Synergistic composition of radix rehmanniae preparata and donkey-hide gelatin as well as preparation method, pharmaceutical preparation and application of synergistic composition

Abstract

The preparation method comprises the following steps: extracting and separating polar parts of radix rehmanniae preparata, screening key parts, mixing a screened and determined key synergistic part extracting solution with colla corii asini to generate a reaction product, and then concentrating and drying to obtain the synergistic composition of radix rehmanniae preparata and colla corii asini. The blood enriching activity of the composition is obviously superior to that of an equivalent single component, the synergistic interaction effect of the hydrophilic part of the prepared rehmannia root and the donkey-hide gelatin is clearly confirmed, and the bottleneck problem that the traditional compatibility activity is limited to be improved is solved.

Description

Technical Field

[0001] This invention relates to the fields of traditional Chinese medicine and pharmaceutical preparations, specifically to a synergistic composition comprising Rehmannia glutinosa extract and donkey-hide gelatin, and to a method for preparing the composition and its application in the preparation of blood-tonifying drugs. Background Technology

[0002] Rehmannia glutinosa (Radix Praeparata) and Asini Corii Colla are core medicinal materials in traditional Chinese medicine for treating blood deficiency syndrome, and their combined use has a long history. However, existing formulation techniques have a significant deficiency: they have failed to explore the nature of the interaction between Rehmannia glutinosa and Asini Corii Colla from the perspective of separating the polar parts of Rehmannia glutinosa, resulting in a lack of clear key material basis and direct activity data to support the synergistic effect of the two.

[0003] Specifically, traditional processes and existing patents (such as application number CN201510520154.8) all use Rehmannia glutinosa whole together with donkey-hide gelatin. This method ignores the essential differences in chemical composition and biological activity between different polar parts of Rehmannia glutinosa (such as hydrophilic and lipophilic parts). Although the oral liquid of Rehmannia glutinosa and its preparation method in application number CN202310902167.6 combines Rehmannia glutinosa and donkey-hide gelatin to make an oral liquid and adds excipients such as Codonopsis pilosula and Lycium barbarum to optimize the efficacy, its preparation process only performs two water decoction extractions and mixed concentrations on raw materials such as Rehmannia glutinosa. It does not separate the polar parts of the Rehmannia glutinosa extract and cannot distinguish the differences in the interaction between different polar components such as polysaccharides and iridoid glycosides and donkey-derived polypeptides in donkey-hide gelatin. In addition, the patent for a traditional Chinese medicine composition for the prevention and treatment of menopausal syndrome in women, authorized by announcement number CN103566020B, combines Rehmannia glutinosa and donkey-hide gelatin with Coptis chinensis, Scutellaria baicalensis and other ingredients to make immediate-release and sustained-release capsules. During preparation, only ethanol solution is used to extract the raw materials, including Rehmannia glutinosa, as a whole. Similarly, no exploration is conducted on the reaction mechanism between specific polar parts of Rehmannia glutinosa and donkey-hide gelatin.

[0004] Although existing research, such as "Study on the Pharmacodynamic Material Basis of Rehmannia glutinosa in Promoting the Proliferation of Aging Neural Stem Cells," suggests that different parts of Rehmannia glutinosa exhibit varying activities, two key issues remain unresolved due to a lack of in-depth exploration of the reaction mechanism between specific polar parts and donkey-hide gelatin: First, the key active components are unclear. Because the interaction between different polar parts of Rehmannia glutinosa and donkey-hide gelatin has not been analyzed in a targeted manner, it is impossible to determine which type of polar component in Rehmannia glutinosa specifically binds to or modifies the core active substances in donkey-hide gelatin. This leaves the material basis of the formulation in a vague state of "overall mixing," making targeted optimization difficult. Second, synergistic effects lack empirical support. The lack of comparison between the activity of the formulated product and individual components means that the mechanism of synergistic effect is not well-documented, making it impossible to enhance blood-tonifying and other related therapeutic effects through targeted regulation of the reaction, thus hindering the precise optimization of the formulation's efficacy.

[0005] In summary, current technologies remain at the level of simple mixing or compound formulation, lacking a systematic strategy of "isolation of polar components - targeted action research - activity verification." There is an urgent need in this field for a refined approach that, by isolating the polar components of Rehmannia glutinosa and screening the key components that have the most significant effect on donkey-hide gelatin, can verify their synergistic effects from both the component and efficacy levels, thereby developing a blood-tonifying composition with clearly defined activity and mechanism. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin, its preparation method, and its application in the preparation of blood-tonifying drugs. This aims to clarify the synergistic effect of specific polar fractions of Rehmannia glutinosa with donkey-hide gelatin, solving the problem of limited activity enhancement in traditional formulations.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin, comprising the following steps: (1) Extraction and separation of polar fractions of Rehmannia glutinosa: Take Rehmannia glutinosa, extract it with water, and then use macroporous resin to separate the Rehmannia glutinosa extract to obtain extracts of different polar fractions of Rehmannia glutinosa. (2) Screening of key components: The extracts of different polar components of Rehmannia glutinosa obtained in step (1) were subjected to directional reactions with donkey-hide gelatin. The content of characteristic polypeptides of donkey-hide gelatin before and after the reaction was detected by LC-MS. The extract of polar component of Rehmannia glutinosa that had the greatest impact on the content of characteristic polypeptides of donkey-hide gelatin was selected as the key synergistic component extract.

[0008] (3) The extract of the key synergistic component selected in step (2) is mixed with the gelatin liquid at a mass ratio of 1:2 to 5:1. The mixture is reacted at 100-120℃ for 16-20 hours to allow the two to fully react and generate a reaction product. The product is then concentrated and dried to obtain the synergistic composition of Rehmannia glutinosa and gelatin.

[0009] Preferably, step (1) includes the following specific steps: The mass-volume ratio of Rehmannia glutinosa and water is 1:2-1:10. Place Rehmannia glutinosa in a round-bottom flask, add water, heat to boiling, and maintain for 60-120 minutes. Pour out the extract, repeat the extraction twice, with the same amount of water and extraction time each time. Combine the extracts to obtain Rehmannia glutinosa extract. The column was packed with Rehmannia glutinosa and macroporous resin at a mass ratio of 1:2 to 1:3. After loading the Rehmannia glutinosa extract at a flow rate of 2-4 BV / h, static adsorption was performed for 25-40 minutes. The first eluent was collected by eluting with 5-7 BV of distilled water. The second eluent was collected by eluting with 3-5 BV of 10% ethanol. The third eluent was collected by eluting with 3-5 BV of 70% ethanol. The fourth eluent was collected by eluting with 3-5 BV of 95% ethanol. The extracts of different polar fractions of Rehmannia glutinosa included the first, second, third, and fourth eluents.

[0010] Preferably, the directed reaction between the extracts of different polar fractions of Rehmannia glutinosa and donkey-hide gelatin in step (2) includes: Extracts of different polar fractions of Rehmannia glutinosa were mixed evenly with gelatin solution to obtain samples before reaction. The samples before reaction were heated at 100-120℃ for 16-22 hours to obtain samples after reaction. The volume ratio of extracts of different polar fractions of Rehmannia glutinosa to gelatin solution was 1:1.

[0011] Preferably, the LC-MS detection of the content of characteristic polypeptides of donkey-hide gelatin before and after the directional reaction in step (2) includes: Take samples before and after the reaction and place them in separate volumetric flasks. The volume ratio of the sample before and after the reaction to the volumetric flask is (0.5-1.5):(4-6). Add the same volume of trypsin solution as the sample before and after the reaction. The trypsin solution is prepared by adding 1 wt.% ammonium bicarbonate solution to trypsin to a solution containing 5 mg of trypsin per ml. Then, add 1 wt.% ammonium bicarbonate solution to dilute to the mark of the volumetric flask, shake well, and incubate at 37°C for 12 hours. Filter, collect the filtrate, and perform component analysis on each filtrate using a liquid chromatograph.

[0012] Preferably, the chromatographic conditions are as follows: Flow rate 0.3 mL / min, gradient elution for 30 min, mobile phase A: 0.1% formic acid, mobile phase B: 100% acetonitrile, gradient elution program as shown in the table: .

[0013] Preferably, the chromatograph is directly connected to the mass spectrometer, using the mass spectrometer as the detector, and the electrospray ionization positive ion mode (ESI+) is used for SRM mode monitoring. The monitored ion pairs are shown in the table below: The mass spectrometer outputs a characteristic spectrum, and the content of donkey-derived polypeptide A2 in the sample before and after the reaction is calculated using the characteristic spectrum.

[0014] Preferably, the screening includes: The rate of change in the content of donkey-derived polypeptide A2 was calculated using the following formula: By comparing the rate of change in the content of donkey-derived polypeptide A2, the sample with the largest rate of change in the content of donkey-derived polypeptide A2 before the reaction was selected, and the extracts of different polar fractions of Rehmannia glutinosa were used as the key synergistic extracts.

[0015] Furthermore, in step (3), the preferred mass ratio of the extract of the key enhancing component to the donkey-hide gelatin is 3.75:1.

[0016] Furthermore, in step (3), it is preferable to react at 110°C for 18 hours.

[0017] In another aspect, the present invention provides a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin, wherein the synergistic composition of Rehmannia glutinosa and donkey-hide gelatin is prepared by the above-mentioned preparation method of the synergistic composition of Rehmannia glutinosa and donkey-hide gelatin.

[0018] Another aspect of the present invention provides a traditional Chinese medicine preparation comprising the above-mentioned synergistic composition of Rehmannia glutinosa and donkey-hide gelatin, and pharmaceutically acceptable excipients. It is understood that the above-mentioned traditional Chinese medicine preparation can be formulated into clinically acceptable dosage forms, including but not limited to tablets, pills, capsules, granules, or oral liquids.

[0019] In another aspect, the present invention provides the application of the above-mentioned synergistic composition of Rehmannia glutinosa and donkey-hide gelatin in the preparation of blood-tonifying drugs.

[0020] The present invention has achieved the following beneficial effects: 1. Precise formulation and clear mechanism. This invention, for the first time, discovers and confirms that the hydrophilic parts of Rehmannia glutinosa are the key material basis for its synergistic effect with donkey-hide gelatin through the separation and directional reaction of polar parts. This breaks through the vague mode of traditional whole-component mixing and provides a scientific basis for achieving precise formulation and quality control.

[0021] 2. Significant activity and synergistic effect. Rigorous pharmacodynamic experiments using a zebrafish anemia model have demonstrated that the hematopoietic activity of the composition of this invention is significantly superior to that of an equal amount of a single component (donkey-hide gelatin or the hydrophilic fraction of Rehmannia glutinosa), clearly confirming the synergistic effect between the hydrophilic fraction of Rehmannia glutinosa and donkey-hide gelatin, thus solving the bottleneck problem of limited activity enhancement in traditional formulations.

[0022] 3. Controllable process and strong applicability. This invention provides a complete process route from separation and screening to preparation, with clear steps and controllable quality. The obtained composition can be easily prepared into various modern dosage forms such as tablets and capsules, showing good prospects for industrial production and market application value. Attached Figure Description

[0023] Figure 1The MRM chromatograms of the test samples before and after the reaction are shown in the donkey-hide gelatin group, where a and b are before the reaction, and c and d are after the reaction. Figure 2 The MRM chromatograms of the test sample before and after the reaction of Rehmannia glutinosa-donkey-hide gelatin group eluted with 0% ethanol are shown, where a and b are before the reaction, and c and d are after the reaction. Figure 3 The MRM chromatograms of the test sample before and after the reaction of Rehmannia glutinosa-donkey-hide gelatin group eluted with 10% ethanol are shown, where a and b are before the reaction, and c and d are after the reaction. Figure 4 The MRM chromatograms of the test sample before and after the reaction of Rehmannia glutinosa-donkey-hide gelatin group eluted with 70% ethanol are shown, where a and b are before the reaction, and c and d are after the reaction. Figure 5 The MRM chromatograms of the test sample before and after the reaction of Rehmannia glutinosa-donkey-hide gelatin group eluted with 95% ethanol are shown, where a and b are before the reaction, and c and d are after the reaction. Figure 6 To investigate the effect of different polar fractions on the detection amount of donkey-derived polypeptide A2 in donkey-hide gelatin.

[0024] Figure 7 The statistical results show the effect of the combination of donkey-hide gelatin and prepared rehmannia root on the staining area of ​​zebrafish erythrocytes.

[0025] Figure 8 The results show the effect of the combination of donkey-hide gelatin and prepared rehmannia root on the number of erythrocytes in zebrafish. Detailed Implementation

[0026] The embodiments of the present invention will now be clearly and completely described in conjunction with examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0027] The main instruments and reagents used in the embodiments of this invention are as follows: (1) Instruments: Analytical balance (ME 204, Mettler Toledo, Switzerland), ultrapure water system (Elixs-Milli-QA, Millipore, France), CNC ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.), triple quadrupole mass spectrometer (TSQ Quantum Access Max, Thermo Fisher Scientific), high performance liquid chromatograph (VanquishFlex, Thermo Fisher Scientific), chromatographic column (Thermo Syncronis C18 (100mm*21mm*1.7um)), electronic balance (CPA 224S, Sartotius, Germany), electric drying oven (BGZ-246, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), ultrasonic cleaner (KQ-800KDE, Kunshan Ultrasonic Instrument Co., Ltd.), image acquisition system (DP2-BSW, Olympus, Japan); stereo fluorescence microscope (AXIO ZOOM.V16, Carl Zeiss, Germany); water-jacketed CO2 incubator (Forma). 3111, Forma Corporation, USA); Zebrafish farming and breeding equipment (Beijing Aisheng Technology Co., Ltd.).

[0028] (2) Reagents: D101 macroporous resin (Anhui Samsung Resin Co., Ltd.), Rehmannia glutinosa and donkey-hide gelatin were collected from the market, water was purified water, trypsin, formic acid and acetonitrile were chromatographic grade, AB line and green fluorescent labeled Tg line immune cells (Lyz:GFP), wild-type AB line zebrafish were provided by the zebrafish drug screening platform of Shandong Academy of Sciences Institute of Biology, phenylhydrazine (PHZ) was purchased from Sigma-Aldrich, vinorelbine ditartrate (VB), salvianic acid A sodium (SAAS), and o-Dianisidine were purchased from MCE Ltd., and donkey-derived polypeptide A2 (batch number: 112041-202102, for content determination, calculated at 95.3%, purchased from the China National Institutes for Food and Drug Control) was used. The remaining reagents were of analytical grade.

[0029] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods; the materials, reagents, equipment, etc. used are all commercially available unless otherwise specified; and unless otherwise specified, the raw materials used in parallel experiments are from the same batch.

[0030] Example 1 Example 1 of the present invention provides a method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin, comprising the following steps: 1. Separation of different polarity parts of Rehmannia glutinosa: 1.1 Preparation of Rehmannia glutinosa extract Place 200g of prepared Rehmannia root in a round-bottom flask, add 1000ml of water, heat to boiling, maintain for 90 minutes, pour out the liquid, and repeat the extraction twice, with the same amount of water and extraction time each time, and combine the extracts.

[0031] 1.2 Separation of different polarity parts of Rehmannia glutinosa Weigh 100 g of D101 macroporous resin and soak it in 500 ml of 95% ethanol for 12 hours to allow it to swell fully. Discard the floating matter and fragments on the upper layer, pack the column using the wet method (φ 15×300 mm), rinse the resin column with water to remove broken resin, and let the washing liquid become clear and allow the resin to settle naturally.

[0032] Wash the resin column twice with alternating amounts of 95% ethanol and water, twice with two column volumes (BV, or Body Volume). Elute with 95% ethanol, then rinse with 3-4 BV of 95% ethanol. Finally, wash with water until there is no alcohol odor.

[0033] The column was packed with a medicinal material to resin ratio of 1:2. After loading the Rehmannia glutinosa extract at a flow rate of 3 BV / h, it was statically adsorbed for 30 minutes. It was then eluted with 6 BV of distilled water, and the eluent was collected (i.e., Rehmannia glutinosa eluted with 0% ethanol). Elution was then performed with 4 BV of 10% ethanol, and the eluent was collected (i.e., Rehmannia glutinosa eluted with 10% ethanol). Elution was then performed with 4 BV of 70% ethanol, and the eluent was collected (i.e., Rehmannia glutinosa eluted with 70% ethanol). Finally, elution was performed with 4 BV of 95% ethanol, and the eluent was collected (i.e., Rehmannia glutinosa eluted with 95% ethanol), thus obtaining extracts of different polar fractions of Rehmannia glutinosa.

[0034] 2. Screening of key components: 2.1 Sample Preparation 2.1.1 Single donkey-hide gelatin group: Take 2 mL of purified water and put it into an ampoule. Add 2 mL of donkey-hide gelatin solution (mass concentration of 30 mg / mL). After mixing evenly, divide it into 2 portions and heat it at 110℃ for 0 h (unreacted group) and 18 h (reacted group), respectively. 2.1.2 Reaction groups of different polar fractions of Rehmannia glutinosa-donkey-hide gelatin: Take 2 ml of extract of different polar fractions of Rehmannia glutinosa and put it into an ampoule. Add 2 ml of donkey-hide gelatin solution (mass concentration of 30 mg / ml). After mixing evenly, divide it into two portions and heat at 110℃ for 0 h (before reaction) and 18 h (after reaction) to obtain the samples before and after reaction, namely the Rehmannia glutinosa-donkey-hide gelatin group eluted with 0% ethanol, the Rehmannia glutinosa-donkey-hide gelatin group eluted with 10% ethanol, the Rehmannia glutinosa-donkey-hide gelatin group eluted with 70% ethanol, and the Rehmannia glutinosa-donkey-hide gelatin group eluted with 95% ethanol.

[0035] 2.1.3 Enzymatic digestion and sample pretreatment Take 1 ml of the above sample solution and place it in a 5 ml volumetric flask. Add 1 ml of trypsin (prepare a solution containing 5 mg per ml by adding 1% ammonium bicarbonate solution to trypsin, and prepare fresh before use). Dilute to the mark with 1% ammonium bicarbonate solution, shake well, and incubate at 37°C for 12 hours. Filter the solution and collect the filtrate. Inject each filtrate into a liquid chromatograph for component analysis.

[0036] 2.2 Analyze the changes in the content of characteristic polypeptides in donkey-hide gelatin before and after the reaction using LC-MS: 2.2.1 Chromatographic conditions The chromatographic column (Thermo Syncronis C18 (100mm*21mm*1.7um) was used. The column temperature was 45℃, the flow rate was 0.3 mL / min, the injection volume was 5μL, the mobile phase A was 0.1% formic acid, and the mobile phase B was 100% acetonitrile. Gradient elution was performed for 60 min, and the gradient elution program was as shown in Table 1.

[0037] Table 1 Gradient elution program according to the table The column is directly connected to a TSQ Quantum Access Max triple quadrupole mass spectrometer.

[0038] 2.2.2 Mass Spectrometry Conditions Using mass spectrometry as a detector, electrospray ionization (ESI) positive ion mode (ESI) + SRM mode monitoring was performed with the following ion source parameters: spray voltage 3500V, capillary temperature 320℃, sheath gas (nitrogen) pressure 35 Arb, auxiliary gas (nitrogen) pressure 10 Arb, collision gas (argon) pressure 1.5 mTorr. Ion pair monitoring is shown below. Table 2 Monitoring Ion Pairs

[0039] 2.2.3 Sample Testing All test solutions (before and after reaction) were divided into two parallel samples and injected for analysis according to the conditions described in sections 2.2.1 and 2.2.2. The output MRM chromatogram is shown below. Figure 1-5 Where a and b represent the peaks before the reaction, and c and d represent the peaks after the reaction, the peak areas of the quantitative ion pairs (618.35→779.40) are recorded.

[0040] 2.2.4 Screening of the polar fractions of Rehmannia glutinosa that have the greatest impact on the content of characteristic polypeptides in donkey-hide gelatin: The rate of change in the content of donkey-derived polypeptide A2 was calculated using the following formula: The magnitudes of the changes in the content of donkey-derived polypeptide A2 are compared in the following figures. Figure 6As shown in Table 3, the rate of change indicates that the highly active small molecules in the polar fraction of Rehmannia glutinosa specifically and efficiently covalently modify the donkey-derived polypeptide A2. The trypsin cleavage sites in the modified donkey-derived polypeptide A2 sequence or its precursor protein are affected, leading to a decrease in the detection content of donkey-derived polypeptide A2. The larger the rate of change, the more significant the effect.

[0041] The sample with the largest change rate of donkey-derived polypeptide A2 content was screened, and the corresponding extracts of different polar fractions of Rehmannia glutinosa were selected as the key synergistic extracts. In this example, the group of Rehmannia glutinosa-donkey-hide gelatin eluted with 0% ethanol had the largest change rate of donkey-derived polypeptide A2 content. Therefore, Rehmannia glutinosa eluted with 0% ethanol was selected as the key synergistic extract.

[0042] Table 3. Results of the change rate of donkey-derived polypeptide A2 content before and after the reaction. 3. Preparation of the composition The extract of Rehmannia glutinosa eluted with 0% ethanol, which was identified as the key synergistic component, was mixed with donkey-hide gelatin solution (mass concentration of 30 mg / ml) at a mass ratio of 1:1. The mixture was reacted at 100-120℃ for 16-20 hours to allow the two components to fully interact and generate a reaction product. The product was then concentrated and dried to obtain the synergistic composition of Rehmannia glutinosa and donkey-hide gelatin.

[0043] Example 2 The difference between this embodiment and Example 1 is that in step (3), 0% ethanol is used to wash the prepared Rehmannia glutinosa and mix it with donkey-hide gelatin in a mass ratio of 3.75:1.

[0044] Example 3 Take 100g of the synergistic composition of Rehmannia glutinosa and donkey-hide gelatin prepared in Example 2, mix it evenly with 900g of dextrin, granulate it with an appropriate amount of ethanol as a wetting agent, dry it, granulate it, and package it to obtain the granules of the composition of the present invention.

[0045] Test case To verify the beneficial effects of the synergistic composition of Rehmannia glutinosa and donkey-hide gelatin of the present invention, a zebrafish blood-tonifying activity test was conducted on Example 2.

[0046] Obtaining zebrafish embryos The culture and breeding of wild-type AB line zebrafish followed the Westerfield method. The day before egg collection, males and females were separated and fed artificial pelleted food and newly hatched Artemia nauplii at regular intervals. Alternating between 14 hours of light and 10 hours of darkness, males and females (1:2 or 1:1) were combined into one tank before the start of light exposure on the second day. Fertilized eggs were obtained after light exposure. The fertilized eggs were disinfected and washed, then transferred to zebrafish embryo culture water (containing 5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.16 mM MgSO4) and cultured under controlled light at 28°C.

[0047] Detection of blood replenishment activity in samples Wild-type AB zebrafish with normal development at 48 hpf (hour post fertilization) were used as experimental animals. An anemia model was constructed using PHZ (phenylhydrazine) as the modeling drug to evaluate the hemolytic activity of the samples.

[0048] Normally developing wild-type AB line zebrafish embryos (2 hpf) were selected. To reduce the influence of melanin in zebrafish on quantitative analysis, an appropriate amount of PTU was added to inhibit melanin production. Zebrafish embryos that developed to 48 hpf were randomly grouped and placed in 24-well plates, with 10 embryos per well.

[0049] The system included a blank control group, a model group (0.175 µg / ml pH), a positive control group (100 μg / ml sodium tanshinone SAAS), and three drug concentration groups, with three replicates for each group. The blank control group received treatment with fish tank water; the model group received the model drug pH; the positive control group received pH and sodium tanshinone; and the drug treatment groups received pH and different concentrations of the test samples.

[0050] Table 4 shows the specific groupings for the zebrafish hemolytic activity test: The dosage regimens for each drug are shown in Table 5.

[0051] Table 5 Dosage concentrations in each treatment group Note: Rehmannia glutinosa is calculated based on the amount of raw medicinal material, and donkey-hide gelatin is calculated based on the amount of medicinal material added. The composition is calculated by converting the amount of raw medicinal material and the amount of donkey-hide gelatin added.

[0052] After 72 hpf, zebrafish embryos in each group were stained, and white light micrographs were acquired to record the erythrocyte staining in the zebrafish heart region. Image-Pro Plus was used for statistical processing, and the significance levels of differences between groups were analyzed and compared. Statistical data are expressed as relative x ± SEM. Statistical differences were assessed through analysis of variance, with p < 0.05 considered statistically significant. Results are as follows: Figure 2 As shown.

[0053] The Control group was a blank control group, in which zebrafish were treated with embryo culture water without any drug treatment; the Model group (PHZ) was a phenylhydrazine treatment group without any drug treatment; the Positive Control Group (SAAS) was a modeling drug + tanshinone sodium combined treatment group, and each drug administration group was a modeling drug + graded concentration drug combined treatment group.

[0054] like Figure 3 As shown, compared with the control group, the erythrocyte staining area in the model group was significantly reduced (p < 0.001), indicating the success of the PHZ-induced anemia model. Compared with the model group, the positive control group SAAS significantly increased the number of erythrocytes in anemic zebrafish, further demonstrating that the model can be used to evaluate the hematopoietic activity of the samples. The experiment found that the combination of Rehmannia glutinosa and donkey-hide gelatin significantly improved the erythrocyte reduction in zebrafish induced by PHZ, exhibiting hematopoietic activity, and the activity was superior to that of donkey-hide gelatin alone or the polar fraction of Rehmannia glutinosa, proving that donkey-hide gelatin and the polar fraction of Rehmannia glutinosa have a synergistic effect.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin, characterized in that, Includes the following steps: (1) Extraction and separation of polar fractions of Rehmannia glutinosa: Take Rehmannia glutinosa, extract it with water, and then use macroporous resin to separate the Rehmannia glutinosa extract to obtain extracts of different polar fractions of Rehmannia glutinosa. (2) Screening and enrichment of key components: The extracts of different polar components of Rehmannia glutinosa obtained in step (1) were subjected to directional reaction with donkey-hide gelatin solution. The content of characteristic polypeptides of donkey-hide gelatin before and after the reaction was detected by LC-MS method. The extract of polar component of Rehmannia glutinosa that has the greatest impact on the content of characteristic polypeptides of donkey-hide gelatin was selected as the key synergistic component extract. (3) The extract of the key synergistic component selected in step (2) is mixed with the gelatin liquid at a mass ratio of 1:2 to 5:

1. The mixture is reacted at 100-120℃ for 16-20 hours to allow the two to fully react and generate a reaction product. The product is then concentrated and dried to obtain the synergistic composition of Rehmannia glutinosa and gelatin.

2. The method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin as described in claim 1, characterized in that, Step (1) includes the following specific steps: The mass-volume ratio of Rehmannia glutinosa and water is 1:2-1:

10. Place Rehmannia glutinosa in a round-bottom flask, add water, heat to boiling, and maintain for 60-120 minutes. Pour out the extract, repeat the extraction twice, with the same amount of water and extraction time each time. Combine the extracts to obtain Rehmannia glutinosa extract. The column was packed with Rehmannia glutinosa and macroporous resin at a mass ratio of 1:2 to 1:

3. After loading the Rehmannia glutinosa extract at a flow rate of 2-4 BV / h, static adsorption was performed for 25-40 minutes. The first eluent was collected by eluting with 5-7 BV of distilled water. The second eluent was collected by eluting with 3-5 BV of 10% ethanol. The third eluent was collected by eluting with 3-5 BV of 70% ethanol. The fourth eluent was collected by eluting with 3-5 BV of 95% ethanol. The extracts of different polar fractions of Rehmannia glutinosa included the first, second, third, and fourth eluents.

3. A method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin as described in claim 1 or 2, characterized in that, The directional reaction between the extracts of different polar fractions of Rehmannia glutinosa and the gelatin solution in step (2) includes: Extracts of different polar fractions of Rehmannia glutinosa were mixed evenly with gelatin solution to obtain samples before reaction. The samples before reaction were heated at 100-120℃ for 16-22 hours to obtain samples after reaction. The volume ratio of extracts of different polar fractions of Rehmannia glutinosa to gelatin solution was 1:

1.

4. The method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin as described in claim 3, characterized in that, In step (2), the LC-MS method was used to detect the content of characteristic polypeptides in donkey-hide gelatin before and after the directional reaction, including: Take the pre-reaction and post-reaction samples and place them in their respective volumetric flasks. The volume ratio of the pre-reaction sample to the volumetric flask is (0.5-1.5):(4-6). Add the same volume of trypsin solution as the pre-reaction and post-reaction samples. The trypsin solution is prepared by adding 1 wt.% ammonium bicarbonate solution to trypsin to a solution containing 5 mg of trypsin per ml. Then, add 1 wt.% ammonium bicarbonate solution to dilute to the mark of the volumetric flask, shake well, and incubate at 37°C for 12 hours. Filter, collect the filtrate, and perform component analysis on each filtrate using a liquid chromatograph.

5. The method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin as described in claim 4, characterized in that, The conditions for the chromatograph are as follows: Flow rate 0.3 ml / min, gradient elution for 30 min, mobile phase A: 0.1% formic acid, mobile phase B: 100% acetonitrile, gradient elution program as shown in the table: 。 6. The method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin as described in claim 5, characterized in that, The chromatograph was directly connected to the mass spectrometer, using the mass spectrometer as the detector. Electrospray ionization (ESI+) mode was used in positive ion mode, and SRM mode was employed for monitoring. The monitored ion pairs are shown in the table below: The mass spectrometer outputs a characteristic spectrum, and the content of donkey-derived polypeptide A2 in the sample before and after the reaction is calculated using the characteristic spectrum.

7. The method for preparing a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin as described in claim 6, characterized in that, The filtering includes: The rate of change in the content of donkey-derived polypeptide A2 was calculated using the following formula: By comparing the rate of change in the content of donkey-derived polypeptide A2, the sample with the largest rate of change in the content of donkey-derived polypeptide A2 before the reaction was selected, and the extracts of different polar fractions of Rehmannia glutinosa were used as the key synergistic extracts.

8. A synergistic composition of Rehmannia glutinosa and donkey-hide gelatin, characterized in that, The synergistic composition of Rehmannia glutinosa and donkey-hide gelatin is prepared by any one of the preparation methods of claims 1-7.

9. A traditional Chinese medicine preparation, characterized in that, It includes the synergistic composition of Rehmannia glutinosa and donkey-hide gelatin as described in claim 8 and pharmaceutically acceptable excipients.

10. The use of a synergistic composition of Rehmannia glutinosa and donkey-hide gelatin, characterized in that, The use of the synergistic composition of Rehmannia glutinosa and donkey-hide gelatin as described in claim 8 in the preparation of blood-tonifying drugs.

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