CN116124940A - Method for establishing finger print of Wei-medicine hard acutus herb - Google Patents

Method for establishing finger print of Wei-medicine hard acutus herb Download PDF

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CN116124940A
CN116124940A CN202310058193.5A CN202310058193A CN116124940A CN 116124940 A CN116124940 A CN 116124940A CN 202310058193 A CN202310058193 A CN 202310058193A CN 116124940 A CN116124940 A CN 116124940A
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establishing
peak
hard
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finger print
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刘永利
朱靖
赵振霞
段琼
孔亚萍
苏建
王常顺
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Hebei Institute for Drug and Medical Device Control
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G—PHYSICS
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Abstract

The invention relates to a method for establishing a finger print of hard-tip vanilla, belonging to the technical field of Min's medicine. In particular to a fingerprint spectrum measuring method for establishing 18 characteristic peaks including components such as new chlorogenic acid, geraniin, rosmarinic acid, salvianolic acid B, linarin and the like in the lysimachia foetida by adopting an HPLC method. In the method for establishing the fingerprint spectrum, 328nm is selected as the measurement wavelength; acetonitrile-0.1% phosphoric acid is used as a mobile phase, and gradient elution is carried out; preferably 70% methanol is used as extraction solvent, and heating reflux is performed for 40 min. The established method can accurately reflect the whole chemical characteristics of the hard-tipped vanilla, is simple and quick, and provides scientific basis for comprehensively improving the quality of the hard-tipped vanilla.

Description

Method for establishing finger print of Wei-medicine hard acutus herb
Technical Field
The invention belongs to the technical field of ethnic medicines, and particularly relates to a method for establishing a finger print of a medicinal herb of hard acutus.
Background
The hard-tipped vanilla is the dry aerial part of the hard-tipped vanilla (Hyssopus cuspidatus Boiss.) belonging to the genus of the family Labiatae, is perennial herb or sub-shrub, is mainly produced in the Archaete area in the North of Xinjiang, is a medicinal material commonly used by Uygur medical science, and is commonly named as Shenxiang, the Uygur-character 'ancestor' of the Shenxiang has the characteristics of dry heat, strong fragrance, has the effects of relieving cough and reducing sputum, relieving asthma and benefiting lung, and is often used for treating cough, cold, asthma and the like. Modern pharmacological researches have shown that the herb of the common sage herb has various pharmacological effects of anti-inflammatory, antioxidant, blood sugar reducing, antibacterial and the like, and is mainly used for treating cough, asthma and other symptoms through the anti-inflammatory and antibacterial effects.
According to the records of modern national medical monograph at home and abroad, the medicinal materials of 'ancestral' are herba schizonepetae and Shenxiang grass plants, the overground parts are mainly used as medicines, and each document mainly uses Shenxiang Hysspous officinais L, shangjianshenxiang Hyssopus cuspidatus Boiss and Nepeta break beta Beth of large-bud herba schizonepetae as genuine products, but the raw materials or substitutes in different areas exist. In Chinese literature such as Xinjiang traditional Chinese medicine, uygur medicine upper book and Xinjiang plant book, the primordium of the 'ancestral' is hard tip herb (Hyssopus cuspidatus Boiss.) which is mainly wild in the initial stage of application, but in recent years, due to shortage of resources, market supply is insufficient, and at present, hard tip herb (Hyssopus cuspidatus Boiss.) which is cultivated by large-area introduction in Xinjiang area of China is mainly introduced into the Xinjiang, and according to investigation, the introduction sources of the Chinese literature are mainly two types of the Xinjiang herb, namely the introduction of the Xinjiang herb, and the introduction of the Chinese herb is foreign type. The existing quality standard of Xinjiang traditional Chinese medicine Uighur medicine decoction piece processing standards (2010 edition) only contains items such as characters, microscopic identification, inspection and the like, the standard is relatively simple, the purpose of effectively controlling the quality of products cannot be achieved, and in view of the problems, in order to better evaluate the quality of the variety, related research work is carried out on the variety.
Disclosure of Invention
The invention aims to provide a determination method for analyzing the finger print of the lysimachia foenum-graecum hance, and establishes a finger print determination method for 18 characteristic peaks comprising components such as chlorogenic acid, dioscin, rosmarinic acid, salvianolic acid B, linarin and the like by adopting an HPLC method.
In order to solve the problems, the invention adopts the following technical scheme: a method for establishing a finger print of a Wei-medicinal hard jianshen herb comprises the following steps:
a. preparing a mixed reference substance solution;
b. preparing a sample solution;
c. assay: respectively precisely sucking the mixed reference substance solution and the sample solution respectively at 10 mu 1, injecting into a liquid chromatograph, and measuring to obtain the final product;
d. adopting traditional Chinese medicine chromatographic fingerprint similarity evaluation system software, taking S1 as a reference spectrum, taking the median as a benchmark, setting the width of a time window to be 0.1, and generating a fingerprint through multi-point correction and full spectrum peak matching;
chromatographic conditions:
chromatographic column: boston Green ODS 4.6X105 mm,5 μm,
column temperature: 30 ℃,
flow rate: 1ml/min of the total volume of the mixture,
detection wavelength: 328nm of the wavelength of the light,
sample injection amount: 10. Mu.l of the total volume of the solution,
acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid aqueous solution is taken as a mobile phase B, the gradient elution procedure is shown in the following table,
mobile phase gradient elution procedure
Figure BDA0004060832090000031
Preferably, in the step a, the preparation of the mixed reference solution: taking reference substances of chlorogenic acid, myrtillin, rosmarinic acid, salvianolic acid B and linarin, weighing, adding 70% methanol to prepare a mixed reference solution containing 1 μg, 3 μg, 10 μg, 8 μg, 2 μg and 3 μg of the reference substances in sequence per 1 ml.
Preferably, in the step b, the sample solution is prepared; sieving the powder with a third sieve, taking 0.3g, weighing, placing into a conical flask with a plug, adding 100ml of 70% methanol, sealing, weighing, heating and refluxing for 40 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
Preferably, in the step c, 10 mu 1 of each of the mixed reference substance solution and the sample solution is precisely sucked, 10 mu l of each sample solution is injected according to chromatographic conditions for analysis, and a chromatogram is recorded.
The beneficial effects are that:
in the method for establishing the fingerprint spectrum, 328nm is selected as the measurement wavelength; acetonitrile-0.1% phosphoric acid is used as a mobile phase, and gradient elution is carried out; preferably 70% methanol is used as extraction solvent, and heating reflux is performed for 40 min. The established method can accurately reflect the whole chemical characteristics of the hard-tipped vanilla, is simple and quick, and provides scientific basis for comprehensively improving the quality of the hard-tipped vanilla.
Drawings
FIG. 1 is an HPLC chromatogram of a mixture of Shenxiang grass and a control sample;
FIG. 2 is an HPLC chromatogram of a Shenxiang herb sample;
FIG. 3 is a diagram showing the durability of a Boston Green ODS (4.6X105 mm,5 μm) column;
FIG. 4 is a chart of the durability of a column of COSMIL C18 (4.6X105 mm,5 μm);
FIG. 5 is a chart of the durability of a waters C18 (4.6X105 mm,5 μm) column;
FIG. 6 is a chart of the durability of the Waters e2695 instrument;
FIG. 7 is a chart of the durability of the Shimadzu LC-20AT instrument;
FIG. 8 is a Ultimate3000 instrument durability map;
FIG. 9 is a superimposed fingerprint of all 14 batches of vanilla samples and a control fingerprint;
FIG. 10 is a superimposed fingerprint of 10 batches of Shenxiang herb samples and a control fingerprint;
wherein: peak 3, neochlorogenic acid in figure 1; peak 9, chlorogenic acid peak; peak 14, pelargonium graveolens; peak 15, rosmarinic acid; peak 16, salvianolic acid B; peak 18, linarin.
Detailed Description
The present application is described in further detail below with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
Examples
1. Instrument and reagent
1.1 instruments
High performance liquid chromatograph (equipped with quaternary pump, DAD detector, waters e 2695); analytical balance: mettler XPE26 (parts per million) (Shanghai mertrehler instruments limited); mettler XS105 (parts per million) (Shanghai mertrehler instruments limited); ultrapure water instrument (Millipore Co., U.S.A.).
1.2 reagents
Chlorogenic acid reference substance (batch No. 110753-202119, content 96.3%);
rosmarinic acid reference (lot number: 111871-202007, content 98.1%);
salvianolic acid B reference substance (batch No. 111562-201917, content of 96.6%);
linarin control (lot number: 111528-202112, content 98.0%);
the above 4 controls were purchased from chinese food and drug assay institute.
New chlorogenic acid reference (batch number: DSTDX001504, content > 98%) was purchased from Lemeitian medical Derster Biotechnology Co.
The dioscin control (lot number: 0518-RD-0011, content 94.5%) was purchased from Guangzhou Jia Tu technology Co., ltd.
14 batches of vanilla samples: all are provided by Xinjiang Uygur autonomous region medicine inspection institute, and detailed information is shown in table 1.
TABLE 1 14 sample information table of Shenxiang grass
Figure BDA0004060832090000061
Reagent: acetonitrile (chromatographic purity, merck, germany), water is ultrapure water; the other reagents were all analytically pure.
2. Method and results
2.1 chromatographic conditions
Chromatographic column: boston Green ODS (4.6X105 mm,5 μm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid aqueous solution is taken as a mobile phase B, and the gradient elution procedure is shown in Table 2; column temperature: 30 ℃, flow rate: 1ml/min; detection wavelength: 328nm; sample injection amount: 10 μl.
TABLE 2 gradient elution procedure for mobile phases
Figure BDA0004060832090000062
Figure BDA0004060832090000071
2.2 preparation of solutions
2.2.1 preparation of the Mixed control solution
3.125mg of chlorogenic acid, 5.879mg of chlorogenic acid, 20.577mg of myrtillin, 16.021mg of rosmarinic acid, 4.127mg of salvianolic acid B and 6.057mg of linarin are precisely weighed and respectively placed into 200mL measuring bottles, an appropriate amount of 70% methanol is added for ultrasonic dissolution, the mixture is cooled to room temperature, 70% methanol is used for constant volume to scale, 10mL of each solution is precisely measured and placed into the same 100mL measuring bottle, and 70% methanol is used for dilution and constant volume to scale, so that the mixed reference solution is obtained.
2.2.2 preparation of sample solutions
Taking sample powder (sieving with a third sieve) with the number of SXC-9, precisely weighing 0.3015g, placing into a conical flask with a plug, precisely adding 100ml of 70% methanol, sealing, weighing, heating and refluxing in a water bath at 85 ℃ for 40 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking a subsequent filtrate.
2.3 assay
Respectively precisely sucking 10 μ1 of the mixed reference solution and the sample solution, respectively injecting 10 μl under the chromatographic condition of "2.1", analyzing, and recording chromatograms (figure 1 and figure 2). In the sample chromatogram, chromatographic peaks which are consistent with the retention time of the reference substances of the chlorogenic acid, the geraniin, the rosmarinic acid, the salvianolic acid B and the linarin are respectively displayed, the peak shape is good, and the separation degree meets the requirements.
2.4 building a map
Adopting traditional Chinese medicine chromatographic fingerprint similarity evaluation system software, taking S1 as a reference spectrum, taking the median as a reference, setting the time window width to be 0.1, and generating a fingerprint through multi-point correction and full spectrum peak matching
The invention is validated in methodology as follows:
3.1 selection of detection wavelength
Taking 6 reference substance solutions, and carrying out spectrum scanning at 200 nm-400 nm, wherein the maximum absorption wavelengths of the 6 reference substances are respectively as follows: 326nm, 345nm, 328nm, 287nm, 333nm. The test is carried out at the maximum absorption wavelength of 6 components, and the result shows that the fingerprint spectrum at 328nm has more chromatographic peaks, good peak shape and separation degree meeting the requirement, so 328nm is selected as the measurement wavelength of the test.
3.2 selection of extraction solvent
The extraction conditions of methanol, 70% methanol, 30% methanol, ethanol, 70% ethanol and 30% ethanol on the samples were examined respectively. The results show that the components in the hard-tipped vanilla can be better embodied in 70% methanol, so 70% methanol is selected as the extraction solvent in the experiment.
3.3 selection of mobile phases
The effect of different mobile phase systems acetonitrile-water, acetonitrile-0.1% phosphoric acid and methanol-0.1% phosphoric acid on chromatographic peaks was examined respectively. The result shows that when the mobile phase system is acetonitrile-0.1% phosphoric acid solution (gradient elution), the obtained chromatogram has stable baseline, good peak shape of chromatographic peaks and good separation effect.
3.4 precision test
A sample was prepared from the powder of Hierochloes Adoratae (No. SXC-9) by the method under item "2.2.2", and the chromatogram was recorded by measuring 6 times successively under the chromatographic conditions under item "2.1". Calculating relative retention time and relative peak area of common peaks (peak 1-peak 8, peak 10-peak 12) by taking chlorogenic acid as reference peak (S1), and calculating RSD value; calculating relative retention time and relative peak area of common peaks (peak 13, peak 14, peak 16-peak 18) by using rosmarinic acid as reference peak (S2), and calculating RSD value; the results are shown in tables 3 and 4. The relative retention time RSD value of each characteristic peak is less than 0.3%, and the RSD value of the relative peak area is less than 1.2%, which indicates that the instrument precision is good.
TABLE 3 precision versus retention time results
Figure BDA0004060832090000091
Figure BDA0004060832090000101
TABLE 4 precision versus peak area results
Figure BDA0004060832090000102
3.5 stability test
Sample solutions (numbered SXC-9) under the item "2.2.2" were taken, and measured under chromatographic conditions under the item "2.1" at 0, 2, 6, 10, 16, 22 and 26h, respectively, and chromatograms were recorded. Calculating relative retention time and relative peak area of common peaks (peak 1-peak 8, peak 10-peak 12) by taking chlorogenic acid as reference peak (S1), and calculating RSD value; calculating relative retention time and relative peak area of common peaks (peak 13, peak 14, peak 16-peak 18) by using rosmarinic acid as reference peak (S2), and calculating RSD value; the results are shown in tables 5 and 6. The relative retention time (RSD) value of each characteristic peak is less than 0.3%, and the RSD value of the relative peak area is less than 2.0%, which shows that the stability of the test sample solution is good at least in 26 hours.
TABLE 5 stability versus retention time results
Figure BDA0004060832090000111
TABLE 6 stability versus peak area results
Figure BDA0004060832090000112
Figure BDA0004060832090000121
3.6 repeatability test
The same batch of vanilla samples (No. SXC-9) is crushed, 0.15g, 0.30g and 0.45g of powder (sieved by a No. four sieve) are taken, 3 parts of powder are taken respectively, the powder is precisely weighed, a sample solution is prepared according to the method under the item "2.2.2", the chromatographic condition under the item "2.1" is measured, and a chromatogram is recorded. Calculating relative retention time and relative peak area of common peaks (peak 1-peak 8, peak 10-peak 12) by taking chlorogenic acid as reference peak (S1), and calculating RSD value; calculating relative retention time and relative peak area of common peaks (peak 13, peak 14, peak 16-peak 18) by using rosmarinic acid as reference peak (S2), and calculating RSD value; the results are shown in tables 7 and 8. The relative retention time RSD value of each characteristic peak is less than 0.3%, and the RSD value of the relative peak area is less than 10.0%, which shows that the method has good repeatability.
TABLE 7 repeatability versus retention time results
Figure BDA0004060832090000131
The sampling amounts of Y1-Y9 are as follows: 0.1504g, 0.1508g, 0.1509g, 0.3019g, 0.3024g,
0.3039g、0.4517g、0.4515g、0.4519g。
TABLE 8 repeatability versus peak area results
Figure BDA0004060832090000132
Figure BDA0004060832090000141
The sampling amounts of Y1-Y9 are as follows: 0.1504g, 0.1508g, 0.1509g, 0.3019g, 0.3024g,
0.3039g、0.4517g、0.4515g、0.4519g。
3.7 durability test
3.7.1 chromatographic column durability inspection
The same batch of hard-tipped vanilla samples (No. SXC-9) are measured by adopting three different brands of chromatographic columns, the relative retention time and the relative peak area result are shown in tables 9 and 10, the chromatograms are shown in fig. 3, 4 and 5, the peak time of each characteristic peak in the chromatogram of the sample to be tested is proper, the separation is good, the relative retention time (RSD is less than 10%) and the relative peak area (RSD is less than 10%) are basically consistent, and the method is indicated that the chromatographic column has good durability.
Table 9 column durability versus retention time results
Figure BDA0004060832090000142
Figure BDA0004060832090000151
Note that: chromatographic column I: boston Green ODS (4.6X105 mm,5 μm)
Ⅱ：COSMOSIL C18(4.6×250mm，5μm)
Ⅲ：waters C18(4.6×250mm，5μm)
TABLE 10 results of column durability versus peak area
Figure BDA0004060832090000152
Figure BDA0004060832090000161
Note that: chromatographic column I: boston Green ODS (4.6X105 mm,5 μm)
Ⅱ：COSMOSIL C18(4.6×250mm，5μm)
Ⅲ：waters C18(4.6×250mm，5μm)
3.7.2 investigation of Instrument durability
The same chromatographic column (Boston Green ODS (4.6X250 mm,5 μm) is adopted, three different brands of high performance liquid chromatographs are respectively used for measuring the same batch of hard-point vanilla samples (No. SXC-9), the relative retention time and the relative peak area result are shown in tables 11 and 12, the chromatograms are shown in fig. 6, 7 and 8, the peak appearance time of each characteristic peak in the chromatograph of the sample is proper, the separation is good, the relative retention time (RSD is less than 3%) and the relative peak area (RSD is less than 10%) are basically consistent, and the method is indicated to have good instrument durability.
Table 11 instrument durability versus retention time results
Figure BDA0004060832090000162
Figure BDA0004060832090000171
Table 12 results of instrument durability versus peak area
Figure BDA0004060832090000172
Figure BDA0004060832090000181
4 establishment of fingerprint
14 batches of hard acutus samples were taken, and test solutions were prepared according to the method under item "2.2.2", respectively, and were measured according to the chromatographic conditions under item "2.1", and chromatograms were recorded.
And (3) adopting software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), taking S1 as a reference spectrum, taking the median as a reference, setting the width of a time window to be 0.1, and generating 14 overlapped batches of Shenxiang herb sample fingerprints and a control fingerprint through multi-point correction and full spectrum peak matching, wherein the figure 9 is shown in the specification. After matching, 18 common peaks are calibrated, and as shown in a similarity result (table 13) of 14 batches of samples, the similarity of fingerprints of different batches is between 0.411 and 0.999, and the similarity of samples of each batch and a generated contrast map is only between 0.703 and 0.977, so that the overall similarity is not high, and the requirements of fingerprints are not met.
Table 13 results of evaluation of finger print similarity of 14 batches of vanilla samples
Figure BDA0004060832090000182
Figure BDA0004060832090000191
By adopting software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), S1 is used as a reference, the same parameter setting is adopted to generate a superimposed 10 batches of wild and cultivated Shenxiang herb sample fingerprint sharing mode and reference fingerprints, and as shown in figure 10, the similarity of fingerprints of different batches is between 0.896 and 0.998 as shown in a 10-batch sample similarity result (table 14), the similarity of each batch of samples and the generated reference fingerprints is larger than 0.9, and the overall similarity is higher, so that the fingerprint requirement is met. 4 batches of foreign introduction cultivars have the similarity between 0.649 and 0.781 with the generated comparison, and the similarity is low, so that the characteristic spectrum can effectively distinguish the produced samples from the foreign introduction cultivars.
TABLE 14 evaluation results of finger print similarity of samples of Shenxiang vanilla produced in batches 10
Figure BDA0004060832090000192
Figure BDA0004060832090000201
Identification of 5-chromatographic peaks
From fig. 1, it can be seen that 6 characteristic peaks are identified based on the retention time of the chromatogram of the sample and the chromatogram of the mixed reference sample and the consistency of the ultraviolet spectrogram, wherein the peak No. 3 is neochlorogenic acid, the peak No. 9 is chlorogenic acid, the peak No. 14 is geranylgeranioside, the peak No. 15 is rosmarinic acid, the peak No. 16 is salvianolic acid B, and the peak No. 18 is linalool.
In summary, 14 batches of the hard-point vanilla are taken as research objects, wherein the research objects comprise 4 batches of wild products, 6 batches of real-time introduction cultivars and 4 batches of foreign introduction cultivars, and the fingerprint of the hard-point vanilla is established by adopting a high performance liquid chromatography method, so that the method is simple and has strong specificity. The fingerprint spectrum is taken as an analysis method capable of comprehensively reflecting the quality of medicines, has an irreplaceable effect in the aspect of establishing a quality control method of the hard-tipped vanilla, and can not only play a role in comprehensively controlling the quality of medicines, but also effectively distinguish samples from different sources, thereby evaluating the quality of products. The HPLC method is used for establishing the fingerprint of the hard-tip vanilla, can reflect the whole chemical characteristics of the hard-tip vanilla, can effectively distinguish the real-time samples from foreign introduction cultivars, is simple and quick, and provides scientific basis for comprehensively improving the quality standard of the hard-tip vanilla.
The foregoing description is only of the preferred embodiments of the present application and is presented as a description of the principles of the technology being utilized. It will be appreciated by persons skilled in the art that the scope of the invention referred to in this application is not limited to the specific combinations of features described above, but it is intended to cover other embodiments in which any combination of features described above or equivalents thereof is possible without departing from the spirit of the invention. Such as the above-described features and technical features having similar functions (but not limited to) disclosed in the present application are replaced with each other.

Claims (4)

1. A method for establishing a finger print of a Wei-medicinal hard jianshen herb is characterized by comprising the following steps: the method comprises the following steps:
a. preparing a mixed reference substance solution;
b. preparing a sample solution;
c. assay: respectively precisely sucking the mixed reference substance solution and the sample solution respectively at 10 mu 1, injecting into a liquid chromatograph, and measuring to obtain the final product;
d. adopting traditional Chinese medicine chromatographic fingerprint similarity evaluation system software, taking S1 as a reference spectrum, taking the median as a benchmark, setting the width of a time window to be 0.1, and generating a fingerprint through multi-point correction and full spectrum peak matching;
chromatographic conditions:
chromatographic column: boston Green ODS 4.6X105 mm,5 μm,
column temperature: 30 ℃,
flow rate: 1ml/min of the total volume of the mixture,
detection wavelength: 328nm of the wavelength of the light,
sample injection amount: 10. Mu.l of the total volume of the solution,
acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid aqueous solution is taken as a mobile phase B, the gradient elution procedure is shown in the following table,
mobile phase gradient elution procedure
Figure FDA0004060832060000011
Figure FDA0004060832060000021
2. The method for establishing the finger print of the Violet herb hard tip of claim 1, which is characterized by comprising the following steps: in the step a, the preparation of the mixed reference substance solution: taking reference substances of chlorogenic acid, myrtillin, rosmarinic acid, salvianolic acid B and linarin, weighing, adding 70% methanol to prepare a mixed reference solution containing 1 μg, 3 μg, 10 μg, 8 μg, 2 μg and 3 μg of the reference substances in sequence per 1 ml.
3. The method for establishing the finger print of the Violet herb hard tip of claim 1, which is characterized by comprising the following steps: in the step b, the sample solution is tested; sieving the powder with a third sieve, taking 0.3g, weighing, placing into a conical flask with a plug, adding 100ml of 70% methanol, sealing, weighing, heating and refluxing for 40 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
4. The method for establishing the finger print of the Violet herb hard tip of claim 1, which is characterized by comprising the following steps: in the step c, respectively and precisely sucking 10 mu 1 of the mixed reference substance solution and the sample solution, respectively injecting 10 mu l of the mixed reference substance solution and the sample solution according to chromatographic conditions for analysis, and recording a chromatogram.
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