TWI788083B - Use of cordyceps cicadae mycelia active substance for preventing or curing macular degeneration - Google Patents

Abstract

A use of Cordyceps Cicadaemycelia active substance is provided. The Cordyceps Cicadaemycelia active substance is for manufacturing a composition for preventing or curing macular degeneration. The Cordyceps Cicadaemycelia active 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)inoculating the mycelium of step(b) to a fermenter tank and culturing it at 15-35℃ with a pH of 2-8 for 3 to 5 days, so as to obtain a Cordyceps Cicadaemycelium fermentation liquid containing said Cordyceps Cicadaemycelium active substance.

Description

Use of active substance of cicada flower mycelium for treating macular degeneration

The invention relates to the use of an active substance of cicada flower mycelium, in particular to a composition prepared by using cicada flower mycelium to prevent or treat macular degeneration.

Age-related macular degeneration (AMD) is a progressive degenerative disease of the macular part of the retina responsible for vision and color vision, mainly caused by the death of central retinal photoreceptors. The retinal pigment epithelium (RPE) cells located between the photoreceptor layer and the choroid are also involved in the pathogenesis of early AMD. As its name "senile" suggests, the prevalence of AMD gradually increases with age, becoming the leading cause of blindness in the elderly.

The pathogenesis of AMD is related to many factors, such as metabolic disorders, immunity, inflammation, reactive oxygen species (reactive oxygen species; ROS) and so on. Recent studies have shown that oxidative stress caused by ROS is the main pathogenic factor of AMD. In the past few decades, the methods used to treat AMD include: (1) photocoagulation laser (laserphotocoagulation), (2) transpupillary thermotherapy (TTT), (3) photodynamic therapy (photodynamictherapy) PDT), (4) antiangiogenic therapy (antiangiogenic therapy), (5) nutritional supplement therapy, (6) gene therapy, (7) antioxidants and (8) combining the above therapies, etc., but surgical therapy has side effects, and surgery Blindness rates are still increasing. Therefore, the development of novel therapeutic agents with less toxicity is quite important for the prevention or treatment of AMD.

Cicadae (Cordyceps cicadae) is a kind of entomogenous fungus, also known as soil cicadae, insect flower, cicadae, cicada, cicadae, cicada pupae, golden cicadae, cicada antler or silkworm antler. 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 is no research to date that cicadae has a curative effect on macular degeneration.

The invention provides an application of the mycelia active substance of cicada ( Cordyceps cicadae) , which is used for preparing a composition for preventing or treating macular degeneration. The preparation method of the cicada flower mycelium 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 in an environment of 15-35°C and pH 2-8 for 3-7 days;

(c) inoculate the cicadae mycelium after step (b) cultivation in a fermenter, stir and cultivate for 3-5 days under the environment of 15-35° C. and pH 2-8, and form the cicadae mycelium containing the One of the body active substances cicadae mycelium fermentation broth.

In one embodiment, the step of preparing the active substance of the cicadae mycelium further includes a step (d): freeze-drying the fermented liquid of the cicadae mycelium and then pulverizing it to form one of the active substances containing the cicadae mycelium Cicada mycelium freeze-dried powder.

In one embodiment, the step of preparing the active substance of the cicada flower mycelium further includes step (e): extracting the freeze-dried powder of the cicada flower mycelium with a solvent to form one of the active substances containing the cicada flower mycelium Cicada mycelium extract.

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

In one embodiment, the solvent in step (e) is water.

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

In one embodiment, the fermentation tank in step (c) is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium or a combination thereof, the tank pressure of the fermentation tank is 0.5-1.0 kg/cm ² and The ventilation rate was 0.01-1.5 VVM.

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 composition is administered orally.

In order to make the above and other aspects of the present invention more clear, the following specific embodiments are described together with the accompanying drawings.

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 bodies are separated to obtain their hyphae, and are subcultured on 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 active substance of the cicadae mycelium described in the present invention is not limited to be obtained from this strain, and other kinds of cicadae strains can also be used.

Mycelium liquid culture

Inoculate the mycelium of Cicada japonica on a plate medium, cultivate it 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.

Freeze-dried powder extraction-water extraction

Take the freeze-dried powder of cicada mycelium and add 20 times the volume of distilled water to dissolve it, heat it at 100°C for 30 minutes, and dry it by freeze-drying after cooling to obtain the water extract of cicada flower mycelium.

The above-mentioned fermented liquid of cicada flower mycelium, freeze-dried powder of cicada flower mycelium and aqueous extract of cicada flower mycelium all contain the active substance of cicada flower mycelium of the present invention. The active substance of the cicadae mycelium is prepared according to the above-mentioned method, and a biological experiment is carried out to evaluate its efficacy. Embodiment one : preparation of cicada flower mycelium active substance

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 the official website of the depository unit BCRC, and is easy to obtain for those with common 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 mycelium on the plate and inoculate it in 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;

Medium formula: Element Content ( weight %) sucrose 3.0 yeast extract 0.5 soy flour 1.0 Protein Potion 1.0 magnesium sulfate 0.05 Glycine 0.1

Fermentation tank culture: the culture medium is the same as above, inoculate the flask culture in the fermentation tank medium, at 25°C, tank pressure 1.0 kg/cm ² , pH 5.5, stirring speed 10 rpm, and air at 1.0 VVM ventilation rate, culture 5 One day, the cicada flower mycelium fermentation liquid was obtained. The cicada flower mycelium fermented liquid can be freeze-dried to obtain the cicada flower mycelium freeze-dried powder.

Extract preparation: take the freeze-dried powder of cicada mycelium and add 20 times the volume of distilled water to dissolve it, heat it at 100°C for 30 minutes, and dry it by freeze-drying after cooling to obtain the water extract of cicada mycelium.

Results: After lyophilization, about 320 kg of freeze-dried powder can be obtained from the fermented broth of cicadae mycelium cultured in 20 metric tons of fermentation tanks, and about 20-30 kg of water extract can be obtained through the extraction step. The following biological experiment was carried out with water extract of cicada flower mycelium. Example 2 Analysis of macular degeneration animal models and related indicators

Establishment of mouse model of macular degeneration

Sodium iodate (NaIO ₃ ), a stable oxidant, has been proven to be an effective method for inducing retinal degeneration. Sodium iodate-induced retinal degeneration is associated with regional loss of the retinal pigment epithelium (RPE), along with some morphological features of regional atrophy. Numerous studies have demonstrated the toxicity of NaIO ₃ to the biological retina using different mammalian species, including sheep, rabbits, rats and mice. The above studies pointed out that in the retina, the main target of NaIO ₃ is RPE cells, which can induce their death, such as necrosis, apoptosis or autophagy, followed by choriocapillaris atrophy. atrophy) and panretinal degeneration.

In recent years, researchers have used low doses of NaIO ₃ (15-35 mg/kg) to induce age-related macular degeneration (Age-related macular degeneration, AMD) models in mice, and found that it is associated with decreased visual function and local RPE loss It is related to external retinal damage and is very similar to the common pathogenesis of dry AMD. Therefore, in recent years, this model has mostly been used to explore the prevention of RPE regeneration and AMD.

In this example, 40 mg/kg NaIO3 was used to induce AMD in mice, so as to evaluate the preventive and therapeutic effects of the active substances of _Cicadae mycelium on macular degeneration.

Experimental animals: Strain Balb/c male mice were purchased from Taiwan National Experimental Animal Center, aged about 6-8 weeks, weighing about 26.21±1.76 g. Mice were raised in the Experimental Animal Center of Sun Yat-Sen Medical University, provided with normal clean feed and drinking water. The feeding environment was 12-hour light and 12-hour dark cycle light, the temperature was controlled at 20±2°C, and the humidity was controlled at 50±5%.

Feeding dosage and experimental procedures

The experiment was carried out for 21 days in total. Before the experiment, the mice were divided into 4 groups, 6 in each group:

(1) Blank control group (Mock): 100 μL of phosphate-buffered saline (PBS) was injected intravenously (i.v.), and 200 μL of PBS was given orally every day.

(2) Negative control group: sodium iodate NaIO ₃ 40 mg/kg was given intravenously (iv) to induce AMD, and PBS 200 μL was given orally every day.

(3) Cicada flower water extract group (experimental group): first give the cicada flower water extract 100 mg/kg prepared in Example 1 for 14 days by oral feeding every day, and then give sodium iodate NaIO by intravenous injection ₃ 40 mg/kg induced AMD, and then fed cicada flower water extract 100 mg/kg daily for 7 days.

(4) Positive control group: Onion extract is known to be used to improve macular degeneration. Here, as a positive control group, 1500 mg/kg of onion extract was administered orally every day for 14 days, and then administered intravenously Sodium iodate (NaIO ₃ ) 40 mg/kg induced AMD, and then fed onion extract 1500 mg/kg daily for 7 days.

The mice in each group were sacrificed 21 days after the experiment to detect the degree of retinal damage and analyze related indicators.

Detection of retinal damage and analysis of related indicators

1. Fundus image detection

Using the Phoenix-Micro IV fundus image detection system, it can detect optical ophthalmoscope, fluorescein fundus angiography and retinal tomography scanner, and use this to evaluate the integrity and smoothness of mouse retinal tissue changes. Figure 1(A) is the fundus photography of mice in each group on the 7th day after injection of sodium iodate to induce macular degeneration (the 21st day of the experiment), and Figure 1(B) is the optical tomography of the position marked by the red line in the corresponding photo (Optical Coherence Tomography) diagram.

The thickness (red line range) of the mouse in Figure 1(B) was measured 6 times in the range of 300 μm to 600 μm on the nasal side and temporal side (indicated by the yellow line) with the optic nerve as the center, and the average value was calculated to obtain the small The retinal thickness of the mouse is shown in Figure 2.

From the fundus photography in Figure 1(A), compared with the blank control group (Mock), the retina of the mouse group (NaIO ₃ ) administered sodium iodate alone 7 days after receiving sodium iodate injection The arrangement is irregular, and the ordering of tissues is not smooth, the boundaries are blurred, and there are obvious retinal distortions. However, compared with the mouse group (NaIO ₃ ) pretreated with onion extract (Onion) and cicadae extract, the retinal arrangement irregularity and blurred situation were significantly different. less phenomenon.

The results of retinal thickness measurement in Fig. 2 show (wherein the * sign indicates that compared with adding NaIO ₃ groups alone, p < 0.05, there is a significant difference), compared with the blank control group (Mock), administering sodium iodate alone The retinal thickness of the mouse group (NaIO ₃ ) was significantly thinner after receiving sodium iodate injection for 7 days. Compared with the mouse group (NaIO ₃ ) pre-treated with onion extract (Onion), it can significantly slow down the thinning of the mouse retina induced by sodium iodate, while Although there was no significant difference in cicada flower extract, the thickness still rebounded. However, the concentration of the positive control group using onion extract to slow down retinal thinning needs to be as high as 1500 mg/kg, while the concentration of cicada flower extract used in this example only needs 100 mg/kg (1/15).

Accordingly, it can be known that the cicadae extract of the embodiment of the present invention can slow down the distortion and thinning of the mouse retina induced by sodium iodate NaIO ₃ , thereby improving the situation of AMD. And compared with onion extract, the effect can be achieved at a lower concentration.

2. Histopathological analysis (H&E stainine)

The mice in each group were sacrificed and blood was collected on the 7th day after the injection of sodium iodate. After the eyes were made into tissue sections, they were stained with hematoxylin and eosin to distinguish the structure and type of RPE. The photos are shown in Figure 3(A) Show. Figure 3(B) is an enlarged view of the red dotted line frame in Figure 3(A), which is used to observe the thickness of the inner nuclear layer (Inner nuclear layer, INL) and outer nuclear layer (Outer nuclear layer, ONL), and to observe the number of inflammatory cells.

Both the INL and ONL layer thickness tests were centered on the optic nerve, and the thickness was measured 6 times (marked by the yellow line in Figure 3(B)) at the nasal and temporal sides of 300 μm to 600 μm, and the average value was calculated. The results were respectively Listed in Figure 4 (A) and Figure 4 (B), where the mark * indicates that compared with the NaIO ₃ group alone, p < 0.05, there is a significant difference.

Compared with the blank control group (Mock), the mouse group (NaIO ₃ ) injected with sodium iodate alone had thinner retinal ONL and INL layers 7 days after sodium iodate injection. Compared with the mouse group (NaIO ₃ ) administered with sodium iodate alone, the group of mice pretreated with onion extract (Onion) and cicadae flower extract can significantly slow down the retinal damage induced by sodium iodate. The ONL and INL layers become thinner.

Accordingly, it can be known that the cicadae flower extract of the embodiment of the present invention can slow down the thinning of the inner inner layer and outer nuclear layer of the mouse retina induced by sodium iodate (NaIO ₃ ), thereby improving the condition of AMD. Embodiment three composition preparation

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

Composition 1: get the lyophilized powder or the water extract (20 wt%) of the active substance of the cicadae mycelium of embodiment one, and magnesium stearate (8wt%) as lubricant, the carbon dioxide as preservative Silicon (7wt%) was mixed thoroughly, dissolved in pure water (65wt%), and 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 the cicadae mycelium in Example 2 is fed to the mice orally, other methods such as drops and suppositories can also be used in practice.

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

Composition 2: get the freeze-dried powder or water extract (20 wt%) of the active substance of cicadae flower mycelium of embodiment one, and benzyl alcohol (8 wt%) as preservative, glycerol (7 wt%) as diluent wt%) 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.

Fig. 1 (A) is the photographic fundus of each group of mice, and Fig. 1 (B) is the optical tomography (Optical Coherence Tomography) map corresponding to the position marked by the red line in the photo;

Fig. 2 is the retinal thickness measurement result of Fig. 1 (B);

Fig. 3 (A) is the photo of hematoxylin & eosin staining (H&E stain) of mouse eyes in each group, and Fig. 3 (B) is the enlarged view of the red dotted line frame of Fig. 3 (A);

Fig. 4(A) is the measurement result of the INL thickness of the inner inner layer in Fig. 3(A), and Fig. 4(B) is the measurement result of the ONL thickness of the outer nuclear layer in Fig. 3(A).

Claims (11)

A kind of cicadae ( Cordyceps cicadae ) mycelium active substance is used to prepare the application of the composition of preventing or treating macular degeneration, wherein the preparation method of the cicadae mycelium active substance comprises the following steps: (a) taking a cicadae The mycelium is cultured at a temperature of 15-35° C. for 2-10 days on a plate culture medium; (b) inoculating the cicadae mycelium after step (a) cultivation into a flask, at 15-35° C. Cultivate in an environment of pH 2-8 for 3-7 days; (c) inoculate the cicadae mycelium cultured in step (b) in a fermentation tank, and culture with stirring at 15-35°C and pH 2-8 After 3-5 days, a cicadae mycelium fermentation liquid containing one of the active substances of the cicadae mycelium is formed, wherein the active substance of the cicadae mycelium does not contain beauveria. The use as described in claim 1, wherein the step of preparing the active substance of the cicadae mycelium further includes step (d): freeze-drying the cicadae mycelium fermentation liquid and then grinding to form a powder containing the cicadae mycelium Freeze-dried powder of cicadae mycelium, one of the body active substances. The use as described in claim 2, wherein the step of preparing the active substance of the cicadae mycelium further includes step (e): extracting the freeze-dried powder of the cicadae mycelium with a solvent to form the cicadae mycelium containing the One of the body active substances cicada mycelium extract. The use as described in claim 3, wherein the step of preparing the active substance of the cicadae mycelium further includes a step (f): drying the extract of the cicadae mycelium to obtain the active substance of the cicadae mycelium. The use as described in claim 3 or 4, wherein in step (e), the solvent is water. Use as described in claim item 1, wherein the flask culture of step (b) is shaking culture, and the rotating speed is 10-250rpm. The use as described in Claim 1, wherein the macular degeneration is age-related macular degeneration. Use as described in claim item 1, wherein in the step (c), this fermenter further feeds a gas, and this gas comprises air, oxygen, carbon dioxide, helium or its combination, and the tank pressure of this fermenter is 0.5-1.0kg /cm ² and the ventilation rate is 0.01-1.5VVM. The use as described in Claim 1, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. The use as described in claim 1, wherein the composition is a food additive. The use as described in Claim 1, wherein the administration methods of the composition include oral administration, drops and suppositories.

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