Indigo plant leaf extract inhibits the binding of SARS‑CoV‑2 spike protein to angiotensin‑converting enzyme 2

Hagiyama,Man; Takeuchi,Fuka; Sugano,Aki; Yoneshige,Azusa; Inoue,Takao; Wada,Akihiro; Kajiyama,Hiroshi; Takaoka,Yutaka; Sasaki,Kenroh; Ito,Akihiko

doi:10.3892/etm.2022.11200

Indigo plant leaf extract inhibits the binding of SARS‑CoV‑2 spike protein to angiotensin‑converting enzyme 2

Authors:
- Man Hagiyama
- Fuka Takeuchi
- Aki Sugano
- Azusa Yoneshige
- Takao Inoue
- Akihiro Wada
- Hiroshi Kajiyama
- Yutaka Takaoka
- Kenroh Sasaki
- Akihiko Ito
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Affiliations: Department of Pathology, Kindai University Faculty of Medicine, Osaka‑Sayama, Osaka 589‑8511, Japan, Center for Clinical Research, Toyama University Hospital, Toyama, Toyama 930‑0194, Japan, Data Science Center for Medicine and Hospital Management, Toyama University Hospital, Toyama, Toyama 930‑0194, Japan, Division of Pharmacognosy, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981‑8558, Japan
Published online on: February 10, 2022 https://doi.org/10.3892/etm.2022.11200
Article Number: 274
Copyright: © Hagiyama et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) uses its S1 spike protein to bind to angiotensin‑converting enzyme 2 (ACE2) on human cells in the first step of cell entry. Tryptanthrin, extracted from leaves of the indigo plant, Polygonum tinctorium, using d‑limonene (17.3 µg/ml), is considered to inhibit ACE2‑mediated cell entry of another type of coronavirus, HCoV‑NL63. The current study examined whether this extract could inhibit the binding of the SARS‑CoV‑2 spike protein to ACE2. Binding was quantified as cell‑bound fluorescence intensity in live cell cultures in which canine kidney MDCK cells overexpressing ACE2 were incubated with fluorescein‑labeled S1 spike protein. When indigo extract, together with S1 protein, was added at 8,650x and 17,300x dilutions, fluorescence intensity decreased in a dose‑ and S1 extract‑dependent manner, without affecting cell viability. When 4.0‑nM tryptanthrin was added instead of the indigo extract, fluorescence intensity also decreased, but to a lesser degree than with indigo extract. Docking simulation analyses revealed that tryptanthrin readily bound to the receptor‑binding domain of the S1 protein, and identified 2‑ and 7‑amino acid sequences as the preferred binding sites. The indigo extract appeared to inhibit S1‑ACE2 binding at high dilutions, and evidently contained other inhibitory elements as well as tryptanthrin. This extract may be useful for the prevention or treatment of SARS‑CoV‑2 infection.

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