CN116381066A - Quality evaluation method for determining optimal processing technology of Mongolian medicine myrobalan processed kusnezoff monkshood root - Google Patents
Quality evaluation method for determining optimal processing technology of Mongolian medicine myrobalan processed kusnezoff monkshood root Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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Abstract
The invention discloses a quality evaluation method for determining an optimal processing technology of Mongolian medicine myrobalan prepared kusnezoff monkshood, belonging to the technical field of traditional Chinese medicine analysis and detection. According to the quality evaluation method, the content change of myrobalan and alkaloid in the myrobalan and radix aconiti kusnezoffii manufactured by the method is taken as an evaluation index, and an orthogonal optimization test is established to establish an optimal processing method of the myrobalan and radix aconiti kusnezoffii manufactured by the method; a method for evaluating the quality of the myrobalan prepared kusnezoff monkshood root in multiple directions is established, the method is also suitable for raw kusnezoff monkshood root, and the quality of the prepared kusnezoff monkshood root is evaluated by various detection means, and comprises impurity, moisture, total ash, thin layer, content measurement related to high performance liquid chromatography and fingerprint. Test examples prove that the method is stable and reliable, can comprehensively evaluate the quality of the myrobalan processed kusnezoff monkshood root, and is suitable for scientific research and production practice.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine analysis and detection, in particular to a quality evaluation method for determining an optimal processing technology of Mongolian medicine myrobalan for preparing kusnezoff monkshood root.
Background
The dried root tuber of the annual herb aconitum kusnezoffii of Ranunculaceae is hard in texture, and is called duck head, sheep wormwood, herba abri, herba baistep, etc., and when the dried root tuber is taken as a medicinal material, the dried root tuber can be called aconitum kusnezoffii. The application of the kusnezoff monkshood root in the traditional Chinese medicine field has a long history. The Chinese medicinal composition is recorded in Shennong Ben Cao Jing at the earliest time, has the effects of dispelling wind and removing dampness, warming meridians and relieving pain, and has the treatment effect and toxic effect coexisting, so that the Chinese medicinal composition must be subjected to attenuation treatment before clinical use. The Mongolian medicine belongs to the special national medicine in China, has rich experience and special characteristics in the aspect of processing the kusnezoff monkshood root, has remarkable attenuation effect and effectively retains the components in the kusnezoff monkshood root due to the processing method of the Mongolian medicine myrobalan kusnezoff monkshood root, and is widely applied to various Mongolian medicine formulas. However, the specific processing methods of the myrobalan processed kusnezoff monkshood root in different medical books are different, and a set of complete quality evaluation system is lacked, so that standardized research is carried out on the processing and attenuation process of the Mongolian medicine myrobalan processed kusnezoff monkshood root by means of modern detection means, and the construction of the quality evaluation system has important significance.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a quality evaluation method for determining the optimal processing technology of the Mongolian medicine myrobalan prepared kusnezoff monkshood root, and the quality evaluation method has comprehensive research content and has important significance on the construction quality evaluation system of the Mongolian medicine prepared kusnezoff monkshood root.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides a quality evaluation method for determining an optimal processing technology of Mongolian medicine myrobalan processed kusnezoff monkshood, which comprises the following steps:
step one: establishing an orthogonal optimization test of the myrobalan processed kusnezoff monkshood root to obtain the myrobalan processed kusnezoff monkshood root with different processing methods;
step two: comparing the changes of alkaloids and tannins in the radix aconiti kusnezoffii processed by the myrobalan with different processing methods by using an Aobitrap LC-MS detector;
step three: testing the myrobalan prepared kusnezoff monkshood root by different processing methods according to the identification method of the impurity, the moisture, the total ash and the thin-layer chromatography of the prepared kusnezoff monkshood root in the part 2020 of Chinese pharmacopoeia;
step four: determining the content of aconite alkaloid in the myrobalan prepared kusnezoff monkshood root by adopting a high performance liquid chromatography method;
step five: and establishing a fingerprint of the myrobalan processed kusnezoff monkshood root, and evaluating the quality to determine the optimal processing technology of the Mongolian medicine myrobalan processed kusnezoff monkshood root.
In the above technical solution, it is preferable that: one specific implementation mode of the first step is as follows:
the preparation method comprises the steps of taking the clean preparation of the raw kusnezoff monkshood root, taking the processing time, the processing temperature, the particle size of the myrobalan and the dosage of the soup as factors, and taking the raw kusnezoff monkshood root every day according to the following steps: the myrobalan is boiled according to the ratio of the myrobalan to the ratio of 10:1, the myrobalan soup is changed every day, and then the myrobalan soup is washed once by clean water, stirring properly, adding new fructus Chebulae soup, soaking, taking out, and drying; obtaining the myrobalan processed kusnezoff monkshood root with different processing methods.
In the above technical solution, it is preferable that: the method for detecting the alkaloid component change in the second step comprises the following steps:
1.0g of myrobalan prepared kusnezoff monkshood powder with different processing methods is taken, the myrobalan prepared kusnezoff monkshood powder is precisely weighed and placed in a conical flask with a plug, 1mL of ammonia test solution is added, 30mL of diethyl ether is precisely added, ultrasonic treatment is carried out for 30min, extraction is carried out twice, the extracting solutions are combined, filtrate is placed in an evaporation dish, after natural volatilizing, 5mL of 50% methanol solution is used for ultrasonic redissolution, a sterile filter membrane with the thickness of 0.22 mu m is used as a test sample solution, and the positive spectrum analysis is carried out by using an Aobitrap LC-MS detector; the analytical process parameters were as follows:
ESI source, positive ion scanning mode, capillary temperature 270 ℃, capillary voltage 30V, spray voltage 5KV, sheath gas (N) 2 ) Flow rate 49psi (1 psi. Apprxeq. 6.895 kPa), auxiliary gas (N 2 ) A flow rate of 10psi; chromatographic column (C18 column Waters-C18,4.6mm x 100mm,1.7 μm;) methanol: acetonitrile=50:50 is mobile phase A,0.35mol/L ammonium acetate ammonia water pH=10.5 is mobile phase B, the column temperature is 30 ℃, the flow rate is 0.3mL/min, the sample injection amount is 10 mu L, and elution is carried out;
the elution conditions were: 0-15 min, 10-45% of A; 15-45 min, 45-60% of A; 45-55 min, 60-85% of A, 55-65 min and 85-90% of A; 65-71 min, 90-10% of A; 71-75 min,10% A.
In the above technical solution, it is preferable that: the detection method of the change of the tannic component in the second step is as follows:
taking 0.5g of myrobalan prepared kusnezoff monkshood powder with different processing methods, precisely weighing and placing the powder into a conical flask with a plug, precisely adding 20mL of 50% methanol aqueous solution containing 0.2% formic acid, standing overnight, performing ultrasonic treatment for 60min, passing through a 0.22 mu m sterile filter membrane, and performing negative spectrum analysis by using an Orbitrap LC-MS detector as a sample solution; the analytical process parameters were as follows:
ESI source, negative ion scanning mode, mass spectrum condition, chromatographic column and positive spectrum condition; acetonitrile is a mobile phase A,0.1% formic acid water is a mobile phase B, the column temperature is 30 ℃, the flow rate is 0.3mL/min, the sample injection amount is 5 mu L, and elution is carried out;
the elution conditions were: 0-3 min,5% A; 3-12 min, 5-13% of A; 12-20 min, 13-25% A; 20-30 min, 25-30% of A; 30-38 min, 30-60% of A; 38-48 min, 60-95% A; 48-49 min, 95-5%A; 49-52 min,5% A.
In the above technical solution, it is preferable that: the aconite alkaloid content determination method in the step four is as follows:
1.0g of raw kusnezoff monkshood root and myrobalan processed kusnezoff monkshood root powder obtained by different processing factors are taken, the raw kusnezoff monkshood root powder and the myrobalan processed kusnezoff monkshood root powder are precisely weighed and placed in a conical flask with a plug, 1mL of ammonia test solution is added, 50mL of diethyl ether is precisely added, the weight is weighed, the ultrasonic treatment is carried out for 30min, the extraction is carried out twice, the extracting solutions are combined, filtrate is placed in an evaporation dish, after natural volatilizing, 5mL of methanol solution containing 0.1% hydrochloric acid is used for ultrasonic redissolution as a test sample solution, and the content of aconite alkaloids in the solution is measured.
In the above technical solution, it is preferable that: one specific implementation mode of the fifth step is as follows:
detecting the liquid to be detected by high performance liquid chromatography, wherein the chromatographic parameters are as follows: mobile phase a:50 mmol.L -1 Is an amine acetate solution; mobile phase B: acetonitrile; the C18 column was a Sunniest-C18 (4.6 mm x 250mm,5 μm); the detection wavelength is 235nm; the flow rate is 1m L min -1 The method comprises the steps of carrying out a first treatment on the surface of the The column temperature is 30 ℃; the sample injection amount is 20 mu L; gradient elution is carried out;
the effective components are qualitatively determined according to the peak time of the obtained chromatogram; quantifying the effective components according to the obtained cover of the chromatogram; in a chromatogram obtained at a detection wavelength of 235 nm: the peak time is 13.720 +/-0.2 min, which is benzoylmesaconine; the peak time is 16.607 +/-0.2 min, which is benzoylaconitine; the peak time is 18.587 +/-0.2 min, which is benzoylmesaconine; the peak time is 35.127 +/-0.2 min, which is aconitine; the peak time is 38.313 +/-0.2 min, which is aconitine; the peak time is 39.340 +/-0.2 min, which is aconitine.
The quality evaluation method for determining the optimal processing technology of the Mongolian medicine myrobalan processed kusnezoff monkshood root provided by the invention is also suitable for raw kusnezoff monkshood root.
And (3) applying the content measurement chromatographic conditions to obtain fingerprint spectrum superposition graphs of nine different processed products, and analyzing the stability and quality consistency of alkaloid components.
The beneficial effects of the invention are as follows:
according to the quality evaluation method for determining the optimal processing technology of the Mongolian medicine myrobalan and the radix aconiti kusnezoffii preparata, the content change of myrobalan and alkaloid in the myrobalan and the radix aconiti kusnezoffii preparata is taken as an evaluation index, and an orthogonal optimization test is established to determine the optimal processing method of the myrobalan and the radix aconiti kusnezoffii preparata; a method for evaluating the quality of the prepared kusnezoff monkshood root by the myrobalan in multiple directions is established, and the method is also suitable for the raw kusnezoff monkshood root, and evaluates the quality of the prepared kusnezoff monkshood root by various detection means, including impurity, moisture, total ash, thin layer, content measurement related to high-performance liquid chromatography and fingerprint. Test examples prove that the method is stable and reliable, can comprehensively evaluate the quality of the myrobalan processed kusnezoff monkshood root, and is suitable for scientific research and production practice.
The invention provides a quality evaluation method for determining an optimal processing technology of a Mongolian medicine myrobalan processed kusnezoff monkshood, which is a relatively comprehensive quality evaluation method for the myrobalan processed kusnezoff monkshood. The method is characterized by identifying impurities, moisture, total ash and thin-layer chromatography on the basis of a part of Chinese pharmacopoeia 2020 edition, establishing a new method for measuring the content of aconite alkaloids by HPLC, and analyzing alkaloids and tannins in the processed product by using an Orbitrap LC-MS detector and analyzing the influence of content changes of different processing factors. Compared with the current evaluation method of pharmacopoeia, the content determination method of the invention can simultaneously determine six alkaloids of aconitine, benzoylaconitine and benzoylaconitine, has better separation degree, does not have false positive and interference peaks, has matrix with liquid inlet quality, and determines the optimal processing technology of the myrobalan prepared kusnezoff monkshood root by combining the results. Has far-reaching significance for preparing the radix aconiti kusnezoffii drug by using the myrobalan.
Drawings
The invention is described in further detail below with reference to the drawings and the detailed description.
FIG. 1 is a diagram showing identification of thin layers of radix Aconiti Kusnezoffii processed with fructus Chebulae by different processing methods.
FIG. 2 is a graph showing the effect of different factors on the total alkaloids of radix Aconiti Kusnezoffii processed by fructus Chebulae.
FIG. 3 is a graph showing the influence of different factors on the total tannins of the myrobalan processed radix aconiti kusnezoffii.
Fig. 4 is a graph of the superposition of radix Aconiti Kusnezoffii processed by different methods.
Fig. 5 is a graph of wild aconite control patterns of different producing places.
Fig. 6 is a superposition of different processes.
Fig. 7 shows control patterns of radix Aconiti Kusnezoffii processed with fructus Chebulae by different processing methods.
Detailed Description
Example 1
The invention provides a quality evaluation method for determining an optimal processing technology of Mongolian medicine myrobalan processed kusnezoff monkshood, which comprises the following steps:
step one: taking raw kusnezoff monkshood root, and establishing an orthogonal optimization test according to four factors of processing time, processing temperature, particle size of myrobalan and dosage of soup to obtain the myrobalan processed kusnezoff monkshood root with different processing methods; the three levels of the four different factors in the first step compare the influence of the different factors on the components in the myrobalan processed kusnezoff monkshood root;
step two: comparing the alkaloid in the processed radix aconiti kusnezoffii with the change of the tannin components in the myrobalan by using an Aobitrap LC-MS detector; the types of the alkaloids in the step two are more than those of the alkaloids in the step two, such as alkaloids without ultraviolet absorption, such as Niaolin and the like; the change of the tannin components in the myrobalan processed kusnezoff monkshood root with different processing methods along with the processing factors can be compared;
step three: the myrobalan prepared kusnezoff monkshood obtained in the first step is identified by referring to the requirements of the first part of Chinese pharmacopoeia 2020 on the identification of impurities, ash, moisture and thin layers of the prepared kusnezoff monkshood;
step four: establishing a high performance liquid chromatography method, and determining the content of aconite alkaloid in the prepared kusnezoff monkshood root medicinal material; the content determination method in the fourth step can be used for simultaneously determining diester alkaloids and monoester alkaloids in the radix aconiti kusnezoffii processed by the myrobalan, and has better separation degree and liquid-feeding matrix; the method for extracting the sample in the fourth step adopts diethyl ether ultrasonic extraction;
step five: establishing a radix aconiti kusnezoffii preparata fingerprint spectrum, and evaluating quality to determine the optimal processing technology of radix aconiti kusnezoffii preparata by using the Mongolian medicine myrobalan; the fingerprint spectrum established in the fifth step can give out comprehensive and reliable information, and objectively reflects the consistency and the authenticity of the myrobalan processed kusnezoff monkshood root.
The quality evaluation method specifically comprises the following steps of:
according to the invention, after the raw kusnezoff monkshood root is cleaned, the raw kusnezoff monkshood root is taken as factors including processing time, processing temperature, particle size of myrobalan and dosage of soup every day according to the following steps: the myrobalan=10:1 ratio is used for boiling the myrobalan soup, the myrobalan soup is changed every day, then the myrobalan soup is cleaned once by clean water, and is properly turned over, then a new myrobalan soup is added for soaking, and the myrobalan soup is taken out and dried, thus obtaining the myrobalan radix aconiti kusnezoffii processed, see the following table 1.
TABLE 1 different processing methods
According to the invention, the impurity of the prepared kusnezoff monkshood root in the part of the 'Chinese pharmacopoeia' 2020 edition is not more than 1% (general rule 2301). The moisture content is less than 12.0% (general rule 0832, second method). The total ash content is less than 6.0 percent (general rule 2302) and the thin layer identification is carried out, the identification is carried out on the radix aconiti kusnezoffii preparata, the inspection results of the moisture and the total ash content are shown in the following table 2, and the thin layer identification is shown in figure 1.
TABLE 2 moisture, total ash examination results
In fig. 1: s3 and s4 are mixed reference substances of benzoylmesaconine, benzoylmesaconine and benzoylhypaconitine sequentially from top to bottom, and 1 is a No. 1 preparation; 2 is the No. 2 processed product; 3 is a No. 3 processed product; 4 is a No. 4 processed product; 5 is a No. 5 processed product; 6 is a No. 6 processed product; 7 is a No. 7 processed product; 8. is a No. 8 processed product; 9 is a No. 9 processed product; spots of the same color appear at positions corresponding to the chromatogram of the control.
According to the invention, 1.0g of myrobalan prepared kusnezoff monkshood root powder with different processing methods is taken, the powder is precisely weighed and placed in a conical flask with a plug, 1mL of ammonia test solution is added, 30mL of diethyl ether is precisely added, ultrasonic treatment is carried out for 30min, extraction is carried out twice, the extracting solutions are combined, filtrate is placed in an evaporation dish, after natural volatilizing, 5mL of 50% methanol solution is used for ultrasonic redissolution, a sterile filter membrane with the thickness of 0.22 mu m is used as a test sample solution, and the positive spectrum analysis is carried out by using an Aobitrap LC-MS detector; ESI source, positive ion scanning mode, capillary temperature 270 ℃, capillary voltage 30V, spray voltage 5KV, sheath gas (N) 2 ) Flow rate 49psi (1 psi. Apprxeq. 6.895 kPa), auxiliary gas (N 2 ) A flow rate of 10psi; column C18 column was Waters-acquisition-UPLC-C18, (2.1 mm x 100mm,1.7 μm;), methanol: acetonitrile=50:50 is mobile phase a,0.35mol/L ammonium acetate aqueous ammonia ph=10.5 is mobile phase B, column temperature 30 ℃, flow rate 0.3mL/min, sample injection amount 10 μl, elution is performed under the following conditions. 0-15 min, 10-45% of A; 15-45 min, 45-60% of A; 45-55 min, 60-85% of A, 55-65 min, 85-90% of A; 65-71 min, 90-10% of A; 71-75 min,10% A. The influence of different processing methods on the alkaloid content change is calculated according to the change of the peak area ratio of the radix aconiti kusnezoffii, and the result is shown in the following table 3.
TABLE 3 alkaloid content variation for nine different processing methods
Accurately weighing 0.5g of myrobalan processed kusnezoff monkshood root and myrobalan powder with different processing methods, placing the myrobalan processed kusnezoff monkshood root and the myrobalan powder into a conical flask with a plug, accurately adding 20mL of 50% methanol aqueous solution containing 0.2% formic acid, standing overnight, performing ultrasonic treatment for 60min, passing through a 0.22 mu m sterile filter membrane to serve as a sample solution, and performing negative spectrum analysis by using an Orbitrap LC-MS detector; ESI source, negative ion scanning mode, mass spectrum condition, chromatographic column and positive spectrum condition; acetonitrile is a mobile phase A,0.1% formic acid water is a mobile phase B, the column temperature is 30 ℃, the flow rate is 0.3mL/min, the sample injection amount is 5 mu L, and the elution is carried out under the following conditions, namely, 0-3 min and 5% A; 3-12 min, 5-13% of A; 12-20 min, 13-25% A; 20-30 min, 25-30% of A; 30-38 min, 30-60% of A; 38-48 min, 60-95% of A; 48-49 min, 95-5%A; 49-52 min,5% A. The influence of different processing methods on the change of tannins in the myrobalan is calculated according to the change of the area ratio of the peak of the myrobalan, and the result is shown in the following table 4.
TABLE 4 nine different processing methods for changing the content of the Chebula tannin components
According to the total component change, an intuitive graph of the influence of different processing factors on the components is made by combining with an orthogonal test, the influence of different factors on the total alkaloids of the myrobalan processed kusnezoff monkshood is shown in fig. 2, the influence of different factors on the total tannins of the myrobalan processed kusnezoff monkshood is shown in fig. 3, and the influence of processing time on the total alkaloids is the largest, and the total alkaloids are greatly reduced based on the prolongation of the processing time; secondly, the processing temperature is greatly influenced, the content of the total alkaloids is also influenced by the temperature rise, and the influence of the particle size of the myrobalan and the dosage of the soup on the total alkaloids is small. Compared with the tanning components in the myrobalan processed kusnezoff monkshood, the particle size of the myrobalan has the greatest influence on the total tanning components, the smaller the particle size, the more tanning components enter the kusnezoff monkshood, the processing time is the processing time, the change of the tanning components is relatively small from 2 days to 4 days, the processing temperature is 50 ℃ and the myrobalan components are more than 4 ℃, the change of the soup is minimum at 25 ℃, the change of the tanning components in the myrobalan is small, and the change of the soup is in negative correlation and the change of the total alkaloid content is positive, and the myrobalan soup soaking method of the Mongolian medicine kusnezoff monkshood is recorded according to the medicine standard (Mongolian medicine division book) of the Ministry of health of the people's republic of China: soaking radix aconiti kusnezoffii in the soup of myrobalan at room temperature for 3-5 days, turning over 3-5 times daily, taking out with slightly tingling sensation, and drying at low temperature. The processing method of the myrobalan processed kusnezoff monkshood root is mentioned in the Mongolian medicine processing Specification: every 100kg of kusnezoff monkshood root, 300kg of myrobalan soup (30 kg of myrobalan is added with 300kg of water to be boiled) is used for recording the process of preparing the kusnezoff monkshood root by using the myrobalan in Mongolian medicine processing science: the kusnezoff monkshood root is soaked in the myrobalan decoction for 1-3 days (10 kg of kusnezoff monkshood root and 3kg of myrobalan decoction are 30L), and the radix aconiti kusnezoffii is prepared according to the Mongolian medicine, and the processing temperature is not easy to be too high in spring and autumn, so that the processing temperature is selected to be 4 days finally according to experimental results and literature reports, the processing temperature is 4 ℃, the myrobalan is selected to be crushed into fragments with the particle size of 0.3cm for boiling the myrobalan decoction, and the radix aconiti kusnezoffii is prepared every day according to the following steps: soaking the myrobalan decoction with the ratio of (radix aconiti kusnezoffii: myrobalan=10:3 decoction) of (1:3) to obtain the optimal processing method.
According to the invention, 1.0g of prepared kusnezoff monkshood root and different batches of raw kusnezoff monkshood root powder are taken, accurately weighed and placed in a conical flask with a plug, 1mL of ammonia water is added, 50mL of diethyl ether is accurately added, weighing is carried out, ultrasonic treatment is carried out for 30min, extraction is carried out twice, the extracting solutions are combined, filtrate is placed in an evaporation dish, and after natural volatilizing, 5mL of methanol solution containing 0.1% hydrochloric acid is used for ultrasonic re-dissolution to be taken as a sample solution. Detecting the liquid to be detected by high performance liquid chromatography, and finally determining the preferable chromatographic conditions according to chromatographic peak separation conditions, and precision, stability, repeatability, accuracy and linearity of detection, wherein the preferable chromatographic conditions are as follows: the chromatographic parameters are: a C18 chromatographic column; mobile phase a:50 mmol.L -1 Is an amine acetate solution; mobile phase B: acetonitrile; gradient elution procedure is described in table 5 below; the C18 chromatographic column is Sunniest-C18, the size is 4.6mm, 250mm and 5 μm; the detection wavelength is 235nm; the flow rate is 1 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the The column temperature is 30 ℃; the sample loading was 20. Mu.L.
TABLE 5 gradient elution procedure
EXAMPLE 2 methodology investigation
1. Linear relationship investigation
Mixed standard substance solutions with different concentrations are prepared, standard curves are drawn by taking peak areas of various standard substances as Y axis and the concentrations as X axis, the results are shown in the following table 6, and the results show that: by adopting the method provided by the invention, each alkaloid component in the kusnezoff monkshood root has good linearity in the corresponding concentration range.
TABLE 6 Linear relationship of six aconite alkaloids
2. Precision experiments
Taking control solution, and continuously sampling for 6 times according to the chromatographic conditions. And calculating RSD of peak areas of the components, and examining precision of the instrument. The RSD (%) of aconitine, benzoylmesaconine, and benzoylmesaconine are 2.876, 2.195, 2.506, 1.331, 2.387, and 1.476, respectively. The instrument has good precision.
3. Stability test
Precisely measuring the solution of the same sample, sealing and standing at room temperature, and measuring under chromatographic conditions for 0,2,4,8, 12, 16, 20 and 24 hours respectively, wherein the results show that RSD (%) of aconitine, neoaconitine, sub-aconitine, benzoylaconitine, benzoylsub-aconitine is 1.449, 0.538, 2.284, 0.863, 0.522 and 2.36 respectively. Indicating that the test solution was substantially stable for 24 hours.
4. Repeatability experiments
Precisely weighing the same batch of test products, preparing test product solution, continuously sampling for 6 times, and detecting according to chromatographic conditions, wherein the results show that RSD (%) of aconitine, neoaconitine, sub-aconitine, benzoylaconitine, benzoylneoaconitine and benzoylsub-aconitine is 0.893, 0.38, 1.301, 1.29, 1.381 and 1.282 respectively. Indicating that the method is well reproducible.
5. Reproducibility experiments
Precisely weighing the same batch of test samples, preparing 6 parts of test sample solutions, and detecting according to chromatographic conditions, wherein the results show that RSD (%) of aconitine, neoaconitine, sub-aconitine, benzoylaconitine, benzoylsub-aconitine is 1.442, 1.502, 2.318, 1.586, 1.459 and 2.175 respectively. The method is shown to have good reproducibility.
6. Sample addition recovery experiment
Precisely measuring 6 parts of a sample with known content, wherein the components are as follows: 1, respectively and precisely adding a certain amount of mixed reference substances into the mixture according to the proportion, and detecting the chromatographic condition, wherein the sample adding and recovering calculation mode is (the content of each compound in the sample after the reference substances are added, the content of each compound in the known test substances)/the content of each component reference substance added is multiplied by 100 percent=the sample adding and recovering rate. The results showed that the average recovery (%) of aconitine, benzoylmesaconine was 97.41%, 91.79%, 98.50%, 106.61%, 99.53%, 93.93%, respectively. Indicating good recovery of each component.
7. Sample content determination
According to the preparation method, each sample was extracted twice as a sample solution, each sample solution was sampled twice, and the content of the components in each sample was calculated by measuring according to the content measuring method, and the results are shown in table 7 below.
TABLE 7 determination of aconite alkaloid content in raw and processed radix Aconiti Kusnezoffii
According to the preparation method, the fingerprint of the myrobalan prepared kusnezoff monkshood root is established, and the difference of the alkaloid types in the kusnezoff monkshood roots in different producing areas is smaller before and after processing, and the fingerprint of the myrobalan prepared kusnezoff monkshood root is established by adopting the raw kusnezoff monkshood roots in a single producing area, so that the fingerprint of 12 batches of raw kusnezoff monkshood roots in inner Mongolian, xinjiang and Shaanxi 3 producing areas is established by adopting the content measuring liquid phase method in the experiment through a common peak establishing fingerprint methodology, and the method can more intuitively see the change of the alkaloid content of the myrobalan prepared kusnezoff monkshood roots before and after processing by different methods. The overlapping chart is shown in fig. 4, the overlapping chart of the kusnezoff monkshood root in different producing places is shown in fig. 5, the reference chart of the kusnezoff monkshood root in different producing places is shown in fig. 5, 9 common peaks are detected in total in 12 batches of kusnezoff monkshood root in fig. 5, and the 9 common peaks are numbered from 1 to 9 in sequence according to the retention time.
1. Finger print precision investigation
Peak No. 2, peak No. 3, peak No. 4, peak No. 5, peak No. 1, peak No. 7, peak No. 8, peak No. 9, peak No. 6, peak retention time and peak area 1 were referenced, and RSD of the relative retention time and relative peak area of the common peaks was calculated. The sample is continuously injected for 6 times, and the result shows that the relative retention time RSD (%) of 9 peaks is respectively 0.00, 0.20, 0.29, 0.53, 0.00, 0.12, 0.08 and 0.10,9 peaks and the relative peak area RSD (%) of the 9 peaks is respectively 0.00, 1.23, 1.86, 1.26, 2.91, 0.00, 1.62, 2.86 and 2.25, the similarity is larger than 0.9, and the instrument precision is good, and meets the fingerprint spectrum measurement requirement.
2. Finger print stability investigation
Taking the solution of the same test sample, sealing and placing under room temperature conditions, respectively measuring according to chromatographic conditions at 0,2,4,8, 12, 16, 20 and 24 hours, wherein the relative retention time RSD (%) of 9 peaks is respectively 0.00, 0.11, 0.21, 0.53, 0.00, 0.15, 0.12 and 0.16,9 peaks, and the relative peak area RSD (%) of the 9 peaks is respectively 0.00, 2.00, 1.67, 1.99, 1.76, 0.00, 0.54, 1.62 and 2.87, and the similarity is greater than 0.9, which indicates that the test sample solution is basically stable when placed for 24 hours.
3. Fingerprint reproducibility investigation
The relative retention times RSD (%) of 9 peaks in the repeatability test were 0.00, 0.11, 0.13, 0.15, 0.26, 0.00, 1.03, 1.74 and 0.24, respectively, and the relative peak areas RSD (%) were 0.00, 0.27, 1.38, 0.85, 1.51, 0.00, 0.51, 1.63 and 1.73, respectively, and the similarity was greater than 0.9, indicating that the method was good in repeatability.
The chromatograms measured by different processing methods are overlapped to obtain a fingerprint overlapping chart of the radix aconiti kusnezoffii processed by the myrobalan of different processing methods, which is shown in fig. 6, and a reference chart of the radix aconiti kusnezoffii processed by the myrobalan of different processing methods, which is shown in fig. 7. The common peaks of different batches of kusnezoff monkshood root and the common peaks of different processing methods are combined, and the common peaks as shown in figure 7 are used as characteristic peaks for evaluating the myrobalan processed kusnezoff monkshood root.
In summary, the quality evaluation method of the myrobalan processed kusnezoff monkshood root can refer to the identification method of ash, moisture and thin layers in the part 2020 of Chinese pharmacopoeia, the content measurement and fingerprint evaluation method can refer to the method established by the invention, and the evaluation method can lay a foundation for the deep research and quality evaluation of chemical components of the subsequent myrobalan processed kusnezoff monkshood root.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (7)
1. A quality evaluation method for determining the optimal processing technology of Mongolian medicine myrobalan prepared kusnezoff monkshood root is characterized by comprising the following steps of:
step one: establishing an orthogonal optimization test of the myrobalan processed kusnezoff monkshood root to obtain the myrobalan processed kusnezoff monkshood root with different processing methods;
step two: comparing the changes of alkaloids and tannins in the radix aconiti kusnezoffii processed by the myrobalan with different processing methods by using an Aobitrap LC-MS detector;
step three: identifying impurities, moisture, total ash and thin layers in the black-bone processed by the myrobalan according to different processing methods;
step four: determining the content of aconite alkaloid in the myrobalan prepared kusnezoff monkshood root by adopting a high performance liquid chromatography method;
step five: and establishing a fingerprint of the myrobalan processed kusnezoff monkshood root, and evaluating the quality to determine the optimal processing technology of the Mongolian medicine myrobalan processed kusnezoff monkshood root.
2. The quality evaluation method according to claim 1, wherein one embodiment of the first step is:
the preparation method comprises the steps of taking the clean preparation of the raw kusnezoff monkshood root, taking the processing time, the processing temperature, the particle size of the myrobalan and the dosage of the soup as factors, and taking the raw kusnezoff monkshood root every day according to the following steps: the myrobalan is boiled according to the ratio of the myrobalan to the myrobalan=10:1, the myrobalan soup is changed every day, then the myrobalan soup is cleaned once by clean water, turned over, added with new myrobalan soup for soaking, taken out and dried; obtaining the myrobalan processed kusnezoff monkshood root with different processing methods.
3. The method for evaluating the quality according to claim 1, wherein the method for detecting the change of the alkaloid component in the second step is as follows:
1.0g of myrobalan prepared kusnezoff monkshood powder with different processing methods is taken, the myrobalan prepared kusnezoff monkshood powder is precisely weighed and placed in a conical flask with a plug, 1mL of ammonia test solution is added, 30mL of diethyl ether is precisely added, ultrasonic treatment is carried out for 30min, extraction is carried out twice, the extracting solutions are combined, filtrate is placed in an evaporation dish, after natural volatilizing, 5mL of 50% methanol solution is used for ultrasonic redissolution, a sterile filter membrane with the thickness of 0.22 mu m is used as a test sample solution, and the positive spectrum analysis is carried out by using an Aobitrap LC-MS detector; the analytical process parameters were as follows:
ESI source, positive ion scanning mode, capillary temperature 270 ℃, capillary voltage 30V, spray voltage 5KV and sheath gas N 2 The flow rate is 49psi, and the auxiliary gas is N 2 A flow rate of 10psi; the chromatographic column is a C18 chromatographic column Waters-C18, the size of which is 4.6mm x 100mm,1.7 μm; methanol: acetonitrile=50:50 is mobile phase A,0.35mol/L ammonium acetate ammonia water pH=10.5 is mobile phase B, the column temperature is 30 ℃, the flow rate is 0.3mL/min, the sample injection amount is 10 mu L, and elution is carried out;
the elution conditions were: 0-15 min, 10-45% of A; 15-45 min, 45-60% of A; 45-55 min, 60-85% of A, 55-65 min and 85-90% of A; 65-71 min, 90-10% of A; 71-75 min,10% A.
4. The method for evaluating quality according to claim 1, wherein the method for detecting change of tannic acid component in the second step is as follows:
taking 0.5g of myrobalan prepared kusnezoff monkshood powder with different processing methods, precisely weighing and placing the powder into a conical flask with a plug, precisely adding 20mL of 50% methanol aqueous solution containing 0.2% formic acid, standing overnight, performing ultrasonic treatment for 60min, passing through a 0.22 mu m sterile filter membrane, and performing negative spectrum analysis by using an Orbitrap LC-MS detector as a sample solution; the analytical process parameters were as follows:
ESI source, negative ion scanning mode, capillary temperature 270 ℃, capillary voltage 30V, spray voltage 5KV and sheath gas N 2 The flow rate is 49psi, and the auxiliary gas is N 2 A flow rate of 10psi; the chromatographic column is C18 chromatographic column Waters-C18 and has the size4.6mm x 100mm,1.7 μm; acetonitrile is a mobile phase A,0.1% formic acid water is a mobile phase B, the column temperature is 30 ℃, the flow rate is 0.3mL/min, the sample injection amount is 5 mu L, and elution is carried out;
the elution conditions were: 0-3 min,5% A; 3-12 min, 5-13% of A; 12-20 min, 13-25% A; 20-30 min, 25-30% of A; 30-38 min, 30-60% of A; 38-48 min, 60-95% A; 48-49 min, 95-5%A; 49-52 min,5% A.
5. The quality evaluation method according to claim 1, wherein the aconite alkaloid content determination method in the fourth step is as follows:
1.0g of raw kusnezoff monkshood root and myrobalan processed kusnezoff monkshood root powder obtained by different processing factors are taken, the raw kusnezoff monkshood root powder and the myrobalan processed kusnezoff monkshood root powder are precisely weighed and placed in a conical flask with a plug, 1mL of ammonia test solution is added, 50mL of diethyl ether is precisely added, the weight is weighed, the ultrasonic treatment is carried out for 30min, the extraction is carried out twice, the extracting solutions are combined, filtrate is placed in an evaporation dish, after natural volatilizing, 5mL of methanol solution containing 0.1% hydrochloric acid is used for ultrasonic redissolution as a test sample solution, and the content of aconite alkaloids in the solution is measured.
6. The quality evaluation method according to claim 1, wherein one specific embodiment of the fifth step is:
detecting the liquid to be detected by high performance liquid chromatography, wherein the chromatographic parameters are as follows: the C18 chromatographic column is Sunniest-C18, the size is 4.6mm, 250mm and 5 μm; mobile phase a:50 mmol.L -1 Is an amine acetate solution; mobile phase B: acetonitrile; the detection wavelength is 235nm; the flow rate is 1 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the The column temperature is 30 ℃; the sample injection amount is 20 mu L; gradient elution is carried out;
the effective components are qualitatively determined according to the peak time of the obtained chromatogram; quantifying the effective components according to the obtained cover of the chromatogram; in a chromatogram obtained at a detection wavelength of 235 nm: the peak time is 13.720 +/-0.2 min, which is benzoylmesaconine; the peak time is 16.607 +/-0.2 min, which is benzoylaconitine; the peak time is 18.587 +/-0.2 min, which is benzoylmesaconine; the peak time is 35.127 +/-0.2 min, which is aconitine; the peak time is 38.313 +/-0.2 min, which is aconitine; the peak time is 39.340 +/-0.2 min, which is aconitine.
7. The quality evaluation method according to claim 1, which is also applicable to raw kusnezoff monkshood root.
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