TWI764520B - Uses of a plant fermentation liquid in preparing composition for improving metabolism - Google Patents

Abstract

Uses of a plant fermentation liquid in preparing composition for reducing lipid. The plant fermentation liquid made from water extract ofPolygonatum kingianum, Euryale ferox andLycium chinense after first fermentation of Yeast and Lactobacillus and second fermentation of Acetic acid bacteria.

Description

Use of plant fermented juice for the preparation of a metabolism-boosting composition

The present invention relates to a fermentation liquid, in particular to the use of a fermentation liquid of Polygonatum chinensis, Gorgon Fructus and Lycium barbarum for preparing a composition for improving metabolism.

The concept of organic and natural diets is becoming more popular due to increasing consumer skepticism about artificial compounds or additives. Biotechnology companies and food companies are actively involved in the research and development of natural products related products.

On the basis of various scientific verifications that are beneficial to health, the active ingredient analysis and efficacy evaluation of different plants have become key projects in product development. The main direction of product development is to improve or enhance body functions through natural plants, such as weight loss, whitening, gastrointestinal health, ketogenic diet, etc.

Among them, too low metabolism, chronic inflammation of the body, and edema are common health hazards in modern people, and too low metabolism may further lead to various diseases such as kidney diseases, fatty liver, arteriosclerosis and/or myocardial weakness.

In some embodiments, the use of plant fermentation juices for the preparation of metabolism enhancing compositions. Among them, the plant fermented juice is obtained from the water extracts of Polygonatum kingianum , Euryale ferox and Lycium chinense after primary fermentation by yeast and lactobacillus, and then secondary fermentation with acetic acid bacteria.

In some embodiments, plant fermentation juice is used to increase the expression of genes related to mitochondrial activity. In some embodiments, mitochondrial activity-related genes include at least one of CCT5 gene, SIRT1 gene and FOXO gene.

In some embodiments, the plant fermentation juice is used to reduce the expression of anti-inflammatory response-related genes. In some embodiments, the anti-inflammatory response-related gene is at least one of IL-1β gene, IL-8 gene, IL-6 gene, IL-18 gene and TNF-α gene.

In some embodiments, the plant fermented juice is used to increase the amount of water metabolism.

In some embodiments, the plant fermented juice is used to increase urine output.

In some embodiments, the plant fermented juice is used to reduce edema.

In some embodiments, plant fermented juice is used to improve cold hands and feet.

In some embodiments, the effective amount of plant fermentation juice is 7 mL/day.

To sum up, the plant fermentation juice according to any one of the embodiments can enhance the activity of mitochondria in cells, and achieve the purpose of enhancing metabolism by enhancing the activity of cells. The plant fermented juice according to any one of the embodiments can reduce the inflammatory response in the body, and achieve the purpose of improving metabolism by maintaining the normal function of cells. The plant fermented juice according to any one of the embodiments can increase the amount of urine, and achieve the purpose of improving metabolism by improving water metabolism in the body. The plant fermented juice according to any one of the embodiments can improve water metabolism in the body, reduce edema, maintain body circulation, and achieve the purpose of increasing metabolism. According to the plant fermented juice of any one of the embodiments, the temperature of the limbs is increased, so as to improve the coldness of hands and feet. Moreover, according to the plant fermented juice of any one of the embodiments, the purpose of improving metabolism can be effectively achieved when 7 mL of the plant fermented juice is taken daily.

In some embodiments, the plant fermented juice is first fermented with yeast and lactobacillus, and then fermented with acetic acid bacteria from water extracts of Polygonatum kingianum , Euryale ferox and Lycium chinense . and made. In some embodiments, the water extract is prepared from Polygonatum raw material, Gorgon raw material, Lycium barbarum raw material and water. In some embodiments, the water extract is prepared from Polygonatum chinensis raw material, Gorgon raw material, Lycium barbarum raw material, water and glucose.

In some embodiments, the Polygonatum kingianum plant is a deciduous shrub of the genus Polygonatum in the Liliaceae family. In some embodiments, the raw material of Polygonatum chinensis is the rhizome of Polygonatum chinensis plant. In some embodiments, Polygonatum chinensis raw material is Polygonatum chinensis dried rhizomes. For example, Polygonatum raw materials may be commercially available.

In some embodiments, the Euryale ferox plant is an aquatic plant of the genus Euryale of the Nymphaeaceae family, also known as Gallantia chinensis. In some embodiments, the raw material of Gorgon Fructus refers to Gorgon fruit, also known as Gorgon Rice or Chicken Head Rice. In some embodiments, the raw material of gorgonian root refers to gorgonian root. In some embodiments, the gorgonian seed kernel is prepared by removing the pericarp of the gorgonian fruit, taking out the seed kernel, removing the hard shell of the seed kernel and drying it in the sun. In some embodiments, the raw material of Gorgon Fructus refers to the Gorgon Radix Seed Kernel obtained by hot frying. For example, the raw material of gorgonian can be commercially available gorgonian kernels.

In some embodiments, the Lycium chinense plant is a deciduous shrub of the genus Lycium ( Lycium ) of the Solanaceae family , and its fruit is commonly called Lycium barbarum or Lycium barbarum, its young leaves are called Lycium barbarum head, and its root bark is called terrestrial Bone skin. In some embodiments, the raw material of wolfberry is wolfberry fruit. In some embodiments, the raw material of wolfberry is the dried fruit of wolfberry. For example, the raw material of Lycium barbarum can be commercially available dried fruits of Lycium barbarum.

In some embodiments, the water extract is prepared from the raw materials of Polygonatum chinensis, the raw materials of Gorgon Fructus, the raw materials of Lycium barbarum and water in a weight ratio of 2:2:0.5~1.5:38~40. In some embodiments, the water extract is prepared from Polygonatum chinensis raw materials, Gorgon Fructus raw materials, Lycium barbarum raw materials and water in a weight ratio of 2:2:1:40. In some embodiments, the water leaching solution is a high-temperature leaching (eg, 90±5 minutes) after mixing the raw materials of Polygonatum japonicus, the raw materials of Gorgon Fructus, the raw materials of Lycium barbarum and water according to the ratio of 2:1:80 for 60 minutes to 70 minutes. ℃) prepared. In some embodiments, the water leaching solution is obtained by mixing the raw materials of Polygonatum japonicus, the raw materials of Gorgon Fructus, the raw materials of Lycium barbarum and water according to the ratio of 2:2:1:40 and then performing high temperature extraction (eg, 95°C) for 60 minutes. be made of. In some embodiments, the water extract has a Brix° greater than or equal to 9.

In some embodiments, the water extract is prepared from Polygonatum chinensis raw material, Gorgon raw material, Lycium barbarum raw material, water and glucose. Among them, the weight ratio of rhizoma rhizoma raw material, gorgonian raw material, wolfberry raw material and water is 2:2:1:40, and the content of glucose is 11% relative to the total weight of rhizoma rhizoma raw material, gorgonian raw material, wolfberry raw material and water (V/V). In some embodiments, the water extract is obtained by mixing the raw materials of Polygonatum japonicus, the raw materials of Gorgon Fructus, the raw materials of Lycium barbarum and water according to the ratio of 2:2:1:40, then adding 11% glucose, and then carrying out a high temperature for 1 hour. Extraction (eg, 95 ° C) prepared. In some embodiments, the water extract is made by mixing the raw materials of Polygonatum japonicus, the raw materials of Gorgon Fructus, the raw materials of Lycium barbarum and water according to the ratio of 2:2:1:40, and then adding glucose to make the Brix degree (Brix°) The amount of glucose greater than or equal to 9. That is, during the process of adding glucose, the Brix content of the solution is simultaneously measured, and when the Brix content reaches 9 or exceeds 9, the addition of glucose is stopped.

In some embodiments, the water extract is directly added to the bacteria for fermentation without filtering out the solids inside it (that is, the raw materials of Polygonatum japonicus/or the raw materials of Gorgon Fructus/or the raw materials of Lycium barbarum), so as to further extract the solids in the solids by using the bacterial strains. Active ingredients, and then obtain plant fermented juice.

In some embodiments, after high temperature leaching, the water extract is cooled down for use in subsequent fermentation processes. In some embodiments, after the high temperature leaching, the water leachate is cooled to less than 40°C for use in the subsequent fermentation process. In some embodiments, after high temperature extraction, the water extract is cooled to 35°C ± 2 for subsequent fermentation process.

In some embodiments, after the water extract is inoculated with the strain, a fermentation process is performed to obtain a plant fermented juice, and the fermentation process is divided into a primary fermentation process and a secondary fermentation process. First, the initial fermentation procedure is to add 0.05wt%~0.15wt% yeast and 0.025%~0.01wt% lactic acid bacteria to the water extract and ferment at room temperature for 2~5 days to form the primary fermentation liquid . In some embodiments, the primary fermentation procedure is to add 0.1 wt % yeast and 0.05 wt % lactic acid bacteria to the water extract and ferment at 25°C to 28°C for 3 days to form primary fermentation broth.

In some embodiments, the yeast may be Saccharomyces cerevisiae . For example, the lactic acid bacteria may be Saccharomyces cerevisiae strain deposited with the Biological Resource Conservation and Research Center (BCRC) of the Institute for Food Industry Development and Deposit No. BCRC 20271 (and International Deposit No. ATCC26602) or other commercially available brewer's yeast.

In some embodiments, the lactic acid bacteria may be Lactobacillus plantarum . For example, the lactic acid bacteria can be the TCI028 strain deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Institute and the deposit number BCRC 910805 strain, and this strain is also deposited in the German Collection of Microorganisms and Cell Cultures (German Collection of Microorganisms and Cell Cultures). , DSMZ) and its International Deposit Number is DSM33108.

Next, the primary fermentation liquid prepared in the primary fermentation process is subjected to a secondary fermentation process. In some embodiments, the secondary fermentation procedure is to add 5wt% of acetic acid bacteria to the initial fermentation broth and let it stand and detect that its Brix content is less than 4, and its pH value (pH value) is 3.8±0.5 to complete the fermentation, A plant fermentation stock solution is formed. In some embodiments, the secondary fermentation procedure is to add 5 wt% acetic acid bacteria to the primary fermentation broth and ferment at room temperature for 3 to 6 days to form a plant fermentation stock solution. In some embodiments, the secondary fermentation procedure is to add 5 wt% acetic acid bacteria to the primary fermentation broth and ferment at 25°C to 28°C for 4 days to form a plant fermentation stock solution. In some embodiments, acetic acid bacteria of the accession number BCRC11688 (International Accession ATCC15973) strain are used.

In some embodiments, the plant fermentation stock solution obtained from the fermentation procedure is the plant fermentation juice. In other embodiments, the plant fermentation stock solution obtained after the fermentation process can be subjected to at least one of the following reprocessing processes: filtration process, reduced pressure concentration process, seasoning process, filling process and sterilization process to form plant fermentation juice. Here, the filtration process, the decompression concentration process, the seasoning process, the filling process or the sterilization process are used to prolong the shelf life, taste and avoid spoilage of the plant fermented juice.

In some embodiments, the filtering procedure is to filter the plant fermentation stock through a 400 mesh mesh screen to form the plant fermentation juice. Here, solids are removed through a filter screen with an appropriate mesh.

In some embodiments, the reduced pressure concentration procedure is to concentrate the plant fermentation stock solution under reduced pressure at 55°C to 65°C to form a plant fermentation juice. For example, the temperature set point for concentration under reduced pressure is 60°C. In some embodiments, the reduced pressure concentration has a pressure setting of 150 bar (Bar). Here, the alcohol content in the plant fermented juice can be removed by concentration under reduced pressure, and the storage volume of the plant fermented juice can be reduced.

In some embodiments, the flavoring procedure is the addition of oligosaccharides to a plant fermentation stock to form a plant fermentation juice. Here, oligosaccharides refer to oligosaccharides formed by the polymerization of 3 to 10 monosaccharide molecules. In some embodiments, the oligosaccharides can be fructooligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomalt-oligosaccharides, and the like. In one embodiment, 40%-70% oligosaccharides are added relative to the plant fermentation stock solution. In one embodiment, 60% oligosaccharides are added relative to the plant fermentation stock. In one embodiment, when oligosaccharides are added to make the Brix° of the plant fermentation stock solution reach 42±2, the addition of oligosaccharides is stopped to obtain plant fermentation juice. Here, the oligosaccharide is added to adjust the osmotic pressure of the plant fermentation juice, so that the water activity in the plant fermentation juice is reduced, and the growth of other microorganisms is avoided to affect the quality of the plant fermentation juice.

In some embodiments, the filling procedure is to dispense the plant fermentation juice and/or plant fermentation stock into appropriately sized storage containers. In some embodiments, the filling procedure is to dispense the plant fermentation juice and/or plant fermentation stock into 7 mL storage containers.

In some embodiments, the sterilization procedure is heating the plant fermentation juice/or plant fermentation stock to 95°C ± 5 for at least 60 minutes. In some embodiments, the sterilization procedure is heating the plant fermentation juice/or plant fermentation stock to 100°C for 70 minutes.

In some embodiments, the plant fermentation juice/or plant fermentation stock has a total polyphenol content of 62 ppm. Polyphenols have anti-oxidative properties, which help to boost the body's metabolism.

In some embodiments, plant fermentation juice is used to increase the expression of genes related to mitochondrial activity. It is generally believed that the higher the mitochondrial activity, the lower the physiological age of the cells. That is to say, the higher the expression level of mitochondrial activity-related genes, the higher the body function and the stronger its metabolic capacity. Therefore, genes related to mitochondrial activity are also called anti-aging genes. High mitochondrial activity can also assist cells to achieve the function of regulating the balance of electrolytes (such as sodium, potassium, and calcium).

In some embodiments, mitochondrial activity-related genes include at least one of CCT5 gene (GeneID: 22948), SIRT1 gene (GeneID: 23411) and FOXO gene (GeneID: 2308). Among them, the increase of CCT5 gene expression indicates that the cells return to a youthful state, the SIRT1 gene is used to assist the mitochondria to repair damaged DNA to delay the occurrence of aging, and the FOXO gene restores mitochondrial activity to maintain the youthful state of the cells.

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

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

Among them, the expression of the IL-8 gene causes neutrophils, macrophages, basophils and T cells to undergo chemotaxis and move to the inflamed site.

Among them, the overexpression of the IL-6 gene causes an inflammatory response and stimulates the production of acute-phase proteins in liver cells.

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

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

In some embodiments, the plant fermented juice is used to increase the amount of water metabolism.

In some embodiments, the plant fermented juice is used to increase urine output.

In some embodiments, the plant fermented juice is used to reduce edema.

In some embodiments, plant fermented juice is used to improve cold hands and feet.

In some embodiments, the effective amount of plant fermentation juice is 7 mL/day.

To sum up, the plant fermentation juice according to any one of the embodiments can enhance the activity of mitochondria in cells, and achieve the purpose of enhancing metabolism by enhancing the activity of cells. The plant fermented juice according to any one of the embodiments can reduce the inflammatory response in the body, and achieve the purpose of improving metabolism by maintaining the normal function of cells. The plant fermented juice according to any one of the embodiments can increase the amount of urine, and achieve the purpose of improving metabolism by improving water metabolism in the body. The plant fermented juice according to any one of the embodiments can improve water metabolism in the body, reduce edema, maintain body circulation, and achieve the purpose of increasing metabolism. According to the plant fermented juice of any one of the embodiments, the temperature of the limbs is increased, so as to improve the coldness of hands and feet. Moreover, according to the plant fermented juice of any one of the embodiments, the purpose of improving metabolism can be effectively achieved when 7 mL of the plant fermented juice is taken daily. Here, in some embodiments, plant fermentation juices can be used to prepare metabolism-enhancing compositions.

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

In some embodiments, the aforementioned medicinal products may 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 product may comprise a pharmaceutically acceptable carrier that is 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, disintegrant ( decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent ), gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. 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 aqueous solution containing alcohol (aqueous solution containing alcohol). alcohol).

In some embodiments, any of the foregoing compositions can be an edible product (ie, a food composition). In other words, the edible product contains a specific amount of fermented plant juice. In some embodiments, the edible product may be a general food, a health food, a dietary supplement, or a food additive.

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

In some embodiments, the plant fermentation juice of any embodiment can be added (ie, as a food additive) during the preparation of raw materials by conventional methods, or the plant fermentation juice of any embodiment can be added during the production process of food (that is, as a food additive), and formulated with any edible material into edible products for human and non-human animals.

example 1 : Preparation of plant fermented juice

First, prepare the raw materials of Polygonatum chinensis, the raw materials of Gorgon, the raw materials of Lycium barbarum and water with a weight ratio of 2:2:1:40. Among them, the raw materials of Polygonatum rhizoma use dried Rhizoma Polygonatifolia purchased from New Century Kampo Bio-Tech Co., Ltd., the raw materials of Gorilla are from New Century Kampo Bio-Tech Co., Ltd. for stir-fried seed kernels, and the raw materials of wolfberry are purchased from New Century Kampo. Dried wolfberry fruit from Biotech Co., Ltd.

Next, the raw materials of Polygonatum Radix, Rhizoma Gorilla, raw materials of Lycium barbarum and water were mixed and added with 11% of the glucose relative to the total weight of the raw materials of Rhizoma Rhizoma Rhizoma Rhizoma, Rhizoma Rhizoma raw materials, Lycium barbarum raw materials and water to form a plant base liquid. Here, the Brix of the vegetable base liquid is greater than 9.

The plant base liquid is heated to 95° C., and the heating is continued for 60 minutes after the heating environment reaches 95° C. to obtain a water extract. In addition, after the temperature of the water extract is cooled to less than 40° C., it can be used as the culture solution of the subsequent fermentation process.

0.1 wt % of Saccharomyces cerevisiae and 0.05 wt % of Lactobacillus plantarum were added to the culture solution, and the culture solution was allowed to stand for three days to form an initial fermentation solution. Herein, S. cerevisiae is the Saccharomyces cerevisiae with the accession number BCRC 20271, and the germ lactic acid bacteria is the TCI028 strain with the accession number BCRC 910805.

Next, 5wt% of acetic acid bacteria was added to the initial fermentation broth and allowed to ferment for four days to form a plant fermentation stock solution. Here, the acetic acid bacteria of accession number BCRC11688 were used. The Brix of the plant fermentation stock solution is less than 4, and its pH value (pH value) is 4.5±0.5.

The plant fermentation stock solution is filtered with a sieve with a pore size of 400 meshes, and the filtered plant fermentation stock solution is subjected to a decompression concentration procedure at 60 ° C and 150 bar, and 60% of the isomalt oligosaccharide relative to the plant fermentation stock solution is added. Then the plant fermentation juice is obtained.

example 2 : Total polyphenol content test

2.1. Test Process

The water extract obtained in Example 1 was used as the control group sample and the plant fermentation stock solution as the experimental group sample. 100 μL of each sample was diluted with water and placed in a centrifuge tube. Next, add 500 μL of Folin-Ciocalteu's phenol reagent (Folin-Ciocalteu's phenol reagent, Merck 1.09001.0100) to the centrifuge tube and mix with the diluted sample and let stand for 3 minutes, then add 400 μL of 7.5% (W/V) Sodium carbonate (Sigma 31432) was mixed and let stand for 30 minutes to obtain the reaction solution to be tested. After confirming that there are no air bubbles in the reaction solution to be tested, 200 μL of the reaction solution to be tested is taken into a 96-well plate, and the absorbance of the reaction solution to be tested at 750 nm is measured. The above samples were tested in triplicate and the rounded average value was taken.

In addition, a standard curve was prepared using gallic acid as a standard. Here, standard solutions of 0 μL/mL, 20 μL/mL, 40 μL/mL, 60 μL/mL, 80 μL/mL, and 100 μL/mL of gallic acid were prepared with gallic acid (powder, Sigma G7384) and pure water. , and respectively take 100 μL of the standard solution of each concentration into a 10 mL centrifuge tube. Add 500 μL of Folin-Ciocalteu phenol reagent to the centrifuge tube and mix with the standard solution and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate and mix and let stand for 30 minutes to obtain a standard reaction solution. Take 200 μL of the standard reaction solution into a 96-well dish, and measure its absorbance at 750 nm. Then, draw a standard curve with the concentration of gallic acid and its corresponding absorbance value.

Next, after using the standard curve to convert the absorbance value of the reaction solution to be tested into the concentration by the inner difference method, multiply the dilution ratio back to obtain the total polyphenol content of each sample.

2.2. Test Results

Here, 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, as shown in FIG. 1 .

It can be seen from the experimental results that the total polyphenol content of the plant fermentation juice is nearly 10 times higher than that of the water extract. Based on this, it can be seen that the fermentation of yeast, lactic acid bacteria and acetic acid bacteria can be more beneficial to the extraction of active ingredients.

example 3 : Cell Mitochondrial Activity Test

Mitochondrion is an intracellular organelle, which mainly synthesizes adenosine triphosphate (ATP) as a source of cellular energy. When the intracellular mitochondria are sufficient, it usually means that the cell is young and elastic, and the cell has a high metabolic rate. . Furthermore, since blood vessels are all over the body, monocytes are produced in the bone marrow and spread in the blood vessels, and can also enter other body cells as needed. Extend the effect of enhancing basal metabolism.

3.1 Materials, instruments and solution configuration

Experimental cell: human monocytic cell THP-1 (hereinafter referred to as monocytic cell, purchased from ATCC company, the deposit number is TBI202).

Medium: RPMI1640 medium (brand Gibco model RPMI medium 1640, purchased from Thermo Fisher Scientific, No. 31800-022) supplemented with 10% fetal bovine serum (FBS, brand Gibco No. 10438-026) and 1% antibiotics- Antimycotic (Antibiotic-Antimycotic, brand Gibco No. 15240-062).

Reagents: RNA extraction reagent set (purchased from Geneaid, Taiwan, Lot No. FC24015-G), KAPA SYBR ^® FAST qPCR reagent set (purchased from Sigma, USA, No. 38220000000).

Reverse transcriptase: SuperScript® III Reverse Transcriptase brand Invitrogen, USA, No. ^18080-051 was used.

Detection equipment: ABI StepOnePlus ^TM real-time PCR system (Real-Time PCR system, purchased from Thermo Fisher Scientific, USA).

3.2. Test Process

2 mL of the above medium was added to each well of a six-well plate and 1×10 ⁶ monocytes were seeded for overnight culture.

The cells cultured overnight were divided into two groups (control group and experimental group). Among them, the control group did not add any test samples. The test sample added to the experimental group was the prepared plant fermentation stock solution of Example 1. Here, the test sample concentration in the culture medium of the experimental group was 1.0% (v/v).

The control group and the experimental group were incubated at 37°C for 24 hours. After 24 hours, centrifuge at 400xg for 5 minutes, remove the supernatant, rinse with 1Xdpbs buffer, and centrifuge at 400xg for 5 minutes, remove the supernatant again, and add 600µL of RB buffer to lyse the cells.

Next, use the RNA extraction reagent kit to collect RNA in the two groups of cell solutions respectively. Next, 2000 nanograms (ng) of the extracted RNA was used as a template for each group, and the corresponding cDNA was generated by reverse transcription with the primers in Table 1 by reverse transcriptase. Subsequently, using the real-time PCR system and the qPCR reagent group, the reverse transcription products of the two groups were subjected to quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain reaction) with the combination primers in Table 1 to observe the experimental group and the control group. Set of monocyte gene expression levels. The instrument setting conditions for quantification of real-time reverse transcription polymerase chain reaction were 95°C for 1 s, 60°C for 20 s, a total of 40 loops, and gene quantification was performed using the 2-ΔCt method. Here, quantitative real-time reverse transcription polymerase chain reaction with cDNA can indirectly quantify the mRNA expression level of each gene, and then infer the expression level of the protein encoded by each gene.

Table I Gene SEQ ID NO. Introductory name Sequence (5'→3') Primer length CCT5 1 CCT5-F ATAAATGTGAGGCTGAATC 19 2 CCT5-R ACTTGTCACTTGTGGCAC 18 SIRT1 3 SIRT1-F TGCTGGCCTAATAGAGTGGCA twenty one 4 SIRT1-R CTCAGCGCCATGGAAAATGT 20 FOXO 5 FOXO-F CGGACAAACGGCTCACTCT 19 6 FOXO-R GGACCCGCATGAATCGACTAT twenty one

And, the relative gene expression of each gene shown in Figure 2 is presented in relative magnification, in which the standard deviation is calculated using the STDEV formula of Excel software, and the one-tailed Student t-test (Student t-test) in Excel software is used 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.

3.3. Test Results

See Figure 2. When the expression level of the CCT5 gene in the control group is regarded as 1 (ie 100%), the expression level of the CCT5 gene in the experimental group relative to the control group is 1.65 (ie 120%), representing the expression level of the CCT5 gene in the experimental group as the control group. 1.65 times the group.

When the expression level of the SIRT1 gene in the control group is regarded as 1 (ie 100%), the expression level of the SIRT1 gene in the experimental group relative to the control group is 1.32 (ie 130%), representing the expression level of the SIRT1 gene in the experimental group as the control group. 1.32 times the group.

When the expression level of the FOXO gene in the control group is regarded as 1 (ie 100%), the expression level of the FOXO gene in the experimental group relative to the control group is 1.21 (ie 150%), and the expression level of the FOXO gene representing the experimental group is the control group. 1.21 times the group.

It can be seen from Figure 2 that after the mononuclear cells are treated with the plant fermented juice prepared from Polygonatum chinensis, Gorgon Fructus and Lycium barbarum, it can effectively achieve the effect of enhancing mitochondrial activity and thus enhancing metabolism.

example 4 : Cell Anti-Inflammation Test

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 spread in blood vessels, and will respond to systemic immune responses in the event of inflammation. Therefore, monocytes were used as experimental cell lines to test the expression of related anti-inflammatory genes. Reducing the expression of inflammation-related genes means that the body is in a normal state, and the extension can improve the efficiency of metabolism.

4.1 Materials, instruments and solution configuration

Experimental cell: human monocytic cell THP-1 (hereinafter referred to as monocytic cell, purchased from ATCC company, the deposit number is TBI202).

Medium: RPMI1640 medium (brand Gibco model RPMI medium 1640, purchased from Thermo Fisher Scientific, No. 31800-022) supplemented with 10% fetal bovine serum (FBS, brand Gibco No. 10438-026) and 1% antibiotics- Antimycotic (Antibiotic-Antimycotic, brand Gibco No. 15240-062).

Reagents: RNA extraction reagent set (purchased from Geneaid, Taiwan, Lot No. FC24015-G), KAPA SYBR ^® FAST qPCR reagent set (purchased from Sigma, USA, No. 38220000000).

Reverse transcriptase: SuperScript® III Reverse Transcriptase brand Invitrogen, USA, No. ^18080-051 was used.

Detection equipment: ABI StepOnePlus ^TM real-time PCR system (Real-Time PCR system, purchased from Thermo Fisher Scientific, USA).

4.2. Test Process

2 mL of the above medium was added to each well of a six-well plate and 1×10 ⁶ monocytes were seeded for overnight culture.

The cells cultured overnight were divided into two groups (control group and experimental group). Among them, the control group did not add any test samples. The test sample added to the experimental group was the prepared plant fermentation stock solution of Example 1. Here, the test sample concentration in the culture medium of the experimental group was 1.0% (v/v).

The control group and the experimental group were incubated at 37°C for 24 hours. After 24 hours, centrifuge at 400xg for 5 minutes, remove the supernatant, rinse with 1Xdpbs buffer, and centrifuge at 400xg for 5 minutes, remove the supernatant again, and add 600µL of RB buffer to lyse the cells.

Next, use the RNA extraction reagent kit to collect RNA in the two groups of cell solutions respectively. Next, 2000 nanograms (ng) of the extracted RNA was used as a template for each group, and the corresponding cDNA was generated by reverse transcription with the primers in Table 2 by reverse transcriptase. Subsequently, using the real-time PCR system and the qPCR reagent group, the reverse transcription products of the two groups were subjected to quantitative real-time reverse transcription polymerase chain reaction with the combination primers in Table 2 to observe the experimental group and the control group. Set of monocyte gene expression levels. The instrument setting conditions for quantification of real-time reverse transcription polymerase chain reaction were 1 sec at 95°C and 20 sec at 60°C for a total of 40 loops, and gene quantification was performed using the 2-ΔCt method. Here, quantitative real-time reverse transcription polymerase chain reaction with cDNA can indirectly quantify the mRNA expression level of each gene, and then infer the expression level of the protein encoded by each gene.

Table II Gene SEQ ID NO. Introductory name Sequence (5'→3') Primer length IL-1β 7 IL-1β-F AGCTACGAATCTCCGACCAC 20 8 IL-1β-R CGTTATCCCATGTGTCGAAGAA twenty two IL-8 9 IL-8-F TTTTGCCAAGGAGTGCTAAAGA twenty two 10 IL-8-R AACCCTCTGCACCCAGTTTTC twenty one IL-6 11 IL-6-F ACTCACCTCTTCAGAACGAATTG twenty three 12 IL-6-R CCATCTTTGGAAGGTTCAGGTTG twenty three IL-18 13 IL-18-F TCTTCATTGACCAAGGAAATCGG twenty three 14 IL-18-R TCCGGGGTGCATTATCTCTAC twenty one TNF-α 15 TNF-α-F GAGGCCAAGCCCTGGTATG 19 16 TNF-α-R CGGGCCGATTGATCTCAGC

Moreover, the relative gene expression of each gene shown in Figure 3 is presented in relative magnification, wherein the standard deviation is calculated using the STDEV formula of Excel software, and the one-tailed Student t-test (Student t-test) in Excel software is used 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.

4.3. Test Results

See Figure 3. When the expression level of the IL-1β gene in the control group is regarded as 1 (ie 100%), the expression level of the IL-1β gene in the experimental group relative to the control group is 0.58 (ie 58%), representing the IL-1β gene of the experimental group. The expression level of the gene was reduced by 42% compared with the control group.

When the expression level of the IL-8 gene in the control group was regarded as 1 (ie 100%), the expression level of the IL-8 gene in the experimental group relative to the control group was 0.59 (ie 59%), representing the IL-8 expression of the experimental group. The expression level of the gene was reduced by 41% compared with the control group.

When the expression level of the IL-6 gene in the control group is regarded as 1 (ie 100%), the expression level of the IL-6 gene in the experimental group relative to the control group is 0.25 (ie 25%), representing the IL-6 expression of the experimental group. The expression level of the gene was reduced by 75% compared with the control group.

When the expression level of the IL-18 gene in the control group was regarded as 1 (ie 100%), the expression level of the IL-18 gene in the experimental group relative to the control group was 0.73 (ie 73%), representing the IL-18 expression of the experimental group. The expression level of the gene was reduced by 27% compared with the control group.

When the expression level of the TNF-α gene in the control group was regarded as 1 (ie 100%), the expression level of the TNF-α gene in the experimental group relative to the control group was 0.48 (ie, 48%), representing the TNF-α expression of the experimental group. The expression level of the gene was reduced by 52% compared with the control group.

It can be seen from Figure 3 that after the mononuclear cells are treated with the plant fermented juice prepared from Polygonatum chinensis, Gorgon Fructus and Lycium barbarum, it can effectively reduce the inflammation of the whole body and improve the metabolism.

example 5 : Human Test One

5-1. Sample Preparation

Plant fermentation juice: the plant fermentation juice prepared in Example 1 was used.

5-2. Subjects: 4 subjects (female subjects aged 25-30). Among them, each subject is a patient prone to edema symptoms. That is to say, subjects with poor water metabolism status and metabolic rate were selected for this test.

5-3. Test item: urine output

5-4. Test method:

On the first day, 4 subjects began to drink 50 mL of water after emptying their body of urine at 9:00 am, and then 180 mL of water every hour, and counted the subjects between 9:00 am and 1:00 pm The total urine output of the subjects was the control group.

On the second day, the same 4 subjects were asked to drank 7mL of plant fermented juice and 43mL of water after emptying their bodies of urine at 9:00 a.m., and then 180mL of water every hour, again counting at 9:00 a.m. The total urine output of each subject between 1:00 pm, this is the experimental group.

5-5. Test Results

Referring to Figure 4, in the case of drinking plant fermented juice, the average total urine volume of the 4 subjects increased from 465mL to 785mL, and the difference between their average total urine volume before and after reached 40%. That is to say, drinking 7mL of plant fermented juice can effectively increase urine output and improve the body's water metabolism rate.

example 6 : Human Test II

6-1. Sample Preparation

Plant fermentation juice: the plant fermentation juice prepared in Example 1 was used.

6-2. Subjects: 3 subjects (female subjects aged between 25 and 30). Among them, each subject is a patient prone to edema symptoms. That is to say, subjects with poor water metabolism status and metabolic rate were selected for this test.

6-3. Test items: lower extremity edema index, ankle circumference and somatosensory questionnaire.

6-4. Test method:

Three subjects were asked to drink 7 mL of plant fermented juice every day for 8 consecutive weeks. Measurements were performed before drinking (ie, the 0th week, also known as the control group) and after 8 weeks of drinking (ie, the 8th week, also known as the experimental group).

Among them, the lower extremity edema index was measured by the body composition analyzer X-SCAN on the lower extremities of the subjects. Edema index is the ratio of extracellular water (ECW)/total body water (TBW). Here, the higher the edema index, the more severe the edema.

Among them, ankle circumference is a common method commonly used by clinicians to determine whether a patient has poor water metabolism. Here, the subject's ankle circumference was measured with a tape measure.

Among them, the subjects filled in the somatosensory questionnaires in the 0th week and the 8th week respectively, and various conditions related to metabolism were investigated in the questionnaires. The investigation and scoring methods are as follows in Table 3. Among them, each score in the first part represents the severity of various symptoms, 1 is no abnormality, 2 is mild, 3 is ordinary, 4 is somewhat severe, and 5 is severe.

Table 3 Symptoms/scores 1 2 3 4 5 Question 1. Sitting or standing for a long time is prone to edema Question 2. It is easy to feel that the pants or shoes are tight at night Question 3. It is easy to feel that the hands are swollen Question 4. Often feel tired and tired Question 5. Poor blood circulation or cold hands and feet

The figures shown in the figure below are presented as the average ankle circumference of the subjects, in which the standard deviation was calculated using the STDEV formula of Excel software, and the one-tailed Student t-test was used in Excel software to analyze whether there was a statistical Significant difference. "*" in the figure means that its p value is less than 0.05, which means there is a statistically significant difference.

6-5. Test Results

See Figure 5. After 8 weeks of daily consumption of plant fermented juice, the mean edema index of the 3 subjects decreased from 0.387ECW/TBW (week 0) to 0.369ECW/TBW (week 8). In addition, according to the reference value of edema index <0.40 is normal, 0.40-0.41 is borderline edema, and >0.41 is edema. From the above reference value, it can be seen that the difference of edema index is 0.01, which means from the critical edema to the occurrence of edema, and drinking the plant fermented juice of the present invention for 8 weeks, in fact, the difference of the average edema index before and after is as high as 0.018. After conversion, the difference between the average edema index before and after reached 4.5%. That is to say, drinking 7mL of plant fermented juice every day can effectively reduce edema and improve metabolism.

See Figure 6. After 8 weeks of daily drinking of plant fermented juice, the average ankle circumference of the 3 subjects decreased from 21.25 cm (week 0) to 20.82 cm (week 8), with a difference of 0.43 cm before and after. That is, drinking 7mL of plant fermented juice a day can significantly reduce ankle edema. Among them, one subject pressed the edema of the ankle at the 0th week, and it took about 3 minutes to restore the sunken skin to the original position, while when the subject pressed the edema of the ankle at the 8th week, it restored the sunken skin. It only takes 4 seconds to return to the original position.

Refer to Figure 7. Among them, for the long-term sitting or standing prone to edema mentioned in question 1, the average score of the subjects dropped from 4.17 points to 0.67 points, which means that the subjects' self-assessment has returned to the condition of no abnormality. Among them, for the situation mentioned in question 2 that it is easy to feel that the pants or shoes are tight at night, the average score of the subjects dropped from 3 points to 1.67 points. Among them, for the situation that the hands are easily swollen as mentioned in question 3, the average score of the subjects dropped from 2.67 points to 1 point, which means that the subjects' self-assessment has returned to the situation of no abnormality. Among them, for the situation of often feeling tired and tired as mentioned in question 4, the average score of the subjects dropped from 4.33 points to 2 points, which means that the subjects' self-evaluation has returned to a slight situation. Among them, for the situation of poor blood circulation or cold hands and feet mentioned in question 5, the average score of the subjects dropped from 4.67 points to 3 points, which means that the subjects' self-assessment was close to the highest score. Ordinary situation.

It can be seen from the above that the average score of each item has decreased, which means that each subject has a significant improvement in the above symptoms on average. The sensitivity of the overall low metabolism decreased, and the subjects could obviously feel the improvement of their metabolic capacity.

It can be seen that long-term use of plant fermented juice can increase mitochondrial activity, reduce inflammation, increase urine output, reduce edema symptoms, improve cold hands and feet, etc., that is, plant fermented juice has a significant effect of improving metabolism.

To sum up, the plant fermentation juice according to any one of the embodiments can enhance the activity of mitochondria in cells, and achieve the purpose of enhancing metabolism by enhancing the activity of cells. The plant fermented juice according to any one of the embodiments can reduce the inflammatory response in the body, and achieve the purpose of improving metabolism by maintaining the normal function of cells. According to the plant fermented juice of any one of the embodiments, it can increase the amount of urine, and achieve the purpose of improving metabolism by improving water metabolism in the body. The plant fermented juice according to any one of the embodiments can improve water metabolism in the body, reduce edema, maintain body circulation, and achieve the purpose of increasing metabolism. According to the plant fermented juice of any one of the embodiments, the temperature of the limbs is increased, so as to improve the coldness of hands and feet. Moreover, according to the plant fermented juice of any one of the embodiments, the purpose of improving metabolism can be effectively achieved when 7 mL of the plant fermented juice is taken daily. Here, in some embodiments, plant fermentation juices can be used to prepare compositions for enhancing metabolism.

Although the technical content of the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person who is familiar with the art, makes some changes and modifications without departing from the spirit of the present invention, should be included in the present invention. Therefore, the protection scope of the present invention should be determined by the scope of the appended patent application.

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Figure 1 is a graph showing the total polyphenol content of water extract and plant fermentation juice. Figure 2 is a graph of the experimental results of gene expression related to mitochondrial activity Fig. 3 is a graph showing the experimental results of cell anti-inflammation-related gene expression. FIG. 4 is a graph showing the results of urine output testing in human experiments. Figure 5 is a graph of the results of human experimental edema index. Figure 6 is a graph of the measurement results of the ankle circumference of the human experiment. Figure 7 is a graph of the results of a questionnaire survey on human experiments.

Claims (10)

A use of a plant fermented juice, which is used to prepare a composition for improving metabolism, wherein the plant fermented juice is obtained from a water extract of Polygonatum kingianum, Euryale ferox and Lycium chinense. Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Lactobacillus plantarum (Lactobacillus plantarum) are first fermented, and then acetic acid bacteria (Acetobacter aceti) secondary fermentation to obtain, wherein the water extract is the Polygonatum chinensis, the Gorgon fruit, the Lycium barbarum and water It is prepared by leaching at 90±5℃ for more than 60 minutes. The use according to claim 1, wherein the fermented juice of the plant is used to increase the expression level of genes related to mitochondrial activity. The use according to claim 2, wherein the mitochondrial activity-related gene is at least one of CCT gene, SIRT1 gene and FOXO gene. The use according to claim 1, wherein the fermented juice of the plant is used to reduce the expression level of genes related to anti-inflammatory response. The use according to claim 4, wherein the anti-inflammatory response-related gene is at least one of IL-1β gene, IL-8 gene, IL-6 gene, IL-18 gene and TNF-α gene. The use according to claim 1, wherein the plant fermentation juice is used to increase the amount of water metabolism. The use of claim 6, wherein the plant fermented juice is used to increase urine output. The use of claim 1, wherein the plant fermented juice is used to reduce edema. The use as claimed in claim 1, wherein the plant fermented juice is used to improve cold hands and feet. The use according to any one of claims 1 to 9, wherein the effective amount of the plant fermented juice is 7 mL/day.

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