ANTIVIRAL ACTIVITY OF EXTRACT AND PURIFIED COMPOUND FROM RED MACROALGAE ASPARAGOPSIS TAXIFORMIS AGAINST H5N1 VIRUS

Aim and objective: The discovery and development of new natural antiviral compounds which exhibit various antiviral activities are required. The aim of this investigation is to assess the potential use of the red seaweed Asparagopsis taxiformis as a new source of anti H5N1 agent. Methods: The seaweed was collected from Marsa Matrouh, Mediterranean Sea, Egypt during spring season, the effects of successive extracts and the pure compounds from the investigated alga on H5N1 virus were performed using plaque reduction assay. In addition, the mechanism of action of promising extract on the virus adsorption and replication was determined. Chromatographic and spectroscopic analyses were used for the identification of chemical structure of active compound(s) isolated from the studied seaweed. Results: The obtained results showed that petroleum ether and water algal extracts exhibited high antiviral activity (>99.9%) and the mode of action of extracts was not correlated with virus replication but with its adsorption process.The isolated pure compound was identified as 6-methyl-Δ-stigmasterol-2, 3 di acetate and its antiviral activity (for H5N1) was tested. Pure compound showed antiviral activity reached 56% at 100 μg/ml. Conclusion: The obtained results suggests that crude extracts and isolated active compound from A. taxiformis has the capacity to protect people against pandemic H5N1preventing virus adsorption to the human host cells. Recommendation for testing the extracts and pure compounds from the studied seaweed as potential inhibitor of COVID-19.


INTRODUCTION
In general, IAV or Influenza A virus is a healthy threat to the human community. This virus has high ability for infecting various hosts e.g: horse's waterfowl, dogs, cats, humans, and other mammals. H 5 N 1 virus induces public health and economic problems because of direct contact with birds and indirect contact with contaminated media transmits the virus to people 1 . New antiviral drugs are needed to nullify the percentage of mortality caused by virus infection. Neuraminidase inhibitors (NALs) drugs were worldwide used in curing the IAV infected people, but its use nowadays were less effective 2, 3 . Therefore, the findings or development of natural anti-influenza virus drugs is recommended. Macroalgal species well known or recognized as ecosystem engineers and/or foundation organisms in different environment or habitats since they convert the simple surfaces into structured environments that support many of living species 4 . Cardozo et al., reported that algal products are essential components in many industries 5 . Algae synthesize many bioactive substances that exhibit different biological activities 6 . In relation to the activity of antiviral and marine seaweed species, the algal species have high ability for producing and providing novel leads against various viruses e,g: H 5 N 1 , H 1 N 1 , hepatitis, HSV etc become less sensitive to the existing drugs as reported by Vo

Collection of alga
The alga was harvested from El-Garam beach of Marsa Matrouh. The alga belong to Bonnemaisoniaceae (Asparagopsis sp, supra littoral and intertidal zones, 11-13 cm). Algal thalli were washed from sand and debris by sea water then by fresh water. After preparation of herbarium specimens of the alga, the alga was identified as A. taxiformis by the phycologist Prof. Dr. Sanaa M. Shanab, Botany and microbiology Department, Faculty of Science, Cairo University.

Quantitative analysis of alga secondary metabolites Total Glycosides
The total glycosides content in A. taxiformis was extracted and spectrophotometrically determined (as glucose) using the method described by Dubois et al., 8 . Total saponin Saponins were estimated by the method used by Ebrahimzadeh and Niknam method 9 . Total Alkaloids Alkaloids were determined by the method used by Sabri et al. 10 . Total organic acids Plant acids in macroalga were determined using titratable acidity method according to Harborne 11 . Phenolic compounds Total phenolics contents in the studied seaweed were estimated by the method reported by Meda et al., and the standard curve was established using Ferulic acid 12 .

Preparation of algal extracts
Fifty grams of the seaweed was extracted by successive organic solvent of increasing polarity (from the nonpolar hexane to the highly polar water). All extracts were dried under vacuum using rotary evaporator and weighed according to Rosenthaler 13 . Antiviral activity Antiviral bioassay was prepared according to the method of Silva et al. 14 . A known weight of each seaweed extract was dissolved in one ml of 10 % DMSO, to give a final concentration of100 µg/µ1 and served as stock solution. These solutions were sterilized by the addition of a commercial antibiotic antimycotic mixture (10,000 U Penicillin sodium or 25 µg amphotericin B, 10.000 µg streptomycin sulphates). Then a sterility test was carried out in nutrient agar. Cells MDBK cells: The cell lines of MDBK were obtained and propagated in Virology Laboratory, National Research Center (NRC).

Media and supplements
Media: The Minimum essential medium and RBMI 1640 medium were prepared from powdered stock and pH was adjusted to 7.3 with NaHCO 3 . The prepared media were sterilized by filtration through nitrocellulose membrane filter (pore size of 0.2 µm). Sterility test was carried out on nutrient agar plates. Supplements Firstly a cell dissociation solution (0.15% Trypsin, 0.04% versene mixture) was prepared as follows: Phosphate buffered saline (0.15 M, pH 7.5, PBS) was sterilized by 0.22 µm nitrocellulose membranes, then used for washing of cell monolayer sheets and in preparation of cell dissociation solution. The dissociation solution was composed of 1.5 g of trypsin powder (1:250, Sigma-Aldrich) dissolved in 500 ml PBS and incubated overnight at 4ºC with stirring. Total 0.04 % Versene solution, Fetal bovine serum (Sigma-Aldrich) and Antibiotic-antimycotic mixture (10,000 U Penicillin sodium or 25 µg amphotericin B, 10.000 µg streptomycin sulphates, Sigma-Aldrich) were also prepared. 0.04 gram tetra sodium salt of ethylene diamine tetra acetic acid (EDTA) was dissolved in 500 ml of 1.5 M PBS (pH 7.5) and mixed with equal volume of trypsin-versene mixture, this solution was adjusted to pH 8.4 by 7.5% NaHCO 3 solutions and sterilized by filtration through 0.22 µm nitrocellulose membrane. All the reagents were stored at -20 ºC until used.

Reference viruses Avian virus (H 5 N 1 ):
The virus was kindly given by Virology Laboratory, NRC. It was propagated and titrated on MDBK cells as indicated by Silva et al. 14 .

Materials for plaque infectivity assays:
Over layer medium was prepared as follows: Double strength concentration of both types of media was prepared and sterilized by filtration. Supplements were added to concentration of 2 % antibiotic-antimycotic. Total 2 % Agarose solution was prepared by cooking 2 % agarose in deionized water and sterilized by autoclaving. Ten % formalin in H 2 O was used as fixative solution. Staining solution was made by dissolving 1% crystal violet in 20% methanol (w/v) and then filtered through Whatman no.1 paper. Plaque infectivity reduction assay Anti-H 5 N 1 assay A 6-well plate was cultivated with MDBK culture (10 5 cell/ml) and incubated for 2 days at 37ºC. The culture of H 5 N 1 virus was diluted to give 10 7 PFU/ml as final concentrations and mixed with the algal extract and incubated overnight at 4ºC. Growth medium was removed from the multiwell plate and the viruscompound mixture was inoculated (100 µl/well). After 1h contact time, the inocula were aspirated on MDBK culture and 3ml of MEM with 1% agarose were overlaid the cell sheets. The plates were left to solidity and incubated at 37ºC until the development of virus plaques. Cell sheets were fixed in 10% formalin solution for 2hr and stained with crystal violet solution. Control virus and cells were treated identically without chemical compounds. Virus plaques were counted and the percentage of inhibition was calculated 14,15 .

Mode of action
Crude algal extracts were used for monitoring virus inhibition mechanisms through both viral replications 15 and viral adsorption assays 16 .

Separation of active gradient
Ten gram of crude petroleum ether extract was analysed using GL column packed with VLC silica gel H. Elution was performed by hexane, chloroform and their combinations. Fractions were separately collected, evaporated then redisolved in 5 ml ethanol and used by TLC chromatogram (elution system was ethyl acetate, 97:3 v/v). Isolated spots were visualized using UV light at 365 and 245 nm then colored by anizaldehyde reagent. Fraction No 10 produce the potent pure compound, which was further identified using chromatographic and spectroscopic analyses as LC/MS, UV-Vis spectrophotometer, FTIR, NMR, CHN analyses.

Secondary metabolites
Different algal phytochemical contents were illustrated in Figure 1. This result revealed that the A. taxiformis extraction contained the high amount from secondary metabolites as Alkaloids followed in descending order by Glycosides, Plant acids, Terpenoids and Phenolic compounds, which were 2.66, 2.15, 0.48, 0.13 and 0.11%, respectively.

Antiviral activity
The antiviral activity of successive Asparagopsis algal extracts was evaluated against avian virus (H 5 N 1 ) virus which used as a model of RNA virus. Table 1 and Figure 2 showed the antiviral activity of different extracts against H 5 N 1 by using plaque reduction assay.
The obtained results showed that the treatment of H5N1 with different extracts at concentration 20 and 40 µg/ml significantly inhibited % of H 5 N 1 virus (ranged 0.0-100%). These means that successive extracts of alga extract exhibited remarkable antiviral activity. Also, the obtained data revealed that the extracts affected viral inhibition in a dose and chemical composition dependent manner (Table 1). Results illustrated that the activity was variable between the extracts according to the polarity of these extracts. In which the maximum inhibition (virus reduction) was occurred in the following extracts: pet ether and water extracts by 100% ethyl acetate, by 55.5% at 40 µg/ml. These results were in agreement with those reported by Bouhlal et al., 17 who illustrated that aqueous extracts of different red seaweeds (including A. armata showed antiviral replication activity against Herpes simplex virus type 1 with EC 50 range from 2.5 to 75.9 μg/ml. Different extracts of air-dried Ulva lactuca (methanol, ethanol, chloroform, ethyl acetate and diethyl ether) were tested for biological activity and analysed by TLC. A complex of 6 components was tested for antiviral activity of influenza virus (H1N1). An inhibitory effect was recorded on both viral reproduction and infectious capacity 18 . Spirulina maxima showed an antiviral activity against herpes simplex virus type 2 as reported by Hernandez-Corona et al., 19 , who mentioned that methanol-water extract (3:1) have the greatest activity which may be due to the polar substances in the extract. It was suggested that the negatively charged sulfated polysaccharides interacted with positively charged cell surface of the virus so preventing its penetration to the host cell 20 .

Mechanism of algal extracts as antiviral activity The effect of algal extract on virus replication
In these experiments the activities of algal extracts against H 5 N 1 and the clinical strain were evaluated by the plaque reduction assay. No effect of algal extract on viral replication was recorded but it affects virus H 5 N 1 adsorption on the host cell ( Figure 3 and Figure  4).  dotted wells=obvious virus growth.

The effect of algal extract on virus adsorption
The inhibitory effect of algal extracts on virus adsorption to host cell was measured by monitoring the attachment of infectious H 5 N 1 virions on to host cells in the presence of extracts. As shown in Figure 3 and Figure 4, extracts inhibited the cell-associated infectivity by 100% of the control levels. These results go parallel with those reported by Carlucci et al., who showed that the sulphated galactan in the red algal extract inhibited the adsorption of herpes simplex virus (HSV-1 and HSV-2). In addition, the cyanovirin-N (CV-N) from the cyanobacterium Nostoc sp inhibited HIV-2 through the interaction with glycoprotein (gp120) of the viral envelope 21 .

Isolation and identification of the bioactive compounds
During the isolation of the active compounds from A. taxiformis alga, the non-polar extract (petroleum ether extract) was more effective than other organic solvent extracts as antiviral activity as shown in Table 1.
Further fractionation of petroleum ether extract yielded pure compound; the obtained compounds were tested for antiviral activity against H 5 N 1 virus. The result showed that this pure compound had antiviral activity by 56% at 100 µg/ml as shown in Figure 5. These results may be attributed to the presence of various active groups in the isolated compound (6-methyl-Δ22-stigmasterol-2, 3 di acetate) such as Acetate group, double bonds in the chemical structure of this compound and its conformational structure that increase from the ability of this compound to react and bind with virus protein and prevent its adsorption into specific receptor. The chemical structure of active ingredients isolated from A. taxiformis Figure 6 presented the suggested chemical structure configuration of the active constituents of the algal petroleum ether. The proposed configuration satisfies and complies with the analytical identification characteristics shown by the CHN Elemental Analyzer, UV, IR, spectroscopic and chromatographic analyses used. Sub-fraction with TLC Rf value of 0.13, was analyzed by HPLC, LC-MS and GC-MS. The results revealed the presence of 3 compounds of which, one major constituent was found as main compounds (> 96%). As can be seen in the IR spectra (Fig.7), the intense bands in region between 2935 and 2850 and at 1660 cm-1 was shows due to presence of -CH 2 -and -CH 3 groups and double bond. The -OH group of steroid has and intense band in region between 3000 and 3360 cm- . This showed the presence of steroidal skeleton. According to the obtained data the chemical structure of isolated compound was elucidated as 6methyl-Δ22-stigmasterol-2, 3 di acetate.

CONCLUSION
The red alga A. taxiformis was evaluated in this study as a new source of antivirus against H 5 N 1 . Successive extractions with organic solvents of increasing polarities were performed [hexane, petroleum ether, ethyl acetate, methylene chloride: methanol (1:1v/v), water] using concentrations 20 and 40 µg/ml. Petroleum ether and water extracts showed the highest antiviral activity (>99.9%) using plaque reduction assay. Fractionation of the nonpolar petroleum ether extract yielded a pure active compound of steroidal skeleton with antiviral activity against H 5 N 1 .It may be due to the presence of different active groups as acetate group and double bonds in the chemical configuration of the compound (6-methyl-422-stigmasterol-2,3di acetate) which increase the ability of the compound to react and bind with the virus protein envelope and so prevent its adsorption on specific receptors. The mode of action of algal extract and the active compound was shown to be through inhibition of virus adsorption and not its replication.

CONFLICT OF INTEREST
No conflict of interest associated with this work.