Method for extracting, separating and purifying peonidin-3-O glucoside

Abstract

The invention belongs to the technical field of biological extraction, and particularly relates to a method for extracting, separating and purifying peonidin-3-O glucoside. The method comprises the following steps: pre-soaking purple corn flour in a methanol solution, then adding into an extraction column, loading an obtained crude extracting solution on an XAD-2 macroporous resin column, and eluting by using an ethanol solution; adjusting the volume fraction of ethanol in the eluent, loading on a spherical C18 silica gel column, carrying out gradient elution by using an ethanol solution as a phase B and a phase A as water, separating by using a rapid purification system to obtain a rapid purification system chromatogram, collecting a main elution peak, and carrying out reduced pressure rotary evaporation to obtain a concentrated product; and further purifying by using the preparative liquid phase to obtain the peonidin-3-O glucoside. The peonidin-3-O glucoside obtained by the method is in a purple fluffy lamellar crystal shape, the purity can reach 97% or above, and the peonidin-3-O glucoside has the advantages of short time consumption, low toxicity and the like, and is suitable for popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of bioextraction technology, specifically relating to a method for extracting, separating and purifying paeoniflorin-3-O glucoside. Background Technology

[0002] Purple corn is an annual plant belonging to the genus *Zea* of the Poaceae family. It has a sweet taste and neutral properties, and possesses hemostatic, diuretic, choleretic, and blood pressure-lowering effects. It is beneficial for premature graying of hair in young people, postpartum weakness in women, post-illness weakness, anemia, and kidney deficiency. Peonidin-3-glucoside is the main anthocyanin in the cob of purple corn, possessing antioxidant, anti-inflammatory, and metabolic regulatory functions, and is of significant value in the prevention of chronic diseases and the development of functional products.

[0003] Currently, the main extraction methods for paeoniflorin-3-O glucoside from purple corn anthocyanins include aqueous solution extraction, organic solvent extraction, ultrasonic-assisted extraction, and microwave-assisted extraction. The main separation methods for paeoniflorin-3-O glucoside include conventional silica gel column chromatography, ultrasonic extraction, macroporous adsorption resin purification, high-speed countercurrent chromatography (HSCLC), and high-performance liquid chromatography (HPLC). Conventional silica gel column chromatography yields high product purity but suffers from drawbacks such as highly toxic elution solvents, cumbersome process, long processing time, and low yield. HSCLC, as an efficient and convenient method, is used in research laboratories to separate the active ingredients of medicinal plants and fungi; however, it consumes a large amount of organic solvents, making it difficult to scale up for industrial separation and limiting its application. Summary of the Invention

[0004] In view of this, the purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for extracting, separating and purifying paeoniflorin-3-O glucoside. This invention employs column chromatography extraction, ethanol extraction, and C18-modified silica gel column chromatography to separate paeoniflorin, which features short extraction time, low toxicity, high extraction rate, and high purity.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: A method for extracting, separating, and purifying paeoniflorin-3-O glucoside includes the following steps: S1. Pre-soak purple corn flour in methanol solution, then add it to the extraction column to obtain crude extract of purple corn anthocyanins; S2. Load the crude extract of purple corn anthocyanins onto an XAD-2 macroporous resin column and elute with ethanol solution. S3. After adjusting the volume fraction of ethanol in the eluent, the solution is loaded onto a spherical C18 silica gel column. The ethanol solution is used as phase B and water is used as phase A. Gradient elution with ethanol solution is used. The product is separated by a rapid purification system. The chromatogram of the rapid purification system is obtained. The main elution peak is collected and the concentrated product is obtained by rotary evaporation under reduced pressure. S4. The concentrated product was further purified using preparative liquid chromatography to obtain paeoniflorin-3-O glucoside.

[0006] Furthermore, in step S1, the ratio of purple corn flour to methanol solution is 1 kg: 20 L.

[0007] Furthermore, in step S1, the methanol volume fraction in the methanol solution is 40%.

[0008] Furthermore, in step S2, the ethanol solution contains 90% ethanol by volume.

[0009] Further, in step S3, the volume fraction of ethanol in the eluent is adjusted to 70%.

[0010] Further, in step S3, the conditions for the C18 silica gel column are: particle size 20-45µm, pore size 100Å, diameter 3.12cm, and length 25.74cm.

[0011] Further, in step S3, gradient elution is performed using an ethanol solution with a volume fraction of 70-90%.

[0012] Further, in step S4, the purification conditions are as follows: the chromatographic column is a Shim-pack GIS C18 preparative column with a packing particle size of 10 μm, a column diameter of 30 mm, and a column length of 250 mm.

[0013] Further, in step S4, the separation conditions for the prepared liquid phase are as follows: phase A is an aqueous solution of formic acid with a volume fraction of 5%, and phase B is an acetonitrile solution of formic acid with a volume fraction of 5%; the elution gradient is: 0-90 minutes, 5% phase B - 22.5% phase B; the flow rate is 5 ml / min, and the detection wavelength is 525 nm.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) Compared with the traditional organic solvent extraction-silica gel column chromatography separation, the extraction method of the present invention produces paeoniflorin-3-O glucoside in the form of purplish-red fluffy crystals with a purity of over 97%.

[0015] 2) The method of extracting, separating and purifying paeoniflorin-3-O glucoside of the present invention yields paeoniflorin-3-O glucoside of high quality.

[0016] 3) The method for extracting, separating and purifying paeoniflorin-3-O glucoside of the present invention uses column chromatography extraction, which effectively reduces the consumption of extraction solvent and saves costs compared with conventional extraction methods.

[0017] 4) This invention uses C18 modified silica gel column chromatography to separate paeoniflorin-3-O glucoside. Compared with traditional silica gel column chromatography, it effectively shortens the separation time, which can be completed in just 90 minutes, and significantly improves the purity of paeoniflorin-3-O glucoside, which can be applied to large-scale production.

[0018] 5) This invention uses C18 modified silica gel column chromatography to separate paeoniflorin-3-O glucoside, and the elution solvent is non-toxic ethanol, which effectively avoids the use of highly toxic and highly volatile organic reagents such as chloroform. Attached Figure Description

[0019] Figure 1 The effect of different extraction solvents on the extraction rate of paeoniflorin-3-O glucoside; Figure 2 The effect of different methanol volume fractions on the extraction rate of paeoniflorin-3-O glucoside; Figure 3 To screen the adsorption capacity of different macroporous resins for paeoniflorin-3-O glucoside; Figure 4 To screen the desorption capacity of different macroporous resins for paeoniflorin-3-O-glucoside; Figure 5 The desorption rates of paeoniflorin-3-O glucoside by four different resins are shown. Figure 6 The desorption rates of paeoniflorin-3-O-glucoside when eluting XAD-2 macroporous resin with methanol and ethanol of different volume fractions are shown. Figure 7 Elution diagram of purple corn anthocyanins purified by macroporous resin XAD-2; Figure 8 The HPLC chromatogram shows the anthocyanin extracted with 40% methanol. Figure 9 The HPLC chromatogram shows the anthocyanins purified by XAD-2 resin. Figure 10 Recovery rate of paeoniflorin-3-O glucoside purified by macroporous resin; Figure 11 The elution amounts of paeoniflorin-3-O glucoside when eluting a C18 silica gel column with different volume fractions of methanol and ethanol; Figure 12 The elution amount of paeoniflorin-3-O glucoside when eluting a C18 silica gel column with different volume fractions of ethanol. Figure 13 Elution diagram for rapid purification system separation of anthocyanins from purple corn; Figure 14 HPLC detection chromatogram for rapid purification system separation of paeoniflorin-3-O glucoside; Figure 15Elution diagram for the preparation of paeoniflorin-3-O glucoside by liquid phase purification Figure 16 HPLC detection chromatogram for the preparation of paeoniflorin-3-O glucoside for liquid phase purification Figure 17 MS preparation of paeoniflorin-3-O glucoside after liquid-phase purification + Detection image; Figure 18 MS preparation of paeoniflorin-3-O glucoside after liquid-phase purification 2+ Detection image; Figure 19 This invention provides a flowchart for the extraction, separation, and purification of paeoniflorin-3-O glucoside. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention. Example

[0021] The selection of extraction solvents, macroporous resins, macroporous resin elution solvents, and C18 silica gel column elution solvents, as well as the analysis of the ethanol gradient elution process on the C18 silica gel column, were conducted as follows: 1. Effect of different extraction solvents on the extraction rate of paeoniflorin-3-O-glucoside Three portions of pulverized purple corn cob powder, each weighing 3g, were added to 60mL of methanol, ethanol, and acetone, respectively. The mixtures were extracted by shaking at 100 rpm for 2 hours, followed by centrifugation at 5000 rpm. The supernatant was collected, and the concentration (mg / mL) of paeoniflorin-3-O glucoside (P3G) in the supernatant was quantitatively determined using HPLC (high performance liquid chromatography). The extraction rate was calculated using the following formula: Extraction rate = (n×v) / m In the formula, n is the concentration of P3G in the extract, in mg / ml; v is the volume of different extracts, in ml; and m is the mass of purple corn cob powder, in g.

[0022] Depend on Figure 1 It is evident that the extraction rate of paeoniflorin-3-O glucoside from purple corn cob powder using methanol was significantly higher than that using ethanol or acetone as extraction solvents. The extraction rate with methanol was approximately 3.1 ± 0.1 mg / g, while the extraction rates with ethanol and acetone were significantly lower, at only 0.7 ± 0.07 and 0.1 ± 0.001 mg / g respectively. In conclusion, methanol is the optimal extraction solvent for paeoniflorin-3-O glucoside extraction.

[0023] Following the above screening, paeoniflorin-3-O glucoside was extracted using methanol as the extraction solvent. The effect of different methanol volume fractions on the extraction rate of paeoniflorin-3-O glucoside was further investigated. 5g of purple corn cob powder was weighed and added to 200ml Erlenmeyer flasks. 100ml of methanol aqueous solutions with volume fractions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% were added, respectively. Extraction was carried out by shaking at 100 rpm for 2 hours. After centrifugation at 5000 rpm, the supernatant was collected, and the concentration of P3G in the supernatant (mg / ml) was quantitatively determined using HPLC (High Performance Liquid Chromatography). The extraction rate of P3G was calculated.

[0024] Depend on Figure 2 It is evident that the extraction rate of paeoniflorin-3-O-glucoside gradually increased and then decreased with increasing methanol volume fraction. The extraction rate of paeoniflorin-3-O-glucoside reached its peak under both 40% and 100% methanol conditions. Under 40% methanol conditions, the extraction rate of paeoniflorin-3-O-glucoside reached 2.62 mg / g. Under 100% methanol conditions, the extraction rate was 3.16 mg / g. Considering the need for subsequent macroporous resin purification, 40% methanol was chosen as the main extraction solvent.

[0025] 2. Screening of the adsorption capacity of different macroporous resins for paeoniflorin-3-O-glucoside Pretreated AB-8, NKA-2, X-5, and XAD-2 resins were packed into chromatography columns with a column volume of 10.5 ml. 40% methanol extracts were directly loaded onto the column. Each resin was loaded 5 times, 10.5 ml each time. The eluent was collected, and 1 ml of the eluent was concentrated by nitrogen purging at 40°C. Then, 1 ml of chromatographic methanol was added to dissolve the eluent. The concentration of paeoniflorin-3-O-glucoside was then detected by HPLC. The adsorption capacity of the resin for anthocyanins was calculated according to the following formula.

[0026] Adsorption capacity = (n1×v1) - (n2×v2) / m1 In the formula, n1 is the concentration of P3G in the macroporous resin loading solution, in mg / ml; v1 is the volume of the loading solution, in ml; n2 is the concentration of P3G in the effluent during loading, in mg / ml; v2 is the volume of the effluent during loading, in ml; and m1 is the mass of the macroporous resin, in g.

[0027] from Figure 3It is evident that for the 40% methanol extract, AB-8, X-5, and XAD-2 resins reached adsorption saturation after three loading cycles, while NKA-2 resin did not reach adsorption saturation even after five loading cycles. NKA-2 showed the highest adsorption capacity for paeoniflorin-3-O-glucoside compared to the other three resins, reaching 0.50 mg / g. XAD-2's adsorption capacity for anthocyanins was second only to NKA-2 resin, with an adsorption capacity of 0.41 mg / g for paeoniflorin-3-O-glucoside.

[0028] Pretreated AB-8, NKA-2, X-5, and XAD-2 resins were packed into chromatography columns with a column volume of 10.5 ml. 40% methanol extract was directly loaded onto the column, with 52.5 ml of sample loaded onto each resin to saturate the anthocyanins. 80% methanol was used as the eluent, and each resin was eluted 5 times with 10.5 ml eluent each time. 1 ml of the eluent was collected, concentrated by nitrogen purging at 40°C, and then dissolved in 1 ml of chromatographic methanol. The concentration of paeoniflorin-3-O-glucoside was then determined by HPLC. The desorption capacity and desorption rate of anthocyanins were calculated using the following formula.

[0029] Desorption capacity = (n³ × v³) / m³ In the formula, n3 is the concentration of P3G in the macroporous resin eluent, in mg / ml; v3 is the volume of the eluent, in ml; and m3 is the mass of the macroporous resin, in g.

[0030] Desorption rate = Desorption amount * 100 / Adsorption amount from Figure 4 It is evident that when eluting the four saturated resins with 80% methanol, the desorption capacity of paeoniflorin-3-O-glucoside gradually increases with the number of elution cycles. XAD-2 and NKA-2 exhibit higher desorption capacities for paeoniflorin-3-O-glucoside compared to the other resins.

[0031] Depend on Figure 5 It is evident that NKA-2 resin exhibits the highest adsorption capacity for paeoniflorin-3-O-glucoside, but its desorption capacity for anthocyanins is weak when eluted with 80% methanol, with a desorption rate of only 43.46%. XAD-2 resin, on the other hand, shows the second highest adsorption capacity for anthocyanins, achieving a desorption rate of 52.53% when eluted with 80% methanol. Therefore, XAD-2 macroporous resin was selected for subsequent application.

[0032] 3. Screening of elution solvents for macroporous resins Twenty portions of XAD-2 pretreated adsorbent were loaded into chromatography columns (column volume 10.5 ml). A 40% methanol extract (without rotary evaporation to remove methanol) was directly loaded onto each column, with a loading volume of 21 ml. At this point, the adsorption of anthocyanins by XAD-2 reached saturation. Each column was rinsed with distilled water (21 ml) and then eluted with methanol and ethanol at volume fractions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, respectively, with an elution volume of 31.5 ml per column. All eluents were collected, and 1 ml of each eluent was concentrated by nitrogen purging at 40°C. The eluent was then redissolved in 1 ml of chromatographic grade methanol. The concentration of paeoniflorin-3-O-glucoside was determined by HPLC, and the desorption capacity was calculated.

[0033] Depend on Figure 6 It is evident that the desorption capacity of paeoniflorin-3-O-glucoside first increases and then decreases with increasing volume fractions of methanol and ethanol. Using 80% methanol provides a better elution effect for paeoniflorin-3-O-glucoside, achieving a desorption capacity of 0.22 mg / g. When using ethanol as the eluent, 90% ethanol exhibits a better elution effect, achieving a desorption capacity of 0.35 mg / g. Based on comprehensive comparison, we selected 90% ethanol as the eluent for purifying paeoniflorin-3-O-glucoside using XAD-2 macroporous resin.

[0034] 4. Scale-up of the process for purifying purple corn anthocyanins using macroporous resin After XAD-2 pretreatment, the resin was packed into columns with a diameter of 37.2 mm, a bed height of 372 mm (diameter-to-height ratio of 1:10), a resin packing amount of 170 g, and a column volume of 404 ml. The 40% methanol extract was directly loaded onto the XAD-2 column at a loading volume of 404 ml. After loading, elution was performed first with distilled water, followed by elution with 90% ethanol at a flow rate of 27 ml / min (1 column volume / hour). Figure 7 It can be seen that when the elution time reached 16 minutes, the anthocyanins were completely eluted, and the total elution volume was 432 ml.

[0035] Collect the elution peak No. 2, take 1 ml of the collected solution, concentrate it by purging with nitrogen at 40℃, add 1 ml of chromatographic methanol to dissolve it, and then detect the concentration of paeoniflorin-3-O glucoside by HPLC and calculate the elution volume. Figure 8 This is an HPLC chromatogram of anthocyanins extracted with 40% methanol. Figure 9 This is an HPLC chromatogram showing the purification of anthocyanins using XAD-2 resin. From... Figure 8 and Figure 9As can be seen from the comparison, after purification with XAD-2, paeoniflorin-3-O glucoside was effectively enriched, and the proportion of paeoniflorin-3-O glucoside in the total anthocyanin extract increased from 5.98% (mg / mg) to 7.63% (mg / mg). Figure 10 To improve the recovery rate of paeoniflorin-3-O glucoside purified by macroporous resin, Figure 10 The results showed that the recovery rate of paeoniflorin-3-O glucoside in purple corn anthocyanins purified by XAD-2 macroporous resin reached 97.91%.

[0036] 5. Screening of elution solvents for C18 silica gel columns Twenty spherical C18 silica gel columns (particle size 20-45µm, pore size 100Å, diameter 3.12cm, length 25.74cm, two columns in series) were used, with a column volume of 393.5ml. XAD-2 eluent was adjusted to 70% ethanol and loaded onto the C18 silica gel columns, with each loading volume being 555.43ml. After loading each silica gel column, 555.43ml of each column was washed with distilled water, followed by elution with methanol and ethanol at concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (v / v) at a flow rate of 18.5ml / min for a total of 393.5ml. One ml of the eluent was concentrated by nitrogen purging at 40℃, dissolved in 1ml of chromatographic methanol, and then analyzed by HPLC to determine the concentration of paeoniflorin-3-O-glucoside. The anthocyanin elution volume and elution rate were calculated by multiplying the anthocyanin concentration by the elution volume.

[0037] Elution volume = (n5 × v5) / m5 In the formula, n5 is the concentration of P3G in the eluent of the C18 silica gel column, in mg / ml; v5 is the volume of the eluent, in ml; and m5 is the mass of the C18 silica gel, in g.

[0038] Figure 11 The effect of different volume fractions of methanol and ethanol on the elution amount of paeoniflorin-3-O-glucoside. Figure 11It is evident that the elution amount of paeoniflorin-3-O-glucoside gradually increases with increasing methanol volume fraction. However, once the methanol volume fraction exceeds 50%, the elution amount of anthocyanins no longer increases with further increases in methanol volume fraction. When using 10%-50% ethanol for elution, the elution amount of anthocyanins gradually increases with increasing ethanol volume fraction. With 60%-70% ethanol, the elution amount of anthocyanins decreases. With 70%-100% ethanol, the elution amount of anthocyanins increases with increasing ethanol volume fraction. Ethanol is more effective than methanol in eluting paeoniflorin-3-O-glucoside, therefore ethanol is chosen as the elution solvent. The elution amount of paeoniflorin-3-O-glucoside with 100% ethanol is 0.37 mg / g, while the elution amount with 70% ethanol is only 0.12 mg / g, a difference of 0.25 mg / g. The elution of paeoniflorin-3-O glucoside by 70% ethanol was low. Based on this finding, we adjusted the ethanol concentration of the XAD-2 eluent containing 90% ethanol to 70% and directly loaded it onto a C18 silica gel column for adsorption. Then, we used a gradient of 70%-100% ethanol to separate the anthocyanins. This reduced the step of rotary evaporation to remove ethanol and saved time.

[0039] 6. Ethanol gradient elution on a C18 silica gel column XAD-2 eluent, adjusted to 70% ethanol concentration, was directly loaded onto a spherical C18 silica column (particle size 20-45µm, pore size 100Å, diameter 3.12cm, length 25.74cm, two columns in series), with a column volume of 393.5ml and a sample loading volume of 555.43ml. Subsequently, 393.5ml was eluted sequentially with 70%, 75%, 80%, 85%, 90%, 95%, and 100% ethanol at a flow rate of 18.5ml / min. 1ml of the eluent was collected, concentrated under nitrogen at 40℃, dissolved in 1ml of chromatographic methanol, and then analyzed by HPLC to determine the concentration of paeoniflorin-3-O-glucoside and calculate the elution volume.

[0040] Figure 12 The effect of sequential elution with different volume fractions of ethanol on the elution rate of paeoniflorin-3-O glucoside is shown. Since paeoniflorin-3-O glucoside was not detected in the 100% ethanol eluate, it is not shown in the figure. Figure 12 It is evident that during sequential elution, elution of paeoniflorin-3-O glucoside can be completed by increasing the ethanol concentration from 70% to 90%. The elution of paeoniflorin-3-O glucoside peaks at 0.20 mg / g with 75% ethanol. After sequential elution reaches 90% ethanol fraction, the total eluted anthocyanin concentration reaches 93.15%.

[0041] 7. Use a rapid purification system to separate paeoniflorin-3-O glucoside XAD-2 eluent, after adjusting the ethanol concentration to 70%, was directly loaded onto a spherical C18 silica column (3.12 cm in diameter, 25.74 cm in length, two columns in series), with a column volume of 393.5 ml and a sample loading volume of 555.43 ml. The anthocyanins purified from XAD-2 were further separated using a rapid purification system (SepaBean machine T, Sante Technology (Changzhou) Co., Ltd.). The separation column used was a spherical C18 silica column (particle size 20-45 µm, pore size 100 Å, diameter 3.12 cm, length 25.74 cm, two columns in series). The elution conditions were: phase A water, phase B ethanol, gradient set to 0-90 min, 70% B-100% B, flow rate of 26.5 ml / min, and detection wavelength of 525 nm. Figure 13 Elution diagram for rapid purification system separation of anthocyanins from purple corn. Figure 13 The results showed that when using a rapid purification system to separate anthocyanins from purple corn, four main elution peaks appeared. After collecting the second elution peak, 1 ml of the collected solution was concentrated by nitrogen purging at 40°C and then dissolved in 1 ml of chromatographic methanol. Subsequently, the concentration of paeoniflorin-3-O glucoside was detected by HPLC, and the elution volume was calculated.

[0042] Figure 14 HPLC chromatogram for the rapid purification system separating paeoniflorin-3-O glucoside. (From...) Figure 14 As can be seen, the content of paeoniflorin-3-O glucoside in peak 2 accounted for 43.56% (mg / mg) of the total anthocyanin extract. This demonstrates that the rapid purification system effectively enriched paeoniflorin-3-O glucoside.

[0043] Preparative liquid chromatography purification of high-purity paeoniflorin-3-O glucoside Peak 2, separated by the rapid purification system, was subjected to rotary evaporation at 40°C under reduced pressure to remove ethanol, yielding a concentrated product. To obtain high-purity paeoniflorin-3-O glucoside, the concentrated product was further purified using a preparative HPLC system. A Shim-pack GIS C18 preparative column (10 μm particle size, 30 mm diameter, 250 mm length) was selected. The separation conditions for the preparative HPLC were: Phase A 5% formic acid aqueous solution, Phase B 5% formic acid acetonitrile solution, 0-90 min, 5% B-22.5% B, flow rate 5 ml / min, and detection wavelength 525 nm.

[0044] Figure 15 Elution chromatogram for the preparation and purification of paeoniflorin-3-O glucoside by liquid chromatography. (From...) Figure 15As can be seen, after preparative liquid chromatography separation, the rapid purification system separated the product into 6 main chromatographic peaks, and peak number 4 was collected. 1 ml of eluent was taken, concentrated by nitrogen purging at 40 °C, and then dissolved in 1 ml of chromatographic methanol. Subsequently, the concentration of paeoniflorin-3-O-glucoside was detected by HPLC, and the elution amount of anthocyanins was calculated by multiplying the anthocyanin concentration by the volume of the collected liquid.

[0045] Figure 16 HPLC detection chromatogram for the preparation of paeoniflorin-3-O glucoside for liquid phase purification. Figure 16 Display of preparative liquid chromatography Figure 4 The purity of peak number 1 reached 97%, and the recovery rate reached 89.62%.

[0046] High-resolution mass spectrometry (HMS) analysis was performed on the prepared paeoniflorin-3-O glucoside after liquid-phase purification. The mass spectrometry analysis was performed in positive ion mode by electrospray ionization (ESI), with the following parameters: fragmentation voltage 4 kV, ion source temperature 300 ℃, drying gas temperature 250 ℃, and drying gas flow rate 15 L / min. Figure 17 MS of paeoniflorin-3-O glucoside after preparative liquid chromatography purification + Detection image. Figure 18 MS of paeoniflorin-3-O glucoside after preparative liquid chromatography purification 2+ Detection image. Figure 17 The results show that the m / z (mass-to-charge ratio) of the molecular ion peak of paeoniflorin-3-O glucoside is 463.12317. Figure 18 The results showed that the second-order mass spectrometry peak of the molecular ion peak with an m / z (mass-to-charge ratio) of 463.12317 had an m / z (mass-to-charge ratio) of 301.07025. High-resolution mass spectrometry analysis confirmed that peak number 4 obtained from preparative liquid phase separation was paeoniflorin-3-O glucoside.

[0047] Based on the above analysis, a method for extracting, separating, and purifying paeoniflorin-3-O glucoside is proposed, such as... Figure 19 As shown, it includes the following steps: S1. The dried purple corn cobs, dried to constant weight, are crushed into coarse powder with a particle size of 60 mesh for later use. Weigh 50g of the crushed purple corn powder from S1, add 1000mL of 40% methanol solution for pre-soaking, then add it to the extraction column, shake at 100 rpm for 2 hours, centrifuge at 5000 rpm, and collect the supernatant to obtain the crude extract of purple corn anthocyanins.

[0048] S2. The crude extract of anthocyanins from purple corn is directly loaded onto an XAD-2 macroporous resin column, and the liquid is used for the extraction of the next batch of anthocyanins. Eluent is eluted with 90% ethanol solution.

[0049] S3. After adjusting the ethanol volume fraction in the eluent to 70%, the solution is loaded onto a spherical C18 silica gel column with a particle size of 20-45µm, a pore size of 100Å, a diameter of 3.12cm, and a length of 25.74cm. The ethanol eluent is used as phase B, and water is used as phase A. Gradient elution is performed using ethanol solutions with volumes of 70-100% ethanol. The solution is then separated using a rapid purification system to obtain a chromatogram of the rapid purification system. The main elution peak is collected, and the ethanol is removed by rotary evaporation under reduced pressure at 40℃ to obtain the concentrated product. S4. The concentrated product was further purified by preparative liquid chromatography to obtain paeoniflorin-3-O glucoside. The purification conditions were as follows: a Shim-pack GIS C18 preparative column (10 μm particle size, 30 mm diameter, 250 mm length); the separation conditions for preparative liquid chromatography were: phase A was a 5% (v / v) formic acid aqueous solution, and phase B was a 5% (v / v) formic acid-acetonitrile solution; the elution gradient was 0-90 min, 5% phase B - 22.5% phase B; the flow rate was 5 ml / min, and the detection wavelength was 525 nm. After purification by preparative liquid chromatography, paeoniflorin-3-O glucoside with a purity greater than 97% was obtained.

[0050] The above description is only for better explaining the embodiments of the present invention and is not intended to limit them. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims

1. A method for extracting, separating, and purifying paeoniflorin-3-O glucoside, characterized in that, Includes the following steps: S1. Pre-soak purple corn flour in methanol solution, then add it to the extraction column to obtain crude extract of purple corn anthocyanins; S2. Load the crude extract of purple corn anthocyanins onto an XAD-2 macroporous resin column and elute with ethanol solution. S3. After adjusting the volume fraction of ethanol in the eluent, the solution is loaded onto a spherical C18 silica gel column. The ethanol solution is used as phase B and water is used as phase A. Gradient elution with ethanol solution is used. The product is separated by a rapid purification system. The chromatogram of the rapid purification system is obtained. The main elution peak is collected and the concentrated product is obtained by rotary evaporation under reduced pressure. S4. The concentrated product was further purified using preparative liquid chromatography to obtain paeoniflorin-3-O glucoside.

2. The method for extracting, separating, and purifying paeoniflorin-3-O glucoside according to claim 1, characterized in that, In step S1, the ratio of purple corn flour to methanol solution is 1 kg: 20 L.

3. The method for extracting, separating, and purifying paeoniflorin-3-O glucoside according to claim 1, characterized in that, In step S1, the methanol volume fraction in the methanol solution is 40%.

4. A method for extracting, separating, and purifying paeoniflorin-3-O glucoside according to claim 1, characterized in that, In step S2, the ethanol solution contains 90% ethanol by volume.

5. A method for extracting, separating, and purifying paeoniflorin-3-O glucoside according to claim 1, characterized in that, In step S3, the volume fraction of ethanol in the eluent is adjusted to 70%.

6. The method for extracting, separating, and purifying paeoniflorin-3-O glucoside according to claim 1, characterized in that, In step S3, the conditions for the C18 silica gel column are: particle size 20-45µm, pore size 100Å, diameter 3.12cm, and length 25.74cm.

7. A method for extracting, separating, and purifying paeoniflorin-3-O glucoside according to claim 1, characterized in that, In step S3, gradient elution is performed using an ethanol solution with a volume fraction of 70-90%.

8. A method for extracting, separating, and purifying paeoniflorin-3-O glucoside according to claim 1, characterized in that, In step S4, the purification conditions are as follows: the chromatographic column is a Shim-pack GIS C18 preparative column with a packing particle size of 10 μm, a column diameter of 30 mm, and a column length of 250 mm.

9. A method for extracting, separating, and purifying paeoniflorin-3-O glucoside according to claim 1, characterized in that, In step S4, the separation conditions for the prepared liquid phase are as follows: phase A is an aqueous solution of formic acid with a volume fraction of 5%, and phase B is an acetonitrile solution of formic acid with a volume fraction of 5%; the elution gradient is: 0-90 minutes, 5% phase B - 22.5% phase B; the flow rate is 5 ml / min, and the detection wavelength is 525 nm.

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