A method for preparing high-purity vitexin from the above-ground parts of Stenoloma chusanum

Abstract

The present invention belongs to the technical field of extraction and purification of traditional Chinese medicine, and specifically relates to a method for preparing high-purity vitexin from the aerial parts of Pteris viticola. The method comprises the preparation of Pteris viticola medicinal material powder, extraction, macroporous resin impurity removal, and preparative liquid phase purification. The present invention adopts a strong acid-ethanol aqueous solution as an extraction solution, fully utilizing the difference in stability of flavonoid carbon glycosides and flavonoid oxygen glycosides to strong acid, hydrolyzing derivatives such as vitexin glucoside and vitexin rhamnoside with a strong acid to generate vitexin. While increasing the content of vitexin, substances with similar polarity can also be hydrolyzed, reducing the difficulty of subsequent preparative liquid phase purification. The obtained vitexin has a high yield, high purity, simple steps, and low cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine extraction and refinement, and particularly relates to a method for preparing high-purity vitexin from the above-ground part of Stenoloma chusanum (L.) Ching. Background Art

[0002] Stenoloma chusanum (L.) Ching Odontosoria chinensis is a perennial herb of the genus Stenoloma in the family Lindsaeaceae, mainly distributed near the Yangtze River Basin. As an ornamental plant, Stenoloma chusanum (L.) Ching has high ornamental value. Stenoloma chusanum (L.) Ching tastes slightly bitter and is cold in nature, and belongs to the liver and large intestine meridians, with the effects of clearing heat, detoxifying, promoting diuresis, and stopping bleeding. Its main active ingredients are mainly flavonoids and phenolic acid compounds. Stenoloma chusanum (L.) Ching is mostly used to treat diseases such as hepatitis, infantile bronchitis, pneumonia, tonsillitis, and gastroenteritis. The chemical components in Stenoloma chusanum (L.) Ching are relatively complex. Its main active ingredients are flavonoids and polyphenolic compounds. In addition, there are a large number of water-soluble pigments and fat-soluble oily components, making the extraction and purification process of compounds in Stenoloma chusanum (L.) Ching relatively difficult.

[0003] As one of the main active ingredients in Stenoloma chusanum (L.) Ching, vitexin, also known as vitexin, is a natural plant flavonoid C-glycoside compound. Its chemical name is 8-β-D-glucopyranosyl-4',7-dihydroxyisoflavone, and its molecular formula is C 21 H 20 O 10 , with a relative molecular mass of 432.4, and its molecular structure is shown in the following formula. Stenoloma chusanum (L.) Ching is rich in vitexin and its derivatives, and its total content is as high as 9 mg / g. Modern pharmacological studies have shown that vitexin has various pharmacological effects such as anti-myocardial infarction, anti-inflammatory and analgesic, blood pressure lowering, antibacterial, and antioxidant. There is already a finished drug, injectable vitexin, used in clinical medicine. The main function of vitexin is to activate blood circulation and remove stasis, regulate qi and dredge meridians, and is used for chest obstruction caused by blood stasis blocking the meridians, with symptoms such as chest tightness and shortness of breath, stabbing pain in the precordial area, palpitations and forgetfulness, dizziness and tinnitus, and patients with symptoms such as coronary heart disease angina, hyperlipidemia, and insufficient blood supply to the heart artery.

[0004]

[0005] Stenoloma chusanum (L.) Ching has rich wild resources and low raw material costs. The existing methods for preparing vitexin from Stenoloma chusanum (L.) Ching mainly include extraction, concentration, organic solvent extraction, re-concentration, recrystallization, alkali-acid treatment, reverse silica gel elution, polyamide inspection, and repeating the above steps. This method has cumbersome steps, low extraction rate of the prepared vitexin, requires treatment with a variety of organic solvents, has low purity, is difficult to purify in the follow-up, and the process is relatively complex, with high costs and is not environmentally friendly. In view of this, it is necessary to develop a simpler, higher extraction rate and purity, environmentally friendly, and industrially applicable method for preparing vitexin, providing technical support for the development and utilization of vitexin in Stenoloma chusanum (L.) Ching and providing a direction for the industrial economy of Stenoloma chusanum (L.) Ching. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a method for preparing high-purity vitexin from the aerial parts of Stenoloma chusanum. This method uses a strong acid extraction system. By utilizing the difference in the stability of flavone C-glycosides and flavone O-glycosides to strong acids, derivatives such as vitexin glucoside and vitexin rhamnoside are hydrolyzed by strong acids to form vitexin, so as to increase the content of vitexin. At the same time, strong acids can degrade pigments and other impurities in Stenoloma chusanum, reduce the difficulty in preparation, simplify the process, and obtain high yield and purity of vitexin.

[0007] The present invention provides a method for preparing high-purity vitexin from the aerial parts of Stenoloma chusanum, comprising the following steps:

[0008] (1) Preparation of Stenoloma chusanum medicinal material powder: Cut and dry the aerial parts of Stenoloma chusanum, pulverize the dried medicinal materials, and sieve through a No. 3 sieve for standby;

[0009] (2) Extraction: Weigh a certain weight of Stenoloma chusanum medicinal material powder and put it into a round-bottom flask, add a certain volume of a mixed solution containing 2 mol / L - 4 mol / L acid and 30% - 80% ethanol, place it in a magnetic stirring water bath reflux constant temperature heating system for extraction. After complete extraction, add an alkaline substance to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to an equal volume, and centrifuge or filter to obtain the Stenoloma chusanum extract; In this step, a high-concentration acid-ethanol solution is used as the extraction system, making full use of the advantage that strong acids can remove most impurities and convert some active ingredients; and a magnetic stirring water bath reflux constant temperature heating system is used to increase the extraction rate and hydrolysis rate, which can not only simplify the extraction process and shorten the reaction time, but also remove most impurities for subsequent purification and further simplify the subsequent process;

[0010] (3) Removal of impurities by macroporous resin: Pack a column with swollen D101 macroporous resin. After loading the Stenoloma chusanum extract, first wash it with water until the effluent is colorless, then elute it with 30% ethanol, collect the 30% ethanol eluate and concentrate it to obtain the crude extract; In this step, 30% ethanol is used as the eluent, which can further remove most impurities and improve the purity of the crude extract;

[0011] (4) Purification: Dissolve the crude extract with 40% methanol, purify it by preparative liquid phase, collect the eluate, and concentrate and dry it to obtain the final product.

[0012] The present invention uses a strong acid-ethanol aqueous solution as the extraction solution, making full use of the difference in the stability of flavone C-glycosides and flavone O-glycosides to strong acids. Derivatives such as vitexin glucoside and vitexin rhamnoside are hydrolyzed by strong acids to form vitexin, increasing the content of vitexin. At the same time, strong acids can hydrolyze substances with similar polarities and some pigments, improving the extraction efficiency and reducing the difficulty of subsequent preparative liquid phase purification. The obtained vitexin has a high yield and high purity, greatly simplifying the process and cost of extracting vitexin from Stenoloma chusanum, and is green and environmentally friendly.

[0013] Further, in step (2) of the above technical solution, the mass-to-volume ratio of the Stenoloma chusanum medicinal material powder to the acid-ethanol aqueous solution is 1:15 - 50; preferably 1:25.

[0014] Further, in step (2) of the above technical solution, the acid is hydrochloric acid with a concentration of 3 mol / L, and the concentration of ethanol is 60%.

[0015] Further, in step (2) of the above technical solution, in the magnetic stirring water bath reflux constant temperature extraction process, the extraction temperature is 70°C - 90°C, preferably 80°C, the extraction time is 1 h - 3 h, preferably 1 h; the magnetic stirring speed is 100 r / min - 200 r / min, preferably 150 r / min.

[0016] Further, in step (3) of the above technical solution, the ratio of the sample loading volume to the column packing volume is 1:1, and the swelling method of the D101 macroporous resin is: soaking the D101 macroporous resin in absolute ethanol overnight to obtain it.

[0017] Further, in step (3) of the above technical solution, the volume of water flushing and elution is 3 BV - 8 BV, preferably 6 BV; the volume of 30% ethanol elution is 10 BV - 16 BV, preferably 14 BV.

[0018] Further, in step (4) of the above technical solution, the parameters of preparative liquid chromatography purification are: using SinoChrom ODS-BP as the chromatographic column, 40% methanol as the elution solvent, the detection wavelength is 360 nm, and the flow rate is 4 mL / min.

[0019] Advantages over the prior art:

[0020] Existing conventional hydrolysis processes all first extract, concentrate, redissolve and then hydrolyze, which requires a large amount of time and solvents. By optimizing the extraction process, impurity removal process and purification process through the single factor combined with the response surface method, the present invention uses a strong acid-ethanol aqueous solution as the extraction solution, and at the same time uses magnetic stirring to increase the extraction rate and promote the hydrolysis reaction, significantly shortening the extraction and hydrolysis time. It is more time-saving, labor-saving and solvent-saving. The present invention makes full use of the stability difference of flavone C-glycoside and flavone O-glycoside to strong acid, and completely hydrolyzes derivatives such as vitexin glucoside and vitexin rhamnoside with strong acid to generate vitexin, doubling the content of vitexin in Stenoloma chusanum. At the same time, impurities with similar polarity can also be hydrolyzed to reduce the difficulty of subsequent preparative liquid chromatography purification; by using macroporous resin to remove impurities, a large amount of salts and pigments are removed, and at the same time, using Elite preparative liquid chromatography purification, the obtained vitexin has a high yield, high purity, simple steps and low cost, and is suitable for industrial production. Description of the Drawings

[0021] Figure 1Chromatogram of the Stenoloma chusanum extract obtained in Example 1 of the present invention, where 1 is vitexin;

[0022] Figure 2 Chromatogram of the crude extract obtained in Example 1 of the present invention, where 1 is vitexin;

[0023] Figure 3 Chromatogram of the purified product obtained in Example 1 of the present invention, where 1 is vitexin;

[0024] Figure 4 Chromatogram of the Stenoloma chusanum extract obtained after extraction without adding acid in Comparative Example 1 of the present invention, where 1 is vitexin, 2 is vitexin glucoside, and 3 is vitexin rhamnoside. Detailed implementation mode

[0025] Any of the above technical features of the present invention can be combined with the technical features specifically described below (such as in the examples) to form new or preferred technical solutions. However, the present invention is not limited to these examples, and these examples do not limit the present invention in any way.

[0026] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the preparations involved in the following examples are all ordinary commercially available products and can be obtained through market purchase.

[0027] Preparative liquid chromatography purification uses an Eclassical 3100 preparative HPLC instrument; high-performance liquid chromatography content detection uses a Waters E2695 high-performance liquid chromatography instrument, and the detection conditions are as follows:

[0028] Welchrom C18 chromatographic column (4.6 mm×250.0 mm, 5 μm), the mobile phase is acetonitrile (A)-water (B), the elution conditions are (0 - 20 min, 16%A; 20 - 50 min, 16% - 50%A; 50.01 min - 55 min, 100%A; 55.01 min - 60 min, 16%A), the detection wavelength is 360 nm, the flow rate is 1.0 mL / min; the column temperature is 30°C; the injection volume is 10 μL.

[0029] The present invention will be further described in detail below with reference to the drawings and examples:

[0030] Example 1

[0031] A method for preparing high-purity vitexin from the above-ground part of Stenoloma chusanum, comprising the following steps:

[0032] (1) Preparation of Stenoloma chusanum medicinal material powder: Cut and dry the above-ground part of Stenoloma chusanum, and pulverize the dried medicinal material for standby;

[0033] (2) Extraction: Weigh 1 g of Pteris viridis powder and place it in a round-bottom flask. Add 3 mol / L hydrochloric acid-60% ethanol solution according to a solid-liquid ratio (g / mL) of 1:25, place it in a magnetic stirring water bath reflux constant temperature heating system, and stir at a speed of 150 r / min. Extract in a water bath at 80°C for 1 h. After the extraction is complete, cool it, add alkaline substances to adjust the pH to neutral, filter it, concentrate the filtrate until there is no alcohol taste, and dilute it with water to an equal volume. Filter it to obtain the Pteris viridis extract.

[0034] (3) Macroporous resin impurity removal: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Load 25 mL of the swollen D101 macroporous resin into the column. Load an equal volume of the fern extract. Rinse with 6 BV of water until the effluent is colorless. Then elute with 14 BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract.

[0035] (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP column (5μm, 10mm×250mm), detection wavelength of 360nm, flow rate of 4mL / min, temperature of 20℃, elution condition of 40% methanol, and the eluate was collected, concentrated, and dried to obtain the final product.

[0036] The content of vitexin in the extract of the black fern obtained in step (2) was 4.52 mg by high performance liquid chromatography, and its chromatogram is shown in FIG. Figure 1 As shown; the content of vitexin in the crude extract obtained by high performance liquid chromatography (HPLC) is 3.48 mg, and its chromatogram is as shown Figure 2 As shown; the content of vitexin in step (4) was also detected by high performance liquid chromatography and was 3.21 mg. The chromatogram is shown as Figure 3 As shown, its liquid phase purity is 98%.

[0037] Example 2

[0038] A method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, comprising the following steps:

[0039] (1) Preparation of Pteris chinensis powder: Cut and dry the above-ground part of Pteris chinensis, crush the dried medicinal material and set aside;

[0040] (2) Extraction: Weigh 1 g of Pteris chinensis powder and put it into a round-bottom flask. Add 4 mol / L hydrochloric acid-30% ethanol solution according to the material-liquid ratio (g / mL) of 1:25, put it into a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and reflux extraction is carried out in a water bath at 80°C for 1 hour. After the extraction is complete, cool it, add alkaline substances to adjust the pH to neutral, filter it, concentrate the filtrate until there is no alcohol taste, and dilute it with water to an equal volume. Filter it to obtain the Pteris chinensis extract;

[0041] (3) Impurity removal by macroporous resin: Soak the refined D101 macroporous resin in absolute ethanol overnight to fully swell it. Pack a 25 mL column with the swollen D101 macroporous resin. After loading an equal volume of Stenoloma chusanum extract, first rinse it with 3 BV of water until the effluent is colorless, and then elute it with 16 BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0042] (4) Purification: Dissolve the crude extract in 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm×250 mm), detection wavelength is 360 nm, flow rate is 4 mL / min, temperature is 20 °C, elution condition is 40% methanol. Collect the eluate, concentrate it, and dry it to obtain the final product.

[0043] The content of vitexin in the extract obtained in step (2) was detected by high performance liquid chromatography to be 4.49 mg; the content of vitexin in the crude extract obtained in step (3) was detected by high performance liquid chromatography to be 3.46 mg; the content of vitexin in step (4) was also detected by high performance liquid chromatography to be 3.19 mg, and its liquid phase purity was 95%.

[0044] Example 3

[0045] A method for preparing high-purity vitexin from the above-ground part of Stenoloma chusanum, comprising the following steps:

[0046] (1) Preparation of Stenoloma chusanum medicinal material powder: Cut and dry the above-ground part of Stenoloma chusanum, and crush the dried medicinal material for standby;

[0047] (2) Extraction: Weigh 1 g of Stenoloma chusanum medicinal material powder and put it into a round-bottom flask. Add 3 mol / L hydrochloric acid - 50% ethanol solution according to the solid-liquid ratio (g / mL) of 1:25. Put it into a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150 r / min, and extract it at 80 °C in a water bath for 1 h. After complete extraction, let it cool, adjust the pH to neutral with an alkaline substance, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to an equal volume, and then filter to obtain the Stenoloma chusanum extract;

[0048] (3) Impurity removal by macroporous resin: Soak the refined D101 macroporous resin in absolute ethanol overnight to fully swell it. Pack a 25 mL column with the swollen D101 macroporous resin. After loading an equal volume of Stenoloma chusanum extract, first rinse it with 8 BV of water until the effluent is colorless, and then elute it with 14 BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0049] (4) Purification: Dissolve the crude extract with 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5μm, 10mm×250mm), detection wavelength is 360nm, flow rate is 4mL / min, temperature is 20°C, elution condition is 40% methanol. Collect the eluate, concentrate and dry it to obtain the final product.

[0050] The content of vitexin in the extract obtained in step (2) was detected by high performance liquid chromatography to be 4.43mg; the content of vitexin in the crude extract obtained in step (3) was detected by high performance liquid chromatography to be 3.41mg; the content of vitexin in step (4) was also detected by high performance liquid chromatography to be 3.15mg, and its liquid phase purity was 98%.

[0051] Example 4

[0052] A method for preparing high-purity vitexin from the aerial parts of Stenoloma chusanum, comprising the following steps:

[0053] (1) Preparation of Stenoloma chusanum medicinal material powder: Cut and dry the aerial parts of Stenoloma chusanum, and pulverize the dried medicinal materials for standby.

[0054] (2) Extraction: Weigh 1g of Stenoloma chusanum medicinal material powder and put it into a round-bottom flask. Add 3mol / L hydrochloric acid-60% ethanol solution according to the material-liquid ratio (g / mL) of 1:15. Put it into a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150r / min, and extract at 80°C in a water bath for 1h. After complete extraction, let it cool, adjust to neutral pH with alkaline substances, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to an equal volume, then filter to obtain the Stenoloma chusanum extract.

[0055] (3) Impurity removal by macroporous resin: Soak the refined type D101 macroporous resin with anhydrous ethanol overnight to make it fully swollen. Pack 15mL of the swollen D101 macroporous resin into a column. After loading an equal volume of Stenoloma chusanum extract, first rinse with 6BV of water until the effluent is colorless, and then elute with 14BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract.

[0056] (4) Purification: Dissolve the crude extract with 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5μm, 10mm×250mm), detection wavelength is 360nm, flow rate is 4mL / min, temperature is 20°C, elution condition is 40% methanol. Collect the eluate, concentrate and dry it to obtain the final product.

[0057] The content of vitexin in the extract obtained in step (2) detected by high performance liquid chromatography was 4.19 mg; the content of vitexin in the crude extract obtained in step (3) detected by high performance liquid chromatography was 3.23 mg; similarly, the content of vitexin in step (4) detected by high performance liquid chromatography was 3.03 mg, and its liquid phase purity was 98%.

[0058] Comparative Example 1

[0059] A method for preparing vitexin from the aerial parts of Stenoloma chusanum (L.) Ching, comprising the following steps:

[0060] (1) Preparation of Stenoloma chusanum (L.) Ching medicinal material powder: Cut and dry the aerial parts of Stenoloma chusanum (L.) Ching, and pulverize the dried medicinal materials for standby;

[0061] (2) Extraction: Weigh 1 g of Stenoloma chusanum (L.) Ching medicinal material powder and put it into a round-bottomed flask. Add 60% ethanol solution according to the solid-liquid ratio (g / mL) of 1:25, place it in a magnetic stirring water bath reflux constant temperature heating system, with the magnetic stirring speed of 150 r / min, reflux extract at 80 °C for 1 h. After complete extraction, let it cool, adjust to neutral pH with alkaline substances, filter, concentrate the filtrate until there is no alcohol smell, and dilute with water to the same volume, then filter to obtain the Stenoloma chusanum (L.) Ching extract;

[0062] (3) Impurity removal by macroporous resin: Soak the refined D101 macroporous resin with absolute ethanol overnight to make it fully swollen. Pack 25 mL of the swollen D101 macroporous resin into a column. After loading an equal volume of Stenoloma chusanum (L.) Ching extract, first rinse with 6 BV of water until the effluent is colorless, then elute with 14 BV of 30% ethanol, collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0063] (4) Purification: Dissolve the crude extract with 40% methanol, and then purify it by preparative liquid phase. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength is 360 nm, flow rate is 4 mL / min, temperature is 20 °C, elution condition is 40% methanol. Collect the eluate, concentrate and dry it to obtain the final product.

[0064] The content of vitexin in the extract obtained in step (2) detected by high performance liquid chromatography was 1.52 mg, and its chromatogram is as Figure 4 shown; the content of vitexin in step (4) detected by high performance liquid chromatography was 1.06 mg, and its liquid phase purity was 45%.

[0065] Comparative Example 2

[0066] A method for preparing vitexin from the aerial parts of Stenoloma chusanum (L.) Ching, comprising the following steps:

[0067] (1) Preparation of the powder of Stenoloma chusanum (L.) Ching medicinal material: Cut and dry the above-ground part of Stenoloma chusanum (L.) Ching, and pulverize the dried medicinal material for standby;

[0068] (2) Extraction: Weigh 1 g of the powder of Stenoloma chusanum (L.) Ching medicinal material and put it into a round-bottom flask. Add 3 mol / L hydrochloric acid solution according to the material-liquid ratio (g / mL) of 1:25. Place it in a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150 r / min, and reflux extract at 80 °C in water bath for 1 h. After complete extraction, let it cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to the same volume, then filter to obtain the Stenoloma chusanum (L.) Ching extract;

[0069] (3) Impurity removal by macroporous resin: Soak the refined D101 macroporous resin in absolute ethanol overnight to fully swell it. Pack 25 mL of the swollen D101 macroporous resin into a column. After loading the same volume of Stenoloma chusanum (L.) Ching extract, first rinse it with 6 BV of water until the effluent is colorless, then elute it with 14 BV of 30% ethanol, collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0070] (4) Purification: Dissolve the crude extract with 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength is 360 nm, flow rate is 4 mL / min, temperature is 20 °C, elution condition is 40% methanol. Collect the eluate, concentrate and dry it to obtain the final product.

[0071] The content of vitexin in the extract obtained in step (2) was detected by high performance liquid chromatography to be 2.29 mg; the content of vitexin in step (4) was detected by high performance liquid chromatography to be 1.62 mg, and its liquid phase purity was 98%.

[0072] Comparative Example 3

[0073] A method for preparing vitexin from the above-ground part of Stenoloma chusanum (L.) Ching, comprising the following steps:

[0074] (1) Preparation of the powder of Stenoloma chusanum (L.) Ching medicinal material: Cut and dry the above-ground part of Stenoloma chusanum (L.) Ching, and pulverize the dried medicinal material for standby;

[0075] (2) Extraction: Weigh 1 g of the powder of Stenoloma chusanum (L.) Ching medicinal material and put it into a round-bottom flask. Add 1 mol / L hydrochloric acid - 60% ethanol solution according to the material-liquid ratio (g / mL) of 1:25. Place it in a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150 r / min, and reflux extract at 80 °C in water bath for 1 h. After complete extraction, let it cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to the same volume, then filter to obtain the Stenoloma chusanum (L.) Ching extract;

[0076] (3)Impurity removal by macroporous resin: Soak the refined D101 macroporous resin in absolute ethanol overnight to fully swell it. Pack 25 mL of the swollen D101 macroporous resin into a column. After loading an equal volume of Stenoloma chusanum extract, first rinse with 6 BV of water until the effluent is colorless, then elute with 14 BV of 30% ethanol, collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0077] (4)Purification: Dissolve the crude extract in 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength is 360 nm, flow rate is 4 mL / min, temperature is 20 °C, elution condition is 40% methanol. Collect the eluate, concentrate it, and dry it to obtain the final product.

[0078] The content of vitexin in the extract obtained in step (2) was detected by high performance liquid chromatography to be 2.68 mg; the content of vitexin in step (4) was detected by high performance liquid chromatography to be 1.90 mg, and its liquid phase purity was 70%.

[0079] Comparative Example 4

[0080] A method for preparing vitexin from the aerial part of Stenoloma chusanum, comprising the following steps:

[0081] (1)Preparation of Stenoloma chusanum medicinal material powder: Cut and dry the aerial part of Stenoloma chusanum, and pulverize the dried medicinal material for standby;

[0082] (2)Extraction: Weigh 1 g of Stenoloma chusanum medicinal material powder and put it into a round-bottom flask. Add 3 mol / L hydrochloric acid - 15% ethanol solution according to the material-liquid ratio (g / mL) of 1:25. Place it in a magnetic stirring water bath reflux constant temperature heating system, with the magnetic stirring speed of 150 r / min, and extract at 80 °C in a water bath for 1 h. After complete extraction, let it cool, adjust to neutral pH with an alkaline substance, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to an equal volume, then filter to obtain the Stenoloma chusanum extract;

[0083] (3)Impurity removal by macroporous resin: Soak the refined D101 macroporous resin in absolute ethanol overnight to fully swell it. Pack 25 mL of the swollen D101 macroporous resin into a column. After loading an equal volume of Stenoloma chusanum extract, first rinse with 6 BV of water until the effluent is colorless, then elute with 14 BV of 30% ethanol, collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0084] (4)Purification: Dissolve the crude extract in 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5μm, 10mm×250mm), detection wavelength is 360nm, flow rate is 4mL / min, temperature is 20°C, elution condition is 40% methanol. Collect the eluate, concentrate it, and dry it to obtain the final product.

[0085] The content of vitexin in the extract obtained in step (2) was detected by high performance liquid chromatography to be 3.38mg; the content of vitexin in step (4) was detected by high performance liquid chromatography to be 2.40mg, and its liquid phase purity was 97%.

[0086] Comparative Example 5

[0087] A method for preparing vitexin from the above-ground part of Stenoloma chusanum, comprising the following steps:

[0088] (1)Preparation of Stenoloma chusanum medicinal material powder: Cut and dry the above-ground part of Stenoloma chusanum, and pulverize the dried medicinal material for later use;

[0089] (2)Extraction: Weigh 1g of Stenoloma chusanum medicinal material powder and put it into a round-bottom flask. Add 3mol / L hydrochloric acid - 60% ethanol solution according to the solid-liquid ratio (g / mL) of 1:25. Place it in a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150r / min, and extract at 80°C in a water bath for 0.5h. After complete extraction, cool it, add an alkaline substance to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to an equal volume, then filter to obtain the Stenoloma chusanum extract;

[0090] (3)Removing impurities by macroporous resin: Soak the refined D101 macroporous resin in absolute ethanol overnight to make it fully swollen. Pack 25mL of the swollen D101 macroporous resin into a column. After loading an equal volume of Stenoloma chusanum extract, first rinse it with 6BV of water until the effluent is colorless, and then elute it with 14BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0091] (4)Purification: Dissolve the crude extract in 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5μm, 10mm×250mm), detection wavelength is 360nm, flow rate is 4mL / min, temperature is 20°C, elution condition is 40% methanol. Collect the eluate, concentrate it, and dry it to obtain the final product.

[0092] The content of vitexin in the extract obtained in step (2) was detected by high performance liquid chromatography to be 2.88mg; the content of vitexin in step (4) was detected by high performance liquid chromatography to be 2.08mg, and its liquid phase purity was 86%.

[0093] Comparative Example 6

[0094] A method for preparing vitexin from the above-ground parts of Stenoloma chusanum (L.) Ching, comprising the following steps:

[0095] (1) Preparation of the powder of Stenoloma chusanum (L.) Ching medicinal materials: Cut and dry the above-ground parts of Stenoloma chusanum (L.) Ching, and pulverize the dried medicinal materials for standby;

[0096] (2) Extraction: Weigh 1 g of the powder of Stenoloma chusanum (L.) Ching medicinal materials and put it into a round-bottom flask. Add 3 mol / L hydrochloric acid - 60% ethanol solution according to the solid-liquid ratio (g / mL) of 1:25. Place it in a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150 r / min, and reflux extraction is carried out in a 60 °C water bath for 1 h. After complete extraction, let it cool, add an alkaline substance to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to an equal volume, and then filter to obtain the Stenoloma chusanum (L.) Ching extract;

[0097] (3) Impurity removal by macroporous resin: Soak the refined type D101 macroporous resin with absolute ethanol overnight to make it fully swollen. Pack 25 mL of the swollen D101 macroporous resin into a column. After loading an equal volume of the Stenoloma chusanum (L.) Ching extract, first rinse with 6 BV of water until the effluent is colorless, and then elute with 14 BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0098] (4) Purification: Dissolve the crude extract with 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm × 250 mm), the detection wavelength is 360 nm, the flow rate is 4 mL / min, the temperature is 20 °C, the elution condition is 40% methanol. Collect the eluate, concentrate it, and dry it to obtain the final product.

[0099] The content of vitexin in the extract obtained in step (2) was detected by high performance liquid chromatography to be 1.89 mg; the content of vitexin in step (4) was detected by high performance liquid chromatography to be 1.34 mg, and its liquid phase purity was 56%.

[0100] Comparative Example 7

[0101] A method for preparing vitexin from the above-ground parts of Stenoloma chusanum (L.) Ching, comprising the following steps:

[0102] (1) Preparation of the powder of Stenoloma chusanum (L.) Ching medicinal materials: Cut and dry the above-ground parts of Stenoloma chusanum (L.) Ching, and pulverize the dried medicinal materials for standby;

[0103] (2) Extraction: Weigh 1 g of the powder of Stenoloma chusanum medicinal material and put it into a round-bottom flask. Add 3 mol / L hydrochloric acid - 60% ethanol solution according to the material-liquid ratio (g / mL) of 1:25. Place it in a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150 r / min, and reflux extraction is carried out in a water bath at 80 °C for 1 h. After complete extraction, let it cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to the same volume, then filter to obtain the Stenoloma chusanum extract;

[0104] (3) Impurity removal by macroporous resin: Soak the refined type D101 macroporous resin in absolute ethanol overnight to fully swell it. Pack 25 mL of the swollen D101 macroporous resin into a column. After loading an equal volume of the Stenoloma chusanum extract, first rinse it with 6 BV of water until the effluent is colorless, and then elute it with 8 BV of 50% ethanol. Collect the 50% ethanol eluate and concentrate it to obtain the crude extract;

[0105] (4) Purification: Dissolve the crude extract with 40% methanol, and then purify it by preparative liquid phase. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm × 250 mm), the detection wavelength is 360 nm, the flow rate is 4 mL / min, the temperature is 20 °C, the elution condition is 40% methanol. Collect the eluate, concentrate it, and dry it to obtain the final product.

[0106] The content of vitexin in the crude extract obtained in step (3) detected by high performance liquid chromatography is 3.74 mg; the content of vitexin in step (4) detected by high performance liquid chromatography is 3.36 mg, and its liquid phase purity is 82%.

[0107] Comparative Example 8

[0108] A method for preparing vitexin from the above-ground part of Stenoloma chusanum, comprising the following steps:

[0109] (1) Preparation of the powder of Stenoloma chusanum medicinal material: Cut and dry the above-ground part of Stenoloma chusanum, and crush the dried medicinal material for standby;

[0110] (2) Extraction: Weigh 1 g of the powder of Stenoloma chusanum medicinal material and put it into a round-bottom flask. Add 3 mol / L hydrochloric acid - 60% ethanol solution according to the material-liquid ratio (g / mL) of 1:25. Place it in a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150 r / min, and reflux extraction is carried out in a water bath at 80 °C for 1 h. After complete extraction, let it cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to the same volume, then filter to obtain the Stenoloma chusanum extract;

[0111] (3) Impurity removal by macroporous resin: Soak the refined D101 macroporous resin in absolute ethanol overnight to fully swell it. Pack a 16 mL column with the swollen D101 macroporous resin. After loading the Stenoloma chusanum extract with a volume 1.5 times that of the column, first rinse with 6 BV of water until the effluent is colorless, and then elute with 14 BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract.

[0112] (4) Purification: Dissolve the crude extract in 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength is 360 nm, flow rate is 4 mL / min, temperature is 20 °C, elution condition is 40% methanol. Collect the eluate, concentrate it, and dry it to obtain the final product.

[0113] It was found that vitexin leaked in step (3). The content of vitexin in the product of step (4) was detected by high performance liquid chromatography to be 2.89 mg, and its liquid phase purity was 97%.

[0114] Comparative Example 9

[0115] A method for preparing high-purity vitexin from the aerial part of Stenoloma chusanum, comprising the following steps:

[0116] (1) Preparation of Stenoloma chusanum medicinal material powder: Cut the aerial part of Stenoloma chusanum and dry it, then crush the dried medicinal material for standby.

[0117] (2) Extraction: Weigh 1 g of Stenoloma chusanum medicinal material powder and put it into a round-bottom flask. Add 3 mol / L hydrochloric acid - 60% ethanol solution according to the solid-liquid ratio (g / mL) of 1:2,5. Place it in a water bath at 80 °C and reflux for 1 h. After complete extraction, let it cool, adjust to neutral pH with an alkaline substance, filter, concentrate the filtrate until the alcohol smell disappears, and dilute it with water to the same volume, then filter to obtain the Stenoloma chusanum extract.

[0118] (3) Impurity removal by macroporous resin: Soak the refined D101 macroporous resin in absolute ethanol overnight to fully swell it. Pack a 25 mL column with the swollen D101 macroporous resin. After loading the same volume of Stenoloma chusanum extract, first rinse with 6 BV of water until the effluent is colorless, and then elute with 14 BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract.

[0119] (4) Purification: Dissolve the crude extract in 40% methanol, and then purify it by preparative liquid chromatography. The specific conditions are as follows: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength is 360 nm, flow rate is 4 mL / min, temperature is 20 °C, elution condition is 40% methanol. Collect the eluate, concentrate it, and dry it to obtain the final product.

[0120] The content of vitexin in the extract obtained in step (2) detected by high performance liquid chromatography was 3.19 mg; the content of vitexin in step (4) detected by high performance liquid chromatography was 2.24 mg, and its liquid phase purity was 82%.

[0121] Comparative Example 10

[0122] A method for preparing high-purity vitexin from the aerial part of Stenoloma chusanum (L.) Ching, comprising the following steps:

[0123] (1) Preparation of Stenoloma chusanum (L.) Ching medicinal material powder: Cut and dry the aerial part of Stenoloma chusanum (L.) Ching, and pulverize the dried medicinal material for standby;

[0124] (2) Extraction: Weigh 1 g of Stenoloma chusanum (L.) Ching medicinal material powder and put it into a round-bottomed flask, add 3 mol / L hydrochloric acid-60% ethanol solution according to the solid-liquid ratio (g / mL) of 1:25, put it into a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 50 r / min, reflux and extract at 80 °C for 1 h. After complete extraction, let it cool, adjust to neutral pH with alkaline substances, filter, concentrate the filtrate until there is no alcohol smell, and dilute with water to the same volume, and filter to obtain the Stenoloma chusanum (L.) Ching extract;

[0125] (3) Impurity removal by macroporous resin: Soak the refined D101 macroporous resin with anhydrous ethanol overnight to fully swell it. Pack 25 mL of the swollen D101 macroporous resin into a column. After loading an equal volume of Stenoloma chusanum (L.) Ching extract, first rinse with 6 BV of water until the effluent is colorless, and then elute with 14 BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract;

[0126] (4) Purification: Dissolve the crude extract with 40% methanol, and then purify it by preparative liquid phase. The specific conditions are: SinoChrom ODS-BP chromatographic column (5 μm, 10 mm × 250 mm), the detection wavelength is 360 nm, the flow rate is 4 mL / min, the temperature is 20 °C, the elution condition is 40% methanol, collect the eluate, concentrate and dry to obtain the final product.

[0127] The content of vitexin in the extract obtained in step (2) detected by high performance liquid chromatography was 3.67 mg; the content of vitexin in step (4) detected by high performance liquid chromatography was 2.59 mg, and its liquid phase purity was 88%.

[0128] Conclusion: By comparing Example 1 and Comparative Example 1, it can be found that when using strong acid-ethanol aqueous solution as the extraction solution, the yield of vitexin is higher, and from the chromatography Figure 1 and Figure 4It can be seen that after magnetic stirring extraction with strong acid-ethanol aqueous solution, the peaks of vitexin glucoside and vitexin rhamnoside disappear, while the vitexin peak becomes more obvious and there are fewer impurity peaks, indicating that derivatives such as vitexin glucoside and vitexin rhamnoside are completely hydrolyzed by strong acid to form vitexin and most other impurities are degraded; by comparing Example 1-Example 4 and Comparative Example 2-Comparative Example 10, it can be seen that high-yield and high-purity vitexin cannot be obtained under all conditions. After optimization, the present invention can significantly improve the yield and purity of vitexin.

[0129] In summary, by optimizing the extraction process, impurity removal process and purification process, the present invention not only improves the yield and purity of vitexin, but also significantly shortens the extraction and hydrolysis time by magnetic stirring. At the same time, due to the strong acid hydrolysis reaction during the extraction process, pigments and other impurities with similar polarities disappear, greatly reducing the difficulty of subsequent purification process, simplifying the process, significantly reducing the time cost and economic cost, and avoiding the operation of a large amount of organic solvent extraction in the prior art, making it more environmentally friendly.

[0130] Finally, it should be emphasized that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing high-purity vitexin from the above-ground part of Stenoloma chusanum, which is characterized in that, It includes the following steps: (1) Preparation of Stenoloma chusanum medicinal material powder: Cut and dry the above-ground part of Stenoloma chusanum, pulverize the dried medicinal material, and sieve it through a No. 3 sieve for standby; (2) Extraction: Weigh a certain weight of Stenoloma chusanum medicinal material powder and put it into a round-bottom flask, add a certain volume of a mixed solution containing 3 mol / L hydrochloric acid and 60% ethanol, place it in a magnetic stirring water bath reflux constant temperature heating system for extraction. After complete extraction, add an alkaline substance to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol smell, and dilute it with water to an equal volume, and centrifuge or filter to obtain the Stenoloma chusanum extract; the mass-volume ratio of the Stenoloma chusanum medicinal material powder to the acid-ethanol aqueous solution is 1:25; the magnetic stirring speed is 150 r / min, the extraction temperature is 80 °C, and the extraction time is 1 h; (3) Removal of impurities by macroporous resin: Pack the swollen D101 macroporous resin into a column. After loading the Stenoloma chusanum extract, first wash it with water until the effluent is colorless, then elute it with 30% ethanol, collect the 30% ethanol eluate and concentrate it to obtain the crude extract; (4) Purification: Dissolve the crude extract with 40% methanol and purify it by preparative liquid chromatography, collect the eluate, and concentrate and dry it to obtain the final product.

2. A method for preparing high-purity vitexin from the above-ground part of Stenoloma chusanum according to claim 1, characterized in that, In step (3), the volume ratio of the sample loading amount to the column volume is 1:1, and the swelling method of the D101 macroporous resin is: soak the D101 macroporous resin in absolute ethanol overnight to obtain it.

3. A method for preparing high-purity vitexin from the above-ground part of Stenoloma chusanum according to claim 1, characterized in that, In step (3), the washing and elution volume with water is 3 BV - 8 BV; the elution volume with 30% ethanol is 10 BV - 16 BV.

4. A method for preparing high-purity vitexin from the above-ground part of Stenoloma chusanum according to claim 3, characterized in that, The washing and elution volume with water is 6 BV; the elution volume with 30% ethanol is 14 BV.

5. A method for preparing high-purity vitexin from the aerial part of Stenoloma chusanum according to claim 1, characterized in that, In step (4), the parameters of preparative liquid chromatography purification: Use SinoChrom ODS-BP as the chromatographic column, 40% methanol as the elution solvent, the detection wavelength is 360 nm, and the flow rate is 4 mL / min.

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