Antimicrobial, Antiviral Activity and GC-MS Analysis of Essential Oil Extracted from Achillea fragrantissima Plant Growing In Sinai Peninsula, Egypt

Medicinal plants are considered new resources for producing agents that could act as alternatives to antibiotics in treatment of antibiotic-resistant bacteria. The present study aimed to evaluate the efficacy of essential oil extracted from Achillea fragrantissima plant growing in Egypt for antimicrobial, antiviral activities and chemical composition analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). We also performed determination of essential oil antimicrobial activity by agar desk diffusion method and Minimal Inhibitory Concentration (MIC). Also, antiviral activity on ORF virus by pock reduction test was performed. It was reduced virus titer from 5.9 to 1.00 at 180 minutes. Detection of beta lactams resistant bacteria (Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis (MRSA) and Gram-negative bacteria Escherichia coli) by PCR with primers of mecA gene and Bela gene. The essential oil obtained by hydrodistillation was analyzed by GC-MS. The major components were found to be Santolina triene (1.97%), 2,5,5-trimethyl-3,6-heptadien-2-ol (8.23%) Eucalyptol 8.17, trans-2,7-Dimethyl4,6octadien-2-ol (24.40%), 1,5-Heptadien-4-one-3,6-trimethyl (7.65%), Artemisia alcohol (3.49%), à Thujone (33.97%), Cissabinol (1.92%), Lavandulol (0.71%), 2-Octen-4-ol, 2-methyl (2.02%), 3-Cyclohexen1ol,4-methyl1 (1 methylethyl) (CAS) (2.15%), à terpineol ( 0.05% ), Estragole (0.71%) Lavandulyl acetate (0.49%), Sabinyl acetate (2.12 %), Germacrened (0.94%). Finally, our study proved that the essential oil has bactericidal effect on some bacterial resistant antibiotic (Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis (MRSA) and Gram-negative bacteria Escherichia coli ) as well as antiviral activity on ORF virus but it is still need further extensive studies for safety and drug interaction. Antimicrobial, Antiviral Activity and GC-MS Analysis of Essential Oil Extracted from Achillea fragrantissima Plant Growing In Sinai Peninsula, Egypt


Introduction
Medicinal plants still constitute one of the major source of drugs in modern as well as traditional medicine throughout the world [1,2]. Since a long period of time, plants have been a valuable source of natural products for maintaining animals and human health [2,3]. The development of multidrug-resistant(MDR) bacteria takes place because of the accumulation of different antibiotic resistance mechanisms inside the same strain which able to live in the presence of antibiotics drugs so that standard treatments become ineffective [4,[14][15][16]. Although, the pharmacological companies have produced a number of new antibiotics recently, but even then microorganism resistance to antibiotics increased throughout the world [5,7]. This situation has attract attention of researchers towards herbal products for developing and improve the antibacterial drugs quality [6,7].
Natural products of higher plants may possess a new source of antimicrobial agents with possibly novel mechanisms for treatment of infectious diseases [8,9]. Medicinal plants contain active principles which can be used as alternative effective herbal drugs against common bacterial infections [10]. They would be the best source to obtain a variety of drugs and active compounds. Therefore, such plants should be investigated to better understand their properties, safety and efficiency [11]. Essential oils are commercially used in different industries such as pharmaceutical, agronomic, food, sanitary, cosmetic and perfume [12]. In medicine they are used antioxidant, antitumor and antifungal [13][14][15][16].
The genera Achillea belongs to Family Asteraceae are widely distributed in the Middle East countries [17]. Ancient people had been used Achillea species in traditional medicine to alley pain, spasms and inflammation [18,3,4]. In Arabian countries all Achillea species were referred to locally names as Qaysum, Gesoom or Bu`eithraan [20,42] Numerous studies describe the various pharmacological effects of the Achillea fragrantissima hydrodistilled volatile oils in management of several diseases, used topically and orally [18][19][20][21].
Researchers studied antibacterial properties of various plants against Gram-negative as well as Gram-positive bacterial strains but only few reports on drug resistant bacteria and antiviral activity. Our study was aimed to evaluate in vitro antibacterial of essential oil extracted from Achillea fragrantissima plant against Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis (MRSA) and Gram-negative bacteria Escherichia coli isolated from bovine

Gas chromatography and mass spectrometry (GC-MS) analysis
The essential oil was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) (NRC-GC/El-MS Lab), according to method [25][26][27] column with 0.25 μm film thickness, Helium was used as carrier gas; the flow through the column was 1.4 ml/min. The column temperature was programmed from 40 to 85°C at 20°C/min, increased from 85 to 300°C at rate of 5°C/min and finally held for 10 min. The MS operating parameters were as follows: ionization potential, 70 V; ionization current, 2 A; ion source temperature, 200°C, resolution, 1000. Mass unit were monitored from 30 to 450 m/z. Determination and identification of each components in the oil was based on RI relatives to n-alkanes and computer matching with library search report, as well as by comparison of the fragmentation patterns of mass spectra [27].

Bacterial strain
Microbial strains: Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis (MRSA) and Gram-negative bacteria Escherichia coli were isolated from bovine mastitis provided by Veterinary Research Division at National Research Center (NRC), Egypt. All bacteria strains were stored in broth containing 25% (v/v) glycerol (Sigma-Aldrich) at -20°C. Prior to use, the culture were propagated twice in the appropriate media with shaking for 24 h at 37°C to physiologically active them.

Virus strain
The egg-adapted ORF virus was provided by Dr. G.S. Zeedan, NRC, Cairo, Egypt. Virus was propagated in the Chorio-Allantoic Membranes (CAM) of Specific Pathogen Free (SPF) embryonated chicken eggs (11 days age) as previously described by [28]. Both CAM and AF were harvested and ground in 0.1 M sterile PBS. The homogenate was frozen at -20°C and thawed three times and then centrifuged at 3000 rpm/15 min. Then supernatant was titrated and stored at -20°C until used.

Animal used
Twenty five clinically healthy Albino rats (130-150 g body weight) were obtained from Animal house of National Research Center Cairo Egypt, and randomly divided into 5 groups for experiment. The Animals were housed in a well-ventilated animal room under standardized conditions of 24°C; relative humidity 50 ± 5% and 12 hours light/dark cycle at the Animal House, National Research Center, Giza, Egypt. Feed and water were supplied adlibitum to meet the requirements of the NRC [29]. Rats were acclimatized for 15 days before the start of the experiment.

Toxicity of essential oil
Essential oil was diluted as the following: 100, 200, 300, 400 and 800 µl/ml then 1ml for each rat was injected S.C/I.P twice dose one week interval. Behavioral alterations, inflammatory effects, illness, and weight changes were recorded for 2 weeks post-treatment. Control animals (Rats). The toxicity of essential oil in ECE was examined as follows: inculcated 0.2 ml from each concentration of essential oil into Chorio-Allantoic Membranes (CAM) and Chorio-Allantoic Sac (CAS) incubated at 37°C for 7 days daily examination. The maximum nonlethal dose was taken as the maximum non-toxic concentration.

Antiviral activity of essential oil determined by Pock Reduction Assay
Virus titers were estimated from cytopathogenicity by tenfold dilution method 0.02 ml of each dilution was inoculated in five CAM of ECE for each dilution and expressed as 50 % Egg Infectious Dose per ml (EID50/ml). The calculation Calculated for each tested were was performed according to [30].

Antimicrobial activity
Essential oils were individually tested against Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis (MRSA) and Gram-negative bacteria Escherichia coli were previously isolated from bovine mastitis by Agar diffusion methods and Minimal Inhibitory Concentration (MIC).
Agar diffusion method: These methods were performed according to Forbes et al. [31]. Bacteria cultures were diluted with sterile physiological saline solution with reference to the MC Farland 0.5 standard to achieve inoculums of approximately 10 6 CFU/ml. A suspension was swab in three directions on 4 mm thick Mueller Hinton Agar (MHA) (Oxoid) with a cotton swap. Modified discs of 6 mm were prepared using a Whitman filter paper. 100 discs were obtained by punching and putting in vials-bottles and sterilizing in an oven at 170°C for 30 min. The discs were impregnating with 20 μl of essential oil. Prepared discs containing the various essential oil were carefully placed on the inoculated plates using a sterilized forceps in each case [31]. The disc with solvent alone used as negative control and antibiotic discs as control positive. The plates were then turned upside-down and incubated at 37°C for 24 h in an incubator by the same manner used rosette with 6 well puncture the agar and 50 μl each well by essential oil, Tween 20 with saline and saline only as control negative. The results were taken by measuring the Diameter of the Inhibition Zone (DIZ) around the disc or well. This was repeated thrice and mean ± SD was calculated.

Minimal inhibitory concentration (MIC) using microdilution method
It was done according to [32,33] guidelines for determination of Minimum Inhibitory Concentration (MIC). The MIC was defined as lowest concentration of essential oils that inhibiting visible bacterial growth after incubation for 20 h at 37°C. Into each well of microplate 100 μl of Mueller-Hinton Broth (Oxoid) inoculated with the bacteria strain. An aliquot (100 μl) of the essential oil was added in first well serial dilutions of the essential oils were prepared in a 96 well micro titer plate, including one growth control and one sterility control. The contents of the wells were mixed and micro plates were incubated Page 3 of 7 at 37°C for 24 h. Micro titer plates were incubated at 37°C for 24 h. The activity was measured as a function of turbidity. Lack of turbidity was further confirmed by pouring suspension aliquot of 0.1 ml into pre-sterilized Petri dishes with nutrient agar medium. The tests were conducted in triplicate.

Estimation of the number of the viable bacterial cells
The time that taken by essential oil to kill staphylococcus and E. coli were evaluated by the time-kill assay method. The Time-kill curves were performed in tube containing nutrient or Muller Hinton broth, using inoculums density of approximately (10 6 CFU/mL) in the presence of essential oil. The tubes were continuously shaken and incubated at 37°C. Samples were obtained at 0, 2, 4, 8 and 16. At each sample time, they were taken and serially diluted log 2 2, 4, 8, 16, 32. 100 µl of undiluted and diluted samples were inoculated on nutrient agar or Muller Hinton agar. The plates were incubated at 37°C for 24 h at the appropriate temperatures, the number of colonies on each plate was counted, and the number of the viable cells in the cultures was calculated.

Polymerase chain reaction (PCR)
Detection of the mec A and Bela A Genes for Staphylococcus epidermidis strains and E. coli were accomplished using PCR amplification. Cells were suspended in a lysis buffer containing 1 M Tris HCl, 5 M NaCl, and 0.1 M EDTA, which was incubated at 95°C for 10 minutes. After incubation, the suspension was centrifugated at 23 000 rpm for 5 min. The supernatant was used as a template in PCR. PCR assay was carried out as described by [34]. Using primers Beta-lactams gene resistant MRSA Mec A F (GTGAAGATATACCAAGTGATT) and R (GTGAAGATATACCAAGTGATT) gave 147 bp PCR product of mecA gene from strains Staphylococcus epidermidis and/also using primer Beta-lactams gene resistant for E. coli bla C (F) TGGCCAGAACTGACAGGCAAA (R) TTTCTCCTGAACGTGGCTGGC 462 [35,34]. The final PCR products were visualized using UV-trans-illuminator after electrophoresis on 1.5% agarose gel containing 50 mg/mL EtBr.

Statistical analysis
All data were subjected to statistical analysis including the calculation of the mean and standard Deviation. Significance between data of control and infected groups was evaluated by the Student t-test at level P<0.05 according to [36] using SPSS for windows version 15 computer program.

Antimicrobial activity by the agar diffusion method
The essential oil extracted from A. fragrantissima inhibited the growth of the microorganisms tested. It was especially active against Staphylococcus aureus, MRSA and E. coli. The Staphylococcus aureus showed the highest sensitivity to essential oil effect ranged from (18-22 mm-24-26 mm) and MRSA from (14-16 mm), (16-18 mm) and followed by E. coli from (8-11 mm) and (12-16 mm) inhibition zone

Discussion
Achillea fragrantissima plant has been used for many years in traditional medicine in Middle East countries for the treatment of some diseases [37]. Several Achillea plants have been found to possess antiseptic and infection preventing properties [38,39]. They are being used as ingredients in medicinal and cosmetic preparations [40]. Recently, it was documented that the essential oil has antiinflammatory, antioxidant and anti-proliferative capacities [41]. The present study showed that the essential oil has wide varieties of volatile oil terpene hydrocarbons (aliphatic and cyclic) and their corresponding isoprenoid derivatives and analogues, mixtures of these substances, terpinen-4-01 demonstrated fraction that strongly antimicrobial effect by acting only in eukaryotic bacteria cell as in Table 1 and Figures 1 and  2 and this finding is in agreement with [42].
The essential oil extracted from A. fragrantissima plant inhibits growth of microorganism to various degree can determined by disc and well diffusion methods according to diameter of inhibition zone. The inhibition zone of Staphylococcus aureus from 18 mm to 22 mm, followed by MRSA from 12 to 16 and followed by Gram negative bacteria Escherichia coli nearly smaller to MRSA inhibition zones, from 7.0 mm to 10.4 mm Staphylococcus aureus was the most sensitive organism to A. fragrantissima essential oil than MRSA and E. coli. However, the sensitivity of Escherichia coli not only similar to that of the MRSA but also required a higher concentration from essential oil to induce the desirable bactericidal effect as in Table 2 and Figures 3 A and 3B this results is in agreement with [43].
Surprising results that was no difference between inhibition zones of methicillin-resistant and methicillin-susceptible strain may be due to the some component of essential oil caused bacterial cell damaged this result is in agreement with [44][45][46]. They found that essential oil caused gross membrane damaged and provoke whole cell lysis of eukaryotic. The mechanism of spread antibiotic resistance from food animals to humans remains controversial and resistance to the same drugs [47]. The most of the bacteria isolated from bovine mastitis showed a multidrug resistant for antibiotics due to the presences of drug resistant gene as beta lactimase which easily detected and evaluated by PCR as in Figure 4 this result is in agreement with [48]. The explanation of antibacterial activity of essential oil on bacterial cell depend on the action of terpenes fraction that involving their action by causing membrane perturbation and marked leakage of cytoplasmic material lead to induce irreversible damage to the cytoplasmic membran but fully action of terpenes still unknown and this explanation is agreement with [48,49].
The mecA and Bel gene coding for methicillin resistance via penicillin binding protein 2a (PBP2a) are well established and is considered as "gold standard" for detection of methicillin resistance in comparison with phenotypic methods [46,47]. Our results revealed that the inhibitory effect of essential oils on Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis (MRSA) and Gram-negative bacteria Escherichia coli bacteria may be due to reduction or modified of β-lactamse (methicillin) resistance but the actual mechanism is unknown. However, is likely explained by some structural change in the resistant bacteria. Firstly, volatile oils fraction can be connected with the perturbation of cell membrane, the second mechanism may be inhibited the production of protein binding protein (PBP2) by inhibition of mecA and bel genes expression and final stage involved to subset synthesis of peptidoglycan of cell wall (transpeptidation reaction), which present outside the cell membrane this explanation is agreement with [50].
The essential oil was observed safe, no effect in the nature and color of embryo fluid comparing with control as well as experimental animals this result is in agreement with [51,52]. It was reduced infectivity titer   of ORF virus from log 10 5.9 to 0.00 within 180 minutes. Antiviral effect of essential oil may be due to block the viral infection by blocking the cell membrane receptor for ORF virus or induce internal changes in the host cells, which in turn affect the virus replication cycle or due to production cytokines which blocked viral infection other health cells as in Table 3 and Figure 5. This result was in agreement with [53]. They found the essential oil caused sharp reduction in small pox viruses infectivity titer. Also, they observed that the concentration of viruses were reduce log 10 5-1 and they infectivity titer were reduced 21 and 25 time more than control group. We found that the essential oil reduced plaque size and number when mixing with ORF virus before inoculation on CAM, Finally, Our results proved that the Achillea fragrantissima essential oil has antiviral and antibacterial activities on B-lactmase resistant

Conclusion
The composition of the essential oil of Achillea fragrantissima growing in Sinai, Egypt has been analyzed by GC-MS and its antimicrobial activity against Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis (MRSA) and Gram-negative bacteria Escherichia coli as well as antiviral activity against ORF virus reduced titer from 5.9 to 1. Detection of B-lactam antibiotic resistance bacteria was done by PCR with primers mec A and Bela genes. The results proved that the essential oil can be used in the treatment of diseases caused by the drug resistant microorganism. But, still need further extensive researches are required to prove safety, drugs interaction and clinical studies are required to prove the safety of the oil as a medicine.