WO2016062125A1 - 还原型辅酶ⅱ的用途 - Google Patents

还原型辅酶ⅱ的用途 Download PDF

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WO2016062125A1
WO2016062125A1 PCT/CN2015/083890 CN2015083890W WO2016062125A1 WO 2016062125 A1 WO2016062125 A1 WO 2016062125A1 CN 2015083890 W CN2015083890 W CN 2015083890W WO 2016062125 A1 WO2016062125 A1 WO 2016062125A1
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fatigue
use according
mice
nadph
control group
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PCT/CN2015/083890
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French (fr)
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秦正红
孙玮
陈曹鑫
寇建群
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苏州人本药业有限公司
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    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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

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  • the present invention relates to the field of biotechnology, and in particular to the use of reduced coenzyme II.
  • Fatigue is a subjective discomfort, mainly characterized by fatigue and tiredness. It can be detected under long-term labor, strenuous exercise or prolonged mental stress. This is a normal physiological protective response, suggesting that the body should Restoring the function by rest, thus avoiding further damage to the body.
  • people often choose to work long hours regardless of fatigue.
  • the present invention provides the use of reduced coenzyme II.
  • NADPH has the advantages of prolonging the weight-bearing swimming time of middle-aged and old mice, increasing the liver glycogen reserve, reducing the content of lactic acid and urea nitrogen, and has the advantages of small dosage, safety, injectability, oral administration, etc., and has great clinical application value.
  • the present invention provides the following technical solutions:
  • the invention provides the use of reduced coenzyme II (NADPH) in the preparation of anti-fatigue drugs, health care products or foods.
  • NADPH reduced coenzyme II
  • the research of the present invention shows that the free radical theory of sports fatigue sports injury indicates that the generation of free radicals caused by exercise is an important factor causing fatigue of the body.
  • Reduced coenzyme II (NADPH) acts as a hydrogen donor in many chemical reactions in the body.
  • NADPH can treat ischemic stroke by anti-oxidation, and its antioxidant effect is mainly reflected in oxygen free radicals. Clear the effect. It was also found that NADPH can participate in mitochondrial energy supply and participate in hormone synthesis in the body, indicating that NADPH may have anti-fatigue effects.
  • Weight-bearing swimming is an important indicator for evaluating the body's exercise fatigue.
  • the length of swimming time can reflect the degree of animal fatigue.
  • the glycogen stored in the body is the main source of energy for the movement of mice. The vigorous movement of the body requires the consumption of a large amount of glycogen. Therefore, the glycogen reserve is also an important indicator for evaluating the body's anti-fatigue.
  • Lactic acid plays an important role in the body's energy supply system. It is the final product of glycolysis energy supply and an important oxygen metabolism matrix in the aerobic metabolic energy supply system, and lactic acid can pass the xenobiotic action of sugar in the liver. Converted to glucose for energy. However, if excessive lactic acid is produced in the body, the concentration of H+ in the muscle will rise.
  • BUN urea nitrogen
  • the anti-fatigue is to correct a sub-health state or to treat chronic fatigue syndrome.
  • the correcting the sub-health state is increasing hepatic glycogen content, increasing whole blood lactate content, or lowering plasma urea nitrogen content.
  • the medicament further comprises a pharmaceutically acceptable adjuvant.
  • the method of preparing the medicament a certain amount NADPH is soluble in physiological saline or deionized water and can be used alone or in combination with other active substances.
  • the pharmaceutical dosage form is an oral instant drug film, an oral liquid, a beverage, a tablet, a capsule, a spray, an injection, or a transdermal absorbent. It has been found that the anti-fatigue characteristics of reduced coenzyme II (NADPH) are not affected by the dosage form, and any pharmaceutically acceptable dosage form can achieve similar effects, which are all within the scope of the present invention, and the invention is not limited thereto. .
  • NADPH reduced coenzyme II
  • the medicament is administered in a manner of from 1 to 3 times a day, from 1 to 100 mg of reduced coenzyme II per kg of animal body weight.
  • the drug is administered by injection or orally. Any of the pharmaceutically acceptable modes of administration can achieve similar effects, and are within the scope of the present invention, and the present invention is not limited thereto.
  • the invention provides the use of reduced coenzyme II (NADPH) in the preparation of anti-fatigue drugs, health care products or foods.
  • NADPH reduced coenzyme II
  • the results showed that intraperitoneal administration of NADPH had a tendency to prolong the weight-bearing swimming time in mice, and this trend was dose-dependent.
  • the weight-bearing swimming time of mice after oral administration of NADPH was dose-dependently extended.
  • the weight-bearing swimming time of the mice in the model group was significantly decreased, and the weight-bearing swimming time of the mice after oral administration of NADPH was significantly longer than that of the model group.
  • the plasma urea nitrogen content of NADPH group was decreased compared with the control group.
  • the dose group and the control group were highly significant.
  • Oral administration was highly significant in the high-dose group and the control group (p ⁇ 0.05).
  • Oral administration was resistant to chronic fatigue.
  • the high-dose group of the syndrome (Fig. 4C) showed a downward trend compared with the model group, but it was not statistically significant.
  • NADPH can significantly prolong the weight-bearing swimming time of mice, increase the liver glycogen reserve of fatigued mice, and reduce the content of lactic acid and urea nitrogen in fatigue mice, suggesting that the present invention has obvious anti-fatigue effects.
  • Figure 1 shows the effect of NADPH on the weight-bearing swimming time in mice;
  • Figure 1A shows the results of the first batch of intraperitoneal injection experiments;
  • Figure 1B shows the results of the second batch of oral experiments, * indicates p ⁇ 0.05 compared with the control group;
  • Figure 1C shows the third Batch oral anti-chronic fatigue syndrome experimental results;
  • Figure 2 shows the effect of NADPH on liver glycogen reserve in mice
  • Figure 2A shows the results of the first batch of intraperitoneal injection experiments, * indicates p ⁇ 0.05, compared with the control group
  • Figure 2B shows the results of the second batch of oral experiments, * indicates p ⁇ 0.05, compared with the control group
  • Figure 2C is the third batch of oral anti-chronic fatigue syndrome experimental results, * indicates p ⁇ 0.05, compared with the model group;
  • Figure 3 shows the effect of NADPH on whole blood lactate levels in mice
  • Figure 3A shows the results of the first batch of intraperitoneal injection experiments, * indicates p ⁇ 0.05, compared with the control group
  • Figure 3B shows the results of the second batch of oral experiments
  • Figure 3C is The third batch of oral anti-chronic fatigue syndrome experimental results, ** said p ⁇ 0.01, compared with the model group;
  • Figure 4 shows the effect of NADPH on plasma urea nitrogen levels in mice;
  • A is the first batch of intraperitoneal injection test results, * indicates p ⁇ 0.05, compared with control group;
  • B is the second batch of oral test results, * indicates p ⁇ 0.05 Compared with the control group;
  • C is the third batch of oral anti-chronic fatigue syndrome experimental results.
  • the invention discloses the use of reduced coenzyme II, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention.
  • the technique of the present invention is applied.
  • the original drug, the auxiliary material, and the preparation used in the use of the reduced coenzyme II provided by the present invention are commercially available.
  • A. First batch 60 male 5-month-old middle-aged ICR mice were randomly divided into 4 groups, 15 in each group.
  • the low-dose group was intraperitoneally administered with 2.5 mg/kg of the drug, the medium-dose intraperitoneal injection of 5 mg/kg of the drug, and the high-dose intraperitoneal injection of 10 mg/kg of the drug.
  • the Control group was given the same volume of physiological saline. It is administered once every day from 10:00 to 11:00 and continuously for 7 days.
  • Second batch 100 male 8-month-old aged C57 mice were randomly divided into 4 groups, 20 in each group.
  • the low-dose group was intragastrically administered with 2.5 mg/kg of the drug, the medium-dose was administered with 5 mg/kg of the drug, and the high-dose was administered with the drug of 10 mg/kg.
  • the Control group was given the same volume of water. Yu It is administered once a day from 10:00 to 11:00 and continuously for 7 days.
  • mice After the last administration of the drug for 30 minutes, the tail of the mouse was given 4% by mass of its body weight.
  • the mice were placed in a swimming pool with a water depth of 35 cm and a water temperature of 21 ⁇ 1 ° C until they were exhausted.
  • the criterion for exhaustion is that the head of the mouse does not rise to the surface within 10 s of sinking into the water, and the exhaustion time is recorded.
  • mice were loaded with weight-bearing swimming, they were rested for 10 minutes, and the mice were sacrificed after the eyeballs were collected.
  • the liver was taken out, rinsed with physiological saline, and then blotted dry with a filter paper, and 50 mg of liver was accurately weighed. Liver liver glycogen was measured according to the kit instructions.
  • mice After the mice were loaded with weight-bearing swimming, rest for 10 minutes, remove the eyeballs and take blood, and measure the whole blood lactic acid according to the kit instructions.
  • mice After the mice were loaded with weight-bearing swimming, the eyes were rested for 10 minutes, blood was collected from the eyeballs, and after centrifugation at 3500 rpm for 10 minutes with anticoagulation of EDTA sodium salt, plasma was separated, and plasma urea nitrogen was measured according to the kit instructions.
  • Figure 1 shows the effect of NADPH on the weight-bearing swimming time in mice.
  • A is the first batch of intraperitoneal injection test results;
  • B is the second batch of oral test results, * indicates p ⁇ 0.05 compared with the control group;
  • C is the third batch of oral anti-chronic fatigue syndrome experimental results.
  • Figure 1A can be seen that intraperitoneal injection of NADPH has a tendency to prolong the weight-bearing swimming time of mice, and this trend is dose-dependent; as can be seen from Figure 1B, compared with the control group, the weight-bearing swimming time of mice after oral administration of NADPH is presented. The dose-dependent prolongation was significantly different between the middle and high dose groups and the control group by analysis of variance (p ⁇ 0.05).
  • Fig. 1C compared with the control group, the weight-bearing swimming time of the mice in the model group was significantly decreased, and the weight-bearing swimming time of the mice after oral administration of NADPH was significantly longer than that of the model group.
  • Figure 2 shows the effect of NADPH on liver glycogen stores in mice, where A is the first batch of intraperitoneal injection results, * indicates p ⁇ 0.05, compared with the control group; B is the second batch of oral results, * indicates p ⁇ 0.05 Compared with the control group; C is the third batch of oral anti-chronic fatigue syndrome experimental results, * indicates p ⁇ 0.05, compared with the model group.
  • A is the first batch of intraperitoneal injection results, * indicates p ⁇ 0.05, compared with the control group
  • B is the second batch of oral results, * indicates p ⁇ 0.05
  • C is the third batch of oral anti-chronic fatigue syndrome experimental results, * indicates p ⁇ 0.05, compared with the model group.
  • the liver glycogen content of the mice increased significantly, wherein the difference between the dose group and the control group was significant.
  • 2A the high-dose group of oral administration was significantly different from the control group (Fig. 2B), which was statistically significant (p ⁇ 0.05).
  • Figure 3 shows the effect of NADPH on whole blood lactate levels in mice.
  • A is the first batch of intraperitoneal injection test results, * indicates p ⁇ 0.05, compared with the control group;
  • B is the second batch of oral test results,
  • C is the third batch of oral anti-chronic fatigue syndrome experimental results, ** indicates p ⁇ 0.01, compared with the model group.
  • Fig. 3A after administration of NADPH, the lactic acid content of the whole blood was significantly decreased compared with the control group, and the medium dose and the large dose were the most significant (p ⁇ 0.05); as can be seen from Fig. 3B, compared with the control group The lactic acid content of whole blood decreased after oral administration of NADPH, but it was not statistically significant (p>0.05).
  • Fig. 3C compared with the model group, the lactic acid content of the whole blood of the mice after oral administration of NADPH was significantly prolonged, which was statistically significant (p ⁇ 0.01).
  • Figure 4 shows the effect of NADPH on plasma urea nitrogen levels in mice.
  • A is the first batch of intraperitoneal injection test results, * indicates p ⁇ 0.05, compared with the control group;
  • B is the second batch of oral test results, * indicates p ⁇ 0.05, compared with the control group;
  • C is the third batch of oral anti-chronic Fatigue syndrome experimental results.
  • the plasma urea nitrogen content of each dose group of NADPH decreased compared with the control group.
  • the dose group and the control group were highly significant.
  • Oral administration (Fig. 4B) was highly significant in the high-dose group and the control group (p ⁇ 0.05).
  • Oral administration was resistant to chronic fatigue.
  • the high-dose group of the syndrome (Fig. 4C) showed a downward trend compared with the model group, but it was not statistically significant.
  • NADPH can significantly prolong the weight-bearing swimming time of mice, increase the liver glycogen reserve of fatigued mice, and reduce the content of lactic acid and urea nitrogen in fatigue mice, suggesting that the present invention has obvious anti-fatigue effects.

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Abstract

一种抗疲劳的制剂、保健品或食品，以还原型辅酶II为活性成分，试验表明，能够明显延长小鼠负重游泳时间、提高疲劳小鼠肝糖原储备、降低疲劳小鼠乳酸和尿素氮含量。

Description

还原型辅酶Ⅱ的用途 技术领域
本发明涉及生物技术领域，特别涉及还原型辅酶Ⅱ的用途。
背景技术
疲劳是一种主观的不适感，主要表现为疲劳感、倦怠感，在进行长时间劳动、剧烈运动或长时间精神压力下会察觉到，这是一种正常的生理性保护反应，提示机体应该通过休息使机能恢复，从而避免对身体的进一步伤害。然而随着社会竞争的加剧，生活节奏的加快，使人们常常不顾疲劳，仍然选择长时间工作。长期处于疲劳状态，会损害人体的体力、体能，身体失衡，使人难以从事或完成某些消耗体力较大或动作细腻、精巧的工作，从而使工作效率下降；另一方面，长期疲劳还会使人皮肤松弛，面色无华，呈现出未老先衰的征兆；长期疲劳得不到恢复，使人体免疫系统功能失调，甚至造成免疫低下，机体抵抗疾病的屏障被打破，使患者患病的几率增加。另外，长期心理压力、激素紊乱、免疫异常等，均是造成慢性疲劳综合征的主要诱因。超负荷的工作、过度的压力与不规律的生活作息使人们常常感觉到疲劳感、倦怠感长时间难以恢复，使人们长期处于亚健康状态，人群中约有70％的人处于这一状态，其中又以中老年人高发。
基于此，需要找到抗疲劳物质来延缓长时间劳动、剧烈运动或过度精神压力下造成的疲劳发生，或使疲劳状态尽快恢复到正常状态，以期使人们亚健康状态恢复正常或对疲劳综合征产生疗效。现有的抗疲劳药物主要以中药为主，特点是注重养生，消费者需长期服用，起效慢。
因此，提供一种新的抗疲劳药物具有现实意义。
发明内容
有鉴于此，本发明提供还原型辅酶Ⅱ的用途。试验结果表明， NADPH具有延长中年与老年小鼠负重游泳时间，增加肝糖原储备，减少乳酸、尿素氮含量，具有用量小、安全、可注射、可口服等优点，具有较大的临床应用价值。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了还原型辅酶Ⅱ(NADPH)在制备抗疲劳药物、保健品或食品中的应用。
本发明研究显示，运动疲劳运动损伤的自由基学说指出，运动引起的自由基的产生是引起机体疲劳的重要因素。还原型辅酶Ⅱ(NADPH)在体内许多化学反应中起递氢体的作用，研究结果显示NADPH可通过抗氧化作用来治疗缺血性脑中风，其抗氧化作用主要体现在对含氧自由基的清除作用。另外还发现NADPH可参与线粒体供能、参与体内激素合成的功能，表明NADPH可能具有抗疲劳作用。
负重游泳是评价机体运动性疲劳的重要指标，游泳时间的长短可以反映动物运动疲劳的程度。体内储备的糖原是小鼠运动的主要能量来源，机体剧烈运动需要消耗大量糖原，因此糖原储备也是评价机体抗疲劳的一个重要指标。乳酸在机体供能体系中占有重要的地位，它是糖酵解供能的最终产物，又是有氧代谢供能系统中的重要氧代谢基质，并且乳酸可在肝脏内通过糖的异生作用转变为葡萄糖供能。但是如果机体内产生过量的乳酸.会使肌肉中H+浓度上升.pH下降，进而引起一系列生化变化，就会引起疲劳。研究显示，尿素氮(BUN)的增加与机体对负荷适应能力呈负相关，机体BUN含量随运动负荷的增加而增加，所以BUN是较为理想、灵敏的疲劳指标。
在本发明的一些实施例中，所述抗疲劳为纠正亚健康状态或治疗慢性疲劳综合症。
在本发明的一些实施例中，所述纠正亚健康状态为增加肝糖原含量、增加全血乳酸含量或降低血浆尿素氮含量。
作为优选，本发明提供的还原型辅酶Ⅱ制备抗疲劳药物的用途中，所述药物还包括药学上可接受的辅料。
在本发明的一些实施例中，所述药物的制备方法：将一定量 NADPH溶于生理盐水或去离子水中，可以单独应用或与其它活性物质组成复合制剂。
在本发明的一些实施例中，所述药物的剂型为口腔速溶药膜、口服液、饮料、片剂、胶囊、喷雾剂、注射剂或透皮吸收剂。经试验发现，还原型辅酶Ⅱ(NADPH)抗疲劳的特点不受剂型的影响，任何药物可接受的剂型均可达到相近的效果，均在本发明的保护范围之内，本发明在此不作限定。
在本发明的一些实施例中，所述药物的给药方式为每日1～3次，每次1～100mg还原型辅酶Ⅱ/kg动物体重。
在本发明的一些实施例中，所述药物的给药方式为注射或口服。任何药物可接受的给药方式均可达到相近的效果，均在本发明的保护范围之内，本发明在此不作限定。
本发明提供了还原型辅酶Ⅱ(NADPH)在制备抗疲劳药物、保健品或食品中的应用。试验表明，腹腔注射给予NADPH具有延长小鼠负重游泳时间的趋势，且此趋势呈剂量依赖性；与control组相比，口服给予NADPH后小鼠负重游泳时间呈现剂量依赖性延长，经方差分析，中、大剂量组与control对照组相比有显著性差异(p<0.05)。与control组相比，模型组的小鼠负重游泳时间明显下降，而口服给予NADPH后小鼠负重游泳时间与模型组相比有明显延长。
与control组相比，不论是注射或是口服给予NADPH，小鼠肝糖原含量均显著增加，其中，注射给药中剂量组与对照组差异达显著性水平(图2A)，口服给药大剂量组与对照组差异达显著水平(图2B)，具有统计学意义(p<0.05)，在抗慢性疲劳综合症模型实验中，口服给药小剂量组与模型组差异达显著水平(图2C)，具有统计学意义(p<0.05)。
注射给予NADPH后，与control组比较，全血乳酸含量明显下降，以中剂量和大剂量最为显著(p<0.05)；与control组相比，口服给予NADPH后全血乳酸含量均有所下降，但无统计学意义(p>0.05)。与model组相比，口服给予NADPH后小鼠全血乳酸含量有明显延长， 有统计学显著意义(p<0.01)。
NADPH各剂量组血浆尿素氮含量较control组相比均有所下降。经方差分析注射给药(图4A)中剂量组与对照组有高度显著性，口服给药(图4B)大剂量组与对照组有高度显著性(p<0.05)，口服给药抗慢性疲劳综合症(图4C)大剂量组与模型组相比有下降趋势，但无统计学意义。
综合上述实验结果，NADPH能明显延长小鼠负重游泳时间、提高疲劳小鼠肝糖原储备、降低疲劳小鼠乳酸和尿素氮含量，提示本发明具有明显的抗疲劳作用。
NADPH抗疲劳综合症的治疗获得类似效果。
附图说明
图1示NADPH对小鼠负重游泳时间的影响；其中图1A为第一批腹腔注射实验结果；图1B为第二批口服实验结果，*表示p<0.05与control组比较；图1C为第三批口服抗慢性疲劳综合症实验结果；
图2表示NADPH对小鼠肝糖原储备的影响；其中图2A为第一批腹腔注射实验结果，*表示p<0.05，与control组比较；图2B为第二批口服实验结果，*表示p<0.05，与control组比较；图2C为第三批口服抗慢性疲劳综合症实验结果，*表示p<0.05，与model组比较；
图3表示NADPH对小鼠全血乳酸水平的影响；其中图3A为第一批腹腔注射实验结果，*表示p<0.05，与control组比较；图3B为第二批口服实验结果，图3C为第三批口服抗慢性疲劳综合症实验结果，**表示p<0.01，与model组比较；
图4表示NADPH对小鼠血浆尿素氮水平的影响；其中A为第一批腹腔注射实验结果，*表示p<0.05，与control组比较；B为第二批口服实验结果，*表示p<0.05，与control组比较；C为第三批口服抗慢性疲劳综合症实验结果。
具体实施方式
本发明公开了还原型辅酶Ⅱ的用途，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的还原型辅酶Ⅱ的用途中所用原药及辅料、制剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
1材料与方法
1.1样品：
1.2实验动物：五月龄中年雄性ICR小鼠；八月龄老年雄性C57小鼠。
1.3仪器与试剂：游泳箱、铅丝、全波长酶标仪、恒温水浴箱、离心机、肝糖原检测试剂盒(南京建成，货号A043)、乳酸检测试剂盒(南京建成，货号A019-1)、尿素氮试剂盒(南京建成，货号C013-1)、SOD检测试剂盒(南京建成，货号A001-1)、MDA检测试剂盒(南京建成，货号A003-1)。
1.4实验方法：
A.第一批：将60只雄性五月龄中年ICR小鼠随机分为4组，每组15只。小剂量组腹腔注射给药2.5mg/kg的药品，中剂量腹腔注射给药5mg/kg的药品，大剂量腹腔注射给药10mg/kg的药品。Control组给予同体积的生理盐水。于每天上午10:00至11:00给药一次，连续给药7天。
B.第二批：将100只雄性八月龄老年C57小鼠随机分为4组，每组20只。小剂量组灌胃2.5mg/kg的药品，中剂量灌胃5mg/kg的药品，大剂量灌胃10mg/kg的药品。Control组给予同体积的水。于 每天上午10:00至11:00给药一次，连续给药7天。
C.第三批：将80只雄性五月龄中年ICR小鼠随机分为4组，每组20只。Control组正常环境下饲养14天，其余三组进行慢性疲劳综合症造模：每天上午9:00至11:00进行束缚，每次一小时，每天下午将小鼠放入水深为35cm，水温为21±1℃的游泳箱内强制游泳1小时，连续造模14天。从第15天开始给药，小剂量灌胃5mg/kg的药品，大剂量灌胃10mg/kg的药品。Control组给予同体积的水。于每天上午10:00至11:00给药一次，连续给药7天。
1.4.1负重游泳实验
末次给予药物30min后，给小鼠尾部负其体重的4％质量的铅丝。将小鼠放入水深为35cm，水温为21±1℃的游泳箱内游泳，直到力竭。力竭的判定标准为小鼠头部沉入水中10s内不浮出水面，记录力竭时间。
1.4.2肝糖原测定
小鼠负重游泳后，休息10分钟，在摘眼球采血后将小鼠处死。取出肝脏，用生理盐水漂洗后再用滤纸吸干，精确称取50mg肝脏。按照试剂盒说明书进行肝脏肝糖原的测定。
1.4.3全血乳酸测定
小鼠负重游泳后，休息10分钟，摘除眼球取血，按照试剂盒说明进行全血乳酸的测定。
1.4.4血浆指标测定
小鼠负重游泳后，休息10分钟，眼球采血，用EDTA钠盐抗凝后3500转离心10分钟后分离出血浆，按照试剂盒说明书进行血浆尿素氮的测定。
2结果
2.1小鼠负重游泳时间测定
图1表示NADPH对小鼠负重游泳时间的影响。其中A为第一批腹腔注射实验结果；B为第二批口服实验结果，*表示p<0.05与control组比较；C为第三批口服抗慢性疲劳综合症实验结果。由图 1A可以看出，腹腔注射给予NADPH具有延长小鼠负重游泳时间的趋势，且此趋势呈剂量依赖性；由图1B可以看出，与control组相比，口服给予NADPH后小鼠负重游泳时间呈现剂量依赖性延长，经方差分析，中、大剂量组与control对照组相比有显著性差异(p<0.05)。由图1C可以看出，与control组相比，模型组的小鼠负重游泳时间明显下降，而口服给予NADPH后小鼠负重游泳时间与模型组相比有明显延长。
2.2小鼠肝糖原测定
图2表示NADPH对小鼠肝糖原储备的影响，其中A为第一批腹腔注射实验结果，*表示p<0.05，与control组比较；B为第二批口服实验结果，*表示p<0.05，与control组比较；C为第三批口服抗慢性疲劳综合症实验结果，*表示p<0.05，与model组比较。由图2可以看出，与control组相比，不论是注射或是口服给予NADPH，小鼠肝糖原含量均显著增加，其中，注射给药中剂量组与对照组差异达显著性水平(图2A)，口服给药大剂量组与对照组差异达显著水平(图2B)，具有统计学意义(p<0.05)，在抗慢性疲劳综合症模型实验中，口服给药小剂量组与模型组差异达显著水平(图2C)，具有统计学意义(p<0.05)。
2.3小鼠全血乳酸测定
图3表示NADPH对小鼠全血乳酸水平的影响。其中A为第一批腹腔注射实验结果，*表示p<0.05，与control组比较；B为第二批口服实验结果，C为第三批口服抗慢性疲劳综合症实验结果，**表示p<0.01，与model组比较。由图3A可以看出，注射给予NADPH后，与control组比较，全血乳酸含量明显下降，以中剂量和大剂量最为显著(p<0.05)；由图3B可以看出，与control组相比，口服给予NADPH后全血乳酸含量均有所下降，但无统计学意义(p>0.05)。由图3C可以看出，与model组相比，口服给予NADPH后小鼠全血乳酸含量有明显延长，有统计学显著意义(p<0.01)。
2.4小鼠血浆尿素氮含量测定
图4表示NADPH对小鼠血浆尿素氮水平的影响。其中A为第一批腹腔注射实验结果，*表示p<0.05，与control组比较；B为第二批口服实验结果，*表示p<0.05，与control组比较；C为第三批口服抗慢性疲劳综合症实验结果。由图4可以看出，NADPH各剂量组血浆尿素氮含量较control组相比均有所下降。经方差分析注射给药(图4A)中剂量组与对照组有高度显著性，口服给药(图4B)大剂量组与对照组有高度显著性(p<0.05)，口服给药抗慢性疲劳综合症(图4C)大剂量组与模型组相比有下降趋势，但无统计学意义。
综合上述实验结果，NADPH能明显延长小鼠负重游泳时间、提高疲劳小鼠肝糖原储备、降低疲劳小鼠乳酸和尿素氮含量，提示本发明具有明显的抗疲劳作用。
NADPH抗疲劳综合症的治疗获得类似效果。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 还原型辅酶Ⅱ在制备抗疲劳药物、保健品或食品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述抗疲劳为纠正亚健康状态或治疗慢性疲劳综合症。
  3. 根据权利要求2所述的应用，其特征在于，所述纠正亚健康状态为增加肝糖原含量、增加全血乳酸含量或降低血浆尿素氮含量。
  4. 根据权利要求1所述的应用，其特征在于，所述药物还包括药学上可接受的辅料。
  5. 根据权利要求1所述的应用，其特征在于，所述药物的剂型为口腔速溶药膜、口服液、、片剂、胶囊、喷雾剂、注射剂或透皮吸收剂。
  6. 根据权利要求1所述的应用，其特征在于，所述药物的给药方式为每日1～3次，每次1～100mg还原型辅酶Ⅱ/kg动物体重。
  7. 根据权利要求6所述的应用，其特征在于，所述药物的给药方式为注射或口服。
  8. 根据权利要求1所述的应用，其特征在于，所述保健品或食品为饮料。
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