Preparation method of tanshinone, salvianolic acid and salvia miltiorrhiza extract

Abstract

The invention discloses a preparation method of tanshinone, salvianolic acid and a salvia miltiorrhiza extract, which comprises the following steps: performing percolation extraction on crushed salvia miltiorrhiza by using an ethanol solution, and collecting percolate; recovering ethanol from the percolate, precipitating and carrying out solid-liquid separation to obtain a water solution and a precipitate, adding a solid dispersant into the precipitate, crushing, and extracting with ethanol to obtain a tanshinone ethanol solution; recovering and concentrating the water solution, adjusting the pH value to be greater than 1 and less than 3 under stirring, carrying out alcohol precipitation to remove impurities, and collecting the supernatant to obtain a salvianolic acid ethanol solution; or adjusting the pH value of the water solution to be greater than 2 and less than 4, then recovering, concentrating, carrying out alcohol precipitation to remove impurities, and collecting the supernate to obtain the salvianolic acid ethanol solution; salvianolic acid ethanol liquid and tanshinone ethanol liquid are respectively used for preparing tanshinone and salvianolic acid or preparing a radix salviae miltiorrhizae extract containing tanshinone and salvianolic acid. The method is simple to operate, free of special equipment, high in production efficiency and low in cost; the yield of the salvianolic acid extract is about 12%, the content of salvianolic acid B is about 30%, and the extraction transfer rate is greater than 80%.

Description

Technical Field

[0001] The invention relates to a novel method for simultaneously extracting and separating tanshinone and salvianolic acid from salvia miltiorrhiza and preparing a salvia miltiorrhiza extract. Background Art

[0002] Danshen is the dried root and rhizome of Salvia miltiorrhiza Bge., a plant of the Lamiaceae family. It mainly contains fat-soluble components, tanshinone, water-soluble components, salvianolic acid, and sugar components. Among them, the main representative active ingredient of tanshinone is tanshinone ⅡA, and the main representative active ingredient of salvianolic acid is salvianolic acid B. Sugar components include monosaccharides, oligosaccharides, polysaccharides, etc. Tanshinone and salvianolic acid are important raw materials or intermediates in the pharmaceutical industry and daily chemical industry. Danshen is a commonly used raw material for Chinese patent medicine. Conventional extraction methods include: single water or ethanol reflux extraction, which cannot extract both tanshinone and salvianolic acid at the same time; or two-step alternating extraction with ethanol and water, which can extract tanshinone and salvianolic acid components well, but the production process is complicated, the amount of solvent used is large, and the production efficiency is low. For example, the preparation method of "Danshen water extract (Danshen total phenolic acid extract)" included in the current edition of the "Chinese Pharmacopoeia" is to extract with water, concentrate to a clear paste, add ethanol to make the alcohol content 70%, precipitate and remove impurities, take the supernatant to recover and concentrate, and dry to obtain the Danshen total phenolic acid extract, and it is stipulated that this product, calculated on a dry basis, contains not less than 5% of tanshinone B; the preparation method of "tanshinone extract" is to extract with ethanol reflux, recover and concentrate to a thick paste, wash with hot water, and dry to obtain the Danshen ketone extract, and it is stipulated that this product, calculated on a dry basis, contains not less than 2.1% of cryptotanshinone and not less than 9.8% of tanshinone ⅡA; the above-mentioned Danshen total phenolic acid extract does not contain tanshinone components, and the tanshinone extract does not contain tanshinone acid components. The effective ingredients of both are not fully utilized, and the utilization rate of Danshen medicinal materials is not high. The preparation method of "Danshen Tablets" included in the current edition of the "Chinese Pharmacopoeia" is to first use ethanol reflux extraction, then decoct the residue in water, recover and concentrate, and dry, which is a two-step extraction method of ethanol and water, which can better extract tanshinone and tanshinone acid components.

[0003] In some applications, the content of the corresponding active ingredients in the total phenolic acid extract and tanshinone extract of salvia miltiorrhiza is required to be high, so many extraction methods use macroporous resin purification. For example, CN102351819B discloses a method for extracting and purifying high-purity salvia miltiorrhiza phenolic acid B, firstly crushing the salvia miltiorrhiza medicinal material and then extracting it with ethanol; the clarified extract obtained after solid-liquid separation, ethanol recovery and concentration is first enriched with a macroporous resin, and then purified by chromatographic column chromatography to obtain a salvia miltiorrhiza phenolic acid fraction, which is dried to obtain a salvia miltiorrhiza phenolic acid content greater than 90%. In the Chinese patent document CN104189073B, the applicant also disclosed a method for preparing total phenolic acid of salvia miltiorrhiza, the process is as follows: Figure 1As shown, the crushed Salvia miltiorrhiza is extracted by percolation with 80±5% ethanol by volume under the condition of temperature ≥20°C, and the percolation liquid is collected. In order to make the salvianolic acid components in the percolation liquid better adsorbed by the macroporous resin, the pH value of the upper column liquid needs to be adjusted. Therefore, the ethanol in the percolation liquid is first removed, and then the pH value is adjusted to ≤4, precipitated and solid-liquid separated. After the obtained aqueous solution is adsorbed on the macroporous resin, it is first washed with water to remove impurities, and then eluted with 40-80% ethanol by volume, and the ethanol eluate is collected and the solvent is removed to obtain the total phenolic acid extract of Salvia miltiorrhiza. However, the adsorption and separation process of macroporous resin is relatively complicated, the production efficiency is low, and a large amount of ethanol is used, and the production cost is high. The Chinese patent document CN1857403A discloses a preparation method for separating and enriching the effective part of salvianolic acid from Salvia miltiorrhiza without using macroporous resin, that is, water decoction extraction, concentration, alcohol precipitation, filtration concentration, acidification, ethyl acetate extraction, separation of organic solvent layer, recovery, and obtaining total phenolic acid of Salvia miltiorrhiza. However, this method uses organic solvent extraction, which is flammable and explosive, has a high safety risk, and has a low yield (4.8%).

[0004] When applying Danshen extract in the fields of Chinese patent medicine and health products, the content of the extract is not required to be very high, such as the Danshen total phenolic acid extract and tanshinone extract included in the above-mentioned "Chinese Pharmacopoeia". Therefore, the macroporous resin purification method is complicated to operate, has low production efficiency and high cost, and is not the preferred option for the factory. In this case, the relatively simple water extraction and alcohol precipitation method or alcohol extraction and water precipitation method is still widely used, but the existing water extraction and alcohol precipitation method or alcohol extraction and water precipitation method still has the defects of low target product content and / or yield. Therefore, improving the water extraction and alcohol precipitation method or alcohol extraction and water precipitation method to increase the content and / or yield of Danshen extract is still the goal pursued by the factory. Summary of the invention

[0005] In view of the above situation, the present invention first provides a method for simultaneously extracting tanshinone and salvianolic acid through one-step alcohol extraction. On this basis, the present invention also provides a purification method for obtaining salvianolic acid by "acidification and alcohol precipitation", and a purification method for obtaining tanshinone by "water precipitation and alcohol extraction" combined with a solid dispersant. Finally, a new method for preparing a danshen extract containing salvianolic acid and tanshinone is provided. The method is simple and feasible to operate, does not require special equipment, has high production efficiency and low cost, and is suitable for large-scale industrial production.

[0006] In a first aspect, the present invention provides a method for preparing tanshinone, comprising the following steps:

[0007] Step 1: extract the crushed Salvia miltiorrhiza by percolation with 80±5% ethanol by volume, and collect the percolation liquid.

[0008] Step 2, recovering ethanol from the percolation liquid, precipitating and performing solid-liquid separation (the water after solid-liquid separation is reserved), collecting the precipitate, first adding a solid dispersant (at least one of micro-powdered silica gel, calcium hydrogen phosphate, light magnesium oxide, diatomaceous earth, starch, and microcrystalline cellulose) equivalent to 0.2%-2% of the weight of Salvia miltiorrhiza, pulverizing the precipitate, and then extracting with 70%-95% ethanol, filtering, and obtaining tanshinone ethanol solution; recovering the solvent from the tanshinone ethanol solution, and then dissolving it with 70%-95% ethanol, precipitating and performing solid-liquid separation, collecting the precipitate, and drying to obtain a tanshinone extract.

[0009] In a second aspect, the present invention provides a method for preparing salvianolic acid, comprising the following steps:

[0010] Step 1, extracting the crushed Salvia miltiorrhiza by percolation with 80±5% ethanol by volume, and collecting the percolation liquid;

[0011] Step 2, recovering ethanol from the percolate, precipitating and separating the solid and liquid, and collecting the water for later use;

[0012] Step 3, recovering and concentrating the aqueous solution, adjusting the pH value to be greater than 1 and less than 3 under sufficient stirring, adding ethanol to make the alcohol content in the liquid ≥70% (both refer to volume content), precipitating and removing impurities, and collecting the supernatant to obtain salvianolic acid ethanol solution; or first adjusting the pH value of the aqueous solution to be greater than 2 and less than 4, then recovering and concentrating, adding ethanol to make the alcohol content in the liquid ≥70% under sufficient stirring, precipitating and removing impurities, and collecting the supernatant to obtain salvianolic acid ethanol solution;

[0013] Step 4, recovering ethanol from the salvianolic acid ethanol solution, concentrating and drying to obtain a salvianolic acid extract.

[0014] In a third aspect, the present invention provides a method for preparing a salvia miltiorrhiza extract, wherein the salvia miltiorrhiza extract contains both tanshinone and salvianolic acid. Figure 2 As shown, the method includes the following steps:

[0015] Step 1, extracting the crushed Salvia miltiorrhiza by percolation with 80±5% ethanol by volume, and collecting the percolation liquid;

[0016] Step 2, recovering ethanol from the percolation liquid, precipitating and solid-liquid separation (the water after solid-liquid separation is reserved), collecting the precipitate, first adding a solid dispersant (at least one of micro-powdered silica gel, calcium hydrogen phosphate, light magnesium oxide, diatomaceous earth, starch, and microcrystalline cellulose) equivalent to 0.2%-2% of the weight of Danshen, pulverizing, then extracting with 70%-95% ethanol, filtering, and obtaining tanshinone ethanol solution;

[0017] Step 3, recovering and concentrating the aqueous solution after solid-liquid separation, adjusting the pH value to be greater than 1 and less than 3 under sufficient stirring, adding ethanol to make the alcohol content in the liquid ≥70%, precipitating and removing impurities, and collecting the supernatant to obtain salvianolic acid ethanol solution; or first adjusting the pH value of the aqueous solution to be greater than 2 and less than 4, then recovering and concentrating, adding ethanol to make the alcohol content in the liquid ≥70% under sufficient stirring, precipitating and removing impurities, and collecting the supernatant to obtain salvianolic acid ethanol solution;

[0018] Step 4, recovering and concentrating the ethanol solution of salvianolic acid, adding the above-mentioned ethanol solution of tanshinone, recovering ethanol, concentrating and drying to obtain a danshen extract containing salvianolic acid and tanshinone;

[0019] Alternatively, the tanshinone ethanol solution is used to prepare the tanshinone extract, and the tanshinone acid ethanol solution is used to prepare the tanshinone acid extract, and the tanshinone extract and the tanshinone acid extract are mixed, crushed, and mixed to obtain the danshen extract.

[0020] In a fourth aspect, the present invention provides a method for the combined preparation of tanshinone and salvianolic acid, comprising the following steps:

[0021] Step 1: extract the crushed Salvia miltiorrhiza by percolation with 80±5% ethanol by volume, and collect the percolation liquid.

[0022] Step 2, recovering ethanol from the percolation liquid, precipitating and performing solid-liquid separation (the water after solid-liquid separation is reserved), collecting the precipitate, first adding a solid dispersant (at least one of micro-powdered silica gel, calcium hydrogen phosphate, light magnesium oxide, diatomaceous earth, starch, and microcrystalline cellulose) equivalent to 0.2%-2% of the weight of Danshen, pulverizing, then extracting with 70%-95% ethanol, filtering, and obtaining tanshinone ethanol solution; recovering the solvent from the tanshinone ethanol solution, then dissolving with 70%-95% ethanol, precipitating and performing solid-liquid separation, collecting the precipitate, and drying to obtain a tanshinone extract;

[0023] Step 3, the aqueous solution after solid-liquid separation is recovered and concentrated, the pH value is adjusted to be greater than 1 and less than 3 under sufficient stirring, ethanol is added to make the alcohol content in the liquid ≥70%, precipitation and impurities are removed, and the supernatant is collected to obtain salvianolic acid ethanol solution; or the aqueous solution is first adjusted to a pH value greater than 2 and less than 4, then recovered and concentrated, ethanol is added to make the alcohol content in the liquid ≥70% under sufficient stirring, precipitation and impurities are removed, and the supernatant is collected to obtain salvianolic acid ethanol solution. Ethanol is recovered from the salvianolic acid ethanol solution, concentrated and dried to obtain a salvianolic acid extract.

[0024] In the above-mentioned preparation methods, the crushing in step 1 is to crush the salvia miltiorrhiza to a particle size not larger than that passing through a 10-mesh sieve. A finer salvia miltiorrhiza particle size is beneficial to improving the extraction efficiency and the utilization rate of the medicinal material extraction. For example, in winter when the room temperature is below 20°C, the salvia miltiorrhiza should be crushed to pass through a 20-mesh sieve or a 40-mesh sieve, which can significantly improve the extraction transfer rate of tanshinone and salvianolic acid. The collection of percolate in step 1 refers to collecting ethanol percolate in an amount of 6 to 10 times the volume-weight ratio of salvia miltiorrhiza, that is, the volume (L) of ethanol percolate: the weight (kg) of salvia miltiorrhiza is 6 to 10 times.

[0025] In the above-mentioned preparation methods, the method for recovering ethanol from the percolate in step 2 is to add water to adjust the concentration of the drug solution to 0.5-1g crude drug / mL after recovering the ethanol, and then let it stand to precipitate and separate the solid and liquid.

[0026] In the preparation method of tanshinone, the method for recovering the solvent of the tanshinone ethanol solution is to use 70%-95% ethanol to dissolve (3-10g crude drug / mL), let it stand for precipitation, and separate the solid and liquid.

[0027] In the preparation method of tanshinone, the preparation method of danshen extract and the combined preparation method of tanshinone and danshen phenolic acid, the percolation liquid described in step 2 recovers ethanol, precipitates and separates the solid and liquid, collects the precipitate, and first adds a solid dispersant (at least one of micro-powder silica gel, calcium hydrogen phosphate, light magnesium oxide, diatomaceous earth, starch, microcrystalline cellulose, etc.) equivalent to 0.2-2% of the weight of danshen for pulverization treatment, especially a solid absorption dispersant that is allowed and accepted in pharmacy (but not soluble in ethanol). Because the above-mentioned precipitate containing tanshinone has a certain oiliness and viscosity, it is not easy to disperse evenly by direct pulverization (there is a clumping and caking phenomenon), so the above-mentioned solid dispersant (micro-powder silica gel, calcium hydrogen phosphate, light magnesium oxide, diatomaceous earth, starch, microcrystalline cellulose, etc.) is added and then pulverized and dispersed evenly, which is conducive to subsequent ethanol reflux, ultrasonic or stirring extraction. After ethanol extraction, it is filtered to remove the above-mentioned dispersant.

[0028] Step 3 is the water liquid concentration, alcohol precipitation, and impurity removal process after solid-liquid separation. The water liquid after solid-liquid separation is recovered and concentrated to about 2-3g crude drug / mL, and then alcohol precipitation is used for impurity removal. Alcohol precipitation and impurity removal refer to adding ethanol so that impurities are precipitated after ethanol reaches a certain concentration, and impurities can be removed by solid-liquid separation. The applicant unexpectedly found through in-depth research that acidification of water liquid (or extract) before alcohol precipitation can significantly reduce the loss of salvianolic acid B during alcohol precipitation. The water liquid after solid-liquid separation is first recovered and concentrated to about 2-3g crude drug / mL, and then the pH value is adjusted, or the pH value is first adjusted, and then recovered and concentrated to about 2-3g crude drug / mL, all are OK. The pH adjusting agent (acidification) used in step 3 is at least one of the acidic components such as hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, etc., especially the acidic components that can be allowed and accepted in pharmacy are acidified and adjusted.

[0029] Optionally, one of the preparation methods of the Salvia miltiorrhiza extract described in the present invention is: the Salvia miltiorrhiza that has been crushed to pass through a 10-mesh sieve is first soaked in ethanol with a volume content of 80% to swell, and then soaked in the same amount of ethanol for 3-5 hours, and then percolated at a rate of 3-4 mL / min·kg, after collecting 8-10 times the amount of percolated liquid, the ethanol is recovered under reduced pressure, diluted with water to 0.5-1.0 g / mL based on the crude drug, allowed to stand for precipitation, solid-liquid separation (the water after solid-liquid separation is reserved for later use), the precipitate is collected, micro-powder silica gel or calcium hydrogen phosphate equivalent to 0.5-1% of the weight of Salvia miltiorrhiza is added, crushed, extracted with 70%-95% ethanol, filtered, and obtained tanshinone ethanol solution. The aqueous solution after solid-liquid separation is adjusted to about pH 3, recovered and concentrated to about 2-3 g / mL based on the crude drug, 85%-95% ethanol is added under sufficient stirring to make the alcohol content in the liquid ≥70%, precipitation is performed to remove impurities, the supernatant is taken to recover ethanol, the above-mentioned tanshinone ethanol solution is added, ethanol is recovered, concentrated and dried to obtain a danshen extract containing tanshinone acid and tanshinone.

[0030] In the present invention, crude drug refers to Danshen crude drug. When the amount of crude drug is used as a reference object in the process of adjusting the concentration of liquid such as recovery and concentration, the Danshen used in step 1 of the above methods is the crude drug referred to in the present invention; recovery and concentration refers to recovering the solvent to concentrate the liquid, wherein when the solvent is an ethanol solution, recovery and concentration means recovering ethanol and concentrating. The alcohol precipitation described in the present invention refers to the conventional method operation according to the current version of the Chinese Pharmacopoeia, such as the pharmacopoeia stipulates that "add ethanol to make the alcohol content 70%, precipitate and remove impurities", which is also a conventional method in the art, generally adding the required ethanol under sufficient stirring and standing to precipitate. According to the conventional understanding of the art, 80±5% ethanol means that the volume fraction of ethanol in the ethanol aqueous solution is 75%-85%, and other similar expressions for ethanol are understood in the same way.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects: the method of the present invention is simple and feasible to operate, does not require special equipment, has high production efficiency and low cost, and is suitable for large-scale industrial production. The yield of salvianolic acid extract is about 12%, the content of salvianolic acid B is about 30%, and the extraction transfer rate is greater than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the process flow of CN104189073B;

[0033] Figure 2 This is the process flow of the present invention. DETAILED DESCRIPTION

[0034] The above contents of the present invention are further described in detail below through the specific implementation methods of the embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. Various substitutions or changes made according to common technical knowledge and customary means in the art without departing from the above technical concept of the present invention should be included in the scope of the present invention.

[0035] Example 1

[0036] Salvia powder (crushed and passed through a 10-mesh sieve) was soaked with an appropriate amount of 80% ethanol (volume content, the same below) according to the conventional percolation method, and placed in a sealed container for 3 hours to make the powder uniformly moistened and expanded, and then placed in a percolation cylinder, soaked in 80% ethanol for 4 hours, and then percolated at a rate of 3 mL / min·kg, and 10 times the amount of percolation liquid was collected, and the ethanol was recovered under reduced pressure, and diluted with water to 0.5 g / mL (based on the crude drug, the same below), and allowed to settle, filtered (the filtrate was reserved), and the precipitate was collected, and micro-powder silica gel equivalent to 0.2% of the weight of Salvia was added, crushed, dried, and extracted twice with 95% ethanol under reflux, filtered, and the filtrates were combined to obtain tanshinone ethanol solution. The tanshinone ethanol solution recovered the solvent, dissolved it with 80% ethanol (5 g crude drug / mL), allowed to settle, and the precipitate was collected and dried to obtain tanshinone extract. The above-mentioned spare filtrate is adjusted to pH 2.5 with phosphoric acid, recovered and concentrated to 2g crude drug / ml, and 3 times the volume of 95% ethanol (so that the alcohol content of the liquid is ≥70%) is added under full stirring, and it is allowed to stand overnight to precipitate and remove impurities, and the supernatant is collected to obtain a salvia miltiorrhiza acid ethanol liquid. Ethanol is recovered from the salvia miltiorrhiza acid ethanol liquid, concentrated and dried to obtain a salvia miltiorrhiza acid extract. The above-mentioned tanshinone extract and salvia miltiorrhiza acid extract are mixed and crushed, and mixed to obtain the salvia miltiorrhiza extract containing salvia miltiorrhiza acid and tanshinone.

[0037] Example 2

[0038] Salvia powder (crushed and passed through a 20-mesh sieve) was soaked with an appropriate amount of 75% ethanol according to the conventional percolation method, and placed in a sealed container for 2 hours to allow the powder to be evenly moistened and expanded. It was then placed in a percolation cylinder, soaked in 75% ethanol for 5 hours, and then percolated at a rate of 4 mL / min·kg. Eight times the amount of percolation liquid was collected, and the ethanol was recovered under reduced pressure. The percolation liquid was diluted with water to 0.75 g of crude drug / mL, and allowed to stand for precipitation. The filtrate was filtered (the filtrate was reserved), and the precipitate was collected. Calcium hydrogen phosphate equivalent to 1% of the weight of Salvia was added, crushed, and extracted twice with 70% ethanol under reflux, filtered, and the filtrates were combined to obtain tanshinone ethanol solution. The tanshinone ethanol solution recovered the solvent, dissolved it with 95% ethanol (10 g of crude drug / mL), allowed to stand for precipitation, and the precipitate was collected and dried to obtain tanshinone extract. The above-mentioned spare filtrate is recovered and concentrated to about 2g crude drug / ml, and the pH is adjusted to 1.5 with hydrochloric acid. 4 times the volume of 95% ethanol (so that the alcohol content of the liquid is ≥70%) is added under full stirring, and it is allowed to stand overnight to precipitate and remove impurities, and the supernatant is collected to obtain a salvia miltiorrhiza acid ethanol liquid. Ethanol is recovered from the salvia miltiorrhiza acid ethanol liquid, concentrated and dried to obtain a salvia miltiorrhiza acid extract. The above-mentioned tanshinone extract and salvia miltiorrhiza acid extract are mixed and crushed, and mixed to obtain the salvia miltiorrhiza extract containing salvia miltiorrhiza acid and tanshinone.

[0039] Example 3

[0040] Salvia powder (crushed and passed through a 40-mesh sieve) was soaked with an appropriate amount of 85% ethanol according to the conventional percolation method, and placed in a sealed container for 4 hours to allow the powder to be evenly moistened and expanded. It was then placed in a percolation cylinder, soaked in 85% ethanol for 3 hours, and percolated at a rate of 2 mL / min·kg. Six times the amount of percolation liquid was collected, and the ethanol was recovered under reduced pressure. It was diluted with water to 1 g of crude drug / mL, allowed to stand for precipitation, centrifuged (the centrifuge liquid was reserved), the precipitate was collected, and microcrystalline cellulose equivalent to 2% of the weight of Salvia was added, crushed, and extracted twice with 80% ethanol under reflux, filtered, and the filtrates were combined to obtain tanshinone ethanol solution. The tanshinone ethanol solution recovered the solvent, dissolved it with 70% ethanol (3 g of crude drug / mL), allowed to stand for precipitation, the precipitate was collected, and dried to obtain tanshinone extract. The above-mentioned standby centrifuge liquid is adjusted to pH 3.5 with citric acid, recovered and concentrated to about 3g crude drug / ml, and 4 times the volume of 80% ethanol (so that the alcohol content in the liquid is ≥70%) is added under full stirring, and the liquid is allowed to stand overnight to precipitate and remove impurities, and the supernatant is collected to obtain a salvia miltiorrhiza acid ethanol liquid. Ethanol is recovered from the salvia miltiorrhiza acid ethanol liquid, and the salvia miltiorrhiza acid extract is obtained by concentration and drying. The above-mentioned tanshinone extract and salvia miltiorrhiza acid extract are mixed and crushed, and mixed to obtain the salvia miltiorrhiza extract containing salvia miltiorrhiza acid and tanshinone.

[0041] Example 4

[0042] Salvia powder (crushed and passed through a 10-mesh sieve) is routinely operated by the percolation method. It is first soaked with an appropriate amount of 80% ethanol, placed in a sealed container for 2 hours to allow the powder to be evenly moistened and expanded, and then placed in a percolation cylinder. After being soaked in 80% ethanol for 5 hours, the percolation is performed at a rate of 3.5 mL / min·kg, and 10 times the amount of percolation liquid is collected. The ethanol is recovered under reduced pressure, diluted with water to 1 g / mL, allowed to stand for precipitation, centrifuged (the centrifuge liquid is reserved), the precipitate is collected, micro-powder silica gel equivalent to 0.5% of the weight of Salvia is added, crushed, and extracted twice with 85% ethanol under stirring, centrifuged, and the centrifuge liquids are combined to obtain tanshinone ethanol liquid. The above-mentioned reserved centrifuge liquid is adjusted to pH 3 with hydrochloric acid, recovered and concentrated to 2.5 g crude drug / ml, and 4 times the volume of 90% ethanol (alcohol content ≥ 70%) is added under sufficient stirring, and allowed to stand overnight to precipitate and remove impurities, and the supernatant is collected to obtain tanshinone acid ethanol liquid. The ethanol solution of salvianolic acid is recovered and concentrated, and the above-mentioned ethanol solution of tanshinone is added, mixed evenly, ethanol is recovered, concentrated and dried to obtain a danshen extract containing salvianolic acid and tanshinone.

[0043] Example 5

[0044] Salvia powder (crushed and passed through a 10-mesh sieve) is soaked with an appropriate amount of 80% ethanol according to the conventional percolation method, and placed in a sealed container for 4 hours to allow the powder to be evenly moistened and expanded, and then placed in a percolation cylinder, soaked in 80% ethanol for 3 hours, and percolated at a rate of 4 mL / min·kg, and 8 times the amount of percolation liquid is collected, and the ethanol is recovered under reduced pressure, diluted with water to 0.5 g of crude drug / mL, and allowed to stand for precipitation, filtered (the filtrate is reserved), and the precipitate is collected, and light magnesium oxide equivalent to 1% of the weight of Salvia is added, crushed, and ultrasonically extracted with 95% ethanol twice, filtered, and the filtrates are combined to obtain tanshinone ethanol solution. The above-mentioned reserved filtrate is recovered and concentrated to 2.5 g of crude drug / ml, and the pH is adjusted to 2 with phosphoric acid, and 3.5 times the volume of 90% ethanol (alcohol content ≥ 70%) is added under sufficient stirring, and allowed to stand overnight to precipitate and remove impurities, and the supernatant is collected to obtain tanshinone acid ethanol solution. The ethanol solution of salvianolic acid is recovered and concentrated, and the above-mentioned ethanol solution of tanshinone is added, mixed evenly, ethanol is recovered, concentrated and dried to obtain a danshen extract containing salvianolic acid and tanshinone.

[0045] Example 6

[0046] Salvia powder (crushed and passed through a 10-mesh sieve) is soaked with an appropriate amount of 80% ethanol according to the conventional percolation method, and placed in a sealed container for 3 hours to allow the powder to be evenly moistened and expanded, and then placed in a percolation cylinder, soaked in 80% ethanol for 4 hours, and percolated at a rate of 3 mL / min·kg, and 10 times the amount of percolation liquid is collected, and the ethanol is recovered under reduced pressure, diluted with water to 0.75 g of crude drug / mL, and allowed to stand for precipitation, filtered (the filtrate is reserved), and the precipitate is collected, and starch equivalent to 2% of the weight of Salvia is added, crushed, and extracted twice with 70% ethanol under reflux, filtered, and the filtrates are combined to obtain tanshinone ethanol solution. The above-mentioned reserved filtrate is recovered and concentrated to about 3 g of crude drug / ml, and the pH is adjusted to 2.5 with citric acid, and 4 times the volume of 85% ethanol (alcohol content ≥ 70%) is added under full stirring, and allowed to stand overnight to precipitate and remove impurities, and the supernatant is collected to obtain tanshinone acid ethanol solution. The ethanol solution of salvianolic acid is recovered and concentrated, and the above-mentioned ethanol solution of tanshinone is added, mixed evenly, ethanol is recovered, concentrated and dried to obtain a danshen extract containing salvianolic acid and tanshinone.

[0047] The following relevant test results further prove the remarkable effect of the method of the present invention.

[0048] Experiment 1: Effect of “acidification and alcohol precipitation” on the extraction transfer rate of salvianolic acid B

[0049] 1000g of Danshen powder (crushed and passed through a 10-mesh sieve) was soaked with an appropriate amount of 80% ethanol according to the conventional percolation method, and placed in a sealed container for 4 hours to allow the powder to be evenly moistened and expanded. It was then placed in a percolation cylinder, soaked in 80% ethanol for 5 hours, and then percolated at a rate of 3mL / min·kg. 10 times the amount of percolation liquid was collected, the ethanol was recovered under reduced pressure, and water was added to dilute to 0.5g crude drug / mL, and the solution was allowed to settle and centrifuged. The water solution after centrifugation was divided into three equal parts for use (the pH value of the water solution after centrifugation was 4.3).

[0050] The first portion of aqueous solution was directly recovered and concentrated to 2 g crude drug / ml, and 4 times the volume of 95% ethanol was added under sufficient stirring. The mixture was allowed to stand overnight to precipitate and remove impurities. The supernatant was collected to obtain salvianolic acid ethanol solution, which was recovered and concentrated, and dried under reduced pressure to obtain salvianolic acid extract (A).

[0051] The second portion of aqueous solution was directly recovered and concentrated to 2 g crude drug / ml, and the pH was adjusted to 2 with phosphoric acid. 4 times the volume of 95% ethanol was added under sufficient stirring, and the mixture was allowed to stand overnight to precipitate and remove impurities. The supernatant was collected to obtain salvianolic acid ethanol solution, which was recovered and concentrated, and dried under reduced pressure to obtain salvianolic acid extract (B).

[0052] The third portion of aqueous solution was adjusted to pH 3 with phosphoric acid, recovered and concentrated to 2 g crude drug / ml, and 4 times the volume of 95% ethanol was added under sufficient stirring. The mixture was allowed to stand overnight to precipitate and remove impurities. The supernatant was collected to obtain salvianolic acid ethanol solution, which was recovered and concentrated, and dried under reduced pressure to obtain salvianolic acid extract (C).

[0053] The above-mentioned salvianolic acid extracts A, B, and C were weighed, and the content of salvianolic acid B was determined by HPLC according to the method under the item of determination of the content of Danshen medicinal materials in the 2010 edition of the Chinese Pharmacopoeia, the extraction transfer rate was calculated, and the content of total phenolic acids in salvianolic acid was determined by ultraviolet spectrophotometry. The results are shown in Table 1.

[0054] Table 1 Yield, content and extraction transfer rate of salvianolic acid extract

[0055] Salvianolic acid extract A B C Yield / % 10.2 12.5 12.4 Salvianolic acid B content / % 24.2 29.8 30.1 Transfer rate of salvianolic acid B / % 58.3 87.8 88.5 Total phenolic acid content / % 33.1 43.6 44.2

[0056] As can be seen from Table 1, the salvianolic acid extract A is a solution directly concentrated and precipitated with alcohol after solid-liquid separation, with low yield, low content of the main active ingredient salvianolic acid B, low transfer rate and large loss; the salvianolic acid extract B is a solution directly concentrated and acidified and precipitated with alcohol after solid-liquid separation, with high yield, high content of the main active ingredient salvianolic acid B, high transfer rate and small loss; the salvianolic acid extract C is a solution first acidified and then concentrated and precipitated with alcohol after solid-liquid separation, with high yield, high content of the main active ingredient salvianolic acid B, high transfer rate and small loss. The above experimental phenomena show that the precipitates after alcohol precipitation of the first, second and third parts of the aqueous solution are dark brown, light yellow and light yellow respectively, indicating that acidification changes the physical and chemical properties of the precipitate, which may reduce the adsorption and / or encapsulation of the precipitate on salvianolic acid B (reduces the loss), thereby improving the yield, content and extraction transfer rate of the salvianolic acid extract. It can be seen that the "acidification and alcohol precipitation" method of the present invention can significantly reduce the loss of salvianolic acid B, increase the yield and content of the extract, and produce unexpected effects.

[0057] Experiment 2: Effect of “water-precipitated alcohol extraction” on the content of tanshinone extract

[0058] 1000 g of Danshen powder (crushed and passed through a 10-mesh sieve) was soaked with an appropriate amount of 80% ethanol according to the conventional percolation method, and placed in a sealed container for 4 hours to allow the powder to be evenly moistened and expanded. The powder was then placed in a percolation cylinder, soaked in 80% ethanol for 5 hours, and then percolated at a rate of 3 mL / min·kg. Ten times the amount of percolation liquid was collected, and the ethanol was recovered under reduced pressure. The percolation liquid was diluted with water to 0.5 g crude drug / mL, and allowed to settle ("water sedimentation"). The percolation was centrifuged and the precipitate was collected. Among them, 1 / 10 of the precipitate was dried under reduced pressure and used as a standby (tanshinone extract A); the other 9 / 10 of the precipitate was added with micro-powder silica gel equivalent to 0.5% of the weight of Danshen, crushed, and divided into 3 equal parts (equivalent to 300 g Danshen), and the following operations were performed:

[0059] The first portion was extracted by heating with 70% ethanol under reflux, filtered to obtain tanshinone ethanol solution, the solvent was recovered under reduced pressure, dissolved by heating with 100 ml of 70% ethanol, allowed to stand for precipitation, filtered, the precipitate was collected, and dried to obtain tanshinone extract B.

[0060] The second portion was extracted by heating with 80% ethanol under reflux, filtered to obtain tanshinone ethanol solution, the solvent was recovered under reduced pressure, dissolved by heating with 60 ml of 80% ethanol, allowed to stand for precipitation, filtered, the precipitate was collected, and dried to obtain tanshinone extract C.

[0061] The third portion was extracted by heating with 95% ethanol under reflux, filtered to obtain tanshinone ethanol solution, the solvent was recovered under reduced pressure, dissolved by heating with 30 ml of 95% ethanol, allowed to stand for precipitation, filtered, the precipitate was collected, and dried to obtain tanshinone extract D.

[0062] According to the method under the item of determination of the content of Danshen medicinal materials in the 2010 edition of the Chinese Pharmacopoeia, the content of tanshinone ⅡA in the above tanshinone extracts A to D was determined by HPLC, and the contents were 11.8%, 20.2%, 22.9%, and 23.7%, respectively; the content of total tanshinone was determined by ultraviolet spectrophotometry, and the contents were 29.1%, 52.6%, 55.8%, and 57.3%, respectively. It can be seen that the addition of a solid dispersant during the "water-precipitated alcohol extraction" method of the present invention can significantly increase the content of tanshinone ⅡA and total tanshinone in the tanshinone extract.

[0063] In summary, the present invention provides a method for simultaneously extracting tanshinone and salvianolic acid by one-step percolation and alcohol extraction. On this basis, the present invention further provides a purification method for obtaining salvianolic acid by "acidification and alcohol precipitation", and a purification method for obtaining tanshinone by "water precipitation and alcohol extraction". Finally, a new method for preparing a danshen extract containing salvianolic acid and tanshinone is provided. The method is simple and feasible to operate, does not require special equipment such as macroporous resin columns and organic solvent extraction, and adopts conventional extraction, recovery, precipitation and other methods. It has high production efficiency and low cost, is suitable for large-scale industrial production, and has broad application prospects.

Claims

1. A method for preparing a salvia miltiorrhiza extract, characterized in that The steps include: Step 1, extracting the crushed Salvia miltiorrhiza by percolation with an ethanol solution, and collecting the percolation liquid; Step 2, recovering ethanol from the percolate, precipitating and performing solid-liquid separation to obtain a water solution and a precipitate, first adding a solid dispersant to the precipitate, crushing it, then extracting it with 70%-95% ethanol by volume, filtering it, and obtaining a tanshinone ethanol solution; Step 3, recovering and concentrating the aqueous solution after solid-liquid separation, adjusting the pH value to be greater than 1 and less than 3 under sufficient stirring, removing impurities by alcohol precipitation, and collecting the supernatant to obtain salvianolic acid ethanol solution; or first adjusting the pH value of the aqueous solution to be greater than 2 and less than 4, and then recovering and concentrating, removing impurities by alcohol precipitation, and collecting the supernatant to obtain salvianolic acid ethanol solution; Step 4, recovering and concentrating the ethanol solution of salvianolic acid, adding the above-mentioned ethanol solution of tanshinone, recovering ethanol, concentrating and drying to obtain a danshen extract containing salvianolic acid and tanshinone; Alternatively, the tanshinone ethanol solution is used to prepare the tanshinone extract, and the tanshinone acid ethanol solution is used to prepare the tanshinone acid extract, and the tanshinone extract and the tanshinone acid extract are mixed, crushed, and mixed to obtain the danshen extract.

2. The method for preparing the Danshen extract according to claim 1, characterized in that The solid dispersant is selected from at least one of micro-powder silica gel, calcium hydrogen phosphate, light magnesium oxide, diatomaceous earth, starch, and microcrystalline cellulose; the amount of the solid dispersant is equivalent to 0.2%-2% of the weight of Salvia miltiorrhiza.

3. The method for preparing the Danshen extract according to claim 1, characterized in that The acid used to adjust the pH value is at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid and other acidic components.

4. The method for preparing the Danshen extract according to claim 1, characterized in that The alcohol precipitation and impurity removal described in step 3 refers to adding ethanol under stirring to make the alcohol content in the liquid ≥ 70%, and precipitating and removing impurities.

5. The method for preparing the Danshen extract according to claim 1, characterized in that The ethanol solution used in the percolation extraction in step 1 is an ethanol aqueous solution with a volume content of 75%-85%.

6. The method for preparing the Danshen extract according to claim 1, characterized in that The method of recovering ethanol from the percolation liquid and then performing precipitation in step 2 is: after recovering ethanol from the percolation liquid, water is added to adjust the concentration to 0.5-1g crude drug / mL, and the concentration is allowed to stand for precipitation; the method of preparing tanshinone extract from tanshinone ethanol solution in step 4 is: recovering the solvent from tanshinone ethanol solution, and then dissolving it with 70%-95% ethanol to 3-10g crude drug / mL, standing for precipitation, collecting the precipitate, and drying to obtain tanshinone extract; the method of preparing tanshinone acid extract from tanshinone acid ethanol solution in step 4 is: recovering ethanol from tanshinone acid ethanol solution, concentrating and drying to obtain tanshinone acid extract.

7. The method for preparing the Danshen extract according to claim 1, characterized in that The recovery and concentration described in step 3 refers to recovering and concentrating the aqueous solution to 2-3 g crude drug / mL.

8. The method for preparing the Danshen extract according to claim 1, characterized in that The volume-to-weight ratio of the percolate collected in step 1 to the salvia miltiorrhiza is 6 to 10 times.

9. A method for preparing tanshinone, characterized in that: The steps are as follows: Step 1, extracting the crushed Salvia miltiorrhiza by percolation with 75%-85% ethanol by volume, and collecting the percolation liquid; Step 2, recovering ethanol from the percolation liquid, precipitating and performing solid-liquid separation to obtain a precipitate, first adding at least one of micro-powdered silica gel, calcium hydrogen phosphate, light magnesium oxide, diatomaceous earth, starch, and microcrystalline cellulose equivalent to 0.2%-2% of the weight of Danshen, and pulverizing, then extracting with 70%-95% ethanol, filtering, and obtaining tanshinone ethanol solution; recovering the solvent from the tanshinone ethanol solution, and then dissolving it with 70%-95% ethanol, precipitating and performing solid-liquid separation, collecting the precipitate, and drying to obtain a tanshinone extract.

10. A method for preparing salvianolic acid, characterized in that: The steps are as follows: Step 1, extracting the crushed Salvia miltiorrhiza by percolation with 75%-85% ethanol by volume, and collecting the percolation liquid; Step 2, recovering ethanol from the percolate, precipitating and separating the solid and liquid, and collecting the water for later use; Step 3, recovering and concentrating the aqueous solution, adjusting the pH value to be greater than 1 and less than 3 under sufficient stirring, adding ethanol to make the alcohol content in the liquid ≥70%, precipitating and removing impurities, and collecting the supernatant to obtain the salvianolic acid ethanol solution; or first adjusting the pH value of the aqueous solution to be greater than 2 and less than 4, then recovering and concentrating, adding ethanol to make the alcohol content in the liquid ≥70% under sufficient stirring, precipitating and removing impurities, and collecting the supernatant to obtain the salvianolic acid ethanol solution; Step 4, recovering ethanol from the salvianolic acid ethanol solution, concentrating and drying to obtain a salvianolic acid extract.

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