CN114152705B - HPLC fingerprint quality evaluation method for rhizoma atractylodis stem and leaf - Google Patents
HPLC fingerprint quality evaluation method for rhizoma atractylodis stem and leaf Download PDFInfo
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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
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Abstract
The application relates to the technical field of agricultural biology, in particular to an HPLC fingerprint quality evaluation method of rhizoma atractylodis stem and leaf extract. The application adopts high performance liquid chromatography to establish a quality control method of rhizoma atractylodis stem and leaf, determines the high performance liquid chromatography analysis method of the rhizoma atractylodis stem and leaf by examining the influence of wavelength, mobile phase, modifier, column temperature, flow rate, analysis time and the like on the quantity and separation degree of chemical components of the rhizoma atractylodis stem and leaf, further verifies the precision, stability and repeatability of the established liquid chromatography analysis method, adopts similarity analysis and chemometry to evaluate the spectral efficiency relationship between HPLC fingerprint and antibacterial activity, and finally determines the mass control point of the rhizoma atractylodis stem and leaf batch, thereby laying a quality foundation for the application of the rhizoma atractylodis stem and leaf.
Description
Technical Field
The application relates to the technical field of agricultural biology, in particular to an HPLC fingerprint quality evaluation method for stems and leaves of rhizoma atractylodis.
Background
The traditional Chinese medicine has the effects of building up body and preventing and curing diseases for people and animals, so the market demand of the traditional Chinese medicine is rapidly increased. In addition, the development of the technology and method for quality control and spectral efficiency evaluation of the traditional Chinese medicine is promoted while the complexity of the traditional Chinese medicine components brings great change. Fingerprint analysis is a well-known and effective quality control method for traditional Chinese medicine. The fingerprint analysis of traditional Chinese medicine is widely applied in the standardized production process of traditional Chinese medicine. The quality standard of the traditional Chinese medicine formulated in the Chinese pharmacopoeia not only accords with the characteristics of the traditional Chinese medicine, but also adopts the method of the fingerprint spectrum of the traditional Chinese medicine to control the quality of the traditional Chinese medicine. Meanwhile, the high performance liquid chromatography plays a vital role in qualitative and quantitative determination of the active ingredients of the Chinese herbal medicines by virtue of the advantages of high efficiency, high sensitivity, high speed, high pressure and the like.
Rhizoma Atractylodis is rhizome part of Atractylodes lancea stem and leaf of Atractylodes lancea (Thunb.) DC or Atractylodes chinensis stem and leaf A.chinensis (DC.) Koidz. Modern pharmacological researches show that rhizoma atractylodis is used as a medicinal material, has unique chemical components and remarkable pharmacological activity, has the effects of resisting ulcer, arrhythmia, inflammation, bacteria, liver protection, blood pressure reduction and the like, and is widely applied. The stem and leaf of the rhizoma atractylodis is used as a non-medicinal part of the rhizoma atractylodis to be discarded as garbage, so that the resource is seriously wasted. Research shows that the stem and leaf of rhizoma atractylodis has the functions of anti-inflammatory, bacteriostasis and antioxidation. The application of stem and leaf of rhizoma atractylodis is promoted, and the standard formulation needs to be advanced. Therefore, it is particularly important to formulate standards for stems and leaves of rhizoma atractylodis.
The chromatographic peak in the HPLC chromatogram is influenced by a plurality of factors, such as detection wavelength, flow velocity of mobile phase, proportion of each component of mixed mobile phase, column temperature and the like, which can influence the HPLC fingerprint of the stems and leaves of rhizoma atractylodis, especially the proportion of each component of mixed mobile phase has larger influence, has uncertainty, and any change of chromatographic conditions means that the overall chromatographic pattern is changed, thus causing difficulty to the establishment work of the HPLC fingerprint.
Disclosure of Invention
The application aims to provide an HPLC fingerprint quality evaluation method for rhizoma atractylodis stem and leaf.
The HPLC fingerprint quality evaluation method for the stems and leaves of the rhizoma atractylodis comprises the following steps:
preparing stem and leaf extract solution of a stem and leaf sample of rhizoma atractylodis to be detected and stem and leaf extract solution of a stem and leaf standard substance of rhizoma atractylodis;
subjecting the obtained extract solutions to HPLC chromatographic analysis, wherein the conditions of the HPLC chromatographic analysis are as follows:
the mobile phase is a methanol-0.1 vol% trifluoroacetic acid system, the flow rate is 1mL/min, the column temperature is 30 ℃, the detection wavelength is 260nm, and the analysis time is 40min
At the detection wavelength of 260nm, the column temperature of 30 ℃ and the flow rate of 0.8mL/min, methanol-0.1 vol% trifluoroacetic acid solution is taken as a mobile phase for gradient elution, the sample injection volume is 10 mu L, and the gradient elution program is as follows: the elution gradient is 0 to 15min,5 percent of methanol to 35 percent of 0.1 percent trifluoroacetic acid-water, 15 to 25min,35 percent of methanol to 45 percent of 0.1 percent trifluoroacetic acid-water, 25 to 30min,45 percent of methanol to 60 percent of 0.1 percent trifluoroacetic acid-water; 30-40 min,60% methanol-95% 0.1% trifluoroacetic acid-water;
comparing the HPLC chromatograms of the stem and leaf extract solution of the stem and leaf sample of the rhizoma atractylodis to be detected and the stem and leaf extract solution of the stem and leaf standard of the rhizoma atractylodis, and judging that the stem and leaf sample of the rhizoma atractylodis to be detected is a qualified product if the similarity of the HPLC chromatograms of the stem and leaf extract solution of the stem and leaf sample of the rhizoma atractylodis to be detected and the stem and leaf extract solution of the stem and leaf standard of the rhizoma atractylodis is more than or equal to 0.998.
According to the HPLC fingerprint quality evaluation method of the rhizoma atractylodis stem and leaf extract, an extract solution of the rhizoma atractylodis stem and leaf is prepared through the following steps:
grinding rhizoma Atractylodis stems and leaves into powder, and sieving with 40 mesh sieve;
adding an ethanol-water solution to the sieved powder;
then ultrasonic extracting, centrifuging to collect supernatant, evaporating and concentrating, and freeze drying to obtain powder;
the powder was dissolved with water.
According to the HPLC fingerprint quality evaluation method of the rhizoma atractylodis stem and leaf extract, the feed liquid ratio of the sieved powder to the ethanol-water solution is 1:40.
According to the HPLC fingerprint quality evaluation method of the rhizoma atractylodis stem and leaf extract, the extraction is carried out for 30min under the condition of 500W.
According to the HPLC fingerprint quality evaluation method of the rhizoma atractylodis stem and leaf extract, the supernatant is concentrated by rotary evaporation at 45 ℃, and is frozen and dried into powder by vacuum, and the powder is preserved at 4 ℃ for standby.
The application adopts high performance liquid chromatography to establish a quality control method of rhizoma atractylodis stem and leaf, determines the high performance liquid chromatography analysis method of the rhizoma atractylodis stem and leaf by examining the influence of wavelength, mobile phase, modifier, column temperature, flow rate, analysis time and the like on the quantity and separation degree of chemical components of the rhizoma atractylodis stem and leaf, further verifies the precision, stability and repeatability of the established liquid chromatography analysis method, adopts similarity analysis and chemometry to evaluate the spectral efficiency relationship between HPLC fingerprint and antibacterial activity, and finally determines the mass control point of the rhizoma atractylodis stem and leaf batch, thereby laying a quality foundation for the application of the rhizoma atractylodis stem and leaf.
Drawings
FIG. 1 shows the separation effect of rhizoma Atractylodis stem and leaf extract at 260nm wavelength;
FIG. 2 shows the separation effect of rhizoma Atractylodis stem and leaf extract at 340nm wavelength;
FIG. 3 shows the full wavelength scan of the extract of stems and leaves of Atractylodes lancea;
FIG. 4 shows the separation effect of methanol-water system on rhizoma Atractylodis stem and leaf extract;
FIG. 5 shows the separation effect of acetonitrile-water system on rhizoma Atractylodis stem and leaf extract;
FIG. 6 shows the separation effect of methanol-water-formic acid modifier on rhizoma Atractylodis stem and leaf extract;
FIG. 7 shows the separation effect of methanol-water-acetic acid modifier on rhizoma Atractylodis stem and leaf extract;
FIG. 8 shows the separation effect of methanol-water-phosphoric acid modifier on rhizoma Atractylodis stem and leaf extract;
FIG. 9 shows the separation effect of methanol-water-trifluoroacetic acid modifier on stem and leaf extract of Atractylodes lancea;
FIG. 10 effect of elution procedure 1 on separation effect of rhizoma Atractylodis stem and leaf extract;
FIG. 11 shows the effect of elution procedure 2 on the separation effect of the stem and leaf extract of Atractylodes lancea;
FIG. 12 shows the effect of elution procedure 3 on the separation effect of the stem and leaf extract of Atractylodes lancea;
FIG. 13 shows the effect of elution procedure 4 on the separation effect of the stem and leaf extract of Atractylodes lancea;
FIG. 14 shows the effect of column temperature 25℃on separation effect of rhizoma Atractylodis stem and leaf extract;
FIG. 15 shows the effect of column temperature 30deg.C on separation effect of rhizoma Atractylodis stem and leaf extract;
FIG. 16 shows the effect of column temperature 35℃on separation of rhizoma Atractylodis stem and leaf extract;
FIG. 17 shows the effect of flow rate 0.8mL/min on the separation effect of rhizoma Atractylodis stem and leaf extract;
FIG. 18 shows the effect of a flow rate of 1.0mL/min on the separation effect of the leaf extract from the stem of Atractylodes lancea;
FIG. 19 shows the effect of a flow rate of 1.2mL/min on the separation effect of the stem and leaf extract of Atractylodes lancea;
FIG. 20 shows characteristic fingerprints of rhizoma Atractylodis stem and leaf extract;
FIG. 21 is a superposition of 10 batches of rhizoma Atractylodis stem and leaf extract samples in HPLC fingerprint;
FIG. 22 is a cluster map of 10 batches of rhizoma Atractylodis stem and leaf extract;
FIG. 23 shows principal component analysis;
FIG. 24 shows the antibacterial activity regression coefficient (PLS) of rhizoma Atractylodis stem and leaf extract;
FIG. 25 shows the bacteriostatic activity VIP (PLS) of rhizoma Atractylodis stem and leaf extract.
Detailed Description
Example 1
1. Reagent(s)
High purity water was obtained using a millipore-Q water purification system (Billerica, MA, USA). Analytical grade ethanol was purchased from beijing chemical plant (beijing, china). Analytical grade phosphoric acid, formic acid and trifluoroacetic acid were all purchased from the technical company of Tianjin Ai Jieer, china. Chromatographic grade methanol and acetonitrile were purchased from the sameimers technology (waltherm, MA, usa).
2. Stem and leaf of Atractylodes lancea
The stem and leaf specimen of rhizoma Atractylodis was collected in inner Mongolia at 9 months of 2020.
3. Sample preparation
Grinding rhizoma Atractylodis stems and leaves into powder, and sieving with 40 mesh sieve. Accurately weighing 20g of the crushed and sieved sample, adding 800mL of 50% (v/v) ethanol-water solution (the feed-liquid ratio is 1:40), and performing ultrasonic extraction for 30min at 500W. Centrifuging at 5000rpm for 10min, collecting supernatant, concentrating by rotary evaporation at 45deg.C, vacuum lyophilizing to obtain powder, and preserving at 4deg.C.
4. Sample solution preparation
Taking rhizoma atractylodis stem and leaf extract fine powder, precisely weighing 100mg, placing in a 15mL centrifuge tube, precisely adding 10mL of ultrapure water, performing vortex dissolution, filtering by a 0.22 μm filter, and taking filtrate for subsequent chromatographic analysis.
5. Instrument conditions
All chromatographic analyses adopted an Shimadzu high performance liquid chromatography system, which consists of an LC-20AD pump, an automatic sampler (SIL-20A), a system controller (CBM-20A), a column greenhouse (CT 0-20A) and ultravioletA visible diode array detector (SPD-M20 a230 v). Agilent ZORBAXSB-C18 column was used for research (4.6X250 mm,5 μm; agilent technologies, santa Clara, calif., U.S.A.). MTT was measured using an enzyme-labeled instrument (Multiskan) TM SkyHigh,ThermoFisherScientific,MA,USA)。
Example 2 optimization of high Performance liquid chromatography conditions
The separation condition of the rhizoma atractylodis stem and leaf extract is optimized on the basis. The sample injection amount of the whole experiment was set to 10. Mu.L.
2.1 selection of detection wavelength
The complexity of the extract components is compared with the separation effect of the rhizoma atractylodis stem and leaf extract under different wavelengths (figures 1 and 2) and the full wavelength scanning is carried out at the wavelength of 200-600 nm (figure 3), and the result shows that the wavelength is 260nm and has the maximum absorption peak, the absorption peak and peak signals are strong, and the number of the obtained peaks is large, so that 260nm is selected as the optimal detection wavelength.
2.2 selection of mobile phase System
After the optimal detection wavelength is determined, the influence of acetonitrile-water and methanol-water mobile phase systems on the separation effect of the stem and leaf extract of the rhizoma atractylodis is compared, and the results are shown in figures 4 and 5, at the wavelength of 260nm, the separation effect and analysis time of the stem and leaf extract of the rhizoma atractylodis by the acetonitrile-water and methanol-water systems are not greatly different, but the number of peaks obtained by the methanol-water system is one more than that of the acetonitrile-water system, so that the methanol-water is selected as the analysis mobile phase of the stem and leaf extract of the rhizoma atractylodis.
2.3 selection of modifier
Under the condition of determining the wavelength and the mobile phase, the influence of the modifier on the separation effect of the stem and leaf extract of the rhizoma atractylodis is examined, and the result is that when the modifier is formic acid, acetic acid and phosphoric acid, the number of peaks is small and the retention time is long compared with a mobile phase system of which the modifier is trifluoroacetic acid as shown in figures 6-9. The modifier is trifluoroacetic acid mobile phase system, which has the best separation effect, the number of the generated peaks is up to 91, the absorption peak signals are obvious, and the peak signal retention time is short. Therefore, a methanol-water-0.1 vol% trifluoroacetic acid mobile phase system is selected as a mobile phase system for chromatographic analysis of rhizoma atractylodis stem and leaf extract.
2.4 selection of elution procedure
Under the condition of determining wavelength, mobile phase and modifier, examining the influence of the elution procedure (table) on the separation effect of rhizoma Atractylodis stem and leaf extract, and the result is shown in fig. 10-13 and tables 1-4, wherein the number of chromatographic peaks obtained in the elution procedure 1 is the largest, but the analysis time is the longest; although the number of chromatographic peaks obtained in the elution procedure 3 is the smallest, the analysis time is the shortest, and only 5 chromatographic peaks with very low content are less than those in the elution procedure 1, so that the overall evaluation of the chemical components of the stem and leaf extract of the rhizoma atractylodis is not affected, and the elution procedure 3 is selected as the elution procedure of the stem and leaf extract of the final rhizoma atractylodis.
TABLE 1 elution procedure 1
TABLE 2 elution procedure 2
TABLE 3 elution procedure 3
TABLE 4 elution procedure 4
2.5 selection of column temperature
HPLC conditions were further optimized and different column temperatures were selected for separation. The results show that when the column temperature was set to 30 ℃ (fig. 15), a greater number of chromatographic peaks and a stronger peak signal were obtained with better separation than at 25 ℃ and 35 ℃ (fig. 14, 16).
2.6 selection of flow Rate
The flow rate is one of the important factors influencing the establishment of the finger print of the rhizoma atractylodis stem and leaf extract. The effect of the flow rate on the separation effect of the stem and leaf extract of Atractylodes lancea was examined, and the results were shown in FIGS. 17 to 19, in which the flow rate was 0.8mL/min, the number of peaks obtained by separation was the largest and the retention time was the shortest, so that the flow rate was determined to be 0.8mL/min.
In summary, the optimal HPLC conditions of the rhizoma atractylodis stem and leaf extract are established: a column chromatography of AgilentZORBAXSB-C18 (4.6X250 mm,5 μm) was used, and gradient elution was carried out with a mobile phase of methanol (A) -0.1vol% trifluoroacetic acid solution at a detection wavelength of 260nm, a column temperature of 30℃and a flow rate of 0.8mL/min, with a sample volume of 10. Mu.L. The gradient elution procedure was: the elution gradient is 0-15 min,5% -35% (A); 15-25 min, 35-45% (A); 25-30 min, 45-60% (A); 30-40 min, 60-95% (A).
Example 3 stability, repeatability and accuracy of high Performance liquid chromatography detection method of Atractylodes lancea Stem and leaf extract
According to the technical scheme of the application, the high performance liquid chromatography analysis condition of the rhizoma atractylodis stem and leaf extract is optimized, and the obtained chromatogram is used as a standard characteristic fingerprint of the rhizoma atractylodis stem and leaf extract (figure 20). Has high resolution and reasonable peak height, and is considered as a common peak for sample identification. The 19 peaks present in all samples within 40min of detection were evaluated based on fingerprint parameters of chromatographic intensity, peak shape and stability.
The peak number 10 with stable content is used as reference peak (S), the other 18 peaks are selected as characteristic peaks with higher absorption intensity of rhizoma Atractylodis stem and leaf extract, and the related difference in the retention time field is evaluated. The formulas of RRT and RPA are rrt=rtpeak/RTpeak 10 and rpa=paseak/paseak 10, respectively.
Preparing rhizoma Atractylodis stem and leaf extract solution, continuously measuring for 6 times according to optimal chromatographic conditions, recording peak area and peak outlet time, and calculating to obtain RSD <0.57% of each common peak relative retention time, and RSD <2.57% of relative peak area, which indicates that the instrument precision is good.
Under the optimal chromatographic conditions, the extract solutions were sampled and tested at 0, 1, 2, 4, 6, 8, 10, 12h, respectively. The solution was kept at room temperature. RSD <0.87% for 19 common peaks and RSD <3.90% for the relative peak area. The results showed that the extract solution was stable over 12 hours.
6 batches of atractylis lancea stem and leaf extracts were prepared at a time and evaluated under optimal chromatographic conditions. RSD <0.34% for 19 common peaks and RSD <4.33% for the relative peak area. The result shows that the method has good repeatability and obvious effect.
The method of the application has stability, repeatability and accuracy, and the similarity of the results proves the effectiveness of the method. The method can obtain relatively stable rhizoma Atractylodis stem and leaf extract, which can be used as the basis for subsequent functional experiments.
3.1 similarity analysis of Atractylodes lancea Stem and leaf extract
Similarity is widely accepted as a traditional Chinese medicine fingerprint evaluation index. The application adopts the data of 19 common peaks in the fingerprint to control the quality, and adopts a similarity evaluation method to control the quality. To determine the consistency of each batch of medicinal materials, HPLC fingerprint analysis is performed on 10 batches of rhizoma Atractylodis stem and leaf extracts. 10 batches of rhizoma atractylodis stem and leaf extracts are prepared and prepared into a solution, sample injection measurement is carried out according to optimal chromatographic conditions, chromatograms are recorded, chromatographic data at 260nm are imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2004A edition) provided by Chinese pharmacopoeia Committee, multipoint correction and chromatographic peak matching are carried out, a superposition spectrum and a comparison spectrum (figure 21) are obtained after Quan Feng matching, similarity values (table 5) among fingerprint spectrums of each sample are obtained through calculation, and the similarity of 10 batches of samples is easy to find, and is equal to or more than 0.998 (table 5).
3.2 bacteriostatic Effect
MTT assay was used to evaluate the inhibition of E.coli by Atractylodes lancea stem and leaf extract. The inhibiting effect of 10 batches of rhizoma Atractylodis stem leaf and leaf extract (10 mg/mL) in Escherichia coli was examined. As shown in Table 5, the inhibition rate of 10 batches of rhizoma Atractylodis stem and leaf extract on Escherichia coli is relatively stable, and there is no statistical difference. The result shows that 10 batches of rhizoma atractylodis stem and leaf extracts have stable inhibition effect on escherichia coli, and the good quality control can ensure the stability of biological activity among the rhizoma atractylodis stem and leaf batches.
Table 510 batch of finger print similarity and antibacterial rate of rhizoma Atractylodis stem and leaf extract
3.3 chemometric analysis
3.3.1 hierarchical clustering analysis
The 10×19-order original data matrix is obtained by taking the peak areas of 19 common peaks in 10 batches of rhizoma atractylodis stem and leaf extracts as variables, and clustering analysis is carried out by adopting an inter-group connection method and taking the Euclidean square distance as a measurement standard by adopting SPSS23.0 software, wherein the result is shown in fig. 22. When the classification distance is 15, 10 batches of atractylis lancea stem and leaf extracts can be gathered into 3 types, S2, S3, S5-S7 and S9 are gathered into A types, S1, S4 and S10 are gathered into B types, and S8 is independently a C type sample. The clustering results indicate that the sample collection may not be at the same time.
3.3.2 principal component analysis
The PCA converts a plurality of variables into a few comprehensive variables (namely main components), is an effective dimension reduction statistical method, eliminates the correlation influence among evaluation indexes, is helpful for describing the relative position of samples more objectively, and has important significance for comprehensive evaluation of the quality of the rhizoma atractylodis stem and leaf extract. The SPSS23.0 statistical software is adopted to form a matrix by the relative peak areas of 19 peaks of 10 batches of samples, the data are subjected to factor analysis by the software, 6 main components are extracted, and the PCA characteristic value and the contribution rate are calculated. As can be seen from Table 6, the characteristic values of the first 6 principal components are all >1, which indicates that the first 6 principal components play a leading role in evaluating the quality of the extract of stems and leaves of rhizoma atractylodis and are the marker components for generating classification difference of samples. The cumulative contribution rate of the 6 main components reaches 93.546% (> 90%), the comprehensive quality of the rhizoma atractylodis stem and leaf extract can be objectively reflected, and the first 6 main components are selected for analysis. In addition, the total peak area was introduced into SIMCA10.0 software to plot a principal component analysis score chart 23, and 10 batches of rhizoma Atractylodis stem and leaf extracts were classified into 3 classes, which substantially agree with the results of the cluster analysis.
Table 13: characteristic value and contribution rate of main component of rhizoma atractylodis stem and leaf extract
| Main component | Eigenvalues | Contribution rate% | Cumulative contribution rate |
| 1 | 7.888 | 41.513 | 41.513 |
| 2 | 3.207 | 16.881 | 58.394 |
| 3 | 2.129 | 11.206 | 69.601 |
| 4 | 1.907 | 10.035 | 79.635 |
| 5 | 1.470 | 7.735 | 87.37 |
| 6 | 1.173 | 6.175 | 93.546 |
3.3.3 spectral Effect relation
The chromatographic fingerprint spectrum is combined with the drug effect evaluation, so that the relevant mark components of the drug effect of the traditional Chinese medicine can be found and identified. The research of the spectrum effect relation converts the visual property of traditional Chinese medicine into the consistency of spectrum and effect. The BCA model is used for establishing the spectral efficiency relation between 19 common peak area data and the inhibition rate of the escherichia coli, and the correlation coefficients are shown in fig. 24, wherein the P1, P3, P5, P11, P13, P14, P15, P16 and P19 have positive effects on inhibiting the escherichia coli. In the PLSR model, the peak areas of 19 common peaks are defined as independent variables, and the dependent variables are the bacteriostatic rates. VIP values were obtained from SIMCA software (version 14.1). As shown in FIG. 25, P1, P5, P3, P6 and P4 are major marker components affecting inhibition of E.coli using VIP >1 as a screening standard.
The combined effect of different chemical components of the traditional Chinese medicine results in the curative effect of most traditional Chinese medicines. The application establishes HPLC fingerprint of the rhizoma atractylodis stem and leaf extract, and fully reflects the types and the amounts of chemical components of the rhizoma atractylodis stem and leaf extract. The chromatographic fingerprint similarity of 10 batches of extracts is as high as 0.998, the peak-to-peak distribution is stable and consistent, which proves that the rhizoma atractylodis stem and leaf extracts in the same production place have enough similarity in chemical composition, and the quality is stable. Meanwhile, the spectrum effect relation between the quality control and the antibacterial effect of the rhizoma atractylodis stem and leaf extract is evaluated by combining a chemometric method. In conclusion, the HPLC fingerprint quality evaluation method of the rhizoma atractylodis stem and leaf extract established by the application has certain reference significance for further perfecting the quality standard of the rhizoma atractylodis stem and leaf extract.
Claims (5)
1. The HPLC fingerprint quality evaluation method for the stems and leaves of the rhizoma atractylodis is characterized by comprising the following steps:
(1) Preparing stem and leaf extract solution of a stem and leaf sample of rhizoma atractylodis to be detected and stem and leaf extract solution of a stem and leaf standard substance of rhizoma atractylodis;
(2) Subjecting the extract solutions obtained in step (1) to HPLC chromatography using a 4.6x mm,5 μm, ZORBAX SB-C18 model column, respectively, wherein the conditions of the HPLC chromatography are:
the mobile phase is a methanol-0.1 vol% trifluoroacetic acid system, the flow rate is 1mL/min, the column temperature is 30 ℃, the detection wavelength is 260nm, the analysis time is 40min, the detection wavelength is 260nm, the column temperature is 30 ℃, the flow rate is 0.8mL/min, the methanol-0.1 vol% trifluoroacetic acid solution is used as the mobile phase for gradient elution, the sample injection volume is 10 mu L, and the gradient elution program is as follows: the elution gradient is 0-15 min, 5-35% methanol, 15-25 min, 35-45% methanol, 25-30 min, 45-60% methanol, 30-40 min and 60-95% methanol;
(3) Comparing the HPLC chromatograms of the stem and leaf extract solution of the stem and leaf sample of the rhizoma atractylodis to be detected and the stem and leaf extract solution of the stem and leaf standard of the rhizoma atractylodis, and judging that the stem and leaf sample of the rhizoma atractylodis to be detected is a qualified product if the similarity of the HPLC chromatograms of the stem and leaf extract solution of the stem and leaf sample of the rhizoma atractylodis to be detected and the HPLC chromatograms of the stem and leaf extract solution of the stem and leaf sample of the rhizoma atractylodis to be detected is equal to or more than 0.998.
2. The method for evaluating the quality of the HPLC fingerprint of the stems and leaves of the rhizoma atractylodis according to claim 1, wherein the extract solution of the stems and leaves of the rhizoma atractylodis is prepared by the following steps:
grinding rhizoma Atractylodis stems and leaves into powder, and sieving with 40 mesh sieve;
adding an ethanol-water solution to the sieved powder;
then ultrasonic extracting, centrifuging to collect supernatant, evaporating and concentrating, and freeze drying to obtain powder;
the powder was dissolved with water.
3. The method for evaluating the quality of the HPLC fingerprints of the stems and leaves of rhizoma atractylodis according to claim 2, wherein the ratio of the feed liquid of the sieved powder to the ethanol-water solution is 1:40.
4. The method for evaluating the quality of the HPLC fingerprint of the stems and leaves of rhizoma atractylodis according to claim 2, wherein the extraction is carried out for 30min under the condition of 500 and W.
5. The method for evaluating the quality of the HPLC fingerprints of the stems and leaves of the rhizoma atractylodis according to claim 2, wherein the supernatant is concentrated by rotary evaporation at 45 ℃.
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