THE ANTI HRSV ACTIVITY OF Ferula halophila Peşmen AQUEOUS AND METHANOL EXTRACT BY MTT ASSAY

: The trend towards natural products in the world is increasing due to the increased drug resistance of infectious diseases, the high prices for drugs and the difficulty of access. Also, bacterial or viral diseases that are difficult to treat and need a long time for proper treatment cause important infections in people. Since effective drugs could not be developed for most viral infections, it is very important to find natural products against viruses to introduce them to the world of science. The antiviral activities of the aqueous and methanol extract from Ferula halophila Peşmen, an endemic species to Turkey, has been investigated


Introduction
Respiratory syncytial virus (RSV) is a negativestranded segmentless RNA virus belonging to the genus Pneumovirus of the family Paramyxoviridae (Falsey & Walsh 2000). RSV is recognized worldwide as one of the leading causes of lower respiratory tract disease in infants and children under the age of 5, and the estimated annual number of human respiratory syncytial virus (HRSV)associated hospitalizations is 3.4 million with at least 66,000 deaths worldwide (Hall et al. 2009, Nair et al. 2010, Lambert et al. 2014. The virus also causes acute respiratory diseases in the elderly (Collins & Melero 2011) and can be destructive in individuals with weakened immune systems (Dudas & Karron 1998). Although most individuals are infected with RSV at an early age of their lifes, susceptibility to recurrent RSV infections continues throughout whole life, also in older ages (Varga & Braciale 2013). RSV can infect the upper respiratory mucosa and may initially replicate in the nasopharynx. It is likely to spread rapidly to the lower respiratory tract by aspiration of secretions and causes morbidity and mortality mainly with the pathology of the lower respiratory tract. Therefore, the management of RSV infection requires an effective strategy to prevent viral infection of both the upper and the lower respiratory tracts (Collins & Crowe 2007). No licensed RSV vaccine has yet been developed, although the disease remains a common viral cause of serious respiratory disorders (Durbin & Karron 2003). Failure to develop specific therapeutic or safe and effective vaccines adversely affects the effort to reduce global mortality and morbidity caused by RSV infections. Ribavirin (RBV) is known as the only drug approved for treatment of RSV infections, and immunoglobulin preparations are also used for prevention from RSV infections. However, none of these methods is cheap, they are difficult to implement (Kneyber et al. 2000, Pelaez et al. 2009) and all potential RSV vaccines as prophylactic and therapeutic candidates are currently under clinical trials (Lindsay et al. 2015).
The genus Ferula L. (Apiaceae) contains about 170 species generally growing in the arid regions of temperate Eurasia, the Canary Islands and North Africa (Downie et al. 2000). The gene centre of the genus is located in West and Central Asia (Drude 1898). Traditionally, some species of this genus are being used both in "the kitchen" and for "medical treatment". The genus is well known in Turkey and aerial or root parts of some species are used as food and aphrodisiac in some parts of the country (Baytop 1984 Considering the medical features mentioned above, F. halophila Peşmen, an endemic species for Turkey, has been chosen to determine its antiviral effects. Although several studies were performed on antiviral properties of extracts of some species of the genus against various viruses, no study was performed on antiviral effects on RSV. In the present study, we tested, cytotoxic and antiviral effects of methanol and aqueous extracts of F. halophila on HRSV cultured in HEP-2 human laryngeal epidermoid carcinoma cell line.

Plant samples
Ferula halophila samples were collected from the Cihanbeyli-Yavşan Salty area Konya province in Central Anatolia, Turkey, and the collected specimens were identified taxonomically by Dr. Osman TUGAY from Selçuk University, Faculty of Pharmacy. A voucher sample is stored at S.Ü. Kon-Fungarium, Konya/Turkey.

Cell and virus
Human larynx epidermoid carcinoma cells [HEp-2; ATCC (the American Type Culture Collection) CCL 23] were used to culture human respiratory syncytial virus (HRSV Long strain: ATCC VR-26). Cells were propagated at 37°C under 5% CO2 in Eagle's minimum essential medium (EMEM) supplemented with 10% foetal bovine serum (FBS, ATCC-30-2020), 10000 U/mL penicillin, 10 mg/mL streptomycin, and 25 µg/mL amphotericin B. HRSV is a medium-sized (120-200 nm) enveloped virus that contains a linear negative-sense RNA genome (must be converted to a positive RNA prior to translation). Virus (coded as ATCC-VR-26) was purchased from American Type Culture Collection (ATCC) and reproduced in Virology Laboratory of Science Faculty of Selçuk University. The virus was propagated on a 90% confluent cell monolayer in EMEM with 1% FBS and antibiotics as described above. Viral titer was determined by 50% tissue culture infectious dose (TCID50) method and expressed as TCID50 per 0.1 mL (Kaerber 1964). The virus was stored at -196°C until use.

Preparation of the extracts
Dried aerial parts (body and leaves) of the plant were used for the experiments. Each 30 g sample in powder form was placed separately in 400 mL of methanol and 400 mL of sterile distilled water and extracted for 1 hour with an ultrasonicator at 45°C. Plant extracts were filtered through Whatman No: 1 filter paper, and then the solvents used were completely evaporated at 45°C under reduced pressure in a rotary evaporator (Heidolph Laboratory 4000, Germany). After evaporation, the plant extracts were lyophilized at -110°C under reduced pressure in the lyophilizer (Labconco, USA). Each 1000 mg of the lyophilized extracts were dissolved in 10 mL of EMEM (serum-free) and stock solutions were prepared at a concentration of 100 mg/mL. The stock solutions were sterilized by a trough in 0.22 µm Millipore filter, then stored in 2 mL tubes and stored at +4°C until use. Ribavirin (RBV, R9644-10 mg, Sigma, USA), a drug approved for the treatment of HRSV infections in humans, was purchased. 10 mg ribavirin was dissolved in 5 mL of EMEM (serum-free). This 2 mg/mL stock concentration was filtered in 0.22 µm Millipore filter, then they were stored at -80°C or +4°C (When stored at +4°C, it was used within 1 week).

Cytotoxicity assay
The cytotoxic effects of methanol and aqueous extracts of F. halophila on HEp-2 cells, and RBV were determined by the ability to reduce Trypan blue dye and Tetrazolium salt (MTT) which was described by Ho et al. (2010). 96-well microtiter plates were used in the experiments. The 1 st column was used as Negative control (Nc), the 2 nd was used as Cell control (Cc), and serial dilutions according to log2 base for both extract solutions with a concentration of 100,000 µg/mL were used from 50,000 to 195.313 µg/mL. A suspension of HEp-2 at a concentration of 0.625 × 10 5 cells/mL was prepared with the cell growth medium, and 100 µL cell suspensions were seeded to all wells of the plate (6250 cells/well) except the 1 st column and all 3 lower wells of columns 3-12 which was used to check whether the extracts had direct chemical interaction with MTT. After 24 hours of incubation at 37°C in 5% CO2, each 100 µg/mL extracts were added on to wells and the plates were incubated for 48 hours. Then, the MTT assay was performed. Mean OD were read via an ELISA reader (Multiskan EX, Lab systems) at a test wavelength of 570 nm and a reference wavelength of 630 nm. The mean OD values of the extracts of the same concentration without cells were subtracted from the mean OD of the different extract concentrations in the cell-containing wells.
Maximum non-toxic concentrations (MNTCs) of the extracts were determined by comparing them with the OD of the Cc. These MNTCs were used to determine the antiviral activity of the extracts.
In determining the cytotoxic effects of RBV on HEp-2 cells, a solution of RBV was prepared at a concentration of 1,000 µg/mL in EMEM from the stock solution (2,000 µg/mL). Subsequent dilutions (from 500 to 1.95 µg/mL concentrations) were prepared from the base of log2. Then the same procedure which was used to determine the cytotoxic effects of the extracts was followed for RBV. Dilutions of the test samples (extracts and RBV) in 2 × MNTCs (MNTCs= 195.313 µg/mL for methanol, 390.625 µg/mL for aqueous and 0.98 µg/mL for ribavirin) were prepared using maintenance medium. Subsequently, twofold dilutions with maintenance medium were prepared from these dilutions. 2.5 × 10 4 cells were seeded on the wells and incubated at 37ºC for 24 hours in a 5% CO2 incubator. When the cells were confluent, the production medium in the wells was evacuated and each 100 µL of RSV suspension (containing 100 TCID50) and each 100 µL of the extract samples were put in the wells, simultaneously. 100 µL of RSV suspension and 100 µL of maintenance medium were put into Virus Control (VC) wells. 200 µL of maintenance medium was added to the Ccwells. Plates were incubated for 2-5 days (more precisely, until 85-90% CPE was seen in VC wells) for the development of CPE at 37°C in 5% CO2. When 85-90% CPE was observed in VC wells, the solutions were removed in the wells, and an MTT assay was performed. The protection percentages were calculated. The EC50 value, which is defined as the concentration of extract (or RBV) that provides protection in 50% of the infected cells, was determined and the SI of the extracts (or RBV) was calculated.

Virus Titration
In the titration of RSV in HEp-2 cell culture by the microtitration method, the power of infectiousness was determined as DCID50 = 10 -4.5 /0.1 mL at the end of the 5 th

Cytotoxicity and Antiviral Assay Results
The cytotoxic effect of the extracts and RBV on Hep-2 cells , were evaluated by colorimetric cell viability test.
In the experiments, the non-toxic dose of the extracts and RBV on HEp-2 cells was determined. Then, starting from non-toxic doses, the protection percentages and SI of the extracts and RBV were determined. The MNTCs and CC50 values of the methanol, aqueous extracts, and RBV against HEp-2 cells are shown in Table 1. Methanol extract is more toxic than aqueous extract (Fig. 3). The MNTCs of methanol was 195.313 µg/mL, while aqueous was 390.625 µg/mL, and for RBV was 0.98 µg/mL (Fig.  4). While methanol extract did not show any activity against RSV, on the contrary, the aqueous extract showed an important antiviral activity. The EC50 value of aqueous extract was 50.69 µg/mL, while the SI value was 97.54 (Fig. 5). The EC50 and SI values of RBV were 2.39 µg/mL and 46.19 µg/mL, respectively (Fig. 6).
In this study, the aqueous extract is even more effective than the standard drug RBV, whereas methanol extract was found to be ineffective against RSV.
Furthermore, it can be seen that the extracts are less toxic than RBV on HEp-2 cells, and CC50 values of aqueous extract and RBV are higher than EC50 values.       This is important for the reliability of an antiviral agent (Schinazi et al. 2009). Besides, Chattopadhyay et al. (2009) reported that if SI values are 10 or greater than 10, antiviral results determined at or above this SI value may indicate that the extracts may have potential antiviral activity. Ferula species are rich in biologically active compounds, some of which are coumarins, sesquiterpenes, sesquiterpene coumarins, sesquiterpene lactones, and daucan esters (Zhou et al. 2017). It was reported in many studies on phytochemicals that these secondary metabolites and extracts obtained from Ferula species can show anti-fertility, antifungal, antiinflammatory, antispasmodic, antitumor, antiviral, antiulcerogenic, cancer chemopreventive, digestive enzyme inhibition and hypotensive effects (Iranshahy & Iranshahi 2011). Ferula genus consists of 170 species in a wide region extending from Central Asia to the Mediterranean part (Mozaffarian 1996, Downie et al. 2000, and is represented in Turkey with 18 species of which 9 are endemic for the country (Davis 1972). Different parts of Ferula species are used in the treatment of various diseases such as neurological diseases, inflammations, dysentery, digestive system disorders, rheumatism, headache, arthritis and dizziness (Tamemoto et al. 2001). The anti-inflammatory, cytotoxicity and P-gp inhibitor, cancer chemo-preventive, antibacterial and antileishmanial activities of the components obtained from the genus Ferula are summarized (Nazari & Iranshahi 2011). The basic contents of essential oils obtained by hydrodistillation from dried roots of F. hormonis Boiss. were analysed by Gas Chromatography-Mass Spectroscopy (GC-MS) and the anti-HSV-1 activity of the essential oil was evaluated with the plaque inhibition test. The results showed that the plaque inhibition percentage of essential oil against HSV-1 was 81.4 (Mohamed et al. 2006).
In a study evaluating the anti-HSV-1 activities of sesquiterpene coumarins (badrakemin acetate, kellerin (2) and samarcandin diastereomer) isolated from the gum resin of F. assa-foetida, it has been shown that kellerin could significantly inhibit the cytopathic effects and reduce the viral titre of theHSV-1 DNA viral strain KOS at concentrations of 10, 5 and 2.5 µg/mL (Ghannadi et al. 2014). Antiviral activity of the methanol and water extracts of F. halophila was investigated against HSV-1(strain HF, ATCC VR-260) by the colorimetric XTT assay in vitro HSV-1/Vero cell systems by Duman (2016). According to his results, EC50 and SI values for methanol extract were 1035.29 µg/mL and 35.56, respectively, and EC50 and SI values for water extract were 264.45 µg/mL and 130.81, respectively. Similarly, EC50 and SI of aqueous extract (EC50: 50.64, SI: 97) in the present study are higher than methanol extract. According to Duman (2016) and our results, we can say that aqueous extracts are more effective than methanol extracts. Several compounds, including glucuronic acid, galactose, arabinose, and rhamnose (Kapoor 1990), sulfurcontaining derivatives (Kajimoto et al. 1998 However, it was determined that F. halophila methanol extract did not have antiviral activity against RSV, whereas the aqueous extract of the same plant had significant antiviral activity. This may be due to the presence of the compound or compounds that are soluble in water, which are insoluble in the methanol extract.

Conclusion
In this study, anti-RSV activities of methanol and aqueous extracts of F. halophila were investigated, and aqueous extract exhibited the highest antiviral effect. To find the compound that causes antiviral activity, it is necessary to analyse the aqueous extract in the following steps and to renew the antiviral tests. In this way, the active substance or substances would be determined and they would be used as new antiviral drugs. However, the methanol extract did not show any antiviral effect. Due to the high toxicity of the methanol extract on HEp-2 cells, the use of lower concentrations in the antiviral test might have reduced the antiviral effect, or the antiviral compounds had not been completely dissolved in the methanol extract.
Ethics Committee Approval: Since it was a systematic review, its approval to the ethics committee was not required.