Fibroin Hydrolysate Research Articles

Development of an enzyme‐linked immunosorbent assay based on the monoclonal antibody of Bombyx mori to detect ancient silk

Identifying the traces and residues of unearthed silk fibres is crucial for further analysis and study. In this study, an enzyme‐linked immunosorbent assay (ELISA) based on the monoclonal antibodies specific to Bombyx mori fibroin was developed in order to for detect ancient silk. Five monoclonal antibodies were successfully prepared based on fibroin hydrolysate, and four had a high specificity for mulberry silk. Furthermore, they could be used to discern B. mori fibre from tussah fibre and eri fibre. The ELISA standard curve of the most sensitive monoclonal antibody, anti‐Silk Fibroin (SF)‐3, was y = 0.022x + 0.177 (R2 = 0.998), and the detection limit was 4.65 ng/ml. Using this antibody, we successfully identified five ancient mulberry silk samples from different time periods. The results suggest that ELISA assays based on monoclonal antibodies can be used as an immunological testing method for unearthed silk fibre.

Neuroprotective effects of silk fibroin hydrolysate against Aβ25–35 induced cytotoxicity in SH-SY5Y cells and primary hippocampal neurons by regulating ROS inactivation of PP2A

Silk fibroin (SF) has been widely used as biomedical materials and the hydrolysate form of SF (SFH) has shown many activities. The purpose of the current study was to explore the neuroprotective effects of SFH against Aβ25–35-induced cytotoxicity in SH-SY5Y cells and primary hippocampal neurons and the possible mechanism. The results showed that SFH could improve the viabilities of SH-SY5Y and primary hippocampal neurons injured by Aβ25–35. The lowered mitochondrial membrane potential and increased cell apoptosis induced by Aβ25–35 were recovered after SFH treatment. Further study indicated that the beneficial effects of SFH were likely due to its interference with reactive oxygen species (ROS). SFH inhibited the phosphorylation of Ser/Thr protein phosphatase 2A (PP2A) by suppressing Aβ25–35-induced ROS, and then suppressed the activation of MAPKs and tau hyperphosphorylation, and protected SH-SY5Y cells and primary neurons from Aβ25–35-induced damages. SFH has the potential to be a nutraceutical for AD intervention.

Improving glycemic control in model mice with type 2 diabetes by increasing superoxide dismutase (SOD) activity using silk fibroin hydrolysate (SFH)

Islet cell dysfunction in type 2 diabetes is primarily attributed to the increased apoptosis of pancreatic beta cells. Silk fibroin hydrolysate (SFH) has an effect on blood in type 2 diabetes model mice (C57BL/KsJ-db/db). However, its exact mechanism is unknown. The type 2 diabetes model mice were randomly divided into non-diabetic mice (ND), diabetic mice (DB), and diabetic mice treated with silk fibroin hydrolysate (DB-SFH). The results showed that SFH significantly decreased fasting blood glucose and hemoglobin A1c (HbA1c). The oral glucose tolerance and insulin tolerance were significantly improved in the DB-SFH group. The DB-SFH group exhibited increased superoxide dismutase (SOD) activity in the plasma, as well as increased Mn-SOD and CuZn-SOD activities in the pancreatic islets. Furthermore, the pancreatic islet cells' death was decreased in the DB-SFH group. In the DB-SFH group, the protein expression of caspase-3 was significantly decreased compared with the DB group. The expression of the Nkx6.1 and Pdx1 proteins were increased in the DB-SFH group. The results suggest that SFH prevents the degeneration of pancreatic islets via increasing SOD while hyperglycemia is alleviated by maintaining beta cell mass in type 2 diabetes model mice.

Anti-apoptotic effects of silk fibroin hydrolysate in RIN5F cell on high glucose condition

Hyperglycemia-induced pancreatic β-cell apoptosis causes serious health complications in diabetic patients. Recently, studies demonstrated that silk and silk-related materials have anti-diabetic effects. We previously reported that silk fibroin hydrolysate (SFH) has anti-diabetic effects through increased pancreatic β-cell mass in type 2 diabetic animals (C57BL/KsJdb/db). However, it is not known whether SFH has anti-apoptotic effects in hyperglycemic conditions. The present study investigates the anti-apoptotic effects of SFH on high glucose-induced apoptosis using RIN5F cells, a rat pancreatic β-cell line. Hyperglycemic conditions decreased RIN5F cell viability in a concentration-dependent manner. High glucose treatment of 33 mM or higher caused a significant decrease in RIN5F cell viability. However, addition of SFH significantly recovered cell viability in the presence of high glucose. Flow cytometry analysis showed that high glucose treatment significantly increased early stage apoptosis in RIN5F cells. This was inhibited by SFH treatment, which significantly decreased not only early stage apoptosis but also decreased the production of nitrite. Additionally, SFH protected RIN5F cells from oxidative stress-induced apoptosis. These results suggest that SFH has anti-apoptotic effects by protecting pancreatic β-cell from high glucose and/or oxidative stress. Our results support in vivo anti-diabetic effects of SFH and validation of the traditional use of silkworm and silkworm materials in the treatment of diabetes.

Effects of silk fibroin hydrolysate on bone metabolism in ovariectomized rats

This study aimed to investigate the effects of silk fibroin on bone metabolism in ovariectomized rats. A total of 30 Sprague-Dawley rats were randomized into sham-operated (SHAM), ovariectomized control (OVX), alendronate (OVX+ALEN, 10 mg/kg body weight/d), low silk fibroin (OVX+SF100, 100 mg/kg body weight/d), and high silk fibroin (OVX+SF300, 300 mg/kg body weight/d) groups. All the rats were fed by gavage for 12 wk. At the end of 12 wk, blood and urine were collected for analysis of bone turnover markers, and bone mineral density (BMD) was measured by micro-computed tomography. The results show that the OVX group (p < 0.05) displayed the highest mean body weight gain. Among the five groups, serum levels of bone alkaline phosphatase (ALP) and urine levels of deoxypyridinoline (DPD) were highest in the OVX group (p < 0.05). Bone ALP levels in the ALEN group were significantly lower than that of the silk-treated groups. On the other hand, DPD levels were not significantly different between the ALEN and silk-fibroin-treated groups ( p < 0.05). The trabecular BMD was significantly higher in the ALEN and silk-treated groups compared to the OVX group ( p < 0.05). In conclusion, this study showed that silk fibroin has similar effects as alendronate, which is used in osteoporosis medication. Therefore silk fibroin might be a new candidate for the prevention and treatment of osteoporosis in patients.

Spider silk fibroin enhances insulin secretion and reduces blood glucose levels in diabetic mice

Fibroin silk proteins make up the cocoons of silkworms and spider webs and are rich in glycine and alanine residues. Recent studies have shown that silk fibroin hydrolysate from silkworms improves blood glucose and lipid metabolism. In the present study, we investigated the anti-diabetic effects of spider silk fibroin protein in type 2 diabetic mice. Recombinant AvMaSp-R, which consists of the 240 amino acid repetitive domain of major ampullate silk protein (AvMaSp) from the spider Araneus ventricosus, was produced in baculovirus-infected insect cells. We tested the effects of oral AvMaSp-R administration on serum insulin and blood glucose levels in diabetic mice and found that AvMaSp-R increases serum insulin levels and reduces blood glucose levels in diabetic mice. Consequently, our results are the first to provide evidence that silk fibroin protein from spiders enhances insulin secretion, which leads to reduced blood glucose levels in type 2 diabetic mice.

Effect of Silk Fibroin Hydrolysate on the Apoptosis of MCF-7 human Breast Cancer Cells

Breast cancer is one of the most common cancers among women worldwide. Recently anticancer agents have been developed using natural substances. To evaluate the anticancer effect of hydrolysates of silk fibroin (HSF), we investigated the effect of HSF on cell viability and apoptosis of a breast cancer cell line, MCF-7, induced through the mitochondrial pathway. The result showed that HSF decreased cell viability in MCF-7 cells in a dose- and time-dependent manner, resulting in an increase in the sub-G1 phase cell population. HSF increased the level of the pro-apoptotic Bax protein and decreased the levels of the anti-apoptotic Bcl-2 protein. In addition, HSF induced apoptosis in MCF-7 cells through a mitochondria-dependent pathway by increasing levels of cytochtome c, and cleavage of PARP. Taken together, these findings suggest that HSF inhibits the proliferation of MCF-7 breast cancer cells through a mitochondria and caspase dependent apoptotic pathway.

Memory-Enhancing Effects of Silk Fibroin-Derived Peptides in Scopolamine-Treated Mice

Although enzyme-hydrolyzed silk fibroin has been reported to enhance cognitive function before, it has been still unknown which peptides can improve memory. Here we report that amino acid sequences of three novel peptides were identified from fibroin hydrolysate. Fibroin hydrolysate was obtained by hydrolysis with protease after partial hydrolysis with 5M CaCl2. Synthesized peptides derived from these sequences improved scopolamine-induced memory impairments in mice. We confirmed this hydrolysate had effects that improved learning and memory abilities by performing the Rey-Kim test. From this hydrolysate of silk fibroin, amino acid sequences of eight peptides were identified by LC-MS/MS. Three peptides (GAGAGTGSSGFGPY, GAGAGSGAGSGAGAGSGAGAGY, and SGAGSGAGAGSGAGAGSGA) were synthesized to investigate whether they could improve memory. Passive avoidance test and Morris water maze test were performed, and all peptides showed memory-enhancing abilities on scopolamine-induced memory impairments in mice. In this study, we identified three novel peptides that could improve memory, and that silk fibroin hydrolysate was a mixture of various active peptides that could enhance memory.

Silk fibroin hydrolysate ameliorates diabetic dyslipidemia in type 2 diabetic model mice

The antidiabetic properties in silk materials, such as silkworm and silk fibroin, have been described in traditional folk medicine. In a previous study, we reported that the silk fibroin hydrolysate (SFH) had a protective effect against glucotoxicity in HIT-15 cells and an antidiabetic effect in a noninsulin-dependent diabetes mellitus (type 2 diabetes, T2DM) mouse model. This study aimed to determine whether SFH ameliorates diabetic dyslipidemia and promotes pancreatic β-cell function in a T2DM animal model (C57BL/KsJdb/db). SFH significantly improved blood glucose levels after 4 weeks of treatment. T2DM animals had a normal serum blood glucose level after 6 weeks of SFH treatment. Hemoglobin A1c (HbA1c) levels were significantly lower in T2DM animals that were treated with SFH for 6 weeks. Glucose tolerance in T2DM animals also significantly improved after 6 weeks of SFH treatment. Furthermore, plasma total cholesterol, high-density lipoprotein cholesterol, nonesterified fatty acid, and triglyceride concentrations significantly decreased in SFH-treated T2DM animals compared to T2DM animals. This result showed that SFH ameliorated diabetic dyslipidemia in the T2DM model mice. Taken together, our results suggest that SFH could be used as a potential agent to treat dyslipidemia in patients who have type 2 diabetes mellitus.

In VivoHypotensive and Physiological Effects of a Silk Fibroin Hydrolysate on Spontaneously Hypertensive Rats

A silk fibroin hydrolysate (SFH) exhibited a pronounced in vivo blood pressure-lowering effect on spontaneously hypertensive rats (SHRs) accompanied with decreases in the plasma angiotensin II, endothelin (ET), TNF-α and NO concentrations. We also observed a markedly decreased LPO level, a parameter of oxidative tissue damage, in the medium- and high-dose SFH-treated groups which was accompanied by an increased SOD level in erythrocytes. Our data suggest ACE inhibition together with an improved antioxidative status as the underlying antihypertensive mechanism for the silk fibroin hydrolysate. Since SFH could markedly lower the blood pressure and improve several physiological parameters involved in the occurrence of hypertension, it could be used as a possible supplement against cardiovascular diseases and as a functional food ingredient.

Silk fibroin hydrolysate inhibits osteoclastogenesis and induces apoptosis of osteoclasts derived from RAW 264.7 cells

Bone disease can be associated with bone resorption by osteoclasts, and interest in the development of antiresorptive agents has recently increased. The hydrolysate of silk fibroin has been studied with respect to such biomedical applications. In a previous study, silk fibroin showed indirect inhibitory effects on the differentiation of osteoclasts. To further evaluate the effect of a hydrolysate of silk fibroin on osteoclasts, we investigated the direct effects of the silk fibroin hydrolysate on osteoclastogenesis and apoptosis of osteoclasts induced by receptor activation of nuclear factor κB ligand (RANKL). The silk fibroin hydrolysate inhibited RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP) in RAW 264.7 cells. The inhibitory effect of the silk fibroin hydrolysate resulted in the decreased expression of osteoclast marker genes, such as matrix metalloproteinase-9 (MMP-9), cathepsin-K and calcitonin receptor (CTR). In addition, the silk fibroin hydrolysate blocked the signaling pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) and expression of transcription factors, such as nuclear factor of activated T cells c1 (NFATc1) and NF-κB. Finally, the silk fibroin hydrolysate induced apoptosis signaling cascades. Taken together, the present results indicate that silk fibroin hydrolysate has antiresorptive activity by both inhibiting osteoclastogenesis and inducing osteoclast apoptosis.

Silk fibroin hydrolysate exerts an anti-diabetic effect by increasing pancreatic β cell mass in C57BL/KsJ-db/db mice

Components of silk including silk fibroin have long been used as anti-diabetic remedies in oriental medicine. However, detailed mechanisms underlying these anti-diabetic effects remain unclear. In this study, we examined the anti-diabetic activity of silk fibroin hydrolysate (SFH) in C57BL/KsJ-db/db (db/db) mice, a well-known animal model of non-insulin dependent diabetes mellitus. When the db/db mice were administered SFH in drinking water for 6 weeks, hyperglycemia in the animals gradually disappeared and the level of glycosylated hemoglobin decreased, indicating that SFH plays important role in reducing the symptoms of diabetes. In addition, SFH-treated db/db mice exhibited improved glucose tolerance with increased plasma insulin levels. Immunohistochemical and morphological analyses showed that SFH up-regulated insulin production by increasing pancreatic β cell mass in the mice. In summary, our results suggest that SFH exerts anti-diabetic effects by increasing pancreatic β cell mass in a non-insulin dependent diabetes mellitus mouse model.

Characterization of tyrosine-rich Antheraea pernyi silk fibroin hydrolysate

Antheraea pernyi silk fibroin (SF) hydrolysate were characterized using UV–VIS spectrometer, amino acid composition and heavy metal contents to explore its potential sources for food or cosmetic additives. The hydrolyzed A. pernyi SF was separated into two parts: (a) SFA, alanine-rich fraction and (b) SFB, tyrosine-rich fraction. SFB exhibited strong absorption peaks at 210 and 280 nm due to the presence of the tyrosine. Heavy metal analysis showed that arsenic and mercury did not detect. Other heavy metals, which includes lead, cadmium, etc., were recorded only a trace amount. Therefore, A. pernyi SF hydrolysate could be safely used as sources of food, cosmetic and pharmaceuticals.

Antihypertensive Effects of Silk Fibroin Hydrolysate by Alcalase and Purification of an ACE Inhibitory Dipeptide

Silk fibroin, which is normally discarded as an industrial byproduct in clothing plants, was hydrolyzed with alcalase. Angiotensin I converting enzyme (ACE) inhibitory activities of the silk fibroin hydrolysates (SFH) were investigated, and the hydrolysate with hydrolysis degree of 17% exhibited the highest ACE inhibitory activity. At the tested 600 mg/kg.d and 1200 mg/kg x d doses, SFH significantly lowered blood pressure of spontaneously hypertensive rats (SHR) after chronic oral administration. SFH was further purified using consecutive chromatographic methods on Sephadex G-15 column and reverse phase high-performance liquid chromatography (RP-HPLC) on an octadecylsilane column. After its purity was confirmed by analytical RP-HPLC and capillary electrophoresis, one ACE inhibitory dipeptide was isolated, and its molecular mass and amino acid sequence were determined as 238.2 Da and Gly-Tyr, respectively, by LC-ESI/MS. The results of this study suggest that silk fibroin byproducts have the possibility to become an effective source for ACE inhibitory peptides.

Even-numbered peptides from a papain hydrolysate of silk fibroin

A protease with broad substrate specificity usually produces a complex peptide mixture. However, even-numbered peptides were obtained at high proportion upon papain hydrolysis of fibroin composed of highly repetitive Ala- and Gly-rich blocks. MALDI-TOF and ESI mass spectrometric analysis revealed that the even-numbered peptides were in the forms of di-, tetra-, hexa-, and octa-peptides with repeating units in combination of Ala–Gly, Ser–Gly, Tyr–Gly, and Val–Gly. Application of tandem mass spectrometry identified the sequences of the tetra-peptides to be in the order of Ala–Gly–X–Gly (X = Tyr or Val). Therefore, the substrate specificity of papain and the unique repetitive sequence of fibroin generated the hydrolysate composed of even number of amino acids at a high percentage. In this work, fibroin hydrolysate was investigated as an example of an end product of protein hydrolysis, which provides a clue to understand the fate of peptides in a protein hydrolysate.

Fractionation of a silk fibroin hydrolysate and its protective function of hydrogen peroxide toxicity

AbstractFractionated components of Bombyx mori silk fibroin, which were hydrolyzed with protease, were prepared with a preparative‐recycling high‐performance‐liquid‐chromatography system to evaluate the protective effects of molecular‐weight‐controlled B. mori silk fibroin components on H2O2‐injured neuronal cells. Three major fractions, having molecular weights less than about 1500, were first collected with the recycling techniques. The highest protective effect of the molecular‐weight‐controlled B. mori silk fibroin components on H2O2‐injured neuronal cells was obtained when a fraction with a molecular weight of approximately 1400 was used. This protective effect of the silk fibroin hydrolysate on H2O2‐injured neuronal cells was suggested to correlate with the contents of aromatic amino acids such as tyrosine and phenylalanine. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 772–776, 2006