摘要

水蛭药用的首次记载出现在汉朝《神农本草经》，水蛭具有悠久入药历史，是我国传统活血化瘀要药^[1]。2020年版《中华人民共和国药典》（以下称《中国药典》）第一部规定水蛭药材为水蛭科动物蚂蟥Whitmania pigra Whitman、水蛭Hirudo nipponica Whitman或柳叶蚂蟥Whitmania acranulata Whitman的干燥全体，具有破血通经、逐瘀消癥功能。近年来，心脑血管疾病患者的数量显著增多，含水蛭的中成药已广泛应用于临床，随着近年来含水蛭中成药的不断开发，水蛭药材需求量逐年递增。市场上的水蛭药材品种良多、质量不一。基原以宽体金线蛭为主，但仍有部分混伪品在药材市场流通。传统鉴定方法难以实现中成药中水蛭成份物种鉴定，因此亟须理清水蛭药材流通情况并进行鉴定。

水蛭药材市场调查及商品规格等级划分

本研究对药材市场、各产地、养殖基地、水蛭药材供应商及药店等多途径进行水蛭现状调研。水蛭药材正品来源中仅宽体金线蛭在国内有较广规模养殖，日本医蛭绝的来源大部分依靠野外捕获。尖细金线蛭则基本未见。水蛭药材商品规格多样，以清水吊干品为主。不同地区水蛭药材个体间有较为明显的形态差异，尤其是东北居群水蛭在外观体色上整体偏黑。水蛭药材价格近年来总体呈上升趋势。市场上流通的水蛭药材除了正品基原物种外，还有习称“金边蚂蟥”、“东北小水蛭”、“小绿条”以及“黑条水蛭”的多个品种。

按2020年版《中国药典》所述长、宽等要求进行测量记录，测定水蛭厚度及称量重量并进行统计分析。市售三种等级的水蛭药材组间长度、宽度、厚度及重量P均<0.05，差异显著。市售以宽体金线蛭为基原的水蛭药材可依据其长度、宽度、厚度及重量，辅以表面、断面特征，划分为一等、二等选货和统货3个等级及等下级、掺杂货。

水蛭本草考证和主要混伪品的基原鉴定

经系统文献梳理与分析，总结发现自古使用的传统中药水蛭的基原动物是一类生于水中、个头较小的吸血蛭类。2020年版《中国药典》规定的水蛭基原动物中满足这三种特征的只有日本医蛭，宽体金线蛭和尖细金线蛭均不完全符合此些特征。建议在水蛭药材基原物种上保留原有日本医蛭。是否需要增补吸血蛭类品种来源，还需对水蛭入药品种做进一步深入研究。对于宽体金线蛭和尖细金线蛭，探究其是否可能成为新的药用品种“蚂蟥”。建议对同属的光润金线蛭进行更有针对性的药理药效及化学成分研究。

市场上存在一定数量的水蛭药材掺伪现象。掺伪蛭类主要有菲牛蛭、光润金线蛭、八目石蛭及日本类蛭等，也均不满足本草考证发现的药用水蛭特征。本研究对这些混伪品蛭类动物进行了形态学观察，获得了更加直观展现蛭类动物形态的彩色生物学特征图谱，发现采用底座背光拍摄法可更加直观看清蛭类微小特征；发现《中国动物志·蛭纲》中未记录的石蛭属蛭类眼点数目新变异。

基于DNA条形码技术的水蛭及其混伪品鉴定

本研究共收集130份宽体金线蛭为基原的样品，测序结果拼接后获得162条COⅠ序列，76个单倍型。经聚类分析，所有样本分为两个subclade。系统发育结果表明东北居群宽体金线蛭和其他地区的分别聚为独立的一支。

共收集22份日本医蛭为基原的样品，获得COI序列22条，构建NJ树结果可见样品分为两个subclade。subclade 1包含17个样品。subclade 2包含5份样品，形态特征与subclade 1相比，整体颜色偏深，背部棕褐色或黑色，5条黄白色的纵纹呈断续的棒状，subclade 2样品为东北来源日本医蛭。

共收集27份水蛭药材混伪品样品，拼接后获得COⅠ序列27条。以三种水蛭药材正品来源为外类群进行聚类分析。结果显示27个混伪品样品不与正品来源聚为一支，分为4个subclade，分别为市场上习称“黑条水蛭”的石蛭属蛭类Erpobdella，习称“东北小水蛭”的八目石蛭Erpobdella octoculata，习称“金边蚂蟥”的菲牛蛭Poecilobdella manillensis和习称“小绿条”或“绿皮水蛭”的光润金线蛭Whitmania laevis。

天目山蛭线粒体基因组分析

本研究首次测定并报道了我国特有蛭类动物天目山蛭的线粒体全基因组。全长14,625 bp，由22个tRNA基因、2个rRNA基因、13个PCGs和三个控制区组成。线粒体全基因组核苷酸碱基含量分别为35.1% A、10.5% C、11.6% G和42.8% T。tRNA基因长度为57 bp-66 bp。系统进化树结果表明天目山蛭是一个单系进化支，与黄蛭科的三种水蛭明显分开为两个独立的分支。通过线粒体基因组理清天目山蛭系统发育关系，丰富了蛭类线粒体基因组数据建库。

基于鸟枪法宏条形码技术的脉血康胶囊水蛭成份物种鉴定

本研究对18份市售脉血康胶囊样品采用基于PCR-free的鸟枪法宏条形码技术进行水蛭成份的物种鉴定。所有样品中均能检出蛭类动物，说明此方法检测中成药中水蛭成份物种具有可行性。16份样品中检出了日本医蛭，其中1份样品中同时还检出了宽体金线蛭。18份样品中均检出了菲牛蛭。未检测到微生物污染。同批样品检测结果具有均一性，说明此方法的可靠性。这是鸟枪法宏条形码技术首次应用于含水蛭成份中成药的研究。基于市售脉血康胶囊中水蛭成份的鉴定，建立了一种中成药中水蛭成份的基原物种鉴定方法。

Abstract

Leeches were recorded for the first time in Shennong's Herbal Classic of Materia Medica, and have a long history of medicinal application. They are the traditional Chinese medicine for promoting blood circulation and removing blood stasis. In recent years, the number of patients with cardiovascular and cerebrovascular diseases has increased significantly, Chinese patent medicine containing leeches has been widely used in clinical practice. With the continuous development of Chinese patent medicine containing leeches in recent years, the demand for leeches has been increasing year by year. The leech medicinal material on the market has many varieties and different quality. But there are still some counterfeit products in the medicinal market circulation. However, traditional identification methods are difficult to identify the components of Chinese patent medicine leeches, so it is urgent to optimize the methods for monitoring the safety and quality of leeches.

HIRUDO market survey and commodity specification grade division

In this study, the current situation of HIRUDO was investigated in the market of medicinal materials and in different producing areas. Among the genuine sources of HIRUDO, only the W. pigra have been widely cultivated in China, and most of the sources of H. nipponica rely on field capture. W. acranulata were almost absent. HIRUDO commodity specifications are diverse, mainly with dry water hanging. There were obvious morphological differences among individuals of leeches from different regions, especially in appearance and body color of HIRUDO from northeast population. The price of leech medicine is rising in recent years. The HIRUDO that circulates on the market besides official source species, still have P. manillensis, W. laevis, E. octoculata and Erpobdella leeches for many varieties.

The length, width, thickness and weight of HIRUDO were measured, recorded and analyzed statistically according to the requirements of the 2020 edition of The Chinese Pharmacopoeia. The length, width, thickness and of the three grades of commercial HIRUDO were all P <0.05, and the difference was significant. According to its length, width, thickness and weight, as well as the characteristics of surface and section, the HIRUDO on the market can be divided into three grades: first class selected goods, second class selected goods and unified goods, and second class and adulterated goods.

Textual research of HIRUDO and primitive identification of main adulterants

After systematic literature combing and analysis, it is concluded that the leech primitive animals used in traditional Chinese medicine since ancient times are small leeches that lives in water and can suck blood. In the leech protozoa stipulated in The Chinese Pharmacopoeia in 2020, only H. nipponica meet these three characteristics, while the W. pigra and W. acranulata do not fully meet these characteristics. It is suggested that the original H. nipponica should be preserved on the original species of leeches. Whether it is necessary to supplement bloodsucking leeches needs further research on leeches used in medicine. For W. pigra and W. acranulata, explore whether they may become a new medicinal variety "MAHUANG". It is suggested to carry out more targeted pharmacological and chemical studies on the same genus of W. laevis.

There are a certain number of adulterated leeches in the market, such as P. manillensis, W. laevis, E. octoculata and Mimobdella japonica, which did not meet the characteristics of medicinal leeches found in textual research. In this study, morphological observations were made on these counterfeits, color micrographia of biological characteristics was obtained, which could display the morphology of leeches more intuitively. It is found that the base backlight photography method can be used to see the tiny characteristics of the leeches more directly. A new variation in the number of eyeches in Erpobdella leeches, which was not recorded in Zoology of China, Clitellata, was found.

Identification of HIRUDO and their adulterants based on DNA barcoding technology

A total of 130 W. pigra samples were collected in this study, and 162 COI sequences and 76 haplotypes were obtained after sequencing results were splined. After cluster analysis, all samples were divided into two subclades. The phylogenetic results showed that the HIRUDO from northeast China and the others clustered into two separate branches.

A total of 22 H. nipponica samples were collected, and 22 COI sequences were obtained. The results of NJ tree construction showed that all samples were divided into two subclades. Subclade 1 contained 17 samples. Subclade 2 contains 5 samples, compared with Subclade 1, the overall color is darker, with brown or black back, and 5 yellow and white longitudinal lines are in the shape of intermittent rods. Subclade 2 samples were from Northeast China.

A total of 27 counterfeit samples were collected, and 27 COI sequences were obtained after splicing. Cluster analysis was conducted to construct NJ tree with three authoritative sources of leeches as outgroup. The results showed that the 27 counterfeit samples were not fused with the genuine ones, and were divided into 4 subclades, which were respectively Erpobdella leeches, E. octoculata, P. manillensis, W. laevis.

Mitochondrial genome analysis of Haemadipsa tianmushana

The complete mitochondrial genome of H. tianmushana endemic to China was reported for the first time. The total length was 14,625 bp. It consists 22 tRNA genes, 2 rRNA genes, 13 PCGs and three A+T regions. The contents of nucleotide bases in the whole mitochondrial genome were 35.1% A, 10.5% C, 11.6% G and 42.8%T, respectively. The length of tRNA gene ranged from 57 bp to 66 bp. Phylogenetic analysis showed that H. tianmushana were a mono-phyletic clade, which was separated with three species of Whitmania into two independent branches obviously. Through mitochondrial genome, the phylogenetic relationship of H. tianmushana was clarified and the database of leech mitochondrial genome data was enriched.

Identification of leech components in Maixuekang Capsules based on shotgun metagenomic sequencing

In this study, 18 samples of Maixuekang capsules were used for species identification by PCR-free shotgun sequencing. Leeches were detected in all the samples, indicating that this method is feasible to detect leeches in Chinese patent medicine. H. nipponica were detected in 16 samples, and one of them also had W. pigra. P. manillensis were detected in all 18 samples. No microbial contamination was detected. The uniformity of the test results of the same batch of samples indicates the reliability of this method. It is the first time that Shotgun metabarcoding has been applied to the study of patent medicine containing leech ingredients. Based on the identification of leeches in Maixuekang capsules, a method for identification of leeches in Chinese patent medicine was established.