TWI666324B - Cicada flower active substance and its use for reducing intraocular pressure - Google Patents

Abstract

The invention provides the use of a cicada flower active substance for preparing an oral composition for reducing intraocular pressure. This cicada flower active substance is prepared by the following steps: (a) taking the cicada flower mycelium on a plate medium and culturing it at 15-35 ° C for 5-14 days; (b) cultivating the cicada flower after step (a) The mycelium was inoculated into the flask, and cultured at 15-35 ° C, pH 2-8 for 3-10 days; (c) inoculating the cicada flower mycelium after the culture in step (b) into the fermentation tank, and at 15- Culture at 35 ° C and pH 2-8 for 3-10 days to form a cicada flower mycelium fermentation liquid; (d) freeze drying the cicada flower mycelium fermentation liquid and grind it to form a lyophilized cicada flower mycelium powder ; And (e) extracting the lyophilized powder of the cicada flower mycelium with a solvent to form a cicada flower mycelium extract containing a cicada flower active substance.

Description

Cicada flower active substance and its use for reducing intraocular pressure

The invention relates to a cicada flower active substance and a composition containing the same, particularly to the use of the cicada flower active substance and its composition for reducing intraocular pressure.

Aqueous humor

The eyes are made of soft and strong tissues and can move lightly and quickly. The aqueous humor circulates in the eyes, carrying oxygen, sugar, and other necessary nutrients to nourish the eyes. The composition of aqueous humor is related to the metabolites produced by the tissues that flow through the route. For example, water crystals consume glucose, potassium, and amino acids in aqueous humor, and produce lactic acid metabolites. The cornea has a similar situation, and its metabolism also consumes Nutrients for water supply. Therefore, compared with the posterior chamber of the eyeball, the aqueous humor of the anterior chamber contains lower concentration of glucose and higher concentration of lactic acid. In order to keep the cornea clear and transparent, the corneal endothelial cells must drain water to the anterior chamber at a rate of 10 mL per hour; under normal circumstances, each eye secretes aqueous humor at a rate of about 150 mL per hour. The ciliary body is responsible for the secretion of aqueous humor. The speed of aqueous humor secretion and the speed of aqueous humor from trabecular meshwork (TM) and uveoscleral outflow are the main factors that determine the level of intraocular pressure. factor.

intraocular pressure

Intraocular pressure is also called intraocular pressure (IOP), which refers to the pressure of the contents of the eyeball on the inner wall of the eyeball. Normal IOP usually falls between 10-21mmHg, the difference between the eyes is not greater than 5mmHg, and the daily fluctuation range is within 8mmHg. High intraocular pressure can easily cause damage to the optic nerve, leading to glaucoma.

Intraocular pressure is maintained by the dynamic balance of aqueous humor production and drainage, which can be affected by three parameters: the rate of aqueous humor generation, the resistance to aqueous humor drainage, and venous pressure. Venous pressure rarely changes, so drugs that regulate intraocular pressure are mainly used to control intraocular pressure by regulating the rate of aqueous humor formation and drainage resistance.

Glaucoma

The cause of glaucoma is that the ciliary body secretes aqueous humor too quickly, or the drainage of aqueous humor from the trabecular tissue and the uveal scleral pathway is blocked, which causes the aqueous humor in the anterior chamber to stay and cause abnormal intraocular pressure Rise. Long-term high intraocular pressure will cause chronic and irreversible degradation of retinal ganglion cells, which will lead to visual field defects, visual nerve damage and blindness. The causes of glaucoma can be divided into congenital glaucoma, secondary glaucoma, chronic open-angle glaucoma, and acute angle-locked glaucoma.

Glaucoma medicine

At present, the drugs commonly used in clinical treatment of glaucoma can be roughly divided into seven types, of which β-sympathetic blockers or prostaglandin derivatives are the drugs of choice if they are not contraindicated. After glaucoma treatment, fixed tracking is needed to determine the efficacy or side effects. If the effect of a single drug is not satisfactory, multiple drugs may be combined. When using more than one kind of eye drops, please pay attention to the interval of five minutes between the drops, the suspension eye drops should be used last, and care should be taken not to touch the mouth of the bottle to maintain the sterility of the eye drops.

β-Adrenergic blockers

These medicines can reduce the secretion of aqueous humor and have a significant effect on reducing intraocular pressure. They are currently the first-line drugs. Its main side effects are eye irritation (tingling, burning), conjunctivitis, and keratitis. In addition, there may be systemic side effects due to absorption through the mucosa. Non-selective β-sympathetic blockers, such as timolol, may cause systemic side effects such as slow heartbeat, arrhythmia, dizziness, and asthma attacks, so patients with arrhythmia and asthma should be avoided.

α 2-Adrenergic agonists

Such drugs can reduce the secretion of aqueous humor and increase the humoral humor outflow of the uveal sclera path to reduce intraocular pressure. If patients have poor tolerance or contraindications to β-sympathetic blockers, they can be used as alternative drugs. Its main side effects are allergic conjunctivitis, conjunctival hyperemia, itchy eyes, burning sensation, blurred vision, and dry mouth. Such drugs should not be used with Monoamine Oxidase Inhibitors (MAOIs) for psychiatric use, which may aggravate their side effects.

Cholinergic agonists

This kind of medicine is the earliest medicine for treating glaucoma. By reducing the pupil and tightening the iris to increase the space of the anterior chamber angle, increase the outflow of the aqueous chamber angle of the anterior chamber angle and reduce intraocular pressure. The side effects of these drugs are significant. For example, they may cause ciliary muscle contraction and cause headaches, eye tingling, burning sensation, and pupil shrinkage after taking the medicine. Patients' vision will be weakened when the light is poor, and the use rate has been reduced.

Carbonic anhydrase inhibitors

These drugs can suppress the secretion of aqueous humor to reduce intraocular pressure, and can be divided into oral and eye drop formulations. The side effects of oral dosage forms are nausea, vomiting, numbness in hands and feet, fatigue, kidney stones, myopia, and abnormal taste. Because of its many side effects, it is now mostly used as an aid to reduce intraocular pressure. Ophthalmic preparations have fewer systemic side effects. Common side effects are conjunctivitis, blurred vision, itchy eyes, burning sensation, and photophobia. Because it is a sulfa drug, patients who are allergic to sulfa drugs should avoid using it. It is also not recommended for patients with severe renal insufficiency (creatinine clearance <30 mL / min).

Prostaglandin analogs

Such drugs can increase the drainage of aqueous humor in the uveal scleral path and reduce intraocular pressure. Because of their long efficacy and good blood pressure reduction effect, they need to be used only once a day. Its drug price is high, although some literature lists it as the first-line medication in treatment recommendations, but the health insurance regulations are limited to β-sympathetic blockers that are not effective or not applicable. Side effects are blurred vision, conjunctival congestion, itchy eyes, dry eyes, keratitis, pigmentation around the eyes, eyelash hyperplasia, and darkening of the color (it is an irregular hyperplasia, which may cause eyelashes to reverse, and can be restored after stopping treatment), macular Edema, iriditis, photophobia (can be administered before bedtime to avoid discomfort), etc.

compound

Many glaucoma patients must order more than two eye drops to achieve the effect of lowering intraocular pressure. The compound eye drops combine two drugs with different action mechanisms, which can reduce the number of daily prescriptions, increase compliance of the prescriptions, and reduce the amount of preservatives. The side effects of the compound preparation are similar to those of the ingredients, and patients can choose based on their antihypertensive effect and tolerance.

Osmotic diuretics

Such drugs, such as intravenous mannitol and oral liquid isosorbide, can increase blood tension and reduce the vitreous volume due to the absorption of water into the blood vessels. Therefore, it can reduce intraocular pressure and cause the iris and crystalline lens to move backwards and increase. Corner depth. Mannitol injection can be used for rapid reduction of intraocular pressure in acute glaucoma, but it is not suitable for patients with dehydration, difficulty urinating, renal dysfunction, and congestive heart disease. These drugs have many systemic side effects, such as headache, frequent urination, vomiting, and electrolyte imbalance.

Cicada flower (Cordyceps cicadae)

Pattern and distribution

Cicada flower is also known as soil cicada flower, insect flower, cicada, cicada, cicada, cicada, cicada flower, cicada or silkworm, etc. It is Ascomycotina, Claricipiyales ), Clavicipitaceae, Cordyceps fungi, infected by Cicada flammate or Cicada flammate, Platypleura kaempferi, Crytotympana pustulata and Platylomia pieli Wait for the larva to die, and then form a bud-like constellation on the front end of the cicada pupa or the head of the worm body, hence the name cicada flower, which is a fungal-worm complex. Cicada flowers can be classified into three types: Cicada flower or C. cicadae, C. sobolifera and Cicada, or C. cicadicola according to different hosts and infectious species. Cicada flowers are mostly produced in tropical and subtropical areas south of the Yangtze River, and in China are Fujian, Zhejiang, Sichuan, Yunnan, and Jiangsu. There are also traces of wild cicada fruit bodies in some mountain areas of Taiwan.

The sexual stage of Paecilomyces cicadae is considered to be Cordyceps cicadae, the common name of the cicada cicadae, the rod is rod-shaped or horn-shaped, and it emerges from the host's head, solitary or clustered, brown. In nature, Paecilomyces cicadae (Cicada flower) is widely distributed, and Cicada is rare.

efficacy

Cicada flower is a precious traditional Chinese medicinal material. It is cold, sweet, and non-toxic. It can be used as medicine after drying. It has the effects of dispersing wind-heat, calming eyesight, eyesight, dysentery, and rash. "Compendium of Materia Medica" states that it treats infantile hangs, epilepsy, palpitations, and night cry.

Cicada flower has been used as medicine for more than one thousand years, and the history of wild cicada flower is 800 years earlier than Cordyceps sinensis. The name of the cicada flower was first seen in the "Lei Gong Potion Theory" during the Liu and Song dynasties of the Northern and Southern Dynasties. It records that anyone who makes the cicada flower want all white flowers. After harvesting, hang it under the house, remove the soil, and cook it with slurry water. Bake dry day and night, use it carefully.

Song Song ’s traditional medical book, “Tu Jing Herbs” records “In the mountains, a cicada has a horn, like a corolla, which is called a cicada flower.”; “Certificate Herbs” written by Tang Shenwei of the Northern Song Dynasty records “Cicadas are sweet and sexual. Cold, non-toxic, with the effect of evacuation of wind-heat, deterrence and relief of spasms, treating symptoms of pediatric night cry, heart palpitations and so on. ";" Compendium of Materia Medica "also recorded" Cicada flower function with cicada cessation, and stop malaria. "; There is also Chinese medicine The book records attending to the diaphragm, covering the eyes, such as: "The Book of Jingyue" "Cicada Flower Powder: cures the liver and the wind, heats the poisonous gas, attacks the eyes, and the eyes are painful, and all internal and external disorders." However, the above effects are still not related to science. Demonstrate or publish.

At present, there are also reports in the literature on the research of traditional prescriptions such as "Cicada Flower Wuwei Powder" and "Wanying Cicada Flower Powder" for eye-related diseases. Peng Guanghua from the Department of Ophthalmology of the First Affiliated Hospital of Henan Medical University has used traditional Chinese medicine Shengye San and "Cicada Wuwei San" with western medicine to treat traumatic low intraocular pressure. Traumatic hypotension is a common complication of ocular trauma, which can seriously affect visual function. The results of the study showed that 14 people in the Chinese and western medicine treatment group achieved normal IOP (> 1.33kPa), with an effective rate of 46.67%, and an average increase of 0.76kPa. Eight of the 30 people in the western medicine treatment group achieved normal IOP, with an effective rate of 26.67%, The average increase in intraocular pressure was 0.41kPa; Xu Damei and others from Xinxiang Hospital of Traditional Chinese Medicine, China observed the clinical observation of 100 cases of spring conjunctivitis with the addition and subtraction of "Wanying Cicada Flower Powder" taken internally and externally, and compared with western medicine. Results The cure rate was 78% in the treatment group and 26% in the control group. After one year, the relapse rate was 22% in the treatment group and 88% in the control group.

The above two cases are both the application of a compound medicine added with the cicada fruit body, which are used to increase the intraocular pressure and the clinical observation of the treatment of spring conjunctivitis. They do not clearly indicate the effect of cicada flower on reducing intraocular pressure or the prevention of glaucoma.

Cicada flower and Cordyceps sinensis are the same entomogenous fungal complex. Functionality and application of cicada flower It is as good as Cordyceps sinensis and Cordyceps sinensis. It has similar health care effects and contains similar chemical ingredients, so it is often used as a substitute for Cordyceps sinensis. However, the output of natural Cordyceps sinensis is decreasing, and there are not many fruit bodies of natural cicadas, which restricts a large number of uses. Therefore, artificial culture can be used as a substitute for natural cicada flower. The main biological active ingredients and pharmacological effects of artificial culture are similar to or exceed those of natural cicada flower. The generation of natural cicada flowers depends on the host, and the host is restricted by the natural environment, especially by climatic factors or human factors. It is an ideal way to replace the increasingly depleted natural resources with artificial culture products. Therefore, the liquid fermentation mycelia of cicada flower has high economic application value.

The object of the present invention is to provide a novel use of a cicada active substance, which can be used to prepare medicines for reducing intraocular pressure or treating glaucoma. Compared with the drugs for treating glaucoma introduced in the prior art, all of them are chemical drugs. The cicada active substance used in the present invention is more natural, safe, and has a simple preparation method.

According to the present invention, the use of a cicada active substance for preparing an oral composition for reducing intraocular pressure is provided. This cicada flower active substance is prepared by the following steps: (a) taking the cicada flower mycelium on a plate medium and culturing it at 15-35 ° C for 5-14 days; (b) cultivating the cicada flower after step (a) The mycelium was inoculated into the flask, and cultured at 15-35 ° C, pH 2-8 for 3-10 days; (c) inoculating the cicada flower mycelium after the culture in step (b) into the fermentation tank, and at 15- Culture at 35 ° C and pH 2-8 for 3-10 days to form a cicada flower mycelium fermentation broth; (d) drying the cicada flower mycelium fermentation broth and grinding to form a lyophilized cicada mycelium powder; And (e) extracting the lyophilized powder of the cicada flower mycelium with a solvent to form a cicada flower mycelium extract containing a cicada flower active substance.

In one embodiment, the cicada mycelium used for preparing the cicada flower active substance is the cicada mycelium deposited at the Institute of Food Industry Development, with the registration number MU30106.

In one embodiment, the step of preparing the cicada flower active substance further includes step (f): drying the cicada flower mycelium extract to obtain the cicada flower active substance.

In an embodiment, the fermentation tank of step (c) is further permeated with a gas, and the gas includes air, oxygen, carbon dioxide, helium, or a combination thereof.

In one embodiment, the tank pressure of the fermentation tank in step (c) is 0.5-1.0 kg / cm ² and the aeration rate is 0.01-1.5 VVM.

In one embodiment, the composition is a food composition or a pharmaceutical composition.

In one embodiment, when the composition is a pharmaceutical composition, the composition further comprises a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.

In one embodiment, the solvent used in step (e) is an alcohol, water or a water-alcohol mixed solution.

In one embodiment, the volume of the alcohol is at least 10 times larger than the volume of the lyophilized powder of the fermented broth of the cicada flower mycelium.

In one embodiment, the use of the cicada active substance is to manufacture an oral composition for treating glaucoma.

In order to make the features and advantages of the present invention clearer, the following describes embodiments of the present invention with reference to the drawings.

Figures 1 to 3 show the results of rabbit IOP changes (corresponding to Table 3 to Table respectively) 6).

Figure 1 shows the intraocular pressure of the normal rabbit intraocular pressure animal model.

Figure 2 shows the intraocular pressure change (Delta IOP) in the normal rabbit intraocular pressure animal model.

Figure 3 shows the delta IOP ratio change in normal rabbit intraocular pressure animal model.

Experimental principle

Normal rabbit intraocular pressure animal model

The New Zealand White (NZW) Rabbit, an adult, 8-12 weeks old, female was used in this test. This line of laboratory animals has a wealth of basic reference materials and data, which can be applied to the eyes of normal rabbits. Animal pattern. The normal intraocular pressure mode is used in the trial, which is less prone to distortion than the induced high intraocular pressure data, which is the current method commonly used in drug development.

Experimental steps

Preparation of cicada flower active substance

Cicada mycelium source

The Cordyceps cicadae hyphae system used in the embodiments of the present invention is obtained from the collected Taiwan wild cicada flower fruiting bodies, and the hyphae are isolated and stored on the plate culture medium. They are studied by the development of the Taiwan food industry. The identification confirmed that the gene sequence is Cordyceps cicadae. This strain is now publicly deposited at the Biological Resource Research Center (BCRC) of the Food Industry Development Research Institute, and the deposit number is MU30106 (this strain is also deposited in Chinese microorganisms. The General Microbiological Center of the Strain Collection Management Committee, No. CGMCC No. 10486). But the invention The active substance of Cicada flower is not limited to those obtained from this strain.

Liquid culture

Liquid fermentation culture can have different degrees of control equipment depending on the scale, including shake flasks, tanks, stirring control, temperature control, pH control, dissolved oxygen control, defoaming control and fermentation process control, etc. It takes a lot of manpower and time cost, and the process can be completely in a sterile environment and can uniformly sample and control the quality, and it has good reproducibility during mass production. The cicada flower active substance of the present invention is obtained by extracting the cicada flower mycelium after liquid culture. In liquid culture, the mycelia are first inoculated on a plate medium, and cultured at an appropriate temperature of 15-35 ° C (preferably 25 ° C) for 5 days to 2 weeks, and then the mycelia are scraped and inoculated into the flask. Incubate at 15-35 ° C (preferably 25 ° C), pH 2-8, preferably pH 4-7, more preferably about pH 4.5, and shaking rate 10-250 rpm for 3-10 days. The flask culture is then inoculated into the fermentation tank culture medium (the composition is shown in Table 1 below, the same as the flask culture medium), at a temperature of 15-35 ° C (preferably 25 ° C), a tank pressure of 0.5-1.0 kg / cm ² , and a pH of 2-8. , 10-150rpm stirring speed or no air lift, 0.01-1.5VVM ventilation rate (into air or air and oxygen, carbon dioxide and nitrogen mixture, preferably air), and culture for 3-10 days , That is to obtain the cicada flower mycelium fermentation broth, including mycelium and clarified liquid. The fermentation broth contains the cicada flower active substance of the present invention. The cicada flower mycelium fermentation liquid can be further prepared into a lyophilized powder of the fermentation liquid by a drying step.

In the above medium formulation, the comprehensive carbon and nitrogen source may be cereals (such as wheat flour) Or beans (such as: soy flour, mung bean flour, soybean flour, etc.); inorganic salts can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, iron sulfate, etc .; sugars can be glucose, fructose, maltose, sucrose, etc. . It is specifically noted that the above-mentioned culture medium formula is only an example, and the ingredients can be adjusted according to requirements during use, or used with commercially available culture medium, and there is no particular limitation.

dry

The drying method used in the present invention includes, but is not limited to, spray drying, hot air drying, drum drying, freeze drying, reduced pressure concentration, or other suitable drying methods to prepare a fermented liquid of cicada flower mycelium into a lyophilized powder of a fermentation liquid.

Alcohol extraction

The fermented liquid of the cicada flower mycelium fermentation liquid prepared by the drying method is lyophilized powder, and the alcohol solvent (1-100% methanol or ethanol, weight percent concentration or volume percent concentration) is added to dissolve, and extracted for several minutes (including However, it is not limited to soaking, stirring, shaking, or ultrasonic extraction), and then drying under reduced pressure concentration method or any one of the above drying methods, to obtain cicada mycelium alcohol extract.

Example 1 Liquid fermentation culture of cicada flower and preparation of active substance

Mycelium strain: BCRC MU30106

Plate culture: Cicada mycelium is inoculated on a plate medium, and the medium is potato dextrin medium (Potato Dextrose Agar, PDA), and cultured at 25 ° C for about 5 days.

Flask culture: The mycelium on the plate was scraped and inoculated into the flask, and the medium in Table 2 below was used, and cultured at about 25 ° C. and pH 4.5 on a shaker at 120 rpm for 3 days.

Fermentation tank culture: The culture medium is the same as in Table 2. The flask culture was inoculated in the fermentation tank culture medium, at 25 ° C, tank pressure 0.5-1.0kg / cm ² , pH 4.5, stirring speed 10-150rpm or not (air lift) In the case, the air was aerated at a ventilation rate of 0.5-1.0 VVM and cultured for 3 days to obtain a mycelium and a clarified liquid, which is called a cicada flower mycelium fermentation broth. The fermentation broth contains the cicada flower active substance of the present invention. The lyophilized fermented liquid of the cicada flower mycelium can be freeze-dried to obtain a lyophilized powder of the fermentation liquid.

Extract preparation-alcohol extraction: take lyophilized powder of cicada flower mycelium fermentation solution and add 20 times the volume of ethanol (95% by volume) to dissolve, extract with ultrasonic shock for 1 hour, extract the suspension and centrifuge the supernatant The liquid was concentrated under reduced pressure to obtain an alcohol extract of cicada flower mycelium (in this case, a thick paste).

Result: After freeze-drying the fermented broth of the cicada flower mycelium cultured in the 20 metric ton fermentation tank, about 110 kg of fermented broth lyophilized powder was obtained. Through the extraction step, a higher concentration of cicada flower active substance can be obtained to reduce intraocular pressure or treat glaucoma. The appearance of the cicada flower active substance includes a cicada flower mycelium fermentation broth (mycelium and clarified liquid), a lyophilized powder of the fermentation broth, an alcohol extract, or other dosage forms. In the second embodiment below, the alcohol extract is used as the active substance of the cicada flower.

Example 2 Analysis of Animal Models and Related Indexes

Experimental animal

The experimental New Zealand White (NZW) Rabbit was used as an adult, 8-12 weeks old, and female, and the test number was marked by ears to distinguish the individual experimental animals, and the animals were kept in a cage. Indicate the cage number, strain, age, animal test number, test group, date of admission and test period. The lighting time of the breeding area is automatically controlled to be 12 hours light, 12 hours dark, room temperature 23 ± 2 ℃, relative humidity 40-70%. Animals have free access to adequate food and water. During the quarantine and test period of the experimental animals, the clinical symptoms are observed and recorded by the veterinarian and the test staff, respectively, to ensure the health status of the experimental animals. After the animal is quarantined and domesticated for one week, the experiment can begin.

Before the experiment (D0), the experimental animals were randomly divided into groups according to the weight of the experimental animals, so that the average weight and weight distribution trend of each group were similar, and the intraocular pressure (IOP) of the right eye of the experimental animals was measured as a reference point.

Solvents and test substances

When the viscous creamy cicada extract is obtained by the above fermentation, it is dissolved in a solvent with a ratio of soybean salad oil: physiological saline = 1: 1 when tube feeding. The positive control group was a commercially available drug for treating glaucoma, Timolol, which could reduce the production of aqueous humor in the eyeball and indirectly reduce the intraocular pressure. The concentration used in this test was 0.5%. Timolol is applied as an eye drop.

Test group

During the test, the animals were divided into 5 groups. Each group had 5 animals, and the test substance was administered once a day. See Table 3 below.

Test method

The experimental animals were administered with a solvent, a test substance (containing the cicada flower extract of the present invention) or a positive control drug (0.5% Timolo) once a day in each group. The intraocular pressure of the right eye was measured 1 hour before, 1 hour after, and 3 hours after the administration of the solvent, test substance or positive control drug. The performance of intraocular pressure, intraocular pressure change (Delta IOP), and delta IOP ratio were compared respectively.

Intraocular pressure: TonoVet Tonometer is used to test the intraocular pressure of the right eye of the test animal at various time points. The advantages are that the test animal does not need to be anesthetized, and it can be detected directly without pain. Bring errors.

Intraocular pressure change (Delta IOP): It is the amount of intraocular pressure change compared to 0hr at each time point

△ IOP = IOP _{time point} -IOP _0hr

Delta IOP ratio change: It is the ratio of delta IOP at each time point compared to the IOP change amount of 0hr

Percentage of △ IOP = △ IOP / IOP _0hr

Test data analysis

The test data is based on the mean (standard) of the experimental results (standard error of mean, S.E.M.). Student's t-test was used to compare the differences between the groups. If the p value is less than 0.05, it means that there is a statistically significant difference between the two test groups.

Example 3 Evaluation of the Effect of Cicada Flower Active Substances on Reducing IOP and Related Indexes

Intraocular pressure (IOP)

The measurement results of intraocular pressure are shown in Table 4 below and Figure 1 of the drawing, wherein an asterisk (*) indicates that there is a significant difference between the test group and the solvent control group. Single asterisk (*) represents P <0.05; double asterisk (**) represents P <0.01; Samsung (***) represents P <0.001.

Compared with the solvent control group, the cicada flower group 2.5mg / kg / b.w. Alcohol extract group 1 hour after the first day of administration [D1 (1)], the intraocular pressure was significantly reduced. Cicada flower 25mg / kg / bw alcohol extract group is acceptable 1 hour [D1 (1)], 3 hours [D1 (3)] and 1 hour after administration [D2 (1)] on the first day Significantly reduces intraocular pressure. The positive control group 0.5% Timolol significantly reduced intraocular pressure at various time points after administration.

Intraocular pressure change (△ IOP)

The measurement results of intraocular pressure changes are shown in Table 5 and Figure 2 below. The meaning of the asterisk mark (*) is the same as that in Table 1 and Figure 1, which indicates that the test group and the solvent control group are significantly different.

Table 5.Intraocular pressure change in normal rabbit intraocular pressure animal model (corresponding to Figure 2)

Compared with the solvent control group, the 25 mg / kg / b.w. Alcohol extract group of Cicada spent 1 and 3 hours after administration on the first and second days could significantly reduce the change in intraocular pressure. The 0.5% Timolol group in the positive control group had no significant decrease in △ IOP at three hours after the second day of administration, and it could significantly decrease △ IOP at other time points.

Percentage of Delta IOP

The measurement results of the intraocular pressure change percentage (proportion change) are shown in Table 6 and Figure 3 of the figure below. The meaning of the asterisk (*) is the same as above, which indicates that the test group and the solvent control group are significantly different.

Compared with the solvent control group, the Cicada 25 mg / kg / b.w. Alcohol extract group can significantly reduce the △ IOP percentage on the first and second days after administration. In the positive control group, 0.5% Timolol group had no significant decrease in the percentage of △ IOP three hours after the second day of administration. Both can significantly reduce the percentage of △ IOP. .

The test of Example 3 above proves that the cicada flower active substance prepared by the preparation method of Example 1 has a significant effect on reducing intraocular pressure (IOP) for normal rabbit intraocular pressure model animals by oral administration, and can even achieve close to proven The therapeutic effect of Timolol drug. Therefore, the active substance of cicada flower can be used in the field of reducing intraocular pressure and preventing glaucoma.

According to the above-mentioned animal low-dose and high-dose group tests, the converted human effective dose is about 52.34 mg / person / day to 523.4 mg / person / day (converted to 70 kg per person).

Although the present invention has been described above by way of examples, these examples are not intended to limit the present invention. Those skilled in the art can perform equivalent implementations or changes to these embodiments without departing from the technical spirit of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of patents attached thereafter.

[Biological Material Storage] Domestic Deposit Information [Please note according to the order of deposit organization, date, and number]

Depository Organization: Biological Resource Research Center (BCRC) of Food Industry Development Institute

Hosting date: November 25, 102

Registration Number: MU30106

Claims (11)

A use of a cicada flower active substance for manufacturing an intraocular pressure-reducing composition, wherein the cicada flower active substance is prepared through the following steps: (a) taking a cicada flower ( Cordyceps cicadae ) mycelium on a flat medium, at 15 Culture at -35 ° C for 5-14 days; (b) inoculate the cicada flower mycelium after the culture in step (a) into a flask, and culture at 15-35 ° C, pH 2-8 for 3-10 days; c) Inoculating the cicada flower mycelium after the culture in step (b) into a fermentation tank, and culturing at 15-35 ° C and pH 2-8 for 3-10 days to form a cicada containing the cicada flower active substance Flower mycelium fermentation broth; (d) drying the cicada mycelium fermentation broth to form a dried cicada mycelium containing one of the cicada flower active substances; and (e) the cicada mycelium The dried product is extracted with an alcohol to form a cicada flower mycelium extract containing one of the cicada flower active substances. The use according to item 1 of the scope of the patent application, wherein the step of preparing the cicada flower active substance further comprises step (f): drying the cicada flower mycelium extract to obtain the cicada flower active substance. For the application described in item 1 of the scope of patent application, wherein the cicada mycelium is the cicada mycelium deposited at the Food Industry Development Institute of the Consortium, and the registration number is MU30106. The application according to item 1 of the scope of the patent application, wherein in the step (c), a gas is further introduced into the fermentation tank, and the gas includes air, oxygen, carbon dioxide, helium, or a combination thereof. The use as described in item 1 of the scope of patent application, wherein the tank pressure of the fermentation tank in step (c) is 0.5-1.0 kg / cm ² and the aeration rate is 0.01-1.5 VVM. The use according to item 1 of the patent application scope, wherein the composition is a food composition or a pharmaceutical composition. The use according to item 6 of the scope of patent application, wherein when the composition is a pharmaceutical composition, the composition further comprises a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. The use according to item 1 of the scope of the patent application, wherein the volume of the alcohol is at least 20 times larger than the volume of the lyophilized powder of the fermented broth of the cicada flower mycelium. The use as described in item 1 of the scope of patent application is for the manufacture of a composition for treating glaucoma. The use according to item 1 of the scope of patent application, wherein the intraocular pressure reducing composition is administered orally. The use as described in item 1 of the scope of patent application, wherein the alcohol in step (d) is ethanol.