In Vitro Suppression Effects of Ephedra przewalskii Stapf-Derived Natural Compounds on SARS-CoV-2

Abstract

Background: Ephedra przewalskii Stapf stems are a traditional Mongolian medicine commonly used to treat infectious diseases. Previous in vitro experiments have shown that the extract powder derived from its stems possesses antiviral activity. However, the active compounds responsible for this activity in E. przewalskii Stapf have not yet been identified or evaluated. This study aimed to identify the active components in E. przewalskii that exhibit antiviral effects against SARS-CoV-2 in vitro and validate their antiviral activity.

Methods: E. przewalskii stem extracts were subjected to high-performance liquid chromatography with varying methanol ratios in the mobile phase to obtain fractions with different polarities. Antiviral activity was assessed by infecting VeroE6/TMPRSS2 cells with the SARS-CoV-2 Delta strain and treating them with the obtained fractions. Infectious titers were measured using the 50% tissue culture infective dose (TCID₅₀) method, and half-maximal inhibitory concentration (IC₅₀) values were calculated for each fraction. The active components in the two fractions with the highest antiviral activity were identified and structurally characterized by nuclear magnetic resonance analysis. The antiviral activity of these compounds was confirmed by adding them to SARS-CoV-2-infected cells and measuring their infectious titers using the TCID₅₀ method. The IC₅₀ values were also calculated. Viral-particle inactivation assays were conducted by mixing the extracts with SARS-CoV-2 and measuring infectious titers.

Results: (-)-Catechin, (+)-epigallocatechin-(2α→O→7,4α→8)-(-)-epicatechin, and ent-epicatechin-(4α→8;2α→O→7)-catechin were isolated from E. przewalskii. These compounds exhibited significant antiviral activity against SARS-CoV-2 but demonstrated minimal direct virucidal effects.

Conclusion: (-)-Catechin, (+)-epigallocatechin-(2α→O→7,4α→8)-(-)-epicatechin, and ent-epicatechin-(4α→8;2α→O→7)-catechin exhibit antiviral activity against SARS-CoV-2 in infected cells.

Keywords: Ephedra przewalskii Stapf; IC50; SARS-CoV-2; natural compounds.

Figure 1

Evaluation of the cytotoxicity of E. przewalskii Fractions 1–7 (A–G) and their antiviral effects against SARS-CoV-2 under multiplicities of infection (MOIs) of 0.05 and 10. (A) Fraction 1, (B) Fraction 2, (C) Fraction 3, (D) Fraction 4, (E) Fraction 5, (F) Fraction 6, (G) Fraction 7. The antiviral effects of the fractions were compared in DMEM(−) and DMEM (1% DMSO). The orange points and lines represent DMEM(−) while the blue ones represent DMEM (1% DMSO). Triangles and circles indicate the cytotoxicity and infectious titer at a given concentration, respectively. Approximate equations, determination coefficients (R²), and half-maximal inhibitory concentrations (IC₅₀) for each MOI are provided.

Figure 2

Structures of the components isolated from E. przewalskii Fractions 3 and 5. (A) Component-1: (−)-catechin, (B) Component-2: (+)-epigallocatechin-(2α→O→7,4α→8)-(−)-epicatechin, and (C) Component-3: ent-epicatechin-(4α→8;2α→O→7)-catechin.

Figure 3

Evaluation of the cytotoxicity of Component-1, Component-2, and Component-3 and their antiviral effects against SARS-CoV-2 under multiplicities of infection (MOIs) of 0.05 and 10. (A) Component-1, (B) Component-2, (C) Component-3. Orange plots represent the condition of MOI 10, and blue plots represent the condition of MOI 0.05. Triangles indicate the cytotoxicity at a given concentration. Circles indicate the infectious titer at a given concentration. Approximate equations, determination coefficients (R²), and half-maximal inhibitory concentrations (IC₅₀) are provided for each MOI. The antiviral effects of all fractions were nearly identical under both conditions.

Figure 4

Viral-particle inactivating effect of Component-1, Component-2, and Component-3. Each group had a sample size of 4. The results for the four groups were analyzed against those of the PBS group using an unpaired t test.