Preparation method of fucoxanthin and application of fucoxanthin in preparation of product with efficacy of relieving xerophthalmia

Abstract

The invention belongs to the technical field of marine natural product extraction and biological medicine, and particularly relates to a preparation method of fucoxanthin and application of the fucoxanthin to preparation of a product with the effect of relieving xerophthalmia. The fucoxanthin disclosed by the invention is prepared by the following steps: crushing cleaned and dried seaweed, performing alcohol extraction, extracting and purifying to obtain a fucoxanthin product. The method has the beneficial effects that the purity of the fucoxanthin prepared by adopting the method disclosed by the invention is higher, in-vivo and in-vitro experiments prove that the extracted fucoxanthin has the effect of relieving the tear secretion deficiency type xerophthalmia, and a new raw material source and theoretical support are provided for treating the xerophthalmia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of marine natural product extraction and biomedicine technology, and particularly relates to a preparation method of fucoxanthin and application of the fucoxanthin in preparation of products with the efficacy of relieving dry eye syndrome, in particular, the application of the fucoxanthin in preparation of health foods and products with the efficacy of relieving dry eye syndrome of the type of insufficient tear secretion. BACKGROUND

[0002] Fucoxanthin is a natural carotenoid mainly existing in brown algae (such as kelp, wakame, sargassum, etc.) and some microalgae. The molecular structure of fucoxanthin is unique, containing a propylene bond and an epoxy group. Its unique structure makes it have stronger antioxidant, anti-inflammatory and photoprotective effects than ordinary carotenoids. Studies have shown that fucoxanthin can protect eye tissues such as retinal pigment epithelial cells (RPE) and optic nerve cells through mechanisms such as free radical scavenging and inhibition of inflammatory factor release, suggesting its potential application value in relieving dry eye syndrome of the type of insufficient tear secretion.

[0003] Dry eye disease (DED) is an eye discomfort caused by long-term eye use, such as electronic screen use, reading, etc. Its symptoms include eye dryness, acid swelling, blurred vision, etc. At present, the products for relieving dry eye syndrome of the type of insufficient tear secretion are mainly artificial tears, lutein or blueberry extracts, but there are problems such as single action, slow effect or limited long-term effect. Therefore, developing new active ingredients with multiple protection mechanisms has become a research hotspot.

[0004] In recent years, fucoxanthin has been widely used in products related to vision protection, such as patents CN117099950A, CN105996027A, CN119523098A, etc. These existing technologies are all used to improve visual fatigue after compounding fucoxanthin with other active ingredients.

[0005] However, there is no reported research on the application of fucoxanthin in dry eye syndrome. In addition, the existing technology for extracting fucoxanthin still has limitations, such as low extraction efficiency, and fucoxanthin is easily degraded by light and heat, affecting the activity of the product. SUMMARY

[0006] In order to solve the above technical problems, the present application provides a preparation method of fucoxanthin and its application, in particular, the application of the fucoxanthin in preparation of health foods and products with the efficacy of relieving dry eye syndrome of the type of insufficient tear secretion.

[0007] The inventors find through in-vivo and in-vitro experiments that feeding on fucoxanthin can improve dry eye induced by scopolamine (SCOP), i.e. dry eye caused by insufficient tear secretion, and that fucoxanthin can increase tear secretion of the body, prevent corneal tissue from apoptosis or necrosis caused by inflammatory damage, and increase secretion of conjunctival mucin (an important component of tear) of the body, so as to keep the eyeball moist, thereby relieving symptoms such as dryness and acid swelling of the eyes caused by insufficient tear secretion, and thus providing a treatment scheme for eye diseases or inflammation problems induced by SCOP, but the present application is not limited to dry eye induced by SCOP.

[0008] The first aspect of the present application provides a preparation method of fucoxanthin, specifically comprising the following steps: (1) drying, crushing, sieving and obtaining seaweed powder after dehydration of seaweed; (2) extracting the seaweed powder with an ethanol aqueous solution with a volume fraction of 70% to 90%, and centrifuging to obtain an ethanol extract of seaweed; (3) concentrating the ethanol extract in (2) to obtain a concentrated solution, extracting the concentrated solution with a non-polar solvent, and collecting the extract; the non-polar solvent comprises at least one of ethyl acetate, dichloromethane and n-hexane; (4) purifying the extract in (3) by using an active carbon column and a silica gel column in sequence, preliminarily adsorbing chlorophyll and fat-soluble impurities by the active carbon column, further purifying by the silica gel column, collecting target effluent, and concentrating and drying to obtain the fucoxanthin; at least two of ethyl acetate, methanol, n-hexane and petroleum ether are used as eluent in the silica gel column chromatography; 2,6-di-tert-butyl-p-cresol is added to the eluent, and the volume ratio of 2,6-di-tert-butyl-p-cresol to the eluent is 1:9000 to 10000.

[0009] In the above preparation method, as a preferred, the seaweed in (1) to (2) is selected from any one of Undaria pinnatifida, Laminaria japonica, Sargassum fusiforme and Sargassum thunbergii.

[0010] As a preferred, the drying temperature of the seaweed in (1) is 70 to 80℃, and the crushing temperature is 20 to 25℃.

[0011] As a preferred, the mass-volume ratio of the seaweed powder to the ethanol aqueous solution in (2) is 1 g:10 to 15 mL, and the extraction temperature is 40 to 55℃.

[0012] As a preferred, the extraction in (2) is performed for 1 to 3 times, and the extraction time is 0.5 to 1.5 h each time.

[0013] As preferred, the volume ratio of the concentrated liquid to the non-polar solvent in (3) is 1:1-1.5, and the extraction temperature is 25-35℃.

[0014] As preferred, the number of times of extraction in (3) is 2-4, and the time of each extraction is 1-1.5 h.

[0015] As preferred, the activated carbon column in (4) is a SPE special column, and the purpose of adsorbing the extraction liquid by the activated carbon in the present application is to remove liposoluble impurities such as chlorophyll, and the SPE column is selected due to high processing efficiency and small loss of fucoxanthin.

[0016] As preferred, the extraction liquid is subjected to vacuum concentration treatment before being purified in (4), the vacuum degree of the vacuum concentration is-0.085--0.095 Mpa, and the temperature of the vacuum concentration is 55-65℃.

[0017] The fucoxanthin prepared by the above method is also the technical content protected by the present application.

[0018] The second aspect of the present application is to provide the above-mentioned fucoxanthin in the preparation of any form of food in liquid, spray, solid powder, tablet.

[0019] The third aspect of the present application is to provide the above-mentioned fucoxanthin in the preparation of products with the effect of relieving dry eye, as preferred, the product is a medicine, and the medicine contains the above-mentioned fucoxanthin and conventional pharmaceutical excipients.

[0020] The fourth aspect of the present application is to provide the above-mentioned fucoxanthin in the preparation of products with the effect of relieving visual fatigue.

[0021] As preferred, the product form described above includes but is not limited to eye mask, eye patch, eye spray.

[0022] As preferred, the dry eye in the above-mentioned application includes but is not limited to tear secretion deficiency type dry eye.

[0023] The present application has the following advantages: (1) The present application uses seaweed as raw material, and through alcohol extraction and non-polar solvent extraction, the extract is further separated and purified by activated carbon and silica gel column chromatography to obtain high-purity fucoxanthin, and the purity of the fucoxanthin is as high as 92.31%. (2) The application develops a new application of fucoxanthin, that is, the application of fucoxanthin in preparation of a product for relieving tear secretion deficiency type dry eye syndrome, and the experimental results show that after the mice are treated with 2.5 μM low-dose fucoxanthin, the tear secretion amount of the mice is increased from 1.64 mm to 8.47 mm, and the fucoxanthin also shows good inhibitory effect on inflammatory factors such as IL-6, IL-1β and TNF-α of HCE-T cells. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 High performance liquid chromatogram of the fucoxanthin prepared in the embodiment 1 of the application; Figure 2 Mass spectrum of the fucoxanthin prepared in the embodiment 1 of the application; Figure 3 Effect of the fucoxanthin in the application example 1 on the survival rate of human corneal epithelial model cells; Figure 4 Effect of the fucoxanthin in the application example 1 on the expression of inflammatory factors in human corneal epithelial model cells; Figure 5 Tear secretion amount improvement of the fucoxanthin in the application example 2; Figure 6 Fluorescein staining result chart of the mouse cornea in the application example 2; Figure 7 Fluorescein staining score result chart of the mouse cornea in the application example 2; Figure 8 High performance liquid chromatogram of the fucoxanthin obtained by the method in the embodiment 2 of the application; Figure 9 High performance liquid chromatogram of the fucoxanthin obtained by the method in the embodiment 3 of the application; Figure 10 High performance liquid chromatogram of the fucoxanthin obtained by the method in the embodiment 4 of the application; Figure 11 High performance liquid chromatogram of the fucoxanthin obtained by the method in the comparative example 1 of the application; Figure 12 High performance liquid chromatogram of the fucoxanthin obtained by the method in the comparative example 2 of the application. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the application, the application will be further described in conjunction with specific embodiments.

[0026] Embodiment 1 A kind of fucoxanthin, the preparation method is as follows: (1) Take Undaria pinnatifida, clean it, dry it in a blast drying oven at 80℃, and then crush it in a crusher at 25℃, pass it through a 40-mesh sieve, and obtain Undaria pinnatifida powder; (2) Add a 70% ethanol aqueous solution to the Undaria pinnatifida powder, and extract it at 45℃ for 2 times, 1h each time, wherein the mass-volume ratio of the Undaria pinnatifida powder to the ethanol aqueous solution is 1g:10mL, and then centrifuge it at a speed of 4000 rpm after the extraction is completed, collect the supernatant, and obtain an ethanol extract of Undaria pinnatifida; (3) Concentrate the ethanol extract in (2) under reduced pressure to obtain a concentrated solution, and extract the concentrated solution with a separatory funnel at 25℃ for 3 times, 1h each time, and collect the extract. (4) First, concentrate the extract in (3) under reduced pressure, with a vacuum degree of -0.095 Mpa and a temperature of 60℃, and then purify the extract in (3) by using an SPE activated carbon column and a silica gel column chromatography in sequence, and obtain the fucoxanthin.

[0027] The eluent for the silica gel column chromatography is petroleum ether: ethyl acetate = 60:40 (v / v).

[0028] When eluting, 2,6-di-tert-butyl-p-cresol (BHT) is added to the eluent, and the volume ratio of the 2,6-di-tert-butyl-p-cresol to the eluent is 1:10000.

[0029] The structure and content of the obtained fucoxanthin are detected by high performance liquid chromatography (HPLC) and electrospray time-of-flight mass spectrometry (ESI-TOF-MS).

[0030] The detection conditions of HPLC are as follows: a chromatographic column: Shim-pack VP-ODS C18 (5μm, 250mm×4.6mm); a flow rate: 1mL / min; a mobile phase: acetonitrile: 0.5% phosphoric acid solution (9:1), isocratic elution; a column temperature: 35℃; a detection wavelength: 450nm; and a sample injection amount: 10μL.

[0031] The detection conditions of ESI-TOF-MS are as follows: the obtained fucoxanthin target sample is blown dry into an orange yellow powder with nitrogen, and is dissolved into a solution with a small amount of chloroform. The electrospray (ESI) mass spectrometry adopts a direct injection method, a positive ion detection mode, and the sample solution is sent into the instrument through an injection valve for detection.

[0032] The high performance liquid chromatogram of the fucoxanthin prepared in this example is shown in Figure 1 , wherein A is a fucoxanthin standard, and B is the fucoxanthin sample prepared in this example. Figure 1

[0033] ​In addition, the ion flow chart of the obtained phlorofucoxylin with one propadiene bond and an epoxy group was analyzed by ESI-TOF-MS, as shown in Figure 2 .

[0034] Figures 1-2 The chromatogram data showed that the phlorofucoxylin was successfully prepared by the method of Example 1 of the present application.

[0035] In addition, the purity of the phlorofucoxylin was as high as 92.31% by high performance liquid chromatography analysis.

[0036] Application Example 1 Cellular evaluation of the phlorofucoxylin prepared in Example 1 for the alleviating effect on dry eye syndrome with insufficient tear secretion.

[0037] 1.1 Cell survival rate determination The cell survival rate was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.

[0038] The hyperosmotic stress (Hyp) model was induced in vitro by placing human corneal epithelial cells (HCE-T) in culture medium supplemented with 69 mM sodium chloride (NaCl) for 24 hours. Then, after treating the cells with different concentrations of phlorofucoxylin (2.5 μM, 5 μM, 10 μM) for 12 hours, 20 μL of 5 mg / mL MTT in phosphate buffered saline (PBS) solution was added to each well, and further incubated at 37°C for 4 hours. Then, the supernatant was removed, 100 μL of DMSO was added to each well to dissolve the purple formazan crystals, and the absorbance of each sample was read at 570 nm using a microplate reader.

[0039] The results of the effect of different concentrations (2.5 μM, 5 μM, 10 μM) of phlorofucoxylin on the cell survival rate in the HCE-T model are shown in Figure 3 .

[0040] Figure 3 It was shown that, compared with the model group, the phlorofucoxylin groups of different concentrations could significantly improve the cell survival rate. Since there was no significant difference in the cell survival rate at the concentrations of 5 μM and 10 μM of phlorofucoxylin, 2.5 μM and 5 μM were used in the subsequent experiments.

[0041] 1.2 Inflammatory factor determination The contents of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture supernatant of HCE-T cells treated with different samples (2.5 μM phlorofucoxylin, 5 μM phlorofucoxylin) were evaluated by enzyme-linked immunosorbent assay (ELISA) kit produced by Dakewe Biotechnology Co., Ltd. All experimental procedures were performed according to the manufacturer's guidelines.

[0042] The change of inflammatory factors in the cells is shown in Figure 4 .

[0043] Figure 4 The results show that the fucoidin prepared in Example 1 can inhibit the inflammatory factors such as IL-6, IL-1β and TNF-α of HCE-T cells, and has good anti-inflammatory effect, and has better effect at a concentration of 2.5 μM.

[0044] The above Figures 1-4 It can be seen from the above that the fucoidin obtained in Example 1 has a significant effect of reducing eye inflammation.

[0045] Application Example 2 Animal evaluation of the fucoidin prepared in Example 1 on the remission effect of the tear secretion deficiency type dry eye.

[0046] 2.1 Tear secretion detection The phenol red thread test (Jingming, Tianjin, China) was used to quantitatively detect the amount of tear secretion.

[0047] The test was divided into a blank group (experimental mice without any treatment Control), a model group (SCOP-induced tear secretion deficiency type dry eye mice Model), an experimental group (a low-dose group FX-LOW of 5 mg / kg / d fucoidin, a high-dose group FX-HIGH of 10 mg / kg / d fucoidin), and each group of mice was treated 12 hours after treatment on the 7th and 14th day. The phenol red cotton thread was placed at the outer third of the lower eyelid margin of the eyeball of the mouse for 60 seconds, and the length of the thread was measured in millimeters.

[0048] The tear secretion data and results are shown in Table 1 and Figure 5 .

[0049] Table 1 Tear secretion results

[0050] The results of the above Table 1 and Figure 5 The results show that the tear secretion of the model group mice is significantly reduced compared with the tear secretion of the blank group mice, and after treatment with low-dose and high-dose fucoidin, the tear secretion is significantly improved, and the tear secretion of the high-dose group is more significantly increased than that of the low-dose group. It can be seen that the high-dose fucoidin treatment has greater advantages than the low-dose fucoidin treatment.

[0051] 2.2 Corneal fluorescein staining To assess the extent of corneal epithelial damage in mice, corneal fluorescein staining was performed on days 7 and 14 of treatment. 1 µL of fluorescein dye (1%) was applied to the mouse ocular surface, and after 90 seconds, the ocular surface was assessed under a slit-lamp microscope using cobalt blue light and scored using the Oxford scoring system. The results of corneal fluorescein staining and scoring are shown below. Figure 6 , Figure 7 .

[0052] Figure 6 It is evident that the model group still exhibited obvious yellow-green fluorescent spots in their eyes after cobalt blue light irradiation on Day 7 and Day 14, indicating corneal damage. In contrast, the yellow-green fluorescent spot area in the fucoxanthin treatment group was significantly reduced compared to the model group.

[0053] Figure 7 for Figure 6 The quantitative scoring results of fluorescent spots in the eyes of each group show that the fluorescence scores of the high- and low-dose fucoxanthin groups were significantly lower than those of the model group. Furthermore, the high-dose group decreased to a level comparable to the blank group on Day 14, proving that fucoxanthin can repair corneal damage caused by insufficient tear secretion.

[0054] Example 2 A fucoxanthin is prepared using the following method: (1) Take wakame seaweed, wash it clean, dry it in a forced-air drying oven at 80°C, then pulverize it in a pulverizer at 25°C and pass it through a 40-mesh sieve to obtain wakame seaweed powder; (2) Add 80% ethanol aqueous solution to wakame powder and extract at 45°C. The extraction is performed twice, for 1 hour each time. The mass-volume ratio of wakame powder to ethanol aqueous solution is 1 g: 10 mL. After extraction, centrifuge at 4000 rpm and collect the supernatant to obtain the ethanol extract of wakame. (3) The ethanol extract in (2) was concentrated under reduced pressure to obtain a concentrate, and the concentrate was extracted three times at 25°C using a separatory funnel for 1 hour each time, and the extract was collected. (4) First, the extract in (3) is concentrated under reduced pressure. The vacuum degree of the reduced pressure concentration is -0.09 MPa and the temperature of the reduced pressure concentration is 60℃. Then, the extract in (3) is purified by SPE activated carbon column and silica gel column chromatography in sequence to obtain fucoxanthin from wakame seaweed.

[0055] The eluent for silica gel column chromatography was n-hexane:ethyl acetate = 60:40 (v / v).

[0056] When eluting, 2,6-di-tert-butyl-p-cresol (BHT) is added in the eluent, and the volume ratio of 2,6-di-tert-butyl-p-cresol to the eluent is 1:10000.

[0057] The structure and content of the obtained fucoxanthin are detected by HPLC and ESI-TOF-MS.

[0058] The high performance liquid chromatogram of the obtained fucoxanthin is shown in Figure 8 , and the purity of the obtained fucoxanthin is 89.467%.

[0059] Example 3 A kind of fucoxanthin, the following method is used to prepare: (1) take kelp, clean up after, using the drying oven at 70 DEG C drying, using the pulverizer at 25 DEG C pulverization treatment, pass 40 mesh screen, obtain kelp powder; (2) to kelp powder, add volume fraction 90% ethanol aqueous solution, at 45 DEG C extraction, extraction frequency is 2 times, 1 h each time, wherein, the mass volume ratio of kelp powder and ethanol aqueous solution is 1 g: 10 mL, after extraction, at 4000 rpm centrifugal, collect supernatant, obtain kelp ethanol extract; (3) the ethanol extract in (2) is concentrated under reduced pressure to obtain a concentrated solution, and the concentrated solution is extracted with a separatory funnel at 25 DEG C for 3 times, each time for 1 h, and the extract is collected. (4) first, the extract in (3) is concentrated under reduced pressure, the vacuum degree of the reduced pressure concentration is-0.085 Mpa, and the temperature of the reduced pressure concentration is 60 DEG C, then the extract in (3) is purified by using SPE activated carbon column and silica gel column chromatography in turn, to obtain fucoxanthin derived from kelp.

[0060] The eluent of the silica gel column chromatography is petroleum ether: ethyl acetate=60:40 (v / v).

[0061] When eluting, 2,6-di-tert-butyl-p-cresol (BHT) is added in the eluent, and the volume ratio of 2,6-di-tert-butyl-p-cresol to the eluent is 1:10000.

[0062] The structure and content of the obtained fucoxanthin are detected by HPLC and ESI-TOF-MS, and the high performance liquid chromatogram of the obtained fucoxanthin is shown in Figure 9 , and the purity of the obtained fucoxanthin is 83.274%.

[0063] Example 4 A kind of fucoxanthin, the following method is used to prepare: (1) Take Sargassum, clean it up, dry it in a blast drying oven at 80℃, then crush it in a crusher at 25℃, pass it through a 40-mesh sieve, and obtain Sargassum powder; (2) Add 70% ethanol aqueous solution by volume to the Sargassum powder, extract it at 45℃, 3 times, 1.5h each time, wherein the mass-volume ratio of Sargassum powder to ethanol aqueous solution is 1g:10mL, centrifuge after extraction, collect the supernatant, and obtain ethanol extract of Sargassum; (3) Concentrate the ethanol extract in (2) under reduced pressure to obtain a concentrated solution, and extract the concentrated solution with a separatory funnel at 25℃, 4 times, 1.5h each time, and collect the extract; (4) First, concentrate the extract in (3) under reduced pressure, with a vacuum degree of -0.09 Mpa and a temperature of 60℃, then purify the extract in (3) with SPE activated carbon column and silica gel column chromatography in sequence, and obtain fucoxanthin derived from Sargassum.

[0064] The eluent for silica gel column chromatography is petroleum ether: ethyl acetate = 60:40 (v / v).

[0065] When eluting, add 2,6-di-tert-butyl-p-cresol (BHT) to the eluent, and the volume ratio of 2,6-di-tert-butyl-p-cresol to eluent is 1:10000.

[0066] Use HPLC and ESI-TOF-MS to detect the structure and content of the obtained fucoxanthin.

[0067] The high-performance liquid chromatogram of the obtained fucoxanthin is shown in Figure 10 , and the purity of the obtained fucoxanthin is 90.049%.

[0068] Comparative Example 1 The difference between Example 1 and Comparative Example 1 is that only column chromatography is used for purification in (4), and the high-performance liquid chromatogram of the obtained fucoxanthin is shown in Figure 11 , and the purity of the obtained fucoxanthin is 62.571%.

[0069] Comparative Example 2 The difference between Example 1 and Comparative Example 2 is that BHT is not added to the eluent in (4), and the high-performance liquid chromatogram of the obtained fucoxanthin is shown in Figure 12 , and the purity of the obtained fucoxanthin is 71.483%.

Claims

1. A method for the preparation of fucoxanthin, characterized by, The method comprises the following steps: (1) drying, crushing, sieving and obtaining seaweed powder after seaweed is dehydrated; (2) using an ethanol aqueous solution with a volume fraction of 70% to 90% to extract the seaweed powder, centrifuging and obtaining an ethanol extract of seaweed; (3) concentrating the ethanol extract in (2) to obtain a concentrated solution, using a non-polar solvent to extract the concentrated solution, and collecting the extract liquid; the non-polar solvent comprises at least one of ethyl acetate, dichloromethane and n-hexane; (4) sequentially using an activated carbon column and a silica gel column to purify the extract liquid in (3), collecting target effluent, concentrating and drying to obtain the fucoxanthin; at least two of ethyl acetate, methanol, n-hexane and petroleum ether are used as the eluent when the silica gel column is used for chromatography; 2,6-di-tert-butyl-p-cresol is added to the eluent, and the volume ratio of 2,6-di-tert-butyl-p-cresol to the eluent is 1:9000 to 10000.

2. The production method according to claim 1, wherein The seaweed in (1) to (2) is selected from any one of Undaria pinnatifida, Laminaria japonica, Sargassum fusiforme and Sargassum thunbergii.

3. The production method according to claim 1, wherein The drying temperature of the seaweed in (1) is 70 to 80 DEG C, and the crushing temperature is 20 to 25 DEG C.

4. The production method according to claim 1, wherein The mass-volume ratio of the seaweed powder to the ethanol aqueous solution in (2) is 1 g:10 to 15 mL, and the extraction temperature is 40 to 55 DEG C.

5. The production method according to claim 1, wherein The volume ratio of the concentrated solution to the non-polar solvent in (3) is 1:1 to 1.5, and the extraction temperature is 25 to 35 DEG C.

6. The production method according to claim 1, wherein The activated carbon column in (4) is an SPE special column.

7. The production method according to claim 1, wherein Before the extract liquid is purified in (4), the extract liquid is subjected to vacuum concentration treatment, the vacuum degree of the vacuum concentration is-0.085 to-0.095 Mpa, and the temperature of the vacuum concentration is 55 to 65 DEG C.

8. The use of the fucoxanthin prepared by any one of the methods in claims 1 to 7 in the preparation of any one of liquid, spray, solid powder and tablet food.

9. The use of the fucoxanthin prepared by any one of the methods in claims 1 to 7 in the preparation of a product with the effect of relieving dry eye.

10. Use of fucoxanthin prepared according to any one of the methods of claims 1 to 7 in the manufacture of a product having an effect of alleviating visual fatigue, characterized in that, The product forms include but are not limited to eye patches, eye patches and eye sprays.

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