Tasar silk fiber waste sericin: New source for anti ...

International Journal of Biological Macromolecules 114 (2018) 1102–1108

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Tasar silk fiber waste sericin: New source for anti-elastase, anti-tyrosinase and anti-oxidant compounds K. Jena ⁎, J.P. Pandey, Ruchi Kumari, A.K. Sinha, V.P. Gupta, G.P. Singh Silkworm Physiology Laboratory, Central Tasar Research and Training Institute (Central Silk Board), P.O. Piska-Nagri, Ranchi 835303, Jharkhand, India

a r t i c l e

i n f o

Article history: Received 25 October 2017 Received in revised form 9 March 2018 Accepted 12 March 2018 Available online 14 March 2018 Keywords: Silk waste Sericin Anti-elastase Anti-tyrosinase Antioxidant

a b s t r a c t The present study investigates the properties of sericin extracted from tasar silk fiber waste (TSFW). The surface morphology of TSFW was observed by scanning electron microscope (SEM). SEM images revealed the removal of residual sericin over the surface of TSFW. The molecular weight distribution of sericin was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results suggested that TSFW sericin represented a family of proteins with wide-ranging molecular weight distribution (11–245 kDa). Structural determination by FTIR revealed the presence of both α-helical and β-sheet structures. The colour was studied by colorimeter indicating less brightness, more red and yellow colour intensities. The carbon: nitrogen ratio (C:N) was studied by CHNS element analyzer and the ratio is 5.15-7.85. Thermal properties of TSFW sericin have been studied by thermogravimetric analysis (TGA) method. TGA curve showed higher thermal stability and variable degradation profiles. Furthermore, TSFW sericin contains 17 amino acids where serine, aspartic acid and glycine are the more significant compounds (54.34–60.49%). In addition, sericin was found to inhibit tyrosinase, elastase and glutathione-S-transferase activity, and had apparent radical scavenging impacts on 2.2 diphenyl 1 picryl hydrazil (DPPH), hydrogen peroxide and inhibition of lipid peroxidation. Result suggested that TSFW sericins might be a valuable ingredient for cosmoceutical products. © 2018 Published by Elsevier B.V.

1. Introduction Silk sericin and fibroin are natural macromolecular proteins derived from the cocoons of silkworm and are synthesized in the silkworms middle and posterior regions of silk gland, respectively. Sericin envelops the fibroin fiber with successive adhesive layers and ensures the cohesion of the cocoon by gluing the silk threads together. Sericin consists of 18 kinds of amino acids; most of which have strong polar side groups. It exhibits various biological functions that make it an ideal component for cosmetic and pharmaceutical field. Chlapanidas et al. [1] found that sericin has ROS-scavenging, anti-tyrosinase and anti-elastase activity, and suggested sericin is an ideal constituent for cosmetic industry. Masahiro et al. [2] reported consumption of sericin enhances the bioavailability of Zn, Fe, Mg and Ca in rats, and suggested that sericin is a valuable natural ingredient for the food industry. Furthermore, hydrophilic natures, activation of collagen production, promotion of attachment and proliferation of fibroblast, osteoblast and keratinocytes of ⁎ Corresponding author at: Central Tasar Research & Training Institute, Nagri, Ranchi 835303, Jharkhand, India. E-mail address: [email protected] (K. Jena).

https://doi.org/10.1016/j.ijbiomac.2018.03.058 0141-8130/© 2018 Published by Elsevier B.V.

sericin have also been reported by several researchers [3–6]. In addition, sericin was found to have anti-tumoral action against colon cancer [7], anticoagulants [8] and cryoprotective [9]. These findings implied that sericin is a valuable component for pharmaceutical industry. Tasar silk is the one of the wild silk exclusively found in India. During the process of reeling, about 30–40% of fiber goes into waste [10]. The fiber wastes were deflossing waste (generated before reeling process for easy operation), reeling waste (feeding after exhaustion and mending breakage) and basin waste (left over residues i.e., unreelable innermost shell layer). In India, the total production of tasar silk during 2016–17 was 3268 MT [11], which produces about 980.4 to 1307.2 MT of TSFW. Fiber wastes are used for producing spun silk and fabrics [10]. In addition, silk effluents contain sericin which is a valuable protein and can be used in several industries such as cosmetics, pharmaceuticals and preparation of biomaterials. Although the extraction of sericin from the mulberry silkworm (Bombyx mori) cocoons is well established; but the extraction of sericin from TSFW has still not been investigated properly. There is also ample of scope to utilize the residual wastes of tasar silk by extracting sericin from silk polymer, which would provide significant benefits and lead to the value-added utilization. In the present study, an attempt has

K. Jena et al. / International Journal of Biological Macromolecules 114 (2018) 1102–1108

1103

been made to characterize the properties of TSFW sericin which may be helpful in exploiting it as a potential biomaterial.

proteins covering wide range molecular weights from 11 to 245 kDa (HiMedia Chemicals, India) were used as standard proteins.

2. Materials and methods

2.4.6. Amino acid analysis Amino acid profiling of sericin samples was determined through Ultra Performance Liquid Chromatography (UPLC) at Sandor Life Sciences Pvt. Ltd, Hyderabad.

2.1. Materials TSFW were collected from Post Cocoon Technology Section, Central Tasar Research and Training Institute, Ranchi, Jharkhand, India. 2.2. Extraction of sericin After collection, the fiber wastes were washed with warm water (40 °C for 5–10 min) for removal of contaminants and then dried properly. Sericin was extracted by boiling the fiber waste with alkali solution (0.2% sodium carbonate) using an autoclave at 120 °C for 1 h. The extracted solution was filtered through Whatmann No. 1 filter paper and concentrated to 1/3rd of its volume at 60 °C. The concentrated solution was mixed with ethanol (1:3) and stored at −20 °C for overnight. The mixed solution was centrifuged at 10000 rpm for 15 min and sericin obtained as precipitant was lyophilized as powder. 2.3. Scanning electron microscopy imaging To obtain scanning electron microscopy (SEM) images of TSFW, the fiber samples were gold coated and observed by using SEM (JEOL JSM6390LV) with incident electron beam energy of 10 kV at the Birla Institute of Technology (BIT), Mesra, Ranchi. 2.4. Physico-chemical and biological properties 2.4.1. FTIR spectral analysis The FTIR spectra of sericin samples were recorded on FTIR spectrophotometer (IR-Prestige-21, at BIT) in order to determine the functional group present in the TSFW sericin. The IR spectra were obtained in the spectral region of 400–4000 cm−1. 2.4.2. Thermogravimetric analysis (TGA) The thermal properties of the samples were measured in a TGA (SDT Q 600, TA Instruments, USA, at Karnataka University, Dharwad) under a nitrogen gas flow of 100 ml min−1. The samples were heated at 10 °C min−1 from 30 °C to 1000 °C. 2.4.3. Elemental composition of recovered sericin To ascertain the elemental composition of samples, the weight percent of carbon (C), hydrogen (H) and nitrogen (N) elements of sericin samples was determined on dry basis using Vario EF III elemental analyzer at BIT, Mesra, Ranchi. 2.4.4. Colour Powder colour was measured using a Color Flex Colorimeter, where any colour can be represented by three colour variables: L*, a* and b*. L* is a measure of brightness from black (L* = 0) to white (L* = 100). The a* value ranged from −60 (green) to 60 (red) and the b* value ranged from −60 (blue) to 60 (yellow). 2.4.5. Molecular weight determination The SDS-PAGE analysis of sericin was performed according to Laemmli [12]. SDS-PAGE of sericin was studied by using 5% stacking gel and 10% separating gel. Samples were prepared in Tris-HCl buffer (pH 6.8) containing 10% SDS, 0.5 mL β mercapto ethanol, 20% glycerol and 1% bromophenol blue. The gel was run in a vertical slab-gel electrophoresis apparatus at 110 V for 1 h by Dual Mini Slab Kit (Mini PROTEAN Bio-Rad). The molecular weight of the peptide bands was detected through Silver stains. A protein standard with pre-stained

2.4.7. Biological properties Free radical scavenging potential was measured by 2,2 diphenyl 1 picry hydrazil (DPPH) following the method of Blois [13]. Tyrosinase-inhibition activity of the TSFW sericin was assayed as per the method of Aramwti et al. [14]. The H2O2 scavenging power of extract was estimated following the method of Mukhopadhyay et al. [15]. Elastase inhibition activity was determined as per the method of Nam et al. [16]. Glutathione-S-transferase was determined according to the method of Habig et al. [17]. Lipid peroxidation, induced by FeCl3 system, was estimated as thiobarbituric acid reacting substances [18]. Results are expressed as mean ± standard deviation (SD). Difference among the mean of doses were analyzed by Students t-test. Differences were considered statistically significant when P b 0.05. 3. Results and discussion In India, the production of TSFW was about 980.4 to 1307.2 MT during 2016–17. There is ample of scope for utilization of this waste for industrial application. A detailed study was conducted in order to isolate the tasar sericin for its potential use and demand in local as well as the international scenario. The degumming ratio was determined from the weight of fiber after degumming comparing with initial fiber weight. The degumming percentage was 2.00 to 2.79% (Fig. 1a). For further elucidation, SEM study was conducted to confirm the removal of sericin from TSFW. The results confirm the removal of residual sericin from fiber waste (Fig. 1b). The elemental profile (nitrogen, carbon, hydrogen and sulfur) of sericin samples are given in Table 1. As seen from Table 1, elemental compositions of standard sericin (B. mori) were 14.61% nitrogen, 40.45% carbon, 6.652% hydrogen and 0.872% sulfur. However, for TSFW sericin, carbon content was about 19.33–22%, nitrogen 2.461–4.273%, sulfur 0.6–1.04% and hydrogen 2.618–3.377% (Table 1). Earlier studies have classified sericin of cocoon shell into two classes: α sericin and β sericin. The outer cocoon shell is made of α sericin while inner layer of β sericin. Solubility of α-sericin in hot water is higher than β sericin [19]. In the present study, the low solubility was recorded for TSFW sericin when compared with standard sericin (B. mori), which may be due high CN ratio (5.15-7.85). The amino acid composition of recovered TSFW sericin was compared with standard sericin (B. mori). Serine, aspartic acid and glycine contents of standard sericin were about 64.63% of the whole amino acid composition which are in conformity with the earlier reports of 64.3% in B. mori cocoon white shell [14]. However, in case of TSFW sericin, the amount was about 55.99, 60.49 and 54.34% in basin, deflossing and reeling waste sericin, respectively (Table 2). In the present analysis, the polar uncharged amino acid for standard sericin (B. mori) was about 49.58%; however, in TSFW, the amount of polar uncharged amino acid was 25.21–29.88% (Table 2) and in waste water, the amount was 36.3% [20]. The non-polar amino acid was 20.95, 27.78, 26.11 and 25.84% for standard sericin, basin, deflossing and reeling waste sericin, respectively and 22.3% in waste water [20]. The aromatic amino acid was 5.95% in std. sericin and 3.92-7.32% in TSFW sericin (Table 2) and 6.6% was reported in silk waste water [20]. The positive charges amino acid was 9.08 in standard sericin and 8.8–10.8% in TSFW sericin. Similarly, the negative charged amino acid was 14.43, 32.06, 28.98 and 29.56% for standard sericin, basin, deflossing and reeling tasar waste sericin, respectively and 26% in waste water [20]. Further, the difference

1104


Degumming ratio (%)

4

3

2

1

0 Basin waste

Reeling waste

Deflossing waste

b

A

B

C

D

E

F

Fig. 1. a. Degumming ratio of fiber waste samples. b. Scanning Electronic Microscopy (SEM) of degummed fiber waste samples, before (A-basin, B-reeling and C-deflossing) and after (Dbasin, E-reeling and F-deflossing) the extraction with Na2CO3 0.2% at 120 °C.

in amino acid composition might be due to the species specificity, purification process as well as the location of the protein. The UV–Vis absorbance spectra of standard sericin and TSFW sericin were determined by wave length scanning from 220 to 350 nm. The maximal absorption of sericin was observed at 220 nm and another minor peak was obtained between 274 and 279 nm. The results were in accordance with Caper and Aygun [21]. However, for standard sericin, clear distinct peak was observed between 275 and 280 nm (Fig. 2a), which is associated to the aromatic amino acid absorption wavelength [20]. Although the exact size of sericin protein is difficult to estimate precisely, it has been reported within the molecular weight (MW) ranging from 20 to 400 kDa [22]. The present study is the first report about the constituent of A. mylitta fiber waste sericin. Broad smear of sericin proteins (11–245 kDa) were observed in all three TSFW sericin samples (Fig. 2b). Dash et al. [23] reported sericin bands of 200 kDa and N200 kDa in the cocoons of A. mylitta. The result indicates the breaking of peptide bonds of sericin at various positions leading to peptide fragmentation with broad range of molecular weight (MW). As reported, the sericins which have been classified as high MW are suitable for making bio-materials and membranes, and low MW are suitable for cosmetic applications [24]. The TSWF sericin has wide MW protein which can be used for both biomaterials as well as cosmetic industry. Table 1 Comparison of elemental composition of tasar fiber waste sericin. Samples

N

C

S

H

Basin waste Deflossing waste Reeling waste Std. sericin (B. mori) Literature Caper and Aygun (2015)

3.13 4.27 2.46 14.61 10.2–16.8

21.48 22 19.33 40.45 36.7–45.3

1.04 0.71 0.6 0.87 –

3.02 3.38 2.62 6.65 5.4–8.8

Table 2 Amino acid profiling of tasar fiber waste sericin. Polar uncharged

Standard sericin (B. mori)

Tasar silk fiber waste Basin

Diflossing

Reeling

Threonine Serine Proline Cysteine Asparagine Gultamine

8.45 40.51 0.59 0.03 ND ND

6.34 15.93 2.94 ND ND ND

6.95 19.45 1.96 ND ND ND

8.46 18.07 3.35 ND ND ND

Non-polar Glycine Alanine Valine Leucine Isoleucine Methionine

12.60 3.28 3.56 1.05 0.34 0.13

16.90 5.20 2.27 2.19 0.80 0.42

19.60 3.68 0.99 1.23 0.43 0.18

15.89 4.10 2.53 2.05 0.99 0.28

Aromatic Phenylalanine Tyrosine Tryptophan

0.53 5.42 ND

1.51 4.64 ND

0.77 6.55 ND

1.35 2.56 ND

Positively charged Lysine Histidine Arginine

2.33 2.05 4.71

1.70 2.86 4.24

1.18 2.91 5.15

1.71 3.44 5.65

Negatively charged Aspartic acid Glutamic acid

11.52 2.91

23.16 8.90

21.44 7.53

20.38 9.18


R D B SS

3

Absorbance

1105

2

1

0 228

246

264

282

300

318

336

354

372

390

b 245 180 135 100 75 63 48 35

Weight loss (%)

100

b

80 60 40

R B D SS

20 0 0

100

200

25 20 11

D

B

R

M

Fig. 2. a. UV–Vis absorption spectra of sericin samples (R-reeling waste, D-deflossing waste, B-basin waste and SS-standard sericin). b. Molecular weight of fiber waste sericin (D-Deflossing waste sericin, B-basin waste and R-reeling waste and M-Molecular marker).

The FTIR spectra of various sericin samples are presented in Fig. 3a. Amide peaks are involved in a polypeptide backbone and therefore are sensitive to the structure and orientation of protein. Sericin extracted from TSFW showed absorption between 1651 and 1654 cm−1 confirming amide I (C\\O stretching) absorption and is the most useful peak for determining secondary structure of proteins. Amide II (1540– 1580 cm−1) absorption contains contributions from N\\H bending; in this case the amide II peak was evident around 1458.18–1462.04 cm−1. Amide II peak seems to have shifted in TSWF sericin samples, suggesting the conformational change occurred. However, FTIR spectra of sericin purified from Antheraea assamensis shows peak at 1660 (for amide-I) and 1540 (for amide-II) [25]. To identify the thermal stability of TSFW sericin, TGA was performed. The thermal degradation of TSFW sericin found to occur in 4 stages (Fig. 3b), characterized by evident mass loss rates. The initial weight loss (1st stage) was detected below 116 °C, which is attributed to the evaporation of water, and is followed by low weight loss (2nd stage) from 116 °C to 250 °C. The low mass loss observed during this stage is attributed to the loss of low temperature volatile compounds. In the next stage (3rd stage), from 250 to 600 °C, TSFW sericin weight loss reached about 75–40%. However, for std. sericin (B. mori) the major thermal degradation was observed at 200–400 °C, clearly suggesting that the thermal stability of B. mori sericin in high temperature regions is weaker than that of TSFW sericin. Similarly, as compared with B. mori sericin higher thermal stability of A. mylitta sericin was observed by Sahu et al. [26]. The fourth stage is characterized by a mass loss about 65 to 30%. The evident mass losses from second to fourth stage is attributed to the breakdown of side chain groups of amino acid residue as well as the cleavage of peptide bonds [27].

300

400

500

600

700

800

900

Temperature (OC)

Fig. 3. a. FTIR Spectra of different silk fiber waste sericin (R-reeling waste, B-basin waste, D-deflossing waste, and SS-standard sericin). b. TGA thermograms of sericin powder recovered from different silk fiber waste (R-reeling waste, B-basin waste, D-deflossing waste, and SS-standard sericin).

Colour values (L*, a* and b*) of TSWF sericin powders are shown in Table 3. Colour parameters from spectra were calculated by using the colorimeter. The L* value indicates brightness ranging from black (L* = 0) to white (L* = 100). The L* value ranged from −60 (green) to 60 (red) and the b* value ranged from −60 (blue) to 60 (yellow). However, sericin generated from the TSWF showed low L* values and high a* and b* values as compared to earlier reports on B. mori sericin [14], indicating less brightness, more red and yellow colour intensities in TSWF sericin (Table 3). Tasar silkworm host plant contains higher phenolic and tannins (earlier observations) which might be responsible for colour variation between the sericins from B. mori and A. mylitta. Melanin is responsible for the natural colour of human skin and hair. However, various dermatological disorders result in excessive accumulations of epidermal melanin which is known as melanogenesis. The process is inhibited by avoiding ultraviolet (UV) exposure, tyrosinase inhibition and/or inhibiting melanocyte metabolism and proliferation [28–30]. However, the majority of skin-lightening agents used tyrosinase inhibitors to inhibit the accumulation of melanin [31]. Tyrosinase is a copper-containing enzyme and plays a central role in melanogenesis. In vitro mushroom tyrosinase inhibition assay is commonly used to assess the direct effect of a given skin lightener on tyrosinase activity [32]. In the present study, TSFW sericin confirms anti-tyrosinase activity (Fig. 4a), and the results are corroborate with earlier finding of Aramwati et al. [14] and Chlapanidas et al. [1]. The anti-tyrosinase activity may be due to the presence of various amino acids and/or flavonoids Table 3 Comparison of colour of tasar fiber waste sericin. Samples

L

a*

b*

Basin waste Deflossing waste Reeling waste B. mori [14]

42.31 48.31 47.925 84.83–94.43

7.72 7.105 8.095 −4.02–0.41

15.09 19.425 20.885 3.34–15.53

1106

K. Jena et al. / International Journal of Biological Macromolecules 114 (2018) 1102–1108 1mg/ml 2mg/ml

75 * 50

*

25

0.125mg/ml 0.25mg/ml

100 Inhibition of GST activity (%)

Inhibition of tyrosinase activity(%)

100

75

50 25 0

0 TBA (100µg)

Basin waste

Deflossing

TA(0.25mg/ml)

Reeling waste

Basin waste

Deflossing waste

b 75 60

*

45

* *

30

0.5mg/ml 1mg/ml

100

0.5mg/ml 1mg/ml

DPPH Scavenging potential (%)

Inhibition of elastase activity (%)

b

Reeling waste

75

50

25

15 0 BHT (100µg/ml)

0 Deflossing

Basin waste

Deflossing

Reeling waste

Reeling waste

Fig. 4. a. Inhibition of tyrosinase activity of fiber waste sericins. Data expressed as mean ± SD (n = 3). Denote indicates significant difference between doses *P b 0.05. b. Inhibition of elastase activity of fiber waste sericins. Data expressed as mean ± SD (n = 3). Denote indicates significant difference between doses *P b 0.05.

and carotenoids, as their removal induces a decrease in the biological property as reported by Aramwit et al. [33]. Elastase is a proteolytic enzyme involved in the degradation of the extracellular matrix (ECM) that includes elastin. Elastin provides much of the elastic recoil properties of skin, arteries, lungs and ligaments [34]. Degradation of elastin is a major visible sign of ageing in skin. As shown in the Fig. 4b, TSFW sericin shows anti-elastase activity. In agreement with our results, Chlapanidas et al. [1] have also reported the anti-elastase activity of sericin. In the present study, the variation of inhibitory activity may be the variation of amino acids or presence of various phenolic compounds in sericin of polyphagus insect [35]. GSTs are a group of detoxification enzymes, mainly localized in the cytosol which catalyzes the conjugation of GSH with a wide range of metabolites bearing electrophilic sites [17]. In the present study, inhibition of GST activity was observed on exposure of TSFW sericins (Fig. 5a). Similarly, the inhibitory effects of plant phenolics against GST activity were also reported by various authors [36,37]. Further, inhibition of GST activity was also observed during in vitro exposure of tannic acid as a positive control (Fig. 5a). The search for novel antioxidants with GST inhibitory capacity has become an important issue because of their role in tumor cells [38,39]. DPPH is a relatively stable free radical and the assay determines the ability of extract to reduce DPPH• to the corresponding hydrogen by converting the unpaired electrons to form pairs, and the solution loses colour stoichiometrically depending on the number of electrons taken up [13]. The decreasing intensity of colour is directly proportional to the inhibition of DPPH. The present study shows the increasing concentration of the extract inhibiting the activity of DPPH. The maximum inhibition was noticed at 1 mg/ml (Fig. 5b). Similar findings were reported by Takechi et al. [40] on free radical scavenging potential of sericin. Scavenging potentials are attributed to the composition of its amino acids, hydroxyl groups of serine and threonine [41], the delocalizing electrons of aromatic acids [42] and the associated polyphenols (secondary metabolites) contributed to its distinct antioxidant (AO) properties.

Fig. 5. a. Inhibition of glutathione-S-transferase activity of fiber waste sericins. Data expressed as mean ± SD (n = 3). b. DPPH scavenging potential of fiber waste sericins. Data expressed as mean ± SD (n = 3).

Hydrogen peroxide is an important reactive oxygen species because it can react with metals such as Fe2+ or Cu2+ as well as superoxide anions in the Haber-Weiss reaction, producing highly reactive hydroxyl radicals [43]. The present study proves the H2O2 scavenging potential (Fig. 6a), while standard (ascorbic acid) showed more effective capacity to scavenge H2O2 due to electron donor which accelerates the conversion of H2O2 to H2O. Similarly, sericin extracted from A. mylitta having H2O2 scavenging potential was also reported by Dash et al. [44]. 2mg/ml 4mg/ml

90

H2O2 Scavenging potential (%)

Basin waste

60 *

30

0 ASA

Basin waste

Deflossing waste

**

*

Reeling waste

b 75

Inhibition of lipid peroxidation (%)

ASA (100µg)

0.5mg/ml 1mg/ml

50

25

0 ASA (100µg)

Basin waste

Deflossing waste

Reeling waste

Fig. 6. a. Hydrogen peroxide scavenging potential of fiber waste sericins. Data expressed as mean ± SD (n = 3). Denote indicates significant difference between doses *P b 0.05. b. Inhibition of lipid peroxidation by fiber waste sericins. Data expressed as mean ± SD (n = 3). Denote indicates significant difference between doses *P b 0.05 and **P b 0.01.


Oxidation of lipids affects the membrane fluidity, reduction potential, increased permeability to ions and leakage of cell molecules. Hence, lipid peroxidation can play crucial role in various diseases such as inflammation, cancer and cardiac diseases [45]. Antioxidant supplementation constitutes important defense against various oxidative stress-related diseases. Our investigations indicated that TSFW sericin inhibited lipid peroxidation (Fig. 6b). Decrease in LPX by test sample is probably due to the scavenging action of OH• radicals, H2O2, other free radicals and/or the reducing potential of the extract. Qian et al. [46] suggested that antioxidative activity of peptide or protein is dependent on various factors i.e., molecular size, chemical properties such as hydrophobicity and electron transferring ability of amino acid residues in the sequence. The data presented in Table 3 indicated that the amino acid compositions of TSFW sericin were different from standard sericin (B. mori). The hydrophobic and aromatic amino acid contents of TSWF sericin were higher than those of standard sericin (B. mori). Earlier report confirmed that the higher contents of hydrophobic and aromatic amino acids facilitated the radical scavenging and metal chelating activities of protein [47]. The hydrophobic amino acids facilitate interactions with hydrophobic targets such as the cell membrane, and thereby enhance the bioavailability [48], protect against macromolecular oxidation by donating photons to reactive radicals [49] and facilitate greater interactions between the peptide and fatty acids [50]. Furthermore, aromatic amino acids increase the antioxidant activities of peptides and protein due to easily donated protons to electron-deficient radicals, maintain their stabilities via resonance structures and enhance their radical scavenging activities [47,51,52]. Therefore, the variation of amino acid contents might be responsible for higher antioxidant activity of tasar fiber waste sericin than standard sericin.

4. Conclusion It is concluded that 0.2% sodium carbonate removes residual sericin from tasar silk fiber wastes. There is no significant colour variation between different tasar silk fiber wastes. The recovered sericin had a wide range of molecular weight 11–245 kDa, which is classified as both low and high-molecular weight. The higher C:N ratio and thermal stability of residual sericin had wide variation with standard sericin. This was attributed to the fact that recovered powder was residual sericin. The variation of amino acid contents might be accountable for free radical scavenging potential, anti-tyrosinase, anti-elastase and anti GST activity, and thus it can be used as a component of cosmetics as well as pharmaceutics. However, in vivo evaluations are needed for the development of natural source to formulate pharmaceutics and skin care cosmetics.

Acknowledgements The authors acknowledge the grant provided by the Department of Biotechnology (DBT), Ministry of Science & Technology, Government of India (BT/PR14004/TDS/121/7/2015). The authors thank Central Instrumentation Facilities Centre, Birla Institute of Technology, Mesra, Ranchi, University Science Instruments Center, Karnataka University, Dharwad and Sandoor Biotech, Hyderabad for instrumental analysis. References [1] T. Chlapanidas, S. Farago, G. Lucconi, S. Perteghella, M. Galuzzi, M. Mantelli, M.A. Avanzini, M.C. Tosca, M. Marazi, D. Vigo, M.L. Torre, M. Faustini, Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase and in vitro immunomodulatory activities, Int. J. Biol. Macromol. 58 (2013) 47–56. [2] S. Masahiro, Y. Hideyuki, K. Norihisa, Consumption of silk protein, sericin elevates intestinal absorption of zinc, iron, magnesium and calcium in rats, Nutr. Res. 20 (2000) 1505–1511. [3] P. Aramwit, A. Sangcakul, The effects of sericin cream on wound healing in rats, Biosci. Biotechnol. Biochem. 71 (2007) 2473–2477.

1107

[4] R. Dash, M. Mandal, S. Ghosh, S. Kundu, Silk sericin protein of tropical tasar silkworm inhibits UVB-induced apoptosis in human skin keratinocytes, Mol. Cell. Biochem. 311 (2008) 111–119. [5] S. Terada, T. Nishimura, M. Sasaki, H. Yamada, M. Miki, Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma, Cytotechnology 40 (2002) 3–12. [6] K. Tsubouchi, Y. Igarashi, Y. Takasu, H. Yamada, Sericin enhances attachment of cultured human skin fibroblasts, Biosci. Biotechnol. Biochem. 69 (2005) 403–405. [7] W. Kaewkorn, N. Limpeanchob, W. Tiyaboonchai, S. Pongcharoen, M. Sutheerawattananonda, Effects of silk sericin on the proliferation and apoptosis of colon cancer cells, Biol. Res. 45 (2012) 45–50. [8] M. Sano, Y. Tamada, K. Niwa, T. Morita, G. Yoshino, Sulfated sericin is a novel anticoagulant influencing the blood coagulation cascade, Aust. J. Biol. Sci. 20 (2009) 773–783. [9] J.H. Wu, S.Y. Wang, Y. Wu, Z.W. Wang, Cryoprotective effect of sericin enzymatic peptides on the freeze-induced denaturation of grass carp surimi, Appl. Mech. Mater. 140 (2012) 291–295. [10] D. Chattopadhyay, G. Mitra, R. Munshi, Modern techniques for utilisation of tasar silk wastes to convert yarn, Ind. J. Nat. Fibre 2 (2015) 41–49. [11] Annual Report. Central Silk Board, Ministry of Textiles, Govt. of India, 2016–17. [12] U.K. Laemmli, Cleavage of structural proteins during the assembly of head of bacteriophage T4, Nature 227 (1970) 680–685. [13] M.S. Blois, Antioxidant determinations by the use of a stable free radical, Nature 181 (1958) 1199–1200. [14] P. Aramwti, S. Damrongsakkul, S. Kanokpanont, T. Srichana, Properties and antityrosinase activity of sericin from various extraction methods, Biotechnol. Appl. Biochem. 55 (2010) 91–98. [15] D. Mukhopadhyay, P. Dasgupta, D.S. Roy, S. Palchoudhuri, I. Chatterjee, S. Ali, S.G. Dastidar, A sensitive in vitro spectrophotometric hydrogen peroxide scavenging assay using 1,10 phenanthroline, Free Radic. Antioxid. 6 (2016) 124–132. [16] K.A. Nam, S.G. You, S.M. Kim, Molecular and physical characteristics of squid (Todarodes pacificus) skin collagens and biological properties of their enzymatic hydrolysates, J. Food Sci. 73 (2008) C249–C255. [17] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S transferases, the first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130–7139. [18] H. Ohkawa, N. Ohisi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [19] F. Sadov, M. Korchagin, A. Matetsky, Chemical Technology of Fibrous Material, Mir Publication, Moscow, 1987. [20] J. Wu, Z. Wang, S.Y. Xu, Preparation and characterization of sericin powder extracted from silk industry wastewater, Food Chem. 103 (2007) 1255–1262. [21] G. Capar, S.S. Aygun, Characterization of sericin protein recovered from silk wastewaters, Turk. Hij. Den Biyol. Derg. 72 (2015) 219–234. [22] M.L. Gimenes, V.R. Silva, M.G.A. Vieira, M.G.C. Silva, A.P. Scheer, High molecular sericin from Bombyx mori cocoons: extraction and recovering by ultrafiltration, Int. J. Chem. Eng. Appl. 5 (3) (2014) 266–271. [23] R. Dash, S.K. Ghosh, D.L. Kaplan, S.C. Kundu, Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta, Comp. Biochem. Physiol. 147 (2007) 129–134. [24] Y.Q. Zhang, Applications of natural silk protein sericin in biomaterials, Biotechnol. Adv. 20 (2002) 91–100. [25] S. Dutta, R. Bharali, R. Devi, D. Devi, Purification and characterization of glue like sericin protein from a wild silkworm Antheraea assamensis Helfer, Glob. J. Bio-Sci. Biotechnol. 1 (2012) 229–233. [26] N. Sahu, S. Pal, S. Sapru, J. Kundu, S. Talukdar, N.I. Singh, J. Yao, S.C. Kundu, Nonmulberry and mulberry silk protein sericins as potential media supplement for animal cell culture, Biomed. Res. Int. 2016 (2016) 7461041. [27] I.C. Um, J.Y. Kweon, Y.H. Park, S. Hudson, Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid, Int. J. Biol. Macromol. 29 (2001) 91–97. [28] C. Cotellessa, K. Peris, M.T. Onorati, M.C. Fargnoli, S. Chimenti, The use of chemical peelings in the treatment of different cutaneous hyperpigmentations, Dermatol. Surg. 25 (1999) 450–454. [29] M. Seiberg, C. Paine, E. Sharlow, P. Andrade-Gordon, M. Costanzo, M. Eisinger, S.S. Shapiro, Inhibition of melanosome transfer results in skin lightening, J. Invest. Dermatol. 115 (2000) 162–167. [30] S.H. Park, D.S. Kim, W.G. Kim, I.J. Ryoo, D.H. Lee, C.H. Huh, S.W. Youn, I.D. Yoo, K.C. Park, Terrein: a new melanogenesis inhibitor and its mechanism, Cell. Mol. Life Sci. 61 (2004) 2878–2885. [31] A. Perez-Bernal, M.A. Munoz-Perez, F. Camacho, Management of facial hyperpigmentation, Am. J. Clin. Dermatol. 1 (2000) 261–268. [32] C. Madhosingh, L. Sundberg, Purification and properties of tyrosinase inhibitor from mushrooms, FEBS Lett. 49 (1974) 156–158. [33] P. Aramwit, S. Kanokpanont, T. Nakpheng, T. Srichana, The effect of sericin from various extraction methods on cell viability and collagen production, Int. J. Mol. Sci. 11 (2010) 2200–2211. [34] T.S.A. Thring, P. Hili, D.P. Naughton, Anti-collagenase, anti-elastase and anti-oxidant activities of extracts from 21 plants, BMC Complement. Altern. Med. 9 (2009) 1–11. [35] K.K. Lee, J.J. Cho, E.J. Park, J.D. Choi, Anti-elastase and anti-hyaluronidase of phenolic substance from Areca catechu as a new anti-ageing agent, Int. J. Cosmet. Sci. 23 (2001) 341–346. [36] M. Das, D.R. Bickers, H. Mukhtar, Plant phenols as in vitro inhibitors of glutathione Stransferase, Biochem. Biophys. Res. Commun. 120 (1984) 427–433. [37] A.R. Bartholomaeus, R. Bolton, J.T. Ahokas, Inhibition of rat liver cytosolic glutathione S-transferase by silybin, Xenobiotica 24 (1994) 17–24. [38] K. Zhang, N.P. Das, Inhibitory effects of plant polyphenols on rat liver glutathione Stransferases, Biochem. Pharmacol. 47 (1994) 2063–2068.

1108


[39] A.M. Gyamfi, I.I. Ohtani, E. Shinno, Inhibition of glutathione-s-transferases by thonningianin A, isolatedfrom the African medicinal herb, Thonningia sanguinea, in vitro, Food Chem. Toxicol. 42 (2004) 1401–1408. [40] T. Takechi, R. Wada, T. Fukuda, K. Harada, H. Takamura, Antioxidant activities of two sericin proteins extracted from cocoon of silkworm (Bombyx mori) measured by DPPH, chemiluminescence, ORAC and ESR methods, Biomed. Rep. 2 (2014) 364–369. [41] N. Kato, S. Sato, A. Yamanaka, H. Yamada, N. Fuwa, M. Nomura, Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity, Biosci. Biotechnol. Biochem. 62 (1998) 145–147. [42] J.B. Fan, L.P. Wu, L.S. Chen, X.Y. Mao, F.Z. Ren, Antioxidant activities of silk sericin from silkworm Bombyx mori, J. Food Biochem. 33 (2009) 74–88. [43] A.C.M. Filho, R. Meneghini, In vivo formation of single-strand breaks in DNA by hydrogen peroxide is mediated by Haber-Weiss reaction, Biochem. Biophys. Acta 781 (1984) 56–63. [44] R. Dash, C. Acharya, P.C. Bindu, S.C. Kundu, Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts, BMB Rep. 41 (2008) 236–241. [45] R.K. Geetha, D.M. Vasudevan, Inhibition of lipid peroxidation by botanical extracts of Ocimum sanctum: in vivo and in vitro studies, Life Sci. 76 (2004) 21–28.

[46] Z.J. Qian, W.K. Jung, S.K. Kim, Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw, Bioresour. Technol. 99 (2008) 1690–1698. [47] T.L. Pownall, C.C. Udenigwe, R.E. Aluko, Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions, J. Agric. Food Chem. 58 (2010) 4712–4718. [48] S.W. Himaya, B. Ryu, D.H. Ngo, S.K. Kim, Peptide isolated from Japanese flounder skin gelatin protects against cellular oxidative damage, J. Agric. Food Chem. 60 (2012) 9112–9119. [49] C.F. Chi, F.Y. Hu, B. Wang, Z.R. Li, H.Y. Luo, Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle, Mar. Drugs 13 (2015) 2580–2601. [50] E. Mendis, N. Rajapakse, S.K. Kim, Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate, J. Agric. Food Chem. 53 (2005) 581–587. [51] O.K. Chang, G.E. Ha, G.S. Han, K.H. Seol, H.W. Kim, S.G. Jeong, M.H. Oh, B.Y. Park, J.S. Ham, Novel antioxidant peptide derived from the ultrafiltrate of ovomucin hydrolysate, J. Agric. Food Chem. 61 (2013) 7294–7300. [52] B.H. Sarmadi, A. Ismail, Antioxidative peptides from food proteins: a review, Peptides 31 (2010) 1949–1956.