TW201634043A - Aqueous extract liquid of water calltrop shell, method of producing the same and applications thereof - Google Patents

Abstract

The present invention is related to an aqueous extract liquid of red water calltrop (Trapa bicornis var. taiwanensis (Nakai) shell, a method of producing the same and applications thereof. The red water calltrop shells are extracted by a thermal reflux extraction in water at pH 7.0 under 100 DEG C for no more than 3 hours, followed by removal of insolubles of the crude extract of the red water calltrop shells, thereby obtaining the aqueous extract liquid of red water calltrop shells. The aqueous extract liquid of red water calltrop shells can inhibit activities of matrix metalloproteinase-2 (MMP-2) and MMP-9, so as to be applied on a composition for external use on the skin or a medicinal composition.

Description

Water chestnut shell water extract, manufacturing method and application thereof

The invention relates to a plant extract and a preparation method thereof, in particular to an aqueous extract of water chestnut shell which inhibits the activity of matrix metalloproteinase, a preparation method and application thereof.

Water chestnut is an important cash crop in southern Taiwan. In the southern part of Taiwan, there are two common varieties of water chestnuts. According to the number of sharp corners of the fruit, they can be divided into double-angled diamonds (also known as two-horned diamonds, wild diamonds) and four-angled diamonds (also known as four-pointed diamonds). In the double-angled ridge, Trapa bicornis and Trapa bicornis var. taiwanensis (Nakai) are the more common varieties. Taiwan Hung Ling Ling, also known as Lythraceae (Lythraceae) Link subfamily (Trapaceae) Trapa (Trapa) of floating annual herb. Currently, there is a large area planted in the Guantian area of Tainan.

It is rich in starch, fat, protein, multivitamins and minerals. It is known to have the effects of diuretic through milk, anti-thirst, clearing heat and qi and hangover. In the production process of the water chestnut kernel, the water chestnut shell is a by-product of the water chestnut, which was widely used as agricultural waste in the past.

In recent years, most studies on the extract of the water chestnut shell have extracted the water chestnut shell with an aqueous solution of alcohol or alcohol, and the biological activity of the obtained water chestnut shell extract such as antioxidation, antibacterial, lipase inhibiting lipase, and angiotensin-converting enzyme (ACE) inhibition effect and liver protection. However, it takes a long time to extract the water chestnut shell with an alcohol or alcohol aqueous solution, and the extracted components are complicated. If further purification of specific components for a specific use is required, the process steps are cumbersome, the process time is long, and the cost is high.

In view of this, it is urgent to provide a method for reusing the water chest shell to simplify the conventional process steps and shorten the process time to improve the economic value of the water chest shell.

Therefore, one aspect of the present invention provides a method for producing a water extract of a water chestnut shell, which extracts a shell of Trapa bicornis var. taiwanensis (Nakai) by a single-step hot reflux extraction step and a solid-liquid separation step. A water extract of the water chestnut shell is obtained.

Next, another aspect of the present invention provides a water extract of a water chestnut shell which is obtained by the above method.

Furthermore, another aspect of the present invention provides a method for inhibiting a matrix metalloproteinase in vitro, which comprises treating a plant cell in vitro with a composition comprising the above-mentioned water extract of water chestnut shell to inhibit a second type matrix of animal cells. Activity of metalloproteinase-2 (MMP-2) and/or ninth matrix metalloproteinase (MMP-9).

According to the above aspect of the invention, a water chestnut shell extraction is proposed. The method of manufacturing the liquid. In one embodiment, the method comprises performing a one-step hot reflux extraction step of treating the red cube shell with water at 100 ° C and pH 7.0 for no more than 3 hours to obtain a crude extract. Next, a solid-liquid separation step is performed to remove the insoluble matter of the above crude extract, thereby obtaining a water extract of the water chestnut shell.

According to an embodiment of the invention, the weight-to-volume ratio (w/v; g/mL) of the red cube shell and the water may be, for example, 1:5 to 1:20, but 1:5 to 1:10. Preferably.

According to an embodiment of the present invention, before the single-step hot reflux extraction step, the red horn shell may be selectively dried, and the dried red horn shell is ground to obtain a water chestnut dry powder.

According to another aspect of the present invention, there is provided a water extract of water chestnut shell which is obtained by the above method. In one example, the resulting water extract of the water chestnut shell has activity to inhibit matrix metalloproteinase-2 (MMP-2) and/or matrix metalloproteinase (MMP-9).

According to still another aspect of the present invention, a method for inhibiting a matrix metalloproteinase in vitro is provided, which comprises treating a plant cell in vitro with a composition comprising the above-mentioned water extract of a water chestnut shell to inhibit a second type of matrix metal of the animal cell. Activity of protease (MMP-2) and/or ninth matrix metalloproteinase (MMP-9).

The water chestnut water extract of the present invention and the method for producing the same are characterized in that the red water chestnut shell is extracted by a single-step hot reflux extraction step and a solid-liquid separation step, and the obtained water extract of the water chestnut shell has been inhibited by an in vitro experiment. The activity of the second type matrix metalloproteinase and/or the ninth type matrix metalloproteinase can be applied to a skin external composition or a pharmaceutical composition.

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; A bar graph of cell viability.

[Fig. 2 to Fig. 4] Fig. 2 to Fig. 4 are diagrams showing gelatin zymogram analysis of the fibroblasts cultured at different concentrations using different concentrations of water chestnut aqueous extracts in several embodiments of the present invention.

[Fig. 5 to Fig. 7] Fig. 5 to Fig. 7 are diagrams showing the analysis of hyaluronic acid spectroscopy electrophoresis after reacting different concentrations of water chestnut aqueous extracts with hyaluronidase at different times in several embodiments of the present invention.

As described above, the present invention provides a water chestnut shell water extract and a method for producing the same, which utilize a single-step hot reflux extraction step and a solid-liquid separation step to extract a water chestnut shell.

The term "water chestnut shell water extract" as used herein refers to a water chestnut shell that has been discarded by the Taiwan native species, Trapa bicornis var. taiwanensis (Nakai), and is subjected to water in a short time. The crude extract of the hot reflux extraction step is removed after the insoluble matter is removed. In one embodiment, the red water chest shell used in the present invention is a harvester of the Tainan Guantian production area, and substantially does not contain the varieties other than the above. Secondly, the present invention uses only the red horn shell for extraction, and does not substantially contain the horn kernel. In another embodiment, the red cube shell used in the present invention may optionally be subjected to conventional drying, grinding, and the like. In still another embodiment, the water content of the red horn shell used in the present invention may be, for example, 3.0% by weight to 10.0% by weight.

It is stated that if a variety other than the above-mentioned production area or a water chestnut shell having a water content outside the above range is used, the water extract of the water chestnut shell obtained by subsequent extraction cannot reach the second type substrate which is required to inhibit animal cells in vitro. The activity of metalloproteinases and/or ninth type matrix metalloproteinases is also low cytotoxic.

The term "low cytotoxicity" as used herein refers to a situation in which the cell viability is still higher than 60% after 24 to 48 hours of culture of the water chestnut water extract prepared from the following water cells.

The aforementioned water extract of the water chestnut shell can be obtained by the following method. In an embodiment, the method may first provide a red horn shell, such as a harvester in the Tainan Guantian production area. In one example, the water content of the aforementioned red water chest shell may be, for example, 3.0% by weight to 10.0% by weight, and the water content of the water chest shell may be dried by a conventional drying method, such as natural air drying, or in an oven of, for example, about 50 ° C. Dry inside to adjust to the desired water content.

Next, a one-step hot reflux extraction step was carried out, and the red water chestnut shell was treated with water at 100 ° C and pH 7.0 for no more than 3 hours to obtain a crude extract. In one embodiment, the single-step hot reflux extraction step has a shorter treatment time of not more than 3 hours, preferably 1 hour to 3 hours. If the processing time of the single-step hot reflux extraction step is less than one hour, then the subsequent The content of the active ingredient of the water extract of the water chestnut shell will be insufficient, and if the treatment time of the single-step hot reflux extraction step exceeds 3 hours, the subsequent water extract of the water chestnut shell will contain other unwanted sub-components, which cannot meet the requirements. The activity of the second type matrix metalloproteinase and/or the ninth type matrix metalloproteinase which inhibits animal cells in vitro is also low in cytotoxicity.

In an example, the weight-to-volume ratio (w/v; g/mL) of the above-mentioned red cube shell to water may be, for example, 1:5 to 1:20, but preferably 1:5 to 1:10, and 1:5 to 1:8 is better.

Before the single-step hot reflux extraction step, the above-mentioned red water chest shell may be selectively dried by a conventional drying step, and then processed into a powder to carry out a subsequent single-step hot reflux extraction step.

It should be added that the above-mentioned single-step hot reflux extraction step only needs to be carried out by using a conventional heat reflux device, and it is not necessary to incorporate other auxiliary methods (for example, stirring, ultrasonic vibration).

After the single-step hot reflux extraction step, the crude extract is subjected to a solid-liquid separation step to remove the insoluble matter of the crude extract, thereby obtaining a water extract of the water chestnut shell, wherein the solid content of the water extract of the water chestnut shell is obtained. It is generally from 4.1% by weight to 6.5% by weight, and preferably from 4.5% by weight to 6.0% by weight.

It should be additionally noted that the solid-liquid separation and drying steps and the like may be carried out by any conventional means, and are not limited to the above, and are well known to those of ordinary skill in the art to which the present invention pertains, so that they are not separately Narration.

In one embodiment, the water extraction is carried out using a water chestnut shell containing the above The composition of the liquid inhibits the activity of the second type matrix metalloproteinase and/or the matrix metalloproteinase type 9 in vitro and has low cytotoxicity when the biological material is treated in vitro. In one example, the biological material may be, for example, an animal cell, an enzyme or a protein, the source of the animal cell may be, for example, a mouse, and the animal cell may be, for example, a fibroblast. In another example, the water extract of the water chestnut shell can indeed inhibit the activity of the second type matrix metalloproteinase and/or the matrix metalloproteinase type 9 in vitro and has low cytotoxicity.

The second type of matrix metalloproteinase (MMP-2) and the ninth type of matrix metalloproteinase (MMP-9) are gelatinases, which are also part of the matrix metalloproteinases (MMPs) family, which have collagen and cells. The ability of an extracelluar matrix (ECM). MMP-2 is abundantly present in normal fibroblasts, endothelial cells, epithelial cells and transformed cells, while MMP-9 is present in white blood cells and transfected cells. The invention utilizes gelatinase electrophoresis analysis method to prove that the water extract of the water chestnut shell can inhibit the enzyme activity of MMP-2 and MMP-9 and has low cytotoxicity.

It is explained that, since the above-mentioned crude extract of the water chestnut shell and the water extract of the water chestnut shell have excellent biological activity and low cytotoxicity, they can be applied to prepare various skin external compositions or pharmaceutical compositions, for example, Cosmetics, lotions, masks, medicated soaps, body washes, ointments, sprays, etc., but the above applications are merely illustrative, and the invention is not limited thereto.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Preparation of water chestnut shell water extract

Example

First, as shown in Table 1, the water chestnut shells that were harvested during the processing of the red diamond harvested in the Tainan Guantian production area were collected, and after natural air drying, the water content of the water chestnut shell was controlled to be 3 to 10 weight percent. After the red horn shell was ground into a powder, 20 g (g) of the red stalk shell powder and 160 mL of water were placed in a round bottom flask of a hot reflux apparatus, and subjected to hot reflux extraction at 100 ° C and water of pH 7.0. After 3 hours, the filtrate was subjected to preliminary filtration, suction filtration, and centrifugation (8000 rpm, 20 minutes) to carry out a solid-liquid separation step to remove the insoluble matter of the above crude extract, thereby obtaining a water extract of the water chestnut shell and storing it in The content of the solid matter of the water extract of the water chestnut shell obtained in the example was 5.7 weight%.

Evaluation method

The effect of inhibiting matrix metalloproteinase by the water extract of the water chestnut shell of the examples was evaluated by several tests. The concentration of the water extract of the water chestnut shell was 100% of the stock solution of the water extract of the water chestnut shell obtained in the examples.

1. Thawing and activation of cells

3T3 cell T3 mouse embryonic fibroblasts (purchased from the Bioresource Conservation and Research Center of the Food Industry Research Institute, registration number BCRC 60071; ATCC accession number CCL-92) frozen vials (containing antifreeze DMSO) The liquid nitrogen drum was taken out and quickly placed in a 37 ° C water bath. After it was fully thawed, the cell suspension was added to a petri dish containing fresh culture solution and placed in a cell culture incubator at 37 ° C (containing 5.0% CO). ₂₎ for 30 minutes. After the cells were pasted, the medium containing DMSO was removed, and 4 mL of fresh medium was added to continue the culture for 2 to 3 days. The above fresh culture solution contains Dulbecco's Modified Eagle's Medium (DMEM; Biowest, USA), 10 volume percent of newborn calf serum (NCS; Gibco, USA) and 1 volume. Percentage of three-in-one antibiotics (containing penicillin, streptomycin and amphotericin, penicillin/streptomycin/amphotericin; pen-strep-ampho; Gibco, USA).

When the cells were grown to about 90% fullness, remove the culture solution, add 1 mL of IX PBS buffer (Biowest, USA) to rinse the cell surface, remove the PBS buffer, and add 1 mL of trypsin (trypsin-EDTA). ; Gibco, USA) causes the cells to fall off. Then, the desired number of cells were added to 4 mL of the above fresh medium, and cultured in a 37 ° C cell culture incubator (containing 5.0% CO ₂ ) for 2 to 3 days, followed by subculture.

Subculture may be carried out using any conventional means, which is well known to those of ordinary skill in the art to which the invention pertains, and will not be further described.

2. Evaluation of cytotoxicity

This evaluation is based on MTT[3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] Test for cytotoxicity. MTT is a tetrazolium salt, which is decomposed by intracellular granule gland succinate dehydrogenase to produce blue formazan crystals from transparent colorless. Dead cells can not produce blue crystals because the intracellular granule dehydrogenase loses activity, so the survival of cells can be judged by the amount of blue crystals.

Prior to the assay, 100 μL of 3T3 cells (cell density 5×10 ⁴ cell/well) were inoculated into 96 well microplates by aseptic technique and placed in a cell culture incubator at 37 ° C to maintain humidity. (containing 5.0% CO ₂ ) for 24 hours. Next, the culture solution was removed, and the cell surface was washed with 100 μL/well of PBS buffer and removed, and then 100 μL/well of the cell culture medium and the water extract containing different concentrations of the water chestnut shell (experimental group) or without the water chestnut were added. Shell water extract (control group), cultured for 24, 48 hours. Then remove the solution, add 100 μL/well of 0.5 mg/mL MTT solution, incubate in a cell culture incubator (containing 5.0% CO ₂ ) maintained at 37 ° C for 3 hours, remove the solution, and add 100 μL/well of DMSO to dissolve. Hyperthyroidism crystallizes. The same volume of DMSO was added to the wells of the blank group (without cells). Thereafter, the absorbance was detected at a wavelength of 540 nm by an ELISA reader, and the absorbance at 540 nm of the control group was taken as 100%, and the results are shown in FIG.

Please refer to FIG. 1 , which is a bar graph showing the cell survival rate of fibroblasts cultured at different times using different concentrations of water chestnut shell water extract according to an embodiment of the present invention. In Figure 1, the number of samples per set of data is at least three (n ≧ 3).

As can be seen from the results of Fig. 1, 3T3 fibroblasts were cultured using 0.25%, 0.50%, 1.00%, 2.50% of the water extract of the water chestnut shell of the examples. After an hour, cell viability was still above 60%. After incubating 3T3 fibroblasts with 0.25%, 0.50%, and 1.00% of the water extract of the water chestnut shell of the example for 48 hours, the cell survival rate was still higher than 60%. The water extract of the water chestnut shell is low in cytotoxicity.

2. To evaluate the inhibitory effect of water extract of water chestnut shell on matrix metalloproteinase

The second type of matrix metalloproteinase (MMP-2) and the ninth type of matrix metalloproteinase (MMP-9) are gelatinases, which are also part of the matrix metalloproteinases (MMPs) family, which have cleavage of collagen and cells. The ability of an extracelluar matrix (ECM). MMP-2 is abundantly present in normal fibroblasts, endothelial cells, epithelial cells and transformed cells, while MMP-9 is present in white blood cells and transformed cells. Using gelatinase electrophoresis analysis, MMP-2 (MW 64kDa) and MMP-9 (MW 86kDa), original MMP-9 (pro-MMP-9, MW 92kDa) can be isolated and the enzyme activities of the three can be detected simultaneously. . The water extract of the water chestnut shell of the example was cultured with the cells, and the gelatinase electrophoresis analysis method was used to evaluate the inhibitory effect of the water extract of the water chestnut shell on the matrix metalloproteinase.

2.1 Treatment of cells with the water extract of the water chestnut shell of the examples

After cultured for 24 hours or 48 hours, 3T3 fibroblasts were treated with different concentrations (0.25%, 0.50%, 1.00%, 2.5%, 5.00%, 10.0%) of the water extract of the water chestnut shell (experimental group) or An equal volume of sterile water (Control Group, C) cell culture medium was incubated at 37 ° C for 0 hours, 24 hours or 48 hours. Then, take the cell culture supernatant of each group and perform gelatin. The gelatin zymography electrophoresis analysis was carried out to evaluate the inhibitory effect of MMP-2 and MMP-9 on the water extract of the water chestnut shell of the examples.

2.2 Electrophoresis analysis of gelatin zymogram

When making the gel, first prepare 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing gelatin-type A (Sigma, USA) at a final concentration of 1.0%. Separating gel solution, injected into the cast rubber model (gel caster, BIO-RAD; spacer thickness 0.75mm), after about eight minutes, slowly add deionized water to the upper layer, and stand overnight to be colloidal solidification. Then, prepare a stacking gel, first remove the deionized water above the separation colloid, and then slowly add a 4% SDS-PAGE coke collecting colloid solution, then insert the electrophoresis tooth comb and let stand at room temperature. To colloid condensation. The formulation of each of the above colloids is well known to those of ordinary skill in the art and will not be described in detail.

After the colloid was completely solidified, the electrophoresis comb was pulled out, and the cast gelatin-SDS-PAGE electrophoresis film was placed in an electrophoresis tank containing 1X tank buffer (containing 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3). Then, the sample to be tested is mixed with an equal volume of 2 times concentrated dyeing agent [2X loading dye, a mixed solution containing Bromophenol Blue (Amresco, USA) and glycerol (JT Baker, USA)], A sample of the coke-colloid colloid was injected with 20 μL of the mixed sample of the dye, and electrophoresis was carried out at a voltage of 80 V. When the trace dye on the film left the stacked colloid, the voltage was raised to 100 V for electrophoresis. After the dye is moved to the bottom of the separation gel for about 40 minutes, the electrophoresis is finished, and the gelatin is taken out. -SDS-PAGE electrophoresis film.

Thereafter, the gelatin-SDS-PAGE electrophoresis film was washed with a washing buffer (containing 0.05 M Tris, 0.1 M NaCl, and 2.5% Triton X-100) at 37 ° C for 1 hour, and then the reaction buffer was sequentially used ( Reaction buffer; containing 0.05M Tris, 0.1M NaCl, 2.5% Triton X-100 and 0.1 mM CaCl ₂ ), shaking at room temperature (10 ° C to 40 ° C) for 20 hours, using fixing buffer (fixation buffer; 50% methanol And 12% acetic acid) fixed at room temperature for 30 minutes, stained with staining buffer (containing 0.25% Coomassie blue, 40% methanol and 7% acetic acid) for 10 minutes to 30 minutes at room temperature, and using the depletion buffer (destaining buffer; containing 40% methanol and 7% acetic acid) was allowed to fade for 1 hour to 2 hours until the transparent band was clearly visible. Then, it was fixed on an acrylic plate with a cellophane sheet, and left to stand at room temperature to be air-dried. The results are shown in Figs. 2 to 4 .

Please refer to FIG. 2 to FIG. 4 , which are diagrams showing the gelatin zymogram analysis of the fibroblasts cultured at different concentrations using different concentrations of water chestnut aqueous extracts in several embodiments of the present invention. In FIG. 2 to FIG. 4, lane 1 represents a protein marker and 9 ^{(PageRuler TM Prestained Protein Ladder # 26616} , Thermo, USA; M), lane 2 representing the control group were cultured 0 hours (C _0), the first 5 lanes represent the results of cell culture for 24 hours (C ₂₄ ), lane 8 represents the results of control group cell culture for 48 hours (C ₄₈ ), lanes 3 and 4 represent experimental group cell culture for 24 hours, phase 6 Seven lanes represent the results of cell culture in the experimental group for 48 hours.

It can be seen from the results of FIG. 2 that the angle of 0.25% of the embodiment is utilized. The shell water extract cultured the fibroblasts for 24 hours, and began to have an effect of inhibiting the activities of MMP-2 and MMP-9. With the increase of the concentration of the water extract of the water chestnut shell and the incubation time, the effect of inhibiting the activity of MMP-2 and MMP-9 is more obvious, as shown by the results of FIGS. 2 to 4. In contrast, MMP-2 and MMP-9 activities in the control group cells were not inhibited. Therefore, the results of FIGS. 2 to 4 demonstrate that the water extract of the water chestnut shell of the examples can inhibit the activity of the second type matrix metalloproteinase and the matrix type metalloproteinase of the cells in vitro.

3. To evaluate the inhibitory effect of water extract of water chestnut shell on hyaluronidase

Hyaluronic acid is a mucopolysaccharide that is important for the protection of cartilage in bone and joint. Hyaluronidase decomposes hyaluronic acid, chondroitin, glucosamine, and the like. The hyaluronidase enzyme activity can be detected by hyaluronidase electrophoresis. After the reaction with the hyaluronidase was carried out by using the water extract of the water chestnut shell of the example, the hyaluronidase aqueous extract was used to evaluate the inhibitory effect of the water extract of the water chestnut shell on the hyaluronidase activity.

3.1 Using the water extract of the water chestnut shell of the embodiment and reacting with hyaluronidase

Different concentrations (0.25%, 0.50%, 1.00%, 2.5%, 5.00%, 10.0%) of the water extract of the water chestnut shell (experimental group) or an equal volume of sterile water (control group, C) and hyaluronic acid After mixing with an acidase (Hyaluronidase, Creative Biomart, USA), it was then reacted at 37 ° C for 0 hours, 24 hours or 48 hours. Then, the reactants of each group were taken and analyzed by hyaluronidase spectroscopy to evaluate the inhibitory effect of the water extract of the water chestnut shell of the examples on hyaluronidase activity.

3.2 Electrophoretic analysis of hyaluronidase

Separating colloids containing 10.0% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) containing a final concentration of 0.017% hyaluronic acid (Sigma-Aldrich, USA) as described in 2.2 After collecting the gel, after extracting the electrophoresis comb, the cast hyaluronic acid-SDS-PAGE electrophoresis film was placed in an electrophoresis tank containing 1X tank buffer (containing 25 mM Tris, 192 mM glycine and 0.1% SDS, pH 8.3). . Then, the sample to be tested is mixed with an equal volume of 2 times concentrated dyeing agent [2X loading dye, a mixed solution containing Bromophenol Blue (Amresco, USA) and glycerol (JT Baker, USA)], A sample of the coke-colloid colloid was injected with 20 μL of the mixed sample of the dye, and electrophoresis was carried out at a voltage of 80 V. When the trace dye on the film left the stacked colloid, the voltage was raised to 100 V for electrophoresis. After about 40 minutes of moving the dye to be traced to the bottom of the separation gel, the electrophoresis was terminated, and the hyaluronic acid-SDS-PAGE electrophoresis film was taken out.

Thereafter, the hyaluronic acid-SDS-PAGE electrophoresis film was washed with a washing buffer (containing 0.05 M Tris, 0.1 M NaCl, and 2.5% Triton X-100) at 37 ° C for 1 hour, and then the reaction buffer was sequentially used. The reaction buffer (containing 0.02 M sodium acetate-acetic acid and 0.135 M NaCl) was shaken at room temperature (ie, 10 ° C to 40 ° C) for 20 hours, and a 0.5% solution of Alcian blue (Sigma, USA) was applied. Dye at room temperature for 2 hours, and fade with a fade buffer (containing 40% methanol and 7% acetic acid) for 1 hour to 2 hours until transparent The ribbon is clearly visible. Then, it was fixed on an acrylic plate with a cellophane sheet, and left to stand at room temperature to be air-dried. The results are shown in Figs. 5 to 7.

Please refer to FIG. 5 to FIG. 7 , which are diagrams showing the analysis of hyaluronic acid zymography after reacting different concentrations of water extract of water chestnut shell with hyaluronidase at different times according to several embodiments of the present invention. In Figure 5, lanes 1 through 4 represent the results of sequence dilution (50X, 100X, 200X) of hyaluronidase, where 1X represents the original concentration of hyaluronidase. Representative of five protein marker ^{(PageRuler TM Prestained Protein Ladder # 26616} , Thermo, USA; M), the first representative of the control group and six hyaluronidase 0 hours reaction (C _0), the control section 11 representative of the hyaluronic group As a result of the acidase reaction for 24 hours (C ₂₄ ), the 16th channel represents the result of the control group reacting with hyaluronidase for 48 hours (C ₄₈ ), and the 7th to 10th channels represent the experimental group reacting with hyaluronidase for 24 hours. As a result, the 12th to 15th rows represent the results of the experimental group reacting with hyaluronidase for 48 hours.

In FIG. 6 to FIG. 7, lane 1 represents a protein marker and 9 ^{(PageRuler TM Prestained Protein Ladder # 26616} , Thermo, USA; M), lane 2 representing the control group and 0 hours reaction hyaluronidase (C ₀ ), the fifth lane represents the result of the control group reacting with hyaluronidase for 24 hours (C ₂₄ ), and the eighth lane represents the result of the control group reacting with hyaluronidase for 48 hours (C ₄₈ ), lanes 3 and 4 Representing the results of the experimental group reacting with hyaluronidase for 24 hours, lanes 6 and 7 represent the results of the experimental group reacting with hyaluronidase for 48 hours.

It can be seen from the results of FIG. 5 that after 0.25% of the water extract of the water chestnut shell of the example was reacted with hyaluronidase for 24 hours, it was significantly inhibited. The effect of hyaluronidase activity. As the concentration of the water extract of the water chestnut shell and the reaction time increase, the effect of inhibiting the hyaluronidase activity is still very significant, as shown by the results of FIGS. 5 to 7.

In contrast, the hyaluronidase activity of the control group was not inhibited. Therefore, the results of Figures 5 to 7 demonstrate that the water extract of the water chestnut shell of the examples does inhibit the hyaluronidase activity in vitro.

In summary, the water extract of the water chestnut shell of the present invention inhibits MMP-2, MMP-9 and hyaluronidase activity in vitro, and can be used as an MMP-2 inhibitor, an inhibitor of MMP-9 and a hyaluronidase inhibitor. Further, it is applied to the preparation of various skin external compositions or pharmaceutical compositions.

However, it should be added that the present invention exemplifies a specific variety and location of the water chestnut shell, specific extraction equipment and conditions, specific concentration of water chestnut shell water extract, specific test cells, specific analytical methods or specific instruments. The water extract of the water chestnut shell of the present invention, a method for producing the same, and the same are applicable as an MMP-2 inhibitor, an inhibitor of MMP-9, and a hyaluronidase inhibitor, but any one of ordinary skill in the art to which the present invention pertains is known. The present invention is not limited to this. The water chestnut shell water extract of the present invention and the method for producing the same can also use other concentrations of water chestnut shell water extract, other extraction equipment and conditions, other test cells, and other analysis without departing from the spirit and scope of the present invention. Method or other instrumentation, and then applied to other uses.

It can be seen from the above embodiments of the present invention that the water extract of the water chestnut shell of the present invention and the method for producing the same have the advantages of using a single-step hot reflux extraction step to treat the red cube shell in water at 100 ° C and pH 7.0 for no more than 3 hours. Obtaining the crude extract, and then removing the insoluble matter, obtaining the water extract of the water chestnut shell liquid. The resulting. The thus obtained water extract of water chestnut shell has been proved to inhibit MMP-2, MMP-9 and hyaluronidase activity in vitro, and can be inhibited as MMP-2 inhibitor, MMP-9 inhibitor and hyaluronidase. The agent is further applied to the preparation of various skin external compositions or pharmaceutical compositions, but is merely illustrative here, and the present invention is not limited thereto.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

Claims (10)

A method for producing a water extract of a water chestnut shell, comprising: performing a single-step hot reflux extraction step, treating a shell of Trapa bicornis var. taiwanensis (Nakai) with water at 100 ° C and pH 7.0 for no more than 3 hours to obtain a crude extract; and performing a solid-liquid separation step to remove one of the crude extract insolubles to obtain the water extract of the water chestnut shell. The method for producing a water chestnut shell water extract according to claim 1, wherein the weight ratio of the red cube shell to the water (w/v; g/mL) is 1:5 to 1:20. The method for producing a water chestnut shell water extract according to claim 1, wherein the weight ratio of the red cube shell to the water (w/v; g/mL) is 1:5 to 1:10. The method for producing a water chestnut shell water extract according to claim 1, wherein the weight ratio of the red cube shell to the water (w/v; g/mL) is 1:5 to 1:8. The method for producing a water extract of a water chestnut shell according to claim 1, wherein before the single-step hot reflux extraction step, at least comprising: drying the red cube shell; and drying the shell The red horn shell is subjected to a grinding treatment to obtain a dry powder of a water chestnut shell. The method for producing a water chestnut shell water extract according to claim 1, wherein the hot reflux extraction step is performed for 1 hour to 3 hours. An aqueous extract of water chestnut shell obtained by the method according to any one of claims 1 to 6, wherein the water extract of the water chestnut shell has a second type of matrix metalloproteinase (MMP-2). And/or one of the ninth type of matrix metalloproteinase (MMP-9) is active. A method for inhibiting a matrix metalloproteinase in vitro, which comprises treating an animal cell in vitro with a composition comprising a water extract of a water chestnut shell as described in claim 7 to inhibit a second type matrix of the animal cell An activity of a metalloproteinase and/or a ninth type matrix metalloproteinase. The method for inhibiting a matrix metalloproteinase in vitro according to the invention of claim 8, wherein one of the animal cells is derived from a mouse, and the animal cell is a fibroblast. The method for inhibiting a matrix metalloproteinase in vitro according to the invention of claim 8, wherein the composition is a skin external composition or a pharmaceutical composition.