TWI740536B - Uses of gynura procumbens extract for manufacturing a composition for improving sleep quality - Google Patents

Abstract

The present disclosure provides a use of Gynura procumbens extract for manufacturing a composition for improving sleep quality, wherein the composition includes Gynura procumbens extract as an active ingredient for regulating the expression of a melatonin synthesis gene. The present disclosure also provides a use of Gynura procumbens extract for manufacturing a composition for cardiovascular health, wherein the composition includes Gynura procumbens extract as an active ingredient for regulating the expression of a cholesterol metabolism gene, a high-density lipoprotein production gene, a thrombosis-related gene, a vasodilator related gene or their combinations.

Description

The use of the extract of P. chinensis and Panax notoginseng for the preparation of a composition for improving sleep quality way

The present invention relates to a use of an extract of P. chinensis and Panax notoginseng, in particular to the use of the extract of P. chinensis and Panax notoginseng in preparing a composition for improving sleep quality and a composition for preparing cardiovascular health care.

Gynura procumbens (Lour.) Merr. (scientific name: Gynura procumbens (Lour.) Merr.) It is a species under the genus Chrysanthemum notoginseng in the Compositae family.

Panax notoginseng is a climbing herb. Its stems are creeping, its leaves are stalked and its leaves are ovoid. Generally speaking, Panax notoginseng is distributed in China's Guangdong, Hainan, Guizhou, Yunnan, Vietnam, Thailand, Indonesia and Africa. It grows on the sandy soil on the slope beside the forest stream and climbs on shrubs or trees.

Panax notoginseng is a non-toxic medicinal and edible plant, and its roots, stems and leaves can be used. Among them, the rhizome of P. chinensis Panax notoginseng can be used as the raw material of Chinese patent medicine, and its fresh leaves and tender stems are rich in nutrition, and can be used as a green food for middle-aged and elderly people to supplement calcium. In addition, the leaves of Panax notoginseng can also be eaten raw, and the fresh leaves can be brewed in boiling water for tea.

In view of this, the present invention provides the use of the extract of Panax notoginseng.

In some embodiments, an extract of P. chinensis and Panax notoginseng is used to prepare a composition for improving sleep quality, wherein the composition includes P. chinensis as an active ingredient for regulating the expression level of melatonin synthesis genes. Seven extracts.

In some embodiments, an extract of P. chinensis Panax notoginseng is used to prepare a composition for cardiovascular health, wherein the composition includes the extract of P. chinensis Panax notoginseng as an active ingredient. This active ingredient is used to regulate the expression of cholesterol metabolism genes, the expression of high-density lipoprotein production genes, the expression of thrombosis-related genes, the expression of vascular health-related genes, or a combination thereof.

To sum up, the Phenix Notoginseng extract of any embodiment can be used to prepare a composition for improving sleep quality or a composition for preparing cardiovascular health. In some embodiments, the composition includes an extract of P. notoginseng as an active ingredient, and this active ingredient can be used to regulate the expression of a melatonin synthesis gene (eg, AANAT gene). In addition, the prepared composition can be used to improve insomnia, increase the proportion of deep sleep, or a combination thereof, thereby achieving the purpose of improving sleep quality. In some embodiments, the composition includes an extract of P. notoginseng as an active ingredient, and this active ingredient can be used to regulate the expression of cholesterol metabolism genes (eg, CEPT gene and SCARB1 gene), high-density lipoprotein production The expression level of genes (for example, ABCA1 gene), the expression level of thrombosis-related genes (for example, EDN1 gene, F3 gene, and SERPINE1 gene), the expression level of vasodilator-related genes (for example, PTGIS gene), or a combination thereof. In addition, the prepared composition has the potential to reduce cardiovascular diseases, and further achieves the purpose of vascular health care.

Figure 1 is the result graph of the relative gene expression magnification of the AANAT gene in the experimental group and the control group; Figure 2 is the result graph of the relative gene expression magnification of the CETP gene and SCARB1 gene of the experimental group and the control group; Figure 3 is the result graph of the relative gene expression magnification of the ABCA1 gene of the experimental group and the control group; Figure 4 is the experimental group and the control group Figure 5 shows the results of the relative gene expression magnifications of the EDN1, F3 and SERPINE1 genes in the group; Figure 5 shows the results of the relative gene expression magnifications of the PTGIS gene in the experimental group and the control group; Figure 6 shows the results of the experimental group and the control group at week 0 and The result graph of the relative gene expression rate of the AANAT gene in the 4th week; Fig. 7 is the result graph of the percentage of deep sleep in the 0th week and the 4th week of the experimental group and the control group; and Fig. 8 is the experimental group and the control group The result graph of the percentage of poor sleep quality in the 0th week and the 4th week.

Gynura procumbens (Gynura procumbens), commonly known as fairy grass, has the functions of clearing heat and detoxifying, stopping bleeding and relieving cough, improving human immunity and antiviral ability, supplementing calcium, fighting colds, and improving immunity.

In some embodiments, Gynura procumbens extract (or Longevity spinach extraction) is a whole plant (whole plant) of Gynura procumbens extract (or Longevity spinach extraction) is ground, extracted, filtered, concentrated, sterilized and combined. Obtained after drying. For example, after the whole plant is chopped up, extracted with an extraction solvent to obtain a preliminary extract, then the preliminary extract is filtered to remove impurities, and then the filtered preliminary extract is concentrated to obtain a concentrated liquid. Next, the concentrated solution is sterilized, and the sterilized concentrated solution is spray-dried to dry into a powder. Here, the dried powder contains an extract of P. chinensis Panax notoginseng as an active ingredient. In some embodiments, the powder containing the extracts of Panax notoginseng can also be purchased from a manufacturer (Changsha Agricultural Construction, China).

In some embodiments, the extract of Panax notoginseng is used to prepare a composition for improving sleep quality. The composition includes an extract of Panax notoginseng as an active ingredient for regulating the expression level of melatonin synthesis genes. In some embodiments, the melatonin synthesis gene is an AANAT gene. For example, a composition prepared with an extract of P. notoginseng as the active ingredient can increase the expression of melatonin synthesis genes. In some embodiments, the composition containing the extract of Panax notoginseng can improve the quality of sleep by increasing the proportion of deep sleep. In some embodiments, the composition containing the extract of P. chinensis Panax notoginseng improves the quality of sleep by improving insomnia.

In some embodiments, the extract of Panax notoginseng is used to prepare a cardiovascular health care composition. The composition includes an extract of P. chinensis and Panax notoginseng as an active ingredient. In addition, this active ingredient is used to regulate the expression of cholesterol metabolism genes, the expression of high-density lipoprotein production genes, the expression of thrombosis-related genes, the expression of vasodilation factor-related genes, or a combination thereof. For example, the composition prepared with the P. chinensis extract as the active ingredient can have at least one of the following functions: increase the expression level of cholesterol metabolism genes, increase the expression level of high-density lipoprotein production genes, and reduce thrombosis. Generate the expression level of related genes and enhance the expression level of vasodilation factor-related genes.

In some embodiments, cholesterol metabolism genes include CEPT gene and SCARB1 gene. When the expression level of CEPT gene and SCARB1 gene (also known as SRB1 gene) increases, it represents an increase in cholesterol metabolism.

In some embodiments, the high-density lipoprotein production gene is ABCA1 gene. When the expression level of ABCA1 gene increases, it means that the production of high-density lipoprotein also increases.

In some embodiments, thrombosis-related genes include EDN1 gene, F3 gene, and SERPINE1 gene. When the expression level of EDN1 gene, F3 gene and SERPINE1 gene decreases, it means that the possibility of thrombosis is reduced.

In some embodiments, the vasodilator-related gene is the PTGIS gene. When the expression level of PTGIS gene increases, it means that the expression level of endothelial-derived vasodilation factor also increases.

In organisms, the extract of Panax notoginseng will affect one or more genes in the organism (e.g., melatonin synthesis gene, cholesterol metabolism gene, high-density Lipoprotein production genes, thrombosis-related genes, vasodilator-related genes or combinations thereof). In some embodiments, the composition prepared by the extract of Panax notoginseng can simultaneously regulate melatonin synthesis genes, cholesterol metabolism genes, high-density lipoprotein production genes, thrombosis-related genes, and vasodilation factor-related genes. In some embodiments, the composition prepared by the extract of Panax notoginseng can simultaneously regulate the expression of cholesterol metabolism genes and the expression of high-density lipoprotein production genes, or simultaneously regulate the expression of genes related to thrombosis and The expression level of vasodilator-related genes. In some embodiments, the composition prepared by the extract of Panax notoginseng can regulate the expression of cholesterol metabolism genes and the expression of genes related to thrombosis at the same time.

In some embodiments, the composition prepared from the extract of Panax notoginseng further includes an adjuvant. For example, the adjuvant may be Maltodextrin, a food additive, or a combination thereof. For example, the food additive may be malic acid, sucralose, citric acid, fruit flavor, honey flavor, steviol glycoside or a combination thereof.

In one example, the extract of Panax notoginseng and Metodextrin were mixed in a weight ratio of 9:1 to form the first composition (purchased from Changsha Agricultural Construction, China). In another exemplary embodiment, the Phenix Notoginseng extract and the adjuvant are mixed to form the second composition. For example, the first composition (purchased from Changsha Nongjian, China), maltodextrin, and food additives are mixed to form a second composition, and this second composition includes 15.75% by weight of P. chinensis extract Ingredients, 72% by weight of maltodextrin, 4.5% by weight of water and 7.75% by weight of food additives.

Herein, in some embodiments, the composition prepared with the P. notoginseng extract as the active ingredient can be processed by extracting the P. notoginseng extract obtained by extracting the P. notoginseng extract. get.

Example 1: Cell gene experiment related to sleep

Melatonin can regulate the operation of the human biological clock, but as we age, the secretion of melatonin will decrease and cause insomnia. Therefore, improving the performance of melatonin helps to improve the quality of sleep. Here, in Example 1, the first composition (purchased from Changsha Agricultural Construction, China) containing 90% by weight of Panax notoginseng extract and 10% maltodextrin was used to test the effect of Panax notoginseng extract on human nerve cells. (SH-SY5Y) (purchased from ATCC) the expression of melatonin-producing genes. For example, the melatonin-producing gene is the AANAT gene (Gene ID: 15), and the protein encoded by the AANAT gene is one of the enzymes involved in the synthesis of melatonin and can catalyze the synthesis of melatonin by serotonin. hormone. Therefore, the AANAT gene was used as the target of analysis in Example 1.

First, culture human nerve cells in EBSS medium (Ham's F12 and DMEM are mixed in a 1:1 volume ratio (Gibc; Cat.11320033). Then, 10 ⁵ human nerve cells are taken to each well to contain 2 ml of EBSS medium Culture the human nerve cells in each well at 37°C for 24 hours in the six-well cell culture dish, and divide the human nerve cells cultured in each well into an experimental group and a control group (two groups in total) according to the following test conditions to treat the human nerves cultured in each well cell.

Experimental group: According to the ratio of 0.25 mg of the first composition per milliliter of EBSS medium (ie, the concentration is 0.25 mg/ml), the first combination was added to the EBSS medium containing cultured human nerve cells.

Control group: No treatment, that is, no additional compounds were added to the EBSS medium containing cultured human nerve cells.

The experimental group and the control group were cultured again at 37°C for 24 hours, so that the first composition in the experimental group treated the cultured human nerve cells, and the control group was used as a control group for comparison with the experimental group.

The treated human nerve cells (ie, the experimental group) and the untreated human nerve-like cells (ie, the control group) are respectively broken through cell membranes with cell lysates to form two sets of cell solutions. Next, RNA extraction reagent kits (purchased from Geneaid, Taiwan, Lot No. FC24015-G) were used to extract RNA in the two sets of cell solutions respectively. Then, each group takes 1000 nanograms (ng) of the extracted RNA as a template, and ^{reverse transcribes the extracted RNA into the corresponding cDNA by SuperScript ®} III reverse transcriptase (purchased from Invitrogene, USA, number 18080-051) . Then by ABI StepOnePlus ^TM real-time PCR system (ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific company, U.S.)), KAPA SYBR FAST (purchased from Sigma company, U.S., No. 38220000000) and the primers in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2) Quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain rcaction) was performed on the cDNA of the two groups to observe the AANAT gene in the human nerve cells of the experimental group and the control group Performance. The instrument setting conditions for quantitative real-time reverse transcription polymerase chain reaction were 95°C for 20 seconds, followed by 95°C for 3 seconds, and 60°C for 30 seconds, 40 cycles were repeated, and the 2-△Ct method was used for gene quantification. Here, the quantitative real-time reverse transcription polymerase chain reaction by cDNA can indirectly quantify the mRNA expression of the AANAT gene, and then infer the expression of the protein encoded by the AANAT gene.

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

Please refer to Figure 1. When the expression level of the AANAT gene in the control group is regarded as 1 (that is, 100%), the expression level of the AANAT gene in the experimental group relative to the control group is 1.36 (that is, 136%), which means that the expression level of the AANAT gene in the experimental group is control 1.36 times of the group. It can be seen that when human nerve cells are treated with the first composition containing 90% by weight of the extract Later, the expression level of the AANAT gene of human nerve cells increased, which represented an increase in the expression level of the melatonin-producing gene. When the expression level of melatonin-producing genes increases, the expression level of melatonin also increases, which has the effect of improving sleep quality.

Example 2: Sleep-related human efficacy experiments

A number of subjects with poor sleep quality were recruited, and a sleep quality test (hereinafter referred to as the test) was performed on the second composition containing 15.75% by weight of the extract of P. chinensis and Panax notoginseng. In addition, before the test, each subject used the Pittsburgh sleep quality index (PSQI) scale to test the Pittsburgh sleep quality index, and the higher the Pittsburgh sleep quality index, the better the subjects thought of their sleep quality. Difference.

There are 7 subjects in total, and the Pittsburgh Sleep Quality Index of each subject is greater than 5. The duration of the test was 4 weeks. During the test, the subjects took 4 grams of the second composition one hour before going to bed every day, in other words, the subjects took 0.7 grams of Phenix Notoginseng extract per day.

The second composition is a mixture of the first composition (purchased from Changsha Agricultural Construction, China) and an adjuvant, and its formula is that every 100 grams of the second composition contains 15.75 grams of Phenix Notoginseng extract , 76.5 grams of maltodextrin mixture (mixed with 72 grams of maltodextrin and 4.5 grams of water), 0.25 grams of sucralose, 0.5 grams of steviol glycosides, 2.25 grams of fruit flavor powder, and 5 grams of malic acid and anhydrous Citric acid.

In addition, three sleep-related experiments were tested before the test (that is, the state where the subject did not take the second composition, which is regarded as week 0, hereinafter referred to as week 0) and after the test (that is, week 4). The three sleep-related experiments are the expression level of the melatonin production-related genes in the blood of the subjects (the AANAT gene is used as the target in Example 2) (as shown in Figure 2), and the smart bracelet is used to detect the depth of the subjects. Sleep percentage (as shown in Figure 3), and assess the subjects’ sleep quality with the Pittsburgh Sleep Quality Index The degree of poor quality (as shown in Figure 4). It should be noted that the relative gene expression of the AANAT gene shown in the diagram is presented in relative magnification, and the percentage of deep sleep and the degree of poor sleep quality shown in the diagram are presented in percentages, which are calculated using the STDEV formula of Excel software Standard deviation, and use one-tailed Student t-test in Excel to analyze whether there is a statistically significant difference.

Experiment (1) The expression level of the AANAT gene of the subjects in the 0th week and the 4th week

Before the test (i.e. week 0) and after the test (i.e. week 4), venous blood collection tubes containing EDTA anticoagulant were used to collect 6 ml of venous blood from 7 subjects, and then the venous blood was collected. speed centrifugation at 300x g for 15 min. Take 2 ml of buffy coat white blood cell layer from the venous blood after centrifugation, and dilute the white blood cell layer with 2 ml phosphate buffer (1X PBS; hereinafter referred to as 1X PBS buffer) to 4 ml, and then The diluted white blood cell layer is slowly added to a centrifuge tube containing 3 ml of cell separation solution (Ficoll-Paque Plus cell separation solution). During the addition, the diluted white blood cell layer and the cell separation solution must be in a layered state and cannot be mixed. Next, centrifuge the centrifuge tube containing the layered and diluted white blood cell layer and cell separation solution at 400 x g for 40 minutes, and remove the supernatant after centrifugation, and take the peripheral blood in the middle layer in the centrifuge tube after centrifugation. Peripheral blood mononuclear cell (PBMC; hereinafter referred to as PBMC) 2 to 3 ml. The PBMC is rinsed with 3 times the volume of 1X PBS buffer and then mixed with the aforementioned 1X PBS buffer to form a PBMC mixture. Then, the PBMC mixture was centrifuged at 300×g for 10 minutes to form a PBMC upper solution and a PBMC precipitate, and the PBMC precipitate was lysed with 600 microliters (μl) of RNA lysis buffer to extract RNA.

Then, as in the step of example 1, after reverse transcription of the extracted RNA into cDNA, the cDNA is quantitatively and instantly reversed with primers (SEQ ID NO: 1 and SEQ ID NO: 2) The polymerase chain reaction was recorded to observe the expression level of the AANAT gene in the blood in the 0th and 4th weeks. Please refer to Figure 2. When the average expression level of the AANAT gene of the 7 subjects in week 0 is regarded as 1 (ie 100%), the average expression level of the AANAT gene in the fourth week is 3.45 (ie 345%), which represents the average expression level of the AANAT gene in the fourth week. The average expression level of AANAT gene was 3.45 times that of week 0. It can be seen that when the user administers the second composition containing 15.75% by weight of Panax notoginseng extract daily for 4 weeks, the expression level of the AANAT gene in the blood of the user increases, which represents the AANAT gene. The expression level of the encoded protein is improved. In addition, the protein encoded by the AANAT gene acts as an enzyme involved in the melatonin production pathway, which can react serotonin to N-Acetylserotonin, which is from serotonin to melatonin. Reaction intermediates for endogenous synthesis. In other words, when the expression level of the AANAT gene increases, the expression level of melatonin also increases, which in turn has the effect of improving sleep quality.

Experiment (2) The proportion of subjects in deep sleep at week 0 and week 4

Under normal conditions, humans will have 4 to 5 sleep cycles every night, and each sleep cycle is divided into four stages. Among them, the "deep sleep" state occurs in the third and fourth stages. "Deep sleep" or deep sleep is also known as slow wave sleep (SWS). When the human body enters a deep sleep state, brain waves change greatly, and their frequency and amplitude increase. In addition, the deep sleep state of the first sleep cycle every night usually lasts 45 to 90 minutes, and the duration of the sleep state will shorten as the number of sleep cycles increases. Generally speaking, the proportion of deep sleep in the sleep cycle (that is, the proportion of deep sleep) can represent the quality of sleep. When the proportion of deep sleep increases, it means that the quality of sleep is also improved. Therefore, experiment (2) takes the proportion of deep sleep as the test item.

Before the test (that is, the 0th week), the sleep status of the subjects was monitored by the smart bracelet, and after the test (that is, the 4th week), the sleep status of the subjects was monitored by the smart bracelet. And observe the proportion of subjects in deep sleep. In experiment (2), the subject wears a Xiaomi smart bracelet, and uses the built-in acceleration sensor and photoelectric heart rate sensor of the bracelet to evaluate deep sleep and light sleep based on the subject’s arm activity variables and heart rhythm during sleep Sleep ratio. In addition, the subjects' arm activity was low and the heart rate was relatively stable in the deep sleep state.

Please refer to Figure 3. At week 0, the average deep sleep ratio of the 7 subjects was 31.3%. At the 4th week, the average deep sleep of 7 subjects accounted for 35.6%. In other words, the average deep sleep proportion of the 7 subjects increased by 4.3%, which means that the time of the deep sleep state in the sleep cycle of the subjects increased after taking the second composition. Herein, it can be seen that taking a composition containing 15.75% by weight of Panax notoginseng extract can improve the quality of sleep by increasing the proportion of deep sleep.

Experiment (3) The degree of poor sleep quality of the subjects in the 0th and 4th weeks

Before the test (that is, week 0), the Pittsburgh Sleep Quality Index was used to test the Pittsburgh Sleep Quality Index of 7 subjects, and after the test (that is, week 4), the Pittsburgh Sleep Quality Index was tested again by the Pittsburgh Sleep Quality Index. Pittsburgh sleep quality index of 7 subjects, and then observe the subjects' poor sleep quality. The Pittsburgh Sleep Quality Index Scale mainly assesses the sleep quality of testers, and its contents include sleep quality, sleep lag period, total sleep hours, habitual sleep efficiency, sleep disturbances (such as insomnia), use of sleeping pills, and daytime dysfunction In addition to seven indicators, it also includes evaluation factors such as whether sleep interruption is caused by physiological factors such as cough, dryness and heat.

Please refer to Figure 4. When the poor sleep quality of the 7 subjects at week 0 was regarded as 100%, the poor sleep quality of the 7 subjects at week 4 was 67.9%. In other words, the poor sleep quality of 7 subjects decreased by 32.1%, which means that the sleep quality of 7 subjects improved.

It can be seen that when the user administers the second composition containing 15.75% by weight of the extract of P. notoginseng every day for 4 weeks, the expression of the AANAT gene in the user’s blood The current amount increased, the proportion of deep sleep increased, and the degree of poor sleep quality decreased. Herein, it is verified that when each individual is administered a composition containing 15.75% by weight of the extract of P. chinensis, the sleep quality of the individual can be improved after continuous administration for 4 weeks.

Example 3: Cell gene experiments related to high- and medium-density cholesterol

The first composition (purchased from Changsha Agricultural Construction, China) containing 90% by weight of the extract of Panax notoginseng was used to test the high level of Panax notoginseng extract in human liver cells (HepG2) (purchased from ATCC). Density lipoprotein production gene and cholesterol metabolism gene. For example, the high-density lipoprotein production gene is ABCA1 gene (Gene ID: 19), and the protein encoded by the ABCA1 gene can promote the production of high-density lipoprotein and cholesterol metabolism. Cholesterol metabolism genes include CETP gene (Gene ID: 1071) and SCARB1 gene (Gene ID: 949). Among them, the protein encoded by the CETP gene is plasma cholesteryl ester transfer protein, which is one of the important transport proteins in lipid metabolism, and the protein encoded by the SCARB1 gene is the receptor for high-density lipoprotein (HDL) in the liver, which can promote The liver metabolizes cholesterol. Therefore, in Example 3, ABCA1 gene, CETP gene and SCARB1 gene are used as the analysis targets.

First, human liver cells were cultured in DMEM (Dulbecco's modified Eagle's) medium (Gibco; Cat. 11965-092). Then, 10 ⁵ human liver cells were respectively taken into a six-well cell culture dish containing 2 ml of DMEM medium per well and cultured at 37° C. for 24 hours. The human liver cells cultured in each well were divided into two groups according to the following test conditions to process the human liver cells cultured in each well.

Experimental group: The first combination was added to the DMEM medium containing cultured human liver cells according to the ratio of 0.5 mg of the first composition per milliliter of DMEM medium (ie, the concentration was 0.5 mg/ml).

Control group: No treatment, that is, no additional compounds were added to the DMEM medium containing cultured human liver cells.

The experimental group and the control group were cultured again at 37° C. for 48 hours, so that the first composition in the experimental group treated the cultured human liver cells, and the control group was used as a control group for comparison with the experimental group.

The treated human liver cells (ie, the experimental group) and the untreated human liver cells (ie, the control group) were respectively broken through cell membranes with cell lysates to form two sets of cell solutions. Then, RNA extraction reagent kits (purchased from Geneaid, Taiwan, Lot No. FC35901-DG) were used to extract RNA in the two sets of cell solutions respectively. Then, the experimental group and the control group each took 1000 nanograms (ng) of the extracted RNA as a template, and used SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) to transfer the extracted RNA Reverse transcription into the corresponding cDNA. Then by ABI StepOnePlus ^TM real-time PCR system (ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific company, U.S.)), KAPA SYBR FAST (purchased from Sigma company, U.S., number 38220000000) and the primers in Table 2 (SEQ ID NO: 3 to SEQ ID NO: 8) Quantitative real-time reverse transcription polymerase chain reaction was performed on the cDNA of the two groups to observe the ABCA1 gene in the human liver cells of the experimental group and the control group , CETP gene and SCARB1 gene expression level. The instrument setting conditions for quantitative real-time reverse transcription polymerase chain reaction were 95°C for 20 seconds, followed by 95°C for 3 seconds, and 60°C for 30 seconds, 40 cycles were repeated, and the 2-△Ct method was used for gene quantification. Here, quantitative real-time reverse transcription polymerase chain reaction using cDNA can indirectly quantify the mRNA expression levels of ABCA1, CETP, and SCARB1 genes, and infer the expression levels of the proteins encoded by ABCA1, CETP, and SCARB1 genes.

It is important to note that the relative gene expressions of ABCA1, CETP and SCARB1 genes shown in the diagrams mentioned below are presented in relative magnification. The STDEV formula of Excel software is used to calculate the standard deviation, and the standard deviation is calculated in Excel software. One-tailed Student t-test analyzes whether there are statistically significant differences. In the diagram, "*" represents the p-value is less than 0.05, "**" represents the p-value is less than 0.01, and "***" represents the p-value is less than 0.001. The more "*", the more significant the statistical difference.

Refer to Figure 5. When the expression level of ABCA1 gene in the control group is regarded as 1 (that is, 100%), the expression level of the ABCA1 gene in the experimental group relative to the control group is 1.58 (that is, 158%), which means that the expression level of the ABCA1 gene in the experimental group is control 1.58 times of the group. In other words, the expression level of ABCA1 gene in the experimental group increased by 58% compared to the control group. It can be seen that when the human liver cells are treated with the first composition containing 90% by weight of the extract of Panax notoginseng, the expression level of the ABCA1 gene of the human liver cells increases, which represents an increase in the expression level of the high-density lipoprotein production gene. . When the expression level of high-density lipoprotein-producing genes increases, the expression of high-density lipoprotein The current amount has also been increased, and it has the effect of protecting the cardiovascular system.

Refer to Figure 6. When the expression level of CETP gene and SCARB1 gene in the control group is regarded as 1, the expression level of the CETP gene in the experimental group relative to the control group is 198.32, and the expression level of the SCARB1 gene is 206.88, which represents the expression level of the CETP gene in the experimental group It was 198.32 times that of the control group, and its expression of SCARB1 gene was 206.88 times that of the control group. In other words, the expression levels of CETP gene and SCARB1 gene in the experimental group increased by about 200% compared with the control group. It can be seen that when the human liver cells are treated with the first composition containing 90% by weight of the extract of Panorax notoginseng, the expression levels of the CETP gene and the SCARB1 gene of the human liver cells increase, which represents the expression level of cholesterol metabolism genes. improve. When the expression level of cholesterol metabolism genes increases, the metabolic power of cholesterol increases. Therefore, the composition containing the extract of P. chinensis and Panax notoginseng has the potential to reduce cardiovascular diseases, thereby achieving the effect of cardiovascular health.

Example 4: Cell gene experiments related to cardiovascular health care

The first composition (purchased from Changsha Agricultural Construction, China) containing 90% by weight of Panax notoginseng extract was used to test the effect of Panax notoginseng extract on human vascular endothelial cells (HUVEC) (purchased from BCRC). Genes related to vascular health care and genes related to thrombosis. For example, the vascular health-related gene is the PTGIS gene (Gene ID: 5740), and the protein encoded by the PTGIS gene is endothelial-derived vasodilator factor. Thrombosis-related genes include EDN1 gene (Gene ID: 1906), F3 gene (Gene ID: 2152) and SERPINE1 gene (Gene ID: 5054). Among them, the proteolytic peptide encoded by the EDN1 gene can be used as a vasoconstrictor, the protein encoded by the F3 gene is related to the coagulation mechanism, and the protein encoded by the SERPINE1 gene is used as a serine protease inhibitor. Therefore, in Example 4, PTGIS gene, EDN1 gene, F3 gene and SERPINE1 gene are used as the analysis targets.

First, human vascular endothelial cells were cultured in EC medium (Gibc; Cat.M-200-500). Then, 10 ⁵ human vascular endothelial cells were respectively taken into a six-well cell culture dish containing 2 ml of EC culture medium per well and cultured at 37° C. for 24 hours. The human vascular endothelial cells cultured in each well were divided into two groups according to the following test conditions to treat the human vascular endothelial cells cultured in each well.

Experimental group: 0.125% by volume of the first composition was added to the EC medium containing cultured human vascular endothelial cells.

Control group: No treatment, that is, no additional compounds were added to the EC medium containing cultured human vascular endothelial cells.

The experimental group and the control group were cultured again at 37°C for 6 hours, so that the first composition in the experimental group would treat the cultured human vascular endothelial cells, and the control group would serve as a control group for comparison with the experimental group.

The treated human vascular endothelial cells (ie, the experimental group) and untreated human vascular endothelial cells (ie, the control group) were respectively broken through cell membranes with cell lysates to form two sets of cell solutions. Next, RNA extraction reagent kits (purchased from Geneaid, Taiwan, Lot No. FC24015-G) were used to extract RNA in the two sets of cell solutions respectively. Then, the experimental group and the control group each took 1000 nanograms (ng) of the extracted RNA as a template, and used SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) to transfer the extracted RNA Reverse transcription into the corresponding cDNA. Then by ABI StepOnePlusTM real time PCR system ^{(ABI StepOnePlus TM Real-Time PCR} system (Thermo Fisher Scientific , USA)), KAPA SYBR FAST (commercially available from Sigma, USA, No. 38220000000) and Table 3 primers (SEQ ID NO : 9 to SEQ ID NO: 20) Quantitative real-time reverse transcription polymerase chain reaction was performed on the cDNA of the two groups to observe the PTGIS gene in the human vascular endothelial cells of the experimental group and the control group , EDN1 gene, F3 gene and SERPINE1 gene expression level. The instrument setting conditions for quantitative real-time reverse transcription polymerase chain reaction were 95°C for 20 seconds, followed by 95°C for 3 seconds, and 60°C for 30 seconds, 40 cycles were repeated, and the 2-△Ct method was used for gene quantification. Here, quantitative real-time reverse transcription polymerase chain reaction using cDNA can indirectly quantify the mRNA expression of PTGIS gene, EDN1 gene, F3 gene and SERPINE1 gene, and then infer the proteins encoded by PTGIS gene, EDN1 gene, F3 gene and SERPINE1 gene. Performance.

It is important to note that the relative gene expression lines of PTGIS gene, EDN1 gene, F3 gene and SERPINE1 gene shown in the scheme mentioned below are presented in relative magnification. Now, the STDEV formula of Excel software is used to calculate the standard deviation, and the one-tailed Student t-test is used to analyze whether there is a statistically significant difference in Excel software. In the diagram, "*" represents the p-value is less than 0.05, "**" represents the p-value is less than 0.01, and "***" represents the p-value is less than 0.001. The more "*", the more significant the statistical difference.

Refer to Figure 7. When the expression level of the PTGIS gene in the control group is regarded as 1 (that is, 100%), the expression level of the PTGIS gene in the experimental group relative to the control group is 1.42 (that is, 142%), which represents the expression level of the PTGIS gene in the experimental group is control 1.42 times of the group. In other words, the expression level of the PTGIS gene in the experimental group was 42% higher than that in the control group. It can be seen that when human vascular endothelial cells are treated with the first composition containing 90% by weight of the extract of Panax notoginseng, the expression level of PTGIS gene of human vascular endothelial cells increases, which represents the expression level of vasodilator-related genes improve. When the expression level of vasodilation factor-related genes increases, the expression level of the protein encoded by the vasodilation factor-related genes also increases, which has the effect of protecting the cardiovascular system.

Refer to Figure 8. When the expression level of the EDN1 gene, F3 gene and SERPINE1 gene of the control group is regarded as 1 (ie 100%), the expression level of the EDN1 gene of the experimental group relative to the control group is 0.72 (ie 72%), the expression level of the F3 gene The expression level of the SERPINE1 gene is 0.61 (61%) and the expression level of the SERPINE1 gene is 0.91 (91%), which means that the expression level of the EDN1 gene in the experimental group is reduced to 0.72 times that of the control group, and the expression level of the F3 gene is reduced to 0.61 times that of the control group. And the expression level of SERPINE1 gene decreased to 0.91 times of the control group. In other words, compared with the control group, the expression level of the EDN1 gene in the experimental group decreased by 28%, the expression level of the F3 gene decreased by 39%, and the expression level of the SERPINE1 gene decreased by 9%. It can be seen that when the human vascular endothelial cells are treated with the first composition containing 90% by weight of Panax notoginseng extract, the expression levels of EDN1, F3 and SERPINE1 genes of human vascular endothelial cells decrease, which represents thrombosis. List of related genes The current volume has dropped. When the expression of thrombosis-related genes decreases, the amount of thrombosis also decreases, which has the effect of health care for the cardiovascular system.

Here, the composition prepared by the extract of P. chinensis Panax notoginseng can be used to improve sleep quality and health care of the cardiovascular system. In addition, the composition has the ability to regulate at least one gene expression of AANAT gene, ABCA1 gene, CEPT gene, SCARB1 gene, PTGIS gene, EDN1 gene, F3 gene and SERPINE1 gene. In some embodiments, the composition includes an extract of P. chinensis Panax notoginseng as an active ingredient, and this active ingredient can be used to increase the expression of AANAT gene. Therefore, the prepared composition can be used to improve insomnia and/or enhance deepness. The proportion of sleep, thereby improving the quality of sleep. In some embodiments, the composition includes an extract of P. notoginseng as an active ingredient, and this active ingredient can be used to increase the expression level of CEPT gene and SCARB1 gene, increase the expression level of ABCA1 gene, and reduce EDN1 gene and F3 gene. And the expression level of SERPINE1 gene, increase the expression level of PTGIS gene, or a combination thereof. Therefore, the prepared composition has the potential to reduce cardiovascular diseases and has the effect of protecting cardiovascular diseases.

To sum up, according to any embodiment of the present invention, the composition with P. notoginseng extract as the active ingredient can regulate AANAT gene, ABCA1 gene, CEPT gene, SCARB1 gene, PTGIS gene, EDN1 gene, F3 gene and The ability to express at least one of the SERPINE1 genes. Therefore, the composition with P. chinensis extract as the active ingredient can have at least one of the following functions: improve insomnia, increase the proportion of deep sleep, improve sleep quality, increase cholesterol metabolism, promote high-density lipoprotein production, and reduce thrombosis Generating, health-care cardiovascular, or a combination thereof.

Although the technical content of the present invention has been disclosed in the preferred embodiment as above, it is not intended to limit the present invention. Anyone familiar with the art can make a little without departing from the spirit of the present invention. The changes and modifications should be included in the scope of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Claims (5)

A use of an extract of P. notoginseng for preparing a composition for improving sleep quality, wherein the composition includes the extract of P. notoginseng as an active ingredient for regulating the expression of a melatonin synthesis gene. The use according to claim 1, wherein the composition containing the P. chinense extract extract improves the quality of sleep by increasing the proportion of deep sleep. The use according to claim 1, wherein the composition containing the Phenix Notoginseng extract improves sleep quality by improving insomnia. The use according to any one of claim 1 to claim 3, wherein the melatonin synthesis gene is an AANAT gene. The use according to any one of claim 1 to claim 3, wherein the composition includes 15.75% by weight of the Phenix Notoginseng extract.

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