WO2020192163A1 - Refined american ginseng total saponin component and method for purifying and preparing american ginseng total saponin - Google Patents

Abstract

A method for extracting and purifying American ginseng total saponin, and American ginseng total saponin prepared using said method; using American ginseng as the raw material, extracting a crude extract solution using ethanol, clarifying using membrane filtration techniques, using high performance liquid chromatography techniques to implement refinement, and using ion exchange gel chromatography to decolourise and remove impurities.

Description

A kind of refined American ginseng total saponins components and a method for purifying and preparing American ginseng total saponins Technical field

The invention relates to a preparation process of traditional Chinese medicine, and mainly relates to a method for preparing panax quinquefolium saponins by applying high-performance liquid phase preparation and ion exchange chromatography technology.

Background technique

American ginseng (scientific name: Panax quinquefolius) is a perennial plant of the genus Araliaceae ginseng. It has the dual effects of nourishing yin and nourishing qi, calming the mind, nourishing the mind and nourishing energy, clearing heat and promoting fluid, reducing heat and relieving heat. It is the special feature of American ginseng to nourish but not dry; Pharmacological studies have shown that the saponins in American ginseng can also protect the cardiovascular system, enhance the function of the central nervous system, and improve immunity. At present, the dosage forms of American ginseng medicinal preparations are liquid and solid. In order to retain the effective ingredients of American ginseng, the medicines are often dosed in the form of fine powder in the solid dosage form and directly added to capsules, tablets, etc.; while liquid preparations require the effective ingredients of American ginseng As far as possible, it is necessary to ensure that the appearance of the preparation is clear, so the purification process of extraction and preparation is the key.

The chemical components in American ginseng are many and complex. Saponins are one of the most important effective components and the most significant physiologically active substances. Among them, triterpene saponins play a major role. Among them, ginsenoside Rb1 has the most content, which has the potential to affect animal testes. , It also affects the embryonic development of mice, has the function of enhancing the choline system, increasing the synthesis and release of acetylcholine and improving memory.

At present, the commonly used preparation and separation methods for total saponins of American ginseng include liquid-liquid extraction, precipitation, and adsorption.

The liquid-liquid extraction method uses dichloromethane, chloroform, benzene and other degreasing methods, which not only easily causes the loss of effective ingredients,

Moreover, due to the toxicity of organic solvents, the production process is not safe, and even the residual solvents in the product reduce the product quality. The commonly used precipitant in the precipitation method is lead salt, which can form insoluble lead salts or complexes with many substances, with poor selectivity; at the same time, lead salts are highly toxic and seriously affect product quality. Common adsorbents in the adsorption method are silica gel, alumina, active polyphthalamide, and diatomaceous earth. At present, the separation of the total saponins of American ginseng mostly adopts column chromatography, and often adopts a large-pored adsorption resin column. However, the macroporous resin adsorption and separation technology has disadvantages: (1) high organic residues, difficult pretreatment; (2) poor strength, severe breakage during use, and short service life; (3) wide particle size distribution and poor separation effect (4) The same type of resin produced by the same manufacturer has large differences in specific surface area and functional group content between batches, and poor reproducibility in the purification of traditional Chinese medicine components.

Summary of the invention

In order to solve the above-mentioned problems, the present invention provides a refined American ginseng total saponins component and a method for purifying and preparing the American ginseng total saponins. The purification preparation method includes the following steps:

(1) Extraction, concentration and drying: Wash, dry, pulverize American ginseng medicinal materials, add 30-80% ethanol for decocting for 0.1-5h, filter out the liquid; add 30-80% ethanol for decocting the medicinal residue for 0.1-3h , Filter the medicinal liquid, discard the medicinal residue, combine the two medicinal liquids to obtain the crude American ginseng extract; perform rotary steaming, concentration and drying to obtain the crude American ginseng dry powder;

(2) Purification: Pass the crude extract of American ginseng through a filter membrane to remove particles, and use high-performance liquid chromatography for purification preparation. Collect the column eluate to obtain the refined total saponins of American ginseng, which is concentrated and dried to obtain refined American ginseng powder;

(3) Decolorization and impurity removal: Ion exchange chromatography is used to decolorize and remove impurities in the refined total saponins of American ginseng, collect the column flow-through liquid to obtain the total saponins with higher purity; condense and dry them to obtain the total saponins of American ginseng Pure powder.

Furthermore, in step (2), the type of the filtration membrane is an organic membrane or an inorganic membrane, the membrane module is a flat membrane, a tubular membrane, a hollow fiber membrane or a roll type, and the pore size is 750kD～1000kD, 0.1-0.65μm; The packing matrix of the reversed-phase chromatographic column used in high performance liquid chromatography technology is silica gel or high molecular polymer; one or more of the normal chain alkyl groups with 1-30 carbon atoms of the packing ligand; the inner diameter of the chromatographic column is 20mm ～1600mm, the height of the chromatographic column bed is 100mm～1000mm; the chromatographic balance liquid contains organic solvent; the sample loading amount of the chromatographic column is 0.01%～60% of the weight of the stationary phase; the sample loading and elution flow rate of the chromatogram is 140cm/h～ 350cm/h; the organic solvent concentration for chromatographic purification and elution of the total saponin target component is 30-90%, and the elution method is isocratic or linear gradient.

Furthermore, in step (3), the matrix of the separation material used in the ion exchange chromatography technique is agarose, silica gel or high molecular polymer; the particle size is 40-100 μm; the structural formula of the ligand is

The loading volume is 10-60 times the column volume; the loading and elution flow rate for chromatographic operation can be 150-500cm/h; the method of chromatographic purification of target components is flow-through or elution mode.

Furthermore, the concentrated drying method used in steps (1)-(3) may be vacuum drying, microwave drying, freeze drying, vacuum drying or spray drying.

A refined total saponin component of American ginseng, the main raw material is American ginseng medicinal materials.

The invention provides a refined Panax quinquefolium saponins component and a method for purifying and preparing Panax quinquefolium saponins, and achieves the following technical effects:

(1) The combined use of high performance liquid chromatography separation technology and ion exchange chromatography separation technology can effectively separate ginsenosides, and can also effectively remove pigments, pesticide residues, heavy metals, endotoxins and other toxic and harmful substances to achieve total saponins of American ginseng High-efficiency purification preparation, more suitable for industrial production;

(2) The retention and improvement of the content of active ingredients can maximize the development of the medicinal value of American ginseng;

(3) Using refined American ginseng total saponins, the refined American ginseng total saponins powder obtained in steps 2 and 3 has a uniform pink color, a smooth surface and a finer powder quality compared to the crude extract obtained in step 1; The product powder dissolves quickly after adding purified water, and the solution is clear and bright, and the taste is sweet and sweet; while the crude dry powder dissolves slowly, the solution is turbid, and the taste is bitter;

(4) In the whole process of the preparation method provided by the present invention, the operating parameters related to the total saponins of American ginseng are safe and controllable, and the external interference factors are small, which can significantly improve the quality of the product;

(5) The preparation process is simple in operation and low in cost, and does not require toxic and harmful organic solvents. The eluent and regenerant can be recycled and reused and are environmentally friendly and can be used for large-scale production.

Description of the drawings

Figure 1 is an HPLC analysis chromatogram of the crude extract of American ginseng prepared in the present invention;

Figure 2 is an HPLC analysis chromatogram of the refined solution of total saponins of Panax quinquefolium prepared in the present invention;

Fig. 3 is an HPLC analysis chromatogram of the pure solution of Panax quinquefolium saponins prepared in the present invention.

detailed description

In the following, the refined and pure Panax quinquefolium saponins of the present invention will be described in more detail in conjunction with the schematic diagram, and the preferred embodiments of the present invention are shown. It should be understood that those skilled in the art can modify the present invention described here. And still achieve the advantageous effects of the present invention. Therefore, the following description should be understood to be widely known to those skilled in the art, and not as a limitation to the present invention.

For clarity, not all features of actual embodiments are described. In the following description, well-known functions and structures are not described in detail because they may confuse the present invention due to unnecessary details. It should be considered that in the development of any actual embodiment, a large number of implementation details must be made to achieve the developer's specific goal, such as changing from one embodiment to another in accordance with related system or related business restrictions. In addition, it should be considered that this kind of development work may be complicated and time-consuming, but it is only routine work for those skilled in the art.

In the present invention, unless expressly stipulated and limited otherwise, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features. Therefore, the definition of “first” and “second” features may explicitly or implicitly include one or more of these features. In the description of the present invention, “at least” means one or more than one, unless otherwise clearly specified. The limit.

In the following paragraphs, the present invention is described in more detail by way of example with reference to the drawings. According to the following description, the advantages and features of the present invention will be clearer. It should be noted that the drawings are in a very simplified form and all use imprecise proportions, which are only used to conveniently and clearly assist in explaining the purpose of the embodiments of the present invention.

Example one

The present invention provides a purification preparation method for extracting panax quinquefolium saponins, and the specific steps are as follows:

Weigh 100g of pretreated American ginseng medicinal materials, add 1L of 65% ethanol water bath and heat reflux extraction (90℃) for 3h, filter with filter paper, add 0.8L 65% ethanol and then heat reflux extraction (90℃) 2h in water bath, gauze Filter and combine the filtrate; after rotary evaporation and concentration, spray-dried to obtain dry powder of the extract. The filtrate was analyzed by HPLC, and the chromatographic analysis result is shown in Figure 1.

Connect a C18 reverse phase chromatography column (filler weight 300g, particle size 60μm) to the high-pressure liquid system, design a flow rate of 60ml/min (190cm/h), flush the column 3BV with 5% ethanol equilibration buffer, and extract the American ginseng crudely The liquid (using a 0.22μm filter membrane to remove particulate insoluble matter) is purified by the system pump and passed through the chromatographic column. The impurities are eluted by 5% ethanol, and the target is eluted by 60% ethanol. The fractions are collected for HPLC analysis, and the qualified fractions are combined. For refined liquid. Rotate the refined liquid to remove the ethanol, vacuum dry until all the water is removed, and collect the dried powder, which is the refined American ginseng total saponin powder. Figure 2 shows the HPLC analysis results of the refined liquid.

Take 20g of refined dry powder and dissolve it in purified water to prepare a concentration of 100mg/mL. Connect the WorkBeads 40TREN ion exchange chromatography column (column volume 20mL) to the chromatography system, with a design flow rate of 12ml/min (360cm/h), and pump the sample through the system through the balanced column. After the sample is loaded, pure water Wash the chromatographic column and use 1M NaOH to elute the impurities bound to the chromatographic column. Collect the column effluent and eluent for HPLC analysis, which is the high-purity Panax quinquefolium saponins component. The fraction is frozen and placed in a freeze dryer, vacuum dried until all the water is removed, and the freeze-dried powder is collected, which is the pure powder of total saponins of American ginseng. Weighing analysis to calculate the total saponin content. The HPLC analysis result of the high-purity Panax quinquefolium total saponin component solution is shown in Figure 3. The saponin content is about 72%, and the yield is about 98%.

Example two

The present invention provides a purification preparation method for extracting panax quinquefolium saponins, and the specific steps are as follows:

Weigh 100g of the pretreated American ginseng medicinal materials, add 2L of 70% ethanol in a water bath and heat reflux (90℃) for 2h, filter with gauze, add 1L of 70% ethanol to the medicinal residue and extract again in a water bath (90℃) for 2h, filter with gauze , Combine the filtrate, spin-evaporate and concentrate, spray-dried to obtain a dry extract powder.

Connect a C18 reverse phase chromatography column (filler weight 300g, particle size 30μm) to the high-pressure liquid system, design a flow rate of 60ml/min (190cm/h), flush the column 3BV with pure water balance buffer, and remove the crude American ginseng extract (Use 0.22μm filter membrane to remove particulate insoluble matter) Purify through the chromatographic column through the system pump, rinse the impurities with 10% ethanol, and elute the target with 65% ethanol, collect each fraction for HPLC analysis, and combine the qualified fractions, that is Refined liquid. The refined liquid is revolved to remove the ethanol and then frozen, vacuum dried until all the water is removed, and the freeze-dried powder is collected, which is the refined American ginseng total saponin powder.

Take 20g of refined dry powder and dissolve it in purified water to prepare a concentration of 100mg/mL. Connect the Q Sepharose 6FF ion exchange chromatography column (column volume 20mL) to the chromatography system, with a design flow rate of 10ml/min (300cm/h), and pump the sample through the system through the balanced column. After the sample is loaded, pure water The chromatographic column is eluted, and the impurities bound to the chromatographic column are eluted with 1M NaOH and 0.1M HCl. Collect the column effluent and eluent for HPLC analysis, that is, the high-purity Panax quinquefolium saponins components. The fraction is frozen and placed in a freeze dryer, vacuum dried until all the water is removed, and the freeze-dried powder is collected, which is the pure powder of total saponins of American ginseng. Weighing analysis calculated that the total saponin content was about 73%, and the yield was about 97%.

Example three

The present invention provides a purification preparation method for extracting panax quinquefolium saponins, and the specific steps are as follows:

Weigh 100g of the pretreated American ginseng medicinal materials, add 1.5L of 55% ethanol in a water bath and heat reflux for extraction (90℃) for 2h, filter with gauze, add 1L of 55% ethanol, and extract again in a water bath (90℃) for 1h. Filter, combine the filtrate, spin-evaporate and concentrate, and spray-dry to obtain a dry powder of the extract.

Connect a C18 reverse phase chromatography column (filler weight 300g, particle size 15μm) to the high-pressure liquid system, design a flow rate of 50ml/min (150cm/h), wash the column 3BV with 10% ethanol equilibration buffer, and extract the American ginseng crudely The solution (using a 0.22μm filter membrane to remove particulate insolubles) is purified by the system pump and passed through the chromatographic column. The target is eluted by a linear gradient (10-65% ethanol, 45min), and each fraction is collected according to the peak type for HPLC After analysis, the qualified fractions are combined to form the refined liquid. After the refined liquid is concentrated by rotary evaporation, it is vacuum dried until all the water is removed, and the freeze-dried powder is collected, which is the refined total saponins powder of American ginseng.

Take 40g of refined dry powder and dissolve it in purified water to prepare a concentration of 50mg/mL. Connect the WorkBeads 100TREN ion exchange chromatography column (column volume 20mL) to the chromatography system with a design flow rate of 12ml/min (360cm/h). Pump the sample through the system through the balanced column. After the sample is loaded, pure water is poured. Wash the chromatographic column, and use 1M NaOH and 0.1M HCl to elute the impurities bound to the chromatographic column. Collect the column effluent and eluent for HPLC analysis, that is, the high-purity Panax quinquefolium saponins components. The fraction is frozen and placed in a freeze dryer, vacuum dried until all the water is removed, and the freeze-dried powder is collected, which is the pure powder of total saponins of American ginseng. Weighing analysis calculates that the total saponin content is about 70%, and the yield is about 95%.

Example four

The present invention provides a method for preparing total saponins in American ginseng medicinal materials, and the specific steps are as follows:

Weigh 100g of pretreated American ginseng medicinal materials, add 1.5L of 65% ethanol in a water bath and heat under reflux for extraction (90℃) for 1h, filter with gauze, add 1L of 55% ethanol and extract again in a water bath with reflux (90℃) for 1h, gauze Filter, combine the filtrate, spin-evaporate and concentrate, and spray-dried to obtain a dry extract powder.

Connect the C18 reverse phase chromatography column (filler weight 300g, particle size 30μm) to the high pressure liquid system, design flow rate 60ml/min (190cm/h), flush the column 3BV with 5% ethanol balance buffer, and extract the American ginseng crudely The liquid (using a 0.22μm filter membrane to remove particulate insolubles) is purified by the system pump and passed through the chromatographic column. The impurities are eluted by 15% ethanol, and the target is eluted with 70% ethanol. The fractions are collected for HPLC analysis, and the qualified fractions are combined. For refined liquid. The refined liquid is concentrated and dried until all the water is removed, and the dried powder is collected, which is the refined American ginseng total saponin powder.

Take 30g of refined dry powder and dissolve it in purified water to prepare a concentration of 100mg/mL. Connect the WorkBeads 40Q ion exchange chromatography column (column volume 20mL) to the chromatography system, with a design flow rate of 12ml/min (360cm/h), and pump the sample through the system through the balanced column. After the sample is loaded, pure water Wash the chromatographic column and use 1M NaOH and 0.1M HCl to elute the impurities bound to the chromatographic column. Collect the column effluent and eluent for HPLC analysis, which is the high-purity Panax quinquefolium saponins component. The fraction is frozen and placed in a freeze dryer, vacuum dried until all the water is removed, and the freeze-dried powder is collected, which is the pure powder of total saponins of American ginseng. Weighing analysis calculated that the total saponin content was about 68%, and the yield was about 97%.

In the above embodiments, the HPLC analysis conditions are as follows:

(1) Chromatographic column packing: Unitary C18 (5μm, 4.6*150mm);

(2) Mobile phase A: water, mobile phase B: acetonitrile;

(3) Flow rate: 1ml/min;

(4) Gradient conditions: 0-25min 20% phase B, 25-50min from 20-46% phase B, 50-60min from 46-55% phase B, 65min 55% phase B.

(5) Column temperature: 25℃;

(6) Detection wavelength: 203nm;

(7) Injection volume: 10μl.

The present invention also provides a refined American ginseng total saponins component, the raw material is mainly composed of American ginseng medicinal materials, refined by the preparation methods in the above embodiments, the refined American ginseng total saponins powder obtained in steps 2 and 3 is compared with that in step 1. The obtained crude dry powder has uniform pink color, smooth surface and fine powder quality; the refined powder of American ginseng and the final pure product powder are quickly dissolved after adding purified water, and the solution is clear and bright, and the taste is sweet and sweet; while the dissolving speed of the crude dry powder is slow and The solution is cloudy and tastes bitter.

As shown in Figures 1, 2, and 3, the HPLC analysis results in each step showed that the target peak in the analysis spectrum of the sample before and after purification did not change significantly. According to the calculation of the total volume, integrated peak area and weight, the total of American ginseng after purification The content of the main components in the refined and pure saponins did not decrease significantly, the yield of total saponins reached more than 95%, and the purity could reach more than 68%. The comparison with the prior art is shown in Table 1. The above-mentioned experimental results show that the obtained Panax quinquefolium saponins have the advantages of high yield and high purity by using the method for preparing the refined and pure Panax quinquefolium saponins of the present invention.

The foregoing are only preferred embodiments of the present invention, and do not play any restrictive effect on the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification and other changes to the technical solution and technical content disclosed by the present invention, which does not depart from the technical solution of the present invention. The content still falls within the protection scope of the present invention.

Table 1 Comparison of total saponin content and yield obtained by the embodiment of the present invention and the prior art preparation method

Preparation	Total saponin content (%)	Total yield of saponin (%)
Example one	72	98
Example two	73	97
Example three	70	95
Example four	68	97
current technology	30-50	75-85

It can be seen from Table 1 that, compared with the prior art, the preparation method provided by the present invention has a much higher total saponin content and yield than the prior art. Obviously, the preparation method provided by the present invention Can get better products and better technical effects.

For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description, and therefore it is intended to fall within the claims. All changes within the meaning and scope of the equivalent elements of are included in the present invention. Any reference signs in the claims should not be regarded as limiting the claims involved.

Claims (7)

1. A purification preparation method for extracting total saponins of American ginseng, which is characterized in that the steps are as follows:

   (1) Extraction, concentration and drying: Wash, dry, pulverize American ginseng medicinal materials, add 30-80% ethanol for decocting for 0.1-5h, filter out the liquid; add 30-80% ethanol for decocting the medicinal residue for 0.1-3h , Filtrate the medicinal solution, discard the medicinal residue, combine the two medicinal solutions to obtain the crude American ginseng extract; perform rotary evaporation and concentration and drying to obtain the crude American ginseng dry powder;

   (2) Purification: Pass the crude extract of American ginseng through a filter membrane to remove particles, and use high-performance liquid chromatography for purification preparation. Collect the column eluate to obtain the refined total saponins of American ginseng, which is concentrated and dried to obtain refined American ginseng powder;

   (3) Decolorization and impurity removal: Ion exchange chromatography is used to decolorize and remove impurities from the refined total saponins of American ginseng, collect the column flow-through to obtain high-purity total saponins; concentrate and dry it to obtain pure saponins品粉。 Product powder.
2. The purification preparation method for extracting total saponins of American ginseng according to claim 1, characterized in that: in step (2), the type of the filter membrane is an organic membrane or an inorganic membrane, and the membrane module is a flat membrane or a tubular membrane. , Hollow fiber membrane or roll type, pore size is 750kD～1000kD, 0.1～0.65μm.
3. The purification preparation method for extracting total saponins of American ginseng according to claim 1, characterized in that, in step (2), the filler matrix of high performance liquid chromatography is silica gel or high molecular polymer; the carbon atoms of the filler ligand One or more of the normal chain alkyl groups of 1-30; the inner diameter of the chromatographic column is 20mm～1600mm, the height of the chromatographic column bed is 100mm～1000mm; the chromatographic balance liquid contains organic solvent; the sample load of the chromatographic column is 0.01% to 60% of the weight of the stationary phase; the loading and elution flow rate of the chromatogram is 140 cm/h to 350 cm/h; the organic solvent concentration of the chromatographic purification and elution total saponin target component is 30 to 90%, the elution method can Is an isocratic or linear gradient.
4. The purification preparation method for extracting total saponins of American ginseng according to claim 3, wherein the organic solvent is methanol, ethanol or acetonitrile, and the content of the organic solvent is 5%-20%.
5. The purification preparation method for extracting total saponins of American ginseng according to claim 1, wherein the matrix of the separation material used in the ion exchange chromatography technique used in step (3) is agarose, silica gel or high molecular polymer; Is 40～100μm; the structural formula of the ligand is

   

   The loading volume is 10-60 times the column volume; the loading and elution flow rate for chromatographic operation can be 150-500 cm/h; the method of chromatographic purification of target components is flow-through or elution mode.
6. The method for purification and preparation of extracting total saponins of American ginseng according to claim 1, wherein the concentration and drying method used in steps (1) to (3) is vacuum drying, microwave drying, freeze drying, vacuum drying or spraying dry.
7. The American ginseng total saponin component refined by the above preparation method is characterized in that the main raw material is American ginseng medicinal materials.

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