CN117607278A - Construction method and application of HPLC fingerprint of ginseng prebiotic particles - Google Patents
Construction method and application of HPLC fingerprint of ginseng prebiotic particles Download PDFInfo
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- 235000003140 Panax quinquefolius Nutrition 0.000 title claims abstract description 108
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- 235000013406 prebiotics Nutrition 0.000 title claims abstract description 87
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- 238000010276 construction Methods 0.000 title claims description 5
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a fingerprint detection method of ginseng prebiotic particles. The quality of the ginseng prebiotic particles can be effectively represented by the method, and the quality stability, consistency and controllability of products can be comprehensively monitored. The fingerprint spectrum of the ginseng prebiotic particles constructed by the invention has 21 peaks in total, contains the effective components belonging to six medicinal materials such as white paeony root, largehead atractylodes rhizome, liquorice, astragalus root, suberect spatholobus stem, ginseng and the like, efficiently characterizes the quality of the ginseng prebiotic particles, and is beneficial to comprehensively monitoring the quality of products. The invention has the characteristics of convenience, rapidness, stability, high precision and good repeatability, and can evaluate the quality of the ginseng prebiotic particles more comprehensively, objectively and scientifically, thereby ensuring the safety and effectiveness of clinical medication.
Description
Technical Field
The invention belongs to the field of medicines, and in particular relates to a method for controlling the quality of ginseng prebiotic particles by establishing a high performance liquid chromatography of ginseng prebiotic particles.
Background
In recent years, the overall incidence of malignant tumors continues to rise, and has become a major disease threatening human health. It has been reported that over 90% of chemotherapeutic agents may cause bone marrow suppression, mainly manifested by a differential drop in peripheral blood leukocytes, platelets, erythrocytes and hemoglobin, which can cause infection, bleeding and anemia, resulting in interruption of chemotherapy, reduced quality of life for the patient, and even endangered patient life. At present, the main medicines for treating bone marrow suppression by Western medicine are colony stimulating factors, recombinant human thrombopoietin, glucocorticoid and the like, but the adverse reactions are more. Although the colony stimulating factor can rapidly increase neutrophils, repeated administration is required because the proliferation of hematopoietic stem cells cannot be promoted, and the risk of aggravating tumor immunosuppression microenvironment exists. In the traditional Chinese medicine, the traditional Chinese medicine single medicine and the classical prescription take qi and blood supplementing as the treatment principle of bone marrow suppression, and clinical researches and reports in recent years show that the combination of the traditional Chinese medicine and the radiotherapy and chemotherapy medicine can play the roles of attenuation and synergy, and can lead patients to complete radiotherapy and chemotherapy in sufficient quantity on time, thereby improving prognosis.
The ginseng qi-tonifying granule has the effects of tonifying qi and enriching blood, and strengthening body resistance and banking up root. Can be used for treating bone marrow depression and immune hypofunction caused by postoperative or malignant tumor chemotherapy. Is prepared from ginseng, astragalus root, white peony root, white atractylodes rhizome, spatholobus stem and honey-fried licorice root. The traditional Chinese medicine has the advantages that the ginseng and the astragalus are compatible, the primordial energy is benefited, the yin is nourished, the middle-jiao is nourished, the qi is benefited, the yang is raised, the exterior is strengthened, and the ginseng and the astragalus are all monarch drugs; the bighead atractylodes rhizome can strengthen the spleen, assist ginseng and astragalus root to tonify qi and strengthen the spleen; white peony root nourishes blood, softens liver and harmonizes nutrient, and is a ministerial drug; caulis Spatholobi has effects of promoting blood circulation and replenishing blood, and can be used together with other medicines to supplement without stagnation, and is adjuvant drug; the honey-fried licorice root is used for regulating the functions of qi and middle energizer, and harmonizing various medicines. The medicines are combined to achieve the effects of tonifying qi and enriching blood, and strengthening body resistance and banking up root.
Before the invention is completed, a method for establishing a fingerprint by taking chemical components contained in ginseng prebiotic particles as indexes is not reported.
Disclosure of Invention
The invention aims to provide a fingerprint detection method of ginseng prebiotic particles and a fingerprint thereof, wherein the fingerprint constructed by the method can comprehensively reflect the whole chemical components of the ginseng prebiotic particles, provides an effective means for the whole quality control and evaluation of the ginseng prebiotic particles, aims to solve the defect that no fingerprint literature report about the ginseng prebiotic particles exists in the prior art, and effectively controls the quality of the ginseng prebiotic particles.
The second purpose of the invention is to comprehensively reflect the current situation of each component in the ginseng prebiotic particles through component identification of the system and the attribution of single medicinal materials with peaks shared by different batches of medicines, and can provide reference basis for the quality of the ginseng prebiotic particles.
The invention adopts the following technical scheme: a fingerprint detection method of Ginseng radix granule comprises measuring its fingerprint by high performance liquid chromatography. Specifically, the fingerprint detection method of the ginseng prebiotic particles comprises the following steps:
extracting ginseng prebiotic particles by adopting 80% methanol, wherein the extracting solution is used as a sample solution; dissolving protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, glycyrrhizic acid and atractylenolide III with methanol to obtain reference solution; and respectively performing high performance liquid chromatography analysis on the reference substance solution and the sample solution, and taking a chromatographic peak of the reference substance solution as a benchmark to obtain the ginseng prebiotic particle fingerprint.
Optimally, the weight of the ginseng prebiotic particles is 0.2-2 g, and the dosage of 80% methanol is as follows: 20-200 mL.
Optimally, the extraction is carried out under the ultrasonic condition, the power of the ultrasonic wave is 200-300W, and the frequency is 30-50 kHz; the extraction time is 20-40 min.
Preferably, when the high performance liquid chromatography analysis is performed, the mobile phase A is acetonitrile, and the mobile phase B is 0.02-0.2% of phosphoric acid aqueous solution; the flow rate of the mobile phase system is 0.7-1.2 mL/min; in the gradient elution mode, the proportion of the mobile phase A, B is changed as follows: 0-10min,3% A;10-15min,3-10% A;15-35min,10-15% A;35-65min,15-30% A;65-90min,30-40% A;90-105min,40-80% A.
Preferably, when the high performance liquid chromatography is performed, octadecylsilane chemically bonded silica is used as a filler, and the specification of the chromatographic column is as follows: 250X 4.60mm; the column temperature is 20-40 ℃; the detection wavelength is 210-360 nm; the sample injection volume is 5-15 mu L.
Preferably, when the high performance liquid chromatography analysis is performed, the theoretical plate number is not less than 5000 as calculated as paeoniflorin peak.
Preferably, the ginseng prebiotic particle fingerprint contains 21 common peaks, wherein the chromatographic peaks of numbers 4, 5, 8, 10, 12, 18, 19 and 21 are respectively identified as protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, glycyrrhizic acid and atractylenolide III.
Preferably, the 21 common peaks respectively belong to six medicinal materials including white paeony root, largehead atractylodes rhizome, liquoric root, membranous milkvetch root, suberect spatholobus stem, ginseng and the like; wherein, the No. 1 peak belongs to ginseng, astragalus root, the No. 2 peak belongs to white peony root, white atractylodes rhizome, the No. 3 peak belongs to white peony root, suberect spatholobus stem, the No. 4 peak belongs to suberect spatholobus stem, the No. 6 peak belongs to liquorice, the No. 7 peak belongs to white peony root, the No. 8 peak belongs to white peony root, the No. 9 peak belongs to liquorice, astragalus root, the No. 10 peak belongs to liquorice, the No. 11 peak belongs to white peony root, the No. 12 peak belongs to astragalus root, the No. 13 peak belongs to ginseng, the No. 14 peak belongs to white peony root, the No. 15 peak belongs to liquorice, the No. 15 peak belongs to suberect spatholobus stem, the No. 17 peak belongs to white peony root, the No. 18 peak belongs to suberect spatholobus stem, the No. 19 peak belongs to liquorice, the No. 20 peak belongs to liquorice and the No. 21 peak belongs to white atractylodes rhizome.
The invention provides the ginseng prebiotic particle fingerprint spectrum obtained by the method for constructing the ginseng prebiotic particle HPLC fingerprint spectrum.
The invention provides an application of the ginseng prebiotic particle fingerprint spectrum in quality control of ginseng prebiotic particles.
The beneficial effects of the invention are as follows:
1. according to the invention, the HPLC fingerprint of 10 batches of ginseng prebiotic particles is established, the similarity is greater than 0.90, 21 common peaks are marked, 7 traditional Chinese medicines are covered, the chemical information contained in the ginseng prebiotic particles can be more comprehensively reflected, and the quality of the ginseng prebiotic particles is better represented.
2. Because the fingerprint is not used for measuring the accurate content of a certain component, but fully reflects the information of chemical components, the invention selects to measure at the wavelength of 230nm, has more peaks, more completely reflected information, good absorption value of each peak and stable base line.
3. The high-efficiency liquid-phase fingerprint method established by the invention realizes the quality control of the whole formula of the ginseng prebiotic particles for the first time, does not identify single compounds or medicinal materials, overcomes the problems of singleness and one-sided property of the existing quality control method, can more effectively guide feeding and strictly standardize production operation, and ensures the safety and effectiveness of clinical medication.
Drawings
FIG. 1 HPLC chromatogram of the mixed control solution
FIG. 2A-ginseng prebiotic particle consensus pattern
FIG. 3 is a superimposed fingerprint image of ginseng prebiotic particles of 10 batches
FIG. 4 sample and each medicinal material chromatogram comparison assignment chart
FIG. 5 Ginseng radix prebiotic particle map under the condition of 360nm detection wavelength
FIG. 6 is a graph of ginseng prebiotic particles at 320nm of wavelength
FIG. 7 is a graph of ginseng prebiotic particles at 280nm detection wavelength
FIG. 8 is a drawing of a spectrum of RENSHENYIYUAN granule under the condition of 254nm detection wavelength
FIG. 9A graph of ginseng prebiotic particles under the condition of 230nm detection wavelength
FIG. 10 is a graph of ginseng prebiotic particles at 210nm detection wavelength
FIG. 11 is a graph of ginseng prebiotic particles under the detection wavelength of 203nm
Detailed Description
The ginseng prebiotic granule is prepared from 160g of ginseng, 480g of astragalus, 240g of bighead atractylodes rhizome, 240g of white paeony root, 240g of suberect spatholobus stem and 120g of honey-fried licorice root. The specific preparation method comprises the following steps: decocting the ginseng and astragalus root with 10 times of water twice for 3 hours for 2 hours, mixing decoctions, standing, filtering, concentrating the filtrate to obtain thick paste with the relative density of 1.30-1.35 (60 ℃); decocting the other four traditional Chinese medicines such as bighead atractylodes rhizome and the like twice by adding 8 times of water for 2 hours each time, filtering, concentrating filtrate to obtain extractum with the relative density of 1.10-1.15 (60 ℃), adding 2 times of ethanol, stirring, standing for 12-24 hours, filtering, concentrating filtrate to obtain thick paste with the relative density of 1.30-1.35 (60 ℃) and mixing with the thick paste, drying, crushing, sieving, adding a proper amount of dextrin-sucrose (1:4), uniformly mixing, granulating with 90% ethanol, drying, granulating, and preparing 1000 g.
The invention provides a construction method of an HPLC fingerprint of ginseng prebiotic particles, which comprises the following steps:
a. extracting ginseng prebiotic particles by adopting 80% methanol, and taking the obtained ginseng prebiotic particle extract as a sample solution;
b. taking methanol solution of protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, glycyrrhizic acid and atractylenolide III as reference substance solution;
c. and respectively performing high performance liquid chromatography analysis on the reference substance solution and the sample solution, and taking a chromatographic peak of the reference substance solution as a benchmark to obtain the ginseng prebiotic particle fingerprint.
The invention adopts 80% methanol to extract the ginseng prebiotic particles, and the obtained ginseng prebiotic particle extract is used as a sample solution. The ginseng prebiotic particles are preferably ground and extracted. The weight of the ginseng prebiotic particles is 0.2-2 g, and the dosage of 80% methanol is as follows: 20-200 mL; more preferably, the weight of the ginseng prebiotic particles is 0.5g, and the dosage of 80% methanol is 50mL; the power of the ultrasonic wave is 200-300W, the frequency is 30-50 kHz, more preferably the power is 250W, and the frequency is 40kHz; the extraction time is 20-40 min, more preferably 30min.
In the embodiment of the invention, ginseng prebiotic particles are specifically taken, ground, 0.5g is taken, precisely weighed, placed in a conical bottle with a plug, 50mL of 80% methanol is precisely added, the weighed weight is weighed, ultrasonic treatment (power 40W, frequency 250 kHz) is carried out for 30 minutes, the cooled product is placed, then the weighed weight is weighed, 80% methanol is used for supplementing the lost weight, the shaking is carried out, filtration is carried out, and subsequent filtrate is taken, thus obtaining the sample solution.
In the embodiment of the invention, a proper amount of protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, ammonium glycyrrhizate and atractylenolide III reference substances are precisely weighed respectively, put into a 10mL measuring flask, and methanol is used as a solvent to prepare reference substance stock solution solutions with mass concentrations of 0.08034, 0.06149, 0.9810, 0.9672, 0.0421, 0.05088, 0.0436 and 0.06214mg/mL respectively; then respectively precisely sucking the reference substance stock solutions of 0.1, 0.5, 0.2, 0.1 and 0.1mL, putting into a same 10mL measuring flask, adding methanol for dissolving and diluting to scale, shaking uniformly, preparing mixed reference substance solutions with mass concentrations of 0.008034, 0.006149, 0.04905, 0.01934, 0.00421, 0.005088, 0.00436 and 0.006214mg/mL respectively, passing through a microporous filter membrane of 0.45 mu m, and taking the subsequent filtrate as the mixed reference substance solution.
After preparing a reference substance solution and a test substance solution, the invention respectively carries out high performance liquid chromatography analysis on the reference substance solution and the test substance solution, and takes the chromatographic peak of the reference substance solution as a benchmark to obtain the ginseng prebiotic particle fingerprint. In the present invention, when the high performance liquid chromatography is performed, the mobile phase a is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution of 0.02 to 0.2%, and the phosphoric acid aqueous solution is more preferably 0.1%; the flow rate of the mobile phase system is 0.7-1.2 mL/min, and more preferably 1.0mL/min; in the gradient elution mode, the proportion of the mobile phase A, B is changed as follows: 0-10min,3% A;10-15min,3-10% A;15-35min,10-15% A;35-65min,15-30% A;65-90min,30-40% A;90-105min,40-80% A.
In the present invention, in the case of performing the high performance liquid chromatography, the filler in the chromatographic column is preferably octadecylsilane chemically bonded silica, and the chromatographic column is preferably an Alphasil C18 column, and has the following specification: 250X 4.60mm; the column temperature is 20-40 ℃, more preferably 30 ℃; the detection wavelength is preferably 210 to 360nm, more preferably 230nm; the sample volume is preferably 5 to 15. Mu.L, more preferably 10. Mu.L.
In the present invention, when the high performance liquid chromatography is performed, the theoretical plate number is preferably not less than 5000, more preferably 6000 to 8000, as calculated as paeoniflorin peaks.
After the high performance liquid chromatography analysis is carried out on the reference substance solution and the sample solution respectively, in the invention, the reference substance chromatogram is obtained after the high performance liquid chromatography analysis is carried out on the reference substance solution; carrying out high performance liquid chromatography on the sample solution to obtain a sample chromatogram; the sample chromatogram is preferably guided into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to perform similarity analysis, so that the ginseng prebiotic particle fingerprint is obtained. In the invention, the traditional Chinese medicine chromatographic fingerprint similarity evaluation system is preferably 2012A edition of the traditional Chinese medicine chromatographic fingerprint similarity evaluation system; the similarity analysis is preferably performed by a median method, and the time window width is preferably set to 0.1min, and 21 common peaks are calibrated in total. In the embodiment of the invention, specifically, high performance liquid chromatography is carried out on 10 batches of ginseng prebiotic particles, and the obtained sample chromatograms are guided into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis, wherein the similarity of the ginseng prebiotic particles in each batch is more than 0.90. The sample chromatogram is preferably compared with the control chromatogram, chromatographic peaks at the same retention time as each control are identified, and the result shows that the 21 common peaks, wherein the 4, 5, 8, 10, 12, 18, 19 and 21 chromatographic peaks are respectively identified as protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, glycyrrhizic acid and atractylenolide III. The invention carries out component attribution on 21 common peaks, wherein the 21 common peaks are respectively attributed to six medicinal materials such as white paeony root, largehead atractylodes rhizome, liquorice, astragalus root, suberect spatholobus stem, ginseng and the like; wherein, the No. 1 peak belongs to ginseng, astragalus root, the No. 2 peak belongs to white peony root, white atractylodes rhizome, the No. 3 peak belongs to white peony root, suberect spatholobus stem, the No. 4 peak belongs to suberect spatholobus stem, the No. 6 peak belongs to liquorice, the No. 7 peak belongs to white peony root, the No. 8 peak belongs to white peony root, the No. 9 peak belongs to liquorice, astragalus root, the No. 10 peak belongs to liquorice, the No. 11 peak belongs to white peony root, the No. 12 peak belongs to astragalus root, the No. 13 peak belongs to ginseng, the No. 14 peak belongs to white peony root, the No. 15 peak belongs to liquorice, the No. 15 peak belongs to suberect spatholobus stem, the No. 17 peak belongs to white peony root, the No. 18 peak belongs to suberect spatholobus stem, the No. 19 peak belongs to liquorice, the No. 20 peak belongs to liquorice and the No. 21 peak belongs to white atractylodes rhizome.
The invention provides the ginseng prebiotic particle fingerprint spectrum obtained by the construction method of the ginseng prebiotic particle fingerprint spectrum.
The invention provides the application of the ginseng prebiotic particle fingerprint spectrum in the quality control of the ginseng prebiotic particles
In order to facilitate understanding of the technical scheme of the invention, the HPLC fingerprint establishment and the fingerprint of the ginseng prebiotic particles are further described below by combining specific test examples. The embodiments are merely some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: fingerprint spectrum of ginseng prebiotic particles in different batches is detected
1. Instrument and reagent
1.1 Agilent 1220 type high performance liquid chromatograph (Agilent, usa); KQ-250B ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); BSA 124S electronic balance (certolis); QUINTIX35-1CN (Saidolite).
1.2 baby ha purified water, acetonitrile (chromatographic purity), other reagents are analytical purity. Protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, ammonium glycyrrhizate, and atractylenolide III controls (lot numbers 110809-202207, 110810-202210, 110736-202145, 111610-202209, 111530-201914, 111704-202104, 110731-202122, 111978-201501, purity 97.5%, 99.9%, 94.6%, 95.2%, 98.8%, 94.4%, 99.9%, respectively, all purchased from China food and drug verification institute). Ginseng radix prebiotic granule (lot number: 220901, 220902, 220903, 221101, 221102, 221103, 230101, 230102, 230103, 230104).
2. Fingerprint measurement
2.1 chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, gradient elution is carried out, the flow rate is 1.0ml/min, the column temperature is 25 ℃, the detection wavelength is 230nm, and the gradient elution program of the mobile phase is as follows: 0-10min,3% A;10-15min,3-10% A;15-35min,10-15% A;35-65min,15-30% A;65-90min,30-40% A;90-105min,40-80% A.
2.2 preparation of test solutions: grinding ginseng prebiotic particles, taking 0.5g, precisely weighing, placing in a conical bottle with a plug, precisely adding 50mL of 80% methanol, weighing, performing ultrasonic treatment (power 40W, frequency 250 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 80% methanol, shaking uniformly, filtering, and collecting subsequent filtrate.
2.3 preparation of control drug and control extract: weighing control extract or control medicinal material according to prescription proportion and preparation method, and preparing according to test sample preparation method.
2.4 preparation of control solution: accurately weighing appropriate amounts of protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, ammonium glycyrrhizate and atractylenolide III reference substances respectively, accurately weighing, placing into a 10mL measuring flask, and respectively preparing reference substance stock solution solutions with mass concentrations of 0.08034, 0.06149, 0.9810, 0.9672, 0.0421, 0.05088, 0.0436 and 0.06214mg/mL by taking methanol as a solvent; respectively precisely sucking control stock solutions of 0.1, 0.5, 0.2, 0.1 and 0.1mL, placing into a same 10mL measuring flask, adding methanol for dissolving and diluting to scale, shaking uniformly to prepare mixed control solutions with mass concentrations of 0.008034, 0.006149, 0.04905, 0.01934, 0.00421, 0.005088, 0.00436 and 0.006214mg/mL, and passing through a microporous filter membrane of 0.45 μm to obtain a subsequent filtrate as the mixed control solution;
2.4 determination: respectively precisely sucking 10 μl of the reference solution and the sample solution, injecting into high performance liquid chromatograph, and recording chromatogram within 105 minutes, respectively shown in fig. 1 and 2.
Example 2: fingerprint analysis of 10 batches of ginseng prebiotic particles
1. Fingerprint similarity analysis
Taking 10 batches of ginseng prebiotic particles, preparing according to a sample preparation method, analyzing by chromatographic condition sample introduction, recording the fingerprints of the ginseng prebiotic particles, carrying out similarity analysis on the fingerprints of 10 batches of ginseng prebiotic particle compositions by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012A edition issued by the national formulary committee, setting S1 as a reference map, setting the time window width for 0.1min by utilizing a median method, calibrating 21 common peaks altogether, wherein the similarity of 10 batches of ginseng prebiotic particles is greater than 0.90, and the similarity evaluation result is shown in table 1. The relative retention times of the 21 consensus peaks were substantially uniform, while there was a large difference in relative peak areas, see table 2. The fingerprint of 10 batches of ginseng prebiotic particles is shown in figure 3 (S1-S10 in figure 3 correspond to batches 220901, 220902, 220903, 221101, 221102, 221103, 230101, 230102, 230103, 230104 respectively).
TABLE 1 similarity evaluation results of samples of ginseng prebiotic particles of 10 batches
Table 2 standard fingerprint data
2. Common peak assignments: comparing the chromatogram obtained by the reference substance with sample finger print, and identifying chromatographic peaks at the same retention time as each reference substance, wherein the chromatogram of the reference substance is shown in figures 1 and 2 (4, 5, 8, 10, 12, 18, 19 and 21, and the chromatographic peaks are respectively identified as protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, verbascoside, genistein, glycyrrhizic acid and atractylenolide III).
3. Belonging to the medicinal materials: the chromatographic peak assignment is carried out according to standard fingerprint condition, and is shown in figure 4 (S1 is sample, and S2-S7 are respectively six medicinal materials including radix Paeoniae alba, atractylodis rhizoma, glycyrrhrizae radix, radix astragali, caulis Spatholobi, ginseng radix, etc.). The 21 common peaks are respectively attributed to six medicinal materials such as white paeony root, largehead atractylodes rhizome, liquorice, astragalus root, suberect spatholobus stem, ginseng and the like; wherein, the No. 1 peak belongs to ginseng, astragalus root, the No. 2 peak belongs to white peony root, white atractylodes rhizome, the No. 3 peak belongs to white peony root, suberect spatholobus stem, the No. 4 peak belongs to suberect spatholobus stem, the No. 6 peak belongs to liquorice, the No. 7 peak belongs to white peony root, the No. 8 peak belongs to white peony root, the No. 9 peak belongs to liquorice, astragalus root, the No. 10 peak belongs to liquorice, the No. 11 peak belongs to white peony root, the No. 12 peak belongs to astragalus root, the No. 13 peak belongs to ginseng, the No. 14 peak belongs to white peony root, the No. 15 peak belongs to liquorice, the No. 15 peak belongs to suberect spatholobus stem, the No. 17 peak belongs to white peony root, the No. 18 peak belongs to suberect spatholobus stem, the No. 19 peak belongs to liquorice, the No. 20 peak belongs to liquorice and the No. 21 peak belongs to white atractylodes rhizome.
Example 3 selection of wavelength
220901 samples were taken for testing, and the overall effect of chromatograms at different wavelengths of 360, 320, 280, 250, 230, 210, 203 and the like was examined, and other chromatographic conditions were the same as in example 1, see fig. 5, 6, 7, 8, 9, 10, 11. As shown in the measured chromatogram, when the wavelength is 360nm, the number of chromatographic peaks is small, and all peaks cannot comprehensively reflect the characteristics of each medicinal material (figure 5); when the wavelength is 320nm, the number of chromatographic peaks is small, and all peaks cannot comprehensively reflect the characteristics of each medicinal material (figure 6); when the wavelength is 280nm, the number of chromatographic peaks is small, and all peaks cannot comprehensively reflect the characteristics of each medicinal material (figure 7); when the wavelength is 254nm, the number of chromatographic peaks is small, and all peaks cannot fully reflect the characteristics of each medicinal material (FIG. 8). When the wavelength is 230nm, the chromatographic peaks are more, each chromatographic peak can comprehensively reflect the characteristics of each medicinal material, and the base line of the chromatographic peak is flat, so that the peak shape is better, thus being the best condition (figure 9). At a wavelength of 210, the chromatogram has uneven baseline and large fluctuation of chromatographic peaks (fig. 10); at a wavelength of 203nm, the chromatogram baseline is not at standard position (FIG. 11). The fingerprint spectrum with 230nm as the detection wavelength is selected comprehensively to reflect the components of the preparation most comprehensively, and the peak shape is the best, so that the 230nm is preferable as the best detection wavelength.
Example 4, methodology investigation
220901 batches of samples were taken for testing, and chromatographic conditions and preparation methods of the test samples were the same as in example 1.
(1) Precision test
Taking ginseng prebiotic particles, preparing a sample solution, continuously injecting the sample for 6 times, and recording 21 main chromatographic peak retention times and peak areas. Paeoniflorin is used as a reference peak, the relative peak area RSD is less than 3.0 percent (n=6), the relative retention time RSD is less than 1.0 percent (n=6), and the instrument precision is proved to be good. The experimental results are shown in Table 3.
(2) Stability test
Taking ginseng prebiotic particles, preparing test solution, testing at 0, 2, 4, 8, 12 and 24 hours respectively, and recording 21 main chromatographic peak retention times and peak areas. Paeoniflorin is used as a reference peak, the relative peak area RSD is less than 3.0 percent (n=6), the relative retention time RSD is less than 1.0 percent (n=6), and the sample has good stability within 24 hours. The experimental results are shown in Table 3.
(3) Repeatability test
Taking ginseng prebiotic particles, preparing 6 parts of sample solution, testing, and recording 21 main chromatographic peak retention times and peak areas. Paeoniflorin is used as a reference peak, the relative peak area RSD is less than 3.0 percent (n=6), the relative retention time RSD is less than 1.0 percent (n=6), and the repeatability is good. The experimental results are shown in Table 3.
TABLE 3 methodological investigation of experimental results
Claims (10)
1. A method for constructing HPLC fingerprint of ginseng prebiotic particles is characterized in that: the method comprises the following steps:
a. extracting ginseng prebiotic particles by adopting 80% methanol, wherein the extracting solution is used as a sample solution;
b. dissolving protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, glycyrrhizic acid and atractylenolide III with methanol to obtain reference solution;
c. and respectively performing high performance liquid chromatography analysis on the reference substance solution and the sample solution, and taking a chromatographic peak of the reference substance solution as a benchmark to obtain the ginseng prebiotic particle fingerprint.
2. The method for constructing the HPLC fingerprint of the ginseng prebiotic particles according to claim 1, wherein the weight of the ginseng prebiotic particles is 0.2-2 g, and the dosage of 80% methanol is as follows: 20-200 mL.
3. The method for constructing the HPLC fingerprint of the ginseng prebiotic particles according to claim 2, wherein the extraction is performed under the ultrasonic condition, the power of the ultrasonic wave is 200-300W, and the frequency is 30-50 kHz; the extraction time is 20-40 min.
4. The method for constructing HPLC fingerprints of ginseng prebiotic particles according to claim 1, wherein when the HPLC analysis is performed, mobile phase A is acetonitrile, and mobile phase B is 0.02-0.2% phosphoric acid aqueous solution; the flow rate of the mobile phase system is 0.7-1.2 mL/min; in the gradient elution mode, the proportion of the mobile phase A, B is changed as follows: 0-10min,3% A;10-15min,3-10% A;15-35min,10-15% A;35-65min,15-30% A;65-90min,30-40% A;90-105min,40-80% A.
5. The method for constructing HPLC fingerprints of ginseng prebiotic particles according to claim 1, wherein octadecylsilane chemically bonded silica is used as a filler in the high performance liquid chromatography, and the chromatographic column has a specification of 250×4.60mm and 5 μm; the column temperature is 20-40 ℃; the detection wavelength is 210-360 nm; the sample injection volume is 5-15 mu L.
6. The method for constructing HPLC fingerprints of ginseng prebiotic particles according to claim 1, wherein the theoretical plate number is not less than 5000 calculated as paeoniflorin peak when the HPLC analysis is performed.
7. The method of constructing a ginseng prebiotic particle HPLC fingerprint according to any one of claims 1 to 6, wherein the ginseng prebiotic particle fingerprint contains 21 common peaks, wherein the chromatographic peaks No. 4, 5, 8, 10, 12, 18, 19, 21 are respectively designated as protocatechuic acid, protocatechuic aldehyde, paeoniflorin, glycyrrhizin, acteoside, genistein, glycyrrhizic acid, and atractylenolide iii.
8. The method for constructing HPLC fingerprints of ginseng prebiotic particles according to claim 7, wherein the 21 common peaks are respectively attributed to six medicinal materials including white peony root, white atractylodes rhizome, licorice, astragalus root, spatholobus stem and ginseng; wherein, the No. 1 peak belongs to ginseng, astragalus root, the No. 2 peak belongs to white peony root, white atractylodes rhizome, the No. 3 peak belongs to white peony root, suberect spatholobus stem, the No. 4 peak belongs to suberect spatholobus stem, the No. 6 peak belongs to liquorice, the No. 7 peak belongs to white peony root, the No. 8 peak belongs to white peony root, the No. 9 peak belongs to liquorice, astragalus root, the No. 10 peak belongs to liquorice, the No. 11 peak belongs to white peony root, the No. 12 peak belongs to astragalus root, the No. 13 peak belongs to ginseng, the No. 14 peak belongs to white peony root, the No. 15 peak belongs to liquorice, the No. 15 peak belongs to suberect spatholobus stem, the No. 17 peak belongs to white peony root, the No. 18 peak belongs to suberect spatholobus stem, the No. 19 peak belongs to liquorice, the No. 20 peak belongs to liquorice and the No. 21 peak belongs to white atractylodes rhizome.
9. The ginseng prebiotic particle fingerprint spectrum obtained by the construction method of the ginseng prebiotic particle HPLC fingerprint spectrum of any one of claims 1 to 8.
10. The application of the ginseng prebiotic particle fingerprint spectrum of claim 9 in quality control of ginseng prebiotic particles.
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