Silymarin Extraction
Mar 02, 2021

silymarin extraction process

Silymarin, a mixture of flavonolignans exhibiting many pharmacological activities, is obtained from the fruits of milk thistle (Silybum marianum L. Gaertner). Due to the high lipid content in thistle fruits, the European Pharmacopoeia recommends a two-step process of its extraction. First, the fruits are defatted for 6 h, using n-hexane; second, silymarin is extracted with methanol for 5 more hours.

The presented data show that this extremely long traditional Soxhlet extraction process can be shortened to a few minutes using pressurized liquid extraction (PLE). PLE also allows to eliminate the defatting stage required in the traditional procedure, thus simplifying the silymarin extraction procedure and preventing silymarin loss caused by defatting. The PLE recoveries obtained under the optimized extraction conditions are clearly better than the ones obtained by the Pharmacopoeia-recommended Soxhlet extraction procedure.

The PLE yields of silychristin, silydianin, silybin A, silybin B, isosilybin A and isosilybin B in acetone are 3.3, 6.9, 3.3, 5.1, 2.6 and 1.5 mg/g of the non-defatted fruits, respectively. The 5-h Soxhlet extraction with methanol on defatted fruits gives only ∼72% of the silymarin amount obtained in 10 min PLE at 125°C.

Silymarin Extraction PROCESS

What is Silybum?

Silybum marianum L. Gaertner, commonly called as milk thistle, blessed milk thistle, Marian Thistle, Mary Thistle or Saint Mary's Thistle, is an annual or biannual plant from the Asteraceae family. The plant, originally growing in Southern Europe and Asia, is now found throughout the world (1). This troublesome weed is presently cultivated as a medicinal plant and is one of the most important medicinal crops in Europe.

Milk thistle has been used for medicinal purposes for over 2000 years, most commonly for the treatment of liver disease (cirrhosis and hepatitis), as well as for the protection of the liver from toxic substances (2–5). Recent research interest in this plant has been stimulated by studies showing its exceptionally high antitumor activity. Extracts from the plant are now under intense study in the experimental chemoprevention of cancer, and in the amelioration of chemotherapy side effects (6). Recent reports have demonstrated that extracts from this plant are also characterized by many other pharmacological activities, such as anti-inflammatory and antifibrotic effects (2, 7, 8).

Silymarin, a mixture of flavonolignans exhibiting many pharmacological activities, is obtained from the seeds of milk thistle (Silybum marianum L. Gaertner). The common procedures used so far for silymarin extraction are based mainly on slow high-temperature solvent-extraction methods. The drawbacks associated with these techniques have led to the search for new alternative extraction processes that additionally could improve silymarin extracts quality. The latter is particularly important as regards silymarin application. The obtained silymarin extracts, in fact, could contain toxic organic residues that need to be removed to meet consumer acceptability.

In the attempt to improve the process of silymarin extraction and make it more effective and environmentally friendly, recently microwave-assisted solvent extraction, ultrasound-assisted solvent extraction, pressurized liquid extraction, enzyme-assisted extraction, supercritical fluid extraction, and especially subcritical water extraction also known as pressurized hot water extraction methods have been developed and applied.

What is Silybum

Materials and Methods

Plant material

Dried fruits of S. marianum L. Gaertner were purchased from a local pharmacy (Lublin, Poland) in autumn 2011. A sufficiently large representative sample of the plant material (ca. 500 g) was ground and sieved to obtain the particle size of 0.4 mm. Precisely weighed portions of the material were used for extractions.

Materials and reagents

The standardized dry extract of S. marianum L. Gaertner and silybin B (with a purity of 98%), applied as standards, was purchased from Sigma-Aldrich, Poland. Acetone, ethyl acetate, phosphoric acid, n-hexane (all of them of analytical reagent grade) and methanol (analytical reagent grade and HPLC grade) were purchased from the Polish Chemical Plant (POCh, Gliwice, Poland). Water was purified using a Milli-Q system from Millipore (Millipore, Bedford, MA, USA). Neutral glass, obtained as a gift from local glassworks (fraction 0.4–0.6 mm), was applied as a dispersing agent in the PLE cell.

Pressurized liquid extraction

PLE was performed with a Dionex ASE200 instrument (Dionex Corp., Sunnyvale, CA, USA). The plant material (0.5 g) was mixed with inert material (neutral glass) and placed into a 22-mL stainless steel extraction cell containing filter paper at the bottom. Another circle of filter paper was placed at the top of the extraction cell. Finally, the cell was tightly closed and placed in the heating oven.

The content of the cell was extracted at the operating pressure of 60 bar. At the end of the process, the extracted sample was flushed using the solvent volume equal to 60% of that of the extraction cell. Finally, the sample was purged for 60 s applying pressurized nitrogen (150 psi.), and the extract was collected into a 60-mL glass vial with a Teflon-coated rubber cap. The volume of the collected extract was between 25 and 31 mL, depending on the packing density of the extraction cell. The obtained extract was transferred into a 50-mL volumetric flask and filled up to its volume using an appropriate solvent type. Three independent extractions were performed under the same conditions. Between the runs, the system was washed with an appropriate extraction solvent.

PLE parameters under study were solvent type (methanol, acetone and ethyl acetate), temperature (50, 75, 100, 125 and 150°C), time (5, 10, 15 and 20 min) and the number of extraction cycles (1–5). For the PLE defatting process, n-hexane was applied as solvent, and parameters under study were temperature (50 and 100°C) and time (5 and 10 min) of lipids removal. Acetone and ethyl acetate extracts were evaporated to dryness under vacuum and redissolved in methanol before chromatographic analysis.

silymarin extraction from milk thistle

Soxhlet extraction

Exhaustive extractions in the Soxhlet apparatus were performed using 2.0 g portions of the material. Precisely weighed samples were transferred to a paper thimble. The loaded thimble was inserted into a 100-mL Soxhlet extractor. Extractions were performed in the two-step process (n = 3). In the first step of the procedure, the plant material was defatted for 6 h using 75 mL of n-hexane. In the second, silymarin was extracted for 5 h with 75 mL of methanol. After cooling to room temperature, the obtained extract was transferred to a 100-mL volumetric flask, which was subsequently filled up to its volume with methanol. Three independent extractions were performed.

Chromatographic analysis of extracts

HPLC measurements were performed on a Dionex Liquid Chromatograph (Dionex Corp.) consisting of a chromatography enclosure (LC20) containing a PEEK automated injection valve equipped with a 10- μL sample loop, a gradient pump (GP50), an absorbance detector (AD25) and a photodiode array detector (PDA100). The whole chromatographic system was under the control of the PeakNet6 data acquisition system. Chromatographic separations were carried out at 40°C using a Prodigy ODS-2 column (5 μm, 250 × 4.6 mm, ID) (Phenomenex, Torrance, CA, USA). Mobile phase A was a mixture of methanol with aqueous phosphoric acid solution containing 0.5 mL of 75% phosphoric acid in 100 mL of solution (35 : 65, v/v). Mobile phase B was a mixture of methanol with the aqueous phosphoric acid solution containing 0.5 mL of 75% phosphoric acid in 100 mL of solution (50 : 50, v/v). The flow rate was 0.8 mL/min. The analyses were performed in a mobile phase gradient with the percentage of B in A varying as follows: initial concentration, 0%B; 28 min, 100%B; 35 min, 100%B, 36 min 0%B. Before the next analysis, the column was equilibrated using the mobile phase containing 0%B for 20 min. Each extract was HPLC-analyzed three times. The wavelength for detecting flavonolignans was set at 288 nm, and the UV-Vis spectra from 210 to 500 nm were also recorded for peak characterization.

The qualitative analysis of the extracts was carried out by comparing the retention times of the peaks and their UV–Vis spectra in the extracts with respect to those of the standardized dry silymarin sample. To prepare the standardized dry silymarin solution, a 0.02-g portion of the sample containing 5.0 mg of silybin A + B was dissolved in 50 mL of methanol. The peaks for silychristin, silydianin, silybin A, silybin B, isosilybin A and B appeared at retention times of 15.1, 17.4, 27.5, 29.1, 33.6 and 34.8 min, respectively. Quantitative analysis was based on silybin B standard, and external standard method was used. A calibration curve was generated from five concentrations of the compound in the concentration range of 0.1–1.0 mg/mL. Three measurements of peak area for each concentration of standard solution were performed. The characteristic parameters of the obtained calibration curve were as follows: slope, 0.516 and intercept, 0.003. The calibration curve was found to be linear in the tested concentration range. The correlation coefficient was found to be >0.995. Because of the difficulty of purchasing silychristin, silydianin, silybin A, isosilybin A and B standards, the amounts of these compounds were calculated by relating their chromatographic responses to the calibration curve for silybin B.

Statistical analysis

All data are expressed as mean ± standard deviation (SD). The analysis of variance (ANOVA) and F-test were used to assess the influence of PLE conditions on silymarin yield. The mean values were considered significantly different when result of compared parameters differed at P = 0.05 significance level. To check the significance of each Fisher coefficient, the P-values were used.


Figure 1a presents typical chromatogram of PLE extract obtained from the fruits of S. marianum L. Gaertner, whereas Figure 1b shows the chromatogram of silymarin extraction from milk thistle solution prepared dissolving the standardized dry extract of S. marianum L. Gaertner in methanol (1 mg/mL). The analysis of chromatograms of PLE or Soxhlet extracts with that for standardized solution (retention times, UV–Vis spectra and peak purity index) proved that the applied chromatographic conditions allow for a sufficient resolution of the examined compounds, peaks numbered from 1 to 6, from sample matrix components. The peaks were identified as: (1) silychristin, (2) silydianin, (3) silybin A, (4) silybin B, (5) isosilybin A and (6) isosilybin B, respectively.

How to extract milk thistle

How to extract milk thistle?

Unlike most herbs, milk thistle seeds do not very extract well in water so don’t try to make a cup of tea out of them! Instead eat the fresh ground seeds, or get a good quality capsule. If using the seeds buy them whole and then grind them as needed (I use a coffee grinder to make them into a coarse powder). The seeds taste oily, sweet and bitter all at once — perhaps they are an acquired taste, but they are not unpleasant at all. Start with taking 1/2 to 1 tablespoon per day sprinkled on your food and see how it feels to you, adjusting the amount as you need.


1 cup lukewarm water

1 cup 100 proof alcohol I use vodka

¾ to 1 cup milk thistle seeds ground if possible


  1. Sterilize the Mason jar being used by placing it in boiling water for approximately 10 minutes, or by placing it on the rack in the oven at about 250 degrees for 15 minutes.

  2. Pour all of the ingredients in a Mason jar.

  3. Place a new lid on the jar, and screw it in place firmly with a ring.

  4. Shake the jar vigorously for about 1 minute.

  5. Put the jar in a cool dark place for five weeks.

  6. Shake the jar daily to ensure the milk thistle seeds do not float to the top, and get fully infused with the alcohol and water.

  7. At the end of the designated number of weeks, strain the milk thistle away from the liquid through a fine mesh strainer – keeping the liquid. If you do not have a fine mesh strainer, use a small colander and/or line a colander with several coffee filters to decrease the size of the openings.

How long for milk thistle to take effect?

Milk thistle seed might protect liver cells from toxic chemicals and drugs. It also seems to have blood sugar-lowering, antioxidant, and anti-inflammatory effects.

Improvement in oxidative stress parameters was consistently noted at six months' duration of treatment with a specialized milk thistle delivery system (2). Improvement in oxidative stress patterns is responsible for improved insulin-signaling and liver cell functioning.

How long should be milk thistle be taken?(Milk thistle DOSAGE)

Milk thistle is considered safe in dosages of 420 mg/day orally in divided doses for up to 41 months. One source suggests daily doses of 12 to 15 g of dry fruits for dyspepsia and disorders of the biliary system, while an extract containing 200 to 400 mg/day of silymarin is considered effective in various liver disorders.


Milk Thistle Benefits

1. Supports liver health

One of the most common uses of milk thistle is to treat liver problems. A 2016 study found that milk thistle improved diet-induced liver damage in mice. More evidence is needed to prove that milk thistle benefits human livers in the same way.However, researchers theorize that it does. The active ingredient in milk thistle, silymarin, acts as an antioxidant by reducing free radical production. Scientists think this creates a detoxifying effect, which is why it is milk thistle may be beneficial for liver problems.

Until more research is carried out, however, milk thistle is not recommended as the primary treatment option for liver problems. But it may be a helpful complementary treatment to try.

2. Promotes skin health

Milk thistle oil may be used topically to improve skin health.

Milk thistle may help to promote healthy skin. A 2015 study found that milk thistle helped improve inflammatory skin conditions when applied to the skin of mice.

Milk thistle was also found to have antioxidant and anti-aging effects on human skin cells in a laboratory environment in another study.

Further research on humans is needed to identify what benefits a person can expect from applying milk thistle to their skin.

3. Reduces cholesterol

High cholesterol can lead to problems with heart health and increase a person’s chance of stroke.

A 2006 study suggests milk thistle may play an important role keeping cholesterol levels down. It found that cholesterol levels were lower in people taking milk thistle to treat diabetes than those taking a placebo.

4. Supports weight loss

Initial animal research conducted in 2016 found that silymarin caused weight loss in mice that were fed a diet intended to cause weight gain.

This suggests milk thistle may be beneficial for those looking to lose weight. More research into the effects of milk thistle on weight loss in humans is needed to confirm this, however.

5. Reduces insulin resistance

A study on mice found milk thistle extract helped to reduce insulin resistance. Insulin resistance is a problem for people with type 2 diabetes.

Although this research suggests milk thistle could play a role in diabetes management, more research is needed to confirm whether milk thistle reduces insulin resistance and supports diabetes management.

6. Improves allergic asthma symptoms

The active ingredient in milk thistle can help to reduce inflammation. A 2012 study found that silymarin helped to protect against inflammation in the airways of mice with allergic asthma.

More research is needed to see if silymarin benefits asthma symptoms in humans.

7. Limits the spread of cancer

Milk thistle may help to stop the spread of certain types of cancer. A 2016 review found that milk thistle extract inhibited the growth of cancerous cells in colorectal cancer.

More research is needed to determine how milk thistle may be used to help fight cancer.

8. Supports bone health

Milk thistle tea may help to prevent bone loss caused by a lack of estrogen. Milk thistle may play an essential role in supporting bone health. A 2013 study found that milk thistle helped to prevent bone loss.The study looked specifically at bone loss caused by a deficiency in estrogen. It is not yet clear whether milk thistle is equally beneficial for bone loss with a different cause. Further studies are needed before it is safe to conclude that milk thistle supports bone health in humans.

9. Improves cognition

A 2015 study found that milk thistle increased resistance to oxidative stress. Oxidative stress is a potential cause of Alzheimer’s disease.

In this way, milk thistle may help improve cognition and treat degenerative conditions that affect the mind. More research on humans is needed to confirm the effects of milk thistle on cognition.

10. Boosts the immune system

Milk thistle may help strengthen a person's immune response and help them fight off infection.

A 2016 study on an animal model found that milk thistle extract improved the immunity when consumed. An older study found that milk thistle extract had a positive effect on immune response in humans.

More studies with human participants are needed before scientists can say with certainty that milk thistle boosts a person’s immune system.

Milk thistle side effects

Milk thistle seems to have few side effects, even when taken for several years. Some people have nausea, diarrhea, itching, and bloating.

For bulk milk thistle powder, please contact us at [email protected]


  • QR Code