A method for simultaneously separating and purifying six flavonoid glycosides from Disporum japonicum

Abstract

This invention belongs to the field of natural product separation and purification technology, specifically relating to a method for simultaneously separating and purifying six flavonoid glycosides from *Erigeron simonii*. The method includes alcohol extraction, liquid-liquid extraction, high-speed countercurrent chromatography enrichment and separation, and preparative liquid chromatography purification steps. This method can simultaneously obtain rutin, isoquercitrin, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside from *Erigeron simonii*, and is suitable for the preparation and quality control research of flavonoid glycosides from *Erigeron simonii*.

Description

Technical Field

[0001] This invention belongs to the field of natural product separation and purification and preparation of traditional Chinese medicine compounds, specifically involving a method for simultaneously separating and purifying six flavonoid glycosides from *Echeveria elegans*. Background Technology

[0002] Small-leaved lotus (Prunella vulgaris) is the dried, mature fruit of the Berberidaceae plant *Prunella vulgaris*, and is a commonly used medicinal material in Tibetan medicine. Small-leaved lotus contains various chemical components, among which flavonoid glycosides are an important part. Due to the complex chemical composition of small-leaved lotus, and the similar structures and polarities of different flavonoid glycosides, problems such as component overlap, insufficient enrichment of target components, and low purification efficiency easily occur during the separation and purification process. Therefore, establishing a method for the simultaneous separation and purification of multiple flavonoid glycosides in small-leaved lotus is of great significance.

[0003] Existing methods for separating natural products typically include solvent extraction, liquid-liquid extraction, column chromatography separation, and preparative liquid chromatography purification. While traditional column chromatography can achieve a certain degree of separation, it suffers from problems such as significant sample loss, long separation cycles, high solvent consumption, and irreversible adsorption of some target components. For the various flavonoid glycosides in *Echeveria elegans* with similar structures and polarities, relying solely on conventional column chromatography often makes it difficult to achieve rapid and efficient simultaneous preparation of multiple components.

[0004] High-speed countercurrent chromatography (HSCLC) is a separation technique based on the liquid-liquid partition principle. It does not use a solid stationary phase, reducing irreversible adsorption and sample loss, making it suitable for the enrichment and separation of target components in natural products. However, the solvent system, elution mode, and pretreatment method of HSCLC need to be specifically optimized for different plant sources, different combinations of target components, and different impurity backgrounds. Insufficient pretreatment enrichment or inappropriate solvent selection can still lead to problems such as low separation efficiency, long elution cycles, overlapping target components, or difficulties in subsequent preparative liquid chromatography purification.

[0005] Therefore, a separation and purification method suitable for the complex matrix of *Echeveria elegans* is still needed, which can obtain a variety of flavonoid glycosides through reasonable pretreatment enrichment, high-speed countercurrent chromatography enrichment and separation, and preparative liquid phase purification, so as to provide a material basis for the study of chemical composition of *Echeveria elegans*, quality control and related product development. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for simultaneously isolating and purifying six flavonoid glycosides from *Echeveria elegans*. This method combines alcohol extraction, two-stage liquid-liquid extraction, high-speed countercurrent chromatography enrichment and separation, and preparative liquid chromatography purification to achieve simultaneous enrichment, separation, and purification of multiple flavonoid glycosides from *Echeveria elegans*.

[0007] This invention provides a method for simultaneously isolating and purifying six flavonoid glycosides from *Echeveria elegans*, comprising the following steps:

[0008] (1) Extraction: The small-leaved lotus was extracted with an alcohol solution with a volume fraction of 50% to 90%, and the extract was concentrated to obtain the small-leaved lotus extract paste;

[0009] (2) Liquid-liquid extraction: The extract of the small-leaved lotus is mixed with an aqueous medium to obtain the extract to be extracted; then the extract to be extracted is extracted sequentially with the first extraction solvent and the second extraction solvent, the extract containing the target flavonoid glycosides is collected, concentrated and dried to obtain crude extract of total flavonoid glycosides.

[0010] Wherein, the first extraction solvent is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 1:1 to 1:5; the second extraction solvent is a mixed solvent of n-butanol, ethyl acetate and water, with a volume ratio of n-butanol, ethyl acetate and water of (1 to 10):(0.1 to 5):(1 to 10).

[0011] (3) Enrichment and separation: The crude extract of total flavonoid glycosides is enriched and separated by high-speed countercurrent chromatography to obtain a component containing the target flavonoid glycosides; wherein the high-speed countercurrent chromatography uses a n-butanol-ethyl acetate-water solvent system for elution, and the volume ratio of n-butanol, ethyl acetate and water in the n-butanol-ethyl acetate-water solvent system is (0.5~5):(5~15):(5~15);

[0012] (4) Purification: The components obtained in step (3) were purified by preparative liquid chromatography. The mobile phase was an organic phase-water system, and the organic phase was selected from acetonitrile, methanol or a combination thereof. The volume fraction of the organic phase in the mobile phase was 5% to 50%. The elution method was isocratic elution and / or gradient elution, to obtain 6 flavonoid glycosides.

[0013] The six flavonoid glycosides mentioned are rutin, isoquercitrin, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside.

[0014] In some embodiments, the alcohol solution is an ethanol solution with a volume fraction of 60% to 80%, for example, 60%, 65%, 70%, 75% or 80%; more preferably 70%.

[0015] In some embodiments, in step (1), 1 to 30 mL of alcohol solution is used for extraction per 1 g of *Echeveria elegans*; preferably, 10 to 20 mL of alcohol solution is used for extraction per 1 g of *Echeveria elegans*; more preferably, 15 mL of alcohol solution is used for extraction per 1 g of *Echeveria elegans*.

[0016] In this invention, the extraction method in step (1) includes, but is not limited to, ultrasonic extraction, heating extraction, reflux extraction, maceration extraction, shaking extraction, stirring extraction, or a combination thereof. Preferably, the extraction method is ultrasonic extraction.

[0017] In some implementations, in step (1), the number of extractions can be 1 to 3 times, for example, 1 time, 2 times or 3 times; the extraction time for each extraction can be 0.5 to 2 hours, for example, 0.5 hours, 1 hour, 1.5 hours or 2 hours.

[0018] In this invention, in step (1), the concentration can be achieved by vacuum concentration, rotary evaporation concentration, atmospheric pressure concentration, low temperature concentration, or a combination thereof. Preferably, the concentration is vacuum concentration.

[0019] In this invention, in step (2), the aqueous medium is water or an aqueous solution. The water includes, but is not limited to, purified water, deionized water, distilled water, or ultrapure water; the aqueous solution includes, but is not limited to, aqueous alcohol solutions, acidic aqueous solutions, or other aqueous solutions capable of forming a liquid-liquid extraction system. Preferably, the aqueous medium is water.

[0020] In this invention, in step (2), the method of mixing the extract of *Echeveria elegans* with the aqueous medium includes, but is not limited to, dissolving, resolving, dispersing, suspending, stirring, ultrasonic mixing, or a combination thereof.

[0021] In some embodiments, in step (2), the volume ratio of petroleum ether to ethyl acetate in the first extraction solvent is 1:2 to 1:4; more preferably, the volume ratio of petroleum ether to ethyl acetate is 1:3.

[0022] Using the first extraction solvent to extract the extract can remove some fat-soluble components, pigments, moderately polar impurities or other non-target components from the extract of *Echeveria elegans*, which is beneficial for the subsequent enrichment and separation of the target flavonoid glycosides.

[0023] In some embodiments, in step (2), the volume ratio of n-butanol, ethyl acetate and water in the second extraction solvent is (2-5):(0.5-1.5):(2-5); preferably, the volume ratio of n-butanol, ethyl acetate and water in the second extraction solvent is (2-5):1:(2-5); more preferably, the volume ratio of n-butanol, ethyl acetate and water is 5:1:5.

[0024] In some embodiments, the extraction is performed 2 to 10 times using a second extraction solvent; preferably, the extraction is performed 3 to 8 times; more preferably, the extraction is performed 8 times.

[0025] In some implementations, after collecting the extract containing the target flavonoid glycosides in step (2), the solvent can be removed by vacuum concentration, rotary evaporation, freeze drying, vacuum drying, low-temperature drying or a combination thereof to obtain the crude extract of total flavonoid glycosides.

[0026] In a preferred embodiment, in step (3), the volume ratio of n-butanol, ethyl acetate and water in the n-butanol-ethyl acetate-water solvent system is (0.5-2):(8-12):(8-12); more preferably, the volume ratio of n-butanol, ethyl acetate and water is 1:10:10.

[0027] In step (3), the n-butanol-ethyl acetate-water solvent system is thoroughly mixed and allowed to stand to separate into layers, forming an upper phase and a lower phase, one of which serves as the stationary phase and the other as the mobile phase.

[0028] In some implementations, in step (3), during the high-speed countercurrent chromatography separation process, the mobile phase is first introduced in the first direction for elution, and after the first 1 to 4 components are separated, the direction of the mobile phase is changed to continue elution; preferably, after the first 2 components are separated, the direction of the mobile phase is changed to continue elution.

[0029] In some implementations, the fraction containing the target flavonoid glycosides obtained in step (3) is a high-speed countercurrent chromatography enrichment fraction;

[0030] The number of components enriched by high-speed countercurrent chromatography is 4 or 5;

[0031] When the number of high-speed countercurrent chromatography enrichment components is 4, they are sequentially named high-speed countercurrent chromatography enrichment component I, high-speed countercurrent chromatography enrichment component II, high-speed countercurrent chromatography enrichment component III, and high-speed countercurrent chromatography enrichment component IV according to the high-speed countercurrent chromatography elution order.

[0032] When the number of high-speed countercurrent chromatography enrichment components is 5, they are sequentially named high-speed countercurrent chromatography enrichment component I, high-speed countercurrent chromatography enrichment component II, high-speed countercurrent chromatography enrichment component III, high-speed countercurrent chromatography enrichment component IV, and high-speed countercurrent chromatography enrichment component V according to the high-speed countercurrent chromatography elution order.

[0033] In this embodiment, at least one of the high-speed countercurrent chromatography enrichment components contains two target flavonoid glycosides.

[0034] In this invention, in step (4), the preparative liquid chromatography can be preparative high performance liquid chromatography, semi-preparative high performance liquid chromatography, or other liquid chromatography methods that can achieve the purification of the target component.

[0035] In some implementations, in step (4), the preparative liquid chromatography is purified using a reversed-phase column; preferably, the reversed-phase column is a C18 column.

[0036] In some implementations, in step (4), the purification conditions for the preparative liquid chromatography include any one of the following items (i) to (iv), depending on the number of components enriched by the high-speed countercurrent chromatography and the mobile phase system:

[0037] (i) When the number of components enriched by the high-speed countercurrent chromatography is 5, the preparative liquid chromatography uses an acetonitrile-water system for elution;

[0038] Among them, high-speed countercurrent chromatography (HSCCC) enrichment component I was eluted using isocratic elution with a volume ratio of acetonitrile to water of 25:75; HSCCC enrichment component II was eluted using gradient elution, with the volume fraction of acetonitrile increasing from 15% to 30% within 0–30 min; HSCCC enrichment component III was eluted using gradient elution, with the volume fraction of acetonitrile increasing from 20% to 35% within 0–30 min; HSCCC enrichment components IV and V were eluted using isocratic elution with a volume ratio of acetonitrile to water of 27:73.

[0039] (ii) When the number of components enriched by the high-speed countercurrent chromatography is 5, the preparative liquid chromatography uses a methanol-water system for elution;

[0040] Among them, high-speed countercurrent chromatography (HSCCC) enrichment fraction I was eluted using isocratic elution with a methanol-to-water volume ratio of 25:75; HSCCC enrichment fraction II was eluted using gradient elution, with the methanol volume fraction increasing from 15% to 30% within 0–30 min; HSCCC enrichment fraction III was eluted using isocratic elution with a methanol-to-water volume ratio of 30:70; HSCCC enrichment fraction IV was eluted using gradient elution, with the methanol volume fraction increasing from 20% to 35% within 0–30 min; and HSCCC enrichment fraction V was eluted using isocratic elution with a methanol-to-water volume ratio of 25:75.

[0041] (iii) When the number of components enriched by the high-speed countercurrent chromatography is 5, the preparative liquid chromatography uses an acetonitrile-water system for elution;

[0042] In this study, high-speed countercurrent chromatography (HSCCC) enrichment fractions I and II were eluted using isocratic elution with an acetonitrile to water volume ratio of 25:75; HSCCC enrichment fraction III was eluted using gradient elution, with the acetonitrile volume fraction increasing from 15% to 30% within 0–30 min; HSCCC enrichment fraction IV was eluted using isocratic elution with an acetonitrile to water volume ratio of 30:70; and HSCCC enrichment fraction V was eluted using gradient elution, with the acetonitrile volume fraction increasing from 15% to 35% within 0–30 min.

[0043] (iv) When the number of components enriched by the high-speed countercurrent chromatography is 4, the preparative liquid chromatography uses an acetonitrile-water system for elution;

[0044] Among them, high-speed countercurrent chromatography (HSCCC) enrichment components I and II were eluted using isocratic elution with a volume ratio of acetonitrile to water of 25:75; HSCCC enrichment components III and IV were eluted using gradient elution, with the volume fraction of acetonitrile increasing from 20% to 35% within 0–30 min.

[0045] After the above elution, the target peaks in each high-speed countercurrent chromatography enrichment fraction were collected to obtain the preparative liquid phase purified product.

[0046] In some implementations, the method satisfies one or more of the following conditions:

[0047] (a) In step (3), the rotation speed of the high-speed countercurrent chromatography is 600 to 1000 rpm;

[0048] (b) In step (3), the column temperature of the high-speed countercurrent chromatography is 30–50 °C;

[0049] (c) In step (3), the flow rate of the mobile phase in the high-speed countercurrent chromatography is 4 to 8 mL / min;

[0050] (d) In step (3), the detection wavelength of the high-speed countercurrent chromatography is 254 nm;

[0051] (e) In step (4), the preparative liquid chromatography is performed using a C18 column for purification;

[0052] (f) In step (4), the water in the organic phase-water system is an acidic aqueous solution, and the acid in the acidic aqueous solution is selected from one or more of formic acid, acetic acid, glacial acetic acid, and phosphoric acid, and the volume fraction of the acid in the acidic aqueous solution is 0.05% to 0.5%;

[0053] In some embodiments, in step (3), the high-speed countercurrent chromatography has a rotation speed of 800 rpm, a column temperature of 40°C, a mobile phase flow rate of 6 mL / min, and a detection wavelength of 254 nm.

[0054] In some embodiments, in step (4), the acidic aqueous solution is an aqueous solution containing 0.1% glacial acetic acid.

[0055] In this invention, the structures of the six flavonoid glycosides obtained were identified. The structural identification methods include, but are not limited to, nuclear magnetic resonance, mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, or combinations thereof.

[0056] In some embodiments, the method meets the following conditions, and the resulting six flavonoid glycosides, after structural confirmation, can be used as chemical reference standards:

[0057] ① In step (1), the alcohol solution is a 70% ethanol solution by volume, and 15 mL of alcohol solution is used to extract 1 g of *Echeveria elegans*.

[0058] ② In step (2), the volume ratio of petroleum ether to ethyl acetate in the first extraction solvent is 1:3; the volume ratio of n-butanol, ethyl acetate and water in the second extraction solvent is 5:1:5, and the number of extractions is 8.

[0059] ③ In step (3), the volume ratio of n-butanol, ethyl acetate and water in the n-butanol-ethyl acetate-water solvent system is 1:10:10, the rotation speed of the high-speed countercurrent chromatography is 800 rpm, the column temperature is 40℃, the mobile phase flow rate is 6 mL / min, and after the first two components are separated, the direction of the mobile phase is changed to continue elution.

[0060] ④ In step (4), the preparative liquid chromatography uses a C18 column for purification and an acetonitrile-water system as the mobile phase, wherein the water is an aqueous solution containing 0.1% glacial acetic acid, and the elution is performed under the conditions described in item (i) above.

[0061] Compared with the prior art, the present invention has at least the following beneficial effects:

[0062] This invention combines alcohol extraction, liquid-liquid extraction, high-speed countercurrent chromatography enrichment and separation, and preparative liquid chromatography purification to simultaneously separate and purify six flavonoid glycosides from *Echeveria elegans*, avoiding the cumbersome and inefficient process of separating individual compounds one by one. The obtained six flavonoid glycosides can be used for component identification, content determination, quality control, or related research of *Echeveria elegans* medicinal materials, processed slices, extracts, or related products. Attached Figure Description

[0063] Figure 1 HPLC chromatogram of the total flavonoid glycoside extract of *Echeveria elegans* prepared in Example 1;

[0064] Figure 2 : High-speed countercurrent chromatogram obtained in Example 1;

[0065] Figure 3 Example 1: Liquid chromatography purification chromatogram of rutin prepared in Example 1;

[0066] Figure 4 Example 1: Liquid chromatography purification chromatogram of isoquercitrin prepared in Example 1;

[0067] Figure 5 Example 1: Liquid chromatography purification chromatogram of quercetin-3-O-(6''-O-malonyl)-β-D-glucoside prepared in Example 1;

[0068] Figure 6 Example 1: Liquid chromatography purification chromatogram of kaempferol-3-O-rutin prepared in Example 1;

[0069] Figure 7 : Preparative liquid chromatogram of astragaloside and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside obtained in Example 1;

[0070] Figure 8 Example 1: Purity test chart of rutin obtained;

[0071] Figure 9 Graph showing the purity of isoquercitrin obtained in Example 1;

[0072] Figure 10 Graph showing the purity of quercetin-3-O-(6''-O-malonyl)-β-D-glucoside prepared in Example 1;

[0073] Figure 11 Graph showing the purity of kaempferol-3-O-rutin obtained in Example 1;

[0074] Figure 12 Example 1: Purity test chart of astragaloside obtained;

[0075] Figure 13 Graph showing the purity of kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside prepared in Example 1. Detailed Implementation

[0076] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All features disclosed in this specification, or steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0078] Unless otherwise stated, the terms used in this invention have the following meanings.

[0079] "Small-leaf lotus" refers to samples derived from the medicinal plant of small-leaf lotus, including but not limited to small-leaf lotus medicinal materials, small-leaf lotus slices, small-leaf lotus powder, small-leaf lotus extract, or other unprocessed samples made from small-leaf lotus.

[0080] "Alcohol solution" refers to a solution containing an alcohol solvent and water, wherein the alcohol solvent includes, but is not limited to, methanol, ethanol or a combination thereof.

[0081] "Volume fraction" refers to the proportion of a liquid component in a liquid mixture by volume. For example, a 70% volume fraction ethanol solution means that ethanol accounts for 70% of the volume of an aqueous ethanol solution.

[0082] "Aqueous medium" refers to a liquid medium with water as the main component that can be mixed with the extract of *Echeveria elegans* to form the extract solution, including but not limited to water, aqueous solutions, aqueous alcohol solutions, or acidic aqueous solutions.

[0083] "Extraction solution" refers to the liquid system obtained by mixing the extract of *Echeveria elegans* with an aqueous medium, which is used for subsequent liquid-liquid extraction.

[0084] "Liquid-liquid extraction" refers to the operation of transferring, removing impurities, or enriching components by utilizing the difference in the partitioning ability of target and non-target components between different liquid phases.

[0085] "First extraction solvent" refers to the petroleum ether-ethyl acetate mixed solvent used for pretreatment extraction of the extract.

[0086] "Second extraction solvent" refers to the n-butanol-ethyl acetate-water mixed solvent used to enrich the target flavonoid glycosides.

[0087] "Target flavonoid glycosides" refers to flavonoid glycosides in *Echeveria elegans* that are to be enriched, separated, or purified, including but not limited to rutin, isoquercitrin, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside.

[0088] "Total flavonoid glycoside crude extract" refers to the crude separation product rich in target flavonoid glycosides obtained by liquid-liquid extraction of *Echeveria elegans* extract.

[0089] "Enrichment separation" refers to the process of increasing the relative content of a target component in a specific component through chromatography or other separation methods, and separating the target component from some impurities or other components.

[0090] "High-speed countercurrent chromatography" refers to a liquid-liquid partition chromatography technique that utilizes a two-phase solvent system that is not completely immiscible, with one phase as the stationary phase and the other as the mobile phase, to achieve separation of target components by means of the difference in distribution between the two phases.

[0091] The “n-butanol-ethyl acetate-water solvent system” refers to a two-phase solvent system composed of n-butanol, ethyl acetate and water, which can be mixed and allowed to stand to form a two-phase system for high-speed countercurrent chromatography separation.

[0092] "Eluting by introducing the mobile phase in the first direction" means that during high-speed countercurrent chromatography separation, the mobile phase enters the chromatographic system in a preset direction and is eluted.

[0093] "Changing the direction of the mobile phase to continue elution" refers to changing the direction of the mobile phase in the high-speed countercurrent chromatography separation process to a direction different from the first direction, so that the remaining components can continue to be eluted.

[0094] "High-speed countercurrent chromatography enrichment fraction" refers to the intermediate fraction or fraction containing one or more target flavonoid glycosides collected after high-speed countercurrent chromatography enrichment and separation of the crude total flavonoid glycoside extract. The high-speed countercurrent chromatography enrichment fraction is not equivalent to the final purified compound; it requires further purification by preparative liquid chromatography to obtain the target compound.

[0095] "High-speed countercurrent chromatography enrichment component I, high-speed countercurrent chromatography enrichment component II, high-speed countercurrent chromatography enrichment component III, high-speed countercurrent chromatography enrichment component IV, and high-speed countercurrent chromatography enrichment component V" refers to the numbering of different high-speed countercurrent chromatography enrichment components according to the elution or collection order of high-speed countercurrent chromatography. The numbering is only used to distinguish different collection parts and does not necessarily indicate a one-to-one correspondence with the final six flavonoid glycoside compounds.

[0096] "Preparative liquid chromatography" refers to liquid chromatography techniques used for the preparation, separation, or purification of target compounds, including but not limited to preparative high-performance liquid chromatography, semi-preparative high-performance liquid chromatography, or other liquid chromatography techniques with preparative separation functions.

[0097] "Organic phase-water system" refers to a liquid chromatography mobile phase system with organic solvent and water as the main components. The organic solvent includes, but is not limited to, acetonitrile, methanol or a combination thereof; wherein the water can be pure water or an acidic aqueous solution.

[0098] "Acetonitrile-water system" refers to a liquid chromatography mobile phase system with acetonitrile and water as the main components, where the water can be pure water or an acidic aqueous solution.

[0099] "Methanol-water system" refers to a liquid chromatography mobile phase system with methanol and water as the main components, where the water can be pure water or an acidic aqueous solution.

[0100] "Isocratic elution" refers to an elution method in which the composition of the mobile phase remains essentially unchanged during the chromatographic elution process.

[0101] "Gradient elution" refers to an elution method in which the composition of the mobile phase changes over time during chromatographic elution.

[0102] "Target peak" refers to the chromatographic peak corresponding to the target flavonoid glycoside during the preparative liquid chromatography purification process.

[0103] "Preparative liquid-phase purified product" refers to the product obtained after high-speed countercurrent chromatography enrichment of components, purification by preparative liquid chromatography, and collection of the target peak. The preparative liquid-phase purified product, after structural identification, can be confirmed as the six flavonoid glycoside compounds described in this invention.

[0104] "Acidic aqueous solution" refers to an aqueous solution containing acidic additives, including but not limited to formic acid, acetic acid, glacial acetic acid, phosphoric acid, or combinations thereof.

[0105] "Volume ratio" refers to the ratio of each liquid component by volume. For example, n-butanol:ethyl acetate:water = 5:1:5 means that the volume ratio of n-butanol, ethyl acetate and water is 5:1:5.

[0106] "Chemical reference standards" refer to compound monomers that, after separation, purification, and structural confirmation, can be used for component identification, content determination, quality control, or related research.

[0107] In this invention, high-performance liquid chromatography (HPLC) can be used to detect the extract of *Erigeron simonii*, crude extract of total flavonoid glycosides, high-speed countercurrent chromatography enriched components, and / or preparative liquid chromatography purified products. Preferably, the HPLC detection uses a C18 column with gradient elution of an aqueous solution containing 0.1% glacial acetic acid and acetonitrile as the mobile phase; wherein the volume fraction of acetonitrile increases from 10% to 55% within 0–30 min, and the detection wavelength is 254 nm.

[0108] "Including" or "comprises" indicates an open-ended limitation, meaning that in addition to the listed technical features, other technical features not explicitly listed may also be included. "Including but not limited to" indicates that the listed content is merely an example and does not exclude other technical means with the same or similar functions.

[0109] Example 1

[0110] 1. Sample extraction

[0111] Dried *Echeveria elegans* herb was pulverized and placed in an ultrasonic extractor. 70% ethanol solution (material-to-liquid ratio 1:15) was added at room temperature, and ultrasonic extraction was performed for 1 hour. Under these conditions, the peak area ratio of the six flavonoid glycosides was 26.5%. After standing and separating into layers, the mixture was filtered. The filtrate was concentrated under reduced pressure until no alcohol odor was detected, yielding *Echeveria elegans* extract.

[0112] 2. Liquid-liquid extraction

[0113] The above-mentioned extract was dispersed in an appropriate amount of water to prepare an aqueous extract solution. First, petroleum ether and ethyl acetate were repeatedly extracted at a ratio of 1:3 until the upper phase showed no obvious color, in order to remove non-polar and moderately polar impurities. The treated aqueous phase was then extracted eight times with a mixed solvent of n-butanol, ethyl acetate, and water at a volume ratio of 5:1:5. Under these conditions, the peak area of ​​the six flavonoid glycosides reached 82% after extraction. The upper phases were combined, concentrated to dryness under reduced pressure, and then freeze-dried to obtain the crude extract of total flavonoid glycosides for later use.

[0114] 3. Enrichment and Separation

[0115] The crude extract of total flavonoid glycosides rich in the target compounds obtained in step 2 was enriched and separated by high-speed countercurrent chromatography (HSCLC) using a combination of forward and reverse rotation. Specifically, the mobile phase was first eluted in forward rotation mode to separate the first two components, then the mobile phase direction was reversed. The stationary phase was pumped into a multilayer spiral tube at a flow rate of 30 mL / min. Under the conditions of a rotation speed of 800 rpm, a column temperature of 40℃, and a flow rate of 6 mL / min, the stationary phase retention rate was 68%. The volume ratio of the solvent in the n-butanol-ethyl acetate-water solvent system was V... 正丁醇 V 乙酸乙酯 V 水 The ratio of flavonoid glycosides to other compounds was 1:10:10, with partition coefficients of 0.34, 2.72, 1.00, 5.73, 6.91, and 3.06, respectively. Using this system, five fractions were obtained, and the elution time was 300 min. Specifically, the peak area of ​​flavonoid glycosides in fraction I was 92%, in fraction II it was 85%, in fraction III the peak areas of the two flavonoid glycosides were 40% and 54%, respectively, in fraction IV it was 75%, and in fraction V it was 89%.

[0116] 4. Purification

[0117] When purifying the components obtained by high-speed countercurrent chromatography (HSCLC) using preparative liquid chromatography (PLC), a C18 column was used with gradient elution of acetonitrile-water (0.1% glacial acetic acid) as the mobile phase. The detection wavelength was 254 nm. Component I, corresponding to compound 1 (rutin), was purified using isocratic elution (acetonitrile:water = 25:75, v / v), resulting in a 99% peak area for the purified flavonoid glycosides. Component II, corresponding to compound 3 (quercetin-3-O-(6''-O-malonyl)-β-D-glucoside), was purified using gradient elution (0–30 min, acetonitrile ratio increasing from 15% to 30%), resulting in a 98% peak area for the purified flavonoid glycosides. Component III, containing compounds 5 (astragalin) and 6 (kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside), was purified using gradient elution (0–30 min). (The acetonitrile ratio was increased from 20% to 35%). Under these conditions, the peak areas of the two flavonoid glycosides purified were 96% and 98%, respectively. Compound 2 (isoquercetin) corresponding to component IV and compound 4 (kaempferol-3-O-rutin) corresponding to component V were purified by isocratic elution (acetonitrile:water = 27:73, v / v). Under these conditions, the peak areas of the flavonoid glycosides purified were 98% and 97%, respectively.

[0118] 5. Structural Identification

[0119] The six isolated compounds were dissolved in DMSO and analyzed by NMR. They were identified by 1H-NMR and 13C-NMR as rutin, isoquercitrin, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside.

[0120] Example 2

[0121] 1. Sample extraction

[0122] Dried *Echeveria elegans* herb was pulverized and placed in an ultrasonic extractor. A 50% ethanol solution (material-to-liquid ratio 1:15) was added at room temperature, and ultrasonic extraction was performed for 1 hour. Under these conditions, the peak area ratio of the six flavonoid glycosides was 21.5%. After standing and separating into layers, the mixture was filtered. The filtrate was concentrated under reduced pressure until no alcohol odor was detected, yielding *Echeveria elegans* extract.

[0123] 2. Liquid-liquid extraction

[0124] The above-mentioned extract was dispersed in an appropriate amount of water to prepare an aqueous extract solution. First, petroleum ether and ethyl acetate were repeatedly extracted at a ratio of 1:3 until the upper phase showed no obvious color, in order to remove non-polar and moderately polar impurities. The treated aqueous phase was then extracted three times with a mixed solvent of n-butanol, ethyl acetate, and water at a volume ratio of 2:1:2. Under these conditions, the peak area of ​​the six flavonoid glycosides accounted for 65% after extraction. The upper phases were combined, concentrated to dryness under reduced pressure, and then freeze-dried to obtain the crude extract of total flavonoid glycosides for later use.

[0125] 3. Enrichment and Separation

[0126] The crude extract of total flavonoid glycosides rich in the target compounds obtained in step 2 was enriched and separated by high-speed countercurrent chromatography (HSCLC) using a combination of forward and reverse rotation. Specifically, the mobile phase was first eluted in forward rotation mode to separate the first three components, then the mobile phase direction was reversed. The stationary phase was pumped into a multilayer spiral tube at a flow rate of 30 mL / min. Under the conditions of a rotation speed of 600 rpm, a column temperature of 30℃, and a flow rate of 6 mL / min, the stationary phase retention rate was 60%. The volume ratio of the solvent in the n-butanol-ethyl acetate-water solvent system was V... 正丁醇 V 乙酸乙酯 V 水 With a ratio of 0.5:8:8, this system yielded five fractions, with a total elution time of 330 min. The peak area of ​​flavonoid glycosides was 85% in fraction I, 88% in fraction II, 84% in fraction III, 45% and 50% for the two flavonoid glycoside peaks in fraction IV, and 86% in fraction V.

[0127] 4. Purification

[0128] The components separated by high-speed countercurrent chromatography were purified by preparative liquid chromatography using a C18 column and a gradient elution with methanol-water (0.1% glacial acetic acid) as the mobile phase. The detection wavelength was 254 nm. nm, wherein, component I corresponds to compound 1 (rutin), and isocratic elution (methanol:water = 25:75, v / v) was used, under which the peak area of ​​the purified flavonoid glycosides was 92%; component II corresponds to compound 4 (kaempferol-3-O-rutin), and isocratic elution (0–30 min, methanol ratio increased from 15% to 30%) was used, under which the peak area of ​​the purified flavonoid glycosides was 95%; component III corresponds to compound 2 (isoquercetin), and isocratic elution (methanol:water = 30:70, v / v) was used, under which the peak area of ​​the purified flavonoid glycosides was 92%; component IV contains compounds 5 (astragalin) and 6 (kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside), and isocratic elution (0–30 min, methanol ratio increased from 15% to 30%) was used, under which the peak area of ​​the purified flavonoid glycosides was 92%; and ... (The methanol ratio was increased from 20% to 35%). Under these conditions, the peak areas of the two flavonoid glycosides purified were 90% and 92%, respectively. Compound 3 (quercetin-3-O-(6''-O-malonyl)-β-D-glucoside) corresponding to component V was purified by isocratic elution (methanol:water = 25:75, v / v). Under these conditions, the peak area of ​​the flavonoid glycosides purified was 94%.

[0129] 5. Structural Identification

[0130] The six isolated compounds were dissolved in DMSO and analyzed by NMR. 1 H-NMR and 13 C-NMR analysis revealed that the compounds were rutin, isoquercitrin, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside.

[0131] Example 3

[0132] 1. Sample extraction

[0133] Dried *Echeveria elegans* herb was pulverized and placed in an ultrasonic extractor. 90% ethanol solution (material-to-liquid ratio 1:15) was added at room temperature, and ultrasonic extraction was performed for 1 hour. Under these conditions, the peak area ratio of the six flavonoid glycosides was 27%. After standing and separating into layers, the mixture was filtered. The filtrate was concentrated under reduced pressure until no alcohol odor was detected, yielding *Echeveria elegans* extract.

[0134] 2. Liquid-liquid extraction

[0135] The above-mentioned extract was dispersed in an appropriate amount of water to prepare an aqueous extract solution. First, petroleum ether and ethyl acetate were repeatedly extracted at a ratio of 1:3 until the upper phase showed no obvious color, in order to remove non-polar and moderately polar impurities. The treated aqueous phase was then extracted five times with a mixed solvent of n-butanol, ethyl acetate, and water at a volume ratio of 5:1:5. Under these conditions, the peak area of ​​the six flavonoid glycosides accounted for 75% after extraction. The upper phases were combined, concentrated to dryness under reduced pressure, and then freeze-dried to obtain the crude extract of total flavonoid glycosides for later use.

[0136] 3. Enrichment and Separation

[0137] The crude extract of total flavonoid glycosides rich in the target compounds obtained in step 2 was enriched and separated by high-speed countercurrent chromatography (HSCLC) using a combination of forward and reverse rotation. Specifically, the mobile phase was first eluted in forward rotation mode to separate the first four components, then the mobile phase direction was reversed. The stationary phase was pumped into a multilayer spiral tube at a flow rate of 30 mL / min. Under the conditions of a rotation speed of 800 rpm, a column temperature of 50℃, and a flow rate of 6 mL / min, the stationary phase retention rate was 65%. The volume ratio of the solvent in the n-butanol-ethyl acetate-water solvent system was V... 正丁醇 V 乙酸乙酯 V 水 The ratio was 2:12:12. Using this system, five fractions were obtained, and the elution time was 380 min. The peak area of ​​flavonoid glycosides in fraction I was 87%, in fraction II it was 89%, in fraction III it was 75%, in fraction IV it was 78%, and in fraction V the peak areas of the two flavonoid glycosides were 40% and 48%, respectively.

[0138] 4. Purification

[0139] The components separated by high-speed countercurrent chromatography were purified by preparative liquid chromatography using a C18 column and gradient elution with acetonitrile-water (0.1% glacial acetic acid) as the mobile phase. The detection wavelength was 254 nm. Component I (corresponding to compound 1, rutin) and component II (corresponding to compound 4, kaempferol-3-O-rutin) were eluted isocratically (acetonitrile:water = 25:75, v / v). Under these conditions, the peak areas of the purified flavonoid glycosides were 97% and 95%, respectively. Component III (corresponding to compound 2, isoquercitrin) was purified using gradient elution (0–30 nm). The acetonitrile ratio was increased from 15% to 30% at 0 min. Under these conditions, the peak area of ​​the purified flavonoid glycosides was 97%. Compound 6 (kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside) of component IV was purified by isocratic elution (acetonitrile:water = 30:70, v / v). Under these conditions, the peak area of ​​the purified flavonoid glycosides was 92%. Component V, containing compound 3 (quercetin-3-O-(6''-O-malonyl)-β-D-glucoside) and compound 5 (astragaloside), was purified by gradient elution (0–30 min, acetonitrile ratio increased from 15% to 35%). Under these conditions, the peak areas of the two purified flavonoid glycosides were 90% and 92%, respectively.

[0140] 5. Structural Identification

[0141] The six isolated compounds were dissolved in DMSO and analyzed by NMR. They were identified by 1H-NMR and 13C-NMR as rutin, isoquercitrin, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside.

[0142] Example 4

[0143] 1. Sample extraction

[0144] Dried *Echeveria elegans* herb was pulverized and placed in an ultrasonic extractor. 70% ethanol solution (material-to-liquid ratio 1:10) was added at room temperature, and ultrasonic extraction was performed for 1 hour. Under these conditions, the peak area ratio of the six flavonoid glycosides was 26.5%. After standing and separating into layers, the mixture was filtered. The filtrate was concentrated under reduced pressure until no alcohol odor was detected, yielding *Echeveria elegans* extract.

[0145] 2. Liquid-liquid extraction

[0146] The above-mentioned extract was dispersed in an appropriate amount of water to prepare an aqueous extract solution. First, petroleum ether and ethyl acetate were repeatedly extracted at a ratio of 1:3 until the upper phase showed no obvious color, in order to remove non-polar and moderately polar impurities. The treated aqueous phase was then extracted five times with a mixed solvent of n-butanol, ethyl acetate, and water at a volume ratio of 4:1:4. Under these conditions, the peak area of ​​the six flavonoid glycosides reached 76% after extraction. The upper phases were combined, concentrated to dryness under reduced pressure, and then freeze-dried to obtain the crude extract of total flavonoid glycosides for later use.

[0147] 3. Enrichment and Separation

[0148] The crude extract of total flavonoid glycosides rich in the target compounds obtained in step 2 was enriched and separated by high-speed countercurrent chromatography (HSCLC) using a combination of forward and reverse elution. Specifically, the mobile phase was first eluted in forward mode to separate the first fraction, and then the direction of the mobile phase was reversed. The stationary phase was pumped into a multilayer spiral tube at a flow rate of 30 mL / min. Under the conditions of a rotation speed of 1000 rpm, a column temperature of 40℃, and a flow rate of 6 mL / min, the retention rate of the stationary phase was 69%. The volume ratio of the solvent in the n-butanol-ethyl acetate-water solvent system was V. 正丁醇 V 乙酸乙酯 V 水 The ratio was 2:10:10. Using this system, four components were obtained, and the elution time was 430 min. Among them, the peak area of ​​flavonoid glycosides in component I was 89%, the peak area of ​​flavonoid glycosides in component II was 85%, the peak areas of the two flavonoid glycosides in component III were 46% and 43%, respectively, and the peak areas of the two flavonoid glycosides in component IV were 40% and 47%, respectively.

[0149] 4. Purification

[0150] When purifying the components obtained by high-speed countercurrent chromatography (HSCLC) using preparative liquid chromatography (PLC), a C18 column was used with gradient elution of acetonitrile-water (0.1% glacial acetic acid) as the mobile phase and a detection wavelength of 254 nm. nm, wherein, component I corresponds to compound 1 (rutin), and isocratic elution (acetonitrile:water = 25:75, v / v) was used, and the peak area of ​​the purified flavonoid glycosides was 97% under these conditions; component II corresponds to compound 4 (kaempferol-3-O-rutin glycoside), and isocratic elution (acetonitrile:water = 25:75, v / v) was used, and the peak area of ​​the purified flavonoid glycosides was 98% under these conditions; component III corresponds to compounds 2 (isoquercetin) and 3 (quercetin-3-O-(6''-O-malonyl)-β-D-glucoside), and isocratic elution (0–30 min, acetonitrile ratio increasing from 20% to 35%) was used, and the peak areas of the two purified flavonoid glycosides were 92% and 94% respectively under these conditions; component IV corresponds to compounds 5 (astragalin) and 6 (kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside), and isocratic elution (0–30 min, acetonitrile ratio increasing from 20% to 35%) was used, ... (The acetonitrile ratio was increased from 20% to 35%). Under these conditions, the peak areas of the two flavonoid glycosides purified were 89% and 93%, respectively.

[0151] 5. Structural Identification

[0152] The six isolated compounds were dissolved in DMSO and analyzed by NMR. They were identified by 1H-NMR and 13C-NMR as rutin, isoquercitrin, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside.

[0153] Comparative Example 1

[0154] The high-speed countercurrent chromatography solvent system was 2:8:1:9 (v:v:v:v) using hexane-ethyl acetate-methanol-water, and the other conditions were the same as in Example 1.

[0155] The results showed that the partition coefficients (K values) of flavonoid glycosides between the two phases in this system were 0.009, 0.189, 0.026, 0.328, 0.046, and 0.168, all of which were far below the selectivity difference threshold required for effective separation. The target flavonoid glycosides were almost completely partitioned in the lower phase in this system, and the effective enrichment and separation of the six target flavonoid glycosides could not be achieved.

[0156] Comparative Example 2

[0157] Ethyl acetate-water = 1:1 (v:v) was used as the high-speed countercurrent chromatography solvent system, and the other conditions were the same as in Example 1.

[0158] The results showed that the partition coefficients (K values) of the six flavonoid glycosides between the two phases in this system were 0.07, 1.32, 1.48, 0.19, 0.40, and 1.26. The K values ​​fell into two categories: those with low K values ​​of 0.07, 0.19, and 0.40 eluted too quickly in HSCCC, merging into an indistinguishable broad peak; while those with K values ​​of 1.48, 1.32, and 1.26, although capable of separation, had small separation factors (α), resulting in peak overlap. This system could not simultaneously obtain all six target flavonoid glycosides.

[0159] Comparative Example 3

[0160] The method is the same as in Example 1, except that HSCCC uses a forward elution mode throughout the process and does not reverse the direction of the mobile phase.

[0161] The results showed that the first two components could be eluted and collected normally, consistent with Example 1; however, the remaining components still exhibited severe peak broadening after elution in forward mode for over 500 min, making collection impossible within a reasonable timeframe. In contrast, Example 1, after separating the first two components, changed the mobile phase direction, resulting in rapid elution of the high-K-value components retained in the stationary phase, allowing for the collection of all five components within 300 min. This comparative example demonstrates that in solvent systems with a wide K-value range (0.34–6.91), unidirectional elution alone is insufficient for effectively eluting high-K-value components; changing the mobile phase direction is a key technique for achieving efficient and complete elution of all six target flavonoid glycosides.

[0162] Comparative Example 4

[0163] The method of Example 1 was followed, except that the second extraction solvent was n-butanol-ethyl acetate-water (volume ratio 1:5:1), and the extraction was performed three times. The results showed that after extraction, the peak area of ​​the six flavonoid glycosides accounted for only 58%, and most of the target flavonoid glycosides remained in the aqueous phase and were not effectively transferred to the organic phase, resulting in a significant decrease in the overall recovery rate.

[0164] Comparative Example 5

[0165] The method was the same as in Example 1, except that isocratic elution (acetonitrile:water = 25:75, v / v) was used in preparative liquid chromatography for the purification of HSCCC component III obtained in step 3. The results showed that the two target components merged into a single broad peak and could not be effectively separated.

[0166] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for simultaneously isolating and purifying six flavonoid glycosides from *Echeveria elegans*, characterized in that, Includes the following steps: (1) Extraction: The small-leaved lotus was extracted with an alcohol solution with a volume fraction of 50% to 90%, and the extract was concentrated to obtain the small-leaved lotus extract paste; (2) Liquid-liquid extraction: The extract of the small-leaved lotus is mixed with an aqueous medium to obtain the extract to be extracted; then the extract to be extracted is extracted sequentially with the first extraction solvent and the second extraction solvent, the extract containing the target flavonoid glycosides is collected, concentrated and dried to obtain crude extract of total flavonoid glycosides. Wherein, the first extraction solvent is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 1:1 to 1:5; the second extraction solvent is a mixed solvent of n-butanol, ethyl acetate and water, with a volume ratio of n-butanol, ethyl acetate and water of (1 to 10):(0.1 to 5):(1 to 10). (3) Enrichment and separation: The crude extract of total flavonoid glycosides is enriched and separated by high-speed countercurrent chromatography to obtain a component containing the target flavonoid glycosides; wherein the high-speed countercurrent chromatography uses a n-butanol-ethyl acetate-water solvent system for elution, and the volume ratio of n-butanol, ethyl acetate and water in the n-butanol-ethyl acetate-water solvent system is (0.5~5):(5~15):(5~15); (4) Purification: The components obtained in step (3) were purified by preparative liquid chromatography. The mobile phase was an organic phase-water system, and the organic phase was selected from acetonitrile, methanol or a combination thereof. The volume fraction of the organic phase in the mobile phase was 5% to 50%. The elution method was isocratic elution and / or gradient elution, to obtain 6 flavonoid glycosides. The six flavonoid glycosides mentioned are rutin, isoquercitrin, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-3-O-(6''-O-malonyl)-β-D-glucoside.

2. The method according to claim 1, characterized in that, In step (1), the alcohol solution is a methanol solution, an ethanol solution, or a combination thereof; preferably, the alcohol solution is an ethanol solution; more preferably, the volume fraction of the ethanol solution is 60% to 80%; more preferably, it is 70%.

3. The method according to claim 1, characterized in that, In step (1), 1-30 mL of alcohol solution is used to extract 1 g of *Echeveria elegans*; preferably, 10-20 mL of alcohol solution is used to extract 1 g of *Echeveria elegans*; more preferably, 15 mL of alcohol solution is used to extract 1 g of *Echeveria elegans*.

4. The method according to claim 1, characterized in that, In step (2): The aqueous medium is water or an aqueous solution, preferably water; And / or, the volume ratio of petroleum ether to ethyl acetate in the first extraction solvent is 1:2 to 1:4, preferably 1:3; And / or, the volume ratio of n-butanol, ethyl acetate and water in the second extraction solvent is (2-5):(0.5-1.5):(2-5); preferably, the volume ratio of n-butanol, ethyl acetate and water is (2-5):1:(2-5); more preferably, the volume ratio of n-butanol, ethyl acetate and water is 5:1:

5.

5. The method according to claim 1, characterized in that, In step (3), the volume ratio of n-butanol, ethyl acetate and water in the n-butanol-ethyl acetate-water solvent system is (0.5-2):(8-12):(8-12), preferably 1:10:

10.

6. The method according to claim 1, characterized in that, In step (3), during the high-speed countercurrent chromatography separation process, the mobile phase is first introduced in the first direction for elution. After the first 1 to 4 components are separated, the direction of the mobile phase is changed to continue elution. Preferably, after the first 2 components are separated, the direction of the mobile phase is changed to continue elution.

7. The method according to claim 6, characterized in that, The fraction containing the target flavonoid glycosides obtained in step (3) is the high-speed countercurrent chromatography enrichment fraction; The number of components enriched by high-speed countercurrent chromatography is 4 or 5; When the number of high-speed countercurrent chromatography enrichment components is 4, they are sequentially named high-speed countercurrent chromatography enrichment component I, high-speed countercurrent chromatography enrichment component II, high-speed countercurrent chromatography enrichment component III, and high-speed countercurrent chromatography enrichment component IV according to the high-speed countercurrent chromatography elution order. When the number of high-speed countercurrent chromatography enrichment components is 5, they are sequentially named high-speed countercurrent chromatography enrichment component I, high-speed countercurrent chromatography enrichment component II, high-speed countercurrent chromatography enrichment component III, high-speed countercurrent chromatography enrichment component IV, and high-speed countercurrent chromatography enrichment component V according to the high-speed countercurrent chromatography elution order. In this embodiment, at least one of the high-speed countercurrent chromatography enrichment components contains two target flavonoid glycosides.

8. The method according to claim 7, characterized in that, In step (4), based on the number of components enriched by high-speed countercurrent chromatography and the mobile phase system, the purification conditions for preparative liquid chromatography include any one of the following items (i) to (iv): (i) When the number of components enriched by the high-speed countercurrent chromatography is 5, the preparative liquid chromatography uses an acetonitrile-water system for elution; Among them, high-speed countercurrent chromatography (HSCCC) enrichment component I was eluted using isocratic elution with a volume ratio of acetonitrile to water of 25:75; HSCCC enrichment component II was eluted using gradient elution, with the volume fraction of acetonitrile increasing from 15% to 30% within 0–30 min; HSCCC enrichment component III was eluted using gradient elution, with the volume fraction of acetonitrile increasing from 20% to 35% within 0–30 min; HSCCC enrichment components IV and V were eluted using isocratic elution with a volume ratio of acetonitrile to water of 27:

73. (ii) When the number of components enriched by the high-speed countercurrent chromatography is 5, the preparative liquid chromatography uses a methanol-water system for elution; Among them, high-speed countercurrent chromatography (HSCCC) enrichment fraction I was eluted using isocratic elution with a methanol-to-water volume ratio of 25:75; HSCCC enrichment fraction II was eluted using gradient elution, with the methanol volume fraction increasing from 15% to 30% within 0–30 min; HSCCC enrichment fraction III was eluted using isocratic elution with a methanol-to-water volume ratio of 30:70; HSCCC enrichment fraction IV was eluted using gradient elution, with the methanol volume fraction increasing from 20% to 35% within 0–30 min; and HSCCC enrichment fraction V was eluted using isocratic elution with a methanol-to-water volume ratio of 25:

75. (iii) When the number of components enriched by the high-speed countercurrent chromatography is 5, the preparative liquid chromatography uses an acetonitrile-water system for elution; In this study, high-speed countercurrent chromatography (HSCCC) enrichment fractions I and II were eluted using isocratic elution with an acetonitrile to water volume ratio of 25:75; HSCCC enrichment fraction III was eluted using gradient elution, with the acetonitrile volume fraction increasing from 15% to 30% within 0–30 min; HSCCC enrichment fraction IV was eluted using isocratic elution with an acetonitrile to water volume ratio of 30:70; and HSCCC enrichment fraction V was eluted using gradient elution, with the acetonitrile volume fraction increasing from 15% to 35% within 0–30 min. (iv) When the number of components enriched by the high-speed countercurrent chromatography is 4, the preparative liquid chromatography uses an acetonitrile-water system for elution; Among them, high-speed countercurrent chromatography (HSCCC) enrichment components I and II were eluted using isocratic elution with a volume ratio of acetonitrile to water of 25:75; HSCCC enrichment components III and IV were eluted using gradient elution, with the volume fraction of acetonitrile increasing from 20% to 35% within 0–30 min. After the above elution, the target peaks in each high-speed countercurrent chromatography enrichment fraction were collected to obtain the preparative liquid phase purified product.

9. The method according to claim 1, characterized in that, The method satisfies one or more of the following conditions: (a) In step (3), the rotation speed of the high-speed countercurrent chromatography is 600 to 1000 rpm; (b) In step (3), the column temperature of the high-speed countercurrent chromatography is 30–50 °C; (c) In step (3), the flow rate of the mobile phase in the high-speed countercurrent chromatography is 4 to 8 mL / min; (d) In step (3), the detection wavelength of the high-speed countercurrent chromatography is 254 nm; (e) In step (4), the preparative liquid chromatography is performed using a C18 column for purification; (f) In step (4), the water in the organic phase-water system is an acidic aqueous solution, and the acid in the acidic aqueous solution is selected from one or more of formic acid, acetic acid, glacial acetic acid, and phosphoric acid, and the volume fraction of the acid in the acidic aqueous solution is 0.05% to 0.5%; Preferably, in step (3), the high-speed countercurrent chromatography has a rotation speed of 800 rpm, a column temperature of 40℃, a mobile phase flow rate of 6 mL / min, and a detection wavelength of 254 nm. Preferably, in step (4), the acidic aqueous solution is an aqueous solution containing 0.1% glacial acetic acid.

10. The method according to claim 8, characterized in that, The method meets the following conditions, and the six flavonoid glycosides obtained can be used as chemical reference standards after structural confirmation: ① In step (1), the alcohol solution is a 70% ethanol solution by volume, and 15 mL of alcohol solution is used to extract 1 g of *Echeveria elegans*. ② In step (2), the volume ratio of petroleum ether to ethyl acetate in the first extraction solvent is 1:3; the volume ratio of n-butanol, ethyl acetate and water in the second extraction solvent is 5:1:5, and the number of extractions is 8. ③ In step (3), the volume ratio of n-butanol, ethyl acetate and water in the n-butanol-ethyl acetate-water solvent system is 1:10:10, the rotation speed of the high-speed countercurrent chromatography is 800 rpm, the column temperature is 40℃, the mobile phase flow rate is 6 mL / min, and after the first two components are separated, the direction of the mobile phase is changed to continue elution. ④ In step (4), the preparative liquid chromatography is purified using a C18 column and uses an acetonitrile-water system as the mobile phase, wherein the water is an aqueous solution containing 0.1% glacial acetic acid, and elution is performed according to the conditions described in claim 8, paragraph (i).

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