Identification of ground beetle sinensis and its preparation by characteristic ions
The ground beetle worm was first recorded in Shennong’s Classic of Materia Medica, and was listed as the middle grade. Ground beetle sinensis Walker or Steleophaga plancyi (Boleny) is the dried female body of Eupolyphaga sinensis Walker or Steleophaga plancyi. The ground beetle beetle mainly contains amino acids, proteins, volatile oil compounds, and other ingredients such as alkaloids, fatty acids, fibrinolytic active ingredients and trace elements. The ground beetle beetle has the effects of dispersing blood stasis, eliminating firm knot, detumescence and pain, lowering the breast and opening the channels, etc. It is used for bruises and injuries, tendon injuries and fractures, blood stasis and amenorrhea, postpartum blood stasis and abdominal pain, signs and swelling. Modern pharmacological research shows that ground beetle beetle has anticoagulant, anti-tumor, blood lipid regulating, analgesic, anti-inflammatory and bactericidal activities, and is even called “the first drug to regulate blood flow”. It is widely used in clinical practice (according to the statistics of the database of Yaozhinet, 151 traditional Chinese patent medicines and simple preparations prescriptions containing ground beetle beetle were collected), and the dosage is also large. At present, there are two kinds of ground beetle beetles sold on the market: wild and artificially raised. However, because the number of wild ground beetle beetles is decreasing year by year, artificially raised products account for a large proportion in clinical application, and the behavior of artificial adulteration and impurity occurs from time to time, which may lead to misjudgment only by identifying their characteristics. On the whole, the test items included under the ground beetle worm in the 2020 edition of the Chinese Pharmacopoeia are relatively simple, which cannot comprehensively and accurately reflect the authenticity and quality of the ground beetle worm.

The ground beetle beetle mainly contains amino acids, proteins, volatile oil compounds, etc. At this stage, its quality related research is also focused on these components. In recent years, some scholars have also used molecular pharmacognosy methods to study the authenticity of ground beetle beetles. For example, Chen et al. have also established the identification method of eupolyphaga beetles and their fake ground beetle Phnom Penh through chromatography and molecular biology. After comparing DNA barcodes with the database, the sequence of the closest species and its comparison can be given, which can clearly determine the source of varieties; Lu et al. used polyacrylamide gel electrophoresis to identify the proteins of three kinds of ground beetle beetles and those from the same and different places. The method has high resolution and good reproducibility. However, molecular pharmacognosy technology has high laboratory requirements and is difficult to promote, with certain limitations. In the Pharmacopoeia of the People’s Republic of China (2020 Edition) (hereinafter referred to as the “Chinese Pharmacopoeia”), there are only microscopic identification and thin layer identification under the item of identification of ground beetle sinensis medicinal materials, and this method is only applicable to the identification of raw materials. For the extracted ground beetle sinensis and the formulated preparations containing ground beetle sinensis, the method has poor transmissibility and cannot be effectively distinguished.
As a biomarker with species difference, characteristic peptides are becoming more and more mature in animal medicine research. Some scholars have realized the research of turtle A, antelope horn, ass hide glue, deer horn gum, centipede, deer horn, etc. through characteristic peptide technology. Its characteristics of good specificity and simple operation make it easier to promote in daily inspection. The characteristic peptide technology based on mass spectrometry detection can simultaneously set multiple species characteristic peptide ion information as detection indicators, which is expected to achieve authenticity identification while further accurately determining adulterated components and relatively quantifying adulterated components. It has broad and bright application prospects. Ion pair determination has been included in the identification items of ass hide glue, antler glue and tortoiseshell glue in the Chinese Pharmacopoeia (2020). The appearance of characteristic peptides as quality indicators in China’s official legal code has also put an end to the situation where animal drugs do not have specific quality control components in legal standards. The extract mostly uses water as the solvent. After the animal drugs such as ground beetle beetle are decocted and boiled, the collagen will undergo non-specific degradation to form water-soluble peptides. Some peptides can be absorbed into the blood through the biofilm to play a variety of biological effects. However, there is no report on the identification of ground beetle sinensis and its preparation by characteristic ions at home and abroad. In this study, by using the above characteristic ion identification technology for reference, it was applied to identify the authenticity of ground beetle beetles, providing a reference for the quality research of ground beetle beetles and their preparations.

Ground beetle sinensis has been proved to have the efficacy of treating various diseases, such as cancer, inhibiting thrombosis, enhancing immunity and treating avascular necrosis of the femoral head. Recently, it was reported that ground beetle beetle, leech and centipede showed obvious clinical efficacy in endometritis. However, it is difficult to correctly identify ground beetle beetles and prescriptions containing ground beetle beetles. At present, the quality testing of animal drugs mainly focuses on methods such as morphology, microscopy, and thin-layer testing. Moreover, most animal drugs contain similar compositions of amino acids and nucleosides, which also leads to ambiguity in the identification and quality analysis of animal drug varieties. Therefore, it is crucial to develop and establish a series of exclusive analytical methods for identifying and evaluating animal drugs for quality control and assessment of animal drugs. In recent years, the quality control model of animal traditional Chinese medicine based on qualitative/quantitative analysis of characteristic peptides has been gradually popularized and applied. The Chinese Pharmacopoeia 2020 contains the exclusive peptide identification methods of ass hide glue, deer horn gum and tortoise shell gum, but there is no identification method of characteristic ions in ground beetle beetle.
The characteristic ions established in this study were detected by protein digestion technology using 1% ammonium bicarbonate solution as the extraction solvent and trypsin for enzymatic hydrolysis of the sample; And the extraction method, enzyme dosage, and extraction time of the samples were investigated, and the optimal extraction conditions were determined to be heating reflux (extraction method), 50 μ L (enzyme dosage), and 30 minutes (extraction time).

Because the quality control method of ground beetle worm recorded in the Pharmacopoeia of the People’s Republic of China is relatively simple, it is not uncommon to produce and sell fake products and pass the inferior products off as the superior ones in the ground beetle worm market. At present, the price of the ground beetle worm continues to rise in the market. For economic benefits, lawbreakers have added alum, salt and other ingredients to ground beetle worm medicine to increase profits, and even used fake products to replace authentic ones. In addition, Phnom Penh ground beetle has also been included in the standard of traditional Chinese medicine in Guangdong Province, and has been used as a local household product for a long time. However, research shows that the composition of Phnom Penh ground beetle is different from that of Eupolyphaga and Eupolyphaga; At the same time, the standard decoction of different animal drugs has lost the shape of decoction pieces and is difficult to identify. Therefore, this study has established a characteristic ion identification technology based on mass spectrometry. It is found that m/z 415.72 (double charge) → 406.72, 415.72 (double charge) → 667.34 can be used as the characteristic ion for the identification of ground beetle sinensis. Through the application and detection of 24 batches of ground beetle worm medicinal materials from different sources, 21 batches of ground beetle worm extracts, 6 batches of ground beetle worm preparations, 6 different animal drugs and 3 kinds of ground beetle worm adulterants, the detection method can be applied to 2 different primitive ground beetle worms, which confirms the specificity of the characteristic ion, even if it has a clear distinction between the intermediates and preparations containing ground beetle worm that have lost the properties of the original medicinal materials.
At the same time, this method is simple to use, highly reproducible, and has no complex preprocessing process, greatly reducing the implementation threshold of this method. Although a large number of studies have shown that the method of identifying animal based traditional Chinese medicine using characteristic peptides is reasonable and feasible, the relevant quality evaluation methods still need to be further enriched and improved, which is also an important direction for future research on quality control of traditional Chinese medicine. In conclusion, the method established in this study will effectively supplement the deficiencies in the inspection standards of ground beetle and its decoction pieces, solve the problem that ground beetle can only identify authenticity through traditional experience for a long time, improve its quality control level, and provide a reference for the safety and effectiveness of clinical drugs related to ground beetle.