CN113876787B - Herba Lupuli extract, its application and preparation - Google Patents

Herba Lupuli extract, its application and preparation Download PDF

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CN113876787B
CN113876787B CN202111196258.XA CN202111196258A CN113876787B CN 113876787 B CN113876787 B CN 113876787B CN 202111196258 A CN202111196258 A CN 202111196258A CN 113876787 B CN113876787 B CN 113876787B
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parts
extract
fibrosis
composition
tannic acid
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CN113876787A (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 relates to a composition for preventing and/or treating organ fibrosis, which is applied for patent application No. 2021101975884, applied for patent application No. 2021, 02 and 22, and the application name of the composition and the application and preparation of the composition. The application provides application of a herba lycopodii extract in preparing a medicine for preventing and/or treating skin fibrosis, and researches show that the herba lycopodii extract has obvious effect of treating skin fibrosis, belongs to a natural plant-derived medicine, has no side effect, is suitable for oral administration and external use, has good application value in developing a medicine product for resisting skin fibrosis, and provides a novel and natural medicine source for treating skin fibrosis diseases.

Description

Herba Lupuli extract, its application and preparation
The application relates to a composition for preventing and/or treating organ fibrosis, which is applied for patent application No. 2021101975884, applied for patent application No. 2021, 02 and 22, and the application name of the composition and the application and preparation of the composition.
Technical Field
The application relates to the technical field of medicines, in particular to application of a dew grass extract in preparing a medicine for resisting skin fibrosis and a preparation thereof.
Background
Any cause can cause tissue cell damage, can cause tissue cells to generate denaturation, necrosis and inflammatory reaction, if the damage is small, normal parenchymal cells around the damaged cells can generate proliferation repair, and the repair can completely restore normal structure and function. However, if the injury is large or repeated beyond the regenerative capacity of parenchymal cells surrounding the injury, the interstitial fibrous connective tissue (extracellular matrix) will proliferate in large amounts to repair the defective tissue, i.e., a pathological change in fibrosis (fibrosis) occurs. Fibrosis is thus essentially a repair reaction of tissue after it has been damaged to preserve the relative integrity of the tissue organ. The proliferated fibrous connective tissue, although repairing the defect, does not possess the structure and function of the original organ parenchymal cells. Fibrosis can occur in various organs such as lung, liver, kidney, peritoneum, blood vessels, pancreas, skin, etc., and the major pathological changes are fibrous connective tissue increase in organ tissues, parenchymal cytopenia, and continued progression can lead to destruction and hypofunction of organ structures, or even failure, directly leading to loss of organ function, severely threatening human health and life. Statistics on the united states show that approximately 45% of patients deaths from various diseases in this country can be attributed to fibroproliferative disease. In addition, there is also a great deal of pulmonary fibrosis in the development of diseases associated with respiratory viral infection, such as severe pulmonary fibrosis caused by severe infection with the novel coronavirus SARS-CoV-2.
Patent publication No. CN107019697A discloses the use of Nidamib in the treatment of fibrotic diseases, and in addition, studies have also shown that tannic acid is specific to CCl 4 The induced liver fibrosis mice have remarkable liver protection effect and can play a certain role in resisting liver fibrosis. However, on the one hand, application is limited because fibrosis occurs in many organs of the body, except liver fibrosis; on the other hand, in order to ensure the drug effect, the dosage of tannic acid is generally larger, the cost is higher, and certain toxic and side effects are unavoidable. Thus, it is a current research focus to seek new drug regimens with reduced costs and improved anti-organ fibrosis effects.
Disclosure of Invention
Aiming at the limitation of the existing anti-skin fibrosis medicines, the application aims to provide the application of the dew grass extract in preparing the medicines for preventing and/or treating the skin fibrosis, and researches find that the dew grass extract has obvious effect of treating the skin fibrosis, belongs to natural plant source medicines, has no side effect, is suitable for oral administration and external use, has good application value in the aspect of developing anti-skin fibrosis medicine products, and provides a high-efficiency and safe medicine source for treating the skin fibrosis diseases.
Another object of the present application is to provide an oral liquid for preventing and/or treating skin fibrosis, comprising a comfrey extract.
It is another object of the present application to provide a tablet for preventing and/or treating skin fibrosis, which comprises a dew grass extract.
It is another object of the present application to provide a spray for preventing and/or treating skin fibrosis, which comprises a dew grass extract.
The above object of the present application is achieved by the following technical scheme:
the present application firstly provides a dew grass composition for preventing and/or treating fibrosis, comprising tannic acid and/or a dew grass extract; the herba Avenae Fatuae extract is herba Avenae Fatuae water extract and/or herba Avenae Fatuae alcohol extract.
In the dew grass composition for preventing and/or treating fibrosis, tannic acid (tannic acid) is a secondary metabolite of some plants, is also called tannic acid in the medical science, is a natural plant polyphenol widely existing in gallnut plants, contains a large amount of hydroxyl and carbonyl, is a soluble polyhydric phenol compound, and has biological functions of diarrhea resistance, bacteriostasis, oxidation resistance, cancer resistance, virus resistance, fibrosis resistance and the like.
The extract of herba Cynomorii is the product extracted from Cyanotis arachnoides C.B.Clarke, which is a Commelinaceae plant, and contains 20-hydroxyecdysterone (beta-ecdysone) as main ingredient, and has effects of promoting cell growth, stimulating dermis cell division, promoting protein synthesis, dispelling pathogenic wind, activating collaterals, promoting diuresis, relieving swelling, removing asthenic fever, dredging channels, relieving pain, etc., and can be used for treating rheumarthritis, limb numbness, etc., promoting production of skin peeling of silkworm, shrimp and crab, reducing cholesterol in human body, and lowering blood sugar concentration.
The research result of the application shows that the dew grass extract has obvious effect of treating skin fibrosis, and after tannic acid is compounded with the dew grass extract, the anti-fibrosis effect is obviously improved, and the compounded composition has obvious promotion and synergy on the aspects of treating pulmonary fibrosis, liver fibrosis, heart fibrosis and skin fibrosis.
As a preferred method, the preparation method of the aqueous extract of dew grass comprises the following steps:
s1, drying and crushing the dew grass, weighing 50g of dew grass powder, adding 500mL of water, and carrying out reflux extraction for 2h.
S2, filtering, adding 500mL of water into filter residues, extracting for 2 hours under reflux, filtering, and combining the two filtrates.
S3, concentrating and drying to obtain the water extract of the dew grass.
As a preferred method, the preparation method of the dew grass alcohol extract comprises the following steps:
drying and crushing the dew grass, weighing 50g of dew grass powder in 500mL of ethanol solution with the concentration of 60vol%, carrying out reflux extraction for 1h, filtering, concentrating and drying the filtrate to obtain the dew grass.
Preferably, the tannic acid and the dew grass extract comprise the following components in parts by weight: 12-45 parts of tannic acid and 12-30 parts of a dew grass extract.
More preferably, the tannic acid and the dew grass extract are in parts by weight: 19 parts of tannic acid and 15 parts of a dew grass extract.
The application also provides application of the dew grass composition in preparing a medicament for preventing and/or treating organ fibrosis.
Use of a subject dew grass composition in the manufacture of a medicament for preventing and/or treating organ fibrosis, including, but not limited to, administering to a patient an effective amount of a subject dew grass composition for the manufacture of a medicament for preventing or treating a fibrosis-induced condition, alleviating a symptom of a fibrosis-induced condition, or delaying the progression or onset of a fibrosis-induced condition.
The composition provided by the application has no toxic or side effect on organisms, and can be used for preparing health products or foods for daily administration so as to prevent organ fibrosis of specific people. Therefore, the application also claims the application of the dew grass composition in preparing health care products or foods for preventing organ fibrosis.
The dew grass composition disclosed by the application is suitable for daily administration of specific people when preparing health-care products or foods for preventing organ fibrosis, has the functions of regulating organism, preventing organ fibrosis, and does not generate any acute, subacute or chronic harm to human bodies.
The disclosed dew grass compositions are useful for veterinary treatment of pets, introduced species of animals and farm animals, including mammals, rodents, and the like, in addition to human treatment. Examples of other animals include horses, dogs, cats, and the like.
Preferably, the organ fibrosis comprises pulmonary fibrosis, liver fibrosis, heart fibrosis, skin fibrosis.
Preferably, the medicament is prepared by adding pharmaceutically acceptable auxiliary materials into the dew grass composition for preventing and/or treating organ fibrosis to prepare different dosage forms. The dosage forms can be oral liquid, tablets, spray and the like.
The application also provides an oral liquid containing the dew grass composition 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 a dew grass extract, 6-28 parts of an antioxidant, 6-30 parts of a sweetener, 6-25 parts of a bacteriostatic agent and 90-200 parts of purified water.
The components of the anti-organ fibrosis oral liquid provided by the application are reasonably compatible, wherein tannic acid can inhibit the expression of TGF-beta induced collagen-1 and smooth muscle alpha-actin (SMA) to play an anti-fibrosis role; the main component of the herba lolii officinalis is 20-hydroxyecdysterone (beta-ecdysone), which has the main effects of promoting cell growth, stimulating dermis cell division and promoting protein synthesis for human body; the antioxidant can prevent the oxidation of the raw medicine, so that the stability of the antioxidant is good; the sweetener can improve taste sensation of a user, so that the sweetener has good adaptability; the bacteriostat can effectively prevent the oral liquid from deteriorating; purified water acts as a solvent for dissolution. The obtained anti-fibrosis oral liquid has remarkable 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 can be any one of rosemary, clove and sage.
As a preferable implementation manner, the preparation method of the oral liquid comprises the following steps:
12-45 parts of tannic acid, 12-30 parts of a dew grass extract, 6-28 parts of an antioxidant, 6-30 parts of a sweetener, 6-25 parts of a bacteriostat and 90-200 parts of purified water, wherein the tannic acid, the dew grass extract, the antioxidant, the sweetener and the bacteriostat are fully dissolved in the purified water according to the parts by weight, the purified water is added until the tannic acid, the dew grass extract, the antioxidant, the sweetener and the bacteriostat are fully dissolved, and the beverage is prepared by subpackaging.
The application 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 a dew grass extract, 9-30 parts of a filling agent, 9-28 parts of an adhesive and 2-25 parts of a lubricant.
The components of the anti-organ fibrosis tablet provided by the application are reasonably compatible, and tannic acid can inhibit TGF-beta induced collagen-1 and smooth muscle alpha-actin (SMA) expression, so as to play an anti-fibrosis role; the main component of the herba lolii officinalis is 20-hydroxyecdysterone (beta-ecdysone), which has the main effects of promoting cell growth, stimulating dermis cell division and promoting protein synthesis for human body; the filler can increase capacity and weight, reduce material cost and improve material performance; the adhesive is capable of joining together two separate materials by virtue of its tackiness such that it effectively adjusts the viscosity; the lubricant can reduce the friction resistance of the friction pair and slow down the abrasion of the friction pair, so that the lubricant has good lubricating performance; the obtained anti-fibrosis tablet has remarkable effects of resisting pulmonary fibrosis, liver fibrosis, heart fibrosis and skin fibrosis, is beneficial to 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 acacia, methylcellulose, hydroxypropyl cellulose.
Preferably, the lubricant may be magnesium stearate or hydrogenated vegetable oil.
As a preferred embodiment, the preparation method of the tablet comprises the following steps:
s1, weighing the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of a dew grass extract, 9-30 parts of a filling agent, 9-28 parts of an adhesive and 2-25 parts of a lubricant.
S2, mixing tannic acid, the dew grass extract and the filling agent uniformly.
S3, adding an adhesive to prepare a soft material (in the form of a block which can be held by hand and can be broken off without being powdery when the fingers are pressed lightly), and squeezing and sieving by hands, wherein the obtained granules are free of long strips, blocks and fine powder. During mass production, the soft material is extruded by a roller (or a scraper) of a granulator to pass through sieve holes so as to prepare particles.
S4, drying at 80 ℃ by adopting a wet grain drying method (drying by using an electric heating oven and the like in the case of small-volume preparation and drying by using a steam drying room and the like in the case of mass production). The dried granules are often agglomerated and adhered, sieving and granulating are needed, finally, a lubricant is added, and tabletting can be carried out after uniform mixing. Obtaining the product.
The application also provides a spray for preventing and/or treating organ fibrosis, which comprises the dew grass composition.
Preferably, the spray comprises the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of a dew grass extract, 6-25 parts of a cosolvent, 6-20 parts of an antioxidant, 3-21 parts of a bacteriostat and 100-180 parts of purified water.
The external spray for resisting organ fibrosis provided by the application has reasonable compatibility of each component, wherein tannic acid can inhibit the expression of TGF-beta induced collagen-1 and smooth muscle alpha-actin (SMA) to play a role in resisting fibrosis; the main component of the herba lolii officinalis is 20-hydroxyecdysterone (beta-ecdysone), which has the main effects of promoting cell growth, stimulating dermis cell division and promoting protein synthesis for human body; the solubility of the medicine can be multiplied by several times or even tens of times after the cosolvent and the medicine form a complex; the antioxidant can prevent the oxidation of the raw medicine, so that the stability of the antioxidant is good; the bacteriostat can effectively prevent the spray from deteriorating; purified water is used as a solvent to play a role in dissolution; the obtained anti-fibrosis spray has remarkable anti-skin fibrosis effect, is beneficial to the treatment and rehabilitation of patients, improves the life quality, and can be used as an effective method for daily protection and prevention of fibrosis diseases of normal people.
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 preparation method of 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 a dew grass extract, 6-25 parts of a cosolvent, 6-20 parts of an antioxidant, 3-21 parts of a bacteriostat and 100-180 parts of purified water.
S2, mixing tannic acid, the dew grass extract, the cosolvent, the antioxidant and the bacteriostatic agent, adding purified water until the tannic acid, the dew grass extract and the bacteriostatic agent are completely dissolved, and packaging into spray bottles.
Compared with the prior art, the application has the beneficial effects that:
the application provides application of the herba lycopodii extract in preparing anti-skin fibrosis medicines, and the first study shows that the herba lycopodii extract has obvious effect of treating skin fibrosis, belongs to natural plant source medicines, has no side effect, is suitable for oral administration and external use, has good application value in developing anti-skin fibrosis medicine products, and provides a novel and natural medicine source for treating skin fibrosis diseases.
In addition, after the combination of the tannic acid compounded by the dew grass extract, the anti-skin fibrosis effect is remarkably improved, the synergistic effect on the anti-skin fibrosis effect is realized, the generated comprehensive anti-skin fibrosis capability is quite different, the compatibility of raw materials is precise, no side effect is generated, the dew grass extract is suitable for oral administration and external use, and the dew grass extract has good application value in the aspect of developing anti-fibrosis medical products.
Detailed Description
The application will be further described with reference to the following specific embodiments, but the examples are not intended to limit the application in any way. Raw materials reagents used in the examples of the present application are conventionally purchased raw materials reagents unless otherwise specified.
Example 1 preparation of aqueous extract of Lushui grass
The preparation of the water extract of the water grass comprises the following steps:
s1, drying and crushing the dew grass, weighing 50g of dew grass powder, adding 500mL of water, and carrying out reflux extraction for 2h.
S2, filtering, adding 500mL of water into filter residues, extracting for 2 hours under reflux, filtering, and combining the two filtrates.
S3.80 ℃ concentrating and drying to obtain the water extract of the dew grass.
Example 2 preparation of aqueous ethanol extract of Lushui grass
The preparation of the lupulus alcohol extract comprises the following steps:
and (3) drying and crushing the dew grass, weighing 50g of dew grass powder, adding 500mL of 60vol% ethanol solution, carrying out reflux extraction for 1h, filtering, concentrating and drying the filtrate at 80 ℃ to obtain the dew grass ethanol extract.
EXAMPLE 3 dew grass composition for preventing and/or treating fibrosis
The present embodiments provide a series of compositions for preventing and/or treating fibrosis:
composition 1: 12 parts of tannic acid and 12 parts of the dew grass extract of example 1;
composition 2: 19 parts of tannic acid and 15 parts of the dew grass extract of example 2;
composition 3: 25 parts of tannic acid and 20 parts of the dew grass extract of example 1;
composition 4: 36 parts of tannic acid and 24 parts of the dew grass extract of example 1;
composition 5: 45 parts of tannic acid and 30 parts of the dew grass extract of example 1.
Comparative example 1 composition
This comparative example provides a series of compositions:
composition 6: 3 parts of tannic acid and 9 parts of the dew grass extract of example 1;
composition 7: 4 parts of tannic acid and 11 parts of the dew grass extract of example 2;
composition 8: 12 parts of tannic acid and 34 parts of the dew grass extract of example 1.
Experimental example 1 acute toxicity test of compositions 1 to 5
(1) Experimental method
The test was performed by the maximum tolerance method for acute toxicity. Six groups of blank groups and compositions 1-5 (1.1 g/mL) were arranged, 60 SPF grade ICR mice qualified in quarantine were selected, male and female mice were divided into half groups according to sex weight by a segment random method, 10 mice in each group were fasted for more than 12 hours before administration, the blank groups were not administered, and 0.5mL/10g of compositions 1-5 were used in the test Weight of body And (5) performing one-time gastric lavage administration. The poisoning performance and characteristics, toxic reaction occurrence and recovery time, death condition and the like of each group of animals are closely observed and recorded on the same day of administration, particularly within 0-4 hours after administration. Then, the observation was carried out 2 times a day, i.e., 1 time each of the morning and afternoon, and the observation was continued for 14 days. The weights of mice were weighed using an electronic balance before and after the administration at 7 th and 14 th days, respectively. All mice were sacrificed after the end of the test by intraperitoneal injection of pentobarbital sodium anesthesia, and were roughly dissected, and the positions, sizes, colors, adhesion, etc. of the organs were visually observed, and abnormal changes in the texture, effusion, tumor, etc. of the surfaces and sections of the organs were examined. If there are non-dying animals (including dying animals) during the test, gross dissection should be performed.
(2) Experimental results
1. General movement status of mice
The mice in the blank group and the six groups of compositions 1 to 5 were active spontaneously as usual, without abnormality and death, before administration and during observation period of 14 days.
2. Toxic symptoms and death in mice
No abnormalities and death of mice were seen throughout the test period.
3. Constitution change condition
The results of weighing before, 7 and 14 days after administration showed no significant difference in weight change of five groups of mice of compositions 1 to 5, and the increase in body mass was within a normal range, indicating that oral drenching of compositions 1 to 5 had no significant effect on the increase in body mass of ICR mice, as shown in table 1.
TABLE 1 influence of compositions 1 to 5 on the weight of mice
4. General anatomical examination results
No obvious abnormalities were seen on the surface and the tangential plane of each organ in both groups of mice.
Experimental example 2 Long-term toxicity test of compositions 1 to 5
(1) Experimental method
Take composition 2 as an example. 80 rats were randomly divided into 20 of 4 groups, control group, composition 2 high dose group (760 mg/kg), medium dose group (380 mg/kg), and low dose group (190 mg/kg). Each group was sacrificed 1 time per day for index detection after 3 months and 2 weeks of withdrawal (recovery period). Composition 1, composition 3, composition 4, composition 5 were grouped as described above. The following experiment was then performed.
The skin surface, mucous membrane, secretions and excretions of the rats were observed daily for normal conditions, while the general manifestations, toxic manifestations and death of the rats were recorded. The water intake, feed intake and body weight of the rats were recorded every 7d. On both day 30 and day 45, a number of rats were sampled and dissected for hematology and blood biochemistry indices, including: differential counts of White Blood Cells (WBC), neutrophils (NE), eosinophils (EO), basophils (BA), lymphocytes (LY), monocytes (MO), red Blood Cells (RBC), hemoglobin (hb), and the like; the biochemical indexes of blood include: alanine aminotransferase (glutamic pyruvic transaminase, ALT), aspartic aminotransferase (glutamic oxaloacetic transaminase, AST), albumin (ALB), urea nitrogen (BUN), and Triglyceride (TG), etc. The weights of the major organs (heart, liver, spleen, lung, kidney, stomach and reproductive organs) of each group of rats were weighed and organ coefficients were calculated for pathological examination of the control group and high dose group of rats.
(2) Experimental results
1. Weight change
During the test, all groups of rats have good mental state, normal drinking water and appetite, normal stool and urine and smooth and bright hair color. There was no significant difference in the body weight gain trend between the test and blank groups.
2. Blood routine and blood biochemical detection
After 3 months of administration, the SD rat blood routine and blood biochemical tests of the high, medium and low dose groups of compositions 1 to 5 were not significantly different from those of the control group.
3. Histopathological changes
The organ coefficients of each group were relatively indistinguishable in significance on day 30 and day 45. The eyes do not have obvious abnormality on the surfaces of heart, liver, spleen, lung, kidney, stomach, small intestine, testis and ovary organs of each group of rats; no significant lesions were seen in each tissue section of the test and blank groups.
Experimental example 3 anti-pulmonary fibrosis experiment
1. Cell experiment
1. Experimental method
The experiment is to carry out an anti-pulmonary fibrosis experiment on the compositions 1-8 prepared in the example 3 and the comparative example 1, tannic acid and the dew grass extract in the example 1, and the specific method is as follows:
(1) Cell culture
Culturing lung fibroblast in high sugar DMEM medium containing 10% foetal calf serum, 1% penicillin and streptomycin double antibody and glucose concentration of 4.5g/L, placing the cells at 37deg.C and containing 5% CO 2 Incubator, 2d change cell culture liquid once. And when the cell density is 80% -90%, adopting pancreatin digestion and resuspending the cells for passage.
(2) Cell grouping processing
After observing that the cell growth state was good, the cells were digested and resuspended, and fibroblasts were seeded on a black 96-well plate at a density of 3000 cells per well. 10ng/mL TGF-beta was added per well 24h after cell inoculation 1 And establishing a cell lung fibrosis model. Culturing was continued for 72 hours.
Drug administration group: after cell attachment, tannic acid, a dew grass extract, composition 1, composition 2, composition 3, composition 4, composition 5, composition 6, composition 7, and composition 8 were administered, respectively. The administration concentrations were set to be low concentration 1.56. Mu.g/mL, medium concentration 6.25. Mu.g/mL and high concentration 25. Mu.g/mL, respectively, and after each group of administration, 96-well plates were placed in a cell incubator in the dark for further culture for 24 hours.
Control group: the difference from the dosing group is that no dosing was performed.
Blank control group: in comparison with the administration group, the difference is that TGF-beta is not added 1 Nor is it administered.
To avoid systematic errors. Each set was designed with 6 duplicate wells and each set of experiments was repeated 3 times.
(3) CCK8 detection
Cell proliferation rate was measured using CCK 8. The OD of the cells was measured by using a microplate reader at a wavelength of 450nm after adding 10. Mu.L of 10% concentration CCK8 reagent per well for 2 hours according to the protocol described in the kit.
(4) Cell viability
Cell viability was calculated for each group of test cell OD values. Cell viability = (dosing group OD value-blank group OD value)/(control group OD value-blank group OD value) ×100%
2. Experimental results
The lung fibroblast viability results are shown in table 2 below.
TABLE 2 survival of abnormally proliferating lung fibroblasts (%)
As can be seen from table 2, in terms of inhibiting survival rate of abnormally proliferating lung fibroblasts, compositions 1 to 5 were lower than compositions 6 to 8, and when tannic acid or the dew grass extract was used alone, indicating that the two had an effect of synergistically reducing survival rate of lung fibroblasts when used in combination by proper ratio.
2. Animal experiment
1. Experimental method
The experiment was conducted on compositions 1 to 8 prepared in example 3 and comparative example 1, tannic acid and the dew grass extract in example 1, and an anti-pulmonary fibrosis experiment was conducted.
The specific method comprises the following steps:
(1) Grouping animals:
SPF-class Wistar rats were randomly divided into 12 groups of 30 animals each based on body weight. Respectively, a sham operation group, a model group, a composition 1 group, a composition 2 group, a composition 3 group, a composition 4 group, a composition 5 group, a composition 6 group, a composition 7 group, a composition 8 group, a tannic acid group, and a dew grass extract group.
(2) And (3) model preparation:
the experimental animals are routinely fed after purchase, and are adapted to the environment of 7d. In the experiment, animals were anesthetized with 3mL/kg intraperitoneal injection of 10% chloral hydrate, and were fixed on the rat plate in the supine position, with shearing at the neck. Skin is conventionally sterilized and an incision is made in the neck of about 0.5-1cm in length under aseptic manipulation. The exposed air pipe is peeled off layer by layer, and then the head end of the rat board is lifted to form an angle of 30-35 degrees with the experimental table. Under direct vision, a 1mL syringe is used for penetrating into the trachea and approaching to the bifurcation of the trachea as much as possible, the needle head is kept consistent with the direction of the airway, 0.2mL of bleomycin hydrochloride (BLOCIN) and physiological saline solution for injection are rapidly pushed and injected (15 mg of BLOCIN for each time), the concentration is 5mg/mL prepared by physiological saline before use, and the drug is administered according to the weight of 5 mg/kg), the needle head is immediately pulled out, a rat plate is erected, the vertical position of the rat is kept, and the left and right directions are rotated back and forth for 3min, so that the liquid medicine reaches the lungs on two sides as much as possible, and the liquid medicine is uniformly distributed. Suturing skin, continuously injecting penicillin solution into abdominal cavity for 3d, preventing wound infection, and placing in cage for conventional feeding after animal naturally wakes up. The operation method of the rat operation in the sham operation group is the same as that of the rats in other groups, except that the same amount of physiological saline is injected into the trachea after the operation to replace bleomycin hydrochloride.
(3) Administration of drugs
Each group of rats was given 1 time per day by intratracheal injection of bleomycin hydrochloride after modeling on day 2. Compositions 1 to 8, each 190 mg/(kg.d), and tannic acid and the dew grass extract alone were administered continuously for 28d.
(4) Specimen collection
10 animals are randomly selected from each group 1h after administration on the 7 th day, the 14 th day and the 28 th day after molding, right lung tissues are dissected after sacrifice, 9mL of physiological saline is added to each gram of lung tissues to prepare 10% tissue homogenate, and centrifugation is carried out for 10min at 3000r/min, and the supernatant is taken.
(5) Index detection method
Spectrophotometry is used to detect the activity of SOD in lung tissue.
2. Experimental results
TABLE 3 animal model of pulmonary fibrosis lung tissue SOD Activity
As can be seen from Table 3, the results of this experiment show that the SOD activity in lung tissue of three animals in the model group was significantly lower than that in the sham operation group after molding. SOD activity decreases with longer molding time. The method shows that the function of a protection system for resisting free radicals in lung tissues of model animals is reduced, and the formation of lung fibrosis is indirectly promoted. The SOD activity in the lung tissues of the animals of the compositions 1 to 5 is obviously increased compared with that of the synchronous model group; it was demonstrated that compositions 1-5 inhibited the progression of pulmonary fibrosis by increasing SOD activity in lung tissue during the process of excessive proliferation and secretion of large amounts of collagen by fibroblasts. The compositions 6 to 8 are better than tannic acid and the dew grass extract, but worse than the compositions 1 to 5, which shows that the tannic acid and the dew grass extract can realize the effect of synergistically enhancing the SOD activity of lung tissue of the pulmonary fibrosis animal model only by proper proportion.
Experimental example 4 anti-hepatic fibrosis experiment
1. Cell experiment
1. Experimental method
The experiment was conducted on the compositions 1 to 8 prepared in example 3 and comparative example 1, tannic acid and the dew grass extract in example 1, and an anti-hepatic fibrosis experiment was conducted.
The specific method comprises the following steps:
(1) Cell culture
Culturing liver fibroblast in high sugar DMEM medium containing 10% foetal calf serum, 1% penicillin and streptomycin double antibody and glucose concentration of 4.5g/L, placing the cells at 37deg.C and containing 5% CO 2 Incubator, 2d change cell culture liquid once. And when the cell density is 80% -90%, adopting pancreatin digestion and resuspending the cells for passage.
(2) Cell grouping processing
After observing that the cell growth state was good, the cells were digested and resuspended, and fibroblasts were seeded on a black 96-well plate at a density of 3000 cells per well. 10ng/mL TGF-beta was added per well 24h after cell inoculation 1 And establishing a cell hepatic fibrosis model. After 72h of cultivation.
Drug administration group: after cell attachment, tannic acid, a dew grass extract, composition 1, composition 2, composition 3, composition 4, composition 5, composition 6, composition 7, and composition 8 were administered, respectively. The administration concentrations were set to be low concentration 1.56. Mu.g/mL, medium concentration 6.25. Mu.g/mL and high concentration 25. Mu.g/mL, respectively, and after each group of administration, 96-well plates were placed in a cell incubator in the dark for further culture for 24 hours.
Control group: the difference from the dosing group is that no dosing was performed.
Blank control group: in comparison with the administration group, the difference is that TGF-beta is not added 1 Nor is it administered.
To avoid systematic errors. Each set was designed with 6 duplicate wells and each set of experiments was repeated 3 times.
(3) CCK8 detection
Cell proliferation rate was measured using CCK 8. The OD of the cells was measured by using a microplate reader at a wavelength of 450nm after adding 10. Mu.L of 10% concentration CCK8 reagent per well for 2 hours according to the protocol described in the kit.
(4) Cell viability
Cell viability was calculated for each group of test cell OD values. Cell viability = (dosing group OD value-blank group OD value)/(control group OD value-blank group OD value) ×100%
2. Experimental results
The hepatic fibroblast viability results are shown in table 4 below.
TABLE 4 survival of abnormally proliferating liver fibroblasts (%)
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As can be seen from table 4, compositions 1 to 5 were better than compositions 6 to 7 in terms of reducing survival rate of abnormally proliferating liver fibroblasts, and tannic acid or the dew grass extract alone had a lower survival rate of abnormally proliferating liver fibroblasts, indicating that the two had an effect of synergistically reducing survival rate of abnormally proliferating liver fibroblasts when used in combination at a proper ratio.
2. Animal experiment
1. Experimental method
The experiment is to conduct anti-liver fibrosis experiments on the compositions 1-8 prepared in the example 3 and the comparative example 1, tannic acid and the dew grass extract in the example 1, and the concrete method is as follows:
(1) Grouping animals
SPF-class mice were randomly divided into 12 groups of 30 mice each, based on body weight. Respectively, a sham operation group, a model group, a composition 1 group, a composition 2 group, a composition 3 group, a composition 4 group, a composition 5 group, a composition 6 group, a composition 7 group, a composition 8 group, a tannic acid group, and a dew grass extract group.
(2) Moulding
Using classical CCl 4 Induction method, establishing a mouse liver fibrosis model: CCl is put into 4 After being prepared into an oil solution with olive oil according to the volume ratio of 1:1, the injection is subcutaneously injected into mice, the injection volume is 3mL/kg, 2 times per week (interval is 3 d), and the injection is continuously carried out for 6 weeks. Mice from the placebo group were injected with an equal volume of olive oil in the same way.
(3) Administration of drugs
Mice in each group were dosed 1 time per day by gavage on day 2 after modeling. Compositions 1 to 8 were administered at 190 mg/(kg.d), followed by 28d.
(4) Specimen collection
10 animals are selected randomly from each group 1h after administration on 7 th day, 14 th day and 28 th day after molding, liver tissues are dissected after sacrifice, 9mL of physiological saline is added to each gram of liver tissues to prepare 10% tissue homogenate, and centrifugation is carried out for 10min at 3000r/min, and the supernatant is taken.
(5) The detection index method comprises the following steps: spectrophotometry is used to detect the activity of SOD in liver tissue.
2. Experimental results
TABLE 5 liver tissue SOD Activity in animal models of liver fibrosis
As can be seen from Table 5, the results of this experiment show that the SOD activity in liver tissue of three animals in the model group was significantly lower than that in the sham operation group after molding. SOD activity decreases with longer molding time. The method proves that the function of a protection system for resisting free radicals in liver tissues of model animals is reduced, and the formation of liver fibrosis is indirectly promoted. The SOD activity of the animal liver tissues of the compositions 1-5 is obviously improved compared with that of the synchronous model group, the effect is obviously better than that of the compositions 6-8 and the tannic acid or the dew grass extract are independently administered, and the effect shows that the compositions 1-5 can inhibit the liver fibrosis process by increasing the SOD activity of the liver tissues in the process of excessive proliferation and secretion of a large amount of collagen of fibroblasts.
Experimental example 5 anti-dermal fibrosis experiment
1. Cell experiment
1. Experimental method
The experiment is to conduct an anti-skin fibrosis experiment on the compositions 1 to 8 prepared in the example 3 and the comparative example 1, tannic acid and the dew grass extract in the example 2, and the specific method is as follows:
(1) Cell culture
Culturing skin fibroblast in high sugar DMEM medium containing 10% foetal calf serum, 1% penicillin and streptomycin double antibody and glucose concentration of 4.5g/L, placing the cells at 37deg.C and containing 5% CO 2 Incubator, 2d change cell culture liquid once. And when the cell density is 80% -90%, adopting pancreatin digestion and resuspending the cells for passage.
(2) Cell grouping processing
After observing that the cell growth state is good, the cell is eliminatedThe skin fibroblasts were seeded on a black 96-well plate at a density of 3,000 cells per well. 10ng/mL TGF-beta was added per well 24h after cell inoculation 1 Establishing a cell skin fibrosis model, and continuously culturing for 24 hours.
Drug administration group: after cell attachment, tannic acid, a dew grass extract, composition 1, composition 2, composition 3, composition 4, composition 5, composition 6, composition 7, and composition 8 were administered, respectively. The administration concentrations were set to be low concentration 1.56. Mu.g/mL, medium concentration 6.25. Mu.g/mL and high concentration 25. Mu.g/mL, respectively, and after each group of administration, 96-well plates were placed in a cell incubator in the dark for further culture for 24 hours.
Control group: the difference from the dosing group is that no dosing was performed.
Blank control group: in comparison with the administration group, the difference is that TGF-beta is not added 1 Nor is it administered.
To avoid systematic errors. Each set was designed with 6 duplicate wells and each set of experiments was repeated 3 times.
(3) CCK8 detection
The OD of the cells was measured by using a microplate reader at a wavelength of 450nm after adding 10. Mu.L of 10% concentration CCK8 reagent per well for 2 hours according to the protocol described in the kit.
(4) Cell viability
Cell viability was calculated for each group of test cell OD values. Cell viability = (dosing group OD value-blank group OD value)/(control group OD value-blank group OD value) ×100%
2. Experimental results
The results of skin fibroblast viability are shown in table 6 below.
TABLE 6 survival of abnormally proliferating skin fibroblasts (%)
As can be seen from table 6, compositions 1 to 5 were better than compositions 6 to 8 in terms of reducing the survival rate of abnormally proliferating skin fibroblasts, and the survival rate of abnormally proliferating skin fibroblasts was low compared to tannic acid or the extract of dew grass alone, indicating that tannic acid and the extract of dew grass, when used in combination in a proper ratio, had an effect of synergistically reducing the survival rate of abnormally proliferating skin fibroblasts.
2. Animal experiment
1. Experimental method
The experiment was conducted on the compositions 1 to 8 prepared in example 3 and comparative example 1, tannic acid and the dew grass extract of example 2, for anti-skin fibrosis.
The specific method comprises the following steps:
(1) Grouping animals
SPF-class 6-week-old male BALB/c mice were randomly divided into 12 groups of 30 animals each by body weight. Respectively, a sham surgery group, a simple Phosphate Buffer (PBS) group, a composition 1 group, a composition 2 group, a composition 3 group, a composition 4 group, a composition 5 group, a composition 6 group, a composition 7 group, a composition 8 group, a tannic acid group and a dew grass extract group.
(2) Model preparation
The mice in the blank group were not treated at all; the back skin of mice in the PBS alone group and the composition group were subcutaneously injected with 100. Mu.L of PBS and bleomycin (1 mg/mL), respectively.
(3) Administration of drugs
Each group of mice was skin-dosed 2 times daily from day 2 after modeling with bleomycin. Compositions 1 to 8 were administered at 190 mg/(kg.d), followed by 28d.
(4) Specimen collection
And (3) 1h after the administration of the medicine on the 7 th day, the 14 th day and the 28 th day after molding, randomly selecting 10 animals for each group, visually observing skin changes of the back of the mice, killing the mice after the observation is finished, taking back skin tissues, adding 9mL of physiological saline into each gram of skin tissues to prepare 10% tissue homogenate, centrifuging for 10min at 3000r/min, and taking the supernatant.
(5) The detection index method comprises the following steps: spectrophotometry is used to detect SOD activity of skin tissue.
2. Experimental results
TABLE 7 animal model of skin fibrosis skin tissue SOD Activity
As can be seen from Table 7, the results of this experiment show that the SOD activity in skin tissue of three animals in the model group was significantly lower than that in the sham group after molding. SOD activity decreases with longer molding time. It is explained that the function of the protection system for resisting free radicals in skin tissues of model animals is reduced, and the formation of skin fibrosis is indirectly promoted. The SOD activity in animal skin tissues of the compositions 1 to 5 is obviously increased compared with that of the synchronous model group; it was demonstrated that compositions 1 to 5 inhibited the progression of skin fibrosis by increasing SOD activity in skin tissue during the process of excessive proliferation and secretion of large amounts of collagen by fibroblasts. The compositions 6 to 8 are better than tannic acid and the dew grass extract, but worse than the compositions 1 to 5, which shows that the tannic acid and the dew grass extract can synergistically enhance the SOD activity of skin tissue of the skin fibrosis animal model only by proper proportion.
Example 4 oral liquid 1 for preventing and/or treating 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 dew grass 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 dew grass, weighing 50g of dew grass powder, adding 500mL of water, and carrying out reflux extraction for 2h; filtering, adding 500mL of water into the filter residue, extracting under reflux for 2h, filtering, and combining the two filtrates; concentrating and drying at 80 ℃ to obtain the water extract of the dew grass.
(2) Mixing tannic acid, the dew grass extract, vitamin C, aspartame and rosemary according to the weight parts, adding purified water until the tannic acid, the dew grass extract, the vitamin C, the aspartame and the rosemary are completely dissolved, and packaging to obtain the oral liquid.
EXAMPLE 5 oral liquid 2 for preventing and/or treating 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 dew grass 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 water extract of the herba mesonae chinensis is the same as that of the example 4;
mixing tannic acid, the dew grass extract, vitamin E, stevioside and clove according to the weight parts, adding purified water until the tannic acid, the dew grass extract, the vitamin E, the stevioside and the clove are completely dissolved, and packaging to obtain the oral liquid.
EXAMPLE 6 oral liquid 3 for preventing and/or treating 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 dew grass 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 water extract of the herba mesonae chinensis is the same as that of the example 4;
mixing tannic acid, the dew grass extract, sodium sulfite, aspartame and sage according to the weight parts, adding purified water until the tannic acid, the dew grass extract, the sodium sulfite, the aspartame and the sage are completely dissolved, and packaging to obtain the oral liquid.
EXAMPLE 7 tablet 1 for preventing and/or treating fibrosis
1. The embodiment provides a tablet, which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of a dew grass extract, 21 parts of starch, 13 parts of Arabic gum and 8 parts of magnesium stearate.
2. Preparation method
(1) The dew grass extract was as in example 1.
(2) Mixing tannic acid, herba Cynomorii extract and starch.
(3) Adding acacia to make soft material (in the form of lump by hand, and breaking off when finger is pressed slightly without forming powder), and sieving by hand to obtain granule without strip, block and fine powder. During mass production, the soft material is extruded by a roller (or a scraper) of a granulator to pass through sieve holes so as to prepare particles.
(4) Drying at 80deg.C (electric heating oven for small amount preparation, and steam drying room for mass production). The dried granules are often conglobated and adhered, the granules are sieved and granulated, finally magnesium stearate is added, and the granules can be pressed into tablets after being uniformly mixed. Obtaining the product.
Example 8 tablet 2 for preventing and/or treating fibrosis
1. The embodiment provides a tablet, which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of a dew grass extract, 22 parts of dextrin, 15 parts of methyl cellulose and 7 parts of magnesium stearate.
2. Preparation method
(1) The dew grass extract was as in example 1.
(2) Mixing tannic acid, herba Cynomorii extract and dextrin.
(3) Adding methyl cellulose to make soft material (in the form of lump by hand, and the powder can be broken when finger is pressed slightly), squeezing with hand, and sieving to obtain granule without long strip, lump and fine powder. During mass production, the soft material is extruded by a roller (or a scraper) of a granulator to pass through sieve holes so as to prepare particles.
(4) Drying at 80deg.C (electric heating oven for small amount preparation, and steam drying room for mass production). The dried granules are often conglobated and adhered, the granules are sieved and granulated, finally magnesium stearate is added, and the granules can be pressed into tablets after being uniformly mixed. Obtaining the product.
EXAMPLE 9 tablet 3 for preventing and/or treating fibrosis
1. The embodiment provides a tablet, which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of a dew grass extract, 23 parts of sugar powder, 16 parts of hydroxypropyl cellulose and 10 parts of hydrogenated vegetable oil.
2. Preparation method
(1) The dew grass extract was as in example 1.
(2) Mixing tannic acid, herba Cynomorii extract and sugar powder.
(3) Adding hydroxypropyl cellulose to make soft material (in the form of lump by hand, and capable of breaking apart without forming powder when finger is pressed slightly), squeezing with hand, and sieving to obtain granule without long strips, blocks and fine powder. During mass production, the soft material is extruded by a roller (or a scraper) of a granulator to pass through sieve holes so as to prepare particles.
(4) Drying at 80deg.C (electric heating oven for small amount preparation, and steam drying room for mass production). The dried granules are often conglobated and adhered, the granules are sieved and granulated, and finally hydrogenated vegetable oil is added, and the granules can be pressed into tablets after being uniformly mixed. Obtaining the product.
EXAMPLE 10 spray 1 for preventing and/or treating fibrosis
1. The embodiment provides a spray, which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of a dew grass 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 dew grass extract was as in example 2;
(2) Mixing tannic acid, the dew grass extract, acetamide, sodium sulfite and clove according to the weight parts, adding purified water until the tannic acid and the dew grass extract are completely dissolved, and packaging into spray bottles.
EXAMPLE 11 spray 2 for preventing and/or treating fibrosis
1. The embodiment provides a spray, which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of a dew grass 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 dew grass extract was as in example 2;
(2) Mixing tannic acid, herba Cynomorii extract, sodium salicylate, vitamin C and herba Rosmarini officinalis according to the above weight parts, adding purified water until completely dissolved, and packaging into spray bottle.
EXAMPLE 12 spray 3 for preventing and/or treating fibrosis
1. The embodiment provides a spray, which comprises the following components in parts by weight: 19 parts of tannic acid, 15 parts of a dew grass extract, 8 parts of sodium benzoate, 14 parts of vitamin E, 17 parts of sage and 100 parts of purified water.
2. Preparation method
(1) The dew grass extract was as in example 2;
(2) Mixing tannic acid, the dew grass extract, sodium benzoate, vitamin E and sage according to the weight parts, adding purified water until the tannic acid and the dew grass extract are completely dissolved, and packaging into spray bottles.
It is to be understood that the above examples of the present application are provided by way of illustration only and not by way of limitation of the embodiments of the present application. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the application are desired to be protected by the following claims.

Claims (3)

1. An oral liquid for preventing and/or treating skin fibrosis is characterized by comprising the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of a dew grass extract, 6-28 parts of an antioxidant, 6-30 parts of a sweetener, 6-25 parts of a bacteriostatic agent and 90-200 parts of purified water; the antioxidant is any one of vitamin C, vitamin E and sodium sulfite, the sweetener is aspartame or stevioside, and the bacteriostatic agent is any one of rosemary, clove and sage; the herba Avenae Fatuae extract is herba Avenae Fatuae water extract and/or herba Avenae Fatuae alcohol extract.
2. A tablet for preventing and/or treating skin fibrosis, comprising the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of a dew grass extract, 9-30 parts of a filling agent, 9-28 parts of an adhesive and 2-25 parts of a lubricant; the filler is any one of starch, dextrin and powdered sugar, the adhesive is any one of acacia, methylcellulose and hydroxypropyl cellulose, and the lubricant is magnesium stearate or hydrogenated vegetable oil; the herba Avenae Fatuae extract is herba Avenae Fatuae water extract and/or herba Avenae Fatuae alcohol extract.
3. A spray for preventing and/or treating skin fibrosis, which is characterized by comprising the following components in parts by weight: 12-45 parts of tannic acid, 12-30 parts of a dew grass extract, 6-25 parts of a cosolvent, 6-20 parts of an antioxidant, 3-21 parts of a bacteriostat and 100-180 parts of purified water; the cosolvent is any one of acetamide, sodium salicylate and sodium benzoate, the antioxidant is any one of sodium sulfite, vitamin C and vitamin E, and the bacteriostatic agent is any one of clove, rosemary and sage; the herba Avenae Fatuae extract is herba Avenae Fatuae water extract and/or herba Avenae Fatuae alcohol extract.
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