Alkaloid monomer in camphor tree seed kernel and its enrichment and separation method

Abstract

The present invention relates to the technical field of extraction and separation of natural products, and discloses an alkaloid monomer in camphor tree seed kernel and a method for enriching and separating the same. The method adopts a water emulsification extraction method to extract the oil in camphor tree seed kernel, that is, the camphor tree seed kernel is mixed with water, ultrafinely ground by a colloid milling method at room temperature, and then centrifuged to obtain an upper emulsified phase, an intermediate aqueous phase, and a lower slag phase. In the process of extracting the oil from the camphor tree seed kernel, the alkaloids are leached to the intermediate aqueous phase. The alkaloids in the intermediate aqueous phase are enriched and purified by a macroporous resin, and a high-purity alkaloid monomer is obtained by preparative liquid phase separation. The method for enriching and separating alkaloids in camphor tree seed kernels by using a water emulsification extraction, a macroporous resin, and a preparative liquid phase coupling technology provided by the present invention has the advantages of simple process flow, easy operation, high extraction and separation efficiency, no use of multiple organic reagents, and the purity of the separated alkaloid monomers can reach more than 95%.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and separation of natural products and relates to an alkaloid monomer in camphor tree seed kernel and an enrichment and separation method thereof. Background Art

[0002] The nutrients in camphor seeds are mainly oil and protein. In addition, camphor seeds are rich in polyphenols, alkaloids and other plant active ingredients. The main component of camphor seed kernel oil is medium-chain fatty acids, which is the only natural medium-chain oil discovered in the world so far, and can completely replace existing synthetic medium-chain oil products. Therefore, camphor seeds are an excellent edible oil resource rich in medium-chain oils, proteins and active ingredients.

[0003] Camphor tree kernels are rich in a variety of isoquinoline alkaloids, such as magnolamine, camphoravine, scutellarine and N-methyldomesticinium. Among them, magnolamine has anti-diabetic, anti-inflammatory, immunomodulatory, antihypertensive and cognitive improvement effects, and is expected to become a candidate drug for the treatment of diabetes, depression and Alzheimer's disease; medicinal plants containing camphoravine have pharmacological effects such as analgesia, antihypertensive, antibacterial, free radical scavenging, anti-epileptic, neuroprotective, antianxiety and hypnotic; the plant scutellaria containing scutellarine has anti-tumor, anti-inflammatory, blood sugar regulation and blood pressure regulation. However, these alkaloids are rarely distributed in plants and are difficult to separate. The iodide of N-methyldomesticinium has only been found and reported at trace levels in Glaucium oxylobum Boiss.et Buhse. At present, the existing literature and patents have not reported the extraction and purification methods of camphoravine, scutellarine and N-methyldomesticinium. The patent with the publication number of CN 109369529 B, the patent name of which is a method for preparing high-purity magnolia alkaloids using the mother liquor of Coptis chinensis as raw material, the high-purity magnolia alkaloids prepared therefrom and their application, and the patent with the publication number of CN 110437152 A, the patent name of which is a method for extracting and separating magnolia alkaloids from bald acne flower, respectively disclose a method for preparing high-purity magnolia alkaloids, but both patents use solvents of different polarity to extract magnolia alkaloids extracts, and then separate magnolia alkaloids by silica gel column chromatography. Although magnolia alkaloids with a purity higher than 90% can be obtained by the method described in the patent, the separation process is complicated and a variety of toxic, flammable and explosive organic reagents (petroleum ether, chloroform, dichloromethane, ethyl acetate, n-butanol, etc.) are used, which limits its industrial production.

[0004] Macroporous resins have been widely used in the enrichment of target compounds in natural products. They have the advantages of good adsorption and desorption effects, simple equipment, easy operation, and reusability. However, other efficient preparative separation technologies are needed to prepare compounds with higher purity. Preparative liquid chromatography is a chromatographic technology that has been widely used in recent years for the separation of natural products. It has the advantages of online monitoring, efficient separation, and automatic control. It has multiple separation modes such as forward, reverse, exclusion, and ion exchange, and is suitable for the purification of various compounds. Therefore, the use of macroporous resins and preparative liquid chromatography can establish a rapid enrichment and separation preparation process for target compounds. It has the advantages of simple operation, short production cycle, safe preparation process, and is suitable for industrial production.

[0005] At present, the existing literature and patents have not reported the method for extracting and purifying alkaloid monomers in camphor tree seed kernels. Therefore, in view of the defects and shortcomings in the above-mentioned prior art, the present invention provides a method for separating high-purity alkaloid monomers in camphor tree seed kernels by using macroporous resin combined with preparative liquid chromatography technology, which realizes the green recovery of the main by-products in the process of extracting camphor tree seed kernel oil by water method, provides technical guidance for the separation of high-purity alkaloid monomers, and improves the comprehensive utilization of camphor tree seed resources. Summary of the invention

[0006] The invention aims to provide an alkaloid monomer in camphor tree seed kernel and an enrichment and separation method thereof, which adopts a macroporous resin in combination with a preparative liquid chromatography technology, simplifies the separation process, reduces environmental pollution, and has a high product purity.

[0007] A method for enriching and separating alkaloid monomers in camphor tree seed kernels, the method comprising the following steps:

[0008] Step S1: Preparation of the middle layer aqueous phase: Mix camphor tree seed kernels and water according to the material-liquid mass ratio, add them into a colloid mill for wet ultrafine grinding, and obtain camphor tree seed kernel slurry; centrifuge the obtained camphor tree seed kernel slurry to separate liquid-liquid-solid, and obtain an upper emulsified phase, a middle layer aqueous phase, and a lower layer slag phase.

[0009] Step S1: During the extraction of camphor tree seed oil, alkaloid monomers are leached to the middle layer of water phase. The colloid mill is mainly composed of a fixed surface and a rotating surface, and there is a fine-tunable gap between the two surfaces. When the material passes through the gap, due to the high-speed rotation of the rotating body (3000-15000r / min), a large velocity gradient is generated between the fixed body and the rotating body, so that the material is subjected to strong shearing to produce a crushing and dispersion effect, and the finished product particle size reaches 2-50μm, so that the material is effectively emulsified, dispersed and crushed, achieving the effect of ultrafine crushing and emulsification of the material; after the material is processed by ultrafine crushing, there is basically no intact cell, its cell wall is broken, and the effective components in the cell are exposed, which improves the release rate and release amount.

[0010] The main components of camphor seed kernels are oil and protein. Camphor seed kernels have the highest oil content, followed by protein and dietary fiber. Camphor seed kernel oil (CCSKO) has a high content of medium-chain fatty acids (MCFA) and is a natural capric lauric acid type (series) medium-chain triglyceride (MCT) that is safe for consumption. The amino acid composition of camphor seed kernel protein is similar to that of peanut protein, making camphor seed kernels a potential alternative resource for plant protein. In addition, camphor seed kernels are an important source of plant active substances such as polyphenols and alkaloids. Camphor seed kernel extracts have a variety of potential biological activities, including antioxidant, diabetes improvement, anti-obesity and anti-inflammatory activities.

[0011] Since the surface of plant oil bodies is hydrophilic, in aqueous solution, oil bodies can be dispersed in the water phase to form a uniform emulsion. Taking advantage of this property, mechanical crushing (such as refining) is usually used to destroy the plant cell wall structure and separate the oil bodies from protein bodies and starch granules. At the same time, the fine particles formed greatly increase the contact area between the oil bodies and water, which is conducive to the dispersion of the oil bodies in aqueous solution; then, the solubility and specific gravity difference between the oil bodies and other components such as proteins, starch, and polysaccharides are used to float the oil bodies with the help of centrifugation to obtain the oil bodies. In addition, the biggest advantage of the water method is that it can extract oil bodies and other beneficial components at the same time. Water-soluble active ingredients (such as polysaccharides, polyphenols, flavonoids, alkaloids, saponins, etc.) are dissolved in the process water of the water emulsification extraction method and are extracted simultaneously during the oil extraction process. These active ingredients can better maintain their natural activity. At present, the process water in the production of water emulsification extraction of plant oils and fats is not effectively utilized, but directly discharged into the environment, which not only causes a waste of resources, but also may cause environmental pollution. Therefore, obtaining valuable active substances from the wastewater of the process of extracting camphor tree seed kernel oil by water emulsification extraction can not only improve the comprehensive utilization rate of camphor tree seeds, but also realize the clean production of camphor tree seed industry.

[0012] The water emulsification extraction method is considered to be a new technology for extracting vegetable oils today. It has a simple extraction process, low equipment requirements, and good quality of the obtained oils. It is safer, more nutritious and economical. It solves the problems of low extraction rate of the pressing method for extracting vegetable oils, flammable and explosive production process of the pre-pressing-organic solvent leaching method for extracting vegetable oils, toxic solvent residues in the vegetable oil products, and reduced activity of plant active ingredients under heat.

[0013] The technology of extracting plant oils and proteins by water (water emulsification extraction-demulsification release technology) has the characteristics of mild conditions, high extraction efficiency, and good quality of oils and proteins. In the extraction process, active ingredients such as polyphenols and alkaloids can be extracted into the aqueous solution at the same time, which largely retains the biological activity of the active ingredients. Compared with the organic solvent leaching method for removing oils, the water extraction technology can solve the low extraction rate of the pressing method for extracting plant oils, reduce the use of toxic, flammable and explosive organic solvents (petroleum ether, n-hexane, etc.) in the production process of the pre-pressing-organic solvent leaching method for extracting plant oils, reduce safety hazards, and reduce the organic solvent residues in plant active ingredients and the reduction of the thermal activity of plant active ingredients.

[0014] The present invention adopts a water emulsification extraction method to separate and prepare oil from camphor tree seed kernels, and at the same time obtains water phase components. Specifically, camphor tree seed kernel slurry is centrifuged and layered, the upper emulsified phase mainly contains camphor tree seed kernel oil bodies, the middle layer water phase is water-soluble components such as camphor tree seed kernel protein, camphor tree seed kernel polysaccharides, camphor tree seed kernel active substances (polyphenols, alkaloids, etc.); the lower layer residue phase mainly contains a mixture of camphor tree seed kernel water-insoluble protein and fiber.

[0015] Alkaloids usually have one or more nitrogen atoms. The lone pairs of electrons on these nitrogen atoms can form hydrogen bonds with water molecules, making the alkaloids have a certain hydrophilicity. Therefore, during the water emulsification extraction process, alkaloids tend to dissolve in the aqueous phase.

[0016] Whether the intermediate aqueous phase contains alkaloid monomers can be verified by the following detection method: take an appropriate amount of aqueous phase solution, add 4 times the amount of anhydrous ethanol solution (the purpose is to precipitate components such as proteins and polysaccharides in the intermediate aqueous phase, so that active ingredients such as alkaloids are dissolved in anhydrous ethanol), mix well, centrifuge at 4800rpm for 10min, take an appropriate amount of supernatant and filter it through a 0.22μm filter membrane, use high performance liquid chromatography to analyze the components in the aqueous phase, and judge whether there are corresponding alkaloid monomers in the aqueous phase solution from the peaks of the liquid chromatogram.

[0017] The polarity and solubility of alkaloids are important bases for their extraction, separation and purification. There are great differences in the polarity and solubility of different types of alkaloids. Most alkaloids are soluble in organic solvents such as ethanol, chloroform, ether, and benzene, but insoluble or poorly soluble in water. Quaternary ammonium alkaloids are easily soluble in water, acid water, and alkaline water, soluble in alcohol solvents, and poorly soluble in lipophilic organic solvents; some small molecule alkaloids are soluble in both water and chloroform. Most of the extraction methods of alkaloids adopt solvent extraction, which can be divided into water extraction, acid water extraction, alkaline water extraction, organic solvent extraction and steam distillation according to the different solvents used. The water extraction method is easy to operate and has low cost, but the extraction times are many and it consumes more water; the acid water extraction method can only extract alkaline alkaloids; the alkaline water extraction method generally extracts alkaloids that are unstable under acidic or neutral conditions; the organic solvent extraction method generally extracts free alkaloids and their salts, and the organic solvent extraction method inevitably has the problem of solvent residue; the steam distillation method is not suitable for components that are easily decomposed by heat; in addition, there are new technologies such as supercritical fluid extraction, ultrasonic extraction, microwave extraction, and two-phase aqueous extraction for the extraction of alkaloids. Each method has its own advantages and limitations. Due to the complexity of plant components, the types and contents of alkaloids extracted by different extraction methods will be significantly different; currently, most reports on the extraction of camphor seed kernel components are mainly camphor seed kernel oil, and there are no reports on the extraction of its alkaloid components.

[0018] Step S2: Enrichment of alkaloid monomers in the intermediate aqueous phase: The alkaloid monomers in the intermediate aqueous phase obtained in step S1 are statically or dynamically adsorbed by a macroporous adsorption resin. For static adsorption, after adsorption, the resin is first washed with distilled water, and then the macroporous resin is desorbed using ethanol solutions of different concentrations, wherein the static adsorption method uses 5%, 20% and 40% ethanol solutions for elution in sequence; a monomer alkaloid qualitative detection method is used to detect whether the eluent contains the corresponding alkaloid monomer, the ethanol eluent containing the corresponding alkaloid monomer is combined, and the ethanol eluent is concentrated and dried under reduced pressure to obtain the corresponding alkaloid monomer enriched product; for dynamic adsorption, after adsorption, the resin column is first washed with distilled water, and then 5BV5%, 5BV20% and 5BV40% ethanol are used for gradient elution in sequence; the eluent is qualitatively detected whether the corresponding alkaloid monomer is contained, the ethanol eluent containing the corresponding alkaloid monomer is combined, and the ethanol eluent is concentrated and dried under reduced pressure to obtain the corresponding alkaloid monomer enriched product; the content of alkaloid monomers in the enriched product is at least 40%.

[0019] The commonly used traditional methods for the separation and purification of alkaloids include recrystallization, sublimation, atmospheric or vacuum fractionation, pH gradient extraction, precipitation, steam distillation, salting out, solid phase extraction, etc. In recent years, new technologies such as silica gel column chromatography, Al 2 O 3Chromatographic methods such as column chromatography, gel chromatography column and high performance liquid chromatography, resin adsorption methods such as macroporous resins and ion exchange resins, molecular imprinting, high-speed countercurrent chromatography, membrane separation, molecular distillation technology and other new separation technologies. The problems of traditional separation methods are low yield and high purification cost. Compared with traditional separation methods, the aforementioned new separation technologies have a positive effect in improving separation quality, efficiency, reducing pollution and saving energy. However, since the separation and purification of alkaloids has always been the difficulty and key to the development and research of plant active ingredients, the application of each new technology in the separation and preparation industrialization of specific types of alkaloids still requires solving many actual and unpredictable problems.

[0020] In step S2, a macroporous adsorption resin is selected to statically or dynamically adsorb the alkaloid monomers in the intermediate layer aqueous phase obtained in step S1. The macroporous resin is a polymer compound with a special pore structure and surface properties. The pore size is usually between 50-1000nm, which is much larger than that of traditional small-pore resins. It has a high specific surface area and a soft physical structure, and can adapt to biological molecules of different shapes to a certain extent. Alkaloids are alkaline organic molecules, and their molecular structure usually contains some nitrogen atoms and aromatic rings. The principle of macroporous resin enrichment of alkaloids is mainly achieved through electrostatic effect and adsorption; alkaloid molecules usually contain some positively charged nitrogen atoms, and the surface of macroporous resin usually carries some negatively charged groups, so electrostatic effect will be generated, so that alkaloid molecules are adsorbed on macroporous resin; macroporous resin has a large pore size and a high specific surface area, which can provide sufficient adsorption sites to achieve adsorption of alkaloids; after adsorption by macroporous resin, it is necessary to select a suitable eluent and concentration according to the strength of the adsorption force. For components with less polarity, an eluent with less polarity needs to be selected; for compounds with medium polarity or greater polarity, an eluent with greater polarity should be used. In step S2, static adsorption or dynamic adsorption adopts gradient elution respectively to improve the collection rate of alkaloid monomers. The alkaloid monomer content of the obtained alkaloid monomer enrichment is detected. If the alkaloid monomer content is too low, it will affect the efficiency of the subsequent step S3 preparative liquid chromatography separation and increase the preparation cost; the content of alkaloid monomers in the enrichment reaches 50%, which can ensure the efficient preparation of high-purity alkaloid monomers.

[0021] Step S3: Preparative liquid chromatography separation to obtain refined alkaloid monomers: the alkaloid monomer concentrate obtained in step S2 is subjected to preparative liquid separation, eluted with formic acid water-methanol solution according to an elution gradient, the eluted fractions are detected with an ultraviolet detector, and the effluent is qualitatively detected to determine whether it contains the corresponding alkaloid monomers, the fractions containing the corresponding alkaloid monomers are combined and concentrated, and after drying, a refined alkaloid monomer with a purity of more than 95% is obtained.

[0022] Preparative HPLC is a chromatographic separation method that achieves high-purity separation through a high-load, high-resolution preparative column. Its characteristics are: ① It uses a high-efficiency chromatographic column with high separation efficiency; ② It has a wide range of applications and has good separation effects on polar and non-polar, ionic and non-ionic, small and large molecules, thermally stable and thermally unstable compounds, and has an irreplaceable position in the field of plant research; ③ It has a large processing capacity and can meet different separation needs; according to the preparation scale, it is divided into semi-preparative chromatography, gram-level preparative chromatography and industrial chromatography, which has the advantage of industrial application; ④ It has the advantages of good reproducibility, low toxicity, economy and environmental protection. The use of preparative HPLC to separate alkaloids often results in peak tailing and extremely low sample loading, so alkaloid separation often has problems such as wide peak width, short peak height, and poor detection sensitivity; increasing the injection volume will also lead to aggravated chromatographic peak tailing, rapid decline in column efficiency, and adjacent components are more likely to be wrapped by tailing peaks, so the use of preparative HPLC to separate alkaloids is a difficult point in the field of natural plant component separation.

[0023] Step S3 is to separate the alkaloid monomers by preparative liquid chromatography on the basis of the enrichment of the alkaloid monomers by static adsorption or dynamic adsorption in step S2, and to optimize the separation conditions of the preparative liquid chromatography to prepare high-purity alkaloid monomers, thereby filling the gap in the high-purity separation and preparation of alkaloid monomers in camphor tree seed kernels, providing a feasible technical solution for the industrial preparation of high-purity alkaloid monomers in camphor tree seed kernels, laying a foundation for the medicinal use of high-purity alkaloid monomers in camphor tree seed kernels, and further enhancing the economic value of camphor tree seed kernels.

[0024] In a preferred technical solution, the mass ratio of the material to the liquid in step S1 is (camphor tree seed kernel: water) 1:3-1:5, the wet ultrafine grinding time is 4-6 minutes; the centrifugal speed is 2000-8000rpm; the centrifugal time is 5-30 minutes; and the content of camphor tree seed kernel oil in the residue phase is less than 5%.

[0025] In a preferred technical solution, the macroporous resin in step S2 is one of ADS-5, D101, HPD-100, HJ-01, and HJ-18.

[0026] In a preferred technical solution, the static adsorption process in step S2 is as follows: adding the pretreated macroporous resin to the intermediate layer aqueous phase obtained in step S1, stirring and adsorbing at room temperature, the adsorption time is 1-2 hours, filtering to obtain the macroporous resin after saturated adsorption, washing the macroporous resin with distilled water, and then adding 5%, 20% and 40% ethanol aqueous solutions in sequence for desorption, the amount of desorption liquid used is 5-10 times (v / w) of the macroporous resin, and the desorption time is 1-2 hours.

[0027] In a preferred technical solution, the dynamic adsorption process in step S2 is: the intermediate layer aqueous phase obtained in step S1 is passed through a macroporous resin column, the height-to-diameter ratio of the macroporous resin column is 15:1, the column volume is 50-500mL, and the loading flow rate is 1.5-3.0BV / h; after the adsorption is completed, the column is first washed with distilled water to remove impurities such as proteins and polysaccharides, and then gradient elution is performed with 5BV5%, 5BV20% and 5BV40% ethanol aqueous solutions in sequence, the flow rate is 3.0-4.5BV / h, and the effluent is collected once every 1BV.

[0028] In a preferred technical solution, the method for qualitatively detecting whether the eluate in step S2 or the effluent in step S3 contains the corresponding alkaloid monomer comprises the following steps:

[0029] a. Preparation of standard solution: Weigh a certain amount of alkaloid monomer standard, accurately weigh it, dissolve it in methanol aqueous solution and make up to volume, filter it with a 0.22 μm microporous filter membrane to obtain a standard solution with a concentration of 200 μg / mL;

[0030] b. Preparation of sample solution: Take the eluate in step S2 or the effluent in step S3 and filter it with a 0.22 μm microporous membrane to obtain a sample solution;

[0031] c. HPLC detection conditions: Agilent 1260 HPLC; Amethyst C18-H reverse phase column (250mm×4.6mm×5μm), column temperature 25℃; mobile phase: phase A is 0.10% by volume formic acid aqueous solution, phase B is methanol; elution gradient: 0-15 min, 5-80% B; 15-18 min, 80-5% B; injection volume 5μL; flow rate 1.0mL / min; detection wavelength 280nm;

[0032] Sample detection: Analyze the standard solution under the above detection conditions and record its retention time; analyze the sample solution under the above conditions and observe whether there is a peak in the liquid chromatogram of the sample that is consistent with the retention time of the standard; if a peak consistent with the retention time of the standard appears in the chromatogram of the sample, it means that the sample contains the target component.

[0033] In a preferred technical solution, the equipment used for the preparative liquid chromatography separation in step S3 is a LC3000 medium-pressure preparative liquid, and the chromatographic column is HPLCONE-5C18A. The chromatographic column of the preparative liquid chromatography is filled with octadecylsilane bonded silica gel, with a column length of 250 mm, an inner diameter of 30.0 mm, and a particle size of 5 μm.

[0034] In a preferred technical solution, the preparation liquid phase method described in step S3 is: the alkaloid monomer enrichment is dissolved in methanol water and then loaded, eluted through the following elution gradient program, the effluent is collected once every 50 mL, the ultraviolet detector is used for online detection, the eluate containing the alkaloid monomer is combined, concentrated under reduced pressure, and vacuum freeze-dried to obtain an alkaloid monomer with a purity higher than 95%; the mobile phase A is a 0.15% by volume formic acid aqueous solution, the mobile phase B is methanol, the flow rate is 10-30 mL / min, the injection volume is 5-30 mg, the detection wavelength is 280 nm, and the elution gradient is: 0-3 min, 20 ~30%B; 3~15min, 30~34%B; 15~17min, 34~100%B; 17~25min, 100~100%B; 25~27min, 100~20%B; 27~45min, 20~20%B; or the elution gradient is: 0~6min, 23~29%B; 6~24min, 29~37%B; 24~28min, 37~100%B; 28~36min, 100~100%B; 36~37min, 100~23%B; 37~45min, 23~23%B.

[0035] In a preferred technical solution, the method for detecting the content of alkaloid monomers in the enrichment obtained in step S2 comprises the following steps:

[0036] d. Solution preparation: Dissolve the alkaloid monomer standard in methanol aqueous solution to obtain a 0.5 mg / mL standard stock solution, and then use methanol aqueous solution as a diluent to dilute to obtain 500 μg / mL, 250 μg / mL, 125 μg / mL, 50 μg / mL, 25 μg / mL, and 10 μg / mL standard solutions; Take the alkaloid monomer enriched product obtained in step S2 and dissolve it in methanol aqueous solution, filter it with a 0.22 μm microporous filter membrane to obtain a sample solution;

[0037] e. Liquid chromatography conditions: Agilent 1260 high performance liquid chromatograph, Amethyst C18-H reverse phase column (250 mm × 4.6 mm × 5 μm), column temperature set at 25 °C, mobile phase A was 0.10% by volume formic acid aqueous solution, mobile phase B was methanol, elution gradient was: 0-15 min, 5-80% B; 15-18 min, 80-5% B, injection volume was 5 μL, flow rate was 1.0 mL / min, detection wavelength was 280 nm;

[0038] f. Injection test: Use an automatic sample injector to quantitatively inject the standard solution and the sample solution into the sample injector of the liquid chromatograph, and obtain the UV absorption peak area of ​​the standard at different concentrations and the UV absorption peak area of ​​the sample solution;

[0039] g. Establishment of standard curve: With the UV absorption peak area as the ordinate and the corresponding alkaloid monomer standard concentration as the abscissa, the standard curve of the standard is obtained after linear fitting;

[0040] h. Calculation of alkaloid monomer content in the sample: Substitute the obtained ultraviolet absorption peak area of ​​the alkaloid monomer enrichment into the standard curve to calculate the concentration of the alkaloid monomer in the sample, and divide it by the concentration of the enrichment sample to obtain the alkaloid monomer content in the enrichment.

[0041] In a preferred technical solution, the method for detecting the purity of the alkaloid monomer obtained in step S3 comprises the following steps:

[0042] i. Sample preparation: accurately weigh a certain amount of the purified alkaloid monomer sample obtained in step S3, dissolve it in methanol aqueous solution to a fixed volume, and filter it through a 0.22 μm microporous membrane to obtain an alkaloid monomer sample solution;

[0043] j. Liquid chromatography conditions: Agilent 1260 high performance liquid chromatograph and Amethyst C18-H reverse phase column (250 mm × 4.6 mm × 5 μm) were used. The column temperature was set at 25°C. The mobile phase A was a 0.10% by volume aqueous formic acid solution, and the mobile phase B was methanol. The elution gradient was: 0-15 min, 5-80% B; 15-18 min, 80-5% B. The injection volume was 5 μL, the flow rate was 1.0 mL / min, and the detection wavelength was 280 nm.

[0044] k. Injection test: Use an automatic sample injector to quantitatively inject the alkaloid monomer sample solution into the sample injector of the liquid chromatograph to obtain a high performance liquid chromatogram of the sample solution;

[0045] 1. Chromatogram analysis: Measure the area of ​​each chromatographic peak on the chromatogram, including the target component peak and the area of ​​other component peaks; the purity of the target component is the ratio of the component peak area to the total peak area multiplied by 100%.

[0046] A camphor tree seed kernel alkaloid monomer product is prepared according to any one of the above-mentioned methods for enriching and separating the camphor tree seed kernel alkaloid monomers, wherein the purity of the alkaloid monomers is above 95%.

[0047] The beneficial effects of the present invention are as follows: the macroporous resin has the advantages of good adsorption performance, high selectivity, high mechanical strength, low price, and the ability to be processed and regenerated, and has a good effect on the enrichment of alkaloid monomers. By combining with preparative liquid chromatography separation technology, high-purity alkaloid monomers (HPLC>95%) can be separated and prepared from camphor tree seed kernels, and the preparation process is safe and stable. Only methanol and ethanol solutions are used in the process, and other low-boiling organic solvents are not used. This is a method for efficiently preparing camphor tree seed kernel alkaloid monomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 is a preparative liquid chromatogram of magnolamine and camphoravine prepared by the method of Example 1 of the present invention;

[0050] Figure 2 is a preparative liquid chromatogram of nicotinic acid and N-methyldomesticinium prepared by the method of Example 1 of the present invention;

[0051] Figure 3 is a liquid chromatogram of magnolia alkaloids prepared by the method of Example 1 of the present invention (HPLC>99%);

[0052] Figure 4 is a liquid chromatogram of camphor tetrandrine prepared by the method of Example 1 of the present invention (HPLC>99%);

[0053] Figure 5 is a liquid chromatogram of sphenanthene prepared by the method of Example 1 of the present invention (HPLC>97%);

[0054] Figure 6 is a liquid chromatogram of N-methyldomesticinium prepared by the method of Example 1 of the present invention (HPLC>97%);

[0055] Figure 7 The structure diagram of each alkaloid prepared by the method of Example 1 of the present invention and some key two-dimensional nuclear magnetic resonance correlations; wherein, the single arrow is HMBC correlation, and the double arrow is COSY correlation;

[0056] Figure 8 is the mass spectrum of magnolia alkaloids prepared by the method of Example 1 of the present invention;

[0057] Fig. 9 It is the magnolia alkaloids prepared by the method of Example 1 of the present invention. 1 H NMR spectra;

[0058] Fig.10 It is the magnolia alkaloids prepared by the method of Example 1 of the present invention. 13 C NMR spectra;

[0059] Fig.11is the HSQC chart of magnolia alkaloids prepared by the method of Example 1 of the present invention;

[0060] Fig.12 is the HMBC chart of magnolia alkaloids prepared by the method of Example 1 of the present invention;

[0061] Fig.13 is the COSY diagram of magnolia alkaloids prepared by the method of Example 1 of the present invention;

[0062] Fig.14 is a mass spectrum of camphor tetrandrine prepared by the method of Example 1 of the present invention;

[0063] Fig.15 It is the camphor tetrandrine prepared by the method of Example 1 of the present invention 1 H NMR spectra;

[0064] Fig.16 It is the camphor tetrandrine prepared by the method of Example 1 of the present invention 13 C NMR spectra;

[0065] Fig.17 HSQC chart of camphor tetrandrine prepared by the method of Example 1 of the present invention;

[0066] Fig.18 is the HMBC chart of camphor tetrandrine prepared by the method of Example 1 of the present invention;

[0067] Fig.19 is a COSY diagram of camphor tetrandrine prepared by the method of Example 1 of the present invention;

[0068] Fig. 20 is a mass spectrum of sphenanthene prepared by the method of Example 1 of the present invention;

[0069] Fig.21 It is the black shell pine prepared by the method of Example 1 of the present invention 1 H NMR spectra;

[0070] Fig. 22 It is the black shell pine prepared by the method of Example 1 of the present invention 13 C NMR spectra;

[0071] Fig.23 is the HSQC chart of sphenanthene prepared by the method of Example 1 of the present invention;

[0072] Fig.24 is the HMBC chart of sphenanthene prepared by the method of Example 1 of the present invention;

[0073] Fig.25 is a COSY diagram of sphenanthene prepared by the method of Example 1 of the present invention;

[0074] Fig.26 is a mass spectrum of N-methyldomesticinium prepared by the method of Example 1 of the present invention;

[0075] Fig. 27 It is N-methyldomesticinium prepared by the method of Example 1 of the present invention 1 H NMR spectra;

[0076] Fig.28 It is N-methyldomesticinium prepared by the method of Example 1 of the present invention 13 C NMR spectra;

[0077] Fig.29 is the HSQC chart of N-methyldomesticinium prepared by the method of Example 1 of the present invention;

[0078] Fig.30 is the HMBC chart of N-methyldomesticinium prepared by the method of Example 1 of the present invention;

[0079] Fig.31 It is the COSY diagram of N-methyldomesticinium prepared by the method of Example 1 of the present invention. DETAILED DESCRIPTION

[0080] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Example 1

[0081] According to a method for enriching and separating alkaloid monomers in camphor tree seed kernels of the present invention, Example 1 provides a method for enriching and separating alkaloids in camphor tree seed kernels, comprising the following steps:

[0082] (1) 500 g of camphor seed kernels were mixed with water at a material-liquid mass ratio (camphor seed kernels: water) of 1:4, and the mixture was added to a colloid mill for wet ultrafine grinding for 4 min to obtain a camphor seed kernel slurry; the camphor seed kernel slurry was centrifuged at 4500 rpm for 10 min to obtain an upper emulsified phase, a middle aqueous phase, and a lower slag phase.

[0083] (2) The process of statically adsorbing each alkaloid monomer in the aqueous phase by the macroporous resin comprises: taking 1000 mL of the intermediate aqueous phase obtained in step (1), adding 100 g of the pretreated ADS-5 macroporous resin, adsorbing with stirring at room temperature for 1 hour, filtering to obtain the macroporous resin after saturation adsorption, washing the macroporous resin with distilled water 3 times the weight of the macroporous resin, adding 5%, 20% and 40% ethanol aqueous solutions in a ratio of 1:5 (m:v) in turn for desorption, desorbing at room temperature for 1 hour, and qualitatively detecting whether the eluent contains the alkaloid monomer. , and recover 20% of the desorption solution (mainly containing magnolamine and camphoravine) and 40% of the desorption solution (mainly containing styracine and N-methyldomesticinium), concentrate under reduced pressure, and obtain enrichment 1 and enrichment 2 after drying. The contents of magnolamine and camphoravine in enrichment 1 are 46.65% and 42.78%, respectively; the contents of styracine and N-methyldomesticinium in enrichment 2 are 41.22% and 47.82%, respectively.

[0084] The method for qualitatively detecting whether the eluate contains each alkaloid comprises the following steps:

[0085] a. Preparation of standard solution: Weigh a certain amount of standard products of magnolamine / camphor tetrandrine / styraxine / N-methyldomesticinium, accurately weigh, dissolve and fix to volume with 20% methanol aqueous solution, filter with a 0.22 μm microporous filter membrane to obtain a standard solution with a concentration of 200 μg / mL;

[0086] b. Preparation of sample solution: Take the eluate in step (2) and filter it with a 0.22 μm microporous filter membrane to obtain a sample solution;

[0087] c. HPLC detection conditions: Agilent 1260 HPLC; Amethyst C18-H reverse phase column (250mm×4.6mm×5μm), column temperature 25℃; mobile phase: phase A is 0.10% by volume formic acid aqueous solution, phase B is methanol; elution gradient: 0-15min, 5-80%B; 15-18min, 80-5%B; injection volume 5μL; flow rate 1.0mL / min; detection wavelength 280nm;

[0088] Sample detection: Analyze the standard solution under the above detection conditions and record the retention time of each standard; analyze the sample solution under the above conditions and observe whether there is a peak in the liquid chromatogram of the sample that is consistent with the retention time of the standard; if a peak consistent with the retention time of the standard appears in the chromatogram of the sample, it means that the sample contains the target component.

[0089] The method for detecting the content of each alkaloid in the enrichment comprises the following steps:

[0090] d. Solution preparation: Dissolve each standard in 20% methanol aqueous solution to obtain a 0.5 mg / mL standard stock solution, and then use 20% methanol aqueous solution as a diluent to dilute to obtain 500 μg / mL, 250 μg / mL, 125 μg / mL, 50 μg / mL, 25 μg / mL, and 10 μg / mL standard solutions; Take the enrichment obtained in step (2) and dissolve it in 20% methanol aqueous solution, filter it with a 0.22 μm microporous filter membrane to obtain a sample solution;

[0091] e. Liquid chromatography conditions: Agilent 1260 high performance liquid chromatograph, Amethyst C18-H reverse phase column (250 mm × 4.6 mm × 5 μm), column temperature set at 25 °C, mobile phase A was 0.10% by volume formic acid aqueous solution, mobile phase B was methanol, elution gradient was: 0-15 min, 5-80% B; 15-18 min, 80-5% B, injection volume was 5 μL, flow rate was 1.0 mL / min, detection wavelength was 280 nm;

[0092] f. Injection test: Use an automatic sample injector to quantitatively inject the standard solution and the sample solution into the sample injector of the liquid chromatograph, and obtain the UV absorption peak area of ​​the standard at different concentrations and the UV absorption peak area of ​​the sample solution;

[0093] g. Establishment of standard curve: UV absorption peak area is the ordinate (y), the corresponding magnolia alkaloid standard concentration (mg / mL) is the abscissa (x), and the standard curve of the standard is obtained after linear fitting;

[0094] h. Calculation of the content of each alkaloid in the sample: Substitute the obtained ultraviolet absorption peak area of ​​the enrichment into the standard curve to calculate the concentration of each alkaloid in the sample, and divide it by the concentration of the enrichment sample to obtain the content of each alkaloid in the enrichment.

[0095] Specifically, the standard curve in this embodiment is established by taking the ultraviolet absorption peak area as The vertical coordinate (y) is the ordinate, the corresponding alkaloid standard concentration (mg / mL) is the horizontal coordinate (x), and the standard curve of the standard is obtained after linear fitting.

[0096]

[0097] Verification of the detection method for the content of each alkaloid in the enrichment:

[0098] ①Precision test:

[0099] The alkaloid standards of equal concentration (50 μg / mL) were selected as the precision determination samples. Under the same liquid phase conditions, the method precision was investigated by parallel determination 3 times a day for 3 consecutive days. The test results within the same day were the intra-day precision, and the results of the measurements every other day were the inter-day precision. The results showed that the inter-day precision of magnolamine, camphoravine, scutellarine and N-methyldomesticinium were 4.596%, 0.675%, 2.572% and 2.011%, respectively, indicating that the method had good precision.

[0100] ②Repeatability test:

[0101] Take 6 portions of each alkaloid sample, each portion is 10 mg, and prepare a sample solution with a concentration of 1 mg / mL. Continuous injection is carried out under the above chromatographic conditions, 5 μL each time. The results show that the RSD values ​​of the peak areas of magnolamine, camphoravine, scutellarine and N-methyldomesticinium are 1.65%, 1.30%, 1.66% and 1.14%, respectively, indicating that the method has good repeatability.

[0102] ③Stability test:

[0103] The same alkaloid standard solution was injected at 0, 2, 4, 8, 10, 12, and 24 hours, with an injection volume of 5 μL. The results showed that the RSD values ​​of the peak areas of magnolamine, camphoravine, scutellarine, and azodimethylnandinoside were 1.34%, 0.83%, 0.97%, and 1.26%, respectively, indicating that the test solution was basically stable within 24 hours.

[0104] ④ Sample recovery test:

[0105] Take 9 samples with known alkaloid content, each about 5 mg, and add each alkaloid standard precisely at 120%, 100%, and 80% of the content, respectively, put them in a 25mL volumetric flask, and dissolve them in 20% methanol aqueous solution. The results show that the average recoveries of magnolamine, camphoravine, scutellarine and N-methyldomesticinium are 99.65%, 99.61%, 99.66% and 99.78%, respectively, and the RSD values ​​are 0.88%, 1.52%, 1.82% and 0.88%, respectively, indicating that the test accuracy of this method is good.

[0106] Taking magnolia alkaloids as an example, the content of magnolia alkaloids in the enrichment 1 obtained in Example 1 was determined as follows:

[0107] Accurately weigh 3 portions of alkaloid enrichment 1 sample, each portion is about 10 mg, and dissolve in a 10 mL volumetric flask with 20% methanol aqueous solution, shake well and dilute to volume, as the test solution for determining the content of magnolia alkaloids in the enrichment. The test solution is detected by liquid chromatography using the above method, and the concentration of magnolia alkaloids in the sample is calculated based on the obtained peak area and standard curve, and then the content of magnolia alkaloids in the enrichment is calculated according to the formula:

[0108] Content of magnolamine in the enrichment (%) = C 木兰花碱 / C 富集物 ×100%

[0109] Where C 木兰花碱 is the concentration of magnolidine in the enrichment, mg / mL; C 富集物 is the concentration of the enrichment, mg / mL.

[0110] According to calculation, the content of magnolia alkaloids in the enrichment 1 obtained in Example 1 is 46.65%.

[0111] The content detection method provided by the present invention mainly adopts high performance liquid chromatography to determine the content of magnolia alkaloids, has good linear range, accuracy, precision and repeatability, and the method is stable and feasible, and is suitable for determining the content of magnolia alkaloids in products.

[0112] (3) The samples were separated by preparative liquid chromatography. The equipment used for preparative liquid chromatography separation was LC 3000 medium-pressure preparative liquid chromatography, which was purchased from Beijing Chuangxin Tongheng Technology Co., Ltd.; the chromatographic column was HPLCONE-5C18A, Cosmosil; a 30×250 mm C18 reversed phase chromatographic column with a particle size of 5 μm was used, phase A was a 0.15% by volume formic acid aqueous solution, and phase B was methanol as the mobile phase.

[0113] The enriched product 1 was dissolved in 20% methanol water and then loaded on the sample with a loading amount of 25 mg. The flow rate was 20 mL / min and the elution program was: 0-3 min, 20-30% B; 3-15 min, 30-34%; 15-17 min, 34-100%; 17-25 min, 100-100%; 25-27 min, 100-20%; 27-35 min, 20-20%.

[0114] The enriched product 2 was dissolved in 20% methanol water and loaded on the sample with a loading amount of 25 mg and a flow rate of 20 mL / min. The elution was performed through a gradient program: 0-6 min, 23-27% B; 6-24 min, 29-37%; 24-28 min, 37-100%; 28-36 min, 100-100%; 36-38 min, 100-23%; 38-50 min, 23-23%.

[0115] Under the above conditions, the alkaloids in the enrichment were prepared and separated, and the peaks were monitored online by an ultraviolet detector (detection wavelength of 280 nm), and the effluent was collected every 50 mL; at the same time, the effluent was analyzed by high performance liquid chromatography in step (2), and the peaks were compared to detect whether each effluent contained the target component, and the components containing each alkaloid were combined ( Figure 1 , magnolamine: 10.5~12.0min, camphoravine: 12.4~13.7min; Figure 2 , domesticaine: 11.0~14.9min, N-methyldomesticinium: 15.9~22.3min), the solvent was recovered and dried to obtain a refined sample.

[0116] (4) Purity identification: The purity detection method of the obtained refined sample includes the following steps:

[0117] i. Sample preparation: accurately weigh a certain amount of each refined sample obtained in step (3), dissolve it in a 20% methanol aqueous solution to a fixed volume, and filter it through a 0.22 μm microporous filter membrane to obtain a sample solution;

[0118] j. Liquid chromatography conditions: Agilent 1260 high performance liquid chromatograph and Amethyst C18-H reverse phase column (250 mm × 4.6 mm × 5 μm) were used. The column temperature was set at 25°C. The mobile phase A was a 0.10% by volume aqueous formic acid solution, and the mobile phase B was methanol. The elution gradient was: 0-15 min, 5-80% B; 15-18 min, 80-5% B. The injection volume was 5 μL, the flow rate was 1.0 mL / min, and the detection wavelength was 280 nm.

[0119] k. Injection test: Use an automatic sampler to quantitatively inject the sample solution into the sampler of the liquid chromatograph to obtain a high performance liquid chromatogram of the sample solution;

[0120] 1. Chromatogram analysis: Measure the area of ​​each chromatographic peak on the chromatogram, including the target component peak and the area of ​​other component peaks; the purity of the target component is the ratio of the component peak area to the total peak area multiplied by 100%.

[0121] Specifically, in this example, 1 mg of the purified sample was weighed, dissolved in 1 mL of 20% methanol aqueous solution, and filtered through a 0.22 μm microporous membrane. The purity of each alkaloid in the sample was calculated according to the peak area normalization method. Figure 3 It can be seen that the peak time of magnolia alkaloids is 8.659min, and the HPLC purity is >99%; Figure 4 It can be seen that the peak time of camphor tetrandrine is 8.686min, and the HPLC purity is >99%; Figure 5It can be seen that the peak time of sphenantheline is 9.929min, and the HPLC purity is >97%; Figure 6 It can be seen that the peak time of N-methyldomesticinium is 9.923 min, and the HPLC purity is >97%.

[0122] (5) Structural identification: Combining high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy ( 1 H. 13 C, HSQC, HMBC, COSY) were used to identify the structure of the alkaloid obtained in step (3).

[0123] High-resolution mass spectrometry method: TripleTOF 5600+ quadrupole time-of-flight tandem mass spectrometer equipped with electrospray ion source was used. Detection was performed in positive and negative ion scanning modes (±ESI), electrospray voltage ±5.5kV, ion source temperature 550℃, air curtain gas pressure 30psi, ion source nebulizer gas pressure 50psi, ion source heating auxiliary gas pressure 50psi, declustering potential ±60 V, primary mass spectrometry collision energy ±10eV, secondary mass spectrometry collision energy ±45eV. The primary mass spectrometry scanning range was 100-1500Da, and the secondary mass spectrometry scanning range was 50-1200Da.

[0124] Nuclear magnetic resonance spectroscopy detection method: One-dimensional and two-dimensional nuclear magnetic resonance experiments were performed on the samples using Swiss Bruker AVANCE III HD 400MHZ, and the data were recorded. The samples were dissolved in deuterated dimethyl sulfoxide (DMSO) with 0.03% tetramethylsilane (TMS) as the internal standard.

[0125] Magnolia alkaloids: The compound has a main primary mass peak ( Figure 8 , m / z=342.1657). Further, the specific results of the compound were identified by NMR. Specifically, according to the hydrogen spectrum ( Fig. 9 ) peaks and integration, showing that there are 21 hydrogen signals. Further, using the HMBC relationship ( Fig.12 ) can be used to convert the methoxyl hydrogen signal δ H 3.70 ppm [HC(2-OMe)] and δ C 151.03 ppm (C-2) was correlated to locate the position of the substituent; δ H 6.56 ppm (H-3) and δ C 150.87 ppm (C-1), δ C 120.09 ppm (C-1b), δ C 23.23 ppm (C-4) has a long-range coupling relationship. The coupling strength shows that the above three are 3J coupling, and the chemical shift determined that (C-1) was the hydroxyl substitution position on the aromatic ring; on the other hand, according to the COSY relationship ( Fig.13 ),δ H There is a difference between 3.16,2.84 ppm (H-4) and 3.66, 3.59 ppm (H-5). 3 J coupling, and through the HSQC relationship ( Fig.11 ) is attributed to δ C 23.23 ppm (C-4) and δ C 60.41 ppm (C-5). The above analysis completed the assignment of ring A and ring B.

[0126] According to the carbon spectrum ( Fig.10 ) peaks, and 21 signal peaks can be observed in the signal range between δC 151.32 and 22.23 ppm. For the carbon in the D ring region, we first use the relationship between HSQC and COSY, combined with basic knowledge of chemical shifts on the benzene ring, to convert δ C 109.76ppm and δ C The carbon number on the aromatic ring at 113.33 ppm is the same as that assigned to δ H 6.66ppm (H-9) and δ H 6.44ppm(H-8); using HMBC relationship, we found δ C 150.17 ppm (C-10) and δ H 3.68ppm (H-10-ome) was correlated, and the carbon (C-10) on the benzene ring was determined to be the methoxy substitution point, and (H-8) and (C-10) were found to exist. 3 J CH Through the above analysis, combined with the subsequent signal attribution of other rings, the carbon and hydrogen signals of the D ring were assigned.

[0127] Finally, using the HMBC relationship, we find that H 6.44 ppm (H-8) and δ C 30.37 ppm (C-7) Long range 3 J coupling relationship. According to the COSY relationship, the hydrogen signal δ H There is a peak between 6.44 ppm (H-8) and 6.66 ppm (H-9) 3 J coupling proves the existence of continuous unsubstituted protons on the benzene ring; δ H 4.36 ppm (H-6a) and δ H There is a difference between 3.14 and 2.61 ppm (H-7) 3 J coupling completes the link between ring C and ring D.

[0128] The above analysis completed the analysis of the compound, which is Magnoflorine (CAS: 2141-09-5), with a molecular formula of C 20 H 24 NO 4 + The molecular mass is 342.1700. All the assignment results are shown in Table 2. The structure is shown in Figure 7 As shown in (a) in .

[0129]

[0130] Camphoravine: The compound has a main primary mass spectrum peak ( Fig.14 , m / z=342.1657). Further, its specific structure was identified by NMR. Specifically, according to the hydrogen spectrum ( Fig.15 ) peaks and integration, showing that there are 21 hydrogen signals in total. The signal at the lowest field δH 8.54 ppm has no obvious integer multiple relationship with other hydrogen signals, and the remaining signals range from δH7.89 to 2.76 ppm. Among them, the three hydrogen signals near δH 7.98 to 6.77 ppm are assigned to the protons on the aromatic ring, and the six hydrogen signals at δH 3.29 ppm and δH 3.75 ppm are assigned to the methoxy protons on the aromatic ring. The signals near δH 3.73 to 2.76 ppm and 4.58 ppm are assigned to the protons on the saturated carbon close to the nitrogen atom. According to the carbon spectrum ( Fig.16 ) peaks, and 21 signal peaks can be observed in the signal range between δC 165.49 and 23.17 ppm. The HMBC relationship found that the carbon number at δC 165.49 ppm had no multi-bond correlation with other hydrogen spectra, and it was attributed to the hydrogen signal of δH8.54 ppm through the HSQC relationship, and there was no integer multiple relationship between the hydrogen signal at this location and other hydrogen signals. Combined with the specific process of separation and purification, as well as the specific chemical shift, it can be judged that δC 165.49 ppm and δH 8.54 ppm are residual formic acid NMR signals in the sample, and formic acid and the alkaloid form a quaternary ammonium salt.

[0131] Furthermore, using HMBC ( Fig.18 ) can be used to convert the methoxyl hydrogen signal δ H 3.84 ppm [HC(2-OMe)] and δ C 148.40 ppm (C-2) was correlated to locate the position of the substituent; δ H 6.77 ppm (H-3) and δ C 142.37 ppm (C-1), δ C119.41 ppm (C-1b), δ C 23.17 ppm (C-4) has a long-range coupling relationship. The coupling strength shows that the above three are 3 J coupling, and the chemical shift indicated that (C-1) was the hydroxyl substitution position on the aromatic ring; on the other hand, according to COSY ( Fig.19 ) relationship, δ H There is a difference between 3.18,2.91 ppm (H-4) and 3.75, 3.64 ppm (H-5). 3 J coupling, and then through HSQC ( Fig.17 ) relationship attributes it to δ C 23.17 ppm (C-4) and δ C 60.45 ppm (C-5). The above analysis completed the assignment of ring A and ring B.

[0132] According to the COSY relationship, the hydrogen signals on the benzene ring have no COSY relationship, proving that there are no continuous unsubstituted protons on the benzene ring. H 4.61 ppm (H-6a) and δ H There is a difference between 2.80 and 3.29 ppm (H-7) 3 J coupling; using the HMBC relationship, it is found that δ H 6.85 ppm (H-8) and δ C 28.25 ppm (C-7), δ H 7.98 ppm (H-11) and δ C 120.45 ppm (C-1a) long range 3 J coupling relationship, combining the long-range molecular coupling between the protons (H-11) and (H-8) on the aromatic ring carbon and their corresponding meta carbon atoms 3 J coupling relationship, successfully assigned the carbon and hydrogen signals of the C and D rings, and connected the C and D rings with the A and B rings. Finally, the HMBC relationship was used again to assign the second methoxy hydrogen signal δ H 3.72 ppm [HC(10-OMe)] and δ C The positions of the substituents on the two D rings were distinguished and located by correlation with 146.35 ppm (C-10).

[0133] The above analysis completed the identification of the compound, which is laurifoline (CAS: 7224-61-5). All the attribution results are shown in Table 3. The structure is shown in Figure 7 As shown in (b) in .

[0134]

[0135] Black shell alkaloids: according to Fig. 20 It can be seen that the m / z of the compound is 569.2622. According to the one-dimensional hydrogen spectrum of the compound ( Fig.21 ) integration shows that there are 31 hydrogen signals, with a signal range of δH 7.22 to 2.74 ppm; the signals at δH 7.22 to 6.53 ppm are assigned to protons on the aromatic ring, the signals near δH 3.71 ppm are assigned to methoxy protons on the benzene ring, and the signals at δH 3.25 to 2.74 and 4.24 ppm are assigned to protons on carbon close to nitrogen atoms. One-dimensional carbon spectrum of the compound ( Fig. 22 ) 21 carbon signals were observed, ranging from δC 156.63 to 26.28 ppm. Fig.23 ), it was found that there was no relevant hydrogen at δC 144.63 ppm (C-7, C-7`), which should be a quaternary carbon, but the peak height was higher than that of other hydrogen-substituted carbons, which did not conform to the basic rule that the quaternary carbon signal was relatively low. It was determined that δC 144.63 ppm was the superposition of quaternary carbon signals on two benzene rings; the two hydrogen signals at δH 7.21 ppm (H-10`, 14`) belonged to a tertiary carbon signal peak δC 130.55 ppm (C-10`, C-14`), so it was determined that δC 130.55 ppm was the superposition of tertiary carbon signals on two benzene rings; similarly, the signal at δC 116.28 ppm (C-11`, C-13`) was determined to be the superposition of tertiary carbon signals on two benzene rings; the broader carbon signal at δC55.68~55.33 ppm should be the superposition of two methoxy primary carbon signals and two aliphatic tertiary carbon signals. In addition, four aliphatic secondary carbon signals δC 39.76 ppm (C-3`), 39.69 ppm (C-3), 39.16 ppm (C-α`), and 39.14 ppm (C-α) were found to be obscured under the DMSO-d6 solvent peak and were attributed to δH 2.85 ppm (H-3`), 3.10 ppm (H-3), 3.18 ppm (H-α`), and 2.98 ppm (H-α), respectively.

[0136] Using HMBC spectra ( Fig.24 ) can convert the two sets of methoxy proton signals δ H 3.72 ppm [HC (6-OMe)] and benzene ring carbon δ C 146.58 ppm (C-6) correlated; at the same time δ H 6.65 ppm (H-5) and δ C146.58 ppm (C-6), 144.61 ppm (C-7), 128.00 ppm (C-8a), 26.28 ppm (C-4) have long-range coupling relationships. Judging from the coupling strength, (H-5) and (C-6) should be 2 J coupling, the rest are 3 J coupling; and δ H 6.63 ppm (H-8) and δ C 146.58 ppm (C-6), 123.65 ppm (C-4a), and 55.33 ppm (C-1) have long-range coupling relationships, and the coupling strengths show that they are 3 J coupling; According to the COSY spectrum ( Fig.25 ),δ H There is a peak between 2.74 ppm (H-4) and 3.10 ppm (H-3) 3 J coupling, δ H 4.21 ppm (H-1) and 2.98 ppm (H-α) 3 J coupling. This molecule is highly symmetrical, and all the above coupling relationships have mirror-image coupling relationships with very similar chemical shifts (indicated by the label with ` in the table below); through all the above HMBC and COSY coupling relationships and chemical shift information, the signal attribution of the A ring and B ring (A` ring and B` ring) of this molecule can be made.

[0137] By COSY spectrum ( Fig.25 ) found two sets of overlapping carbon peaks in the benzene ring region δ C 116.28 ppm (C-11`, C-13`) and δ C 130.55 ppm (C-10`, C-14`), the hydrogen on the corresponding carbon has a COSY relationship, which is a typical spectrum of disubstituted para-position on the benzene ring; on the other hand, δ H 6.80 ppm (C-13`, C-11`) and δ C 131.41 ppm (C-9`) has HMBC relationship, judging by the coupling intensity, it is 3 J coupling, and δ H 3.18 ppm (C-α`) and δ C There is an HMBC relationship at 130.55 ppm (C-10`, C-14`), and the C` ring was successfully assigned through the above relationship.

[0138] The above analysis completed the analysis of the compound, which is Lindoldhamine (CAS: 60342-37-2), with a molecular formula of C34 H 36 N 2 O 6 , molecular weight is 568.2573. All the assignment results are shown in Table 4, the structure is as follows Figure 7 As shown in (c) in .

[0139]

[0140] N-methyldomesticinium: Fig.26 It can be seen that m / z = 340.1533. According to the hydrogen spectrum of the compound ( Fig. 27 ), there are 22 hydrogen signals in total, with a signal range of δ H 7.88~2.80 ppm. H The two hydrogen signals at 6.04 ppm were assigned to benzodioxole (-O-CH 2 -O-) group. Carbon spectrum ( Fig.28 ) 21 signal peaks can be observed, and the signal range is δ C The hydrogen signals on the benzene ring have no COSY relationship, proving that there are no continuous unsubstituted protons on the benzene ring.

[0141] Using the HMBC relationship ( Fig.30 ) can be used to convert the methoxyl hydrogen signal δ H 3.86 ppm [HC(2-OMe)] and δ C 148.73 ppm (C-2) was correlated to locate the substituent position; δ H 6.85 ppm (H-3) and δ C 142.97ppm (C-1), δ C 119.99 ppm (C-1b), δ C 23.55 ppm (C-4) has a long-range coupling relationship. The coupling strength shows that the above three are 3 J coupling, and the chemical shift determined that (C-1) was the hydroxyl substitution position on the aromatic ring; the HSQC relationship ( Fig.29 ) is attributed to δ C 23.55 ppm (C-4) and δ C 60.86 ppm (C-5). After the above analysis, the assignment of ring A and ring B was completed.

[0142] According to the COSY relationship ( Fig.31 ),δ HThere is a difference between 4.61 ppm (H-6a) and 2.80,3.29 ppm (H-7) 3 J coupling, while using the HMBC relationship ( Fig.30 ), and found that δ H 7.88 ppm (H-11) and δ C 120.06 ppm (C-1a), δ H 6.95ppm (H-8) and δ C 29.11 ppm (C-7) has a long-range coupling relationship, and the coupling relationship between the protons (H-11) and (H-8) on the benzene ring carbon and their corresponding meta-carbon atoms can be found. The relevant coupling strengths are all 3 J coupling, combining benzodioxole (-O-CH 2 -O-) group on the proton δ H 6.04 ppm and δ C 146.62 ppm (C-9), δ C 146.31 ppm (C-10) 3 J long-range coupling relationship, successfully attributed the C and D rings and connected them to the A and B rings.

[0143] Finally, through all the above analyses, the substance was identified as N-methyldomesticinium, CAS: 780712-00-7, with a molecular formula of C 20 H 22 NO 4 , molecular mass is 340.1549.

[0144] All the attribution results are shown in Table 5, and the structure is as follows Figure 7 As shown in (d) in .

[0145]

[0146] Example 2

[0147] According to a method for enriching and separating alkaloid monomers in camphor tree seed kernels of the present invention, Example 2 provides a method for enriching and separating alkaloids in camphor tree seed kernels, comprising the following steps:

[0148] (1) 500 g of camphor seed kernels were mixed with water at a material-liquid mass ratio (camphor seed kernels: water) of 1:3, and the mixture was added to a colloid mill for wet ultrafine grinding for 5 min to obtain a camphor seed kernel slurry; the camphor seed kernel slurry was centrifuged at 2000 rpm for 30 min to obtain an upper emulsified phase, a middle aqueous phase, and a lower slag phase.

[0149] (2) The process of dynamic adsorption of each alkaloid monomer in the aqueous phase by the macroporous resin includes: the height-to-diameter ratio of the macroporous resin column is 15:1, the column volume is 300 mL, the intermediate layer aqueous phase obtained in step (1) is flowed through the ADS-5 macroporous resin column at a flow rate of 1.5 BV / h and a sample volume of 5.0 BV. After the adsorption is completed, the column is first washed with 3.0 BV of distilled water to remove impurities such as proteins and polysaccharides, and then washed with 5 BV 5%, 5 BV 20% and 5 BV in sequence. The product was eluted with 40% ethanol aqueous solution at a flow rate of 3.0 BV / h. The effluent was collected every 1 BV, and the eluent was qualitatively detected for the presence of alkaloid monomers. 20% of the desorption solution (mainly containing cynaroline and camphoravine) and 40% of the desorption solution (mainly containing cynaroline and N-methyldomesticinium) were recovered, concentrated under reduced pressure, and vacuum freeze-dried to obtain enrichment 1 and enrichment 2. The contents of cynaroline and camphoravine in enrichment 1 were 52.77% and 45.79%, respectively; the contents of cynaroline and N-methyldomesticinium in enrichment 2 were 47.14% and 52.43%, respectively.

[0150] (3) Separating by preparative liquid chromatography, the enriched product 1 and the enriched product 2 were respectively dissolved in 20% methanol water and loaded onto the sample, the loading amount for preparative liquid chromatography separation was 5 mg, and the flow rate was 10 mL / min; the remaining operation method was the same as step (3) of Example 1; after purity identification, the HPLC purity of magnolamine and camphoravine was greater than 99%; the HPLC purity of scutellarine and N-methyldomesticinium was greater than 97%.

[0151] Example 3

[0152] According to a method for enriching and separating alkaloid monomers in camphor tree seed kernels of the present invention, Example 2 provides a method for enriching and separating alkaloids in camphor tree seed kernels, comprising the following steps:

[0153] (1) 500 g of camphor seed kernels were mixed with water at a material-liquid mass ratio (camphor seed kernels: water) of 1:5, and the mixture was added to a colloid mill for wet ultrafine grinding for 6 min to obtain a camphor seed kernel slurry; the camphor seed kernel slurry was centrifuged at 8000 rpm for 5 min to obtain an upper emulsified phase, a middle aqueous phase, and a lower slag phase.

[0154] (2) During the dynamic adsorption of each alkaloid monomer in the aqueous phase by the macroporous resin, the column volume was 500 mL, the flow rate of the intermediate layer aqueous phase was 1.5 BV / h, the elution flow rate of the ethanol aqueous solution was 4.5 BV / h, and the remaining operation method was the same as step (2) of Example 1. The contents of cynaroline and camphoravine in the obtained enrichment 1 were 49.50% and 44.13%, respectively; the contents of cynaroline and N-methyldomesticinium in the enrichment 2 were 44.85% and 50.37%, respectively.

[0155] (3) Separating by preparative liquid chromatography, the enriched product 1 and the enriched product 2 were respectively dissolved in 20% methanol water and loaded onto the sample, the loading amount for preparative liquid chromatography separation was 50 mg, and the flow rate was 30 mL / min; the remaining operation method was the same as step (3) of Example 1; after purity identification, the HPLC purity of magnolamine and camphoravine was greater than 99%; the HPLC purity of scutellarine and N-methyldomesticinium was greater than 97%.

[0156] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for enriching and separating alkaloid monomers in camphor tree seed kernels, characterized in that: The following steps are involved: Step S1: Preparation of the middle layer aqueous phase: Mix camphor seed kernels and water according to the material-liquid mass ratio, add them into a colloid mill for wet ultrafine grinding, and obtain camphor seed kernel slurry; centrifuge the obtained camphor seed kernel slurry to separate liquid-liquid-solid, and obtain an upper emulsified phase, a middle layer aqueous phase, and a lower layer slag phase; Step S2: Enrichment of alkaloid monomers in the intermediate aqueous phase: using a macroporous adsorption resin to statically or dynamically adsorb the alkaloid monomers in the intermediate aqueous phase obtained in step S1, and washing with distilled water, and then eluting the macroporous resin with ethanol solutions of different concentrations, wherein the static adsorption method uses 5%, 20% and 40% ethanol for elution in sequence; the dynamic adsorption method uses 5BV5%, 5BV20% and 5BV40% ethanol for gradient elution in sequence; using a monomer alkaloid qualitative detection method to qualitatively detect whether the eluent contains the corresponding alkaloid monomer, combining the ethanol eluents containing the corresponding alkaloid monomers, the ethanol eluents include 20% and 40% ethanol eluents, and concentrating and drying them to obtain enrichment 1 and enrichment 2; the content of magnolamine and camphoravine in enrichment 1 is at least 40%; the content of scutellarine and N-methyldomesticinium in enrichment 2 is at least 40%; Step S3: Preparative liquid chromatography to obtain refined alkaloid monomers: The alkaloid monomer enriched product obtained in step S2 is subjected to preparative liquid separation, and eluted with formic acid water-methanol solution according to an elution gradient, with a flow rate of 10-30 mL / min, a sample load of 5-50 mg, and the elution procedure of enriched product one is: 0-3 min, 20-30% B; 3-15 min, 30-34%; 15-17 min, 34-100%; 17-25 min, 100-100%; 25-27 min, 100-20%; 27-35 min, 20-20%; the elution procedure of enriched product two is: 0-6 min, 23- 27%B; 6-24min, 29-37%; 24-28min, 37-100%; 28-36min, 100-100%; 36-38min, 100-23%; 38-50min, 23-23%; The eluted fractions were detected by ultraviolet detector, and the effluent was qualitatively detected to determine whether it contained the corresponding alkaloid monomers, the fractions containing the corresponding alkaloid monomers were combined and concentrated, and after drying, magnolamine and camphoravine with a purity of more than 95% purified from the enrichment one, and scutellarine and N-methyldomesticinium with a purity of more than 95% purified from the enrichment two were obtained.

2. The method for enriching and separating alkaloid monomers in camphor tree seed kernels according to claim 1, characterized in that: In step S1, the mass ratio of camphor tree seed kernel to water liquid is 1:3-1:5, the wet ultrafine grinding time is 4-6 minutes, the centrifugal speed is 2000-8000rpm, the centrifugal time is 5-30 minutes, and the content of camphor tree seed kernel oil in the lower layer residue phase is less than 5%.

3. The method for enriching and separating alkaloid monomers in camphor tree seed kernels according to claim 2, characterized in that: The static adsorption process in step S2 is as follows: adding the pretreated macroporous resin to the intermediate layer aqueous phase obtained in step S1, stirring for adsorption at room temperature, the adsorption time is 1-2 hours, filtering to obtain the macroporous resin after saturation adsorption, washing the macroporous resin with distilled water, and then sequentially adding 5%, 20% and 40% ethanol aqueous solutions for desorption, the volume mass ratio of the desorption liquid to the macroporous resin is 5-10, and the desorption time is 1-2 hours.

4. The method for enriching and separating alkaloid monomers in camphor tree seed kernels according to claim 2, characterized in that: The dynamic adsorption process in step S2 is as follows: the intermediate layer aqueous phase obtained in step S1 is passed through a macroporous resin column, the aspect ratio of the macroporous resin column is 15:1, the column volume is 50-500 mL, and the loading flow rate is 1.5-3.0 BV / h; after the adsorption is completed, the column is first washed with distilled water, and then eluted with 5BV5%, 5BV20% and 5BV40% ethanol aqueous solutions in sequence, the flow rate is 3.0-4.5 BV / h, and the effluent is collected once every 1 BV.

5. The method for enriching and separating alkaloid monomers in camphor tree seed kernels according to claim 1, characterized in that: The method for qualitatively detecting whether the eluate in step S2 or the effluent in step S3 contains the corresponding alkaloid monomer comprises the following steps: a. Preparation of standard solution: Weigh a certain amount of alkaloid monomer standard, accurately weigh it, dissolve it in methanol aqueous solution and make up to volume, filter it with a 0.22 μm microporous filter membrane to obtain a standard solution with a concentration of 200 μg / mL; b. Preparation of sample solution: Take the eluate in step S2 or the effluent in step S3 and filter it with a 0.22 μm microporous membrane to obtain a sample solution; c. HPLC detection conditions: Agilent 1260 HPLC; Amethyst C18-H reverse phase column size 250mm×4.6mm×5μm, column temperature 25℃; mobile phase: phase A is 0.10% by volume formic acid aqueous solution, phase B is methanol; elution gradient: 0-15min, 5-80%B; 15-18min, 80-5%B; injection volume 5μL; flow rate 1.0mL / min; detection wavelength 280nm; Sample detection: Analyze the standard solution under the above detection conditions and record its retention time; analyze the sample solution under the above conditions and observe whether there is a peak in the liquid chromatogram of the sample that is consistent with the retention time of the standard; if a peak consistent with the retention time of the standard appears in the chromatogram of the sample, it means that the sample contains the target component.

6. The method for enriching and separating alkaloid monomers in camphor tree seed kernels according to claim 1, characterized in that: The equipment used for the preparative liquid chromatography separation in step S3 is LC 3000 medium-pressure preparative liquid, and the chromatographic column is HPLCONE-5C18A. The chromatographic column of the preparative liquid chromatography uses octadecylsilane bonded silica gel as a filler, with a column length of 250 mm, an inner diameter of 30.0 mm, and a particle size of 5 μm.

7. The method for enriching and separating alkaloid monomers in camphor tree seed kernels according to claim 6, characterized in that: In the step S3, the mobile phase A of the preparative liquid chromatography separation is a 0.15% by volume aqueous formic acid solution, and the mobile phase B is methanol.

8. The method for enriching and separating alkaloid monomers in camphor tree seed kernels according to claim 1, characterized in that: The method for detecting the content of alkaloid monomers in the enrichment obtained in step S2 comprises the following steps: d. Solution preparation: Dissolve the alkaloid monomer standard in methanol aqueous solution to obtain a 0.5 mg / mL standard stock solution, and then use methanol aqueous solution as a diluent to dilute to obtain 500 μg / mL, 250 μg / mL, 125 μg / mL, 50 μg / mL, 25 μg / mL, and 10 μg / mL standard solutions; Take the alkaloid monomer enriched product obtained in step S2 and dissolve it in methanol aqueous solution, filter it with a 0.22 μm microporous filter membrane to obtain a sample solution; e. Liquid chromatography conditions: Agilent 1260 high performance liquid chromatograph, Amethyst C18-H reverse phase column size 250mm×4.6mm×5μm, column temperature set at 25°C, mobile phase A was 0.10% by volume formic acid aqueous solution, mobile phase B was methanol, elution gradient was: 0-15min, 5-80%B; 15-18min, 80-5%B, injection volume was 5μL, flow rate was 1.0mL / min, detection wavelength was 280nm; f. Injection test: Use an automatic sample injector to quantitatively inject the standard solution and the sample solution into the sample injector of the liquid chromatograph, and obtain the UV absorption peak area of ​​the standard at different concentrations and the UV absorption peak area of ​​the sample solution; g. Establishment of standard curve: With the UV absorption peak area as the ordinate and the corresponding alkaloid monomer standard concentration as the abscissa, the standard curve of the standard is obtained after linear fitting; h. Calculation of alkaloid monomer content in the sample: Substitute the obtained ultraviolet absorption peak area of ​​the alkaloid monomer enrichment into the standard curve to calculate the concentration of the alkaloid monomer in the sample, and divide it by the concentration of the enrichment sample to obtain the alkaloid monomer content in the enrichment.

9. The method for enriching and separating alkaloid monomers in camphor tree seed kernels according to claim 1, characterized in that: The method for detecting the purity of the alkaloid monomer obtained in step S3 comprises the following steps: i. Sample preparation: accurately weigh a certain amount of the purified alkaloid monomer sample obtained in step S3, dissolve it in methanol aqueous solution to a fixed volume, and filter it through a 0.22 μm microporous membrane to obtain an alkaloid monomer sample solution; j. Liquid chromatography conditions: Agilent 1260 high performance liquid chromatograph, Amethyst C18-H reverse phase column size 250mm×4.6mm×5μm, column temperature set at 25°C, mobile phase A was 0.10% by volume formic acid aqueous solution, mobile phase B was methanol, elution gradient: 0-15min, 5-80%B; 15-18min, 80-5%B, injection volume 5μL, flow rate 1.0mL / min, detection wavelength 280nm; k. Injection test: Use an automatic sample injector to quantitatively inject the alkaloid monomer sample solution into the sample injector of the liquid chromatograph to obtain a high performance liquid chromatogram of the sample solution; 1. Chromatographic analysis: Measure the area of ​​each chromatographic peak on the chromatogram, including the target component peak and the areas of other component peaks; the purity of the target component is the ratio of the component peak area to the total peak area multiplied by 100%.

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