Antiviral activity of Humulus lupulus (HOP) aqueous extract against MERS-CoV and SARS-CoV-2: in-vitro and in-silico study

Figures & data

Figure 1. Cytotoxicity and inhibitory effect of H. lupulus crude extract on Vero E6 cells. EC₅₀ for MERS-CoV and SARS-CoV-2.

Table 1. Inhibitory effect of Hop extract against MERS-CoV and SARS-CoV-2 in VERO E6 cell line.

		SARS-CoV-2		MERS-CoV
Agent	CC50	IC50	SI (CC50/IC50)	IC50	SI (CC50/IC50)
HOP extract	23.255 µg/µL	0.941 µg/µL	24.7	0.186 µg/µL	125.0
Hydroxychloroquine	193.6 ng/µL	8.909 ng/µL	21.7	10.72 ng/µL	18.06
Favipiravir	83.3 ng/µL	>CC50	<1	37.11 ng/µL	2.24

Figure 2. Inhibitory effect of Hop extract against MERS-CoV and SARS-CoV-2 by plaque reduction assay.

Table 2. Inhibitory effect of Hop extract against MERS-CoV and SARS-CoV-2.

Virus	Agent	Concentration (µg/µL)	Virus control	Viral count after treatment	Viral inhibition (%)
			(PFU/mL)	(PFU/mL)
MERS-CoV	Hop extract	3.000	5.2 × 10^4	8 × 10^4	84.60
		1.500		20 × 10^4	61.5
		0.750		28 × 10^4	46.2
		0.375		40 × 10^4	23.1
	Hydroxychloroquine	0.007	8 × 10^6	1.2 × 10^6	85.0
		0.003		5.3 × 10^6	34.0
		0.002		8.5 × 10^6	0.0
		0.001		9.7 × 10^6	0.0
	Favipiravir	0.980	90 × 10^5	70 × 10^5	70.0
		0.490		70 × 10^5	33.0
		0.245		60 × 10^5	22.0
		0.123		27 × 10^5	22.0
SARS-CoV-2	Hop extract	3.000	1.0 × 10^4	0.2 × 10^4	80.0
		1.500		0.3 × 10^4	70.0
		0.750		0.4 × 10^4	60.0
		0.375		0.6 × 10^4	40.0
	Hydroxychloroquine	0.007	7 × 10^5	0.0 × 10^5	100.0
		0.003		0.0 × 10^5	100.0
		0.002		1.0 × 10^5	85.7
		0.001		3.0 × 10^5	57.1
	Favipiravir	0.980	4.7 × 10^4	3.4 × 10^4	27.0
		0.490		3.5 × 10^4	25.0
		0.245		3.9 × 10^4	17.0
		0.123		4.7 × 10^4	0.0

Figure 3. Mechanism of the anti-SARS-CoV-2 activity targeting the viral replication mechanism.

Figure 4. 3 D poses and 2 D intermolecular contact between the top 5 compounds and MERS-CoV RBD. Note: In the 3D figures, the RBD protein is presented as a carton model and the ligands are presented as green sticks, and in the 2D plot, the black ball indicates the carbon atom, the red ball indicates the oxygen atom, the blue ball indicates the nitrogen atom, the red eyelash lines indicate the hydrophobic interaction between ligands and, and the green dashed string indicates the hydrogen bond formed between ligands and the amino acid residues of MERS-CoV RBD.

Figure 5. 3 D poses and 2 D intermolecular contact between the top 5 compounds and SARS-CoV 2 RBD. Note: In the 3D figures, the RBD protein is presented as a carton model and the ligands are presented as green sticks, and in the 2D plot, the black ball indicates the carbon atom, the red ball indicates the oxygen atom, the blue ball indicates the nitrogen atom, the red eyelash lines indicate the hydrophobic interaction between ligands and, and the green dashed string indicates the hydrogen bond formed between ligands and the amino acid residues of SARS-CoV 2 RBD.

Figure 6. SARS-CoV-2 and MERS-CoV RBD protein multiple sequence alignment.

Table 3. Binding affinity, hydrogen bonds and hydrophobic interactions of compounds isolated from H. lupulus docked into SARS-CoV-2 and MERS-CoV RBD protein.

MERS-CoV		SARS-CoV-2
Compounds	Binding affinity (Kcal/mol)	Compounds	Binding affinity (Kcal/mol)
5′-Prenylxanthohumol	−7.3	Catechin gallate	−7.4
Dehydrocycloxanthohumol hydrate	−7.2	Quercitrin	−7.2
Epicatechin gallate	−7.1	Rutin	−7
Catechin gallate	−6.9	8-Prenylnaringenin	−7
Xanthogalenol	−6.8	Epicatechin gallate	−6.9
Adlupulone	−6.7	Dehydrocycloxanthohumol hydrate	−6.9
6-Prenylnaringenin	−6.7	Xanthohumol	−6.9
Quercitrin	−6.6	5′-Prenylxanthohumol	−6.8
Isoxanthohumol	−6.6	Isoxanthohumol	−6.8

Table 4. Candidate compounds analyzed utilizing the ProTox-II server for their toxicity properties.

Compound	LD50 (mg/kg)	Class
Quercitrin	5000	5
Rutin	5000	5
5′-Prenylxanthohumo	3800	5
Dehydrocycloxanthohumol hydrate	3800	5
Xanthohumol	3800	5
6-prenylnaringenin	2000	4
Isoxanthohumol	2000	4
8-prenylnaringenin	2000	4
Xanthogalenol	1000	4
Catechin gallate	1000	4
Epicatechin gallate	1000	4
Adlupulone	100	3

Data availability statement

Data sharing not applicable: All data that support the findings reported in this study are available from the corresponding author [T. B.] upon reasonable request.