CN114441662A - Characteristic spectrum construction method and identification method of poria cocos, cassia twig, rhizoma atractylodis and rhizoma atractylodis decoction - Google Patents
Characteristic spectrum construction method and identification method of poria cocos, cassia twig, rhizoma atractylodis and rhizoma atractylodis decoction Download PDFInfo
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Abstract
The invention discloses a construction method and an identification method of a characteristic spectrum of a poria cocos, cassia twig, rhizoma atractylodis and rhizoma atractylodis decoction, which are characterized by comprising the following steps: (1) respectively preparing a test solution of the tuckahoe, cinnamon, atractylodes rhizome and licorice decoction and a test solution of the gan ginger, tuckahoe and atractylodes decoction; (2) adding methanol to a constant volume to obtain a reference substance solution, wherein the reference substance solution comprises liquiritin, ammonium glycyrrhizinate, cinnamic acid, cinnamaldehyde and protocatechuic acid; (3) and high performance liquid chromatography conditions: liquid chromatography column: a C18 chromatography column; column temperature: 25-35 ℃; detection wavelength: 190-400 nm; flow rate: 0.8mL/min to 1.2 mL/min; feeding amount: 5-20 mul; the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and gradient elution is carried out; (4) and (3) respectively carrying out high performance liquid chromatography detection on the test solution prepared in the step (1) and the reference solution prepared in the step (2) to obtain corresponding characteristic maps.
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 characteristic spectrums of a poria cocos-cassia twig-rhizoma atractylodis-rhizoma glycyrrhizae decoction and a rhizoma zingiberis-rhizoma atractylodis-rhizoma decoction.
Background
The formula of Ling Gui Zhu gan Tang is from Zhang Zhongjing treatise on exogenous febrile diseases. Item 67 cloud of the book: for vomiting and diarrhea, heart-downward fullness, qi-upward rushing to the chest, rising head , deep and tense pulse, and sweating, meridian movement, it is the main herb of "Fu Ling Gui Zhi Bai Zhi gan Cao Tang". The 'golden lack essentials' also carries the prescription, which is recorded as: the recipe is mainly based on Lianggui Zhu gan Tang, which is marked by phlegm-fluid retention in the lower heart, fullness in the chest and hypochondrium, and dizziness. The recipe is designed for the syndrome of insufficiency of middle-jiao yang and retention of water and fluid, and the pathogenic factors of the syndrome of cold vomiting after discharge or retention of phlegm and fluid in the body caused by miscellaneous diseases are the yang deficiency of the middle-jiao, the dysfunction of the spleen in transportation and the retention of water and fluid in the body. The decoction prescription of gan Jiang Ling Zhu is from the syndrome of accumulation of wind-cold in the five zang organs and the syndrome of deficiency of the jin Kui Yao (slightly deficient), and the combination treatment of diseases and pulses. Original book cloud: for kidney-attachment diseases, the body is heavy, the waist is cold, such as sitting in water, the body is water-like, the body is not thirst, the urine is free, the diet is so marked, the disease belongs to the lower energizer, the body is overstrain and sweats, the clothes are cold and wet, the waist is cold and painful, the abdomen is heavy, such as with five thousand of money, and gan Jiang Zhi Shu Tang is mainly used. The recipe is designed to treat kidney stagnation caused by the invasion of pathogenic cold-dampness and obstruction of the waist. Compared with the traditional Chinese medicine, the gan Jiang Ling Zhu Tang is poor in medicine, but the Ling Gui Zhu Gancao Tang is mainly a prescription for warming yang and promoting diuresis and resolving dampness, and is mainly a water-dispelling decoction, while the gan Jiang Zhi Zhu Tang is mainly a prescription for warming spleen and stomach and dispelling cold and dampness, and is mainly a prescription for dispelling cold and dampness. At present, the establishment of characteristic spectrums of the tuckahoe, cinnamon, atractylodes, rhizoma atractylodis macrocephalae and the gan-ginger-poria, atractylodes, rhizoma atractylodis macrocephalae soup and the identification method of the tuckahoe, cinnamon, rhizoma atractylodis macrocephalae and the gan-ginger-poria, atractylodes, rhizoma atractylodis macrocephalae soup are lacked.
Disclosure of Invention
In order to solve the problems, the invention provides a construction method and an identification method of a characteristic spectrum of a poria cocos-cassia twig-rhizoma atractylodis-macrocephalae decoction and a rhizoma zingiberis-rhizoma atractylodis-macrocephalae decoction.
The purpose of the invention is realized by the following technical scheme:
a method for constructing a characteristic spectrum of a poria cocos, cassia twig, rhizoma atractylodis macrocephalae and rhizoma zingiberis, rhizoma atractylodis macrocephalae soup is characterized by comprising the following steps:
(1) and preparing a test solution: respectively preparing a test solution of the LINGGUIZHGAN decoction and a test solution of the GANJIANGLINGZHU decoction;
(2) and preparing a reference substance solution: taking liquiritin, ammonium glycyrrhizinate, cinnamic acid, cinnamaldehyde and protocatechuic acid reference substances, adding methanol to a constant volume to obtain a reference substance solution;
(3) and high performance liquid chromatography conditions: liquid chromatography column: a C18 chromatography column;
column temperature: 25-35 ℃;
detection wavelength: 190-400 nm;
flow rate: 0.8mL/min to 1.2 mL/min;
feeding amount: 5-20 mul;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and gradient elution is carried out;
(4) and high performance liquid chromatography detection: and (3) respectively carrying out high performance liquid chromatography detection on the test solution prepared in the step (1) and the reference solution prepared in the step (2) to obtain corresponding characteristic maps.
Preferably, the preparation of the sample solution of the linggui zhu gan decoction 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 by using a 200-mesh screen, collecting liquid medicine, and freeze-drying by using a vacuum freeze-drying machine to obtain freeze-dried powder of poria cocos, cassia twig, rhizoma atractylodis macrocephalae and licorice root decoction;
(2) accurately weighing 0.5g of the lyophilized powder of the Linggui Atractylodes sweet soup, adding 20ml of 70 percent methanol, treating for 30min by ultrasonic waves (power 600W and frequency 40 kHz), cooling to room temperature, shaking up, and filtering to obtain the test solution of the Linggui Atractylodes sweet soup.
Preferably, the preparation of the sample solution of gan jiang ling shu 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 by using a 200-mesh screen, collecting liquid medicine, and freeze-drying by using a vacuum freeze-drying machine to obtain freeze-dried powder of the decoction of the rhizoma zingiberis zeylanicae and the rhizoma atractylodis macrocephalae;
(2) accurately weighing 0.5g of the gan-ginger-poria-surgery-decoction freeze-dried powder, adding 20ml of 70% methanol, treating for 30min by using ultrasonic waves (power 600W and frequency 40 kHz), cooling to room temperature, shaking uniformly, and filtering to obtain a gan-ginger-poria-surgery-decoction test solution.
Preferably, the vacuum freeze dryer freeze drying procedure comprises prefreezing, sublimation drying and analytical drying.
Preferably, the preparation method of the mixed control solution in the step (2) comprises the following steps: precisely weighing liquiritin, ammonium glycyrrhizinate, cinnamic acid, cinnamaldehyde and protocatechuic acid reference substances respectively, placing in a volumetric flask, adding methanol to dissolve, shaking uniformly, and making into reference substance solutions containing 50 μ g of each of 1ml of methanol solution.
Preferably, the high performance liquid chromatography conditions of step (3) are as follows:
column temperature: 30 ℃;
detection wavelength: 270 nm;
flow rate: 1.0 mL/min;
feeding amount: 10 mu L of the solution;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and the gradient elution process comprises the following steps:
0-12 min, 3-20% of mobile phase A, 97-80% of mobile phase B,
12-36 min, 20-30% of mobile phase A, 80-70% of mobile phase B,
36-45 min, the mobile phase A is 30-60%, the mobile phase B is 70-40%,
45-60 min, the mobile phase A is 60-85%, and the mobile phase B is 40-15%.
The identification method of the tuckahoe, cinnamon, rhizoma atractylodis and rhizoma atractylodis soup is characterized by comprising the following steps:
(1) determining the characteristic maps of the tuckahoe, cassia twig, atractylodes rhizome, liquorice root, rhizoma zingiberis, rhizoma atractylodis macrocephalae and rhizoma atractylodis macrocephalae soup;
(2) determining a characteristic map of the decoction to be detected;
(3) and comparing the characteristic spectrum of the decoction to be detected with the characteristic spectrums of the tuckahoe, cinnamon, atractylodes rhizome and licorice decoction and the gan ginger, tuckahoe and atractylodes rhizome decoction.
Preferably, the characteristic map of the linggui zhu gan tang has 17 characteristic peaks, the peak corresponding to the liquiritin reference substance is the 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 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 map of the gan Jiang Ling Zhu Tang has 15 characteristic peaks, the peak corresponding to the cinnamic acid reference substance 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 as follows: 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 maps of the tuckahoe, cinnamon, rhizoma atractylodis and rhizoma atractylodis soup, and realizes the quick identification of the tuckahoe, cinnamon, rhizoma atractylodis and rhizoma atractylodis soup through the method of the characteristic maps, and the method is simple.
(2) The invention firstly provides the identification of the characteristic spectrum methods of the two formulas, and particularly distinguishes the characteristic spectrums of the tuckahoe, cinnamon, atractylodes rhizome and licorice root, poria cocos and atractylodes rhizome soup, so that the quick identification of the two formulas can be realized.
(3) The method adopts gradient elution for determination by the high performance liquid chromatography, and has the advantages of simple operation, good separation degree, high precision, good repeatability, good stability and the like.
(4) The method firstly provides the identification method of the poria, cassia, rhizoma atractylodis and rhizoma zingiberis, rhizoma atractylodis and rhizoma atractylodis soup, and under the specific detection condition, the characteristic spectrum method adopted by the poria, cassia, rhizoma atractylodis and rhizoma zingiberis, rhizoma atractylodis and rhizoma atractylodis soup can be identified by taking the liquiritin chromatographic peak as a reference substance S peak.
Drawings
FIG. 1 is a chromatogram of Linggui shugan decoction at different wavelengths;
FIG. 2 is a chromatogram of Linggui shugan decoction at different feed rates;
FIG. 3 is a chromatogram of Linggui shugan decoction at different column temperatures;
FIG. 4 is a chromatogram of Linggui shugan decoction at different flow rates;
FIG. 5 shows the identification of chromatographic peak of Linggui Shugan Tang;
FIG. 6 is a chromatogram of different batches of LINGGUISHUGAN decoction;
FIG. 7 is a chromatogram of different batches of Ganjang Lingzhu decoction;
FIG. 8 is a chromatogram of LINGGUIZHANG decoction and GANJIANGLINGZHU decoction.
Detailed Description
Laboratory instruments and materials
The following examples relate to the following test instruments and materials (all in mass concentration):
high performance liquid chromatograph: agilent 1260 type high performance liquid chromatograph
An electronic balance: ME204E/02, XP26 (Mettler-Tollido instruments, Inc.);
an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);
an ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);
a chromatographic column: agilent 5TC-C18(2) 250X 4.6mm 5 um;
acetonitrile and phosphoric acid are chromatographically pure, water is ultrapure water, and other reagents are analytically pure;
liquiritin (China institute for testing and testing food and drug, lot number: 111610-;
ammonium glycyrrhizinate (China institute for testing and testing food and drug; batch No. 110731-containing 202021, content is 96.2%);
cinnamic acid (China food and drug testing institute, batch number 110786-201604, content 98.8%);
protocatechuic acid (China institute for testing and testing food and drug; lot number 110809-;
cinnamaldehyde (China institute for testing and drug testing of food and drug, batch number 110710-201821, content in 99.6%);
poria cocos (batch No.: 010145-1912001);
ramulus Cinnamomi (batch No. 010182- > 2008001);
atractylodes macrocephala Koidz (batch No. 010022-1905001);
radix Glycyrrhizae Preparata (batch No. 010154-;
zingiberis rhizoma (batch: XLS 20200807);
5 batches of LINGGUIZHUGAN decoction (LGZGTBT 01, LGZGTBT02, LGZGTBT03, LGZGTBT04, LGZGTBT 05)
Gan Jiang Ling Zhu Tang (GJLZTBT01, GJLZTBT02, GJLZTBT03, GJLZTBT04, GJLZTBT 05) in 5 batches.
Example 1
The present embodiment relates to linggui zhu gan tang and gan jiang ling zhu tang.
(1) The preparation method of the 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 by using a 200-mesh screen, collecting liquid medicine, and freeze-drying by using a vacuum freeze-drying machine to obtain freeze-dried powder of poria cocos, cassia twig, rhizoma atractylodis macrocephalae and licorice root decoction;
the freezing procedure for freeze-drying to lyophilized powder is shown in the following table
Precisely weighing 0.5g of the lyophilized powder of the Linggui shugan decoction, adding 20ml of 70% methanol, treating for 30min by ultrasonic treatment (power 600W and frequency 40 kHz), cooling to room temperature, shaking up, and filtering to obtain the sample solution of the Linggui shugan decoction.
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 for 1 time, filtering by using a 200-mesh screen, collecting liquid medicine, and freeze-drying by using a vacuum freeze-drying machine to obtain freeze-dried ganjiang and poria cocos decoction powder;
the freezing procedure for freeze-drying to lyophilized powder is shown in the following table
Accurately weighing 0.5g of rhizoma Zingiberis recens-rhizoma Atractylodis decoction lyophilized powder, adding 20ml of 70% methanol, treating with ultrasound (power 600W, frequency 40 kHz) for 30min, cooling to room temperature, shaking, and filtering to obtain rhizoma Zingiberis recens-rhizoma Atractylodis decoction sample solution.
(2) Preparation of a reference solution: precisely weighing liquiritin, ammonium glycyrrhizinate, cinnamic acid, cinnamaldehyde and protocatechuic acid reference substances respectively, placing in a volumetric flask, adding methanol to dissolve, shaking uniformly, and making into reference substance solutions containing 50 μ g of each of 1ml of methanol solution.
(3) And (3) respectively carrying out high performance liquid chromatography detection on the test solution prepared in the step (1) and the reference solution prepared in the step (2) to obtain corresponding characteristic maps.
The detection conditions of the high performance liquid chromatography meet the following conditions:
liquid chromatography column: a C18 chromatography column;
column temperature: 30 ℃;
detection wavelength: 270 nm;
flow rate: 1.0 mL/min;
feeding amount: 10 mu L of the solution;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and the gradient elution process comprises the following steps:
0-12 min, 3-20% of mobile phase A, 97-80% 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, the mobile phase A is 60-85%, and the mobile phase B is 40-15%.
(4) And (4) taking the characteristic map of the reference substance solution in the step (3) as a reference map, selecting common peaks from the characteristic maps of the test substance solution, and constructing the characteristic maps of the poria cocos, cassia twig, rhizoma atractylodis and rhizoma atractylodis decoction and the rhizoma zingiberis, rhizoma atractylodis decoction.
The constructed characteristic spectrum of the tuckahoe, cinnamon, atractylodes and licorice decoction has 17 characteristic peaks, wherein the peak corresponding to the liquiritin reference substance is the S peak. The relative retention time of each characteristic peak to the S peak is calculated and should be within ± 10% of the specified value. The specified values are: 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 gan Jiang Ling Zhu Tang has 15 characteristic peaks, wherein the peak corresponding to the ferulic acid reference substance is the S peak. The relative retention time of each characteristic peak to the S peak is calculated and should be within ± 10% of the specified value. The specified values are: 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 present embodiment relates to the identification of lingzi zhu gan tang and gan jiang lingzhu tang.
(1) Taking the poria, cassia, rhizoma atractylodis and rhizoma atractylodis decoction and the gan, rhizoma zingiberis and rhizoma atractylodis decoction, and preparing sample solutions of the poria, cassia, rhizoma atractylodis and rhizoma atractylodis decoction and the gan, rhizoma atractylodis and rhizoma atractylodis decoction respectively according to the preparation method of the test solution in the step (1) of the example 1;
(2) respectively carrying out high performance liquid chromatography detection on the sample solution prepared in the step (1) according to the detection conditions of the step (3) in the embodiment 1 to obtain corresponding characteristic maps to be identified;
(3) and (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 the characteristic spectrum to be identified 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 linggui zhu gan tang described in example 2 as an object, performing full-wavelength scanning in the range of 190-400 nm by using a DAD detector, and finding that the chromatographic peak information amount is larger when the detection wavelength is 270nm and the chromatogram baseline is more stable, so that the detection wavelength is determined to be 270nm, see the characteristic map shown in fig. 1.
Example 4
Investigation of sample size
By taking the sample solution of the linggui zhu gan tang described in example 2 as an object, comparing the characteristic maps obtained by the sample volumes of 5 microliters, 10 microliters and 20 microliters, the chromatogram peaks are symmetrical under each sample volume condition, and the separation degree is good, so that the sample volume of 10 microliters is finally selected as the sample volume of the characteristic map method, which is shown in fig. 2.
Example 5
Investigation of column temperature
Taking the sample solution of the linggui zhu gan tang described in example 2 as the target, comparing the characteristic maps obtained at 25 ℃, 30 ℃ and 35 ℃, the chromatogram peaks are symmetrical and the separation degree is good under each column temperature condition, so that 30 ℃ is finally selected as the column temperature of the characteristic map method, see the characteristic map shown in fig. 3.
Example 6
Investigation of flow Rate
By comparing the characteristic maps obtained at flow rates of 0.8ml/min, 1.0ml/min and 1.2 ml/min with the sample solution of the LINGGUISHUGAN decoction described in example 2, the results show that the chromatogram has a good peak shape and a moderate degree of separation at a flow rate of 1.0 ml/min. Therefore, the flow rate was determined to be 1.0ml/min, see the characteristic map shown in FIG. 4.
Example 7
Identification of chromatographic peaks
Taking the sample solution of the linggui zhu gan tang described in example 2 as the subject, different reference substances (liquiritin reference substance solution, ammonium glycyrrhizinate reference substance solution, cinnamic acid reference substance solution, cinnamaldehyde reference substance solution, protocatechuic acid reference substance solution) were used for comparison, and it was found that peak No. 5 was protocatechuic acid, peak No. 8 was liquiritin, peak No. 11 was cinnamic acid, peak No. 12 was cinnamaldehyde, and peak No. 14 was ammonium glycyrrhizinate, as shown in the characteristic map shown in fig. 5.
Example 8
Examination of precision
0.5g of poria cocos, cassia twig and rhizoma atractylodis decoction freeze-dried powder is precisely weighed, a sample solution is prepared according to the method in the embodiment 2, the sample solution is injected into a liquid chromatograph for 6 times of continuous sample injection, and the RSD% of each characteristic peak relative retention time is calculated to be less than 2%, so that the instrument precision is good.
Example 9
Examination of repeatability
Accurately weighing 0.5g of 6 parts of poria, cassia twig and rhizoma atractylodis and licorice decoction freeze-dried powder respectively, preparing a test solution according to the method in the embodiment 2, injecting the test solution into a liquid chromatograph, and calculating the relative retention time RSD% of each characteristic peak to be less than 5%, which indicates that the method has good repeatability.
Example 10
Investigation of stability
0.5g of poria cocos, cassia twig, rhizoma atractylodis and licorice root decoction freeze-dried powder is precisely weighed, the test solution is prepared according to the method in the embodiment 2, the test solution is respectively measured in 0h, 2h, 4h, 8h, 12h and 24h, and the RSD% of each characteristic peak is calculated to be less than 5%, so that the test solution of the test sample is stable in 24 hours.
Example 11
Investigation of durability
(one) different personnel and time investigation
Different persons (A, B) precisely weigh 0.5g of the Lianggui zhu gan tang lyophilized powder at different times (T1, T2), and prepare two parts of the powder to be tested for determination. And calculating the relative retention time RSD% of each characteristic peak to be less than 5%, which indicates that the method has better stability.
(II) verification and investigation of multiple batches of LINGGUIZHANG decoction and GANJIANGLINGZHU decoction
Identifying the characteristic map method of each batch of the poria cocos, cassia twig, rhizoma atractylodis macrocephalae and rhizoma zingiberis cocos, rhizoma atractylodis macrocephalae soup to obtain corresponding characteristic maps, namely fig. 6 and fig. 7, wherein the fig. 6 is numbered LGZGTBT01, LGZGTBT02, LGZGTBT03, LGZGTBT04 and LGZGTBT05 from bottom to top, and the fig. 7 is numbered GJLZTBT01, GJLZTBT02, GJLZTBT03, GJLZTBT04 and GJLZTBT05 from bottom to top.
HPLC comparison of (III) Linggui-Zhu-gan decoction with gan Jiang-Ling-Zhu decoction
0.5g of the LINGGUIZHANG decoction and 0.5g of the GANJIANGLIZHI decoction are precisely weighed respectively, the corresponding test sample solutions are prepared according to the method shown in the example 2, and are injected into a high performance liquid chromatograph, and the characteristic map shown in the figure 8 is referred.
As can be seen from the above fig. 1 to 8, the present invention establishes a method for establishing a characteristic chromatogram of the linggui zhu gan tang and the gan jiang zhu tang by analyzing HPLC chromatogram, detects 17 characteristic peaks in the linggui zhu gan tang chromatogram, and identifies the difference between the linggui zhu gan tang and the gan jiang zhu tang by the method. As can be seen from FIG. 8, the identification of the 2 prescriptions is due to the difference of one herb between the two prescriptions, wherein the LINGGUIZHANG decoction is prepared from Poria, ramulus Cinnamomi, Atractylodis rhizoma, and radix Glycyrrhizae Preparata, and the GANJIANGLINGZHU decoction is prepared from Poria, Zingiberis rhizoma, Atractylodis rhizoma, and radix Glycyrrhizae Preparata. Compared with Ganyang Lingzhu decoction, the chromatogram of Linggui Ganyu decoction has 11-peak at about 33.882min and 12-peak at about 37.111min, and the 11-peak is identified as cinnamic acid, the 12-peak is cinnamaldehyde, and the cinnamic acid and cinnamaldehyde belong to the ingredients in ramulus Cinnamomi; in addition, around 47.050min, gan Jiang Ling Zhu Tang has 13-th peak more than Ling Gui Zhu Gan Tang, and is identified as 6-gingerol as the component in dried ginger. Therefore, the characteristic map is constructed to identify the difference point between the Guizhu Ganju decoction and the gan Jiangzi Ganju decoction, so that the difference of only one medicine in the composition of different prescriptions in the prior art is compensated, the identification is carried out by a characteristic map method, the prospect of the prior prescriptions in clinical application is improved, and the time is saved.
The above-mentioned embodiments are further described in detail for the purpose of illustrating the invention, the technical solutions and the advantages, it should be understood that the above-mentioned embodiments are only exemplary of the invention, and are not intended to limit the invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the invention should be included in the protection scope of the invention.
Claims (9)
1. A method for constructing a characteristic spectrum of a poria cocos, cassia twig, rhizoma atractylodis macrocephalae and rhizoma zingiberis, rhizoma atractylodis macrocephalae soup is characterized by comprising the following steps:
(1) and preparing a test solution: respectively preparing a test solution of the LINGGUIZHGAN decoction and a test solution of the GANJIANGLINGZHU decoction;
(2) and preparing a reference substance solution: taking liquiritin, ammonium glycyrrhizinate, cinnamic acid, cinnamaldehyde and protocatechuic acid reference substances, adding methanol to a constant volume to obtain a reference substance solution;
(3) and high performance liquid chromatography conditions: liquid chromatography column: a C18 chromatography column;
column temperature: 25-35 ℃;
detection wavelength: 190-400 nm;
flow rate: 0.8mL/min to 1.2 mL/min;
feeding amount: 5-20 mu L;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and gradient elution is carried out;
(4) and detecting by high performance liquid chromatography: and (3) respectively carrying out high performance liquid chromatography detection on the test solution prepared in the step (1) and the reference solution prepared in the step (2) to obtain corresponding characteristic maps.
2. The method for constructing the characteristic maps of the poria cocos, cassia twig, rhizoma atractylodis macrocephalae and the rhizoma zingiberis, rhizoma atractylodis macrocephalae soup as claimed in claim 1, is characterized in that: the preparation of the sample solution of the tuckahoe, cinnamon, atractylodes and licorice decoction 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 for 1 time, filtering by using a 200-mesh screen, collecting liquid medicine, and freeze-drying by using a vacuum freeze-dryer to obtain freeze-dried powder of poria cocos, cassia twig, rhizoma atractylodis macrocephalae and licorice root decoction;
(2) precisely weighing 0.5g of the poria cocos, cassia twig and rhizoma atractylodis macrocephalae decoction freeze-dried powder, adding 20ml of 70% methanol, carrying out ultrasonic treatment for 30min, cooling to room temperature, shaking up, and filtering to obtain the poria cocos, cassia twig, rhizoma atractylodis macrocephalae decoction test solution.
3. The method for constructing the characteristic spectrums of the Linggui Zhigan Tang and the gan Jiang Zhi Tang according to claim 1 is characterized in that: the method for preparing the sample solution of the gan ginger and poria cocos-rhizoma atractylodis soup 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 by using a 200-mesh screen, collecting liquid medicine, and freeze-drying by using a vacuum freeze-drying machine to obtain freeze-dried powder of the decoction of the rhizoma zingiberis zeylanicae and the rhizoma atractylodis macrocephalae;
(2) accurately weighing 0.5g of the freeze-dried powder of the gan-ginger-poria-bighead atractylodes rhizome decoction, adding 20ml of 70% methanol, carrying out ultrasonic treatment for 30min, cooling to room temperature, shaking uniformly, and filtering to obtain a test solution of the gan-ginger-poria-bighead atractylodes rhizome decoction.
4. The method for constructing the feature maps of the LINGGUIZHANG decoction and the GANJIANGLIZHI decoction according to the claims 2 or 3, which is characterized in that: the freeze-drying procedure of the vacuum freeze-drying machine comprises prefreezing, sublimation drying and analytical drying.
5. The method for constructing the characteristic maps of the poria cocos, cassia twig, rhizoma atractylodis macrocephalae and the rhizoma zingiberis, rhizoma atractylodis macrocephalae soup as claimed in claim 1, is characterized in that: the preparation method of the mixed reference solution in the step (2) comprises the following steps: precisely weighing liquiritin, ammonium glycyrrhizinate, cinnamic acid, cinnamaldehyde and protocatechuic acid reference substances respectively, placing in a volumetric flask, adding methanol to dissolve, shaking uniformly, and making into reference substance solutions containing 50 μ g of each of 1ml of methanol solution.
6. The method for constructing the characteristic maps of the poria cocos, cassia twig, rhizoma atractylodis macrocephalae and the rhizoma zingiberis, rhizoma atractylodis macrocephalae soup as claimed in claim 1, is characterized in that: the conditions of the high performance liquid chromatography in the step (3) are as follows:
column temperature: 30 ℃;
detection wavelength: 270 nm;
flow rate: 1.0 mL/min;
feeding amount: 10 mu L of the solution;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 phosphoric acid, and the gradient elution process comprises the following steps:
0-12 min, 3-20% of mobile phase A, 97-80% of mobile phase B,
12-36 min, 20-30% of mobile phase A, 80-70% of mobile phase B,
36-45 min, the mobile phase A is 30-60%, the mobile phase B is 70-40%,
45-60 min, the mobile phase A is 60-85%, and the mobile phase B is 40-15%.
7. The identification method of the tuckahoe, cinnamon, rhizoma atractylodis and rhizoma atractylodis soup is characterized by comprising the following steps:
(1) determining the characteristic maps of the tuckahoe, cassia twig, atractylodes rhizome, liquorice root, rhizoma zingiberis, rhizoma atractylodis macrocephalae and rhizoma atractylodis macrocephalae soup;
(2) determining a characteristic map of the decoction to be detected;
(3) and comparing the characteristic spectrum of the decoction to be detected with the characteristic spectrums of the tuckahoe, cinnamon, atractylodes rhizome and licorice decoction and the gan ginger, tuckahoe and atractylodes rhizome decoction.
8. The method for identifying Linggui shugan Tang and gan Jiang ling shu Tang as claimed in claim 7, wherein: the characteristic map of the tuckahoe, cassia twig, atractylodes rhizome and licorice root decoction has 17 characteristic peaks, the peak corresponding to the liquiritin reference substance 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 the peaks are as follows: 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.
9. The method for identifying Linggui shugan Tang and gan Jiang ling shu Tang as claimed in claim 7, wherein: the characteristic spectrum of the gan Jiang Ling Zhu Tang has 15 characteristic peaks, the peak corresponding to the cinnamic acid reference substance 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 the peaks 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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