TWI772201B - Use of extract of fruit body of cordyceps militaris for manufacturing medication for improving anti-blue light damage effect - Google Patents

Abstract

A use of an extract of fruit body of Cordyceps militaris is for manufacturing a medication of improving anti-blue light damage effect. The extract of fruit body of Cordyceps militaris is obtained by extracting a sample of fruit body of Cordyceps militaris using an extractant being an aqueous ethanol solution with a concentration of 62.9% for 88.3 minutes.

Description

Cordyceps militaris fruiting body extract is used to manufacture anti-blue light damage effect Use of the drug

The present invention relates to the use of a fruiting body extract of Cordyceps militaris, especially the use of a fruiting body extract of Cordyceps militaris to manufacture a medicine for enhancing the anti-blue light damage effect.

Visible light refers to electromagnetic radiation visible to the human eye, with wavelengths ranging from 380 to 750 nm. Blue light generally refers to visible light with wavelengths ranging from 380 to 500 nm. violet light with a wavelength of about 410-455 nm, blue-violet light with a wavelength of about 455-500 nm, and blue-turquoise light with a wavelength of about 455-500 nm. Violet and blue-violet light have shorter wavelengths, so they have higher energy, which can cause damage to the eyes, so they are often referred to as harmful blue light.

Blue light can pass through the cornea and lens, and then reach the retina, and research has confirmed that blue light not only affects vision, but may also lead to premature aging of the eye, for example, overexposure Exposure to blue light may cause sore or irritated eyes, digital eye strain such as difficulty focusing, and age-related macular degeneration. retinal damage, etc.

Generally speaking, the sources of blue light in life include sunlight, fluorescent lamps, LED lamps and screens of TVs, tablets and smart phones. The distance of the screen is relatively short, and the time of staring at the screen is also long. Therefore, many anti-blue light products such as screen filters, computer glasses, and anti-reflective lenses have been developed on the market. It can help to avoid the damage caused by blue light to the eyes.

Cordyceps militaris ( Cordyceps militaris ) is a fungus of the family Cordycipitaceae and is a type species of the genus Cordyceps . It is currently known that the active ingredients of Cordyceps militaris are beneficial to promote libido, anti-inflammatory, antioxidant and anti-aging. , Anti-tumor / anti-cancer / anti-leukemia, anti-proliferation, anti-metastasis, immune regulation, anti-bacteria, fungi, viruses and other microorganisms, anti-fibrosis, hypoglycemic, hypolipidemic, anti-angiogenesis, anti-fatigue, active ingredients of Cordyceps militaris It also helps to protect organs such as nerves, liver, kidneys and lungs.

At present, a variety of culture technologies for Cordyceps militaris have been developed, such as the Republic of China Announcement No. I459953 Patent Case, which is to inoculate Cordyceps militaris in a compound containing added animal proteins (milk, gravy extract, fetal bovine serum, fish meal, fish essence, silkworm chrysalis, red Earthworms), vegetable protein, probiotic fermentation broth (lactic acid bacteria fermentation broth, fungal fermentation broth), Chinese herbal medicine extracts, etc. PCB (plate count broth) medium, at 20~24 ℃ temperature, at 90 rpm After 3~7 days of shaking flask culture, inoculate it on the grain medium containing white rice, brown rice or oats, etc., first in a dark environment with a temperature of 18~25°C and a humidity of 70~90% (light intensity is about 0Lux) Cultivated for 5~7 days, and then cultivated for about 50 days in a lighting environment with a temperature of 18~25°C and a humidity of 70~90% (the light intensity is more than 500Lux, and the lighting time is 8~15 hours per day), so that the obtained The fruiting body of Cordyceps militaris not only grows faster, but also the content of cordycepin contained in the fruiting body extract of Cordyceps militaris obtained by extraction is also higher. However, it is still unknown whether the fruiting body extract of Cordyceps militaris thus obtained can be used to enhance the anti-blue light damage effect. In view of this, it is still necessary to provide a The use of Cordyceps fruiting body extract in the manufacture of a drug for enhancing the anti-blue light damage effect.

In order to solve the above problems, the purpose of the present invention is to provide a use of the fruiting body extract of Cordyceps militaris, which is used to manufacture a medicine for enhancing the anti-blue light damage effect.

The use of the Cordyceps militaris fruiting body extract of the present invention can be used to manufacture a medicine for enhancing the effect of anti-blue light damage; wherein, the Cordyceps militaris fruiting body extract is extracted from an ethanol aqueous solution with a concentration of 62.9%. Cordyceps militaris fruiting body The sample was obtained in 88.3 minutes.

Accordingly, the use of the Cordyceps militaris fruiting body extract of the present invention can protect the desired individual with the active ingredients contained in the Cordyceps militaris fruiting body extract (for example, including Cordyceps militaris carotenoids such as Cordyceps militaris flavin III) The photoreceptor cells located in the retina prevent apoptosis of these photoreceptor cells, thus preventing retinal degeneration caused by the irradiation of harmful blue light (generally referring to high-energy visible light with a wavelength of 380~500nm), and then It can be applied to the manufacture of medicines for enhancing the anti-blue light damage effect, which is the effect of the present invention. And, by extracting the Cordyceps militaris fruit body sample with a concentration of 62.9% ethanol aqueous solution for 88.3 minutes, the Cordyceps militaris carotenoids contained in the Cordyceps militaris fruit body extract can be effectively dissolved in the ethanol aqueous solution, so that each kilogram of The Cordyceps militaris fruiting body extract can contain more than 690 mg of Cordyceps militaris carotenoids, which achieves the effect of optimizing the process for obtaining the Cordyceps militaris fruiting body extract.

The use of the Cordyceps militaris fruit body extract of the present invention, wherein the Cordyceps militaris fruit body extract is administered to a desired individual by oral feeding. In this way, the user can ingest the Cordyceps militaris fruiting body extract in a convenient manner, which helps to improve the user's drug compliance.

The use of the Cordyceps militaris fruit body extract of the present invention, wherein, the Cordyceps militaris fruit body extract can be continuously administered to the desired individual at a dose of 10-100 mg/kg/day for 2-30 days. like Therefore, the Cordyceps militaris fruit body extract has a better ability to protect photoreceptor cells, and can prevent the decline of visual acuity and visual contrast sensitivity.

The use of the Cordyceps militaris fruit body extract of the present invention, wherein the Cordyceps militaris fruit body extract can be continuously administered to the desired individual at a dose of 20 mg/kg/day for 7 to 22 days. In this way, the Cordyceps militaris fruit body extract has better ability to protect photoreceptor cells, and can prevent the decline of visual acuity and visual contrast sensitivity.

The use of the Cordyceps militaris fruit body extract of the present invention, wherein the Cordyceps militaris fruit body extract can be continuously administered to the desired individual at a dose of 1-10 mg/kg/day for 7-60 days. In this way, the Cordyceps militaris fruit body extract has better ability to protect photoreceptor cells, and can prevent the decline of visual acuity and visual contrast sensitivity.

The use of the Cordyceps militaris fruit body extract of the present invention, wherein the Cordyceps militaris fruit body extract can be continuously administered to the desired individual at a dose of 1-3 mg/kg/day for 7-22 days. In this way, the Cordyceps militaris fruit body extract has better ability to protect photoreceptor cells, and can prevent the decline of visual acuity and visual contrast sensitivity.

The use of the Cordyceps militaris fruit body extract of the present invention, wherein the Cordyceps militaris fruit body extract can be continuously administered to the desired individual at a frequency of 1 to 3 times/day. In this way, the Cordyceps militaris fruit body extract has better ability to protect photoreceptor cells, and can prevent the decline of visual acuity and visual contrast sensitivity.

Use of the Cordyceps militaris fruit body extract of the present invention, wherein the Cordyceps militaris fruit body extract can be continuously administered to the desired individual at a frequency of 2 times/day. In this way, the Cordyceps militaris fruit body extract has better ability to protect photoreceptor cells, and can prevent the decline of visual acuity and visual contrast sensitivity.

[Figure 1] The chemical structure of Cordyceps militaris III.

[Fig. 2] In test (A), a line graph showing the change of the total content of carotenoids in Cordyceps militaris fruit body extracts extracted under different extraction times.

[FIG. 3] In test (B), a line graph showing the change of the total content of carotenoids in Cordyceps militaris in fruit body extracts of Cordyceps militaris extracted with ethanol aqueous solutions of different ethanol concentrations.

[Fig. 4] In the test (C), the ethanol concentration of the ethanol aqueous solution and the extraction time were used as the reaction variation, and the total content of the Cordyceps militaris carotenoids in the obtained Cordyceps militaris fruit body extract was used as the reaction value. 3D surface response plots obtained from model predictions of the response surface method.

[FIG. 5] Analysis results of the liquid chromatography tandem mass spectrometer in the test (E).

[FIG. 6] In the test (F), the spectrum diagram of white light generated by the LED light source used to form the retinal photodamage model mouse.

[Fig. 7] In test (G), a bar graph showing the number of photoreceptor cells that entered apoptosis in the outer nuclear membrane region of mice in groups F1 to F5 on day 1 of the test. Mann-Whitney U test was used for analysis, in which, "#" represents a significant difference compared with the F1 group ( p <0.05);"*" represents a significant difference compared with the F2 group ( p <0.05);"$" represents a significant difference compared with the F5 group ( p < 0.05).

[Fig. 8] In test (G), on day 1 of the test, the results of TUNEL assay of mice in group F3, where "ONL" represents the outer nuclear layer region, and "INL" represents the inner nuclear layer The inner nuclear layer region.

[Fig. 9] In test (G), on day 1 of the test, the results of TUNEL assay analysis of mice in group F5, where ONL represents the outer shell membrane region and INL represents the inner shell membrane region.

[Fig. 10] In the test (H), between the -5th day and the 15th day of the test, the change line of the threshold value of visual acuity of mice in groups F1 to F5.

[Fig. 11] In test (H), the histogram of the threshold value of visual acuity of mice in groups F1 to F5 at day 15 of the test. Mann-Whitney U test was used for analysis, in which, "#" represents a significant difference compared with the F1 group ( p <0.05);"*" represents a significant difference compared with the F2 group ( p <0.05);"$" represents a significant difference compared with the F5 group ( p < 0.05).

[Fig. 12] In test (I), on the 15th day of the test, the change curve of the visual contrast sensitivity of the mice in Figs. F1 to F5.

[Fig. 13] In test (I), on the 15th day of the test, the histogram of the visual contrast sensitivity and visibility index of the mice in Figs. F1 to F5. Mann-Whitney U test was used for analysis, in which, "#" represents a significant difference compared with the F1 group ( p <0.05);"*" represents a significant difference compared with the F2 group ( p <0.05);"$" represents a significant difference compared with the F5 group ( p < 0.05).

[FIG. 14] A bar graph showing the number of nuclei of photoreceptor cells in the outer nuclear membrane region of mice in groups F1 to F5 on day 16 of the experiment in test (J). Mann-Whitney U test was used for analysis, in which, "#" represents a significant difference compared with the F1 group ( p <0.05);"*" represents a significant difference compared with the F2 group ( p <0.05);"$" represents a significant difference compared with the F5 group ( p < 0.05).

In order to make the above-mentioned and other purposes, features and advantages of the present invention more obvious and easy to understand, the preferred embodiments of the present invention are exemplified below, and the accompanying drawings are described in detail as follows: The system can be obtained by extracting a fruiting body sample of Cordyceps militaris with an aqueous ethanol solution. For example, the worker can take 10 grams of Cordyceps militaris fruiting body sample and mix it with 100~1000 ml of ethanol aqueous solution (eg, ethanol aqueous solution with an ethanol concentration of more than 20%), and reflux extraction at a temperature of 40~70 ℃ for 20~1000 ml. For 100 minutes, ultrasonic vibration (frequency is 40kHz) can be performed at the same time during reflux extraction to improve extraction efficiency. The crude extract of Cordyceps militaris fruiting body obtained according to the aforementioned process can be obtained after being filtered, concentrated under reduced pressure and freeze-dried. Cordyceps militaris fruiting body extract.

Specifically, the Cordyceps militaris fruiting body sample can be obtained by culturing the fruiting body of Cordyceps militaris by the method disclosed in the Patent Case No. 1459953 of the Republic of China Announcement. , fetal bovine serum, fish meal, fish essence, silkworm chrysalis, red earthworm), vegetable protein, probiotic fermentation broth (lactic acid bacteria fermentation broth, fungal fermentation broth), Chinese herbal medicine extract, etc. PCB (plate count broth) medium, in 20 At a temperature of ~24°C, shake-flask culture was carried out at a rotating speed of 90 rpm for 3 to 7 days to obtain a cordyceps militaris mycelium, which was inoculated on a grain medium comprising white rice or brown rice or oat, etc., Incubate for 5~7 days in a dark environment with a temperature of 18~25°C and a humidity of 70~90% (light intensity is about 0Lux), and then in a light environment with a temperature of 18~25°C and a humidity of 70~90% (The light intensity is more than 500Lux, and the light time is 8 to 15 hours per day) After culturing for about 50 days, the Cordyceps militaris fruit body sample can be obtained.

Preferably, before extracting with the ethanol aqueous solution, the worker can pre-dry the Cordyceps militaris fruit body sample to obtain a Cordyceps militaris dried product (the water content of the Cordyceps militaris dried product is lower than 15%); in addition, The fruiting body sample of Cordyceps militaris can also be crushed into powder (eg, powder with a particle size of less than 0.4 mm) in advance, so as to increase the contact surface area between the fruiting body sample of Cordyceps militaris and the ethanol aqueous solution, thereby improving the extraction efficiency of subsequent extraction. .

In this example, 100 grams of the fruiting body sample of Cordyceps militaris obtained as above was mixed with 1000 ml of aqueous ethanol solution (an aqueous ethanol solution with an ethanol concentration of 62.9%), and refluxed for 88.3 minutes at a temperature of 70° C., and At the same time of reflux extraction, ultrasonic vibration (frequency is 40 kHz) was carried out with ultrasonic equipment (DC-100H, purchased from Delta Ultrasonic Co., Ltd.), after filtration, concentration under reduced pressure and freeze-drying, about 5 g was finally obtained. Cordyceps militaris fruiting body extract.

The fruiting body extract of Cordyceps militaris obtained according to the aforementioned process is rich in carotenoids (carotenoid, hereinafter referred to as Cordyceps militaris carotenoids). Also contains water-soluble carotenoids (water-soluble carotenoid), among which cordyxanthin III (cordyxanthin III, 2,3,2',3'-tetradehydro-18,17',18'-trinor having the chemical formula shown in Figure 1) -ε,ε-carotene-5,5',-diol) is the bulk (about 35% of the total Cordyceps militaris carotenoids in the Cordyceps militaris fruiting body extract), which is the same as the carotenoid lutein Compared with (β,ε-carotene-3,3'-diol, lutein), the number of methyl groups in the aforementioned cordycepin III is less, so that cordycepin III has good water solubility, so it can be used in When administered to a desired individual, it can be easily absorbed by the desired individual, thereby exerting the bioactivity of cordycepin III in the body of the desired individual.

The fruiting body extract of Cordyceps militaris obtained according to the aforementioned process can be administered to the desired individual, so that the active ingredients in the fruiting body extract of Cordyceps militaris (for example, containing Cordyceps militaris carotenoids such as Cordyceps militaris flavin III) protect the desired individual. The photoreceptor cells located in the retina of the required individual can prevent apoptosis of these photoreceptor cells, thus preventing harmful blue light (generally referring to high-energy visible light with a wavelength between 380 and 500 nm). Retinal degeneration (retinal degeneration) caused by the irradiation of ), and then can be used to manufacture medicines to enhance the anti-blue light damage effect. The Cordyceps militaris fruiting body extract is combined with a pharmaceutically acceptable carrier or excipient to form a medicine Composition, the Cordyceps militaris fruit body extract can also be prepared into any convenient edible form, such as lozenges, capsules, powders, granules or liquids, etc., or the Cordyceps militaris fruit body extract can be combined with other food or beverages , in a form suitable for consumption for oral administration by the desired individual.

Also, when the desired individual is a mouse individual, the Cordyceps militaris fruit body extract can be administered to the desired individual at a dose of 10-100 mg/kg/day, preferably 20 mg/day A kg/day dose is administered to the desired individual. Moreover, the Cordyceps militaris fruit body extract can be continuously administered to the desired individual for 2-30 days, preferably, the desired individual can be continuously administered to the desired individual for 7-22 days. In addition, the Cordyceps militaris fruit body extract can be continuously administered to the desired individual at a frequency of 1-3 times/day, preferably, the desired individual can be continuously administered at a frequency of 2 times/day.

The formula for dose translation based on body surface area (BSA) (refer to the journal paper "Dose translation from animal to human studies revisited", Reagan-Shaw et al., published in FASEB Journal in 2008 Periodical), further convert the aforementioned dosage, it can be known that in the case that the required individual is a human individual, the dosage of the Cordyceps militaris fruit body extract can be 1 ~ 10 mg/kg/day, preferably About 1~3 mg/kg/day. Moreover, the Cordyceps militaris fruit body extract can be continuously administered to the desired individual for 7-60 days, preferably, the desired individual can be continuously administered for 7-22 days. In addition, the Cordyceps militaris fruit body extract can be continuously administered to the desired individual at a frequency of 1-3 times/day, preferably, the desired individual can be continuously administered at a frequency of 2 times/day.

In order to confirm that the Cordyceps militaris fruiting body extract of the present invention is indeed rich in Cordyceps militaris carotenoids, and also contains Cordyceps militaris carotenoids such as the aforementioned Cordyceps militaris flavin III, the following tests were carried out:

(A) Optimization of extraction parameters (1)

In this experiment, the ethanol aqueous solution with an ethanol concentration of 65% was used as the extraction solvent, and the fruiting body samples of Cordyceps militaris obtained as described above were refluxed at a temperature of 55 ° C. The extraction time was 20, 40, 60, 80 or 100 minutes, respectively. And while refluxing extraction, carry out ultrasonic vibration (frequency is 40kHz) with ultrasonic equipment, after filtering, decompression concentration and freeze-drying, measure the absorbance value under the wavelength of 445nm with spectrophotometer, and then convert the The total content of Cordyceps carotenoids (refer to the journal paper "Metabolic Responses of Carotenoid and Cordycepin Biosynthetic Pathways in Cordyceps militaris under Light-Programming Exposure through Genome-Wide Transcriptional Analysis", Thananusak et al., published in "Biology" in 2020).

Please refer to Figure 2, the extraction time is between 20 and 100 minutes, the carotenoids of Cordyceps militaris can be extracted from the fruiting body sample of Cordyceps militaris. Among them, the extraction time of 60-100 minutes Room is better.

(B) Optimization of extraction parameters (2)

In this experiment, ethanol aqueous solutions with ethanol concentrations of 20%, 40%, 60%, 80% and 95% were used as the extraction solvent, and the fruiting body samples of Cordyceps militaris obtained as described above were refluxed at a temperature of 60 °C. The extraction time For 50 minutes, and while refluxing extraction, carry out ultrasonic vibration (frequency is 40kHz) with ultrasonic equipment, after filtering, concentrating under reduced pressure and freeze-drying, analyze the Cordyceps militaris obtained by the spectrophotometer method as described above Total content of Cordyceps militaris carotenoids in fruiting body extracts.

Please refer to Figure 3, the carotenoids of Cordyceps militaris can be extracted from the fruiting body sample of Cordyceps militaris when the ethanol concentration is above 20%, and the ethanol aqueous solution with the ethanol concentration above 40% is the best.

(C) Optimization of extraction parameters (3)

Next, the ethanol concentration of the ethanol aqueous solution used and the extraction time were used as the response variable, and the total content of the Cordyceps militaris carotenoids in the obtained Cordyceps militaris fruit body extract was set as the response value (response), The model prediction of the response surface methodology (RSM) was carried out, the statistical results of the variance of each response were obtained by analysis of variance (ANOVA) as shown in Table 1, and the obtained three-dimensional surface response The figure (three-dimensional response surface plot) is shown in Figure 4.

¹ : X ₁ : ethanol concentration of the ethanol aqueous solution; ² : X ₂ : extraction time.

Referring to the results in Table 1 and Figure 4, it can be seen from the software analysis of Design Expert 8.0 that the extraction time is 88.3 minutes with an ethanol aqueous solution with an ethanol concentration of 62.9%, which contains the most Cordyceps militaris. Cordyceps militaris fruiting body extract of carotene, and the concentration of Cordyceps militaris carotenoid in the Cordyceps militaris fruiting body extract (total content of Cordyceps militaris carotenoids/total amount of Cordyceps militaris fruiting body extract) is predicted to be 690.8 mg /kg.

(D) Optimization of extraction parameters (4)

In this experiment, an ethanol aqueous solution with an ethanol concentration of 62.9% was used as the extraction solvent, and the fruiting body samples of Cordyceps militaris obtained as described above were refluxed at a temperature of 65 °C. The extraction time was 88.3 minutes. The sonic equipment carries out ultrasonic vibration (frequency is 40kHz), after filtration, decompression concentration and freeze-drying, the total content of Cordyceps militaris carotenoids in the obtained Cordyceps militaris fruiting body extract is analyzed by spectrophotometer, and it can be seen that The carotenoid concentration of Cordyceps militaris in the fruiting body extract of Cordyceps militaris obtained under the aforementioned optimal parameters was 691.7±1.5 mg/kg, which was similar to the aforementioned prediction result.

(E) Identification of Cordyceps militaris carotenoids

In this experiment, the ethanol aqueous solution with an ethanol concentration of 62.9% was taken as the extraction solvent, and the reflux extraction was carried out at a temperature of 65 ° C for 88.3 minutes, and while the reflux extraction was carried out, ultrasonic vibration was carried out with ultrasonic equipment (frequency is 40kHz). ), the Cordyceps militaris fruit body extract obtained after filtration, concentration under reduced pressure and freeze-drying, the Cordyceps militaris fruit body extract is redissolved in methanol, so that the concentration of the Cordyceps militaris fruit body extract is 1 mg/mL. , and then identified by liquid chromatography-tandem mass spectrometry (LC-MS).

Specifically, after collecting the fractions containing the carotenoids of Cordyceps militaris, the separated liquid obtained is analyzed by a mass spectrometry detector (MSD-TRAP-XCT) in a positive charge mode. The parameters of the mass spectrometer detector were: ion spray voltage (IS) of 4,500 V; nebulizer gas pressure (nebulizer gas pressure) of 12 psi; nitrogen curtain gas pressure of 10 psi; heater temperature ( heater temperature) was 450°C; collision induced dissociation (CID) gas pressure: 6 psi.

Please refer to Figure 5, the analysis result of the Cordyceps militaris fruiting body extract shows that there is a signal at 523 m/z, it is inferred that the water-soluble Cordyceps militaris carotenoids in the Cordyceps militaris fruiting body extract contain at least molecular weight (molecular weight) is a water-soluble Cordyceps militaris carotenoid of 522Da, compared with the known Cordyceps militaris (refer to the journal paper "Composition and Characterization of Cordyxanthins from Cordyceps militaris fruit bodies", Dong et al., published in "Journal of Functional Functionality" in 2013). Foods” journal), it can be known that the most important water-soluble carotenoids in the Cordyceps militaris fruiting body extract is Cordyceps militaris xanthophylls III.

Next, in order to confirm that the Cordyceps militaris fruiting body extract of the present invention can indeed protect the photoreceptor cells located in the retina in vivo, it can prevent retinal degeneration caused by the irradiation of harmful blue light, and then can be used in manufacturing to improve anti-blue light damage The effect of the drug was then carried out with the fruiting body extract of Cordyceps militaris obtained under the aforementioned optimal parameters, and 24 mg of the fruiting body extract of Cordyceps militaris was dissolved in 800 μL of a carrier solution containing 50% by volume percentage. water, 20% polyethylene glycol 400 (PEG400) and 30% glycerol, and then add 10 μL of aqueous sodium hydroxide solution (concentration 12N) to adjust the pH to Above 11, after shaking overnight, add about 35~40μL of hydrochloric acid aqueous solution (concentration is 4N) to adjust the pH value to be between 7.0~7.5 for use.

(F) Establishment of retinal photodamage model mice

The following experimental system selected 8-14-week-old ICR (BLTW: CD1) mice as experimental animals (purchased from Taiwan Lester Biotechnology Co., Ltd.), and the aforementioned mice were raised at a temperature of 23±2°C and a humidity of In 55±7% of the animal rooms, and can eat and drink freely, and according to the order As shown in Table 2, the mice were divided into groups F1 to F5 (4 mice each).

¹ : BID (bis in die): 2 times a day.

From the first 5 days of the experiment, the carrier solution (groups F1, F2), low dose of Lutein powder (group F3), high-dose lutein powder (group F4) and the Cordyceps militaris fruit body extract (group F5) were orally administered to the aforementioned mice, respectively, and were administered on the 0th day of the test. On the day of the day, the mice in the F2~F5 groups were placed in a treatment box, the treatment box was wrapped with tin foil, and there was an LED light source on the top. The spectrogram is shown in Figure 6 (the harmful blue light with wavelength between 380~500nm accounts for about 27.1% of the luminous flux of visible light, that is, about 3,800~5,500Lux), with the white light generated by the LED light source The mice in groups F2 to F5 were treated with light for 4 to 5 hours to induce apoptosis in the retinal photoreceptor cells of these mice, thereby forming a light-induced retinal damage model. damage model) mice.

In addition, from day 0 to day 15 of the trial, still at a frequency of 2 times a day, The carrier solution (groups F1, F2), the low-dose lutein powder (group F3), the high-dose lutein powder (group F4) and the Cordyceps militaris fruit body extract (group F5) The aforementioned mice were administered orally, respectively.

(F) Analysis results of apoptosis

On the first day of the experiment (24 hours after the light treatment of mice in groups F2 to F5), the mice in groups F1 to F5 were sacrificed, and their eyes were quickly removed, followed by fixation with fixative. The fixed eyes were embedded in paraffin and sliced vertically, and the slices containing the outer nuclear layer (ONL) region were selected for TUNEL assay (terminal deoxynucleotidyl transferase (TdT) dUTP nick and labeling assay), That is, terminal deoxynucleotidyl transferase (terminal deoxynucleotidyl transferase, TdT), deoxyuridine labeled with bromodeoxyridine (BrdU) or green fluorescent protein (green fluorescent protein, GFP) and other markers 2'-deoxyuridine 5'-triphosphate (dUTP) is incorporated into the 3'-OH sticky end produced by fragmented DNA, and the label is recognized by an antibody for staining , and then the DNA fragmentation in the slices of each group of mice can be marked to calculate the number of photoreceptor cells in the outer nuclear membrane region that have entered apoptosis.

Please refer to Figure 7, the light treatment can cause a large number of photoreceptor cells to enter apoptosis (group F2), pre-feeding the Cordyceps militaris fruiting body extract helps to reduce the photoreceptor cells that enter apoptosis The amount of lutein (group F5), and its effect was significantly better than pre-feeding low-dose or high-dose lutein (groups F3, F4). The results of TUNEL staining analysis of groups F3 and F5 are shown in Figures 8 and 9, respectively.

The visual acuity (VA) and visual contrast sensitivity function (VCSF) tests described below are all based on the optomotor reflex (OMR) of animals, and the thresholds of each test are determined by Visual field of a test mouse The visual field was determined by the reflexive head movement of the test mice when the stimulus grating appeared. Specifically, the test mouse was placed on a high platform, a monitor was set in front of the high platform, the distance between the test mouse and the screen was 15 cm, and the field of view was 110×90°. The display shows that it is the stimulus grating of the optokinetic reflex (the stimulus grating is a number of vertical gratings of equal width and equal spacing to each other) used to excite the subject mouse. The operator can record the reflex movements of the test mouse's head and body (ie, whether the test mouse's head and body move with the stimulus grating), thereby obtaining the test mouse's threshold.

(G) Visual acuity test

Visual acuity tests were performed on the first 5 days (day -5) of the test, on the 0th day of the test (after light treatment), and on the 5th, 10th, and 15th days.

In the visual acuity test, the operator sets the contrast ratio of the stimulus grating on the display to 100%, and the display uses a space of 0.033, 0.055, 0.082, 0.164, 0.328 and 0.437 cycles per decree (cpd) Spatial frequency, and horizontal drift at a constant rotational speed of 12 degrees (°) per second, displaying an episodic stimulus consisting of a full-screen square wave . After starting the stimulus grating, the operator can record the reflex movement of the subject's head and body (ie, whether the subject's head and body move with the stimulus grating), until the aforementioned reflex movements no longer exist. This is then coordinated with the stimulus grating, whereby the threshold of the subject mouse at 100% contrast is known.

Please refer to Figure 10. After the light treatment, the visual acuity of the mice in groups F2 to F5 was impaired, but the visual acuity of the mice in group F5, which was pre-fed with the Cordyceps militaris fruiting body extract The degree of acuity impairment was lower (day 0), after 15 days, the mice in groups F3 and F4 were either pre-fed with low-dose or high-dose lutein, or pre-fed the Cordyceps militaris fruiting body extract The thresholds of visual acuity of the mice in the F5 group of animals increased, but the effect was still limited by the prediction. The F5 group mice fed with the Cordyceps militaris fruit body extract first had the best recovery.

Continued, please refer to Figure 11, taking the test results on the 15th day as an example, the light treatment will destroy the visual acuity of the test mice (F2 group), pre-feeding the Cordyceps militaris fruiting body extract helps. In restoring the visual acuity of the test mice (group F5), and its effect was significantly better than pre-feeding low-dose or high-dose lutein (groups F3, F4).

(H) Visual contrast sensitivity test

On day 15 of the experiment, a visual contrast sensitivity test was performed.

In the visual contrast sensitivity test, the operator set the contrast ratio of the stimulus grating on the display to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively. and 100% and other different levels, and repeat the above test to know that the tested mice are at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% According to the threshold value under the same contrast, the visual contrast sensitivity curve (VSCF curve) shown in Figure 12 can be drawn, and then the area under the visual contrast sensitivity curve can be converted into the visual contrast shown in Figure 13. Sensitivity visibility index (VSCF visibility index).

Please refer to Figures 12 and 13, the light treatment will reduce the visual contrast sensitivity curve of the test mice, and reduce the visual contrast sensitivity visibility index (group F2), feeding the Cordyceps militaris fruiting body extract Helps to restore the visual contrast sensitivity and visibility index of the test mice (group F5), and its effect is significantly better than pre-feeding low-dose or high-dose lutein (groups F3, F4).

(I) Analysis of tissue staining results

On the 16th day of the experiment, after the mice in groups F1 to F5 were sacrificed, their eyes were quickly removed, and then fixed with a fixative. The fixed eyes were embedded in paraffin and vertically sectioned (5 μm thick), and the sections containing the outer and inner nuclear membrane regions were selected for hematoxylin and eosin stain, H&E stain ), computing and vision The number of nuclei at a distance of about 0.4-0.6 mm from the optic nerve head (ONH).

Please refer to Figure 14, the light treatment will cause the death of photoreceptor cells and reduce the number of nuclei that can be stained (group F2). Pre-feeding the Cordyceps militaris fruiting body extract helps to slow down the photoreceptor The death of somatic cells resulted in an increase in the number of stained nuclei (group F5), which was significantly better than pre-feeding low-dose lutein (group F3), but compared with pre-feeding high-dose lutein (Group F4) No significant difference.

To sum up, the use of the Cordyceps militaris fruiting body extract of the present invention is to protect the active ingredient (such as Cordyceps militaris carotenoids such as Cordyceps militaris) contained in the Cordyceps militaris fruiting body extract. It is necessary for the individual's photoreceptor cells in the retina to prevent apoptosis of these photoreceptor cells, thus preventing retinal degeneration caused by the irradiation of harmful blue light (generally refers to high-energy visible light with a wavelength of 380~500nm). , and then can be applied to the manufacture of medicines for enhancing the anti-blue light damage effect, which is the effect of the present invention.

Although the present invention has been disclosed by the above-mentioned preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes and modifications relative to the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the patent application attached hereto.

Claims (10)

The use of a Cordyceps militaris fruit body extract is used to manufacture a medicine for improving the effect of anti-blue light damage; wherein, the Cordyceps militaris fruit body extract is extracted from an ethanol aqueous solution with a concentration of 62.9% of a Cordyceps militaris fruit body sample for 88.3 minutes obtained. The use of the Cordyceps militaris fruit body extract according to claim 1, wherein the Cordyceps militaris fruit body extract is administered to a desired individual by oral feeding. The use of the fruiting body extract of Cordyceps militaris as claimed in claim 2, wherein the desired individual is a mouse individual, and the fruiting body extract of Cordyceps militaris is continuously administered to the animal at a dose of 10-100 mg/kg/day. It takes 2 to 30 days for individuals. The use of the Cordyceps militaris fruit body extract as claimed in claim 3, wherein the Cordyceps militaris fruit body extract is continuously administered to the desired individual at a dose of 20 mg/kg/day for 7 to 22 days. The use of the fruiting body extract of Cordyceps militaris as claimed in claim 3, wherein the fruiting body extract of Cordyceps militaris is continuously administered to the desired individual at a frequency of 1 to 3 times/day. The use of the Cordyceps militaris fruit body extract according to claim 5, wherein the Cordyceps militaris fruit body extract is continuously administered to the desired individual at a frequency of 2 times/day. The use of the fruiting body extract of Cordyceps militaris as claimed in claim 2, wherein the desired individual is a human individual, and the fruiting body extract of Cordyceps militaris is continuously administered at a dose of 1-10 mg/kg/day to the desired individual Individuals 7 to 60 days. The use of the Cordyceps militaris fruit body extract as claimed in claim 7, wherein the Cordyceps militaris fruit body extract is continuously administered to the desired individual at a dose of 1-3 mg/kg/day for 7-22 days. As claimed in claim 7, the use of the Cordyceps militaris fruit body extract, wherein the Cordyceps militaris fruit body extract is continuously administered to the desired individual at a frequency of 1 to 3 times/day. The use of the Cordyceps militaris fruit body extract according to claim 9, wherein the Cordyceps militaris fruit body extract is continuously administered to the desired individual at a frequency of 2 times/day.

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