Identification of potential antiviral compounds from Egyptian sea stars against seasonal influenza A/H1N1 virus

Abstract

Background

One of the most dangerous problems that the world faced recently is viral respiratory pathogens. Marine creatures, including Echinodermata, specially Asteroidea class (starfish) have been extensively studied due to their miscellaneous bioactivities, excellent pharmacological properties, and complex secondary metabolites, including steroids, steroidal glycosides, anthraquinones, alkaloids, phospholipids, peptides, and fatty acids. These chemical constituents show antiviral activities against a wide range of viruses, including respiratory viruses.

Results

The present study aimed at the identification of potential antiviral compounds from some starfish species. The bioactive compounds from Pentaceraster cumingi, Astropecten polyacanthus, and Pentaceraster mammillatus were extracted using two different solvents (ethyl acetate and methanol). The antiviral activity against influenza A/H1N1 virus showed that ethyl acetate extract from Pentaceraster cumingi has the highest activity, where the selective index was 150.8. The bioactive compounds of this extract were identified by GC/MS analysis. The molecular docking study highlighted the virtual mechanism of binding of the identified compounds towards polymerase basic protein 2 and neuraminidase for H1N1 virus. Interestingly, linoleic acid showed promising binding energy of −10.12 Kcal/mol and −24.20 Kcal/mol for the selected two targets, respectively, and it formed good interactive modes with the key amino acids inside both proteins.

Conclusion

The molecular docking analysis showed that linoleic acid was the most active antiviral compound from P. cumingi. Further studies are recommended for in-vitro and in-vivo evaluation of this compound against influenza A/H1N1 virus.

1. Introduction and Background

Respiratory and airborne viruses are the most recurrent causative agents of disease in humans, with significant impact on mortality and morbidity worldwide.¹ Influenza A viruses are the source of all influenza pandemics and consist structurally of hemagglutinin (HA) and neuraminidase (NA) on the surface, M2 transmembrane ion channel protein, M1 that is lining the core of the virus particle where the viral ribonucleoprotein complex (a complex of viral RNA, NP, PB2, PB1 and PA protein) is residing.² Influenza A/H1N1 virus is classified as a seasonal respiratory tract pathogen. In 2009, the H1N1 influenza virus was generated in Mexico as the product of natural genetic reassortment of influenza virus from human, swine, and birds, and declared in few weeks the first global threat pandemic of this century.³

Since the emergence of the first H1N1 pandemic of influenza in 1918, progress in developing anti-influenza drugs is limited. Recently, the drug-resistance rate of circulating influenza viruses increasingly reported for the M2-channel blockers (amantadine and rimantadine).⁴ This minimized the treatment options in moderate to severe influenza infections to neuraminidase inhibitors (Oseltamivir and Zanamivir) and recently developed RdRp inhibitors (Baloxavir marboxil). Therefore, patients with H1N1 infection receive one of these three antivirals: Oseltamivir, Baloxavir marboxil and Zanamivir.⁵ But all of these antivirals must be taken within 48 h of the onset of flu symptoms,⁶ and the rate of drug resistance started recently to increase against treatments with these limited medications.⁴ A study performed by Hu et al.,⁴ found the presence of genetic variations in H1N1/2009, in which substitutions take place in the PB2 and HA proteins.⁷ The evolution of these viruses would require developing effective tools for controlling them worldwide. The marine environment is very important source of bioactive compounds. There are many natural products extracted from many organisms, especially echinoderms,⁸ that showed antiviral activity against a wide range of viruses. Sea stars are star-shaped organisms that belong to phylum echinoderms and class Asteroidea, as these organisms are exposed to relatively high concentrations of viruses, so it’s surviving depending on the synthesis of antiviral peptides⁹ and other natural compounds such as saponins, peptides, polysaccharides, lipids, and proteins which also possess activity against human viruses.⁸ A previous study performed by Peng et al.,⁴⁵ reported that eight steroid compounds were identified from the ethanol extract of the whole body of starfish Asterina pectinifera. Four of these compounds showed antiviral activity against HSV-1 virus. These compounds were identified as (25S)-5a -cholestane-3b, 4b, 6a, 7a, 8, 15a, 16b, 26-octol, (25S)-5a cholestane-3b, 4b, 6a, 7a, 8, 15b, 16b, 26-octol, cholest-7-en-3-sodium sulfate, and (24S)-5acholestane 3b, 6a, 8, 15a, 24-pentol. So, the aim of the present study was to extract and identify potential antiviral compounds from Egyptian sea stars against influenza A/H1N1 viruses. The identified compounds were docked against influenza H1N1-PB2 and H1N1-NA targets.

2. Methods

2.1. Samples collection and preparation

Three different samples of starfish (sea stars) were collected by fisher by hands from Ras Shuker, Ras Ghareb, and Gulf of Suez, Egypt, in February 2021. The collected samples were taxonomically identified as Astropecten polyacanthus, Pentaceraster cumingi and Pentaceraster mammillatus by Dr. Mohamed Abu El Regal (Professor of Biological Oceanography, Marine Science Department, Faculty of Science, Port Said University). The collected starfish samples were washed with fresh water to remove associated debris. The internal organs were removed using a sharp knife. After that, the dorsal surface of the starfish’s body known as (Ossicles) were broken into small pieces using bone grinder and stored at temperature −20 °C until use.

2.2. Preparation of starfish crude extracts

Hundred grams of grinded Ossicles from each type were added in a flask and sequentially extracted with 100 ml of ethyl acetate (HPLC grade, Daejung Chemicals & Metals Co., Korea) and methanol (HPLC grade, Advent, India) for 24 h under shaking. The crude extracts were filtered using Whatman filter paper No. 1. The filtrate was concentrated to dryness by a rotary evaporator (BIBBY STERILIN LTD STONE STAFFORDSHIRE ENGLAND ST15 0SA, RE200). The paste of each type was collected in a brown bottle and stored at −20 °C until use.

2.3. Determination of antiviral activity

2.3.1. Determination of half-maximal cytotoxic concentration (CC₅₀)

To assess the half maximal cytotoxic concentration (CC₅₀), stock solutions of the extracts were prepared in 10 % DMSO in ddH₂O and diluted further to the working solutions with DMEM. The cytotoxic activity of the extracts was tested in MDCK/Vero E6 cells by using CPE reduction assay as described by Mahmoud et al.⁴¹ Briefly, the cells were seeded in 96 well-plates (100 µl/well at a density of 3 × 10⁵ cells/ml) and incubated for 24 h at 37 °C in 5 % CO₂. After 24 h, cells were treated with various concentrations of the extracts in triplicates. 72 h later, the supernatant was discarded, and cell monolayers were fixed with 10 % formaldehyde for 1 h at room temperature (RT). The fixed monolayers were then dried and stained with 50 µl of 0.1 % crystal violet for 20 min on bench rocker at RT. The monolayers were then washed, dried and the crystal violet dye in each well was then dissolved with 200 µl methanol for 20 min on bench rocker at RT. Absorbance of crystal violet solutions was measured at λmax 570 nm as a reference wavelength using a multi-well plate reader (Anthos Zenyth 200rt plate reader). The percentage of cytotoxicity compared to the untreated cells was determined with the following equation. The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50 % cytotoxicity.

$$\%\mathrm{c}\mathrm{y}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{(\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{o}\mathrm{u}\mathrm{t}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}-\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t})\mathrm{X}100}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{o}\mathrm{u}\mathrm{t}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}$$

2.3.2. Determination of half-maximal inhibitory concentration (IC₅₀)

The IC₅₀ values for extracts were determined as described by Mostafa et al.⁴⁴ Briefly, in 96-well tissue culture plates, 2.4 × 10⁴ MDCK/Vero E6 cells were distributed in each well and incubated overnight at a humidified 37 °C incubator under 5 % CO² condition. The cell monolayers were then washed once with 1x PBS. An aliquot of the influenza A/PR/8/34 (H1N1) virus containing 100 TCID₅₀ was incubated with serial diluted concentrations of the tested compounds and kept at 37 °C for 1 h. The MDCK/Vero E6 cells were treated with virus/compounds mix and co-incubated at 37 °C in a total volume of 200 µl per well. Untreated cells infected with virus represent virus control, however cells that were not treated and not infected were cell control. Following incubation at 37 °C in 5 % CO₂ incubator for 72 h, the cells were fixed with 100 μl of 10 % paraformaldehyde for 20 min and stained with 0.5 % crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 μl absolute methanol per well and the optical density of the color is measured at 570 nm using Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC₅₀ of the extract is that required to reduce the virus-induced cytopathic effect (CPE) by 50 %, relative to the virus control and calculated by the following equation.

$$\%\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\frac{\mathrm{v}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}-\mathrm{v}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{\mathrm{v}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\mathrm{x}100$$

2.3.3. Statistical analysis and calculation of CC₅₀ and IC₅₀

All antiviral experiments were presented in three replicates. Statistical analysis, calculations, and graphical data presentation were performed using GraphPad Prism 5.01 software. The data is presented as the average of the means. The half maximal of cytotoxicity (CC₅₀) and the half-maximal of inhibitory concentration (IC₅₀) curves symbolize the nonlinear fit of “normalize” of “transform” of the obtained area; their values were assessed using GraphPad prism as “best fit values”.

2.4. TLC analysis

TLC analysis was carried out as a primary comparison between the components of starfish extracts. The precoated silica TLC plates were used. The mobile phase was a mixture of ethyl acetate: methylene chloride with different percentages: 100%: 0%, 75%: 25%, 50%:50%, 25%: 75%, and 0%: 100%). The TLC plate was then air-dried and observed under the UV-TLC reader.

2.5. GC/MS analysis

Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) equipped with capillary column TG-5MS (30 m x 0.25 mm x 0.25 m film thickness) was used to evaluate the crude extracts. The column oven temperature was primarily held at 50 °C and then increased by 5 °C /min to 230 °C keep up for 2 min. increased to the final temperature 290 °C by 30 °C /min and keep up for 2 min. The injector and MS transfer line temperatures were kept at 250, 260 °C, respectively; Helium was used as a carrier gas at a constant flow rate of 1 ml/min. The solvent delay was 3 min and diluted samples of 1 µl were injected automatically using Autosampler AS1300 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 40–1000 in full scan mode. The ion source temperature was set at 200 °C. The retention times and mass spectra of the components were compared to those in the NIST 11 and WILEY 09 mass spectral databases, allowing for their identification. Mass spectrum patterns were compared to databases in ChemSpider to determine structures.

2.6. Molecular docking

Chimera-UCSF and AutoDock Vina were used for molecular modelling investigations on Linux-based machines at Suez Canal University. Binding sites within proteins were identified by measuring the dimensions of grid boxes surrounding the co-crystallized ligands, a process that was facilitated by Maestro's structural preparation and optimization of both proteins and compounds. Using AutoDock Vina, the compounds under study were docked against the protein structures of H1N1 virus polymerase basic protein 2 (PB2) (PDB = 4P1U) and neuraminidase (PDB = 6PH0)¹⁰ following routine work.¹¹ The protein and ligand structures were optimized and energetically favored Maestro. Molecule docking data were evaluated by binding activities in terms of binding energy and ligand-receptor interactions. The data was then visualized using Chimera-UCSF.¹²

3. Results

3.1. Antiviral activity against influenza a (subtype H1N1)

The ethyl acetate extracts from the three species of starfish showed higher selective index values than the methanol extracts of the same starfishes (Table 1). The CC₅₀ and IC₅₀ values for the methanol and ethyl acetate extracts of the three starfishes are shown in Fig. 1, Fig. 2, respectively. The highest selective index (SI) (150.833) was obtained from ethyl acetate extract of Pentaceraster cumingi. This was followed by (42.3) and (28.1) obtained from the ethyl acetate extracts of A. polyacanthus and P. mammillatus, respectively. This high value indicates the selectivity and safety of the antiviral components of this extract.

Table 1.

CC₅₀, IC₅₀, and selective index (SI) of the three species of starfish extracts.

Organism	Solvents	CC50 (mg/ml)	IC50 (mg/ml)	SI
Pentaceraster mammillatus	Ethyl acetate	10.4	0.37	28.108
	Methanol	5.87	1.2	4.89
Pentaceraster cumingi	Ethyl acetate	9.05	0.06	150.833
	Methanol	9.03	6.13	1.473
Astropecten polyacanthus	Ethyl acetate	1.27	0.03	42.333
	Methanol	1.3	1	1.3

Fig. 1.

CC₅₀ and IC₅₀ for the methanol extracts of three starfish species: A. polyacanthus, P. cumingi, and P. mammillatus.

Fig. 2.

CC₅₀ and IC₅₀ for the ethyl acetate extracts of the three starfish species: A. polyacanthus. P. cumingi and P. mammillatus.

3.2. TLC analysis

The best separation was obtained using ethyl acetate: methylene chloride with a ratio of 75%:25% and 50%:50% as mobile phases. At the first mobile phase, A. Polyacanthus showed four bands at Rf values 0.91, 0.75, 0.5, and 0.06; P. cumingi showed three bands at Rf values 0.98, 0.83, and 0.01, while P. mammillatus showed six bands at Rf values 0.91, 0.83, 0.81, 0.38, 0.16 and 0.01. Although similar Rf values appeared in the different starfish extracts, these compounds are different since they showed different fluorescent colors under Uv light (Fig. 3).

Fig. 3.

TLC analysis of the ethyl acetate extract of the three starfishes (Ap: A. Polyacanthus, Pc: P. cumingi and Pm: P. mammillatus) eluted with different mixtures of ethyl acetate: methylene chloride as mobile phases: A: 100%:0%, B: 75%:25%, C: 50%:50%, D: 25%:75% and E: 0%:100%, respectively.

3.3. Gas chromatography

The presence of the antiviral compounds in the ethyl acetate extract rather than the methanol extract of P. cumingi indicate the non-polar nature of the bioactive compounds. Therefore, the crude extract was analyzed by GC/MS. The analysis showed the presence of free fatty acids, fatty acids methyl ester, and steroids, where twelve compounds were identified (Table 2).

Table 2.

Identified compounds in the ethyl acetate extract of P. cumingi by GC/MS analysis.

Peak No	Rt (min)	Compound Name	Molecular Formula	MW
1	7.31	2-Phenyltridecane	C19H32	260
2	7.47	Methyl 14-methylpentadecanoate	C17H34O2	270
3	8.40	Estra-1,3,5(10)-trien-17á-ol	C18H24O	256
4	9.26	Octadecanoic acid, methyl ester	C19H38O2	298
5	9.66	Linoleic acid	C18H32O2	280
6	11.17	12-Methyl-E,E-2,13-octadecadien-1-ol	C19H36O	280
7	11.81	9,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester	C21H38O4	354
8	12.70	3′,8,8′-Trimethoxy-3-piperidyl-2,2′-binaphthalene-1,1′,4,4′-tetrone	C28H25NO7	487
9	13.63	Ethyl iso-allocholate	C26H44O5	436
10	16.55	Cholesta-8,24-dien-3-ol,4-methyl-, (3á,4à)-	C28H46O	398
11	17.27	25-Norcholesterol, 25-oxo	C26H42O2	386
12	17.65	Ergosta-7,22-dien-3-ol, (3á,22e)-	C28H46O	398

3.4. Molecular docking

Molecular docking studies highlighted the promising virtual activity of identified compounds, with good binding affinities with good binding energies towards both proteins of polymerase basic protein 2 and neuraminidase. As summarized in Table 3, most compounds exhibited good interactions with the key amnio caids, like the co-crystallized ligands of two proteins.

Table 3.

Ligand-receptor interactions of the docked compounds towards polymerase basic protein 2 (PB2) and neuramindase using AutoDock Vina software.

Compound structure	Polymerase basic protein 2 (PB2) (PDB = 4P1U)		Neurminidase (NA) (PDB = 6PH0)
	Binding energy (Kcal/ mol)	H-bonds*	Binding energy (Kcal/ mol)	H-bonds*
Co-crystallized ligand		3H-bonds with Lys 76, Arg 55, Glu 61		7H-bonds with Arg 118, Asp 151, Arg 152, Trp 179, Arg 293 and Arg 368
	−14.64	–	−17.72	–
	−11.42	2H-bonds with Arg 55	−19.90	2H-bonds with Arg 118 and Arg 368
	48.43	2H-bonds with Lys 76 and Phe 104	−17.75	2H-bonds with Arg 293 and Arg 368
	−3.86	2H-bonds with Arg 55	−18.49	1H-bond with Asp 151
	−0.12	2H-bonds with (2 HB with Lys 76 and 1 HB with Glu 61)	−24.20	3H-bonds (2 HB Arg 368 and 1 HB Arg 118)
	−13.63	1H-bond with Arg 55	−25.13	2H-bonds with Arg 293 and Arg 368
	−2.17	1H-bond with Glu 61	−15.90	2H-bonds with Arg 118 and Asp 151
	−21.71	1H-bond with Arg 55	−6.86	3H-bonds (2 HB Arg118 and 1 Arg 368)
	−11.96	1H-bond with Arg 55	−15.46	3H-bonds (2 HB with and 1 HB with Arg 368 Arg 293)
	−16.07	1H-bond with Arg 55	−17.91	2H-bonds with Arg 293 and Arg 368
	5.22	1H-bond with Arg 55	−15.97	1H-bond with Asp 151
	−10.11	1H-bond with Arg 55	40.94	1H-bond with Trp 179

^*Docking calculations are carried out using AutoDock Vina and interactions were visualized using Chimera-UCSF.

As seen in Fig. 4A, Linoleic acid showed promising interactions when docked inside Polymerase basic protein 2 with binding energy − 0.12 Kcal/mol, with three H-Bonds as two hydrogen bond acceptors with Lys 76 and one H-bond donor with Glu 61 as key amino acids. As seen in Fig. 4B, Linoleic acid showed promising interactions when docked inside Neuraminidase with binding energy 24.20 Kcal/mol, with three H-Bonds as hydrogen bond acceptors Arg 368 and Arg 118.

Fig. 4.

Binding disposition and ligand-receptor interactions of the co-crystallized ligand and the docked compound (Linoleic acid) inside A: Polymerase basic protein 2 (PB2) (PDB = 4P1U) and B: Neurminidase (PDB = 6PH0) using Chimera-UCSF software.

4. Discussion

In the present study a high selective index value (150.8) obtained from ethyl acetate extract of Pentaceraster cumingi indicates the high safety and selectivity of this extract components. This value is more greater than previously tested antiviral natural compounds or extracts. Previous study performed by Maria John et al.,⁴² in which, the authors screened the ethnic medicinal plants of South India against influenza (H1N1) showed that the highest selective index (21.97) was obtained from methanol extract of Strychnos minor in comparison with Oseltamivir as a control drug with a selective index of 15.51. The study performed by You et al.,⁴⁷ on testing the catechin and gallic acid as antiviral against H1N1 showed that the selective index was 5.6 and 8.6, respectively. Among the identified compounds, 2-Phenyltridecane has also been found in many plants like Acca sellowiana (PubChem, 2022) Ipomoea tricolor and Ipomoea fistulosa, which showed antioxidant activity.¹³ 14-Methylpentadecanoic acid, was also found in Actinomadura macrotermitis,¹⁴ Streptomyces endophyticus, Streptomyces indicus and Petrosia pellasarca (PubChem, 2022), and according to Siddiqui et al.,⁴⁸ the 14-Methylpentadecanoic acid compound was identified in the ethanolic extract of Azadirachta indica (neem) and showed pesticidal activity against Anopheles stephensi Liston. Estra-1,3,5(10)-triene-3,17-diol) was also found in Arabidopsis thaliana and showed inhibition of human aldehyde oxidase–catalyzed 6-deoxypenciclovir oxidation.¹⁵ Stearic acid was found in various animals and plants fats and are major component of cocoa butter and shea butter (Pubchem, 2022). Jubie et al.,⁴⁰ extracted stearic acid from microalga Spirulina platensis, which showed antidepressant and antimicrobial activities against Escherichia coli and Staphylococcus aureus in addition to moderate antibacterial activities against Bacillus subtilis and P. aeruginosa. Linoleic acid, was also found in general in plant oil. Linoleic acid is used in the biosynthesis of prostaglandins and cell membranes. It is also found in dairy products or beef and exerts specific effects on adipocytes, in particular, reducing the uptake of lipid by inhibiting the activities of lipoprotein lipase and stearoyl–CoA desaturase also, appear to be active in inhibiting carcinogenesis in animal models.¹⁶ 12-Methyloctadeca-2,13-dien-1-ol was also found in Citrullus colocynthis and used as a biodiesel.¹⁷ A study by Shiva et al.,⁴⁶ showed that 9,12-Octadecadienoic acid is from the component of Vajra kandi maathirai (VKM) drug, which possess activity against SARS-CoV-2. The compound 3′,8,8′-Trimethoxy-3-piperidyl-2,2′-binaphthalene-1,1′,4,4′-tetrone was also identified in the ethyl acetate extract of P. crustosum, which possess effect against some pathogenic bacteria. It showed a broad spectrum effect and had potential activity against cancer.¹⁸ Ethyl iso-allocholate was previously extracted from Karungkavuni rice and possess antimicrobial activity.¹⁹ It was also found in T. foenum-graecum L. seeds and tested against A549 lung cancer cells in vitro and in vivo. This compound rendered the highest cytotoxicity potential.²⁰ The compound 4-Methylcholesta-8,24-dien-3-ol was found also in Phytophthora Spp.²¹ The compound 25-Norcholesterol, 25-oxo was previously found in the extract of Skeletonema costatum,²² which is used as a substitute in edible oils.²³ Finally, Ergosta-7,22-dien-3-ol, (3á,22E)-) was previously found in the methanolic extract of Chenopodium’s leaves.²⁴

PB1, PB2, and PA are the three viral proteins that make up the RdRp. PB2 has endonuclease activity,²⁵ which binds to the 5′ methylated caps of cellular mRNAs and cleaves the cellular mRNAs’ 10 to 15 nucleotides 3′ to the cap structure. The viral RdRp used cellular capped RNA fragments to prime viral transcription²⁶ by a “cap-snatching” mechanism.²⁷ Supplement for the importance of the role of the PB-2, in addition to its nuclear localization, interestingly accumulates in the mitochondria of influenza viruses which represent 56 % of cells expressing the PB2 protein in subtype H1N1.²⁸ PB2 protein reduces MAVS-mediated beta interferon (IFN-β) expression and interacts with MAVS (also known as IPS-1, VISA, or Cardif), which is found on the outer membrane of the mitochondria.29, 30, 31, 32 This interaction results in an increase in virulence of viral load.²⁸

According to McAuley et al.,⁴³ neuraminidase (NA) is involved in several steps of the infection process including but not limited to virus invasion, receptor binding, virus internalization, catalytic activity, and substrate specificity; its primary role is in the late stages of infection. Sialic acids are removed by viral NA from cellular receptors and from newly generated HA and NA on nascent virions as part of the host cell's glycosylation processes.³³ By cleaving sialic acids, NA prevents virion aggregation and blocks the virus from binding back to the dying host cell via the HA, allowing for the effective release of virion offspring and propagation to other cell targets.³⁴ It has been speculated that the sialidase activity of NA promotes the virus in entering cells by accelerating the breakdown of sialic acids provided by decoy receptors, such as mucins,³⁵ giving NA a possible pivotal function in viral entry. Additional evidence for an involvement of NA in a virus entry phase comes from research indicating a decrease in infection of cells in the presence of NA-blocking medications.36, 37, 38, 39

5. Conclusion

The sea star Pentaceraster cumingi ethyl acetate extract contains antiviral compounds that showed activity against influenza H1N1 virus. Among the identified compounds, linoleic acid showed the best molecular docking results toward polymerase basic protein 2 and neuraminidase of H1N1 virus. Further studies are required for in vitro and in vivo evaluation of linoleic acid against H1N1 virus.

Funding

This research was partially funded by Egyptian National Research Centre (NRC)-funded projects (TT110801 and 12,010,126 to AM). The funders had no role in study design, data collection, analysis, publication decision, or manuscript preparation.

Authors' contributions

Nadia I. Okasha (Corresponding author).

Extraction of antiviral compounds, docking analysis, paper writing and submission

Mohamed Abdel Rahman.

Experiment design, samples collection and identification.

Mohammed S. Nafie.

Molecular docking analysis and data interpretation, paper writing and revision

Noura M. Abo Shama.

Antiviral assay.

Ahmed Mostafa.

Antiviral assay.

Dalia A. El-Ebeedy.

Experiment design, antiviral assay data interpretation, paper revision

Ahmed Z. Abdel Azeiz.

Experiment design, mass spectra data interpretation, paper writing and revision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.