CN116173134A - Application of traditional Chinese medicine composition in preparation of medicine for treating organ fibrosis - Google Patents
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- CN116173134A CN116173134A CN202210985854.4A CN202210985854A CN116173134A CN 116173134 A CN116173134 A CN 116173134A CN 202210985854 A CN202210985854 A CN 202210985854A CN 116173134 A CN116173134 A CN 116173134A
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Abstract
The invention relates to the technical field of traditional Chinese medicines, and in particular discloses an application of a traditional Chinese medicine composition in preparation of a medicine for treating organ fibrosis. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 150-350 parts of semen cuscutae, 100-200 parts of medlar, 30-60 parts of shizandra berry, 20-50 parts of fructus cnidii, 20-50 parts of cherokee rose fruit, 20-50 parts of semen allii tuberosi, 20-50 parts of morinda officinalis, 20-50 parts of cistanche salsa, 30-60 parts of radix rehmanniae, 20-50 parts of radix cyathulae, 40-100 parts of epimedium, 20-50 parts of raspberry, 15-35 parts of ginseng, 10-25 parts of hairy antler, 15-35 parts of sea horse and 15-35 parts of szechwan chinaberry fruit. The traditional Chinese medicine composition provided by the invention can be applied to preparation of medicines for treating organ fibrosis, and the obtained medicines have the effects of inhibiting the pulmonary fibrosis degree, reducing the myocardial fibrosis degree and reducing the kidney fibrosis degree, and have the advantages of remarkable effect, safe application and low price.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to an application of a traditional Chinese medicine composition in preparing a medicine for treating organ fibrosis.
Background
Fibrosis (fibrosis) is an important pathological change of various diseases, the cause of which is complex, and which can lead to impairment or even loss of normal tissue function. The major pathological changes of fibrosis are increased fibrous connective tissue in organ tissue, reduced parenchymal cells, and continued progression can lead to destruction and hypofunction of organ structures, and even failure. Fibrosis can be caused by chronic inflammatory reactions caused by factors such as infection, autoimmune reactions, allergic reactions, and chemo-radiation stimulation, the common route of which is fibroblast activation proliferation. Fibrosis can occur in various organs, such as the lung, liver, kidneys, heart, etc., resulting in fibrosis of the viscera.
Organ fibrosis is a pathological change of fibrous connective tissue increase and parenchymal cytopenia in organ tissues caused by various acute and chronic lesions, and excessive remodeling, hardening and scarring of various organ tissues, and a common pathological basis is abnormal increase and excessive deposition of extracellular matrix (extracellular matrix, ECM). Among them, kidney fibrosis is a common pathway for various chronic kidney diseases to progress to end-stage renal failure, the essence of which is "scarring" repair following injury to kidney tissue; pulmonary fibrosis is the final outcome of the development, evolution, and scarring of pulmonary diseases; liver fibrosis is a repair response of the liver to chronic injury, a pathological change common to chronic liver disease; myocardial fibrosis is an important marker for decompensated myocardial hypertrophy and heart failure, and is involved in myocardial remodeling caused by hypertension, hypertrophic cardiomyopathy, heart failure and myocardial infarction.
At present, fibrosis of parenchymal organs such as heart, liver, lung and kidney is a main cause of organ failure and disability and death of patients, and when important organs such as heart, lung and liver are fibrosed, the important organs are not treated timely and effectively, so that the organs are hardened or the organs are failure, physiological and psychological health of human beings is endangered, and even life is endangered. However, due to the lack of in-depth knowledge of the pathogenesis of organ fibrosis, there is a clinical lack of biomarkers for early diagnosis and early warning, and few therapeutic drugs specifically directed to fibrosis are available.
Although organ transplantation is the most remarkable clinical solution for the final stage of organ fibrosis, most patients cannot benefit due to the problems of scarcity, low survival rate, high cost, rejection after transplantation, high infection risk and the like of the donor, so that the finding of other treatment solutions with more remarkable curative effects, safer application and simpler operation is still a clinical urgent problem to be solved.
Disclosure of Invention
Aiming at the technical problems existing in the prior art of organ fibrosis of the aged, the invention provides application of a traditional Chinese medicine composition in preparing a medicine for treating organ fibrosis.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
the application of a traditional Chinese medicine composition in preparing a medicine for treating organ fibrosis is characterized in that the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 150-350 parts of semen cuscutae, 100-200 parts of medlar, 30-60 parts of shizandra berry, 20-50 parts of fructus cnidii, 20-50 parts of cherokee rose fruit, 20-50 parts of semen allii tuberosi, 20-50 parts of morinda officinalis, 20-50 parts of cistanche salsa, 30-60 parts of radix rehmanniae, 20-50 parts of radix cyathulae, 40-100 parts of epimedium, 20-50 parts of raspberry, 15-35 parts of ginseng, 10-25 parts of hairy antler, 15-35 parts of sea horse and 15-35 parts of szechwan chinaberry fruit.
Eight sub-drugs of the Chinese medicinal composition, namely medlar and semen cuscutae, tonify kidney and replenish essence, and shizandra berry, raspberry and cherokee rose, tonify kidney and arrest spontaneous emission, and fructus cnidii and semen allii tuberosi, invigorate yang and replenish essence. Chuan Lian Zi is a herb which is not tonifying but is a herb for promoting qi, and in qi regulating herbs, chuan Lian Zi is good at regulating qi of lower jiao liver and kidney and tonifying kidney essence, it is combined with tonifying and not stagnating. The traditional Chinese medicine composition can be applied to preparing medicines for treating organ fibrosis, and the obtained medicines have the effects of inhibiting the pulmonary fibrosis degree, reducing the myocardial fibrosis degree and reducing the kidney fibrosis degree, and have the advantages of remarkable effect, safe application and low price.
Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 150 of semen cuscutae, 200 of medlar, 30 of shizandra berry, 50 of fructus cnidii, 20 of cherokee rose fruit, 20 of Chinese chive seed, 50 of morinda root, 20 of cistanche salsa, 30 of radix rehmanniae, 50 of medicinal cyathula root, 40 of epimedium herb, 50 of raspberry, 15 of ginseng, 25 of hairy antler, 15 of sea horse and 35 of szechwan chinaberry fruit.
Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: semen Cuscutae 350, fructus Lycii 100, fructus Schisandrae 60, fructus Cnidii 20, fructus Rosae Laevigatae 50, semen Allii Tuberosi 50, radix Morindae officinalis 20, cistanchis herba 50, radix rehmanniae 60, radix Cyathulae 20, herba Epimedii 100, rubi fructus 20, ginseng radix 35, cornu Cervi Pantotrichum 10, hippocampus 35, and fructus Toosendan 15.
Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: semen Cuscutae 250, fructus Lycii 138, fructus Schisandrae 46, fructus Cnidii 35, fructus Rosae Laevigatae 35, semen Allii Tuberosi 35, radix Morindae officinalis 35, cistanchis herba 35, radix rehmanniae 46, radix Cyathulae 35, herba Epimedii 70, rubi fructus 35, ginseng radix 25, cornu Cervi Pantotrichum 16, hippocampus 21, and fructus Toosendan 23.
Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: semen Cuscutae 250, fructus Lycii 150, fructus Schisandrae 45, fructus Cnidii 30, fructus Rosae Laevigatae 30, semen Allii Tuberosi 30, radix Morindae officinalis 30, cistanchis herba 30, radix rehmanniae 45, radix Cyathulae 30, herba Epimedii 70, rubi fructus 30, ginseng radix 20, cornu Cervi Pantotrichum 19, hippocampus 20, and fructus Toosendan 20.
Further, the medicament is in the form of decoction, capsules, tablets, granules, powder or pills.
Further, the preparation method of the capsule comprises the following steps:
a. weighing fructus Cnidii, semen Cuscutae, fructus Schisandrae chinensis, and herba Epimedii in proportion, adding 6-10 times of 70% ethanol, reflux extracting for 1-3 times each for 1-3 hr, filtering, mixing filtrates, and recovering ethanol under reduced pressure until no ethanol smell exists to obtain ethanol extract;
b. mixing the residue obtained after the ethanol extraction in step a with fructus Lycii, radix rehmanniae, rubi fructus, fructus Rosae Laevigatae, semen Allii Tuberosi, radix Morindae officinalis and fructus Toosendan in proportion, decocting with 7-12 times of water for 1-3 times each time for 1-3 hours, filtering, mixing filtrates, mixing with the ethanol extract obtained in step a, concentrating under reduced pressure until the relative density is 1.25-1.30 at 60deg.C to obtain extract;
c. weighing Ginseng radix, cornu Cervi Pantotrichum, hippocampus, cistanchis herba, and radix Cyathulae at a certain proportion, pulverizing into fine powder, mixing with the extract obtained in step b, drying at 60-70deg.C, pulverizing, sieving, and making into capsule.
Further, the organ fibrosis includes at least one of myocardial fibrosis, liver fibrosis, lung fibrosis, or kidney fibrosis.
The invention also provides application of the pharmaceutical composition in medicines for reducing liver inflammation.
The traditional Chinese medicine composition provided by the invention can be applied to preparation of medicines for treating organ fibrosis including at least one of myocardial fibrosis, liver fibrosis, lung fibrosis or kidney fibrosis, and has the advantages of obvious effect, safe application and low price, and the degree of organ fibrosis is reduced.
Drawings
FIG. 1 is a map of the Masson staining of lung tissue in a test example of the present invention;
FIG. 2 is a map of Masson staining of heart tissue at various magnifications in accordance with an embodiment of the present invention;
FIG. 3 is a chart of immunofluorescence staining in a test example of the present invention;
FIG. 4 is a graph of staining of kidney tissue in a test example of the invention;
FIG. 5 is a graph of HE staining of liver in a test example of the invention;
FIG. 6 is a map of liver Masson staining in a test example of the invention;
fig. 7 is a graph of HAI (Hepatic Activity Index) liver histological activity index.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1: preparation of capsules
The formula comprises the following components: 250g of semen cuscutae, 138g of medlar, 46g of shizandra berry, 35g of fructus cnidii, 35g of cherokee rose, 35g of semen allii tuberosi, 35g of morinda officinalis, 35g of herba cistanches, 46g of radix rehmanniae, 35g of radix cyathulae, 70g of herba epimedii, 35g of raspberry, 25g of ginseng, 16g of hairy antler, 21g of sea horse and 23g of szechwan chinaberry fruit.
The preparation method comprises the following steps:
a. weighing fructus Cnidii, semen Cuscutae, fructus Schisandrae chinensis and herba Epimedii in proportion, adding 3208ml70% ethanol, reflux extracting for 3 times each for 2 hr, filtering, mixing filtrates, and recovering ethanol under reduced pressure until no ethanol smell exists to obtain ethanol extract;
b. mixing the residue obtained after the ethanol extraction in step a with fructus Lycii, radix rehmanniae, rubi fructus, fructus Rosae Laevigatae, semen Allii Tuberosi, radix Morindae officinalis and fructus Toosendan in proportion, decocting with 6732ml water for 2 times each time for 2 hours, filtering, mixing filtrates, mixing with the ethanol extract obtained in step a, concentrating under reduced pressure until the relative density is 1.28 at 60deg.C to obtain extract;
c. weighing Ginseng radix, cornu Cervi Pantotrichum, hippocampus, cistanchis herba, and radix Cyathulae at a certain proportion, pulverizing into fine powder, mixing with the extract obtained in step b, drying at 65deg.C, pulverizing, sieving, and making into capsule 757 granule.
Example 2: preparation of tablets
The formula comprises the following components: 250g of semen cuscutae, 150g of medlar, 45g of shizandra berry, 30g of fructus cnidii, 30g of cherokee rose fruit, 30g of semen allii tuberosi, 30g of morinda officinalis, 30g of herba cistanches, 45g of radix rehmanniae, 30g of radix cyathulae, 70g of herba epimedii, 30g of raspberry, 20g of ginseng, 19g of hairy antler, 20g of sea horse and 20g of szechwan chinaberry fruit.
The preparation method comprises the following steps:
a. weighing fructus Cnidii, semen Cuscutae, fructus Schisandrae chinensis, and herba Epimedii in proportion, adding 2765ml70% ethanol, reflux extracting for 3 times each for 1 hr, filtering, mixing filtrates, and recovering ethanol under reduced pressure until no ethanol smell exists to obtain ethanol extract;
b. mixing the residue obtained after the ethanol extraction in the step a with a certain proportion of fructus Lycii, radix rehmanniae, rubi fructus, fructus Rosae Laevigatae, semen Allii Tuberosi, radix Morindae officinalis and fructus Toosendan, adding 5110ml of water, decocting for 1 time and 3 hours, filtering, mixing the filtrates, mixing with the ethanol extract obtained in the step a, concentrating under reduced pressure until the relative density is 1.30 at 60deg.C, and obtaining extract;
c. weighing Ginseng radix, cornu Cervi Pantotrichum, hippocampus, cistanchis herba, and radix Cyathulae at a certain proportion, pulverizing into fine powder, mixing with the extract obtained in step b, drying at 70deg.C, pulverizing, sieving, adding 1% magnesium stearate, and tabletting to obtain 760 tablet.
Example 3: preparation of pill
The formula comprises the following components: 350g of semen cuscutae, 100g of medlar, 60g of shizandra berry, 20g of fructus cnidii, 50g of cherokee rose fruit, 50g of semen allii tuberosi, 20g of morinda officinalis, 50g of herba cistanches, 60g of radix rehmanniae, 20g of radix cyathulae, 100g of epimedium, 20g of raspberry, 35g of ginseng, 10g of hairy antler, 35g of sea horse and 15g of szechwan chinaberry fruit.
The preparation method comprises the following steps:
a. weighing fructus Cnidii, semen Cuscutae, fructus Schisandrae chinensis, and herba Epimedii in proportion, adding 5300ml70% ethanol, reflux extracting for 1 time and 3 hours, filtering, mixing filtrates, and recovering ethanol under reduced pressure until no ethanol smell exists to obtain ethanol extract;
b. mixing the residue obtained after the ethanol extraction in step a with fructus Lycii, radix rehmanniae, rubi fructus, fructus Rosae Laevigatae, semen Allii Tuberosi, radix Morindae officinalis and fructus Toosendan in proportion, decocting with 10140ml water for 3 times each for 1 hr, filtering, mixing filtrates, mixing with the ethanol extract obtained in step a, concentrating under reduced pressure until the relative density is 1.25 at 60deg.C to obtain extract;
c. weighing Ginseng radix, cornu Cervi Pantotrichum, hippocampus, cistanchis herba, and radix Cyathulae at a certain proportion, pulverizing into fine powder, mixing with the extract obtained in step b, drying at 60deg.C, pulverizing, sieving, and making into pill 320g.
Example 4: preparation of granule
The formula comprises the following components: 150g of semen cuscutae, 200g of medlar, 30g of shizandra berry, 50g of fructus cnidii, 20g of cherokee rose fruit, 20g of semen allii tuberosi, 50g of morinda officinalis, 20g of herba cistanches, 30g of radix rehmanniae, 50g of radix cyathulae, 40g of herba epimedii, 50g of raspberry, 15g of ginseng, 25g of hairy antler, 15g of sea horse and 35g of szechwan chinaberry fruit.
The preparation method comprises the following steps:
a. weighing fructus Cnidii, semen Cuscutae, fructus Schisandrae chinensis and herba Epimedii in proportion, adding 2430ml70% ethanol, reflux-extracting for 2 times each for 1.5 hr, filtering, mixing filtrates, and recovering ethanol under reduced pressure until no ethanol smell exists to obtain ethanol extract;
b. mixing the residue obtained after the ethanol extraction in the step a with the rest medicinal materials in a proportion, adding 8800ml of water, decocting for 3 times each for 1 hour, filtering, mixing the filtrates, mixing with the ethanol extract obtained in the step a, concentrating under reduced pressure until the relative density is 1.29 at 60 ℃ to obtain extract;
c. mixing the extract obtained in the step b with 300g of dextrin and 200g of sucrose powder uniformly, drying at 70 ℃, crushing, sieving, and granulating to obtain 700g of granules.
Example 5: preparation of powder
The formula comprises the following components: 250g of semen cuscutae, 150g of medlar, 45g of shizandra berry, 30g of fructus cnidii, 30g of cherokee rose fruit, 30g of semen allii tuberosi, 30g of morinda officinalis, 30g of herba cistanches, 45g of radix rehmanniae, 30g of radix cyathulae, 70g of herba epimedii, 30g of raspberry, 20g of ginseng, 19g of hairy antler, 20g of sea horse and 20g of szechwan chinaberry fruit.
The preparation method comprises the following steps: weighing the traditional Chinese medicinal materials in proportion, mixing and crushing into fine powder to obtain 823g of powder.
Example 6: preparation of decoction
The formula comprises the following components: 250g of semen cuscutae, 150g of medlar, 45g of shizandra berry, 30g of fructus cnidii, 30g of cherokee rose fruit, 30g of semen allii tuberosi, 30g of morinda officinalis, 30g of herba cistanches, 45g of radix rehmanniae, 30g of radix cyathulae, 70g of herba epimedii, 30g of raspberry, 20g of ginseng, 19g of hairy antler, 20g of sea horse and 20g of szechwan chinaberry fruit.
The preparation method comprises the following steps: weighing the traditional Chinese medicinal materials in proportion, adding water until the traditional Chinese medicinal materials are soaked for 15 minutes, heating until the water is boiled, continuously decocting for 30 minutes with slow fire, filtering, adding water into the residues again until the traditional Chinese medicinal materials are soaked, decocting for 30 minutes again, filtering, and combining the filtrates to obtain 4320g decoction.
Test example:
to elucidate the effect of the drug of the present invention on improving the degree of visceral fibrosis weakness, the following experiment was conducted using example 1 (hereinafter referred to as the drug of the present invention or BZBS):
1. animal experiment for pulmonary fibrosis
A single tracheal injection of bleomycin was used to model pulmonary fibrosis in mice. Male ICR mice (SPF grade) were divided into a blank group, a model group, and a pirfenidone group (manufacturing company: beijing Kanti pharmaceutical Co., ltd.) and BZBS group at random according to body weight of 22-25 g. The animals were subjected to tracheal injection surgery, the blank group was injected with physiological saline, and the remaining groups were injected with bleomycin (manufacturer: japanese chemical Co., ltd.). BZBS group administration dose is folded crude drug dose of 2g crude drug/kg/d, pirfenidone group administration dose is 300mg/kg/d, corresponding volume solvent (CMC) is given to blank group animals and model group animals, stomach irrigation administration is carried out, administration is started on the molding day, and continuous administration is carried out for 21 days.
After the administration, the weight change of the animals is observed by weighing, the liquid nitrogen is placed after the whole lung is weighed, the lung coefficient is calculated, the result is shown in table 1, and the result is shown in table 2. Part of the animals were stained for Masson's lung perfusion fixation and the lung tissue fibrosis was examined and the results are shown in table 3 and fig. 1 (lung tissue Masson staining pattern (20×), including blank, model, pirfenidone and BZBS groups).
From the above data, the model group showed significantly increased lung coefficient (P < 0.01) compared with the blank group, and significantly decreased lung coefficient (P < 0.05) compared with the BZBS group and pirfenidone group; meanwhile, compared with a blank group, the hydroxyproline content of the model group is obviously increased (P is less than 0.05); compared with the model group, the BZBS group and the pirfenidone group have obviously reduced hydroxyproline content (P < 0.05); furthermore, the BZBS and pirfenidone groups have relatively low lung fibrosis scores compared to the model group. The BZBS has the effects of inhibiting the pulmonary fibrosis degree of mice caused by bleomycin and preventing and treating the pulmonary fibrosis.
2. Animal experiment for myocardial fibrosis
50 male SD rats (SPF grade) were 2 months old, and had a weight of 200+ -10 g, and purchased from Beijing Vetong Lihua laboratory animal technology Co., ltd. The animal raising conditions are 12 hours/day of illumination, the temperature is 20-26 ℃, the relative humidity is 40-70%, the animals are fed with maintenance feed (supplied by Australian feed Co., beijing family), clean water prepared by a ROA50 experimental animal drinking system is drunk, animals are quarantined for 3 days, the animals are adaptively raised for 2 days, and drinking water, ingestion and health conditions of the animals and whether disease and death symptoms exist or not are observed in the period.
The 50 SD rat random number table method was divided into 5 groups: the drug intervention was performed in the normal group (normal diet + isovolumetric solvent gavage), in the model group (normal diet + isovolumetric solvent gavage), in the BZBS low dose group (normal diet + BZBS 1g/kg/d gavage), in the BZBS high dose group (normal diet + BZBS 4g/kg/d gavage), and in the Sha Kuba troxatan control group (normal diet + 20mg/kg/2d gavage), as shown in table 4. The administration modes of the test substances are BZBS high-dose group, BZBS low-dose group and control group, wherein the daily gastric lavage drug solution is 10mL/kg, and the normal group and the model group are the daily gastric lavage of the same volume solvent CMC.
The model making method comprises the following steps: SD rats were fed adaptively for 1 week, weighed, injected intraperitoneally with 10% chloral hydrate (0.35 g/kg), fixed on the rat plates in right lateral recumbent position after anesthesia, shaved in right quaternary rib area, and left kidney area exposed; sterilizing the kidney region with 75% alcohol, making a longitudinal incision along the free edge of the left twelve ribs of the spine, taking a length of about 1-2cm, performing blunt stripping to remove the muscle layer, finding out the kidney, performing extrusion exposure, stripping fat around the kidney, and exposing the artery and vein of the kidney and ureter; the hemostatic forceps fix the kidney, ligate the whole arteriovenous and ureter with the 4-0 operation suture line, and cut off the kidney; sequentially suturing the visceral layer and the muscular layer, performing abdominal cavity injection with 10000u row of penicillin, wiping off excessive blood, placing the rats on an electric blanket for heat preservation, and placing the rats back into a squirrel cage for feeding after observing the stable vital signs, wherein 1% saline water is used for drinking and free diet. After 4 weeks, the heart function of the rat is detected by using a small animal ultrasonic instrument, and EF is more than 50%, and the E/A ratio is reduced, so that the establishment of the chronic heart failure model is successful.
After 2 months of administration, the rat is anesthetized with isoflurane, the rat is waited for losing consciousness, the abdominal aorta is taken for blood after the rat is anesthetized, after standing for 2 hours at normal temperature, the serum is taken after centrifugation for 15 minutes at 3000r, and the rat is preserved at-80 ℃ for standby. The heart tissue is taken and fixed in 4% paraformaldehyde solution, and one part of the heart tissue is placed in a freezing tube and stored at-80 ℃ for standby. Part of the heart was subjected to Masson and HE staining to observe the morphological changes of heart tissue. The results are shown in fig. 2 (same group in each column, different groups in each column), and Masson staining patterns at different magnifications (10×, 20×, and 40×) for the normal group, model group, control group, BZBS low dose group, and BZBS high dose group. Masson staining showed that the cardiomyocytes of the normal group of rats were ordered, the cells were not necrotic, and no significant collagen fibroplasia was observed. The model group can be used for treating a large number of myofiber cracks, atrophy and degeneration, the blue dyeing degree of collagen fiber is heavy, the fibrosis degree of myocardial cells is obviously increased, and the myocardial cells are stiff. The administration groups (control group, BZBS low dose group and BZBS high dose group) lighten the myocardial fibrosis degree in different degrees.
Simultaneously, the cross-sectional area of myocardial cells is measured by immunofluorescence, myocardial tissue sections are taken, dewaxing, antigen repair and endogenous peroxidase blocking are performed, 3% BSA is blocked for 30 min at room temperature, primary antibody is incubated overnight at 4 ℃, secondary antibody is incubated for 1 h in dark, DAPI (4', 6-diamidino-2-phenylindole) is used for dying cell nuclei, and neutral resin is used for sealing. 3 discrete fields were randomly selected under a microscope, and staining results were observed, as shown in fig. 3 (each column is the same group, and different columns correspond to different groups respectively), wheat Germ Agglutinin (WGA) appears green, alpha smooth muscle actin (alpha-SMA) appears red, DAPI appears blue, and amber particles are positive cells in an immunofluorescent staining chart (20×). WGA staining showed an increase in cardiomyocyte cross-sectional area in the model group over the normal group, morphologically confirming the presence of myocardial hypertrophy. The cardiomyocyte size (P < 0.05) was significantly restored in each of the dosing groups (control, BZBS low dose, BZBS high dose). There was no significant difference in the perivascular α -SMA expression levels for each of the dosing groups.
From the data, BZBS can obviously reduce the collagen content of myocardial tissues of rats with chronic heart failure, reduce the cross-sectional area of myocardial cells, reduce the degree of myocardial fibrosis, inhibit the reconstruction of the left ventricle, and further improve the heart function of the rats.
3. Animal experiment of kidney fibrosis
15 male BKS-DB (WT) mice (weighing about 19-30 g) were used as a blank group, 45 male BKS-DB mice (weighing about 40-69 g) were used as an experimental group, and the animal source was Jiangsu Jiuzhikang Biotech Co. Animals in the experimental group begin to be fed with high-fat high-protein diet at the age of 8 weeks, and blood sugar and urine biochemical (mAlb/CREA) indexes of the animals are detected after continuous feeding for 8 weeks, so that diabetic nephropathy symptoms are achieved. Animals in the experimental group were divided into a model group, a BZBS group and a dapagliflozin group according to blood sugar, 15 animals in each group were administered on the day of grouping, 1 time/day, and 6 weeks continuously. BZBS group administration dose is 1.44g crude drug/kg/d, dapagliflozin group administration dose is 1mg/kg/d, and the blank group and model group animals are given equal volume of water. After the completion of the administration, each group was examined for renal fibrosis and urine biochemistry, and the results are shown in fig. 4 (renal tissue staining chart (10×)) and tables 5 and 6. Compared with the model group, the urine biochemical index of the BZBS group is obviously reduced (P < 0.05).
From the above data, it can be seen that the urine biochemical (mAlb/CREA) index of the test animals is significantly improved and the degree of renal fibrosis is significantly reduced after BZBS administration for 6 weeks.
4. Animal experiment of liver fibrosis
Purpose of test
The regulation effect of the pharmaceutical composition on the liver inflammation and the fibroplasia of the mice with natural aging is studied.
1 Experimental materials
1.1 Test article
1.1.1 name: the invention relates to a pharmaceutical composition (BZBS).
1.1.2 routes and amounts: oral administration
1.1.3 sources and lot numbers: the Shijia was XB2103001, a lot number of Shijia Kao Co., ltd.
1.2 tool medicine and main reagents and materials
Sodium carboxymethyl cellulose (CMC-Na): the Tianjin market metallocene chemical reagent plant, lot number: 20181006, expiration date: by 2021, 12 months and 30 days.
1.3 experiment System
1.3.1 The animal species: c57BL/6J mice.
1.3.2 Animal grade: SPF stage.
1.3.3 Sex and number of animals: 60 male mice were purchased, and 30 (30/male) were selected for the test. In addition, 12 mice of the same strain and sex and manufacturer with the age of 23 weeks are singly selected and directly taken as a control.
1.3.4 Age of animals at the start of dosing: 52 weeks of age.
1.3.5 Animal body weight at the start of dosing: actual 20-40g.
1.3.6 Animal origin: purchased from beijing vernalia laboratory animal technology limited.
1.3.7 Animal pass number, issuing unit, date of receipt: the certificate number 1100112011043405, the license number SCXK 2016-0006, the certification unit was Beijing Vertolaccording laboratory animal technology Co., ltd, and the date of receipt was 2020, month 07, and 15.
1.3.7 feeding conditions: mice are raised in cages and raised in Hebei Kagaku Co., ltd, and the new medicine evaluation center is illuminated for 12 hours/day, the temperature is 20-26 ℃, the relative humidity is 40-70%, and the use license number of experimental animals is as follows: SYXK (Ji) 2020-003.
1.3.8 Quarantine process: animals were quarantined for 3 days and acclimatized for 2 days during which time animals were observed for drinking, ingestion, and health status, as well as for the presence of disease and mortality symptoms.
1.3.9 Feed: SPF-size mice maintained feed, supplied by Australian feed Co., ltd. Feed production license number: jinggao (2018) 06073, feed lot number: 20103213, 20113213.
1.3.10 And (3) drinking water: clean water prepared by an ROA50 experimental animal drinking system (instrument number YL-LE-A01) is drunk, a drinking bottle is filled for free drinking, and the water quality is detected to meet the sanitary standard of drinking water.
1.3.11 Padding: the common-grade corncob padding is used after being sterilized at high temperature and high pressure, and is provided by Beijing Australian feed Co-Ltd, and the padding batch number is as follows: 20079811.
1.3.12 Identification: ear tag of experimental animal.
2 test method
2.1 Test design basis
2.1.1 The design basis is as follows: and related literature data.
2.1.2 Experimental system selection description:
aged C57BL/6J mice: research applications include immunology, cancer, long-lived interventions, and biomarker studies. The balance and stability of stem cells in 13 month old C57 mice was destroyed and animals began to age rapidly, so the naturally aging C57BL/6J mice were used to simulate the premature aging state of humans.
2.2 doses and groupings
The mice are divided into model groups (equal volume distilled water), the pharmaceutical composition group (2 g/kg/d) of the invention, and the mice with the age of 23 weeks are young groups, and the mice are directly obtained without any treatment.
The daily dosage of the medicine composition is 2.4g crude drug (60 kg weight of human body), and the mice BZBS group is defined as 2g crude drug/kg. 30C 57 mice were randomly divided into 2 groups according to body weight, including model group and pharmaceutical composition group according to the present invention.
2.3 method of administering test substances
The stomach is irrigated, which is consistent with the intended application diameter.
2.4 preparation and preservation of samples
The pharmaceutical composition (BZBS) of the invention: 1.2g of the superfine powder +12ml of CMC of the pharmaceutical composition of the invention, which is prepared and used at present
According to the dosage of 10ml/kg and the dosage setting, the preparation concentration is calculated (see Table 7), the pharmaceutical composition of the invention is weighed according to the number of animals, the solvent is added for grinding and suspending, the volume is fixed, and the test sample is prepared at present. The solvent is CMC-Na with concentration of 0.5 percent, evenly spread on the surface of pure water for full swelling, constant volume and preservation at 2-8 ℃ for standby.
2.5 administration of test substances
The test sample was administered by syringe in a volume of 10ml/kg, animals in model group, BZBS group were given the corresponding volume of solvent, and the replacement was performed every 48 hours.
2.6 model making: and naturally breeding the C57 mice to 13 months of age to obtain the natural aging animals.
2.7 detection index: histopathological analysis
Liver samples were fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin. A5 μm thick section was prepared, hematoxylin-eosin (HE) staining and Masson staining were performed, and histological analysis was performed under a light microscope. We slightly improved this scoring criteria based on the widely accepted Histological Activity Index (HAI) (Knodell et al, 1981) scoring criteria. Briefly, HAI represents the sum of scores of necrosis-inflammatory lesions, i.e. perioral and bridging necrosis (0-10), intralobular degeneration and focal necrosis (0-4), inflammation (0-4), fibroplasia (0-4). Liver-stained sections of each group were scored together by two pathologists according to HAI scoring criteria.
3 pathological results
3.1 As a result, as shown in FIG. 5, the liver HE staining, the natural aging model group showed inflammation in the central vein region and the peripheral part of portal vein, the pharmaceutical composition group of the present invention showed little inflammation, and the young group showed no inflammation.
3.2 Results As shown in FIG. 6, liver masson staining, partial fibroplasia was seen in the natural aging model group, and the pharmaceutical composition of the present invention group, young group, had no fibroplasia.
3.3 results as in FIG. 7 HAI (Hepatic Activity Index) liver histological activity index model group was 10 points, the pharmaceutical composition of the present invention group was 3 points, and the young group was 0 points. HAI index (n=10)
4. Conclusion: the pharmaceutical composition can reduce inflammation and hyperplasia of liver in the aging process.
In summary, the BZBS provided by the embodiment of the invention can inhibit the pulmonary fibrosis degree of mice caused by bleomycin, can reduce the myocardial cell cross-sectional area of rats with chronic heart failure, can reduce the myocardial fibrosis degree of the mice with diabetic nephropathy symptoms, can reduce the kidney fibrosis degree of the mice with diabetic nephropathy symptoms, and can reduce the inflammation and the hyperplasia of liver in the aging process, so that the BZBS can be used for treating organ fibrosis including myocardial fibrosis, pulmonary fibrosis, kidney fibrosis and liver fibrosis. The medicaments provided in examples 2 to 6 of the present invention have substantially equivalent effects to those of the capsule of example 1.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (9)
1. The application of the traditional Chinese medicine composition in preparing the medicine for treating the organ fibrosis is characterized in that the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 150-350 parts of semen cuscutae, 100-200 parts of medlar, 30-60 parts of shizandra berry, 20-50 parts of fructus cnidii, 20-50 parts of cherokee rose fruit, 20-50 parts of semen allii tuberosi, 20-50 parts of morinda officinalis, 20-50 parts of cistanche salsa, 30-60 parts of radix rehmanniae, 20-50 parts of radix cyathulae, 40-100 parts of epimedium, 20-50 parts of raspberry, 15-35 parts of ginseng, 10-25 parts of hairy antler, 15-35 parts of sea horse and 15-35 parts of szechwan chinaberry fruit.
2. The use of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 150 of semen cuscutae, 200 of medlar, 30 of shizandra berry, 50 of fructus cnidii, 20 of cherokee rose fruit, 20 of Chinese chive seed, 50 of morinda root, 20 of cistanche salsa, 30 of radix rehmanniae, 50 of medicinal cyathula root, 40 of epimedium herb, 50 of raspberry, 15 of ginseng, 25 of hairy antler, 15 of sea horse and 35 of szechwan chinaberry fruit.
3. The use of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: semen Cuscutae 350, fructus Lycii 100, fructus Schisandrae 60, fructus Cnidii 20, fructus Rosae Laevigatae 50, semen Allii Tuberosi 50, radix Morindae officinalis 20, cistanchis herba 50, radix rehmanniae 60, radix Cyathulae 20, herba Epimedii 100, rubi fructus 20, ginseng radix 35, cornu Cervi Pantotrichum 10, hippocampus 35, and fructus Toosendan 15.
4. The use of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: semen Cuscutae 250, fructus Lycii 138, fructus Schisandrae 46, fructus Cnidii 35, fructus Rosae Laevigatae 35, semen Allii Tuberosi 35, radix Morindae officinalis 35, cistanchis herba 35, radix rehmanniae 46, radix Cyathulae 35, herba Epimedii 70, rubi fructus 35, ginseng radix 25, cornu Cervi Pantotrichum 16, hippocampus 21, and fructus Toosendan 23.
5. The use of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: semen Cuscutae 250, fructus Lycii 150, fructus Schisandrae 45, fructus Cnidii 30, fructus Rosae Laevigatae 30, semen Allii Tuberosi 30, radix Morindae officinalis 30, cistanchis herba 30, radix rehmanniae 45, radix Cyathulae 30, herba Epimedii 70, rubi fructus 30, ginseng radix 20, cornu Cervi Pantotrichum 19, hippocampus 20, and fructus Toosendan 20.
6. The use according to any one of claims 1 to 5, wherein the medicament is in the form of a decoction, a capsule, a tablet, a granule, a powder or a pill.
7. The use according to claim 6, wherein the method of preparing the capsule comprises the steps of:
a. weighing fructus Cnidii, semen Cuscutae, fructus Schisandrae chinensis, and herba Epimedii in proportion, adding 6-10 times of 70% ethanol, reflux extracting for 1-3 times each for 1-3 hr, filtering, mixing filtrates, and recovering ethanol under reduced pressure until no ethanol smell exists to obtain ethanol extract;
b. mixing the residue obtained after the ethanol extraction in step a with fructus Lycii, radix rehmanniae, rubi fructus, fructus Rosae Laevigatae, semen Allii Tuberosi, radix Morindae officinalis and fructus Toosendan in proportion, decocting with 7-12 times of water for 1-3 times each time for 1-3 hours, filtering, mixing filtrates, mixing with the ethanol extract obtained in step a, concentrating under reduced pressure until the relative density is 1.25-1.30 at 60deg.C to obtain extract;
c. weighing Ginseng radix, cornu Cervi Pantotrichum, hippocampus, cistanchis herba, and radix Cyathulae at a certain proportion, pulverizing into fine powder, mixing with the extract obtained in step b, drying at 60-70deg.C, pulverizing, sieving, and making into capsule.
8. The use according to any one of claims 1 to 5, wherein the organ fibrosis comprises at least one of myocardial fibrosis, liver fibrosis, lung fibrosis or kidney fibrosis.
9. The use according to any one of claims 1 to 5, wherein the pharmaceutical composition of the invention is for use in a medicament for reducing liver inflammation.
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