Studies On Improved Quality Standard And Preliminary Pharmacologic Effects Of QiZao Oral Liquid

ObjectiveQiZao Oral Liquid(QZOL),a Chinese patent medicine,is composed of four medicinal herbs including Radix Astragali,Fructus Jujubae,Poria and Helms Mucuna sempervirens.It has effect to supplement qi to invigorate spleen and nourish blood to activative blood circulation.In clinic,it is mainly used for leukopenia,body asthenia after illness and liver damage caused by immune suppression,etc.Recently QZOL quality standard is not complete enough due to its single and out-dated evaluation method,lack of quality control for Helms Mucuna sempervirens,and no pharmacodynamic studies on QZOL.It was put forward by China Food and Drug Administration to improve the quality standard of QZOL.Therefore,this project aims to establish quality standards of mucuna sempervirens extract with more indicators,set up comprehensive evaluation system to evaluate its quality more objectively and reasonably,laying the foundation for the quality control of QZOL;Besides,by improving the content determination method of Astragaloside by HPLC-ELSD and establishing the HPLC fingerprint of the QZOL,this topic expect to optimize the quality standards of QZOL systematically,which is providing scientific basis for its security,effectiveness and stability.Preliminary pharmacologic effects investigation on the function of supplementing qi and nourishing blood of QZOL is also carried out.Methods1.Establishment of the quality standards of mucuna sempervirens extractBy investigating kinds of factors,such as eight different extraction methods,two inspect way,temperature and humidity,the TLC method was established to identify mucuna sempervirens which Chosed the formononetin as control.With reference to the 2015 edition of China pharmacopoeia,the extract content,total ash and heavy metal residues were detected by the relevant provisions.The HPLC fingerprints of mucuna sempervirens was established by adopting PAD detector wavelength scanning to choose the best detector wavelength,and inspecting different factors,such as extraction method,chromatographic column,mobile phase,Column temperature,flow velocity and gradient elution,with corresponding methodological investigation;With traditional Chinese medicine(TCM)fingerprint database software,ten batches of fingerprints were analyzed,in which characteristic peaks were labeled with reference substance.The mutual pattern was set up from fingerprints,which was carried on recognizing pattern with principal components analysis and cluster analysis in SIMCAP-17.The coupling technique of HPLC-DAD was employed to determine the content of protocatechuic acid,genistein and formononetin.2.Improvement of quality standards of QZOLAstragalus glycosides and formononetin being chosen as the control,TLC method was adopted to identify Radix Astragali and Mucuna sempervirens of QZOL,by investigating kinds of factors,such as eight different extraction methods,two inspect way,temperature and humidity.The coupling technique of HPLC-ELSD was employed to determine the content of Astragaloside of QZOL,inspecting different factors,such as extraction method,chromatographic column,mobile phase,Column temperature,flow velocity and gradient elution,with corresponding methodological investigation;20 batches of the content of Astragaloside were determined with this method,the content limit was formulated according to the results.The determination with HPLC-DAD was employed to establish the fingerprints of QZOL,by adopting PAD detector wavelength scanning to choose the best detector wavelength,and inspecting different extraction conditions and chromatographic conditions as well as the system methodology.With traditional Chinese medicine(TCM)fingerprint database software,ten batches of fingerprints were analyzed,in which characteristic peaks were labeled with reference substance.The mutual pattern was set up from fingerprints,which was carried on recognizing pattern with principal components analysis and cluster analysis in SIMCAP-17.0.3.Studies on the pharmacologic effects of QZOL3.1 Blood defieieney in mice was mimicked by directly drawing blood once 5 mL/kg consceutively for 12 day.The treatment group was given the QZOL,positive control group was given the WeiXueKang syrup.The amount of RBC,WBC,HGB and PLT was dectected after treatment.3.2 Blood defieieney in mice was copyed by injecting cyclophosphamide,the treatment group was given the QZOL,the positive control group was treated the Transfer Factor Ord Solution.The amount of RBC,WBC,HGB and PLT was dectected during the 16th and 30th treatment day.At last,a complete femur of mice of all of the bone marrow was washed out with 3%acetic acid solution.A single cell suspension was produced so that the bone marrow cells can be counted.3.3 The 80 BALB/c mice were randomized into normal control group,Transfer Factor Ord Solution group and QZOL high-dose,medium-dose and low-dose groups,which were given relevant medicines intragastrically for consecutive 30 days.The visceral index and phagocytic index were determined,investigate the effect of QZOL on the visceral index and phagocytic index of BALB/c mice;3.4 The 108 SD rats were randomized into normal control group,model group,Positive control group p and QZOL high-dose,medium-dose and low-dose groups,which were given relevant medicines intragastrically for consecutive 15 days.The qi deficiency model was induced by forcing rats to swim until they were exhausted.The red cell immune function was determinated with Yeast erythrocyte garland test method,and Hemoglobin,blood lactic acid and ATP enzyme activity were determinated by blood analyzer,which are providing foundation to evaluating the immunologic function of QZOL.Results1.Establishment of the quality standards of mucuna sempervirens extractThe 10 batches of mucuna sempervirens extract met the Chinese Pharmacopoeia 2015 requirements in extract content,total ash and heavy metal.The extract content was 9.28%～18.27%,the total ash was 1.45%～2.58%,the heavy metal of Pb was less than 10 ppm,the Cd was less than 10 ppm,the As was less than 5 ppm,the Hg was ionreference substances with good resolution in the corresponding position.This method was stable with good repeatability and durability.The HPLC-DAD analysis methods were established to determine 3 components including protocatechuic acid,genistein and formononetin,which showed good linearity(r>0.999)in the range of the test concentration,and the recoveries of protocatechuic acid was 97.82%～104.99%(RSD=2.59%),the recoveries of genistein was 98.61%～104.42%(RSD=2.34%).the recoveries of formononetin was 95.52%～99.34%(RSD=1.79%).The content of protocatechuic acid is between 0.0506-5.4343mg/g,genistein is between 0.0169～1.8115 mg/g,and formononetin is between 0.0837～0.7814mg/g.The fingerprint chromatogram of HPLC-DAD was built up,which was established on a SHISEIDO-CAPCELL PAK-C18(4.6 mm×250 mm,5 μm),and the column temperature was 30 ℃,the mobile phase consisted of acetonitrile and 0.2%phosphoric acid in gradient elution and the flow rate was 0.8 mL/min.In the fingerprint chromatogram,thirteen common peaks were labeled,and three compentents were identified,among which formononetin was used as reference compound.What is more,the relative retention times of characteristic peaks were confirmed.The RSD of relative retention time of samples were less than or equal to 3.55%,the mean similarity of those samples was higher than 80%2.Improvement of quality standards of QZOLTLC method was adopted to identify Radix Astragali and Mucuna sempervirens.The TLC spots showed the same colors to the reference substances with good resolution and clear spots in the corresponding position.And the negative control showed no interference.The HPLC-ELSD analysis methods were established to determine the content of Astragaloside of QZOL,which was established on a Agilent-TC-C18(4.6mmwas est,5 μm),and the column temperature was 30℃,the mobile phase consisted of acetonitrile and water in a rate of 36:64,and the flow rate was 1.0 mL/min.which showed good linearity(r>0.9999)in the range of the test concentration,and the recoveries of Astragaloside were 95.05%～104.93%(RSD=3.60%),the average recoveries was 99.12%.The content of Astragaloside is between 0.1258～0.2623mg/mL,The average content was 0.2026 mg/ml((RSD<4.29%).Meanwhile,the specific chromatogram of HPLC-DAD was set up,which was established on a Agilent-TC-C 18(4.6 mmwas est,5 6 mmwas established on a Agilent-TC-C 18℃,the mobile phase consisted of 50%acetonitrile and 0.2%phosphoric acid in gradient elution,and the flow rate was 0.6 mL/min.From the specific chromatogram,twelve common peaks were labeled.Five competents were identified,amomg which ononin was chosen as the reference compound.The relative retention times of characteristic peaks were confirmed.The RSD of relative retention time of samples were less than or equal to 5%.The mean similarity of those samples was higher than 92%,which met the requirement of the fingerprint chromatogram technology.3.Studies on the pharmacologic effects of QZOL3.1 In the experiment of blood deficiency model mice induced by ossaorbitals,compared with model group,the hemoglobin content are obviously increased of the positive group(P<0.01),there is no statistical difference in the rest of group(P>0.05).3.2 In the experiment of blood deficiency model mice induced by injecting of cyclophosphamide,compared with model group,the WBC,PLT,bone marrow nucleated cells were increased(P<0.05)in the positive group,the Medium and high dose group mice of the process I.In the while,the WBC,RBC,HGB,PLT,bone marrow nucleated cells were increased(P<0.05)in the Medium and high dose group mice of the process II.3.3 compared with the normal group,the phagocytic index of the positive control group was increased(P<0.05),however,the rest of treatment groups did not show any Statistical differences(P>0.05).3.4 compared with the blank group,the Red blood cell immune garland rate and the ATPase content of the model were diminished(P<0.05);compared with model group,the swimming time and increase the content of ATPase of the positive control group were increased(P<0.05),so were the each dose of the treatment group.Conclusion1.With the perspective of qualitative and quantitative,this project established and enhanced the quality standards of QZOL and mucuna sempervirens extract,which is evaluated from three inspects named Thin-layer identification,Content determination and HPLC fingerprint.This study provide a scientific basis to evaluate QZOL’s effectiveness,safety and stability reasonably.2.This study confirmed that two different process of QZOL both have certain effects on nourishing blood and tonifying qi;at first,one sample of QZOL called the process Ⅰ can improve the blood deficient model in mouse,two samples of QZOL namely the process Ⅰand the process Ⅱ both can enhance the hematopoietic function of mouse aplastic anemia model by using cyclophosphamide.secondly,the process Ⅰ and the process Ⅱ neither can strengthen monocyte-macrophages phagocytosis function of the normal mice significantly,while they both can prolong the swimming time and increase the content of ATPase of the qi deficiency model in mouse,showing that QZOL can improve the anti-fatigue function.