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

水飞蓟素（0-120 μg/mL；24 小时）可抑制 AGS 细胞的活力。 AGS 细胞活力在 20 μg/mL、40 μg/mL 和 60 μg/mL 时分别为 71.5% 和 59.8%。在 80 μg/mL 时，检测结果为 44.5%、35.3% 和 33.9% [1]。稀释后，水飞蓟素（40–80 μg/mL；24 小时）可抑制 AGS 细胞。在 40 μg/mL 和 80 μg/mL 时，它分别抑制 AGS 细胞迁移 59.4% 和 21.7% [1]。

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细胞凋亡被认为是一种治疗靶点，因为它在人类癌症中通常受到干扰。据报道，水飞蓟中的水飞蓟素（Silybum marianum）通过调节细胞凋亡、抗炎、抗氧化和保护肝脏而表现出抗癌特性。本研究考察水飞蓟素对人胃癌细胞增殖和凋亡的抑制作用。MTT法测定AGS人胃癌细胞的生存能力。采用伤口愈合实验研究AGS细胞的迁移情况。水飞蓟素能显著降低AGS细胞的活力和迁移能力，且呈浓度依赖性。DAPI染色和Annexin V/碘化丙啶双染色结果显示，凋亡小体数量和凋亡率呈剂量依赖性增加。采用western blotting方法研究水飞蓟素诱导的人胃癌细胞凋亡蛋白的表达变化。水飞蓟素增加Bax的表达，磷酸化(p) JNK和p - p38，切割聚ADP核糖聚合酶，并以浓度依赖的方式降低Bcl - 2和p - ERK1/2的水平。［2］

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2019年底，发生了一场全球大流行。病原体被确定为冠状病毒科的一员，称为严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）。在这项研究中，我们对人类代谢组中鉴定的能够结合SARS-CoV-2主要蛋白酶（Mpro）活性位点的物质进行了分析。存在于人体代谢组中的物质既有内源性的，也有外源性的。这项研究的目的是找到已知的生物化学和毒理学特征的分子，这可能是开发抗病毒疗法的起点。我们的分析揭示了许多代谢产物——包括外源物——与这种蛋白酶结合，这对病毒的生命周期至关重要。其中，特别值得注意的是水飞蓟素的主要活性成分、黄烷脂素类化合物水飞蓟宾。水飞蓟素是水飞蓟（Silybum marianum）的标准提取物，已被证明具有抗氧化、保护肝脏、抗肿瘤和抗病毒活性。我们在硅和体外获得的结果分别证明水飞蓟宾和水飞蓟素能够抑制Mpro，这代表了一种可能的食物来源的天然化合物，可用于治疗COVID-19。［４］

在强迫游泳测试 (FST) 期间，水飞蓟素（口服灌胃；10、20、50、100 和 200 毫克/千克）可缩短舞蹈模式的停止时间。此外，它还降低了尾悬试验（TST）中水飞蓟素的 ED50，大约为 10 mg/kg。在这两项试验中，发现 100 mg/kg 的剂量是最有效的剂量 [3]。

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水飞蓟素Silymarin (SM)有效剂量为10、20、50和100 mg/kg时，FST中静止时间呈剂量依赖性减少（p < 0.01, p < 0.05, p < 0.05和p < 0.001）。SM（10、20、50和100 mg/kg）也呈剂量依赖性地降低了TST的静止度（分别为p < 0.01、p < 0.05、p < 0.01和p < 0.001）。此外，SM的50%的最大响应（ED50）在10 mg/kg左右。在两次试验中，100 mg/kg的剂量被证明是最有效的剂量。此外，这种影响与运动活动的变化无关。此外，L-NAME逆转了SM（20和100 mg/kg）对FST和SM （100 mg/kg）对TST的作用。然而，AG并没有影响这种影响。 结论：水飞蓟素(SM)的抗抑郁样作用可能至少部分通过NO介导，SM可增加NO的调节。［3］

体外分析[4]
酶分析基本上按照我们以前的工作进行。简单地说，我们在制造商提供的反应缓冲液中使用纯化的SARS-CoV-2 Mpro Untagged，终浓度为0.5 ng/µL。水飞蓟素(SM)和杉木素。实验在Tecan微孔板阅读器中进行，室温下使用内淬荧光FRET底物（DABCYL-KTSAVLQSGFRKME-EDANS）作为底物，浓度为40µM。据报道，该肽在Mpro上的Km为17µM， Kcat为1.9 s−1。以实验兽药GC376为阳性对照，浓度为100µM。后者能够抑制SARS-CoV-2 Mpro， IC50约为0.42µM。实验在制造商提供的反应缓冲液中进行，在从酶的储存溶液中提取的0.1µM DTT（无DTT条件）或存在1mm DTT的情况下进行。

细胞活力测定[2]
细胞类型： AGS 细胞
测试浓度： 20 µg/ml，迁移 40 µg。 /ml、80 µg/ml、100 µg/ml 和 120 µg/ml
孵育时间： 24 小时
实验结果： 从 20 µg/ml 开始，对 AGS 细胞表现出显着的浓度依赖性抑制作用。

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细胞活力测定[2]
MTT法研究水飞蓟素(SM)对AGS人胃癌细胞增殖的影响。将AGS细胞接种于96孔板，密度为2×104 cells/ml，在rpm -1640培养基中，37℃，5% CO2培养箱内培养~24 h。然后用浓度为0、20、40、60、80、100和120µg/ml的Silymarin (SM)处理细胞。24 h后，将MTT[3-（4,5-二甲基噻唑-2-酰基）-2,5-二苯基溴化四唑]溶液加入到含有AGS细胞的96孔板中，体积为40µl/孔，培养2 h。去除MTT溶液后，加入100µl/孔的二甲基亚砜（DMSO）溶解孔中形成的所有甲醛，用ELISA-reader在595 nm处测定吸光度。与未处理的对照细胞相比，估计活细胞的百分比。

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伤口愈合实验 [2]
将AGS人胃癌细胞接种于60毫米培养皿中，培养24小时。使用无菌1毫升蓝色移液枪头划痕形成均匀伤口。更换含水飞蓟素(SM)的培养基（浓度分别为0、40和80 µg/ml），继续培养24小时。分别在划痕后0小时和24小时，通过相差显微镜(×200)拍摄图像，检测40和80 µg/ml 水飞蓟素(SM)处理组与未处理组的细胞伤口愈合率。

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DAPI染色 [2]
采用4′,6-二脒基-2-苯基吲哚(DAPI)染色观察细胞核凋亡特征性形态变化。将AGS人胃癌细胞以1×105个细胞/毫升密度接种于60毫米培养皿，稳定24小时后，分别用0、40和80 µg/ml的水飞蓟素(SM)处理，并在培养箱中孵育24小时。PBS冲洗细胞两次，4%多聚甲醛溶液固定15分钟，再次PBS冲洗后，加入1:10稀释的DAPI溶液(2毫升)，于暗室环境中通过荧光显微镜(×200)观察。 流式细胞分析[2]
采用FITC-Annexin V细胞凋亡检测试剂盒检测细胞凋亡。在膜联蛋白v -碘化丙啶（PI）染色中，AGS人胃癌细胞分别用浓度为0、40和80µg/ml的Silymarin (SM)/水水蓟素 处理。培养24 h的细胞用PBS洗涤，悬浮于胰蛋白酶- edta中，离心（260 × g, 5 min, 4°C）得到细胞颗粒。然后用冷PBS洗涤两次，离心得到细胞颗粒。然后，将它们悬浮在1X结合缓冲液中，浓度为1×106 cells/ml。然后加入异硫氰酸荧光素（FITC）偶联的膜联蛋白V和藻红蛋白（PE）偶联的PI，反应15分钟后进行流式细胞术检测。

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Western blot分析[2]
Western blot检测Silymarin (SM)处理后 蛋白表达的变化。AGS人胃癌细胞培养于175 cm2的瓶中，37℃，5% CO2培养箱中，用浓度为0、40和80µg/ml的Silymarin (SM)处理，培养24 h，加入胰蛋白酶- edta，悬浮，离心（260 × g, 5 min, 4℃）。将细胞裂解缓冲液加入细胞球中，4℃反应20 min， 15000 × g离心5 min得到的上清作为细胞裂解液。用Bradford蛋白法测定提取蛋白的浓度。蛋白质通过12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，并转移到硝化纤维素膜上。用5%脱脂牛奶阻断膜2小时，然后加入一抗。

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.

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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.

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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
Silybin has known human metabolites that include O-demethylated-silybin.

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.

[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 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.

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Drug Indication
Currently being tested as a treatment of severe intoxications with hepatotoxic substances, such as death cap (Amanita phalloides) poisoning.

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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]

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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]

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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.

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

155.14

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

配方 1 中的溶解度: ≥ 3 mg/mL (Infinity mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 30.0 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80 +，混匀；然后加入450 μL 生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。