Method for extracting harpagoside from magic grass

Abstract

The invention relates to a method for extracting a magic grass extract, in particular to a method for extracting harpagoside from magic grass, and aims to overcome the defect of long process period in the existing technology for extracting harpagoside. The method comprises the steps of raw material crushing and enzymolysis, organic solvent impurity removal, extraction, graded sedimentation, membrane separation, membrane concentration, extraction, crystallization and the like, the whole preparation process is mild in condition, the loss of the harpagoside under a high heat condition is avoided, the influence of temperature on the instability of the harpagoside is effectively avoided, and the method is suitable for industrial production. The activity and the purity of the harpagoside can be maintained, and the harpagoside with relatively high quality can be obtained.

Description

Technical Field

[0001] This invention relates to a method for extracting extracts from *Gnaphalium affine*, specifically a method for extracting harpaoside from *Gnaphalium affine*. Background Technology

[0002] Devil's Claw (Harpagophytum procumbens), also known as devil's claw or South African hook vine, is a perennial herbaceous plant belonging to the genus Harpagophy in the family Pedaliaceae. Its flowers are red, and its fruits are covered with claw-like protrusions. The roots are rhizomatous. Native to southern Africa, it is mainly distributed in the Kalahari Desert, shared by Namibia, Botswana, South Africa, and Angola. It gets its name from the many strangely shaped small claws attached to its fruits.

[0003] The secondary tubers of the roots of the devil's claw herb can be collected, sliced, and dried for medicinal use. Locals in Africa use it to treat skin cancer, colds and fevers, indigestion, malaria, allergies, rheumatism, and arthritis. Since its introduction to Europe in the 18th century, it has been widely used in North America and Europe and is listed in the British Pharmacopoeia and the German Pharmacopoeia as an analgesic, sedative, and diuretic. Modernly, it is mainly used to treat various inflammations, with arthritis being a prominent example.

[0004] The main compounds in *Polygonum multiflorum* are iridoid glycosides (0.5%–3%), which are found in the plant's tubers and are considered to be the most promising therapeutic components. Iridoid glycosides are a class of phenolic glycosides that have been widely used to treat inflammatory diseases. One of the main iridoid glycosides in *Polygonum multiflorum* is harpagoside, with a content of approximately 1%–3%. Harpagoside is one of the most significant and extensively studied active ingredients in *Polygonum multiflorum*, believed to have anti-inflammatory and analgesic effects, and is therefore widely used to treat arthritis and other inflammatory diseases. Harpagoside is also known as harpagoside, harpagoside glycoside, harpagoside glycoside, harpagoside glycoside, etc. Harpagoside has a molecular weight of 494.49 and a molecular formula of C2. 24 H 30 O 11 It is a white crystalline powder, readily soluble in methanol and water, and is one of the main active ingredients of *Gnaphalium affine*. The structural formula of harpagoside is shown below.

[0005]

[0006] In traditional medicine, *Polygonum multiflorum* (also known as devil's claw herb) has been used to treat menstrual problems, reduce inflammation and fever, treat syphilis, and even loss of appetite. Modern medicine has linked chronic inflammation to various diseases, such as Alzheimer's disease, obesity, rheumatoid arthritis, type 2 diabetes, cancer, and cardiovascular and pulmonary diseases. Furthermore, chronic inflammatory diseases account for 60% of deaths from chronic diseases worldwide. Inflammation and pain-related diseases have garnered significant attention as a major global health challenge. Therefore, *Polygonum multiflorum* has excellent development potential.

[0007] Research revealed few reports on the extraction and separation techniques of harpagoside from Salvia miltiorrhiza. The literature mainly describes the separation and purification of harpagoside using macroporous resin enrichment combined with silica gel column chromatography, which involves a long process cycle and is primarily used for the extraction and separation of Salvia miltiorrhiza raw materials.

[0008] Chinese patent CN 112675186A discloses a pharmaceutical composition and its application, which mainly includes harpagoside, harpagoside, verbascoside, cinnamic acid, etc. It directly uses harpagoside for pharmaceutical composition and application, without involving the extraction and separation technology of harpagoside's active ingredients.

[0009] Chinese patent CN 115350224A discloses a uric acid-lowering composition and its preparation method, the active ingredients of which include celery seed extract, sour cherry extract, creeping fig extract, poplar bud extract, sage extract, and devil's claw extract. However, this patent does not disclose the separation technology of devil's claw extract and harpagoside from devil's claw. Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of existing technologies for extracting harpagoside, which have long processing cycles, and to provide a method for extracting harpagoside from *Gnaphalium affine*.

[0011] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0012] A method for extracting harpagoside from *Gnaphalium affine* is characterized by including the following steps:

[0013] Step 1. Raw material crushing and enzymatic hydrolysis, and organic solvent removal: Disperse the compound enzyme in water, crush the raw material of *Polygonum multiflorum* and add it to the compound enzyme, hydrolyze and inactivate the enzyme, remove impurities using n-hexane or petroleum ether at room temperature, filter, collect the filter residue and dry it for later use.

[0014] The compound enzyme is a mixture of cellulase and pectinase, and the amount of compound enzyme added is 3-5‰ of the weight of the raw material of *Polygonum multiflorum*.

[0015] Step 2. Extraction: Extract the above filter residue using an ethanol-water solution or a methanol-water solution, collect the extract, filter, collect the filtrate and concentrate it to obtain the first concentrated paste;

[0016] Step 3. Sedimentation: The first concentrated paste is subjected to sedimentation once, and filtered to obtain a first supernatant. Gelatin solution is added to the first supernatant for a second sedimentation, and filtered again to obtain a second supernatant. Finally, an adsorbent is used to adsorb and remove impurities from the second supernatant.

[0017] Step 4. Membrane separation and membrane concentration: The secondary supernatant after adsorption and impurity removal is passed through an ultrafiltration membrane and a nanofiltration membrane in sequence to remove small molecule impurities such as salts and monosaccharides and to concentrate the solution to obtain a concentrated solution.

[0018] Step 5. Extraction: Adjust the pH of the concentrate to 8-9 with ammonia water to deprotonate it, then extract the concentrate with an organic solvent, collect the extract, and concentrate it until there is no solvent to obtain the second concentrated paste.

[0019] Step 6. Crystallization: Dissolve the second concentrated paste in anhydrous ethanol, filter and concentrate the filtrate, crystallize, filter and dry the crystals to obtain harpaoside.

[0020] Furthermore, in step 1, the amount of water is 1-3 times the mass of the devil's claw herb raw material;

[0021] In the compound enzyme, the mass ratio of cellulase to pectinase is 1-1.5:1, the enzymatic hydrolysis temperature is 45-50℃, and the enzymatic hydrolysis time is 4-6h; the enzyme inactivation temperature is 79-81℃, the heating method is water bath heating, and the enzyme inactivation time is 5-8min.

[0022] The specific method for removing impurities using n-hexane or petroleum ether at room temperature is as follows: use n-hexane or petroleum ether to remove impurities 2-3 times, with each application being 6-10 times the mass of the raw material, and each removal process taking 1-2 hours.

[0023] Further, in step 2, the amount of the ethanol aqueous solution or methanol aqueous solution used is 8-10 times the mass of the filter residue, the mass concentration is 90-95%, the specific gravity of the first concentrated paste is 1.02-1.03, and the number of extractions is at least 2-4.

[0024] In step 3, the first settling is static settling, with a settling time of 2-4 hours and a temperature of room temperature, and the second settling time is 0.5-2 hours;

[0025] The amount of gelatin solution added is 10%-20% of the volume of the first supernatant.

[0026] Further, step 3 specifically involves: adding 10-25% by volume of adsorbent to the secondary supernatant, stirring and adsorbing at 50-70°C for 0.5-1.5 hours, filtering, and collecting the filtrate;

[0027] The adsorbent is a composite adsorbent, which is composed of activated carbon and bentonite, wherein activated carbon accounts for 50-70% and bentonite accounts for 30-50%.

[0028] Further, step 5 specifically involves: adjusting the pH of the concentrate to 9 with ammonia water, then adding an extraction solvent to repeatedly extract the above concentrate, collecting the extract obtained from each extraction, and concentrating it until there is no solvent to obtain a second concentrated paste;

[0029] The extraction solvent is petroleum ether, or ethyl acetate, or a mixture of petroleum ether and ethyl acetate, wherein the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is 1:1.

[0030] The extraction is performed 3-6 times, and the amount of extraction solvent used in each extraction is 1-2 times the volume of the concentrated liquid.

[0031] Furthermore, in step 6, the amount of anhydrous ethanol used is 15-20 times the volume of the second concentrated paste, the filtrate is concentrated to Brix = 49-52°, the crystallization temperature is 2-8°C, and the crystallization time is 48-72h.

[0032] Furthermore, in step 1, the amount of water is 1 times the mass of the *Gnaphalium affine* raw material; in the compound enzyme, the mass ratio of cellulase to pectinase is 1.4:1, the enzymatic hydrolysis temperature is 45℃, the enzymatic hydrolysis time is 4h; the enzyme inactivation temperature is 80℃, and the enzyme inactivation time is 5min.

[0033] In step 2, the amount of ethanol aqueous solution or methanol aqueous solution used is 8 times the mass of the filter residue, the extraction is performed twice, the extraction time is 2 hours per extraction, and the specific gravity of the first concentrated extract is 1.02.

[0034] In step 3, the first settling time is 2 hours and the second settling time is 0.5 hours; the amount of gelatin solution added is 10% of the volume of the first supernatant.

[0035] Furthermore, in step 3, the amount of composite adsorbent is 10% of the secondary supernatant, and adsorption is carried out by stirring at 60°C for 1 hour. The composite adsorbent has 50% activated carbon and 50% bentonite.

[0036] In step 5, the extraction is performed 5 times, and the extraction solvent is ethyl acetate. The amount of extraction solvent used in each extraction is 1 times the volume of the concentrate.

[0037] In step 6, the amount of anhydrous ethanol used is 20 times the volume of the second concentrated paste. The filtrate is concentrated to Brix = 50°, the crystallization temperature is 6°C, and the crystallization time is 48 hours.

[0038] Furthermore, in step 3, the gelatin solution is prepared by mixing gelatin and purified water, with a mass ratio of gelatin to purified water of 1:99.

[0039] Furthermore, in step 4, the ultrafiltration membrane includes membranes with molecular weights of 100 kDa and 1 kDa arranged sequentially, and the nanofiltration membrane has a molecular weight of 200-300 Da.

[0040] The beneficial effects of this invention are:

[0041] 1. In this invention, a compound enzyme hydrolysis process is first selected, which improves the extraction recovery rate by utilizing its high selectivity. At the same time, it lays a good foundation for the removal of small polar impurities with hexane or petroleum ether. Combined with membrane filtration and membrane separation technology, the overall preparation process of this invention is mild, avoiding the loss of harpagoside under high heat conditions, effectively avoiding the influence of temperature on the instability of harpagoside, and helping to maintain the activity and purity of harpagoside, so as to obtain high-quality harpagoside.

[0042] 2. This invention is highly efficient and energy-saving, has low cost, and is easy to operate and control, making it suitable for industrial production.

[0043] 3. This invention abandons the traditional process of macroporous adsorption resin and takes a new approach by using staged sedimentation to remove macromolecular impurities such as proteins and starch, as well as some small molecule impurities such as sugars and heavy metals, thus avoiding the contamination of harmful substances in the final product.

[0044] 4. In this invention, the membrane concentrate is deprotonated by adjusting the pH value before extraction, which can increase the solubility of the active ingredients in organic solvents.

[0045] 5. This invention employs a combination of multiple technologies, including enzymatic hydrolysis, impurity removal, extraction, fractionation and sedimentation, membrane separation, membrane concentration, extraction, and crystallization, to maximize the extraction and purification of active ingredients in the raw material of *Gnaphalium affine*, resulting in a high content of harpagoside. In the final product, the content of harpagoside can reach up to 97%, achieving a harpagoside recovery rate of over 70% in the raw material of *Gnaphalium affine*. Attached Figure Description

[0046] Figure 1 This is a process flow diagram of Embodiment 1 of the present invention;

[0047] Figure 2 This is the HPLC chromatogram of the *Gnaphalium affine* raw material in Example 1 of this invention;

[0048] Figure 3 This is the HPLC chromatogram of harpagoside in the concentrate obtained in step S4 of Example 1 of the present invention;

[0049] Figure 4 This is the HPLC chromatogram of harpagoside in the final product obtained in step S6 of Example 1 of the present invention. Detailed Implementation

[0050] Example 1

[0051] This invention provides a method for extracting harpagoside from *Gnaphalium affine*. Figure 1 As shown, the specific steps include:

[0052] S1. Raw material crushing and enzymatic hydrolysis, followed by impurity removal with organic solvents;

[0053] Take 10 kg of *Gnaphalium affine* raw material, crush it to 60 mesh, weigh out 3‰ of the compound enzyme of the *Gnaphalium affine* raw material and disperse it in water with an amount equal to the weight of the *Gnaphalium affine* raw material, add the *Gnaphalium affine* raw material and stir evenly, then inactivate the enzyme after enzymatic hydrolysis at 45℃ for 4 hours. The inactivation time is 5 minutes and the inactivation temperature is 80℃. The heating method is water bath heating to obtain the enzymatic hydrolysis residue.

[0054] Add n-hexane to the enzymatic hydrolysis residue for extraction and impurity removal three times, filter, collect the filter residue and dry it for later use; the amount of n-hexane added each time is 10 times the mass of the enzymatic hydrolysis residue, the extraction time is 1 hour / time, and the extraction is carried out with stirring at room temperature.

[0055] In this embodiment, the raw material of *Gnaphalium affine* was analyzed by HPLC, such as... Figure 2 As shown, the content of harpagoside was 1.47%. The complex enzyme was a mixture of cellulase and pectinase in a mass ratio of 1.4:1.

[0056] The cell walls of *Polygonum multiflorum* are composed of cellulose, hemicellulose, and pectin, forming a dense structure that restricts the release of target substances. This invention utilizes cellulase and pectinase to specifically degrade cell wall polysaccharides by hydrolyzing the glycosidic bonds in pectin, disrupting their integrity and improving both the efficiency of hexane removal and the extraction rate of harpagoside. Harpagoside is a polar glycoside compound, while lipid-soluble impurities (such as waxes and chlorophyll) are non-polar substances. Therefore, this invention uses hexane as a non-polar solvent, selectively dissolving non-polar impurities based on the principle of "like dissolves like" after enzymatically disrupting the cell wall.

[0057] In other embodiments, petroleum ether can also be used for extraction and impurity removal.

[0058] S2. Ethanol extraction;

[0059] The filter residue obtained in step S1 was extracted twice with an ethanol aqueous solution of 8 times its mass and a mass concentration of 95%, with an extraction time of 2 hours per extraction. The residue was filtered, and the filtrate was concentrated to a specific gravity of 1.02 to obtain the first concentrated paste.

[0060] This invention first removes small polar impurities through enzymatic hydrolysis and n-hexane extraction, and then uses an ethanol aqueous solution to extract the effective components (mainly harpagoside) from the filter residue, which can improve the content and yield of the final product harpagoside and lay the foundation for subsequent purification steps.

[0061] In other embodiments, the active ingredient can also be extracted using a methanol-water solution, with a mass concentration of 90%, and the extraction can be performed 3 or 4 times to fully extract the active ingredient.

[0062] S3. Staged settlement;

[0063] The first concentrated paste obtained from S2 was allowed to stand for 2 hours for sedimentation, filtered, and the first supernatant was collected. 10% of the volume of gelatin solution with a mass concentration of 1% was added to the first supernatant and stirred evenly. After standing for 0.5 hours for sedimentation, the second supernatant was obtained and filtered to remove impurities through adsorption.

[0064] The gelatin solution was prepared by mixing gelatin and purified water in a mass ratio of 1:99.

[0065] The adsorption and impurity removal process involves adding 10% (by volume) of the composite adsorbent to the secondary supernatant, heating to 60°C, stirring for 1 hour, filtering, and collecting the filtrate. The composite adsorbent comprises 50% activated carbon and 50% bentonite by mass. The optimal adsorption capacity of the composite adsorbent is 15-25%.

[0066] In other embodiments, the stirring adsorption temperature can also be 50°C or 70°C, and the stirring adsorption time can be adjusted according to the temperature, ranging from 0.5h to 1.5h.

[0067] Large molecular impurities (such as proteins and starches) have large particle sizes and settle quickly, thus being removed through a single sedimentation step. Small molecular impurities (small sugars and heavy metals) remain suspended and are accelerated to settle during a second sedimentation step with the aid of an adsorbent. Finally, a small amount of pigments and other small molecular impurities in the secondary supernatant are adsorbed by an adsorbent. This step can remove over 90% of large molecular impurities, reducing the risk of subsequent membrane fouling. No complex equipment is required, making it suitable for industrial pretreatment.

[0068] S4. Membrane separation, membrane concentration;

[0069] The filtrate obtained from adsorption and purification was sequentially passed through ultrafiltration membranes with molecular weights of 100 kDa and 1 kDa for further impurity removal. Small molecule impurities such as salts and monosaccharides were retained and removed by the ultrafiltration membranes. The liquid that passed through the ultrafiltration membrane was then concentrated by passing it through a nanofiltration membrane with a molecular weight of 300 Da to obtain a concentrate. A sample of the concentrate was analyzed by HPLC, revealing a harpagoside content of 43.04%. Figure 3 As shown.

[0070] This invention removes small molecule impurities such as salts and monosaccharides through membrane separation. Membrane separation and membrane concentration enable closed-loop production, meaning that separation and concentration can be completed using only membrane equipment composed of ultrafiltration and nanofiltration membranes, without the need for other concentration equipment.

[0071] S5. Extraction;

[0072] The concentrate was adjusted to pH 9 with ammonia, and then extracted five times with ethyl acetate of equal volume. The ethyl acetate extract was collected and concentrated until solvent-free to obtain the second concentrated paste.

[0073] The partition coefficient (K) of harpao glycosides in ethyl acetate and / or petroleum ether increases with increasing pH. Adjusting the pH to 8-9 with ammonia deprotonates the target compound (R-OH→RO-), enhancing its solubility in the organic phase.

[0074] S6. Crystallization;

[0075] Add 20 times its volume of anhydrous ethanol to the second concentrated paste obtained from S5 to dissolve it completely. Filter the solution, then concentrate the filtrate to Brix = 50°C. Crystallize the solution at 6°C for 48 hours. Filter and dry the crystals to obtain 110g of the final product. HPLC analysis showed that the harpagoside content was 96.84% (see [reference]). Figure 4 The final product was 1.10% of the weight of the raw material of *Gnaphalium affine* (i.e., the yield was 1.10%), which is equivalent to 1.07% of the harpagoside content in the raw material of *Gnaphalium affine*, and the recovery rate of harpagoside in the raw material of *Gnaphalium affine* was 72.4%.

[0076] In other embodiments, the crystallization temperature may also be any temperature between 2 and 8°C.

[0077] This invention incorporates hexane purification after enzymatic hydrolysis to better remove small polar impurities, laying the foundation for extracting higher concentrations of harpagoside. Then, it removes large molecules such as polysaccharides through fractional sedimentation and small molecule impurities such as pigments through adsorption. In step S4, membrane separation and concentration techniques are simple and gentle, achieving separation and purification while avoiding harpagoside degradation. Step S5 uses extraction to lay the foundation for further crystallization, making crystals easier to precipitate. This invention sequentially performs crude extraction (enzymatic hydrolysis - removal of small polar impurities - ethanol extraction) and purification (fractional sedimentation - membrane separation and concentration - extraction - crystallization), with each step specifically removing particular impurities, gradually increasing the purity of the final product. By combining these technologies, a high-purity harpagoside monomer is ultimately obtained.

[0078] Example 2

[0079] The present invention discloses a method for extracting harpagoside from *Gnaphalium affine*, which specifically includes the following steps:

[0080] S1. Take 10 kg of *Gnaphalium affine* raw material (harpagoside content approximately 1.50%), pulverize to 60 mesh, weigh out 4‰ of the *Gnaphalium affine* raw material's compound enzyme, disperse it in water equal to twice the weight of the *Gnaphalium affine* raw material, add the *Gnaphalium affine* raw material, stir evenly, and enzymatically hydrolyze at 45℃ for 5 hours, then inactivate the enzyme at 80℃ for 5 minutes. The compound enzyme is a mixture of cellulase and pectinase in a 1:1 mass ratio. Add 10 times the weight of n-hexane to the enzymatic hydrolysis residue, extract twice at room temperature with stirring, each extraction lasting 1 hour. Filter, collect the filter residue, and dry for later use.

[0081] S2. The above filter residue is extracted twice with an ethanol aqueous solution of 9 times its mass and a mass concentration of 95%, with an extraction time of 2 hours each time. After filtration, the filtrate is concentrated to a specific gravity of 1.02 to obtain the first concentrated paste.

[0082] S3. The first concentrated paste is allowed to settle for 3 hours, filtered, and a first supernatant is obtained. A gelatin solution with a volume of 15% and a mass concentration of 1% is added to it. After settling for 0.5 hours, the mixture is filtered again to obtain a second supernatant, which is then adsorbed to remove impurities.

[0083] During adsorption and impurity removal, the amount of composite adsorbent used is 10% of the volume of the secondary supernatant. The composite adsorbent contains 60% activated carbon and 40% bentonite. The adsorption temperature is 60℃ and the stirring adsorption time is 1 hour.

[0084] S4. The secondary supernatant after adsorption and impurity removal is passed through 100 kDa and 1 kDa ultrafiltration membranes in sequence, and then through a nanofiltration membrane with a molecular weight of 300 Da to obtain a concentrated solution. The solution is sampled and tested, and the content of harpagoside is 42.11%.

[0085] S5. The pH of the concentrate is adjusted to 8 with ammonia, and then twice the volume of petroleum ether is added for extraction three times. The extract is collected and concentrated until there is no solvent to obtain a concentrated paste.

[0086] S6. Add 15 times its volume of anhydrous ethanol to the second concentrated paste to dissolve it completely, filter, concentrate the filtrate to Brix = 49°, and let it stand at 6°C for 48 hours to crystallize. Filter and dry the crystals to obtain 115g of the final product, of which the harpagoside content is 95.35%, the yield is 1.15%, which is equivalent to 1.10% of the harpagoside content in the raw material of *Gnaphalium affine*, and the harpagoside recovery rate is 74.6%.

[0087] Example 3

[0088] The present invention discloses a method for extracting harpagoside from *Gnaphalium affine*, which specifically includes the following steps:

[0089] S1. Take 10 kg of *Happathena scabra* raw material (harpagoside content approximately 1.50%), pulverize to 60 mesh, weigh out 5‰ of the *Happathena scabra* raw material's compound enzyme, disperse it in water equal to twice the weight of the *Happathena scabra* raw material, add the *Happathena scabra* raw material, stir evenly, and enzymatically hydrolyze at 45℃ for 6 hours, then inactivate the enzyme at 80℃ for 6 minutes. The compound enzyme is a mixture of cellulase and pectinase in a mass ratio of 1.5:1. Add 6 times the weight of n-hexane to the enzymatic hydrolysis residue, extract twice at room temperature with stirring, 2 hours each time, filter, collect the filter residue, and dry for later use.

[0090] S2. Extract the above filter residue twice with 10 times its mass of 95% ethanol aqueous solution, with an extraction time of 2 hours each time. Filter and concentrate the filtrate to a specific gravity of 1.02 to obtain a concentrated paste.

[0091] S3. The first concentrated paste was allowed to settle for 4 hours, then filtered to obtain a primary supernatant. A gelatin solution with a volume of 20% (1% by mass) was added to the supernatant, and the mixture was allowed to settle for 1 hour. After filtration, a secondary supernatant was obtained. A composite adsorbent with a volume of 15% of the secondary supernatant was then added to the supernatant. The mixture was heated to 60°C and stirred for 1 hour to adsorb the adsorbent. The filtrate was then collected. The composite adsorbent consisted of 70% activated carbon and 30% bentonite.

[0092] S4. The filtrate obtained in S3 is passed through 100 kDa and 1 kDa ultrafiltration membranes to remove impurities, and then concentrated through a nanofiltration membrane with a molecular weight of 300 Da to obtain a concentrate. The concentrate is sampled and tested, and the content of harpagoside is 41.05%.

[0093] S5. The pH of the concentrate is adjusted to 8 with ammonia, and then twice its volume of ethyl acetate:petroleum ether = 1:1 is added for extraction 4 times. The extract is collected and concentrated until there is no solvent to obtain a concentrated paste.

[0094] S6. Add 20 times its volume of anhydrous ethanol to the second concentrated paste to dissolve it completely, filter, concentrate the filtrate to Brix = 52°, and let it stand at 6°C for 60 h to crystallize. Filter and dry the crystals to obtain 109 g of the final product. HPLC analysis showed that the harpagoside content was 96.35%, the yield was 1.09%, which is equivalent to 1.05% harpagoside content in the raw material of *Gnaphalium affine*, and the harpagoside recovery rate was 71.4%.

[0095] Example 4

[0096] The present invention discloses a method for extracting harpagoside from *Gnaphalium affine*, which specifically includes the following steps:

[0097] S1. Take 10 kg of *Gnaphalium affine* raw material (harpagoside content approximately 1.50%), pulverize to 60 mesh, weigh out 5‰ of the *Gnaphalium affine* raw material's compound enzyme, disperse it in water equal to twice the weight of the *Gnaphalium affine* raw material, add the *Gnaphalium affine* raw material to the water and stir evenly. Inactivate the enzyme at 50℃ for 6 hours, then inactivate it for 7 minutes at 80℃. The compound enzyme is a mixture of cellulase and pectinase in a mass ratio of 1.3:1. Add 7 times the weight of n-hexane to the enzymatic hydrolysis residue, extract twice at room temperature with stirring, 1 hour per extraction. Filter, collect the residue, and dry for later use.

[0098] S2. Extract the filter residue twice with 10 times its mass of 95% ethanol aqueous solution, with an extraction time of 2 hours each time. Filter the residue and concentrate the filtrate to a specific gravity of 1.02 to obtain a concentrated paste.

[0099] S3. The first concentrated paste is allowed to settle for 2 hours, then filtered to obtain a primary supernatant. A gelatin solution with a volume of 20% (1% by mass) is added to this supernatant, and the mixture is allowed to settle for 1 hour. After filtration, a secondary supernatant is obtained. A composite adsorbent with a volume of 20% of the secondary supernatant is then added to this supernatant. The mixture is heated to 60°C and stirred for 1 hour to adsorb the adsorbent. The filtrate is then collected. The composite adsorbent consists of 70% activated carbon and 30% bentonite.

[0100] S4. The filtrate obtained in S3 is passed through 100 kDa and 1 kDa ultrafiltration membranes to remove impurities, and then concentrated through a 300 Da nanofiltration membrane to obtain a concentrate. The concentrate is sampled and tested, and the harpagoside content is 42.25%.

[0101] S5. The pH of the concentrate is adjusted to 8 with ammonia, and then twice the volume of ethyl acetate is added for extraction four times. The extract is collected and concentrated until there is no solvent to obtain a concentrated paste.

[0102] S6. Add 20 times its volume of anhydrous ethanol to the second concentrated paste to dissolve it completely, filter, concentrate the filtrate to Brix = 52°, and let it stand at 6°C for 60 h to crystallize. Filter and dry the crystals to obtain a final product of 112 g. HPLC analysis showed that the harpagoside content was 96.43%, the yield was 1.12%, which is equivalent to 1.08% harpagoside content in the raw material of *Gnaphalium affine*, with a recovery rate of 73.5%.

[0103] Example 5

[0104] The present invention discloses a method for extracting harpagoside from *Gnaphalium affine*, which specifically includes the following steps:

[0105] S1. Take 10 kg of *Happathena scabra* raw material (harpagoside content approximately 1.50%), pulverize to 60 mesh, weigh out 5‰ of the *Happathena scabra* raw material's compound enzyme, disperse it in 3 times the weight of the raw material's water, add the *Happathena scabra* raw material to the water and stir evenly. Inactivate the enzyme at 50℃ for 6 hours, then inactivate it for 8 minutes at 80℃. The compound enzyme is a mixture of cellulase and pectinase in a 1:1 mass ratio. Add 9 times the weight of n-hexane to the enzymatic hydrolysis residue, extract twice at room temperature with stirring, 1 hour per extraction. Filter, collect the residue, and dry for later use.

[0106] S2. Extract the above filter residue twice with 10 times its mass of 95% ethanol aqueous solution, with an extraction time of 2 hours each time. Filter and concentrate the filtrate to a specific gravity of 1.02 to obtain a concentrated paste.

[0107] S3. The first concentrated paste is allowed to settle for 2 hours, then filtered to obtain a primary supernatant. A gelatin solution with a volume of 20% (1% by mass) is added to this supernatant, and the mixture is allowed to settle for another 2 hours, then filtered again to obtain a secondary supernatant. A composite adsorbent with a volume of 25% is then added to this supernatant, and the mixture is heated to 60°C and stirred for 1 hour to adsorb the adsorbent. The supernatant is then filtered, and the filtrate is collected. The composite adsorbent consists of 70% activated carbon and 30% bentonite.

[0108] S4. The filtrate obtained in S3 is passed through 100 kDa and 1 kDa ultrafiltration membranes in sequence, and then through a nanofiltration membrane with a molecular weight of 300 Da for concentration to obtain a concentrate. The concentrate is sampled and tested, and the harpagoside content is 40.87%.

[0109] S5. The concentrated solution is adjusted to pH 9 with ammonia water, and then extracted 6 times with a mixed solvent of petroleum ether and ethyl acetate in a 1:1 ratio, with an equal volume of the concentrated solution. The extract is collected and concentrated until no solvent is present to obtain a concentrated paste.

[0110] S6. Add 20 times its volume of anhydrous ethanol to the second concentrated paste to dissolve it completely, filter, concentrate the filtrate to Brix = 52°, and let it stand at 6°C for 60 h to crystallize. Filter and dry the crystals to obtain a final product of 108 g. HPLC analysis showed that the harpagoside content was 96.81%, the yield was 1.08%, which is equivalent to 1.05% of the harpagoside content in the raw material of *Gnaphalium affine*, and the recovery rate was 71.1%.

[0111] Example 6

[0112] The present invention discloses a method for extracting harpagoside from *Gnaphalium affine*, which specifically includes the following steps:

[0113] S1. Take 10 kg of *Gnaphalium affine* raw material (harpagoside content approximately 1.50%), pulverize to 60 mesh, weigh out 5‰ of the *Gnaphalium affine* raw material's compound enzyme, disperse it in 3 times the weight of the raw material in water, add the *Gnaphalium affine* raw material to the water and stir evenly. Inactivate the enzyme at 50℃ for 4 hours, then inactivate it for 8 minutes at 80℃. The compound enzyme is a mixture of cellulase and pectinase in a 1:1 mass ratio. Add 10 times the weight of n-hexane to the enzymatic hydrolysis residue, extract twice at room temperature with stirring, 1 hour per extraction. Filter, collect the residue, and dry for later use.

[0114] S2. Extract the above filter residue twice with 10 times its mass of 95% ethanol aqueous solution, with an extraction time of 2 hours each time. Filter and concentrate the filtrate to a specific gravity of 1.03 to obtain a concentrated paste.

[0115] S3. The first concentrated paste is allowed to settle for 3 hours, then filtered to obtain a primary supernatant. A gelatin solution with a volume of 20% (1% by mass) is added to this supernatant, and the mixture is allowed to settle for 0.5 hours, then filtered again to obtain a secondary supernatant. A composite adsorbent with a volume of 20% of the secondary supernatant is then added to this supernatant. The mixture is heated to 60°C, stirred, and adsorbed for 1 hour. The supernatant is then filtered, and the filtrate is collected. The composite adsorbent consists of 70% activated carbon and 30% bentonite.

[0116] S4. The filtrate obtained in S3 is passed through 100 kDa and 1 kDa ultrafiltration membranes in sequence, and then through a nanofiltration membrane with a molecular weight of 300 Da for concentration to obtain a concentrate. The concentrate is sampled and tested, and the content of harpagoside is 41.33%.

[0117] S5. The pH of the concentrate is adjusted to 9 with ammonia, and then twice the volume of petroleum ether is added for extraction three times. The extract is collected and concentrated until there is no solvent to obtain a concentrated paste.

[0118] S6. Add 15 times its volume of anhydrous ethanol to the second concentrated paste to dissolve it completely, filter, concentrate the filtrate to Brix = 51°, and let it stand at 6°C for 72 hours to crystallize. Filter and dry the crystals to obtain a final product of 111g. HPLC analysis showed that the harpagoside content was 97.03%, the yield was 1.11%, which is equivalent to 1.08% of the harpagoside content in the raw material of *Gnaphalium affine*, and the harpagoside recovery rate was 73.3%.

Claims

1. A method for extracting harpagoside from *Gnaphalium affine*, characterized in that, Includes the following steps: Step 1. Raw material crushing and enzymatic hydrolysis, and organic solvent removal: Disperse the compound enzyme in water, crush the raw material of *Polygonum multiflorum* and add it to the compound enzyme, hydrolyze and inactivate the enzyme, remove impurities using n-hexane or petroleum ether at room temperature, filter, collect the filter residue and dry it for later use. The compound enzyme is a mixture of cellulase and pectinase, and the amount of compound enzyme added is 3-5‰ of the weight of the raw material of *Gnaphalium affine*. Step 2. Extraction: Extract the above filter residue using an ethanol-water solution or a methanol-water solution, collect the extract, filter, collect the filtrate and concentrate it to obtain the first concentrated paste; Step 3. Sedimentation: The first concentrated paste is subjected to sedimentation once, and filtered to obtain a first supernatant. Gelatin solution is added to the first supernatant for a second sedimentation, and filtered again to obtain a second supernatant. Finally, an adsorbent is used to adsorb and remove impurities from the second supernatant. Step 4. Membrane separation and membrane concentration: The secondary supernatant after adsorption and impurity removal is passed through an ultrafiltration membrane and a nanofiltration membrane in sequence to remove small molecule impurities such as salts and monosaccharides and to concentrate the solution to obtain a concentrated solution. Step 5. Extraction: Adjust the pH of the concentrate to 8-9 with ammonia water to deprotonate it, then extract the concentrate with an organic solvent, collect the extract, and concentrate it until there is no solvent to obtain the second concentrated paste. Step 6. Crystallization: Dissolve the second concentrated paste in anhydrous ethanol, filter and concentrate the filtrate, crystallize, filter and dry the crystals to obtain harpaoside.

2. The method for extracting harpagoside from *Gnaphalium affine* according to claim 1, characterized in that: In step 1, the amount of water should be 1-3 times the mass of the devil's claw herb raw material; In the compound enzyme, the mass ratio of cellulase to pectinase is 1-1.5:1, the enzymatic hydrolysis temperature is 45-50℃, and the enzymatic hydrolysis time is 4-6h; the enzyme inactivation temperature is 79-81℃, the heating method is water bath heating, and the enzyme inactivation time is 5-8min. The specific method for removing impurities using n-hexane or petroleum ether at room temperature is as follows: use n-hexane or petroleum ether to remove impurities 2-3 times, with each application being 6-10 times the mass of the raw material, and each removal process taking 1-2 hours.

3. The method for extracting harpagoside from *Gnaphalium affine* according to claim 2, characterized in that: In step 2, the amount of the ethanol aqueous solution or methanol aqueous solution used is 8-10 times the mass of the filter residue, the mass concentration is 90-95%, the specific gravity of the first concentrated paste is 1.02-1.03, and the number of extractions is at least 2-4. In step 3, the first settling is static settling, with a settling time of 2-4 hours and a temperature of room temperature, and the second settling time is 0.5-2 hours; The amount of gelatin solution added is 10%-20% of the volume of the first supernatant.

4. The method for extracting harpagoside from *Gnaphalium affine* according to claim 3, characterized in that: Step 3 specifically involves adding 10-25% by volume of adsorbent to the secondary supernatant, stirring and adsorbing at 50-70°C for 0.5-1.5 hours, filtering, and collecting the filtrate. The adsorbent is a composite adsorbent, which is composed of activated carbon and bentonite, wherein activated carbon accounts for 50-70% and bentonite accounts for 30-50%.

5. The method for extracting harpagoside from *Gnaphalium affine* according to claim 4, characterized in that: Step 5 specifically involves adjusting the pH of the concentrate to 9 with ammonia water, then adding an extraction solvent to repeatedly extract the concentrate, collecting the extract obtained from each extraction, and concentrating it until there is no solvent to obtain the second concentrated paste. The extraction solvent is petroleum ether, or ethyl acetate, or a mixture of petroleum ether and ethyl acetate, wherein the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is 1:

1. The extraction is performed 3-6 times, and the amount of extraction solvent used in each extraction is 1-2 times the volume of the concentrated liquid.

6. The method for extracting harpagoside from *Gnaphalium affine* according to claim 5, characterized in that: In step 6, the amount of anhydrous ethanol used is 15-20 times the volume of the second concentrated paste. The filtrate is concentrated to Brix = 49-52°, the crystallization temperature is 2-8°C, and the crystallization time is 48-72h.

7. The method for extracting harpagoside from *Gnaphalium affine* according to claim 6, characterized in that: In step 1, the amount of water is 1 times the mass of the raw material of *Gnaphalium affine*; in the compound enzyme, the mass ratio of cellulase to pectinase is 1.4:1, the enzymatic hydrolysis temperature is 45℃, the enzymatic hydrolysis time is 4h; the enzyme inactivation temperature is 80℃, and the enzyme inactivation time is 5min. In step 2, the amount of ethanol aqueous solution or methanol aqueous solution used is 8 times the mass of the filter residue, the extraction is performed twice, the extraction time is 2 hours per extraction, and the specific gravity of the first concentrated extract is 1.

02. In step 3, the first settling time is 2 hours and the second settling time is 0.5 hours; the amount of gelatin solution added is 10% of the volume of the first supernatant.

8. The method for extracting harpagoside from *Gnaphalium affine* according to claim 7, characterized in that: In step 3, the amount of composite adsorbent is 10% of the secondary supernatant, and adsorption is carried out by stirring at 60°C for 1 hour. The composite adsorbent has 50% activated carbon and 50% bentonite. In step 5, the extraction is performed 5 times, and the extraction solvent is ethyl acetate. The amount of extraction solvent used in each extraction is 1 times the volume of the concentrate. In step 6, the amount of anhydrous ethanol used is 20 times the volume of the second concentrated paste. The filtrate is concentrated to Brix = 50°, the crystallization temperature is 6°C, and the crystallization time is 48 hours.

9. The method for extracting harpagoside from *Gnaphalium affine* according to claim 1, characterized in that: In step 3, the gelatin solution is prepared by mixing gelatin and purified water, with a mass ratio of gelatin to purified water of 1:

99.

10. The method for extracting harpagoside from *Gnaphalium affine* according to claim 1, characterized in that: In step 4, the ultrafiltration membrane includes membranes with molecular weights of 100 kDa and 1 kDa arranged sequentially, and the nanofiltration membrane has a molecular weight of 200-300 Da.

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