Method for separating and preparing hemsleya amabilis saponin, hemsleya amabilis element and oleanolic acid in hemsleya amabilis

Abstract

The invention discloses a method for separating and preparing hemsleya amabilis saponin, hemsleya amabilis element and oleanolic acid in hemsleya amabilis, and relates to the technical field of deep processing of hemsleya amabilis. The method comprises the following steps: heating, refluxing and extracting hemsleya amabilis powder with an ethanol solution or absolute ethyl alcohol, filtering, combining filtrate, concentrating, and recovering ethanol to obtain a concentrated solution; adsorbing the concentrated solution by using macroporous adsorption resin, sequentially eluting by using 50%, 70% and 95% ethanol solutions, respectively collecting eluents, concentrating, extracting the 50% ethanol concentrated eluent by using a water saturated n-butyl alcohol solution, concentrating, and drying to obtain hemsleya amabilis saponin; heating and dissolving the 70% ethanol concentrated eluent with a 60% ethanol solution, and recrystallizing to obtain the hemsleya amabilis element; and heating and dissolving the 95% ethanol concentrated eluent with a 90% ethanol solution, and recrystallizing to obtain the oleanolic acid. According to the method provided by the invention, the purity of hemsleya amabilis saponin exceeds 70%, the purity of hemsleya amabilis element exceeds 80%, and the purity of oleanolic acid exceeds 70%; and the yield exceeds 80%. The method is coherent and simple in process, low in cost, safe, environment-friendly and suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of deep processing of trichosanthes, in particular to a method for separating and preparing trichosanthes saponins, trichosanthin and oleanolic acid from trichosanthes. BACKGROUND

[0002] Trichosanthes is a plant of Cucurbitaceae, and it is recorded in the National Chinese Herbal Medicine Compilation that it has the effects of clearing heat and resolving toxicity, and strengthening stomach and relieving pain. Modern research shows that trichosanthes contains trichosanthin, saponins and oleanolic acid and other active ingredients. Pharmacological research shows that trichosanthes has various biological activities such as antibacterial, anti-inflammatory, antioxidant, antitumor and anti-gastric ulcer, and can treat various diseases such as sore throat, toothache, red and swollen eyes, bacillary dysentery, enteritis, stomach disease, hepatitis, urinary tract infection and tumor.

[0003] In the prior art, Chinese patent application CN101974054A discloses an improved method for extracting trichosanthin, which uses trichosanthes medicinal materials as raw materials, and obtains trichosanthin through ethanol extraction, macroporous adsorption resin elution and recrystallization. This method only obtains one type of active ingredient of trichosanthes. Chinese patent application CN104910246A discloses a preparation method of trichosanthin A, which uses trichosanthes as raw materials, and obtains trichosanthin through supercritical carbon dioxide extraction, ethanol dissolution, petroleum ether extraction and recrystallization. This method needs to use flammable and explosive organic reagents. Chinese authorized patent CN118652297B discloses a method for extracting trichosanthin A by biological enzyme pretreatment, which pretreats trichosanthes by amylase, glucoamylase and pectinase in turn, greatly reduces the dissolution rate of gum in the extraction process, and increases the extraction rate of trichosanthin A. This method can only obtain one type of active ingredient of trichosanthes.

[0004] The above-mentioned prior art is often the extraction and utilization of single component in trichosanthes resources. In the current actual production, the industrialized production and utilization of trichosanthes mainly focuses on the extraction and separation of trichosanthin. In the purification process of trichosanthin, trichosanthes saponins and oleanolic acid components are often discarded, causing serious waste of resources.

[0005] Therefore, it is urgent to develop a method for green and efficient utilization of trichosanthes raw materials, to realize the simultaneous separation and preparation of trichosanthin, trichosanthes saponins and oleanolic acid components. The obtained products have high purity and economic value. This has important practical significance for environmental protection, promoting the development of trichosanthes industry as a whole, and promoting the development of agricultural economy in planting areas. SUMMARY

[0006] The application provides a method for separating and preparing snow cardin saponins, snow cardin and oleanolic acid in Hemsleya chinensis, which can simultaneously separate and extract the snow cardin saponins, snow cardin and oleanolic acid, has high purity and high recovery rate of the extracted products, is simple in process, low in production cost, safe in separation material, green and pollution-free, and suitable for industrialized production, and is a high-value comprehensive utilization method for the natural resource Hemsleya chinensis.

[0007] The application provides a method for separating and preparing snow cardin saponins, snow cardin and oleanolic acid in Hemsleya chinensis, which comprises the following steps.

[0008] The Hemsleya chinensis powder is added into an ethanol solution with a volume fraction of not less than 70% or anhydrous ethanol, heated to reflux and extracted, and the extraction is repeated at least twice; the filtrates are filtered and combined, concentrated, and the ethanol is recovered to obtain a concentrated solution;

[0009] The concentrated solution is added into a macroporous adsorption resin column, adsorbed, and sequentially eluted with ethanol solutions with volume fractions of 50%, 70% and 95%, and the eluates are collected and concentrated under reduced pressure to obtain 50% ethanol concentrated eluate, 70% ethanol concentrated eluate and 95% ethanol concentrated eluate, respectively;

[0010] The 50% ethanol concentrated eluate is extracted with water-saturated n-butanol solution for several times, concentrated and dried to obtain snow cardin saponins;

[0011] The 70% ethanol concentrated eluate is dissolved by heating with an ethanol solution with a volume fraction of 60%, and recrystallized by standing at room temperature; the crystals are filtered and dried to obtain snow cardin;

[0012] The 95% ethanol concentrated eluate is dissolved by heating with an ethanol solution with a volume fraction of 90%, and recrystallized by standing at room temperature; the crystals are filtered and dried to obtain oleanolic acid.

[0013] Further, when the first heating reflux extraction is performed, the mass-volume ratio of the Hemsleya chinensis powder to the ethanol solution with a volume fraction of not less than 70% or anhydrous ethanol is 1 / 15-1 / 20 kg / L.

[0014] Further, from the beginning of the second heating reflux extraction, the mass-volume ratio of the Hemsleya chinensis powder to the ethanol solution with a volume fraction of not less than 70% or anhydrous ethanol is 1 / 10-1 / 15 kg / L.

[0015] Further, the temperature of the heating reflux extraction is 60-80 DEG C.

[0016] Further, the macroporous adsorption resin in the macroporous adsorption resin column is selected from D101B, D101C, LX-11 or LX-60.

[0017] Further, the loading amount of the concentrated solution into the macroporous adsorption resin column is 5 times of the volume of the macroporous adsorption resin.

[0018] Further, the elution volume is 2-5 BV, and the elution flow rate is 1-3 BV / h.

[0019] Further, the temperature for concentrating the eluate under reduced pressure is 60-80 DEG C.

[0020] Further, the amount of snow-borne crude drug in the concentrated solution is 1.2-1.5 g / mL, and the volume fraction of alcohol content is 30-40%.

[0021] Further, the recrystallization time is 10-12 h.

[0022] It should be noted that in the method of the present application, 1 BV=1 column volume.

[0023] The purpose of the fractionated extraction in the above method for separating and preparing snow-borne saponins, snow-borne elements and oleanolic acid from snow-borne is to fully leach the snow-borne saponins, snow-borne elements and oleanolic acid in the snow-borne powder. If the extraction temperature is too low, or the amount of ethanol solution / anhydrous ethanol is too small, or the extraction time is too short, the above purpose cannot be achieved. If the extraction temperature is too high, or the amount of ethanol solution / anhydrous ethanol is too large, or the extraction time is too long, energy or materials will be wasted.

[0024] Compared with the prior art, the present application has the following advantages:

[0025] 1. The present application uses multiple ethanol extractions and macroporous adsorption resin gradient elution to achieve the full and rapid leaching and separation of snow-borne saponins, snow-borne elements and oleanolic acid in the snow-borne powder. The purity of the snow-borne saponins obtained by the method of the present application is more than 70%, the purity of the snow-borne elements is more than 80%, and the purity of the oleanolic acid is more than 70%. The yield is more than 80%, and the economic value is very high.

[0026] 2. The method of the present application can simultaneously and rapidly separate multiple active ingredients, can comprehensively utilize snow-borne resources, has a coherent and simple process, is highly operable, has low production cost, can reduce the use of toxic and harmful chemical reagents, is safe and environmentally friendly, and is suitable for industrial production. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0028] In some embodiments of the present application, the provided method for separating and preparing scillanin, scillin and oleanolic acid from Hemsleya chinensis includes the following steps:

[0029] The Hemsleya chinensis powder is added to an ethanol solution with a volume fraction of not less than 70% or anhydrous ethanol, heated to reflux and extracted, and the extraction is repeated at least twice. The filtrates are combined, concentrated, and after recovery of ethanol, a concentrated solution is obtained.

[0030] The concentrated solution is added to a macroporous adsorption resin column, adsorbed, and sequentially eluted with ethanol solutions with volume fractions of 50%, 70% and 95%, respectively. The eluates are collected and concentrated under reduced pressure to obtain 50% ethanol concentrated eluate, 70% ethanol concentrated eluate and 95% ethanol concentrated eluate, respectively.

[0031] The 50% ethanol concentrated eluate is extracted with water-saturated n-butanol solution for several times, concentrated and dried to obtain scillanin.

[0032] The 70% ethanol concentrated eluate is dissolved by heating with an ethanol solution with a volume fraction of 60%, and recrystallized by standing at room temperature. The crystals are collected by filtration and dried to obtain scillin.

[0033] The 95% ethanol concentrated eluate is dissolved by heating with an ethanol solution with a volume fraction of 90%, and recrystallized by standing at room temperature. The crystals are collected by filtration and dried to obtain oleanolic acid.

[0034] Optionally, when performing the first heating reflux extraction, the mass-volume ratio of the Hemsleya chinensis powder to the ethanol solution with a volume fraction of not less than 70% or anhydrous ethanol is 1 / 15-1 / 20 kg / L.

[0035] Optionally, from the beginning of the second heating reflux extraction, the mass-volume ratio of the Hemsleya chinensis powder to the ethanol solution with a volume fraction of not less than 70% or anhydrous ethanol is 1 / 10-1 / 15 kg / L.

[0036] In the following specific embodiments, a method of twice heating reflux extraction is generally adopted.

[0037] Optionally, the temperature of the heating reflux extraction is 60-80°C.

[0038] Optionally, the macroporous adsorption resin in the macroporous adsorption resin column is selected from D101B, D101C, LX-11 or LX-60.

[0039] Optionally, the loading amount of the concentrated solution into the macroporous adsorption resin column is 5 times the volume of the macroporous adsorption resin.

[0040] Optionally, the elution volume is 2-5 BV, and the elution flow rate is 1-3 BV / h.

[0041] Optionally, the temperature for concentrating the eluate under reduced pressure is 60-80℃.

[0042] Optionally, the amount of snow-borne crude drug in the concentrated solution is 1.2-1.5 g / mL, and the volume fraction of alcohol content is 30-40%.

[0043] Optionally, the time for recrystallization is 10-12 h.

[0044] It should be noted that in the method of the present application, 1 BV = 1 column volume.

[0045] In the following specific embodiments of the present application, the content of scaphigenin in the snow-borne medicinal material is 1.81 wt%, the content of saponin is 2.53 wt%, and the content of oleanolic acid is 0.83 wt%. The macroporous adsorption resin used is purchased from Dacheng Ruixing Chemical Building Material Co., Ltd. Ethanol, n-butanol and other reagents are purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Other auxiliary materials or chemical reagents used are obtained through conventional commercial channels unless otherwise specified.

[0046] In the following specific embodiments of the present application, vanillin-perchloric acid method is used to determine the content of snow-borne saponin, and high performance liquid chromatography (HPLC) is used to detect the content of scaphigenin and oleanolic acid. The specific method is as follows;

[0047] (1) Snow-borne saponin content determination: prepare 2 mg / mL sample solution and 1 mg / mL ginsenoside Re standard solution, take 1 mg / mL ginsenoside Re standard solution 20, 40, 80, 120, 160, 200 μL and sample solution 100 μL, respectively, in a test tube with a plug, and evaporate the solvent at low temperature; add 5 %(w / v) vanillin glacial acetic acid solution 0.2 mL, add perchloric acid 0.8 mL under ice bath, mix well, place in 60℃ constant temperature water bath for 15 min, immediately cool with ice water for 2 min; add acetic acid solution 5 mL, shake well, and measure the absorbance at 540 nm.

[0048] (2) Scaphigenin content determination: prepare 1 mg / mL sample solution and 0.2, 0.4, 0.6, 0.8, 1 mg / mL scaphigenin A standard solution, and detect different concentrations of standard and sample solution by high performance liquid chromatography under the following conditions: reverse phase C-18 chromatographic column, mobile phase A is water, mobile phase B is acetonitrile (elution program is 0-10 min, 75%-50% A; 10-25 min, 50%-20% A; 25-35 min, 20%-75% A; 35-40 min, 75% A), flow rate 1 mL / min, column temperature 30℃, injection volume 20 μL, detection wavelength 210 nm.

[0049] (3) Oleanolic acid content determination: prepare 1 mg / mL sample solution and 0.2, 0.4, 0.6, 0.8, 1 mg / mL oleanolic acid standard solution, and detect different concentrations of standard and sample solutions by high performance liquid chromatography under the following conditions: reverse phase C-18 chromatographic column, mobile phase is methanol and 0.1% phosphoric acid aqueous solution (85:15, V / V) isocratic elution, flow rate 1 mL / min, column temperature 30°C, sample size 10 μL, detection wavelength 210 nm.

[0050] Example 1

[0051] The method for separating and preparing snow cardin, hemsleyadonin and oleanolic acid in Hemsleya chinensis provided in the embodiment has the advantages that the separation and preparation of snow cardin, hemsleyadonin and oleanolic acid in Hemsleya chinensis is simple, the yield is high, and the quality of the products is good.

[0052] S1, take 5 kg of Hemsleya chinensis powder, mix with 70% ethanol solution at a mass-volume ratio (kg / L) of 1:20, heat to reflux at 80°C for the first time for 2 h, and filter; take the filter residue, add 70% ethanol solution (the mass-volume ratio (kg / L) of Hemsleya chinensis powder to 70% ethanol solution is 1:15), heat to reflux at 80°C for the second time for 2 h, and filter;

[0053] Combine the filtrates of the two times of filtration, concentrate under reduced pressure, recover ethanol, and obtain a concentrated solution containing 40% of alcohol.

[0054] S2, load the concentrated solution obtained in step S1 into a D101B macroporous adsorption resin column, the loading amount is 5 times the column volume (BV), the loading flow rate is 2 BV / h, stand for adsorption for 60 min, then elute with 5 BV of 50%, 70% and 95% ethanol solution in sequence, the elution flow rate is 2 BV / h, and collect the eluate.

[0055] Concentrate the three eluates under reduced pressure at 80°C respectively, and obtain 50% ethanol concentrated eluate, 70% ethanol concentrated eluate and 95% ethanol concentrated eluate respectively. These ethanol concentrated eluates mainly contain snow cardin, hemsleyadonin and oleanolic acid components respectively.

[0056] S3, add water-saturated n-butanol solution to the 50% ethanol concentrated eluate (snow cardin component concentrated solution) in step S2 according to a volume ratio of 1:10, extract 3 times, concentrate under reduced pressure, freeze-dry, and obtain 139.77 g of snow cardin.

[0057] Add 60% ethanol solution to the 70% ethanol concentrated eluate (hemsleyadonin component concentrated solution) according to a volume ratio of 1:10, heat to dissolve, stand for recrystallization at room temperature for 12 h, filter, dry the crystals, and obtain 108.67 g of hemsleyadonin.

[0058] 95% ethanol concentrated eluent (oleanolic acid component concentrated solution), according to the volume ratio 1:10, 90% ethanol solution was added to heat and dissolve, and recrystallization was carried out at room temperature for 12 h, and then the crystal was obtained by filtration and drying, and oleanolic acid 46.02 g was obtained.

[0059] The content of the obtained scchidan saponin was 75.1% by vanillin-perchloric acid method, and the yield was 82.98%. The content of the obtained scchidan was 82.60% by high performance liquid chromatography, and the yield was 83.50%; the content of the obtained oleanolic acid was 74.30%, and the yield was 82.39%.

[0060] Example 2

[0061] The method for separating and preparing scchidan saponin, scchidan and oleanolic acid in scchidan provided in the embodiment is as follows:

[0062] S1, 5 kg of scchidan powder was mixed with anhydrous ethanol according to the mass-volume ratio (kg / L) 1:15, and was heated to reflux at 60°C for the first time for 1 h, and then was filtered. The residue was filtered, and anhydrous ethanol was added (the mass-volume ratio (kg / L) of scchidan powder to anhydrous ethanol was 1:10), and was heated to reflux at 60°C for the second time for 1 h, and then was filtered.

[0063] The filtrates of the two times of filtration were combined, and were concentrated under reduced pressure to recover ethanol, and a concentrated solution with an alcohol content of 40% was obtained.

[0064] S2, the concentrated solution obtained in step S1 was loaded onto a D101C macroporous adsorption resin column, and the loading amount was 5 times the column volume (BV), and the loading flow rate was 2 BV / h. After standing for 40 min, 2 BV of 50%, 70% and 95% ethanol was used to elute in sequence, and the elution flow rate was 1 BV / h, and the eluate was collected.

[0065] The three eluates were concentrated under reduced pressure at 60°C, respectively, and 50% ethanol concentrated eluent, 70% ethanol concentrated eluent and 95% ethanol concentrated eluent were obtained, respectively. These ethanol concentrated eluents mainly contained scchidan saponin, scchidan and oleanolic acid components, respectively.

[0066] S3, the 50% ethanol concentrated eluent (scchidan saponin component concentrated solution) in step S2 was added with water-saturated n-butanol solution according to the volume ratio 1:5, and was extracted for 3 times, and was concentrated under reduced pressure, and was freeze-dried to obtain scchidan saponin 137.65 g.

[0067] The 70% ethanol concentrated eluent (scchidan component concentrated solution) was added with 60% ethanol solution according to the volume ratio 1:5 to heat and dissolve, and recrystallization was carried out at room temperature for 10 h, and then the crystal was obtained by filtration and drying, and scchidan 106.51 g was obtained.

[0068] 95% ethanol concentrated eluent (oleanolic acid component concentrated solution) was added with 90% ethanol solution at a volume ratio of 1:5, heated and dissolved, recrystallized at room temperature for 10 h, filtered to obtain crystals, and dried to obtain oleanolic acid 45.53 g.

[0069] The content of the obtained scyllitol saponin was 73.52% by vanillin-perchloric acid method, and the yield was 80.0%; the content of the obtained scyllitol was 80.76% by high performance liquid chromatography, and the yield was 80.02%; and the content of the obtained oleanolic acid was 72.96%, and the yield was 80.05%.

[0070] Example 3

[0071] The method for separating and preparing scyllitol saponin, scyllitol and oleanolic acid in scylylphora provided in the example has the following steps:

[0072] S1, 5 kg of scylylphora powder was mixed with 90% ethanol solution at a mass-volume ratio (kg / L) of 1:18, heated and refluxed at 60°C for the first time for 2 h, and filtered; the filter residue was taken, 90% ethanol solution was added (the mass-volume ratio (kg / L) of the scylylphora powder to the 90% ethanol solution was 1:12), heated and refluxed at 60°C for the second time for 1 h, and filtered;

[0073] The filtrates of the two times of filtration were combined, concentrated under reduced pressure, and ethanol was recovered to obtain a concentrated solution with an alcohol content of 36%.

[0074] S2, the concentrated solution obtained in step S1 was loaded onto an LX-11 macroporous adsorption resin column, the loading amount was 5 times the column volume (BV), the loading flow rate was 2 BV / h, and after standing for 50 min, 4 BV of 50%, 70% and 95% ethanol were sequentially eluted, the elution flow rate was 3 BV / h, and the eluates were collected respectively.

[0075] The three eluates were respectively concentrated under reduced pressure at 70°C to obtain 50% ethanol concentrated eluent, 70% ethanol concentrated eluent and 95% ethanol concentrated eluent. These ethanol concentrated eluents respectively mainly contained scyllitol saponin, scyllitol and oleanolic acid components.

[0076] S3, the 50% ethanol concentrated eluent (scyllitol saponin component concentrated solution) in step S2 was added with water-saturated n-butanol solution at a volume ratio of 1:8, extracted for 3 times, concentrated under reduced pressure, and freeze-dried to obtain scyllitol saponin 138.13 g.

[0077] The 70% ethanol concentrated eluent (scyllitol component concentrated solution) was added with 60% ethanol solution at a volume ratio of 1:8, heated and dissolved, recrystallized at room temperature for 12 h, filtered to obtain crystals, and dried to obtain scyllitol 107.56 g.

[0078] 95% ethanol concentrated eluent (oleanolic acid component concentrate), 90% ethanol solution was added according to the volume ratio of 1:8 to heat and dissolve, and recrystallized at room temperature for 12 h, filtered, dried, and oleanolic acid 45.68 g was obtained.

[0079] The content of the obtained scchidan saponin was 74.83% by vanillin-perchloric acid method, and the yield was 81.71%; the content of the obtained scchidan was 81.54% by high performance liquid chromatography, and the yield was 81.59%; and the content of the obtained oleanolic acid was 73.26%, and the yield was 80.64%.

[0080] Example 4

[0081] The method for separating and preparing scchidan saponin, scchidan and oleanolic acid in scchidan provided in the embodiment has the following steps:

[0082] S1, 5 kg of scchidan powder was mixed with 80% ethanol solution according to the mass-volume ratio (kg / L) of 1:16, and was heated to reflux at 78°C for the first time for 2 h, and was filtered; the filter residue was taken, 80% ethanol solution was added (the mass-volume ratio (kg / L) of scchidan powder to 80% ethanol solution was 1:14), and was heated to reflux at 60°C for the second time for 2 h, and was filtered;

[0083] The filtrates of the two times of filtration were combined, concentrated under reduced pressure, and ethanol was recovered to obtain a concentrated solution with an alcohol content of 32%.

[0084] S2, the concentrated solution obtained in step S1 was loaded into an LX-60 macroporous adsorption resin column, the loading amount was 5 times the column volume (BV), the loading flow rate was 2 BV / h, and after standing for 60 min, 3 BV of 50%, 70% and 95% ethanol were sequentially eluted, the elution flow rate was 2 BV / h, and the eluates were collected respectively.

[0085] The three eluates were respectively concentrated under reduced pressure at 78°C to obtain 50% ethanol concentrated eluent, 70% ethanol concentrated eluent and 95% ethanol concentrated eluent. These ethanol concentrated eluents respectively mainly contained scchidan saponin, scchidan and oleanolic acid components.

[0086] S3, the 50% ethanol concentrated eluent (scchidan saponin component concentrate) in step S2 was added with water-saturated n-butanol solution according to the volume ratio of 1:6, extracted for 3 times, concentrated under reduced pressure, and freeze-dried to obtain scchidan saponin 138.54 g.

[0087] 70% ethanol concentrated eluent (concentrated solution of jujuboside), 60% ethanol solution was added according to the volume ratio of 1:6, heated to dissolve, recrystallized at room temperature for 10 h, filtered to obtain crystallization and dried to obtain jujuboside 107.98 g.

[0088] 95% ethanol concentrated eluent (concentrated solution of oleanolic acid), 90% ethanol solution was added according to the volume ratio of 1:6, heated to dissolve, recrystallized at room temperature for 10 h, filtered to obtain crystallization and dried to obtain oleanolic acid 45.96 g.

[0089] The content of the obtained jujuboside was 74.95% by vanillin-perchloric acid method, and the yield was 82.08%; the content of the obtained jujuboside was 81.68% by high performance liquid chromatography, and the yield was 82.04%; the content of the obtained oleanolic acid was 73.57%, and the yield was 81.48%.

[0090] Comparative Example 1

[0091] The method for separating and preparing jujuboside, jujuboside and oleanolic acid in jujube provided in the comparative example has the same conditions and operations as example 1, except that the 50% ethanol concentrated eluent, 70% ethanol concentrated eluent and 95% ethanol concentrated eluent prepared in step S2 are directly freeze-dried (i.e. without water-saturated n-butanol extraction, or recrystallization with ethanol solution).

[0092] Finally, jujuboside 210.61 g, jujuboside 180.58 g and oleanolic acid 72.52 g were obtained. The content of the obtained jujuboside was 53.58% by vanillin-perchloric acid method, and the yield was 89.20%; the content of the obtained jujuboside was 54.20% by high performance liquid chromatography, and the yield was 91.05%; the content of the obtained oleanolic acid was 51.79%, and the yield was 90.50%.

[0093] Comparative Example 2

[0094] The method for separating and preparing jujuboside, jujuboside and oleanolic acid in jujube provided in the comparative example has the same conditions and operations as example 1, except that the comparative example only has 1 heating reflux extraction; that is, the jujube powder is mixed with 70% ethanol solution according to the mass-volume ratio (kg / L) of 1:35, heated at 80℃ for 2 h, and filtered; concentrated under reduced pressure to recover ethanol to obtain a concentrated solution with an alcohol content of 40%.

[0095] Finally, 132.78 g of jujuboside, 103.25 g of jujubogenin and 43.73 g of oleanolic acid were obtained. The content of jujuboside was 75.05% and the yield was 78.78% by vanillin-perchloric acid method. The content of jujubogenin was 82.56% and the yield was 79.30% by high performance liquid chromatography. The content of oleanolic acid was 74.32% and the yield was 78.31%.

[0096] Comparative Example 3

[0097] The method for separating and preparing jujuboside, jujubogenin and oleanolic acid in Jujubae fructus provided by the present comparative example has the same conditions and operations as those of Example 1, except that:

[0098] In step S2, the elution was performed by using 95% ethanol solution first, then 70% ethanol solution and finally 50% ethanol solution, and the eluates were collected respectively. The three eluates were concentrated under reduced pressure at 80℃ respectively, and 95% ethanol concentrated eluate, 70% ethanol concentrated eluate and 50% ethanol concentrated eluate were obtained respectively.

[0099] In step S3, the 95% ethanol concentrated eluate in step S2 was added with water-saturated n-butanol solution at a volume ratio of 1:10, and extracted for 3 times. The extract was concentrated under reduced pressure, and freeze-dried to obtain extract A 420.85 g.

[0100] The 70% ethanol concentrated eluate was directly dried to obtain jujubogenin 1.15 g.

[0101] The 50% ethanol concentrated eluate was directly dried to obtain oleanolic acid 1.65 g.

[0102] It was detected that extract A was a mixture of jujuboside, jujubogenin and oleanolic acid, wherein the content of jujuboside was 35.48%, the content of jujubogenin was 30.15% and the content of oleanolic acid was 11.64%. No jujuboside, jujubogenin and oleanolic acid were detected in extract B and extract C.

[0103] It can be seen that when the gradient elution sequence was changed in Comparative Example 3, the results showed that jujuboside, jujubogenin and oleanolic acid could not be separated. It can be seen that in the present application, the gradient elution sequence of macroporous adsorption resin cannot be changed, otherwise the purpose of the present application cannot be achieved.

[0104] The above specific embodiments describe the implementation of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the present application, the technical solutions of the present application can be modified and changed in many ways, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A method for separating and preparing genistein, genistein, and oleanolic acid from genistein, characterized in that, Includes the following steps: Add the snow gall powder to an ethanol solution with a volume fraction of not less than 70% or anhydrous ethanol, heat and reflux to extract, repeat at least twice, filter and combine the filtrates, concentrate, and recover the ethanol to obtain the concentrated solution. The concentrated solution was added to a macroporous adsorption resin column for adsorption. It was then eluted sequentially with ethanol solutions of 50%, 70%, and 95% by volume. The eluents were collected and concentrated under reduced pressure to obtain 50% ethanol concentrated eluent, 70% ethanol concentrated eluent, and 95% ethanol concentrated eluent, respectively. The 50% ethanol concentrated eluent was extracted several times with water-saturated n-butanol solution, concentrated, and dried to obtain sucrose saponins. The 70% ethanol concentrated eluent was dissolved in a 60% ethanol solution by heating, allowed to stand at room temperature for recrystallization, filtered, and dried to obtain sucralose. The 95% ethanol concentrated eluent was dissolved by heating with a 90% ethanol solution, allowed to stand at room temperature for recrystallization, filtered, and dried to obtain oleanolic acid.

2. The method for separating and preparing genistein, genistein, and oleanolic acid from genistein according to claim 1, characterized in that, During the first heating reflux extraction, the mass-to-volume ratio of the *Gynostemma pentaphyllum* powder to an ethanol solution or anhydrous ethanol with a volume fraction of not less than 70% is 1 / 15-1 / 20 kg / L.

3. The method for separating and preparing genistein, genistein, and oleanolic acid from genistein according to claim 1, characterized in that, Starting from the second heating reflux extraction, the mass-to-volume ratio of the snow gall powder to an ethanol solution or anhydrous ethanol with a volume fraction of not less than 70% is 1 / 10-1 / 15 kg / L.

4. The method for separating and preparing genistein, genistein, and oleanolic acid from genistein according to claim 1, characterized in that, The temperature for the heating and reflux extraction is 60-80℃.

5. The method for separating and preparing genistein, genistein, and oleanolic acid from genistein according to claim 1, characterized in that, The macroporous adsorption resin in the macroporous adsorption resin column is selected from D101B, D101C, LX-11 or LX-60.

6. The method for separating and preparing genistein, genistein, and oleanolic acid from genistein according to claim 1, characterized in that, The amount of the concentrated solution added to the macroporous adsorption resin column is 5 times the volume of the macroporous adsorption resin.

7. The method for separating and preparing genistein, genistein, and oleanolic acid from genistein according to claim 1, characterized in that, The elution volume is 2-5 BV, and the elution flow rate is 1-3 BV / h.

8. The method for separating and preparing genistein, genistein and oleanolic acid from genistein according to claim 1, characterized in that, The eluent is concentrated under reduced pressure at a temperature of 60-80°C.

9. The method for separating and preparing genistein, genistein, and oleanolic acid from genistein according to claim 1, characterized in that, The concentrate contains 1.2-1.5 g / mL of raw *Gentiana scabra* and has an alcohol content of 30-40% by volume.

10. The method for separating and preparing genistein, genistein and oleanolic acid from genistein according to claim 1, characterized in that, The recrystallization time is 10-12 h.