Fingerprinting, Antimicrobial, Antioxidant, Anticancer, Cyclooxygenase and Metabolic Enzymes Inhibitory Characteristic Evaluations of Stachys viticina Boiss. Essential Oil

The present study aimed to identify the chemical constituents and to assess the in-vitro, antimicrobial, anticancer, antioxidant, metabolic enzymes and cyclooxygenase (COX) inhibitory properties of essential oil (EO) of Stachys viticina Boiss. leaves. The S. viticina EO was isolated and identified using microwave-ultrasonic and GC-MS techniques, respectively. Fifty-two compounds were identified, of which endo-borneol was the major component, followed by eucalyptol and epizonarene. The EO was evaluated against a panel of in-vitro bioassays. The EO displayed antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Epidermophyton floccosum, with MIC values of 0.039, 0.078 and 0.78 mg/mL, respectively. The EO exhibited cytotoxicity against HeLa (cervical adenocarcinoma) and Colo-205 (colon) cancer cell lines with percentages of inhibition of 95% and 90%, for EO concentrations of 1.25 and 0.5 mg/mL, respectively. Furthermore, it showed metabolic enzyme (α-amylase, α-glucosidase, and lipase) inhibitory (IC50 = 45.22 ± 1.1, 63.09 ± 0.26, 501.18 ± 0.38 µg/mL, respectively) and antioxidant activity, with an IC50 value of 19.95 ± 2.08 µg/mL. Moreover, the S. viticina EO showed high cyclooxygenase inhibitory activity against COX-1 and COX-2 with IC50 values of 0.25 and 0.5 µg/mL, respectively, similar to those of the positive control (the NSAID etodolac). Outcomes amassed from this investigation illustrate that S. viticina EO represents a rich source of pharmacologically active molecules which can be further validated and explored clinically for its therapeutic potential and for the development and design of new natural therapeutic preparations.


Introduction
People since ancient times have been using plant secondary metabolites in their daily life for the treatment of a wide range of diseases, as food preservatives and flavoring, and to annihilate insects.The World Health Organization has estimated that around 80% of the world population rely on plant extracts for their medication [1].With time, synthetic chemicals have replaced plant secondary metabolites as the former provide early results.However, most synthetic drugs have various side effects and may cause many serious human health problems, therefore strategies have been implemented to replace synthetic drugs with plant secondary metabolites due to their ability to protect human body against oxidative damage and their great positive impact on human health [1][2][3].Nowadays, due to the growing public awareness about the probable harmful effects of synthetic additives and the tendency of consumers to use natural foods, the use of natural bioactive compounds is witnessing significant growth [4].Due to the wide prevalence of cancer worldwide, there is an essential and urgent need for a search for new anticancer medications [5].In fact, majorities of used antitumor drugs are derived from natural sources [6].
The essential oils extracted from plants have been used in traditional medicine for the treatment and prevention of various diseases, in cosmetics, foods, and food supplements in addition to dental practice and hygiene products [7,8].
Stachys (Lamiaceae family) is a large genus of herbs and shrubs containing around 300 species, widely spread in the temperate regions, especially in the Irano-Turanian and Mediterranean areas.Recently, several studies have documented anti-inflammatory, cytotoxic, antioxidant, immune system booster and antimicrobial properties of the extracts from a number of Stachys species [9].
Stachys viticina Boiss. is a perennial herbaceous shrubby plant that can reach a height of 100 cm, with multiple branches, grey-colored and erect stems.The upper leaves are crenate, while the lower ones are large, oblong-ovate and verticillaster.In traditional medicine, various Stachys species have been utilized as anti-inflammatory, antimicrobial, anti-diarrheal, wound healing and astringent remedies.In addition, they were used to treat ulcers, cough, sclerosis of the spleen and genital tumors [10,11].
It is worth mentioning that the chemical constituents of S. viticina EO, as well as its biological and pharmacological properties, haven't been reported.Thus, the current study was designed to investigate the chemical composition of the EO extracted from S. viticina leaves.Additionally, the antioxidant, anticancer, antimicrobial, metabolic enzyme (α-amylase, α-glucosidase, and lipase) and cyclooxygenase (COX) inhibitory properties of S. viticina EO was assessed.

Phytochemical Composition of S. viticina Essential Oil
In accord with our ability and experience in the isolation and identification of natural products from plants, as well as in assessing their biological activities, we decided to study the chemical components of S. viticina EO, as well as their biological effects, in the hope to finding new drug leads from natural sources.The isolation procedure was carried out using a microwave-ultrasonic apparatus.The phytochemical composition was identified and estimated using GC-MS.The obtained yield of the EO was 1.72 ± 0.97%.GC-MS analyses indicated the presence of 52 phytochemical compounds in S. viticina EO, of which endo-borneol was the major component, followed by eucalyptol and epizonarene, as revealed in Table 1 and Figure S1.

Antioxidant and Metabolic Enzymes Inhibitory Activity
The inhibitory activity of S. viticina EO against oxidation and metabolic enzymes (α-amylase and α-glucosidase and lipase) was assessed using standard biomedical assays.The results showed that S. viticina essential oil has α-amylase and α-glucosidase inhibitory activities, with IC 50 values of 45.22 ± 1.1 and 63.09 ± 0.26 µg/mL, respectively.
As indicated in

Antimicrobial Capacity
The microdilution assay was used to determine the antimicrobial potential of S. viticina essential oil against Methicillin-resistant Staphylococcus aureus (MRSA), S. aureus, E. faecium, S. sonnie, P. aeruginosa, E. coli, E. floccosum, and C. albicans.The EO inhibited the growth of all of the studied bacterial and fungal strains to different degrees.The highest antibacterial activity was recorded against MRSA, followed by E. coli with MIC values of 0.039 and 0.078 mg/mL, respectively, while the highest antifungal activity observed was against E. floccosum with MIC value of 0.78 mg/mL as presented in Table 3.

Cyclooxygenase Inhibitory Effect
The cyclooxygenase enzymatic inhibitory activity assay of S. viticina EO was conducted utilizing Cayman Chemical ELISA kit No. 560131.The calculated percentage inhibitions of COX-2 as well as for COX-1 were 98% and 99% at EO concentrations of 0.25 and 0.5 µg/mL, respectively.The results showed that the S. viticina EO displayed a strong, but not selective inhibition activity towards both COX-2 and COX-1 enzymes.

Cytotoxic Effect
As shown in Figure 1, treatment of Colo-205 cells with 1, 0.5 and 0.25 mg/mL of EO induced cytotoxicity significantly (p ≤ 0.0001) by approximately 90%, 90%, and 55%, respectively.Treatment of HeLa cells with 2.5, 1.25 and 0.625 mg/mL of EO induced cytotoxicity significantly (p ≤ 0.0001) by approximately 95%, 95%, and 80%, respectively, while at a concentration of 0.1875 mg/mL did not exhibit a significant effect as presented in Figure 2.

Discussion
The study of plants secondary metabolites represents a worldwide strategy for the search of new therapeutic agents since plants contain a wide range of biologically active compounds.Medicinal plants are extensively used in traditional medicine due to their healing properties.In order to set up an inventory of their biological activities, it is important to link scientific studies with clinical observations and traditional knowledge.This investigation is part of this scenario and was aimed to screen the biological activity of Stachys viticina essential oil.To the best of the authors' knowledge, the full chemical constituents, as well as the antimicrobial, anticancer, antioxidant, metabolic enzymes and cyclooxygenase inhibitory properties of S. viticina EO have not been reported to date.

Phytochemical Components of S. viticina Essential Oil
The S. viticina EO was isolated using an eco-friendly microwave-ultrasonic technique and the components of the EO were identified using GC-MS technology.The primary components were determined to be endo-borneol, eucalyptol, epizonarene, α-cadinene, bornyl chloride, and benzylin with percentage areas of 29.06 ± 1.24, 21.26 ± 1.97, 7.89 ± 0.91, 6.09 ± 0.1, 5.79 ± 0.09, and 5.29 ± 0.31, respectively.The chromatogram of S. viticina EO is shown in Figure S1.The retention times and mass fragmentation data of the 52 compounds identified are listed in Table 1.
Recently, Gören et al. reported the identification of only 20 compounds from EO chemical composition of S. viticina aerial parts (flowering tops, stems, and leaves) growing in Turkey, of which β-caryophyllene (62.3%), farnesyl acetate (8.9%) and α-bisabolol (4.4%) were the major components [12].The difference in the results could be attributed to the difference in the area of plant growth, extraction conditions and/or to the fact that only the leaves were used in this investigation, while in Goren et al. study the aerial parts (flowers, leaves and stems) were used.

Metabolic Enzymes Inhibitory Activity
The metabolic pancreatic α-amylase and the intestinal α-glucosidase enzymes play a vital role in the digestion of carbohydrates such as starch, dextrin, maltotriose, maltose, glucose and table sugar.In fact, diabetes, overweight and obesity can be controlled by the inhibition of these enzymes due to their ability to retard the carbohydrate's digestion process and to improve glucose tolerance in patients suffering from type II of diabetes [13].
There are many reports on the inhibitory effect of EO extracted from different plant species on the α-amylase and α-glycosidase.Loizzo et al. reported that the Cedrus libani wood EO exhibited an α-amylase inhibitory effect with IC 50 of 140 µg/mL [14].Ma et al. found that the EOs extracted from Pyrola calliantha entire plant, Senecio scandens flowers and of Schisandra chinensis roots have α-glycosidase inhibitory activity with IC 50 values of 22.11, 130.4 and 19.25 µg/mL, respectively [15].Another study conducted by Dang et al. revealed that the Citrus medica var.sarcodactylis (Buddha's hand) fruits EO inhibited an α-glucosidase enzyme with an IC 50 value of 412.2 µg/mL [16].
However, the IC 50 values showed that S. viticina EO displayed a stronger inhibitory activity on α-amylase than on α-glucosidase activity, and therefore this finding is therapeutically important especially in preventing some of the side effects associated with the use of chemical α-glucosidase and α-amylase inhibitory agents [17].The observed α-amylase and α-glucosidase inhibitory activity of the S. viticina EO gives credence to the fact that S. viticina EO could be promising antidiabetic medicine after in vivo and clinical trials.
Therapeutic agents that can inhibit the pancreatic lipase enzyme have gained much importance in recent years.Obviously, the inhibition of pancreatic lipase enzyme retards the lipids digestion and consequently lower the rate of fats absorption and decrease the levels of triglyceride in blood serum which may also cause a reduction of body weight.The EO of S. viticina revealed weak porcine pancreatic lipase inhibitory activity with an IC 50 value of 501.18 ± 0.38 µg/mL compared to that of antiobesity available drug Orlistat (IC 50 = 12.3 ± 0.33 µg/mL).Salameh et al. reported that EO of Micromeria fruticosa serpyllifolia leaves (Lamiaceae) exhibited potent antilipase activity with an IC 50 value of 39.81 µg/mL, which is better than that of S. viticina EO (IC 50 = 501.18± 0.38 µg/mL) [18].

Antioxidant Activity
Due to its simplicity and high sensitivity, the widely used DPPH assay for radical scavenging was employed to assess the antioxidant activity of S. viticina EO [19].The results of the present work showed that the EO exhibited antioxidant activity with an IC 50 value of 19.95 ± 2.08 µg/mL, less than that of the positive control (Trolox) which has an IC 50 value of 2. 23

Cyclooxygenase Inhibitory Assay
Non-steroidal anti-inflammatory molecules have the potentials to reduce inflammation and relieve pains in the human body which is associated with the increase of prostaglandin levels.However, several traditional herbal products have been used to reduce pain sensation, inflammations and fever [23].The current cyclooxygenase inhibitory assay revealed that S. viticina EO caused a 98% and 99% inhibition of COX-1 and COX-2 enzymes activity at concentrations of 0.25 and 0.5 µg/mL, respectively.S. viticina EO exhibited cyclooxygenase inhibitory activity against COX-1 and Cox-2 with IC 50 values of 0.25 and 0.5 µg/mL, respectively, similar to those IC 50 values of the positive control (the NSAID etodolac).

Cytotoxic Activity
Cytotoxicity assays have gained increasing interest over recent years due to their great value in the biological screening of insecticide, herbicides and anticancer agents [24].These assays are proved to be reliable, quick, cheap and reproducible.Various types of cytotoxic tests, such as luminometric, fluorometric, dye exclusion and colorimetric assays, are used in the fields of pharmacology and toxicology [25].The significant antimicrobial activity of S. viticina EO encouraged us to evaluate its cytotoxicity.The potential cytotoxicity of the S. viticina EO towards HeLa (cervical adenocarcinoma) and Colo-205 (colon) cancer cells was evaluated using the MTS assay.As shown in Figure 1; Figure 2, treatment of Colo-205 cells with 0.5 mg/mL of S. viticina EO induced the best cytotoxic effect (90%), while treatment of HeLa cells with 2.5 mg/mL of EO induced the highest cytotoxicity (95%).The S. viticina EO displayed very strong cytotoxicity against HeLa and Colo-205 cancer cells.
In a previous study conducted in our laboratory, Jaradat et al. reported that Teucrium pruinosum EO at a dose of 7.67 mg/mL induced cytotoxic activity on HeLa cancer cells by 90-95%.The cytotoxicity of S. viticina EO against HeLa cancer cells is triple that induced by Teucrium pruinosum EO (Lamiaceae) [26].

Antimicrobial Capacity
The microdilution assay was used to assess the antimicrobial activity of S. viticina EO against MRSA, S. aureus, E. faecium, S. sonnie, P. aeruginosa, E. coli, E. floccosum, and C. albicans.The S. viticina EO inhibited the growth of all the screened pathogens.In particular, the S. viticina EO exhibited potent antibacterial activity against MRSA with MIC value of 0.039 mg/mL, which is two folds more potential than the inhibition activity against E. coli (MIC = 0.078 mg/mL).Moreover, the EO of S. viticina plant inhibited the growth of all the studied fungal strains, especially inhibited potentially the growth of E. floccosum with MIC value of 0.78 mg/mL.

Chemical Reagents
All the experiments in the current study were carried out utilizing commercially available chemicals and reagents unless otherwise stated.

Herbal Material
Stachys viticina leaves were collected in March 2018 from the Nablus area, Palestine.The taxonomical characterization of the plant was established at the Pharmacy Department at An-Najah National University and deposited under the voucher specimen number (Pharm-PCT-2341).Later on, S. viticina leaves were cleaned, rinsed at least three times with distilled water and then dried in the shade at 25 ± 2 • C and 55 ± 5 RH of humidity for three weeks.The dried leaves were cut into small pieces which were kept in special paper bags for farther experimental work [27].

Isolation of S. viticina Essential Oil
The EO of S. viticina plant was extracted using the microwave and ultrasonic apparatus as reported by Jaradat et al. [28].Dried S. viticina powder (100 g) and distilled water (0.5 L) were placed in a round bottomed flask and exposed to micro-and ultrasonic-waves at a fixed power of 1000 W at 100 • C for 10 min.The extracted EO was collected into an amber glass bottle and kept in the refrigerator at 2-8 • C for further experiments.

GC-MS Characterization of S. viticina Essential Oil
The separation was achieved on a Perkin Elmer Elite-5-MS fused-silica capillary column (30 m × 0.25 mm, film thickness 0.25 µm), using helium as carrier gas at a standard flow rate of 1.1 mL/min.The temperature of the injector was adjusted at 250 • C with an initial temperature of 50 • C, initial hold 5 min, and ramp 4.0 • C/min to 280 • C. The total running time was 62.50 min and the solvent delay was from 0 to 4.0 min.MS scan time was from 4 to 62.5 min, covering mass range 50.00 to 300.00 m/z.The chemical ingredients of the EO were characterized by comparing their mass spectra with the reference spectra in the MS Data Centre of the National Institute of Standards and Technology, and by matching their Kovats and retention indices with values reported in the literature [29,30].In addition, the EOs Kovats and retention indices with values were compared with 20% of HPLC grade of reference EOs including α-pinene, eucalyptol, caryophyllene, γ-terpinene, ocimene, endo-borneol, α-terpineol, jasmone, α-cadinene, p-menthane that were purchased from Sigma-Aldrich, Hamburg, Germany) [31].

Porcine Pancreatic Lipase Inhibitory Assay
A porcine pancreatic lipase inhibition assay was conducted in order to assess the anti-obesity activity of S. viticina EO.The drug Orlistat, an anti-obesity, and anti-lipase agent, was used as a positive control.The porcine pancreatic lipase inhibitory method was performed according to the protocol described by Zheng et al. with slight modifications [32].A 500 µg/mL stock solution from the EO was dissolved in dimethyl sulfoxide (DMSO): methanol (1:9), and five different dilutions (50, 100, 200, 300 and 400 µg/mL) were prepared.Then, a 1 mg/mL stock solution of porcine pancreatic lipase was freshly prepared before use and was dispersed in Tris-HCl buffer.The substrate used was p-nitrophenyl butyrate (PNPB) (Sigma-Aldrich, Hamburg, Germany), prepared by dissolving 20.9 mg of it in 2 mL of acetonitrile.0.1 mL of porcine pancreatic lipase (1 mg/mL) and 0.2 mL of the EO from each of the concentration series were placed in 5 different working test tubes and mixed.The resulting mixture was adjusted to 1 mL by adding Tri-HCl solution and incubated at 37 • C for 15 min.thereafter, 0.1 mL of p-nitrophenyl butyrate solution was added to each test tube and the mixture was incubated for 30 min.at 37 • C. The pancreatic lipase activity was determined by measuring the hydrolysis of PNPB into p-nitrophenolate ions at 410 nm using a UV-Vis spectrophotometer.The same procedure was repeated for the positive control sample (Orlistat, Sigma-Aldrich).The inhibitory percentage of the anti-lipase activity was calculated using the following equation: where AB is the recorded absorbance of the blank solution and Ats is the recorded absorbance of the tested sample solution.

α-Amylase Inhibition Assay
The α-amylase inhibitory activity of S. viticina EO was assessed according to the standard method reported by Nyambe-Silavwe et al. with minor modifications [33].The EO was dissolved in DMSO (Riedel-de-Haen, Hamburg, Germany) and then diluted with a buffer ((Na 2 HPO 4 /NaH 2 PO 4 (0.02 M), NaCl (0.006 M) at pH 6.9) to a concentration of 1000 µg/mL.A concentration series of 10, 50, 70, 100 and 500 µg/mL were prepared.0.2 mL of porcine pancreatic α-amylase enzyme solution (Sigma-Aldrich, St. Louis, MO, USA) with a concentration of 2 units/mL was mixed with 0.2 mL of the EO and incubated at 30 • C for 10 min.Thereafter, 0.2 mL of freshly prepared starch solution (1%) was added and the mixture was incubated for at least 3 min.The reaction was stopped by addition of 0.2 mL dinitrosalicylic acid (DNSA) (Alf aAesar, Lancashire, UK), then the mixture was diluted with 5 mL of distilled water and heated in a water bath at 90 • C for 10 min.The mixture was left to cool down to room temperature and the absorbance was measured at 540 nm.A blank was prepared following the same procedure by replacing the S. viticina EO with 0.2 mL of the buffer.
Acarbose (Sigma-Aldrich, USA) was used as a positive control and prepared to adopt the same procedure described above.The α-amylase inhibitory activity was calculated using the following equation: where A b is the absorbance of the blank and A S is the absorbance of the tested sample or control.

α-Glucosidase Inhibitory Activity Assay
The α-glucosidase inhibitory activity of S. viticina EO was performed according to the standard protocol with a slight modification [34].A mixture of 50 µL of phosphate buffer (100 mM, pH 6.8), 10 µL α-glucosidase (1 U/mL) (Sigma-Aldrich, USA) and 20 µL of varying concentrations of S. viticina EO (100, 200, 300, 400 and 500 µg/mL) were placed in 5 different test tubes.After 15 min.incubation at 37 • C, 20 µL of pre-incubated 5 mM PNPG (Sigma-Aldrich, USA) was added as the substrate to each test tube and the reaction mixtures were incubated at 37 • C for additional 20 min.The reaction was terminated by adding 50 µL of aqueous Na 2 CO 3 (0.1 M).The absorbance of the released p-nitrophenol was measured by a UV/Vis spectrophotometer at 405 nm.Acarbose at similar concentrations as the plant EO was used as the positive control.The inhibition percentage was calculated using the following equation: where A b is the absorbance of the blank and A S is the absorbance of the tested sample or control [35].

Antioxidant Assay
For estimation of S. viticina EO antioxidant potential, a solution of EO (1 mg/mL) in methanol was serially diluted with methanol to obtain concentration of 1, 2, 3, 5, 7, 10, 20, 30, 40, 50, 80 and 100 µg/mL.Then, DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent (Sigma, New York, NY, USA) was dissolved in 0.002% w/v methanol and mixed with the previously prepared working concentrations in a 1:1 ratio.The same procedures were repeated for Trolox (Sigma-Aldrich, Darmstadt, Denmark) which was used as a positive control.All of the solutions were kept in a dark chamber for 30 min at ordinary temperature.
Then, their absorbance values were measured at a wavelength of 517 nm utilizing a UV-Visible spectrophotometer.The DPPH inhibition potential by S. viticina EO and Trolox were determined employing the following equation: DPPH inhibition (%) = (abs blank − abs sample )/abs blank 100% (4) where abs blank is the blank absorbance and abs sample is the absorbance of the samples.The antioxidant half-maximal inhibitory concentration (IC 50 ) of S. viticina EO and Trolox were assessed using BioDataFit-E1051 program [36].

Antimicrobial Activities of S. viticina Essential Oil
The fungal and bacterial isolates used in this study were from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from selected MRSA species (), that were obtained from the Palestinian area at clinical settings and exhibited multi-antibiotic resistance.The screened microorganisms included three Gram-positive bacteria (MRSA, Enterococcus faecium (ATCC 700221) and S. aureus (ATCC 25923)), three of Gram-negative bacteria (Shigella sonnie (ATCC 25931), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) and two fungal strains (Epidermophyton floccosum (ATCC 10231) and Candida albicans (ATCC 90028)).
The microdilution assay was utilized to assess the antimicrobial activity of S. viticina EO against bacterial and yeast strains.A total of 8.8 g of Mueller-Hinton broth were dissolved in 400 mL of distilled water under heat and boiled for 1 min.The obtained solution was autoclaved and kept at 4 • C until use. 100 µL from the broth was placed in each well of the microdilution tray.Then, 100 µL of the EO was added to the first well and the mixture was shacked.Thereafter, serial dilutions up to well #11 were performed.Well #12 did not contain any EO and was considered as a positive control for microbial growth.A fresh bacterial colony was picked from an overnight agar culture and was prepared to match the turbidity of the 0.5 McFarland standards to provide a bacterial suspension of 1.5 × 10 8 CFU (colony forming unit)/mL.The suspension was diluted with broth by a ratio of 1:3 to a final concentration of 5 × 10 7 CFU.Then, 1 µL of the bacterial suspension was added to each well except well #11, which was a negative control for microbial growth.Finally, the plate was incubated at 35 • C for 18 h.
The microdilution method for the yeast C. albicans was performed as described above, except that after matching the yeast suspension with the McFarland standard, it was diluted with NaCl by a ratio of 1:50, followed by 1:20, and 100 µL was placed in the wells.The plate was incubated for 48 h instead of 18 h.
To investigate anti-Epidermatophyton floccosum mold activity of the S. viticina EO, an agar dilution method was performed.Sabouraud dextrose agar (SDA) was prepared, of which 1 mL was placed in each tube and kept in a 40 • C water bath. 1 mL of the EO was mixed with 1 mL of SDA in the first tube and serial dilutions were performed in six tubes, except tube #6, which did not contain any plant material and considered as a positive control for microbial growth.
After the SDA was solidified, the spores of the mold culture were dissolved using distilled water containing 0.05% Tween 80 and scratched from the plate for comparison with McFarland turbidity.From that suspension, 20 µL was pipetted into the six tubes, except tube #5, which was considered as a negative control.The tubes were incubated at 25 • C for 14 days.The MIC is the lowest concentration of an antimicrobial agent that inhibited the visible growth of a microorganism [37,38].

Determination of COX Inhibition
The S. viticina EO inhibitory activity of human recombinant COX-2 and ovine COX-1 enzymes was assessed using a COX inhibitor screening method (Cayman Chemical, kit No.560131, Ann Arbor, MI, USA).The yellow color of the enzymatic reaction was evaluated using UV spectrophotometer in a Microplate Reader at a wavelength of 415 nm.Cyclooxygenase inhibitory assay was performed for two concentrations of EO (0.25 and 0.5 µg/mL) with celecoxib (a commercial NSAID marker).The anti-inflammatory activity of the tested EO was determined by calculating the percentage production inhibition of prostaglandin E2 (PGE2).The tested compounds concentration causing 50% inhibition (IC 50 ) of the formation of prostaglandin PGE2 by COX1 and COX2 enzymes were determined from the curve of concentration inhibition response by regression analysis [39].

Cell Culture and Cytotoxicity Assay
The HeLa (cervical adenocarcinoma) and Colo-205 (colon) cancer cells were cultured in media (RPMI-1640) and supplemented with 10% fetal bovine serum, 1% Penicillin/Streptomycin antibiotics and 1% l-glutamine.Cells were grown in a humidified atmosphere with 5% CO 2 at 37 • C. Cells were seeded in 96-well plates (2.6 × 10 4 cells).After 48 hrs, the cells were incubated with EO at various concentrations for 24 h.Cell viability was assessed by Cell-Tilter 96 ® Aqueous One Solution Cell Proliferation (MTS) Assay following the instructions of the manufacturer (Promega Corporation, Madison, WI, USA).At the end of the treatment, 20 µL of MTS solution per 100 µL of media was added to each well and incubated at 37 • C for 2 h and finally, the absorbance was spectrophotometrically estimated at 490 nm [40].

Statistical Examination
All the data on α-amylase, α-glucosidase and porcine pancreatic enzymes inhibitory activity as well as on the antioxidant, COX inhibitory and cytotoxicity activities were the average of triplicate analyses.The outcomes were presented as means ± standard deviation (SD).Statistical analysis was established employing GraphPad Prism software version 6.01 (GraphPad, San Diego, CA).

Conclusions
The GC-MS analysis revealed the presence of fifty-two compounds in the S. viticina EO, of which endo-borneol was the major component, followed by eucalyptol and epizonarene.The EO of S. viticina showed an ability to scavenge the free radical DPPH with an IC 50 value of 19.95 µg/mL and displayed cytotoxic activity against colon and HeLa cancer cells at 0.5 mg/mL and 1.25 mg/mL, respectively.It also showed very strong antimicrobial (against three Gram-positive, three Gram-negative bacteria and two fungi) effects, of which the strongest was against MRSA, with a MIC value of 0.039 mg/mL.Moreover, the S. viticina EO showed high cyclooxygenase inhibitory activity against COX-1 and COX-2, with IC 50 values of 0.25 and 0.5 µg/mL, respectively, similar to those of the positive control (the NSAID etodolac).In addition, the EO showed potential in inhibiting the activity of α-amylase (56.52% at 45.22 µg/mL) and of α-glucosidase (69.82% at 63.09 µg/mL).Briefly, S. viticina EO contains pharmacologically active molecules which could be further validated and explored clinically for their therapeutic potential and for the development and design of new natural therapeutic preparations.

Figure 1 .Figure 2 .
Figure 1.The effect of S. viticina EO on the cytotoxicity of Colo-205 cells.Results were depicted as relative quantities (RQs) compared to the control (only media).# where P < 0.0001.Error bars represent SD.

Figure 1 .Figure 1 .Figure 2 .
Figure 1.The effect of S. viticina EO on the cytotoxicity of Colo-205 cells.Results were depicted as relative quantities (RQs) compared to the control (only media).# where P < 0.0001.Error bars represent SD.

Figure 2 .
Figure 2. The effect of S. viticina EO on the cytotoxicity of HeLa cells.Results were depicted as relative quantities (RQs) compared to the control (only media).# Where P < 0.0001.Error bars represent SD.

Table 2 .
The metabolic enzymes inhibitory and the antioxidant activities IC 50 values of S. viticina essential oil compared with the required positive controls. α-Amylase
± 1.57 µg/mL.Previous studies demonstrated that various Stachys species exhibited good antioxidant activity.A study conducted by Kukić et al. revealed that S. anisochila, S. beckeana, S. plumose, and S. alpina ssp.dinarica have antioxidant potential with IC 50 values of 17.9, 20.9, 101.61, and 26.14 µg/mL, respectively [20].The EO of S. inflate, growing Iran, exhibited antioxidant activity with an IC 50 value of 89.50 ± 0.65 µg/mL [21].Furthermore, A study performed by Conforti et al. on the antioxidant activity of the EOs extracted from various Stachys species from different regions of the Mediterranean area revealed that S. palustris, S. cretica, and S. hydrophila displayed the highest antiradical effect, with IC 50 values of 48.2, 65.2 and 66.4 µg/mL, respectively [22].