CN114428130B - Method for obtaining potential lipid-lowering markers and metabolic pathways of walnut green seedcase polyphenol extract - Google Patents
Method for obtaining potential lipid-lowering markers and metabolic pathways of walnut green seedcase polyphenol extract Download PDFInfo
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- CN114428130B CN114428130B CN202111572448.7A CN202111572448A CN114428130B CN 114428130 B CN114428130 B CN 114428130B CN 202111572448 A CN202111572448 A CN 202111572448A CN 114428130 B CN114428130 B CN 114428130B
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- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
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Abstract
The invention discloses a liver potential metabolism marker for reducing blood fat of a walnut green seedcase polyphenol extract and a metabolic pathway obtaining method thereof, wherein the marker is obviously influenced by the walnut green seedcase polyphenol extract, 18 potential markers are screened from liver metabolites, and a metabolic fingerprint is obtained from the liver metabolites; and (3) analyzing the relevant metabolic pathways of the two groups of differential metabolites through MetPA to obtain 12 liver metabolic pathways which are interfered by the green walnut peel polyphenol extract. The method comprises the steps of detecting liver metabolites of a rat with hyperlipidemia induced by high fat diet by utilizing UPLC/Q-TOF-MS technology, obtaining related liver potential markers of walnut green seedcase polyphenol extract lipid reduction, analyzing content changes of the liver potential markers of walnut green seedcase polyphenol extract lipid reduction by the technology, and analyzing metabolic pathways of the potential markers to obtain metabolic pathways of walnut green seedcase polyphenol extract lipid reduction. The lipid-lowering action mechanism of the walnut green seedcase polyphenol extract is comprehensively and comprehensively evaluated from the whole level, and a basis is provided for development and utilization of the walnut green seedcase.
Description
Technical Field
The invention belongs to the technical field of biological resource utilization, and particularly relates to a potential lipid-lowering marker of a walnut green seedcase polyphenol extract and a method for obtaining a metabolic pathway.
Background
Abnormal metabolism or transport of human body fat can lead to excessive high total cholesterol TC or triglyceride TG in blood or low high density lipoprotein cholesterol. The damage to the body is hidden, gradual, progressive and systemic, and if not timely intervening, a series of complications are further caused, which seriously jeopardize the health of the human body. Therefore, it is very important to find drugs that interfere with the abnormal processes of lipid metabolism and elucidate their mechanism of action. Metabonomics is the science of studying the collection of all small molecule metabolites in a cell, tissue or organ. The purpose is to quantitatively analyze the content of the metabolites in the organism so as to assist in explaining the functions of genes, reveal the association among various metabolic networks and help researchers to know the organism more systematically. The comprehensive and integral characteristics of metabonomics can be used for clarifying the occurrence of diseases and the lipid-lowering action mechanism of bioactive substances by examining the change of endogenous micromolecular metabolites after the biological system is stimulated or disturbed.
The green walnut shell is Juglans regia (Juglandaceae) plant JuglandaceaeJuglans regiaL, the epicarp of the fruit is mostly thrown away as waste in the production and processing process, which causes resource waste and environmental pollution. The walnut green seedcase mainly contains quinones, flavonoids, phenols, fatty acids, steroids, diaryl heptanes, terpenes, polysaccharides, volatile oil and the like. The medical book records that the walnut green seedcase has the effects of clearing heat and detoxicating, dispelling wind and treating tinea, relieving pain and dysentery and the like, and also has the effects of resisting tumor, resisting oxidation, inhibiting bacteria, relieving pain and the like. Research on walnut green seedcase by applying modern biotechnologyThe active ingredients and the action mechanism thereof are beneficial to the development and utilization of agricultural and forestry waste resources. Along with the development of ecological industry, the cultivation area and yield of the walnut are rapidly increased, and the green seedcase resource of the walnut is very rich, but the resource utilization level is low, so that huge waste is caused. Therefore, as a resource with good medicinal value, how to better extract and treat the effective components in the walnut green seedcase and effectively utilize the effective components is worth intensive research.
Disclosure of Invention
The first aim of the invention is to provide a potential marker for reducing blood fat of a walnut green seedcase polyphenol extract.
The invention further aims at providing a potential marker for reducing blood fat of the walnut green seedcase polyphenol extract and a method for obtaining metabolic pathways.
The first object of the present invention is achieved by a liver potential marker for lipid-lowering of walnut green seedcase polyphenol extract, which is significantly affected by walnut green seedcase polyphenol extract, from among 18 potential markers selected from the liver, 13 in positive ion mode and 5 in negative ion mode, including riboflavin, picolinic acid, tyrosine, L-methionine, lysine, L-leucone, hypoxanthine, glycerophosphole, glutathione, gamma-Glu-Leu, cytidine, 9 h-xanthone, R-carnitine, UDP-glucose, ocular acid, N-actylglutamic acid, glucose 6-phosphate, ADP;
the marker obtains a metabolic fingerprint from liver metabolites of rats with hyperlipidemia induced by high fat diet and the dry prognosis of the green tangerine peel polyphenol extract, and the potential lipid-lowering markers of 18 green tangerine peel polyphenol extracts are screened out by utilizing multivariate variable statistical analysis and identified;
the relative metabolic pathways of the two groups of different metabolites are analyzed by MetPA, and 12 liver metabolic pathways after intervention of walnut green seedcase polyphenol extract are respectively D-glutamine and D-glutamic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, glutathione metabolism, taurine and hypotaurine metabolism, biosynthesis of valine, leucine and isoleucine, interconversion of pentose and gluconate, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism, starch and sucrose metabolism.
The invention also discloses a method for realizing potential markers and metabolic pathways for lipid-lowering of the walnut green seedcase polyphenol extract, which is characterized by comprising the following specific operation steps:
(1) Preparing a walnut green seedcase polyphenol extract: crushing walnut green Pi Shaigan, sieving with an 80-mesh sieve, weighing a certain amount of walnut green husk powder, adding 50-70% ethanol according to a liquid-to-material ratio of 20:1, reflux-extracting for 80-100 min, controlling the temperature to be 50-60 ℃, filtering, extracting for 2 times, combining the filtrates, concentrating in vacuum, and freeze-drying to obtain a walnut green husk polyphenol extract;
(2) And (3) preparing a high-fat feed: the high-fat feed is prepared from, by weight, 60-75 parts of a basic feed, 5-15 parts of egg yolk powder, 5-15 parts of lard, 1-4 parts of cholesterol, 0.6-1.0 part of pig bile salt, 5-15 parts of sucrose and 0.1-0.3 part of salt, and by uniformly mixing, preparing and drying at 45-55 ℃;
(3) Preparation of animal model: 72 healthy male SD rats, 130-150 g in weight, were randomly divided into 2 groups after one week of adaptive feeding: one group is 60 model groups, and the other group is 12 normal control groups; normal control rats were fed with normal feed and model rats were fed with self-hyperlipidemic feed to construct a hyperlipidemic rat model. After 4 weeks of modeling, 6 rats in a model group and a control group are randomly extracted, blood is collected, total cholesterol TC and triglyceride TG in the blood are detected, and whether the modeling of the hyperlipidemic rat model is successful is judged; taking a hyperlipidemic rat with successful modeling, and randomly dividing the hyperlipidemic rat into a model group, a positive medicine group and a walnut green seedcase polyphenol extract intervention group;
(4) Rat liver collection and pretreatment: at the end of the test, the rats are fasted and not forbidden for 12 hours, the rats are dissected after being anesthetized by 10% chloral hydrate, the livers are rapidly taken out, the livers are rinsed by 0.9% normal saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rats are sucked dry, a sample is divided into small blocks with about 1g by using sterile scissors, the small blocks are split into sterile centrifuge tubes, and the sterile centrifuge tubes are immediately placed in liquid nitrogen for quick freezing for at least 15 minutes and then are stored at the temperature of minus 80 ℃; taking a liver sample to obtain a sample to be detected, and detecting liver metabolic products by using UPLC/Q-TOF-MS technology;
(5) LC-MS detection: chromatographic conditions: the instrument adopts WatersaCQUITYUPLC, uses an ACQUITYUPLC chromatographic column with a BEH C of 18.7 [ mu ] m (2.1X100 mm), the temperature of an automatic sampler is set to 4 ℃, the temperature of the column is set to be 0.25mL/min, the temperature of the column is set to be 40 ℃, 10 mu L of the column is sampled for gradient elution, and the mobile phase is 0.1% formic acid water of A phase to 0.1% formic acid acetonitrile of B phase; the gradient elution procedure is 0-1 min,2% phase B; 1-9.5 min, 2-50% of phase B; 9.5-14 min, 50-98% of phase B; 14-15 min,98% of phase B; 15-15.5 min, 98-2% of phase B; 15.5-17 min,2% phase B; mass spectrometry conditions: the instrument uses a thermoLTQorbitrapXL electrospray ion source ESI, a positive and negative ion ionization mode, a positive ion spray voltage of 4.80kV, a negative ion spray voltage of 4.50kV, a sheath gas of 45arb and an auxiliary gas of 15arb; the temperature of the capillary tube is 325 ℃, the capillary tube voltage is 35V/-15V, the tube lens voltage is 50V/-50V, the full scanning is carried out at the resolution ratio 60000, the scanning range is 50-1000, the CID is adopted for carrying out secondary cracking, the collision voltage is 30eV, meanwhile, the dynamic exclusion (the repeated count is 2) is adopted for removing unnecessary MS/MS information, and the dynamic exclusion time is set to be 15s;
(6) Data preprocessing: the obtained original data is converted into mzXML format (XCMS input file format) by Proteowizard software (v3.0.8789), and the XCMS program package of R (v3.3.2) is used for carrying out peak identification, peak filtration and peak alignment; the main parameters are bw=5, ppm=15, peakwidth=c (10, 120), mzwid=0.015, mzdiff=0.01, method= "centrwave". Obtaining a data matrix comprising information such as mass-to-nuclear ratio (m/z), retention time, peak area and the like; 4535 precursor molecules are obtained in a positive ion mode, 4188 precursor molecules are obtained in a negative ion mode, and data are led out to excel for subsequent analysis; to enable comparison of data of different magnitudes, batch normalization of peak areas is performed on the data;
(7) Screening and pathway analysis of potential biomarkers: first, adaptive scaling (UV) was applied to the sample data, followed by multivariate statistical analysis.
According to the invention, the UPLC/Q-TOF-MS technology is utilized to detect liver metabolites of the rat with hyperlipidemia induced by high fat diet, so as to obtain related liver potential markers of the walnut green husk polyphenol extract for reducing the lipid, the content change of the liver potential markers of the walnut green husk polyphenol extract for reducing the lipid is further analyzed by the technology, and metabolic pathway analysis is carried out on the potential markers, so as to obtain the metabolic pathway of the walnut green husk polyphenol extract for reducing the lipid. The lipid-lowering action mechanism of the walnut green seedcase polyphenol extract is comprehensively and comprehensively evaluated from the whole level, and a basis is provided for development and utilization of the walnut green seedcase.
Drawings
FIG. 1 is a graph of total basal peak flow in liver of a control group, a hyperlipidemic rat model group, and a walnut green seedcase polyphenol extract intervention group based on UPLC/Q-TOF-MS technology of the present invention, wherein: 1-1 is a positive ion mode; 1-2 is negative ion mode, CD is normal group, HFD is model group, WGH is walnut green seedcase polyphenol extract group.
FIG. 2 is a diagram of metabolic pathways analyzed using the metaboanalyst platform of the present invention, wherein: a is D-glutamine and D-glutamic acid metabolism, B is biosynthesis of phenylalanine, tyrosine and tryptophan, C is glutathione metabolism, D is taurine and hypotaurine metabolism, E is biosynthesis of valine, leucine and isoleucine, F is interconversion of pentose and gluconate, G is alanine, aspartic acid and glutamic acid metabolism, H is nicotinic acid and nicotinamide metabolism, I is arginine and proline metabolism, J is tryptophan metabolism, K is tyrosine metabolism, L is starch and sucrose metabolism.
Detailed Description
The invention is further described below with reference to the drawings and examples, but is not limited in any way, any changes or substitutions made in accordance with the technical teachings and teachings of the invention are within the scope of the invention.
The method for obtaining the liver potential marker for reducing blood fat by the walnut green seedcase polyphenol extract and the metabolic pathway is realized by the following specific operation steps:
preparing a walnut green seedcase polyphenol extract, namely crushing walnut green Pi Shaigan, sieving with an 80-mesh sieve, weighing a certain amount of walnut green seedcase powder, adding 50% ethanol with the volume fraction of 50% in a liquid-to-material ratio of 20:1, carrying out reflux extraction for 80min, controlling the temperature at 50 ℃, extracting for 2 times, merging filtrate, carrying out vacuum concentration, and freeze-drying to obtain the walnut green seedcase polyphenol extract.
The high-fat feed is prepared by uniformly mixing 67 parts of basic feed, 10 parts of yolk powder, 10 parts of lard, 2 parts of cholesterol, 0.8 part of pig bile salt, 10 parts of sucrose and 0.2 part of salt, preparing and molding, and drying at 45-55 ℃.
The preparation of the animal model comprises the steps of randomly dividing 72 healthy male SD rats with weight of 130-150 g into 2 groups after feeding for one week: one group is 60 model groups, and the other group is 12 normal control groups; normal control rats were fed with normal feed and model rats were fed with self-hyperlipidemic feed to construct a hyperlipidemic rat model. After 4 weeks of modeling, 6 rats in a model group and a control group are randomly extracted, blood is collected, total cholesterol TC and triglyceride TG in the blood are detected, and whether the modeling of the hyperlipidemic rat model is successful is judged; after the modeling is successful, the model groups are randomly divided into model groups, and the model groups are fed with high-fat feed and filled with normal saline with the same amount of stomach; the positive medicine group is fed with high-fat feed and irrigates 40mg/kg.d of simvastatin; feeding high-fat feed to the intervention group of the walnut green seedcase polyphenol extract, and filling the high-fat feed with different doses of the stomach-filling walnut green seedcase polyphenol extract, wherein the high dose is 600mg/kg.d, the medium dose is 300mg/kg.d, and the low dose is 150mg/kg.d; normal control group is fed with normal feed, and normal saline with the same amount of stomach is infused; free diet during the period, raising the environment temperature (242) deg.C, alternating day and night for 12h, and continuously administering for 11 weeks.
The method comprises the steps of collecting and preprocessing the livers of the rats, at the end of a test, taking out the rats after fasted and water-forbidden rats are not treated for 12 hours, anesthetizing the rats by using 10% chloral hydrate, rapidly taking out the livers, rinsing the livers by using 0.9% physiological saline, removing blood stains and dirt, removing connective tissues, adipose tissues and the like, sucking the livers, dividing the samples into small blocks of about 1g by using sterile scissors, split charging the small blocks into a sterile centrifuge tube, immediately quick-freezing the small blocks in liquid nitrogen for at least 15 minutes, and then preserving the small blocks at the temperature of minus 80 ℃; taking liver sample 100mg in a 2mL tube, adding 1000 μl of methanol-water 4:1 mixed solution, and adding 5 steel balls at-20deg.C; placing in a high-throughput tissue grinder, 70Hz,1min; grinding, placing into an ultrasonic machine, treating at room temperature for 30min, placing on ice for 30min, centrifuging at 14000RPM and 4 ℃ for 10min, taking 800 mu L of supernatant, transferring into a new 1.5mL centrifuge tube, and concentrating the sample by using a vacuum centrifugal concentrator; the sample was dissolved with 400. Mu.L of 1:1 aqueous methanol at 4℃and filtered through a 0.22 μm membrane to give a sample to be tested, which was assayed for liver metabolites using UPLC/Q-TOF-MS technology.
The LC-MS detection, chromatographic conditions: the instrument adopts WatersaCQUITYUPLC, uses an ACQUITYUPLC chromatographic column with a BEH C18.7 [ mu ] m (2.1X100 mm), the temperature of an automatic sampler is set to 4 ℃, the temperature of the column is set to be 0.25mL/min, the temperature of the column is set to be 40 ℃, 10 mu L of the column is sampled for gradient elution, and the mobile phase is 0.1% of formic acid water in the A phase and 0.1% of acetonitrile formate in the B phase; the gradient elution procedure is 0-1 min,2% phase B; 1-9.5 min, 2-50% of phase B; 9.5-14 min, 50-98% of phase B; 14-15 min,98% of phase B; 15-15.5 min, 98-2% of phase B; 15.5-17 min,2% phase B; mass spectrometry conditions: the instrument uses a thermoLTQorbitrapXL electrospray ion source ESI, a positive and negative ion ionization mode, a positive ion spray voltage of 4.80kV, a negative ion spray voltage of 4.50kV, a sheath gas of 45arb and an auxiliary gas of 15arb; the temperature of the capillary tube is 325 ℃, the capillary tube voltage is 35V/-15V, the tube lens voltage is 50V/-50V, the full scanning is carried out at the resolution ratio 60000, the scanning range is 50-1000, the CID is adopted for carrying out secondary cracking, the collision voltage is 30eV, meanwhile, the dynamic exclusion (the repeated count is 2) is adopted for removing unnecessary MS/MS information, and the dynamic exclusion time is set to be 15s.
The data preprocessing is carried out, the obtained original data is converted into mzXML format (XCMS input file format) through Proteowizard software (v3.0.8789), and the XCMS program package of R (v3.3.2) is utilized for carrying out peak identification, peak filtration and peak alignment; the main parameters are bw=5, ppm=15, peakwidth=c (10, 120), mzwid=0.015, mzdiff=0.01, method= "centrwave". Obtaining a data matrix comprising information such as mass-to-nuclear ratio (m/z), retention time, peak area and the like; 4535 precursor molecules are obtained in a positive ion mode, 4188 precursor molecules are obtained in a negative ion mode, and data are led out to excel for subsequent analysis; to enable comparison of data of different magnitudes, batch normalization of peak areas was performed on the data.
Screening and channel analysis of potential biomarkers, firstly adopting self-adaptive conversion (UV) to sample data, and then carrying out multivariate statistical analysis to screen differential metabolites under the condition that p-value is less than or equal to 0.05+fold_change is less than or equal to 1.5 or less than or equal to 0.667 and one-way ANOVA p-value is less than or equal to 0.05, so as to find the differential metabolites; the identification of the metabolites was first confirmed based on the exact molecular weight (molecular weight error <20 ppm), followed by comparison Human Metabolome Database (HMDB) based on MS/MS fragmentation pattern, metlin, massbank, lipidMaps, mzclound and Smart nucleic from the established standard database confirmation notes to obtain the metabolites; the 18 potential markers selected from the liver were significantly affected by the walnut green husk polyphenol extract, 13 in positive ion mode and 5 in negative ion mode, including riboflavin, picolinic acid, tyrosine, L-methionine, lysine, L-leucone, hypoxanthine, glycylphosholine, glutathione, gamma-Glu-Leu, cytidine, 9 h-xanthone, (R) carnitine, UDP-glucose, ocular acid, N-Acetylglutamic acid, glucose 6-phosphate, ADP.
Analyzing the relevant metabolic pathways of two groups of differential metabolites through a MetPA database, wherein the adopted data analysis algorithm is supergeometric inspection, and the path topology structure adopts Relative-betweeness Centrality; data between groups are expressed as S+ -D, and statistical analysis is performed by using software GraphPad Prism 7.0, and each group of data is expressed as mean+ -standard deviation ± 0%SD) indicates that ONE-WAY anov et was used for comparison between multiple recombinations; p is p<0.05 is a standard with statistical difference, p<0.01 is a very significant standard of variation; the liver samples obtain 12 paths after intervention of the walnut green husk polyphenol extract, namely D-glutamine and D-glutamic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, glutathione metabolism, taurine and hypotaurine metabolism, biosynthesis of valine, leucine and isoleucine, interconversion of pentose and gluconate, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism, starch and sucrose metabolism.
Example 1
Crushing walnut green Pi Shaigan, sieving with an 80-mesh sieve, weighing a certain amount of walnut green husk powder, adding 50% ethanol with a volume fraction of 20:1, reflux-extracting for 80min at 50 ℃ for 2 times, mixing filtrates, vacuum concentrating, and freeze-drying to obtain a walnut green husk polyphenol extract; the high-fat feed is prepared by uniformly mixing 60 parts of basic feed, 15 parts of yolk powder, 15 parts of lard, 4 parts of cholesterol, 1.0 part of pig bile salt, 15 parts of sucrose and 0.3 part of salt in parts by weight, preparing and shaping, and drying at 55 ℃; the high fat diet was fed to rats according to the procedure of animal model preparation to construct a hyperlipidemic rat model. After molding for 4 weeks, blood is collected, total cholesterol TC and triglyceride TG in the blood are detected, and after the successful molding of the rat model with hyperlipidemia is determined; the stomach-filling green tangerine peel polyphenol extract comprises a high amount of 600mg/kg.d, a medium amount of 300mg/kg.d and a low amount of 150mg/kg.d; normal control group is fed with normal feed, and normal saline with the same amount of stomach is infused; free diet during the period, raising the environment temperature (242) deg.C, alternating day and night for 12h, and continuously administering for 11 weeks. At the end of the test, the rats are fasted and not forbidden for 12 hours, the rats are dissected after being anesthetized by 10% chloral hydrate, the livers are rapidly taken out, the livers are rinsed by 0.9% normal saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rats are sucked dry, a sample is divided into small blocks with about 1g by using sterile scissors, the small blocks are split into sterile centrifuge tubes, and the sterile centrifuge tubes are immediately placed in liquid nitrogen for quick freezing for at least 15 minutes and then are stored at the temperature of minus 80 ℃; taking liver sample 100mg in 2mL tube, adding 1000 μl methanol-water mixed solution (4:1, -20deg.C), and adding 5 steel balls; placing in a high-throughput tissue grinder, 70Hz,1min; grinding, placing into an ultrasonic machine, treating at room temperature for 30min, placing on ice for 30min, centrifuging at 14000RPM and 4 ℃ for 10min, taking 800 mu L of supernatant, transferring into a new 1.5mL centrifuge tube, and concentrating the sample by using a vacuum centrifugal concentrator; the sample was dissolved with 400. Mu.L of 1:1 aqueous methanol at 4℃and filtered through a 0.22 μm membrane to give a sample to be tested, which was assayed for liver metabolites using UPLC/Q-TOF-MS technology. Through LC-MS detection, data pretreatment and screening and pathway analysis of potential biomarkers, 18 potential markers which are obviously influenced by the walnut green seedcase polyphenol extract are obtained, wherein 13 of the potential markers are in a positive ion mode, 5 of the potential markers are in a negative ion mode, and the potential markers comprise riboflavin, picolinic acid, tyrosine, L-methionine, lysine, L-leucone, hypoxanthine, glycerophosphole, glutathione, gamma-Glu-Leu, cytidine, 9 h-xanthone, (R) carnitine, UDP-glucose, ocular acid, N-acetylglutmic acid and glucose 6-phosphate, ADP. Meanwhile, 12 liver sample paths are obtained after intervention of the walnut green seedcase polyphenol extract, namely D-glutamine and D-glutamic acid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, glutathione metabolism, taurine and hypotaurine metabolism, valine, leucine and isoleucine biosynthesis, pentose and gluconate interconversion, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism, starch and sucrose metabolism.
Example 2
Crushing walnut green Pi Shaigan, sieving with an 80-mesh sieve, weighing a certain amount of walnut green husk powder, adding 70% ethanol with a volume fraction of 20:1, reflux-extracting for 100min, controlling the temperature to 60 ℃, extracting for 2 times, mixing filtrates, vacuum concentrating, and freeze-drying to obtain a walnut green husk polyphenol extract; the high-fat feed is prepared by uniformly mixing 75 parts of basic feed, 5 parts of yolk powder, 5 parts of lard, 1 part of cholesterol, 0.6 part of pig bile salt, 5 parts of sucrose and 0.1 part of salt in parts by weight, preparing and shaping, and drying at 45 ℃; the high fat diet was fed to rats in the same manner as in example 1 to construct a model of hyperlipidemic rats. After molding for 4 weeks, blood is collected, total cholesterol TC and triglyceride TG in the blood are detected, and after the successful molding of the rat model with hyperlipidemia is determined; the stomach-filling green tangerine peel polyphenol extract comprises a high amount of 600mg/kg.d, a medium amount of 300mg/kg.d and a low amount of 150mg/kg.d; normal control group is fed with normal feed, and normal saline with the same amount of stomach is infused; free diet during the period, raising the environment temperature (242) deg.C, alternating day and night for 12h, and continuously administering for 11 weeks. At the end of the test, the rats are fasted and not forbidden for 12 hours, the rats are dissected after being anesthetized by 10% chloral hydrate, the livers are rapidly taken out, the livers are rinsed by 0.9% normal saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rats are sucked dry, a sample is divided into small blocks with about 1g by using sterile scissors, the small blocks are split into sterile centrifuge tubes, and the sterile centrifuge tubes are immediately placed in liquid nitrogen for quick freezing for at least 15 minutes and then are stored at the temperature of minus 80 ℃; taking a liver sample to obtain a sample to be detected, and detecting liver metabolic products by using UPLC/Q-TOF-MS technology. Through LC-MS detection, data pretreatment and screening and channel analysis of potential biomarkers, 18 potential markers which are obviously influenced by the walnut green seedcase polyphenol extract are obtained. And 12 liver sample passages are obtained after intervention of the walnut green seedcase polyphenol extract.
Example 3
Crushing walnut green Pi Shaigan, sieving with an 80-mesh sieve, weighing a certain amount of walnut green husk powder, adding 60% ethanol with a volume fraction of 20:1, reflux-extracting for 90min, controlling the temperature to 55 ℃, extracting for 2 times, mixing filtrates, vacuum concentrating, and freeze-drying to obtain a walnut green husk polyphenol extract; the high-fat feed is prepared by uniformly mixing 72 parts of basic feed, 10 parts of yolk powder, 10 parts of lard, 2 parts of cholesterol, 0.8 part of pig bile salt, 10 parts of sucrose and 0.21 part of salt in parts by weight, preparing and shaping, and drying at 50 ℃; the high fat diet was fed to rats in the same manner as in example 1 to construct a model of hyperlipidemic rats. After molding for 4 weeks, blood is collected, total cholesterol TC and triglyceride TG in the blood are detected, and after the successful molding of the rat model with hyperlipidemia is determined; the stomach-filling green tangerine peel polyphenol extract comprises a high amount of 600mg/kg.d, a medium amount of 300mg/kg.d and a low amount of 150mg/kg.d; normal control group is fed with normal feed, and normal saline with the same amount of stomach is infused; free diet during the period, raising the environment temperature (242) deg.C, alternating day and night for 12h, and continuously administering for 11 weeks. At the end of the test, the rats are fasted and not forbidden for 12 hours, the rats are dissected after being anesthetized by 10% chloral hydrate, the livers are rapidly taken out, the livers are rinsed by 0.9% normal saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rats are sucked dry, a sample is divided into small blocks with about 1g by using sterile scissors, the small blocks are split into sterile centrifuge tubes, and the sterile centrifuge tubes are immediately placed in liquid nitrogen for quick freezing for at least 15 minutes and then are stored at the temperature of minus 80 ℃; taking a liver sample to obtain a sample to be detected, and detecting liver metabolic products by using UPLC/Q-TOF-MS technology. Through LC-MS detection, data pretreatment and screening and channel analysis of potential biomarkers, 18 potential markers which are obviously influenced by the walnut green seedcase polyphenol extract are obtained. And 12 liver sample passages are obtained after intervention of the walnut green seedcase polyphenol extract.
Table 1 is a table of liver sample detailed OPLS-DA model validation parameters based on UPLC/Q-TOF-MS technology of the invention. In the table, CD is a normal group, HFD is a model group, and WGH is a walnut green seedcase polyphenol extract group. Wherein: re, principal component fraction; R2X, model (for X variable dataset) interpretable degree; R2Y, model (for Y variable dataset) interpretable degree; q2, model predictability.
TABLE 1 liver sample detailed OPLS-DA model verification parameter Table based on UPLC/Q-TOF-MS technology
Table 2 is a table of potential biomarkers for liver samples based on UPLC/Q-TOF-MS technology of the invention. Mz in the table: mass-to-nuclear ratio; and rt: retention time, units s; identity: the result of the identification; form: ionization mode, [ M+H ] + is positive ion mode, [ M-H ] -is negative ion mode; exact mass: accurate molecular weight; formula: a theoretical molecular formula; KEGG is KEGG compound number.
TABLE 2 liver sample potential biomarker Table based on UPLC/Q-TOF-MS technology
。
Claims (5)
1. A method for obtaining potential markers and metabolic pathways for lipid-lowering of walnut green seedcase polyphenol extract is characterized by comprising the following specific operation steps:
(1) Preparation of walnut green seedcase polyphenol extract
Crushing walnut green Pi Shaigan, sieving with an 80-mesh sieve, weighing a certain amount of walnut green husk powder, adding 50-70% ethanol according to a liquid-to-material ratio of 20:1, carrying out reflux extraction for 80-100 min, controlling the temperature to be 50-60 ℃, extracting for 2 times, combining filtrates, carrying out vacuum concentration, and freeze-drying to obtain a walnut green husk polyphenol extract;
(2) Preparation of high-fat feed
The high-fat feed is prepared from, by weight, 60-75 parts of a basic feed, 5-15 parts of egg yolk powder, 5-15 parts of lard, 1-4 parts of cholesterol, 0.6-1.0 part of pig bile salt, 5-15 parts of sucrose and 0.1-0.3 part of salt, and by uniformly mixing, preparing and drying at 45-55 ℃;
(3) Preparation of animal models
72 healthy male SD rats, 130-150 g in weight, were randomly divided into 2 groups after one week of adaptive feeding: one group is 60 model groups, and the other group is 12 normal control groups; normal control rats were fed with normal feed and model rats were fed with self-hyperlipidemic feed to construct a hyperlipidemic rat model; after 4 weeks of modeling, 6 rats in a model group and a control group are randomly extracted, blood is collected, total cholesterol TC and triglyceride TG in the blood are detected, whether the modeling of a hyperlipidemic rat model is successful is judged, and the hyperlipidemic rat with successful modeling is randomly divided into a model group, a positive medicine group and a walnut green seedcase polyphenol extract intervention group;
(4) Rat liver harvesting and pretreatment
At the end of the test, the rats are fasted and not forbidden for 12 hours, the rats are dissected after being anesthetized by 10% chloral hydrate, the livers are rapidly taken out, the livers are rinsed by 0.9% normal saline, blood stains and dirt are removed, connective tissues and adipose tissues are removed, the rats are sucked dry, a sample is divided into 1g small pieces by sterile scissors, the small pieces are split into sterile centrifuge tubes, and the small pieces are immediately placed in liquid nitrogen for quick freezing for at least 15 minutes and then are stored at the temperature of minus 80 ℃; taking a liver sample to obtain a sample to be detected, and detecting liver metabolic products by using UPLC/Q-TOF-MS technology;
(5) LC-MS detection
Chromatographic conditions: waters ACQUITYUPLC, using a chromatographic column with a volume of 2.1X100 mm and BEH C of 18.7 mu m, an automatic sampler temperature of 4 ℃ and a column temperature of 40 ℃ at a flow rate of 0.25mL/min, and performing gradient elution by injecting 10 mu L, wherein the mobile phase is 0.1% of formic acid water of A phase and 0.1% of acetonitrile formate of B phase; the gradient elution procedure is 0-1 min,2% phase B; 1-9.5 min, 2-50% of phase B; 9.5-14 min, 50-98% of phase B; 14-15 min,98% of phase B; 15-15.5 min, 98-2% of phase B; 15.5-17 min,2% phase B; mass spectrometry conditions: the instrument uses Thermo LTQ Orbitrap XL electrospray ion source ESI, positive and negative ion ionization modes, the positive ion spray voltage is 4.80kV, the negative ion spray voltage is 4.50kV, the sheath gas is 45arb, and the auxiliary gas is 15arb; the temperature of the capillary is 325 ℃, the capillary voltage is 35V/-15V, the tube lens voltage is 50V/-50V, the full scanning is carried out at the resolution ratio 60000, the scanning range is 50-1000, the CID is adopted for carrying out secondary cracking, the collision voltage is 30eV, meanwhile, the dynamic elimination is adopted for removing unnecessary MS/MS information, the repeated counting is 2, and the dynamic elimination time is set to 15s;
(6) Data preprocessing
Converting the obtained original data into mzXML format by Proteowizard software, and carrying out peak identification, peak filtration and peak alignment by using an XCMS program package of R; the main parameters are bw=5, ppm=15, peak width=c10, 120, mzwid=0.015, mzdiff=0.01, method= "centWave", and a data matrix comprising information such as mass-nuclear ratio m/z, retention time and peak area is obtained; 4535 precursor molecules are obtained in a positive ion mode, 4188 precursor molecules are obtained in a negative ion mode, and data are led out to excel for subsequent analysis; to enable comparison of data of different magnitudes, batch normalization of peak areas is performed on the data;
(7) Screening and pathway analysis of potential biomarkers
Firstly, adopting self-adaptive conversion UV for sample data, and then carrying out multivariate statistical analysis;
screening differential metabolites under the condition that p-value is less than or equal to 0.05+fold_change is more than or equal to 1.5 or less than or equal to 0.667 and one-way ANOVA p-value is less than or equal to 0.05, and searching differential metabolites; the identification of the metabolites is firstly confirmed according to the accurate molecular weight, the molecular weight error is less than 20ppm, and then the metabolites are obtained by comparing Human Metabolome Database and Metlin, massbank, lipidMaps, mzclound according to MS/MS fragment patterns and confirming and annotating the Smart nucleic self-built standard database; the 18 potential markers selected from the liver are significantly affected by the walnut green husk polyphenol extract, 13 in positive ion mode and 5 in negative ion mode, including riboflavin, picolinic acid, tyrosine, L-methionine, lysine, L-leucone, hypoxanthine, glycylphosholine, glutathione, gamma-Glu-Leu, cytidine, 9 h-xanthone, R-carnitine, UDP-glucose, ophtalmic acid, N-Acetylglutamic acid, glucose 6-phosphate, ADP;
analyzing the relevant metabolic pathways of two groups of differential metabolites through a MetPA database, wherein the adopted data analysis algorithm is supergeometric inspection, and the path topology structure adopts Relative-betweeness Centrality; data among groups are expressed by S+/-D, and are statistically analyzed by using software GraphPad Prism 7.0, each group of data is expressed by mean+/-standard deviation x+/-S, and ONE-WAY ANOVET is adopted for comparison among multiple recombinations; p < 0.05 is a standard with statistical difference, and p < 0.01 is a standard with extremely obvious difference; the liver samples obtain 12 paths after intervention of the walnut green husk polyphenol extract, namely D-glutamine and D-glutamic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, glutathione metabolism, taurine and hypotaurine metabolism, biosynthesis of valine, leucine and isoleucine, interconversion of pentose and gluconate, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism, starch and sucrose metabolism.
2. The method of claim 1, wherein the preparation of the walnut green seedcase polyphenol extract in the step (1) comprises the steps of crushing the walnut green seedcase Pi Shaigan, sieving the crushed walnut green seedcase Pi Shaigan with a sieve of 80 meshes, weighing a certain amount of walnut green seedcase powder, adding ethanol with the volume fraction of 50% into the powder in a liquid-to-material ratio of 20:1, carrying out reflux extraction for 80 minutes, controlling the temperature to 50 ℃, filtering, extracting for 2 times, combining the filtrates, carrying out vacuum concentration, and freeze-drying to obtain the walnut green seedcase polyphenol extract.
3. The method of claim 1, wherein the high-fat feed in the step (2) is prepared by uniformly mixing 67 parts by weight of basic feed, 10 parts by weight of yolk powder, 10 parts by weight of lard, 2 parts by weight of cholesterol, 0.8 part by weight of pig bile salt, 10 parts by weight of sucrose and 0.2 part by weight of salt, and drying at 45-55 ℃ after preparation and molding.
4. The method of claim 1, wherein after the modeling in step (3), the model groups are randomly divided into model groups, and the model groups are fed with high-fat feed and filled with normal saline with the same amount as the stomach; the positive medicine group is fed with high-fat feed and irrigates 40mg/kg.d of simvastatin; feeding high-fat feed to the intervention group of the walnut green seedcase polyphenol extract, and filling the high-fat feed with different doses of the stomach-filling walnut green seedcase polyphenol extract, wherein the high dose is 600mg/kg.d, the medium dose is 300mg/kg.d, and the low dose is 150mg/kg.d; normal control group is fed with normal feed, and normal saline with the same amount of stomach is infused; free diet during the period, raising the environment temperature to 242 ℃, and carrying out daily alternation for 12 hours, and carrying out uninterrupted administration for 11 weeks.
5. The method according to claim 1, wherein in step (4), 100mg of liver sample is taken in a 2mL tube, 1000 μl of methanol-water 4:1 mixed solution is added, and 5 steel balls are added at-20deg.C; placing in a high-throughput tissue grinder, 70Hz,1min; grinding, placing into an ultrasonic machine, treating at room temperature for 30min, placing on ice for 30min, centrifuging at 14000RPM and 4 ℃ for 10min, taking 800 mu L of supernatant, transferring into a new 1.5mL centrifuge tube, and concentrating the sample by using a vacuum centrifugal concentrator; the sample was dissolved with 400. Mu.L of 1:1 aqueous methanol at 4℃and filtered through a 0.22 μm membrane to give a sample to be tested, which was assayed for liver metabolites using UPLC/Q-TOF-MS technology.
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