| Cordyceps is a type of traditional Chinese medicinal herb and has been wildly usedas a general tonic since ancient times in China. As the rapid development of China’spharmaceutical research after the reform and opening up, the effects of Cordyceps andits artificially fermented alternatives were gradually revealed. Currently, the domesticand international research has shown that Cordyceps and its extracted materials oralternatives possess many important pharmacological activities, such as anti-fatigue,antioxidant, immunomodulatory, anti-tumor and anti-hyperglycaemic[1,2]. However,there are wide varieties about the wild Cordyceps and more than400species ofCordyceps around the world[3]. The qualities of Cordyceps and its alternatives arevastly different, it is more difficult to identify for ordinary patients or normalconsumers. In addition, due to the harsh growing conditions of wild Cordyceps andthe depletion of natural resource, Cordyceps has an excessive demand and expensiveprice in the market. And now, wild Cordyceps has become endangered naturalresources, it should be protected urgently and used rationally. Therefore, it isincreasingly important for the research and development of efficacy and stabilityCordyceps alternatives, which is also an effective way to solve above problems.The Paecilomyces hepiali mycelium (PHM), studied by us in this experiment arePaecilomyces hepiali strains isolated from wild Cordyceps, which is collected fromTibetan in China. By the way of artificial and fermented cultivation, it could beproduced and purified in a large scale, and then we can get a large number of PHM atthe same time. By the studying of its efficacy, its potential as an alternative ofCordyceps could be evaluated and the market demand could be relieve. In addition, itis positive to the protection of endangered wild Cordyceps resources, and will providetheoretical and experimental basis for its development as a therapeutic drug.Fatigue is a widespread physiological phenomenon, it include physical fatigue andmental fatigue. Not only it has a seriously affect on our work efficiency, sports ability,but also on our family life, and even the social relations were be effected[4]. Fatigue isassociated with the occurrence of various diseases such as HIV, hepatitis and tumor[5,6], and it could cause some health problems such as immunity disorder, endocrine disorders, infections, diabetes[7,8]. The antifatigue and antihypoxia effects of PHMare evaluated in this research, and the mechanism of which were simply discussed.Objectives:The main work of this study is researching the anti-chronic fatigue,prophylactically anti-fatigue, anti-anoxia effect and the mechanisms of fermentedPaecilomyces hepiali mycelium. It may provide some rationales and experimentbasises for the researching and development of the substitutive materials of wildCordyceps.Research methods:1. Effects of PHM on the physical fatigue of CF mice:Seventy-two adult male Kunming mice were randomly divided into six groups:One home cage control group and five chronic fatigue (CF) groups. All groups weretreated with vehicle, vehicl,13mg/kg modafinil, and140,280,560mg/kg PHM,respectively. Home group is not participated in the climbing training run, the CF micewere forced to receive a climb-running training for30min daily after the drugs orvehicles administrating for40min over an18-day period by the YLS-10B mice wheelrunning fatigue apparatus. The climb-running speed was10.2m/min on the first dayand gradually increased to10.8m/min,11.4m/min,12m/min, and12.6m/min on the5th,9th,13th, and17th day, respectively. On the19th day, CF mice were exposed toan exhaustive exercise for60min at the speed of12.6m/min. If the mice got fatiguedand stopped running or slowed down, they would slid to the bottom of the movingwheels and be shocked by a1.2mA current. So, they had to keep running to avoid theelectric shock. For each trial, the number of electric shock was recorded as theevaluation indicator of exercise tolerance. The more number of electric shocks meansthe more severe physical fatigue of the mice.On the19th day, after the exhaustive exercise, all mice were placed in a quietenvironment to rest for40minutes. The blood samples were collected from mice orbitto a2mL plastic centrifuge tubes and placed at a45degrees slope for30min. Theserum was prepared by centrifugation at4000×g for15minutes. After mice werekilled by cervical luxation, liver and leg muscle tissues were removed rapidly on theice plate. They were then washed with cold saline, and blotted with filter papers, andstored at-20°C until used. The fatigue-related biochemical parameters includingmuscle glycogen (MG), liver glycogen (LG), serum urea nitrogen (SUN), serum lactic acid (SLA), and serum creatine kinase (CK) were detected according to theinstructions of commercially available kits.2. The preventive anti-fatigue effect of PHM:72male mice were randomly divided into six groups by the same method. Exceptfor the modafinil group, which is administrated once a day, the other wereadministrated with vehicle or PHM twice a day during14days, and the dosage issame to the first experiment. After the last administering of14th day, except for thehome cage group, all the other groups were forced to receive the climb-runningexercise tests induced by wheel running apparatus at the40min,8h,24h,48h, andthe exhaustive exercise tests at the72h. The number of electric shock was recorded.The exercise time was30min and the exhaustive exercise time was60min. Thespeed was set at12.6m/min and the current is1.2mA in each experiments.After the exhaustive exercise, all mice were placed in a quiet environment to restfor40minutes, the blood were collected by the way of eyeball removal, and then theblood samples were divided into two groups. One of the blood samples isanticoagulated by heparin, and stored at4℃for the detecting of Hb and blood ATPlevels. The another blood samples is normal blood and used to prepare serum for thedetecting of SUN, SLA, CK in a same method with the first experiment. The liversand leg muscle tissues of mice were removed rapidly and used to detect the levels ofLG, MG by the same method.3. The antihypoxia effect of PHM:48male Kunming mice were divided into4groups, and administrated with vehicle,140mg/kg,280mg/kg and560mg/kg PHM twice a day during a14day period at8am and7pm, respectively. After the40min in last administration, mice were put in a250mL jar where is filled with15g soda limes, and then immediately closed the jarwith rubber stopper, which is coated with white petrolatum, tightly and start timing. Inorder to ensure its tightness, white petrolatum was coated at the junction part betweenthe jar and rubber stopper rapidly and uniformly. The survival time of mice wererecorded according to the indicator of breathing stopping. As the same grouping andadministration methods, the other48male Kunming mice were treated with400mg/kg NaNO2after the last administration, and recorded the survival timeimmediately according the indicator of breathing stopping. In order to exclude thesubjective timing error, the program of administration and timing were done bydifferent experimenters in above experiments.The other40male Kunming mice were randomly divided into four groups and administered with the same way for14days. Then collected there anticoagulatedblood samples to detect the effects of PHM on the hemoglobin levels in normal mice.Results:1. Effects of PHM on the physical fatigue of CF mice:The data indicated that PHM had no effect on the body weight of CF mice. Overthe18-day training period, CF mice that treated with PHM showed a decrease in thenumber of electric shock (Con:137.08±53.01versus PHM280and560mg/kg:93.67±40.45and79.00±33.48) when compared with those treated with vehicle onthe17th day. On the19th day, the results also showed significant benefits in reducingthe number of electric shock (Con:240.08±88.80versus PHM280and560mg/kg:133.17±55.07and115.08±40.81) in CF mice treated with PHM at the doses of280,560mg/kg during the exhaustive exercise. Compared with the Con group, the positivedrug modafinil have a significant effect of decreasing the number of electric shock ofmice on the first day (Con:105.00±59.19versus modafinil13mg/kg:51.75±26.49),and this effect can be last to the19th day (Con:240.08±88.80versus modafinil13mg/kg:85.17±45.04) and there is no any phenomenon of tolerence.The MG and LG (MG:0.91±0.26; LG:22.24±2.92) levels of CF control groupwere significantly lower than home cage control group and the SUN and CK (SUN:7.50±2.09; CK:0.85±0.27) levels were significantly higher than it. These resultsalso suggested that the energy material of CF mice was decreased and theaccumulation of metabolites, which could induce fatigue, was increased. However,these abnormal changings of CF mice could be reversed by PHM and even relieved tothe normal levels. The CF mice treated with PHM560mg/kg showed a significantlyeffect on increasing the levels of MG and LG (MG:1.28±0.24; LG:40.86±10.19),and decreasing the levels of SUN, SLA and CK (SUN:5.77±1.31; SLA:5.67±1.66;CK:0.61±0.19). Those treated with PHM280mg/kg showed significantly effect ondecreasing the levels of SLA and CK (SLA:5.56±1.99; CK:0.62±0.34), and thosetreated with PHM140mg/kg significantly increased the level of MG and LG (MG:1.19±0.29; LG:32.84±9.5). The positive control drug modafinil could decrease thenumber of electric shock significantly, but failed to improve the levels of thesefatigue-related biochemical parameters.2. The preventive anti-fatigue effect of PHM:After the administration of vehicle or PHM during14days, the increase of bodyweight has no any significant difference, it indicates that PHM has no significant effect on the increase of mice body weight. Compared with control group, the numberof electric shock of mice administered with PHM was markedly decreased at differenttime points after the last administration. It showed a markedly effect in decreasing theshock times at40min (Con:209.82±20.65versus PHM280and560:122.91±21.61and135.33±22.18),8h (Con:247.00±35.02versus PHM560:159.18±22.64),48h(Con:172.83±25.77versus PHM560:96.00±18.00), and in the exhaustive exerciseat72h (Con:296.18±37.36versus PHM140,280and560:206.20±26.72,203.73±29.98and171.73±27.61). Compared with control group, the number of electricshock of mice administered with the positive drug modafinil was markedly decreasedat40min (Con:209.82±20.65versus modafinil13mg/kg：122.18±104.97) and8h(Con:247.00±35.02versus modafinil13mg/kg：149.67±93.07), but this effect wasvanished after the24h.Compared with the Con group, the detecting results of biochemical parameterindicated that PHM280and560mg/kg could lead to a marked increase of MG (Con:1.26±0.06versus PHM280and560:1.49±0.10and1.48±0.07) and LG (Con:18.72±1.59versus PHM280and560:26.85±2.06and28.01±2.04) levels and to asignificant decrease of SLA (Con:9.13±0.4versus PHM280and560:7.5±0.32and7.38±0.34) and CK (Con:0.87±0.06versus PHM280and560:0.69±0.06and0.66±0.56) levels. PHM140mg/kg could induce a significant decrease of SLA level(PHM140:7.45±0.33); PHM280mg/kg could induce a significant decrease of SUNlevels (PHM280:5.70±0.30). In addition, the blood ATP levels (Con:3.65±0.50versus PHM560:5.17±0.46) could be increased markedly by PHM560mg/kgadministration.3. The antihypoxia effect of PHM:After the administration of PHM at a frequency of twice a day during two weeks,the survival times of PHM group mice were markedly increased in the experiment ofatmospheric hypoxia (Con:23.63±2.01versus PHM140mg/kg:27.29±1.76; PHM280mg/kg:28.64±2.11; PHM560mg/kg:29.51±2.34) and the experiment ofNaNO3poisoning (Con:7.99±0.67versus PHM140mg/kg:9.89±1.09; PHM280mg/kg:10.40±0.58; PHM560mg/kg:10.28±0.82). Compared with each controlgroups, three dosages of PHM could increase the survival time of mice in these twoexperiments. However, after the administration with PHM in two weeks, the levels ofHb were not be effected markedly. Conclusion:In this study, PHM showed significant effects on anti-fatigue and antihypoxia. Thedetecting results of biochemical parameters, which is related to the fatigue andhypoxia closely, indicated that the mechanism of anti-fatigue and antihypoxia of PHMmay increase the energy reserve, and reduce the accumulation of the inducing fatiguemetabolites. And it need another experiments to demonstrate whether the othermechanism of antifatigue and antihypoxia are similar to the cordyceps sinensis, whichis related to reduce the superoxide radicals, and enhance the body’s ability to scavengefree radicals. The PHM, as the cordyceps alternatives in the areas of anti-fatigue andhypoxia, maybe has a good prospect and a great value for the drug research anddevelopment. |
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