TWI725298B - Use of imperata cylindrica fermented extracts in a composition for regulating the expression of ldlr gene, abca1 gene, ptgis gene, serpine1 gene and igf2bp3 gene - Google Patents

Abstract

The present invention provides a use of Imperata cylindrica fermented extracts in a composition for regulating the expression of LDLR gene, ABCA1 gene, PTGIS gene, SERPINE1 gene and IGF2BP3 gene, so as to effectively increase the metabolism of LDL and to promote the production of HDL, in order to maintain the balance of LDL and HDL in the blood, and also to effectively prevent thrombosis and vasodilatation, and to reduce the occurrence of cardiovascular diseases such as obesity and diabetes. The Imperata cylindrica fermented extracts is prepared by fermenting the Imperata cylindrica extracts to yeast, lactic acid bacteria and acetic acid bacteria for three-stage fermentation.

Description

Use of the fermented product of Imperata cylindrica for regulating the expression of LDLR gene, ABCA1 gene, PTGIS gene, SERPINE1 gene and IGF2BP3 gene expression level

The present invention relates to the use of a fermented Imperata cylindrica root, especially a pharmaceutical composition for the use of a fermented Imperata cylindrica root for regulating the expression levels of LDLR gene, ABCA1 gene, PTGIS gene, SERPINE1 gene and IGF2BP3 gene.

Related literature statistics show that about one-third of the world’s population die of cardiovascular disease (CVD). Cardiovascular disease, also known as circulatory system disease, refers to diseases caused by organs and tissues that transport blood in the human body. Therefore, all diseases of the heart and blood vessels can be called cardiovascular diseases, such as stroke and myocardial infarction.

Nowadays, the drugs used to treat cardiovascular diseases are mainly derived from chemical synthesis, and can be divided into two categories: one is drugs to prevent thrombosis and anti-platelet coagulation, and the other is drugs to control high blood pressure, cholesterol, and diabetes. However, these two types of drugs are often used. Accompanied by strong side effects. For example, angiotensin-related antagonists may cause hyperkalemia, causing symptoms such as muscle weakness or slow heartbeat; or powerful cholesterol-lowering drugs Statins (HMG-CoA reductase inhibitors) may increase liver function index Rise, or have headaches, nausea, tiredness and other side effects. Moreover, these drugs must be taken for a long time to control the disease. However, the side effects of long-term use of drugs on the individual cannot fundamentally improve the health of the individual.

Based on the above, in response to the high incidence of cardiovascular diseases, and based on the improvement of modern people’s living standards and the improvement of health care concepts, we have developed a natural plant that can effectively prevent cardiovascular diseases and avoid the side effects of synthetic drugs on the human body. A pharmaceutical composition composed of active ingredients is indeed necessary.

For this reason, one purpose of the present invention is to provide a fermented Imperata cylindrica root for the preparation of low-density lipoprotein receptor (LDLR) gene, adenosine triphosphate binding cassette transporter A1 (ATP binding cassette transporter). A1, ABCA1) gene, Prostaglandin I synthase (PTGIS) gene, Serpin family E member 1, SERPINE1 gene, and insulin-like growth factor 2 mRNA binding protein 3 (Insulin -like growth factor 2 mRNA-binding protein 3, IGF2BP3) gene expression level of the pharmaceutical composition.

Another object of the present invention is to provide a method for producing the fermented Imperata cylindrica roots, which comprises: combining a water extract of Imperata cylindrica roots and a microbial group at a ratio of material to water of 1:15~35, and the temperature is between 20~35°C, It is obtained through three-stage fermentation. The microbial community is composed of a yeast, a lactobacillus, and an acetic acid bacterium. The yeast is added in the first stage, the lactobacillus is added in the second stage, and the third The acetobacter is added in stages to obtain a fermented cypress root.

In an embodiment of the present invention, the low-density lipoprotein receptor gene, the adenosine triphosphate binding cassette transporter A1 gene, and the prostaglandin I synthase gene are up-regulated; and the serine protease inhibitor E family 1 Genes, and insulin-like growth factor 2 mRNA binding protein 3 genes are down-regulated.

In an embodiment of the present invention, the microbial community is composed of a yeast ( Saccharomyces cerevisiae ), a Lactobacillus ( Lactobacillus plantarum TCI028), and an Acetobacter aceti , and the added amount of the yeast 0.01~0.5%(v/v), the addition amount of the lactobacillus is 0.01~0.25%(v/v), and the addition amount of the acetobacter is 1~20%(v/v), and the three-stage fermentation In the three stages, the temperature is 20~35℃, and the incubation time ratio is 1~2:1~3:14~21.

In an embodiment of the present invention, the water extract of the Imperata cylindrica root is obtained by mixing the Imperata cylindrica root with water at a solid-to-liquid ratio of 1:18-30, and sterilized and extracted at 50-100° C. for 0.5-3 hours.

In an embodiment of the present invention, the total polysaccharide of the fermented Imperata cylindrica is at least 20%.

At the same time, the pharmaceutical composition of the present invention for increasing the LDLR gene, ABCA1 gene and PTGIS gene, and reducing the expression of SERPINE1 gene and IGF2BP3 gene may also include an effective amount of the fermented cypress root and a pharmaceutically acceptable carrier. The composition is in the form of powder, granules, liquid, gel or paste.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with the art will not depart from the spirit and spirit of the present invention. Within the scope, some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Fig. 1 is a bar graph showing how the fermented Imperata cylindrica roots of an embodiment of the present invention can increase the expression of Low-Density Lipoprotein Receptor (LDLR) genes.

Fig. 2 is a bar graph showing the increase of ATP binding cassette transporter A1 (ABCA1) gene expression by the fermented Imperata cylindrica roots of an embodiment of the present invention.

Fig. 3 is a bar graph showing the use of fermented cypress roots of the embodiment of the present invention to increase the expression of Prostaglandin I synthase (PTGIS) gene.

Fig. 4 is a bar graph showing the reduction of Serpin family E member 1 (Serpin family E member 1, SERPINE1) gene expression by the fermented cypress roots of the embodiment of the present invention.

Fig. 5 is a bar graph showing the reduction of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) gene expression by the fermented Imperata cylindrica root of an embodiment of the present invention.

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

Example 1 Preparation method of the fermented product of Imperata cylindrica root

Imperata cylindrica (Imperata cylindrica) is a perennial herb of the Gramineae genus Imperata , also known as thatch, Imperata cylindrica, and thatch. The plant height is 20 to 80 cm. The rhizome is white. It runs across the ground and has its nodes. The scales have a sweet taste at the tip. The single leaves are alternate, concentrated at the base. In old age, there are often broken and fibrous leaf sheaths at the base. The leaves are flat, strip or strip-lanceolate. They bloom in summer, and the flowers are silvery white with densely branched branches. . At present, it is known that the root of Imperata cylindrica has the effects of clearing away heat and moisturizing the lung, promoting body fluid and detoxification, strengthening the liver and nourishing the spleen, reducing fire and generating fluid, cooling blood, and diuresis.

In an embodiment of the present invention, the Imperata cylindrica roots are thoroughly cleaned, and the washed Imperata cylindrica roots are mixed with water at a solid-liquid ratio of 1:18-30, and sterilized and extracted at 50-100°C for 0.5-3 hours to obtain Imperata cylindrica root water extract, which is cooled to room temperature for subsequent three-stage fermentation. Firstly, it is colonized with 0.01~0.5% (v/v) yeast ( Saccharomyces cerevisiae) , purchased from the Biological Resources Conservation and Research Center , Taiwan, numbered BCRC20271) After fermenting the water extract of Imperata cylindrica root for 1~2 days, then directly add 0.01~0.25% (v/v) Lactobacillus plantarum TCI028, the patent is deposited in the preservation and research of biological resources Center, Taiwan, number BCRC910805) Ferment for 1~3 days, then add 1~20% (v/v) Acetobacter aceti (purchased from Biological Resources Conservation and Research Center, Taiwan, number BCRC11688) for fermentation 14~21 Day; Among them, the Lactobacillus TCI028 system has been patented in the Republic of China Patent Application No. 106145146. The fermentation sequence of these three bacteria is: yeast, lactobacillus, acetobacter, and cannot be reversed. Finally, do not remove these three In the case of inoculating bacteria, use the set sugar content range 35~45°, pH2~4, alcohol <3% and other specifications. If the test meets the specifications, the fermentation is determined to be completed and the fermentation broth is obtained, and the total polysaccharide content is at least 20%. Next, the fermentation broth is concentrated under reduced pressure at 45-70°C, and then filtered through a 200-400 mesh screen, followed by adding 1 to 3% citric acid and 40 to 70% isomalto-oligosaccharides to adjust the specifications, and then sterilize it The fermented product of the Imperata cylindrica root of the present invention is produced by microbial fermentation to release a large amount of the effective substances of the Imperata cylindrica root.

Example 2 The effect of the fermented product of Imperata cylindrica root of the present invention on enhancing LDLR and ABCA1 gene expression

In the present invention, human umbilical vascular endothelial cells (HUVEC) are used to test the expression of the Imperata cylindrica root fermentation product of the present invention on the LDLR gene and ABCA1 gene. The human vascular endothelial cell line was purchased from the Biological Resources Preservation and Research Center (Taiwan), numbered No.H-UV001. The cells were cultured in a medium containing 10% Fetal Bovine Serum and 90% DMEM (Dulbecco's Modified Eagle Medium, purchased from Gibco, USA, 12100-046), and 0.5 mM sodium pyruvate ( sodium pyruvate) and 15mM 4-hydroxyethyl piperazineethanesulfonic acid (HEPES) buffer solution.

Human vascular endothelial cells were divided into three groups: (1) the control group added with the aforementioned water extract of Imperata cylindrica, (2) the experimental group added 0.25% of the fermented cypress of the present invention, and (3) a blank containing only the culture solution In the control group, the vascular endothelial cells were then recovered with cell lysate (RB buffer, purchased from Genaid Company, Taiwan, Lot No. FC24015-G), and then RNA extraction reagent kit (purchased from Geneaid Company, Taiwan, Lot No.) was used to recover the cells. .FC24015-G) Collect the RNA in three groups of vascular endothelial cells, and then use SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA, number 18080-051) to use 2000ng of extracted RNA as a template and use primers to generate mRNA. Transcribe the corresponding cDNA products, and then use ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific company, the United States), and KAPA SYBR FAST (purchased from Sigma company, the United States, number 38220000000) to the three groups of reverse transcription products were listed in the table One combination of primers was used to perform a quantitative real-time reverse transcription polymerase chain reaction test under the conditions of 95°C for 1 second, 60°C for 20 seconds, and a total of 40 cycles. It is used to quantify the mRNA expression level of LDLR gene and ABCA1 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 the equation 2 ^-△Ct , where △Ct=Ct _{target gene-} Ct _ACTB (β-actin, beta-actin), then use Excel software to perform unpaired one-tailed student t-test to determine whether the coefficient of variation is statistically significant (*p value<0.05; **p Value<0.01; ***p value<0.001).

The results of the fermented Imperata cylindrica root of the present invention on enhancing the expression of LDLR gene and ABCA1 gene in vascular endothelial cells are shown in Figure 1 and Figure 2. Previous studies have indicated that low-density lipoprotein receptor (LDLR) can increase blood low-density lipoprotein Metabolism, reducing the proportion of low-density lipoprotein in the blood, And high-ATP binding cassette transporter A1 (ABCA1) is pointed out to promote the synthesis of high-density lipoprotein (HDL) to maintain blood lipid balance. After the vascular endothelial cells were treated with the Imperata cylindrica root fermentation product in this case, the expression level of LDLR gene was about 51% higher than that of the blank control group and the Imperata cylindrica root water extract group, and the ABCA1 gene was also higher than that of the blank control group and the Imperata cypress root water extract group, respectively. It is 31% and 15% higher. This result shows that the fermented Imperata cylindrica root of the present invention has a better ability to enhance the expression of LDLR gene and ABCA1 gene than the water extract of Imperata cylindrica after fermentation, and can increase the low density more effectively Lipoprotein metabolism and promotion of high-density lipoprotein production to maintain the balance of LDL and HDL in the blood, and achieve the effect of reducing blood lipids and preventing cardiovascular diseases.

Example 3 The effect of the fermented Imperata cylindrica root of the present invention on regulating the expression of PTGIS gene, SERPINE1 gene and IGF2BP3 gene

The human vascular endothelial cells were used to test the expression of the fermented cypress root of the present invention on the PTGIS gene, SERPINE1 gene and IGF2BP3 gene. The human vascular endothelial cells were divided into three groups: (1) a control group added with water extract of Imperata cylindrica root, (2) an experimental group added with the fermented product of Imperata cylindrica of the present invention, and (3) a blank control group containing only the culture solution. The expressions of PTGIS gene, SERPINE1 gene and IGF2BP3 gene in vascular endothelial cells treated with the fermented product of Imperata cylindrica of the present invention were analyzed by the method of Example 2.

The results of the fermented material of Imperata cylindrica of the present invention on enhancing the expression of PTGIS gene, SERPINE1 gene and IGF2BP3 gene in vascular endothelial cells are shown in Figure 3, Figure 4 and Figure 5. Previous studies have indicated that prostaglandin I synthase (PTGIS) is a prostaglandin ring The synthetase of Prostaglandin I2 (PGI2), PGI2 can inhibit platelet aggregation and prevent the formation of thrombus; Serine protease inhibitor E family 1 (SERPINE1) will promote plasminogen activator inhibitor-1 (Plasminogen activator inhibitor-1) , PAI-1). Excessive expression of PAI-1 is related to obesity, type 2 diabetes and cardiovascular disease; and insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) can promote Vascular endothelial growth factor, VEGF), VEGF can promote angiogenesis, and is related to cancer, heart Muscle infarction, cerebral apoplexy, and degenerative macular degeneration in the elderly are related. After the vascular endothelial cells were treated with the fermented Imperata cylindrica root in this case, the expression level of the PTGIS gene was about 72% higher than that of the blank control group and the Imperata cylindrica root water extract group; the SERPINE1 gene was about 40% lower than that of the blank control group and the Imperata cylindrica root water extract group. %, the IGF2BP3 gene is also about 90% and 70% lower than the blank control group and the Imperata cylindrica root water extract group respectively. This result shows that the IGF2BP3 gene of the present invention is more excellent than the Imperata cylindrica root water extract after fermentation. The ability to enhance the PTGIS gene and reduce the expression level of the SERPINE1 gene and IGF2BP3 gene can more effectively prevent thrombosis and vasodilation. It can also effectively reduce the occurrence of obesity and diabetes and other related cardiovascular diseases, so as to regulate blood pressure and prevent cardiovascular disease. effect.

To sum up, the present invention uses the water extract of Imperata cylindrica to perform three-stage fermentation with yeast, Lactobacillus, and Acetobacter. The fermentation product of Imperata cylindrica root can effectively enhance the expression of LDLR gene, ABCA1 gene and PTGIS gene in vascular endothelial cells. It also reduces the expression of SERPINE1 gene and IGF2BP3 gene, and has a better effect than the water extract of Imperata cylindrica. It can more effectively increase the metabolism of low-density lipoprotein and promote the production of high-density lipoprotein to maintain LDL and LDL in the blood. The balance of HDL can also more effectively prevent thrombosis and vasodilation, reduce obesity and diabetes and other related cardiovascular diseases, and achieve the effects of reducing blood lipids, vasodilation, and thrombosis to prevent cardiovascular diseases.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of the fermented product of Imperata cylindrica for regulating the expression of LDLR gene, ABCA1 gene, PTGIS gene, SERPINE1 gene and IGF2BP3 gene expression level

<130> 106B0621-I1

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Claims (7)

A fermented Imperata cylindrica root is used to prepare and regulate Low-Density Lipoprotein Receptor (LDLR) gene, adenosine triphosphate binding cassette transporter A1 (ATP binding cassette transporter A1, ABCA1) gene, and prostaglandin I synthesis Enzyme (Prostaglandin I synthase, PTGIS) gene, Serpin family E member 1, SERPINE1 gene, and insulin-like growth factor 2 mRNA-binding protein 3 (Insulin-like growth factor 2 mRNA-binding protein 3) , IGF2BP3) the use of a medicinal composition for gene expression, wherein the Imperata cylindrica root fermented product is obtained by a three-stage fermentation of a water extract of Imperata cylindrica root and a microbial group, wherein the microbial group is derived from a yeast ( Saccharomyces cerevisiae) ), a Lactobacillus plantarum , and Acetobacter aceti ; wherein the three-stage fermentation system is formed by the fermentation of the yeast, the Lactobacillus and the Acetobacter in sequence, and the microbial group In the three-stage fermentation, the first-stage fermentation is fermented with the yeast, the second-stage fermentation is fermented with the Lactobacillus, and the third-stage fermentation is fermented with the Acetobacter, wherein the yeast is added The amount is 0.01~0.5% (v/v), the addition amount of the Lactobacillus is 0.01~0.25% (v/v), and the addition amount of the Acetobacter is 1~20% (v/v); the first stage The ratio of the culture time between the second stage and the third stage is 1~2:1~3:14~21, so that the alcohol content of the fermented Imperata cylindrica is less than 3%. The use described in item 1 of the scope of patent application, wherein the low-density lipoprotein receptor gene, adenosine triphosphate binding cassette transporter A1 gene, and prostaglandin I synthase gene are up-regulated. The use as described in item 1 of the scope of patent application, wherein the serine protease inhibitor E family 1 gene and the insulin-like growth factor 2 mRNA binding protein 3 gene are down-regulated. The use as described in item 1 of the scope of patent application, wherein the total polysaccharide of the fermented Imperata cylindrica is at least 20%. A manufacturing method of fermented Imperata cylindrica root, which includes: The water extract of Imperata cylindrica root and a microbial group are obtained through three-stage fermentation. The microbial group is composed of a yeast, a lactobacillus, and an acetobacter. Yeast, the Lactobacillus, and the Acetobacter are fermented, and the yeast is added in the first stage, the Lactobacillus is added in the second stage, and the Acetobacter is added in the third stage to obtain a fermented cypress root, The addition amount of the yeast is 0.01~0.5% (v/v), the addition amount of the Lactobacillus is 0.01~0.25% (v/v), and the addition amount of the Acetobacter is 1~20% (v/v) ); The culture time ratio of the first stage, the second stage and the third stage is 1~2:1~3:14~21; so that the alcohol content of the fermented Imperata cylindrica is less than 3%. The manufacturing method as described in item 5 of the scope of patent application, wherein the water extract of Imperata cylindrica root is a mixture of Imperata cylindrica root in a solid-liquid ratio of 1:18-30 with water, and sterilized and extracted at 50-100℃ for 0.5-3 Earned in hours. According to the manufacturing method described in item 5 of the scope of patent application, the temperature of the first stage, the second stage and the third stage is 20~35°C.

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