Food Sci. Biotechnol.
21(2): 389-398 (2012)
DOI 10.1007/s10068-012-0050-0
RESEARCH ARTICLE
Phenolic Composition and Antioxidant Activity of Thai and
Eri Silk Sericins
Luchai Butkhup, Montree Jeenphakdee, Sujitar Jorjong, Supachai Samappito, Wannee Samappito, and
Jirapa Butimal
Received: 5 September 2011 / Revised: 20 December 2011 / Accepted: 22 December 2011 / Published Online: 30 April 2012
© KoSFoST and Springer 2012
Abstract The present study investigates the phenolic
contents and antioxidant activities of silk sericin extracted
(SSE) from Thai mulberry silkworms (Bombyx mori) and
non-mulberry silkworm (Samia ricini). The SSE from all
strains revealed the mainly presence of flavonoid
compounds including (+)-catechin (20.63-145.64 mg/100
g), quercetin (7.89-35.95 mg/100 g), and (-)-epicatechin
(3.36-44.19 mg/100 g). The antioxidant activity of the SSE
with water from Chokumnoui 1 was found to have low
EC50 values (0.96 mg/mL), the concentrations of the SSE
that exhibit 50% reduction in DPPH, and highest
scavenging of ABTS• + radicals (1.73 mg TEAC/g) and
highest reduce TPRZ-Fe (III) complex to TPTZ-Fe (II)
(8.03 mg Fe(II)/g), thus indicating high antioxidant
activities. Total phenolic content and total flavonoid
content of the SSE showed positive correlated to the
scavenging activity of DPPH and ABTS• + radicals and the
ferric reducing ability (FRAP assay).
Keywords: Bombyx mori, Samia ricini, silk cocoon,
phenolic compound, antioxidant activity
Luchai Butkhup ( ), Montree Jeenphakdee, Sujitar Jorjong, Supachai
Samappito
Department of Biotechnology, Faculty of Technology, Mahasarakham
University, Mahasarakham 44000, Thailand
Tel: +66 43 754238; Fax: +66 43 754238
E-mail: Tak_biot2000@yahoo.com
Wannee Samappito
Department of Food Technology, Faculty of Technology, Mahasarakham
University, Mahasarakham 44000, Thailand
Jirapa Butimal
Silk Innovation Center, Division of Research Supporting and Extension
Services, Mahasarakham University, Mahasarakham 44000, Thailand
Introduction
Mulberry silkworms (Bombyx mori) and non-mulberry
silkworms (Samia ricini) synthesize and secrete 2 classes
of silk protein, silk fibroin (SF) and silk sericin (SS). SF is
a fibrous protein that has been widely investigated for
biomedical purposes owing to its physiochemical properties
and relatively inert immune response (1). SS, on the other
hand, was long considered to simply be a waste product in
the silk industry until it was found to have important
biochemical functions such as antioxidant activity (2,3),
antityrosinase activity (2), and effects on tumor progression
(4,5). SS also can be used as a serum-free freezing medium
for mammalian cells (6). SS alone or in combination with
SF has been used in skin, hair, and nail cosmetics. Sericin
when used in the form of lotion, cream, and ointment
shows increased skin elasticity, antiwrinkle, and antiaging
effects (7). Silks differ widely in composition, structure,
and biochemical properties depending on the specific
source and strain. Thai silk, particularly the yellow
Nangnoi silk, contains a significant amount of pigments,
which are primarily associated with carotenoids and
flavonoids (8,9). These phenolic compounds are most
commonly known for their antioxidant properties and other
diverse biochemical functions, such as antityrosinase,
antiallergy, or anti-inflammatory activities (10,11).
The mulberry silkworm spins silk threads to make a
cocoon shell, where some pigments coexist and
accumulate in the layers of cocoon sericin. The coloring
components are associated with phenolic compounds in the
leaves of mulberry (Morus alba Linn.), the sole food for
mulberry silkworm larvae. The two main kinds of pigments
responsible for cocoon color are ether-soluble yellowish
carotenoids and water-soluble green flavonoids (8,12).
Recently, Tamura et al. (8) isolated quercetin 5-glucoside,
390
quercetin 5,40-diglucoside, and quercetin 5,7,4'-triglucoside
from the yellow-green cocoon shell of a race Multi-Bi.
These phenolics were not found to be present in leaves of
its host plant, the mulberry tree, in which quercetin-3-
glycosides (rutin, isoquercitrin) and quercetin aglycon are
naturally occurring (13). This suggests that phenolics from
the dietary mulberry leaves are modified within the
silkworm by a glucosyltransferase that can transfer glucose
residue to the C-5 hydroxy position of quercetin, and
accumulate in the silk fiber. The content of cocoon
coloring components varies depending on the mulberry
silkworm strain. Some strains produce green cocoon shells
containing at least 9 fluorescent yellow compounds, 5 of
which have been identified as flavonoid related compounds
(14). There are several native strains of silkworm in
Thailand with various colors of cocoon shells. The most
common native Thai silk is Nangnoi (yellow cocoon shell),
which has been found to contain several flavonoids such as
c-prolinylquercetins, while other Thai silk cocoons have
white and yellow-green shells. Flavonoid compounds, in
addition to SS, are also responsible for the antioxidant
properties of mulberry silkworm cocoons (2,12). Although
the patterns of phenolics accumulation in some yellow
cocoons were found, little is known about the phenolics
and antioxidant activity of the white and yellow cocoon of
Thai and Eri silk. The objectives of the present study were
to investigate the phenolic composition and antioxidant
activity of SS extracted from different Thai mulberry and
non-mulberry silkworm strains via aqueous and ethanolic
extraction.
Materials and Methods
Reagents and chemicals ABTS, DPPH, Trolox, and
BHT were obtained from Fluka (Buchs, Switzerland).
Ethanol, acetonitrile, and phosphoric acid were of HPLC
grade (Tedia Company-Fairfield, Fairfield, OH, USA).
Deionized water was prepared by a Milli-Q water purification
system (Millipore, MA, USA). (+)-Catechin, (−)-epicatechin,
rutin, tran-resveratrol, procyanidin B1, procyanidin B2,
myricetin, quercetin, naringenin, luteolin, and kaempferol
standards were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Standard stock solvents of phenolic compounds
were prepared in methanol at concentration of 0.50 g/L. All
sample solvents were filtered through 0.45-mm membrane
filter (Millipore), and injected directly.
Silkworm cocoons Fresh cocoon shells of the mulberry
silkworm and Eri silkworm were kindly supplied by the
Silk Innovation Center, Mahasarakham University,
Mahasarakham province, Thailand. Silkworm cocoons
were produced in a controlled environment from 3 Thai
Butkhup et al.
native silk strains [Nangnoi (bivoltine, yellow shell),
Chokumnoui 1 (bivoltine, yellow shell), and Chokumnoui
2 (bivoltine, white shell)] and 1 Thai Eri silk strain (white
shell).
Preparation of SS powder Clean fresh cocoon shells of
the mulberry silkworm and Eri silkworm were chopped
into square pieces (approx. 5 mm2), and 1 g of silk cocoons
was extracted with 40 mL of 70% ethanol or 40 mL of
distilled water by autoclaving (SX-700; Tomy Seiko,
Tokyo, Japan) at 121oC and 103.5 kPa for 60 min. The
aqueous solution obtained from autoclaving the silk cocoon
was collected, and the residue was repeatedly extracted 3
times with the same solvent until it was colorless and
centrifuged (10 min, 5,000×g). The extracts were filtrated
through Whitman No. 1 paper under vacuum to remove
insoluble material, which is fibroin. After this filtering
process, the filtrate was frozen and freeze-dried using a
Powerdry PL 3,000 lyophilizer (Thermo Electron Corporation-
Site Thermo Electron CZ a.s. Tehovec 107, Mukarov, 251
62, Czech Republic) to obtain SS powder.
Determination of total phenolic content The total
phenolic contents of the extracts were determined using
Folin-Ciocalteu reagent as described by Singleton and
Rossi (15). Briefly, 12.5 µL of the samples were mixed
thoroughly with 12.5 µL of distilled water and 12.5 µL of
Folin-Ciocalteu reagent (previously pre-diluted 10 times
with distilled water). After 6 min, 125 µL of 7% sodium
carbonate (Na2CO3) and 100 µL of distilled water were
added and allowed to react for 90 min at room temperature.
The absorbance was measured at 760 nm using microplate
reader spectrophotometers (Synergy HT; BioTek Instruments,
Winooski, VT, USA). Samples were measured in 3
replicates. A standard curve of gallic acid solution (6.25,
12.5, 25, 50, 100, 200, and 400 mg/mL) was prepared
using the similar procedure. The results were expressed as
mg gallic acid equivalents (GAE)/100 g.
Determination of total flavonoid content A colorimetric
assay (16) with some modifications was used to quantify
total flavonoid content. Briefly, 25 µL of the diluted
sample or (+)-catechin was added to 125 µL of ddH2O.
Subsequently, 7.5 µL of NaNO2 solution (5%) was added
to the mixture and allowed to stand for 5 min, 15 µL of
AlCl3 (10%) was added. The mixture was incubated at
ambient temperature (25oC) for an additional 5 min.
Following that 50 µL of 1 M NaOH solution was then
added to the mixture. The mixture was immediately diluted
by the addition of 27.5 µL of ddH2O, thoroughly mixed,
and the absorbance of the mixture was determined at 510
nm against a blank prepared with ddH2O using a microplate
reader (Synergy HT; BioTek Instruments). Total flavonoids
Phenolic Composition of Thai and Eri Silk
were expressed as mg (+)-catechin equivalent (CE)/100 g,
through the calibration curve of (+)-catechin. All samples
were analyzed in 3 replications.
Determination of free radical scavenging using DPPH
method The antioxidant activities of all extracts were
evaluated through the free radical scavenging effect on
DPPH radical. The determination was based on the method
proposed by Akowuah et al. (17). Briefly, 100 µL of
0.2 mM DPPH methanolic solution was added to 100 µL
of the sample extracts. The mixture was thoroughly mixed
and kept in the dark for 1 h. The control was prepared by
mixing 100 µL of DPPH and 100 µL of methanol. The
absorbance was measured at 520 nm using microplate reader
spectrophotometers (Synergy HT: BioTek Instruments).
The capability of extracts to scavenge the DPPH radical
was calculated using the following equation:
Scavenging effect (%)
Absorbance of sample at 520 nm
= 1 – ⎛-------------------------------------------------------------------------------⎞ × 100
⎝Absorbance of control at 520 nm⎠
EC50 value was determined from the plotted graph of
scavenging activity versus the concentration of extracts,
which is defined as the amount of antioxidant necessary to
decrease the initial DPPH radical concentration by 50%
(18). Triplicate measurements were carried out and the
EC50 value (mg/mL) was determined for all the extracts.
Ascorbic acid and BHT were used as the reference
compounds (EC50 =0.01 and 0.09 mg/mL, respectively).
Determination of ferric reducing/antioxidant power
assay (FRAP) FRAP assay was carried out with some
modification to the method of Benzie and Strain (19). The
FRAP reagent was prepared from 300 mM acetate buffer
(pH 3.6), 20 mM ferric chloride and 10 mM TPTZ in 40
mM HCl. All 3 solutions were mixed together in the ratio
of 10:1:1 (v/v/v). The FRAP reagent was prepared fresh
daily and warmed to 37oC in an oven prior to use. A total
of 30 µL samples extract was added to 270 µL of the
FRAP reagent and mixed well. The absorbance was measured
at 595 nm using microplate reader spectrophotometers
(Synergy HT; BiotTek Instruments) after 30 min. Samples
were measured in 3 replicates. A standard curve of iron (II)
sulfate solution (6.25, 12.5, 25, 50, 100, 200, and 400 mg/
mL) was prepared using a similar procedure. The results
were expressed as mg Fe(II)/g.
ABTS• + scavenging assay For the ABTS cation radical
scavenging assay, the assay was carried out with some
modification to the method of previously reported (20).
Briefly, ABTS diammonium salt (ABTS• +) radical cations
were prepared by adding potassium persulphate (2.6 mM)
391
to a 7.4 mM aqueous stock solution of ABTS• + in
proportion of 1:1 (v/v). The mixture was thoroughly mixed
and allowed to stand for 12 h prior to use. Trolox, a water-
soluble analogue of vitamin E, was used as an antioxidant
standard. A standard calibration curve was constructed for
Trolox at 6.25, 12.5, 25, 50, 100, 200, and 400 mg/mL
concentrations. A total of 10 µL samples extract were
added to 190 µL of ABTS• + solution and mixed well. The
absorbance was measured at 595 nm using microplate
reader spectrophotometers (Synergy HT; BioTek Instruments)
after 2 h. Samples were assayed in 3 replicates. Trolox
equivalent antioxidative capacity (TEAC) values were
calculated from the Trolox standard curve and expressed in
mg TEAC/g extract.
Determination of phenolic compounds by reversed-
phase HPLC with diode array detector (RP-HPLC-
DAD) Determination of phenolic compounds was
carried out as described in detail elsewhere (21) and 20 µL
of the clear samples were analyzed by RP-HPLC-DAD.
HPLC apparatus consisting of Shimadzu (Kyoto, Japan)
LC-20AD series pumping system, SIL-10AD Series auto
injector system and SPD-M20A Series DAD was used to
record online UV spectra of the phenolic compounds in the
samples. The data were collected and analyzed with a
Shimadzu computing system. The column used was an
Apollo C18 (Alltech Associates, Deerfield, IL, USA) (ø
4.6×250 mm, 5 µm) protected with guard column Inertsil
ODS-3 (ø 4.0×10 mm, 5 µm; GL Science Inc., Tokyo,
Japan). Twenty µL of each sample were analyzed using an
HPLC system. The mobile phase for phenolic compounds
determination was acetonitrile/deionized water (2/97.8, v/
v) containing 0.2% phosphoric acid (solvent A) and
acetonitrile/deionized water (97.8/2, v/v) containing 0.2%
phosphoric acid (solvent B) at a flow rate of 0.6 mL/min,
and column temperature was at 40oC. The UV-Vis spectra
were recorded from 190 to 400 nm, with detection at
254 nm. The linear gradient started with 20% solvent B,
50% solvent B at 30 min, 60% solvent B at 35 min, 20%
solvent B at 40 min at isocratic elution until 55 min.
Quantification was carried out by comparing the retention
times and the spectra as well as by the addition of standards.
The concentrations of phenolic compounds were calculated
with the help of a corresponding external standard.
Statistical analysis All the compounds and parameters
reported below were evaluated in triplicate, in each of the
samples. The statistical analysis of the data was carried out
by analysis of the variance (ANOVA) and the Duncan’s
multiple range test (DMRT) to show measurements which
can be considered statistically different. p-Value of <0.05
was regarded as significant.
392 Butkhup et al.

Table 1. Yield of silk sericin extracted by different solvents

Color of Extraction
Silk strain Yield (%)
cocoon shell solvent
Nangnoi Yellow Water 27.35±1.33
Yellow Ethanol 23.18±1.46
Chokumnoui 1 Yellow Water 13.04±2.05
Yellow Ethanol 10.18±1.87
Chokumnoui 2 White Water 31.54±2.10
White Ethanol 26.19±1.36
Eri White Water 9.20±1.24
White Ethanol 7.48±1.03

Fig. 1. Physical appearance of Thai mulberry and non-
mulberry silk cocoons from 4 different strains. A, Nangnoi; B,
Chokumnoui 1; C, Chokumnoui 2; and D, Eri
Results and Discussion
Yields of SS extracts Fig. 1 displays the physical
appearance of Thai mulberry and non-mulberry silk
cocoons from 4 different strains: Nangnoi, Chokumnoui 1,
Chokumnoui 2, and Eri. SS yield using the different
extraction solutions listed in Table 1. For all silk strains, SS
extracted by water at high-temperature and high-pressure
had higher yields compared with that extracted with
ethanol solution.
Quantitative of phenolic compounds in SSE The
results of previous studies (22) showed that some of the
phenolic components such as quercetin and (+)-catechin
had anticancer activities, and these components were able
to inhibit cancer cell growth. The suppression colon
carcinogenesis of silk sericin was also substantiated (4).
Results of the current research (Table 2 and 3), both of
water and ethanolic SS extract, showed that phenolics are
found in higher content in the mulberry silk cocoons
(Nangnoi, Chokumnoui 1, and Chokumnoui 2) than in Eri
silk cocoon. The SS extracted by ethanol revealed the
presence of many kinds of phenolics such as (+)-catechin,
(-)-epicatechin, rutin, procyanidin B1 [(-)-epicatechin-(4β
→8)-catechin], procyanidin B2 [(-)-epicatechin-(4β→8)-(-)-
epicatechin], quercetin, myricetin, trans-resveratrol, luteolin,
naringenin, and kaempferol. However, the SS extracted
with water from Chokumnoui 1, Nangnoi, Chokumnoui 2,
and Eri had higher content of sum phenolics than the SS
extracted with ethanol with average content of 196.15±
2.23, 146.86±4.22, 131.89±2.06, and 117.64±1.51 mg/100
g, respectively. The SS extracted with ethanol exhibited
significant result in (+)-catechin, quercetin, (-)-epicatechin,
rutin, and trans-resveratrol. (+)-Catechin and quercetin
were the major phenolics presented in all the samples after
HPLC analysis was conducted. The (+)-catechin had the
highest content in the SS extracted with water from

Table 2. Composition of phenolics (mg/100 g) of water silk sericin extracted from Nangnoi, Chokumnoui 1, Chokumnoui 2, and
Eri strains
Silk strains
Phenolics
Nangnoi Chokumnoui 1 Chokumnoui 2 Eri
b1) a c
(+)-Catechin 102.34±11.06 128.51±16.82 068.73±10.07 59.86±3.35d
(–)-Epicatechin 3.36±0.17d 18.42±4.95b 07.12±0.48c 32.29±4.30a
Rutin 5.37±0.76c 06.79±2.67b 10.62±2.01a 06.48±1.89b
Procyanidin B1 1.41±0.16b 02.22±0.46a 00.94±0.08c 00.60±0.11d
Procyanidin B2 1.14±0.03c 07.26±1.31b 10.11±1.16a 01.27±0.09c
Quercetin 12.86±1.25b0 19.52±0.54a 20.83±1.08a ND
Myricetin ND 02.45±0.23a 02.20±0.01a 02.17±0.13a
trans-Resveratrol ND 10.37±0.05b 10.59±0.20b 14.97±0.35a
Luteolin 0.38±0.01c 00.61±0.01b 00.75±0.02a ND
Naringenin ND ND ND ND
Kaempferol ND ND ND ND
Σ Phenolics 146.86±4.22b00 196.15±2.23a0 131.89±2.06c0 117.64±1.51d
1)
Mean of triplicate measurements±SD; Means not sharing a common letter within each row were significantly different at p<0.05 as measured
by the DMRT; ND, not detected
Phenolic Composition of Thai and Eri Silk 393

Table 3. Composition of phenolics (mg/100 g) of ethanol silk sericin extracted from Nangnoi, Chokumnoui 1, Chokumnoui 2, and
Eri strains
Silk strains
Phenolics
Nangnoi Chokumnoui 1 Chokumnoui 2 Eri
(+)-Catechin 058.87±16.19b1) 62.22±5.25a 043.30±12.11c 20.63±0.40d
(–)-Epicatechin 4.16±1.64d 15.36±3.23c 24.24±4.23b 44.19±5.56a
Rutin 4.13±0.81b 8.15±0.29a 4.01±0.55b 4.80±0.31b
Procyanidin B1 2.22±0.24b 5.20±1.89a 3.37±1.33b 1.56±0.07c
Procyanidin B2 7.36±0.26a 6.99±0.45b 5.76±0.40c 1.71±0.05d
Quercetin 35.95±5.21a0 30.82±5.07a0 7.89±1.26b ND
Myricetin 2.29±0.23b 3.16±0.02a 2.54±0.19b 2.27±0.05b
trans-Resveratrol 11.12±0.65b0 10.58±0.12b0 11.63±0.15b0 18.25±0.11a0
Luteolin 1.09±0.03b 0.45±0.02c 1.64±0.04a ND
Naringenin ND 0.43±0.03b 0.62±0.07a ND
Kaempferol 1.16±0.16a 1.38±0.02a 1.18±0.03a ND
Σ Phenolics 128.35±2.12b00 144.74±1.04a00 106.18±5.04c00 93.41±1.14d0
1)
Mean of triplicate measurements±SD; Means not sharing a common letter within each row were significantly different at p<0.05 as
measured by the DMRT; ND, not detected

Chokumnoui 1 (128.51±16.82 mg/100 g) followed by
Nangnoi (102.34±11.06 mg/100 g), Chokumnoui 2 (68.73±
10.07 mg/100 g), and Eri (59.86±3.35 mg/100 g), respectively
(Table 2). The (+)-catechin exhibited highest content in the
SS extracted with ethanol from Chokumnoui 1 (62.22±
5.25 mg/100 g) followed by Nangnoi (58.87±16.19 mg/
100 g), Chokumnoui 2 (43.30±12.11 mg/100 g), and Eri
(20.63±0.40 mg/100 g), respectively (Table 3). The quercetin
had the highest content in the SS extracted with water from
Chokumnoui 2 (20.83±1.08 mg/100 g) and Chokumnoui 1
(19.52±0.54 mg/100 g) followed by Nangnoi (12.86±1.25
mg/100 g) while the Eri was not detected. The quercetin
exhibited highest content in the SS extracted with ethanol
from Nangnoi (35.95±5.21 mg/100 g) and Chokumnoui 1
(30.82±5.07 mg/100 g) followed by Chokumnoui 2 (7.89±
1.26 mg/100 g) while the Eri was not detected. The
extraction of quercetin in the SS from Nangnoi and
Chokumnoui 1 with ethanol using a high-temperature and
high-pressure had higher content than with water. The
quercetin was detected only in the cocoon shell of the
mulberry silkworm. Of all the phenolics found in silk,
quercetin, kaempferol, and glucosides of kaempferol were
reported to be major components found in silk (12).
Tamura et al. (8) and Kurioka and Yamazaki (12) reported
that the quercetin glucosides, quercetin-5,4'-di-O-β-D-
glucopyranoside, quercetin-7,5,4'-tri-O-β-D-glucopyranoside,
quercetin-5-O-β-D-glucoside, quercetin 7-O-β-D-glucoside,
and quercetin-4'-O-β-D-glucoside were isolated from the
cocoon shell of the mulberry silkworm. These glucosides
were not present in mulberry leaves, the silkworm’s only
food; they are considered to be metabolites produced by
the silkworm. Kurioka and Yamazaki (12) reported that
quercetin and quercetin 4'-O-β-D-glucoside content in the
cocoon shell of the silkworm was in the amounts of 13.4
and 15.3%, respectively. Similarly, the results of this study
show that the quercetin content in the cocoon shell of the
Chokumnoui 2 is 15.79% of the sum of individual phenolics.
In addition, Kurioka and Yamazaki (12) reported that the
kaempferol was also present in the cocoon shell of the
silkworm in the amount of 1.2%. This study found that the
kaempferol content in the cocoon shell of the Nangnoi,
Chokumnoui 1, and Chokumnoui 2 were 0.90, 0.95, and
0.92% of sum individual phenolics, respectively.
Total phenolic content and total flavonoid content in
the studied SSE The Folin-Ciocalteu method is a rapid
and widely-used assay to investigate the total phenolic
content. This method is based on reducing the power of
phenolic hydroxyl groups; however, it is known that
different phenolic compounds have different responses to
the Folin-Ciocalteu reagent (23). Total phenolic content is
expressed as mg GAE/g, which was determined from
known concentrations of gallic acid prepared similarly.
Table 4 shows the effect of different extraction solutions on
total phenolic content in the cocoon shell of the Nangnoi,
Chokumnoui 1, Chokumnoui 2, and Eri. The Chokumnoui
1 shows significantly (p<0.05) higher total phenolic
content, when compared to those of the studied silk strains,
with an average value of 230.45±13.87 and 532.07±12.25
mg GAE/100 g for water and ethanol extraction at high-
temperature and high-pressure, respectively. The total
phenolic content of Eri was the lowest. The results of
ANOVA analysis reveal a significant difference (p<0.05)
between the mean of total phenolic content of the water
and ethanol extracts of the studied silk strains. The SS
extracted with water had a higher content of total phenolic
content than the SS extracted with ethanol. Total phenolic
content of ethanolic extracted of Chokumnoui 1, Nangnoi,
394 Butkhup et al.

Table 4. Total phenolic content and total flavonoid content of water and ethanolic silk sericin extracted
Total phenolic content Total flavonoid content
Silk strains (mg GAE/100 g) (mg CE/100 g)
Ethanol Water Ethanol Water
Nangnoi 0108.52±11.09bB1) 501.10±10.56bA 62.46±1.57bB 107.62±3.12bA
Chokumnoui 1 230.45±13.87aB 532.07±12.25aA 93.75±2.28aB 127.89±2.82aA
Chokumnoui 2 084.65±12.58cB 393.04±15.67cA 49.03±1.86cB 077.03±4.23cA
Eri 56.26±4.32dB 266.58±11.61dA 28.46±2.14dB 064.45±3.67dA
1)
Mean of triplicate measurements±SD; Means not sharing a lowercase within each column or capital letter within each row were significantly
different at p<0.05 as measured by the DMRT.

Chokumnoui 2, and Eri was 230.45±13.87, 108.52±11.09,
84.65±12.58, and 56.26±4.32 mg GAE/100 g, respectively.
Total phenolic content of water extracted of Chokumnoui
1, Nangnoi, Chokumnoui 2, and Eri was 532.07±12.25,
501.10±10.56, 393.04±15.67, and 266.58±11.61 mg GAE/
100 g, respectively. These quantities are regarded as higher
content, compared to the amount of total phenols in other
medicinal plants such as Cyphostemma digitatum leaf
(1.56 mg GAE/100 g dry weight) (24), ginger (101.6 mg
GAE/100 g extracts), and turmeric (67.9 mg GAE/100 g
extracts) (25).
Total flavonoid content in the cocoon shell of Nangnoi,
Chokumnoui 1, Chokumnoui 2, and Eri is shown in Table
4. Among the studied silk strains, Chokumnoui 1 had the
(p<0.05) highest total flavonoid content with average value
of 93.75±2.28, and 127.89±2.82 mg CE/100 g for water
and ethanol extracts respectively. The results of ANOVA
analysis reveal a significant difference (p<0.05) in the
mean of total flavonoid content between the water and
ethanol extractions of the studied silk strains. The SS
extracted with water had higher total flavonoid content
than those in the SS extracted with ethanol which indicated
that water was more effective solvent for flavonoid extraction
than ethanol. The reasons that flavonoid has higher
solubility in subcritical water than in ethanol may be due to
the presence of many polar hydroxyl groups within the
compounds. In addition, flavonids are usually present in
glycosidic forms, in which the different hydroxyl groups of
the flavonoids molecules bind to different glycosides, thus
resulting in increased polarity (12,26). The total avonoid
content in the ethanolic extract of Chokumnoui 1, Nangnoi,
Chokumnoui 2, and Eri was found to be 93.75±2.28,
62.46±0.57, 49.03±1.86, and 28.46±2.14 mg CE/100 g,
respectively. The total avonoid content in the water extracted
of Chokumnoui 1, Nangnoi, Chokumnoui 2, and Eri was
found to be 127.89±2.82, 107.62±3.12, 77.03±4.23, and
64.45±3.67 mg CE/100 g, respectively. These quantities
are regarded as moderate content, compared to the amount
of avonoids in other fruits and vegetables such as onion
skin (96 mg/100 g d.w.) (27) broccoli (304 mg/100 g d.w.),
Italian kale (1,127 mg/100 g d.w.) (28).
Antioxidant activity of SSE using DPPH, FRAP, and
ABTS assays In recent years, there has been a growing
interest in obtaining biologically active compounds from
natural sources. The potential dangers of some synthetic
antioxidants, such as butylated hydroxytoluene (BHT) and
butylated hydroxy-anisole (BHA), have been documented,
and this has stimulated the substitution of synthetic
antioxidants by natural ones (20). Flavonoids modied by
mulberry silkworm may be useful as medicinal or cosmetic
materials because the extracts of yellow-green colored
cocoon shells of a range of strains of mulberry silkworm
have potent antibacterial activity (29) and strong
antioxidant activity (30). In a continuing search among
studied silk strains and extraction techniques for
antioxidant activity, we found that the water and ethanolic
extracts of the yellow and white cocoon shell of studied
silk strains reveal a significant difference (p<0.05)
antioxidant activity. Table 5 shows the comparison of the
mean concentration for 50% free radical scavenging
activity (EC50) of water and ethanolic extracts of silk
cocoons against 50.62 µg/mL of DPPH radical. The EC50
of vitamin C and BHT were 0.01 and 0.09 mg/mL
respectively, which is stronger than other water and
ethanolic extracts of cocoon shells. The EC50 values of
ethanol extracts of Chokumnoui 1, Nangnoi, Chokumnoui
2, and Eri were 1.03±0.02, 1.11±0.03, 1.10±0.02, and
1.88±0.14 mg/mL, respectively. For water extracts, the
EC50 values of Chokumnoui 1, Nangnoi, Chokumnoui 2,
and Eri were 0.96±0.02, 1.05±0.02, 1.04±0.02, and
7.59±0.12 mg/mL, respectively. The highest mean EC50
value was found in water extract of Chokumnoui 1
(0.96±0.02 mg/mL). Thus, Chokumnoui 1 exhibited the
highest ability of free radical scavenging activity among
the studied silk strains. The results of ANOVA analysis
reveal a significant difference (p<0.05) between the mean
of EC50 values of water and ethanolic extracts of
Chokumnoui 1. Water extracts of Chokumnoui 1 exhibited
the higher ability of free radical scavenging activity than
ethanolic extracts. Water and ethanolic extracts of cocoon
shells of the non-mulberry silkworm, Eri, in which had
mean EC50 values of 7.59±0.12 and 1.88±0.14 mg/mL,
Phenolic Composition of Thai and Eri Silk
Table 5. Antioxidant activity of water and ethanolic silk sericin extracted
DPPH assay (EC50, mg/mL)
Silk strains
Ethanol Water
bA1) bB
Nangnoi 01.11±0.03 1.05±0.02
Chokumnoui 1 1.03±0.02cA 0.96±0.02cB
Chokumnoui 2 1.10±0.02bA 1.04±0.03bB
Eri 1.88±0.14aA 7.59±0.12aB
1)
Mean of triplicate measurements±SD; Means not sharing a lowercase within each column or capital letter within each row are significantly
different at p<0.05 as measured by the DMRT.
respectively, showed the lower ability as DPPH radical
scavengers than the non-mulberry silkworm. However, the
finding presented no significant difference (p>0.05) in the
means of free radical scavenging activity between water
and ethanolic extracts of Nangnoi (yellow cocoon) and
Chokumnoui 2 (white cocoon).
In this study, the antioxidant activity is also determined
on the basis of the ability of antioxidant in these SS
extracts to reduce ferric (III) iron to ferrous (II) iron in
FRAP reagent (31). Generally, FRAP assay was used due
to its simplicity and reproducibility. The ferric reducing
ability (FRAP assay) is widely used in the evaluation of the
antioxidant component in dietary polyphenols (18,24). The
comparison of the FRAP activity of water and ethanolic
extracts of silk cocoons is shown in Table 5. The FRAP
values of ethanolic extracts of Chokumnoui 1, Nangnoi,
Chokumnoui 2, and Eri were 6.21±0.18, 3.93±0.07,
2.65±0.18, and 0.34±0.01 mg Fe(II)/g respectively. For
water extracted, the values of Chokumnoui 1, Nangnoi,
Chokumnoui 2, and Eri were 8.03±0.36, 6.83±0.56,
5.09±0.07, and 2.75±0.05 mg Fe(II)/g, respectively. The
highest FRAP activity was found in water extract of
Chokumnoui 1. Thus, Chokumnoui 1 exhibited significantly
(p<0.05) highest antioxidant activity among the studied
silk strains. The results of ANOVA analysis reveal a
significant difference (p<0.05) between the FRAP activity
of water and ethanolic extracts of studied silk strains. Water
extracts of Chokumnoui 1, Nangnoi, Chokumnoui 2, and
Eri exhibited a higher ability of FRAP activity than
ethanolic extracts. Water and ethanolic SS extracts of the
non-mulberry silkworm Eri, which had mean values of
2.75±0.05 and 0.34±0.01 mg Fe(II)/g, respectively, showed
the lower ability as DPPH radical scavengers than the non-
mulberry silkworm. In addition, the result demonstrated
that antioxidant activity of SS extracts by FRAP assay had
similar trend with DPPH assay.
One of the most commonly used organic radicals for the
evaluation of antioxidant efficiency of pure compounds,
and a complex mixture, is the radical cation derived from
ABTS. These radical cations could be generated by
enzymatic, chemical, and electrochemical means. The
quench of ABTS radical cations by polyphenols is the
395
FRAP assay (mg Fe(II)/g) ABTS• + (mg TEAC/g)
Ethanol Water Ethanol Water
bB bA bA
3.93±0.07 6.83±0.56 1.19±0.04 1.42±0.01bB
6.21±0.18aB 8.03±0.36aA 1.41±0.06aA 1.73±0.03aB
2.65±0.18cB 5.09±0.07cA 1.04±0.02cB 1.23±0.03bA
0.34±0.01dB 2.75±0.05cA 1.00±0.05cA 1.14±0.04cA
criteria used to assess their relative antioxidant capacity. In
these ABTS• +-based methods, it is assumed that the
antioxidants simply reduce the radicals back to the parent
substrate, ABTS• + (32). To save time and reagents, a
microplate reader was used to measure total antioxidant
activity of the fluid samples in this experiment. All samples
could be measured simultaneously, and it required only 1/
3 the amount of reagents as compared to the conventional
spectrophotometric method. The antioxidant activity
measurements of the water and ethanolic extracts from
Chokumnoui 1, Nangnoi, Chokumnoui 2, and Eri, expressed
as Trolox equivalent antioxidant capacity (TEAC) are
presented below. All extracts show antioxidant activities
proving their capacity to scavenge the ABTS radical
cation. ABTS• + scavenging ability of ethanolic extracted
from Chokumnoui 1, Nangnoi, Chokumnoui 2, and Eri
was 1.41±0.06, 1.19±0.04, 1.04±0.02, and 1.00±0.05 mg
TEAC/g, respectively. ABTS• + scavenging ability of water
extracted from Chokumnoui 1, Nangnoi, Chokumnoui 2,
and Eri was 1.73±0.03, 1.42±0.01, 1.23±0.03, and 1.14±
0.04 mg TEAC/g, respectively (Table 5). These quantities
are regarded as moderate content, compared to the amount
of ABTS•+ values (in mg TEAC/g) in other vegetables such
as sugar beet root (2.03±0.07), red cabbage leaf (1.22±
0.23), edible rape leaf (0.71±0.10), lettuce leaf (0.45±
0.06), mungbean sprout (0.24±0.05), bitter melon fruit
(0.23±0.03), carrot root (0.17±0.02), and tomato fruit
(0.143±0.01) (33). In the present study, the highest ABTS+
scavenging ability was found in water extracted of
Chokumnoui 1. Thus, Chokumnoui 1 exhibited a significantly
(p<0.05) highest antioxidant activity among the studied
silk strains. These results showed that water extract exhibited
significantly (p<0.05) higher ABTS• + scavenging activity
than ethanolic extracted.
Correlation between total phenolic and flavonoid
contents and antioxidant activity Antioxidant activity
is found to be linearly proportional with phenolic content.
Oktay et al. (34) reported a strong positive relationship
between total phenolic content and antioxidant activity,
which appears to be the trend in many plant species. The
relationship between total phenolic content and antioxidant
396
Fig. 2. Correlation between total phenolic content and antioxidant activity of water and ethanolic silk sericin extracted.
Fig. 3. Correlation between total flavonoid content and antioxidant activity of water and ethanolic silk sericin extracted.
properties of many plants (e.g., common vegetables, fruits,
spices, and medicinal herbs) has been investigated in
previous studies (31,35), and reported that phenolic
compounds in many plants significantly contributed to
their antioxidant properties. Figure 2 shows the correlation
between total phenolic content and DPPH assay, total
phenolic content and FRAP assay, and total phenolic
content and ABTS• + assay of water and ethanolic extracted
from Chokumnoui 1, Nangnoi, Chokumnoui 2, and Eri.
Correlation between DPPH radical scavenging activity (Y)
and total phenolic content (X) was established as an
equation (Y=0.0009X+0.5528), and a highly significant
linear correlation (R2 =0.838) was obtained. Correlation
between ferric reducing ability (FRAP assay) (Y) and total
phenolic content (X) was established as an equation
(Y=0.0114X+1.3749), and a high significant linear
correlation (R2=0.706) was obtained. Correlation between
ABTS• + radical scavenging activity (Y) and total phenolic
content (X) was established as an equation (Y=
0.0011X+0.9834), and a high significant linear correlation
(R2 =0.674) was obtained. Results show a positive
correlation coefficient between the total phenolic content
and DPPH assay (r=0.92), total phenolic content and
FRAP assay (r=0.84), and total phenolic content and
ABTS• + assay (r=0.82) of water and ethanolic extracted
from studied silk strains which is highly significant
(p<0.05). Correlation between total flavonoid content and
Butkhup et al.
DPPH assay, total flavonoid content and FRAP assay, and
total flavonoid content and ABTS•+ assay of water and
ethanolic extracted from Chokumnoui 1, Nangnoi,
Chokumnoui 2, and Eri were obtained (Fig. 3). Correlation
between DPPH radical scavenging activity (Y) and total
flavonoid content (X) was established as an equation (Y=
0.0049X+0.4249), and a highly significant linear
correlation (R2 =0.736) was obtained. Correlation between
ferric reducing ability (FRAP assay) (Y) and total flavonoid
content (X) was established as an equation (Y=0.0771X−
1.4038), and a high significant linear correlation (R2 =
0.959) was obtained. Correlation between ABTS•+ radical
scavenging activity (Y) and total flavonoid content (X) was
established as an equation (Y=0.0072X+0.72), and a high
significant linear correlation (R2 =0.938) was obtained.
Results show a positive correlation coefficient between the
total flavonoid content and DPPH assay (r=0.86), total
flavonoid content and FRAP assay (r=0.98), and total
flavonoid content and ABTS• + assay (r=0.97) of water and
ethanolic extracted from the studied silk strains which is
highly significant (p<0.05). The increased antioxidant
activity of SS extracted from silk strains is related to an
accumulation of phenolics in the cocoon shells. Phenolic
compounds from the dietary mulberry leaves are modified
within the silkworm and accumulated in the silk fiber. The
accumulation of phenolics in the cocoon shells is
responsible for the antioxidant properties of mulberry
Phenolic Composition of Thai and Eri Silk
silkworm cocoons (2,12). A significant and linear
relationship existed between the antioxidant activity and
phenolic content of SS extracted thus indicating that
phenolic compounds are major contributors to antioxidant
activity.
Such high R2 value suggested that the DPPH and ABTS
radical scavenging activity and FRAP activity could be
credibly predicted on the basis of the Folin-Ciocalteu assay
for total phenolic content or a colorimetric assay for
flavonoid content and directly confirmed that the phenolic
compounds in the SS extracts were responsible for their
antioxidant capacity. The results emphasized the importance
of phenolic compounds in the antioxidant behavior of SS
extracts and also indicated that the phenolic compounds
contributed significantly to the total antioxidant capacity.
In conclusion, the SS extracted with water by autoclave
(121oC, 103.5 kPa, 60 min) resulted in significant (p<0.05)
gains in the sum of phenolic compounds, total phenolic
content, total flavonoid content, and antioxidant activities.
Different silk strains contain SS with various phenolic
compositions, which are significantly influenced by the
extraction method used. Phenolics are found in higher
content in the mulberry silk cocoons (Chokumnoui 1,
Nangnoi, and Chokumnoui 2) than in non-mulberry silk
cocoon (Eri). Among the phenolics studied, (+)-catechin
and quercetin were the major flavonoid presented in the
extracted SS. The SS extracted from Chokumnoui 1 had
the highest content in the sum of phenolic compounds, total
phenolic content, total flavonoid content and antioxidant
activities. The results obtained demonstrated that the SS
extracts with high antioxidant activity exhibited relatively
high total phenolic content and total flavonoid content.
There was good correlation between total phenolic content,
total flavonoid content, and antioxidant activity (DPPH,
ABTS, and FRAP assays) that supports the idea of
phenolics as contributor of the antioxidant power of SS
extracts. The SS extracted by water at a high-temperature
and high-pressure was superior. It is useful as medicinal or
cosmetic material for all antioxidant properties studied.
Water extraction appears to be a sound method for
producing SS extracted from silk cocoon shells. Due to its
operational cost, water extraction can be applied to produce
high-value SS.
Acknowledgments The authors would like to thank the
Office of the Higher Education Commission (OHEC) for
the financial support (OHEC grant-2554A10952003) for
this research. We also thank Mr. Paul Alexander Dulfer
from the Division of Research Supporting and Extension
Services, Mahasarakham University, for reading the
manuscript, suggestions, and corrections.
397
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