Method for preparing paeoniflorin and albiflorin by utilizing supercritical chromatography

Abstract

The invention provides a method for preparing paeoniflorin and albiflorin by utilizing supercritical chromatography, and belongs to the field of natural products. The invention provides a method for preparing paeoniflorin and albiflorin by using supercritical chromatography, which comprises the following steps: carrying out supercritical chromatography analysis on a solution of a sample to obtain a chromatogram of the sample; if the chromatogram shows that the sample contains paeoniflorin and albiflorin, carrying out supercritical chromatographic separation on the solution of the sample to obtain pure paeoniflorin and pure albiflorin; a chromatographic column used for the supercritical chromatographic analysis and the supercritical chromatographic separation is silica. The method is simple, convenient and efficient, simple in pretreatment, high in analysis and separation speed, small in used solvent amount, easy to recover and reusable, time, manpower and material resources are saved, the purity of paeoniflorin and albiflorin is improved by selecting silica as a chromatographic column, the single sample loading amount is large, and high-purity, rapid and large-scale preparation of paeoniflorin and albiflorin can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of natural products, and in particular to a method for preparing paeoniflorin and paeoniflorin by utilizing supercritical chromatography. Background Art

[0002] Peony ( Paeonia lactiflora Pall.) is a genus of the family Paeoniaceae in the order Pentacarales. Paeonia Paeonia lactiflora L. is a perennial herbaceous plant also known as Biejiangli, Licao, and Huaxiang. It enjoys the reputation of being the "Prime Minister of Flowers" and the "Fairy of Flowers." Modern pharmacological research indicates that peony has significant antispasmodic, analgesic, anti-inflammatory, hepatoprotective, and immunomodulatory effects. It also improves blood rheology, lowers blood pressure, acts as an antioxidant, reduces free radicals, and has anti-tumor properties. The main active ingredients in peony are monoterpenoid glycosides, particularly paeoniflorin and paeoniflorin, which are highly soluble in both water and alcohol.

[0003] Currently, research on the extraction and separation of peony chemical components primarily focuses on faster and more efficient extraction, separation, and purification of components such as total paeoniflorin, paeoniflorin, and paeoniflorin lactones. Extraction methods typically rely on solvents (water or alcohol), such as decoction, reflux, and leaching. In recent years, new extraction methods have been reported, such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), accelerated solvent extraction (ASE), and subcritical water extraction (SubWE). Regarding separation and purification, conventional column chromatography remains the mainstream technology for purifying paeoniflorin components. Other separation methods, such as preparative chromatography and high-speed countercurrent chromatography, have also been reported. However, these methods are associated with high equipment costs and consumables, resulting in low resolution between paeoniflorin and paeoniflorin lactones, making it difficult to prepare large quantities of both components simultaneously and rarely used on a large scale. Summary of the Invention

[0004] The present invention provides a method for preparing paeoniflorin and paeoniflorin by utilizing supercritical chromatography. The method of the present invention realizes the simultaneous analysis, separation and purification of paeoniflorin and paeoniflorin.

[0005] The present invention provides a method for preparing paeoniflorin and paeoniflorin using supercritical fluid chromatography, comprising the following steps: The solution of the sample is subjected to supercritical chromatography analysis to obtain a chromatogram of the sample; If the chromatogram shows that the sample contains paeoniflorin and paeoniflorin, the sample solution is subjected to supercritical fluid chromatography to obtain pure paeoniflorin and pure paeoniflorin; The chromatographic column used for the supercritical chromatography analysis and supercritical chromatography separation is silica.

[0006] Preferably, the mobile phase used in the supercritical chromatography analysis and supercritical chromatography separation is a mixture of a supercritical fluid and a modifier; The supercritical fluid comprises one of supercritical CO2, supercritical NO2 and supercritical NH3; The modifier includes one or more of methanol, ethanol, acetonitrile and isopropanol.

[0007] Preferably, during the supercritical chromatography analysis: The silica has an inner diameter of 3-4.6 mm, a length of 100-250 mm, and a normal phase silica gel particle size of 3-5 μm; The concentration of the sample solution is 0.5-3 mg / mL, and the injection volume of the sample solution is 3-20 μL; The total flow rate of the mobile phase is 0.5~5mL / min, the BPR pressure is 5~50MPa, the volume proportion of the modifier is 2~60%, or the volume proportion of the modifier is 10% from 0 to 1 min, the volume proportion of the modifier increases uniformly from 10% to 40% from 1 to 8 min, the volume proportion of the modifier is 40% from 8 to 9 min, and the volume proportion of the modifier decreases uniformly from 40 to 10% from 9 to 10 min.

[0008] Preferably, during the supercritical chromatography analysis and supercritical chromatography separation: The inner diameter of the silica is independently 4.6-10 mm, the length is independently 100-250 mm, and the normal phase silica gel particle size is independently 3-50 μm; The concentration of the sample solution is independently 0.5-10 mg / mL, and the injection volume of the sample solution is independently 10-1000 μL; The total flow rate of the mobile phase is independently 3 to 100 mL / min, the BPR pressure is independently 50 to 200 MPa, and the volume proportion of the modifier is independently 5 to 60%.

[0009] Preferably, the temperature of the chromatographic column during the supercritical fluid chromatography analysis and supercritical fluid chromatography separation is independently 25-50°C, and the BPR temperature is independently 30-60°C.

[0010] Preferably, the detector used for the supercritical fluid chromatography analysis is a PDA, and the detection wavelength during the supercritical fluid chromatography analysis is 190-800 nm.

[0011] Preferably, the method for preparing the sample solution comprises the following steps: Mixing the medicinal material containing paeoniflorin and paeoniflorin with an alcohol aqueous solution for extraction, and then concentrating the obtained extract under reduced pressure to obtain a solution of the sample; or, The peony root is mixed with an aqueous alcohol solution for extraction, and then the obtained extract is concentrated under reduced pressure to obtain a crude extract; The crude extract is extracted, and the obtained extract is concentrated to obtain a crude extract of paeoniflorin and paeoniflorin; the crude extract is segmented with silica gel and eluted, and the obtained eluate is concentrated to obtain a solution of the sample.

[0012] Preferably, the volume fraction of alcohol in the alcohol aqueous solution is 60-100%; the alcohol includes methanol and / or ethanol; The extraction method includes one of maceration extraction, percolation extraction, ultrasonic extraction and reflux extraction.

[0013] Preferably, the extraction agent used in the extraction comprises one or more of dichloromethane, chloroform and ethyl acetate; The eluent used for the elution includes one or more of petroleum ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol and ethanol.

[0014] Preferably, after the separation, the method further comprises: concentrating the obtained paeoniflorin fraction and the obtained paeoniflorin lactone glycoside fraction under reduced pressure and drying them respectively to obtain the pure paeoniflorin and pure paeoniflorin lactone glycoside respectively.

[0015] The method provided by the present invention is simple and efficient, with simple pretreatment, fast analysis and separation speeds, a small amount of solvent used, easy recovery and reusability, saving time, manpower and material resources, and improving the purity of paeoniflorin and paeoniflorin by selecting silica as a chromatographic column. The single sample loading capacity is large, and high-purity, rapid and large-scale preparation of paeoniflorin and paeoniflorin can be achieved.

[0016] Furthermore, the present invention controls the analysis and preparation parameters by supercritical chromatography technology, thereby further improving the purity and analysis and preparation efficiency of paeoniflorin and paeoniflorin.

[0017] The results of the examples show that the present invention can simultaneously obtain paeoniflorin and paeoniflorin with a purity of more than 99.80% by only subjecting the peony extract to supercritical chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The HPLC chromatogram and MS total ion current spectrum of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1; Figure 2 This is an SFC chromatogram of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1; Figure 3 This is the SFC chromatogram of the refined product containing paeoniflorin and paeoniflorin prepared in Example 2; Figure 4The HPLC chromatogram and MS total ion current spectrum of the paeoniflorin prepared in Example 2; Figure 5 The HPLC chromatogram and MS total ion current spectrum of the paeoniflorin prepared in Example 2; Figure 6 The SFC chromatogram preparation diagram and preparation stacking diagram of the refined product containing paeoniflorin and paeoniflorin of Example 1 in Example 3; Figure 7 The HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 3; Figure 8 The HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 3; Figure 9 The SFC chromatogram preparation diagram and preparation stacking diagram of the refined product containing paeoniflorin and paeoniflorin of Example 1 in Example 4; Figure 10 The HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 4 are shown; Figure 11 The HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 4; Figure 12 This is an SFC chromatogram of the crude extract containing paeoniflorin and paeoniflorin prepared in Example 1 of Example 5; Figure 13 This is an SFC chromatogram of the crude extract containing paeoniflorin and paeoniflorin prepared in Example 1 of Example 6; Figure 14 The H NMR spectrum of the high-purity paeoniflorin prepared in Example 6 ( 1 H-NMR); Figure 15 The carbon-1NMR spectrum of the high-purity paeoniflorin prepared in Example 6 ( 13 C-NMR); Figure 16 Mass spectrum (ESI+) of high-purity paeoniflorin prepared in Example 6; Figure 17 The H NMR spectrum of the high-purity paeoniflorin prepared in Example 6 ( 1 H-NMR); Figure 18 The carbon NMR spectrum of the high-purity paeoniflorin prepared in Example 6 ( 13 C-NMR); Figure 19 Mass spectrum (ESI+) of high-purity paeoniflorin prepared in Example 6; Figure 20This is an SFC chromatogram of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1 of Comparative Example 1; Figure 21 This is the SFC chromatographic analysis diagram of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1 in Comparative Example 2. DETAILED DESCRIPTION

[0019] The present invention provides a method for preparing paeoniflorin and paeoniflorin using supercritical fluid chromatography, comprising the following steps: The solution of the sample is subjected to supercritical chromatography analysis to obtain a chromatogram of the sample; If the chromatogram shows that the sample contains paeoniflorin and paeoniflorin, the sample solution is subjected to supercritical fluid chromatography to obtain pure paeoniflorin and pure paeoniflorin; The chromatographic column used for the supercritical chromatography analysis and supercritical chromatography separation is silica.

[0020] The present invention performs supercritical chromatography analysis on a sample solution to obtain a chromatogram of the sample.

[0021] In the present invention, before the ultrasonic critical mass chromatography analysis or the ultrasonic critical mass chromatography separation, the sample solution is preferably filtered, and the pore size of the filter membrane used for the filtration is preferably 0.25 μm.

[0022] In the present invention, there are preferably two methods for preparing the sample solution, and the first preparation method is described below: The method for preparing the sample solution preferably comprises the following steps: The medicinal material containing paeoniflorin and paeoniflorin is mixed with an alcohol aqueous solution for extraction, and then the obtained extract is concentrated under reduced pressure and freeze-dried to obtain the sample solution.

[0023] In the present invention, the medicinal materials preferably include red peony root and / or white peony root.

[0024] In the present invention, the volume fraction of the alcohol in the alcohol aqueous solution is preferably 60-100%. In a specific embodiment of the present invention, the volume fraction of the alcohol in the alcohol aqueous solution may be 65%, 70%, 75%, 80%, 85%, 90% or 95%. The alcohol preferably includes methanol and / or ethanol. The extraction method preferably includes one of maceration extraction, percolation extraction, ultrasonic extraction and reflux extraction.

[0025] The present invention has no particular limitation on the reduced pressure concentration, and the reduced pressure concentration may be performed until the alcohol taste is eliminated.

[0026] The second preparation method is described below: The method for preparing the sample solution preferably comprises the following steps: The peony root is mixed with an aqueous alcohol solution for extraction, and then the obtained extract is concentrated under reduced pressure to obtain a crude extract; The crude extract is extracted, and the obtained extract is concentrated to obtain a crude extract of paeoniflorin and paeoniflorin; the crude extract is segmented with silica gel and eluted, and the obtained eluate is concentrated to obtain a solution of the sample.

[0027] The present invention mixes peony with an alcohol aqueous solution for extraction, and then concentrates the obtained extract under reduced pressure to obtain a crude extract. In the present invention, the volume fraction of the alcohol in the alcohol aqueous solution is preferably 60-100%. In a specific embodiment of the present invention, the volume fraction of the alcohol in the alcohol aqueous solution may be 65%, 70%, 75%, 80%, 85%, 90% or 95%. The alcohol preferably includes methanol and / or ethanol. The extraction method preferably includes one of maceration extraction, percolation extraction, ultrasonic extraction and reflux extraction.

[0028] After obtaining the crude extract, the present invention extracts the crude extract, and then concentrates the obtained extract to obtain a crude extract of paeoniflorin and paeoniflorin; the crude extract is segmented with silica gel and eluted, and then the obtained eluate is concentrated to obtain a solution of the sample.

[0029] In the present invention, the sample solution preferably includes the crude extracts of paeoniflorin and paeoniflorin.

[0030] In the present invention, the extractant used for the extraction preferably includes one or more of dichloromethane, chloroform and ethyl acetate; the eluent used for the elution preferably includes one or more of petroleum ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol and ethanol.

[0031] In the present invention, the mobile phase used in the supercritical chromatography analysis is a mixture of a supercritical fluid and a modifier; the supercritical fluid preferably includes one of supercritical CO2, supercritical NO2 and supercritical NH3; the modifier preferably includes one or more of methanol, ethanol, acetonitrile and isopropanol.

[0032] In the present invention, the temperature of the chromatographic column during the supercritical chromatography analysis is preferably 25-50°C. In a specific embodiment of the present invention, the temperature of the chromatographic column may be 30°C, 35°C, 40°C or 45°C; the BPR temperature is preferably 30-60°C. In a specific embodiment of the present invention, the BPR temperature may be 35°C, 40°C, 45°C, 50°C or 55°C.

[0033] In the present invention, during the supercritical chromatography analysis: The inner diameter of the silica is preferably 3-4.6 mm, the length is preferably 100-250 mm, and the normal phase silica particle size is preferably 3-5 μm. In a specific embodiment of the present invention, the inner diameter of the silica can be 3.2 mm, 3.5 mm, 3.8 mm, 4 mm, 4.2 mm or 4.5 mm, and the length can be 120 mm, 150 mm, 180 mm, 200 mm or 240 mm. The concentration of the sample solution is preferably 0.5-3 mg / mL. In a specific embodiment of the present invention, the concentration of the sample solution may be 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or 2.5 mg / mL. The injection volume of the sample solution is preferably 3-20 μL. In a specific embodiment of the present invention, the injection volume of the sample solution may be 5 μL, 7 μL, 9 μL, 11 μL, 13 μL, 15 μL, 17 μL, or 19 μL. The total flow rate of the mobile phase during the supercritical fluid chromatography analysis is preferably 0.5-5 mL / min. In a specific embodiment of the present invention, the total flow rate of the mobile phase may be 1 mL / min, 2 mL / min, 3 mL / min or 4 mL / min. The BPR pressure is preferably 5-50 MPa. In a specific embodiment of the present invention, the BPR pressure may be 10 MPa, 15 MPa, 20 MPa, 25 MPa, 30 MPa, 35 MPa, 40 MPa or 45 MPa. The volume proportion of the modifier is preferably 2-60%. In a specific embodiment of the present invention, the volume proportion of the modifier may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or the volume proportion of the modifier is 10% at 0-1 min, and the volume proportion of the modifier increases uniformly from 10% to 40% at 1-8 min, the volume proportion of the modifier is 40% at 8-9 min, and the volume proportion of the modifier decreases uniformly from 40% to 10% at 9-10 min. Or during the supercritical fluid chromatography analysis: The inner diameter of the silica is preferably 4.6-10 mm, the length is preferably 100-250 mm, the normal phase silica particle size is preferably 5-50 μm, in a specific embodiment of the present invention, the inner diameter of the silica can be 5 mm, 5.5 mm, 6.5 mm, 7 mm, 7.5 mm, 8.5 mm, 9 mm or 9.5 mm, the length can be 120 mm, 150 mm, 180 mm, 200 mm or 240 mm, the normal phase silica particle size can be 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm or 45 μm; the total flow rate of the mobile phase is preferably 3-100 mL / min, in a specific embodiment of the present invention, the total flow rate of the mobile phase can be 10 mL / min, 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, 60mL / min, 70mL / min, 80mL / min or 90mL / min; the BPR pressure is preferably 50-200MPa. In a specific embodiment of the present invention, the BPR pressure may be 80MPa, 120MPa, 150MPa, 180MPa or 200MPa; the volume proportion of the modifier is preferably 5-60%. In a specific embodiment of the present invention, the volume proportion of the modifier may be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or 55%; the injection volume of the sample solution is preferably 10-1000μL. In a specific embodiment of the present invention, the injection volume of the sample solution may be 100μL, 200μL, 300μL, 400μL, 500μL, 600μL, 700μL, 800μL or 900μL. After analysis using the above parameters, increasing the liquid inlet volume can achieve the preparation of high-purity paeoniflorin and high-purity paeoniflorin.

[0034] In the present invention, the detector used for the supercritical chromatography analysis is preferably a PDA, and the detection wavelength during the supercritical chromatography analysis is preferably 190-800 nm. In a specific embodiment of the present invention, the detection wavelength can be 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm or 750 nm.

[0035] After obtaining the chromatogram of the sample, if the chromatogram shows that the sample contains paeoniflorin and paeoniflorin, the present invention performs supercritical chromatography on the sample solution to obtain pure paeoniflorin and pure paeoniflorin.

[0036] In the present invention, during the supercritical chromatography separation: The inner diameter of the silica is preferably 4.6-10 mm, the length is preferably 100-250 mm, the particle size of the normal phase silica gel is preferably 5-50 μm. In a specific embodiment of the present invention, the inner diameter of the silica can be 5 mm, 5.5 mm, 6.5 mm, 7 mm, 7.5 mm, 8.5 mm, 9 mm or 9.5 mm, the length can be 120 mm, 150 mm, 180 mm, 200 mm or 240 mm, and the particle size of the normal phase silica gel can be 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm or 45 μm. In the present invention, the mobile phase used for supercritical chromatography analysis and supercritical chromatography separation is a mixture of a supercritical fluid and a modifier; the supercritical fluid preferably includes one of supercritical CO2, supercritical NO2 and supercritical NH3; the modifier preferably includes one or more of methanol, ethanol, acetonitrile and isopropanol.

[0037] In the present invention, the temperature of the chromatographic column during supercritical chromatography separation is preferably 25-50°C. In a specific embodiment of the present invention, the temperature of the chromatographic column can be independently 30°C, 35°C, 40°C or 45°C; the BPR temperature is preferably independently 30-60°C. In a specific embodiment of the present invention, the BPR temperature can be independently 35°C, 40°C, 45°C, 50°C or 55°C. The concentration of the sample solution is preferably 0.5-10 mg / mL. In a specific embodiment of the present invention, the concentration of the sample solution may be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL or 9 mg / mL. The injection volume of the sample solution is preferably 10-1000 μL. In a specific embodiment of the present invention, the injection volume of the sample solution may be 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL or 900 μL. The total flow rate of the mobile phase during the supercritical chromatography separation is preferably 3-100 mL / min. In a specific embodiment of the present invention, the total flow rate of the mobile phase may be 10 mL / min, 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min or 90 mL / min; the BPR pressure is preferably 50-200 MPa. In a specific embodiment of the present invention, the BPR pressure may be 80 MPa, 120 MPa, 150 MPa, 180 MPa or 200 MPa; the volume proportion of the modifier is preferably 5-60%. In a specific embodiment of the present invention, the volume proportion of the modifier may be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or 55%.

[0038] After the separation, the present invention preferably further comprises: concentrating the obtained paeoniflorin fraction and the obtained paeoniflorin lactone glycoside fraction under reduced pressure and drying them, respectively, to obtain the pure paeoniflorin and pure paeoniflorin lactone glycoside, respectively.

[0039] The following is a detailed description of the method for preparing paeoniflorin and paeoniflorin using supercritical fluid chromatography provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1 S1. Red peony root ( P. veitchii Lynch ) The rhizomes were crushed (80-100 mesh), and extracted 3 times at room temperature (18°C) (adding red peony root ( P. veitchii Lynch ) 8 times the weight of the rhizome) for 2 days each time, and the obtained alcohol extracts are combined and concentrated under reduced pressure to obtain the peony extract; S2. An equal volume of dichloromethane was added to the peony extract and extracted three times, the organic phases were combined and concentrated under reduced pressure to obtain a crude extract containing paeoniflorin and paeoniflorin; S3. The crude extract is concentrated to dryness under reduced pressure and then fractionated by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1 (volume ratio)), and the paeoniflorin and paeoniflorin fractions are collected and concentrated under reduced pressure to obtain a refined product containing paeoniflorin and paeoniflorin.

[0041] The purified product containing paeoniflorin and paeoniflorin was analyzed by HPLC using an Agilent ZORBAX SB-C18 5μm 4.6*150 mm column; time program: B-acetonitrile 0→100% (1-12 min)→100% (15 min)→0% (18 min); A-water; column temperature: 30°C.

[0042] Figure 1These are the HPLC chromatogram and MS total ion current spectrum of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1.

[0043] from Figure 1 It can be seen that it is difficult to achieve baseline separation of paeoniflorin and paeoniflorin in HPLC.

[0044] S4. Analysis by supercritical fluid chromatography The purified product containing paeoniflorin and paeoniflorin was filtered through a 0.25 μm filter membrane and then passed through a supercritical fluid chromatography instrument for analysis; Supercritical liquid chromatography: Shimadzu supercritical liquid chromatography system SFC-LC (DAD); Chromatographic column: silica (3 μm, 4.6*150 mm); Mobile phase: A-supercritical CO2 fluid; B-modifier (methanol); Time program: B Conc. 10% (0–1 min), 10%→40% (1→8 min), 40% (8–9 min), 40%→10% (9→10 min); Flow rate: 2.0 mL / min; Column temperature: 40°C; Back pressure regulator (BPR): 50 MPa, 50°C; Injection volume: 5 μL.

[0045] Figure 2 This is the SFC chromatographic analysis diagram of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1.

[0046] from Figure 2 It can be seen that paeoniflorin and paeoniflorin have good separation in SFC.

[0047] Example 2 The refined product containing paeoniflorin and paeoniflorin of Example 1 was filtered through a 0.25 μm filter membrane and then passed into a supercritical fluid chromatograph for analysis and separation. The chromatogram was recorded and the paeoniflorin and paeoniflorin fractions were collected and concentrated under reduced pressure to obtain high-purity paeoniflorin and high-purity paeoniflorin.

[0048] Supercritical liquid chromatography: Shimadzu supercritical liquid chromatography system SFC-LC (DAD); Chromatographic column: silica (3 μm, 4.6*150 mm); Mobile phase: A-supercritical CO2 fluid; B-modifier (methanol); Time program: B Conc. 20% methanol, 6.5 min / injection Flow rate: 3 mL / min; Column temperature: 40°C; Back pressure regulator (BPR): 50 MPa, 50°C; Injection volume: 10 μL; Figure 3 This is the SFC chromatogram of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1 in Example 2.

[0049] Figure 4 The high performance liquid chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 2 are shown.

[0050] from Figure 4 It can be seen that the content of the prepared high-purity paeoniflorin is 97.59%.

[0051] Figure 5 These are the HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 2.

[0052] from Figure 5 It can be seen that the content of the prepared high-purity paeoniflorin is 88.39%.

[0053] Example 3 The refined product containing paeoniflorin and paeoniflorin of Example 1 was filtered through a 0.25 μm filter membrane and then passed into a supercritical fluid chromatograph for analysis and separation. The chromatogram was recorded and the paeoniflorin and paeoniflorin fractions were collected and concentrated under reduced pressure to obtain high-purity paeoniflorin and high-purity paeoniflorin.

[0054] Supercritical liquid chromatography: Buchi SFC-50; Chromatographic column: silica (5 μm, 9.4*150 mm); Mobile phase: A-supercritical CO2 fluid; B-modifier (methanol); Time program: B Conc. 35% methanol, 2.7 min / injection, overlapping injection 2 min / injection; Flow rate: 20 mL / min; Column temperature: 40°C; Back pressure regulator (BPR): 100 MPa, 40°C; Injection volume: 500 μL; Figure 6 The SFC chromatogram preparation diagram and preparation stacking diagram of the refined product containing paeoniflorin and paeoniflorin of Example 1 in Example 3; from Figure 6It can be seen that under the SFC chromatographic conditions, the preparation of each sample injection only takes 2.5 minutes, and high-purity paeoniflorin and high-purity paeoniflorin can be prepared by stacked injection at 2 minutes per injection.

[0055] Figure 7 The HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 3; from Figure 7 It can be seen that the content of the prepared high-purity paeoniflorin is 99.90%.

[0056] Figure 8 The HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 3; from Figure 8 It can be seen that the content of the prepared high-purity paeoniflorin is 88.45%.

[0057] Example 4 The refined product containing paeoniflorin and paeoniflorin of Example 1 was filtered through a 0.25 μm filter membrane and then passed into a supercritical fluid chromatograph for analysis and separation. The chromatogram was recorded and the paeoniflorin and paeoniflorin fractions were collected and concentrated under reduced pressure to obtain high-purity paeoniflorin and high-purity paeoniflorin.

[0058] Supercritical liquid chromatography: Buchi SFC-50; Chromatographic column: Silica (5 μm, 9.4*150 mm); Mobile phase: A-supercritical CO2 fluid; B-modifier (methanol); Time program: B Conc. 35% methanol, 3.5 min / injection, overlapping injection 2 min / injection; Flow rate: 20 mL / min; Column temperature: 40°C; Back pressure regulator (BPR): 100 MPa, 40°C; Injection volume: 500 μL; Figure 9 This is the SFC chromatographic preparation diagram and preparation stacking diagram of the refined product containing paeoniflorin and paeoniflorin in Example 1 in Example 4.

[0059] from Figure 9 It can be seen that under the SFC chromatographic conditions, the preparation of each sample injection only takes 3.5 minutes, and high-purity paeoniflorin and high-purity paeoniflorin can be prepared by stacked injection at 3 minutes per injection.

[0060] Figure 10 The HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 4 are shown; from Figure 10It can be seen that the content of the prepared high-purity paeoniflorin is 98.48%.

[0061] Figure 11 The HPLC chromatogram and MS total ion current spectrum of the high-purity paeoniflorin prepared in Example 4; from Figure 11 It can be seen that the content of the prepared high-purity paeoniflorin is 92.97%.

[0062] Example 5 The only difference from Example 1 is that the crude extract containing paeoniflorin and paeoniflorin of Example 1 was used for analysis. Figure 12 shown.

[0063] Figure 12 This is the SFC chromatographic analysis chart of the crude extract containing paeoniflorin and paeoniflorin prepared in Example 1 in Example 5.

[0064] Depend on Figure 12 It can be seen that both paeoniflorin and paeoniflorin in the crude extract can be detected and have good separation from other impurities.

[0065] Example 6 The only difference from Example 2 is that the crude extract containing paeoniflorin and paeoniflorin from Example 1 was used for analysis and preparation. The results are as follows: Figure 13 shown.

[0066] Figure 13 This is the SFC chromatogram of the crude extract containing paeoniflorin and paeoniflorin prepared in Example 1 in Example 6.

[0067] Depend on Figure 13 It can be seen that both paeoniflorin and paeoniflorin in the crude extract can be monitored and baseline-separated from other impurities, and paeoniflorin and paeoniflorin can be directly prepared from the crude extract by supercritical chromatography.

[0068] Figure 14 The H NMR spectrum of the high-purity paeoniflorin prepared in Example 6 ( 1 H-NMR).

[0069] from Figure 14 It can be seen that the hydrogen spectrum information of paeoniflorin is: 1 H-NMR (600 MHz, CDCl3) δ : 8.06 (2H, d, J =7.0 Hz, H-2″ / 6″), 7.61 (1H, t, J = 7.5 Hz, H-4″), 7.49 (2H, t, J= 7.7 Hz, H-3″ / 5″), 5.44 (1H, s, 9-H), 4.75 (2H, s, H-8), 4.55 (1H, d, J = 7.5 Hz, H-Glc-1'),3.85 (1H, d, J = 12.0 Hz, H-Glc-6'a), 3.61 (1H, dd, J = 7.5, 4.0 Hz, H-Glc-6'b), 3.21-3.30 (4H, m, H-Glc-2' / 3' / 4' / 5'), 2.60 (1H, d, J = 6.0 Hz, H-5), 2.49 (1H,dd, J = 13.0, 7.0 Hz, H-7 β ), 2.20 (1H, d, J = 13.0 Hz, H-3 β ), 1.96 (1H, d, J = 11.0Hz, H-7 α ), 1.82 (1H, d, J = 12.0 Hz, H-3 α ), 1.37 (3H, s, H-10).

[0070] Figure 15 The carbon-1NMR spectrum of the high-purity paeoniflorin prepared in Example 6 ( 13 C-NMR).

[0071] from Figure 15 It can be seen that the carbon spectrum information of paeoniflorin is: 13 C-NMR (150 MHz, CDCl3) δ : 168.2 (C-7″), 134.7 (C-4″), 131.3 (C-1″), 130.9 (CH-2″ / 6″), 129.9 (CH-3″ / 5″), 106.6 (C-4), 102.5 (CH-9), 100.3 (CH-1’), 89.5 (C-1), 87.5 (C-2), 78.2 (CH-3'), 78.1 (CH-5'), 75.2 (CH-2'), 72.4 (C-6), 71.9 (CH-4'), 63.0 (CH2-6'), 61.9 (CH2-8), 44.7(CH2-3), 44.1 (CH-5), 23.6 (CH2-7), 19.8 (CH3-10).

[0072] Figure 16 Mass spectrum (ESI+) of high-purity paeoniflorin prepared in Example 6; Figure 17 The H NMR spectrum of the high-purity paeoniflorin prepared in Example 6 ( 1 H-NMR).

[0073] from Figure 17 It can be seen that the hydrogen spectrum information of paeoniflorin is: 1 H-NMR (600 MHz, CDCl3) δ : 8.04 (2H, d, J =7.0 Hz, H-2″ / 6″), 7.61 (1H, t, J = 7.5 Hz, H-4″), 7.48 (2H, t, J = 7.7 Hz, H-3″ / 5″), 4.79 (1H, d, J = 12.0 Hz, H-8a), 4.68 (1H, d, J = 12.0 Hz, H-8b), 4.54 (1H,d, J = 7.5 Hz, H-Glc-1'), 3.86 (1H, d, J = 12.0 Hz, H-Glc-6'a), 3.60 (1H, dd, J =7.5, 4.0 Hz, H-Glc-6'b), 3.21-3.35 (4H, m, H-Glc-2' / 3' / 4' / 5'), 2.91 (1H, d, J =6.0 Hz, H-5), 2.69 (1H, dd, J = 13.0, 7.0 Hz, H-7 β ), 2.41 (1H, d, J = 13.0 Hz, H-3 β ), 2.04 (1H, d, J = 11.0 Hz, H-7 α ), 2.02 (1H, d, J = 12.0 Hz, H-3 α ), 1.52 (3H,s, H-10).

[0074] Figure 18 The carbon NMR spectrum of the high-purity paeoniflorin prepared in Example 6 ( 13 C-NMR).

[0075] from Figure 18 It can be seen that the carbon spectrum information of paeoniflorin is: 13 C-NMR (150 MHz, CDCl3) δ : 178.1 (C-9),168.1 (C-7″), 134.5 (C-4″), 131.2 (C-1″), 130.8 (CH-2″ / 6″), 129.8 (CH-3″ / 5″), 100.2 (CH-1’), 93.6 (C-1), 88.8 (C-2), 78.1 (CH-3'), 78.0 (CH-5'), 74.9 (CH-2'), 71.6 (CH-4'), 68.4 (CH-4), 62.8 (CH2-6'), 62.1 (CH2-8), 56.9 (C-6), 41.7(CH2-3), 41.6 (CH-5), 28.5 (CH2-7), 20.7 (CH3-10).

[0076] Figure 19 This is the mass spectrum (ESI+) of the high-purity paeoniflorin prepared in Example 6.

[0077] Comparative Example 1 The only difference from Example 2 is that a Diol column was used and the optimized time program was B Conc. 10% methanol and a flow rate of 2 mL / min. The SFC analysis results are shown in Figure 20 shown.

[0078] Figure 20 This is the SFC chromatogram of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1 in Comparative Example 1.

[0079] Depend on Figure 20 It can be seen that when using Diol column for analysis and / or preparation, the paeoniflorin (4.40-4.90 min) obviously contains impurities, resulting in a shoulder peak, which cannot achieve the purpose of separation and purification.

[0080] Comparative Example 2 The only difference from Example 2 is that a C18 column was used and the optimized time program was B Conc. 30% methanol and a flow rate of 3 mL / min. The SFC analysis results are shown in Figure 21 shown.

[0081] Figure 21 This is the SFC chromatogram of the refined product containing paeoniflorin and paeoniflorin prepared in Example 1 in Comparative Example 2.

[0082] Depend on Figure 21It can be seen that when using a C18 column for analysis and / or preparation, both paeoniflorin and paeoniflorin (0.90-1.60 min) showed severe tailing peaks and poor separation, which could not achieve the purpose of separation and purification.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing paeoniflorin and paeoniflorin using supercritical fluid chromatography, characterized in that: The following steps are involved: The solution of the sample is subjected to supercritical chromatography analysis to obtain a chromatogram of the sample; If the chromatogram shows that the sample contains paeoniflorin and paeoniflorin, the sample solution is subjected to supercritical fluid chromatography to obtain pure paeoniflorin and pure paeoniflorin; The chromatographic column used for the supercritical chromatography analysis and supercritical chromatography separation is silica.

2. The method according to claim 1, characterized in that The mobile phase used in the supercritical chromatography analysis and supercritical chromatography separation is a mixture of a supercritical fluid and a modifier; The supercritical fluid comprises one of supercritical CO2, supercritical NO2 and supercritical NH3; The modifier includes one or more of methanol, ethanol, acetonitrile and isopropanol.

3. The method according to claim 2, characterized in that During the supercritical fluid chromatography analysis: The silica has an inner diameter of 3-4.6 mm, a length of 100-250 mm, and a normal phase silica gel particle size of 3-5 μm; The concentration of the sample solution is 0.5-3 mg / mL, and the injection volume of the sample solution is 3-20 μL; The total flow rate of the mobile phase is 0.5-5 mL / min, the BPR pressure is 5-50 MPa, the volume proportion of the modifier is 2-60%, or the volume proportion of the modifier is 10% from 0 to 1 min, the volume proportion of the modifier increases uniformly from 10% to 40% from 1 to 8 min, the volume proportion of the modifier is 40% from 8 to 9 min, and the volume proportion of the modifier decreases uniformly from 40 to 10% from 9 to 10 min.

4. The method according to claim 2, characterized in that During the supercritical chromatography analysis and supercritical chromatography separation: The inner diameter of the silica is independently 4.6-10 mm, the length is independently 100-250 mm, and the normal phase silica gel particle size is independently 3-50 μm; The concentration of the sample solution is independently 0.5-10 mg / mL, and the injection volume of the sample solution is independently 10-1000 μL; The total flow rate of the mobile phase is independently 3-100 mL / min, the BPR pressure is independently 50-200 MPa, and the volume proportion of the modifier is independently 5-60%.

5. The method according to claim 1, 3 or 4, characterized in that During the supercritical fluid chromatography analysis and supercritical fluid chromatography separation, the temperature of the chromatographic column is independently 25-50° C., and the BPR temperature is independently 30-60° C.

6. The method according to claim 5, characterized in that The detector used for the supercritical fluid chromatography analysis is PDA, and the detection wavelength during the supercritical fluid chromatography analysis is 190-800 nm.

7. The method according to claim 1, characterized in that The method for preparing the sample solution comprises the following steps: Mixing the medicinal material containing paeoniflorin and paeoniflorin with an alcohol aqueous solution for extraction, and then concentrating the obtained extract under reduced pressure to obtain a solution of the sample; or, The peony root is mixed with an aqueous alcohol solution for extraction, and then the obtained extract is concentrated under reduced pressure to obtain a crude extract; The crude extract is extracted, and the obtained extract is concentrated to obtain a crude extract of paeoniflorin and paeoniflorin; the crude extract is segmented with silica gel and eluted, and the obtained eluate is concentrated to obtain a solution of the sample.

8. The method according to claim 7, characterized in that The volume fraction of alcohol in the alcohol aqueous solution is 60-100%; the alcohol includes methanol and / or ethanol; The extraction method includes one of maceration extraction, percolation extraction, ultrasonic extraction and reflux extraction.

9. The method according to claim 7 or 8, characterized in that The extraction agent used in the extraction includes one or more of dichloromethane, chloroform and ethyl acetate; The eluent used for the elution includes one or more of petroleum ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol and ethanol.

10. The method according to claim 1, characterized in that After the separation, the method further comprises: concentrating the obtained paeoniflorin fraction and the obtained paeoniflorin lactone glycoside fraction under reduced pressure and drying them respectively to obtain the pure paeoniflorin and the pure paeoniflorin lactone glycoside respectively.

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