CN109991341B - Quality detection method of radix polygoni multiflori preparata - Google Patents
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Abstract
The invention belongs to the technical field of traditional Chinese medicine quality standard detection, and discloses a quality detection method of radix polygoni multiflori preparata. Establishing radix Polygoni Multiflori Preparata fingerprint by high performance liquid chromatography; the activity of blood and essence can be evaluated by erythrocyte count, hemoglobin content, erythrocyte pressure and platelet count. Performing spectrum-effect correlation analysis by using a grey correlation degree analysis method, determining that the main effective components in the alcohol-soluble part and the micromolecular sugar part of the radix polygoni multiflori preparata are respectively 16 and 5, and establishing the quality standard of the alcohol-soluble part and the micromolecular sugar part of the radix polygoni multiflori preparata, wherein the ratio of the main active component peak of the alcohol-soluble part is not less than 61.93 percent, and the ratio of the main active component peak of the micromolecular sugar part is not less than 62.58 percent. The method provided by the invention establishes a quality control mode capable of directly judging the activity of the vital essence and blood of the radix polygoni multiflori preparata through the fingerprint spectrum based on the spectrum effect relationship of the radix polygoni multiflori preparata, has the advantages of simplicity, rapidness, objectivity, accuracy and the like, and ensures that the quality control system of the radix polygoni multiflori preparata is more scientific and perfect.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine quality standard detection, and particularly relates to a quality detection method of prepared fleece flower root, in particular to a quality detection method of the activity of benefiting essence and blood of prepared fleece flower root based on a spectrum effect relationship.
Background
Polygonum multiflorum (Polygonum multiflorum Thunb.) also known as Polygonum multiflorum, Stephania delavayi and Tuber fleeceflower stem is a perennial winding vine plant of Polygonum multiflorum of Polygonaceae, and has thick root tuber, oblong shape and dark brown color. The tuber root of Polygonum multiflorum Thunb, also called raw Polygonum multiflorum Thunb, can be used directly as a medicine. Raw fleece-flower root is bitter, sweet and colored in taste and slightly warm in nature, enters liver, heart and kidney channels, has the effects of detoxifying, resolving carbuncle, preventing malaria and relaxing bowel, and is clinically commonly used for sores and carbuncles, scrofula, rubella pruritus, chronic malaria, weak constitution, intestinal dryness and constipation and the like.
The raw fleece-flower root is deeply processed to obtain the prepared fleece-flower root. Steaming raw radix Polygoni Multiflori to brown color inside and outside to obtain radix Polygoni Multiflori Preparata. The surface of the prepared fleece-flower root is dark brown or tan and uneven. Hard, keratotic, tan or black. Light smell, slightly sweet and bitter taste. The prepared fleece-flower root has the effects of tonifying liver and kidney, benefiting essence and blood, blackening beard and hair, strengthening muscles and bones, eliminating turbid pathogen and reducing blood fat, and is a common medicament for treating blood deficiency, slight yellow, dizziness and tinnitus, early white beard and hair and soreness and weakness of waist and knees clinically. Radix Polygoni Multiflori Preparata is slightly warm and not dry, tonify but not greasy, Yun from Ben Cao gang mu: blood nourishing and liver benefiting, essence securing and kidney tonifying, bone and muscle strengthening, beard and hair blackening, which are good tonics, even reputed as a good medicine which is not cold and dry and has the functions of being above the medicines of rehmannia root and asparagus.
However, the quality of prepared fleece flower root in the current market is not uniform, so that the efficacy of the prepared fleece flower root is influenced by different degrees, the qualitative analysis of a few active ingredients is only focused on the quality standard of the prepared fleece flower root, and the research on the spectrum effect relationship of the prepared fleece flower root is few. Therefore, an effective and scientific quality assessment method for the advantages and disadvantages of the radix polygoni multiflori preparata medicinal materials, in particular to a method for detecting the advantages and disadvantages of the blood and essence activities of the radix polygoni multiflori preparata is not available at present.
Disclosure of Invention
In view of the above, the present invention provides a quality detection method for radix polygoni multiflori preparata, which uses a grey correlation analysis method to perform spectrum effect correlation study on a fingerprint of radix polygoni multiflori preparata and activity of blood and essence to obtain a result that the activity of blood and essence can be directly predicted through the fingerprint, so as to judge the quality of radix polygoni multiflori preparata.
In order to achieve the above object, the present invention provides the following technical solutions:
a quality detection method of radix polygoni multiflori preparata comprises the following steps:
(1) preparing alcohol-soluble part and micromolecular sugar part of radix Polygoni Multiflori Preparata;
(2) establishing fingerprint of alcohol-soluble part and micromolecular sugar part of radix Polygoni Multiflori Preparata;
(3) the pharmacodynamics research of the essence and blood benefiting part and the micromolecular sugar part of the prepared fleece flower root,
(4) determining the correlation degree of the common peak in the radix Polygoni Multiflori Preparata fingerprint and the effect on replenishing essence and blood by using a grey correlation analysis method, thereby determining the influence of different common peaks of alcohol-soluble parts and small molecular sugar parts in radix Polygoni Multiflori Preparata on the effect on replenishing essence and blood;
(5) establishing quality standard according to the influence of different common peaks of alcohol-soluble part and micromolecular sugar part in radix Polygoni Multiflori Preparata on its effect of benefiting essence and blood, and directly evaluating the quality of radix Polygoni Multiflori Preparata by using the established quality standard.
Preferably, the preparation method of the alcohol-soluble part of the prepared fleece flower root in the step 1) comprises the steps of adding water into prepared fleece flower root powder for extraction, concentrating an extracting solution, adding absolute ethyl alcohol until the alcohol content is 50%, and centrifuging; adding absolute ethanol into the supernatant, concentrating until the alcohol content is 80%, and centrifuging; concentrating the supernatant, enriching with D101 type macroporous resin, eluting with water, and eluting with 95% ethanol.
Preferably, the preparation method of the small molecular sugar fraction of prepared fleece flower root in the step 1) comprises the steps of adding water into prepared fleece flower root powder for extraction, concentrating the extract, adding absolute ethyl alcohol, and centrifuging; adding absolute ethyl alcohol into the supernatant, concentrating, and centrifuging; collecting precipitate, dissolving, adding anhydrous ethanol, concentrating, and centrifuging; drying the supernatant to obtain the final product.
Preferably, the alcohol soluble part fingerprint spectrum establishment in the step 2) comprises the following steps: 1) determining HPLC chromatographic conditions; 2) establishing multiple batches of alcohol-soluble part fingerprint spectrums of prepared fleece flower root, determining a common chromatographic peak by a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, and analyzing the similarity among samples; 3) and (3) qualitatively analyzing the alcohol-soluble part of the prepared fleece flower root by using HPLC-ESI-MS to identify the active ingredients in the alcohol-soluble part.
Further, preferably, the HPLC chromatographic condition of the alcohol-soluble part fingerprint is that an Agela Venusil MP C18(250 multiplied by 4.6mm, 5 mu m) chromatographic column is adopted, the mobile phase consists of acetonitrile-0.1% phosphoric acid water, the flow rate is 1.0mL/min, the sample injection amount is 10 mu L, the column temperature is 20 ℃, and the detection wavelength is 254 nm; the detector is an SPD-M20A diode array detector, and the gradient elution procedure is as follows:
preferably, the establishment of the fingerprint of the small molecule sugar part in the step 2) comprises the following steps: determining HPLC chromatographic conditions; establishing fingerprint spectrums of micromolecular sugar parts of a plurality of prepared fleece-flower roots, determining common chromatographic peaks through a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, and analyzing the similarity among samples; thirdly, a standard curve is prepared by the logarithmic value of the molecular weight of the standard substance to the corresponding retention time so as to obtain the molecular weight distribution condition of the small molecular sugar part of the radix polygoni multiflori preparata.
Further, preferably, the HPLC chromatographic conditions of the fingerprint of the small molecule sugar part are that a TSK gel GMPWxl (300X 7.8mm, 13 μ M) chromatographic column is adopted, 0.05M ammonium acetate is used for isocratic elution, the flow rate is 0.4mL/min, the sample injection amount is 3 μ L, and the column temperature is 40 ℃; the detector is an agent 1260 definition ELSD.
The quality detection method provided by the invention comprises the step 3) of researching the pharmacodynamics of the essence and blood benefiting of the alcohol-soluble part and the micromolecular sugar part of the prepared fleece-flower root.
Preferably, the pharmacodynamic study of the essence and blood benefiting effect of the alcohol-soluble part and the small molecule sugar part of the prepared fleece flower root in the step 3) is to detect the influence of a plurality of batches of alcohol-soluble parts and small molecule sugar parts of the prepared fleece flower root on the number of red blood cells, the hematocrit, the number of platelets and the content of hemoglobin.
The quality detection method comprises the step 4) of determining the correlation degree of the common peak in the radix polygoni multiflori preparata fingerprint and the effect of benefiting essence and blood by using a grey correlation analysis method to carry out spectrum-effect correlation analysis, thereby determining the influence of different common peaks of alcohol-soluble parts and small molecular sugar parts in the radix polygoni multiflori preparata on the effect of benefiting essence and blood.
Wherein the determination of the correlation degree is carried out by taking the medicinal effect data of benefiting essence and blood of different radix Polygoni Multiflori Preparata batches as reference series, taking the areas of 32 common peaks and peaks of different radix Polygoni Multiflori Preparata batches as comparison series, and analyzing the correlation coefficient r (X) of the common peaks and the medicinal effect index through dimensionless processing0(k),Xi(k) And degree of correlation r (X)0,Xi) The calculation formula is as follows:
in the above formula, reference number sequence X0(k) Wherein k is radix Polygoni Multiflori Preparata of different batches, and comparing the number series Xi(k) Wherein i is each common peak of the fingerprint,
and
respectively recording the minimum value and the maximum value in the difference values of the reference number array and the comparison number array in all batches as delta min and delta max; i X0(k)-Xi(k) I is the absolute difference between the reference and comparison series at k batches, marked by Delta0,i(k) (ii) a In order to attenuate the distortion caused by too large Δ k, a resolution coefficient ρ, ρ ∈ (0,1) is introduced, and in practical application, 0.5 is taken.
In the above formula, reference number sequence X0(k) Wherein k is radix Polygoni Multiflori Preparata of different batches, and comparing the number series Xi(k) Wherein i is each common peak of the fingerprint; the closer the r value is to 1, the greater the correlation.
The result shows that the main effective components with high influence correlation degree on the efficacy of the essence and blood of the alcohol-soluble part of the prepared fleece flower root are 16, mainly comprising stilbene glucoside and a few flavonoids and anthraquinones.
The main effective components of the small molecular sugar part of the prepared fleece-flower root which have higher influence on the efficacy of benefiting essence and blood are 5, and the molecular weight range is mainly concentrated below 3000 Da.
The quality detection method comprises the step 5) of establishing a quality standard according to the influence of different common peaks of an alcohol-soluble part and a small molecular sugar part in the prepared fleece-flower root on the efficacy of the alcohol-soluble part and the small molecular sugar part in the fleece-flower root, and directly evaluating the quality of the prepared fleece-flower root by utilizing the established quality standard.
Preferably, the quality standard of the alcohol-soluble part in the radix polygoni multiflori preparata is that octadecylsilane chemically bonded silica is used as a filling column, acetonitrile is used as a mobile phase A, 0.1% phosphoric acid water is used as a mobile phase B, gradient elution is carried out according to the table 1, and the flow rate is 1.0 mL/min; the detection wavelength was 254 nm. The theoretical plate number is not less than 2000 calculated according to the peak of 2,3,5, 4' -tetrahydroxystilbene-2-O-beta-D-glucoside. The content of 16 main active component peaks of the alcohol-soluble part is not less than 61.93%, and the similarity between the fingerprint of the sample and the fingerprint of the reference is not less than 0.90 according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprints.
Preferably, the mass standard of the small molecular sugar part in the radix polygoni multiflori preparata in the step 5) is that porous methacrylate is used for forming a filling column; isocratically eluting with 0.05M ammonium acetate at 40 deg.C and flow rate of 0.4 mL/min; the ELSD detector parameters were: the drift tube temperature is 45 ℃, the carrier gas flow rate is 1.2L/min, the ratio of 5 main active ingredient peaks at the small molecular sugar part is not less than 62.58%, and the similarity of the fingerprint of the sample to the fingerprint of the reference sample is not less than 0.90 according to the traditional Chinese medicine chromatogram fingerprint similarity evaluation system.
The quality detection method of the prepared fleece flower root adopts a high performance liquid chromatography to establish a prepared fleece flower root fingerprint; the activity of blood and essence can be evaluated by erythrocyte count, hemoglobin content, erythrocyte pressure and platelet count. Performing spectrum-effect correlation analysis by using a grey correlation degree analysis method, determining that the main effective components in the alcohol-soluble part and the micromolecular sugar part of the radix polygoni multiflori preparata are respectively 16 and 5, and establishing the quality standard of the alcohol-soluble part and the micromolecular sugar part of the radix polygoni multiflori preparata, wherein the ratio of the main active component peak of the alcohol-soluble part is not less than 61.93 percent, and the ratio of the main active component peak of the micromolecular sugar part is not less than 62.58 percent. The method provided by the invention establishes a quality control mode capable of directly judging the activity of the vital essence and blood of the radix polygoni multiflori preparata through the fingerprint spectrum based on the spectrum effect relationship of the radix polygoni multiflori preparata, has the advantages of simplicity, rapidness, objectivity, accuracy and the like, and ensures that the quality control system of the radix polygoni multiflori preparata is more scientific and perfect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a flow chart of the extraction and preparation of each part of radix Polygoni Multiflori Preparata;
FIG. 2 is HPLC-DAD fingerprint of alcohol-soluble part of radix Polygoni Multiflori Preparata;
FIG. 3 shows the common peaks of alcohol-soluble portion fingerprints of radix Polygoni Multiflori Preparata;
FIG. 4 is an ion flow diagram of an alcohol-soluble part of radix Polygoni Multiflori Preparata;
FIG. 5 is a chromatogram peak spectrum of known structure of alcohol-soluble part of radix Polygoni Multiflori Preparata;
FIG. 6 is HPLC-ELSD fingerprint of small molecule sugar fraction of radix Polygoni Multiflori Preparata;
FIG. 7 shows the common peaks of the fingerprint of the small molecular sugar of radix Polygoni Multiflori Preparata.
Detailed Description
The invention discloses a quality detection method of radix polygoni multiflori preparata. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1. preparation of active site of radix Polygoni Multiflori Preparata:
1) alcohol-soluble part: heating and reflux-extracting a certain amount of radix Polygoni Multiflori Preparata powder with 10 times of deionized water twice, and rotary-steaming under reduced pressure to concentrate the extractive solution; adding anhydrous ethanol into the concentrated solution until the ethanol content is 50%, and centrifuging; adding absolute ethanol into the supernatant, concentrating until the alcohol content is 80%, and centrifuging; concentrating the supernatant, enriching with D101 type macroporous resin, eluting with water, and eluting with 95% ethanol to obtain ethanol soluble part of radix Polygoni Multiflori Preparata, with the flow chart shown in figure 1;
2) small molecule sugar site: extracting a certain amount of radix Polygoni Multiflori Preparata powder with 10 times of deionized water twice, and concentrating the extractive solution under reduced pressure by rotary evaporation; adding anhydrous ethanol into the concentrated solution until the ethanol content is 50%, and centrifuging; adding absolute ethanol into the supernatant, concentrating until the alcohol content is 80%, centrifuging, and dissolving the precipitate in water to obtain crude polysaccharide solution; adding absolute ethanol into the crude polysaccharide solution, concentrating until the alcohol content is 50%, and centrifuging; freeze drying the supernatant to obtain the final product, which is shown in figure 1.
Example 2 alcohol-soluble portion fingerprint creation and analysis
1) Test solution: weighing appropriate amount of 16 batches of alcohol-soluble part of radix Polygoni Multiflori Preparata (each batch is prepared into alcohol-soluble part by the method of example 1), and adding diluted ethanolAdding 529mL of alcohol, diluting with water to 1000mL to obtain diluted ethanol solution containing C at 20 deg.C2H5OH should be 49.5% -50.5%) to obtain test solution with crude drug content of 0.5g/mL, and filtering with 0.45 μm microporous membrane. Wherein the information of each batch of samples is shown in table 1;
TABLE 1 radix Polygoni Multiflori Preparata sample information
Note: different numbers under the same blasting method represent the blasting products produced in different batches under each blasting method
The method for preparing the polygonum multiflorum by using the black beans comprises the following steps: taking 20.0kg of polygonum multiflorum decoction pieces, putting the polygonum multiflorum decoction pieces into a container, adding 5.0L of black bean juice according to the general rule of steaming method (0213) of 2015 version of Chinese pharmacopoeia, stirring uniformly, continuously stirring to enable the polygonum multiflorum decoction pieces to uniformly adsorb the black bean juice, putting the polygonum multiflorum decoction pieces into a stainless steel barrel (without a cover) after the black bean juice is absorbed completely (about 9h), continuously steaming for 32h, taking samples at different time points, and continuously steaming the rest samples after the rest samples are turned over while being hot. Taking out 2 barrels each time, cooling, mixing with the steam, air drying surface water, and drying in a 50 deg.C oven until the water content is less than 12%.
The method for high-pressure production of polygonum multiflorum comprises the following steps: taking 20.0kg of polygonum multiflorum decoction pieces, placing in a container, adding 5.0L of water according to the general rule of steaming method (0213) of 2015 version of Chinese pharmacopoeia, stirring uniformly, stirring continuously to ensure that the polygonum multiflorum decoction pieces absorb water uniformly, placing the polygonum multiflorum decoction pieces in a stainless steel barrel (without covering) after the water is absorbed completely (about 9h), steaming for 32h continuously, sampling at different time points, stirring the rest samples while the rest samples are hot, and continuing steaming. Taking out 2 barrels each time, cooling, mixing with the steam, air drying surface water, and drying in a 50 deg.C oven until the water content is less than 12%.
The method for preparing the polygonum multiflorum by using the black bean and yellow wine comprises the following steps: taking 20.0kg of polygonum multiflorum decoction pieces, placing in a container, adding 5.0L of bean juice yellow wine according to the general rule of steaming method (0213) of the 2015 version of Chinese pharmacopoeia, stirring uniformly, continuously stirring to enable the polygonum multiflorum decoction pieces to uniformly adsorb the bean juice yellow wine, placing in a stainless steel barrel (without a cover) after the bean juice yellow wine is completely absorbed, continuously steaming for 32 hours, sampling at different time points, stirring the rest samples while the samples are hot, and continuously steaming. Taking out 2 barrels each time, cooling, mixing with the steam, air drying surface water, and drying in a 50 deg.C oven until the water content is less than 12%.
2) Chromatographic conditions are as follows: an Agela Venusil MP C18 (250X 4.6mm, 5 μm) chromatographic column is adopted, the mobile phase consists of acetonitrile-0.1% phosphoric acid water, the gradient elution procedure is shown in Table 2, the flow rate is 1.0mL/min, the sample injection amount is 10 μ L, the column temperature is 20 ℃, and the detection wavelength is 254 nm; the detector is an SPD-M20A diode array detector;
TABLE 2 gradient elution procedure of HPLC fingerprint of alcohol-soluble part of prepared fleece flower root
3) And (3) spectrum analysis:
firstly, fingerprint spectrum: sampling different batches of sample solutions, respectively, and establishing alcohol soluble part fingerprint (figure 2) of radix Polygoni Multiflori Preparata according to the above chromatographic conditions, wherein the common peak is shown in figure 3, and the peak area is shown in table 3;
table 316 batch shouwu alcohol soluble part common peak area
Similarity analysis: inputting the chromatograms of the test solution of different batches into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, setting a time window, and selecting a chromatographic peak with better separation degree and larger peak area for matching. Finally, determining that chromatograms of alcohol-soluble parts of the prepared fleece-flower root in different batches contain 32 common peaks, wherein the non-common peak area of each sample accounts for less than 10% of the total peak area, and the similarity values of the non-common peak areas are all greater than 0.90 (see table 4), which indicates that the prepared fleece-flower root medicinal material in 16 batches has good similarity and meets the requirement of a fingerprint;
TABLE 4 chromatogram similarity of alcohol-soluble part of radix Polygoni Multiflori Preparata by different processing methods
| Numbering | Degree of similarity | Numbering | Degree of similarity |
| S01 | 0.998 | S09 | 0.997 |
| S02 | 0.999 | S10 | 0.998 |
| S03 | 0.999 | S11 | 0.997 |
| S04 | 0.998 | S12 | 0.997 |
| S05 | 0.998 | S13 | 0.999 |
| S06 | 0.999 | S14 | 0.997 |
| S07 | 0.998 | S15 | 0.998 |
| S08 | 0.998 | S16 | 0.998 |
③ chromatographic peak qualitative analysis: ESI-MS/MS method, Agilent 1260-6530HPLC-1-TOF, anion mode, mobile phase 0.1% formic acid. Ion source parameters: drying N2Temperature of 350 ℃, flow rate of 9.0L/min, capillary voltage of 3300V, atomization pressure of 40psi, and scanning range m/z of 100-1000. Data Acquisition and processing were done using Agilent HPLC-Q-TOF-MS MassHunter Acquisition Software Version b.04.00 Software (Agilent, USA) and MassHunter Workstation quantization Analysis Software Version b.06.00 Software (Agilent, USA), respectively. Wherein the parameters for measuring the element composition are set as follows: c, 0-100; h, 0-200; o, 0-100; n,0-3, preliminarily assuming that all compounds do not contain elements such as nitrogen, phosphorus, sulfur, bromine, chlorine and the like; the maximum allowable error range is + -5 ppm.
The total ion flow diagram of alcohol-soluble part of radix Polygoni Multiflori Preparata is shown in figure 4, 25 chromatographic peaks are assigned by sorting retention time, ultraviolet spectral characteristics and MS fragment ion information of each chromatographic peak, possible structures of the chromatographic peaks are shown in table 5, the chromatographic peaks are compared with HPLC-UV chromatograms, and identified compounds are marked (shown in figure 5).
TABLE 5 Mass Spectrometry data and cracking method of alcohol-soluble part components of radix Polygoni Multiflori Preparata
Through qualitative analysis of alcohol-soluble parts of prepared fleece flower root, 25 components are identified, mainly including stilbene and anthraquinone components, and also including flavone and phenolic acid.
Example 3 establishment and analysis of Small molecule sugar site fingerprints
1) Test solution: weighing appropriate amount of 16 batches of radix Polygoni Multiflori Preparata small molecule sugar parts (prepared by the method of example 1 in each batch), ultrasonically dissolving with 30% ethanol to obtain test solution with crude drug content of 0.5g/mL, and filtering with 0.45 μm microporous membrane; wherein the information of each batch of samples is the same as that of example 2;
2) chromatographic conditions are as follows: eluting with TSK gel GMPWxl (300 × 7.8mm, 13 μ M) chromatographic column and 0.05M ammonium acetate at 0.4mL/min flow rate, 3 μ L sample introduction rate and 40 deg.C; the detector is an agent 1260 definition ELSD;
3) and (3) spectrum analysis:
firstly, fingerprint spectrum: sampling different batches of sample solutions, respectively, and establishing fingerprint (figure 6) of small molecule sugar part of radix Polygoni Multiflori Preparata according to the above chromatographic conditions, wherein the common peak is shown in figure 7, and the peak area is shown in table 6;
table 616 batch processed fleece-flower root small molecule sugar part common peak area
| S01 | S02 | S03 | S04 | S05 | S06 | S07 | S08 | S09 | S10 | S11 | S12 | S13 | S14 | | S16 | |
| Peak | ||||||||||||||||
| 1 | 2070 | 1387 | 1118 | 989 | 616 | 1005 | 995 | 2440 | 1683 | 2480 | 832 | 714 | 1821 | 1673 | 871 | 1837 |
| |
9098 | 12020 | 12427 | 8590 | 6362 | 17594 | 20232 | 19567 | 10862 | 12271 | 10994 | 9972 | 19663 | 9695 | 8153 | 19798 |
| |
2977 | 6152 | 8218 | 3926 | 5432 | 4801 | 20814 | 12238 | 6768 | 4777 | 4653 | 6386 | 7270 | 3842 | 3978 | 7394 |
| |
6583 | 8245 | 9307 | 6272 | 5336 | 12548 | 15209 | 13091 | 8381 | 7756 | 5371 | 5782 | 13616 | 6123 | 5630 | 13669 |
| |
471 | 464 | 463 | 455 | 341 | 725 | 985 | 523 | 296 | 349 | 311 | 323 | 655 | 282 | 317 | 663 |
Similarity analysis: inputting the chromatograms of the test solution of different batches into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, setting a time window, and selecting a chromatographic peak with better separation degree and larger peak area for matching. Finally, determining that chromatograms of the small molecular sugar parts of the prepared fleece-flower roots in different batches contain 6 common peaks, wherein the non-common peak area of each sample accounts for less than 10% of the total peak area, and the similarity values of the samples are all greater than 0.90 (see table 7), which indicates that the prepared fleece-flower roots in 16 batches have good similarity and meet the requirement of a fingerprint;
TABLE 7 chromatogram similarity of alcohol-soluble part of radix Polygoni Multiflori Preparata by different processing methods
(iii) molecular weight distribution: a calibration curve (see Table 8) was prepared from the logarithmic value of the molecular weight (Mp) of the polysaccharide standard substance versus the corresponding retention time (tR), and then the molecular weight distribution of the small-molecule sugar site of radix Polygoni Multiflori Preparata was estimated from the calibration curve as shown in Table 9.
TABLE 8 measurement and calculation of molecular weight distribution
TABLE 9 measurement and calculation of molecular weight distribution
| Chromatographic | tR | Mp | |
| 1 | 20.919 | 60402 | |
| 2 | 24.694 | 2226 | |
| 3 | 25.413 | 1187 | |
| 4 | 27.544 | 184 | |
| 5 | 31.099 | 8 | |
| 6 | 36.434 | / |
EXAMPLE 4 study of drug Effect of alcohol-soluble part
1) Animal grouping and drug intervention: the method comprises the following steps of randomly and averagely dividing mice into 19 groups, wherein each group comprises 10 mice, feeding physiological saline to a blank group and a model group through intragastric administration, feeding donkey-hide gelatin solution (crude drug amount is 5g/kg) to a positive drug group, and feeding corresponding drugs to 16 alcohol-soluble radix polygoni multiflori groups through intragastric administration (alcohol-soluble parts of 16 batches of radix polygoni multiflori in example 2, crude drug amount is 4g/kg), wherein the intragastric administration amount is 0.1mL/10g, and the continuous 9 days are carried out;
2) preparation of blood deficiency model: injecting cyclophosphamide salt solution (40mg/kg) into abdominal cavity of mice in the model group, the positive drug group and 16 ethanol-soluble fractions of radix Polygoni Multiflori Preparata, injecting equal volume of normal saline into mice in the blank group, wherein the dosage is 0.1mL/10g, and continuously administering for 5 days while molding;
3) the experimental results are as follows: as shown in Table 10, the number of erythrocytes, the hemoglobin content, the hematocrit and the platelet number were all significantly reduced (P < 0.01 or P < 0.001) in the model group mice as compared with the blank group; compared with the model group, the alcohol-soluble part of the prepared fleece-flower root in each batch has the improvement effect on peripheral hemogram of a blood-deficiency mouse to different degrees, wherein the number of red blood cells, the number of platelets and the content of hemoglobin are better improved (P is less than 0.05 or P is less than 0.01 or P is less than 0.001).
TABLE 10 influence of alcohol-soluble fraction of radix Polygoni Multiflori Preparata in different batches on peripheral hemogram of blood-deficiency model mouse
| Group of | Erythrocyte count (10)12/L) | Hemoglobin (g/L) | Packed cell volume (%) | Platelet count (10)9/L) |
| Blank group | 9.23±0.47 | 144.10±4.18 | 57.22±3.02 | 1254.50±162.90 |
| Model set | 6.79±0.49### | 108.40±5.93### | 45.46±3.77### | 1014.30±160.70## |
| Positive drug group | 7.60±0.26*** | 122.40±5.44*** | 47.51±2.24 | 1226.80±154.08** |
| S01 | 7.75±0.59*** | 123.90±8.12*** | 50.91±3.80** | 1243.90±285.27* |
| S02 | 7.48±0.44** | 119.90±7.49** | 49.24±2.78* | 1456.50±237.39*** |
| S03 | 7.48±0.91* | 118.60±9.25** | 49.34±6.65 | 1244.00±283.04* |
| S04 | 7.31±0.40* | 118.00±6.33** | 47.35±2.62 | 1233.60±284.59* |
| S05 | 7.43±0.32** | 120.20±5.18*** | 46.74±1.75 | 1332.60±131.77*** |
| S06 | 7.66±1.03* | 125.60±16.91** | 49.07±6.85 | 1274.40±217.44** |
| S07 | 7.86±0.62*** | 126.60±10.02*** | 48.96±3.49* | 1302.50±248.93** |
| S08 | 7.19±0.51 | 115.40±8.59* | 47.66±3.97 | 1175.80±299.97 |
| S09 | 7.32±0.49* | 116.70±7.17* | 48.76±3.28 | 1223.70±225.00* |
| S10 | 7.76±0.75** | 125.20±11.59*** | 51.23±4.14** | 926.90±320.26 |
| S11 | 7.09±0.43 | 114.20±5.37* | 45.22±1.85 | 1253.80±148.33** |
| S12 | 7.21±0.40 | 116.90±7.75* | 45.96±1.83 | 1284.40±223.40** |
| S13 | 7.39±0.73* | 120.00±10.73** | 46.49±2.93 | 1251.70±336.83* |
| S14 | 7.38±0.53* | 121.00±10.04** | 46.95±3.90 | 1348.10±249.93** |
| S15 | 7.42±0.39** | 120.20±6.37*** | 47.24±2.49 | 1321.50±221.79** |
| S16 | 7.51±0.37** | 122.30±6.55*** | 47.57±2.56 | 1295.00±192.87** |
Note: in comparison to the blank set, the data is,##P<0.01，###P<0.001; in comparison to the set of models,*P<0.05，**P<0.01，***P<0.001
example 5 study of the pharmacodynamics of Small molecule sugar moieties
1) Animal grouping and drug intervention: the mice are randomly and evenly divided into 19 groups, 10 mice in each group, physiological saline is fed into the blank group and the model group through gastric lavage, donkey-hide gelatin solution (crude drug amount is 5g/kg) is fed into the positive drug group, corresponding drugs are fed into the 16 fleece-flower root small molecular sugar part groups through gastric lavage (the small molecular sugar parts of the 16 batches of fleece-flower root in the embodiment 3, the crude drug amount is 4g/kg), the gastric lavage amount is 0.1mL/10g, and the continuous 9 days are carried out;
2) preparation of blood deficiency model: injecting cyclophosphamide salt solution (40mg/kg) into abdominal cavity of mice of the model group, the positive drug group and 16 radix Polygoni Multiflori Preparata micromolecule sugar part groups, injecting equal volume of normal saline into blank mice, wherein the dosage is 0.1mL/10g, continuously administering for 5 days, and molding and simultaneously administering;
3) the experimental results are as follows: as shown in Table 11, the number of erythrocytes, the hemoglobin content, the hematocrit and the platelet number were all significantly reduced (P < 0.01 or P < 0.001) in the model group mice as compared with the blank group; compared with the model group, the small molecular sugar parts of the prepared fleece-flower roots in each batch have different degrees of improvement effects on peripheral hemogram of a blood-deficiency mouse, wherein the number of red blood cells, the content of hemoglobin and the hematocrit are better improved (P is less than 0.05 or P is less than 0.01 or P is less than 0.001).
TABLE 11 influence of different batches of small-molecule sugar fraction of radix Polygoni Multiflori Preparata on peripheral hemogram of blood deficiency model mouse
| Group of | Erythrocyte count (10)12/L) | Hemoglobin (g/L) | Packed cell volume (%) | Platelet count (10)9/L) |
| Blank group | 8.97±0.58 | 141.80±6.76 | 49.67±7.67 | 1489.50±291.47 |
| Model set | 6.46±0.52### | 112.70±7.24### | 38.73±4.66## | 1178.90±137.31## |
| Positive drug group | 7.90±0.75*** | 128.20±8.89*** | 46.38±6.87** | 1400.40±244.84* |
| S01 | 7.75±0.67*** | 125.00±11.57** | 42.83±4.39* | 1355.70±295.08 |
| S02 | 8.26±1.25*** | 134.30±17.66** | 52.71±6.42*** | 1372.00±188.25** |
| S03 | 7.40±0.82* | 120.70±13.22 | 43.42±6.49 | 1433.80±338.43* |
| S04 | 7.44±0.22*** | 121.00±5.29** | 44.25±6.18* | 1561.10±366.56** |
| S05 | 7.90±1.05** | 123.90±17.06* | 47.35±7.09** | 1417.10±255.11** |
| S06 | 7.85±0.92** | 122.40±10.39* | 44.59±8.39* | 1370.50±271.99* |
| S07 | 7.74±0.92** | 123.30±14.45* | 45.77±6.54** | 1371.20±219.46* |
| S08 | 7.40±0.53** | 121.10±6.33** | 40.14±4.61 | 1438.80±304.90* |
| S09 | 7.21±0.41* | 117.30±8.26 | 38.60±4.28 | 1451.80±227.73** |
| S10 | 7.79±0.87** | 125.40±13.40* | 42.99±3.93* | 1076.60±168.78 |
| S11 | 7.82±0.84** | 126.90±12.47** | 45.73±4.79** | 1269.20±278.02 |
| S12 | 7.92±1.20** | 127.20±17.61* | 45.54±8.72* | 1281.20±394.48 |
| S13 | 7.80±0.68*** | 127.10±11.80** | 46.86±5.19*** | 1453.20±321.10* |
| S14 | 7.81±1.01** | 123.50±17.09* | 46.35±7.50** | 1279.60±216.66 |
| S15 | 7.84±0.63*** | 125.80±8.57** | 48.54±5.58*** | 1343.40±223.62* |
| S16 | 7.95±1.27** | 127.10±19.35* | 47.49±9.41** | 1334.30±184.10* |
Note: in comparison to the blank set, the data is,##P<0.01，###P<0.001; in comparison to the set of models,*P<0.05，**P<0.01，***P<0.001
example 6 study of the spectral efficiency relationship of radix Polygoni Multiflori Preparata
1) Data processing: using 16 batches of medicinal effect data of radix Polygoni Multiflori Preparata for replenishing essence and blood as reference series, using 32 common peak-peak areas of 16 batches of alcohol-soluble parts of radix Polygoni Multiflori Preparata or 6 common peak-peak areas of small molecule sugar parts as comparison series, and analyzing common peak and medicinal effect finger by dimensionless treatmentTarget correlation coefficient r (X)0(k),Xi(k) And degree of correlation r (X)0,Xi) The calculation formula is as follows:
in the above formula, reference number sequence X0(k) Wherein k is radix Polygoni Multiflori Preparata of different batches, and comparing the number series Xi(k) Wherein i is each common peak of the fingerprint,
and
In the above formula, reference number sequence X0(k) Wherein k is radix Polygoni Multiflori Preparata of different batches, and comparing the number series Xi(k) Wherein i is each common peak of the fingerprint; the closer the r value is to 1, the greater the correlation.
In order to realize the comprehensive quantitative evaluation of the efficacy of the radix polygoni multiflori preparata for benefiting essence and blood, the weight coefficients of all indexes are introduced. Searching a PubMed database, and mining documents related to blood deficiency, erythrocyte number, hemoglobin, hematocrit, platelet number diseases and drug effect indexes. The retrieval keywords are respectively 'Blood specificity', 'Red Blood cell' or 'erythrocyte', 'Hemoglobin', 'Packed cell volume' or 'PCV' and 'Blood plain', the first 1000 document abstracts are obtained through respective mining and retrieval, protein keyword names related to the physiological and pathological processes are extracted from the document abstracts, and the protein target point sets related to the keywords are obtained through sorting. And further performing ontology function annotation on the protein obtained by searching each keyword by means of a DAVID (http:// DAVID-d.ncifcrf.gov) database, and analyzing to obtain the ontology function related to each keyword. The coincidence quantity of the four drug effect related bodies, namely the erythrocyte number, the hemoglobin, the erythrocyte pressure and the platelet number, and the blood deficiency body is analyzed through mapping, and the functional coincidence quantity of each drug effect index and the blood deficiency body is normalized to be used as the weight coefficient of each drug effect index, which is shown in table 12.
TABLE 12 weight coefficient of each efficacy index
| Index (I) | Number of red blood cells | Hemoglobin | Hematocrit of red blood cells | Number of platelets |
| Weight coefficient | 0.260982 | 0.273601 | 0.227427 | 0.237990 |
2) As a result: the correlation between the common peaks of alcohol-soluble part of radix Polygoni Multiflori Preparata and the effects of replenishing essence and blood is shown in Table 13, and the correlation between the common peaks of small molecule sugar part and the effects of replenishing essence and blood is shown in Table 14.
For alcohol-soluble part of radix Polygoni Multiflori Preparata, the first 16 of 32 common peaks with high correlation degree with blood and essence-replenishing drug effect are main active ingredients of the part for playing blood and essence-replenishing drug effect, and qualitative analysis shows that the main active ingredients are stilbene glycosides and also include a few flavonoids and anthraquinones.
TABLE 13 correlation degree and sequence between alcohol-soluble part common peak and medicinal effects of replenishing essence and blood
For the part of the small molecular sugar of the radix polygoni multiflori preparata, except the No. 4 peak, other chromatographic peaks have larger association degree with the efficacy of replenishing vital essence and blood, and the molecular weight range is mainly concentrated below 3000Da as can be known from molecular weight analysis.
TABLE 14 correlation degree and sequence between common peaks of small molecule sugar sites and drug effects of replenishing essence and blood
| Peak mark number | Red blood cell | Hemoglobin | Hematocrit of red blood cells | Blood platelet | Total drug effect | Order of |
| Peak | ||||||
| 1 | 0.7523 | 0.7558 | 0.6923 | 0.7307 | 0.7345 | 4 |
| |
0.7700 | 0.7541 | 0.6906 | 0.7721 | 0.7481 | 2 |
| |
0.7294 | 0.7291 | 0.6757 | 0.7217 | 0.7153 | 5 |
| |
0.7466 | 0.7365 | 0.6791 | 0.7845 | 0.7375 | 3 |
| |
0.7808 | 0.7623 | 0.7100 | 0.7673 | 0.7564 | 1 |
Example 7 establishment of quality Standard of radix Polygoni Multiflori Preparata
1) Quality control standard of alcohol-soluble part:
liquid chromatograph: type SHIMADZU LC-2010C; a detector: SPD-M20A diode array detector; using octadecylsilane chemically bonded silica as a packed column (250X 4.6mm, 5 μm), acetonitrile as a mobile phase A, and 0.1% phosphoric acid water as a mobile phase B, and performing gradient elution according to Table 1 at a flow rate of 1.0 mL/min; the detection wavelength was 254 nm. The theoretical plate number is not less than 2000 calculated according to the peak of 2,3,5, 4' -tetrahydroxystilbene-2-O-beta-D-glucoside.
According to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprints, the similarity between the fingerprint of the test sample and the reference fingerprint is not less than 0.90; establishing alcohol-soluble part fingerprint of effective parts of the blood and essence of prepared fleece-flower root to obtain 32 common peaks, taking emodin with Peak 31 as a reference Peak, and taking the relative retention time of each common Peak as shown in table 15, wherein 80% of the ratio average value of the main active component peaks (Peak3,4,5,6,10,11,12,14,15,17,18,20,21,22,26 and 31) in different batches is taken as the lower quality control limit, and according to the result in table 3, the ratio is not lower than 61.93%.
TABLE 15 relative retention time of alcohol soluble fraction common peak
| Peak mark number | Relative retention time | Peak mark number | Relative |
| Peak | |||
| 1 | 0.123 | Peak 17 | 0.516 |
| |
0.153 | |
0.531 |
| |
0.159 | |
0.554 |
| |
0.164 | |
0.573 |
| |
0.246 | |
0.599 |
| |
0.267 | |
0.613 |
| Peak 7 | 0.276 | |
0.624 |
| Peak 8 | 0.305 | |
0.641 |
| |
0.312 | |
0.681 |
| |
0.323 | |
0.714 |
| Peak 11 | 0.330 | |
0.777 |
| |
0.337 | |
0.844 |
| |
0.355 | |
0.879 |
| |
0.359 | |
0.882 |
| |
0.388 | |
1.000 |
| |
0.401 | |
1.133 |
2) The quality control standard of the small molecular sugar part is as follows:
liquid chromatograph: agent 1260 infnity; a detector: agent 1260infnity ELSD; a packed column (300X 7.8mm, 13 μm) was made of porous methacrylate; isocratically eluting with 0.05M ammonium acetate at 40 deg.C and flow rate of 0.4 mL/min; the ELSD detector parameters were: the drift tube temperature was 45 ℃ and the carrier gas flow rate was 1.2L/min.
According to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprints, the similarity between the fingerprint of the test sample and the reference fingerprint is not less than 0.90; establishing fingerprint of effective part of prepared fleece flower root for replenishing vital essence and blood-micromolecule sugar part to obtain 6 common peaks, taking the No. 5 Peak as a reference Peak, taking the relative retention time of each common Peak as shown in Table 16, taking 80% of the average value of the main active ingredient peaks (Peak1,2+3,5,6) of the micromolecule sugar part in different batches as the lower limit of quality control, and according to the result of Table 6, not less than 62.58%.
TABLE 16 Small molecule carbohydrate site consensus peak relative retention time
| Peak mark number | Relative |
| Peak | |
| 1 | 0.671 |
| |
0.793 |
| |
0.817 |
| |
0.885 |
| |
1.000 |
| |
1.172 |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A quality detection method of radix polygoni multiflori preparata comprises the following steps:
(1) preparing alcohol-soluble part and micromolecular sugar part of radix Polygoni Multiflori Preparata; the alcohol-soluble part of radix Polygoni Multiflori Preparata is prepared by extracting radix Polygoni Multiflori Preparata powder with water, concentrating the extractive solution, adding anhydrous ethanol until the alcohol content is 50%, and centrifuging; adding absolute ethanol into the supernatant, concentrating until the alcohol content is 80%, and centrifuging; concentrating the supernatant, enriching with D101 type macroporous resin, eluting with water, and eluting with 95% ethanol;
the preparation method of the small molecular sugar part of the prepared fleece flower root comprises the steps of adding water into prepared fleece flower root powder for extraction, concentrating an extracting solution, adding absolute ethyl alcohol, and centrifuging; adding absolute ethyl alcohol into the supernatant, concentrating, and centrifuging; collecting precipitate, dissolving, adding anhydrous ethanol, concentrating, and centrifuging; drying the supernatant to obtain the final product;
(2) establishing fingerprint of alcohol-soluble part and micromolecular sugar part of radix Polygoni Multiflori Preparata; the HPLC chromatographic condition of the alcohol-soluble part fingerprint spectrum adopts an Agela Venusil MP C18250 multiplied by 4.6mm and 5 mu m chromatographic column, the mobile phase consists of acetonitrile-0.1 percent phosphoric acid water, the flow rate is 1.0mL/min, the sample amount is 10 mu L, the column temperature is 20 ℃, and the detection wavelength is 254 nm; the detector is an SPD-M20A diode array detector, and the gradient elution procedure is as follows:
The HPLC chromatographic conditions of the fingerprint of the small molecular sugar part are that TSK gel GMPPWxl 300 multiplied by 7.8mm, a 13 mu M chromatographic column and 0.05M ammonium acetate are adopted for isocratic elution, the flow rate is 0.4mL/min, the sample injection amount is 3 mu L, and the column temperature is 40 ℃; the detector is an agent 1260 definition ELSD;
(3) the pharmacodynamic study of the essence and blood benefiting of the alcohol-soluble part and the micromolecular sugar part of the prepared fleece-flower root is to detect the influence of a plurality of batches of alcohol-soluble parts and micromolecular sugar parts of the prepared fleece-flower root on the number of red blood cells, the hematocrit, the number of platelets and the content of hemoglobin;
(4) determining the correlation degree of the common peak in the radix Polygoni Multiflori Preparata fingerprint and the effect on replenishing essence and blood by using a grey correlation analysis method, thereby determining the influence of different common peaks of alcohol-soluble parts and small molecular sugar parts in radix Polygoni Multiflori Preparata on the effect on replenishing essence and blood;
(5) establishing a quality standard according to the influence of different common peaks of an alcohol-soluble part and a small molecular sugar part in the prepared fleece-flower root on the efficacy of the alcohol-soluble part and the small molecular sugar part in the prepared fleece-flower root, and directly evaluating the quality of the prepared fleece-flower root by using the established quality standard, wherein the quality standard of the alcohol-soluble part in the prepared fleece-flower root is that octadecylsilane chemically bonded silica is used as a filling column, acetonitrile is used as a mobile phase A, 0.1% phosphoric acid water is used as a mobile phase B, gradient elution is carried out according to the table 1, and the flow rate is 1.0 mL/min; the detection wavelength is 254 nm;
the quality standard of the small molecular sugar part in the prepared fleece-flower root is that porous methacrylate is used for forming a packed column; isocratically eluting with 0.05M ammonium acetate at 40 deg.C and flow rate of 0.4 mL/min; the ELSD detector parameters were: the drift tube temperature was 45 ℃ and the carrier gas flow rate was 1.2L/min.
2. The quality inspection method according to claim 1, wherein the step 2) of establishing the alcohol soluble part fingerprint comprises the following steps: 1) determining HPLC chromatographic conditions; 2) establishing multiple batches of alcohol-soluble part fingerprint spectrums of prepared fleece flower root, determining a common chromatographic peak by a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, and analyzing the similarity among samples; 3) and (3) qualitatively analyzing the alcohol-soluble part of the prepared fleece flower root by using HPLC-ESI-MS to identify the active ingredients in the alcohol-soluble part.
3. The quality detection method according to claim 1, wherein the establishment of the fingerprint of the small molecule sugar part in the step 2) comprises the following steps: determining HPLC chromatographic conditions; establishing fingerprint spectrums of micromolecular sugar parts of a plurality of prepared fleece-flower roots, determining common chromatographic peaks through a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, and analyzing the similarity among samples; thirdly, a standard curve is prepared by the logarithmic value of the molecular weight of the standard substance to the corresponding retention time so as to obtain the molecular weight distribution condition of the small molecular sugar part of the radix polygoni multiflori preparata.
4. The quality detection method according to claim 1, wherein in the alcohol-soluble part of radix Polygoni Multiflori Preparata of step 4), the main effective components with high influence on the effect of replenishing essence and blood are stilbene glycosides, flavonoids, and anthraquinones; the molecular weight of the main effective components with higher influence correlation degree on the efficacy of benefiting essence and blood in the small molecular sugar parts in the prepared fleece-flower root is below 3000 Da.
5. The method of claim 1, wherein the theoretical plate number in step 5) is not less than 2000 calculated from the peak of 2,3,5, 4' -tetrahydroxystilbene-2-O- β -D-glucoside; the content of 16 main active component peaks of the alcohol-soluble part is not less than 61.93%, and the similarity between the fingerprint of the sample and the fingerprint of the reference is not less than 0.90 according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprints.
6. The quality detection method according to claim 1, wherein 5) the ratio of 5 main active ingredient peaks at the small molecule sugar part is not less than 62.58%, and the similarity between the fingerprint of the sample and the fingerprint of the reference sample is not less than 0.90 according to the similarity evaluation system of the chromatographic fingerprint of traditional Chinese medicine.
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