CN116808131A - Pharmaceutical composition, preparation method thereof and application of pharmaceutical composition in preventing and treating arteriosclerosis - Google Patents
Pharmaceutical composition, preparation method thereof and application of pharmaceutical composition in preventing and treating arteriosclerosis Download PDFInfo
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- CN116808131A CN116808131A CN202310744999.XA CN202310744999A CN116808131A CN 116808131 A CN116808131 A CN 116808131A CN 202310744999 A CN202310744999 A CN 202310744999A CN 116808131 A CN116808131 A CN 116808131A
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Abstract
The invention relates to the technical field of traditional Chinese medicines, in particular to a pharmaceutical composition, a preparation method thereof and application of the pharmaceutical composition in preventing and treating arteriosclerosis. The pharmaceutical composition for preventing and treating arteriosclerosis comprises the following raw materials: 20-30 parts of spatholobus stem, 20-30 parts of glabrous greenbrier rhizome and 2-10 parts of astragalus root. The invention also discloses a preparation method of the pharmaceutical composition and application of a preparation containing the pharmaceutical composition in preparation of drugs for preventing and treating arteriosclerosis. The anti-arteriosclerosis pharmaceutical composition adopts suberect spatholobus stem, glabrous greenbrier rhizome and astragalus root, has multiple components and multiple targets for resisting arteriosclerosis, can obviously improve the arteriosclerosis symptoms caused by high fat, has good efficacy, takes effect quickly and has a definite action mechanism; the pharmaceutical composition can improve and reduce the haemorheology index of blood, not only can prevent hyperviscosity induced by high-fat diet, but also can improve metabolic disorders of heart and liver amino acid, lipid and the like, and prevent the damage of heart and liver functions induced by high-fat diet.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to a pharmaceutical composition for preventing and treating arteriosclerosis, and a preparation method and application thereof.
Background
Atherosclerosis belongs to the category of qi stagnation and blood stasis in traditional Chinese medicine, and pulse obstruction in the category of arthralgia in traditional Chinese medicine diseases is a main pathological basis and a lethal factor of cardiovascular and cerebrovascular diseases; meanwhile, the traditional Chinese medicine composition is also a traditional Chinese medicine pattern common in menstrual diseases such as irregular menstruation, dysmenorrhea, amenorrhea and the like, and diseases such as diabetes, internal rheumatic arthritis, gout, hyperosteogeny, lumbar intervertebral disc protrusion and the like; the global mortality rate of diseases caused by coronary atherosclerotic diseases alone is about 31%, so atherosclerosis (qi stagnation and blood stasis) seriously affects people's health and quality of life.
At present, in clinically used western medicines, chemical biological medicines such as simvastatin, aspirin, metformin, allopurines, probenecid, colchicine, fenpride, glucocorticoid, beta-estradiol, estrone and the like have an improvement effect on biochemical indexes such as blood pressure, blood fat, hemorheology, in-vivo hormone level and the like caused by atherosclerosis, and also have a certain pain relieving effect, but the effect of the western medicines on improving clinical symptoms is poor, and long-term single use of the single medicines has large side effect and is easy to cause damage to functions of organs.
The Zhuang nationality is the minority with the largest population in China, the application history of the Zhuang nationality medicine is long, the Zhuang nationality medicine is simple and effective, and some Zhuang nationality medicine formulas are peculiar to difficult and complicated diseases. According to the theory of Zhuang nationality medicine, atherosclerosis belongs to two paths (dragon road and fire road correspond to blood system and nervous system respectively) of diseases, the main treatment of atherosclerosis is "through", the prescription of Zhuang nationality medicine has clear characteristics, and the prescription is combined according to main medicines, auxiliary medicines, guiding medicines and antidotes; the prescription-strengthening preparation usually contains main drugs and is matched with main drugs, and the main drugs in the case generally belong to the category of 'public drugs' and 'mother drugs', and have no major and minor components, so that the combination of the prescription can obviously enhance the curative effect. The raw materials of the medical diet prescription for treating arthralgia comprise 25g of suberect spatholobus stem, 25g of glabrous greenbrier rhizome and 500g of pig leg bones, and the medical diet prescription belongs to a typical compatibility method of main medicine and guiding medicine; in the prescription, spatholobus stem is a common drug, glabrous greenbrier rhizome is a mother drug, pig leg bones are guiding drugs, and the prescription belongs to a prescription of a typical prescription strengthening agent, namely a traditional Chinese medicine simple prescription; however, the traditional soup making method has low extraction rate of medicinal substances, and is not convenient to use compared with the use of medicinal materials and difficult to guarantee the quality of products.
In view of the above, it is of great importance to provide a novel pharmaceutical composition for preventing and treating arteriosclerosis.
Disclosure of Invention
The invention provides a pharmaceutical composition, a preparation method thereof and application of the pharmaceutical composition in preventing and treating arteriosclerosis, and solves the technical problems of poor drug effect and low extraction rate of drug effect substances of the anti-arteriosclerosis drugs in the prior art.
The technical scheme for solving the technical problems is as follows:
the invention provides a pharmaceutical composition for preventing and treating arteriosclerosis, which comprises the following raw materials:
20-30 parts of spatholobus stem, 20-30 parts of glabrous greenbrier rhizome and 2-10 parts of astragalus root.
In the invention, the spatholobus stem belongs to the root and stem of the spatholobus stem belonging to the genus spatholobus of the family Leguminosae, because the Chinese medicinal composition has the effects of nourishing blood, activating blood, dredging meridian passage and the like, is used for treating anemia in the clinic of the Chinese medicine, menstrual disorder, hypomenorrhea, amenorrhea, dysmenorrhea, and other menstrual diseases, atherosclerosis, and cardiovascular and cerebrovascular diseases; modern pharmacological research results show that the ethanol extract has anti-inflammatory efficacy through anti-cyclooxygenase-1, 5-lipoxygenase, phospholipase A2 and 12-lipoxygenase activities, and has neuroprotection through reducing the number of apoptosis of excitatory toxic injury cells induced by NMDA; caulis Spatholobi also has effects of promoting blood circulation and replenishing blood by resisting Streptococcus mutans Srt A, and has anticancer effect by increasing signal transducer and transcriptional activator, and p53, E2F1 and p21mRNA expression and reducing apoptosis regulator Bcl-2 (BCL-2) mRNA expression; the main active ingredients of the composition are flavonoid substances such as 7-hydroxy-6-methoxy flavanone, formononetin, isoliquiritigenin, genistein, 7-hydroxy flavanone, fisetin (butin) and the like;
rhizoma Smilacis Glabrae belongs to root of rhizoma Smilacis Glabrae of Smilax of Liliaceae, and is used for treating syphilis, heavy metal poisoning, muscular tension increase, nephritis, cardiovascular disease, diabetic vascular complications, etc.; modern pharmacological studies have shown that they have anti-inflammatory and antioxidant effects by inhibiting interleukin-1, interleukin-6, NO and NF-Bp-p65 protein expression, and can treat heart failure by down-regulating NF-Bp-p65 mRNA, RAC-alpha serine/threonine protein kinase, protein phosphatase 2A, cell division controlling protein 42 homolog, c-Jun amino-terminal kinase, extracellular signal-regulated kinase, bax and Caspase3 expression, and up-regulating mRNA expression of transcription factors AP-1, IKK, sirt1, p38MAPK and BCL-2; the main active ingredients of the composition are flavonoid ingredients and polysaccharides, such as 2 (S) -5-hydroxy-6, 8-dimethoxy Huang Tongtong-7-O-beta-D-glucopyranosyl- (1- & gt 6) -O-beta-D glucopyranoside, 5-hydroxy-3, 8-dimethoxy flavone-7-O-beta-D-glucopyranosyl- (1- & gt 6) -O-beta-D glucopyranoside, 3, 7-dihydroxy-8-methoxy flavone-6-O-beta-D-glucopyranosyl- (1- & gt 6) -O-beta-D-desglucopyranoside, isoabscisin, neo-astilbin and the like;
astragalus membranaceus is the root of Astragalus membranaceus belonging to the genus Astragalus of the family Leguminosae, and has good efficacy and low cost, and is widely used for enhancing human immunity in the clinic of traditional Chinese medicine, and is mainly used for enhancing and recovering human immunity, and enhancing cardiovascular function and vitality in modern medicine. Modern pharmacological studies have shown that astragalus increases cardiac function, promotes angiogenesis, regulates blood glucose, inhibits cancer, enhances immunomodulatory activity, etc. by increasing NO content, nitric oxide synthase activity and cyclic adenosine monophosphate levels and inhibiting matrix metalloproteinase-9 activation; the main medicinal components are flavonoids, triterpenoid saponins and polysaccharides.
Preferably, the method comprises the following raw materials:
22-26 parts of spatholobus stem, 22-26 parts of glabrous greenbrier rhizome and 3-6 parts of astragalus root.
Preferably, the method comprises the following raw materials:
23.0 parts of suberect spatholobus stem, 23.0 parts of glabrous greenbrier rhizome and 4.0 parts of astragalus root.
The invention also provides a preparation method of the pharmaceutical composition, which comprises the following steps:
respectively extracting caulis Spatholobi, rhizoma Smilacis Glabrae and radix astragali with extractive solution, mixing, or mixing, extracting with extractive solution, concentrating, and evaporating to dryness to obtain pharmaceutical composition.
Preferably, the extracting solution is ethanol and water.
Preferably, the extraction is carried out for 2-4 times by adopting ethanol and water in sequence; the ethanol is used in an amount of 88-92vol% each time and the water is used in an amount of 50vol% each time.
The invention also provides a preparation comprising the pharmaceutical composition, wherein the pharmaceutical composition is added with conventional auxiliary materials and is prepared into clinically acceptable tablets, capsules, powder, mixture, pills, granules, syrup, soft extract, emplastrum, suppository, aerosol or ointment according to the conventional process.
The conventional auxiliary materials are as follows: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, matrices, and the like. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, and the like; the disintegrating agent comprises starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, and crosslinked sodium carboxymethyl cellulose; lubricants include magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, agar, hydroxypropyl propyl cellulose, and the like; the binder comprises starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; the sweetener comprises: saccharin sodium, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; the flavoring agent comprises: sweetener and various flavors; the preservative comprises: nipagin, benzoic acid, sodium benzoate, sorbic acid and salts thereof, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil and the like; the matrix comprises: PEG6000, PEG4000, insect wax, and the like.
The invention also provides the application of the pharmaceutical composition or the preparation of the pharmaceutical composition in preparing medicines for preventing and treating arteriosclerosis.
Compared with the prior art, the invention has the beneficial effects that:
1. the anti-arteriosclerosis pharmaceutical composition adopts suberect spatholobus stem, glabrous greenbrier rhizome and astragalus root, has multiple components and multiple targets for anti-arteriosclerosis, can obviously improve the arteriosclerosis symptoms caused by high fat, and has good efficacy, quick response and definite action mechanism.
2. In the anti-arteriosclerosis pharmaceutical composition, spatholobus stem and glabrous greenbrier rhizome are used as monarch drugs, and astragalus root is used as adjuvant drug and guiding drug; the invention can improve and reduce blood rheological indexes such as blood viscosity, blood plasma viscosity, erythrocyte aggregation index, whole blood high-cut index, whole blood low-cut index and the like, and the pharmaceutical composition can prevent hyperviscosity induced by high-fat diet; the invention can improve the metabolic disturbance of a plurality of amino acids, lipids and the like in blood, heart and liver, and can prevent the heart and liver function from being damaged induced by high-fat diet.
3. The invention also optimizes the extraction method of the pharmaceutical composition, adopts a double extraction mode of ethanol and water, can extract and obtain compounds with more anti-arteriosclerosis effect, and improves the utilization value of the traditional Chinese medicine raw materials.
Drawings
FIG. 1 is a diagram of the pathological morphology of the normal group aortic arch of the present invention;
FIG. 2 is a diagram of the pathology of the aortic arch of the model set of the present invention;
FIG. 3 is a diagram showing the pathological morphology of the aortic arch of the positive drug group of the present invention;
FIG. 4 is a diagram showing the pathological morphology of the aortic arch of the suberect spatholobus stem group of the present invention;
FIG. 5 is a diagram showing the pathological morphology of aortic arch in the prescription of the medical diet for treating arthralgia according to the present invention;
FIG. 6 is a diagram showing the pathological morphology of aortic arch in the prescription of the medical diet for treating arthralgia according to the present invention;
fig. 7 is a principal component analysis score chart of a jugular vein serum quality control sample of the spatholobus stem group, the arthralgia-syndrome medicated diet prescription minus prescription group and the arthralgia-syndrome medicated diet prescription plus prescription group of the invention;
FIG. 8 is a principal component analysis score chart of heart quality control samples of the spatholobus stem group, the arthralgia-syndrome medicated diet prescription minus group and the arthralgia-syndrome medicated diet prescription plus group;
fig. 9 is a principal component analysis score chart of liver quality control samples of the spatholobus stem group, the arthralgia-syndrome medicated diet prescription minus group and the arthralgia-syndrome medicated diet prescription plus group of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Example 1
The pharmaceutical composition for preventing and treating arteriosclerosis of the embodiment comprises the following raw materials: 3.68kg of spatholobus stem, 3.68kg of glabrous greenbrier rhizome and 0.64kg of astragalus root.
The preparation method of the pharmaceutical composition comprises the following steps: respectively weighing caulis Spatholobi, rhizoma Smilacis Glabrae and radix astragali in selected weight, and mixing well; extracting with 90vol% ethanol at 50deg.C for 12 hr, filtering, extracting the residue with 90vol% ethanol for 3 times; extracting the residue with 50vol% water at 100deg.C for 30min, and extracting with water for 2 times; combining the filtrates of 5 times, concentrating under reduced pressure, and evaporating to dryness to obtain 2.35kg of medicinal composition.
Example 2
The preparation method of this example is different from that of example 1, and includes the following steps: respectively weighing caulis Spatholobi, rhizoma Smilacis Glabrae and radix astragali in selected weight; extracting each raw material respectively, extracting each raw material with 90vol% ethanol at 50deg.C for 12 hr, and extracting with ethanol for 3 times; extracting the residue with 50vol% water at 100deg.C for 30min, and extracting with water for 2 times; mixing the three materials, extracting for 15 times, concentrating under reduced pressure, and evaporating to dryness to obtain 2.16kg of medicinal composition.
Example 3
The pharmaceutical composition for preventing and treating arteriosclerosis of the embodiment comprises the following raw materials: 3.28kg of spatholobus stem, 3.83kg of glabrous greenbrier rhizome and 0.82kg of astragalus root.
The preparation method of this example is the same as that of example 1.
Example 4
The pharmaceutical composition for preventing and treating arteriosclerosis of the embodiment comprises the following raw materials: 3.83kg of spatholobus stem, 3.41kg of glabrous greenbrier rhizome and 0.55kg of astragalus root.
The preparation method of this example is the same as that of example 1.
Comparative example 1
This comparative example differs from example 1 only in that: extracting only chemical components in caulis Spatholobi, concentrating under reduced pressure, and evaporating to dryness to obtain concentrated extract.
Comparative example 2
This comparative example differs from example 1 only in that: extracting only chemical components in caulis Spatholobi and rhizoma Smilacis Glabrae, concentrating under reduced pressure, and evaporating to dryness to obtain concentrated extract.
Comparative example 3
This comparative example differs from example 1 only in that: the three materials of example 1 were mixed and then extracted with water at 100℃for 30min, extracted with water 2 times, and the extracted solutions were mixed and concentrated under reduced pressure and evaporated to dryness.
This comparative example gave 1.23kg of a paste-like substance.
Comparative example 4
This comparative example differs from example 2 only in that: the three materials of experimental example 2 were extracted with water at 100 ℃ for 30min, respectively, and extracted with water for 2 times, and all the solutions obtained by the extraction were mixed and concentrated under reduced pressure to dryness.
This comparative example gave 1.20kg of a paste-like substance.
To further demonstrate the effect of the pharmaceutical composition for preventing and treating arteriosclerosis of the present invention in preventing and treating arteriosclerosis, the following animal test was provided:
animal test
1. Reagents and animals
Cholesterol (lot. No.531F102; beijing Soy Bao technology Co., ltd.);
total cholesterol (No. 201012; meikang Biotech Co., ltd.);
low density lipoprotein cholesterol (No. 200928; meikang Biotech Co., ltd.);
triglycerides (No. 201020; meikang Biotech Co., ltd.);
high density lipoprotein (No. 200928; meikang Biotech Co., ltd.);
superoxide dismutase (No. 20010910228; meikang Biotechnology Co., ltd.);
uratam (urethane, HY1223-500g; shanghai Hengyuan Biotech Co., ltd.);
rabbits (No. 0008757; male and female halves; 4.5 months old; body weight 2.00-2.50kg; guangxi Tian Donglong lucky rabbit Co., ltd.);
high-fat feed: self-made, which comprises cholesterol, lard, yolk powder and basic feed with the mass ratio of 1:8:4:8.
2. Test drug
Test drug: the pharmaceutical composition obtained by the preparation method of example 1; the concentrated extracts prepared in comparative examples 1 and 2.
3. Sample collection
The method comprises the steps of feeding 48 rabbits in a single cage, randomly dividing the rabbits into a normal group, a model group, a positive medicine group, a spatholobus stem group, a arthralgia disease medicated diet prescription reducing group and an arthralgia disease medicated diet prescription increasing group after one week, wherein 6 groups are adopted, the rabbits are randomly allocated, and each group is a male half and a female half; wherein, the normal group orally administers basal feed; the model group orally administered high fat diet; orally taking high-fat feed and Xuezhikang into a positive medicine group; administering high-fat feed and concentrated extract of comparative example 1 to caulis Spatholobi group; the prescription of the medical diet prescription for treating arthralgia is orally administered with high-fat feed and concentrated extract of comparative example 2; the prescription of the medical diet prescription for treating arthralgia is added with high-fat feed and the pharmaceutical composition of the example 1; the daily dosage of the Xuezhikang is 0.36g/kg, and the daily dosage of the Xuezhikang is 3.5g/kg.
Feeding for 60 days, wherein the blood fat recovery group is prepared by taking crude drug with water according to dosage every day, and the spatholobus stem group, the arthralgia-syndrome medicated diet prescription and the arthralgia-syndrome medicated diet prescription adding prescription are prepared by taking extract quality with water according to the calculation of dosage multiplied by extract yield; feeding all the prepared solutions to rabbits orally on the same day; weighing 1 time per week;
feeding for 30 days, and taking the blood of the ear margin vein after feeding on the same day, wherein the fasting is not carried out for 12 hours without water control;
after feeding is finished until the 60 th day, all rabbits are fasted and not forbidden for 12 hours, and the venous blood at the edge of the ear is collected; from the ear margin of rabbits, ulatin-water (25:100, m/v;4 mL/kg) was intravenously injected, and jugular vein serum was collected; after the collection is completed, the rabbits are sacrificed, and heart and liver, heart, liver and jugular vein serum are collected for metabonomics research; aortic arch samples were collected for case slice analysis.
4. Detection analysis
4.1 blood test analysis
4.1.1, measuring haemorheological indicators of venous blood at the ear margin, including whole blood viscosity (1.00 s -1 、5.00s -1 、30.00s -1 、200.00s -1 ) Plasma viscosity, erythrocyte aggregation index, whole blood high cut index, whole blood low cut index, and superoxide dismutase index.
The data of the blood rheology indexes are expressed by mean value + -standard deviation, and the statistics of the results are shown in tables 1-9.
TABLE 1 Whole blood viscosity of venous blood at the ear margin (1.00 s -1 ) Statistics of detection results
TABLE 2 Whole blood viscosity of venous blood at the ear margin (5.00 s -1 ) Statistics of detection results
TABLE 3 Whole blood viscosity of venous blood at the ear margin (30.00 s -1 ) Statistics of detection results
TABLE 4 Whole blood viscosity of venous blood at the ear margin (200.00 s -1 ) Statistics of detection results
TABLE 5 statistics of blood plasma viscosity measurements of ear vein blood
TABLE 6 statistics of erythrocyte aggregation index detection results for venous blood at the ear margin
TABLE 7 statistics of Whole blood high cut index detection results for ear margin venous blood
TABLE 8 statistics of Whole blood low cut index detection results for venous blood at the ear margin
TABLE 9 statistics of superoxide dismutase detection results of venous blood at the ear margin
As can be seen from tables 1 to 9, the values of the model groups are significantly increased, in addition to the plasma viscosity index and the whole blood high cut index, compared with the normal group, indicating that administration of the high fat feed affects the blood whole blood viscosity (1.00 s -1 、5.00s -1 、30.00s -1 、200.00s -1 ) A red blood cell aggregation index, a whole blood hypocut index, and a superoxide dismutase index; compared with the model group, the prescription of the medical diet prescription for treating arthralgia can reduce the viscosity of whole blood in hemorheology (1.00 s) -1 、5.00s -1 、30.00s -1 、200.00s -1 ) The blood plasma viscosity, the erythrocyte aggregation index, the whole blood high-cut index and the whole blood low-cut index are used for increasing the superoxide dismutase index of hemorheology; the results show that the rabbit hyperlipoidemia can be induced by the rabbit hyperlipoidemia, and the efficacy of the prescription of the medical diet prescription for improving blood hyperviscosity syndrome is superior to that of the prescription of the medical diet prescription for improving blood hyperviscosity syndrome.
4.1.2, serum lipid indicators of blood from the ear margin veins, including triglyceride, total cholesterol, low density lipoprotein and high density lipoprotein indicators.
The data for the serum lipid index measurements are shown as mean ± standard deviation, and the statistics are shown in tables 10-13.
TABLE 10 statistics of triglyceride measurements in blood from the ear margin veins
TABLE 11 statistics of total cholesterol measurements in ear margin venous blood
TABLE 12 statistics of results of low density lipoprotein measurements on venous blood at the ear margin
TABLE 13 high density lipoprotein results statistics of ear margin venous blood
As can be seen from tables 1-13, the values of the model group are significantly greater and the differences are significant compared to the normal group, indicating that administration of high fat feed affects the blood triglyceride, total cholesterol, low density lipoprotein and high density lipoprotein index; compared with the model group, the indexes of triglyceride, total cholesterol, low density lipoprotein and high density lipoprotein of the positive medicine group, the spatholobus stem group, the arthralgia-syndrome medicated diet prescription reducing prescription and the arthralgia-syndrome medicated diet prescription increasing prescription are lower than those of the model group, the order of the medicine effect from strong to weak is that of the arthralgia-syndrome medicated diet Fang Zengfang, the arthralgia-syndrome medicated diet prescription reducing prescription and the spatholobus stem, which shows that the high-fat diet of the arthralgia-syndrome medicated diet prescription increasing prescription can cause hyperlipidemia diseases, and the immunity of the arteriosclerosis resistance can be enhanced by using a small amount of astragalus to replace the spatholobus stem and the glabrous greenbrier rhizome with the quality, and the astragalus plays roles of adjuvant and medicament in the arthralgia-syndrome medicated diet prescription increasing prescription.
4.1.3, detecting aortic arch sample intima thickness, intima/media, plaque area/aortic arch area index; the pathological morphology of the aortic arch was examined.
The pathological morphology of the aortic arch is shown in figures 1-6.
Aortic arch test data are expressed as mean ± standard deviation, and the statistics of the results are shown in tables 10-13.
TABLE 14 results statistics of intima-media and intima/media index measurements of aortic arch
TABLE 15 results statistics of aortic arch sample intima thickness and intima/media index measurements
As can be seen from tables 14-15 and FIGS. 1-6, the endothelial cells of the normal group of aortic arch intima are relatively intact, the alignment is clean, the internal elastic membrane is clear, the alignment and volume of cells in the media smooth muscle are normal, and no foam cells and lipid deposition are found from intima to media; compared with the normal group, the endothelial cells of the aortic arch intima of the model group are swelled and denatured, a plurality of foam cells are gathered, and some of the internal elastic membranes are broken; smooth muscle damage and alignment turbidity, lipid deposition was found extracellular from intima to media; the aortic arch of the positive medicine group, the spatholobus stem group, the medical diet prescription reducing group and the medical diet prescription increasing group have lesions similar to those of the aortic arch of the model group, but the degree is lighter than that of the model group, and the sequence from heavy to light of the lesions is the positive medicine group, the spatholobus stem group, the medical diet prescription reducing group and the medical diet prescription increasing group; the results show that the efficacy of the prescription of the medical diet for treating the arthralgia-syndrome is superior to that of the prescription of the medical diet for treating the arthralgia-syndrome, and the results are consistent with the results of the thickness value of the intima, the ratio of the intima to the media thickness and the area of the atheromatous plaque.
5. Metabonomics study
5.1 Metabolic analysis
The method comprises the following steps: respectively taking 80mg heart and liver samples collected in each group, adding 200 mu L of water, homogenizing, swirling for 60s, adding 800 mu L of methanol-acetonitrile (1:1, v/v), and performing treatments for 2 times in the case of swirling for 60s and low-temperature ultrasonic for 30min to obtain heart samples and liver samples; 400. Mu.L of methanol-acetonitrile (1:1, v/v) was added to 100. Mu.L of heart sample, liver sample and each group of collected jugular vein serum samples, respectively; all samples were vortexed for 60s and left at-20℃for 1h to precipitate proteins, then centrifuged at 14000rpm for 10min, the supernatant filtered using a 0.22 μm filter membrane and each sample was analyzed for differential metabolism foreign bodies using UPLC-Q-TOF-MS.
Parameters analyzed by UPLC-Q-TOF-MS are specifically as follows: Q-TOF-MS configured electrospray; UPLC configured chromatographic column (packing: waters, ACQUITY UPLC HSS T3.8 μm, column gauge 2.1 mm. Times.100 mm; packing: waters, ACQUITY UPLC BEH Amide 1.7.7 μm, column gauge 2.1 mm. Times.100 mm); the chromatographic conditions were as follows: all samples were placed in a 4 ℃ autosampler at 25 ℃ during analysis, sample injection amount: 2 μl, flow rate: 0.3mL/min, mobile phase A-B (A is an aqueous solution of 25mM ammonium acetate and 25mM ammonia, B is acetonitrile, 0-0.5min is 5:95,0.5-7min is 5:95 to 35:65,7-8min is 35:65 to 60:40,8-9min is 60:40,9-9.1min is 60:40 to 5:95,9.1-12min is 5:95; v/v); the mass spectrometry uses positive ion and negative ion modes, and the instrument parameter conditions are air temperature: 250 ℃, dry gas: 16L/min, sprayer: 20psig, sheath air temperature 400 ℃, sheath air flow rate: 12L/min, voltage: 3000V, nozzle voltage: 0V, fragments: 175V, a mass range of 50-1200, a collection rate of 4Hz and a cycle time of 250ms.
5.2 statistical analysis
Activity data were analyzed using SPSS software 26.0 and GraphPad Prism 8.0;
principal Component Analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed on the poor metabolic foreign bodies using SIMCA-P14.1 software.
5.3 Metabolic analysis results
5.3.1 reliability and stability study of model
In order to more comprehensively clarify the drug effect and the drug effect mechanism of the medical diet prescription for the arthralgia, the metabonomics of the jugular vein serum, the heart and the liver of the rabbit are researched, and the overlapping property of the retention time of a Quality Control (QC) sample UPLC-Q-TOF-MS total ion flow diagram and the response intensity of chromatographic peaks are better, so that the change caused by instrument errors in the whole experimental process is smaller; the number of ion peaks extracted from jugular vein serum, heart and liver respectively in positive/negative ion mode using XCMS software was 11489/8516, 16286/11321 and 16147/11150 respectively, the number of positive ion peaks was higher than the negative ion peaks; this result shows that the positive ion pattern is superior to the negative ion pattern in studying rabbit serum, heart and liver metabonomics.
And establishing a Principal Component Analysis (PCA) model by using UPLC-Q-TOF-MS data to identify the total difference between a normal group, a model group, a spatholobus stem group, a arthralgia disease medicated diet prescription minus group and a arthralgia disease medicated diet prescription plus group.
The principal component analysis score results of each group of jugular vein serum quality control samples are shown in fig. 7;
the principal component analysis score results of each group of heart quality control samples are shown in fig. 8;
the principal component analysis score results of each group of liver quality control samples are shown in fig. 9;
the principal component analysis statistics between the different groups are shown in table 17; the statistics of partial least squares discriminant analysis between different groups are shown in Table 18; the results of the orthorhombic least squares discriminant analysis statistics between the different groups are shown in table 19.
TABLE 17
Note that: on the left and right sides of "/", respectively, in positive and negative ion modes.
TABLE 18
Note that: on the left and right sides of "/", respectively, in positive and negative ion modes.
TABLE 19
Note that: on the left and right sides of "/", respectively, in positive and negative ion modes.
As can be seen from fig. 7-9, the ion peaks of all quality control samples in the positive and negative ion modes are tightly aggregated, which shows that the experiment has good repeatability; the score diagram shows that the aggregation among different groups can be well separated except a normal group and a model group in serum in a negative ion mode and a medical diet formula subtracting group and a model group for arthralgia, and the result shows that differential metabolism foreign matters in serum in a heart, liver and positive ion mode are feasible; as can be seen from tables 17 to 19, the values of the other components except the heart in the positive ion mode were 0.473 cm, which is greater than 0.50 cm; the model stability of the prescription and model group of the medical diet prescription for treating the arthralgia-syndrome of the heart in the positive ion mode is good.
5.3.2 study of foreign matter with Metabolic Difference
In the metabonomics study herein, the poor metabolic profile (P < 0.05) between the normal and model groups is referred to as poor disease metabolic profile; the positive drug group has a value closer to that of the normal group than the model group, i.e., a foreign substance with a poor metabolism that achieves a callback after administration is referred to herein as a foreign substance with a poor metabolism (pharmacodynamic metabolite, PM).
Differential metabolism foreign bodies (p < 0.05) recognized between different groups in jugular vein serum, heart and liver; the statistics of the differential metabolic disease identified from jugular vein serum, heart and liver are shown in Table 20.
Table 20
The 39, 56 and 97 different metabolic substances are identified from heart, blood serum and liver respectively, and the different metabolic substances relate to amino acid, sugar, lipid, organic acid and the like, which indicate that the metabolism of amino acid, sugar, lipid, organic acid and the like in the blood serum, heart and liver of a human body is disturbed due to high-fat diet.
According to table 20, in jugular vein serum, 25, 22 and 41 drug effect metabolism difference foreign matters are respectively identified in a suberect spatholobus stem group, a arthralgia disease medicated diet prescription addition prescription; when half of the spatholobus stem is replaced by the same mass of rhizoma smilacis glabrae, namely, the spatholobus stem group is compared with the medical diet prescription minus prescription group of the arthralgia disease, the pharmacodynamic metabolites of No.7, no.18, no.19, no.24, no.28, no.31, no.33, no.36, no.37 and No.45 disappear, but the pharmacodynamic metabolites of No.5, no.11, no.21, no.23, no.32, no.35 and No.47 are found; in addition, the fold changes of pharmacodynamic metabolites of No.29, no.30 and No.34 were also significantly changed; when the medicine was changed from the arthralgia-syndrome medicated diet prescription to the arthralgia-syndrome medicated diet prescription, no.4, no.7, no.8, no.9, no.13, no.17, no.18, no.19, no.20, no.24, no.28, no.31, no.36, no.38, no.39, no.41, no.42, no.44, and No.45 were also identified, and the fold change of the pharmacodynamic metabolites of No.12 and No.29 was found to be changed. The results show that compared with the prescription of the medical diet prescription for treating the arthralgia, the prescription of the medical diet for treating the arthralgia has the maximum drug effect metabolites; the medicinal diet prescription for treating the arthralgia disease has the best effect of preventing metabolic disorder in body serum caused by high fat induction.
In the jugular vein serum, the spatholobus stem group and the medical diet prescription adding group respectively have 2 and 11 unique medical effect metabolism difference foreign matters, and the spatholobus stem group and the medical diet prescription adding prescription group identify 8 common medical effect metabolism difference foreign matters; the common 10 foreign matters with poor drug effect and metabolism are identified by the prescription of the medical diet prescription and the prescription of the medical diet prescription; the spatholobus stem group, the arthralgia-syndrome medicated diet prescription minus prescription group and the arthralgia-syndrome medicated diet prescription plus prescription group identify 15 common foreign matters with poor drug effect and metabolism. The result shows that the regulation and control of the medical food formula for treating the arthralgia-syndrome can change the metabolic profile of the metabolic disorder of the organism caused by the high-fat diet by utilizing the compatibility rule of the traditional Chinese medicine formulas.
The method comprises the steps of identifying 40 medical herb prescription with the increased efficacy and metabolism difference foreign matters from heart samples, and identifying 63 medical herb prescription with the increased efficacy and metabolism difference foreign matters from liver samples; the result shows that the prescription of the medical diet for treating the arthralgia has better drug effect for preventing metabolic disorder in the heart and the liver of a human body caused by high fat induction.
A total of 4 foreign substances having poor pharmacodynamic metabolism of No.5, no.13, no.14 and No.29 were identified from carotid serum, heart and liver, and a total of 10 foreign substances having poor pharmacodynamic metabolism of No.8, no.11, no.16, no.17, no.25, no.26, no.38, no.41, no.44 and No.45 were also identified from carotid serum and liver; a total of 10 foreign substances with poor pharmacodynamic metabolism were also identified from the heart and liver, no.3, no.48, no.49, no.54, no.57, no.65, no.66, no.74, no.75 and No. 78; from carotid serum and liver, 9 pharmacodynamic differential foreign bodies were also identified, no.1, no.7, no.15, no.18, no.31, no.32, no.35, no.36 and No. 47. The result shows that the metabolic profiles of different tissues and organs in the organism are different, and the efficacy of the arthralgia prescription for recovering the metabolic profiles of different tissues and organs is also different.
The metabolic pathway analysis and the pathway enrichment analysis are carried out by using MetaboAnalyst 5.0 to clarify key metabolic pathways, and the results show that the metabolic difference foreign matters No.7, no.11, no.15, no.23 and No.35 in the spatholobus stem group, the metabolic difference foreign matters No.11, no.15, no.23, no.25 and No.35 in the medical diet prescription subtraction group, and the metabolic difference foreign matters No.7, no.11, no.15, no.22 and No.35 in the medical diet prescription subtraction group are medical effect metabolic markers; no.11, no.15 and No.35 are three groups of potentially common pharmacodynamic poor foreign substances; no.11, no.74, no.128, no.131, no.114, no.75 and No.141, respectively, are considered as potentially critical pharmacodynamic poor-metabolizing impurities in the heart and liver, respectively. For the serum of modified jugular vein, arginine, proline and histidine metabolism and glycerophospholipids biosynthesis are important pharmacodynamic metabolic pathways of the spatholobus stem group, the arthralgia-treating medicated diet prescription and the arthralgia-treating medicated diet prescription; alanine, aspartic acid, glutamic acid, arginine, proline and glutathione metabolism are important pharmacodynamic metabolic pathways in the heart as a medical diet for arthralgia, and glutathione, D-glutamine, alanine, aspartic acid, glutamic acid and arginine are important pharmacodynamic metabolic pathways in the liver.
5.3.3 network pharmacological analysis
In order to more clearly and comprehensively reveal the action mechanism of the prescription of the medical diet for treating the arthralgia-syndrome, the network pharmacology and molecular docking technology are applied to analyze regulated genes and targets, and the results of metabonomics, network pharmacology, molecular docking and metabonomics research are subjected to associated analysis, so that the action mechanism of the prescription of the medical diet for treating the arthralgia-syndrome is further deeply explained.
2044, 4481 and 228 targets associated with arteriosclerosis were collected from OMIM, disGeNET and GeneCards databases, respectively, and the compounds belonging to astragalus, spatholobus stem and smilax glabra were 87, 69 and 79, respectively, including 20, 24 and 15 compounds meeting the conditions of oral availability (OB) greater than 30% and drug class (DL) greater than 0.18, respectively; collecting 217, 205 and 262 drug efficacy targets of the three drugs for resisting arteriosclerosis through TCMSP, drugBank, OMIM, disGeNET and GeneCards databases respectively; of these anti-AS pharmacodynamic targets, 355 were non-duplicated targets.
The gene chips GSE9820 (severe three coronary artery lesions), GSE18608 (coronary artery diseases) and GSE48964 (severe obesity) related to the atherosclerosis are found from the GEO database, and 144 targets are identified in the more reliable medical diet prescription increasing group of the arthralgia, and the 144 targets are regarded as targets of interest in the following study.
Apoptosis (Apoptosis, ASP), choline metabolism in cancer (choline metabolism in cancer, CMC), insulin resistance (insulin resistance, IR), neuroactive ligand-receptor interactions (neuroactive ligand-receptor interaction, NLRI), sphingolipid signaling pathway (sphingolipidsignaling pathway, SSP), cAMP signaling pathway (cAMP signaling pathway, cAMPSP), central carbon metabolism in cancer (central carbon metabolism in cancer, CCMC), cGMP-PKG signaling pathway (cGMP-PKG signaling pathway, cGMP-pkgspp), parkinson's disease (Parkinson disease, PD, PDs), phospholipase D signaling pathway (phospholipase D signaling pathway, PDSP), renal cancer cells (renal cell carcinoma, RCC), vascular smooth muscle contraction (vascular smoothmuscle contraction, VSMC), diabetic cardiomyopathy (diabetic cardiomyopathy, DC), lipid and atherosclerosis (lipid and atherosclerosis, LA), parathyroid hormone synthesis (parathyroid hormone synthesis, PHS), pertussis (pssis) and Tuberculosis (TSPs) are interesting signals for TSP-channel smoothing when the anti-arteriosclerosis target and poor pharmacodynamic metabolism of the prescription is diffracted into the KEGG database for pathway analysis.
The cytoscape 3.9.1 software is used for constructing a correlation network among targets, targets-medicinal materials, targets-passages, targets-compounds and metabolic difference foreign matters-passages related to arteriosclerosis resistance of the medical diet prescription of the arthralgia disease.
Adenosine participates in 5 signal pathways, and metabolic difference foreign substances No.18, no.74, no.112 and No.137 participate in two signal pathways, and metabolites No.1, no.2, no.114 and No.133 participate in only one signal pathway, and these pharmacodynamic metabolic difference foreign substances, particularly metabolites No.74, no.75 and No.114, are very important pharmacodynamic metabolic difference foreign substances.
Of SSP, VSMC, CCMC, CMC, NLRI and PDS pathways, not less than 2 catabolic foreign substances participate, and thus these signaling pathways, particularly the Sphingolipid Signaling Pathway (SSP) and Vascular Smooth Muscle Contraction (VSMC) signaling pathway, are very important signaling pathways in enhancing the metabolic mechanism of anti-atherosclerosis in the medical prescription of arthralgia, because both sphingolipid and vascular smooth muscle are closely related to atherosclerosis.
The specific targets 38, 23 and 78 of the spatholobus stem, the glabrous greenbrier rhizome and the astragalus root are found respectively, and the targets include 17, 11 and 26 focused targets; 46. the 30 and 21 targets comprise 18, 11 and 13 targets concerned as common targets between astragalus and glabrous greenbrier rhizome, suberect spatholobus stem and astragalus and suberect spatholobus stem and glabrous greenbrier rhizome respectively; the 116 targets comprise 47 targets of interest as common targets of three medicinal materials; among these targets of interest, the connectivity of IL6, TP53, CASP3, JUN, etc. is being a potentially important target due to its potential of the first 20; LA, ASP, DC, NLRI, TSP, IR, cGMP-PKGSP and SSP signal paths, especially LA and SSP, are contemplated.
Molecular docking was performed using AMBER18 and pymol2.5 software, autodock vina1.2, and several other tools, according to connectivity order, structural features and sources of the compounds; it was found that taxifolin was derived from smilax glabra, naringenin and beta-sitosterol were derived from spatholobus stem and smilax glabra, quercetin was derived from smilax glabra and astragalus, formononetin and cap fungus were derived from spatholobus stem and astragalus, diosgenin, 7-O-methyl isofagomine, kaempferol and isomouse Li Sulai were derived from astragalus, and others were derived from spatholobus stem.
The result shows that the medicine composition has various chemical components for resisting arteriosclerosis, correspondingly has foreign matters with poor metabolism, and the astragalus root is added into the prescription of the medical diet prescription for treating the arteriosclerosis, so that the medicine composition has various compounds for treating the arteriosclerosis, correspondingly has more targets, can obviously improve the limitation of independently using the spatholobus stem and the glabrous greenbrier rhizome for improving the arteriosclerosis, and improves the effect of the traditional Chinese medicine for resisting the arteriosclerosis.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (8)
1. A pharmaceutical composition for preventing and treating arteriosclerosis, which is characterized by comprising the following raw materials:
20-30 parts of spatholobus stem, 20-30 parts of glabrous greenbrier rhizome and 2-10 parts of astragalus root.
2. The pharmaceutical composition for preventing and treating arteriosclerosis according to claim 1, comprising the following raw materials:
22-26 parts of spatholobus stem, 22-26 parts of glabrous greenbrier rhizome and 3-7 parts of astragalus root.
3. The pharmaceutical composition for preventing and treating arteriosclerosis according to claim 1, comprising the following raw materials:
23.0 parts of suberect spatholobus stem, 23.0 parts of glabrous greenbrier rhizome and 4.0 parts of astragalus root.
4. A process for the preparation of a pharmaceutical composition according to any one of claims 1 to 3, comprising the steps of:
respectively extracting caulis Spatholobi, rhizoma Smilacis Glabrae and radix astragali with extractive solution, mixing, or mixing, extracting with extractive solution, concentrating, and evaporating to dryness to obtain pharmaceutical composition.
5. The method according to claim 4, wherein the extract is ethanol or water.
6. The preparation method according to claim 4, wherein the extraction is carried out for 2-4 times by sequentially adopting ethanol and water in a ladder manner; the ethanol is used in an amount of 88-92vol% each time and the water is used in an amount of 50vol% each time.
7. A formulation comprising the pharmaceutical composition of any one of claims 1-3, wherein the pharmaceutical composition is formulated into clinically acceptable tablets, capsules, powders, mixtures, pills, granules, syrups, ointments, pastes, suppositories, aerosols or ointments by conventional techniques by adding conventional adjuvants.
8. Use of a pharmaceutical composition according to any one of claims 1 to 3 or a formulation of a pharmaceutical composition according to claim 7 for the preparation of a medicament for the prevention and treatment of arteriosclerosis.
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