CN112255324B - Method for establishing fingerprint of wolfberry fruit and kudzuvine root solid beverage and fingerprint thereof - Google Patents

Abstract

The invention provides a method for establishing a wolfberry fruit-kudzu root solid beverage fingerprint and a fingerprint thereof, wherein the establishing method comprises the following steps: step one, preparation of a test solution: preparing a test solution from a wolfberry fruit and kudzu root solid beverage product; step two, preparing a reference substance solution: taking puerarin and oleanolic acid as reference substances, and respectively preparing puerarin reference substance solution and oleanolic acid reference substance solution; and step three, constructing a high performance liquid chromatography determination method. The wolfberry fruit-pueraria root solid beverage liquid phase fingerprint established by the method provided by the invention can effectively represent the quality of the wolfberry fruit-pueraria root solid beverage, and is beneficial to comprehensively monitoring the quality of the product.

Description

Method for establishing fingerprint of wolfberry fruit and kudzu vine root solid beverage and fingerprint thereof

Technical Field

The invention relates to the technical field of solid beverages, in particular to a method for establishing a wolfberry fruit-radix puerariae solid beverage fingerprint and a fingerprint thereof.

Background

The wolfberry fruit and kudzuvine root solid beverage is one of the products developed independently by me, and the main raw materials comprise: the medlar, the kudzuvine root, the dried ginger, the peach seed, the Chinese date, the longan pulp, the double-petal red rose, the peanut skin and the auxiliary materials have the effects of tonifying the kidney and strengthening the spleen, tonifying qi and nourishing the blood, promoting blood circulation and removing blood stasis, dispelling qi and soothing the nerves and the like. At present, the quality control of the product is only limited to the detection of physical and chemical indexes, microbial limits and other macroscopic characteristics, the quality control is single, the indexes are few, and the quality condition of the wolfberry fruit and kudzu root solid beverage is difficult to be comprehensively reflected. In order to more comprehensively and effectively control the quality control level of the product, ensure the safety and the curative effect of the product and meet the increasingly severe requirements of sellers on high-quality products, a more advanced quality control means needs to be adopted for the product.

The traditional Chinese medicine fingerprint spectrum technology is regarded as an effective means for the quality control research of traditional Chinese medicines, and as a quality control technology, the traditional Chinese medicine fingerprint spectrum technology uses the modern analysis technology to represent and describe chemical information of traditional Chinese medicines in a graphic mode, and is a spectrum or a chromatogram spectrum of certain components which are shared by certain traditional Chinese medicines and have characteristics. The traditional Chinese medicine fingerprint can be relatively comprehensively, quickly and accurately controlled, the quality of the traditional Chinese medicine and related products can be reflected, and the method is mainly used for evaluating the authenticity, the excellence and the stability of the quality of the traditional Chinese medicine and traditional Chinese medicine preparations. At present, no literature is published and reported on the research of the fingerprint of the wolfberry fruit and radix puerariae solid beverage, and no fingerprint of the wolfberry fruit and radix puerariae solid beverage is established, so that a method for controlling the quality of the wolfberry fruit and radix puerariae solid beverage is urgently needed in the field, the variety and the quantity of chemical components contained in the wolfberry fruit and radix puerariae solid beverage can be comprehensively reflected, and the quality of the wolfberry fruit and radix puerariae solid beverage can be effectively controlled.

Disclosure of Invention

In view of the above, the invention aims to provide a method for establishing a fingerprint of a wolfberry fruit-pueraria lobata solid beverage and a fingerprint thereof, which aims to comprehensively reflect the types and the amounts of chemical components contained in the wolfberry fruit-pueraria lobata solid beverage and effectively control the quality of the wolfberry fruit-pueraria lobata solid beverage.

Based on the purpose, the invention provides a method for establishing a wolfberry fruit and radix puerariae solid beverage fingerprint, which comprises the following steps:

step one, preparation of a test solution: preparing a test solution from a wolfberry fruit and kudzu root solid beverage product;

step two, preparing a reference substance solution: taking puerarin and oleanolic acid as reference substances, and respectively preparing puerarin reference substance solution and oleanolic acid reference substance solution;

step three, constructing a high performance liquid chromatography determination method:

a. the method comprises the steps of performing 190-400nm full-wavelength scanning on a test solution, and determining the required scanning wavelength through chromatogram contrast analysis;

b. respectively taking methanol-water, acetonitrile-water and methanol-0.1% phosphoric acid as elution mobile phases, carrying out sample injection analysis under the same liquid phase condition, and determining that the methanol-0.1% phosphoric acid is taken as the elution mobile phase according to the result;

c. determining a proper chromatographic column by analyzing obtained spectrum information of chromatographic columns of different brands and the separation effect of a test solution;

d. determining elution conditions of the fingerprint spectrum by selecting different sample volumes of the mobile phase at different time periods;

e. introducing the test solution into a high performance liquid chromatograph, introducing the obtained high performance liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for analysis, and obtaining the fingerprint of the wolfberry fruit and kudzu root solid beverage.

Preferably, the wolfberry fruit and kudzuvine root solid beverage comprises the following raw materials in parts by weight:

the preparation method of the test solution comprises the following steps: dissolving the solid beverage of the medlar and the kudzuvine root with purified water according to the material-liquid ratio of 1 (5-15) g/mL, and then filtering the solution by a water-washing filter membrane to obtain the medlar and kudzuvine root solid beverage.

The method for determining the required scanning wavelength in the step a comprises the following steps: scanning the test solution with DAD detector at 190-400nm full wavelength, analyzing the absorption chart, extracting chromatogram under five wavelengths with multiple absorption peaks, and comparing to give a comprehensive response to chemical components in the solid beverage, wherein the detection wavelength of 254nm has good peak separation degree and stable baseline.

D, gradient selection is carried out according to the following four experiments, and then the gradient of the experiment four is selected as the elution condition of the fingerprint spectrum according to the experiment result, wherein methanol is A, and 0.1% phosphoric acid is B;

experiment one: 0-10min:5-10% by weight A, 95-90% by weight B;10-15min:10-32% by weight A, 90-68% by weight B;15-30min:32-40% by weight, A, 68-60% by weight, B;30-55min:40-65% by weight, A, 60-35% by weight, B;

experiment two: 0-10min:10-15% by weight A, 95-90% by weight B;10-15min:10-30% by weight A, 90-60% by weight B;15-30min:30-35% by weight A, 70-65% by weight B;30-45min:35-60% A, 65-40% B;

experiment three: 0-10min:5-10% by weight A, 95-90% by weight B;10-45min:10-70% by weight A, 90-30% by weight B;

experiment four: 0-10min:5-10% by weight A, 95-90% by weight B;10-15min:10-25% by weight A, 90-75% by weight B;15-45min:25-65% by weight A, 75-35% by weight B.

The chromatographic column determined in the step c is Inertsil ODS-3.

The fingerprint is analyzed by taking methanol-phosphoric acid as a mobile phase and injecting samples according to the elution gradient under the wavelength of 254nm, wherein the concentration of the phosphoric acid is 0.1 percent, the flow rate is 1.0mL/min, the chromatographic column is Inertsil ODS-3 (4.6 mm multiplied by 250mm,5 mu m), the column temperature is 35 ℃, and the sample injection amount is 10uL.

The fingerprint obtained by the method for establishing the fingerprint of the wolfberry fruit-kudzu root solid beverage has 13 common peaks, wherein the 5 peak is puerarin and the 10 peak is oleanolic acid.

And calculating the relative retention time and the relative peak area of each common peak and the reference peak by taking the No. 10 peak as the reference peak, and obtaining that the RSD of the relative retention time is less than or equal to 1.5 percent and the RSD of the relative peak area is less than or equal to 5.0 percent.

Preferably, the wolfberry fruit and kudzu vine root solid beverage comprises the following raw materials in parts by weight:

15-16 parts of wolfberry fruit, 13-14 parts of kudzu root, 4.5-5.5 parts of peach kernel, 4.5-5.5 parts of Chinese date, 2-3 parts of longan aril, 2-3 parts of double-petal red rose, 1-2 parts of peanut coat, 2.5-3.5 parts of dried ginger HE powder and 45-55 parts of maltodextrin.

The invention also provides a preparation method of the wolfberry fruit and kudzu root solid beverage, which comprises the steps of crushing and sieving kudzu root, rose and peanut coat according to the formula amount, adding water into wolfberry fruit, peach kernel, chinese date and longan pulp, crushing and sieving, and then carrying out primary extraction, centrifugation, secondary extraction, secondary centrifugation, concentration, filtration, drying and granulation on the crushed and sieved raw materials.

Preferably, the preparation method comprises the following steps:

(1) Treating raw materials: weighing the kudzu root, the double red rose and the peanut coat according to the formula ratio, crushing in a crusher, and sieving; weighing medlar, peach kernel, chinese date and longan pulp, adding 6-8 times of purified water, crushing and sieving, wherein the sieved aperture is 0.9-1.5mm;

(2) Primary extraction: adopting high-efficiency high-pressure-difference low-temperature continuous extraction equipment, wherein the mass ratio of the medicine material liquid is 1:17-20, adding water for extraction at 20-30 ℃ to obtain primary extract;

(3) Primary centrifugation: centrifuging the primary extractive solution at 3500-5000r/m to obtain centrifugate and centrifugate residue;

(4) Secondary extraction and secondary centrifugation: adding 13-15 times of purified water into the centrifugal slag obtained in the step (3), extracting by adopting high-efficiency high-pressure-difference low-temperature continuous extraction equipment, and centrifuging, wherein the extraction pressure is 20-60Mpa;

(5) Concentration and filtration: performing microfiltration on the centrifugate obtained in the steps (3) and (4), wherein the aperture of the microfiltration is 300-600nm, and then sequentially performing membrane concentration/vacuum concentration and plate type filtration to obtain a concentrated solution; the concentration temperature is less than or equal to 65 ℃;

(6) And (3) drying: the concentrated solution enters a universal clean area (more than or equal to 10 ten thousand grade), and auxiliary materials are added for drying to obtain dry powder;

(7) Granulating: and adding an adhesive into the dry powder and the weighed HE dry powder of the dried ginger for granulation, wherein the granulation process is wet granulation/dry granulation.

Preferably, the auxiliary material is maltodextrin/fructo-oligosaccharide, and the addition amount of the auxiliary material is 30-50%.

The main components and effects of the raw materials of the invention are as follows:

fructus Lycii, dried mature fruit of Lycium barbarum (Lycium barbarum) of Solanaceae, and medicinal and edible Chinese medicinal materials with mild nature and taste, which enter liver and kidney channels, have effects of nourishing liver and kidney, replenishing vital essence and improving eyesight, and can be used for treating consumptive disease and essence deficiency, blood deficiency and chlorosis, soreness of waist and knees, etc. Contains nutrients such as fat, protein, vitamins (B1, B2, E, and C) and minerals (potassium, sodium, calcium, magnesium, ferrum, copper, manganese, zinc, and selenium), and effective components such as lycium barbarum polysaccharide, taurine, and betaine. Modern medicine proves that the medlar has the functions of promoting hematopoiesis, protecting liver, regulating immunity, improving eyesight, beautifying, strengthening brain, expelling toxin and the like.

Kudzu root is a dry root of Pueraria lobata (Willd.) Ohwi) which is a leguminous plant, is a medicine-food homologous traditional Chinese medicine, has sweet, pungent and cool nature and taste, and has the effects of invigorating spleen and stomach channels, relieving muscles and reducing fever, promoting the production of body fluid to quench thirst, invigorating yang and relieving diarrhea, puerarin, daidzein and the like are main effective components in kudzu root, wherein the puerarin is a specific component of the kudzu root and has the effects of nourishing ovary and preventing and treating cardiovascular and cerebrovascular diseases. In addition, researches show that the kudzuvine root also has the effects of regulating female endocrine, expanding blood vessels, dispelling the effects of alcohol, protecting liver and the like.

Dried ginger, dried rhizome of Zingiber officinale Rosc of Zingiberaceae, also known as white ginger and Yujiang, is a medicine and food homologous Chinese medicine, has pungent and hot taste, enters spleen, stomach, kidney, heart and lung channels, and has the effects of warming spleen and stomach for dispelling cold, restoring yang and dredging collaterals, etc. It is indicated for wind-cold type common cold, cold pain in the heart and abdomen, and dizziness and vomiting due to yang deficiency. Modern researches find that the dried ginger contains various chemical components such as volatile oil, diphenyl heptane, gingerol and the like, and has various pharmacological effects such as antioxidation, antipyretic and anti-inflammatory, pathogen resistance, liver protection and cholagogue, tumor resistance, ulcer resistance, local blood circulation improvement, digestive system function and the like; has obvious curative effect on dizziness, vomiting of pregnancy, weakness of spleen and stomach and other aspects.

Peach kernel, which is dry mature seed of Prunus persica (L.) Batsch) or Prunus davidiana (P.davidiana (Carr.) Franch.) belonging to Rosaceae family, and can be used as medicine and food homologous Chinese medicine. It has mild nature and bitter and sweet taste, and has the effects of invigorating blood circulation, removing blood stasis, loosening bowel to relieve constipation, relieving cough and asthma, and can be used for treating amenorrhea, dysmenorrhea, traumatic injury, constipation due to intestinal dryness, etc. Semen Persicae contains glycosides effective components such as amygdalin and prunasin, and has high protein content. Pharmacological research shows that peach kernel has good effects of promoting blood circulation to remove blood stasis, improving cardiovascular and cerebrovascular diseases, protecting liver and kidney, enhancing immunity and the like.

Fructus Jujubae, the mature fruit of Zizyphi fructus (Zizp Husjujuba Miil) of Rhamnaceae, is sweet in taste and neutral in nature, is a common medicine and food homologous Chinese medicine, and has effects of invigorating qi and spleen, nourishing heart and tranquilizing mind. Contains nutrients such as protein, fat, saccharide, vitamins, minerals, etc., especially saccharide and vitamin C, and is rich in various bioactive components such as polysaccharide, polyphenol, organic acid, saponin, etc. Many researches show that the red dates have multiple effects of tonifying qi and nourishing blood, resisting aging, resisting fatigue, protecting livers, enhancing immunity and the like.

Arillus longan is aril of arillus longan (Dimocarpus longan Long, lour.) of Sapindaceae, is a medicinal and edible Chinese medicine, has sweet taste and mild nature, and enters heart and spleen meridians. It is not hot, peaceful and expensive, and it can help heart and develop intelligence. The arillus longan is nutritious, sweet, and rich in sugar, protein, vitamins (B1, B2, C) and minerals (phosphorus, calcium, and iron). Modern medicine considers that longan pulp has the functions of tonifying heart and spleen, nourishing blood and tranquillizing mind, resisting aging and cancer.

The red rose with double petals enters liver and spleen channels, can promote qi circulation to relieve depression, and disperse blood stasis to relieve pain, and can be used for improving chest distress, depression, gastrointestinal discomfort, breast distending pain, abnormal leucorrhea, etc. caused by stagnation of liver qi. And has effects of astringing and removing blood stasis, and can be used for caring skin, preventing wrinkle, healing wound, and treating carbuncle, sore and pain. Modern researches find that roses contain abundant vitamins and tannin, and can improve endocrine dyscrasia.

Peanut coat (Arachi hypogaea Linn.), also known as peanut coat, is a traditional Chinese medicine, has sweet, astringent and mild nature and taste, enters lung, spleen and liver meridians, and has the functions of strengthening spleen and stomach, nourishing blood and stopping bleeding, and dissipating blood stasis and relieving swelling. The peanut coat contains various flavonoid components including functional components such as quercetin, proanthocyanidin oligomer and the like, is rich in polyphenol substances such as resveratrol, catechin, proanthocyanidin and the like, has the effect of resisting rancidity and oxidation of peanut kernels, is known as 'seventh nutrient', and has remarkable effects of protecting blood vessels, resisting oxidation, preventing thrombocytopenia and promoting coagulation.

The invention has the beneficial effects that:

1. the wolfberry fruit-pueraria root solid beverage liquid phase fingerprint established by the method provided by the invention can effectively represent the quality of the wolfberry fruit-pueraria root solid beverage, and is beneficial to comprehensively monitoring the quality of the product.

2. The invention carries out extraction, concentration, drying and granulation by high-efficiency high-pressure-difference low-temperature continuous extraction equipment (ZL200920304058. X), the high-pressure-difference continuous extraction can ensure higher extraction rate (more than or equal to 90%), the low-temperature extraction can ensure complete structure of effective components, the combined process can shorten the preparation time of the product, the process is stable, and stable and reliable raw materials can be provided for development of the wolfberry fruit and radix puerariae solid beverage product.

3. By optimizing chromatographic conditions such as a mobile phase system, wavelength, chromatographic column, mobile phase elution gradient and the like, methodology investigation, common mode establishment and similarity evaluation of multiple batches of products are carried out; the established fingerprint spectrums are used for analyzing a plurality of batches of Chinese wolfberry fruit and kudzu vine root solid beverage product spectrograms, and the similarity of the Chinese medicine chromatographic fingerprint spectrum similarity evaluation software (2012 edition) is used for analyzing the similarity, so that the product quality is objectively, comprehensively and scientifically evaluated and controlled.

4. The membrane filtration sterilization is used in the extraction and concentration process, and the terminal process is implemented in a universal clean area, so that the microbial qualification of the product can be fully ensured, the product preparation process is further simplified, and other sterilization processes do not need to be developed.

5. The medlar and kudzu root solid beverage has pure taste: has strong peculiar smell and taste of the medlar and the red dates, is instant and clear when meeting water, and is not added with food additives such as preservatives, flavoring agents and the like. The traditional Chinese medicine composition has a unique formula, mellow taste and pure nature without additives, has the effect of assisting in treating dysmenorrhea, and can further enhance certain health-care effects of products through compound compatibility.

Drawings

In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only one or more embodiments of the present disclosure, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a chromatogram of the solid beverage of wolfberry fruit and kudzu vine root in different wavelengths;

FIG. 2 is a chromatogram of the wolfberry fruit-kudzu root solid beverage of the present invention using different elution mobile phases;

FIG. 3 is a chromatogram of the solid beverage of wolfberry fruit and kudzu vine root in different chromatographic columns;

FIG. 4 is a chromatogram of the solid beverage of wolfberry fruit and kudzu vine root obtained by different elution procedures;

FIG. 5 shows a fingerprint of the precision test of the present invention;

FIG. 6 is a fingerprint of a repeatability test of the present invention;

FIG. 7 is a fingerprint of the stability test of the present invention;

FIG. 8 is the high performance liquid chromatogram (common peak) and characteristic peak calibration of the solid beverage of wolfberry fruit and kudzu vine root;

FIG. 9 shows the fingerprint of 9 solid wolfberry fruit-kudzu vine root beverages of the present invention.

Detailed Description

To make the objects, technical solutions and advantages of the present disclosure more apparent, the present disclosure is further described in detail below with reference to specific embodiments.

It should be noted that technical terms or scientific terms used in the embodiments of the present specification should have a general meaning as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined.

The experimental materials selected by the invention are as follows;

equipment: agilenggt 1100 series high performance liquid chromatograph (including DAD array detector, agilent, USA), inertsil ODS-3 chromatographic column (4.6 mm. Times.250mm, 5 μm), DV215CD type electronic balance (Aohaus, USA), KQ3200B type ultrasonic cleaner (Kunshan ultrasonic instruments, inc.)

Reagent: acetonitrile, methanol (chromatography, fisher corporation, USA), phosphoric acid (analytical purity, chengdu Kelong chemical reagent factory), reference puerarin (Beijing Yihuatong technology Limited, batch No. 21H20010, gallic acid (Beijing Yihuatong technology Limited, batch No. 16042708), and super pure water

Example 1

A preparation method of a wolfberry fruit and kudzuvine root solid beverage product comprises the following steps:

(1) Weighing the following raw materials in proportion: 15.3 parts of barbary wolfberry fruit, 13.77 parts of kudzuvine root, 5.1 parts of peach kernel, 5.1 parts of Chinese date, 2.55 parts of longan aril, 2.55 parts of double-petal red rose, 1.53 parts of peanut coat, 3.17 parts of dried ginger HE powder and 50.915 parts of maltodextrin.

(2) Pulverizing radix Puerariae, flos Rosae Rugosae and peanut coat in the raw materials according to the above formula ratio in a universal pulverizer, and sieving by 1.2mm; adding 6-8 times of purified water into the medlar, the peach kernel, the Chinese date and the longan pulp, crushing and sieving by 1.2mm;

(3) Primary extraction: adopting a high-efficiency high-pressure-difference low-temperature continuous extraction device (HHLSE) which is a proprietary device of China, supplementing an extraction medium by 17 times for low-temperature instantaneous extraction (20-30 ℃), wherein the extraction pressure is not lower than 20Mpa, and the extraction medium is water;

(4) Centrifuging: (3) Centrifuging the extractive solution at 3500-5000r/m to obtain fructus Lycii flos Rosae Rugosae centrifugate and fructus Lycii flos Rosae Rugosae centrifugate residue;

(5) Secondary extraction and centrifugation: (4) Adding 14 times of purified water into the medium centrifugal slag, performing high-efficiency high-pressure-difference low-temperature continuous extraction under a certain pressure (20-60 Mpa), and centrifuging;

(6) Concentration and plate-frame filtration: carrying out a series of procedures of microfiltration (300-600 nm), membrane concentration/vacuum concentration (less than or equal to 65 ℃) and plate-frame filtration (sterilization) on the centrifugate obtained in the steps (5) and (6) to obtain concentrated liquor;

(7) And (3) drying: (6) Allowing the concentrated solution to enter a universal clean area, adding appropriate amount of adjuvants, and spray drying/vacuum drying to obtain dry powder, wherein the adjuvants are maltodextrin/fructo-oligosaccharide powder;

(8) Granulating: (7) Adding a proper amount of adhesive into the dry powder obtained in the step (1) and the dried powder of the dried ginger HE weighed in the step (1) for granulation, wherein the granulation process is wet granulation/dry granulation.

2. Optimizing the dissolving times of the test solution:

the product is dissolved according to the material-liquid ratio of 1.

3. The method for establishing the liquid phase fingerprint spectrum of the medlar and kudzuvine root solid beverage comprises the following steps:

preparing a test solution: precisely weighing 1.0g of sample, placing the sample in a beaker, adding purified water, carrying out ultrasonic dissolution at room temperature, transferring the sample to a 10mL volumetric flask, carrying out constant volume to a scale mark, shaking up, and filtering the sample in a 0.45-micron water system filter membrane to a sample injection bottle to obtain the product.

Preparation of a control solution: accurately weighing 0.7147g of puerarin and 13mg of oleanolic acid in a 10mL volumetric flask, adding methanol to a constant volume to obtain puerarin reference solution with the concentration of 0.7004mg/mL and oleanolic acid reference solution with the concentration of 1.278 mg/mL.

The specific chromatographic condition optimization process is as follows:

(1) Optimization of wavelength: 190-400nm full-wavelength scanning is carried out on the product by using a DAD detector, an equivalent absorption chart is analyzed, chromatograms with more absorption peaks under 5 wavelengths (210 nm, 254nm, 260nm, 280nm and 300 nm) are extracted for comparison, chemical components in the wolfberry fruit and radix puerariae solid beverage can be comprehensively reflected by the 254nm detection wavelength, the separation degree of each chromatographic peak is good, a base line is stable, and therefore the 254nm wavelength is selected for scanning in later experiments.

(2) Selection of elution system: methanol-water, acetonitrile-water and methanol-0.1% phosphoric acid are taken as a mobile phase system, and the sample injection analysis is carried out under the same liquid phase condition, and the result shows that the chromatogram obtained by the methanol-0.1% phosphoric acid has rich information content, so the later experiment of the invention takes the methanol-0.1% phosphoric acid as the mobile phase.

(3) Optimization of the chromatographic column: different brands of columns were examined, diamonsi C18 (4.6 mm. Times.250mm, 5 μm), platisil C18 (4.6 mm. Times.250mm, 5 μm) and Inertsil ODS-3 (4.6 mm. Times.250mm, 5 μm), respectively. The results show that the three obtained spectral peaks are similar in shape and rich in information, but Inertsil ODS-3 (4.6 mm multiplied by 250mm,5 mu m) has a good separation effect on samples, so that the chromatographic column is used for sample injection analysis in later experiments of the invention.

(4) Optimization of elution gradient: gradient optimization was performed according to the following 4 experiments

(1) Methanol (a) -0.1% phosphoric acid (B): 0-10min:5-10% of A;10-15min:10-32% of A;30-50min:32-40% of A;40-60min:40-65% by weight A;

(2) methanol (a) -0.1% phosphoric acid (B): 0-10min:10-15% of A;10-15min:10-30% of A;15-30min:30-35% by weight of A;30-45min:35-60% of A;

(3) methanol (a) -0.1% phosphoric acid (B): 0-10min:5-10% of A;10-45min:10-70% by weight of A;

(4) methanol (a) -0.1% phosphoric acid (B): 0-10min:5-10% by weight of A;10-15min:10-25% by weight of A;15-45min:25 to 65 percent.

According to the test result, the gradient of the elution procedure (4) is selected as the fingerprint elution condition, and under the condition, the separation degree of each chromatographic peak is good, the content is high, and the used time is short, so that the gradient condition is selected for sample analysis in the later stage experiment of the invention.

(5) Methodology survey validation

And (3) precision test: taking the wolfberry fruit and kudzuvine root solid beverage of the batch number 20200401, preparing a test solution according to the preparation condition of the test solution, repeatedly injecting the sample for 6 times according to the optimized liquid phase condition, recording each chromatogram, and investigating the relative retention time and the relative peak area of the common peak. The result shows that the RSD of the relative retention time is less than or equal to 1.0 percent (shown in table 1), the RSD of the relative peak area is less than or equal to 2.0 percent (shown in table 2), and the similarity of each spectrogram is over 0.99, which indicates that the precision of the instrument is good and meets the technical requirements of the fingerprint, and the figure 5 shows that the instrument has good precision.

TABLE 1 evaluation results of similarity in precision test (common Peak relative Retention time)

TABLE 2 evaluation results of similarity in precision test (common peak relative peak area)

And (3) repeatability test: taking the wolfberry fruit and kudzu root solid beverage of 20200401 batch, respectively preparing 6 parts of test solution according to the preparation conditions of the test solution, sequentially injecting samples according to the optimized liquid phase conditions, recording each chromatogram, and inspecting the relative retention time and the relative peak area of the common peak. The results show that the RSD of the relative retention time is less than or equal to 1.0 percent (shown in Table 3), the RSD of the relative peak area is less than or equal to 2.5 percent (shown in Table 4), and the similarity of each spectrogram is more than 0.98, which indicates that the method has good repeatability and meets the technical requirements of the fingerprint, and the figure 6 shows that the method has good repeatability.

TABLE 3 evaluation results of the degree of similarity in the reproducibility test (relative retention time of common peaks)

TABLE 4 evaluation results of the degree of similarity in the reproducibility test (common Peak relative Peak area)

And (3) stability test: taking the wolfberry fruit and kudzu root solid beverage of the batch number 20200401, preparing a test solution according to the preparation conditions of the test solution, injecting samples at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours respectively according to the optimized liquid phase conditions, recording a fingerprint chromatogram, measuring 6 time points in total, and inspecting the relative retention time and the relative peak area of a common peak. As a result, the RSD of the relative retention time was not more than 1.0% (shown in Table 5), the RSD of the relative peak area was not more than 2.0% (shown in Table 6), and the similarity of each chromatogram was 0.99 or more, indicating that the stability of the test solution was good in 24-hour test at room temperature, as shown in FIG. 7.

TABLE 5 evaluation results of similarity in stability test (common Peak relative Retention time)

TABLE 6 evaluation results of similarity in stability test (common Peak relative Peak area)

(7) Establishment of fingerprint spectrum of wolfberry fruit and radix puerariae solid beverage product and identification of common peak

Taking the wolfberry fruit and radix puerariae solid beverages with the batch numbers of 20200401, 20200402, 20200403, 20200404, 20200405, 20200406, 20200407, 20200408 and 20200409 respectively, preparing 9 parts of test solution according to the preparation conditions of the test solution, respectively injecting the test solution into a high performance liquid chromatograph to obtain finger prints, analyzing the print data of 9 batches of samples by using software of a traditional Chinese medicine chromatogram finger print similarity evaluation system (version 2.0), selecting S1 as a reference print, adopting a median and a time window of 0.1S, matching the prints of 9 batches of samples, and calibrating 13 common peaks in total, as shown in figures 8 and 9. Precisely absorbing the reference substance for determination, comparing the retention time of the reference substance, and identifying the 5 th peak as puerarin and the 10 th peak as oleanolic acid in 13 common peaks of the fingerprint, as shown in FIG. 8.

(8) Similarity evaluation of multi-batch wolfberry fruit and radix puerariae solid beverage products

By carrying out fingerprint analysis on 9 batches of wolfberry fruit and radix puerariae solid beverages, the 10 th peak has better peak type and separation degree, so that the peak is selected as a reference peak, and the relative retention time and the relative peak area of each common peak and the reference peak are calculated. The results show that the RSD of the relative retention time is less than or equal to 1.5 percent, the RSD of the relative peak area is less than or equal to 5.0 percent, and the similarity of each spectrogram is more than 0.98, which indicates that the product has stable process and good product quality control in the production process, and also indicates that a rapid and accurate quality standard can be provided for the quality control of the product by using a fingerprint technology, and the results are shown in tables 7 and 8.

TABLE 7.9 batch products fingerprint chromatogram common peak relative retention time

TABLE 8.9 batch of products having peak area relative to the fingerprint chromatogram common peak

In the present embodiment, the liquid chromatography conditions are as follows: agilent 1100 liquid phase instrument, inertsil ODS-3 (4.6 mm. Times.250mm, 5 μm) chromatographic column, sample introduction amount 10uL, flow rate 1.0mL/min, column temperature 35 ℃, mobile phase: methanol-0.1% phosphoric acid, elution gradient as follows:

and (3) sample determination: respectively and precisely sucking 10uL of the test solution and the reference solution, and injecting into a high performance liquid chromatograph for determination to obtain the fingerprint.

The test results show that the chromatogram similarity of 9 batches of wolfberry and radix puerariae solid beverage products (20200401-20200409) produced in 4 months in 2020 is over 0.98, the RSD of the relative retention time is less than or equal to 1.5%, and the RSD of the relative peak area is less than or equal to 5.0%, so that the method meets the internal control standard of enterprises and can provide reference for quality evaluation and quality control of wolfberry and radix puerariae solid beverage products.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the present disclosure, also technical features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present description as described above, which are not provided in detail for the sake of brevity.

The embodiments of the present description are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalents, improvements, and the like that may be made without departing from the spirit or scope of the embodiments of the present disclosure are intended to be included within the scope of the disclosure.

Claims (3)

1. The method for establishing the fingerprint spectrum of the wolfberry fruit and kudzu root solid beverage is characterized by comprising the following steps of:

step one, preparation of a test solution: dissolving the solid beverage of the medlar and the kudzuvine root with purified water according to the material-liquid ratio of 1 (5-15) g/mL, and then filtering the solution by a water washing filter membrane to obtain the medlar and kudzuvine root solid beverage;

step two, preparing a reference substance solution: taking puerarin and oleanolic acid as reference substances, and respectively preparing puerarin reference substance solution and oleanolic acid reference substance solution;

step three, the high performance liquid chromatography determination method comprises the following steps:

a. scanning the test solution with 190-400nm full wavelength by using a DAD detector, analyzing the equivalent absorption chart, extracting chromatograms with more absorption peaks under five wavelengths, and comparing to comprehensively react chemical components in the solid beverage of the medlar and the radix puerariae, wherein the 254nm detection wavelength with good separation degree of each chromatographic peak and stable base line is used as the scanning wavelength;

b. the elution conditions of the fingerprint are as follows: 0-10min:5-10% A, 95-90% B;10-15min:10-25% A, 90-75% B;15-45min:25-65% by weight of A, 75-35% by weight of B, wherein methanol is A and 0.1% phosphoric acid is B;

c. the chromatographic column is Inertsil ODS-3;

d. introducing the test solution into a high performance liquid chromatograph, introducing the obtained high performance liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for analysis, and obtaining the fingerprint of the wolfberry fruit and kudzu root solid beverage.

2. The method for establishing the fingerprint of the solid beverage with wolfberry and kudzuvine root according to claim 1, wherein the total peaks in the fingerprint are 13, wherein the 5 peak is puerarin and the 10 peak is oleanolic acid.

3. The method for establishing fingerprint of solid beverage containing wolfberry fruit and pueraria lobata as claimed in claim 2, wherein the relative retention time and relative peak area of each common peak and the reference peak are calculated using peak 10 as the reference peak, and RSD of relative retention time is less than or equal to 1.5% and RSD of relative peak area is less than or equal to 5.0%.

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