Method for extracting and separating flavonoids from fennel

Abstract

The invention discloses a method for extracting and separating takakin‑8‑O‑β‑D‑glucopyranoside from Herba Cibotii, which comprises the steps of total flavonoid extraction, macroporous adsorption resin purification, silica gel column chromatography, semi-preparative liquid phase separation, etc. The invention prepares the flavonoid compound from Herba Cibotii for the first time, solves the practical problem that the prior art can only analyze and identify but cannot prepare the compound, thus laying a foundation for exploring the pharmacological activity of the compound. The invention also has the advantages of high yield and purity, low pollution, etc.

Description

Technical Field

[0001] The invention relates to the extraction of active ingredients of traditional Chinese medicine, and in particular to a method for extracting and separating takakin-8-O-β-D-glucopyranoside from scutellaria baicalensis. Background Art

[0002] Dracocephalum moldavica L, known as Badiranjibuya in Xinjiang, China, is the dried aerial part of the plant Dracocephalum moldavica of the Lamiaceae family. It is also the key raw material for preparing the Chinese patent medicine Yixin Badiranjibuya granules. Dracocephalum moldavica is mainly used clinically to treat cardiovascular and cerebrovascular diseases such as coronary heart disease, angina pectoris, cerebral thrombosis, and hypertension. It has multiple pharmacological effects such as protecting myocardial ischemia, lowering blood lipids, resisting lipid peroxidation damage, and scavenging free radicals.

[0003] There are a large number of saponin compounds in Xiangqinglan, which are divided into triterpenoid saponins and steroidal saponins. Representative ingredients include rosmarinic acid, oleanolic acid, betulinic acid, etc. In addition, there are a variety of phenylpropanoid compounds in Xiangqinglan, including phenylpropionic acid, coumarins, and lignans, all of which have the characteristics of C6-C3 carbon skeleton structure. Its representative ingredients are chlorogenic acid, rosmarinic acid, syringaresinol, caffeic acid, coumarin, etc. Plant polysaccharides are also one of the important active components in Xiangqinglan. Guo Chunmei et al. extracted and determined the content of polysaccharides in Xiangqinglan and found that the content of polysaccharides in Xiangqinglan was 6.38%. The whole plant of Xiangqinglan contains a variety of volatile oils, which gives it a unique smell and can be used to prepare essential oils. The volatile oils in Xiangqinglan mainly include limonene, geraniol, nerol, citronellol, thymol and other ingredients.

[0004] In addition, flavonoids are an important component of the chemical composition of radix scutellariae, with a typical structure of C6-C3-C6 carbon skeleton structure, mainly including flavonoids, flavonols, isoflavones, dihydroflavones, flavonoid glycosides, etc. Representative components include rutin, quercetin, luteolin, isorhamnetin, acaciatin, and scutellariae.

[0005] In order to explore the mechanism of action of traditional Chinese medicine and reveal the material basis of its effect in treating diseases, the applicant used modern traditional Chinese medicine extraction technology, combined with gas phase, liquid phase, mass spectrometry, nuclear magnetic resonance and other detection and identification methods, to systematically study the chemical components in Xiangqinglan, and finally identified a variety of compounds. In order to conduct high-throughput activity screening of these compounds, it is necessary to extract and separate them. In this process, the applicant identified a flavonoid compound from Xiangqinglan. In order to explore its pharmacological activity, the applicant designed an experiment to extract and separate the compound from Xiangqinglan, thus completing the present invention. Summary of the invention

[0006] The purpose of the present invention is to provide a method for extracting and separating the following flavonoid compounds from Herba Cibotii.

[0007]

[0008] takakin-8-O-β-D-glucopyranoside

[0009] The method comprises the following steps:

[0010] 1) crushing the fragrant blue orchid, adding an extraction solvent, heating at 80-100° C. for reflux extraction, collecting the extract and concentrating it into an extract;

[0011] 2) applying the extract to a macroporous adsorption resin column, eluting with 70% (volume) ethanol after adsorption equilibrium, and concentrating the eluate to obtain a purified total flavonoid;

[0012] 3) The purified total flavonoids were mixed with silica gel powder and then dry-loaded for silica gel column chromatography. The mixture was eluted with methanol-dichloromethane in different volume ratios, and the eluted fractions containing the target compound were collected;

[0013] 4) The eluted fraction containing the target product is concentrated and dried, then diluted with a diluent, and then subjected to chromatographic separation on a semi-preparative high performance liquid chromatograph, wherein the stationary phase of the semi-preparative high performance liquid chromatograph is C18, the mobile phase is acetonitrile-water, and the gradient elution is performed. The eluate containing the target product is detected at a certain wavelength and collected, and the pure product of takakin-8-O-β-D-glucopyranoside is obtained after drying.

[0014] Wherein, the extraction solvent is alcohol, preferably methanol, and most preferably 80% (volume) methanol.

[0015] The solid-liquid ratio of the reflux extraction is 1:40 (g / ml), the extraction temperature is 80° C., and the extraction time is 1 h.

[0016] Wherein, the macroporous adsorption resin is a polar resin, preferably HPD-500 macroporous adsorption resin.

[0017] The elution with methanol-dichloromethane in different volume ratios refers to the elution using methanol-dichloromethane in 11 different ratios, namely 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0, and the target object is present in the 4:6 eluent.

[0018] Wherein, the gradient elution program is shown in the following table:

[0019]

[0020]

[0021] Wherein, the sample loading volume of the chromatographic separation is 0.5 ml, and the flow rate is 6 ml / min.

[0022] Wherein, the detection wavelength of the semi-preparative high performance liquid chromatograph is 328 nm.

[0023] Wherein, the diluent is methanol.

[0024] The beneficial effects of the present invention are:

[0025] The present invention prepares takakin-8-O-β-D-glucopyranoside from Scutellaria baicalensis for the first time, solving the practical problem that the prior art can only analyze and identify the compound from Scutellaria baicalensis but cannot prepare it, thereby laying a foundation for exploring the pharmacological activity of the compound.

[0026] The invention optimizes the process conditions so that the prepared compound has higher yield and purity.

[0027] Compared with chemical synthesis methods, the present invention has the advantages of less pollution, energy saving and environmental protection. DETAILED DESCRIPTION

[0028] The present invention is described in detail below with reference to specific embodiments.

[0029] The Herba Citri Reticulatae used in the experiment was from Wuhan Jianmin Zhongwei Pharmaceutical Co., Ltd., batch number: 20210315. Other reagents, materials, and instruments are shown in Tables 1 and 2.

[0030] Table 1 Reagents and materials used in the experiment

[0031]

[0032]

[0033] Table 2 Main experimental instruments and equipment

[0034]

[0035] Example 1 Study on the extraction process of total flavonoids

[0036] This study used a single factor experiment to determine the key variables that affect the extraction process, and on this basis, an orthogonal test was conducted. The total flavonoids content was determined using the UV method with rutin as the standard. Finally, the optimal extraction process for total flavonoids from Rhizoma Cibotii was screened out.

[0037] 1. Extraction process single factor determination

[0038] Take the powder of Scutellaria baicalensis, weigh it, place it in a round-bottom flask, add an appropriate amount of methanol solution with a corresponding volume fraction, heat it under reflux at a corresponding temperature, filter it, and concentrate it to obtain the extract. The single factor selection and level setting are as follows:

[0039] Extraction solvent: fixed solid-liquid ratio 1:30, extraction time 1h, extraction temperature 60℃, extraction 1 time. The effects of 7 different extraction solvents (30% methanol, 70% methanol, 95% methanol, 30% ethanol, 70% ethanol, 95% ethanol, water) on the extraction of total flavonoids from Scutellaria baicalensis were investigated.

[0040] Solid-liquid ratio: fixed extraction solvent 70% methanol, extraction time 1h, extraction temperature 60℃, extraction 1 time. The effects of three different solid-liquid ratios (1:10, 1:30, 1:50) on the extraction of total flavonoids from Scutellaria baicalensis were investigated.

[0041] Extraction time: The extraction solvent was fixed at 70% methanol, the solid-liquid ratio was 1:30, the extraction temperature was 60°C, and the extraction was repeated once. The effects of three different extraction times (0.5h, 1h, and 1.5h) on the extraction of total flavonoids from Scutellaria baicalensis were investigated.

[0042] Extraction temperature: The extraction solvent was fixed at 70% methanol, the solid-liquid ratio was 1:30, the extraction time was 1h, and the extraction was repeated once. The effects of three different extraction temperatures (50℃, 70℃, and 90℃) on the extraction of total flavonoids from Scutellaria baicalensis were investigated.

[0043] Extraction times: The extraction solvent was fixed at 70% methanol, the solid-liquid ratio was 1:30, the extraction time was 1h, and the extraction temperature was 60°C. The effects of three different extraction times (1, 2, and 3 times) on the extraction of total flavonoids from Scutellaria baicalensis were investigated.

[0044] The extraction effect of total flavonoids was evaluated by thin layer chromatography: rutin was used as the reference substance, the extract was filtered and concentrated at the same ratio, and then spotted on GF 254 Silica gel plate, using BAW system (n-butanol: glacial acetic acid: water = 4:1:5) as developing solvent, observed under 254nm ultraviolet light. The extraction conditions were preliminarily determined based on the number of spots and color depth of the thin layer chromatography.

[0045] Extraction solvent: There were more spots and darker colors in the 70% methanol extract, so three volume fraction levels of 60%, 70%, and 80% were set.

[0046] Solid-liquid ratio: The number of spots in the extract with a solid-liquid ratio of 1:10 is relatively small, while the number of spots in the extracts with a solid-liquid ratio of 1:30 and 1:50 is similar, and the color depth is roughly the same, indicating that the dissolution of flavonoids and their glycosides reaches equilibrium at a solid-liquid ratio of 1:30. Therefore, 1:30 is selected as the base value of the solid-liquid ratio, and three solid-liquid ratio levels are set at 1:20, 1:30, and 1:40, respectively.

[0047] Extraction time: The spots in the extracts after 0.5h, 1h, and 1.5h of extraction were similar in color and the difference was not obvious, so 1h was selected as the optimal extraction time.

[0048] Extraction temperature: At 90°C, there were obviously more spots in the extract and the spots were darker in color, so 90°C was selected as the base value, and three temperature levels were set at 80°C, 90°C, and 100°C.

[0049] Number of extractions: The UV colorimetric results showed that the spots in the extract after the first extraction were very dark in color, while there were almost no spots in the extracts after the second and third extractions. Therefore, one extraction was selected as the fixed number of extractions.

[0050] 2. Orthogonal test to select the best process

[0051] 2.1 Based on the single factor experiment, the extraction time and extraction times were fixed, and the solid-liquid ratio (A), extraction temperature (B), and methanol volume fraction (C) were used as horizontal factors to design L9 (3 4 ) was used as an orthogonal experimental scheme to optimize the total flavonoids content in the extract of Scutellaria baicalensis to obtain the optimal extraction process.

[0052] 2.2 Determination of total flavonoid content (sodium nitrite-aluminum nitrate method)

[0053] 2.2.1 Principle and method

[0054] Metal ions can complex with carbonyl and hydroxyl groups in adjacent positions in the flavonoid structure to produce a color reaction, and then the total flavonoid content is determined according to the absorbance value of the solution. This method uses rutin as the standard, ultraviolet spectrophotometry, sodium nitrite-aluminum nitrate as the color developer, and the detection wavelength is 510nm.

[0055] 2.2.2 Preparation of standard solution

[0056] Place the rutin reference substance in a vacuum drying oven and continue drying for 24 hours. Accurately weigh 1.6544 mg and place it in a 100 ml volumetric flask. Add methanol and dilute to the scale to prepare a standard solution containing 16.544 μg / ml of rutin for use.

[0057] 2.2.3 Rutin standard curve drawing

[0058] Accurately measure 0.0, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 ml of 16.544 μg / ml rutin standard solution into a 25 ml volumetric flask and shake well. First add 0.3 ml of 5% sodium nitrite solution, shake well and let stand for 6 minutes, add 0.3 ml of 10% aluminum nitrate solution, shake well and let stand for 6 minutes, add 4 ml of 4% sodium hydroxide solution, add distilled water to volume, shake well, and let stand for 15 minutes.

[0059] Each standard solution is numbered 0, 1, 2, 3, 4, 5, and 6 in sequence, and No. 0 is the blank control group. 510nm is selected as the ultraviolet absorption wavelength for detection, and the absorbance of each solution is measured. With rutin concentration as the horizontal coordinate and absorbance as the vertical coordinate, the least squares method is used for linear regression to obtain the rutin standard curve. The equation of the rutin standard curve is y=0.0128x-0.0033, R 2 =0.9995. The results showed that the total flavonoids had a good linear relationship in the range of 1.6544μg / ml to 16.544μg / ml.

[0060] 2.2.4 Sample content determination

[0061] After the extract of Scutellaria baicalensis is concentrated and dried, it is re-dissolved in 70% methanol. 1 ml is accurately measured in a 25 ml volumetric flask, and the absorbance is determined according to the above method. The total flavonoids standard curve is substituted to calculate the total flavonoids content in the extract.

[0062] 2.3 Orthogonal experimental results

[0063] Through the single factor experiment, it was found that the extraction time and the number of extractions under the preset experimental conditions had little effect on the extraction of total flavonoids. Therefore, the extraction time was fixed to 1h, the number of extractions was 1, and the solid-liquid ratio (A), extraction temperature (B), and methanol volume fraction (C) that mainly affected the extraction of total flavonoids were selected as three factors, and three different levels were selected for each factor. The factor and level settings are shown in Table 3.

[0064] Table 3 Extraction process factors and levels

[0065]

[0066] The results of the orthogonal experiment and variance analysis are shown in Tables 4 and 5. It can be seen from the range that the factors affecting the extraction of total flavonoids from Scutellaria baicalensis are A>C>B. Different solid-liquid ratios have a significant effect on the total flavonoids content of Scutellaria baicalensis. The extraction temperature and the volume fraction of methanol in the extraction solvent have no significant effect. The optimal process is A3B1C3, that is, the solid-liquid ratio is 1:40, the extraction temperature is 80℃, the volume fraction of methanol is 80%, reflux extraction is once, and the extraction time is 1h.

[0067] Table 4L9(3 4 )Orthogonal experimental scheme and results

[0068]

[0069] Table 5 Analysis of variance

[0070]

[0071] F 0.05 (2,2)=19; F0.01 (2,2)=99;f=2.

[0072] 2.4 Validation of the optimal process for total flavonoid extraction

[0073] Three process verification tests were carried out under the optimal process of solid-liquid ratio of 1:40, extraction temperature of 80°C, methanol volume fraction of 80%, reflux extraction once and extraction time of 1h. The results showed that the average content of total flavonoids in 1g of Scutellaria baicalensis extract was 12.56%.

[0074] Example 2 Study on the purification and separation process of the target

[0075] In this study, macroporous adsorption resin purification, silica gel column chromatography and semi-preparative liquid phase separation were used as technical methods to purify and separate the total flavonoids of Herba Cinerariae.

[0076] 1. Screening of macroporous adsorption resin

[0077] The type of macroporous adsorption resin used for purification was determined through static adsorption elution experiments. The main performance parameters of the four macroporous adsorption resins investigated in the experiment are shown in Table 6.

[0078] Table 6 Performance of macroporous adsorption resin for total flavonoid purification

[0079]

[0080] Accurately weigh 2.0g of each of HPD-500, HP-20, AB-8, and D101 resins, place them in a 50ml conical flask, and add 10ml of the extract of 23.54mg / ml of total flavonoids from the fragrant blue orchid. Place it in a constant temperature oscillator for static adsorption, set the temperature to 25°C, the oscillation frequency to 150rpm, and fully adsorb for 12h. Use a pipette to draw 2ml, and use the method under "2.2 of Example 1" to determine the total flavonoid concentration of the sample after adsorption equilibrium. Calculate the adsorption amount of the resin at adsorption equilibrium by formula (1-1), and calculate the total flavonoid adsorption rate by formula (1-2).

[0081] After adsorption equilibrium, transfer the resin to a 150 ml conical flask, add 10 ml 70% ethanol, put it in a constant temperature oscillator, set the temperature to 25 ° C, the oscillation frequency to 150 rpm, and oscillate fully for 12 hours. Determine the total flavonoid concentration of the sample solution after elution equilibrium, and calculate the total flavonoid resolution rate by formula (1-3).

[0082] Resin adsorption capacity: Q (mg / g) = V (C0-C1) / M (1-1)

[0083] Resin adsorption rate: E(%) = [(C0-C1) / C0]*100% (1-2)

[0084] Resin resolution: D (%) = [C2 / (C0-C2)] * 100% (1-3)

[0085] Where: C0: extract concentration before adsorption; C1: sample solution concentration after adsorption equilibrium; C2: sample solution concentration after desorption equilibrium; V: sample solution volume; M: resin mass.

[0086] Static adsorption and elution tests were carried out on the total flavonoids in the extract using different types of macroporous adsorption resins. The results are shown in Table 7.

[0087] Table 7 Adsorption and desorption results of total flavonoids by different types of macroporous adsorption resins

[0088]

[0089] According to the results in Table 7, the adsorption and desorption effects of the four types of macroporous adsorption resins on the total flavonoids of Scutellaria baicalensis are ranked from large to small as follows:

[0090] Adsorption capacity: HPD-500>AB-8>HP-20>D101

[0091] Desorption capacity: HPD-500>AB-8>D101>HP-20

[0092] Among the four resins, HPD-500 has the strongest static adsorption capacity for total flavonoids from Scutellaria baicalensis, with an adsorption capacity of 78.64 mg / g and an adsorption rate of 67.33%, which is better than the other three resins in adsorption of total flavonoids from Scutellaria baicalensis. The static desorption rate reaches 80% under 70% ethanol elution, and the desorption effect is also the best. Therefore, HPD-500 is selected as the resin for purification of total flavonoids from Scutellaria baicalensis.

[0093] 2. Purification by macroporous adsorption resin

[0094] Collect the total flavonoids extract of the herb, concentrate it into an extract, weigh 1000g of the pretreated HPD-500 macroporous adsorption resin, and wet-load it into the column. Measure 500ml of the total flavonoids extract of the herb and slowly load it. After adsorption for 24h, discard the effluent, use 10L of 70% ethanol to elute the macroporous adsorption resin, and concentrate the eluate by rotary evaporation to a relative density of 1.6 to obtain the purified total flavonoids of the herb.

[0095] 3. Silica gel column chromatography separation

[0096] Weigh 200g of purified total flavonoids, take twice the amount of silica gel powder (200-300 mesh) in an evaporating dish, place in a water bath and stir thoroughly, and put into an oven for use after the sample is completely absorbed by the silica gel. Measure 2000g of silica gel powder in a 5L beaker, add dichloromethane, stir evenly, wet-load the column, and dry-load the sample. The column volume is about 4L. Use 15L of methanol-dichloromethane in 11 different ratios (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0) to elute in sequence. The order of eluents is shown in Table 8. Each ratio takes 1L as a sample, and is concentrated under reduced pressure for use. A total of 165 samples are obtained.

[0097] Table 8 Total flavonoids elution order

[0098]

[0099]

[0100] 165 samples were tested by HPLC method, samples with similar peak results in the same ratio were merged, and samples with high content, complete peaks and good separation were selected for semi-preparative separation. Finally, 3-4 to 3-6, 4-2 to 4-4, 5-3 to 5-4, 6-3 to 6-5 were merged and marked as 1, 2, 3, and 4 respectively. The target compound takakin-8-O-β-D-glucopyranoside was present in the 3rd mark, and the next step of separation was carried out.

[0101] The HPLC sample detection conditions are as follows: the mobile phase is methanol-0.4% phosphoric acid water (55%-45%), the total flow rate is 1 ml / min, the column temperature is 40°C, the detection wavelength is 328 nm, and the injection volume is 10 μl.

[0102] 4. Semi-preparative liquid phase separation

[0103] The combined No. 3 sample was concentrated under reduced pressure and dried, then weighed, diluted to 0.01 g / ml by adding methanol, and then ultrasonicated for 30 min for use.

[0104] Semi-preparative separation method development: Take 0.5 ml of sample solution, select flow rate 6 ml / min, detection wavelength 328 nm, and investigate the separation effects of two different mobile phase systems acetonitrile-water and methanol-water. It can be seen from the semi-preparative liquid phase chromatogram that with acetonitrile-water as the mobile phase system, the peak shapes in the sample are more complete, the separation degree is high, and it is conducive to collection. Gradient elution is used to adjust the mobile phase ratio in different time periods to improve the separation degree of each peak. The gradient elution method is shown in Table 9.

[0105] Table 9 Semi-preparative separation of total flavonoids from Lantana odorata by gradient elution

[0106]

[0107]

[0108] A total of four peaks were separated from sample No. 3 and collected separately. The four samples were detected by high performance liquid chromatography, and the purity of the compounds in the samples was determined by area normalization method. The compounds were structurally analyzed by NMR, MS and IR. The data were compared with literature reports, and one of the samples was determined to be takakin-8-O-β-D-glucopyranoside with a sample purity of 96.77%.

[0109] Example 3

[0110] 1) 5 kg of radix scutellariae was crushed and then added with 80% methanol, and refluxed under heating at 80°C, with a solid-liquid ratio of 1:40 and a time of 1 hour. The extract was collected and concentrated into an extract with a relative density of 1.1;

[0111] 2) The extract was loaded onto a HPD-500 macroporous adsorption resin column with a loading amount of 1 column volume. After adsorption equilibrium, 5 column volumes were eluted with 70% ethanol at a flow rate of 2 column volumes / h. The eluates were combined and concentrated to a relative density of 1.6 to obtain a purified total flavonoid product.

[0112] 3) The purified total flavonoids were mixed with twice the weight of silica gel powder and then dry-loaded for silica gel column chromatography. The mixture was eluted with methanol-dichloromethane in different volume ratios. The amount of eluent in each ratio was 4 times the volume of the silica gel column. Dichloromethane was used first for elution, followed by 1:9, 2:8, 3:7, and 4:6 methanol-dichloromethane elutions. After the elution was completed, the elution component containing the target substance in the 4:6 eluent was collected;

[0113] 4) The eluted fraction containing the target product was concentrated and dried, and then diluted with methanol to 0.01 g / ml. After ultrasonication for 30 min, the fraction was subjected to chromatographic separation on a semi-preparative HPLC, wherein the stationary phase of the semi-preparative HPLC was C18, the mobile phase was acetonitrile-water, and the gradient elution was performed. The eluate containing the target product was detected at a wavelength of 328 nm and collected. After freeze drying, 8.76 mg of pure takakin-8-O-β-D-glucopyranoside was obtained with a purity of 98.13%.

[0114]

[0115] Example 4

[0116] 1) 5 kg of radix scutellariae was crushed and added with 70% methanol, and then heated at 90°C for reflux extraction, with a solid-liquid ratio of 1:30 and a time of 2 hours. The extract was collected and concentrated into an extract with a relative density of 1.1;

[0117] 2) The extract was loaded onto a HPD-500 macroporous adsorption resin column at a loading rate of 2 column volumes. After adsorption equilibrium, 6 column volumes of 50% ethanol were used for elution at a flow rate of 3 column volumes / h. The eluates were combined and concentrated to a relative density of 1.6 to obtain a purified total flavonoid product.

[0118] 3) The purified total flavonoids were mixed with twice the weight of silica gel powder and then dry-loaded for silica gel column chromatography. The elution was performed with methanol-dichloromethane in different volume ratios. The amount of eluent in each ratio was 3 times the volume of the silica gel column. Dichloromethane was used first for elution, followed by 1:9, 2:8, 3:7, and 4:6 methanol-dichloromethane elutions. After the elution was completed, the elution component containing the target substance in the 4:6 eluent was collected;

[0119] 4) The eluted fraction containing the target product was concentrated and dried, and then diluted with methanol to 0.02 g / ml. After ultrasonication for 30 min, the fraction was subjected to chromatographic separation on a semi-preparative HPLC, wherein the stationary phase of the semi-preparative HPLC was C18, the mobile phase was acetonitrile-water, and the elution was gradient eluted. The eluate containing the target product was detected at a wavelength of 328 nm and collected. After freeze drying, 7.89 mg of pure takakin-8-O-β-D-glucopyranoside was obtained with a purity of 97.69%.

[0120]

[0121] Example 5 Structural analysis of the compound

[0122] This product is a yellow powder; melting point: 234℃-236℃; magnesium hydrochloride powder shows a positive reaction (red), indicating that it may be flavonoids, flavonols, dihydroflavonoids and dihydroflavonols; AlCl3 ethanol solution sprayed on a thin layer shows green fluorescent spots under ultraviolet light at 254nm, indicating that it is a flavonoid component.

[0123] 3400cm in infrared absorption spectrum -1 、1650cm -1 The absorption at 40° indicated that the product contained hydroxyl and carbonyl groups. m / z(%): 463[M+1]+(10), 321(100), 284(25), 265(35), 136, 115(30), 93(75). The molecular weight was determined to be 462 by mass spectrometry. 13C-NMR (DMSO-d6, 150M) also showed the carbon spectrum signal characteristics of flavonoids. The HMBC spectrum showed that the proton signal of the sugar terminal δ4.67 (1H, d, J = 8.7 Hz, H-1") was correlated with the 8-position carbon atom δ125.3 on the A ring, and the proton signal of the methoxy group δ3.84 (3H, s, -OMe) was connected to the carbon atom at the 4' position on the C ring. Combined with the carbon spectrum, the molecular formula C 22 H 22 O 11 The hydrogen spectrum shows obvious hydrogen spectrum signal characteristics of flavonoids: 1 H-NMR (DMSO-d6, 300MHz) δ: 12.75 (1H, s, 5-OH), 8.25 (2H, d, J = 8.7Hz, 2', 6'-H), 7.10 (2H, d, J = 8.7Hz, 3', 5'-H), 6.91 (1H, s, 3-H), 6.28 (1H, s, 6-H), 4.67 (1H, d, J = 7.8Hz, 1'-H), indicating that the compound is a glycoside compound, 3.84 (3H, s, OMe). The above data combined with the literature confirmed that this product is takakin-8-O-β-D-glucopyranoside, and its structural formula is:

[0124]

Claims

1. A method for extracting and separating takakin-8-O-β-D-glucopyranoside from safflower, characterized in that The following steps are involved: 1) crushing the fragrant blue orchid, adding 80% methanol, heating at 80°C for reflux extraction, wherein the solid-liquid ratio of the reflux extraction is 1:40, the extraction time is 1h, and the extract is collected and concentrated into an extract; 2) applying the extract to a HPD-500 macroporous adsorption resin column with a loading amount of 1 column volume, eluting with 70% ethanol for 5 column volumes after adsorption equilibrium, with an eluent flow rate of 2 column volumes / h, and concentrating the eluate to obtain a purified total flavonoid; 3) The purified total flavonoids were mixed with silica gel powder and then dry-loaded for silica gel column chromatography. The mixture was eluted with methanol-dichloromethane in different volume ratios. The amount of eluent in each ratio was 4 times the volume of the silica gel column. Dichloromethane was used first for elution, followed by 1:9, 2:8, 3:7, and 4:6 methanol-dichloromethane elutions, and the eluted fractions containing the target compound were collected. 4) The eluted fraction containing the target product was concentrated and dried, and then diluted with methanol to 0.01 g / ml, and then chromatographically separated on a semi-preparative high performance liquid chromatograph, wherein the stationary phase of the semi-preparative high performance liquid chromatograph was C18, the mobile phase was acetonitrile-water, and gradient elution was performed. The eluate containing the target product was detected at a wavelength of 328 nm and collected. After drying, the pure product of takakin-8-O-β-D-glucopyranoside was obtained. The gradient elution procedure was as follows: 。 2. The extraction and separation method according to claim 1, wherein: The sample loading volume for the chromatographic separation was 0.5 ml, and the flow rate was 6 ml / min.

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