Size | Price | Stock | Qty |
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250mg |
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500mg |
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Other Sizes |
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Targets |
SARS-CoV-2 main protease (Mpro); natural flavone
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ln Vitro |
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] |
ln Vivo |
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] |
Enzyme Assay |
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 Assay |
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. |
Animal Protocol |
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). |
ADME/Pharmacokinetics |
Metabolism / Metabolites
Silybin has known human metabolites that include O-demethylated-silybin. |
Toxicity/Toxicokinetics |
Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation Milk thistle (Silybum marianum) contains silymarin, which is a mixture of flavonolignans, mainly silibinin (also known as silybin), as well as silycristine, silydianin, quercetin and taxifolin. Silymarin is a standardized preparation extracted from the fruits (seeds) of milk thistle. Milk thistle is a purported galactogogue, and is included in some proprietary mixtures promoted to increase milk supply; however, no scientifically valid clinical trials support this use. Although a study on the high potency purified milk thistle component, silymarin, and a phosphatidyl conjugate of silymarin indicated some galactogogue activity, this does not necessarily imply activity of milk thistle itself. Galactogogues should never replace evaluation and counseling on modifiable factors that affect milk production. Limited data indicate that the silymarin components are not excreted into breastmilk in measurable quantities. Additionally, because silymarin components are poorly absorbed orally, milk thistle is unlikely to adversely affect the breastfed infant. Milk thistle and silymarin are generally well tolerated in adults with only mild side effects such as diarrhea, headache, and skin reactions. Mothers taking milk thistle to increase milk supply reported weight gain, nausea, dry mouth and irritability occasionally. Milk thistle might increase the metabolism of some drugs. Rarely, severe allergies and anaphylaxis are reported. Avoid in patients with known allergy to members of the aster (Compositea or Asteraceae) family, such as daisies, artichokes, common thistle, and kiwi because cross-allergenicity is possible. Dietary supplements do not require extensive pre-marketing approval from the U.S. Food and Drug Administration. Manufacturers are responsible to ensure the safety, but do not need to prove the safety and effectiveness of dietary supplements before they are marketed. Dietary supplements may contain multiple ingredients, and differences are often found between labeled and actual ingredients or their amounts. A manufacturer may contract with an independent organization to verify the quality of a product or its ingredients, but that does not certify the safety or effectiveness of a product. Because of the above issues, clinical testing results on one product may not be applicable to other products. More detailed information about dietary supplements is available elsewhere on the LactMed Web site. ◉ Effects in Breastfed Infants A study compared a commercial product containing silymarin 252 mg (BIO-C) to placebo every 12 hours in mothers of preterm (<32 weeks) infants. No adverse effects were observed in any of the infants. In a study of galactogogue containing 5 grams of a mixture of silymarin, phosphatidylserine and galega (goat's rue) in an unspecified proportion and from an unspecified source, none of the typical adverse effects of silymarin were noted in the breastfed infants. ◉ Effects on Lactation and Breastmilk No human data are available on the effect of milk thistle or its components on serum prolactin. A study in gilts (female domestic pigs) found that silymarin 4 grams twice daily during pregnancy and lactation found that serum prolactin levels were increased compared to gilts given placebo. The slight increase in prolactin had no effect on mammary gland development, nor on plasma progesterone or estradiol. A study was performed on 50 medically normal postpartum mothers with milk production judged to be less than normal for patients in the hospital in Lima, Peru where the study was conducted. Mothers were divided non-randomly into 2 groups of 25 women who had identical ages, weights, number of children and newborn's age, although ages were not reported. The group that was given micronized silymarin (BIO-C brand) 420 mg daily for 63 days had a baseline milk production of 602 mL daily. The milk volumes and composition (water, fats, carbohydrate and protein) of the 2 groups were not significantly different on day 0. The group given an identical placebo had a baseline milk production of 530 mL daily. Milk production was measured on day 30 and day 63 by infant weighing before and after nursing followed by emptying the breasts with a breast pump. The composition of the milk was also determined. Statistically significant differences in average milk production were found on day 30 (990 grams in the silymarin group and 650 grams in the placebo group) and on day 63 (1119 grams in the silymarin group and 701 grams in the placebo group). Milk composition was not different between the groups at the two time points. Deficiencies in this study include the lack of randomization, no investigator blinding, and no optimization of breastfeeding technique prior to study enrollment. Also, breastfeeding duration and long-term infant growth were not studied. In a randomized, double blind study, a placebo (5 grams of lactose) or a commercial product (Piùlatte Plus, Milte) containing 5 grams of a mixture of silymarin, phosphatidylserine and galega (goat's rue) was given once daily to mothers of preterm infants. Phosphatidylserine purportedly improves the bioavailability of silymarin. The medication or placebo was given from day 3 to day 28 postpartum. Mothers pumped using a breast pump every 2 to 3 hours during the day and as desired at night. Milk production was measured on days 7, 14 and 28 postpartum. Daily milk production averaged 200 mL in the treated group and 115 mL in the control group. The total amount of milk produced during the study period and the proportion of women producing more than 200 mL daily was greater in the treated group than controls on days 7 and 28. Mothers were contacted at 3 and 6 months postpartum concerning breastmilk production. Of the 89 mothers who responded satisfactorily at 3 months, more mothers who had received silymarin-galega were exclusively breastfeeding than those who received placebo (22/50 vs 12/50). Also, more mothers were feeding more than 50% breastmilk to their infants in the treatment group than the placebo group (29/50 vs 18/50). At 6 months postpartum, more mothers were feeding more than 50% breastmilk to their infants in the treatment group than in the placebo group (22/50 vs 12/50). These differences were statistically significant. A randomized study compared a commercial product containing micronized silymarin 252 mg (BIO-C) to placebo every 12 hours in mothers of preterm (<32 weeks) infants, beginning at 10 days postpartum. Mothers used a breast pump 6 times daily and measured milk output before beginning, 5 times during the 28 days of treatment, and on days 36 and 45. No difference in milk production was observed between the two groups at any time point. The mothers' guesses of whether they had taken placebo or silymarin were no better than chance. In a survey of 188 nursing women from 27 states (52% from Louisiana), 24 had used milk thistle as a galactogogue. Of those who used it, 52% were not sure that it increased their milk supply and 4 reported unspecified side effects. In a survey of nursing mothers in Australia, 40 mothers were taking milk thistle as a galactogogue. On average, mothers rated milk thistle as being between “slightly effective” and “moderately effective” on a Likert scale. Ten percent of mothers taking milk thistle reported experiencing adverse reactions, most commonly weight gain, nausea, dry mouth and irritability. A retrospective study was performed in a Greek hospital on 161 mothers who were given Silitidil (a standardized extract consisted of 33% silymarin, 33% lecithin and 33% phosphatidylserine supplied as Piùlatte by Humana) 5 grams daily for 14 days. Mothers who were given Siltidil had twins or premature newborns, or whose neonates had weight loss greater than 10% of body weight, needed phototherapy, or required transport to a tertiary intensive care unit, and mothers unable to breastfeed due to any other reason. Telephone follow-up was done at 10 days, 1, 4 and 6 months. Breastfeeding rates (exclusive and nonexclusive) were 100% during their first week, 98.8% during the first month, 87% during the first 4 months, 56.5% at 6 months, 41% at 1 year and 19.3% over 1 year of age. The retrospective nature of this study and lack of a control group, blinding, and characterization of breastfeeding, among other problems, make this paper impossible to interpret. A double-blind placebo-controlled trial randomized mothers of preterm (32 weeks or less) infants to a product that contained 120 mg silymarin and 120 mg of phosphatidylserine (Silitidil, The Netherlands) or placebo. Of the 91 randomized mother-infant-dyads, 68 (35 Siltidil, 46 placebo) completed the study per protocol. Their mean daily milk production at 21 days was 506 mL with the Siltidil and 523 mL for placebo, which was not statically significant. Pumping frequency and duration did not differ at any visit. There was no difference in the urinary prolactin/creatinine ratio before and after pumping and no correlation with milk production. The authors concluded that the silymarin product does not increase mean daily milk volume in mothers of premature infants of 32 weeks or less of gestation compared to placebo. |
References |
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Additional Infomation |
Silibinin is a flavonolignan isolated from milk thistle, Silybum marianum, that has been shown to exhibit antioxidant and antineoplastic activities. It has a role as an antioxidant, an antineoplastic agent, a hepatoprotective agent and a plant metabolite. It is a flavonolignan, a polyphenol, an aromatic ether, a benzodioxine and a secondary alpha-hydroxy ketone.
Silibinin is the major active constituent of silymarin, a standardized extract of the milk thistle seeds, containing a mixture of flavonolignans consisting of silibinin, isosilibinin, silicristin, silidianin and others. Silibinin is presented as a mixture of two diastereomers, silybin A and silybin B, which are found in an approximately equimolar ratio. Both in vitro and animal research suggest that silibinin has hepatoprotective (antihepatotoxic) properties that protect liver cells against toxins. Silibinin has also demonstrated in vitro anti-cancer effects against human prostate adenocarcinoma cells, estrogen-dependent and -independent human breast carcinoma cells, human ectocervical carcinoma cells, human colon cancer cells, and both small and nonsmall human lung carcinoma cells. Silibinin has been reported in Aspergillus iizukae, Silybum eburneum, and other organisms with data available. Silymarin is a mixture of flavonolignans isolated from the milk thistle plant Silybum marianum. Silymarin may act as an antioxidant, protecting hepatic cells from chemotherapy-related free radical damage. This agent may also promote the growth of new hepatic cells. (NCI04) The major active component of silymarin flavonoids extracted from seeds of the MILK THISTLE, Silybum marianum; it is used in the treatment of HEPATITIS; LIVER CIRRHOSIS; and CHEMICAL AND DRUG INDUCED LIVER INJURY, and has antineoplastic activity; silybins A and B are diastereomers. Drug Indication Currently being tested as a treatment of severe intoxications with hepatotoxic substances, such as death cap (Amanita phalloides) poisoning. In chronic liver diseases caused by oxidative stress (alcoholic and non-alcoholic fatty liver diseases, drug- and chemical-induced hepatic toxicity), the antioxidant medicines such as silymarin can have beneficial effect. Liver cirrhosis, non-alcoholic fatty liver and steatohepatitis are risk factors for hepatocellular carcinoma (HCC). Insulin resistance and oxidative stress are the major pathogenetic mechanisms leading the hepatic cell injury in these patients. The silymarin exerts membrane-stabilizing and antioxidant activity, it promotes hepatocyte regeneration; furthermore it reduces the inflammatory reaction, and inhibits the fibrogenesis in the liver. These results have been established by experimental and clinical trials. According to open studies the long-term administration of silymarin significantly increased survival time of patients with alcohol induced liver cirrhosis. Based on the results of studies using methods of molecular biology, silymarin can significantly reduce tumor cell proliferation, angiogenesis as well as insulin resistance. Furthermore, it exerts an anti-atherosclerotic effect, and suppresses tumor necrosis factor-alpha-induced protein production and mRNA expression due to adhesion molecules. The chemopreventive effect of silymarin on HCC has been established in several studies using in vitro and in vivo methods; it can exert a beneficial effect on the balance of cell survival and apoptosis by interfering cytokines. In addition to this, anti-inflammatory activity and inhibitory effect of silymarin on the development of metastases have also been detected. In some neoplastic diseases silymarin can be administered as adjuvant therapy as well. [1] Context: Silymarin (SM) is extracted from milk thistle Silybum marianum L. [Asteraceae (Compositae)] and known for antioxidative and anti-inflammatory effects. Objective: The potential antidepressant-like effect of acute SM and possible involvement of nitric oxide (NO) were determined in male mice. Material and methods: SM was administered orally (5, 10, 20, 50, 100, and 200 mg/kg; p.o.) 60 min before the tests. After assessment of locomotor activity, the immobility time was measured in forced swimming test (FST) and tail suspension test (TST). To assess the possible involvement of NO, a non-specific NO synthase inhibitor, L-NAME (10 mg/kg, i.p.), and a specific iNOS inhibitor, aminoguanidine (AG) (50 mg/kg, i.p.), were administered separately 30 min before SM (20 and 100 mg/kg). Results: 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 SM is probably mediated at least in part through NO and SM may increase NO tune.[3] In recent years (2020, in particular), several studies have focused on the research of natural food-derived compounds exhibiting antiviral activities both in silico and in vitro. Among these substances, flavonoids are particularly noteworthy. One of the first papers exploring the antiviral effects of flavonoids on coronaviruses was conducted in 1990. Here, the authors showed that quercetin, at a concentration value of 60 μg/mL, reduced infectivity of human and bovine coronaviruses, OC43, and NCDCV by 50%. Quercetin may be considered a promising candidate for further preclinical studies as its ability to influence the thermal stability of SARS-CoV-2 Mpro, interact with SARS-CoV-2 Mpro, and bind to its active site has recently been demonstrated. Based on the results obtained in silico, our group decided to test, by a series of in vitro experiments, the effect of a natural compound known as silymarin on SARS-CoV-2 Mpro. Silymarin exerts a remarkable inhibitory action, as the EC50 observed by our research group is in the micromolar range. In addition, an interesting parameter is the residual activity of the Mpro because of its very low value. We also analyzed the potential effect of taxifolin, a component of the silymarin complex. Docking has shown that taxifolin is not an excellent protease ligand (calculated binding energy −7.7 kcal mol−1) and this was further confirmed by the experimental analysis (see Figure 4). These data confirm our hypothesis that the active component of silymarin is silybin. The choice of using the silymarin complex and not silybin (investigated in silico) is because it is more readily accessible to clinicians and patients, because it is commercially available in the form of supplements containing 51–78% w/w of silymarin. However, a study conducted using computational and experimental approaches has delineated the ability of silybin to target the virus replication machinery by targeting RdRp/nsp12, a central component of a multi-subunit RNA-synthesis complex. Silymarin, and its derivative silybin, present another interesting property as reactive oxygen species (ROS) scavengers and modulators of glutathione levels in various organs. Thus, despite our analysis showed that the silymarin inhibitory action decreases in the presence of DTT, its efficacy may not be reduced in cells or tissues containing high concentrations of glutathione. Finally, pharmacokinetic studies have shown that silymarin is absorbed by the oral route and distributes into the alimentary tract. It is subject to enterohepatic circulation, ensuring that low doses of intake could be sufficient. Acute, subacute, and chronic toxicity is very low. Silymarin can also be consumed in pregnancy because it is devoid of embryotoxic potential. Moreover, silymarin is safe at therapeutic doses and is well tolerated at high doses. For these reasons, we hypothesize that it can be used not only as a therapeutic strategy, but also as a preventive measure against SARS-CoV-2 infection, because of a possible maintenance of its circulating levels. Surely, to confirm this hypothesis, future clinical trials are needed. In conclusion, our study proves that silymarin, as a natural food-derived compound, whose pharmacological, toxicological, and therapeutic profiles are known, can be considered a promising and safe therapeutic strategy against COVID-19. Obviously, these data obtained in silico and in vitro should be confirmed by further in vivo studies, to set the optimal dosages, and assess the efficacy of this compound in inhibiting SARS-CoV-2 Mpro in humans. |
Molecular Formula |
C25H22O10
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Molecular Weight |
482.44
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Exact Mass |
482.121
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Elemental Analysis |
C, 62.24; H, 4.60; O, 33.16
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CAS # |
65666-07-1
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Related CAS # |
Silybin A;22888-70-6;Silybin B;142797-34-0
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PubChem CID |
31553
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Appearance |
Light yellow to yellow solid powder
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Density |
1.5±0.1 g/cm3
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Boiling Point |
793.0±60.0 °C at 760 mmHg
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Flash Point |
274.5±26.4 °C
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Vapour Pressure |
0.0±2.9 mmHg at 25°C
|
Index of Refraction |
1.684
|
LogP |
2.59
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Hydrogen Bond Donor Count |
5
|
Hydrogen Bond Acceptor Count |
10
|
Rotatable Bond Count |
4
|
Heavy Atom Count |
35
|
Complexity |
750
|
Defined Atom Stereocenter Count |
4
|
SMILES |
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
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InChi Key |
SEBFKMXJBCUCAI-HKTJVKLFSA-N
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InChi Code |
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
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Chemical Name |
(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
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Synonyms |
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
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
DMSO : ~100 mg/mL
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Solubility (In Vivo) |
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.) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.0728 mL | 10.3640 mL | 20.7280 mL | |
5 mM | 0.4146 mL | 2.0728 mL | 4.1456 mL | |
10 mM | 0.2073 mL | 1.0364 mL | 2.0728 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.