Inhibitory Activity of Saussurea costus Extract against Bacteria, Candida, Herpes, and SARS-CoV-2

Medicinal herbs have long been utilized to treat various diseases or to relieve the symptoms of some ailments for extended periods. The present investigation demonstrates the phytochemical profile, molecular docking, anti-Candida activity, and anti-viral activity of the Saussurea costus acetic acid extract. GC-MS analysis of the extract revealed the presence of 69 chemical compounds. The chemical compounds were alkaloids (4%), terpenoids (79%), phenolic compounds (4%), hydrocarbons (7%), and sterols (6%). Molecular docking was used to study the inhibitory activity of 69 identified compounds against SARS-CoV-2. In total, 12 out of 69 compounds were found to have active properties exhibiting SARS-CoV-2 inhibition. The binding scores of these molecules were significantly low, ranging from −7.8 to −5.6 kcal/mol. The interaction of oxatricyclo [20.8.0.0(7,16)] triaconta-1(22),7(16),9,13,23,29-hexaene with the active site is more efficient. Furthermore, the extract exhibited significant antimicrobial activity (in vitro) against Candida albicans, which was the most susceptible microorganism, followed by Bacillus cereus, Salmonella enterica, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, respectively. On the other hand, its antiviral activity was evaluated against HSV-1 and SARS-CoV-2, and the results showed a significant positive influence against HSV-1 (EC50 = 82.6 g/mL; CC50 = 162.9 g/mL; selectivity index = 1.9). In spite of this, no impact could be observed in terms of inhibiting the entry of SARS-CoV-2 in vitro.


Introduction
Since antiquity, what we now refer to as folk, traditional, or alternative medicine has been the major source of remedies, which were mostly reliant on medicinal plants. On the other hand, modern medicine is now confronting a problem as a result of its inability to prevent the onset of disease. As a result, there is a pressing need to return to Mother Nature and its wealth of natural medicines [1,2].
In the scientific literature, numerous studies on the bioactive properties of medicinal plants have been published. For example, there are claims that Allium sativum, Trigonella foenum-graecum, Ferula assa-foetida, Carthamus tinctorius, and Mangifera indica are all plants with antidiabetic activity [3]. Pistacia lentiscus, Diospyros abyssinica, Sargentodoxa cuneata, Acacia auriculiformis, Ficus microcarpa, Salvia officinalis, and the Lamiaceae species contain potent antioxidants [4]. Aloe Figure S1 displays the GC-MS chromatograms of Saussurea costus extracted using acetic acid. We identified 109 peaks from the chromatograms that correspond to chemical substances by comparing their peak retention times, peak area percentages, height percentages, and mass spectral fragmentation features to those of known compounds in the National Institute of Standards and Technology (NIST) library [41]. As indicated in Table S2, phytochemical studies of the acetic acid extraction of Saussurea costus roots indicated the existence of 69 compounds. The percentages of the phytochemicals were as follows: alkaloids, 5%; terpenoids, 79%; phenolic compounds, 4%; hydrocarbons, 7%; and sterols, 6%. According to these findings, terpenoids accounted for the highest percentage of organic chemical compounds (78%), whereas alkaloids and phenolic compounds accounted for the lowest percentages (4%) of chemical compounds, as depicted in Figure 1. Terpenoids included 0.02% (4-Terpinenyl acetate) and 13.23% Vanillosmin. Phenolic compounds ranged between 0.01 (Phenol, 2-methoxy-4-(2-propenyl)-, acetate) and 0.06% (Phenol, 2,6-dimethoxy). Alkaloids ranged from 0.51% (Piperine) to 0.04% (Di (1,2,5-oxadiazolo) [3,4- hexadecadien-1-ol acetate). Sterols ranged from 0.26% (Ergost-5-en-3-ol, (3.beta.)) to 3.51% (9,19-Cycloergost-24(28)-en-3-ol, 4,14-dimethyl-, acetate (3.beta.,4.alpha.,5.alpha)). Through a comparison of the chemical profile obtained in the current study with other published data, we found a difference in the nature and number of compounds and their concentrations [42,43]. Various studies have shown that chemical compound concentrations vary depending on different factors, which include the solvent used in plant extracts, the area of plants grown, and the quantity of the plant used [44]. The active constituents of this well-known medicinal plant are mostly terpenes, with various levels of flavonoids, anthraquinones, alkaloids, tannins, and inulin, as described in earlier research [45]. Several laboratory experiments and animal model studies have indicated that Saussurea costus possesses anti-inflammatory, antitrypanosomal, and anti-malignant tumor effects, confirming its extended use in medicine [46,47]. Costsunolide, dehydrocostus lactone, and cynaropicrin are some of the phytochemicals found in this plant that have shown promise in producing bioactive molecules [48]. Because Saussurea costus has displayed solid anticancer, anti-inflammatory, antibacterial, antifungal, and antiviral action, it will be possible to improve its use as a medication soon. According to several studies, sterol compounds act as cancer inhibitors, anti-inflammatory medications, immunomodulatory agents, and antiviral agents [49,50]. Various investigations have shown that terpenoids have a high proclivity for acting as SARS-CoV-2 blockers [51]. Based on a new study integrating quantum chemistry, molecular docking, and dynamic dynamics, phenolic chemicals have potential therapeutic implications for SARS-CoV-2. As a result, the mixture of active components present in each plant may be more effective than a single separated molecule in terms of treatment efficacy [52]. As reported in some investigations, researchers have estimated that the quinoline-2-carboxylic acids identified in Ephedra sinica might be employed as COVID-19 medicinal agents [53].
Plants 2023, 12, x FOR PEER REVIEW compound concentrations vary depending on different factors, which include the used in plant extracts, the area of plants grown, and the quantity of the plant us The active constituents of this well-known medicinal plant are mostly terpenes, w ious levels of flavonoids, anthraquinones, alkaloids, tannins, and inulin, as descr earlier research [45]. Several laboratory experiments and animal model studies hav cated that Saussurea costus possesses anti-inflammatory, antitrypanosomal, and a lignant tumor effects, confirming its extended use in medicine [46,47]. Costsunoli hydrocostus lactone, and cynaropicrin are some of the phytochemicals found in th that have shown promise in producing bioactive molecules [48]. Because Saussure has displayed solid anticancer, anti-inflammatory, antibacterial, antifungal, and a action, it will be possible to improve its use as a medication soon. According to studies, sterol compounds act as cancer inhibitors, anti-inflammatory medicatio munomodulatory agents, and antiviral agents [49,50]. Various investigations have that terpenoids have a high proclivity for acting as SARS-CoV-2 blockers [51]. Bas new study integrating quantum chemistry, molecular docking, and dynamic dyn phenolic chemicals have potential therapeutic implications for SARS-CoV-2. As a the mixture of active components present in each plant may be more effective than a separated molecule in terms of treatment efficacy [52]. As reported in some investig researchers have estimated that the quinoline-2-carboxylic acids identified in E sinica might be employed as COVID-19 medicinal agents [53].

Molecular Docking
This study had a particular focus on the important residue GLU 166 and the c dyad CYS 145, as well as HIS 41 of the main protease (M pro ). All of the 69 comp identified were tested against M pro . Among these, 12 were docked to the active sit M pro ( Figure 2). As shown in Table 1, they had low energy scores and interacted w active sites in the pocket of the M pro enzyme. The binding scores of these molecule significantly low, ranging from −7.8 to −5.6 kcal/mol. The interaction of oxa [20.8.0.0(7,16)]triaconta-1(22),7(16),9,13,23,29-hexaene with the active site has ge been shown to be more efficient. As sterols interact with the active site, they can be ising candidates, particularly oxygenous tetrahydroxy sterol, which has a high te

Molecular Docking
This study had a particular focus on the important residue GLU 166 and the catalytic dyad CYS 145, as well as HIS 41 of the main protease (M pro ). All of the 69 compounds identified were tested against M pro . Among these, 12 were docked to the active site of the M pro ( Figure 2). As shown in Table 1, they had low energy scores and interacted with the active sites in the pocket of the M pro enzyme. The binding scores of these molecules were significantly low, ranging from −7.8 to −5.6 kcal/mol. The interaction of oxatricyclo [20.8.0.0(7,16)]triaconta-1(22),7(16), 9,13,23,29- been shown to be more efficient. As sterols interact with the active site, they can be promising candidates, particularly oxygenous tetrahydroxy sterol, which has a high tendency to form hydrogen bonds. Schiff base ligands have been shown to inhibit M pro in previous studies [54] as well as caffeine [54,55], nethylxanthines [56], natural product isolates [57], glycyrrhizin [58], ML188 [59], pyrimidonic and pyridonic pharmaceuticals [60,61], kaempferol [62], fungal natural products [63], marine natural compounds [64], Ceftazidime [65], hepatitis C virus protease drugs [66], and other inhibitors [67][68][69][70][71][72]. In our molecular docking study, these natural compounds were identified as potential M pro inhibitors.
Plants 2023, 12, x FOR PEER REVIEW 5 of 20 molecular docking study, these natural compounds were identified as potential M pro inhibitors.

Antimicrobial Activity
In the present study, the antimicrobial activity of Saussurea costus extracts (aqueous and acetic acid extracts) was analyzed qualitatively and quantitatively. The results of the disc-diffusion test are represented in Table 2. According to the results, the acetic acid extract revealed significant antimicrobial activity against all tested microorganisms. The most susceptible microorganism was Candida albicans with 38.5 ± 1.5 mm, followed by Bacillus cereus with 25.0 ± 2.0 mm, Salmonella enterica with 18.0 ± 0.0 mm, Staphylococcus aureus with 16.5 ± 0.5 mm, Escherichia coli with 14.0 ± 1.0 mm, and Pseudomonas aeruginosa with 13.5 ± 0.5 mm. Our results are in agreement with previous published studies which showed that the methanol and ethanol extracts of Saussurea lappa showed significant antibacterial and antifungal activity [73]. Essential oils of Saussurea costus roots were also cited to have high antimicrobial efficacy [74]. Our results revealed noticeable anti-Candida activity higher than the reference antibiotic (clotrimazole, 5 mg/mL), which was in agreement with previously published reports showing that Candida albicans was highly susceptible to the semi-polar and non-polar extracts [75]. The antimicrobial activity can be attributed to some phytochemical molecules in the acetic acid extract. Table 2. The zones of inhibition (in mm) of Saussurea costus aqueous and acetic acid extracts against examined microorganisms (mean ± standard deviation).

Microorganisms
Acetic Acid Extract (100 mg/mL)  Table 3. These results support those of the disc-diffusion test, which showed that the MIC, which was the lowest concentration of Saussurea costus acetic acid root extract that inhibited the visible growth of microorganisms after overnight incubation, was 25 mg/mL for the bacterial stains and 6.25 mg/mL for Candida albicans. The results suggest that this yeast is highly susceptible to Saussurea costus and indicate that this plant contains significant organic active ingredients for treating pathogenic Candida spp. Diseases caused by Candida have become much more common around the world, and in some patient groups, the death rate is over 70% [76]. Candida spp. causes cutaneous, gastrointestinal, and vaginal infections, as well as severe vaginitis, endophthalmitis, and candidiasis for medically compromised patients [77]. Moreover, previous research showed that dehydro-costus lactone is the major anti-candida component of this plant [54]. The values of MBC (for bacteria) and MFC (for the fungus), which show the minimum concentrations of Saussurea costus acetic acid extract that killed 99.9% of the examined microorganisms, were 50 mg/mL for all bacteria except for 25 mg/mL for Staphylococcus aureus and 12.5 mg/mL for Candida albicans (MFC). Additionally, the MBC/MIC was less than four, leading us to conclude that the extract had a bactericidal effect on the bacterial strains, and the MFC/MIC ratio was also less than four, which means that it demonstrated fungicidal activity against Candida albicans. When an extract's MBC/MIC ratio is less than four, the extract is regarded as bactericidal, and if the ratio is more than or equal to four, the extract is deemed bacteriostatic [78]. Furthermore, a compound is classified as fungistatic when the MFC/MIC ratio is greater than or equal to four and as fungicidal when the MFC/MIC ratio is less than four [79]. Therefore, we concluded that Saussurea costus acetic acid extract has bactericidal and fungicidal properties.

Antiviral Activity by Saussurea costus Acetic Extract
The inhibitory activity of the S. costus acetic extract against SARS-CoV2 was evaluated using the pseudovirus method. This system allows the investigation of inhibitors of virus entry into the cell [80]. To evaluate the effect of the extract on variants of concern (VOCs) of SARS-CoV2, the antiviral activity was observed using D614G, Gamma, and Delta variants of the pseudotyped virus. No antiviral effect of the acetic extract from Saussurea costus was observed with any SAR-CoV2 variants (Figure 3), and no differences between different variants were observed using one-way ANOVA. These results are in agreement with previous results indicating that the aqueous extract of S. costus lacks entry inhibitory activity against SARS-CoV2 [54]. Furthermore, the antiviral activity of S. costus acetic extract against HSV-1, another enveloped virus, was evaluated. For this purpose, serial dilutions of the extract were added post-virus-adsorption, and the amount of virus produced was evaluated by qPCR. The S. costus acetic extract showed antiviral activity with an effective concentration 50 (EC50) of 82.6 µg/mL, a cytotoxic concentration (CC50) of 162.9 µg/mL, and a selectivity index of 1.9 (Figures 4 and 5).
Plants 2023, 12, x FOR PEER REVIEW 10 of 20 than four [79]. Therefore, we concluded that Saussurea costus acetic acid extract has bactericidal and fungicidal properties..

Antiviral Activity by Saussurea costus Acetic Extract
The inhibitory activity of the S. costus acetic extract against SARS-CoV2 was evaluated using the pseudovirus method. This system allows the investigation of inhibitors of virus entry into the cell [80]. To evaluate the effect of the extract on variants of concern (VOCs) of SARS-CoV2, the antiviral activity was observed using D614G, Gamma, and Delta variants of the pseudotyped virus. No antiviral effect of the acetic extract from Saussurea costus was observed with any SAR-CoV2 variants (Figure 3), and no differences between different variants were observed using one-way ANOVA. These results are in agreement with previous results indicating that the aqueous extract of S. costus lacks entry inhibitory activity against SARS-CoV2 [54]. Furthermore, the antiviral activity of S. costus acetic extract against HSV-1, another enveloped virus, was evaluated. For this purpose, serial dilutions of the extract were added post-virus-adsorption, and the amount of virus produced was evaluated by qPCR. The S. costus acetic extract showed antiviral activity with an effective concentration 50 (EC50) of 82.6 µg/mL, a cytotoxic concentration (CC50) of 162.9 µg/mL, and a selectivity index of 1.9 (Figures 4 and 5).  A time-of-infection study was performed to identify the extract's effect on the viral cycle. The extract was added at different times during infection. In the pre-infection condition, the acetic extract was added 2 h before the addition of the virus, then it was removed, and the cells were infected. In the adsorption condition, the virus was added at the same time as the extract and incubated for one hour and then replaced with the medium. Under the post-entry condition, the extract was added after the virus entered the cell. The Saussurea costus acetic extract showed pre-infection and post-entry antiviral activities, indicating that its compounds act by different antiviral mechanisms.
cycle. The extract was added at different times during infection. In the pre-infection condition, the acetic extract was added 2 h before the addition of the virus, then it was removed, and the cells were infected. In the adsorption condition, the virus was added at the same time as the extract and incubated for one hour and then replaced with the medium. Under the post-entry condition, the extract was added after the virus entered the cell. The Saussurea costus acetic extract showed pre-infection and post-entry antiviral activities, indicating that its compounds act by different antiviral mechanisms. In a previous study, the antiviral activity of the aqueous extract of S. costus (EC50 = 1.35 mg/mL, CC50 = 4.92 mg/mL, and SI = 3.6) was reported. In the present work, a similar behavior was observed with the S. costus acetic extract (EC50 = 82.6 mg/mL, CC50 = 162.9 mg/mL, and SI = 1.9). However, a difference in the mechanisms of action of the extracts was observed. The aqueous extract only showed post-entry antiviral activity, whereas the acetic extract also inhibited the virus in the pre-infection condition. This result suggests that the acetic extract also induced an antiviral state in cells. In a previous study, the antiviral activity of the aqueous extract of S. costus (EC50 = 1.35 mg/mL, CC50 = 4.92 mg/mL, and SI = 3.6) was reported. In the present work, a similar behavior was observed with the S. costus acetic extract (EC50 = 82.6 mg/mL, CC50 = 162.9 mg/mL, and SI = 1.9). However, a difference in the mechanisms of action of the extracts was observed. The aqueous extract only showed post-entry antiviral activity, whereas the acetic extract also inhibited the virus in the pre-infection condition. This result suggests that the acetic extract also induced an antiviral state in cells.  The percentage of inhibition was determined as the ratio between treated and untreated infected cells. Data are expressed as mean +/− SD for n = 3. Statistical analysis was performed using one-way ANOVA, followed by multiple comparisons testing with significance indicated as * p < 0.05, ** p < 0.01.

Chemicals
All chemicals used (acetic acid, dimethyl sulfoxide, ethanol, anhydrous sodium sulfite) were of analytical grade, were used as received without any further purification.

Sample Preparation and Extraction
Roots of Saussurea costus of high quality were purchased from "the attar shop", which is a trusted herbal store in Riyadh, Saudi Arabia, with a license from the government to Figure 5. Time-of-addition assay results for Saussurea costus acetic extract. Vero cells were infected at MOI 1.5 and incubated with the extract at different times during the infection, as described in the Materials and Methods. After 48 h post-infection, the virus genome was quantitated in the supernatant via qPCR. The percentage of inhibition was determined as the ratio between treated and untreated infected cells. Data are expressed as mean +/− SD for n = 3. Statistical analysis was performed using one-way ANOVA, followed by multiple comparisons testing with significance indicated as * p < 0.05, ** p < 0.01.

Chemicals
All chemicals used (acetic acid, dimethyl sulfoxide, ethanol, anhydrous sodium sulfite) were of analytical grade, were used as received without any further purification.

Sample Preparation and Extraction
Roots of Saussurea costus of high quality were purchased from "the attar shop", which is a trusted herbal store in Riyadh, Saudi Arabia, with a license from the government to sell herbs; the source of Saussurea costus is Kashmir in the northwestern region of India. The root of Saussurea costus was air-dried at room temperature for many days. The dried roots were ground using an electric grinder. Dried and powdered samples of Saussurea costus acetic acid solvent (99.8%) extract (Scharlau Sentmenat, Barcelona, Spain) underwent filtration, and the filtrate was allowed to dry on a glass Petri dish at room temperature. Fifty grams of dried Saussurea roots were extracted in 150 milliliters of acetic acid at room temperature for four days with an Orbital Shaker (BioSan PSU-20i, Riga Latvia).

Gas Chromatography-Mass Spectrometry (GC/MS) Analysis
A GM/MS system from Shimadzu Kyoto Japan, with serial number 020525101565SA and a capillary column (Rtx-5MS 30 m 0.25 mm × 0.25 mm), was employed for the analysis of samples. The solution of the samples was passed through a 0.45 mm syringe filter and into a 1.5 mL GC-MS vial, where it was prepared for injection. The specimen was injected utilizing split mode with helium serving as the carrier gas, which had a 1.61 mL/min flow rate and moved inside the injector. According to the manufacturer's instructions, the temperature program started at 50 • C at a rate of 10 • C per minute and ended at 300 • C with a hold duration of 10 min. The injection port temperature was 300 degrees Celsius; the ion source was 200 • C, and the interface was 250 • C. The sample required 35 min for the analysis in scan mode in the m/z 40-500 mass-to-charge ratio range, with a 40-min run time. (GC-MS analysis conditions are shown in Table S1). The sample's contents were identified by comparing the retention index and mass fragmentation patterns of the sample to those in the National Institute of Standards and Technology library (NIST). The relative quantities of the different components were determined without correction factors based on the peak area of the GC (FID response).

Molecular Docking
As described in PubChem, Canonical SMILES was used to prepare the 3D models for molecular docking, along with Open Babel. Using the Protein Data Bank database, we downloaded the M pro of SARS-CoV-2's crystal structure (PDB ID: 6Y2E). Then, water residues were removed, and their energy was minimized using the Molecular Modeling Toolkit plugin UCSF Chimera [60,[81][82][83]. Molecular docking was accomplished using AutoDock Vina with a grid box of (−16.5 × −24.0 × 16.5) Å, and it was centered at (35.0, 65.0, 65.0) Å. UCSF Chimera was used for the visualization of images.

Microorganisms
In this investigation, six American type culture collection (ATCC) microbial isolates were utilized, including Gram-positive bacteria (Staphylococcus aureus ATCC BAA 1026 and Bacillus cereus ATCC 10876), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 10145, Salmonella enterica ATCC 14028, and Escherichia coli ATCC 9637), and a yeast (Candida albicans ATCC 10231). All of the isolated microorganisms were provided by the Department of Laboratory Sciences at Al-Rass, Qassim University in Saudi Arabia.

Disc-Diffusion Test
The disc-diffusion test was used to evaluate the initial antimicrobial activities of the extract against the selected microbial strains, using the previously reported procedure with minor modifications [84]. Briefly, the dried acetic acid extract of Saussurea costus roots was re-constituted by dissolving 500 mg of the dry extract in 10% dimethyl sulfoxide (DMSO) and mixed well using a shaker for up to 1 h. According to our pre-experimental evaluation and the literature, 10% DMSO has no inhibitory effect on microbial growth [85][86][87]. A fresh microbial suspension adjusted to 0.5 McFarland turbidity (10 8 CFU/mL) (CFU: colonyforming units) was streaked over sterile plates containing Mueller-Hinton Agar for bacteria or Sabouraud Dextrose Agar for the yeast (microbial media were obtained from Oxoid, UK). Under aseptic conditions, sterile paper discs (6 mm) were carefully saturated with 10 µL of the reconstituted extract (100 mg/mL) using an Eppendorf pipette. Saturated papers were immediately loaded over the streaked plates and left to settle for up to 15 min, and then the seeded plates were incubated upside-down at about 37 • C for 24 hours for bacteria and for 48 hours for Candida albicans. A disc containing 10 µL of 10% DMSO served as a negative control, whereas a disc containing chloramphenicol (2.5 mg/mL) for bacteria and clotrimazole (5 mg/mL) for yeast served as a reference drug (positive control). The inhibitory diameter was then measured in millimeters (disc included) and reported as the mean ± standard deviation for two trials.

Minimum Inhibitory Concentration MIC Assay
The minimum inhibitory concentration (MIC) values were determined using the microwell dilution method for the microorganisms determined to be sensitive to the extract in the disc diffusion experiment [88]. The microorganism inoculum was taken from stock culture (in slant tubes containing general-purpose nutrient agar, Oxoid, UK) for bacteria or Sabouraud dextrose agar (Oxoid, UK) for the yeast and inoculated in nutrient broth (general-purpose growth medium, Oxoid, UK for bacteria or Sabouraud dextrose broth for the yeast and incubated for up to 12 h and up to 24 h for the yeast to reach the exponential phase. Then, microbiological suspensions were adjusted to a turbidity standard of 0.5 McFarland units. The dry crude extract was diluted in 10% dimethyl sulfoxide (DMSO) to obtain a concentration of 100 mg/mL, and then two-fold dilutions were prepared in test tubes containing nutrient broth over a concentration range of 1.5 to 100 mg/mL. In order to prepare the 96-well plates, 100 µL of the previously prepared serial dilutions were transferred into each of seven successive wells. Then, 95 µL of nutrient broth for bacteria or Sabouraud dextrose broth for the yeast and 5 µL of microbial inoculum were added to each well, bringing the total amount to 200 µL. As a negative control in another well series, nutrient broth or Sabouraud dextrose broth with sterile normal saline instead of extract and 5 mL of the inoculum were applied to each well. Additionally, successive dilutions of the standard antimicrobial drugs (chloramphenicol or clotrimazole) were used as positive controls. The 96-well plates were covered using a sterile sealant. The contents of each well were shaken on a plate shaker at 300 rpm for 20 s, before being incubated for 24 h for bacteria of 48 h for yeast at the specified temperatures. Microbial growth was estimated by measuring the absorbance at 595 nm using an iMark Absorbance microplate reader (BIO-RAD Inc., Hercules, California, USA). In this investigation, the extract was screened twice against each organism. The MIC is the lowest concentration of a substance that inhibits the growth of microorganisms.

Minimum Bactericidal Concentration or Minimum Fungicidal Concentration MBC or MFC Assay
The minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) can be defined as the lowest concentration at which an antimicrobial agent will eradicate a certain microorganism. The agar diffusion test was used here with minor modifications [89]. After the MIC test was completed, the MBC or MFC test was conducted. In a nutshell, 50 µL from each MIC tube was taken using an Eppendorf pipette and spotted onto nutritional agar plates for bacteria or Sabouraud dextrose agar for the yeast. These plates were then incubated overnight at 30 • C -35 • C. MBC was defined as the lowest MIC that showed no detectable growth. Additionally, MBC/MIC values for bacteria and MBC/MFC values for Candida albicans were calculated to classify the extract as bactericidal/fungicidal (4 or less) or bacteriostatic/fungistatic (more than 4).
3.9. Virological Assessments 3.9.1. Cytotoxicity Assays Cytotoxicity assays were performed as previously described [90,91]. A stock solution of 100 mg/mL of the re-constituted acetic acid extract of Saussurea costus in DMSO was made. Human ACE2 Stable Cell Line and HEK293T: is a popular derivative of the original HEK293 parent cell line (HEK293T-ACE2) or Vero cells were cultured at 1 × 10 4 cells/well in a 96-well plate in the presence of different concentrations of the S. costus extract in DMEM 10% FBS (Foetal Bovine Serum) for HEK293T-ACE2 cells and in DMEM 2% FBS in the case of Vero cells. DMSO-treated cells were used as a control and normalizer. After 48 h, resazurin was added to a final concentration of 0.0015% per well. Absorbance was measured on the Multiskan TM GO (Thermo Scientific, USA) at 570 and 630 nm.

Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Entry Assay
The assay was performed in HEK293-ACE2 cells, as described previously [95]. Briefly, 1 × 10 4 cells in suspension were added to each well of 96-well plates and infected in the presence of 62.5 µg/mL of the re-constituted acetic acid extract of Saussurea costus in DMEM 10% FBS. After 48 h, the firefly luciferase activity was measured using Dual-Luciferase Reporter Assay System kit (Promega, USA) and Fluoroskan FL (Thermo Scientific). HEK293T-ACE2 cells transduced with the pseudotyped virus without the extract were used as control untreated cells. The following formula was used to calculate the percentage of inhibition: 100 − (RLUs of treated cells/RLUs of control untreated cells) × 100.
3.9.4. Antiviral Activity against Herpes Simplex Virus Type-1 (HSV-1) Antiviral activity assays were performed as previously described [96]. Briefly, Vero cells were seeded in 96-well plates at 1 × 10 4 cells/well in DMEM 7.5% FBS. The next day, the cells were infected at MOI 1.5 in DMEM 2% FBS. After 1 h of virus adsorption, the medium was replaced, and dilutions of the extract in DMEM 2% FBS were added. Control-infected cells were incubated with DMSO. Forty-eight hours later, the virus in the supernatant was quantified via qPCR, using 5 µL of SYBR ® Green Master Mix buffer (Bio-Rad) 2X, 500 pM of each primer, and 1 µL of sample as a template. Amplifications were carried out on a StepOnePlus™ thermocycler (Applied Biosystems). A two-step program of 10 min at 95 • C, 35 cycles of 15 s at 95 • C, and 30 s at 60 • C, and a final melting curve was used. The viral genome copies (VGC) were calculated using a calibration curve.

Time-of-Addition Assay of HSV-1
The time-of-addition assay was carried out under three different experimental conditions according to the time at which the extract was added. In the pre-infection condition, the extract was added two hours before viral adsorption. Subsequently, cells were washed with PBS, the virus was added, and the infection was performed as described previously. Under the adsorption conditions, the extract was added with the virus and was incubated for 1 h to 37 • C. After that, the viral inoculum was removed, cells were washed with PBS and replaced with DMEM 2% FBS, and the infection was performed as previously described.
In the post-entry condition, the viral inoculum was added and incubated for 1 h at 37 • C and washed with PBS, and the extract in DMEM 2%FBS was added. Forty-eight hours later, the virus genome in the supernatant was quantitated via qPCR, and the antiviral activity was quantified as previously described [96].

Statistical Analysis
In this study, the data are shown as the mean standard error of the mean. The one-way ANOVA (analysis of variance) method and SPSS 14.0 (SPSS Inc., Chicago, IL, USA) were used to determine whether there were statistically significant differences between the microorganisms that were tested.

Research Limitations
Experiments carried out on Saussurea costus roots grown in India and those carried out on the same plant grown in other tropical and subtropical regions may show some variations in results. The antimicrobial experiments were repeated twice, and the mean was calculated.

Conclusions
In summary, GC-MS analytical screening of Saussurea costus extract using acetic acid was investigated. The survey outcomes resulted in the detection of 69 phytochemical substances in the acetic acid extract. The order of phytochemicals showed the following trend: terpenoids > hydrocarbons > sterols > alkaloids = phenolic compounds. The crude root extract of Saussurea costus obtained using acetic acid displayed significant antibacterial activity. The inhibitory activity of sterols and terpenoids against SARS-CoV-2 was studied using molecular docking. The examined sterols and terpenoids were found to be active. Furthermore, sterols generally exhibited a more efficient interaction with the active site. Candida albicans was the most vulnerable organism, followed by Bacillus cereus, Salmonella enterica, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, respectively. Evaluations of antiviral activity against HSV-1 and SARS-CoV2 revealed a considerable beneficial effect against HSV-1 (EC50 = 82.6 g/mL; CC50 = 162.9 g/mL; selectivity index = 1.9). However, no effect was detected in terms of the inhibition of SARS-CoV2 entry.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data will be available upon suitable request.