Method for extracting effective active ingredients from euonymus

Abstract

The application discloses a method for extracting effective active components from Radix Cinnamomi, which comprises the following steps: firstly, extracting Radix Cinnamomi with ethanol; then, extracting the ethanol extract with petroleum ether, ethyl acetate and n-butanol respectively; detecting whether there are obvious peaks by TLC; collecting different fractions by elution and washing of the ethyl acetate extract with obvious peaks through an alumina column; and obtaining target products by crystallization and drying treatment of the fractions, and then identifying the target products by infrared, hydrogen spectrum, carbon spectrum, mass spectrum and ultraviolet combined detection, wherein the target products are identified as monomer compounds of berberine hydrochloride, jatrorrhizine hydrochloride and palmatine hydrochloride. The method can further separate monomer compounds of Radix Cinnamomi, can better control the quality standard of Radix Cinnamomi, can lay a good foundation for further research on the pharmacological action of Radix Cinnamomi, and can lay a foundation for the research on new drugs with antitumor, antioxidant, immunoregulatory activity and anti-inflammatory effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant active ingredient extraction, and specifically relates to a method for extracting effective active ingredients from Mahoniae Caulis. BACKGROUND

[0002] Mahoniae Caulis is first recorded in the Plant Name and Actual Figure, and is derived from the dried stems of Mahonia bealaei (Fort.) Carr. and Mahonia fortunei (Lindl.) Fedde of Berberidaceae, and mainly distributed in the southwest region (Sichuan, Yunnan, Guizhou, Guangxi and eastern Tibet). Mahoniae Caulis has various pharmacological activities, such as antioxidant, anti-inflammatory, antibacterial, analgesic, antidiarrheal, liver-protecting and anti-tumor, acetylcholinesterase inhibitory activity, and blood lipid-lowering effect. Clinically, it is mainly used for treating breast cancer, breast duct dilatation with inflammation, acne, chronic hepatitis B, chronic pharyngitis, chronic tonsillitis, acute eczema and other diseases, and has the effects of clearing heat and drying dampness, purging fire and detoxification. Studies have found that Mahoniae Caulis contains various effective components, such as alkaloids, volatile oils, flavonoids, polysaccharides and the like, and the alkaloid components are currently the most studied. In the extraction of Mahoniae Caulis components, the reflux extraction method and the ultrasonic-assisted method are commonly used.

[0003] For example, Chinese patent 201610133474.2 discloses a method for separating alkaloids from Mahoniae Caulis by high-speed counter-current chromatography. The separation conditions are as follows: the solvent system is chloroform:methanol:0.5 mol.L -1 HCl solution = 3.5-4.5:1-2:1.5-2.5, the column volume of the high-speed counter-current chromatograph is 200-400, the loading amount is 200-500, the rotation speed is 300-1000 rpm, the upper phase is the stationary phase, the lower phase is the mobile phase, the flow rate is 1.5-2.5 mL / min, and the detection wavelength is 250-260 nm.

[0004] For example, Chinese patent 201710136039.X discloses a method for extracting alkaloids from Mahoniae Caulis based on the response surface method. The cellulase enzymolysis method is used to extract Mahoniae Caulis, and the cellulase dosage, enzymolysis time, enzymolysis temperature, and enzymolysis pH value and other process parameters in the extraction process are reasonably determined and selected, so that the yield of total alkaloids of Mahoniae Caulis reaches 8.4% or more, and the total alkaloid extraction cost of Mahoniae Caulis is effectively reduced.

[0005] At present, the extraction of effective components of Mahonia bealei is limited to the extraction of total alkaloids or total berberine substances, and there is no further separation of single compounds of Mahonia bealei. Therefore, the method can further separate single compounds of Mahonia bealei, better control the quality standard of Mahonia bealei, lay a good foundation for further research on the pharmacological action, and lay a foundation for the research of new drugs with anti-tumor, antioxidant, immune regulation activity and anti-inflammatory in clinical development. SUMMARY

[0006] The present application aims at a method for extracting effective active components from Mahonia bealei. The method first extracts Mahonia bealei with ethanol, then extracts with petroleum ether, ethyl acetate and n-butanol, respectively, detects whether there is an obvious peak by TLC, collects different fractions by elution and washing of the ethyl acetate extract with an alumina column, and then crystallizes, dries and processes the fractions to obtain target products. The target products are identified by infrared, hydrogen spectrum, carbon spectrum, mass spectrum and ultraviolet combined detection. The identified target products are respectively berberine hydrochloride, jatrorrhizine hydrochloride and palmatine hydrochloride single compounds. The method can further separate single compounds of Mahonia bealei, better control the quality standard of Mahonia bealei, lay a good foundation for further research on the pharmacological action, and lay a foundation for the research of new drugs with anti-tumor, antioxidant, immune regulation activity and anti-inflammatory in clinical development.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] A method for extracting effective active components from Mahonia bealei, comprising the following steps:

[0009] S1: After crushing Mahonia bealei, add ethanol for heating reflux extraction, repeat the extraction for 3 times, combine the filtrate, filter the ethanol extract with filter cloth to remove residues, and obtain the filtrate by reducing pressure concentration to remove alcohol to obtain Mahonia bealei extraction concentrate;

[0010] S2: After diluting the Mahonia bealei extraction concentrate with water, extract with petroleum ether, ethyl acetate and n-butanol respectively, reduce pressure concentration of the organic phase to paste, and reduce pressure concentration of the water phase to remove organic reagents, respectively obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water phase extract. After TLC chromatographic analysis, the ethyl acetate extract has obvious characteristic peaks, and other components have no obvious characteristic peaks;

[0011] S3: Dissolve the ethyl acetate extract with methanol, and then chromatograph with an alumina column, and elute with dichloromethane-methanol gradient to collect three different fractions;

[0012] S4: Filter the different fractions, reduce pressure concentration to dryness, dissolve, adjust pH, filter, dissolve the solid with methanol, and crystallize;

[0013] S5: the three target products GLM1, GLM2 and GLM3 obtained after drying the crystalline body are respectively subjected to structure identification, and the structure of the target product is finally determined.

[0014] The preferred technical scheme of the present application: the ethanol reflux extraction in step S1 is 90% ethanol reflux extraction for 3 times, 1 h each time, and concentration under reduced pressure at 50-60 DEG C.

[0015] The preferred technical scheme of the present application: the water dilution in step S2 is 2-3 times, and the amount of petroleum ether, ethyl acetate and n-butanol is respectively 1-3 times of the extraction concentrated solution.

[0016] The preferred technical scheme of the present application: the temperature of petroleum ether in the reduced pressure concentration in step S2 is 60-90 DEG C, the temperature of ethyl acetate is 70 DEG C, and the temperature of n-butanol and water is 90 DEG C.

[0017] The preferred technical scheme of the present application: the volume ratio of dichloromethane-methanol in the gradient elution in step S3 is 20:1 to 0:1.

[0018] The preferred technical scheme of the present application: step S4 is dissolved with chloroform, and the pH is adjusted to 2-3 with 10% HCl.

[0019] The preferred technical scheme of the present application: the drying is air drying at 50-60 DEG C and reduced pressure drying at 40-50 DEG C.

[0020] The preferred technical scheme of the present application: the structure identification method is infrared, hydrogen spectrum, carbon spectrum, mass spectrum and ultraviolet combined detection to identify the target product. The target products GLM1, GLM2 and GLM3 separated by the method are respectively berberine hydrochloride, jatrorrhizine hydrochloride and palmatine hydrochloride monomer compounds, and the structural formula of the three compounds is as follows:

[0021] Berberine hydrochloride:

[0022]

[0023] Jatrorrhizine hydrochloride:

[0024]

[0025] Palmatine hydrochloride:

[0026]

[0027] Compared with the prior art, the present application has the advantages and beneficial effects including:

[0028] 1. The method of this invention can further isolate berberine hydrochloride, berberine hydrochloride and palmatine hydrochloride monomer compounds from Mahonia japonica, which can lay the groundwork for better control of Mahonia japonica quality standards, more in-depth research on its pharmacological effects, and lay the foundation for the clinical development of new drugs with anti-tumor, antioxidant, immunomodulatory and anti-inflammatory activities.

[0029] 2. The method of this invention utilizes different organic solvents, namely petroleum ether, ethyl acetate, and n-butanol, for extraction. The remaining aqueous phase is then concentrated under reduced pressure, and the peaks are detected by TLC before further extraction. This method is more conducive to the separation of the target product of Mahonia betel nut and also helps to improve the extraction rate and purity of the monomeric compounds. According to high performance liquid chromatography, the purity of the monomeric compounds of berberine hydrochloride, berberine hydrochloride, and palmatine hydrochloride reaches more than 98.5%. Attached Figure Description

[0030] Figure 1 Infrared spectrum of berberine hydrochloride from the tree;

[0031] Figure 2 The hydrogen spectrum of berberine hydrochloride from the tree;

[0032] Figure 3 Carbon spectrum of berberine hydrochloride from Merbau tree;

[0033] Figure 4 Mass spectrum of berberine hydrochloride from Merbau tree;

[0034] Figure 5 The ultraviolet detection spectrum of berberine hydrochloride from the tree;

[0035] Figure 6 The high-performance liquid chromatogram of berberine hydrochloride from Mahonia japonica in Example 1;

[0036] Figure 7 This is the high-performance liquid chromatogram of berberine hydrochloride from Mahonia japonica in Example 2.

[0037] Figure 8 The hydrogen spectrum of the root alkaloid of Mahonia hydrochloride;

[0038] Figure 9 Carbon spectrum of the root alkaloid of Mahonia hydrochloride;

[0039] Figure 10 Mass spectrum of the root alkaloid hydrochloride of Mahonia japonica;

[0040] Figure 11 The high-performance liquid chromatogram of the root alkaloid hydrochloride of Mahonia japonica in Example 1;

[0041] Figure 12 This is a high-performance liquid chromatogram of the root alkaloid hydrochloride of Mahonia japonica in Example 2.

[0042] Figure 13 The infrared detection spectrum of the picroside II hydrochloride;

[0043] Figure 14 The hydrogen spectrum of the picroside II hydrochloride;

[0044] Figure 15 The carbon spectrum of the picroside II hydrochloride;

[0045] Figure 16 The mass spectrum of the picroside II hydrochloride;

[0046] Figure 17 The ultraviolet detection spectrum of the picroside II hydrochloride;

[0047] Figure 18 The high performance liquid chromatogram of the picroside II hydrochloride of Example 1;

[0048] Figure 19 The high performance liquid chromatogram of the picroside II hydrochloride of Example 2. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0050] Example 1

[0051] A method for extracting effective active ingredients from the Mahonia bealei Fort. includes the following steps:

[0052] S1: The Mahonia bealei Fort. is crushed and then added with 90% ethanol according to a solid-liquid ratio of 1:5, and heated to reflux at 60℃ for 3 times, each time for 1.5h. The filtrates are combined, and the ethanol extract is filtered through filter cloth to remove residues, and the obtained filtrate is concentrated at 55℃ under reduced pressure until no alcohol is left, to obtain a Mahonia bealei Fort. extraction concentrate;

[0053] S2: The Mahonia bealei Fort. extraction concentrate is diluted with 3 times water, and then extracted with 3 times petroleum ether, ethyl acetate and n-butanol respectively. The organic phases are concentrated under reduced pressure to a paste at 80℃, 70℃ and 90℃ respectively, and the water phase is concentrated under reduced pressure at 90℃ until no organic reagent is left, to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water phase extract respectively. After TLC chromatographic analysis, the ethyl acetate extract has obvious characteristic peaks, and the other components have no obvious characteristic peaks;

[0054] S3: The ethyl acetate extract was dissolved in methanol and then subjected to column chromatography on an alumina column, eluted with a gradient of dichloromethane-methanol (20:1 to 0:1 by volume), and three different fractions were collected according to the color and fractionation time;

[0055] S4: The different fractions were filtered and concentrated to dryness under reduced pressure at 60°C, dissolved in chloroform, adjusted to pH 2 with 10% HC1, filtered, and the solid was dissolved in methanol and crystallized;

[0056] S5: The three target products GLM1, GLM2, and GLM3 obtained after drying the crystallized product were subjected to structural identification by infrared, hydrogen spectrum, carbon spectrum, mass spectrum, and ultraviolet combined detection.

[0057] The detection results of the target product GLM1 are shown in the following table and Figure 1: Figures 1-5 and the following table data, and the GLM1 target product was identified as berberine hydrochloride, with a molecular formula of C 20 H 18 NO4Cl, a molecular weight of 371.81, and a structural formula as follows:

[0058]

[0059] Structural identification results of the target product GLM1:

[0060]

[0061] Nuclear Magnetic Resonance NMR

[0062] 1 H-NMR

[0063]

[0064]

[0065] 13 C-NMR

[0066]

[0067] The berberine hydrochloride isolated in this example was subjected to high performance liquid chromatography to test the product purity, and the high performance liquid chromatography detection results are shown in the following table and Figure 2: Figure 6 The product purity reached 99.38%.

[0068]

[0069] Name Retention Time Area Peak Height % Area USP Separation USP Theoretical Plates USP Tailing 1 10.776 125834 6378 0.30 6.810332e+003 1.476368e+000 2 12.642 7572 524 0.02 3.922847e+000 1.388936e+004 1.273407e+000 3 13.875 14940 753 0.04 2.609539e+000 1.157633e+004 4 14.415 96442 5207 0.23 1.076964e+000 1.399090e+004 1.072823e+000 5 15.394 11724 537 0.03 1.842078e+000 1.146826e+004 1.198343e+000 6 20.920 41371675 1467650 99.38 8.297451e+000 1.221618e+004 1.587293e+000 7 22.366 3341 248 0.01 2.595655e+000 6.284936e+004 8.262877e-001

[0070] The detection results of the target product GLM2 are shown in the following table and Figure 3: Figures 8-10The GLM2 target product is identified as sanguinarine hydrochloride, with a molecular formula of C 20 H 14 O4N·Cl, a molecular weight of 367.79, and a structural formula as follows:

[0071]

[0072] The structural identification result of the target product GLM2 is as follows:

[0073] Mass Spectrometry MS

[0074] ESI-MS m / z: 333.0 [M+H] + ;

[0075] The conclusion is consistent with the molecular weight.

[0076] Nuclear Magnetic Resonance NMR

[0077] 1 H-NMR

[0078]

[0079] 13 C-NMR

[0080]

[0081]

[0082] The sanguinarine hydrochloride isolated in this example is subjected to high performance liquid chromatography to test the product purity, and the high performance liquid chromatography detection result is as shown in the following table and Figure 11 the product purity reaches 99.54%.

[0083]

[0084] Retention Time Area Peak Height % Area 1 4.635 2530647 267797 99.54 2 5.728 1120 105 0.04 3 6.773 10586 1006 0.42

[0085] The detection result of the target product GLM3 is as Figures 13-17 follows, and the data in the following table show that the GLM1 target product is identified as palmatine hydrochloride, with a molecular formula of C 21 H 22 ClNO4, a molecular weight of 387.86, and a structural formula as follows:

[0086]

[0087] The structural identification result of the target product GLM3 is as follows:

[0088]

[0089]

[0090] Nuclear Magnetic Resonance NMR

[0091] 1 H-NMR

[0092]

[0093] 13 C-NMR

[0094]

[0095] The isolated sanguinarine hydrochloride in this example is tested for product purity by high performance liquid chromatography. The results of the high performance liquid chromatography test are shown in the following table and in Figure 1, and the product purity is 98.69%. Figure 18

[0096]

[0097] Example 2

[0098] A method for extracting effective active ingredients from Mahonia bealei includes the following steps:

[0099] S1: Mahonia bealei is ground and then added to 90% ethanol at a solid to liquid ratio of 1:7. The mixture is heated to reflux at 55°C for 3 hours, and the filtrate is combined. The ethanol extract is filtered using filter cloth to remove residue, and the filtrate is concentrated at 60°C under reduced pressure until no ethanol remains. A Mahonia bealei extract concentrate is obtained.

[0100] S2: The Mahonia bealei extract concentrate is diluted with 2 times the amount of water, and then extracted with 2 times the amount of petroleum ether, ethyl acetate, and n-butanol, respectively. The organic phases are concentrated under reduced pressure at temperatures of 75°C, 70°C, and 90°C, respectively, until they become pasty. The water phase is concentrated under reduced pressure at 90°C until no organic reagents remain. Petroleum ether extract, ethyl acetate extract, n-butanol extract, and water phase extract are obtained. TLC chromatographic analysis shows that the ethyl acetate extract has a distinct characteristic peak, and the other components have no distinct characteristic peaks.

[0101] S3: The ethyl acetate extract is dissolved in methanol and then chromatographed using an alumina column. The column is eluted with dichloromethane-methanol (20:1 to 0:1 by volume) in a gradient. Three different fractions are collected based on color and fractionation time.

[0102] S4: The different fractions are filtered and then concentrated under reduced pressure at 50°C until dry. The residue is dissolved in chloroform, and the pH is adjusted to 3 using 10% HCl. The mixture is filtered, and the solid is dissolved in methanol and crystallized.

[0103] ​S5: The three target products GLM1, GLM2 and GLM3 obtained after drying the crystalline body are respectively subjected to infrared, hydrogen spectrum, carbon spectrum, mass spectrum and ultraviolet combined detection for structure identification (the method is the same as that in Example 1).

[0104] The target products GLM1, GLM2 and GLM3 separated in the example are respectively monomer compounds of berberine hydrochloride, jatrorrhizine hydrochloride and palmatine hydrochloride. The monomer compounds of berberine hydrochloride, jatrorrhizine hydrochloride and palmatine hydrochloride are subjected to high performance liquid chromatography detection, and the detection results are shown in the following table and Figure 7 、 Figure 12 、 Figure 19 The product purities are respectively 99.47%, 99.39% and 98.58%.

[0105] High performance liquid chromatography result of berberine hydrochloride in Mahonia bealei

[0106]

[0107]

[0108] High performance liquid chromatography result of jatrorrhizine hydrochloride in Mahonia bealei

[0109]

[0110]

[0111] Retention Time Area Peak Height % Area 1 4.544 516 118 0.02 2 4.847 557 113 0.02 3 5.074 2276521 406386 99.39 4 6.949 10140 1796 0.44 5 9.723 2820 711 0.12

[0112] High performance liquid chromatography result of palmatine hydrochloride in Mahonia bealei

[0113]

[0114] Example 3

[0115] A method for extracting effective active ingredients from Mahonia bealei, comprising the following steps:

[0116] S1: After Mahonia bealei is crushed, 90% ethanol is added according to a solid-liquid ratio of 1:6, and the mixture is heated to reflux at 50°C for 3 times, each time for 1 h. The filtrate is obtained by filtering the ethanol extract with filter cloth, removing the residue, and concentrating the filtrate at 60°C under reduced pressure until no alcohol is left, thereby obtaining a Mahonia bealei extraction concentrate;

[0117] S2: The Mahonia aquifolium extract is concentrated and diluted with 3 times water, then extracted with 1 times petroleum ether, ethyl acetate and n-butanol respectively. The organic phase is concentrated to paste under reduced pressure at 75℃, 70℃ and 90℃ respectively, and the water phase is concentrated to no organic reagent under reduced pressure at 90℃. The ethyl acetate extract has obvious characteristic peaks, and other components have no obvious characteristic peaks through TLC chromatographic analysis;

[0118] S3: The ethyl acetate extract is dissolved in methanol and then subjected to alumina column chromatography, eluted with dichloromethane-methanol (volume ratio 20:1 to 0:1) gradient, and three different fractions are collected according to color and fractionation time;

[0119] S4: The different fractions are filtered and concentrated to dryness under reduced pressure at 50℃, dissolved in chloroform, adjusted to pH 3 with 10% HCl, filtered, and the solid is dissolved in methanol and crystallized;

[0120] S5: The three target products GLM1, GLM2 and GLM3 obtained after drying the crystalline are subjected to infrared, hydrogen spectrum, carbon spectrum, mass spectrum and ultraviolet combined detection for structure identification (the same as method and example 1).

[0121] The target products GLM1, GLM2 and GLM3 separated in this example are monomer compounds of berberine hydrochloride, jatrorrhizine hydrochloride and palmatine hydrochloride respectively. The monomer compounds of berberine hydrochloride, jatrorrhizine hydrochloride and palmatine hydrochloride are detected by high performance liquid chromatography, and the product purity reaches 99.06%, 98.96% and 98.61% respectively.

[0122] In summary, this method can further separate monomer compounds of berberine hydrochloride, jatrorrhizine hydrochloride and palmatine hydrochloride from Mahonia aquifolium, which can better control the quality standard of Mahonia aquifolium, further study its pharmacological action, lay a good foundation for the research of new drugs with anti-tumor, antioxidant, immune regulation activity and anti-inflammatory activity in clinical development.

[0123] The above is a further detailed description of the present application in combination with specific / preferred embodiments, and cannot be regarded as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, they can make several alternatives or modifications to the described embodiments without departing from the concept of the present application, and these alternatives or modifications shall be regarded as falling within the protection scope of the present application.

Claims

1. A method for extracting effective active ingredients from Mahonia japonica, characterized in that: Includes the following steps: S1: After crushing the Mahonia wood, add ethanol and heat and reflux to extract. Repeat the extraction 3 times, combine the filtrates, filter the ethanol extract with filter cloth to remove the residue, and concentrate the obtained filtrate under reduced pressure until it is alcohol-free to obtain Mahonia wood extract concentrate. S2: After diluting the concentrated extract of Mahonia japonica with water, it was extracted with petroleum ether, ethyl acetate and n-butanol respectively. The organic phases of the extracts were concentrated under reduced pressure to a paste, and the aqueous phases were concentrated under reduced pressure until no organic reagents were obtained, respectively, to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and aqueous extract. After TLC chromatographic analysis, the ethyl acetate extract had obvious characteristic peaks, while other components did not have obvious characteristic peaks. S3: The ethyl acetate extract was dissolved in methanol and then subjected to alumina column chromatography with dichloromethane-methanol gradient elution, and three different fractions were collected. S4: After filtering different fractions, concentrate them to dryness under reduced pressure, dissolve them, adjust the pH, filter them, dissolve the solids in methanol, and crystallize them. S5: The structures of the three target products GLM1, GLM2, and GLM3 obtained after drying the crystals were identified, and the structures of the target products were finally determined. The isolated target products GLM1, GLM2, and GLM3 were berberine hydrochloride, cypermethrin hydrochloride, and palmatine hydrochloride monomer compounds, respectively. In step S2, dilute with water 2-3 times, and use petroleum ether, ethyl acetate, and n-butanol in amounts 1-3 times that of the extract concentrate, respectively. In step S3 gradient elution, the volume ratio of dichloromethane to methanol is 20:1 to 0:1; Step S4 involves dissolving the substance in chloroform and adjusting the pH to 2-3 with 10% HCl.

2. The method for extracting effective active ingredients from Mahonia japonica according to claim 1, characterized in that: The ethanol reflux extraction in step S1 involves reflux extraction with 90% ethanol three times, each time for 1 hour, followed by concentration under reduced pressure at 50-60°C.

3. The method for extracting effective active ingredients from Mahonia japonica according to claim 1, characterized in that: In step S2, the temperature of petroleum ether during vacuum concentration is 60-90℃, the temperature of ethyl acetate is 70℃, and the temperature of n-butanol and water is 90℃.

4. The method for extracting effective active ingredients from Mahonia japonica according to claim 1, characterized in that: The drying process involves drying the crystalline solid with forced air at 50-60°C and then with reduced pressure at 40-50°C.

5. The method for extracting effective active ingredients from Mahonia japonica according to claim 1, characterized in that: The structural identification method uses a combination of infrared, proton, carbon, mass spectrometry and ultraviolet detection to identify the target product.

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