Method for preparing high-purity vitexin from overground part of stenoloma chusanum

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction and refining, and particularly relates to a method for preparing high-purity vitexin from the overground part of stenoloma chusanum, which comprises the following steps: preparing stenoloma chusanum medicinal material powder, extracting, removing impurities by macroporous resin, and purifying a prepared liquid phase. According to the method, a strong acid-ethanol water solution is adopted as an extraction solution, the stability difference of flavone C-glycoside and flavone oxygen glycoside to strong acid is fully utilized, vitexin glucoside, vitexin rhamnoside and other derivatives are hydrolyzed through strong acid to generate vitexin, the content of vitexin is increased, meanwhile, substances with similar polarity can be hydrolyzed, and the content of vitexin is increased. The difficulty of subsequent preparation liquid phase purification is reduced, the obtained vitexin is high in yield and purity, the steps are simple, and the cost is low.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and purification of traditional Chinese medicines, and particularly relates to a method for preparing high-purity vitexin from the aerial part of Pteris chinensis. Background Art

[0002] Pteris chinensis Odontosoria chinensis ) is a perennial herb of the genus Pteris of the family Lepidopteris, mainly distributed near the Yangtze River basin. As an ornamental plant, Pteris has a high ornamental value. Pteris tastes slightly bitter and is cold in nature. It enters the liver and large intestine meridians. It has the effects of clearing heat, detoxifying, promoting dampness and stopping bleeding. Its main active ingredients are flavonoids and phenolic acid compounds. Pteris is mostly used to treat hepatitis, pediatric bronchitis, pneumonia, tonsillitis and gastroenteritis. The chemical composition of Pteris is relatively complex. Its main active ingredients are flavonoids and polyphenol compounds. There are also a large number of water-soluble pigments and fat-soluble oily components, which make the extraction and purification process of compounds in Pteris difficult.

[0003] Vitexin, one of the main active ingredients in Pteris chinensis, is also known as vitexin. It is a natural plant flavonoid carbon glycoside compound. Its chemical name is 8-β-D-glucose pyranose-4',7-dihydroxyisoflavone, and its molecular formula is C 21 H 20 O 10 , relative molecular mass 432.4, molecular structure as shown below. Black fern contains rich vitexin and its derivatives, with a total content of up to 9 mg / g. Modern pharmacological studies have shown that vitexin has multiple pharmacological effects such as anti-myocardial infarction, anti-inflammatory analgesia, blood pressure reduction, antibacterial, and antioxidant. Vitexin for injection has been used in clinical medicine. The main function of vitexin is to promote blood circulation and remove blood stasis, regulate qi and dredge meridians. It is used for chest pain caused by blood stasis and meridian obstruction, with symptoms of chest tightness, shortness of breath, stabbing pain in the precordial area, palpitations and forgetfulness, dizziness and tinnitus, angina pectoris of coronary heart disease, hyperlipidemia, and insufficient blood supply to the heart arteries.

[0004]

[0005] The wild resources of Pteris chinensis are rich, and the cost of raw materials is low. The main process of the method for preparing vitexin in the existing Pteris chinensis includes extraction, concentration, organic solvent extraction, re-concentration, recrystallization, alkali acid treatment, reverse silica gel elution, polyamide inspection and repeating the above steps. The method has cumbersome steps, the extraction rate of the prepared vitexin is low, and a variety of organic solvents are required for treatment, the purity is not high, the subsequent purification is difficult and the process is relatively complicated, the cost is high and it is not green and environmentally friendly. In view of this, it is necessary to develop a more simple, high extraction rate and purity, green and environmentally friendly, and suitable for industrial production Vitexin preparation method, which provides technical support for the development and utilization of vitexin in Pteris chinensis and provides direction for the Pteris chinensis industrial economy. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for preparing high-purity vitexin from the aerial part of Pteris cretica. The method adopts a strong acid extraction system and utilizes the difference in stability of flavonoid carbon glycosides and flavonoid oxygen glycosides to strong acid, and hydrolyzes vitexin glucoside, vitexin rhamnoside and other derivatives with strong acid to generate vitexin, so as to increase the content of vitexin. At the same time, the strong acid can degrade the pigment and other impurities in the Pteris cretica, thereby reducing the difficulty in preparation, simplifying the process, and obtaining the vitexin with high yield and purity.

[0007] The present invention provides a method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, comprising the following steps: (1) Preparation of Pteris chinensis medicinal material powder: Cut and dry the above-ground part of Pteris chinensis, crush the dried medicinal material, pass it through a No. 3 sieve, and set aside; (2) Extraction: Weigh a certain weight of the powder of the medicinal material of the black fern and put it into a round-bottom flask, add a certain volume of a mixed solution containing 2mol / L-4mol / L acid and 30%-80% ethanol, and put it into a magnetic stirring water bath reflux constant temperature heating system for extraction. After the extraction is complete, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute it with water to an equal volume, and centrifuge or filter to obtain the black fern extract. In this step, a high concentration of acid-ethanol solution is used as the extraction system, making full use of the advantage that concentrated acid can remove most impurities and convert some effective ingredients. 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, further simplifying the subsequent process. (3) Macroporous resin impurity removal: The swollen D101 macroporous resin was loaded into the column, and the extract of Pteris crenata was loaded. The column was first rinsed with water until the effluent was colorless, and then eluted with 30% ethanol. The 30% ethanol eluate was collected and concentrated to obtain the crude extract. In this step, 30% ethanol was used as the eluent to further remove most impurities and improve the purity of the crude extract. (4) Purification: The crude extract was dissolved in 40% methanol and purified by preparative liquid phase. The eluate was collected and concentrated to dryness to obtain the final product.

[0008] The invention adopts a strong acid-ethanol aqueous solution as an extraction solution, fully utilizes the difference in stability of flavonoid carbon glycoside and flavonoid oxygen glycoside to strong acid, hydrolyzes vitexin glucoside, vitexin rhamnoside and other derivatives with strong acid to generate vitexin, thereby increasing the content of vitexin, and simultaneously utilizing concentrated acid to hydrolyze substances with similar polarity and some pigments, thereby increasing the extraction efficiency and reducing the difficulty of subsequent preparation of liquid phase purification, and the obtained vitexin has high yield and high purity, greatly simplifies the process and cost of extracting vitexin from fern, and is green and environmentally friendly.

[0009] Furthermore, in step (2) of the above technical solution, the mass volume ratio of the Pteris crenata medicinal material powder to the acid-ethanol aqueous solution is 1:15-50; preferably 1:25.

[0010] Furthermore, 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%.

[0011] Furthermore, 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 1h-3h, preferably 1h; the magnetic stirring speed is 100r / min-200r / min, preferably 150r / min.

[0012] Furthermore, in step (3) of the above technical solution, the ratio of the sample volume to the column volume is 1:1, and the swelling method of the D101 macroporous resin is: soaking the D101 macroporous resin in anhydrous ethanol overnight.

[0013] Furthermore, in step (3) of the above technical solution, the elution volume of water washing is 3BV-8BV, preferably 6BV; the elution volume of 30% ethanol is 10BV-16BV, preferably 14BV.

[0014] Furthermore, in step (4) of the above technical solution, the parameters for the preparative liquid phase purification are: SinoChromODS-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.

[0015] Advantages compared to the prior art: The existing conventional hydrolysis process is to extract first, concentrate, and then dissolve and hydrolyze, which requires a lot of time and solvent. The present invention optimizes the extraction process, impurity removal process and purification process by single factor combined with response surface method, adopts strong acid-ethanol aqueous solution as the extraction solution, and adopts magnetic stirring at the same time, increases the extraction rate to promote the hydrolysis reaction, and significantly shortens 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 between flavonoid carbon glycosides and flavonoid oxygen glycosides to strong acid, and completely hydrolyzes the derivatives such as vitexin glucoside and vitexin rhamnoside with strong acid to generate vitexin, so that the content of vitexin in the black fern is doubled, and impurities with similar polarity can also be hydrolyzed, reducing the difficulty of subsequent preparation of liquid phase purification; by using macroporous resin for impurity removal, a large amount of salt and pigment are removed, and elite is used for preparation of liquid phase purification, the obtained vitexin has high yield, high purity, simple steps, low cost, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a chromatogram of the extract of Pteris chinensis obtained in Example 1 of the present invention, wherein 1 is vitexin; Figure 2 is a chromatogram of the crude extract obtained in Example 1 of the present invention, wherein 1 is vitexin; Figure 3 This is a chromatogram of the purified product in Example 1 of the present invention, wherein 1 is vitexin; Figure 4 This is a chromatogram of the extract of Pteris crenata obtained after extraction without adding acid in Comparative Example 1 of the present invention, wherein 1 is vitexin, 2 is vitexin glucoside, and 3 is vitexin rhamnoside. DETAILED DESCRIPTION

[0017] The above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0018] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0019] The preparative liquid phase purification was performed using an Eclassical 3100 preparative HPLC instrument; the HPLC content was detected using a Waters E2695 HPLC, and the detection conditions were as follows: Welchrom C18 chromatographic column (4.6 mm×250.0 mm, 5 μm), the mobile phase was acetonitrile (A)-water (B), the elution conditions were (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 was 360 nm, the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 10 μL.

[0020] The present invention is further described in detail below in conjunction with the drawings and embodiments: Example 1 A method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol. The eluate was collected, concentrated and dried to obtain the final product.

[0021] The content of vitexin in the extract of Pteris chinensis obtained in step (2) was 4.52 mg by high performance liquid chromatography. The chromatogram is shown in FIG. Figure 1 As shown; the content of vitexin in the crude extract obtained in step (3) was 3.48 mg by high performance liquid chromatography, and its chromatogram was 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 in Figure 3 As shown, the liquid phase purity is 98%.

[0022] Example 2 A method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 4 mol / L hydrochloric acid-30% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern extract, first rinse with 3 BV of water until the effluent is colorless, then elute with 16 BV of 30% ethanol. Collect the 30% ethanol eluate and concentrate it to obtain the crude extract. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0023] The content of vitexin in the extract obtained in step (2) was 4.49 mg by high performance liquid chromatography. The content of vitexin in the crude extract obtained in step (3) was 3.46 mg by high performance liquid chromatography. Similarly, the content of vitexin in step (4) was 3.19 mg by high performance liquid chromatography, and its liquid phase purity was 95%.

[0024] Example 3 A method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-50% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern extract, first rinse with 8 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0025] The content of vitexin in the extract obtained in step (2) was 4.43 mg by high performance liquid chromatography. The content of vitexin in the crude extract obtained in step (3) was 3.41 mg by high performance liquid chromatography. Similarly, the content of vitexin in step (4) was 3.15 mg by high performance liquid chromatography, and its liquid phase purity was 98%.

[0026] Example 4 A method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:15, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 15 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0027] The content of vitexin in the extract obtained in step (2) was 4.19 mg by high performance liquid chromatography. The content of vitexin in the crude extract obtained in step (3) was 3.23 mg by high performance liquid chromatography. Similarly, the content of vitexin in step (4) was 3.03 mg by high performance liquid chromatography, and its liquid phase purity was 98%.

[0028] Comparative Example 1 A method for preparing vitexin from the aerial parts of Pteris crenata, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0029] The content of vitexin in the extract obtained in step (2) was 1.52 mg by high performance liquid chromatography. The chromatogram is shown in FIG. Figure 4 As shown; the content of vitexin in step (4) was 1.06 mg by high performance liquid chromatography, and its liquid phase purity was 45%.

[0030] Comparative Example 2 A method for preparing vitexin from the aerial parts of Pteris crenata, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of the powder of the medicinal material of Pteris crenata and put it into a round-bottom flask. Add 3 mol / L hydrochloric acid solution at a solid-liquid ratio (g / mL) of 1:25, and place it in a magnetic stirring water bath reflux constant temperature heating system. The magnetic stirring speed is 150 r / min. Extract in a water bath reflux 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 crenata extract. (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0031] The content of vitexin in the extract obtained in step (2) was 2.29 mg by high performance liquid chromatography. The content of vitexin in step (4) was 1.62 mg by high performance liquid chromatography, and its liquid phase purity was 98%.

[0032] Comparative Example 3 A method for preparing vitexin from the aerial parts of Pteris crenata, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 1 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0033] The content of vitexin in the extract obtained in step (2) was 2.68 mg by high performance liquid chromatography. The content of vitexin in step (4) was 1.90 mg by high performance liquid chromatography, and its liquid phase purity was 70%.

[0034] Comparative Example 4 A method for preparing vitexin from the aerial parts of Pteris crenata, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-15% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0035] The content of vitexin in the extract obtained in step (2) was 3.38 mg by high performance liquid chromatography. The content of vitexin in step (4) was 2.40 mg by high performance liquid chromatography, and its liquid phase purity was 97%.

[0036] Comparative Example 5 A method for preparing vitexin from the aerial parts of Pteris crenata, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a 80°C water bath reflux for 0.5 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0037] The content of vitexin in the extract obtained in step (2) was 2.88 mg by high performance liquid chromatography. The content of vitexin in step (4) was 2.08 mg by high performance liquid chromatography, and its liquid phase purity was 86%.

[0038] Comparative Example 6 A method for preparing vitexin from the aerial parts of Pteris crenata, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a 60°C water bath reflux for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0039] The content of vitexin in the extract obtained in step (2) was 1.89 mg by high performance liquid chromatography. The content of vitexin in step (4) was 1.34 mg by high performance liquid chromatography, and its liquid phase purity was 56%.

[0040] Comparative Example 7 A method for preparing vitexin from the aerial parts of Pteris crenata, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern extract, first rinse with 6 BV of water until the effluent is colorless, then elute with 8 BV of 50% ethanol. Collect the 50% ethanol eluate and concentrate it to obtain the crude extract. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0041] The content of vitexin in the crude extract obtained in step (3) was 3.74 mg by high performance liquid chromatography. The content of vitexin in step (4) was 3.36 mg by high performance liquid chromatography, and its liquid phase purity was 82%.

[0042] Comparative Example 8 A method for preparing vitexin from the aerial parts of Pteris crenata, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 150 r / min, and extract in a water bath reflux at 80°C for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it, and then load 16 mL of the swollen D101 macroporous resin into the column. After loading 1.5 times the column volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP chromatographic column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol, and the eluent was collected, concentrated, and dried to obtain the final product.

[0043] It was found that there was leakage of vitexin in step (3). The content of vitexin in the product of step (4) was 2.89 mg by high performance liquid chromatography, and its liquid phase purity was 97%.

[0044] Comparative Example 9 A method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, put into a water bath, and reflux extract at 80°C for 1 h. After extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol. The eluate was collected, concentrated and dried to obtain the final product.

[0045] The content of vitexin in the extract obtained in step (2) was 3.19 mg by high performance liquid chromatography. The content of vitexin in step (4) was 2.24 mg by high performance liquid chromatography, and its liquid phase purity was 82%.

[0046] Comparative Example 10 A method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, comprising the following steps: (1) Preparation of the powder of the medicinal material of Pteris chinensis: Cut and dry the above-ground part of Pteris chinensis, and crush the dried medicinal material for later use; (2) Extraction: Weigh 1 g of Pteris chinensis powder into a round-bottom flask, add 3 mol / L hydrochloric acid-60% ethanol solution at a solid-liquid ratio (g / mL) of 1:25, place in a magnetic stirring water bath reflux constant temperature heating system, the magnetic stirring speed is 50 r / min, and extract at 80°C water bath reflux for 1 h. After the extraction is complete, cool, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute with water to an equal volume, filter to obtain the Pteris chinensis extract; (3) Removal of impurities from macroporous resin: Soak the refined D101 macroporous resin in anhydrous ethanol overnight to fully swell it. Use the swollen D101 macroporous resin to load 25 mL of the column. Load an equal volume of the black fern 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. (4) Purification: The crude extract was dissolved in 40% methanol and then purified by preparative liquid phase. The specific conditions were: SinoChromODS-BP column (5 μm, 10 mm × 250 mm), detection wavelength of 360 nm, flow rate of 4 mL / min, temperature of 20 °C, elution condition of 40% methanol. The eluate was collected, concentrated and dried to obtain the final product.

[0047] The content of vitexin in the extract obtained in step (2) was 3.67 mg by high performance liquid chromatography. The content of vitexin in step (4) was 2.59 mg by high performance liquid chromatography, and its liquid phase purity was 88%.

[0048] Conclusion: By comparing Example 1 with Comparative Example 1, it can be found that when a strong acid-ethanol aqueous solution is used as the extraction solution, the yield of vitexin is higher, and the chromatographic Figure 1 and Figure 4 It can be seen that after magnetic stirring extraction with a strong acid-ethanol aqueous solution, the peaks of vitexin glucoside and vitexin rhamnoside disappeared, while the vitexin peak was more obvious and there were fewer impurity peaks, indicating that vitexin glucoside, vitexin rhamnoside and other derivatives were completely hydrolyzed by a strong acid to generate vitexin and degrade most of the other impurities; it can be seen from comparative examples 1 to 4 and comparative examples 2 to 10 that high-yield and high-purity vitexin can not be obtained under all conditions, and the yield and purity of vitexin can be significantly improved after optimization.

[0049] In summary, the present invention not only improves the yield and purity of vitexin by optimizing the extraction process, the impurity removal process and the purification process, but also significantly shortens the extraction and hydrolysis time by using magnetic stirring. At the same time, due to the strong acid hydrolysis reaction in the extraction process, pigments and other impurities with similar polarity disappear, which greatly reduces the difficulty of subsequent purification process treatment, simplifies the process, and significantly reduces the time cost and economic cost. At the same time, it avoids the operation of a large amount of organic solvent extraction in the prior art, making it more green and environmentally friendly.

[0050] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing high-purity vitexin from the aerial parts of Pteris chinensis, characterized in that: The following steps are involved: (1) Preparation of Pteris chinensis medicinal material powder: Cut and dry the above-ground part of Pteris chinensis, crush the dried medicinal material, pass it through a No. 3 sieve, and set aside; (2) Extraction: Weigh a certain weight of the powder of the medicinal material of Pteris chinensis and put it into a round-bottom flask, add a certain volume of a mixed solution containing 2mol / L-4mol / L acid and 30%-80% ethanol, and put it into a magnetic stirring water bath reflux constant temperature heating system for extraction. After the extraction is complete, add alkaline substances to adjust the pH to neutral, filter, concentrate the filtrate until there is no alcohol taste, and dilute it with water to an equal volume, centrifuge or filter to obtain the Pteris chinensis extract; (3) Macroporous resin impurity removal: The swollen D101 macroporous resin was loaded into the column. After loading the extract of Pteris crenata, it was first rinsed with water until the effluent was colorless, and then eluted with 30% ethanol. The 30% ethanol eluate was collected and concentrated to obtain the crude extract. (4) Purification: The crude extract was dissolved in 40% methanol and purified by preparative liquid phase. The eluate was collected and concentrated to dryness to obtain the final product.

2. The method for preparing high-purity vitexin from the aerial parts of Pteris cretica according to claim 1, characterized in that: In step (2), the mass volume ratio of the Pteris crenata medicinal material powder to the acid-ethanol aqueous solution is 1:15-50.

3. A method for preparing high-purity vitexin from the aerial parts of Pteris crenata according to claim 2, characterized in that: The mass volume ratio of the Pteris crenata medicinal material powder to the acid-ethanol aqueous solution is 1:

25.

4. The method for preparing high-purity vitexin from the aerial parts of Pteris crenata according to claim 1, characterized in that: In step (2), the acid is hydrochloric acid with a concentration of 3 mol / L, and the concentration of ethanol is 60%.

5. The method for preparing high-purity vitexin from the aerial parts of Pteris crenata according to claim 1, characterized in that: In step (2), in the magnetic stirring water bath reflux constant temperature extraction process, the magnetic stirring speed is 100r / min-200r / min, the extraction temperature is 70°C-90°C, and the extraction time is 1h-3h.

6. The method for preparing high-purity vitexin from the aerial parts of Pteris crenata according to claim 5, characterized in that: The rotation speed is 150 r / min, the extraction temperature is 80° C., and the extraction time is 1 h.

7. The method for preparing high-purity vitexin from the aerial parts of Pteris crenata according to claim 1, characterized in that: In step (3), the ratio of the sample volume to the column volume is 1:1, and the swelling method of the D101 macroporous resin is: soaking the D101 macroporous resin in anhydrous ethanol overnight.

8. The method for preparing high-purity vitexin from the aerial parts of Pteris crenata according to claim 1, characterized in that: In step (3), the elution volume of water washing is 3BV-8BV; the elution volume of 30% ethanol is 10BV-16BV.

9. The method for preparing high-purity vitexin from the aerial parts of Pteris crenata according to claim 8, characterized in that: The elution volume for water washing is 6 BV; the elution volume for 30% ethanol is 14 BV.

10. The method for preparing high-purity vitexin from the aerial parts of Pteris crenata according to claim 1, characterized in that: In step (4), the parameters for the preparative liquid phase purification were: SinoChromODS-BP as the chromatographic column, 40% methanol as the elution solvent, the detection wavelength was 360 nm, and the flow rate was 4 mL / min.

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