Simpliﬁed Procedure of Silymarin Extraction from Silybum marianum L. Gaertner
Dorota Wianowska* and Mariusz Wis
Faculty of Chemistry, Department of Chromatographic Methods, Maria Curie-Sklodowska University, Pl. Maria Curie-Sklodowska 3,
Lublin 20-031, Poland
*Author to whom correspondence should be addressed. Email: firstname.lastname@example.org
Received 19 September 2013; revised 16 April 2014
Silymarin, a mixture of ﬂavonolignans exhibiting many pharmacolog-
ical 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; sec-
ond, silymarin is extracted with methanol for 5 more hours. The pre-
sented data show that this extremely long traditional Soxhlet
extraction process can be shortened to a few minutes using pressur-
ized liquid extraction (PLE). PLE also allows to eliminate the defatting
stage required in the traditional procedure, thus simplifying the sily-
marin 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/gof
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 12588888C.
Silybum marianum L. Gaertner, commonly called as milk this-
tle, 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
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 exception-
ally 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
The therapeutic effects of milk thistle are closely connected
with the presence of the ﬂavonoid complex called silymarin.
The mixture consists of silybin A and B, isosilybin A and B, sily-
christin and silydianin. The highest amount of the complex is
present in the fruits of the plant (9–11). The medicinal
properties of milk thistle explain why the importance of the ﬂa-
vonolignans analysis has been recognized by researchers, who, so
far, have most frequently used high-performance liquid chroma-
tography (HPLC) for this purpose.
The separation of compounds to be analyzed from the plant
matrix is the ﬁrst step in any analysis of medicinal plant constit-
uents. Due to the high contents of lipids in the thistle fruits
(25%), the silymarin extraction procedure from the matrix in-
volves a two-step process (12). First, the fruits are defatted for
6 h, using n-hexane; second, silymarin is extracted with metha-
nol for 5 more hours. However, the application of the mentioned
procedure as sample preparation prior to the chromatographic
analysis of silymarin would hardly be economical. Not only
does it last too long but also uses large amounts of toxic solvents
and generates too much waste. Researchers, therefore, have been
focused on alternative methods of plant sample preparation that
allow for elimination of the drawbacks of the traditional ap-
proach. The pressurized liquid extraction (PLE) is one of such
emerging methods applied in an increasing number of newer an-
alytical studies (13–17), as it presents important advantages over
traditional extraction techniques.
PLE allows us to use extractants at elevated pressure and,
hence, at temperatures above their boiling point. High temper-
ature increases the rate of analyte diffusion through a cell wall,
its solubility into extractant, and decreases the solvent’s viscos-
ity and surface tension. These factors improve the contact of the
analytes with the solvent and enhance extraction efﬁciency
(13). The possibility of PLE application for silymarin isolation
from milk thistle was mentioned by Benthin et al. (16).
However, there are no literature reports on the inﬂuence of
PLE extraction conditions on the extraction effectiveness of
these compounds. (The effect of hot water extraction condi-
tions on the silymarin extraction effectiveness is known from
the literature (18).) As PLE is recognized as one of the most ef-
fective extraction techniques used for the isolation of biologi-
cally active compounds from plants, the question appears
whether its application allows for full isolation of silymarin
from the thistle fruits in one-step extraction process (without
a defatting step).
The present paper discusses the effectiveness of the PLE pro-
cess applied for silymarin extraction from the defatted and non-
defatted fruits of S. marianum L. Gaertner. The effects of solvent
type, temperature of the process, duration of static extraction
and the number of extraction cycles on the yield of silymarin
from the fruits are examined. The temperature and the time ef-
fect of defatting by PLE using n-hexane on the change of sily-
marin yield are also discussed. The PLE results are compared
with the data obtained using Soxhlet extraction.
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Journal of Chromatographic Science 2014;1– 7
Journal of Chromatographic Science Advance Access published June 3, 2014
Materials and Methods
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
Materials and reagents
The standardized dry extract of S. marianum L. Gaertner and
silybin B (with a purity of 98%), applied as standards, was pur-
chased from Sigma-Aldrich, Poland. Acetone, ethyl acetate, phos-
phoric 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 ap-
plied 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 ex-
traction 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 vol-
ume of the collected extract was between 25 and 31 mL, de-
pending 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, ac-
etone and ethyl acetate), temperature (50, 75, 100, 125 and
1508C), time (5, 10, 15 and 20 min) and the number of extraction
cycles (1–5). For the PLE defatting process, n-hexane was ap-
plied as solvent, and parameters under study were temperature
(50 and 1008C) 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 chromato-
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 per-
formed in the two-step process (n¼3). 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,
ﬂask, which was subsequently ﬁlled up to its volume with meth-
anol. 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- mL sample loop, a gradient pump (GP50),
an absorbance detector (AD25) and a photodiode array detector
(PDA100). The whole chromatographic system was under the con-
trol of the PeakNet6 data acquisition system. Chromatographic
separations were carried out at 408C using a Prodigy ODS-2 col-
umn (5 mm, 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 mix-
ture of methanol with the aqueous phosphoric acid solution con-
taining 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 per-
formed 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 wave-
length for detecting ﬂavonolignans was set at 288 nm, and the
UV-Vis spectra from 210 to 500 nm were also recorded for peak
The qualitative analysis of the extracts was carried out by com-
paring the retention times of the peaks and their UV– Vis spectra
in the extracts with respect to those of the standardized dry sily-
marin sample. To prepare the standardized dry silymarin solu-
tion, a 0.02-g portion of the sample containing 5.0 mg of silybin
AþB was dissolved in 50 mL of methanol. The peaks for sily-
christin, 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, re-
spectively. 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 con-
centration range of 0.1 –1.0 mg/mL. Three measurements of
peak area for each concentration of standard solution were per-
formed. The characteristic parameters of the obtained calibra-
tion curve were as follows: slope, 0.516 and intercept, 0.003.
The calibration curve was found to be linear in the tested con-
centration range. The correlation coefﬁcient was found to be
.0.995. Because of the difﬁculty of purchasing silychristin, sily-
dianin, silybin A, isosilybin A and B standards, the amounts of
these compounds were calculated by relating their chromato-
graphic responses to the calibration curve for silybin B.
All data are expressed as mea n +standard deviation (SD). The
analysis of variance (ANOVA) and F-test were used to assess the
2Wianowska and Wis
inﬂuence of PLE conditions on silymarin yield. The mean values
were considered signiﬁcantly different when result of compared
parameters differed at P¼0.05 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 solution prepared
dissolving the standardized dry extract of S. marianum
L. Gaertner in methanol (1 mg/mL). The analysis of chromato-
grams of PLE or Soxhlet extracts with that for standardized solu-
tion (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 num-
bered 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.
The results in Table Ipresent the effect of solvent type (meth-
anol, acetone and ethyl acetate in the case of non-defatted fruits
and methanol and acetone in the case of defatted ones) on the
cumulative yield of silymarin. The effect of solvent type on the
individual silymarin detection is presented in Figure 2. The ex-
periments were performed under a set of preliminary conditions
(temperature, 1008C; pressure, 60 bar; static extraction time,
10 min; ﬂush volume, 60%, purge time 60 s and one extraction
cycle). Ten minutes PLE at 508Cwithn-hexane was applied to
remove lipids from the fruits. Moreover, Table Ipresents the sily-
marin yields obtained with methanol, which is the solvent rec-
ommended by the European Pharmacopoeia.
The effect of temperature increases on individual components
of the silymarin complex extracted from defatted and non-
defatted fruits is presented in Table II. The last row of the table
contains the total silymarin amount obtained at a given extrac-
tion temperature using acetone. The results in Table III present
the effect of extraction time on silymarin yields obtained from
defatted and non-defatted fruits, using PLE with acetone at
1258C. In this series of experiments, the fruits were defatted
by 10 min preliminary PLE at 508C using n-hexane. The impor-
tance of the experimental factors determined according to the
F-value is listed in Table IV.
Figure 3presents the inﬂuence of various conditions of defat-
ting process on the silymarin yield. To estimate the inﬂuence, dif-
ferent extraction temperatures (50 and 1008C) and times (5 and
10 min) of lipids removal, using n-hexane as solvent, were tested.
To isolate the ﬂavonolignans (the second step of the procedure),
the same PLE conditions were applied—10 min extraction at
1258C using acetone as solvent. For a better comparison of the
impact of defatting process onthe silymarin yield, the results pre-
sented in Figure 3are compared with the data obtained, under
the same PLE conditions, for the sample not subjected to the pro-
cess of defatting.
The recovery of silymarin from defatted and non-defatted
fruits of S. marianum L. Gaertner was determined by consecu-
tive extractions of the same sample under the same PLE condi-
tions (at 1258C for 10 min using acetone, defatting at 508Cfor
Effect of Solvent Type on the Silymarin Yield from the Non-Defatted and Defatted Milk Thistle Fruits Obtained by PLE and the Recommended Soxhlet Procedure
Amount of silymarin (in mg/g) estimated in milk thistle fruits by
Methanol Acetone Ethyl acetone Methanol after n-hexane Acetone after n-hexane Methanol after n-hexane
16.01 +1.14 19.10+2.08 10.82 +1.12 17.04+0.98 19.65 +1.75 16.40 +1.35
Data expressed as mean values +SD (n¼3).
PLE conditions: 1008C, 60 bar, 10 min, defatting at 508C for 10 min.
Soxhlet conditions: the boiling point temperature for 5 h, defatting for 6 h.
Figure 1. Exemplary chromatograms of methanolic extracts from the fruits of S.
marianum L. Gaertner: (a) typical chromatogram of PLE extract; (b) the chromatogram
of silymarin solution prepared dissolving the standardized dry extract of the fruits in
methanol. Peaks: (1) silychristin; (2) silydianin; (3) silybin A; (4) silybin B; (5) isosilybin
A and (6) isosilybin B (chromatographic conditions—see experimental part).
Silymarin Extraction from Silybum marianum L. Gaertner 3
Figure 2. Inﬂuence of extracting solvent type on the PLE yield of individual ﬂavonolignans from milk thistle fruits.
Effect of Extraction Temperature on the Silymarin Yield from the Defatted and Non-Defatted Milk Thistle Fruits Obtained by PLE
Silymarin constituent Amount of silymarin constituents (in mg/g) obtained at a given temperature from
Defatted material Non-defatted material
508C758C 1008C 1258C 1508C508C758C 1008C 1258C 1508C
Silychristin 1.55 +0.12 2.46 +0.18 2.98 +0.23 3.18 +0.26 2.50 +0.35 1.57 +0.07 2.44 +0.17 3.14 +0.09 3.59 +0.05 2.70 +0.28
Silydianin 3.36 +0.29 5.66 +0.34 7.03 +0.31 6.60 +0.35 2.81 +0.18 3.40 +0.23 5.65 +0.25 7.28 +0.19 6.98 +0.20 3.12 +0.27
Silybin A 1.41 +0.12 2.17 +0.11 2.54 +0.08 2.96 +0.19 1.84 +0.21 1.41 +0.05 2.18 +0.06 2.69 +0.07 3.11 +0.12 2.01 +0.21
Silybin B 2.32 +0.21 3.61 +0.21 4.23 +0.14 4.61 +0.30 3.30 +0.38 2.33 +0.18 3.62 +0.14 4.42 +0.16 5.10 +0.25 3.58 +0.35
Isosilybin A 1.01 +0.09 1.72 +0.13 2.10 +0.08 2.42 +0.13 1.59 +0.20 1.07 +0.05 1.74 +0.08 2.26 +0.09 2.51 +0.08 1.76 +0.17
Isosilybin B 0.53 +0.03 0.95 +0.09 1.20 +0.05 1.12 +0.11 1.11 +0.16 0.56 +0.02 1.01 +0.06 1.22 +0.10 1.42 +0.18 1.24 +0.17
Total amount 10.18 +0.84 16.57 +1.07 20.08 +0.35 21.38 +0.85 13.14 +1.47 10.34 +0.53 16.63 +0.68 20.99 +0.58 22.70 +0.87 14.41 +1.41
Data expressed as mean values +SD (n¼3).
PLE for 10 min with acetone, defatting at 508C for 10 min.
4Wianowska and Wis
5 min) until no ﬂavonolignans were detected by HPLC. Five inde-
pendent series of multiple PLE of silymarin were performed. The
results are collected in Table V. Moreover, Table Vpresents the
silymarin yield obtained during the recommended extraction
procedure in the Soxhlet apparatus. As shown in the table, the
extraction in the Soxhlet apparatus gives only 67% yield of
that obtained during multiple PLE and only 72% of the amount
obtained in one-cycle PLE.
Effect of extraction solvent type
The selection of the proper solvent is known to be a prerequisite
to obtaining high yields of analytes from plant material. Although
in the last decade the application of subcritical water extraction
has been reported for the isolation of biologically active com-
pounds from the milk thistle fruits (18–20), it is organic solvents
that are most often applied for silymarin extraction.
As results from Table I, the extraction efﬁciency of acetone is
the highest. Methanol extracts a slightly lower amount of the sily-
marin mixture than acetone, and ethyl acetate isolates the small-
est amount of silymarin. The effect of extraction solvent type on
the silymarin yield obtained from non-defatted fruits is con-
ﬁrmed by the F-value presented in the last row of Table IV
). It is evident from the results in Figure 2that
the inﬂuence of the solvent type on the yield of individual sily-
marin components is more complex. Acetone and methanol ex-
tract comparable amounts of the majority of the investigated
ﬂavonolignans. Their amounts are higher than those obtained
by means of ethyl acetate. However, in the case of silydianin,
Effect of Extraction Time on the Silymarin Yield from the Non-Defatted and Defatted Milk Thistle Fruits Obtained by PLE
Silymarin constituent Amount of silymarin constituents (in mg/g) obtained after a given extraction time (in min) from
Defatted material Non-defatted material
5 10 15 20 5 10 15 20
Silychristin 3.68 +0.12 3.26 +0.07 2.65 +0.12 2.39 +0.15 3.05 +0.09 3.32 +0.18 2.96 +0.06 2.66 +0.16
Silydianin 7.43+0.21 6.59 +0.19 4.91 +0.26 4.66 +0.38 6.61 +0.12 6.89 +0.25 5.84 +0.10 4.91 +0.43
Silybin A 3.11 +0.05 3.03 +0.17 2.23 +0.05 2.25 +0.15 2.39 +0.05 3.34 +0.17 2.76 +0.08 2.28 +0.18
Silybin B 5.19 +0.16 4.72 +0.23 3.72 +0.09 3.38 +0.21 4.51 +0.13 5.14 +0.19 4.27 +0.21 3.63 +0.28
Isosilybin A 2.49 +0.02 2.36 +0.10 1.83 +0.04 1.57 +0.03 2.11 +0.04 2.58 +0.11 2.17 +0.09 1.94 +0.21
Isosilybin B 1.32 +0.03 1.31 +0.06 0.99 +0.02 0.81 +0.06 0.99 +0.06 1.50 +0.11 1.19 +0.06 1.04 +0.11
Total amount 23.22 +0.53 21.27 +0.61 16.33 +0.25 15.06 +0.68 19.66 +0.48 22.76 +0.96 19.19 +0.38 16.47 +0.87
Data are expressed as mean values +SD (n¼3).
PLE at 1258C with acetone, defatting at 508C for 10 min.
F- and P-Values Obtained During Variance Analysis for the Effects of PLE Conditions on the Silymarin Yield from the Non-Defatted and Defatted Milk Thistle Fruits
Silymarin constituent Effect of solvent type for Effect of temperature for Effect of time for
Defatted fruits extracted with Non-defatted fruits
Silychristin 3.32 0.10 0.15 0.70 72.37 6.3 10
20.56 8.2 10
73.39 2.3 10
72.20 3.9 10
12.59 2.1 10
Silydianin 5.12 0.10 0.52 0.50 73.36 6.1 10
121.63 2.0 10
211.64 1.3 10
73.99 3.6 10
34.28 6.5 10
Silybin A 3.44 0.10 0.04 0.90 104.10 2.2 10
46.97 1.9 10
96.62 6.0 10
49.63 1.6 10
39.21 3.9 10
Silybin B 0.75 0.40 0.02 0.90 394.26 2.3 10
33.93 8.6 10
61.70 5.2 10
66.07 5.5 10
26.59 1.6 10
Isosilybin A 0.01 0.90 0.02 0.90 89.63 3.4 10
48.20 1.7 10
89.78 8.6 10
169.24 1.4 10
13.16 1.8 10
Isosilybin B 0.02 0.90 0.01 0.90 141.79 8.9 10
21.63 6.6 10
20.01 6.1 10
82.13 2.4 10
20.41 4.2 10
Silymarin mixture 8.14 0.10 0.42 0.60 152.84 7.1 10
63.70 4.5 10
97.51 5.7 10
155.16 2.0 10
38.80 4.1 10
Figure 3. Effect of different defatting conditions on the silymarin yield from: (A) sample
not subjected to a prior defatting step, (B) sample defatted for 5 min at 508C, (C)
sample defatted for 10 min at 508C and (D) sample defatted for 10 min at 1008C.
Silymarin Extraction from Silybum marianum L. Gaertner 5
the main ﬂavonolignan in the silymarin mixture, the yields ob-
tained with acetone are appreciably greater than those obtained
with any other solvents. Besides, the yields of the compound ob-
tained with methanol and ethyl acetate are almost the same
(within the experimental error). The presented effect of extrac-
tion solvent type on the silydianin yield is consistent with the lit-
erature data (21). In the cited work, acetone gives the highest
silydianin yield for shorter extraction times.
The comparison of the silymarin yields obtained using methanol
and acetone, without and with preliminary defatting, supports the
conclusion that, in PLE, lipids removal has no essential effect on
the yield ofthe silymarin mixture. The obtained silymarin amounts
from fat-free fruits are only slightly higher in comparison with
those found for non-defatted material. Yet, the F-value shows
that the differences between the yields are statistically insigniﬁ-
, see Table IV). The only advantage of defatting
prior to the silymarin extraction by more polar solvents is a slightly
greater precision of the analytical method. The extracts obtained
from fat-free fruits are more transparent, making the chromato-
graphic analysis easier. Lipids elimination from the sample subject-
ed to the HPLC analysis in the reversed-phase mode undoubtedly
prolongs the analytical column lifetime.
In the light of the above, the most powerful extraction solvent
for silymarin isolation from the milk thistle fruits is acetone.
There is no essential difference in the silymarin amount estimat-
ed in non-defatted and defatted fruits. The silymarin yield esti-
mated by long-lasting Soxhlet extraction conﬁrms that defatting
process has no signiﬁcant effect on the yield of these compounds
in PLE conditions.
Effect of extraction temperature
To estimate the optimum temperature for the extraction of ﬂavo-
nolignans from the defatted and non-defatted milk thistle fruits by
PLE, the extraction efﬁciency in the temperature range from 50 to
1508C, using acetone (extraction time, 10 min), was examined.
Limiting of the extraction temperature range up to 1508Cwas
due to the fact that at higher temperatures cloudy extracts were
obtained in PLE. The presence of sediment may cause undesirable
effects, e.g., loss of analytes as a result of adsorption, etc.
As results from Table II, a signiﬁcant increase of silymarin
amount is observed when extraction temperature is increased
from 508Cupto100 – 1258C, regardless of whether the fruits
were defatted or not (see the last row of Table II). The observed
increase is connected with the improvement of PLE efﬁciency
through the increase of silymarin diffusion rate from the matrix
to the solvent and through the increase of silymarin solubility in
the solvent. Furthermore, temperature increase to 1508C dimin-
ishes the silymarin yield. The observed decrease probably results
from the thermal degradation of silymarin. It should be noted,
however, that the effect is smaller in the case of the non-defatted
fruits. The correctness of the hypothesis about the thermal deg-
radation of silymarin is supported by the literature data (19,22).
Although the thermal degradation of silymarin compounds in the
cited work was discovered in subcritical water extraction, the ef-
fect of high extraction temperature lowering the extraction efﬁ-
ciency of biologically active compounds from plants, using
organic solvents, is well known from other reports (17,23). A
smaller degradation of the silymarin compounds at high temper-
atures for the non-defatted fruits (see Table II) can be explained
by a protective effect of lipids.
Taking the presented data into account, 1258C was selected as
optimal temperature for PLE of silymarin from the defatted and
non-defatted milk thistle fruits.
Effect of static extraction time
The efﬁciency of the PLE process depends on the sample extrac-
tion time. For the defatted fruits (see Table III), it is observed that
the yield of silymarin is diminished when the extraction time in-
creases from 5 to 20 min. When the extraction was performed
for only 5 min, the yields of the silymarin compounds were great-
er than those obtained in the course of 10 min extraction. In the
case of the non-defatted fruits, the increase of extraction time re-
sults in a small increase and then gradual decrease of the sily-
marin yield. The smallest amounts of ﬂavonolignans were
obtained during the longest static extraction time of 20 min.
This ﬁnding supports correctness of the conclusion about the
thermal degradation of silymarin and suggests that thermal deg-
radation of ﬂavonolignans occurs even at 1258C; however, it be-
comes visible for longer extraction times. It cannot be, therefore,
excluded that a higher optimum temperature will occur at a
shorter extraction times, and a lower optimum temperature
may occur with longer extraction times.
The observed differences in the extraction behavior of sily-
marin from the defatted and non-defatted fruits (see Table III)
can be explained by the presence of lipids protecting silymarin
from degradation and hindering silymarin diffusion into the
extractant. The results presented in ref. (21) conﬁrm that lipids
removal helps to release silymarin from the fruits without affect-
ing the release of the individual silymarin constituents. Clearly,
smaller the F-values obtained for the non-defatted fruits (see
Table IV) also conﬁrm the correctness of the conclusion. The
high value of Fobtained for the defatted fruits, however, shows
that an eventual silymarin loss during the fruit defatting process
also cannot be excluded.
Effect of defatting process
The defatting process of the fruits decreases the silymarin con-
centration in the plant material (Figure 3). The longer extraction
time and the higher lipids extraction temperature the greater
silymarin loss (F
¼4.1). The observed loss of
the silymarin yield after the lipids removal is consistent with
the research reported previously, in which the loss of other
Silymarin Amount Estimated in Milk Thistle Fruits Using Different Sample Preparation Methods
Sample preparation method Silymarin amount (mg/g) in milk thistle fruits
First cycle 22.58 +0.67 (93.24%)
21.77 +0.50 (92.67)
Second cycle 0.88 +0.08 (3.65%)
1.22 +0.03 (5.20)
Third cycle 0.47 +0.09 (1.93%)
0.42 +0.02 (1.81)
Fourth cycle 0.20 +0.06 (0.84%)
0.08 +0.02 (0.32)
Fifth cycle 0.08 +0.03 (0.34%)
S24.21 +0.71 23.49 +0.51
Recommended Soxhlet procedure
Data expressed as mean value +SD (n¼5).
Recovery in %.
PLE with acetone, each extraction cycle lasted 10 min at 1258C, defatting at 508C for 5 min.
5-h extraction with methanol on defatted fruits, defatting for 6 h.
6Wianowska and Wis
polar compounds (toxoids) after preliminary PLE of non-polar
ballast substances from yew twigs using n-hexane was found
(24). In PLE, the preliminary extraction of ballast substances
from plant samples apparently leads to the loss of analytes.
In the light of the obtained results, the optimal PLE conditions
for analysis of silymarin in milk thistle fruits are as follows: extrac-
tion solvent—acetone; temperature—1258Candtime—10min
without the preliminary defatting process.
Recovery of silymarin during consecutive PLE
Quantitative isolation of silymarin mixture from the non-defatted
milk thistle fruits requires ﬁve successive extraction cycles on
the same sample, whereas four cycles are required for the defat-
ted fruits. The greater number of cycles for the quantitative ex-
traction of silymarin from the non-defatted material results from
the presence of lipids hindering silymarin diffusion.
As shown in Table V, the extraction efﬁciency of PLE is much
higher than that of Soxhlet extraction recommended for sily-
marin isolation from milk thistle fruits.
Due to the high content of lipids in the thistle fruits, European
Pharmacopoeia recommends a two-step process of silymarin ex-
traction from the matrix: ﬁrst, fruits defatting for 6 h, using
n-hexane; second, silymarin extraction with methanol for 5
more hours. The obtained results show that PLE is a very effective
sample preparation method for silymarin extraction from milk
thistle fruits. The PLE yields of silychristin, silydianin, silybin A, sily-
bin 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 PLE
silymarin yield is higher than that obtained using the Soxhlet ap-
paratus. Moreover, PLE application for silymarin extraction signiﬁ-
cantly reduces the extraction time and volumes of solvents used.
The PLE technique allows for the effective isolation of silymarin
mixture in a one-step extraction process (without defatting).
The presented data also demonstrate that the elimination of defat-
ting from the PLE extraction of silymarin prevents its loss.
The research carried out in the framework of own research of
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Silymarin Extraction from Silybum marianum L. Gaertner 7