WO2009000168A1 - An antrodia camphorata cyclohexenone compound used for decreasing physiological fatigue - Google Patents

Abstract

Antrodia camphorata cyclohexenone compounds used for decreasing physiological fatigue, in particular 4-hydroxy-2,3-dimethoxy-6-methy-5[3,7,11-trimethy- dodeca-2,6,10-trienyl]-cyclohex-2-enone isolated from extract of Antrodia camphorata, are capable of decreasing physiological fatigue effectively. In the present invention, administration of said compounds immediately after highly intensive prostrate physical exercises with 80% maximal oxygen uptake can increase the metabolism of creatine kinase and blood ammonia and militate to recover their concentration, thus to achieve the effect of decreasing physiological fatigue.

Description

 Anthraquinone cyclohexenone compound for relieving physiological fatigue

Technical field

 The present invention relates to a compound for use in anti-fatigue, and more particularly to an anthraquinone

(Antrodia camphorata) extracts are purified and prepared to reduce physiological fatigue of cyclohexenone compounds. Background technique

 Antrodia camphorata (also known as Antrodia camphorata, burdock or red locust, etc.) is a perennial fungus belonging to the family Polyporaceae of Aphyllophorales. It is a endemic fungus of Taiwan and only grows in The inner wall of the hollow decayed heartwood of Taiwan's conservation tree species, Cinnamoum kanehirai Hay. Due to the extremely rare distribution of burdock trees, and the artificial piracy, the number of wild burdocks that can grow in it is even rarer. Because the fruiting bodies grow quite slowly, the growth period is only between June and October, so the price is very expensive.

 The fruit body of Antrodia camphorata is perennial, sessile, with cork to wood. It has a strong aroma of eucalyptus, and its morphology varies, with plate, bell, horseshoe or tower. It is flat and bright red at the beginning of life, and then it radiates and rewinds around it, and grows to the surrounding area. The color also changes to reddish brown or yellowish brown, and has many fine pores. It is also a medicinal herb of Antrodia camphorata. The most valuable part.

 In Taiwanese folk medicine, Niobium has the functions of detoxification, alleviating diarrhea, anti-inflammatory, treating liver-related diseases and anti-cancer. Antrodia camphorata has many complex ingredients, such as triterpenoids, polysaccharides (such as β-D-glucan), and glands. Adenosine, vitamins (such as vitamin B, nicotinic acid), proteins (including immunoglobulins), superoxide dismutase (SOD), trace elements (eg calcium, phosphorus, wrong), nucleic acids, solid Alcohols and blood pressure stabilizing substances (such as antodia acid), these physiologically active ingredients are considered to have anti-tumor, immunity, anti-allergy, anti-bacteria, anti-hypertensive, hypoglycemic, cholesterol-lowering, liver protection and anti-fatigue Kind of effect.

Among the many components of Antrodia camphorata, triterpenoids are the most studied. Triterpenoids are a general term for the combination of thirty carbon elements into hexagonal or pentagonal natural compounds. The bitterness of Antrodia camphorata is mainly derived from triterpenoids. In 1995, Cherng et al. discovered the extract of Antrodia camphorata fruit body. Contains three new triterpenoids based on ergostane: antcin A, antcin B and antcin C (Chemg, IH, and Chiang, HC 1995. Three new triterpenoids from Antrodia cinnamomea. J. Nat. Prod. 58:365-371). Chen et al. extracted three kinds of triterpenoids such as zhankuic acid A, zhankuic acid B and zhankuic acid C after extracting the fruit body of Antrodia camphorata by ethanol (Chen, C. Η·, and Yang, SW 1995. New steroid acids from Antrodia cinnamomea) , - a fungus parasitic on Cinnamomum micranthum. J. Nat. Prod. 58: 1655-1661 ). In addition, in 1995, Chiang et al. also found three other new triterpenoids, sesquiterpene lactone and two bisphenol derivatives, from the fruit extract, which is antrocin, 4, 7-dimethoxy-5-indenyl-1,3-benzodioxane

(4,7-dimethoxy-5-methy-l,3-benzodioxole) with 2,2',5,5'-tetradecyloxy-3,4,3',4'-bis-arylenedioxy -6,6'-dimercaptobiphenyl (2,2',5,5'-teramethoxy-3,4,3 ^, ,4 ^, -bi- methylenedioxy-6,6'- dimethylbiphenyl ) (Chiang, H. C, Wu, DP, Chemg, I. W" and Ueng, CH 1995. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. Phytochemistry. 39: 613-616). By 1996, Chemg et al. used the same analytical method. Four new triterpenoids were discovered again: antcin E, antcin F, methyl antcinate G, methyl antcinate H (Chemg, IH, Wu, DP, and Chiang, HC 1996. Triteroenoids from Antrodia cinnamomea. Phytochemistry. 41 :263- 267 ); and Yang et al. discovered two new compounds, zhankuic acid D, zhankuic acid E, and three new compounds based on lanostane: 15 α - - argon-colored sulphuric acid (15 a-acetyl-dehydrosulphurenic acid), de-argon-doped acid

( dehydroeburicoic acid ) and dehydrated stone acid ( dehydrasulphurenic acid X Yang, SW, Shen, Y. C, and Chen, CH 1996. Steroids and triterpenoids of Antrodia cinnamomea - a fungus parasitic on Cinnamomum micranthum. Phytochemistry. 41 : 1389-1392 ).

Although it is known from many experiments at present that the extract of Antrodia camphorata has the aforementioned effects, and the components thereof are gradually analyzed, what kind of active ingredients in the extract can contribute to the effects of Antrodia camphorata, and there is no specific correlation. The active ingredients are published, and the anti-fatigue ingredients contained in the extract of Antrodia camphorata are still to be further studied and studied, so if we can find out that the extract contains The anti-fatigue ingredients will help to study the mechanism of anti-fatigue of Antrodia camphorata, and it will greatly help the application of Niobium in the human body to slow down the physiological fatigue. Summary of the invention

 In order to clarify which component of the extract of Antrodia camphorata has anti-fatigue effect, the present invention separates and purifies a compound of the formula (1) from the extract of Burdock;

Wherein X is oxygen (0) or sulfur (S), Y is oxygen or sulfur; is hydrogen (H), sulfhydryl (CH ₃ ) or (CH ₂ )m-CH ₃ , and R ₂ is hydrogen, sulfhydryl or (CH ₂ )m-CH ₃ , R ₃ is hydrogen, fluorenyl or (CH ₂ )m-CH ₃ , m = 1-12; n = 1-12.

 Among the compounds of the formula (1), a compound represented by the following formula (2) is preferred:

a compound of formula (2), chemical name 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene) 4-hydroxy-2,3-dimethoxy-6-methy-5 (3,7,11-trimethyl-dodeca-2,6, 10-trienyl)-cyclohex-2-enone ) , the molecular formula is C ₂₄ H ₃ 80 ₄ , the appearance is light yellow powder, and the molecular weight is 390.

 The cyclohexenone compound of the formula (1) and the formula (2) in the present invention is isolated and purified from an aqueous extract or an organic solvent extract of Antrodia camphorata, and the organic solvent may include an alcohol (for example, decyl alcohol, ethanol or propanol), an ester. (e.g., ethyl acetate), an alkane (e.g., hexane) or an alkylene (e.g., chlorodecane, ethyl chloride), but not limited thereto, preferably an alcohol, more preferably ethanol.

By the cyclohexenone compound of the above formula (1), formula (2), the present invention applies the compound to the reduction In the physiological fatigue, immediately after the high-intensity exhaustion exercise of 80% maximal oxygen uptake (80% V0 ₂ max), the immediate addition of the anthraquinone cyclohexenone compound contributes to the in vivo creatine phosphate kinase (CPK) and blood. The metabolism of ammonia (Ammonia) and the recovery of its concentration slow down muscle cell damage caused by exercise, and improve the central fatigue and peripheral fatigue caused by blood ammonia accumulation in the blood, thereby achieving anti-fatigue effect.

 The embodiments of the present invention will be further described in conjunction with the accompanying drawings. The embodiments of the present invention are set forth to illustrate and not to limit the scope of the present invention. In the meantime, the scope of the invention is defined by the appended claims. DRAWINGS

 1 is a result of in vivo creatine kinase concentration at each time point after supplementation with a placebo or anthraquinone cyclohexenone compound in a subject with or without exercise in the embodiment of the present invention; Placebo (PR); er: exercise and placebo (PE); Δ; no exercise and administration of anthraquinone cyclohexenone compound (DR); A: exercise and administration of anthraquinone cyclohexenone compound (DE);

2 is a result of in vivo blood glucose concentration at each time point after supplementation with a placebo or anthraquinone cyclohexenone compound after performing a depletion exercise or no exercise in the embodiment of the present invention; the mouth of the figure: no exercise and a placebo (PR); Country: exercise with placebo (PE); Δ; no exercise and administration of anthraquinone cyclohexenone compound (DR); A: exercise and administration of anthraquinone cyclohexenone compound (DE); In the case of the subject of the present invention, after performing a depletion exercise or no exercise, the placebo or the burdock cyclohexenone compound was respectively added to the blood ammonia concentration result at each time point; the mouth of the figure: no exercise and a placebo ( PR); Country: exercise and placebo (PE); Δ; no exercise and administration of anthraquinone cyclohexenone compound (DR); A: exercise and administration of anthraquinone cyclohexenone compound (DE); In the embodiment of the present invention, after the subject undergoes exhaustive exercise or no exercise, the placebo or the burdock cyclohexenone compound is respectively added to the blood lactate concentration result at each time point; the mouth is: no exercise and a placebo (PR) ); Country: Yes And be given a placebo (PE); Δ; no motion and give a cyclohexenone compound of Antrodia (DR); A: sports and administering a cyclohexenone compound of Antrodia (DE); Figure 5 is a graph showing the results of in vivo free fatty acid concentration at various time points after supplementation with placebo or burdock cyclohexenone compound in a subject with or without exercise in the embodiment of the present invention; Agent (PR); Country: exercise and placebo (PE); Δ; no exercise and administration of anthraquinone cyclohexenone compound (DR); A: exercise and administration of anthraquinone cyclohexenone compound (DE). detailed description

 First, Antrodia camphorata mycelium, fruiting body or a mixture of the two is taken, and extracted by water or an organic solvent by a known extraction method to obtain an aqueous extract of Antrodia camphorata or an organic solvent extract. Wherein, the organic solvent may include an alcohol (such as decyl alcohol, ethanol or propanol), an ester (such as ethyl acetate), an alkane (such as hexane) or an alkyl halide (such as chlorodecane, ethyl chloride), but Not limited to this. Among them, preferred are alcohols, and more preferably ethanol.

 The extracted aqueous extract of Antrodia camphorata or the organic solvent extract can be further separated and purified by high performance liquid chromatography, and then each fraction is subjected to a fatigue-resistant biochemical test. Finally, component analysis is performed on the liquid separation with anti-fatigue effect, and the components that may have anti-fatigue effects are further subjected to biochemical tests related to fatigue resistance. Finally, it was found that the compound of the formula (1) / formula (2) of the present invention has an effect of alleviating physiological fatigue.

For convenience of description of the present invention, 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-) of the formula (2) will be exemplified below. The dodecanetriene)-2-cyclohexenone compound will be described. Further, in order to confirm 4-hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-tridecyl-2,6,10-dodecatriene)-2-ring cyclohexenone compound having an anti-fatigue effect of the present invention, by detecting over 80% of the maximal oxygen uptake (80% V0 ₂ max) creatine subject after exercise kinase failure, blood lactate, glucose, free ammonia and A fatigue index such as a fatty acid is used to measure the fatigue resistance of the anthraquinone cyclohexenone compound. From these biochemical test results, 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene) was confirmed. The 2-cyclohexenone compound has the effect of slowing down physiological fatigue caused by exercise. The foregoing embodiment is described in detail as follows:

Example 1

4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexanone Separation Put about 100 grams of Astragalus membranaceus mycelium, fruiting body or a mixture of the two into a triangular conical flask, add appropriate proportion of water and alcohol (for example, more than 70% aqueous alcohol solution), at 20-25 ° The mixture was stirred and extracted at C for at least 1 hour, and then filtered through a filter paper and a 0.45 μηι filter to collect the extract.

 The collected Antrodia camphorata extract was analyzed by high performance liquid chromatography using a column of RP18, and decyl alcohol (A) and 0.1%-0.5% aqueous acetic acid solution (B). As the mobile phase (the ratio of the solution is: 0~10 minutes, B ratio is 95%~20%; 10~20 minutes, B ratio is 20%~10%; 20-35 minutes, B ratio is 10 %~10%; 35-40 minutes, B ratio is 10%~95%), eluted at a rate of 1 ml per minute, and analyzed by UV-visible full wavelength detector.

The eluate from 25 minutes to 30 minutes is collected and concentrated to obtain a pale yellow powdery solid product, which is 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11) - Tridecyl-2,6,10-dodecatriene)-2-cyclohexenone. After analysis, the molecular formula is C ₂₄ H ₃₈ 0 ₄ , the molecular weight is 390, and the melting point (mp ) is 48 ° C - 52 ° C. The nuclear magnetic resonance (NMR) analysis values are as follows: lH-NMR (CDC13) 5 (ppm): 1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.68, 4.05, 5.07 and 5.14. 13C-NMR (CDC13) 5 (ppm): 12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 39.71, 39.81, 4.027, 43.34, 59.22, 60.59, 120.97, 123.84, 124.30, 131.32, 135.35, 135.92 , 138.05, 160.45 and 197.12. Example 2: 80% maximal oxygen uptake (80% V0 ₂ max) exercise load test

 In order to measure the effect of supplementing Antrodia camphora cyclohexenone on anti-fatigue ability after exhaustive exercise, this experiment uses 80% maximal oxygen uptake exercise load for subsequent depletion exercise, so the maximum oxygen uptake should be measured first to further Estimate the speed of 80% of the maximum oxygen uptake. Among them, oxygen uptake refers to the product of cardiac output and arteriovenous blood oxygen concentration difference, and the maximum oxygen uptake refers to a person at sea level, when engaged in the most intense exercise, tissue cells can be consumed or utilized every minute. The highest value of oxygen, which is the best index for evaluating cardiorespiratory endurance. The present invention uses a direct measurement method in the laboratory, using an in-situ treadmill to directly increase the exercise load to the maximum exercise load, and directly measures with a gas analyzer. The maximum oxygen uptake, this progressive incremental exercise load sustained exercise, can induce the actual maximum oxygen uptake capacity, so it is a method to directly and accurately measure the maximum oxygen uptake.

(1) Basic data collection of subjects The invention adopts 15 males who are 20 years old and healthy and voluntary, and selects those who have not taken drugs, have normal liver and kidney function, no cardiovascular disease, and have no smoking, drinking, and using nutritional supplements on weekdays, and measure the records. Basic data for each subject included: age, height, weight, and BMI. Subjects need to have an empty stomach for at least eight hours before the formal test, and maintain a normal diet during the test period and avoid taking nutritional supplements or other medications to avoid affecting the experimental data.

(2) 80% maximal oxygen uptake exercise load test

Subjects must undergo a second pre-test before formal testing to determine the maximum oxygen uptake (V0 ₂ max ) and estimate the exercise load of 80% of the maximum oxygen uptake, followed by an 80% maximal oxygen uptake. The test is confirmed to ensure the exercise intensity of the subject's 80% maximal oxygen uptake at this particular load, and the test procedure is detailed below.

 First, the maximum oxygen uptake test is performed. Before the subject arrives, the oxygen analyzer of the gas analyzer (Vmax Spectra, SensorMedics) and the standard gas calibration of different concentrations are required. After the subject arrives, put on the heartbeat test table (Polar810i) and record the resting heart rate, place the heartbeat test strip close to the subject's heart, and place the test table within 1 meter of the test strip. Subjects warmed up for 3-5 minutes on a local treadmill (Vision, T8600) and exercised on their own, then placed the subject on the treadmill, put on a gas mask, and gas The mask is connected to the breathing tube, and the breathing tube is connected to the gas analyzer, so that the gas exhaled by the subject can be transmitted to the gas analyzer through the gas mask and the breathing tube. Then, the running speed of the treadmill is fixed at 9.6 km/hr, and the treadmill is set to 0% slope within 0~3 minutes from the start of the test. After starting the test for 3 minutes, the treadmill should be increased by 3% every 3 minutes until Subjects stop exercising and stop exercising. After analyzing the data of the gas analyzer, the oxygen uptake 1 minute before each slope and before the exercise failure was obtained, and the maximum oxygen uptake obtained in the test was the maximum oxygen uptake. In addition, the maximum oxygen uptake criteria must meet at least two of the following conditions to determine the maximum oxygen uptake: (a) The subject has tried his best to not continue the exercise test (the subject's footsteps are slow) (b) The heart rate is (220 - age) ± 10 beats / min; (c) The respiratory quotient (RQ) must be greater than 1 · 1 or more; (d) Self-conscious scale ( Rating perceived exertion, RPE) has reached the stage of 18 or 19.

The 80% maximal oxygen uptake rate can be further obtained by the regression equation of the maximum oxygen uptake and load intensity obtained from the pre-test. This calculation process uses the oxygen consumption and the speed obtained in the maximal oxygen uptake test. The data of the degree, and the regression line of the speed (ordinate) and oxygen intake (abscissa), and then multiply the individual's maximum oxygen intake by 80%, and obtain the oxygen uptake at 80% of the maximum oxygen uptake. Draw a line to find the relative speed of this point. It is necessary to exercise for 10 minutes at this load intensity, and measure the oxygen uptake at 5-6 minutes and the last 1 minute to determine the true 80% maximal oxygen uptake exercise load. The results are shown in Table 1.

 Table 1. Basic data of the subjects and their maximum oxygen uptake

 Item Measurement value (mean ± standard deviation)

 Age (years) 22.8±0.89

 Height (m) 1.75±0.01

 Weight (kg) 67·73±1·81

BMI (kgxm" ² ) 22.14±0.63

 Maximum oxygen uptake (ml/min/kg) 50.22±0.17

 80% maximal oxygen uptake rate 7.61±1.87

 η = 15 Example 3: Anti-fatigue test of anthraquinone cyclohexenone compound

 The extensive definition of muscle fatigue during exercise is that the individual's physiological processes cannot maintain their functions at a certain level, or the organs cannot maintain the predetermined exercise intensity. The causes of physical activity fatigue may include psychological, physiological and biochemical aspects, among which, biochemical fatigue The underlying mechanisms include both central and peripheral fatigue. The mechanisms that cause central fatigue include hypoglycemia, changes in the concentration of key amino acids in the blood, and changes in the concentration of neurotransmitters in the brain. The mechanism of peripheral fatigue is phosphoric acid in the muscle. Exhaustion of phospocreatine (PC) leads to energy shortages such as increased blood ammonia, hepatic glucose depletion in muscles, and insufficient oxygen supply, as well as increased lactic acid due to accumulation of hydrogen ions in muscles and accumulation of phosphate in muscles. Stacking factors.

 Therefore, the present invention provides a subject with a placebo containing an anatase ring cyclohexenone compound and an anthrax-free cyclohexenone compound, and subjecting the subject to a depletion exercise having an intensity of 80% of the maximum oxygen uptake exercise load. At the same time, before and after exercise, the content of fatigue indexes such as acid kinase, blood lactic acid, blood sugar, blood ammonia and free fatty acid in the subject was analyzed, and the anti-fatigue of the anthraquinone cyclohexenone compound was measured after the exhaustion exercise. efficacy.

Randomly administered to the subject 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-ten a dicarbotriene)-2-cyclohexenone compound and a placebo of a non-burdock cyclohexenone compound, and the placebo component may be any non-burdock cyclohexenone compound such as corn starch, and This test uses a double-blind crossover design, and neither the subject nor the tester knows the substance to be administered. Following this, based on the principle of order balance, each subject is required to undergo the following tests: no exercise and placebo (PR), exercise and placebo (PE), no exercise and given burdock The cyclohexenone compound (DR) and the subject who is exercised and given the anthraquinone cyclohexenone compound (DE), and who are tested in different groups, need to rest after a week after completing the one-week trial of the group. The next set of tests is continued until each subject completes the above four sets of tests. Among them, the exercise is performed on the treadmill with the intensity of the 80% maximal oxygen uptake exercise load (7.61±1.87) measured above, and runs until the subject is exhausted, and is given after exercise. The subject was anthraquinone cyclohexenone compound or placebo; in addition, the placebo was administered in an amount of 0.2 g/kg (kg) per day, and the anthraquinone cyclohexenone compound was administered in an amount of 0.2 g/kg (kg) per day. The test was conducted for seven days, and venous blood was collected at 0, 0.5, 1, 2, 24, 48, 72, 120, and 168 hours before exercise and after exhaustive exercise. Blood was collected with anticoagulant (eg, EDTA). Tube collection, centrifugation at 3000 xg for 10 minutes, taking the plasma fraction, and performing subsequent biochemical analysis of fatigue indexes such as creatine kinase, blood lactate, blood glucose, blood ammonia, and free fatty acids, and comparing different supplements before and after exercise. Changes in the physiology and biochemical values of the substance; all biochemical analysis values are expressed as mean and standard deviation (Mean soil SEM), and the analytical values are analyzed by the number of repetition factors. Ted measurement two way ANOVA) The difference between the blood collection points in the group and the group was compared, and the post-mortem comparison was performed by Tukey's honestly significant difference. The critical value of the significant level after the statistics was determined as α= 0.05, and the analysis of these biochemical values and their results are detailed: 3⁄4 mouth:

 (1) Analysis of creatine phosphate kinase (CPK)

Serum creatine kinase is produced in human skeletal muscle, myocardium, brain and prostate, among which skeletal muscle is the most abundant, accounting for 96% of the total body; under normal circumstances, serum creatine kinase activity is very low. When muscle contraction during exercise, creatine kinase can catalyze the high energy riding acid bond between adenosine triphosphate (ATP) and phosphate creatine (PC). The chemical reaction formula is: _CpK

ADP + PC ► ATP + Creatine To ensure rapid muscle movement, the energy required for muscle contraction and the re-synthesis of ATP are promoted. The reason why the increase of creatine kinase activity during exercise is caused by hypoxia during exercise, accumulation of metabolites, constant imbalance of intracellular and extracellular calcium ions, increase of permeability of myocyte membrane, or damage of myocyte membrane, for example. Muscle pulls mechanical damage or produces a hematoma, which in turn causes creatine kinase to release into the blood circulation, so creatine kinase is often used as an indicator of exercise intensity and muscle cell damage.

 The method for measuring the concentration of creatine kinase in blood according to the present invention is measured by a dry automatic blood analyzer (Johnson & Johnson DT-60 II) and by the principle of enzyme action and colorimetric assay. After adding the creatine phosphate glucose oxidase reaction to the quantified plasma, 4-aminoantipyrine and 1,7-dihydroxynaphthalene were added. After the action of peroxidase, a white compound was produced, and the absorbance was measured at a wavelength of 680 nm, and then the concentration of creatine kinase was converted. The results are shown in Table 2 and Figure 1. Table 2. Creatine kinase concentration (U/L) at various time points after exercise or no exercise and supplementation with placebo or burdock cyclohexenone compound, exercise and post-exercise exercise after exercise After exercise, after exercise, after exercise, after exercise

 First 0 hours 0.5 hours 1 hour 2 hours 24 hours 48 hours 72 hours 120 hours 168 hours Ann No movement 205.40 204.80 206.27 198.33 194.67 194.07 193.40 171.73

S (PR) ±42.49 ±40.28 ±40.54 ±42.99 ±40.77 ±25.81 ±18.00 ±25.92 ±22.44 ±22.75 with motion 152.00 232.13 219.20 205.53 188.47 383.53 321.60 360.20 279.73 231.13

(PE) ±32.06 ±38.98* ^ac ±33.56* ^acd ±29.88* ^acd ±32.25* ^c ±134.86° ±99.33 ^ac ±111.71 ^{ac d} ±69.06 ±70.20 Burdock

Zhihuan no movement 167.27 150.20 166.53 167.40 146.33 158.47 133.87 165.73 243.00 183.27 Hexene (DR) ±19.5 ±21.71* ±19.71 ±23.16 ±23.16 ±21.63 ±16.51 ±28.07 ±48.21 ±38.37 Ketone

Compound

 There are sports 226.64 259.57 221.86 232.93 208.07 332.50 244.00 193.86 193.86 204.83

±37.20 ±38.83*° ±37.74 ±32.98 ±30.53 ±81.75 ±49.65 ±34.44 ±25.49 ±31.89

(DE)

 n=15, the value is expressed as "mean ± standard deviation"

 * indicates p<.05: there is a difference from the pre-test ratio; a indicates p<.05: PR group significantly larger than the simultaneous point

b indicates p<.05: significantly greater than the PE group at the same time; c indicates p<.05: significantly greater than the same time Point DR group

d indicates p<.05: DE group significantly larger than the contemporaneous point. As shown in Table 2 and Figure 1, after the high-intensity exhaustive exercise with 80% maximal oxygen uptake, the group with exercise and placebo (PE) was At 0, 0.5, 1, 2, and 24 hours after exercise, the concentration of creatine kinase continued to increase and was significantly (p < 0.05) higher than the concentration of creatine kinase before exercise, which also indicates exercise intensity of the subject. The degree of muscle damage has been achieved; the concentration of creatine kinase in the group with exercise and placebo (PE) was significantly (p < 0.05) higher than no exercise and placebo (PR) with no exercise and given Antrodia The values shown in the two groups of cyclohexenone compounds (DR), and the groups of untreated movements (PR and DR) of the two groups did not differ until 120 hours after exercise.

 Exercise and give the group of Antrodia camphora cyclohexanone compound (DE). After high-intensity exhaustion exercise, the concentration of the acid kinase increases only immediately after exercise, and returns to the pre-exercise after 0.5 hours after exercise. Concentration level. In addition, the concentration of creatine kinase was significantly (p < 0.05) higher in the group with exercise and placebo (PE) than in the group with exercise and administration of the anthraquinone cyclohexenone compound (DE). And this difference in concentration is continued from the exercise of exhaustion to 1 hour after exercise, and this phenomenon can still be observed 72 hours after exercise. This result shows that the addition of the anthraquinone cyclohexenone compound immediately after the exhaustion exercise can effectively reduce The concentration of creatine kinase, which represents an indicator of muscle damage, helps to slow the physical fatigue that occurs after exercise.

(2) Analysis of blood glucose before meals

 During exercise, with the release of calcium ions during muscle contraction and the secretion of epinephrine, phosphorylase can be activated to increase the rate of hepatic glycolytic decomposition in the muscle, and the increased adrenaline accelerates the liver glycolytic reaction. (glycogenolysis), which in turn increases blood glucose levels. The fatigue caused by high-intensity exhaustion exercise with 80% maximal oxygen uptake is often caused by the continuous use of blood sugar in the oxidative metabolism process, resulting in hypoglycemia, which leads to the depletion of glycogen in the muscle. Therefore, detection The utilization of sugar in the body during exercise can be used as one of the indicators for observing physiological fatigue.

The method for measuring blood glucose concentration according to the present invention is measured by a dry automatic blood analyzer (Johnson & Johnson DT-60 II) and by the principle of enzyme action and colorimetric measurement. After adding glucose oxidase to the quantitative plasma, 4-aminoantipyrine and 1,7-dihydroxynaphthalene are added to the plasma. 4 substance enzyme Consolation

 Material loss

 (peroxidase) produces a red compound with the following reaction formula: glucose oxidase

β-D glucose + 0 ₂ + 3⁄40 ► D-gluconic acid + 3⁄40 ₂ peroxidase

2 3⁄40 ₂ + 4-aminoantipyrine + 1,7-dihydroxynaphthalene ► Red dye (Red dye) and its absorbance measured at 555 nm, then converted to blood glucose concentration, the results are shown in Table 3 and Figure 2 Show.

 Table 3. Blood glucose concentration (mg/dl) at various time points after subjects undergoing exhaustive exercise or no exercise and supplemented with placebo or anthrax Cyclohexenone compound, respectively.

 After exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise,

 0 hours 0.5 hours 1 hour 2 hours 24 hours 48 hours 72 hours 120 hours 168 hours

90.27 90.73 92.87 92.27 97.20 86.87 87.73 90.20 88.47 87.47 ±1.80 ±1.90 ±2.62 ±1.71 ±4.84 ±1.85 ±1.65 ±1.08 ±0.90 ±2.25

89.67 106.60 90.00 97.73 99.13 92.47 90.33 91.47 88.13 91.07 ±2.50 ±4.25* ^ac ±3.34 ±5.15 ±5.90 ±3.16 ^a ±2.1 1 ±1.57 ±1.90 ±1.75

91.73 91.93 93.33 95.53 103.20 90.00 90.40 93.40 94.00 92.20 ±1.65 ±1.95 ±2.99 ±3.23 ±4.35* ±1.91 ±2.56 ±2.34 ±2.65 ^a ±1.98

89.21 103.43 88.71 93.86 101.07 94.43 89.21 93.00 93.29 92.93 ±2.02 ±4.49* ^ac ±1.74 ±2.03 ±6.03* ±1.57; ±1.69 ±1.42 ±1.42'±1.34;

11=15, the value is expressed as "mean ± standard deviation"

 * indicates p<.05: there is a difference from the pre-test ratio; a indicates p<.05: PR group significantly larger than the simultaneous point

 b indicates p<.05: PE group significantly larger than the simultaneous point; c indicates p<.05: DR group significantly larger than the simultaneous point

 d indicates p<.05: DE group significantly larger than the simultaneous point

As can be seen from Table 3 and Figure 2, the group with exercise and placebo (PE) and exercised and given the anthraquinone cyclohexenone compound (DE), after 80% maximal oxygen uptake, after high-intensity exhaustive exercise, Blood sugar There was a significant (p < 0.05) difference between the concentration values and no exercise and placebo (PR) and no exercise and administration of the anthraquinone cyclohexenone compound (DR) group; in addition, exercise and placebo (PE) and Exercise and give the group of burdock ring cyclohexenone (DE), the blood glucose concentration of 0.5 hours after high-intensity exhaustion exercise of 80% maximal oxygen uptake will return to the level before the exercise and close to the quiet value. This result shows that supplementation with anthocyanin cyclohexenone after exercise has no effect on the metabolism of blood glucose before meals.

 (3) Analysis of blood ammonia (Ammonia)

 Blood ammonia is a metabolite of protein, which is mainly caused by the decomposition of amino acids in the purine nucleotide cycle (PNC) and the deamination of adenosine monophosphate (AMP). Depletion of creatine phosphate causes adeninenucleotide in the tissue to decompose to accelerate the re-synthesis of ATP, which produces a large amount of blood ammonia, and an increase in blood ammonia concentration changes the center. The role of the nervous system changes the extracellular pH, electrolyte concentration, and neurotransmitter concentration, and interferes with the Krebs cycle, causing fatigue. Therefore, blood ammonia accumulation is also causing central fatigue and peripheral fatigue. One of the factors, blood ammonia is often used as one of the indicators of fatigue.

 The method for measuring blood ammonia concentration according to the present invention uses a dry automatic blood analyzer (Johnson & Johnson DT-60 II) and adds a coloring agent bromophenol blue (bromphenol) to the quantitative plasma by the principle of colorimetric measurement. Blue), after the action, produces a blue compound with the following reaction formula:

NH ₃ + bromophenol blue ► blue dye (Blue dye ) and its absorbance measured at 605 nm wavelength, and then converted to blood ammonia concentration, the results are shown in Table 4 and Figure 3

Table 4. Blood ammonia concentration at each time point after subjects undergoing exhaustive exercise or no exercise and supplemented with placebo or Antrodia camphora compound respectively ^g/dl) After exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise, group

 First 0 hours 0.5 hours 1 hour 2 hours 24 hours 48 hours 72 hours 120 hours 168 hours No movement 49.93 61.67 64.53 74.00 69.07 39.27 28.73 30.73 21.27 32.14 安

(PR) ±5.71 ±7.61* ±4.68* ±6.55* ^d ±8.37* ^d ±4.0 ±3.15* ±4.94* ±2.86* ±3.22* s with motion 52.73 153.27 75.27 64.40 73.20 40.27 27.73 27.43 24.71 29.60

±4.40 ±16.38* ^ac ±5.42* ±5.85 ±10.49 ^d ±6.01 ±3.28* ±2.92* ±3.63* ±3.53*

(PE)

Burdock

 Movement 39.20 55.60 52.53 57.47 54.07 30.33 26.86 25.20 27.80 28.57 Zhihuan No

 ±6.87 ±9.72 ±8.16 ±7.83* ±7.05 ±4.51 ±2.48 ±*4.45 ±4.63* ±3.61 hexene (DR)

Ketolation has exercise 45.86 118.07 57.64 44.86 37.53 27.93 20.93 30.07 23.21 24.86 compound

(DE) ±7.28 ±11.06* ^ac ±7.96* ±7.23 ±6.09 ±2.96* ±2.28* ±5.56* ±2.28* ±4.04* n=15, the value is expressed as "mean ± standard deviation"

 * indicates p<.05: there is a difference from the pre-test ratio; a indicates p<.05: PR group significantly larger than the simultaneous point

b indicates p<.05: PE group significantly larger than the simultaneous point; c indicates p<.05: DR group significantly larger than the simultaneous point

d indicates p<.05: DE group significantly larger than the simultaneous point

As can be seen from Table 4 and Figure 3, the group with exercise and placebo (PE) and exercised and given the anthraquinone cyclohexenone compound (DE), after 80% maximal oxygen uptake, after high-intensity exhaustive exercise, The blood ammonia concentration value was significantly (p < 0.05) higher than that without exercise and given placebo (PR) and no exercise and given to the group of R. chinensis cyclohexenone compounds (DR). The blood ammonia concentration of exercise group and placebo (PE) and exercise and administration of anthraquinone cyclohexenone compound (DE) gradually recovered 1 hour after exercise, and the blood ammonia concentration value at this time point was There was no significant difference (p > 0.05) between the blood ammonia concentrations measured before exercise; and 2 hours after exercise, there was exercise and given the group of O. chinensis cyclohexenone (DE) with exercise and comfort. There was a statistically significant (p < 0.05) difference in blood ammonia concentration values between the agent (PE) groups. In addition, as can be observed from Table 4 and Figure 3, the blood ammonia concentration shown by exercise and given to the Antrodia camphora cyclohexanone compound (DE) group was significantly (p < 0.05) lower than that at 24 hours after exercise. Before exercise, at the same time point, the blood concentration of the same exercise group and the placebo (PE) group was similar to the value before the exercise, and there was no significant decrease in the blood ammonia concentration. Therefore, the above results show that supplementing the anthraquinone cyclohexenone compound after exercise contributes to the metabolism of blood ammonia, so that the blood ammonia accumulated in the body after exercise can be rapidly metabolized and thus slowed down. Physiological fatigue caused by exhaustive exercise.

 (4) Analysis of blood lactate (Lactate)

 Lactic acid is a product of the metabolism of glycogen and glucose in muscle and liver through anaerobic glycolysis. In a quiet state, the amount of lactic acid produced is small. When it is intense or prolonged, the tissue hypoxia is more obvious. The rate of oxygen metabolism is increased, and when the rate of lactic acid production is higher than the rate of oxidative metabolism of lactic acid by mitochondria, increased lactic acid is gradually accumulated in the muscle. The accumulation of lactic acid causes the concentration of hydrogen ions in the muscle to rise and the pH to decrease, which in turn inhibits the activity of the phospholyzed phosphofructokinase (PFK), resulting in a decrease in the rate of glycolytic reaction and a decrease in the rate of ATP re-synthesis. As the concentration of hydrogen ions increases, it affects the release of calcium ions in the sarcoplasmic reticulum and reduces the contraction ability of muscle fibers. Therefore, a large accumulation of lactic acid will accelerate the occurrence of fatigue reaction. Therefore, blood lactic acid is often used as one of the indicators of fatigue.

 The method for measuring blood lactic acid concentration according to the present invention uses a dry automatic blood analyzer (Johnson & Johnson DT-60 II), and adds lactate oxygenase to the quantitative plasma by the principle of enzyme action and colorimetric determination. After (lactate oxidase) reaction, 4-aminoantipyrine and 1,7-dihydroxynaphthalene are added to the solution, which is red by the action of peroxidase. a compound having the following reaction formula: lactate oxidase

Lactic acid + 0 ₂ ► pyruvate + 3⁄40 ₂

Peroxidase

2 3⁄40 ₂ + 4-aminoantipyrine + 1 ,7-dihydroxynaphthalene ► Red dye (Red dye ) and its absorbance measured at 540 nm, then converted to blood lactate concentration, the results are shown in Table 5 and Figure 4 Shown.

Table 5. Blood lactate concentration (mmol/1) at each time point after subjects undergoing exhaustive exercise or no exercise and supplemented with placebo or Antrodia camphora After exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise, before exercise

0 hours 0.5 hours 1 hour 2 hours 24 hours 48 hours 72 hours 120 hours 168 hours no motion 2.43 2.62 2.80 2.50 2.43 2.71 2.41 2.15 2.30 2.68 ( 0.12 ± 0.11 ± 0.19 ± 0.17 ± 0.14 ± 0.20 ± 0.12 ± 0.21 ± 0.17 ± 0.12 ^Bd安PR) ±

s has motion 2.68 7.79 3.45 2.64 2.68 2.69 2.45 2.17 2.27 2.09 (PE) ±0.17 ±0.80* ^ac ±0.28* ^c ±0.23 ±0.13 ±0.16 ±0.21 ±0.19* ±0.17* ±0.14* Burdock no motion 2.54 2.39 2.25 3.00 2.52 2.26 2.71 2.22 2.41 2.41 芝 ring ±0.19 ±0.19* ±0.53 ±0.20 ±0.16 ±0.12 ±0.18 ±0.30 ±0.19 hexene (DR) ±0.18

Ketolation

 Have exercise

Compound 2.50 7.07 3.20 2.96 2.9 2.39 2.77 2.18 2.53 2.23 (DE) ±0.17 ±0.97* ^ac ±0.3 l* ^c ±0.18 ±0.15 ±0.20 ±0.20 ±0.19 ±0.25 ±0.16 n=15, the value is "mean ± Standard deviation"

 * indicates p<.05: there is a difference from the pre-test ratio; a indicates p<.05: PR group significantly larger than the simultaneous point

 b indicates p<.05: PE group significantly larger than the simultaneous point; c indicates p<.05: DR group significantly larger than the simultaneous point

 d indicates p<.05: DE group significantly larger than the simultaneous point

 As can be seen from Table 5 and Figure 4, the group with exercise and placebo (PE) and exercise and given the anthraquinone cyclohexenone compound (DE), 0.5 hours after high-intensity exhaustion exercise with 80% maximal oxygen uptake Within, the blood lactate concentration value was significantly (p < 0.05) higher than that without exercise and given placebo (PR) and without exercise and given to the group of the cow's anthraquinone cyclohexenone compound (DR), and the group with exercise There was also a significant difference in the concentration of blood lactate between the interventions of exercise intensity (PE and DE) and the non-sports group (PR and DR) (p < 0.05). The blood ammonia concentration of exercise group and placebo (PE) and exercise and administration of anthraquinone cyclohexenone compound (DE) gradually recovered and approached the quiet value before exercise at 1 hour after exercise, and at this time There was no significant difference between the blood ammonia concentration value of the point and the blood lactate concentration measured before exercise (p > 0.05). This result showed that after the exercise, the metabolism of the blood lactic acid was supplemented with the anthraquinone cyclohexenone compound. no effect.

 (5) Analysis of free fatty acids

The production of free fatty acids is derived from the hydrolysis of adipose tissue between fibers, or intracellular triglycerides. During endurance exercise, the glycogen stored in the muscle will be depleted, and as the duration of exercise increases, the rate of glycolytic will gradually decrease, and the utilization of energy in the body tends to utilize fatty acids. At this time, the production of ATP is reduced, thus causing fatigue; in addition, when the rate of fat decomposition is increased, and the concentration of free fatty acids in plasma is increased, free fatty acids compete with tryptophan in blood for albumin (Albumin). The binding position causes an increase in free tryptophan in the blood. Tryptophan is a precursor of serotonin. When tryptophan enters the brain, the rate of serotonin synthesis increases. When the serotonin in the brain increases, it will damage some aspects of the central nervous system. For example, reducing the activity of dopamine can also lead to fatigue and sleep reaction. Therefore, the concentration of free fatty acids in the blood can be used as a lipolysis. And indicators of fatigue.

 The method for measuring the concentration of free fatty acids of the present invention, using the principle of enzyme action and colorimetric determination, adding acyl-CoA synthetase to acyl-CoA synthetase, acyl-CoA synthase (acyl) CoA oxidase), after the action of peroxidase, produces a purple compound with the following reaction formula:

N EFA + ATP + CoA ^Α ° ^Υ ί ^CoA _A cyl CoA + AMP + PPi

Acyl CoA

Acyl CoA + 0 ₂ ^ ^ ► 2,3-trans-enoyl-CoA + H ₂ 0 ₂ peroxidase

H ₂ 0 ₂ + TOOS + 4-AAP ^ purple product + 4 3⁄40

4-AAP: 4-aminoantipyrine

 TOOS: N-ethyl-N-(2-hydroxy-3-sphingylpropyl) m-nonanilide

 (N-ethyl-N-(2-hydroxy-3-sulphopropyl) m-toluidine) and its absorbance was measured at a wavelength of 550 nm, and then the blood lactate concentration was converted. The results are shown in Table 6 and Figure 5.

Table 6. Free fatty acid concentrations (μηιοΐ/ΐ) at various time points after subjects undergoing exhaustive exercise or no exercise and supplemented with placebo or Antrodia camphora respectively. After exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise, after exercise, before exercise

 0 hours 0.5 hours 1 hour 2 hours 24 hours 48 hours 72 hours 120 hours 168 hours An 0.33 0.26 0.31 0.28 0.30 0.37 0.38 0.32 0.29 0.27 No movement

Comfort ±0.04 ±*0.03 ±0.04 ±0.04 ±0.05 ±0.05 ±0.04 ±0.03 ±0.03 ±0.04 dose (PR)

Exercise 0.35 0.94 0.61 0.62 0.63 0.47 0.39 0.47 0.39 0.40 ±0.05 ±0.12* ^ac ±0.08* ^ac ±0.11 * ^ac ±0.08* ^ac ±0.06 ^c ±0.05 ±0.06 ^ac ±0.05 ±0.06 (PE)

Burdock

Zhihuan no movement 0.38 0.33 0.34 0.30 0.23 0.28 0.29 0.31 0.48 0.28 hexene ±0.04 ±0.04 ±0.04 ±0.05 ±0.03* ±0.05 ±0.04 ±0.05 ±0.10 ^a ±0.03 Ketolation (DR)

The compound has motion 0.34 0.91 0.60 0.61 0.59 0.41 0.28 0.44 0.30 0.32 (DE) ±0.03 ±0.14* ^ac ±0.08* ^ac ±0.09* ^ae ±0.08* ^ae ±0.06 ^c ±0.04 ±0.05 ^c ±0.04 ±0.06

n=15, the value is expressed as "average ± standard deviation"

 * indicates p<.05: there is a difference from the pre-test ratio; a indicates p<.05: PR group significantly larger than the simultaneous point

 b indicates p<.05: PE group significantly larger than the simultaneous point; c indicates p<.05: DR group significantly larger than the simultaneous point

 d indicates p<.05: the DE group significantly larger than the contemporaneous point is shown in Table 6 and Figure 5, with exercise and placebo (PE) and exercise and given to the group of O. chinensis cyclohexenone compounds (DE), Within 2 hours after high-intensity exhaustion exercise with 80% maximal oxygen uptake, the concentration of free fatty acids in the blood was significantly (p < 0.05) higher than no exercise and placebo (PR) and no exercise and given to Antrodia camphorata Group of ketene compounds (DR). There was no significant difference in blood free fatty acid concentration values between exercise and placebo (PE) and exercise and administration of Antrodia camphora (DE) at various time points after exercise. > 0.05), this result shows that supplementation with anthocyanin cyclohexenone after exercise has no effect on the metabolism of free fatty acids in the blood.

In summary, the results of the invention show that the anthocyanin compound is supplemented immediately after the high-intensity exhaustion exercise with 80% maximal oxygen uptake, and the concentration of creatine kinase rises only immediately after exercise, and the muscle is 0.5 hours later. The acid kinase concentration returned to pre-exercise levels, and this result showed that the addition of Antrodia camphorata immediately after exhaustive exercise had a significant (P < 0.05) effect on the metabolism of creatine kinase; whereas after exercise, the metabolism of blood ammonia, immediately after exhaustive exercise The group supplemented with anthocyanin compound (DE) was added 2 hours after exercise. The recovery effect was significantly better (p < 0.05) than the group without supplemented with anthocyanin compound (PE); in addition, fatigue indicators such as blood sugar, blood lactate and free fatty acids were in exercise and given placebo (PE) There was no significant difference between the two groups with exercise and given the anthraquinone cyclohexenone compound (DE). Therefore, supplementation with anthocyanin cyclohexenone after the high-intensity exhaustion exercise of 80% of the maximum oxygen uptake contributes to the recovery of creatine kinase and blood ammonia in the body, thereby achieving the effect of slowing down physiological fatigue.

Claims

Claim An anthraquinone cyclohexenone compound for slowing physiological fatigue, the compound having the following structural formula:

Wherein X is oxygen (0) or sulfur (S), Y is oxygen or sulfur; hydrogen (H), methyl (CH ₃ ) or (CH ₂ )m-CH ₃ , R ₂ is hydrogen, methyl Or (CH ₂ )m-CH ₃ , R ₃ is hydrogen, methyl or (CH 2 ) m-CH ₃ , m = 1-12; n = l-12.  The burdock cyclohexenone compound for relieving physiological fatigue according to claim 1, wherein the compound is obtained by isolating an organic solvent extract of Antrodia camphorata.  The burdock cyclohexenone compound for slowing physiological fatigue according to claim 2, wherein the organic solvent is selected from the group consisting of esters, alcohols, alkanes or alkanes.  The burdock cyclohexenone compound for slowing physiological fatigue according to claim 3, wherein the alcohol is ethanol.  The burdock cyclohexenone compound for use in relieving physiological fatigue according to claim 1, wherein the compound is obtained by separating an aqueous extract of Antrodia camphorata.  The burdock cyclohexenone compound for slowing physiological fatigue according to claim 1, wherein the compound is 4-hydroxy-2,3-dimethoxy-6-methyl-5 (3, 7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexanone (4-hydroxy-2,3-dimethoxy-6-methy-5(3,7,ll- Trimethyl-dodeca- 2,6,10-trienyl)-cyclohex-2-enone ).  The anthraquinone cyclohexenone compound for use in relieving physiological fatigue according to claim 1 or 6, wherein the compound is resistant to fatigue by promoting metabolism of a creatine phosphate kinase in the body after exercise. 8. The B. angustifolia cyclohexenone compound for slowing physiological fatigue according to claim 7, wherein the exercise is a high intensity exhaustion exercise of 80% maximal oxygen uptake. The B. angustifolia cyclohexenone compound for relieving physiological fatigue according to claim 1 or 6, wherein the compound is resistant to fatigue by promoting metabolism of ammonia in the body after exercise.  10. The burdock cyclohexenone compound for use in mitigating physiological fatigue according to claim 9, wherein the exercise is a high-intensity exhaustive exercise of 80% maximal oxygen uptake.