Active Components Of Ethanol Extract From Silkworm Cocoon Shell And Its Inhibition To DMBA/UVB-induced Skin Damage And Tumor Promotion In BALB/C Mouse

The cocoon shell of the silkworm consists mainly of two proteins, fibroin and sericin. In addition to these proteins, the cocoon shell contains small amounts of pigments, waxes and carbohydrates. There were some studies showed that the non-sericin components primarily consist of an ethanol extract from the sericin layer of the silkworm cocoon, which was found to mainly contain flavonoids and free amino acids. Flavonoids are commonly present in their glycosylated forms and mostly exist as quercetin and kaempferol glycosides in the sericin layers of silkworm Bombyx mori cocoons. Here, an efficient procedure of hydrolysis-assisted extraction(HAE) was first established to estimate the level of the total flavonoids through the determination of their aglycones, quercetin and kaempferol, in silkworm(Daizo) cocoons. These aglycones were released from flavonol glycosides by HAE and then detected quantitatively by HPLC-DAD. A 250 mg sample of the cocoon shell was suspended in 10 ml of a methanol-HCl-water(7/2/1, v/v/v) solution and maintained for the hydrolysis of the flavonol glycosides at 75°C for 4 h. The average contents of quercetin and kaempferol from the cocoon after 5 repeated measurements were 1.98 mg/g and 0.42 mg/g, with a relative standard diversity(RSD) of 3.01% and 2.38%, respectively. Their recoveries were 99.56% and 99.17%, respectively. The total sum of quercetin and kaempferol was detected to be 2.40 ±0.07 mg/g in the cocoon by HAE-HPLC, while the total flavonoids(2.59 ±0.48 mg/g) estimated by the traditional colorimetric method were only equivalent to 1.28 ±0.04 mg/g of quercetin. There was a great difference in the amount of total flavonoids using various silkworm cocoons. This may because the non-flavonoids have maximum absorption at 500 nm. These results show that the evaluation method for the total flavonoids reported here through detection of these aglycones in the cocoons is accurate and far superior to the traditional colorimetric method using rutin as a standard. Therefore, this study has profound significance for the comprehensive utilization of silkworm cocoons and recycling of flavonoids.Exposure of the skin to ultraviolet B(UVB) radiation causes oxidative stress which results in sunburn, photoaging, and skin cancer. We aimed to investigate the antioxidant, anti-inflammatory, and anticancer effects of a flavonoid extract(EE) from the sericin layer of Daizo silkworm cocoons. In a skin tumour mouse model, the topical application of EE reduced the number of tumours per mouse from 5.1 tumours per mouse in the negative control group to 3.2(EE1) and 2.6(EE2) tumours per mouse prior to induction with UVB/DMBA. UVB/DMBA can destroy the antioxidant system in the skin; however, the topical application of EE can increase the T-AOC to 1.63 U/mg prot equal to normal levels. The EE decreases the level of NF-κB from 1.16 ng/mg prot to 0.53 ng/mg prot. Our in vivo experimental results also showed that the EE could reduce the phototoxicity of UVB at different cellular and molecular levels by decreasing the severity of dermal inflammation and keratinocyte proliferation through the modulation of oxidative stress and NF-κB. EE could increase antioxidase activity and decrease the levels of the inflammatory cytokines IL6 and TNF-α. These results suggest that the flavonoid extract might be a potential drug for the prevention and treatment of skin cancer or a potential additive to skin cosmetics to protect against sun damage.