Method for simultaneously preparing ginseng flavonoid glycoside and quercetin-3-O-beta-D-glucopyranose-(1-> 2)-beta-D-galactopyranoside from pseudo-ginseng stems and leaves

Abstract

The invention relates to a method for extracting and preparing high-purity ginseng flavonoid glycoside and quercetin-3-O-beta-D-glucopyranose-(1-> 2)-beta-D-galactopyranoside from stems and leaves of panax notoginseng. The method comprises the following specific steps: coarsely crushing dry stems and leaves of pseudo-ginseng, extracting with ethanol and water, and carrying out chromatography twice with different types of macroporous adsorption resin columns, so as to simultaneously prepare high-purity ginseng flavonoid glycoside and quercetin-3-O-beta-D-glucopyranose-(1-> 2)-beta-D-galactopyranoside (the purities are respectively greater than 97% and 93%). The process is simple, convenient and feasible, the transfer rates are high (the transfer rates are respectively greater than 90% and 86%), the high-purity ginseng flavonoid glycoside and quercetin-3-O-beta-D-glucopyranose-(1-> 2)-beta-D-galactopyranoside can be stably obtained, large-scale preparation and industrial production can be realized, and the used solvent is non-toxic, easy to recover, reusable, low in cost and suitable for industrial production. The environment is not polluted, and the obtained final product does not contain organic solvent residues.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for extracting and preparing high-purity ginsenoside and quercetin-3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside from Panax notoginseng stem and leaf. BACKGROUND

[0002] Panax notoginseng (Buck.) FH. Chen is a perennial plant of Araliaceae Panax. Panax notoginseng was first recorded in Compendium of Materia Medica, and its root is used as medicine, which is warm in nature and pungent in taste, and has the functions of activating blood circulation, resolving stasis, relieving swelling and relieving pain. It is the main drug for treating sprains and injuries, and is known as "gold does not change" and "the god of medicine in the south". The rhizome and fleshy root are precious traditional Chinese medicines, and Panax notoginseng is one of the earliest plants with medicinal and edible properties in China. According to Compendium of Materia Medica, "Panax notoginseng leaf can treat sprains and injuries, and can stop bleeding, disperse swelling and dissipate blood stasis, and has the same functions as the root". This shows that the traditional medicine Panax notoginseng stem and leaf has similar effects to the root.

[0003] Panax notoginseng stem and leaf mainly contains saponins and flavonoid glycosides, and the total saponins account for 4%~6% of the total components. Compared with other parts of Panax notoginseng, the content of flavonoids in Panax notoginseng stem and leaf is higher, and the two main flavonoids are ginsenoside [kaempferol-3-O-glucosyl (1→2) galactoside] and quercetin-3-O-glucosyl (1→2) galactoside. The chemical structures of the two are very similar, and the latter has an additional phenolic hydroxyl group at the 3' position. Flavonoid chemical components have good therapeutic effect on many diseases, especially on cardiovascular and cerebrovascular diseases. Modern pharmacological studies have also confirmed that Panax notoginseng stem and leaf has pharmacological effects on the blood system, cardiovascular system, nervous system and metabolic system, which may be related to the flavonoid components in Panax notoginseng stem and leaf. In recent years, with the widespread attention on the medicinal and edible value of Panax notoginseng stem and leaf, the research and development of Panax notoginseng stem and leaf has become one of the hotspots. Further research on the pharmacological activity and clinical application of Panax notoginseng stem and leaf, and the pharmacodynamic activity mechanism of the main flavonoid compounds ginsenoside and quercetin-3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside have become one of the main research directions.

[0004] According to Scifinder search, more than 20 articles have reported the separation and purification of ginsenoside, and more than 10 articles have reported the separation and purification of quercetin-3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside. However, the methods used are all small-scale separation and purification methods in the laboratory, which involve a large amount of organic harmful solvents and silica gel, gel, MCI, C18, and preparative HPLC chromatographic methods. The purpose of separation and purification is only to obtain the compounds at the laboratory level for compound structure identification and limited cell activity screening, and the yield and purity of the compounds are not reported.

[0005] Chinese invention patent: A method for preparing ginseng flavonoid glycosides from Panax notoginseng stems and leaves (CN117964671B), which involves coarsely pulverizing dried Panax notoginseng stems and leaves, extracting with high alcohol, combining macroporous adsorption resin and silica gel column chromatography, followed by a single column chromatography using a mixed column of polyamide and reversed-phase chromatographic materials to achieve hydrogen bonding adsorption of polyamide and chromatographic purification of the reversed-phase materials, thus preparing high-purity ginseng flavonoid glycosides; A method for preparing ginseng flavonoid glycosides (CN11454827A) uses an online HPLC-SP-NMR-MS system, which can directly and rapidly prepare and identify ginseng flavonoid glycoside monomers from ginseng leaf powder, but its technical conditions are demanding and can only achieve... Preparation and purification of small amounts of monomers; Preparation method and application of total flavonoids from Panax notoginseng leaves (CN1609114A), which discloses a method for extracting total flavonoids from Panax notoginseng leaves, but does not describe the preparation of monomeric flavonoid compounds; a method for obtaining flavonoid aglycones from ginseng stems and leaves (CN104784231B), a method for extracting flavonoid compounds from American ginseng leaves (CN107595909A), and a method for simultaneously preparing total saponins, total flavonoids, and unsaponifiable oils from ginseng stems and leaves (CN110917228B). These methods use large amounts of organic reagents such as petroleum ether and ethyl acetate during extraction and preparation, and the final products are all mixtures of flavonoid aglycones. No patents related to the preparation of quercetin-3-O-β-D-glucopyranose-(1→2)-β-D-galactopyranoside were found. Summary of the Invention

[0006] This invention provides a method for simultaneously preparing ginsenosides and quercetin-3-O-β-D-glucopyranoside (1→2)-β-D-galactopyranoside from Panax notoginseng stems and leaves. After coarsely pulverizing dried Panax notoginseng stems and leaves, extraction with ethanol and water is performed, followed by two chromatography sessions using macroporous adsorption resins of different types. This allows for the simultaneous preparation of high-purity ginsenosides and quercetin-3-O-β-D-glucopyranoside (purities greater than 97% and 93%, respectively). The process of this invention is simple and feasible, with a high transfer rate (greater than 90% and 86%, respectively). It can stably obtain high-purity ginseng flavonoid glycosides and quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside, enabling large-scale preparation and industrial production. Furthermore, the solvents used are non-toxic, easy to recycle, and reusable, resulting in low cost, no environmental pollution, and no organic solvent residue in the final product.

[0007] This invention relates to a method for simultaneously preparing ginsenosides and quercetin-3-O-β-D-glucopyranoside (1→2)-β-D-galactopyranoside from Panax notoginseng stems and leaves, comprising the following steps:

[0008] 1) Coarsely crush the dried stems and leaves of Panax notoginseng, extract with 40%~70% ethanol solution, with a material-to-liquid ratio of 1:6~1:20. Filter the extract and concentrate it until there is no alcohol odor. Set aside for later use.

[0009] 2) The concentrate obtained in step 1) is loaded onto macroporous adsorption resin No. 1 and eluted sequentially with 2 to 6 column volumes of water, 10% ethanol and 30% ethanol solution. The 30% ethanol eluent is collected and concentrated until there is no alcohol odor, and then set aside for later use.

[0010] 3) The concentrate obtained in step 2) was loaded onto macroporous adsorption resin No. 2 and eluted sequentially with 2 to 10 times column lengths of 10% ethanol, 20% ethanol and 30% ethanol solutions. The 30% ethanol eluent was collected in two segments and concentrated until no alcohol odor was found. The eluents were then freeze-dried to obtain quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside and ginsenoside, respectively.

[0011] The extraction method in step 1) is one of percolation, impregnation, or reflux.

[0012] In step 2), the macroporous adsorption resin is one of HP20, HP21 and HPD100.

[0013] In step 3), the macroporous adsorption resin is one of LK1300S, SP825L and SP850.

[0014] 4) The contents of ginsenosides and quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside were detected by high performance liquid chromatography. The detection method included:

[0015] Chromatographic conditions and system suitability tests: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile:0.1% phosphoric acid water = 17:83 was used as the mobile phase; the detection wavelength was 365 nm; the column temperature was 30℃; and the flow rate was 1.0 ml / min. The theoretical plate number, calculated based on ginseng flavonoid glycosides, should not be less than 3000.

[0016] Preparation of reference solution: Dilute the test solution 1000 times to obtain (0.01 mg / ml).

[0017] Preparation of the test solution: Take an appropriate amount of the powder of this product, accurately weigh it, and add it to 50% methanol solution to prepare a solution containing 10mg per ml.

[0018] Assay: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0019] Step 4) HPLC analysis: The sample prepared in step 3) was analyzed by HPLC using a high performance liquid chromatograph.

[0020] The method described above for simultaneously preparing ginsenosides and quercetin-3-O-β-D-glucopyranoside from Panax notoginseng stems and leaves yields samples with a purity of 93%~97% and a transfer rate of 86%~91% as determined by HPLC. Attached Figure Description

[0021] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. The drawings, as part of the present invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the present invention are used to explain the invention, but do not constitute an improper limitation of the invention. Obviously, the drawings described below are merely some embodiments. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The HPLC detection results of quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside obtained by the preparation method provided in Example 1 are shown in the figure.

[0023] Figure 2 The HPLC detection results of ginsenosides obtained by the preparation method provided in Example 1 are shown in the figure.

[0024] Figure 3 The HPLC detection results of quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside obtained by the preparation method provided in Example 2 are shown in the figure.

[0025] Figure 4 The HPLC detection results of ginsenosides obtained by the preparation method provided in Example 2 are shown in the figure.

[0026] Figure 5 The HPLC detection results of quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside obtained by the preparation method provided in Example 3 are shown in the figure.

[0027] Figure 6 The HPLC detection results of ginsenosides obtained by the preparation method provided in Example 3 are shown in the figure. Detailed Implementation

[0028] The present invention will be further described in detail below through embodiments. Those skilled in the art can refer to the content of the present invention and appropriately modify the relevant parameters according to the specific actual equipment to implement it. The method of the present invention has been described in detail for the preparation of ginseng flavonoid glycosides and quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside. Those skilled in the art can modify or appropriately change and combine the method described in the present invention without departing from the content, spirit and scope of the present invention to realize and apply the method of the present invention. However, the scope of protection of the present invention is not limited to the content described. Unless otherwise specified, the methods in the embodiments are conventional methods, and the reagents used are all conventional commercial reagents or reagents prepared according to conventional methods.

[0029] Example 1

[0030] 1) Coarsely crush 10kg of Panax notoginseng stems and leaves, and extract by percolation with 55% ethanol solution at a material-to-liquid ratio of 1:20 and a flow rate of 40L / h. Extract once at room temperature (20℃). Filter the extract and concentrate it until there is no alcohol taste.

[0031] 2) The concentrate obtained in step 1) is loaded onto a macroporous adsorption resin (HPD100) and eluted sequentially with 2 column volumes of water, 6 column volumes of 10% ethanol and 2 column volumes of 30% ethanol. The 30% ethanol eluent is collected and concentrated until there is no alcohol odor, and then set aside for later use.

[0032] 3) The concentrate obtained in step 2) was loaded onto a macroporous adsorption resin (LK1300S) and eluted sequentially with 2 column volumes of 10% ethanol, 10 column volumes of 20% ethanol, and 10 column volumes of 30% ethanol. The 30% ethanol eluent was collected in fractions, with the first 5 column volumes as one fraction and the last 5 column volumes as another fraction. The fractions were concentrated until no alcohol odor was detected and then freeze-dried to obtain quercetin-3-O-β-D-glucopyranose-(1→2)-β-D-galactopyranoside and ginseng flavonoid glycoside, respectively. Quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside weighed 32.51g, with a transfer rate of 86.49%. HPLC analysis showed a purity of 94.38% and a moisture content of 1.27%. Ginsenosides weighed 42.11g, with a transfer rate of 91.38%. HPLC analysis showed a purity of 98.78% and a moisture content of 1.13%.

[0033] Example 2

[0034] 1) Coarsely crush 10kg of Panax notoginseng stems and leaves, reflux extract with 70% ethanol solution, material-to-liquid ratio 1:10, extract 3 times, extraction temperature 100℃, extraction time 60min each time, filter the extract and concentrate until there is no alcohol taste, and set aside.

[0035] 2) The concentrate obtained in step 1) is loaded onto a macroporous adsorption resin (HP20) and eluted sequentially with 6 column volumes of water, 2 column volumes of 10% ethanol and 6 column volumes of 30% ethanol. The 30% ethanol eluent is collected and concentrated until there is no alcohol odor, and then set aside for later use.

[0036] 3) The concentrated solution obtained in step 2) was loaded onto a macroporous adsorption resin (SP825L) and eluted sequentially with 10 column volumes of 10% ethanol, 2 column volumes of 20% ethanol, and 8 column volumes of 30% ethanol. The 30% ethanol eluent was collected in fractions, with the first four column volumes constituting one fraction and the last four column volumes constituting another. Each fraction was concentrated until no alcohol odor remained, and then freeze-dried to obtain quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside and ginseng flavonoid glycoside, respectively. Quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside weighed 32.45 g, with a transfer rate of 86.76%, and a purity of 94.77% and a moisture content of 1.19% as determined by HPLC. Ginseng flavonoid glycoside weighed 41.95 g, with a transfer rate of 91.04%, and a purity of 98.92% and a moisture content of 1.26% as determined by HPLC.

[0037] Example 3

[0038] 1) Coarsely crush 10kg of Panax notoginseng stems and leaves, extract with 40% ethanol solution, material-to-liquid ratio 1:6, extract 5 times, extraction temperature room temperature (28℃), each extraction 180min, filter the extract and concentrate until there is no alcohol taste, and set aside.

[0039] 2) The concentrate obtained in step 1) is loaded onto a macroporous adsorption resin (HP21) and eluted sequentially with 4 column volumes of water, 4 column volumes of 10% ethanol and 4 column volumes of 30% ethanol. The 30% ethanol eluent is collected and concentrated until there is no alcohol odor, and then set aside for later use.

[0040] 3) The concentrated solution obtained in step 2) was loaded onto a macroporous adsorption resin (SP825L) and eluted sequentially with 5 column volumes of 10% ethanol, 6 column volumes of 20% ethanol, and 9 column volumes of 30% ethanol. The 30% ethanol eluent was collected in fractions, with the first 4 column volumes constituting one fraction and the last 5 column volumes constituting another. Each fraction was concentrated until no alcohol odor remained, and then freeze-dried to obtain quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside and ginseng flavonoid glycosides, respectively. Quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside weighed 32.28 g, with a transfer rate of 86.46%, and a purity of 94.82% and a moisture content of 1.08% as determined by HPLC. Ginseng flavonoid glycosides weighed 42.42 g, with a transfer rate of 91.97%, and a purity of 98.73% and a moisture content of 1.17% as determined by HPLC.

[0041]

[0042]

Claims

1. A process for the simultaneous preparation of panaxifolin and quercetin-3-0-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside from the stems and leaves of Panax notoginseng characterized in that The method comprises the following steps: 1) The dried stems and leaves of Panax notoginseng are coarsely crushed, and extracted with 40%-70% ethanol solution at a material-to-liquid ratio of 1:6-1:

20. The extract is filtered and concentrated to remove the alcohol taste, and is prepared for use; 2) The concentrated solution obtained in step 1) is subjected to column chromatography on No. 1 macroporous adsorption resin, and is eluted with water, 10% ethanol and 30% ethanol successively in an amount of 2-6 times the column volume. The 30% ethanol eluate is collected, concentrated to remove the alcohol taste, and prepared for use; 3) The concentrated solution obtained in step 2) is subjected to column chromatography on No. 2 macroporous adsorption resin, and is eluted with 10% ethanol, 20% ethanol and 30% ethanol successively in an amount of 2-10 times the column volume. The 30% ethanol eluate is collected in two sections, and is concentrated to remove the alcohol taste, and is freeze-dried to obtain quercetin-3-O-beta-D-glucopyranosyl-(1→2)-beta-D-galactopyranoside and ginsenoside, respectively.

2. The process for simultaneous preparation of panaxifolin and quercetin-3-0-β-D- glucopyranosyl-(1→2)-β-D-galactopyranoside from leaves of Panax notoginseng as claimed in claim 1, wherein, The extraction method in step 1) is one of percolation, immersion and reflux. ​ 3. The process for simultaneous preparation of panaxifolin and quercetin-3-0-β-D- glucopyranosyl-(1→2)-β-D-galactopyranoside from leaves of Panax notoginseng as claimed in claim 1, wherein, The macroporous adsorption resin in step 2) is one of HP20, HP21 and HPD100. ​ 4. The process for simultaneous preparation of ginsenoside and quercetin-3-0-β-D- glucopyranosyl-(1→2)-β-D-galactopyranoside from leaves of Panax notoginseng as claimed in claim 1, wherein, The macroporous adsorption resin in step 3) is one of LK1300S, SP825L and SP850.

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