Studies On The Materials Basis Of Medicinal Effectiveness In Kang'ai Injection

It is significant to study the materials basis of medicinal effectiveness in Chinese injection,not only for the quality control,but also for the medication safety and secondary development.Kang'ai injection is a kind of Chinese medicine injection,which is made from the extract of Panax ginseng C.A.Mey,the extract of Astragalus membranaceus?Fisch.?Bge.var.mongholicus?Bge.?Hsiao and oxymatrine.It can strengthen physique and enhance immunity.It also can treat malignant tumor such as liver cancer,lung cancer,adenocarcinoma cell of rectum,malignant lymphoma and gynecological malignant tumors.Accordingly,the chemical constituents and the relevant content in the Kang'ai injection was investigated in this paper.Meanwhile,the experiments of anti-tumor activity were conducted to investigate the correlation between the functional components and major function of Kang'ai injection.First,the extract of Panax ginseng C.A.Mey and the extract of Astragalus membranaceus were obtained,isolated and purified.The results show that there are11 kinds of the major ginsenosides in the extract of Panax ginseng C.A.Mey,including ginsenoside Rg₁,Re,Rf,Rg₂,Rb₁,Rc,Rb₂,Rd,Ra₁,Ra₂ and Ra₃;and there are also 10 kinds of the rare ginsenosides isolated and identified from the extract of Panax ginseng C.A.Mey,including ginsenoside 20?S?-Rh₁,20?R?-Rh₁,Rg₆,F₄,Rk₃,Rh₄,20?S?-Rg₃,20?R?-Rg₃,Rk₁ and Rg₅;8 kinds of flavonoids and 8kinds of astragalosides in the extract of Astragalus membranaceus were isolated and identified as,?3R?-7,2'-dihydroxy-3',4'-dimethoxyisoflavanone,?6aR,11aR?-3-hydroxy-9,10-dimethoxypterocarpan,?3R?-2'-dihydroxy-3',4'-dimethoxyisoflavanone-7-O-?-D-glucoside,?6aR,11a R?-9,10-dimethoxypterocarpan-3-O-?-D-glucoside,formononetin,ononin,calycosin,calycosin-7-O-?-D-glucoside,cycloastragenol,astragaloside I,astragaloside II,astragaloside III,astragaloside IV,isoastragaloside I,isoastragaloside II and isoastragaloside IV.Based on the above study,it provided the basis for the chemical component analysis and the tests of antitumor activity.Next,the constituents in the Kang'ai injection were confirmed according to the HPLC method.The results showed that there are 19 kinds of ginsenosides from Panax ginseng C.A.Mey and 5 kinds of flavonoids and 2 kinds of astragalosides from Astragalus membranaceus in the injection.Then different analytical methods coupled with the novel sample preparation techniques were established to determine the contents of 20 kinds of chemical compounds in the Kang'ai injection,which could be determine by HPLC.Based on deep-eutectic solvents?DES?,aqueous two-phase system coupled with high-performance liquid chromatography has been developed and applied.The proposed method was used to determine the major ginsenosides,including ginsenoside Rg₁,Re,Rf,Rg₂,Rb₁,Rc,Rb₂ and Rd.Single factors were investigated and the conditions of this experiment were optimized.Experiment conditions were as follows:choline chloride/1,4 butanediol?molar ratio of 1:1?was used as the type of DES,and the amount of DES was 0.5 g;K₂HPO₄ was used as the inorganic salt,and the amount of inorganic salt was 3.5 g;the ultrasonic time was 2 min.Under the appropriate optimal conditions,the limits of detection?LODs?were in the range from 0.34 to 1.45?g/mL.The intra-and interday precisions were in the range from1.3%to 5.2%in the optimal conditions.The satisfactory recoveries of analytes were in range from 92.7 to 110.2%with the relative standard deviations?RSDs?ranging from 1.2 to 3.9%.The experimental results show the contents of the major ginsenosides?ginsenoside Rg₁,Re,Rf,Rg₂,Rb₁,Rc,Rb₂,Rd?are 87.89⁹3.51?g/mL,68.38⁶9.43?g/mL,25.02³4.87?g/mL,4.86⁵.21?g/mL,9.48¹0.63?g/mL,14.57¹5.68?g/mL,3.22³.52?g/mL and 7.91⁸.56?g/mL,respectively.Air assisted liquid-liquid microextraction was used in this work for the first time to determine the rare ginsenosides in Kang'ai injection,including ginsenoside20?S?-Rh₁,20?R?-Rh₁,Rg₆,F₄,Rk₃,Rh₄,20?R?-Rg₃ and Rk₁.Single factors were investigated and the conditions of this experiment were optimized.Experiment conditions were as follows:DES?ChCl:phenol,1:3?was used as the extraction solvent,and the volume of DES was 400?L;THF was used as the emulsified solvent,and the volume of the emulsified solvent was 500?L;the concentrations of NaCl was 3.5%;the pH value was 6.0;the amount of Fe₃O₄ was 150 mg;the gas flow was 0.8 L/min,the ventilation time was 5 min.Under the optimum extraction condition,the validation parameters showed the limits of detection?LODs?were in the range from 10.2 ng/mL to 137.8 ng/mL.The recoveries of analytes were in the range from 91.3%to 106.7%with the RSDs ranging from 1.2%to 4.5%.The experimental results show the contents of the rare ginsenosides?ginsenoside20?S?-Rh₁,20?R?-Rh₁,Rg₆,F₄,Rk₃,Rh₄,20?R?-Rg₃,Rk₁?are 11.25¹2.21?g/mL,4.59⁷.78?g/mL,0.85¹.76?g/mL,2.00³.23?g/mL,0.73¹.42?g/mL,0.90¹.97?g/mL,3.94⁷.28?g/mL and 0.81⁰.97?g/mL,respectively.The contents of flavonoid glycosides and astragaloside III in Kang'ai injection was determined by using the magnetic dispersive solid phase extraction method coupled HPLC/ELSD.Single factors were investigated and the conditions of this experiment were optimized.Experimental conditions were described as follows:200mg Fe₃O₄@MWCNT-I was used as the solid phase extraction material;the concentration of NaCl was 3.5%;the pH value was 6.0;methanol was used as the eluent,and 1.5 mL was used every time for three times.Under the appropriate optimal conditions,the LODs of the analytes ranged from 0.16?g/mL to 0.60?g/mL.Moreover,the intra-and interday precisions ranged from 1.9%to 4.5%.Meanwhile,the recoveries of spiked samples were measured,which ranged from91.1%to 96.2%with RSDs ranging from 2.8%to 3.8%.The experimental results show the contents of the analytes??3R?-2'-dihydroxy-3',4'-dimethoxyisoflavanone-7-O-?-D-glucoside,?6aR,11a R?-9,10-dimethoxypterocarpan-3-O-?-D-glucoside,astragaloside III?are 18.85±0.37?g/mL,7.07±0.18?g/mL,10.73±0.32?g/mL,respectively.In addition,astragaloside IV was determined directly by HPLC/ELSD.The experimental results show the content of astragaloside IV is 58.53±1.37?g/mL.Kang'ai injection can strengthen physique and enhance immunity.It also can treat malignant tumor such as liver cancer,lung cancer,adenocarcinoma cell of rectum,malignant lymphoma and gynecological malignant tumors.The aim is to study the correlation between the functional components and the major function of Kang'ai injection.The composition solutions were prepared according to the experimental results of chapter 3.Then MTT assay was used to investigate the bioactivity of the cell dealt with the above solutions.The results showed that the flavonoids from Astragalus membranaceus?Fisch.?Bge.var.mongholicus?Bge.?Hsiao and the rare ginsenoside have an inhibitory effect on HepG2 and MCF-7 cell.The IC₅₀ values of the flavonoids on HepG2 and MCF-7 cell in 48h were 70.59?g/mL and 13.71?g/mL,respectively.?3R?-2'-Dihydroxy-3',4'-dimethoxy isoflavanone-7-O-?-D-glucosideand?6aR,11aR?-9,10-dimethoxypterocarpan-3-O-?-D-glucoside both have a markedly inhibitory effect on HepG2 and MCF-7cell.The results from experiments indicated that flavonoids from Astragalus membranaceus?Fisch.?Bge.var.mongholicus?Bge.?Hsiao and the rare ginsenosides are the major effective substances.In conclusion,the chemical constituents and the relevant contents in the Kang'ai injection were investigated in this paper.Meanwhile,the correlation between the functional components and the major function of Kang'ai injection were also studied in this paper.The results from experiments indicated that flavonoids from Astragalus membranaceus?Fisch.?Bge.var.mongholicus?Bge.?Hsiao and the rare ginsenosides are the major effective substances.These results can provide valuable reference,improve quality standards and ensure the medication safety of Kang'ai injection.