Study on Quality Evaluation of Bombyx mori by Chemical Pattern Recognition Combined with HPLC Fingerprint
Bombyx mori is one of the commonly used animal medicines in clinical practice, mainly used to treat diseases such as liver wind phlegm, seizures, infantile startle, tetanus, etc. Its source is the dried body of the 4th to 5th instar larvae of the silkworm Bombyx mori Linnaeus, which were infected with Beauveria bassiana (Bals.) Vuillart and died. Chemical studies have shown that its main active ingredients are proteins, amino acids, flavonoids, and other components. Modern pharmacological studies have shown that the protein rich extract of Bombyx mori has anti epileptic, immunomodulatory, and inhibitory effects The tumor growth of G7 tumor bearing mice has enhanced protective effects, promotes the recovery of motor impairment induced by MPTP in Parkinson’s disease mice, prevents dopamine depletion, and so on.
As a silkworm with a single diet, its characteristic of only feeding on mulberry leaves determines the rich and diverse absorption, transformation, and metabolism of the main flavonoids in mulberry leaves in its body. Research has shown that after infection with Fusarium oxysporum, flavonoids in silkworms undergo biotransformation. For example, flavonoids such as quercetin and kaempferol are converted into quercetin-7-O – β – D-4-O-methylglucoside and kaempferol-7-O – β – D-4-O-methylglucoside through 4-O-methylglucosylation reaction. Xing et al. found that as the time of death increases, the content and transformation of flavonoids in the body also differ, and speculated that quercetin 7-O – β – D-4-O-methylglucoside and kaempferol-7-O – β – D-4-O-methylglucoside can be used as one of their quality control indicators. Therefore, flavonoids can serve as important indicators for evaluating the quality of silkworms.

The combination of fingerprint spectrum and chemical pattern recognition is an effective method for evaluating the quality of traditional Chinese medicine, which has been widely used in the quality evaluation of traditional Chinese medicine, Chinese medicine decoction pieces, formula granules, etc. At present, there are various studies on fingerprint spectra related to silkworms, including ultraviolet fingerprint, infrared fingerprint, HPLC, HPCE, and protein fingerprint spectra. The composition categories cover water-soluble components, lipid soluble components, alcohol soluble components, etc. However, there are issues with poor separation efficiency and limited identification peaks, making it difficult to characterize their quality characteristics. Researchers have shown that Sichuan produced silkworms have better quality and higher levels of Beauveria bassiana compared to other regions such as Guangxi, Yunnan, and Zhejiang. Although there have been some research reports on the chemical composition and quality control of Bombyx mori, there is currently no good quality control method due to its complex composition and low content. Therefore, this study used high-performance liquid chromatography to establish fingerprint spectra of 20 batches of silkworm medicinal materials from different origins, and conducted differentiation research by combining chemical identification modes such as similarity analysis, cluster analysis, and principal component analysis. Referring to the requirements of the silkworm item in the 2020 edition of the Chinese Pharmacopoeia, the moisture content, total ash content, acid insoluble ash content, and extract were determined. The effective ingredient Beauveria bassiana content was determined using the previous method of the research group, aiming to comprehensively evaluate the quality of medicinal materials from different origins, provide scientific basis for quality control and standard improvement of silkworm medicinal materials, and provide reference for the research of other medicinal materials.

This experiment investigated the pre-treatment conditions such as extraction method, extraction solvent, and extraction time, and determined that the extraction method (reflux extraction, ultrasonic extraction) was optimal for ultrasonic extraction, the extraction solvent (methanol, 80% methanol, acetonitrile, and water) was optimal for 80% methanol, and the extraction time (15, 30, 45 minutes) was optimal for 30 minutes. The established fingerprint of the silkworm has a similarity between 0.92 and 0.99, which is relatively high and provides a basis for the quality evaluation of the silkworm.
The fingerprint spectrum established by our research institute mainly focuses on the flavonoids in the medicinal materials of Bombyx mori. Wang et al. used network pharmacology and molecular docking techniques to study the results, which showed that quercetin, kaempferol, and other components may be the main material basis components for Bombyx mori’s ability to relieve wind and spasms. Multiple studies have also shown that quercetin and kaempferol are one of the main active ingredients in the treatment of epilepsy. As a key herb for relieving wind and spasms, one of the substance bases for the action of Bombyx mori may be flavonoids. In addition, this study also conducted a correlation analysis between the peak areas of each common peak and the characteristics, extracts, total ash content, and white muscardine content of the medicinal materials. The results showed that the cross-sectional characteristics were positively correlated with the surface characteristics, medicinal material length, and white muscardine content, and positively correlated with the peak areas of peaks 1, 2, 3, 4, 6, and 7. After comparison, peaks 1, 2, 4, 6, and 7 were identified as rutin, hyperoside, astragaloside, quercetin, and kaempferol, which are the main flavonoids in mulberry leaves. This indicates that the transformation of flavonoids during the formation of silkworms is related to the “vitreous body” of the cross-section. There is a strong correlation between the quality of medicinal herbs and the formation, which also confirms the quality characteristic point of “white color, straight stripes, and black and bright cross-sections are preferred for silkworms” since ancient times.

In summary, the fingerprint spectrum established in this article not only includes the possible material basis components of the “Xifeng Zhijing” pharmacological effect of the silkworm, but also has a certain correlation with the quality indicators of the silkworm medicinal material itself, which can comprehensively reflect the quality of the silkworm medicinal material and provide scientific basis for the quality control of the silkworm medicinal material. In the quality control standards for Cordyceps sinensis medicinal materials, it should be fully considered that it is a complex of “bacteria insect leaf”, and multiple indicators should be used to comprehensively evaluate its quality, establishing a more scientific and reasonable quality standard for medicinal materials.