WO2021195904A1

WO2021195904A1 - Use of composition containing astragali radix, puerariae lobatae radix and erigerontis herba

Info

Publication number: WO2021195904A1
Application number: PCT/CN2020/082231
Authority: WO
Inventors: 孙克宏; 秦少容; 白礼西; 段俊国; 杨巧巧; 郑飞鸣; 卿玉玲; 黄静; 彭涛; 金思岑
Original assignee: 重庆太极实业（集团）股份有限公司; 重庆太极医药研究院有限公司; 成都中医药大学
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2021-10-07

Abstract

Provided is the use of a composition containing Astragali radix, Puerariae lobatae radix and Erigerontis herba in the preparation of a drug for treating glaucoma and ocular hypertension in individuals.

Description

Use of a composition containing astragalus, kudzu root and breviscapine

Technical field

This application relates to a composition containing astragalus, kudzu root and breviscapine and the use thereof. Specifically, the application relates to the use of a composition containing astragalus, kudzu root and breviscapine in the treatment of ophthalmic diseases.

Background technique

Glaucoma is a group of diseases characterized by atrophy and depression of the optic papilla, visual field defects, and decreased vision. Pathological increase in intraocular pressure and insufficient blood supply to the optic nerve are the primary risk factors for its onset. The tolerance of the optic nerve to pressure damage is also similar to that of glaucoma. The occurrence is related to development. Obstruction of any link in the aqueous humor circulation can lead to increased intraocular pressure and cause pathological changes. However, some patients have normal intraocular pressure glaucoma. Glaucoma is one of the three major blindness diseases that cause blindness in humans, with an incidence of 1% in the total population and 2% after the age of 45. Clinically, glaucoma is divided into primary, secondary, congenital, and mixed types based on the cause, angle of the chamber, and intraocular pressure.

Pathological increase in intraocular pressure is the main risk factor for glaucoma. Increased intraocular pressure leads to optic nerve damage through two mechanisms: mechanical compression and optic nerve ischemia. The longer the increase in intraocular pressure lasts, the more severe the damage to visual function. The reason for the increased intraocular pressure in glaucoma is that the dynamic balance of aqueous humor circulation is disrupted. A few are due to excessive secretion of aqueous humor, but most of them still have obstacles to the outflow of aqueous humor, such as narrow or even closed anterior chamber angle, hardening of the trabeculae, etc.

Primary glaucoma is divided into angle-closure glaucoma and open-angle glaucoma according to the state of the anterior chamber angle when intraocular pressure is increased. Angle-closure glaucoma is divided into acute angle-closure glaucoma and chronic angle-closure glaucoma according to the rapid onset of disease. glaucoma.

Glaucoma is one of the main causes of blindness in my country, and the damage to visual function caused by glaucoma is irreversible and the consequences are extremely serious. Generally speaking, glaucoma cannot be prevented, but if it is detected early and treated appropriately, most patients can maintain useful visual function throughout their lives. Therefore, the prevention of blindness in glaucoma must emphasize early detection, early diagnosis and early treatment. The main purpose of treatment is to reduce intraocular pressure, reduce eye tissue damage, and protect visual function.

Traditional Chinese medicine is gradually used for the treatment of various ophthalmic diseases due to its high safety. However, due to the complex composition of natural medicines, the compatibility of medicines may produce different effects. At present, there is no Chinese medicine as raw materials for effective treatment of glaucoma or high eyes. Related reports of stress disorder.

Summary of the invention

In the first aspect, the present application provides the use of a composition comprising astragalus, pueraria lobata and breviscapine in the preparation of a medicament for treating glaucoma in an individual.

In the second aspect, the present application provides a method for treating glaucoma, which comprises administering to an individual a therapeutically effective amount of a composition comprising astragalus, kudzu root and breviscapine.

In the third aspect, the present application provides a composition comprising astragalus, kudzu root and breviscapine, which is used for the treatment of glaucoma.

In a fourth aspect, the present application provides a composition comprising astragalus, kudzu root and breviscapine.

In certain embodiments, the aforementioned glaucoma is ocular hypertension glaucoma. In certain embodiments, the glaucoma is selected from primary glaucoma, secondary glaucoma, congenital glaucoma, or mixed glaucoma.

In a preferred embodiment, the glaucoma is open-angle glaucoma or closed-angle glaucoma.

In certain embodiments, the above-mentioned composition and a pharmaceutically acceptable carrier are formulated into a pharmaceutically acceptable dosage form, preferably a suspension, powder, tablet, granule, pill, syrup or eye drops, more Preferably it is a suspension.

In certain embodiments, the above-mentioned composition is administered by gavage, oral administration, eye drops, enema, subcutaneous, intramuscular, or intraperitoneal administration; preferably, the composition is administered by gavage.

In some embodiments, the dosage of the composition is 100 to 1000 mg/kg body weight, preferably 300 to 600 mg/kg body weight per day.

In certain embodiments, the composition is for administration 1 to 3 times a day, for example, once a day by gavage.

In some embodiments, the above composition includes the following raw materials in the following weight ratio: 10-40 parts of Astragalus, 10-30 parts of Pueraria lobata, and 10-30 parts of Erigeron breviscapus.

In some embodiments, the above-mentioned composition contains the following active ingredients in the following weight ratio: 1-10 parts of Astragalus extract, 50-125 parts of Pueraria lobata extract, and 20-60 parts of Dengzhan extract.

In some embodiments, the medicament of the present application is prepared from raw materials of Astragalus, Pueraria lobata and Erigeron breviscapus or water or organic solvent extracts of Astragalus, Pueraria lobata and Erigeron breviscapus as active ingredients, plus a pharmaceutically acceptable carrier. Become. In some embodiments, the raw materials of Astragalus, Pueraria lobata and Erigeron breviscapus in a certain weight ratio are weighed to prepare extracts of Astragalus, Pueraria lobata and Erigeron breviscapus, respectively, and other active ingredients or pharmaceuticals are added according to the preparation method of conventional Chinese medicine formulations. The above-acceptable carriers are made into different dosage forms, such as suspensions, powders, tablets, granules, pills, eye drops and other dosage forms.

In addition, the application also provides the use of a composition comprising astragalus, pueraria lobata and breviscapine in the preparation of a medicament for the treatment of ocular hypertension in an individual, as well as a method for the treatment of ocular hypertension in an individual, which comprises administering to the individual A therapeutically effective amount of a composition comprising astragalus, kudzu root and breviscapine.

Description of the drawings

Figure 1 shows the individual data of each group of animal eye examinations.

Figure 2 shows the individual data of OCT and HRT examination of the anterior segment of each group of animals.

Figures 3A-D show individual data of intraocular pressure measurement (mmHg) of animals in each group.

Figures 4A-D show individual data on the percentage reduction in intraocular pressure (ΔIOP%) of each group of animals.

Figure 5 shows statistical data of the percentage decrease in intraocular pressure (ΔIOP%) in male animals.

Figure 6 shows statistical data of the percentage decrease in intraocular pressure (ΔIOP%) of female animals.

Detailed ways

The inventor of the present application unexpectedly discovered that a composition comprising astragalus, kudzu root and breviscapine can treat glaucoma, such as high intraocular pressure glaucoma.

Unless otherwise specified, all terms in this application have the meanings commonly understood by those skilled in the art.

The application provides the use of a composition comprising astragalus, kudzu root and breviscapine in the preparation of a medicament for the treatment of glaucoma.

In certain embodiments, the glaucoma is ocular hypertension glaucoma. In certain embodiments, the glaucoma is selected from primary glaucoma, secondary glaucoma, congenital glaucoma, or mixed glaucoma.

In certain embodiments, the glaucoma is open-angle glaucoma or closed-angle glaucoma.

"Treatment" as used herein includes inhibiting, curing, alleviating, alleviating or delaying glaucoma and related symptoms.

In some embodiments, the composition of the present application is formulated into a pharmaceutically acceptable dosage form together with a pharmaceutically acceptable carrier, preferably a suspension, eye drops, oral liquid, capsule, tablet or granule, More preferably, it is a suspension suitable for gastric gavage.

The "pharmaceutically acceptable carrier" mentioned in this application refers to a carrier that does not interfere with the effectiveness of the biological activity of the active ingredient, including those conventionally used in the pharmaceutical field. The pharmaceutically acceptable carrier of the present application can be solid or liquid, and includes pharmaceutically acceptable excipients, buffers, emulsifiers, stabilizers, preservatives, diluents, encapsulants, fillers, and the like. For example, pharmaceutically acceptable buffers include phosphate, acetate, citrate, borate, and carbonate.

The composition of the present application can be presented in a unit dosage form, and can be prepared by any method known in the pharmaceutical art. All methods include the step of combining the active ingredient of the present application with one or more pharmaceutically acceptable carriers. Generally, the composition is prepared by combining the active ingredient with a liquid carrier, a solid carrier, or both, and then the prepared product is shaped as needed. The specific formulation method depends on the chosen route of administration. In some embodiments, the composition can be manufactured by conventional mixing, dissolving, granulating, tableting, grinding, emulsifying, encapsulating, capturing, or freeze-drying methods.

In some embodiments, the composition is administered by gavage, oral, eye drops, enema, subcutaneous, parenteral, intravenous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular administration . In a specific embodiment, the combination is administered by gavage or orally.

In certain embodiments, the administration dose of the composition of the present application is 50 to 1000 mg/kg body weight per day, preferably 100-800 mg/kg body weight, more preferably 300-600 mg/kg body weight, such as 300, 350, 400, 450, 500, 550, 600 mg/kg body weight or a dose between any two of the above values.

In some embodiments, the composition is administered 1, 2, 3, 4 or more times a day. The composition may also be administered less than once a day, for example, every 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the number of daily administrations is constant (e.g., once or twice daily). In other embodiments, the number of administrations is variable. The number of administrations may vary according to the effectiveness of the composition, observed side effects, specific routes of administration, specific characteristics of the individual, individual health history and risk factors (such as age, weight, general health, etc.), or dosage form.

In some embodiments, the duration of administration of the composition is 1-12 weeks, such as 1, 2, 3, 4, 6, 8, 10, 12 weeks, or the number of weeks between any two of the above values, Or about the time period. In some embodiments, the composition is continuously administered for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or more weeks. In some embodiments, administration of the composition is continued until the individual no longer requires treatment.

In another aspect, the present application provides a method for treating glaucoma, which comprises administering to an individual a therapeutically effective amount of a composition comprising astragalus, kudzu root and breviscapine.

In yet another aspect, a therapeutically effective amount of a composition comprising astragalus, kudzu root and breviscapine for treating glaucoma in an individual is provided.

The "therapeutically effective dose" used herein can be determined according to specific conditions, and those of ordinary skill in the art can easily grasp the actual required drug dose, for example, it can be determined according to the patient's weight, age, and disease conditions. When the composition contains pharmaceutically acceptable carriers, they can be mixed according to conventional methods in the pharmaceutical field to prepare the desired drug.

In some embodiments, the composition of the application comprising astragalus, kudzu root and breviscapine can improve the intraocular pressure of glaucoma patients. In a specific embodiment, the composition significantly reduces the intraocular pressure of glaucoma patients without significant side effects. In a specific embodiment, the composition of the present application has a dose-effect relationship in reducing intraocular pressure.

In some embodiments, the composition of the present application comprising Astragalus, Pueraria lobata and Erigeron breviscapus can reduce the ocular hypertension induced by dexamethasone in SD rats. In a specific embodiment, only 3-4 days after administration, it exhibits a significant effect of reducing intraocular pressure, which is significantly different from the control group. In a specific embodiment, the effect of reducing intraocular pressure of the above composition is independent of the sex of the affected individual, for example, it is effective for both female rats and male rats.

In some embodiments, the composition of the application comprising Astragalus, Pueraria lobata and Erigeron breviscapus has no significant difference in the incidence of adverse events and adverse reactions in the glaucoma used to treat individuals compared with the negative control treatment group, indicating that this The applied drug has no obvious side effects in the treatment of glaucoma and has high safety.

The "individual" mentioned in this application refers to mammals, including primates and non-primates, such as humans, apes, monkeys, cows, horses, pigs, sheep, goats, dogs, cats, and such as rats And mice, rodents, etc.

In yet another aspect, the present application provides a composition comprising Astragalus, Pueraria lobata and Erigeron breviscapus. In a specific embodiment, the composition described above is used to treat or prevent glaucoma in an individual.

In some embodiments, the composition of the present application is prepared from raw materials containing the following weight ratios: 10-40 parts of Astragalus, 10-30 parts of Pueraria lobata, and 10-30 parts of Erigeron breviscapus.

In some embodiments, the composition of the present application is prepared from raw materials containing the following weight ratios: 20-30 parts of Astragalus, 20-40 parts of Pueraria lobata, and 20-30 parts of Erigeron breviscapus.

In some embodiments, the composition of the present application is prepared from raw materials containing the following weight ratios: 20 parts of Astragalus, 30 parts of Pueraria lobata, and 20 parts of Erigeron breviscapus.

In some embodiments, the composition of the present application is prepared from raw materials or water or organic solvent extracts of Astragalus, Pueraria lobata, Erigeron breviscapus as active ingredients, plus pharmaceutically acceptable excipients or auxiliary ingredients.

In some embodiments, the composition comprises the following components in a weight ratio: 1-10 parts of Astragalus extract, 50-125 parts of Pueraria lobata extract, and 20-60 parts of Dengzhan extract.

In some embodiments, the composition comprises astragalus extract, pueraria lobata extract, breviscapine extract, wherein the pueraria lobata extract contains not less than 60% total isoflavones calculated as puerarin, and contains no less than puerarin 20%; Astragalus extract contains not less than 50% total saponins calculated as astragaloside IV, and not less than 5% astragaloside IV; Erigeron breviscapus extract contains not less than 35% total flavonoids calculated as rutin, including wild Baicalin is not less than 3%.

In some embodiments, the weight ratio of the composition is: 1-10 parts of Astragalus extract, 50-125 parts of Pueraria lobata extract, and 20-60 parts of Dengzhan extract.

In some embodiments, the weight ratio of the composition is: 1 part of Astragalus extract, 70 parts of Pueraria lobata extract, and 25 parts of Dengzhan extract.

In a specific embodiment, the preparation method of the composition comprising Astragalus, Pueraria lobata and Erigeron breviscapus in this application includes the following steps:

a. Weigh the raw materials with the following weight ratio:

10-40 parts of Astragalus, 10-30 parts of Pueraria, 10-30 parts of Erigeron;

b. Pueraria lobata pulverized into coarse powder, extracted with 60-95% ethanol, filtered, combined the filtrate, recovered ethanol, concentrated, passed through a macroporous adsorption resin column, eluted with water, discarded the water eluate, and reused 10-95 % Ethanol elution, collect ethanol eluate, recover ethanol, concentrate into thick paste, dry under reduced pressure, pulverize into fine powder to obtain Pueraria lobata extract;

c. Astragalus is crushed into coarse powder, extracted with 60-95% ethanol under heating and refluxing, filtered, combined with the filtrate, recovered ethanol, concentrated, sodium hydroxide is added to the alkali content of 2-5%, and n-butanol is added for dynamic extraction 2 -5 times, combine the n-butanol solution, wash with 2-5% sodium hydroxide solution, then wash with water, discard the lotion, combine the n-butanol solution, concentrate under reduced pressure to near dryness, add acetone to grind and wash until the color is light, take The precipitate was dried under reduced pressure and pulverized into fine powder to obtain an Astragalus extract;

d. Decoction of Erigeron breviscapus with water, combine the decoction, filter, concentrate and pass through a macroporous adsorption resin column, eluate with water, discard the water eluate, continue to elute with 10-95% ethanol, collect ethanol for elution Liquid, recovered ethanol, concentrated into a thick paste, dried under reduced pressure, pulverized into fine powder, to obtain Erigeron breviscapus extract;

e. Mix the astragalus extract, pueraria lobata extract and breviscapine extract prepared in steps b, c, and d, and add a pharmaceutically acceptable carrier to prepare a commonly used pharmaceutical preparation.

In some embodiments, in step a, the weight ratio of the raw materials may be 20-30 parts of Astragalus, 20-40 parts of Pueraria lobata, and 20-30 parts of Erigeron breviscapus. Among them, Astragalus can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and Pueraria can be 20, 22, 24, 26, 28, 30, 32, 34, 36, 38. Or 40 parts, Erigeron breviscapus can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 parts. In a specific embodiment, the weight ratio of the raw materials may be 20 parts of Astragalus, 30 parts of Pueraria lobata, and 20 parts of Erigeron breviscapus.

Within the reasonable range of the selection of the raw materials of the drug in this application, according to the drug extraction and processing method of this application, it can be prepared into a pharmaceutical commonly used preparation. For example, according to the aforementioned method, the obtained dry fine powder with the three herbs as the main component is added with auxiliary materials according to the preparation method of traditional Chinese medicine dosage forms to make different dosage forms, such as suspensions, tablets, granules, pills, capsules and eye drops. Agents and other dosage forms.

In some embodiments, 30 kg of Astragalus, 40 kg of Pueraria lobata, and 30 kg of Erigeron breviscapus are weighed, and powdered directly to obtain a powder. Before use, add a suitable solvent and mix to obtain a suspension for later use.

In some embodiments, 1 kg of the aforementioned astragalus extract, 50 kg of Pueraria lobata extract, and 30 kg of Erigeron breviscapus extract are taken, mixed, starch is added, granulated, granulated, magnesium stearate is added, and tablets are compressed to obtain tablets.

In some embodiments, 10 kg of the above-mentioned astragalus extract, 125 kg of Pueraria lobata extract, and 60 kg of Erigeron breviscapus extract are mixed, and starch is added, granulated, sized, and directly filled into capsules to obtain capsules.

In some embodiments, 2 kg of the above-mentioned astragalus extract, 80 kg of Pueraria lobata extract, and 40 kg of Erigeron breviscapus extract are mixed, and starch is added, granulated, granulated, and directly encapsulated to obtain pharmaceutical granules.

In some embodiments, 30 kg of Astragalus, 40 kg of Pueraria lobata, and 30 kg of Erigeron breviscapus are extracted according to the above method, and 20 kg of Rehmannia glutinosa, 20 kg of wolfberry, 15 kg of cassia, and 20 kg of Fuwei seed are decocted and concentrated. Mixing, adding starch, granulating, granulating, adding magnesium stearate, and compressing to obtain tablets.

In some embodiments, the composition containing Astragalus, Pueraria lobata, Erigeron breviscapus is first made into powder, and then a suitable solvent is added and mixed to obtain a suspension for later use.

The preparation method of the composition of the present application can also refer to the method in Chinese Patent CN100594913C, the entire content of which is incorporated herein by reference in its entirety.

In the specification and claims of this application, the words "including", "including" and "containing" mean "including but not limited to", and are not intended to exclude other parts, additives, components, or steps.

It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of this application can be applied to any other aspect, embodiment or example described herein unless contradictory thereto.

Unless otherwise stated, all numbers of characteristics, items, quantities, parameters, properties, terms, etc. used in this specification and claims should be understood as being modified by the term "about" in all cases. As used herein, the term "about" means that the defined characteristic, item, quantity, parameter, property, or term encompasses plus or minus percentages of the value of the characteristic, item, quantity, parameter, property, or term that is higher or lower The scope of tenth. Therefore, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximate values that can be varied.

The above disclosure describes the application as a whole, and the application is further illustrated by the following examples. The description of these embodiments is only to illustrate the application, but not to limit the scope of the application. Although special terms and values are used herein, these terms and values are also understood to be exemplary and do not limit the scope of the application. Unless otherwise specified, the experimental methods and techniques in this specification are methods and techniques known to those skilled in the art.

Example

The following examples are provided only to illustrate some embodiments of the present application, without any restrictive purpose.

Unless otherwise specified, the methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: Preparation of a composition containing Astragalus, Pueraria lobata and Erigeron breviscapus

Weigh 30 kg of Pueraria lobata, 20 kg of Erigeron breviscapus 20 kg, and 20 kg of Astragalus membranaceus to prepare fine powders as follows.

Pueraria lobata was crushed into coarse powder, extracted twice with 60% ethanol at reflux for 1 hour each time, filtered, combined the filtrate, recovered ethanol, concentrated appropriately, added to the treated macroporous adsorption resin column, and eluted with water. Discard the water eluent, continue to elute with 70% ethanol, collect the ethanol eluent, concentrate under reduced pressure into a thick paste, dry under reduced pressure, and pulverize into fine powder to obtain Pueraria lobata extract;

Astragalus was pulverized into coarse powder, extracted with 90% ethanol under heating and refluxing for three times, 1 hour each time, filtered, combined the filtrates, recovered ethanol, concentrated appropriately, added sodium hydroxide to 5% alkali content, and dynamically extracted with n-butanol Twice, combine the n-butanol solution, wash with 5% sodium hydroxide solution once, and wash twice with water, discard the lotion, combine the n-butanol solution, concentrate under reduced pressure to near dryness, add acetone to grind and wash until the color is light, take The precipitate was dried under reduced pressure and pulverized into fine powder to obtain an Astragalus extract;

Decoction of Erigeron breviscapus with water for three times, 1 hour each time, combine the decoction, filter, and concentrate appropriately, add to the treated macroporous adsorption resin column, elute with water, discard the water eluent, and continue to use 70 % Ethanol elution, collect the ethanol eluate, recover the ethanol, concentrate into a thick paste, dry under reduced pressure, and pulverize into fine powder to obtain the Erigeron breviscapus extract;

Mix the above three kinds of fine powders with sodium starch glycolate, hydroxypropyl cellulose, micronized silica gel, and magnesium stearate to obtain yellow-brown to light brown powders containing astragalus, kudzu root and breviscapine for use.

Example 2: The therapeutic effect of the composition of the present application on glaucoma

2.1 Test plan design

1) Experimental animals

40 Sprague-Dawley (SD) rats were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., with half males and half males. The animal grade is SPF grade. Qualified feed (purchased from Beijing Keao Xieli Feed Co., Ltd.) is provided every day. Animals eat and drink freely. Beijing Keyao Xieli Feed Co., Ltd. and Pony tested the nutrient components (main test components: crude protein, crude fat, crude fiber, moisture, calcium, total phosphorus, crude ash) and physical and chemical indicators (mainly tested) of each batch of feed. Indicators: arsenic, lead, mercury, cadmium, BHC, DDT, aflatoxin B1) for testing, refer to China's national standards GB14924.2-2001 and GB14924.3-2010 and GB5749-2006 to ensure that there is no influence or interference test The resulting contaminants are present. The starting age (D1) of the experimental animals was 10-12 weeks of age. Before the administration (D1 day), the weight of male animals was 298-358g, and the weight of female animals was 174-239g.

2) Animal modeling

For the 40 SD rats mentioned above, dexamethasone (purchased from Chongqing Taiji Pharmaceutical Research Institute Co., Ltd., batch number 20180812, specification 5mL:5mg) was used to create a model, twice a day, 15μL each time, and continue until the test Finish.

3) Animal grouping and dosing schedule

After 16 days of modeling, according to the intraocular pressure measured before the administration, 30 animals that passed the quarantine and had no abnormal eyes were selected for testing and grouping. Using the computer system, the animals were randomly divided into 3 groups (groups 1-3) according to gender. , 10 per group. The order of administration is: normal saline (10mL/kg), 3% test substance suspension (300mg/kg), 6% test substance suspension (600mg/kg), which will be in accordance with Example 1. Add 3 g of the powder prepared by the method to 100 mL of physiological saline and shake to obtain a 3% suspension of the test substance, and add 6 g of the above powder to 100 mL of physiological saline and shake to obtain a 6% suspension of the test substance.

All groups were given intragastric administration once a day for 30 consecutive days.

Administration method: animals in the negative control group were given physiological saline, and animals in the low-dose and high-dose groups of the test article were given different concentrations of the test article. Use disposable 1mL, 2mL or 5mL sterile syringes and 16 gauge gavage needle for oral administration. The dosage of each animal needs to be adjusted according to the body weight recently measured before the administration. The volume of administration should be 1 or 2 digits after the decimal point. The test product is in a suspended state during the administration.

2.2 Indices for live animal detection

A. General clinical observation

During the quarantine period and test period of all animals, observe the death, illness, respiration, secretions, feces, diet, and drinking water once a day.

B. Local eye observation

During the test, local eye observations were performed once a day. The eyelids, conjunctiva, cornea, anterior chamber, iris, pupil and their reflection on the pupil and the pupillary penlight were observed with the naked eye.

C. Weight

The animals are weighed when they are received, before grouping, and once a week after grouping. They are planned to be weighed before euthanasia, and they are also measured when they are found dead or dying.

D. Eye examination

Inspection time: 1 time before modeling and after the last IOP measurement.

Inspection method: After the animal is dilated, use a slit lamp to observe the animal’s eyelids, conjunctiva, sclera, cornea, anterior chamber, iris, pupil and its posterior refractive interstitium, and use ophthalmoscope to observe the fundus. Use a slit lamp microscope system to acquire images. Use standardized language to describe and classify ocular abnormalities, and classify ocular abnormalities according to the 4-level method (mild, mild, moderate, and severe).

E. Measurement of intraocular pressure

One week before modeling (not less than 4 days), all animals were trained to measure intraocular pressure in the awake state, once a day in the morning and in the afternoon (the measurement time should be controlled at 9:00 in the morning and 14:00 in the afternoon). After the pressure is stabilized, the basic intraocular pressure is calculated and the model is made.

After the model is made, the intraocular pressure is measured at least 3 times a week (in the morning, once every other day), until the model is divided into groups.

After group administration, 3 times a week, namely D1, D3, D5, D8, D10, D12, D15, D17, D19, D22, D24, D26, D29, D31, the intraocular pressure was measured once a day in the morning.

Measurement method: Put the animal in a fixed box, drop 1 to 2 drops of 4% oxybucaine hydrochloride eye drops for epithelial anaesthesia, and then use a rebound tonometer to measure the binocular intraocular pressure, and get each eye each time After taking the average of the 2 valid data, get the intraocular pressure of the eye.

F. OCT and HRT examination of the anterior segment

Inspection time: 3 times a week after administration, up to 30 days (D31)

Check animals: all animals in the low-dose group and high-dose group of the test product

Check the eyes: both eyes

Use 1 to 2 drops of 0.5% compound tropikamide eye drops to dilate the eyes of all animals, and intramuscularly inject Shutai TM 50 (40mg/kg, 50mg/mL) to anesthetize the animals. If the animals are not anesthetized within 5 minutes , According to the dose of 5 to 10%, additional anesthesia 1 to 2 times. Use optical coherence tomography (OCT) to examine the animal's cornea, and perform Heidelberg retinal tomography (HRT) to examine the full-thickness cornea.

2.3 Test indicators after animal death

A. Euthanasia of animals

According to the 2013 version of the AVMA Animal Euthanasia Guidelines (the American Veterinary Medical Association, 2013), all animals were euthanized by carbon dioxide inhalation anesthesia after the end of the experiment, bloodletting of the abdominal aorta and open thoracic cavity.

B. General observation

General observation: All animals in groups 1 to 3 (including animals found dead and euthanized animals) were observed in general. It was found that animals died after non-working hours were stored in a refrigerator at 2-8°C, and dissected as soon as possible within 24 hours. After dissecting and collecting the tissue, the animal carcass is treated as medical waste.

During the dissection, observe whether the animal's body surface, orifices, head, chest, abdomen, pelvis, etc. and its contents are abnormal. Take the animal’s eyeballs and place them in Davidson’s solution for fixation.

C. Histopathology

Take the fixed animal eyeballs for routine paraffin embedding, sectioning, and HE staining, and observe the morphological changes of each part of the eyeball, especially the cells of each layer of the retina under a light microscope. Use standard terminology to diagnose and classify, and use four-level method (mild, mild, moderate, severe) to classify the symptoms seen under the microscope.

2.4 Data collection and statistics

Data collection: Write down the measured and observed data results required by the test plan on an appropriate table or collect data directly through a computer.

Statistical analysis: In this experiment, statistical software SPSS13.0 was used to process the data. All statistical analysis adopts two-tailed analysis, and the statistical level is set at p≤0.05. Calculate the mean (Mean) and standard deviation (SD) of the intraocular pressure of animals in different groups. The above data will be analyzed according to the following process: First, use Levene Test to test the homogeneity of the data, if the data is uniform (p>0.05), then perform a one-way analysis of variance; if the analysis of variance is significant (p≤0.05), select the appropriate statistics Learn how to make multiple comparisons. If the result of Levene Test is significant (p≤0.05), perform Kruskal-wallis non-parametric test. If the Kruskal-wallis non-parametric test result is significant (p≤0.05), then the Mann-Whitney U test is further used for pairwise comparison.

2.5. Test results

1) Eye examination

The individual data of each group of animal eye examination is shown in Figure 1. There was no abnormality in the ophthalmological examinations of all animals before and after the administration.

2) OCT and HRT examination of the anterior segment

The individual data of the OCT and HRT examination of the anterior segment of the animals in each group is shown in Figure 2. The animals in the low-dose and high-dose groups of the test product showed no abnormal changes 30 days after the first administration.

3) intraocular pressure

The Tonovet Lab rebound tonometer of Icare was used to measure the intraocular pressure (IOP) of animals.

The individual data of the intraocular pressure of each group of animals are shown in Figures 3A-D, the individual data of the intraocular pressure reduction percentage (ΔIOP%) are shown in Figures 4A-D, and the statistical data are shown in Tables 1A-C and Figures 5 and 6.

A. Male animals:

Test product low-dose group: Compared with the negative control group in the same period, ΔIOP% at all checkpoints decreased after administration, and the difference was statistically significant at 7, 11, 14, 18, 21, and 23 days after the first administration Significance (P values are p≤0.05, p≤0.001, p≤0.01, p≤0.05, p≤0.001).

Test product high-dose group: Compared with the negative control group in the same period, ΔIOP% at all checkpoints decreased after administration, among which 4, 7, 9, 11, 14, 16, 18, 21, 23 after the first administration , 25, and 28 days, the difference was statistically significant (P values were p≤0.05, p≤0.01, p≤0.05, p≤0.001, p≤0.001, p≤0.05, p≤0.01, p≤0.001, p≤ 0.001, p≤0.05, p≤0.05); Compared with the low-dose group of the test product during the same period, ΔIOP% at all checkpoints decreased after administration, and only 9 days after the first administration, the difference was statistically significant (p ≤0.05).

B. Female animals:

Test product low-dose group: Compared with the negative control group in the same period, ΔIOP% at all checkpoints decreased after administration, among which 4, 7, 11, 14, 16, 18, 21, 23, 25 after the first administration , 28 days, the difference was statistically significant (P values were p≤0.001, p≤0.001, p≤0.01, p≤0.001, p≤0.01, p≤0.01, p≤0.001, p≤0.001, p≤0.01, p≤0.05).

Test product high-dose group: Compared with the negative control group in the same period, ΔIOP% at all checkpoints decreased after administration, among which 4, 7, 9, 11, 14, 16, 18, 21, 23 after the first administration , 25, 28, and 30 days, the difference was statistically significant (P values were p≤0.001, p≤0.001, p≤0.001, p≤0.01, p≤0.001, p≤0.001, p≤0.001, p≤0.001, p≤0.001, p≤0.001, p≤0.01, p≤0.05); Compared with the low-dose group of the test product in the same period, except for 4, 7, 14, 16 days after the first dose, the remaining days after the administration checkpoint ΔIOP% Both decreased, only 9 days after the first administration, the difference was statistically significant (p≤0.05).

Table 1A Summary Table of Intraocular Pressure Reduction Percentage (ΔIOP%)

Note: n represents the number of animal eyes included in the statistical analysis.

Compared with the negative control group in the same period, ^a p≤0.05, ^aa p≤0.01, and ^aaa p≤0.001.

Compared with the low-dose group of the test product during the same period, ^b p ≤ 0.05, ^bb p ≤ 0.01, and ^bbb p ≤ 0.001.

Table 1B Summary Table of Intraocular Pressure Reduction Percentage (ΔIOP%)

Table 1C Summary of intraocular pressure reduction percentage table (ΔIOP%)

4) Other

In general clinical observation, eye local observation, body weight, gross and histopathological examination, etc., there were no statistically significant differences between the groups of animals.

2.6. Conclusion

Under the above test conditions, the test product (300mg/kg, 600mg/kg) was administered once a day to SD rats with high intraocular pressure by intragastric administration for 30 consecutive days, all of which showed the effect of lowering intraocular pressure. Among them, the 600mg/kg dose group The effect of lowering intraocular pressure is stronger than that of the 300mg/kg dose group.

Based on the above experimental results, it can be seen that the composition of the present application has a good effect in treating glaucoma, can significantly reduce the intraocular pressure of model animals in a short time, and this improvement effect presents a certain dose-effect relationship.

It can be understood that although the application is described in a certain form, the application is not limited to the content shown and described in the specification. It is obvious to those skilled in the art that various changes can be made without departing from the scope of the present application. These changes are all within the scope of protection claimed by this application.

Claims

Use of a composition containing astragalus, kudzu root and breviscapine in the preparation of a medicament for treating glaucoma in an individual.
The use according to claim 1, wherein the glaucoma is high intraocular pressure glaucoma.
The use according to claim 1 or 2, wherein the glaucoma is selected from primary glaucoma, secondary glaucoma, congenital glaucoma or mixed glaucoma.
The use according to any one of claims 1 to 3, wherein the glaucoma is open-angle glaucoma or closed-angle glaucoma.
Use of a composition comprising astragalus, kudzu root and breviscapine in the preparation of a medicament for treating ocular hypertension in an individual.
The use according to any one of claims 1-5, wherein the composition comprises raw materials of Astragalus, Pueraria lobata and Erigeron breviscapus in the following weight ratios: 10-40 parts of astragalus, 10-30 parts of puerariae and breviscapine 10-30 parts; preferably, the astragalus is 20 parts, the kudzu root is 30 parts, and the breviscapine is 20 parts.
The use according to any one of claims 1 to 5, wherein the composition is prepared from active ingredients comprising astragalus extract, kudzu root extract and breviscapine extract; preferably, the weight of the active ingredient The mixing ratio is: 1-10 parts of Astragalus extract, 50-125 parts of Pueraria lobata extract, and 20-60 parts of Dengzhan extract.
The use according to any one of claims 1-7, wherein the composition and a pharmaceutically acceptable carrier are formulated into a pharmaceutically acceptable dosage form, preferably a suspension, powder, tablet, granule, Pills, syrups or eye drops are more preferably suspensions.
The use according to any one of claims 1-8, wherein the composition is administered by gavage, oral administration, eye drops, enema, subcutaneous, intramuscular or intraperitoneal administration; preferably, the composition is administered by gavage Administration.
The use according to any one of claims 1-8, wherein the composition is administered 1-3 times a day, preferably once a day by intragastric administration.