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Adulteration of Milk Thistle (Silybum marianum)

By Allison McCutcheon, PhD

American Botanical Council, PO Box 144345, Austin, TX 78714

Keywords: Adulterant, adulteration, milk thistle, Silybum marianum, silymarin, silybin, silychristin, silydianin

Goal: The goal of this bulletin is to provide timely information on issues of adulteration of milk thistle (Silybum marianum, Asteraceae) fruit and its extracts to the international herbal industry and extended natural products community in general, by presenting data on the type and occurrence of adulteration, the market situation, and consequences for the consumer and the industry.

1                 General Information

1.1 Common name: Milk thistle¹

1.2 Other common names:

English:^1-3 Mary’s thistle, blessed milk thistle, spotted milk thistle,² bull thistle, gundagai thistle, holy thistle, lady's thistle, variegated artichoke, variegated milk thistle,³ St. Mary's thistle
Arabic:^3-4 shawk el-gamal (also written as shuk aljamal) (شوك الجمل), shuk al-halib (شوك الحليب ), alsalabayn almurimi (السلبين المريمي )
Armenian:⁵ kat’ ughtap’ush  
Chinese:⁵ nai ji (奶薊), shui fei ji (水飛薊)
Danish:^5-6 marietidsel, mælk tidsel
Dutch:⁶ mariadistel
Finnish:⁶ maarianoh dake, hedelmä
French:^3,7,8 chardon argenté, chardon Marie, lait de Notre Dame, silybe de Marie, chardon Notre Dame, épine blanche
German:^3,8 Mariendistel, Frauendistel
Greek:⁵ gaïdouránkatho gála (γαϊδουράγκαθο γάλα)
Hebrew:⁵ dilan mazui
Hindi:⁵ dugdh rom
Italian:^6,7 cardo mariano, cardo di Maria
Japanese:⁵ ooazami (オオアザミ), mariazami (マリアアザミ)
Norwegian:⁶ marietistel
Pharmacopoeial:⁸ fructus silybi Mariae, fructus cardui Mariae
Polish:⁶ ostropest plamisty
Portuguese:³ cardo-leiteiro
Russian:³ ostro-pestro (остро-пестро), rastoropša pyatnistaja (расторопша пятнистая)
Spanish:^3,7 cardo de María, cardo lechero, cardo mariano, cardo asnal, cardo blanco, cardo santo, poma
Swedish:³ mariatistel

1.3 Accepted Latin binomial: Silybum marianum (L.) Gaertn.

1.4 Synonyms: Carduus marianus L., Carthamus maculatus (Scop.) Lam., Cirsium maculatum Scop., Mariana mariana (L.) Hill.,⁸ Silybum maculatum (Scop.) Moench., Silybum mariae (Crantz) Grey, Silybum pygmaeum Cass.⁹

The synonyms S. marianum var. marianum and S. marianum var. albiflorum have been used to differentiate respectively, the commercial, purple-flowered variety from the white-flowered variety of milk thistle.

1.5 Botanical family: Asteraceae

1.6 Distribution range: Indigenous to North Africa, Asia Minor, southern Europe and southern Russian Federation; naturalized in North and South America, Australia, China, and Central Europe.^3,8 

1.7 Plant part: The plant part used is the dried mature fruit devoid of the pappus. Characteristic of the Asteraceae family, the one-ovule, achene-like fruit is called a cypsela (pl. cypselae). However, in common parlance (and in many scientific articles) the fruit is often referred to as a seed. To facilitate communication and maintain continuity with the literature, the generic terms fruit or seed are used in this document. The fruit is comprised of four parts: pericarp, integument, albumen (storage protein), and embryo (two large cotyledons with fat as storage material). Collectively, the albumen and embryo are described as the kernel, while the pericarp and integument may be called the seed coat or hull.^10-12

1.8 Key constituents and markers: Silymarin, the main pharmacologically active fraction of milk thistle,^13-15 comprises 1.5–3.5% of the fruit by dry weight and 30–65% by high-performance liquid chromatography with ultraviolet detection (HPLC-UV),¹⁶ corresponding to 65–80% by UV/Vis of the extract.^16-20 Silymarin is a complex mixture of 3-hydroxyflavonolignans (Figure 1), of which the primary components are silybin (syn. silibin, silibinin), silychristin (syn. silichristin), silydianin (syn. silidianin), isosilybin (syn. isosilibin), and the flavanonol taxifolin (syn. dihydroquercetin).^21-24 Silybin, isosilybin, and silychristin are each present as a pair of diastereoisomers. The seven flavonolignans and taxifolin are the primary compounds used to assess milk thistle quality. Silymarin is mainly found in the seed coat.^25,26

The most abundant component of silymarin, silybin (the mixture of silybins A and B), was the first constituent that was identified,^23,24 and it has been reported as the constituent with the highest bioactivity.^5,27,28 Additional minor flavonolignans have been identified, which together with a mixture of unknown flavonolignan oligomers and polymers constitute the remaining 20–35% of the silymarin fraction.^5,28

The foregoing data pertain to purple-flowered milk thistle. In addition to the main silymarin constituents, a white-flowered milk thistle variety also contains the 3-deoxyflavonolignans silandrin, isosilandrin, silymonin, silyhermin, and neosilyhermin.^29-31

The fruits also have a relatively high content of fatty oils (20–25%) that consist of fatty acids such as linoleic acid (60–66%), oleic acid (21%), palmitic acid (13%), phospholipids, and vitamin E.^32-34

1.9 General uses and forms: The principal medicinal product is the fruit extract standardized to contain a minimum of 45% silymarin by HPLC.¹⁶ The European Pharmacopoeia specifies ethyl acetate, acetone, ethanol, methanol, and mixtures of water with acetone, ethanol, and/or methanol as appropriate solvents for extraction.¹⁶ Standardized milk thistle extract has been used for supportive treatment of acute or chronic hepatitis and cirrhosis induced by alcohol, drugs, or toxins, treatment of dyspeptic complaints, and gallstones.^8,35 The dried extract is most commonly sold in the form of capsules or tablets. Traditional aqueous alcohol tinctures and extracts are also marketed but lack scientific data to support healthy liver claims. Silybin isolated from the silymarin extract is sold as a pharmaceutical treatment for toxic liver injury and hepatitis. The disodium salt of silybin C-2,3-dihydrogen succinate is approved for intravenous infusion in cases of acute death cap mushroom (Amanita phalloides, Amanitaceae) poisoning in some European countries, and approved for conditional use (physicians need to submit an application to the National Institutes of Health to gain access to it) for the same indication in the United States.³⁶

The fruits (whole, pulverized, and powdered) are variously sold in bulk, tea bags, capsules, or tablets. A separate ingredient, the cold-pressed fruit oil, is sold in bulk as an edible oil and in capsules and soft gels as a dietary supplement.³⁷ The seed cake, flour, and aerial plant parts may be sold as food or fodder supplements.^5,37

In addition to the medicinal uses, the photoprotective silymarin extract is also used as cosmeceutical ingredient.³⁷

Numerous methods for extracting silymarin have been described/patented.^{38,40,41,45,47-60}  Most of these methods are processes involving defatting, followed by silymarin extraction. After silymarin extraction, an additional defatting step using liquid-liquid extraction is often performed to further reduce the oil content.⁶¹

Defatting may be accomplished by cold pressing the oil from the fruit or solvent extraction. Cold pressing removes 60–65% of the oil. The cold pressed oil contains ~0.8–1.0 % silymarin and after further processing, it may be sold as an edible oil, dietary supplement, or biodiesel.^5,62 Another common process to remove the milk thistle seed oil is the extraction with hexane or petroleum ether, which may be followed by secondary defatting with di-isopropyl ether. On the industrial scale, defatting using solvents is associated with longer processing times, consumes relatively large amounts of organic solvents, and carries the risk of excessive residual solvent presence in the silymarin extract.⁶³ Alternative methods that preclude the need for defatting include hot water extraction, pressurized liquid extraction (PLE) with either acetone, ethyl acetate, or methanol, and mechanical separation. Hot water extraction offers a green alternative with low costs for solvent purchase and disposal; however, the high temperatures may degrade the silymarin.^{58 ,59} PLE or accelerated solvent extraction (ASE) utilizes increased pressure to optimize extraction conditions by keeping the liquid solvent below its boiling point at higher temperatures, and thus increasing extraction efficiency and reducing costs for solvent purchase and disposal.⁵⁶ AbouZid et al. described a mechanical separation of the silymarin-bearing seed coat from the oil- and protein-rich kernel, which permits the extraction of the seed coat with an appropriate solvent to produce a silymarin extract with a high silymarin content.⁶³

The European Pharmacopoeia (Ph. Eur.) specifies that silymarin extract is produced from the defatted fruit, using one or more of the following solvents: ethyl acetate; acetone or mixture of acetone and water; ethanol or mixture of ethanol and water; and methanol or mixture of methanol and water.¹⁶ Several studies suggest that acetone extraction may be the most cost-effective and result in the least toxic solvent residues.^50,56,61 Isolated silymarin is an important ingredient for the herbal drug (pharmaceutical) market and the silybin-depleted marc which still contains some milk thistle flavonolignans reportedly is sometimes offered for sale as silymarin extract (or simply as milk thistle seed) to the dietary supplement industry.^64-66 The sale of products with little or no flavonolignans labeled as milk thistle seed, silymarin, or milk thistle extract is misleading and considered fraudulent. Most all scientific literature demonstrating the benefits of milk thistle fruit extract are based on products characterized to contain 30–65% (by HPLC) of the silymarin complex.⁶⁷

By-products of milk thistle extraction, such as the seed flour (containing ~22% protein, 7% oil, and some residual silymarin), may be sold as a food and fodder supplement.³⁷ However, data on therapeutic benefits of milk thistle seed flour are lacking.

1.11 Other sources of silymarin/flavonolignans: The fruits of the related species, S. eburneum, have been reported to contain ~10% silymarin.⁶⁸ While there are limited data on the flavonolignan composition of this species, S. eburneum can be distinguished from milk thistle by the presence of high concentrations of isosilychristin.⁶⁹ Flavonolignans that differ in their chemical structure form silymarin compounds have been identified in members of the plant families Asteraceae, Berberidaceae, Chenopodiaceae, Flacourtiaceae, Fabaceae, Poaceae, and Scrophulariaceae.^22,28,70,71

Silybin has been isolated from the dried whole plant of Gentiana apiata (Gentianaceae) collected in Shaanxi Province, China,⁷² and from the fungal endophyte, Aspergillus iizukae (Trichocomaceae), isolated from the surface-sterilized leaves of milk thistle.⁷³

2          Market

2.1 Importance in the trade: In 2018, milk thistle held the eighth position among the top-forty best selling herbal dietary supplements in the US natural channel with sales of $10,419,926, an increase of 3.5% compared to 2017.⁷⁴ However, retail sales fell slightly in 2019 to the tenth position in this channel to $10,010,699, a drop of 4.7% from 2018. Milk thistle dietary supplements ranked 23rd in the mainstream market retail channel in 2019, selling a measured total of $16,244,188.⁷⁵

2.2 Market dynamics: Over the past three decades, sales have remained strong, with milk thistle consistently ranking among the forty top-selling herbal dietary supplements in the United States, particularly in the natural sales channel. However, when comparing the annual sales data from 2016–2019, the market has remained flat (Table 1). Both the type of milk thistle label claims and the content declared has broadened. Silymarin content claims range from 30–85% based on spectrophotometric ("UV/Vis") or high-performance liquid chromatography (HPLC) assessments. There is potential confusion with respect to levels of chemical standardization for silymarin and/or its constituents as different analytical methods produce lower or higher readings of constituents, respectively. Alternatively, some manufacturers specify silybin content (35–45%), some report flavonolignan content, and others do not make any marker content claims.

Table 1: Sales data for milk thistle dietary supplements in the United States from 2016-2019.^74-77

Channel	2016		2017		2018		2019
	Rank	Sales [US$]	Rank	Sales [US$]	Rank	Sales [US$]	Rank	Sales [US$]
Natural	6	9,968,995	7	9,960,892	8	10,419,926	10	10,010,699
Mainstream Multi-Outlet	16	17,077,481	17	16,799,553	20	16,596,226	23	16,244,188

The scientific research on milk thistle over the past three decades also has had significant impacts on market dynamics, including advances in plant breeding and agricultural research which have led to the development of silymarin-enriched varietals and improved agronomic techniques to increase the silymarin content of milk thistle fruit; processing and extraction optimization (especially the differential extraction of only the seed coat and the resultant increases in silymarin yield and reduction in financial and environmental costs); and research and development of markets for milk thistle by-products.^5,37 The adaptation of these advances has allowed manufacturers to produce milk thistle extracts with higher silymarin contents at lower cost, and access new revenue streams by marketing milk thistle by-products.

Costs for bulk milk thistle extracts vary depending on the origin of the material. Authentic European milk thistle extract is sold as an ingredient in bulk quantities for a wholesale price of US $130-150/kg. (C. Bewicke [Ethical Naturals] email to S. Gafner, August 11, 2020; G. Ris [Indena] email to S. Gafner, August 14, 2020) Authentic milk thistle extracts from outside Europe complying with United States Pharmacopeia (USP) specifications for powdered milk thistle extract²⁰ are sold at the wholesale level for US $90-110 (Cal Bewicke email to S. Gafner, August 11, 2020). An informal review by S. Gafner of bulk prices for ingredients sold as containing milk thistle extracts labeled to contain 80% silymarin on retail and e-commerce company Alibaba (Hangzhou, China) showed costs being generally in the range of US $30-35/kg, although some suppliers claim to offer such extracts for as little as US $1/kg.

2.3 Supply sources: Teuscher et al. stated that milk thistle comes "from cultivated plants grown to a limited extent in northern Germany but mainly imported from Argentina, Austria, China, Hungary, Poland, Romania, and Venezuela."³⁵ Vereš and Týr reported that milk thistle is grown commercially as a medicinal plant in Europe, Egypt, China, and Argentina.⁷⁸

Some manufacturers purchase milk thistle extracts rather than raw milk thistle fruit. As outlined in section 1.11, extraction methods significantly impact silymarin composition and content, as well as the cost of production. In addition to pharmacopeial 30–70% silymarin extracts of milk thistle fruit, milk thistle by-products are also known to be sold as "milk thistle extract." Products that do not contain the concentration and composition of silymarin as supported by the scientific literature may not deliver the liver-supportive benefits documented in the pharmacological and clinical research literature and expected by consumers and/or health professionals. Additionally, as noted, extracts prepared from by-products claiming to provide “silymarin” and not meeting the standard definition as established by pharmacopeial monographs and the scientific literature are considered to be adulterated.

There have also been significant shifts in the geographical origin of the commercial supply, and the analytical methods used to assess silymarin content. In the 1990s and early 2000s, Europe was the primary supplier of raw milk thistle and the analysis of silymarin content was largely based on spectrophotometry. Since then, the market has diversified with Asia now providing the majority of the international supply, and analytical methods have advanced considerably. However, concerns have been expressed that companies which market milk thistle products with label claims based only on spectrophotometric analysis may accidentally or intentionally be selling products that are silybin-depleted or contain <30% silymarin.^65,79

3          Pharmacopeial Standards

The World Health Organization (WHO) and the USP specify the dried mature fruit contains not less than 1.5% silymarin, calculated as silybin.^8,18 The USP defines milk thistle extract as containing 90–110% of the claimed amount of silymarin, calculated as silybin, consisting of the sum of silychristin and silydianin (20–45%), silybin A and B (40–60%), and isosilybin A and B (10–20%).²⁰ The Ph. Eur. defines the dried standardized extract as containing "30–65% silymarin, expressed as silibinin, and corresponding to the sum of silychristin and silydianin (20–45% of silymarin), silybin A and B (40–60% of silymarin), and isosilybin A and B (10–20% of silymarin)."¹⁶

4          Adulteration

4.1 Known Adulterants: Depleted Silybum marianum extracts, Silybum eburneum, and synthetic colorants. Globe thistle (Echinops echinatus, Asteraceae) reportedly has been confused with milk thistle in Pakistan due to the use of the same vernacular name “untkatara” for both milk and globe thistle in Hindi and Urdu,^80,81 but this appears to be a geographically limited issue.

4.2 Sources of information confirming adulteration

4.2.1 Chemical evidence: According to Langhammer,⁸² it is difficult to discriminate between the species S. marianum and S. eburneum based on morphological characteristics and hence, wildcrafted material may contain the fruits of S. eburneum.

Numerous studies have found that commercial milk thistle products do not meet their label claims for silymarin content or pharmacopeial specifications for silymarin content.

Frommenwiler analyzed 31 milk thistle products which were acquired from the internet, local shops, and pharmacies in the United Kingdom (UK), by high-performance thin-layer chromatography (HPTLC).⁸³ Among these products, 10 had a Traditional Herbal Registration while the remaining 21 products were sold as food supplements. The 10 THR products, and seven food supplements were deemed to be of good quality; seven additional food supplements were consistent with the milk thistle fingerprint, except that these products were missing the zone for taxifolin. However, four food supplements contained a weak chromatographic fingerprint, and two did not show any of the characteristic milk thistle zones.

Fenclova et al. used a validated ultrahigh-performance liquid chromatography mass spectrometry (UHPLC-MS) method to assess the quality of 26 milk thistle supplements sold as capsules* in the United States (n = 19) and Czech markets (n = 7). Of the 24 products with silymarin label claims, total silymarin (TS) content ranged from 35–125% of the declared content, with four products containing <50% of the declared content and four containing more than the declared content. The sum of flavonoid/flavonolignan content ranged from 5–393 mg/g of capsule content, the maximum recommended daily intake (RDI, based on product label recommendations) ranged from 18–600 mg/day, and the sum of silybin A and B ranged from 36–66% of total silymarin (i.e., all of the products contained silybin).⁸⁴

Fibigr et al. also used a validated UHPLC-UV method to evaluate the silymarin content of four teas and seven supplements sold in the Czech Republic. The total silymarin content of the teas ranged from 3.4 mg/g to 7.1 mg/g of tea. The supplements contained 39.5–77.6% of the claimed silymarin content; the two registered pharmaceutical preparations had the highest content (72.9% and 77.6% of the label claims). The seven supplement samples contained 42.9–59.4% silybin, calculated as the percentage of total silymarin.⁸⁵ While some of the food supplements had much lower than declared silymarin content, this may be partly due to different standardization methods rather than using spent milk thistle fruit.

Pendry et al. evaluated the silymarin content in 11 milk thistle tinctures purchased in the United UK using HPLC-UV. The tinctures varied in their extraction ratios (4.54–18.37 mg/mL w/v) and the percentage of alcohol (25–75%). Silymarin could not be detected in the seven samples extracted with 25% ethanol (regardless of the extraction ratio which ranged from 1:5 to 1:1), possibly due to the instability of flavonolignans in low alcohol tinctures.⁸⁶ Of the four samples with detectable amounts of silymarin, only one sample with an extraction ratio of 1:1 and an alcohol content of 70% provided an effective therapeutic dose and one sample did not exhibit the characteristic silymarin fingerprint, suggesting the sample was derived from an adulterant.⁸⁷

Scientists at the University College of London (UCL) evaluated seven milk thistle products sold on the European market for a television show by the British Broadcasting Corporation (BBC) using high-performance thin-layer chromatography (HPTLC); six of the products were legally sold in the EU as food supplements with no silymarin label claims and one was a registered traditional herbal medicine (THM). Two of the supplements (36%) did not contain any milk thistle; one of these produced a faint fingerprint suggesting the presence of an unidentified synthetic adulterant. Another two supplements contained only low levels of milk thistle. Three products (one traditional herbal remedy and two food supplements) produced fingerprints comparable to the standard.⁸⁸ Further testing by UCL researchers showed that of 18 commercial supplements, products sold under a traditional herbal registration (THR) were of good quality, while 22% of food supplements were of poor quality with little or no detectable milk thistle.⁸⁹

Eight of the 12 companies ConsumerLabs tested failed testing for silymarin content in commercial milk thistle products. Among milk thistle extract supplements claiming to provide 80% silymarin, amounts in five products actually ranged between 51.7–60.4% silymarin. Three other milk thistle supplements which were tested did not list their amount of silymarin but failed to meet minimum standards.⁹⁰ Some of the discrepancies observed are likely due to milk thistle supplement manufacturers using spectrophotometric methods for standardization, which, as noted above, produces a higher value for silymarin. ConsumerLab used a HPLC-UV method for the product analysis, which produces a reading of lower numerical value, and thus, perceptually of lesser chemical concentration.

Stranska and Hajslova used UHPLC-high resolution mass spectroscopy (HRMS) analysis to assess the silymarin content of milk thistle products sold in the EU. They determined that three out of seven milk thistle powdered extract products contained ≤ 50% of the declared silymarin content. Of the six milk thistle oil products assessed, three contained no detectable silymarin and three contained less than 15 mg/capsule; two of the milk thistle oils contained less than 30 mg/kg silymarin. Of the 10 milk thistle teas analyzed, five contained <30% silymarin and only two contained ˃60% silymarin.⁹¹

Anthony and Saleh assessed 45 commercial milk thistle products from the United States (24) and international markets (21, purchased in Cairo, Egypt) using HPLC-MS and HPTLC. Silymarin label claims ranged from 51.8 to 1358.0 mg/tablet. One product from the United States and three international products did not contain any silymarin, and 23 contained <30% silymarin (11 from the United States and 12 international). Four products from the United States and 17 international products showed non-silymarin bands at retention factors (Rfs) above silybin. Two US products stood out because of their relatively high concentration of silybin, representing 78% and 93% of the total silymarin, well above the 40–60% specified by the European and US pharmacopeias. It is not clear how such silybin-enriched extracts are obtained, but it could point to the addition of silybin made by chemical synthesis (a process frequently referred to as spiking), which was published as early as 2000.⁹² Another explanation is the use of an extraction process that selectively enriches silybin.^93,94 Eight samples contained <100 mg/g, nine samples contained 100–200 mg/g, six contained 200–300mg/g, eight contained 300–400 mg/g, five samples contained 400–500 mg/g, and five samples contained ˃500 mg/g.⁹⁵

Lee et al. evaluated the content of silychristin, silydianin, silybin A, silybin B, isosilybin A, and isosilybin B in seven commercial milk thistle extracts purchased in Philadelphia, PA using HPLC-MS. Six of the products were manufactured in the United States and one was manufactured in China.⁹⁶ Silydianin was the least abundant constituent, and the content of this constituent did not vary significantly among the samples. There was considerable heterogeneity among the samples in the content of silychristin, silybin A, silybin B, isosilybin A, isosilybin B, and total silymarin. Of the six US products, one had substantially higher levels of total silymarin, and two products had significantly lower contents of total silymarin, while the remaining three were intermediate and very similar in content. The Chinese extract had the lowest contents of total silymarin. Since the authors did not divulge the exact sample preparation procedure, only a relative comparison of silymarin contents among the products can be made. The relative content in silybin (39–46% of total flavonolignans) in the six US samples was relatively consistent, while the relative silybin content in the sample from China (33.8%) was a bit lower.⁹⁶

Kvasnicka et al. used HPLC-UV to analyze the silymarin content of six commercial silymarin extracts purchased in the EU. They reported that the total silymarin content of the silymarin extracts ranged from 17.21–63.96%; among three different batches of the same product, the content ranged from 41.6–63.96%. In all of the samples, silybin made up between 40–60% of the total flavonolignans.⁹⁷

Three additional publications detailed results from quantitative analysis of commercial milk thistle products using HPLC-UV.^98-100 In two publications, all of the analyzed products contained significantly less silymarin than the amount claimed on the label; however, the silybin content was between 40–60% of total flavonolignans.^98,99 In the third paper, Schulz reported that the nine commercial milk thistle products tested contained 58–116% of the silymarin label claim, when measured by HPLC-UV.¹⁰⁰ These findings can be attributed in most cases to the manufacturers’ use of a spectrophotometric method for determining silymarin content (per section 4.7 below).

A consistent theme of all these published data on milk thistle quality is the market presence of products with lower than declared contents in silymarin. In addition, four papers provided evidence of milk thistle adulteration based on the absence of any of the characteristic silymarin signals, or additional peaks/zones from undeclared adulterants.^87,88,95,101 The identity of these adulterants was not determined in any of the four investigations.

Before the advent of official HPLC-UV methods for the flavonolignan quantification, most manufacturers relied on spectrophotometric methods. Some manufacturers use a direct UV-spectrophotometric method (absorbance at 288 nm; without derivatization) for quantitative estimation of silymarin in the extracts using silybin as the reference standard, while others use a formerly official method which calculates the total flavonolignan content based on the absorption of a milk thistle extract solution after a reaction with 2,4-dinitrophenylhydrazine in alkaline conditions at 490 nm,¹⁰² or variations thereof. Many suppliers still use spectrophotometric methods for standardization.⁶⁶ Fraudsters have reportedly taken advantage of the lack of specificity of UV/Vis tests by adding undisclosed colorants to milk thistle extracts in order to provide an appearance of compliance with standardization requirements.¹⁰³

4.2.2 Other evidence of adulteration: There is ample evidence (see section 4.2.1) of commercial products containing lower amounts of silymarin. While such data do not provide direct evidence for milk thistle adulteration, as it is possible that a manufacturer uses low-quality starting material, leading to an extract with low amounts of flavonolignans, there is consensus from industry experts that these low-quality products are most often due to intentional silymarin depletion of milk thistle fruit, constituting the fraudulent sale of a low-quality product to unsuspecting buyers.^64-66,104  

4.3 Accidental or intentional adulteration: The sale of products that do not meet their silymarin label claims could be attributed to the use of the spectrophotometric method to determine silymarin content; however, the sale of products that do not contain any detectable silymarin (and in some cases, contain foreign substances) cannot be accidental unless the manufacturer(s) did not have adequate quality assurance programs. The sale of depleted milk thistle fruit extracts is done on purpose, since such extracts can be sold at a lower price and thus provide a competitive advantage to fraudulent suppliers and manufacturers.^64-66 Regarding other forms of adulteration, as most of the commercial milk thistle supply is produced under cultivation, the risk of accidental adulteration is very low; however, the misidentification of other thistle species may occur when milk thistle fruits are collected in the wild.⁸⁰

4.4 Frequency of occurrence: The milk thistle monograph in Wichtl (2016) stated that adulteration "practically never occurs," and in the milk thistle fruit supply chain that may largely remain true.³⁵ However, as summarized in section 4.2 the evidence accumulated since 1994 suggests that 30–50% of commercial products that are labeled to contain milk thistle extract do not meet their label claims for silymarin content. The occurrence of products that did not contain silymarin ranged from 0–28.6% and the incidence of products with evidence of an adulterant ranged from 0–46%, demonstrating that the trade of substandard extracts as an ingredient is extensive.

4.5 Possible safety/therapeutic issues: While the sale of milk thistle products with sub-therapeutic levels of silymarin pose no immediate safety concerns, the therapeutic impacts are substantial, considering that consumers who use milk thistle may have serious liver disease or liver dysfunction and may not obtain the benefits expected. Additionally, the presence of unidentified adulterants may potentially pose safety issues. As Spink and Moyer point out, the act of adulteration, although carried out with economic or financial motivation, can have an effect that can lead unintentionally to a public health concern.¹⁰⁵

4.6 Analytical methods to detect adulteration: For whole fruit, botanical identity may be confirmed by a qualified analyst based on organoleptic and macroanatomical characteristics. The identity of powdered material may be determined using a combination of organoleptic, microscopic, and chemical techniques. Both the Ph. Eur. and USP specify HPTLC methods for the identification of milk thistle fruits,^18,19 powdered milk thistle fruit,¹⁰⁶ and powdered extracts.^16,20 Langhammer reported a simple chemical test with sulfuric acid to differentiate S. marianum and S. eburneum.⁸² but the sensitivity to identify mixed batches may be lacking.

There are numerous chemical techniques that can be used to authenticate milk thistle extracts, including TLC,¹⁰⁷ HPTLC,²⁰ HPLC-UV,^44,85,86,108 HPLC-MS,^{84,86,96,109,110} mid- and near-infrared spectroscopy (MIR and NIR),^111,112 nuclear magnetic resonance (NMR),¹⁰⁸ and various combinations of these methods. Quantitation of silymarin in a given extract is complicated by the fact that part of what is considered to be silymarin is a mixture of flavonolignans. Though they differ slightly, the Ph. Eur. and USP provide official HPLC-UV methods for authentication of milk thistle extracts and quantification of silymarin content. Silymarin depletion and silybin spiking can be determined by measuring the individual flavonolignan concentrations, e.g., using HPLC-UV. However, other forms of adulteration such as the presence of dyes may not be detected unless the methods have been specifically developed to do so.^113,114 Genetic approaches, such as random amplified polymorphic DNA (RAPD) analysis,^115-117 have focused mainly on the distinction among milk thistle genotypes, but such methods could be used as well to differentiate milk thistle and closely related species. Since the same DNA is found in all parts of a specific plant species, DNA-based identification methods cannot distinguish between genuine and depleted milk thistle extracts.

4.7 Perspectives: A number of hypotheses have been put forth to explain the frequent occurrence of milk thistle products containing <30% silymarin, including silybin depletion, the use of spectrophotometry to quantify silymarin content, milk thistle by-products, dyes, and excipients. Silybin depletion is a reasonable explanation, because silybin is easily isolated from silymarin extract by precipitation, isolated silybin has a much higher market value than silymarin, and the silybin-depleted marc might be sold inappropriately as silymarin extract. However, none of the studies described in section 4.2.1 reported evidence supporting this hypothesis (i.e., all of the products that contained silymarin also contained silybin in appropriate proportions to the other marker constituents). Another explanation is the sale of silymarin as a whole to the pharmaceutical industry, while the leftover, post-extract milk thistle fruit mass (marc) might be re-extracted and sold to the dietary supplement and cosmetic industries.

When the label in commercial products claims 80% silymarin, it is indicative that the number is based on analysis by UV/Vis spectrophotometry and not HPLC-UV. The high incidence of milk thistle products containing <30% silymarin reported in the published literature has also been attributed to the use of spectrophotometry to analyze silymarin content. The 2,4-dinitrophenylhydrazine method assesses the content of all ketones/flavones in milk thistle samples, not only the flavonolignans that constitute the silymarin complex. Hence, this technique typically overestimates silymarin content.^65,79,118 Direct analysis at 288 nm usually provides even higher “silymarin” contents as many compounds absorb at this wavelength and thus interfere with the assay. In addition, spectrophotometric analysis may not detect other forms of adulteration, such as the presence of milk thistle by-products, other herbs with high flavone/flavonolignan content, or dyes. For these reasons, HPLC-UV analysis superseded spectrophotometry for the authentication of milk thistle extracts.

The substitution/addition of non-functional plant parts and the deliberate mislabeling of these products is one of the oldest forms of adulteration.¹¹³ The entry of milk thistle by-products into the commercial supply chain, together with the increase in the complexity of supply networks, has created an environment where these inexpensive, "near-identical" milk thistle materials that may pass both spectrophotometric and cursory chromatographic analyses can effectively be sold as " milk thistle extracts."¹¹⁹ The presence of milk thistle by-products in commercial milk thistle preparations may also account for the appearance of HPTLC bands that do not occur with authentic fruit extracts. 

The increasing incidence of unauthorized colorants/dyes in herbs and spices has been ranked as a serious hazard.¹²⁰ In the absence of analytical methods specifically designed for their detection, this form of adulteration may go undetected,^{101,114,121-123} especially when combined with milk thistle by-products. 

A largely unexplored explanation of finding such a large array of substandard products is the presence of large amounts of excipients in finished milk thistle products. Silymarin has poor oral bioavailability and a number of novel formulations specifically designed to enhance bioavailability have been developed.^124-135 However, the impact of these excipients/processes on the efficiency of silymarin extraction in the gastrointestinal tract has not been investigated.

Considering the issue from a geographical perspective, China has become a leading supplier of milk thistle and several authors have reported that "Chinese" milk thistle extracts contain <30% silymarin. However, this constitutes weak evidence when the low number of samples that have been analyzed (<10) are considered in relation to the high volume of milk thistle that China produces. More rigorous research with representative sampling is needed before this postulate can be given credence. 

Unfortunately, most of the investigators reporting the occurrence of milk thistle products containing <30% silymarin did not conduct in-depth analyses of the actual chemical composition of the suspect products. In the absence of further evidence, it is unclear which of these hypotheses may be correct; however, considering the current domination of HPLC analysis in the industry, it strains credibility to accept that the recently reported deficiencies may be blamed on spectrophotometric overestimation of silymarin content.

Further, none of these postulates can explain the alarmingly high incidence of milk thistle products that do not contain any silymarin.

5          Conclusions

Regardless of the underlying cause, the relatively high incidence of materials labeled as milk thistle that contain little or no silymarin is clearly an ongoing problem, a problem that is legally defined within some countries as food fraud.^104,136,137 Lack of adequate therapeutic benefit in patients believing that they can utilize milk thistle for serious liver disease, and loss of consumer confidence due to products that do not meet their label claims are serious concerns. The sale of products labeled as containing milk thistle but lacking the claimed ingredient and containing unidentified adulterants instead potentially may also pose a health concern. However, it is a risk which may be prevented with appropriate supply chain qualification and adequate quality control, and quality assurance protocols.

* The RDIs for milk thistle issued by the European Medicines Agency⁶ varies depending on the extraction solvent and the drug-extract ratio. However, the lowest RDI is generally above 200 mg. For comparison, the RDI for the most extensively clinically tested milk thistle product, Legalon® (Meda Pharma AB, Solna, Sweden) is standardized to 420 mg silymarin per day.

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