TWI774046B - Use of solanum muricatum fermented liquid for preparing a composition for promoting skin anti-aging ability, promoting anti-glycation ability, reducing melanin content, increasing skin moisturization, reducing skin texture, reducing skin wrinkles, reducing skin redness and/or reducing fat - Google Patents

Abstract

A use of cantaloupe tomato fermented juice for preparing a composition. The composition has one or more of the following functions: enhancing glutathione synthesis, inhibiting the formation of glycation end products, enhancing anti-aging related genes, inhibiting melanin production, enhancing moisturizing-related gene secretion, inhibiting fat accumulation, promoting lipolysis, reducing receptors body fat and improve skin condition. In addition, the cantaloupe and eggplant fermentation juice is obtained by performing a fermentation process with a cantaloupe and eggplant culture medium and a plurality of bacterial species.

Description

Cantaloupe tomato fermented juice is used to prepare skin to improve anti-aging ability and anti-sugar Use of a composition for reducing skin pigmentation, reducing melanin content, enhancing skin hydration, reducing skin texture, reducing skin wrinkles, reducing skin redness and/or fat loss

The present invention relates to cantaloupe tomato fermented juice, in particular to the use of cantaloupe tomato fermented juice for preparing skin condition and/or fat-reducing compositions.

Since the rise of the concept of organic and natural diets, biotechnology companies and food companies have been actively investing in the research and development of natural plant-related products. In order to make plant-related products have a scientifically proven basis for their health benefits, active ingredient analysis and efficacy evaluation of plants have become key items in product development.

Pepino Dulce (scientific name Solanum muricatum), also known as ginseng fruit, is a perennial herb of the nightshade family Solanaceae. Cantaloupe is native to South America and has become one of the natural plants researched and developed in recent years.

In one embodiment, a kind of purposes of cantaloupe tomato fermented juice, it is for preparing The composition for improving antioxidant capacity, wherein, the cantaloupe and eggplant fermentation juice is obtained by performing a fermentation procedure with a cantaloupe tomato culture medium and a plurality of bacterial species, and the cantaloupe tomato culture liquid comprises cantaloupe tomato juice and water, and cantaloupe tomato juice and water The ratio is 1:2-5. Compared with the cantaloupe and eggplant culture medium, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In one embodiment, the use of a cantaloupe tomato fermented juice is used to prepare a composition for improving skin condition. Wherein, the cantaloupe and eggplant fermentation juice is obtained by performing a fermentation procedure with a cantaloupe and eggplant culture medium and a plurality of bacterial species. The cantaloupe and eggplant culture solution includes cantaloupe tomato juice and water, and the ratio of cantaloupe and eggplant to water is 1:2-5. Compared with the cantaloupe and eggplant culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% Lactic acid bacteria and 1%-15% acetic acid bacteria.

In one embodiment, a use of cantaloupe fermented juice for preparing a composition for reducing fat. Wherein, the cantaloupe and eggplant fermentation juice is obtained by performing a fermentation procedure with a cantaloupe and eggplant culture medium and a plurality of bacterial species. The cantaloupe and eggplant culture solution includes cantaloupe tomato juice and water, and the ratio of cantaloupe and eggplant to water is 1:2-5. Compared with the cantaloupe and eggplant culture medium, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In another embodiment, the antioxidant capacity of the cantaloupe tomato fermented juice is increased by increasing the total antioxidant capacity (Total Anti-oxidative Capacity), reducing the peroxidation product MDA (Malondialdehyde), increasing glutathione, or a combination thereof.

In yet another embodiment, the fermented juice of cantaloupe to improve skin condition is to improve skin anti-aging ability, improve anti-glycation ability, reduce melanin content, improve skin moisturization, reduce skin texture, reduce skin wrinkles, reduce skin redness or a combination thereof.

In yet another embodiment, the fermented juice of cantaloupe tomato to enhance skin anti-aging ability is to regulate CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene or UBL5 gene.

In yet another embodiment, the fermented juice of cantaloupe tomato to enhance skin moisturizing is the regulation of HAS2 gene or KRT1 gene.

In yet another embodiment, the cantaloupe fermented juice reduces fat by inhibiting fat accumulation, promoting lipolysis, or a combination thereof.

In yet another embodiment, the cantaloupe and eggplant fermentation juice is prepared through a fermentation process, wherein the fermentation process includes adding the yeast to the cantaloupe tomato culture solution; fermenting the cantaloupe and eggplant culture solution with the yeast for 1 to 3 days to form a first initial fermentation juice, wherein the yeast is preferably Saccharomyces cerevisiae; adding the lactic acid bacteria to the first initial fermentation juice; fermenting the first initial fermentation juice and the lactic acid bacteria for 1 to 5 days forming a second primary fermentation juice, wherein the lactic acid bacteria is preferably Streptococcus thermophilus; adding the acetic acid bacteria into the second primary fermentation juice; fermenting the second primary fermentation juice and the acetic acid bacteria for 3 to 10 days to form a third primary fermentation juice, wherein the acetic acid bacteria is preferably Acetobacter aceti; and filtering the third primary fermentation juice.

In yet another embodiment, the pH of the cantaloupe fermented juice is 4.0±1.0, and the sugar content of the cantaloupe fermented juice is 40±2.

In yet another embodiment, the polyphenol content of the cantaloupe tomato fermented juice is 88 ppm.

To sum up, according to the fermented juice of cantaloupe tomato according to any embodiment of the present invention, a composition for improving skin condition and/or reducing fat can be prepared. In other words, the aforementioned composition has one or more of the following functions: enhancing glutathione synthesis, inhibiting the formation of glycation end products, enhancing anti-aging-related genes, inhibiting melanin production, enhancing moisturizing-related gene secretion, inhibiting fat accumulation, promoting lipolysis, Reduces body fat in receptors and improves skin condition.

Embodiments of the present invention will be further described below, with the following examples It is used to illustrate the present invention, not to limit the scope of the present invention. Anyone who is familiar with this technique can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be regarded as The scope of the attached patent application shall prevail.

FIG. 1 is a bar graph showing the change of the total polyphenol content of the cantaloupe and tomato culture broth (before fermentation) and the cantaloupe fermented juice (after fermentation).

Figure 2 is a bar graph showing the relative glutathione content of each group.

Figure 3 is a bar graph showing the relative amounts of glycation end products formed for each group.

FIG. 4 is a bar graph showing the relative expression levels of anti-aging genes in each group.

Figure 5 is a bar graph showing the relative melanin content of each group.

FIG. 6 is a bar graph showing the relative moisturizing gene expression of each group.

FIG. 7 is a bar graph showing the effect of inhibiting fat accumulation in each group.

FIG. 8 is a bar graph showing the promotion of lipolysis for each group.

Figure 9 is a bar graph showing the relative expression of fat-burning genes in each group.

FIG. 10 is a bar graph showing the change in body weight before and after use of the cantaloupe tomato fermented juice of an example.

FIG. 11 is a bar graph showing the change of the whole body fat percentage before and after use of the cantaloupe tomato fermented juice of an example.

FIG. 12 is a bar graph showing the change of the trunk body fat percentage before and after use of the cantaloupe tomato fermented juice of an example.

FIG. 13 is a graph showing the change of hip circumference of the cantaloupe tomato fermented juice before and after use of an example. Transformed bar chart.

Fig. 14 is a bar graph showing the change of the total antioxidant capacity of the fermented juice of cantaloupe and eggplant before and after use.

FIG. 15 is a bar graph showing the change of the peroxidation product MDA of the cantaloupe tomato fermented juice before and after use of an exemplary example.

FIG. 16 is a bar graph showing the change in relative skin texture (roughness) of the cantaloupe tomato fermented juice before and after use.

Fig. 17 is a bar graph showing changes in relative skin wrinkles before and after use of the cantaloupe and eggplant fermented juice.

FIG. 18 is a bar graph showing changes in relative skin redness (red pigment) before and after use of the cantaloupe and eggplant fermented juice of an example.

FIG. 19 is a bar graph showing the change of relative skin moisture content before and after use of the cantaloupe and eggplant fermented juice.

The concentration symbol "%" as used herein generally refers to weight percent concentration, while the concentration symbol "vol%" generally refers to volume percent concentration. As used herein, "cantaloupe" generally refers to the fruit of cantaloupe.

The terms "cantaloupe and eggplant culture liquid" and "cantaloupe and eggplant water extract" used in this article can be used interchangeably, and both refer to the mixture of cantaloupe tomato juice and water evenly mixed.

According to the present invention, the operation procedures and parameter conditions related to the microbial fermentation reaction fall within the professional quality and routine technical scope of those who are familiar with the technology.

In some embodiments, the cantaloupe and tomato fermented juice is composed of the cantaloupe and tomato culture broth and Obtained by performing a fermentation procedure with several strains. Wherein, the cantaloupe and eggplant culture solution includes cantaloupe and eggplant and water. In the cantaloupe medium, the ratio of cantaloupe to water is 1:2-5. In the fermentation process, these strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria relative to the cantaloupe medium.

In some embodiments, the yeast may be Saccharomyces cerevisiae. In some embodiments, the lactic acid bacteria may be Streptococcus thermophilus or Lactobacillus plantarum. In some embodiments, the acetic acid bacteria can be Acetobacter aceti.

In some embodiments, in the fermentation process, 0.01%-0.5% yeast is added to the cantaloupe tomato culture solution, and the cantaloupe tomato culture solution and yeast are fermented for 1 to 3 days to form the first primary fermentation juice. In other words, the first mixed liquid obtained by mixing 0.01%-0.5% of the yeast with the cantaloupe and eggplant culture liquid is fermented for 1 to 3 days to form the first initial fermentation juice. In some embodiments, the first mixed liquor is fermented at 28°C-37°C.

After the first primary fermentation juice is formed, 0.01%-0.25% lactic acid bacteria are added to the first primary fermentation juice, and the first primary fermentation juice and lactic acid bacteria are fermented for 1 to 5 days to form the second primary fermentation juice. In other words, the second mixed liquor obtained by mixing 0.01%-0.25% lactic acid bacteria and the first primary fermentation juice is fermented for 1 to 5 days to form the second primary fermentation juice. In some embodiments, the second mixed liquor is fermented at 28°C-37°C.

After the second primary fermentation juice is formed, 1%-15% acetic acid bacteria are added to the second primary fermentation juice, and the second primary fermentation juice and acetic bacteria are fermented for 3 to 10 days to form the third primary fermentation juice. In other words, the third mixed liquor obtained by mixing 1%-15% of acetic bacteria and the second primary fermentation juice is fermented for 3 to 10 days to form the third primary fermentation juice. In some embodiments Among them, the third mixed liquor is fermented at 28°C-37°C.

After the third primary fermentation juice is formed, the third primary fermentation juice is filtered to obtain the cantaloupe tomato fermented juice. In the first exemplary example, the filtering step of the third primary fermentation juice includes filtering the third primary fermentation juice with a filter screen having a mesh of 200 to 400 mesh. In the second exemplary example, the filtering step of the third primary fermentation juice includes concentrating under reduced pressure at 40°C to 70°C and filtering the third primary fermentation juice with a mesh of 200 to 400 mesh. In the third exemplary example, the filtering step of the third primary fermentation juice includes concentrating under reduced pressure at 40°C to 70°C and filtering the third primary fermentation juice with a mesh of 200 to 400 mesh to obtain a Fermentation stock solution, and adjusting the sugar content (Degrees Brix) of fermentation stock solution to form cantaloupe tomato fermented juice.

In some embodiments, the sugar content of the fermentation stock can be adjusted by adding 55%-70% excipients. In some embodiments, the excipient used to adjust the sugar content may be isomalt-oligosaccharide.

In some embodiments, the pH of the cantaloupe fermented juice is 3.7±1.0, and the sugar content of the cantaloupe fermented juice is 40±2.

In some embodiments, the polyphenol content of the cantaloupe fermented juice is 88 ppm.

The cantaloupe tomato culture solution is obtained by mixing cantaloupe tomato juice and water in a ratio of 1:2-5. In the first example, the cantaloupe culture solution is obtained by mixing cantaloupe juice selected from South American sources with water in a ratio of 1:2-5. In the second example, the cantaloupe and eggplant culture solution is mixed with water in a ratio of 1:2-5 from the cantaloupe and eggplant juice selected from Agro international business a&c sac company in Peru, and then a glucose solution is added to adjust the sugar content to 9 to obtain . In the third example, the cantaloupe and eggplant culture solution is a mixture of cantaloupe and eggplant juice selected from Peru Agro international business a&c sac company and water in a ratio of 1:5, and then a glucose solution is added to increase the sugar content Adjust to 9 to get.

In some embodiments, the sterilization procedure of the raw material mixture may be sterilizing the raw material mixture at 80° C. to 100° C. for 0.2 hour to 1 hour, and cooling the sterilized raw material mixture to room temperature (ie, 25° C. to 30° C.). °C) to form a cantaloupe eggplant culture solution. In some embodiments, the sterilized raw material mixture can be cooled to room temperature by natural cooling.

In some embodiments, the cantaloupe tomato fermented juice has the effect of improving skin condition and/or reducing fat. In some examples, improving the skin condition of the recipient can be improved skin anti-aging ability, improved anti-glycation ability, reduced melanin content, improved skin hydration, reduced skin texture, reduced skin wrinkles, reduced skin redness, or a combination thereof. In some embodiments, cantaloupe tomato fermented juice can improve skin condition and/or reduce fat through one or more of the following cellular-level actions: enhancing glutathione synthesis, inhibiting the formation of end-products of glycation, and enhancing anti-aging related Genes, inhibit melanin production, enhance moisturizing-related gene secretion, inhibit fat accumulation, promote lipolysis, reduce receptor body fat, and improve skin condition. Wherein, the receptor can be human.

In some embodiments, the cantaloupe tomato fermented juice can be used to prepare a composition for enhancing antioxidant capacity, wherein the enhancement of antioxidant capacity can be to increase the total antioxidant capacity (Total Anti-oxidative Capacity), reduce the peroxidation product MDA ( Malondialdehyde), boosting glutathione, or a combination thereof.

In some embodiments, cantaloupe fermented juice can be used to prepare a composition for improving skin condition, wherein improving skin condition can be to improve skin anti-aging ability, improve anti-glycation ability, reduce melanin content, improve skin moisturization, reduce skin condition Texture, skin wrinkle reduction, skin redness reduction or a combination thereof, wherein improving skin anti-aging ability is regulating at least one of CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene or UBL5 gene and improving skin protection. Wet is to regulate at least one of HAS2 gene or KRT1 gene.

In some embodiments, cantaloupe fermented juice can be used to prepare a composition for reducing fat, wherein reducing fat is inhibiting fat accumulation, promoting lipolysis, or a combination thereof.

In some embodiments, any of the foregoing compositions can be a pharmaceutical. In other words, the medicinal product contains an effective content of fermented juice of cantaloupe and tomato.

In some embodiments, the aforementioned pharmaceutical products can be manufactured for parenterally, parenterally, orally, or topically using techniques well known to those skilled in the art Dosage form.

In some embodiments, the dosage form for parenteral or oral administration can be, but is not limited to, a tablet, troche, lozenge, pill, capsule , dispersible powder or granule, solution, suspension, emulsion, syrup, elixir, slurry or the like. In some embodiments, the dosage form for parenteral or topical administration can be, but is not limited to, an injection, sterile powder, external preparation, or the like. In some embodiments, the injection can be administered by subcutaneous injection, intraepidermal injection, intradermal injection or intralesional injection.

In some embodiments, the aforementioned pharmaceutical products may include pharmaceutically acceptable carriers that are widely used in pharmaceutical manufacturing techniques. In some embodiments, the pharmaceutically acceptable carrier can be one or more of the following carriers: solvent, buffer, emulsifier, suspending agent agent), decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, dilution Diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like thing. Regarding the type and quantity of the carrier selected, it falls within the scope of the professional quality and routine skills of those who are familiar with the technology. In some embodiments, the solvent as a pharmaceutically acceptable carrier can be water, normal saline, phosphate buffered saline (PBS), or an alcohol containing aqueous solution ).

In some embodiments, any of the foregoing compositions may be edible compositions. In other words, the edible composition contains a specific amount of cantaloupe fermented juice. In some embodiments, the aforementioned edible composition may be a food product or a food additive. In some embodiments, the food products can be, but are not limited to, beverages, fermented foods, bakery products, health foods, or dietary supplements.

In some embodiments, the aforementioned edible compositions can be administered to a recipient orally. Wherein, the form of the edible composition can be powder, granule, solution, colloid or paste.

In some embodiments, any of the foregoing compositions can be a cosmetic or skin care product. In other words, cosmetics or skin care products contain a specific amount of cantaloupe tomato fermented juice.

In some embodiments, the aforementioned cosmetics or skin care products can be in any of the following forms: lotion, gel, jelly, mud film, lotion, cream, lipstick, foundation, powder, powder, Cleansing Oil, Cleansing Milk, Facial Cleanser, Body Wash, Shampoo, Conditioner, Sunscreen, Hand Cream, Nail Polish, Perfume, Serum and Mask. In some embodiments, the aforementioned cosmetics or skin care products may further include externally acceptable ingredients as needed. In some embodiments, topical acceptable ingredients may be, for example, emulsifiers, penetration enhancers, emollients, solvents, excipients, antioxidants, or combinations thereof.

Embodiment 1: the preparation of cantaloupe tomato fermented juice

First, the cantaloupe tomato juice (product name Pepino Dulce, Nature Pulp, the origin of cantaloupe and tomato is the Peruvian Andes) purchased from Peru, Agro international business a&c sac company is mixed with water in a ratio of 1:5 (that is, the cantaloupe tomato juice is The ratio of one time weight and five times the weight of water) was mixed evenly, and then the glucose solution was added to adjust the sugar content to 9.0 to obtain the cantaloupe eggplant culture solution. However, the present invention does not limit the source of the cantaloupe tomato juice, for example, the cantaloupe tomato juice provided by Peruvian Nature or Naike Company from suppliers in South America.

Then, 0.1% brewer's yeast (Saccharomyces cerevisiae) (purchased from the Biological Resource Conservation and Research Center (BCRC) of the Institute of Food Industry Development, accession number BCRC20271) was propagated into the cantaloupe eggplant culture solution and fermented at about 30°C for 1 day, to obtain the first primary fermentation juice. Next, 0.05% Streptococcus thermophilus (purchased from BCRC, accession number BCRC910636) was propagated into the first primary fermentation juice and fermented at about 30° C. for 1 day to obtain a second primary fermentation juice. Finally, 10% Acetobacter aceti (purchased from BCRC, accession number BCRC11688) was propagated in the second primary fermentation juice and fermented at about 30°C for 5 days to obtain the third primary fermentation juice.

The third primary fermentation juice is then concentrated under reduced pressure at about 60° C. and filtered through a mesh filter with a mesh number of 200 to obtain a fermentation stock solution. Finally, add 60% isomalt oligosaccharide to After fermenting the stock solution, it is sterilized at about 100° C. for 2 hours to obtain the cantaloupe tomato fermented juice.

Here, the sugar content of the cantaloupe and tomato culture liquid, the fermentation stock solution and the cantaloupe tomato fermented juice are detected respectively. Among them, the sugar content of the cantaloupe and eggplant raw material mixture is about 9.0, the sugar content of the fermentation stock solution is about 4.0, and the sugar content of the cantaloupe and eggplant fermentation juice is about 40.

Embodiment 2: the preparation of cantaloupe eggplant water extract (without fermentation)

Mix the cantaloupe tomato juice purchased from Peru, Agro international business a&c sac company (product name Pepino Dulce, Nature Pulp, cantaloupe tomato from the Peruvian Andes) and water in a ratio of 1:5, then add glucose solution The sugar content was adjusted to 9, and the water extract of cantaloupe and eggplant was obtained, and its pH value was 6.3.

Embodiment 3: total polyphenol content test

10.0 mg of gallic acid was weighed into a 10 mL volumetric flask, and then quantified to 10 mL with water (H ₂ O) to obtain a stock solution of gallic acid. The stock solution of gallic acid was diluted 10-fold, ie, 100 μL of the stock solution of gallic acid plus 900 μL of water, to obtain an initial solution of 100 μg/mL gallic acid (ie, containing 1000 ppm of gallic acid). Then, prepare standard solutions of gallic acid of 0 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL according to Table 1, and take 100 μL of the standard solutions of each concentration. into a glass test tube. Add 500 μL of Folin-Ciocalteu's phenol reagent (purchased from Merck) to each glass test tube and mix it with the standard solution and let it stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix well, and react for 30 minutes. A standard reaction solution was obtained. Take 200 μL of the standard reaction solution into a 96-well plate, and measure its absorbance at 750 nm to obtain a standard curve.

Table I

The test samples of the experimental group (ie, the fermented juice of cantaloupe and eggplant in Example 1) and the test sample of the control group (ie, the water extract of cantaloupe and eggplant in Example 2) were diluted 10 times with water, respectively. 100 mL of each sample was taken into a glass test tube. Next, add 500 μL of Folin phenol reagent to the glass test tube and mix with the sample evenly and let it stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix well, and react for 30 minutes to obtain the reaction solution to be tested. After shaking the glass test tube containing the reaction solution to be tested to ensure no air bubbles, take 200 μL of the reaction solution to be tested into a 96-well plate, and measure the absorbance of the reaction solution to be tested at 750 nm.

Next, the absorbance value of the reaction solution to be tested corresponding to each sample is firstly divided by the sugar content of the sample, and then converted into the total polyphenol content by the interpolation method of the standard curve. Here, the total polyphenol content of the water extract of cantaloupe tomato was 27 ppm and the total polyphenol content of the cantaloupe tomato fermented juice was 88 ppm, as shown in FIG. 1 . It can be seen that the total polyphenol content of cantaloupe can be increased by 3.2 times after microbial fermentation. That is, compared with the water extract of cantaloupe and tomato, the fermented juice of cantaloupe and tomato can enhance the antioxidant activity.

Example 4: Assessing Cellular Antioxidant Efficacy - Detection of Glutathione Content

A. Experimental materials

1. Cell line: human peripheral blood mononuclear cell (human peripheral blood mononuclear cell, hPBMC).

2. Medium: X-VIVO10 medium (purchased from Lonza, model 04-380Q)

3. GSH detection reagent (purchased from abcam, model Ab112132).

4. Water extract of cantaloupe and eggplant: The water extract of cantaloupe and eggplant used in this experiment was obtained through the second embodiment.

5. Cantaloupe and eggplant fermented juice: The cantaloupe and eggplant water extract used in this experiment was obtained through Example 1 above.

B. Experimental Procedure

1. In a 6-well culture dish, 2×10 ⁶ human peripheral blood mononuclear cells (abbreviated as cells in the following steps) were implanted in each well of culture medium.

2. Incubate at 37°C for 24 hours.

3. Divide the cells into three groups: blank group, experimental group A, and experimental group B. The first group was the blank group (Mock); the second group (experimental group A) added the water extract of cantaloupe tomato, so that after adding the cantaloupe tomato fermented juice, the concentration of the cantaloupe tomato fermented juice in the overall solution was 0.0625%; the third group (Experimental group B) added the cantaloupe tomato fermented juice, so that after adding the cantaloupe tomato fermented juice, the concentration of the cantaloupe tomato fermented juice in the overall solution was 0.0625%; then the cells of the three groups of N were cultured at 37°C for 24 hours.

4. Harvest cells.

5. Rinse once with Phosphate-Buffered Saline (PBS) (purchased from Gibco).

6. Resuspend cells in 1 ml of PBS.

7. Stain cells with GSH reagent (1:1000) for 15 minutes.

8. Rinse once with PBS.

9. Resuspend cells in 200 μl of PBS.

10. Analyze relative fluorescent isothiocyanate (FITC) signal by flow cytometry (available from BD Accuri, model C6 Plus).

11. Using Microsoft EXCEL software, the detected values were analyzed for statistical significance by Student's t test. The results are shown in Figure 1. Wherein, *** means p < 0.001 compared to blank group.

It can be seen from the results in Figure 2 that the cells of the second group (experimental group A) added with the water extract of cantaloupe and eggplant, the GSH content did not increase, but decreased by 7.41% compared with the blank group; the third group ( In experimental group B) cells, the GSH content was significantly increased by 8.34% compared to the blank group, making its relative GSH content 1.08 times that of the cells in the first group (blank group), and was higher than that in the second group (experimental group). group) cells 1.17 times. GSH is a tripeptide composed of glutamic acid, 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. harm. Therefore, it can also be confirmed from the results in FIG. 1 that adding the cantaloupe tomato fermented juice obtained in Example 1 of the present case can indeed increase the GSH content in cells and increase the ability of intracellular redox, so as to achieve the effect of anti-oxidation in cells.

Example 5: Anti-glycation test

200mM sodium phosphate buffer (Sodium phosphate buffer, pH 7.4), sodium azide (Sodium azide, NaN ₃ ) and bovine serum albumin (BSA, brand: Gibco) were prepared with 0.06% NaN ₃ 60 mg/mL BSA solution.

A 1.5M solution of D-fructose was prepared in 200 mM sodium phosphate buffer and D-fructose (D-( _- ) _- Fructose, _C6H12O6 ).

A 3 mM AG solution was prepared with 200 mM sodium phosphate buffer and aminoguanidine hydrochloride (AG, CH ₆ N ₄ ·HCl).

Take 0.25 mL of the water extract of cantaloupe and eggplant obtained in Example 2, add 0.25 mL of BSA solution and D-fructose solution and mix them uniformly to obtain the test solution of experimental group A.

Take 0.25 mL of the cantaloupe tomato fermented juice obtained in Example 1, add 0.25 mL of BSA solution and D-fructose solution and mix them uniformly to obtain the test solution of experimental group B.

Take 0.25mL of 3mM AG solution, add 0.25mL of BSA solution and D-fructose solution and mix them uniformly to obtain the test solution of blank group.

Take 0.1 mL of the solution to be tested in each group as the zero point solution for each group. Using a spectrophotometer (Thermo Fisher Scientific), the fluorescence values of 0.1 mL of each group of zero point solutions were measured with excitation light of 360 nm and emission light of 460 nm to obtain the fluorescence value of zero point before the reaction.

Take 0.45mL of the test solution of each group and incubate at 50°C for 24 hours to obtain the end point solution of each group. Take 0.1mL of the end point solution of each group and use a spectrophotometer to measure the fluorescence value of 0.1mL of the end point solution of each group with excitation light of 360nm and emission light of 460nm to obtain the fluorescence value of the end point of the reaction .

Then, the relative formation amount (%) of Advanced Glycation End products (AGEs) of each group was calculated according to the following formula (1) to know its antiglycative activity. In other words, here, the relative amount (%) of AGEs formed in each group was calculated by considering the amount of AGEs formed in the blank group as 1 (ie, the relative amount of AGEs formed in the blank group was 100%).

Among them, Fluorescence sample _24hr represents the fluorescence value of the endpoint of the experimental group, Fluorescence sample _0hr represents the fluorescence value of the zero point of the experimental group, Fluorescence control _24hr represents the fluorescence value of the endpoint of the blank group, and Fluorescence control _0hr represents the zero point of the blank group. Fluorescence value.

Referring to Figure 3, compared with the blank group, experimental group A (water extract) did not affect the formation of saccharification end products, while experimental group B (fermentation juice) significantly reduced the formation of saccharification end products by 24.24%. It can be seen that the cantaloupe tomato fermented juice can effectively inhibit the formation of end products of glycation, that is, it has the effect of anti-glycation.

Example 6: Anti-aging gene detection

This example uses RNA extraction kit, reverse transcriptase, KAPA SYBR® FAST qPCR reagent kit and quantitative PCR instrument to determine the anti-aging-related genes in human fibroblasts treated with cantaloupe water extract or cantaloupe fermented juice The change.

For example, skin anti-aging CCT5 gene (Gene ID: 22948), FOXO gene (Gene ID: 2308), MRPS5 gene (Gene ID: 64969), Pink1 gene (Gene ID: 65018), Ubl-5 gene (Gene ID: 65018) : 59286).

Among them, CCT5 gene is a member of the CCT gene family, which is related to the process of mature cells returning to a young state; Pink1 gene is related to the process of restoring young state of aging mitochondria; FOXO gene, MRPS5 gene, and Ubl-5 gene are all related to mitochondrial mitochondria. It is related to the process of body activation, and mitochondria are the power plants in the living body. If the activity of mitochondria is insufficient, it is easy to cause cell aging. phenomenon.

Therefore, in Example 6, CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene, and Ubl-5 gene are used as anti-aging gene analysis targets.

Materials and Instruments

1. Cell line (hereinafter referred to as cell): human fibroblasts (CCD-996SK, from ATCC).

2. Medium: Minimum essential medium (MEM) (Gibco; model: 11095080), added with 10% fetal bovine serum (FBS) (Gibco; model: 10437-028), 1% penicillin-streptomycin (Gibco; model: 15140122), 1 mM sodium pyruvate (Gibco; model: 11360-070), 1.5 g/l sodium bicarbonate (Sigma; model: S5761-500G), and 0.1 mM Non-essential amino acids (Gibco; Cat. 11140050).

3. RNA extraction reagent kit (purchased from Geneaid, Taiwan, model: .FC24015-G)

4. SuperScript® III Reverse Transcriptase (Invitrogen, USA, No. 18080-051)

5. Measure target gene primers, which include FOXO gene, MRPS5 gene, Pink1 gene, Ubl-5 gene, and an internal control group (GAPDH gene).

6. KAPA SYBR® FAST qPCR reagent set (purchased from Sigma, USA, No. 38220000000)

7. ABI StepOnePlus ^™ real-time PCR system (ABI StepOnePlus ^™ Real-Time PCR system (Thermo Fisher Scientific, USA)).

8. Cantaloupe tomato fermented juice: The cantaloupe tomato fermented juice used in this experiment was obtained through Example 1 above.

9. Water extract of cantaloupe and eggplant: The water extract of cantaloupe and eggplant used in this experiment was obtained through the second embodiment.

First, human fibroblasts (referred to as cells in this example) were cultured at 1×10 ⁵ cells per well in a six-well culture dish containing 2 mL of the above-mentioned culture medium, and cultured at 37° C. for 16 hours, and then The cells were divided into the following three groups: experimental group A, experimental group B and blank group.

Test Conditions

In detail, in experimental group A, human fibroblasts were cultured for 24 hours with 2 ml of the 2.0% concentration medium of the water extract of cantaloupe and eggplant prepared in the above example. In the experimental group B, human fibroblasts were cultured for 24 hours with 2 ml of the medium containing the 2.0% concentration of the cantaloupe tomato fermented juice prepared in the above example.

In the blank group, human fibroblasts were simply cultured with 2 ml of culture medium, and no other additives were added. Human fibroblasts were cultured for 24 hours and used as the experimental blank group.

The above experimental group A, experimental group B and blank group, each group was repeated four times.

The treated human fibroblasts (ie experimental group A and experimental group B) and the human fibroblasts in the blank group were respectively ruptured cell membranes with cell lysate to form three groups of cell solutions. Next, the RNAs in the cell solutions of the three groups were extracted with an RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G). Next, 1000 nanograms (ng) of the extracted RNA was used as a template for each group, and the extracted RNA was reverse transcribed into corresponding cDNA by ^{SuperScript®} III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051). . Then by ABI StepOnePlus ^TM real-time PCR system (ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific Company, USA)), KAPA SYBR FAST (purchased from Sigma Company, USA, No. 38220000000) and the primer (SEQ ID of Table 1) NO: 1 to SEQ ID NO: 12) Quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain reaction) was performed on the cDNA of six groups to observe the CCT5 gene, FOXO in human fibroblasts of three groups Gene, MRPS5 gene, Pink1 gene, UBL5 gene expression. The instrument setting conditions for quantitative real-time reverse transcription polymerase chain reaction were 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, and repeated 40 cycles, and the 2-ΔCt method was used for gene quantification. Here, quantitative real-time reverse transcription polymerase chain reaction with cDNA can indirectly quantify the mRNA expression levels of CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene, and UBL5 gene, and then infer CCT5 gene, FOXO gene, MRPS5 gene, and Pink1 gene. , the expression of the protein encoded by the UBL5 gene.

It should be noted that the relative gene expression of each gene shown in the figures described below is presented in relative magnification, wherein the standard deviation was calculated using the STDEV formula of Excel software, and the one-tailed Student's t test ( Student t-test) to analyze whether there is a statistically significant difference. In the graph, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*", the more significant the statistical difference.

See Figure 4. When the expression level of the CCT5 gene in the blank group was regarded as 1 (ie 100%), the expression level of the CCT5 gene in the experimental group A relative to the blank group was 0.22 (ie 22%), and the CCT5 gene in the experimental group B relative to the blank group was 0.22 (ie 22%). The expression level of CCT5 gene was 1.46 (ie, 146%), which represented that after the cells were treated with cantaloupe tomato fermented juice, the expression level of CCT5 gene increased, which was 1.46 times that of the blank group.

See Figure 4. When the expression level of the FOXO gene in the blank group was regarded as 1 (ie 100%), the expression level of the FOXO gene in the experimental group A relative to the blank group was 0.25 (ie 25%), and the FOXO gene expression in the experimental group B relative to the blank group was 0.25 (ie 25%). The performance amount of 1.58 (ie 158%), After the representative cells were treated with cantaloupe tomato fermented juice, the expression level of FOXO gene increased, which was 1.58 times that of the blank group.

See Figure 4. When the expression level of the MRPS5 gene in the blank group was regarded as 1 (ie 100%), the expression level of the MRPS5 gene in the experimental group A relative to the blank group was 0.17 (ie 17%), and the expression level of the MRPS5 gene in the experimental group B relative to the blank group was 0.17 (ie 17%). The expression level of MRPS5 gene was 1.12 (ie, 112%), which represented that after the cells were treated with cantaloupe tomato fermented juice, the expression level of MRPS5 gene increased, which was 1.12 times that of the blank group.

See Figure 4. When the expression level of the Pink1 gene in the blank group is regarded as 1 (ie 100%), the expression level of the Pink1 gene in the experimental group A relative to the blank group is 0.30 (ie 30%), and the experimental group B is relative to the Pink1 gene in the blank group. The expression level of Pink1 gene was 1.35 (ie, 135%), which represented that the expression level of Pink1 gene increased after the cells were treated with cantaloupe tomato fermented juice, which was 1.35 times that of the blank group.

See Figure 4. When the expression level of the UBL5 gene in the blank group was regarded as 1 (ie 100%), the expression level of the UBL5 gene in the experimental group A relative to the blank group was 0.25 (ie 25%), and the UBL5 gene in the experimental group B relative to the blank group was 0.25 (ie 25%). The expression level of UBL5 gene was 1.48 (ie 148%), which represented that after the cells were treated with cantaloupe tomato fermented juice, the expression level of UBL5 gene in the experimental group was increased, which was 1.48 times that of the blank group.

It can be seen that when human fibroblasts are treated with cantaloupe tomato fermented juice, the expression of various genes related to anti-aging and rejuvenation in the cells increases, which means that the cells return from the aging state to the state before the non-aging, effectively delaying the cell regeneration. Ageing.

Embodiment 7: Detection of melanin content

Here, the cell culture medium used was supplemented with 1 vol% penicillin/streptomycin (brand name: Gibco) and 10 vol% fetal bovine serum (fetal calf serum). bovine serum, FBS, brand: Gibco) of Dulbecco's Modified Eagle's Medium (DMEM, brand: Gibco).

First, the mouse melanoma cell line B16F10 (purchased from the American Type Culture Collection (ATCC), accession number CRL-6475) was inoculated into 6 wells containing 3 mL of cell culture medium at a cell number of 1.5×10 ⁵ cells per well. in each well of the culture plate and incubated at 37°C for 24 hours.

After culturing for 24 hours, the B16F10 cells were divided into 3 groups: an experimental group (ie, the experimental group (cantaloupe tomato fermented juice group)), a control group, and a blank group. The cell culture medium of each group was removed and replaced with 3 mL of experimental medium per well, and then placed at 37° C. for 48 hours. Wherein, the experimental medium of the experimental group was a cell culture medium containing 0.03125 vol% of the cantaloupe tomato fermented juice obtained in Example 1. The experimental medium of the control group was a cell culture medium containing 0.03125 vol% kojic acid (the experimental medium of the blank group was a simple cell culture medium (that is, without the fermented juice of cantaloupe and without kojic acid).

After 48 hours of incubation, the experimental medium in each well was removed and rinsed twice with 1x (1x) phosphate buffered saline (PBS, brand: Gibco). After washing, trypsin was added to each well to treat the cells for 3 minutes. After 3 minutes of treatment, the suspended cells in each well were individually collected in 15 mL centrifuge tubes, and then centrifuged at 400×g for 5 minutes to separate the cell pellet and the supernatant. After the cell pellet was resuspended in 1xPBS and centrifugation was repeated twice, the cell pellet was resuspended with 200 μL of 1xPBS to obtain a cell solution. Next, the cell solution was placed in liquid nitrogen for 10 minutes, and then stood at room temperature for 30 minutes to thaw. After complete thawing, each centrifuge tube was centrifuged at 12,000 xg for 30 minutes. After 30 minutes of centrifugation, the supernatant in each centrifuge tube was removed and 120 μL of 1N _NaOH (in ddH2O) was added to mix with the pellet in each centrifuge tube. After mixing uniformly, each centrifuge tube containing the mixed solution was left to stand in a 60°C dry bath for 1 hour. Then, take 100 μL of the mixed solution from each centrifuge tube into a 96-well culture plate and read it with an ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay) reader (brand: BioTek) at a wavelength of 450 nm. Absorbance value (OD450) of each well in a 96-well plate.

After the measurement, the relative melanin content (%) was calculated by substituting the measured absorbance values into the following formula (2). In other words, here, the relative melanin content (%) of each group is calculated by considering the melanin content of the blank group as 1 (ie, the relative melanin content of the blank group is 100%). Also, statistically significant differences between groups were statistically analyzed by student t-test, as shown in FIG. 5 . In Figure 5, "*" represents that the p-value is less than 0.05 when compared with the blank group, "**" represents that the p-value is less than 0.01 when compared with the blank group, and "***" represents the comparison with the blank group. Its p-value is less than 0.001.

Relative melanin content (%)=(OD450 sample/OD450 control)×100%

Among them, OD450 sample represents the absorbance value of the group to be converted, and OD450 control represents the absorbance value of the blank group.

Referring to Figure 5, compared with the blank group, the melanin content of the experimental group was significantly reduced, and it could reduce the production of melanin by 24.71%; the melanin content of the control group was also significantly reduced, which reduced the production of melanin by 14.45%, and koji acid was Known whitening ingredients, it can be seen that the cantaloupe tomato fermented juice can effectively inhibit the formation of melanin, that is, effectively inhibit the formation of tyrosinase, and has the effect of whitening.

Example 8: Moisturizing gene detection

In this example, RNA extraction kit, reverse transcriptase, KAPA SYBR® FAST qPCR reagent kit and quantitative PCR instrument are used to determine the moisturizing-related genes in primary human skin keratinocytes treated with cantaloupe water extract or cantaloupe fermented juice. The change.

For example, the moisturizing genes are Krt1 gene (Gene ID: 3848) and HAS2 gene (Gene ID: 3037)

Among them, the Krt1 gene is related to keratin, which is the main structure of the skin. When it is closely arranged, it has the effect of reducing water evapotranspiration and locking water. Therefore, the promotion of Krt1 gene can affect the skin moisturizing. The HAS2 gene is a hyaluronic acid synthase-related gene , It is related to the synthesis of hyaluronic acid, and the improvement of HAS2 gene is related to the content of hyaluronic acid, so it can also affect the moisture and elasticity of the skin.

Therefore, in Example 8, Krt1 gene and HAS2 gene were used as the analysis targets.

Materials and Instruments

1. Cell line: Cell line: Human primary epidermal keratinocytes (CELLnTEC (Switzerland), HPEK-50).

2. Medium: Keratinocyte-SFM (1X) (Thermo).

3. RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No.FC24015-G)

4. SuperScript® III Reverse Transcriptase (Invitrogen, USA, No. 18080-051)

5. Measure the target gene primer, which includes Krt1 gene and HAS2 gene, and also includes internal control group (TBP gene).

6. KAPA SYBR® FAST qPCR reagent set (purchased from Sigma, USA, No. 38220000000)

7. ABI StepOnePlus ^™ real-time PCR system (ABI StepOnePlus ^™ Real-Time PCR system (Thermo Fisher Scientific, USA)).

8. Cantaloupe tomato fermented juice: The cantaloupe tomato fermented juice used in this experiment was obtained through Example 1 above.

9. Water extract of cantaloupe and eggplant: The water extract of cantaloupe and eggplant used in this experiment was obtained through Example 2 above.

Experimental procedure

First, take 1.5x10 ⁵ human primary skin keratinocytes into a six-well cell culture dish containing 2 ml of the above-mentioned medium in each well, and culture at 37°C for 24 hours. The cultured primary human skin keratinocytes in each well were tested according to the following The conditions were divided into blank group and experimental group (two groups in total) to deal with the human primary skin keratinocytes cultured in each well.

Test Conditions

In detail, the blank group simply used 2 ml of culture medium to culture primary human skin keratinocytes, without adding other ingredients, and cultured primary human skin keratinocytes for 6 hours, which was used as the experimental blank group.

In the experimental group, human primary skin keratinocytes were cultured for 6 hours with 2 ml of culture medium containing 1% concentration of cantaloupe tomato fermented juice prepared in Example 1 above.

The blank group and the experimental group above were repeated four times in each group.

The treated human primary skin keratinocytes (ie, blank group and experimental group) were respectively ruptured cell membranes with cell lysate to form two groups of cell solutions. Next, RNAs in four groups of cell solutions were extracted with RNA extraction reagent kits (purchased from Geneaid, Taiwan, Lot No. FC24015-G). Next, 1000 nanograms (ng) of the extracted RNA was used as a template for each group, and the extracted RNA was reverse transcribed into corresponding cDNA by ^{SuperScript®} III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051). . Then by ABI StepOnePlus ^TM real-time PCR system (ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific Company, USA)), KAPA SYBR FAST (purchased from Sigma Company, USA, No. 38220000000) and the primer (SEQ ID of Table 1) NO: 13 and SEQ ID NO: 18) Quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain reaction) was performed on the cDNAs of the two groups to observe the Krt1 gene, Expression of the HAS2 gene. The instrument setting conditions for quantitative real-time reverse transcription polymerase chain reaction were 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, and repeated 40 cycles, and the 2-ΔCt method was used for gene quantification. Here, quantitative real-time reverse transcription polymerase chain reaction with cDNA can indirectly quantify the mRNA expression levels of Krt1 and HAS2 genes, and then infer the expression levels of proteins encoded by Krt1 and HAS2 genes.

It should be noted that the relative gene expression of Krt1 gene and HAS2 gene shown in the diagrams described below is presented in relative magnification, wherein the standard deviation is calculated using the STDEV formula of Excel software, and the one-tailed student is used in Excel software. Student t-test was used to analyze whether there were statistically significant differences. In the graph, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*", the more significant the statistical difference.

Referring to Figure 6, when the expression level of HAS2 gene and KRT1 gene in the blank group is regarded as 1 (ie, 100%), the expression level of the HAS2 gene in the experimental group relative to the blank group is 1.43 (ie, 143%), and the expression level of the KRT1 gene is 1.43 (ie, 143%). The expression level was 2.27 (ie, 227%), representing that the expression level of the HAS2 gene in the experimental group was 1.43 times higher than that in the blank group, and the expression level of KRT1 gene was 2.27 times higher than that in the blank group.

In other words, compared with the blank group, the expression of HAS2 gene in the experimental group increased by 43%, and the expression of KRT1 gene increased by 127%.

The results are shown in Figure 6. When the primary human skin keratinocytes were treated with cantaloupe tomato fermented juice, the expression of HAS2 gene and KRT1 gene in the primary human skin keratinocytes increased, indicating that the cantaloupe tomato fermented juice could regulate the content of keratin and hyaluronic acid. It has the potential to enhance the skin's moisturizing ability.

Example 9: fat accumulation detection

Here, the pre-adipocyte expansion medium used is the minimum essential medium α (Minimum Essential Medium Alpha, MEMα, 20 vol% FBS (brand name: Gibco) and 1 vol% penicillin-streptomycin) supplemented with Brand: Gibco). The differentiation medium used was MEMα (brand: Gibco) supplemented with 20 vol% FBS (brand: Gibco) and 1 vol% penicillin-streptomycin. And, Oil-Red O staining reagent (brand: Sigma) was thoroughly dissolved in 100% isopropanol (isopropanol, supplier: ECHO) to prepare a 3 mg/mL stock solution of Oil-Red O staining reagent. To obtain a ready-to-use oil-red O working solution, dilute the stock solution of oil-red O staining reagent with secondary water (ddH2O) to a concentration of 1.8 mg/mL immediately before use, This is a stock solution of 60% Oil-Red O staining reagent.

First, the mouse bone marrow stromal cell line OP9 (purchased from ATCC, No. CRL-2749) was inoculated into each well of a 24-well culture plate containing 500 μL of preadipocyte proliferation medium at a cell number of 8×10 ⁴ cells per well. and cultured at 37°C for 7 days. During the 7-day culture period, fresh 500 μL differentiation medium was replaced every 3 days. After 7 days of culture, the formation of lipid droplets in the cells in each well was observed using a microscope (brand: ZEISS) to confirm that the cells were fully differentiated into adipocytes for subsequent experiments.

After 24 hours of culture, the adipocytes were divided into 3 groups: two experimental groups (ie, experimental group A (water extract of cantaloupe) and experimental group B (fermented juice of cantaloupe) and blank group. The differentiation medium of each group was removed and replaced with 500 μL of experimental medium per well, and then cultured at 37° C. for 7 days. During the 7-day culture period, fresh 500 μL experimental medium was replaced every 3 days. Among them, the experimental medium of experimental group A was obtained in Example 1 containing 0.0625vol% Differentiation medium of cantaloupe water extract. The experimental medium of experimental group B was a differentiation medium containing 0.0625 vol% of the cantaloupe tomato fermented juice obtained in Example 1. The experimental medium of the blank group was a simple differentiation medium (that is, without the water extract of cantaloupe and the fermented juice of cantaloupe).

Next, the experimental medium in each well was removed and rinsed twice with IxPBS. Then, 1 mL of 10% formaldehyde (supplier: ECHO) was added to each well and incubated at room temperature for 30 minutes to fix the cells. Afterwards, the formaldehyde in each well was removed and each well was rinsed twice with 1 mL of PBS. After re-rinsing, 1 mL of 60% isopropanol was added to each well and allowed to act for 1 minute. Next, the isopropanol was removed and an additional 1 mL of Oil-Red O working solution was added and allowed to act at room temperature for 1 hour.

After 1 hour of exposure, the Oil-Red O working solution was removed and rapidly destained with 1 mL of 60% isopropanol for 5 seconds. After destaining, the stained cells were rinsed with 1×PBS, 100% isopropanol was added to each well, and the cells were placed on a shaker to react for 10 minutes to dissolve the dye. Then, 100 μL of the aforementioned dye-isopropanol solution was taken from each well to a 96-well culture plate and the absorbance (OD510) of each well was read with an ELISA reader (brand: BioTek) at a wavelength of 510 nm. .

After the measurement, the relative fatty oil droplet amount (%) was calculated by substituting the measured absorbance value into the following formula (3). In other words, here, the relative fatty oil droplet amount (%) of each group is calculated by considering the amount of fatty oil droplets in the blank group as 1 (ie, the relative amount of fatty oil droplets in the blank group is 100%). Also, statistically significant differences between groups were statistically analyzed by Student's t-test, as shown in FIG. 4 . In Figure 4, "**" represents that the p-value is less than 0.01 in comparison with the blank group.

Relative fat droplet amount (%)=(OD510 sample/OD510 control)×100% (3)

Among them, OD510 sample represents the absorbance value of the group to be converted, and OD510 control represents the absorbance value of the blank group.

Referring to FIG. 4 , compared with the blank group, the relative amount of fatty oil droplets in the experimental group A was significantly reduced, and it could reduce the amount of oil droplets by 11.72%. Compared with the blank group, the relative amount of oil droplets in experimental group B was significantly reduced, and it could reduce the amount of oil droplets by 32.29%. It can be seen that the cantaloupe tomato fermented juice can effectively inhibit the accumulation of fat and has the effect of reducing fat. Moreover, the cantaloupe eggplant may produce more fat-reducing active ingredients than the cantaloupe eggplant culture medium after microbial fermentation.

Example ten: fat-reducing efficacy test

Fat loss refers to the decomposition of fat, and lipolysis refers to the process in which triglyceride (TG) stored in fat cells is gradually degraded into fatty acid (FA) and glycerol (Glycerol). . Based on this, in this experiment, the content of glycerol (Glycerol) in adipocytes was analyzed as a quantitative index to observe whether there is a lipolytic effect.

Mouse bone marrow stromal cells (hereinafter referred to as OP9 cells) were used in this experiment, and the OP9 cell line (ATCC CRL-2749) was purchased from the American Type Culture Collection (ATCC ^® ).

First, take a 24-well culture plate and inoculate each well with 8×10 ⁴ OP9 cells and 500 μL of medium (Medium), which contains 80% MEMAM (Minimum Essential Medium Alpha Medium, purchased from Gibco, USA) cell culture medium, 20% Fetal Bovine Serum (Fetal Bovine Serum, available from Gibco, USA, Cat# 10437-028) and added with 0.1% Penicillin-streptomycin (available from Gibco, USA) at 37°C Cultured for 7 days. The medium was changed every 3 days during this 7-day cell culture period. After 7 days, intracellular oil droplet formation was observed under a microscope (ZEISS; magnification 400x) to confirm that the cells had fully differentiated into adipocytes.

Then, the differentiated adipocytes were divided into two groups: two experimental groups (that is, experimental group A (adding the cantaloupe and eggplant water extract prepared in Example 2) and experimental group B (adding the cantaloupe eggplant prepared in Example 1) fermented juice)) and blank group.

Experimental group A: According to the ratio of 500 μL medium containing 0.156 μL of cantaloupe water extract per well (ie, the concentration is 0.03125%), the water extract of cantaloupe and eggplant was added to the differentiated medium, and cultured at 37°C for 7 days . The medium was changed every 3 days during the 7 days of cell treatment.

Experimental group B: The cantaloupe fermented juice was added to the differentiated medium according to the ratio of 0.156 μL of cantaloupe tomato fermented juice per well of 500 μL medium (ie, the concentration was 0.03125%), and cultured at 37° C. for 7 days. The medium was changed every 3 days during the 7 days of cell treatment.

Blank group: no treatment, that is, no additional components were added to the differentiated medium, and cultured at 37°C for 7 days. The medium was changed every 3 days during the 7 days of cell treatment.

After 7 days of cell treatment, glycerol content was measured using the Glycerol cell-based assay kit (available from Cayman, USA, product number 10011725) according to the following procedure. Collect the supernatant of each group, and transfer 25 μL of each to a new 96-well culture plate, and add 100 μL of Reconstituted free glycerol assay reagent to each well, and then at room temperature After 15 minutes of action, the absorbance at OD _540nm of each group was read with an ELISA reader to quantify the amount of glycerol decomposed and released into cell culture medium by adipocytes in each group, as shown in FIG. 6 . Here, the amount of glycerin is proportional to the amount of decomposition of fat. Among them, use Excel software to perform student t-test to determine whether there is a statistically significant difference between the two sample groups ("*" in the diagram represents p-value less than 0.05, "**" represents p-value less than 0.01, and "***" means p-value is less than 0.001. When there are more "*", it means the statistical difference is more significant).

Refer to Figure 8. When the lipolysis of the blank group was regarded as 100%, the lipolysis of experimental group A was 96.07%, and the lipolysis of experimental group B was 104.85%. % of lipolysis. The results show that the cantaloupe tomato fermented juice can directly and effectively promote the decomposition of fat in adipocytes, so as to reduce the content of fat in adipocytes, and achieve the effect of burning fat and losing weight.

Example 11 - Cell Experiment of Fat Metabolism Gene

This example uses RNA extraction kit, reverse transcriptase, KAPA SYBR® FAST qPCR reagent kit and quantitative PCR instrument to determine the lipid metabolism genes in mouse bone marrow stromal cells treated with cantaloupe water extract or cantaloupe fermented juice genetic changes.

For example, the fat metabolism gene gene is the UCP2 gene (Gene ID: 7351).

Among them, the protein encoded by the UCP2 (Uncoupling Protein 2) gene is the uncoupling protein (UCP), which is one of the members of the mitochondrial anion carrier protein (Mitochondrial anion carrier proteins, MACP) family. Nucleoside (Adenosine triphosphate, ATP) reduction, and promote the transfer of anions from the inner membrane of mitochondria to the outside, and promote the return transfer of protons from the outside to the inner membrane of mitochondria, and release the energy generated in the process as heat; UCP2 gene It is expressed in many tissues, with the highest expression in skeletal muscle, and is considered to be related to nonshivering thermogenesis. Therefore, the expression level of the UCP2 gene increases, which can promote the decomposition of fat and accumulate fat. amount decreased.

Therefore, in Example 11, the UCP2 gene was used as the analysis target.

Materials and Instruments

10. Cell line: mouse bone marrow stromal cell OP9 (BCRC; No. CRL-2749).

11. Culture medium: containing 80% MEMAM (Minimum Essential Medium Alpha Medium, purchased from Gibco, USA) cell culture medium, 20% Fetal Bovine Serum (purchased from Gibco, USA, Cat#10437-028), And 0.1% penicillin/streptomycin (Penicillin-streptomycin, purchased from Gibco, USA) was added.

12. RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No.FC24015-G)

13. SuperScript® III Reverse Transcriptase (Invitrogen, USA, No. 18080-051)

14. The measurement target gene primer, which contains the UCP2 gene, and the internal blank group (m-ACTB gene).

15. KAPA SYBR® FAST qPCR reagent set (purchased from Sigma, USA, No. 38220000000)

16. ABI StepOnePlus ^™ real-time PCR system (ABI StepOnePlus ^™ Real-Time PCR system (Thermo Fisher Scientific, USA)).

17. Cantaloupe and eggplant fermentation juice: The cantaloupe and eggplant water extract used in this experiment was obtained through the above Example 1.

18. Water extract of cantaloupe and eggplant: The water extract of cantaloupe and eggplant used in this experiment was obtained through the second embodiment.

Experimental procedure

First, take 1.5x10 ⁵ mouse bone marrow stromal cells OP9 into a six-well cell culture dish containing 2 ml of the above medium in each well, and culture at 37°C for 24 hours. The following test conditions were divided into blank group and experimental group (three groups in total) to treat the cultured mouse bone marrow stromal cells in each well.

Test Conditions

In detail, the blank group simply used 2 ml of culture medium to culture mouse bone marrow stromal cells, without adding other additives, and cultured for 6 hours, which was used as the experimental blank group.

In the experimental group A, 2 ml of the culture medium of 0.0625% concentration of the water extract of cantaloupe and eggplant prepared in the above example was used to culture the mouse bone marrow stromal cells for 6 hours.

In the experimental group B, 2 ml of the culture medium of 0.0625% concentration of cantaloupe tomato fermented juice prepared in the above example was used to culture mouse bone marrow stromal cells for 6 hours.

The above blank group, experimental group A, and experimental group B were repeated four times in each group.

The treated mouse bone marrow stromal cells (ie, blank group, experimental group A, and experimental group B) were broken into cell membranes with cell lysate to form cell solutions of three groups. Next, the RNAs in the cell solutions of the three groups were extracted with an RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G). Next, 1000 nanograms (ng) of the extracted RNA was taken as a template for each group, and the extracted RNA was reverse transcribed into corresponding cDNA through ^{SuperScript®} III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051). . Then by ABI StepOnePlus ^TM real-time PCR system (ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific Company, USA)), KAPA SYBR FAST (purchased from Sigma Company, USA, No. 38220000000) and the primer (SEQ ID of Table 1) NO: 19 to SEQ ID NO: 22) Quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain reaction) was performed on the cDNAs of the three groups to observe the UCP2 gene expression in the mouse bone marrow stromal cells of the three groups. performance. The instrument setting conditions for quantitative real-time reverse transcription polymerase chain reaction were 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, and repeated 40 cycles, and the 2-ΔCt method was used for gene quantification. Here, quantitative real-time reverse transcription polymerase chain reaction with cDNA can indirectly quantify the mRNA expression level of the UCP2 gene, thereby inferring the expression level of the protein encoded by the UCP2 gene.

It should be noted that the relative gene expression of UCP2 shown in the figures described below is presented in relative magnification, in which the standard deviation was calculated using the STDEV formula of Excel software, and the one-tailed Student t test (Student t-test) was used in Excel software. t-test) to analyze whether there is a statistically significant difference. In the figure, "*" represents p-value less than 0.05, "**" represents p-value less than 0.01, And "***" means p-value less than 0.001. The more "*", the more significant the statistical difference.

Referring to Figure 9, when the expression level of the UCP2 gene in the blank group is regarded as 1 (ie 100%), the expression level of the UCP2 gene in the experimental group A relative to the blank group is 1.26 (ie 126%), and the experimental group B is relative to the blank group. The expression level of UCP2 gene in the blank group was 4.75 (ie, 475%), which means that the expression level of UCP2 gene in experimental group A increased to 1.26 times that of the blank group, and the expression level of UCP2 gene in experimental group B increased to that of the blank group. 4.75 times.

In other words, the expression of UCP2 gene in experimental group A increased by 26% compared with the blank group, and the expression of UCP2 gene in experimental group B increased by 375% compared with the blank group.

From this, it was found that when the mouse bone marrow stromal cells were treated with the fermented juice containing cantaloupe, the expression level of UCP2 in the mouse bone marrow stromal cells increased.

The results are shown in FIG. 9 , the cantaloupe tomato fermented juice of the present invention can increase the gene expression level of UCP2 in cells, which means that the cantaloupe tomato fermented juice has an effect on promoting the decomposition of fat and reducing the accumulation of fat.

Example 12: Human Body Detection for Fat Loss and Slimming

Make 8 obese subjects (that is, their body fat rate is greater than 25% or BMI value greater than 24) to drink a bottle of 50mL cantaloupe fermented beverage (which contains 12vol% of the cantaloupe and tomato fermented juice obtained in Example 1 and 88vol% water) for 4 consecutive weeks. And, before drinking (ie, the 0th week) and after drinking for 4 weeks (ie, the 4th week), the body weight of these subjects was measured with a body weight machine, and these subjects were measured with a body fat meter (brand: TANITA BC-601FS). The subject's total body fat percentage, and the waist circumference of these subjects was measured with a cloth ruler. Also, the statistically significant differences between the measurements at Week 0 and those at Week 4 were statistically analyzed by Student's t-test, as shown in Figures 10-13. in Figure 10 In Fig. 13, "*" represents that the p-value is less than 0.05 in comparison with the 0th week; "**" represents that the p-value is less than 0.01 in comparison with the blank group.

Referring to FIG. 10 , compared to before consumption (week 0), drinking the cantaloupe and eggplant fermented beverage for 4 weeks significantly reduced body weight by about 1.0 kg.

Referring to FIG. 11 , compared to before drinking (week 0), drinking the cantaloupe and eggplant fermented beverage for 4 weeks can significantly reduce the total body fat rate by about 1.0%.

Referring to FIG. 12 , compared to before drinking (week 0), drinking the cantaloupe and eggplant fermented beverage for 4 weeks significantly reduced the trunk body fat rate by about 1.2%.

Referring to FIG. 13 , compared to before drinking (week 0), drinking cantaloupe and eggplant fermented beverage for 4 weeks can significantly reduce waist circumference by about 1.1 cm.

It can be seen that the long-term use of cantaloupe tomato fermented juice can improve fat accumulation and body weight in obese people, that is, cantaloupe tomato fermented juice has the effect of slimming and reducing fat.

Example 13: Antioxidant human detection

Make 8 obese subjects (that is, their body fat rate is greater than 25% or BMI value greater than 24) to drink a bottle of 50mL cantaloupe fermented beverage (which contains 12vol% of the cantaloupe and tomato fermented juice obtained in Example 1 and 88vol% water) for 4 consecutive weeks. In addition, blood was collected before drinking (ie, the 0th week) and after 4 weeks of drinking (ie, the 4th week), and the total anti-oxidative capacity (Total Anti-oxidative Capacity) and peroxidation products (MDA) in the blood were detected, among which The total antioxidant capacity and peroxidation products were tested by Realan Medical Laboratory (Taiwan).

In general, total antioxidant capacity, or TAC for short, represents the ability of the body to reduce oxides. Many studies have shown that free radicals can cause some diseases in the human body, such as: cardiovascular disease and various inflammatory diseases, etc., and antioxidants in the body have the effect of protecting cells and can prevent Therefore, the imbalanced state of the body can be monitored by detecting the state of antioxidant capacity in the body. The higher the total antioxidant capacity, the better the antioxidant capacity in the body; the peroxide product (MDA, Malondialdehyde), representing the Malondialdehyde is the product of lipid peroxidation in the body, and is widely accepted as one of the targets for the determination of oxidative damage. The higher the peroxidation product (MDA, Malondialdehyde), the higher the oxidative stress in the body.

After the measurement, the relative antioxidant-related value (%) of the fourth week was calculated by taking the value in the 0th week as the benchmark (ie, the antioxidant-related value in the 0th week was 100%). Also, statistically significant differences between the measurements at week 0 and those at week 4 were statistically analyzed by Student's t-test, as shown in Figures 14 and 15 . In Fig. 14 and Fig. 15, "*" represents that the p-value is less than 0.05 when compared with the 0th week; "***" represents that the p-value is less than 0.001 when compared with the blank group.

Referring to Figure 14, compared with before drinking (week 0), drinking cantaloupe and eggplant fermented beverage for 4 weeks can significantly increase the total antioxidant capacity by about 11.0%; the original value of the total antioxidant capacity in week 0 is 0.74 nmol/ L, the original value of total antioxidant capacity at week 4 was 0.83 nmol/L.

Referring to Figure 15, compared to before drinking (week 0), drinking cantaloupe and eggplant fermented beverage for 4 weeks can significantly reduce peroxide products (MDA) by about 26.1%; the original value of MDA in week 0 is 1.1 nmol/ml , the original value of MDA in the fourth week was 0.9nmol/ml.

Example 14: Human testing to improve skin condition

Make 8 obese subjects (that is, their body fat rate is greater than 25% or BMI value greater than 24) to drink a bottle of 50mL cantaloupe fermented beverage (which contains 12vol% of the cantaloupe and tomato fermented juice obtained in Example 1 and 88vol% water) for 4 consecutive weeks. And, before drinking (ie the 0th week) and after drinking for 4 weeks (ie the 4th week), use the VISIA Skin Tester (VISIA Complexion Analysis System, purchased from Canfield Company, USA) and skin surface humidity tester (Corneometer CM825, purchased from C+K Company, Germany) were used to detect skin condition. Here, skin texture, skin wrinkles, and skin redness (red pigment) were measured with a VISIA skin tester, and skin moisture content was measured with a skin surface humidity tester.

After the measurement, the skin condition in the 0th week was used as the benchmark (ie, the relative skin condition in the 0th week was 100%), and the relative skin condition (%) in the 4th week was calculated. Also, the statistically significant difference between the relative skin condition at week 0 and the relative skin condition at week 4 was statistically analyzed by Student's t-test, as shown in Figures 16-19. In Figures 16 to 19, "*" represents a p-value less than 0.05 in comparison with Week 0.

Referring to Figure 16 to Figure 19, compared to before drinking (week 0), drinking cantaloupe eggplant fermented beverage for 4 weeks can significantly reduce skin texture by about 4.3%, reduce skin wrinkles by about 5.3%, and reduce skin red pigment by about 4.3%. 7.6%, and significantly increased skin moisture content by about 9.6%. It can be seen that the long-term use of the cantaloupe tomato fermented juice can improve the skin condition, that is, the cantaloupe tomato fermented juice has the effect of improving the skin condition.

To sum up, according to the fermented juice of cantaloupe tomato according to any embodiment of the present invention, a composition for improving skin condition and/or reducing fat can be prepared. In other words, the aforementioned composition has one or more of the following functions: enhancing glutathione synthesis, inhibiting the formation of glycation end products, enhancing anti-aging-related genes, inhibiting melanin production, enhancing moisturizing-related gene secretion, inhibiting fat accumulation, promoting lipolysis, Reduces body fat in receptors and improves skin condition.

Claims (8)

A use of cantaloupe tomato fermented juice, which is used to prepare a method for improving skin anti-aging ability, improving anti-glycation ability, reducing melanin content, improving skin moisturizing, reducing skin texture, reducing skin wrinkles, reducing skin redness or a combination thereof. The composition, wherein the cantaloupe tomato fermented juice is obtained by a fermentation process carried out by a cantaloupe tomato culture liquid and a plurality of bacterial species, wherein the cantaloupe tomato culture liquid comprises cantaloupe tomato juice and water, and the ratio of the cantaloupe tomato juice to the water is: 1:2-5, and relative to the cantaloupe and eggplant culture solution, the plurality of strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria. The use according to claim 1, wherein the skin anti-aging ability is improved by regulating at least one of CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene or UBL5 gene. The use according to claim 1, wherein the improving skin moisturizing is regulating at least one of HAS2 gene or KRT1 gene. A kind of purposes of cantaloupe tomato fermented juice, it is used to prepare the composition for reducing fat, wherein this cantaloupe tomato fermented juice is obtained by a cantaloupe tomato culture liquid and a plurality of bacterial species to carry out a fermentation procedure, wherein the cantaloupe tomato cultured The liquid includes cantaloupe tomato juice and water, the ratio of the cantaloupe to the water is 1:2-5, and relative to the cantaloupe and tomato culture liquid, the plurality of strains include 0.01%-0.5% yeast, 0.01%-0.25% % lactic acid bacteria and 1%-15% acetic acid bacteria. The use of claim 4, wherein the reducing fat is inhibiting fat accumulation, promoting lipolysis, or a combination thereof. The use according to any one of claims 1 to 5, wherein the fermentation procedure comprises: adding the yeast to the cantaloupe culture solution; The cantaloupe eggplant culture liquid and the yeast are fermented for 1 to 3 days to form a first primary fermentation juice, wherein the yeast is Saccharomyces cerevisiae; the lactic acid bacteria are added to the first primary fermentation juice; the first primary fermentation juice is added. The primary fermentation juice and the lactic acid bacteria are fermented for 1 to 5 days to form a second primary fermentation juice, wherein the lactic acid bacteria are Streptococcus thermophilus; the acetic acid bacteria are added to the second primary fermentation juice; the second primary fermentation juice is mixed with The acetic acid bacteria are fermented for 3 to 10 days to form a third primary fermentation juice, wherein the acetic acid bacteria are Acetobacter aceti; and the third primary fermentation juice is filtered to obtain the cantaloupe tomato fermented juice. The use according to any one of claims 1 to 5, wherein the pH value of the cantaloupe fermented juice is 4.0±1.0, and the sugar content of the cantaloupe fermented juice is 40±2. The use according to any one of claims 1 to 5, wherein the polyphenol content of the cantaloupe tomato fermented juice is 88 ppm.

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