Chemical Composition, in Vitro Digestibility and
Antioxidant Activity of Solid Wastes from the Fruits
of Silybum Marianum
Fang Li, Xiangyang Wu, Ting Zhao, Jiangli Zhao
School of Chemistry & Chemical Engineering,
Jiangsu University, UJS
Zhenjiang, Jiangsu, China
AbstractʊThe chemical composition of solid wastes from
Silybum marianum fruits (WSS) and the contents of total
polyphenols, total Àavonoids and antioxidant activity of the
extracts from WSS with distilled water, ethanol (40%, 70%
and 95%) and methanol was investigated. Chemical analysis
indicated that WSS contained high amounts of crude protein
(25.58%), calcium, potassium and magnesium. The content of
plumbum, selenium and arsenium in WSS was below the
maximum level established by the Commission of the
European Communities. Essential amino acids in WSS
favorably compared with the recommended level
pre-school children. Nitrogen release of WSS
sequential pepsin and trypsin digestion was 70.60%. The 70%
ethanol extract exhibited the highest antioxidant activity. The
nutritional compositions and bioactive compounds in WSS
made it appropriate for applications in feedstuffs as
supplemental sources in animal feeds and food additives.
Keyword-Silybum marianum, Solid wastes, Chemical
composition, Amino acids composition, Antioxidant activity
ĉINTRODUCTION
Milk thistle Silybum marianum L. Gaernt is an annual or
biennial plant, native to the Mediterranean, which has now
spread to other warmer and drier regions. Various
preparations of the fruits, had been used medicinally for
over 2000 years to treat liver disorders [1]. Recently, the
increased consumption of silymarin has eventually made
farmers expand the harvest area of S. marianum. The
growing area in Qinghai, Shanxi, Hubei, Jiangsu and
Guangdong Provinces in China has increased annually [2].
The production of silymarin is accompanied by generation
of vast quantities of solid wastes from S. marianum fruits.
The wastes are rich in proteins, minerals, vitamins,
polyphenols and flavonoids, etc. It could be used as an
*Corresponding author. Email: yangliuqing@ujs.edu.cn
978-1-4244-9171-1/11/$26.00 ©2011 IEEE
Feng Li, Liang Han, Liuqing Yang*
School of Pharmacy,
Jiangsu University, UJS
Zhenjiang, Jiangsu, China
alternative in food and feedstuffs industrial applications.
However, there were few reports exclusively aimed at
evaluating the nutritional quality of the solid wastes.
Therefore, in order to utilize the solid waste, it is necessary
to investigate to evaluate the nutritional quality of the solid
wastes. The present study was to investigate the proximate
composition, amino acid compositions and mineral
contents of the solid wastes and to estimate the content of
total polyphenols and flavonoids and radical-scavenging
activity of the extracts from the solid wastes with distilled
for water, ethanol (40%, 70% and 95%) and methanol.
during
Ċ MATERIALS AND METHODS
A. Chemicals and Materials
Folin-Ciocalteu reagent and gallic acid were obtained from
Sigma-Aldrich. Rutin was obtained from National institute for
control of pharmaceutical and biological products. All other
chemicals and solvents were analytical reagent grade and
obtained from Sinopharm Chemical Reagent Co., Ltd. Solid
wastes from Silybum marianum fruits generated during the
production of silymarin were obtained from Zhongxing
Pharmaceutical Co., Ltd.). The solid wastes were oven-dried
at 60 ć for 24 h and then crushed to the average particle size
of 300 ȝm.
B. Proximate Analysis
The recommended methods of the Association of Official
Analytical Chemists (AOAC) were adopted to determine the
levels of crude protein, crude fat, moisture and total ash. The
crude fiber content was determined by the ceramic fiber filter
method. The carbohydrate contents of the samples were
determined as nitrogen free extract (NFE) by the following
formula: NFE % =100 - (moisture + crude fat + crude protein
+ crude fiber + total ash)
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C. Amino Acid Analysis
Amino acid compositions of WSS were determined by a
Hitachi L-8800 automatic amino acid analyzer (Tokyo, Japan).
The amino acid composition was reported as proportion of the
single amino acid to total amino acids. Tryptophan was not
determined.
D. Mineral Analysis
WSS was treated by wet digestion, then the digested
products were used for the determination of the contents of
calcium, copper, iron, magnesium, manganum, potassium and
zinc by TAS-986 atomic absorption spectrometer (Purkinje
General Instrument Co., Ltd, Beijing, China), whilst the
contents of chromium, phosphonium, plumbum and sodium
were determined by Vista-MPX Simultaneous ICP (Varian,
Inc. USA). In addition, the contents of asenium and selenium
were determined by AFS-930 hydride generation atomic
fluorescence spectrometry (Titan instruments Co., Ltd.).
I. Statistical Analysis
Data were reported as mean ± SD (standard deviation) of
triplicate determinations. Statistical calculations were carried
out by SPSS version 16.0. One-way ANOVA of the data was
performed.
ċRESULTS AND DISSCUSSION
A. Chemical Composition and in Vitro Digestibility
The proximate analysis of WSS showed that the samples
contained 9.23% moisture, 25.58% crude proteins, 9.85% ash,
22.09% crude fiber, 32.46% NFE and very little fat (0.3%).
The amino acid compositions of WSS were shown in Table 1.
The percentages of histidine, valine, isoleucine, phynylalanine
+ tyrosine of the WSS were higher than their counterparts
stated in the FAO/WHO standard. Some mineral contents of
WSS were shown in Table 2. Potassium content was high
comparison to sodium, and the Na/K ratio was very low.

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Digestibility of WSS protein in vitro was determined
according to the method described by Yousif et al. [3]. Amino acid WSS (%) FAO/WHO
Asx 9.85
Glx 22.72
F. Preparation the WSS Extracts Serine (Ser) 5.65
Histidine (His) 2.37 1.9
Samples of the WSS (15 g) were extracted with 300 mL of Glycine (Gly) 6.16
Threonine (Thr) 4.15 3.4
distilled water, ethanol (40%, 70% and 95%) and methanol at Arginine(Arg) 8.23
75 ć for 2 h, followed by filtration, and the residues were Alanine (Ala) 4.59
Tyrosine (Tyr) 3.71
re-extracted with 300 mL of the same solvent as described Cystine (Cys) 1.18
Valine (Val) 5.08 3.5
above. The combined supernatants were concentrated under Methionine (Met) 1.38
Phenylalanine (Phe) 4.31
vacuum in a rotary evaporator, and lyophilized. Isoleucine (Ile) 4.08 2.8
Leucine (Leu) 6.86 6.6
G. Total Polyphenol (TP) and Total Flavonoid (TF) Lysine (Lys) 4.63 5.8
Proline (Pro) 5.06
Determiration Total sulfur-containing 2.56 2.5
amino acids (Met and Cys)
TP and TF were determined by the Folin-Ciocalteu method Total aromatic amino acids 8.02 6.3
[4]
(Phe and Tyr)
and the method described by Makris et al , respectively. 
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Gallic acid and rutin was used as a calibration standard. )58,76

H. Radical Scavenging Assay
Mineral WSS (mg/kg)
DPPH free radical-scavenging activity was determined Arsenium (As) 0.02 ± 0.01
Calcium (Ca) 13480.1 1 ± 105.81
according to the method described by Scherer el al [5]. ABTS Chromium (Cr) 1.33 ± 0.04
Copper (Cu) 21.74 ± 0.35
assay was performed according to the method described by Iron (Fe) 328.87 ± 4.01
Potassium (K) 9092.40 ± 105.8
[6]
Katalinic et al . Ascorbic acid was used as a standard Magnesium (Mg) 8677.32 ± 240.47
Manganum (Mn) 58.69 ± 1.27
antioxidant to validate the assay. The IC50 was calculated Sodium (Na) 155.01 ± 2.70
Phosphonium (P) 7128.17 ± 68.10
graphically using a calibration curve in the linear range by Plumbum (Pb) 0.08 ± 0.03
Selenium (Se) 0.08 ± 0.00
plotting the extract concentration versus the corresponding Zinc (Zn) 97.22 ± 1.22

scavenging effect.
It was considered as an advantage from the nutritional point

2999

of view, since the intake of sodium chloride and diets with a
high Na/K ratio had been related to the incidence of
hypertension. Plumbum, selenium and arsenium content in
WSS was below the maximum levele stablished by the
Commission of the European Communities. The in vitro
digestibility of WSS was evaluated by nitrogen release during
digestion of pepsin and trypsin, in simulated gastric fluid.
Nitrogen release of WSS during sequential pepsin and trypsin
digestion was 70.60%. which was higher than that (66.8%) of
Napier grass [7]. These results were the indications that WSS
appropriated for applications in feedstuffs industry such as
supplemental source in animal feeds.
B. Extraction Yields and Contents of TP and TF
It was evident that the aqueous extract exhibit highest
yields. The results might be explained that WSS had relatively
higher protein content where water-soluble proteins were the
most dominant. The contents of TP and TF of five different
extracts were presented in Table 3. It was shown that ethanol
and methanol extracts of WSS exhibited relatively higher TP
[8]
content. It was reported by Lafka et al that ethanol was
selected as the most appropriate solvent for the extraction of
phenolic compounds from winery wastes for production of
extracts with high TP and high antioxidant activity. Our
results were in agreement with the above-mentioned ¿nding.
Correlation analysis was performed on the polyphenolic
content analysis for the five extracts. The correlations
between TP and TF assays were 0.7862, which was
significant at the 0.05 level. The results indicated that the
Àavonoids were the main phenolic groups in WSS.
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The extract samples
IC50 (μg/mL)
DPPH A BTS
Aqueous extract 347.58 ± 2.84d 258.05± 2.36e
40% ethanol extract 228.05 ± 1.76c 222.89±1.82d
70% ethanol extract 191.00 ± 1.36a 190.12±0.89a
95% ethanol extract 217.23 ± 2.33b 199.88±0.63c
Methanol extract 219.80 ± 1.49b 195.07 ±2.64b
Values in the same column followed by a different letter were significantly
different (p<0.05) according to least significant difference (LSD) test.
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different (p<0.05) according to least significant difference (LSD) test.
C. Radical Scavenging Activity
DPPH radical scavenging abilities of the extracts were
depicted in Fig. 1B.The standard ascorbic acid showed a high
activity of 93% at a concentration of 40 ȝg/mL, with IC50
value of 10.91 ± 0.15 μg/mL, confirmed the validity of this
method. The IC50 values indicated that the antioxidant
effectiveness decreased in following order (Table 4): 70%
ethanol extract > 95% ethanol extract = methanol extract >
40% ethanol extract > aqueous extract. The different
antioxidant activities of the extracts might be attributed to the
type and polarity of the extracting solvent, the isolation
procedures, as well as the test system, as these affected the
content and purity of the bioactive compounds. ABTS radical
scavenging abilities of the extracts were depicted in Fig. 1B.
IC50 values indicated that ABTS radical scavenging activity of
the extracts of WSS decreased in following order (Table 4):
70% ethanol extract > methanol extract > 95% ethanol
extract > 40% ethanol extract > aqueous extract.
D. Correlations between Antioxidant Activity and Contents
of TP and TF
Correlations were studied between the antioxidant
activities and the contents of TP and TF, and the results were
shown in Table 5. There was a significant linear correlation
between the antioxidant activity and the content of TP in the
extracts of WSS. The results indicated that polyphenolic
compounds significantly contributed to the antioxidant
capacity of the WSS. The observation was consistent with the
findings of many research groups who reported such
significant correlation between antioxidant activity and
[9-12]
content of TP . The antioxidant activity also increased
proportionally to the total flavonoid content of the extracts
and the correlation was also significant. Whereas the
correlation coefficient between the total Àavonoids and FRAP
values of 68 Chinese herbals was determined to be r2=0.4555
[12]
.

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ČCONCLUSION
The TP TF Yield
extract samples (g GAE/kg) (g RE/kg) (g/kg)
Aqueous extract 0.36 ± 0.01a 0.29 ± 0.01a 2.596 ± 0.19e
Chemical analysis showed that WSS contained high
40% 0.58 ± 0.02 b 0.51 ± 0.01b 1.162 ± 0.11d amounts of crude fiber, protein, calcium, potassium and
ethanol extract
70% 0.63 ± 0.14c 0.65 ± 0.07c 0.593 ± 0.03b magnesium, and low in asenium, plumbum and sodium.
ethanol extract
95% 0.65 ± 0.01c 0.51 ± 0.04c 0.441 ± 0.03a Essential amino acids in WSS favorably compared with the
ethanol extract
c c c
Methanol extract 0.62 ± 0.02 0.52 ± 0.02 0.809 ± 0.04 recommended level for pre-school children. Nitrogen release
Values in the same column followed by a different letter were significantly

3000
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Figure 1 Antioxidant activities of the solid wastes extracts in different
solvent in DPPH (A) and ABTS (B) free radical-scaveng ing assays.
Ascorbic acid was used as a positive control.

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9$/8(6$1'7+(&217(1762)73$1'7)

Value Equationa
TP TF
IC50 of the Y= -37.367x + 465.91 Y= -45.65x + 467.16,
2 2
WSS DPPH R = 0.9094, p < 0.05 R = 0.9248, p < 0.05
assay
IC50 of the Y = -22.489x + 341.02, Y = -19.689x + 310.84,
WSS ABTS R2 = 0.9095, p < 0.05 R2 = 0.8223, p < 0.05
assay
a
x represents the content of total polyphenol, flavonoids (g/kg extract), Y
represents IC50 (μg/mL).

and the total polyphenol or flavonoid content. Thus,

Bioactive compounds and rich nutritional composition in
WSS makes it appropriate for applications as food
antioxidant and supplemental source in animal feeds.

ACKNOWLEDGMENT
The authors gratefully acknowledge Zhongxing
Pharmaceutical Co., Ltd. for positively providing industrial
solid wastes of Silybum marianum fruits.

3001