Qualitative tests for active compounds from Methanolic extract of
The wild silkworm Antheraea mylitta is grown and cultivated in several parts of India ranging from Bihar to West Bengal and several parts of Telangana. The wild silkworm rearing has been a source of income for the tribal populations who rely on it as income source; the intervention of government agencies has increased the cultivation. Our research involves understanding the secondary metabolites in the silkworm Cocoons and elucidating how the pupa survives the harsh environment during pupal diapause of the insect. We have realized the role of insect repellent compounds and other metabolites and their interaction with the insect. Wild silkworm Cocoons are the specialized natural structures constructed by Antheraea mylitta silkworms. They are the protein composites of sericin and fibroin as a structural material. The silkworm cocoons are presumed to be evolved structures through the course of evolution over millions of years. This chapter focuses on Biophysical analysis of chemical compounds, proteins and other secondary metabolites traced in the Wild Antheraea mylitta Tasar cocoons which are predicted to be the key factors to achieve the unique structural and chemical barriers to protect the pupa within the cocoons.
- Antheraea mylitta
- Bioactive compounds
- Tasar Silkworms
In wild silkworms host plant specificity is achieved due to the co-evolution of host plants and their monophagous or oligophagous specific herbivorous insect’s leads to the accretion of host plant derived allelochemicals in the specific insect cocoons. The plant derived chemicals, play a vital role in the life cycle of the respective phytophagous insects. These bioactive compounds affect the growth, survival, fecundity including behavior of the insects. The economically significant insect cocoons of Tasar silkworm also revealed for biological functions by their secondary metabolites like saponins, flavonoids, terpenoids, tannins and phytosterols sequestered from plant into the larvae to cocoons . The feeding habit and the growing conditions of the silkworms directly influence the chemical composition of the cocoons and the phytochemicals from the host plants of silkworms might be sequestered to cocoons . The secondary metabolites consumed by the silkworms from the host plants are sequestered with silk proteins and play significant role in cocoon formation . In the cocoons of mulberry silkworms three flavonoid 5-glucosides and many other flavonoids of host plant were identified in the sericin layer in yellow-green cocoon of the Sasammayu silkworms. These flavonoids from silkworm cocoons are proved effective for free radical scavenging, antioxidation, inhibition of hydrolytic and oxidative enzymes, and anti-inflammatory action  Recently along with mulberry silkworms, the wild non-mulberry silkworms also emerged as commercially significant in textile industry  Hence, in present study we focused on the extraction of non-protein active chemical compounds
As compared to domesticated mulberry silkworm cocoons, the wild silkworms and their cocoons show slightly different combination of morphological, chemical properties and are adapted to cope with harsh natural conditions. By considering this, we selected commercially exploited silkworms of domesticated and wild silkworms to screen the active chemical components from their cocoons and their sequestration patterns from feeding plants to the cocoons by qualitative methods.
The cocoons of most insect larvae are complex structures potentially serving various synchronized functions. The silkworm cocoon is generally presumed to provide the protection to inactive pupa against predation, biodegradation, dehydration etc. Among the most extensively studied cocoons
The wild sericigenous Indian tropical Tasar Silkworm
2. Bioactive silk proteins
The cocoons of the mulberry silkworm are composed of two types of proteins: fibroins and sericins. The fibroin is the core protein constitutes 70% of the cocoon and is a hydrophobic glycoprotein secreted from the posterior part of the silk gland. The expression of fibroin protein and P25 genes are transcriptionally regulated during larval development in both
Indian tropical wild
The silk of domesticated and wild silkworms has a core shell type structure, it is composed of a complex of 3 proteinaceous components: a large heavy chain fibroin (350 kDa) that is linked to a light chain fibroin (25 kDa) by disulfide bonds and another glycoprotein P25 protein (30 kDa) are linked with non-covalent hydrophobic interactions . The molar ratios of Heavy chain, Light chain and P25 are 6:6:1. The heavy chain is hydrophobic in nature and makes crystalline features to the silk fiber, but the Light chain is more hydrophilic and comparatively elastic. The P25 protein is supposed to play a crucial role to maintain the integrity of the complex . Prior to silk fiber formation, the solution of all the three silk proteins secreted from silk glands assembling into double filaments that come out from an exit tube in its spinneret and dry after exposure to air. Consequently core contains anisotropic β-sheet-rich nanocrystals are loosely aligned with the fiber axis and dispersed in an unstructured matrix . Another pair of silk glands secrete glue-like sericins (serine-rich glycoproteins) that coat the fibroin filaments for the cohesion of the cocoon by sticking the twin filaments together. The silk fibers coated with several other proteins are presumed to protect the cocoon against microorganisms and other predators [31, 32].
2.1 Biomedical applications of silk proteins
The silk proteins are biologically versatile molecules in the context of biomedical applications. The silk fibers have been used as sutures for wounds since many centuries, because of its strength, biocompatibility and low immunogenicity . Even the silk fibers spun into yarns and consequently textured via permanent deformation may be used as non-load-bearing spacers in tissue grafts where tissue in-growth is desirable. The cabled yarns have great tunable mechanical properties and therefore potential in load-bearing tissue engineering applications . In addition to these silk foams prepared from fibroin has been used as scaffolds for the attachment and proliferation of fibroblasts
Silk protein-based materials have found application as solid supports for potentially expensive enzyme and organometallic catalysts. Silk proteins are capable of forming functional complexes with metal ions. The enzymes can be successfully immobilized by means of covalent linking of the silk protein to the enzyme using conventional cyanogen bromide, azide, diazo or glutaraldehyde methodologies. The aspartate aminotransferase enzyme, calf intestine alkaline phosphatase enzyme and ribonuclease enzymes have been covalently linked to
The silk sericin has many biomedical applications as antioxidant, anticancer drug and anticoagulant. The study on macrophage response of silk proteins concludes that silk sericin does not allow inflammatory response when supply in soluble form. But the macrophage activation study of silk sericin reveals that, when attached to fibers induce inflammatory responses . The silk sericin in presence of lipopolysaccharides shows inflammatory reaction by initiating the synthesis of tumor necrosis factor and which is connected with native silk fiber-induced immune responses [42, 43, 44]. The explanation could be that coated sericin proteins either provide better adhesion to macrophages or the structural changes of sericin after binding to silk fibers prime the macrophage for consequent stimulation.
The bioconjugation (e.g. polymer-protein) is beneficial because it leads to minimize immunogenicity and improve stability. The biopolymer conjugations with anticancer drug candidates promote tumor targeting efficiency through superior permeability and increase drug retention time. Many investigators have utilized sericin as a natural biopolymer for bioconjugation with various therapeutic proteins, enzymes and polysaccharides. The sericin is the best natural biopolymer which can be conjugated effectively due to the presence of functional surface-active groups (-OH, -COOH, -NH2), which can form covalent linkage with the conjugates. The success of best performance of sericin is due to its hydrophilic nature and low antigenicity and other immunogenic properties and higher half-life period
The two-dimensional films and three-dimensional matrices of hydrogels and porous scaffolds of sericin proteins have been reported for their better performance. The membranes of sericin are naturally fragile in the dry state. The blending of sericin with water-soluble polymers like polyvinyl alcohol for making films has been investigated. The sericin hydrogels blend with polyvinyl alcohol by irradiation at 40 kGy has been reported. The blended hydrogels show an excellent moisture adsorbing tendency, desorbing and elastic properties with potential applications as soil conditioners to biomaterials for biomedical applications including wound dressings [50, 51].
3. Bioactive Tasar cocoon secondary metabolites
In present research the qualitative analysis of phytochemicals were tested for alkaloids, saponins, steroids, phenols, flavonoids, terpenoids, tannins, fatty acids, carboxylic acids, volatile oils, fixed oils and aldehydes in the methanolic extracts of Tasar cocoons by using standard methods  and compiled in Table 1. The alkaloids and volatile oils are present in Tasar cocoons whereas terpenoids and tannins are absent. The tests for phenols, fatty acids, carboxylic acids and fixed oils have shown positive reports.
The Tasar cocoon extract contained 14 characteristic GC–MS peaks emerged, which represents various respective chemical components in the extract (Figure 6). The Tasar cocoon methanolic extract revealed fourteen characteristic GC–MS peaks which are correlated with FT-IR (Figure 7), which represent their respective chemical components in the extract. The chromatogram maximum peak area percentage was observed for 26-Nor-5-cholesten-3.beta-ol-25-one (65%), Oleic acid (9.47%), n-Hexadecanoic acid (7.24%), Stegmasterol (5.93%) and Octadecanoic acid (5.51%). The FT-IR spectral analysis of the compounds of Tasar cocoons are presented in Table 2 and chemical structures are presented (Figure 8). These compounds are reported as potent antimicrobial, anti-inflammatory, anticancer, antioxidant, Hypocholesterolemic in nature respectively [53, 54, 55, 56, 57, 58, 59]. Other compounds shown minimum peak area percentage it directly dictates less in concentration. The complete GC–MS analysis and the biological activity of the compounds are presented in Table 3.
|FT-IR spectra of Tasar cocoons (Wavenumbers cm-1)||Bonds and Structures||Description|
|O-H stretch, free hydroxyl, H-bonded||Alcohols and Phenols|
|3447.64||N-H stretch||1˚, 2˚ amines, amides|
|C=O stretch||Amide I|
|1477.35||C-N Stretching, N-H bending||Amide II|
|1348.31||N-O symmetric stretch||Nitro compounds|
|1318.50||C-O Stretch||Alcohols, carboxylic acids, esters, ethers|
|C-N Stretching, N-H bending||Amide III (Aliphatic amines)|
|862.14||O-H bending||Carboxylic acids|
|801.48||C-Cl Stretch||Alkyl halides|
|4-Methyltridecane||11.30||0.45||C14H30||198||Alkane||Insect predator and Pheromone|
|Isobutyl alcohol||12.00||0.51||C4H10O||74||Alcohol||Alcohol detoxicant, disinfectant|
|Decanoic acid, methyl ester||13.59||1.38||C11H22O2||186||Fatty acid ester||Food additive and lubricant, Insecticide|
|n-Hexadecanoic acid||14.05||7.24||C16H32O2||256||Palmitic acid||Antioxidant, nemeticide and Hypocholesterolemic|
|13-Docosenoic acid , methyl ester||15.29||0.84||C22H44O2||352||Fatty acid ester||Lubricant and surfactant|
|Pentanoic acid, 4-methyl, methyl ester||15.49||1.30||C7H14O2||130||Fatty acid ester||Flavouring agent|
|Oleic acid||15.72||9.47||C18H34O2||282||Fatty acid||Anticancer, anti-aging and Anti-inflammatory|
|Octadecanoic acid||15.91||5.51||C18H36O2||284||Fatty acid||Adhesive and sealant|
|Oxalic acid, allylpentadecyl ester||17.03||0.42||C20H36O4||340||Carboxylic acid ester||-|
|1-Chlorododecane||17.20||0.54||C12H25Cl||204||Alkyl halide||Antitumor, Increase natural killer cell activity|
|Stigmasterol||19.35||5.93||C29H48O||412||Steroid||Antioxidant, hypoglycemic and thyroid inhibiting properties,|
Precursor of progesterone, Antimicrobial, Anticancer,
Antiarthritic, Antiasthama, Anti-inflammatory, Diuretic.
3.1 4-Methyltridecane and isobutyl alcohol
4-methyltridecane is a branched alkane consisting of tridecane bearing a single methyl substituent at position 4. It has a role as a plant metabolite. It is reported for antioxidant properties .
Isobutanol is an alkyl alcohol substituted by a methyl group at position 2. In
3.2 Decanoic acid, methyl ester and N-hexadecanoic acid
Methyl decanoate is a fatty acid methyl ester and a decanoate ester, it is reported for Antioxidant activity, Antibacterial, antiviral, antifungal activity .
The hexadecanoic acid is a straight-chain, sixteen-carbon, saturated long-chain fatty acid. N-hexadecanoic acid and it showed significant cytotoxicity against human colorectal carcinoma cells. It is also reported as anti-inflammatory compound . Hexadecanoic acid, methyl ester exhibited antioxidant, hypocholesterolemic, anti-androgenic, hemolytic, alpha reductase inhibitor activities . The major saturated fatty acid hexadecanoic acid has recently been shown to be neutral in its cholesterolaemic effect. The Palm oil rich with hexadecanoic acid, the consumption has been reported to reduce blood cholesterol in comparison with the traditional sources of saturated fats such as coconut oil, dairy and animal fats .
3.3 13-Docosenoic acid, methyl ester
13-Docosenoic acid, methyl ester is a fatty acid methyl ester, that is a flavor-active, volatile, and aromatic compound found in cooked commercial shrimp waste. It is a component of biodiesel formed from
3.4 Oleic acid
Oleic acid has been reported to have hypocholesterolemic, antioxidant and lubricating activity . Oleic acid is commonly found in diet. It is a monounsaturated fat which on consumption has been linked with decreased lowdensity lipoprotein cholesterol, and possibly increased high-density lipoprotein cholesterol .
3.5 Octadecanoic acid and oxalic acid, allylpentadecyl ester
A C18 straight-chain saturated fatty acid component of many animal and vegetable lipids. As well as in the diet, it is used in hardening soaps, softening plastics and in making cosmetics, candles and plastics. It is a stearic acid ester reported for its antioxidant and anti-inflammatory activity . The alcoholic compound of oxalic acid is reported for antimicrobial preservative .
1-Chlorododecane belonging to the family of organic halogen compounds. It is hard to dissolve in water but can be mixed with alcohol and ether. This chemical is less health hazard substance than short chain alkyl chlorides. It is used as a solvent, as chemical intermediate to make photographic chemicals, pharmaceuticals, organometallic compounds, surfactants .
3.7 2, 5-Dimethyl-3,4-hexanediol and stigmasterol
2, 5-Dimethyl-3, 4-hexanediol extracted from
The GC–MS analysis of essential oils obtained from the peel of
The steroid compound of cholesten reported for Antimicrobial, Diuretic, Anti-inflammatory, anti-asthmain acetone extract of
The primary host plant for Tasar silkworm is
The silk fibers and insect extracts have been comprehensively used in folk medicines from thousands of years. In Chinese traditional medicines insects and insect-based products are used for various diseases and ailments. The insects and their products have evolved individually over the track of evolution; naturally insects face numerous biotic and abiotic challenges in their life cycles. Due to microorganisms infested habitats they occupied their success in the survival, infinite numbers and diversity specify the presence of extremely effective immune systems produce powerful antimicrobial, cytotoxic compounds for the parasites and other medicinally valuable chemical compounds. The chemical defense strategies of insects have evolved, including odorous repellents to avoid or to kill or inactivate the defending individual predatory organisms including microorganisms. By considering this, we designated the commercially exploited lepidopteran insect wild Tasar silkworm cocoons to screen the active non-protein chemical components .
Naturally, silkworm pupa is enclosed within the cocoons during the metamorphosis from pupa to adult. This is the most susceptible stage for the insects because the immobile pupa is not able to respond to biotic and abiotic threats. Therefore, cocoon provides or modifies the microenvironment of the pupa to ensure the optimal conditions for successful pupation and possesses antimicrobial properties. In comparison to wild silkworms, domesticated mulberry silkworms are more privileged due to their domestication and indoor rearing practices. Hence the structural and chemical composition of wild silkworms is strong enough to face threats during their life cycle. The diverse chemical compounds with strong biological activities were identified in the wild silkworm cocoons compared to mulberry silkworms. The comprehensible mechanism of bioactive compound synthesis and their sequestration from specific host plants to the silkworm cocoons and their function for the silkworm physiology is yet to be explained. The insect specific chemical products including antimicrobial compounds, their biosynthesis strategies and the mechanism of action may take part in the discovery of new drug candidates in the field of biomedical science.
The Bioactive components in addition to the Silk proteins from Tasar cocoons were identified by various methods and their biomedical application was compiled. The qualitative analysis of the extracts was performed to validate the active chemical compounds. The conclusion of our results drawn as the phytochemical of the host plants sequestrated to cocoons and the biosynthesis of bioactive compounds by the silkworm larvae during spinning of the cocoons to protect pupae during metamorphosis. But the molecular mechanisms or metabolic pathway for phytochemical sequestration or the biosynthesis of the bioactive compounds in the insects need further research. In comparison to mulberry cocoons, non-mulberry cocoons possess the antimicrobial and insect repellent agents, which are presumed to be involved in the direct protection of wild cocoons by microbial decomposition and other insect predators in wild environmental conditions. We conclude the physico-chemical interactions of the cocoons are responsible to protect the inactive pupae during metamorphosis. Further exploration is required for strategic isolation of cocoon protecting active compounds from mulberry and non-mulberry cocoons for biomedical applications.
Prof. V. Shyam Kumar thanks to Department of Science and Technology- Science and Engineering Research Board (DST-SERB), New Delhi. India for funding of this project (SB/EMEQ-154/2013) and Authors are thankful to the University Science Instruments Center (USIC), DST-FIST, Dept of Biotechnology & Microbiology, Karnatak University Dharwad for GC–MS, FT-IR studies.
Conflict of interest
“The authors declare no conflict of interest.”