TWI734435B - Use of Pennisetum extract for improving muscle mass and improving muscle wasting symptoms - Google Patents

Abstract

The present invention provides a use of Pennisetum extract, which is used to prepare a composition for improving muscle mass. The improving muscle mass includes improving muscle endurance, improving muscle strength and promoting muscle cell proliferation. The Pennisetum extract is Dried Pennisetum is extracted with alcohol. It can be separated by column chromatography through a macroporous resin column, and extracted with 50%-99% alcohol and ethyl acetate, and its fractions are collected; in addition, the Pennisetum Grass extracts can also be used to prepare pharmaceutical compositions for the treatment and improvement of muscle wasting symptoms. The treatment and improvement of muscle wasting symptoms include promoting muscle regeneration and repair, improving muscle weakness, improving muscle atrophy, repairing muscle cell damage, and improving pre-cachexia The symptoms.

Description

Use of Pennisetum extract for improving muscle mass and improving muscle wasting symptoms

The present invention relates to the use of Pennisetum extract for improving muscle mass and improving muscle wasting symptoms.

Muscle is an important component of the human body. When the rate of muscle protein degradation is greater than the rate of muscle protein synthesis, it will cause muscle loss. At present, factors known to cause muscle loss include: lack of exercise, aging, neurodegeneration, disease, and cachexia. ), etc., muscle loss can even lead to symptoms of muscle atrophy, including: decreased muscle fiber cross-sectional area, decreased muscle fiber type-related proteins, decreased neuromuscular junction (motor unit), and significantly decreased muscle weight, which ultimately lead to dyskinesias , Metabolic disorders, decreased vitality, etc., serious conditions will affect daily work and life functions, so how to prevent muscle loss and improve muscle mass is a topic that modern people pay attention to.

Muscle atrophy refers to rhabdomyo dystrophy, muscle volume reduction, thinning or disappearance of muscle fibers, etc. Aging, lack of exercise, and disease can cause muscle atrophy, which in turn leads to muscle weakness, and makes patients lose their ability to live. The muscle atrophy caused is different from pathological muscle loss, including: sepsis, organ failure, Cushing's syndrome (Cushing's syndrome, CS), AIDS, sarcopenia (sarcopenia), pre-cachexia, Cancer, heart failure, trauma, etc., which directly induce symptoms such as decreased muscle mass and decreased muscle strength, and then Causes various body functions to decrease and obstacles, bring a high risk of falls or fractures, and the risk of diabetes and cardiovascular myopathy will also increase significantly.

The treatment of muscle wasting symptoms is mainly to delay and stop muscle atrophy, and supplemented with regular visits, physical therapy, instrument assistance, use of assistive devices, etc. to maintain muscle state, while monitoring and tracking to reduce the occurrence of complications, there is currently no effective The drug or course of treatment can completely cure muscular dystrophy, so how to treat or improve the symptoms of muscular dystrophy is an important issue facing this field.

Pennisetum (Pennisetum) is a perennial gramineous plant that grows in tropical and warm-temperate regions around the world. It has a high yield and can be one of the main green medicinal materials for herbivores. The inventors of the present case related previous proposals TW 201136600 and TW 201336504 The anti-cancer Pennisetum extract is obtained through acidic solution, alkaline solution and solvent treatment; and the inventor of this case further discloses a fermented Pennisetum extract for the treatment or prevention of cancer through TW 201803580 Purpose: TW 201924710 discloses a method of Pennisetum extract and its use in the treatment of osteoporosis. However, there is no relevant previous case discussing the effect of Pennisetum extract on muscle cells, and there is no research on the effect of pennisetum extract on muscle wasting symptoms. reveal.

In view of the shortcomings of the prior art, the inventor of the present application hopes to provide a component that prevents muscle loss, improves muscle mass, and improves muscle wasting symptoms, and provides an important research direction for the future development of drugs to cure muscle wasting symptoms.

One object of the present invention is to provide a composition comprising a Pennisetum extract, which is obtained by extracting dried Pennisetum with alcohol, wherein the Pennisetum extract can be further processed with macroporous Resin column for column chromatography separation, with 50%-99% alcohol and ethyl acetate Extract, and collect its distinguished objects.

Among them, the alcohol concentration of the extraction with alcohol is 65-69%.

Among them, the macroporous resin column is a styrene resin column, preferably an HP-20 resin column.

Wherein, the composition is a pharmaceutical composition, a dietary supplement composition, a food composition or a health food composition.

Wherein, the aforementioned Pennisetum extract can be made into dosage forms including liquid nutritional supplements, oral liquids, beverages, powders, capsules, tablets, pills, granules, jellies, chewing gums, injections or other foods and pharmaceuticals. Acceptable carrier.

Another object of the present invention is to provide a use of Pennisetum extract, which is used to prepare a medicinal composition for improving muscle mass. The Pennisetum extract is obtained by extracting dried Pennisetum with alcohol, wherein The Pennisetum extract can be further separated by column chromatography with a macroporous resin column, extracted with 50%-99% alcohol and ethyl acetate, and the fractions are collected.

Another object of the present invention is to provide a health food containing Pennisetum extract for improving muscle mass. The Pennisetum extract is obtained by extracting dry Pennisetum with alcohol, wherein the Pennisetum The grass extract can be further separated by column chromatography with a macroporous resin column, extracted with 50%-99% alcohol and ethyl acetate, and the fractions are collected.

Among them, the improvement of muscle mass includes improvement of muscle endurance, improvement of muscle strength and promotion of muscle cell proliferation.

Another object of the present invention is to provide a use of Pennisetum extract, which is used to prepare a pharmaceutical composition for the treatment and improvement of muscle wasting symptoms. The Pennisetum extract is extracted from dried Pennisetum with alcohol. Among them, the Pennisetum extract can be further used in a macroporous resin tube The column is separated by column chromatography, extracted with 50%-99% alcohol and ethyl acetate, and the fractions are collected.

Among them, the muscle wasting symptoms are caused by at least one of the following: lack of exercise, aging, neurodegeneration, disease, and cachexia.

Among them, the treatment and improvement of muscle wasting symptoms include promoting muscle regeneration and repair, improving muscle weakness, improving muscle atrophy, repairing muscle cell damage, and improving symptoms of pre-cachexia.

Wherein, the applicable dose of the Pennisetum extract to human body is estimated to be 1-50 mg/kg, preferably 10-40 mg/kg.

The present invention studies the effects of Pennisetum extract on muscle cells, and then reveals the use of a natural plant extract for improving muscle mass and improving muscle wasting symptoms, and provides further development of related medications for the treatment of muscle wasting symptoms in the future. The direction of potential is also rich in industrial application value.

Fig. 1 is the preparation flow chart of Pennisetum extract; Fig. 2 is the experimental results of Pennisetum extract (PP2018) promoting the proliferation of C2C12 muscle blasts; Fig. 3 is the effect of each Pennisetum extract on C2C12 muscle blasts Figure 4 shows the experimental results of the effect of Pennisetum active fraction ZP-H123 on the muscle strength of lung cancer cell (LLC) tumor-bearing mice; Figure 5 shows the effect of Pennisetum active fraction ZP-H123 on lung cancer The experimental results of the weight of the gastrocnemius muscle of the tumor-bearing mice with cell (LLC); Figure 6 shows the experimental results of the Pennisetum grass active fraction ZP-H123 on the weight of paratestinal adipose tissue in mice bearing lung cancer cells (LLC); Figure 7 shows the average (A) (B) food intake of mice during the experiment And (C)(D) a statistical chart of dietary intake.

All technical and scientific terms described in this specification, unless otherwise defined, are meanings that can be understood by those with ordinary knowledge in the field; the materials used in the present invention, unless otherwise specified, are all marketable. Sell easily available materials.

The term "column chromatography" in this manual refers to the use of a macroporous resin column for chromatography, extraction with alcohol and/or ethyl acetate, and collection of sample fractions.

The present invention provides a composition comprising a Pennisetum extract. The Pennisetum extract is obtained by extracting dried Pennisetum with alcohol, wherein the Pennisetum extract can be further processed with a macroporous resin column Column chromatographic separation, extraction with 50%-99% alcohol and ethyl acetate, and collecting the distinguished substances; wherein the macroporous resin column is a styrene resin column, preferably an HP-20 resin column ; Wherein, the composition is a pharmaceutical composition, a dietary supplement composition, a food composition or a health food composition.

According to Figure 1, the preparation method of the Pennisetum extract in the present invention is as follows: S1 extracts dried Pennisetum grass with 20 times the volume of 65-69% alcohol to obtain the Pennisetum extract PP2018; S2 system uses Pennisetum extract PP2018 to pass through a macroporous resin (HP-20) column for column chromatography. The macroporous resin column is filled with a dry packing method; S3 is washed with 20% alcohol. Extract, and collect its distinguished objects; S4 is to extract with 50% alcohol, and collect its distinguished objects; S5 is extracted with 80% alcohol and collected its distinguished objects; S6 is extracted with 95% alcohol and its distinguished objects are collected; S7 is extracted with ethyl acetate and collected its distinguished objects.

In the embodiment of the present invention, dried Pennisetum is extracted with 66.5% alcohol, and the obtained Pennisetum extract sample is named PP2018.

In order to further explore the effects of the various components of the Pennisetum extract, the present invention performs column chromatography on the Pennisetum extract sample PP2018, extracts it with 18%-99% alcohol and ethyl acetate, and collects it. Separate objects for subsequent activity determination.

According to the above steps of different extraction conditions, different partitions of the Pennisetum extract sample PP2018 can be prepared. The partition sample extracted in step S3 is named H1, the partition sample extracted in step S4 is named H2, the partition sample extracted in step S5 combined with step S6 is named H4, and the partition sample extracted in step S7 is named H4. The sample is named H5. The combined eluted product from steps S3-S5 is named H123, the combined eluted product from steps S4-S6 is named H234, and the combined eluted product from steps S4-S7 is named H2345.

In the present invention, the above-mentioned Pennisetum extract samples were tested to explore the effect of Pennisetum extract on muscles.

The following embodiments are only to illustrate the technical ideas and features of the present invention, and their purpose is to enable those with ordinary knowledge in the field to understand the content of the present invention and implement them accordingly. When they cannot be used to limit the patent scope of the present invention, That is, all equal changes or modifications made in accordance with the spirit of the present invention should still be covered by the patent scope of the present invention.

Example 1. Pennisetum extract promotes the proliferation of C2C12 muscle blasts

Musculoskeletal formation is a series of complex processes, composed of loose interstitial cells (mesenchymal cell), forming myoblast. Muscle mother cells will proliferate, elongate, and fuse to form myotubes, which in turn form muscle fibers.

Since the proliferation of muscle blasts is a key factor for muscle production, this example uses MTS assay to test the effect of Pennisetum extract on C2C12 muscle blasts. The C2C12 muscle blasts were cultured at 10%. The cultivation process of FBS/DMEM is as follows:

(1) Inoculate the cells in a T75 flask and incubate in a cell incubator at 37°C and 5% CO ₂ .

(2) When the cells grow to 8 to 90% full (to avoid letting the cells grow to fullness and cause differentiation), wash the cells twice with PBS, then remove the PBS, add trypsin-EDTA and act at 37°C for a few minutes. Observe the cell peeling from the bottle wall under a microscope, add 5% FBS/DMEM and mix well, terminate the trypsin enzyme reaction, collect the cell suspension in a centrifuge tube, remove the supernatant after centrifugation, and re-dissolve the cells in the culture containing 5% FBS In the solution, calculate the cell concentration with the hemocytometer.

^{(3) Seed 6x10 3} /well cells in a 96-well cell culture dish with a volume of 180 μL/well, and incubate in a cell culture incubator for 4 hours to allow them to be mounted on the plate for later use.

The experimental design of this embodiment is as follows:

(1) Add the Pennisetum extract sample to the cells (20 μL/well), the final volume reaches 200 μL/well, each group is 3 replicates, and the cells are treated in a cell incubator for 24 hours.

(2) Remove the cell culture medium containing the test sample, dilute the MTS reagent in 5% FBS culture medium at a volume ratio of 1:10, add cells (100 μL/well), and place in a cell incubator to react for 1 hour.

(3) Use a spectrophotometer to read the absorbance value of 490nm and calculate the cell proliferation rate of the cells relative to the control group.

(4) One-way ANOVA was used to perform multiple comparison analysis (Dunnett’s test) to calculate differences between groups (* p<0.05; ** p<0.005).

1. Pennisetum extract promotes the proliferation of C2C12 muscle blasts

The preparation method of the Pennisetum extract sample PP2018 is as follows: Dissolve the PP2018 sample with 100% DMSO to make the concentration 50mg/mL. The test samples were diluted with 5% FBS/DMEM to 10 times the final concentration, respectively 2000, 1500, 1000, 500, 250, 125, 62.5 and 31.25μg/mL.

The above-mentioned experimental steps were performed on samples of various concentrations. The results are shown in Figure 2. The 6.25, 12.5, 25, 50 and 100μg/mL Pennisetum extract PP2018 can significantly promote the proliferation of C2C12 cells, and the effect of promoting cell proliferation gradually increases to 50μg /mL is the maximum value.

1. Pennisetum active fraction promotes the proliferation of C2C12 muscle blasts

The preparation method of Pennisetum active fraction samples is as follows: H4, H234, and H2345 samples are dissolved in 100% DMSO at a concentration of 50 mg/mL; H1 samples are re-dissolved with 100% secondary water at a concentration of 50 mg/mL. Dilute the test sample with a culture medium containing 5% FBS to 10 times the final concentration, which is 500, 100, and 20 μg/mL, respectively.

The Pennisetum active fraction samples of various concentrations were subjected to the above experimental steps, and the results are shown in Figure 3. H4 (10μg/mL), H234 (2μg/mL) and H2345 (2μg/mL) Pennisetum active fractions Can significantly promote the proliferation of C2C12 cells (Figure 3B, 3C and 3D). On the other hand, no significant cell proliferation was observed in the active classifier H1 (Figure 3A).

In summary, the results of this example show that the Pennisetum extract PP2018 and the active fractions H4, H234 and H2345 have the ability to promote the proliferation of mouse muscle blasts C2C12.

Example 2. Effect of Pennisetum extract on lung cancer cell LLC tumor-bearing mice

In this example, mouse lung cancer cells Lewis lung carcinoma (LLC) were implanted into immune-normal mice to obtain symptoms similar to cancer cachexia, such as decreased muscle strength. Construct an animal model of muscle atrophy mice, and apply Pennisetum extract samples for evaluation to explore the improvement of related symptoms.

The experimental design of this embodiment is as follows:

(1) When the tumor size of the mouse reaches 100~200mm ³ , each Pennisetum species is used as the test substance, and the tube feeding doses are 400mg/kg and 200mg/kg. Feed continuously for 12 days, during which the tensile test is performed and recorded Indicators such as body weight, food intake, drinking water and tumor size.

(2) After the sacrifice, the accessory testicular fat and gastrocnemius muscle were removed and weighed to evaluate its efficacy.

2. 1. The effect of Pennisetum extract on muscle strength of lung cancer cell (LLC) tumor-bearing mice (tension test)

Tensile tests were carried out on the 0th, 6th and 12th days of the test period. The mice were naturally laid flat on the mesh frame connected to the tensile tester, and the tail was gently pulled and continued to force the mice backwards until the forelimbs were free from the net. Until the shape frame is loosened, the peak force of the resistance output from the instrument is the resistance force generated by the mouse during the period, which is used to evaluate the muscle strength of the mouse.

The results of the experiment are shown in Figure 4. On the 6th and 12th day, the muscle strength of the tumor control group (Tumor control) was significantly lower than that of the control group (Control), confirming that the lung cancer cell (LLC) tumor-bearing mice did Symptoms of muscle atrophy with decreased muscle strength.

On day 12, the muscle strength values of the tumor control group (Tumor control) mice were 0.57±0.09N, and the muscle strength values of the 400mg/kg ZP-H123 and 200mg/kg ZP-H123 groups were 1.02±0.11, respectively N and 0.78±0.12N. Compared with the Tumor control group, the ZP-H123 group administered with the Pennisetum grass classifier showed a significant increase in muscle strength, and a significant difference was reached ( p <0.01). It can be seen that the administration of 400 mg/kg ZP-H123 and 200 mg/kg ZP-H123 has a significant effect on improving the symptoms of decreased muscle strength.

2. The effect of Pennisetum extract on the weight of gastrocnemius in lung cancer cell (LLC) tumor-bearing mice

The gastrocnemius muscle weight is an important cachexia evaluation index for evaluating the degree of muscle atrophy symptoms in mice. In this example, the animal was sacrificed at the end of the experiment, and the gastrocnemius muscle was surgically removed and weighed to observe changes in the muscle mass of the mice.

The results of the experiment are shown in Figure 5. The gastrocnemius weight of the tumor control group (Tumor control) was significantly lower than that of the control group (Control).

The gastrocnemius weight of the Tumor control group was 4.29±0.39mg/g, while the gastrocnemius weight of the 400mg/kg ZP-H123 and 200mg/kg ZP-H123 groups were 6.24±0.39mg/g and 6.58±0.32mg, respectively /g, compared with the Tumor control group (Tumor control), the gastrocnemius content of the pennisetum divider ZP-H123 group was significantly increased ( p <0.01), which was consistent with the results of the tensile test. It is obvious that the Pennisetum extract The substance has the effect of improving the symptoms of muscle atrophy and increasing muscle mass.

2. The effect of Pennisetum extract on the weight of paratestinal adipose tissue in lung cancer cell (LLC) tumor-bearing mice

Today's health care products must not only have the effect of enhancing muscle strength and physiological functions, but also must emphasize that they will not cause the formation and accumulation of fat. Therefore, this example further evaluates the effect of the active divider of Pennisetum on the adipose tissue of mice. Experiments The results are shown in Figure 6. The sample ZP-H123 has no significant increase in adipose tissue content, indicating that it will not cause the formation and stacking of fat, and has the potential to become a health care product.

2. 4. The effect of Pennisetum extract on food intake and drinking water in mice

In this example, the average food intake and water consumption of the mice were counted during the test period to explore whether the application of Pennisetum extract affects the eating behavior of the mice, and it was regarded as a safety indicator.

As shown in Figure 7, the groups administered with the Pennisetum species ZP-H2345, ZP-H345 and ZP-H123 did not change the food intake (Figures 7A and 7B), nor did it cause the water intake of the mice. The changes (Figures 7C and 7D) show that the administration of Pennisetum extract does not affect the eating habits of mice, and it is safe in use.

In summary, according to the results of this example, the Pennisetum extract not only has the effect of increasing muscle strength, but also can improve the symptoms of muscle atrophy and increase muscle mass without causing fat formation and stacking.

The method of calculating the dosage of Pennisetum extract in this case: According to the actual recommended supplementation design of the human body, due to the different metabolic rates of humans and experimental animals, the dosage used in humans needs to be appropriately converted, according to the United States Food and Drugs The "Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" standard proposed by the Administration in 2005, the conversion factor between humans and mice is approximately 12.3 times. According to the experimental data, the human body is suitable The dosage of Pennisetum extract is about 16.26-32.52mg/kg after coefficient conversion, so the applicable dosage of Pennisetum extract for human body is estimated to be 1-50mg/kg, preferably 10-40mg/kg.

The present invention provides a use of Pennisetum extract, which is used to prepare a composition for improving muscle mass. The Pennisetum extract is obtained by extracting dry Pennisetum with alcohol, and the improving muscle mass includes improving muscle mass. Muscle endurance, enhancement of muscle strength and promotion of muscle cell proliferation, the composition is a pharmaceutical composition, a dietary supplement composition, a food composition or a health food composition Things.

The present invention also provides a use of Pennisetum extract, which is used to prepare a pharmaceutical composition for treating and improving muscle wasting symptoms, wherein the Pennisetum extract is obtained by extracting dried Pennisetum with alcohol, wherein The Pennisetum extract can be further separated by column chromatography with a macroporous resin column, extracted with 18%-99% alcohol and ethyl acetate, and the fractions are collected. The muscle wasting symptoms are caused by at least one of the following: lack of exercise, aging, neurodegeneration, disease, and cachexia. The treatment and improvement of muscle wasting symptoms include promoting muscle regeneration and repair, improving muscle weakness, and improving muscles. Atrophy, repair muscle cell damage and improve the symptoms of pre-cachexia.

The pennisetum extract as described above can be made into dosage forms including liquid nutritional supplements, oral liquids, beverages, powders, capsules, tablets, pills, granules, jellies, chewing gums, injections or other foods and pharmaceuticals. Acceptable carrier.

The Pennisetum extract disclosed in the present invention can increase muscle tension and endurance, and has the effect of promoting muscle cell proliferation, helping to increase muscle mass and improve muscle atrophy symptoms, and is safe in clinical use and has no adverse side effects. It has urgent development value in the medical field; in addition, the Pennisetum extract of the present invention does not cause the formation and stacking of fat, and is suitable for food and supplements for athletes, as well as health food and weight loss for the general public. Products, etc., have urgent industrial use value.

The above-mentioned multiple functions have fully met the requirements of novel and progressive statutory invention patents. You file an application in accordance with the law, and I implore your office to approve this invention patent application to encourage invention and make it easy.

Claims (10)

A use of Pennisetum extract, which is used to prepare a medicinal composition for improving muscle mass. The Pennisetum extract is obtained by extracting dry Pennisetum with alcohol. Among them, the Pennisetum extract can be The column chromatography is further performed with a macroporous resin column, and extracted with 50%-99% alcohol and ethyl acetate, and the fractions are collected. A use of Pennisetum extract, which is used to prepare health foods for improving muscle mass. The Pennisetum extract is obtained by extracting dried Pennisetum with alcohol. Among them, the Pennisetum extract can be further Column chromatographic separation is carried out with a macroporous resin column, extracted with 50%-99% alcohol and ethyl acetate, and the fractions are collected. The use according to claim 1 or 2, wherein the improvement of muscle mass includes improvement of muscle endurance, improvement of muscle strength, and promotion of muscle cell proliferation. For the use described in claim 1 or 2, the applicable dosage of the Pennisetum extract for humans is 1-50 mg/kg. For the use described in claim 1 or 2, the applicable dose of the Pennisetum extract for humans is 10-40 mg/kg. A use of Pennisetum extract, which is used to prepare a medicinal composition for the treatment and improvement of muscle wasting symptoms, wherein the Pennisetum extract is obtained by extracting dried Pennisetum with alcohol, wherein the Pennisetum The grass extract can be further separated by column chromatography with a macroporous resin column, extracted with 18%-99% alcohol and ethyl acetate, and the fractions are collected. The use according to claim 6, wherein the muscle wasting symptoms are caused by at least one of the following: lack of exercise, aging, neurodegeneration, disease, and cachexia. The use according to claim 6, wherein the treatment and improvement of muscle wasting symptoms Including the promotion of muscle regeneration and repair, improvement of muscle weakness, improvement of muscle atrophy, repair of muscle cell damage and improvement of the symptoms of pre-cachexia. For the use described in claim 6, the applicable dose of the Pennisetum extract for humans is 1-50 mg/kg. For the use described in claim 6, the applicable dosage of the Pennisetum extract for human body is 10-40mg/kg.

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