Method for improving dry eye syndrome using ethanol extract of soybean seeds and combination thereof with ethanol distillate of soybean seeds

Abstract

Disclosed in the present invention are an ethanol extract of soybean seeds and a combination containing the ethanol extract of soybean seeds and an ethanol distillate, which can be used to improve dry eye syndrome.

Description

Method for improving dry eye using an ethanol extract of soybean seeds and a combination thereof with an ethanol distillation product of soybean seeds Technical Field

[0001] The present invention relates to a method for improving dry eye syndrome using an ethanol extract of Glycine max seeds and a combination thereof with an ethanol-distilled product. Background Art

[0002] Dry eye syndrome (DES) is primarily caused by insufficient tear secretion and excessive tear evaporation. Symptoms include dryness, redness, itching, photophobia, and pain. In severe cases, it may even cause visual disturbances and ocular surface damage.

[0003] Clinically, artificial tears are primarily used to treat mild dry eye, while severe cases require anti-inflammatory drugs (SAIDs). However, both artificial tears and SAIDs can only temporarily alleviate eye discomfort and do not increase tear production.

[0004] Soybean (Glycine max) is a plant of the genus Glycine in the family Fabaceae. Its seeds are primarily used and are considered a good source of protein. In TW I640318 B (corresponding to US 10543243 B2), the applicant disclosed an extract composition comprising an extract obtained by extracting soybean seeds using water or 90% or less ethanol by weight, and a distillation product obtained by distilling the soybean seeds using water or 15% or less ethanol by weight. This extract composition has been shown to promote skin wound healing and nerve cell proliferation, as well as treat dementia and breast cancer. In TW I724417 B (corresponding to US 11083766 B2), the applicant disclosed the use of this extract composition to alleviate pain and skin inflammation caused by cancer radiation therapy.

[0005] As far as the applicant is aware, there has been no literature or prior patent application that has ever disclosed that soybean seed extracts or distilled products can be used to improve dry eye syndrome. Summary of the Invention

[0006] In the present invention, the applicants have experimentally discovered that the ethanol extract of Glycine max seeds, alone or in combination with an ethanol-distilled product, can effectively increase tear secretion in mice with scopolamine-induced dry eye syndrome, and is therefore believed to have the effect of improving dry eye syndrome.

[0007] Therefore, in a first aspect, the present invention provides a method for improving dry eye, comprising administering a medicament comprising the soybean seed ethanol extract as described above to a subject in need thereof.

[0008] In a second aspect, the present invention provides a method for improving dry eye, comprising administering a medicament comprising the combination of the soybean seed ethanol extract and the ethanol distillation product as described above to a subject in need thereof.

[0009] In a third aspect, the present invention provides a use of an ethanol extract of soybean seeds for preparing a medicine for improving dry eye.

[0010] In a fourth aspect, the present invention provides a use of the above-mentioned combination for preparing a medicament for improving dry eye.

[0011] In a fifth aspect, the present invention provides a medicine for improving dry eye, comprising the soybean seed ethanol extract as described above.

[0012] In a sixth aspect, the present invention provides a pharmaceutical for improving dry eye, comprising the combination described above.

[0013] Preferably, the ethanol extract is prepared by extracting soybean seeds using 70 wt % to 90 wt % ethanol.

[0014] Preferably, the ethanol distillation product is obtained by distilling soybean seeds using 0.01 wt % to 15 wt % of ethanol.

[0015] Preferably, the ethanol distillation product is obtained by distilling soybean seeds using 0.13 wt % to 15 wt % of ethanol.

[0016] Preferably, the combination comprises 0.05 wt % to 1 wt % of the ethanol extract and 70 wt % to 90 wt % of the ethanol distillate.

[0017] Preferably, the medicine further comprises a pharmaceutically acceptable carrier.

[0018] Preferably, the pharmaceutical product is in a dosage form for oral administration, topical administration or parenteral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and other objects, features and advantages of the present invention will become apparent after referring to the following detailed description and preferred embodiments and the accompanying drawings, in which:

[0020] FIG1 shows the wet lengths measured for each group of mice in Example 1, wherein “*” and “**” respectively indicate p<0.05 and p<0.01 when compared with the pathological control group.

[0021] FIG2 shows the wet lengths measured for each group of mice in Example 2, wherein “**” indicates that p<0.01 when compared with pathological control groups 1 to 3. DETAILED DESCRIPTION

[0022] For the purposes of this specification, it will be expressly understood that the word "comprising" means "including, but not limited to," and that the word "comprises" has a corresponding meaning.

[0023] The present invention provides a soybean seed (Glycine max seed) ethanol extract for use in preparing a pharmaceutical for improving dry eye syndrome. The present invention also provides a combination of the soybean seed ethanol extract and an ethanol-distilled product for use in preparing a pharmaceutical for improving dry eye syndrome.

[0024] In addition, the present invention provides a medicine for improving dry eye, comprising the soybean seed ethanol extract as described above. The present invention also provides a medicine for improving dry eye, comprising the combination as described above.

[0025] As used herein, the terms "dry eye syndrome," "xerophthalmia," "sclerophthalmia," "keratoconjunctivitis sicca," and "dysfunctional tear syndrome" are used interchangeably and are intended to encompass at least one of the following forms: aqueous tear-deficient dry eye, mucin-deficient dry eye, lipid-deficient dry eye, and evaporative dry eye.

[0026] Dry eye, to which the present invention is applicable, can be caused by a variety of factors, including, but not limited to: a deficiency in the tear-flow system; sleep disorders, such as insomnia and sleep apnea syndrome; the natural aging process, particularly menopause; side effects of medications (e.g., antidepressants, blood pressure medications, Parkinson's medications, antihistamines, and birth control pills); diseases that affect tear production, such as Sjogren's syndrome, rheumatoid arthritis, and collagen vascular disease; contact lens wear; smoking; and particulate matter (PM), such as PM. 2.5; dry climate; insufficient blinking; and structural problems that prevent the eyelid from closing properly.

[0027] As used herein, the terms "soybean (Glycine max)", "edamame (Glycine max)", "soybean (Glycine max)", and "soybean" are used interchangeably and are intended to encompass soybeans that are readily available to those skilled in the art or that are collected from natural sources.

[0028] According to the present invention, the methods for ethanol extraction and ethanol distillation from soybean seeds can be performed using techniques well known and commonly used by those skilled in the art. In this regard, reference may be made, for example, to TW 1724417 B (corresponding to US 11083766 B2) and TW 1640318 B (corresponding to US 11452753 B2, US 11400128 B2, and US 10543243 B2).

[0029] It is understood that the operating conditions for ethanol extraction and ethanol distillation of soybean seeds will vary depending on factors such as the soybean seed processing method and the ratio of soybean seed to ethanol to achieve optimal extraction results. The selection of these operating conditions is a matter of routine discretion for those skilled in the art.

[0030] According to the present invention, ethanol extraction and ethanol distillation of soybean seeds can be performed using fresh soybean seeds, or can be performed using soybean seeds that have previously undergone a processing selected from the group consisting of: drying, grinding, chopping, pulverizing, and combinations thereof.

[0031] According to the present invention, the ethanol extraction and ethanol distillation can be performed using soybean seeds and ethanol solution at a weight ratio of 1:1 to 1:100, respectively. In a preferred embodiment of the present invention, the weight ratio is 1:10.

[0032] According to the present invention, the ethanol extraction can be performed by using 70 wt % to 90 wt % ethanol, preferably, 70 wt % to 80 wt % ethanol. In a preferred embodiment of the present invention, the ethanol extraction is performed by using 70 wt % ethanol.

[0033] According to the present invention, the ethanol extraction can be performed at a temperature of 22° C. to 45° C. In a preferred embodiment of the present invention, the temperature is 45° C.

[0034] According to the present invention, the ethanol distillation can be performed by using 0.01 wt% to 15 wt% ethanol, preferably, 0.13 wt% to 15 wt% ethanol. In a preferred embodiment of the present invention, the ethanol distillation is performed by using 0.13 wt% ethanol.

[0035] According to the present invention, the ethanol distillation can be carried out at a temperature of 55° C. to 98° C. In a preferred embodiment of the present invention, the temperature is 90° C.

[0036] According to the present invention, the combination may comprise 0.05 wt % to 5 wt % of an ethanol extract of soybean seeds and 20 wt % to 90 wt % of an ethanol distillation product of soybean seeds, preferably, 0.05 wt % to 1 wt % of an ethanol extract of soybean seeds and 70 wt % to 90 wt % of an ethanol distillation product of soybean seeds, and more preferably, 0.05 wt % to 0.3 wt % of an ethanol extract of soybean seeds and 70 wt % to 90 wt % of an ethanol distillation product of soybean seeds.

[0037] According to the present invention, the pharmaceutical product may be in a dosage form suitable for parenteral administration, oral administration, or topical administration.

[0038] According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, thickener, and the like. The selection and amount of these agents are within the professional knowledge and routine skills of those skilled in the art.

[0039] According to the present invention, the pharmaceutical product can be manufactured into a dosage form suitable for parenteral administration (including injection, for example, a sterile aqueous solution or dispersion) using techniques well known to those skilled in the art, and administered by a route selected from the group consisting of intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration.

[0040] According to the present invention, the pharmaceutical product can be manufactured into a dosage form suitable for oral administration using techniques well known to those skilled in the art, including, but not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0041] According to the present invention, the pharmaceutical product can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to, emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, salve and bandages.

[0042] According to the present invention, the external preparation is prepared by mixing the pharmaceutical of the present invention with a base well known to those skilled in the art.

[0043] According to the present invention, the base may contain one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons (such as petroleum jelly and white petrolatum), waxes (such as paraffin and yellow wax), preservatives, antioxidants, surfactants, absorption enhancers, stabilizers, gelling agents (such as Microcrystalline cellulose and carboxymethylcellulose, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents, emollients, solubilizing agents, penetration enhancers, thickeners, anti-irritants, preservatives, colorants, and propellants. The selection and amount of these additives are within the professional knowledge and routine skills of those skilled in the art.

[0044] More preferably, the medicine is an ointment comprising 0 wt % to 10.15 wt % of a thickener, 0 wt % to 13.75 wt % of an emulsifier, and 0.28 wt % to 0.35 wt % of a preservative.

[0045] In a preferred embodiment of the present invention, the thickener is polyacrylic acid (carbomer), glyceryl polyacrylate, and hyaluronic acid.

[0046] According to the present invention, the emulsifier can be selected from the group consisting of hydrogenated polydecene (Parleam), borneol, propylene glycol, polysorbate 60, trideceth-6 (Mihacol 139), isoceteth-20 (Mihacol 250), stearic acid, stearyl alcohol, polysorbate 80, and potassium hydroxide.

[0047] In a preferred embodiment of the present invention, the preservative is benzyl alcohol or chloroxylenol.

[0048] In another aspect, the present invention provides a method for improving dry eye, comprising administering a medicament comprising the soybean seed ethanol extract as described above to a subject in need thereof.

[0049] The present invention also provides a method for improving dry eye, comprising administering a medicament comprising the combination described above to a subject in need thereof.

[0050] As used herein, the terms "administration" and "administration" are used interchangeably and mean introducing, providing or delivering a predetermined active ingredient to a subject by any appropriate route to perform its intended effect.

[0051] As used herein, the term "subject" means any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats.

[0052] According to the present invention, the dosage and frequency of administration of the soybean seed ethanol extract and the combination will vary depending on the severity of the condition being treated, the route of administration, and the age, physical condition, and response of the individual being treated. Generally, the soybean seed ethanol extract and the combination can be administered as a single dose or divided into multiple doses. For example, the soybean seed ethanol extract and the combination can be administered twice daily.

[0053] The present invention will be further described with reference to the following examples. However, it should be understood that these examples are for illustration only and should not be construed as limitations on the implementation of the present invention.

[0054] <Example>

[0055] General experimental materials:

[0056] 1. Preparation of Glycine max seed ethanol extract and ethanol-distilled product:

[0057] The soybean seed ethanol extract and ethanol distillation product used in this example were prepared according to the method described in the examples of TW I640318 B (corresponding to US 10543243 B2).

[0058] Briefly, soybean seed powder collected from Tainan City was mixed with 70 wt% ethanol at a weight ratio of 1:10, followed by heat extraction at 45°C. The resulting mixture was then filtered to remove solids, vacuum filtered, and dried to obtain an ethanol extract of the soybean seeds.

[0059] In addition, soybean seed powder was mixed with 0.13 wt % ethanol at a weight ratio of 1:10, and then distilled at 90° C. and the condensate was collected to obtain a soybean seed ethanol distillation product.

[0060] 2. Experimental Animals

[0061] Male C57BL / 6 mice (weighing approximately 24 g) used in this example were purchased from BioLasco Taiwan Co., Ltd. All experimental animals were housed in an animal room with a 12-hour light and dark cycle, maintained at 24-26°C and a relative humidity of 55%-65%, and provided with ample water and feed. Animal handling and experimental procedures complied with the standards of the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) International.

[0062] General experimental methods:

[0063] 1. Statistical analysis:

[0064] The experimental data were expressed as mean ± standard error of the mean (SEM) and analyzed using Student's t-test to assess differences between groups. Statistical significance was indicated if p < 0.05.

[0065] Example 1. Evaluation of the efficacy of soybean seed ethanol extract in improving dry eye

[0066] Experimental Materials:

[0067] 1. Preparation of ointment containing ethanol extract of soybean seeds:

[0068] Ointments 1 and 2 used in this example had the formulations shown in Table 1 below. These ointments were prepared according to the following steps: An ethanol extract of soybean seeds was dissolved in water and heated to a temperature between 95°C and 100°C. Polyacrylic acid (carbomer), glyceryl polyacrylate, and hyaluronic acid were then added and stirred. After the resulting mixture cooled, benzyl alcohol and chloroxylenol were added.

[0069] Table 1. Formulations of Ointments 1 and 2

[0070] Experimental methods:

[0071] A. Induction and medication of dry eye:

[0072] First, mice (14 weeks old) were randomly divided into four groups: a normal control group (n=6), a pathological control group (n=6), experimental group 1 (n=6), and experimental group 2 (n=5). Mice in the pathological control group, the comparative group, and experimental groups 1 and 2 were each given subcutaneous injections of scopolamine (Sigma, Catalog No. S0929) (0.5 mg / mouse, dissolved in 0.2 mL of saline) twice daily to induce dry eye. Mice in the normal control group received no treatment.

[0073] On the first day after the initial scopolamine injection, ointments 1 and 2 were applied to the skin around the eyes of mice in experimental groups 1 and 2, respectively (both doses: 50 mg of ointment per 1 square centimeter), twice daily for a total of 21 days. Mice in the normal control and pathological control groups received no treatment.

[0074] result:

[0075] Figure 1 shows the measured wet lengths of each group of mice. As shown in Figure 1, the wet lengths of the pathological control group were significantly lower than those of the normal control group, indicating that scopolamine successfully induced dry eye and tear deficiency in the mice. Compared to the pathological control group, the wet lengths of experimental groups 1 and 2 were significantly increased, and this increase became more pronounced with increasing concentrations of soybean seed ethanol extract. In particular, the wet length of experimental group 2 was significantly higher than that of the pathological control group.

[0076] This experimental result shows that both ointments 1 and 2 can effectively increase tear secretion. Therefore, the soybean seed ethanol extract can improve dry eye symptoms in a dose-related manner. Therefore, the soybean seed ethanol extract is considered to have high potential for the preparation of a pharmaceutical product to improve dry eye symptoms.

[0077] In addition, the applicant also prepared an ointment containing 1 wt% of an ethanol extract of soybean seeds and 90 wt% of an ethanol distillate of soybean seeds according to the above method and conducted the same test. The results showed that the measured wet length was significantly longer than that of Ointments 1 and 2 (data not shown). Therefore, the applicant further tested the effectiveness of the combination of an ethanol extract of soybean seeds and an ethanol distillate of soybean seeds in the following examples.

[0078] Example 2. Evaluation of the efficacy of a combination of an ethanol extract of soybean seeds and an ethanol distillation product in improving dry eye

[0079] Experimental Materials:

[0080] 1. Preparation of an ointment containing an ethanol extract of soybean seeds and an ethanol distillation product:

[0081] Ointments 3 to 5 used in this example had the formulations shown in Table 2 below. These three ointments were prepared generally according to the method described in Example 1, except that the ethanol extract of soybean seeds was dissolved in water together with the ethanol distillation product, and the polyacrylic acid, polyacrylate glycerides, and hyaluronic acid in Ointment 4 were replaced with hydrogenated polydecene (Parleam), borneol, propylene glycol, polysorbate 60, trideceth-6 (Mihacol 139), and isoceteth-20 (Mihacol 250). In Ointment 5, the polyacrylic acid, polyacrylate glycerides, and hyaluronic acid were replaced with borneol, stearic acid, stearyl alcohol, polysorbate 80, and potassium hydroxide, which were added after heating.

[0082] Table 2. Formulations of Ointments 3 to 5

[0083] Experimental methods:

[0084] A. Induction and medication of dry eye:

[0085] First, mice (8 weeks old) were randomly divided into 9 groups, including 3 normal control groups (i.e., normal control groups 1 to 3, n=6 per group), 3 pathological control groups (i.e., pathological control groups 1 to 3, n=6 per group), and 3 experimental groups (i.e., experimental groups 1 to 3, n=7 per group). Following the method described in Example 1, scopolamine was injected into the mice in pathological control groups 1 to 3 and experimental groups 1 to 3 four times daily and exposed to a dry environment with fan-generated airflow for a total of 7 days to induce dry eye. Mice in normal control groups 1 to 3 did not receive any treatment.

[0086] On the eighth day after the initial scopolamine injection, ointments 3 to 5 (50 mg per 1 square centimeter) were applied to the skin around the eyes of mice in experimental groups 1 to 3, respectively, twice daily for a total of seven days. Mice in normal control groups 1 to 3 and pathological control groups 1 to 3 received no treatment.

[0087] B. Schirmer's tear test

[0088] On the 14th day after the start of scopolamine injection, the wet length of each group of mice was measured and recorded according to the method described in Example 1.

[0089] result:

[0090] Figure 2 shows the measured wet lengths for each group of mice. As shown in Figure 2, the wet lengths of pathological control groups 1 to 3 were significantly lower than those of normal control groups 1 to 3, indicating that scopolamine and the dry environment successfully induced dry eye and tear deficiency in the mice. Furthermore, compared to pathological control groups 1 to 3, the wet lengths of experimental groups 1 to 3 were significantly increased. These results demonstrate that ointments 3 to 5 are all effective in increasing tear secretion. Therefore, the combination of soybean seed ethanol extract and ethanol distillate is considered to have high potential for the development of pharmaceuticals to improve dry eye.

[0091] All patents and publications cited in this specification are hereby incorporated by reference in their entirety. In the event of any conflict, the detailed description of this specification (including definitions) will prevail.

[0092] Although the present invention has been described with reference to the specific embodiments above, it is apparent that many modifications and variations can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only as indicated by the appended claims.

Claims

1. An ethanol extract of soybean seeds is used for preparing a medicine for improving dry eye syndrome.

2. A combination of an ethanol extract of soybean seeds and an ethanol distillation product for use in preparing a pharmaceutical product for improving dry eye syndrome.

3. The method according to claim 1 or 2, characterized in that The ethanol extract is prepared by extracting soybean seeds using 70 wt % to 90 wt % of ethanol.

4. The method according to claim 2, characterized in that The ethanol distillation product is prepared by distilling soybean seeds using 0.01 wt % to 15 wt % of ethanol.

5. The method according to claim 2, characterized in that The ethanol distillation product is prepared by distilling soybean seeds using 0.13 wt % to 15 wt % of ethanol.

6. The method according to claim 2, characterized in that The combination comprises 0.05 wt % to 1 wt % of an ethanol extract and 70 wt % to 90 wt % of an ethanol distillation product.

7. The method according to claim 1 or 2, characterized in that The pharmaceutical product further comprises a pharmaceutically acceptable carrier.

8. The method according to claim 1 or 2, characterized in that The pharmaceutical product is in a dosage form for oral administration, topical administration or parenteral administration.

9. A method for improving dry eye, characterized in that: The method comprises administering a medicament comprising an ethanol extract of soybean seeds to a subject in need thereof.

10. A method for improving dry eye, characterized in that: The method comprises administering a medicament comprising a combination of an ethanol extract of soybean seeds and an ethanol distillation product to an individual in need thereof.

11. The method according to claim 9 or 10, characterized in that The ethanol extract is prepared by extracting soybean seeds using 70 wt % to 90 wt % of ethanol.

12. The method according to claim 10, characterized in that The ethanol distillation product is prepared by distilling soybean seeds using 0.01 wt % to 15 wt % of ethanol.

13. The method according to claim 10, characterized in that The ethanol distillation product is prepared by distilling soybean seeds using 0.13 wt % to 15 wt % of ethanol.

14. The method according to claim 10, characterized in that The combination comprises 0.05 wt % to 1 wt % of an ethanol extract and 70 wt % to 90 wt % of an ethanol distillation product.

15. The method according to claim 9 or 10, characterized in that The pharmaceutical product further comprises a pharmaceutically acceptable carrier.

16. The method according to claim 9 or 10, characterized in that The pharmaceutical product is in a dosage form for oral administration, topical administration or parenteral administration.

17. A pharmaceutical product for improving dry eye syndrome, characterized in that: The drug contains an ethanol extract of soybean seeds.

18. A pharmaceutical product for improving dry eye syndrome, characterized in that: The medicine comprises a combination of an ethanol extract of soybean seeds and an ethanol distillation product.

19. The pharmaceutical product according to claim 17 or 18, characterized in that The ethanol extract is prepared by extracting soybean seeds using 70 wt % to 90 wt % of ethanol.

20. The pharmaceutical product according to claim 18, characterized in that The ethanol distillation product is prepared by distilling soybean seeds using 0.01 wt % to 15 wt % of ethanol.

21. The pharmaceutical product according to claim 18, characterized in that The ethanol distillation product is prepared by distilling soybean seeds using 0.13 wt % to 15 wt % of ethanol.

22. The pharmaceutical product according to claim 18, characterized in that The combination comprises 0.05 wt % to 1 wt % of an ethanol extract and 70 wt % to 90 wt % of an ethanol distillation product.

23. The pharmaceutical product according to claim 17 or 18, characterized in that The pharmaceutical product further comprises a pharmaceutically acceptable carrier.

24. The pharmaceutical product according to claim 17 or 18, characterized in that The pharmaceutical product is in a dosage form for oral administration, topical administration or parenteral administration.

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