| Purpose:Juglans mandshurica Maxim,also known as hickory and walnut tincture,is a walnut plant of Juglandaceae.Its roots(skins),branches(skins),leaves and green peel all have good anti-tumor effects.At present,Juglans mandshurica is not included in Chinese Pharmacopoeia,and the research on its quality standard and quality evaluation is still incomplete.Therefore,the anti-tumor activities of the main compounds and their combinations isolated from Juglans mandshurica were studied in order to provide scientific basis for quality evaluation.Furthermore,the quality evaluation of roots,branches,leaves and green peel of Juglans mandshurica was studied by fingerprint,which provided scientific basis for the application and quality standard research of Juglans mandshurica.Methods:1.Characterization of chemical components in various parts: UHPLC-Q-TOF-MS/MS analysis was used to characterize the chemical components in stems,roots,leaves and pericarp of Juglans mandshurica.2.Isolation and identification of main chemical components: The chemical components in stems and branches of Juglans mandshurica were extracted with ethanol under reflux.The column chromatography was used for preliminary separation,and the high performance liquid chromatography was used for further separation and purification.The structure of the separated compounds was identified by MS,NMR and other spectral methods.3.Main monomer components and anti-tumor activity in groups: MTT method was used to measure the growth inhibition rate and IC50 of the isolated monomer compounds and their combinations on human liver cancer cells(Hep G-2),and Compusyn method was used to calculate the combined index CI of monomer compound combinations.4.Fingerprint study of various parts: HPLC was used to establish the fingerprints of stems,branches,roots,leaves and green peel of Juglans mandshurica from different producing areas.SPSS and SIMCA-P software were used to carry out cluster analysis,principal component analysis and partial least squares discriminant analysis on the obtained fingerprints.5.Determination of the content of main components in each part: Using Agilent Poroshell 120 SB-C18 chromatographic column,gradient elution was carried out with formic acid water(A)-formic acid acetonitrile(B)as mobile phase.Flow rate was 0.3 m L·min-1.Column temperature was 30℃.Detection wavelength was 270 nm.And retrograde gallic acid,gallic acid,1,6-di-O-galloyl-β-Dglucose,1,2,6-tri-O-galloyl-β-D-glucose,1,2,3,6-tetra-O-galloyl-β-Dglucose were determined in stems and roots of Juglans mandshurica.Agilent Poroshell 120 SB-C18 column was used for gradient elution with formic acid water(A)-acetonitrile(B)as mobile phase.The flow rate was 0.3 m L·min-1.The column temperature was 30℃.And the detection wavelengths were 325 nm and 350 nm.The contents of chlorogenic acid,hyperin and isoquercitrin in Juglans mandshurica leaves and chlorogenic acid in Juglans mandshurica green peel were determined.The content of total flavonoids in Juglans mandshurica leaves was determined by ultraviolet spectrophotometry with rutin as reference substance at 504 nm wavelength.Results:1.Characterization of chemical components in various parts: 83,75,39 and 18 chemical components were identified from stems,roots,leaves and pericarp of Juglans mandshurica.The compounds identified from 4 parts of Juglans mandshurica can be divided into 8 categories,namely tannin,organic acid,naphthalene derivative,anthracene derivative,flavone,diarylheptane,lignan and saccharide.The chemical compositions of different parts of Juglans mandshurica are quite different,and the types of main components are different.Stems and branches mainly contain gallic tannins,organic acids and naphthalene derivatives,while the amount of reverse gallic tannins is relatively small.The root mainly contains reverse gallic tannin,gallic tannin,organic acid and naphthalene derivatives,and the quantity of reverse gallic tannin and gallic tannin are more.Leaves mainly contain flavonoids,organic acids and naphthalene derivatives,with a large number of flavonoids and a small number of tannins.The green pericarp of Juglans mandshurica mainly contains naphthalene derivatives,organic acids and flavonoids,and the number of naphthalene derivatives is relatively large while the number of tannins is relatively small.2.Isolation and identification of main chemical components: Five compounds were isolated from stems and branches of Juglans mandshurica and identified as retrogallic acid(1),gallic acid(2),1,6-di-O-galloyl-β-D-glucose(3),1,2,6-tri-O-galloyl-β-D-glucose(4)and 1,2,3,6-tetra-O-galloyl-β-D-glucose(5).3.Main monomer components and anti-tumor activity of grouped components:3.1 Antitumor activities of main monomer components: Five compounds: gallic acid,retro-gallic acid,1,6-di-O-galloyl-β-D-glucose,1,2,6-tri-O-galloyl-β-D-glucose and 1,2,3,6-tetra-O-galloyl-β-D-glucose,all have different growth inhibition effects on Hep G-2 tumor cells at concentrations of 3.125 ~ 100μg·m L-1,with IC50 of 41.8105,83.3612,244.588,48.6312 and 49.49 respectively.3.2 Anti-tumor activity in groups: Gallic acid,retrogallic acid,1,6-di-O-galloyl-β-D-glucose,1,2,6-tri-O-galloyl-β-D-glucose and 1,2,3,6-tetra-O-galloyl-β-D-glucose in different ratios or in the same ratio all have certain anti-tumor activity.The interaction among the components is mainly antagonistic,and the structures of 1,2,6-tri-O-galloyl-β-D-glucose and 1,2,3,6-tetra-O-galloyl-β-D-glucose are similar.4.Fingerprint study of each part:4.1 Fingerprint of Juglans mandshurica stems and branches: Harvest time is one of the important factors that affect the quality of Juglans mandshurica stems and branches,while the producing areas in the three northeastern provinces have little or no obvious rule on the quality of Juglans mandshurica stems and branches.The contents of each component in the samples collected in the two harvest periods are quite different,and the contents of each component in the samples collected in summer are obviously lower than those collected in spring and winter.4.2 Study on roots fingerprint: The clustering results of Juglans mandshurica roots are not related to the origin and harvest time,which indicates that the origin and harvest time have little or no regular influence on the root quality of Juglans mandshurica in the three northeastern provinces.4.3 Study on leaves fingerprint: The clustering results of Juglans mandshurica leaves are not correlated with the producing areas,indicating that the producing areas have little or no obvious effect on the quality of Juglans mandshurica leaves in the three northeastern provinces..4.4 Study on fingerprint of green peel: Samples taken from the northeast region and samples purchased from Hebei region are divided into 2 categories.The results show that there are some differences between samples taken from the northeast region and samples purchased from Hebei region.5.Determination of main components in each part:5.1 Contents of 5 components in stem branches and roots: In 40 batches of Juglans mandshurica stem and branch samples,the contents of gallic acid,retrogallic acid,1,6-di-O-galloyl-β-D-glucose,1,2,6-tri-O-galloyl-β-Dglucose and 1,2,3,6-tetra-O-galloyl-β-D-glucose are 0.1386 ~ 1.6004,0.0360 ~ 0.9559,0.0158 ~ 1.1174,0.1120 ~ 10.8878,0.1168 ~ 9.8768 mg·g-1.The contents of 5 components in 23 batches of Juglans mandshurica root samples are respectively 1.0968 ~ 4.9590,0.2355 ~ 3.6041,0.2995 ~ 5.5486,0.3784 ~ 17.3004,0.9556 ~ 31.3878 mg·g-1.5.2 Contents of 3 components in leaves: The contents of chlorogenic acid,hyperin and isoquercitrin in 30 batches of Juglans mandshurica leaves are 0.266-5.614,0.113-3.774 and 0.439-6.537 mg·g-1 respectively;5.3 Content of total flavonoids in leaves: The content of total flavonoids in 30 batches of Juglans mandshurica leaves ranges from 50.12 to 232.88 mg·g-1.5.5 Content of chlorogenic acid in fruits: The content of chlorogenic acid in 23 batches of Juglans mandshurica green peel samples is between 0.0104 and 0.1723 mg·g-1.Conclusion:1.132 components in roots,stems,branches,leaves and green pericarp of Juglans mandshurica were characterized by LC-MS.It mainly included tannin,organic acid,naphthalene derivative and flavone.The chemical composition of different parts was quite different.2.The isolated organic acid,tannin monomer and their combination all had anti-tumor activity in vitro.Under the test ratio and concentration,each combination mainly showed antagonism and additive effects.3.Fingerprint-based quality evaluation showed that the quality of Juglans mandshurica stem and branch samples varied greatly in different harvest periods.The quality of Juglans mandshurica root,leaf and green peel samples from different harvest periods and producing areas(the three northeastern provinces)is relatively stable.4.PCA and PLS-DA analysis based on fingerprint showed that tannin contributed significantly to the difference in fingerprint of Juglans mandshurica roots,stems and branches.Flavone contributed significantly to the difference in fingerprint of Juglans mandshurica leaves.Naphthalene and organic acid contributed significantly to the difference in fingerprint of Juglans mandshurica fruits. |
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