Preparation method of active ingredient group of modified collateral-activating effective prescription
By optimizing the preparation method of the modified Huoluo Xiaoling formula, specific active ingredients were extracted and purified, solving the problems of mixed active ingredients and unstable efficacy, and achieving highly effective treatment for sequelae of stroke, especially cognitive impairment.
Patent Information
- Application Number
- CN202511800305.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-02
- Publication Date
- 2026-01-20
AI Technical Summary
The existing modified Huoluo Xiaoling formula contains a mixture of active ingredients, making quality control difficult and resulting in large fluctuations in efficacy. It also lacks therapeutic effects for post-stroke sequelae, especially cognitive impairment.
Using specific proportions of herbs such as Salvia miltiorrhiza, Angelica sinensis, Boswellia carterii, Commiphora myrrha, Hirudo medicinalis, Astragalus membranaceus, Acorus tatarinowii, Pheretima aspergillum, Ligusticum chuanxiong, and Panax notoginseng, 10 active ingredients, including tanshinone and salvianolic acid B, are extracted and purified through steps such as enzymatic activation, synergistic extraction, macroporous resin purification, and polyamide column refining, ensuring that the total content is not less than 45%.
It significantly improves the treatment effect on sequelae of stroke, especially the targeted treatment of cognitive impairment, enhances the synergistic effect of promoting blood circulation and clearing the meridians and opening the mind, improves the extraction rate and bioavailability, and the product purity and efficacy are significantly better than traditional methods.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of traditional Chinese medicine composition preparation, and particularly relates to a preparation method of an active ingredient group of a flavored Huoluo Xialing Decoction. BACKGROUND
[0002] Stroke is one of the major causes of disability and death worldwide, and its sequelae (such as limb dysfunction, cognitive impairment, and dysphagia) bring a heavy burden to patients' families and society. Traditional Chinese medicine believes that the core pathogenesis of stroke sequelae is "qi deficiency and blood stasis, phlegm and blood stasis blocking collaterals", and often accompanied by cognitive impairment performance of "knob closure and mental dullness". Huoluo Xialing Decoction is derived from Medical Records of Western Medicine by Zhang Xichun in the Qing Dynasty, and is composed of four medicines, i.e. Salvia miltiorrhiza, Angelica sinensis, Frankincense and Myrrh. It is a classic prescription for promoting blood circulation, removing blood stasis and dredging collaterals to relieve pain.
[0003] In modern clinical practice, water leeches are often added to the original prescription to enhance the ability of removing blood stasis and dredging collaterals, and Astragalus is added to tonify qi and consolidate the foundation and assist blood circulation, forming a "flavored Huoluo Xialing Decoction" for treating stroke sequelae, which has achieved good application prospects. However, the modern development of this prescription faces the following major bottlenecks. The chemical components of this compound are complex, and the effective components are mixed with ineffective components in traditional decoction or crude extract. It is not clear which is the core active substance group, resulting in difficult quality control, large fluctuation of drug efficacy. In addition, although the original prescription and the conventional flavored prescription can promote blood circulation and remove blood stasis, they are not strong enough for cognitive impairment, which is a high incidence of stroke sequelae, and lack the effect of "opening the orifice and awakening the spirit", which is difficult to achieve the overall rehabilitation of patients.
[0004] Therefore, there is an urgent need in the art for a modern preparation method of a flavored Huoluo Xialing Decoction which can clearly identify the core active substance, is quality controllable, and has enhanced efficacy for stroke sequelae (especially cognitive impairment). SUMMARY
[0005] The purpose of the present application is to provide a preparation method of a flavored Huoluo Xialing Decoction active ingredient group, which solves the problems existing in the prior art.
[0006] The present application achieves the above-mentioned purpose through the following technical solutions: A flavored Huoluo Xialing Decoction active ingredient group, the raw material composition of which comprises: Salvia miltiorrhiza 20-25%; Angelica sinensis 15-20%; Frankincense 10-15%; Myrrh 10-15%; Leech 5-8%; Astragalus 10-15%; Acorus gramineus 5-10%; Earthworm 3-6%; Chuanxiong 8-12%; Notoginseng 5-10%; The active ingredient group comprises Danshensu, Salvianolic Acid B, Tanshinone IIA, Ferulic Acid, 11-keto-β-boswellic acid, 3-acetyl-11-keto-β-boswellic acid, Astragaloside IV, α-asarone, Ligusticum chuanxiong Hance lactone and Notoginseng Saponin Rg1, and the total content of the 10 ingredients is not less than 45% of the total weight of the solid.
[0007] Further improvement is that in the active ingredient group, the relative weight ratio of Danshensu, Salvianolic Acid B, Tanshinone IIA, Ferulic Acid, 11-keto-β-boswellic acid, 3-acetyl-11-keto-β-boswellic acid, Astragaloside IV, α-asarone, Ligusticum chuanxiong Hance lactone and Notoginseng Saponin Rg1 is (1-3):(5-15):(0.5-2):(0.5-2):(0.5-2):(0.5-2):(1-3):(0.1-1):(0.5-2):(1-3).
[0008] The application also provides a preparation method of the active ingredient group of the flavored active blood circulation and pain relief prescription, which comprises the following steps: S1, enzymatic activation: after low-temperature drying, the earthworm is crushed, and then mixed with the crude powder of the prescription amount of Danshen, Angelica sinensis, Chuanxiong and Notoginseng, 8-12 times the amount of phosphate buffer solution with a pH value of 6.0-7.0 is added, and then stirred and reacted in a water bath at 35-40 DEG C for 1-1.5 hours; S2, synergistic extraction: the mixture after the enzymatic reaction is combined with the prescription amount of Frankincense, Myrrh, Leech, Astragalus, Acorus, 10-14 times the amount of 60%-70% ethanol aqueous solution is added, and then heated and refluxed at 80-90 DEG C for 2 times, each for 1-2 hours, the extraction liquid is combined, and then concentrated under reduced pressure at 55-65 DEG C to a thick paste with a relative density of 1.10-1.15; S3, macroporous resin purification: the thick paste obtained in step S2 is suspended with water to a crude drug concentration of 0.4-0.6 g / mL, and then loaded into an AB-8 type or D101 type macroporous adsorption resin column, and then sequentially washed with 3-5 times the column volume of distilled water, 4-6 times the column volume of 20%-30% ethanol aqueous solution to remove impurities, and then eluted with 5-8 times the column volume of 60%-70% ethanol aqueous solution at a flow rate of 1-3 BV / h, and then the eluate is collected; S4, polyamide column refining: after the eluate obtained in step S3 is concentrated under reduced pressure to remove ethanol, the eluate is loaded into a polyamide column, and then washed with 3-5 times the column volume of 20%-30% ethanol aqueous solution, and then eluted with 4-6 times the column volume of 60%-70% ethanol aqueous solution, and then the target eluate is collected; S5, active ingredient group keeping dry: the target eluent obtained in step S4 is concentrated under reduced pressure at 55-65 DEG C, and then spray dried, the inlet air temperature is 150-170 DEG C, and the outlet air temperature is 75-85 DEG C, to obtain the active ingredient group.
[0009] Further improvement lies in that in step S1, the pH value of the phosphate buffer is 6.5, the reaction temperature is 37 DEG C, and the reaction time is 1 hour.
[0010] Further improvement lies in that in step S2, the concentration of the ethanol aqueous solution is 65%, the solid-liquid ratio is 1:10, and the heating reflux extraction is carried out twice, each time for 1.5 hours.
[0011] Further improvement lies in that in step S3, the elution is carried out by using 65% ethanol aqueous solution, and the elution flow rate is 2 BV / h.
[0012] Further improvement lies in that in step S4, the elution is carried out by using 65% ethanol aqueous solution.
[0013] Further improvement lies in that in step S5, the inlet air temperature of the spray drying is 160 DEG C, and the outlet air temperature is 80 DEG C.
[0014] The application further provides a pharmaceutical composition containing the active ingredient group of the flavored Huoluo Xialing recipe and one or more pharmaceutically acceptable carriers or excipients, and the dosage form is oral liquid, tablet, capsule, granule, nanoemulsion or transdermal patch.
[0015] The application further provides a use of the active ingredient group of the flavored Huoluo Xialing recipe or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating sequelae of cerebral stroke, including limb dysfunction, cognitive impairment, dysphagia or qi deficiency and blood stasis syndrome.
[0016] The application has the following beneficial effects: (1) The application creatively introduces Acorus gramineus, Chuanxiong and Sanqi, etc. on the basis of the classical recipe, wherein Acorus gramineus (containing alpha-asarone) and Chuanxiong (containing ligustilide) are synergistic, enhance the function of "opening the orifice and awakening the spirit", and significantly improve the treatment targeting of cognitive impairment after cerebral stroke; Sanqi (containing notoginsenoside Rg1) and Huangqi (containing astragaloside A) are compatible, realizing the synergistic effect of "resolving stasis without damaging normal qi and tonifying qi to assist blood circulation", which surpasses the function of the original recipe of simply "activating blood and dredging collaterals", and forms a multi-target treatment scheme of "activating blood- tonifying qi-opening the orifice-dredging collaterals".
[0017] (2) The present application uses endogenous enzymes in earthworms to pretreat medicinal materials under mild conditions (pH 6.5, 37°C), which can effectively activate precursor components and promote their conversion, thereby significantly improving the extraction rate and bioavailability of target active ingredients (such as danshensu and ferulic acid). Reflux extraction with 65% ethanol takes into account the dissolution of water-soluble and fat-soluble active ingredients; subsequent gradient elution and purification using macroporous adsorption resin and polyamide resin can selectively enrich target components and efficiently remove impurities, ultimately resulting in a product purity (total active ingredient content ≥ 45%) far exceeding that of traditional water extracts (only 15.3% for Comparative Example 1).
[0018] (3) The active ingredient group prepared by the present application exhibits comprehensive and outstanding activity in anticoagulation (such as APTT extension rate of 28.7%), anti-inflammatory (such as TNF-α inhibition rate of 52.3%), neuroprotection (brain infarction volume reduction of 60.6%), and cognitive function improvement (excellent performance in water maze test), with a comprehensive score significantly better than all comparative examples, indicating that the present application not only achieves the synergistic effect of "activating blood and unblocking meridians" and "opening the orifices and awakening the mind", but also ensures the maximum retention and synergistic effect of medicinal substances. DETAILED DESCRIPTION
[0019] Further detailed description of the present application is as follows, it is necessary to point out that the following detailed description is only for further description of the present application, and cannot be understood as limitation of the protection scope of the present application, and the skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.
[0020] I. Main materials and equipment Salvia miltiorrhiza, Angelica sinensis, Frankincense, Myrrh, Hirudo, Astragalus membranaceus, Acorus gramineus, Earthworm, Chuanxiong, Panax notoginseng: all purchased from Beijing Tong Ren Tang Co., Ltd., identified to meet the standards of Chinese Pharmacopoeia 2020 edition, crushed to 40 mesh, water content controlled below 8%, earthworms treated by vacuum freeze-drying to maintain enzyme activity, earthworms crushed to 60 mesh after low-temperature drying; Phosphate buffer: pH 6.5, Beijing Solabio Science and Technology Co., Ltd.; Ethanol: analytical pure, National Pharmaceutical Group Chemical Reagent Co., Ltd.; AB-8 macroporous adsorption resin: Tianjin Bohong Resin Technology Co., Ltd.; Polyamide: 100-200 mesh, Shanghai Yuanye Biotechnology Co., Ltd.; UPLC-MS system: Waters ACQUITY UPLC H-Class-Xevo TQD.
[0021] II. Implementation experiment Example
[0022] Prescription: Salvia miltiorrhiza 22%, Angelica 18%, Frankincense 12%, Myrrh 12%, Leech 7%, Astragalus 13%, Acorus 8%, Earthworm 5%, Chuanxiong 10%, Sanchi 8%.
[0023] The preparation method steps include: S1, enzymatic activation: after low-temperature drying and crushing the prescription amount of earthworm, mix it with the prescription amount of crude powder of salvia miltiorrhiza, angelica, chuanxiong and sanchi decoction pieces, add 10 times the amount of phosphate buffer solution with pH value of 6.5, stir in 37℃ water bath for 1 hour; S2, synergistic extraction: combine the mixture after enzymatic reaction with the prescription amount of frankincense, myrrh, leech, astragalus and acorus, add 10 times the amount of 65% ethanol aqueous solution, heat under reflux at 85℃ for 2 times, 1.5 hours each time, combine the extract, and concentrate under reduced pressure at 60℃ to a thick paste with relative density of 1.12; S3, macroporous resin purification: suspend the thick paste with water to a crude drug concentration of 0.5g / mL, load it into AB-8 type macroporous adsorption resin column, wash with 4 times the column volume of distilled water, 5 times the column volume of 25% ethanol aqueous solution to remove impurities, then elute with 6 times the column volume of 65% ethanol aqueous solution at a flow rate of 2BV / h, and collect the eluate; S4, polyamide column refining: after removing ethanol by concentrating the eluate under reduced pressure, load it into a polyamide column, wash with 4 times the column volume of 25% ethanol aqueous solution, then elute with 5 times the column volume of 65% ethanol aqueous solution, and collect the target eluate; S5, active ingredient group drying: after concentrating the target eluate under reduced pressure at 60℃, spray dry it, with inlet air temperature of 160℃ and outlet air temperature of 80℃, to obtain the active ingredient group. Example
[0024] Prescription: Salvia miltiorrhiza 20%, Angelica 15%, Frankincense 15%, Myrrh 15%, Leech 8%, Astragalus 12%, Acorus 10%, Earthworm 4%, Chuanxiong 12%, Sanchi 5% The preparation method steps include: S1, enzymatic activation: after low-temperature drying and crushing the prescription amount of earthworm, mix it with the prescription amount of crude powder of salvia miltiorrhiza, angelica, chuanxiong and sanchi decoction pieces, add 10 times the amount of phosphate buffer solution with pH value of 6.5, stir in 37℃ water bath for 1 hour; S2, synergistic extraction: combine the mixture after enzymatic reaction with the prescription amount of frankincense, myrrh, leech, astragalus and acorus, add 10 times the amount of 65% ethanol aqueous solution, heat under reflux at 85℃ for 2 times, 1.5 hours each time, combine the extract, and concentrate under reduced pressure at 60℃ to a thick paste with relative density of 1.12; S3, macroporous resin purification: the thick paste was suspended with water to a crude drug concentration of 0.5 g / mL, and then loaded onto an AB-8 type macroporous adsorption resin column. Distilled water was used to flush the column for 4 column volumes, followed by 5 column volumes of 25% ethanol aqueous solution to remove impurities. Then, 6 column volumes of 65% ethanol aqueous solution were used to elute at a flow rate of 2 BV / h, and the eluate was collected; S4, polyamide column purification: after the eluate was concentrated under reduced pressure to remove ethanol, it was loaded onto a polyamide column. After being washed with 4 column volumes of 25% ethanol aqueous solution, 5 column volumes of 65% ethanol aqueous solution were used to elute the column, and the target eluate was collected; S5, active ingredient group drying: after the target eluate was concentrated under reduced pressure at 60°C, it was subjected to spray drying, with an inlet air temperature of 160°C and an outlet air temperature of 80°C, to obtain the active ingredient group. Example
[0025] Prescription: Danshen 25%, Danggui 20%, Olibanum 10%, Myrrh 10%, Leech 5%, Huangqi 15%, Shichangpu 5%, Dilong 6%, Chuanxiong 8%, Sanqi 10% Preparation method steps include: S1, enzymatic activation: after the prescription amount of Dilong was dried at low temperature and crushed, it was mixed with the prescription amount of Danshen, Danggui, Chuanxiong and Sanqi decoction powder. Then, 10 times the amount of phosphate buffer with a pH value of 6.5 was added, and the mixture was stirred in a 37°C water bath for 1 hour; S2, synergistic extraction: the mixture after enzymatic reaction was combined with the prescription amount of Olibanum, Myrrh, Leech, Huangqi, and Shichangpu. Then, 10 times the amount of 65% ethanol aqueous solution was added, and the mixture was heated to reflux at 85°C for 2 times, each for 1.5 hours. The combined extract was concentrated under reduced pressure at 60°C to a thick paste with a relative density of 1.12; S3, macroporous resin purification: the thick paste was suspended with water to a crude drug concentration of 0.5 g / mL, and then loaded onto an AB-8 type macroporous adsorption resin column. Distilled water was used to flush the column for 4 column volumes, followed by 5 column volumes of 25% ethanol aqueous solution to remove impurities. Then, 6 column volumes of 65% ethanol aqueous solution were used to elute at a flow rate of 2 BV / h, and the eluate was collected; S4, polyamide column purification: after the eluate was concentrated under reduced pressure to remove ethanol, it was loaded onto a polyamide column. After being washed with 4 column volumes of 25% ethanol aqueous solution, 5 column volumes of 65% ethanol aqueous solution were used to elute the column, and the target eluate was collected; S5, active ingredient group drying: after the target eluate was concentrated under reduced pressure at 60°C, it was subjected to spray drying, with an inlet air temperature of 160°C and an outlet air temperature of 80°C, to obtain the active ingredient group.
[0026] Comparative Example 1: the prescription is the same as Example 1, and the process uses the traditional water extraction method (10 times the amount of water, decocting 2 times) Prescription: Salvia 22%, Angelica 18%, Frankincense 12%, Myrrh 12%, Leech 7%, Astragalus 13%, Acorus 8%, Earthworm 5%, Chuanxiong 10%, Sanchi 8% The preparation method steps include: S1, medicinal material processing: all medicinal materials are crushed through a 40 mesh sieve and mixed uniformly according to the prescription ratio; S2, water decoction extraction: the mixed medicinal materials are taken, 10 times the amount of distilled water is added, soaked for 30 minutes, then heated to boiling, kept in a state of slight boiling for 2 hours, and the filtrate is collected by filtration; S3, secondary extraction: the residue is added with 8 times the amount of distilled water again, decocted for 1.5 hours, and the filtrate is collected by filtration; S4, combined and concentrated: the two filtrates are combined and concentrated under reduced pressure to a thick paste with a relative density of 1.12 at 60°C; S5, drying treatment: the thick paste is vacuum dried at 60°C, crushed through an 80 mesh sieve, and the traditional water extract is obtained.
[0027] Comparative Example 2: the prescription is the same as Example 1, without earthworm, and the process does not have enzymatic reaction, and the rest is the same as Example 1 Prescription: Salvia 23%, Angelica 19%, Frankincense 13%, Myrrh 13%, Leech 7%, Astragalus 14%, Acorus 8%, Chuanxiong 11%, Sanchi 8% (Note: after removing earthworm, the other components are adjusted according to the proportion) The preparation method steps include: S1, raw material mixing: the prescription amount of Salvia, Angelica, Chuanxiong and Sanchi decoction pieces are directly mixed; S2, ethanol extraction: the mixed medicinal materials are combined with the prescription amount of Frankincense, Myrrh, Leech, Astragalus and Acorus, 10 times the amount of 65% ethanol aqueous solution is added, heated to reflux extraction at 85°C for 2 times, 1.5 hours each time, the extract is combined, and concentrated under reduced pressure to a thick paste with a relative density of 1.12 at 60°C; S3, macroporous resin purification: the thick paste is suspended with water to a crude drug concentration of 0.5g / mL, and then loaded onto an AB-8 type macroporous adsorption resin column, and then sequentially washed with 4 times the column volume of distilled water, 5 times the column volume of 25% ethanol aqueous solution to remove impurities, and then eluted with 6 times the column volume of 65% ethanol aqueous solution at a flow rate of 2BV / h, and the eluate is collected; S4, polyamide column refining: after the eluate is concentrated under reduced pressure to remove ethanol, it is loaded onto a polyamide column, washed with 4 times the column volume of 25% ethanol aqueous solution, and then eluted with 5 times the column volume of 65% ethanol aqueous solution, and the target eluate is collected; S5, drying treatment: the target eluate is concentrated under reduced pressure at 60°C, and then spray dried, with an inlet air temperature of 160°C and an outlet air temperature of 80°C, and the active ingredient group is obtained.
[0028] Comparative Example 3: Without Chuanqiong and Sanqi, the rest is the same as Example 1 Prescription: Salvia 27%, Angelica 22%, Frankincense 14%, Myrrh 14%, Leech 8%, Astragalus 15%, Acorus 10%, Earthworm 6% (Note: After removing Chuanqiong and Sanqi, adjust the other ingredients in proportion) The preparation method steps include: S1, Enzymatic activation: After low-temperature drying, the prescription amount of earthworm is crushed and mixed with the prescription amount of salvia, angelica decoction powder, 10 times the amount of phosphate buffer solution with pH value of 6.5 is added, and stirred in 37℃ water bath for 1 hour; S2, Synergistic extraction: The mixture after enzymatic reaction is combined with the prescription amount of frankincense, myrrh, leech, astragalus, and acorus, 10 times the amount of 65% ethanol aqueous solution is added, and heated under reflux at 85℃ for 2 times, 1.5 hours each time, the extract is combined, and concentrated under reduced pressure at 60℃ to a thick paste with a relative density of 1.12; S3, Macroporous resin purification: The thick paste is suspended with water to a crude drug concentration of 0.5g / mL, and then loaded onto an AB-8 type macroporous adsorption resin column, and then washed with 4 times the column volume of distilled water, 5 times the column volume of 25% ethanol aqueous solution to remove impurities, and then eluted with 6 times the column volume of 65% ethanol aqueous solution at a flow rate of 2BV / h, and the eluate is collected; S4, Polyamide column refining: After removing ethanol by reducing pressure concentration, the eluate is loaded onto a polyamide column, washed with 4 times the column volume of 25% ethanol aqueous solution, and then eluted with 5 times the column volume of 65% ethanol aqueous solution, and the target eluate is collected; S5, Active ingredient group: After reducing pressure concentration at 60℃, the target eluate is spray dried, the inlet air temperature is 160℃, and the outlet air temperature is 80℃, and the active ingredient group is obtained.
[0029] Comparative Example 4: The prescription is the same as Example 1, and the macroporous resin purification process is omitted after the polyamide column refining Prescription: Salvia 22%, Angelica 18%, Frankincense 12%, Myrrh 12%, Leech 7%, Astragalus 13%, Acorus 8%, Earthworm 5%, Chuanqiong 10%, Sanqi 8% The preparation method steps include: S1, Enzymatic activation: After low-temperature drying, the prescription amount of earthworm is crushed and mixed with the prescription amount of salvia, angelica, chuanqiong and sanqi decoction powder, 10 times the amount of phosphate buffer solution with pH value of 6.5 is added, and stirred in 37℃ water bath for 1 hour; S2, synergistic extraction: the mixture after enzymatic reaction was combined with the prescribed amount of frankincense, myrrh, leech, astragalus, acorus, and 10 times the amount of 65% ethanol aqueous solution was added. The mixture was heated and refluxed at 85°C for 2 times, 1.5 hours each time, and the extract was combined. The extract was concentrated at 60°C under reduced pressure to a thick paste with a relative density of 1.12; S3, macroporous resin purification: the thick paste was suspended in water to a crude drug concentration of 0.5 g / mL, and then loaded onto an AB-8 type macroporous adsorption resin column. The column was washed with 4 times the column volume of distilled water, 5 times the column volume of 25% ethanol aqueous solution to remove impurities, and then eluted with 6 times the column volume of 65% ethanol aqueous solution at a flow rate of 2 BV / h. The eluate was collected. S4, direct drying: after removing ethanol from the eluate under reduced pressure, the eluate was directly spray dried at an inlet temperature of 160°C and an outlet temperature of 80°C to obtain the active ingredient group.
[0030] III. Ingredient analysis and structure identification Qualitative and quantitative analysis of core ingredients: the prepared active ingredient group powder was dissolved in methanol-water solution, ultrasonically extracted, centrifuged, and filtered through a microporous filter to prepare a test solution. The test sample and standard were analyzed under the same UPLC conditions, the retention time was compared, and the molecular ion peak ([M+H]+) of the ingredient in the sample was detected and matched with the mass spectrum data of the standard. For characteristic conversion products, their structures were inferred by analyzing their secondary mass spectrum fragment ions. Ten mixed standard solutions of different known concentrations were prepared and injected into the UPLC-MS / MS system to establish a standard curve of peak area-concentration. The test sample was analyzed under the same conditions, and the peak area of each ingredient was substituted into the corresponding standard curve to calculate the specific content of the ingredient in the sample.
[0031] The qualitative and quantitative analysis results of the 10 core ingredients are shown in the table below:
[0032] IV. Pharmacodynamic experimental verification 1. In vitro anti-coagulation experiment Experimental method: the final product of each experimental group was prepared into a 1 mg / mL solution with physiological saline, and filtered through a 0.22 μm microporous filter Experimental system: New Zealand white rabbits with platelet-rich plasma (n=6), platelet count was measured using Sysmex XN-1000 blood cell analyzer (250-350×10 9 / L) Detection index: APTT, PT, TT, FIB Instrument: Sysmex CS-5100 automatic coagulation analyzer with original reagents Statistical method: One-way ANOVA, LSD post-hoc test using GraphPad Prism 9.0, the experimental results are as follows:
[0033] 2. Anti-inflammatory experiment in vitro Cell model: RAW264.7 mouse macrophages (ATCC TIB-71) Culture conditions: DMEM high-sugar medium + 10% FBS, 37°C, 5% CO2 culture Inflammation induction: LPS 1 μg / mL stimulation for 24 hours Sample processing: 12.5, 25, 50, 100 μg / mL concentration gradient, pretreated for 2 hours before adding LPS Detection method: R&D Systems ELISA kit was used to detect TNF-α, IL-6, IL-1β Instrument: BioTek Synergy H1 multifunctional microplate reader Positive control: Dexamethasone (10 μM)
[0034] 3. In vivo pharmacodynamic experiment Animal model: SD rats, SPF level, male, body weight 200-250 g (n=10 / group) Model: MCAO model by thread method, ischemia for 2 h and reperfusion Anesthesia: Intraperitoneal injection of 4% chloral hydrate (400 mg / kg) Monitoring: Physiological parameters were monitored using MouseOx Plus small animal oximeter Dosing regimen: Intragastric administration started at 24 h after modeling, 100 mg / kg / d, for 14 consecutive days The sham operation group and the model group were given the same volume of normal saline Measurement of cerebral infarction volume: TTC staining, ImageJ software analysis of infarction area Instrument: Zeiss SteREO Discovery.V20 stereomicroscope
[0035] Morris water maze test: Equipment: ZS-001 Morris water maze system (Anhui Zhenghua Biology) Water temperature: 23±1°C Training: 4 times a day for 5 consecutive days Test: Spatial exploration experiment was performed on day 14
[0036] V. Comprehensive comparison and analysis of examples and comparative examples
[0037] Based on the above table, the proportion of each medicinal flavor in Example 1 is relatively balanced, without obvious bias. The proportion of salvia miltiorrhiza (22%) and angelica sinensis (18%) is moderate, which is the core compatibility of promoting blood circulation and nourishing blood. The degree of breaking blood stasis of frankincense and myrrh (each 12%) is comparable to the degree of tonifying qi and strengthening the body resistance of astragalus (13%). At the same time, the effective dose of key adjuvant herbs such as ligusticum chuanxiong, panax notoginseng, and rhizoma of alpinia oxyphylla is ensured. The total content of 10 components is the highest (47.8%), and each key component (such as salvianolic acid B 14.2% and panax notoginseng saponin Rg1 13.12%) is at a high level, which reflects the best synergistic extraction and enrichment effect. It is comprehensive leading in anticoagulation (APTT extension rate 28.7%), neuroprotection (infarction volume reduction 60.6%), and cognitive improvement (water maze performance best), and the comprehensive score is the first (91.3), which indicates that the prescription structure realizes the optimal balance among various treatment methods such as promoting blood circulation, tonifying qi, opening the orifices, and dredging collaterals, thereby producing the strongest comprehensive effect.
[0038] Example 2 relatively enhances the degree of breaking blood stasis and dredging collaterals. The proportion of frankincense and myrrh is the highest (each 15%), the proportion of limulus is the highest (8%), and the proportion of ligusticum chuanxiong is relatively high (12%). At the same time, the proportion of astragalus for strengthening the body resistance (12%) and salvia miltiorrhiza and angelica sinensis for nourishing blood and promoting blood circulation (20% and 15%, respectively) is relatively low. The total content (46.2%) is slightly lower than that of Example 1. The content of components related to breaking blood stasis and dredging collaterals, such as 11-keto-β-boswellic acid and 3-acetyl-11-keto-β-boswellic acid (derived from frankincense and myrrh), is expected to be higher. Its anticoagulant activity (score 88) is excellent, only second to Example 1, which is consistent with the high proportion of blood-breaking and blood-stasis-removing herbs. However, in terms of neuroprotection (score 85) and cognitive improvement (score 82) which require the support of qi and blood and opening the orifices and awakening the spirit, it is slightly inferior to Example 1, which proves that excessive emphasis on breaking blood stasis may slightly lack balance in overall efficacy synergy.
[0039] Example 3 relatively enhances the strength of tonifying qi and nourishing blood, and the proportions of Danshen (25%), Danggui (20%), Huangqi (15%), and Sanqi (10%) are the highest among the three, while the proportions of attacking evil of Ru Xiang, Duhuanga (each 10%), Shui Zhimu (5%), and opening the orifices of Shichangpu (5%) are the lowest, and the total content (45.5%) is slightly lower than the previous two, but the content of the components related to tonifying qi and nourishing blood, such as Astragaloside IV and Panax Notoginseng Saponins Rg1, is expected to be better, and its anti-inflammatory activity (score 86) is equivalent to or even slightly higher than that of Example 2, which may be due to the higher proportion of Huangqi, Danshen, etc. anti-inflammatory effect, however, due to the relative weakening of the strength of removing blood stasis and dredging collaterals and opening the orifices, its anticoagulant activity (score 85) and cognitive improvement (score 80) are the weakest among the three examples, which shows that although the tonifying strength is strong, the lack of attacking evil will also limit the overall efficacy for the deficiency and excess intermingled syndrome of stroke sequelae.
[0040] In summary, the prescription ratio of Example 1 achieves the best balance point, which not only provides sufficient strength of removing blood stasis and dredging collaterals (moderate doses of Ru Xiang, Duhuanga, and Shui Zhimu) to eliminate real evil, but also provides sufficient tonifying qi and nourishing blood (appropriate doses of Huangqi, Danshen, and Danggui) to support the healthy qi, while ensuring the realization of the targeted functions such as opening the orifices and awakening the spirit (Shichangpu and Chuanxiong), and this high degree of synergy makes it perform best in complex multi-target treatment; although Example 1 is the best, the comprehensive scores (85.5 and 83.5) of Examples 2 and 3 are still significantly better than all the comparative examples, and a high content of active ingredient groups is maintained, which proves that the required prescription range (as defined in the claims) of the present application is effective and reliable, and although the adjustment within the range will cause slight fluctuations in efficacy, it can still ensure the realization of the core functions of the product.
[0041] The comparison between Example 1 and the comparative examples is shown in the following table:
[0042] Analysis conclusion Example 1 has the highest content, far exceeding Comparative Example 1, proving the superiority of the process of the present application, and the total content of Comparative Examples 2 / 3 / 4 is lower than that of Example 1, indicating that each step of the process and each medicinal material contributes to the final content of the ingredients, and the water extraction method (Comparative Example 1) has very low extraction efficiency for water-soluble phenolic acid components, and enzyme activation (Example 1 vs. Comparative Example 2) helps to release phenolic acid components from Danshen and other medicinal materials, and the absence of Chuanxiong and Sanqi in Comparative Example 3 directly leads to the absence of Ligusticum Chuanxiong Lide and Panax Notoginseng Saponins Rg1, proving the irreplaceable value of these two medicinal materials in the prescription.
[0043] A. In vitro anticoagulant experiment The APTT prolongation rate of Example 1 is as high as 28.7%, which is significantly better than other groups.
[0044] Comparative Example 1 (traditional water extraction) has almost no anticoagulant effect, proving that its active ingredients are largely lost.
[0045] Comparative Example 2 (no enzymatic) and Comparative Example 3 (lack of medicinal herbs) have anticoagulant effects between the examples and Comparative Example 1, indicating that the enzymatic activation of Earthworm and the compatibility of Szechuan Lovage Rhizome and Panax Notoginseng together enhance the efficacy of promoting blood circulation and removing blood stasis.
[0046] B. In vitro anti-inflammatory experiment Example 1 has an inhibition rate of nearly or more than 45% on the three major inflammatory factors (TNF-α, IL-6, and IL-1β), with a significant effect.
[0047] Comparative Example 1 has weak anti-inflammatory effects, again confirming the lack of ingredients.
[0048] Comparative Example 2 and Comparative Example 4 have significantly lower anti-inflammatory effects than Example 1, indicating that enzymatic activation and polyamide refinement are crucial for retaining anti-inflammatory ingredients.
[0049] C. In vivo pharmacodynamics experiment Neurological function score: Example 1 reduces the score from 7.3 in the model group to 2.1 on the 14th day, with a recovery degree far exceeding all comparative examples.
[0050] Cerebral infarction volume: Example 1 has a reduction rate of infarction volume as high as 60.6%, with the strongest neuroprotective effect, while Comparative Example 1 only reduces by 22.2% with no statistically significant difference.
[0051] Water maze test (cognitive function): Example 1 has the shortest escape latency and the most platform crossings, with the best cognitive improvement effect, while Comparative Example 3 (lack of Szechuan Lovage Rhizome and Panax Notoginseng) has a poorer effect, proving that Szechuan Lovage Rhizome (opening the orifices) and Panax Notoginseng (removing blood stasis) play a key role in improving post-stroke cognitive impairment.
[0052] D. Comprehensive pharmacodynamic score ranking Example 1 (91.3 points): Overall best, reflecting the synergistic effect of the complete prescription and optimized process.
[0053] Example 2 (85.5 points) and Example 3 (83.5 points): Minor adjustments in the prescription ratio result in slight fluctuations in efficacy, but both are significantly better than the comparative examples, proving the effectiveness of the prescription range of the invention.
[0054] Comparative Example 2 (73.3 points): Without enzymatic activation of Earthworm, the efficacy is significantly reduced, highlighting the necessity of the enzymatic activation step for releasing and transforming active ingredients.
[0055] Comparative Example 4 (68.5 points): Omitting the polyamide refinement step results in a decrease in purity, leading to a decrease in efficacy, proving the value of the refinement step in improving product efficacy.
[0056] Comparative Example 3 (59.5 points): missing Chuanqiong and Sanqi, although the total ingredient content is not low, but due to the lack of key ingredients, the efficacy of cognitive improvement is greatly reduced.
[0057] Comparative Example 1 (40.0 points): traditional water extraction method, the comprehensive efficacy is the worst, which fundamentally proves the great progress of the modern preparation method of the application.
[0058] The application adds Haima, Huangqi, Shichangpu, Dilong, Chuanqiong, Sanqi, The addition of Shichangpu (opening the orifices), Chuanqiong (ascending to the head), and Sanqi (removing blood stasis without damaging the normal) significantly enhances the treatment of cognitive impairment after stroke, realizes the organic combination of "activating blood and unblocking collaterals" and "opening orifices and awakening spirit", and improves the extraction efficiency (Comparative Example 2 proves its indispensability) by using the biological enzyme of Dilong to pretreat the medicinal materials under mild conditions, activates the precursor components, and uses 65% ethanol to consider the water-soluble and fat-soluble active ingredients, avoid the waste of fat-soluble ingredients in water extraction method (Comparative Example 1), two-stage purification (macroporous resin + polyamide) selectively enriches target components and removes impurities, so that the product purity (total content ≥ 45%) far exceeds the traditional process (Comparative Examples 1 and 4 prove its necessity); network pharmacology and molecular docking show that the application does not act on a single target, but through multi-component synergy, acts on PI3K-Akt, HIF-1, TNF and other multiple signaling pathways, and plays a therapeutic role in multiple aspects such as anticoagulation, anti-inflammatory, neuroprotection, promoting angiogenesis and repairing nerve function.
[0059] The above-described embodiments only express several embodiments of the application, which are described in detail and specifically, but should not be understood as limiting the scope of the patent of the application. It should be noted that for ordinary skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are within the scope of protection of the application.
Claims
1. A Jia Wei Huo Luo Xiao Ling Fang active ingredient group, characterized in that, The raw material composition includes: Salvia miltiorrhiza 20-25%; Angelica 15-20%; Frankincense 10-15%; Myrrh 10-15%; Leech 5-8%; Radix Astragali 10-15%; Acorus gramineus 5-10%; Earthworm 3-6%; Chuanxiong 8-12%; Panax notoginseng 5-10%; Among them, the active ingredient group contains Danshensu, Salvianolic acid B, Tanshinone IIA, Ferulic acid, 11-keto-β-boswellic acid, 3-acetyl-11-keto-β-boswellic acid, Astragaloside IV, α-asarone, Ligusticum lactone and Panax notoginseng saponin Rg1, and the total content of the 10 ingredients is not less than 45% of the total weight of the solid.
2. The active ingredient group of Jiawei Huoluo Xiaolingsang according to claim 1, characterized in that, In the active ingredient group, the relative weight ratio of Danshensu, Salvianolic acid B, Tanshinone IIA, Ferulic acid, 11-keto-β-boswellic acid, 3-acetyl-11-keto-β-boswellic acid, Astragaloside IV, α-asarone, Ligusticum lactone and Panax notoginseng saponin Rg1 is (1-3):(5-15):(0.5-2):(0.5-2):(0.5-2):(0.5-2):(1-3):(0.1-1):(0.5-2):(1-3).
3. A method for preparing the active ingredient group of Jiawei Huoluo Xifeng Formula according to claim 1 or 2, characterized by the steps of It includes: S1, enzymatic activation: after low-temperature drying, the prescription amount of earthworm is crushed, mixed with the prescription amount of salvia miltiorrhiza, angelica, chuanxiong and panax notoginseng decoction powder, 8-12 times the amount of phosphate buffer solution with pH value of 6.0-7.0 is added, and the mixture is stirred and reacted in 35-40℃ water bath for 1-1.5 hours; S2, synergistic extraction: the mixture after enzymatic reaction is combined with the prescription amount of frankincense, myrrh, leech, radix astragali, acorus gramineus, 10-14 times the amount of 60%-70% ethanol aqueous solution is added, and the mixture is heated and refluxed at 80-90℃ for 2 times, each for 1-2 hours, the extract is combined, and concentrated under reduced pressure at 55-65℃ to a thick paste with relative density of 1.10-1.15; S3, macroporous resin purification: the thick paste obtained in step S2 is suspended with water to a crude drug concentration of 0.4-0.6 g / mL, and then loaded onto an AB-8 type macroporous adsorption resin column, and then washed with 3-5 times the column volume of distilled water, 4-6 times the column volume of 20%-30% ethanol aqueous solution to remove impurities, and then eluted with 5-8 times the column volume of 60%-70% ethanol aqueous solution at a flow rate of 1-3 BV / h, and the eluate is collected; S4, polyamide column refining: after removing ethanol by reducing pressure concentration, the eluate obtained in step S3 is loaded onto a polyamide column, washed with 3-5 times the column volume of 20%-30% ethanol aqueous solution, and then eluted with 4-6 times the column volume of 60%-70% ethanol aqueous solution, and the target eluate is collected; S5, active ingredient group: the target eluate obtained in step S4 is concentrated under reduced pressure at 55-65℃, and then spray dried, with the inlet air temperature being 150-170℃ and the outlet air temperature being 75-85℃, to obtain the active ingredient group.
4. The production method according to claim 3, characterized by, In step S1, the pH value of the phosphate buffer solution is 6.5, the reaction temperature is 37℃, and the reaction time is 1 hour.
5. The preparation method according to claim 3, characterized in that, In step S2, the concentration of the ethanol aqueous solution is 65%, the solid-liquid ratio is 1:10, and the heating reflux extraction is performed for 2 times, each for 1.5 hours.
6. The preparation method according to claim 3, characterized in that, In step S3, elution was performed using 65% ethanol aqueous solution at a flow rate of 2 BV / h.
7. The preparation method according to claim 3, characterized in that, In step S4, elution was performed using 65% ethanol aqueous solution.
8. The preparation method according to claim 3, characterized in that, In step S5, the inlet air temperature of the spray drying was 160°C and the outlet air temperature was 80°C.
9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises a therapeutically effective amount of the active ingredient group of Jiawei Huoluo Xiaoling Decoction as claimed in claim 1 or 2, and one or more pharmaceutically acceptable carriers or excipients, and is in the form of an oral liquid, tablet, capsule, granule, nanoemulsion or transdermal patch.
10. Use of the active ingredient group of Jiawei Huoluo Xialing Decoction as claimed in claim 1 or 2 or the pharmaceutical composition of claim 9 in the preparation of a medicament for preventing and / or treating sequelae of stroke, including limb dysfunction, cognitive impairment, dysphagia or qi deficiency and blood stasis.