| Purpose: Juglans mandshurica Maxim.,a tree belonged to Juglandaceae.In the folk,the decoction of barks,branches and leaves have been using for treating cancer with an impressive effect.In clinic,it mixed with other traditional Chinese medicinal material as a compound preparation has been applied to treat cancer.Due to the widely used and impressive effect,Juglans mandshurica Maxim.earned extensive attention.With further research,more than one hundred constituents were separated and authenticated from it,and part of them have the function on reducing growth of cancer cells,which had been proved by experiments in vitro.However,besides Zhi Sun,who come from Shenyang Pharmaceutical University,researched on the pharmacokinetic of eight consistuents in Juglans mandshurica Maxim.Extraction in rat plasma,few focus on its physiological disposition.Therefore,tissue distribution,metabolism and pharmacokinetic of its decoction would be researched in this paper,which could lay the foundation for making certain the active components in Juglans mandshurica Maxim..Material and method: 1.Materials 1.1 The branches of Juglans mandshurica were collected in Lianshan town of Dalian at April,2016 and were identified by professor Zhai.Then,they were stored at teaching and research section of Chinese medicine identification with the specimen No.of ZHI20160406.1.2 ICR mouse and SD rats of SPF grade were purchased from Liaoning Changsheng Biotech Co.,LTD.Certification No.of animals is SCXK(?)2015-0003.2.Methods 2.1 A refluxing extraction method was used for extracting Juglans mandshurica with water.A UHPLC-Q-TOF-MS/MS method was applied to identify the consistuents in Juglans mandshurica Maxim.extract.2.2 A maximum asministration dasage method for indagation acute toxicity test of Juglans mandshurica.2.3 A precipitation protein method for preparation biological samples,including plasma,heart,liver,spleen,lung,bile,urine,and feces.Identification constituents and metabolites of Juglans mandshurica in biological samples to indagate its tissue distribution and metabolism in mouse.2.4 Application of a HPLC method to research pharmacokinetic of gallic acid and syringic acid in the water extraction of Juglans mandshurica in rat plasmaResults: 1.The Juglansmandshurica decoction was extracted according to standard operation,and the yield is 12.5 %,dose of raw medicine is 8 g/g.105 compounds were identified,which are consist of 54 tannins,30 organic acid,8 naphthalene derivative,8 oligosaccharides,4 flavonoids and 1 diarylhetanoids derivatives.2.The mouse were given orally with 9.6 g/m L Juglansmandshurica decoction under dosage of 0.5 m L/20 g.General physiological condition is normal during 14 days.With no pathological changes occur in tissues.Maximum tolerance dose is 226 g/kg.3.A recipitation protein method with methanol and phosphate buffer(p H 7.8~8.0)for preparation of samples.The extract recoveries of most constituents weregreater than 75 percentages.4.33 compounds were identified in plasma,including 12 constituents and 21 metabolites.Constituents can be classified into 4 species,containing tannins,organic acid,naphthalene derivative and diarylhetanoids derivatives;metabolites can be categorized into 4 kinds,including glucuronidation,sulfation,sulfoglucuronidation and isomerization.28 compounds were identified in bile,including 7 constituents and 21 metabolites.Metabolites can be categorized into 4 kinds,including glucuronidation,sulfation,sulfo-glucuronidation and isomerization 33 compounds were identified in urine,including 20 constituents and 13 metabolites.Constituents can be classified into 4 species,containing tannins,organic acid,oligosaccharides and flavonoids;metabolites can be categorized into 3 kinds,including glucuronidation,sulfation and sulfo-glucuronidation.71 compounds were identified in urine,including 67 constituents and 4 metabolites.Constituents can be classified into 6 species,containing tannins,organic acid,naphthalene derivative,oligosaccharides,diarylhetanoids derivatives and flavonoids;metabolites can be categorized into 2 kinds,including sulfation and isomerization.Malic acid and citric acid were identified in liver;citric acid was identified in spleen and lung;constituents and metabolites were not found in heart and kidney.5.Gallic acid had great linear at the range of 0.04-5 ?g/m L,LOD = 0.04?g/m L,LOQ = 0.006?g/m L;the RSDs of precision were from 3.5 to 9.4% for intra-day and from 1.8 to 11.0% for inter-day assay.The accuracy were at the range of-0.5-2.1% and-0.7-1.3%,respectively;the extraction recoveries were from 81.2% to 94.4%;Cmax and Tmax were 0.64±0.05?g/m L and 61.8±8.03 min,T1/2were 184.21±12.73 min,AUC0-t and AUC0-? were 96.37±10.33?g?min/m L and 121.59±8.87?g?min/m L.Springic acid had great linear at the range of 0.04-5 ?g/m L,LOQ = 0.008?g/m L,LOD = 0.04?g/m L;the RSDs of precision were from 4.0 to 6.9% for inter-day assay and from 2.1 to 8.8% for intra-day.The accuracy were at the range of-1.2-4.9% and-4.8-0.8%,respectively;the extraction recoveries were from 84.9% to 93.6%;Cmaxand Tmax were 0.43±0.04?g/m L and 30.67±0.92 min,T1/2 were 184.21±14.73 min,AUC0-t and AUC0-? were 40.33±2.42?g?min/m L and 47.02±3.29?g?min/m L.Conclusion: 1.Tannins and organic acids are major compounds in Juglansmandshurica decoction than contains several naphthalene derivative,oligosaccharides,diarylhetanoids derivatives and flavonoids? 2.Juglansmandshurica decoction is non-acute toxicity.3.In plasma,metabolites are more than constituents in quantitative terms.Tannins and organic acids are major compounds in constituents.Most naphthalene derivative,flavonoids and diarylhetanoids derivatives would be metabolized by combining with sulfate and glucopyranosiduronic acid group.A little bite of them was isomerizated.In bile,constituents are much more than metabolites in quantitative terms.Tannins and organic acids are major compounds in constituents.Most naphthalene derivative and flavonoids would be metabolized by combining with sulfate and glucopyranosiduronic acid group.In urine,constituents are much more than metabolites in quantitative terms.Organic acids are major compounds in constituents.4.Tannins,organic acids,naphthalene derivative and flavonoids can be absorbed into blood.The major excretion path of aphthalene derivative and flavonoids was bile;tannins and organic acids were most excreted within urine.Mostnaphthalene derivative and organic acids would be metabolized by combining with sulfate and glucopyranosiduronic acid group.A mount of constituents were identified in feces,and only 4 metabolites in it,concluding sulfation and insomerization.Only 2 organic acids were distributed into part of tissues.None was found in heart and kidney.5.A HPLC method for analysis the pharmacokinetic of gallic acid and syringic acid in rat plasma.This method,with acceptable accuracy,met all requirements in bioanalysis.The research might be helpful to explain the mechanism of drugs and give guidance on medication. |
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