Method for extracting multiple components of traditional Chinese medicine compound based on supercritical fluid chromatography technology

Abstract

The invention provides a supercritical fluid chromatography technology-based traditional Chinese medicine compound multi-component extraction method, which comprises: (1) carrying out pretreatment on traditional Chinese medicine compound Xiaoyao powder to obtain a sample to be detected; (2) preparing standard solutions of apiose liquiritin, apiose isoliquiritin, paeoniflorin, albiflorin, saikoside A and saikoside D; and (3) carrying out supercritical fluid chromatographic analysis on the to-be-detected sample, and extracting. According to the extraction method of the traditional Chinese medicine compound multi-component based on the supercritical fluid chromatography technology, the simultaneous preparation of the multi-component (apiose liquiritin, apiose isoliquiritin, paeoniflorin, albiflorin, saikoside A and saikoside D) in the traditional Chinese medicine compound preparation Xiaoyao powder can be realized.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and in particular relates to a method for extracting multiple components of a traditional Chinese medicine compound based on supercritical fluid chromatography technology. Background Art

[0002] At present, the most commonly used methods for preparing components of traditional Chinese medicine are to use preparative liquid phase and dynamic axial technology, which can realize the preparation of components of complex systems of traditional Chinese medicine. However, there are expensive instruments and equipment, large consumption of mobile phase for sample preparation, and huge workload for mobile phase recovery and processing in the later stage of preparation, which greatly consumes manpower and material resources. Supercritical fluid chromatography (SFC) technology uses CO2 as mobile phase, which greatly reduces the consumption of solvents. The solvent is green and environmentally friendly, and reduces the sample post-processing process of component preparation. At the same time, SFC technology has good separation and can achieve rapid separation. However, at present, SFC technology is only used for the separation and purification of chemical drugs, or the preparation of single components, and the multi-component preparation of complex systems of traditional Chinese medicine extracts has not yet been studied and developed. Summary of the invention

[0003] In view of this, the present invention aims to overcome the defects in the prior art and proposes a method for extracting multiple components of a traditional Chinese medicine compound based on supercritical fluid chromatography technology.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] A method for extracting multiple components of a traditional Chinese medicine compound based on supercritical fluid chromatography technology comprises the following steps:

[0006] (1) Pre-treating the traditional Chinese medicine compound Xiaoyaosan to obtain a sample to be tested;

[0007] (2) preparing standard solutions of apigenin, apigenin isoliquiritigenin, paeoniflorin, paeoniflorin, saikosaponin A and saikosaponin D;

[0008] (3) The sample to be tested is subjected to supercritical fluid chromatography analysis and extracted.

[0009] Furthermore, the pretreatment in step (1) specifically includes the following steps: extracting the traditional Chinese medicine compound Xiaoyaosan, and subjecting the obtained extract to alcohol precipitation to obtain the sample to be tested.

[0010] Furthermore, the extraction step is specifically as follows: soaking the various component medicinal materials of Xiaoyao Powder in deionized water, decocting them once, filtering to obtain a primary filtrate, placing the filtered medicinal materials into deionized water for a second decoction, filtering to obtain a secondary filtrate, and mixing the primary filtrate with the secondary filtrate.

[0011] Furthermore, the soaking step lasts for 1-60 minutes; the first decoction lasts for 1-60 minutes; and the second decoction lasts for 1-60 minutes.

[0012] Furthermore, in the alcohol precipitation step, the volume ratio of the extract to ethanol is 1:3-3.5.

[0013] Furthermore, the mass concentration of ethanol in the sample to be tested is 70-80%.

[0014] Furthermore, the mobile phase of the supercritical fluid chromatography analysis in step (3) is CO2 and methanol, and the volume ratio of CO2 to methanol is 4-5:1; the flow rate of CO2 is 8 mL / min.

[0015] Furthermore, the parameters of the supercritical fluid chromatography analysis in the step (3) are specifically as follows: supercritical fluid chromatography: SFC-Lab20-1 machine; chromatographic column: SI 10*250mm; wavelength: 210-280nm; column temperature: 30-35°C; back pressure: 10-12Mpa; injection volume: 50-60μL; CO2 pump back pressure: 10-15Mpa.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The method for extracting multiple components of a traditional Chinese medicine compound based on supercritical fluid chromatography technology can realize the simultaneous preparation of multiple components (apigenin, isoliquiritigenin, paeoniflorin, paeoniflorin, saikosaponin A and saikosaponin D) in the traditional Chinese medicine compound preparation Xiaoyaosan; the multiple components prepared simultaneously have high purity and good separation, which greatly reduces the solvent consumption in the preparation process and the sample solvent post-treatment process, greatly reduces the production cost, and saves manpower and material resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The chromatogram is of the Chinese medicine compound Xiaoyaosan described in Example 3 of the present invention;

[0019] Figure 2 is a chromatogram of the paeoniflorin standard product described in Example 4 of the present invention;

[0020] Figure 3 The chromatogram determined for the paeoniflorin described in Example 4 of the present invention;

[0021] Figure 4 is a chromatogram of the standard product of paeoniflorin described in Example 4 of the present invention;

[0022] Figure 5 The chromatogram determined for the paeoniflorin described in Example 4 of the present invention;

[0023] Figure 6 This is a chromatogram of the standard product of apigenin liquiritin described in Example 4 of the present invention;

[0024] Figure 7 The chromatogram determined for the apigenin liquiritin described in Example 4 of the present invention;

[0025] Figure 8 is a chromatogram of the standard product of saikosaponin A described in Example 4 of the present invention;

[0026] Fig. 9 The chromatogram determined for saikosaponin A described in Example 4 of the present invention;

[0027] Fig.10 is the standard curve of paeoniflorin described in Example 5 of the present invention;

[0028] Fig.11 is the standard curve of paeoniflorin described in Example 5 of the present invention;

[0029] Fig.12 The standard curve of apigenin described in Example 5 of the present invention;

[0030] Fig.13 The standard curve of apigenin isoliquiritigenin described in Example 5 of the present invention;

[0031] Fig.14 is the standard curve of saikosaponin A described in Example 5 of the present invention;

[0032] Fig.15 This is the standard curve of saikosaponin D described in Example 5 of the present invention. DETAILED DESCRIPTION

[0033] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0034] The present invention will be described in detail below with reference to the embodiments.

[0035] Example 1 Pretreatment of samples

[0036] Preparation of the extract of the traditional Chinese medicine compound Xiaoyao Powder: Xiaoyao Powder is composed of Bupleurum, Angelica, White Peony, Atractylodes, Poria, Ginger, Licorice and Mint, and the weight ratio is 5; 5: 5: 5: 5: 5: 4: 1. Soak each medicinal material in water (solid-liquid ratio of 1: 8) for 30 minutes, heat to boiling and boil for 30 minutes, pour out the first filtrate for later use; then add water (solid-liquid ratio of 1: 8) to the medicinal material and boil for 30 minutes, pour out the filtrate; finally, combine the two filtrates to obtain the extract of Xiaoyao Powder.

[0037] The Xiaoyaosan extract was precipitated by spray alcohol precipitation, and the volume ratio of the Xiaoyaosan extract to ethanol was 1:3, so that the concentration of ethanol in the extract was finally 80%.

[0038] After the Xiaoyao Powder alcohol precipitate was allowed to stand for 24 hours, the supernatant was taken for rotary evaporation to finally prepare the Xiaoyao Powder extract sample to be tested, which was used for sample component preparation and stored in a -20°C refrigerator.

[0039] Example 2 Preparation of standard solution

[0040] Weigh out 6 standard products (peony lactone glycosides, paeoniflorin, apigenin liquiritigenin, apigenin isoliquiritigenin, saikosaponin A, saikosaponin D) and prepare a standard solution with a concentration of 1 mg / ml using methanol solvent.

[0041] Example 3 Supercritical Fluid Chromatography Extraction

[0042] Sample method: entrainer sample

[0043] Flow cell type: analytical flow cell

[0044] Chromatographic column: SI 10*250mm

[0045] Wavelength: 254nm

[0046] Column temperature: 35°C

[0047] Back pressure: 10Mpa

[0048] Injection volume: 50μl (extract dissolved in methanol, 1:3) Sample injection dilution ratio

[0049] CO2 pump pressure: 12.51Mpa

[0050] Cooling temperature: 3.72℃

[0051] Mobile phase: A: CO2, B: methanol; A = 80%, B = 20%

[0052] Flow rate: 8mL

[0053] Under the above conditions, the sample to be tested is measured. According to the chromatographic peak, each chromatographic peak is collected as a component. According to the retention time, 10 components are numbered 1-10, and 10 components are collected (one injection of sample is measured, and 10 components can be prepared at the same time). The spectrum is as follows: Figure 1 The retention time of each component is shown in Table 1.

[0054] Table 1 Retention schedule

[0055] project Time (min) project Time (min) No. 1 3.9-6.4 No. 6 18.2-20.6 No. 2 6.7-8.1 No.7 22.7-24 No.3 10-12 No. 8 24.2-25.9 No. 4 13-14.7 No. 9 28.8-31.5 No. 5 15-16.7 No. 10 46.8-50

[0056] Example 4 Component Confirmation

[0057] The 10 components and the 6 prepared standard solutions were measured under the same conditions, and each component was identified by the retention time of the standards.

[0058] Collect components 1-10 for testing:

[0059] Instrument: Waters UPCC

[0060] Chromatographic column: DIOL 5um 4.6*250 Chromatographic column number:

[0061] Wavelength: 210 / 254nm

[0062] Back pressure: 1750psi

[0063] Injection volume: 10 μl

[0064] Mobile phase: A:CO2 B:Methanol

[0065] Table 2 Flow rate

[0066] Time / min Flow rate / ml A / ％ B / ％ 0 2 90 10 20 2 50 50 30 2 50 50

[0067] Chromatogram Figure 1 As shown, the assay spectrum of the standard peony inoside is as follows Figure 2 As shown, wavelength 254nm, No. 5 collection bottle-peonidin confirmation as Figure 3 As shown; the determination spectrum of the standard product of peony lactone glycosides is as shown Figure 4 As shown, wavelength 254nm, collection bottle No. 6 - confirmation of paeoniflorin Figure 5 As shown; the determination spectrum of apigenin liquiritin standard is as shown Figure 6 As shown, wavelength 254nm, collection bottle No. 10 - confirmation of apigenin liquiritin as shown Figure 7 As shown; the determination spectrum of saikosaponin A standard is as shown Figure 8 As shown, wavelength 210nm, collection bottle No. 10 - confirmation of saikosaponin A as shown Fig. 9 By comparing the samples collected simultaneously by supercritical fluid technology with the standard substances, it can be found that the constructed supercritical fluid methodology can realize the possibility of simultaneously preparing multiple components of different structural types in the complex system of traditional Chinese medicine compound.

[0068] In addition, supercritical fluid chromatography technology has very high requirements for sample pretreatment. If the Xiaoyaosan compound is directly decocted according to the standards of the Chinese Pharmacopoeia using conventional methods to obtain the Xiaoyaosan extract, and then directly injected, the supercritical fluid chromatography will experience a sudden increase in pressure and will be unable to inject samples. Because the extract sample is too complex, it cannot be injected normally. Therefore, we constructed a spray alcohol precipitation method to pre-treat the Xiaoyaosan extract sample to purify it, which can reduce the pressure of the supercritical fluid system and allow injection to operate normally.

[0069] Example 5 Purity calculation of preparation components

[0070] The purity of the six components (apiosylliquiritigenin, apiosylliquiritigenin, paeoniflorin, paeoniflorin, saikosaponin A and saikosaponin D) collected simultaneously by supercritical fluid chromatography was analyzed.

[0071] Chromatographic conditions: ultra performance liquid chromatography was used for purity detection, chromatographic column: Waters T3, column temperature: 35°C, A was 0.1% formic acid in water, B was acetonitrile, and the elution gradient was 0-3 min, 95% A-95% A, 3-12 min, 95% A-77% A, 12-17 min, 77% A-71% A, 17-25 min, 71% A-55% A, 25-30 min, 55% A-5% A.

[0072] Preparation of sample solution: Weigh 1 mg of each of the 6 prepared components, add 1 ml of methanol solution to dissolve, and filter to obtain the sample solution of each component.

[0073] Preparation of standard solution: 1 mg of six standard substances, i.e., apigenin, isoliquiritigenin, paeoniflorin, peony lactone glycosides, saikosaponin A and saikosaponin D, were accurately weighed and dissolved in 1 ml of methanol to prepare a 1 mg / ml standard stock solution. The points of the standard curve were obtained by diluting the standard stock solution by 2 times in sequence, and the standard curve was measured.

[0074] The standard curves of the six components are as follows Figure 10-15 The specific methodology and purity content are shown in Table 3.

[0075] Table 3 Data

[0076]

[0077] Finally, the purity of the six components is: 75% for paeoniflorin, 93% for paeoniflorin, 75% for apigenin, 65% for isoliquiritigenin, 45% for saikosaponin A, and 53% for saikosaponin D. Through quantitative analysis, we found that the purity of multi-component components of different structures prepared simultaneously by the constructed supercritical fluid chromatography method is high. At the same time, this method uses CO2 as the mobile phase, which is green and environmentally friendly. It can also realize the simultaneous preparation of six components, greatly improving the efficiency of preparation and saving costs.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting multiple components of a traditional Chinese medicine compound based on supercritical fluid chromatography, characterized in that: The steps include: (1) Pre-treating the traditional Chinese medicine compound Xiaoyaosan to obtain a sample to be tested; (2) preparing standard solutions of apigenin, apigenin isoliquiritigenin, paeoniflorin, paeoniflorin, saikosaponin A and saikosaponin D; (3) The sample to be tested is subjected to supercritical fluid chromatography analysis and extracted.

2. The method for extracting multiple components of a Chinese medicinal compound based on supercritical fluid chromatography according to claim 1, characterized in that: The pretreatment in step (1) specifically includes the following steps: extracting the traditional Chinese medicine compound Xiaoyaosan, and subjecting the obtained extract to alcohol precipitation to obtain the sample to be tested.

3. The method for extracting multiple components of a Chinese medicinal compound based on supercritical fluid chromatography according to claim 2, characterized in that: The extraction steps are specifically as follows: soaking the various component medicinal materials of Xiaoyao Powder in deionized water, decocting them once, filtering them to obtain a primary filtrate, putting the filtered medicinal materials into deionized water for a second decoction, filtering them to obtain a secondary filtrate, and mixing the primary filtrate with the secondary filtrate.

4. The method for extracting multiple components of a Chinese medicinal compound based on supercritical fluid chromatography according to claim 3, characterized in that: The soaking step lasts for 1-60 minutes; the first decoction lasts for 1-60 minutes; and the second decoction lasts for 1-60 minutes.

5. The method for extracting multiple components of a Chinese medicinal compound based on supercritical fluid chromatography according to claim 2, characterized in that: In the alcohol precipitation step, the volume ratio of the extract to ethanol is 1:3-3.

5.

6. The method for extracting multiple components of a Chinese medicinal compound based on supercritical fluid chromatography according to claim 5, characterized in that: The mass concentration of ethanol in the sample to be tested is 70-80%.

7. The method for extracting multiple components of a Chinese medicinal compound based on supercritical fluid chromatography according to claim 1, characterized in that: The mobile phase of the supercritical fluid chromatography analysis in step (3) is CO2 and methanol, and the volume ratio of CO2 to methanol is 4-5:1; the flow rate of CO2 is 8 mL / min.

8. The method for extracting multiple components of a Chinese medicinal compound based on supercritical fluid chromatography according to claim 1, characterized in that: The parameters of the supercritical fluid chromatography analysis in the step (3) are specifically as follows: supercritical fluid chromatography: SFC-Lab20-1 machine; chromatographic column: SI 10*250mm; wavelength: 210-280nm; column temperature: 30-35°C; back pressure: 10-12Mpa; injection volume: 50-60μL; CO2 pump back pressure: 10-15Mpa.

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