Semen lepidii-poria cocos-based cardiac glycoside compound

Abstract

The invention discloses a semen lepidii-poria cocos-based cardiac glycoside compound, and relates to the technical field of traditional Chinese medicine pharmacy, the compound comprises the following components: semen lepidii-poria cocos extract powder, a complex enzyme preparation, and an ethanol aqueous solution with a volume fraction of 50%; the compound enzyme preparation is prepared from pectinase, cellulase and beta-glucosidase; according to the method, semen lepidii and poria cocos raw material combination and a compound enzyme two-phase enzymolysis system are combined with ultrasonic-assisted extraction, membrane separation purification and vacuum freeze-drying technologies, efficient extraction and high-purity enrichment of cardiac glycoside compounds are achieved, the raw material utilization rate and product purity are synergistically optimized in all links, meanwhile, energy consumption and pollution are reduced, the process is standard and controllable, and the method is suitable for industrial production. The method provides a reliable path for large-scale preparation of high-activity and high-purity cardiac glycoside, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, specifically to cardiac glycoside compounds based on Lepidium apetalum and Poria cocos. Background Technology

[0002] Cardiac glycosides, as a class of natural products with important biological activities, have become core raw materials for the research and development of many products due to their unique physiological effects. The technology for extracting cardiac glycosides from natural products has always attracted much attention. Traditional extraction methods mostly rely on single raw materials or conventional extraction processes. However, Lepidium apetalum and Poria cocos, as common natural raw materials, each contain abundant active ingredients. Their synergistic effect provides new possibilities for the efficient extraction of cardiac glycosides. With the industry's increasing demands for the purity and activity of effective components in natural products, how to achieve efficient enrichment and purification of cardiac glycosides through scientific raw material matching and process optimization has become a key direction for current technological research and development. Based on modern biological extraction and separation technologies, constructing a mild and efficient extraction system to fully explore the application value of natural raw materials and meet the market demand for high-quality cardiac glycosides has become a development trend in this field.

[0003] Traditional extraction techniques for cardiac glycosides have several limitations. First, in terms of raw material utilization, single-raw-material extraction methods fail to fully leverage the synergistic effects between different raw materials, resulting in low extraction efficiency of the target component and underutilization of raw material resources. Second, traditional extraction processes often employ high-temperature reflux and single-solvent extraction, which not only easily lead to the loss of activity in cardiac glycosides but also suffer from long extraction cycles and high energy consumption. In the purification stage, traditional techniques often rely on complex operations such as multiple recrystallizations or column chromatography, which are not only cumbersome and time-consuming but may also result in product purity failing to meet standards due to the use of large amounts of chemical reagents, while also generating numerous environmental problems. Furthermore, traditional processes lack precise control over extraction conditions, leading to poor batch-to-batch quality stability and failing to meet the requirements for product uniformity and reliability in large-scale production. These shortcomings severely restrict the industrial application and market expansion of cardiac glycosides. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide cardiac glycoside compounds based on Lepidium apetalum and Poria cocos. By combining biphasic enzymatic hydrolysis with ultrasonic extraction technology, and through steps such as raw material pretreatment, enzymatic hydrolysis, ultrasonic-assisted extraction, membrane separation purification, and vacuum freeze-drying, a high-purity extract of total cardiac glycosides is obtained, which improves extraction efficiency and purity and solves the problems of low efficiency and insufficient purity in traditional processes. This invention has a cardiac stimulating effect, the process is stable and controllable, it is suitable for industrial production, has broad market application prospects, and is in line with the development direction of modern Chinese medicine extraction technology.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On the one hand, a cardiac glycoside compound based on Lepidium apetalum-Poria cocos is composed of the following components: Lepidium apetalum-Poria cocos extract powder, a compound enzyme preparation, and a 50% ethanol aqueous solution; the compound enzyme preparation is composed of pectinase, cellulase, and β-glucosidase.

[0006] Furthermore, the Lepidium apetalum-Poria cocos extract powder is obtained by mixing Lepidium apetalum powder and frozen Poria cocos powder, followed by enzymatic hydrolysis, ultrasonic extraction, membrane separation, and drying. The total cardiac glycoside purity of the Lepidium apetalum-Poria cocos extract powder is ≥85%, and the content of Lepidium apetalum glycosides is ≥60%.

[0007] Furthermore, the amounts of pectinase, cellulase, and β-glucosidase added to the compound enzyme preparation are 0.5%, 1.0%, and 0.2% respectively, based on the mass of the mixed raw material powder.

[0008] Furthermore, the amount of the 50% ethanol aqueous solution added is 20%-30% of the total volume of the complex enzyme biphasic enzymatic hydrolysis system.

[0009] On the other hand, a method for preparing cardiac glycosides based on Lepidium apetalum and Poria cocos includes the following steps:

[0010] S100. Raw material pretreatment: Select Lepidium apetalum seeds, remove impurities, stir-fry, cool, crush, and sieve to obtain Lepidium apetalum seed pretreatment powder; Select Poria cocos seeds, remove the outer skin, cut into pieces, freeze-dry and crush at low temperature to obtain Poria cocos pretreatment powder; Mix the two pretreatment powders in a certain proportion to obtain mixed raw material powder.

[0011] S200, compound enzyme biphasic enzymatic hydrolysis: Add deionized water to the mixed raw material powder, stir and disperse, adjust the pH with buffer solution, add compound enzyme preparation and 50% ethanol aqueous solution, incubate and enzymatically hydrolyze to obtain enzymatic hydrolysis mixture;

[0012] S300, Ultrasonic-assisted extraction: Transfer the enzymatic hydrolysis mixture to an ultrasonic extraction tank for ultrasonic extraction. After extraction, centrifuge and collect the ultrasonic extract.

[0013] S400, Membrane separation purification and concentration: The ultrasonic extract is sequentially processed by an ultrafiltration device and a nanofiltration device to obtain a concentrated solution;

[0014] S500, Vacuum freeze drying: After spreading the concentrate evenly, place it in a vacuum freeze dryer to dry, and obtain the extract.

[0015] Furthermore, the stir-frying of the northern lepidium seeds is carried out using a drum-type stir-frying device, with a stir-frying temperature of 120℃, a stir-frying time of 10 minutes, and a drum rotation speed of 15-20 r / min; the pulverization is carried out using a universal pulverizer, with a pulverization speed of 2000-2500 r / min and a pulverization time of 3-5 minutes; and the sieving is carried out using an 80-mesh standard inspection sieve.

[0016] Furthermore, the amount of deionized water added is 8-12 times the mass of the mixed raw material powder; the pH is adjusted using a 0.1 mol / L citric acid-sodium citrate buffer solution, and the pH of the system after adjustment is 4.4-4.6; the incubation temperature for enzymatic hydrolysis is 50℃, and the enzymatic hydrolysis time is 2 hours.

[0017] Furthermore, the ultrasonic extraction frequency is 40kHz, the extraction temperature is 60℃, the power density is 0.3-0.5W / cm², the ultrasonic mode is intermittent, with a cycle of 5s ultrasonic operation followed by a 2s pause, and the cumulative ultrasonic duration is 1h; the centrifugation uses a horizontal spiral centrifuge with a centrifugation speed of 4000-6000r / min, a centrifugal force of 3000-3500×g, and a centrifugation time of 10-15min.

[0018] Furthermore, the ultrafiltration device uses a polyvinylidene fluoride ultrafiltration membrane with a molecular weight cutoff of 3000 Da, an operating pressure of 0.1-0.2 MPa, and a cross-flow velocity of 1.0-1.2 m / s; the nanofiltration device uses a polyamide nanofiltration membrane with a molecular weight cutoff of 1000 Da, an operating pressure of 0.3-0.5 MPa, and a concentration factor of 5-8 times.

[0019] Furthermore, the concentrated liquid is spread on the freeze-drying tray to a thickness of 5-8 mm; the pre-freezing temperature of the vacuum freeze-drying is -40℃ to -30℃, the pre-freezing holding time is 2 hours, the vacuum degree is 10-30 Pa, the heating rate is 1℃ / h, and the temperature is held at -10℃ for 8 hours, with a total drying time of 12-24 hours.

[0020] Compared with existing technologies, this cardiac glycoside compound based on Lepidium apetalum and Poria cocos has the following beneficial effects:

[0021] I. This invention achieves efficient extraction of cardiac glycosides by rationally combining the raw materials of Lepidium apetalum and Poria cocos with the synergistic effect of a compound enzyme biphasic enzymatic hydrolysis system. Multiple enzymes in the compound enzyme preparation work together to disrupt the cell wall structure and macromolecular binding state of the raw materials, promoting the full release of active ingredients. Simultaneously, the addition of an ethanol-water solution optimizes the extraction environment and improves the dissolution efficiency of the target components. Subsequent ultrasound-assisted extraction enhances the mass transfer process, and precise purification using membrane separation technology effectively removes impurities and enriches cardiac glycosides. Vacuum freeze-drying then preserves the activity of the components, ultimately yielding a high-purity extract. The entire preparation process is tightly integrated, achieving simultaneous improvement in raw material utilization and product purity. This provides a reliable pathway for the large-scale preparation of cardiac glycosides, meeting the demand for high-activity, high-purity raw materials in relevant application scenarios.

[0022] II. This invention optimizes the synergistic design of raw material pretreatment and preparation processes, balancing extraction efficiency and product stability. The raw material pretreatment process, through methods such as stir-frying and low-temperature freeze-drying, improves the physicochemical properties of the raw materials, creating favorable conditions for subsequent enzymatic hydrolysis and extraction. The combination of biphasic enzymatic hydrolysis with ultrasound-assisted extraction overcomes the limitations of traditional extraction methods, shortening the extraction cycle and reducing the loss of effective components. The precise application of membrane separation technology achieves targeted purification, avoiding the loss and property changes of effective components in traditional purification methods. Vacuum freeze-drying maximizes the preservation of the extract's bioactivity. This preparation method is standardized and controllable, ensuring product quality stability and uniformity while reducing energy consumption and pollution during production. It provides new technical support for the efficient preparation of cardiac glycosides from natural products, possessing broad application prospects and practical value.

[0023] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0025] Figure 1 The flowchart shows the cardiac glycosides based on Lepidium apetalum and Poria cocos.

[0026] Figure 2 This is a schematic diagram of data transmission of cardiac glycosides based on Lepidium apetalum and Poria cocos.

[0027] Figure 3 This diagram illustrates the pretreatment process and data transmission preparation of Lepidium apetalum seeds according to the present invention. Detailed Implementation

[0028] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0029] Example 1:

[0030] S100. Raw Material Pretreatment: Select *Lepidium apetalum* seeds free from mold and impurities, manually removing stones, twigs, and other foreign objects to ensure raw material purity. Place the treated *Lepidium apetalum* seeds into a drum-type roasting device, set the roasting temperature, start the device and adjust the drum speed, and continue roasting for the preset time. After roasting, remove the seeds and allow them to cool naturally to room temperature. Then, place the cooled *Lepidium apetalum* seeds into a universal pulverizer, set the pulverizing speed and time, and after pulverizing, screen them through an 80-mesh standard inspection sieve. Collect the undersized particles, which is the *Lepidium apetalum* seed pretreatment powder. Unpulverized particles on the sieve are returned to the pulverizer for further pulverization until all particles pass through the sieve.

[0031] Select fresh, unrotten Poria cocos, peel off the surface skin with a knife, cut the peeled Poria cocos into uniform pieces, put them into a low-temperature freeze pulverizer, and pulverize them in a low-temperature environment to obtain pre-treated Poria cocos powder. This pulverization method can avoid the destruction of active ingredients in Poria cocos due to high temperature.

[0032] The pre-treated Lepidium seed powder and the pre-treated Poria cocos powder prepared above were placed in a mixer at a weight ratio of 1:1 and stirred for 15 minutes to ensure that the two powders were mixed evenly, thus obtaining a mixed raw material powder, as shown below. Figure 3 As shown.

[0033] S200, Compound Enzyme Biphasic Hydrolysis: Weigh 100g of mixed raw material powder and place it in an enzymatic hydrolysis vessel. Add deionized water to the vessel and stir to fully disperse the mixed raw material powder in the water, forming a uniform suspension. Then add citric acid-sodium citrate buffer solution to the suspension while stirring, and monitor the pH value of the system in real time with a pH meter until the pH value reaches the set range.

[0034] According to the mass ratio of the mixed raw material powder, weigh out pectinase, cellulase and β-glucosidase respectively, mix the three enzymes evenly to obtain a compound enzyme preparation, and slowly add it to the above suspension, stirring evenly. Then add 50% ethanol aqueous solution to the enzymatic hydrolysis tank, the amount of which is 20% of the total volume of the compound enzyme biphasic enzymatic hydrolysis system, and stir to fully mix the ethanol aqueous solution with the suspension.

[0035] Adjust the temperature of the enzymatic hydrolysis tank to the set value and maintain this temperature for incubation and enzymatic hydrolysis. Stir the tank every 20 minutes for 5 minutes each time to ensure that the enzymatic hydrolysis reaction proceeds evenly. After the preset time has elapsed, the enzymatic hydrolysis mixture is obtained.

[0036] S300, Ultrasonic-Assisted Extraction: First, clean and dry the ultrasonic extraction tank to ensure no impurities remain. Slowly transfer the enzymatic hydrolysis mixture into the ultrasonic extraction tank through a clean delivery pipe, avoiding leakage or contamination during the transfer process. After transfer, close the tank door and check the sealing performance to ensure the tank is airtight. According to process requirements, precisely set the ultrasonic extraction frequency, extraction temperature, and power density in the equipment control system, select the intermittent ultrasonic mode, and define the ultrasonic working time and pause time as a complete cycle. Start the ultrasonic extraction equipment, and the equipment operates according to the set parameters. The cavitation and mechanical effects of ultrasound enhance the dissolution of active ingredients, and the pause phase facilitates uniform diffusion of substances within the system. Once the cumulative ultrasonic time reaches the set value, the equipment automatically stops extraction.

[0037] After extraction, wait for the temperature inside the tank to drop slightly, then open the tank door and carefully transfer the material to a pre-cleaned horizontal screw centrifuge. Set the preset centrifugal speed and centrifugal force in the centrifuge control system, start the equipment, and begin centrifugation once it stabilizes. During centrifugation, solid impurities settle under centrifugal force, forming a clear liquid on the surface. After centrifugation for the preset time, turn off the centrifuge and slowly collect the supernatant, which is the ultrasonic extract. Use a clean container during collection to avoid contamination. Collect the lower sediment and process it according to standard procedures.

[0038] S400, Membrane Separation, Purification and Concentration: The ultrasonic extract is passed into an ultrafiltration device. This ultrafiltration device uses a polyvinylidene fluoride ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The operating pressure and cross-flow velocity of the ultrafiltration device are adjusted to make the ultrasonic extract form a stable cross-flow state on the membrane surface. Through the retention effect of the ultrafiltration membrane, large molecular impurities such as proteins and polysaccharides in the extract are removed, and the ultrafiltration permeate is collected.

[0039] The ultrafiltration permeate is passed into a nanofiltration device, which uses a polyamide nanofiltration membrane with a molecular weight cutoff of 1000 Da. The operating pressure of the nanofiltration device is adjusted to concentrate the ultrafiltration permeate until the concentration factor reaches the set value, thus obtaining a concentrated solution.

[0040] S500 Vacuum Freeze-Drying: Evenly spread the concentrated liquid on a freeze-drying tray, controlling the thickness. Place the tray into the vacuum freeze dryer. Close the dryer door, start the equipment, and first lower the chamber temperature to the pre-freezing temperature. Maintain this temperature for pre-freezing and insulation. After pre-freezing, adjust the vacuum to the set range, and then heat according to the set heating rate. When the temperature reaches -10℃, maintain this temperature for insulation. After insulation, continue heating to room temperature. After the total set drying time is reached, remove the tray and collect the dried solid material, which is the cardiac glycoside compound based on Lepidium apetalum and Poria cocos. Figure 1 As shown.

[0041] Example 2:

[0042] S100. Raw Material Pretreatment: Select qualified *Lepidium apetalum* seeds, remove impurities by screening, and then place them in a drum-type roasting device. Roast them according to the set roasting temperature, drum speed, and roasting time. After roasting, cool them to room temperature. Put the cooled *Lepidium apetalum* seeds into a universal pulverizer, set the corresponding pulverizing speed and pulverizing time, and pulverize them. The pulverized product passes through an 80-mesh standard inspection sieve, and the *Lepidium apetalum* seed pretreatment powder that passes through the sieve is collected.

[0043] High-quality Poria cocos was selected, peeled, and cut into pieces. It was then pulverized using a low-temperature cryogenic pulverizer to obtain pre-treated Poria cocos powder. The pre-treated Lepidium apetalum powder and the pre-treated Poria cocos powder were then placed in a mixing device at a weight ratio of 1:2 and thoroughly stirred to ensure uniform mixing, resulting in a mixed raw material powder.

[0044] S200, Compound Enzyme Biphasic Hydrolysis: Weigh 100g of mixed raw material powder and place it in an enzymatic hydrolysis container. Add deionized water and stir to disperse evenly. Add citric acid-sodium citrate buffer solution to adjust the pH of the system to the set range. Weigh pectinase, cellulase, and β-glucosidase according to the ratio, mix them to prepare a compound enzyme preparation, and add it to the enzymatic hydrolysis system. Stir evenly.

[0045] Add 50% ethanol aqueous solution to the enzymatic hydrolysis system, the amount of which is 25% of the total volume of the compound enzyme biphasic enzymatic hydrolysis system. Stir to mix the system evenly. Place the enzymatic hydrolysis container in a constant temperature water bath and maintain the set enzymatic hydrolysis temperature for incubation. Stir regularly during the enzymatic hydrolysis process to ensure complete enzymatic hydrolysis. After the set enzymatic hydrolysis time is reached, the enzymatic hydrolysis mixture is obtained.

[0046] S300, Ultrasonic-Assisted Extraction: Thoroughly clean the ultrasonic extraction tank beforehand, removing any residual impurities and drying it to ensure a clean extraction environment. Slowly transfer the prepared enzymatic hydrolysis mixture into the ultrasonic extraction tank using a sterile conveyor, avoiding spillage or introduction of contaminants during the transfer. After closing the tank door and confirming a secure seal, precisely set the ultrasonic extraction frequency, extraction temperature, and power density parameters according to the process requirements on the equipment control panel. Select the intermittent ultrasonic mode, specifying the duration of ultrasonic operation and pauses; these alternating periods constitute a complete extraction cycle. After starting the equipment, ultrasound accelerates the dissolution of active ingredients through cavitation and mechanical vibration. The pause phase facilitates uniform diffusion of substances within the system. Once the cumulative ultrasonic duration reaches the set value, the extraction program automatically terminates.

[0047] After extraction, once the temperature inside the tank has dropped to near room temperature, the material is smoothly transferred to a pre-treated horizontal screw centrifuge. The preset centrifugal speed and centrifugal force are set in the centrifuge control system. The equipment is started and centrifuged until it stabilizes. During centrifugation, solid impurities settle under centrifugal force, forming a clear liquid on the surface. After centrifugation for the set time, the equipment is stopped, and the supernatant is carefully collected in a clean container; this is the ultrasonic extract.

[0048] S400, Membrane Separation, Purification and Concentration: The ultrasonic extract is passed into an ultrafiltration device. The ultrafiltration membrane of this device is made of polyvinylidene fluoride with a molecular weight cutoff of 3000 Da. The operating pressure and crossflow velocity are adjusted to perform ultrafiltration treatment, remove macromolecular impurities, and collect the ultrafiltration permeate.

[0049] The ultrafiltration permeate is passed into a nanofiltration device. The nanofiltration membrane is made of polyamide with a molecular weight cutoff of 1000 Da. The operating pressure is adjusted to concentrate the permeate until the set concentration factor is reached, thus obtaining a concentrated solution.

[0050] S500, Vacuum Freeze-Drying: Spread the concentrated liquid evenly on a freeze-drying tray, controlling the thickness of the spread, and place it in a vacuum freeze dryer. First, lower the temperature inside the dryer to the pre-freezing temperature, hold it at this temperature for a period of time, then adjust the vacuum level and raise the temperature according to the set heating rate. When the temperature reaches -10℃, hold it at this temperature, then continue to raise the temperature to room temperature. After the total drying time reaches the set value, remove the dried product, which is a cardiac glycoside compound based on Lepidium apetalum and Poria cocos, such as... Figure 2 As shown.

[0051] Example 3:

[0052] S100. Raw material pretreatment: The seeds of Lepidium apetalum are screened to remove impurities and then placed in a drum-type frying equipment. They are fryed according to the specified frying temperature, drum speed and frying time. After frying, they are cooled to room temperature and then pulverized by a universal pulverizer. The appropriate pulverizing speed and pulverizing time are set. After pulverization, they are screened through an 80-mesh standard inspection sieve to obtain Lepidium apetalum pretreated powder.

[0053] After peeling and cutting, Poria cocos was cryogenically pulverized to obtain pre-treated Poria cocos powder. The pre-treated Lepidium apetalum powder and the pre-treated Poria cocos powder were then mixed evenly at a weight ratio of 2:1 to obtain a mixed raw material powder.

[0054] S200, Compound Enzyme Biphasic Hydrolysis: Place 100g of mixed raw material powder into an enzymatic hydrolysis tank, add deionized water, stir to disperse, and adjust the pH value to the set range with citrate-sodium citrate buffer solution. Add a compound enzyme preparation composed of pectinase, cellulase and β-glucosidase in a certain proportion, stir evenly, and then add 50% ethanol aqueous solution, the amount of which is 30% of the total volume of the compound enzyme biphasic hydrolysis system. Continue stirring until evenly mixed.

[0055] Place the enzymatic hydrolysis vessel in a constant temperature environment and maintain the set enzymatic hydrolysis temperature for incubation. Stir regularly during the enzymatic hydrolysis process to ensure that the enzymatic hydrolysis reaction is complete. After the set time is reached, an enzymatic hydrolysis mixture is obtained.

[0056] S300, Ultrasonic-Assisted Extraction: First, clean and dry the ultrasonic extraction tank to ensure no impurities remain. Smoothly transfer the enzymatic hydrolysis mixture into the tank through a clean pipeline, close the tank door, and confirm a good seal. In the equipment control system, precisely set the ultrasonic extraction frequency, extraction temperature, and power density according to process requirements, and start extraction using intermittent ultrasonic mode. The cavitation and mechanical effects of ultrasound accelerate the dissolution of active ingredients. Once the cumulative ultrasonic duration meets the set requirements, the equipment automatically stops. After extraction, transfer the material to a pre-treated horizontal spiral centrifuge, set the preset centrifugal speed and centrifugal force, and start the equipment for stable operation. During centrifugation, solid impurities settle. After centrifugation for the specified time, stop the machine and collect the clear liquid at the top in a clean container; this is the ultrasonic extract.

[0057] S400, Membrane Separation, Purification, and Concentration: The ultrasonic extract is first processed by an ultrafiltration unit. The ultrafiltration membrane is made of polyvinylidene fluoride with a molecular weight cutoff of 3000 Da. The operating pressure and cross-flow rate are adjusted to remove large molecular impurities, and the ultrafiltration permeate is collected. The ultrafiltration permeate then enters a nanofiltration unit. The nanofiltration membrane is made of polyamide with a molecular weight cutoff of 1000 Da. The operating pressure is adjusted to concentrate the solution to the set concentration factor, yielding the concentrated solution.

[0058] S500, Vacuum Freeze-Drying: Spread the concentrated liquid evenly on a freeze-drying tray, controlling the thickness, and place it in a vacuum freeze dryer. Perform pre-freezing, vacuuming, and heating and holding operations in sequence, strictly controlling the pre-freezing temperature, pre-freezing and holding time, vacuum degree, heating rate, and total drying time. After drying, collect the product, which is the cardiac glycoside compound based on Lepidium apetalum and Poria cocos.

[0059] Comparative example:

[0060] S100. Raw material pretreatment: Select Lepidium apetalum and Poria cocos of the same quality as in Example 1, and prepare Lepidium apetalum pretreated powder and Poria cocos pretreated powder respectively according to the raw material pretreatment method in Example 1. Then mix them evenly according to the same weight ratio to obtain mixed raw material powder.

[0061] S200, Single Solvent Extraction: Weigh 100g of mixed raw material powder and put it into the extraction tank. Add 70% ethanol aqueous solution. The amount of ethanol aqueous solution added is 10 times the mass of the mixed raw material powder. After stirring evenly, place the extraction tank in a constant temperature water bath at 80℃ and reflux for 2 hours. Stir once every 30 minutes during the extraction process.

[0062] S300. Filtration and Concentration: After extraction, the extract is filtered through ordinary filter paper, and the filtrate is collected. The residue is then refluxed once with an ethanol-water solution of the same concentration and volume, and the two filtrates are combined. The combined filtrate is placed in a rotary evaporator and concentrated under reduced pressure at 60℃ and 0.08MPa until the concentrate reaches a certain concentration.

[0063] S400, Drying: Place the concentrate in a forced-air drying oven and dry it at 60°C until constant weight to obtain the extract.

[0064] To visually demonstrate the technical effects of the embodiments of the present invention and the comparative examples, the test results are summarized in the table below:

[0065] Group Total cardiac glycoside purity Lepidium glycoside content Example 1 86% 61% Example 2 88% 63% Example 3 87% 62% Comparative Example 65% 40%

[0066] The comparison of the above examples and comparative examples shows that the process of combining compound enzyme biphasic enzymatic hydrolysis with ultrasound-assisted extraction, membrane separation purification, and vacuum freeze-drying can effectively improve the purity of cardiac glycosides and the content of lepidoside. The cardiac glycosides based on Lepidium apetalum and Poria cocos prepared in Examples 1 to 3 all achieved a total cardiac glycoside purity of over 85% and a lepidoside content of over 60%, significantly superior to the products obtained by the comparative examples using the traditional single-solvent reflux extraction process. This is because the raw material pretreatment process of the present invention can improve the characteristics of the raw materials, laying a good foundation for subsequent extraction; the synergistic effect of multiple enzymes in the compound enzyme biphasic enzymatic hydrolysis system can destroy the cell wall structure of the raw materials, promoting the full release of effective components; ultrasound-assisted extraction can enhance the mass transfer process and improve extraction efficiency; membrane separation technology can accurately remove impurities and achieve enrichment of effective components; and vacuum freeze-drying can maximize the preservation of the activity of effective components. The synergistic cooperation of each process step forms a complete technical system, ensuring the high quality of the product. In contrast, the traditional extraction process used in comparison involves harsh conditions that easily lead to the loss or destruction of active ingredients, and the purification effect is limited, resulting in low product purity and low content of target components. The technical solution of this invention is standardized, highly controllable, and can stably prepare high-purity cardiac glycosides, possessing significant practical application value.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A cardiac glycoside compound based on Lepidium apetalum and Poria cocos, characterized in that, It consists of the following components: The ingredients include Lepidium apetalum-Poria cocos extract powder, a compound enzyme preparation, and a 50% (v / v) ethanol aqueous solution; the compound enzyme preparation consists of pectinase, cellulase, and β-glucosidase.

2. The cardiac glycoside compound based on Lepidium apetalum and Poria cocos according to claim 1, characterized in that, The Lepidium apetalum-Poria cocos extract powder is obtained by mixing Lepidium apetalum powder and frozen Poria cocos powder, followed by enzymatic hydrolysis, ultrasonic extraction, membrane separation, and drying. The total cardiac glycoside purity of the Lepidium apetalum-Poria cocos extract powder is ≥85%, and the content of Lepidium apetalum glycosides is ≥60%.

3. The cardiac glycoside compound based on Lepidium apetalum and Poria cocos according to claim 1, characterized in that, The amounts of pectinase, cellulase, and β-glucosidase added to the compound enzyme preparation are 0.5%, 1.0%, and 0.2% respectively, based on the mass of the mixed raw material powder.

4. The cardiac glycoside compound based on Lepidium apetalum and Poria cocos according to claim 1, characterized in that, The amount of the 50% ethanol aqueous solution added is 20%-30% of the total volume of the compound enzyme biphasic enzymatic hydrolysis system.

5. A method for preparing cardiac glycosides based on Lepidium apetalum and Poria cocos, the method being used to prepare the cardiac glycosides based on Lepidium apetalum and Poria cocos as described in any one of claims 1-4, characterized in that, The specific steps of this method are as follows: S100. Raw material pretreatment: Select Lepidium apetalum seeds, remove impurities, stir-fry, cool, crush, and sieve to obtain Lepidium apetalum seed pretreatment powder; Select Poria cocos seeds, remove the outer skin, cut into pieces, freeze-dry and crush at low temperature to obtain Poria cocos pretreatment powder; Mix the two pretreatment powders in a certain proportion to obtain mixed raw material powder. S200, compound enzyme biphasic enzymatic hydrolysis: Add deionized water to the mixed raw material powder, stir and disperse, adjust the pH with buffer solution, add compound enzyme preparation and 50% ethanol aqueous solution, incubate and enzymatically hydrolyze to obtain enzymatic hydrolysis mixture; S300, Ultrasonic-assisted extraction: Transfer the enzymatic hydrolysis mixture to an ultrasonic extraction tank for ultrasonic extraction. After extraction, centrifuge and collect the ultrasonic extract. S400, Membrane separation purification and concentration: The ultrasonic extract is sequentially processed by an ultrafiltration device and a nanofiltration device to obtain a concentrated solution; S500, Vacuum freeze drying: After spreading the concentrate evenly, place it in a vacuum freeze dryer to dry, and obtain the extract.

6. The method for preparing cardiac glycoside compounds based on Lepidium apetalum and Poria cocos according to claim 5, characterized in that, In step S100, the raw material pretreatment, the stir-frying of *Lepidium apetalum* seeds is carried out using a drum-type stir-frying device, with a stir-frying temperature of 120℃, a stir-frying time of 10 min, and a drum rotation speed of 15-20 r / min; the pulverization is carried out using a universal pulverizer, with a pulverization speed of 2000-2500 r / min and a pulverization time of 3-5 min; and the sieving is carried out using an 80-mesh standard inspection sieve.

7. The method for preparing cardiac glycoside compounds based on Lepidium apetalum and Poria cocos according to claim 5, characterized in that, In step S200, the amount of deionized water added is 8-12 times the mass of the mixed raw material powder; the pH is adjusted using a 0.1 mol / L citric acid-sodium citrate buffer solution, and the pH of the system after adjustment is 4.4-4.6; the incubation temperature for enzymatic hydrolysis is 50℃, and the hydrolysis time is 2 hours.

8. The method for preparing cardiac glycoside compounds based on Lepidium apetalum and Poria cocos according to claim 5, characterized in that, In step S300, ultrasonic-assisted extraction, the ultrasonic extraction frequency is 40kHz, the extraction temperature is 60℃, the power density is 0.3-0.5W / cm², the ultrasonic mode is intermittent, and one cycle consists of 5 seconds of ultrasonic operation followed by a 2-second pause, with a cumulative ultrasonic duration of 1 hour; the centrifugation uses a horizontal spiral centrifuge with a centrifugation speed of 4000-6000 r / min, a centrifugal force of 3000-3500 × g, and a centrifugation time of 10-15 min.

9. The method for preparing cardiac glycoside compounds based on Lepidium apetalum and Poria cocos according to claim 5, characterized in that, In step S400, membrane separation, purification, and concentration, the ultrafiltration device uses a polyvinylidene fluoride ultrafiltration membrane with a molecular weight cutoff of 3000 Da, an operating pressure of 0.1-0.2 MPa, and a cross-flow velocity of 1.0-1.2 m / s; the nanofiltration device uses a polyamide nanofiltration membrane with a molecular weight cutoff of 1000 Da, an operating pressure of 0.3-0.5 MPa, and a concentration factor of 5-8 times.

10. The method for preparing cardiac glycoside compounds based on Lepidium apetalum and Poria cocos according to claim 5, characterized in that, In step S500, during vacuum freeze drying, the concentrated liquid is spread on the freeze drying tray to a thickness of 5-8 mm; the pre-freezing temperature of the vacuum freeze drying is -40℃ to -30℃, the pre-freezing holding time is 2 hours, the vacuum degree is 10-30 Pa, the heating rate is 1℃ / h, and the temperature is held at -10℃ for 8 hours after heating, with a total drying time of 12-24 hours.

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