阅读说明：本技术 荷叶清轻茶指纹图谱的检测方法 (Method for detecting fingerprint spectrum of light lotus leaf tea ) 是由 金俊杰 李国维 刘爱国 于 2021-07-31 设计创作，主要内容包括：本发明公开了荷叶清轻茶指纹图谱的检测方法,它包括步骤1、荷叶清轻茶供试品溶液的制备；步骤2、混合对照品溶液的制备：步骤3、分别精密吸取混合对照品溶液和供试品溶液注入液相色谱仪,记录色谱图；步骤4,荷叶清轻茶指纹图谱仪器导出,导入中药色谱指纹图谱相似度评价系统,选择不同批次荷叶清轻茶的色谱图中均存在的色谱峰作为共有峰；用平均值计算法生成荷叶清轻茶的对照指纹图谱；计算各共有峰的相对保留时间、相对峰面积；将荷叶清轻茶指纹图谱和混合标准品图谱进行比对,指认主要成分峰。本发明所提供的荷叶清轻茶指纹图谱,能全面,客观地表征荷叶清轻茶的质量。且检测方法具有方法简便、稳定、精密度高、重现性好等优点。(The invention discloses a method for detecting a fingerprint of light lotus leaf tea, which comprises the following steps of 1, preparing a test solution of the light lotus leaf tea; step 2, preparation of a mixed reference solution: step 3, respectively and precisely sucking the mixed reference solution and the test solution to be injected into a liquid chromatograph, and recording a chromatogram; step 4, guiding out a fingerprint instrument of the light lotus leaf clear tea, guiding the fingerprint instrument into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and selecting chromatographic peaks existing in chromatograms of different batches of light lotus leaf clear tea as common peaks; generating a comparison fingerprint of the light lotus leaf tea by using an average value calculation method; calculating the relative retention time and the relative peak area of each common peak; and comparing the fingerprint spectrum of the light lotus leaf tea with the spectrum of the mixed standard substance, and identifying the peak of the main component. The fingerprint spectrum of the light lotus leaf tea provided by the invention can comprehensively and objectively represent the quality of the light lotus leaf tea. And the detection method has the advantages of simple and stable method, high precision, good reproducibility and the like.)

1. A method for detecting a fingerprint of a light lotus leaf tea is characterized by comprising the following steps:

step 1, preparing a test solution of the light lotus leaf tea:

weighing different batches of light lotus leaf tea samples respectively, powdering, placing in a round-bottomed bottle, adding a methanol solution, carrying out ultrasonic extraction, filtering, placing the filtrate in a volumetric flask, adding methanol to a constant volume, and filtering through a 0.45-micrometer microporous filter membrane to obtain a sample solution;

step 2, preparation of mixed reference solution:

precisely weighing chlorogenic acid and chrysophanol reference substances, placing in a volumetric flask, adding methanol to constant volume to scale, shaking up to obtain mixed reference substance solution;

step 3, precisely absorbing the test solution in the step 1 and the reference solution in the step 2 respectively, injecting the test solution and the reference solution into a high performance liquid chromatograph, and recording a chromatogram;

step 4, exporting the fingerprint of the light lotus leaf tea test sample solution obtained in the step 3, and importing the fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012A; selecting chromatographic peaks existing in chromatograms of different batches of the light lotus leaf tea as common peaks; generating a comparison fingerprint of the light lotus leaf tea by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed reference solution chromatogram;

and 5, comparing the fingerprint spectrum of the light lotus leaf clear tea obtained in the step 3 with the spectrum of the mixed standard substance, and identifying that the peak 1 in the light lotus leaf clear tea is chlorogenic acid and the peak 13 in the light lotus leaf clear tea is chrysophanol.

2. The method for detecting the fingerprint of the light lotus leaf tea as claimed in claim 1, wherein the method for preparing the test solution of the light lotus leaf tea in step 1 comprises the following steps: taking 5-50 g of 8 batches of light lotus leaf tea samples, powdering, placing in a round-bottom flask, carrying out ultrasonic extraction and filtration, placing filtrate in a volumetric flask of 100-1000 mL, adding methanol to a constant volume, and filtering through a 0.45-micrometer microporous membrane to obtain a test solution.

3. The method for detecting the fingerprint of the light lotus leaf tea as claimed in claim 1, wherein the step 2 of preparing the mixed reference solution comprises the following steps: precisely weighing chlorogenic acid and chrysophanol, placing in a volumetric flask, adding methanol to constant volume to scale, shaking, and making into mixed reference solution containing 0.1019mg/mL chlorogenic acid and 0.1048mg/mL chrysophanol.

4. The method for detecting the fingerprint of the light lotus leaf tea as claimed in claim 1, wherein in the step 3, the liquid chromatography conditions are as follows: a chromatographic column: purospher STAR LP RP-18 endclamped, mobile phase: acetonitrile and 0.1% phosphoric acid water, gradient elution, diode array detector, detection wavelength: 245nm, column temperature 30 ℃, flow rate 1.0mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:

Procedure time (min) Acetonitrile volume (%) 1 0 5 2 10 5 3 18 13 4 118 23 5 125 35 6 190 100

。

5. The method for detecting the fingerprint of the light lotus leaf tea as claimed in claim 1, wherein the fingerprint contains 13 peaks, the retention time of the 13 peaks is 27.094min for peak 1, 44.868min for peak 2, 52.306min for peak 3, 55.153min for peak 4, 69.471min for peak 5, 81.816min for peak 6, 85.479min for peak 7, 88.331min for peak 8, 108.954min for peak 9, 139.753min for peak 10, 155.801min for peak 11, 157.283min for peak 12 and 162.082min for peak 13, wherein the peak 1 is chlorogenic acid and the peak 13 is chrysophanol.

6. The method for detecting the fingerprint of the light lotus leaf tea as claimed in any one of claims 1 to 5, wherein the components and the proportions of the medicinal ingredients of the light lotus leaf tea are as follows: 8-16 parts of lotus leaves, 7-14 parts of cassia seeds, 7-14 parts of hawthorn, 4-87 parts of mulberry leaves, 5-10 parts of radish seeds, 4-8 parts of fructus cannabis and 5-10 parts of mulberries.

7. The method for detecting the fingerprint of the light lotus leaf tea as claimed in claim 6, wherein the components and the proportions of the medicinal ingredients of the light lotus leaf tea are as follows: 8 parts of lotus leaf, 7 parts of cassia seed, 7 parts of hawthorn, 4 parts of mulberry leaf, 5 parts of radish seed, 4 parts of fructus cannabis and 5 parts of mulberry.

Technical Field

The invention relates to a detection method of traditional Chinese medicine substituted tea, in particular to a detection method of fingerprint spectrum of light lotus leaf tea.

Background

With the improvement of living standard and the change of living style, the obese people are obviously increased. Obesity is an independent chronic disease widely prevalent worldwide with excess fat accumulation in the body being a major feature. It not only affects the beauty of the human appearance, but also obesity and many chronic diseases such as: diabetes, hypertension, cardiovascular and cerebrovascular diseases, etc. The lotus leaf light tea concentrates the unique taste and the food therapy function of medicinal and edible medicinal materials in the tea bag, so that people with irregular diet and work can also drink a cup of functional substitute tea by themselves, moisten intestines, remove fat and keep a healthy life.

From the perspective of traditional Chinese medicine, the lotus leaf light-weight tea aims at clearing heat and removing fat, adopts a method of removing water and sweating, mainly adopts heat-clearing and fire-purging medicinal materials, and is supplemented with dampness-resolving, qi-moving and food stagnation-removing medicines to moisten intestines and remove fat. The lotus leaf has the effects of clearing summer heat, raising hair, clearing yang, cooling blood and stopping bleeding, and has the functions of regulating fat, losing weight, resisting oxidation, resisting aging and inhibiting bacteria; the cassia seed can clear liver and improve vision, and moisten intestines to relieve constipation; haw can invigorate spleen, resolve dampness, relieve summer-heat, warm middle-jiao, promote qi circulation and promote digestion; the mulberry leaves can disperse wind and heat, clear away the lung-heat and moisten dryness, calm the liver and improve eyesight, cool blood and stop bleeding; the radish seeds have the effects of promoting digestion, relieving distension, descending qi and reducing phlegm; fructus Cannabis is used for treating blood deficiency and body fluid deficiency, constipation due to intestinal dryness, and has effects of loosening bowel to relieve constipation; the tea has the effects of clearing heat and purging fire, relaxing bowel, reducing pathogenic fire, tonifying liver and kidney, reducing swelling and promoting urination, nourishing liver and improving eyesight and the like by daily drinking.

At present, the quality detection methods of the light lotus leaf tea are few. The invention adopts the high performance liquid chromatography to establish the fingerprint spectrum detection method of the light lotus leaf tea, and has important significance for ingredient identification, quality evaluation and quality standard formulation of the light lotus leaf tea.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art and provides the fingerprint spectrum detection method of the light lotus leaf tea, the detection method can objectively, comprehensively and accurately evaluate the quality of the light lotus leaf tea, and the method has important significance for controlling the quality of the light lotus leaf tea and ensuring the clinical curative effect.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:

a method for detecting a fingerprint of a light lotus leaf tea comprises the following steps:

step 1, preparing a test solution of the light lotus leaf tea:

accurately weighing different batches of light folium Nelumbinis samples, pulverizing, placing in round-bottomed bottles, adding methanol solution, reflux extracting, filtering, placing filtrate in volumetric flask, adding methanol to desired volume, and filtering with 0.45 μm microporous membrane to obtain sample solution;

step 2, preparation of mixed reference solution:

precisely weighing chlorogenic acid and chrysophanol reference substances, placing in a volumetric flask, adding methanol to constant volume to scale, shaking up to obtain mixed reference substance solution;

step 3, precisely absorbing the test solution in the step 1 and the reference solution in the step 2 respectively, injecting the test solution and the reference solution into a high performance liquid chromatograph, and recording a chromatogram;

step 4, exporting the fingerprint of the light lotus leaf tea test sample solution obtained in the step 3, and importing the fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012A; selecting chromatographic peaks existing in chromatograms of different batches of the light lotus leaf tea as common peaks; generating a comparison fingerprint of the light lotus leaf tea by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed reference solution chromatogram;

and 5, comparing the fingerprint spectrum of the light lotus leaf clear tea obtained in the step 3 with the spectrum of the mixed standard substance, and identifying that the peak 1 in the light lotus leaf clear tea is chlorogenic acid and the peak 13 in the light lotus leaf clear tea is chrysophanol.

As a preferred scheme, the above method for detecting the fingerprint of the light lotus leaf tea comprises the following steps of 1: taking 5-50 g of light lotus leaf tea sample powder of 8 batches, placing the light lotus leaf tea sample powder in a round-bottomed bottle of 100-1000 mL, adding 50-500 mL of methanol solution, carrying out ultrasonic extraction for 30-60 min, filtering, placing the filtrate in a volumetric flask of 100-500 mL, adding methanol to a constant volume, and filtering through a microporous filter membrane of 0.45 mu m to obtain a sample solution.

As a preferred scheme, the method for detecting the fingerprint of the light lotus leaf tea comprises the following steps of 2, preparation of a mixed reference solution: precisely weighing chlorogenic acid and chrysophanol, placing in a volumetric flask, adding methanol to constant volume to scale, shaking, and making into mixed reference solution containing 0.1019mg/mL chlorogenic acid and 0.1048mg/mL chrysophanol.

As a preferred scheme, in the above method for detecting the fingerprint of the light lotus leaf tea, in step 3, the liquid chromatography conditions are as follows: a chromatographic column: purospher STAR LP RP-18 endclamped, mobile phase: acetonitrile and 0.1% phosphoric acid water, gradient elution, diode array detector, detection wavelength: 245nm, column temperature 30 ℃, flow rate 1.0mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:

Procedure	time (min)	Acetonitrile volume (%)
			1	0	5
2	10	5
			3	18	13
4	118	23
			5	125	35
6	190	100

Preferably, in the method for detecting the fingerprint of the light lotus leaf tea, 13 peaks are shared in the fingerprint.

The detection method of the fingerprint of the light lotus leaf tea comprises the following steps of: 8-16 parts of lotus leaves, 7-14 parts of cassia seeds, 7-14 parts of hawthorn, 4-87 parts of mulberry leaves, 5-10 parts of radish seeds, 4-8 parts of fructus cannabis and 5-10 parts of mulberries.

As a preferred scheme, the method for detecting the fingerprint of the light lotus leaf tea comprises the following steps: 8 parts of lotus leaf, 7 parts of cassia seed, 7 parts of hawthorn, 4 parts of mulberry leaf, 5 parts of radish seed, 4 parts of fructus cannabis and 5 parts of mulberry.

Optimizing fingerprint detection conditions:

1. in the aspect of preparation optimization of sample solution

According to the invention, through experimental comparison of different extraction methods (ultrasonic extraction, reflux extraction, percolation extraction and the like) and different extraction solvents (methanol, water, 50% ethanol water solution, 95% ethanol and absolute ethanol), the results show that the spectrogram obtained by ultrasonic extraction has more comprehensive components, high extraction efficiency and good separation degree, so that the ultrasonic extraction method is adopted; in the investigation of the extraction solvent, the chromatogram of the methanol extract is found to have the most information content and the highest component content, so that methanol is selected for extraction.

2. In the aspect of optimizing chromatographic conditions

According to the invention, a diode array detector is adopted to examine the detection wavelength, chromatograms at 205nm, 225nm, 245nm and 280nm are extracted, and when the detection wavelength is 245nm, the information content contained in the chromatograms is most comprehensive and the base line is stable, so 245nm is selected as the detection wavelength.

The invention screens the flow rate (1mL/min, 0.9mL/min, 0.8mL/min, 0.6mL/min), and finally selects the gradient condition with the flow rate of 1.0mL/min because the components in the light lotus leaf tea have better peak shape and better separation effect under the flow rate of 1 mL/min.

The invention compares the elution effects of 5 different elution systems of methanol-water, acetonitrile-0.1% formic acid, acetonitrile and 0.05% phosphoric acid water, and acetonitrile-0.1% phosphoric acid water under different gradients. As a result, it was found that when acetonitrile and 0.1% phosphoric acid water were used as the mobile phase, the separation effect of each component in the light lotus leaf tea was good, and therefore acetonitrile and 0.1% phosphoric acid water were finally selected as the mobile phase.

After the optimal fluidity composition is determined, the optimal gradient elution procedure is screened through a large number of experiments, and the experiment shows that when acetonitrile with the volume of 5-5% is adopted for 0-10 min; 5-13% of acetonitrile in 10-18 min; 13-23% of acetonitrile in 18-118 min; the volume of the acetonitrile is 23-35% in 118-125 min; and when the volume of acetonitrile is 35-100% in 125-190 min, good separation degree of each spectrum peak in the fingerprint can be realized.

Has the advantages that:

1. according to the structural property characteristics of active ingredients contained in the light lotus leaf tea, the optimal mobile phase composition is screened out through a large number of experiments, and analysis conditions such as gradient elution procedures, flow rate, detection wavelength, chromatographic column, column temperature and the like are verified through a plurality of experiments.

2. The fingerprint spectrum detection method of the light lotus leaf tea can comprehensively, objectively and accurately detect and evaluate the quality of the light lotus leaf tea, and has important significance for ensuring the curative effect of the light lotus leaf tea.

3. The fingerprint spectrum of the light lotus leaf tea established by the method provided by the invention can effectively represent the quality of the light lotus leaf tea, objectively reflect the front and back sequence and the mutual relation of each formed fingerprint characteristic peak, pay attention to the overall facial features, avoid the one-sidedness of determining the quality of the light lotus leaf tea due to the determination of individual chemical components, and reduce the possibility of manual treatment for reaching the quality standard.

4. The method for detecting the fingerprint of the light lotus leaf tea provided by the invention has the advantages of simplicity, convenience, good stability, high precision, good reproducibility and the like.

Drawings

FIG. 1 is a chromatogram of a mixed control according to the present invention.

FIG. 2 is a comparison fingerprint of the light lotus leaf tea sample of the present invention.

FIG. 3 shows the fingerprint of 8 batches of test samples of the light lotus leaf tea.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not specified, according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The following examples used the following instruments and reagents:

experimental equipment

1.1 instruments

A high-performance liquid chromatography system with full-wave-band scanning (200- & 800nm) of Shimadzu corporation in Japan comprises a full-automatic online degassing system, a full-automatic sample introduction system promience SIL-20A, a diode array detector SPD-M20A, an automatic temperature control column oven CTO-20A, a KQ3200DB type numerical control ultrasonic cleaner (ultrasonic instruments Co., Ltd., Kunshan city) and a BP121S electronic analysis balance (SARTORIUS).

1.2 drugs and reagents

The light lotus leaf tea sample is from Jiangsu Haihong pharmaceutical Co Ltd; chlorogenic acid reference (batch No. 110753-201314) was purchased from China food and drug Biometrics institute; chrysophanol controls (batch No. 19011601) were purchased from gongyi biotechnology limited; methanol (analytically pure); phosphoric acid (analytically pure); acetonitrile (chromatographically pure); water (ultrapure water).

Embodiment 1 a method for detecting fingerprint of a light lotus leaf tea, comprising the following steps:

step 1, preparing a test solution of the light lotus leaf tea:

taking 20g of 8 batches of light lotus leaf tea samples (4 g of lotus leaves, 3.5g of cassia seeds, 3.5g of hawthorn, 2g of mulberry leaves, 2.5g of radish seeds, 2g of fructus cannabis and 2.5g of mulberry), pulverizing, putting into a 500mL round-bottomed bottle, adding 100mL of methanol solution, carrying out ultrasonic extraction for 30min, filtering, putting the filtrate into a 100mL volumetric bottle, adding methanol to fix the volume, and passing through a 0.45-micrometer microporous filter membrane to obtain a sample solution;

step 2, preparation of mixed reference solution:

precisely weighing chlorogenic acid and chrysophanol, placing in a volumetric flask, adding methanol to constant volume to scale, shaking, and making into mixed reference solution containing 0.1019mg/mL chlorogenic acid and 0.1048mg/mL chrysophanol.

Step 3, precisely absorbing 8 batches of light lotus leaf tea test sample solutions and mixed reference substance solutions respectively, injecting the solutions into a high performance liquid chromatograph, and recording chromatograms; the liquid chromatography conditions were: a chromatographic column: purospher STAR LP RP-18 endclamped, mobile phase: acetonitrile and 0.1% phosphoric acid water, gradient elution, diode array detector, detection wavelength: 245nm, column temperature 30 ℃, flow rate 1.0mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:

Procedure	time (min)	Acetonitrile volume (%)
			1	0	5
2	10	5
			3	18	13
4	118	23
			5	125	35
6	190	100

Step 4, exporting the fingerprints of the 8 batches of the light lotus leaf tea test sample solutions obtained in the step 3, and importing the fingerprints into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012A; selecting chromatographic peaks existing in chromatograms of 8 batches of light lotus leaf tea as common peaks; generating a control fingerprint of 1 batch of light lotus leaf tea by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; as a result, 13 common peaks were observed in 1 batch of the raw light lotus leaf tea, and the reference fingerprint is shown in FIG. 2, and the fingerprint of 8 batches of the test sample is shown in FIG. 3. The retention time of chlorogenic acid is 27.094min, and the retention time of chrysophanol is 162.082 min.

Step 5, comparing the fingerprint spectrum of the light lotus leaf tea obtained in the step 3 with the spectrum of the mixed standard substance (figure 1), identifying the main components, and comparing the number 1 and 13 chromatographic peaks in the light lotus leaf tea to obtain the following chromatographic peaks: chlorogenic acid (retention time 27.094min), and chrysophanol (retention time 162.082 min).

Meanwhile, the invention uses the automatically generated contrast HPLC fingerprint spectrum R to generate a common chromatographic peak mode, and the common chromatographic peaks of 8 batches of the lotus leaf light weight tea traditional Chinese medicines of Jiangsu Haoyangyao pharmaceutical manufacturers are analyzed and calculated to have relatively good similarity, which shows that the fingerprint spectrum established by the lotus leaf light weight tea traditional Chinese medicine established by the method can well detect the quality of a plurality of batches of lotus leaf light weight tea in Jiangsu Haoyangyao pharmaceutical industries, and the results are shown in Table 1.

TABLE 1 similarity between batches of samples and consensus patterns

Example 2 methodological studies of fingerprint detection:

1. methodology investigation

1.1 precision investigation

Taking 20g of a light lotus leaf tea sample (4 g of lotus leaf, 3.5g of cassia seed, 3.5g of hawthorn fruit, 2g of mulberry leaf, 2.5g of radish seed, 2g of fructus cannabis and 2.5g of mulberry), preparing a sample solution according to the sample preparation method of the example 1, continuously sampling for 6 times, wherein the sampling amount is 10 mu L each time, detecting according to the chromatographic condition of the example 1, measuring the HPLC chromatogram, and inspecting 13 common fingerprint peaks in the chromatogram, wherein the result shows that the retention time RSD of the common fingerprint peaks is less than 0.8%, the area RSD of the common fingerprint peaks is less than 0.9%, and the precision of instruments is better.

1.2 stability Studies

Taking 20g of a light lotus leaf tea sample (4 g of lotus leaf, 3.5g of cassia seed, 3.5g of hawthorn fruit, 2g of mulberry leaf, 2.5g of radish seed, 2g of fructus cannabis and 2.5g of mulberry), preparing a test solution according to the preparation method of the test sample in the example 1, injecting samples according to the chromatographic conditions in the example 1 for 0, 2, 4, 8, 12 and 24 hours respectively, recording a chromatogram, and inspecting 13 common fingerprint peaks in the chromatogram, wherein the result shows that the retention time RSD of the common fingerprint peaks is less than 1.1%, the area RSD of the common fingerprint peaks is less than 1.5%, and the test solution has better stability in 24 hours.

1.3 repeatability test

Taking 6 parts of light lotus leaf tea sample, each part being 20g, preparing a sample solution according to the sample preparation method of the example 1, respectively measuring, recording a chromatogram, and inspecting 13 common fingerprint peaks in the chromatogram, wherein the results show that the retention time RSD of the common fingerprint peaks is less than 2.1 percent, and the area RSD of the common fingerprint peaks is less than 1.9 percent, which indicates that the method has good repeatability.

The experimental results show that the fingerprint spectrum detection method for the light lotus leaf tea provided by the invention has the advantages of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the light lotus leaf tea, and has important significance for ensuring the clinical curative effect.

Example 3

20 people with obesity, irregular stool and abdominal discomfort and sub-health are selected, the age is 21-45 years old, 2 tea generations (8 g of lotus leaves, 7g of cassia seeds, 7g of hawthorn, 4g of mulberry leaves, 5g of radish seeds, 4g of fructus cannabis and 5g of mulberry fruits) are drunk every day, and after the tea is continuously taken for 7 days, the stool is smooth and excellent in morning hours, and the effective rate is 100%.

The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.