Method for extracting and separating sodium aescinate from plants

Abstract

The invention discloses a method for extracting and separating sodium aescinate from plants, and belongs to the technical field of biological medicines. Comprising the following steps: 1, slicing a dry plant, crushing, screening with a screen, and collecting filtered powder; 2, weighing the collected filtered plant powder, adding the plant powder into an acidic buffer solution, then adding an organic phase solution, and slightly stirring and extracting at room temperature; 3, after the extraction is completed, collecting an ethyl acetate phase, and concentrating and drying under reduced pressure to obtain an aescin extract; and 4, carrying out precipitation decoloration treatment on the aescin extract obtained in the step (3) by using acetone, carrying out rotary evaporation concentration to obtain a solid, dissolving the solid by using an ethanol solution, and carrying out one-step purification by using reversed-phase chromatography to obtain sodium aescinate. Compared with a traditional ethanol / water extraction method, the method has the advantages that the target product can be selectively extracted into an organic phase, the extraction efficiency is higher, impurities contained in main components are fewer, and subsequent purification treatment is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for extracting and separating sodium aescinate from plants. Background Art

[0002] Aescin from the seeds of Salvia officinalis has sweet and warm properties, and enters the liver and stomach meridians. It has the effects of regulating qi, relieving stomach discomfort, and relieving pain. Among them, aescin A, B, C, and D are the main active medicinal ingredients, and are mostly used to treat chest and abdominal distension and stomach pain. It is one of the few known pure natural herbal medicines with stable and definite efficacy and fewer side effects. Its clinical application range is constantly expanding, and it has broad application prospects.

[0003] The extraction process of aescin is divided into two steps: initial extraction and purification. At present, the main methods for extracting aescin in China are as follows:

[0004] (1) Patent No. CN 102309529 A discloses a microwave extraction method for aescin from Sphaerocarpus chinensis. That is: ① After the Sphaerocarpus chinensis is dried and crushed, it is soaked in an ethanol aqueous solution (50% to 95%), extracted with the assistance of microwaves, and the temperature of the extract is maintained at 25 to 75°C while stirring continuously. ② After the extraction is completed, the extract is filtered out, concentrated and dried under reduced pressure to obtain an aescin extract. However, given the easy breakage of glycosidic bonds, the actual effect of microwave-assisted extraction is difficult to verify.

[0005] (2) Patent No. CN 107602641 A discloses an ultra-high pressure device-assisted extraction method; Patent No. CN106491672A discloses a method for extracting aescin using supercritical carbon dioxide extraction. However, the process conditions are relatively complex and the equipment requirements are high, which will significantly increase the production cost and lack practical application value.

[0006] (3) Most of the extraction of aescin from the seeds of Sphaerocarpus rotundus uses 20% to 80% ethanol / water as the extraction solvent and a hot reflux method, among which CN 104402963 A, CN 102659897 A, CN 106589045 A and CN 104804060A and other patent documents all use this type of method. Alternatively, different concentrations of ethanol / water are used for soaking, mechanical stirring is used to assist extraction, and the extraction temperature is controlled at the same time, among which CN 109096351 A, CN 106589045 A and CN 107759656 A and other patent documents all use this type of method. The main problem is that aescin has poor heat resistance, and a temperature exceeding 60°C is likely to cause glycosidic bond breakage, so the extraction temperature cannot be too high, resulting in a long extraction time.

[0007] The main purification methods of aescin extract in China are as follows:

[0008] (1) Patent No. CN 106554384 A discloses a method for purifying aescin extract. The crude aescin extract extracted by ultrasound-assisted extraction is subjected to acetone precipitation, macroporous resin adsorption purification, acetone washing and decolorization, and reduced pressure concentration and drying, and the product purity reaches more than 85%.

[0009] (2) Patent No. CN 112724192 A discloses a method for extracting and purifying aescin. The seeds of Sauvignon ovata are extracted with acidic ethanol water (ethanol concentration 50%, solution pH 5.0-6.9) at 40°C for 3-5 hours and centrifuged to obtain a primary extract. The primary extract is concentrated to an appropriate volume by rotary evaporation, and dichloromethane is added to extract and remove oil. A high molecular polymer surfactant is added to the aqueous phase for rotary evaporation concentration to obtain a concentrated solution. The concentrated solution is decolorized by activated carbon, ultrafiltered, and the filtrate is collected for sodium cation exchange resin chromatography. The target chromatographic solution is concentrated and repeatedly crystallized 3 times with anhydrous ethanol to prepare high-purity sodium aescinate (purity 95%).

[0010] (3) CN102532241A discloses that after the seeds of Sauvignon ovata are crushed, they are extracted by ethanol reflux, the extract is separated by cation exchange resin, purified by alumina column chromatography, and then recrystallized by ethanol to obtain sodium aescinate, with a yield of about 2% and a purity of about 96%.

[0011] It can be known from the above-mentioned prior art analysis that in the existing extraction step, the extraction process of aescin from the seeds of Sauerkraut mostly adopts ethanol / water solution of different concentrations as extraction solvent to obtain a crude extract of aescin. In addition to containing saponin components, the crude extract also contains a large amount of other components, such as flavonoids, flavonoid glycosides, tannins and organic acids, etc., and also contains pigment components, which makes the color of the crude extract darker, resulting in greater difficulty in subsequent purification. In view of this, the present invention provides a technical solution, which can avoid the disadvantages of the above-mentioned technology, selectively extract the target product, and complete a single extraction purification at the same time, and the impurities contained in the extract are less, which is conducive to subsequent purification treatment.

[0012] The existing purification method for crude sodium aescinate is complicated and consumes a lot of solvents, and the purity of sodium aescinate in the final product is generally not high. In view of this, the present invention provides a purification technology scheme, in which the primary extract is subjected to simple extraction, impurity removal and decolorization treatment, and then only one step of reverse phase purification is performed to obtain high-purity sodium aescinate (purity greater than 96%). Summary of the invention

[0013] In view of the defects existing in the above-mentioned prior art, the object of the present invention is to design and provide a method for extracting and separating sodium aescinate from plants (such as Shorea seeds). The three-phase extraction and purification method of the present invention can treat the plant to be extracted, such as Shorea seeds, as a solid phase, with ethyl acetate as an organic liquid phase, and an acidic buffer as an inorganic liquid phase. Its extraction and purification principle is a three-phase distribution extraction and purification method between a solid phase and a liquid phase, and between a liquid phase and a liquid phase, which can be applied to the extraction of most plant natural products. Compared with the traditional ethanol / water extraction method, the target product can be selectively extracted into the organic phase, the extraction efficiency is higher, and the impurities contained in the main component are less, which is conducive to subsequent purification treatment.

[0014] In order to achieve the above object, the present invention adopts the following technical solutions:

[0015] In one aspect, the present invention provides a method for extracting and separating sodium aescinate from plants, comprising the following steps:

[0016] (1) Slice the dried plant, crush it, pass it through a sieve, and collect the filtered powder;

[0017] (2) Weigh the filtered plant powder collected in step (1), add it to an acidic buffer solution, then add the organic phase solution, and extract by slight stirring at room temperature to prevent emulsification and mix the three phases evenly;

[0018] (3) After the extraction is completed, the ethyl acetate phase is collected, concentrated under reduced pressure and dried to obtain aescin extract; the extract is dissolved in 60% ethanol solution, and the total amount of sodium aescinate A, B, C, and D in the extract is determined by high performance liquid chromatography.

[0019] (4) The aescin extract obtained in step (3) is subjected to precipitation and decolorization treatment with acetone, and then concentrated by rotary evaporation to a solid. The solid is dissolved with an ethanol solution, and then purified by reverse phase chromatography in one step to obtain sodium aescinate.

[0020] In the method described, the sieve in step (1) is 60 mesh.

[0021] In the method, the plant is selected from one of the following: sago palm seeds, liquorice, cockscomb and beet, and preferably the plant is sago palm seeds.

[0022] In the method described, the acidic buffer in step (2) is selected from the group consisting of hydrochloride buffer, sulfate buffer, phosphate buffer, potassium salt buffer and sodium salt buffer, preferably the acidic buffer is hydrochloride buffer; the pH of the acidic buffer is 4.0-7.0, preferably the pH is 6.0±0.5.

[0023] In the method, in step (2), the mass-to-volume ratio of the plant powder to the acidic buffer solution is 1:20-100 g / mL, and the volume of the organic phase solution is the same as that of the acidic buffer solution.

[0024] In the method, the organic phase solution in step (2) is one of ethyl acetate, methyl acetate or dichloromethane; preferably ethyl acetate.

[0025] In the method described, the conditions of the reverse phase chromatography in step (4) are: phase A is 5.5% phosphoric acid, pH 1.85-2.10; phase B is acetonitrile, and gradient elution is performed, phase B, 37%-39%, 150 min; wherein phase B, 37%-39% means that the acetonitrile concentration increases from 37% at 0 min to 39% after 150 min; 150 min is the gradient elution time.

[0026] Alternatively, the volume ratio of phase A to phase B is 38:62, and the elution is performed for 60 min.

[0027] In the method, the sodium aescinate in step (4) includes sodium aescinate A, sodium aescinate B, sodium aescinate C and sodium aescinate D.

[0028] In a second aspect, the present invention provides the use of any of the methods described above in extracting, separating and purifying sodium aescinate from plants.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The extraction and separation method of the present invention can selectively extract the target product into the organic phase. Compared with the traditional ethanol / water extraction method, the extraction efficiency is higher and the main component contains fewer impurities, which is beneficial to the subsequent purification process.

[0031] 2. The three-phase extraction and purification method of the present invention can be applied to the extraction of most natural plant products.

[0032] 3. The present invention only uses one step of reverse phase purification, and the purity of sodium aescinate is greater than 96%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The chromatogram is the extraction result of aescin from Semen Sinensis in Example 1;

[0034] Figure 2 The chromatogram is the purification result of aescin extract in Example 2;

[0035] Figure 3 The chromatogram is the purification result of aescin extract of Example 3;

[0036] Figure 4 The chromatogram is the purification result of aescin extract in Example 4. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0038] Example 1: Extraction of Sodium Aescinate from Semen Sinensis

[0039] 1. Pretreatment of Salvia officinalis

[0040] The dried Salacia chinensis medicinal material was sliced, crushed, passed through a 60-mesh sieve, and the filtered powder was collected for extraction.

[0041] 2. Determination of total content of sodium aescinate A, B, C and D

[0042] Take 1.0 g of the powder of the seeds of Sauvignon ovata in step 1 above, and determine the content of saponin compounds in the medicinal material by referring to the high performance liquid chromatography method (Appendix VD of Part II of the Chinese Pharmacopoeia 2005 edition), and calculate the total content of aescin A, B, C, and D in the medicinal material.

[0043] 3. Extraction of Aescin

[0044] Weigh 5.0g of the seed powder of Shorea in the above step 1, in 100mL hydrochloride buffer, pH=6.0±0.5, then add 100mL ethyl acetate into the water, gently stir the extraction (prevent emulsification) at room temperature, and mix the three phases evenly. After extraction is completed, collect the ethyl acetate phase, concentrate under reduced pressure and dry (the vacuum degree of reduced pressure concentration is as low as possible, the rotary evaporation concentration temperature is 30°C, and the concentration end point is that no liquid flows out), and obtain the aescin extract. Use 60% ethanol / water to dissolve the extract, and measure the total amount and purity of sodium aescinate A, B, C, and D in the extract by high performance liquid chromatography, and calculate the total extraction rate of sodium aescinate A, B, C, and D. The results are as shown in Table 1, wherein, total saponins refers to all saponin substances including aescin A, B, C, and D. Figure 1 This is the chromatogram of the extraction results of aescin from Salacia seeds.

[0045] Example 2: Purification of Sodium Aescinate Extract

[0046] The extract of Example 1 was decolorized with acetone and concentrated by rotary evaporation to a solid. The solid was dissolved with 60% ethanol / water and purified by reverse phase chromatography in one step. Phase A was 5.5% phosphoric acid (pH adjusted to 1.85 with sodium hydroxide) and phase B was acetonitrile. Gradient elution (phase B, 37% to 39%, 150 min) was performed. The purified sodium aescinate (including aescin A, B, C, and D) had a purity of 96.6%. Figure 2 The chromatogram is the purification result of aescin extract in Example 2.

[0047] Example 3: Purification of Sodium Aescinate Extract

[0048] The extract of Example 1 was decolorized with acetone and concentrated by rotary evaporation to a solid. The solid was dissolved with 60% ethanol / water and purified by reverse phase chromatography in one step. Phase A was 5.5% phosphoric acid (pH adjusted to 2.1 with sodium hydroxide) and phase B was acetonitrile. Gradient elution (phase B, 37% to 39%, 150 min) was performed. The purified sodium aescinate (including aescin A, B, C, and D) had a purity of 96.4%. Figure 3 The chromatogram is the purification result of aescin extract in Example 3.

[0049] Example 4: Purification of Sodium Aescinate Extract

[0050] The extract of Example 1 was decolorized with acetone and concentrated by rotary evaporation to a solid. The solid was dissolved with 60% ethanol / water and purified by reverse phase chromatography in one step. Phase A was 5.5% phosphoric acid (pH adjusted to 2.1 with sodium hydroxide) and phase B was acetonitrile. Phase A: phase B (38:62) were isocratically eluted for 60 min. The purity of the purified sodium aescinate (including aescin A, B, C, and D) was 96.2%. Figure 4 The chromatogram is the purification result of aescin extract in Example 3.

[0051] Comparative Example 1: Microwave Extraction of Aescin from Seeds of Smilax china

[0052] The medicinal material of Sauerkraut was dried and crushed, and the content of saponin compounds in the medicinal material was determined by referring to the method in the "Chinese Pharmacopoeia" and the high performance liquid chromatography method (Appendix VD of Part II of the Chinese Pharmacopoeia 2005 edition), and the total content of aescin A, B, C, and D in the medicinal material was calculated.

[0053] Weigh 10g of dried and crushed Sauerkraut seeds, add 200mL of 50% ethanol aqueous solution, soak for 30 minutes, irradiate with microwave power 600W for 3 minutes, keep the temperature of the extract at 30-60°C, stir continuously during extraction, filter out the extract after microwave irradiation, concentrate and dry under reduced pressure to obtain aescin extract. Weigh the total amount of the extract, and determine the total amount of aescin A, B, C, and D in the extract by high performance liquid chromatography. The results are shown in Table 1.

[0054] Comparative Example 2: Extraction and purification of sodium aescinate from seeds of Sauvignon ovata by ethanol / hydrothermal reflux method

[0055] Weigh 10g of dried and crushed Sauvignon ovata seeds, add 200mL of 60% ethanol aqueous solution, soak for 30 minutes, heat and reflux in a 70°C water bath for extraction for 30 minutes, filter out the extract after extraction, concentrate and dry under reduced pressure to obtain aescin extract. Weigh the total amount of the extract, and determine the total amount of aescin A, B, C, and D in the extract by high performance liquid chromatography. Concentrate the extract, remove impurities and decolorize it with acetone precipitation, and then purify it with macroporous adsorption resin AB-8, collect the eluate, concentrate it, and vacuum dry it to obtain off-white aescin, and the product purity is more than 85%.

[0056] Comparative Example 3: Extraction and purification of sodium aescinate from seeds of Sauvignon ovata by ethanol / water soaking

[0057] Slice the dried Salacia sutchuenensis medicinal material, crush it, pass it through a 60-mesh sieve, and collect the powder. Weigh 10g of powder, add 200mL of 60% ethanol / acidic water as the extraction solvent, stir and assist the extraction at 45°C, and the extraction time is 180min. After the extraction is completed, filter out the extract, concentrate and dry under reduced pressure to obtain aescin extract. Weigh the total amount of the extract, and determine the total amount of aescin A, B, C, and D in the extract by high performance liquid chromatography. The above concentrate is eluted with dichloromethane for oil and fat, decolorized with activated carbon, chromatographed with sodium type cation exchange resin, and the eluent is collected; after crystallization and recrystallization, dry to obtain sodium aescinate with a purity greater than 95%.

[0058] Table 1 Product extraction rate and purity results of Example 1 and Comparative Examples 1-3

[0059]

Claims

1. A method for extracting and separating sodium aescinate from plants, characterized in that: The following steps are involved: (1) Slice the dried plant, crush it, pass it through a sieve, and collect the filtered powder; (2) Weighing the filtered plant powder collected in step (1), adding it to an acidic buffer solution, then adding the organic phase solution, and extracting by slight stirring at room temperature; (3) After the extraction is completed, the ethyl acetate phase is collected, concentrated under reduced pressure and dried to obtain aescin extract; (4) The aescin extract obtained in step (3) is subjected to precipitation and decolorization treatment with acetone, and then concentrated by rotary evaporation to a solid. The solid is dissolved with an ethanol solution, and then purified by reverse phase chromatography in one step to obtain sodium aescinate.

2. The method according to claim 1, characterized in that The sieve in step (1) is 60 mesh.

3. The method according to claim 1, characterized in that The plant is selected from one of the group consisting of sago asiatica, liquorice, cockscomb and beet, and preferably the plant is sago asiatica.

4. The method according to claim 1, characterized in that The acidic buffer in step (2) is selected from one of hydrochloride buffer, sulfate buffer, phosphate buffer, potassium salt buffer and sodium salt buffer, preferably the acidic buffer is hydrochloride buffer; the pH of the acidic buffer is 4.0-7.0, preferably pH 6.0±0.

5.

5. The method according to claim 1, characterized in that The mass-to-volume ratio of the plant powder to the acidic buffer solution in step (2) is 1:20-100 g / mL, and the volume of the organic phase solution is the same as that of the acidic buffer solution.

6. The method according to claim 1, characterized in that The organic phase solution in step (2) is one of ethyl acetate, methyl acetate or dichloromethane; preferably ethyl acetate.

7. The method according to claim 1, characterized in that The conditions of the reverse phase chromatography in step (4) are as follows: phase A is 5.5% phosphoric acid, pH 1.85-2.10; phase B is acetonitrile, gradient elution is performed, phase B, 37%-39%, 150 min; Alternatively, the volume ratio of phase A to phase B is 38:62, and the elution is performed for 60 min.

8. The method according to claim 1, characterized in that The sodium aescinate in step (4) includes sodium aescinate A, sodium aescinate B, sodium aescinate C and sodium aescinate D.

9. The method according to claim 1, characterized in that The volume ratio of ethanol to water in the ethanol solution in step (4) is 60:

40.

10. Use of the method according to any one of claims 1 to 9 in extracting, separating and purifying sodium aescinate from plants.

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