TW202308672A - Use of extract in manufacturing a pharmaceutical or non-pharmaceutical composition for enhancing the activity of mitochondria - Google Patents

Abstract

The embodiment according to the present disclosure provides a use of extract in manufacturing a pharmaceutical or non-pharmaceutical composition for enhancing the activity of mitochondria. The activity-enhanced mitochondria can maintain its function and activity when encountering stress to ensure normal work of cells without affecting by external or internal stress.

Description

Use of extract for preparing medicinal or non-medicinal composition for improving mitochondrial activity

The present invention relates to the use of the extract for preparing a composition for improving mitochondrial activity, in particular to the use of sea grape extract for preparing a medicinal or non-medicine composition for improving mitochondrial activity.

Mitochondria is the main site for oxidative phosphorylation and synthesis of adenosine triphosphate (ATP) in cells. Since adenosine triphosphate is the energy source for cell activities, mitochondria are also known as "cell energy factories". In addition to providing energy for cells, mitochondria are also involved in processes such as cell differentiation, cell message transmission, and cell apoptosis, and have the ability to regulate cell growth cycles.

However, some of the by-products produced by mitochondria during oxidative phosphorylation reactions are harmful to mitochondria. Damaged mitochondria will have adverse effects on cellular energy supply, cell growth, etc. Accumulated over time, severely damaged mitochondria will trigger mitochondrial disintegration, which in turn triggers apoptosis. Therefore, how to improve the ability of mitochondria to face stress, protect and repair mitochondria to maintain its function and slow down the breakdown of mitochondria has become an important topic.

The embodiment of the present invention provides the use of extracts to increase the activity of mitochondria, so as to enhance the ability of mitochondria to face stress, and then maintain the function and activity of mitochondria in the face of stress.

An embodiment of the present invention provides a use of an extract for preparing a medicinal or non-medical composition for enhancing mitochondrial activity, wherein the extract is a sea grape extract.

The use of the extract provided according to the embodiment of the present invention for preparing a medicinal or non-pharmaceutical composition for improving mitochondrial activity, wherein the extract is sea grape extract, and sea grape extract can improve the maximum oxygen consumption capacity of mitochondria , Improve the pre-stored oxygen consumption capacity of mitochondria, improve the bioenergy health index of mitochondria, and improve the ability of mitochondria to cope with stress. Mitochondria whose activity is enhanced by sea grape extract can maintain their functions in the face of stress and activity to ensure that cells function properly without adverse effects from external or internal stress.

The detailed features and advantages of the present invention are described in detail in the following embodiments, the content of which is sufficient to enable anyone familiar with the relevant art to understand the technical content of the present invention and implement it accordingly, and according to the content disclosed in this specification, the scope of the patent application and the drawings , anyone skilled in the art can easily understand the purpose and advantages of the present invention. The following examples are to further describe the concept of the present invention in detail, but not to limit the scope of the present invention in any way.

Caulerpa lentillifera is a kind of edible seaweed. It is called "sea grape" because of its upright stem globules that are crystal clear, moist and plump like grapes. It tastes as rich and juicy as caviar, so it is also Known as "green caviar". In this article, the common name "sea grape" is used to refer to Botrytis elongatus. Sea grapes are often used as food in Asia-Pacific regions such as Japan, Vietnam, the Philippines, Malaysia and Indonesia. Studies have pointed out that about 80% of the composition of sea grapes is mainly composed of seaweed polysaccharides, collagen and dietary fiber, which are rich in nutritional value. Sea grapes are rich in micro-mineral elements and vitamins, and contain unsaturated fatty acids, amino acids and algae polysaccharides. Of the 16 amino acids detected from sea grapes, 11 are essential amino acids for the human body, including 7 amino acids that cannot be synthesized by the human body. Moreover, the polysaccharides contained in sea grapes contribute to biological activities, such as anti-oxidation, anti-coagulation, anti-tumor, anti-virus, and immune-stimulating activities.

The extract used in one embodiment of the present invention is sea grape extract, which is obtained by extraction in the following manner. First, place 100 grams of sea grapes in 400 grams of water and cook for 3 hours at 100°C. Filter the boiled sea grapes after they are cooled, and centrifuge the filtrate at 1000 g for 10 minutes to get the supernatant. The supernatant was frozen at -80°C for one night, and freeze-dried the next day to obtain sea grape extract. 100 grams of sea grapes can be made into about 1.8 grams of sea grape extract, with a yield of about 1.8%.

In the sea grape extract extracted by the above method, it contains 16.87% to 18.65% by weight of sugar, 5.99% to 6.63% of protein and 1.9% to 2.1% of lipid, wherein the weight percentage of sugar is 4.84% to 6.52% polysaccharides, lipids contain unsaturated fatty acids.

In one embodiment of the present invention, providing the sea grape extract with a concentration of 250 μg/ml to 1000 μg/ml to the cells can improve the activity of mitochondria, specifically manifested in improving the maximum oxygen consumption capacity of mitochondria, Improve the pre-stored oxygen consumption capacity of mitochondria, improve the bioenergy health index of mitochondria, and improve the ability of mitochondria to cope with stress. In another embodiment, the concentration of the sea grape extract may be 500 μg/ml to 1000 μg/ml. In other embodiments, the concentration of the sea grape extract may be 250 μg/ml to 500 μg/ml.

The method of providing the sea grape extract to cells is, for example, eating the sea grape extract orally. When the sea grape extract is provided to the cells in the form of food, the effective dose of the sea grape extract is 2.703 grams to 10.812 grams. The effective dose here is calculated according to the conversion formula between the effective dose of the cell experiment and the kilogram of the human body. The conversion formula is as follows: effective dose for human body = effective dose for cell experiment × mouse weight × conversion factor × human kilogram. The conversion coefficient is obtained by looking up the conversion coefficient table of the dose per kilogram of body weight for animals and humans. When the weight of the mouse is 20 grams and the kilograms of the human body is 60 kilograms, the conversion coefficient is 9.01. In other embodiments, the effective dose of sea grape extract is 5.406 grams to 10.812 grams.

In order to conveniently ingest the sea grape extract by mouth, the sea grape extract can be made into liquid, solid, granular, powder, pasty or gel processed sea grape extract. In some embodiments of the present invention, sea grape extract processed products may also contain other ingredients or additives, such as carriers, diluents, adjuvants, without affecting the efficacy and purpose of the present invention. , excipient or flavoring agent. Excipients can make preparations convenient and practical, while flavoring agents can enhance the flavor of preparations.

Examples of excipients are wheat starch, rice starch, corn starch, potato starch, dextrin, cyclodextrin and other starches; crystalline cellulose; lactose, glucose, granulated sugar, reduced maltose, maltose, fructooligosaccharides, emulsified oligosaccharides Sugars such as sorbitol, erythritol, xylitol, lactitol, mannitol and other sugar alcohols.

Taste agents are, for example, various fruit juice extracts such as longan extract, lychee extract, and grapefruit extract; various fruit juices such as apple juice, orange juice, and lemon juice; various spices such as peach flavors, plum flavors, and yoghurt flavors; Various sweeteners such as potassium phosphate, sucralose, erythritol, oligosaccharides, mannose, xylitol, and isomerized sugars; various sour agents such as citric acid, malic acid, tartaric acid, and gluconic acid; green tea, oolong tea, Various tea ingredients such as Banaba tea, Eucommia tea, Tieguanyin tea, coix tea, seven-leaf bile tea, wild rice stem tea, and kelp tea.

Furthermore, the composition of the sea grape extract according to the embodiment of the present invention can be a pharmaceutical composition or a non-pharmaceutical composition, and can also be a health food. The sea grape extract or the composition comprising the sea grape extract can also be wrapped in a capsule to facilitate oral intake of the sea grape extract. Sea grape extract or a composition containing sea grape extract can be coated in hard capsules in the form of dry powder, or in the form of solution, suspension, paste, powder or granules. in soft capsules.

Oils used to dissolve or disperse sea grape extract in soft capsules, such as calyx pear oil, almond oil, linseed oil, cumin oil, white sage oil, olive oil, olive squalene, sweet orange oil, breast Orange roughy oil, sesame oil, garlic oil, cocoa butter, pumpkin seed oil, chamomile oil, carrot oil, cucumber oil, tallow fatty acid, macadamia nut oil, lingonberry oil, brown rice germ oil, rice oil, wheat Germ oil, safflower oil, shea butter, liquid shea butter, perilla oil, soybean oil, evening primrose oil, camellia oil, corn oil, rapeseed oil, saw palmetto extract oil, coix seed oil , peach kernel oil, celery seed oil, castor oil, sunflower oil, grapeseed oil, borage oil, macadamia oil, meadowfoam oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg butter , fish oil, palm oil, palm kernel oil, wood wax, coconut oil, long-chain/medium-chain/short-chain fatty acid triglycerides, diglycerides, tallow, lard, squalene, squalane, Sparane, and the hydrogenated products of these fats and oils, etc. Among them, borage oil and evening primrose oil contain a large amount of Gamma-Linolenic Acid (GLA). Gamma-Linolenic Acid is an essential fatty acid in the human body. It has the functions of moisturizing, promoting cell regeneration and enhancing the activity of brown fat. Degree to promote the function of fat burning.

In addition, colorants, preservatives, tackifiers, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH value regulators, etc. are added in accordance with government regulations It can also be added to sea grape extract processed products in accordance with the dosage standards stipulated by government units and the needs of processing and production.

The experiment of using the sea grape extract of the embodiment of the present invention to improve mitochondrial activity is described below.

The cells used in the experiment are skeletal muscle cells (C2C12). Cultured in DMEM medium containing 10% fetal bovine serum (FBS). The method of cell subculture is described below. First, the cells are cultured to a certain amount, and then the culture medium is removed, and the cells are rinsed twice with phosphate buffered saline (PBS). Next, add trypsin (trypsin) and react with the cells at 37°C for 5 minutes, then add culture medium to stop the trypsin. Then, centrifuge at 300 g (relative centrifugal force, RCF) for 5 minutes, remove the supernatant, and redissolve with culture medium. Finally, the cells were transferred to a cell culture flask (175T flask), and the cell count was 1×10 ⁶ cells.

[Experiment 1] Cytotoxicity of sea grape extract

First, the cytotoxicity test of the sea grape extract was carried out. Alamar blue (Alamar blue) is a detection reagent used to detect cell viability. The resazurin in the detection kit is a redox indicator, which is a dark blue dye that is non-toxic, can penetrate the cell membrane and has low fluorescence. When resazurin enters healthy cells, it will be reduced to pink and highly fluorescent resorufin due to the reducing environment in living cells. The cell viability can be evaluated by measuring the light absorption value or fluorescence value of Lufin. The higher the light absorption value or fluorescence value of resorufin, the higher the cell viability. The higher the cell survival rate, the healthier the cells and the stronger their proliferative ability. The stronger the cell proliferation ability, the more cells. Therefore, alamar blue can be used as an indicator of cytotoxicity, so as to know the cell survival rate and cell proliferation rate.

The following is a detailed test procedure for the cytotoxicity of sea grape extract. On the first day, the cells were cultured for one day in a 96-well dish with a total volume of cells and medium of 200 microliters and 10,000 cells per well. The next day, sea grape extract was added so that the concentration of sea grape extract in the wells was 50, 100, 200, 250, 500, 1000 μg/ml, and the cells were cultured at 37°C for one day. On the third day, Alamar blue (Alamar blue) was used for cytotoxicity detection. In detail, alamar blue was prepared into a solution with a concentration of 10% by weight in a light-proof environment, and it was added to the well plate at a volume of 100 microliters per well, and it was mixed with the cells at 37°C. Cultivate for 3 to 4 hours, and finally measure the absorbance and fluorescence values (OD530/590) with a spectrophotometer (ELISA reader). Obtain the cell viability after treatment with the sea grape extract to represent the toxicity of the sea grape extract to the cells.

The experimental results are shown in Figure 1, which shows the cytotoxicity test results of different concentrations of sea grape extracts. The control group is the cells not treated with sea grape extract, and the vertical axis is the cell viability relative to the control group.

As shown in Figure 1, the sea grape extract with a concentration below 1000 μg/ml has no effect on cell viability, indicating that the sea grape extract with a concentration below 1000 μg/ml has no cytotoxicity. Accordingly, sea grape extracts at 250, 500, and 1000 μg/ml were selected as Examples 1 to 3 of the present invention for subsequent experiments.

[Experiment 2] Sea grape extract improves mitochondrial activity

Next, experiments were performed to increase mitochondrial activity using sea grape extract. In this experiment, tert-butyl hydroperoxide (tBHP) was used as a substance to induce cell oxidative stress damage, aging and inhibit mitochondrial activity.

The following is a detailed description of the experimental procedure for sea grape extract to improve mitochondrial activity. On the first day, incubate the cells in a 24-well hippocampal dish with a total volume of 100 microliters of cells and culture medium and 25,000 cells per well for 4 hours, then add 150 microliters of culture medium and incubate for one day . The next day, add sea grape extract so that the concentration of sea grape extract in the well is 250, 500, 1000 μg/ml and the total volume of the solution in the well is 250 μl, and the cells are incubated with sea grape extract under this condition one day. On the third day, replace the culture medium and add 100 μM tBHP to each well to react with the cells for 1 hour, then replace the culture medium in the well plate with 675 microliters of the machine culture medium (DMEM culture medium without fetal bovine serum) ), after culturing in an incubator without carbon dioxide for 1 hour, the oxygen consumption of the cells in the wells was measured with a hippocampal bioenergy meter.

The measurement principle and process of the Hippocampus Bioenergy Meter are as follows. First, the basal oxygen consumption of the cells in the wells is detected. Next, an inhibitor of netoside triphosphate synthase was added to inhibit mitochondrial production of netoside triphosphate, and the reduced oxygen consumption at this time was the oxygen consumption for synthesizing netoside triphosphate. Then, an appropriate concentration of anti-coupling agent was added to evaluate the maximum oxygen consumption of mitochondria without damaging the electron transport chain of the inner mitochondrial membrane, allowing the mitochondria to idle at the limit. Finally, an electron transport chain inhibitor was added to completely shut down mitochondrial oxygen consumption, thereby confirming the measured background value, ie non-mitochondrial oxygen consumption. Mitochondrial basal oxygen consumption equals cellular basal oxygen consumption minus non-mitochondrial oxygen consumption. The basal oxygen consumption of mitochondria minus the oxygen consumption for synthesizing hetanoside triphosphate is equal to the oxygen consumption for overcoming hydrogen ion leakage. The maximal mitochondrial oxygen consumption minus the mitochondrial basal oxygen consumption equals the mitochondrial prestored oxygen consumption. The ratio of maximum mitochondrial oxygen consumption is equal to the maximum mitochondrial oxygen consumption divided by the mitochondrial basal oxygen consumption.

The experimental results are shown in Table 1 and Figures 2 to 4, Figure 2 is the measurement result of the maximum oxygen consumption of mitochondria, Figure 3 is the measurement result of the pre-stored oxygen consumption of mitochondria, Figure 4 is the measurement result of mitochondria The body's maximum oxygen consumption ratio. Control group is cells not treated with tBHP and not treated with sea grape extract, control group is cells treated with tBHP but not treated with sea grape extract, example is cells treated with tBHP and treated with sea grape extract . The vertical axis of Fig. 2 and Fig. 3 is the oxygen consumption in picomoles per minute, and the vertical axis of Fig. 4 is the maximum oxygen consumption ratio represented by the oxygen consumption rate. *(P<0.05) means there is a significant difference relative to the control group, **(P<0.01) means there is a very significant difference compared to the control group, #(P<0.05) means there is a significant difference compared to the control group, ##(P<0.01 ) indicates a very significant difference compared to the control group.

Table 1 Sea Grape Extract (µg/mL) Basal oxygen consumption (pmol/min) Oxygen consumption to overcome hydrogen ion leakage (pmol/min) Oxygen consumption for the synthesis of lineoside triphosphate (pmol/min) Maximum oxygen consumption (pmol/min) Stored oxygen consumption (pmol/min) Non-mitochondrial oxygen consumption (pmol/min) Maximum oxygen consumption ratio (%) control group - 87.45 ±1.13 14.20±0.85 73.25 ±1.08 228.39 ±7.39 140.94±7.48 12.55±1.13 261.19 ±9.04 control group - 83.94±1.92 14.25 ±1.98 69.69 ±0.51 214.81 ±12.41 130.86 ±10.98 16.06 ±1.92 255.80 ±10.67 Embodiment one 250 81.15 ±2.22 12.25±1.03 68.89 ±1.59 224.69 ±11.91 143.54 ±11.68 18.85±2.22 276.98 ±15.08 Embodiment two 500 82.44 ±1.09 13.70±0.55 68.74±1.13 247.99 ±20.63 165.55 ±19.60 17.56±1.09 300.64 ±21.38 Embodiment three 1000 83.73 ±1.35 14.58±0.72 69.15 ±1.05 248.10 ±10.96 164.36 ±9.98 16.27±1.35 296.23 ±10.00

As shown in Figure 2, in the measurement results of the maximum oxygen consumption, the control group is lower than the control group, and the embodiment 1 is slightly equal to the control group, indicating that the treatment of 250 μg/ml sea grape extract can make the cells maintain the same level as the control group. Group-like maximum oxygen consumption capacity. The results of Examples 2 and 3 were higher than those of the comparison group and the control group, indicating that the maximum oxygen consumption capacity of the cells could be improved after treatment with 500 μg/ml or 1000 μg/ml of sea grape extract.

As shown in FIG. 3 , in the measurement result of the prestored oxygen consumption, a result similar to the maximum oxygen consumption was obtained. Cells treated with 250 μg/ml sea grape extract can maintain the pre-existing oxygen consumption capacity of the cells as in the control group, and treatment with 500 μg/ml or 1000 μg/ml sea grape extract can make the cells’ pre-existing oxygen consumption capacity Capabilities are enhanced.

As shown in Fig. 4, in the calculation result of the maximum oxygen consumption ratio, a result similar to the maximum oxygen consumption and the stored oxygen consumption is obtained. Among them, the maximum oxygen consumption ratio is defined as the maximum oxygen consumption of mitochondria divided by the basal oxygen consumption of mitochondria, which further takes into account the basal oxygen consumption of mitochondria to reduce the mitochondrial oxygen consumption in different cells. The difference in prestored oxygen consumption, which is more representative of the ability of cells to cope with stress. Cells treated with 250 μg/ml sea grape extract can maintain the maximum oxygen consumption ratio of the cells as in the control group, and treatment with 500 μg/ml or 1000 μg/ml sea grape extract can make the cells maximize oxygen consumption ratio has been increased.

According to the measurement results of the hippocampal bioenergy analyzer, the bioenergetic health index (Bioenergetic Healthy Index, BHI) can be calculated from the oxygen consumption of mitochondria. Bioenergy health index is an energy metabolism evaluation index calculated by using mitochondrial energy metabolism data as a parameter. BHI=[Oxygen consumption for the synthesis of hetanoside triphosphate×prestored oxygen consumption]/[Oxygen consumption for overcoming hydrogen ion leakage×Non-mitochondrial oxygen consumption]. A high bioenergy health index of a cell means that the activity of mitochondria in the cell is high, and it also means that the cell has a strong ability to cope with stress. Therefore, the bioenergy health index can be used as an index to evaluate the health of mitochondria and cells.

The biological health energy index calculated from the above experimental results is disclosed in Table 2. As shown in Table 2, compared with the control group, the treatment with the sea grape extract of the embodiment can improve the bioenergy health index, indicating that the health of mitochondria and cells is improved.

Table 2 Sea Grape Extract (µg/mL) BHI control group - 1.78±0.03326 control group - 1.60±0.04003 Embodiment one 250 1.63±0.06886 Embodiment two 500 1.67±0.07962 Embodiment Three 1000 1.68±0.05661

From the above experimental results, it can be seen that the sea grape extract with a concentration below 1000 μg/ml has no toxicity to cells. Moreover, the sea grape extract can improve the activity of mitochondria, which is specifically manifested in improving the maximum oxygen consumption capacity of mitochondria, improving the pre-stored oxygen consumption capacity of mitochondria, improving the bioenergy health index of mitochondria, and increasing the The ability of the thread body to cope with pressure.

The use of the extract provided according to the embodiment of the present invention for preparing a medicinal or non-pharmaceutical composition for improving mitochondrial activity, wherein the extract is sea grape extract, and sea grape extract can improve the maximum oxygen consumption capacity of mitochondria , Improve the pre-stored oxygen consumption capacity of mitochondria, improve the bioenergy health index of mitochondria, and improve the ability of mitochondria to cope with stress. Mitochondria whose activity is enhanced by sea grape extract can maintain their functions in the face of stress and activity to ensure that cells function properly without adverse effects from external or internal stress.

Although the present invention is disclosed by the aforementioned embodiments, they are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, all changes and modifications are within the scope of patent protection of the present invention. For the scope of protection defined by the present invention, please refer to the appended scope of patent application.

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Figure 1 shows the cytotoxicity test results of different concentrations of sea grape extracts.

Fig. 2 is the measurement result of the maximum oxygen consumption of mitochondria.

Fig. 3 is the measurement result of mitochondrial prestored oxygen consumption.

Figure 4 shows the maximum oxygen consumption ratio of mitochondria.

Claims (10)

A use of an extract for preparing a medicinal or non-medicinal composition for improving mitochondrial activity, wherein the extract is sea grape extract. The use according to claim 1, wherein increasing the mitochondrial activity comprises increasing the pre-stored oxygen consumption capacity of the mitochondria. The use as claimed in item 1, wherein increasing the mitochondrial activity comprises increasing the maximum oxygen consumption ratio of the mitochondria. The use according to claim 1, wherein increasing the activity of mitochondria comprises increasing the bioenergy health index of the mitochondria. The use according to claim 1, wherein increasing the activity of mitochondria comprises improving the ability of the mitochondria to cope with stress. The use as described in Claim 1, wherein the sea grape extract contains 4.84% to 6.52% polysaccharide by weight. The use as described in Claim 1, wherein the sea grape extract contains 16.87% to 18.65% by weight of carbohydrates, 5.99% to 6.63% of proteins and 1.9% to 2.1% of lipids. The use as described in Claim 1, wherein the sea grape extract is obtained by immersing sea grapes in water at 100°C for extraction, collecting the extracted supernatant, and freeze-drying the supernatant. The use according to claim 1, wherein the mitochondria are mitochondria of skeletal muscle cells. The use as described in Claim 1, wherein the concentration of the sea grape extract is 250 μg/ml to 1000 μg/ml.

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