| Objective:Hirudo are dried whole bodies of Whitmania pigra Whitman,Hirudo nipponica Whitman and Whitmania acaranulata Whitman,which have the effects of removing blood stasis and dredging meridian.They are mainly used in the treatment of blood stasis and amenorrhea,abdominal mass,apoplectic hemiplegia,injuries due to falling and so on.At present,Whitmania pigra Whitman are the main hirudo on the market of medicinal materials while Hirudo nipponica Whitman and Whitmania acaranulata Whitman are rare.Also,plenty of counterfeit products exist.Furthermore,hirudo marketed are mostly from wild resources,resulting in quality difference and heavy metals over the limit.The chemical components of hirudo are mainly proteins,polypeptides,polysaccharides and other macromolecular compounds,and amino acids and other small molecular compounds.However,the Pharmacopoeia of the people’s Republic of China(2015 edition,hereinafter referred to as the Chinese Pharmacopoeia)has regulations on morphological identification,thin-layer chromatography(TLC)identification,quantitative methods of antithrombin activity and routine examination terms of hirudo,which makes it difficult to comprehensively evaluate the quality of hirudo and guarantee its effectiveness and safety of clinical medication.Considering the wide clinical application of hirudo,based on the current quality standard,this study established the quality control method of hirudo,including the identification,characteristic and fingerprint spectrum,heavy metal test,and the quantification of protein,amino acid,polypeptide and polysaccharide,and draft the quality standard of hirudo,so as to provide some reference for the quality control and quality standard research of hirudoMethods:(1)The TLC method of hirudo recorded in Chinese Pharmacopoeia(2015 edition)was difficult to identify the authenticity of the collected medicinal materials.Therefore,DNA of hirudo were extracted by kits and the DNA barcoding identification method was established.Furthermore,near-infrared spectrometer was applied to establish near-infrared spectrum consistency test model.(2)The characteristic spectrum of protein electrophoresis and the high performance liquid chromatography(HPLC)fingerprint of free amino acids were developed by vertical plate twelve alkyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and HPLC method,respectively.(3)Routine examinations and harmful substance tests listed in Chinese pharmacopoeia(2015 edition)were performed,including the moisture,total ash,acid insoluble ash,pH,heavy metal and aflatoxin investigations,to determine the contents of moisture,total ash,acid insoluble ash,pH,lead(Pb),cadmium(Cd),arsenic(As),mercury(Hg),aflatoxin G1,aflatoxin G2,aflatoxin B1 and aflatoxin Toxin B2.(4)Quantitative study on antithrombin activity in vitro was carried out according to Chinese pharmacopoeia(201 5 edition).The contents of hydrolytic and free amino acids,total protein,polysaccharide and polypeptide were determined by HPLC,semi-automatic nitrogen analyzer,UV spectrophotometer and enzyme standard instrument.(5)Based on the studies of in vitro antithrombin activity and in vivo adrenaline hydrochloride-induced zebrafish thrombus model,the antithrombotic effects of polysaccharide and enzymatic extracts of hirudo were preliminarily evaluated.Results:(1)The DNA barcoding identification method of hirudo was established.The barcoding length of 18 batches of hirudo samples was 617 bp based on mitochondrial cytochrome oxidase I(COI)gene,and their guanine cytosine(GC)content was in the range of 3 1.6-36.75%.There were 140 variation sites between hirudo and two batches of Hirudinaria Manillensis,and there were 195 variation sites between hirudo and eight batches of commercial counterfeit.Hirudinaria.Thus,Manillensis and the commercial counterfeit differed in sequence length and content.The genetic N-J tree showed that the genuine hirudo,Hirudinaria Manillensis and other counterfeit were clustered,respectively,and the support rate was 100%.The established near-infrared consistency test model could distinguish hirudo and the counterfeit with the Cl value set at 6.0.Through these two methods,we could achieve the true or false identification of the collected medicinal materials.And DNA barcoding method could be applied to fulfill the origin identification of the collected medicinal materials.(2)SDS-PAGE spectrums of protein and HPLC fingerprint spectrums of free amino acids were respectively established to perform the qualitative analysis of hirudo.16 Common peaks of the fingerprints were identified,and the similarity between the batches was higher than 0.960.Also,the results of cluster analysis and principal component analysis were consistent.(3)The moisture content,total ash,acid insoluble ash,pH value,and aflatoxin of hirudo all met the requirements of Chinese pharmacopoeia(2015 edition).Among the heavy metals,Pb and Hg of all batches met the standards,however,10 batches of medicinal materials of Cd and As exceed the limit standards.(4)Accurate and effective quantitative methods of free and hydrolyzed amino acids,total proteins,total polysaccharides and peptides in hydrolysates were respectively established and applied for the determination of 18 batches of hirudo.The mean value of hydrolytic amino acids in 18 batches of hirudo was ranked as follows:aspartic acid>glutamic acid>lysine>leucine>alanine>histidine>glycine>valine>arginine>threonine>serine>proline>tyrosine>phenylalanine>isoleucine>methionine,and the mean value of free amino acids was ranked as follows:glutamic acid>alanine>aspartic acid>leucine>glycine>valine>serine>proline>isoleucine>threonine>phenylalanine>methionine>lysine>tyrosine>arginine>histidine.The mean values of free and hydrolyzed amino acids in 18 batches of leeches were 9.9195±2.1025 mg/g and 101.2436±23.2982 mg/g,respectively.The content of total free and hydrolyzed amino acids could be considered as the quality evaluation indexes of hirudo;The average content of total protein in 18 batches of hirudo was 70.78±1.55%,and that reached the highest in S7(76.34%).The average content of polysaccharide in 18 batches of of hirudo was 6.14±1.50 mg/g,the polysaccharide content of S10 was as high as 13.22 mg/g.The average content of polypeptide in 18 batches of hirudo was 318.06 mg/g,and that of S8 was the highest as 354.54 mg/g.The contents of total protein,polysaccharide and polypeptide in different batches of hirudo were significantly different,which suggested that the content of total protein,polysaccharide and polypeptide could be considered as the quality evaluation indexes of hirudo.(5)Based on the study of in vitro antithrombin activity and in vivo zebrafish thrombus model induced by adrenaline hydrochloride,the polysaccharide and enzymatic hydrolysis extracts of hirudo exhibited obvious anticoagulant activity in vitro and antithrombotic activity in vivo.When the exposure concentration of polysaccharide and enzymatic hydrolysate was 300 and 260 μg/mL,the thrombus inhibition rate was as high as 73.72 and 78.72%,respectively.Moreover,the thrombus inhibition rate was dependent on the concentration of polysaccharide and enzymatic hydrolysate extracts,and the difference between groups was statistically significant.Conclusion:Hirudo has strong antithrombotic effect and is widely used in clinic.Therefore,it is very important to systematically evaluate and control the quality of hirudo.In this study,based on the established DNA barcoding and NIR spectrum methods,the origin and true or false identification of hirudo was performed,the protein characteristic spectrums and free amino acid fingerprint spectrums of hirudo were established,the routine and harmful substances tests of hirudo were assessed,the content of free and hydrolyzed amino acids,total protein,total polysaccharides and polypeptides were determined,and the in vitro and in vivo antithrombus activity of hirudo polysaccharides and enzymatic hydrolysate were evaluated.The quality control methods of hirudo were concluded to improve from many aspects.And the draft of quality standard is put forward.The polysaccharides and enzymatic hydrolysate in hirudo showed obvious antithrombotic activity in vitro and in vivo.And the polypeptide was found to be the main component of the enzymatic hydrolysate according to reference,therefore,the content of polysaccharide and polypeptide could be considered as the quality evaluation indexes of hirudo. |
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