TWI811009B - Use of cordyceps cicadae active substance for improving visual acuity - Google Patents

Abstract

A use of Cordyceps Cicadaeactive substance is provided. The Cordyceps Cicadaeactive substance is for manufacturing a composition for improving visual acuity. The Cordyceps Cicadaeactive substance is prepared by the following steps: (a)culturing a Cordyceps Cicadaemycelium in a plate media at 15 to 35℃ for 2-10 days; (b)inoculating the mycelium of step(a) to a flask and culturing it at 15 to 35℃ with a pH of 2-8 for 3-7 days; (c-1)inoculating the mycelium of step(b) to a fermenter tank so as to obtain a Cordyceps Cicadaemycelium fermentation liquid containing said Cordyceps Cicadaeactive substance; or (c-2) inoculating the mycelium of step(b) to a solid medium so as to obtain a Cordyceps Cicadaefruiting body containing said Cordyceps Cicadaeactive substance.

Description

Use of Cicada Flower Active Substances for Improving Vision

The present invention relates to the application of an active substance of cicadae, in particular to a composition prepared by using cicadae mycelium to improve eyesight.

There are many causes of eye fatigue, including overuse of eyes, partial eclipse, presbyopia, astigmatism, lack of sleep or stress, etc. In modern society, the popularity of 3C products and the fast pace of life have greatly increased the chance of eye fatigue. If the eyes are tired, the eyes will feel sore and painful, and if you don't pay attention to the improvement, your eyesight will decrease.

Generally speaking, the ways to relieve eye fatigue include eye hot compress or acupoint massage to relieve vision loss and blurred vision. If the eyesight degeneration caused by fatigue cannot be relieved for a long time, refractive glasses must be worn to correct the eyesight.

Cicadae (Cordyceps cicadae) is a kind of entomogenous fungus, also known as soil cicadae, insect flower, cicadae, cicada, cicadae, cicada pupa, cicadae, cicada or silkworm etc. Cicada is a fungus of Ascomycotina , Claricipiyales , Clavicipitaceae , Cordyceps , which infects cicada pupae or Cicada flammate , cicada ( Platypleura kaempferi) , black grasshopper (Crytotympana pustulata) and bamboo cicada (Platylomia pieli) and other larvae make them die, and form flower bud-like sub-seats at the front of the cicada pupae or the head of the insect body, hence the name cicadae. Natural wild cicadas are mostly produced in the tropical and subtropical regions south of the Yangtze River, and there are also traces of wild cicadas in some mountainous areas of Taiwan.

Cicada is a precious traditional Chinese medicinal material, which has been used as medicine for more than one thousand years. Modern pharmacological experiments have shown that cicadae and its artificial culture have obvious immune-regulating, nervous system regulation, anti-fatigue, sedative, analgesic, antipyretic, improving kidney function, lowering blood sugar, lowering blood pressure, slowing heart rate, and inhibiting atherosclerosis. Formation, anti-tumor, anti-radiation and nourishing and strengthening. However, there are currently no studies showing that cicadae can improve eyesight.

The invention provides an application of the active substance of cicadae (Cordyceps cicadae), which is used for preparing a composition for improving eyesight. The preparation method of this cicadae active substance comprises the following steps:

(a) Take a cicadae mycelium on a plate culture medium and cultivate it at a temperature of 15-35° C. for 2-10 days;

(b) Inoculate the cicadae mycelium cultivated in step (a) into a flask, and cultivate for 3-7 days in an environment of 15-35° C. and pH 2-8; and

(c-1) Inoculate the cicadae mycelium cultivated in step (b) in a fermentation tank, and culture it with stirring at 15-35° C. and pH 2-8 for 2-10 days to form a product containing the cicadae activity One of the substances is cicadae mycelium fermentation broth; or

(c-2) Inoculate the cicada mycelium cultivated in step (b) into a solid-state culture medium to avoid light and isolate the air for 3-7 days, and then cultivate it for 7-40 days under a 12-hour light-dark cycle, so that the cicada The flower mycelium is covered with the solid culture medium; after the cicada flower mycelium is covered with the solid culture medium, it is exposed to the air for 7-14 days to form a cicada flower fruiting body containing one of the cicada flower active substances.

In one embodiment, the cicadae mycelium is selected from the cicadae mycelium stored in the Biological Resource Conservation and Research Center (BCRC) with the deposit number MU30106.

In one embodiment, the step of preparing the cicadae active substance further includes step (d): after drying the cicadae mycelium fermentation broth in step (c-1) or the cicadae fruiting body in step (c-2) Grinding to form a cicadae active substance powder.

In one embodiment, the method for drying the mycelia fermentation liquid of Cicadae or the fruiting body of Cicadae includes spray drying, hot air drying, drum drying and freeze drying.

In one embodiment, the flask culture in step (b) is shaking culture, and the shaking speed is 10-250 rpm.

In one embodiment, in step (c-1), the fermentation tank is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium or a combination thereof, and the tank pressure of the fermentation tank is 0.5-2.0 kg/cm ² And the ventilation rate is 0.01-1.5 VVM.

In one embodiment, the solid-state culture in step (c-2) is carried out under the conditions of relative humidity 60-100%, temperature 15-35°C, and pH 2-8.

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

In one embodiment, the composition is a food additive.

In one embodiment, the administration methods of the composition include oral administration, drops and suppositories.

In one embodiment, the composition is used to prepare a composition for improving distance vision. In order to make the above and other aspects of the present invention more clear, the following specific examples are described for illustration.

Cicada mycelium source

The mycelium system of the cicadae ( Cordyceps cicadae ) used in this example is collected from the fruiting bodies of wild cicadae in Taiwan, not the fruit. The fruiting body is separated to obtain its mycelium, and is subcultured on the plate medium. The gene sequence of this strain was identified as Cordyceps cicadae by the Taiwan Institute of Food Industry Development, and it has been deposited in the Bioresource Research Center (BCRC) of the Institute of Food Industry Development, Taiwan, with deposit number MU30106. This strain is also deposited in the China General Microorganisms Collection and Management Center, and the preservation number is CGMCC No.10486. But the cicadae active substance described in the present invention is not limited to be obtained from this strain, and other types of cicadae strains can also be used. Preparation of Active Substances of Cicada Flower - Liquid Fermentation Mycelia

The active substance of cicadae used in the present invention includes two kinds: mycelium fermented liquid and fruiting body powder, which are all obtained from mycelium culture. The preparation method of mycelium fermented liquid is briefly introduced here.

Inoculate the mycelia of cicadae on a plate culture medium, culture at an appropriate temperature of 15-35° C. (preferably 25° C.) for 2-10 days, scrape the mycelium and inoculate it in a flask. Cultivate for 3-7 days at 15-35°C (preferably 25°C), pH 2-8 (preferably pH 4-7, more preferably pH 5.5), shaking rate 10-250 rpm, and then The flask culture is inoculated in the fermenter medium (same as the flask medium), at 15-35°C (preferably 25°C), tank pressure 0.5-2.0 kg/cm ² , pH 2-8, stirring speed 10-150 rpm or Without stirring (air lift), feed air, oxygen, carbon dioxide, nitrogen or a mixture of the above gases (preferably air) with a ventilation rate of 0.01-1.5 VVM, and the cultivation time is within 2-10 days to obtain cicadae. Mycelium Fermentation Broth. This fermented liquid includes mycelia and clarified liquid. The aforementioned culture conditions are only examples, and users can adjust them according to the situation.

The flask culture medium that the present invention uses, fermenter culture medium formula can be as follows: Element Content (weight%) Comprehensive Carbon and Nitrogen Sources 0.01-5 Animal and plant source protein and its hydrolyzate 0.01-2 Yeast or malt extract (powder, paste) 0.001-2 Inorganic salts 0.0001-0.05 carbohydrate 0.01-10

Wherein the comprehensive carbon and nitrogen source can be cereals (such as: wheat flour) or beans (such as: soybean flour, mung bean flour, soybean flour, etc.). The inorganic salts can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, iron sulfate and the like. The sugars can be glucose, fructose, maltose, sucrose and the like. In particular, the culture medium used in the present invention is not limited to the above-mentioned composition or ratio, and the user can adjust it according to the actual situation.

fermentation broth drying

The cicadae mycelium fermentation broth can be further prepared into other dosage forms such as freeze-dried powder through a drying step. Drying methods include, but are not limited to: spray drying, hot air drying, drum drying, freeze drying or other methods. Preparation of Active Substances of Cicada Flower - Solid Culture Fruiting Bodies

As mentioned above, the active substances of cicadae used in the present invention include two types: mycelium fermentation liquid and fruiting body powder, both of which are obtained from the cultivation of mycelium. The preparation method of fruiting body powder is introduced here.

The preparation method of part of the fruiting body powder is similar to the mycelia fermentation liquid, and the mycelium needs to be cultured on plates and flasks first. Firstly, inoculate the mycelium of cicadae on a plate culture medium, culture at an appropriate temperature of 15-35° C. (preferably 25° C.) for 2-10 days, scrape the mycelium and inoculate it in a flask. Cultivate for 3-7 days at 15-35°C (preferably 25°C), pH 2-8 (preferably pH 4-7, more preferably pH 5.5), shaking rate 10-250 rpm,

Next, inoculate the cultured mycelium in the flask into the solid medium, the amount of the solid medium is 20%-200% of the volume or weight of the mycelium. Then, under the condition of relative humidity 60-100%, temperature 15-35 ℃, pH 2-8, avoid light and isolate air for 3-7 days, and then cultivate 7-40 days under 12 hours light and dark cycle, so that The mycelium is overgrown in the solid medium. After the mycelium is covered with the solid medium, make it contact with the air and maintain a 12hr light-dark cycle. After 7-14 days, the fruiting body of the cicadae is formed, and the fruiting body of the cicadae is harvested, dried and ground to form the fruiting body powder of the cicadae.

Drying methods include, but are not limited to: spray drying, hot air drying, drum drying, freeze drying or other methods.

The above-mentioned cicadae mycelium fermentation liquid, cicadae mycelium fermentation liquid powder (freeze-dried powder) and cicadae fruiting body (powder) all contain the cicadae flower active substance of the present invention. Cicada flower mycelium powder (freeze-dried powder) and Cicada flower fruit body (powder) can be further extracted to increase the concentration of Cicada flower active substances. The active substance of cicadae is prepared according to the above-mentioned method, and a biological experiment is carried out to evaluate its efficacy of improving eyesight. Embodiment one : the preparation of cicada flower active substance - liquid state fermentation culture

Bacterial strain: deposited in the Bioresource Research Center (BCRC) of the Food Industry Development Research Institute of the foundation, the registration number is MU30106 Cicada mycelium. This bacterial strain can be purchased from depository institutions such as the official website of BCRC, and is easily obtained by those with ordinary knowledge in the technical field.

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

Flask culture: Scrape the mycelia on the plate and inoculate into the flask, use the following medium formula, at 25°C, pH 5.5, shake and culture on a shaker at a speed of 120 rpm for three days;

Flask medium recipe: Element Content (weight%) Comprehensive Carbon and Nitrogen Sources 0.01-5 Animal and plant source protein and its hydrolyzate 0.01-2 Yeast or malt extract (powder, paste) 0.001-2 Inorganic salts 0.0001-0.05 carbohydrate 0.01-10

Fermentation tank culture: the medium is the same as the flask medium, inoculate the flask culture into the fermentation medium, at 25°C, tank pressure 1.0 kg/cm ² , pH 5.5, stirring speed 10 rpm, and air at 1.0 VVM aeration rate, Cultivate for 5 days to obtain the cicadae mycelium fermentation liquid. The cicada flower mycelium fermentation liquid is freeze-dried to obtain the cicada flower mycelium freeze-dried powder.

In this example, 20 metric tons of fermentation tanks were used for fermentation tank cultivation. After the obtained cicadae mycelium fermentation liquid was freeze-dried, about 320 kg of fermentation liquid freeze-dried powder could be obtained, which is the active substance of cicadae flower that can be used to improve eyesight. Embodiment two: the preparation of cicada flower active substance - solid-state culture

Bacterial strain: deposited in the Bioresource Research Center (BCRC) of the Food Industry Development Research Institute of the foundation, the registration number is MU30106 Cicada mycelium. This bacterial strain can be purchased from depository institutions such as the official website of BCRC, and is easily obtained by those with ordinary knowledge in the technical field.

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

Flask culture: Scrape the mycelia on the plate and inoculate into the flask, use the following medium formula, at 25°C, pH 5.5, shake and culture on a shaker at a speed of 120 rpm for three days;

Flask medium recipe: Element Content ( weight %) sucrose 3.0 yeast extract 1.0 soy flour 1.0

The steps of above-mentioned plate culture and flask are basically the same as in Example 1. The difference is that in this embodiment, the flask culture is inoculated in the solid medium prepared according to the following table

Solid medium formula: Element Content (weight%) solid matrix 10g (50%) - barley kernels 5g -oat 5g liquid culture medium 10g (50%) -sucrose 2.0% - soy flour 1.0% -yeast extract 0.5% - magnesium sulfate 0.4% -Histidine 0.10%

In this solid medium, the materials of the liquid culture medium are firstly dissolved in the liquid and homogenized, then mixed with the solid matrix, and then sterilized together. After sterilization, water will be lost, and finally a solid medium will be formed.

Culture conditions: First, the solid-state culture of the inoculated flask culture was based on a relative humidity of 75%, a temperature of 18°C, and a pH of 5.0. The culture was protected from light and air for 3 days; then cultured for 30 days under a 12-hour light-dark cycle, Make the mycelia overgrow in the solid medium. After the mycelium is covered with solid-state culture medium, it is opened to the air and maintained in a 12-hour light-dark cycle. After 7 days, the cicadae fruiting body is formed.

Fruiting body harvest: Use tweezers to collect rod-shaped to bouquet-shaped fruiting bodies on the plane of the solid substrate, excluding the part of the cereal base covered with hyphae. The collected artificially cultivated fruiting body is dried by freeze-drying method, which is the active substance of cicadae which can be used to improve eyesight. Embodiment 3: visual acuity measurement experiment

Vision Measurement Principles

Vision scale: The vision scale is designed according to the principle of visual angle. The so-called angle of view is the angle formed by the light emitted by two external points and the node inside the eye. Under normal circumstances, the human eye can distinguish the angle of view formed by the minimum distance between two points as the minimum angle of view, that is, one arc minute. The eye chart is designed in units of one corner.

In this embodiment, the Snellen's illiterate E (Snellen's illiterate E; test distance is 6 meters) eye chart is used to measure the visual acuity (distance vision) of the subject. This table is composed of 12 rows of "E" characters with different sizes and opening directions; the measured visual acuity ranges from 0.1 to 1.5. Each line has a label, and the line of sight of the subject should be parallel to the line of 1.0, and the distance from the eye chart should be 6 meters. Cover one eye before performing the test, and identify the direction of the "E" gap from top to bottom with one eye until it is unrecognizable, and record it. The two openings of the E symbol at the visual acuity of 1.0 in Snellen's simple E vision chart are 1 angular minute. Therefore, when the subject can distinguish the symbol of visual acuity 1.0, it means that the minimum discrimination angle can reach 1 angular angle of a normal person. point.

Normal vision should be above 1.0. If the subject can't see 0.1, move forward until he can see 0.1, his vision is "0.1 x distance / 5 = vision"; if he still can't see 0.1 within half a meter, he can Let the testees identify the index, measure the manual, light perception and so on. Visual acuity was recorded by inspection.

The eye chart is used to measure visual acuity (Visual acuity), so vision is also called visual acuity. It mainly checks "central vision", which is the visual acuity of the "fovea of the macular part of the retina", so that it can be easily and quickly understood It is of great significance to the clinical diagnosis and treatment of eye diseases.

Cicada mycelium fermentation broth test (using the sample of Example 1)

A total of 11 subjects were recruited, aged between 20-40 years old, 9 females and 2 males, all with myopia or astigmatism, the degree of myopia was between 50-700 degrees, of which 2 were less than 100 degrees, 100- There are 7 people with 500 degrees, 2 people with 500-700 degrees; 9 people with astigmatism, 50-200 degrees, 2 people with less than 100 degrees, and 7 people with 100-200 degrees. Eye diseases without presbyopia, hyperopia or other obvious symptoms. When measuring, 2 people used naked vision, and 9 people used refractive glasses to correct their vision.

Cicada flower fruiting body powder test (using the sample of embodiment two)

A total of 5 subjects were recruited, aged between 20-40 years old, 3 females and 2 males, all with myopia or astigmatism, the degree of myopia was between 50-700 degrees, of which 1 was less than 100 degrees, 100- 3 for 500 degrees, 1 for 500-700 degrees; 4 for astigmatism, 50-200 degrees, 1 for less than 100 degrees, and 3 for 100-200 degrees. Eye diseases without presbyopia, hyperopia or other obvious symptoms. When measuring, 1 person used naked vision, and 4 people used refractive glasses to correct their vision.

Test method

Visual acuity was assessed using the Snellen's illiterate E (Snellen's illiterate E) vision scale. When performing the test, first cover one eye, identify the direction of the "E" gap from top to bottom with one eye, and record it until it cannot be recognized. Then cover the other eye and perform the test with the same steps. Then give the subject 0.85±0.05 g of the cicadae active substance sample of Embodiment 1 or 2, and take it with boiled water. After 90 minutes, test again with the same visual inspection method, and the recorded value is the post-measurement value.

Test data analysis

The test data is represented by the mean (Mean) ± standard deviation (Standard Deviation, SD) of the test results. The Paired Sample t test (Paired Sample t test) was used to compare whether there is a difference between the groups before and after. If the p value is less than 0.05, it means that there is a statistically significant difference between the groups. The results are listed in Table 1 (corresponding to Figure 1) and Table 2 (corresponding to Figure 2) below.

Table 1 Cicada flower mycelium fermentation liquid group (sample of embodiment 1) visual acuity test result Pretest Posttest p-value left eye 1.12 ± 0.20 1.25 ± 0.21* 0.01 right eye 1.12 ± 0.16 1.31 ± 0.15** 0.00

Table 2 Cicada flower fruiting body powder group (sample of embodiment 2) vision test results Pretest Posttest p-value left eye 1.18 ± 0.20 1.32 ± 0.16* 0.04 right eye 1.08 ± 0.11 1.38 ± 0.16** 0.00

From Table 1 and Table 2, it can be seen that the vision of the left eye and the right eye of the subject improved after taking the cicadae sample of Example 1 or 2 for 90 minutes. Therefore, the cicadae active substance of the present invention does have the effect of improving eyesight. Embodiment four: composition preparation

If the cicadae active substance of this embodiment is used in medicine, the following composition 1 is used as an illustrative example.

Composition 1: get the freeze-dried powder or fruiting body powder (20 wt%) of the cicadae active substance of embodiment one or two, and magnesium stearate (8wt%) as lubricant, silicon dioxide as preservative (7wt%) mixed thoroughly, and dissolved in pure water (65wt%), stored at 4 ° C for use. The aforementioned wt% refers to the ratio of each component to the total weight of the composition.

However, although the active substance of cicadae in Example 3 is administered orally, other methods such as drops and ointment can also be used in practical applications.

If the active substance of cicadae flower disclosed in this disclosure is applied to food in a liquid dosage form, the following composition 2 is used as an illustrative example.

Composition 2: get the lyophilized powder or fruit body powder (20 wt%) of the cicadae active substance of embodiment one or two, and benzyl alcohol (8 wt%) as preservative, glycerin (7 wt%) as diluent %) and sucrose (10 wt%) as a diluent were thoroughly mixed, dissolved in pure water (55 wt%), and stored at 4°C for later use. The aforementioned wt% refers to the ratio of each component to the total weight of the composition.

The above-mentioned embodiments are specific descriptions of several feasible embodiments of the present invention, but these embodiments are not intended to limit the present invention. Those skilled in the art may perform equivalent implementations or changes to these embodiments without departing from the technical spirit of the present invention. Therefore, the protection scope of the present invention shall be determined by the scope of the appended patent application.

Fig. 1 shows cicadae mycelium fermentation broth group (sample of embodiment 1) vision test result;

Fig. 2 shows cicadae fruiting body powder group (embodiment 2 sample) vision test result;

Among them, * indicates that there is a significant difference before and after the test (P<0.05); ** indicates that there is an extremely significant difference before and after the test (P<0.01).

TW Republic of China Food Industry Development Institute Biological Resource Conservation and Research Center 2013/11/25 MU30106

Claims (10)

A kind of cicadae ( Cordyceps cicadae ) active substance is used to prepare the application of the pharmaceutical composition of improving eyesight, wherein the preparation method of this cicadae active substance comprises the following steps: (a) take a cicadae mycelium on the plate culture medium, in Cultivate at a temperature of 15-35°C for 2-10 days; (b) inoculate the cicadae mycelium after the cultivation in step (a) into a flask, and cultivate in an environment of 15-35°C and pH 2-8 for 3- 7 days; and (c-1) inoculating the cicadae mycelium cultivated in step (b) in a fermenter, stirring and cultivating for 2-10 days at 15-35°C and pH 2-8, forming a mixture containing One of the cicadae active substances is a cicadae mycelium fermentation broth; or (c-2) inoculating the cicadae mycelium cultivated in step (b) into a solid medium to avoid light and isolate the air for 3-7 days, Then cultivate it for 7-40 days under a 12-hour light-dark cycle, so that the cicada flower mycelium is covered with the solid medium; after the cicada flower mycelium is covered with the solid medium, it is exposed to the air for 7-14 days, forming a mixture containing the cicada. One of the flower active substances is the fruiting body of cicadae. The use as described in Claim 1, wherein the cicadae mycelium is selected from the cicadae mycelium stored in the Biological Resource Conservation and Research Center (BCRC) with deposit number MU30106. The use as described in claim 1, wherein the step of preparing the cicadae active substance further includes step (d): the step (c-1) of the cicadae mycelium fermentation liquid or the step (c-2) of the The cicadae fruiting body is dried and ground to form a cicadae active substance powder. The use as described in claim 3, wherein the method of drying the cicadae mycelium fermentation liquid or the cicadae fruiting body includes spray drying, hot air drying, drum drying and freeze drying. The use as described in claim 1, wherein the flask culture in step (b) is shaking culture, and the shaking speed is 10-250rpm. purposes as described in claim item 1, wherein in step (c-1), this fermenter further feeds a gas, and this gas comprises air, oxygen, carbon dioxide, helium or its combination, and the cell pressure of this fermenter is 0.5- 2.0kg/cm ² with a ventilation rate of 0.01-1.5VVM. The use as described in Claim 1, wherein the solid-state culture in step (c-2) is carried out under the conditions of relative humidity 60-100%, temperature 15-35°C, and pH 2-8. The use as described in Claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. The use as described in claim 1, wherein the pharmaceutical composition is administered orally. The use as described in claim 1, which is used to prepare a pharmaceutical composition for improving distance vision.