Study On HPLC Fingerprint Of Polygonati Rhizoma Dispensing Granules And The Effect On Cyclophosphamid Injury In Mice

1.BackgroundTraditional Chinese medicine（TCM）is the precious wealth of China.With the development of TCM research,TCM has been recognized and accepted by more and more people because of its efficacy and low side effects in the prevention and treatment of common diseases,frequently encountered diseases,chronic diseases and significant diseases.However,traditional Chinese medicine has its own shortcomings:inconvenience in decocting,cooking and carrying when we are traveling.These are the inevitable problems that we will encounter when using traditional Chinese medicine.Dispensing granules,as productions of modernization of traditional Chinese medicine,are designed to solve the above problems.Traditional Chinese medicine dispensing granules are made of medicinal slices with guidance of TCM theory,which are refined by water extraction,concentration,drying,granulation and packaging for clinical prescription with modern pharmaceutical technology.Traditional Chinese medicine dispensing granules partly overcome the shortcomings of traditional Chinese medicine.However,as supplements to traditional Chinese medicinal slices,most dispensing granules still lack quality standards which are supplements to medicinal slices.It still needs further studies on the pharmaceutical efficacy of dispensing granules if that is consistent with medicinal slices.In this study,we preliminarily studied the quality standard and pharmacodynamic evaluation of Polygonati Rhizoma dispensing Granules.Firstly,TLC and HPLC fingerprints of Polygonati Rhizoma dispensing granules were established by TLC and high performance liquid chromatography.Secondly,the contents of polysaccharides,total phenols and 5-hydroxymethyl furfural（5-HMF）in Polygonati Rhizoma dispensing granules were determined quantitatively.In addition,the pharmacodynamic evaluation of Polygonati Rhizoma dispensing granules was carried out by establishing mice cyclophosphamide immunodeficiency model,which provided the basis for the development of Polygonati Rhizoma dispensing granules in the future.Method1.TLC was used to qualitatively identify 12 batches of Rhizoma Polygonatum dispensing granules from different regions.2.The content of Polysaccharide and Total Phenol in 12 batches of Polygonati Rhizoma dispensing granules from different regions were determined by UV spectrophotometry.3.The content of 5-hydroxymethylfurfural in 12 batches of Polygonati Rhizoma dispensing granules from different origins was determined by HPLC.Chromatographic conditions:column was Intersil C₁₈（250 mm×4.6 mm,5μm）;mobile phase:acetonitrile-0.085%phosphoric acid aqueous solution（5:95）;flow rate:0.8 mL/min;column temperature:35℃;detection wavelength:274 nm;Injection volume:10μL.4.HPLC was used to establish the fingerprint of Rhizoma Polygonatum dispensing.Chromatographic conditions:Kromaisil C₁₈（250 mm×4.6 mm,5μm）;mobile phase:methanol-0.085%phosphoric acid aqueous solution gradient elution;flow rate:0.4 mL/min;column temperature:26℃;detection wavelength:254 nm;Injection volume:20μL;acquisition time is 70 min.5.Immunosuppressive mice model were constructed by intraperitoneal injection of cyclophosphamide into mice.Mice were given different doses of Polygonati Rhizoma dispensing granules（750 mg/kg,1500 mg/kg,3000 mg/kg）or positive drug levamisole（20 mg/kg）.The effects of Polygonati Rhizoma dispensing granules on the body weight of immunosuppressed mice were measured by recording the body weight of mice from the first day to the 16th day;thymus index and spleen index of mice were measured;the concentration of TNF-α,IL-2 and IL-10 in serum and spleen of mice were determined by Elisa.The expressions of CD4⁺T and CD8⁺T in the spleen of mice were determined by immunofluorescence method.Results1.The spots on the TLC plate were clear,specific and separated well.The Rf value was moderate.2.At 582 nm,anhydrous glucose showed a good linear relationship between 0.03328and 0.19968 mg/mL with regression equation y₁=4.034x₁+0.028,r₁=0.9993（n=6）.The average recovery was 98.24%and the RSD was 2.23%.Polygonatum polysaccharide contents of 12 batches of the dispensing granules were ranged from 22.51 to 48.14 mg/g.At 765 nm,phenolic acid showed a good linear relationship between 0.0052 and 0.052mg/mL with regression equation y₂=12.967x₂+0.14,r₂=0.9996（n=9）.The average recovery was 98.96%and the RSD was 0.68%.3.The contents of total phenolic acid were ranged from 11.80 to 18.95 mg/g;5-HMF showed a good linear relationship between 0.020 and 2.000 mg/mL with regression equation y₃=15468610x₃-663735（r₃=0.9991）（n=6）.The average recovery of 5-HMF was 97.16%and the RSD was 1.07%.The contents of 5-HMF were ranged from 0.5736 to0.8916 mg/g.4.The fingerprints of 12 batches of Polygonati Rhizoma dispensing granules from different regions were established,and their stability,repeatability,specificity,resolution were good and the similarities were all greater than 0.9.A total of 8 common peaks were determined and the number 7 peak is 5-HMF.5.Polygonati Rhizoma dispensing granules had no significant effect on the weight of immunocompromised mice,but can improve the thymus index and spleen index of immunosuppressed mice.It can up-regulate the concentration of serum and spleen TNF-α,IL-2 and reduce the concentration of IL-10 and promote the expression of CD4⁺T and CD8⁺T cells.Conclusion1.The established TLC and content determination method of Rhizoma Polygonatum dispensing granules were simple,feasible,specific and reproducible,and can be used as the quality control standard for Polygonati Rhizoma dispensing granules.2.The HPLC fingerprint of the established Polygonati Rhizoma dispensing granules is simple,stable and reliable,and can comprehensively evaluate the Polygonati Rhizoma dispensing granules from different regions.3.Polygonati Rhizoma dispensing granules had no significant effect on the weight of immunocompromised mice,but promoted the expression of CD4⁺T and CD8⁺T cells by regulating various inflammatory cytokines to enhance immune function.4.In summary,this study comprehensively evaluates the quality of Polygonati Rhizoma dispensing granules from different origins by establishing the content determination of Polygonati Rhizoma dispensing granules and HPLC fingerprints method.By evaluating the immunomodulatory function of Rhizoma Polygonatum dispensing granules,we provided the experimental basis for further development of Polygonati Rhizoma and that was beneficial for the development of Polygonati Rhizoma dispensing granules.