key: cord-0915558-o8y82efg
authors: Fang, Shisong; Xu, Benhong; Song, Xiangrong; Liu, Wukun; Xie, Yongmei; Yang, Xifei
title: A novel fermented Yi traditional medicine efficiently suppresses the replication of SARS-CoV-2 in vitro
date: 2020-12-30
journal: bioRxiv
DOI: 10.1101/2020.12.29.424534
sha: 7e6fe4f371703c23911fc4971862355bbb3290c8
doc_id: 915558
cord_uid: o8y82efg

Currently, the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a worldwide epidemic, causing more than 80 million infections and more than 1.7 million deaths. The pandemic has led to the closure of enterprises and schools in many countries, resulting in serious disruption of the global economy and social activities. Remdesivir is currently approved by the FDA for the treatment of COVID-19, but the WHO declared that Remdesivir is almost ineffective against COVID-19. The research and development of vaccines has made great progress, but it will take at least several months for safe and effective vaccines to be widely used clinically. Clinical studies revealed that some Traditional Chinese Medicines, such as Lianhua Qingwen Capsule and Huoxiang Zhengqi Water, exhibited excellent therapeutic effect on COVID-19. However, until now, there is still no cure for COVID-19. Therefore, there is an urgent need to find medicines that can effectively fight against the SARS-CoV-2. In this study, JIE BEN No. 10 (JB10), a fermentation broth produced by Yi traditional medicine fermentation technology, was explored for its anti-coronavirus activity. The in vitro data showed that JB10 could significantly suppresses the replication of the SARS-CoV-2 with an EC50 of 769.1 times dilution and a selection index of 42.68. Further studies indicated that JB10 had significant anti-inflammatory and antioxidant activities. The analysis of active components suggested that JB10 contained a large amount of superoxide dismutase (SOD), flavones, polyphenols, crude polysaccharide, etc. which may explain the anti-coronavirus activity, anti-inflammatory and antioxidant effects. Our study provides a new potentially therapeutic strategy for COVID-19.

Coronavirus disease 2019 (COVID- 19) is an acute respiratory infection caused by SARS-CoV-2. The main symptoms of patients are fever, fatigue, tussiculation, rhinobyon, runny nose, etc. About half of the patients will have difficulty breathing within a week, and severe cases will rapidly progress to acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis and coagulopathy. 1 The SARS-CoV-2 belongs to the β genus of coronaviruses. It has an envelope and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. 2 At present, the SARS-CoV-2 has caused more than 80 million infections and more than 1.7 million deaths in the worldwide according to Johns Hopkins University data. The epidemic has caused many countries to close enterprises and schools, causing severe damage to the global economy. At present, Remdesivir has been approved by the FDA for the treatment of patients with COVID-19, 3 but the WHO claims that Redesivir is almost ineffective against COVID-19. 4 The research and development of vaccines has made great progress, but safe and effective vaccines are widely used in clinical practice, and it will take at least several months.

At present, the COVID-19 epidemic has been well controlled in China. In this process, traditional Chinese medicine has made great contributions. For example, Lianhua Qingwen granules and Huoxiang Zhengqi dropping pills can significantly improve the symptoms of fever, cough, sputum and breathing difficulties caused by the novel coronavirus. 5 However, there is still no specific medicine to treat the COVID-19. Therefore, the discovery of medicines that can significantly inhibit the replication of the novel coronavirus is vital to the prevention and control of the COVID-19 epidemic.

Yi traditional medicine is an important part of Traditional Chinese Medicine (TCM) and has made great contributions to the development of Chinese civilization.

Among them, the world-famous Yunnan Baiyao is derived from the ancient Yi prescription. 6 JB10, a fermentation broth, is fermented from Phyllanthus emblica, Rosa roxburghii Tratt., Lemon, Gastrodia elata Bl. and honey according to Yi traditional medical theory. 7 In this work, in the process of screening anti-SARS-CoV-2 candidate drugs, we found that JB10 can significantly inhibit the replication of the novel coronavirus. Further research shows that JB10 has excellent antioxidant and anti-inflammatory effects. This research will provide new methods for anti-novel coronavirus treatment.

Standard assays were carried out to measure the cytotoxicity effects of JB10. The cytotoxicity of JB10 in Vero E6 cells was determined by the CCK8 assay. As can be seen from Figure 

Oxidative stress, characterized by excess levels of reactive oxygen species (ROS), plays a vital role in the progression of COVID-19. 9 Many evidences support therapeutic counterbalancing of ROS by antioxidants can prevent COVID-19 from becoming severe. 10, 11 In order to evaluate the antioxidant activities of JB10, DPPH free radical and hydroxyl radical scavenging assay were performed. As shown in Figure 2A , JB10 exhibited very strong and significant dose-dependent DPPH radical scavenging activities. When JB10 was diluted to 800, 1600, 6400, and 12800 times, the DPPH free radicals were removed 82.99%, 65.23%, 53.09% and 25.84%, respectively. As can be seen from Figure 2B , JB10 also displayed strong hydroxyl radical scavenging activities. After diluted to 20, 30, 40, and 50 times, the hydroxyl radicals were removed 67.42%, 59.92%, 53.91% and 51.64%, respectively. A. The JB10 was diluted for 800, 1600, 6400, and 12800 times.

B. The JB10 was diluted for 20, 30, 40, and 50 times.

Pulmonary hyper-inflammation and potentially life-threatening "cytokine storms" are the characteristics of COVID-19, which cause disease severity and death in infected patients. 12 According to the publication, controlling the local and systemic inflammatory response in COVID-19 may be as important as anti-viral therapies. 13 The anti-inflammatory activity was determined according to Liu 14 with some modifications. Before starting the experiment of anti-inflammatory activity, the cytotoxicity of JB10 to RAW 264.7 cell line was tested for 24 h by MTT assay. As shown in Figure 3A , there was no significant toxicity up to diluted 160 times on RAW 264.7 cells.

The anti-inflammatory activity of JB10 was evaluated in LPS-induced RAW264.7 cells under the following concentrations (diluted to 500, 1000, 2000, 4000, 8000, and 16000 times). As can be seen from Figure 3B , JB10 could effectively suppress the NO production. When the JB10 was diluted to 2000 times, the inhibition rate of NO rate was still close to 80%. These results suggested that JB10 has potent activity against inflammation. 

Since JB10 has potent antiviral activities against SARS-CoV-2, antioxidant activities and anti-inflammatory activity, we analyzed its components to provide evidences for JB10. As can be seen from Table 1 casualties occurred, in part due to the use of TCM. 15 Yi traditional medicine is a vital part of TCM and has its unique medical theory and practical experience. 16 for their antiviral activity and pleiotropic effects. In a previous report, polyphenols was shown to inhibit SARS-CoV-2 fusion/entry, disrupt SARS-CoV-2 replication and suppress the host inflammatory response. 21 From Table 1 , the content of polyphenols in JB10 is abundant. This may also contribute to the antiviral activity of JB10.

Polysaccharides have broad applications in anti-virus, especially in anti-coronavirus due to the good safety, immune regulation and antiviral activity. 22 Song et al. found that polysaccharides exhibited potent inhibitory activity against SARS-CoV-2. A further study suggested that polysaccharides could bind to the S glycoprotein to prevent SARS-CoV-2 host cell entry. 23 The synthesized coumarin analog (1m) also showed considerable inhibitory ability, and the binding energy to the main protease of COVID-19 (containing α-ketoamide) was -7.1kcal/mol. JB10 contains high amount of coumarin, which might contribute to the inhibitory effects of the virus replication as we observed. 26 In summary, given the urgency of the pandemic around the world, such safe and cheap liquor might provide help for prevention or treatment of COVID-19 patients.

Our data support that the JB10 goes into the clinical stage for further validation of the efficiency for anti-SARS-CoV-2 infection and therapy. 

The antioxidant activities of JB10 were evaluated by DPPH free radical scavenging assay and hydroxyl radical scavenging assay according to the slightly modified method by Lim et al. 27 

Where, Z0 is the absorbance value of the blank control, Zi is the absorbance value of the sample, Zi0 is the absorbance value of the H2O2 without the color agent.

Before starting the experiment of anti-inflammatory activity, the cytotoxicity of SOD is the sample optical density. The IC50 value was defined as the concentration at which the nitrite radicals were reduced by 50%.

The active components were determined by China National Research Institute of Food & Fermentation Industries (Beijing, China) according to relevant standards.

Characteristics of SARS-CoV-2 and COVID-19

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Repurposed antiviral drugs for COVID-19--Interim WHO solidarity trial results

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This work was financially supported by the Shenzhen Bay Laboratory Opening Fund