an acute bout of physical exercise depending on duration and intensity is known to induce changes of the immune response of both the innate and the adaptive immune system .
changes of leukocytes are regularly accompanied by an exercise - induced increase of inflammatory cytokines such as il-6 , il-10 , and il-1ra .
eccentric exercise like downhill running is a special exercise mode which is known to cause substantial muscle damage followed by a pronounced inflammatory response .
this muscle damage is accompanied by muscle soreness , which regularly occurs 1248 hours after the eccentric exercise bout and which was termed delayed onset of muscle soreness ( doms ) .
doms is furthermore accompanied by stiffness or tenderness on palpation and a loss of muscle force [ 2 , 3 ] . actually , there are two different mechanisms that explain the induction of doms .
thereby , the increased tension per individual cross bridge and the stretching forces on sarcomeres induce microtrauma in muscle fibers .
microscopically , observations show ruptures of z - discs and a - bands and the subsequent dissolution of sarcomere structures in individual muscle fibers [ 5 , 6 ] .
structural damage is accompanied by increased cytosolic calcium concentrations , which activate proteolytic enzymes and increase cell membrane permeability .
the result is release of muscle enzymes such as creatine kinase ( ck ) into the interstitial fluid .
secondly , it is suggested that doms is based on inflammatory responses after eccentric exercise .
thereby , several studies described the occurrence of a local inflammation in muscle tissue . within this inflammation , migration of neutrophils and macrophages into the damaged tissue several hours after exercise is observed .
neutrophils eliminate cell fragments and perform reorganization processes via reactive oxygen species . both neutrophils and macrophages
are involved in the release of cytokines such as il-1 , tnf- , and il-6 [ 3 , 8 ] .
some studies investigated the effect of nonsteroidal anti - inflammatory drugs ( nsaids ) or antioxidants [ 911 ] .
others examined the application of various physical therapy methods [ 12 , 13 ] . regarding the use of antioxidants ( e.g. , vitamin c and vitamin e ) , no clear effect on reduction of muscle damage was found .
similarly , neither cryotherapy nor stretching exercise was effective in treating exercise - induced muscle damage [ 12 , 13 ] .
while some studies showed no changes after treatment with nsaids [ 9 , 10 ] , others demonstrated lower symptoms of doms [ 14 , 15 ] .
however , athletes have to consider several side effects of the regular use of nsaids such as upper gastrointestinal bleeding and perforation , renal injury , liver injury , and heart failure . therefore , the current study intended to test the effect of the well tolerated natural combination medicine tr14 on doms .
it is composed mainly of plants of the compositae family ( chamomile , common daisy , common marigold , common yarrow , mountain arnica , narrow - leaved purple coneflower , and purple coneflower ) .
for example , for both chamomile and coneflower , anti - inflammatory effects have been shown [ 17 , 18 ] .
the residual plant components of tr14 are composed of different plant families ( comfrey , common witch - hazel , deadly nightshade , st .
john 's wort , anti - inflammatory effects have been shown in an in vivo study .
tr14 also consists of two mineral components ( calcium sulfide and mercurio - amidonitrate ) .
evidence for immunomodulatory effects of tr14 comes from humans [ 1921 ] , animals [ 22 , 23 ] , and in vitro [ 21 , 24 ] studies .
gonzlez de vega et al . demonstrated in a human clinical trial that tr14 decreased pain and improved joint function in acute ankle sprain when topically applied .
recently , we observed in a human clinical trial that tr14 has effects on various immune parameters of the exercise - induced immune response . here , we showed that tr14 reduced the inflammatory response of the innate immune system while the response of proinflammatory cytokines was promoted .
the animal study of oberbaum et al . demonstrated that tr14 increased il-1 in an in vivo sepsis model in rats .
furthermore , lussignoli et al . showed that tr14 significantly reduced il-6 production in rats with edema .
study that tr14 inhibits the secretion of proinflammatory cytokines such as il-1 , cxcl8 , and tnf- in resting as well as activated immune cells .
the present double - blind , randomized , placebo - controlled study follows up our research on effects of tr14 in strenuous exercise paradigm , this time examining exercise - induced muscle soreness .
we hypothesized that tr14 is effective in limiting exercise - induced muscle damage and inflammatory response .
the results might be helpful in optimizing load - recovery cycles during various training regimes thereby improving the training effectivity .
the present study was conducted as a double - blind , randomized , placebo - controlled clinical trial .
this monocenter study was performed at the department of sports medicine , university of giessen .
the study protocol was approved by the local ethics committee and the federal institute for drugs and medical devices ( eudract 2009 - 010898 - 21 ) .
initially , 110 subjects were examined for their medically approved unrestricted participation in sport using an incremental protocol on a treadmill . after the test
, 14 subjects were not included into the study because they did not meet the inclusion criteria of the study ( see below ) .
hence , a total of 96 subjects were enrolled in the study and randomized either to tr14 ( n = 48 ) or to placebo ( n = 48 ) . a total of 95 subjects were analyzed because of one dropout due to a study related adverse event in the placebo group before intake of study medication .
the screening was performed within 8 days prior to study entry . on day one ,
the subjects came fasting to the study center and got a standardized breakfast and the following procedure was performed : inclusion / exclusion criteria rechecking , subject randomization , acute bout of eccentric exercise , data collection at three different time points , and dispensing of study medication ( figure 2 ) . before , immediately after , and three hours after exercise , various analyses were performed ( blood samples , strength measurement , and sf - mcgill pain questionnaire ) .
follow - up visits were performed at 24  h , 48  h , and 72  h after exercise .
the subjects were requested 48  h prior to study participation and during the study to refrain from strenuous physical activity .
the subject characteristics showed only marginal differences between the two groups ( table 1 ) .
subjects were included if they met all inclusion criteria and none of the exclusion criteria .
subjects were eligible for the study if they were male , were aged  18 and 40 years , had bmi  18.5 and < 27.5  kg / m , had maximum relative oxygen uptake ( vo2max ) < 53  ml / kgmin , were healthy , were nonsmokers , had medically approved unrestricted sports participation as shown by diagnostic performance test conducted on treadmill for no longer than three months prior to study entry , and were willing to provide informed consent . if any of the following exclusion criteria existed , the subjects were not included in the study : regular eccentric exercise training , weekly training volume  6 hours , use of dietary supplements , chronic immune deficiency , current infection , heart and/or circulation disorders , musculoskeletal disorders , nothing that requires a systemic therapy , hypersensitivity to botanicals of the compositae family , lactose intolerance , illicit drug or alcohol abuse , and participation in another clinical trial within four weeks prior to study entry .
the subjects were instructed to run downhill ( 10% incline ) on a treadmill ( h / p / cosmos quasar med 4.0 , h / p / cosmos sports & medical gmbh , nussdorf , traunstein , germany ) as a means to eccentric exercise at a heart rate ( hr ) corresponding to 80% of vo2max .
the duration of the eccentric exercise was set to 45  min unless the subjects were forced to quit earlier due to exhaustion .
afterwards , the treadmill speed increased to 8  km / h and the downhill running started .
the study medication was manufactured by biologische heilmittel heel gmbh , baden - baden , according to the german homeopathic pharmacopoeia ( hab ) , current eu - gmp guidelines , and pertinent national regulations .
participants and investigators were blinded to the study medication by identical color , smell , taste , and size .
the 1-to-1 randomization with a block size of eight was conducted by datamap gmbh , freiburg , germany , according to the company internal sop .
the subjects were randomized on day one directly before the first study related procedure was carried out .
tr14 consists of the following active components ( d stands for dilutions in increments , amounts per 300  mg ) : achillea millefolium ( d3 , 15  mg ) , aconitum napellus ( d3 , 30  mg ) , arnica montana ( d2 , 15  mg ) , atropa belladonna ( d4 , 75  mg ) , bellis perennis ( d2 , 6  mg ) , calendula officinalis ( d2 , 15  mg ) , chamomilla recutita ( d3 , 24  mg ) , echinacea angustifolia ( d2 , 6  mg ) , echinacea purpurea ( d2 , 6  mg ) , hamamelis virginiana ( d2 , 15  mg ) , hepar sulfuris ( d8 , 30  mg ) , hypericum perforatum ( d2 , 3  mg ) , mercurius solubilis hahnemanni ( d8 , 30  mg ) , and symphytum officinale ( d8 , 24  mg ) ; and it also consists of the following inactive components : lactose ( 6  mg ) and magnesium stearate ( 1.5  mg ) .
the placebo tablet consists of 300  mg lactose monohydrate and 1.5  mg magnesium stearate .
the subjects received one tablet every 15 minutes until eight tablets were taken ( dissolved in the mouth ) after the exercise test .
in addition , the subjects ingested two tablets 6  h and 10  h after the exercise test . on the following two days
, they received two tablets three times a day and on the fourth day they took two tablets in the morning .
the total study dose was 26 tablets . at each data collection point , approximately 19  ml of blood was taken from the antecubital vein using safety - multifly needle ( sarstedt , nmbrecht , germany ) .
the tube with edta blood ( 2.7  ml edta k tubes , sarstedt , nmbrecht , germany ) was analyzed for white blood cell count using the optoelectronic principle ( xe2100 , sysmex , norderstedt , germany ) .
for the determination of apoptosis and surface markers , the peripheral blood mononuclear cells ( pbmcs ) were isolated by using lithium - heparinized blood and density gradient centrifugation .
subsequently , the cells were labeled with fitc - conjugated ( annexin v , anti - human cd62l , anti - human cd69 , and anti - human cd95r ; immunotools , friesoythe , germany ) and pe - conjugated ( anti - human cd95l ; biolegend , san diego , usa ) antibodies .
next , the lymphocytes were gated by flow cytometry and measured ( coulter epics xl - mcl , beckman - coulter , brea , usa ) . for more details , see works of our own group .
the following parameters were measured using multiplex elisa ( myriad rbm , austin , texas ) in stimulated whole - blood cultures : bdnf , ccl2 , ccl3 , ccl4 , cxcl8 , ccl11 , factor vii , gm - csf , icam-1 , ifn- , il-2 , il-3 , il-4 , il-5 , il-6 , il-7 , il-10 , il-12p40 , il-12p70 , il-15 , il-17 , il-18 , il-23 , il-1ra , il-1 , il-1 , mmp-3 , mmp-9 , scf , tnf- , tnf- , and vegf . here
, 1  ml of whole blood was withdrawn in blood collection tubes ( truculture , edi gmbh , reutlingen , germany ) by safety - multifly needle ( sarstedt , nmbrecht , germany ) .
the collection tubes contained the stimulants lipopolysaccharide ( lps ) from escherichia coli serotype o55:b5 and staphylococcus enterotoxin b ( se - b ) in a concentration of 0.1  g / ml . each tube was incubated for 24  h in a dry block incubator ( vlm gmbh , bielefeld , germany ) at 37c .
after 24  h , a valve separator was inserted into the tube and the samples were stored at 80c until further analyses . for more details , see work of our own group . additionally , six inflammatory mediators were measured in serum samples using multiplex elisa : ccl2 , cxcl8 , il-1ra , il1-  , il-6 , and tnf-. the maximum isometric strength of the anterior ( extension ) and posterior ( flexion ) thigh muscles was used as a marker of exercise - induced muscle damage and recovery . the schnell m3-diagnos multifunctional training and analysis station ( schnell gmbh , peutenhausen , germany ) was utilized for the measuring of the muscle strength .
furthermore , creatine kinase ( ck ) and lactate dehydrogenase ( ldh ) in serum ( 1.2  ml serum z tubes , sarstedt , nmbrecht , germany ) were additionally used as markers for exercise - induced muscle damage . the short form mcgill pain questionnaire ( sf - mpq )
the sf - mpq contains ten questions about relating to the sensory level of pain experience and four questions about relating to the affective status .
in addition , the sf - mpq contains a 5-value likert scale for the overall pain experience and a visual analogue scale ( vas ) for the momentary pain intensity .
statistical analyses were conducted on the intention - to - treat ( itt ) principle and were performed using sas v 9.3 ( sas institute , cary , nc ) .
all randomized and treated subjects were analyzed according to the intention - to - treat principle .
auci was calculated using the trapezoidal rule . in order to identify a potential difference between the two groups , a median test of auci
compliance with exercise protocol was assessed using the following variables : completion of exercise protocol over 45  min ( tr14 : n = 48 , 100% ; placebo : n = 44 , 93.6% ) , time to exhaustion if the subject did not accomplish the exercise test ( tr14 : 45  0  min ; placebo : 40.3  4.6  min ) , and average hr throughout the exercise protocol ( tr14 : 164  12.9  bpm ; placebo : 161.8  10.4  bpm ) .
the maximum isometric strength ( flexion and extension ) decreased after exercise in both groups ( supplemental table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/1693918 ) . the muscle strength ( flexion ) showed a minimum immediately after exercise in the tr14 group and 24  h after exercise in the placebo group .
in contrast , the muscle strength ( extension ) indicated a minimum immediately after exercise in the tr14 group and 3  h after exercise in the placebo group and a slight increase 24  h after exercise in both groups .
additionally , an exercise - induced increase of ck , ldh , and pain score was observed in both groups ( supplemental table 1 ) .
in contrast , ck and the pain score peaked at 24  h after exercise in both groups .
ck levels , which were one of the primary outcome measures , showed borderline statistical significance ( p = 0.05 ) and ldh ( p = 0.06 ) with lower values in the tr14 group ( table 2 ) .
additionally , the subjects were classified into three groups according to their peak ck values : low responders ( < 500 
u / l ) , medium responders ( 5002000  u / l ) , and high responders ( > 2000 
similarly , no group differences were found for either total or vas pain score or for the strength measurements ( extension and flexion ) ( table 2 ) .
thereby , neutrophils and monocytes increased three hours after exercise while lymphocytes increased one hour after exercise and subsequently decreased in both groups ( supplemental table 1 ) .
the tr14 group indicated less pronounced postexercise lymphopenia ( p = 0.02 ) ( table 2 ) .
also , the lymphocyte activation marker cd69 count showed a trend towards a lower level in the tr14 group ( p = 0.07 ) ( table 2 ) .
also , a trend towards lower level neutrocytosis was observed in the tr14 group ( p = 0.10 ) ( table 2 ) .
for all other immune cell numbers and apoptosis and surface markers , no treatment effects could be shown ( table 2 ) .
various anti- ( e.g. , il-1ra and il-10 ) and proinflammatory ( e.g. , il-1 , il-6 , and tnf- ) cytokines reached their maximum three hours after exercise and subsequently decreased to baseline values within 72 hours in both groups ( supplemental table 1 ) .
in contrast , other inflammatory mediators such as icam-1 , il-18 , and il-12p70 showed group differences .
the tr14 group indicated a lower expression of antigen - stimulated icam-1 ( p = 0.01 ) ( table 2 ) .
furthermore , antigen - stimulated cytokine il-18 showed a trend towards a lower level in the tr14 group ( p = 0.09 ) ( table 2 ) . on the contrary
, we found a lower decrease of antigen - stimulated il-12p70 in the verum group ( p = 0.03 ) ( table 2 ) .
for all other inflammatory mediators , no treatment effects could be shown ( table 2 ) .
the antigen - stimulated growth factors ( bdnf , gm - csf , il-3 , scf , and vegf ) were enhanced by exercise in both groups ( supplemental table 1 ) .
there was a lower expression of antigen - stimulated bdnf ( p = 0.03 ) and a trend towards lower values of antigen - stimulated scf ( p = 0.09 ) in the tr14 group ( table 2 ) . on the contrary ,
antigen - stimulated gm - csf demonstrated a trend towards a higher expression in the tr14 group ( p = 0.09 ) ( table 2 ) .
no treatment effects could be found for either il-3 or vegf ( table 2 ) .
the result of the current study demonstrated that muscle damage and inflammatory response were slightly modulated by tr14 after acute bout of eccentric exercise .
while trends towards lower levels on parameters of muscle damage were observed , tr14 affected lymphopenia , cellular and soluble activation markers , lowered exercise - induced neutrocytosis , and reduced the expression of selected growth factors such as bdnf .
whether these effects on the cellular recovery in the postexercise period might have any relevance for the adaptational training response remains to be shown .
the eccentric exercise protocol was effective in inducing distinct muscle damage as indicated by a loss of muscle strength and release of intracellular proteins such as ck and ldh in both groups [ 3 , 7 ] . however , there were no group differences in muscle strength , which is one of the most reliable markers of exercise - induced muscle damage .
we assume that the lack of differences is due to the large variability of muscle damage .
however , our data indicated a trend towards lower levels of serum muscle damage markers following ingestion of tr14 after the eccentric exercise suggesting a protective effect on muscle integrity .
it can be speculated that this effect could be even more pronounced if tr14 ingestion had started before and not after the exercise bout .
previous studies focused on individual components of tr14 on the inflammatory response after exercise [ 31 , 32 ] .
application of isolated arnica demonstrated no effects on muscle damage [ 31 , 32 ] .
they found a more rapid decrease of paw edema in rats treated with tr14 , associated with an improved process of healing .
accordingly , it is concluded that the effect of tr14 seems not to depend on an individual component , but the synergy of all components of tr14 .
regarding the use of ck , it has to be recognized that a single marker of muscle damage is anyway a limitation of our and other studies because there is a large interindividual variability of ck .
therefore , there is still a discussion on whether ck is a reliable marker for muscle damage . in many studies
, the subjects were divided into low , medium , and high responders based on ck activity [ 34 , 35 ] .
paulsen et al . proposed to make this classification for a better presentation and interpretation .
although the ck response showed high interindividual variations , we can not conclude any differences from the p value between the groups ( supplemental table 2 ) .
in addition to muscle damage , the consecutive inflammatory process is considered to be a major cause of doms .
it is known that the expression of various chemokines in damaged muscle tissue is followed by mobilization of leukocytes into the circulation and eventually their infiltration into muscle tissue . in line with previous studies , an exercise - induced increase of circulating leukocytes
it is proposed that lymphocytes initially increase due to redistribution processes from lymphatic and nonlymphatic organs followed by apoptotic cell death and/or redistribution into tissues [ 38 , 39 ] . in this
regard , adhesion molecules such as selectins and icam-1 play an important role in transmigration of lymphocytes to the inflammation site .
ingestion of tr14 induced a lower decrease of lymphocytes , a reduced expression of antigen - stimulated icam-1 , and a trend towards a lower expression of absolute cd69 in the tr14 group .
since cd69 is known to stimulate cell proliferation and cytokine secretion , all of these effects of tr14 are suggested to represent less pronounced activation of the adaptive immune system .
thereby , the lower levels of cell activation and adhesion molecule expression might be responsible for lower emigration of lymphocytes into muscle thereby contributing to the lower lymphopenia .
indirectly , this might also reflect a lower level of damaged muscle tissue . besides components of the adaptive immune system , also the innate
immune system plays an important role in muscle damage . in accordance with previous studies ,
, we showed that the exercise - induced increase of neutrophils was lower by trend after ingestion of tr14 .
in general , it is assumed that neutrophils are the first cells which infiltrate the damaged tissue .
. the lower peripheral increase of neutrophils in the tr14 group is the result of lower mobilization into the blood which might be the result of a lower expression of hematopoietic factors . in line with this data ,
showed a lower level of neutrophils in the tr14 group after a concentric exercise test .
possibly , tr14 affects the number of mobilized neutrophils by inhibition of gm - csf , which leads to reduced recruitment of neutrophils into circulation .
however , such an underlying mechanism could not be confirmed in the current study . in response to exercise , cells of the innate immune system are also known to produce several proinflammatory cytokines . in this regard
, we showed that the proinflammatory antigen - stimulated cytokine il-12p70 showed a higher level after exercise in the tr14 group .
it plays an important role in the regulation of t helper 1 cell ( th1 ) responses . a higher level of il-12p70 indicates a higher type 1 t cell response after exercise .
it was demonstrated in a stimulated human whole - blood model that the production of il-12p70 is inhibited by various stress hormones ( dexamethasone , norepinephrine , and epinephrine ) .
possibly , tr14 caused a reduced stress response and lower production of stress hormones resulting in an extenuated t cell mediated immune response .
demonstrated in an in vivo sepsis model in rats a higher expression of proinflammatory cytokine il-1 in the tr14 group .
similarly , pilat et al . demonstrated a higher expression of the proinflammatory cytokines il-1 and il-1 after a concentric exercise test in the tr14 group .
it is suggested that the higher levels of proinflammatory cytokines have a protective effect   because a previous injection of il-1 showed improved survival in a murine sepsis model .
in contrast , in a study of porozov et al . , a lower expression of proinflammatory cytokines il-1 ,
this study examined the effects of tr14 in an in vitro model , so the methodological component might have an impact on the results .
however , we suggest that the contradictory findings on tr14 might also be caused by a pleiotropic effect induced by variable effects on different target cells . altered expression of proinflammatory cytokines might also be due to redox disturbances which are known to affect mapk and nf-b signaling [ 48 , 49 ] . in this
they suggested that the attenuation of nf-b / mapk by antioxidants might lower proinflammatory response .
however , in the current study , no markers of oxidative stress or related signaling pathways have been analyzed .
it is widely accepted that the exercise - induced muscle damage is part of a muscular adaptation process .
this adaptation is visible in the so - called  repeated bout effect ,  a term which describes lower level of muscle damage and doms after a repeated eccentric exercise bout . in this regard
, it is assumed that in response to muscle damage growth factors are expressed which play a role in the regeneration and adaptation of skeletal muscle . in this
regard , menetrey et al . showed that basic fibroblast growth factor ( b - fgf ) , insulin growth factor type 1 ( igf-1 ) , and at a less intensity nerve growth factor ( ngf ) amplify muscle regeneration in vivo .
in general , growth factors play a role in exercise - induced stimulation of cellular growth , proliferation , and differentiation . in our study
, we analyzed various growth factors like bdnf , scf , gm - csf , il-3 , and vegf .
bdnf belongs to the neuropathic family as well as ngf . in the tr14 group ,
a lower expression of bdnf and a trend towards lower expression of scf were found .
there is some evidence that bdnf plays a role during myogenic differentiation if it is expressed by satellite cells .
similarly , scf is suggested to be involved in the stimulation of muscle - derived stem cell   and plays a major role in hematopoiesis .
the reduced expression of bdnf and scf in the tr14 group might indicate a reduced need for tissue regeneration .
however , if the reduction of muscle damage is the reason for lower growth factors , expression remains to be shown . for the interpretation of the current results , it has to be considered that a stimulated in vitro whole - blood culture was used for analysis of the cytokines and growth factors .
an advantage of using whole - blood culture is that the cellular environment is similar to in vivo blood conditions .
however , the included stimulants lps and seb stimulate primarily monocytes and lymphocytes , respectively [ 58 , 59 ] .
the complete synergy of the cytokines in all compartments is not shown in this model , so that no conclusions can be concluded about the exact mechanism of the effect of tr14 . in our study , tr14 was ingested after the exercise bout .
possibly , the ingestion of tr14 before the eccentric exercise may have a more pronounced preventive effect .
accordingly , further studies including the analysis of detailed mechanisms of the effects of tr14 still need to be performed . besides this
, a potential negative effect of anti - inflammatory drugs on adaptation processes has to be considered and investigated in future studies . in summary
, these results indicate that tr14 affects exercise - induced muscle damage leading to reduced activation of the innate and the adaptive immune system in response to eccentric exercise .
 the present double - blind , randomized , placebo - controlled clinical trial intended to test whether ingestion of a natural combination medicine ( tr14 tablets ) affects serum muscle damage and inflammatory immune response after downhill running . 
 96 male subjects received tr14 tablets , which consist of 14 diluted biological and mineral components , or a placebo for 72  h after the exercise test , respectively . 
 changes in postexercise levels of various serum muscle damage and immunological markers were investigated . 
 the area under the curve with respect to the increase ( auci ) of perceived pain score and creatine kinase ( ck ) were defined as primary outcome measures . while for ck the p value of the difference between the two groups is borderline , the pain score and muscle strength were not statistically significant . however , 
 a trend towards lower levels of muscle damage ( ck , p = 0.05 ; ldh , p = 0.06 ) in the tr14 group was shown . 
 less pronounced lymphopenia ( p = 0.02 ) , a trend towards a lower expression of cd69 count ( p = 0.07 ) , and antigen - stimulated icam-1 ( p = 0.01 ) were found in the verum group . 
 the tr14 group showed a tendentially lower increase of neutrophils ( p = 0.10 ) , bdnf ( p = 0.03 ) , stem cell factor ( p = 0.09 ) , and gm - csf ( p = 0.09 ) to higher levels . 
 the results of the current study indicate that tr14 seems to limit exercise - induced muscle damage most likely via attenuation of both innate and adaptive immune responses . 
 this study was registered with clinicaltrials.gov ( nct01912469 ) . 
1. Introduction
2. Methods
3. Results
4. Discussion