Dissolution of Philosamia ricini Silk Film: Properties and Functions in Different Solutions

MATERIALS AND METHODS

Materials: Both Nang Lai variety(B. mori) and Eri (Philosamia ricini) silk cocoons were kindly supplied from Silk Innovation Center (SIC), Mahasarakham University, Thailand. The cocoons were strips and then degummed twice using 0.5 g mL^-1 Na₂CO₃ and thoroughly rinsed 2 times in warm distilled water. They were then dried at room temperature before dissolving. All of dissolving solution including LiBr, Ca(NO₃)₂, ZnCl₂, LiSCN, 85% phosphate, CaCl₂ and ethanol in analytical grade were used.

Methods
Dissolution of silk: The Eri silk was tested with several systems: 9M LiBr, 9M Ca(NO₃)₂, 9M ZnCl₂, 9M LiSCN, 85% phosphate and 1:2:8 mole ratios of CaCl₂/Ethanol/H₂O (mixture solution). Time for dissolving of the silk was determined and compared between each solution. All silk hydrolysate were then centrifuged at 1000 g for 30 min. The silk solution was then separated and the precipitate part was taken in air-dried, calculated silk remaining (%) as follow equation;

Finally, the best solution was chosen to dissolve Eri silk for film preparation step.

Eri silk was dissolved with the 85% phosphate follow the modified of earlier reported by Wang et al. (2000). Briefly, dried Eri silk fibroin and the solution were mixed at the rate of 1 g fibroin to 10 mL of solution. The solution was firstly warmed to about 100°C, then gradually added fibroin into the solution and stirred with controlled temperature at 100-105°C until silk already hydrolyzed. The hydrolysate was neutralized, filtrated, dialyzed in cellulose tube against distilled water for 3 days at room temperature and concentrated.

Nang Lai variety: The Nang Lai variety was dissolved by using the mixture solution of CaCl₂/Ethanol/H₂O (1:2:8 mole ratios) follow with earlier reported by Ajisawa (1998). In addition, the solution used for dissolving of Eri silk was also used.

Preparation of silk film: The silk solution was stirred and then 10 mL solution was cast on the polystyrene plates. The plates were left air dried at room temperature for about 2-3 days. Finally, the films with a thickness of 20-30 μm were removed.

Investigation of silk films: The silk films were analyzed for their structure with fourier transform infrared spectroscopy (FTIR, Perkin Elmer-Spectrum Gx) in the spectral range of 4000-400 cm^-1 at 4 cm^-1 spectral resolution and 32 scans. FTIR was used to measure the absorption bands which represented the silk structure. A TA-Instrument TG SDT Q600 thermogravimetric analyzer was used to determine of the thermal decomposition pattern of the silk films. The analysis condition was 50-100°C for heating sample at 20°C min^-1 rates under nitrogen atmosphere.

Peptide bond measurements: To confirm the hydrolysis of silk, biuret reaction was performed as earlier reported by Wang et al. (2000). In brief, 1 mL of 20% of NaOH was added into 1 mL of 1% silk solution, then added 0.5% CuSO₄. The solution was then mixed until color became violet. The depth of color was correlated with the number of peptide bonds in the reaction. After placing at 37°C for about 10 min, the solution was cooled at room temperature and measured at 540 nm. In the same time, ninhydrin reaction was also tested. Briefly, 1 mL of silk solution was diluted with phosphate buffer pH 7.0 to obtain 5 dilutions at 10 folds for each. One milliliter of ninhydrin solution was added, mixed together and then boiled at 100°C for 10 min. The color became blue-violet, then observed and measured at 540 nm.

RESULTS AND DISCUSSION

Solubility of the wild silk in the different systems: As shown in the Table 1 % silk remaining was highest when used calcium nitrate, but lowest and fastest for 85% phosphate solution. Thus, the suitable solution for dissolving Eri silk was 85% phosphate. It quite similar with earlier reported by Wang et al. (2000) that Eri silk was dissolved by this solution but different in time used.

Nang Lai variety was dissolved by using CaCl₂/Ethanol/H₂O (1:2:8 mole ratios) and quite different when compared to the Eri silk. However, the solution from 85% phosphate was similar both physical appearance and biuret reaction as shown in Table 2. However, both biuret and ninhydrin reactions were supported that the current method is validity.

Results analyzed from Table 1 and 2 showed that the 85% phosphate solution could be hydrolyzed both of silk fibroin with related to the early studied of peptide bond remain (Wang et al., 2000). Time used in this study was shorter than earlier reported but result of biuret reaction was the same. In addition, silk fibroin of both silks could be hydrolyzed at the room temperature in the same condition (data not shown). This might be suggesting that the experiment time could be performed as shorter and more safety. However, it might be difference in the film appearances.

FTIR spectra of regenerated silk fibroin films: Generally, analysis of secondary structures of protein are indicated by the amide I (1700-1600 cm^-1) amide II (1600-1500 cm^-1) and amide III (1300-1200 cm^-1) bands (Kweon et al., 2000; Hino et al., 2003). The pattern of the absorptions in each film was observed.

Table 1:	Effect of salts solution on Eri silk
Remarks: — (not dissolve), + (less), 2+ (little), 4+ (good), 5+ (excellent)

Table 2:	Characteristics of Eri and Nang Lai silk solution

Table 3:	FTIR absorption bands of Eri and Nang Lai silk films

Figure 1 shows the FTIR spectra of the native Eri (A), Nang Lai (B) silk films and blended film of both silk solutions (C), respectively. The amide I (C = O stretching) band of the Eri silk strongly shown at 1629 cm^-1, while the Nang Lai variety were 1650 and 1620 cm^-1 with a shoulder at 1690 cm^-1 (Table 3). The results suggest that the Eri silk composed of more peptide bonds than the Nang Lai variety since the spectral region at 1700 to 1620 cm^-1 is the vibration of peptide bond, but the band at wave number below 1620 is amino acid side chain vibration (Wang et al., 2000). It is clearly revealed that 85% phosphate solution could be hydrolyzed the Nang Lai variety to obtain smaller amino acids than Eri silk. These results were related to the previous results of biuret reaction.

The amide II (-NH₂ stretching) of Eri silk film was absorbed at 1533 cm^-1 with small peak at 1448 cm^-1, while Nang Lai variety film showed at least 4 peaks: 1575, 1527, 1490 and 1450 cm^-1. IR spectra showed that the Eri silk film composed of the higher β-sheet (1533 cm^-1) structure than Nang Lai variety film (1575, 1527 cm^-1). Amide III of Eri silk film (1238 cm^-1) which was β-sheet form contrasted Nang Lai film (1243 cm^-1) was irregular structure (Hino et al., 2003). Both silk films were similar of C-O stretching (~1068 cm^-1) bands (Kweon et al., 2001). The absorption band at 3286 cm^-1 was N-H stretching band of Eri silk film. This band was different for Nang Lai variety film which showed multi-bands at 3386, 3344, 3289 and 3236 cm^-1.

The IR spectra revealed that the Eri silk film strongly appeared with stable profiles of absorption bands contrasted to Nang Lai variety film. In addition, the absorption bands of Eri silk was higher indicated of the β-sheet structure than Nang Lai variety which composed of many structures including α-helix, β-sheet and unordered structures. With compare to previously report that fibroin film of wild silks are partly rich in α-helical structure than those of B. mori (Kweon and Park, 1999; Li et al., 2003). However, some researchers demonstrated that poly (L-alanine) is stable conformations of parallel-chain pleated sheet structure (Moore and Krimm, 1976; Simmond et al., 1996). Furthermore, glycine units related to different conformation (Miyazawa et al., 2002). Thus, it may be concerned to the structural changes in several fragment of Nang Lai variety fibroin since glycine are dominant amino acids component of B. mori silk. These results might be suggested that the Eri silk film stronger than Nang Lai variety film in mechanical properties.

Figure 2 indicated the thermogravimetric curves of the films. The results showed that the Nang Lai variety film little stronger than the Eri silk film due to the break point was higher.

Fig. 1:	IR spectra of (A) Eri, (B) Nang Lai and (C) combined of Eri and Nang Lai silk films

Fig. 2:	Thermogravimetric curves of regenerated Eri and Nang Lai silk films

Fig. 3:	DTG curves of Eri and Nang Lai silk films

However, the break points of both silk were closed at about 300°C with reveal by the DTG curves (Fig. 3). More details were the maximum tolerances to the temperature of the Nang Lai variety at about 313°C. The obtained results were arranged value in comparison to earlier report that the maximum tolerance of the B. mori was 280-308°C (Zhang et al., 2002). However, for wild silk (A. pernyi and other belonging to the family Saturniidae) thermal decomposition underwent several steps (Kweon et al., 2000). Eri silk film found that the thermal decomposition was single step at 312°C. The result suggests that the components of silk were different profiles depend on the types or silk strains. In the cases of the silk film in this study, the compositions of both silk may be the same but different in the compositional ratio.

CONCLUSION

The solution of Eri and Nang Lai variety silks could be prepared by dissolving in 85% phosphate and could then use as samples for regenerating silk films. In general of the both silk films characteristics were similar compared with the earlier reported. Moreover, the thermal decomposition of the prepared films was also closed to the other reports. FTIR results were also confirmed that the method used in this study was practically created for silk solution preparation both domesticated and wild silks.

ACKNOWLEDGMENTS

We would like to thank Instrumental Center and Department of Chemistry, Faculty of Science and Silk Innovation Center, Mahasarakham University for conveniences analysis facilities.