CN112083087A - Bletilla striata extracting solution obtaining method and operation parameter selecting method thereof - Google Patents
Bletilla striata extracting solution obtaining method and operation parameter selecting method thereof Download PDFInfo
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- CN112083087A CN112083087A CN202010835120.9A CN202010835120A CN112083087A CN 112083087 A CN112083087 A CN 112083087A CN 202010835120 A CN202010835120 A CN 202010835120A CN 112083087 A CN112083087 A CN 112083087A
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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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
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
Abstract
The invention provides a method for selecting operation parameters for obtaining rhizoma bletillae extracting solution, which comprises the following steps: carrying out chromatographic determination; analyzing and measuring; and (5) factor analysis optimization. The invention also provides a method for obtaining the bletilla striata extracting solution; heating in water bath at 40-60 deg.c for 2.5-3.5 hr with 80% concentration methanol in bletilla tuber powder of 80 mesh size. The invention belongs to the technical field of foodstuff processing, and is simple and easy to implement, low in operation cost, stable and reliable; for the extraction of the bletilla striata, the extraction of the bletilla striata can be effectively realized as far as possible at the lowest cost, and the efficiency is high through optimization, the rejection rate is low, and the quality of a finished product is good; for the optimization of the operation parameters for obtaining the rhizoma bletillae extracting solution, the dependence on human experience is avoided, and meanwhile, excessive invalid experiments in a conventional method can be avoided, so that the extraction process can be optimized and adjusted quickly and efficiently, the whole process does not depend on a single specific scene or expensive equipment, and the operation is easy, stable and reliable.
Description
Technical Field
The invention relates to a method for obtaining rhizoma bletillae extract and a method for selecting operating parameters of the rhizoma bletillae extract, and belongs to the technical field of food processing.
Background
The Bletilla striata is a dry tuber of a Bletilla striata (Bletilla striata) of the family Orchidaceae, has the effects of astringing to stop bleeding, reducing swelling and promoting tissue regeneration, and is used for treating internal and external bleeding syndromes, carbuncle swelling, scald, rhagadia manus et pedis and the like. Mainly produced in Guizhou, Sichuan, Hubei and other places, and the Guizhou has the highest yield and the best quality, and only Guizhou Anlong and Xingzhou are the most suitable areas. Rhizoma bletillae is a rare hemostatic material, and the pharmacological effects of the rhizoma bletillae mainly comprise antibiosis, blood circulation promotion and hemostasis, wound healing promotion, gastrointestinal tract mucous membrane injury repair promotion, bone marrow hematopoiesis promotion, virus resistance, tumor resistance and the like. The chemical components of the bletilla striata mainly comprise bibenzyl, dihydrophenanthrene, diphenanthrene ether, phenanthropyran, bibenzyl glucoside, polysaccharide, steroid, triterpene, lignin, anthraquinone and the like, and the bletilla striata is rich in chemical components and various in types. However, the existing extraction process research on the traditional Chinese medicine bletilla striata mainly focuses on the extraction of polysaccharide components of the bletilla striata, neglects the influence of other components in the bletilla striata on the whiteness and the quality, regarding non-polysaccharide components in the bletilla striata, bibenzyl, dihydrophenanthrene and the like are considered as main effective components of the bletilla striata, and the ethanol part, the ethyl acetate part and the chloroform part of the bletilla striata are found to have good pharmacological activity.
The inventor of the invention finds that in the prior art, most of the common bletilla pseudobulb extraction processes are empirical methods, namely, the steps and the operating parameters of the common bletilla pseudobulb extraction processes are designed, adjusted and optimized according to the past operating experience of a designer, the method is long in time consumption and high in dependence on the experience of personnel, so that the methods, namely the extraction processes and the methods for adjusting and optimizing the extraction processes, face greater probability, are unstable and unreliable and are troublesome in actual operation in the process of leaving a laboratory.
Disclosure of Invention
In order to solve the technical problems, the invention provides a rhizoma bletillae extracting solution obtaining method and an operating parameter selecting method thereof, and the rhizoma bletillae extracting solution obtaining method and the optimization method thereof can greatly improve the certainty with extremely low cost, so that the stability, reliability, simplicity, feasibility and the like of the whole operating process and the operating result are ensured.
The invention is realized by the following technical scheme.
The invention provides a method for selecting operation parameters for obtaining rhizoma bletillae extracting solution, which comprises the following steps:
and (3) chromatographic determination: carrying out chromatographic separation analysis on the bletilla striata extracting solution extracted by methanol under various operating parameters to obtain a corresponding fingerprint spectrum;
analysis and determination: selecting a fingerprint spectrum of the rhizoma bletillae extracting solution for statistical verification;
factor analysis optimization: and carrying out statistical analysis on the fingerprint, and selecting the optimal extraction operation parameters based on the statistical analysis result.
Before the step of chromatographic determination, the method also comprises the following steps:
preparing materials: and (5) supplementing the white and the extracting solution to loss, fixing the volume to 50ml, and filtering with a filter membrane for later use.
After the factor analysis optimization step, the method also comprises the following steps:
and (3) verification: and verifying the statistical difference among the various operating parameters by adopting single-factor analysis of variance or multi-factor analysis of variance and/or a minimum significant difference method for the fingerprint.
The statistical check is to calculate the relative standard deviation of the peak area and/or retention time of the characteristic peak in the fingerprint.
The rhizoma bletillae extract is obtained by heating rhizoma bletillae powder in methanol water bath; the operating parameters include methanol concentration, water bath temperature, heating time, and powder particle size.
The chromatographic separation and analysis adopts a C18 liquid chromatographic column.
In the chromatographic separation analysis, the mobile phase is acetonitrile-water, and the gradient elution procedure is as follows:
0-15 min, 10% -28% acetonitrile;
15-20 min, 28-36% acetonitrile;
20-25 min, 36% -39% acetonitrile;
25-30 min, 39-45% acetonitrile;
30-50 min, 45% -70% acetonitrile;
50-55 min, 70% -10% acetonitrile.
The statistical verification respectively verifies the precision, stability and repeatability of the fingerprint.
The invention also provides a method for obtaining the bletilla striata extract, which is to heat bletilla striata powder which is finer than 80 meshes in water bath at 40-60 ℃ for 2.5-3.5 hours by using 80% methanol.
The powder of bletilla striata sieved through a six-mesh sieve is heated in a water bath at 55 ℃ for 3 hours with 80% methanol.
The invention has the beneficial effects that: the method is simple and easy to implement, low in operation cost, stable and reliable; for the extraction of the bletilla striata, the extraction of the bletilla striata can be effectively realized as far as possible at the lowest cost, and the efficiency is high through optimization, the rejection rate is low, and the quality of a finished product is good; for rhizoma bletillae extraction optimization, dependence on human experience is avoided, meanwhile, excessive invalid experiments in a conventional method can also be avoided, so that optimization and adjustment of an extraction process can be achieved quickly and efficiently, the whole process does not depend on a single specific scene or expensive equipment, and therefore operation is easy, stable and reliable.
Drawings
FIG. 1 is a schematic view of an operation flow of a method for selecting bletilla striata extract acquisition operation parameters in the present invention;
FIG. 2 is an HPLC fingerprint of bletilla striata in an embodiment of the present invention;
FIG. 3 is a fingerprint after feature peaks are selected in accordance with an embodiment of the present invention;
FIG. 4 is a data diagram illustrating the influence of various factors on the total area of the characteristic peaks after the characteristic peaks are selected according to an embodiment of the present invention.
Detailed Description
The technical solution of the present invention is further described below, but the scope of the claimed invention is not limited to the described. The invention mainly provides a method for selecting operation parameters for obtaining rhizoma bletillae extracting solution, which comprises the following steps:
and (3) chromatographic determination: carrying out chromatographic separation analysis on the bletilla striata extracting solution extracted by methanol under various operating parameters to obtain a corresponding fingerprint spectrum;
analysis and determination: selecting a fingerprint spectrum of the rhizoma bletillae extracting solution for statistical verification;
factor analysis optimization: and carrying out statistical analysis on the fingerprint, and selecting the optimal extraction operation parameters based on the statistical analysis result.
Before the step of chromatographic determination, the method also comprises the following steps:
preparing materials: and (5) supplementing the white and the extracting solution to loss, fixing the volume to 50ml, and filtering with a filter membrane for later use.
After the factor analysis optimization step, the method also comprises the following steps:
and (3) verification: and verifying the statistical difference among the various operating parameters by adopting single-factor analysis of variance or multi-factor analysis of variance and/or a minimum significant difference method for the fingerprint.
The statistical verification is that the relative standard deviation of the peak area and/or retention time of the characteristic peak in the fingerprint is calculated. The rhizoma bletillae extract is obtained by heating rhizoma bletillae powder in methanol water bath; the operating parameters included methanol concentration, water bath temperature, heating time, powder particle size.
The chromatographic separation and analysis adopts a C18 liquid chromatographic column.
In the chromatographic separation analysis, the mobile phase is acetonitrile-water, and the gradient elution procedure is as follows:
0-15 min, 10% -28% acetonitrile;
15-20 min, 28-36% acetonitrile;
20-25 min, 36% -39% acetonitrile;
25-30 min, 39-45% acetonitrile;
30-50 min, 45% -70% acetonitrile;
50-55 min, 70% -10% acetonitrile.
The statistical verification respectively verifies the precision, stability and repeatability of the fingerprint.
The method for obtaining the bletilla striata extracting solution comprises the following steps: heating in water bath at 40-60 deg.C for 2.5-3.5 hr with 80% methanol.
Specifically, the ideal state without considering practical operation conditions and the extraction solution acquisition method are as follows: rhizoma bletilla powder sieved through a six-mesh (based on pharmacopeia sieve criteria) was heated in a water bath at 55 ℃ for 3 hours with 80% methanol.
Example 1
The basic acquisition scheme of the rhizoma bletillae extract is set as follows: heating rhizoma Bletillae powder in methanol water bath. The operating parameters in the extraction process are therefore mainly methanol concentration, water bath temperature, heating time, powder particle size.
Putting rhizoma bletilla powder into a round-bottom flask, performing water bath reflux extraction according to the factor conditions in Table 1, and sequentially inspecting the influence of the concentration (X1) of an extraction solvent, the crushing particle size (X2) of a medicinal material, the extraction time (X3) and the extraction temperature (X4) on the extraction effect. And (5) complementing the extract for loss, diluting to 50ml, and filtering with a 0.22 micron filter membrane for later use.
TABLE 1 Process optimization factors and levels
In the chromatographic separation and analysis, the chromatographic column is an American Agilent chromatographic column (Agilent 5 HC-18250 multiplied by 4.6mm), the mobile phase is acetonitrile-water, and the gradient elution procedure is as follows: 0-15 min, 10% -28% acetonitrile; 15-20 min, 28-36% acetonitrile; 20-25 min, 36% -39% acetonitrile; 25-30 min, 39-45% acetonitrile; 30-50 min, 45% -70% acetonitrile; 50-55 min, 70% -10% acetonitrile. The sample injection amount is 10 mu L; the flow rate is 1.0 mL/min; the detection wavelength is 270 nm; the column temperature was 25 ℃.
According to the factor levels in Table 1, B, C, D is given first, the optimal condition of the A factor is determined, then the determination of the B factor is performed after C, D is determined and the optimal condition of A is obtained, and so on, and finally the optimal condition is determined, as shown in Table 2.
TABLE 2 Experimental procedures for various factors
The finally obtained rhizoma bletilla fingerprint spectrum is shown in FIG. 2, wherein S1-S5 is the investigation of extraction methanol concentration, S6-S10 is the investigation of extraction temperature, S11-S15 is the investigation of extraction time, and S16-S20 is the investigation of rhizoma bletilla particle size.
From this comparison, it can be seen that, in the basic extraction scheme of this example, the optimal operating parameters are 80%, 6, 3h, and 55 ℃, i.e., the bletilla striata powder passing through a six-mesh sieve (based on pharmacopeia sieve standards) is heated in a water bath at 55 ℃ for 3 hours with 80% methanol.
Considering that the chemical industry environment is probably adopted during the final batch production, the industrial standard is applied, and the deviation is probably existed in the actual operation, therefore, the optimal scheme in the practical aspect is that bletilla striata powder which is finer than 80 meshes (based on the industrial sieve standard) is heated in water bath with the concentration of 80% for 2.5-3.5 hours at the temperature of 40-60 ℃.
And (3) checking precision: after the test sample is prepared by the method, the sample is continuously fed for 6 times, and the RSD% of the peak area of the characteristic peak in the obtained atlas is 0.06-0.09. RSD% of characteristic peak retention time is: 0.15 to 2.31. And (4) stability checking: after the test sample is prepared by the method, sample injection is carried out for 0h, 2h, 4h, 6h, 8h and 12h respectively, and the RSD% of the characteristic peak area in the obtained atlas is 0.08-0.66. RSD% of characteristic peak retention time is: 0.26 to 2.71.
And (3) repeatability checking: 6 parts of samples are prepared in parallel according to the method, and the RSD% of the peak area of the characteristic peak is 0.04-0.26 in the obtained atlas after sample injection. RSD% of characteristic peak retention time is: 0.26 to 1.77. The peak shapes with high separation degree are selected as characteristic peaks of the rhizoma bletillae for further analysis and verification of the extraction process of the rhizoma bletillae, the 16 characteristic peaks are shown in figure 3, the influence results of all factors (including methanol concentration, concentration in figure 4 for short, water bath temperature, temperature in figure 4 for short, heating time in figure 4 for short, time in figure 4, powder particle size, particle size in figure 4 for short) on the total area of the characteristic peaks are shown in figure 4, and the average number and standard deviation of the total area of the characteristic peaks of all factors are shown in table 3.
TABLE 3 mean and standard deviation of the total area of the peaks for each factor
It can be seen that the characteristic peak area corresponding to the temperature is the lowest, but the standard deviation is larger than other factors, which indicates that the intra-group variation of the characteristic peak corresponding to the temperature is larger, the influence of the change of the characteristic peak area on the characteristic peak area is larger, while the standard deviation of the characteristic peak corresponding to the time is much smaller than that of other groups, which indicates that the intra-group variation of the characteristic peak corresponding to the time is smaller, i.e. the influence of the extraction time on the peak area is smaller.
The above determinations were verified by one-way analysis of variance, and the results obtained using the total area of each fingerprint characteristic peak obtained under different processing conditions as a dependent variable are shown in table 4.
TABLE 4 results of one-way ANOVA
According to the results, the F value is 1.782, the P value is 0.191 and is more than 0.05, which indicates that the several factors have no significant difference, and indicates that the extraction concentration, the extraction time, the extraction temperature and the particle size have no statistical difference to influence the final extraction result to a certain extent.
The results of the LSD test are shown in table 5.
TABLE 5 LSD test results
It can be seen that there was no significant difference between the groups except that the P-value between time and temperature and time and particle size was close to 0.05.
It should be noted that, in this embodiment, the optimal extraction operation parameters are selected in a manner of item-by-item determination, the calculation is relatively simple, and one way of automatic calculation is to arrange the operation parameters according to the calculation result of the variance or the standard deviation; and secondly, sequentially selecting operation parameters according to the arrangement sequence. In practical application, however, the optimal extraction operation parameter may be selected in other manners, for example, clustering is a typical method.
From the results, the bletilla striata extraction process is influenced by a plurality of factors, and is a comprehensive multi-factor final result, based on the scheme of the invention, a large amount of experiment time can be saved, the optimal bletilla striata extraction scheme can be rapidly obtained based on the fingerprint spectrum and the statistical method, and optimization of the bletilla striata and the extraction method is realized.
It can be seen from the above embodiment that the technical scheme of the present invention is not limited to the basic scheme of heating rhizoma bletillae powder in methanol water bath in practical application, and the optimal selection of the operation parameters of the extraction method can be realized for other rhizoma bletillae extraction schemes with various operation parameters.
Claims (10)
1. A bletilla striata extracting solution obtaining operation parameter selection method is characterized by comprising the following steps: the method comprises the following steps:
and (3) chromatographic determination: carrying out chromatographic separation analysis on the bletilla striata extracting solution extracted by methanol under various operating parameters to obtain a corresponding fingerprint spectrum;
analysis and determination: selecting a fingerprint spectrum of the rhizoma bletillae extracting solution for statistical verification;
factor analysis optimization: and carrying out statistical analysis on the fingerprint, and selecting the optimal extraction operation parameters based on the statistical analysis result.
2. The method for extracting operational parameters from rhizoma bletillae extract as claimed in claim 1, wherein the method comprises: before the step of chromatographic determination, the method also comprises the following steps:
preparing materials: and (5) supplementing the white and the extracting solution to loss, fixing the volume to 50ml, and filtering with a filter membrane for later use.
3. The method for extracting operational parameters from rhizoma bletillae extract as claimed in claim 1, wherein the method comprises: after the factor analysis optimization step, the method also comprises the following steps:
and (3) verification: and verifying the statistical difference among the various operating parameters by adopting single-factor analysis of variance or multi-factor analysis of variance and/or a minimum significant difference method for the fingerprint.
4. The method for extracting operational parameters from rhizoma bletillae extract as claimed in claim 1, wherein the method comprises: the statistical check is to calculate the relative standard deviation of the peak area and/or retention time of the characteristic peak in the fingerprint.
5. The method for extracting operational parameters from rhizoma bletillae extract as claimed in claim 1, wherein the method comprises: the rhizoma bletillae extract is obtained by heating rhizoma bletillae powder in methanol water bath; the operating parameters include methanol concentration, water bath temperature, heating time, and powder particle size.
6. The method for extracting operational parameters from rhizoma bletillae extract as claimed in claim 1, wherein the method comprises: the chromatographic separation and analysis adopts a C18 liquid chromatographic column.
7. The method for extracting operational parameters from rhizoma bletillae extract as claimed in claim 1, wherein the method comprises: in the chromatographic separation analysis, the mobile phase is acetonitrile-water, and the gradient elution procedure is as follows:
0-15 min, 10% -28% acetonitrile;
15-20 min, 28-36% acetonitrile;
20-25 min, 36% -39% acetonitrile;
25-30 min, 39-45% acetonitrile;
30-50 min, 45% -70% acetonitrile;
50-55 min, 70% -10% acetonitrile.
8. The method for extracting operational parameters from rhizoma bletillae extract as claimed in claim 1, wherein the method comprises: the statistical verification respectively verifies the precision, stability and repeatability of the fingerprint.
9. A method for obtaining rhizoma bletillae extract is characterized by comprising the following steps: heating in water bath at 40-60 deg.c for 2.5-3.5 hr with 80% concentration methanol in bletilla tuber powder of 80 mesh size.
10. The method for obtaining rhizoma bletillae extract as claimed in claim 9, wherein the method comprises the following steps: the powder of bletilla striata sieved through a six-mesh sieve is heated in a water bath at 55 ℃ for 3 hours with 80% methanol.
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| US20030152651A1 (en) * | 2000-12-21 | 2003-08-14 | Tianjin Tasly Pharmaceutical Co., Ltd., China | Herbal composition for angina pectoris, method to prepare same and uses thereof |
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| CN107340348A (en) * | 2017-06-07 | 2017-11-10 | 贵阳中医学院 | A kind of method for building up of Bletilla striata medicinal materials HPLC finger-prints |
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2020
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| US20030152651A1 (en) * | 2000-12-21 | 2003-08-14 | Tianjin Tasly Pharmaceutical Co., Ltd., China | Herbal composition for angina pectoris, method to prepare same and uses thereof |
| CN103344717A (en) * | 2013-06-25 | 2013-10-09 | 广州中医药大学 | Method for establishing rhizoma bletillae high performance liquid chromatography (HPLC) fingerprint spectrum and standard fingerprint spectrum thereof |
| CN107340348A (en) * | 2017-06-07 | 2017-11-10 | 贵阳中医学院 | A kind of method for building up of Bletilla striata medicinal materials HPLC finger-prints |
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