ABSTRACT
OBJECTIVE:To establish the quality standard of Hirudo nipponica freeze-dried powder(called"freeze-dried powder"for short),and to provide reference for controlling its quality. METHODS:A total of 3 batches of freeze-dried powder were collected,identified and tested according to the requirements of H. nipponica stated in 2015 edition of Chinese Pharmacopoeia(part Ⅰ)(shorted for pharmacopoeia);the antithrombin activity was also analyzed. The maximum tolerated dose (MTD)was used to investigate the toxicity. The stability was determined by designing temperature,humidity and strong light exposure tests. RESULTS:In the TLC of test sample,the same red spots were found in the corresponding location of the control drug chromatogram,and the same orange-red fluorescence spots were shown under the UV light(365 nm). Average content of moisture in 3 batches of samples was 2.61%,and the levels of total ash,acid-insoluble ash,pH aflatoxin and antithrombin activity were 2.83%,0.38%,6.92,0.28 μg/kg and 257.0 U/g,respectively. The content of Pb,Cd,As and Cu were in line with the requirements of pharmacopoeia except that the content of Hg was slightly higher than lower limit of H. nipponica in pharmacopoeia. Results of MTD showed that no death and ADR was found in mice after giving 26.4 g/kg freeze-dried powder by the amount of crude drug,which was 58 times as large as the maximum dosage that the pharmacopoeia described. Under the condition of 20, 40 ℃ and strong light exposure [(4 500±500)Lx],the anticoagulase activity of freeze-dried powder decreased significantly over time,while the anticoagulase activity of freeze-dried powder stored at 40 ℃ for 6 months was in line with the requirements of pharmacopoeia. Under the condition of high humidity(relative humidity were 90%,75%),freeze-dried powder showed a strong hygroscopicity. CONCLUSIONS:Established quality evaluation standard for freeze-dried powder according to pharmacopoeia standard could be used to control its quality.
ABSTRACT
Leeches are not only important medicinal animals worldwide but also are endangered. We aimed to (i) explore the level of genetic diversity within/among populations of three leeches, (ii) assess genetic differentiation among these three leeches, and (iii) discuss an appropriate strategy for conserving leech germplasm. A total of 315 individuals of Whitmania pigra, Hirudo nipponica and Poecilobdella manillensis from 21 populations were collected in China and Vietnam. The genetic structure and genetic diversity among and within the 21 populations were evaluated using target region amplified polymorphism (TRAP) and simple sequence repeat (SSR) markers. Sixteen pairs of TRAP primers generated a total of 398 fragments, of which 396 (99.50%) were polymorphic; fourteen pairs of SSR primers generated a total of 60 fragments, of which 59 (98.33%) were polymorphic. Shannon's index (I) and Nei's gene diversity index (H) for the three leeches were high at the species level (I=0.4980 and H=0.3323 for TRAPs, I=0.4487 and H=0.2969 for SSRs in W. pigra; I=0.4147/0.3769, H=0.2788/0.2566 for H. nipponica; and I=0.4616/0.4717, H=0.3099/0.3203 for P. manillensis). However, low genetic diversity was determined at the population level; the average genetic diversity measures within populations were H=0.1767/0.1376, I=0.2589/0.2043 for W. pigra, H=0.2149/0.2021, I=0.3184/0.3000 for H. nipponica and H=0.2850/0.2724, I=0.4152/0.3967 for P. manillensis. We conclude that there was limited gene exchange within/among populations and species, as the gene flow number (Nm) was 0.5493/0.5807. However, for all three species, the genetic diversity was different at the population level. Gene differentiation (Gst) and Nm were 0.4682 /0.5364 and 0.5678/0.4321 for W. pigra, 0.2294/0.2127 and 1.6797/1.8512 for H. nipponica and 0.1214/0.1496 and 3.6202/2.8412 for P. manillensis. STRUCTURE analysis, Unweighted Pair-Group Method with Arithmetic means (UPGMA) cluster analysis and Principal Coordinates Analysis (PCOA) all yielded similar results. The isolation-by-distance pattern was not significant for any of the three species by the Mantel test. These data emphasize the need for management, conservation, and rehabilitation of this animal species. Finally, an appropriate strategy for conserving leech is proposed.
Subject(s)
Genetic Variation , Leeches/genetics , Microsatellite Repeats/genetics , Phylogeny , Animals , China , Endangered SpeciesABSTRACT
Hirudo nipponica Whitman,a Chinese medicine which has a strong effect on platelet aggregation and antithrombin activity,is involved in the studies.The current situation is adverse to the cultivation,development and utilization of H.nipponica.Prior to summarizing the current research status and the unresolved problems and the prospective of H.nipponica,domestic and foreign literatures over the H.nipponica was discussed and judged,aiming at providing a reference for the comprehensive development and utilization of H.nipponica.
