CN110243988B - Establishment method of Zhuang medicine white flower murray HPLC fingerprint - Google Patents
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Abstract
The invention belongs to the field of traditional Chinese medicine fingerprint spectrums, and particularly relates to a method for establishing a Zhuang medicine-Baihua-muriming HPLC fingerprint spectrum. The establishment method of the Zhuang medicine white flower murray HPLC fingerprint uses caffeic acid which is a main component of the white flower murray as a reference substance, and accordingly, the Zhuang medicine white flower murray common mode fingerprint is obtained. The fingerprint spectrum has 10 chromatographic peaks, can fully reflect chemical components in the white flower murray jasminorange medicinal material, has rich information quantity and good method reproducibility, provides more powerful theoretical basis for controlling the quality of the medicinal material and identifying the quality of the medicinal material, improves the quality control and the authenticity identification level of the Zhuang medicine white flower murray jasminorange, and realizes the rapid and accurate identification of the authenticity of the white flower murray jasminorange.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the field of traditional Chinese medicine fingerprint spectrums, and in particular relates to a method for establishing a Zhuang medicine-Baihua-muriming HPLC fingerprint spectrum.
[ background of the invention ]
The flos Hibisci Murrayae is aerial part of Blumea megacephala (Randeria) Chang et Tseng of Compositae, and is produced in provinces such as Yunnan, Sichuan, Guizhou, Guangxi, Guangdong, Hunan south, Jiangxi, Fujian and Taiwan. Baihua Jiuliming is decocted in water for treating postpartum excessive bleeding in Guangxi Zhuang nationality.
The fingerprint (fingerprint) of Chinese medicine is developed by means of DNA fingerprint. The first developed is the chromatographic fingerprint of chemical components of Chinese medicine, especially the High Performance Liquid Chromatography (HPLC) fingerprint. HPLC has high resolution, and can separate complex chemical components to form peaks with different heights to form a chromatogram, and the heights and peak areas of the chromatogram peaks respectively represent various chemical components and contents thereof. The research and establishment of the traditional Chinese medicine fingerprint spectrum have important significance for improving the quality of the traditional Chinese medicine and promoting the modernization of the traditional Chinese medicine.
At present, the Zhuang medicine white flower murray jasminoides elline is mainly subjected to character identification, microscopic identification, physicochemical identification, content determination and the like, and the methods have the problems of small information amount, incomplete sample chemical component embodiment, insufficient identification component quantity and the like, so that the whole quality of the Zhuang medicine white flower murray jasminoides elline cannot be expressed. For example, the research on the chemical components of the Zhuang medicine-Baihuajiuliming [ J ] research on the chemical components of the Baihuajiuliming [ Chinese medicinal materials 2016,39(11): 2536) and 2538 ] is carried out on the effective components of the Baihuajiuliming, and the contents of the catechin, the chlorogenic acid and the caffeic acid are measured. It only uses the contents of several components as indexes to determine the quality of the medicinal materials. However, Zhuang nationality herbs (Chinese herbs) have complicated chemical components, many effective components, many unknown components, and different components or efficacies affecting each other, and any active component cannot comprehensively reflect the overall quality and curative effect of the herb, so that it is difficult to evaluate the effectiveness and specificity of Chinese herbs by only depending on one or more components.
The U.S. Food and Drug Administration (FDA) has introduced fingerprinting into quality control in the area of botanical drug substances and botanical drug products; the World Health Organization (WHO) points out in the herbal medicine evaluation guide that the mass spectrum of the botanical medicine and the effective components of the final product which cannot be identified can adopt the chromatographic fingerprint to identify the characteristic components or the mixed components, thereby ensuring the consistency of the product quality.
At present, the research on the Zhuang medicine Baihua Jiuliming fingerprint spectrum at home and abroad is not reported at all, the invention carries out high performance liquid phase fingerprint spectrum research on the Zhuang medicine Baihua Jiuliing, fills the blank under the Baihua Jiuliing quality standard item, provides basis for the quality evaluation of the Baihua Jiuliing medicinal material, and provides reference for more scientific, reasonable and normative use of the Baihua Jiuliing and improving the safety of clinical medication.
[ summary of the invention ]
The invention aims to provide a method for establishing HPLC fingerprint spectrum of Zhuang medicine white flower murrayon. The method can provide reliable basis for the identification and quality control of the white flower murraya paniculata.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for establishing HPLC fingerprint of Zhuang medicine white flower murray includes the following steps:
(1) preparation of control solutions: dissolving caffeic acid reference substance in methanol to obtain reference substance solution;
(2) preparation of a test solution: precisely weighing coarse powder of Zhuang medicine Baihua Jiuliming medicinal material, adding water, performing ultrasonic extraction, cooling to room temperature, supplementing the weight loss with water, shaking up, filtering, and taking the subsequent filtrate;
(3) high performance liquid chromatography determination: respectively and precisely sucking 10-20 mu l of each of the reference solution in the step (1) and the test solution in the step (2), injecting into a high performance liquid chromatograph, measuring, recording a chromatogram, determining a characteristic common peak by taking a caffeic acid chromatographic peak as a reference peak through analysis and comparison, and obtaining the white flower murray HPLC fingerprint;
wherein, the chromatographic conditions are as follows: c18 alkyl bonded silica gel is used as a filling agent; gradient elution is carried out by using acetonitrile as a mobile phase A and using a phosphoric acid solution as a gradient eluent consisting of a mobile phase B; the detection wavelength is 300-350 nm.
Further, the step (1) is performed as follows: the caffeic acid control substance is dissolved in methanol to obtain a control solution containing caffeic acid 20 μ g per 1 ml.
Further, the step (2) is performed as follows: precisely weighing 1g of coarse powder of the medicinal material of the Murrayae Koehne, adding 20-40ml of water, sealing, weighing, ultrasonically extracting for 20-40min, cooling to room temperature, weighing again, supplementing the weight loss with water, shaking up, filtering, and taking the subsequent filtrate.
Preferably, the step (2) is performed as follows: precisely weighing 1g of coarse powder of the medicinal material of the Murrayae Koehne, adding 20ml of water, sealing, weighing, ultrasonically extracting for 30min, cooling to room temperature, weighing again, supplementing the weight loss with water, shaking up, filtering with a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the composition.
Further, the chromatographic conditions of the step (3) are as follows: c18 alkyl bonded silica gel is used as a filling agent; gradient elution is carried out by taking acetonitrile as a mobile phase A and taking 0.1 percent phosphoric acid solution as a gradient eluent consisting of a mobile phase B; the column temperature was 25 ℃; the detection wavelength is 300-350 nm; the flow rate is 1.0 ml/min; the analysis time is 60min, and the sample amount is 10-20 μ l.
Further, elution was carried out using the following gradient elution:
TABLE 1 gradient elution schedule
The invention establishes a fingerprint of a white flower murray medicinal material, which consists of 10 common fingerprint peaks, and the relative retention time and standard deviation of the 10 common fingerprint peaks of the fingerprint are shown in the following table 2.
TABLE 2 retention time and relative standard deviation of 10 common fingerprint peaks of fingerprint
In order to improve the quality control level of the white flower muraming medicine, the inventor adopts a high performance liquid chromatography and takes caffeic acid which is a main effective component in the white flower muraming as a reference substance to develop a white flower muraming HPLC fingerprint establishment method, so that a Zhuang medicine white flower muraming common mode fingerprint is obtained, the fingerprint has 10 common chromatographic peaks, the white flower muraming medicine common mode fingerprint can be fully reflected, the fingerprint has 10 common chromatographic peaks, the chemical components in the white flower muraming can be fully reflected, the information is rich, the method reproducibility is good, and theoretical bases are provided for controlling the medicine quality and identifying the medicine quality.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
(1) the fingerprint method of the Murrayamine white flower medicinal material has the advantages of simple treatment method of a test sample, complete retention of characteristic components and stable solution of the test sample.
(2) The high performance liquid chromatography has high precision, good reproducibility and strong specificity.
(3) The standard fingerprint spectrum established by the invention has 10 common peaks, has larger information quantity and completely reserves chemical components in the test solution.
(4) Through similarity evaluation, the similarity of the fingerprint spectrums of the selected white flower muramin in different prefectures of Guangxi is greater than 0.90, and the correlation is good.
(5) The invention discloses a method for determining a fingerprint of a white flower murray yamami medicinal material, which can obtain a standard fingerprint of the white flower murray yamami medicinal material, compare whether a common peak exists or not, effectively control the quality of the white flower murray yamami medicinal material, perfect a quality evaluation system of the white flower murray yamami medicinal material and provide a theoretical and time basis for comprehensive and effective control of the quality of the white flower murray yamami medicinal material.
[ description of the drawings ]
FIG. 1 is an HPLC fingerprint of a control solution, in which: 6 is caffeic acid;
FIG. 2 is an HPLC fingerprint of a Murrayamine drug material;
FIG. 3 is an integrated overlay of HPLC fingerprints of Zhuang medicine and Murrayamine medicinal material in each batch;
FIG. 4 is an overlay chart of HPLC fingerprint precision investigation of a white flower muramin test sample;
FIG. 5 is an overlay chart of HPLC fingerprint stability investigation of white flower muramin test sample;
FIG. 6 is an overlay chart of HPLC fingerprint repeatability inspection of white flower muramin sample.
[ detailed description ] embodiments
The inventor establishes fingerprint spectrums of 13 batches of medicinal materials of the Kyunming with different sources under the condition of the same fingerprint spectrum respectively, then fuses the fingerprint spectrums, determines a common fingerprint characteristic diagram, and increases the information content of the fingerprint spectrum, thereby improving the fingerprint identification capability and realizing the comprehensive and integral evaluation of the fingerprint spectrum. The present invention is further illustrated by the following specific examples, which are provided by way of illustration only and are not intended to limit the scope of the invention.
Example 1
1 Experimental materials and instruments
1.1, medicinal materials: the production area and the recovery time are shown in Table 3.
TABLE 3 Baihua Murrayamine herb producing area and harvesting time
| Medicinal material numbering | Medicinal material source (producing area) | Time of harvest |
| 1 | Meng Shan | 2014.11 |
| 2 | Nanning peak | 2014.11 |
| 3 | Bama | 2014.11 |
| 4 | Nanning peak | 2014.03 |
| 5 | Heaven, etc | 2015.11 |
| 6 | Bama | 2015.11 |
| 7 | North current | 2015.11 |
| 8 | That slope | 2013.06 |
| 9 | Chong left | 2013.11 |
| 10 | South Ning tiger Ling | 2015.11 |
| 11 | North current | 2016.10 |
| 12 | City-proof | 2015.11 |
| 13 | Lu Chuan | 2016.10 |
1.2 Instrument: agilent1260 hplc (including G1311C quaternary pump, G1329B autosampler, G1316A column oven, G1315D DAD detector, Agilent1260 chromatography workstation (Agilent technologies, usa)); medium ultrasonic cleaners (Elma Schmidbauer GmbH, P300H); electronic balance (SQP, sydow scientific instruments ltd.); chinese medicine chromatogram fingerprint similarity evaluation system 2004 edition A (State Committee for pharmacopoeia).
1.3 reagent: caffeic acid control (purchased from the national institute for food and drug testing) lot number: 5 WZE-RZUW; mobile phase acetonitrile (available from seimer feishell science & ltd. (china)) batch No.: a998-4; phosphoric acid (purchased from shin & Fine chemical research institute in Tianjin) lot number: 25447-33-0.
2 high performance liquid chromatography
2.1 chromatographic conditions: the column was packed with octadecylsilane chemically bonded silica (Agilent column (4.6 mm. times.150 mm, 5 μm)); gradient elution was used, the elution procedure being given in Table 4
Table 4 mobile phase elution procedure
Mobile phase A: acetonitrile; mobile phase B: 0.1% phosphoric acid; the detection wavelength is 325nm, and the flow rate is 1 ml/min; the column temperature is 25 ℃; the sample amount is 10 mul; the detection time is 60min, and the theoretical plate number is not less than 3000 based on caffeic acid.
2.2 preparation of control solutions: precisely weighing 0.2mg caffeic acid into a 10ml volumetric flask, dissolving with methanol and diluting to scale, and performing ultrasonic treatment for 5 min.
2.3 preparation of test solution: precisely weighing 1g of coarse powder of the Baizhuang Baihuajiuliming for each generation, respectively placing in conical bottles with stoppers, precisely adding 20ml of water, sealing the conical bottles, weighing, ultrasonically extracting for 30min, cooling to room temperature, weighing, supplementing with water, shaking uniformly, filtering with a 0.45 mu m microporous membrane, and taking the subsequent filtrate.
2.4 determination: precisely sucking 10 μ l of each of the sample solution and the reference solution, injecting into high performance liquid chromatograph, measuring by high performance liquid chromatography, and recording 60min chromatogram (FIG. 1 and FIG. 2).
Example 2
Taking 13 batches of the Murrayamine white flower medicinal materials (medicinal material numbers 1-13), preparing a test solution according to the method under item 2.3, and measuring according to the chromatographic condition under item 2.1 and the measuring method under item 2.4 to obtain an HPLC fingerprint chromatogram superposition chart of the 13 batches of samples, which is shown in figure 3. Similarity evaluation was performed by comparing the 13 HPLC profiles to determine characteristic consensus peaks: the fingerprint has 10 characteristic common peaks, and the retention time, the average value of the peak area and the proportion of the peak area to the total peak area are summarized as follows:
peak 1, retention time 2.726min, RSD% 0.66%, peak area 156.555, RSD% 47.01%; 1.520% of the total peak area;
peak 4, retention time 10.375min, RSD% 0.31%, peak area 1340.542, RSD% 36.52%; 13.011% of the total peak area;
therefore, 10 characteristic peaks in the fingerprint of Zhuang medicine Baihua Jiuliming medicinal material, No. 6 is caffeic acid contrast peak, and the total length of the fingerprint is 60 min. Wherein, 10 peak areas are 1% of the area of a single peak and are peaks 1-13; the peak with a single peak area exceeding 5 percent of the total peak area has 4 peaks which are No. 2 peak, No. 4 peak, No. 5 peak and No. 6 peak; the peak having a single peak area exceeding 10% of the total peak area has 3 peaks, which are peak No. 4, peak No. 5, and peak No. 6.
And (3) similarity evaluation: the HPLC chromatogram is comprehensively evaluated by adopting Chinese pharmacopoeia committee recommended Chinese medicine chromatogram fingerprint similarity evaluation system software 2004A, and the similarity evaluation result is shown in a figure 3 and a table 5.
TABLE 5 evaluation of similarity of HPLC finger prints
Remarking: S1-S13 respectively represent the serial numbers of the test samples of 13 Zhuang medicine white flower murray medicinal materials.
As a result: the HPLC fingerprint similarity of 13 Zhuang medicine and Murrayamine is more than 0.90.
Example 3
3 methodology examination
3.1 precision test
Precisely sucking the same sample solution (prepared from powder of Murrayamine (1) by the method under the item "2.3"), introducing sample for 6 times under the chromatographic condition under the item "2.1" and the determination method under the item "2.4", and recording fingerprint chromatogram, as shown in FIG. 4. The relative retention time RSD value of each peak is calculated to be 0.01-0.48% by taking the No. 6 peak (caffeic acid) as a reference peak, and the relative peak area RSD value of each peak is 0.3-2.04%. The correlation coefficients are shown in Table 6.
TABLE 6 results of similarity of precision experiments
As can be seen from Table 6, the precision experiment correlation coefficients are all larger than 0.95, and the similarity measurement result shows that the instrument has good precision and meets the fingerprint detection requirement.
3.2 stability test
Precisely sucking the same test solution (prepared from the powder of the white flower muramin medicinal material with the number of No. 1 according to the method under the item '2.3'), respectively measuring for 0, 4, 6, 8, 12, 16 and 24 hours after preparation according to the chromatographic conditions under the item '2.1' and the measuring method under the item '2.4', and inspecting the repeatability of the experimental method, wherein the repeatability is shown in figure 5. Calculating to obtain the relative retention time RSD of each chromatographic peak at 0.01-0.19% and the relative peak area RSD of each peak at 0.05-1.96% by taking the No. 6 peak (caffeic acid) as a reference peak. The correlation coefficients are shown in Table 7.
Table 7 stability test similarity results
As can be seen from Table 7, the correlation coefficient of each fingerprint is greater than 0.95, and the result shows that the sample solution has stable components within 24 hours and meets the fingerprint detection requirements.
3.3 repeatability test
Taking the powder of the white flower murrayon medicinal material with the number of No. 1, preparing 6 parts according to the method under the item "2.3", measuring according to the chromatographic condition under the item "2.1" and the measuring method under the item "2.4", and inspecting the repeatability of the experimental method, as shown in figure 6. Calculating to obtain the relative retention time RSD of each chromatographic peak at 0.04-0.21% and the relative peak area RSD of each peak at 0.66-1.74% by taking the No. 6 peak (caffeic acid) as a reference peak. The correlation coefficients are shown in Table 8.
TABLE 8 repeatability test similarity results
As can be seen from Table 8, the correlation coefficients of the repeatability tests are all larger than 0.95, and the similarity calculation result shows that the method has good repeatability and meets the requirement of fingerprint detection.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (2)
1. The establishment method of the HPLC fingerprint of Zhuang medicine white flower murrayon is characterized by comprising the following steps:
(1) preparation of control solutions: dissolving caffeic acid control in methanol to obtain control solution containing caffeic acid 20 μ g per 1 ml;
(2) preparation of a test solution: precisely weighing 1g of coarse powder of the Murraya koenigii, adding 20-40ml of water, sealing, weighing, ultrasonically extracting for 20-40min, cooling to room temperature, weighing again, supplementing the weight loss with water, shaking up, filtering, and taking the subsequent filtrate;
(3) high performance liquid chromatography determination: respectively and precisely sucking 10-20 mu l of each of the reference solution in the step (1) and the test solution in the step (2), injecting into a high performance liquid chromatograph, measuring, recording a chromatogram, determining a characteristic common peak by taking a caffeic acid chromatographic peak as a reference peak through analysis and comparison, and obtaining the white flower murray HPLC fingerprint;
wherein, the chromatographic conditions are as follows: c18 alkyl bonded silica gel is used as a filling agent; gradient elution is carried out by taking acetonitrile as a mobile phase A and taking 0.1 percent phosphoric acid solution as a gradient eluent consisting of a mobile phase B;
the gradient elution program is set as a time gradient of 0min → 5min → 10min → 25min → 40min → 60 min; mobile phase a volume percent gradient 8% → 11% → 10% → 21% → 24% → 30%, mobile phase B volume percent gradient 92% → 89% → 90% → 79% → 76% → 70%;
the column temperature was 25 ℃; the detection wavelength is 300-350 nm; the flow rate is 1.0 ml/min; the analysis time is 60min, and the sample amount is 10-20 μ l;
the fingerprint comprises 10 common peaks, and retention time is 2.726min, 3.483min, 4.599min, 10.375min, 17.489min, 19.26min, 25.687min, 34.223min, 36.628min and 37.756 min.
2. The method for establishing HPLC fingerprint of Zhuang medicine white flower murimine as claimed in claim 1, wherein the step (2) is performed according to the following operations: precisely weighing 1g of coarse powder of the medicinal material of the Murrayae Koehne, adding 20ml of water, sealing, weighing, ultrasonically extracting for 30min, cooling to room temperature, weighing again, supplementing the weight loss with water, shaking up, filtering with a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the composition.
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