TWI801738B - Use of seed coat extracts of fagopyrum tataricum for reducing the loss of elastin - Google Patents

Abstract

A use of seed coat extract of tartary buckwheat (Fagopyrum tataricum) seed coat for a preparation of a composition for reducing the loss of elastin in the body.

Description

Use of tartary buckwheat seed coat extract for reducing elastin loss

The present invention relates to the use of a plant extract, in particular to the use of a tartary buckwheat seed coat extract for preparing a composition for slowing down skin aging.

The skin provides the first-stage protection for human individuals against environmental factors such as ultraviolet (ultraviolet, UV) radiation in the sun, pathogens, and friction. The skin consists of the epidermis, the dermis, which is mainly composed of connective tissue, and the subcutaneous tissue from the outside to the inside. Among them, the dermis contains molecules such as collagen, elastin, and hyaluronic acid. Collagen is like a "scaffold" in the skin, and elastin is like a "lock" in the skin. Collagen is connected by elastin, which can give the skin support and elasticity.

Collagen is an important matrix protein in the skin. It is a long-chain structure with a triple helix. It occupies a relatively high weight ratio in the dermis and provides structural stability for the skin. According to recent studies, one of the possible factors for the breakage of collagen chains is the glycosylation of collagen (Non-enzymatic glycation, adding sugar molecules to protein molecules). Since the glycation of protein will lead to the loss of protein function, the collagen chain will be broken and the loss rate will be accelerated, which will promote skin aging or even other related diseases.

In addition to collagen, which has been more researched in the past, elastin also plays an important role in the overall skin Body status has a significant impact. Elastin is a protein that maintains the elasticity of connective tissue, allowing many tissues in the body to regain their shape after being stretched or contracted. With the increase of age and the external damage received, the content of elastin in the skin will gradually decrease. When elastin is lost, even if there is still a certain proportion of collagen, the skin will also lose its elasticity and cause sagging and wrinkles.

In addition, when elastin and/or collagen are lost, it will indirectly lead to thinning of the skin, making the epidermis closer to the blood vessels in the dermis, making the skin appear red overall and affecting its appearance. Skin in this state is also more sensitive than healthy skin, and is more prone to other skin symptoms such as inflammation.

In view of this, there is a need to research or develop a composition that can reduce the loss of collagen and/or elastin to slow down skin aging, so as to maintain or improve the appearance of the skin.

Accordingly, one object of this case is to provide a use of tartary buckwheat seed coat extract for preparing a composition for reducing elastin loss in vivo, wherein the tartary buckwheat seed coat extract is obtained by extracting tartary buckwheat seed coat with water.

According to some embodiments of the present application, the composition is used to reduce the concentration of desmosin in blood.

According to some embodiments of the present application, the composition is administered to an individual in need daily for at least 2 weeks, wherein the composition contains 5 grams to 10 grams of tartary buckwheat seed coat extract.

According to some embodiments of the present application, the composition is further prepared into a pharmaceutical composition, a food composition, or a health food composition.

Figure 1 shows that tartary buckwheat seed coat extract reduces the production of collagen end-glycation products.

Figure 2 shows that tartary buckwheat seed coat extract reduces the content of Desmosine in the blood of the subjects. **p-value<0.01; ***p-value<0.001.

Fig. 3 shows the improvement rate of the skin texture of the subject's facial skin after applying tartary buckwheat seed coat extract for four consecutive weeks.

Figures 4 to 6 are real photos of the subjects' facial skin at the 0th week, the 2nd week, and the 4th week (the texture part is marked by software).

Fig. 7 shows the changes in the relative amount of red spots on the skin of the subject after applying the tartary buckwheat seed coat extract to the facial skin for four consecutive weeks. *p-value < 0.05.

Figures 8 to 10 are the images taken by the tester at the 0th week, the 2nd week, and the 4th week of the subject's facial skin.

Some specific implementation aspects of this case will be described below. Without departing from the spirit of this case, this case can still be practiced in many different forms, and the scope of protection should not be limited to the conditions specifically stated in the specification.

In this case, Excel software was used for statistical analysis. Data are presented as mean ± standard deviation (SD), and differences among groups were analyzed by student's t - test.

Common and edible species of buckwheat ( Fagopyrum ) include, for example, common buckwheat ( Fagopyrum esculentum , also known as buckwheat) and tartary buckwheat ( Fagopyrum tataricum , also known as Tartar buckwheat). Common buckwheat is commonly grown on all continents, while tartary buckwheat, which originates from eastern Tibet or northwestern Yunnan, is only grown in Asia, Europe and North America. Compared with rice, buckwheat has a shorter growth period, can grow in poor acidic soil, does not need too much nutrients and nitrogen, but needs sufficient water, and can bloom in cooler climates. Therefore, in addition to being used as food, it can be used as replanting after floods, and can also be used as green manure, feed or cover plants to prevent soil erosion.

Generally speaking, the seeds of plants are generally composed of three parts: seed coat, embryo and endosperm, and the seeds of buckwheat are also the same. In this case, it was found that the seed coat extract of tartary buckwheat in the buckwheat genus has the effect of reducing the loss rate of elastin and collagen in the body, and can be used to prepare a composition for reducing the loss of elastin and collagen in the body. And because tartary buckwheat seed coat extract has the aforementioned effects, it can be used to improve skin wrinkles, skin redness and other skin problems caused by the loss of elastin and collagen, thereby improving the overall appearance of the skin.

The term "tartary buckwheat seed coat extract" used herein is obtained by extracting tartary buckwheat seed coats with a solvent (such as water) for a specific time and temperature.

The tartary buckwheat seed coat extract in this case can be used to prepare a composition for reducing the loss of elastin in the body or a composition for inhibiting saccharification of collagen, and the composition can be a pharmaceutical composition, a food composition, or a health food composition. Alternatively, the pharmaceutical composition of this case may be included in food or skin care products. Or the tartary buckwheat seed coat extract of this case can be further prepared into a pharmaceutical composition, a skin care product composition, a food composition, or a health food composition.

Pharmaceutical compositions can be manufactured into dosage forms suitable for parenterally or topically administered by techniques well known to those skilled in the art, including, but not limited to: injections [eg, sterile aqueous solution or dispersion], sterile powder, external preparation, and the like.

The pharmaceutical composition may further contain a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may contain one or more agents selected from the group consisting of: solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes and the like. The selection and quantity of these reagents can be selected by those skilled in the art depending on the situation.

In some embodiments, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS) ), aqueous solution containing alcohol (aqueous solution containing alcohol), and combinations thereof.

In some embodiments, the pharmaceutical composition may be administered by a parenteral route selected from the group consisting of subcutaneous injection, intraepidermal injection , intradermal injection and intralesional injection.

In some embodiments, the pharmaceutical composition may be formulated as an external formulation suitable for topical application to the skin using techniques well known to those skilled in the art. preparation), which includes, but is not limited to: emulsion, gel, ointment, cream, patch, liniment, powder, gas aerosol, spray, lotion, serum, paste, foam, drop, suspension, salve, and bandage.

In some embodiments, topical formulations are prepared by mixing the pharmaceutical composition with a base well known to those skilled in the art.

In some embodiments, the substrate may contain one or more additives (additives) selected from the group consisting of water, alcohols, glycols, hydrocarbons [such as petroleum jelly (petroleum, jelly) ) and white petroleum jelly (white petrolatum,)], wax (wax) [such as paraffin (paraffin) and yellow wax (yellow wax)], preservatives (preserving agents), antioxidants (antioxidants), surfactants (surfactants), absorption Absorption enhancers, stabilizing agents, gelling agents (such as carbopol® 974P, microcrystalline cellulose, and carboxymethylcellulose )], active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers , occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants And propellants (propellants), etc. The selection and quantity of these additives are subject to familiarity with It is within the scope of the professionalism and routine skills of the people who use this technology.

In some embodiments, the skin care product may contain an acceptable adjuvant that is widely used in the manufacturing technology of skin care products. For example, the acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, dyes Coloring agents, thickening agents, fillers, fragrances and odor absorbers. The selection and quantity of these reagents can be appropriately adjusted according to actual needs.

In some embodiments, skin care products can be manufactured into a form suitable for skin care or makeup using techniques well known to those skilled in the art, including, but not limited to: aqueous solutions, Aqueous-alcohol solution or oily solution, emulsion in the form of oil-in-water type, water-in-oil type or compound type, Gels, ointments, creams, masks, patches, packs, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, dispersions, drops, mousses (mousse), sunblock, tonic water, foundation, makeup remover products, soap and other body cleansing products.

In some embodiments, skin care products can also be used in combination with one or more known active external use agents selected from the following: whitening agents (such as tretinoin, pediatric Catechin, kojic acid, arbutin and vitamin C], humectants, bactericides, UV absorbers (ultraviolet absorbers), plant extracts (such as aloe extract), skin nutrients, anesthetics, anti-acne agents, antipruritics ( antipruritics), analgesics, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antiperspirants (antipsoriatic agents), anti-aging antiaging agents, antiwrinkle agents, antiseborrheic agents, wound-healing agents, corticosteroids, and hormones. The selection and amount of these topical agents are within the professionalism and routine skill of those skilled in the art.

In some embodiments, the pharmaceutical composition can be used as a food additive, which can be added during the preparation of raw materials or during the production of food by known methods, and can be combined with any edible material Prepared as a food product intended for consumption by humans and non-human animals.

In some embodiments, the types of food include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

The extraction method of the tartary buckwheat seed coat extract and the detection method related to the buckwheat seed coat extract will be described in detail below.

[Example 1] The preparation method of tartary buckwheat seed coat extract

In this embodiment, the preparation steps of tartary buckwheat seed coat extract are as follows:

1. Crush the seed coat of tartary buckwheat (origin: Taiwan) and sieve it through a 10-mesh sieve to remove oversized particles to obtain tartary buckwheat seed coat powder. In other embodiments, the mesh size of the screen can be 8 mesh or 12 mesh.

2. Mix water and buckwheat seed coat powder at a weight ratio of 11:1, and soak at 85±5° C. for about 1.0 hour to extract the tartary buckwheat seed coat powder to form a first extract containing solids. In some other embodiments, the weight ratio of the solvent to the tartary buckwheat seed coat powder may also be 10:1 to 12:1, the temperature during soaking may be 80°C-90°C, and the extraction time may be 0.5 to 3.0 hours. If the solvent is too small or the extraction time is too short, the extraction efficiency will be significantly reduced; if the extraction time is too long, the active ingredients in the extract may be degraded.

3. Cool the first extract to room temperature (25°C-30°C).

4. Centrifuge the cooled first extract with solids in a centrifuge (brand/model: Thermo Scientific Heraeus Fresco 17) for about 10 minutes, and take the supernatant as the first extraction supernatant.

5. Filter the first extraction supernatant with a 400-mesh filter to remove fine solids.

6. Concentrate the filtered first extraction supernatant to the Brix value of the solution (Degrees Brix ) is 2.0 ± 0.5, the concentration is stopped to obtain the tartary buckwheat seed coat extract. In other embodiments, concentration under reduced pressure can be carried out at 45°C-70°C.

[Example 2] Collagen glycosylation detection

As mentioned previously, one of the factors in the breakdown of collagen chains is the glycation of collagen. After the protein is glycosylated, a variety of advanced glycation end products (AGEs) will be generated, which are irreducible substances and will change and affect normal function of the protein. Therefore, if the glycosylation of collagen can be inhibited, the collagen chain can be prevented from breaking, and the loss rate of collagen can be effectively reduced. In order to confirm whether the tartary buckwheat seed coat extract has the effect of inhibiting the glycation of collagen, the amount of glycation of collagen was tested as follows.

Drug preparation:

1. Use the tartary buckwheat seed coat extract obtained in previous example 1, with phosphate-buffered saline (Phosphate-Buffered Saline, PBS, NaH ₂ PO ₃ (Honeywell, #04269), Na ₂ HPO ₄ (Sigma, #V900061) and water, its pH value is 7.4) as a solvent, the concentration of 0.1mg/mL, 0.5mg/mL, 1.0mg/mL, and 5mg/mL tartary buckwheat seed coat extract solution.

2. Using the aforementioned phosphate-buffered saline at a concentration of 200 mM and collagen powder (collagen was purchased from Rousselot, model #P2000HD), a collagen solution with a concentration of 60 mg/mL was prepared. In addition, sodium azide was added to the collagen solution, so that the collagen solution contained 0.06 wt% sodium azide. (Sodium azide was purchased from Sigma, #S2002)

3. Using the aforementioned 200 mM phosphate-buffered saline and fructose (fructose was purchased from Sigma, #F0127), a fructose solution with a concentration of 1.5 M was prepared.

Collagen glycation test:

1. Mix 0.2 mL of tartary buckwheat seed coat extract solutions of the aforementioned four concentrations with 0.2 mL of the aforementioned collagen solution and 0.2 mL of the aforementioned fructose solution respectively to prepare 4 groups of sample solutions.

2. Mix 0.2 mL of deionized water with 0.2 mL of the aforementioned collagen solution and 0.2 mL of the aforementioned fructose solution to prepare a blank solution as a control group.

3. Measure the fluorescence of the 4 groups of sample solutions and the blank solution using a spectrofluorometer (manufacturer/model: BioTek FLx 800) before the collagen saccharification reaction. Light intensity (excitation wavelength 360nm, emission wavelength 460nm).

4. Place the 4 groups of sample solutions and the blank solution at 50° C. for 24 hours to react for saccharification of collagen.

5. Measure the fluorescence intensity of the four groups of sample solutions and the blank solution after the reaction with a spectrofluorometer (excitation wavelength 360nm, emission wavelength 460nm).

6. Calculate the relative production rate of protein glycation end products according to the following formula: [( _{after the fluorescence intensity of the sample is reacted} - _{before the fluorescence intensity of the sample is reacted} )/( _{after the fluorescence intensity of the control group is reacted} - _{before the fluorescence intensity of the control group is reacted} )]× 100%

The relative production rate of protein glycation end products of each sample relative to the control group is shown in Fig. 1 .

As shown in Figure 1, the tartary buckwheat seed coat extracts of various concentrations (0.1mg/mL, 0.5mg/mL, 1.0mg/mL, and 5mg/mL) all have the effect of inhibiting the production of glycation end products of collagen, and compared with The concentration of tartary buckwheat seed coat extract has correlation. In 0.1mg/mL, 0.5mg/mL, 1.0mg/mL, and 5mg/mL tartary buckwheat seed coat extract sample solutions, the relative production rates of protein glycation end products were only 92%, 48%, and 31% of those in the control group, respectively. %, 19%. It shows that tartary buckwheat seed coat extract can indeed inhibit the glycation reaction of collagen, reduce the formation of glycation end products, and then reduce the loss of collagen in the body to achieve the effect of slowing down skin aging.

[Example 3] Desmosin content detection in vivo

There is a special component in elastin - desmosine (Desmosine), which is a cross-linked body formed by three aldehyde lysine side chains and one lysine side chain, which can make more than two of the collagen molecules Covalent crosslinks occur between polypeptide chains. The special structure and properties of desmosin endow elastin with elasticity and connectivity, so that elastin can act as a "lock" between collagens in the skin.

When elastin is decomposed, the desmosin contained in it is released into the blood, which increases the concentration of desmosin in the blood; conversely, if the decomposed amount of elastin decreases, the desmosin concentration in the blood will decrease. Therefore, the concentration of desmosin in the blood can be used as an indicator of the amount of elastin breakdown. In order to confirm whether the tartary buckwheat seed coat extract can inhibit the decomposition of elastin, the desmosin content in the body was detected as follows.

The tartary buckwheat seed coat extract prepared in Example 1 and water are formulated into a tartary buckwheat seed coat extract drink. And ask 8 test subjects (general women aged 30-55) to drink the prepared tartary buckwheat seed coat extract drink with a daily intake dose of 5g of tartary buckwheat seed coat extract for a total of 4 weeks. And before the start of the experiment (the 0th week, not yet drinking), the 2nd week and the 4th week, blood was collected from the subjects, and the Desmosine ELISA Kit (CUSABIO, CSB-E12871h) was used to measure the concentration of desmosin in the blood ( The drugs used in the following steps, unless otherwise specified, are provided by Desmosine ELISA Kit).

Desmosin concentration in the blood is measured by:

1. Separate the serum sample from the collected whole blood sample.

2. Pipette 100 μl of each serum sample, a set of serially diluted desmosin standards with known concentrations (used to make a calibration line), and a blank solution into a 96-well plate dedicated to the Desmosine ELISA Kit .

3. Place the aforementioned 96-well plate in an oven at 37°C for 2 hours to react.

4. Remove the liquid from the 96-well plate.

5. Pipette 100 μl of Biotin-antibody (1x) into each well of the 96-well plate with a micropipette.

6. Put the 96-well plate back into the oven at 37°C to react for 1 hour.

7. Remove the liquid from the 96-well plate.

8. Wash the 96-well plate 3 times with Wash buffer (200mM phosphate-buffered saline, pH 7.4), each time as follows:

(1) Add 200 μl of Wash buffer to each well.

(2) Let stand for 2 minutes.

(3) Remove the liquid in the 96-well plate.

9. Pipet 100 μl of HRP-avidin (1x) into each well of the 96-well plate with a micropipette.

10. Put the 96-well plate back into the oven at 37°C for 1 hour.

11. Wash the 96-well plate 3 times with Wash buffer, that is, repeat step 8. Three times.

12. Use a micropipette to pipette 90 μl of TMB (3,3',5,5'-Tetramethylbenzidine) Substrate into each well of the 96-well plate.

13. Put the 96-well plate back into the oven at 37°C for 15 minutes.

14. Pipette 50 μl of Stop Solution into each well of the 96-well plate with a micropipette.

15. Use ELISA reader (BioTek, FLx 800) to measure the absorbance of wavelength 450nm and 540nm.

16. Use Excel to establish a standard curve, and correct the absorbance value at 450nm with the absorbance value at 540nm (use the absorbance value at 450nm and 540nm to detect the absorbance value of each well, then subtract the absorbance value at 450nm from the absorbance value at 540nm), and Carry out quantitative conversion of samples.

17. Use the Student-t test in Excel to analyze the samples statistically.

Taking the concentration at week 0 as 100%, the detection results of the relative concentration of desmosin in the blood are shown in Figure 2. In the figure, ** means p<0.01 relative to the reference group; *** means relative p<0.001 for the baseline group.

As shown in Figure 2, after the subject continued to drink the tartary buckwheat seed coat extract for 2 weeks, the content of desmosin in the body decreased significantly, only about 78.1% of the level before intake. After 4 weeks of continuous drinking of tartary buckwheat seed coat extract, the content of desmosin in the body can be reduced to about 65.7% of that before intake. It shows that after subjects ingest tartary buckwheat seed coat extract, the amount of elastin damage in the body is greatly reduced, and as the intake period increases, the reduction effect can be more obvious. Therefore, it is confirmed that tartary buckwheat seed coat extract can effectively slow down the loss of elastin in the body.

[Example 4] Skin texture improvement test / relative amount of skin red spots detection

The tartary buckwheat seed coat extract prepared in Example 1 and water were formulated into a tartary buckwheat seed coat extract drink to provide a suitable effective dose for the subject. In this example, 8 subjects (general women aged 30-55) were asked to drink the prepared tartary buckwheat seed coat extract drink at a daily intake of 5g of tartary buckwheat seed coat extract for 4 weeks. And before the start of the experiment (the 0th week, not yet drinking), the 2nd week and the 4th week, the cleansed facial skin of the subjects was tested with a full-face skin quality detector (7th Generation VISIA Complexion Analysis System; Canfield, USA ) to measure the skin texture of the whole face and the number and area of red spots of these test subjects.

The detection results of the skin texture and the actual changes in appearance are shown in Figure 3 and Figures 4 to 6, respectively. Figure 3 takes the skin roughness of the subject before taking the tartary buckwheat seed coat extract as 100%, and records the relative skin roughness of the subject after taking the tartary buckwheat seed coat extract for 2 weeks and 4 weeks. Figures 4 to 6 are real photos of the facial skin of one of the subjects at week 0, week 2, and week 4, in which the texture part is marked by software processing.

As shown in Figure 3, when the subject ingested the tartary buckwheat seed coat extract for 2 weeks, the The improvement of skin texture was observed, and after 4 weeks of continuous intake, the relative roughness of skin texture dropped to 87.6%. It can also be found from the real photos in Figure 4 to Figure 6 that as the period of intake of tartary buckwheat seed coat extract increases, the fine lines on the skin are significantly reduced or lightened.

The results of the number and area of red spots on the subjects' skin and the actual changes in appearance are shown in Figure 7 and Figures 8 to 10, respectively. Figure 7 shows that before the subject ingested the tartary buckwheat seed coat extract, the value measured by the instrument according to the number and area of red spots was 100%, and the subject continued to ingest the tartary buckwheat seed coat extract for 2 weeks and 4 weeks. The value measured according to the number/area of red spots. Figures 8 to 10 are images of the facial skin of one of the subjects taken by the detector at week 0, week 2, and week 4.

As shown in Figure 7, when the subject ingested the tartary buckwheat seed coat extract for 2 weeks, the reduction in the number/area of red spots could be observed, and after 4 weeks of continuous intake, the number/area of red spots on the facial skin was Dropped to 95.1% of the original. It can also be found in the real images of Fig. 8 to Fig. 10 that the number/area of red spots on the skin is significantly reduced or lightened as the period of ingestion of the tartary buckwheat seed coat extract increases.

From the results of the skin texture improvement test and the relative amount of red spots on the skin, as well as the other experiments mentioned above, it can be known that tartary buckwheat seed coat extract can inhibit collagen glycation and reduce the concentration of desmosin in the blood, thereby inhibiting the loss of collagen in the body and inhibiting the elastin in the body loss and can be used to slow down skin aging and/or improve skin appearance.

The above descriptions are illustrative only, not restrictive. Any equivalent modification or change made without departing from the spirit and scope of the present invention shall be included in the scope of the appended patent application.

Claims (3)

A tartary buckwheat seed coat extract is used to prepare a composition for reducing the loss of elastin in the body, wherein the tartary buckwheat seed coat extract is obtained by extracting a tartary buckwheat seed coat with water, and the composition is used to reduce desmosin concentration in blood , the weight ratio of the water to the tartary buckwheat seed coat is 10:1 to 12:1. The use as described in claim 1, wherein the composition is a pharmaceutical composition. The use as described in claim 1, wherein the composition is further prepared into a skin care composition, a food composition, or a health food composition.

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