CN111257481B

CN111257481B - Method for establishing fingerprint of rhizoma gastrodiae medicinal material and fingerprint thereof

Info

Publication number: CN111257481B
Application number: CN201811456287.3A
Authority: CN
Inventors: 尹春萍; 马元春; 赵佳鑫
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2020-12-08
Anticipated expiration: 2038-11-30
Also published as: CN111257481A

Abstract

The invention provides a method for establishing UPLC fingerprint spectrums of a gastrodia elata medicinal material and an extract thereof, and simultaneously provides the UPLC fingerprint spectrums of the gastrodia elata medicinal material and the extract thereof. The fingerprint has 8 characteristic peaks: characteristic peak No. 1 corresponds to gastrodin; characteristic peak p-hydroxybenzyl alcohol No. 2; the characteristic peak No. 3 corresponds to the balaneboside E; the No. 4 characteristic peak corresponds to the balaneboside B; the characteristic peak No. 5 corresponds to the balaneboside C; the No. 6 characteristic peak corresponds to the balaneboside A; the characteristic peak No. 7 corresponds to hydroxybenzaldehyde; characteristic peak No. 8 corresponds to vanillin. The quality of the gastrodia elata medicinal material can be evaluated by the existence of the common peak in the fingerprint, and the stability of the quality of the medicinal material and the effectiveness and safety of clinical medication are ensured.

Description

Method for establishing fingerprint of rhizoma gastrodiae medicinal material and fingerprint thereof

Technical Field

The invention belongs to the field of medicines, relates to a detection technology of active ingredients of traditional Chinese medicinal materials, and particularly relates to a rapid qualitative and quantitative method for eight active ingredients in gastrodia elata and extracts thereof and preparations containing the gastrodia elata.

Background

The 2015 version of the chinese pharmacopoeia specifies that Gastrodia elata is a dried tuber of Gastrodia elata (Gastrodia elata BI.) of the family orchidaceae [ see: chinese pharmacopoeia [ S ]. department. 2015 ]. In China, the distribution is mainly in Sichuan, Yunnan, Guizhou, Shaanxi and other places, and the distribution of Heilongjiang and Jilin is small. The current research on active ingredients in the gastrodia elata shows that the gastrodia elata has the following pharmacological effects, namely sedation [ see: zou N, Jian-Tao L V, Xue R Y, et al. effects of gases on Sedation and Hypnosis of Mice [ J ]. Lishizhen Medicine & Material Medical Research,2011,22(4):807-809.Cao H Z, Chi H, Qian Y. effects of gases on the expression TH and DBH mRNA in hydrothoramus and advanced gland of rates [ J ]. Medical Journal of Chinese needles Health,2012 ], anticonvulsions [ see: ha J H, Lee D U, Lee J T, et al, 4-Hydroxybenzaldehyde from Gastrodia elata B1.is active in the antibiotic and GABAergic neurological modulation of the rat damage [ J ]. Journal of Ethnopharmacology,2000,73(1): 329) anxiolytic and antidepressant [ see: jung J W, Yoon B H, Oh H R, et al, antioxidant-lipid compositions in microorganisms [ J ]. Biological & Pharmaceutical Bulletin,2006,29(2):261.ZHou B H, Li X J, Liu M, et al, antioxidant-lipid activity of the gastodia alcohol extract in microorganisms [ J ]. Fitoterapia,2006,77(7):592-594 ], neuroprotection [ see: kim H J, Lee S R, Moon K D. ether fraction of methane extrusions of gateway element, media here technical protection against library neural cell transfer colloidal chemistry in gels [ J ]. Phytootherapy Research Ptr,2003,17(8):909. LeGH, Kim HR, Han SY, et al, gateway element blank and its pure compounds technical-2 micro-mechanical-derived cell lines aggregate β -amino acid: the organic solvent of GRP78 and CHCHK [ J ]. Biological Research,2012,45(4): Lee Y K, O M H, Kim C, K. molecular H. K. yeast and K. genome J. molecular DNA, et al.: gradient I, DNA, et al. [ 1. nucleotide J.: flower 5, et al.: DNA, molecular strain, DNA, et al. [ 1. nucleotide, 5J.: DNA, et al.: flower 5, DNA, K, M H, K. In the course of disease treatment, not one component acts, but a plurality of active ingredients act synergistically on the disease, thereby achieving a therapeutic effect. In addition, the situation that the content of the traditional Chinese medicine components is different due to different production places and different batches is common, and the quality of the medicinal materials cannot be really reflected only by using one or two index components as quality standards in pharmacopoeia. In addition, gastrodia elata is also one of the common medicinal materials in many classical famous prescriptions, decoctions and compound preparations [ see: 2862-2865, Kailan H E, Zhang P, Liu X.Tianma Injection in the Treatment of vertical basic ingredient Group Study [ J ] Journal of Practical Traditional Chinese Medicine 2014. Therefore, the simultaneous determination of various active ingredients in the gastrodia elata can provide reference basis for the quality control and application of the medicinal material and also provide identification reference basis for true and false products and compound preparations containing the gastrodia elata. At present, the determination of the components of the gastrodia elata in China mainly adopts an HPLC method to detect the gastrodin [ see: the determination of Gastrodin Contents in Wild and diversified Gastrodia elata from differential Regions of Chongqin by RP-HPLC [ J ]. Medicinal Plant,2012(5) has the disadvantages of long useful time, much loss of organic solution and the like, so that the establishment of a Gastrodia elata detection method which can be fast, stable and environment-friendly and can be suitable for qualitative and quantitative analysis research of Gastrodia elata and extracts thereof or compound preparations containing Gastrodia elata in Different production places is an important subject in the field. At present, a complete and efficient gastrodia elata medicinal material quality evaluation system is lacked, and effective control on the quality of gastrodia elata is difficult to achieve.

Disclosure of Invention

One of the tasks of the invention is to provide a method for rapidly detecting the gastrodia elata, the gastrodia elata extract and various components in the gastrodia elata-containing preparation, and the method can rapidly and simultaneously determine the contents of various active components in the gastrodia elata, the gastrodia elata extract and the gastrodia elata-containing preparation and detect the gastrodia elata components in the preparation.

The technical scheme for realizing the invention is as follows:

the invention provides a method for rapidly detecting various components in gastrodia elata, gastrodia elata extracts and gastrodia elata-containing preparations, which comprises the following steps:

the method comprises the following steps: preparing a sample solution to be tested: taking a gastrodia elata medicinal material or a gastrodia elata extract or powder containing a gastrodia elata preparation, adding ethanol, carrying out ultrasonic extraction, and filtering to obtain a sample solution;

step two: ultra performance liquid chromatograph detection (UPLC): taking 1 mu L of the sample solution obtained in the first step, carrying out sample injection analysis, recording a chromatogram, and measuring the peak area of a component to be detected, wherein the component to be detected is determined to be one or more than two or all of gastrodin, p-hydroxybenzene methanol (gastrodin), barban E, barban B, barban C, barban A, p-hydroxybenzaldehyde and vanillin according to the retention time of eight components of a mixed standard substance contrast map;

step three: substituting the peak area (y) of the component to be detected obtained in the second step into the following standard equation corresponding to the component to be detected, and calculating the concentration (x, mu g. mL) of each corresponding compound^-1) And obtaining the content of the active ingredients in the detected gastrodia elata:

in the formulae: y is the peak area; x is the concentration of each corresponding compound.

The specific method for obtaining the sample solution by taking the gastrodia elata medicinal material or the gastrodia elata extract or the powder containing the gastrodia elata preparation in the step one, adding ethanol, carrying out ultrasonic extraction and filtering comprises the following steps: accurately weighing 0.2g of rhizoma Gastrodiae material or rhizoma Gastrodiae extract or powder containing rhizoma Gastrodiae preparation which has passed through No. 3 sieve, diluting with 70% ethanol to 10ml, and weighing; ultrasonic extracting at 250W and 53kHz for 60min, supplementing 70% ethanol to the original weight, shaking up, and filtering with 0.22 μm organic filter membrane to obtain sample solution.

The conditions of the high performance liquid chromatograph in the second step are as follows: adopts a chromatographic column Luna C₁₈-HST, gauge 2.5 μm, 100 × 3.0 mm; mobile phase: the mobile phase A is CH₃CN and mobile phase B is H₂O and 0.05% H₃PO₄Gradient elution; the elution ratio is 0-2min, and the volume fraction is 97-90% B; 2-5min, volume fraction is 90-78% B; 5-6min, volume fraction is 78-76% B; 6-6.5min, the volume fraction is 76-72%; 6.5-10min, volume fraction 72-60% B; detection wavelength: 220 nm; column temperature: 35 ℃; flow rate 0.4 mL/min^-1(ii) a The sample volume is 1 mu L; the analysis time is 10 min.

In the second step, substances detected and analyzed by an Ultra Performance Liquid Chromatograph (UPLC) are sequentially gastrodin, p-hydroxybenzene methanol (gastrodin), barbaloin E, barbaloin B, barbaloin C, barbaloin a, p-hydroxybenzaldehyde and vanillin according to the arrangement of the peak appearance time. The components reach baseline separation and the linear relation is good. The average sample adding recovery rate is between 99.79 and 101.45 percent, and the RSD is between 1.10 and 1.75 percent. The experimental result shows that the method is stable, rapid and reliable, and can be suitable for qualitative and quantitative detection of the gastrodia elata and the extract thereof or the compound preparation containing the gastrodia elata in different producing areas.

The invention also aims to provide a method for establishing the fingerprint spectrum of the gastrodia elata medicinal material, which comprises the following steps of:

s1: preparing a gastrodia elata medicinal material test solution: taking different batches of gastrodia elata medicinal materials and/or different producing areas, respectively weighing different batches of Mg and/or different gastrodia elata medicinal material test samples, placing the different batches of Mg and/or different producing areas in a container, adding 40-80% by volume of ethanol, preferably 70% by volume of ethanol, fixing the volume to Vml, and satisfying M, V, 0.2:10-0.4:10, preferably M, 0.2, V, 10, then carrying out ultrasonic extraction and filtration, and taking filtrate to obtain different batches of gastrodia elata medicinal material test sample solutions; the solution of the reference peak is ethanol solution of known mass of gastrodin, parahydroxybenzene methanol (gastrodiadin), parahydroxybenzaldehyde, barban E, barban B, barban C, barban A and vanillin;

s2: respectively and precisely absorbing the solution of the reference peak and the solution of each test sample, injecting the solution into an ultra-high performance liquid chromatograph, and recording a chromatogram;

s3: exporting the rhizoma gastrodiae medicinal material fingerprint obtained in the step S2, and importing the rhizoma gastrodiae medicinal material fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system; taking chromatographic peaks existing in chromatograms of different batches of gastrodia elata medicinal materials as common peaks; generating a reference fingerprint of the rhizoma gastrodiae medicinal material by using an average value calculation method; the relative retention time and the relative peak area of each common peak were calculated.

In step S1, the ultrasonic extraction time is 15min or more, preferably 60min, and more preferably 60min by using ultrasonic equipment of 250W and 53 kHz.

The filtration in step S1 is performed with a 0.22 μm organic filter membrane.

In step S2, specifically: precisely absorbing 1 μ l of reference peak solution and each sample solution, injecting into ultra high performance liquid chromatograph, and recording chromatogram for 10 min. Wherein the ultra-high performance liquid chromatography conditions are as follows: adopts a chromatographic column Luna C₁₈-HST, gauge 2.5 μm, 100 × 3.0 mm; mobile phase: the mobile phase A is CH₃CN and mobile phase B is H₂O and 0.05% H₃PO₄Gradient elution; the elution ratio is 0-2min, and the volume fraction is 97-90% B; 2-5min, volume fraction is 90-78% B; 5-6min, volume fraction is 78-76% B; 6-6.5min, the volume fraction is 76-72%; 6.5-10min, volume fraction 72-60% B; detection wavelength: 220 nm; column temperature: 30-40 ℃, preferably 35 ℃; the flow rate is 0.4mL min < -1 >; the sample volume is 1 mu L; the analysis time is 10 min.

The invention also aims to provide a rhizoma gastrodiae medicinal material fingerprint spectrum, which is shown as the attached figure 5, has 8 characteristic peaks, and sequentially comprises the following corresponding active ingredients and retention time:

peak gastrodin No. 1: 3.9-4.1min

Peak p-hydroxybenzol No. 2: 5.2-5.4 min;

peak 3, balamin E: 5.9-6.1min

Peak 4, balamin B: 6.9-7.1min

Peak 5, Balisin C: 7.2-7.4min

6 peak balamin a: 7.9-8.1min

Peak 7 p-hydroxybenzaldehyde: 8.0-8.2min

Peak 8 vanillin: 8.8-9.0 min.

The invention provides a method for establishing a fingerprint, provides a fingerprint of gastrodia elata, and can evaluate the quality of gastrodia elata medicinal materials by the existence of common peaks in the fingerprint, so that the stability of the quality of the medicinal materials and the effectiveness and safety of clinical medication are ensured. The invention also provides a method for rapidly, stably and reliably detecting the rhizoma gastrodiae medicinal material, the true and false product and the compound preparation thereof. The method can perform qualitative and quantitative analysis on 8 active ingredients in rhizoma Gastrodiae, its extract or its preparation in 10 min. The methodological verification proves that the method has good stability, repeatability and precision, can provide reference basis for qualitative and quantitative detection of 8 components in the gastrodia elata medicinal material, the gastrodia elata extract and the compound preparation containing the gastrodia elata, and can quickly detect whether the gastrodia elata is added into the compound preparation.

Drawings

FIG. 1: is a standard chromatogram, wherein the chromatogram comprises 1-gastrodine, 2-p-hydroxybenzyl alcohol, 3-barban E, 4-barban B, 5-barban C, 6-barban A, 7-p-hydroxybenzaldehyde and 8-vanillin.

FIG. 2: is a chromatogram of a Gastrodia elata medicinal material G6a, wherein the chromatogram comprises 1-gastrodin, 2-p-hydroxybenzene methanol, 3-barban E, 4-barban B, 5-barban C, 6-barban A, 7-p-hydroxybenzaldehyde and 8-vanillin.

FIG. 3: is a self-made gastrodia elata extract chromatogram, wherein 1-gastrodin, 2-p-hydroxybenzyl alcohol, 3-balsamoside E, 4-balsamoside B, 5-balsamoside C, 6-balsamoside A, 7-p-hydroxybenzaldehyde and 8-vanillin are adopted.

FIG. 4: the chromatogram map of the headache tablet containing gastrodia elata contains 1-gastrodine, 2-p-hydroxybenzene methanol, 3-balsamoside E, 4-balsamoside B, 5-balsamoside C, 6-balsamoside A, 7-p-hydroxybenzaldehyde and 8-vanillin.

FIG. 5: the fingerprint spectrum of the gastrodia elata medicinal material provided by the invention has 1-8 peaks, wherein 1-gastrodine, 2-p-hydroxybenzyl alcohol, 3-barban E, 4-barban B, 5-barban C, 6-barban A, 7-p-hydroxybenzaldehyde and 8-vanillin are contained.

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. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1

1 Material

1.1 sources of medicinal materials

Rhizoma Gastrodiae purchased from different producing areas and purchasing areas is identified as dry tuber of Gastrodia elata (Gastrodia elata BI.) of Orchidaceae by Duckweed subsidiary professor of medical college of Tongji medical college of Huazhong university of science and technology. Gastrodia elata of different producing areas, purchasing areas and lots is numbered sequentially according to the collecting time, ab represents medicinal materials of different lots of the same manufacturer, and is specifically shown in table 1-1. And purifying a batch of self-made extracts by ethanol reflux macroporous resin in a laboratory and a batch of rhizoma gastrodiae compound preparation rhizoma gastrodiae headache tablets purchased in the market.

TABLE 1-1 Gastrodia elata medicinal material sources

Tab.1-1 The herbs source of Gastrodia elata BI.

1.2 Main test Instrument

1.3 test reagents

2 experimental methods and results

2.1 establishment of analytical methods

In order to meet the analysis requirements, the analysis method is optimized from the conditions of detection wavelength, chromatographic column, mobile phase, column temperature and the like, and the following analysis methods are finally established:

adopts a chromatographic column Luna C₁₈-HST (2.5 μm, 100 × 3.0 mm); mobile phase: mobile phase A (CH)₃CN) -mobile phase B (H)₂O+0.05％H₃PO₄) Gradient elution; the elution ratio is 0-2min, and B is 97-90%; 2-5min, 90-78% B; 5-6min, 78-76% B; 6-6.5min, 76-72%; 6.5-10min, 72-60% B; detection wavelength: 220 nm; the column temperature is 35 ℃; flow rate 0.4 mL/min^-1(ii) a The sample volume is 1 mu L; the analysis time is 10 min.

The substances to be analyzed are sequentially gastrodin, p-hydroxybenzene methanol (gastrodin), barban E, barban B, barban C, barban A, p-hydroxybenzaldehyde and vanillin according to the peak emergence time arrangement. The specific elution gradient is shown in tables 1-2.

TABLE 1-2 gradient elution procedure

Tab.1-2 The gradient elution program

2.2 sample pretreatment

Selecting an extraction solvent, and inspecting the three aspects of ultrasonic time and medicinal material quantity. When the extraction solvent was selected, the extraction effect was examined when water and ethanol with a volume fraction of 40%, 50%, 60%, 70%, 80% were used as the extraction solvent, and 70% ethanol was finally selected as the extraction solvent. When the ultrasonic time is selected, the extraction effects of the medicinal materials when the ultrasonic time is 15min, 30min, 45min and 60min are examined. The result shows that the longer the ultrasonic time is, the better the extraction effect of the medicinal materials is, when the ultrasonic time is 60min, various components of the gastrodia elata reach the peak value, and in consideration of the time benefit relation, 60min is preferably used as the ultrasonic time for the pretreatment of the gastrodia elata.

Ultrasonic time selection of gastrodia elata

When the amount of the medicinal materials for preparing the sample solution is selected, the influence effect on the medicinal material atlas and the peak area when 0.2g, 0.3g and 0.4g of medicinal materials with different weights are weighed is examined. When the amount of the medicinal materials is 0.2g, the peak area of each target component is appropriate, the interference of magazine peaks is less, and the peak area is smaller or none; when the amount of the drug is 0.3g or 0.4g, the peak area of the target component increases, but the number of impurity peaks increases, which disturbs the chromatogram of the target component. Therefore, 0.2g of the amount of the test solution is preferably used as the amount of the drug to be prepared. Determination of column temperature in ultra-high performance liquid chromatography: according to the van's equation, the column temperature affects the column efficiency, the degree of separation, the stability of the chromatographic column, etc., to a certain extent. In this experiment, the separation conditions at 30 deg.C, 35 deg.C, and 40 deg.C were tested as shown in the figure. The chromatographic peak of each component at 30 ℃ is more compact than 35 ℃, the retention time of each component at 40 ℃ is integrally shifted backwards, and the final column temperature is preferably 35 ℃ in consideration of the service life of the chromatographic column.

2.3 preparation of the solution

Preparation of control stock solutions: respectively weighing 2.71mg of gastrodin, 1.11mg of p-hydroxybenzene methanol (gastrodin), 1.23mg of p-hydroxybenzaldehyde, 2.2mg of barbasonin E, 2.28mg of barbasonin B, 1.49mg of barbasonin C, 2.94mg of barbasonin A and 1.08mg of vanillin. In a 10mL volumetric flask; adding 10mL 70% ethanol, ultrasonic dissolving to clarify, and filtering with 0.22 μm organic filter membrane.

Preparation of a medicinal material test solution: accurately weighing 0.2G of rhizoma Gastrodiae powder material with number G1 and passing through No. 3 sieve, placing in a 10ml volumetric flask, adding 70% ethanol, and weighing; ultrasonic extracting for 60min, supplementing to original weight, and shaking.

2.4 method validation

This experiment will verify the method according to the procedure for ICH assay verification.

2.4.1 stability of the solution

Filtering the test solution with 0.22 μm organic filter membrane, and analyzing by injecting 1 μ L sample according to the chromatographic condition under the term of "2.1", to obtain 0h peak area. Respectively taking 1 mu L of sample injection when the sample is placed at room temperature for 1,2, 4, 8, 12 and 24 hours, wherein the RSD of the peak areas of each component of gastrodin, p-hydroxybenzene methanol, barbadensin E, barbadensin B, barbadensin C, barbadensin A, p-hydroxybenzaldehyde and vanillin is respectively 1.1%, 1.3%, 0.88%, 0.87%, 1.2%, 1.7% and 1.8%, and the total area is less than 2.0%. The sample solution was shown to be stable at room temperature for 24 h.

2.4.2 specificity

Sequentially filtering and sampling 1 μ L of blank solvent (70% ethanol solution by volume fraction), control solution and test solution, parallel for 3 times, and recording chromatogram. In detail, see FIGS. 1 and 2, which illustrate that the solvent does not interfere with the assay components.

2.4.3 Linear, quantitative and detection limits

Diluting the stock solution of the reference substance by 2, 5, 10, 25, 50, 100, 250, 625 times, filtering with 0.22 μm organic filter membrane, sampling 1 μ L sample according to the chromatographic conditions under "2.1", and recording chromatogram. Peak area versus compound concentration (. mu.g.mL)^-1) Linear regression was performed to obtain a regression equation, and the experimental results are shown in tables 1-8. KnotThe results show that the linear relationship of the 8 target components is good in the linear range determined by the experiment. The signal-to-noise ratio (S/N) results of the detection limit and the quantification limit of the 8 components are respectively above 3:1 and 10: 1.

2.4.4 precision

For 3 consecutive days, 3 portions of the test solution were prepared in parallel according to the method under "2.3". Sampling 1 μ L of sample, recording chromatogram and peak area, and calculating precision in and during day.

TABLE 1-10 precision within and during days

Tab.1-10 The results of the intra-and inter-day precision

2.4.5 repeatability test

Accurately weighing 0.2G of rhizoma Gastrodiae powder with 60 mesh sieve number G1, preparing test solution according to the method under item "2.3", parallel 6 parts, sampling 1 μ L respectively according to the chromatographic conditions under item "2.1", analyzing, and recording chromatogram and peak area. The RSD value of each target component peak area is calculated to be less than 2.0 percent, which indicates that the established method has good repeatability.

2.4.6 sample recovery

Accurately weighing 0.1G of rhizoma gastrodiae medicinal material powder which is sieved by a 60-mesh sieve and is numbered as G1, and taking 9 parts and 3 parts as 1 group. Test solutions were prepared under the heading "2.3". And precisely adding a single reference substance solution with the amount of 80%, 100% and 120% of the known reference substance into each group, respectively, taking 3 parts of the reference substance solution at each mass concentration, and filtering the solution by using a 0.22-micron microporous membrane to obtain a rhizoma gastrodiae sample solution. According to the chromatographic conditions under the item of 2.1, 1 μ L of sample is injected for analysis, a chromatogram is recorded, and the average recovery rate of each component is calculated. (Note: recovery rate of sample addition ═ (amount detected-content in sample)/amount of control added) × 100%. The sample recovery rate of each target component was calculated. As shown in tables 1-11 and tables 1-12, the average sample recovery rate of Gastrodia elata is between 99.79% and 101.45%, and the RSD is between 1.10% and 1.75%. Experiments prove that the method has good recovery rate. (Note: recovery rate (detection amount-content in drug solution)/addition of control substance amount 100%)

TABLE 1-12 recovery rate of rhizoma Gastrodiae sample

Tab.1-12 The recovery of Gastrodia elata BI

2.5 sample assay

21 batches of sieved rhizoma gastrodiae medicinal powder with different production places, four batches of sieved small rhizoma gastrodiae powder with different production places, self-made extract and rhizoma gastrodiae preparation are respectively prepared according to the preparation method under 2.3, and 3 parts are in parallel. After filtration, 1 μ L of sample was injected and chromatograms are recorded as shown in FIG. 2, FIG. 3, and FIG. 4. The measurement results of 25 batches of Gastrodia elata Blume and Gastrodia elata Blume of different origin are shown in the following table. The results show that the content difference of the gastrodia elata in different producing areas and different batches is large. Generally speaking, the content of each component of the gastrodia elata in the Yunnan producing area is high.

Table 1-14 content of each target component of gastrodia elata in different regions (n ═ 25)

Tab.1-14 The contents of Gastrodia elata BI from different habitats(n＝25)

Note: "-" indicates detection below the limit of quantitation.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A method for establishing a UPLC fingerprint spectrum of a gastrodia elata medicinal material is characterized by comprising the following steps:

s1: preparing a gastrodia elata medicinal material test solution: taking gastrodia elata medicinal materials of different batches and/or different producing areas, respectively weighing M g gastrodia elata medicinal material test products of different batches and/or different producing areas, placing the weighed gastrodia elata medicinal material test products in containers, adding 40-80% ethanol by volume fraction into the containers, fixing the volume to V ml, satisfying M: V ═ 0.2:10-0.4:10, then carrying out ultrasonic extraction and filtration, and taking filtrate to obtain gastrodia elata medicinal material test product solutions of different batches and/or different producing areas; the solution of the reference peak is ethanol solution of a mixture of known mass of gastrodin, parahydroxybenzyl alcohol, parahydroxybenzaldehyde, barban E, barban B, barban C, barban A and vanillin;

s2: respectively and precisely absorbing the solution of the reference peak and the solution of each test sample, injecting the solution into an ultra-high performance liquid chromatograph, and recording a chromatogram; the conditions of the ultra-high performance liquid chromatography are as follows: adopts a chromatographic column Luna C18-HST with the specification of 2.5 μm and the thickness of 100 multiplied by 3.0 mm; mobile phase: the mobile phase A is CH3CN, the mobile phase B is H2O and H3PO4 with the volume fraction of 0.05 percent, and gradient elution is carried out; elution ratio: 0-2min, the volume fraction is 97-90% B; 2-5min, volume fraction is 90-78% B; 5-6min, volume fraction is 78-76% B; 6-6.5min, the volume fraction is 76-72%; 6.5-10min, volume fraction 72-60% B; detection wavelength: 220 nm; column temperature: 30-40 ℃; flow rate 0.4 mL/min^-1(ii) a The sample volume is 1 mu L; the analysis time is 10 min;

s3: exporting the rhizoma gastrodiae medicinal material fingerprint obtained in the step S2, and importing the rhizoma gastrodiae medicinal material fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system; taking chromatographic peaks existing in chromatograms of different batches and/or different places of origin of the gastrodia elata medicinal materials as common peaks; generating a reference fingerprint of the rhizoma gastrodiae medicinal material by using an average value calculation method; the relative retention time and the relative peak area of each common peak were calculated.

2. The method for establishing the UPLC fingerprint of the gastrodia elata medicinal material as claimed in claim 1, wherein the column temperature in step S2 is 35 ℃.