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Simplified Procedure of Silymarin Extraction from Silybum marianum L. Gaertner


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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.
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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:
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 (25). 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 (911). 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 (1317), 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.
#The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email:
Journal of Chromatographic Science 2014;1– 7
doi:10.1093/chromsci/bmu049 Article
Journal of Chromatographic Science Advance Access published June 3, 2014
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
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 (15). 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-
graphic analysis.
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 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.
Statistical analysis
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, UVVis 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
Table I
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
using Soxhlet
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.
Table II
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 (1820), 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
.. F
). 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,
Table III
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.
Table IV
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
Defatted fruits
Non-defatted fruits
Defatted fruits
Non-defatted fruits
P-value F
P-value F
P-value F
P-value F
P-value F
P-value F
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-
cant (F
, 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
¼26.54, 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
Table V
Silymarin Amount Estimated in Milk Thistle Fruits Using Different Sample Preparation Methods
Sample preparation method Silymarin amount (mg/g) in milk thistle fruits
Non-defatted Defatted
Multiple PLE
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
16.40 +0.70
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—1258Candtime10min
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
the Department.
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Silymarin Extraction from Silybum marianum L. Gaertner 7
... Several extraction techniques have been applied to extract SLY from Silybum marianum seeds such as pressurized liquid extraction [18] , subcritical water extraction [ 19 , 20 ], supercritical fluid extraction [21][22][23] , ultrasound assisted extraction [ 24 , 7 ] and microwave assisted extraction [ 25 , 26 ]. Usually, the SLY extraction process from milk thistle seeds requires a first step defatting pretreatment using hazardous and contaminant solvent such as hexane or petroleum ether [27][28][29] . ...
... In this sense, it is clear that none of them can obtain the same levels of flavonolignans of the silymarin complex than those obtained using GXL. For example, Wianowska et al. [18] developed a single step approach but based on PLE with organic solvent whose best extraction rate was 24.21 mg Silymarin per g of extract. Or Andrzejewska et al. [56] who developed a single step method based on ultrasounds and obtained up to 43.6 mg Silymarin per g dry weight of extract in their best conditions. ...
... As can be seen in Figure S1 A, using GXL in the present study led to 545.73 (mg/g of extract) of SB a + b , more than 63 fold the recovery of UAE reported in literature. Further, according to the reported values for the content of SC in S. marianum seeds (3 -6.2 mg/g of DW) [18,26,57] it seems that in the present work, the GXL-Extraction using ternary mixture of CO 2 :EtOH:H 2 O provided a higher extraction rate. It is worth mentioning that the content of SD in any of the GXL extracts was higher than that pre-viously reported by Wianowska et al. using acetone for 10 min at 125 °C [18] . ...
This work reports the application of Gas Expanded Liquid (GXL) extraction to concentrate the flavonolignan fraction (silymarin) and taxifolin from Silybum marianum seeds, which have proven to be highly valuable health-promoting compounds. GXL using green solvents was used to isolate silymarin with the objective of replacing conventional methods. In one hand, the effect of different compositions of solvents, aqueous ethanol (20 %, 50 % or 80 % (v/v)) at different CO2/liquid (25, 50 and 75 %) ratios, on the GXL extraction was investigated. The obtained extracts have been chemically and functionally characterized by means of UHPLC-ESI-MS/MS (triple quadrupole) and in-vitro assays such as anti-inflammatory, anti-cholinergic and antioxidant. Results revealed that the operating conditions influenced the extraction yield, the total phenolic content and the presence of the target compounds. The best obtained yield was 55.97 % using a ternary mixture of solvents composed of CO2:EtOH:H2O (25:60:15) at 40°C and 9 MPa in 160 min. Furthermore, the results showed that obtained extracts had significant antioxidant and anti-inflammatory activities (with best IC50 value of 8.80 μg/mL and 28.52 μg/mL, respectively) but a moderate anti-cholinesterase activity (with best IC50 value of 125.09 μg/mL). Otherwise, the concentration of silymarin compounds in extract can go up to 59.6 % using the present one-step extraction method without further purification, being silybinA+B the predominant identified compound, achieving value of 545.73 (mg silymarin/g of extract). The obtained results demonstrate the exceptional potential of GXL to extract high-added values molecules under sustainable conditions from different matrices.
... Due to the high lipid content (20-30%) in milk thistle seeds, extracting silymarin using a single-step procedure is quite a difficult task. Hence, the European Pharmacopoeia suggests a two-step solvent extraction (SE): first, the defatting of the seeds for 6 h in n-hexane, followed by silymarin extraction using methanol for five additional hours [117]. ...
... Therefore, alternative methods for silymarin extraction have been explored. A significant shortening of the process can be achieved by using the technique of pressurized liquid extraction (PLE) [117], which permits the avoidance of the preliminary defatting, otherwise required by the traditional method. Additionally, new technologies such as microwave-assisted, ultrasound-assisted, and enzyme-assisted extraction have been studied to increase the extraction yield of silymarin [118]. ...
Full-text available
Milk thistle (Silybum marianum (L.) Gaertn.) is a versatile crop that has adapted to the broadly different soil and environmental conditions throughout all continents. To date, the fruits (“seeds”) of the plant are the only reliable source of silymarin, which, given its recognized therapeutic effects and its many present and potential uses, has led to a significant re-discovery and enhancement of the crop in recent years. Overall, although many studies have been carried out globally on the bioactivity, phytochemistry, and genetics of milk thistle, few and discontinuous research activity has been conducted on its basic agronomy as well as on the farm opportunities offered by the cultivation of this species. However, the multiple potential uses of the plant and its reduced need for external inputs suggest that milk thistle can perfectly fit among the most interesting alternative crops, even for marginal environments. The growing interest in natural medicine, the increasing popularity of herbal dietary supplements, and the multiple possibilities for livestock feeding are all arguments supporting the idea that in many rural areas, this crop could represent a significant tool for enhancing and stabilizing farm income. However, several issues still have to be addressed. The species retains some morphological and physiological traits belonging to non-domesticated plants, which make the application of some common agronomic practices challenging. Furthermore, the lack of reliable field data devoted to the definition of suitable cropping protocols represents a major constraint on the spread of this crop among farmers. This review has therefore focused on updating information on the main morphological and phytochemical traits of the crop and its agronomic characteristics and novel uses. Several gaps in technical knowledge have been addressed, and further goals for experimental activity have been outlined in order to guide farmers eager to cope with the cultivation of such a challenging and resource-rich crop.
... It is also relatively easy to automate, without requiring a lot of supervision (Raynie 2019). However, exceptionally long extraction times (12 to 24 h), high energy consumption, and problems in selectivity and efficiency limit the scope of this technique (Weggler et al. 2020;Mussatto 2015;Wianowska and Wiśniewski 2015). ...
... Baicalein, a flavone obtained from the dried roots of S. baicalensis Georgi, is known for its anti-cancer and antiinflammatory activities and has been used to treat several gastrointestinal ailments such as gastric ulceration, liver fibrosis, and so on(Xie et al. 2019). Comparable properties have been observed in silymarin (treatment of liver disorders as well as antitumor activity), extracted from Silybum marianum L. Gaertner(Wianowska and Wiśniewski 2015). The extracts of Anthemis cotula L. (stinking chamomile) were found to have potential in the treatment of Alzheimer's disease and skin hyperpigmentation conditions(Sut et al. 2019). ...
Full-text available
Bioactive compounds refer to secondary metabolites extracted from plants, fungi, microbes, or animals. Besides having pharmacological or toxicological effects on organisms leading to utilization in food and pharmaceutical industries, the discovery of novel properties of such compounds has led to the diversification of their applications, ranging from cosmetics and functionalized biomaterials to bioremediation and alternate fuels. Conventional time-consuming and solvent-intensive methods of extraction are increasingly being replaced by green solvents such as ionic liquids, supercritical fluids, and deep eutectic solvents, as well as non-conventional methods of extraction assisted by microwaves, pulse electric fields, enzymes, ultrasound, or pressure. These methods, along with advances in characterization and optimization strategies, have boosted the commercial viability of extraction especially from agrowastes and organic residues, promoting a sustainable circular economy. Further development of microfluidics, optimization models, nanoencapsulation, and metabolic engineering are expected to overcome certain limitations that restrict the growth of this field, in the context of improving screening, extraction, and economy of processes, as well as retaining biodiversity and enhancing the stability and functionality of such compounds. This review is a compilation of the various extraction and characterization methods employed for bioactive compounds and covers major applications in food, pharmacy, chemicals, energy, and bioremediation. Major limitations and scope of improvement are also discussed. Graphical abstract
... The pet ether extract displayed the highest hepatoprotective activity at concentrations of 5 and 10 mg/ml, respectively (Table 3), followed by CH2Cl2 & then MeOH extracts. The hepatoprotective effect of pet ether extract was not significantly dissimilar from that of silymarin generally used as hepatoprotective natural drug from milk thistle fruits of Silybum marianum (Wianowsk and Wisniewski, 2014). The results of this assay will lead to consideration of the different extracts from the stems of Euphorbia tithymaliodes as a source for hepatoprotective compounds. ...
... S. marianum is from the Mediterranean Mountains, Asia, and North Africa, but today grows in several parts of the world [1]. ...
... Newly hatched Seeds of S. marianum L. were obtained from the Genetics and Genetic Engineering Department, Faculty of Agriculture, Benha University. Extraction and determination of Flavonolignans from S. marianum L. dry seeds Flavonolignans (silychristin, silydianin, silybin A and B, iso silybin A and B, and taxifolin) was performed following the methodology described by [16]. All the standards of flavonolignans were purchased from Sigma-Aldrich, St. Louis, MO, USA. ...
Full-text available
The effect of feeding on diets supplemented with Silybum marianum L. dry seeds (SMS) on growth performance, mortality percentage, biochemical parameters, the expression profile of related genes, and genotoxic effect in Muscovy ducklings was evaluated during a brooding period of 4 weeks. Two hundred and forty one-day-old Muscovy ducks were randomly assigned to four treatment groups (60 ducklings/group), the first group fed on basal diet with no additives (control), and the second (4 g kg−1), third (8 g kg−1), and fourth (12 g kg−1) groups fed the basal diet supplemented with 0, 4, 8, and 12 g kg−1 diet SMS, respectively. A substantial improvement in live body weight (LBW), body weight gain (BWG), and growth rate (GR), and a decrease in feed conversion ratios (FCR) and mortality rate were shown in ducks fed a diet supplemented with either 8 g kg−1 or 12 g kg−1 SMS compared to the other groups. Relevant improvements in liver function, oxidative stress markers, purinergic cell energy, and brain appetite were recorded on ducklings fed diets supplemented with SMS. Moreover, diets which included 8 or 12 g kg−1 SMS positively upregulated the expression of growth hormone gene (GH) and antioxidant genes (SOD1, SOD2, and CAT). These results are consistent with the increase in liver activity SOD and CAT enzymes, resulting in less DNA fragmentation. Consequently, all the aforementioned improvements in biochemical parameters and gene expression profiling may explain the superiority of the treated ducklings compared with the control group. Conclusively, the SMS could be used as a natural feed additive to promote health status and improve the growth performance of small grower ducks during the brooding period.
... This led to the isolation of some important early drugs i.e. digitoxin, quinine, pilocarpine, cocaine, codeine, etc. In the modern era, few other important natural compounds have been isolated from different plants such as Paclitaxel from Taxus brevifolia, Artemisinin from Artemisia annua (Su and Miller 2015) and Silymarin from Silybum marianum (Wianowska and Wiśniewski 2015). Some of the natural products of these plants are available in the local herbal medicine markets, such as Castor oil, an effective natural laxative derived from Ricinus communis, and Hab-e-Munir tablet, a natural product derived from Withania somnifera to treat arthritis, rheumatism and muscular pain. ...
Full-text available
Medicinal plants are the primary and effective source to cure various human ailments throughout the globe. The remote area of the Pashat valley at the Pakistan–Afghanistan border is still unexplored to document ethnomedicinal knowledge and practices. In the current study, the contemporary investigation recorded the native acquaintance of remedial plants. A total of 73 plant species belonging to 68 genera and 46 families were used by traditional communities for the treatment of 40 different kinds of human diseases including digestive, respiratory, urinary, skeletal, muscular systems, etc. The family Lamiaceae was found dominant with 9 species followed by Leguminosae and Solanaceae with 4 species each, Euphorbiaceae, Moraceae, Rhamnaceae and Rosaceae with three species each. The most frequently used parts of plants were leaves (35.7%) followed by fruits (22.4%), seeds (12.2%), roots (7.14%) and plants as a whole (6.12%). Whereas the remedy preparation approaches were in powder form (28.46%), decoction (26.15%), extract (20%) and a paste (11.54%). The quantitative ethnobotanical indices i.e. used value (UV), relative frequency citation (RFC), family importance value (FIV) and Jaccard index (JI) were calculated for all plant species. The used value (UV = 0.04) was found highest for Berberis lyceum, Myrtus communis, Punica granatum and Dodonea viscosa. Similarly, the maximum RFC value of 0.25 was investigated for Foeniculum vulgare and Olea ferruginea each. Besides, the highest FIV of 96.08% and 58.17% values were calculated for the family Lamiaceae and Solanaceae. This study shows that the corresponding area is rich in medicinal plants and could contribute to ethnomedicinal knowledge. The collected data will establish a baseline for the comprehensive investigation of active molecular constituents of the medicinal flora of the area. Soon, the exploration of these medicinal plants could discover new herbal drugs in the field of pharmacology.
... The SFE process was also studied for the separation of highlyvaluable compounds from defatted seeds (Csupor et al., 2016;Wianowska & Wiśniewski, 2014). For this purpose, a pretreatment step involving Soxhlet extraction with petroleum ether was performed separating 23.0 and 21.8 g/100 g of oil from 8-years-old and 1-year-old seeds, respectively. ...
Full-text available
Extracts from Silybum marianum seeds have high industrial potential due to their application as phytopharmaceuticals and food. In order to optimize its production, conventional extraction (in Soxhlet apparatus using solvents ethanol and n-hexane) and supercritical fluid extraction (using CO2 at the pressure of 30 MPa and temperature of 40 °C with and without co-solvent ethanol, from oil-rich and defatted seeds) were compared. In addition, the effect of plant cultivation (years 2011 and 2019) and the effect of seeds and extract storage on the yield and quality of extracts were assessed. It was shown that unsaturated fatty acids (linoleic and oleic acids being most dominant) constituted 64–87% of obtained extracts. Content of α-tocopherol varied from 0.01 to 13 g/100 g, while total phenolic content was in the range from 13.2 to 104.2 gGAE/100 gextract. In vitro cytotoxic activity analysis confirmed that extracts obtained from defatted seeds showed activity against cancer cells. This study pointed out the important role of the cultivation year, selection of extraction technique and solvent, as well as storage on the extraction yield, chemical profile, and cytotoxic activity of extracts.
Plant cell cultures of Silybum marianum L. are studied under different classes of elicitors including heavy metal ions (Ag+), polysaccharides (yeast extract and chitosan), and plant response signaling compounds (salicylic acid) in order to enhance silybin production. Remarkably, all elicitors enhanced the accumulation of silybin compared to the control experiment but the highest total silybin content was achieved in cell suspensions elicited with yeast extract at 0.5 µg.mL‐1 producing a total silybin yield at 0.15 mg.g‐1 cells dry weight after two days of elicitation for a total cell dry weight of 1.49 g. The lowest silybin accumulation belongs to chitosan treatment producing maximum silybin content and dry weight of 0.038 mg.g‐1 DW and 1.19 g respectively. These results are promising to establish a proof of concept using yeast extract as a viable elicitor option to produce silybin in tissue culture.
Full-text available
Four types of solvent extraction methods (ultrasound and microwave assisted extraction, pressurized liquid extraction, and extraction in the Soxhlet apparatus) for paclitaxel, cephalomannine, and 10-deacetylbaccatin, taxoids recovered from common yew twigs, were compared. By use of pressurised liquid extraction (PLE), the most effective extractant of taxoids was determined. HPLC was used for the analysis of the extracts. Comparison of the obtained results revealed differences in the extraction power of the applied methods. The greatest yields were obtained by multiple PLE, which can be recommended as the best sample preparation method for taxoids analysis in yew twigs.
Full-text available
The accelerated solvent extraction (ASE) procedure has been examined as a sample preparation method for the HPLC analysis of rutin and isoquercitrin in Sambucus nigra L. The experimental extraction parameters (solvent composition, temperature, operation pressure, and static extraction time) have been optimized in order to receive the best recovery of both analytes from the flowers, leaves, and berries of black elder. The ASE recoveries obtained under the optimal extraction conditions were compared with analogous ones obtained by means of the maceration technique.
Full-text available
In an effort to increase revenues from a given feedstock, valuable co-products could be extracted prior to biochemical or thermochemical conversion with subcritical water. Although subcritical water shows significant promise in replacing organic solvents as an extraction solvent, compound degradation has been observed at elevated extraction temperatures. First order thermal degradation kinetics from a model system, silymarin extracted from Silybum marianum, in water at pH5.1 and 100, 120, 140, and 160°C were investigated. Water pressure was maintained slightly above its vapor pressure. Silymarin is a mixture of taxifolin, silichristin, silidianin, silibinin, and isosilibinin. The degradation rate constants ranged from 0.0104min−1 at 100°C for silichristin to a maximum of 0.0840min−1 at 160°C for silybin B. Half-lives, calculated from the rate constants, ranged from a low of 6.2min at 160°C to a high of 58.3min at 100°C, both for silichristin. The respective activation energies for the compounds ranged from 37.2kJ/gmole for silidianin to 45.2kJ/gmole for silichristin. In extracting the silymarin with pure ethanol at 140°C, no degradation was observed. However, when extracting with ethanol/water mixtures at and 140°C, degradation increased exponentially as the concentration of water increased.
Full-text available
A method of subcritical water extraction (SCWE) of biologically active substances from milk thistle seed (Silybum marianum L.) that makes it possible to obtain aqueous extracts without using organic solvents is suggested. The contents of hepatoprotective biologically active substances (taxifolin, silychristin, silydianin, and silybin) in milk thistle seed extracts obtained by SCWE and by standardized liquid extraction procedures are compared. Keywordsextraction–subcritical water extraction–biological active substances–milk thistle–taxifolin–silychristin–silydianin–silybin–flavolignans–high-performance liquid chromatography
Conference Paper
Seeds from milk thistle (Silybum marianum Gaert L.) contain flavanolignan and dihydroflavanol compounds that have interesting and important therapeutic activities. The recovery of these silymarin compounds generally involves a two-step defatting and extraction process using organic solvents. This study examined the batch, single-stage extraction of whole and defatted seeds using ethanol, methanol, acetonitrile, and acetone as the solvents. In extracting defatted milk thistle seeds with organic solvents, extraction with ethanol resulted in the highest silymarin yield, although some potential degradation was observed. The maximum yields of taxifolin, silychristin, silydianin, silybinin A, and silybinin B in ethanol were 0.6, 4.0, 0.4, 4.0, and 7.0 mg/g of defatted seed, respectively. However, if silybinin A were the diastereoisomer of choice, methanol would be the preferred extraction solvent because it yielded the highest silybinin A to silybinin B ratio. Interestingly, lipid removal is an important extraction step, because defatted material yields twice the silymarin concentration.
Objectives: During recent years, increasing side effects for syntactic drugs have been motivated by more researchers for finding new compounds of the plant with antifungal activity. Dried fruits extract of Silybum marianum contain flavonoid compounds and until now, no studies have been conducted on the antifungal activates of methanolic extract of this plant. In this study, inhibitory potential of S. marianum methanolic extract on dermatophytes and saprophytes fungi was investigated in vitro compare to clotrimazol. Method: Antifungal activities of S. marianum seeds extract were evaluated against pathogens (Trichophyton mentagrophytes, Epidermaphyton folocosom, Microsporum canis) and saprophytes (Aspergillus niger, Candida albicans) fungi with different methods such as, disc diffusion (60, 30, 15, 7.5, 3.2 and 1.6 mg extract per disc ), pour plate and broth (50, 25,12.5, 6.2, 3.1, 1.5 mg ml-1 extract in medium). Concentration of cloterimazol as a control was 10 μg ml-1. Results: Our results showed that, S. marianum seeds extract prevents the growth of dermatophytes more than saprophytes fungi. The best inhibitory effects of extract (6.2 mg ml-1) in Microsporum canis and Epidermaphyton folocosom cultures were achieved by porplate and broth methods that were similar to our results with 10 μg ml-1 cloterimazol. No inhibitory effects were observed in Aspergillus niger and Candida albicans cultures. Conclusion: With attention to our finding, components of the Silybum marianum extract have antifungal effects on the growth of dermatophytes.
The yields of oxygenated and non-oxygenated flavour and fragrance compounds from savory (Satureja hortensis) and peppermint (Mentha piperita) were compared using subcritical water extraction, supercritical carbon dioxide extraction (SFE) and hydrodistillation. Extraction rates with subcritical water increased with temperature (100-175°C), but some desired organics (linalool and γ-terpinene) showed substantial degradation at temperatures >150°C. However, subcritical water did not expose extracted compounds to atmospheric oxygen (as occurs in hydrodistillation) and thus may avoid the degradation of compounds like thymoquinone. Extraction of savory with subcritical water at 100°C for 40 min gave ca. 100% recoveries (compared to hydrodistillation) for thymol and carvacrol, and >150% recoveries of borneol and linalool. Recoveries with 60 min of SFE (pure CO2 at 400 bar and 50°C) were similar to hydrodistillation for borneol and linalool, but only ca. 50% for thymol and carvacrol. For peppermint, 30 min (at 150°C) or 12 min (at 175°C) of subcritical water extraction and 1 h of SFE gave good quantitative agreement with 4 h of hydrodistillation for carvone, pulegone, piperitone, eucalyptol, menthone, neomenthol and menthol, but the short subcritical water extractions only recovered ca. 40% of the less polar menthyl acetate. Subcritical water preferentially extracts more polar (oxygenated) flavour compounds, and ca. 80% extraction of oxygenated flavour compounds could be achieved under conditions which only extracted ca. 10-15% of the monoterpenes and <5% of the sesquiterpenes. In contrast, SFE had the lowest degree of selectivity and SFE extracts included plant alkane waxes as well as the same flavour compounds recovered by hydrodistillation.
Fast centrifugal partition chromatography was used to separate a class of flavonolignans called silymarins from both a purchased silymarin powder and a crude pressurized hot water extract of milk thistle (Silybum marianum L.). Initially, a purchased power of a mixture of the six silymarin compounds was separated with a two-phase solvent system consisting of heptane/ethyl acetate/methanol/water (1:4:3:4 v/v/v/v) in order to verify elution times of the compounds by fast centrifugal partition chromatography. Next, a crude pressurized hot water extract from 10 g of ground seeds of Silybum marianum was separated with the same solvent system. The separation from the hot water extract gave yields of silychristin at 70.2% purity, silydianin at 93.7% purity, and a mixture of silybinin and isosilybinin at 96.1% purity.
In the present work, the antimicrobial activity of different pressurized liquid extracts obtained from Haematococcus pluvialis microalga was tested against several microorganisms of importance for the food industry (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus niger). Extractions were performed with hexane and ethanol at four different temperatures (50, 100, 150 and 200 °C) for 20 min. The results showed that extracts obtained with both solvents (hexane and ethanol) from the green motile cells of the microalgae (green phase) presented a low antimicrobial activity against all the microorganisms tested. However, the antimicrobial activity of the extracts obtained from the red hematocysts without flagella (red phase) was totally different depending on the solvent used for the extraction. Hexane extracts showed an antimicrobial activity quite similar to that obtained with the green microalgae, while the antimicrobial activity of ethanol extracts was much higher. This fact seems to indicate that compounds related to antimicrobial activity of this microalga are found in higher quantities in the red phase of the microalgae and could be relatively polar compounds. Moreover, ethanol extracts from the red phase obtained at 100 °C presented the highest antimicrobial activity. In order to identify the compounds responsible for the antimicrobial activity, a GC–MS characterization of the extracts obtained with both hexane and ethanol at 100 °C, for Haematococcus pluvialis in the green and red phases was also performed. Therefore, the highest antimicrobial activity of the ethanol extract corresponding to red Haematococcus can be associated with the presence in this extract of short-chain fatty acids, which have been previously described to possess antimicrobial activity.
The suitability of pressurized liquid extraction (PLE) in medicinal plant analysis was investigated. PLE extracts from a selection of representative herbs were compared with extracts obtained according to Pharmacopoeia monographs with respect to yield of relevant plant constituents, extraction time and solvent consumption. In all cases a significant economy in time and solvents was realized, while extraction yields of the analytes were equivalent or higher.