Bioactive Secondary Metabolites of Wild Antheraea mylitta Silkworm Cocoons

1. Introduction

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 [1]. 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 [2]. The secondary metabolites consumed by the silkworms from the host plants are sequestered with silk proteins and play significant role in cocoon formation [3]. 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 [4] Recently along with mulberry silkworms, the wild non-mulberry silkworms also emerged as commercially significant in textile industry [5] Hence, in present study we focused on the extraction of non-protein active chemical compounds Antheraea mylittacocoons qualitatively and validated by using Fourier Transform-Infrared spectroscopy (FT-IR) and Gas chromatography–Mass spectrometry (GC–MS). The biological activity of the compounds was screened by compared; the phytochemicals from the cocoons were further confirmed in their respective host plants from the earlier reports to elucidate the arthropod-host plant interactions to predict the sequestration of allelochemicals from host plants to the insect cocoons.

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 Bombyx moricocoons take the lead. Silk fabric has been valued in numerous cultures for many millennia [6]. As per commercial production of silk Bombyx morisilk has been comprehensively investigated. As representative of many holometabolous insects, the silkworm life cycle start as a larva, passing through five larval instars and after the completion of fifth larval instar, the reduction of the juvenile hormone permit the neurosecretory hormone ecdysone to initiate metamorphosis and activate the initiation of the prepupal stage. The prepupa locates the suitable place for cocoon formation, then start to spin the cocoons. The silkworms spun the cocoons around the constricted body of the pupa which uses silk strands secreted from labial glands [7]. Silk strands themselves are polypeptide polymers composed of multiple components-microfilaments of insoluble proteins (fibroin), covered with a soluble adhesive protein (sericin) that provides structural support for the cocoon [8]. Other minor components include small proteins, lipids, and carbohydrates [9].

The wild sericigenous Indian tropical Tasar Silkworm Antheraea mylittais a polyphagous insect feeding on variety of leaves; it has rich genetic resources of forty four races acclimatized to diverse ecological zones. In the course of evolution it has been evolved with many advanced qualities such as disease resistance, silk quality, fecundity and tolerance to various environmental conditions (Figure 1). The wild Tasar cocoons are exposed to various biotic and abiotic environmental conditions, hence the quality of the silk, silk proteins and other secondary metabolites of the cocoons are differing than of mulberry silkworm cocoons [10, 11]. Comparatively the domesticated Bombyx moricocoons are soft and delicate, only the hot water treatment swells and partly dissolves the sericin gum, which coats and cements the fibroin filaments together inthe cocoon. But Wild silkworm species including Tasar silkworms are heavily mineralized with calcium oxalates (Figure 2) [12]. In addition to this wild cocoon are additionally stabilized by oxidative phenolic tanning, dityrosine cross-linking, and tannins derived from the caterpillar’s food plant. Mineralization is probably more important than the tanning in making Wild Silk cocoons difficult to soften and reel (Figure 3) [13]. The remarkable contrast in the composition of cocoons, the methods of obtaining silk from the cocoons of wild Silkworm with those domesticated Bombyx morimakes it complicated. The most important difference lay in the mineralization that very much common in wild Silkworms but absent in Bombyx moriand the difference arise from the gluing together of the fibers in mineralized matrix of wild silk cocoons made them moisture resistant (Figure 4) [14].

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 Bombyx moriand Antheraea mylitta. The fibroin protein is semi-crystalline in nature, showing of two phases, i. e greatly crystalline β-sheeted phase and a lesser or non-crystalline phase. The hydrophilic sericin proteins constitute about 20–30% of the cocoon which is hot water-soluble glycoproteins and hold the silk fibers together form the ecologically stable sericin-fibroin composite cocoon structure. The glue like sericin protein biosynthesized and secreted in the middle region of the silk gland. It comprises diverse polypeptides ranging from 24 to 400 kDa with high serine content (40%) with considerable amount of glycine (16%). There are three major polypeptides of sericin have been isolated from the silkworm cocoons with the molecular weights ranges from 150, 250 and 400 kDa. The sericin residues are partially unfolded with 35% β-sheet and 63% random coil, without α-helical structures. In addition to these major proteins, the low molecular weight hydrophilic proteins are also reported in the cocoons. The seroin protein is the product of a discrete gene that is expressed exclusively in the middle and the posterior part of the silk glands [15, 16, 17, 18, 19, 20].

Indian tropical wild Antheraea mylittasilkworms are of Saturniidae respectively. Silkworms produce delicate twin thread of silk protein fibroin, which is coated by glue like hydrophilic sericin protein (Figure 5). During pupation silkworms spin the cocoons to protect the inactive pupae. The silk proteins are synthesized by silk gland cells and stored in the lumen of the glands. The sericin protein is biosynthesized in the middle silk gland of the mature silkworm larvae, which constitutes 25–30% of silk proteins [21]. It is a water-soluble globular protein family whose molecular mass ranges from 10 to 310 kDa [22]. Naturally sericin is responsible for adhere both the fibroin filaments to maintain the structural integrity of the cocoon. The cocoon of Antheraea mylittahas three major fractions of sericin of which the lower fraction is around 70 kDa, the middle fraction is approximately 200 kDa, and the higher fraction is more than 200 kDa. The peduncle of Tasar silkworm Antheraea mylittahas a 200-kDa sericin protein, possesses serine, glutamic acid, glycine, tyrosine and threonine as predominant amino acid residues. The Serine (∼39%) is the principal amino acid. The Antheraea mylittasilk sericin is biochemically distinct from Bombyx morihaving lower percentages of serine and tyrosine. During degumming process of silk textile industry, sericin is removed as waste from fibroin to make silk fibers more lustrous, soft, smooth, white, and dye able. The global discarded sericin constitutes approximately 50,000 tons out of the 1 million tons of fresh cocoons annually [15]. Silk sericin of Bombyx moriis one of the most researched proteins. Presently sericin can be used in food, cosmetics, pharmaceutical products and the preparation of biomaterials. The sericin proved as antioxidant, anticoagulant and anti-wrinkle agent. It is also reported to suppress tumor growth and to reduce oxidative stress [23, 24, 25].

The domesticated Bombyx morisilk sericin contains 18 amino acids including polar amino acids such as 32% serine and 17% aspartic acid gives higher hydrophilic property and processing ability [26]. In contrast to this Tasar silk sericin contains 19% serine contents. The mulberry and Tasar sericins are biochemically distinct due to differences in their amino acid compositions, leading to differences in the immunological responses. Being non-domesticated and wild, Tasar cocoons are more resistant towards environmental stresses such as heat and drying, the sericin coat may contributed for toughness and resistance properties [27].

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 [28]. 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 [29]. 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 [30]. 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].

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 [52] 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.

4. Conclusion

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 [80].

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.

Acknowledgments

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.”