SARS-CoV-2 main protease (Mpro); natural flavone

Silymarin (0-120 μg/mL; 24 hours) suppresses the viability of AGS cells. At 20 μg/mL, 71.5% at 40 μg/mL, and 59.8% at 60 μg/mL, AGS cell viability is measured. At 80 μg/mL, 44.5%, 35.3%, and 33.9% were found [1]. At dilution, silymarin (40–80 μg/mL; 24 hours) inhibits AGS cells. At 40 μg/mL and 80 μg/mL, it inhibits AGS cell migration by 59.4% and 21.7%, respectively [1].

---

Apoptosis is regarded as a therapeutic target because it is typically disturbed in human cancer. Silymarin from milk thistle (Silybum marianum) has been reported to exhibit anticancer properties via regulation of apoptosis as well as anti‑inflammatory, antioxidant and hepatoprotective effects. In the present study, the effects of silymarin on the inhibition of proliferation and apoptosis were examined in human gastric cancer cells. The viability of AGS human gastric cancer cells was assessed by MTT assay. The migration of AGS cells was investigated by wound healing assay. Silymarin was revealed to significantly decrease viability and migration of AGS cells in a concentration‑dependent manner. In addition, the number of apoptotic bodies and the rate of apoptosis were increased in a dose‑dependent manner as determined by DAPI staining and Annexin V/propidium iodide double staining. The changes in the expression of silymarin‑induced apoptosis proteins were investigated in human gastric cancer cells by western blotting analysis. Silymarin increased the expression of Bax, phosphorylated (p)‑JNK and p‑p38, and cleaved poly‑ADP ribose polymerase, and decreased the levels of Bcl‑2 and p‑ERK1/2 in a concentration‑dependent manner. [2]

---

In late 2019, a global pandemic occurred. The causative agent was identified as a member of the Coronaviridae family, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we present an analysis on the substances identified in the human metabolome capable of binding the active site of the SARS-CoV-2 main protease (Mpro). The substances present in the human metabolome have both endogenous and exogenous origins. The aim of this research was to find molecules whose biochemical and toxicological profile was known that could be the starting point for the development of antiviral therapies. Our analysis revealed numerous metabolites—including xenobiotics—that bind this protease, which are essential to the lifecycle of the virus. Among these substances, silybin, a flavolignan compound and the main active component of silymarin, is particularly noteworthy. Silymarin is a standardized extract of milk thistle, Silybum marianum, and has been shown to exhibit antioxidant, hepatoprotective, antineoplastic, and antiviral activities. Our results—obtained in silico and in vitro—prove that silybin and silymarin, respectively, are able to inhibit Mpro, representing a possible food-derived natural compound that is useful as a therapeutic strategy against COVID-19. [4]

During the forced swim test (FST), silymarin (oral gavage; 10, 20, 50, 100, and 200 mg/kg) shortens the stopping time in a dance pattern. Additionally, it lowers silymarin's ED50 in the tail suspension test (TST), which is roughly 10 mg/kg. In both trials, a dose of 100 mg/kg was found to be the most efficacious dose [3].

---

Silymarin (SM) at its effective doses 10, 20, 50, and 100 mg/kg decreased the immobility time in a dose-dependent manner (p < 0.01, p < 0.05, p < 0.05, and p < 0.001, respectively) in FST. SM (10, 20, 50, and 100 mg/kg) also lowered the immobility measure dose dependently in TST (p < 0.01, p < 0.05, p < 0.01, and p < 0.001, respectively). In addition, 50% of maximum response (ED50) of SM was around 10 mg/kg. The dose 100 mg/kg proved the most effective dose in both the tests. Further, this effect was not related to changes in locomotor activity. Moreover, L-NAME reversed the effect of SM (20 and 100 mg/kg) in FST and SM (100 mg/kg) in TST. However, AG did not influence this impact. Conclusion: The antidepressant-like effect of Silymarin (SM) is probably mediated at least in part through NO and SM may increase NO tune. [3]

In Vitro Analyses [4]
Enzymatic assays were performed essentially as described in our previous work. Briefly, we used the purified SARS-CoV-2 Mpro Untagged at a final concentration value of 0.5 ng/µL in the reaction buffer supplied by the manufacturer. Silymarin (SM) and taxifolin were used. Experiments were performed at room temperature in a Tecan microplate reader using an internally quenched fluorogenic FRET substrate (DABCYL-KTSAVLQSGFRKME-EDANS) as substrate at a concentration value of 40 µM. For this peptide, a Km of 17 µM and a Kcat of 1.9 s−1 on the Mpro have been reported. The experimental veterinary drug GC376 was used at a concentration value of 100 µM as a positive control. The latter is capable of inhibiting SARS-CoV-2 Mpro with an IC50 of approximately 0.42 µM. The assays were carried out in the reaction buffer supplied by the manufacturer, in the presence of 0.1 µM of DTT derived from the storage solution of the enzyme (DTT free condition) or in the presence of 1 mM of DTT.

Cell Viability Assay[2]
Cell Types: AGS cells
Tested Concentrations: 20 µg/ml, migration of 40 µg. /ml, 80 µg/ml, 100 µg/ml and 120 µg/ml
Incubation Duration: 24 hrs (hours)
Experimental Results: Demonstrated significant concentration-dependent inhibitory effect on AGS cells starting from 20 µg/ml.

---

Cell viability assay [2]
An MTT assay was performed to investigate the effect of Silymarin (SM) on proliferation of AGS human gastric cancer cells. AGS cells were seeded in 96-well plate at a density of 2×104 cells/ml and cultured in the RPMI-1640 culture medium for ~24 h in an incubator at 37°C and 5% CO2. The cells were then treated with Silymarin (SM) at concentrations of 0, 20, 40, 60, 80, 100 and 120 µg/ml. After 24 h, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution was added to the 96-well plates containing AGS cells in a volume of 40 µl/well and cultured for 2 h. After removing the MTT solution, 100 µl/well of dimethyl sulfoxide (DMSO) was added to dissolve all formazan formed in the well, and the absorbance was measured at 595 nm with an ELISA-reader. The percentage of viable cells was estimated in comparison to the untreated control cells.

---

Wound healing assay [2]
AGS human gastric cancer cells were seeded in a 60-mm dish and cultured for 24 h. A uniform wound was created by scratching cells with a sterile 1-ml blue-pipette tip. The culture medium was replaced with that containing Silymarin (SM) at concentrations of 0, 40 and 80 µg/ml, followed by culture for 24 h. After 24 h, the wound healing rates of cells treated with Silymarin (SM) at concentrations of 40 and 80 µg/ml and those without silymarin were examined on images captured under a phase contrast microscope (×200) at 0 and 24 h after wound incision.

---

DAPI staining [2]
4′,6-Diamidino-2-phenylindole (DAPI) staining was performed to examine the specific morphological changes in the nuclei with induction of apoptosis. AGS human gastric cancer cells were seeded in a 60-dish at 1×105 cells/ml, stabilized for 24 h, treated with Silymarin (SM) at 0, 40 and 80 µg/ml, and cultured in an incubator for 24 h. The cells were then washed twice with PBS and fixed with the 4% paraformaldehyde solution for 15 min. Subsequently, they were washed again with PBS, treated with 1:10 diluted DAPI solution (2 ml), and observed under a fluorescence microscope at an ×200 magnification in a dark room.

---

Flow cytometric analysis [2]
Apoptosis was measured using an FITC-Annexin V apoptosis detection kit. For Annexin V-propidium iodide (PI) staining, AGS human gastric cancer cells were treated with Silymarin (SM) at concentrations of 0, 40 and 80 µg/ml. Cells cultured for 24 h were washed with PBS, suspended in trypsin-EDTA, and centrifuged (260 × g, 5 min, 4°C) to obtain the cell pellet. They were then washed twice with cold PBS and centrifuged to obtain the cell pellet. Next, they were suspended in 1X binding buffer at a concentration of 1×106 cells/ml. Fluorescein isothiocyanate (FITC)-conjugated Annexin V and phycoerythrin (PE)-conjugated PI were then added and reacted for 15 min followed by flow cytometry.

---

Western blot analysis [2]
Western blot analysis was performed to determine the changes in protein expression associated with Silymarin (SM) treatment. AGS human gastric cancer cells cultured in 175-cm2 flasks in an incubator at 37°C and 5% CO2 were treated with Silymarin (SM) at concentrations of 0, 40 and 80 µg/ml and cultured for 24 h. Trypsin-EDTA was added to the cells, which were then suspended and centrifuged (260 × g, 5 min, 4°C). Cell lysis buffer was added to the cell pellet, and allowed to react at 4°C for 20 min. The supernatant obtained by centrifugation at 15,000 × g for 5 min was used as the cell lysate. The concentration of the extracted protein was determined by Bradford protein assay. Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% skim milk for 2 h, followed by the addition of the primary antibodies.

In vivo xenograft tumor model [2]
Ten BALB/c nude mice (Four-week-old, male, 20 g) were housed in isolated and ventilated cages (≤3 mice per cage). Mice were maintained under a 12-h light/dark cycle, and housed under controlled temperature (23±3°C) and humidity (40±10%) conditions. Mice were allowed access to laboratory pelleted food and water ad libitum. Cervical dislocation was used to sacrifice the mice. AGS human gastric cancer cells were cultured in an incubator at 37°C and 5% CO2 in RPMI-1640 culture medium containing 5% FBS. When the cell density reached approximately 80–90%, they were transferred into 175-cm2 flasks and suspended by addition of trypsin-EDTA, followed by centrifugation (260 × g, 5 min, 4°C). They were then washed with PBS and centrifuged again (260 × g, 3 min, 4°C) to obtain the cell pellet, which was divided into aliquots in culture medium at a concentration of 1×107 cells/ml. AGS cells were injected in a volume of 200 µl (1:1 Matrigel mixture) into the backs of male BALB/c nude mice. One week later, after tumors had formed, the mice were anesthetized with diethyl ether and the tumor tissue was extracted, cut into blocks ~1 mm3, and then reinjected into nude mice. Diethyl ether was provided as inhalant. They were grouped according to uniform tumor size. The injected group received oral administration of 100 mg/kg of Silymarin diluted in ethanol five times per week, at the same time of day in each session, for 2 weeks. The control group received oral administration of a mixture of ethanol and distilled water according to the same schedule for 2 weeks. During the administration period, the general conditions of the mice were examined, and tumor size was measured twice a week with Vernier calipers and calculated as follows: Size (mm3) = [0.5 × (length + width)]3.

---

Animals and experimental groups [3]
Male NMRI (National Medical Research Institute) mice weighing 20–27 g were used throughout the study. Animals were allowed free access to food and water. All behavioral experiments were conducted during the period between 10:00 and 14:00 A.M. with normal room light (12 h regular light/dark cycle) and temperature (22 ± 1 °C). We handled the mice as indicated in the criteria proposed by the Guide for the Care and Use of Laboratory Animals (NIH US publication, no. 23-86, revised 1985).

Mice (288) were divided into 36 groups of 8. Randomly, 18 groups were assigned for FST and 18 groups for TST. Control groups received only the vehicle (saline; i.p. and p.o.). Fluoxetine (20 mg/kg, i.p.) (Owolabi et al., Citation2014) was applied as a reference drug. To assess the antidepressant-like effect of Silymarin (SM)/SM, six groups were assigned as treatment groups and given Silymarin (SM)/SM orally (5, 10, 20, 50, 100, and 200 mg/kg; p.o.), 60 min prior to the behavioral tests. Ten groups were determined for antagonist administration and possible involvement of NO synthesis on the antidepressant-like activity of SM was studied using administration of two effective doses of Silymarin (SM)/SM (20 and 100 mg/kg; p.o.) with a non-effective dose of l-NAME (10 mg/kg, i.p.) (Sadaghiani et al., Citation2011) or a non-effective dose of AG (50 mg/kg; i.p.) (Sadaghiani et al., Citation2011). Both l-NAME and AG were administered 90 min before the tests. Moreover, one group received only l-NAME or AG. All drugs were dissolved in saline and prepared immediately before the experiments.

---

Silymarin (SM) toxicology [3]
The 50% lethal dose (LD50) values for Silymarin (SM)/SM are 400 mg/kg in mice and 385 mg/kg in rats. However, these values are only approximate, as they depend on the infusion rate. When the compound is given by slow infusion (over 2–3 h), values of 2000 mg/kg may be recorded in rats. Tolerance is even higher after oral administration, with values over 10 000 mg/kg (Lecomte, Citation1975). Similar results have also been obtained by Vogel et al. (Citation1975). The LD50 was 1050 and 970 mg/kg in male and female mice, respectively, and 825 and 920 mg/kg in male and female rats, respectively (Desplaces et al., Citation1975). Recently, in animal studies, SM has been reported to be non-toxic and symptom free with the maximum oral doses of 2500 and 5000 mg/kg. It has also been illustrated that SM is not teratogen and had no post-mortem toxicity (Rana et al., Citation2006).

Metabolism / Metabolites
Silymarin's known metabolites include O-demethylsilymarin.

Effects during pregnancy and lactation
◉ Overview of use during lactation
Milk thistle (Silybum marianum) contains silymarin, a mixture of flavonoid lignans, primarily composed of Silibinin (also known as silymarin), and also contains silymarin, silymarinine, quercetin, and taxine. Silymarin is a standardized preparation extracted from the fruit (seeds) of milk thistle. Milk thistle is considered a galactagogue and has been added to some patented formulations claiming to increase milk production; however, there are currently no scientifically valid clinical trials to support this use. While one study on high-purity milk thistle component silymarin and its phosphatidyl conjugates showed some galactagogue activity, this does not necessarily mean that milk thistle itself possesses this activity. Lactagogues should never replace assessment and consultation regarding controllable factors affecting milk production. Limited data suggest that silymarin is not secreted into breast milk in measurable amounts. Furthermore, due to the low oral absorption rate of silymarin, milk thistle is unlikely to have adverse effects on breastfed infants. Adults generally tolerate milk thistle and silymarin well, experiencing only mild side effects such as diarrhea, headache, and skin reactions. Mothers who take milk thistle to increase milk production occasionally experience symptoms such as weight gain, nausea, dry mouth, and irritability. Milk thistle may accelerate the metabolism of certain medications. In rare cases, there have been reports of severe allergic reactions and anaphylactic shock. Patients with known allergies to plants in the Asteraceae family (Compositea or Asteraceae) such as daisies, artichokes, thistles, and kiwifruit should avoid use due to the possibility of cross-reactivity. Dietary supplements do not require extensive premarket approval from the U.S. Food and Drug Administration (FDA). Manufacturers are responsible for ensuring product safety but are not required to prove the safety and effectiveness of dietary supplements before they are marketed. Dietary supplements may contain multiple ingredients, and the ingredients listed on the label often differ from the actual ingredients or amounts. Manufacturers may commission independent agencies to verify the quality of their products or their ingredients, but this does not guarantee the product's safety and efficacy. Given the above issues, clinical trial results for one product may not be applicable to other products. For more detailed information on dietary supplements, please visit other pages on the LactMed website.
◉ Effects on Breastfed Infants
One study compared the effects of a commercial product (BIO-C) containing 252 mg of silymarin every 12 hours administered to mothers of preterm infants (<32 weeks) with a placebo. No adverse reactions were observed in any infants.
In a study of a galactagogue containing a mixture of 5 g silymarin, phosphatidylserine, and goat's bean (goat's bean) (ratio and source not specified), no typical adverse reactions to silymarin were observed in breastfed infants.
◉ Effects on Lactation and Breast Milk
There are currently no human data on the effects of silymarin or its components on serum prolactin. A study in sows found that twice-daily administration of 4 g silymarin during pregnancy and lactation resulted in higher serum prolactin levels in sows than in sows receiving a placebo. A slight increase in prolactin has no effect on mammary gland development or plasma progesterone or estradiol levels. A study conducted in a Lima hospital in Peru included 50 medically normal postpartum women with below-normal milk production. These women were non-randomized into two groups of 25 each, with identical age, weight, number of offspring, and neonatal age (although specific ages were not reported). The group receiving micronized silymarin (BIO-C brand) 420 mg/day for 63 days had a baseline daily milk production of 602 ml. There were no significant differences in milk volume and composition (water, fat, carbohydrates, and protein) between the two groups on day 0. The group receiving the same placebo had a baseline daily milk production of 530 ml. Milk production was measured on days 30 and 63 by weighing the infants before and after breastfeeding, followed by emptying the breasts with a breast pump. Milk composition was also measured. The results showed a statistically significant difference in mean milk production between the two groups on day 30 (990g in the silymarin group, 650g in the placebo group) and day 63 (1119g in the silymarin group, 701g in the placebo group). There were no differences in milk composition between the two groups at either time point. Limitations of this study included the lack of randomization, the absence of blinding, and the failure to optimize breastfeeding techniques before enrollment. Furthermore, the duration of breastfeeding and long-term infant growth and development were not investigated.
In a randomized, double-blind study, mothers of preterm infants received either a placebo (5g lactose) or a commercially available product (Piùlatte Plus, Milte) containing 5g of silymarin, phosphatidylserine, and a mixture of goat's bean extract once daily. Phosphatidylserine is said to enhance the bioavailability of silymarin. From day 3 to day 28 postpartum, mothers received either the medication or the placebo. Breast pumping was used every 2 to 3 hours during the day and as needed at night. Milk production was measured on days 7, 14, and 28 postpartum. The average daily milk production was 200 ml in the treatment group and 115 ml in the control group. Throughout the study, the total milk production and the proportion of women producing more than 200 ml of milk per day on days 7 and 28 were significantly higher in the treatment group than in the control group. Researchers contacted mothers at 3 and 6 months postpartum to assess their breastfeeding status. At 3 months postpartum, among the 89 mothers who reported satisfactory results, the proportion of mothers receiving silymarin-goat bean treatment who exclusively breastfed was higher than that of mothers receiving a placebo (22/50 vs 12/50). Furthermore, the proportion of mothers in the treatment group who breastfed more than 50% of the time was also higher than that in the placebo group (29/50 vs 18/50). At 6 months postpartum, the proportion of mothers in the treatment group who breastfed more than 50% of the time remained higher than that in the placebo group (22/50 vs 12/50). These differences were statistically significant. A randomized study compared the effects of a commercial product (BIO-C) containing 252 mg of micronized silymarin, administered every 12 hours to mothers of preterm infants (<32 weeks) starting 10 days postpartum, versus a placebo. Mothers used breast pumps six times daily, and milk production was measured five times before treatment began, five times during the 28-day treatment period, and on days 36 and 45. At any time point, there was no difference in milk production between the two groups. Mothers' guesses about whether they were taking a placebo or silymarin were indistinguishable from random guesses. A survey of 188 breastfeeding women from 27 states (52% from Louisiana) showed that 24 had used milk thistle as a lactation stimulant. Among those who took the drug, 52% were unsure whether it increased their milk production, and four reported unspecified side effects. In an Australian survey of breastfeeding mothers, 40 mothers used milk thistle as a lactation stimulant. On average, mothers rated the efficacy of milk thistle as "slightly effective" to "moderately effective" according to the Likert scale. 10% of mothers taking milk thistle reported adverse reactions, the most common being weight gain, nausea, dry mouth, and irritability. A retrospective study conducted in a Greek hospital included 161 mothers who took 5 grams of Silitidil (a standardized extract composed of 33% silymarin, 33% lecithin, and 33% phosphatidylserine, supplied by Humana under the name Piùlatte) daily for 14 days. Mothers receiving cetirizine who gave birth to twins or premature infants, or whose newborns had a weight loss exceeding 10% of their body weight, required phototherapy, required transfer to a level 3 intensive care unit, or were unable to breastfeed for other reasons were excluded. Follow-up by telephone was conducted at 10 days, 1 month, 4 months, and 6 months postpartum. Breastfeeding rates (exclusively breastfed and not exclusively breastfed) were 100% in the first week after birth, 98.8% in the first month, 87% in the first four months, 56.5% at 6 months, 41% at 1 year, and 19.3% after 1 year. The retrospective nature of this study, the lack of a control group, blinding, and the description of breastfeeding characteristics make this study difficult to interpret. A double-blind, placebo-controlled trial randomized mothers of preterm infants (≤32 weeks of gestation) to either a product containing 120 mg of silymarin and 120 mg of phosphatidylserine (Silitidil, Netherlands) or a placebo. Of the 91 randomized mother-infant pairs, 68 pairs (35 in the Silitil group and 46 in the placebo group) completed the study according to the protocol. At 21 days, the mean daily milk production was 506 ml in the Silitil group and 523 ml in the placebo group, with no statistically significant difference. There were no differences in the frequency and duration of breastfeeding between the two groups at any visit. There was no difference in the urinary prolactin/creatinine ratio before and after breastfeeding, and it was not correlated with milk production. The authors concluded that, compared with placebo, silymarin products did not increase the average daily milk production of mothers of preterm infants at 32 weeks of gestation or shorter.

[1]. Silymarin in the prevention and treatment of liver diseases and primary liver cancer. Curr Pharm Biotechnol. 2012 Jan;13(1):210-7.

[2]. Silymarin induces inhibition of growth and apoptosis through modulation of the MAPK signaling pathway in AGS human gastric cancer cells. Oncol Rep. 2019 Nov;42(5):1904-1914.

[3]. Possible involvement of nitric oxide in antidepressant-like effect of silymarin in male mice. Pharm Biol. 2015 May;53(5):739-45.

[4]. SARS-CoV-2 Main Protease Active Site Ligands in the Human Metabolome. Molecules 2021, 26(5), 1409.

Silibinin is a flavonoid lignan isolated from milk thistle (Silybum marianum) and has been shown to possess antioxidant and antitumor activities. It has multiple functions including antioxidant, antitumor, hepatoprotective, and phytometabolism-enhancing effects. Silibinin is a flavonoid lignan, polyphenol, aromatic ether, benzodioxin, and secondary α-hydroxyketone. Silibinin is the main active ingredient of Silibininin, a standardized extract of milk thistle seeds containing various flavonoid lignans, including Silibinin, isoSilibinin, Silibininine, and Silibininine. Silibinin exists as a mixture of two diastereomers—Silibinin A and Silibinin B—in approximately equal molar ratios. In vitro and animal studies have shown that Silibinin possesses hepatoprotective (anti-hepatotoxic) properties, protecting hepatocytes from toxin damage. Silibinin has also been shown to have in vitro anticancer effects, inhibiting human prostate adenocarcinoma cells, estrogen-dependent and non-essential breast cancer cells, human cervical os cancer cells, human colon cancer cells, and small cell and non-small cell lung cancer cells. Silibinin has been reported to be found in Aspergillus iizukae, Silybum eburneum, and other organisms with relevant data. Silibinin is a mixture of flavonoid lignans isolated from Silybum marianum. Silibinin may have antioxidant effects, protecting hepatocytes from chemotherapy-related free radical damage. This substance may also promote the growth of new hepatocytes. (NCI04) Silibinin is the main active ingredient extracted from the seeds of Silybum marianum, belonging to the flavonoid class of compounds; it is used to treat hepatitis, cirrhosis, and chemical and drug-induced liver injury, and has antitumor activity; Silibinin A and B are diastereomers.

---

Drug Indications
Currently being tested as a treatment for severe hepatotoxic poisoning (e.g., Amanita phalloides poisoning). Antioxidant drugs such as silymarin may have beneficial effects on chronic liver disease caused by oxidative stress (alcoholic and non-alcoholic fatty liver, drug and chemical-induced hepatotoxicity). Cirrhosis, non-alcoholic fatty liver, and steatohepatitis are risk factors for hepatocellular carcinoma (HCC). Insulin resistance and oxidative stress are the main pathogenic mechanisms leading to hepatocellular damage in these patients. Silymarin has membrane-stabilizing and antioxidant activity, promoting hepatocyte regeneration; in addition, it can reduce inflammatory responses and inhibit liver fibrosis. These results have been confirmed through experimental and clinical trials. Open-label studies have shown that long-term use of silymarin can significantly prolong the survival of patients with alcoholic cirrhosis. Based on molecular biology methods, studies have shown that silymarin can significantly reduce tumor cell proliferation, angiogenesis, and insulin resistance. In addition, it also has anti-atherosclerotic effects and can inhibit the production of adhesion molecules and mRNA expression induced by tumor necrosis factor-α. Multiple in vitro and in vivo studies have confirmed that silymarin has chemopreventive effects on hepatocellular carcinoma (HCC); it can have a beneficial effect on the balance of cell survival and apoptosis by interfering with cytokines. In addition, silymarin also has anti-inflammatory activity and can inhibit the occurrence and development of tumor metastasis. In some neoplastic diseases, silymarin can also be used as an adjuvant therapy. [1]

---

Background: Silymarin (SM) is extracted from milk thistle (Silybum marianum L.) [Asteraceae] and is known for its antioxidant and anti-inflammatory effects.
Objective: This study aims to investigate the potential antidepressant-like effects of acute administration of SM to male mice and its possible association with nitric oxide (NO).
Materials and Methods: SM (5, 10, 20, 50, 100 and 200 mg/kg; po) was administered orally to mice 60 minutes before the experiment. After assessing the motor activity of mice, immobility time was measured in both the forced swimming test (FST) and the tail suspension test (TST). To assess the potential involvement of NO, the nonspecific NO synthase inhibitor L-NAME (10 mg/kg) and the specific iNOS inhibitor aminoguanidine (AG) (50 mg/kg) were administered intraperitoneally 30 minutes before administration of SM (20 and 100 mg/kg). Results: SM at effective doses of 10, 20, 50, and 100 mg/kg significantly reduced immobility time in the forced swimming test (FST) (p < 0.01, p < 0.05, p < 0.05, and p < 0.001). SM (10, 20, 50, and 100 mg/kg) also significantly reduced immobility time in the tail suspension test (TST) (p < 0.01, p < 0.05, p < 0.01, and p < 0.001). Furthermore, the half-maximal response dose (ED50) of SM was approximately 10 mg/kg. In both tests, the dose of 100 mg/kg was shown to be the most effective. Moreover, this effect was independent of changes in motor activity. Furthermore, L-NAME reversed the effects of SM (20 and 100 mg/kg) in the forced swimming test (FST) and the effect of SM (100 mg/kg) in the tail suspension test (TST). However, AG had no effect on this. Conclusion: The antidepressant-like effect of SM may be mediated at least in part by NO, and SM may increase NO levels. [3] In recent years (especially in 2020), some studies have focused on natural food-derived compounds with antiviral activity that have shown antiviral activity in both computer simulations and in vitro experiments. Among these substances, flavonoids are of particular interest. One of the earliest papers exploring the antiviral effects of flavonoids against coronaviruses was published in 1990. This study showed that quercetin at a concentration of 60 μg/mL reduced the infectivity of human coronavirus, bovine coronavirus, OC43, and NCDCV by 50%. Quercetin can affect and interact with the thermostability of SARS-CoV-2 Mpro and bind to its active site, thus it is considered a promising candidate drug worthy of further preclinical research. Based on the results of computer simulations, our research group decided to test the effect of a natural compound called silymarin on SARS-CoV-2 Mpro through a series of in vitro experiments. Silymarin showed a significant inhibitory effect, with EC50 values observed by our group in the micromolar range. Furthermore, the residual activity of Mpro is also a parameter of interest due to its very low value. We also analyzed the potential role of taxine, a component of the silymarin complex. Molecular docking results showed that taxine is not a good protease ligand (calculated binding energy of -7.7 kcal mol⁻¹), which was further confirmed by experimental analysis (see Figure 4). These data confirm our hypothesis that the active ingredient of silymarin is Silibinin. The silymarin complex was chosen over Silibinin (through computer simulation studies) because the former is more readily available to clinicians and patients, as it is marketed as a supplement containing 51-78% (w/w) silymarin. However, a computational and experimental study showed that Silibinin inhibits viral replication by targeting RdRp/nsp12 (a core component of the multi-subunit RNA synthesis complex). Silymarin and its derivative Silibinin possess significant properties for scavenging reactive oxygen species (ROS) and modulating glutathione levels in multiple organs. Therefore, although our analysis indicates that the inhibitory effect of silymarin is reduced in the presence of dithiothreitol (DTT), its efficacy may not be diminished in cells or tissues with high glutathione concentrations. Finally, pharmacokinetic studies show that silymarin is absorbed orally and distributed in the digestive tract. It undergoes enterohepatic circulation, thus achieving the desired effect with low-dose intake. Its acute, subacute, and chronic toxicity are all extremely low. Since silymarin is not embryotoxic, it can be taken by pregnant women. Furthermore, silymarin is safe at therapeutic doses and well-tolerated at high doses. Based on these reasons, we hypothesize that silymarin could be used not only as a treatment strategy but also as a preventative measure against SARS-CoV-2 infection, as it may maintain circulating levels. Of course, further clinical trials are needed to confirm this hypothesis. In conclusion, our study demonstrates that silymarin, as a compound from a natural food source, has well-defined pharmacological, toxicological, and therapeutic properties and can be considered a promising and safe treatment strategy for COVID-19. Clearly, these data obtained through computer simulations and in vitro experiments need to be validated through further in vivo studies to determine the optimal dosage and evaluate the efficacy of this compound in inhibiting SARS-CoV-2 Mpro in humans.

C25H22O10

482.44

482.121

C, 62.24; H, 4.60; O, 33.16

65666-07-1

Silybin A;22888-70-6;Silybin B;142797-34-0

31553

Light yellow to yellow solid powder

1.5±0.1 g/cm3

793.0±60.0 °C at 760 mmHg

274.5±26.4 °C

0.0±2.9 mmHg at 25°C

1.684

2.59

5

10

4

35

750

4

COC1=C(C=CC(=C1)[C@@H]2[C@H](OC3=C(O2)C=C(C=C3)[C@@H]4[C@H](C(=O)C5=C(C=C(C=C5O4)O)O)O)CO)O

SEBFKMXJBCUCAI-HKTJVKLFSA-N

InChI=1S/C25H22O10/c1-32-17-6-11(2-4-14(17)28)24-20(10-26)33-16-5-3-12(7-18(16)34-24)25-23(31)22(30)21-15(29)8-13(27)9-19(21)35-25/h2-9,20,23-29,31H,10H2,1H3/t20-,23+,24-,25-/m1/s1

(2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one

Legalon 70; Milk thistle; SILYMARIN; 65666-07-1; Legalon; 84604-20-6; (2R,3R)-3,5,7-trihydroxy-2-[(2R)-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one; 142796-20-1; Apihepar; Silimarin; Silymarin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL

Solubility in Formulation 1: ≥ 3 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)