Study on the Quality of Different Primitive Leeches and Their Confused Products
The 2020 edition of the Pharmacopoeia of the People’s Republic of China, hereinafter referred to as the Chinese Pharmacopoeia, stipulates that leeches are the dried whole of the leech family animals, such as the leech Whitman pigra Whitman, leech Hirudo nipponica Whitman, or willow leaf leech Whitman acranulata Whitman. Its taste is salty, bitter, and flat; There is a small toxin, which belongs to the liver meridian. It has the function of breaking blood and promoting circulation, removing blood stasis and eliminating stagnation. It is commonly used for blood stasis and meridian obstruction, treating masses, stroke hemiplegia, and falls and injuries. Studies have shown that the pharmacological effects of leeches mainly include anticoagulation, antithrombotic, anti-tumor, hypolipidemic and anti-inflammatory effects, and have very wide clinical applications, including the treatment of thrombosis, hyperlipidemia, hypertension, diabetes, prostatitis, cancer, varicose veins, infertility, cirrhosis and other diseases. The main components of leeches are macromolecular compounds, mainly containing proteins and 17 types of amino acids, including 8 essential amino acids for the human body. The hydrolyzed amino acid content is as high as 49.4%; In addition, it also contains various substances such as hirudin, heparin, histamine, hirudin, glycolipids, sphenoids, steroids, and carboxylic esters.
Through literature review, it has been found that current research on leeches focuses on anticoagulant mechanisms and amino acid determination, with limited comprehensive studies on the quality of different primitive leeches and their mixtures. Identification studies between different primitive leeches and their mixtures are mostly based on trait identification under the 2020 edition of the Chinese Pharmacopoeia. The market for leeches is expensive and chaotic, and traditional appearance identification alone cannot quickly and effectively evaluate their quality. Moreover, appearance identification cannot be applied to leech extracts and preparations, which has certain limitations. Therefore, establishing a rapid, scientific, and effective evaluation method is of great significance. This study used HPLC to establish the characteristic spectrum and content determination method of leeches. This method comprehensively evaluates leeches and their mixed products, providing a scientific basis for the quality evaluation of leeches and their mixed products.

Optimization of chromatographic conditions. On the basis of establishing a characteristic spectrum, this study used the same method to determine the content of two characteristic components of leech, leech amine C carboxyl derivative and leech amine B. A diode array detector was used to perform full wavelength scanning of the test solution in the wavelength range of 200-400nm. The results showed that the chromatographic peaks of each main component had strong UV absorption at 254nm wavelength. Therefore, the same conditions were used to analyze the characteristic spectrum and content of leech samples.
Analysis of feature map results. The leech is the dried whole body of the leech family animal, the leech Whitmania pigra Whitman. Its medicinal properties are flat spindle shaped. The 2020 edition of the Chinese Pharmacopoeia only controls its quality through moisture, total ash content, acidity and alkalinity tests, and antithrombin activity, and has not yet made differential characterization between different substrates. In this study, HPLC characteristic maps of hirudo with different origins and its adulterants were established. Through comparison of reference substances, it was confirmed that hirudo contains five components: uracil, hypoxanthine, xanthine, hirudine C-carboxyl derivatives, hirudine B. According to literature reports, the above pteridine compounds have similar structures with antiplatelet aggregation drugs such as clopidogrel, and it is believed that these components may be the active components of hirudo to play antiplatelet aggregation. The pharmacological effects of hirudo reported more are mainly anticoagulant, antithrombotic, and antiatherosclerotic effects. In addition, there are anti-tumor, anti inflammatory, anti fibrosis, and lipidemic activities, which indicate that the selected characteristic peaks in this study are related to It is of great significance to clarify the functional substances of leeches that play pharmacological activities and promote the secondary research and development of drugs. At the same time, the established method can identify different primitive leeches and their mixed products. The next step is to continue the application research of this method in extracts and preparations, providing reference for the development and utilization of leech medicinal materials.
Analysis of content determination results. Based on the study of characteristic maps, this study quantitatively determined five components in leeches, including three nucleoside compounds, uracil, hypoxanthine, and two sphenoid compounds, leechamine C-carboxyl derivatives and leechamine B. According to existing literature reports, nucleoside compounds and sphenoid compounds can be used as quality evaluation indicators for leeches. The experimental results showed that there were certain differences in the content of uracil and hypoxanthine among different primitive leeches and their mixtures. The average content of uracil and hypoxanthine was in the following order: leech>leech>willow leech>Philippine leech; The average content of xanthine is as follows: leech>leech>Philippine leech>willow leaf leech; The average content of C-carboxyl derivatives of leech amine is as follows: leaf leech>leech>leech>Philippine leech; The average content of leechamine B is as follows: leaf leech>leech>leech>Philippine leech. The Philippine leech does not contain leechamine B and the content of hypoxanthine is significantly lower than the other three substrates. Therefore, determining the content of the above components can be used to distinguish between water leeches (including leeches, leeches, and leaf leeches) and medical leeches (Philippine leeches); In addition, PLS-DA analysis uses the peak areas of 8 characteristic peaks in the sample as variables, and the peak areas of characteristic peaks are closely related to the content of their corresponding indicator components. Combined with the PLS-DA analysis results in “2.4.5”, the VIP values of the characteristic peaks corresponding to hypoxanthine and xanthine are greater than 1, and the willow leaf leech does not contain xanthine. The average content of the other three primitive xanthines also shows significant differences, further indicating that selecting these five components as content determination indicators has certain scientific validity.
The method established by our research institute is accurate, sensitive, and has good specificity. It can simultaneously analyze the characteristic spectra and evaluate the content of leech medicinal materials. It can be used as a quality evaluation method for different primitive leeches and their mixtures, providing a reference for the quality evaluation of leech medicinal materials.