Jinhua LIU, Shuang PAN

Jining Center for Food and Drug Control, Jining 272025, China

Abstract This article reviews the species, identification, chemical composition and pharmacological effects of hirudo. Hirudo is widely sourced, difficult to identify, contains a variety of active ingredients, and has a variety of pharmacological effects. In-depth research on hirudo is of great significance to the quality control and clinical application of hirudo.

Key words Hirudo, Species, Identification, Chemical composition, Pharmacological effect

1 Introduction

Hirudo is the dried whole body ofWhitmaniapigraWhitman,HirudonipponicaWhitman, orWhitmaniaacranulataWhitman, which is captured in summer and autumn, scalded to death with boiling water, sun-dried or dried at low temperature[1]. Hirudo was first recorded in theShennong’sClassicofMateriaMedica, tastes salty, and is neutral in nature[2]. According to the record ofCompendiumofMateriaMedica, the saltiness and bitterness of hirudo can be used to remove blood stasis[3]. According to literature, hirudo is neutral in nature, slight toxic, and metabolized through liver, tastes salty and bitter, has the effects of removing blood stasis and regulating bodily functions, and is mainly used for treating menostasis, abdominal mass, apoplexy, hemiplegia and traumatic injury.

Hirudo has a significant effect in various blood stasis diseases, and physicians of the past dynasties have given high evaluations. In particular, the discovery of hirudin in recent years has aroused widespread concern in the pharmaceutical industry. Many scholars have thought that hirudin is the main active ingredient for hemolysis of hirudo. This article reviews the species, identification, chemical composition, and pharmacological effects of hirudo, in order to provide a theoretical basis for the systematic research of hirudo.

2 Species

At present, the following hirudo species in China are considered to have medicinal value:WhitmaniapigraWhitman,HirudonipponicaWhitman,WhitmaniaacranulataWhitman,PoecilobdellamanillensisLesson,PoecilobdellajavanicaWahlberg,PoecilobdellahubeiensisYang,HaemadipsahainanaSong, Zhang et Tan,HaemadipsatianmushanaSong,HaemadipsajaponicaWhitman,WhitmanialaevisBaird andErpobdellaoctoculataLinnaeus (Fig.1). Among them,H.nipponicaWhitman,P.manillensisLesson,P.javanicaWahlberg andDinobdellaferoxare hematophagous. In current domestic medicinal material market,W.pigraWhitman has the largest sales volume, followed byH.nipponicaWhitman,W.acranulataWhitman andP.manillensisLesson.

Fig.1 Taxonomy of hirudo

3 Identification

According to theChinesePharmacopoeia(2020 edition), hirudo has three sources. However, in a broad sense, hirudo is a general term for animals in the class Annelida. At present, there are many similar hirudo materials on the market, and it is difficult to distinguish. The identification methods of medicinal materials include traditional character identification method. Whitmania has a shape of flat spindle with many links, 4-10 cm in length and 0.5-2.0 cm in width. The back is dark brown and slightly bulging. After soaking in water, the black spots arranged in five vertical stripes can be seen. The ventral surface is flat and brownish-yellow. Abdomen is brownish-yellow on both the sides, slightly pointed in the front end and blunt in the back end. There is one suction cup at each end. The front suction cup is not prominent, and the rear suction cup is larger. The texture is brittle, and easy to break, and the section is gelatinous. The taste is slightly fishy. Hirudo is oblate and cylindrical. The body is mostly bent and twisted, with a length of 2-5 cm and a width of 0.2-0.3 cm.W.acranulataWhitman is long and flat, 5-12 cm long and 0.1-0.5 cm wide. With the widespread application of hirudo in preparations for cardiovascular and cerebrovascular diseases, many fake products have appeared on the market, seriously affecting the safety and effectiveness of clinical medication. Therefore, it is particularly necessary to accurately identify the genuine origin of hirudo. In traditional identification methods, hirudo is mainly identified by characters and thin-layer chromatography, with weak specificity. In Chinese patent medicines containing hirudo, the characters of hirudo are invisible, and traditional identification methods are more difficult to meet actual work needs. With the rapid development of molecular biology and the improvement of PCR technology, methods for species identification of hirudo have emerged, and DNA barcoding technology is one of the most widely used. DNA barcode technology is a new biological species identification technology developed in the world in recent years. Since the "DNA barcode" was proposed, a large number of relevant studies and reports have appeared in top international academic journals and media. A mitochondrial gene COI (or cox1) with a length of about 650 bp was selected as the animal DNA barcode (Hebertetal., 2003a, 2003b). In February, 2005, the first session of International Conference of Life Barcode Association hosted by the International Barcode Union and the British Natural History Museum was held in London. In September, 2007, the second session of life barcode conference was held in Taipei, China. These two sessions of conference established COI as a universal animal barcode sequence. Many studies have explored the possibility of using mitochondrial cytochrome c oxidase subunit I (COI), internal transcribed spacer (ITS), ITS2, 12S rRNA and 16S rRNA to classify and identify hirudinous animals. The results show that the above-mentioned genes can effectively identify hirudinous animals to different extents. To a certain extent, they can meet the urgent needs of relevant industries for species identification.

4 Chemical composition

4.1 HirudinIn 1884, Haycraft first discovered an anticoagulant substance from the extract of medical hirudo. In 1904, Jacoby named the substance hirudin (hirudin). Markwardt isolated pure hirudin from the peripharyngeal glands of medical hirudo in 1955. Hirudin is a single-chain polypeptide composed of 66 amino acid residues. Its relative molecular mass is about 7 000. There are 3 pairs of disulfide bonds (Cys6-Cysl4, Cys16-Cys28, Cys32-Cys39) at the N-terminus, which make the peptide chain wrap into a dense core ring peptide structure and stabilize the protein structure. The N-terminal contains an active center, enabling it to recognize and bind to the enrichment site of the basic amino acid of the substrate-thrombin. There is a special sequence composed of Pro-Lys-Pro in the middle of the peptide chain of hirudin. It will not be degraded by ordinary proteases. This structure helps maintain the stability of the hirudin molecule and guides the hirudin molecule to bind to the thrombin molecule in the correct direction[4]. It is reported that hirudin only exists in the salivary glands of hirudo, and its content is very low and activity is extremely sensitive[5]. Some literatures have shown that recombinant hirudin can be synthesized by genetic engineering. Hirudin is a highly effective, specific, and strongest thrombin inhibitor currently known. As a blood activating and removing blood stasis medicine, it can affect blood coagulation and promote fibrinolysis in multiple links. It is widely used clinically for anticoagulation and antithrombosis.

4.2 Other macromolecular substancesThe main components of hirudo are macromolecular compounds. It contains 17 kinds of amino acids, which account for about 49% of hirudo, including 7 essential amino acids, tryptophan, valine, lysine, leucine, isoleucine, threonine and phenylalanine. Qu Xinyan[6]speculated that free amino acids may be the main effective components for anticoagulation of hirudo. In recent years, a variety of polypeptides have been isolated from hirudo, including an anticoagulant substance named "whitmanin", which has a relative molecular mass of 8 608, and a structure that a carbohydrate chain with a relative molecular mass of 1 898 is bound to a peptide chain formed by 63 amino acid residues[7-8].

4.3 Small molecule substancesThe current research shows that hirudo contains 14 kinds of trace elements, including Co, Cu, Zn and Fe. Studies have shown that Ca and Me in hirudo are essential macroelements for the human body. Yan Jidong[9]found that hirudo can treat diseases caused by changes in trace elements in the body’s physiological functions. In addition, hirudo also contains pteridines, glycolipids, carboxylic acid esters and steroids and other small molecules.

5 Pharmacological effects

5.1 Anticoagulant and antithromboticHirudin is considered to be the most specific thrombin preparation at present. It exerts anticoagulant effect by combining with thrombin. The combination of hirudin and thrombin inhibits the activation of thrombin on platelets in the process of thrombosis, thereby inhibiting the formation of thrombus. Zhai Xinyan’s research shows that the extract of hirudo can prolong the time of blood clotting and bleeding in mice, indicating that the extract of hirudo has anticoagulant effect. Li Yanlingetal.[10]showed that the ethyl acetate layer of hirudo had the best anticoagulation effect, and the n-butanol layer and n-hexane layer also had different degrees of anticoagulation effect.

5.2 Anti-inflammatory and improving cerebral hemorrhage

Cerebral hemorrhage seriously endangers people’s health. It has a high fatality rate, but there is still no specific treatment for it. Studies have shown that hirudin extract contains anti-inflammatory enzymes and has anti-inflammatory effect. Wu Yingetal.[11]confirmed in research reports that the addition of hirudin on the basis of controlling cerebral hemorrhage in the early stage of cerebral hemorrhage can reduce acute brain tissue damage.

5.3 Anti-tumorA lot of research shows that hirudin can inhibit the growth of tumor cells by inhibiting the action of thrombin in the body. Cui Meiyueetal.[12]found that the active factor of hirudin has a significant inhibitory effect on S180 tumor. Yu Ruifaetal.[13]showed that the extract of hirudo has a strong inhibitory effect on transplanted tumors in mice.

6 Conclusions

With the deepening of research on hirudo, significant results have been achieved in terms of species identification, chemical composition and pharmacological effects. However, the identification of the source of hirudo, the research of hirudin, and the in-depth research on the ingredients of hirudo for promoting blood circulation are still in the preliminary stage, and there is still a lack of more extensive application data on its pharmacological effects. Therefore, the research on hirudo will not stop here, and it requires the continuous efforts of the majority of scholars to solve many problems that exist in reality, and make hirudo give full play to a wide range of curative effects.