Method for simultaneously preparing ginsenoside and quercetin-3-o-beta-d-glucopyranosyl-(1→2)-beta-d-galactopyranoside from panax notoginseng stem and leaf

Abstract

The present application relates to a kind of from Panax stem leaf extraction, preparation high purity ginseng flavonoid glycoside and quercetin-3-O-β-D-glucopyranose-(1→2)-β-D-galactopyranoside method.The specific steps of the method are as follows: after Panax stem leaf is coarsely crushed, ethanol water extraction is carried out, and high purity ginseng flavonoid glycoside and quercetin-3-O-β-D-glucopyranose-(1→2)-β-D-galactopyranoside (purity is greater than 97% and 93% respectively) can be prepared simultaneously by two times different type macroporous adsorption resin column chromatography.The present application is simple and feasible, and the transfer rate is high (the transfer rate is greater than 90% and 86% respectively), and high purity ginseng flavonoid glycoside and quercetin-3-O-β-D-glucopyranose-(1→2)-β-D-galactopyranoside can be stably obtained, mass production and industrial production can be realized, and the solvent used is non-toxic, easy to recover, reusable, low cost, no pollution to the environment, and the final product obtained does not contain organic solvent residue.

Description

Technical Field

[0001] This invention relates to a method for extracting and preparing high-purity ginsenosides and quercetin-3-O-β-D-glucopyranoside from the stems and leaves of Panax notoginseng. Background Technology

[0002] Panax notoginseng (Buck.) FH. Chen is a perennial plant belonging to the genus Panax in the family Araliaceae. First recorded in the *Compendium of Materia Medica*, the root of Panax notoginseng is used medicinally. It is warm in nature and pungent in taste, possessing the effects of promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. It is a primary medicine for treating injuries from falls and blows, and is known as "priceless" and "the divine medicine of the South." The rhizome and fleshy root are precious traditional Chinese medicines, and it is one of the earliest plants in my country to be used both as food and medicine. According to the *Compendium of Materia Medica*, "Panax notoginseng leaves treat fractures and bleeding from falls; applying them stops the bleeding immediately, and bruises and swellings subside overnight; the other effects are the same as the root," indicating that the effects of the stems and leaves of Panax notoginseng in traditional medicine are similar to those of its root.

[0003] The stems and leaves of Panax notoginseng mainly contain saponins and flavonoids, with total saponins accounting for 4% to 6% of the total composition. Compared with other parts of Panax notoginseng, the stems and leaves have a higher content of flavonoids. The two main flavonoid components are ginsenoside [kaempferol-3-O-glucosyl(1→2)galactoside] and quercetin-3-O-glucosyl(1→2)galactoside, which have very similar chemical structures, with the latter having an additional phenolic hydroxyl group at the 3' position. Flavonoids have good therapeutic effects on various diseases, especially cardiovascular and cerebrovascular diseases. Modern pharmacological studies have also confirmed that the stems and leaves of Panax notoginseng have medicinal effects on the blood system, cardiovascular system, nervous system, and metabolic system, which may be closely related to the flavonoid components in the stems and leaves. In recent years, with the widespread attention paid to the medicinal and edible value of Panax notoginseng stems and leaves, research and development on Panax notoginseng stems and leaves has become a hot topic. Further research into the pharmacological activities and clinical applications of Panax notoginseng stems and leaves, and clarifying the pharmacodynamic mechanisms of its main flavonoid compounds, ginsenosides and quercetin-3-O-β-D-glucopyranoside (1→2)-β-D-galactopyranoside, has become one of the main research directions.

[0004] A SciFinder search revealed over 20 articles reporting the isolation and purification of ginseng flavonoid glycosides, and over 10 articles reporting the isolation and purification of quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside. However, all of these were small-scale laboratory-scale isolation and purification methods involving large amounts of harmful organic solvents and chromatographic methods such as silica gel, gel electrophoresis, MCI, C18, and preparative HPLC. The purpose of the isolation and purification was merely to obtain the compound at the laboratory level for compound structure identification and limited cell activity screening. The yield and purity of the compound were 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 column volumes 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-glucopyranose-(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 method for simultaneously preparing ginsenosides and quercetin-3-O-β-D-glucopyranoside (1→2)-β-D-galactopyranoside from Panax notoginseng stems and leaves, characterized in that... Includes the following steps: 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. 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. 3) The concentrate obtained in step 2) was loaded onto macroporous adsorption resin No. 2 and eluted sequentially with 2 to 10 column volumes 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 detected. The eluents were then freeze-dried to obtain quercetin-3-O-β-D-glucopyranoside-(1→2)-β-D-galactopyranoside and ginsenoside, respectively. In step 2), the No. 1 macroporous adsorption resin is one of HP20, HP21 or HPD100; in step 3), the No. 2 macroporous adsorption resin is one of LK1300S, SP825L or SP850.

2. The method for simultaneously preparing ginsenosides and quercetin-3-O-β-D-glucopyranoside from Panax notoginseng stems and leaves according to claim 1, characterized in that, Step 1) The extraction method is one of percolation, impregnation, or reflux.

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