TWI757586B - Plant ferment, preparation method thereof and use thereof for liver health care - Google Patents

Abstract

The present invention provides a plant ferment, preparation method thereof and use thereof for liver health care. The plant ferment can effectively improve the antioxidant activity of liver cells to prevent the damage caused by the oxidative stress, and can simultaneously effectively improving the gene expression level of CCT7, CCT8, Pink1, and MRPS5 to enhance the anti-aging activity of liver cells. The plant burning ferment is prepared by fermenting the Dolichos lobatus Willd. extract and the Hovenia dulcis Thunb. extract to yeast, lactic acid bacteria, and acetic acid bacteria for three-stage fermentation.

Description

Plant fermented product and its preparation method and use for liver health care

The present invention relates to a plant fermented product, a preparation method thereof, and the use of a composition for preparing liver health care, in particular, a plant fermented product is used to improve the antioxidant activity of liver cells, and to improve the CCT7 gene, CCT8 gene, Expression of Pink1 gene and MRPS5 gene; wherein, the plant fermentation product is a three-stage fermentation of a mixture consisting of a pueraria root extract and a Fructus aurantium extract with yeast, Lactobacillus, and Acetobacter in sequence. and obtained.

In biology and medicine, aging refers to the phenomenon that the physiological state of an organism deteriorates with time. Aging will cause the function of various parts of the body or organs to decline or weaken, making the organism's health function worse. In the end, it leads to the death of the organism, and the pressure of modern people's life is high. The accumulation of long-term stress will accelerate the aging of body functions, and also lead to many chronic diseases.

In addition, with the rise of health awareness and the advancement of medical technology, the average life expectancy is increasing, but the functional degradation caused by aging is still exacerbated with aging. Therefore, in addition to the treatment and prevention of diseases, aging prevention is also an aging society. one of the important topics.

Although many aging-related genes and related mechanisms have been studied, there is still no drug or method that can be effectively applied to delay aging. Clinically, it is only recommended to take more foods with antioxidant active substances in the diet, and Reduce bad eating habits such as drinking, and maintain proper exercise and maintain a normal routine and a happy mood.

The liver is the largest organ in the human body and has various physiological functions such as digestion and detoxification. Therefore, aging of the liver will cause problems in the physiological functions of the whole body. However, because liver cells have strong regeneration ability and the liver is a silent organ, it is difficult to detect liver discomfort or aging on its own.

To sum up, in order to achieve the effect of delaying aging conveniently and effectively, it is especially aimed at maintaining the liver, which is very important for the normal function of the human body, in a young and energetic state. A comprehensive liver health care composition for the antioxidant capacity of liver cells is indeed necessary.

Therefore, an object of the present invention is to provide a plant fermentation product, wherein the plant fermentation product is an extract obtained by extracting a mixture of a pueraria root and a citrus aurantium through a solvent, and then through a yeast ( Saccharomyces cerevisiae ), A Lactobacillus plantarum ( Lactobacillus plantarum ) and an Acetobacter aceti ( Acetobacter aceti ) are sequentially obtained by fermentation.

Another object of the present invention is to provide the use of the aforementioned plant fermentation product for preparing a liver health care composition; wherein the effective concentration of the plant fermentation product is at least 0.5% (v/v).

Another object of the present invention is to provide a method for producing a plant fermented product, which comprises: passing a mixture of a pueraria root extract and a Fructus aurantium extract through a yeast, a lactobacillus, and an acetic acid bacillus It is obtained by performing three-stage fermentation in sequence.

In one embodiment of the present invention, the mixture of the pueraria root and the aurantium seed is obtained by extracting the pueraria root, the aurantium seed, and the water system at 50-100° C. for 0.5-3 hours; Mix in a ratio of 1-3:1:25-35 (w/w).

In another embodiment of the present invention, the addition amount of the yeast is 0.01-0.5% (w/w); the addition amount of the lactobacillus is 0.01-0.25% (w/w); the addition amount of the acetic acid bacteria and the yeast strain is a strain of BCRC20271; the lactobacillus strain is a strain of BCRC910805; the acetic acid bacillus strain is a strain of BCRC11688.

In yet another embodiment of the present invention, the plant fermentation product enhances the antioxidant activity of a liver cell.

In yet another embodiment of the present invention, the plant fermentation product enhances the anti-aging activity of a liver cell; and the plant fermentation product enhances the mitochondrial activity of the liver cell, the ability to clear misfolded proteins, and/or Increases the concentration of NAD ⁺ in cells.

In yet another embodiment of the present invention, the plant fermented product enhances a TCP1-containing folded protein ( CCT1 ) gene, a PTEN-induced putative kinase 1 (PTEN-induced putative kinase 1, Pink1 ) gene in a liver cell, and The expression level of mitochondrial ribosomal protein 5 (mitochondrial ribosomal protein S5, MRPS5 ) gene; and the CCT gene is the folded protein 7 (Chaperonin containing TCP1 7, CCT7 ) gene of TCP1, the folded protein 8 of TCP1 (Chaperonin containing TCP1 8, CCT8 ) gene, or a combination thereof.

In another embodiment of the present invention, the addition amount of the yeast is 0.01-0.5% (w/w); the addition amount of the lactobacillus is 0.01-0.25% (w/w); the addition amount of the acetic acid bacteria It is 1-20% (w/w); and the fermentation time ratio of the yeast, the lactobacillus, and the acetic acid bacteria is 1-5:1-5:3-10.

In the present invention, the extract of the mixture of pueraria and Fructus Aurantii is fermented in three stages by yeast, lactobacillus and acetic acid bacteria, and the plant fermentation product can directly and effectively improve the antioxidant activity of liver cells, and can improve the clearance of liver cells. The ability of active oxides can prevent damage from oxidative stress; the plant fermentation product of the present invention can also effectively increase the expression of CCT7 gene, CCT8 gene, Pink1 gene, and MRPS5 gene in liver cells, so as to improve the mitochondria of liver cells. Activity, the ability to clear misfolded proteins, and increase the concentration of NAD ⁺ in cells, which can enhance the anti-aging activity of liver cells, make liver cells younger in physiological condition and prolong life. The five elements and meridians of traditional Chinese medicine are the channels for the circulation of qi and blood. If the meridians are open, all diseases will be eliminated. The plant fermented product of the present invention can remove free radicals and detoxify, achieve the effect of improving spirit and energy, and can also improve the expression of rejuvenation-related genes. It can enhance liver resistance and rejuvenate health effects to regulate liver meridians. Therefore, the plant fermented product of the present invention can be used for the preparation of a liver health care composition, and the composition is a medicine or a food, which can be administered to an individual by oral administration, smearing and the like.

The embodiments of the present invention will be further described below in conjunction with the drawings. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Within the scope, some changes and modifications can be made, so the protection scope of the present invention should be defined by the appended patent application scope.

FIG. 1 is a bar graph showing that the proportion of liver cells containing active oxides is reduced by the plant fermentation product according to an embodiment of the present invention. ***p-value < 0.001.

Figure 2 is a bar graph showing that the plant fermentation product according to one embodiment of the present invention enhances the expression levels of CCT7 gene, CCT8 gene and Pink1 gene in liver cells. **p-value < 0.01.

FIG. 3 is a bar graph showing that the expression level of the MRPS5 gene in liver cells is increased by the plant fermentation product according to an embodiment of the present invention.

Numerical values used herein are approximations and all experimental data are expressed within 20%, preferably within 10%, and most preferably within 5%.

Statistical analysis was performed using Excel software. Data are presented as mean ± standard deviation (SD), and differences between them were analyzed by Student's t -test .

Pueraria is the dry root of Pueraria lobata , which is a perennial deciduous entwining large vine of the Fabaceae ( Fabaceae ) genus Pueraria . , Huang Jin and other names. The roots of kudzu are tuberous and thick in appearance, up to 1 meter long and 15 cm thick, which can be used for food or medicine.

Hovenia dulcis Thunb. ( Hovenia dulcis Thunb. ) is a deciduous tree plant of the genus Hovenia of the Rhamnaceae family, also known as jujube, jujube, tree density, dragon claw, golden hook pear, chicken claw, and manzi pear. , Sweet Midnight, Qi Jiu Zai and other names. Native to south-central China, southwest China and Japan, Taiwan was introduced and planted in Xitou and Ruili in the 1960s. The fruit stalk of A. quincea is sweet and edible, commonly known as jujube or pear; its leaves and seeds can be used for medicinal purposes, with thirst-quenching, hangover-relieving and anti-emetic effects; its root bark or stem bark It has the effect of promoting blood circulation and relaxing tendons; its fruit can strengthen the stomach and nourish blood.

As used herein, the term "plant fermented product" means an extract obtained by extracting pueraria root and aurantia aurantium with a solvent in a ratio of 1-5:1:25-50 (w/w) for a specific time and temperature , and then perform a three-stage fermentation with yeast, lactobacillus, and acetic acid bacteria in sequence to obtain, wherein the addition amount of the yeast is 0.01-0.5% (w/w); the addition amount of the lactobacillus is 0.01- 0.25% (w/w); the amount of Acetobacter is 1-20% (w/w).

The "effective concentration" described herein refers to the concentration of the plant fermentation product of the present invention required to effectively enhance the antioxidant activity of liver cells, and/or to effectively enhance the expression levels of CCT7 gene, CCT8 gene, Pink1 gene, and MRPS5 gene in liver cells. . The effective concentration may vary depending on the target, but can be determined experimentally by, for example, dose escalation.

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

According to the present invention, pharmaceutical products can be manufactured into a dosage form suitable for parenterally or topically administration using techniques well known to those skilled in the art, including, but not limited to: injections (injection) [eg, sterile aqueous solution or dispersion], sterile powder, external preparation, and the like.

According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier can include one or more of, but not limited to, the following reagents: solvent, buffer (buffer), emulsifier (emulsifier), suspending agent (suspending agent), decomposer (decomposer), disintegrating agent (disintegrating agent), dispersing agent (dispersing agent), binding agent (binding agent), excipient (excipient) ), stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, Absorption delaying agents, liposomes and the like. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.

According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), Aqueous solutions containing alcohol and combinations thereof.

According to the present invention, the medicinal product may be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection Intradermal injection and intralesional injection.

According to the present invention, the food product can be regarded as a food additive, which is added during the preparation of raw materials by conventional methods, or added during the production process of the food, and is formulated with any edible material for Food products consumed by humans and non-human animals.

According to the present invention, types of food products include, but are not limited to, beverages, fermented foods, bakery products, health foods, and dietary supplements.

The present invention provides a plant fermented product, a preparation method thereof, and its use in protecting liver and health care. The plant fermented product of the present invention is a mixture of pueraria, Fructus Aurantii and a solvent in a ratio of 1-5:1:25-50 (w/w) The ratio is obtained by extracting the extract at a specific time and temperature, and then performing a three-stage fermentation with yeast, Lactobacillus, and Acetobacter in sequence to obtain, wherein the yeast is the strain of BCRC20271, and the lactobacillus is BCRC910805 The strain of Acetobacter is the strain of BCRC11688. The plant fermented product of the present invention can effectively enhance the antioxidant activity of liver cells, and enhance the expression levels of CCT7 gene, CCT8 gene, Pink1 gene and MRPS5 gene in liver cells.

At the same time, the composition for protecting liver and health care of the present invention may also comprise an effective amount of plant fermentation product and a pharmaceutically acceptable carrier, and the composition is a medicine or a food.

The detailed preparation method of the plant fermentation product of the present invention, the test of the effect of the plant fermentation product in enhancing the antioxidant activity of liver cells, and the test of the efficacy of the plant fermentation product in enhancing the expression of anti-aging genes in liver cells will be described in detail below, so as to confirm the The plant fermented product of the present invention can effectively enhance the antioxidant activity of liver cells, and enhance the expression levels of CCT7 gene, CCT8 gene, Pink1 gene and MRPS5 gene in liver cells.

Example 1 The preparation method of the plant fermentation product of the present invention

In one embodiment of the present invention, after mixing the roots of Pueraria lobata and the fruit of Aurantium chinensis, they are uniformly mixed with water in a ratio of 1-5:1:25-50, and simultaneously sterilized and extracted at 50-100°C for 0.5- After 3 hours, the extraction solution was cooled to room temperature for subsequent three-stage fermentation, and the following three bacteria were fermented in sequence: yeast, lactobacillus, and acetic acid bacteria. The fermentation order of these three bacteria could not be reversed, and their The fermentation time ratio is 1-5:1-5:3-10. In a preferred embodiment of the present invention, firstly, 0.01-0.5% (w/w) yeast ( Saccharomyces cerevisiae , purchased from the Biological Resource Conservation and Research Center, Taiwan, No. BCRC20271) is implanted in the extraction solution, Pre-fermentation at 25-35°C for 1-5 days, the actual time varies depending on the fermentation state. Then, 0.01-0.25% (w/w) Lactobacillus plantarum TCI028 ( Lactobacillus plantarum TCI028, purchased from the Biological Resource Conservation and Research Center, Taiwan, No. BCRC910805) was directly implanted and post-fermented at 25-35°C for 1-3 days , the actual time varies depending on the fermentation state. Then directly implant 1-20% (w/w) Acetobacter aceti (purchased from Biological Resource Conservation and Research Center, Taiwan, No. BCRC11688), and carry out submerged fermentation at 25-35°C for 5-15 days , the actual time varies depending on the fermentation state. Finally, without removing the three bacteria, the set sugar content range of 2-4°, pH<4, alcohol>5% and other specifications are used. If the inspection meets the specifications, it will be judged. Fermentation is completed and fermentation broth is obtained. Next, the fermentation broth was concentrated under reduced pressure at 45-70°C, filtered through a 200-400 mesh screen, and 40-70% isomalt oligosaccharide was added to adjust the specifications, and then heated at 95-105°C for 90 minutes. After sterilization, the plant fermentation product of the present invention is obtained.

Example 2 The effect of the plant fermented product of the present invention in enhancing the antioxidant activity of liver cells

In one embodiment of the present invention, human hepatoma cells (Human hpatocellular carcinoma, HepG2) were used as Dichloro-dihydro-fluorescein diacetate (DCFH-DA, purchased from Sigma, USA, No. SI-D6883-50MG, 5 mg/mL stored in DMSO) The efficacy test of the plant fermented product of the present invention in enhancing the antioxidant activity of liver cells was carried out. The HepG2 cell line was purchased from the American Type Culture Collection (USA), number ATCC ^® HB-8065 ^™ , and the cell line was cultured in 10% Fetal Bovine Serum (from Gibco, USA) and 90% Fetal Bovine Serum % DMEM (Dulbecco's Modified Eagle Medium, purchased from Gibco, USA) in the cell culture medium, which was added with 1% penicillin-streptomycin.

DCFH-DA is a stable non-polar compound that can freely permeate cell membranes. When DCFH-DA enters cells, it will be hydrolyzed by intracellular lipid enzymes to form polar DCFH, which stays in cells and cannot come out. The reactive oxygen species (ROS) in it reacts with DCFH to form a redox reaction to form Dichloro-fluorescin (DCF). After excitation at a wavelength of 450-490nm, the resulting green fluorescence can be detected at a wavelength of 510-550nm. Therefore, detecting the fluorescence intensity of cells treated with DCFH-DA can reflect the content of reactive oxygen species in the cells.

In order to test the efficacy of the three-stage fermentation process of the present invention to improve the antioxidant activity of plant fermentation products in liver cells, firstly, 1×10 ⁵ HepG2 cells were cultured in a 6-well culture plate containing 2 mL of the above-mentioned cell culture medium at 37° C. After 24 hours of culture, new cell culture medium was replaced without disturbing the attached cells, and then the cells were divided into the following five groups: (1) blank control group, (2) positive control group, (3) added 1% (v/v) the experimental group of the plant fermented product of the present invention, (4) the comparative group to which 1% (v/v) Aurantium chinensis water extract was added, and (5) 1% (v/v) Pueraria lobata water For the comparison groups of extracts, each group was reacted at 37 °C for 1 hour, and then 5 μg/mL DCFH-DA was added to each group for 15 minutes at 37 °C to label ROS in cells. -(5) group was treated with 0.5mM H ₂ O ₂ (purchased from Sigma, USA, the concentration of stock solution was 35%) at 37°C for 1 hour, and the group without H ₂ O ₂ effect was the blank control group , then the culture medium was removed without disturbing the attached cells, and the cells were washed twice with 1 mL of 1x phosphate buffered saline (1x phosphate buffered saline, 1xPBS), and then 200 μL of trypsin was added to react in the dark For 5 minutes, the cells were detached from the culture plate, and the appropriate culture medium was added to stop the reaction. The cells and the culture medium were collected in a 15 mL centrifuge tube in the dark, centrifuged at 300 g for 5 minutes, and the supernatant was removed. After washing the cells once with 1xPBS, the supernatant was removed after centrifugation at 300g for 5 minutes, then the cells were resuspended in 1mL of 1xPBS in the dark, and the cells were detected by flow cytometry (BD Accuri ^™ C6 Plus, purchased from the United States). The fluorescence values of excitation wavelength 450-490nm and emission wavelength 510-550nm were used to calculate the number of cells containing fluorescent signal. Then use Excel software to perform student t-test to determine whether the (1), (3)-(5) have statistically significant differences compared to the (2) group (*p value<0.05; ** p-value <0.01; ***p-value < 0.001).

The experimental results of the plant fermentation product of the present invention enhancing the antioxidant activity of liver cells are shown in FIG. 1 . In the blank control group without H ₂ O ₂ treatment, only about 0.67% of HepG2 cells contained reactive oxides; while in the positive control group treated with H ₂ O ₂ only, about 61.97% of HepG2 cells contained reactive oxides. Active oxides, the results show that H ₂ O ₂ treatment can indeed produce active oxides in HepG2 cells. In the experimental group pretreated with the plant fermentation product of the present invention, only about 11.87% of HepG2 cells contained active oxides, which could significantly reduce the production of active oxides by 50.1% of HepG2 cells compared with the positive control group; In the comparison group pretreated with Pueraria lobata water extract or Citrus aurantium water extract, about 41.57% and 28.43% of HepG2 cells still contained active oxides, respectively, which was only significantly reduced by about 20.4% and 20.4%, respectively, compared with the positive control group. 33.54% of HepG2 cells produced reactive oxides. This result shows that the plant fermentation product of the present invention can more effectively reduce the active oxides produced by liver cells stimulated by H ₂ O ₂ after going through the three-stage fermentation step of the microorganisms of the present invention, and can effectively enhance the antioxidant activity of liver cells , prevents liver cells from being damaged by oxidative stress.

Example 3 The effect of the plant fermentation product of the present invention in regulating the expression of anti-aging genes in liver cells

One embodiment of the present invention is to carry out the efficacy test of the three-stage fermentation step of the present invention to improve the regulation of the expression of anti-aging genes in liver cells by plant fermentation products. First, 1×10 ⁵ HepG2 cells were cultured in a 6-well culture dish containing 2 mL of the above-mentioned cell culture medium, and cultured at 37°C for 24-48 hours, and then the cells were divided into the following four groups: (1) Only the cell culture medium was added The control group, (2) the experimental group added with 0.5% (v/v) of the plant fermented product of the present invention, (3) the comparative group added with 0.5% (v/v) Fructus Auricularia water extract, and (4) Add 0.5% (v/v) pueraria water extract to the comparison group, and after the cells of these groups were treated at 37°C for 24 hours, the expression levels of target genes in HepG2 cells of each group were tested. First, the HepG2 cells were collected with cell lysate (RB buffer, purchased from Geanaid, Taiwan, Cat No.RBD300), and then collected using RNA extraction reagent kits (purchased from Geneaid, Taiwan, Cat No.RBD300). The RNAs in the four groups of cells were then reversed using ^{SuperScript®} III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) with 2000 ng of the extracted RNA as a template, and the combination of primers and reverse transcriptase in Table 1 was used for reverse transcriptase. Transcription to generate cDNA products corresponding to the mRNAs of these genes, followed by ABI StepOnePlus ^™ Real-Time PCR system (Thermo Fisher Scientific, USA), and KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000) The four groups of reverse-transcribed products were subjected to quantitative real-time polymerase chain reaction (qPCR) tests using the combination primers in Table 1. The conditions were 95°C for 1 second and 60°C for 20 seconds. 40 cycles. To quantify CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin1 gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, PARP1 gene, PARP2 gene, NADSYN gene, MRPS5 gene, UBL- mRNA expression levels of 5 genes, SOD3 gene, TERT gene, TERC gene, and RTEL1 gene, where the quantitative value is taken from the threshold cycle number (Ct), and the relative amount of mRNA of the target gene is derived from Equation 2 ^{- △△Ct} , where △ _CT = _{CT comparison group or target gene in experimental group/target gene in control group} -C _TGAPDH (Glyceraldehyde-3-phosphate dehydrogenase); △△ _CT = _{CT comparison Group or experimental group target gene -CT control group target gene} ; the fold change of each gene in each group is 2- ^△△Ct . Next, use the Excel software to determine whether the coefficient of variation is statistically significant (*p-value<0.05;**p-value<0.01;***p-value<0.001).

CCT gene is a kind of folded protein (Chaperonin), its main function is to correct the misfolded protein, and send the protein that cannot be successfully repaired to the proteasome for hydrolysis. Previous studies have pointed out that if this protein regulates the The failure of the reuse system will lead to the accumulation of misfolded proteins in cells, and cause the decline of cell function and accelerate the aging and death of cells, so it is considered to be related to the regulation of aging in the individual. Pink1 is a mitochondrial-located serine/threonine protein kinase that protects mitochondria from malfunctioning during cellular stress. Mutations in this gene are known to be associated with Parkinson's disease related, and previous studies have pointed out that the overexpression of this gene can increase the lifespan of mice, so it is also considered to be related to the regulation of individual aging. Parkin gene is an enzyme present in the ubiquitin-proteasome system and acts as a regulator of protein breakdown. Mutations in this gene are known to be associated with Parkinson's disease, and previous studies have also pointed out that increasing this gene The amount of expression can delay the aging of cells, so it is considered to be related to the aging of cells. Atg gene is an autophagy-related gene, which is involved in waste degradation and circulating autophagy in cells. Previous studies have pointed out that the overexpression of this gene can prolong the lifespan of mice, so it is considered to be related to cell aging. Telomeres are DNA repeat sequences at the ends of biological chromosomes. The main function is to maintain the integrity of chromosomes and control the cycle of cell division. Each time the DNA is replicated, the telomeres will be shortened. Once the telomeres are exhausted, the cells will move towards Apoptosis, so the length of telomeres is related to the age of cells, and it is also known that TERT gene, TERC gene and RTEL1 gene are related to the regulation of telomere length. Previous studies have pointed out that young mice contain more NAD ⁺ than old mice; and increasing the concentration of NAD ⁺ in old mice can make them physiologically younger, so NAD ⁺ is It is considered to be related to the delay of individual aging, and the research results have known that the SIRT1 gene, FOXO3 gene, NADSYN gene, UBL-5 gene, SOD3 gene, and MRPS5 gene are related to the regulation of NAD ⁺ .

Figure 2 shows the experimental results that the plant fermentation product of the present invention enhances the expression levels of CCT7 gene, CCT8 gene and Pink1 gene in liver cells. After the action of the plant fermentation product of the present invention, compared with the control group, the expression levels of CCT7 gene, CCT8 gene, and Pink1 gene in HepG2 cells can be significantly increased by 1.31 times, 1.2 times, and 1.32 times, respectively. The expression levels of the remaining genes The expression levels of CCT7 gene, CCT8 gene, and Pink1 gene were similar to or lower than those of the control group after being acted by Pueraria lobata water extract or Citrus aurantium water extract. . This result shows that the plant fermentation product of the present invention can effectively increase the expression levels of CCT7 gene, CCT8 gene, and Pink1 gene in liver cells, and can effectively improve the mitochondrial activity of liver cells after going through the three-stage fermentation step of the microorganism of the present invention. And the ability to clear misfolded proteins to enhance the anti-aging activity of liver cells and make liver cells more youthful.

Figure 3 shows the experimental results that the plant fermentation product of the present invention enhances the expression level of the MRPS5 gene. Compared with the control group, the expression of the MRPS5 gene in HepG2 cells can be increased after the action of the plant fermentation product of the present invention, and the expression of the remaining genes related to the regulation of NAD ⁺ is similar to or not different from the control group (data not shown). ); and after the water extract of Pueraria lobata or Citrus aurantium, the expression of MRPS5 gene will be reduced instead. This result shows that the plant fermented product of the present invention can effectively increase the expression of MRPS5 gene and the concentration of NAD ⁺ in liver cells after going through the three-stage fermentation step of the microorganisms of the present invention, so as to enhance the anti-aging activity of liver cells. Extend the lifespan of liver cells

To sum up, the present invention can directly and effectively enhance the antioxidant activity of liver cells by fermenting the extract of the mixture of Pueraria lobata and Fructus Aurantii with yeast, Lactobacillus, and Acetobacter in three-stage fermentation. It can improve the ability of liver cells to remove active oxides to prevent damage from oxidative stress; the plant fermentation product of the present invention can also effectively increase the expression of CCT7 gene, CCT8 gene, Pink1 gene, and MRPS5 gene in liver cells, so as to improve the liver Mitochondrial activity of cells, the ability to clear misfolded proteins, and increase the concentration of NAD ⁺ in cells can enhance the anti-aging activity of liver cells, making liver cells younger in physiological condition and prolonging lifespan. The five elements and meridians of traditional Chinese medicine are the channels for the circulation of qi and blood. If the meridians are open, all diseases will be eliminated. The plant fermented product of the present invention can remove free radicals and detoxify, achieve the effect of improving spirit and energy, and can also improve the expression of rejuvenation-related genes. It can enhance liver resistance and rejuvenate health effects to regulate liver meridians. Therefore, the plant fermented product of the present invention can be used for the preparation of a liver health care composition, and the composition is a medicine or a food, which can be administered to an individual by oral administration, smearing and the like.

<110> Dajiang Biomedical Co., Ltd.

<120> Plant fermented product and its preparation method and use for liver health care

<130> 107B0591-I1

<160> 44

<170> PatentIn version 3.5

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Claims (7)

Use of a plant fermented product for preparing a composition for enhancing the anti-aging activity of a liver cell, enhancing the mitochondrial activity of the liver cell, and enhancing the ability to clear misfolded proteins so as to make the physiological condition of the newborn liver cell younger, And the plant fermented product is by enhancing the folded protein containing TCP1 (Chaperonin containing TCP1, CCT) gene, PTEN-induced kinase (PTEN-induced putative kinase 1, Pink1) gene, and mitochondrial ribosomal protein 5 ( The expression level of mitochondrial ribosomal protein S5, MRPS5) gene is achieved to enhance the anti-aging activity of the liver cell, enhance the mitochondrial activity of the liver cell, and enhance the ability to clear the misfolded protein, wherein the plant fermentation product is composed of A mixture consisting of a pueraria root and a Citrus aurantium is extracted with water, and then pre-fermented by a yeast with an addition amount of 0.01-0.5% (w/w), and then through an addition amount of 0.01-0.25% ( w/w) of a lactobacillus to carry out post-fermentation, and then to obtain by submerged fermentation of an acetic acid bacillus added in an amount of 0.01-0.25% (w/w), and the fermentation of the yeast, the lactobacillus and the acetic acid bacillus The order cannot be reversed. The use according to claim 1, wherein the plant fermented product enhances the antioxidant activity of a liver cell. The use as described in claim 1, wherein the CCT gene is a TCP1 folded protein 7 (Chaperonin containing TCP1 7, CCT7 ) gene, a TCP1 folded protein 8 (Chaperonin containing TCP1 8, CCT8 ) gene, or its combination. The use as described in item 1 of the claimed scope, wherein the effective concentration of the plant fermentation product is at least 0.5% (v/v). A method for producing a plant fermented product, comprising: a mixture consisting of a Pueraria lobata extract and a Citrus aurantium extract is obtained by performing three-stage fermentation in sequence with a yeast, a lactobacillus, and an acetobacter, Wherein the mixture is prepared by mixing the pueraria, the aurantium, and water, and the yeast is the yeast with the preservation number of BCRC20271, and the pre-fermentation is carried out under the addition amount of 0.01-0.5% (w/w). ; The lactobacillus is preservation number BCRC910805 The lactobacillus is fermented at an addition amount of 0.01-0.25% (w/w); the acetic acid bacillus is the acetic acid bacillus with the preservation number of BCRC11688, and the addition amount is 1-20% (w/w). During fermentation, the fermentation order of the yeast, the lactobacillus and the acetic acid bacteria cannot be reversed. The manufacturing method as described in item 5 of the claimed scope, wherein the pueraria, the aurantium, and the water system are mixed in a ratio of 1-3:1:25-35 (w/w). The manufacturing method as described in claim 5, wherein the fermentation time ratio of the yeast, the Lactobacillus, and the Acetobacter is 1-5:1-5:3-10.

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