A method for extracting and purifying apigenin 7-O-glucoside from peony leaves

Abstract

The present invention belongs to the technical field of methods for providing apigenin 7-O-glucoside, and specifically relates to a method for extracting and purifying apigenin 7-O-glucoside from peony leaves. A method for extracting and purifying apigenin 7-O-glucoside from peony leaves, comprising the following steps: S1. ethanol extraction; S2. extraction; S3. macroporous resin separation; S4. gel column separation; S5. HPLC preparation and purification. The present invention selects an ethanol concentration with higher extraction efficiency for extraction, and the extract is separated by LX-38 macroporous resin and Sephadex LH-20 gel column respectively. The separation process is mainly based on thin layer chromatography identification, and the standard and the sample are developed under the same conditions, and the components with the same ratio shift value are combined, impurities are removed, and the target compound is retained. Finally, apigenin 7-O-glucoside with higher purity is obtained by semi-preparative high performance liquid chromatography.

Description

Technical Field

[0001] The invention belongs to the technical field of apigenin 7-O-glucoside extraction methods, and specifically relates to a method for extracting and purifying apigenin 7-O-glucoside from originally discarded peony leaves and application thereof. Background Art

[0002] Apigenin 7-O-glucoside, also known as cosmos glycoside, is mainly found in many plants of oleander, Asteraceae, Ericaceae, Caprifoliaceae, Leguminosae and Rosaceae. Its CAS: 578-74-5, molecular formula: C 21 H 20 O 10 , the structural formula is as follows:

[0003]

[0004] Apigenin 7-O-glucoside has good antioxidant, antibacterial, antiviral and anti-dermatitis effects, and can also be used as a raw material for synthesizing other active derivatives. The prior art discloses a method for extracting apigenin 7-O-glucoside.

[0005] Application No. CN200910233834.6 discloses a process for extracting cosmos glycosides from chrysanthemum, which uses chrysanthemum as raw material, and undergoes crushing, soaking, macroporous resin treatment, membrane concentration, acid adjustment, polyamide treatment, and freezing crystallization to obtain the finished product. The extraction process disclosed in the patent uses separation methods such as macroporous resin, membrane and polyamide, and the purity of the extracted cosmos glycosides is 92.1-96.7%.

[0006] Application No. CN201110143974.1 discloses a method for extracting cosmos glycoside from Agrimoniae Herba, the process steps are: removing the aboveground part of Agrimoniae Herba, crushing, adding to a CO2 supercritical extractor, obtaining an extract, adding to a polyamide chromatography column, eluting with a 2:1 acetone-methanol mixed solvent as an eluent, recovering the solvent, adding ethanol for crystallization, separating the crystals, washing, and drying to obtain the cosmos glycoside. According to PHLC detection, the purity of the extracted cosmos glycoside is 93.8-96.3%.

[0007] Although the process disclosed in the above patent can realize the extraction of apigenin 7-O-glucoside, the maximum purity of the extraction is about 96%. The inventors have improved the existing extraction process, and the purity of apigenin 7-O-glucoside extracted from discarded peony leaves can reach more than 98%, which can further improve the extraction purity of apigenin 7-O-glucoside. At present, there is no relevant report on the extraction and purification of apigenin 7-O-glucoside from peony leaves.

[0008] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0009] The object of the present invention is to provide a method for extracting and purifying apigenin 7-O-glucoside from peony leaves, which can further improve the extraction purity of apigenin 7-O-glucoside, and the extraction process is simple and efficient.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] A method for extracting and purifying apigenin 7-O-glucoside from peony leaves comprises the following steps:

[0012] S1. Ethanol extraction: weigh the raw material, extract it with 60% by volume ethanol under reflux, and concentrate the extract to obtain a crude extract;

[0013] S2. Extraction: dispersing the crude extract with water, defatting, and extracting to obtain an extract;

[0014] S3. Macroporous resin separation: dissolve the extract with 20% ethanol by volume, apply the macroporous resin, and obtain the adsorbate;

[0015] S4. Gel column separation: dissolve the adsorbate with 80% ethanol by volume, load the gel column, and elute to obtain an eluate;

[0016] S5.HPLC preparation and purification: The eluate was dissolved in methanol, and 35% by volume acetonitrile was used as the mobile phase, and the extracted and purified apigenin 7-O-glucoside was separated by semi-preparative HPLC.

[0017] Preferably, in step S1, the raw material is peony leaves, and the solid-liquid ratio during reflux extraction is 1:15.

[0018] Preferably, in step S2, n-hexane is used for degreasing 3 times, and ethyl acetate is used for extraction 3 times.

[0019] Preferably, in step S3, the macroporous resin model is LX-38.

[0020] Preferably, in step S4, the gel column model is Sephadex LH-20, the flow rate is 0.5 mL / min, and the eluent is ethanol with a volume fraction of 80%.

[0021] Preferably, in step S5, the flow rate of semi-preparative HPLC is 8 mL / min, and the detection wavelength is 206 nm.

[0022] The present invention also provides application of the method for extracting and purifying apigenin 7-O-glucoside from peony leaves in preparing apigenin 7-O-glucoside.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention selects an ethanol concentration with a higher extraction efficiency for extraction, and the extract is separated by LX-38 macroporous resin and Sephadex LH-20 gel column respectively. The separation process is mainly based on thin layer chromatography identification. The standard and the sample are developed under the same conditions, the components with the same ratio shift value are combined, the impurities are removed, the target compound is retained, and finally the apigenin 7-O-glucoside with high purity is obtained by semi-preparative high performance liquid chromatography. The product obtained by HPLC is identified by NMR structure and HPLC analysis purity. The purity of the apigenin 7-O-glucoside extracted and purified by the present invention is above 98%, which can further improve the extraction purity of apigenin 7-O-glucoside compared with the prior art, and the extraction process is simple and efficient.

[0025] (2) The extraction process of the present invention highlights that the extracted and purified apigenin 7-O-glucoside can meet the characteristics and requirements of a practical API in the pharmaceutical field. In the entire extraction and purification process, only solvents or reagents permitted for use in the pharmaceutical industry are used to ensure that the API meets the standard of a pharmaceutical API free of chemical residues.

[0026] (3) The present invention selects peony leaves as the source of raw materials, which is a very rich resource in my country. More than 10,000 tons of peony leaves are discarded every year. The present invention has the great significance of green environmental protection, resource conservation and turning waste into treasure.

[0027] (4) The purity of apigenin 7-O-glucoside prepared by the extraction and purification process of the present invention is significantly higher than that of the currently disclosed invention patent technology, and the extraction and purification process and its industrial production cost are significantly lower than that of the currently disclosed invention patent technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the extraction process flow chart of the present invention;

[0029] Figure 2 This is the thin layer chromatogram of the macroporous resin eluate 4BV-21BV sample;

[0030] Figure 3 This is the thin layer chromatography of the samples in tubes 20-62 of the gel column;

[0031] Figure 4 The purity of the compound obtained by gel analysis was HPLC;

[0032] Figure 5Preparation of monomeric apigenin 7-O-glucoside for HPLC;

[0033] Figure 6 Sample preparation for HPLC 1 H NMR spectrum (800 MHz, C5D5N);

[0034] Figure 7 The purity of the obtained compound was analyzed by HPLC.

[0035] Description of reference numerals:

[0036] Figure 4-7 Where a is the standard and b is the sample. DETAILED DESCRIPTION

[0037] The following is a clear and complete description of the technical solution of the patent of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1 :

[0039] 1. Experimental Materials

[0040] 1.1 Reagents and instruments

[0041] See Table 1.

[0042] Table 1 Reagents and instruments

[0043]

[0044]

[0045] 1.2 Experimental plants

[0046] Peony leaves were collected from the Chinese-funded Peony Garden in Heyang, Weinan City, Shaanxi Province.

[0047] 2. Experimental Procedure

[0048] 2.1 Ethanol extraction

[0049] The preliminary experimental results showed that when 20%, 40%, 60% and 80% ethanol were used to extract apigenin 7-O-glucoside from peony leaves, the extraction efficiency of 60% ethanol was the highest under the same other conditions (40% ethanol extracted more impurities), so 60% ethanol was selected for extraction.

[0050] Weigh 200 g of peony leaves, extract with 60% ethanol (solid-liquid ratio 1:15) under reflux for 1 h, extract twice, concentrate the extract, and obtain 52.49 g of crude extract.

[0051] 2.2 Extraction

[0052] The crude extract was dispersed with water, defatted with n-hexane, and extracted with ethyl acetate three times to obtain 15.42 g of ethyl acetate extract, wherein the volume ratio of n-hexane and ethyl acetate to the crude extract dispersion was 1:2.

[0053] 2.3 Macroporous resin separation

[0054] Select LX-38 macroporous resin (good for adsorption and analysis of flavonoid glycosides), dissolve the obtained ethyl acetate extract in 20% ethanol by volume, and load the macroporous resin. 20% ethanol elutes 4BV, 30% ethanol elutes 19BV, and according to thin layer chromatography (TLC) identification, combine the 4BV of macroporous resin 20% eluate and the 1BV-17BV of 30% ethanol eluate, totaling 3.80g. Among them, the thin layer chromatogram of the 4BV-21BV sample of the macroporous resin eluate is shown in Figure 2 .

[0055] 2.4 Gel column separation

[0056] Dissolve with 80% ethanol by volume, load on Sephadex LH-20 gel column (4 cm × 150 cm), flow rate 0.5 mL / min, eluent 80% ethanol, 10 mL per tube, according to TLC detection, combine the 37th to 49th tubes where the target component appears (see Figure 3 ), spin-dried to obtain 60 mg.

[0057] The purity of the obtained compound was analyzed by HPLC, specifically: 4.4 mg of the standard and 5.9 mg of the sample were weighed and dissolved in a 25 mL volumetric flask, the injection volume was 10 μL, the flow rate was 0.8 mL / min, the column temperature was 25°C, and 0.1% acetonitrile (A)-formic acid water (B) was used as the mobile phase for gradient elution. The elution gradient was 0-10 min, 97-95% B; 10-20 min, 95-88% B; 20-30 min, 88-80% B; 30-60 min, 80-79% B (the purity analysis method is the same below). The final sample purity was ≥90% (see Figure 4 ).

[0058] 2.5 HPLC preparation and purification

[0059] The obtained sample was dissolved in methanol and separated by semi-preparative HPLC (flow rate 8 mL / min, detection wavelength 206 nm) with 35% acetonitrile as the mobile phase to obtain compound 1 (38.6 mg, t R =12.1min, see Figure 5 ). It was determined to be apigenin 7-O-glucoside by NMR detection (see Figure 6 ), HPLC analysis showed a purity of ≥98% (see Figure 7 ).

[0060] Comparative Example 1 : The extraction process in patent 200910233834.6 is adopted, and the tribute chrysanthemum is replaced by peony leaves. Other operations are the same.

[0061] Comparative Example 2 : The extraction process in patent 201110143974.1 was used, and the herb was replaced with peony leaves. Other operations were the same.

[0062] Table 2 Determination of the purity and extraction rate of apigenin 7-O-glucoside extracted by the extraction method of the present invention

[0063]

[0064] As shown in Table 2, the purity, extraction rate and extraction cost of apigenin 7-O-glucoside extracted and purified by the extraction method of the present invention, i.e., Example 1, are better than those of Control Example 1 and Control Example 2.

[0065] In summary, the purity of apigenin 7-O-glucoside extracted and purified from peony leaves in the present invention is above 98%, which can further improve the extraction purity of apigenin 7-O-glucoside compared with the prior art, and the extraction process is simple and efficient.

[0066] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is obvious that many changes and variations can be made in accordance with the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes.

Claims

1. A method for extracting and purifying apigenin 7-O-glucoside from peony leaves, characterized in that: The following steps are involved: S1. Ethanol extraction: weigh the raw material, extract it with 60% ethanol by volume, and concentrate the extract to obtain a crude extract; S2. Extraction: dispersing the extract with water, defatting, extracting, and obtaining an extract; S3. Macroporous resin separation: The extract was dissolved in 20% ethanol by volume, and loaded onto a macroporous resin to obtain a crude extract containing the target compound to obtain an adsorbent; S4. Gel column separation: dissolving the adsorbate with 80% ethanol by volume, loading the gel column, and eluting to obtain apigenin 7-O-glucoside with a purity greater than 90% to obtain an eluate; S5. HPLC preparation and purification: The eluate was dissolved in methanol, and 35% by volume acetonitrile was used as the mobile phase, and apigenin 7-O-glucoside with a purity greater than 98% was separated by semi-preparative HPLC; Wherein, in step S1, the raw material is peony leaves, and the solid-liquid ratio during reflux extraction is 1:15; In step S2, n-hexane is used for degreasing 3 times, and ethyl acetate is used for extraction 3 times; In step S3, the macroporous resin model is LX-38; In step S4, the gel column model is Sephadex LH-20, the flow rate is 0.5 mL / min, and the eluent is ethanol with a volume fraction of 80%; In step S5, the flow rate of semi-preparative HPLC is 8 mL / min, and the detection wavelength is 206 nm.

2. Use of the method for extracting and purifying apigenin 7-O-glucoside from peony leaves according to claim 1 in the preparation of apigenin 7-O-glucoside.

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