CN110376310B - Method for detecting quality of angelica sinensis - Google Patents
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Abstract
The invention discloses a method for detecting the quality of angelica, which refers to the selection standards of pharmacokinetic parameters such as lipid-water distribution coefficient, oral bioavailability, drug forming property and the like of each matched compound, secondarily selects compound groups with excellent pharmacokinetics and higher safety, and takes the compounds as 'drug effect component groups' related to the efficacy of angelica. Then according to the 'substance-efficacy' interaction relation of the angelica, the measured in vitro/in vivo biological activity of the angelica is used as a monitoring factor Y, the 'pharmacodynamic component group' of the angelica is used as an observation factor X, a partial least squares regression model of the biological activity of the angelica and the 'pharmacodynamic component group' is established, the components which are positively correlated with the biological activity of the angelica and have higher correlation coefficient are found, and the components are used as 'quality markers' for the quality evaluation of the angelica. And finally, establishing a content determination method of the Chinese angelica quality marker by using a liquid chromatography-mass spectrometry technology, and providing an industrialized path for the whole quality tracing and control of Chinese angelica medicinal materials or decoction pieces.
Description
Technical Field
The invention relates to a method for detecting the quality of Chinese angelica, in particular to a method for detecting the quality evaluation of Chinese angelica based on a substance-effect research idea, and belongs to the technical field of medicine detection.
Background
The Chinese medicinal materials are used as basic raw materials for producing Chinese medicinal materials and Chinese patent medicines, the quality of the Chinese medicinal materials and the Chinese patent medicines is key and fundamental for ensuring the quality of the Chinese medicinal materials and the Chinese patent medicines, the standardization of the Chinese medicinal materials is an important measure for ensuring the quality of the Chinese medicinal materials, and the standardization of the quality of the Chinese medicinal materials is an important precondition for the standardization of the Chinese medicinal materials.
When the plant is classified as the dried root of Angelica sinensis (Oliv.) Diels, a 2-year-old herbaceous plant of Umbelliferae, the medical record is originally found in Shen nong Ben Cao Jing, which has been 2000 years old to date. The angelica has strong fragrance, sweet, pungent and slightly bitter taste, and has the effects of enriching blood and activating blood, regulating menstruation and relieving pain, and relaxing bowel. It is mainly used for treating blood deficiency and sallow complexion, vertigo and palpitation, irregular menstruation, amenorrhea and dysmenorrhea, deficiency cold and abdominal pain, rheumatic arthralgia, traumatic injury, superficial infection, pyocutaneous disease, intestinal dryness and constipation. The angelica and the main chemical components thereof have pharmacological effects on a plurality of systems of the body, such as a hematopoietic system, a circulatory system, a nervous system, an immune system and the like; the main biological activities of the medicine include hematopoiesis, platelet aggregation resistance, arrhythmia resistance, radiation resistance, tumor resistance, pain relief, smooth muscle regulation and organ protection.
The quality evaluation of medicinal materials is a difficult and hot problem in the field of traditional Chinese medicine research, and is concerned by a plurality of scholars and researchers. Because the angelica is widely applied, many counterfeit products and defective products are not avoided. The quality standard of the Chinese angelica medicinal material at the early stage is mainly based on the traditional Chinese materia medica and the determined commodity specification summarized, but the quality standard of the Chinese angelica medicinal material is based on the Chinese pharmacopoeia which is issued in 1953. At present, the inspection standard of angelica is one part of the 2015 edition of Chinese pharmacopoeia, and the specifications of angelica tablets and wine angelica are recorded. For a long time, except that the appearance of the angelica medicinal material is evaluated according to the commodity specification, the character, the micro and the thin layer chromatography identification and the component analysis are mainly evaluated according to the classic method of Chinese pharmacopoeia, the internal quality of the angelica is evaluated according to the contents of alcohol-soluble extract, ferulic acid and volatile oil, the quality of the angelica is evaluated only by taking the ferulic acid as a single index component in the Chinese pharmacopoeia of 2015 edition, and the index control components of the ligusticum wallichii and the ligusticum sinensis are also the ferulic acid, so that the single component cannot scientifically, comprehensively and accurately reflect the internal quality of the medicinal material. Therefore, in practice, the quality of angelica sinensis is evaluated by adopting a certain component content, but actually, the pharmacodynamic value of the traditional Chinese medicine is not reflected on a single component, but the effect of the combined action of a plurality of effective components is still present, and the situation that objective reflection is difficult exists.
The Chinese medicine quality Marker (Q-Marker) is a chemical substance which is inherent in Chinese medicinal materials and Chinese medicinal products (such as Chinese medicinal decoction pieces, Chinese medicinal decoction, Chinese medicinal extract and Chinese patent medicine preparation) or is formed in the processing and preparation process and closely related to the functional attributes of the Chinese medicines, and is used as a marking substance for reflecting the safety and the effectiveness of the Chinese medicines for quality control. Rather than the chemicals they absorb and produce through in vivo processes (e.g., human metabolites, digestive enzymes or microbial conversion chemicals), the specific chemical components that require structural analysis to determine their chemical structure and that can be qualitatively and quantitatively determined.
At present, no research on the quality marker of angelica has been reported. The invention creatively establishes the fingerprint spectrums of the angelica medicinal materials in different producing areas by adopting a high resolution liquid chromatography-mass spectrometry (UPLC/Q-TOF-MS) technology based on the compatibility of the traditional Chinese medicine and the complex active ingredients and innovatively combining the current scientific analysis and evaluation means. And secondly screening compound groups with excellent pharmacokinetics and higher safety by referring to pharmacokinetic parameter screening standards of lipid-water distribution coefficients, oral bioavailability, drug forming properties and the like of all matched compounds, and taking the compounds as 'drug effect component groups' related to the efficacy of the angelica. Then according to the 'substance-efficacy' interaction relation of the angelica, the measured in vitro/in vivo biological activity of the angelica is used as a monitoring factor (Y), the angelica 'pharmacodynamic component group' is used as an observation factor (X), a partial least squares regression model of the angelica biological activity and the angelica 'pharmacodynamic component group' is established, the components which are positively correlated with the angelica biological activity and have higher correlation coefficient are found, and the components are used as 'quality markers' for the angelica quality evaluation. Therefore, the invention actually provides a brand-new detection method for evaluating the quality of the angelica.
Disclosure of Invention
The invention aims to provide an angelica quality marker evaluation method based on a substance-effect research idea, and further solves the technical problem that only a single component is used for evaluating the quality of angelica in practice.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
a method for detecting the quality of Chinese angelica comprises the following steps: radix Angelicae sinensis or radix Angelicae sinensis decoction pieces; the detection method is established by establishing a partial least squares correlation model of the angelica biological activity and the main components in the angelica.
Further, the detection method of the quality of the angelica sinensis comprises the following steps:
(1) establishing fingerprint spectra of radix Angelicae sinensis in different production places by liquid mass spectrometry, and screening medicinal component groups of radix Angelicae sinensis by combining with traditional Chinese medicine database;
(2) taking the in vitro antioxidant and in vivo anti-blood deficiency efficacy indexes of the angelica as a monitoring factor Y of the angelica quality, taking the angelica active ingredient group in the step (1) as an observation factor X, establishing a partial least squares regression model of the angelica bioactivity and the active ingredient group, determining angelica quality detection ingredients with positive correlation and higher correlation coefficient of the angelica bioactivity, and determining the ingredients as angelica quality markers;
(3) and (3) establishing a content determination methodology for the angelica quality marker determined in the step (2) and verifying the method.
Further, the detection method of the quality of the angelica sinensis comprises the following conditions of liquid phase and mass spectrum methods in the step (1):
A. liquid phase process conditions: by C18A chromatographic column; the mobile phase is the volume ratio of 0.1 percent formic acid-water solution to acetonitrile; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, the detection wavelength is 323nm, and the gradient elution parameters are as follows: the elution time is 0-2min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent; the elution time is 2-5min, and the volume ratio of 0.1 percent formic acid-water solution is from 98 percent to 85 percent; the elution time is 5-8min, and the volume ratio of 0.1 percent formic acid-water solution is from 85 percent to 70 percent; the elution time is 8-12min, and the volume ratio of 0.1 percent formic acid-water solution is from 70 percent to 50 percent; the elution time is 12-15min, and the volume ratio of 0.1 percent formic acid-water solution is from 50 percent to 33 percent; the elution time is 15-18min, and the volume ratio of 0.1 percent formic acid-water solution is from 33 percent to 20 percent; the elution time is 18-19min, and the volume ratio of 0.1 percent formic acid-water solution is from 20 percent to 0 percent; the elution time is 19-21min, and the volume ratio of 0.1 percent formic acid-water solution is 0 percent; the elution time is 21-22min, and the volume ratio of 0.1 percent formic acid-water solution is from 0 percent to 98 percent; the elution time is 22-25min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent;
B. mass spectrum conditions: the ion source is an electrospray ion source, data are acquired in a positive and negative ion mode, the injection voltage of the positive and negative ion sources is 5500V, -4500V respectively, the atomization temperature is 550 ℃, the atomization gas and the auxiliary gas are nitrogen, the pressure is 50psi, and the pressure is 35 psi. The scanning range of the parent ion is 50-1600 m/z, and the MS is carried out on 8 strongest peaks exceeding 100cps2And collecting data, wherein the scanning range of the sub-ions is 50-1500 m/z.
Still further, the method for detecting the quality of angelica sinensis, according to the method conditions in the step (1), common characteristic peaks of 22 fingerprint spectra in angelica sinensis are determined, and are angelicin A, ligustilide, senkyunolide A, 5-methoxypsoralen, chlorogenic acid, isoanisidide, isopimpinellin, ferulic acid, dihydrocarveol angelate, senkyunolide I, 6-hydroxy-7, 8-dimethoxycoumarin, ethyl ferulate, scopoletin, 8-hydroxypsoralen, psoralen, rutin, imperatorin, formononetin, scoparone, methyl linoleate, quercetin dihydrate and curcumenol.
Still further, in the method for detecting the quality of the angelica sinensis, in the step (1), the parameter indexes for screening the angelica sinensis active ingredient groups are lipid-water distribution coefficient, oral bioavailability and drug forming property.
Still further, according to the method for detecting the quality of the angelica sinensis, the medicinal ingredient group of the angelica sinensis screened by the method is 18 ingredients, which are angelicin A, ligustilide, senkyunolide A, 5-methoxypsoralene, isoanetholide, ferulic acid, dihydrooroselol angelate, senkyunolide I, 6-hydroxy-7, 8-dimethoxycoumarin, ethyl ferulate, scopoletin, 8-hydroxypsoralene, psoralen, imperatorin, formononetin, scoparone, quercetin dihydrate and curcumenol.
Furthermore, in the method for detecting the quality of the angelica sinensis, in the step (2), the method for detecting the in-vitro antioxidant activity of the angelica sinensis is ABTS+·Radical scavenging% DPPH·Radical scavenging rate%, hydroxyl radical scavenging ability; the detection index of the blood deficiency resisting effect of the angelica in vivo is glutathione peroxidase activity.
Still further, in the method for detecting angelica sinensis quality of the present invention, in the step (2), the Variable Projection Importance indicator VIP of the angelica sinensis quality marker is greater than 1, and the VIP is a multivariate statistical term and is fully called Variable immunity in project.
Still further, in the detection method of the quality of the angelica, the angelica quality markers are angelica sinensis lactone A, ligustilide and ferulic acid.
Furthermore, in the method for detecting the quality of angelica sinensis, the establishment and verification of the content determination methodology of the quality marker comprise the following conditions:
the preparation method of the test solution comprises the following steps: weighing 0.2g of angelica powder, precisely weighing, placing in a conical flask, precisely adding 20mL of 70% methanol, weighing, refluxing in water bath at 85 ℃ for 30min, standing, cooling, weighing, adding a solvent to supplement the reduced weight, uniformly shaking, standing, taking supernatant, filtering, and taking a subsequent filtrate for later use;
preparation method of mixed control solution: precisely weighing ferulic acid, levisticide A and ligustilide in a 10mL measuring flask, adding methanol, shaking, and fixing volume to obtain mixed control solutions with mass concentrations of 0.105, 0.212 and 0.112mg/mL respectively;
liquid phase conditions: by C18A chromatographic column; the mobile phase is the volume ratio of 0.1 percent formic acid-water solution to acetonitrile; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, the detection wavelength is 323nm, and the gradient elution parameters are as follows: the elution time is 0-2min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent; the elution time is 2-5min, and the volume ratio of 0.1 percent formic acid-water solution is from 98 percent to 85 percent; the elution time is 5-8min, and the volume ratio of 0.1 percent formic acid-water solution is from 85 percent to 70 percent; the elution time is 8-12min, and the volume ratio of 0.1 percent formic acid-water solution is from 70 percent to 50 percent; the elution time is 12-15min, and the volume ratio of 0.1 percent formic acid-water solution is from 50 percent to 33 percent; the elution time is 15-18min, and the volume ratio of 0.1 percent formic acid-water solution is from 33 percent to 20 percent; the elution time is 18-19min, and the volume ratio of 0.1 percent formic acid-water solution is from 20 percent to 0 percent; the elution time is 19-21min, and the volume ratio of 0.1 percent formic acid-water solution is 0 percent; the elution time is 21-22min, and the volume ratio of 0.1 percent formic acid-water solution is from 0 percent to 98 percent; the elution time is 22-25min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent;
mass spectrum conditions: scanning is carried out on quantitative mass spectrometry by adopting an ESI positive ion mode, wherein the detection mode is multi-reaction monitoring, the ionization parameter is ion spray voltage, and the positive ion mode is +5500 v; the atomization temperature is 550 ℃, the atomization gas and the auxiliary gas are nitrogen, both are 50psi, and the gas curtain gas is 35 psi;
the linear range of the 3 angelica quality marker detection methods is as follows: 0.38-380.00 μ g/mL.
The invention has the beneficial effects that:
1. the invention determines Q-markers influencing the quality of the angelica medicinal material by constructing a quality marker (Q-markers for English short) -target-disease network. And finally, a UPLC-MS/MS technology is adopted to establish a determination method for simultaneously determining the content of the Q-marker component in the angelica, and a scientific evaluation method is provided for the quality control of the angelica medicinal material.
2. The scheme of the invention adopts the concept of integrated omics, is based on the overall research thought of system biology and Chinese medicine, takes the single-point component radiation overall drug effect and the overall effect analysis mechanism as the research key point, and establishes the method for screening the angelica quality marker by using the system drug effect component group interaction network. Compared with the existing method, the detection method reflects the quality of the traditional Chinese medicine through biological activity, and is more scientific and accurate.
3. The invention establishes an in vitro and in vivo antioxidant evaluation model by taking the antioxidant effect in the mechanism of enriching and activating blood of angelica as a key point, evaluates key compounds by establishing a related network of angelica-in vitro/in vivo antioxidant activity-chemical component groups, determines corresponding quality markers and fully embodies the integral efficacy representation of the traditional Chinese medicine.
4. The angelica quality detection method based on the research idea of 'substance-effect' provides better scientific data support for the whole quality control of angelica, meets the trade requirement of 'price by quality' of angelica medicinal material circulation, and also ensures the safety and effectiveness of the clinical medication of angelica decoction pieces.
Drawings
FIG. 1 shows the measurement result of ligustilide content in 16 batches of Chinese angelica medicinal material samples according to one embodiment of the present invention;
FIG. 2 shows the determination of the amount of angelicalactone in a sample of 16 batches of an Angelica sinensis medicinal material according to an embodiment of the present invention;
FIG. 3 shows the determination result of ferulic acid content in 16 samples of Angelica sinensis Diels according to one embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example A method for detecting the quality of Chinese angelica
(1) Sample preparation: referring to the method for preparing the test solution under the content measurement item of the angelica in the first edition of 'Chinese pharmacopoeia' 2015 edition, weighing 0.2g of angelica powder (sieved by a third sieve), precisely weighing, placing in a conical flask, precisely adding 20mL of 70% methanol, weighing, refluxing in water bath at 85 ℃ for 30min, standing, cooling, weighing, adding the solvent to supplement the reduced weight, uniformly shaking, standing, taking the supernatant, filtering, and taking the subsequent filtrate for later use.
(2) Fingerprint spectrum determination and component characterization:
and (3) acquiring data by adopting a liquid chromatography-mass spectrometry method, and importing the spectrum data obtained by UPLC/Q-TOF-MS analysis and detection into spectrum processing software to identify the compound. And comparing, analyzing and screening the total ion flow diagram of each sample to obtain a common characteristic peak with certain representativeness on the fingerprint. And identifying the characteristic compound according to the Chinese medicinal component database and by combining the fragment information of the primary mass spectrum and the secondary mass spectrum and the literature, and roughly deducing the possible structure information of the characteristic compound.
1) Selection of measurement wavelength: the detection wavelength is subjected to three-dimensional atlas analysis of PDA detector detection, and according to the characteristic wavelength of main components in the angelica medicinal material, the detection wavelength 323nm representing the angelica medicinal material is selected from the wavelength range of DAD 210-400 nm for comprehensive fingerprint evaluation.
2) Mass spectrum conditions: the Ion Source is an electrospray Ion Source (ESI), data are collected in a positive and negative Ion mode, the Source injection voltage (IonSpray Voltage flowing) in the positive and negative Ion mode is 5500V, -4500V, respectively, the atomization Temperature (TEM) is 550 ℃, the atomization Gas (Ion Source Gas1, GS1) and the auxiliary Gas (Ion Source Gas2, GS2) are nitrogen, both are 50psi, and the Curtain Gas (Curtain Gas, CUR) is 35 psi. Data are acquired by adopting an Information Dependent Acquisition (IDA), a Dynamic Background Subtraction (DBS) and a high-sensitivity mode, the scanning range of a parent ion (TOF-MS) is 50-1600 m/z, the MS2 data acquisition is carried out on 8 strongest peaks exceeding 100cps, and the scanning range of a child ion is 50-1500 m/z.
3) The liquid phase method comprises the following steps: using Acquity UPLC BEH C18Column (50 mm. times.2.1 mm, 1.7 μm) chromatography column; the mobile phase is 0.1% formic acid-water solution A-acetonitrile B; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, and the detection wavelength is 323 nm; gradient elution conditions are shown in table 1:
TABLE 1 gradient elution conditions
4) Screening common peaks: and (3) importing the map data obtained by UPLC/Q-TOF-MS analysis and detection into PeakView 2.2 software to identify the compound. Comparing, analyzing and screening UPLC/Q-TOF-MS total ion flow graphs under positive ion modes of all samples to obtain common characteristic peaks on 22 fingerprint spectrums, wherein the sum of peak areas accounts for more than 90% of the total peak area. Has certain representativeness. Analyzing PeakView 2.2 software according to a spectrogram, searching a traditional Chinese medicine component database of a MasterView module according to the database, identifying 22 compounds by combining first-level mass spectrum fragment information, second-level mass spectrum fragment information and literature, and mainly generating [ M + H ] in a positive mode]+，[M+NH3]+，[M+Na]+And molecular ion peaks are equispaced, and the response value is extremely high. The element composition of the angelica sinensis is analyzed according to PeakView 2.2 software, and the common peaks in the fingerprint of the angelica sinensis are identified by combining the fragment information of primary and secondary mass spectrums and documents, and the possible structure information of 22 compounds is deduced to be shown in Table 2.
TABLE 2 common component information Table of Angelica sinensis
(3) TCMSP chemical component evaluation and pharmacodynamic component group screening:
the traditional chinese medicine database TCMSP (http:// lsp. nwsuaf. edu. cn/TCMSP. php) collected component data from 499 herbs in the chinese pharmacopoeia (2015 edition), covering 13144 molecules and 29384 compounds, which were added by several years of manual validation by the members of the study group, is one of the largest databases of non-commercial chinese medicine molecules in the world. And (3) adopting a TCMSP library as a secondary screening ligand library of a fingerprint spectrum common peak, and carrying out second screening on matched compounds for predicting pharmacokinetic parameters and toxicology parameters, wherein the screening standard is shown in a table 3.
TABLE 3 ADME/T parameter recommendation screening criteria
According to the screening principle of table 3, 18 compounds were obtained with the lipid-water partition coefficient (LogP), Oral Bioavailability (OB) and pharmaceutical potency (DL) as key screening indices, and the results are shown in table 4.
TABLE 4 TCMSP screening results
(4) In vitro bioactivity assay: determining ABTS of radix Angelicae sinensis medicinal material/decoction piece extract by in vitro antioxidant activity evaluation method commonly used for medicinal materials·+The radical scavenging rate, DPPH radical scavenging rate, hydroxyl radical inhibiting ability, and the specific results are shown in Table 5.
1) Clearing ABTS from angelica extract·+Free radical detection method
Firstly, preparing a standard curve for measurement: the standard was diluted with 70% methanol according to the detection method of the Total antioxidant Capacity detection kit (ABTS Rapid method). 10mM Trolox standard solution was diluted to 0.15mM, 0.3mM, 0.6mM, 0.9mM and 1.2 mM.
Preparing a solvent for determination: 1/1000 hydrogen peroxide solution, peroxidase working solution and ABTS working solution were prepared according to the kit (ABTS Rapid method) with the instructions attached.
Measuring total oxidation resistance: the samples were applied according to SOP, and were incubated at 27 ℃ for 6min after application of the sample because the sample was applied under dark conditions, and OD values at 414nm and 734nm were measured using a microplate reader.
2) Method for detecting hydroxyl radical inhibition capability of angelica sinensis extracting solution
Preprocessing a sample: volatilizing the prepared angelica sinensis sample solution by using a low-temperature centrifugal concentrator, and dissolving the solution in distilled water for later use.
Preparing a reagent for inhibiting hydroxyl radical determination: the reagents in the hydroxyl radical kit are respectively prepared into 0.03 percent of standard substance application liquid, substrate application liquid, reagent three application liquid and color developing agent according to the instruction method.
Third, the best sampling concentration is obtained by the ability test of inhibiting free hydroxyl radicals: mixing the sample solution: the sample solutions with different concentrations were diluted with distilled water at a ratio of 1:1, 1:3, 1:5, 1:7, 1:9, 1:19, 1:49, 1:99, etc., and 0.2mL of each sample solution with different concentration was taken and subjected to the operation according to the operation table.
The prepared application liquid is pre-heated in a water bath at 37 ℃ for 3min, and the following operations are carried out under the condition of the water bath at 37 ℃. And immediately starting timing after adding the third reagent, and immediately adding the color developing agent after 1min to terminate the reaction. Mixing, standing at room temperature for 20min, transferring 200 μ L of each detection tube solution to 96-well plate with pipette, and measuring absorbance at 550nm with microplate reader. The dilution ratio between 20% and 55% is the optimal dilution ratio.
According to the pre-test results, it can be seen that the sample is diluted by 1:7 to be the optimal sampling concentration, and each batch of angelica sinensis samples are diluted by 1:7 and then tested according to the operation table.
The optimal dilution multiple of the angelica sinensis sample is measured to be 8 times in the pre-test result, and the hydroxyl inhibition free is determined according to the formula
3) Detection method for removing DPPH free radicals from angelica sinensis extract
Preparation of DPPH solution: 0.2mM DPPH solution is prepared by methanol and stored away from light for standby, and 2.0mg/mL Vc solution is prepared for standby.
② the DPPH free radical clearance rate determination method: the experiment adopts a micro method to load sample according to the surface, the sample is uniformly mixed after being loaded, the mixture is kept stand for 30min in a dark place at room temperature, and the absorbance value at 517nm is measured by a microplate reader. Calculating the DPPH free radical clearance rate of each batch of angelica according to a formula:
150 μ L of DPPH solution +150 μ L of Angelica sinensis sample OD
Ab 150. mu.L of Angelica sinensis sample + 150. mu.L of 70% methanol solution OD value
Ac 150. mu.L of 70% methanol solution + 150. mu.L of DPPH solution OD
(5) In vivo biological activity assay:
1) research on anti-blood deficiency effect of Chinese angelica aqueous extract on fatigue rats
Preparing a model: SPF grade SD male rats, 6-8 weeks old, with a body weight of 200 + -20 g 0. The groups were randomly divided into a blank control group, a model group and a dosing group, and 6 were each group. The administration group is administered with the Chinese angelica aqueous extract for intragastric administration 1 week before the model building, the intragastric administration dosage is 5g/kg/d, and the model group and the control group are administered with distilled water with the same volume for intragastric administration. And (5) intervening the angelica aqueous extract during the molding for 2 weeks. A combined fatigue rat model is established by adopting a exhaustive running and exhaustive swimming alternating method.
The swimming model comprises: after the rats are adapted for 1 week in an environment with the temperature of 18-22 ℃ and the humidity of 40-60%, the rats of the model group and each administration group are placed in an original glass water tank with the diameter of 52cm and the height of 1.2m to carry out adaptive swimming for 1 week, the water temperature is 25 +/-2 ℃ every time, after the adaptive swimming is completed, each rat is weighed, the swimming weight is calculated according to 5% of the weight, the weight is fixed on the body of the rat, the weight swimming is started for 3 weeks, if the rat cannot be kept firmly again during swimming, the action is obviously disordered, the rat is driven to maintain the motion state, and when the rat sinks into the water bottom for 3s, the rat is considered to be fatigued, the rat is fished, and the water is wiped. The normal control group and the model group did not adopt any treatment.
A running model: the electric running table for rats is adopted to carry out one-time high-intensity exercise. 1d before the formal experiment, the rat performs adaptive running table movement for 15-20 min at the speed of 10-15 m/min, and the slope of the running table is 0 degree; the following procedure was followed for the formal experiments: gradient 0 ° × 15m/min × 5min → gradient 5 ° × 20m/min × 10min → gradient 10 ° × 25m/min × 10min → gradient 20 ° × 25m/min × 10min → gradient 30 ° × 25m/min to exhaustion. The model group and the dosing group of rats both perform continuous horizontal running exercise, the exercise intensity is determined according to the maximum oxygen uptake of Bedford, and the exercise intensity is equivalent to that of 60-70% of the maximum oxygen uptake is moderate intensity exercise. The tail of the rat was electrically stimulated with the channel ends during the exercise to maintain the rat in continuous motion. The standard of exhaustion is that the tail of the rat is electrically stimulated and can not keep up with the preset speed, the hip is pressed on the back wall of the treadmill, the rat can still do powerless movement by electric stimulation, the hind limb is dragged for 30s along with the rotating belt, and the rat is taken out and placed on the platform to do powerless walking.
Observation indexes:
and (4) behavioral observation: observing the dead time of tail suspension, exhaustion swimming time and weight weighing of each group at the end of 3 weeks of molding; and (3) detecting the antioxidation index: after treatment, 0.5mL of blood is collected from rat orbit, serum is prepared, and the activity of glutathione peroxidase (GSH-PX) of each group is detected.
2) Research on blood deficiency resisting effect of angelica sinensis aqueous extract on acute blood stasis model rat
Preparing a model: SPF grade SD male rats, 6-8 weeks old, with a body weight of 200 + -20 g. The groups were randomly divided into a blank control group, a model group and a dosing group, and 6 were each group. The administration group is administered with the Chinese angelica aqueous extract for intragastric administration 1 week before the model building, the intragastric administration dosage is 5g/kg/d, and the model group and the control group are administered with distilled water with the same volume for intragastric administration for 2 weeks. After the administration, the rats of the model group and each administration group are respectively injected with 0.08mL of epinephrine hydrochloride per 100g of body mass subcutaneously, the rats are immersed in ice water for 5min after 2h, and the epinephrine is injected again subcutaneously after 2 h. The normal control group was injected with the same amount of physiological saline without ice water treatment. After treatment, fasting was not prohibited. After 12h, blood was taken for detection.
Observation indexes: and (3) detecting the antioxidation index: after treatment, 0.5mL of blood was collected from the orbit of the rat, and serum was prepared and the activity of glutathione peroxidase (GSH-PX) of each group was examined, as shown in Table 5.
TABLE 516 measurement results of in vitro antioxidant and in vivo bioactivity of Angelica sinensis
(6) Analysis of "ingredient-efficacy" association: and introducing the in vitro antioxidant activity and in vivo biological activity data of the UPLC-TOF-MS/MS component identification data of the collected 16 batches of Chinese angelica aqueous extracts into simca-p 14.1 software for characteristic peak evaluation and PLS calculation. And establishing a PLS model by taking in-vitro antioxidant activity and in-vivo biological activity data as monitoring factors and measuring integral areas of all components as observation factors. After abnormal values are screened, the system extracts the first two main components according to the cross validity index (>0.0975), the orthogonal verification value is 81.7%, and the model fitting is better.
(7) Mass markers (Q-markers) determine: in order to find information on the components causing differences among the groups, information on compounds having large differences among the groups was extracted based on the generated PLS results and identified based on their retention times and PDA spectral characteristics. Meanwhile, the Importance of each compound in the system analysis is described by a Variable Projection Importance index (VIP). Compounds with VIP greater than 1 were selected as potential Q-markers. Of all compounds, 9 compounds with VIP values greater than 1 were: levisticide A (VIP: 5.67), ligustilide (VIP: 3.77), senkyunolide A (VIP: 1.83), cytisine (VIP: 1.78), scopolamine (VIP: 1.03), ferulic acid (VIP: 1.03), chlorogenic acid (VIP: 1.03), senkyunolide I (VIP: 1.03) and cryptochlorogenic acid (VIP: 1.03).
The correlation analysis of partial least squares regression is carried out on the compounds, the antioxidant scavenging activity and the GSH-Px activity, wherein the angelicin A, the ligustilide and the ferulic acid are in positive correlation with the biological activity and have higher correlation (more than 0.50), which shows that the substances have important influence on the antioxidant effect, and the specific experimental results are shown in Table 6. Therefore, the levistilide A, the ligustilide and the ferulic acid are selected as quality markers Q-markers for quality control of the angelica medicinal material.
TABLE 6 partial least squares regression correlation analysis results (inclusion rule: correlation > 0.50)
(8) Establishment of quality marker content determination methodology:
1) preparation of a test solution: referring to the method for preparing the test solution under the content measurement item of the angelica in the first edition of 'Chinese pharmacopoeia' 2015 edition, weighing 0.2g of angelica powder (sieved by a third sieve), precisely weighing, placing in a conical flask, precisely adding 20mL of 70% methanol, weighing, refluxing in water bath at 85 ℃ for 30min, standing, cooling, weighing, adding the solvent to supplement the reduced weight, uniformly shaking, standing, taking the supernatant, filtering, and taking the subsequent filtrate for later use.
2) Preparation of mixed control solution: taking appropriate amount of ferulic acid, levistilide A and ligustilide reference substances, precisely weighing, placing in brown measuring flask, and adding methanol to obtain mixed reference solutions with mass concentrations of 0.1, 0.2 and 0.1mg/mL respectively.
3) Quantitative mass spectrum conditions: quantitative mass spectrometry was performed by scanning in ESI positive ion mode, and detection mode was Multiple Reaction Monitoring (MRM). The ionization parameter is ion spray voltage, and the positive ion mode is +5500 v; the atomization Temperature (TEM) was 550 deg.C, the atomization Gas (Ion Source Gas1, GS1) and the assist Gas (Ion Source Gas2, GS2) were nitrogen, both at 50psi, and the Curtain Gas (Curtain Gas, CUR) was 35 psi. Compound ion pair optimization acquisition parameters: the results of the Declustering Potential (DP), Collision Energy (CE), and Collision Cell Exit Potential (CXP) information are shown in table 7.
TABLE 7 Ferulic acid, ligustilide, levistilide A multiple reaction monitoring MRM parameters
4) The liquid phase method comprises the following steps: using Acquity UPLC BEH C18Column (50 mm. times.2.1 mm, 1.7 μm) chromatography column; the mobile phase is 0.1% formic acid-water solution A-acetonitrile B; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, and the detection wavelength is 323 nm; gradient elution conditions, as shown in table 8:
TABLE 8 gradient elution conditions
5) Linear relationship, detection limit and quantitation limit: precisely absorbing 3Q-markers mixed reference substance working solutions, determining according to optimized analysis conditions, and drawing a standard working curve by taking a peak area as a vertical coordinate (Y) and a sample injection solution mass concentration (X, mu g/mL) as a horizontal coordinate. The linear range of the obtained 3 compounds is as follows: 0.38-380.00 μ g/mL, and good linear relationship between X and Y.
And (3) taking the negative sample as a detection object, adding mixed reference substance solutions with different concentrations, and calculating a detection Limit (LOD) and a quantification Limit (LOQ). The detection limit of the method is between 0.06 and 3.79 mu g/mL, and the quantification limit is between 0.19 and 5.05 mu g/mL. The linear equation, correlation coefficient, method detection limit and quantification limit of 3Q-markers are shown in Table 9.
TABLE 9 Linear equation, Linear Range, r, LOD and LOQ of 3 quality markers from Angelica sinensis
6) Specificity test: and (3) precisely absorbing a blank control solution, mixing the control solution, the test solution and the negative sample solution respectively to perform sample injection analysis by optimizing chromatographic conditions, wherein the active components are not interfered with each other, and the theoretical plate number meets the requirement, so that the specificity of each component is good.
7) And (3) precision test: precisely sucking the mixed reference substance solution, continuously measuring for 6 times according to chromatographic conditions, and recording peak area. The result shows that the retention time RSD of 3 components is 0.14-0.21%, and the peak area RSD is 1.28% -2.03%, which indicates that the method has good precision.
8) And (3) repeatability test: preparing 6 parts of test solution according to the test preparation method, wherein the label is 1-6, precisely absorbing each test solution, carrying out sample injection measurement according to chromatographic conditions, recording peak area and calculating mass fraction. The result shows that the retention time RSD of 3 components is 0.07-0.28%, and the peak area RSD is 0.31% -1.98%, which indicates that the method has good repeatability.
9) And (3) stability test: taking appropriate amount of test solution, standing at room temperature for 0, 3, 6, 12, 18, 24, 36, and 48h, measuring by sample injection under chromatographic condition, and recording peak area. The result shows that the retention time RSD of 3 components is 0.01-0.36%, the peak area RSD is 1.56% -2.11%, and the sample solution is basically stable when placed at room temperature for 48 hours.
10) Sample recovery rate test: precisely sucking 1mL of the concentrated extract with the determined index component content, respectively placing 6 parts of the concentrated extract into 10mL measuring bottles, precisely adding 1mL of mixed reference substance solution containing a certain amount of the concentrated extract, respectively, performing sample injection determination according to chromatographic conditions, recording peak area, and calculating sample injection recovery rate. The sample recovery range is ferulic acid: 98.6-102.1%; ligustilide: 97.4-103.7%; levistilide a: 98.47-101.3%.
And (3) testing each batch of samples: taking 16 batches of angelica sinensis medicinal material samples, respectively preparing 3 parts of test solution, precisely absorbing 2 mu L of reference solution and 2-5 mu L of test solution, injecting the test solution into a liquid chromatograph, measuring, recording peak area, and calculating the content of each component in each batch of samples according to an external standard method, wherein the specific result is shown in figures 1-3. According to the results, the method comprises the following steps: the scheme for determining the three quality markers of ligustilide, angelica archang lica lactone, ferulic acid and the like is accurate and feasible.
Claims (9)
1. A method for detecting the quality of Chinese angelica is characterized by comprising the following steps: the detection method comprises the following steps:
(1) establishing fingerprint spectra of radix Angelicae sinensis in different production places by using UPLC/Q-TOF-MS, and screening medicinal component groups of radix Angelicae sinensis by combining with traditional Chinese medicine database; the liquid phase process conditions: using a C18 chromatographic column; the mobile phase is 0.1 percent formic acid-water solution and acetonitrile which are composed according to the volume ratio; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, the detection wavelength is 323nm, and the gradient elution parameters are as follows: the elution time is 0-2min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent; the elution time is 2-5min, and the volume ratio of 0.1 percent formic acid-water solution is from 98 percent to 85 percent; the elution time is 5-8min, and the volume ratio of 0.1 percent formic acid-water solution is from 85 percent to 70 percent; the elution time is 8-12min, and the volume ratio of 0.1 percent formic acid-water solution is from 70 percent to 50 percent; the elution time is 12-15min, and the volume ratio of 0.1 percent formic acid-water solution is from 50 percent to 33 percent; the elution time is 15-18min, and the volume ratio of 0.1 percent formic acid-water solution is from 33 percent to 20 percent; the elution time is 18-19min, and the volume ratio of 0.1 percent formic acid-water solution is from 20 percent to 0 percent; the elution time is 19-21min, and the volume ratio of 0.1 percent formic acid-water solution is 0 percent; the elution time is 21-22min, and the volume ratio of 0.1 percent formic acid-water solution is from 0 percent to 98 percent; the elution time is 22-25min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent;
(2) taking the in vitro antioxidant and in vivo anti-blood deficiency efficacy indexes of the angelica as a monitoring factor Y of the angelica quality, taking the angelica active ingredient group in the step (1) as an observation factor X, establishing a partial least squares regression model of the angelica bioactivity and the active ingredient group, determining angelica quality detection ingredients with positive correlation and higher correlation coefficient of the angelica bioactivity, and determining the ingredients as angelica quality markers;
(3) and (3) establishing a content determination methodology for the angelica quality marker determined in the step (2) and verifying the method.
2. The method for detecting the quality of angelica according to claim 1, wherein: the mass spectrometry conditions in the detection method step (1) are as follows:
B. mass spectrum conditions: the ion source is an electrospray ion source, data are acquired in a positive and negative ion mode, the injection voltage of the positive and negative ion sources is 5500V, -4500V respectively, the atomization temperature is 550 ℃, the atomization gas and the auxiliary gas are nitrogen gas, the pressure is 50psi, the gas curtain gas is 35psi, the scanning range of the parent ions is 50-1600 m/z, and MS is carried out on 8 strongest peaks exceeding 100cps2And collecting data, wherein the scanning range of the sub-ions is 50-1500 m/z.
3. The method for detecting the quality of angelica according to claim 2, wherein: the method conditions in the detection method step (1) determine common characteristic peaks of 22 fingerprint spectra in the angelica, which are respectively levistilide A, ligustilide, senkyunolide A, 5-methoxypsoralen, chlorogenic acid, isoanisidide, isopimpinellin, ferulic acid, dihydrocarveol angelate, senkyunolide I, 6-hydroxy-7, 8-dimethoxycoumarin, ethyl ferulate, scopoletin, 8-hydroxypsoralen, psoralen, rutin, imperatorin, formononetin, scoparone, methyl linoleate, quercetin dihydrate and curcumenol.
4. The method for detecting the quality of angelica according to claim 2 or 3, wherein: in the detection method, in the step (1), the parameter indexes for screening the angelica sinensis active ingredient group are lipid-water distribution coefficient, oral bioavailability and druggability.
5. The method for detecting the quality of angelica according to claim 4, wherein: the effective component groups of the angelica screened by the method are 18 components, which are respectively levistilide A, ligustilide, senkyunolide A, 5-methoxypsoralen, isoanisidide, ferulic acid, dihydrocarveol angelate, senkyunolide I, 6-hydroxy-7, 8-dimethoxycoumarin, ethyl ferulate, scopoletin, 8-hydroxypsoralen, psoralen, imperatorin, formononetin, scoparone, quercetin dihydrate and curcumenol.
6. The method for detecting the quality of angelica according to claim 5, wherein: in the step (2), the detection index of the in-vitro antioxidant activity of the angelica sinensis is ABTS+·Radical scavenging% DPPH·Radical scavenging rate%, hydroxyl radical scavenging ability; the detection index of the blood deficiency resisting effect of the angelica in vivo is glutathione peroxidase activity.
7. The method for detecting the quality of angelica according to claim 6, wherein: in the step (2), the variable projection importance index VIP value of the angelica quality marker is more than 1.
8. The method for detecting the quality of angelica according to claim 7, wherein: in the detection method, the angelica quality markers are angelica sinensis lactone A, ligustilide and ferulic acid.
9. The method for detecting the quality of angelica according to claim 8, wherein: the establishment and verification of the quality marker content determination methodology comprises the following conditions:
the preparation method of the test solution comprises the following steps: weighing 0.2g of angelica powder, precisely weighing, placing in a conical flask, precisely adding 20mL of 70% methanol, weighing, refluxing in water bath at 85 ℃ for 30min, standing, cooling, weighing, adding a solvent to supplement the reduced weight, uniformly shaking, standing, taking supernatant, filtering, and taking a subsequent filtrate for later use;
preparation method of mixed control solution: taking appropriate amount of ferulic acid, levistilide A and ligustilide reference substances, precisely weighing, placing in brown measuring flask, adding methanol to obtain mixed reference solutions with mass concentrations of 0.1, 0.2 and 0.1mg/mL respectively;
liquid phase conditions: by C18A chromatographic column; the mobile phase is 0.1 percent formic acid-water solution and acetonitrile which are composed according to the volume ratio; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, the detection wavelength is 323nm, and the gradient elution parameters are as follows: the elution time is 0-2min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent; the elution time is 2-5min, and the volume ratio of 0.1 percent formic acid-water solution is from 98 percent to 85 percent; the elution time is 5-8min, and the volume ratio of 0.1 percent formic acid-water solution is from 85 percent to 70 percent; the elution time is 8-12min, and the volume ratio of 0.1 percent formic acid-water solution is from 70 percent to 50 percent; the elution time is 12-15min, and the volume ratio of 0.1 percent formic acid-water solution is from 50 percent to 33 percent; the elution time is 15-18min, and the volume ratio of 0.1 percent formic acid-water solution is from 33 percent to 20 percent; the elution time is 18-19min, and the volume ratio of 0.1 percent formic acid-water solution is from 20 percent to 0 percent; the elution time is 19-21min, and the volume ratio of 0.1 percent formic acid-water solution is 0 percent; the elution time is 21-22min, and the volume ratio of 0.1 percent formic acid-water solution is from 0 percent to 98 percent; the elution time is 22-25min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent;
mass spectrum conditions: scanning is carried out on quantitative mass spectrometry by adopting an ESI positive ion mode, wherein the detection mode is multi-reaction monitoring, the ionization parameter is ion spray voltage, and the positive ion mode is +5500 v; the atomization temperature is 550 ℃, the atomization gas and the auxiliary gas are nitrogen, both are 50psi, and the gas curtain gas is 35 psi;
the linear range of the 3 angelica quality marker detection methods is as follows: 0.38-380.00 μ g/mL.
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