Method for extracting apigenin from leaves of siberian cocklebur

Abstract

The invention discloses a method for extracting apigenin from leaves of siberian cocklebur. The extraction method comprises the following steps: (1) digesting the leaves of pseudoxanthium sibiricum by using an extracting agent to obtain crude extract of the active substances of the leaves of pseudoxanthium sibiricum; (2) suspending the crude extract of the active substance of the leaves of the siberian cocklebur and water to obtain a suspension, performing gradient extraction on the suspension by using petroleum ether and ethyl acetate in sequence, and collecting an ethyl acetate extract; (3) carrying out separation and purification on the ethyl acetate extract by using a silica gel chromatographic column to obtain a crude product of coronastachys sieboldii; and (4) purifying the crude product. According to the method, extraction solvents are petroleum ether, dichloromethane and ethyl acetate, the solvent residual quantity is small, environment friendliness is achieved, waste liquid generated in the extraction process can be recycled, the environmental protection load and the extraction cost are reduced, petroleum ether and ethyl acetate are used for gradient extraction, silica gel column chromatography is combined, a high-purity product is obtained, and the method is suitable for industrial production. The method has the advantages of simple process flow, environment friendliness, large-scale application and the like.

Description

Technical Field

[0001] The present invention relates to a method for extracting plant active substances, and particularly to a method for extracting coronopilin from Xanthium strumarium leaves, belonging to the field of extraction of plant active substances. Background Art

[0002] Coronopilin is a sesquiterpene lactone compound, and its chemical structural formula is shown as formula (I):

[0003]

[0004] Coronopilin mainly comes from Compositae plants and has potential biological activities such as anti-inflammatory, antibacterial and anti-cancer. The existing materials for extracting coronopilin are mainly Ambrosia artemisiifolia or Parthenium hysterophorus of Compositae, and their contents are relatively low.

[0005] The existing production processes mainly include solvent extraction method, supercritical extraction method, resin method and chemical synthesis method, etc. The solvent extraction method is simple, but there are also many impurities extracted. The supercritical extraction method requires a large investment in equipment, and the current industrialization level is low. The resin adsorption method has a complex process and a low yield. The chemical synthesis method has high equipment energy consumption and high cost investment.

[0006] At present, there is no relevant research on extracting coronopilin from Xanthium strumarium leaves, and there is no extraction method that can extract high-purity coronopilin. Summary of the Invention

[0007] The main object of the present invention is to provide a method for extracting coronopilin from Xanthium strumarium leaves, which has the advantages of simple process flow, environmental friendliness and high product purity.

[0008] To achieve the above object, the technical solutions adopted by the present invention include:

[0009] A method for extracting coronopilin from Xanthium strumarium leaves, comprising:

[0010] (1) Extracting Xanthium strumarium leaves with an extractant to obtain a crude extract of Xanthium strumarium leaf active substances; (2) Suspending the crude extract of Xanthium strumarium leaf active substances with water to obtain a suspension, and subjecting the suspension to gradient extraction with petroleum ether and ethyl acetate in sequence, and collecting the ethyl acetate extract paste; (3) Separating and purifying the ethyl acetate extract paste with a silica gel chromatography column to obtain a crude product of coronopilin; (4) Purifying the crude product of coronopilin to obtain the product.

[0011] In a preferred specific embodiment of the present invention, in step (1), the extraction of Xanthium italicum Moretti leaves with an extractant is carried out by drying and pulverizing the Xanthium italicum Moretti leaves, sieving to obtain Xanthium italicum Moretti leaf powder, and then mixing it with an extraction solvent; wherein, the drying is preferably carried out by drying in the shade; the sieving is carried out using a 40-mesh sieve.

[0012] In a preferred specific embodiment of the present invention, the extractant in step (1) is absolute ethanol; wherein, calculated by g:mL, the ratio of Xanthium italicum Moretti leaves to the extractant is preferably 1:(10 - 50); more preferably, the ratio of Xanthium italicum Moretti leaf powder to the extractant is 1:25.

[0013] In a preferred specific implementation method of the present invention, the method for extracting the crude extract of Xanthium italicum Moretti leaf active substances by extracting Xanthium italicum Moretti leaves with an extractant in step (1) includes: mixing Xanthium italicum Moretti leaves with an extractant and then extracting to obtain an extraction solution; extracting the extraction solution under ultrasonic conditions to obtain an extract; removing impurities from the extract and then evaporating the extractant to obtain the crude extract of Xanthium italicum Moretti leaf active substances.

[0014] In a preferred specific implementation method of the present invention, the extraction of the extraction solution under ultrasonic conditions to obtain an extract is carried out by placing it in an ultrasonic water bath for ultrasonic extraction to obtain an extract; wherein, the power of ultrasonic extraction is preferably 300 W, and the temperature in the ultrasonic water bath is preferably 30 °C.

[0015] In a preferred specific implementation method of the present invention, the impurity removal is preferably carried out by suction filtration with a circulating water vacuum pump.

[0016] In a preferred specific implementation method of the present invention, the method for evaporating the extractant is preferably to evaporate the extractant in a rotary evaporator to obtain the crude extract of Xanthium italicum Moretti leaf active substances.

[0017] In a preferred specific implementation method of the present invention, calculated by g:mL, in step (2), the crude extract of Xanthium italicum Moretti leaf active substances and water are suspended in a ratio of 1:(5 - 20) to obtain a mixed solution; preferably, the crude extract of Xanthium italicum Moretti leaf active substances and water are suspended in a ratio of 1:10 to obtain a mixed solution.

[0018] In a preferred specific implementation method of the present invention, when the suspension is successively extracted with petroleum ether and ethyl acetate in step (2), the volume ratio of the suspension to petroleum ether or ethyl acetate is 1:1.

[0019] In a preferred specific implementation method of the present invention, the number of extraction times is 1 - 20 times, preferably 10 times.

[0020] A preferred specific implementation method of the present invention. The method for separating and purifying the ethyl acetate extract paste with a silica gel chromatography column in step (3) includes: dissolving the ethyl acetate extract paste with dichloromethane and then loading it onto the chromatography column by the dry method.

[0021] A preferred specific implementation method of the present invention. The method for obtaining the crude coronopilin by separating and purifying the ethyl acetate extract paste with a silica gel chromatography column in step (3) includes: selecting a chromatography column for column packing, mixing silica gel with the ethyl acetate extract paste and then loading it onto the chromatography column, using a dichloromethane-ethyl acetate gradient elution system or a petroleum ether-ethyl acetate gradient elution system for gradient elution, and using TLC to qualitatively and quantitatively analyze the purity of each elution component.

[0022] A preferred specific implementation method of the present invention. The chromatography column is preferably a glass chromatography column; more preferably, the glass chromatography column is packed with 200-300 mesh silica gel, and 100-300 mesh silica gel is mixed with the ethyl acetate extract paste and then loaded onto the column.

[0023] A preferred specific implementation method of the present invention. The height-to-diameter ratio of the glass chromatography column is 5:1-10:1.

[0024] A preferred specific implementation method of the present invention. By volume, the gradient ratio of dichloromethane to ethyl acetate in the dichloromethane-ethyl acetate elution system is as follows: 15:1, 12:1, 10:1, 9:1, 8:2, 7:3.

[0025] A preferred specific implementation method of the present invention. By volume, the gradient ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate elution system is as follows: 9:1, 8:2, 7:3, 6:4, 5.5:4.5.

[0026] A preferred specific implementation method of the present invention. The purification method in step (4) includes: eluting the crude coronopilin with a new silica gel column to remove impurities and then performing vacuum concentration; heating the concentrated solution in a water bath and then standing and cooling to crystallize to obtain the pure product.

[0027] A preferred specific implementation method of the present invention. After eluting the crude coronopilin with a new silica gel column to remove impurities, vacuum concentration is carried out using a rotary evaporator to obtain a concentrated solution; the concentrated solution is heated in a water bath at a temperature of 40°C - 50°C and then placed at 4°C to stand and cool to crystallize, the liquid and impurities are removed by suction filtration, and after drying, it is placed in a low-temperature oven together with the filter paper to dry and separate the product from the filter paper; the crystallization time is 4-10 days; preferably, the crystallization time is 7 days.

[0028] The invention provides a method for extracting and separating gynogynocyanine from pseudo-Xanthium sibiricum leaves. The extraction solvent in the invention is petroleum ether, dichloromethane and ethyl acetate, the solvent residue is small and environmentally friendly, and the waste liquid generated in the extraction process can be recycled, which reduces the environmental load and the extraction cost. Gradient extraction with petroleum ether and ethyl acetate is combined with silica gel column chromatography to obtain a high-purity product, and gynogynocyanine with a purity of more than 98% is obtained by reduced pressure concentration and water bath heating followed by cooling and crystallization. The extraction method of the invention has the advantages of simple process flow, environmental friendliness, high product purity, etc., and has application prospects in large-scale extraction and application of gynogynocyanine. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The purity HPLC detection spectrum of gymnospermum officinale obtained in Example 1 of the present invention;

[0030] Figure 2 This is the nuclear magnetic resonance detection spectrum of gymnospermum officinale obtained in Example 1 of the present invention;

[0031] Figure 3 This is the HPLC detection spectrum of the purity of gymnospermum officinale obtained in Example 2 of the present invention;

[0032] Figure 4 This is the nuclear magnetic resonance detection spectrum of the crown gymnospermum in Example 2 of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the scope of protection of the present invention.

[0034] Example 1 Extraction of Gymnoglossum glomerulone

[0035] (1) Taking Xanthium sibiricum leaf raw material, drying and crushing it, and then sieving it to obtain Xanthium sibiricum leaf powder.

[0036] (2) 125 g of Xanthium sibiricum leaf powder was thoroughly mixed with anhydrous ethanol at a solid-liquid ratio of 1 g:25 mL, and the extract was extracted at room temperature for 48 h. The extract was then placed in an ultrasonic water bath for ultrasonic extraction for 30 min, and filtered out impurities with a circulating water vacuum pump. The extraction was repeated three times, and the filtrate was transferred to a rotary evaporator to evaporate the extractant, concentrated and dried until there was no alcohol taste, to obtain a crude extract of the active substance of Xanthium sibiricum leaf.

[0037] (3) The crude extract of the active substances from Iva xanthifolia leaves was suspended with water at a ratio of 1 g:10 mL. The suspension was successively extracted 10 times with petroleum ether and ethyl acetate (1:1, V / V). The organic solvents were recovered by concentration under reduced pressure to obtain 5 g of the ethyl acetate extract paste.

[0038] (4) The 5 g of the ethyl acetate extract paste was separated and purified by silica gel chromatography column. The sample was mixed with silica gel of 100 - 200 mesh. A glass chromatography column with a diameter of 5 cm was selected, and the column was filled with silica gel of 200 - 300 mesh. Anhydrous sodium sulfate was used as the buffer layer. A gradient elution was carried out using a dichloromethane - ethyl acetate system (15:1, 12:1, 10:1, 9:1, 8:2, 7:3, V / V). TLC was used to qualitatively and purity - detect and analyze each eluted fraction.

[0039] (5) The target component was eluted with a new silica gel column to remove impurities, and concentrated under reduced pressure using a rotary evaporator. The concentrated solution was heated in a water bath, left to stand and cool at 4°C for one week, filtered by suction through a Buchner funnel to remove the liquid and impurities. After suction - drying, it was placed in a low - temperature oven together with the filter paper and dried to separate the product from the filter paper.

[0040] In this example, 1.2 g of ambrosin was obtained, with an extraction rate of 0.96% and a purity of over 98%.

[0041] The HPLC detection spectrum and nuclear magnetic resonance detection spectrum of the ambrosin extracted in this example are respectively as Figure 1 or Figure 2 shown.

[0042] Example 2 Extraction of ambrosin

[0043] The difference from Example 1 is that: in step (2), the Iva xanthifolia leaf powder is 600 g; in step (3), 30 g of the ethyl acetate extract paste is obtained; in step (4), the 30 g of the ethyl acetate extract paste is separated and purified by silica gel chromatography column. The sample is mixed with silica gel of 200 - 300 mesh. A glass chromatography column with a diameter of 10 cm is selected, and a gradient elution is carried out using a petroleum ether - ethyl acetate system (9:1, 8:2, 7:3, 6:4, 5.5:4.5 V / V); the rest are the same as in Example 1.

[0044] In this example, 6.6 g of ambrosin was obtained, with an extraction rate of 1.1% and a purity of over 98%.

[0045] The HPLC detection spectrum and nuclear magnetic resonance detection spectrum of the ambrosin extracted in this example are respectively as Figure 3 or Figure 4 shown.

[0046] Example 3 Extraction of ambrosin

[0047] The difference from Example 1 is that: in step (2), the material-liquid ratio of the false Xanthium leaves powder to absolute ethanol is 1 g: 10 mL; in step (3), the crude extract of the active substances from the false Xanthium leaves is suspended with water at a ratio of 1 g: 5 mL; the rest are the same as in Example 1.

[0048] This example obtained 1.1 g of ambrosin, with an extraction rate of 0.88% and a purity of over 98%.

[0049] Extraction of ambrosin in Example 4

[0050] The difference from Example 1 is that: in step (2), the material-liquid ratio of the false Xanthium leaves powder to absolute ethanol is 1 g: 50 mL; in step (3), the crude extract of the active substances from the false Xanthium leaves is suspended with water at a ratio of 1 g: 20 mL; the rest are the same as in Example 1.

[0051] This example obtained 1.3 g of ambrosin, with an extraction rate of 1.04% and a purity of over 98%.

[0052] In Experimental Examples 1 - 4, the content of ambrosin in the dry matter is 0.88 - 1.1%, and the purity of the ambrosin product can reach over 98%. Therefore, the method provided by the present invention can extract high-purity ambrosin from false Xanthium leaves, and the process flow of the method provided by the present invention is simple and the cost is low.

Claims

1. A method for extracting gynoglossum from Xanthium sibiricum leaves, characterized in that: include: (1) extracting the Xanthium sibiricum leaves with an extractant to obtain a crude extract of active substances from the Xanthium sibiricum leaves; (2) suspending the crude extract of active substances from the leaves of Xanthium sibiricum with water to obtain a suspension, extracting the suspension with petroleum ether and ethyl acetate in turn, and collecting the ethyl acetate extract; (3) separating and purifying the ethyl acetate extract with a silica gel chromatography column to obtain a crude product of gymnospermum officinale; and (4) purifying the crude product of gymnospermum officinale to obtain the product.

2. The method according to claim 1, characterized in that The step (1) of extracting the Xanthium sibiricum leaves with an extractant comprises drying the Xanthium sibiricum leaves, crushing them, sieving them to obtain Xanthium sibiricum leaf powder, and then mixing it with the extraction solvent; wherein the drying is preferably performed in the shade; and the sieving is performed using a 40-mesh sieve.

3. The method according to claim 1, characterized in that The extractant in step (1) is anhydrous ethanol; wherein, in terms of g:mL, the ratio of Xanthium sibiricum leaves to the extractant is preferably 1:(10-50); more preferably, the ratio of Xanthium sibiricum leaf powder to the extractant is 1:

25.

4. The method according to claim 1, characterized in that: The method of extracting the Xanthium sibiricum leaves with an extractant to obtain a crude extract of the Xanthium sibiricum leaves active substance in step (1) comprises: mixing the Xanthium sibiricum leaves with the extractant and then leaching to obtain an extract; extracting the extract under ultrasonic conditions to obtain an extract; and removing impurities from the extract and evaporating the extractant to obtain a crude extract of the Xanthium sibiricum leaves active substance.

5. The method according to claim 4, characterized in that The extracting of the extract under ultrasonic conditions to obtain the extract is performed by placing the extract in an ultrasonic water bath for ultrasonic extraction to obtain the extract; wherein the power of the ultrasonic extraction is preferably 300W, and the temperature in the ultrasonic water bath is preferably 30°C; the impurity removal is preferably performed by filtering and removing impurities using a circulating water vacuum pump; and the method of evaporating the extractant is preferably performed by evaporating the extractant in a rotary evaporator to obtain a crude extract of the active substance of the pseudo-Xanthium sibiricum leaves.

6. The method according to claim 1, characterized in that In terms of g:mL, in step (2), the crude extract of the active substance of the leaves of Xanthium sibiricum and water are suspended in a ratio of 1:(5-20) to obtain a mixed solution; preferably, the crude extract of the active substance of the leaves of Xanthium sibiricum and water are suspended in a ratio of 1:10 to obtain a mixed solution; In step (2), when the suspension is extracted with petroleum ether and ethyl acetate in sequence, the volume ratio of the suspension to petroleum ether or ethyl acetate is 1:1; the number of extractions is 1-20 times, preferably 10 times.

7. The method according to claim 1, characterized in that The method for separating and purifying the ethyl acetate extract using a silica gel chromatography column in step (3) comprises: dissolving the ethyl acetate extract using dichloromethane and then applying the solution to the chromatography column by a dry method.

8. The method according to claim 1, characterized in that The step (3) of separating and purifying the ethyl acetate extract using a silica gel chromatography column to obtain a crude product of gymnospermum officinale comprises: selecting a chromatography column, mixing the silica gel and the ethyl acetate extract and applying the mixture to the chromatography column, performing gradient elution using a petroleum ether-ethyl acetate gradient elution system or a dichloromethane-ethyl acetate gradient elution system, and performing qualitative and purity detection and analysis of each eluted component using TLC; Preferably, the chromatography column is a glass chromatography column; more preferably, the glass chromatography column is filled with 200-300 mesh silica gel; 100-300 mesh silica gel is mixed with the ethyl acetate extract and then applied to the chromatography column; Preferably, the height-to-diameter ratio of the glass chromatography column is 5:1-10:1; Preferably, based on volume ratio, the gradient ratio of dichloromethane to ethyl acetate in the dichloromethane-ethyl acetate gradient elution system is as follows: 15:1, 12:1, 10:1, 9:1, 8:2, 7:3; the gradient ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate gradient elution system is as follows: 9:1, 8:2, 7:3, 6:4, 5.5:4.

5.

9. The method according to claim 1, characterized in that: The purification method described in step (4) comprises: eluting the crude product of crown-gymnostomycin with a new silica gel column to remove impurities and then concentrating under reduced pressure; heating the concentrate in a water bath and then standing to cool and crystallize to obtain a pure product.

10. The method according to claim 9, characterized in that The crude product of crown-studded ambrosia glutinosin is eluted with a new silica gel column to remove impurities, and then concentrated under reduced pressure using a rotary evaporator to obtain a concentrated solution; the concentrated solution is heated in a water bath at a temperature of 40°C-50°C, and then allowed to stand at 4°C for cooling and crystallization, and the liquid and impurities are removed after filtration. After drying, the concentrate is placed in a low-temperature oven together with the filter paper to separate the product from the filter paper; the crystallization time is 4-10 days; preferably, the crystallization time is 7 days.

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