WO2017121333A1 - 管花肉苁蓉萃取物及异类叶升麻苷于保护肌肉的用途 - Google Patents

管花肉苁蓉萃取物及异类叶升麻苷于保护肌肉的用途 Download PDF

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WO2017121333A1
WO2017121333A1 PCT/CN2017/070862 CN2017070862W WO2017121333A1 WO 2017121333 A1 WO2017121333 A1 WO 2017121333A1 CN 2017070862 W CN2017070862 W CN 2017070862W WO 2017121333 A1 WO2017121333 A1 WO 2017121333A1
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food
muscle
extract
cistanche tubulosa
use according
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PCT/CN2017/070862
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English (en)
French (fr)
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王昭日
叶艾灵
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杏辉天力（杭州）药业有限公司
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Priority to MYPI2018702407A priority Critical patent/MY194024A/en
Priority to JP2018533780A priority patent/JP6887433B2/ja
Priority to AU2017206332A priority patent/AU2017206332B2/en
Priority to EP17738147.2A priority patent/EP3403664B1/en
Priority to KR1020187022614A priority patent/KR102195547B1/ko
Priority to CA3010907A priority patent/CA3010907C/en
Publication of WO2017121333A1 publication Critical patent/WO2017121333A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/64—Orobanchaceae (Broom-rape family)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/316—Foods, ingredients or supplements having a functional effect on health having an effect on regeneration or building of ligaments or muscles

Definitions

  • the present invention relates to the use of Cistanche tubulosa extract and isoacteoside (i.e., a component of Cistanche tubulosa extract) or a pharmaceutically acceptable salt of heterologous lutein, especially It relates to the use of medicinal salts of Cistanche tubulosa extract, isoforms, or isoforms to protect muscles, including anti-myocyte damage to regulate, treat and/or delay muscle loss, especially It is due to muscle loss caused by aging, disease and/or cachexia.
  • Cistanche tubulosa extract and isoacteoside i.e., a component of Cistanche tubulosa extract
  • a pharmaceutically acceptable salt of heterologous lutein especially It relates to the use of medicinal salts of Cistanche tubulosa extract, isoforms, or isoforms to protect muscles, including anti-myocyte damage to regulate, treat and/or delay muscle loss, especially It is due to muscle loss caused by aging, disease and/or cache
  • Muscle tissue is the highest quality tissue in a mammal, and its main function is to generate force to pull the movements of various parts of the body. Muscle can be divided into three types: skeletal muscle, myocardium and smooth muscle. Skeletal muscle can be further divided into slow muscle and fast muscle according to its own metabolic type and characteristics. The former is composed of slow muscle fibrin, its contraction lasts for a long time, but the strength is small; the latter is composed of fast muscle fibrin Composition, shrinking faster, greater strength, but also easier to feel tired.
  • muscle loss is caused. Severe muscle loss leads to muscle atrophy, and muscle weight is significantly reduced, muscle fiber cross-sectional area is reduced, and muscle fiber is reduced. Characteristic changes such as selective reduction of type-related proteins (ie, slow muscle fibrin and fast muscle fibrin) cause symptoms such as decreased muscle strength, dyskinesia, fatigue, and metabolic disorders, which seriously affect daily work and life functions.
  • type-related proteins ie, slow muscle fibrin and fast muscle fibrin
  • muscle loss It is known that some physiological conditions or special diseases can cause muscle cell damage, leading to imbalance of muscle protein metabolism or apoptosis of muscle cells, resulting in muscle loss.
  • factors that cause muscle loss include, for example, neurodegeneration, prolonged bed rest, aging, disease, and cachexia (such as cancer cachexia), among which diseases include, for example, septicaemia, AIDS, Kidney failure, Cushing's syndrome (CS), sarcopenia, cancer, chronic obstructive pulmonary disease (COPD), congestive heart failure (CHF), trauma, etc. .
  • Cistanche tubulosa is a perennial parasitic herb of the genus Cistanche of Orobanchaceae. It is distributed in dry areas such as deserts and deserts. It is a rare and valuable medicinal material that relies on the nutrients of the host plant. . Cistanche tubulosa has been used in the Chinese Pharmacopoeia in 2005 to improve renal function, enhance memory, regulate immune function, resist senile dementia, anti-aging, and anti-fatigue.
  • Cistanche tubulosa has a protective effect on muscle atrophy. Therefore, in order to develop a more effective method for treating or delaying muscle loss, the inventors of the present invention have traditional Chinese herbal medicines. Chosen Cistanche is selected for its feasibility in protecting muscles (anti-muscle cell damage, avoiding muscle loss). The present inventors have found that the extract of Cistanche tubulosa and its isoformeoside are effective against myocyte damage and can be used to regulate, treat and/or delay muscle loss, especially because of aging. Caused by disease, and/or cachexia Muscle loss, which can be used to provide a pharmaceutical composition, drug or food that protects muscles.
  • Cistanche tubulosa extract is a polar solvent extract of Cistanche tubulosa, the polar solvent being selected from the group consisting of water, C1-C4 alcohols, and combinations thereof. More preferably, the Cistanche tubulosa extract contains heterophylloside.
  • the drug is for use in anti-myocyte injury, or for treating and/or delaying muscle loss caused by at least one of: aging, disease, and cachexia;
  • the food is used to regulate at least one of the following: Muscle loss: aging, disease and cachexia, help normal muscle contraction, maintain normal muscle physiology, maintain neuromuscular function, maintain normal energy metabolism or enhance energy, and the food is health food, nutritional supplement food Or special nutritious food.
  • Another object of the present invention is to provide a use of an active ingredient for the preparation of a medicament or food for protecting muscles, wherein the active ingredient is a heterophylloside and/or a pharmaceutically acceptable salt thereof.
  • the active ingredient is used in the form of a plant extract; more preferably, in the form of an extract of Cistanche tubulosa, especially in the form of a polar solvent extract of Cistanche tubulosa, the polar solvent It is any one selected from the group consisting of water, a C1-C4 alcohol, and a combination of the foregoing.
  • the drug is for use in anti-myocyte injury, or for treating and/or delaying muscle loss caused by at least one of: aging, disease, and cachexia;
  • the food is used to regulate at least one of the following: Muscle loss: aging, disease and cachexia, help normal muscle contraction, maintain normal muscle physiology, maintain normal function of nerves and muscles, maintain normal energy metabolism or enhance energy, and the food is health food, nutritional supplement or special nutritious food .
  • the Cistanche tubulosa extract is a polar solvent extract of Cistanche tubulosa
  • the polar solvent is selected Any one of water, C1-C4 alcohols, and combinations of the foregoing.
  • the Cistanche tubulosa extract contains heterophylloside.
  • the method is for anti-myocyte injury, for regulating, treating and/or delaying muscle loss caused by at least one of: aging, disease and cachexia, or for helping normal muscle contraction and maintaining normal muscle physiology Maintain normal neuromuscular function, maintain normal energy metabolism or enhance energy.
  • a further object of the present invention is to provide a method of protecting muscles comprising administering an effective amount of an active ingredient to an individual in need thereof, wherein the active ingredient is a heterologous leaf amygdalin and/or its medicinal Accepted salt.
  • the active ingredient is used in the form of a plant extract; more preferably, in the form of an extract of Cistanche tubulosa, especially in the form of a polar solvent extract of Cistanche tubulosa, the polar solvent It is any one selected from the group consisting of water, a C1-C4 alcohol, and a combination of the foregoing.
  • the method is for anti-myocyte injury, for regulating, treating and/or delaying muscle loss caused by at least one of: aging, disease and cachexia, or for helping normal muscle contraction and maintaining normal muscle physiology Maintain normal neuromuscular function, maintain normal energy metabolism or enhance energy.
  • Still another object of the present invention is to provide a muscle-protecting composition which is a pharmaceutical or food product and which comprises an effective amount of Cistanche tubulosa extract.
  • the Cistanche tubulosa extract is a polar solvent extract of Cistanche tubulosa, the polar solvent being selected from the group consisting of water, C1-C4 alcohols, and combinations thereof. More preferably, the Cistanche tubulosa extract contains heterophylloside.
  • the composition is for use in anti-myocyte injury, for regulating, treating and/or delaying muscle loss caused by at least one of: aging, disease and cachexia, or for helping muscles to contract normally and maintaining muscle normal Physiological, maintain normal function of the nerves, maintain normal energy metabolism or enhance energy.
  • Still another object of the present invention is to provide a composition for protecting muscles, wherein the composition is a pharmaceutical or food product and comprises an effective amount of active active ingredient, and
  • the active ingredient is isoformin and/or a pharmaceutically acceptable salt thereof.
  • the active ingredient is used in the form of a plant extract; more preferably, in the form of an extract of Cistanche tubulosa, especially in the form of a polar solvent extract of Cistanche tubulosa, the polar solvent It is any one selected from the group consisting of water, a C1-C4 alcohol, and a combination of the foregoing.
  • the composition is for use in anti-myocyte injury, for regulating, treating and/or delaying muscle loss caused by at least one of: aging, disease and cachexia, or for helping muscles to contract normally and maintaining muscle normal Physiological, maintain normal function of the nerves, maintain normal energy metabolism or enhance energy.
  • FIG. 1A and Figure 1B show the effect of TNF- ⁇ on the mitochondrial membrane potential and intracellular reactive oxygen species levels of C2C12 cells
  • 3A, 3B, and 3C show the effects of Cistanche tubulosa extract on cell viability, mitochondrial membrane potential, and intracellular reactive oxygen species levels of C2C12 cells induced by TNF- ⁇ ;
  • Figure 4A and Figure 4B show the effects of Cistanche tubulosa extract and branched-chain amino acids (positive control group) on intracellular reactive oxygen species levels of C2C12 cells induced by TNF- ⁇ ;
  • 5A, 5B-1 and 5B-2 show the effects of Cistanche tubulosa extract on the glycolysis ability of C2C12 cells induced by TNF- ⁇ ;
  • Figure 6A, Figure 6B-1, Figure 6B-2 and Figure 6B-3 show the effect of Cistanche tubulosa extract on the mitochondrial respiration capacity of C2C12 cells induced by TNF- ⁇ ;
  • Figure 7A-1, Figure 7A-2 and Figure 7A-3 show the effect of echinacoside on cell survival rate, mitochondrial membrane potential and intracellular reactive oxygen species in C2C12 cells induced by TNF- ⁇ . ;
  • Figure 7B-1, Figure 7B-2 and Figure 7B-3 show the effect of flavonoids on cell viability, mitochondrial membrane potential and intracellular reactive oxygen species in C2C12 cells induced by TNF- ⁇ . ;
  • Figure 7C-1, Figure 7C-2, and Figure 7C-3 show the effects of heterologous aglycone on cell viability, mitochondrial membrane potential, and intracellular reactive oxygen species in C2C12 cells induced by TNF- ⁇ . ;as well as
  • Figure 8A, Figure 8B, Figure 8C and Figure 8D show the mTOR/AMPK signaling pathway and the expression of related proteins in the NFkB/p-JNK signaling pathway in C2C12 cells induced by TNF- ⁇ induced by Cistanche tubulosa extract. The impact of quantity.
  • the terms "a”, “the”, and ⁇ RTIgt; ⁇ / RTI> ⁇ RTIgt; ⁇ / RTI> ⁇ RTIgt; ⁇ / RTI> ⁇ RTIgt; refers to the amount of a compound that is effective at least partially to improve the condition of a suspected individual when administered to an individual; the so-called “individual” refers to a human or a non-human mammal; the so-called “treatment” includes preventing a specific disease or symptom, and ameliorating a specific disease. Or symptoms and / or prevent or eliminate the condition; the unit “mg / kg body weight” refers to the amount of administration required per kg of body weight.
  • the term "pharmaceutically acceptable salt” refers to a pharmacological activity which, when administered to a living organism, produces the same or similar pharmacological activity as the parent compound, and is physiologically tolerable (ie, has as little toxicity as possible). The role of the salt.
  • Cistanche tubulosa extract can effectively protect against muscle damage, so it can be used to protect muscles and has the effect of treating and/or delaying muscle loss. Without being bound by theory, it is believed that the Cistanche tubulosa extract used in the present invention is effective in regulating, treating, and/or delaying muscle loss due to aging, disease, and/or cachexia.
  • the present invention provides an application for protecting muscles using Cistanche tubulosa extract, comprising using a Cistanche tubulosa extract to prepare a muscle-protecting drug or food, and administering a Cistanche tubulosa extract to an individual in need thereof to protect A method of muscle and a food or pharmaceutical composition comprising an extract of Cistanche tubulosa.
  • the Cistanche tubulosa extract provided by the method comprising the steps of: (a) extracting Cistanche tubulosa using a polar solvent to obtain an extract; and (b) drying the extract as needed.
  • the polar solvent may be water and/or a C1-C4 alcohol.
  • water, ethanol or a combination thereof is used as the polar solvent.
  • the ratio of the polar solvent used for extraction to the Cistanche tubulosa can be adjusted as needed.
  • the volume ratio of the polar solvent to Cistanche tubulosa can be from about 1:1 to about 50:1, preferably about 5 : 1 to 20:1.
  • any part of Cistanche tubulosa can be used to prepare Cistanche tubulosa extract.
  • stems, flowers or whole plants of Cistanche tubulosa can be used as an extraction raw material.
  • the fleshy stem portion of Cistanche tubulosa is used as an extraction material.
  • the extraction is carried out for a period of time to achieve the desired degree of extraction.
  • the polar solvent for example, it is usually at least 15 minutes, preferably at least 30 minutes, more preferably at least 60 minutes, and may be supplemented, for example, by boiling, cooling, or the like.
  • Other operations such as filtration, concentration under reduced pressure, and resin column chromatography.
  • a plurality of extraction steps (a) may be repeated with the same or different polar solvents before the step (b), and the extract obtained by the multiple extractions may be combined to perform the step (b); or
  • the cycles of extraction step (a), extraction step (b), and other operations as desired are repeated to maximize the extraction benefit.
  • a method for preparing Cistanche tubulosa extract provided in an embodiment of the present invention.
  • the present inventors further studied and found that among the components contained in the extract of Cistanche tubulosa, the isoform can be effectively inhibited from muscle cell damage, so it can be used to protect muscles and has the effect of treating and/or delaying muscle loss. . Without being bound by theory, it is believed that heterophylloside can effectively modulate, treat, and/or delay muscle loss due to aging, disease, and/or cachexia.
  • the present invention also provides an application for protecting muscles using a heterophylloside and/or a pharmaceutically acceptable salt thereof, comprising the use of heterophylloside and/or a pharmaceutically acceptable salt thereof for the preparation of a a muscle-protecting drug or food, a method of administering a heterophylloin and/or a pharmaceutically acceptable salt thereof to an individual in need thereof to protect muscles, and a method comprising providing a heterophylloside and/or a medicinal thereof A food or pharmaceutical composition of the accepted salt.
  • the heterophylloside and/or a pharmaceutically acceptable salt thereof is used in the form of a plant extract; more preferably, in the form of an extract of Cistanche tubulosa, especially Cistanche tubulosa Used in the form of a polar solvent extract.
  • the pharmaceutical composition or medicament provided in the application according to the present invention may be in any convenient form, without particular limitation, and may be in a correspondingly suitable dosage form depending on the intended use.
  • the pharmaceutical composition or medicament may be administered orally or non-orally (eg, subcutaneous, intravenous, intramuscular, intraperitoneal or nasal) to a subject in need thereof.
  • a suitable carrier may be selected to provide the pharmaceutical composition or medicament, wherein the carrier comprises an excipient, a diluent, an adjuvant, a stabilizer, an absorption delaying agent, a disintegrating agent. , solubilizer, emulsifier, antioxidant Agents, binders, binders, tackifiers, dispersants, suspending agents, lubricants, moisture absorbers, and the like.
  • the pharmaceutical composition or medicament provided in the application according to the present invention may contain any desired ingredients which do not adversely affect the active ingredient (i.e., Cistanche tubulosa extract or heterophylloside)
  • a pharmaceutically acceptable carrier for benefit such as water, saline, dextrose, glycerol, ethanol or the like, cellulose, starch, sugar bentonite, and combinations of the foregoing.
  • the pharmaceutical composition or medicament may be provided in a dosage form suitable for oral administration by any convenient method, for example, a tablet (for example, a dragee), a pill, a capsule, a granule, a powder, a flow extract, a solution, a syrup Agents, suspensions, tinctures, and the like.
  • a tablet for example, a dragee
  • a pill for example, a capsule
  • a granule a powder
  • a flow extract a solution
  • a syrup Agents suspensions, tinctures, and the like.
  • one or more, for example, isotonic solutions, salt buffers may be included in the pharmaceutical compositions or medicaments provided in the use according to the invention (such as phosphate buffer or citrate buffer), solubilizer, emulsifier, 5% sugar solution and other carriers, such as intravenous infusion, emulsion intravenous infusion, dry powder injection, suspension injection or dry powder
  • a pharmaceutical composition or a drug is provided in a dosage form such as a suspension injection.
  • the pharmaceutical composition or medicament can be prepared as a pre-injection solid, the pre-injection solid is provided in a dosage form or emulsifiable dosage form which is soluble in the other solution or suspension, and prior to administration to an individual in need thereof, The pre-injection solid is dissolved in another solution or suspension or emulsified to provide the desired injection.
  • a pharmaceutical composition or drug provided according to the application of the present invention may additionally contain a suitable amount of an additive, for example, a flavoring agent which enhances the mouthfeel and visual sensation of the pharmaceutical composition or drug when administered, A toner, a coloring agent, or the like, and a buffering agent, a preservative, a preservative, an antibacterial agent, an antifungal agent, and the like which can improve the stability and storage property of the pharmaceutical composition or drug.
  • the pharmaceutical composition or medicament may additionally contain one or more other active ingredients (eg, vitamin D, vitamin B1, vitamins).
  • the flexibility of application and the degree of formulation of the formulation may be increased as long as the other active ingredient does not adversely affect the effectiveness of the active ingredient of the present invention (i.e., Cistanche tubulosa extract or heterophylloside).
  • the pharmaceutical composition or medicament provided in the application according to the present invention may be administered at different administration times, such as once a day, several times a day or once a day, depending on the needs, age, weight and health conditions of the individual to be administered.
  • the amount of Cistanche tubulosa extract is from about 0.5 mg/kg body weight to about 1000 mg/kg body weight per day, preferably about 2.5 mg per day.
  • / kg body weight to about 1000 mg / kg body weight more preferably about 5 mg / kg body weight to about 500 mg / kg body weight per day.
  • the unit "mg / kg body weight” refers to the amount of administration required per kg of body weight.
  • the food provided in the application according to the present invention may be a health food, a nutritional supplement or a special nutritious food, and may be made into, for example, dairy products, processed meats, breads, noodles, biscuits, buns, capsules, Juices, teas, sports drinks, nutritional drinks and other products, but not limited to this.
  • the food product according to the application of the present invention is provided in the form of a health food.
  • the health food, nutritional supplement food and special nutritious food provided by the application according to the present invention can be eaten at different frequencies once a day, once a day or several times a day, depending on the age, weight and health status of the individual.
  • the recommended intake varies. It is also possible to adjust the content of Cistanche tubulosa extract or heterologous leaf asparagine in the health food, nutritional supplement food and special nutritious food provided by the present invention for a specific ethnic group, preferably The amount that should be taken daily.
  • the recommended dosage, the use standard and conditions of a specific ethnic group (such as pregnant women, cancer patients, heart failure patients) or the use of other foods or medicines may be indicated in the outer packaging of the health food, nutritional supplement food and/or special nutritious food of the present invention.
  • the recommendations are for the user to take it at home without the guidance of a physician, pharmacist or related deacon without any security concerns.
  • the invention also provides a method of protecting muscles in a body comprising administering to the individual an effective amount of an active ingredient, wherein the active ingredient is Cistanche tubulosa extract, heterologous leaf asparagine and/or heterologous leaves A pharmaceutically acceptable salt of ricin.
  • the administration route, the administration form, the applicable dose, and the application of the relevant treatment regarding the active ingredient are as described above.
  • the crude extract is dissolved by heating with 1 volume of water of the crude extract, and the solution is injected into the macroporous adsorption resin column, and sequentially washed with 4 volumes of water and 10 volumes of 40% ethanol of the column. Dissolve, and then inject the water eluent into the macroporous adsorption resin column, sequentially elute with 3 times volume of water in the column and 4 times volume of 40% ethanol in the column, and discard the water to elute. The solution was collected twice with a 40% ethanol eluate and concentrated and dried to obtain about 1.1 kg of Cistanche tubulosa extract (CIS).
  • CIS Cistanche tubulosa extract
  • the components and contents of the extract of Cistanche tubulosa obtained from the above (1-1) were analyzed by high performance liquid chromatography (HPLC) and a photodiode array (PDA) detector.
  • HPLC high performance liquid chromatography
  • PDA photodiode array
  • Example 2 Establishment of a model of myocyte injury
  • Tumor necrosis factor alpha is a pro-inflammatory cytokine with a molecular weight of 17,000.
  • Human clinical data show that in some special diseases (such as cancer, AIDS, chronic obstructive pulmonary disease, etc.), anticancer drugs and the elderly, the concentration of TNF- ⁇ will increase, accompanied by muscle dissimilation (ie, muscle Decomposition consumption) or increased muscle cell death.
  • muscle dissimilation ie, muscle Decomposition consumption
  • the present inventors induced TNF- ⁇ to establish a model of myocyte injury.
  • C2C12 cells i.e., mouse myocytes, purchased from ATCC
  • H-DMEM medium purchased from Sigma
  • C2C12 cells were cultured in H-DMEM medium (purchased from Sigma) until C2C12 cells were grown to 80% confluence (i.e., mixed monolayers accounted for After 80% area, the cells were divided into four groups, and the medium of each group was changed to a differentiation medium of 2% horse serum.
  • TNF- ⁇ purchased from Sigma was added to achieve a final concentration of 0, 2, 5 or 10 ng/ml (ng/mL) in each medium.
  • the Cistanche tubulosa extract obtained in Example 1 was prepared as a Cistanche tubulosa extract solution by using dimethyl sulfoxide (DMSO; purchased from Sigma). After C2C12 cells were grown to 80% confluence in H-DMEM medium, they were divided into eight groups, and each group of medium was changed to 2% horse serum differentiation medium, and the above different concentrations of tube flowers were separately added.
  • the Cistanche extract solution is allowed to have a final concentration of 0, 1, 5, 10, 50, 100, 500 or 1000 ⁇ g/ml ( ⁇ g/mL) in the medium for co-treatment with the differentiation medium for 24 hours. .
  • the survival rate of the C2C12 cells (tested by the MTT assay) and the mitochondrial membrane potential were measured and treated with the extract of Cistanche tubulosa extract (ie, the concentration of Cistanche tubulosa extract was 0 ng. /ml) results, the relative survival rate and intracellular ROS levels of other groups were calculated to evaluate the cytotoxicity of Cistanche tubulosa extract to C2C12 cells, and to determine the rational use concentration of Cistanche tubulosa extract Range and maximum dose.
  • the survival rate of C2C12 cells and the mitochondrial membrane potential were significantly decreased in the group treated with 500 ⁇ g/ml of Cistanche tubulosa extract, so the reasonable use concentration of Cistanche tubulosa extract It should be 1 to 500 ⁇ g/ml, preferably 1 to 100 ⁇ g/ml.
  • C2C12 cells were grown to 80% confluence in H-DMEM medium, they were divided into eleven groups. Seven groups were added to Cistanche tubulosa extract (CIS) solution (formulated in DMSO) to give final concentrations of 0, 1, 5, 10, 50, 100, and 500 ⁇ g/ml in each medium. The pretreatment was carried out for 6 hours; the other three groups were added branched chain amino acid (BCAA; as a positive control group) to a final concentration of 0.1, 1 or 10 ⁇ g in each medium.
  • CIS Cistanche tubulosa extract
  • BCAA branched chain amino acid
  • the active oxygen level was measured to determine the effective concentration of the anti-TNF- ⁇ -induced myocyte injury induced by Cistanche tubulosa extract.
  • C2C12 cells were grown to 80% confluence in H-DMEM medium, they were divided into four groups and subjected to the following treatments:
  • Control group cultured in H-DMEM medium for 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum.
  • TNF- ⁇ group cultured in H-DMEM medium for 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum, and TNF- ⁇ was added to give a final concentration of 10 ng/ml in the medium.
  • TNF- ⁇ +10CIS group a solution of Cistanche tubulosa extract (CIS) (formed in DMSO) was added to the H-DMEM medium to give a final concentration of 10 ⁇ g/ml in the medium. Pretreatment was carried out for 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum, and TNF- ⁇ was added to give a final concentration of 10 ng/ml in the medium.
  • CIS Cistanche tubulosa extract
  • TNF- ⁇ +50CIS group Add Cistanche tubulosa extract solution (formulated in DMSO) to H-DMEM medium, and make it to a final concentration of 50 ⁇ g/ml in the medium for pretreatment. It lasted 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum, and TNF- ⁇ was added to give a final concentration of 10 ng/ml in the medium.
  • ECAR can indirectly reflect the glycolysis ability of cells, and pyruvate produced by glycolysis is reflected in ECAR reading.
  • glucose was added (sampling point was 0, 9, 18 minutes), the cell glycolysis reaction was lower, so ECAR showed a lower reading value; after adding glucose (sampling point was 27, 36, 45 minutes), the cell The glycolysis reaction is elevated, so the ECAR reading is also increased; since oligomycin is an ATP synthase inhibitor, after the addition of oligomycin (sampling point is 54, 63, 72 minutes), intracellular ATP oxidation Phosphorylation is inhibited, and cells can only rely on glycolysis for energy supply, so ECAR is greatly enhanced, and the increased readings represent the potential for glycolysis (ie, the additional glycolysis of the cells compared to the previous stage).
  • the total value represents the maximum glycolysis ability of the cells; after adding 2-deoxyglucose (sampling points are 81, 90, 99 minutes), since 2-deoxyglucose competes with glucose, it blocks glycolysis.
  • the reaction, the ECAR reading at this time reflects the acid produced by the acidogenic mechanism of cells other than glycolysis.
  • C2C12 damage caused by TNF- ⁇ includes a decrease in glycolysis ability, compared to C2C12 cells pretreated with Cistanche tubulosa extract (ie, TNF- ⁇ ). Group), C2C12 cells pretreated with 10 ⁇ g/ml of Cistanche tubulosa extract (ie, TNF- ⁇ +10 CIS group) had higher glycolysis ability.
  • the results show that the extract of Cistanche tubulosa can effectively improve the ability of TNF- ⁇ to reduce the glycolysis of muscle cells, have a protective effect on muscle cells, and effectively resist muscle cell damage.
  • Example 6 Effect of Cistanche tubulosa extract on improving mitochondrial respiration ability of injured muscle cells
  • C2C12 cells were grown to 80% confluence in H-DMEM medium, they were divided into four groups (ie, control group, TNF- ⁇ group, TNF- ⁇ +10CIS group, TNF- ⁇ +50 CIS group). It was treated in the manner described in Example 5 to a differentiation medium in which 2% horse serum was replaced (and TNF- ⁇ was added or not in the group).
  • Each group was sampled once after changing to 2% horse serum differentiation medium (with or without TNF- ⁇ added to the group), and then sampled every 9 minutes, and after the third sampling, in the medium. Oligomycin (oligo) was added for co-treatment. Next, after the sixth sampling, carbonylcyanide-p-trifluoromethoxuphenylhydrazone (FCCP) was added to the medium for co-treatment. Thereafter, after the ninth sampling, the respiratory chain (electron transfer chain) inhibitor antimycin A (anti-A) was added to the culture medium, and after the addition of anti-mycin A (anti-A) Three more samples were taken. Finally, the mitochondrial oxygen consumption rate (OCR) of the C2C12 cells at each sampling point was examined, and the results are shown in Fig. 6A and Fig. 6B-1 to Fig. 6B-3.
  • FCCP carbonylcyanide-p-trifluoromethoxuphenylhydrazone
  • the OCR readings shown before the addition of oligomycin represent the oxygen consumption of the cells in the basal state (reflecting the basic respiratory capacity of the cells), including mitochondrial oxidative phosphorylation and Proton leak (oxygen leak); because oligomycin inhibits ATP synthase, after addition of oligomycin (sampling point 27, 36, 45 minutes),
  • the reduced oxygen consumption after the addition of oligomycin is the oxygen consumption of the ATP added to the oligomycin pre-cell, which indirectly reflects the ATP production of the cells under the basal state; due to the carbonyl-cyano-p-trifluoromethoxy group
  • FCCP Benzoquinone
  • FCCP is an uncoupler that acts as a proton carrier to carry large amounts of protons back into the mitochondrial matrix, neutralizing the pH gradient while consuming large amounts of oxygen, but this proton reflux does not pass ATP synthase, Will drive ATP synthesis, so after adding FCCP (sampling point 54,
  • the C2C12 damage caused by TNF- ⁇ includes a decrease in the mitochondrial respiratory ability.
  • C2C12 cells pretreated with 10 ⁇ g/ml of Cistanche tubulosa extract (ie, TNF- ⁇ +10CIS group) compared to C2C12 cells pretreated with C. tubulosa extract (ie, TNF- ⁇ group) Granules have higher respiratory capacity.
  • the results show that the extract of Cistanche tubulosa can effectively improve the mitochondrial respiration ability caused by TNF- ⁇ , has a protective effect on muscle cells, and can effectively resist muscle cell damage.
  • Example 7 Effect of echinacoside, flavonoids, and isoforms on anti-myocyte injury
  • echinacoside Ech
  • verbascoside VB
  • isoacteoside Iso
  • dimethyl sulfoxide will be used as echinacein, flavonoids, and isoforms. It was purchased from ChromaDex Co., USA) and formulated into echinacoside, flavonoids, and isoforms.
  • C2C12 cells were grown to an 80% confluence in H-DMEM medium, they were divided into seventeen groups and subjected to the following treatments:
  • Control group cultured in H-DMEM medium for 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum for culture for 4 days.
  • TNF- ⁇ group cultured in H-DMEM medium for 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum, and TNF- ⁇ (final concentration of 10 ng/ml) was added for co-treatment for 4 days.
  • TNF- ⁇ + echinacoside group add the echinacoside solution to the H-DMEM medium to make the final concentration in the medium 5, 10, 50, 100 or 500 ⁇ g/ml for pretreatment for 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum, and TNF- ⁇ (final concentration of 10 ng/ml) was added for co-treatment for 4 days.
  • TNF- ⁇ + class of leaf gonadoside group five groups: adding the leaf amygdalin solution to the H-DMEM medium, the final concentration in the medium was 1, 5, 10, 50 or 100 ⁇ g/ml for pretreatment for 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum, and TNF- ⁇ (final concentration of 10 ng/ml) was added for co-treatment for 4 days.
  • TNF- ⁇ + heterophylloside group (five groups): adding heterologous leaf asparagine solution to H-DMEM medium, the final concentration in the medium was 1, 5, 10, 50 or 100 ⁇ g/ml for pretreatment for 6 hours. Next, the medium was changed to a differentiation medium of 2% horse serum, and TNF- ⁇ (final concentration of 10 ng/ml) was added for co-treatment for 4 days.
  • C2C12 cells were pretreated with echinacoside solution at a concentration of 1 to 500 ⁇ g/ml, and the protective effect was not significant at a lower concentration, when the concentration reached 100 ⁇ g/ml. Only significant. However, echinacoside has no protective effect on mitochondrial membrane potential and active oxygen level.
  • C2C12 cells were pretreated with a concentration of 1 to 100 ⁇ g/ml of a leaf amygdalin solution, and it was found that the flavonoids could not protect TNF- ⁇ . Damage to C2C12 cells.
  • pretreatment of C2C12 cells with a concentration of 5 to 100 ⁇ g/ml of a heterologous amygdalin solution can increase the survival rate of C2C12 cells induced by TNF- ⁇ , indicating heterologous leaves.
  • Cimicin can effectively reduce the damage caused by TNF- ⁇ to C2C12 cells.
  • pretreatment of C2C12 cells with a concentration of 100 ⁇ g/ml of a heterologous leaf aglycone solution can significantly improve the mitochondrial membrane potential decrease caused by TNF- ⁇ .
  • Example 8 Molecular mechanism of action of Cistanche tubulosa extract and its components on muscle protection
  • C2C12 cells were grown to 80% confluence in H-DMEM medium, they were divided into four groups (ie, control group, TNF- ⁇ group, TNF- ⁇ +10CIS group, TNF- ⁇ +50 CIS group), and After treatment to the differentiation medium in which 2% horse serum was replaced (and TNF- ⁇ was added or not) in the manner described in Example 5, the culture was continued for 4 days.
  • TNF- ⁇ causes protein degradation, which is thought to be TNF- ⁇ -induced I ⁇ B ⁇ degradation, activates NF ⁇ B, and allows it to enter the nucleus to bind to the ubiquitin-proteasome pathway-associated protein gene and promote its transcription, thereby enabling ubiquitin proteasome Increased synthesis of related proteins, which in turn promotes extensive degradation of muscle proteins.
  • Fig. 8A the inhibition of inflammatory factor NF ⁇ B by Cistanche tubulosa extract was not significant.
  • the protein expression levels of mTOR, AMPK, PGC-1 ⁇ , MFN2 and mitochon complex I in the TNF- ⁇ group decreased; among them, the mitochondrial Complex I is important on the mitochondrial respiratory chain.
  • PGC-1 ⁇ is a transcription factor that promotes mitochondrial synthesis and energy oxidative metabolism in skeletal muscle cells
  • MFN2 is involved in mitochondrial fusion
  • both MFN2 and PGC-1 ⁇ maintain mitochondrial membrane potential and promote oxidative phosphorylation.
  • Aspects have a synergistic effect. The foregoing results show that TNF- ⁇ -induced myocyte injury affects the mTOR/AMPK signaling pathway and disrupts the cellular energy metabolism system.
  • Cistanche tubulosa extract ie, TNF- ⁇ +10CIS group and TNF- ⁇ +50CIS group
  • the performance of I was significantly improved, indicating that C. tubulosa extract can initiate the mTOR/AMPK information transmission pathway in muscle cells, and stabilize the number and activity of mitochondria by expressing proteins such as PGC-1 ⁇ and MFN2, and repair cell energy metabolism. Damage to the system (this function is similar to branched-chain amino acids). Therefore, the protective mechanism of Cistanche tubulosa extract and its components on myocytes is related to anti-myocyte autophagy and promotion of myocyte mitochondrial production.
  • Cistanche tubulosa and the heterologous leaf asparagine contained therein can effectively protect damaged muscle cells, increase the survival rate of muscle cells, and inhibit the decrease and activity of mitochondrial membrane potential.
  • the oxygen level rises and maintains the mitochondrial activity in the cells. Therefore, Cistanche tubulosa extract and heterologous leaf asparagine have improved oxidative stress and maintain mitochondrial activity on injured muscle cells. Good biological effect.
  • Cistanche tubulosa extract can reactivate the mTOR/AMPK signaling pathway in injured myocytes, allowing downstream mitochondria to synthesize related proteins (eg PGC-1 ⁇ ), mitochondrial fusion-related molecules (eg MFN2), The amount of mitochondria involved in the respiratory chain (such as Complex I) rebounds, which restores the energy metabolism of muscle cells.
  • PGC-1 ⁇ mitochondrial fusion-related molecules
  • MFN2 mitochondrial fusion-related molecules

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Abstract

一种使用管花肉苁蓉(Cistanche tubulosa)萃取物、异类叶升麻苷及/或异类叶升麻苷的医药上可接受的盐于制备一药物或一食品的用途，其中，该药物或食品是用于保护肌肉。该药物尤其是用于抗肌细胞损伤，以调节、治疗及/或延缓肌肉流失，特别是因为老化、疾病及/或恶病质所引起的肌肉流失。该食品为保健食品、营养补充食品或特殊营养食品。

Description

管花肉苁蓉萃取物及异类叶升麻苷于保护肌肉的用途 技术领域
本发明涉及管花肉苁蓉(Cistanche tubulosa)萃取物与异类叶升麻苷(isoacteoside)(即，管花肉苁蓉萃取物的一成分)或异类叶升麻苷的医药上可接受的盐的应用，尤其是涉及使用管花肉苁蓉萃取物、异类叶升麻苷或异类叶升麻苷的医药上可接受的盐以保护肌肉的应用，包括抗肌细胞损伤以调节、治疗及/或延缓肌肉流失，特别是因为老化、疾病及/或恶病质所引起的肌肉流失。
背景技术
肌肉组织是哺乳动物体内质量最高的组织，其主要功能是产生力量以牵引身体各部位运动。肌肉可区分为骨骼肌、心肌及平滑肌三种。骨骼肌按照其本身的代谢类型及特性可进一步区分为慢肌和快肌，前者由慢肌纤维蛋白所组成，其收缩可持续较长的时间，但力量较小；后者则由快肌纤维蛋白所组成，收缩较快、力量较大，但也较容易感到疲劳。
在正常生理状态下，肌蛋白的合成与降解呈动态平衡。然而，当发生肌蛋白降解速度大于肌蛋白合成速度的肌蛋白代谢失衡情形时，会造成肌肉流失，严重的肌肉流失则会导致肌肉萎缩，且出现肌肉重量明显减轻、肌纤维横截面积减少、肌纤维类型相关蛋白(即，慢肌纤维蛋白与快肌纤维蛋白)选择性减少等特征性的变化，造成肌力下降、运动障碍、易疲劳、代谢紊乱等症状，严重影响日常工作和生活机能。
已知有些生理状况或特殊疾病会引起肌细胞损伤，导致肌蛋白代谢失衡或肌细胞凋亡，造成肌肉流失。其中，会导致肌肉流失的因素包括例如：神经退化、长期卧床、衰老、疾病和恶病质(cachexia；例如癌症恶病质)等，其中的疾病则包括例如：败血病、艾滋病、 肾脏衰竭、库欣氏症候群(Cushing’s syndrome，CS)、肌少症(sarcopenia)、癌症、慢性阻塞性肺病(chronic obstructive pulmonary disease，COPD)、郁血性心脏衰竭(congestive heart failure，CHF)、创伤等。
老化是肌少症最主要的成因，有统计数据指出，60～70岁的老年人发生肌少症的机率为13％至24％，高于80岁的老年人发生肌少症的机率则为约50％。此外，在美国每年因为肌少症所造成的医疗花费约为118至262亿美元。恶病质则与其他高发生率的疾病相关，举例言之，约有50％的癌症患者、20％至40％的COPD患者及50％至70％的CHF患者会出现恶病质的症状(例如营养不良)。
中药肉苁蓉(Herba Cistanche)最早被记载于《神农本草经》中，并且被《神农本草经》列为上品，具有补肾阳、益精血、润肠通便等功效，是历代医家补肾壮阳处方中使用频度最高的补益中药。管花肉苁蓉(Cistanche tubulosa)是列当科(Orobanchaceae)肉苁蓉属多年生寄生草本植物的一种，其分布于沙漠、荒漠等干燥地带，依赖吸取寄主植物红柳的养分维生，为罕见而贵重的药材。管花肉苁蓉用于提高肾功能、增强记忆力、调节免疫功能、抗老年痴呆、抗衰老、抗疲劳等多方面的功效，已经于2005年被列入《中国药典》中。
目前临床上尚无有效治疗或延缓肌肉流失的方法，且迄今并无文献表示管花肉苁蓉对肌肉萎缩有保护作用，故为开发更有效治疗或延缓肌肉流失的方法，本发明人由传统中药材中选择肉苁蓉，探讨其应用于保护肌肉(抗肌细胞损伤、避免肌肉流失)的可行性。本发明人研究发现，管花肉苁蓉萃取物及其所含的异类叶升麻苷(isoacteoside)，都可有效抗肌细胞损伤，故可用于调节、治疗及/或延缓肌肉流失，特别是因为老化、疾病及/或恶病质所引起的 肌肉流失，从而可用于提供保护肌肉的医药组合物、药物或食品。
发明内容
本发明的一个目的，在于提供一种使用管花肉苁蓉萃取物于制备一保护肌肉的药物或食品的用途。较佳地，该管花肉苁蓉萃取物为管花肉苁蓉的极性溶剂萃取物，该极性溶剂为选自水、C1-C4醇类及前述的组合中的任一种。更佳地，该管花肉苁蓉萃取物含有异类叶升麻苷。其中，该药物是用于抗肌细胞损伤，或者用于治疗及/或延缓以下的至少一者所引起的肌肉流失：老化、疾病及恶病质；该食品是用于调节以下的至少一者所引起的肌肉流失：老化、疾病及恶病质，有助于肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能(neuromuscular function)、维持能量正常代谢或增强能量，且该食品为保健食品、营养补充食品或特殊营养食品。
本发明的另一目的，在于提供一种使用一活性成分于制备一保护肌肉的药物或食品的用途，其中，该活性成分为异类叶升麻苷及/或其医药上可接受的盐。较佳地，该活性成分是以植物萃取物的形式使用；更佳地，以管花肉苁蓉萃取物的形式使用，尤其是以管花肉苁蓉的极性溶剂萃取物的形式使用，该极性溶剂为选自水、C1-C4醇类及前述的组合中的任一种。其中，该药物是用于抗肌细胞损伤，或者用于治疗及/或延缓以下的至少一者所引起的肌肉流失：老化、疾病及恶病质；该食品是用于调节以下的至少一者所引起的肌肉流失：老化、疾病及恶病质，有助于肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能、维持能量正常代谢或增强能量，且该食品为保健食品、营养补充食品或特殊营养食品。
本发明的又一目的，在于提供一种保护肌肉的方法，其包括对一有需要的个体施用一有效量的管花肉苁蓉萃取物。较佳地，该管花肉苁蓉萃取物为管花肉苁蓉的极性溶剂萃取物，该极性溶剂为选 自水、C1-C4醇类及前述的组合中的任一种。更佳地，该管花肉苁蓉萃取物含有异类叶升麻苷。其中，该方法是用于抗肌细胞损伤，用于调节、治疗及/或延缓以下的至少一者所引起的肌肉流失：老化、疾病及恶病质，或用于帮助肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能、维持能量正常代谢或增强能量。
本发明的再一目的，在于提供一种保护肌肉的方法，其包括对一有需要的个体施用一有效量的活性成分，其中，该活性成分为异类叶升麻苷及/或其医药上可接受的盐。较佳地，该活性成分是以植物萃取物的形式使用；更佳地，以管花肉苁蓉萃取物的形式使用，尤其是以管花肉苁蓉的极性溶剂萃取物的形式使用，该极性溶剂为选自水、C1-C4醇类及前述的组合中的任一种。其中，该方法是用于抗肌细胞损伤，用于调节、治疗及/或延缓以下的至少一者所引起的肌肉流失：老化、疾病及恶病质，或用于帮助肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能、维持能量正常代谢或增强能量。
本发明的又再一目的，在于提供一种保护肌肉的组合物，该组合物为一药物或食品，且包含一有效量的管花肉苁蓉萃取物。较佳地，该管花肉苁蓉萃取物为管花肉苁蓉的极性溶剂萃取物，该极性溶剂为选自水、C1-C4醇类及前述的组合中的任一种。更佳地，该管花肉苁蓉萃取物含有异类叶升麻苷。其中，该组合物是用于抗肌细胞损伤，用于调节、治疗及/或延缓以下的至少一者所引起的肌肉流失：老化、疾病及恶病质，或用于帮助肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能、维持能量正常代谢或增强能量。
本发明的又再一目的，在于提供一种保护肌肉的组合物，其中，该组合物为一药物或食品且包含一有效量的活性成活性成分，且其 中，该活性成分为异类叶升麻苷及/或其医药上可接受的盐。较佳地，该活性成分是以植物萃取物的形式使用；更佳地，以管花肉苁蓉萃取物的形式使用，尤其是以管花肉苁蓉的极性溶剂萃取物的形式使用，该极性溶剂为选自水、C1-C4醇类及前述的组合中的任一种。其中，该组合物是用于抗肌细胞损伤，用于调节、治疗及/或延缓以下的至少一者所引起的肌肉流失：老化、疾病及恶病质，或用于帮助肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能、维持能量正常代谢或增强能量。
附图说明
图1A、图1B所示为TNF-α对C2C12细胞的粒线体膜电位与细胞内活性氧水平的影响；
图2A、图2B所示为管花肉苁蓉萃取物对C2C12细胞的存活率与粒线体膜电位的影响；
图3A、图3B、图3C所示为管花肉苁蓉萃取物对经TNF-α诱导损伤的C2C12细胞的细胞存活率、粒线体膜电位及细胞内活性氧水平的影响；
图4A、图4B所示为管花肉苁蓉萃取物与支链氨基酸(正控制组)对经TNF-α诱导损伤的C2C12细胞的细胞内活性氧水平的影响；
图5A、图5B-1及图5B-2所示为管花肉苁蓉萃取物对经TNF-α诱导损伤的C2C12细胞的糖酵解能力的影响；
图6A、图6B-1、图6B-2及图6B-3所示为管花肉苁蓉萃取物对经TNF-α诱导损伤的C2C12细胞的粒线体呼吸能力的影响；
图7A-1、图7A-2及图7A-3所示分别为松果菊苷对经TNF-α诱导损伤的C2C12细胞的细胞存活率、粒线体膜电位及细胞内活性氧水平的影响；
图7B-1、图7B-2及图7B-3所示为类叶升麻苷对经TNF-α诱导损伤的C2C12细胞的细胞存活率、粒线体膜电位及细胞内活性氧水平的影响；
图7C-1、图7C-2及图7C-3所示为异类叶升麻苷对经TNF-α诱导损伤的C2C12细胞的细胞存活率、粒线体膜电位及细胞内活性氧水平的影响；以及
图8A、图8B、图8C及图8D所示为管花肉苁蓉萃取物对经TNF-α诱导损伤的C2C12细胞的mTOR/AMPK信号传递路径以及NFkB/p-JNK信号传递路径的相关蛋白质的表现量的影响。
具体实施方式
本发明的详细技术及较佳实施例，将描述于以下内容中，以供本发明所属领域的技术人员据以明了本发明的特征；但，在不背离本发明精神下，本发明还可以多种不同形式的实施例来实践，不应将本发明保护范围解释为限于说明书所陈述的。此外，除非文中有另外说明，于本说明书中(尤其是在权利要求书中)所使用的“一”、“该”及类似用语应理解为包括单数及复数形式；所谓“有效量”，是指投予至个体时，可有效至少部分改善怀疑个体的病情的化合物数量；所谓“个体”是指人类或非人的哺乳动物；所谓“治疗”是包括预防特定疾病或症状、减轻特定的疾病或症状及/或防止或消除该病症；单位“毫克/公斤体重”是指每公斤体重个体所需的投与量。
本说明书中所使用的数值范围(例如5至100)应理解为也包括在该范围中的所有有理数以及在该范围中的任何有理数所组成的范围，因此，本说明书中所使用的数值范围包括介于所列举的最低值与最高值之间的数值的所有可能组合。另外，本申请所使用的“约”、“大约”或“近乎”等词，实质上代表与所述数值相差在20％ 以内的，较佳在10％以内的，且更佳在5％以内的数值。
于本说明书中，所谓“医药上可接受的盐”是指投予至生物体后，可产生与母体化合物相同或类似的药理活性，且为生理上可忍受(即，具有尽可能小的毒性作用)的盐。
本发明人研究发现，管花肉苁蓉萃取物可有效地抗肌细胞损伤，故可用于保护肌肉，具有治疗及/或延缓肌肉流失的功效。于不受理论限制下，相信本发明所使用的管花肉苁蓉萃取物可有效调节、治疗及/或延缓因为老化、疾病及/或恶病质所引起的肌肉流失。因此，本发明提供一种使用管花肉苁蓉萃取物于保护肌肉的应用，包括使用管花肉苁蓉萃取物于制备一保护肌肉的药物或食品、对有需要的个体投予管花肉苁蓉萃取物以保护肌肉的方法以及提供一包含管花肉苁蓉萃取物的食品或医药组合物。
根据本发明，可采用包括如下步骤的方法所提供的管花肉苁蓉萃取物：(a)使用一极性溶剂以萃取管花肉苁蓉，获得一萃取液；以及(b)视需要干燥该萃取液。其中，该极性溶剂可为水及/或C1-C4醇类。较佳地，使用水、乙醇或其组合作为该极性溶剂。可视需要调整用于萃取的极性溶剂与管花肉苁蓉的用量比率，一般而言，极性溶剂与管花肉苁蓉的体积比可为约1∶1至约50∶1，较佳为约5∶1至20∶1。
根据本发明，可使用管花肉苁蓉的任何部位以制备管花肉苁蓉萃取物。举例言之，可使用管花肉苁蓉的茎部、花或全株植物作为萃取原料。于根据本发明的一实施例中，使用管花肉苁蓉的肉质茎部分作为萃取原料。
于上述步骤(a)中，进行该萃取一段时间以达到所欲的萃取程度。以采用水作为该极性溶剂为例，通常为至少15分钟，较佳为至少30分钟，更佳为至少60分钟，且可视需要辅以例如煎煮、冷却、 过滤、减压浓缩、树脂管柱层析等其它操作。视需要地，可于进行步骤(b)之前，以相同或不同极性溶剂重复进行多次萃取步骤(a)，并合并该多次萃取所得的萃取液以进行步骤(b)；或者，可重复进行萃取步骤(a)、萃取步骤(b)以及前述视需要的其他操作的循环，以尽可能达到最大的萃取效益。例如本发明的一实施例中所提供的管花肉苁蓉萃取物的制备方法。
本发明人进一步研究发现，于管花肉苁蓉萃取物所含有的成分中，异类叶升麻苷本身即可有效地抗肌细胞损伤，故可用于保护肌肉，具有治疗及/或延缓肌肉流失的功效。于不受理论限制下，相信异类叶升麻苷可有效调节、治疗及/或延缓因为老化、疾病及/或恶病质所引起的肌肉流失。因此，本发明也提供一种使用异类叶升麻苷及/或其医药上可接受的盐于保护肌肉的应用，包括使用异类叶升麻苷及/或其医药上可接受的盐于制备一保护肌肉的药物或食品、对有需要的个体投予异类叶升麻苷及/或其医药上可接受的盐以保护肌肉的方法以及提供一包含异类叶升麻苷及/或其医药上可接受的盐的食品或医药组合物。
较佳地，该异类叶升麻苷及/或其医药上可接受的盐是以植物萃取物的形式使用；更佳地，以管花肉苁蓉萃取物的形式使用，尤其是以管花肉苁蓉的极性溶剂萃取物的形式使用。
于根据本发明的应用所提供的医药组合物或药物可呈任何合宜的型式，并无特殊限制，端视所欲的用途而呈对应的合宜剂型。举例言之，但不以此为限，该医药组合物或药物可以口服或非经口服(例如：皮下、静脉内、肌肉、腹腔或鼻腔)的投药方式施用至有需要的个体上。其中，视使用形式及用途而定，可选用合宜的载剂以提供该医药组合物或药物，其中，该载剂包括赋形剂、稀释剂、辅助剂、安定剂、吸收延迟剂、崩散剂、增溶剂、乳化剂、抗氧化 剂、黏合剂、结合剂、增黏剂、分散剂、悬浮化剂、润滑剂、吸湿剂等。
以适于口服的剂型为例，于根据本发明的应用所提供的医药组合物或药物可含有任何不会不利影响活性成分(即，管花肉苁蓉萃取物或异类叶升麻苷)的所欲效益的医药上可接受的载剂，例如：水、食盐水、葡萄糖(dextrose)、甘油、乙醇或其类似物、纤维素、淀粉、糖膨润土(sugar bentonite)及前述的组合。可利用任何合宜的方法，以适于口服投药的剂型提供该医药组合物或药物，例如：锭剂(例如糖衣锭)、丸剂、胶囊剂、颗粒剂、散剂、流浸膏剂、溶液剂、糖浆剂、悬液剂、酊剂等。
至于适于皮下、静脉内、肌肉或腹腔注射的针剂或点滴剂，则可于根据本发明的应用所提供的医药组合物或药物中含有一或多种例如等张溶液、盐类缓冲液(如磷酸盐缓冲液或柠檬酸盐缓冲液)、增溶剂、乳化剂、5％糖溶液以及其他载剂等成分，以静脉输注液、乳剂静脉输注液、干粉注射剂、悬液注射剂或干粉悬液注射剂等剂型提供该医药组合物或药物。或者，可将该医药组合物或药物制备成一注射前固体，以可溶于其他溶液或悬浮液中的剂型或可乳化的剂型提供该注射前固体，并于投予至有需要的个体之前，将该注射前固体溶于其他溶液或悬浮液中或将其乳化，提供所欲的注射剂。
视需要地，可于根据本发明的应用所提供的医药组合物或药物中另外含有合宜用量的添加剂，例如可提高该医药组合物或药物于服用时的口适感及视觉感受的调味剂、调色剂、着色剂等，以及可改善该医药组合物或药物的稳定性及储存性的缓冲剂、保存剂、防腐剂、抗菌剂、抗真菌剂等。此外，该医药组合物或药物可视需要另外含有一或多种其他活性成分(例如维生素D、维生素B1、维生 素B2、烟碱素、生物素、泛酸、钙、碘、镁、锌、蛋白质等)，或者与含有该一或多种其他活性成分的药物并用，以进一步加强该医药组合物或药物的功效或增加制剂配方的运用灵活性与调配度，只要该其他活性成分对本发明活性成分(即，管花肉苁蓉萃取物或异类叶升麻苷)的效益没有不利的影响即可。
于根据本发明的应用所提供的医药组合物或药物可以一日一次、一日多次或数日一次等不同投药频率施用，端视投予个体的需求、年龄、体重及健康况状而异。举例言之，当以口服方式施用至一个体以保护肌肉时，以管花肉苁蓉萃取物计，其用量为每天约0.5毫克/公斤体重至约1000毫克/公斤体重，较佳为每天约2.5毫克/公斤体重至约1000毫克/公斤体重，更佳为每天约5毫克/公斤体重至约500毫克/公斤体重。或者，以异类叶升麻苷计，其用量为每天约0.01毫克/公斤体重至约100毫克/公斤体重，较佳为每天约0.03毫克/公斤体重至约70毫克/公斤体重，更佳为每天约0.05毫克/公斤体重至约50毫克/公斤体重。其中，该单位“毫克/公斤体重”是指每公斤体重个体所需的投与量。
于根据本发明的应用所提供的食品可以是保健食品、营养补充食品或特殊营养食品，且可以制成例如乳制品、肉类加工品、面包类、面食品、饼干、口含锭、胶囊、果汁类、茶类、运动饮料、营养饮料等产品，但不以此为限。较佳地，根据本发明的应用的食品以保健食品的型式提供。
于根据本发明的应用所提供的保健食品、营养补充食品及特殊营养食品可以一日一次、一日多次或数日一次等不同频率食用，端视投予个体的年龄、体重及健康状况而采用的建议摄取量而异。也可针对特定族群调整本发明所提供的保健食品、营养补充食品及特殊营养食品中管花肉苁蓉萃取物或异类叶升麻苷的含量，较佳为调 整至每日应服用的量。
可于本发明保健食品、营养补充食品及/或特殊营养食品的外包装标示建议使用量、特定族群(例如孕妇、癌症患者、心脏衰竭患者)的使用标准及条件或与其他食品或医药共同服用的建议事项，以利用户在无医师、药师或相关执事人员指导下可在家自行服用而无安全疑虑。
本发明也提供一种于一个体中保护肌肉的方法，其包括对该个体施用一有效量的活性成分，其中，该活性成分为管花肉苁蓉萃取物、异类叶升麻苷及/或异类叶升麻苷的医药上可接受的盐。于根据本发明的保护肌肉的方法中，有关该活性成分的投予途径、投予形式、适用剂量以及相关治疗的应用，均如上述说明。
现以下列实施例进一步例示说明本发明。其中，这些实施例仅提供作为说明，而非用以限制本发明的保护范围。本发明的保护范围应以权利要求书所界定。
实施例1：管花肉苁蓉萃取物(Cistanche tubulosa extract，CIS)的制备与成分分析
(1-1)
取管花肉苁蓉的肉质茎部分10公斤，切片后加入其体积的8倍的水，浸泡1小时后，再煎煮2小时，过滤并收集滤液。加入药渣体积的6倍的水，煎煮2次，每次1小时，并过滤。合并三次滤液，于50℃下减压浓缩至比重1.10，添加乙醇至浓度60％，冷藏12小时后倾出上清液，于50℃减压浓缩病回收乙醇至比重1.10，获得粗萃取物6公斤。接着，以粗萃取物的1倍体积的水加热溶解粗萃取物，将溶液注入大孔吸附树脂柱内，依序以管柱4倍体积的水及管柱5倍体积的40％乙醇进行洗脱，再将水洗脱液注入大孔吸附树脂柱中，依序以管柱3倍体积的水及管柱4倍体积的40％乙醇洗脱，弃去水洗脱 液，收集两次40％乙醇洗脱液，浓缩干燥后，即获得管花肉苁蓉萃取物(CIS)约1.1公斤。
(1-2)
以高效液相色谱法(high performance liquid chromatography，HPLC)及光电二极管数组(PDA)检测器分析由上述(1-1)所获得的管花肉苁蓉萃取物所含成分及其含量。结果显示，管花肉苁蓉萃取物含有松果菊苷(echinacoside)、类叶升麻苷(acteoside)及异类叶升麻苷(isoacteoside)等成分，且前述三种成分于管花肉苁蓉萃取物中所占的重量百分比分别为25.4％、3.8％及4.1％。
实施例2：肌细胞损伤模型的建立
肿瘤坏死因子-α(tumor necrosis factor alpha，TNF-α)是一种促进发炎反应(pro-inflammatory)的细胞激素(cytokine)，分子量为17,000。人体临床数据显示，在一些特殊疾病(如癌症、艾滋病、慢性阻塞性肺病等)患者、施用抗癌药物者及老年人的体内，TNF-α的浓度会上升，伴随肌肉异化作用(即，肌肉分解消耗)或肌细胞死亡增加的现象。研究显示，将TNF-α注射至实验动物体内或投予药物使实验动物体内TNF-α浓度增加会诱发肌细胞损伤(包括肌蛋白代谢失衡、肌细胞凋亡等)，进而导致肌肉的流失或萎缩。为了探讨管花肉苁蓉萃取物及其中的成分于保护肌肉的功效与作用机制，本发明人以TNF-α进行诱导，建立肌细胞损伤模型。
首先，以H-DMEM培养基(购自Sigma公司)对C2C12细胞(即，小鼠的肌细胞，购自ATCC)进行培养，待C2C12细胞生长至80％汇合度(即，混合单层细胞占80％面积)后，将细胞分成四组，并将各组的培养基更换为2％马血清的分化培养基。接着，加入TNF-α(购自Sigma公司)使其在各组培养基中的最终浓度达到0、2、5或10纳克/毫升(ng/mL)。在历时4天的分化培养基与TNF-α的共 处理之后，测定C2C12细胞的粒线体膜电位(mitochondrial membrane potential，MMP)以及细胞内活性氧水平(reactive oxidative stress，ROS)作为模型评价指针，建立肌细胞损伤模型。最后，以未经TNF-α处理的组别(即，TNF-α的使用浓度为0纳克/毫升)的结果为基准，计算其他各组的相对的粒线体膜电位及细胞内的活性氧水平，结果示于图1A、图1B(数据均为平均数±SEM，n＝6，由t检验分析得到，*p＜0.05，***p＜0.001)。
由图1A、图1B可知，当使用5纳克/毫升的TNF-α，C2C12细胞的粒线体膜电位明显下降，且活性氧水平略微升高；此外，当使用10纳克/毫升的TNF-α，C2C12细胞粒线体膜电位明显下降，且活性氧水平明显升高。因此，后续实验以10纳克/毫升作为TNF-α诱导肌细胞损伤的浓度。
实施例3：管花肉苁蓉萃取物的使用浓度
取实施例1提供的管花肉苁蓉萃取物，以二甲基亚砜(dimethyl sulfoxide，DMSO；购自Sigma公司)配制成管花肉苁蓉萃取物溶液。待C2C12细胞在H-DMEM培养基中生长至80％汇合度后，将其分成八组，并将各组培养基更换为2％马血清的分化培养基，且分别加入前述不同浓度的管花肉苁蓉萃取物溶液使其于培养基中的最终浓度为0、1、5、10、50、100、500或1000微克/毫升(μg/mL)，以与分化培养基进行共处理，历时24小时。其后，测试C2C12细胞的存活率(以MTT assay进行测试)及粒线体膜电位，并以未经管花肉苁蓉萃取物处理的组别(即，管花肉苁蓉萃取物的使用浓度为0纳克/毫升)的结果为基准，计算其他各组的相对的存活率及细胞内的活性氧水平，以评估管花肉苁蓉萃取物对C2C12细胞的细胞毒性，并确定管花肉苁蓉萃取物的合理使用浓度范围及最大剂量。结果示于图2A、图2B(数据均为平均数±SEM，n＝6)。
由图2A、图2B可知，经500微克/毫升的管花肉苁蓉萃取物处理的组别，其C2C12细胞的存活率及粒线体膜电位都明显下降，故管花肉苁蓉萃取物的合理使用浓度应为1至500微克/毫升，较佳为1至100微克/毫升。
实施例4：管花肉苁蓉萃取物于抗肌细胞损伤的效果
待C2C12细胞在H-DMEM培养基中细胞生长至80％汇合度后，将其分成十一组。其中七组加入管花肉苁蓉萃取物(CIS)溶液(配制于DMSO中)，使其于各组培养基中的最终浓度分别为0、1、5、10、50、100、500微克/毫升，以进行预处理，历时6小时；另外三组则加入支链氨基酸(branched chain amino acid，BCAA；作为正控制组)，使其于各组培养基中的最终浓度分别为0.1、1或10微克/毫升，以进行预处理，历时6小时；接着，将前面提到的十组(加入CIS的七组与加入BCAA的三组)的培养基更换为2％马血清的分化培养基，并加入TNF-α使其于培养基中的最终浓度为10纳克/毫升，以与分化培养基进行共处理，历时4天；此外的最后一组作为对照组，其并未以管花肉苁蓉萃取物溶液、BCAA或TNF-α进行处理。最后，测定各组C2C12细胞的存活率、粒线体膜电位及细胞内的活性氧水平，并以对照组的结果为基准，计算其他各组的相对的存活率、粒线体膜电位以及细胞内的活性氧水平，以测定管花肉苁蓉萃取物抗TNF-α诱导的肌细胞损伤的有效浓度，结果示于图3A、图3B、图3C及图4A、图4B(数据均为平均数±SEM，n＝6，由t检验分析得到，*p＜0.05，**p＜0.01，***p＜0.001)。
由图3A、图3B、图3C可知，在经TNF-α诱导细胞损伤的组别中，若以0至50微克/毫升的管花肉苁蓉萃取物进行预处理，C2C12细胞的存活率随着管花肉苁蓉萃取物浓度的升高而呈逐渐上升的趋势，粒线体膜电位则没有明显的变化。另外，在细胞内的活性氧水 平的方面，若以10微克/毫升的管花肉苁蓉萃取物进行欲处理，活性氧水平下降至与对照组(未经TNF-α诱导损伤)相当，若以50或100微克/毫升的管花肉苁蓉萃取物进行欲处理，活性氧水平下降的幅度又更为明显。
由图4A、图4B可知，以活性氧水平进行评估，针对经TNF-α诱导损伤的C2C12细胞，10至50微克/毫升之管花肉苁蓉萃取物所能提供的保护效果系与正控制组(即，BCAA)相当。
上述结果显示，浓度10至50微克/毫升的管花肉苁蓉萃取物对肌细胞有显著的保护效果，可有效降低TNF-α所造成的肌细胞损伤。因此，后续实验以10及50微克/毫升作为管花肉苁蓉萃取物预处理细胞的浓度。
实施例5：管花肉苁蓉萃取物于改善损伤的肌细胞的糖酵解潜力(glycolytic capacity)的效果
待C2C12细胞在H-DMEM培养基中生长至80％汇合度后，将其分成四组，并进行以下处理：
(1)控制组：以H-DMEM培养基进行培养，历时6小时。接着，将培养基更换为2％马血清的分化培养基。
(2)TNF-α组：以H-DMEM培养基进行培养，历时6小时。接着，将培养基更换为2％马血清的分化培养基，并加入TNF-α使其于培养基中的最终浓度为10纳克/毫升。
(3)TNF-α+10CIS组：于H-DMEM培养基中加入管花肉苁蓉萃取物(CIS)溶液(配制于DMSO中)，使其于培养基中的最终浓度为10微克/毫升，以进行预处理，历时6小时。接着，将培养基更换为2％马血清的分化培养基，并加入TNF-α使其于培养基中的最终浓度为10纳克/毫升。
(4)TNF-α+50CIS组：于H-DMEM培养基中加入管花肉苁蓉萃取物溶液(配制于DMSO中)，使其于培养基中的最终浓度为50微克/毫升，以进行预处理，历时6小时。接着，将培养基更换为2％马血清的分化培养基，并加入TNF-α使其于培养基中的最终浓度为10纳克/毫升。
各组于更换为2％马血清分化培养基(依组别加入或不加入TNF-α)后取样一次，其后，每隔9分钟取样一次，且于第3次取样后，于培养基中加入葡萄糖以进行共处理。接着，于第6次取样后，于培养基中加入寡霉素(oligomycin，oligo；购自Sigma公司)以进行共处理。其后，于第9次取样后，于培养基中加入2-脱氧葡萄糖(2-deoxy-glucose，2-DG；购自Sigma公司)以进行共处理，并于加入2-脱氧葡萄糖后再进行三次取样。最后，检测各取样点的C2C12细胞的细胞外产酸能力(extracellular acidification rate，ECAR)，结果示于图5A及图5B-1、图5B-2。
其中，ECAR可间接反映出细胞的糖酵解能力，糖酵解产生的丙酮酸反映于ECAR读值。未加入葡萄糖前(取样点为0、9、18分钟)，细胞的糖酵解反应较低，故ECAR呈现较低读值；加入葡萄糖后(取样点为27、36、45分钟)，细胞的糖酵解反应升高，故ECAR读值也升高；由于寡霉素是一种ATP合成酶抑制剂，加入寡霉素后(取样点为54、63、72分钟)，细胞内ATP的氧化磷酸化会被抑制，此时细胞只能依赖糖酵解供能，故ECAR大幅提升，所增加的读值代表糖酵解潜力(即，相较于先一阶段，细胞所额外具有的糖酵解能力)，总数值则代表细胞的最大糖酵解能力；加入2-脱氧葡萄糖后(取样点为81、90、99分钟)，由于2-脱氧葡萄糖会与葡萄糖竞争，因而阻断糖酵解反应，此时的ECAR读值所反映的是通过糖酵解以外的细胞产酸机制所产生的酸。
由图5A及图5B-1、图5B-2可知，TNF-α造成的C2C12损伤包括糖酵解能力的下降，相较于未经管花肉苁蓉萃取物预处理的C2C12细胞(即，TNF-α组)，经10微克/毫升的管花肉苁蓉萃取物预处理的C2C12细胞(即，TNF-α+10CIS组)的糖酵解能力较高。此结果显示管花肉苁蓉萃取物能有效改善TNF-α所引起的肌细胞糖酵解能力下降，对肌细胞具有保护效果，可有效地抗肌细胞损伤。
实施例6：管花肉苁蓉萃取物于改善损伤的肌细胞的粒线体呼吸能力的效果
待C2C12细胞在H-DMEM培养基中细胞生长至80％汇合度后，将其分成四组(即，控制组、TNF-α组、TNF-α+10CIS组、TNF-α+50CIS组)，并以实施例5所述的方式处理至更换2％马血清的分化培养基(且依组别加入或不加入TNF-α)。
各组于更换为2％马血清分化培养基(依组别加入或不加入TNF-α)后取样一次，其后，每隔9分钟取样一次，且于第3次取样后，于培养基中加入寡霉素(oligomycin，oligo)以进行共处理。接着，于第6次取样后，于培养基中加入羰基-氰-对-三氟甲氧基苯肼(carbonylcyanide-p-trifluoromethoxuphenylhydrazone，FCCP)以进行共处理。其后，于第9次取样后，于培养基中加入呼吸链(电子传递链)抑制剂抗霉素A(antimycin A，anti-A)，并于加入抗霉素A(anti-A)后再进行三次取样。最后，检测各取样点的C2C12细胞的粒线体耗氧率(oxygen consumption rate，OCR)，结果示于图6A及图6B-1至图6B-3。
其中，加入寡霉素前(取样点0、9、18分钟)显示的OCR读值代表细胞在基础状态下的耗氧量(反映出细胞的基础呼吸能力)，包括粒线体氧化磷酸化及质子渗漏(proton leak)的耗氧；由于寡霉素会抑制ATP合成酶，加入寡霉素后(取样点27、36、45分钟)， 加入寡霉素后所减少的耗氧量就是加入寡霉素前细胞用在合成ATP的耗氧量，间接反映出基础状态下细胞的ATP产量；由于羰基-氰-对-三氟甲氧基苯肼(FCCP)是一种解偶联剂，可作为质子载体载运大量质子回流进入粒线体基质，而中和pH梯度，同时消耗大量氧气，但是这种质子回流不通过ATP合成酶，不会驱动ATP合成，故加入FCCP后(取样点54、63、72分钟)耗氧的增加代表粒线体的最大耗氧能力，间接反映出细胞的最大呼吸能力；加入抗霉素A后(取样点81、90、99分钟)，粒线体的呼吸链会被阻断，此时测得的数据即为背景值。
由图6A及图6B-1至图6B-3可知，TNF-α造成的C2C12损伤包括粒线体呼吸能力的下降。相较于未经管花肉苁蓉萃取物预处理的C2C12细胞(即，TNF-α组)，经10微克/毫升的管花肉苁蓉萃取物预处理的C2C12细胞(即，TNF-α+10CIS组)的粒线体呼吸能力较高。此结果显示管花肉苁蓉萃取物能有效改善TNF-α所引起的肌细胞粒线体呼吸能力下降，对肌细胞具有保护效果，可有效地抗肌细胞损伤。
实施例7：松果菊苷、类叶升麻苷、异类叶升麻苷于抗肌细胞损伤的效果
根据实施例1的结果，松果菊苷(echinacoside，Ech)、类叶升麻苷(verbascoside，VB)、异类叶升麻苷(isoacteoside，Iso)为管花肉苁蓉萃取物的主要成分之一。为探讨松果菊苷、类叶升麻苷、异类叶升麻苷是否具有保护肌肉的功效，以二甲基亚砜将松果菊苷、类叶升麻苷、异类叶升麻苷(都购自美国ChromaDex公司)配制成松果菊苷、类叶升麻苷、异类叶升麻苷溶液。另一方面，待C2C12细胞在H-DMEM培养基中细胞生长至80％汇合度后，将其分成十七组，并进行以下处理：
(1)控制组：以H-DMEM培养基进行培养，历时6小时。接着，将培养基更换为2％马血清的分化培养基，以进行培养，历时4天。
(2)TNF-α组：以H-DMEM培养基进行培养，历时6小时。接着，将培养基更换为2％马血清的分化培养基，并加入TNF-α(最终浓度为10纳克/毫升)进行共处理，历时4天。
(3)TNF-α+松果菊苷组(共五组)：于H-DMEM培养基中加入松果菊苷溶液，使其于培养基中的最终浓度为5、10、50、100或500微克/毫升，以进行预处理，历时6小时。接着，将培养基更换为2％马血清的分化培养基，并加入TNF-α(最终浓度为10纳克/毫升)进行共处理，历时4天。
(4)TNF-α+类叶升麻苷组(共五组)：于H-DMEM培养基中加入类叶升麻苷溶液，使其于培养基中的最终浓度为1、5、10、50或100微克/毫升，以进行预处理，历时6小时。接着，将培养基更换为2％马血清的分化培养基，并加入TNF-α(最终浓度为10纳克/毫升)进行共处理，历时4天。
(5)TNF-α+异类叶升麻苷组(共五组)：于H-DMEM培养基中加入异类叶升麻苷溶液，使其于培养基中的最终浓度为1、5、10、50或100微克/毫升，以进行预处理，历时6小时。接着，将培养基更换为2％马血清的分化培养基，并加入TNF-α(最终浓度为10纳克/毫升)进行共处理，历时4天。
其后，测试C2C12细胞的存活率、粒线体膜电位及细胞内活性氧水平，并以控制组的结果为基准，计算其他各组的相对的存活率、粒线体膜电位及细胞内的活性氧水平，以评估松果菊苷、类叶升麻苷、异类叶升麻苷对损伤的肌细胞所能提供的保护效果。结果示于图7A-1至图7A-3、图7B-1至图7B-3及图7C-1至图7C-3(数据均为平均数±SEM，n＝6，由t检验分析得到。###p＜0.001，*p＜0.05， **p＜0.01，***p＜0.001)。
由图7A-1至图7A-3可知，以浓度1至500微克/毫升的松果菊苷溶液预处理C2C12细胞，在较低浓度时保护效果并不显著，当浓度达100微克/毫升时，才具有显著性。但松果菊苷在粒线体膜电位及活性氧水平都不具保护作用。另一方面，由图7B-1至图7B-3可知，以浓度1至100微克/毫升的类叶升麻苷溶液预处理C2C12细胞，结果发现，类叶升麻苷并无法保护TNF-α对C2C12细胞所造成的损伤。
由图7C-1至图7C-3可知，以浓度5至100微克/毫升的异类叶升麻苷溶液预处理C2C12细胞，能够增加经TNF-α诱导损伤的C2C12细胞的存活率，表示异类叶升麻苷可有效降低TNF-α对C2C12细胞所造成的损伤。另一方面，以浓度100微克/毫升的异类叶升麻苷溶液预处理C2C12细胞，可明显改善TNF-α所引起的粒线体膜电位降低现象。再一方面，以10至100微克/毫升的异类叶升麻苷溶液预处理C2C12细胞，能使经TNF-α诱导损伤的C2C12细胞的细胞内活性氧水平明显降低。前述结果显示，异类叶升麻苷可有效降低TNF-α对肌细胞所造成的损伤，具有显著的保护作用，肉苁蓉萃取物中可保护肌肉的有效成分。
实施例8：管花肉苁蓉萃取物及其所含成分于保护肌肉的分子作用机制
待C2C12细胞在H-DMEM培养基中生长至80％汇合度后，将其分成四组(即，控制组、TNF-α组、TNF-α+10CIS组、TNF-α+50CIS组)，并以实施例5所述的方式处理至更换2％马血清的分化培养基(且依组别加入或不加入TNF-α)后，继续进行培养，历时4天。
其后，萃取各组细胞的蛋白质，并以西方墨点法检测mTOR/AMPK信息传导路径(与维持细胞内能量平衡有关)以及NFκB/p-JNK信息传导路径(与发炎反应、蛋白质降解有关)的相 关蛋白质的表现，结果示于图8A至图8D。
研究发现，TNF-α会引起的蛋白质降解，认为是TNF-α引起IκBα降解，活化NFκB，使其进入细胞核内与泛素蛋白酶体途径相关蛋白基因结合并促进其转录，从而使泛素蛋白酶体相关蛋白合成增加，进而促使肌肉蛋白大量降解。但由图8A结果得知，管花肉苁蓉萃取物对于炎症因子NFκB的抑制并不显著。
由图8B至图8D可知，TNF-α组的mTOR、AMPK、PGC-1α、MFN2及粒线体Complex I的蛋白质表现量下降；其中，粒线体Complex I是粒线体呼吸链上重要的酵素，PGC-1α是促进骨骼肌细胞粒线体合成和能量氧化代谢的转录因子，MFN2则与粒线体融合有关，MFN2和PGC-1α两者在维持粒线体膜电位和促进氧化磷酸化方面具有协同作用。；前述结果显示，TNF-α诱导的肌细胞损伤会影响mTOR/AMPK信息传导路径，破坏细胞能量代谢系统。
另一方面，相较于TNF-α组，经管花肉苁蓉萃取物预处理的C2C12细胞(即，TNF-α+10CIS组与TNF-α+50CIS组)的PGC-1α、MFN2及粒线体Complex I的表现量都明显提升，显示管花肉苁蓉萃取物能够启动肌细胞内的mTOR/AMPK信息传导路径，通过表现PGC-1α及MFN2等蛋白质稳定粒线体的数量与活性，同时修复细胞能量代谢系统受到的破坏(此功能类似于支链氨基酸)。因此，管花肉苁蓉萃取物及其所含成分对肌细胞的保护机制系与抗肌细胞自噬反应及促进肌细胞粒线体生成有关。
由以上实验结果可知，管花肉苁蓉萃取物及其所含的异类叶升麻苷都可有效保护损伤的肌细胞，使肌细胞的存活率上升，同时可抑制粒线体膜电位的降低及活性氧水平的上升，并维持细胞内的粒线体活性。因此，管花肉苁蓉萃取物及异类叶升麻苷对于损伤的肌细胞具有改善氧化压力(oxidative stress)与维持粒线体活性的良 好生物功效。此外，管花肉苁蓉萃取物能够重新启动损伤的肌细胞内的mTOR/AMPK信息传导路径，使得下游粒线体生合成相关蛋白(例如PGC-1α)、粒线体融合相关分子(例如MFN2)、参与呼吸链的粒线体(例如Complex I)的表现量回升，进而恢复肌细胞的能量代谢能力，此再次说明，管花肉苁蓉萃取物及其所含成分可有效抗肌细胞损伤，故可用于保护肌肉，具有治疗及/或延缓肌肉流失或萎缩的功效，并且有助于肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能(neuromuscular function)、维持能量正常代谢或增强能量。

Claims (15)

  1. 一种使用管花肉苁蓉萃取物于制备一药物或一食品的用途，其特征在于，该药物或食品用于保护肌肉。
  2. 如权利要求1所述的用途，其特征在于，该管花肉苁蓉萃取物为管花肉苁蓉的极性溶剂萃取物，该极性溶剂为选自水、C1-C4醇类及前述的组合中的任一种。
  3. 如权利要求1所述的用途，其特征在于，该管花肉苁蓉萃取物含有异类叶升麻苷。
  4. 如权利要求1所述的用途，其特征在于，该药物或食品是用于抗肌细胞损伤。
  5. 如权利要求1所述的用途，其特征在于，该药物是用于治疗及/或延缓以下的至少一者所引起的肌肉流失：老化、疾病及恶病质。
  6. 如权利要求1所述的用途，其特征在于，该食品是用于调节以下的至少一者所引起的肌肉流失：老化、疾病及恶病质。
  7. 如权利要求1所述的用途，其特征在于，该食品有助于肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能、维持能量正常代谢或增强能量。
  8. 如权利要求1所述的用途，其特征在于，该食品为保健食品、营养补充食品或特殊营养食品。
  9. 一种使用一活性成分于制备一药物或食品的用途，其特征在于，该活性成分为异类叶升麻苷及/或其医药上可接受的盐，且该药物或食品用于保护肌肉。
  10. 如权利要求项9所述的用途，其特征在于，该活性成分是以植物萃取物的形式使用。
  11. 如权利要求9所述的用途，其特征在于，该药物或食品是用于抗肌细胞损伤。
  12. 如权利要求9所述的用途，其特征在于，该药物是用于治 疗及/或延缓以下的至少一者所引起的肌肉流失：老化、疾病及恶病质。
  13. 如权利要求9所述的用途，其特征在于，该食品是用于调节以下的至少一者所引起的肌肉流失：老化、疾病及恶病质。
  14. 如权利要求9所述的用途，其特征在于，该食品有助于肌肉正常收缩、维持肌肉正常生理、维持神经肌肉正常功能、维持能量正常代谢或增强能量。
  15. 如权利要求9所述的用途，其特征在于，该食品为保健食品、营养补充食品或特殊营养食品。
PCT/CN2017/070862 2016-01-12 2017-01-11 管花肉苁蓉萃取物及异类叶升麻苷于保护肌肉的用途 WO2017121333A1 (zh)

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MYPI2018702407A MY194024A (en) 2016-01-12 2017-01-11 Use of cistanche tubulosa extract and isoacteoside in protection of muscles
JP2018533780A JP6887433B2 (ja) 2016-01-12 2017-01-11 筋肉の保護におけるカンカニクジュヨウ抽出物およびイソアクテオシドの使用
AU2017206332A AU2017206332B2 (en) 2016-01-12 2017-01-11 Use of Cistanche tubulosa extract and isoacteoside in protection of muscles
EP17738147.2A EP3403664B1 (en) 2016-01-12 2017-01-11 Use of cistanche tubulosa
KR1020187022614A KR102195547B1 (ko) 2016-01-12 2017-01-11 근육의 보호에서 시스탄케 투불로사 추출물 및 이소악테오사이드의 용도
CA3010907A CA3010907C (en) 2016-01-12 2017-01-11 Use of cistanche tubulosa extract and isoacteoside in protection of muscles

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EP3403664A1 (en) 2018-11-21
TWI678211B (zh) 2019-12-01
JP2019501912A (ja) 2019-01-24
AU2017206332A1 (en) 2018-07-26
CN106955297B (zh) 2021-01-12
AU2017206332B2 (en) 2019-07-25
JP6887433B2 (ja) 2021-06-16
CN106955297A (zh) 2017-07-18
US9931367B2 (en) 2018-04-03
KR20180101460A (ko) 2018-09-12
CA3010907C (en) 2023-03-14
EP3403664A4 (en) 2019-09-18
CA3010907A1 (en) 2017-07-20
US20170196926A1 (en) 2017-07-13

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