Naoxinqing capsule and preparation method of extract of Naoxinqing capsule

Abstract

The invention provides a Naoxinqing capsule and a preparation method of an extract of the Naoxinqing capsule, belongs to the technical field of medicines, and aims to solve the technical problems of insufficient quality control and curative effect stability in the aspect of treating cardiovascular and cerebrovascular diseases in the conventional traditional Chinese medicine preparation. The Naoxinqing capsule comprises a persimmon leaf extract, and a preparation method of the persimmon leaf extract comprises the following steps: S1, taking a dried and crushed persimmon leaf raw material; s2, obtaining an extracting solution by adopting a water extraction method or an enzyme-assisted extraction method; s3, concentrating and adding ethanol until the alcohol content reaches 80%-90%; s4, washing the precipitate with an ethanol solution, collecting a washing solution, standing, and combining with the supernatant; s5, carrying out rotary evaporation on the filtrate to recover the ethanol to obtain an extract; s6, dissolving in water, filtering, and extracting filtrate for multiple times by using ethyl acetate; step S7, performing rotary evaporation to recover ethyl acetate to obtain thick paste; and step S8, low-temperature drying. According to the invention, the purity, the stability and the bioavailability of the medicine are high, the content of effective components can be accurately controlled, and the stable and reliable quality of the medicine is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and particularly relates to a Naoxinqing capsule and a preparation method of an extract thereof. BACKGROUND

[0003] Traditional Chinese medicines have unique advantages in treating cardiovascular and cerebrovascular diseases. The multi-component and multi-target mechanism can comprehensively regulate the body function, improve blood circulation, protect vascular endothelium, reduce blood viscosity, etc., and has relatively small side effects. For example, traditional Chinese medicinal materials such as Danshen and Chuanxiong have accumulated rich experience in the prevention and treatment of cardiovascular and cerebrovascular diseases in clinical application. However, the existing Chinese medicine preparations have defects in quality control, efficacy stability, and convenience for taking, etc. The content of effective components of many Chinese medicine preparations fluctuates greatly, and the preparation process lacks standardization, so that the quality of different batches of products is uneven, which affects the reliability of clinical efficacy. At the same time, some Chinese medicine dosage forms (such as pills and decoctions) are inconvenient to take, and the patient compliance is poor, which is not conducive to long-term treatment. SUMMARY

[0004] In view of the defects of the prior art, the present application provides a Naoxinqing capsule and a preparation method of an extract thereof, so as to solve the problems of insufficient quality control and efficacy stability of the existing Chinese medicine preparations in treating cardiovascular and cerebrovascular diseases. The preparation method of the extract can improve the content of quercetin and kaempferol in the extraction process of Naoxinqing, and improve the utilization rate of persimmon leaves. The prepared Naoxinqing capsule is convenient to carry and take, has high bioavailability, reliable quality, and the whole process is more complete, which is suitable for large-scale production.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] A preparation method of a persimmon leaf extract, the extract being used for preparing a Naoxinqing capsule for treating cardiovascular and cerebrovascular diseases; the preparation method comprising the following steps:

[0007] Step S1, taking dry and crushed persimmon leaf raw materials;

[0008] Step S2, obtaining an extract liquid by using a water extraction method or an enzyme-assisted extraction method;

[0009] Step S3, concentrating the extract liquid, adding ethanol to make the alcohol content reach 80%-90%, and standing to obtain supernatant and precipitate;

[0010] Step S4, washing the precipitate with an ethanol solution, collecting the washing liquid, standing, and then sucking the supernatant to combine with the supernatant in step S3;

[0011] Step S5, recovering ethanol by rotary evaporation after the combined filtrate, and obtaining an extract;

[0012] Step S6, the obtained extract is dissolved with water and filtered, and the filtrate is extracted with ethyl acetate for multiple times;

[0013] Step S7, the ethyl acetate liquid extracted for multiple times is combined, and then ethyl acetate is recovered by rotary evaporation to obtain a thick extract;

[0014] Step S8, low-temperature drying.

[0015] The main raw material of the brain heart clear capsule is derived from natural plants, can play the advantages of comprehensive regulation of traditional Chinese medicine, overcome the shortcomings of existing treatment drugs, through the preparation process, can effectively improve the purity, stability and bioavailability of the drug, accurately control the content of effective components, and ensure the stable and reliable quality of the drug.

[0016] Optionally, the water extraction method in step S2 comprises the following process:

[0017] Step S21, first extraction: 12 times the amount of water is added, soaked for 3 hours and then decocted for 2 hours, and then filtered to obtain the first decoction;

[0018] Step S22, second extraction: the filter residue after the first extraction is added with 10 times the amount of water, and then decocted for 1 hour, and then filtered to obtain the second decoction;

[0019] Step S23, the decoctions extracted for two times are mixed to obtain the extraction liquid.

[0020] The water extraction process is stable and feasible, and the content of quercetin in the water layer is only 0.0006%, which indicates that most of the quercetin has been extracted by ethyl acetate, and the extraction is complete.

[0021] Optionally, the enzyme-assisted extraction method in step S2 is selected from a complex enzyme method, and the specific steps of the complex enzyme method comprise:

[0022] Step T1, a complex enzyme with a weight of 5% of persimmon leaves is added, wherein the mass ratio of cellulase to pectinase is 1:1, and the enzyme is hydrolyzed in a sodium phosphate dibasic-citric acid buffer solution;

[0023] Step T2, after enzyme hydrolysis, heating is performed for inactivation, and water is added for decoction and extraction.

[0024] Optionally, in step T1, the amount of the sodium phosphate dibasic-citric acid buffer solution is 10 times the volume of the weight of the persimmon leaves, and the enzyme is hydrolyzed at a pH of 3.5-3.8 and a temperature of 45-55℃ for 1-3 hours.

[0025] Optionally, in step T2, after enzyme hydrolysis, heating is performed to 90-100℃ for 5-15 minutes for decoction, and then the first decoction liquid is obtained after filtration, 10 times the volume of the sodium phosphate dibasic-citric acid buffer solution is added to the filter residue after the first extraction, and then decoction is performed for 1 hour, and then the extraction liquid is obtained after the two decoctions are combined.

[0026] The present application further compares the complex enzyme method with the original water extraction method on persimmon leaves. The rutin yield of the complex enzyme method is 18% higher than that of the original process, reaching 0.026% (p<0.05), and the yield of kaempferol is doubled to 0.002%. This may be related to the specific opening of β-1, 4 glycosidic bond by complex enzyme through enzymatic hydrolysis, releasing the bound flavonoids.

[0027] Optionally, the enzyme-assisted extraction method in step S2 is selected from the Aspergillus niger crude enzyme solution enzymolysis method, and the specific steps of the Aspergillus niger crude enzyme solution enzymolysis method include:

[0028] Step X1, crude enzyme solution preparation: inoculate Aspergillus niger spore solution into enzyme-producing medium to obtain crude enzyme solution;

[0029] Step X2, enzymolysis treatment: add 5 mL of crude enzyme solution per gram of persimmon leaves for enzymolysis;

[0030] Step X3, after enzymolysis, add water for decocting and extracting.

[0031] Optionally, in step X1, the method for obtaining Aspergillus niger spore solution is as follows: inoculate Aspergillus niger strain into PDA medium, incubate at 30°C for 7-9 days, then scrape the spores into sterile normal saline, stir to suspend the spores, and obtain Aspergillus niger spore solution.

[0032] Optionally, in step X2, inoculate Aspergillus niger spore solution into enzyme-producing medium containing 1-3% corn starch and 0.2-0.6% protein peptone, incubate at 28-32°C and 150 rpm for 48-68 h, and collect the filtrate by suction filtration as crude enzyme solution.

[0033] Optionally, in step X2, react at 30°C for 8-12 hours.

[0034] Optionally, the enzyme-producing medium contains 2% corn starch, 0.4% protein peptone, 0.6% (NH4)2SO4, 0.5% KH2PO4, 0.1% MgSO4·7H2O, 0.05% CaCl2, 0.01% FeSO4·7H2O, and pH 6.0.

[0035] The present application further has the promoting effect on the release of part of active components of persimmon leaves by the crude enzyme solution of Aspergillus niger through enzymatic hydrolysis, and the detection sensitivity of flavonoids is improved: the enzymatic hydrolysis treatment makes rutin (0.001%), quercetin (0.0001%) and kaempferol (0.0002%) reach the quantifiable level, while the original process leads to the peak area of HPLC lower than the detection limit due to low extraction efficiency; the enzymatic hydrolysis specifically decomposes plant cell wall polysaccharides to promote the dissociation and release of bound flavonoids; the extraction liquid after enzymatic hydrolysis treatment maintains the color characteristics equivalent to the original process, proving that the process improves the yield of effective components without affecting the appearance stability of the product. In addition, compared with the complex enzyme method (directly adding enzymes is higher in cost for industrial production), the crude enzyme solution of Aspergillus niger can significantly reduce the production cost. Therefore, the optimized preparation process can reduce the production cost, improve the production efficiency, and is conducive to the large-scale production and wide application of the product, providing safer, more effective and convenient treatment options for patients with cardiovascular and cerebrovascular diseases, and has significant social and economic benefits, and positive significance for promoting the modernization development of traditional Chinese medicine.

[0036] Optionally, in the step S3, the extraction liquid is concentrated and ethanol is added to make the alcohol content reach 85%;

[0037] Optionally, the concentrated extraction liquid is placed in a water bath and concentrated to a relative density of 1.12-1.15, and the concentration conditions are as follows: temperature 60-65 DEG C, vacuum degree 0.06-0.08 Mpa;

[0038] Optionally, the amount of ethanol added is 3-5 times the volume of the concentrated liquid.

[0039] Optionally, the standing time is 24 h.

[0040] Optionally, in the step S4, the concentration of the ethanol washing liquid is 60-70%, the amount of use is 3-4 times the amount of the precipitate, and the washing time is twice, and the stirring time of each time is 20-40 min.

[0041] Optionally, in the step S5, the combined filtrate is recovered by rotary evaporation at a water bath temperature of 55 DEG C to obtain the extract.

[0042] Optionally, in the step S6, the amount of water added for dissolving the extract is 2 times the volume of the extract, and the filtrate is extracted with ethyl acetate four times.

[0043] Optionally, in the step S7, the combined ethyl acetate liquid is recovered by rotary evaporation at a water bath temperature of 55 DEG C to obtain the thick paste.

[0044] A preparation method of Naoxinjing capsules, comprising: preparing persimmon leaf extract by any one of the above methods; mixing the extract with pharmaceutical excipients at a ratio of 1:2 to granulate; and packaging the capsules.

[0045] The brain heart clear capsule for treating cardiovascular and cerebrovascular diseases is prepared from persimmon leaf extract obtained by adopting the aspergillus niger crude enzyme liquid enzymolysis method (carrying out enzymolysis on persimmon leaves by using the crude enzyme liquid generated in the aspergillus niger culture process and then carrying out water extraction) and the compound enzyme method (adding 5% compound enzyme (cellulase-pectinase) to persimmon leaves to carry out enzymolysis and then carrying out water extraction), the aspergillus niger crude enzyme liquid enzymolysis method and the compound enzyme method can improve the content of quercetin and kaempferol, the preparation process of the brain heart clear capsule is stable and reliable, and is suitable for large-scale production. In addition, the aspergillus niger crude enzyme liquid enzymolysis is optimal, and the aspergillus niger crude enzyme liquid enzymolysis can save cost.

[0046] Compared with the prior art, the brain heart clear capsule has the following beneficial effects:

[0047] 1. The brain heart clear capsule is mainly prepared from natural plants, can play the advantages of comprehensive regulation of traditional Chinese medicine, and overcomes the shortcomings of the existing treatment drugs. Through the preparation process, the purity, stability and bioavailability of the drug can be effectively improved, the content of the effective components can be accurately controlled, and the quality of the drug is stable and reliable.

[0048] 2. The brain heart clear capsule is further prepared by carrying out the water extraction step after the pretreatment (including the aspergillus niger crude enzyme liquid enzymolysis method and the compound enzyme method) on the persimmon leaves. Compared with the original water extraction method, the rutin yield of the compound enzyme method is increased by 18% and reaches 0.026% (p<0.05), and the kaempferol yield is doubled to 0.002%. This may be related to the specific opening of the beta-1,4 glycosidic bond by the compound enzyme through the enzymolysis effect to release the combined flavonoids. The aspergillus niger crude enzyme liquid enzymolysis method promotes the release of part of the active components of the persimmon leaves, and the detection sensitivity of the flavonoids is improved. The enzymolysis treatment makes the rutin (0.001%), quercetin (0.0001%) and kaempferol (0.0002%) reach the quantifiable level, while the HPLC peak area of the original process is lower than the detection limit due to the low extraction efficiency. The enzymolysis effect specifically decomposes the polysaccharides in the plant cell wall to promote the dissociation and release of the combined flavonoids. The extraction liquid after the enzymolysis treatment maintains the color characteristics as the original process, which proves that the process improves the yield of the effective components while not affecting the appearance stability of the product. The direct enzyme addition method has high industrialization cost, therefore, preferably, the aspergillus niger crude enzyme liquid is used to significantly reduce the production cost. Therefore, the optimized preparation process can reduce the production cost, improve the production efficiency, is beneficial to the large-scale production and wide application of the product, provides safer, more effective and more convenient treatment options for patients with cardiovascular and cerebrovascular diseases, has significant social and economic benefits, and has a positive significance for promoting the modernization development of traditional Chinese medicine.

[0049] 3、The brain heart clear capsule for treating cardiovascular and cerebrovascular diseases is prepared from persimmon leaf extract obtained by using Aspergillus niger crude enzyme solution enzymolysis (carrying out enzymolysis on persimmon leaves by using crude enzyme solution produced in the process of culturing Aspergillus niger and then water extraction) and compound enzyme method (carrying out enzymolysis on persimmon leaves by adding 5% compound enzyme (cellulase-pectinase) and then water extraction). The Aspergillus niger crude enzyme solution enzymolysis method and the compound enzyme method can both improve the content of quercetin and kaempferol, the preparation process of the brain heart clear capsule is stable and reliable, and is suitable for large-scale production. In addition, the Aspergillus niger crude enzyme solution enzymolysis is optimal, and the Aspergillus niger crude enzyme solution enzymolysis can save cost. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0051] Figure 1 The standard HPLC spectrum of rutin, quercetin, isoquercitrin and gallic acid is shown.

[0052] Figure 2 The HPLC spectrum of the persimmon leaf extraction product dry extract is shown.

[0053] Figure 3 The HPLC spectrum of the water extraction method process is shown.

[0054] Figure 4 The HPLC spectrum of the Aspergillus niger crude enzyme solution enzymolysis group is shown. DETAILED DESCRIPTION

[0055] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0056] Embodiment 1

[0057] The present embodiment provides a preparation method of persimmon leaf extract, which comprises the following steps:

[0058] Step S1, taking dry and crushed persimmon leaf raw materials.

[0059] Step S2, obtaining an extraction solution by using a water extraction method, which specifically comprises the following flow:

[0060] Step S21, first extraction: 12 times the amount of water is added, soaked for 3 hours, and then boiled for 2 hours. After filtration, the first decoction is obtained.

[0061] Step S22, second extraction: the residue after the first extraction is added with 10 times the amount of water, and boiled for 1 hour. After filtration, the second decoction is obtained.

[0062] Step S23, the first decoction of the first extraction and the second decoction of the second extraction are mixed to obtain the extraction liquid.

[0063] Step S3, the extraction liquid is concentrated, and ethanol is added to reach an alcohol content of 80%-90%. Then, the supernatant and precipitate are obtained by standing.

[0064] As an implementation scenario, in this scenario, the relative density of the concentrated liquid is 1.12-1.15, the amount of ethanol added is 3-5 times the volume of the concentrated liquid, the amount of anhydrous ethanol added is 85%, and the standing time is 24 hours. The supernatant and precipitate are obtained, and the supernatant is filtered and reserved.

[0065] Optionally, the combined extraction liquid is concentrated in a water bath to a relative density of 1.12-1.15, and the concentration conditions are: temperature 60-65℃, vacuum degree 0.06-0.08Mpa.

[0066] Step S4, the precipitate is washed with an ethanol solution, and the washing liquid is collected. After standing, it is combined with the supernatant in step S3.

[0067] As an implementation scenario, in this scenario, the concentration of the ethanol washing liquid is 60-70%, the amount of the precipitate is 3-4 times, the washing times is 2, the stirring time is 20-40 minutes, and then the washing liquid is collected. After standing, the supernatant and the washing liquid are combined.

[0068] Step S5, the combined filtrate in step S4 is rotary evaporated to recover ethanol to obtain the extract.

[0069] Step S6, the obtained extract is dissolved in water and filtered, and the filtrate is extracted with ethyl acetate multiple times.

[0070] Optionally, the amount of water added to dissolve the extract is 2 times the volume of the extract, and the number of ethyl acetate extractions is 4 times.

[0071] Step S7, the multiple times of ethyl acetate liquid are combined, and then rotary evaporated to recover ethyl acetate to obtain the thick paste.

[0072] Step S8, low-temperature drying.

[0073] Example 2

[0074] The present embodiment provides a preparation method of persimmon leaf extract, which comprises the following steps:

[0075] Step S1, persimmon leaf treatment: clean the persimmon leaves, put the cleaned persimmon leaves in an oven at a temperature of 50 ℃, dry for 5 h, use a pulverizer to pulverize the persimmon leaves, and then put the pulverized persimmon leaves in a bag with good sealing property, and store in a cool and dry place for standby.

[0076] Step S2, water extraction: take 200 g of the pulverized persimmon leaves to a round-bottom flask, add 12 times of water, and soak for 3 h. Set up a condensation reflux device, and use an electric heating jacket to heat and decoct for 2 h. Filter, take the filtrate to a beaker; after the round-bottom flask is cooled, continue to add 10 times of water to the filter residue to decoct for 1 h, filter, take the filtrate to a beaker, and combine the filtrates.

[0077] Step S3, concentrated alcohol extraction: take the combined filtrate, concentrate to a relative density of 1.12-1.15 in a 60 ℃ water bath, add anhydrous ethanol (about 4 times the volume) to reach an alcohol content of 85%, and stand for 24 h; obtain the supernatant and the precipitate, and filter the supernatant for standby.

[0078] Step S4, precipitate washing: take the precipitate, wash twice with 4 times the amount of 65% ethanol, each time for 30 min, collect the washing liquid, and after standing, combine the supernatant and the washing liquid.

[0079] Step S5, recovery of ethanol: take the filtrate in step S4, recover the ethanol in a water bath at a temperature of 55 ℃, and obtain the extract after rotary evaporation.

[0080] Step S6, ethyl acetate extraction: take the extract in step S5, dissolve in 2 times the amount of water, filter, and extract the filtrate with ethyl acetate four times, and combine the ethyl acetate liquid.

[0081] Step S7, recovery of ethyl acetate: take the combined ethyl acetate liquid, recover the ethyl acetate in a water bath at a temperature of 55 ℃, and obtain the thick extract after rotary evaporation.

[0082] Step S8, low-temperature drying at 60 ℃.

[0083] Example 3

[0084] Component analysis of the persimmon leaf extract prepared in Example 2

[0085] 1. Instruments and reagents

[0086] 1.1 Reagents

[0087] Quercetin, rutin, gallic acid, methanol (analytical grade), phosphoric acid, anhydrous ethanol, purified water.

[0088] 1.2 Instruments

[0089] 20A high-pressure pump + 7725i manual sampler + 20A dual-wavelength detector

[0090] 2. Experimental method

[0091] 2.1 Rutin, Quercetin Chromatographic Conditions

[0092] Chromatographic column: Waters T3 150mm x 4.6mm, 5µm; mobile phase: methanol: 0.1% phosphoric acid = 45:55; flow rate: 1ml / min; detection wavelength: 360nm

[0093] 2.2 Gallic Acid Chromatographic Conditions

[0094] Chromatographic column: Waters T3 150mm x 4.6mm, 5µm; mobile phase: methanol: 0.1% phosphoric acid = 5:95; flow rate: 1ml / min; detection wavelength: 272nm

[0095] 3 Experimental Results

[0096] Table 1:

[0097]

[0098] According to the extraction process result verification of Naoxinjing, the extraction process is stable and feasible, and the quercetin content in the water layer is only 0.0006%, which indicates that most of the quercetin has been extracted by ethyl acetate, and the extraction is complete. The main components in the obtained dry extract are rutin, gallic acid, quercetin and isoquercetin, and the spectrum results are recorded as shown in Figure 1 and Figure 2 .

[0099] Example 4

[0100] The present embodiment provides a preparation method of persimmon leaf extract, which comprises the following steps:

[0101] Step S1, taking dry and crushed persimmon leaf raw materials.

[0102] Step S2, obtaining an extraction liquid by using enzyme-assisted extraction method; the enzyme-assisted extraction method is selected from composite enzyme method or Aspergillus niger crude enzyme solution enzymolysis method.

[0103] Step S3, concentrating the extraction liquid and adding ethanol to make the alcohol content reach 80%-90%, and then standing to obtain supernatant and precipitate.

[0104] As an implementation scenario, in the present scenario, the relative density of the concentrated liquid is 1.12-1.15, the amount of added ethanol is 3-5 times the volume of the concentrated liquid, the amount of added anhydrous ethanol is 85%, and the standing time is 24h, to obtain supernatant and precipitate, and the supernatant is filtered and used.

[0105] Optionally, the combined extraction liquid is concentrated to a relative density of 1.12-1.15 in a water bath, and the concentration conditions are: temperature 60-65℃, vacuum degree 0.06-0.08Mpa.

[0106] Step S4, the precipitate is washed with ethanol solution and the washing liquid is collected, and after standing, it is combined with the supernatant in step S3.

[0107] As an implementation scenario, in this scenario, the concentration of the ethanol washing liquid is 60-70%, the amount of use is 3-4 times the weight of the precipitate, the washing times are 2, and the stirring time of each washing is 20-40 minutes, then the washing liquid is collected, and after standing, the supernatant and the washing liquid are combined.

[0108] Step S5, the filtrate combined in step S4 is recovered by rotary evaporation after ethanol is recovered, and then the extract is obtained.

[0109] Step S6, the extract obtained is dissolved in water and filtered, and the filtrate is extracted with ethyl acetate for multiple times.

[0110] Optionally, the amount of water added for dissolving the extract is 2 times the volume of the extract, and the extraction times of ethyl acetate are 4 times.

[0111] Step S7, the ethyl acetate liquid extracted for multiple times is combined, and then the thick paste is obtained after ethyl acetate is recovered by rotary evaporation.

[0112] Step S8, low-temperature drying at 60°C.

[0113] In an embodiment, the complex enzyme method uses a complex enzyme composed of cellulase and pectinase, and the crude enzyme liquid enzymolysis method uses a crude enzyme liquid obtained by fermentation of Aspergillus niger. In other embodiments, semi-bionic method and semi-bionic enzyme method can also be used.

[0114] Embodiment 5

[0115] Based on embodiment 4, in this embodiment, the specific steps of the complex enzyme method in step S2 include:

[0116] Step T1, adding 5% of the weight of persimmon leaves of complex enzyme, wherein the mass ratio of cellulase to pectinase is 1:1, and the enzyme is hydrolyzed in a sodium phosphate dibasic-citric acid buffer solution.

[0117] Step T2, after enzyme hydrolysis, heating and inactivation, water is added for decoction and extraction.

[0118] In an embodiment, in step T1, the amount of sodium phosphate dibasic-citric acid buffer solution is 10 times the volume of the weight of persimmon leaves.

[0119] In an embodiment, in step T1, the enzyme is hydrolyzed at pH 3.5-3.8 and temperature 45-55°C for 1-3 hours.

[0120] In one embodiment, in step T2, after enzymolysis, heat to 90-100°C for 5-15 minutes to decoct, filter to obtain the first decoction, add 10 times the volume of disodium hydrogen phosphate-citric acid buffer solution to the residue after the first extraction, decoct for 1 hour, filter, and combine the two decoctions to obtain the final extract. Preferably, after enzymolysis, heat to 95°C for 10 minutes.

[0121] Example 6

[0122] In this embodiment based on Example 4, the specific steps of the enzyme hydrolysis method in step S2 include:

[0123] Step X1, preparation of crude enzyme solution: inoculate the Aspergillus niger spore solution into enzyme production medium containing 1-3% corn starch and 0.2-0.6% proteose peptone, and culture at 28-32°C for 48-68 hours, then filter to obtain the crude enzyme solution;

[0124] Step X2, enzymolysis treatment: add 5 mL of crude enzyme solution per gram of persimmon leaves for enzymolysis;

[0125] Step X3, decocting extraction after enzymolysis.

[0126] In one embodiment, in step X1, the method for obtaining the Aspergillus niger spore solution is as follows: inoculate the Aspergillus niger strain into PDA medium, incubate at 30°C for 7-9 days, then scrape the spores into sterile normal saline, and stir with a inoculation loop to suspend the spores, thereby obtaining the Aspergillus niger spore solution.

[0127] In one embodiment, in step X1, the enzyme production medium contains 2% corn starch, 0.4% proteose peptone, 0.6% (NH4)2SO4, 0.5% KH2PO4, 0.1% MgSO4·7H2O, 0.05% CaCl2, and 0.01% FeSO4·7H2O, and has a pH of 6.0.

[0128] In one embodiment, inoculate the spore solution into the enzyme production medium at an inoculation amount of 5-10%, and culture at 30°C and 150 rpm for 48-68 hours, then filter to collect the filtrate as the crude enzyme solution.

[0129] In one embodiment, in step X2, grind the persimmon leaves to pass through a 40-mesh sieve, inoculate 5 mL of crude enzyme solution per gram of persimmon leaves, stir uniformly, and then enzymolyze at 30°C for 8-12 hours, and add the same amount of water to the blank group.

[0130] In one embodiment, in step X3, after enzymolysis, add water, and perform water bath at 95°C for 1 hour.

[0131] Example 7

[0132] Experiments on improving the active components of Naoxinqing by enzyme method

[0133] 1. Materials and reagents

[0134] 1.1 Reagents

[0135] Sodium phosphate dibasic-citric acid, cellulase, pectinase, PDA medium, corn starch, peptone, (NH4)2SO4, KH2PO4, MgSO4·7H2O, CaCl2, FeSO4·7H2O.

[0136] 2. Experimental methods

[0137] 2.1 Water extraction method

[0138] Take the crushed persimmon leaves into a round-bottom flask, add 12 times the amount of water, and soak for 3 hours. Set up a condensation reflux device and use an electric heating jacket to heat and decoct for 2 hours. Filter and take the filtrate into beaker 1. Measure the volume. After the round-bottom flask cools down, continue to add 10 times the amount of water to the filter residue and decoct for 1 hour. Filter and take the filtrate into beaker 2. Measure the volume. Combine the filtrates in beaker 1 and beaker 2, measure the volume, take sample A, and prepare for liquid phase detection.

[0139] 2.2 Compound enzyme method

[0140] Take the crushed persimmon leaves into a round-bottom flask, add 5% compound enzyme (cellulase-pectinase 1:1), add 10 times the volume of sodium phosphate dibasic-citric acid buffer solution, adjust the pH to about 3.6, and enzymatically hydrolyze in a 50°C water bath for 2 hours. Decoct for 10 minutes to inactivate the enzyme. Filter and record the extract 1. Add 10 times the volume of sodium phosphate dibasic-citric acid buffer solution to the filter residue and decoct for 1 hour. Filter and record the extract 2. Combine the extracts 1 and 2 and record B.

[0141] 2.3 Aspergillus niger crude enzyme solution enzymolysis method

[0142] (1) Preparation of crude enzyme solution

[0143] Inoculate the Aspergillus niger strain on PDA medium and incubate at 30°C for 7-9 days. Scrape the spores into sterile saline and stir with an inoculation loop to suspend the spores. Obtain the Aspergillus niger spore solution. Inoculate the spore solution into the enzyme production medium at a 5%-10% inoculation amount, incubate at 30°C and 150 rpm for 48-68 hours, and collect the filtrate by suction filtration as the crude enzyme solution. Take the persimmon leaves (crushed and passed through a 40-mesh sieve), inoculate with 5 mL of crude enzyme solution per gram of persimmon leaves, stir evenly, and then enzymatically hydrolyze at 30°C for 8-12 hours. Add the same amount of water to the blank group. After the enzymatic hydrolysis is complete, add water and incubate in a 95°C water bath for 1 hour.

[0144] Enzyme production medium: corn starch 2%, proteose peptone 0.4%, (NH4)2SO4 0.6%, KH2PO4 0.5%, MgSO4·7H2O 0.1%, CaCl2 0.05%, FeSO4·7H2O 0.01%, pH 6.0.

[0145] 2.4 Chromatographic conditions

[0146] (1) Rutin, quercetin chromatographic conditions

[0147] Chromatographic column: Waters T3 150mm x 4.6mm, 5µm; mobile phase: methanol: 0.1% phosphoric acid = 45:55; flow rate: 1ml / min; detection wavelength: 360nm

[0148] (2) Gallic acid chromatographic conditions

[0149] Chromatographic column: Waters T3 150mm x 4.6mm, 5µm; mobile phase: methanol: 0.1% phosphoric acid = 5:95; flow rate: 1ml / min; detection wavelength: 272nm.

[0150] 2.5 Preparation of standard

[0151] Take quercetin, kaempferol, rutin reference substance each appropriate amount, respectively, prepare the standard stock solution with a concentration of 0.500 mg / mL, 0.500 mg / mL, 0.500 mg / mL, 0.800 mg / mL, respectively, precisely take the stock solution and add methanol to prepare the mixed control solution with a concentration of 0.250 mg / mL, 0.250 mg / mL, 0.250 mg / mL and 0.400 mg / mL, namely.

[0152] 3 Experimental results

[0153] 3.1 Water extraction method process

[0154] Table 2:

[0155]

[0156] 3.2 Compound enzyme method

[0157] Table 3:

[0158]

[0159] From the experimental results, the yield of rutin by compound enzyme method is increased by 18% compared with water extraction method process, reaching 0.026% (p<0.05), and the yield of kaempferol is doubled to 0.002%. This may be related to the specific opening of β-1, 4 glycosidic bond by compound enzyme through enzymolysis, releasing the bound flavonoids.

[0160] 3.3 Enzymatic hydrolysis of crude Aspergillus niger enzyme solution

[0161] Table 4:

[0162]

[0163] The present embodiment is based on the process of synergistically improving the active ingredients of Naoxinqing by enzyme method. The spectrum results are shown in Figure 3 and Figure 4 , Figure 3 The HPLC spectrum of the water extraction process (without Aspergillus niger crude enzyme solution treatment group) is shown in Figure 4 The HPLC spectrum of the Aspergillus niger crude enzyme solution enzymolysis group is shown in. By comparing the liquid chromatogram of the sample group after the enzymolysis of the crude Aspergillus niger enzyme solution with the original process (water extraction process) Figure 3 , Figure 4 It is found that the enzyme solution promotes the release of some active ingredients of persimmon leaves, and the detection sensitivity of flavonoids is improved: (1) Enzyme treatment makes rutin (0.001%), quercetin (0.0001%) and kaempferol (0.0002%) reach the quantifiable level, while the original process leads to a peak area lower than the detection limit due to low extraction efficiency; (2) Enzyme treatment specifically decomposes plant cell wall polysaccharides, promoting the dissociation and release of bound flavonoids; (3) The extraction solution after enzyme treatment maintains the color characteristics comparable to the original process, proving that the process improves the yield of active ingredients while maintaining the appearance stability of the product. Since direct enzyme addition is relatively high in industrial production cost, the use of crude enzyme solution of Aspergillus niger for enzymolysis can save cost.

[0164] Example 8

[0165] The present embodiment provides a preparation method of Naoxinqing capsules, which comprises the following steps:

[0166] Step S1, persimmon leaf treatment

[0167] The persimmon leaves are washed clean, and the washed clean persimmon leaves are placed in an oven with a temperature of 50 ℃. The drying time is 5 h. The persimmon leaves are crushed with a crusher. The crushed persimmon leaves are packed in a bag with good sealing performance and placed in a cool and dry place for standby.

[0168] Step S2, extraction

[0169] There are three preparation methods for persimmon leaf extract, which are described in detail as follows:

[0170] Water extraction method: 200 g of crushed persimmon leaves are taken into a round-bottom flask, and 12 times of water is added for soaking for 3 h. A condensation reflux device is set up, and an electric heating jacket is used for heating and decocting for 2 h. Filtration is performed, and the filtrate is taken in a beaker. After the round-bottom flask is cooled, the filter residue is continuously added with 10 times of water for decocting for 1 h. Filtration is performed, and the filtrate is taken in a beaker. The filtrates are combined.

[0171] Compound enzyme method: Take the crushed persimmon leaves into a round-bottom flask, add 5% compound enzyme (cellulase-pectinase 1:1), add 10 times volume of sodium phosphate-citric acid buffer solution, adjust pH to about 3.6, enzyme hydrolysis in 50°C water bath for 2h, decoction for 10min to inactivate the enzyme, filter to obtain the first filtrate; add 10 times volume of sodium phosphate-citric acid buffer solution to the residue, decoction for 1h, filter to obtain the second filtrate. Combine the two filtrates.

[0172] Aspergillus niger crude enzyme solution hydrolysis method: Aspergillus niger strain was inoculated on PDA medium and incubated at 30°C for 7-9 days. The spores were scraped into sterile saline and stirred with a inoculation loop to make the spores suspended to obtain Aspergillus niger spore solution; the spore solution was inoculated into enzyme production medium at a 5%-10% inoculation amount, and incubated at 30°C, 150rpm for 48-68h, and the filtrate was collected by suction filtration as crude enzyme solution; persimmon leaves (crushed and passed through a 40 mesh sieve) were inoculated with 5mL of crude enzyme solution per gram of persimmon leaves, stirred uniformly, and then enzyme hydrolysis was carried out at 30°C for 8-12h, and the blank group was added with the same amount of water. After enzyme hydrolysis, water was added and water bath was carried out at 95°C for 1h. Enzyme production medium: corn starch 2%, peptone 0.4%, (NH4)2SO4 0.6%, KH2PO4 0.5%, MgSO4·7H2O 0.1%, CaCl2 0.05%, FeSO4·7H2O 0.01%, pH 6.0.

[0173] Step S3, concentration of alcohol extraction

[0174] The combined filtrate was taken to a 60°C water bath and concentrated to a relative density of 1.12-1.15, and anhydrous ethanol (about 4 times the volume) was added to reach an alcohol content of 85%, and then it was left to stand for 24h; the supernatant and the precipitate were obtained, and the supernatant was filtered and prepared for use.

[0175] Step S4, washing of the precipitate

[0176] The precipitate was washed with 4 times the amount of 65% ethanol twice, each time stirring for 30min, and the washing liquid was collected, and after standing, the supernatant and the washing liquid were combined.

[0177] Step S5, recovery of ethanol

[0178] The filtrate in step S4 was taken, and the ethanol was recovered by rotary evaporation at a water bath temperature of 55°C, and then the extract was obtained.

[0179] Step S6, ethyl acetate extraction

[0180] The extract in step S5 was taken, dissolved in 2 times the amount of water, filtered, and the filtrate was extracted with ethyl acetate four times, and the ethyl acetate liquid was combined.

[0181] Step S7, recovery of ethyl acetate

[0182] The ethyl acetate liquid was combined, and the ethyl acetate was recovered by rotary evaporation at a water bath temperature of 55°C to obtain a thick paste.

[0183] Step S8, drying

[0184] Low-temperature drying at 60°C.

[0185] Step S9, capsule preparation

[0186] For each 50 g of dry extract, 100 g of starch was added, and the mixture was uniformly mixed to form soft material with starch slurry, and granules were prepared, which were dried at 60°C and then packaged into capsules. For example, for 1000 capsules, 50 g of persimmon leaf dry extract and 100 g of starch were uniformly mixed to form soft material with starch slurry, and granules were prepared, which were dried at 60°C and then packaged into capsules.

[0187] In the preparation method of the above Naoxinqing capsules, the reagent is persimmon leaf, and the instruments include an electronic balance, a temperature-controlled electric heating jacket, a constant-temperature water bath, a rotary evaporator, a circulating water vacuum pump, a low-temperature cooling circulating pump, a vacuum drying box, and an ultrasonic cleaner. When water extraction is used, the composition analysis of the persimmon leaf extraction product is shown in Example 3. The detection indexes (mainly the content of quercetin and kaempferol) of the complex enzyme method and the Aspergillus niger crude enzyme solution enzymolysis method are shown in Example 7. Both the Aspergillus niger crude enzyme solution enzymolysis method and the complex enzyme method can improve the content of quercetin and kaempferol.

[0188] Naoxinqing capsules have important research and development value as a traditional Chinese medicine preparation for treating cardiovascular and cerebrovascular diseases. The main raw materials are derived from natural plants, aiming to exert the advantages of comprehensive regulation of traditional Chinese medicine and overcome the shortcomings of existing treatment drugs. Through in-depth research on the preparation process, the present application can effectively improve the purity, stability and bioavailability of the drug, precisely control the content of effective components, and ensure the stable and reliable quality of the drug. The optimized preparation process can reduce production costs, improve production efficiency, and is conducive to large-scale production and wide application of the product, providing safer, more effective and more convenient treatment options for patients with cardiovascular and cerebrovascular diseases, and having significant social and economic benefits. The present application has a positive significance for promoting the modernization of traditional Chinese medicine. The Naoxinqing capsules obtained by the present application have the advantages of convenient carrying, high bioavailability, easy absorption, and few adverse reactions. The preparation process is reasonable and feasible, the quality is stable and controllable, and the capsules can be applied to large-scale production.

[0189] In the foregoing, only certain exemplary embodiments have been described. As will be apparent to those of ordinary skill in the art, the embodiments described can be modified in various different ways without departing from the spirit or scope of the application. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature and not as restrictive.

[0190] In the description of the application, it is to be understood by the terms "longitudinal", "transverse", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "inner", "outer", "end", "side" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship commonly used when the product of the application is used, or the orientation or positional relationship commonly understood by those skilled in the art, and are only for the convenience of describing the application and simplifying the description, and do not indicate or imply that the devices or elements indicated must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the application.

[0191] In addition, the terms "first", "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. In the description of the embodiments of the present application, the meaning of "a plurality of" is two or more, unless otherwise specifically limited.

[0192] The terms "mounting", "connecting", "connecting", "fixing" and the like should be broadly understood, for example, can be fixedly connected, can be detachably connected, or integrated; can be directly connected, or indirectly connected through an intermediate medium; can be the internal communication of two elements or the interaction relationship of two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

Claims

1. A method for preparing a persimmon leaf extract, the method comprising the following steps: S1, taking dried and powdered persimmon leaf material; S2, obtaining an extract by water extraction or enzyme-assisted extraction; S3, concentrating the extract and adding ethanol to achieve an alcohol content of 80-90%, and then standing to obtain supernatant and precipitate; S4, washing the precipitate with an ethanol solution, collecting the washing liquid, and then standing to obtain supernatant, which is combined with the supernatant obtained in step S3; S5, recovering ethanol from the combined filtrate by rotary evaporation to obtain an extract; S6, dissolving the extract in water, filtering, and then extracting the filtrate with ethyl acetate multiple times; S7, combining the ethyl acetate extracts obtained in step S6, and then recovering ethyl acetate by rotary evaporation to obtain a thick extract; and S8, drying at low temperature. 2.The method of claim 1, wherein the water extraction in step S2 comprises the following steps: S21, first extraction: adding 12 times the amount of water, soaking for 3 hours, and then boiling for 2 hours to obtain a first extract; S22, second extraction: adding 10 times the amount of water to the residue after the first extraction, boiling for 1 hour, and then filtering to obtain a second extract; and S23, mixing the two extracts to obtain the extract. 3.The method of claim 1 or 2, wherein the enzyme-assisted extraction in step S2 is a complex enzyme method, and the complex enzyme method comprises the following steps: T1, adding 5% of the weight of the persimmon leaf material of a complex enzyme, wherein the mass ratio of cellulase to pectinase is 1:1, and the enzyme is hydrolyzed in a sodium phosphate dibasic-citric acid buffer solution; and T2, after the enzyme hydrolysis, heating to inactivate the enzyme, and then adding water to extract. 4.The method of claim 3, wherein in step T1, the sodium phosphate dibasic-citric acid buffer solution is used in an amount of 10 times the volume of the weight of the persimmon leaf material, and the enzyme is hydrolyzed at a pH of 3.5-3.8 and a temperature of 45-55℃ for 1-3 hours; and / or in step T2, after the enzyme hydrolysis, heating to 90-100℃ for 5-15 minutes to extract, filtering to obtain a first extract, adding 10 times the volume of a sodium phosphate dibasic-citric acid buffer solution to the residue after the first extraction, boiling for 1 hour, and then combining the two extracts to obtain the extract. 5.The method of claim 1 or 2, wherein the enzyme-assisted extraction in step S2 is a crude enzyme solution hydrolysis method of Aspergillus niger, and the crude enzyme solution hydrolysis method comprises the following steps: X1, preparing a crude enzyme solution by inoculating Aspergillus niger spore solution into a culture medium to obtain the crude enzyme solution; X2, adding 5 mL of the crude enzyme solution per gram of the persimmon leaf material to perform enzyme hydrolysis; and X3, after the enzyme hydrolysis, adding water to extract. 6.The method of claim 5, wherein in step X1, the Aspergillus niger spore solution is obtained by inoculating an Aspergillus niger strain into a PDA culture medium, incubating at 30℃ for 7-9 days, scraping the spores from the culture medium into sterile normal saline, and then stirring to suspend the spores to obtain the Aspergillus niger spore solution. ​ ​ ​ ​ ​ 2. The method of claim 1, wherein the persimmon leaf extract is prepared by the steps of: ​ ​ ​ ​ 3. The method for preparing persimmon leaf extract according to claim 1, characterized in that, ​ ​ ​ ​ ​ ​ 5. The method for preparing persimmon leaf extract according to claim 1, characterized in that, ​ ​ ​ ​ ​ ​ And / or, in the step X2, the spore solution of Aspergillus niger is inoculated into an enzyme production medium containing 1-3% corn starch and 0.2-0.6% proteose peptone, and cultured at 28-32°C and 150 rpm for 48-68 hours, and the filtrate collected by suction filtration is a crude enzyme solution; And / or, in the step X2, the reaction is carried out at 30°C for 8-12 hours.

7. The method for preparing persimmon leaf extract according to claim 5 or 6, characterized in that, The enzyme production medium contains 2% corn starch, 0.4% proteose peptone, 0.6% (NH4)2SO4, 0.5% KH2PO4, 0.1% MgSO4·7H2O, 0.05% CaCl2, 0.01% FeSO4·7H2O, and has a pH of 6.

0.

8. The method of claim 1, wherein the persimmon leaf extract is prepared by the steps of: In the step S3: ​ The extract is concentrated and ethanol is added to achieve an alcohol content of 85%; And / or, the combined extract is concentrated to a relative density of 1.12-1.15 in a water bath at a temperature of 60-65°C and a vacuum degree of 0.06-0.08 Mpa; And / or, the amount of ethanol added is 3-5 times the volume of the concentrated solution; And / or, the standing time is 24 hours.

9. The preparation method of the persimmon leaf extract according to claim 1, characterized in that: In the step S4, the concentration of the ethanol washing solution is 60-70%, the amount of the ethanol washing solution is 3-4 times the amount of the precipitate, and the washing is performed twice, with each stirring time being 20-40 minutes; And / or, in the step S5, the combined filtrate is subjected to rotary evaporation to recover ethanol at a water bath temperature of 55°C, and then the extract is obtained; And / or, in the step S6, the amount of water added for dissolving the extract is 2 times the volume of the extract, and the filtrate is extracted with ethyl acetate four times; And / or, in the step S7, the combined ethyl acetate solution is subjected to rotary evaporation to recover ethyl acetate at a water bath temperature of 55°C, and then the thick extract is obtained.

10. A method for preparing Naoxingtong capsules, characterized in that, The preparation method comprises the following steps: The persimmon leaf extract is prepared by any one of the methods of claims 1-9; The extract is mixed with pharmaceutical excipients at a ratio of 1:2 to prepare granules; The granules are packaged into capsules.

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