Effects Of Vaccinium Bracteatum Thunb. Leaf Extract On Anti-mental Fatigue In Rats And Its Mechanism

INTRODUCTIONMental fatigue is a common phenomenon that either results from sustained mental effort or from psychological and somatic disorders.Important symptoms of mental fatigue comprise cognitive and attentional difficulties,which have adverse effects on task performance and on everyday activities.It is important to find a way to promote the mental fatigue recovery immediately.Pharmaceutical chemicals and biological products can effectively relief fatigue,however,have some side and addictive effects,which couldn't been used without permission from government.Traditional Chinese medicine views the body as an energy system and emphasizes abnormalities in the balance or flow of energy as the sources of disease.Thus,the basic principle of the traditional Chinese medicine therapy is to regulate the homeostasis of the whole body and to normalize the physical disorders.Medicinal plant use has been practiced for centuries,which has allowed for evaluations on a huge portion of the population.Therefore it has its own superiority to relieve fatigue.Many traditional Chinese medicines,such as ginseng,American ginseng, Astragalus mongholicus and integripetal rhodiola herb,and so on,have the anti-fatigue effect.They can strengthen immunity,anti-stress and improve learning and memory ability. But all of them are expensive and rare,also cann't be used in a large area.Vaccinium bracteatum thumb has gained many honors in strengthening immunity since the Song Dynasty.Huge of studies in its fruit blueberry found that,blueberry had excellent effect in decreasing oxidative stress,improving the faculty of memory in aged rats,curing chronic fatigue syndrome and so on,while there are no report in relative studies in vaccinium bracteatum thumb leaf.Vaccinium bracteatum thumb leaf rich in antioxygen components such as anthocyanidin,flavonoids,triterpenoid,and so on.Effects of physical fatigue relief activity has been found by other researchers,but there is no report upon its mental fatigue relief activity,so we investigate its mental fatigue relief activity, relative mechanisms,and screened effective dosage and components in this study.METHODS1.The mental fatigue rat model:40 normal female SD rats are randomized into 4 groups equally based on body weight:24h,48h,and 72h sleep-deprived groups and control group.After a 7d acclimatization period to the facility,Flower-pot technique is used to induce REMs deprivation for 24h,48h and 72h respectively.Then spontaneous movement activity,learning and memory ability and loaded swimming are measured by spontaneous movement box,Morris Water Maze(MWM) and swimming tank respectively to identify mental status of rats after sleep deprivation,in order to assure the best time to induce mental fatigue.2.Preparation of vaccinium bracteatum thumb leaf extrat:20 kg vaccinium bracteatum thumb leaf is collected in Hangzhou,Zhejiang province,in April,and identified by department of phytochemistry,SMMU.Leaves are cleaned and cutted into pieces,then are extracted by 55%ethanol under 55℃for three times.Filtrate are merged and condensed through rotary evaporation technique,in the mean time VLEE are collected. Then we use chloroform,acetic ether,n-butanol and aqua to extract the VLEE in subsequence.All the extracts are kept in -80℃in dark.3.Sixty female SD rats,weight-matched,and randomly assigned to one of six groups: Normal Control group(groupⅠ),negtive control group(groupⅡ),positive control group (groupⅢ),high dose VLEE group(groupⅣ),medium dose VLEE group(groupⅤ),and low dose VLEE group(groupⅥ).All the groups are fed through intragastric administration for 27 days.Then we implement 72h S-D onto groupⅡ,Ⅲ,Ⅳ,ⅤandⅥ. After that,spontaneous movement activity,Morris water maze and loaded swimming test are employed to assess the anti-fatigue effects of VLEE.Kits are used to test activity of MDA,T-AOC and SOD in brain stem.High performance liquid chromatography(HPLC) is used to measure the monoamine in rat brain stems and MAO kits to test activity of MAO.4.Eighty-four female SD rats,weight-matched,and randomly assigned to one of seven groups:Normal Control group(groupⅠ),negtive control group(groupⅡ),positive control group(groupⅢ),aqua extract group(groupⅣ),chloroform extract group(groupⅤ),and acetic ether extract group(groupⅥ) and n-butanol extrat group(groupⅦ).All the groups are fed through intragastric administration for 27 days.Then we implement 72h S-D onto groupⅡ,Ⅲ,Ⅳ,Ⅴ,ⅥandⅦ.After that,spontaneous movement activity, Morris water maze and loaded swimming test are employed to assess the anti-fatigue effects of VLEE.Kits are used to test activity of MDA,T-AOC and SOD in brain stem.5.Statistical analysis:Data are expressed as means±S.E.M.The statistical analysis of the data was carried out by one-way ANOVA followed by Student's Newman Keuls test. In all comparisons,p＜0.05 was considered significant.RESULTS 1.The results of rat mental fatigue model(1) Spontaneous movement activityAfter different period of time S-D,compared with the control group,the locomotor activity of the 48h S-D and 72h S-D group are significantly reduced,and the spontaneous movement frequence is notably lower than the control group(p＜0.05),72h S-D group is even lower;Average velocity in all S-D groups is reduced,only 72h S-D group has statistical difference with the control group(p＜0.05).(2)Morris water mazeAfter different period of time S-D,compared with the control group,the latency time and total path of the 24h S-D group and 48h S-D group are longer than that of the control group,but still have no statistical difference;While the latency time,total path and swimming velocity to find the hidden platform in water maze of the 72h S-D group are significantly longer than that of the control group(p＜0.05),and the latency time have statistical difference with its time before S-D.(3) Loaded swimming timeCompared with the control group,loading swim time in all S-D groups decreased after different time of S-D,and 48h S-D and 72h S-D have statistical difference(p＜0.01).2.The results of solvent extractionAfter different solvent extraction,we get 455g,80g,3g,23g and 163g extract of VLEE, chloroform,acetic ether,n-butanol and aqua extract respectively.3.Dose-response effect of VLEE on rat mental fatigue(1) Rat weight between different VLEE dosage groups have no notable change.(2) Impacts on praxiology test of rats:Compared with groupⅡ,the locomotor activity of the other groups are increased,and the OFT scores in these groups are better than groupⅡ,and have dose-response effect in VLEE,while groupⅢ,ⅣandⅤhave statistical difference with groupⅡ(p＜0.05);OFT score of groupⅣandⅤhave no statistical difference with groupⅢ.The latency time of groupⅡis longer than that of the groupⅠ,Ⅲ,Ⅳ,ⅤandⅥ,while groupⅢ,ⅣandⅤhave statistical difference with groupⅡ(p＜0.05);The latency time of the groupⅣandⅤhave no statistical difference with groupⅢ. The swimming time of the groupⅡis shorter than that of the other groups,and have dose-response effect among the VLEE intervention groups,the swimming time of groupⅢ,ⅣandⅤare significantly longer than that of the groupⅡ(p＜0.05).Swimming time of groupⅣandⅤhave no statistical difference with groupⅢ.OFT scores,the latency period in Morris water maze and loaded swimming time in groupⅢ,ⅣandⅤhave no statistical difference with groupⅠ.(3) Impacts on brain stem anti-oxidative ability:After sleep deprivation,MDA concentration in brain stem of groupⅡ,ⅤandⅥare significantly higher than groupⅠ(p＜0.05),in the mean time T-AOC and SOD activity are notably increased accordingly(p＜0.05);While groupⅢandⅣcan reduce MDA concentration sharply(p＜0.05),and remedy the SOD level to normal when compared with groupⅡ(p＜0.05).(4)Impacts on brain stem monoamine level:Compared with Normal control group,NE concentration in rat's brain stem of negative and high dose group are reduced dramatically(p＜0.05),and 5-HT concentration have no statistical difference among the three groups,MAO concentration also have no statistical difference among the three groups.4.Studies on Fraction with Mental fatigue relief Activity from Vaccinium bracteatum Thunb.Leaf extract(1) Rat weight between different extracts groups have no notable change.(2) Impacts on praxiology test of rats:After 72h S-D,compared with normal control (groupⅠ),the locomotor activity frequency and reference memory ability in Morris water maze decrease dramatically(p＜0.05),and the time of loaded swimming reduces significantly(p＜0.05) in negative control group;Compared with negative control group, the locomotor activity frequency in positive control and all the dissolvant extract group have dramatical improvement(p＜0.05),reference memory ability in Morris water maze in positive control,aqua extract and chloroform extract group are improved significantly(p＜0.05);There is no difference in locomotor activity,reference memory ability and loaded swimming time between positive group and aqua extract group.(3) Impacts on brain stem anti-oxidative ability:After 72h S-D,MDA concentration in brain stem of negative control group(groupⅡ) is significantly increased compared with normal control group(groupⅠ)(p＜0.05),in the mean time T-AOC and SOD activity are notably increased accordingly(p＜0.05);While positive control(groupⅢ) and aqua extract groups(groupⅣ) can reduce MDA concentration sharply(p＜0.05),and remedy the SOD,T-AOC level to normal when compared with negative control group(groupⅡ)(p＜0.05).CONCLUSIONS1.After 72h S-D,locomotor activity frequency,reference memory ability and loaded swimming time decrease sharply in rats,it means mental fatigue can be induced by long time of S-D.2.Oxidative stress and monoamine transmitters imbalance can be induced by long time of S-D in rat's brain,while high dosage of VLEE could significantly elevate spontaneous movement frequence,shorten latency period in MWM and prolonged loaded swimming time.In the mean time,it can relieve oxidative stress level,facilitate monoamine transmittrers level into the normal one.3.VLE's aqua and chloroform extract could notably prolonged its latency to sleep while shorten the sleeping time caused by sodium pentobarbital.In the mean time,they can shorten latency period in MWM,elevate spoteneous movement frequency and decrease oxidative stress level.So we suppose that there may be include some active components in aqua and chloroform extract of VLE to against mental fatigue.