key: cord-0836721-dfinv0po
authors: Sa-ngiamsuntorn, Khanit; Suksatu, Ampa; Pewkliang, Yongyut; Thongsri, Piyanoot; Kanjanasirirat, Phongthon; Manopwisedjaroen, Suwimon; Charoensutthivarakul, Sitthivut; Wongtrakoongate, Patompon; Pitiporn, Supaporn; Chaopreecha, Jarinya; Kongsomros, Supasek; Jearawuttanakul, Kedchin; Wannalo, Warawuth; Khemawoot, Phisit; Chutipongtanate, Somchai; Borwornpinyo, Suparerk; Thitithanyanont, Arunee; Hongeng, Suradej
title: Anti-SARS-CoV-2 Activity of Andrographis paniculata Extract and Its Major Component Andrographolide in Human Lung Epithelial Cells and Cytotoxicity Evaluation in Major Organ Cell Representatives
date: 2021-04-12
journal: J Nat Prod
DOI: 10.1021/acs.jnatprod.0c01324
sha: 715ee75dbdc894f4fb00ec0047e624682765832f
doc_id: 836721
cord_uid: dfinv0po

[Image: see text] The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID(50) was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC(50) of 0.036 μg/mL and 0.034 μM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC(50) of >100 μg/mL for A. paniculata extract and 13.2–81.5 μM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

T he outbreak of coronavirus disease 2019 (COVID-19) is an emergent global health crisis that requires urgent solutions. Since severe acute respiratory syndrome coronavirus 2 (SAR-CoV-2) emerged in Wuhan, Hubei, China, at the end of 2019, 1 the total confirmed cases are approaching 70 million, with more than one million deaths globally at the end of December 2020. SARS-CoV-2 is a positive sense, singlestranded, enveloped RNA virus belonging to the Coronaviridae family and categorized as a new member of the Betacoronavirus genus together with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012. 2−4 The host range of SARS-CoV-2 is very broad partly due to the relative conservation of the cellular receptor, angiotensin-converting enzyme 2 (ACE2), among mammals. This phenomenon could explain the interspecies transmission of the virus from animals to cause disease in humans. 5 Even though the majority of infections were asymptomatic, clinical manifestations of COVID-19 varies widely, ranging from low-grade fever to severe pneumonia and eventually death. The outcome of the infection depended largely on host factors, e.g., age, previous health problems, and immunological status. 6−8 Among critical manifestations, acute respiratory distress syndrome, cytokine storm, and multiorgan failure are the leading causes of death in COVID-19. 9, 10 Lacking effective antivirals against SARS-CoV-2 is undoubtedly one of the main reasons of poor clinical outcomes in patients with severe COVID-19. Drug discovery and repurposing strategies are being pursued to identify potential therapeutic agents. 11−13 A beneficial instance of this effort was repositioning of remdesivir (that was originally developed for Ebola virus infection) for COVID-19 treatment. 14, 15 Unfortunately, subsequent well-conducted clinical trials revealed that remdesivir had marginal clinical efficacy, 15, 16 while the costeffectiveness and accessibility are still a huge concern. 17 Therefore, further efforts should be made to identify new compounds with potent anti-SAR-CoV-2 activity. Among all promising candidates, natural products have been recognized as the major source for new drug discovery for decades. 18 Ethnobotanical evidence suggests plant-derived natural compounds are worth investigating to identify potent antivirals against coronaviruses, 19 while computational approaches have been applied for phytochemicals in order to define their targetspecific antiviral potential against SARS-CoV-2. 20 One prominent medicinal plant with various pharmacological activities is Andrographis paniculata, known as "king of The optimal dilutions of SAR-CoV-2 TCID 50 , 0.25-, 2.5-, and 25-fold, were evaluated in Calu-3 cells, in which the 25TCID 50 showed the maximal infectivity and was used throughout this study. FITC-labeled anti-SARS-CoV nucleoprotein mAb was used to detect the degree of SAR-CoV-2 viral replication. The positive control, neutralizing serum, demonstrated a dose-dependent effect, whereas the negative control, human IgG, had no antiviral activity. (B) Representative fluorescent images show SARS-CoV-2 infectivity at different TCID 50 (upper row) and anti-SAR-CoV-2 activity of neutralizing serum as compared to human-IgG (lower row). Fluorescent signals: green, anti-SARS-CoV NP mAb; blue, Hoechst. (C) Hydroxychloroquine exhibited no effect against SARS-CoV-2 in Calu-3 cells with IC 50 > 50 μM. bitters", which belongs to the Acanthaceae family. 21 A. paniculata is currently used in traditional medicine to treat the common cold, diarrhea, and fever due to several infectious causes and as a health tonic. 22 A major bioactive component of A. paniculata is andrographolide, 23 a diterpene lactone in the isoprenoid family, which is known for its broad-spectrum antiviral properties. 24 Andrographolide was recently predicted in silico to have a potent anti-SARS-CoV-2 activity through specific targeting of the host ACE2 receptor and viral factors, i.e., RNA-dependent RNA polymerase, main protease, 3-CL protease, PL protease, and spike protein. 25−28 Recently, Shi et al. applied an enzyme-based assay to demonstrate an inhibitory effect of andrographolide against SARS-CoV-2 main protease (M pro ). 29 Furthermore, our group has utilized a phenotypic cell-based immunofluorescent assay (IFA) to reveal the anti-SARS-CoV-2 effect of A. paniculata extract and andrographo-lide in African green monkey kidney cells (Vero E6). 30 Notably the anti-SARS-CoV-2 activity of A. paniculata extract and andrographolide has never been elucidated in infected human lung epithelial cells.

This study aimed to evaluate the anti-SARS-CoV-2 activity of A. paniculata extract and its major component, andrographolide, by using a legitimate model of infected human lung epithelial cell, Calu-3. 31 Cytotoxic profiles of A. paniculata extract and andrographolide over five major human organs, including lung, brain, liver, kidney, and intestine, were achieved by a panel of cell line representatives. The results demonstrated that A. paniculata extract and andrographolide have a potent anti-SARS-CoV-2 activity with a high safety margin for major organs in cell culture models. ■ RESULTS AND DISCUSSION Optimization of Human Lung Epithelial Cells (Calu-3)-Based Anti-SARS-CoV-2 Assay. Urgent demands for effective anti-SARS-CoV-2 agents have drawn much attention from the scientific community to search for new antiviral candidates. In this study, we aimed to document the anti-SARS-CoV-2 activity of A. paniculata extract and andrographolide ( Figure 1A ) for further drug development against COVID-19. Therefore, a robust anti-SARS-CoV-2 screening platform established on a legitimate model is required to facilitate this process. To serve this purpose, we optimized our antiviral assay (which was previously established upon Vero E6 cells) 30 to use Calu-3 human lung epithelial cells as the legitimate host cell for SARS-CoV-2 infection 31 and then validated the assay applicability by hydroxychloroquine and niclosamide ( Figure 1B and 1C, respectively), two FDAapproved drugs with anti-SAR-CoV-2 activities in vitro. 31−33 Calu-3 cells were cultured until they reached confluence and then infected with various concentrations of SARS-CoV-2, 0.25TCID 50 , 2.5TCID 50 , and 25TCID 50 , for 2 h. The cells were washed twice to remove the excess inoculum and further incubated in fresh culture medium for 48 h in the absence or presence of the compounds of interest. To demonstrate the degree of SARS-CoV-2 infectivity, the infected Calu-3 cells were stained by anti-SARS-CoV2 nucleoprotein rabbit monoclonal antibody, followed by goat anti-rabbit IgG Alexa Fluor 488, and the percentage of fluorescent positive cells was counted by a high-content imaging platform. The percentage of viral infectivity in Calu-3 cells at 48 h postinfection achieved approximately 1%, 18%, and 95%, upon infection at 0.25TCID 50 , 2.5TCID 50 , and 25TCID 50 , respectively ( Figure  2A and B, upper panel). Since SARS-CoV-2 at 25TCID 50 was able to reach maximal infectivity, it served as the standard infectious dosage throughout all experiments. To establish the positive control, the neutralizing serum derived from COVID-19 patients with negative SARS-CoV-2 RNA was added to the infected Calu-3 cells. The high-content imaging revealed viral suppression in Calu-3 cells with a percentage of SARS-CoV-2 infectivity of 0%, 10%, 60%, and 80% at 1:100, 1:500, 1:2500, and 1:25 000 dilutions of the neutralizing serum, respectively ( Figure 2A and B, lower panel). In this study, anti-human IgG served as the negative control.

As aforementioned, hydroxychloroquine and niclosamide 31−33 were applied to evaluate the validity of Calu-3based anti-SARS-CoV-2 assay. Hydroxychloroquine, a classical antimalarial drug, had no inhibitory effect against SARS-CoV-2 infection in human lung epithelial cells (IC 50 > 50 μM) ( Figure  2C and D), while niclosamide, a classical antihelminthic drug, inhibited SARS-CoV-2 infection with an IC 50 of 0.90 μM ( Figure 2E and F). Of note, hydroxychloroquine can exhibit antiviral effects in Vero E6 cells 30 but not Calu-3 human lung epithelial cells. 31 These results were consistent with previous reports 31, 32 and supported the validity of the Calu-3-based anti-SARS-CoV-2 assay used in this study.

Dose−Response Relationship of A. paniculata Extract and Andrographolide in SARS-CoV-2-Infected Human Lung Epithelial Cells. To investigate whether A. paniculata extract and its major component andrographolide have potential as anti-SARS-CoV-2 agents, SARS-CoV-2-infected Calu-3 cells were treated for 48 h with 4-fold dilutions of A. paniculata extract (0.05−50 μg/mL) or andrographolide (0.05−50 μM), respectively. The results demonstrated both A. paniculata extract ( Figure 3A and B) and andrographolide ( Figure 3C and D) inhibited SARS-CoV-2 replication in a dose-dependent manner.

To confirm anti-SARS-CoV-2 activity of A. paniculata extract and andrographolide, analysis of viral output using the plaque reduction assay was performed. At 48 h postinfection in the absence or presence of compounds of interest, the culture supernatants were harvested to determine the number of infectious virions produced from SARS-CoV-2-infected Calu-3 cells by the plaque assay. From the result, the evaluation of viral output was consistent with that of the high-content imaging study ( Figure 3A −D), in which A. paniculata extract and andrographolide again demonstrated a dose−response relationship ( Figure 3E and F) with an IC 50 of 0.036 μg/mL for A. paniculata and 0.034 μM for andrographolide ( Figure 3E and F). Similarly, evaluation of viral RNA output in the harvested supernatants by qRT-PCR also showed the reduction of the extracellular viral RNA in a dose-dependent manner following the treatment (Supplementary Figure 1) . This information confirmed the significance of A. paniculata extract and andrographolide in suppressing SARS-CoV-2 infectivity in Calu-3 cells.

It was interesting that the IC 50 values of A. paniculata extract and andrographolide varied between the high-content imaging IFA and viral output study using the plaque assay. Taking our previous study 30 into account, the IC 50 of A. paniculata extract and andrographolide by measurement type and cell host is summarized in Table 1 . The IC 50 values of remdesivir 30 served as the comparators.

As shown in Table 1 , a consistent pattern has been detected for A. paniculata extract, andrographolide, and remdesivir: (i) the IC 50 values measured from the assays using Calu-3 were lower than those of Vero E6; (ii) the IC 50 values measured by plaque assay were lower than those of high-content imaging IFA. The deviation of drug response between two cells of different species and organs highlighted the importance of using Calu-3 human lung epithelial cells, but not Vero E6 African green monkey kidney cells, as the host cells for in vitro SARS-CoV-2 infection experiments. 31 The dissimilarity of IC 50 between the IFA and the plaque assay could be explained by their different principle of SARS-CoV-2 detection. The IFA requires a specific antibody to detect the expression of SARS-CoV-2 nucleoprotein derived from complete virions and subviral particles within the host cells. On the other hand, the plaque assay measures the infectivity of complete virions released from the host cells. Compared to remdesivir, which showed similar IC 50 values obtained from the IFA and plaque assay, treatment of Calu-3 cells with A. paniculata extract and andrographolide exhibited distinct IC 50 values examined by Journal of Natural Products pubs.acs.org/jnp Article both methods. The lower IC 50 evaluated by the plaque assay revealed that both the extract and the compound were more potent in interfering with the production of the infectious viral progeny than inhibiting the early phase of viral genome replication and protein expression in Calu-3 cells.

Cytotoxicity Profiles of A. paniculata Extract and Andrographolide. One of the main concerns in medicinal plant-derived drug development is herb-induced injury of the vital organs, especially the liver. To address this issue, six human cell lines that represent five major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), were applied to evaluate the cytotoxicity profiles of A. paniculata extract and andrographolide by MTT assay. The results showed that the A. paniculata extract had no cytotoxicity to all cell lines examined, with a CC 50 of >100 μg/mL ( Figure 4A This finding pointed out that further development of A. paniculata extract and andrographolide in preclinical models of COVID-19 should pay attention to the neurologic side effects as well as the amount of andrographolide that passes through the blood−brain barrier. In this regard, the boiled-egg plot using the SwissADME program was performed for in silico prediction of the probability of compounds being gastrointestinal absorbable and barrier penetrating. 34, 35 Based on this prediction, andrographolide is a gastrointestinal absorbable compound without an ability to pass through the blood−brain barrier (Supplementary Figure 2) . The SwissADME prediction also reveals that andrographolide is a P-glycoprotein (P-gp) substrate, 36 suggesting that andrographolide would be "pumped out" from central nervous system (CNS) tissues and may not possess significant neurotoxicity if used in further studies.

Potential Clinical Applications of A. paniculata Extract and Andrographolide. A. paniculata has been classified as an essential plant for traditional medicine in various Asian countries for centuries. 37 In Thailand, the Ministry of Public Health has registered this plant, so-called Fah Talai Jone, on The National List of Essential Drugs A.D. 1999 (List of Herbal Medicinal Products). 38 A. paniculata extract has long been available in Thailand's markets as herbal nutraceuticals in several recipes and brands, which the general population can access and use to treat diarrhea, fever, the common cold, and viral infection. It is postulated that beneficial effects of A. paniculata extract largely depend on its major component, andrographolide, the bicyclic diterpene lactone with multifunctionalities including anticancer, antioxidant, anti-inflammatory, immunomodulatory, cardiovascular protection and hepatoprotection, antimicrobial, and antiprotozoal effects. 37,39−42 Andrographolide is also well known for its broad-spectrum antiviral properties. 24 Studies showed andrographolide is effective against influenza A, 43 hepatitis C virus, 44 Chikungunya virus, 40 HIV, 45 hepatitis B virus, 46 Herpes simplex virus 1, 47 Epstein−Barr virus, 48 and human papillomavirus. 49 This study showed that both A. paniculata extract and its active component andrographolide had a potent inhibitory effect against SARS-CoV-2. This finding opens the possibility to develop A. paniculata extract and andrographolide in the context of COVID-19 treatment. In comparison to our previous study, 30 A. paniculata extract and andrographolide exhibited the equivalent IC 50 against SARS-CoV-2 infection to remdesivir 30 (Table 1) . This is an additional rationale to support A. paniculata extract and especially andrographolide for further antiviral development.

Previous studies suggested andrographolide targeted nonstructural proteins of SARS-CoV-2 as the mechanism of action. An enzyme-based assay and in silico modeling prediction showed andrographolide could inhibit the main protease (M pro ) activities of SARS-CoV-2 with an IC 50 of 15 μM. 26, 29 When compared to our finding, the IC 50 from the M pro enzyme-based assay was 10 times higher, implying that andrographolide probably functions through multiple targets as previously predicted by in silico models. 25−28 It has been proposed that andrographolide is involved in multiple steps of the viral life cycle including viral entry, genetic material replication, and protein synthesis and inhibits the expression or function of the mature proteins. 24 Maurya et al. 50 showed that andrographolide has significant binding affinity toward the spike glycoprotein of both SARS-CoV-2 and ACE2 receptors and could be developed as a prophylactic agent for limiting viral entry into the host cells.

In this study, we applied two assays to evaluate the anti-SARS-CoV-2 activity in Calu-3 cells. Since both IFA and plaque assays investigate the distinct steps in the SARS-CoV-2 life cycle (the expression of viral protein vs the production of infectious progenies), interpretation of IC 50 values of the compound obtained from such assays could be made differently. This depends on the potency of the extract or compound in perturbing a specific step of the SARS-CoV-2 life cycle, either the viral protein expression or the particle release. Therefore, the IC 50 value obtained from IFA could be interpreted as the half-maximal concentration of the compound able to inhibit SARS-CoV-2 NP expression. On the other hand, the IC 50 value from the plaque assay could be indicated as the half-maximal concentration of the compound able to inhibit infectious virion production. From our data, the Journal of Natural Products pubs.acs.org/jnp Article IC 50 values measured by the plaque assay were lower than those of the high-content imaging IFA. This can be interpreted as both A. paniculata extract and andrographolide are more potent in interfering at the late phases of the viral life cycle in Calu-3 cells than those of the early steps of viral genome replication and protein expression. The late phases of the SARS-CoV-2 life cycle include viral assembly and maturation, transportation along the secretory pathway, or particle release. Also, we performed pre-entry treatment of andrographolide in Vero E6 cells (Supplementary Figure 3) , in which the compound was added into the cells together with the 25TCID50 of SARS-CoV-2 during the viral adsorption step. Then the inoculum was removed, and the cells were washed before adding fresh medium without the compound into the cells. In this pre-entry treatment, the IC 50 of andrographolide evaluated by IFA was 19.03 μM, compared with the IC 50 value of 6.58 μM in the post-treatment condition. 51 This suggested that the compound might target the postentry event or the late phase of the viral life cycle. Since andrographolide exerted a stronger anti-SARS-CoV-2 effect than the extract, this finding highlighted this compound as a potential monotherapy, even though the combinational regimens with other compounds that interfere with the early phase of the viral life cycle, such as remdesivir, should be prioritized to increase the efficacy and minimize the side effects/toxicities. This study was associated with several limitations. Although our findings support andrographolide as a promising candidate for further anti-SARS-CoV-2 development, the low bioavailability of andrographolide might pose a limitation for clinical applications. 52, 53 Several strategies have been developed to improve andrographolide solubility and bioavailability, i.e., forming complexes with hydroxypropyl-β-cyclodextrin (HP-β-CD), 54 solid dispersion using a spray-drying technique, 55 and loading into the nanoemulsion. 56 Also, it should be noted that this study was conducted using an in vitro cellular model. The safety and efficacy of andrographolide should be further investigated in preclinical animal models and clinical studies.

In conclusion, this study demonstrated anti-SARS-CoV-2 activity of A. paniculata and andrographolide using a Calu-3based anti-SARS-CoV-2 assay. Potent anti-SAR-CoV-2 activities, together with the favorable cytotoxicity profiles, support further development of A. paniculata extract and especially andrographolide as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection. ). An immortalized hepatocyte-like cell line (imHC) was established in-house as previously described, 57 and its hepatic phenotypes were characterized previously. 58, 59 Vero cells were cultured in minimum essential medium (MEM) (Gibco, Detroit, MI, USA). Vero E6 and Caco-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco). HepG2, imHC, Calu-3, and SH-SY5Y cells were cultured in DMEM:nutrient mixture F-12 (Gibco). HK-2 cells were cultured in Dulbecco's low glucose modified Eagle's medium (DMEM low glucose) (HyClone, Logan, UT, USA). The culture media was supplemented with 10% fetal bovine serum (FBS) (Thermo Scientific Fisher, Waltham, MA, USA) and 100 μg/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 1% GlutaMAX (Gibco). Cells were incubated at 37°C in a humidified incubator with 5% CO 2 .

Preparation of SARS-CoV-2 Virus. SARS-CoV-2 virus was isolated from nasopharyngeal swabs of a confirmed COVID-19 patient in Thailand (SARS-CoV-2/01/human/Jan2020/Thailand). The virus was propagated in Vero E6 cells as previously described 30 and stored at −80°C. Viral titration as TCID 50 titer/mL was performed in a 96-well plate. In brief, the virus stock was titrated in quadruplicate in 96-well plates on Vero E6 cells in serial dilution to obtain 50% tissue culture infectious dose (TCID 50 ) using the Reed− Muench method. 60 All the experiments with live SARS-CoV-2 viruses were performed at a certified biosafety level 3 facility at Department of Microbiology, Faculty of Science, Mahidol University.

Preparation of Andrographis paniculata Extract and Andrographolide. Plant material in this study was common herbs in Thailand, and it was listed in Thai Herbal Pharmacopoeia 2019 (https://bdn.go.th/th/sDetail/10/34/). The plant was identified, authenticated by Chao Phya Abhaibhubejhr Hospital, Prachin Buri, Thailand, and deposited at the herbarium unit. The powder of A. paniculata was weighed and soaked in 95% ethanol in a ratio of 1:4. After 24 h, the liquid fraction was separated using a thin straining cloth, then filtered through filter paper by a vacuum pump. The extract obtained was concentrated using a rotary evaporator at a temperature of 45°C and then concentrated in a water bath at 70°C until it became a concentrated solution. The crude extract was stored at 4°C and protected from light until use. 61 The andrographolide concentration in the crude extract was measured by the HPLC method following the Thai Herbal Pharmacopoeia 2019 protocol. The andrographolide content in the crude extract was 7.9% (w/w). 62 The analytical standard andrographolide was used as a reference (Sigma, St. Louis, MO, USA).

In Vitro Antiviral Assay. Calu-3 cells were seeded at 1 × 10 4 cells per well in a 96-well black plate (Corning, NY, USA) and incubated for 24 h at 37°C in a 5% CO 2 atmosphere. Then, culture medium was discarded and washed once with phosphate-buffered saline (PBS). Cells were infected with SARS-CoV-2 at 25TCID 50 for 2 h at 37°C. After viral adsorption, the cells were washed twice with PBS to remove the excess inoculum, and the fresh culture medium (DMEM/ F12 supplemented with 10% FBS and 100 μg/mL penicillin/ streptomycin) was added. Each concentration of drugs, crude extract, or active compound was inoculated into the culture medium. Infected cells were then maintained at 37°C in a 5% CO 2 incubator for 48 h. Positive convalescent serum (heat-inactivated at 56°C for 30 min) of a COVID-19 patient and anti-human IgG (Santa Cruz Biotechnology, Dallas, TX, USA) were used for viral inhibition as positive control and negative control, respectively. The experiment was performed in triplicate.

High-Content Imaging for SARS-CoV Nucleoprotein Detection. The cells in the 96-well plate were fixed and permeabilized with 50% (v/v) acetone in methanol on ice for 20 min. The fixed cells were washed once with PBS with 0.5% Tween detergent (PBST) and blocked with 2% (w/v) BSA in PBST for 1 h at room temperature. Next, the cells were incubated with a 1:500 dilution ratio of primary antibody specific for SARS-CoV nucleoprotein 56 (rabbit mAb) (Sino Biological Inc. China), which cross-reacted with the NP protein of SARS-CoV-2 as well, for 1 h at 37°C. After incubation, cells were washed with PBST three times. Then, the goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA) was used as the secondary antibody at 1:500 dilution. Hoechst dye (Thermo Fisher Scientific) was applied for nuclei staining. The fluorescent signal was detected and analyzed by the Operetta high-content imaging system (PerkinElmer, Waltham, MA, USA) as previously described. 30 Percentage of infected cells in each well was automatically obtained from 13 fields per well using Harmony software (PerkinElmer). Data were normalized to the infected control, and the IC 50 value was calculated by GraphPad Prism 7.

Plaque Assay. The viral output in culture supernatants obtained from SARS-CoV-2-infected Calu-3 cells was determined by plaque assay by using a Vero cell monolayer. In brief, Vero cells were seeded Journal of Natural Products pubs.acs.org/jnp Article into a six-well plate 24 h prior to infection. A serial dilution of the virus-containing supernatants was prepared for inoculation into the Vero cell monolayer. The cells were incubated for viral adsorption for 1 h in a 37°C incubator and then overlaid with 3 mL/well of MEM medium supplemented with 5% FBS and 1% agarose. The culture was incubated at 37°C in 5% CO 2 for 3 days. Thereafter, plaque phenotypes were visualized by staining with 0.33% Neutral Red solution (Sigma-Aldrich) for 5 h. Plaque numbers were counted as plaque-forming units per milliliter and reported as the percentage of plaque reduction in comparison to supernatant obtained from the infected Calu-3 cells without any treatment. Cell Cytotoxicity Assay. All human cell lines were plated in 96well plates at 5 × 10 4 cells/well and treated with various concentrations of A. paniculata extract (0−100 μg/mL) and andrographolide (0−100 μM) for 48 h. Cell viability was examined by an MTT colorimetric assay. In brief, the medium was replaced with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma-Aldrich) at a final concentration of 0.5 mg/mL and incubated for 4 h at 37°C in a humidified incubator with 5% CO 2 . The rest of the MTT solution was removed, and formazan crystals were then dissolved with DMSO (Merck, Schuchardt, Darmstadt, Germany). Absorbance was measured at a wavelength of 570 nm by an EnVision multilabel reader (PerkinElmer). Data were normalized to the solvent control, and then CC 50 ■ REFERENCES

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