Anticancer Activity of Micromeria fruticosa and Teucrium polium Growing in Lebanon

Medicinal plants have been widely utilized for tumor and cancer prevention1,2. Over 60% of today’s anticancer chemotherapeutic drugs originate from natural sources as plants3,4. Family Lamiaceae (or Labiatae), is a flowering plant that has roughly 236 genera and more than7000 species5, of which the genus Micromeria and Teucrium. Micromeria fructicosa6 and Teucrium polium7 (Lamiaceae) are perennial herbs or shrubs that dwells in rocky and dry-open territories in the Mediterranean areas like Turkey, Syria, and Lebanon8,9.


INTRODUCTION
Medicinal plants have been widely utilized for tumor and cancer prevention 1,2 . Over 60% of today's anticancer chemotherapeutic drugs originate from natural sources as plants 3,4 . Family Lamiaceae (or Labiatae), is a flowering plant that has roughly 236 genera and more than7000 species 5 , of which the genus Micromeria and Teucrium. Micromeria fructicosa 6 and Teucrium polium 7 (Lamiaceae) are perennial herbs or shrubs that dwells in rocky and dry-open territories in the Mediterranean areas like Turkey, Syria, and Lebanon 8,9 . Plants of Family Lamiaceae, including that of genus Micomeria and Teucrium, are well known for their therapeutic potential and have been widely used to treat many ailments as migraines, gastrointestinal disorders, upper respiratory tract infections, and some cardiovascular diseases 10 . Micromeria plants was reported to possess antimicrobial activities beneficial for the treatment of common cold, wounds, and skin infections [11][12][13] . Other studies suggested its protective effect against hepatotoxicity and pain management [14][15][16] . On the other hand, Teucrium plant were utilized to treat abscesses, conjunctivitis, gout, inflammation, parasitic infections, and diabetes 17 . Both plants (Micromeria and Teucrium) are considered as important source of phenolic, terpenoidal, and flavonoidal compounds 18 . This abundance of polyphenolic components stands behind the antioxidant quality and the wide range of reported pharmacological activities of both genus 19 .
Numerous studies have elaborated the importance of phenolic compounds and the distinct anticancer effects of Teucrium plants. Extracts from T. polium were capable of diminishing cell invasion and metastasis of human prostate cancer cells 20 , promoting cell apoptosis of human lung carcinoma cells 21 , and inhibiting breast adenocarcinoma cancerous cell growth 22  In Lebanon, twelve Teucrium and eleven Micromeria species were reported to grow widely in the mount region 26 . However, none of these plants were evaluated for its anticancer or other biological effect. In our previous work, the volatile oil of Micromeria fruticosa was analyzed and evaluated for its antimicrobial activity. Accordingly, this study aimed to evaluate the in vitro anticancer activities of M. fruticosa and T. polium, grown in Lebanon, against human breast cancer cell lines (MCF7) and human lung carcinoma cell lines (A549). In addition, we also aimed to determine their beneficial use as adjuvant to anticancer drugs.

Plant materials
The aerial parts of Micromeria fruticosa and Teucrium polium were collected from a rocky mountain in the Lebanese Bekaa valley (1300m above sea level) during their full flowering period on July 2017.The plants were authenticated by Dr. Ali Chakas (Botanist from the Lebanese University, Faculty of Science). A voucher specimen of each plant was deposited in the herbarium of the Faculty of Pharmacy at the Beirut Arab University and give the specimen number Mf-9-17 &Tp-10-17 respectively.
The residues were removed by filtration. M. fruticosa extract (A) and that of T. polium (B) were concentrated using a rotary evaporator under reduced pressure at 35-40°C and then lyophilized into powders.

Chemicals
All organic solvents for column chromatography (CC) and thinlayer chromatography (TLC) (petroleum ether, ethyl acetate, and methanol) were analytical grade and purchased from Sigma-Aldrich® (Germany). The alumina used was Neutral alumina 507 (for CC) and Silica-gel GF254 (for preparativeTLC) that were purchased from Fluka® (Switzerland). TLC was performed on pre-coated silica gel 60 F254 purchased from ALUGRAM® SIL G (Germany). Methanol was used for recrystallization and LC-MS, while UV analysis was HPLC-grade purchased from Sigma-Aldrich® (Germany).

Separation of crude extract
Crude extracts of A and B were separated using column chromatography (silica gel, 350 g, column diameter 3.5 cm). Elution was carried out with gradient mixtures of petroleum ether (PE) and ethyl acetate (eluents from 20% to 100% EtOAc), and then continued with EtOAc and methanol (from 5% to 60% methanol) to give 22 fractions (A1-A22) for Micromeria fruticosa(extract A) and 43 fractions (B1-B43) for Toucrium polium(extract B). Each fraction was analyzed using TLC analysis, detected by UV lamp, and sprayed with different spray reagents to be visualized and to suggest the chemical class. Based on TLC analysis (Rf, shape, and spot color), similar fractions were combined together for further separation through CC to yield the sub fractions. The physical and chemical class of each fraction are shown in Table 1.

Cell culture
Human breast cancer cell lines (MCF7) and human lung carcinoma cell lines (A549) were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and maintained in DMEM high glucose media (Sigma Chemical Company) supplemented with 0.1mg/ml streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum (FBS). Cell lines were maintained at 37°C under an atmosphere containing 5% CO 2 .

Treatment of cells
A 1mg/mL stock solution of lyophilized plant extracts in DMSO was prepared, while the stock solution of the chemotherapeutic drug Cisplatin (CDDP) was diluted in NaCl. Solutions were immediately sterilized by filtration through a sterile membrane filter with a porosity of 0.2 micron. Different concentrations were prepared by diluting the stock solution with DMSO. Cells were plated in 96-well micro titer plates, at a concentration of 105 cells/well, and incubated in a humidified environment of 37°C with a 5% CO2 incubator for cell adhesion. The anti proliferative activity was carried out by measuring the cell viability of the MCF7 and A549 cell lines 72 hours after the treatment with increasing concentrations of total extracts A and B (10,20,30,40,50, and 100 μg/ml), their separated fractions (100, 150, 200, and 250 μg/ ml), and cisplatin (2,4,8,10,20, and 40 μg/ml). Similarly, MCF-7 and A549 cells were treated with combinations of total extracts A and B (20 μg/ml) and cisplatin (2 μg/ ml). Untreated cells (the control) for plant extracts received DMSO, while cisplatin received NaCl.

Cell viability assay
Cells were plated in 96-well plates at a density of 10,000 cells/well and treated with tested compounds and total extracts at different concentrations for 72 hours. A 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution (5 mg/ml) was added to evaluate mitochondrial viability 27 . After 4hrs.of incubation at 37°C, the supernatants were removed and 100 μl of isopropanol-HCL was added to each well to solubilize the formazan crystals. The optical density, determined at 595 nm, measured the signal and the background. The experiment was repeated three times independently, each time in triplicates. The percentage of viability for each sample was calculated using the obtained OD values within the following formula: Percentage of viability (%) = OD sample/OD control × 100. The cytotoxicity of each compound is expressed as an IC50 value. The IC50 value is the concentration of test agents that caused a 50% inhibition or cell death averaged from at least three separate experiments. This value was obtained by plotting percentage inhibition versus concentration of compounds.

Statistical analysis
All results were presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPadPrism 8 (GraphPad Software Inc., CA, USA). Two-way ANOVA was used to calculate sample probability values (p); p ≤ 0.05 was considered statistically significant. Groups that are significantly different from the control are indicated in the Figures as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and p ≤ 0.0001.

RESULTS
Both the total extracts of Micromeria fruticosa (A) and Teucrium polium (B) and their separated fractions were found to reduce cell survival of MCF7 and A549 cancer cells.
The MTT assay was used to determine the proliferation rate of cancerous cell lines (MCF7 and A549) treated with different concentrations of total plant extracts A and B and their fractions. Results revealed that the cytotoxic activity of the total extract of the two plants were higher than their separated fractions. IC50 of extract A was 28.52 ± 1.455 and 26.47 ± 1.423 μg/ml on MCF7 and A549 cell lines, respectively.While IC50 of extract B was 41.07 ± 1.614 and 27.97 ± 1.447 μg/ml on MCF7 and A549 cell lines, respectively (Table 2 and Figure 1). Most of the separated fractions from A and B showed dose-dependent inhibitory activity on the cancerous cell lines. The most effective fraction isolated from extract A was ' A15' (IC50=186.67 ± 2.271 on MCF7), and the most effective fraction separated from extract B 'was 'B5' (IC50=170.8 ±2.232 and 141.9 ± 2.152 on MCF7 and A549 cells, respectively) (    represent the mean ± SEM (standard error of the mean) of n=3 independent experiments. The resultant P-value was expressed as * P <0.05; ** P < 0.01; P*** <0.001 was considered to be statistically highly significant and **** P < 0.0001 extremely significant (Two-way ANOVA).Ctr, control; Ext A,Micromeria fruticosa; Ext B,Teucrium polium.
Co-treatment with cisplatin and plant extracts resulted in potentiation of the inhibitory effects on MCF7 and A549 cell viability. In the same method, the anti-proliferative activity of cisplatin on MCF7 and A549 cell lines was performed after 72 hours of treatment. The results showed that cisplatin inhibited the proliferation of MCF7 and A549 cancer cells in a concentration-dependent manner ( Figure 2C). It was more effective against A549 cancer cells (IC50=5.299 ± 0,7242μg/ml) than MCF7 cancer cells (IC50=14.74 ± 1.168 μg/ml). To investigate whether the combination of cisplatin and plant extracts may have a greater anticancer effect on the cell lines (A549 and MCF7) than single treatment, cells were treated with a low dose of cisplatin 2 μg/ml and low concentrations (20 μg/ml) of total plant extracts. The doses for cisplatin and plant extracts were based on results that revealed a low level of toxicity on cell lines. Results demonstrated that the combination treatment significantly enhanced the inhibitory effects on cell viability compared to total extract or cisplatin treatments alone in the MCF7 and A549 cell lines ( Figure 3A and 3B). Taken together, these results showed that total extracts A and B boosted the cytotoxic effect of cisplatin against the two cancer cell lines.

DISCUSSION
Due to the damaging side effects of chemotherapy, alternative modalities to prevent and treat malignancies are highly demanded and desired. Screening of medicinal plants have been a promising approach to complement or possibly reduce the adverse side effects of chemotherapy 22  Accordingly, the inhibition effect of Lebanese plant species (Micormeria &Tocorium) were much potent than other plants like Salvia (IC50=57-76 μg/ml) against human melanoma cell lines (A375) and human foreskin fibroblast (HFF) cell lines 30 . The differences in IC50 between current results and previously reported studies may be attributed to the methods of extraction, different ecological factors, and cell type specificity. The augmentation of anti-proliferative activity of Cisplastin when co-administered with Micromeria or Teucrium extract suggested the plant as a potential adjuvant remedy in cancer therapy. Further studies are needed to explore the bioactive components in both plants responsible for this cytotoxic activity and possible mechanism of interactions.

CONCLUSION
This is the first study to report the anticancer activity of Micromeria fruticosa and Teucrium polium plants in Lebanon. Both plants showed high inhibitory activity against MCF7 and A549 cancer cell lines with IC50 below 30μg/ml. More interestingly, both plants significantly boosted the activity of cisplatin at low doses, suggesting the plants as new potential remedies in the cancer therapy. Represents the different concentrations (0-40μg/ml) of cisplatin in MCF7 and in A549 cell lines. Experiments were conducted in triplicates and results represent the mean ± SEM (standard error of the mean) of n = 3 independent experiments. The resultant P-value was expressed as * P <0.05; ** P < 0.01; P*** <0.001 was considered to be statistically highly significant and **** P < 0.0001 extremely significant (Two-way ANOVA). Cisp, cisplatin; Ext A,Micromeria fruticosa; Ext B,Teucriu Polium.