Method for preparing tanshinol

Abstract

The invention provides a method for preparing tanshinol, and relates to the technical field of plant extraction. The method comprises the following steps: carrying out hydrolysis reaction on a raw material containing rosmarinic acid in an acidic alcohol solution to obtain a hydrolysis solution which only contains tanshinol and does not contain caffeic acid, and sequentially extracting and purifying the hydrolysis solution to obtain the high-content tanshinol. According to the preparation method of the tanshinol provided by the invention, firstly, the rosemary extract is used as a raw material and is easy to obtain and low in cost; secondly, the method has low requirements on the content of rosmarinic acid in the raw materials, and the tanshinol with the content of 90% or above can be finally prepared through a simple purification process; thirdly, almost 100% conversion of the rosmarinic acid can be realized; the whole process route is simple, easy to operate, high in reaction efficiency, environment-friendly and suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to a method for preparing tanshinone. Background Technology

[0002] Tanshinone, CAS number 76822-21-4, chemical formula C9H 10 O5 is one of the main water-soluble active components of the traditional Chinese medicine Danshen. As an acidic phenolic compound, tanshinone is a white crystal at room temperature, with good water solubility but slightly soluble in organic solvents. Modern pharmacological studies have shown that tanshinone has a variety of significant pharmacological activities, including antioxidant, anti-inflammatory, anti-platelet aggregation, and coronary artery dilation, thus showing broad application prospects in the treatment of cardiovascular and cerebrovascular diseases, kidney diseases, and other related conditions.

[0003]

[0004] The stable and efficient supply of tanshinone has always been a bottleneck restricting its further research and development and clinical application. Currently, the industrial preparation of tanshinone mainly involves the following methods: plant extraction, chemical synthesis, microbial fermentation, and enzymatic conversion. Each of these four methods has its own limitations in the preparation of tanshinone.

[0005] For example, the plant extraction method is a traditional method for directly extracting tanshinone from the Salvia miltiorrhiza plant. The prior art of Chinese patent document number CN114907184B and Chinese patent document number CN105541602A both disclose methods for extracting tanshinone from Salvia miltiorrhiza. However, this method is limited by the extremely low content of tanshinone in the plant, resulting in low extraction efficiency, high cost, and is affected by factors such as medicinal resources, place of origin, and season, making it difficult to meet large-scale clinical needs.

[0006] Chemical synthesis methods, such as the prior art disclosed in Chinese Patent Document No. CN102863328B which uses D-tyrosine as a starting material to chemically synthesize tanshinone, although not dependent on plant resources, usually face problems such as low yield, demanding reaction conditions such as the need for expensive catalysts, and the product being a racemic mixture. If chiral synthesis is chosen, the cost will also increase significantly. In addition, chemical synthesis methods often use toxic and harmful reagents, which does not meet the requirements of green chemistry and sustainable development.

[0007] The prior art disclosed in Chinese Patent Document No. CN104003866B, a method for extracting tanshinone using ultrafine combined solid-state fermentation, i.e., microbial fermentation, suffers from drawbacks such as excessively long fermentation cycles and low production intensity. During the lengthy fermentation process, the tanshinone product is easily oxidized by oxygen in the culture medium, resulting in poor stability. Furthermore, the fermentation broth produces numerous byproducts, significantly increasing the difficulty and cost of subsequent separation and purification. While enzymatic conversion methods proposed in recent years, such as the enzymatic synthesis of tanshinone disclosed in Chinese Patent Document No. CN108424937B, offer the advantage of reaction specificity, they generally suffer from insufficient production intensity, hindering efficiency improvements and limiting the industrial-scale application of tanshinone.

[0008] Although there are many existing methods for preparing tanshinone, each has its own limitations, resulting in the high price of tanshinone, especially high-purity tanshinone.

[0009] The comparative fingerprint spectrum of the Danshen (Salvia miltiorrhiza) formula granules in the national drug standard YBZ-PFKL-2021035 issued by the National Medical Products Administration and reviewed by the National Pharmacopoeia Commission shows that the content of rosmarinic acid in Danshen extract is almost at the same level as that of tanshinone. Figure 4 As shown, peak 1 is tanshinone, peak 2 is protocatechuic aldehyde, peak 3 is caffeic acid, peak 4 is salvianolic acid E, peak 5 is rosmarinic acid, peak 6 is shikonin, peak 7 (S) is salvianolic acid B, and peak 8 is salvianolic acid L. In the process of extracting tanshinone from tanshinone, other components such as rosmarinic acid, protocatechuic acid, and caffeic acid are often separated and sold separately. Rosmarinic acid, as a byproduct of various plant processing methods, has relatively abundant sources; besides being found in tanshinone, it is also abundant (approximately 3%–8%) in rosemary. Rosmarinic acid is also a natural phenolic acid compound. As the main product of the water-soluble extraction route of rosemary, rosmarinic acid has been commercialized on a large scale in health foods, beverages, and cosmetics due to its natural antioxidant, anti-inflammatory, and antibacterial effects.

[0010] However, the price of rosmarinic acid, whether isolated from tanshinone or rosemary, is significantly lower than that of tanshinone. For example, some reagent companies sell 98% pure tanshinone (sodium) for 2500-4000 yuan per 10mg, while 98% pure rosmarinic acid costs only 400-500 yuan per 10mg, a price difference of about 6-8 times. Low-purity rosmarinic acid is even cheaper and is often sold directly as rosemary extract. For instance, rosemary extract with a rosmarinic acid content of around 20% obtained during the rosemary extraction process is generally used directly in beverages, braised products, and pet food as an additive with antioxidant, anti-inflammatory, and antibacterial effects, costing only about 100 yuan per kilogram.

[0011] From a molecular structure perspective, rosmarinic acid is composed of one molecule of tanshinone (R configuration) and one molecule of caffeic acid linked by an ester bond; rosmarinic acid, CAS number 20283-92-5, has the following structural formula:

[0012] Theoretically, hydrolyzing rosmarinic acid under acidic conditions can efficiently and specifically yield one molecule of tanshinone (R configuration) and one molecule of caffeic acid. However, no research or patent reports have been found on a technical route for preparing tanshinone using rosmarinic acid as a starting material through chemical hydrolysis, which provides a potential possibility for developing a novel and efficient tanshinone preparation process. Summary of the Invention

[0013] 1. The problem to be solved Based on the above-mentioned ideas, this invention addresses the technical problems of high cost and difficulty in industrial application of existing methods for preparing tanshinone using plant extraction, chemical synthesis, microbial fermentation, and enzymatic conversion. It proposes a method for preparing tanshinone; this method obtains tanshinone with a content of up to 90% through a one-step hydrolysis process. Compared with existing technologies, this method has a wide range of raw material sources, simple reaction process and conditions, and a short preparation cycle. It not only has high production efficiency and is suitable for industrial application, but also realizes the conversion of the low-value product rosmarinic acid into the high-value product tanshinone.

[0014] 2. Technical Solution The technical solution adopted in this invention is as follows: This invention proposes a method for preparing tanshinone, which involves hydrolyzing a raw material containing rosmarinic acid in an acidic alcohol solution to obtain tanshinone.

[0015]

[0016] Preferably, the rosmarinic acid content in the raw material containing rosmarinic acid is 10% to 100%, more preferably 20% to 90%.

[0017] As a preferred technical solution, the raw material containing rosmarinic acid can be rosmarinic acid of high purity, such as rosmarinic acid with a content of 90% to 100%.

[0018] As a preferred technical solution, the raw material containing rosmarinic acid can be medium to high purity rosmarinic acid, such as rosmarinic acid with a content of 60% to 90%.

[0019] As a preferred technical solution, the raw material containing rosmarinic acid can be rosmarinic acid of medium purity, such as rosmarinic acid with a content of 50% to 60%.

[0020] As a preferred technical solution, the raw material containing rosmarinic acid can be rosmarinic acid of medium to low purity, such as rosmarinic acid with a content of 10% to 50%. More preferably, the raw material containing rosmarinic acid is rosmarinic acid with a purity of 20% to 50%.

[0021] Particularly preferred is that the rosmarinic acid content of 10%~50% or 20%~50% is rosemary extract.

[0022] More preferably, the rosemary extract is an extract obtained by using a eutectic solvent extraction method or an ethanol-macroporous resin combined method.

[0023] Preferably, the reaction conditions for hydrolysis in the acidic alcohol solution are: hydrolysis temperature of 50~100℃ and reaction time of 2~8h.

[0024] It is particularly important to note that when rosemary extract with a rosmarinic acid content of 10%–90% is used as a reaction raw material, under the aforementioned hydrolysis conditions, the hydrolysis product contains almost no caffeic acid; simultaneously, the conversion rate of rosmarinic acid reaches nearly 100%. Rosmarinic acid is hydrolyzed in an acidic alcoholic solution, with the acid catalyzing the ester hydrolysis reaction to equilibrium. Because caffeic acid is consumed during ester hydrolysis, the hydrolysis reaction proceeds in the forward direction. Ultimately, all rosmarinic acid is hydrolyzed, leaving no rosmarinic acid residue in the hydrolysis product, further promoting the conversion of the low-value product rosmarinic acid into the high-value product tanshinone.

[0025] Furthermore, the acid in the acidic alcohol solution is selected from hydrochloric acid, sulfuric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, p-toluenesulfonic acid, trifluoroacetic acid, formic acid, and acetic acid, with a mass percentage of 5% to 15%, and the alcohol is selected from methanol, ethanol, isopropanol, benzyl alcohol, allyl alcohol, and trifluoroethanol; preferably, the acid in the acidic alcohol solution is selected from hydrochloric acid with a mass percentage of 5% to 15%, and the alcohol is selected from ethanol; ethanol mainly serves as a solvent, which can effectively dissolve the water-insoluble components of the raw material containing rosmarinic acid, so the amount of ethanol added is at least enough to completely dissolve the raw material.

[0026] The method for preparing tanshinone according to the present invention specifically includes the following steps: The process involves hydrolyzing a raw material containing rosmarinic acid in an acidic alcoholic solution. The steps for adjusting the pH of the solution after hydrolysis; The steps for extracting and separating organic phases containing tanshinone; The steps for concentrating and purifying tanshinone.

[0027] Furthermore, when the raw material containing rosmarinic acid undergoes hydrolysis in an acidic alcohol solution, the ratio of the raw material containing rosmarinic acid to the acidic alcohol solution is 1 g : (1~5) mL; preferably, the ratio of the raw material containing rosmarinic acid to the acidic alcohol solution is 1 g : (1~3) mL.

[0028] Furthermore, the pH of the hydrolyzed solution is adjusted to be 2-6.

[0029] Preferably, the process of controlling the pH of the hydrolysis solution before extraction is as follows: adjusting the pH of the hydrolysis solution with an alkali, wherein the mass percentage of the alkali is 20% to 50%; preferably, sodium hydroxide is used to adjust the pH of the hydrolysis solution to 3 to 5; optionally, pH affects the extraction efficiency of tanshinone. When the pH of the hydrolysis solution is too high, tanshinone, which is an acidic phenolic acid compound, will undergo a neutralization reaction and be lost. At the same time, ethyl acetate, which is used as the extraction solvent, will also undergo hydrolysis in an alkaline environment, reducing the yield of tanshinone.

[0030] Furthermore, the step of extracting and separating the organic phase containing tanshinone is as follows: extraction is carried out in two phases of water and ethyl acetate, with a material-to-liquid ratio of (1~2):(1~5), and the number of extractions is 1~5 times; the above material-to-liquid ratio represents the volume ratio of the material to be extracted to the extraction solvent. Preferably, the material-to-liquid ratio is 2:1~1:3. During concentration, the organic phase layer after extraction and settling is taken for concentration to obtain crude tanshinone; optionally, the extraction solvent can also be selected from n-butanol; the solubility of tanshinone and impurities in the hydrolysis product differs greatly in water and ethyl acetate. The reason for choosing ethyl acetate as the extraction solvent is to remove water-soluble impurities such as sugars and flavonoids in the hydrolysis solution and reduce the loss of the target product tanshinone.

[0031] Furthermore, the step of concentrating and purifying tanshinone is as follows: the organic phase containing tanshinone is concentrated to obtain crude tanshinone, and the crude tanshinone is then subjected to resin purification and washing to obtain tanshinone with a content of not less than 90%.

[0032] Preferably, the organic solvent used for washing is selected from one or more of methanol, ethanol, ethyl acetate, and n-butanol.

[0033] Preferably, the feed-to-liquid ratio of the column liquid in the resin purification process is 1:(5~20), the eluent used is an alcohol solution with a volume percentage of 0~50%, and the eluent volume is 1~4 BV; preferably, the eluent is methanol or ethanol, preferably ethanol.

[0034] 3. Beneficial effects Compared with existing technologies, the method for preparing tanshinone from rosmarinic acid provided by this invention has the following significant advantages: (1) The method for preparing tanshinone disclosed in this invention involves hydrolyzing a raw material containing rosmarinic acid in an acidic alcohol solution at a temperature of 50-100°C for 2-8 hours to obtain tanshinone in one step. The method abandons the traditional approach of extracting tanshinone from salvia miltiorrhiza in very low amounts, and instead uses a processed product containing rosmarinic acid as the starting material. This raw material is abundant, readily available, and much cheaper than salvia miltiorrhiza, thus fundamentally solving the bottleneck problem of scarce raw materials and high costs in plant extraction methods. The reaction process and reaction conditions of this method are simple, which not only realizes the transformation of low-value products into high-value products, but also lays a solid foundation for the large-scale industrial production of tanshinone.

[0035] (2) The method for preparing tanshinone disclosed in this invention is based on the direct hydrolysis reaction of rosmarinic acid under acidic conditions to fully hydrolyze rosmarinic acid with a content of 10% to 100%. The hydrolysis product contains almost no caffeic acid and only tanshinone is obtained. The conversion rate of rosmarinic acid is almost 100%. The reaction steps are simple, the reaction conditions are mild and easy to control, avoiding the complex multi-step reaction, expensive catalysts and harsh reaction conditions in chemical synthesis. At the same time, it avoids the problems of long cycle and complex process control in microbial fermentation, which greatly simplifies the entire production process of tanshinone, makes it highly operable, and is easy to scale up and promote.

[0036] (3) The method for preparing tanshinone disclosed in this invention theoretically requires the hydrolysis of rosmarinic acid to obtain an equal amount of caffeic acid. However, the reaction conditions disclosed in this invention can efficiently and specifically obtain only a single configuration of tanshinone. The crude tanshinone prepared can be purified to obtain a tanshinone product with a content of more than 90%, resulting in high yield. Furthermore, compared with the chemical synthesis method for obtaining racemic tanshinone, this method produces a single configuration of tanshinone due to the structural characteristics of rosmarinic acid, and the purification process is simple. Compared with the prior art, the method of this invention effectively solves the problems of low yield and low purity of chemical synthesis method and the problems of many by-products and easy oxidation and degradation of products in microbial method, which lead to unstable yield and quality.

[0037] (4) The method for preparing tanshinone disclosed in this invention has a fast reaction rate and short cycle in the rosmarinic acid hydrolysis process. Compared with the fermentation process of microbial fermentation that takes several days and the low production intensity of enzyme conversion method, the production efficiency and production intensity of tanshinone per unit time are greatly improved, which can meet the huge clinical demand for tanshinone in the market.

[0038] (5) The method for preparing tanshinone disclosed in this invention does not use toxic reagents and expensive metal catalysts. Its process route is clean, and most of the organic solvents used can be recycled, effectively reducing environmental pollution and health hazards to operators during the production process, which meets the requirements of sustainable development. That is, through ingenious raw material selection and process design, a new route for preparing tanshinone with low raw material cost, simple process, high product quality, high efficiency and environmental friendliness is provided, which comprehensively solves the core defects of various existing preparation methods and has huge industrial production potential and market application prospects. Attached Figure Description

[0039] Figure 1 This is a high-performance liquid chromatogram of the tanshinone product obtained after purification in Example 1 of the present invention; Figure 2 This is a high-performance liquid chromatogram of the hydrolysis solution after 4 hours of hydrolysis reaction in Example 1 of the present invention; Figure 3 This is a high-performance liquid chromatogram of the hydrolysis solution after 8 hours of hydrolysis reaction in Example 1 of the present invention; Figure 4 This is a reference fingerprint for the Danshen formula granules in the national drug standard YBZ-PFKL-2021035. Detailed Implementation

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0041] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0042] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0043] Molecular weights and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as “less than about 4.5,” which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0044] Current methods for preparing tanshinone, such as plant extraction, suffer from low yield and high cost; chemical synthesis is environmentally unfriendly; and microbial fermentation and enzymatic conversion methods have low production intensity. These issues limit the industrial-scale application of tanshinone. This invention aims to address these problems by proposing a method for preparing tanshinone using rosmarinic acid as a raw material, which enables the industrial-scale application of tanshinone. The process route involves only one hydrolysis reaction, resulting in low reaction cost, environmental friendliness, and suitability for industrial production.

[0045] Rosmarinic acid, chemical formula C 18 H 16 O8, with a molecular weight of 360.315 and CAS number 20283-92-5, is a natural phenolic acid compound formed by the condensation of caffeic acid and tanshinone. It was initially isolated from the medicinal plant rosemary and named accordingly, but it is widely found in various plants belonging to the Lamiaceae, Boraginaceae, Cucurbitaceae, Tiliaceae, and Apiaceae families. Rosmarinic acid is a white to brown solid with a melting point of 171–175°C and a density of 1.5 ± 0.1 g / cm³. It is hygroscopic at room temperature, highly water-soluble, and requires storage away from light and contact with calcium and magnesium ions. Rosmarinic acid is widely used in pharmaceuticals, food preservation, and cosmetic antioxidants. Currently, microbial fermentation has achieved an industrial-scale production of 5780.6 mg / L.

[0046] Theoretically, hydrolysis of one molecule of rosmarinic acid yields one molecule of caffeic acid and one molecule of tanshinone. Therefore, the preparation of tanshinone by hydrolysis of rosmarinic acid does not suffer from a lack of raw materials, as any source of rosmarinic acid can be used as a source for tanshinone preparation. Secondly, based on the molecular structure of rosmarinic acid, the tanshinone obtained by its hydrolysis is a single R-configuration tanshinone. Compared with the chemical synthesis of racemic tanshinone products, this not only reduces the complex reaction steps to a one-step hydrolysis, but also eliminates the need for chiral purification. Therefore, high-content tanshinone can be obtained through simple process steps. With the combination of a wide range of raw material sources and a simple process route, the industrial-scale, efficient, and high-quality production of single R-configuration tanshinone can be fully realized.

[0047] The method for preparing tanshinone disclosed in this invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0048] Example 1 The specific steps for preparing tanshinone in this embodiment are as follows: 200g of a raw material containing rosmarinic acid (20% rosmarinic acid content) was added to a three-necked flask. 760mL of 5% hydrochloric acid and 40mL of ethanol were added sequentially to the flask, the ethanol being just enough to completely dissolve the rosmarinic acid-containing raw material. The reaction was carried out at 50℃ under reflux conditions, with the rosmarinic acid-containing raw material and acidic alcohol solution in the three-necked flask undergoing hydrolysis for 8 hours to obtain a hydrolysate. 30% sodium hydroxide was added to the hydrolysate to adjust the pH to 3, and ethyl acetate was added for liquid-liquid extraction at a solid-liquid ratio of 1:1. The ethyl acetate layer was separated. The aqueous layer was then subjected to a second liquid-liquid extraction with ethyl acetate at a solid-liquid ratio of 1:1, and the ethyl acetate layer was separated again. The ethyl acetate layers from both liquid-liquid extractions were concentrated under reduced pressure to obtain crude tanshinone. The crude tanshinone was dissolved in water and loaded onto a D613 polar macroporous adsorption resin column at a preset flow rate of 2 BV / h for purification. After loading, the D613 column was washed with 5% ethanol at a flow rate of 1 BV / h. A gradient elution was performed using four column volumes (BV) of 10% ethanol as the eluent at a flow rate of approximately 1 BV / h to obtain a tanshinone-rich eluent. The eluent was concentrated under reduced pressure in a water bath at 40–45°C. When the volume of the concentrate was significantly reduced to near dryness or solid crystals precipitated, it was transferred to a vacuum drying oven for further drying to obtain the final tanshinone product. The tanshinone content in the obtained tanshinone product was determined by high-performance liquid chromatography (HPLC), and the results are shown below. Figure 1As shown, the content of tanshinone in the tanshinone product is 91.0%; the chromatographic conditions of high performance liquid chromatography are as follows: using a Diamonsil C18(2) column, 250*4.6mm, 5μm or equivalent column; the mobile phase is acetonitrile and 0.1% phosphoric acid aqueous solution by mass percentage, wherein: the phosphoric acid aqueous solution and acetonitrile are eluted by gradient according to the parameters shown in Table 1 below, the column temperature is 25℃; the detection wavelength is 280nm; and the flow rate is 1.0ml / min.

[0049] Table 1 shows the mobile phase gradient elution parameters for determining tanshinone products by high performance liquid chromatography.

[0050] The hydrolysis reaction in the three-necked flask was sampled and analyzed after 4 h and 8 h of reaction, respectively. The product components were analyzed by combining the peak positions of rosmarinic acid, tanshinone, and caffeic acid standards under the same chromatographic conditions. The retention times of the peaks of the standards rosmarinic acid, tanshinone, and caffeic acid were 23.30 min (peak 5), 6.42 min (peak 1), and 14.85 min (peak 3), respectively. Figure 2 The high-performance liquid chromatography (HPLC) chromatogram of the hydrolysate solution after half the reaction time is shown. The chromatogram shows peaks at the corresponding positions for rosmarinic acid, tanshinone, and caffeic acid, indicating that rosmarinic acid was not completely hydrolyzed after 4 hours of hydrolysis at 50℃, and that caffeic acid and tanshinone coexisted in the hydrolysate products. Figure 3 The high-performance liquid chromatogram of the hydrolysate solution at the reaction endpoint is shown. The peak corresponding to the peak of the standard tanshinone is only present in the figure. This indicates that the rosmarinic acid at the reaction endpoint is fully hydrolyzed and 100% converted. Caffeic acid in the hydrolysis product is also hydrolyzed and consumed, while tanshinone is fully retained. That is, the hydrolysis reaction of rosmarinic acid in acidic alcohol solution proposed in this invention can avoid the further hydrolysis of the generated tanshinone. Rosmarinic acid can be efficiently hydrolyzed and only tanshinone is retained. Example 2 This embodiment provides a method for preparing tanshinone, the steps of which are as follows: 200g of a raw material containing rosmarinic acid (50% rosmarinic acid content) was added to a three-necked flask. 900mL of 10% hydrochloric acid and 100mL of ethanol were added sequentially to the flask. The reaction was carried out at 50℃ under reflux conditions, with the rosmarinic acid-containing raw material and acidic alcohol solution in the flask undergoing hydrolysis for 8 hours to obtain a hydrolysate. 30% sodium hydroxide was added to the hydrolysate to adjust the pH to 3. Ethyl acetate was then added for liquid-liquid extraction at a solid-liquid ratio of 1:1, and the ethyl acetate layer was separated. The aqueous layer was then subjected to a second liquid-liquid extraction with ethyl acetate at a solid-liquid ratio of 1:1, and the ethyl acetate layer was separated again. The ethyl acetate layers from both liquid-liquid extractions were concentrated under reduced pressure to obtain crude tanshinone. The crude tanshinone was dissolved in water and loaded onto a polar macroporous adsorption resin D613 column at a preset flow rate of 2 BV / h for purification. After loading, the column was washed with 5% ethanol at a flow rate of 1 BV / h. A gradient elution was performed using four column volumes (BV) of 15% ethanol solution at a flow rate of 1 BV / h to obtain a tanshinone-rich eluent. The eluent was concentrated using a vacuum concentration device in a water bath at 40–45°C. When the volume of the concentrate was significantly reduced to near dryness or solid crystals precipitated, it was transferred to a vacuum drying oven for further drying to finally obtain the tanshinone product. The content of tanshinone in the obtained tanshinone product was determined by high performance liquid chromatography (HPLC). The results showed that the content of tanshinone in the tanshinone product was 92.5%. The HPLC chromatographic conditions were as follows: a Diamonsil C18(2) column, 250*4.6mm, 5μm or equivalent column was used; the mobile phase was acetonitrile and 0.1% (w / w) of phosphoric acid aqueous solution, wherein the phosphoric acid aqueous solution and acetonitrile were eluted by gradient according to the parameters shown in Table 1 below; the column temperature was 25℃; the detection wavelength was 280nm; and the flow rate was 1.0ml / min.

[0051] Example 3 This embodiment provides a method for preparing tanshinone, the steps of which are as follows: 200g of a raw material containing rosmarinic acid (30% rosmarinic acid content) was added to a three-necked flask. 380mL of 15% hydrochloric acid and 20mL of ethanol were added sequentially to the flask. The reaction was carried out at 90℃ under reflux with cooling water, allowing the rosmarinic acid-containing raw material and acidic alcohol solution to hydrolyze for 5 hours, yielding a hydrolysate. 30% sodium hydroxide was added to the hydrolysate to adjust the pH to 3. Ethyl acetate was then added for liquid-liquid extraction at a 1:1 ratio, and the ethyl acetate layer was separated. The aqueous layer was then subjected to a second liquid-liquid extraction with ethyl acetate at a 1:1 ratio, and the ethyl acetate layer was separated again. The ethyl acetate layers from both liquid-liquid extractions were concentrated under reduced pressure to obtain crude tanshinone. The crude tanshinone was dissolved in water and loaded onto a polar macroporous adsorption resin D613 column at a preset flow rate of 2 BV / h for purification. After loading, the column was washed with 5% ethanol at a flow rate of 1 BV / h. A gradient elution was performed using four column volumes (BV) of 10% ethanol solution as eluent at a flow rate of approximately 1 BV / h to obtain a tanshinone-rich eluent. The eluent was concentrated using a vacuum concentration device in a water bath at 40–45°C. When the volume of the concentrate was significantly reduced to near dryness or solid crystals precipitated, it was transferred to a vacuum drying oven for further drying to finally obtain the tanshinone product. The content of tanshinone in the obtained tanshinone product was determined by high performance liquid chromatography (HPLC). The results showed that the content of tanshinone in the tanshinone product was 90.7%. The HPLC chromatographic conditions were as follows: a Diamonsil C18(2) column, 250*4.6mm, 5μm or equivalent column was used; the mobile phase was acetonitrile and 0.1% (w / w) of phosphoric acid aqueous solution, wherein the phosphoric acid aqueous solution and acetonitrile were eluted by gradient according to the parameters shown in Table 1 below; the column temperature was 25℃; the detection wavelength was 280nm; and the flow rate was 1.0ml / min.

[0052] Example 4 This embodiment provides a method for preparing tanshinone, the steps of which are as follows: 200g of a raw material containing rosmarinic acid (90% rosmarinic acid content) was added to a three-necked flask. 280mL of 15% hydrochloric acid and 120mL of ethanol were added sequentially to the flask. The reaction was carried out at 100℃ under reflux conditions, with the rosmarinic acid-containing raw material and acidic alcohol solution in the flask undergoing hydrolysis for 3 hours to obtain a hydrolysate. 30% sodium hydroxide was added to the hydrolysate to adjust the pH to 3. Ethyl acetate was then added for liquid-liquid extraction at a solid-liquid ratio of 1:1, and the ethyl acetate layer was separated. The aqueous layer was then subjected to a second liquid-liquid extraction with ethyl acetate at a solid-liquid ratio of 1:1, and the ethyl acetate layer was separated again. The ethyl acetate layers from both liquid-liquid extractions were concentrated under reduced pressure to obtain crude tanshinone. The crude tanshinone was dissolved in water and loaded onto a polar macroporous adsorption resin D613 column at a preset flow rate of 2 BV / h for purification. After loading, the column was washed with 5% ethanol at a flow rate of 1 BV / h. A gradient elution was performed using four column volumes (BV) of 10% ethanol solution as eluent at a flow rate of approximately 1 BV / h to obtain a tanshinone-rich eluent. The eluent was concentrated using a vacuum concentration device in a water bath at 40–45°C. When the volume of the concentrate was significantly reduced to near dryness or solid crystals precipitated, it was transferred to a vacuum drying oven for further drying to finally obtain the tanshinone product. The content of tanshinone in the obtained tanshinone product was determined by high performance liquid chromatography (HPLC). The results showed that the content of tanshinone in the tanshinone product was 95.5%. The HPLC chromatographic conditions were as follows: a Diamonsil C18(2) column, 250*4.6mm, 5μm or equivalent column was used; the mobile phase was acetonitrile and 0.1% (w / w) of phosphoric acid aqueous solution, wherein the phosphoric acid aqueous solution and acetonitrile were eluted by gradient according to the parameters shown in Table 1 below; the column temperature was 25℃; the detection wavelength was 280nm; and the flow rate was 1.0ml / min.

[0053] Example 5 This embodiment provides a method for preparing tanshinone, the steps of which are as follows: 1 kg of rosmarinic acid-containing raw material (90% rosmarinic acid content) was added to a 10 L beaker. 4 L of 15% hydrochloric acid and 1 L of ethanol were added sequentially to a three-necked flask. The reaction was carried out at 100°C under reflux conditions, with the rosmarinic acid-containing raw material in the three-necked flask reacting with the acidic alcohol solution for 3 hours to obtain a hydrolysate. 30% sodium hydroxide was added to the hydrolysate to adjust the pH to 3. Ethyl acetate was then added for liquid-liquid extraction at a 1:1 ratio, and the ethyl acetate layer was separated. The aqueous layer was then subjected to a second liquid-liquid extraction with ethyl acetate at a 1:1 ratio, and the ethyl acetate layer was separated again. The ethyl acetate layers from both liquid-liquid extractions were concentrated under reduced pressure to obtain crude tanshinone. The crude tanshinone was dissolved in water and loaded onto a polar macroporous adsorption resin D613 column for purification at a preset flow rate of 2 BV / h. After loading, the column was washed with 5% ethanol at a flow rate of 1 BV / h. A gradient elution was performed using four column volumes (BV) of 10% ethanol solution as eluent at a flow rate of approximately 1 BV / h to obtain a tanshinone-rich eluent. The eluent was concentrated using a vacuum concentration device in a water bath at 40–45°C. When the volume of the concentrate was significantly reduced to near dryness or solid crystals precipitated, it was transferred to a vacuum drying oven for further drying to finally obtain the tanshinone product. The content of tanshinone in the obtained tanshinone product was determined by high performance liquid chromatography (HPLC). The results showed that the content of tanshinone in the tanshinone product was 95.0%. The HPLC conditions were as follows: a Diamonsil C18(2) column, 250*4.6mm, 5μm or equivalent column; the mobile phase was acetonitrile and 0.1% (w / w) of phosphoric acid aqueous solution, wherein the phosphoric acid aqueous solution and acetonitrile were eluted by gradient according to the parameters shown in Table 1 below; the column temperature was 25℃; the detection wavelength was 280nm; and the flow rate was 1.0ml / min.

[0054] Example 6 This embodiment provides a method for preparing tanshinone, the steps of which are as follows: 3 kg of rosmarinic acid-containing raw material (50% rosmarinic acid content) was added to a 10 L beaker. 4250 mL of 15% hydrochloric acid and 750 mL of ethanol were added sequentially to a three-necked flask. The reaction was carried out at 80°C under reflux with cooling water, allowing the rosmarinic acid-containing raw material in the three-necked flask to undergo hydrolysis with the acidic alcohol solution for 6 hours to obtain a hydrolysate. 30% sodium hydroxide was added to the hydrolysate to adjust the pH to 3. Ethyl acetate was then added for liquid-liquid extraction at a 1:1 ratio, and the ethyl acetate layer was separated. The aqueous layer was then subjected to a second liquid-liquid extraction with ethyl acetate at a 1:1 ratio, and the ethyl acetate layer was separated again. The ethyl acetate layers from both liquid-liquid extractions were concentrated under reduced pressure to obtain crude tanshinone. The crude tanshinone was dissolved in water and loaded onto a polar macroporous adsorption resin D613 column at a preset flow rate of 2 BV / h for purification. After loading, the column was washed with 5% ethanol at a flow rate of 1 BV / h. A gradient elution was performed using four column volumes (BV) of 10% ethanol solution as eluent at a flow rate of approximately 1 BV / h to obtain a tanshinone-rich eluent. The eluent was concentrated using a vacuum concentration device in a water bath at 40–45°C. When the volume of the concentrate was significantly reduced to near dryness or solid crystals precipitated, it was transferred to a vacuum drying oven for further drying to finally obtain the tanshinone product. The content of tanshinone in the obtained tanshinone product was determined by high performance liquid chromatography (HPLC). The results showed that the content of tanshinone in the tanshinone product was 92.0%. The HPLC chromatographic conditions were as follows: a Diamonsil C18(2) column, 250*4.6mm, 5μm or equivalent column; the mobile phase was acetonitrile and 0.1% (w / w) of phosphoric acid aqueous solution, wherein the phosphoric acid aqueous solution and acetonitrile were eluted by gradient according to the parameters shown in Table 1 below; the column temperature was 25℃; the detection wavelength was 280nm; and the flow rate was 1.0ml / min.

[0055] Comparative Examples 1-5 show the reaction results outside the reaction conditions claimed in this invention, such as hydrolysis conditions including reaction temperature, reaction time, and reaction environment, and extraction conditions such as extraction solvent and extraction environment. Specific implementation parameters and product detection results are shown in Table 2 below. The reaction conditions of Comparative Examples 1-5 below are all different from those of Example 1.

[0056] Table 2 shows the real-time parameters and product detection results for Example 1 and Comparative Examples 1-5.

[0057] The hydrolysis solution of Comparative Example 5 was further extracted and purified to obtain tanshinone product. The tanshinone content was determined by high performance liquid chromatography. The results showed that the tanshinone content in the tanshinone product was 85.1%.

[0058] Based on Examples 1-6 above and Comparative Examples 1-5 shown in Table 1, the proposed method for hydrolyzing rosmarinic acid to produce tanshinone in this invention has several drawbacks. Incomplete rosmarinic acid reaction occurs when the reaction time is too short or the reaction temperature is too low, resulting in the coexistence of rosmarinic acid, caffeic acid, and tanshinone in the hydrolysis solution, meaning 100% conversion of rosmarinic acid cannot be achieved. Changes in the reaction environment, such as alkaline alcohol solutions, can lead to deprotonation of phenolic hydroxyl groups, hydrolysis of ester bonds, oxidation, and polymerization of rosmarinic acid, ultimately resulting in structural damage and loss, thus preventing the acquisition of the target product, tanshinone. Therefore, an acidic environment is necessary. When the extraction solvent is changed to n-butanol, the total mass of the extracted tanshinone product decreases because the solubility of tanshinone in n-butanol is lower than its solubility in ethyl acetate. Furthermore, excessively high pH adjustments before extraction are detrimental to the structural integrity of the extraction solvent, ethyl acetate, thus affecting the yield of tanshinone.

[0059] The above description provides an illustrative overview of the present invention and its embodiments. This description is not restrictive, and the embodiments shown are merely one example of the invention's implementation. Actual implementations are not limited to these examples. Therefore, if those skilled in the art are inspired by this description and design similar implementations and examples without departing from the spirit of the invention, such designs should fall within the scope of protection of the present invention.

Claims

1. A method for preparing tanshinone, characterized in that, Tanshinone was prepared by hydrolyzing a raw material containing rosmarinic acid in an acidic alcohol solution. 。 2. The method for preparing tanshinone according to claim 1, characterized in that, The hydrolysis conditions in the acidic alcohol solution are: hydrolysis temperature of 50~100℃ and reaction time of 2~8h.

3. The method for preparing tanshinone according to claim 1, characterized in that, The rosmarinic acid content in the raw material is 10% to 100%.

4. The method for preparing tanshinone according to claim 1, characterized in that, The acid in the acidic alcohol solution is selected from hydrochloric acid, sulfuric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, p-toluenesulfonic acid, trifluoroacetic acid, formic acid, and acetic acid, with a mass percentage of 5% to 15%. The alcohol is selected from methanol, ethanol, isopropanol, benzyl alcohol, allyl alcohol, and trifluoroethanol.

5. The method for preparing tanshinone according to claim 1, characterized in that, Includes the following steps: The process involves hydrolyzing a raw material containing rosmarinic acid in an acidic alcoholic solution. The steps for adjusting the pH of the solution after hydrolysis; The steps for extracting and separating organic phases containing tanshinone; The steps for concentrating and purifying tanshinone.

6. The method for preparing tanshinone according to claim 5, characterized in that, When the raw material containing rosmarinic acid is hydrolyzed in an acidic alcohol solution, the ratio of the raw material containing rosmarinic acid to the acidic alcohol solution is 1 g : (1~5) mL.

7. The method for preparing tanshinone according to claim 5, characterized in that, The pH of the hydrolyzed solution is adjusted to be 2-6.

8. The method for preparing tanshinone according to claim 5, characterized in that, The steps for extracting and separating the organic phase containing tanshinone are as follows: extraction is carried out in two phases of water and ethyl acetate, with a material-to-liquid ratio of (1~2):(1~5), and the number of extractions is 1~5.

9. The method for preparing tanshinone according to claim 5, characterized in that, The acid in the acidic alcohol solution is hydrochloric acid, which accounts for 5% to 15% by mass, and the alcohol is ethanol.

10. The method for preparing tanshinone according to claim 5, characterized in that, The steps for concentrating and purifying tanshinone are as follows: the organic phase containing tanshinone is concentrated to obtain crude tanshinone, and the crude tanshinone is then subjected to resin purification and washing to obtain tanshinone with a content of not less than 90%.

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