A method for synchronous preparation of alkaloids and flavonoids in lotus plumule extract

Abstract

The application relates to a synchronous preparation method of alkaloids and flavonoids in lotus plumule extract, characterized by the following steps: using a silica gel matrix filler with a mixed mode of hydrophobicity + cation exclusion, realizing synchronous enrichment and separation of the two types of components by using the difference between the hydrophobicity and charge properties of the alkaloids and the flavonoids, and having a higher yield. The preparation method is fast, simple, environment-friendly, has high comprehensive utilization rate of medicinal materials and can be mass-produced, and provides a technical scheme for purifying and preparing active components in lotus plumule.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Chinese herbal medicine separation and purification, in particular to a synchronous preparation method of alkaloids and flavonoids in lotus plumule extract. BACKGROUND

[0002] Lotus plumule is the dried young leaves and radicles of mature seeds of Nelumbo nucifera Gaertn. It has the pharmacological effects of strengthening heart and lowering blood pressure, inhibiting tumor cells, etc. Lotus plumule contains alkaloids, flavonoids, polysaccharides, volatile oils and other substances, among which alkaloids and flavonoids are the main active ingredients. In the 2020 edition of Chinese Pharmacopoeia, methyl lotus plumule alkaloid is used as the quality control index of lotus plumule.

[0003] Alkaloids are one of the main components of lotus plumule, mainly including lotus plumule alkaloids and lotus plumule quaternary ammonium alkaloids, which have the pharmacological effects of lowering blood pressure, anti-arrhythmia, lowering blood sugar, anti-tumor, etc. Meanwhile, lotus plumule also contains a high content of flavonoids, mainly including syringin and rutin, which have the effects of antioxidant, free radical scavenging, inhibition of tumor cells, etc. This fully shows that alkaloids and flavonoids in lotus plumule have different biological activities.

[0004] There are many methods for purifying alkaloids in lotus plumule, such as solvent extraction, macroporous resin, ion exchange resin, etc. For example, the commonly used purification method uses solvent extraction and crystallization method to separate and purify alkaloid components, such as patent CN98121706.0, which has the problems of complex process and low yield; for example, CN201810822128.4 uses macroporous resin and silica gel column chromatography for two-step purification to prepare effective components in lotus plumule, which has a complex preparation process, uses more toxic organic solvents, and has greater harm to the environment and human body, which is not suitable for large-scale industrial production; for example, CN201110170851.7 uses cation exchange resin to separate and enrich alkaloids and flavonoids, which has low yield and salt in the eluent, and complex post-treatment. At the same time, the current purification means is mostly for the preparation of alkaloids in lotus plumule, and the flavonoids are lost, and the synchronous preparation of the two types of active ingredients is not realized, resulting in low comprehensive utilization rate of medicinal materials, so it is necessary to develop a technical scheme for enriching and separating alkaloids and flavonoids from lotus plumule, which is fast, simple, environment-friendly, has high comprehensive utilization rate of medicinal materials and can be mass-produced. SUMMARY

[0005] The application mainly provides a synchronous preparation method of alkaloids and flavonoids in lotus plumule extract, which uses a silica gel matrix filler with a mixed mode of hydrophobicity and cation exclusion, realizes synchronous enrichment and separation of the two components based on the difference in hydrophobicity and charge properties of alkaloids and flavonoids, improves the yield and comprehensive utilization rate of medicinal materials, and provides a technical scheme for the preparation of flavonoids and alkaloids in lotus plumule.

[0006] The synchronous preparation method of alkaloids and flavonoids in lotus plumule extract in the application comprises the following steps: selection of a silica gel matrix filler, preparation of a lotus plumule extract solution, enrichment of the sample on the silica gel matrix filler, elution and washing, post-treatment of the eluate, and analysis.

[0007] The specific operation steps are as follows:

[0008] a. Selection of a silica gel matrix filler: a silica gel matrix filler with a mixed mode of hydrophobicity and cation exclusion is selected; the silica gel matrix filler refers to a group with hydrophobic properties and a cation on a silica gel matrix; in the application, the group with hydrophobic properties adopts one or more of normal alkanes with a carbon atom number of 1-30; the group with a cation adopts one or more than two of ligands with positive charges such as secondary amine groups, tertiary amine groups, quaternary amines, nitrogen-containing heterocyclic rings, etc.; the particle size is 8-100 μm, the pore size is 100-300 A, and the surface area is 300-800 m2 / g. The inner diameter of the chromatographic column is 10-100 mm, and the bed height of the chromatographic column is 150-250 mm.

[0009] b. Preparation of a lotus plumule extract solution: dissolve the lotus plumule extract in an acid aqueous solution with a volume fraction of 0-2% of a reverse-phase organic solvent, and prepare a solution with a concentration of 150 mg / mL-250 mg / mL;

[0010] Further, the lotus plumule extract in step b includes a crude extract obtained by one or more than two of extraction methods such as decoction, reflux, ultrasonic or microwave-assisted extraction using one or more than two solvents of water, methanol or ethanol, or a refined extract obtained by column chromatography from the crude extract;

[0011] Further, the acid in step b is one or more than two of formic acid, acetic acid, hydrochloric acid or phosphoric acid, etc., and the concentration is 0%-2%;

[0012] c. Enrichment, rinsing, and elution of samples on silica-based packing material: The sample solution was loaded onto the silica-based packing material at a loading volume of 20-300 g / L. Under a reversed-phase organic solvent-acid-water system, rinsing, elution, and column flushing were performed sequentially. Based on the differences in hydrophobicity and charge between alkaloids and flavonoids, elution was performed sequentially at different reversed-phase organic solvent-acid-water ratios with increasing reversed-phase organic solvent content to obtain eluent A containing flavonoids and eluent B containing alkaloids.

[0013] Further, the rinsing operation in step c is to use a reversed-phase organic solvent with a volume fraction of 0-20% acid aqueous solution to rinse impurities in 2-4 column volumes;

[0014] Further, the elution of the flavonoid components in step c is performed by using a reversed-phase organic solvent with a volume fraction of 15%-30% acidic aqueous solution to elute the flavonoid components with 2-4 column volumes to obtain eluent A;

[0015] Further, the elution of the alkaloid components in step c is performed by using a reversed-phase organic solvent with a volume fraction of 50%-70% acid aqueous solution to elute the alkaloid components with 2-4 column volumes to obtain eluent B;

[0016] Further, the column flushing operation in step c is to flush the column with 2-4 column volumes using a 95%-100% acid aqueous solution of a reversed-phase organic solvent.

[0017] Furthermore, the reversed organic solvent mentioned in steps b and c is one or more of acetonitrile, ethanol, or methanol;

[0018] d. Post-processing and analysis of the eluent: Eluent A and eluent B from the above steps were combined and collected separately; after concentration, deacidification, lyophilization or spray drying, dry powder samples of alkaloids and flavonoids were obtained, respectively. The samples were then analyzed by HPLC. The obtained dry powders of alkaloids and flavonoids were prepared into aqueous solutions and analyzed by HPLC. The peak areas were compared with those of lotus seed heart extract solution to obtain data on purity and yield. Specific HPLC analysis conditions are as follows:

[0019] Instrument: Huapu Scientific Instruments S6000 (DAD Detector)

[0020] Column: C18HD, 4.6*250mm, 5μm

[0021] Detection wavelength: 270nm

[0022] Flow rate: 1 mL / min

[0023] Column temperature: 30℃

[0024] Injection volume: 10 μL

[0025] Gradient condition:

[0026]

[0027] The composition and purity of the alkaloids and flavonoids obtained in this invention can be analyzed by HPLC, and the purity and yield data can be obtained by calculation. HPLC analysis and calculation show that the purity of the alkaloids can reach 15%-45%, and the yield is 90%-100% (calculated using lotus seed quaternary ammonium alkaloids as a standard); the purity of the flavonoids can reach 10%-30%, and the yield is 90%-100% (calculated using shampoosine as a standard).

[0028] The present invention has the following advantages:

[0029] 1. The method involved in this invention utilizes the difference in hydrophobic and charge properties between alkaloids and flavonoids to achieve the simultaneous preparation of alkaloids and flavonoids in lotus seeds on a single separation matrix. The preparation method is rapid, simple, environmentally friendly, has a high comprehensive utilization rate of medicinal materials, and can be mass-produced, providing a technical solution for the preparation of active ingredients of alkaloids and flavonoids in lotus seeds.

[0030] 2. The eluent used in the method of the present invention involves only ethanol, water and acid solution. The eluent composition is simple, which eliminates the complicated elution and post-processing process. The overall elution conditions are easy to implement and post-process, which is conducive to large-scale production.

[0031] 3. The method for enrichment and separation of alkaloids and flavonoids in lotus seed cores according to the present invention can achieve the following results: the purity of alkaloids can reach 15%-45%, and the yield is 90%-100% (calculated using lotus seed core quaternary ammonium alkaloids as a standard); the purity of flavonoids can reach 10%-30%, and the yield is 90%-100% (calculated using shampooside as a standard), with high recovery rate of target components. Attached Figure Description

[0032] Figure 1 Elution curves of the Hanbang liquid chromatography system;

[0033] Figure 2 HPLC analysis results of eluent A (flavonoids) and eluent B (alkaloids) and their comparison chromatograms with the original extract of lotus seed heart.

[0034] Figure 3 Images of freeze-dried alkaloid and flavonoid powders, as well as spray-dried lotus seed heart extract powder; Detailed Implementation

[0035] The present invention will be further illustrated below with examples. These examples are merely illustrative and not intended to limit the scope of the invention.

[0036] Example 1:

[0037] The sample was an aqueous extract of lotus seed hearts, purchased from Xi'an Qiancao Biotechnology Co., Ltd. The extract was prepared into a 170 mg / mL 0.1% formic acid aqueous solution, filtered through a 0.45 μm membrane, and then used. C18CH-W silica gel matrix packing material purchased from Dalian Sipu Co., Ltd. was used; the packing material specifications were a particle size of 10 μm and a pore size of [missing information]. The chromatographic column was 30*250mm with a column volume of 180mL. A Hanbang liquid chromatography system was used for preparation. First, the column was equilibrated with 0.1% FA. After equilibration, 30mL of sample was injected at a flow rate of 20mL / min, with a sample loading of 28g / L (the loading was calculated as the ratio of the sample mass to the column volume). Then, at a flow rate of 20mL / min, the column was sequentially eluted with 0.1% formic acid (v / v) for two column volumes to remove impurities, eluted with 20% ethanol-0.1% formic acid (v / v) for two column volumes to elute flavonoids, eluted with 50% ethanol-0.1% formic acid (v / v) for alkaloids, and finally rinsed with 95% ethanol-0.1% formic acid (v / v) for two column volumes until no peak was observed. The eluents of flavonoids and alkaloids were collected, concentrated in a rotary evaporator, and then deacidified using C18 packing material from Dalian Sipu Company. The fractions were concentrated in a rotary evaporator and freeze-dried in a freeze dryer to obtain freeze-dried powders of flavonoids and alkaloids.

[0038] Example 2:

[0039] The sample was an aqueous extract of lotus seed hearts, purchased from Xi'an Qiancao Biotechnology Co., Ltd. The extract was prepared into a 200 mg / mL 0.5% formic acid aqueous solution, filtered through a 0.45 μm membrane, and then used. XCharge C18 silica gel matrix packing material purchased from Dalian Sipu Co., Ltd. was used, with a particle size of 10 μm and a pore size of [missing information]. The chromatographic column was 30*250mm with a column volume of 180mL. A Hanbang liquid chromatography system was used for preparation. First, the column was equilibrated with 0.5% FA. After equilibration, 60mL of sample was injected at a flow rate of 20mL / min, loading 67g / L. Then, the column was eluted sequentially at a flow rate of 20mL / min with 2 column volumes of 0.5% formic acid (v / v) for impurity removal, 2 column volumes of 30% ethanol-0.5% formic acid (v / v) for flavonoids, 70% ethanol-0.5% formic acid (v / v) for alkaloids, and 2 column volumes of 95% ethanol-0.5% formic acid (v / v) for flushing until no peak was observed. The eluents of flavonoids and alkaloids were collected, concentrated using a rotary evaporator, and then deacidified using C18 packing material from Dalian Sipu Company. The fractions were further concentrated using a rotary evaporator and then freeze-dried to obtain freeze-dried powders of flavonoids and alkaloids. The preparative HPLC chromatograms from Hanbang are attached in this case study. Figure 1 The HPLC chromatograms of the eluent containing flavonoids and alkaloids are attached. Figure 2 The lyophilized powder of flavonoid and alkaloid eluent is attached. Figure 3 .

[0040] Implementation Case 3:

[0041] The sample was a spray-dried powder of lotus seed heart aqueous extract, purchased from Xi'an Qiancao Biotechnology Co., Ltd. The spray-dried extract powder was prepared into a 233 mg / mL 1% formic acid aqueous solution, filtered through a 0.45 μm membrane, and then used. XCharge C18 silica gel matrix packing material purchased from Dalian Sipu Co., Ltd. was used, with a particle size of 10 μm and a pore size of [missing information]. The chromatographic column was 10*150mm with a column volume of 12mL. A Hanbang liquid chromatography system was used for preparation. First, the column was equilibrated with 1% FA. After equilibration, 10mL of sample was injected at a flow rate of 4mL / min, loading 194g / L. Then, the column was eluted sequentially at a flow rate of 4mL / min for two column volumes of 1% formic acid (v / v) to remove impurities, eluted with 15% methanol-1% formic acid (v / v) for two column volumes to elute flavonoids, eluted with 60% methanol-1% formic acid (v / v) for alkaloids, and finally rinsed with 100% methanol-1% formic acid (v / v) for two column volumes until no peak was observed. The eluents of flavonoid and alkaloid components were collected, concentrated in a rotary evaporator, and then deacidified using C18 packing material from Dalian Sipu Company. The fractions were then concentrated in a rotary evaporator and freeze-dried to obtain freeze-dried powders of flavonoid and alkaloid components.

[0042] HPLC analysis was performed on the lyophilized powders obtained in Examples 1-3: Analytical method:

[0043] Instrument: Huapu Scientific Instruments S6000 (DAD Detector)

[0044] Column: C18HD, 4.6*250mm, 5μm

[0045] Detection wavelength: 270nm

[0046] Flow rate: 1 mL / min

[0047] Column temperature: 30℃

[0048] Injection volume: 10 μL

[0049] Gradient condition:

[0050]

[0051] Using the method provided in Example 4, the purity and yield of the relevant alkaloids and flavonoids in Examples 1 to 3 were analyzed and calculated by HPLC, as shown in the table below:

[0052]

[0053] Through three implementation cases, it can be seen that the enrichment and separation methods for alkaloids and flavonoids in lotus seeds can achieve the following results: the purity of alkaloids can reach 15%-45%, and the yield is 90%-100% (calculated using lotus seed quaternary ammonium alkaloids as a standard); the purity of flavonoids can reach 10%-30%, and the yield is 90%-100% (calculated using shampooside as a standard).

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing alkaloids and flavonoids from lotus seed heart extract, characterized in that, Includes the following steps: Lotus seed heart extract was prepared into a 170 mg / mL solution of 0.1% formic acid, filtered through a 0.45 μm membrane, and set aside for use. A C18CH-W silica gel matrix was used as the packing material, with a particle size of 10 μm and a pore size of 100 Å. The chromatographic column was 30*250 mm with a volume of 180 mL. The preparation was performed using a Hanbang liquid chromatography system: first, the column was equilibrated with 0.1% formic acid. After equilibration, 30 mL of sample was injected at a flow rate of 20 mL / min, with a sample loading of 28 g / L. The sample loading was calculated as the ratio of the sample mass to the column volume. Then, the column was washed sequentially with 0.1% formic acid water (v / v) at a flow rate of 20 mL / min for two column volumes to remove impurities, followed by 20% ethanol (v / v). The column was eluted with 2 column volumes of % formic acid water v / v to remove flavonoids, 50% ethanol-0.1% formic acid water v / v to remove alkaloids, and 95% ethanol-0.1% formic acid water v / v to flush the column until no peaks were observed. The eluents of flavonoids and alkaloids were collected, concentrated in a rotary evaporator, and then deacidified using C18 packing material. The fractions were concentrated in a rotary evaporator and freeze-dried to obtain freeze-dried powders of flavonoids and alkaloids.

2. A method for preparing alkaloids and flavonoids from lotus seed heart extract, characterized in that, Includes the following steps: Lotus seed heart extract was prepared into a 200 mg / mL solution of 0.5% formic acid, filtered through a 0.45 μm membrane, and set aside for use. An XCharge C18 silica gel matrix was used as the packing material, with a particle size of 10 μm and a pore size of 100 Å. The chromatographic column was 30*250 mm with a column volume of 180 mL. The preparation was performed using a Hanbang liquid chromatography system: first, the column was equilibrated with 0.5% formic acid. After equilibration, 60 mL of sample (67 g / L) was injected at a flow rate of 20 mL / min. Then, the sample was eluted sequentially at a flow rate of 20 mL / min with 0.5% formic acid solution (v / v) for two column volumes to remove impurities, followed by elution with 30% ethanol-0.5% formic acid solution (v / v) for two columns. Elute the column with one column volume of flavonoids, elute the column with 70% ethanol-0.5% formic acid water v / v to alkaloids, and rinse the column with 95% ethanol-0.5% formic acid water v / v until no peaks are observed. Collect the eluents of flavonoids and alkaloids, concentrate them in a rotary evaporator, and then deacidify them using C18 packing. The fractions are concentrated in a rotary evaporator and freeze-dried to obtain freeze-dried powders of flavonoids and alkaloids.

3. A method for preparing alkaloids and flavonoids from lotus seed heart extract, characterized in that, Includes the following steps: The lotus seed heart extract was spray-dried into a 233 mg / mL 1% formic acid aqueous solution, filtered through a 0.45 μm membrane, and set aside for later use. XCharge C18 silica gel matrix packing material was used, with a particle size of 10 μm and a pore size of 100 μm. The chromatographic column was 10*150mm with a volume of 12mL. Preparation was performed using a Hanbang liquid chromatography system: First, the column was equilibrated with 1% formic acid. After equilibration, 10mL of sample was injected at a flow rate of 4mL / min, with a sample loading of 194g / L. Then, the column was eluted sequentially at a flow rate of 4mL / min for two column volumes of 1% formic acid water v / v for impurity removal, followed by elution with 15% methanol-1% formic acid water v / v for flavonoids, 60% methanol-1% formic acid water v / v for alkaloids, and finally eluted with 100% methanol-1% formic acid water v / v for flushing until no peaks were observed. The eluents of flavonoids and alkaloids were collected, concentrated using a rotary evaporator, and then deacidified using C18 packing material. The fractions were concentrated again using a rotary evaporator and then lyophilized to obtain lyophilized powders of flavonoids and alkaloids.

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