CN111358855A

CN111358855A - Application of radix physochlainae extract in preparing medicine for preventing and treating myopia

Info

Publication number: CN111358855A
Application number: CN202010243204.3A
Authority: CN
Inventors: 毕宏生; 郭俊国; 吴建峰; 宋继科; 解孝锋; 赵海强
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-07-03

Abstract

The invention provides an application of a physochlaina extract in preparing a medicament for preventing and treating myopia. The invention obtains the effective composition of anisodamine, scopolamine, scopoletin and the like in the radix physochlainae extract through tests, can achieve the purpose of preventing and treating myopia, and can effectively avoid the defect that the anticholinergic drug cannot be clinically applied due to potential side effects caused by single use for a long time. The physochlaina extract has obvious treatment effect on myopia and high safety, and is expected to be popularized and applied clinically. The invention also proves that the physochlaina extract can improve myopia by regulating and controlling scleral fiber layer and retinal pigment cells, influencing the expression of matrix metalloproteinase 2, matrix metalloproteinase inhibitor 2 and the like, regulating the spasm of ciliary muscle and the like through experiments. The invention also provides a composition, which comprises the effective components of the physochlaina extract; the composition provides a new path for preventing and treating myopia.

Description

Application of radix physochlainae extract in preparing medicine for preventing and treating myopia

Technical Field

The invention relates to application of a physochlaina extract in preparing a medicine for preventing and/or treating myopia and related application, and belongs to the technical field of biological medicines.

Background

The prevalence rate of myopia of children, teenagers and the like in China is continuously rising and develops towards high-rise and low-age, the population of the affected people is high at the first position of the world, and the prevention and treatment of the myopia belong to a worldwide problem. The existing myopia treatment comprises surgical intervention, drug therapy, optical correction and the like, wherein the surgical therapy is interventional therapy, is suitable for middle-low myopia and has certain damage; the drug therapy comprises mydriatic, miotic, vasodilator spasmolytic and the like, is mainly used for preventing and treating pseudomyopia, has large potential toxic and side effects and has large risk to children and teenagers; wearing glasses is the most common means, and causes inconvenience to activities such as sports and reading in life. At home and abroad, the research finds that the M choline receptor anticholinergic can completely relax the ciliary muscle of eyes, so that the ciliary muscle loses the original regulating effect, and the progress of the myopia is relieved. When the tension state of ciliary muscles is effectively relieved, the muscles which are always in the tension adjusting state can be completely relaxed, so that the myopia condition of eyes can be effectively relieved and improved, but atropine cannot completely cure myopia and can only control the further development of myopia, so that the atropine is required to be taken for a long time in clinical application, has no exact curative effect less than one year, and can cause certain damage to the eyes after long-term use.

Although the traditional Chinese medicine has advantages and characteristics for treating myopia, the traditional Chinese medicine decoction can fully meet the requirements of traditional Chinese medicine syndrome differentiation treatment, has the advantages of quick curative effect, easy absorption, strong effect and the like, and also has the defects of great influence by the quality of raw medicinal materials, processing technology, decoction conditions and the like, easy mildewing and deterioration after long-term placement, inconvenient carrying, large taking volume, difficult taking by children and the like. At present, no Chinese patent medicine for treating myopia exists in the market, effective formulas, Chinese medicine monomers and the like are researched and developed into innovative eye Chinese medicine preparations, so that the medicine compliance can be improved, and the eye Chinese medicine preparation has wide market prospect.

According to the traditional Chinese medicine, normal physiological function activities have a close relationship with the smooth abundance and insufficiency of qi and blood, and if the eyes are used for too long time in a short distance, the phenomenon of 'qi and blood stasis' in the traditional Chinese medicine is generated on the local part of the eyes, and the shortsightedness is generated due to ciliary muscle spasm caused by the 'overuse of eyesight, liver qi disharmony, spasm and urgency of tendons and vessels, the loss of marrow and sea and the loss of nourishment of eyes'. The non-selective M receptor blocker atropine can effectively delay the myopia progress of teenagers, but the eye local and general side effects limit wide clinical application, and discomfort symptoms mainly comprise near vision difficulty, photophobia, string light, eyelid allergy and the like. Due to concerns over the long-term use of atropine, researchers have begun to focus on the selective Ml receptor blocker pirenzepine, common complications of which are mydriasis, red spots on the eyelid and ocular stinging, and pupillary responses remain after instillation of pirenzepine. Therefore, the active research and exploration of other drugs with good safety have very important significance.

Radix Physochlainae is root of Physochlaina acinosa (Physochlaina fundibularis) of Solanaceae, and is sweet, slightly bitter and warm in nature; has effects of warming lung, eliminating phlegm, relieving asthma and cough, tranquilizing mind, and relieving convulsion; it also has anticholinergic effect, and can relax bronchial smooth muscle and relieve bronchial smooth muscle spasm. The physochlaina japonica directly acts on smooth muscles to relieve the smooth muscles in a tense state, has the most obvious effect of relieving asthma, and has the effects of resisting histamine and expanding bronchi.

In the prior art, the main research on the physochlaina japonica is focused on nourishing, relieving cough and asthma. So far, the research related to the myopia improvement effect of the physochlaina huashanensis has not been seen.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an application of a radix physochlainae extract in preparing a medicament for preventing and/or treating myopia, the invention firstly provides a new application of the radix physochlainae extract in preventing and/or treating myopia, and further obtains the influence or effect of the radix physochlainae extract on cells, genes and proteins related to myopia through a series of tests, and the radix physochlainae extract has a regulating effect on ciliary spasm of eyes; and according to the effective components in the physochlaina extract, a composition and an ophthalmic preparation are provided, and the composition and the ophthalmic preparation can effectively prevent and treat myopia.

The technical scheme adopted by the invention is as follows:

according to a first aspect of the application, the application of the physochlaina extract in preparing the medicine for preventing and/or treating myopia is provided. The invention innovatively discovers that the radix physochlainae extract has a new application of preventing and/or treating myopia, and contains alkaloid, coumarins, snores, retained alcohol, amino acid, oil and the like. The alkaloid part mainly comprises atropine alkaloids and derivatives thereof, wherein the tropane alkaloids comprise atropine, hyoscyamine, anisodamine, scopolamine, etc., and the water-soluble alkaloids comprise five kinds, mainly choline. The extract of the physochlaina huashanensis is used for preventing and/or treating myopia and has the advantages of good treatment effect and high safety.

The radix physochlainae extract comprises 0.01-200 mg/g of anisodamine, 0.01-200 mg/g of scopolamine, 0.01-200 mg/g of atropine and 0.01-200 mg/g of scopoletin; preferably, the radix physochlainae extract comprises 0.1-50 mg/g of anisodamine, 0.1-50 mg/g of scopolamine, 0.1-50 mg/g of atropine and 0.1-50 mg/g of scopoletin; more preferably, the radix physochlainae extract comprises 0.5-30 mg/g of anisodamine, 0.5-30 mg/g of scopolamine, 0.5-30 mg/g of atropine and 0.5-30 mg/g of scopoletin. When the contents of anisodamine, scopolamine and scopoletin in the radix physochlainae extract are too low, the prevention and improvement effect of the radix physochlainae extract on myopia is not obvious; when the content of atropine in the radix physochlainae extract is too high, the radix physochlainae extract has obvious irritation.

The radix physochlainae extract is obtained by extracting radix physochlainae with a solvent. Wherein said Physochlaina japonica is dried root of Physochlaina indica (Physochlaina japonica) belonging to Solanaceae from the middle to the east of Qinling mountains in Shaanxi, the west and south of Henan, and the south of Shanxi. Collected in spring, removed fibrous root and cleaned. Wherein the radix Physochlainae of Henan origin has brownish surface, yellowish white transverse long skin hole-like protrusions, ring lines, stem marks and warty protrusions at the upper part, yellowish white cross section, narrow skin part, wide wood part, and fine radial texture. In the specific embodiment of the invention, the physochlaina huashanensis from Henan province is selected and ground according to a conventional method to obtain physochlaina huashanensis powder for use.

Preferably, the solvent is selected from at least one of water, ethanol, petroleum ether, n-hexane, acetone, dichloromethane and ethyl acetate; preferably, the solvent is ethanol; more preferably, the solvent is ethanol with a volume fraction of 70%. Preferably, the proportion of the warrior mountain participating in the organic solvent is 1 g: 1-50 mL.

Preferably, the extraction is at least one selected from the group consisting of heating reflux extraction, ultrasonic extraction, percolation extraction and decoction extraction.

The preparation method of the radix physochlainae also comprises the steps of extracting the radix physochlainae by using an organic solvent, concentrating and purifying. Wherein, the purification adopts macroporous resin adsorption to remove impurities.

According to another aspect of the application, the application of the physochlaina extract in regulating and controlling the growth of scleral fibroblasts and/or retinal pigment cells is provided. The invention improves myopia by regulating the growth of the scleral fibroblasts and the retinal pigment cells, thereby achieving the purpose of prevention and treatment.

Preferably, the selected modulation of scleral fibroblast and/or retinal pigment cell growth is inhibition of scleral fibroblast and/or retinal pigment cell growth. Inhibiting the growth of scleral fibroblasts and retinal pigment cells can effectively inhibit the occurrence and development of myopia.

According to another aspect of the application, the application of the physochlaina extract in regulating and controlling the expression of matrix metalloproteinase 2, matrix metalloproteinase inhibitor 2 and/or transforming growth factor β is provided, when myopia occurs, the expression of the matrix metalloproteinase 2 and the transforming growth factor β is up-regulated, the activity is increased, the expression of the matrix metalloproteinase inhibitor 2 is down-regulated, and the activity is reduced.

Preferably, the regulation of the expression of the gene of matrix metalloproteinase 2 and/or transforming growth factor β 2 is to down-regulate the expression of the gene of matrix metalloproteinase 2 and/or transforming growth factor β 2, and the regulation of the expression of the gene of matrix metalloproteinase inhibitor 2 is to up-regulate the expression of the gene of matrix metalloproteinase inhibitor 2.

Preferably, the expression of the matrix metalloproteinase 2 and/or transforming growth factor β 2 protein is regulated to inhibit the expression of the matrix metalloproteinase 2 and/or transforming growth factor β 2 protein, and the expression of the matrix metalloproteinase inhibitor 2 protein is regulated to promote the expression of the matrix metalloproteinase inhibitor 2 protein.

According to another aspect of the present application, there is provided a composition for preventing and/or treating myopia, comprising the following components in parts by mass: 0.01-200 parts of anisodamine, 0.01-200 parts of scopolamine, 0.01-200 parts of atropine and 0.01-200 parts of scopoletin; preferably, the composition comprises the following components in parts by mass: 0.1-50 parts of anisodamine, 0.1-50 parts of scopolamine, 0.1-50 parts of atropine and 0.1-50 parts of scopoletin; more preferably, the composition comprises the following components in parts by mass: 0.5-30 mg/g anisodamine, 0.5-30 mg/g scopolamine, 0.5-30 mg/g atropine and 0.5-30 mg/g scopoletin.

According to another aspect of the application, an ophthalmic preparation containing one of the compositions is provided, wherein the ophthalmic preparation further comprises water and auxiliary materials; the mass ratio of the composition to the auxiliary materials to the water is 0.01-10: 0.1-10: 10 to 100. Preferably, the ophthalmic formulation is selected from at least one of eye drops, ophthalmic gel and ophthalmic cream; more preferably, the ophthalmic formulation is a self-eyedrop. When the ophthalmic preparation is an ophthalmic cream, the auxiliary material can be at least one selected from carbomer, chitosan, hyaluronic acid, poloxamer, propylene glycol and carboxymethyl cellulose; when the ophthalmic preparation is an ophthalmic gel, the adjuvant may be selected from at least one of hyaluronic acid, poloxamer and propylene glycol.

According to a further aspect of the present application, there is provided the use of said composition or said ophthalmic formulation in the manufacture of a medicament for the prevention and/or treatment of myopia.

The invention has the beneficial effects that:

(1) the invention provides a new application of radix physochlainae extract in preventing and treating myopia for the first time, the radix physochlainae extract comprises anisodamine, scopolamine, scopoletin and atropine, and the drug effect that the anisodamine, the scopolamine and the scopoletin can partially replace the atropine is obtained through tests, so that the purpose of preventing and treating myopia is achieved, and the defect that the clinical application cannot be realized due to the serious side effect caused by the simple use of the atropine is effectively avoided. The physochlaina extract has obvious treatment effect on myopia and high safety, and is expected to be popularized and applied clinically.

(2) The invention proves that the physochlaina extract prevents and improves myopia by regulating the growth of scleral fibroblasts and retinal pigment cells and influencing the expression of matrix metalloproteinase 2, matrix metalloproteinase inhibitor 2 and/or transforming growth factor β 2 through tests, and provides a new strategy for preventing and treating myopia in mechanism.

(3) The invention provides a composition for preventing and treating myopia, which contains the effective components of the physochlaina extract and can effectively delay the myopia progress; the physochlaina extract and the composition provided by the invention provide a new path for preventing and treating myopia.

Drawings

FIG. 1 is an HPLC chromatogram of a mixed control of the present invention;

FIG. 2 is an HPLC chromatogram of a radix Physochlainae extract sample of the present invention;

FIG. 3 is a graph showing the effect of the extract of Panax wallichiana on proliferation of scleral fibroblasts;

FIG. 4 is a graph showing the effect of the extract of Panax heminseng on the proliferation of retinal pigment cells according to the present invention

FIG. 5 is a graph showing the effect of the extract of Panax wallichiana on the expression of matrix metalloproteinase inhibitor 2;

FIG. 6 is a graph showing the effect of the extract of Panax wallichiana of the present invention on the expression of transforming growth factor β 2;

FIG. 7 is a graph showing the effect of the extract of Panax wallichiana on the expression of matrix metalloproteinase 2;

FIG. 8 is a Western blot diagram of the protein expression of scleral cell by the extract of Panax schinseng C.A. Meyer of the present invention;

FIG. 9 shows the light reflex in rabbit eyes before and after intervention with the extract of Physochlaina sinica Diels, wherein A is before intervention and B is after intervention;

FIG. 10 is a graph showing the effect of the extract of Panax wallichiana on cell viability.

Detailed Description

The present invention is described in detail with reference to specific examples, which are provided to facilitate the understanding of the technical solutions of the present invention by those skilled in the art, and the implementation or use of the present invention is not limited by the description of the present invention.

In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, if not specified. The methods in the examples are conventional in the art unless otherwise specified.

Example 1Ultrasonic extraction method is adopted to prepare the physochlaina extract

The preparation method of the radix physochlainae extract comprises the following steps:

(1) extraction of

Grinding radix Physochlainae to obtain radix Physochlainae powder, weighing 2g of radix Physochlainae powder, placing into a conical flask, adding 70% ethanol as extractant according to the material-liquid ratio of 1:25(g/mL), and performing ultrasonic treatment (350W, 50Hz) at 30 deg.C for 30 min; vacuum filtering while hot, and mixing filtrates to obtain extractive solution.

(2) Concentrating

Concentrating the extractive solution under reduced pressure until no alcohol smell is present.

Example 2The preparation method of radix Physochlainae extract by heating reflux extraction

(1) extraction of

Grinding radix Physochlainae to obtain radix Physochlainae powder, weighing 200g of radix Physochlainae powder, placing into a 2L round-bottom flask, adding 70% ethanol solution until the sample can be completely soaked, extracting in water bath at constant temperature (100 deg.C) for 3 hr, filtering the obtained extractive solution with gauze, and collecting filtrate; repeating the above operation once again; and finally, combining the filtrates to obtain an extracting solution.

(2) Concentrating

Example 3Decocting to obtain radix Physochlainae extract

(1) extraction of

Grinding the physochlaina huashanensis to prepare physochlaina huashanensis powder, weighing 200g of physochlaina huashanensis, adding water for extraction for 2 times, extracting for 2 hours each time, collecting filtrate, concentrating under reduced pressure to obtain clear paste with the relative density of 1.25-1.35 (60 ℃), adding ethanol for precipitation, adjusting the ethanol content to 70%, standing, and taking supernatant.

(2) Concentrating

Concentrating the supernatant under reduced pressure until no alcohol smell is available.

Example 4Percolating to obtain radix Physochlainae extract

(1) extraction of

Grinding radix Physochlainae to obtain radix Physochlainae powder, weighing 200g of radix Physochlainae powder, adding 70% ethanol solvent, wetting, standing in a sealed conical percolator for a certain period of time, adding 70% ethanol solvent, removing air in the gaps of the decoction pieces as much as possible, soaking above the solvent, percolating overnight, collecting the first percolate of 85% decoction pieces, storing in another container, concentrating at low temperature, and mixing with the first percolate to obtain extractive solution.

(2) Concentrating

Experimental example 1

The main components of the physochlaina extract prepared by different extraction methods were detected by High Performance Liquid Chromatography (HPLC), and the detection results are shown in table 1.

Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; elution with acetonitrile-0.1% formic acid solution gradient, 0 min: 90% -0.1% formic acid solution; 7 min: 90% -0.1% formic acid solution; 15 min: 80% -0.1% formic acid solution; 17min 90% -0.1% formic acid solution; 20min 90% -0.1% formic acid solution; the detection wavelength was 210 nm. The number of theoretical plates is not less than 6000 calculated according to the paeoniflorin peak.

Preparation of mixed control solution: accurately weighing control substances such as anisodamine, scopolamine, atropine and scopoletin, dissolving in methanol, and making into mixed solution containing 34.95 μ g of anisodamine, 34.425 μ g of scopolamine, 39.55 μ g of atropine and 34.45 μ g of scopoletin per 1 mL.

Preparation of a test solution: precisely weighing 0.2g of radix Physochlainae extract, placing in a 25mL volumetric flask, adding methanol solution, sealing, performing ultrasonic treatment for 30min (ultrasonic frequency 40kH, power 150W), standing to room temperature, supplementing methanol to lose mass, and filtering with 0.45 μm filter membrane.

Precisely sucking 5 μ L of each of the reference and sample solutions, and injecting into a liquid chromatograph for determination, wherein the HPLC chromatogram of the reference is shown in FIG. 1, and the HPLC chromatogram of the radix Physochlainae extract sample prepared by percolation in example 4 is shown in FIG. 2, to obtain the content of main components in the radix Physochlainae extract, as shown in Table 1.

TABLE 1 content of Main ingredient (mg/g) of radix Physochlainae extract

	Anisodamine	Scopolamine	Atropine	Scopolactone
					Example 1	29.66	17.88	26.13	6.40
Example 2	39.10	37.37	47.82	14.04
					Example 3	8.56	4.88	4.60	1.82
Example 4	73.55	52.94	97.19	21.07

As can be seen from the results in table 1, the main ingredients of the radix physochlainae extract obtained by different preparation methods include anisodamine, scopolamine, atropine and scopoletin, and the contents of the effective ingredients are different in the different preparation methods; wherein, the percolation extraction method has the best extraction effect. The following examples were studied using the extract of physochlaina obtained by the percolation method of example 4.

Example 5

A preparation method of radix physochlainae eye drops comprises the following steps:

weighing 5g of the physochlaina huashanensis extract obtained in the example 4, adding 800ml of sterile water for injection, stirring to completely dissolve the physochlaina huashanensis extract, adding the sterile water for injection to 1000ml, filtering by a 0.22 mu m filter membrane under positive pressure, filling, packaging by using a brown bottle, and obtaining the physochlaina huashanensis eye drops with the concentration of 0.5 percent by 5ml or 8ml per bottle.

Example 6

weighing 10g of the physochlaina huashanensis extract obtained in the example 4, adding 800ml of sterile water for injection, stirring to completely dissolve the physochlaina huashanensis extract, adding the sterile water for injection to 1000ml, filtering with a 0.22 mu m filter membrane under positive pressure, filling, packaging with a brown bottle, and obtaining the physochlaina huashanensis eye drops with the concentration of 1% by 5ml or 8ml per bottle.

Example 7

weighing 20g of the physochlaina huashanensis extract obtained in the example 4, adding 800ml of sterile water for injection, stirring to completely dissolve the physochlaina huashanensis extract, adding the sterile water for injection to 1000ml, filtering with a 0.22 mu m filter membrane under positive pressure, filling, packaging with a brown bottle, and obtaining the physochlaina huashanensis eye drops with the concentration of 2 percent by 5ml or 8ml per bottle.

Example 8

weighing 40g of the physochlaina huashanensis extract obtained in the example 4, adding 800ml of sterile water for injection, stirring to completely dissolve the physochlaina huashanensis extract, adding the sterile water for injection to 1000ml, filtering with a 0.22 mu m filter membrane under positive pressure, filling, packaging with a brown bottle, and obtaining the physochlaina huashanensis eye drops with the concentration of 4 percent by 5ml or 8ml per bottle.

Example 9Use of radix Physochlainae extract in regulating and controlling scleral fibroblast and its preparation methodApplication in retinal pigment cell growth

The specific operation is as follows:

cell proliferation assay comprises preparing cell suspension from 5-7 generations of sclera fibroblast or retina pigment cell, and mixing with 5 × 10³Per well concentration the cell suspension was inoculated into a 96 well plate, the volume of the cell suspension per well was 100. mu.L, each group of cells was repeated in 6 wells, after all the cell suspensions were inoculated, the 96 well plate was placed in a constant temperature incubator and incubated for 24 hours, the extract of Panax wallichiana prepared in example 4 was added with 1640 medium containing 5% fetal bovine serum to prepare respective extracts of Panax wallichiana at concentrations of 0.2496ng/mL, 1.248ng/mL, 6.24ng/mL, 31.2ng/mL, 156ng/mL, 1.56. mu.g/mL, 15.6. mu.g/mL, 0.156mg/mL, 1.56mg/mL, and the experimental groups were added with respective extracts containing different Panax wallichiana (0.2496ng/mL, 1.248ng/mL, 6.24ng/mL, 31.2ng/mL, 156ng/mL, 1.56. mu.g/mL, 15.6. mu.g/mL, 0.156mg/mL, 1.24 mg/mL), 1.56mg/mL), an equal volume of the culture medium (1640 medium containing 5% fetal bovine serum) was added to the blank control group, and the 96-well plate was placed again at 37 ℃ and 5% CO₂Performing cell culture in a constant temperature incubator, adding 10 mu L of CCK8 solution into each hole after 48h, continuously incubating the cells for 2h and 4h, detecting the absorbance value at the wavelength of 450nm by using a microplate reader, and calculating the cell proliferation rate of the scleral fibroblasts and the retinal pigment cell proliferation rate, wherein the results are shown in fig. 3 and 4.

As shown by the results of fig. 3, the lower concentration of the extract of physochlaina huashanensis has no significant effect on the proliferation of scleral fibroblasts, and the greater the concentration of the extract of physochlaina huashanensis at the higher concentration, the significant effect on the proliferation of scleral fibroblasts exists. Therefore, the physochlaina extract can effectively inhibit the growth of scleral fibroblasts.

As shown by the results of fig. 4, various concentrations of the extract of physochlaina huashanensis affect the proliferation of retinal pigment cells, and the effect on the proliferation of retinal pigment cells is greater as the concentration is increased. Therefore, the physochlaina extract can effectively inhibit the growth of retinal pigment cells.

Cell cycle assay: stimulating each concentration of cell growth factor for 24 hours by flow cytometryThe later growth cycle of scleral fibroblast is determined by preparing cell suspension from 5-7 generations of scleral fibroblast, and culturing at 5 × 10³Concentration per well the cell suspension was seeded in 6-well plates and placed in a thermostatted incubator for 24 cultures. The cells were randomly divided into a control group and a pretreatment group, the control group was administered with 3mL of a culture medium (1640 medium containing 5% fetal bovine serum), the intervention group was administered with 3mL of a concentration of 0.2425mg/mL, 0.7275mg/mL, 0.156mg/mL, 1.56mg/mL, 2.91mg/mL of a concentration of a Panax huashanensis extract, respectively, 24 hours later, the cells were decanted from the culture medium, 0.25% pancreatin was digested for 3 minutes, 100. mu.L of the culture medium was added to each well, the cells were blown up with the culture medium, after being completely suspended, the cells were centrifuged at 1500 rpm in a plate rotor centrifuge for 15 minutes, and the supernatant was discarded. PBS was washed 2 times, 300. mu.L of PBS was added each time and blown up to completely suspend the cells, 1500 rpm was applied, centrifugation was carried out for 15 minutes, and the procedure was repeated once. The cells were collected in a 5ml syringe and forced into 5ml of pre-chilled 70% ethanol, sealed with a sealing membrane and fixed overnight at 4 ℃. The next day, cells were centrifuged at 1500 rpm for 15 minutes, fixed cells were collected and washed 2 times with PBS. The cells were resuspended in 300. mu.L PBS and transferred to an EP tube for gentle pipetting. RNase-A was added to a final concentration of about 50% mL in a water bath at 37 ℃ for 30 minutes. About 50. mu.L of PI staining solution was added to a final concentration of about 65% mL and the cells were stained in ice bath for 30 minutes in the absence of light. Filtering with 300 mesh nylon net, and detecting on machine with the detection results shown in Table 2.

TABLE 2 cell cycle test results

	Stage G1 (%)	S period (%)	G2, M period (%)
				Control group	33.55±2.37	39.67±2.86	24.90±1.18
0.2425mg/mL	32.77±2.41	40.13±3.01	27.10±1.29
				0.7275mg/mL	31.27±2.34	41.81±2.79	26.92±1.26
0.156mg/mL	29.12±2.14	45.16±3.11	25.72±1.23
				1.56mg/mL	23.16±1.97	48.03±3.25	28.81±1.42
2.91mg/mL	17.12±1.68	51.23±4.12	31.64±1.53

As shown in table 2, the extract of physochlaina japonica reduces scleral fibroblast division at stage G1 and promotes scleral fibroblast division at stage S. Therefore, the physochlaina extract can influence the growth of scleral fibroblasts in stage G2.

DAPI staining: the cell density before staining was about 10⁵Well, remove complete medium and wash once with PBS; fixing with 10% formaldehyde solution or 4% paraformaldehyde PBS at room temperature for 20 min; rinsing three times with PBS for 10 minutes each time at room temperature; permeabilization with PBS containing 0.5% Triton-X-100 for 15 minutes at room temperature; rinsing three times with PBS for 10 minutes each time at room temperature; blocking with PBS containing 10% NGS for 1 hour at room temperature, or blocking overnight at 4 ℃; incubating with the specific primary antibody for half an hour at 37 ℃, or incubating for more than one hour at room temperature or incubating overnight at 4 ℃; rinsing with PBS containing 0.1% Tween-20 three times for 10 minutes each at room temperature; coating with aluminum foil, and incubating with secondary antibody at 37 deg.C for more than half an hour, or incubating at room temperature for more than one hour; removing the secondary antibody, adding DAPI staining solution, and reacting for more than 15 minutes at room temperature; the experimental results were stored photographically, rinsed three times with PBS at room temperature for 10 minutes each, observed under a fluorescent microscope, excited with the appropriate band.

The results show that scleral fibroblasts are karyotype intact and chromatin is uniform. Therefore, the physochlaina extract has no influence on the nucleus of the scleral fibroblast.

Example 10Application of radix Physochlainae extract in regulating expression of matrix metalloproteinase 2(MMP-2), matrix metalloproteinase inhibitor 2(TIMP-2) and/or transforming growth factor β 2(TGF- β 2)

Preparing 5-7 generation scleral fibroblast into cell suspension, and mixing with 5 × 10³The cell suspension is inoculated in a 96-well plate according to the concentration of each well, the volume of the cell suspension in each well is 100 mu L, each group of cells is repeated in 6 wells, the 96-well plate is placed in a constant temperature incubator after all the cell suspensions are inoculated, the 96-well plate is cultured for 24h, 100 mu L of culture solution containing different concentrations of physochlaina japonica extracts (the concentration is 5.82mg/mL, 2.91mg/mL, 1.56mg/mL, 0.7275mg/mL and 0.2425mg/mL) is respectively added in experimental groups, equal volume of culture solution (1640 culture medium containing 5% fetal calf serum) is added in blank control groups, the 96-well plate is placed at 37 ℃ again, and 5% CO is added in₂And (5) carrying out cell culture in a constant-temperature incubator.

RT-PCR: split with 600. mu.LLysate RLT Plus bleeds the scleral fibroblasts from the plate and the lysate is transferred to a 1.5mL centrifuge tube and thoroughly lysed by vigorous shaking by hand for 20 s. The lysate was then applied to a DNA clean-up column in its entirety. The mixture was centrifuged at 12,000rpm for 60 seconds, and the filtrate was retained. The volume of the filtrate was estimated more accurately using a micropipette, and an equal volume of 590. mu.L 70% ethanol was added, at which time precipitation may occur, but without affecting the extraction process, and immediately whipped into a uniform mix without centrifugation. The mixture was immediately added to the adsorption column RA, centrifuged at 12,000rpm for 30s, and the waste liquid was discarded. Add 700. mu.L of deproteinizing solution RW1, stand at room temperature for 1min, centrifuge at 12,000rpm for 30s, and discard the waste solution. 500. mu.L of the rinsing solution RW was added, and the mixture was centrifuged at 12,000rpm for 30 seconds, and the waste liquid was discarded. Add 500. mu.L of the rinse RW and repeat. The adsorption column RA was returned to the empty collection tube and centrifuged at 12,000rpm for 2min to remove the rinse as much as possible to avoid ethanol remaining in the rinse inhibiting downstream reactions. Taking out the adsorption column RA, placing into RNase Free centrifuge tube, and adding 30 μ L RNase Free H at the middle part of the adsorption membrane according to the expected RNA yield₂O, standing at room temperature for 1min, and centrifuging at 12,000rpm for 1 min. Agarose gel electrophoresis: mu.L of the extracted total RNA was subjected to 1.0% agarose gel electrophoresis at 200V for 10 min.

Reagents used for reverse transcription (AG0304-B) are shown in Table 3.

TABLE 3 reverse transcription reagents and amounts

Real-time PCR (AH0104-B) was performed using SYBR Green I Real-time fluorescent PCR system using the reagents shown in Table 4. The PCR program settings are shown in Table 5, and the primer-related information is shown in Table 6.

TABLE 4 PCR System reagents and amounts

TABLE 5 PCR procedure

And (3) data analysis: and (3) deriving the information such as the original data, the amplification curve, the melting curve and the like from quantitative software for analysis to obtain a relative expression map of the sample gene, as shown in figures 5-7.

TABLE 6 primer sequences

As shown in the results of FIGS. 5 to 7, the extract of radix physochlainae of the present invention with different concentrations can up-regulate the expression of the gene of matrix metalloproteinase inhibitor 2 and down-regulate the expression of the gene of matrix metalloproteinase 2 and transforming growth factor β 2.

Western Blot: scraping cells with a culture medium, collecting cells, collecting 3000g for 15min, removing the culture medium, and leaving cell precipitate; washing with PBS for 1 time; adding 100 μ L RIPA lysate (containing little precipitate in 3 wells and almost no precipitate in 2 wells, respectively adding 50 μ L RIPA lysate) into each well of cells, blowing to make the cells contact with the lysate sufficiently, placing on ice for 30min, and shaking for 2-3 times; 12000rpm, 15min, 4 ℃ centrifugation, supernatant collection, preservation at-20 ℃.

Adding a proper amount of concentrated SDS-PAGE protein loading buffer, such as 5 × or 2 × SDS-PAGE protein loading buffer, into the collected protein samples, reducing the loading volume by using 5 × SDS-PAGE protein loading buffer, loading more protein samples into loading holes with the same volume, and heating at 100 ℃ or in a boiling water bath for 5-10 min to fully denature the protein.

The preparation of the sealing liquid (8% of the defatted milk powder) comprises the steps of weighing 8g of the defatted milk powder in a beaker by using an electronic balance, sucking 100mL of membrane washing liquid (TBST 1 ×) in the beaker, stirring until the milk powder is completely dissolved, placing the PVDF membrane printed with the protein in the sealing liquid, placing the PVDF membrane on a shaking table at room temperature, shaking and sealing for 1.5 hours, and (3) pouring the defatted milk powder after sealing is finished, washing the PVDF membrane on the shaking table by using TBST (1 ×) once every 5min, and washing for 3-5 times.

The method comprises the steps of cutting a PVDF membrane, placing the PVDF membrane containing target protein fragments obtained by cutting into a box, adding diluted antibodies, incubating overnight, removing the primary antibodies after incubation is finished, washing the PVDF membrane with TBST (1 ×) on a shaking table, washing for 3-5 times every 5min, diluting a secondary antibody with 8% skimmed milk powder or a secondary antibody diluent for 5000 times, adding 8% skimmed milk powder containing a secondary antibody into the cleaned box containing the PVDF membrane, slowly incubating for 1h at room temperature, removing the secondary antibody after incubation is finished, washing the PVDF membrane with ST (1 ×) on the shaking table, washing for 3-5 times every 5 min.

The membrane was placed on a luminescent plate, TBST on the membrane was blotted off with filter paper, and a suitable amount of luminescent solution was added to completely cover the PVDF membrane. The results of exposure with the chemiluminescence imager are shown in fig. 8 and table 7.

TABLE 7

	TGF-β/GAPDH	TIMP-2/GAPDH	MMP-2/GAPDH
				Control group	0.82±0.05	0.63±0.07	1.02±0.04
Radix Physochlainae extract (5.82mg/ml)	0.19±0.02	1.25±0.09	0.51±0.03
				Radix Physochlainae extract (2.91mg/ml)	0.26±0.06	1.06±0.08	0.67±0.04
Radix Physochlainae extract (1.56mg/ml)	0.44±0.07	0.97±0.07	0.76±0.05
				Radix Physochlainae extract (0.7275mg/ml)	0.56±0.05	0.88±0.05	0.83±0.07
Radix Physochlainae extract (0.2425mg/ml)	0.72±0.11	0.81±0.03	0.86±0.09

As can be seen from the results in fig. 8 and table 7, the extract of physochlaina japonica of the present invention with different concentrations can promote the expression of matrix metalloproteinase inhibitor 2 protein, and reduce the expression of matrix metalloproteinase 2 and transforming growth factor β 2 protein, and is dose-dependent.

Example 11Effect of Panax schinseng extract on diopter

The head of the rabbit was fixed, the lower eyelid was lifted, the conjunctival sac was pulled into a ring shape, the left eye of the rabbit was used as the eye to be tested, the eyedrops of the extract of physochlaina sinica diels (4%, 2%, 1% and 0.5%), 3 subjects/group, 1 d/time, and 14d were administered continuously and observed at 7d and 14d, respectively, in the darkroom, with a strip-shaped optical microscope for examination (the working distance was kept at 0.5m), and the OD (right-eye vision) and OS (left-eye vision) results, which were measured using the average of the measured values of the two major meridians, i.e., vertical and horizontal, are shown in table 8. The light reflection by the rabbit eyes before and after the intervention is shown in FIG. 9.

TABLE 8

As can be seen from the results in Table 8 and FIG. 9, the extract of radix physochlainae (4%, 2%, 1% and 0.5%) has mydriasis effect at different concentrations, and the 1% and 0.5% of the extract of radix physochlainae can affect the diopter of rabbit eyes with a difference of about 2D before and after intervention.

Example 12Cytotoxicity study of extract of radix physochlainae

Human ARPE-19 cells were cultured in DMEM/high glucose medium. The medium contained 10% fetal bovine serum, 0.4% penicillin and streptomycin. The culture conditions were 37 ℃ and 5% carbon dioxide. The cytotoxicity of the extract of the mountain ginseng was examined using the human ARPE-19 cell line. ARPE-19 cells in logarithmic growth phase were seeded at 8000/well in 96-well plates, cultured overnight, and then different concentrations of the extract of Physochlaina sinica Stapf were added and the culture was continued for 72 hours. Three replicates per treatment concentration were incubated. After the culture time is over, 20 mu L of MTT is added for continuous culture for 4 hours, then the solution is discarded in each hole, 150 mu L of DMSO is added for dissolution, optical density detection is carried out by a microplate reader, and the cell survival rate is calculated. The experiment was repeated three times, and the measurement results are shown in fig. 10. As shown in FIG. 10, the concentration of the extract of Physochlaina warrior was not toxic to the cells, and the in vitro IC50 experiment showed that no significant cytotoxicity was observed in the 200. mu.g cell study (2%).

Example 13Studies on irritation of extract of mountain ginseng

Irritation test:

taking 6 New Zealand white rabbits, half female and half male, adopting a self-contrast method of the same body, dripping 1 drop of radix physochlainae eye drops with different concentrations (2%, 1% and 0.5%) into the left eye, lightly closing the eyelid for 10 seconds, dripping 1 drop of physiological saline into the right eye as a control, and observing the local reaction condition of the eye 6h, 24h, 48h and 72h to 168h after administration.

After administration for 6h, 24h, 48h, 72h to 168h, no damage is seen to cornea, conjunctiva and iris, no degeneration, necrosis, desquamation, local ulcer or erosion of rabbit conjunctiva epithelial cells is caused after administration, no congestion, edema and obvious inflammatory cell infiltration of subcutaneous tissues are caused, and the radix physochlainae eye drops have no eye irritation. Therefore, the experiment proves that the radix physochlainae extract has good tolerance.

Example 14

The composition for preventing and/or treating myopia comprises the following components in parts by mass: 0.01 part of anisodamine, 0.1 part of scopolamine, 0.5 part of atropine and 200 parts of scopoletin.

Example 15

The composition for preventing and/or treating myopia comprises the following components in parts by mass: 200 parts of anisodamine, 0.01 part of scopolamine, 0.1 part of atropine and 0.5 part of scopoletin.

Example 16

The composition for preventing and/or treating myopia comprises the following components in parts by mass: 0.1 part of anisodamine, 200 parts of scopolamine, 0.01 part of atropine and 0.1 part of scopoletin.

Example 17

The composition for preventing and/or treating myopia comprises the following components in parts by mass: 0.5 part of anisodamine, 0.5 part of scopolamine, 200 parts of atropine and 0.01 part of scopoletin.

Example 18

The composition for preventing and/or treating myopia comprises the following components in parts by mass: 30 parts of anisodamine, 50 parts of scopolamine, 30 parts of atropine and 50 parts of scopoletin.

Example 19

The composition for preventing and/or treating myopia comprises the following components in parts by mass: 50 parts of anisodamine, 30 parts of scopolamine, 50 parts of atropine and 30 parts of scopoletin.

Comparative example 1

The composition comprises the following components in parts by mass: 80 parts of scopolamine, 50 parts of atropine and 30 parts of scopoletin.

Comparative example 2

The composition comprises the following components in parts by mass: 80 parts of anisodamine, 50 parts of atropine and 30 parts of scopoletin.

Comparative example 3

The composition comprises the following components in parts by mass: 50 parts of anisodamine, 60 parts of scopolamine and 50 parts of atropine.

Experimental example 2Study on effect of radix physochlainae extract on treatment of myopia

Selecting healthy British three-color short-hair guinea pigs of 2 weeks old, carrying out adaptive breeding for 3D, randomly grouping a model group and an experimental group, 10 animals/group, starting from the 1 st week, wearing 6.0D lenses on the right eyes of the three-color short-hair guinea pigs of the experimental group except the model group, taking the left eyes as self contrast and not carrying out any treatment on the model group, respectively measuring the diopter and the length of the axis of the eye of each group after wearing the glasses for 2 weeks, and observing the molding condition of the myopia. Beginning at week 3, the guinea pigs in the test group were administered with the eyedrops of the extract of mountain ginseng in the right eye for 1 time/day for 4 weeks, the contralateral eyes were used as self-control without any treatment, while the eyes were continuously worn, and the diopter and the length of the axis of the eye were measured after 4 weeks, with the results shown in Table 9.

TABLE 9 therapeutic Effect of the embodiments of the present invention on myopia

As shown in Table 9, the radix physochlainae extract eye drops can reduce the diopter of the myopic guinea pig and the length of the ocular axis, and have better treatment effect.

The above description is only an example of the present application, and the protection scope of the present application is not limited by these specific examples, but is defined by the claims of the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.

Claims

1. Application of radix Physochlainae extract in preparing medicine for preventing and/or treating myopia is provided.

2. The use according to claim 1, wherein the radix physochlainae extract comprises 0.01-200 mg/g anisodamine, 0.01-200 mg/g scopolamine, 0.01-200 mg/g atropine and 0.01-200 mg/g scopoletin;

preferably, the radix physochlainae extract comprises 0.1-50 mg/g of anisodamine, 0.1-50 mg/g of scopolamine, 0.1-50 mg/g of atropine and 0.1-50 mg/g of scopoletin;

more preferably, the radix physochlainae extract comprises 0.5-30 mg/g of anisodamine, 0.5-30 mg/g of scopolamine, 0.5-30 mg/g of atropine and 0.5-30 mg/g of scopoletin.

3. The use according to claim 1 or 2, wherein the extract of physochlaina is obtained by subjecting physochlaina to solvent extraction;

preferably, the solvent is selected from at least one of water, methanol, ethanol, petroleum ether, n-hexane, acetone, dichloromethane and ethyl acetate;

preferably, the proportion of the mountain warrior attendant solvent is 1 g: 1-50 mL;

preferably, the extraction is at least one selected from the group consisting of percolation extraction, heating reflux extraction, ultrasonic extraction and decoction extraction.

4. Application of radix Physochlainae extract in regulating and controlling growth of scleral fibroblast and/or retinal pigment cell is provided.

5. The use of claim 4, wherein the selected modulation of scleral fibroblast and/or retinal pigment cell growth is inhibition of scleral fibroblast and/or retinal pigment cell growth.

6. Application of radix Physochlainae extract in regulating expression of matrix metalloproteinase 2, matrix metalloproteinase inhibitor 2 and/or transforming growth factor β 2 is provided.

7. The use of claim 6, wherein said modulating matrix metalloproteinase 2, matrix metalloproteinase inhibitor 2, and/or transforming growth factor β 2 expression is modulating matrix metalloproteinase 2, matrix metalloproteinase inhibitor 2, and/or transforming growth factor β 2 gene and/or protein expression;

preferably, the expression of the genes of the matrix metalloproteinase 2 and/or the transforming growth factor β 2 is regulated to down regulate the expression of the genes of the matrix metalloproteinase 2 and/or the transforming growth factor β 2, and the expression of the genes of the matrix metalloproteinase inhibitor 2 is regulated to up regulate the expression of the genes of the matrix metalloproteinase inhibitor 2.

8. The composition for preventing and/or treating myopia is characterized by comprising the following components in parts by mass: 0.01-200 parts of anisodamine, 0.01-200 parts of scopolamine, 0.01-200 parts of atropine and 0.01-200 parts of scopoletin;

preferably, the composition comprises the following components in parts by mass: 0.1-50 parts of anisodamine, 0.1-50 parts of scopolamine, 0.1-50 parts of atropine and 0.1-50 parts of scopoletin;

more preferably, the composition comprises the following components in parts by mass: 0.5-30 mg/g anisodamine, 0.5-30 mg/g scopolamine, 0.5-30 mg/g atropine and 0.5-30 mg/g scopoletin.

9. An ophthalmic formulation comprising the composition of claim 8, wherein said ophthalmic formulation further comprises water and an adjuvant; the mass ratio of the composition to the auxiliary materials to the water is 0.01-10: 0.1-10: 10 to 100 parts;

preferably, the ophthalmic formulation is selected from at least one of eye drops, ophthalmic gel and ophthalmic cream;

more preferably, the ophthalmic formulation is an eye drop.

10. Use of the composition of claim 8 or the ophthalmic formulation of claim 9 for the preparation of a medicament for the prevention and/or treatment of myopia.