Bombyx Mori Cocoons Research Articles

Effect of crude extract of Bombyx mori coccoons in hyperlipidemia and atherosclerosis

The silkworm is the larva or caterpillar of the domesticated silkmoth, Bombyx mori and being a primary producer of silk is an economically important insect. These days the silk is emerging as a resource for solving a broad range of biological problems. The silk (Abresham) is popularly known as Abresham muqriz (muqriz means cut) in Unani medicine. Its cocoons are extensively used as an ingredient of various Unani formulations like Khameer-E- Abresham Sada, Khameere Abresham Hakeem Arshad Wala, Khameere Abresham Ood Mastagi Wala etc. and are used to treat many cardiac and nervous disorders. The hypolipidemic activity of this drug, along with Nepata Hindostana (Badranjboya) and Terminalia Arjuna (Arjan) has been documented. But action of extract of Bombyx mori cocoons as a single drug is not documented. That's why; it was decided to study its effect on hyperlipidemia and atherosclerosis. The Male New Zealand White rabbits all of 1.5kgs were selected for the study. After stabilization period (2 weeks) the rabbits were divided into 3 groups (Group I - Control, Group II Lesion Control and Group III treated with extract of Bombyx mori silk cocoon). Hyperlipidemia and atherosclerosis were induced with 1% cholesterol diet. After induction of hyperlipidemia and atherosclerosis for twelve weeks, Group III rabbits were treated with Bombyx mori for 6 weeks (45 days). A significant decrease in hyperlipidemia was seen within 4 weeks of treatment. Histopathologically, the atherosclerotic plaques showed reduction in size. The third group showed a significant increase in the body weight and also an increase in the HDL cholesterol levels. The study concludes that extract of Bombyx mori cocoons has a significant effect on hypercholesterolemia and atherosclerosis probably because of its antioxidant and hypolipidemic effect.

Correlation of the tenacity and elongation in relation to the size of thread for filament from cocoons of Bombyx mori L.

Correlation of the tenacity and elongation in relation to the size of thread for filament from cocoons of Bombyx mori L.

Different properties of electrospun fibrous scaffolds of separated heavy-chain and light-chain fibroins of Bombyx mori

This study is the first to report on the fabrication and properties of electrospun scaffolds derived from separated light-chain fibroin and heavy-chain fibroin, two major proteins of silk fibroin. Among seven different extraction conditions, which were commonly used to extract fibroin from cocoons of Bombyx mori, only Ajisawa's reagent and 9 M lithium thiocyanate could extract both heavy-chain fibroin and light-chain fibroin, while the other conditions could yield only the light-chain fibroin. Mixed fibroin, light-chain fibroin, and heavy-chain fibroin were fabricated using electrospinning methods. Average diameters of the fibers were 658+/-208, 517+/-162, and 518+/-171 nm, respectively and their sizes after treatment with 50% methanol for 60 min were slightly increased to 747+/-244, 556+/-164 and 521+/-201 nm, respectively. FTIR spectra showed similar predominant beta-sheet conformation of mixed fibroin and heavy-chain fibroin scaffolds after treated with methanol, whereas the predominant structure of light-chain fibroin was random coil conformation. Although, scaffolds derived from mixed fibroin and heavy-chain fibroin showed similar properties, the light-chain fibroin scaffold clearly exhibited different properties, including more hydrophilic character, water uptake ability, degradation rate, and cell adhesion capability.

Fine-tuning scaffolds for tissue regeneration: effects of formic acid processing on tissue reaction to silk fibroin

Formic acid (FA) plays a key role in the preparation of silk fibroin (SF) scaffolds from cocoons of Bombyx mori and is used for fibre distribution. In this study, we used a subcutaneous implantation model in Wistar rats to examine SF scaffolds prepared by treating the degummed cocoon with FA for either 30 or 60 min. The tissue reaction and inflammatory response to SF was assessed by qualitative histology at intervals from 3 to 180 days. Additionally, dynamic biomaterial-induced vascularization and biomaterial degradation were quantified using a technique for analysing an image of the entire implanted biomaterial. Varying the FA treatment time led to different scaffold morphologies and resulted in two distinct peri-implant tissue reactions. The 30 min-treated scaffold was integrated into the surrounding tissue beginning at day 3 after implantation and vascularization increased 10-fold from 15 to 180 days, while the scaffold was continuously degraded throughout the first 90 days. In contrast, the 60 min-treated SF scaffold appeared as bulk for the first 90 days after implantation, after which a rapid degradation and vascularization process began. After 180 days, the tissue response was similar for both scaffolds, with eventual formation of a well vascularized connective tissue integrating the SF fibres. This study indicates that by modifying the FA treatment time, the tissue reaction to SF scaffolds can be tailored for different tissue-engineering applications. The tunability and biocompatibility of SF make it an attractive scaffold for exploration in regenerative medicine and clinical tissue engineering.

Preparation and characterization of regenerated fiber from the aqueous solution of Bombyx mori cocoon silk fibroin

The regenerated silk fibers with high strength and high biodegradability were prepared from the aqueous solution of Bombyx mori silk fibroin from cocoons with wet spinning method. Although the tensile strength of the regenerated silk fibroin fiber, 210 MPa is still half of the strength of native silk fiber, the diameter of the fiber is about 100 μm which is suitable for monofilament of suture together with high biodegradability. The high concentration (30%, w/v) of the aqueous solution of the silk fibroin which corresponds to the high concentration in the middle silkgland of silkworm was obtained. This was performed by adjusting the pH of the aqueous solution to 10.4 which corresponds to p K a value of the OH group of Tyr residues in the silk fibroin. The mixed solvent, methanol/acetic acid (7:3 in volume ratio) was used as coagulant solvent for preparing the regenerated fiber. The structural change of silk fibroin fiber by stretching was monitored with both 13C solid state NMR and X-ray diffraction methods, indicating that the high strength of the fiber is related with the long-range orientation of the silk fibroin chain with β-sheet structure.

Post-spinning modification of electrospun nanofiber nanocomposite from Bombyx mori silk and carbon nanotubes

Electrospinning is an effective procedure for fabricating submicron to nanoscale fibers from synthetic polymer as well as natural proteins. We successfully electrospun regenerated silk protein from cocoons of Bombyx mori to produce random as well as aligned fibers with diameter less than 100nm. The fibers were characterized using field emission environmental scanning electron microscope (ESEM), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy and wide angle X-ray diffraction (WAXD) studies. Post-spinning treatment with methanol and/or stretching and co-electrospinning with single walled carbon nanotubes (CNT) were carried out to alter the strength, toughness, crystallinity and conductivity of silk nanofibers. Addition of just 1% CNT along with post-spinning treatments resulted in 7-fold increase in the strength and 35-fold increase in the modulus of silk nanofibers. Raman spectroscopy confirmed that CNTs were incorporated in the silk fibers. FT-IR spectroscopy and WAXD studies proved that silk–CNT nanofibers had more crystallinity compared to silk nanofibers without CNT. Four-probe method demonstrated that silk–CNT nanofibers had 4 times higher electrical conductivity compared to silk nanofibers without CNT.

The behavior of aged regenerated Bombyx mori silk fibroin solutions studied by 1H NMR and rheology

As part of a project to utilize the regenerated silk fibroin (RSF) membranes as a supporting matrix for the attachment and growth of corneal stem/progenitor cells in the development of tissue engineered constructs for the surgical restoration of the ocular surface, the behavior of the aged RSF solutions has been investigated. The solutions were produced from domesticated silkworm ( Bombyx mori) cocoons according to a protocol involving successive dissolution steps, filtration and dialysis. The solutions were kept at 4 °C in a refrigerator for a certain period of time until near the gelation time. The changes in molecular conformation were studied by solution-state 1H NMR, while the flow of the solutions was characterized by rheological method. Upon ageing turbidity developed in solutions and the viscosity continuously decreased prior to a drastic increased near the gelation time. The 1H resonances of aged solutions showed a consistent downfield shift as compared to the 1H resonances of the fresh solution. Shear thinning with anomalous short recovery within a certain range of low shear rates occurred in both fresh and aged solutions. While the solutions behave as pseudo-plastic materials, the chain conformation in aged solutions adopted all secondary configurations with β-strand being predominant.

Mapping of the yellow inhibitor gene <I>I</I> in silkworm <I>Bombyx mori</I> using SSR markers

The yellow color of silkworm (Bombyx mori) cocoon is mainly controlled by three genes, Y (yellow blood), I (yellow inhibitor) and C (out-layer yellow cocoon) genes. I gene locates on the 9th chromosome of silkworm and prevents the transport of carotenoid from epithelia of midgut into hemolymph. Owning to a lack of crossing over in females, reciprocal backcrossed F1(BC1) progenies were used for linkage analysis and mapping of the I gene based on the SSR linkage map using silkworm strains Baghdad (Ba), which express white hemolymph (II+Y+Y), and KY, which express yellow hemolymph (+I+IYY). The gene of interest was linked to three (S0904, S0905, and S0906) SSR markers. All the individuals with white hemolymph in the BC1F (BC1 was generated using F1 as female) showed heterozygous profile of (BaxKY) F1, and the yellow ones in BC1F showed the homozygous profile of the strain KY. Using a reciprocal BC1M cross, we con-structed a linkage map of 38.4 cM, and the distance between I gene and the nearest marker S0904 is 7.4 cM.

Silkworm and spider silk scaffolds for chondrocyte support

To create scaffolds with silkworm cocoon, spider egg sac and spider dragline silk fibres and examine their use for chondrocyte attachment and support. Three different kinds of scaffolds were developed with Bombyx mori cocoon, Araneus diadematus egg sac and dragline silk fibres. The attachment of human articular cartilage cells were investigated on these bioprotein matrices. The chondrocytes produced an extracellular matrix which was studied by immunostaining. Moreover, the compression behaviour in relation to the porosity was studied. The compression modulus of a silkworm silk scaffold was related to its porosity. Chondrocytes were able to attach and to grow on the different fibres and in the scaffolds for several weeks while producing extracellular matrix products. Porous scaffolds can be made out of silkworm and spider silk for cartilage regeneration. Mechanical properties are related to porosity and pore size of the construct. Cell spreading and cell expression depended on the porosity and pore-size.

Influência de genótipos de amoreira (<em>Morus</em> sp.) e substratos no peso e características de casulos do bicho-da-seda (<em>Bombyx mori</em> L.)

The effect of three genotypes of mulberry, Morus sp. (Moraceae), namely, Miura, FM Shima Miura and IZ 56/4, and three types of rearing substrate comprising compact soil, concrete and plastic screen were estimated on weight and on certain industrial characteristics of Bombyx mori (Lepidoptera) cocoons. Genotype interactions with rearing substrates affected weight of cocoons produced. Analysis showed the best treatments for manufacturer parameters were Miura / compact soil, FM-Shima Miura / plastic screen, IZ 56/4 / compact soil and IZ 56/4 / plastic screen.

Properties of an Emulsion Prepared from Silk Sericin and Vitamin E

An emulsion (SR-EM) was prepared from vitamin E and aqueous sericin solution obtained from cocoons of Bombyx mori. Properties such as emulsifying degree, heat stability and radical scavenging activity of SR-EM were compared with those of CS-EM and AL-EM, vitamin E emulsions prepared from casein and bovine serum albumin, respectively. The degree of emulsifying for SR-EM was three times higher than that for CS-EM. CS-EM and AL-EM were a heat-stable emulsion, whereas SR-EM was a heat-labile emulsion because absorbance spectra changed exclusively in SR-EM after treated from 115 to 127deg. These results indicated that the oil-water interface of SR-EM was unstable under the high pressure-high temperature treatments compared to those of CS-EM and AL-EM. Each emulsion showed lower radical scavenging activity than a vitamin E solution,suggesting that the interaction of vitamin E and a radical molecule may be interfered by a protective protein colloid. SR-EM and CS-EM were more stable in 60% ethanol solution at ambient temperature than AL-EM because the radical scavenging activity of SR-EM and CS-EM was some 50% lower than that of AL-EM.

Silk as Substratum for Cell Attachment and Proliferation

Silk fibroin (SF), isolated from silkworm (Bombyx mori) cocoons, is a natural biodegradable polymer. Over the past decade, there was some interest in using SF as a biomedical material. As part of a project to develop tissue-engineered constructs for the surgical restoration of the ocular surface (cornea, conjunctiva), we have investigated the capacity of SF to function as a substratum for the attachment and growth of corneal stem/progenitor cells harvested from the corneoscleral limbus of donor human corneal tissue. SF membranes were produced from cocoons following a protocol involving successive dissolution steps, filtration, dialysis, evaporation, and methanol treatment. Human limbal epithelial cells were harvested from donor tissue and seeded onto SF membranes. After 5 days, the culture was fixed and stained with specific agents to visualize the cells. The study indicated profuse cellular attachment and growth. SF membranes appear to be suitable as a substratum for the repair of damaged ocular surface.

Role of Hydroxyl Side Chains in Bombyx mori Silk Sericin in Stabilizing Its Solid Structure

It is important to study the hydrogen-bonding character in hydrophilic polymers in detail to create new hydrophilic synthetic polymers controlled by hydration. In this paper, the role of hydroxyl side chains in sericin, a gluelike hydrophilic protein of Bombyx mori cocoon, in forming stable β-sheet aggregates is studied on the basis of 13C solid-state NMR analyses. To clarify the molecular mechanism to stabilize the solid structure of sericin, 13C CP/MAS and DD/MAS NMR measurements of native sericin and a model peptide of its crystal domain were performed at hydrated state. There are hydroxyl side chains of Ser and Thr residues in β-sheet structure which remain rigid after hydration. These hydroxyl side chains were assumed to clump adjacent β-sheets by hydrogen bonding at their OH groups. We inferred that Ser and Thr linkage sequences in the crystal domain are responsible for forming β-sheet aggregates and generating the structural stability of sericin.

Orientation-Insensitive Spectra for Raman Microspectroscopy

Separating effects due to molecular conformation from those due to orientation in the spectra of oriented samples obtained by Raman microspectroscopy is a complex issue. To solve this problem, we propose a procedure to calculate an orientation-insensitive spectrum (so-called isotropic spectrum) from polarized spectra obtained by Raman microspectroscopy that is valid for systems that exhibit a uniaxial symmetry. The method has first been tested on highly oriented samples of high-density polyethylene (HDPE). Polarized and isotropic spectra of a highly oriented HDPE cylindrical rod and an isotropic HDPE sample have been compared. The differences in the relative intensities, which occur in the polarized spectra and are due to orientation of the polyethylene chains, are nearly cancelled in the isotropic spectra, showing that the orientation-insensitive spectrum adequately represents the molecular conformation without contributions of orientation. Second, spectra of silk fibroins have been compared in the amide I region for Bombyx mori cocoon silk fibers and methanol-treated regenerated fibroin films. The similarity of the shape of the amide I band of the isotropic spectra indicates that the secondary structure of the fibroins is very close in both samples. These experimental results support the conclusion that the molecular conformation can be efficiently characterized from the intensity and the shape of Raman bands in the orientation-insensitive spectrum.

Construction and analysis of an AFLP molecular linkage map of the silkworm (Bombyx mori)

AbstractAn amplification fragment length polymorphism (AFLP) molecular linkage map with a relatively high density for location of quantitative trait loci (QTLs) controlling the quantitative traits of silkworm (Bombyx mori) cocoons, was constructed using 91 individuals of the F2generation. Among the 692 effective loci, 550 were allocated to subgroups a and b, of which 21 linkage groups in subgroup a had 233 molecular markers and 28 linkage groups in subgroup b had 317 markers. The number of markers on each linkage group in subgroups a and b ranged from 4 to 43 and 3 to 35, respectively. The total length of linkage groups for subgroup a was 1868.10 cM, and 2677.50 cM for subgroup b. The length of linkage group varied from 22.3 to 424.3 cM in subgroup a and from 2.4 to 366.5 cM in subgroup b. The average variation in the distance between markers was 3.39–17.43 cM in subgroup a and 0.8–26.96 cM in subgroup b. The average distance between the markers was 8.81 cM in subgroup a and 9.26 cM in subgroup b. There were 14 linkage groups, with an average distance below 10 cM, in subgroup a and 18 linkage groups in subgroup b. There were seven linkage groups with an average distance between 10 and 20 cM in subgroup a and ten linkage groups in subgroup b. Each linkage group in subgroup a had 11.1 loci on average, while there were 11.31 loci on each linkage group on average in subgroup b. The mean length for linkage groups in subgroups a and b was 89 and 95.6 cM, respectively. The total average length for both a and b subgroups was 2272.8, and 9.06 cM for the average marker distance, fulfilling the basic known requirements for locating QTLs.

Quantitative Mapping of the Orientation of Fibroin β-Sheets in B. mori Cocoon Fibers by Scanning Transmission X-ray Microscopy

The spatial distribution of the linear dichroic signal associated with aligned beta-sheets in a microtomed section of a Bombyx mori cocoon silk fiber was derived from scanning transmission X-ray microscopy (STXM). The intense C 1s --> pi(amide) peak at 288.25 eV was found to have negligible dichroic signal in transverse sections but a large dichroic signal in longitudinal sections. This is consistent with other measurements of the orientation of the aligned beta-sheets in silk fibers, in particular with those obtained by polarized Raman microspectroscopy to which our results are compared. When the dichroic signal strength is mapped at better than 100 nm spatial resolution, microscopic variations are found. Although the magnitude of the dichroic signal changes over a fine spatial scale, the direction of the maximum signal at any position does not change. We interpret the spatial variation of the intensity of the dichroic signal as a map of the quality of local orientation of the beta-sheets in the fiber. At sufficiently high magnification and resolution, this technique should image individual beta-sheet crystallites, although the present implementation does not achieve that. A map of the orientation parameter P(2) is derived. The average value of P(2) (-0.20 +/- 0.04) from STXM is smaller than that derived from the analysis of the amide I band in polarized Raman spectra (-0.41 +/- 0.03). This deviation is attributed to the fact that the STXM results also include the signal from unaligned regions of the protein.

Tensile behavior and morphology of differently degummed silkworm (Bombyx mori) cocoon silk fibres

Tensile tests have been performed on silkworm cocoon silk fibres degummed using five different methods: distilled water, boracic acid–sodium borate buffer, sodium carbonate, urea and succinic acid. Using an electronic single-fibre tensile instron, the force–displacement curves were obtained for each condition. Effects of degumming on silk include a decrease in the initial elastic modulus and a decrease in the proportional limit (i.e., yield point). These results suggest that degumming weakens at least one type of non-covalent interaction of core fibrion, such as hydrogen bonds and Van der waal's bonds. The stress–strain curve determined from a force–displacement curve as rescaled by the corresponding sample cross-sectional area failed to reproduce the actual mechanical properties of two fibroin core fibres because of variable degradation of the sericin coating. The two important factors associated with degumming could affect the tensile properties of silkworm silk: 1) the change in the microstructure of two core fibroin, and 2) the degumming ratio. SEM was used to observe visually the morphology and fractography of differently degummed cocoon silk fibres. Effects of different degumming treatments on the tensile behavior, mechanical properties, morphology and fractography of cocoon silk are clearly visible.

Thermally Induced α-Helix to β-Sheet Transition in Regenerated Silk Fibers and Films

The structure of thin films cast from regenerated solutions of Bombyx mori cocoon silk in hexafluoroisopropyl alcohol (HFIP) was studied by synchrotron X-ray diffraction during heating. A solid-state conformational transition from an alpha-helical structure to the well-known beta-sheet silk II structure occurred at a temperature of approximately 140 degrees C. The transition appeared to be homogeneous, as both phases do not coexist within the resolution of the current study. Modulated differential scanning calorimetry (DSC) of the films showed an endothermic melting peak followed by an exothermic crystallization peak, both occurring near 140 degrees C. Oriented fibers were also produced that displayed this helical molecular conformation. Subsequent heating above the structural transition temperature produced oriented beta-sheet fibers very similar in structure to B. mori cocoon fibers. Heat treatment of silk films at temperatures well below their degradation temperature offers a controllable route to materials with well-defined structures and mechanical behavior.

Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells

We have previously shown that a biomaterial consisting of a non-woven fibroin net produced from silk ( Bombyx mori) cocoons is an excellent scaffolding material for a wide variety of human cells of different tissue types. Endothelialization must take place for a biomaterial to be successful after implantation. Therefore, primary human endothelial cells and the human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS-1, were examined for adherence and growth patterns on the fibroin nets by confocal laser scanning microscopy after vital staining of the cells and by electron microscopy. Endothelial cells adhered and spread along individual fibers of the nets and did not fill the gaps between individual fibers. Higher attachment and growth coverage was obtained if nets were first coated with gelatin, fibronectin or collagen type I. Proinflammatory markers of endothelial cells on the fibers exhibited a non-activated state and LPS-stimulated cells exhibited activation of these markers. Furthermore, a typical PECAM-1 localization at cell–cell contacts was observed. Scanning electron microscopic examination of fibroin nets after removal of cells did not demonstrate any changes to the fibroin structure. HUVEC and HDMEC on fibroin nets embedded in collagen type I gels formed microvessel-like structures. Thus, silk fibroin nets are a highly endothelial cell-compatible scaffolding material that support the growth, normal and inducible cell functions and angiogenesis potential of human endothelial cells in vitro similar to that observed in vivo.

“Design Rules” of Silk Production Unraveled in Studies of Bombyx mori Cocoons

“Design Rules” of Silk Production Unraveled in Studies of Bombyx mori Cocoons