent manner against most of the tested cell lines, but potent effects were only predicted against MCF-7 cells with IC50 values of 2.17 ± 0.10 and 26.65 ± 3.09 μg/mL for T. aphylla AE and EE, respectively. T. aphylla AE demonstrated a comparable cytotoxic effect with that offered by the control drug cisplatin (IC50 value of 1.17 ± 0.13 μg/mL), even with higher safety profile against normal fibroblast cells (IC50 values of T. aphylla AE versus cisplatin: 79.99 ± 4.90 versus 9.08 ± 0.29 μg/mL). T. aphylla extracts could be a valuable source for cytotoxic agents with high safety and selective cytotoxicity profiles. Unfortunately, no antiproliferative potential against Caco-2 or Panc-1 cancer cell lines was detected at a concentration less than 30 μg/mL.


Introduction
Jordan's flora is rich in a wide variety of medicinal plants and hence is widely utilized by Jordanians for health maintenance. It is believed that the ease of access to herbal remedies alongside their wide safety margins encourages their usage by patients [1]. Recently, many efforts have been exerted to find out the potential roles of different medicinal plants in cancer treatment. Despite the fact that many of the widely grown plants in Jordan have been screened for their antiproliferative activities [2], many others either are under current investigation or are still unevaluated.
Tamarix aphylla (L.), which is known as 'Tamarisk' or 'Athel' in Jordan, is an evergreen tree with tiny, triangular, and scale-like leaves. It grows in dense groves and it flourishes in high alkalinity-salinity soils. Tamarisk is native throughout the Middle East across East, North, and Central Africa and to a little extent into parts of Western and Southern Asia [3]. Many communities have used T. aphylla in traditional medicine. Its leaves were used for wounds and abscess healing, as astringent, and for rheumatism and joint pain [4,5]. Several studies have recognized the various types of secondary metabolites present in T. aphylla. Reports had identified the presence of flavonoids [6], phenolics [7], hydrolysable tannins [3], and alkaloids [7,8] in the plant various extracts. Earlier investigations on the effects of T. aphylla on biological systems had revealed its insect growth inhibitory activity due to presence of ellagic acid [9]. The isolated isoferulic acid derivative, aphyllin, was also shown to exhibit a distinct radical scavenging activity and to improve the viability of human keratinocytes [10]. In Saudi Arabia, alcohol extract from leaves of T. aphylla was shown to possess antioxidant, anti-inflammatory, and wound-healing activities. The authors suggested that the presence of known active 2 Evidence-Based Complementary and Alternative Medicine phytochemicals like flavonoids and polyphenols explains these reported effects [11]. The plant was also reported to exhibit analgesic and antipyretic activities [12].
Several previous studies have investigated the potential roles of various T. aphylla extracts in the prevention and/or treatment of many ailments [5,11,12]; nevertheless, only few researches have focused on investigating its volatile essential oil (EO) composition or evaluating the plant's antiproliferative effects against selected cancer cell lines. In this present study, the EO hydrodistilled from aerial parts of wild grown Jordanian species of T. aphylla was analyzed by gas chromatography-mass spectrometry (GC-MS) for purpose of chemical composition analysis. In vitro cytotoxic activities of the aqueous extract (AE) and ethanol extract (EE) against human colorectal adenocarcinoma (Caco-2), breast adenocarcinoma (MCF-7), and pancreatic carcinoma (Panc-1) cancer cell lines alongside normal human fibroblasts were also assessed. Interestingly, this is the first time that the EO composition and the cytotoxic activities of T. aphylla aerial parts are evaluated.

Phytochemical Analysis
About 650 g of the aerial parts of T. aphylla (L.) H.Karst. was collected from north Amman in May 2016. Collected parts were taxonomically identified by Professor Khaled Tawaha (School of Pharmacy, the University of Jordan) and then set to air-dry under shade in a cool place for further study. Voucher specimen has been deposited in the Department of Pharmaceutical Sciences, School of Pharmacy, the University of Jordan, and given a specimen ID (TA-Hudaib-MAY16-001).

Preparation of Crude Plant Extracts.
Both the aqueous and ethanol extracts of the dried plant aerial parts were prepared by maceration. Separately, a powdered 100 g quantity was placed in round-bottomed flask and either a 1 L of 70% ethanol (EtOH) or 1 L distilled water was added in a ratio of 1:10. Flasks were kept for one week in a cool place; the filtrates were collected and then dried using rotary evaporator at 40 ∘ C. The dried extracts were kept in tightly closed flasks, at 4 ∘ C, for subsequent analysis.

TLC.
Thin layer chromatography (TLC) was performed (in duplicate) to obtain qualitative fingerprinting of the prepared crude extracts. TLC was achieved on precoated TLC silica gel plates (ALUGRAM SIL G/UV254, Macherey-Nagel GmbH & Co., Germany), using different mobile phases. Detection of chemical constituent was conducted as reported by Wagner and Bladt [14].

Essential Oil
Extraction. 300 g of dried aerial parts of T. aphylla was soaked in 2.5 L of distilled water and then hydrodistilled for 2 hours using Clevenger-type apparatus. The hydrodistilled EO was collected using GC grade nhexane, dried over anhydrous sodium sulphate, and then kept in tightly closed vial, at 4 ∘ C, until analysis.

GC-MS Analysis.
GC-MS analysis of EO was performed in duplicate after appropriate dilution of the hydrodistilled oil in GC grade n-hexane. Around 1 L aliquot of the diluted oil was injected into a split-splitless injector of a Varian Chrompack CP 3800 GC/MS/MS-200 (Saturn, Netherlands) GC-MS outfitted with DB-5 (5% diphenyl, 95% dimethyl polysiloxane) capillary column (30 m length x 0.25 mm ID, 0.25 m film thickness). The injector temperature was kept at 250 ∘ C with a split ratio of 1:10 and Helium was used as a carrier gas with a flow rate of 1 mL/minute. The column temperature was programmed to be initially isothermal at 60 ∘ C for 1 minute and then to increase up to 246 ∘ C at a rate of 3 ∘ C/minute and then to be kept isothermal at 246 ∘ C for 3 minutes for a total run time of about 66 min. The MS ionization source temperature was 180 ∘ C with an ionization voltage of 70 eV.

GC-FID Analysis.
Quantitative analysis of the hydrodistilled oil (performed in duplicate) was carried out using a Focus GC (Thermo Electron Corporation) gas chromatograph equipped with fused silica capillary column OPTIMA-5 (5% diphenyl 95% dimethyl polysiloxane; 30 m length × 0.25 mm ID, 0.25 m film thickness). The column was coupled to an injector (split-splitless type) and flame-ionization detector (FID). The same temperature program was used as mentioned above in GC-MS analysis section. Injector temperature was maintained at 250 ∘ C with split ratio of 1:50, while FID temperature was held at 300 ∘ C. Percent content (% w/w) of each component was calculated using its corresponding normalized relative area obtained by FID and assuming a unity response by all components [15].

EO Component Identification.
Qualitatively, identification of volatile components was carried out using built-in libraries (e.g., Wiley, Terpenes, NIST, and Adams' libraries). A comparison of the calculated Arithmetic Retention Index (RI) of each identified component with literature reference value measured with a column of identical polarity alongside MS spectrum matching helped to confirm the identification [15]. RIs of EO components were calculated relative to nalkane hydrocarbons (C8-C20) analyzed under the same chromatographic conditions, as above, using the modified arithmetic equation by Van Den Dool and Kratz [16]. Quantitatively, the % content of each EO component was measured as mentioned above in GC-FID section.   [17], plates were incubated for 72 hours. Once the exposure time had finished, cells growth was analyzed using tetrazolium reduction (MTT) assay as described by Riss [18]. Absorbance was read by multiwell plate reader (Bio-Tek Instrument, USA) at 570 nm using a reference wavelength of 630 nm.

IC 50 Value Calculation.
As described by (1) and (2), percent cytotoxicity at each concentration was calculated from the obtained optical density (OD) by the plate reader. Before any further interpretation, all data were blankadjusted. As described by (3)

Cytotoxicity Evaluation.
The American National Cancer Institute (NCI) guidelines set the limit of activity for crude plants extracts at 50% inhibition (IC 50 ) of proliferation to be < 30 g/mL after the exposure time of 72 hours [19]. Accordingly, it appears that T. aphylla AE and EE are potentially potent cytotoxic extracts against MCF-7 cell line, with IC 50 values ( g/mL) of 2.17 ± 0.10 and 26.65 ± 3.09, respectively. Doxorubicin and cisplatin's respective IC 50 values against MCF-7 cells are 0.01 ± 0.001

Discussion
Preliminary TLC test revealed the presence of flavonoids in both T. aphylla AE and EE, which was previously described by other researches [6]. The main identified volatile principle, 6,10,14-trimethyl-2-pentadecanone, is a nonaromatic oxygenated hydrocarbon (ketone) and has a slightly fatty aroma with reported antimicrobial [20] and antioxidant [21] properties. Other main identified compounds wereionone (13.74%) and dodecanoic acid (6.00%). Oxygenated sesquiterpenes account for almost 27% of the identified oil.  Different compositions of the EO from different Tamarix species were also reported in literature. As described by Orfali [22], bicyclo[2.2.2]octan-2-one was found to be the major compound (46.09%) in T. nilotica of Saudi Arabia. Hexadecanoic acid methyl ester was reported as the major principle in T. chinensis fruit [23]. Hexadecanoic acid (in aerial parts and stems), 2,4-nonadienal (in flowers), and germacrene D (in leaves) were, however, reported as majors of T. boveana [24]. Like in T. chinensis, nonaromatic hydrocarbons resembled the abundant group of T. aphylla aerial parts, while fatty acids and fatty esters are the majors in T. boveana leaves [24]. On the other hand, among terpenes, oxygenated and hydrocarbon sesquiterpenes are prevalent in T. aphylla and T. boveana [24], respectively. Biologically, several previous studies were also performed on the different species of Tamarix. In the current study, both the AE and EE of T. aphylla showed an interesting cytotoxic effect against MCF-7 cells. According to NCI guidelines [19], 6 Evidence-Based Complementary and Alternative Medicine the selective cytotoxicity of T. aphylla AE (IC 50 value of 2.17 ± 0.10 g/mL) against MCF-7 cells proved to be comparable to that of cisplatin (IC 50 value of 1.17 ± 0.13 g/mL) and even with higher safety as revealed by their relative IC 50 values on fibroblast cells (IC 50 ( g/mL): 79.99 ± 4.90 for AE versus 9.08 ± 0.29 for cisplatin). Despite the higher IC 50 values on fibroblast cells compared with doxorubicin and cisplatin, both extracts unfortunately lacked cytotoxic potential against Panc-1 or Caco-2 cell lines in the tested concentration range.
On the other hand, recent study on methanol extract of T. aphylla had investigated its potential cytotoxicity using brine shrimp method and revealed 70% mortality rate at concentration of 500 g/ml [25]. In other studies, different extract of T. nilotica was tested against mouse lymphoma, rat hepatoma, and rat glioma cell lines. Interestingly, it was the presence of potent anticancer agents in the alcohol extract that could result in a powerful cytotoxic effect with % survival of 58.9 at 10 g/mL [22]. Also, T. africana shoot extract inhibited the growth of A-549 lung carcinoma cells, with an IC 50 value of 34 g/mL [26]. Previous reports of phytochemical screening of T. aphylla revealed its high content of flavonoids, polyphenols, and tannins [3,6,8]. Several phenolic compounds isolated from Tamaricaceae were tested for their cytotoxic effects against different cancer cell lines and were shown to exhibit cytotoxic potentials [27]. Of these compounds are ferulic acid derivatives. Aphyllin, the isolated glycosylated isoferulic acid, exhibited a distinct radical scavenging activity and was found to improve the viability of human keratinocytes [10]. Ellagitannins were also reported to exhibit significant host-mediated antitumor and remarkable antiangiogenic activities [28]. Tamarixetin was also found to be cytotoxic against leukemia cells [29]. This latter compound inhibited proliferation in a concentrationand time-dependent manner, induced apoptosis, and blocked cell cycle progression. Syringic acid, a natural phenolic compound with selective antimitogenic activity on human malignant melanoma cells, was recently isolated from the methanol extract of T. aucheriana [30]. Methyl ferulate from T. aucheriana demonstrated cytotoxic and chemo-sensitizing effects [31], while glucuronosylated flavonoids from T. gallica aerial part were shown to prevent amyloid aggregation [32]. The potent cytotoxic activity observed for the tested extracts of T. aphylla under study is potentially attributed to presence of such compounds belonging principally to the phenolic fraction and other secondary metabolites as revealed by the TLC tests (Table 1).

Conclusion
The presence of different secondary metabolites with different biological activities renders medicinal plants valuable in drug discovery. Many biological activities have been linked to specific chemical entities present in the plants, such as flavonoids, tannins, polyphenols, and other compounds. The data obtained in this study suggest that different extracts of T. aphylla have potential antiproliferative activities against MCF-7 cancer cell line, which could be a source of promising lead compounds for the development of new treatments for some cancer types.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest regarding the publication of this manuscript.