Use of plant fermented juice for the preparation of a composition for boosting the water metabolism rate of the body

Abstract

The present application discloses a use of plant fermented juice for preparing a composition for improving the water metabolism rate of the body. The plant fermented juice is prepared by initial fermentation of water extracts of rhizoma polygonati, euryale ferox and medlar with yeast and lactobacillus, and then secondary fermentation with acetic acid bacteria.

Description

[0001] This application is a divisional application of Chinese national patent application No. 202110054088.5, filed on January 15, 2021, entitled "Use of plant fermented juice in the preparation of a composition to enhance metabolism". Technical Field

[0002] This invention relates to a fermentation broth, and more particularly to the use of a fermentation broth of Polygonatum sibiricum, Euryale ferox and Lycium barbarum for preparing a composition that enhances metabolism. Background Technology

[0003] With consumers increasingly concerned about artificial compounds or additives, the concept of organic and natural diets is gaining popularity. Biotechnology companies and food manufacturers are actively investing in the research and development of products related to natural products.

[0004] Based on scientific verification of the health benefits of various plants, the analysis and efficacy evaluation of the active ingredients in different plants have become key aspects of product development. The main direction of product development is to improve or enhance bodily functions through natural plants, such as weight loss, skin whitening, gastrointestinal health, and ketogenic diets.

[0005] Among them, low metabolism, chronic inflammation, and edema are common health hazards for modern people. Low metabolism may further lead to various diseases such as kidney disease, fatty liver, arteriosclerosis / or myocardial weakness. Summary of the Invention

[0006] In some embodiments, the plant fermented juice is used to prepare compositions that enhance the body's water metabolism rate. The plant fermented juice is obtained by primary fermentation of an aqueous extract of Polygonatum kingianum, Euryale ferox, and Lycium chinense with yeast and lactobacillus, followed by secondary fermentation with acetic acid bacteria.

[0007] In some embodiments, plant fermentation juice is used to regulate cellular electrolyte balance. In some embodiments, plant fermentation juice is used to increase the expression levels of mitochondrial activity-related genes. In some embodiments, mitochondrial activity-related genes include at least one of the CCT5 gene, SIRT1 gene, and FOXO gene.

[0008] In some embodiments, the plant fermentation juice is used to modulate immune cells. In some embodiments, the plant fermentation juice is used to reduce the expression levels of anti-inflammatory response-related genes. In some embodiments, the anti-inflammatory response-related genes are at least one of the following: IL-1β gene, IL-8 gene, IL-6 gene, IL-18 gene, and TNF-α gene.

[0009] In some embodiments, plant fermentation juice is used to increase water metabolism.

[0010] In some embodiments, plant fermentation juice is used to increase urine output.

[0011] In some embodiments, plant fermentation juice is used to reduce edema.

[0012] In some embodiments, plant fermentation juices are used to improve cold hands and feet.

[0013] In some embodiments, the effective dosage of plant fermentation juice is 7 mL / day.

[0014] In summary, the plant fermented juice according to any embodiment can enhance intracellular mitochondrial activity, thereby regulating cellular electrolyte balance. The plant fermented juice according to any embodiment can reduce inflammatory responses in the body, thereby regulating immune cells by maintaining normal cellular function. The plant fermented juice according to any embodiment can increase urine output, thereby enhancing the body's water metabolism. The plant fermented juice according to any embodiment can enhance water metabolism in the body, reducing edema and maintaining bodily circulation to enhance metabolism. The plant fermented juice according to any embodiment can increase the temperature of extremities, thereby improving cold hands and feet. Furthermore, the plant fermented juice according to any embodiment, when consumed at a daily dose of 7 mL, can effectively enhance metabolism.

[0015] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the present invention. Attached Figure Description

[0016] Figure 1 This is a graph showing the total polyphenol content of water extracts and plant fermentation juices.

[0017] Figure 2 This is a diagram showing the experimental results of gene expression levels related to mitochondrial activity.

[0018] Figure 3 This is a graph showing the experimental results of the expression levels of anti-inflammatory genes in cells.

[0019] Figure 4 This is a graph showing the results of urine output testing in a human experiment.

[0020] Figure 5 This is a graph showing the results of edema index in human experiments.

[0021] Figure 6 This is a diagram showing the results of ankle circumference measurements in human experiments.

[0022] Figure 7This is a graph showing the results of a human experiment questionnaire survey. Detailed Implementation

[0023] In some embodiments, the plant fermentation juice is obtained by primary fermentation of aqueous extracts of Polygonatum kingianum, Euryale ferox, and Lycium chinense with yeast and lactic acid bacteria, followed by secondary fermentation with acetic acid bacteria. In some embodiments, the aqueous extract is obtained from Polygonatum kingianum, Euryale ferox, Lycium chinense, and water. In some embodiments, the aqueous extract is obtained from Polygonatum kingianum, Euryale ferox, Lycium chinense, water, and glucose.

[0024] In some embodiments, *Polygonatum kingianum* is a deciduous shrub belonging to the genus *Polygonatum* in the family Liliaceae. In some embodiments, the raw material is the rhizome of the *Polygonatum* plant. In some embodiments, the raw material is the dried rhizome of *Polygonatum*. For example, the raw material can be commercially available.

[0025] In some embodiments, *Euryale ferox* is an aquatic plant belonging to the genus *Euryale* of the family Nymphaeaceae, also known as foxnut or water caltrop. In some embodiments, *Euryale ferox* raw material refers to the fruit of the *Euryale* plant, also known as foxnut rice or water caltrop. In some embodiments, *Euryale ferox* raw material refers to the kernel of the *Euryale ferox* seed. In some embodiments, *Euryale ferox* kernel is obtained by removing the pericarp from the *Euryale ferox* fruit, extracting the kernel, removing the hard shell from the kernel, and then drying it. In some embodiments, *Euryale ferox* raw material refers to *Euryale ferox* kernel obtained by stir-frying. For example, *Euryale ferox* raw material can be commercially available *Euryale ferox* kernel.

[0026] In some embodiments, the wolfberry (Lycium chinense) plant is a deciduous shrub belonging to the genus Lycium in the family Solanaceae. Its fruit is commonly known as wolfberry or wolfberry seed, its young leaves are called wolfberry shoots, and its root bark is called lycium bark. In some embodiments, the wolfberry raw material is the wolfberry fruit. In some embodiments, the wolfberry raw material is dried wolfberry fruit. For example, the wolfberry raw material can be commercially available dried wolfberry fruit.

[0027] In some embodiments, the aqueous extract is prepared from Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water in a weight ratio of 2:2:0.5~1.5:38~40. In some embodiments, the aqueous extract is prepared from Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water in a weight ratio of 2:2:1:40. In some embodiments, the aqueous extract is prepared by mixing Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water in a ratio of 2:1:80 and then subjecting the mixture to high-temperature extraction (e.g., 90±5°C) for 60 to 70 minutes. In some embodiments, the aqueous extract is prepared by mixing Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water in a ratio of 2:2:1:40 and then subjecting the mixture to high-temperature extraction (e.g., 95°C) for 60 minutes. In some embodiments, the Brix degree of the aqueous extract is greater than or equal to 9.

[0028] In some embodiments, the aqueous extract is prepared from Polygonatum sibiricum, Euryale ferox, Lycium barbarum, water, and glucose. The weight ratio of Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water is 2:2:1:40, and the glucose content is 11% (V / V) relative to the total weight of the Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water. In some embodiments, the aqueous extract is prepared by mixing Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water in a ratio of 2:2:1:40, then adding 11% glucose, and then subjecting the mixture to high-temperature extraction (e.g., 95°C) for 1 hour. In some embodiments, the aqueous extract is prepared by mixing Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water in a ratio of 2:2:1:40, then adding glucose to achieve a Brix degree (Brix°) greater than or equal to 9. This means that the Brix level of the solution is measured simultaneously during the addition of glucose, and the addition of glucose is stopped when the Brix level reaches 9 or above.

[0029] In some embodiments, the aqueous extract is added directly to the microbial culture for fermentation without further filtering out the solids (i.e., Polygonatum sibiricum raw material / or Euryale ferox raw material / or Lycium barbarum raw material), so as to utilize the microbial culture to further extract the active ingredients in the solids, thereby obtaining plant fermented juice.

[0030] In some embodiments, after high-temperature extraction, the aqueous extract is cooled for use in subsequent fermentation processes. In some embodiments, after high-temperature extraction, the aqueous extract is cooled to below 40°C for use in subsequent fermentation processes. In some embodiments, after high-temperature extraction, the aqueous extract is cooled to 35°C ± 2°C for use in subsequent fermentation processes.

[0031] In some embodiments, after inoculating the aqueous extract with a bacterial strain, a fermentation process is performed to obtain a plant fermented juice. This fermentation process is divided into a primary fermentation process and a secondary fermentation process. First, the primary fermentation process involves adding 0.05wt% to 0.15wt% yeast and 0.025% to 0.01wt% lactic acid bacteria to the aqueous extract and allowing it to ferment at room temperature for 2 to 5 days to form a primary fermentation broth. In some embodiments, the primary fermentation process involves adding 0.1wt% yeast and 0.05wt% lactic acid bacteria to the aqueous extract and allowing it to ferment at 25°C to 28°C for 3 days to form a primary fermentation broth.

[0032] In some embodiments, the yeast may be *Saccharomyces cerevisiae*. For example, the lactic acid bacteria may be *Saccharomyces cerevisiae* strain BCRC 20271 (international accession number ATCC26602) or other commercially available brewer's yeasts.

[0033] In some embodiments, the lactic acid bacteria may be *Lactobacillus plantarum*. For example, the lactic acid bacteria may be strain TCI028, which is deposited at the Bioresource Conservation and Research Center of the Food Industry Development Institute with accession number BCRC910805, and is also deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) with international accession number DSM33108.

[0034] Next, the primary fermentation broth obtained in the primary fermentation process undergoes a secondary fermentation process. In some embodiments, the secondary fermentation process involves adding 5 wt% acetic acid bacteria to the primary fermentation broth, allowing it to stand, and determining that its Brix content is less than 4 and its pH value is 3.8 ± 0.5, at which point fermentation is complete, forming a plant fermentation stock broth. In some embodiments, the secondary fermentation process involves adding 5 wt% acetic acid bacteria to the primary fermentation broth and allowing it to ferment at room temperature for 3 to 6 days to form a plant fermentation stock broth. In some embodiments, the secondary fermentation process involves adding 5 wt% acetic acid bacteria to the primary fermentation broth and allowing it to ferment at 25°C to 28°C for 4 days to form a plant fermentation stock broth. In some embodiments, acetic acid bacteria strain BCRC11688 (International Depositary ATCC 15973) is used.

[0035] In some embodiments, the plant fermentation liquid obtained from the fermentation process is the plant fermentation juice. In other embodiments, the plant fermentation liquid obtained after the fermentation process may undergo at least one of the following further processing procedures: filtration, vacuum concentration, flavoring, filling, and sterilization to form plant fermentation juice. Here, the filtration, vacuum concentration, flavoring, filling, or sterilization processes are used to extend the shelf life and taste of the plant fermentation juice and prevent spoilage.

[0036] In some embodiments, the filtration process involves filtering the plant fermentation liquid through a 400-mesh screen to form a plant fermented juice. Here, solids are removed by using a screen with an appropriate mesh size.

[0037] In some embodiments, the vacuum concentration process involves concentrating the plant fermentation broth under reduced pressure at 55°C to 65°C to form a plant fermented juice. For example, the temperature setting for vacuum concentration is 60°C. In some embodiments, the pressure setting for vacuum concentration is 150 bar. Here, vacuum concentration removes alcohol from the plant fermented juice and reduces its storage volume.

[0038] In some embodiments, the flavoring process involves adding oligosaccharides to the plant fermentation broth to form a plant fermentation juice. Here, oligosaccharides refer to low-molecular-weight sugars composed of 3 to 10 monosaccharide molecules. In some embodiments, oligosaccharides may be fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, etc. In one embodiment, 40% to 70% of the oligosaccharides are added relative to the plant fermentation broth. In another embodiment, 60% of the oligosaccharides are added relative to the plant fermentation broth. In one embodiment, the addition of oligosaccharides is stopped when the Brix degree of the plant fermentation broth reaches 42 ± 2, thus obtaining the plant fermentation juice. Here, the addition of oligosaccharides is to adjust the osmotic pressure of the plant fermentation juice, reducing the water activity within the juice and preventing the growth of other microorganisms that could affect the quality of the juice.

[0039] In some embodiments, the filling process involves aliquoting the plant fermented juice / or plant fermented stock solution into appropriately sized storage containers. In some embodiments, the filling process involves aliquoting the plant fermented juice / or plant fermented stock solution into 7 mL storage containers.

[0040] In some embodiments, the sterilization process involves heating the plant fermentation juice / or plant fermentation stock solution to 95°C ± 5°C for at least 60 minutes. In some embodiments, the sterilization process involves heating the plant fermentation juice / or plant fermentation stock solution to 100°C for 70 minutes.

[0041] In some embodiments, the total polyphenol content of the plant fermentation juice / or plant fermentation concentrate reaches 62 ppm. Polyphenols have antioxidant properties, thereby helping to boost human metabolism.

[0042] In some embodiments, plant fermentation juice is used to enhance the expression of genes related to mitochondrial activity. It is generally believed that higher mitochondrial activity corresponds to a lower physiological age of the cell. That is, higher expression levels of genes related to mitochondrial activity indicate higher bodily function and stronger metabolic capacity. Therefore, genes related to mitochondrial activity are sometimes referred to as anti-aging genes. High mitochondrial activity can also help cells regulate electrolyte balance (such as sodium, potassium, and calcium).

[0043] In some embodiments, mitochondrial activity-related genes include at least one of the following: CCT5 gene (GeneID: 22948), SIRT1 gene (GeneID: 23411), and FOXO gene (GeneID: 2308). Specifically, increased expression of the CCT5 gene indicates a return to a youthful cellular state; the SIRT1 gene assists mitochondria in repairing damaged DNA to delay aging; and the FOXO gene restores mitochondrial activity to maintain cellular youthfulness.

[0044] In some embodiments, plant fermentation juice is used to reduce the expression levels of genes related to anti-inflammatory responses. In some embodiments, the genes related to anti-inflammatory responses are at least one of the following: IL-1β gene (GeneID: 3553), IL-8 gene (GeneID: 3576), IL-6 gene (GeneID: 3569), IL-18 gene (GeneID: 3606), and TNF-α gene (GeneID: 7124).

[0045] Among them, the IL-1β gene is an anti-inflammatory cytokine that can activate vascular endothelial cells and lymphocytes, and destroy local tissues to make it easier for immune cells to enter.

[0046] The expression of the IL-8 gene induces chemotaxis in neutrophils, macrophages, basophils, and T cells, causing them to migrate to sites of inflammation.

[0047] Overexpression of the IL-6 gene can cause an inflammatory response and stimulate hepatocytes to produce acute-phase proteins.

[0048] Among them, the IL-18 gene can induce T cells to produce interferon-γ (IFNγ).

[0049] Among them, the TNF-α gene (tumor necrosis factor-α) has the function of regulating immune cells, and is also involved in systemic inflammation cytokines. It is also one of the many cytokines that cause acute phase reactions.

[0050] In some embodiments, plant fermentation juice is used to increase water metabolism.

[0051] In some embodiments, plant fermentation juice is used to increase urine output.

[0052] In some embodiments, plant fermentation juice is used to reduce edema.

[0053] In some embodiments, plant fermentation juices are used to improve cold hands and feet.

[0054] In some embodiments, the effective dosage of plant fermentation juice is 7 mL / day.

[0055] In summary, the plant fermented juice according to any embodiment can enhance mitochondrial activity within cells, thereby improving metabolism by increasing cell activity. The plant fermented juice according to any embodiment can reduce inflammation in the body, thereby improving metabolism by maintaining normal cell function. The plant fermented juice according to any embodiment can increase urine output, thereby improving metabolism by enhancing water metabolism. The plant fermented juice according to any embodiment can reduce edema by enhancing water metabolism and maintaining blood circulation, thereby improving metabolism. The plant fermented juice according to any embodiment can increase peripheral temperature, thereby improving cold hands and feet. Furthermore, the plant fermented juice according to any embodiment can effectively improve metabolism when taken at a daily intake of 7 mL. In some embodiments, the plant fermented juice can be used to prepare compositions that enhance metabolism.

[0056] In some embodiments, any of the foregoing compositions may be a pharmaceutical product. In other words, the pharmaceutical product comprises an effective amount of plant fermentation juice.

[0057] In some embodiments, the aforementioned pharmaceutical products may be manufactured using techniques known to those skilled in the art into dosage forms suitable for oral, parenterally, oral, or topically administration.

[0058] In some embodiments, enteral or oral dosage forms may be, but are not limited to, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, or the like. In some embodiments, non-enteral or localized dosage forms may be, but are not limited to, injections, sterile powders, external preparations, or the like. In some embodiments, injections may be administered via subcutaneous injection, intraepidermal injection, intradermal injection, or intralesional injection.

[0059] In some embodiments, the aforementioned pharmaceutical products may include pharmaceutically acceptable carriers widely used in pharmaceutical manufacturing techniques. In some embodiments, a pharmaceutically acceptable carrier may be one or more of the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The type and quantity of carriers selected fall within the scope of professional competence and routine techniques of those skilled in this art. In some embodiments, the solvent that serves as a pharmaceutically acceptable carrier may be water, normal saline, phosphate-buffered saline (PBS), or an aqueous solution containing alcohol.

[0060] In some embodiments, any of the foregoing compositions may be an edible product (i.e., a food composition). In other words, the edible product contains a specific amount of plant fermentation juice. In some embodiments, the edible product may be a general food, a health food, a dietary supplement, or a food additive.

[0061] In some embodiments, the aforementioned edible products may be manufactured into dosage forms suitable for oral administration using techniques known to those skilled in the art. In some embodiments, general food may be, but is not limited to, beverages, fermented foods, bakery products, or seasonings.

[0062] In some embodiments, the plant fermentation juice of any embodiment (i.e., as a food additive) can be added during the preparation of raw materials by conventional methods, or the plant fermentation juice of any embodiment (i.e., as a food additive) can be added during the production of food, and thus formulated with any edible material into an edible product for human and non-human animal consumption.

[0063] Example 1: Preparation of plant fermentation juice

[0064] First, prepare raw materials of Polygonatum rhizome, Euryale ferox seed, and wolfberry in a weight ratio of 2:2:1:40, along with water. The Polygonatum rhizome used is dried Polygonatum rhizome purchased from New Century Hanfang Biotechnology Co., Ltd.; the Euryale ferox seed used is roasted Euryale ferox seed kernel purchased from New Century Hanfang Biotechnology Co., Ltd.; and the wolfberry used is dried wolfberry fruit purchased from New Century Hanfang Biotechnology Co., Ltd.

[0065] Next, the raw materials of Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water are mixed, and glucose is added at 11% of the total weight of the raw materials of Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water to form a plant-based liquid. The Brix content of this plant-based liquid is greater than 9.

[0066] The plant-based liquid was heated to 95°C and then maintained at 95°C for 60 minutes to obtain an aqueous extract. Once the aqueous extract has cooled to below 40°C, it can be used as a culture medium for subsequent fermentation processes.

[0067] 0.1 wt% of Saccharomyces cerevisiae and 0.05 wt% of Lactobacillus plantarum were added to the culture medium and the mixture was allowed to stand for three days to form the initial fermentation broth. The Saccharomyces cerevisiae used was registered under license number BCRC 20271, and the Lactobacillus plantarum strain used was TCI028, registered under license number BCRC 910805.

[0068] Next, 5 wt% acetic acid bacteria were added to the initial fermentation broth and allowed to ferment for four days to form the plant fermentation stock broth. The acetic acid bacteria used were those with the registration number BCRC11688. The Brix content of the plant fermentation stock broth was less than 4, and its pH value was 4.5 ± 0.5.

[0069] The plant fermentation broth was filtered through a sieve with a pore size of 400 mesh. The filtered plant fermentation broth was then concentrated under reduced pressure at 60°C and 150 bar. Isomaltooligosaccharides were added at a concentration of 60% relative to the plant fermentation broth to obtain the plant fermentation juice.

[0070] Example 2: Total polyphenol content test

[0071] 2.1. Testing Process

[0072] The aqueous extract obtained in Example 1 was used as the control group sample, and the plant fermentation broth was used as the experimental group sample. Each sample was diluted with water, and 100 μL was transferred to a centrifuge tube. Next, 500 μL of Folin-Ciocalteu's phenol reagent (Merck 1.09001.0100) was added to the centrifuge tube, mixed with the diluted sample, and allowed to stand for 3 minutes. Then, 400 μL of 7.5% (w / v) sodium carbonate (Sigma 31432) was added, mixed well, and allowed to stand for 30 minutes to obtain the test reaction solution. After confirming that no air bubbles were present in the test reaction solution, 200 μL of the test reaction solution was transferred to a 96-well plate, and the absorbance of the test reaction solution at 750 nm was measured. Each sample was tested in triplicate, and the average value after rounding was taken.

[0073] Furthermore, a standard curve was prepared using gallic acid as a standard. Gallic acid (powder, Sigma G7384) was used to prepare standard solutions of 0 μL / mL, 20 μL / mL, 40 μL / mL, 60 μL / mL, 80 μL / mL, and 100 μL / mL with pure water. 100 μL of each concentration of standard solution was then transferred to a 10 mL centrifuge tube. 500 μL of Folin-Ciocalteu phenol reagent was added to the centrifuge tube, mixed with the standard solution, and allowed to stand for 3 minutes. Then, 400 μL of 7.5% sodium carbonate was added, mixed, and allowed to stand for 30 minutes to obtain the standard reaction solution. 200 μL of the standard reaction solution was transferred to a 96-well plate, and its absorbance was measured at 750 nm. A standard curve was then plotted using the concentration of gallic acid and its corresponding absorbance value.

[0074] Next, the absorbance of the test reaction solution was converted into concentration using the interpolation method based on the standard curve, and then multiplied back by the dilution factor to obtain the total polyphenol content of each sample.

[0075] 2.2. Test Results

[0076] Thus, the total polyphenol content of the water extract is only 6 ppm and the total polyphenol content of the plant fermentation juice is 62 ppm. Figure 1 As shown.

[0077] The experimental results show that the total polyphenol content of the plant fermentation juice is nearly 10 times higher than that of the water extract. Therefore, it can be concluded that fermentation by yeast, lactic acid bacteria, and acetic acid bacteria is more conducive to the extraction of active ingredients.

[0078] Example 3: Mitochondrial activity test

[0079] Mitochondria are organelles within cells that primarily synthesize adenosine triphosphate (ATP) as the cell's energy source. Sufficient mitochondria in cells generally indicate younger, more elastic cells and a higher metabolic rate. Furthermore, since blood vessels are distributed throughout the body, and monocytes originate in the bone marrow and are distributed within blood vessels, they can also enter other body cells as needed. Therefore, monocytes are used as experimental cell lines to test the expression levels of related genes, thereby extending the effect of enhancing basal metabolism.

[0080] 3.1 Materials, Instruments and Solution Preparation

[0081] Experimental cells: Human mononuclear spheroid cells THP-1 (hereinafter referred to as mononuclear spheroid cells, purchased from ATCC, with storage number TBI202).

[0082] Culture medium: RPMI 1640 medium (Gibco brand, model RPMI medium 1640, purchased from ThermoFisher Scientific, serial number 31800-022) supplemented with 10% fetal bovine serum (FBS, Gibco brand, serial number 10438-026) and 1% antibiotic-antimycotic (Gibco brand, serial number 15240-062).

[0083] Reagents: RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G), KAPASYBR® FAST qPCR reagent kit (purchased from Sigma, USA, catalog number 38220000000).

[0084] Reverse transcriptase: SuperScript® III Reverse Transcriptase was used from Invitrogen, USA, product number 18080-051.

[0085] Testing instrument: ABI StepOnePlus™ Real-Time PCR system (purchased from ThermoFisher Scientific, USA).

[0086] 3.2. Testing Process

[0087] Add 2 mL of the above culture medium to each well of a six-well plate and inoculate with 1 x 10⁻⁶ cells / well. 6 Overnight culture of mononuclear spheroids.

[0088] The cells cultured overnight were divided into two groups (control group and experimental group). The control group received no test sample. The experimental group received the plant fermentation stock solution prepared as shown in Example 1 as the test sample. The concentration of the test sample in the culture medium of the experimental group was 1.0% (v / v).

[0089] The control and experimental groups were cultured at 37°C for 24 hours. After 24 hours, the cells were centrifuged at 400xg for 5 minutes to remove the supernatant, washed with 1X dpbs buffer, centrifuged at 400xg for 5 minutes again, removed the supernatant, and lysed with 600µL of RB buffer.

[0090] Next, RNA was collected from the cell solutions of the two groups using an RNA extraction reagent kit. Then, 2000 ng of the extracted RNA from each group was used as a template, and reverse transcription was performed using reverse transcriptase with the primers listed in Table 1 to produce the corresponding cDNA. Subsequently, a real-time PCR system and qPCR reagent kit were used to perform quantitative real-time reverse transcription polymerase chain reaction (qPCR) on the reverse transcribed products of the two groups using the primer combinations listed in Table 1 to observe the gene expression levels in the monocytes of the experimental and control groups. The instrument settings for the qPCR were 95°C for 1 second, 60°C for 20 seconds, for a total of 40 cycles, and gene quantification was performed using the 2-ΔCt method. Thus, by using cDNA for qPCR, the mRNA expression level of each gene can be indirectly quantified, thereby inferring the expression level of the protein encoded by each gene.

[0091] Table 1

[0092]

[0093] and, Figure 2 The relative gene expression of each gene shown is presented as a relative ploidy rate. The standard deviation was calculated using the STDEV formula in Excel, and a one-tailed Student's t-test was used in Excel 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 "*" symbols, the more significant the statistical difference.

[0094] 3.3. Test Results

[0095] Please see Figure 2When the expression level of the CCT5 gene in the control group is considered as 1 (i.e., 100%), the expression level of the CCT5 gene in the experimental group relative to the control group is 1.65 (i.e., 120%), which means that the expression level of the CCT5 gene in the experimental group is 1.65 times that of the control group.

[0096] When the expression level of the SIRT1 gene in the control group is considered as 1 (i.e., 100%), the expression level of the SIRT1 gene in the experimental group relative to the control group is 1.32 (i.e., 130%), which means that the expression level of the SIRT1 gene in the experimental group is 1.32 times that of the control group.

[0097] When the expression level of the FOXO gene in the control group is considered as 1 (i.e., 100%), the expression level of the FOXO gene in the experimental group relative to the control group is 1.21 (i.e., 150%), which means that the expression level of the FOXO gene in the experimental group is 1.21 times that of the control group.

[0098] Depend on Figure 2 It is evident that after mononuclear spherocytes are treated with plant fermentation juice made from Polygonatum sibiricum, Euryale ferox, and Lycium barbarum, they can effectively enhance mitochondrial activity and thus improve metabolism.

[0099] Example 4: Cellular anti-inflammatory test

[0100] There is a growing consensus in the medical community that chronic inflammation is one of the causes of metabolic syndrome. Monocytes are produced in the bone marrow and distributed in blood vessels. In cases of inflammation, they trigger a corresponding systemic immune response. Therefore, experiments using monocytes as an experimental cell line to measure the expression levels of related anti-inflammatory genes have shown that reducing the expression of inflammation-related genes indicates a normal state of the body, which may extend to improving metabolic efficiency.

[0101] 4.1 Materials, Instruments and Solution Preparation

[0102] Experimental cells: Human mononuclear spheroid cells THP-1 (hereinafter referred to as mononuclear spheroid cells, purchased from ATCC, with storage number TBI202).

[0103] Culture medium: RPMI 1640 medium (Gibco brand, model RPMI medium 1640, purchased from ThermoFisher Scientific, serial number 31800-022) supplemented with 10% fetal bovine serum (FBS, Gibco brand, serial number 10438-026) and 1% antibiotic-antimycotic (Gibco brand, serial number 15240-062).

[0104] Reagents: RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G), KAPASYBR® FAST qPCR reagent kit (purchased from Sigma, USA, catalog number 38220000000).

[0105] Reverse transcriptase: SuperScript® III Reverse Transcriptase was used from Invitrogen, USA, product number 18080-051.

[0106] Testing instrument: ABI StepOnePlus™ Real-Time PCR system (purchased from ThermoFisher Scientific, USA).

[0107] 4.2. Testing Procedure

[0108] Add 2 mL of the above culture medium to each well of a six-well plate and inoculate with 1 x 10⁻⁶ cells / well. 6 Overnight culture of mononuclear spheroids.

[0109] The cells cultured overnight were divided into two groups (control group and experimental group). The control group received no test sample. The experimental group received the plant fermentation stock solution prepared as shown in Example 1 as the test sample. The concentration of the test sample in the culture medium of the experimental group was 1.0% (v / v).

[0110] The control and experimental groups were cultured at 37°C for 24 hours. After 24 hours, the cells were centrifuged at 400xg for 5 minutes to remove the supernatant, washed with 1X dpbs buffer, centrifuged at 400xg for 5 minutes again, removed the supernatant, and lysed with 600µL of RB buffer.

[0111] Next, RNA was collected from the cell solutions of the two groups using an RNA extraction reagent kit. Then, 2000 ng of the extracted RNA from each group was used as a template, and reverse transcription was performed using reverse transcriptase with the primers listed in Table 2 to produce the corresponding cDNA. Subsequently, a real-time PCR system and qPCR reagent kit were used to perform quantitative real-time reverse transcription polymerase chain reaction (qPCR) on the reverse transcribed products of the two groups using the primer combinations listed in Table 2 to observe the gene expression levels in the monocytes of the experimental and control groups. The instrument settings for the qPCR were 95°C for 1 second, 60°C for 20 seconds, for a total of 40 cycles, and gene quantification was performed using the 2-ΔCt method. Thus, by using cDNA for qPCR, the mRNA expression level of each gene can be indirectly quantified, thereby inferring the expression level of the protein encoded by each gene.

[0112] Table 2

[0113]

[0114] and, Figure 3 The relative gene expression of each gene shown is presented as a relative ploidy rate. The standard deviation was calculated using the STDEV formula in Excel, and a one-tailed Student's t-test was used in Excel 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 "*" symbols, the more significant the statistical difference.

[0115] 4.3. Test Results

[0116] Please see Figure 3 When the expression level of the IL-1β gene in the control group is considered as 1 (i.e., 100%), the expression level of the IL-1β gene in the experimental group relative to the control group is 0.58 (i.e., 58%), which means that the expression level of the IL-1β gene in the experimental group is 42% lower than that in the control group.

[0117] When the expression level of the IL-8 gene in the control group is considered as 1 (i.e., 100%), the expression level of the IL-8 gene in the experimental group relative to the control group is 0.59 (i.e., 59%), which means that the expression level of the IL-8 gene in the experimental group is 41% lower than that in the control group.

[0118] When the expression level of the IL-6 gene in the control group is considered as 1 (i.e., 100%), the expression level of the IL-6 gene in the experimental group relative to the control group is 0.25 (i.e., 25%), which means that the expression level of the IL-6 gene in the experimental group is reduced by 75% compared with the control group.

[0119] When the expression level of the IL-18 gene in the control group is considered as 1 (i.e., 100%), the expression level of the IL-18 gene in the experimental group relative to the control group is 0.73 (i.e., 73%), which means that the expression level of the IL-18 gene in the experimental group is 27% lower than that in the control group.

[0120] When the expression level of the TNF-α gene in the control group is considered as 1 (i.e., 100%), the expression level of the TNF-α gene in the experimental group relative to the control group is 0.48 (i.e., 48%), which means that the expression level of the TNF-α gene in the experimental group is 52% lower than that in the control group.

[0121] Depend on Figure 3 It is evident that monocytes, after being treated with the plant fermentation juice made from Polygonatum sibiricum, Euryale ferox, and Lycium barbarum, can effectively reduce systemic inflammation and thus enhance metabolism.

[0122] Example 5: Human Body Test 1

[0123] 5-1. Sample Preparation

[0124] Fermented plant juice: The fermented plant juice prepared in Example 1 was used.

[0125] 5-2. Subjects: Four subjects (females aged 25-30). All subjects were prone to edema. That is, this test selected subjects with poor water metabolism and metabolic rate.

[0126] 5-3. Test Item: Urine Output

[0127] 5-4. Testing Method:

[0128] On the first day, four subjects emptied their bladders at 9:00 AM and then drank 50 mL of water. They then drank 180 mL of water every hour. The total amount of urine produced by each subject between 9:00 AM and 1:00 PM was recorded. This group was the control group.

[0129] On the second day, the same four subjects emptied their bladders at 9:00 a.m. and then began to drink 7 mL of plant fermentation juice and 43 mL of water. They then drank 180 mL of water every hour. The total amount of urine produced by each subject between 9:00 a.m. and 1:00 p.m. was recorded. This was the experimental group.

[0130] 5-5. Test Results

[0131] Please see Figure 4 When participants consumed fermented plant juice, their average total urine output increased from 465 mL to 785 mL, a difference of 40%. This means that drinking 7 mL of fermented plant juice can effectively increase urine output and improve the body's water metabolism rate.

[0132] Example 6: Human Body Test II

[0133] 6-1. Sample Preparation

[0134] Fermented plant juice: The fermented plant juice prepared in Example 1 was used.

[0135] 6-2. Subjects: 3 subjects (females aged 25-30). All subjects were prone to edema. This means that subjects with poor water metabolism and metabolic rate were selected for this test.

[0136] 6-3. Test items: lower limb edema index, ankle circumference and body sensation questionnaire.

[0137] 6-4. Testing Method:

[0138] Three subjects were instructed to drink 7 mL of fermented plant juice daily for eight consecutive weeks. Measurements were taken before drinking (week 0, also known as the control group) and after eight weeks of drinking (week 8, also known as the experimental group).

[0139] The lower extremity edema index was measured using an X-SCAN body composition analyzer on the subjects' lower extremities. This measurement employs bioelectrical impedance analysis (BIA), utilizing microcurrents to measure the lower limbs. The edema index is the ratio of extracellular water (ECW) to total body water (TBW). A higher edema index indicates more severe edema.

[0140] Ankle circumference is a common clinical method used by physicians to determine whether patients have poor water metabolism. Therefore, ankle circumference was measured using a tape measure for each subject.

[0141] Participants completed a body sensation questionnaire at week 0 and week 8, which surveyed various metabolic-related conditions. The survey and scoring methods are shown in Table 3 below. In the first part, each score represents the severity of various symptoms: 1 for no abnormality, 2 for mild, 3 for moderate, 4 for somewhat severe, and 5 for severe.

[0142] Table 3

[0143]

[0144] The graph below shows the average ankle circumference of the participants. The standard deviation was calculated using the STDEV formula in Excel, and a one-tailed Student's t-test was used in Excel to analyze whether there was a statistically significant difference. In the graph, "*" indicates a p-value less than 0.05, representing a statistically significant difference.

[0145] 6-5. Test Results

[0146] Please see Figure 5 After 8 weeks of daily consumption of the plant fermented juice, the average edema index of the three subjects decreased from 0.387 ECW / TBW (week 0) to 0.369 ECW / TBW (week 8). Furthermore, according to the reference values ​​for the edema index, <0.40 is considered normal, 0.40-0.41 is borderline edema, and >0.41 indicates edema has occurred. From the above reference values, a difference of 0.01 in the edema index means the transition from borderline edema to actual edema. However, after 8 weeks of drinking the plant fermented juice of this invention, the average difference in the edema index was as high as 0.018. Converted, the average edema index difference reached 4.5%. This means that drinking 7mL of plant fermented juice daily can effectively reduce edema and improve metabolism.

[0147] Please see Figure 6 After 8 weeks of daily consumption of plant-fermented juice, the average ankle circumference of the three subjects decreased from 21.25 cm (week 0) to 20.82 cm (week 8), a difference of 0.43 cm. This means that drinking 7 mL of plant-fermented juice daily can significantly reduce ankle edema. In one subject, at week 0, it took approximately 3 minutes for the indented skin to return to its original position when pressing on the edematous area of ​​the ankle, while at week 8, it only took 4 seconds.

[0148] refer to Figure 7Regarding question 1, which describes edema caused by prolonged sitting or standing, the average score from participants decreased from 4.17 to 0.67, indicating that participants' self-reported symptoms had returned to normal. Regarding question 2, which describes feeling that pants or shoes are too tight at night, the average score from participants decreased from 3 to 1.67. Regarding question 3, which describes feeling swelling in the hands, the average score from participants decreased from 2.67 to 1, indicating that participants' self-reported symptoms had returned to normal. Regarding question 4, which describes frequent fatigue, the average score from participants decreased from 4.33 to 2, indicating that participants' self-reported symptoms had returned to mild. Regarding question 5, which describes poor blood circulation or cold hands and feet, the average score from participants decreased from 4.67 to 3, indicating that participants' self-reported symptoms had returned from near the highest score (severe) to normal.

[0149] As shown above, the average scores for each question decreased, meaning that, on average, all subjects experienced a significant improvement in the aforementioned symptoms. The overall perception of a slowed metabolism decreased, and all subjects could clearly feel an improvement in their metabolic capacity.

[0150] Therefore, it can be seen that long-term use of plant fermented juice can enhance mitochondrial activity, reduce inflammation, increase urine output, reduce edema symptoms, and improve cold hands and feet, indicating that plant fermented juice has a significant effect on improving metabolism.

[0151] In summary, the plant fermented juice according to any embodiment can enhance mitochondrial activity within cells, thereby improving metabolism by increasing cell activity. The plant fermented juice according to any embodiment can reduce inflammatory responses in the body, thereby improving metabolism by maintaining normal cell function. The plant fermented juice according to any embodiment can increase urine output, thereby improving metabolism by enhancing water metabolism. The plant fermented juice according to any embodiment can reduce edema by enhancing water metabolism and maintain body circulation, thereby improving metabolism. The plant fermented juice according to any embodiment can increase peripheral temperature, thereby improving cold hands and feet. Furthermore, the plant fermented juice according to any embodiment can effectively improve metabolism when taken in 7 mL daily. In some embodiments, the plant fermented juice can be used to prepare compositions for improving metabolism.

[0152] Although the technical content of the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and refinements made by those skilled in the art without departing from the spirit of the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

[0153] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

[0154] [Sequence List]

[0155] <110> TCI Biotech Co., Ltd.

[0156] <120> Uses of plant fermentation juice in the preparation of compositions that enhance the body's water metabolism rate.

[0157] <150> 62 / 961,742

[0158] <151> 2020-01-16

[0159] <160> 16

[0160] <170> PatentIn version 3.5

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Claims

1. The use of a plant fermentation juice for preparing a composition that enhances the body's water metabolism rate, characterized in that... The plant fermentation juice is prepared by primary fermentation of water extracts of Polygonatum sibiricum, Euryale ferox, and Lycium barbarum with 0.05wt% to 0.15wt% yeast and 0.025% to 0.01wt% lactic acid bacteria, followed by secondary fermentation with 5wt% acetic acid bacteria. The weight ratio of Polygonatum sibiricum, Euryale ferox, Lycium barbarum, and water is 2:2:0.5~1.5:38~40.

2. The use according to claim 1, characterized in that, The plant fermentation juice is used to regulate the electrolyte balance of cells.

3. The use according to claim 2, characterized in that, The plant fermentation juice is used to increase the expression level of mitochondrial activity-related genes, which are at least one of the CCT gene, SIRT1 gene, and FOXO gene.

4. The use according to claim 1, characterized in that, The plant fermentation juice is used to regulate immune cells.

5. The use according to claim 4, characterized in that, The plant fermentation juice is used to reduce the expression level of anti-inflammatory response-related genes, which are at least one of the following: IL-1β gene, IL-8 gene, IL-6 gene, IL-18 gene, and TNF-α gene.

6. The use according to claim 1, characterized in that, The plant fermentation juice is used to increase urine output.

7. The use according to claim 1, characterized in that, The plant fermentation juice is used to reduce edema.

8. The use according to claim 1, characterized in that, The plant fermentation juice is used to improve cold hands and feet.

9. The use according to any one of claims 1 to 8, characterized in that, The Polygonatum is the dried rhizome of Polygonatum, the Euryale seed is the roasted seed kernel of Euryale, and the wolfberry is the dried fruit of wolfberry.

10. The use according to claim 9, characterized in that, The extraction step includes: mixing the Polygonatum sibiricum, Euryale ferox, Lycium barbarum and water in a weight ratio of 2:2:0.5-1.5:38-40, adding glucose to form a plant base liquid, and heating the plant base liquid and maintaining it at 95°C for 60 minutes to obtain the aqueous extract.

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