3. Results and discussion3.1. Prote

3. Results and discussion
3.1. Protein concentration and antioxidant activity of CSE

Protein concentrations of CSE with different extraction time (15, 30, 60, 90 and 120 min) were determined by Bradford assay. The protein concentration showed an obvious increase with increased extraction time as shown in Fig. 1. The longest time of extraction of 120 min showed the highest protein concentration (879 µg/ml).

Antioxidant activity of CSE with different extraction time (15, 30, 60, 90 and 120 min) was evaluated by ABTS assay and DPPH assay as shown in Fig. 1. Free radical scavenging activities of CSE with different extraction time were reported as IC50 values in which lower IC50 value indicates higher antioxidant activity. CSE from 60 min showed the highest antioxidant activity (ABTA assay=18.27 µg/ml, DPPH assay=66.56 µg/ml) while CSE from 120 min showed the lowest antioxidant activity. Studies reported that heat treatment induced conformational and functional changes of proteins, including antioxidant activity (Plancken et al., 2006, Tang et al., 2009, Raikos, 2010 and Rondeau et al., 2007). In this study we found that CSE lose their activity with increased extraction time. Similar finding was reported by Tang et al. (2012) modification of soy protein using heat treatment decreased antioxidant activity with increased treatment time. This result suggested that antioxidant activity of CSE might be related to its conformational changing according to extraction times. Additionally, many studies have reported that sericin exhibited a potent natural antioxidant (Kato et al., 1998, Wu et al., 2007 and Fan et al., 2009). Monosroi et al. (2010) extracted sericin from silk cocoons of five Thai native silkworms by autoclaving. They have found that the sericin extract from the five Thai native silkworms showed potent antioxidant activity, this was lower than that the standard antioxidant (vitamin C). Similar result was reported by Chlapanidas et al. (2013) the sericin extract exhibited antioxidant activity and the strain of silkworms significantly influenced antioxidant activity. Also Dash et al. (2008) reported that the sericin from Antheraea mylitta cocoons can serve as a valuable antioxidant.

3.1.1. Effect of treatment with dithiothreitol (DTT) and β-mercaptoethanol (β-ME)

Antioxidant activity of CSE treated with 0.5–20 mM of DTT measured by ABTS assay and DPPH assay are shown in Fig. 2. Antioxidant activities of CSE treated with different concentration of DTT were compared based on the value of IC50. Untreated CSE showed the lowest IC50 value (ABTS assay=11.12 µg/ml, DPPH assay=79.25 µg/ml) of antioxidant activity when compared to CSE treated with different concentration of DTT, indicating that untreated CSE had the strongest antioxidant activity. Also similarly with the result from DTT treatment, the antioxidant activity of CSE treated with 1 to 30 mM of β-ME were lower than untreated CSE as shown in Fig. 3.

DTT and β-ME are stressors and introduce instability into the core of a protein that promotes the transition between disulfide and SH groups leading to free sulfhydryl in protein ( Cleland, 1994 and Weston et al., 1995). Medina-Navarro et al. (2010) found that DTT treatment increased antioxidant capacity of albumin. Human serum albumin is composed of 35 cysteines, among which 34 are engaged in 17 disulfide bond cysteines, leaving only Cys-34 available for reaction (Peters, 1996). It has been suggested that Cys-34 is one of the most reactive sulfhydryl groups in serum (Narazaki et al., 1997). In contrast, in this study we found that DTT treatment decreased antioxidant activity of CSE. The sericin protein was very low content of Cystiene (∼0.53%), while serine, glycine, tyrosine, theonine and hydroxyl groups were higher ( Kato et al., 1998, Mondal and Trived, 2007 and Wu et al., 2008). Therefore, it is possible to see firstly, no correlation between antioxidant activity and free sulfhydryl of sericin protein and secondly, the native structure of sericin was important to their activity.

3.1.2. Effect of treatment with UV light

Antioxidant activity by ABTS assay and DPPH assay of CSE after exposure to UV light for 10 min are shown in Fig. 4. Both methods showed that antioxidant activities of CSE after exposure UV light were lower than CSE before exposure to UV light. Kristo et al. (2012) found that UV irradiation could change tertiary and quaternary structure of whey protein. The amino acid side chains affected in UV range are tryptophan (Trp), tyrosine (Tyr), histidine (His) and cysteine (Cys) (Mondal and Trived, 2007 and Pattison et al., 2012). UV excitation of aromatic residues can transfer an electron to breakage of intra-molecular disulfide bridges in protein (Hoffman and Hanyon, 1972). This result suggested that exposure to UV light may cause structural changes that could affect antioxidant activity of sericin protein.