Study On Quality Standard Of Hirudo Freeze-dried Powder And Its Therapeutic Effect On Acute Blood Stasis Rats

Objective:Hirudo freeze-dried powder is a new kind of processed product obtained by freeze-drying.This study established the producing technology and exclusive Polymerase Chain Reaction（PCR）identification system of Hirudo freeze-dried powder,studied the acute toxicity and its effect on acute blood stasis rats,and established the quality standards of Hirudo freeze-dried powder.Finally,the quality control of Hirudo freeze-dried powder can be carried out comprehensively through production process-toxicology-pharmacodynamics-quality standards,which could ensure its quality and safety and provide scientific basis for its industrial manufacture and clinical practice.Methods:1.The pre-freezing temperature and sublimation drying temperature of Hirudo freeze-drying process were determined by differential scanning calorimeter.Single factor test and L₉（3⁴）orthogonal test were used to optimize the pre-freezing time,working pressure,drying time,end point of analytical temperature:time and other parameters,so as to determine the best parameters of Hirudo freeze-drying process.Three batches of laboratory samples were prepared in parallel,and their properties,lyophilization rate and antithrombin activity were investigated.2.The PCR identification system of Hirudo was constructed:specific primers were designed and synthesized for Whitmania pigra Whitman,Hirudonip ponica Whitman and Whitmania acranulate Whitman.The PCR conditions were optimized,and the DNA template of 7 different varieties of leech were amplified to investigate the specificity of the primers.Stability test,sensitivity test and specificity test were used to verify the PCR methodology.3.The acute toxicity of Hirudo freeze-dried powder was investigated through the maximum dose test,and the effects of Hirudo freeze-dried powder on coagulation function,hemorheology,platelet index,histopathology and oxidative stress level in acute blood stasis rats were investigated to evaluate the therapeutic effect.4.Identify Hirudo freeze-dried powder by characteristics,TLC and polymerase chain reaction,and control the quality by determining moisture,total ash and other examination items,antithrombin activity,and finally the quality standard was established.Results:1.The optimal process of Hirudo freeze-dried powder:pre-freezing for6 h（-20℃）,sublimation drying for 9 h（20 Pa,-5℃）,analytical drying for 10 h（20 Pa,rising from-5℃to 45℃at a rate of 5℃/h）,heat preservation drying for 2 h（20 Pa,45℃）,and complete freeze-drying period for 27 h.The color of the samples was uniform,the framework was intact,the lyophilization rate was 96.4%～97.1%,and the antithrombin activity was 30 U/g（Whitmania pigra Whitman freeze-dried powder）and220 U/g（Hirudonip ponica Whitman freeze-dried powder）.The results of different batches of samples were stable.2.The primer sequences of Whitmania pigra Whitman,Hirudonip ponica Whitman and Whitmania acranulate Whitman are WP5’-GAGGCTACCAACCTG AACGATTA,WP3’-AGCCGAAGACCTAAATCCATGAG,HN5’-GCCGGGGTT TGGAGCAATTTCA,HN3’-TCGCCGTGGTATTCCTCTTAGCC,WA5’-GCCCC CGAACGAACCTCTCCC,WA3’-TGCCATCGCAGCTGGTAGTCCA,respecti-ve ly.The annealing temperatures were 60℃,61℃,59℃,and the target strip le ngths were 1000～2000 bp,1000～2000 bp,and 3000 bp,respectively.Three p airs of primers could distinguish the corresponding Hirudo varieties from othert wo Hirudo varieties and four non-pharmacopoeial leech varieties,respectively,with strong specificity.Under the same PCR condition,similar and bright ban-ds could be stably amplified from multiple batches of Hirudo samples,which showed good stability.The detection limits of the three pairs of primers were0.1 ng,1 ng and 10 ng,respectively,indicating high sensitivity.Three pairs of primers were able to accurately identify leech samples mixed in different pro-portions,with strong specificity.3.In the acute toxicity test,there was no acute toxicity phenomenon in the freeze-dried powder of Whitmania pigra Whitman and Hirudonip ponica Whitman,when the dosage reached 56 and 68 times of the clinical safe dose for adults,respectively.It was indicated that Hirudo freeze-dried powder had no risk of acute poisoning.In the study of the effect of acute blood stasis rats,Hirudo freeze-dried powder group can extend plasma PT,APTT,TT value,lower levels of WBV and PV,reduce serum TXB 2,GPIIb/IIIa,CD62p,v WF levels,increase SOD and GSH-Px,reduce the MDA content,improve heart,pneumonic sex cells infiltration,hyperplasia of fibre pathological damage,among them,the effect of Hirudonip ponica Whitman is superior to Whitmania pigra Whitman.4.In the study of quality standard of Hirudo freeze-dried powder,PCR system can be used for the identification of Hirudo freeze-dried powder;The moisture,total ash,acid-insoluble ash content should not exceed 4.0%,8.0%and 2.0%respectively;p H value should be in the range of 5.0～7.5;lead,cadmium,arsenic and mercury shall not exceed 10 mg/kg,1 mg/kg,5 mg/kg and 1 mg/kg respectively;aflatoxin B₁ shall not exceed 1 g,and the total amount of aflatoxin G₁,G₂,B₁ and B₂ shall not exceed 3 g;antithrombin activity of Whitmania pigra Whitman freeze-dried powder should not be less than 15.0 U/g,which of Hirudonip ponica Whitman freeze-dried powder should not be less than 110.0 U/g.Conclusion:The results of laboratory test showed that the process of Hirudo freeze-drying has good feasibility and reproducibility,which could provide reference for the large-scale production of leech freeze-dried powder.The PCR system could be used for the accurate identification of Hirudo freeze-dried powder.The methodology verification test showed that the system was stable and feasible.The acute toxicity experiment proved that there was no risk of acute poisoning after Hirudo was freeze-dried into freeze-dried powder.The preliminary pharmacodynamics experiment confirmed that the freeze-dried leech powder had a better therapeutic effect on acute blood stasis syndrome than leech medicinal materials,which indicated that the new processing method of freeze-drying had a significant advantage in the application of Hirudo.Finally,the quality control of Hirudo freeze-dried powder can be carried out comprehensively through production process-toxicology-pharmacodynamics-quality standards,which can provide scientific basis and research ideas for the production and application of Hirudo freeze-drying powder.