KR20240143642A - Medical use of cordyceps militaris pa102 with anti-blue-light effect in slowing down retinal cell aging and retinal cytopathy, and repairing retinal damage - Google Patents

Abstract

The present disclosure discloses the use of a pharmaceutical composition comprising Cordyceps Militaris for resisting blue light, wherein Cordyceps Militaris comprises a mixture of cordyceps I, cordyceps II, cordyceps III, and cordyceps IV. The present disclosure uses as an active ingredient a deep culture product of Cordyceps Militaris PA102 which has 10 to 15 times higher anti-blue light effect than marigold extract (containing 20% lutein) commonly used in the market for protecting eyes. The pharmaceutical composition comprising Cordyceps Militaris of the present disclosure can have a protective effect against eye damage induced by blue light at a low dose and is medically used for delaying retinal cell aging and retinal cytopathy, repairing retinal damage, and preventing macular degeneration.

Description

MEDICAL USE OF CORDYCEPS MILITARIS PA102 WITH ANTI-BLUE-LIGHT EFFECT IN SLOWING DOWN RETINAL CELL AGING AND RETINAL CYTOPATHY, AND REPAIRING RETINAL DAMAGE

The present disclosure relates to a method for deep cultivation of Cordyceps Militaris, and particularly to a method for deep cultivation of Cordyceps Militaris PA102 for improving the content of cordyxanthin.

In modern society, with the change of lifestyle, people often use 3C (computer, communication, home appliance) products at close range for a long time while ignoring the damage to the eyes. In particular, in the case of long-term contact with 3C products including consumer electronic devices such as computers, mobile phones, tablet personal computers, etc., the eyes are used involuntarily, the axis of the eyes is extended, the strength of glasses increases rapidly, and in severe cases, macular damage is caused, and the risk of retinal detachment and blindness increases.

Among the eye damage caused by 3C products, the main damage is blue light reaching the eyes. Blue light with a wavelength of 400 to 500 nm has a short wavelength and strong energy in visible light. Blue light will pass through the cornea and reach the retina through the lens. The energy of blue light is absorbed by the lens and retina. Free radicals are generated by oxidation. If free radicals accumulate excessively, cells will be damaged, cataracts and macular damage will occur, and object distortion and deformation in the field of vision, or abnormal color perception may appear. If blue light damage is not properly avoided or treated in a timely manner, it can eventually cause permanent vision damage.

The retinal pigment epithelium (RPE) is a single layer of cells located between the photoreceptor cells of the neural retina and the choroid plexus. The RPE functions functionally in the external blood-retinal barrier and forms tight junctions with endothelial cells that are important for transporting fluids or substances across the barrier. Thus, the RPE has a function in maintaining the integrity of the retinal layers. Retinal cells will produce deposits called drusen. Drusen are deposited on the basal layer of the retina and contain a lipofuscin mixture that is highly sensitive to light. After stimulation by light, the lipofuscin mixture is susceptible to photooxidative damage by generating reactive oxygen molecules and superoxide anions. Photooxidative damage can cause dysfunction of the RPE, including differentiation, inflammation, and apoptosis.

Cordyceps militaris belongs to the family Cordyceps , Cordycipitaceae , and has an orange oval fruiting body. Since Cordyceps militaris has a wide range of hosts and grows under less harsh conditions, its fruiting body has been artificially cultivated. Cordyceps militaris is one of the important active ingredients. Cordyceps militaris has at least four cordyxanthins, namely, cordyceps I, cordyceps II, cordyceps III, and cordyceps IV. Cordyceps militaris has the effects of improving fertility, resisting fatigue, improving sports performance, resisting oxidation and inflammation, regulating immunity, controlling blood sugar, dissolving thrombosis, and resisting tumors.

Codicxanthin I:

Codicxanthin II:

Codicxanthin III:

Codicxanthin IV:

However, in the technical field of existing anti-blue light Cordyceps militaris, it is difficult to obtain an anti-blue light component containing codicoxanthin I, codicoxanthin II, codicoxanthin III, and codicoxanthin IV simultaneously from Cordyceps militaris, and therefore, the anti-blue light component has poor health-care effects. Currently, in order to further improve the anti-blue light effect of Cordyceps militaris, a Cordyceps militaris strain capable of simultaneously producing a large amount of codicoxanthin I, codicoxanthin II, codicoxanthin III, and codicoxanthin IV and a method for producing the same are urgently required.

In view of the above, the inventors of the present invention have extensively and thoroughly understood the shortcomings and deficiencies of the previous cases, and need to think about improvements and innovations. After years of research, they have successfully discovered a strain of Cordyceps militarius, which can simultaneously produce large quantities of cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV, and a method for deep cultivation thereof. Cordyxanthin has a protective effect against eye damage induced by blue light at low doses, and is used to delay retinal cell aging and retinal cytopathy, repair retinal damage, and prevent macular degeneration. A novel and low-cost health-care strategy for the anti-blue light use of cordyxanthin is also provided.

The present disclosure aims to provide a use of a pharmaceutical composition comprising Cordyceps militaris in resisting blue light, wherein Cordyceps militaris comprises the following: Codicxanthin I, Codicxanthin II, Codicxanthin III, and Codicxanthin IV:

Codicxanthin I:

Codicxanthin II:

Codicxanthin III:

Codicxanthin IV:

In one example of the present disclosure, the use in resisting blue light is to slow down retinal cell aging and retinal cytopathies, repair retinal damage, repair retinal damage, or prevent macular edema.

In one example of the present disclosure, the Cordyceps militaris is Cordyceps militaris PA102 having the registration number BCRC 930232.

In one example of the present disclosure, the pharmaceutical composition comprises a deep culture product of Cordyceps militaris PA102.

In one example of the present disclosure, a method for producing a deep culture product comprises the steps of: culturing Codyceps militaris PA102 in a culture medium; irradiating the culture medium with blue light or red light; and collecting a fermentation product of Codyceps militaris PA102 to obtain a deep culture product.

In one example of the present disclosure, the deep culture product has an effective amount of greater than or equal to 0.25 μg/mL.

In one example of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle, excipients and diluents.

In conclusion, the present disclosure provides a deep culture product containing a large amount of codicanthin I, codicanthin II, codicanthin III, and codicanthin IV simultaneously from Cordyceps militaris PA102. Codicanthin has a protective effect against eye damage induced by blue light at a small amount, and is medically used to delay retinal cell aging and retinal cytopathy, repair retinal damage, and prevent macular degeneration.

The technology of the present invention will be further understood from the detailed description provided herein below, and the accompanying drawings are provided for better illustration, and therefore, the description and drawings do not limit the present invention.
Figure 1 illustrates the effect of different doses of deep culture products of Cordyceps militaris on blue light damage to the retinal pigment epithelium.
Figure 2 shows the effect of different amounts of marigold extract on blue light damage to the retinal pigment epithelium.

[Definition of terms]

In this description, many technical and scientific terms commonly used in the technical field of biotechnology are used extensively. In the description below, the following definitions are provided to ensure a clear and consistent understanding of the scope of the description and claims, as well as the terms to which they are titled. Other terms not specifically defined below have the meanings generally understood by those skilled in the art.

Materials used in the present disclosure are commercially readily available unless otherwise specified. Cordyceps militaris PA102 used in the examples of the present disclosure was deposited with the Institute for Food Industry Development, Hsinchu Incorporated Foundation, Taiwan under the accession number BCRC930232.

The terms "or", "also", "and" as used in the description, unless otherwise indicated, all refer to "or/and". Also, the terms "including" and "comprising" should be construed in an open-ended manner and not as limiting. The preceding paragraphs are merely a systematic reference and should not be construed as limiting the subject matter of the present disclosure.

As used herein, "%" refers to "weight percent (wt %)" unless otherwise specified; a numerical range (e.g., A from 10% to 11%) includes the upper and lower limits (i.e., 10%≤A≤11%) unless otherwise specified; a numerical range that does not define a lower limit (e.g., B less than or B below 0.2%) means that the lower limit can be zero (i.e., 0%≤B≤0.2%); and the ratio relationship of "weight percent" of each component can be replaced with the ratio relationship of "parts by weight".

All numbers disclosed in this description may have a standard technical measurement error (standard deviation) of ±10%. The term "about" is intended to mean ±10%, ±5%, ±2.5%, or ±1% of a given value, i.e., "about 20%" means 20±2%, 20±1%, 20±0.5%, or 20±0.25%.

The term "deep culture" used in this description is different from the conventional term "extraction". Specifically, deep culture is very effective in producing a target compound in a required yield by culturing a strain in a specific culture medium and culture conditions to create an environment suitable for the growth and reproduction of the strain, thereby rapidly proliferating and producing (e.g., fermenting) the target compound. In another aspect, extraction is a method of extracting a compound using a specific solvent or extractant, comprising a step of contacting the extractant with the fungus for a sufficient time to extract the target compound.

The term “pharmaceutically acceptable” as used herein refers to a substance or composition being compatible with the other ingredients of a pharmaceutical formulation and not aggravating the patient’s symptoms.

The pharmaceutical compositions provided by the present disclosure can be prepared in dosages suitable for the compositions of the present disclosure by using the active ingredients or compositions provided in the case and at least one pharmaceutically acceptable vehicle according to techniques known to those skilled in the art to which the present disclosure pertains. The dosages include, but are not limited to, solutions, emulsions, suspensions, powders, lozenges, oral lozenges, tablets, chewing gums, capsules, and other similar or suitable dosages for the present disclosure.

The term "pharmaceutically acceptable vehicle" as used herein includes one or more types of ingredients selected from solvents, emulsifiers, suspending agents, disintegrants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, and other similar or suitable vehicles for the present disclosure.

In the composition, one or more of a dissolution aid, buffer, colorant, flavoring agent, etc. commonly used in the manufacturing field may be appropriately added as needed.

As used herein, “pharmaceutically acceptable excipient” includes, but is not limited to, at least one of a polymer, a resin, a plasticizer, a filler, a lubricant, a diluent, a binder, a disintegrant, a solvent, a cosolvent, a surfactant, a preservative, a sweetener, a flavoring agent, a pharmaceutical grade dye or pigment, and a thickener.

Suitable routes of administration for the pharmaceutical compositions of the present disclosure include, but are not limited to, oral, intravenous, rectal, aerosol, gastrointestinal, ocular, pulmonary, mucosal, dermal, vaginal, auricular, nasal, or topical administration. In addition, examples of gastrointestinal administration include, but are not limited to, intramuscular, subcutaneous, intravenous, and intramedullary injections, and intramedullary, intraventricular, intraperitoneal, intralymphatic, or intranasal injections. The dosage of the pharmaceutical compositions provided in the present disclosure can be appropriately adjusted as needed and is not particularly limited. Oral administration is preferred.

Edible substances may be additionally added to the pharmaceutical composition of the present disclosure to prepare a foodstuff or health-care product. Edible substances include, but are not limited to, water, a fluid milk product, milk, condensed milk, yogurt, sour milk, frozen yogurt, a lactic acid bacteria-fermented beverage, powdered milk, ice cream, cheese, dried cheese, soy milk, fermented soy milk, vegetable-fruit juice, juice, sports drinks, confectionery, jelly, candy, infant formula, health food, animal feed, Chinese herbs or dietary supplements.

The term "treatment" as used herein includes alleviating, alleviating, or ameliorating at least one disease symptom or physiological condition in a curative or preventive manner, preventing new symptoms, inhibiting the disease or physiological condition, arresting or slowing the development of the disease, causing reversal of the disease or physiological condition, slowing a physiological condition caused by the disease, and stopping a disease symptom or physiological condition.

The term "effective amount" as used herein refers to an adequate amount of an effective active ingredient and an amount that prevents or alleviates one or more disease symptoms or physiological conditions in a person to whom it is administered. The result is a reduction and/or alleviation of the signs, symptoms, or causes of the disease, or other intended changes in physiological systems.

[Codyseps Militaris Material]

The Cordyceps militaris used in the description can be obtained from nature by a person skilled in the art to which the present disclosure belongs. An example of the present disclosure exemplifies Cordyceps militaris PA102. However, the present disclosure is not limited by Cordyceps militaris PA102. Any Cordyceps militaris strain capable of producing cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV are included in the scope of the present disclosure. However, from the viewpoint of improving the anti-blue light effect, Cordyceps militaris PA102 is preferably used in the present disclosure, and was deposited with the Research Institute for Food Industry Development, Hsinchu Incorporated Foundation, Taiwan on February 8, 2023 under the accession number BCRC930232. Compared with other Cordyceps militaris strains, richer yields of codicanthin I, codicanthin II, codicanthin III, and codicanthin IV can be obtained from Cordyceps militaris PA102.

[Deep cultivation]

The present disclosure's method for obtaining cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV from Cordyceps militaris is not limited. Any production method for obtaining cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV from Cordyceps militaris should all be included within the scope of the present disclosure. However, from the viewpoint of yield improvement, the present disclosure preferably performs deep cultivation on Cordyceps militaris and takes a fermentation product (deep cultivation product). In an example, the deep cultivation method of the present disclosure includes a step of culturing Cordyceps militaris in a culture medium, and a step of irradiating the culture medium with blue light or red light; Specifically, the deep culture method of the present disclosure can sequentially be solid-state fermentation culture of Cordyceps militarius, preparation of an inoculum source for liquid inoculation, solid-state fermentation culture and inoculation, dark culture (culture without illumination, e.g., dark culture for 10 days), and illuminated culture (culture in an irradiation phase (illumination cycle), e.g., illuminated culture for 20 days).

In the investigation phase of deep culture, the investigation time is not limited, i.e. the specific range of the light cycle is not limited, and the light cycle can provide a continuous light period followed by a continuous dark period. The light period can be, for example, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours per day, or any two intervals therebetween. Correspondingly, the dark period can be, for example, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or any two intervals therebetween. However, considering further enhancing the yields of codicanthin I, codicanthin II, codicanthin III, and codicanthin IV, the light cycle is preferably illuminated for 16 hours or 20 hours per day. That is, the light cycle is preferably illuminated for 16 hours per day and darkened for 8 hours per day, or illuminated for 20 hours per day and darkened for 4 hours; and more preferably illuminated for 20 hours per day and darkened for 4 hours.

In the example, the blue light has a wavelength of 400 to 500 nm, for example, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, or a wavelength in the range of any two of said wavelengths. However, 450 nm is preferred, considering that it further enhances the yields of cordixanthin I, cordixanthin II, cordixanthin III, and cordixanthin IV.

In the example, the red light has a wavelength of 620 to 760 nm, for example, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, or an interval of any two of the above wavelengths. However, 660 nm is preferred, considering that it further enhances the yields of cordixanthin I, cordixanthin II, cordixanthin III, and cordixanthin IV.

In the culturing step, the culturing temperature is not limited and may be, for example, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C or an interval of any two of the above temperatures. However, considering further increasing the yields of codicanthin I, codicanthin II, codicanthin III, and codicanthin IV, 18 to 26°C is preferred, and 21 to 24°C is more preferred.

The culture time in the culture step is not limited and may be, for example, 25 days, 27 days, 29 days, 31 days, 33 days, 35 days, 37 days, 39 days, 41 days, 43 days, 45 days, 47 days, 49 days, 51 days, 53 days, 55 days, 57 days, 59 days, 61 days, or an interval of any two of the above periods. However, considering further increasing the yields of codicanthin I, codicanthin II, codicanthin III, and codicanthin IV, 30 to 60 days is preferred, and 45 days is more preferred.

In the example, considering the increase in the yield of codicanthin I, codicanthin II, codicanthin III, and codicanthin IV, in the irradiation step, the irradiation with blue light is preferably performed for 20 hours per day, and preferably, the cultivating is performed for 45 days, wherein the cultivating is performed in complete darkness for the first 10 days and in a light cycle for the last 35 days; or preferably, the cultivating is performed for 30 days, wherein the cultivating is performed in complete darkness for the first 10 days and in a light cycle for the last 20 days.

[Medical Uses of Cordyceps Militaris]

The present disclosure discloses a method of using Cordyceps militaris to resist blue light for protecting the eye. The method comprises administering to a patient (particularly a human) in need thereof a therapeutically effective amount of a Cordyceps militaris deep culture product (comprising cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV) as an effective ingredient for preventing or treating ophthalmic diseases by slowing down retinal cell aging and retinal cytopathies, repairing retinal damage, and preventing macular degeneration.

[Administration of Cordyceps Militaris]

Targeted administration of Cordyceps militarius to the eye can be accomplished by a variety of methods, including intravenous, intradermal, subcutaneous, oral (e.g., by inhalation), transdermal (i.e., topical), transmucosal, intraperitoneal, intraarterial, and rectal administration. Other suitable routes include administering compositions such as: oral, buccal, nasal, nasopharyngeal, parenteral, enteral, gastric, topical, transdermal, subcutaneous, and intramuscular, in the form of lozenges, solids, powders, liquids, and aerosols, intralesional injection into the eye, intralesional injection near the eye, and intravenous and intraarterial infusions. Topical or systemic administration can be done with or without the addition of excipients. Administration can additionally be done via sustained-release mode into the eye or around the eye of the individual. Preferably, oral administration into the body of the patient is preferred.

[amount]

In the treatment method of the present disclosure, an effective amount of Cordyceps militaris is administered to a subject in need thereof. In particular, the effective amount will vary depending on the purpose of administration, the health and physical condition of the subject to be treated, age, the classification of the subject to be treated (e.g., human, non-human primate, primate, etc.), the dosage of Cordyceps militaris, the assessment of the medical condition by the treating clinician, and other relevant factors. The amount is expected to be within a relatively wide range, which can be determined by routine testing. For example, to sufficiently obtain an eye-protecting effect, the effective amount of the deep culture product of Cordyceps militaris PA102 is about 0.25 μg/mL or more.

In the example, different dosages of the deep culture product of Cordyceps militaris PA102 can be used, depending on the need. The amount is generally at least about 0.1 μg/mL, for example, at least about 0.15 μg/mL, at least 0.2 μg/mL, at least 0.25 μg/mL, at least 0.3 μg/mL, at least 0.35 μg/mL, at least 0.4 μg/mL, at least 0.45 μg/mL, and a range between any two of the aforementioned numerical values. The deep culture product of Cordyceps militaris PA102 of the present disclosure has an eye-protective effect at low dosages. Therefore, the upper limit of the dosage is not particularly limited, and can be, for example, at most about 100 μg/mL, at most 500 μg/mL, at most 1,000 μg/mL, or at most 5,000 μg/mL.

In the example, different administration periods of the deep culture product of Cordyceps militaris PA102 can be used, depending on the need. The administration period is typically from 1 day to 4 months or more, for example, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 days. In another example, the administration period can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, and 18 weeks or more.

In the example, different dosing frequencies of the deep culture product of Cordyceps militarius PA102 can be used, depending on the need, for example, 1 to 3 times per day, 1 to 6 times every 2 days, 1 to 9 times every 3 days, 1 to 14 times per week, and 1 to 60 times per month.

The medical uses of Cordyceps militarius PA102 having anti-blue light effects in delaying retinal cell aging and retinal cytopathies and in repairing retinal damage are described by enumerating several examples and comparative examples, which are not intended to limit the present disclosure. Various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

[Example 1 Strain activation and cultivation]

The strain was isolated from the fruiting body of Codyceps militarius PA102, the strain cryotube was taken out from the refrigerator at -80℃, and the strain was cultured on yeast malt agar (YMA) plate culture medium as an inoculum for solid-state fermentation culture tests.

[Example 2 Liquid inoculation and preparation of inoculum]

The strain was grown on a plate culture medium for 14 days, and after the colonies reached a diameter of 10 to 15 mm, a fungal block of about 3 ㎟ was cut and placed in a liquid culture medium containing 1.8% glucose and 0.3% yeast extract at a temperature of 21°C and a shaking speed of 125 rpm for shake-flask culture for 7 days.

[Example 3 Solid-state culture inoculation and culture conditions]

The cultured fungal liquid in the shake flask was used as an inoculum, and inoculated onto solid culture media containing five grains at an inoculum size of 20 ml/box and a temperature of 21 to 24°C for 45 days.

[Example 4 Dark culture and light culture]

After solid-state culture inoculation, the strain was first cultured in the dark for 10 days and then transferred to light conditions for cultivation. After 35 days, the fungus changed from white to yellow or orange yellow, and was then harvested and dried to obtain Cordyceps militaris deep culture product containing cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV simultaneously.

[Example 5 Analysis of cordixanthin]

The content of codicanthin in the fermentation products in each test group was analyzed using high-performance liquid chromatography (HPLC). The analysis method was as follows:

(1) Preparation of test solution: Take 5 g of solid-state fermentation product powder each, add 100 mL of 80% ethanol, vibrate the mixture ultrasonically, extract continuously for 30 minutes, filter using a 0.45-㎛ filter paper, and then analyze the filtrate by HPLC.

(2) Conditions for HPLC analysis: Chromatography column: Inertsil ODS-2.5 μm, 4.6 I.D. × 25 mm, UV wavelength: 447 nm, flow rate of 1.0 mL/min, injection volume of 10 μl, and mobile phase as follows:

The experimental results are shown in Table 1.

[Table 1] HPLC analysis map (absorption value)

From the above table, it can be seen that when Cordyceps militaris PA102 was deeply cultured by the method of the present disclosure, the deep culture product was simultaneously rich in codicanthin I, codicanthin II, codicanthin III, and codicanthin IV. Therefore, codicanthin I, codicanthin II, codicanthin III, and codicanthin IV can be produced in large quantities at low cost, which can greatly improve the anti-blue light effect of Cordyceps militaris.

[Example 6 Synthesis and purification of A2E]

A2E is lipofuscin, named N-retinylidene-N-retinylethanolamine (also called N-retinylidene-phosphatidylethanolamine), the main component of drusen, with a maximum absorption wavelength of 460 nm, which is in the blue light range. Because of its high energy and penetrating properties, blue light can reach the retina of the fundus, activate the photo-oxidation reaction of lipofuscin, and damage the retinal epithelial cells. Lipofuscin is an important marker for ophthalmic research related to ophthalmic diseases (e.g., macular edema).

100 mg of all-trans-retinal was dissolved in 6 mL of dichloromethane, 140 μl of ethanolamine was added, mixed, 10 μl of acetic acid was added, and the mixture was stirred in the dark at room temperature for 3 days. After the reaction, dichloromethane was removed by concentration, and the residue was preliminarily purified using a C18 silica gel glass column, wherein the eluting solution was 0.05% trifluoroacetic acid (TFA) in acetonitrile (ACN): 0.05% TFA in water (75:25). After collecting the fractions containing A2E, they were purified by semi-preparative HPLC under the following conditions: column of 1 cm RP-C18, temperature controlled at 40°C, flow rate of 1 mL/min, mobile phase A: 0.1% TFA in water, mobile phase B: 0.1% formic acid (FA) in ACN, and ratio of mobile phase A: mobile phase B of 1:9. After collecting the purified fractions, they were analyzed by analytical HPLC under the following conditions:

Chromatography column: Inertsil 250×4.6 mm i.d. 5 ㎛

Detection wavelength: 440 nm

Column temperature: 35℃

Mobile phase: A-0.05% trifluoroacetic acid aqueous solution and B-acetonitrile

Flow rate: 1 ml per minute

The elution gradients are presented in the table below:

[Example 7 Protection of Cordyceps Militaris in Retinal Pigment Epithelium]

Human retinal pigment epithelium (ARPE-19) was cultured in 24-well microplates to ensure a cell density of 6 × 10 ⁴ cells/well. Culture was performed at 37°C for 24 h in a 5% CO ₂ incubator until ARPE-19 attached to the plate; After that, the old culture solution was removed, 0.4 mL of cell culture solution containing different concentrations of test samples (Cordyceps militarius and marigold) was added, the culture solution containing the test samples was removed after 24 h of incubation, 0.4 mL of cell culture solution containing 40 μM A2E was added, the microplate was irradiated with blue light (illuminance: 9,000 lumens for 45 min) to induce photodamage to ARPE-19, the blue light irradiation was removed, the culture solution was removed after 24 h of continuous incubation, 0.3 mL of culture medium containing 1 mg/mL MTT (should be in the dark) was added to each well, and the reaction was performed in a _CO2 incubator for 2 h; finally, the culture medium containing the MTT reagent was removed, 0.4 mL of DMSO was added, and shaken evenly to dissolve the crystals. The absorbance of the mixture was measured by an ELISA reader at a wavelength of 570 nm, and the cell viability was calculated.

Refer to Fig. 1 and Table 2 below. Fig. 1 shows the effect of different amounts of deep culture product of Cordyceps militaris on blue light damage to retinal pigment epithelium. The deep culture product of Cordyceps militaris PA102 was evaluated for reducing blue light-induced retinal cell damage at test concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 ㎍/mL, where * indicates P < 0.05. As illustrated in the figure, blue light irradiation induced A2E to produce photodamage to retinal cells. The cell viability of the control group (untreated) was 57%, and the cell viability of the groups treated with 0.25, 0.5, and 1 μg/mL of the deep culture product of Cordyceps militaris PA102 (containing cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV simultaneously) was 68%, 71%, and 86%, respectively. Therefore, the deep culture product can significantly reduce retinal cell damage induced by blue light, and the correlation is in an amount-effect manner.

[Table 2] Cell survival rate of groups treated with deep culture products of Cordyceps militaris PA102 at various concentrations under blue light damage

From the results, it can be seen that the deep culture product of Cordyceps militarius PA102 of the present disclosure can effectively improve the cell viability of ARPE-19. The deep culture product rich in cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV has a protective effect against eye damage induced by blue light, and can be medically used to delay retinal cell aging and retinal cytopathy, repair retinal damage, and prevent macular edema.

[Example 7: Test using marigold extract (containing 20% lutein) as a control group]

Refer to Fig. 2 and Table 3 below. Fig. 2 shows the effect of different amounts of marigold extract on blue light damage to retinal pigment epithelium. Marigold extract (containing 20% lutein) was evaluated for reducing retinal cell damage induced by blue light at test concentrations of 0.625, 1.25, 2.5, 5, and 10 μg/mL, where * indicates P < 0.05. As shown in the figure, marigold extract could reduce retinal cell damage induced by blue light at higher concentrations (5 and 10 μg/mL). The cell viability was 65%, 63%, 65%, 71%, and 83%, respectively.

[Table 3] Cell viability of marigold extract at different concentrations for blue light damage

From the above results, it can be seen that the commercially available marigold extract (containing 20% lutein) for eye protection can also improve the cell viability of ARPE-19, but the marigold extract had an obvious protective effect only when the amount was as high as 5 μg/mL or more. In addition, referring to Tables 2 and 3, the deep culture product of Cordyceps militaris PA102 of the present disclosure showed a significant effect only at a low amount (about 0.25 μg/mL), i.e., the deep culture product of Cordyceps militaris PA102 of the present disclosure had an anti-blue light effect as high as 10 to 15 times.

In conclusion, by using A2E together with blue light irradiation, we tested whether the deep culture product of Cordyceps militaris PA102 has the effect of protecting retinal pigment epithelium and reducing blue light damage, thereby preventing macular edema. From the experimental results, compared with the commercially available marigold extract (containing 20% lutein) for eye protection, the pharmaceutical composition comprising Cordyceps militaris of the present disclosure can have a protective effect against eye damage caused by blue light at a small amount, and can be medically used for delaying retinal cell aging and retinal cytopathy, repairing retinal damage, and preventing macular edema.

The above detailed description is not intended to limit the scope of the present disclosure, but rather to provide specific examples of possible embodiments of the present disclosure. All equivalent implementations or modifications that do not depart from the spirit of the present disclosure will all be included within the scope of this case. Multiple effects are statutory patentable elements that are fully endowed with novelty and originality. This application has been filed in accordance with the law. An application for an invention patent will be reviewed and approved to claim the invention.

Accordingly, it should be understood that the embodiments of the invention described herein are merely illustrative of the application of the principles of the invention. Reference to details of the embodiments illustrated herein is not intended to limit the scope of the claims, which themselves recite features deemed essential to the invention.

[Deposit of living organisms]

1. The research institute for food industry development of Hsinchu Incorporated Foundation of Taiwan, February 8, 2023, and deposit number BCRC930232

Syi Biotechnology Co., Ltd. TTSY-PA102-1111BCRC930232 20230208

Claims (7)

Use of a pharmaceutical composition comprising cordyceps militaris in resisting blue light, wherein cordyceps militaris comprises the following: cordyxanthin I, cordyxanthin II, cordyxanthin III, and cordyxanthin IV.
Codicxanthin I:

Codicxanthin II:

Codicxanthin III:

Codicxanthin IV:
Use of a pharmaceutical composition comprising Cordyceps militaris in resisting blue light, for slowing down retinal cell aging and retinal cytopathy, repairing retinal damage, or preventing maculopathy in claim 1. Use of a pharmaceutical composition comprising Cordyceps militaris in resisting blue light, wherein the Cordyceps militaris is Cordyceps militaris PA102 having the deposit number BCRC 930232, in claim 1. Use of a pharmaceutical composition comprising Cordyceps militaris in resisting blue light, wherein the pharmaceutical composition comprises a deep culture product of Cordyceps militaris PA102 in claim 3. In the fourth paragraph, a method for producing a deep culture product
Step of culturing Cordyceps militarius PA102 in a culture medium;
A step of irradiating the culture medium with blue light or red light; and
Use of a pharmaceutical composition comprising Cordyceps militaris in resisting blue light, comprising the step of collecting a fermentation product of Cordyceps militaris PA102 to obtain a deep culture product. Use of a pharmaceutical composition comprising Cordyceps militaris for resistance to blue light, wherein the deep culture product has an effective amount of 0.25 ㎍/mL or more in claim 4. Use of a pharmaceutical composition comprising Cordyceps militarius for resisting blue light, wherein the pharmaceutical composition comprises a pharmaceutically acceptable vehicle, excipients and diluents in claim 1.