Molecular Mechanism of Anti-SARS-CoV-2 Activity of Ephedra Herb Macromolecule Condensed-Tannin Contained in Ephedrine Alkaloids-Free Ephedra Herb Extract

Authors

Masashi Hyuga^a, Nahoko Uchiyama^a, Morio Yoshimura^b, Yoshiaki Amakura^b, Sumiko Hyuga^c, Masashi Uema^a, Genichiro Tsuji^a, Akinori Nishi^d, Hiroshi Odaguchi^c, Yosuke Demizu^a, and Yukihiro Goda^a

Organizations

a. Division of Biological Chemistry & Biologicals, National Institute of Health Sciences, 3–25–26 Tonomachi, Kawasaki-ku, Kawasaki 210–9501, Japan
b. College of Pharmaceutical Sciences, Matsuyama University, 4–2 Bunkyo-cho, Matsuyama, Ehime 790–8578, Japan
c. Oriental Medicine Research Center, School of Pharmacy, Kitasato University, 5–9–1 Shirokane, Minato-ku, Tokyo 108–8641, Japan
d. TSUMURA Advanced Technology Research Laboratories, TSUMURA & CO., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300–1192, Japan

Abstract

Ephedrine alkaloids-free Ephedra Herb extract (EFE) and its component, Ephedra Herb macromolecule condensed-tannin (EMCT), have been shown to exhibit anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity in VeroE6/TMPRSS2 cells. Therefore, it is expected that EFE will be developed as a new natural product drug that exhibits anti-SARS-CoV-2 effects. In this study, we analyzed the molecular mechanism of EMCT’s anti-SARS-CoV-2 activity, namely, inhibition of the binding of the viral spike (S) protein to angiotensin-converting enzyme 2 (ACE2), to confirm that EMCT is an active component of the anti-SARS-CoV-2 effect. Furthermore, matrix-assisted laser desorption/ionization time-of-flight-MS of EMCT was performed to determine its molecular mass distribution, resulting in a mass spectrum that exhibited a broad single peak at approximately 60000 and a mass range of m/z 30000–120000. In a binding assay of the receptor-binding domain of the S protein to ACE2, EMCT inhibited this interaction with an IC50 of 48 nM. According to surface plasmon resonance analysis, EMCT binds to both the S protein and ACE2 with KD values of 39 and 44 nM, respectively. Furthermore, the interaction between the predicted substructure of EMCT, flavan-3-ol tetramers, and the S protein was evaluated in silico, indicating that the possible binding site is the ACE2-binding region of the S protein. These results suggest that the anti-SARS-CoV-2 activity of EMCT is exerted through inhibition of S protein/ACE2-mediated viral infection. Therefore, EMCT is thought to be the most useful ingredient for quality control of EFE as an investigational extract drug.

CovalX Technology Used

High-Mass MALDI MS
HM5