CN114441662B - Construction method and identification method of poria, cassia, rhizoma polygonati and sweet soup and Gan Jiangling, rhizoma polygonati and Shang Tezheng atlas - Google Patents
Construction method and identification method of poria, cassia, rhizoma polygonati and sweet soup and Gan Jiangling, rhizoma polygonati and Shang Tezheng atlas Download PDFInfo
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Abstract
The invention discloses a construction method and an identification method of a poria, cassia, rhizoma dioscoreae and licorice decoction and a Gan Jiangling, rhizoma dioscoreae and Shang Tezheng map, which are characterized by comprising the following steps: (1) Preparing a test solution of the poria, cassia bark and rhizoma coptidis decoction and a test solution of the Gan Jiangling rhizoma atractylodis decoction respectively; (2) Adding methanol to constant volume to obtain reference solution; (3), high performance liquid chromatography conditions: liquid chromatographic column: a C18 chromatographic column; column temperature: 25-35 ℃; detection wavelength: 190-400 nm; flow rate: 0.8 mL/min-1.2 mL/min; feed amount: 5 mu L-20 mu L; the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and the gradient elution is carried out; (4) And (3) respectively carrying out high performance liquid chromatography detection on the sample solution prepared in the step (1) and the reference substance solution prepared in the step (2) to obtain corresponding characteristic patterns.
Description
Technical Field
The invention belongs to the technical field of detection of traditional Chinese medicine formulas, and particularly relates to a construction method and an identification method of poria, cassia, rhizoma atractylodis and sweet soup and Gan Jiangling, rhizoma atractylodis Shang Tezheng atlas.
Background
The poria and cassia bark, rhizoma Atractylodis Gan Shangfang, out of Zhang Zhongjing, shang Han Lian Bing Lun. The book, cloud 67: if the typhoid fever is caused, the heart is overfull, the qi is upwards rushed to the chest, the head is reversed, the pulse is deep and tight, the sweating is followed by the movement of the channel, the body is the person who shakes, and the poria, cassia twig, bighead atractylodes rhizome and liquorice are the main decoction. "jin Kui Yao Lv" also carries this formula, as follows: phlegm-fluid retention in the heart, fullness in the chest and hypochondrium, dizziness, and poria, cortex Cinnamomi and rhizoma Atractylodis decoction. The recipe is designed for the symptoms caused by the deficiency of middle-jiao and internal retention of water-retention, and is caused by yang deficiency of middle-jiao, spleen failing to transport and internal retention of water-retention, both after vomiting due to typhoid fever and after vomiting due to phlegm-retention due to miscellaneous diseases. Gan Jiangling the decoction is prepared from the main points of jin Kui, the syndrome of wind-cold accumulation of five zang organs and treatment. Original book cloud: kidney-attached disease, cold in the waist, such as sitting in water, water-like shape, thirst-quenching, self-help urination, and good appetite, the disease is lower in the focus, body overstrain and sweating, cold and dampness in the clothing, long-lasting, cold pain below the waist, and abdominal heaviness such as five thousand-money, gan Jiangling decoction. The recipe is designed to treat kidney attachment caused by cold-dampness attacking the exterior and blocking the waist. Compared with the decoction, the decoction of Gan Jiang Lingshu has only one bad medicine, but the poria cassia twig Gan Shangjun is poria cocos, ministerial cassia twig and is a prescription taking the water-dispelling decoction as the main ingredient, and the Gan Jiang Lingshu Shang Zejun dried ginger and the ministerial poria cocos are used for warming middle-jiao and dispelling cold and dampness and are the prescription taking the cold-dispelling dampness as the main ingredient. At present, the establishment of characteristic maps of the poria, cassia, sweet soup and Gan Jiangling and the identification method of the poria, cassia, sweet soup and Gan Jiangling and the identification method of the poria, cassia, sweet soup and the Gan Jiangling and the identification method of the poria, cassia, sweet soup are lack.
Disclosure of Invention
In order to solve the problems, the invention provides a construction method and an identification method of a poria, cassia, rhizoma polygonati and sweet soup and Gan Jiangling, shang Tezheng atlas.
The invention aims at realizing the following technical scheme:
the construction method of the poria, cassia and rhizoma polygonati soup and Gan Jiangling and Shang Tezheng atlas is characterized by comprising the following steps:
(1) Preparing a sample solution: preparing a test solution of the poria, cassia, rhizoma atractylodis and sweet soup and a test solution of the Gan Jiangling rhizoma atractylodis soup respectively;
(2) Preparing a reference substance solution: adding methanol to constant volume to obtain reference solution;
(3) Conditions of high performance liquid chromatography: liquid chromatographic column: a C18 chromatographic column;
column temperature: 25-35 ℃;
detection wavelength: 190-400 nm;
flow rate: 0.8 mL/min-1.2 mL/min;
feed amount: 5 mu L-20 mu L;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and the gradient elution is carried out;
(4) High performance liquid chromatography detection: and (3) respectively carrying out high performance liquid chromatography detection on the sample solution prepared in the step (1) and the reference substance solution prepared in the step (2) to obtain corresponding characteristic patterns.
Preferably, the preparation of the test solution of the ling gui zhu gan soup in the step (1) comprises the following steps:
(1) Weighing 55g of poria cocos, 41g of cassia twig, 27g of bighead atractylodes rhizome and 27g of honey-fried licorice root, adding 2 liters of water, extracting for 60 minutes, extracting for 1 time, filtering with a 200-mesh screen, collecting liquid medicine, and freeze-drying by a vacuum freeze dryer to obtain freeze-dried powder of poria cocos, cassia twig and sweet soup;
(2) Precisely weighing 0.5g of the freeze-dried powder of the poria, cassia, rhizoma atractylodis and sweet soup, adding 20ml of 70% methanol, processing for 30min by ultrasonic (power 600W, frequency 40 kHz), cooling to room temperature, shaking uniformly, and filtering to obtain the sample solution of the poria, cassia, rhizoma atractylodis and sweet soup.
Preferably, the preparation of the test solution of Gan Jiangling decoction in the step (1) comprises the following steps:
(1) Weighing 55g of poria cocos, 55g of dried ginger, 27g of bighead atractylodes rhizome and 27g of honey-fried licorice root, adding 2 liters of water, extracting for 60 minutes, extracting for 1 time, filtering with a 200-mesh screen, collecting liquid medicine, and freeze-drying by a vacuum freeze dryer to obtain sweet Jiang Lingshu soup freeze-dried powder;
(2) Precisely weighing 0.5g of the sweet Jiang Lingshu soup freeze-dried powder, adding 20ml of 70% methanol, processing for 30min by ultrasonic (power 600W, frequency 40 kHz), cooling to room temperature, shaking uniformly, and filtering to obtain a sweet Jiang Lingshu soup sample solution.
Preferably, the vacuum freeze dryer lyophilization process includes prefreezing, sublimation drying, and analytical drying.
Preferably, the preparation method of the mixed reference substance solution in the step (2) comprises the following steps: the glycyrrhizin, ammonium glycyrrhetate, cinnamic acid, cinnamaldehyde and protocatechuic acid reference substances are respectively precisely weighed, placed in a volumetric flask, dissolved by adding methanol, and uniformly shaken to prepare reference substance solutions containing 50 mug of each 1ml of methanol solution.
Preferably, the high performance liquid chromatography conditions in the step (3) are as follows:
column temperature: 30 ℃;
detection wavelength: 270nm;
flow rate: 1.0mL/min;
feed amount: 10. Mu.L;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and the gradient elution is carried out, wherein the gradient elution process is as follows:
0 to 12min, 3 to 20 percent of mobile phase A, 97 to 80 percent of mobile phase B,
12-36 min, 20-30% of mobile phase A, 80-70% of mobile phase B,
36-45 min, 30-60% of mobile phase A, 70-40% of mobile phase B,
45-60 min, 60-85% of mobile phase A and 40-15% of mobile phase B.
The identifying method of the poria, cassia, rhizoma atractylodis and sweet soup and Gan Jiangling rhizoma atractylodis soup is characterized by comprising the following steps of:
(1) Measuring characteristic patterns of the poria Gui Shugan decoction and the Gan Jiangling rhizoma Atractylodis decoction;
(2) Measuring a characteristic map of the decoction to be measured;
(3) And comparing the characteristic spectrum of the decoction to be detected with the characteristic spectrum of the poria, cassia, rhizoma polygonati and rhizoma polygonati decoction and Gan Jiangling rhizoma polygonati decoction.
Preferably, the feature spectrum of the ling gui zhu gan decoction has 17 feature peaks, the peak corresponding to the glycyrrhizin reference object is an S peak, the relative retention time of each feature peak and the S peak is calculated, the relative retention time is within +/-10% of the specified value, and the specified value is: 0.225 (peak 1), 0.296 (peak 2), 0.390 (peak 3), 0.471 (peak 4), 0.567 (peak 5), 0.724 (peak 6), 0.972 (peak 7), 1.000 (peak 8,S), 1.216 (peak 9), 1.561 (peak 10), 1.682 (peak 11), 1.842 (peak 12), 2.113 (peak 13), 2.207 (peak 14), 2.260 (peak 15), 2.464 (peak 16), 2.603 (peak 17).
Preferably, the characteristic spectrum of the Gan Jiangling decoction has 15 characteristic peaks, the peak corresponding to the cinnamic acid reference object is an S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is: 0.224 (Peak 1), 0.297 (Peak 2), 0.391 (Peak 3), 0.470 (Peak 4), 0.627 (Peak 5), 0.722 (Peak 6), 0.972 (Peak 7), 1.000 (Peak 8,S), 1.216 (Peak 9), 1.560 (Peak 10), 2.189 (Peak 11), 2.241 (Peak 12), 2.314 (Peak 13), 2.444 (Peak 14), 2.580 (Peak 15).
The beneficial effects of this technical scheme are as follows:
(1) The invention adopts high performance liquid chromatography to construct the characteristic spectrum of the tuckahoe-cassia twig-rhizoma Gan Shangji Gan Jiangling-rhizoma decoction, and realizes the rapid identification of the tuckahoe-cassia twig-rhizoma Gan Shangji Gan Jiangling-rhizoma decoction by the characteristic spectrum method, and the method is simple.
(2) The invention provides the identification of the characteristic spectrum method of the two prescriptions for the first time, particularly the characteristic spectrum of the tuckahoe-cassia twig-rhizoma Gan Shangji Gan Jiangling-rhizoma atractylodis decoction is distinguished, and the rapid identification of the two prescriptions can be realized.
(3) The method adopts the high performance liquid chromatography to carry out the measurement by gradient elution, and has the advantages of simple operation, good separation degree, high precision, good repeatability, good stability and the like.
(4) The method of the invention provides the identification method of the poria cassia bark and rhizoma Gan Shangji Gan Jiangling and rhizoma Atractylodis decoction for the first time, and under the specific detection condition, the identification of the poria cassia bark and rhizoma Gan Shangji Gan Jiangling and rhizoma Atractylodis decoction by adopting the characteristic spectrum method can be realized by taking the chromatographic peak of glycyrrhizin as the reference S peak.
Drawings
FIG. 1 is a chromatogram of LINGGUIZHUGAN decoction at different wavelengths;
FIG. 2 is a chromatogram of LINGGUIZHUGAN decoction at different feed rates;
FIG. 3 is a chromatogram of LINGGUIHUANGGAN decoction at different column temperatures;
FIG. 4 is a chromatogram of LINGGUIHUANGGAN decoction at different flow rates;
FIG. 5 is a graph showing the identification of chromatographic peaks of LINGGUIZHENGAN decoction;
FIG. 6 is a chromatogram of different batches of Poria, cortex Cinnamomi, and rhizoma Atractylodis decoction;
FIG. 7 is a chromatogram of different batches of glycerol Jiang Lingshu soup;
fig. 8 is a chromatogram of the ling gui zhu gan decoction and Gan Jiangling zhu decoction.
Detailed Description
Experimental instrument and materials
The test instruments and materials referred to in the following examples are as follows (% each in mass concentration):
high performance liquid chromatograph: agilent 1260 type high performance liquid chromatograph
An electronic balance: ME204E/02, XP26 (Metrele Tolyduo instruments Co., ltd.);
ultrapure water machine: cell type 1810A (Shanghai mueller scientific instruments limited);
ultrasonic cleaner: KQ5200DB model (600W, 40KHz; kunshan ultrasonic instruments Co., ltd.);
chromatographic column: agilent 5TC-C18 (2) 250 x 4.6mm 5um;
acetonitrile and phosphoric acid are chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity;
glycyrrhizin (Chinese food and drug assay institute, lot number: 111610-201908, content of 95.0%);
ammonium glycyrrhizate (Chinese food and drug administration institute, lot number: 110731-202021, content of 96.2%);
cinnamic acid (China food and drug inspection institute, batch No. 110786-201604, content of 98.8%);
protocatechuic acid (national food and drug verification institute, lot number: 110809-201205, content of 99.9%);
cinnamic aldehyde (Chinese food and drug verification institute, batch No. 110710-201821, content of 99.6%);
poria (lot number: 010145-1912001);
ramulus Cinnamomi (lot number: 010182-2008001);
atractylodis rhizoma (lot number: 010022-1905001);
radix Glycyrrhizae Preparata (lot number: 010154-1901001);
dried ginger (lot number: XLS 20200807);
5 batches of poria, cassia, and sweet soup (LGZGTBT 01, LGZGTBT02, LGZGTBT03, LGZGTBT04, LGZGTBT 05)
5 batches of sweet Jiang Lingshu soup (GJLZTBT 01, GJLZTBT02, GJLZTBT03, GJLZTBT04, GJLZTBT 05).
Example 1
The embodiment relates to a poria, cassia, rhizoma atractylodis Gan Shangji Gan Jiangling and rhizoma atractylodis decoction.
(1) The preparation method of the poria cassia bark and rhizoma Gan Shangji Gan Jiangling and rhizoma Atractylodis decoction comprises the following steps:
weighing 55g of poria cocos, 41g of cassia twig, 27g of bighead atractylodes rhizome and 27g of honey-fried licorice root, adding 2 liters of water, extracting for 60 minutes, extracting for 1 time, filtering with a 200-mesh screen, collecting liquid medicine, and freeze-drying by a vacuum freeze dryer to obtain freeze-dried powder of poria cocos, cassia twig and sweet soup;
the procedure for freeze drying to lyophilized powder is shown in the following table
Precisely weighing 0.5g of the freeze-dried powder of the poria, cassia, rhizoma atractylodis and sweet soup, adding 20ml of 70% methanol, processing for 30min by ultrasonic (power 600W, frequency 40 kHz), cooling to room temperature, shaking uniformly, and filtering to obtain the sample solution of the poria, cassia, rhizoma atractylodis and sweet soup.
Weighing 55g of poria cocos, 55g of dried ginger, 27g of bighead atractylodes rhizome and 27g of honey-fried licorice root, adding 2 liters of water, extracting for 60 minutes, extracting for 1 time, filtering with a 200-mesh screen, collecting liquid medicine, and freeze-drying by a vacuum freeze dryer to obtain sweet Jiang Lingshu soup freeze-dried powder;
the procedure for freeze drying to lyophilized powder is shown in the following table
Precisely weighing 0.5g of sweet Jiang Lingshu soup freeze-dried powder, adding 70% methanol 20ml, processing with ultrasound (power 600W, frequency 40 kHz) for 30min, cooling to room temperature, shaking, and filtering to obtain sweet Jiang Lingshu soup sample solution.
(2) Preparing a reference substance solution: the glycyrrhizin, ammonium glycyrrhetate, cinnamic acid, cinnamaldehyde and protocatechuic acid reference substances are respectively precisely weighed, placed in a volumetric flask, dissolved by adding methanol, and uniformly shaken to prepare reference substance solutions containing 50 mug of each 1ml of methanol solution.
(3) And (3) respectively carrying out high performance liquid chromatography detection on the sample solution prepared in the step (1) and the reference substance solution prepared in the step (2) to obtain corresponding characteristic patterns.
The high performance liquid chromatography detection conditions are as follows:
liquid chromatographic column: a C18 chromatographic column;
column temperature: 30 ℃;
detection wavelength: 270nm;
flow rate: 1.0mL/min;
feed amount: 10. Mu.L;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and the gradient elution is carried out, wherein the gradient elution process is as follows:
0 to 12min, 3 to 20 percent of mobile phase A, 97 to 80 percent of mobile phase B,
12-36 min, 20-30% of mobile phase A, 80-70% of mobile phase B,
36-45 min, 30-60% of mobile phase A, 70-40% of mobile phase B,
45-60 min, 60-85% of mobile phase A and 40-15% of mobile phase B.
(4) And (3) taking the characteristic spectrum of the reference substance solution in the step (3) as a reference spectrum, selecting a common peak from the characteristic spectrum of the sample solution, and constructing the characteristic spectrum of the poria, cassia, rhizoma coptidis, sweet soup and Gan Jiangling rhizoma atractylodis soup.
The constructed characteristic spectrum of the poria, cassia and rhizoma coptidis decoction has 17 characteristic peaks, wherein the peak corresponding to the glycyrrhizin reference substance is an S peak. The relative retention time of each characteristic peak and the S peak was calculated to be within + -10% of the prescribed value. The specified value is: 0.225 (peak 1), 0.296 (peak 2), 0.390 (peak 3), 0.471 (peak 4), 0.567 (peak 5), 0.724 (peak 6), 0.972 (peak 7), 1.000 (peak 8,S), 1.216 (peak 9), 1.561 (peak 10), 1.682 (peak 11), 1.842 (peak 12), 2.113 (peak 13), 2.207 (peak 14), 2.260 (peak 15), 2.464 (peak 16), 2.603 (peak 17).
The constructed characteristic spectrum of the sweet Jiang Lingshu soup has 15 characteristic peaks, wherein the peak corresponding to the ferulic acid reference substance is an S peak. The relative retention time of each characteristic peak and the S peak was calculated to be within + -10% of the prescribed value. The specified value is: 0.224 (Peak 1), 0.297 (Peak 2), 0.391 (Peak 3), 0.470 (Peak 4), 0.627 (Peak 5), 0.722 (Peak 6), 0.972 (Peak 7), 1.000 (Peak 8,S), 1.216 (Peak 9), 1.560 (Peak 10), 2.189 (Peak 11), 2.241 (Peak 12), 2.314 (Peak 13), 2.444 (Peak 14), 2.580 (Peak 15).
Example 2
The embodiment relates to the identification of the poria, cassia, rhizoma dioscoreae and licorice root decoction and Gan Jiangling and rhizoma dioscoreae decoction.
(1) Taking the poria, cassia and sweet soup and Gan Jiangling and preparing sample solutions of the poria, cassia and sweet soup and Gan Jiangling and sweet soup according to the preparation method of the sample solution in the step (1) of the example 1;
(2) Performing high performance liquid chromatography detection on the sample solution prepared in the step (1) according to the detection conditions in the step (3) in the embodiment 1 respectively to obtain a corresponding characteristic spectrum to be identified;
(3) Comparing the characteristic spectrum to be identified obtained in the step (2) with the corresponding characteristic spectrum constructed in the step (4) in the embodiment 2, and identifying as qualified if the characteristic spectrum to be identified is consistent with the corresponding characteristic spectrum constructed in the step (4).
Example 3
Investigation of wavelength
Taking the sample solution of the poria cassia bark and sweet soup as described in the embodiment 2 as an object, full-wavelength scanning is carried out in the range of 190-400 nm through a DAD detector, and as a result, the detection wavelength is determined to be 270nm as a result, the detection wavelength is shown in the characteristic spectrum shown in the figure 1, and the chromatographic peak information amount is larger and the chromatographic base line is smoother when the detection wavelength is 270 nm.
Example 4
Investigation of sample injection quantity
The sample solutions of the poria, cassia, rhizoma coptidis and sweet soup in example 2 are taken as targets, characteristic patterns obtained by comparing sample injection amounts of 5 microliter, 10 microliter and 20 microliter are compared, and under the condition of each sample injection amount, the peak shapes of the chromatograms are symmetrical, and the separation degree is good, so that the sample injection amount of 10 microliter is finally selected as the sample injection amount of the characteristic pattern method, and the characteristic pattern shown in fig. 2 is obtained.
Example 5
Investigation of column temperature
The sample solution of the poria, cassia, rhizoma polygonati and sweet soup described in example 2 is taken as an object, characteristic patterns obtained at the column temperature of 25 ℃, 30 ℃ and 35 ℃ are compared, and under the conditions of each column temperature, the peak shapes of the chromatograms are symmetrical, and the separation degree is good, so that the column temperature of 30 ℃ is finally selected as the characteristic pattern method, and the characteristic patterns are shown in figure 3.
Example 6
Investigation of flow Rate
The characteristic patterns obtained by comparing the sample solutions of the poria, cassia and sweet soup in the embodiment 2 with the flow rates of 0.8ml/min, 1.0ml/min and 1.2 ml/min are taken as targets, and the result shows that the chromatogram has better peak shape and moderate separation degree when the flow rate is 1.0 ml/min. The flow rate was thus determined to be 1.0ml/min, see the profile shown in FIG. 4.
Example 7
Identification of chromatographic peaks
The comparison of the test solutions of the ling gui zhu gan soup described in example 2 was performed using different controls (glycyrrhizin control solution, ammonium glycyrrhizinate control solution, cinnamic acid control solution, cinnamaldehyde control solution, protocatechuic acid control solution), and it was found that peak 5 was protocatechuic acid, peak 8 was glycyrrhizin, peak 11 was cinnamic acid, peak 12 was cinnamaldehyde, peak 14 was ammonium glycyrrhizinate, see the characteristic map shown in fig. 5.
Example 8
Investigation of precision
0.5g of the freeze-dried powder of the poria, cassia, rhizoma polygonati and sweet soup is precisely weighed, a test solution is prepared according to the method described in the example 2, and is injected into a liquid chromatograph, and is continuously injected for 6 times, and the relative retention time RSD% of each characteristic peak is calculated to be less than 2%, so that the precision of the instrument is good.
Example 9
Inspection of repeatability
6 parts of the freeze-dried powder of the poria, cassia, rhizoma polygonati and sweet soup are precisely weighed, 0.5g of each freeze-dried powder of the poria, cassia, rhizoma polygonati and sweet soup is prepared according to the method described in the example 2, and the solution is injected into a liquid chromatograph, and the relative retention time RSD% of each characteristic peak is calculated to be less than 5%, so that the method has good repeatability.
Example 10
Investigation of stability
0.5g of the poria, cassia and sweet soup freeze-dried powder is precisely weighed, a test solution is prepared according to the method described in the example 2, and the relative retention time RSD% of each characteristic peak is calculated and is less than 5% after measurement at 0h,2h,4h,8h,12h and 24h respectively, so that the test solution of the test sample is stable within 24 hours.
Example 11
Investigation of durability
(one) different personnel and time investigation
Different persons (A, B) respectively and precisely weigh 0.5g of the poria, cassia and sweet soup freeze-dried powder at different times (T1 and T2), and prepare test samples for measurement. The relative retention time RSD% <5% for each characteristic peak was calculated, indicating better process stability.
(II) verification and investigation of multiple batches of Linggui Shugan Tang and Gan Jiangling Shu Tang
The characteristic spectrum method is identified for the poria cassia bark sweet soup and Gan Jiangling sweet soup of each batch, and corresponding characteristic spectrums are obtained, namely, fig. 6 and 7, wherein the serial numbers of the characteristic spectrums are LGZGTBT01, LGZGTBT02, LGZGTBT03, LGZGTBT04 and LGZGTBT05 from bottom to top, and the serial numbers of the characteristic spectrums are GJLZTBT01, GJLZTBT02, GJLZTBT03, GJLZTBT04 and GJLZTBT05 from bottom to top.
HPLC comparison of (three) Linggui Shugan Tang and Gan Jiangling Shu Tang
Respectively precisely weighing Poria, cortex Cinnamomi, rhizoma Atractylodis and rhizoma Atractylodis decoction (Gan Jiangling, rhizoma Atractylodis decoction) 0.5g, preparing corresponding test solution according to the method shown in example 2, and injecting into high performance liquid chromatograph, see characteristic diagram shown in figure 8.
As can be seen from the above-mentioned figures 1-8, the present invention establishes a method for analyzing HPLC patterns to obtain characteristic patterns of the Linggui art Gantn decoction and Gan Jiangling art Gantn decoction, 17 characteristic peaks are detected in the Linggui art Gantn decoction chromatogram, and the distinction between the Linggui art Gantn decoction and Gan Jiangling art Gantn decoction is identified by the method. As can be seen from FIG. 8, the identification between the 2 prescriptions is due to the difference of one medicine between the two prescriptions, the Ling Gui Zhu Gan Tang is composed of Poria cocos, cassia twig, rhizoma Atractylodis Macrocephalae and radix Glycyrrhizae Preparata, and the Ganzhen Jiang Lingshu Tang is composed of Poria cocos, rhizoma Zingiberis, rhizoma Atractylodis Macrocephalae and radix Glycyrrhizae Preparata. In the chromatographic chart of the poria, cassia and rhizoma coptidis decoction, the number 11 peak is more than the Gan Jiangling rhizoma to the position of about 33.882min, the number 12 peak is more than the position of about 37.111min, and the number 11 peak is identified as cinnamic acid, the number 12 peak is identified as cinnamaldehyde, and the cinnamic acid and the cinnamaldehyde belong to components in cassia twig; in addition, about 47.050min, the decoction of Ga Jiang Lingshu has 13 # more peaks than the decoction of Poria, cortex Cinnamomi and rhizoma Atractylodis, and is identified as 6-gingerol, which belongs to the ingredient in rhizoma Zingiberis. Therefore, the distinguishing points of the poria, cassia and sweet soup and the Gan Jiangling soup can be identified by constructing the characteristic spectrum, so that the defect that in the prior art, different formulas have only one medicine in composition is overcome, and the identification is performed by a characteristic spectrum method, thereby improving the prospect of the existing formulas in clinical application and saving time.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (8)
1. The construction method of the poria, cassia and rhizoma polygonati soup and Gan Jiangling and Shang Tezheng atlas is characterized by comprising the following steps:
(1) Preparing a sample solution: preparing a test solution of the poria, cassia, rhizoma atractylodis and sweet soup and a test solution of the Gan Jiangling rhizoma atractylodis soup respectively;
(2) Preparing a reference substance solution: adding methanol to constant volume to obtain reference solution;
(3) Conditions of high performance liquid chromatography: liquid chromatographic column: a C18 chromatographic column;
column temperature: 30 ℃;
detection wavelength: 270nm;
flow rate: 1.0mL/min;
feed amount: 10. Mu.L;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and the gradient elution is carried out, wherein the gradient elution process is as follows:
0 to 12min, 3 to 20 percent of mobile phase A, 97 to 80 percent of mobile phase B,
12-36 min, 20-30% of mobile phase A, 80-70% of mobile phase B,
36-45 min, 30-60% of mobile phase A, 70-40% of mobile phase B,
45-60 min, 60-85% of mobile phase A and 40-15% of mobile phase B;
(4) High performance liquid chromatography detection: and (3) respectively carrying out high performance liquid chromatography detection on the sample solution prepared in the step (1) and the reference substance solution prepared in the step (2) to obtain corresponding characteristic patterns.
2. The method for constructing the poria, cassia, rhizoma dioscoreae and sweet soup and Gan Jiangling, rhizoma dioscoreae and Shang Tezheng map according to claim 1, which is characterized in that: the preparation method of the test solution of the poria, cassia, rhizoma atractylodis and sweet soup in the step (1) comprises the following steps:
(1) Weighing 55g of poria cocos, 41g of cassia twig, 27g of bighead atractylodes rhizome and 27g of honey-fried licorice root, adding 2 liters of water, extracting for 60 minutes, extracting for 1 time, filtering with a 200-mesh screen, collecting liquid medicine, and freeze-drying by a vacuum freeze dryer to obtain freeze-dried powder of poria cocos, cassia twig and sweet soup;
(2) Precisely weighing 0.5g of the freeze-dried powder of the poria, cassia, rhizoma atractylodis and sweet soup, adding 20ml of 70% methanol, carrying out ultrasonic treatment for 30min, cooling to room temperature, shaking uniformly, and filtering to obtain the sample solution of the poria, cassia, rhizoma atractylodis and sweet soup.
3. The method for constructing the poria, cassia, rhizoma dioscoreae and sweet soup and Gan Jiangling, rhizoma dioscoreae and Shang Tezheng map according to claim 1, which is characterized in that: the step (1) is to prepare a test solution of Gan Jiangling decoction, which comprises the following steps:
(1) Weighing 55g of poria cocos, 55g of dried ginger, 27g of bighead atractylodes rhizome and 27g of honey-fried licorice root, adding 2 liters of water, extracting for 60 minutes, extracting for 1 time, filtering with a 200-mesh screen, collecting liquid medicine, and freeze-drying by a vacuum freeze dryer to obtain sweet Jiang Lingshu soup freeze-dried powder;
(2) Precisely weighing 0.5g of the sweet Jiang Lingshu soup freeze-dried powder, adding 20ml of 70% methanol, performing ultrasonic treatment for 30min, cooling to room temperature, shaking uniformly, and filtering to obtain a sweet Jiang Lingshu soup sample solution.
4. A method for constructing a decoction of poria, cassia bark, rhizoma paridis and sweet potato and Gan Jiangling, shang Tezheng map according to claim 2 or 3, wherein the method comprises the following steps: the vacuum freeze dryer lyophilization process includes pre-freezing, sublimation drying, and analytical drying.
5. The method for constructing the poria, cassia, rhizoma dioscoreae and sweet soup and Gan Jiangling, rhizoma dioscoreae and Shang Tezheng map according to claim 1, which is characterized in that: the preparation method of the mixed reference substance solution in the step (2) comprises the following steps: the glycyrrhizin, ammonium glycyrrhetate, cinnamic acid, cinnamaldehyde and protocatechuic acid reference substances are respectively precisely weighed, placed in a volumetric flask, dissolved by adding methanol, and uniformly shaken to prepare reference substance solutions containing 50 mug of each 1ml of methanol solution.
6. The identifying method of the poria, cassia, rhizoma atractylodis and sweet soup and Gan Jiangling rhizoma atractylodis soup is characterized by comprising the following steps of:
(1) Measuring characteristic patterns of the poria Gui Shugan decoction and the Gan Jiangling rhizoma Atractylodis decoction;
(2) Measuring a characteristic map of the decoction to be measured; taking the poria, cassia and sweet soup and Gan Jiangling and preparing sample solutions of the poria, cassia and sweet soup and Gan Jiangling and sweet soup respectively according to the preparation method of the sample solution in the step (1) in claim 1; respectively performing high performance liquid chromatography detection on the prepared sample solution according to the detection conditions of the step (3) in claim 1 to obtain a corresponding characteristic spectrum of the decoction to be detected;
(3) And comparing the characteristic spectrum of the decoction to be detected with the characteristic spectrum of the poria, cassia, rhizoma polygonati and rhizoma polygonati decoction and Gan Jiangling rhizoma polygonati decoction.
7. The method for identifying the poria, cassia bark and rhizoma coptidis decoction and the Gan Jiangling rhizoma to be used for treating the diarrhea according to claim 6, which is characterized in that: the characteristic spectrum of the poria, cassia and sweet soup has 17 characteristic peaks, the peak corresponding to the glycyrrhizin reference object is an S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified values of each peak are as follows in sequence: 0.225, 0.296, 0.390, 0.471, 0.567, 0.724, 0.972, 1.000, 1.216, 1.561, 1.682, 1.842, 2.113, 2.207, 2.260, 2.464, 2.603.
8. The method for identifying the poria, cassia bark and rhizoma coptidis decoction and the Gan Jiangling rhizoma to be used for treating the diarrhea according to claim 6, which is characterized in that: the characteristic spectrum of Gan Jiangling decoction contains 15 characteristic peaks, the peak corresponding to the cinnamic acid reference substance is S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of the specified value, and the specified values of each peak are as follows: 0.224, 0.297, 0.391, 0.470, 0.627, 0.722, 0.972, 1.000, 1.216, 1.560, 2.189, 2.241, 2.314, 2.444, 2.580.
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