CN111595986A - Quality control method of heart-benefiting pulse-restoring granules - Google Patents
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Abstract
A quality control method of heart-benefiting pulse-restoring granules comprises the following specific steps: preparing a reference substance solution; preparing a test solution; selecting chromatographic conditions; measuring by accurately sucking 20 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring; linear range investigation, precision investigation, repeatability investigation, stability investigation, recovery rate test, thin layer identification of salvia miltiorrhiza and thin layer identification of ligusticum wallichii. The corresponding supplement and improvement are carried out on the basis of the original quality standard, and the thin-layer identification of the salvia miltiorrhiza and the ligusticum wallichii is added; meanwhile, a content measurement item of the ginseng is established. The qualitative method is mature, simple and convenient to operate, reliable in result and free of negative interference; the quantitative method is accurate, stable and good in reproducibility.
Description
Technical Field
The invention relates to a quality control method of heart-benefiting pulse-restoring granules, and particularly relates to the technical field of quality control of the heart-benefiting pulse-restoring granules.
Background
The heart-benefiting pulse-restoring granule has the main functions of supplementing qi, nourishing yin, promoting blood circulation and restoring pulse; can be used for treating deficiency of both qi and yin, heart blood internal resistance, thoracic obstruction, cardiodynia, chest distress, palpitation, and intermittent pulse. Has the characteristics of high drug loading, quick response, no peculiar smell, convenient carrying and the like.
The heart benefiting and pulse restoring granule is a compound preparation consisting of 6 medicinal materials of sun-dried ginseng, astragalus, dwarf lilyturf tuber, Chinese magnoliavine fruit, salvia miltiorrhiza and szechuan lovage rhizome, wherein the sun-dried ginseng: has effects in invigorating primordial qi, nourishing blood, nourishing heart, and tranquilizing mind.
The sun-dried ginseng can improve the activity and the immunologic function of brain and physical strength, enhance the anti-stress, anti-fatigue, anti-tumor, anti-aging, anti-radiation, heart-benefiting, pulse-recovering, nerve-calming, body-fluid-generating, lung-tonifying and spleen-invigorating functions.
Radix ophiopogonis: has sweet, cold, clear and moist properties, and is good at clearing heat of heart and lung to nourish yin and relieve restlessness, and also has the actions of clearing and moistening stomach and intestine to quench thirst and moisten dryness. Resisting experimental arrhythmia, and increasing coronary blood flow.
Schisandra chinensis: is five medicinal properties of pungent, sweet, sour, bitter and salty. The quality is excellent by selecting the traditional original five-ingredient northern herb as the medicine. Astringe to arrest discharge, benefit qi and promote the production of body fluid, tonify kidney and calm heart. Can be used for treating chronic cough, asthma, nocturnal emission, enuresis, frequent micturition, chronic diarrhea, spontaneous perspiration, night sweat, thirst due to body fluid consumption, short breath, weak pulse, internal heat, diabetes, palpitation, and insomnia.
Astragalus root: the astragalus root has the effects of tonifying qi, invigorating yang, promoting blood production and removing stagnation, and pharmacological researches show that the astragalus root can also eliminate oxygen free radicals and relieve lipid peroxidation injury of organisms. Red sage root: has effects in promoting blood circulation, dispelling blood stasis, inhibiting platelet aggregation and thrombosis, inhibiting cholesterol synthesis and resisting cell oxidation.
Ligusticum wallichii: promote blood circulation and move qi, dispel wind and alleviate pain. Can increase cAMP content in blood platelet and reduce TXA2 activity; reducing surface activity of platelet, inhibiting platelet aggregation, and disaggregating aggregated platelet.
The active ingredients are numerous, so a sensitive detection method is needed for detecting the active ingredients of the heart-benefiting pulse-restoring granules, but the existing detection method only comprises related detection items under characters, 2 chromogenic identification and examination items, does not carry out qualitative or quantitative detection on medicinal materials, and cannot comprehensively reflect the quality condition of products.
Disclosure of Invention
The invention aims to provide a quality control method of heart-benefiting pulse-restoring granules.
The invention relates to a quality control method of heart-benefiting pulse-restoring granules, which comprises the following steps: the first step,Preparing reference solution by collecting ginsenoside Rg1Accurately weighing appropriate amount of reference substance, and adding methanol to obtain solution containing 0.5mg per 1 ml;
step two, preparing a sample solution, namely taking 45g of heart-benefiting pulse-restoring particles, precisely weighing, precisely adding 300ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing, complementing the weight, filtering, precisely weighing 200ml of subsequent filtrate, evaporating to dryness, adding 40ml of water into residues to dissolve the residues, extracting with dichloromethane for 3 times, 25ml each time, discarding dichloromethane solution, shaking and extracting with water-saturated n-butanol for 3 times, 40ml each time, combining n-butanol solutions, washing with ammonia test solution for 4 times, 20ml each time, combining ammonia washing solutions, extracting with ammonia-saturated n-butanol for 2 times, 20ml each time, combining the n-butanol solutions, combining with the n-butanol solutions, evaporating to dryness under reduced pressure, adding 10ml of water to dissolve the water solution, passing the water solution through D, and filtering to obtain a filtrate101Macroporous adsorbent resin, wherein the inner diameter of the adsorbent resin is 1.5cm, the length of the adsorbent resin is 15cm, water is used for eluting by 50ml, water solution is discarded, then 20% ethanol is used for eluting by 50ml, 20% ethanol solution is discarded, then 70% ethanol is used for eluting by 100ml, eluent is collected and evaporated to dryness, residues are dissolved by proper amount of methanol and transferred to a 5ml measuring flask, the methanol is added to the scale, the mixture is shaken up and filtered, and then the subsequent filtrate is taken, thus obtaining the adsorbent resin;
selecting chromatographic conditions, and taking octadecylsilane chemically bonded silica as a filling agent; acetonitrile-0.05% phosphoric acid solution (19.5:80.5) is used as a mobile phase; the detection wavelength is 203 nm; the column temperature is 40 ℃; the theoretical plate number is determined according to ginsenoside Rg1Calculating to be not lower than 6000;
step four, measuring, namely precisely sucking 20 mu l of reference substance solution and 20 mu l of test substance solution respectively, injecting the reference substance solution and the test substance solution into a liquid chromatograph, and measuring to obtain the test solution;
step five, examining the linear range, and taking the ginsenoside Rg1Precisely weighing about 20mg of a reference substance, placing the reference substance into a 25ml brown volumetric flask, adding methanol to dilute the reference substance to a scale, precisely sucking the reference substance solution, preparing the reference substance solution into reference substance solutions with the concentrations of 48.75 mu g/ml, 97.50 mu g/ml, 195 mu g/ml and 390 mu g/ml respectively, shaking up the reference substance solutions, respectively injecting 20 mu l of the reference substance solution according to the chromatographic conditions of the third step, and drawing a standard curve by taking the peak area Y as the ordinate and the concentration (X, mu g/ml) as the abscissa, wherein the regression equation is as follows: 18166 or Y.1174+1223252.1760X(r＝0.9998)；
Step six, examining precision, and precisely absorbing ginsenoside Rg with C being 0.3016mg/ml1Repeatedly injecting 20 μ l of the reference substance solution for 6 times under the chromatographic condition of the step three, measuring the peak area, calculating the precision, wherein the RSD value is less than 3%, and the precision is good;
step seven, repeatability inspection, namely precisely weighing samples of the same batch number, preparing 6 parts of test sample solutions in parallel, respectively injecting 20 mu l of sample solution, measuring peak area values, calculating relative standard deviation, wherein RSD is less than 3%, and the repeatability is good;
step eight, stability investigation, namely precisely weighing a batch of samples, preparing a sample solution according to the step two, injecting 20 mu l of sample solution for 0h, 6h, 12h and 24h respectively, and measuring peak area values, wherein the samples have good stability within 24 hours;
step nine, recovery rate test, namely, taking six test sample solutions with known content, precisely weighing the test sample solutions, and adding a certain amount of ginsenoside Rg1Respectively adding the reference substance solution into six samples, operating according to the preparation items of the sample solutions, performing HPLC analysis by using the test conditions, calculating the recovery amount, and calculating the recovery rate according to the following formula, wherein the recovery rate is 95-105%, and the RSD is less than 5%, which indicates that the method has good accuracy;
step ten, identifying the salvia miltiorrhiza by a thin layer, taking 10G of heart-benefiting pulse-restoring particle test sample, grinding, adding 20ml of 0.1mol/L hydrochloric acid solution, stirring to dissolve, centrifuging, taking supernatant, adding ether to extract for 2 times, extracting 20ml each time, combining ether extract, volatilizing, adding 1ml of ethanol to residue to dissolve, taking 0.5G of salvia miltiorrhiza control drug as the test sample solution, adding a proper amount of water, decocting for 0.5 hour, filtering, concentrating the filtrate to be nearly dry, according to the preparation method of the test sample solution, taking 10 mu L of the test sample solution and 5 mu L of the control drug solution according to the thin layer chromatography test, respectively dropping the test sample solution and the control drug solution on the same silica gel G thin layer plate, taking trichloromethane-acetone-formic acid (25:10:4) as a developing agent, taking out, drying in the air, spraying 10% ferric trichloride ethanol solution on the test sample chromatogram, at the position corresponding to the control drug chromatogram, spots with the same color are displayed, the separation degree and the reproducibility are good, and the negative is free from interference;
step eleven, identifying a thin layer of ligusticum wallichii according to a preparation method of a sample solution, taking 10g of a heart-benefiting pulse-restoring particle sample, adding 50ml of 70% ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, drying the filtrate by distillation on a water bath, adding 20ml of water into the residue to dissolve the residue, extracting for 3 times by using ethyl acetate, 20ml each time, combining ethyl acetate solutions, drying by distillation, adding 5ml of methanol into the residue to dissolve the residue, adding the residue onto a neutral alumina column (100 plus 200 meshes, 2g, the inner diameter of 1-1.5 cm), eluting by using 20ml of methanol, discarding the methanol solution, eluting by using 100ml of 70% methanol, collecting the eluent, drying by distillation, adding 1ml of methanol into the residue to dissolve the residue to serve as the sample solution, taking 1g of ligusticum wallichii as a reference drug material, adding water to decoct for 1 hour, filtering, concentrating the filtrate to 5ml, and. And (3) performing thin-layer chromatography, sucking 10 μ l of each of the two solutions, respectively dropping on the same silica gel G thin-layer plate, developing with toluene-ethyl acetate-formic acid 10:5:0.5 as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and observing under ultraviolet lamp 365nm to show main fluorescence spots with the same color in the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
Compared with the prior art, the invention has the following beneficial effects: the corresponding supplement and improvement are carried out on the basis of the original quality standard, and the thin-layer identification of the salvia miltiorrhiza and the ligusticum wallichii is added; meanwhile, a content measurement item of the ginseng is established. The qualitative method is mature, simple and convenient to operate, reliable in result and free of negative interference; the quantitative method is accurate, stable and good in reproducibility.
Drawings
FIG. 1 shows ginsenoside Rg in the examples1Preparing a standard curve;
FIG. 2 shows ginsenoside Rg in the example1Precision test result chart;
FIG. 3 is a graph showing the results of the reproducibility test in the examples;
FIG. 4 is a graph showing the results of the stability test in examples;
FIG. 5 is a graph showing the results of recovery measurements in examples;
FIG. 6 is a graph showing the results of measuring the contents of 3 lots of samples in example;
FIG. 7 is a thin layer chromatogram of Salvia miltiorrhiza Bunge in example;
FIG. 8 is a thin layer chromatogram of Ligusticum chuanxiong Hort in example;
FIG. 9 is a scanning scan of a control of ginsenoside Rg1 in the examples;
FIG. 10 shows ginsenoside Rg in the examples1A control chromatogram;
FIG. 11 is a chromatogram of a sample of heart-benefiting pulse-restoring granule in an example;
FIG. 12 is a negative control chromatogram of an example;
FIG. 13 is a chromatogram of a control of Panax ginseng in the example.
Detailed Description
The technical solutions of the present invention will be described clearly and completely in the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b): the quality control method of the heart-benefiting pulse-restoring granules specifically comprises the following steps: step one, preparing a reference substance solution, namely taking ginsenoside Rg1Accurately weighing appropriate amount of reference substance, and adding methanol to obtain solution containing 0.5mg per 1 ml;
step two, preparing a sample solution, taking 45g of the product under different loading quantity items, precisely weighing, precisely adding 300ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing, complementing the weight, filtering, precisely weighing 200ml of filtrate, evaporating to dryness, adding 40ml of water into residues for dissolving, extracting 3 times with dichloromethane, 25ml each time, discarding dichloromethane solution, shaking and extracting 3 times with water saturated n-butanol, 40ml each time, combining n-butanol solutions, washing 4 times with ammonia test solution, 20ml each time, combining ammonia washing solutions, extracting 2 times with ammonia solution saturated n-butanol, 20ml each time, combining n-butanol solutions, combining with the n-butanol solutions, evaporating to dryness under reduced pressure, adding 10ml of water for dissolving, and passing the water solution through D101Macroporous adsorbent resin with inner diameter of 1.5cm and length of 15cm) Eluting with 50ml of water, discarding water solution, eluting with 50ml of 20% ethanol, discarding 20% ethanol solution, eluting with 100ml of 70% ethanol, collecting eluate, evaporating to dryness, dissolving residue with appropriate amount of methanol, transferring to 5ml measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate;
selecting chromatographic conditions, and taking octadecylsilane chemically bonded silica as a filling agent; acetonitrile-0.05% phosphoric acid solution (19.5:80.5) is used as a mobile phase; the detection wavelength is 203 nm; the column temperature is 40 ℃; the theoretical plate number is determined according to ginsenoside Rg1Calculating to be not lower than 6000; detection of wavelength selection for ginsenoside Rg1The methanol solution was subjected to UV scanning in the wavelength range of 190-300nm, and the results are shown in FIG. 9. Discovery of ginsenoside Rg1Has strong absorption at 203nm wavelength, so 203nm wavelength is selected as ginsenoside Rg1The detection wavelength of (1). Under the condition, the baseline can be separated without obvious interference, and the ginsenoside Rg1The separation from other components is good, and the result is shown in ginsenoside Rg1Reference figure 10 and heart benefiting pulse restoring granule test figure 11; the negative test sample of heart-benefiting pulse-restoring granule without ginseng has no absorption peak in the retention time, which shows no negative interference.
Step four, measuring, namely precisely sucking 20 mu l of reference substance solution and 20 mu l of test substance solution respectively, injecting the reference substance solution and the test substance solution into a liquid chromatograph, and measuring to obtain the test solution;
step five, examining the linear range, and taking the ginsenoside Rg1Precisely weighing about 20mg of a reference substance, placing the reference substance into a 25ml brown volumetric flask, adding methanol to dilute the reference substance to a scale, precisely sucking the reference substance solution, preparing the reference substance solution into reference substance solutions with the concentrations of 48.75 mu g/ml, 97.50 mu g/ml, 195 mu g/ml and 390 mu g/ml respectively, shaking up, injecting 20 mu l of the reference substance solution respectively under the chromatographic conditions, and drawing a standard curve by taking a peak area (Y) as an ordinate and a concentration (X, mu g/ml) as an abscissa, wherein a regression equation is as follows: y18166.1174 +1223252.1760X (r 0.9998); the results show that the ginsenoside Rg1The linear relationship was good in the range of 48.75-780. mu.g/ml, and the results are shown in FIG. 1.
Step six, precisely absorbing ginsenoside Rg1Control solution (C: 0.3016mg/ml) 20. mu.l, as described aboveSample introduction is repeated for 6 times under the chromatographic condition, peak area is measured, precision is calculated, the result is shown in figure 2, and the result shows that: the method has good precision.
Seventhly, repeatedly inspecting, precisely weighing the same batch of samples (20160101), parallelly preparing 6 test solution samples, respectively injecting 20 mu l of the test solution samples, measuring peak area values, calculating relative standard deviation, and showing that the result is shown in figure 3, and the RSD of the samples is less than 3%, which indicates that the method has good repeatability.
Step eight, stability investigation, precisely weighing samples of batch numbers (20160101), preparing test solution according to the method under the content determination item, injecting 20 mul of sample at 0h, 6h, 12h and 24h respectively, and measuring peak area values, wherein the result is shown in figure 4. The results show that: the samples were stable well within 24 hours under the conditions tested.
And step nine, recovery rate test, namely, taking six parts of test sample solutions with known contents, precisely weighing, adding a certain amount of ginsenoside Rg1 reference sample solutions into the six parts of test samples respectively, operating according to the preparation items of the test sample solutions, performing HPLC analysis under the test conditions, calculating the recovery rate, and calculating the recovery rate according to the following formula, wherein the result is shown in figure 5. The recovery rate (%) - (measured amount-preparation content)/added control amount × 100% was between 95% and 105%, and RSD was less than 5%, indicating good method accuracy.
And (3) determination of sample content: three batches of heart invigorating and pulse restoring granules (batch numbers: 20160301, 20160302, 20160303) were taken, 3 test solutions were treated in parallel for each batch, and the results were determined according to the method, and are shown in FIG. 6.
The results show that: ginsenoside Rg in the three provided samples1The average content of the ginseng is 0.9354 mg/bag, calculated by pressing the floater to 30%, each bag of the product contains ginseng and ginsenoside Rg1(C42H72O14) Calculated, the content of the active ingredient should not be less than 0.65 mg.
Step ten, identifying the salvia miltiorrhiza by a thin layer, taking 10G of heart-benefiting pulse-restoring particle test sample, grinding, adding 20ml of 0.1mol/L hydrochloric acid solution, stirring to dissolve, centrifuging, taking supernatant, adding ether to extract for 2 times, extracting 20ml each time, combining ether extract, volatilizing, adding 1ml of ethanol to residue to dissolve, taking 0.5G of salvia miltiorrhiza control drug as the test sample solution, adding a proper amount of water, decocting for 0.5 hour, filtering, concentrating the filtrate to be nearly dry, according to the preparation method of the test sample solution, taking 10 mu L of the test sample solution and 5 mu L of the control drug solution according to the thin layer chromatography test, respectively dropping the test sample solution and the control drug solution on the same silica gel G thin layer plate, taking trichloromethane-acetone-formic acid (25:10:4) as a developing agent, taking out, drying in the air, spraying 10% ferric trichloride ethanol solution on the test sample chromatogram, at the position corresponding to the control drug chromatogram, spots with the same color are shown, the separation degree and the reproducibility are good, and the negative effect is not interfered. In the process of establishing the thin-layer method of the salvia miltiorrhiza, ethyl acetate is adopted for extraction, the salvia miltiorrhiza contrast medicinal material is used as a contrast, chromatography is carried out according to the development method of pharmacopeia, the result effect is poor, and the spots of the test sample are not obvious. Considering that the process is carried out by decocting radix Salviae Miltiorrhizae with water, hydrolyzing with acid water, extracting with diethyl ether, performing chromatography with radix Salviae Miltiorrhizae reference material as reference, developing, and displaying spots with same color at corresponding positions in the chromatogram of the test sample, wherein the method is mature, the spots are clear, the resolution and reproducibility are good, and the negative is interference-free (see fig. 7).
Step eleven, identifying a thin layer of ligusticum wallichii, taking 10g of heart-benefiting pulse-restoring particle sample, adding 50ml of 70% ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, drying the filtrate by distillation on a water bath, adding 20ml of water into the residue for dissolving, extracting for 3 times by using ethyl acetate, 20ml each time, combining ethyl acetate solutions, drying by distillation, adding 5ml of methanol into the residue for dissolving, adding the residue on a neutral alumina column (100 meshes 200, 2g, the inner diameter of 1-1.5 cm), eluting by using 20ml of methanol, discarding the methanol solution, eluting by using 100ml of 70% methanol, collecting the eluent, drying by distillation, adding 1ml of methanol into the residue for dissolving, and taking the residue as the sample solution. Decocting rhizoma Ligustici Chuanxiong 1g with water for 1 hr, filtering, concentrating the filtrate to 5ml, and making into control solution according to the preparation method of the sample solution. And (3) performing thin-layer chromatography, sucking 10 μ l of each of the two solutions, respectively dropping on the same silica gel G thin-layer plate, developing with toluene-ethyl acetate-formic acid 10:5:0.5 as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution, heating at 105 deg.C until the spots develop clearly, inspecting under ultraviolet lamp (365nm), and displaying the main fluorescent spots with the same color in the chromatogram of the sample at the position corresponding to the chromatogram of the control medicinal material. In the process of establishing the thin-layer method, as the ligusticum wallichii is decocted and extracted by water in the process, the water-soluble component ferulic acid is alkalized and then extracted by ethyl ether, an alkali liquid layer is acidified again and then extracted by the ethyl ether to prepare a test solution, the ferulic acid and the ligusticum wallichii are used as a reference medicinal material, petroleum ether (60-90 ℃) and ethyl acetate-glacial acetic acid (12:4:1) are used as a developing agent, and as a result, the spot of the ferulic acid can be detected, but the trailing phenomenon is serious, no improvement is caused after retry, and the method has poor reproducibility. The method is improved, and the spot is clear and the separation degree is good. The method is mature, has good reproducibility and no interference in negativity (see figure 8).
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (1)
1. A quality control method of heart-benefiting pulse-restoring granules is characterized by comprising the following steps: the specific control method comprises the following steps: step one, preparing a reference substance solution, namely taking ginsenoside Rg1Accurately weighing appropriate amount of reference substance, and adding methanol to obtain solution containing 0.5mg per 1 ml;
step two, preparing a sample solution, namely taking 45g of heart-benefiting pulse-restoring particles, precisely weighing, precisely adding 300ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing, complementing the weight, filtering, precisely weighing 200ml of subsequent filtrate, evaporating to dryness, adding 40ml of water into residues to dissolve the residues, extracting with dichloromethane for 3 times, 25ml each time, discarding dichloromethane solution, shaking and extracting with water-saturated n-butanol for 3 times, 40ml each time, combining n-butanol solutions, washing with ammonia test solution for 4 times, 20ml each time, combining ammonia washing solutions, extracting with ammonia-saturated n-butanol for 2 times, 20ml each time, combining the n-butanol solutions, combining with the n-butanol solutions, evaporating to dryness under reduced pressure, adding 10ml of water to dissolve the water solution, passing the water solution through D, and filtering to obtain a filtrate101Macroporous adsorbent resin, adsorptionThe inner diameter of the resin is 1.5cm, the length of the resin is 15cm, the resin is eluted by 50ml of water, water solution is discarded, then 50ml of 20% ethanol is used for elution, 20% ethanol solution is discarded, then 100ml of 70% ethanol is used for elution, eluent is collected and evaporated to dryness, residues are dissolved by proper amount of methanol and transferred to a 5ml measuring flask, methanol is added to the scales, the mixture is shaken up and filtered, and the subsequent filtrate is taken, so that the resin is obtained;
selecting chromatographic conditions, and taking octadecylsilane chemically bonded silica as a filling agent; acetonitrile-0.05% phosphoric acid solution (19.5:80.5) is used as a mobile phase; the detection wavelength is 203 nm; the column temperature is 40 ℃; the theoretical plate number is determined according to ginsenoside Rg1Calculating to be not lower than 6000;
step four, measuring, namely precisely sucking 20 mu l of reference substance solution and 20 mu l of test substance solution respectively, injecting the reference substance solution and the test substance solution into a liquid chromatograph, and measuring to obtain the test solution;
step five, examining the linear range, and taking the ginsenoside Rg1Precisely weighing about 20mg of a reference substance, placing the reference substance into a 25ml brown volumetric flask, adding methanol to dilute the reference substance to a scale, precisely sucking the reference substance solution, preparing the reference substance solution into reference substance solutions with the concentrations of 48.75 mu g/ml, 97.50 mu g/ml, 195 mu g/ml and 390 mu g/ml respectively, shaking up the reference substance solutions, respectively injecting 20 mu l of the reference substance solution according to the chromatographic conditions of the third step, and drawing a standard curve by taking the peak area Y as the ordinate and the concentration (X, mu g/ml) as the abscissa, wherein the regression equation is as follows: y18166.1174 +1223252.1760X (r 0.9998);
step six, examining precision, and precisely absorbing ginsenoside Rg with C being 0.3016mg/ml1Repeatedly injecting 20 μ l of the reference substance solution for 6 times under the chromatographic condition of the step three, measuring the peak area, calculating the precision, wherein the RSD value is less than 3%, and the precision is good;
step seven, repeatability inspection, namely precisely weighing samples of the same batch number, preparing 6 parts of test sample solutions in parallel, respectively injecting 20 mu l of sample solution, measuring peak area values, calculating relative standard deviation, wherein RSD is less than 3%, and the repeatability is good;
step eight, stability investigation, namely precisely weighing a batch of samples, preparing a sample solution according to the step two, injecting 20 mu l of sample solution for 0h, 6h, 12h and 24h respectively, and measuring peak area values, wherein the samples have good stability within 24 hours;
step nine, recovery rate test, taking sixWeighing known content of test solution, precisely weighing, and mixing with a certain amount of ginsenoside Rg1Respectively adding the reference substance solution into six samples, operating according to the preparation items of the sample solutions, performing HPLC analysis by using the test conditions, calculating the recovery amount, and calculating the recovery rate according to the following formula, wherein the recovery rate is 95-105%, and the RSD is less than 5%, which indicates that the method has good accuracy;
step ten, identifying the salvia miltiorrhiza by a thin layer, taking 10G of heart-benefiting pulse-restoring particle test sample, grinding, adding 20ml of 0.1mol/L hydrochloric acid solution, stirring to dissolve, centrifuging, taking supernatant, adding ether to extract for 2 times, extracting 20ml each time, combining ether extract, volatilizing, adding 1ml of ethanol to residue to dissolve, taking 0.5G of salvia miltiorrhiza control drug as the test sample solution, adding a proper amount of water, decocting for 0.5 hour, filtering, concentrating the filtrate to be nearly dry, according to the preparation method of the test sample solution, taking 10 mu L of the test sample solution and 5 mu L of the control drug solution according to the thin layer chromatography test, respectively dropping the test sample solution and the control drug solution on the same silica gel G thin layer plate, taking trichloromethane-acetone-formic acid (25:10:4) as a developing agent, taking out, drying in the air, spraying 10% ferric trichloride ethanol solution on the test sample chromatogram, at the position corresponding to the control drug chromatogram, spots with the same color are displayed, the separation degree and the reproducibility are good, and the negative is free from interference;
step eleven, identifying a thin layer of ligusticum wallichii according to a preparation method of a sample solution, taking 10g of a heart-benefiting pulse-restoring particle sample, adding 50ml of 70% ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, drying the filtrate by distillation on a water bath, adding 20ml of water into the residue to dissolve the residue, extracting for 3 times by using ethyl acetate, 20ml each time, combining ethyl acetate solutions, drying by distillation, adding 5ml of methanol into the residue to dissolve the residue, adding the residue onto a neutral alumina column (100 plus 200 meshes, 2g, the inner diameter of 1-1.5 cm), eluting by using 20ml of methanol, discarding the methanol solution, eluting by using 100ml of 70% methanol, collecting the eluent, drying by distillation, adding 1ml of methanol into the residue to dissolve the residue to serve as the sample solution, taking 1g of ligusticum wallichii as a reference drug material, adding water to decoct for 1 hour, filtering, concentrating the filtrate to 5ml, and. And (3) performing thin-layer chromatography, sucking 10 μ l of each of the two solutions, respectively dropping on the same silica gel G thin-layer plate, developing with toluene-ethyl acetate-formic acid 10:5:0.5 as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and observing under ultraviolet lamp 365nm to show main fluorescence spots with the same color in the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
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