阅读说明：本技术 一种从白桐树中提取酚类化合物的方法 (Method for extracting phenolic compounds from tung tree ) 是由 王志尧 常霞 王伟 王韬 马艳妮 姜晓 陈飞 罗远情 于 2021-09-01 设计创作，主要内容包括：本发明提供一种从白桐树中提取酚类化合物的方法,所述方法包括提取、浓缩、萃取、聚酰胺柱层析、C18柱层析、凝胶柱层析、半制备型高效液相色谱法等步骤,采用本发明方法首次从白桐树中分离得到cleomiscosin C、cleomiscosinA、莨菪亭、6-姜酚和6-姜烯酚5个酚类化合物,且化合物纯度均可达98％以上。该方法操作简单,易于实现,且柱层析所用填料均可重复利用,经济高效。(The invention provides a method for extracting phenolic compounds from a tung tree, which comprises the steps of extraction, concentration, extraction, polyamide column chromatography, C18 column chromatography, gel column chromatography, semi-preparative high performance liquid chromatography and the like, wherein 5 phenolic compounds including cleomiscosin C, cleomiscosin A, hyoscyami, 6-gingerol and 6-shogaol are separated from the tung tree for the first time by adopting the method, and the purity of the compounds can reach more than 98%. The method is simple to operate and easy to realize, and the filler used for column chromatography can be recycled, so that the method is economical and efficient.)

1. A method for extracting phenolic compounds from a tung tree, the method comprising the steps of:

step 1: adding a solvent into the raw materials of the erythrina indica lam to extract to obtain an extracting solution, and concentrating the extracting solution under reduced pressure to obtain an extract, wherein the solvent is any one or the combination of at least two of methanol, ethanol or water;

step 2: adding water into the extract obtained in the step 1 to disperse the extract until the relative density is 1.05-1.20 (measured at room temperature), obtaining a water dispersion, adding petroleum ether to extract, collecting a water phase part, extracting with ethyl acetate, collecting an ethyl acetate part, and concentrating under reduced pressure to obtain an ethyl acetate extract;

and step 3: separating and purifying the ethyl acetate extract obtained in the step 2 by polyamide column chromatography to obtain a crude product of the total polyphenol of the idesia polycarpa;

and 4, step 4: separating and purifying the total polyphenol crude product of the idesia polycarpa obtained in the step 3 by C18 column chromatography to obtain a column chromatography sample I;

and 5: separating and purifying the column chromatography sample I obtained in the step 4 by gel column chromatography to obtain a target I section component, a target II section component and a target III section component;

step 6: separating and purifying the target I section component and the target II section component by semi-preparative high performance liquid chromatography respectively to obtain phenolic compounds of cleomiscosin C, cleomiscosin A, 6-shogaol and 6-shogaol, wherein the separation conditions of the semi-preparative chromatography are as follows: the chromatographic column is an acid-resistant C18 chromatographic column, 0.1-0.3 wt% phosphoric acid aqueous solution and acetonitrile are used as mobile phases, the detection wavelength is 300-365 nm, the flow rate is 2-5 ml/min, eluents corresponding to the chromatogram characteristic peak of the target component are respectively collected, and the phenol compound is obtained through concentration and drying;

recrystallizing the target III-section component to obtain the phenolic compound hyoscyamine, wherein the recrystallization solvent is any one or the combination of at least two of methanol, ethanol and chloroform.

2. The method according to claim 1, wherein the mass-to-volume ratio of the raw material of the erythrina indica lam to the solvent in the extraction in the step 1 is 1: 8-20 in g/ml; the extraction method is any one or the combination of at least two of thermal reflux extraction, ultrasonic extraction or ultrasonic-assisted thermal reflux extraction; extracting for 1-4 times, combining the multiple extracting solutions, and performing reduced pressure concentration to recover the solvent to obtain an extract;

preferably, the ultrasonic extraction is carried out, wherein the ultrasonic power is 20 KHz-40 KHz, and the ultrasonic time is 1-3 h;

preferably, the hot reflux extraction is carried out at the temperature of 50-100 ℃ for 1-3 h;

preferably, the ultrasonic-assisted hot reflux extraction is carried out, wherein the ultrasonic power is 20 KHz-40 KHz, the ultrasonic time is 1-3 h, and the temperature is 50-100 ℃;

preferably, the concentration under reduced pressure is carried out until the organic solvent smell is eliminated and the relative density is less than 1.20 (measured at room temperature).

3. The method according to claim 1, wherein the volume ratio of the aqueous dispersion to the petroleum ether in the petroleum ether extraction in the step 2 is 1: 1-3; in the ethyl acetate extraction, the volume ratio of the water phase part to the ethyl acetate is 1: 1-3.

4. The method according to claim 1, wherein the polyamide column packing in step 3 is polyamide with a particle size of 30-200 meshes; the column chromatography elution solvent is ethanol water solution, the elution flow rate is 5-20 ml/min, and the elution mode is gradient elution: eluting 5-8 column volumes with 25-30 vol% ethanol to remove impurities, eluting 3-6 column volumes with 60-75 vol% ethanol, collecting the eluent, and finally washing the column with 95 vol% ethanol;

preferably, the elution is carried out under medium and low pressure chromatographic conditions of 5 to 20 bar.

5. The method according to claim 4, wherein the mass ratio of the ethyl acetate extract to the polyamide filler in the step 3 is 1:50 to 100; the height-diameter ratio of the polyamide column is 5-10: 1.

6. The method of claim 1, wherein the C18 column packing in step 4 is RP-C18 with pore sizeThe particle size is 40-63 μm; the column chromatography elution solvent is methanol water solution, the elution flow rate is 5-20 ml/min, and the elution mode is as follows: eluting 4-8 column volumes with 25-30 vol% methanol to remove impurities, eluting 4-8 column volumes with 60-70 vol% methanol, collecting the eluent, and finally washing the column with 95-100 vol% methanol;

preferably, the elution is carried out under medium and low pressure chromatographic conditions of 5-20 bar;

preferably, the mass ratio of the jatropha curcas total polyphenol crude product to the C18 filler is 1: 100-150, and the height-diameter ratio of the C18 column is 20-40: 1.

7. The method of claim 1, wherein the gel column packing in step 5 is sephadex and derivatives thereof with a degree of crosslinking of 25 or less; the elution solvent is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol is 1: 0.2-5; the elution flow rate is 3-5 seconds per drop;

preferably, the mass ratio of the column chromatography sample I to the gel filler is 1: 1500-2000, and the height-diameter ratio of the gel column is 50-120: 1.

8. The method as claimed in claim 7, wherein the gel column packing is any one of Sephadex G-25, Sephadex G-10, Sephadex LH-20.

9. The method according to claim 1, wherein in step 6, the 0.1-0.3 wt% phosphoric acid aqueous solution is mobile phase A, the acetonitrile is mobile phase B, and the elution mode is isocratic elution: 20 to 23 percent of mobile phase B, and the balance is complemented to 100 percent by the mobile phase A.

10. The method according to claim 1, wherein the recrystallization of the target group III component in the step 6 is performed by standing at 0-4 ℃ to slowly volatilize the solvent and separate out crystals.

Technical Field

The invention relates to the technical field of extraction and separation of chemical components of natural plants, in particular to a method for extracting phenolic compounds from a tung tree.

Background

Baitong Claoxylon indicum (Reinw. ex Bl.) Hassk, also named Zusan, Bingfeng, Dunyang, Bingfengchang, Zhufenggen, Meiqiao Hua, is a plant of the genus Jatropha (Claoxylon A. juss.) of the family Euphorbiaceae, mainly produced in Guangdong, Hainan, Guangxi, Yunnan, etc., has pungent and slightly bitter taste, small toxicity, mild nature, spleen and kidney channels, and has the effects of dispelling wind, eliminating dampness, and relieving swelling and pain, and can be used for treating rheumatic arthritis, rheumatoid arthritis, lumbocrural pain, traumatic injury, dermatophytosis edema, burn, scald, traumatic hemorrhage, serving as a national medicine, and is mainly used for treating rheumatoid bone diseases among rheumatism and bone diseases. The baitong trees are accepted as clinical medicines in legal norms such as Chinese pharmacopoeia (1977 edition), Guangxi autonomous region Zhuang medicine quality standard (Guangxi Zhuang medicine quality standard), Hunan Chinese medicine decoction piece processing standard (2010 edition), Gansu province Chinese medicine decoction piece processing standard (1980 edition) and the like, and are used in the aspects of treating rheumatic and rheumatoid arthritis and bone diseases, such as Chinese traumatic pills (2010 edition of Chinese pharmacopoeia), Chinese traumatic wine (nineteenth volume of standard Chinese medicine prescription preparation of Ministry of health department), injured tendon and bone-setting tincture (itening of orthopedics and traumatology compiled by national standards of Chinese patent medicine), new strength and bone-setting spray (elementary volume of compiled orthopedics and traumatology), bone-setting water (eleventh volume of standard Chinese medicine prescription preparation of Ministry of health department) and a plurality of Zhuang medicine prescriptions.

At present, few chemical research reports about the erythrina are reported, and the research results of documents show that the erythrina plants contain chemical components such as phenols, alkaloids, diterpenes, triterpenes, saponins and the like, wherein the phenols (mainly lignans, neolignans and coumarins) are characteristic components of the plants and have various activities of resisting cancers, regulating immunity and the like. At present, the research on phenolic components of the aleurites fordii is less, and no clear method is provided for extracting the phenolic components from the aleurites fordii plants, which seriously restricts the wide clinical application of the traditional Zhuang medicine aleurites fordii, so that the research and development of the method for extracting and separating the phenolic compounds from the aleurites fordii plants are carried out, so that various phenolic compounds can be simply and quickly prepared from the aleurites fordii, and the method has important significance for the full development and utilization of the Zhuang medicine aleurites fordii resources.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for extracting phenolic compounds from a tung tree, the extraction method can simply and quickly extract and separate 5 phenolic compounds of cleomiscosin C (1), cleomiscosin A (2), scopoletin (3), 6-gingerol (4) and 6-gingerol (5) from the tung tree, and the chemical structural formula of the phenolic compounds is shown in figure 1.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for extracting phenolic compounds from a tung tree, which comprises the following steps:

step 1: adding a solvent into the raw materials of the erythrina indica lam to extract to obtain an extracting solution, and concentrating the extracting solution under reduced pressure to obtain an extract, wherein the solvent is any one or the combination of at least two of methanol, ethanol or water;

step 2: adding water into the extract obtained in the step 1 to disperse the extract until the relative density is 1.05-1.20 (measured at room temperature) to obtain a water dispersion, adding petroleum ether to extract, collecting a water phase part, extracting with ethyl acetate, collecting an ethyl acetate part, and concentrating under reduced pressure to obtain an ethyl acetate extract;

and step 3: separating and purifying the ethyl acetate extract obtained in the step 2 by polyamide column chromatography to obtain a crude product of the total polyphenol of the idesia polycarpa;

and 4, step 4: separating and purifying the total polyphenol crude product of the idesia polycarpa obtained in the step 3 by C18 column chromatography to obtain a column chromatography sample I;

and 5: separating and purifying the column chromatography sample I obtained in the step 4 by gel column chromatography to obtain a target I section component, a target II section component and a target III section component;

step 6: separating and purifying the target I section component and the target II section component by semi-preparative high performance liquid chromatography respectively to obtain phenolic compounds of cleomiscosin C, cleomiscosin A, 6-shogaol and 6-shogaol, wherein the separation conditions of the semi-preparative high performance liquid chromatography are as follows: the chromatographic column is an acid-resistant C18 chromatographic column, 0.1-0.3 wt% of phosphoric acid aqueous solution and acetonitrile are used as mobile phases, the detection wavelength is 300-365 nm, the flow rate is 2-5 ml/min, eluents corresponding to the chromatogram characteristic peak of the target component are respectively collected, and the phenolic compound is obtained after concentration and drying;

recrystallizing the target III-section component to obtain the phenolic compound hyoscyamine, wherein the recrystallization solvent is any one or the combination of at least two of methanol, ethanol and chloroform.

As a preferential scheme of the invention, the method also comprises a pretreatment step before the extraction of the raw materials of the Chinese erythrina tree in the step 1, wherein the pretreatment step is to take the raw materials of the Chinese erythrina tree, dry or dry the raw materials and crush the raw materials to 20-80 meshes; the plant raw material of the erythrina variegata is any one or the combination of at least two of the roots, stems or branches of the erythrina variegata.

Preferably, the mass-to-volume ratio of the raw material of the erythrina indica lam to the solvent in the extraction in the step 1 is 1: 8-20, more preferably 1: 8-13, in g/ml, and the mass-to-volume ratio of the raw material to the solvent is the ratio of the mass of the raw material to the volume of the solvent, for example, 8-20 ml of the solvent is used for extraction per g of the raw material.

Preferably, the solvent in the extraction in the step 1 is 70 vol% to 100 vol% of methanol or ethanol aqueous solution;

preferably, the extraction method in step 1 is selected from one or a combination of at least two of thermal reflux extraction, ultrasonic extraction or ultrasonic-assisted thermal reflux extraction;

preferably, the ultrasonic extraction is carried out, wherein the ultrasonic power is 20 KHz-40 KHz, preferably 20 KHz-30 KHz, the ultrasonic time is 1-3 h, preferably 1.5-2.5 h, and the extraction times are 1-4 times;

preferably, the hot reflux extraction is carried out at the temperature of 50-100 ℃ for 1-3 h, preferably 2-3 h, and the extraction is carried out for 1-4 times;

preferably, the ultrasonic-assisted hot reflux extraction is carried out, the ultrasonic power is 20 KHz-40 KHz, preferably 20 KHz-30 KHz, the ultrasonic time is 1-3 h, preferably 1.5-2 h, the temperature is 50-100 ℃, and the extraction times are 1-4 times.

Preferably, in step 1, the concentration under reduced pressure is carried out until the organic solvent smell is eliminated and the relative density is 1.20 or less (measured at room temperature).

Preferably, in the step 2, in the petroleum ether extraction, the volume ratio of the aqueous dispersion to the petroleum ether is 1: 1-3, and more preferably 1:1, and most of the small polar fat-soluble impurity components can be removed by petroleum ether extraction.

Preferably, in the step 2, in the ethyl acetate extraction, the volume ratio of the water phase part to the ethyl acetate is 1: 1-3, more preferably 1:1, the phenol component is retained by the ethyl acetate extraction, and the water-soluble highly polar impurity component is retained in the water layer and removed.

Preferably, in the step 3, the polyamide column is polyamide with a particle size of 30-200 meshes, preferably 60-100 meshes; the mass ratio of the ethyl acetate extract to the polyamide filler is 1: 50-100, such as 55 times, 60 times, 70 times, 75 times, 80 times, 85 times, 90 times or 95 times, but not limited to the recited values, and other values not recited in the range of the recited values are also applicable; the aspect ratio of the polyamide column is 5 to 10:1, such as 6:1, 7:1, 8:1 or 9:1, but is not limited to the recited values, and other values not recited within the range of values are also applicable.

Preferably, the elution solvent for polyamide column chromatography separation and purification in step 3 is an ethanol aqueous solution, the elution flow rate is 5-20 ml/min, and the elution mode is gradient elution: eluting 5-8 column volumes with 25-30 vol% ethanol to remove impurities, eluting 3-6 column volumes with 60-75 vol% ethanol, collecting the eluent, and finally washing the column with 95 vol% ethanol.

Preferably, the elution in the step 4 is carried out under medium and low pressure chromatographic conditions of 5-20 bar.

The adsorption of the amino groups of the polyamide filler and the phenolic hydroxyl groups of the phenolic components of the tung tree is realized by hydrogen bonds, and the strength of the adsorption is mainly determined by the number and the position of the hydroxyl groups in the compound and the association capacity of the solvent and the compound or the solvent and the polyamide for forming the hydrogen bonds. Through the polyamide column chromatography in the step 3, the impurities of the non-hydroxyl components can be quickly removed, and the phenolic compounds in the idesia polycarpa can be effectively enriched. And after the column is washed by 95 vol% ethanol, the polyamide filler can be regenerated and reused.

Preferably, the C18 column packing in the step 4 is RP-C18 with pore diameterA particle size of 40 to 63 μm, for example Merck Lichroprep RP-C18; the mass ratio of the jatropha curcas total polyphenol crude product to the C18 filler is 1:100 to 150 times, such as 105 times, 110 times, 120 times, 130 times, 140 times, etc., but not limited to the recited values, and other values not recited within the range of values are also applicable; the ratio of height to diameter of the C18 column is 20 to 40:1, such as 22:1, 25:1, 30:1, 35:1, or 38:1, but is not limited to the recited values, and other values not recited within the range of values are also applicable.

Preferably, the elution solvent for the C18 column chromatography separation and purification in the step 4 is a methanol water solution, the elution flow rate is 5-20 ml/min, and the elution mode is as follows: firstly eluting 4-8 column volumes with 25-30 vol% methanol to remove impurities, then eluting 4-8 column volumes with 60-70 vol% methanol, collecting eluent, finally washing the column with 95-100 vol% methanol, and the washed C18 column can be reused.

Preferably, the elution in the step 4 is carried out under medium and low pressure chromatographic conditions of 5-20 bar.

Through C18 column chromatography separation and purification, other components with larger polarity difference with the target phenolic compound can be effectively removed.

Preferably, the gel column filler in the step 5 is crosslinked Sephadex gel with the crosslinking degree less than or equal to 25 and derivatives thereof, and further preferably any one of Sephadex G-25, Sephadex G-10 and Sephadex LH-20. The sephadex has the function of molecular sieve, and can separate compounds with different molecular weights by separating and purifying through sephadex column chromatography, and other components with similar polarity but different molecular weights with phenolic compounds in the idesia polycarpa in a sample can be separated by selecting the sephadex with the crosslinking degree less than or equal to 25 and derivatives thereof.

Preferably, the mass ratio of the column chromatography sample I to the gel filler in the step 5 is 1: 1500-2000, such as 1:1600, 1:1700, 1:1800, 1:1900, etc.; the ratio of the height to the diameter of the gel column is 50 to 120:1, such as 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, etc., but the ratio is not limited to the above-mentioned values, and other values not shown in the above-mentioned range are also applicable.

Preferably, the elution solvent for separation and purification by gel column chromatography in the step 5 is a chloroform-methanol mixed solvent, the volume ratio of chloroform to methanol is 1: 0.2-5, preferably 1:1, the elution flow rate is 3-5 seconds per drop, the eluate is collected in sections, one fraction is obtained per 0.1-0.5 column volume, the eluate is detected by a silica gel thin layer plate after elution is finished, the eluents with the same components are combined, and the target I-section component, the target II-section component and the target III-section component are obtained through reduced pressure concentration.

The detection method of the silica gel thin layer plate comprises the following steps: the normal phase silica gel thin layer high efficiency precast slab comprises a developing agent which is a chloroform-methanol mixed solvent, wherein the volume ratio of chloroform to methanol is 10-2: 1, such as 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1, etc., but is not limited to the recited values, and other unrecited values within the numerical range are equally applicable. The color development method comprises the following steps: taking 5-15 vol% ethanol sulfate solution as color developing agent, heating at 105-115 deg.C for color development, and comparing with target compound to obtain the target compound_fThe values and colors are combined.

Preferably, the semi-preparative high performance liquid chromatography column in step 6 is a 10mm X250 mm, 10 μm standard acid-resistant C18 column, commercially available, and may be, for example, Waters X-Bridge C18 or Waters Sunfire C18.

Preferably, in step 6, the 0.1 wt% to 0.3 wt% phosphoric acid aqueous solution is a mobile phase a, the acetonitrile is a mobile phase B, and the elution mode is isocratic elution: 20 to 23 percent of mobile phase B, and the balance of the mobile phase B is complemented to 100 percent by the mobile phase A for 20 to 30 min.

The phenolic components of the jatropha curcas are weakly acidic, and the acidic mobile phase can inhibit ionization of the phenolic components, improve the peak type of a chromatographic peak and is more beneficial to separation.

Preferably, in the step 6, in the semi-preparative high performance liquid chromatography separation, the concentration of the sample to be loaded is 10-100 mg/ml (methanol solution), preferably 30-50 mg/ml, and the sample loading amount is 10-100 μml, preferably 30-50 μml each time.

The chromatogram characteristic peaks of the semi-preparative high performance liquid chromatography separation target component are respectively based on the peak emergence time of the target compound reference substance under the same chromatographic conditions.

Preferably, the step 6 of recrystallizing the target III-stage component is to stand at 0-4 ℃ so that the solvent is slowly volatilized to separate out crystals.

In the invention, the temperature of the reduced pressure concentration in the step is 50-65 ℃, such as 50 ℃, 55 ℃,60 ℃ or 65 ℃, but the temperature is not limited to the recited values, and other unrecited values in the numerical range are also applicable; the vacuum degree of the vacuum concentration is-0.08 to-0.1 MPa, such as-0.08 MPa, -0.085MPa, -0.09MPa or-0.095 MPa, but not limited to the values listed, and other values not listed in the numerical range are also applicable.

The technical scheme provided by the invention has the following beneficial effects:

the method provided by the invention is utilized for simply and quickly extracting and separating the phenol compounds such as cleomiscosin C, cleomiscosin A, 6-shogaol, 6-gingerol, hyoscyamine and the like from the idesia polycarpa for the first time. The extraction method has high total polyphenol extraction rate, and can effectively extract polyphenol compounds from plant raw materials; petroleum ether and ethyl acetate are used for extraction in sequence, so that most of low-polarity fat-soluble components and high-polarity water-soluble impurities in the extract can be quickly removed; then polyamide, C18, sephadex and other column chromatography with different separation principles are combined, phenolic compounds in the idesia polycarpa can be simply and rapidly separated from other components, and finally 5 monomer compounds of cleomiscosin C, cleomiscosin A, 6-shogaol, 6-gingerol and hyoscyami can be obtained by semi-preparation of new high and new liquid chromatography or recrystallization for further refining, wherein the purity is more than or equal to 98 percent, and fillers used for column chromatography can be repeatedly utilized, and the method is economical and efficient.

Drawings

FIG. 1 is the chemical structural formula of 5 phenolic compounds prepared by the invention

FIG. 2 is a semi-preparative high performance liquid chromatogram of the target fraction I of example 1

FIG. 3 is a semi-preparative high performance liquid chromatogram of the target fraction II of example 1

FIG. 4 shows Compound 1 prepared in example 1¹H NMR spectrum

FIG. 5 shows Compound 1 prepared in example 1¹³C NMR spectrum

FIG. 6 shows Compound 2 prepared in example 1¹H NMR spectrum

FIG. 7 shows Compound 2 prepared in example 1¹³C NMR spectrum

FIG. 8 is a photograph of Compound 3 prepared in example 1¹H NMR spectrum

FIG. 9 shows Compound 3 prepared in example 1¹³C NMR spectrum

FIG. 10 shows Compound 4 prepared in example 1¹H NMR spectrum

FIG. 11 is a photograph of Compound 4 prepared in example 1¹³C NMR spectrum

FIG. 12 shows Compound 5 prepared in example 1¹H NMR spectrum

FIG. 13 shows Compound 5 prepared in example 1¹³C NMR spectrum

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.

The raw material of the erythrina variegata was purchased from Kunming plant technology Co.

The detection and analysis method of the sample in the embodiment of the application is as follows:

1. during the gel column chromatography in the step 5, the TCL (thin layer chromatography) detection method for detecting the components of the eluent is as follows: the normal phase silica gel thin layer high efficiency precast slab has developing agent chloroform methanol mixed solvent in the volume ratio of 8 to 1. The color development method comprises the following steps: taking 10 vol% ethanol sulfate solution as developer, heating at 105 deg.C for developing color, and comparing with target compound to obtain spots R_fThe values and colors are combined.

2. The compound sample structure identification method in the examples of the present application is as follows:

by means of hydrogen spectra¹H NMR and carbon Spectroscopy¹³C NMR analysis with Bruker DPX 400 nuclear magnetic resonance spectrometer and deuterated dimethyl sulfoxide (DMSO-d) as solvent₆Sigma) or deuterated chloroform (CDCl)₃,sigma)。

3. The purity of compound samples in the examples of this application was determined as follows:

the purity of each compound sample was determined by high performance liquid chromatography under the following chromatographic conditions:

the analytical chromatographic column is Zorbax Eclipse XDB-C18(Agilent, 4.6cm multiplied by 150cm), the methanol aqueous solution of mobile phase chromatography is eluted by gradient by 5-95%, the detection wavelength is 300nm in 0-30min, the purity of each compound is calculated by adopting a peak area normalization method, and the calculation formula is as follows:

in the formula A_iIs the peak area of the compound to be tested, A_{General assembly}Is the sum of the peak areas of the total chromatographic peaks within 5-30min of retention time. (Note that if there is no obvious impurity peak, the defined purity is more than or equal to 98%)

Example 1

The embodiment provides a method for extracting phenolic compounds from a tung tree, which specifically comprises the following steps:

(1) extracting dry branches of Jatropha curcas 7.6kg with 100L water under 100 deg.C under reflux for 3 times for 2 hr, mixing extractive solutions, concentrating under reduced pressure (55 deg.C and-0.09 MPa), and recovering solvent to obtain extract (226 g).

(2) Adding water into the extract obtained in the step (1) to disperse the extract until the relative density is 1.09 (the measurement temperature is 27 ℃) to obtain a water dispersion, adding 1 volume of petroleum ether to extract and degrease, collecting a water phase part, extracting for 5 times by using 1 volume of ethyl acetate, combining ethyl acetate parts, and concentrating under reduced pressure (the temperature is 55 ℃ and the pressure is-0.09 MPa) to obtain an ethyl acetate extract (83.3 g);

(3) subjecting the ethyl acetate extract obtained in the step (2) to polyamide column chromatography (the mass ratio of the ethyl acetate extract to polyamide filler is 1:80, the type is 60-100 meshes, the column height-diameter ratio is 10: 1), the pressure is 5-10 ba, the flow rate is 5ml/min, ethanol-water solution is used for gradient elution, 30 vol% ethanol is used for elution for 6 column volumes, 70 vol% ethanol water solution is used for elution for 5 column volumes, finally 95 vol% ethanol is used for washing the column, 70 vol% ethanol eluent is collected, and the solvent is recovered through reduced pressure concentration (the temperature is 55 ℃ and the pressure is-0.09 MPa) to obtain a crude product (16.9g) of the total polyphenols of the tung trees;

(4) performing C18 column chromatography (C18 is Merck Lichroprep RP-18, pore diameter) on the crude product of the total polyphenol of the idesia polycarpa obtained in the step (3)The particle size is 40-63 mu m, and the mass ratio of the column chromatography sample I to the C18 filler is 1:100, the height-diameter ratio of the C18 column is 20:1), performing gradient elution by using methanol-water solution according to the following procedures, wherein 30 vol%, 70 vol% and 95 vol% are performed, 6 column volumes are eluted in each gradient, 70 vol% methanol eluent is collected, and a solvent is recovered under reduced pressure to obtain a column chromatography sample I (8.9 g);

(5) performing Sephadex HP-20 gel column chromatography (the mass ratio of the sample I to a gel filler is 1:2000, and the height-diameter ratio of the gel column is 120:1) on 100mg of the column chromatography sample I obtained in the step (4) in the example 1, eluting with chloroform-methanol (1: 1) at the flow rate of 3-5 seconds/drop, collecting eluates in sections, collecting one part per 10ml, performing TLC detection after elution, and combining the eluates with the same components and performing reduced pressure concentration to obtain a target I section component, a target II section component and a target III section component;

(6) the fraction of the target stage I was purified by semi-preparative high performance liquid chromatography (Waters X-Bridge C18,10mm X250 mm, 10 μm) using an acetonitrile-0.1 wt% aqueous phosphoric acid system at a flow rate of 4ml/min, according to the following elution procedure (in volume%): 0.1 wt% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, isocratic elution: 22% of mobile phase B, and the balance of mobile phase A is complemented to 100% for 30 min; the detector is a DAD detector, the detection wavelength is 300nm, chromatographic peak eluents (chromatogram shown in figure 2) of 20.8min and 25.4min are respectively collected, and the compound 1(11.8mg, purity 98%) and the compound 2(8.6mg, purity more than or equal to 98%) are obtained by reduced pressure concentration and drying;

separating and purifying target II-stage component by semi-preparative high performance liquid chromatography, collecting chromatographic peak eluents (chromatogram shown in figure 3) of 23.8min and 25.6min respectively under the same chromatographic conditions as the target I-stage component, concentrating under reduced pressure, and drying to obtain compound 4(10.5mg, purity greater than or equal to 98%) and compound 5(16.5mg, purity greater than or equal to 98%), wherein the purity is determined by peak area normalization method;

dissolving the target group III with methanol, placing into colorless transparent hard borosilicate bottle, sealing with sealing film, pricking 5 holes with 0.5mm capillary at the sealing position, placing in 4 deg.C refrigerator, and slowly volatilizing solvent to obtain compound 3 crystal (13.6mg, purity greater than or equal to 98%).

Structural characterization of compounds 1-5:

compound (1): a white powder of a white color, a white powder,¹H NMR(400MHz,DMSO-d₆)δ_H:9.20(1H,br s,OH-4′),7.97(1H,d,J＝9.6Hz,H-4),6.84(1H,s,H-5),7.03(1H,d,J＝1.6Hz,H-2′),6.88(1H,dd,J＝8.4,1.6Hz,H-6′),6.84(1H,s,H-5),6.83(1H,d,J＝8.4Hz,H-5′),6.34(1H,d,J＝9.6Hz,H-3),5.10(1H,br s,OH-9′),4.99(1H,d,J＝8.0Hz,H-7′),4.25(1H,m,H-8′),3.82(3H,s,OCH₃-6),3.81(3H,s,OCH₃-3′),3.45(1H,m,H-9′a),3.78(1H,m,H-9′b),0.89(3H,t,J＝6.8Hz,H-10).¹³C-NMR(100MHz,DMSO-d₆)δ_C:160.0(C-2),113.2(C-3),144.8(C-4),100.7(C-5),145.2(C-6),137.0(C-7),131.7(C-8),138.0(C-9),111.2(C-10),126.2(C-1′),112.0(C-2′),147.6(C-3′),147.2(C-4′),115.3(C-5′),120.8(C-6′),76.2(C-7′),77.8(C-8′),59.8(C-9′),55.7(OCH₃-6),55.8(OCH₃-5'). The above data and literature (Kan S, Chen G Y, Han C R, et al chemical compositions from the roots of Xanthium sibiricum [ J ]]Natural Product Research,2011,25(13): 1243-,¹h NMR and¹³the C NMR spectrum is shown in FIGS. 4-5.

Compound (2): a white powder of a white color, a white powder,¹H NMR(400MHz,DMSO-d₆)δ_H:8.50(1H,s,OH-4′),7.88(1H,d,J＝9.6Hz,H-4),6.74(2H,s,H-2′,6′),6.81(1H,s,H-5),6.28(1H,d,J＝9.6Hz,H-3),5.12(1H,br s,OH-9′),5.00(1H,d,J＝8.0Hz,H-7′),4.25(1H,m,H-8′),3.83(3H,s,OCH₃-6),3.82(6H,s,OCH₃-3′,5′),3.78(1H,m,H-9′b),3.45(1H,m,H-9′a),3.51(1H,s,).¹³C-NMR(100MHz,DMSO-d₆)δ_C:160.0(C-2),114.0(C-3),144.8(C-4),100.7(C-5),145.2(C-6,4′),137.0(C-7),131.6(C-8),138.0(C-9),111.2(C-10),125.7(C-1′),105.6(C-2′,6′),147.9(C-3′,5′),136.2(C-4′),76.5(C-7′),77.8(C-8′),59.8(C-9′),55.8(OCH₃-6),56.4(OCH₃-3',5'). The above data and literature (Ray AB, Chattopthdhyay S K.Structure of Cleomis sin A, a corparinolignoid of Cleome viscose seeds [ J ]]Tetrahedron Letters,1980,21: 4477-4480.) the control was identical, so that compound (2) was identified as Cleomis cosin A, the formula is shown in FIGS. 1(2),¹h NMR and¹³the C NMR spectrum is shown in FIGS. 6 to 7.

Compound (3): a white powder of a white color, a white powder,¹H NMR(400MHz,CDCl₃)δ_H:3.95(3H,s,7-OCH₃),6.27(1H,d,J＝9.5Hz,H-3),6.85(1H,s,H-8),6.92(1H,s,H-5),7.61(1H,d,J＝9.5Hz,H-4).¹³C-NMR(100MHz,CDCl₃)δ_C:161.5(C-2),113.4(C-3),143.3(C-4),107.5(C-5),149.7(C-6),150.3(C-7),103.2(C-8),144.0(C-9),111.5(C-10),56.4(6-OCH₃) The above data and literature (Wang Z Y, He W J, Zhou W B, et al. two new phenyl propanoids from Micromelum integerrimum [ J ]].Chinese Journal of Natural Medicines,2014,12(8):619 and 622) consistent, compound (3) was identified as scopoletin (scopoletin), and the structural formula is shown in FIGS. 1(3),¹h NMR and¹³the C NMR spectrum is shown in FIGS. 8 to 9.

Compound (4): a colorless and transparent oily substance is obtained,¹H NMR(400MHz,CDCl₃)δ_H:6.82(1H,dt,J＝16.0,8.0Hz,H-4),6.81(1H,d,J＝8.0Hz,H-5′),6.70(1H,d,J＝2.0Hz,H-2′),6.68(1H,dd,J＝2.0,8.0Hz,H-6′),6.09(1H,dt,J＝16.0,1.6Hz,H-5),3.84(3H,s,-OCH₃),2.82(2H,m,H-1),2.72(2H,m,H-2),2.53(2H,m,H-4),1.23～1.56(8H,m,H-6,H-7,H-8,H-9),0.89(3H,t,J＝6.8Hz,H-10).¹³c-and DEPT NMR (100MHz, CDCl)₃)δ_C:29.9(C-1),42.0(C-2),199.9(C-3),130.3(C-4),147.9(C-5),32.5(C-6),23.8(C-7),31.3(C-8),22.4(C-9),14.0(C-10),133.2(C-1′),111.1(C-2′),146.4(C-3′),143.9(C-4′),114.3(C-5′),120.8(C-6′),55.9(-OCH₃) Study of chemical composition of ginger [ J ] on the above data and literature (Bayu, Deng Anyu, Li Shihong, etc.)]The comparison of Chinese traditional medicine journal 2010, 35(5):598-,¹H-¹All NMR signals are analyzed in detail and completely attributed by 1D and 2D NMR technologies such as H COSY, HSQC, HMBC and the like, so that the compound (4) is identified as 6-gingerol, the structural formula is shown in figure 1(4),¹h NMR and¹³the C NMR spectrum is shown in FIGS. 10-11.

The compound (5) is a colorless transparent oily substance,¹H NMR(400MHz,CDCl₃)δ_H:6.81(1H,d,J＝8.0Hz,H-5′),6.70(1H,d,J＝2.0Hz,H-2′),6.68(1H,dd,J＝2.0,8.0Hz,H-6′),3.86(3H,s,-OCH₃),2.81～2.89(4H,m,H-1,H-2),2.19(2H,dt,J＝7.2,1.4Hz,H-6),1.44(2H,m,H-4),1.23～1.36(4H,m,H-8,H-9),0.89(3H,t,J＝6.8Hz,H-10).¹³C-NMR(100MHz,CDCl₃)δ_C:36.4(C-1),31.7(C-2),211.5(C-3),49.3(C-4),67.7(C-5),45.4(C-6),25.1(C-7),29.2(C-8),22.6(C-9),14.0(C-10),132.6(C-1′),111.1(C-2′),146.6(C-3′),144.0(C-4′),114.5(C-5′),120.7(C-6′),55.8(-OCH₃). The above data and literature (Sun Fengjiao, Li Zheng Lin, Qianshui, etc.. research on chemical composition of dried ginger [ J)]The Chinese wild plant resource, 2016,35(5):20-24,60) was identical to the control and was open toPassing through DEPT,¹H-¹All NMR signals of H COSY, HSQC, HMBC and other 1D and 2D NMR technologies are analyzed in detail and completely attributed, so that the compound (5) is identified as 6-shogaol, the structural formula is shown in the figure 1(5),¹h NMR and¹³the C NMR spectrum is shown in FIGS. 12-13.

Example 2

The embodiment provides a method for extracting phenolic compounds from a tung tree, which specifically comprises the following steps:

(1) 8.0kg of dried branches of the idesia polycarpa, adding 105L of 70 vol% methanol aqueous solution for ultrasonic-assisted hot reflux extraction, extracting for 3 times at the ultrasonic power of 20KHz and the extraction temperature of 85 ℃, combining the extracting solutions, concentrating under reduced pressure (the temperature is 60 ℃ and the pressure is-0.08 MPa), and recovering the solvent to obtain 290.5g of extract.

(2) Adding water into the extract obtained in the step (1) to disperse the extract until the relative density is 1.13 (the measurement temperature is 26 ℃) to obtain water dispersion, adding 1-time volume of petroleum ether to extract and degrease, collecting a water phase part, extracting by using 2-time volume of ethyl acetate for 3 times, combining ethyl acetate parts, and concentrating under reduced pressure (the temperature is 60 ℃ and the pressure is-0.085 MPa) to obtain an ethyl acetate extract (89.9 g);

(3) subjecting the ethyl acetate extract obtained in the step (2) to polyamide column chromatography (the mass ratio of the ethyl acetate extract to polyamide filler is 1:60, the type is 60-100 meshes, the column height-diameter ratio is 8: 1), the pressure is 5-15 ba, the flow rate is 15ml/min, ethanol-water solution is used for gradient elution, 25 vol% ethanol is used for eluting 5 column volumes, 65 vol% ethanol water solution is used for eluting 6 column volumes, finally 95 vol% ethanol is used for washing the column, 65 vol% ethanol eluent is collected, and the solvent is recovered through reduced pressure concentration (the temperature is 60 ℃ and the pressure is-0.085 MPa) to obtain a crude product of the total polyphenols of the idesia polycarpa;

(4) performing C18 column chromatography (C18 is Merck Lichroprep RP-18, pore diameter) on the crude product of the total polyphenol of the idesia polycarpa obtained in the step (3)The particle size is 40-63 mu m, and the mass ratio of the column chromatography sample I to the C18 filler is 1: 150 said C18 column having an aspect ratio of 35:1), eluting with methanol-water solution according to the following procedure gradient, 25 vol%, 65 vol%, per stepEluting by 6 column volumes, finally washing the column by 100 vol% methanol, collecting 65 vol% methanol eluent, and recovering the solvent under reduced pressure to obtain a column chromatography sample I (9.2 g);

(5) performing SephadexG-10 gel column chromatography (the mass ratio of the column chromatography sample I to a gel filler is 1:1700, and the height-diameter ratio is 80:1) on 100mg of the column chromatography sample I obtained in the step (4), eluting with chloroform methanol (1: 1) at the flow rate of 3-5 seconds/drop, collecting each 15ml of eluent in sections, detecting by TLC after elution, and combining the eluents with the same components and concentrating under reduced pressure to obtain a target I section component, a target II section component and a target III section component;

(6) the target fraction I was purified by semi-preparative high performance liquid chromatography (Waters Sunfire C18,10 mm. times.250 mm, 10 μm) using an acetonitrile-0.1 wt% aqueous phosphoric acid system at a flow rate of 5ml/min, according to the following elution procedure (in volume%): 0.1 wt% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, isocratic elution: 20% of mobile phase B, and the balance of mobile phase A is complemented to 100% for 30 min; the detector is a DAD detector, the detection wavelength is 300nm, chromatographic peak eluents of 19.6min and 25.8min are respectively collected, and the compound 1(11.3mg, the purity is more than or equal to 98%) and the compound 2(9.0mg, the purity is more than or equal to 98%) are obtained by decompression, concentration and drying;

separating and purifying the target II-stage component by a semi-preparative high performance liquid chromatography, collecting chromatographic peak eluates of 23.5min and 26.3min respectively under the same chromatographic conditions as the target I-stage component, and concentrating and drying under reduced pressure to obtain a compound 4(9.8mg, the purity is more than or equal to 98%) and a compound 5(14.2g, the purity is more than or equal to 98%);

the target fraction III was treated as in example 1 to give Compound 3(11.9mg, purity. gtoreq.98%).

Determined, of Compounds 1-5¹H NMR and¹³the C NMR data agree with those of example 1.

Example 3

The embodiment provides a method for extracting phenolic compounds from a tung tree, which specifically comprises the following steps:

(1) extracting dry branches of idesia polycarpa 8.0kg with 65L 75% ethanol under ultrasonic condition for 4 times at 30KHz for 2 hr, mixing extractive solutions, concentrating under reduced pressure (60 deg.C and-0.095 MPa), and recovering solvent to obtain 269.4g extract.

(2) Adding water into the extract obtained in the step (1) to disperse until the relative density is 1.15 (the measurement temperature is 26 ℃) to obtain water dispersion, adding 1 time volume of petroleum ether to extract and degrease, collecting a water phase part, extracting by using 3 times volume of ethyl acetate for 1 time, combining ethyl acetate parts, and concentrating under reduced pressure (the temperature is 60 ℃ and the pressure is-0.090 MPa) to obtain ethyl acetate extract (73.2 g);

(3) subjecting the ethyl acetate extract obtained in the step (2) to polyamide column chromatography (the mass ratio of the ethyl acetate extract to polyamide filler is 1:100, the type is 60-100 meshes, the column height-diameter ratio is 10: 1), the pressure is 5-15 ba, the flow rate is 10ml/min, ethanol-water solution is used for gradient elution, firstly 28 vol% ethanol is used for eluting 8 column volumes, then 60 vol% ethanol is used for eluting 3 column volumes, finally 95% ethanol is used for washing the column, 60 vol% ethanol eluent is collected, and the solvent is recovered through reduced pressure concentration (the temperature is 60 ℃ and the pressure is-0.090 MPa) to obtain a tung tree total polyphenol crude product (15.3 g);

(4) performing C18 column chromatography (C18 is Merck Lichroprep RP-18, pore diameter) on the crude product of the total polyphenol of the idesia polycarpa obtained in the step (3)The particle size is 40-63 mu m, and the mass ratio of the column chromatography sample I to the C18 filler is 1: 150, the aspect ratio of the C18 column is 40:1), performing gradient elution with methanol-water solution according to the following procedure, performing gradient elution with 28 vol%, 60 vol% and 95 vol% methanol, each gradient elution having 6 column volumes, collecting the 60 vol% methanol eluate, and recovering the solvent under reduced pressure to obtain column chromatography sample I (7.39 g);

(5) and (3) performing SephadexG-25 gel column chromatography on the column chromatography sample I obtained in the step (4) by taking 100mg (the mass ratio of the sample I to the gel filler is 1: 1500, and the height-diameter ratio is 60:1), eluting with chloroform-methanol (1: 1) at the flow rate of 3-5 seconds/drop, collecting the eluent in sections, detecting by TLC (thin layer chromatography) after eluting every 8min, and combining the eluents with the same components and concentrating under reduced pressure to obtain a target I section component, a target II section component and a target III section component.

(6) The fraction of the target stage I was purified by semi-preparative high performance liquid chromatography (Waters X-Bridge C18,10mm X250 mm, 10 μm) using an acetonitrile-0.1 wt% aqueous phosphoric acid system at a flow rate of 3ml/min, according to the following elution procedure (in volume%): 0.1 wt% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, isocratic elution: the balance of 24% of mobile phase B and 3 is complemented to 100% by the mobile phase A for 30 min; the detector is a DAD detector, the detection wavelength is 300nm, chromatographic peak eluents of 21.1min and 26.2min are respectively collected, and the compound 1(10.7mg, the purity is more than or equal to 98%) and the compound 2(8.3mg, the purity is more than or equal to 98%) are obtained by decompression, concentration and drying;

separating and purifying the target II-stage component by a semi-preparative high performance liquid chromatography, collecting chromatographic peak eluates of 23.6min and 25.1min respectively under the same chromatographic conditions as the target I-stage component, and concentrating and drying under reduced pressure to obtain a compound 4(8.2mg, the purity is more than or equal to 98%) and a compound 5(14.9g, the purity is more than or equal to 98%);

the target fraction III was treated as in example 1 to give compound 3(10.1 mg; purity. gtoreq.98%)).

Determined, of Compounds 1-5¹H NMR and¹³the C NMR data agree with those of example 1.

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.