Quality Evaluation And Attenuated Mechanism Research On Exocarp Of Juglans Mandshurica Based On Metabonomics

ObjectiveThis study was to characterize and identify chemical components,develop the quality evaluation method and determine the appropriate harvest time in exocarp of Juglans mandshurica(BQLY).Then,in order to access the quality and safety of BQLY,we investigated the changes in the chemical profiles between fresh-raw and dry-processed Juglans Mandshurica,researched on the changes in the metabolic profiles of toxicity,and elucidated the toxic mechanisms induced by fresh-raw BQLY and its processed detoxification mechanism with metabonomics method based on the evaluation of clinical chemicals and histopathology.Methods(1)The multiple constituents in BQLY were analyzed by UPLC-Q-TOF/MS.Before data processing,an in-house formula database was firstly established with 367 components reported in the literature of genus Juglans.The Natural Products HR-MS/MS Spectral Library 1.0 software and ChemSpider database were used for analysis.The formula of compounds was determined by accurate mass and isotopic abundance ratio.The structures were tentatively identified by data obtained in reference substances or inferred through mass spectrometry fragment ion analysis and literature data.(2)Extract peaks with total ions(compounds)of seventy-eight batches obtained from thirteen different regions in Heilongjiang province of China were used to establish the fingerprint and build MS/MS database.The compounds were blocked using the one-dimensional retention time of UPLC.The authenticity was determined by relative retention time and MS/MS fragment.The quality could be holistic evaluated by relative peak area.The PCA was used to find out the characteristic compounds and access the differences between samples derived from various regions.(3)The contents of total naphthoquinones,main active constituents and active ingredients group in BQLY obtained from three regions at six different harvest times were determined by UV,HPLC and UPLC-Q-TOF/MS,respectively.The appropriate harvest time was confirmed by dynamic changes in the metabolite accumulation and yield.(4)The main components between crude and processed BQLY samples were contrasted and analyzed by using UPLC-Q-TOF/MS coupled with multivariate data analysis(PCA)to find the differences.This approach should be applied to understand the relationship between the changing rule of content and the processing mechanism of reducing the toxicity and increasing the activity.(5)Firstly,acute toxicity study in mice was applied to evaluate the toxicity of fresh-raw,dry-processed Juglans Mandshurica,and juglone.Secondly,the toxicity in rats was evaluated by clinical biochemical parameters in blood and histopathologic changes in organs.Then,UPLC-Q-TOF/MS method and pattern recognition analysis(PCA,OPLS-DA)were used to study the metabolic profiles changes and to find the biomarkers of toxicity dosed.Subsequently,the related metabolic pathways of toxic biomarkers were found to illuminate the toxic mechanism of resh-raw BQLY and juglone.The toxic characters of fresh-raw and dry-processed Juglans Mandshurica were studied by comparing with their toxicities using metabolomics method.Finally,sub-chronic toxicity study of 3 months and long-term toxicity of 6 months were applied to evaluate drug safety of dry-processed Juglans Mandshurica.Results1.Characterization and Identification of Multiple Constituents in BQLYA total of 193 compounds,including 68 naphthalenequinones,20 diarylheptanoids,29 flavonoids,20 triterpenes,28 phenolic acids,5 coumarins,8 aliphatics and 15 other compounds,was rapidly tentatively identified or inferred in the exocarp of Juglans mandshurica,2.Development of Fingerprinting for quality evaluation of BQLYThirty-six peaks were selected as common peaks in this fingerprint and thirty-one of these common peaks were tentatively identified.Chromatogram of common ions was established by extraction of common ions within 0.1S retention time window.The relative retention times were calculated by twentieth peak of 12.94 min named Juglanin.The average areas of common ions in 78 samples are the important values for the quality of BQLY.The BQLY derived from Fangzheng,Jiayin,Bin xian may be relatively good quality herbs because of the general high peak area,while BQLY derived from Wuchang,Harbin,Huana,Heihe may be relatively poor quality herbs because of the general low peak area.A total of 36 compounds was detected in each sample of authentic BQLY and used to assess the inferior or false samples from various species,regions and harvest time because of naphthoquinones and theirs derivatives were not detected or detected to be a low content in these samples.The diversity and similarity among different samples analyzed by principal component analysis(PCA)indicated that the samples from WC,JX,JY,BQ,BX,HL,TH,TL were clustered into one domain,but those from other regions deviated from the domain.32 components were identified or tentatively characterized.3.Determination of appropriate harvest time of BQLYThe contents of total naphthoquinones,juglone and regiolone were gradually reduced in exocarp of Juglans mandshurica at six different harvest times from three regions,especially after August 15.The changes in effective components of Juglans mandshurica at different harvest times were analyzed by UPLC-Q-TOF-MS/MS with PCA.The Naphthoquinone were the major markers in samples of Juglans mandshurica obtained at different harvest times.Thirty-eight kinds of naphthalenequinones were identified or inferred,and the content of naphthalenequinones declined gradually.The dynamic changes in the metabolite accumulation were similar among three regions as Harbin,Fangzheng and Wuchang.The content of naphthoquinones was higher in July,but the yield was low.The active ingredients accumulated gradually from July to August,but declined gradually after August,and fell sharply after September.The appropriate harvest time was determined in the middle of July to early August.4.Comparison of the chemical profiles of fresh-raw and dry-processed BQLYEighty-one compounds were rapidly discovered and identified whose contents have significant differences between fresh and dry samples,including thirty-five naphthalene-quinones,eleven diarylheptanoids,nine flavonoids,eight triterpenes,twelve phenolic acids and six aliphatics.Further study indicated that toxic naphthoquinone compounds with 5-hydroxy or 1,4-naphthoquinone structure were transformed from fresh samples to dry samples which may be the important mechanism of reduction toxicity.The generation of Regiolone,diarylheptanoids and triterpenes and the increased contents of them may be the third important mechanism on increasing the activity.5.Toxicity assessment of BQLYThe acute toxicity study showed that fresh-raw BQLY has slight neurotoxicity in mice.The toxic dosage of fresh-raw BQLY is equivalent to the 31 times or 4 times(body surface area calculation)of clinical dosage of people(the scales algorithm).There was no any toxic effect in dry-processed BQLY.The dosage of dry-processed BQLY is equivalent to the 83 times or 11 times(body surface area calculation)of clinical dosage of people(the scales algorithm).The LD50 of juglone were 221.54mg/Kg(oral)and 5.4696mg/Kg(intraperitone-al injection)in mice.In chronic toxicity study,the body weight,biochemical parameters and histopathology showed no significant changes in dry-processed BQLY after 4 weeks in rats.But,fresh-raw BQLY and juglone leaded to weight loss,and a few rats died during experimental period.The levels of ALT,AST,BUN and CREA in serum and urine protein in urine were elevated significantly.The pathological changes in liver,kidney and brain were observed in fresh-raw BQLY and juglone groups after 4 weeks,which means that the liver,kidney and brain of rats were intoxicated.The metabonomics analysis of the body fluids and tissues samples under positive and negative ions showed that the dry samples and control group clustered into the same area,but fresh or samples juglone kept away from control group.The results indicated that metabolic profiles of rats were disturbed slightly by dry samples and disturbed intensely by fresh samples or juglone.24 urine potential markers(8 in common),11 blood potential markers(9 in common),33 liver potential markers(13 in common),55 kidney potential markers(18 in common),78 brain potential markers(54 in common)were identified in fresh-raw BQLY group rats and juglone group rats.The key metabolic markers of KYDS model rats were gradually focused through metabolic pathway and literatures as follows:Phenylalanine metabolism(Phenylpyruvic acid,L-Tyrosine),Tryptophan metabolism(Tryptophan,Kynurenine),Linoleic acid metabolism(Linoleic acid),Linolenic acid metabolism(Alpha-Linolenic acid),Sphingolipid metabolism(S1P,Sphingosine),Steroid hormone biosynthesis(Aldosterone),Retinol metabolism(Vitamin A,Retinyl ester),Purine metabolism(cAMP,Xanthine,Xanthosine),Primary bile acid biosynthesis(Taurocholic acid,Taurochenodesoxycholic acid,Glycocholic acid),Glutathione metabolism(GSH,GSSG,Pyroglutamic acid),Glycolysis or Gluconeogenesis(S-Acetyldihy-drolipoamide),Nicotinate and nicotinamide metabolism(Niacinamide),Sphingolipid metabolism(LysoPC16:0,Glycerol 3-phosphate),Cysteine and methionine(S-Adenosy-lhomocysteine,L-Methionine),Arginine and proline metabolism(L-4-Hydroxyglutamate,L-Arginine),Histidine metabolism(L-Histidine),Citrate cycle(Citric acid,Isocitric acid),Tryptophan metabolism(Tryptophan),Galactose metabolism(Galactose).All these biomarkers were related to the liver,kidney and brain damage.The level of 24 markers had no significant difference between dry groups and control group which indicated relative safety by metabonomics method.The body weight,blood biochemistry,blood routine,urine biochemical indicators,viscera index,and histopathological of rats has no obvious changes after administrated with dry-processed BQLY for six months,which indicated that long-term administration of dry-processed BQLY is safe.ConclusionUPLC-Q-TOF/MS method can identify the chemical constituents from BQLY rapidly and accurately,and main chemical constituents can be used for the quality evaluation.The developed fingerprint was demonstrated to be a valuable strategy for quality identification and comprehensive evaluation of BQLY.The authenticity could be quickly determined by relative retention time and MS/MS fragment.The quality could be integrally evaluated by relative peak area.Analysis and characterization of the differences in ingredient will help to understand the causes of genuine medicine.The appropriate harvest time could be confirmed by dynamic changes in the metabolite accumulation and yield.This study provides a novel methodology and approach to identify the complicated components from various complex mixtures between fresh-raw and dry-processed BQLY.It can be used as a valid analysis method for further understanding the processing mechanism of TCM,along with the intrinsic quality control of TCM and its processed product.The toxic mechanism of fresh-raw BQLY and juglone along with the mechanism of reducing the toxicity could be illustrated by relevant research results of Pathological index,physiological and biochemical indexes,toxic markers and metabolic pathways of organs.