阅读说明：本技术 一种大麦苗中活性黄酮成分群鉴定及其快速检测方法 (Method for identifying active flavone component group in barley seedling and rapidly detecting active flavone component group ) 是由 李静 刘成洪 孙连娜 陈万生 张述伟 陈志伟 宗营杰 陆瑞菊 于 2021-01-04 设计创作，主要内容包括：本发明涉及农业与食品技术和药品研发领域,具体是一种大麦苗中活性黄酮成分群鉴定及其快速检测方法。本发明采用化学系统分离与活性部位追踪分离,首次明确了大麦苗中大麦黄苷、皂草苷、异荭草素-7-O-[6-阿魏酰基]-葡萄糖苷、异牡荆素-7-O-[6-阿魏酰基]-葡萄糖苷、异荭草素-2”-O-(6-阿魏酰基)-葡萄糖苷这5种活性黄酮成分作为抗氧化功能性成分评价指标,并建立5种活性黄酮成分HPLC同步检测方法,弥补了现有评价体系和质控方法的不足,为大麦苗的进一步加工利用和质量控制提供科学依据。(The invention relates to the fields of agriculture, food technology and drug research and development, in particular to a method for identifying active flavone component groups in barley seedlings and quickly detecting the active flavone component groups. The invention adopts the separation of a chemical system and the tracking separation of active sites, firstly defines 5 active flavone components of the barley grass, the saponin, the isoorientin-7-O- [ 6-feruloyl ] -glucoside, the isovitexin-7-O- [ 6-feruloyl ] -glucoside and the isoorientin-2' -O- (6-feruloyl) -glucoside as the evaluation indexes of the antioxidant functional components, establishes the HPLC synchronous detection method of the 5 active flavone components, makes up the defects of the existing evaluation system and quality control method, and provides scientific basis for the further processing utilization and quality control of the barley grass.)

1. A high performance liquid chromatography method for synchronously detecting active flavone components in barley seedlings in a mixed standard mode is characterized in that the active flavone components are 5 of barley xanthosine, saponin, isoorientin-7-O- [ 6-feruloyl ] -glucoside, isovitexin-7-O- [ 6-feruloyl ] -glucoside and isoorientin-2' -O- (6-feruloyl) -glucoside, and the method comprises the following steps:

a. preparation of a control stock solution: taking the control substances of the barley xanthosine, the saponin, the isoorientin-7-O- [ 6-feruloyl ] -glucoside, the isovitexin-7-O- [ 6-feruloyl ] -glucoside and the isoorientin-2' -O- (6-feruloyl) -glucoside, adding a solvent, and dissolving to prepare a control substance solution with a required concentration;

b. preparation of sample solution: sieving barley seedling powder, precisely weighing, adding 30% ethanol solution, ultrasonic extracting for 15min, filtering, and filtering the filtrate with 0.45 μm microporous membrane to obtain sample solution;

c. and (3) measuring the content of 5 active flavone components in the barley seedlings: analyzing the reference solution and the sample solution by a high performance liquid chromatograph under the same chromatographic condition, and calculating the content of the 5 flavone components by a peak area external standard method.

2. The HPLC method for synchronously detecting the content of active flavonoids in barley grass according to claim 1, wherein in step b, the ratio of barley grass powder to 30% ethanol solution is 1:100 (g/mL).

3. The HPLC method for synchronously detecting the content of active flavonoids in barley seedlings according to claim 1, wherein in step b, the sample solution is prepared by the following steps: sieving barley seedling powder 0.2g, adding 20mL 30% ethanol solution precisely, weighing, ultrasonic extracting for 15min, cooling, supplementing with 30% ethanol, shaking, filtering, and filtering with 0.45 μm microporous membrane to obtain sample solution.

4. The HPLC method for synchronously detecting the content of active flavonoids in barley seedlings according to claim 1, wherein the type of the column used in step c isT3, 4.6X 250mm,5 μm, detection wavelength 345nm, column temperature of chromatographic column set at 30 deg.C, and sample amount of 10 μ L.

5. The HPLC method for synchronously detecting the content of active flavone in barley seedlings according to claim 1, wherein in step c, the mobile phase in the liquid chromatography is A% acetonitrile/B% 0.2% formic acid water, the gradient elution procedure is 0-25min, 12% A, and the flow rate is 1.2 mL/min; 25-55min, 12-20% A, and flow rate of 1.2-1.5 mL/min; 55-60min, 20-25% A, and flow rate of 1.5-1.0 mL/min.

6. A quality evaluation method of an active flavone component group in barley seedlings is characterized in that the quality evaluation method takes the following 5 active flavone monomer components as quality evaluation indexes of flavonoid components with antioxidant activity in the barley seedlings: the homotaxin is selected from the group consisting of homotaxin, saponin, isoorientin-7-O- [ 6-feruloyl ] -glucoside, isovitexin-7-O- [ 6-feruloyl ] -glucoside, and isoorientin-2' -O- (6-feruloyl) -glucoside.

7. A method for identifying and rapidly detecting active flavone components in barley seedlings is characterized by comprising the following steps:

A. extracting 80% alcohol extract of barley seedling with n-butanol to obtain n-butanol fraction rich in active flavone;

B. dissolving n-butanol fraction with water, subjecting to D101 macroporous resin column chromatography, and collecting 20-50% ethanol eluate as active fraction;

C. performing activity tracking by adopting various separation methods of sephadex, ODS, C18, MCI gel column chromatography and high performance liquid chromatography in combination with DPPH thin-layer biological self-development technology, separating and identifying main antioxidant activity flavone monomer components, and determining the main antioxidant activity flavone monomer components as quality evaluation indexes of flavone functional component groups in barley seedlings;

D. detecting the content of flavone component by high performance liquid chromatography method of synchronously detecting the mixed standard of active flavone component in barley seedling according to any claim 1-5 with the above identified active flavone monomer component as standard.

8. The method for identifying and rapidly detecting the active flavone component groups in the barley seedlings according to claim 7, wherein in the step C, the active sites are determined by a DPPH antioxidant activity tracking method; then, adopting a plurality of separation methods of MCI reverse phase column chromatography, ODS column chromatography, sephadex column chromatography and high performance liquid chromatography, and combining a DPPH thin-layer biological self-development technology to carry out activity screening, separation and purification to obtain a monomer compound; and (3) evaluating the DPPH antioxidant activity of the obtained monomeric compound, and selecting the monomeric flavone compound with high content and good activity as a quality evaluation index of the flavone functional component group in the barley seedlings.

9. The method for identifying and rapidly detecting active flavone component groups in barley seedlings according to claim 8, wherein in the step C, the barley glucosides, the saponarins, the isoorientin-7-O- [ 6-feruloyl ] -glucosides, the isovitexin-7-O- [ 6-feruloyl ] -glucosides and the isoorientin-2 "-O- (6-feruloyl) -glucosides are quality evaluation indexes of the flavonoid functional component groups in barley seedlings.

Technical Field

The invention relates to the technical field of quality evaluation and detection methods of plant functional components, in particular to a quality evaluation index establishment of 5 active flavone component groups in barley seedlings and a five-in-one mixed standard High Performance Liquid Chromatography (HPLC) rapid detection method thereof.

Background

Barley seedlings are young shoots of Hordeum vulgare L. of Hordeum barley of the genus Hordeum of the family Graminae, Gramineae. Barley is one of the main crops planted in China and is also a traditional medicinal and edible plant. The efficacy of wheat seedlings is recorded in Puji Fang and Ben Cao gang mu. In recent years, barley seedlings are developed into various functional foods such as barley seedling powder, malvidin tablets, barley grass green juice and the like, and the barley seedlings are developed rapidly and have good market prospects.

In recent years, researchers at home and abroad have more and more intensively studied the components and the biological activity of the barley seedlings, and the research shows that the barley seedlings contain various chemical components, such as flavonoids, polyphenols, lignans, polysaccharides, various trace elements and the like. The effects of resisting oxidation, reducing blood sugar and the like of barley seedlings are widely concerned and accepted, but the material basis of active ingredients of the barley seedlings is still lack of systematic research, and no clear evaluation index is found for the functional ingredients of the barley seedlings at present, so that the quality control of the breeding of new barley varieties special for barley chlorophylls and related products thereof is limited to a great extent. The flavonoid compounds are a functional component in the barley seedlings, and the barley flavonosides and saponin are reported to be main active flavone components in the barley seedlings, but an evaluation index and a rapid quantitative detection method capable of evaluating the active flavone in the barley seedlings are still lacked at present.

Disclosure of Invention

The invention aims to provide an identification method and a rapid detection method for active flavone components in barley seedlings, aiming at the problem that an evaluation index capable of evaluating the active flavone in the barley seedlings and a rapid quantitative detection method are lacked at present.

The invention combines chemical system separation and active site tracking, firstly defines 5 active flavone components in the barley seedling as the evaluation indexes of the antioxidant functional components, establishes an HPLC synchronous detection method of the 5 active flavone components, makes up the defects of the existing evaluation system and quality control method, and provides scientific basis for further processing utilization and quality control of the barley seedling.

In order to achieve the purpose, the invention adopts the following technical scheme:

A. systematically separating chemical components in the barley seedlings, and tracking the antioxidant active flavone position by adopting a DDPH in-vitro experiment.

B. Separating and purifying main flavonoid components of the active site, and identifying the structure of the main flavonoid components.

C. And (4) evaluating the antioxidant activity of the separated flavone monomer component.

D. 5 establishment of HPLC synchronous detection method of active flavone component.

Extracting the dry sample of the barley seedling by using 80% ethanol by using a normal temperature percolation method, extracting the total extract by using extraction solvents with different polarities step by step, and determining the n-butyl alcohol extraction part of the barley seedling as an antioxidant active flavone enrichment part by combining a DPPH antioxidant activity experiment. And separating and purifying the active flavone monomer component of the n-butanol active site by using various separation technologies of silica gel column chromatography, MCI gel column chromatography, ODS, C18 reverse phase silica gel column chromatography, sephadex column chromatography and high performance liquid chromatography. By physical and chemical methods and various modern spectral analysis techniques (including ultraviolet, infrared, mass, and infrared spectroscopy),¹H-NMR、¹³C-NMR, DEPT, 2D-NMR) to identify the structure of the compound, and 5 active flavone components in the barley seedlings were determined, respectively: barley xanthosine, saponin, isoorientin-7-O- [ 6-feruloyl]-glucoside, isovitexin-7-O- [ 6-feruloyl]-glucoside and isoorientin-2 "-O- (6-feruloyl) -glucoside. 5 active flavone components which are prepared by separation are taken as standard substances, and an HPLC method for synchronously detecting 5 components is established.

The first aspect of the present invention provides a quality evaluation method of an active flavone component group in barley seedlings, wherein the quality evaluation method comprises the following 5 active flavone monomer components as quality evaluation indexes of flavonoid components having antioxidant activity in barley seedlings: the homotaxin is selected from the group consisting of homotaxin, saponin, isoorientin-7-O- [ 6-feruloyl ] -glucoside, isovitexin-7-O- [ 6-feruloyl ] -glucoside, and isoorientin-2' -O- (6-feruloyl) -glucoside.

In a second aspect of the present invention, there is provided a high performance liquid chromatography method for synchronously detecting active flavone components in barley grass in a mixed standard manner, wherein the active flavone components are 5 of barley xanthosine, saponin, isoorientin-7-O- [ 6-feruloyl ] -glucoside, isovitexin-7-O- [ 6-feruloyl ] -glucoside and isoorientin-2 ″ -O- (6-feruloyl) -glucoside, the method comprising the steps of:

a. preparation of a control stock solution: taking the control substances of the barley xanthosine, the saponin, the isoorientin-7-O- [ 6-feruloyl ] -glucoside, the isovitexin-7-O- [ 6-feruloyl ] -glucoside and the isoorientin-2' -O- (6-feruloyl) -glucoside, adding a solvent, and dissolving to prepare a control substance solution with a required concentration;

b. preparation of sample solution: sieving barley seedling powder, precisely weighing, adding 30% ethanol solution, ultrasonic extracting for 15min, filtering, and filtering the filtrate with 0.45 μm microporous membrane to obtain sample solution;

c. and (3) measuring the content of 5 active flavone components in the barley seedlings: analyzing the reference solution and the sample solution by a high performance liquid chromatograph under the same chromatographic condition, and calculating the content of the 5 flavone components by a peak area external standard method.

Further, in the step b, the feed-liquid ratio of the barley grass powder to the added 30% ethanol solution is 1:100 (g/mL).

In a preferred embodiment of the present invention, in the step b, the sample solution is prepared by: sieving barley seedling powder 0.2g, adding 20mL 30% ethanol solution precisely, weighing, ultrasonic extracting for 15min, cooling, supplementing with 30% ethanol, shaking, filtering, and filtering with 0.45 μm microporous membrane to obtain sample solution.

Further, in the step c, the type of the chromatographic column is(4.6X 250mm,5 μm), a detection wavelength of 345nm, a column temperature of a column set at 30 ℃ and a sample volume of 10 μ L.

Further, in the step c, the mobile phase in the liquid chromatography analysis is A% acetonitrile/B% 0.2% formic acid water, the gradient elution procedure is 0-25min, 12% A, and the flow rate is 1.2 mL/min; 25-55min, 12-20% A, and flow rate of 1.2-1.5 mL/min; 55-60min, 20-25% A, and flow rate of 1.5-1.0 mL/min.

The third aspect of the invention provides a method for identifying and rapidly detecting active flavone component groups in barley seedlings, which comprises the following steps:

A. extracting 80% alcohol extract of barley seedling with n-butanol to obtain n-butanol fraction rich in active flavone;

B. dissolving n-butanol fraction with water, subjecting to D101 macroporous resin column chromatography, and collecting 20-50% ethanol eluate as active fraction;

C. performing activity tracking by adopting various separation methods of sephadex, ODS, C18, MCI gel column chromatography and high performance liquid chromatography in combination with DPPH thin-layer biological self-development technology, separating and identifying main antioxidant activity flavone monomer components, and determining the main antioxidant activity flavone monomer components as quality evaluation indexes of flavone functional component groups in barley seedlings;

D. the content of flavone component is detected by using the identified active flavone monomer component as a standard substance and adopting the high performance liquid chromatography method for synchronously detecting the active flavone component in the barley seedling by mixing the standard substance.

Further, in the step C, an active site is determined by adopting a DPPH antioxidant activity tracking method; then, adopting a plurality of separation methods of MCI reverse phase column chromatography, ODS column chromatography, sephadex column chromatography and high performance liquid chromatography, and combining a DPPH thin-layer biological self-development technology to carry out activity screening, separation and purification to obtain a monomer compound; and (3) evaluating the DPPH antioxidant activity of the obtained monomeric compound, and selecting the monomeric flavone compound with high content and good activity as a quality evaluation index of the flavone functional component group in the barley seedlings.

Further, in the step C, the barley xanthosine, the saponin, the isoorientin-7-O- [ 6-feruloyl ] -glucoside, the isovitexin-7-O- [ 6-feruloyl ] -glucoside and the isoorientin-2' -O- (6-feruloyl) -glucoside are determined and are used as quality evaluation indexes of flavonoid functional component groups in barley seedlings.

The invention has the advantages that:

1. the method adopts solvents with different polarities to extract and carry out systematic separation aiming at chemical components in the barley seedlings, selects DPPH in-vitro experiments to carry out antioxidant activity site tracking, determines an n-butanol extraction site as an antioxidant activity flavone enrichment site, and provides an extraction and separation technical route and a quality evaluation reference index of the active flavone in the barley seedlings;

2. the invention defines that the barley seedling contains 5 main antioxidant active flavone components: the composition comprises barley xanthosine, saponin, isoorientin-7-O- [ 6-feruloyl ] -glucoside, isovitexin-7-O- [ 6-feruloyl ] -glucoside and isoorientin-2' -O- (6-feruloyl) -glucoside, and is determined as quality evaluation index of flavonoid component with antioxidant activity in barley seedling;

3. the invention establishes an HPLC mixed standard synchronous detection method for 5 active flavone components by accurately limiting the separation preparation and high performance liquid chromatography conditions of 5 flavone compound standard substances, and has good chromatographic separation effect, the separation degrees are all more than 1.5, and the theoretical plate number is not low than 7000.

4. The method realizes simultaneous detection of 5 flavone components in the barley grass for the first time, has simple and rapid operation, high accuracy, high precision and good repeatability, can be used for synchronously measuring the content of 5 flavone components in different barley grass samples in a mixed standard manner, provides a basis for screening barley grass varieties rich in active flavone components and developing and controlling the quality of barley grass products, provides a scientific basis for variety breeding of barley grass with high active flavone content and further developing and controlling the quality of barley grass related products, and has higher application value.

Drawings

FIG. 1 shows DPPH clearance at various extraction sites of "flower 30" barley shoots.

FIG. 2 shows DPPH clearance of each eluted fraction in n-butanol fraction of "flower 30" barley grass.

FIG. 3 is a chromatogram of 5 mixed flavone standards.

FIG. 4 is a chromatogram of a sample solution of "flower 30" barley grass.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example (b): identification and rapid detection of active flavone component in 'flower 30' barley seedling

1 test Material

The test material is barley seedling of 'flower 30' variety, provided by plant cell engineering research institute of biotechnology institute of academy of agricultural sciences of Shanghai city. Sowing in experimental field of Shanghai city agricultural science institute in 11 months in 2018, sowing in drill, performing normal water and fertilizer management, harvesting overwintering seedlings when the length of the overwintering seedlings reaches 20-25cm (6-7 leaf period), and drying at 80 ℃ for fixation.

2 method

2.1 extraction of barley grass and determination of active extraction sites

Soaking barley seedling (7.86kg) in 25L 80% ethanol for 24 hr, percolating and extracting with 75L 80% ethanol to obtain ethanol extract of barley seedling, concentrating to obtain 1.629kg of concentrated solution of barley seedling, extracting the concentrated solution of barley seedling with petroleum ether, ethyl acetate and n-butanol respectively, and determining active fraction by DPPH antioxidant activity tracking method.

The DPPH free radical scavenging assay was as follows:

the DPPH solution is prepared for use, and is prepared into a standard reagent with the concentration of 0.2mM by using 95% ethanol, and the standard reagent is stored in a dark place. The experimental group takes a sample with a concentration gradient, 1mL of the sample is fully mixed with 4mL of DPPH solution, the mixture is placed for 30min in a dark place at room temperature, and then the absorbance value is measured at 517nm of an ultraviolet spectrophotometer to obtain the group A of the sample. The control group was a control group A, obtained by mixing 1mL of 95% ethanol with 4mL of DPPH solution and measuring the absorbance. The solvent component is a solvent component A which is obtained by taking 80% ethanol and measuring the absorbance value at 517nm of an ultraviolet spectrophotometer, and three groups of samples are measured in parallel. Clearance was calculated as DPPH clearance (%) [ a control group- (a sample group-a solvent group) ]/a control group x 100%.

2.2 separation and purification of the extract of barley grass

And (3) taking the n-butanol part, completely dissolving the n-butanol part with water, and carrying out D101 macroporous resin column chromatography elution by using ethanol solutions with different concentrations. After elution was complete, TLC thin layer chromatography was used to detect and collect and combine the similar fractions for a total of 4 fractions. Determining active site by DPPH antioxidant activity tracking method. And then, carrying out activity screening, separation and purification by adopting various separation methods of MCI reverse phase column chromatography, ODS column chromatography, sephadex column chromatography and high performance liquid chromatography and combining with DPPH thin-layer biological self-development technology to obtain the monomer compound.

2.3 determination of flavone Standard

And (3) evaluating the DPPH antioxidant activity of the obtained monomer compound, and selecting the monomer flavone compound with high content and good activity as a standard substance.

2.4 preparation of stock solutions of standards

Taking a proper amount of standard substance, precisely weighing, and adding 30% ethanol to obtain a mixed reference substance stock solution with a certain mass concentration.

2.5 preparation of test sample solutions

Taking barley seedling powder, inspecting the extraction method, and performing single-factor inspection on the extraction time, the solvent volume fraction and the feed-liquid ratio on the basis. The optimal preparation method of the sample solution is determined by comparing the peak area sizes of the components.

2.6 measurement of content of active flavone component

The control solution and the test sample solution were analyzed by high performance liquid chromatography under the same chromatographic conditions. The conditions of the liquid chromatography were as follows,

a chromatographic column:(4.6×250mm 5μm)；

detection wavelength: 345 nm;

mobile phase: acetonitrile (a)/0.2% formic acid (B);

the column temperature was 30 ℃ and the amount of sample was 10. mu.L.

The gradient elution procedure was: 0-25min, 12% A, flow rate 1.2 mL/min; 25-55min, 12-20% A, and flow rate of 1.2-1.5 mL/min; 55-60min, 20-25% A, and flow rate of 1.5-1.0 mL/min.

3 results

3.1 extraction of barley grass and determination of active extracted sites

The antioxidant activity of the barley seedling alcohol extract at different solvent extraction parts is evaluated by a DPPH free radical scavenging experiment, and the result (figure 1) shows that the n-butanol part is an active extraction part.

3.2 separation and purification of the extract of barley grass

And (3) eluting the n-butanol part by macroporous resin column chromatography with ethanol solutions of different concentrations to obtain 4 parts. The DPPH antioxidant activity tracking method is adopted to determine the 20-50% ethanol part as the active site (figure 2). And separating and purifying the active site by adopting various chromatographic separation methods to obtain the monomer flavone compound.

3.3 determination of flavone Standard

In the monomer flavone compounds, the contents of the barley xanthosine, the saponin, the isoorientin-7-O- [ 6-feruloyl ] -glucoside, the isovitexin-7-O- [ 6-feruloyl ] -glucoside and the isoorientin-2' -O- (6-feruloyl) -glucoside are high, the activities of the barley xanthosine and the saponin are high, the contents of the other three components are high, and a certain removing effect is realized on DPPH (deoxyribose nucleic acid).

3.4 preparation of stock solutions of standards

Taking appropriate amount of control substances of the barley xanthosine, the saponin, the isoorientin-7-O- [ 6-feruloyl ] -glucoside, the isovitexin-7-O- [ 6-feruloyl ] -glucoside and the isoorientin-2' -O- (6-feruloyl) -glucoside, precisely weighing, and adding 30% ethanol to prepare mixed control substance stock solutions with mass concentrations of 0.5000mg/mL, 0.2500mg/mL, 0.5000mg/mL and 0.5000mg/mL respectively.

3.5 preparation of test sample solutions

3.5.1 examination of extraction methods

Weighing 0.2g and 3 parts of barley seedling powder, precisely weighing, precisely adding 20mL of 30% ethanol solution, weighing, respectively ultrasonically extracting for 15min, reflux extracting at 80 ℃ for 15min, soaking for 24h, cooling after extraction is finished, supplementing weight with 30% ethanol, shaking uniformly, sampling, filtering, and filtering the subsequent filtrate through a 0.45-micrometer microporous membrane to obtain a sample solution. Each sample was prepared in parallel in 3 parts. The peak areas of the 5 components were recorded and the average was calculated, as determined by the chromatographic conditions of 2.6. As shown in table 1, the ultrasonic extraction method is the most suitable method in combination with the highest content and the simplicity of the actual operation.

Table 1 measurement of peak area of two components by different extraction methods (n-3,)

3.5.2 extraction time study

Weighing 0.2g and 3 parts of barley seedling powder, precisely weighing, precisely adding 20mL of 30% ethanol solution, and weighing. Ultrasonic extracting for 15min, 30min and 45min respectively. Taking out, cooling, supplementing with 30% ethanol, shaking, sampling, filtering, and filtering the filtrate with 0.45 μm microporous membrane to obtain sample solution. Each sample was prepared in parallel in 3 parts. The peak areas of the 5 components were recorded and the average was calculated, as determined by the chromatographic conditions of 2.6. As shown in Table 2, the 15min ultrasonic treatment time has a higher extraction rate, so the ultrasonic treatment time is 15 min.

Table 2 peak area measurements of two components at different extraction times (n-3,)

3.5.3 volume fraction of ethanol

Weighing 0.2g 5 parts of barley seedling powder, precisely weighing, precisely adding 20mL of 30%, 60% and 90% ethanol solution, weighing, ultrasonically extracting for 15min, taking out, cooling, supplementing weight, shaking, sampling, filtering, and filtering the filtrate with 0.45 μm microporous membrane to obtain sample solution. Each sample was prepared in parallel in 3 parts. The peak areas of the 5 components were recorded and the average was calculated, as determined by the chromatographic conditions of 2.6. As shown in Table 3, 30% ethanol volume fraction has a higher extraction rate, so 30% ethanol volume fraction is selected.

Table 3 peak area measurements for two components with different ethanol volume fractions (n-3,)

3.5.4 investigation of ratio of liquid to feed

Precisely weighing 0.2g and 3 parts of barley seedling powder, precisely adding 5mL, 10mL and 20mL of 30% ethanol solution respectively, ultrasonically extracting for 15min, taking out, cooling, filtering, placing in a 100mL volumetric flask, fixing the volume to a scale line by using 30% ethanol, sampling, and filtering with a 0.45-micrometer microporous filter membrane to obtain a sample solution. Each sample was prepared in parallel in 3 parts. The peak areas of the 5 components were recorded and the average was calculated, as determined by the chromatographic conditions of 2.6. As shown in table 4, the extract liquor ratio was determined from the results to be 1: 100.

table 4 peak area measurements for the two components with different feed-to-liquid ratios (n-3,)

according to the above investigation results, the preparation method of the sample solution for final determination and measurement comprises sieving barley seedling powder 0.2g, adding 20mL 30% ethanol solution precisely, weighing, ultrasonic extracting for 15min, cooling, supplementing with 30% ethanol, shaking, filtering, and filtering the filtrate with 0.45 μm microporous membrane to obtain the sample solution.

3.6 methodological investigation

3.6.1 specialization examination

Taking the standard solution and the sample solution, measuring according to the liquid chromatography condition under item 2.6, and recording the chromatogram. Chromatograms of the standard and barley seedling sample solutions are shown in FIGS. 3 and 4. In the obtained spectrum, no interference peak exists around, the spectrum is completely separated from other peaks, the separation degree is more than 1.5, the purity of 5 reference substances is more than 98%, and the theoretical plate number is not low 7000.

3.6.2 Linear relationship investigation

Precisely measuring a proper amount of the reference substance mixed stock solution, sequentially diluting step by step, fixing the volume to prepare 6 reference substance mixed solutions with concentration gradients, measuring according to 2.6 chromatographic conditions, and drawing a regression curve by taking the mixed reference substance solution X (mg/mL) as a horizontal coordinate and the corresponding peak area Y as a vertical coordinate. The results are shown in Table 5, and the respective control samples are in good linear relationship within the corresponding concentration ranges.

TABLE 5 regression equation, correlation coefficient and Linear Range

3.6.3 precision test

The sample solution to be tested is precisely sucked by 10 mu L, and is measured according to the chromatographic condition of 2.6, and the sample introduction is carried out for 6 times, the results are shown in Table 6, the areas RSD values of the barley xanthosine, the saponin, the isoorientin-7-O- [ 6-feruloyl ] -glucoside, the isovitexin-7-O- [ 6-feruloyl ] -glucoside and the isoorientin-2' -O- (6-feruloyl) -glucoside are 0.17%, 0.47%, 0.65%, 1.99% and 0.79%, and the precision of the instrument is good.

Table 6 results of precision examination (n ═ 6)

3.6.4 repeatability test

Taking 30 barley seedling samples of the same batch of flowers, preparing a test solution sample, determining according to 2.6 chromatographic conditions, and calculating the RSD value of the sample injection peak area of 5 components of each sample. The results are shown in table 7, the RSD values of the areas of the barley xanthosine, the saponin, the isoorientin-7-O- [ 6-feruloyl ] -glucoside, the isovitexin-7-O- [ 6-feruloyl ] -glucoside and the isoorientin-2' -O- (6-feruloyl) -glucoside are 0.77%, 0.84%, 1.52%, 0.99% and 2.10%, which indicates that the repeatability of simultaneously measuring 5 components by the high performance liquid chromatography established in the experiment is good.

Table 7 repeatability test results (n ═ 6)

3.6.5 stability test

Preparing a sample solution from the same batch of barley seedling samples, respectively standing for 0 hour, 2 hours, 4 hours, 8 hours, 16 hours and 24 hours, determining according to chromatographic conditions of 2.6, and calculating RSD values of sample injection peak areas of 5 components of each sample. The results are shown in Table 8, with RSD values of 0.95%, 1.20%, 1.04%, 0.86%, 1.09%, indicating that the test sample solutions are stable over 24 hours.

Table 8 stability test results (n ═ 6)

3.6.6 sample recovery test

A control sample is added into the same batch of barley seedling samples according to 100 percent of the content of the samples, and 6 parts of test sample solution is prepared in parallel. The peak area was recorded and the recovery was calculated according to the chromatographic conditions of 2.6, and the results are shown in tables 9 to 13. The average recovery rates of the five standards are 100.69%, 99.78%, 99.06%, 102.40% and 98.12%, respectively, and the results show that the detection method has high accuracy.

TABLE 9 inspection of sample application and recovery of barley xanthosine

TABLE 10 examination of saponin sample application recovery

TABLE 11 examination of isoorientin-7-O- [ 6-feruloyl ] -glucoside sample application recovery rate

TABLE 12 isovitexin-7-O- [ 6-feruloyl ] -glucoside sample recovery Studies

TABLE 13 isoorientin-2' -O- (6-feruloyl) -glucoside sample application recovery rate investigation

While the preferred embodiments of the present invention have been described in detail, it is to be understood that the invention is not limited thereto, and that various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present invention, and the equivalents thereof are intended to be encompassed by the scope of the appended claims.