Are alterations needed in Silybum marianum (Silymarin) administration practices? A novel outlook and meta-analysis on randomized trials targeting liver injury

Abstract

It is widely believed that Silybum marianum (Silymarin) alleviates liver injury arising from various etiologies with different degrees of damage through its anti-inflammatory and antioxidant activities. This meta-analysis investigated the effects of silymarin administration on serum levels of liver enzymes including AST, ALT and ALP. From inception to November, 2023, a comprehensive literature search was conducted. Inclusion criteria for this study were randomized trials that provided sufficient data for each group at the beginning and end of the follow-up period. Ultimately, 55 studies with a total of 3545 patients were included. Comprehensive Meta-Analysis (CMA) V4 software was used for meta-analysis. Begg's funnel plot symmetry status, Begg's rank correlation, and Egger's weighted regression tests were used to examine potential publication bias. According to the findings of this meta-analysis silymarin administration showed a significant reduction in AST (SMD [95% CI]: - 0.670 [- 0.931, - 0.408], p-value = 0.000), and ALT (SMD [95% CI]: - 0.912 [- 1.177, - 0.646], p-value = 0.000) levels. While it had no statistically significant effect on ALP level (SMD [95% CI]: - 0.236 [- 1.929, 1.458], p-value = 0.159). Meta-regression analysis showed that there is no significant association between dose, age, BMI, treatment duration and hepatoprotective effects of silymarin. In subgroup analysis, a greater reduction in liver enzymes levels was observed in patients under 50 years old. The subgroup analysis was also showed significant decrease in AST and ALT levels for patients with BMI less than 30, while silymarin treatment had no significant effects on AST and ALT levels in patients with BMI ≥ 30. Silymarin at a dose of less than 400 mg and treatment duration ≤ 2 months showed greater decreasing effects on AST and ALT levels compared to its high doses and longer treatment duration. AST and ALT levels significantly decreased in patients with NAFLD and viral hepatitis, while it had no significant hepatoprotective effects in patients with drugs induced liver injury and alcohol-related liver disease. Modifying the dose and treatment duration with silymarin is recommended in patients with various causes of liver damage.

Graphical Abstract

Introduction

Hepatic injury come in a wide range of forms and can be caused by a variety of diseases, including viral infections (Hepatitis A, B and C), primary biliary cholangitis, autoimmune hepatitis, alcohol-related hepatic injuries, Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), toxins- and drugs-induced liver injury. In the initial phases, the majority of liver diseases do not usually show any symptoms, while serious complications of liver disorders such as acute liver failure and cirrhosis show several symptoms including jaundice, ascites, edema, itchy skin, nausea or vomiting and persistent fatigue [1,2,3]. Every year, about two million people in the world die due to liver disorders; Half of these deaths are caused by complications of cirrhosis, and the rest are due to viral liver infections and hepatocellular carcinomas [4]. Two major and prevalent causes of chronic liver diseases are alcohol-related liver disease (ALD) and MASLD, which have now become public health concerns worldwide and are primarily caused by steatosis in the liver. Globally, the prevalence of both ALD and MASLD is rising. ALD is caused by long-term and excessive alcohol consumption, while metabolic syndrome such as insulin resistance, type II diabetes, obesity, and dyslipidemia are widely recognized as the major causes of MASLD, both of which may progress into liver fibrosis, and ultimately if left untreated, develop into cirrhosis [5,6,7,8].

Herbal medicines and medicinal plants have long been used to treat liver disorders worldwide and are now recognized as an effective treatment for liver diseases. Meanwhile, Silybum marianum (Silymarin) is one of the most widely used herbal medicine for patients with liver diseases [9,10,11]. S. marianum is an annual or biennial plant that is also known as milk thistle and belongs to the Asteraceae family. The therapeutic effects of S. marianum have been of interest to people for more than 2000 years. S. marianum grows widely throughout the world from the Mediterranean, Europe, Africa, China to Australia. The growth of this medicinal plant in the United States and South America began in the 9 th century [12,13,14].

Numerous preclinical studies demonstrated that S. marianum has various biological activities such as antimicrobial, antiaging, anticancer, antidiabetic, neuroprotective, antioxidant, anti-inflammation activities and especially hepatoprotective effects [15,16,17,18,19,20,21,22,23,24]. Hepatoprotective effects of S. marianum extract have been extensively investigated in patients with various liver disorders such as viral hepatitis, ALD and MASLD, poisonous mushroom and drug-induced liver injuries [25,26,27,28]. It seems that the hepatoprotective effect of S. marianum extract is due to the presence of silymarin which consists of a mixture of different flavonolignans including silybin, silychristin, silydianin, isosilychristin, and several other flavonolignans as minor biological compounds [12, 29,30,31,32,33,34,35,36]. According to the findings of clinical trials, silymarin is safe in humans at higher doses (over 1,500 mg/day) and no serious side effects have been reported [37]. The purpose of this current study was to conduct a meta-analysis on 55 randomized clinical trials with a total of 3545 patients that examined the impact of silymarin on the levels of liver enzymes such as aspartate aminotransferase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP).

Methods

Search strategy

The 2020 Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement standards were followed in the design of this study [38]. The keywords utilized in the titles and abstracts of the literature were employed from the beginning up to November 22, 2023, during the systematic search conducted via electronic databases such as PubMed, Scopus, and Web of Science: ("Silymarin"OR"Silybum marianum"OR"Milk Thistle") AND (("transaminase") OR ("aminotransferase") OR ("alanine transaminases") OR ("alkaline phosphatase") OR ("gamma-glutamyltransferase") OR ("lactate dehydrogenase") OR ("liver"). In addition, we looked through published systematic and narrative reviews'reference lists to locate any possible papers that our computerized search had missed. Only research published in English and Farsi was included in the search, and the keywords used are listed in Supplementary Materials Table S1.

Study selection

The aim of the present systematic review and meta-analysis study was to investigate the potential hepatoprotective activity of Silybum marianum (Silymarin) and the effects of its consumption on serum liver enzymes including AST, ALT and ALP. The meta-analysis included clinical trials that met specific inclusion criteria, such as being randomized trials with parallel design, with or without blinding, conducted on patients with any type of disease, and providing adequate data for each group at the beginning and end of the follow-up period, or presenting net values of change. Exclusion criteria encompassed non-randomized clinical trials, case studies, cross-over studies, observational investigations with cross-sectional, case–control or cohort design, and failure to provide necessary data at baseline or at the end of the follow-up time.

Data extraction

To reduce bias and errors in data collection, following the removal of duplicate studies, two researchers conducted independent reviews of the remaining records. The eligibility of articles was initially assessed by screening the titles and abstracts of the studies. The remaining articles were screened based on the full text of the article, adhering to predetermined exclusion and inclusion criteria. Studies were not initially excluded based on poor design or data quality. Upon completion of the evaluation process, any discrepancies in the findings between the two authors were identified and resolved through discussion until a consensus was reached. Clinical data from all eligible studies were carefully extracted and recorded in a structured form. This form included details such as the first author's name, publication year, study design, daily dose administered, treatment duration, type of control group, type of disease, and final clinical outcomes.

Quality assessment

The risk of bias in the clinical trials chosen for this meta-analysis study was evaluated using the revised Cochrane risk-of-bias tool for randomized trials (RoB 2) [39]. Various types of bias, including bias related to randomization, deviations from intended interventions, missing outcome data, outcome measurement, and selection of reported results, were categorized as low risk, some concerns, or high risk of bias across the included studies.

Data synthesis and primary analysis

A meta-analysis was performed utilizing the Comprehensive Meta-Analysis (CMA) V4 software developed by Biostat in New Jersey [40]. The findings were reported in various units of measurement. In order to conduct the statistical analysis, the levels of liver enzymes, including ALT, AST, and ALP, were computed as continuous variables. For each group, the mean or mean change was presented along with its corresponding standard deviation (SD). Additionally, if a study provided medians and interquartile ranges, these values were converted to means and their related SD based on the methods outlined by Luo et al. [41] and Wan et al. [42]. In cases where a study presents adjusted mean values along with a 95% confidence interval, the SD can be computed using a specific equation based on the provided confidence interval:

$$CL=\overline X\pm Z\times\frac S{\sqrt n}$$

where CL, X̄, S and n denote confidence level, sample mean, standard deviation, sample size respectively. Z-value for 95% confidence interval is 1.960 [43].

In this study, standardized mean differences (SMDs) were employed due to the various metrics used to evaluate outcomes. Therefore, the sample size, mean and standard deviation of each group (intervention and control group) for each relevant outcome were collected to calculate the SMDs. A random-effects model was used to pool standardized mean differences (SMDs) and 95% confidence intervals (CIs) because individual trials were conducted in the different populations. Statistical heterogeneity of the present study was assessed using I² (high heterogeneity ≥ 50% and low heterogeneity < 50%) [44]. To determine the possible publication bias, the examination of the symmetry of Begg's funnel plot, Begg’s rank correlation, and Egger’s weighted regression tests were performed [45, 46]. The one-study exclusion method was employed to conduct a sensitivity analysis, with the aim of assessing the reliability of the findings and ensuring that they were not influenced by any single study.

Meta-regression

Meta-regression analysis was conducted to evaluate the relationship between the SMD in AST, ALT, and ALP values with various factors such as dose, age, body mass index (BMI), and duration of treatment across the studies included in the analysis. The purpose of this analysis was to investigate how these factors affect the results reported in the studies. Meta-regression was carried out under a random-effects models.

Results

Study characteristics

A total of 11,255 studies were initially identified through a comprehensive search in databases including PubMed, Scopus, and Web of Science. After a rigorous screening process that included reviewing the title and abstract of each study, 8,501 studies were excluded due to various reasons including duplication, review articles, preclinical studies, case studies, letter to editor, note, or lack of relevance to the research question. After that, 88 articles were selected for further evaluation by full text review, of which 56 studies met the pre-defined inclusion criteria for systematic review and meta-analysis. The selection process was carefully documented in a flow chart, presented as Fig. 1. All 55 studies with a total of 3545 patients provided data for serum AST, ALT and/or ALP levels. A detailed description of each eligible study is provided in Table 1 and provides an overview of data sources and key findings.

Fig. 1

Flow diagram of study selection for meta-analysis

Table 1 The main characteristics of the included studies

Effect of Silymarin on serum AST levels

Meta-analysis of 54 arms with a total of 3188 patients showed a significant reduction in serum AST levels with SMD (95% CI): − 0.670 (− 0.931, − 0.408), prediction interval of PI (95% CI): − 0.670 (− 2.545, 1.206) and p-value of 0.000 (Fig. 2). The meta-analysis was conducted using the random-effects model (I² = 92.69%, p-value = 0.000). The decrease in serum AST levels following silymarin treatment demonstrated significant robustness even after performing a sensitivity analysis through the one-study remove method (Figure S1). Given the asymmetric funnel plot, Egger’s linear regression test (intercept = − 3.88, standard error = 1.58; 95% CI = − 7.06, − 0.70, t = 2.45, df = 52, two-tailed p-value = 0.017) and Begg’s rank correlation test (Kendall’s Tau with continuity correction = − 0.27, z = 2.87, two-tailed p-value = 0.0039) represent publication bias in the meta-analysis of the impact of silymarin administration on AST levels. Employing the"trim and fill"method, 11 potentially missing studies were estimated, resulting in an adjusted effect size (SMD) of − 0.262 (95% CI: − 0.54, 0.01) (Figure S2).

Fig. 2

Forest plot displaying standardized mean difference and 95% confidence intervals for the effect of silymarin treatment on serum AST level

Effect of Silymarin on serum ALT levels

The pooled results from the 59 arms including 3313 patients showed a significant reduction in serum ALT levels following the administration of silymarin, with SMD (95% CI): − 0.912 (− 1.177, − 0.646), prediction interval of PI (95% CI): − 0.912 (− 2.906, 1.082) and p-value of 0.000 (Fig. 3). The meta-analysis was conducted with the random-effects model (I² = 93.24%, p-value = 0.000). Following the implementation of sensitivity analysis through the leave-one-out method, it was observed that the decrease in serum ALT levels as a result of silymarin treatment was significance (Figure S3). According to the asymmetric funnel plot, Egger’s linear regression test (intercept = − 6.41, standard error = 1.31; 95% CI = − 9.04, − 3.78, t = 4.88, df = 57, two-tailed p-value = 0.0000) and Begg’s rank correlation test (Kendall’s Tau with continuity correction = − 0.38, z = 4.34, two-tailed p-value = 0.0000) represent publication bias in the meta-analysis of the impact of silymarin administration on AST levels. Employing the"trim and fill"method, 18 potentially missing studies were estimated, resulting in an adjusted effect size (SMD) of − 0.207 (95% CI: − 0.50, 0.08) (Figure S4).

Fig. 3

Forest plot displaying standardized mean difference and 95% confidence intervals for the effect of silymarin treatment on serum ALT level

Effect of Silymarin on serum ALP levels

The meta-analysis results from the 26 studies with a total of 1876 patients that measured ALP levels, showed no significant changes in serum ALP levels after silymarin treatment, with SMD (95% CI): − 0.236 (− 0.564, 0.092), prediction interval of PI (95% CI): − 0.236 (− 1.929, 1.458) and p-value of 0.159 (Fig. 4). The meta-analysis was conducted using the random-effects model (I² = 92.22%, p-value = 0.000). After performing sensitivity analysis and removing each individual study, it was found that there were no substantial changes in the overall findings and silymarin administration did not yield any noteworthy impact on the levels of ALP (Figure S5). Given the asymmetric funnel plot, Egger’s linear regression test (intercept = − 3.52, standard error = 1.49; 95% CI = − 6.60, − 0.44, t = 2.35, df = 24, two-tailed p-value = 0.026) represent publication bias in the meta-analysis of the impact of silymarin administration on ALP levels. Employing the"trim and fill"method, 8 potentially missing studies were estimated, resulting in an adjusted effect size (SMD) of 0.234 (95% CI: − 0.10, 0.57) (Figure S6).

Fig. 4

Forest plot displaying standardized mean difference and 95% confidence intervals for the effect of silymarin treatment on serum ALP level

Meta-regression analysis

Potential associations between the hepatoprotective effects of silymarin with dose, age, body mass index (BMI) and duration of treatment were examined using meta-regression analysis by random-effects model. With respect to AST and ALT, the results of meta-regression analysis showed no significant association between dose (AST (slope: 0.0003; 95% CI: − 0.0010, 0.0016; p = 0.6112) and ALT (slope: 0.0011; 95% CI: − 0.0001, 0.0024; p = 0.0804) (Fig. 5A and B)), age (AST (slope: 0.0103; 95% CI: − 0.0068, 0.0275; p = 0.2370) and ALT (slope: 0.0110; 95% CI: − 0.0061, 0.0282; p = 0.2059) (Fig. 5C and D)), BMI (AST (slope: 0.0038; 95% CI: − 0.0511, 0.0586; p = 0.8925) and ALT (slope: 0.0004; 95% CI: − 0.0421, 0.0429; p = 0.9853) (Fig. 6A and B)) and treatment duration (AST (slope: 0.0338; 95% CI: − 0.0297, 0.0972; p = 0.2971) and ALT (slope: 0.0120; 95% CI: − 0.0550, 0.0791; p = 0.7246) (Fig. 6C and D)) and hepatoprotective effects of silymarin.

Fig. 5

Random-effects meta-regression plots of the association between standard differences in means in AST values (A and C) and ALT values (B and D) after silymarin supplementation with silymarin dose (A and B) and age of patients (C and D)

Fig. 6

Random-effects meta-regression plots of the association between standard differences in means in AST values (A and C) and ALT values (B and D) after silymarin supplementation with BMI (A and B) and treatment duration (C and D)

Subgroup analysis

Subgroup analysis was also conducted based on age (< 50 or ≥ 50 years), BMI (< 30 or ≥ 30), various doses of silymarin (140 and 400 mg), treatment duration (≤ 2 or > 2 month) and type of disease including non-alcoholic fatty liver disease (NAFLD), thalassemia, T2 diabetes Mellitus, viral hepatitis, alcoholic liver cirrhosis and anti-tuberculosis drugs induced liver injury. The subgroup analysis results were listed in Table 2. In subgroup analysis, a greater reduction in liver enzymes levels was observed in patients under 50 years old (AST (SMD: − 0.703, 95% CI: − 1.054, − 0.352, p-value: 0.000) (Figure S7) and ALT (SMD: − 1.186, 95% CI: − 1.577, − 0.795, p-value: 0.000)) (Figure S8) compare to patients older than ≥ 50 years old (AST (SMD: − 0.545, 95% CI: − 0.852, − 0.237, p-value: 0.001) (Figure S7) and ALT (SMD: − 0.258, 95% CI: − 0.566, − 0.148, p-value: 0.001)) (Figure S8).

Table 2 Subgroup analyses of the effects of silymarin consumption on liver enzymes

The subgroup analysis was also represented substantial decrease in AST (SMD: − 0.889, 95% CI: − 1.428, − 0.350, p-value: 0.001) and ALT (SMD: − 0.700, 95% CI: − 1.074, − 0.327, p-value: 0.000) (Figure S9 A and B) levels for patients with BMI less than 30, while silymarin treatment had no significant effects on AST and ALT levels in patients with BMI more than 30 (Figure S9 A and B). Furthermore, administration of silymarin significantly reduced ALP levels (SMD: − 0.292, 95% CI: − 0.482, − 0.102, p-value: 0.003) in patients with a BMI of less than 30.

Administration of silymarin at a dose of less than 400 mg showed greater decreasing effects on AST (SMD: − 0.905, 95% CI: − 1.396, − 0.415, p-value: 0.000) (Figure S10) and ALT (SMD: − 1.507, 95% CI: − 2.028, − 0.987, p-value: 0.000) (Figure S11) levels compared to a dose of ≥ 400 mg (AST (SMD: − 0.494, 95% CI: − 0.764, − 0.224, p-value: 0.000) (Figure S10) and ALT (SMD: − 0.514, 95% CI: − 0.963, − 0.484, p-value: 0.000)) (Figure S11). It is interesting to note that the results of the subgroup analysis demonstrated that silymarin administration at a dose of ≤ 140 mg had greater decreasing effects on AST (SMD: − 0.988, 95% CI: − 1.883, − 0.093, p-value: 0.031) level compared to silymarin administration at higher doses.

Administration duration ≤ 2 months showed more decreasing effects on AST (SMD: − 0.664, 95% CI: − 1.040, − 0.289, p-value: 0.001) (Figure S12) and ALT (SMD: − 0.964, 95% CI: − 1.423, − 0.504, p-value: 0.000) (Figure S13) levels compared to duration of treatment with silymarin more than 2 months. Treatment with silymarin significantly decreased AST (SMD: − 0.916, 95% CI: − 1.437, − 0.394, p-value: 0.001) (Figure S14) and ALT (SMD: − 0.847, 95% CI: − 1.261, − 0.432, p-value: 0.000) (Figure S15) levels in patients with non-alcoholic fatty liver diseases (NAFLD), while it had no substantial effects on liver enzymes in patients with alcoholic liver cirrhosis (Figure S14 and S15). Furthermore, silymarin treatment showed significant effects on AST (SMD: − 0.655, 95% CI: − 1.021, − 0.289, p-value: 0.000) (Figure S14) and ALT (SMD: − 0.399, 95% CI: − 0.629, − 0.168, p-value: 0.001) (Figure S15) levels in patients with viral hepatitis, while it had no significant hepatoprotective effects in patients with anti-tuberculosis drugs induced liver injury (AST (SMD: 0.037, 95% CI: − 0.362, 0.436, p-value: 0.856) (Figure S14). and ALT (SMD: − 0.026, 95% CI: − 0.582, 0.531, p-value: 0.928)) (Figure S15). Diabetic patients also benefit from the reducing effects of silymarin consumption on AST (SMD: − 0.719, 95% CI: − 1.123, − 0.315, p-value: 0.000) (Figure S14). and ALT (SMD: − 1.291, 95% CI: − 2.395, − 0.186, p-value: 0.022) levels (Figure S15).

Interestingly, treatment of patients with a dose of ≤ 140 mg of silymarin and a treatment duration of ≤ 2 months showed a significant reduction in AST (SMD: − 1.142, 95% CI: − 1.548, − 0.736, p-value: 0.000) and ALT (SMD: − 0.831, 95% CI: − 1.269, − 0.393, p-value: 0.000) levels. The subgroup analysis based on type of study showed a greater significant decrease in AST levels for randomized double blind clinical studies (SMD: − 0.781, 95% CI: − 1.120, − 0.441, p-value: 0.000) than randomized studies (SMD: − 0.506, 95% CI: − 0.933, − 0.079, p-value: 0.020). Additionally, the findings showed significant reduction in ALT levels for randomized double blind clinical studies (SMD: − 1.221, 95% CI: − 1.582, − 0.860, p-value: 0.000), while there was no considerable change in ALT levels for randomized studies (SMD: − 0.222, 95% CI: − 0.739, 0.295, p-value: 0.400). ALP levels did not change significantly after treatment with silymarin in double-blind randomized clinical trials or randomized studies (Table 2). The presence of heterogeneity across studies is apparent through differences in treatment duration, dose levels, and frequency of silymarin intake. Based on subgroup analysis, greater heterogeneity was observed in the group of patients with age less than 50 years, BMI less than 30, and prescribed dose less than 400 mg.

Type I error was initially set at 0.05; however, in our meta-analysis, we used a more stringent threshold for type I error, setting it at 0.01. Interestingly, a significant decrease in AST (SMD: − 0.670, 99% CI: − 1.013, − 0.326, p-value: 0.000) (Figure S16) and ALT (SMD: − 0.912, 99% CI: − 1.261, − 0.563, p-value: 0.000) (Figure S17) levels was observed after treatment with silymarin (Table 2).

Risk of bias assessment of clinical trials

Insufficient data on random sequence generation and allocation concealment was found in the fifteen studies included in the meta-analysis. Seven studies raised some concerns and six clinical trials indicated a significant risk of bias from the method of outcome measurement due to insufficient information or the open-label design of the trial. Three studies demonstrated high risk of bias arising from deviations from the intended interventions. Finally, overall bias was high for eight studies and eleven studies showed some concerns for total bias. Information regarding the evaluation of risk of bias in the chosen clinical trials are depicted in Fig. 7. If the risk of bias is high in the included studies, the meta-analysis results may be biased and should be interpreted with caution. To increase the accuracy of the research, the meta-analysis was conducted by excluding studies identified as having a high risk of bias. After excluding of these studies, the pooled results showed a significant reduction in serum AST (SMD [95% CI]: − 0.647 [− 0.950, − 0.345], and p-value of 0.000) (Figure S18) and ALT (SMD [95% CI]: − 0.885 [− 1.182, − 0.588], and p-value of 0.000) levels (Figure S19) following the administration of silymarin, while no significant change was observed in ALP levels (SMD [95% CI]: − 0.087 [− 0.419, 0246], and p-value of 0.610) (Figure S20).

Fig. 7

Risk of bias assessment in each study

Discussion

Liver disorders are one of the main reasons for mortality worldwide, resulting in approximately 2 million deaths annually. Liver injury may range from acute hepatitis to hepatocellular carcinoma. Furthermore, ALD and MASLD are considered as the major and prevalent causes of chronic liver diseases, which have raised global health concerns. This meta-analysis represents a pioneering effort to investigate the effect of silymarin treatment on liver enzymes in the context of randomized clinical trials in patients with a various range of diseases with different degrees of damage and etiologies. [101]. ALT and AST are the most common and specific indicators used for hepatocellular necrosis. Although the ratio of AST to ALT has more clinical utility than assessing each enzyme individually [102]. Changes in serum levels of ALT, AST, and ALP would signify the progression or improvement of cholestatic or hepatocellular diseases.

The pooled results from the 54 and 59 arms with 3188 and 3313 patients showed a considerable reduction in serum AST and ALT levels after treatment with silymarin, respectively. It is noteworthy to emphasize that taking into account a considerable reduction in serum levels of AST and ALT, even patients who received a lower dose of silymarin (less than 140 mg/day) for a limited period of time (less than 2 months) benefited greatly from silymarin administration in the context of hepatic disorders. The findings of the subgroup analysis indicated that patients with a BMI greater than 30 did not benefit from the administration of silymarin in the treatment of hepatic disorders. Furthermore, silymarin administration demonstrated significant lowering effects on AST and ALT levels in patients with MASLD, Type 2 Diabetes Mellitus, and viral hepatitis; although, it had no considerable impact on serum levels of these enzymes in patients with ALD-induced cirrhosis, thalassemia, or hepatotoxicity caused by anti-tuberculosis drugs.

Based on our findings, ALP showed no significant changes in patients taking silymarin, whereas subgroup analysis showed that patients with a BMI of less than 30 benefited from a reduction in ALP levels. There is not much evidence that silymarin has therapeutic effects on cholestatic diseases. According to previous studies, the use of silymarin considerably decreased the concentration of phospholipids and cholesterol in the bile because it inhibited the synthesis of liver cholesterol, while total bile salt concentration and bile flow remained unchanged [103]. Another study demonstrated that silymarin's ability to scavenge ROS improved cholemic nephropathy in rats caused by bile duct obstruction [104].

MASLD includes a spectrum of liver damage from mild steatosis to progressive fibrosis, cirrhosis, and inflammation of the liver, which is worsened by increased mitochondrial-related elements, decreased β-oxidation, impaired electron transport chain, decreased ATP, overexpression of reactive oxygen species (ROS), oxidative stress-induced cellular damage and resulting in obvious changes in the mitochondrial ultrastructure. mitochondrial dysfunction leads to exacerbate liver fat accumulation and thereby progressive fibrogenesis and inflammation [105,106,107,108,109]. The hepatoprotective properties of silymarin are believed to be significantly attributed to its antioxidant capabilities and its ability to scavenge free radicals. This compound enhances the activity of DNA polymerase within hepatocytes, leading to an increase in the synthesis of ribosomal RNA and proteins, thereby promoting the regeneration of liver cells [110,111,112,113]. Additionally, silymarin exerts an inhibitory effect on beta-glucuronidase, which is thought to mitigate the conversion of glucuronides into harmful metabolites in both the liver and the intestinal tract [114]. Silymarin prevents hepatocyte necrosis by maintaining biological membranes, promoting protein synthesis, scavenging free radicals, and raising glutathione levels. Silymarin also reduces ROS production by raising superoxide dismutase (SOD) activity in lymphocytes and erythrocytes. According to the findings, silymarin also inhibits the formation of collagen fibers by preventing stellate hepatocytes from becoming myofibroblasts, which prevents the progression of MASLD to non-alcoholic steatohepatitis (NASH) or cirrhosis [115]. Hepatic fibrosis and its progression to liver cirrhosis are clearly associated with chronic inflammation, which is also accompanied by nuclear factor kappa B (NF-κB) activation. Several preclinical investigations have proven the anti-inflammatory properties of silymarin through inhibiting NF-κB activation, reduction of hepatic tumor necrosis factor alpha (TNF-α), malondialdehyde (MDA) levels and transforming growth factor-β (TGF-β) mRNA expression, as well as preventing the generation of inflammatory metabolites such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) [116,117,118,119,120].

MASLD frequently manifests only as abnormalities in laboratory tests. According to the results of our investigation, silymarin treatment significantly reduced AST and ALT levels, indicating a pattern of reduced liver necrosis before patients crossed the line of liver cirrhosis. The findings derived from this research fail to support the initial hypothesis that silymarin treatment would lead to improvements in liver function tests and ameliorate the clinical manifestations of liver dysfunction in patients diagnosed with alcoholic liver cirrhosis. This is consistent with the findings of Pares et al. [50], who similarly found that silymarin treatment had no significant effect on liver enzyme levels. These clinical trials focused on patients with alcoholic liver cirrhosis to investigate the impact of silymarin administration on their clinical outcomes. The inability of this type of study to identify abstinence from alcohol during follow-up is one of its limitations. Many of the patients in Pares et al.'s study resumed drinking, but the majority did so sparingly and with a significant reduction in alcohol consumption. In contrast to our findings, a placebo-controlled trial using silymarin in patients with liver cirrhosis of various etiologies showed that treatment of patients with silymarin had a higher 4-year survival rate than patients who received placebo. However, the severity of hepatic injury was different among the subgroups of this clinical trial, and the subgroups of patients were not balanced for the severity of liver failure [121]. Hepatitis C virus infection is very common in alcoholic patients with cirrhosis and is another complicating factor in the treatment of liver cirrhosis in this type of patients [50, 122, 123]. Pares et al. [50] performed a randomized controlled trial on 125 alcoholic patients with liver cirrhosis and evaluated survival rate and clinical outcomes over 2 years. The finding of this clinical study showed that silymarin treatment had no impact on survival rate and the clinical outcomes in the silymarin group did not improve significantly and were similar to the placebo group during the trial.

Based on the findings, diabetic patients have a higher frequency of MASLD with more severe histological manifestations compared to the healthy people. Peripheral insulin resistance is associated with an elevation in the release of fatty acids from adipose tissue and the encouragement of their uptake by the liver. An increase in liver lipogenesis and a decrease in fatty acid oxidation are observed in prediabetic and diabetic patients. In addition, impaired secretion of very low-density lipoprotein, which usually occurs with insulin resistance, contributes to the accumulation of fat in the liver [124,125,126]. The results of an in vivo study on high-fat diet-induced insulin resistance and fatty liver in mice showed that silymarin reduced insulin resistance, serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein-cholesterol (LDL), improved fat accumulation and ameliorated liver damage caused by a high-fat diet. Furthermore, silymarin significantly decreased cytokine levels including TNF-α, interleukin- 6 (IL- 6) and IL- 1β, and also reduced AST and ALT levels, leading to improved liver function [127]. In a diabetic rat model, silymarin significantly decreased oxidative stress biomarkers in the liver tissue and increased PGC- 1α, FNDC5 gene expression and irisin concentration as multifunctional regulatory transcription factors in diabetic rat liver tissues. Antioxidant effects of silymarin seem to be one of the main mechanisms of its hepatoprotective effects [128]. The findings of a randomized clinical trial in diabetic patients showed that silymarin administration significantly decreased fasting blood glucose (FBG), Hemoglobin A1c (HbA1c), fasting serum insulin (FSI) and the homeostasis model assessment-estimated insulin resistance (HOMA-IR), which indicated a decrease in insulin resistance. In addition, patients in the silymarin group had substantially lower levels of TG, LDL, very low density lipoprotein (VLDL), MDA, and C-reactive protein (CRP) in comparison with the placebo group [129]. Mohammadi et al. also found that treatment with silymarin in diabetic patients showed a significant improvement in insulin resistance and serum insulin levels compared to the placebo group [130]. In the present study, the results of subgroup analysis confirmed the beneficial effects of silymarin consumption in diabetic patients.

Silymarin treatment showed significant effects on AST and ALT levels in patients with viral hepatitis (Table 2). Based on the findings of several studies silymarin and its derivatives showed antiviral activity against hepatitis C virus (HCV) infection through inhibiting viral entry, viral pseudoparticles fusion to liposomes, RNA polymerase activity, microsomal triglyceride transfer protein activity, virus transmission as well as apolipoprotein B generation. Furthermore, silymarin also inhibited the proliferation and proinflammatory cytokine (TNF-α, Interferon-γ (IFN-γ), and IL- 2) secretion of T cells, intrahepatic expression of inducible nitric oxide synthase (iNOS) and intrahepatic NF-κB activation [131,132,133,134,135]. In a clinical trial, Ferenci et al. evaluated the effects of intravenous injection of silibinin in patients with chronic hepatitis C who did not respond to treatment with pegylated interferon/ribavirin (PegIFN/RBV). They found that monotherapy with intravenous injection of silibinin significantly decreased serum HCV RNA and viral load in a dose-dependent manner. Treatment with pegylated interferon/ribavirin and higher dose of silibinin (15 and 20 mg/kg/day) reduced HCV RNA levels and were undetectable at week 12 in seven patients [136]. In another study, the effects of intravenous silibinin (20 mg/kg for 14 or 21 days) were evaluated in 27 patients who received a full dose of PegIFN/RBV and did not respond to treatment. 23 patients had undetectable HCV RNA after completing silibinin IV therapy. At the end of PegIFN/RBV treatment, 17 patients were still HCV RNA negative, which decreased to 12 patients after 24 weeks of treatment-free follow-up. Finally, 5 patients experienced viral relapse [137]. Silibinin and its derivatives directly suppress HCV replication by inhibiting HCV RNA-dependent RNA polymerase activity [134].

In a randomized controlled trial of 11 HCV-infected patients awaiting liver transplantation, intravenous silibinin monotherapy demonstrated significant antiviral activity while effectively reducing viral load before liver transplantation and preventing HCV reinfection after receiving graft. In this study, 4 patients had HCV RNA levels below than the limit of quantification (2 patients) and detection (2 patients) after liver transplantation in silibinin monotherapy group. Overall, HCV RNA levels were significantly (p = 0.002) lower in monotherapy with silibinin compared to the placebo group [138]. Several other studies have well documented the antiviral activity of intravenous silibinin in HCV-infected patients [139,140,141,142,143,144]. Collectively, these studies demonstrate the clinical efficacy of silymarin treatment in patients with viral hepatitis.

Hepatotoxicity caused by drugs or their active metabolites is an important problem because many drugs undergo liver metabolism. Liver damage caused by anti-tuberculosis drugs rises the rate of morbidity and mortality. Hepatotoxicity may require discontinuation of treatment or drug, and replacement or adjustment of the dose. Our research did not show a significant decrease in serum AST and ALT levels in cases of hepatotoxicity caused by anti-tuberculosis drugs. Several clinical studies evaluated the possible effectiveness of silymarin in preventing liver damage caused by anti-tuberculosis drugs. Consistent with our findings, randomized clinical trials conducted by Heo et al. [76] (121 patients with tuberculosis) and Marjani et al. [83] (70 patients with tuberculosis) showed no significant protective effect of silymarin against liver injury caused by anti-tuberculosis drug. Additionally, Heo et al. found that silymarin prevented the rise in total bilirubin during the first two months of taking anti-tuberculosis drugs, thus reducing the incidence of hepatotoxicity caused by anti-tuberculosis drugs. However, it showed no clinically significant protective effect against elevated AST or ALT. In another clinical study conducted on 565 patients with tuberculosis, there was no significant difference between patients receiving silibinin and the control group after 8 weeks of treatment, while fewer patients in the treatment group had symptoms of liver damage such as anorexia and nausea compared to the control group [145]. The findings of a clinical trial demonstrated that silymarin administration diminished the incidence of anti-tuberculosis drugs induced-liver injury which can be attributed to a lower reduction in the level of superoxide dismutase compared to the control group [146].

In line with Hagag et al. [62], our data showed a negligible decline in AST and ALT levels; however, we did not measure liver iron concentration, which is a direct predictor of outcome in patients with Thalassemia. Patients with thalassemia usually have abnormal liver enzymes, mostly due to hepatocyte injury from iron excess. Studies have revealed that silybin, the most physiologically active silymarin component, is a tiny lipophilic molecule with significant potential for chelating intracellular iron [147]. According to Gharagozloo et al. [56], silymarin may have preferential access to intracellular iron in organs like the liver and pancreas because of its tiny molecular size and high lipophilicity. Additionally, Gharagozloo et al. discovered that silymarin differs from other iron chelators in its biological effects. This is most likely because of silymarin's antioxidant activity, which promotes oxidation via iron-catalyzed oxidation and the consequent production of reactive oxygen species. The in vivo iron chelating properties of silymarin and silybin were validated by Moayedi et al. However, our research categorically refuted the possibility that silymarin lowers AST or ALT in thalassemic patients, which could have a variety of causes. The AST and ALT readings of several of the included studies that looked at thalassemic patients were normal at baseline. It's possible that these patients did not have thalassemia-related liver damage. An alternative to assessing body iron overload is to consider liver iron, since it is correlated with total body iron.

Silymarin is commonly used as a complementary or alternative treatment for liver disorders due to its antioxidant and anti-inflammatory properties which may help protect liver cells from damage caused by toxins, oxidative stress, and inflammation. Furthermore, silymarin may promote liver cell regeneration and stabilize liver cell membranes [148]. Based on several clinical studies, the suggested dosage of silymarin was 420 mg/day [121, 149]. The findings of this meta-analysis demonstrated that silymarin at a dose of less than 400 mg showed greater decreasing effects on AST and ALT levels compared to its high doses. It is suggested to be used alongside conventional treatments to support liver health, particularly in mild to moderate liver conditions. For conditions like NAFLD, lifestyle modifications (e.g., weight loss, diet, exercise) remain the primary treatment, and silymarin potentially serves as an effective supplement. Therefore, silymarin is better recommended as a complementary treatment rather than a first-line treatment.

The results of this study suggest that the optimal daily dosage of silymarin ranges from 140 to 400 mg, depending on the specific liver condition being considered. Evidence indicates that silymarin is beneficial for short-term therapeutic interventions. After a two-month treatment course, it is recommended that patients undergo assessments of liver enzyme levels or other diagnostic evaluations to monitor the effectiveness of the treatment.

Silymarin is considered safe for human consumption at therapeutic dosages and is generally well tolerated, even at elevated doses of 700 mg administered three times daily up to 48 weeks. However, some individuals may experience gastrointestinal side effects, including nausea and diarrhea [150]. Clinical studies indicate that milk thistle extract has the potential to reduce blood glucose levels and glycated hemoglobin (HbA1c) in individuals diagnosed with type 2 diabetes, who are currently undergoing treatment with antidiabetic medications, should take it with caution [49, 53, 151].

This meta-analysis represents is the first attempt to consolidate the outcomes of prior investigations regarding the impact of silymarin treatment on liver enzyme levels. Consequently, it is imperative to acknowledge certain limitations associated with this meta-analysis while interpreting findings and final conclusions. The participants in these research studies exhibited a variety of illnesses and metabolic disorders. Nevertheless, this meta-analysis possessed adequate statistical power to recognize the significant impact of silymarin on the overall study population, regardless of the specific type of illnesses. This meta-analysis also faced a limitation in the form of varying metrics used to assess outcomes. Therefore, the decision to employ SMD as a summary statistic for the combined effect size in this meta-analysis is deemed appropriate. The inclusion of studies with different sample sizes and populations, as well as the consideration of language restrictions in the study selection process added another layer of limitation to the analysis. Moreover, the presence of heterogeneity across studies is apparent through differences in treatment duration, dose levels, and frequency of silymarin intake. Therefore, it is important to interpret the findings with caution. Although there are certain limitations, our meta-analysis provided valuable perspectives on the impact of silymarin administration on liver enzyme levels. Nevertheless, it is important to be cautious in interpreting the results due to the established limitations. Moreover, further studies are needed to discover the exact mechanism by which silymarin affects liver function.

Conclusion

This meta-analysis provides compelling evidence supporting the effectiveness of silymarin as an herbal remedy for reducing levels of the liver enzymes AST and ALT. However, it does not show a significant effect on ALP levels. The investigation revealed no significant associations between factors such as age, BMI, dosage, or duration of treatment and the hepatoprotective effects of silymarin. In subgroup analyses, silymarin exhibited enhanced effectiveness in reducing liver enzyme levels among individuals younger than 50 years and those with a BMI under 30. Additionally, lower doses of silymarin (below 400 mg) and shorter treatment durations (two months or less) were associated with more pronounced reductions in AST and ALT levels. The findings indicated that silymarin significantly lowered AST and ALT levels in patients suffering from NAFLD and viral hepatitis, while its hepatoprotective effects were not significant in cases of drug-induced liver injury. Treatment regimens involving low doses of silymarin (≤ 140 mg) and short treatment courses (≤ 2 months) resulted in significant decreases in AST and ALT levels. The primary mechanisms underlying the hepatoprotective effects of silymarin are believed to be its antioxidant and anti-inflammatory properties. Further investigation is warranted to elucidate the specific role of silymarin in various liver disorders.