Highly efficient concentrated extraction of salvia miltiorrhiza medicinal material and preparation method thereof

Abstract

The application provides a high-efficiency concentrated extraction of salvia miltiorrhiza medicinal materials and a preparation method thereof, which utilizes a two-phase eutectic solvent extraction system and combines nanofiltration concentration by a composite nanofiltration membrane. Salvia miltiorrhiza powder is mixed with a two-phase eutectic solvent aqueous solution, ultrasonic-assisted extraction and centrifugation are performed, and a hydrophobic phase rich in fat-soluble components and a hydrophilic phase rich in water-soluble components are obtained; then the two-phase extraction liquid is filtered by using an acrylic acid modified graphene oxide composite nanofiltration membrane, and a hydrophobic retention liquid and a hydrophilic retention liquid are obtained, and the two liquids are combined to obtain the extract. The method realizes one-step high-efficiency extraction and separation of fat-soluble and water-soluble active components in salvia miltiorrhiza, has less solvent consumption, is green and environmentally friendly, and has a mild nanofiltration concentration process; the obtained product has high purity and high yield, the two-phase eutectic solvent can be recycled and reused, and the method is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, specifically to a highly efficient method for concentrating and extracting Salvia miltiorrhiza and its preparation. Background Technology

[0002] Danshen, the dried root and rhizome of the plant *Salvia miltiorrhiza* (Lamiaceae family), is a commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis. It possesses effects such as promoting blood circulation, removing blood stasis, regulating menstruation, relieving pain, and clearing the mind and relieving irritability. Modern pharmacological studies have shown that the salvianolic acid, tanshinone, and polysaccharide components in Danshen are the main material basis for its efficacy. Currently, the extraction methods for Danshen mainly include water extraction, alcohol extraction, water extraction and alcohol precipitation, and macroporous resin purification. However, these methods often only target single-category components, resulting in cumbersome procedures, significant component loss, and high solvent consumption, making it difficult to achieve comprehensive and efficient enrichment of different polar active components in Danshen.

[0003] Beijing University of Chinese Medicine and other institutions have made technological innovations, employing a stepwise reflux extraction process involving ethanol and water, which effectively balances the extraction of salvianolic acids, tanshinones, and polysaccharides. However, this method still has shortcomings: the extract requires vacuum concentration and freeze-drying after extraction, which is energy-intensive and high temperatures can easily damage heat-sensitive components; furthermore, lipid-soluble and water-soluble components need to be extracted separately.

[0004] Therefore, based on existing technology, this invention introduces a biphasic eutectic solvent (DES) system to achieve one-step simultaneous extraction and separation of lipid-soluble and water-soluble components. It also combines a modified graphene oxide composite nanofiltration membrane to concentrate the extract at room temperature, thereby reducing energy consumption, protecting heat-sensitive components, and realizing solvent recovery and reuse, thus improving the comprehensive utilization efficiency of Danshen medicinal materials. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a highly efficient method for concentrating and extracting Salvia miltiorrhiza and its preparation.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza, the method comprising the following steps: Step (1): Prepare hydrophobic DES by mixing tetrabutylammonium chloride with n-hexanol and heating and stirring, and dilute with water to obtain hydrophobic DES aqueous solution; prepare hydrophilic DES by mixing choline chloride with ethylene glycol and heating and stirring, and dilute with water to obtain hydrophilic DES aqueous solution. Step (2): Pretreated graphene oxide is dispersed in water and ultrasonically prepared to obtain a pretreated graphene oxide dispersion; the pretreated graphene oxide dispersion, ammonium persulfate, and acrylic acid are mixed, heated and stirred to obtain a modified graphene oxide solution; the pretreated polysulfone ultrafiltration membrane is placed in a vacuum filter, and the initiator solution and the modified graphene oxide solution are filtered sequentially, washed with deionized water and vacuum dried to obtain a composite nanofiltration membrane; Step (3): Mix the danshen powder with hydrophobic and hydrophilic DES aqueous solution, and obtain the hydrophobic phase extract and the hydrophilic phase extract by ultrasonic extraction and centrifugation; respectively, perform dead-end filtration and concentration using composite nanofiltration membrane to obtain the hydrophobic phase retentate and the hydrophilic phase retentate, and combine them to obtain the danshen medicinal material extract.

[0007] Preferably, in step (1), the molar ratio of tetrabutylammonium chloride to n-hexanol in the hydrophobic DES aqueous solution is 1:(1.5~2.5), the heating and stirring temperature is 55~65℃, and the volume ratio of hydrophobic DES to deionized water when diluted with water is 1:(0.54~0.82). The molar ratio of choline chloride to ethylene glycol in the hydrophilic DES aqueous solution is 1:(1.5~2.5), the heating and stirring temperature is 55~65℃, and the volume ratio of hydrophilic DES to deionized water when diluted with water is 1:(0.54~0.82).

[0008] Preferably, in step (2), the preparation method of the pretreated graphene oxide is as follows: graphene oxide and ethanol are mixed at a mass ratio of 1:(110~120), and ultrasonically treated at 100W for 10~20min to obtain a graphene oxide dispersion; the graphene oxide dispersion, vinyltriethoxysilane and ethanol are mixed at a mass ratio of 1:(0.05~0.08):(0.16~0.17), stirred at 55~65℃ for 10~20min, ammonia water with a mass ratio of 0.01~0.02 times that of the graphene oxide dispersion is added, and stirring is continued for 22~26h. The mixture is then washed by centrifugation with deionized water to obtain the pretreated graphene oxide; the ammonia content of the ammonia water is 25wt%~28wt%.

[0009] Preferably, in step (2), the concentration of the pretreated graphene oxide dispersion is 8~9 mg / mL; the mass ratio of the pretreated graphene oxide dispersion, ammonium persulfate, and acrylic acid is 1:(0.003~0.0035):(0.075~0.105); the heating and stirring temperature is 60~70℃; and the heating and stirring time is 3~5 h.

[0010] Preferably, in step (2), the pretreated polysulfone ultrafiltration membrane is prepared as follows: the polysulfone ultrafiltration membrane is soaked in deionized water for 5-6 hours, with the water changed every hour during the soaking period, and then vacuum dried at 40°C; the polysulfone ultrafiltration membrane has a molecular weight cutoff of 100 kDa.

[0011] Preferably, in step (2), the initiator solution is an aqueous solution containing 0.03 mol / L ammonium persulfate and 0.03 mol / L sodium metabisulfite; the specific steps of the vacuum filtration are as follows: place the pretreated polysulfone ultrafiltration membrane in a vacuum filter, pour in the initiator solution to a height of 0.5 cm to 1 cm above the surface of the ultrafiltration membrane and then vacuum dry it, continue to pour in the modified graphene oxide solution to a height of 4 cm to 5 cm above the surface of the ultrafiltration membrane, vacuum dry it, wash it with deionized water, and vacuum dry it at 40 to 50°C to obtain the composite nanofiltration membrane.

[0012] Preferably, in step (3), the mass ratio of the danshen powder, the hydrophobic DES aqueous solution, and the hydrophilic DES aqueous solution is 1:(7.5~12.5):(7.5~12.5); the specific process steps of ultrasonic extraction and centrifugation are as follows: after stirring at 35~45℃ for 10~20min, ultrasonic treatment is performed at 35~45℃ and 160W for 20~40min, followed by centrifugation for 5~10min. After centrifugation, the upper layer is the hydrophobic phase extract and the lower layer is the hydrophilic phase extract; the danshen powder is purchased from Hubei Shian Pharmaceutical Co., Ltd.

[0013] Preferably, in step (3), the process parameters for dead-end filtration concentration are: filtration at a stirring speed of 300~400r / min and a pressure of 2~2.2bar for 7~9h; and pre-pressing with deionized water at a pressure of 2.5~3bar for 30~60min before dead-end filtration concentration.

[0014] This invention also discloses a Danshen extract prepared using the above-described method for efficient concentration and extraction of Danshen medicinal materials.

[0015] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) The efficient concentrated extraction method for preparing Salvia miltiorrhiza medicinal material disclosed in this invention uses a hydrophobic eutectic solvent prepared by tetrabutylammonium chloride and n-hexanol and a hydrophilic eutectic solvent prepared by choline chloride and ethylene glycol to construct a two-phase extraction system. In the one-step extraction process, the fat-soluble components (tanshinones) in Salvia miltiorrhiza are enriched in the hydrophobic phase and the water-soluble components (tanshinones and polysaccharides) are enriched in the hydrophilic phase. This overcomes the defects of the prior art, which is that the components are mixed and difficult to separate due to the first alcohol extraction and then water extraction. It realizes the one-step simultaneous extraction and in-situ separation of different polar active components, which greatly simplifies the process flow. At the same time, the eutectic solvent can be recycled and reused, which solves the problems of difficult solvent recovery and high production cost in the prior art, and meets the requirements of green and environmentally friendly industrial production.

[0016] (2) The preparation method of the highly efficient concentrated extract of Salvia miltiorrhiza disclosed in this invention uses an acrylic acid grafted crosslinked graphene oxide composite nanofiltration membrane to concentrate the extract. The active ingredients are efficiently retained by the electrostatic repulsion and size sieving effect of the membrane surface, achieving mild concentration under normal temperature conditions. This effectively avoids the degradation loss of heat-sensitive components during traditional vacuum concentration or freeze drying. At the same time, the nanofiltration process has low energy consumption, no phase change, and can realize the recovery and recycling of low eutectic solvent, which significantly reduces production costs and meets the requirements of green and environmentally friendly industrial production. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] To more clearly illustrate the method provided by the present invention, the following embodiments will be described in detail.

[0019] Example 1: A method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza mainly includes the following steps: (1) Tetrabutylammonium chloride and n-hexanol were mixed in a molar ratio of 1:1.5 and stirred at 55°C for 20 min to obtain a hydrophobic eutectic solvent; the hydrophobic eutectic solvent was mixed with deionized water in a volume ratio of 1:0.67 and stirred at 25°C for 10 min to obtain an aqueous solution of the hydrophobic eutectic solvent; choline chloride and ethylene glycol were mixed in a molar ratio of 1:1.5 and stirred at 55°C for 20 min to obtain a hydrophilic eutectic solvent; the hydrophilic eutectic solvent was mixed with deionized water in a volume ratio of 1:0.67 and stirred at 25°C for 10 min to obtain an aqueous solution of the hydrophilic eutectic solvent. (2) Graphene oxide and ethanol were mixed at a mass ratio of 1:110 and ultrasonicated at 100W for 10 min to obtain a graphene oxide dispersion. The graphene oxide dispersion, vinyltriethoxysilane and ethanol were mixed at a mass ratio of 1:0.05:0.16 and stirred at 55℃ for 10 min. Ammonia water with a mass ratio of 0.01 times that of the graphene oxide dispersion was added and stirred for 22 h. The mixture was then washed by centrifugation with deionized water to obtain pretreated graphene oxide. The pretreated graphene oxide was mixed with deionized water at a mass ratio of 1:110 and ultrasonicated at 100W for 10 min. n. A pretreated graphene oxide dispersion was obtained. The pretreated graphene oxide dispersion, ammonium persulfate, and acrylic acid were mixed at a mass ratio of 1:0.003:0.075 and stirred at 60℃ for 3 hours to obtain a modified graphene oxide solution. A polysulfone ultrafiltration membrane was soaked in deionized water for 5 hours, with the water changed every hour during this period. It was then vacuum dried at 40℃ to obtain a pretreated polysulfone ultrafiltration membrane. The pretreated polysulfone ultrafiltration membrane was placed in a vacuum filter, and an initiator solution was poured in until it exceeded the surface of the ultrafiltration membrane by 0.5 cm. The membrane was then dried, and the modified graphene oxide solution was poured in until it exceeded the surface of the ultrafiltration membrane by 4 cm. The membrane was then dried, washed with deionized water, and vacuum dried at 40℃ to obtain a composite nanofiltration membrane. (3) Mix the Salvia miltiorrhiza powder, the hydrophobic eutectic solvent aqueous solution and the hydrophilic eutectic solvent aqueous solution at a mass ratio of 1:10:10, stir at 40℃ for 10 min, sonicate at 40℃ and 160W for 20 min, centrifuge for 5 min, and obtain the upper layer as the hydrophobic phase extract and the lower layer as the hydrophilic phase extract after centrifugation; install the composite nanofiltration membrane in the membrane tank of the dead end filtration device, pre-press it with deionized water at 2.5 bar for 30 min, pour in the hydrophobic phase extract and the hydrophilic phase extract respectively, filter at 300 r / min stirring speed and 2 bar pressure for 7 h, and obtain the hydrophobic phase retentate and the hydrophilic phase retentate respectively, and combine them to obtain the Salvia miltiorrhiza extract.

[0020] Example 2: A method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza mainly includes the following steps: (1) Tetrabutylammonium chloride and n-hexanol were mixed in a molar ratio of 1:2 and stirred at 60°C for 30 min to obtain a hydrophobic eutectic solvent; the hydrophobic eutectic solvent and deionized water were mixed in a volume ratio of 1:0.67 and stirred at 27°C for 15 min to obtain an aqueous solution of the hydrophobic eutectic solvent; choline chloride and ethylene glycol were mixed in a molar ratio of 1:2 and stirred at 60°C for 30 min to obtain a hydrophilic eutectic solvent; the hydrophilic eutectic solvent and deionized water were mixed in a volume ratio of 1:0.67 and stirred at 27°C for 15 min to obtain an aqueous solution of the hydrophilic eutectic solvent. (2) Graphene oxide and ethanol were mixed at a mass ratio of 1:115 and ultrasonicated at 100W for 15 min to obtain a graphene oxide dispersion. The graphene oxide dispersion, vinyltriethoxysilane and ethanol were mixed at a mass ratio of 1:0.065:0.165 and stirred at 60℃ for 15 min. Ammonia water with a mass ratio of 0.015 times that of the graphene oxide dispersion was added and stirred for 24 h. The mixture was washed by centrifugation with deionized water to obtain pretreated graphene oxide. The pretreated graphene oxide was mixed with deionized water at a mass ratio of 1:118 and ultrasonicated at 100W for 15 min. A pretreated graphene oxide dispersion was obtained. The pretreated graphene oxide dispersion, ammonium persulfate, and acrylic acid were mixed at a mass ratio of 1:0.0033:0.09 and stirred at 65℃ for 4 hours to obtain a modified graphene oxide solution. A polysulfone ultrafiltration membrane was soaked in deionized water for 5.5 hours, with the water changed every hour, and then vacuum dried at 40℃ to obtain a pretreated polysulfone ultrafiltration membrane. The pretreated polysulfone ultrafiltration membrane was placed in a vacuum filter, and an initiator solution was poured in until it exceeded the surface of the ultrafiltration membrane by 0.7 cm. The membrane was then dried, and the modified graphene oxide solution was continued to be poured in until it exceeded the surface of the ultrafiltration membrane by 4.5 cm. The membrane was then dried, washed with deionized water, and vacuum dried at 45℃ to obtain a composite nanofiltration membrane. (3) Mix the Salvia miltiorrhiza powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution at a mass ratio of 1:10:10, stir at 40℃ for 15 min, sonicate at 40℃ and 160W for 30 min, centrifuge for 8 min, and obtain the upper layer as hydrophobic phase extract and the lower layer as hydrophilic phase extract after centrifugation; install the composite nanofiltration membrane in the membrane tank of the dead end filtration device, pre-press it with deionized water at 2.8 bar for 45 min, pour in the hydrophobic phase extract and the hydrophilic phase extract respectively, filter at 350 r / min stirring speed and 2.1 bar pressure for 8 h, obtain the hydrophobic phase retentate and the hydrophilic phase retentate respectively, and combine them to obtain the Salvia miltiorrhiza extract.

[0021] Example 3: A method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza mainly includes the following steps: (1) Tetrabutylammonium chloride and n-hexanol were mixed in a molar ratio of 1:2.5 and stirred at 65°C for 40 min to obtain a hydrophobic eutectic solvent; the hydrophobic eutectic solvent was mixed with deionized water in a volume ratio of 1:0.67 and stirred at 30°C for 20 min to obtain an aqueous solution of the hydrophobic eutectic solvent; choline chloride and ethylene glycol were mixed in a molar ratio of 1:2.5 and stirred at 65°C for 20-40 min to obtain a hydrophilic eutectic solvent; the hydrophilic eutectic solvent was mixed with deionized water in a volume ratio of 1:0.67 and stirred at 30°C for 20 min to obtain an aqueous solution of the hydrophilic eutectic solvent. (2) Graphene oxide and ethanol were mixed at a mass ratio of 1:120 and ultrasonicated at 100W for 20 min to obtain a graphene oxide dispersion. The graphene oxide dispersion, vinyltriethoxysilane and ethanol were mixed at a mass ratio of 1:0.08:0.17 and stirred at 65℃ for 20 min. Ammonia water with a mass ratio of 0.02 times that of the graphene oxide dispersion was added and stirred for 26 h. The mixture was washed by centrifugation with deionized water to obtain pretreated graphene oxide. The pretreated graphene oxide was mixed with deionized water at a mass ratio of 1:124 and ultrasonicated at 100W for 20 min. n. A pretreated graphene oxide dispersion was obtained. The pretreated graphene oxide dispersion, ammonium persulfate, and acrylic acid were mixed at a mass ratio of 1:0.0035:0.105 and stirred at 70℃ for 5 hours to obtain a modified graphene oxide solution. A polysulfone ultrafiltration membrane was soaked in deionized water for 6 hours, with the water changed every hour during this period, and then vacuum dried at 40℃ to obtain a pretreated polysulfone ultrafiltration membrane. The pretreated polysulfone ultrafiltration membrane was placed in a vacuum filter, and an initiator solution was poured in until it was 1 cm above the surface of the ultrafiltration membrane. The membrane was then dried, and the modified graphene oxide solution was poured in until it was 5 cm above the surface of the ultrafiltration membrane. The membrane was then dried, washed with deionized water, and vacuum dried at 50℃ to obtain a composite nanofiltration membrane. (3) Mix the Salvia miltiorrhiza powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution at a mass ratio of 1:10:10, stir at 40℃ for 20 min, sonicate at 40℃ and 160W for 40 min, centrifuge for 10 min, and obtain the upper layer as hydrophobic phase extract and the lower layer as hydrophilic phase extract after centrifugation; install the composite nanofiltration membrane in the membrane tank of the dead end filtration device, pre-press it with deionized water at 3 bar for 60 min, pour in the hydrophobic phase extract and the hydrophilic phase extract respectively, filter at 400 r / min stirring speed and 2.2 bar pressure for 9 h, and obtain the hydrophobic phase retentate and the hydrophilic phase retentate respectively, and combine them to obtain the Salvia miltiorrhiza extract.

[0022] Comparative Example 1: The only difference from Example 2 is the difference in step (1). The phrase "mixing the hydrophobic eutectic solvent and deionized water at a volume ratio of 1:0.67" is changed to "mixing the hydrophobic eutectic solvent and deionized water at a volume ratio of 1:0.43"; and the phrase "mixing the hydrophilic eutectic solvent and deionized water at a volume ratio of 1:0.67" is changed to "mixing the hydrophilic eutectic solvent and deionized water at a volume ratio of 1:0.43".

[0023] Comparative Example 2: The only difference from Example 2 is the step (1). The phrase "mixing the hydrophobic eutectic solvent and deionized water at a volume ratio of 1:0.67" is changed to "mixing the hydrophobic eutectic solvent and deionized water at a volume ratio of 1:0.54"; and the phrase "mixing the hydrophilic eutectic solvent and deionized water at a volume ratio of 1:0.67" is changed to "mixing the hydrophilic eutectic solvent and deionized water at a volume ratio of 1:0.54".

[0024] Comparative Example 3: The only difference from Example 2 is the difference in step (1). The phrase "mixing the hydrophobic eutectic solvent and deionized water at a volume ratio of 1:0.67" is changed to "mixing the hydrophobic eutectic solvent and deionized water at a volume ratio of 1:0.82"; and the phrase "mixing the hydrophilic eutectic solvent and deionized water at a volume ratio of 1:0.67" is changed to "mixing the hydrophilic eutectic solvent and deionized water at a volume ratio of 1:0.82".

[0025] Comparative Example 4: The only difference from Example 2 is the difference in step (1). The phrase "mixing the hydrophobic eutectic solvent and deionized water at a volume ratio of 1:0.67" is changed to "mixing the hydrophobic eutectic solvent and deionized water at a volume ratio of 1:1"; and the phrase "mixing the hydrophilic eutectic solvent and deionized water at a volume ratio of 1:0.67" is changed to "mixing the hydrophilic eutectic solvent and deionized water at a volume ratio of 1:1".

[0026] Comparative Example 5: The only difference from Example 2 is the difference in step (3), where “mixing danshen powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:10:10” is changed to “mixing danshen powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:5:5”.

[0027] Comparative Example 6: The only difference from Example 2 is the difference in step (3), where “mixing danshen powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:10:10” is changed to “mixing danshen powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:7.5:7.5”.

[0028] Comparative Example 7: The only difference from Example 2 is the difference in step (3), where “mixing danshen powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:10:10” is changed to “mixing danshen powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:12.5:12.5”.

[0029] Comparative Example 8: The only difference from Example 2 is the step (3), which changes "mixing danshen powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:10:10" to "mixing danshen powder, hydrophobic eutectic solvent aqueous solution and hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:15:15".

[0030] Comparative Example 9: The only difference from Example 2 is step (3), which changes "ultrasonic treatment at 40°C and 160W for 30 minutes" to "ultrasonic treatment at 20°C and 160W for 30 minutes".

[0031] Comparative Example 10: The only difference from Example 2 is step (3), which changes "ultrasonic treatment at 40°C and 160W for 30 minutes" to "ultrasonic treatment at 30°C and 160W for 30 minutes".

[0032] Comparative Example 11: The only difference from Example 2 is step (3), which changes "ultrasonic treatment at 40°C and 160W for 30 minutes" to "ultrasonic treatment at 50°C and 160W for 30 minutes".

[0033] Comparative Example 12: The only difference from Example 2 is the step (1), which changes "mixing tetrabutylammonium chloride and n-hexanol in a molar ratio of 1:2" to "mixing tetrabutylammonium chloride and n-butanol in a molar ratio of 1:2".

[0034] Comparative Example 13: The only difference from Example 2 is the step (1), where “mixing tetrabutylammonium chloride and n-hexanol in a molar ratio of 1:2” is changed to “mixing 1-butyl-3-methylimidazolium chloride and n-hexanol in a molar ratio of 1:2”.

[0035] Comparative Example 14: The only difference from Example 2 is the step (1), where “mixing choline chloride and ethylene glycol in a molar ratio of 1:2” is changed to “mixing choline chloride and lactic acid in a molar ratio of 1:2”.

[0036] Comparative Example 15: The only difference from Example 2 is the step (1), where “mixing choline chloride and ethylene glycol in a molar ratio of 1:2” is changed to “mixing choline chloride and glycerol in a molar ratio of 1:2”.

[0037] Comparative Example 16: A method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza mainly includes the following steps: (1) Tetrabutylammonium chloride and n-hexanol were mixed in a molar ratio of 1:2 and stirred at 60°C for 30 min to obtain a hydrophobic eutectic solvent; the hydrophobic eutectic solvent and deionized water were mixed in a mass ratio of 1:0.67 and stirred at 27°C for 15 min to obtain an aqueous solution of the hydrophobic eutectic solvent; choline chloride and ethylene glycol were mixed in a molar ratio of 1:2 and stirred at 60°C for 30 min to obtain a hydrophilic eutectic solvent; the hydrophilic eutectic solvent and deionized water were mixed in a mass ratio of 1:0.67 and stirred at 27°C for 15 min to obtain an aqueous solution of the hydrophilic eutectic solvent. (2) Mix the Salvia miltiorrhiza powder, the hydrophobic eutectic solvent aqueous solution and the hydrophilic eutectic solvent aqueous solution in a mass ratio of 1:10:10, stir at 40℃ for 15 min, sonicate at 40℃ and 160W for 30 min, centrifuge for 8 min, and obtain the upper layer as the hydrophobic phase extract and the lower layer as the hydrophilic phase extract. Combine them and concentrate under reduced pressure at 60℃ to obtain the Salvia miltiorrhiza extract.

[0038] To further illustrate the advantages of the present invention, the beneficial effects will be demonstrated through the following experimental methods.

[0039] I. Determination of optimal extraction conditions (moisture content, solid-liquid ratio, ultrasonic temperature) Based on the total yield and the extraction amounts of total tanshinone, total tanshinone, and total polysaccharides, the determination was made. 1. Total yield = (dry weight of Salvia miltiorrhiza extract / mass of Salvia miltiorrhiza powder) × 100% 2. Total Salvia miltiorrhiza extract: Weigh 200 mg of Salvia miltiorrhiza extract, add 40 mL of 80% methanol aqueous solution, sonicate at 80℃ for 30 min, centrifuge, take 0.3 mL of supernatant into a 10 mL brown volumetric flask, add 0.5 mL of 5% sodium nitrite solution and 0.5 mL of 10% aluminum nitrate solution in sequence, react for 5 min, and then make up to volume with 1 mol / L sodium hydroxide solution. Measure the absorbance at a wavelength of 496 nm. Establish a standard curve using Salvia miltiorrhiza B standard as reference (Y=0.0337X+0.0095, R²=0.9990). Substitute the measured absorbance of the sample into the standard curve to calculate the total Salvia miltiorrhiza concentration. Calculate the mass fraction of total Salvia miltiorrhiza in the Salvia miltiorrhiza extract based on the total Salvia miltiorrhiza concentration C.

[0040] Total tanshinone extraction yield: Weigh 500 mg of the highly concentrated extract of tanshinone from the tanshinone medicinal material, add 40 mL of methanol, sonicate at 80℃ for 30 min, centrifuge, take 0.2 mL of the supernatant and dilute to 10 mL with methanol, and directly measure the absorbance at a wavelength of 268 nm. Establish a standard curve (Y=0.0989X-0.0538, R²=0.9997) using tanshinone IIA standard as the reference standard, calculate the total tanshinone concentration, and calculate the mass fraction of total tanshinone in the tanshinone extract from the tanshinone medicinal material based on the total tanshinone concentration.

[0041] Total polysaccharide extraction: Weigh 200 mg of the highly concentrated extract of Salvia miltiorrhiza, add 40 mL of ultrapure water, sonicate at 70 °C for 30 min, centrifuge, take 1 mL of the supernatant, add 95% ethanol to a final concentration of 70%, let stand for 12 h, centrifuge to collect the crude polysaccharide precipitate, dissolve in 1 mL of water, add 2 mL of L'Sevage reagent (chloroform: n-butanol = 4:1), centrifuge, take 0.4 mL of the supernatant, add water to make up to 2 mL, add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid in sequence, boil in a water bath for 15 min, measure the absorbance at 490 nm, establish a standard curve with glucose standard as reference (Y = 0.0142X + 0.0029, R² = 0.9996), calculate the total polysaccharide concentration, and calculate the mass fraction of total polysaccharides in the Salvia miltiorrhiza extract based on the total polysaccharide concentration.

[0042] The results are shown in Table 1. Example 2 40% 1:20 40℃ 15.31% 40.83% 10.16% 11.24% Comparative Example 1 30% 1:20 40℃ 13.67% 38.63% 8.09% 9.05% Comparative Example 2 35% 1:20 40℃ 14.18% 39.73% 8.87% 9.94% Comparative Example 3 45% 1:20 40℃ 14.23% 39.41% 9.06% 10.03% Comparative Example 4 50% 1:20 40℃ 13.02% 38.75% 7.96% 9.62% Comparative Example 5 40% 1:10 40℃ 13.99% 38.14% 8.29% 9.13% Comparative Example 6 40% 1:15 40℃ 14.20% 39.61% 9.05% 10.11% Comparative Example 7 40% 1:25 40℃ 14.20% 39.69% 9.04% 10.02% Comparative Example 8 40% 1:30 40℃ 13.78% 38.72% 8.13% 9.45% Comparative Example 9 40% 1:20 20℃ 13.02% 36.97% 7.36% 8.99% Comparative Example 10 40% 1:20 30℃ 13.34% 37.61% 7.63% 9.03% Comparative Example 11 40% 1:20 50℃ 13.42% 38.23% 8.73% 9.02% As shown in Table 1, when extracting Danshen extract from Danshen powder, the water content of DES, the solid-liquid ratio, and the ultrasonic temperature significantly affect the total yield and mass fraction of each component. Excessive water content in DES (above 40% v / v) disrupts the hydrogen bonds between DES and the target compound, dilutes the hydrophobic phase, and reduces the distribution and extraction rate of lipid-soluble tanshinone. Conversely, excessively low water content leads to excessively high DES viscosity, hindering mass transfer. Therefore, a volume ratio of 1:0.67 (approximately 40% water content) is suitable in this scheme, balancing viscosity and hydrogen bonding. A low solid-liquid ratio results in incomplete extraction, while a high ratio does not significantly improve the extraction rate and wastes solvent. A ratio of 1:20 ensures sufficient contact and is economical in terms of solvent usage. Appropriately increasing the ultrasonic temperature can reduce DES viscosity and promote diffusion, thereby increasing the total yield. However, excessively high temperatures accelerate the degradation of heat-sensitive components; therefore, 40℃ is within a safe range. Total tanshinone and total polysaccharides are mainly concentrated in the hydrophilic phase. Appropriate water content promotes their dissolution, but excessive water will disrupt hydrogen bonds, leading to a decrease in extraction rate. Total tanshinone is concentrated in the hydrophobic phase. Increased water content dilutes the hydrophobic phase and reduces its polarity, which is detrimental to the dissolution and distribution of tanshinone. Therefore, the parameters determined experimentally in this scheme are the optimal parameters.

[0043] II. The Influence of DES Chemical Composition and Composite Nanofiltration Membrane Concentration on Extraction The extraction conditions were determined based on the total yield and the extraction amounts of total salvianolic acid, total tanshinone, and total polysaccharides. The experimental method was the same as that in "I. Determination of Optimal Extraction Conditions".

[0044] The results are shown in Table 2. Example 1 15.06% 40.16% 9.85% 11.16% Example 2 15.31% 40.83% 10.16% 11.24% Example 3 15.24% 40.45% 10.07% 11.13% Comparative Example 12 14.26% 39.27% 6.94% 10.89% Comparative Example 13 14.88% 39.80% 7.68% 11.04% Comparative Example 14 14.63% 40.85% 7.15% 10.86% Comparative Example 15 14.79% 40.81% 7.57% 10.92% Comparative Example 16 59.32% 6.23% 2.43% 3.06% By comparing Examples 1-3 and Comparative Examples 12-13, it can be found that when the hydrophobic DES component in step (1) is changed from tetrabutylammonium chloride and n-hexanol to tetrabutylammonium chloride and n-butanol or 1-butyl-3-methylimidazolium chloride and n-hexanol, the total yield of Tanshinone extract decreases, and the total mass fraction of tanshinone decreases significantly. The reason is that the original hydrophobic DES has a polarity that matches the fat-soluble tanshinone and has a moderate viscosity. Changing it to n-butanol will increase the polarity of DES and is not conducive to the extraction of fat-soluble components. Although the polarity of 1-butyl-3-methylimidazolium chloride is similar, the π-π and hydrogen bonding with tanshinone is weakened, which ultimately leads to a decrease in the total yield and a decrease in the proportion of fat-soluble components.

[0045] By comparing Examples 1-3 and Comparative Examples 14-15, it can be found that when the hydrophilic DES component in step (1) is changed from choline chloride and ethylene glycol to choline chloride and lactic acid or choline chloride and glycerol, the total yield of Tanshinone extract will decrease and the total mass fraction of tanshinone will decrease significantly. The reason is that choline chloride ethylene glycol DES has moderate polarity and viscosity, which can take into account the extraction of fat-soluble tanshinone and water-soluble phenolic acids and polysaccharides. However, choline chloride lactic acid has stronger acidity and higher polarity, which inhibits the extraction of fat-soluble components. Choline chloride glycerol has a high viscosity, which reduces the mass transfer efficiency and is not conducive to the extraction of tanshinone. In the end, the total yield decreases and the proportion of fat-soluble components decreases.

[0046] By comparing Examples 1-3 and Comparative Example 16, it can be found that the composite nanofiltration membrane concentration step can significantly increase the mass fraction of total tanshinone and total salvianolic acid. The reason is that the composite nanofiltration membrane can efficiently retain small-molecule fat-soluble tanshinone and water-soluble salvianolic acid. The membrane concentration is carried out at low temperature and low pressure to avoid thermal degradation of tanshinone and salvianolic acid. In addition, the membrane filtration can directionally separate the effective components and impurities, realize the enrichment of effective components, and ultimately improve the total yield and the content of effective components.

[0047] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended technical solutions rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the technical solutions are intended to be included within the present invention.

Claims

1. A method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza, characterized in that, The preparation method includes the following steps: Step (1): Tetrabutylammonium chloride and n-hexanol were mixed and heated to prepare a hydrophobic eutectic solvent (DES), and diluted with water to obtain a hydrophobic DES aqueous solution; choline chloride and ethylene glycol were mixed and heated to prepare a hydrophilic DES, and diluted with water to obtain a hydrophilic DES aqueous solution. Step (2): Pretreated graphene oxide is dispersed in water and ultrasonically prepared to obtain a pretreated graphene oxide dispersion; the pretreated graphene oxide dispersion, ammonium persulfate, and acrylic acid are mixed, heated and stirred to obtain a modified graphene oxide solution; the pretreated polysulfone ultrafiltration membrane is placed in a vacuum filter, and the initiator solution and the modified graphene oxide solution are filtered sequentially, washed with deionized water and vacuum dried to obtain a composite nanofiltration membrane; Step (3): Mix the danshen powder with hydrophobic and hydrophilic DES aqueous solution, and obtain the hydrophobic phase extract and the hydrophilic phase extract by ultrasonic extraction and centrifugation; respectively, perform dead-end filtration and concentration using composite nanofiltration membrane to obtain the hydrophobic phase retentate and the hydrophilic phase retentate, and combine them to obtain the danshen medicinal material extract.

2. The method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza medicinal material according to claim 1, characterized in that, In step (1), the molar ratio of tetrabutylammonium chloride to n-hexanol in the hydrophobic DES aqueous solution is 1:(1.5~2.5), the heating and stirring temperature is 55~65℃, and the volume ratio of hydrophobic DES to deionized water when diluted with water is 1:(0.54~0.82). The molar ratio of choline chloride to ethylene glycol in the hydrophilic DES aqueous solution is 1:(1.5~2.5), the heating and stirring temperature is 55~65℃, and the volume ratio of hydrophilic DES to deionized water when diluted with water is 1:(0.54~0.82).

3. The method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza medicinal material according to claim 1, characterized in that, In step (2), the preparation method of the pretreated graphene oxide is as follows: graphene oxide and ethanol are mixed at a mass ratio of 1:(110~120), and ultrasonically treated at 100W for 10~20min to obtain a graphene oxide dispersion; the graphene oxide dispersion, vinyltriethoxysilane and ethanol are mixed at a mass ratio of 1:(0.05~0.08):(0.16~0.17), stirred at 55~65℃ for 10~20min, and ammonia water with a mass ratio of 0.01~0.02 times that of the graphene oxide dispersion is added, and stirring is continued for 22~26h. The mixture is then washed by centrifugation with deionized water to obtain the pretreated graphene oxide; the ammonia content of the ammonia water is 25wt%~28wt%.

4. The method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza medicinal material according to claim 1, characterized in that, In step (2), the concentration of the pretreated graphene oxide dispersion is 8~9 mg / mL; the mass ratio of the pretreated graphene oxide dispersion, ammonium persulfate, and acrylic acid is 1:(0.003~0.0035):(0.075~0.105); the heating and stirring temperature is 60~70℃; and the heating and stirring time is 3~5 h.

5. The method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza medicinal material according to claim 1, characterized in that, In step (2), the pretreated polysulfone ultrafiltration membrane is prepared as follows: the polysulfone ultrafiltration membrane is soaked in deionized water for 5-6 hours, with the water changed every hour during the soaking period, and then vacuum dried at 40°C; the polysulfone ultrafiltration membrane has a molecular weight cutoff of 100 kDa.

6. The method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza medicinal material according to claim 1, characterized in that, In step (2), the initiator solution is an aqueous solution containing 0.03 mol / L ammonium persulfate and 0.03 mol / L sodium metabisulfite; the specific steps of the vacuum filtration are as follows: the pretreated polysulfone ultrafiltration membrane is placed in a vacuum filter, the initiator solution is poured in until it is 0.5 cm to 1 cm above the surface of the ultrafiltration membrane, and then the membrane is dried. The modified graphene oxide solution is then poured in until it is 4 cm to 5 cm above the surface of the ultrafiltration membrane, and the membrane is dried. The membrane is then washed with deionized water and vacuum dried at 40 to 50 °C to obtain the composite nanofiltration membrane.

7. The method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza medicinal material according to claim 1, characterized in that, In step (3), the mass ratio of the danshen powder, the hydrophobic DES aqueous solution and the hydrophilic DES aqueous solution is 1:(7.5~12.5):(7.5~12.5); the specific process steps of ultrasonic extraction and centrifugation are as follows: after stirring at 35~45℃ for 10~20min, ultrasonic treatment is performed at 35~45℃ and 160W for 20~40min, followed by centrifugation for 5~10min. After centrifugation, the upper layer is the hydrophobic phase extract and the lower layer is the hydrophilic phase extract.

8. The method for preparing a highly efficient concentrated extract of Salvia miltiorrhiza medicinal material according to claim 1, characterized in that, In step (3), the process parameters for dead-end filtration concentration are: filtration at a stirring speed of 300~400r / min and a pressure of 2~2.2bar for 7~9h; and pre-pressing with deionized water at a pressure of 2.5~3bar for 30~60min before dead-end filtration concentration.

9. A Danshen extract prepared by the method for efficient concentration and extraction of Danshen medicinal material as described in any one of claims 1 to 8.