{'fraction_non_alphanumeric': 0.044955784990045836, 'fraction_numerical': 0.028612435760914857, 'mean_word_length': 4.764611689351481, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Broad‐spectrum antiviral activity of Spatholobus suberectus Dunn against SARS‐CoV‐2, SARS‐CoV‐1, H5N1, and other enveloped viruses

Abstract The current COVID‐19 pandemic caused by SARS‐Cov‐2 is responsible for more than 6 million deaths globally. The development of broad‐spectrum and cost‐effective antivirals is urgently needed. Medicinal plants are renowned as a complementary approach in which antiviral natural products have been established as safe and effective drugs. Here, we report that the percolation extract of Spatholobus suberectus Dunn (SSP) is a broad‐spectrum viral entry inhibitor against SARS‐CoV‐1/2 and other enveloped viruses. The viral inhibitory activities of the SSP were evaluated by using pseudotyped SARS‐CoV‐1 and 2, HIV‐1ADA and HXB2, and H5N1. SSP effectively inhibited viral entry and with EC50 values ranging from 3.6 to 5.1 μg/ml. Pre‐treatment of pseudovirus or target cells with SSP showed consistent inhibitory activities with the respective EC50 value of 2.3 or 2.1 μg/ml. SSP blocked both SARS‐CoV‐2 spike glycoprotein and the host ACE2 receptor. In vivo studies indicated that there was no abnormal toxicity and behavior in long‐term SSP treatment. Based on these findings, we concluded that SSP has the potential to be developed as a drug candidate for preventing and treating COVID‐19 and other emerging enveloped viruses.


| INTRODUCTION
The Coronavirus disease 2019  pandemic is a significant threat to global health caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since December 2019 (Wu, Leung, & Leung, 2020). The COVID-19 pandemic has led to over 477 million laboratory-confirmed cases and about 6 million deaths worldwide. Although there has been limited data about the virus, present evidence shows that the public gets severe illnesses from COVID-19 irrespective of their gender, age, ethnicity, and health status (Hu, Guo, Zhou, & Shi, 2021). Being greatly transmissible, this novel COVID-19 has spread fast globally and overwhelmingly surpassed severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) in terms of both the number of infected persons and the range of epidemic areas . No effective treatment for this disease was substantiated, thus the morbidity and death rate would be always greater than common respiratory infection. Hence, it requires immediate clinical interventions and great efforts are needed to find effective drugs.
The epidemic outbreaks caused by the emerging and re-emerging viruses represent an extraordinary threat to global public health (Chan et al., 2020). Re-emerging viruses associated with emerging infectious diseases have been increasing nowadays and high mortality occurred especially when healthcare resources were overwhelmed (Dong, Du, & Gardner, 2020). As a novel beta coronavirus, SARS-CoV-2 shares 79% genome sequence character with SARS-CoV and 50% with MERS-CoV (Lu et al., 2020). In addition to SARS-CoV-2, other re-emerging viruses such as the influenza A virus (IAV), and the human immunodeficiency virus (HIV) are clinically important. The genome organization of SARS-CoV-2 shares homology with other beta coronaviruses. The viral replication cycle generally involves multiple steps, including viral entry, genome transcription and translation, virion assembly in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and finally virion budding by exocytosis (Fehr & Perlman, 2015). The entry of CoVs and other viruses are initiated by viral receptor-binding domain (RBD) and subsequent membrane fusion, where the spike (S) proteins of CoVs or envelope (ENV) protein of other viruses and viral receptors expressed on target cells play critical roles (Matsuyama et al., 2020).
In the RBD of S protein, the amino acid similarity between SARS-CoV-2 and SARS-CoV is about 73%. The S protein present in SARS-CoV-2 plays as a viral antigen and is accountable for binding to the host receptor eliciting humoral and cell-mediated immune responses during infection. A specific genomic feature of SARS-CoV-2 is the insertion of four amino acid residues at the junction of subunits of the S glycoprotein (Andersen, Rambaut, Lipkin, Holmes, & Garry, 2020).
This insertion creates a polybasic cleavage site, which permits effective cleavage by furin and other proteases (Coutard et al., 2020). A structural study suggested that the furin-cleavage site can reduce the stability of SARS-CoV-2 S glycoprotein and facilitate the conformational adaption that is essential for the binding of the RBD to its receptor (Wrobel et al., 2020). The binding of SARS-CoV-2 with angiotensin-converting enzyme 2 (ACE2) enables virus entry into the host cells . Obstructing viral entry is one of the effective strategies to inhibit viral infection. Several studies have successfully targeted this mechanism for antiviral treatment. For instance, structurally distinctive novel fusion inhibitors FA-583 and FA-617 inhibit the fusion of IAV by blocking the low-pH-induced hemagglutinin (Lai et al., 2015). Similarly, Maraviroc , enfuvirtide (Ikeda, Tennyson, Walker, Harris, & McNaughton, 2019), and Ibalizumab (Blair, 2020) inhibit HIV entry by blocking viral receptor binding. Only limited antiviral drugs are licensed for clinical practice for these diseases . The situation is further exacerbated by the potential development of drug-resistant mutants Weinreich et al., 2021). Hence, there is an urgent need to find broad-spectrum antiviral agents that are highly efficient and costeffective to combat the current COVID-19 pandemic and emerging variants of related viruses.
Nowadays, structure-based drug design strategies have been implemented to explicate the antiviral activity of active constituents present in traditional medicinal plants. Recently, several natural products are recognized as adjuvant therapy for COVID-19 treatment, not only for direct antiviral therapy (Islam et al., 2020), but also as an immune enhancer (Shah et al., 2021). Notably, natural products/ medical herbs also have potential therapeutic effects for COVID- Spatholobi caulis) belongs to the Rosales, Leguminosae family, which is widely distributed in Yunnan, Guangxi, Guangdong, and Fujian provinces of China. Its dried vine stem is well-recognized as Ji Xue Teng in the Chinese Pharmacopoeia, which is widely used in Traditional Chinese Medicine to treat blood stasis syndrome, abnormal menstruation, blood deficiency, chlorosis, and rheumatism (Mei et al., 2019;Qin et al., 2019). SS contains various classes of bioactive rich compounds viz., proanthocyanidins (PACs), flavonoids, phenolic compounds, quinones, and saponins (Cheng, Wan et al., 2011;Yoon, Sung, Park, & Kim, 2004), which exhibits various pharmacological activities including, antiinflammatory, antioxidant, antiplatelet, anticancer, and antiviral activity, affecting hemagglutination and lipid regulation (Chen et al., 2004;Chen et al., 2016;Im et al., 2014;Lee et al., 2011;Wang et al., 2013). Earlier studies suggested that extract of SS have potent antiviral activities against hepatitis C virus (Chen et al., 2016), coxsackievirus B3 (Pang et al., 2011), and enteroviruses, including coxsackievirus B5, Poliovirus I, Echovirus 9. and Echovirus 29 (Guo et al., 2006).
Hence, we aimed to investigate the broad-spectrum viral entry inhibitory activity and underlying mechanisms of SSP against SARS-CoV-2, SARS-CoV-1, HIV-1 ADA , HIV-1 HXB2 , H5N1, and Indiana vesiculovirus. Further extraction and isolation of SSP were also performed to determine active constituents. In addition, in vivo studies and quality control assays were also performed to analyze the drug-likeness, and toxicity to ensure the safety and efficacy of SSP.

| Extraction of Spatholobus suberectus Dunn
Dried SS stems were purchased from KangMei Pharmaceutical Company Ltd (Guang Xi Province, China) and the plants were authenticated by inspectors in the School of Chinese Medicine, the University of Hong Kong, Hong Kong. Briefly, SS was ground into coarse powder and extracted using a percolating device with 10 times volume (v/w) of 60% ethanol. The extract was then concentrated by reduced pressure to obtain the percolation powder, named SSP. SSP was dissolved in dimethyl sulfoxide (DMSO) to reach a concentration of 40 mg/ml for future use. About 200 g SSP extract was weighed and dispersed evenly in an appropriate amount of water to obtain the aqueous solution, followed by extraction with equal volumes of petroleum ether (PE), ethyl acetate (EA), and nbutanol (n-BuOH) to obtain the following parts: 2 g petroleum ether part (SSP-PE), 12.8 g ethyl acetate part (SSP-EA), 8.2 g EA insoluble part (SSP-EAin), 62 g n-butanol part (SSP-n-BuOH) and 113 g water layer residue (SSP-W). Then the n-butanol part (SSP-n-BuOH) was subjected to macroporous resin columns for gradient elution ethanol-water to obtain fraction A $ J (Figure 1).
The contents of PACs in SSP were determined by the vanillinhydrochloric acid method (Data S1) with minor modification (Cheng, Fu, et al., 2011). UHPLC of SSP was performed by Ultimate 3,000 system (Thermo Fisher Scientific Inc., Waltham, Massachusetts, U.S.) with a diode array detector (DAD). Before injection, the samples were filtrated through a 0.22 μm PTFE filter. An ACE Excel 2 C 18 column (100 mm Â 2.1 mm id, Scotland, UK) was used for the chromatographic separation, and the mobile phase was composed of acetonitrile (solvent A) and Milli-Q water with 0.1% formic acid (solvent B), using a gradient elution of 0 min 95% B, 3 min 92% B, 20 min 85% B, 26 min 70% B, 30 min, 40% B, 35 min 5% B, and keep at 5% B for five more minutes. The solvent flow rate was set as 0.4 ml/min, and the wavelength for analysis was conducted at 280 nm ( Figure 2a). This method was validated and shown in Data S2. F I G U R E 1 Flow chart of the study. Preparation and quality-controlled of SSP which is used to determine the broad-spectrum antiviral activity and its underlying mechanism. The powder SSP was further extracted to determine the effective active parts

| The antiviral assays
The inhibitory activities of the SSP against viruses are evaluated as previously described (Liu et al., 2007;Lu et al., 2012). Briefly, serially diluted SSP was tested against 100TCID50 viral infection. HEK293T-ACE2 were used for SARS-CoV-1/2; MDCK was used for H5N1; GHOST (3)-CD4-CCR5/CXCR4 were used for HIV ADA and HIV HXB2 infection. On day three, the viral infection was determined by measuring the reporter luciferase activity in target cells post-infection using commercially available kits (Promega, Wisconsin, USA). Antiviral data are reported as the concentration of drugs required to inhibit viral replication by 50% (EC 50 ).

| The cytotoxicity assays
Cells were incubated in the presence or absence of serially diluted SSP at 37 C in 5% CO 2 for 48 hours. The viability of the cells was then measured using the CellTiter-Glo ® Luminescent Cell Viability

| UPLC-DAD analysis and UPLC-ESI-QTOF-MS/MSn detection
The UPLC-DAD analysis was conducted with the same instrument as described above, but the gradient elution (name as "SSP isolation was injected into the UPLC system (same as "SSP isolation method") for chromatography separation and further ESI source for mass detection. The mass spectra were recorded from m/z 150 to 1,200 in negative-ion mode. The other instrumental parameters of the ESI-MS analysis were as follows: capillary voltage, 3,500 V; nebulizer gas (N 2 ), 10 psi; and source temperature, 350 C.

| Thiolysis and UPLC analysis
Thiolysis and UHPLC analysis were performed according to the method of Li et al. (2015) with slight changes, with details as follows: 32.5 min, 10% B. The sample injection volume was 5 μl, the flow rate was 0.4 ml/min, and the 280 nm UV absorption was chosen for analysis. The mDP values were calculated by using this equation (Li et al., 2015): mDP = (total area of benzyl-thioethers)/(total area of catechin and epicatechin) + 1.

| Toxicity study of SSP in SD rats
Sprague-Dawley rats (150-180 g) were used to determine a longtime adverse effect of SSP. Twenty-four male and female SD rats were purchased from Guangzhou University of Traditional Chinese Medicine. The toxicity study was evaluated as per OECD guidelines. No. HTSW211215). The animals were randomly divided into four groups, with three males and three females per group. SSP was dissolved in water and administered via gavage once a day at doses of 2, 4, 6 g/kg, or water. Their general behavior, signs of toxicity, food intake, and mortality were recorded at an interval of every 2 days.
Body weights were recorded at an interval of every 3 or 4 days. After 25 days, the animals were sacrificed by excessive anesthesia. Gross necropsy, calculation of organ index (such as brain, heart, lung, liver, kidney, etc.), pathological examinations of the organs, blood collection, and analysis were performed (Data S3). The experiments were not randomized/blinded. The requirements are considered to be relevant in recent guidelines for best practice of natural products in pharmacological research that has been taken into the account (Heinrich et al., 2020;Izzo et al., 2020).

| Data analysis
The EC 50 values and statistical analysis were performed using PRISM 6.0. Data were presented as mean ± SED or representative of three independent experiments, and significant difference (*) was considered if a p-value of <.05 was seen.

| SSP inhibits SARS-CoV-2 entry
To determine the antiviral activity of SSP, SSP was weighed and dissolved in DMSO to reach a concentration of 40 mg/ml. We previously developed a single-cycle reporter virus pseudotyped with a functional S-glycoprotein of SARS-CoV-2 to capture inhibitors targeting at entry step. As previously described, the pseudoviruses were generated by co-transfection of HEK293T cells with S-glycoprotein expression plasmids and a commonly used HIV backbone vector pNL4-3.Luc. R À EÀ (Liang et al., 2013;Liu et al., 2007;Liu et al., 2019;Lu et al., 2012;Yi et al., 2005). To examine the antiviral activity of SSP against SARS-CoV-2, HE293T-ACE2 cells were then infected with 100 TCID50 SARS-CoV-2 pseudoviruses in the presence of serially diluted SSP.
Virus pseudotyped with a second, unrelated viral envelope glycoprotein of VSV-G was included as a control. As shown in Figure  3.4 | SSP blocks SARS-CoV-1 and H5N1 entry by binding to viral envelope and its target cells To determine whether SSP inhibits viral entry by binding to the SARS-

COV-1 envelope or its receptor on target cells, SSP-virus binding and
SSP-cell binding assays were performed. In the SSP-virus binding assay, pseudovirus was initially pre-treated with 50 μg/ml of SSP. The viruses were then recovered by ultracentrifugation and subjected to infect target cells. We found that SSP inhibited SARS-CoV-1 pseudovirus infection at a similar degree when the virus and SSP were added into cells simultaneously ( Figure 5). Meantime, the SSP-cell binding assays were performed by pre-treatment of the target cells with SSP for 1 hr at 37 C. After washing by PBS, cells were incubated with SARS-CoV-1 at 37 C for 48 hr (Rapista et al., 2011). We found that pre-treatment of SSP on the target cells inhibited virus infection ( Figure 5). The same approach was also used to test the inhibitory effect of SSP against H5N1 with a single high dose of 50 μg/ml. Similar to SARS-CoV-1, SSP treatment on virus particles or their target cells efficiently blocked H5N1 virus entry (Figure 5b).

| SSP blocks HIV-1 entry by binding to the viral envelope
We tested HIV-1 with a similar but slightly modified experimental pro- antagonist JM2987 for 2 hr at 37 C. After washing by PBS, cells were subjected to HIV-1 ADA or HIV-1 HXB2 infection and cultivated at 37 C for 48 hr (Rapista et al., 2011). We found that pre-treating the target cells with SSP had no antiviral effect, while MVC and JM2987 showed potent viral inhibition against the respective ADA and HXB2 pseudoviruses at 1 μM as expected (Figures 5e-f). This evidence demonstrated that SSP inhibited HIV-1 infection by action on the viral envelope glycoprotein gp160, which mediates the viral entry into the host target cells.

| Antiviral activity of SSP and its effective constituents
To investigate the fractions containing SSP, a feasible UPLC method was used to separate SSP. SSP was divided into three parts, namely Part I, II, and III (Figure 6a). UPLC-ESI-MS/MSn

| Toxicity study of SSP in vivo
To evaluate the safety of SSP, the cytotoxicity in multiple cell lines was determined by cell toxicity assay (CC 50 = 181.2 $ 339.8 μg/ml as shown in Figure 8a-d). To further examine the toxicity in vivo, 24 male and female SD rats were divided into four groups: water (blank control), 2, 4, and 6 g/kg SSP. Drug or water was administered via oral gavage once a day for 25 days. No mortality was observed in any Levels of glucose, cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein were observed normal after SSP administration. No abnormality was observed in levels of blood biochemical parameters such as urea, uric acid, creatine kinase suggesting hepatic, renal, and heart functions were not harmed, indicating that SSP has no apparent toxicity to the experimental rats (Data S3).

| DISCUSSION
Over the past two decades, there has been an increased number of viruses causing unexpected illnesses and epidemics among humans.
Emerging and re-emerging virus diseases have become a major cardiac, epithelial type II, intestinal, and renal cells and SARS-CoV-2 enters into the cell, releases RNA genome, and replicates (Liskova et al., 2021). When the cell is invaded by the high viral load, the complete protein synthesis mechanism is undertaken to the replication of the virus. Consequently, the assembling of new viral particles leads to exocytosis and apoptotic cell death of the host cell resulting in energetic-metabolic chaos of the virus that spread and infects adjacent cells and organs (Shree et al., 2022).
SARS is caused by SARS-CoV and turned into a pandemic with 37 countries in early 2003 (van Doremalen et al., 2020). Although the disease was successfully contained, SARS-related CoVs still exist in animal host reservoirs, which cause reoccurrence of the outbreakrelated disease. Highly pathogenic avian influenza A H5N1, known as bird flu, also infected human and other animal species (Fusade-Boyer et al., 2019), and resulted in about 60% mortality (Kirtipal, Bharadwaj, & Kang, 2020). There is grave concern that H5N1 may mutate and lead to an influenza pandemic among humans (Fusade- replication and to prevent virus spread between humans (Doshi, Heneghan, & Jefferson, 2016;Jacob et al., 2016). However, generation of oseltamivir, amantadine, and rimantadine resistance H5N1 during treatment was reported (Jacob et al., 2016;Singh & Soliman, 2015). As to SARS-CoV, although some targets for therapeutic prevention have been investigated such as SARS-CoV S-protein, protease, and other proteins concerning the viral life cycle, there are currently no effective vaccines or drugs available for the prevention/ treatment of SARS infection (Althwanay et al., 2020).
Blocking of virus entry is a critical strategy for both prevention and treatment Kang, Guo, & Chen, 2013 Critically, the lack of antiviral effects against VSV rules out the possibility that SSP inactivates VSV by acting like a disinfectant. In support of our findings, several active principles isolated from the plant have also been reported to have some moderate inhibitory ability against HIV-1 entry (Au et al., 2001;Lam et al., 2000). Our results are also consistent with an earlier report that tea polyphenols found in green tea can interrupt membrane fusion between HIV-1 and the host cell plasma membrane (Liu et al., 2005). Further investigations, however, are necessary to reveal the mode of SSP action, which may lead to new drug targets identification on the HIV-1 envelope.
Based on the findings, SSP can be suitable for the prevention and treatment of viral pathogens. The powder SSP was characterized by HPLC spectrum, which was reported in a previous paper from the same lab , and further extracted to determine the effective active parts. The Fraction F, G showed better activity than We, however, found that SSP is a potent SARS-CoV-2 entry inhibitor, which has not been previously reported.
Furthermore, PACs inhibited IL-17-stimulated IL-6 productions in human pulmonary epithelial cells by inhibiting MAPK and NF-κBmediated signaling pathways (Kim et al., 2011). PACs and their derivatives can directly target the preS1 region of the hepatitis B virus large surface protein, thereby inhibiting HBV infection (Tsukuda et al., 2017). Zhang et al. (2018) have also reported that PACs A2 could inhibit the replication of porcine reproductive and respiratory syndrome virus in vitro. Procyanidin trimer C1 is reported to have synergistic anti-HIV effects when combined with kansui and JQ1 by activating latent HIV (Cary & Peterlin, 2018b). Overall, designing antiviral derivatives from the inspiration of proanthocyanidin compounds are highly promising.
Hence, SSP has a broad spectrum of antiviral activities and safe profiles, which warrants treating COVID-19 and other viral infections.
In general, this study is designed to investigate the antiviral activities of SSP on SARS-CoV-2, SARS-CoV-1, HIV-1 ADA , HIV-1 HXB2 , and H5N1. Results indicated that SSP effectively inhibited all these viral envelopes with EC 50 values ranging from 3.6 to 5.1 μg/ml. Also, SSP