Milk thistle [
For this experiment, we used representative samples of fruit collected in 2015 and 2016 and distributed by the company Agrofos (Slovakia). The samples were analysed according to the guidelines of the European Pharmacopoeia (Ph. Eur. 10), which were partially modified by the methodological procedure. High-performance liquid chromatography method (HPLC) was applied to evaluate the silymarin complex of Silybi mariani fructus, using Agilent 1200 Infinity system. The research was carried out at the Department of Sustainable Agriculture and Herbology of Slovak University of Agriculture in Nitra.
The samples were ground in a grinder and 5 g was placed in the apparatus for continual extraction. We added 100 ml of petroleum ether and let it heat in a water bath for 8 hours. We added 100 ml of methanol to the sample and placed it in the apparatus where it was extracted in a water bath for another 5 hours. After the evaporation of the methanol extract, the sample was concentrated to a volume of about 30 ml. The extraction flask and filter were washed with methanol, and the extract was filled in up to 50 ml. Consequently, we prepared the reference solution by dissolving the dried thistle extract and by diluting the sample to 100 ml with the same solvent.
The following were the HPLC conditions: column with length l = 0.125 m and with diameter = 4 mm; stationary phase – 5 μm silica gel end-capping octadecylsilyl; mobile phase – a mixture of phosphoric acid, methanol and water (0.5:35:65), and a mixture of phosphoric acid, methanol and water (0.5:50:50) with the flow 0.8 ml.min−1. The detection was carried out by spectrophotometer at 288 nm and injection volume 10 μl.
Concerning the identification of silymarin complex fractions, we used the record from the chromatogram of the dried milk thistle's extract and from the chromatogram of the reference solution to correctly identify the area and height of the peaks for silychristin, silydianin, silybin A, silybin B and isosilybin A + B (Fig. 1). The peak areas of the respective diastereomers were calculated by the percentage of the total peak area of a known concentration of the isomeric flavonolignan mixture. To obtain the results, we calculated the specific weights of flavonolignans presenting the proportional relations between the individual peak areas and their corresponding applied quantity.
The statistical evaluation of data was carried out by using the program STATISTICA CZ version 10 and ANOVA main effects by means of Fisher's LSD test at the statistical significance level α = 0.05.
Concerning the experimental part of presented study, two parallel analyses and measurements were performed in four repetitions. There were 6 major flavonolignans (silychristin, silydianin, silybin A, silybin B, isosilybin A and isosilybin B) in the silymarin complex of the analysed samples. Individual components of the silymarin complex were identified by the size of the peaks detected by HPLC analysis (Fig. 2) and converted to g.kg−1. The results are reported as mean ± standard deviation (SD) and median. In each observed year, the differences between the representations of individual components of the silymarin complex were statistically significant (
Fractions of silymarin complex (g.kg−1) in Silybi mariani fructus, 2015 (n = 4).
Silychristin | 3.18 ± 0.06a | 3.18 |
Silydianin | 0.91 ± 0.01b | 0.90 |
Silybin A | 3.06 ± 0.06c | 3.07 |
Silybin B | 7.04 ± 0.07d | 7.07 |
Isosilybin A + B | 1.09 ± 0.05e | 1.10 |
Summary | 15.28 ± 0.06A | 15.27 |
Fractions of silymarin complex (g.kg−1) in Silybi mariani fructus, 2016 (n = 4).
Silychristin | 4.03 ± 0.24a | 3.97 |
Silydianin | 1.76 ± 0.14b | 1.78 |
Silybin A | 2.96 ± 0.15a | 2.99 |
Silybin B | 5.92 ± 0.08c | 5.92 |
Isosilybin A + B | 1.98 ± 0.05d | 2.00 |
Summary | 16.65 ± 0.09B | 16.66 |
In a scientific study carried out by Wianovska and Wiśniewski (2015), a pressurized liquid extraction to prepare samples was used. This isolated the silymarin mixture in a one-step extraction process and, therefore, reduced the extraction time and volumes of solvents used. Using this method, the total content of the silymarin complex was 22.7 g.kg−1 (2.27%). Compared to values in presented study, there was a difference of 0.74% concerning fruit collected in 2015 and the difference of 0.6% regarding the fruit collected in 2016. However, the whole process of analysis is time-consuming. Nevertheless, calibration models are currently being developed for highly efficient and fast determination methods to substitute the HPLC method (Ashie et al., 2021). A promising alternative is a near-infrared (NIR) spectroscopy, a fast and non-destructive method for the analysis of samples without the need of sample pretreatment (Vagnerova et al., 2016). This NIR technique gives information about structural and physical qualities of materials based on the radiation transmittance or reflectance at wavelengths in various ranges (Rodriguez-Saona et al., 2000). The proposed method offers a promising approach to the determination of the quality and quantity of active ingredients for its rapid and non-polluting properties and low costs (Ashie et al., 2021). Using NIR technique, Vagnerova et al. (2016) found out that the varieties of milk thistle (Silyb and Mirel) have different ratio of the main silymarin complex components. They also proved that the calibration model of studied varieties can be used to identify an unknown sample. This model is able to classify these varieties. Drouet et al. (2019) used ultrasound extraction to determine the content of the silymarin complex, which was at least 1.80 g.kg−1, and a liquid extraction to quantify the flavonolignans. They observed that silybin B presented the highest representation (7.52–1.29 g.kg−1), followed by silydianin (4.21–0.40 g.kg−1), isosilybin A (2.49–0.45 g.kg−1), silychristin (1.52–0.01 g.kg−1) and silybin A (1.09–0.01 g.kg−1). In another study, Ghafor et al. (2014) analysed the amount of flavonolignans and determined that the highest content within the silymarin complex was reached by silybin A. In all our measurements, we recorded the predominance of silybin B. Wallace et al. (2005) compared the yield of silymarin in the extraction with ethanol boiling at 78.3 °C and ethanol heated to 60 °C. They found out that the average value of silymarin in the fruit in the extraction with boiling ethanol was 5.0 mg.g−1 = 5.0 g.kg−1, while a 60 °C ethanol caused an increase in yield by 1.7 times. From this study, we can conclude that the preparation of mash or decoction of Silybi mariani fructus can degrade the whole silymarin complex and, at the same time, reduce the effects of the ingredients. The most studied substance from silymarin complex is silybin, which exists in the form of two stereoisomeric compounds: silybin A and silybin B. Poppe and Petersen (2016) presented in their study that the content of these components represents 10%–20% of the silymarin content, while isosilybin B represents only about 5% of its content. According to another study, using the same assay procedure as we used in our study, the silybin A content ranged from 0.44 g.kg−1 to 11.77 g.kg−1, while the silychristin content varied from 2.05 g.kg−1 to 15.11 g.kg−1. The average content of isosilybin A was 4.70 g.kg−1 and 3.67 g.kg−1 (Arampatzis et al., 2018). Khan et al. (2015) reported that the concentrations of various silymarin components strongly depend on growth conditions, and stress conditions have a strong impact on its biosynthesis. According to the European Pharmacopoeia, which sets the minimum content of silymarin in the dried drug to 1.5%, we can state that both samples meet the requirements of Pharmacopoeia (1.5% in 2015 and 1.7% in 2016).