TW202112388A - Lotus seed coat extracts for preventing cell damage and the usage thereof - Google Patents

Abstract

The present invention provides a lotus seed coat extract for preventing cell damage and the usage thereof. The lotus seed coat extract is obtained through an extraction method. The extraction method includes the following steps: mixing the lotus seed coat with water at a solid-liquid weight ratio of 1-5: 10-30, and then homogenizing the mixture, and then the mixture is reacted at 30-100 °C for 0.5-3 hours. The lotus seed coat extract can prevent cell damage caused by UV and reactive oxygen species (ROS).

Description

Lotus seed shell extract for preventing cell damage and its use

This case is about a plant extract and its use, especially a lotus seed shell extract for preventing cell damage and its use.

The production of oxidative stress is the result of the imbalance between the reactive oxygen species (ROS) and the antioxidant defense system in the body. A large amount of ROS production will increase the oxidative pressure in the cell, which will cause cell damage or death, and even lead to The formation of many diseases.

Ultraviolet (Ultraviolet, referred to as UV) after passing through the atmosphere, the remaining main ultraviolet can be subdivided into UVA (wavelength 320 to 400 nm) and UVB (wavelength 280 to 320 nm) according to its wavelength. Among them, UVA has strong penetrating power, and even general transparent glass cannot block the penetration of UVA. It is the most easily exposed ultraviolet light in daily life. The absorption of the skin can cause damage to DNA or generate ROS in cells, which leads to Cell damage or death will mainly cause the deterioration of freckles and dark spots, and cause skin tanning. It is also an accomplice of skin aging.

In view of this, there is a great need to develop a composition for preventing/avoiding cell damage, so that the cells have antioxidant capacity and can prevent them from being damaged by ultraviolet rays.

In view of the problems faced by the prior art, one of the objectives of this case is to provide a lotus seed shell extract for preventing cell damage, wherein the lotus seed shell extract is obtained through an extraction method. This extraction method includes the lotus seed shell and water With a solid-liquid weight ratio of 1-5:10-30 (1 to 5:10 to 30), the mixture is mixed into a mixture, and the mixture is homogenized, and then reacted at 30-100°C for 0.5-3 hours.

In an embodiment of this case, the mixture contains pectinase in addition to the lotus seed shell and water, and the pectinase accounts for less than 1 wt% of the mixture (before the reaction).

In an embodiment of this case, the extraction method further includes cooling the reacted mixture to 20-30°C; sequentially filtering with a 400 mesh and 5 micron filter; and concentrating under reduced pressure at 45-70°C.

In an embodiment of this case, the solid-liquid weight ratio of lotus seed shell to water is 1:20.

In an example of this case, after the mixture is homogenized, it is treated at 80-90°C for 1 hour.

In an example of this case, after the mixture is homogenized, it is firstly treated at 35-45°C for 1 hour, and then at 80-90°C for 1 hour.

In an embodiment of the present case, the lotus seed shell extract contains polyphenolic compounds at a concentration of at least 3 mg/ml, and preferably contains polyphenols at a concentration of 3-7 mg/ml.

In an embodiment of this case, the lotus seed shell extract contains polyphenol compounds, flavonoid compounds and trace elements.

In an embodiment of this case, the polyphenol compound is catechin, epicatechin, hyperoside or isoquercitrin.

In an embodiment of this case, the flavonoid compound is quercetin, myricetin or kaempferol; the trace element is calcium, iron, zinc or phosphorus.

This case also provides a use of lotus seed husk extract to prepare an antioxidant composition.

In an embodiment of this case, the antioxidant effect of the antioxidant composition is through increasing the content of glutathione (Glutathione, GSH) in the cell, or reducing the content of reactive oxygen species (ROS) in the cell And reached.

This case also provides a use of lotus seed shell extract to prepare a composition for preventing cells from being damaged by ultraviolet rays.

In an embodiment of this case, the dosage form of the anti-oxidant composition and the composition for preventing cell damage by ultraviolet rays is powder, granule, liquid, gel, paste, capsule or lozenge.

The lotus seed husk extract provided in this case can effectively increase the content of glutathione (Glutathione, GSH) in the cell, and reduce the content of reactive oxygen species (ROS) in the cell, thereby preventing/avoiding cell oxidative damage , Can also effectively resist cell damage caused by ultraviolet rays. Therefore, the lotus seed shell extract in this case can be used to prepare a composition for anti-oxidation or to prevent cells from being damaged by ultraviolet rays.

In the following, a plurality of embodiments of this case will be disclosed in conjunction with the drawings. For the sake of clarity, many practical details will be described in the following description. However, these practical details should not be used to limit the case. In other words, in some implementations of this case, these practical details are unnecessary. The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

In this article, unless the article is specifically limited in the context, "一" and "the" can generally refer to one or more. It can be further understood that the terms "include", "include", "have" and similar words used in this text indicate the recorded features, regions, integers, steps, operations, elements and/or components, but do not exclude The described or additional one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

From the foregoing, it can be known that the absorption of ultraviolet rays through the skin will cause damage to DNA, and a large amount of ROS production will also increase the oxidative pressure in the cells, which will lead to cell damage or death, and cause many diseases.

In view of this, this case provides a lotus seed shell extract, which can be used to prevent cells from being damaged by ultraviolet rays, and has the function of anti-oxidation, which can effectively inhibit the generation of ROS, reduce the oxidative pressure in the cells, and protect the cells from damage.

The following will explain in detail the extraction method of the lotus husk extract in this case, and its anti-oxidation and anti-ultraviolet function tests to verify that the lotus husk extract in this case does have the ability to protect cells from UV damage and anti-oxidation.

In this case, glutathione (Glutathione, GSH) and reactive oxygen species (ROS) were used as indicators of antioxidant function. GSH is a tripeptide composed of glutamine, cystine and glycine, and its thiol group (-SH) is related to redox; its main function is the defense of cellular endogenous antioxidants, which can resist the oxidation of ROS Therefore, if the content of GSH in the cell is increased, it will increase the ability of redox in the cell, so that the cell has a better antioxidant function; in addition, if the production of ROS in the cell decreases, it will reduce the intracellular Oxidative stress can also make cells have a better antioxidant effect.

Example 1: Preparation process of lotus seed shell extract A 1. Wash the lotus seed shell for subsequent extraction; the lotus seed shell is derived from the outer shell of the Nelumbo plant seed. 2. Mix the washed lotus shell and water at a solid-liquid weight ratio of 1-5:10-30 to obtain a mixture of lotus shell and water. The mixture of lotus shell and water is homogenized at 30-100°C for 0.5 -3 hours; in some embodiments, the water may be reverse osmosis (RO) water; in some embodiments, lotus seed shells and water are mixed at a solid-to-liquid weight ratio of 1:20, and the lotus seed shells obtained after mixing The mixture with water is homogenized at 80-90°C for 1 hour. 3. Cool the mixture of lotus seed shell and water after homogenization to room temperature (in some embodiments, about 20-30° C., for example, 25° C.). 4. Sequentially use 400 mesh and 5 micron filters to filter the lotus seed shell and water mixture obtained in step 3. 5. The filtrate obtained in step 4 is concentrated under reduced pressure at 45-70°C, for example, concentrated under reduced pressure at 60±5°C to obtain the lotus seed shell extract. In some embodiments, the extracted lotus seed shell extract contains polyphenolic compounds (such as catechin, epicatechin, hyperoside or isoquercitrin, etc.), flavonoid compounds (such as quercetin, quercetin, etc.) Bayberry flavonoids or kaempferol, etc.) and trace elements (such as calcium, iron, zinc, or phosphorus, which account for less than 0.02% of the total human mass). In some embodiments, the concentration of polyphenol compounds in the lotus husk extract is 3 milligrams/ml (mg/mL) or more, or can be 3-7 mg/ml; in some embodiments, the lotus husk extract The concentration of polyphenolic compounds in 5.9 mg/ml.

Example 2: Preparation process B of lotus seed shell extract 1. Wash the lotus seed shell for subsequent extraction; the lotus seed shell is derived from the shell of the Nelumbo plant seed. 2. Mix the washed lotus seed shell with water at a solid-liquid weight ratio of 1-5:10-30, and further add pectinase (purchased from Hengzhou Industrial Co., Ltd., Viscozyme L) (after adding, Pectinase does not exceed 1wt% of the mixture of whole lotus seed shell and water), homogenize at 35-45°C for 1 hour, and then homogenize at 80-90°C for 1 hour; in some embodiments, lotus seed shell and water After the water system is mixed with a solid-liquid weight ratio of 1:20, homogenization is performed at 35-45°C for 1 hour, and then at 80-90°C for 1 hour; the water can be reverse osmosis (RO) water. 3. Cool the post-reaction mixture obtained in step 2 to room temperature (in some embodiments, about 20-30°C, for example, 25°C). 4. Sequentially use 400 mesh and 5 micron filters to filter the mixed solution obtained in step 3. 5. The filtrate obtained in step 4 is concentrated under reduced pressure at 45-70°C to obtain the lotus seed shell extract. In some embodiments, it may be concentrated under reduced pressure at 55-65°C. In some embodiments, the extracted lotus seed shell extract contains polyphenol compounds, flavonoid compounds and trace elements. In some embodiments, the concentration of polyphenol compounds in the lotus husk extract is 3 milligrams/ml (mg/mL) or more, or can be 3-7 mg/ml; in some embodiments, the lotus husk extract The concentration of polyphenolic compounds in 5.9 mg/ml.

Example 3: Evaluation of Cell Antioxidant Efficacy-Detection of GSH Content of Peripheral Blood Mononuclear Cells A. Experimental Materials 1. Cell line: human peripheral blood mononuclear cell (hPBMC). 2. Medium: Osteoclast differentiation medium (osteoclast differentiation medium), which is supplemented with 10% fetal bovine serum (FBS), 1x penicillin/streptomycin (Penicillin/Streptomycin), human Nuclear factor κ-B ligand receptor activator (human RANKL) (40 ng/mL) and human colony stimulating factor (human M-CSF) (25 ng/mL) α-minimum essential medium (α-minimum essential) medium, α-MEM). 3. GSH detection reagent (purchased from abcam, model Ab112132). B. Experimental steps 1. In a 6-well culture dish, implant 2x10 ⁵ human peripheral blood mononuclear cells (abbreviated as cells in the following steps) into each well of culture medium. 2. Incubate at 37°C for 24 hours. 3. Divide the cells into two groups: the first group is the blank group (Mock); the second group (experimental group) is added with lotus seed shell extract, so that after adding the lotus seed shell extract, the concentration of the lotus seed shell extract in the overall solution It was 0.0625 mg/mL; then the two groups of cells were cultured at 37℃ for 24 hours. 4. Collect the cells. 5. Rinse once with Phosphate-Buffered Saline (PBS) (purchased from Gibco). 6. Resuspend cells in 1 ml of PBS. 7. Stain the cells with GSH reagent (1:1000) for 15 minutes. 8. Rinse once with PBS. 9. Resuspend cells in 200 microliters of PBS. 10. Analyze the relative fluorescent isothiocyanate (FITC) signal by flow cytometry (purchased from BD Accuri, model C6 Plus). 11. The detected values are analyzed by the Microsoft EXCEL software using Student t test to analyze the statistical significance between the values. The results are shown in Figure 1. Among them, compared with the blank group, *** means p >0.001.

From the results in Figure 1, it can be seen that the second group of cells (experimental group) added with lotus seed shell extract can significantly increase their GSH content, making the relative GSH content (relative GSH content) 3.5 of that of the first group (blank group). Times. GSH is a tripeptide composed of glutamine, cystine and glycine. Its thiol group (-SH) is related to redox, and its main function is the defense of cellular endogenous antioxidants, which can resist the oxidation of ROS. hurt. Therefore, the results in Fig. 1 can also confirm that adding the lotus seed shell extract extracted from Example 1 or Example 2 of this case can indeed increase the GSH content in cells and increase the ability of redox in cells to achieve cellular anti-oxidation. The effect of.

Example 4: Evaluation of cellular anti-oxidation efficacy-detection of ROS damage to peripheral blood mononuclear cells A. Experimental materials and equipment 1. Human peripheral blood mononuclear cells (hPBMC). 2. Medium: X-VIVO™ 10 medium (purchased from Lonza/product number: Cat#04-380Q). 3. Phosphate buffered saline (PBS) (purchased from Gibco). 4. 2',7'-Dichlorofluorescin diacetate (2',7'-Dichlorofluorescin diacetate, DCFH-DA) stock solution (stock solution): DCFH-DA (purchased from Sigma-Aldrich, product Model SI-D6883-50MG) is stored in dimethyl sulfoxide (DMSO, purchased from Sigma-Aldrich) at a concentration of 5 mg/mL. 5. Hydrogen peroxide (H ₂ O ₂ ) (purchased from Sigma-Aldrich, 30wt% aqueous solution). 6. Flow cytometer (purchased from BD Accuri). B. Experimental steps 1. In a 6-well culture dish, implant ^{2 ml of culture medium containing 2x10 5} human peripheral blood mononuclear cells (abbreviated as cells in the following steps) in each well. 2. Incubate at 37°C for 24 hours. 3. Remove the medium. 4. Divide the cells into three groups: the first group is a blank group (Mock); the second group (experimental group) is added with hydrogen peroxide, so that after adding hydrogen peroxide, the concentration of hydrogen peroxide in the overall solution is 1 mM; The third group (experimental group) added hydrogen peroxide and lotus husk extract so that the concentration of hydrogen peroxide in the overall solution was 1 mM, and the concentration of lotus husk extract in the overall solution was 0.03125 mg/mL; The three groups of cells were cultured at 37°C for 1 hour. 5. Add 5 μg/ml (μg/mL) DCFH-DA preservation solution to each well, and then place it at 37°C for 15 minutes for further reaction. 6. Add hydrogen peroxide to the cells at 37°C (after adding, the concentration of hydrogen peroxide in the whole solution is 1 mM), and let stand for 1 hour. 7. Wash each well twice with 1 ml of 1X PBS. 8. Add 200 μl trypsin (purchased from Gibco, model 15400-054), and react for 5 minutes in the dark. 9. Collect the medium with the cells in a 1.5 ml test tube and centrifuge at 400xg for 10 minutes. 10. Remove the supernatant and wash once with 1X PBS. 11. Centrifuge at 400xg for 10 minutes. 12. Remove the supernatant and resuspend the cell pellet with 1 ml of 1X PBS. 13. Use a flow cytometer to detect the fluorescent signal of DCFH-DA at the excitation wavelength (450-490 nm) and emission wavelength (510-550 nm). 14. The detected values are analyzed with the use of Microsoft EXCEL software and Student t test to analyze the statistical significance between the values. The results are shown in Figure 2. Among them, the second group (hydrogen peroxide) is compared with the first group (blank group), ### indicates p >0.001; the third group (hydrogen peroxide + lotus seed shell extract) is compared with the second group (over Hydrogen oxide), *** means p >0.001.

It can be seen from the results in Figure 2 that if hydrogen peroxide is provided to the cells (the second group of cells) alone, the relative ROS production (relative ROS production) is 1.6 times that of the blank group (the first group of cells). Hydrogen can increase the production of ROS in cells; however, if the lotus seed shell extract (third group of cells) described in Example 1 or Example 2 is administered together with the hydrogen peroxide to the cells, it can be found to be relative to ROS. The amount of ROS produced in the blank group (the first group of cells) was 0.8 times that of the blank group (the first group of cells), effectively reducing the relative ROS production promoted by hydrogen peroxide, and even less than the amount of ROS produced in the blank group (the first group). It can be seen that the lotus seed shell extract provided in this case can reduce the generation of ROS in cells, reduce oxidative stress, and make cells have antioxidant functions.

Example 5: Ability to resist and defend against ultraviolet rays A. Experimental materials and equipment 1. Cell line: Human skin fibroblasts (CCD-966sk) (ATCC, CRL-1881). 2. Medium: minimum essential medium (MEM), which contains 10% FBS, 1% penicillin/streptomycin and 1 mM sodium pyruvate (purchased from Gibco). 3. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide, MTT) (AMERSCO 0793-5G) is dissolved in PBS, and the concentration is 4 mg/mL. 4. DMSO (ECHO / DA1101-000000-72EC). 5. Enzyme immunoassay (ELISA reader) (purchased from BioTek). 6. Ultraviolet radiation chamber (purchased from Vilber). B. Procedure 1. 96-well culture plate, each well containing 5x10 ³ implanted cells 200-microliter ([mu] L) medium. 2. Incubate at 37°C for 24 hours. 3. Remove the culture medium without disturbing the attached cells. 4. Divide the cells into three groups, the first group is a blank group (Mock), and no ultraviolet light is applied; the second group (experimental group) is only irradiated with ultraviolet light (preferably UVA); the third group (experimental group) The lotus seed shell extract is added first to make the final concentration of the lotus seed shell extract 0.125 mg/mL, and then irradiated with ultraviolet rays (preferably UVA); The above-mentioned culture plate ^{is irradiated with 15 J/cm 2} of ultraviolet light for 1 hour, preferably 15 J/cm ² of UVA for 1 hour. At this time, it will cause LD50 (50% lethal dose), which represents ionizing radiation that causes half of the cells to die ( ionizing radiation) dose. 5. Then the three groups of cells were cultured at 37°C for 24 hours. 6. Add 15 microliters of MTT solution to each well, and then incubate at 37°C for 4 hours. 7. After removing the medium, add 50 microliters of DMSO to each well to dissolve the formazan crystals. 8. Put the culture plate on the shaker and incubate for 10 minutes. 9. Measure the absorbance (OD value) at a wavelength of 570nm. 10. MTT analysis of cell survival rate: cell survival rate (%) = (experimental group OD value / blank group OD value) × 100%, the aforementioned "experimental group" refers to the second and third groups of cells in Example 5 , And the "blank group" refers to the first group of cells in Example 5. 11. Using Microsoft EXCEL software, the Student t test was used to analyze the statistical significance between the values. The results are shown in Figure 3. Among them, the second group (ultraviolet rays, preferably UVA) is compared with the first group (blank group), ## means p >0.01; the third group (ultraviolet rays (preferably UVA) + lotus seed shell extract) is compared In the second group (ultraviolet rays, preferably UVA), *** means p >0.001.

Most of the damage caused by ultraviolet rays to cells comes from UVA (about 95% of ultraviolet rays), and the wavelength of UVA is longer than other UV, between 320-400 nm, which can penetrate the epidermis and damage the cells of the dermis. The cells produce ROS, which in turn increases the oxidative pressure in the cells, which promotes skin aging, relaxation and melanin precipitation.

Therefore, it can be seen from the results in Fig. 3 that when the cells are irradiated with only ultraviolet light (preferably UVA) (the second group of cells), the cell survival rate is 96.57%. It can be seen that ultraviolet light (preferably UVA) will Causes cell damage or death; however, if the cells are irradiated with ultraviolet rays (preferably UVA), the lotus seed shell extract (the third group of cells) described in Example 1 or 2 of this case is administered together, and the cells can be found The survival rate was 127.77%, which effectively improved the damage or death of cells caused by ultraviolet rays (preferably UVA), and the cell survival rate of the cells added with the lotus seed shell extract in this case was even higher than that of the blank group (the first group of cells) ), it can be seen that the lotus seed shell extract provided in this case can resist cell damage caused by ultraviolet rays (especially UVA) and prevent radiation damage to cells, thereby avoiding skin cells from aging, relaxation and melanin precipitation. .

It can be seen from the above implementations or examples of this case that the lotus seed shell extract and its extraction method and application provided in this case can increase the content of glutathione (Glutathione, GSH) in the cell, or/and reduce the reactive oxygen species in the cell. The content of substances (reactive oxygen species, ROS) to achieve the function of cellular anti-oxidation; it can also make cells (especially skin cells) resist or defend against damage caused by ultraviolet rays (especially UVA), so as to prevent aging and relaxation And melanin precipitation and other issues.

In addition, the composition containing lotus seed husk extract described in this case may further include carriers or other adjuvants well known to those skilled in the art; and the dosage form of the composition may be, but not limited to, powders and granules. , Liquid, gel, paste, capsule or lozenge; and the composition can also be added to foods, health foods or dietary supplements, or further applied to products such as beauty, skin care products or facial masks.

The foregoing content has summarized the characteristics of several implementations/examples. Those who are familiar with this technology should understand that this disclosure can be easily used as a basis for designing or modifying other processes and structures to achieve the same purpose and/or the same advantages as the implementation described in this case. Those who are familiar with this technology should also understand that the equivalent structure does not deviate from the spirit and scope of this disclosure, and various changes, substitutions and alterations can be made in this case without departing from the spirit and scope of this disclosure.

no.

The following will further illustrate the implementation of this case in conjunction with the drawings. The following examples are used to illustrate the characteristics and applications of the case, not to limit the scope of the case. Anyone familiar with the relevant technology will not deviate from the spirit of the case. Within the scope and scope, some changes and modifications can be made. Therefore, the scope of protection in this case shall be subject to the scope of the attached patent application. Figure 1 shows the effect of lotus seed shell extract on the content of Glutathione (GSH) in cells in an example of this case. Figure 2 shows the effect of lotus seed shell extract on the production of reactive oxygen species (ROS) in cells in an example of this case. Figure 3 shows the effect of lotus seed shell extract on cell resistance/defense against ultraviolet rays in an example of this case.

no.

Claims (14)

A lotus seed shell extract for preventing cell damage, wherein the lotus seed shell extract is obtained through an extraction method, the extraction method comprising: a lotus seed shell and water in a solid-liquid weight ratio of 1-5:10-30 , Mixed into a mixture; let the mixture perform a homogenization at 30-100°C for 0.5-3 hours. The lotus seed shell extract according to claim 1, wherein the mixture further comprises a pectinase, wherein the pectinase accounts for less than 1 wt% of the mixture (before the homogenization). The lotus seed shell extract according to claim 1 or claim 2, wherein the extraction method further comprises: Cooling the mixture after the homogenization to 20-30°C; Filter the mixture obtained in the previous step using a 400 mesh and 5 micron filter in sequence to obtain a filtrate; and The filtrate obtained in the previous step was concentrated under reduced pressure at 45-70°C. The lotus seed shell extract according to claim 1 or claim 2, wherein the solid-liquid weight ratio of the lotus seed shell to water is 1:20. The lotus seed shell extract according to claim 1, wherein the mixture is subjected to the homogenization at 80-90°C for 1 hour. The lotus seed shell extract according to claim 2, wherein the mixture is first subjected to the homogenization at 35-45°C for 1 hour, and then at 80-90°C for 1 hour. According to the lotus seed husk extract of claim 1 or claim 2, the lotus husk extract contains a polyphenol compound, and the concentration of the polyphenol compound is at least 3 mg/ml. According to the lotus seed husk extract of claim 7, the lotus seed husk extract further contains a flavonoid compound and a trace element. The lotus seed shell extract according to claim 7, wherein the polyphenol compound is catechin, epicatechin, hyperoside or isoquercitrin. The lotus seed shell extract according to claim 8, wherein the flavonoid compound is quercetin, myricetin or kaempferol; the trace element is calcium, iron, zinc or phosphorus. A use of the lotus seed shell extract as described in any one of claim 1 to claim 10 for preparing an antioxidant composition. The use according to claim 11, wherein the antioxidant is achieved by increasing the content of glutathione (Glutathione, GSH) in the cell or reducing the content of reactive oxygen species (ROS) in the cell. A use of the lotus seed shell extract as described in any one of claims 1 to 10 for preparing a composition for preventing cells from being damaged by ultraviolet rays. The use according to claim 11 or claim 13, wherein the dosage forms of the antioxidant composition and the composition for preventing cell damage by ultraviolet rays are powder, granule, liquid, gel, paste, capsule or lozenge.

Images