| PSP(Polysaccharopeptide)is a new drug at the national level,which can improve immune fuction,inhibit tumor cells,analgesia and anti-inflammatory effects.Its active omponents include polysaccharides,petides and nucleosides.From the quality control and pharmacological effects,we need to the monosaccharide,amino acid and nucleoside of PSP.Objective:The composition of polysaccharide peptides and nucleosides in different batches of ganoderma lucidum were investigated.Establish versicolor monosaccharide sugar peptide,amino acids,nucleosides HPLC feature maps,through the traditional Chinese medicine(TCM)chromatographic fingerprint similarity evaluation system(2012)edition;similarity calculation software,select multiple indexes were analyzed by applying the versicolor glycopeptide quality control study,evaluate the quality of different batches of raw material,for scientific and reasonable evaluation versicolor glycopeptide quality to provide the reference.Methods:Study on monosaccharide composition of PSP.PSP were acidolyzed for6 h with 110?,0.1mol/LTFA solution,and then derived by PMP in front of chromatographic column.Chromatographic condition:Chromatographic column:phenomenex Luna C18 chromatographic column(250mm×4.6mm,5?m);Mobile phase:acetonitrile(B)-0.05 mol/L phosphate solution(pH was adjusted to 7.1 with 2 mol/L Na OH solution)(A).Elution at equal degrees.Constant elution conditions:18%Acetonitrile.Flow rate : 0.8 m L/min.The detective wavelength : 254 nm.Column temperature :30?;The "similarity evaluation system of TCM fingerprint chromatogram" was used to establish the control characteristic atlas and evaluate the similarity.The relative retention time and relative peak area of each chromatographic peak were the indexes,methodology was examined with galactose as the reference peak.Study on the amino acid composition of glycopeptide from PSP.PSP were acidolyzed for 2h with 140?,4mol/LHCL solution,and then derived by OPA on line.Chromatographic condition:Chromatographic column: phenomenex Gemini C18 chromatographic column(250mm×4.6mm,5?m);Mobile phase: Mobile phase A:20mmol/L Na H2PO4(pH was adjusted to 6.6 with 2 mol/L Na OH solution);Mobile phase B: methanol: acetonitrile: water =(45:45:10,V/V/V).Gradient elution.Gradient elution conditions:Mobile phase B:0-4 2%-10%;4-20 10%-14%;20-30 14%-31%;30-55 31%-57%.Flow rate : 1.5m L/min.The detective wavelength : 338 nm.Column temperature :30?;The "similarity evaluation system of TCM fingerprint chromatogram" was used to establish the control characteristic atlas and evaluate the similarity.The relative retention time and relative peak area of each chromatographic peak were the indexes,methodology was examined with alanine as the reference peak.Study on nucleoside composition in glycopeptide of PSP.The nucleosides in the PSP were extracted by ultrasound for 30 min.Chromatographic condition:Chromatographic column: Welch AQ-C18 chromatographic column(250mm×4.6mm,5?m);Mobile phase: Methanol(B)-water(A).Gradient elution.Gradient elution conditions:Mobile phase B:0-10 0%;10-15 0%-5%;15-305%;30-45 5%-15%.Flow rate : 1m L/min.The detective wavelength : 260 nm.Column temperature:30?;The "similarity evaluation system of TCM fingerprint chromatogram" was used to establish the control characteristic atlas and evaluate the similarity.The relative retention time and relative peak area of each chromatographic peak were the indexes,methodology was examined with hypoxanthine as the reference peak.Results:The HPLC characteristic spectrum of monosaccharide in the peptides of ganoderma lucidum was established and seven characteristic peaks of monosaccharide were identified.Galactose was the reference peak.The polysaccharide peptides of ganoderma lucidum were composed of 7 monosaccharides,mannose:rhamnose:glucose:galactose:xylose:arabinose:fucose=82.4±0.4:6.3±0.9:193.3±2.8:100:5.3±1.1:4.0±0.9:42.9±2.9.The chromatographic fingerprint similarity of 31 batches of PSP samples was more than 0.989.The study of analytical methodology proved that the linear range of mannose,glucose,galactose fucose was 0.044-0.800,0.072-1.440 0.052-1.040,0.016-0.320mg·m L-1.Linear relationships good,which r was more than 0.9997.The recovery of low,medium,high ratio was mannose101.15% ±0.55%?99.45%±2.05%?100.35%±1.25%;glucose101.45%±0.45%?98.30%±2.50%?98.20%±0.50%;galactose99.50%±2.10%?101.50%±0.40%?101.85%±0.25%;fucose102.25%±1.65%?99.80%±1.10%?99.70%±0.20%.The relative retention time and peak area of each monosaccharide:the RSD of the Intraday precision tset was 0.04%-0.43%?0.10%-3.29%.The RSD of the stability test was0.05%-0.86% ? 0.09%-3.66%.The RSD of the repetitive test was 0.05%-0.51% ?1.76%-4.35%.In addition,it was found that there were significant differences between PSP and Polystictus Glycopeptide,Lentinus Edodes Mycelia Polysacharide,Ginseng glycopeptideand Cordyceps mycelia.from the aspects of the species and similarity of monosaccharide and the molar ratio of monosaccharide.The HPLC characteristic spectrum of amino acid in the PSP was established and sixteen characteristic peaks of monosaccharide were identified.Alanine was the reference peak.The polysaccharide peptides of ganoderma lucidum are composed of 16 amino acids,aspartic acid,glutamate,serine,histidine,threonine,arginine,alanine,tyrosine,valine,methionine,phenylalanine,leucine,lysine hydrochloride =204.6±5.7:145.6±5.5:137.2±3.3:28.6±14.9:218.7±14.8:63.8±0.8:78.0±8.8:100.0:12.7±1.2:5.3±1.3:29.2±2.0:5.7±1.6:10.1±1.8:15.0±2.5:35.4±7.9:116.2±5.7.The chromatographic fingerprint similarity of 31 batches of PSP samples was more than 0.991.The study of analytical methodology proved that the linear range of aspartate,glutamate,serine,glycine,threonine,arginine,alanine,lysine,hydrochloride was 0.009-0.180,0.007-0.14,0.005-0.100,0.005-0.100,0.003-0.060,0.004-0.080,0.003-0.060,0.006-0.120 mg · m L-1.Linear relationships good,which r was more than 0.9995.The recovery of low,medium,high ratio was aspartic acid 99.90% ± 2.40% ? 101.55% ± 0.25% ?100.95%±0.95%;glutamate 102.10%±0.80%?99.35%±1.75%?97.15%±1.95%;serine 100.40%±1.90%?101.05%±2.45%?100.30%±2.20%;glycine 95.8%±1.00%?94.40%±2.40%?96.75%±2.15%;threonine 93.75%±1.55%?95.65%±2.45%?94.00%±1.40%;arginine 101.60%±2.60%?103.80%±2.10%?103.45%±0.35%;alanine93.70%±1.30%?95.15%±2.35%?96.60%±1.10%;lysine hydrochloride95.05%±1.15%,101.80%±2.00%?99.15%±1.55%.The relative retention time and peak area of each amino acid:The RSD of the Intraday precision tset was 0.02%-0.09% ? 0.28%-4.78%.The RSD of the stability test was0.11%-0.42% ? 0.34%-4.5%.The RSD of the repetitive test was 0.01%-0.05% ?0.42%-4.39%.In addition,it was found that there were significant differences between PSP and Polystictus Glycopeptide,Lentinus Edodes Mycelia Polysacharide,Ginseng glycopeptideand Cordyceps mycelia.from the aspects of the species and similarity of amino acid and the molar ratio of amino acid.The HPLC characteristic spectrum of nucleoside in the PSP was established and eight characteristic peaks of monosaccharide were identified.The reference peaks were hypoxanthine.The nucleoside component of ganoderma lucidum glycopeptide is composed of 8nucleosides.In the ganoderma lucidum glycopeptide produced in 2018,cytosine:uracil: cytidine: hypoxanthine: uridine: adenine:guanosine: adenosine ==147.0±19.7:73.1±16.1:68.0±10.6:100:62.9±5.3:135.4±1.4:42.5±8.8:60.5±10.0,in 2017,97.8±14.7:136.9±5.3:14.1±2.7:100:10.3:108.9±5.5:4.9±0.1:8.7±1.0.The chromatographic fingerprint similarity of 2 batches of PSP samples which was producted in 2018 was 0.649 and 0.67.The chromatographic fingerprint similarity of29 batches of PSP samples which was producted in 2018 was between 0.92-0.995.The study of analytical methodology proved that the linear range of Cytosine uracil cytidine hypoxanthine uridine adenine guanosine adenosine was 0.270-5.400 ?0.275-5.500?0.600-12.000?0.900-18.000?1.000-20.000?1.200-24.000?1.750-35.000?0.800-16.000?g·m L-1.Linear relationships good,which r was more than 0.9997.The recoveryof low,medium,high ratio was cytosine 95.35%±1.75%?95.20%±1.00%?97.30%±1.00%;uracil 93.10%±1.00%?101.65%±4.15%?102.35%±0.85%;cytidine 99.15%±0.45%?101.20%±3.40%?103.00%±0.90%;hypoxanthine92.50%±0.50%?97.30%±2.00%?98.95%±0.95%;uridine 98.05%±2.95%?99.20%±0.20%?101.80%±0.60%;adenine 99.25%±3.95%?97.75%±1.15%?99.85%±1.95%;guanosine 96.25%±4.35%?101.75%±0.75%?105.65%±0.95%;adenosine 96.25% ± 2.85% ? 100.00% ± 0.10% ? 104.00% ± 0.30%.The relative retention time and peak area of each nucleoside:The RSD of the Intraday precision tset was 0.06%-0.27% ? 0.68%-2.81%.The RSD of the stability test was0.04%-0.36% ? 0.21%-2.08%.The RSD of the repetitive test was 0.06%-0.20% ?0.64%-3.19%.In addition,it was found that there were significant differences between PSP and Polystictus Glycopeptide,Cordyceps mycelia and Isaria cicadae Miquel.from the aspects of the species and similarity of nucleoside and the molar ratio of nucleoside.Conclusions:The method of HPLC characteristic fingerprint analysis of PSP was simple,stable,reliable.The HPLC characteristic fingerprint of PSP can be evaluated comprehensively.It provides reference for improving the quality control of PSP.And it can be used to distinguish with other glycopeptide products. |
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