A method for preparing icariin

Abstract

The present invention provides a method for preparing icariin, comprising the following steps: (1) mixing icariin extract with organic acid for acid hydrolysis to obtain acid hydrolysis product; wherein, the icariin extract includes icariin and / or icariin C; the organic acid includes citric acid and / or oxalic acid; (2) mixing the acid hydrolysis product with an enzyme for enzymatic hydrolysis to obtain icariin. Icariin and / or icariin C from Epimedium and Epimedium wushanense were converted into icariin using a combined organic acid hydrolysis-enzymatic hydrolysis method. The yield of icariin obtained was high, with the yield of icariin from Epimedium reaching 5.116%, an increase of 44.1 times, and the yield of icariin from Epimedium wushanense reaching 1.441%, an increase of 35.2 times. Moreover, the organic acids used in the acid hydrolysis process were all edible acids and were not harmful to the human body.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a preparation method of icariin. BACKGROUND

[0002] Icariin is a traditional Chinese herbal medicine in China, which has the effects of "tonifying kidney yang, strengthening sinews and bones and dispelling wind-dampness". The 2015 edition of the People's Republic of China Pharmacopoeia records five kinds of icariin, which are Icariin, Icariin, Icariin, Icariin and Icariin. The above-mentioned five kinds of icariin contain a large amount of icariin total flavonoids, including icariin, icariin and chomoforrestin A-C. Studies have shown that icariin has excellent anti-tumor effect, which can inhibit liver cancer, prostate cancer and gastric cancer to varying degrees, especially for liver cancer, which can significantly inhibit the malignant growth of liver cancer cells. However, the quality components of Icariin, Icariin, Icariin and Icariin are icariin (content greater than 0.50%), and the quality control component of Icariin is chomoforrestin C (content > 1%), that is, the amount of icariin in Icariin is small.

[0003] Icariin can be obtained by the conversion of icariin and chomoforrestin C, so the method for converting icariin and chomoforrestin C into icariin has attracted widespread attention from many scholars. In the prior art, acid hydrolysis, enzymatic hydrolysis or acid hydrolysis-enzymatic hydrolysis combined method is often used to convert icariin and chomoforrestin C into icariin, but acid hydrolysis mostly uses hydrochloric acid or sulfuric acid, which results in the residual of Cl - and SO4 2- in the final icariin, which will cause the residual in the human body when used for preparing liver cancer drugs, and is not conducive to human safety. In addition, the yield of icariin prepared by using the above-mentioned icariin medicinal materials as raw materials needs to be improved. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a preparation method of icariin. The preparation method uses organic acid (citric acid and / or oxalic acid) for acid hydrolysis, which is edible acid and will not cause harm to the human body after being taken into the body, and the yield of icariin prepared from icariin medicinal materials is greatly improved by using the organic acid acid hydrolysis-enzymatic hydrolysis combined method.

[0005] To achieve this purpose, the present application adopts the following technical solutions:

[0006] In a first aspect, the present application provides a preparation method of icariin, comprising the following steps:

[0007] (1) mixing icariin extract and organic acid for acid hydrolysis to obtain acid hydrolysis product;

[0008] The icariin and / or damianin C in the Epimedium extract are converted into icariin by acid hydrolysis and enzymatic hydrolysis.

[0009] The organic acid comprises citric acid and / or oxalic acid.

[0010] The acid hydrolysis product is mixed with the enzyme for enzymatic hydrolysis to obtain icariin.

[0011] Preferably, the organic acid is oxalic acid.

[0012] Preferably, the enzyme comprises any one or more of cellulase, beta-glucosidase, snailase or naringinase, and more preferably, the enzyme is cellulase.

[0013] Preferably, the molar ratio of the Epimedium extract to the organic acid in the acid hydrolysis is 1:(100-300).

[0014] Further preferably, the molar ratio of the Epimedium extract to the organic acid in the acid hydrolysis is 1:(200-300).

[0015] Preferably, the temperature of the acid hydrolysis is 50-65 DEG C, and the time of the acid hydrolysis is 24-48 h.

[0016] Further preferably, the temperature of the acid hydrolysis is 55-65 DEG C, and the time of the acid hydrolysis is 36-48 h.

[0017] Preferably, the mass-volume ratio of the Epimedium extract to the buffer solution is 1:(2-4) mg / mL.

[0018] Preferably, the mass ratio of the Epimedium extract to the enzyme is 1:(1.5-3).

[0019] Preferably, the temperature of the enzymatic hydrolysis is 45-55 DEG C, and the time of the enzymatic hydrolysis is 20-36 h.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] The icariin and / or damianin C in the Epimedium extract are converted into icariin by acid hydrolysis and enzymatic hydrolysis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The standard curve of damianin C is shown in the figure;

[0023] Figure 2Standard curve of icariin;

[0024] Figure 3 Standard curve of icariin;

[0025] Figure 4 Development result of icariin in the spot plate process;

[0026] Figure 5 Development result of icariin and doriden C in the spot plate process (from left to right, icariin and doriden C;

[0027] Figure 6 Spot plate result of different systems at 48h of acidolysis in Example 3;

[0028] Wherein, ① icariin + citric acid, ② icariin + oxalic acid, ③ icariin + acetic acid, ④ doriden C + citric acid, ⑤ doriden C + oxalic acid, ⑥ doriden C + acetic acid;

[0029] Figure 7 Liquid phase of icariin standard;

[0030] Figure 8 Liquid phase of doriden C standard;

[0031] Figure 9 Liquid phase of product obtained after reaction of icariin + 1:100 molar ratio oxalic acid system for 24h;

[0032] Figure 10 Liquid phase of product obtained after reaction of icariin + 1:200 molar ratio oxalic acid system for 24h;

[0033] Figure 11 Liquid phase of product obtained after reaction of icariin + 1:300 molar ratio oxalic acid system for 24h;

[0034] Figure 12 Liquid phase of product obtained after reaction of icariin + 1:100 molar ratio oxalic acid system for 48h;

[0035] Figure 13 Liquid phase of product obtained after reaction of icariin + 1:200 molar ratio oxalic acid system for 48h;

[0036] Figure 14 Liquid phase of product obtained after reaction of icariin + 1:300 molar ratio oxalic acid system for 48h;

[0037] Figure 15 Liquid phase of product obtained after reaction of doriden C + 1:100 molar ratio oxalic acid system for 24h;

[0038] Figure 16LC-MS of the product obtained after 24 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system;

[0039] Figure 17 LC-MS of the product obtained after 24 h reaction of icariexin + 1 : 300 molar ratio oxalic acid system;

[0040] Figure 18 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system;

[0041] Figure 19 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 300 molar ratio oxalic acid system;

[0042] Figure 20 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 300 molar ratio oxalic acid system;

[0043] Figure 21 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (positive ion mode);

[0044] Figure 22 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (negative ion mode);

[0045] Figure 23 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (positive ion mode);

[0046] Figure 24 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (negative ion mode);

[0047] Figure 25 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (negative ion mode);

[0048] Figure 26 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (negative ion mode);

[0049] Figure 27 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (negative ion mode);

[0050] Figure 28 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (negative ion mode);

[0051] Figure 29 LC-MS of the product obtained after 48 h reaction of icariexin + 1 : 200 molar ratio oxalic acid system (negative ion mode);

[0052] Figure 30 Liquid chromatogram of the product obtained from the acid hydrolysis + cellulase enzymolysis system of Epimedium brevicornum Morren in Example 6;

[0053] Figure 31 Liquid chromatogram of the product obtained from the alcohol-water system extraction of Epimedium herb in Example 7;

[0054] Figure 32 Liquid chromatogram of the product obtained from the alcohol-water system + cellulase enzymolysis system extraction of Epimedium herb in Example 7;

[0055] Figure 33 Liquid chromatogram of the product obtained from the alcohol-acid system extraction of Epimedium herb in Example 7;

[0056] Figure 34 Liquid chromatogram of the product obtained from the alcohol-acid system + cellulase enzymolysis system extraction of Epimedium herb in Example 7;

[0057] Figure 35 Liquid chromatogram of the product obtained from the alcohol-water system extraction of Epimedium sagittatum (Sweet) Maxim. in Example 7;

[0058] Figure 36 Liquid chromatogram of the product obtained from the alcohol-water system + cellulase enzymolysis system extraction of Epimedium sagittatum (Sweet) Maxim. in Example 7;

[0059] Figure 37 Liquid chromatogram of the product obtained from the alcohol-acid system extraction of Epimedium sagittatum (Sweet) Maxim. in Example 7;

[0060] Figure 38 Liquid chromatogram of the product obtained from the alcohol-acid system + cellulase enzymolysis system extraction of Epimedium sagittatum (Sweet) Maxim. in Example 7. DETAILED DESCRIPTION

[0061] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. 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 the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0062] All the raw materials involved in the present application are not particularly limited in source, and can be purchased from the market or prepared according to the conventional technical means well known to those skilled in the art.

[0063] The present application provides a preparation method of icariin, comprising the following steps:

[0064] (1) mixing Epimedium extract with organic acid to perform acidolysis, to obtain acidolysis product;

[0065] wherein the Epimedium extract comprises icariin and / or epimediumin C;

[0066] the organic acid comprises citric acid and / or oxalic acid;

[0067] (2) mixing the acid hydrolysis product with enzymes for enzymolysis to obtain icariin.

[0068] According to the present application, the Epimedium extract is first mixed with organic acid for acid hydrolysis to obtain an acid hydrolysis product. The Epimedium extract preferably comprises icariin and / or epimediumin C, which can be purchased from the market or obtained by natural extraction from Epimedium herb or Epimedium wushanense herb. The solvent for extraction includes any one or more of water, aqueous ethanol or anhydrous ethanol. The present application does not have special limitations on the way of extraction, which can be performed according to the conventional technical means well known to those skilled in the art. The organic acid preferably comprises citric acid and / or oxalic acid, more preferably oxalic acid. In the present application, the molar ratio of the Epimedium extract to the organic acid is preferably 1:(100-300), the temperature for acid hydrolysis is 50-65℃, and the time for acid hydrolysis is preferably 24-48h. It has been found through research that, during acid hydrolysis, the acid hydrolysis product of icariin and epimediumin C preferentially generates icariin with one molecule of glucose connected to position 7, and then generates anhydric icariin with one molecule of glucose connected to position 7, but the anhydric icariin with one molecule of glucose connected to position 7 is unstable, and under the conditions of increased acid dosage and acid hydrolysis time, the anhydric icariin with one molecule of glucose connected to position 7 can generate icariin with one molecule of glucose connected to position 7 through hydration, i.e. the target product of acid hydrolysis. Therefore, in the present application, the molar ratio of the Epimedium extract to the organic acid is more preferably 1:(200-300), and most preferably 1:300, and the time for acid hydrolysis is more preferably 36-48h, and most preferably 48h.

[0069] According to the present application, after obtaining the acid hydrolysis product, the acid hydrolysis product is subjected to enzymatic hydrolysis. In order to ensure the reaction activity of the enzyme in the enzymatic hydrolysis process, after obtaining the acid hydrolysis product, the acid hydrolysis product is preferably mixed with a buffer solution to adjust the pH of the system to 5-7, the buffer solution is preferably an acetic acid / sodium acetate buffer, and the mass / volume ratio of the Epimedium extract to the buffer solution is preferably 1:(2-4) mg / mL, and more preferably 1:(2-3) mg / mL. In the present application, the enzyme used in the enzymatic hydrolysis process preferably includes any one or more of cellulase, β-glucosidase, snailase or naringinase, and more preferably cellulase from the perspective of cost. The cellulase is used to act on the acid hydrolysis product, and the liquid phase detection results show that icariin can be obtained. In the present application, the mass ratio of the Epimedium extract to the enzyme is preferably 1:(1.5-3), and more preferably 1:(2-3). The temperature of the enzymatic hydrolysis is preferably 45-55°C, and more preferably 50-55°C, and the time of the enzymatic hydrolysis is preferably 20-36 h, and more preferably 24-36 h.

[0070] It should be noted that in the whole preparation process of icariin, the present application uses spot plate to monitor the reaction progress, and determines the yield of the final icariin through a standard curve.

[0071] In the present application, the mobile phase for developing epimedin C and icariin in the spot plate is preferably chloroform:methanol:water:glacial acetic acid=7:3:1:0.3 (volume ratio), and the mobile phase for developing icariin is preferably dichloromethane:ethyl acetate:glacial acetic acid=8:1:0.3 (volume ratio), so as to obtain suitable R f values of each component for qualitative analysis of the components in the system during the monitoring process.

[0072] In the present application, the drawing process of the standard curve is not particularly limited, and can be performed according to the conventional technical means familiar to those skilled in the art. The chromatographic conditions are preferably as follows: octadecylsilane-bonded silica gel is used as the filler, acetonitrile (A)-water (B) is used as the mobile phase for gradient elution, the elution conditions are as follows: 0-30 min, 25% A; 30-42 min, 25%-45% A; 42-55 min, 45% A; 55-65 min, 45%-75% A; 65-77 min, 75% A; 77-90 min, 75%-25% A. The flow rate is 0.8 mL / min, the detection wavelength is 270 nm, the column temperature is 30°C, and the injection amount is 20 μL.

[0073] The preparation method of icariin provided by the present application is to meet the actual application needs, therefore, the present application refers to the above-mentioned method, and icariin is extracted by using Epimedium herb and Epimedium wushanense herb as raw materials. The present application preferably performs acid extraction on Epimedium herb or Epimedium wushanense herb with an alcohol-acid mixed solution to directly obtain an acid extraction product in one step. The present application does not have special restrictions on the acid extraction mode, and the acid extraction mode can be obtained according to the conventional technical means familiar to those skilled in the art. The present application preferably reflux extracts at 70-90 ℃ for 2-4 h, and more preferably reflux extracts at 80-90 ℃ for 3-4 h. In the alcohol-acid mixed solution, the alcohol preferably includes 50 vol% ethanol aqueous solution and / or anhydrous ethanol, and more preferably anhydrous ethanol. The acid preferably includes citric acid and / or oxalic acid, and more preferably oxalic acid. Most preferably, the acid is a saturated oxalic acid solution. The volume ratio of the acid to the alcohol is preferably 1:(1-2), and more preferably 1:1. In order to improve the yield of icariin, after the acid extraction is completed, the acid extraction liquid is preferably suction filtered to obtain a first filtrate. The filter residue is subjected to repeated acid extraction according to the above-mentioned mode. The number of repetitions is not less than 1. The filtrate obtained by subsequent suction filtration is combined with the first filtrate to obtain a combined filtrate.

[0074] According to the present application, after the acid extraction is completed, enzymolysis is performed. The combined filtrate is preferably adjusted to a pH of 6-7. After the precipitates are filtered off, the combined filtrate is concentrated and dried, and then methanol is added for dissolution. After the insoluble substances are filtered off, the concentrated and dried product is subjected to subsequent enzymolysis treatment. The present application preferably adds a buffer solution to the finally obtained concentrated and dried product, and then adds an enzyme for enzymolysis. The buffer solution is preferably an acetic acid / sodium acetate buffer solution with a pH of 5-6. The enzyme preferably includes any one or more of cellulase, β-glucosidase, snailase or naringinase, and more preferably cellulase. The enzymolysis temperature is preferably 45-55 ℃, and more preferably 50-55 ℃. The enzymolysis time is preferably 20-36 h, and more preferably 24-36 h.

[0075] According to the present application, after the enzymolysis is completed, the enzymolysis product is preferably subjected to post-treatment. The post-treatment includes the following steps: the enzymolysis product is concentrated and dried, methanol is added for dissolution, the insoluble substances are filtered off to obtain a filtrate, and the filtrate contains the target product icariin.

[0076] In order to further illustrate the present application, the following examples are used for detailed description. The experimental raw materials used in the following examples of the present application are not particularly limited, and can be purchased on the market or obtained according to the conventional technical means familiar to those skilled in the art.

[0077] Example 1

[0078] This example is used to draw standard curves of damsin C, icariin and icariin. The steps are as follows:

[0079] Precisely take 25.3 mg of D. C, 25.4 mg of Icariin and 25.2 mg of Icaritin, add methanol to dissolve, and then dilute to 50 mL in a volumetric flask to prepare a mixed reference solution with a concentration of 0.5 mg / mL. Precisely take the reference solution, and dilute it by 2, 4, 8, 16, 32, 64, 128 and 256 times respectively to obtain solutions with different concentrations. Take 20 μL of each solution, and record the peak area. Draw a standard curve with the peak area integral value (Y) as the vertical coordinate and the concentration of the reference (X) as the horizontal coordinate.

[0080] Chromatographic conditions: octadecylsilane-bonded silica gel as the filler, acetonitrile (A)-water (B) as the mobile phase for gradient elution, elution conditions: 0-30 min, 25% A; 30-42 min, 25%-45% A; 42-55 min, 45% A; 55-65 min, 45%-75% A; 65-77 min, 75% A; 77-90 min, 75%-25% A. The flow rate is 0.8 mL / min, the detection wavelength is 270 nm, the column temperature is 30°C, and the injection volume is 20 μL.

[0081] The standard curve of D. C is shown in the following formula: Figure 1 y = 44.1585x - 60.74261, R 2 = 0.99857;

[0082] The standard curve of Icariin is shown in the following formula: Figure 2 y = 53.37934x - 61.15418, R 2 = 0.99897;

[0083] The standard curve of Icaritin is shown in the following formula: Figure 3 y = 81.24921x + 226.36158, R 2 = 0.99905.

[0084] Example 2

[0085] In this example, D. C, Icariin and Icaritin are subjected to thin layer chromatography, and the steps are as follows:

[0086] Take 5 mg of D. C, 5 mg of Icariin and 5 mg of Icaritin, add 5 mL of methanol to dissolve, and then use for spotting. According to the spotting results and the pharmacopoeia, the mobile phase for developing D. C and Icariin is chloroform:methanol:water:glacial acetic acid = 7:3:1:0.3, and the mobile phase for developing Icaritin is dichloromethane:ethyl acetate:glacial acetic acid = 8:1:0.3.

[0087] The developing results of Icaritin are shown in the following formula: Figure 4 R f= 2.3 / 3.4 = 0.676;

[0088] The development results of icariin and dammarane-type saponins are shown in Figure 1 (from left to right, icariin and dammarane-type saponins, respectively). Figure 5 f = 2.3 / 3.5 = 0.686, R f = 1.7 / 3.5 = 0.486.

[0089] The thin layer conditions described above were used in the subsequent examples to monitor the progress of the reaction.

[0090] Example 3

[0091] This example was used to determine the hydrolysis conditions of organic acids, and the steps were as follows:

[0092] Icaritin and icariin, 10 mg each, were precisely weighed and dissolved in 10 mL of methanol to obtain stock solutions. Three portions of 1 mL of each of the stock solutions of icariin and icaritin were prepared, and 1 mL of oxalic acid, citric acid and acetic acid solutions were added in a 1:100 molar ratio, respectively. Hydrolysis was carried out in a water bath at 60°C, and samples were taken at 12 h, 24 h, 36 h and 48 h to monitor the progress of the reaction.

[0093] The results of the 48 h sample plate are shown in Figure 2. Figure 6 From left to right in the figure, they are: ① icariin + citric acid, ② icariin + oxalic acid, ③ icariin + acetic acid, ④ icaritin + citric acid, ⑤ icaritin + oxalic acid, and ⑥ icaritin + acetic acid. Figure 6 It can be seen that under the action of the three acids, no new spots appeared on the sample plate, indicating that icaritin did not hydrolyze under the action of organic acids; under the action of citric acid and oxalic acid, new spots appeared on the sample plate, and oxalic acid was stronger than citric acid, and acetic acid was the weakest, and no new spots appeared under the action of acetic acid. Based on the above results, oxalic acid was selected for subsequent acid hydrolysis.

[0094] Example 4

[0095] This example was used to determine the hydrolysis conditions of oxalic acid, and the steps were as follows:

[0096] ​Reference Example 3, prepare stock solutions of the matatabiin C and icariin, take 3 portions of each of the two stock solutions, each 1 mL, add oxalic acid solution 1 mL at a molar ratio of 1:100, 1:200, 1:300 respectively, conduct water bath at 60°C, take samples at 24h, 48h respectively, liquid phase analysis (chromatographic conditions: use octadecylsilane bonded silica gel as filler, use acetonitrile (A)-water (B) as mobile phase for gradient elution, elution conditions: 0-30 min, 25% A; 30-42 min, 25%-45% A; 42-55 min, 45% A; 55-65 min, 45%-75% A; 65-77 min, 75% A; 77-90 min, 75%-25% A. Flow rate 0.8 mL / min, detection wavelength 270 mn, column temperature 30°C, injection volume 20 μL), monitor the reaction progress.

[0097] In addition, take stock solutions of the matatabiin C and icariin, add oxalic acid solution 1 mL at a molar ratio of 1:200, conduct liquid phase analysis (ion source ESI, both positive and negative ion scanning modes are used; ion source voltage 3 kV; ion source temperature 250°C; mass spectrometry interface temperature 300°C; scanning range 50-1000 m / z; scanning mode multiple reaction monitoring MRM) on the liquid of the reaction after 48h, as a reference for the results of the liquid phase analysis.

[0098] The liquid phase chart of the icariin standard is shown in Figure 7 The liquid phase chart of the matatabiin C standard is shown in Figure 8 The liquid phase chart of the icariin+1:100 molar ratio oxalic acid+reaction 24h is shown in Figure 9 The liquid phase chart of the icariin+1:200 molar ratio oxalic acid+reaction 24h is shown in Figure 10 The liquid phase chart of the icariin+1:300 molar ratio oxalic acid+reaction 24h is shown in Figure 11 The liquid phase chart of the icariin+1:100 molar ratio oxalic acid+reaction 48h is shown in Figure 12 The liquid phase chart of the icariin+1:200 molar ratio oxalic acid+reaction 48h is shown in Figure 13 The liquid phase chart of the icariin+1:300 molar ratio oxalic acid+reaction 48h is shown in Figure 14 The liquid phase chart of the matatabiin C+1:100 molar ratio oxalic acid+reaction 24h is shown in Figure 15 The liquid phase chart of the matatabiin C+1:200 molar ratio oxalic acid+reaction 24h is shown in Figure 16 The liquid phase chart of the matatabiin C+1:300 molar ratio oxalic acid+reaction 24h is shown in Figure 17 The liquid phase chart of the matatabiin C+1:100 molar ratio oxalic acid+reaction 48h is shown in Figure 18 The liquid phase chart of the matatabiin C+1:200 molar ratio oxalic acid+reaction 48h is shown in Figure 19As shown, the liquid phase diagram of the reaction of oxalic acid + C+ in a 1:300 molar ratio for 48 hours is as follows. Figure 20 As shown, the liquid chromatography-mass spectrometry (LC-MS) chromatogram of the reaction of icariin + oxalic acid (1:200 molar ratio) for 48 h is as follows. Figures 21-22 As shown in the figure, the liquid chromatography-mass spectrometry (LC-MS) curve of oxalic acid + C+ with a molar ratio of 1:200 for 48 hours is as follows. Figures 23-24 As shown.

[0099] Based on the liquid chromatography-mass spectrometry (LC-MS) results, combined with the literature (Yang Yishun, Zhang Tong, Ding Yue, Chen Jiawen, Zhou Yijia, Zhao Riji, Wu Huayan, Song Zejia. Preparation and HPLC determination of three icariin secondary products [J]. Chinese Traditional and Herbal Drugs, 2020, 42(03):779-782) and patents CN200710179673.8 and CN200680044755.5), the acid hydrolysis products of the two standards showed a peak at 44 min, which was determined to be dehydrated icariin with a glucose molecule linked at the 7-position (product 1), and a peak at 38 min, which was determined to be icariin with a glucose molecule linked at the 7-position (product 2). Combining the LC results and the structures of the two substrates, it can be seen that the acid hydrolysis process first generates product 2, and then product 1 is hydrated under the action of acid to obtain product 2. Moreover, the greater the amount of acid and the longer the reaction time, the more obvious the trend of product 1 converting to product 2. Therefore, we plan to use 48h and 1:300 molar acid hydrolysis conditions as the conditions for subsequent experiments.

[0100] Example 5

[0101] This example is used to determine the enzymatic hydrolysis conditions under the premise of oxalic acid hydrolysis. The steps are as follows:

[0102] Accurately weigh 10 mg each of icariin C and epimedium extract, prepare a 2 mg / mL methanol solution, take two aliquots of each solution, 1 mL each, add 1 mL of oxalic acid solution at a molar ratio of 1:300 to each aliquot, and react at 60℃ for 48 h. After the reaction is complete, adjust the pH to 5-6, add 5 mL of 0.5 M acetate / sodium acetate buffer, and then add 4 mg of dextranase to one aliquot and 4 mg of cellulase to the other aliquot of each of the two identical reaction solutions. React at 50℃ for 24 h, take samples, and perform liquid chromatography analysis.

[0103] The liquid phase diagram of icariin hydrolysis + dextranase hydrolysis is shown below. Figure 25 As shown, the liquid phase diagram of the acid hydrolysis and dextranase enzymatic hydrolysis of Astragalus membranaceus is as follows. Figure 26 As shown, the liquid phase diagram of icariin hydrolysis + cellulase enzymatic hydrolysis is as follows. Figure 27 As shown, the liquid phase diagram of acid hydrolysis + cellulase hydrolysis of Astragalus membranaceus is as follows. Figure 28 As shown.

[0104] Combination Figures 25-28It can be seen that the glucanase acts on the acid hydrolysate, and the liquid phase detection result shows that no peak is detected at the peak position of icariin (about 65 min), indicating that it cannot enzymatically hydrolyze the acid hydrolysate to obtain icariin. The cellulase acts on the acid hydrolysate, and the liquid phase detection result shows that a peak is detected at the peak position of icariin (about 65 min), indicating that it can enzymatically hydrolyze the acid hydrolysate to obtain icariin, and the icariin yield converted from desmodidin C is 9.9%, and the icariin yield converted from icariin is 14.9%. However, there is a very strong peak at 74 min in the reaction product of icariin, which is speculated to be the aglycone of icariin, indicating that the conversion of the aglycone to icariin is not complete, and the amount of acid needs to be increased.

[0105] Example 6

[0106] In this example, the amount of acid is increased, and the icariin yield is calculated, and the steps are as follows:

[0107] Take 1 mL of the 2 mg / mL methanol solution of desmodidin C and icariin prepared in Example 5, respectively, add 2 mL of saturated oxalic acid solution, and react at 60°C for 48 h. After the reaction is completed, adjust the pH to 5-6, add 5 mL of 0.5M acetic acid / sodium acetate buffer, and then add 4 mg of cellulase, respectively, and react at 50°C for 24 h. After the reaction is completed, take samples for liquid phase detection.

[0108] The liquid phase diagram of icariin acid hydrolysis + cellulase enzymolysis is shown in Figure 29 The liquid phase diagram of desmodidin C acid hydrolysis + cellulase enzymolysis is shown in Figure 30 .

[0109] It is calculated that the icariin yield converted from desmodidin C is 15.72%, and the icariin yield converted from icariin is 18.54%. It can be seen that increasing the amount of acid increases the icariin yield converted from desmodidin C and icariin.

[0110] Example 7

[0111] In this example, epimedium and Wushan epimedium are used as raw materials to prepare icariin, and the steps are as follows:

[0112] The Epimedium and the Epimedium koreanum are crushed into powder by using a pulverizer. 5.00g of the Epimedium and the Epimedium koreanum are respectively taken, two for each, and placed in four 250mL round bottom flasks. 100mL of 50vol% ethanol solution is added to one of the flasks, and 50mL of anhydrous ethanol and 50mL of a saturated oxalic acid solution are added to the other flask. The extraction is carried out at 80℃ under oil bath for 4h. After the extraction is completed, the extraction liquid is filtered to obtain filtrate 1. The residue is extracted again in the above-mentioned manner, and the extraction liquid is filtered to obtain filtrate 2. The two times of extraction liquid are combined, and the pH is adjusted to 6-7. Then, the precipitate is filtered out. The rotary evaporation is used to concentrate the filtrate to dryness. Then, 50mL of methanol is added, and the filtrate is ultrasonically dissolved. The insoluble substances are filtered out. A small amount of the filtrate is taken, and liquid phase detection is carried out. Then, the rotary evaporation is used to concentrate the filtrate to dryness again. 100mL of pH=5 acetic acid / sodium acetate solution is added to the concentrated product, and ultrasonic dispersion is carried out. Then, 200mg of cellulase is added, and reaction is carried out at 50℃ under oil bath for 24h. After the reaction is completed, the rotary evaporation is used to concentrate the reaction liquid to dryness. After the concentration, 50mL of methanol is added, and the reaction liquid is ultrasonically dissolved. The insoluble substances are filtered out. A small amount of the filtrate is taken, and liquid phase detection is carried out again.

[0113] The liquid chromatogram of the Epimedium (alcohol-water system) extraction result is shown in Figure 31 The liquid chromatogram of the Epimedium (alcohol-water system+ cellulase enzymolysis) is shown in Figure 32 The liquid chromatogram of the Epimedium (alcohol-acid system) extraction result is shown in Figure 33 The liquid chromatogram of the Epimedium (alcohol-acid system+ cellulase enzymolysis) is shown in Figure 34 The liquid chromatogram of the Epimedium koreanum (alcohol-water system) extraction result is shown in Figure 35 The liquid chromatogram of the Epimedium koreanum (alcohol-water system+ cellulase enzymolysis) is shown in Figure 36 The liquid chromatogram of the Epimedium koreanum (alcohol-acid system) extraction result is shown in Figure 37 The liquid chromatogram of the Epimedium koreanum (alcohol-acid system+ cellulase enzymolysis) is shown in Figure 38 The liquid chromatogram of the Epimedium koreanum (alcohol-acid system+ cellulase enzymolysis) is shown in

[0114] As can be seen from Figures 31-34 , the Epimedium (alcohol-water system) cannot extract icariin; the Epimedium (alcohol-water system+ cellulase enzymolysis) can extract icariin, and the peak area is 1172.1. The yield of icariin is calculated to be 0.116%; the Epimedium (alcohol-acid system) can extract icariin, and the peak area is 4395.8. The yield of icariin is calculated to be 0.513%; the Epimedium (alcohol-acid system+ cellulase enzymolysis) can extract icariin, and the peak area is 41791.8. The yield of icariin is calculated to be 5.116%. As can be seen from Figures 35-38It can be seen that icariin cannot be extracted from Wushan Epimedium (alcohol-water system); icariin can be extracted from Wushan Epimedium (alcohol-water system + cellulase enzymolysis), the peak area of which is 553.4, and the yield of icariin is calculated to be 0.040%; icariin can be extracted from Wushan Epimedium (alcohol-acid system), the peak area of which is 8197.7, and the yield of icariin is calculated to be 0.981%; icariin can be extracted from Wushan Epimedium (alcohol-acid system + cellulase enzymolysis), the peak area of which is 11932.6, and the yield of icariin is calculated to be 1.441%.

[0115] According to the liquid phase results of the two Epimedium medicinal material extraction treatment liquids, compared with alcohol-water system extraction, alcohol-acid water system extraction can increase the yield of icariin, and after cellulase enzymolysis, the yield of icariin is significantly higher than that of the former.

[0116] The above description of disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing icariin, characterized in that, The method comprises the following steps: (1) mixing raw materials with an alcohol-acid mixed solution to perform acid hydrolysis extraction to obtain an acid extraction solution; (2) adjusting the pH of the acid extraction solution to 6-7, filtering, mixing the obtained filtrate with methanol, filtering and drying again, and performing enzymatic reaction on the obtained product and cellulase to obtain icariin; wherein the raw materials are Herba Epimedii or Herba Epimedii from Wushan; the acid in the alcohol-acid mixed solution is oxalic acid; the temperature of the acid hydrolysis extraction is 70-90 DEG C.

2. The production method according to claim 1, characterized by, in the alcohol-acid mixed solution, the alcohol includes 50vol% ethanol aqueous solution and / or anhydrous ethanol.

3. The production method according to claim 1 or 2, characterized by, in the alcohol-acid mixed solution, the volume ratio of the acid to the alcohol is 1:(1-2).

4. The method of claim 1, wherein, the time of the acid hydrolysis extraction is 2-4 h.

5. The preparation method according to claim 1, characterized in that, the temperature of the acid hydrolysis extraction is 80-90 DEG C, and the time of the acid hydrolysis extraction is 3-4 h.

6. The method of claim 1, wherein, the temperature of the enzymatic reaction is 45-55 DEG C, and the time of the enzymatic reaction is 20-36 h.

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