the world health organization ( who ) introduced the global recommendations on physical 
 activity for health in 20101 , which 
 encourages people of all ages to carry out moderate levels of physical activity .
however , 
 after their first session of moderate - intensity exercise , individuals often experience 
 muscle soreness because they do not have sufficient time to adapt to various factors , such 
 as increased mitochondrial activity leading to malondialdehyde ( mda ) production from lipid 
 peroxidation of the plasma membrane2 , 3 .
this phenomenon can cause muscle damage , 
 leakage of creatine kinase ( ck)2 , 4 into the circulation , and inflammation5 , 6 . 
 then
, c - reactive protein ( crp ) is generated to stimulate the influx of inflammatory cells to 
 repair muscular damage7 .
neutrophils , 
 lymphocytes , and monocytes are inflammatory cells with important roles in the damage / repair 
 of muscle tissue due to exercise - induced injury .
neutrophils and monocytes repair muscle 
 via oxidative or proteolytic modification to remove tissue debris in 
 injured muscle8 .
this process (  acute 
 inflammatory response  ) causes delayed onset of muscle soreness after exercise9 .
the inflammation seems to be important for 
 healing , but rapid recovery from previous exercise allows continuation of exercise training . 

therefore , the search for an antioxidant supplement that can scavenge excess 
 exercise - induced free radicals , resulting in reduced muscle soreness after a single session 
 of moderate - intensity exercise , is important so that novice trainers can continue to 
 exercise to improve their health10 .
phyllanthus amarus ( pa ) is a plant that grows in tropical / subtropical 
 areas , including thailand .
it comprises phytochemicals such as alkaloids , flavonoids , 
 hydrolyzable tannins , major lignans , and polyphenols5 , 
 11 .
several studies have revealed that pa 
 has anti - inflammatory12 and analgesic 
 properties13 .
recently , we reported on 
 the antioxidant and antinociceptive effects of pa on recovery after a single session of 
 high - intensity exercise in sedentary male subjects5 .
however , a single session of moderate - intensity exercise was also 
 shown to produce lipid peroxidation , inflammation , and damage in muscle2 , 4 .
we suspected that 
 antioxidant supplementation is needed to reduce the risk of these adverse processes . 

accordingly , we hypothesized that pa may confer beneficial effects on recovery after a 
 single session of moderate - intensity exercise ( which is recommended for health promotion in 
 sedentary individuals ) .
hence , we investigated the acute effect of pa on oxidative stress 
 and damage , inflammation , and soreness in muscles after a single session of 
 moderate - intensity exercise .
subjects provided written consent to participate in the study after receiving a written and 
 verbal explanation of its requirements .
the study protocol was approved by the ethics 
 committee of khon kaen university ( khon kaen , thailand ) .
the study conformed to the 
 standards set by the declaration of helsinki in 2010 ( he531029 ) .
twelve healthy , sedentary , male participants aged 222.90 
 ( meansd ) years were the study cohort .
they were blinded to the composition of the pa or 
 placebo control ( ctl ) they were given and to which treatment they would receive .
supplements 
 were prepared by a researcher who was not involved in the remainder of the study .
none of 
 the participants were smokers ; had neuromuscular , cardiovascular , orthopedic , renal , or 
 liver diseases ; had taken antioxidant supplements or medications ; or undertook exercise 
 regularly .
subjects underwent a physical examination and electrocardiography before 
 enrollment in the study . to ascertain health status before participation in this study
, 
 blood samples were collected after a 12-h overnight fast to measure levels of glucose , 
 creatinine , lipid profiles , and alanine aminotransferase .
winpepi was used to calculate the sample size of the present study based on the method 
 described in the study of fenercioglu et al14 .
previously , we reported that antioxidant supplementation prevented 
 increases in mda levels after exercise .
aerial parts of pa were collected from the campus of khon kaen province 
 ( thailand ) during august and september .
, aerial 
 parts were washed thoroughly under distilled water , oven - dried at 50  c , and powdered in a 
 mechanical grinder .
one - hundred 
 milligrams of dried pa powder was placed into each capsule containing excipients .
each 
 placebo control capsule contained avicel ph101 ( microcrystalline cellulose ; fmc 
 biopolymer , philadelphia , pa , usa ) , aerosil ( desiccant ; evonik industries , 
 beijing , pr china ) , and artificial colors .
the center for research and development of herbal 
 health products of khon kaen university manufactured and controlled the contents of pa and 
 ctl capsules .
pa dose was based on 
 recommendations for the product in thailand ( g 357/42 ; khaolaor laboratories co. ltd .
each pa capsule contained total polyphenol compounds ( 33  mg / g ) and 
 vitamin c ( 1.60  mg/100 g ) as measured by the central laboratory ( thailand ) co. , ltd . 

participants undertook an incremental exercise test on a electromagnetic cycle ergometer 
 ( corival , lode , the netherlands ) fitted with a gas analysis system ( ml206 ; adinstruments , 
 bella vista , nsw , australia ) .
peak oxygen uptake ( vo2 , peak ) was determined if 
 any of the following was achieved : vo2 reached a plateau with 
 increased workload ; a respiratory gas exchange ratio > 1.15 ; heart rate ( hr ) reached a 
 maximum hr ( calculated using the equation : 220age ) ; and maximal symptoms of dyspnea and 
 fatigue using the ratings of perceived dyspnea and perceived exertion scales .
also , 
 vo2 , peak was determined if the participant could not maintain a cycling speed 
 of 60  rpm .
the relationship between workload and vo2 from the test was used to 
 determine the workload for the subsequent exercise session . room temperature and humidity 
 were 250.5  c and 481.8% , respectively .
one week after the vo2 , peak test , participants returned to the laboratory to 
 participate randomly in three - day phases at one - week apart to avoid the carryover effect .
on 
 the first day of both phases , they consumed two capsules of pa or ctl 20  min before 
 undertaking exercise with 3-min of warming up on the cycle ergometer .
then , they carried out 
 moderate - intensity exercise ( 65%vo2 , peak ) for 20  min .
subsequently they consumed 
 four capsules of the same supplement ( two capsules each after lunch and dinner ) on the same 
 day of the exercise .
this supplementation regimen was continued for the next two days after 
 exercise ( two capsules / time ; t.d.s . ) . at each visit ,
a 12-ml blood sample was collected from 
 the antecubital vein of each participant immediately before and after exercise , and 12-ml 
 blood samples were also collected at 24  h and 48  h after exercise .
one week before and 
 during the experiment , subjects were asked to drink no more than one glass of fruit juice 
 and two cups of coffee / tea per day .
fruit juices especially rich in antioxidants ( e.g. , 
 orange juice ) were to be avoided .
each blood sample was divided into three parts : 6  ml into ethylenediaminetetraacetic acid 
 ( edta)-lined tubes , 2  ml into lithium heparin - lined tubes , 4  ml into clotting tubes .
mol / l ) was then added to one edta tube 
 ( 2  ml ) to precipitate protein for the measurement of vitamin c levels .
all tubes were 
 centrifuged at 3,000  g for 15  min at 4  c .
the upper layer was 
 transferred to a microcentrifuge tube and stored at 80  c until assay .
levels of ck and 
 high - sensitivity c reactive protein ( hs - crp ) and a complete blood count were ascertained in 
 the clinical laboratory of srinagarind hospital ( faculty of medicine , khon kaen university ) . 

levels of mda , vitamin c , and nitric oxide radicals ( no ) were measured by our 
 research team .
mda levels were measured using 
 the thiobarbituric acid ( tba ) test according to the method of draper15 .
the method of zhang et al.16 was used to measure plasma levels of vitamin c. for measurement of 
 plasma levels of no , the inno nitric oxide measuring system and sensor 
 ( innovative instruments inc . , tampa , fl , usa ) was used .
ck activity in serum was assessed as 
 an indicator of muscle damage using standard automated laboratory methods with a 
 cobas c 502 analyzer ( roche , basel , switzerland ) .
hscrp level was determined 
 using a particle - enhanced immunoturbidimetric assay . in this assessment ,
levels of human 
 hscrp agglutinated with latex particles coated with monoclonal anti - crp antibodies were 
 determined by turbidimetric means . a mechanical algometer ( fpk / fpn ;
wagner instruments , 
 riverside , ct , usa ) was used to measure the pressure - pain tolerance of the quadriceps muscle 
 at the mid - thigh point . during measurement , participants were asked to indicate verbally 
 when the pressure became intolerable .
they completed food records for the three days before the 
 experiment ( two weekdays and one weekend day ) .
records were analyzed for dietary composition 
 using inmucal ( mahidol university , bangkok , thailand ) .
participants were instructed to keep 
 records of physical activities for three days before the experimental ( two weekdays and one 
 weekend day ) .
two - way analysis 
 of variance with repeated measures was used to compare the pa and ctl groups at each time 
 point during recovery . a paired t test with bonferroni correction
statistical analyses were carried out using pasw 
 statistics v18 ( spss , chicago , il , usa ) .
anthropometric and physiological characteristics are summarized in table 1table 1.anthropometric and physiologic characteristics of participantsmean  sdage ( yrs)22.02.90height ( m)1.700.06body weight ( kg)63.610.7bmi ( kg / m)22.83.3bf ( % ) 19.59.0fm ( kg)12.57.3ffm ( kg)48.46.6waist circumference ( cm)76.77.5hip circumference ( cm)94.86.1waist : hip ratio0.810.04vo2 , peak ( ml / kg body weight / min)36.95.2work loadmax ( w)136.132.4% vo2 , peak during exercise in ctl 
 treatment70.810.9% vo2 , peak during exercise in pa 
 treatment68.418.9values are the mean  sd ; n=12 for each group . bmi : body mass index ; % bf : percentage 
 of body fat ; fm : fat mass ; ffm : fat free mass ; vo2 , peak : peak oxygen 
 consumption ; ctl : placebo control ; pa : p. amarus . with regard to vo2 , peak ,
physiological 
 parameters , dietary composition , and energy expenditure were not significantly different 
 between the pa and ctl groups ( table 
 2table 2.daily dietary composition and energy expenditure of participantsctlpaprotein ( g)122.930.6125.435.6fat ( g)89.330.278.017.4carbohydrate ( g)254.371.7239.262.9fiber ( g)5.62.07.12.1thiamine ( mg)1.30.41.20.5riboflavin ( mg)1.91.01.40.5niacin ( mg)20.76.618.66.5vitamin c ( mg)23.218.427.112.2vitamin a ( re)746.11388.8710.41382.4energy intake ( kcal / day)2289.2475.82112.0423.2energy expenditure ( kcal / day)2292.1388.12127.9654.6values are the mean  sd ; n=12 for each group .
ctl : placebo control ; pa : p. 
 amarus ; vitamin a 1 re = 12 g beta carotene ) .
baseline blood parameters were within normal ranges ( table 3table 3.baseline blood parameters of participantsmean  sdblood glucose ( mm ) 4.60.3creatinine ( mol / l ) 0.010.001alt ( u / l ) 23.124.9tg ( mm)1.10.3tc ( mm)2.61.4ldl ( mm)2.80.9hdl ( mm)1.60.4values are the mean  sd ; n=12 . alt : alanine transferase ; tg : triglycerides ; tc : 
 total cholesterol ; ldl : low - density lipoprotein ; hdl : high - density lipoprotein ) .
bmi : body mass index ; % bf : percentage 
 of body fat ; fm : fat mass ; ffm : fat free mass ; vo2 , peak : peak oxygen 
 consumption ; ctl : placebo control ; pa : p. amarus values are the mean  sd ; n=12 for each group .
ctl : placebo control ; pa : p. 
 amarus ; vitamin a 1 re = 12 g beta carotene values are the mean  sd ; n=12 .
alt : alanine transferase ; tg : triglycerides ; tc : 
 total cholesterol ; ldl : low - density lipoprotein ; hdl : high - density lipoprotein vitamin c levels in the pa group were significantly higher than those in the ctl group 
 immediately after moderate - intensity exercise ( p<0.05 ) and 24  h ( p<0.01 ) and 48  h 
 after moderate - intensity exercise ( p<0.05 , table 
 4table 4.levels of mda , vitamin c , no , ck , and hs - crp before exercise ( pre - exercise ) , as 
 well as immediately after moderate - intensity exercise ( post - exercise ) and 24  h 
 ( post-24 h ) and 48  h ( post-48 h ) after moderate - intensity exercisetimepre - exercisepost - exercisepost-24 hpost-48 hmda ( nm)ctl1.740.453.090.823.480.792.870.95pa1.720.402.590.642.230.742.080.63vitamin c ( g / ml)ctl7.971.217.181.057.451.207.351.06*pa7.821.3710.981.8211.911.9610.921.93*no ( nm)ctl141.058.4134.862.0131.527.3113.848.4pa145.452.1165.370.0134.616.8144.364.2ck ( u / l)ctl146.224.3158.625.8 * 167.228142.321.4pa149.324.4157.924.3 * 151.723.7161.3432.8hs - crp ( mg / l)ctl0.600.190.650.20.740.250.660.28pa0.530.170.520.170.710.280.810.25values are the mean  se ; n=12 for each group .
ctl : placebo control ; pa : p. 
 amarus ; mda : malondialdehyde ; no : nitric oxide radical . * significantly 
 different from pre - exercise ( p<0.05 ) , # significantly different from the ctl group 
 at the same time point ( p<0.05 ) )
when vitamin c levels at 48  h after moderate - intensity exercise were 
 compared with those before exercise , they were significantly higher in the pa group but 
 lower in the ctl group ( p<0.05 ) . however , mda levels and no levels were not 
 significantly different between the pa and ctl groups at any time point ( table 4 ) .
ctl : placebo control ; pa : p. 
 amarus ; mda : malondialdehyde ; no : nitric oxide radical .
* significantly 
 different from pre - exercise ( p<0.05 ) , # significantly different from the ctl group 
 at the same time point ( p<0.05 ) plasma levels of ck were increased significantly immediately after exercise compared with 
 those before exercise ( p<0.05 , table 4 ) and 
 returned to pre - exercise levels in both supplementation groups . moreover , hscrp levels were 
 not significantly different between the pa and ctl groups at any time point ( table 4 ) .
neutrophil counts were significantly 
 higher immediately after exercise than before exercise in the ctl group ( p<0.05 ) . 
 however , the lymphocyte count and monocyte count were not significantly different between 
 the supplement treatments at any time point ( table 
 5table 5.counts of neutrophils , lymphocytes , and monocytes before exercise ( pre - exercise ) , 
 as well as immediately after moderate - intensity exercise ( post - exercise ) and 24  h 
 ( post-24 h ) and 48  h ( post-48 h ) after moderate - intensity exercisetimepre - exercisepost - exercisepost-24 hpost-48 hneutrophils ( 10/l)ctl3.650.314.110.36 * 3.560.303.410.46pa3.230.473.460.433.400.223.580.41lymphocytes ( 10/l)ctl2.060.182.580.342.050.182.160.17pa2.080.212.330.221.950.182.110.21monocyte ( 10/l)ctl0.460.040.570.070.470.060.420.06pa0.520.070.590.080.450.070.520.08values are the mean  se ; n=12 for each group .
the pain tolerance of both legs was not significantly different between the 
 pa and ctl groups at any time point ( table 
 6table 6.levels of muscle soreness in the right leg and left leg before exercise 
 ( pre - exercise ) , as well as immediately after moderate - intensity exercise 
 ( post - exercise ) and 24  h ( post 24 h ) and 48  h ( post 48 h ) after moderate - intensity 
 exercisetimepre - exercisepost - exercisepost-24 hpost-48 hright legctl4.70.44.80.45.00.65.30.6pa4.40.24.50.54.90.64.80.5left legctl4.70.44.60.45.00.55.40.5pa4.60.44.40.44.90.55.10.5values are the mean  se ; n=12 for each group .
ctl : placebo control ; pa : p. 
 amarus . * significantly different from pre - exercise ( p<0.05 ) values are the mean  se ; n=12 for each group .
this is the first study to show that acute supplementation of pa increased plasma levels of 
 vitamin c during the recovery period after a single session of moderate - intensity exercise 
 in healthy , sedentary participants .
however , pa supplementation did not alter levels of 
 lipid peroxidation , damage , inflammation , or soreness in muscle .
a single session of moderate - intensity exercise was shown to contribute to muscle damage as 
 reflected by increased levels of ck .
this exercise - induced muscle damage is in agreement 
 with two previous studies2 , 4 . both studies reported increased levels of ck after running 
 on a treadmill for 2030  min at 60% vo2 , peak .
( 2012 ) demonstrated 
 muscle damage with increased lipid peroxidation , but seifi - skishahr et al .
we found that the neutrophil count 
 increased immediately after exercise in the control group .
exercise also decreased vitamin c 
 levels a few days later in the control group .
this observation is supported by a study that 
 found decreased vitamin c levels after a single session of exercise without prior 
 supplementation in untrained , healthy subjects18 .
the mechanical forces occur 
 at the sarcomere level and induce structural damage to contractile proteins and plasma 
 membranes19 . however , the muscle damage 
 observed in the present study appeared to be small because ck leakage was minimal and the 
 increases in neutrophil count were low , and it only occurred immediately after exercise 
 without changes in inflammation .
the unaltered inflammation may explain why there was no 
 change in pressure pain tolerance when the ck was increased after exercise .
our research team has examined , using a similar protocol , these affects after 
 high - intensity exercise5 .
our results in 
 that study confirmed the beneficial effect of pa supplementation in subjects after exercise 
 that produced high oxidative stress .
we found that pa supplementation increased plasma 
 levels of vitamin c immediately after high - intensity exercise , which might result in 
 decreased lipid peroxidation and muscle soreness two days later5 .
vitamin c levels increased over pre - exercise values 
 throughout the recovery period in the present study but increased only immediately after 
 exercise in our previous study5 . this 
 disparity in results may suggest that moderate - intensity exercise requires ( as antioxidant 
 activity ) less vitamin c than high - intensity exercise .
increases in levels of other 
 antioxidants ( e.g. , glutathione peroxidase , glutathione reductase , catalase ) may be 
 responsible for free radicalscavenging20 , 21 .
further research investigating the effect 
 of pa supplementation on these antioxidants during recovery from moderate - intensity exercise 
 is needed to confirm this hypothesis . in the present study ,
pa supplementation increased antioxidant levels as reflected by 
 increased vitamin c levels in plasma after moderate - intensity exercise .
this finding may 
 show pa supplementation to be an additional source of vitamin c that can not be synthesized 
 by the human body .
the pa - induced increase in plasma vitamin c level is supported by 
 previous studies in the human body5 and 
 rat22 .
although the vitamin c content 
 in the pa in the present study may have been very small ( 1.60  mg/100  g ) , a previous study 
 reported that supplementation with less than 200  mg of vitamin c increased the absorption 
 rate to 98%23 .
this may have contributed 
 to the increased levels of plasma vitamin c in the pa group in the present study .
taking 
 this together with the lack of inflammation and soreness in muscle , it is appropriate to 
 recommend pa supplementation before 20  min of moderate - intensity exercise for health 
 promotion . exercise for 20  min is practical for healthy , sedentary thai participants who , 
 under normal circumstances , do not like to exercise for longer .
importantly , elderly 
 individuals24 , patients ( e.g. , those 
 with diabetes mellitus25 , cancer26 , or pre - hypertension27 , and cigarette smokers3 who have high oxidative stress even at rest may need antioxidant 
 supplementation during exercise at this intensity . the effect of pa supplementation on 
 oxidative stress and damage , inflammation , and soreness in muscle after a longer - duration of 
 exercise at moderate intensity in elderly individuals , cigarette smokers , and patients with 
 metabolic problems should be studied further . in this study ,
the pa in each capsule also contained 33  mg / g of total polyphenol compounds 
 ( 19.8  mg / day ) .
the effects of polyphenol compounds on biomarkers of oxidative stress and 
 muscle damage induced by exercise have been studied28 .
intra and kuo29 
 suggested that , even though the physiologic level ( 0.11 m ) of dietary catechins ( which 
 contain polyphenol compounds ) is much lower than that of vitamins , dietary catechins 
 contribute to total antioxidant capacity even in the presence of vitamins .
we did not 
 measure levels of polyphenol compounds in blood in the present study .
together with other 
 antioxidants in the pa11 , polyphenol 
 compounds may have played a part as antioxidants during exercise recovery in the present 
 study .
their actions may have preserved vitamin c levels after exercise throughout the 
 recovery period . in accordance with a review article examining the impact of antioxidant supplementation on 
 high - intensity exercise30
, we prepared 
 and stored the pa in the present study carefully to prevent oxidation and degradation of pa . 

moreover , we assessed the diet composition and physical activity associated with both 
 treatments , and the results confirmed that there was no difference between them .
a one - week 
 washout period was sufficient to eliminate the effect of the previous treatment because 
 supplementation took only three days .
this was confirmed by data at the start of each 
 treatment , which were not significantly different .
a limitation of our study was that we measured only plasma levels of mda and vitamin c to 
 determine oxidative status , but we stored and analyzed them carefully within 3 months . 

moreover , one might question whether vitamin c , which is located in aqueous compartments , 
 can be used to scavenge lipophilic radicals .
in fact , there is a well - described dependency 
 between these two antioxidants : vitamin c recycles vitamin e via the 
 tocopheroxyl radical31 .
importantly , unaltered levels 
 of hscrp suggest that pa supplementation did not produce harmful effects in the male 
 sedentary participants .
acute pa 
 supplementation increased plasma levels of vitamin c throughout the two days after 
 moderate - intensity exercise .
however , pa supplementation did not affect lipid peroxidation 
 or inflammation , damage , and soreness in muscle during the recovery period .
 [ purpose ] we aimed to evaluate the effects of phyllanthus amarus ( pa ) on 
 oxidative stress and damage , inflammation , and soreness in muscle after a single session 
 of moderate - intensity exercise . 
 [ subjects and methods ] twelve men randomly participated in 
 2 , three - day phases with a one - week washout period . on the first day , participants 
 consumed two capsules of pa or placebo control ( ctl ) before 20  min of cycling . 
 they then 
 consumed four capsules on the same day after exercise and six capsules / day for the next 
 two days . 
 blood samples were obtained before , immediately after exercise and 24  h and 48  h 
 after exercise . 
 the pain tolerance was measured at both legs . 
 [ results ] plasma vitamin c 
 levels in the pa group were higher than those in the ctl group after exercise . at 48  h 
 after exercise , 
 vitamin c levels were higher in the pa group , but those in the ctl group 
 were lower than the pre - exercise levels . 
 however , plasma levels of creatine kinase were 
 increased in both groups after exercise compared with the pre - exercise levels . 
 the 
 neutrophil count was higher immediately after exercise than the pre - exercise levels in the 
 ctl group . [ conclusion ] 
 acute supplementation with pa improves antioxidant status after a 
 single session of moderate - intensity exercise . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION