CN113876787A - Cyanotis extract and application and preparation thereof - Google Patents

Cyanotis extract and application and preparation thereof Download PDF

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Publication number
CN113876787A
CN113876787A CN202111196258.XA CN202111196258A CN113876787A CN 113876787 A CN113876787 A CN 113876787A CN 202111196258 A CN202111196258 A CN 202111196258A CN 113876787 A CN113876787 A CN 113876787A
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parts
extract
fibrosis
group
composition
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CN113876787B (en
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付军
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Guangdong Shengpu Life Technology Co ltd
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Guangdong Shengpu Life Technology Co ltd
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Abstract

The application is a divisional application with the application number of 2021101975884, application date of 22/02/2021 and the title of "a composition for preventing and/or treating organ fibrosis and its application and preparation". The invention provides an application of a herba dewettiae extract in preparing a medicament for preventing and/or treating skin fibrosis.

Description

Cyanotis extract and application and preparation thereof
The application is a divisional application with the application number of 2021101975884, application date of 22/02/2021 and the title of "a composition for preventing and/or treating organ fibrosis and its application and preparation".
Technical Field
The invention relates to the technical field of medicines, in particular to application of a herba Cymbopogonis Citrari extract in preparing an anti-skin fibrosis medicine and a preparation thereof.
Background
Any cause can cause the tissue cells to be damaged, and can cause the tissue cells to generate degeneration, necrosis and inflammatory reaction, if the damage is small, the normal parenchymal cells around the damaged cells can generate hyperplasia repair, and the repair can completely restore the normal structure and function. However, if the damage is large or the repeated damage exceeds the regeneration capacity of the parenchymal cells around the damage, the interstitial fibrous connective tissue (extracellular matrix) is proliferated to repair the defective tissue, i.e., the pathological change of fibrosis (fibrosis) occurs. Fibrosis is thus essentially a repair response after tissue has been damaged to preserve the relative integrity of the tissue and organs. The hyperplastic fibrous connective tissue, although repairing the defect, does not possess the structure and function of the parenchymal cells of the original organ. Fibrosis can occur in various organs such as lung, liver, kidney, peritoneum, blood vessel, pancreas, skin, etc., and the main pathological changes are that fibrous connective tissue in organ tissues is increased, parenchymal cells are reduced, and continuous progress can cause structural damage and hypofunction of organs and even failure, directly causes the loss of organ functions and seriously threatens human health and life. As shown by the relevant statistics in the United states, nearly 45% of the patients fatal to various diseases in this country can be attributed to the tissue fibroproliferative disease. In addition, the development of respiratory virus infection related diseases is also accompanied by pulmonary fibrosis, for example, severe pulmonary fibrosis can be caused by severe infection of novel coronavirus SARS-CoV-2.
Application of Nintedanib in treating fibrotic diseases is disclosed in the patent publication CN107019697A, and it has been shown that tannic acid acts on CCl4The induced hepatic fibrosis mouse has obvious liver protection effect and can play a certain hepatic fibrosis resistance effect. However, on the one hand, the application is limited because apart from liver fibrosis, fibrosis occurs in many organs of the body; on the other hand, in order to ensure the drug effect, the dosage of the tannic acid is usually large, the cost is high, and certain toxic and side effects are inevitable. Thus, the search for new drug regimens with reduced cost and improved anti-organ fibrosis effects is the current focus of research.
Disclosure of Invention
The invention aims at the limitation of the existing anti-skin fibrosis drugs, and aims to provide the application of the herba oenoprasis extract in the preparation of the drugs for preventing and/or treating skin fibrosis.
Another object of the present invention is to provide an oral liquid for preventing and/or treating skin fibrosis comprising the extract of dewy grass.
Another object of the present invention is to provide a tablet for preventing and/or treating skin fibrosis comprising the extract of dewy grass.
Another object of the present invention is to provide a spray for preventing and/or treating skin fibrosis comprising the extract of dewy grass.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides a dew grass composition for preventing and/or treating fibrosis, which comprises tannic acid and/or a dew grass extract; the herba Cymbopogonis Citrari extract is herba Cymbopogonis Citrari water extract and/or herba Cymbopogonis Citrari alcohol extract.
In the dewy herb composition for preventing and/or treating fibrosis, tannic acid (tannic acid) is a secondary metabolite of some plants, is also called tannic acid in pharmaceutical medicine, is a natural plant polyphenol, widely exists in gallnut plants, contains a large amount of hydroxyl and carbonyl, is a soluble and polyphenol compound, and has biological functions of diarrhea resistance, bacteriostasis, oxidation resistance, cancer resistance, virus resistance, fibrosis resistance and the like.
The herba Eragrostidis extract is extracted from herba Eragrostidis Cyanotis arahnoids C.B.Clarke of Commelinaceae, and mainly contains 20-hydroxyecdysterone (beta-ecdysone), which has effects of promoting cell growth, stimulating dermal cell division, promoting protein synthesis, dispelling pathogenic wind, activating collaterals, promoting diuresis, relieving swelling, clearing away asthenic fever, dredging channels, relieving pain, and can be used for treating rheumatic arthritis, limbs anesthesia, promoting silkworm, shrimp, and crab production desquamation, reducing cholesterol in human body, and reducing blood sugar concentration.
The research result of the invention shows that the herba dewettiae extract has obvious effect of treating the skin fibrosis, the anti-fibrosis effect is obviously improved after the tannic acid is compounded with the combination of the herba dewettiae extract, and the compound composition is integrally and obviously improved and synergized in the aspect of treating the pulmonary fibrosis, the hepatic fibrosis, the cardiac fibrosis and the skin fibrosis.
As a preferred implementable method, the preparation method of the aqueous extract of the dewy grass comprises the following steps:
s1, drying and crushing the dewed grass, weighing 50g of dewed grass powder, adding 500mL of water, and performing reflux extraction for 2 hours.
S2, filtering, adding 500mL of water into filter residues, performing reflux extraction for 2h, filtering, and combining the two filtrates.
And S3, concentrating and drying to obtain the aqueous extract of the dewy grass.
As a preferable implementable method, the preparation method of the alcohol extract of the dewed grass comprises the following steps:
drying and crushing the dewed grass, weighing 50g of dewed grass powder in 500mL of 60 vol% ethanol solution, performing reflux extraction for 1h, filtering, and concentrating and drying the filtrate to obtain the dewed grass extract.
Preferably, the tannin and the extract of the dewed grass comprise the following components in parts by weight: 12-45 parts of tannic acid and 12-30 parts of a herba Cymbopogonis Citrari extract.
More preferably, the tannin and the extract of the herba oenoprasis comprise the following components in parts by weight: 19 parts of tannic acid and 15 parts of herba Cymbopogonis Citrari extract.
The invention also provides application of the dewy grass composition in preparing a medicament for preventing and/or treating organ fibrosis.
The application of the dewet grass composition claimed by the invention in preparing a medicament for preventing and/or treating organ fibrosis includes but is not limited to the application of an effective amount of the dewet grass composition in the preparation of a medicament for preventing or treating fibrosis-induced diseases, relieving the symptoms of the fibrosis-induced diseases or delaying the development or onset of the fibrosis-induced diseases by administering the effective amount of the dewet grass composition to a patient.
The composition has no toxic or side effect on organisms, and can be used for preparing health care products or food for daily use so as to prevent organ fibrosis of specific people. Therefore, the invention also claims the application of the dew grass composition in preparing health care products or foods for preventing organ fibrosis.
The herba rhodiolae composition claimed by the invention is suitable for being daily taken by specific people when preparing health care products or foods for preventing organ fibrosis, has the functions of regulating the organism and preventing the organ fibrosis, and does not produce any acute, subacute or chronic harm to human bodies.
The dew grass compositions claimed in this invention may be applied to veterinary treatment of pets, animals of the introduced species and animals of farms, including mammals, rodents, etc., in addition to being beneficial for human treatment. Other examples of animals include horses, dogs, cats, and the like.
Preferably, the organ fibrosis comprises pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, skin fibrosis.
Preferably, the medicament is prepared into different formulations by adding pharmaceutically acceptable auxiliary materials into the dewy grass composition for preventing and/or treating organ fibrosis. The dosage form can be oral liquid, tablet, spray, etc.
The invention also provides an oral liquid containing the dew grass composition and used for preventing and/or treating fibrosis.
Preferably, the oral liquid comprises the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of herba Cymbopogonis Citrari extract, 6-28 parts of antioxidant, 6-30 parts of sweetener, 6-25 parts of bacteriostatic agent and 90-200 parts of purified water.
The anti-organ fibrosis oral liquid provided by the invention has reasonable compatibility of all components, wherein the tannic acid can inhibit the expression of collagen-1 and smooth muscle alpha-actin (SMA) induced by TGF-beta, and plays a role in anti-fibrosis; the main component of the herba Cymbopogonis Citrari is 20-hydroxyecdysterone (beta-ecdysone), which has the main effects of promoting cell growth, stimulating dermal cell division, and promoting protein synthesis for human body; the antioxidant can prevent the oxidation of the original medicine, so that the stability of the antioxidant is good; the sweetener can improve the taste feeling of a user, so that the adaptability of the sweetener is good; the bacteriostatic agent can effectively prevent the oral liquid from deteriorating; purified water acts as a solvent for dissolution. The obtained anti-fibrosis oral liquid has obvious effects of resisting pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis and skin fibrosis, is beneficial to treatment and rehabilitation of patients, and improves the life quality.
Preferably, the antioxidant may be any one of vitamin C, vitamin E, sodium sulfite.
Preferably, the sweetener may be aspartame or stevioside.
Preferably, the bacteriostatic agent may be any one of rosemary, clove and sage.
As a preferable embodiment, the method for preparing the oral liquid comprises the steps of:
the antibacterial agent is prepared by fully dissolving tannic acid, a dew grass extract, an antioxidant, a sweetening agent and a bacteriostatic agent in purified water according to the weight parts of 12-45 parts of tannic acid, 12-30 parts of dew grass extract, 6-28 parts of an antioxidant, 6-30 parts of a sweetening agent, 6-25 parts of a bacteriostatic agent and 90-200 parts of purified water, adding the purified water until the tannin, the dew grass extract, the antioxidant, the sweetening agent and the bacteriostatic agent are completely dissolved, and subpackaging.
The invention also provides a tablet for preventing and/or treating organ fibrosis, which comprises the dew grass composition.
Preferably, the tablet comprises the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of herba Cymbopogonis Citrari extract, 9-30 parts of filler, 9-28 parts of binder and 2-25 parts of lubricant.
The anti-organ fibrosis tablet provided by the invention has reasonable compatibility of all components, and tannic acid can inhibit the expression of collagen-1 and smooth muscle alpha-actin (SMA) induced by TGF-beta and plays a role in resisting fibrosis; the main component of the herba Cymbopogonis Citrari is 20-hydroxyecdysterone (beta-ecdysone), which has the main effects of promoting cell growth, stimulating dermal cell division, and promoting protein synthesis for human body; the filler can increase the volume and weight, reduce the cost of the material and play a role in improving the performance of the material; the adhesive is capable of joining two separate materials together by virtue of its adhesive properties, such that it effectively adjusts viscosity; the lubricant can reduce the friction resistance of the friction pair and slow down the abrasion of the friction pair, so that the friction pair has good lubricating performance; the obtained anti-fibrosis tablet has obvious effects of resisting pulmonary fibrosis, hepatic fibrosis, heart fibrosis and skin fibrosis, is helpful for treatment and rehabilitation of patients, inhibits loss of organ functions, and protects health and life of people.
Preferably, the filler may be any one of starch, dextrin, and powdered sugar.
Preferably, the binder may be any one of gum arabic, methylcellulose, and hydroxypropylcellulose.
Preferably, the lubricant may be magnesium stearate or hydrogenated vegetable oil.
As a preferable mode of execution, the above-mentioned tablet preparation method comprises the steps of:
s1, weighing the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of herba Cymbopogonis Citrari extract, 9-30 parts of filler, 9-28 parts of binder and 2-25 parts of lubricant.
S2, uniformly mixing the tannic acid, the herba Cymbopogonis Citrari extract and the filler.
S3, adding an adhesive to prepare a soft material (the soft material can be agglomerated when being held by hands and can be scattered but not be powdery when being lightly pressed by fingers), and extruding and sieving the soft material by hands to obtain particles without strips, blocks or fine powder. In mass production, the soft material is passed through the mesh of the sieve by squeezing with a granulator roller (or a rubbing plate) to obtain granules.
S4, drying at 80 ℃ by adopting a wet particle drying method (drying in an electric heating oven and the like in small-scale preparation and drying in a steam drying room and the like in large-scale production). The dried granules are often agglomerated and adhered, and the granules are sieved and granulated, finally added with a lubricant and uniformly mixed to be tabletted. And (5) obtaining the product.
The invention also provides a spray containing the dew grass composition and used for preventing and/or treating organ fibrosis.
Preferably, the spray comprises the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of herba Cymbopogonis Citrari extract, 6-25 parts of cosolvent, 6-20 parts of antioxidant, 3-21 parts of bacteriostatic agent and 100-180 parts of purified water.
The anti-organ fibrosis external spray provided by the invention has reasonable compatibility of all components, wherein the tannic acid can inhibit the expression of TGF-beta induced collagen-1 and smooth muscle alpha-actin (SMA), and plays a role in anti-fibrosis; the main component of the herba Cymbopogonis Citrari is 20-hydroxyecdysterone (beta-ecdysone), which has the main effects of promoting cell growth, stimulating dermal cell division, and promoting protein synthesis for human body; after the cosolvent and the drug form a complex, the solubility of the drug can be increased by times or even tens of times; the antioxidant can prevent the oxidation of the original medicine, so that the stability of the antioxidant is good; the bacteriostatic agent can effectively prevent the spray from deteriorating; the purified water is used as a solvent to play a dissolving role; the obtained anti-fibrosis spray has obvious effect of resisting skin fibrosis, is beneficial to the treatment and rehabilitation of patients, improves the life quality, and can be used as an effective method for preventing normal people from daily protection and fibrotic diseases.
Preferably, the cosolvent may be any one of acetamide, sodium salicylate, and sodium benzoate.
Preferably, the antioxidant may be any one of sodium sulfite, vitamin C, and vitamin E.
Preferably, the bacteriostatic agent can be any one of clove, rosemary and sage.
In a preferred embodiment, the method for preparing the spray comprises the following steps:
s1, weighing the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of herba Cymbopogonis Citrari extract, 6-25 parts of cosolvent, 6-20 parts of antioxidant, 3-21 parts of bacteriostatic agent and 100-180 parts of purified water.
S2, mixing the tannic acid, the herba Cymbopogonis Citrari extract, the cosolvent, the antioxidant and the bacteriostatic agent, adding purified water until the mixture is completely dissolved, and subpackaging the mixture in a spray bottle to obtain the pesticide.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides application of a herba dewettiae extract in preparing an anti-skin fibrosis medicament, and the first research of the invention shows that the herba dewettiae extract has an obvious effect of treating skin fibrosis, belongs to a natural plant-derived medicament, has no side effect, is suitable for oral administration and external use, has good application value in the aspect of developing anti-skin fibrosis medicinal products, and provides a novel and natural medicament source for treating skin fibrosis diseases.
In addition, after the dewy herb extract is compounded with the tannin, the skin fibrosis resistance of the dewy herb extract is obviously improved, the synergistic effect on the skin fibrosis resistance is realized, the generated comprehensive skin fibrosis resistance is completely different, the raw material compatibility is precise and appropriate, no side effect is generated, the dewy herb extract is suitable for oral administration and external use, and the dewy herb extract has good application value in the aspect of developing anti-fibrosis medicine products.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.
Example 1 preparation of aqueous extract of Cyanotis arachnoidea
The preparation of the water extract of the dewed grass comprises the following steps:
s1, drying and crushing the dewed grass, weighing 50g of dewed grass powder, adding 500mL of water, and performing reflux extraction for 2 hours.
S2, filtering, adding 500mL of water into filter residues, performing reflux extraction for 2h, filtering, and combining the two filtrates.
S3.80 ℃, concentrating and drying to obtain the aqueous extract of the dewy grass.
Example 2 preparation of an alcohol extract of Cyanotis arachnoidea
The preparation method of the alcohol extract of the herba oenoprasis comprises the following steps:
drying and crushing the dewed grass, weighing 50g of dewed grass powder, adding 500mL of ethanol solution with the concentration of 60 vol%, extracting for 1h under reflux, filtering, concentrating and drying the filtrate at 80 ℃ to obtain the dewed grass alcohol extract.
EXAMPLE 3A dew grass composition for the prevention and/or treatment of fibrosis
The present practice provides a range of compositions for the prevention and/or treatment of fibrosis:
composition 1: 12 parts of tannic acid, 12 parts of the herba Cymbopogonis Citrari extract in example 1;
composition 2: 19 parts of tannic acid, 15 parts of the extract of the dewy grass in example 2;
composition 3: 25 parts of tannin, 20 parts of the extract of the dewy grass in example 1;
composition 4: 36 parts of tannic acid, 24 parts of the herba Cymbopogonis Citrari extract in example 1;
composition 5: 45 parts of tannic acid and 30 parts of the herba Cymbopogonis Citrari extract in example 1.
Comparative example 1 composition
This comparative example provides a series of compositions:
composition 6: 3 parts of tannin, 9 parts of the extract of the dewy grass in example 1;
composition 7: 4 parts of tannin, 11 parts of the extract of the dewy grass in example 2;
composition 8: 12 parts of tannic acid and 34 parts of the herba Cymbopogonis Citrari extract in example 1.
Experimental example 1 acute toxicity test of compositions 1 to 5
(1) Experimental methods
The test was carried out by the maximum tolerance to acute toxicity method. Setting six groups of blank groups and compositions 1-5 (1.1g/mL), selecting 60 quarantine qualified SPF grade ICR mice, each half of male and female, grouping according to sex and body weight by adopting a section random method, wherein each group comprises 10 mice, fasting is carried out for more than 12h before administration, the blank groups do not administer drugs, and the compositions 1-5 are 0.5mL/10g during experimentBody weightPerforming disposable intragastric administration. On the day of administration, particularly, the poisoning performance and characteristics, the occurrence and recovery time of toxic reaction, the death condition and the like of each group of animals are closely observed and recorded within 0-4 h after administration. The observation was then carried out 2 times a day, i.e., 1 time each in the morning and afternoon, for 14 consecutive days. The body mass of the mice was weighed before and on the 7 th and 14 th days after the administration, respectively, using an electronic balance. All mice were anesthetized and sacrificed by pentobarbital sodium intraperitoneal injection after the experiment was finished, and the general dissection was performed, the position, size, color, adhesion and other conditions of the organs were visually observed, and abnormal changes of the texture, effusion, tumor and the like of the surfaces and sections of the organs were examined. In thatGross dissection should be performed if there are animals (including moribund animals) that are not scheduled to die during the test.
(2) Results of the experiment
1. General activity status of mice
Before administration and during 14 days of observation, mice in the blank group and the composition 1-5 six groups have normal autonomous activities and have no abnormal or dead phenomena.
2. Toxic symptoms and death in mice
No abnormality and death of the mice were observed during the whole experimental period.
3. Changes in physical constitution
The weight of the composition is weighed before administration, at the 7 th day and at the 14 th day after administration respectively, compared with the blank group, the results show that the weight change of five groups of mice of the composition 1-5 has no significant difference, and the body mass growth is within a normal range, which shows that the body mass growth of ICR mice is not obviously influenced by oral drench of the composition 1-5, and the concrete contents are shown in Table 1.
TABLE 1 Effect of compositions 1-5 on mouse body weight
Figure BDA0003303074230000081
4. Gross anatomical examination results
No obvious abnormal condition was observed on the surface and section of each organ in the two groups of mice.
Experimental example 2 Long-term toxicity test of compositions 1 to 5
(1) Experimental methods
Take composition 2 as an example. 80 rats were randomly divided into 4 groups of 20 rats, a control group, a composition 2 high dose group (760mg/kg), a medium dose group (380mg/kg), and a low dose group (190 mg/kg). The administration was 1 time per day, and 10 rats were sacrificed separately for each fraction after 3 months of administration and 2 weeks of withdrawal (recovery period) for index testing. Compositions 1, 3, 4 and 5 were prepared in the same manner as described above. The following experiment was performed thereafter.
The skin, mucous membranes, secretions and excretions of the rats were observed daily for normality, while the general manifestations, toxic manifestations and death of the rats were recorded. Rats were recorded every 7d for water intake, feed intake and body weight. Collecting blood and performing cesarean examination on a certain number of rats on both day 30 and day 45, and detecting hematology and blood biochemical indexes, wherein the hematology indexes comprise: differential counting of White Blood Cells (WBC) and Neutrophils (NE), Eosinophils (EO), Basophils (BA), Lymphocytes (LY), Monocytes (MO), Red Blood Cells (RBC) and hemoglobin (hb), etc.; the biochemical indexes of the blood comprise: alanine aminotransferase (alanine aminotransferase, ALT), aspartate aminotransferase (aspartate aminotransferase, AST), Albumin (ALB), urea nitrogen (BUN), and Triglyceride (TG), and the like. The weight of the major organs (heart, liver, spleen, lung, kidney, gastrointestinal and reproductive organs) of each group of rats was weighed and the organ coefficient was calculated, and pathological examination was performed on the control group and the high dose group of rats.
(2) Results of the experiment
1. Weight change
During the test period, rats in all groups had good mental status, normal drinking water and appetite, normal defecation and urination, and smooth and clean hair color. The weight growth trend of the test group and the blank group has no significant difference.
2. Blood routine and blood biochemical detection
After 3 months of administration, the SD rats with high, medium and low doses of the compositions 1-5 have no obvious difference in blood routine and blood biochemical detection compared with a control group.
3. Histopathological changes
The organ coefficients of each group on the 30 th day and the 45 th day have no difference significance. No obvious abnormality is found on the surfaces of organs such as heart, liver, spleen, lung, kidney, stomach, small intestine, testis and ovary of rats in each group of eye observation; no obvious lesions were observed in each tissue section of the test and blank groups.
Experimental example 3 anti-pulmonary fibrosis experiment
First, cell experiment
1. Experimental methods
The experiment is to perform anti-pulmonary fibrosis experiment on the compositions 1-8 prepared in the example 3 and the comparative example 1, the tannic acid and the dew grass extract in the example 1, and the specific method comprises the following steps:
(1) cell culture
Culturing lung fibroblast in high-glucose DMEM medium containing 10% fetal calf serum, 1% penicillin and streptomycin double antibody and glucose concentration of 4.5g/L, and placing the cell at 37 deg.C and containing 5% CO2Incubator, 2d change once cell culture fluid. When the cell density is 80-90%, the cells are digested by pancreatin and resuspended for passage.
(2) Cell grouping process
After observing that the cells had good growth status, the cells were digested, resuspended, and fibroblasts were seeded on a black 96-well plate at a density of 3000 cells per well. 24h after cell inoculation, 10ng/mL TGF-beta is added into each hole1And establishing a cell pulmonary fibrosis model. The culture was continued for 72 h.
Administration group: after the cells adhered, tannic acid, dewet grass extract, composition 1, composition 2, composition 3, composition 4, composition 5, composition 6, composition 7, composition 8 were administered, respectively. The administration concentrations were set to 1.56. mu.g/mL at low concentration, 6.25. mu.g/mL at medium concentration and 25. mu.g/mL at high concentration, and after administration, the 96-well plate was placed in a cell incubator in the dark and the culture was continued for 24 hours.
Control group: compared with the administration group, the administration is not carried out.
Blank control group: compared with the administration group, the difference is that TGF-beta is not added1Nor administered.
To avoid systematic errors. Each set was designed with 6 replicate wells, and each set of experiments was replicated 3 times.
(3) CCK8 detection
Cell proliferation rate was measured using CCK 8. According to the kit specification operation, 10 microliter of CCK8 reagent with the concentration of 10% is added into each well, and after 2 hours, the OD value of the cells is detected by adopting a microplate reader with the wavelength of 450 nm.
(4) Cell survival rate
Cell viability was calculated as OD values of cells detected in each group. The cell survival rate (administration group OD value-blank control group OD value)/(control group OD value-blank control group OD value) × 100%
2. Results of the experiment
The results of lung fibroblast survival are shown in table 2 below.
TABLE 2 survival rate of abnormally proliferating lung fibroblasts (%)
Figure BDA0003303074230000101
As can be seen from table 2, in terms of inhibiting the survival rate of abnormally-proliferating lung fibroblasts, the survival rate of the abnormally-proliferating lung fibroblasts is lower when the compositions 1 to 5 are compared with the compositions 6 to 8 and the tannin or the extract of the herba eriocauli is used alone, which indicates that the compositions and the extract have the effect of synergistically reducing the survival rate of the lung fibroblasts when used together in a proper ratio.
Second, animal experiment
1. Experimental methods
The experiment is to perform anti-pulmonary fibrosis experiment on the compositions 1-8 prepared in the example 3 and the comparative example 1, the tannic acid and the dew grass extract in the example 1.
The specific method comprises the following steps:
(1) animal grouping:
SPF grade Wistar rats, randomly divided into 12 groups of 30 rats by body weight. Respectively, sham surgery group, model group, composition 1 group, composition 2 group, composition 3 group, composition 4 group, composition 5 group, composition 6 group, composition 7 group, composition 8 group, tannic acid group, and herba Eragrostidis Nigrae extract group.
(2) Preparing a model:
the experimental animals are bred regularly after being purchased and are adaptive to the environment for 7 d. In the experiment, 3mL/kg of chloral hydrate of 10% is injected into the abdominal cavity to anaesthetize the animal, so that the animal is fixed on a rat board in a supine position, and the neck is sheared. The skin is sterilized conventionally and an incision is made in the neck of about 0.5-1cm in length under sterile conditions. And stripping the exposed trachea layer by layer, and then lifting the head end of the rat board to form an angle of 30-35 degrees with the experiment table board. The method comprises the steps of penetrating a trachea with a 1mL syringe under direct vision, enabling the syringe to be as close to the bifurcation of the trachea as possible, keeping the direction of a needle head consistent with the direction of an airway, quickly injecting and injecting bleomycin hydrochloride (BLOCIN) and 0.2mL of physiological saline solution (15 mg of each bleOCIN, the concentration of the prepared physiological saline solution is 5mg/mL before use, and administering according to the weight of 5 mg/kg), immediately pulling out the needle head, erecting a rat plate, keeping the rat in an upright position, and rotating the rat plate back and forth for 3min to enable liquid medicine to reach the lungs on two sides as far as possible and to be uniformly distributed. Suturing skin, continuously injecting penicillin solution into abdominal cavity for 3 days to prevent wound infection, and placing into cage for conventional breeding after animal naturally revives. The operation method of the rat operation in the sham operation group is the same as that in other groups, except that the same amount of physiological saline is injected into the trachea after the operation to replace the bleomycin hydrochloride.
(3) Administration of drugs
Each group of rats was administered 1 intragastrically daily from day 2 after intratracheal injection of bleomycin hydrochloride for modeling. 190 mg/(kg. d) of the composition 1-8, tannic acid alone, and herba Cymbopogonis Citrari extract were administered continuously for 28 d.
(4) Specimen collection
Respectively at 7 days, 14 days and 28 days after the model is made, 1h after the drug administration, 10 animals are randomly selected from each group, right lung tissues are dissected out after the sacrifice, 9mL of physiological saline is added according to each gram of lung tissues to prepare 10% tissue homogenate, the homogenate is centrifuged at 3000r/min for 10min, and supernatant is taken.
(5) Index detection method
Detecting the activity of the SOD in the lung tissue by spectrophotometry.
2. Results of the experiment
TABLE 3 pulmonary tissue SOD Activity in animal models of pulmonary fibrosis
Figure BDA0003303074230000121
As can be seen from Table 3, the results of this experiment indicate that the SOD activity in the lung tissues of three batches of animals in the model group is significantly lower than that in the sham operation group after the model is made. The SOD activity decreased with the increase of molding time. The results show that the function of the anti-free radical protection system in the lung tissue of the model animal is reduced, and the formation of pulmonary fibrosis is indirectly promoted. The SOD activity in the lung tissues of the animals of the composition 1-5 is obviously improved compared with that of a contemporary model group; the compositions 1-5 inhibit the progress of pulmonary fibrosis by increasing SOD activity in lung tissues during the process of excessive proliferation and large amount of collagen secretion of fibroblasts. The effect of the compositions 6-8 is better than that of the tannic acid and the extract of the herba dewettiae, but is worse than that of the compositions 1-5, which indicates that the tannic acid and the extract of the herba dewettiae can realize the effect of synergistically enhancing the SOD activity of lung tissues of the pulmonary fibrosis animal model by proper proportion.
EXAMPLE 4 anti-hepatic fibrosis test
First, cell experiment
1. Experimental methods
The experiment is to perform anti-hepatic fibrosis experiment on the compositions 1-8 prepared in the example 3 and the comparative example 1, the tannic acid and the extract of the dewy grass in the example 1.
The specific method comprises the following steps:
(1) cell culture
Culturing hepatic fibroblast in high-glucose DMEM medium containing 10% fetal calf serum, 1% penicillin and streptomycin double antibody and glucose concentration of 4.5g/L, and placing the cells at 37 deg.C containing 5% CO2Incubator, 2d change once cell culture fluid. When the cell density is 80-90%, the cells are digested by pancreatin and resuspended for passage.
(2) Cell grouping process
After observing that the cells had good growth status, the cells were digested, resuspended, and fibroblasts were seeded on a black 96-well plate at a density of 3000 cells per well. 24h after cell inoculation, 10ng/mL TGF-beta is added into each hole1And establishing a cellular hepatic fibrosis model. After 72h of incubation.
Administration group: after the cells adhered, tannic acid, dewet grass extract, composition 1, composition 2, composition 3, composition 4, composition 5, composition 6, composition 7, composition 8 were administered, respectively. The administration concentrations were set to 1.56. mu.g/mL at low concentration, 6.25. mu.g/mL at medium concentration and 25. mu.g/mL at high concentration, and after administration, the 96-well plate was placed in a cell incubator in the dark and the culture was continued for 24 hours.
Control group: compared with the administration group, the administration is not carried out.
Blank control group: compared with the administration group, the difference is that TGF-beta is not added1Nor administered.
To avoid systematic errors. Each set was designed with 6 replicate wells, and each set of experiments was replicated 3 times.
(3) CCK8 detection
Cell proliferation rate was measured using CCK 8. According to the kit specification operation, 10 microliter of CCK8 reagent with the concentration of 10% is added into each well, and after 2 hours, the OD value of the cells is detected by adopting a microplate reader with the wavelength of 450 nm.
(4) Cell survival rate
Cell viability was calculated as OD values of cells detected in each group. The cell survival rate (administration group OD value-blank control group OD value)/(control group OD value-blank control group OD value) × 100%
2. Results of the experiment
The results of hepatic fibroblast survival are shown in table 4 below.
TABLE 4 survival rate of abnormally proliferating hepatic fibroblasts (%)
Figure BDA0003303074230000131
Figure BDA0003303074230000141
As can be seen from table 4, in terms of reducing the survival rate of the abnormally-proliferating hepatic fibroblasts, the compositions 1 to 5 have better effects than the compositions 6 to 7 when tannin or the extract of the herba aquaticae is used alone, and the survival rate of the abnormally-proliferating hepatic fibroblasts is low, which indicates that the compositions and the extracts have the effect of synergistically reducing the survival rate of the abnormally-proliferating hepatic fibroblasts when used together in a proper ratio.
Second, animal experiment
1. Experimental methods
The experiment is to perform anti-hepatic fibrosis experiment on the compositions 1-8 prepared in the example 3 and the comparative example 1, the tannic acid and the extract of the dewy grass in the example 1, and the specific method comprises the following steps:
(1) grouping animals
SPF grade mice were randomly divided into 12 groups of 30 mice per group by body weight. Respectively, sham surgery group, model group, composition 1 group, composition 2 group, composition 3 group, composition 4 group, composition 5 group, composition 6 group, composition 7 group, composition 8 group, tannic acid group, and herba Eragrostidis Nigrae extract group.
(2) Molding die
Using classical CCl4An induction method, establishing a mouse hepatic fibrosis model: mixing CCl4The injection is prepared into an oil solution with olive oil according to the volume ratio of 1: 1, and then the subcutaneous injection is carried out on mice, the injection volume is 3mL/kg, 2 times per week (interval of 3d), and the continuous injection is carried out for 6 weeks. The blank control group mice were injected with an equal volume of olive oil by the same method.
(3) Administration of drugs
Each group of mice was dosed 1 time daily by gavage from day 2 after modeling. 190 mg/(kg. d) of the compositions 1 to 8 were administered for 28 days continuously.
(4) Specimen collection
Respectively 1h after the drug administration on 7 th day, 14 th day and 28 th day after the model building, randomly selecting 10 animals in each group, dissecting and taking liver tissues after the death, adding 9mL of physiological saline into each gram of liver tissues to prepare 10% tissue homogenate, centrifuging at 3000r/min for 10min, and taking supernatant.
(5) The index detection method comprises the following steps: detecting the activity of the SOD in the liver tissue by adopting a spectrophotometry.
2. Results of the experiment
TABLE 5 hepatic fibrosis animal model hepatic tissue SOD Activity
Figure BDA0003303074230000151
As can be seen from Table 5, the results of this experiment indicate that the SOD activity in the liver tissues of three animals in the model group is significantly lower than that in the sham operation group after the model is made. The SOD activity decreased with the increase of molding time. The results show that the function of the anti-free radical protection system in the liver tissue of the model animal is reduced, and the formation of hepatic fibrosis is indirectly promoted. Compared with a contemporary model group, the SOD activity in the liver tissues of the animals of the compositions 1 to 5 is obviously improved, the effect is obviously better than that of the compositions 6 to 8 and the tannin or the extract of the herba Cymbopogonis Citrari is singly administered, which indicates that the compositions 1 to 5 inhibit the hepatic fibrosis process by increasing the SOD activity in the liver tissues in the process of excessive proliferation of fibroblasts and secretion of a large amount of collagen.
Experimental example 5 anti-dermal fibrosis experiment
First, cell experiment
1. Experimental methods
The experiment is to perform an anti-skin fibrosis experiment on the compositions 1-8 prepared in the example 3 and the comparative example 1, the tannic acid and the dew grass extract in the example 2, and the specific method comprises the following steps:
(1) cell culture
Culturing skin fibroblast in high-glucose DMEM medium containing 10% fetal calf serum, 1% penicillin and streptomycin double antibody and glucose concentration of 4.5g/L, and placing the cell at 37 deg.C and containing 5% CO2Incubator, 2d change once cell culture fluid. When the cell density is 80-90%, the cells are digested by pancreatin and resuspended for passage.
(2) Cell grouping process
After observing that the cells had good growth status, the cells were digested, resuspended, and skin fibroblasts were seeded at a density of 3000 cells per well in a black 96-well plate. 24h after cell inoculation, 10ng/mL TGF-beta is added into each hole1Establishing a cell skin fibrosis model, and continuously culturing for 24 h.
Administration group: after the cells adhered, tannic acid, dewet grass extract, composition 1, composition 2, composition 3, composition 4, composition 5, composition 6, composition 7, composition 8 were administered, respectively. The administration concentrations were set to 1.56. mu.g/mL at low concentration, 6.25. mu.g/mL at medium concentration and 25. mu.g/mL at high concentration, and after administration, the 96-well plate was placed in a cell incubator in the dark and the culture was continued for 24 hours.
Control group: compared with the administration group, the administration is not carried out.
Blank control group: compared with the administration group, the difference is that TGF-beta is not added1Nor administered.
To avoid systematic errors. Each set was designed with 6 replicate wells, and each set of experiments was replicated 3 times.
(3) CCK8 detection
According to the kit specification operation, 10 microliter of CCK8 reagent with the concentration of 10% is added into each well, and after 2 hours, the OD value of the cells is detected by adopting a microplate reader with the wavelength of 450 nm.
(4) Cell survival rate
Cell viability was calculated as OD values of cells detected in each group. The cell survival rate (administration group OD value-blank control group OD value)/(control group OD value-blank control group OD value) × 100%
2. Results of the experiment
The results of the skin fibroblast survival rate are shown in table 6 below.
TABLE 6 abnormal proliferation of skin fibroblast survival rate (%)
Figure BDA0003303074230000161
Figure BDA0003303074230000171
As can be seen from table 6, compositions 1 to 5 were more effective than compositions 6 to 8 in reducing the survival rate of the skin fibroblasts abnormally proliferating, and the survival rate of the skin fibroblasts abnormally proliferating was lower than that of the skin fibroblasts abnormally proliferating alone, indicating that the combination of tannic acid and the extract of herba dewoniae has the synergistic effect of reducing the survival rate of the skin fibroblasts abnormally proliferating when used in a suitable ratio.
Second, animal experiment
1. Experimental methods
The experiment is to perform an anti-skin fibrosis experiment on the compositions 1-8 prepared in example 3 and comparative example 1, tannic acid and the extract of the dewy grass in example 2.
The specific method comprises the following steps:
(1) grouping animals
SPF grade 6 week old male BALB/c mice were randomly divided into 12 groups of 30 mice per group by body weight. The group of sham surgery, the group of simple Phosphate Buffered Saline (PBS), the group of composition 1, the group of composition 2, the group of composition 3, the group of composition 4, the group of composition 5, the group of composition 6, the group of composition 7, the group of composition 8, the group of tannic acid and the group of the extract of the dewweed were respectively.
(2) Preparation of the model
The placebo mice were not treated at all; the back skin of mice in the PBS only group and the composition group were injected subcutaneously with 100. mu.L of PBS and bleomycin (1mg/mL), respectively.
(3) Administration of drugs
Each group of mice was skin dosed 2 times daily from day 2 after modeling with bleomycin. 190 mg/(kg. d) of the compositions 1 to 8 were administered for 28 days continuously.
(4) Specimen collection
Respectively 1h after the drug administration on 7 th day, 14 th day and 28 th day after the model building, randomly selecting 10 animals in each group, observing the change of the back skin of the mouse by naked eyes, killing the mouse after the observation, taking back skin tissue, adding 9mL of physiological saline into each gram of the skin tissue to prepare 10% tissue homogenate, centrifuging at 3000r/min for 10min, and taking supernatant.
(5) The index detection method comprises the following steps: detecting the activity of SOD in skin tissue by spectrophotometry.
2. Results of the experiment
TABLE 7 skin tissue SOD Activity in animal models of skin fibrosis
Figure BDA0003303074230000181
As can be seen from Table 7, the results of this experiment indicate that the SOD activity in the skin tissues of three batches of animals in the model group after molding is significantly lower than that in the sham operation group. The SOD activity decreased with the increase of molding time. The results show that the function of the anti-free radical protection system in the skin tissue of the model animal is reduced, and the formation of skin fibrosis is indirectly promoted. The SOD activity in the animal skin tissues of the administration compositions 1-5 is obviously increased compared with that of a contemporary model group; the compositions 1-5 inhibit the process of skin fibrosis by increasing SOD activity in skin tissues during the process of excessive proliferation and large amount of collagen secretion of fibroblasts. The effect of the compositions 6-8 is better than that of the tannic acid and the extract of the herba dewettiae, but is worse than that of the compositions 1-5, which shows that the tannic acid and the extract of the herba dewettiae can realize the effect of synergistically enhancing the SOD activity of the skin tissue of the skin fibrosis animal model by proper proportion.
Example 4 oral liquid 1 for prevention and/or treatment of fibrosis
1. The embodiment provides an oral liquid which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of herba Cymbopogonis Citrari extract, 12 parts of vitamin C, 9 parts of aspartame, 10 parts of rosemary and 200 parts of purified water.
2. Preparation method
(1) Drying and crushing the dewed grass, weighing 50g of dewed grass powder, adding 500mL of water, and performing reflux extraction for 2 hours; filtering, adding 500mL of water into filter residue, extracting for 2h under reflux, filtering, and combining the two filtrates; concentrating and drying at 80 ℃ to obtain the aqueous extract of the dewy grass.
(2) Mixing tannic acid, herba Eragrostidis extract, vitamin C, aspartame and herba Rosmarini officinalis, adding purified water, dissolving completely, and packaging to obtain oral liquid.
EXAMPLE 5 oral liquid 2 for prevention and/or treatment of fibrosis
1. The embodiment provides an oral liquid which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of herba Cymbopogonis Citrari extract, 18 parts of vitamin E, 8 parts of stevioside, 11 parts of clove and 150 parts of purified water.
2. The preparation method of the aqueous extract of the dewy grass is the same as that of the example 4;
mixing tannic acid, herba Eragrostidis extract, vitamin E, steviosin, and flos Caryophylli in the above weight parts, adding purified water to dissolve completely, and packaging to obtain oral liquid.
Example 6 oral liquid 3 for prevention and/or treatment of fibrosis
1. The embodiment provides an oral liquid which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of a herba Cymbopogonis Citrari extract, 15 parts of sodium sulfite, 6 parts of aspartame, 12 parts of sage and 90 parts of purified water.
2. The preparation method of the aqueous extract of the dewy grass is the same as that of the example 4;
mixing tannic acid, herba Eragrostidis extract, sodium sulfite, aspartame and herba Salvia officinalis, adding purified water, dissolving completely, and packaging to obtain oral liquid.
Example 7 tablet for prevention and/or treatment of fibrosis 1
1. The embodiment provides a tablet which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of herba Cymbopogonis Citrari extract, 21 parts of starch, 13 parts of Arabic gum and 8 parts of magnesium stearate.
2. Preparation method
(1) The same procedure as in example 1 was repeated.
(2) Mixing tannin, herba Eragrostidis Pendulae extract, and starch uniformly.
(3) Adding acacia to make into soft mass (with a degree of forming into block when held by hand and breaking into powder when pressed by finger), squeezing with hand, and sieving to obtain granule without strip, block or fine powder. In mass production, the soft material is passed through the mesh of the sieve by squeezing with a granulator roller (or a rubbing plate) to obtain granules.
(4) Drying at 80 deg.C by wet granule drying method (drying in electric oven for small-scale preparation, or drying in steam oven for mass production). The dried granules are often conglobated and adhered, and sieving and finishing are needed, and finally magnesium stearate is added, and tabletting can be carried out after uniform mixing. And (5) obtaining the product.
EXAMPLE 8 tablet 2 for the prevention and/or treatment of fibrosis
1. The embodiment provides a tablet which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of herba Cymbopogonis Citrari extract, 22 parts of dextrin, 15 parts of methyl cellulose and 7 parts of magnesium stearate.
2. Preparation method
(1) The same procedure as in example 1 was repeated.
(2) Mixing tannin, herba Eragrostidis Pendulae extract, and dextrin.
(3) Adding methylcellulose to make into soft material (with a degree of conglomeration when held by hand and spalling but not powdering when lightly pressed by fingers), squeezing with hand, and sieving to obtain granule without strip, block and fine powder. In mass production, the soft material is passed through the mesh of the sieve by squeezing with a granulator roller (or a rubbing plate) to obtain granules.
(4) Drying at 80 deg.C by wet granule drying method (drying in electric oven for small-scale preparation, or drying in steam oven for mass production). The dried granules are often conglobated and adhered, and sieving and finishing are needed, and finally magnesium stearate is added, and tabletting can be carried out after uniform mixing. And (5) obtaining the product.
Example 9 tablet 3 for the prevention and/or treatment of fibrosis
1. The embodiment provides a tablet which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of herba Cymbopogonis Citrari extract, 23 parts of powdered sugar, 16 parts of hydroxypropyl cellulose and 10 parts of hydrogenated vegetable oil.
2. Preparation method
(1) The same procedure as in example 1 was repeated.
(2) Mixing tannin, herba Eragrostidis Pendulae extract, and sugar powder.
(3) Adding hydroxypropyl cellulose to make into soft material (with a degree of forming into mass when held by hand and breaking into powder when pressed by finger), squeezing with hand, and sieving to obtain granule without strip, block and fine powder. In mass production, the soft material is passed through the mesh of the sieve by squeezing with a granulator roller (or a rubbing plate) to obtain granules.
(4) Drying at 80 deg.C by wet granule drying method (drying in electric oven for small-scale preparation, or drying in steam oven for mass production). The dried granules are often conglobated and adhered, and sieving and grading are needed, and finally hydrogenated vegetable oil is added, and tabletting can be carried out after uniform mixing. And (5) obtaining the product.
Example 10 spray for prevention and/or treatment of fibrosis 1
1. The embodiment provides a spray which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of herba Cymbopogonis Citrari extract, 13 parts of acetamide, 13 parts of sodium sulfite, 11 parts of clove and 180 parts of purified water.
2. Preparation method
(1) The same procedure as in example 2 was repeated to obtain a extract of Cyanotis arachnoidea;
(2) mixing tannic acid, herba Eragrostidis extract, acetamide, sodium sulfite and flos Caryophylli in the above weight parts, adding purified water to dissolve completely, and packaging into spray bottle.
Example 11 spray for prevention and/or treatment of fibrosis 2
1. The embodiment provides a spray which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of herba Cymbopogonis Citrari extract, 15 parts of sodium salicylate, 21 parts of vitamin C, 12 parts of rosemary and 150 parts of purified water.
2. Preparation method
(1) The same procedure as in example 2 was repeated to obtain a extract of Cyanotis arachnoidea;
(2) mixing tannic acid, herba Cymbopogonis Citrari extract, sodium salicylate, vitamin C, and herba Rosmarini officinalis, adding purified water to dissolve completely, and packaging into spray bottle.
Example 12 spray for prevention and/or treatment of fibrosis 3
1. The embodiment provides a spray which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of herba Cymbopogonis Citrari extract, 8 parts of sodium benzoate, 14 parts of vitamin E, 17 parts of herba Salvia officinalis and 100 parts of purified water.
2. Preparation method
(1) The same procedure as in example 2 was repeated to obtain a extract of Cyanotis arachnoidea;
(2) mixing tannic acid, herba Cymbopogonis Citrari extract, sodium benzoate, vitamin E, and herba Salvia officinalis, adding purified water, dissolving completely, and packaging into spray bottle.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (9)

1. Application of herba Cymbopogonis Citrari extract in preparing medicine for preventing and/or treating skin fibrosis is provided.
2. The use of claim 1, wherein the skin fibrosis is bleomycin-induced skin fibrosis.
3. The use of claim 1, wherein the extract of the aquatic weed is an aqueous extract of the aquatic weed and/or an alcoholic extract of the aquatic weed.
4. An oral liquid for preventing and/or treating skin fibrosis, which comprises the extract of the dewy grass of claim 1.
5. The oral liquid according to claim 4, which comprises the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of herba Cymbopogonis Citrari extract, 6-28 parts of antioxidant, 6-30 parts of sweetener, 6-25 parts of bacteriostatic agent and 90-200 parts of purified water.
6. A tablet for preventing and/or treating skin fibrosis, which contains the extract of the dew grass of claim 1.
7. The tablet according to claim 6, comprising the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of herba Cymbopogonis Citrari extract, 9-30 parts of filler, 9-28 parts of binder and 2-25 parts of lubricant.
8. A spray for preventing and/or treating skin fibrosis, which comprises the dew grass extract of claim 1.
9. The spray according to claim 8, which is characterized by comprising the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of herba Cymbopogonis Citrari extract, 6-25 parts of cosolvent, 6-20 parts of antioxidant, 3-21 parts of bacteriostatic agent and 100-180 parts of purified water.
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