significantly increased mortality rates and decreased life quality are observed in patients who have experienced fractures .
owing to their high socioeconomic relevance and increasing incidence , apart from the estimated costs of around 5.4 billion euros a year , therapeutic alternatives need to be investigated .
sarcopenia , a loss of skeletal muscle mass , emerges during the development of osteoporosis [ 36 ] .
this results in a higher risk of falls and , consequently , fall - related injuries [ 7 , 8 ] . ninety percent of all fractures in elderly people arise from falls .
a variety of endocrine substances synthesised by muscle which influence the bone are reduced in sarcopenia .
for instance , levels of muscle - derived humoral bone anabolic factors , which activate osteoblasts , are decreased .
this implies that a sarcopenic muscle is an accompaniment of osteoporosis and the occurrence of osteoporotic fractures .
histologically , sarcopenia leads to a reduction in size of both muscle fibre types i and ii , whereas the fast type ii fibres are chiefly affected [ 1214 ] .
former studies suggested that a low grade inflammation contributes to sarcopenia ; however , the detailed underlying mechanisms remain unclear .
the endocrine axis between muscle and bone , consisting of insulin - like growth factor 1 , interleukins ( ils ) 6 , 7 , and 15 , and osteoglycin , and vice versa for sclerostin with its muscle counterparts myostatin and osteocalcin , is impaired in osteoporosis and , therefore , in sarcopenia  they seem to be  two sides of the same coin  [ 1619 ] .
recent studies also showed a significant loss of number , thickness , and capillarization of muscle fibres with a majority of type ii fibre loss [ 2022 ] .
therefore , therapeutic possibilities reside in pharmacological intervention which target impaired regenerative capacity , elevated reactive oxygen species production , and inflammation observed in sarcopenic muscle .
several of these inflammation reactions are mediated by lipoxygenases , enzymes of arachidonic acid metabolism .
baicalein , as a phytochemical agent , is extracted from the plant scutellaria baicalensis georgi .
it acts as a lipoxygenase ( especially cyclooxygenase ( cox - i ) inhibitor ) and is also a potent inhibitor to 12-lipoxygenase ( 12-lox ) and 15-lox and , therefore , has an antioxidative effect [ 2427 ] .
furthermore , it inhibits nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) and suppresses the function of tumor necrosis factor - alpha 8 ( tnf- ) and/or il-6 , all of them being mediators of inflammation cascades .
an effect of baicalein on inflammatory degenerative bone diseases , such as rheumatoid arthritis , through its suppression of apoptosis and secretion of matrix metalloproteinases , has been recently described [ 28 , 29 ] .
baicalein , as a common flavonoid , also inhibits eotaxin production , probably having a positive effect on asthma .
additionally , 12-lox is inhibited by baicalein , where the former increases the activity of hif-1 under hypoxic conditions , causing the upregulation of the vascular endothelial growth factor , a major factor in promoting prostate cancer progression and metastasis . 
 proposal . as the effect of baicalein on sarcopenia
has not been clarified in previous studies , we aimed to decipher whether baicalein could reduce sarcopenic symptoms in muscles of osteoporotic rats .
outcome measures included the number and diameter of muscle fibres , the relation with type i , type iia , and type iib muscle fibres , and the number of capillaries in muscles ( the relation of capillaries to muscle fibre ) .
furthermore , serum levels of calcium , magnesium , and creatine kinase , a possible biomarker for sarcopenia , were measured to estimate the general muscle condition .
for the study , 61 female , 3-month - old sprague dawley rats ( winkelmann company ) were kept in cages at 20c and at a relative humidity of 55% in makrolon iv. after 1 week of acclimatization , the experiments were conducted in accordance with the ethical standards of animal care and approved by the local district government ( application number : g14/1530 ) . at 13 weeks of age
, the rats underwent bilateral ovariectomy ( ovx ) or were left intact , as previously described .
surgical procedures were carried out under ketamine / domitor anaesthesia ( 0.1  ml/100  g body weight ( bw ) , intraperitoneal ( i.p . ) ) . in brief , after shaving , anaesthesia , and disinfection , the skin was incised on both sides of the lower abdomen .
after 8 weeks , osteotomy of tibia metaphysis with plate osteosynthesis was performed for different studies , according to .
if the standardized tibia osteotomy , performed bilaterally in all rats , would have any effect on muscles , it would be similar in all treatment groups .
on the basis of previous studies [ 35 , 36 ] , we assumed that , at around this time point , that is , 8 weeks after ovx , the rats would have developed osteoporosis with sarcopenia .
baicalein ( 98% , sigma - aldrich chemie gmbh , munich ) was dissolved in 100% dimethyl sulfoxide ( dmso ) , while both control groups ( non - ovx and ovx , each n = 10 ) received dmso alone .
baicalein was injected subcutaneously at different concentrations as previously described [ 37 , 38 ] ( c1 : 1  mg / kg  bw , c2 : 10  mg / kg  bw , and c3 : 100  mg / kg  bw , resp . ) in three groups with 10 animals each .
m. gastrocnemius , m. soleus , and m. longissimus were removed , and m. gastrocnemius and m. soleus were weighed .
afterwards , m. gastrocnemius and m. soleus were sectioned transversely in half across the whole muscle to make cryotome - cutting easier .
subsequently , 12  m thick frozen sections were prepared by cutting the muscle samples orthogonally with a cryotome ( cm 1900 ; leica microsystems ) . to identify the distribution and changes in types i and ii fibres ,
atpase staining was carried out on muscle sections , as described by hork . moreover ,
periodic acid - schiff ( pas ) staining to visualize capillaries in muscle and subsequent counting was performed according to the method described by andersen et al .
for capillary counting , two sections of each muscle in pas staining were chosen and , in a 0.25  mm square , every capillary and muscle fibre were counted , which taken together delivered the ratio of capillary / muscle fibre . for fibre measurements , in three different image sections of each muscle in atpase staining , 30 type i , 30 type iia , and 30 type iib fibres were edged and area and diameter were determined by the program nis - elements ar 4.0 ( nikon instruments europe , amsterdam , netherlands ) .
after that , in 1  mm fibre , types were counted and their relation calculated . after the rats were sacrificed , blood samples of 0.5  ml were collected and analysed with the creatine kinase assay using a c16000 analyser ( abbott , wiesbaden , germany ) for quantitative measurement of serum values .
calcium was measured with the same analyser at 660  nm using arsenazo iii dye and magnesium at 572  nm after the calcium was complexed and eliminated with a chelating agent so as to not interfere with the measurements .
statistical analyses were conducted using graphpad prism ( version 5.04 , graphpad software , inc .
, san diego , ca ) . for detecting differences between the different groups , one - way anova ( f test ,
differences between individual means were estimated using tukey 's test (  = 0.05 ) . in all figures ,
the mean values and the standard error of the mean ( sem ) are displayed .
to test the effects of ovariectomy and different concentrations of baicalein on body weight , the rats were weighed on the day of receipt ( figure 2(a ) ) and at postmortem ( figure 2(b ) ) .
there was no significant difference between groups in body weight on the day of receipt .
however , a significant difference in postmortem weight was noted between the non - ovx and ovx groups .
the postmortem weights of m. gastrocnemius ( figure 2(d ) ) and m. soleus ( figure 2(e ) ) were not statistically different ; the weight of m. longissimus was not detected . to test the effects of ovariectomy on the uterus ,
the uteri of non - ovx rats weighed approximately 0.6  g and were significantly heavier than those collected from the other groups ( on average , 0.10.2  g ) .
these findings indicate successful ovariectomy with resulting atrophy of the uterus in all ovx groups . in a previous study ,
since sarcopenia is associated with lower skeletal muscle capillarization , the effect of baicalein on muscle capillarization was interesting ; however , the effect of baicalein on muscle capillarization has not been described yet .
we analysed the number of capillaries per 0.5  mm muscle tissue in m. soleus ( 3a ) , m. gastrocnemius ( figure 3(b ) ) , and m. longissimus ( figure 3(c ) ) .
although we could not see differences between non - ovx and ovx animals , rats treated with baicalein showed significantly higher numbers of capillaries per muscle fibre when compared with the untreated groups . on average , the rate of capillarization
moreover , the group treated with the highest concentration of baicalein ( c3 ) showed significantly more capillaries per fibre when compared with the group treated with the lowest concentration ( c1 ) .
after atpase staining , muscle fibres were analysed for their diameter and area . in total ,
90 fibres of each type for each sample were assessed , and 90% of all cells were type i ; therefore , no further differentiation was performed . to assess characteristics of sarcopenia
, we determined the diameters and areas of the muscle fibres . for m. soleus ,
although the diameter in non - ovx rats was on average 63  m , it was reduced in the ovx cohort ( approximately 57  m ; figure 4(a ) ) . compared with ovx group ,
baicalein c2- and c3-treated groups possessed single fibres with significantly increased diameters ( 6568  m ) .
muscle fibre areas were calculated after preparing muscle sections ( figure 4(b ) ) ; 90 fibres per muscle were assessed .
the ovx group showed a decrease in area ( 2,511  m ) when compared with the non - ovx control group ( 3,161  m ) . upon comparing the baicalein - treated groups to ovx groups ,
significantly greater areas were found in the c2 and c3 groups ( 3,408  m and 3,630  m , resp . ) .
effects of baicalein on area and diameter were confirmed when observing the results in relation to body weight ( figures 4(c ) and 4(d ) ) .
assessment of single muscle weight in relation to muscle diameter showed no significant differences ( data not shown ) . when relating muscle fibre diameter and area to muscle weight , no significant differences , but similar tendencies to the relation to body weight , were seen .
this is perhaps because of relatively low muscle weight , which could lead to measurement inaccuracy ( figures 4(b ) and 4(e ) ) . in m. gastrocnemius ,
type i fibres showed an increased diameter of approximately 20% when treated with baicalein compared with the non - ovx group ( figure 5(a ) ) . a similar effect
could be observed in type iia fibres , with the strongest effect in the c2 group ( figure 5(b ) ) . by contrast , type iib fibres showed no significant differences among all groups ( data not shown ) . regarding the size of the area of different fibre types , in type
i ( figure 5(c ) ) , compared with the non - ovx control group , all ovx cohorts ( treated and untreated ) showed an increase in fibre area .
compared with the non - ovx group , ovx rats showed an increase in area of 18% , and baicalein - treated animals showed an increase of up to 41% .
c2 cohort showed the highest effect , but only c2 and c3 were statistically significant .
a similar tendency was observed in type iia fibres ; however , only c2 group possessed a significantly greater area ( figure 5(d ) ) .
no significant differences were observed in the area of single type iib muscle fibres ( data not shown ) .
thereafter , we calculated the diameter to body weight ratio and found no significant differences in types i ( figure 5(e ) ) , iia , and iib fibres ( data not shown ) .
similar results were obtained when relating diameter to muscle weight for types i ( figure 5(f ) ) , iia , and iib fibres ( data not shown ) , where no significant differences could be determined .
moreover , we calculated the relation of fibre area to body weight . in type iia fibres , we found a slight increase in m. gastrocnemius area in group c2 when compared with ovx rats ; however , other groups did not show significant differences ( figure 5(g ) ) .
overall findings for types i and iib fibres indicated no striking differences between the groups ( data not shown ) . when relating area to muscle weight , we found significant differences in type i fibres , where non - ovx , ovx , and c1 groups did not show significant differences ( figure 5(h ) ) .
however , when comparing their values to those of the c3 group , baicalein c3 treatment could increase the area by approximately 60% .
similar effects were observed for type iia fibres , where the highest baicalein concentration increased the area by approximately 44% ( figure 5(i ) ) . in iib fibres ,
a similar tendency was observed ; however , the differences were not significant ( figure 5(j ) ) .
comparable with m. gastrocnemius , the three fibre types could also be differentiated for m. longissimus . when observing the diameter in m. longissimus type
i fibres , we did not detect significant differences between non - ovx and ovx rats ; however , high doses of baicalein ( c2 ) could raise the diameter by approximately 13% ( figure 6(a ) ) .
when comparing the effects of different baicalein concentrations , the strongest effects were recorded in the c2 group .
similar effects were observed in type iia fibres , where baicalein c2 could increase the diameter to 86.7  m when compared with 77.6  m in non - ovx and 74.8  m in ovx cohorts ( figure 6(b ) ) .
the iib fibre analysis showed no significant differences among the different groups ( data not shown ) .
we measured the area of muscle fibres types i and iia and , analogous to the diameter , similar values were obtained for non - ovx and ovx animals ( figures 6(c ) and 6(d ) ) . by contrast , baicalein treatment could significantly increase the muscle fibre area .
while all baicalein concentrations increased the area compared with the control animals , c2 rats showed the highest values .
type iib fibres showed no significant differences among the cohorts ( data not shown ) . unlike the analysis of m. soleus and gastrocnemius , we related the fibre diameter and area in m. longissimus to animal body weight because single muscle weight was not detected .
type i fibre diameter was significantly decreased after ovariectomy ( figure 6(e ) ) ; however , after baicalein treatment ( c2 ) , this effect could be rescued .
the same phenomenon was observed for types iia ( figure 6(f ) ) and iib ( figure 6(g ) ) fibres .
when relating the areas of types i and iia fibres to body weight , a similar decrease could be observed in ovx rats when compared with the non - ovx control group ( figures 6(h ) and 6(i ) ) .
when treating the animals with low ( c1 ) or high ( c3 ) baicalein concentrations , the effect of the ovariectomy was dampened ; however , fibre areas were lower than those in the non - ovx cohort .
in contrast , medium baicalein concentrations ( c2 ) could not only rescue the consequences of the ovariectomy but also increase the fibre areas when compared with the non - ovx rats .
for iib fibres , no significant differences between the groups could be measured ( data not shown ) .
finally , the proportions of different fibre types ( type i , iia , and iib ) were assessed in m. longissimus ( figure 7 ) . in general , no striking differences were observed among the different groups .
the strongest effects were noted in animals treated with the medium concentration of baicalein ( c2 ) .
notably , when comparing the non - ovx and c2 cohorts , we detected that c2 rats tended to have less type i and type iib fibres but contained significantly more type iia fibres . in m. soleus and m. gastrocnemius ,
creatine kinase ( u / l ) , calcium ( ca ; mmol / l ) , and magnesium ( mg ; mmol / l ) levels were measured in the groups .
creatine kinase levels were significantly lower in the c3 group than in the ovx group ( figure 8(a ) ) .
a higher serum magnesium value could be detected in the c2 group when compared with the ovx group ( figure 8(c ) ) .
sprague dawley rats are a valuable model for the induction of osteoporosis after hormonal depletion via ovariectomy [ 43 , 44 ] . here , the rats were 13 weeks old at the time of ovariectomy , which is reportedly acceptable for the osteoporosis model [ 45 , 46 ] . furthermore , francisco et al .
compared the effects of ovariectomy in rats of different ages and found no related differences in bone mineral response .
the effects on muscle in rats seem to be similar to those in humans : they develop sarcopenia [ 48 , 49 ] . to determine the effects of baicalein on sarcopenia
independent of baicalein treatment , body weight of the rats after ovariectomy was significantly greater for the ovx ( control ) group than for the non - ovx group .
interestingly , in m. gastrocnemius , an increasing dose of baicalein led to slightly reduced muscle weight , supporting the hypothesis of estrogen deficiency - induced muscle degeneration .
however , the reduced muscle weight of m. gastrocnemius could also be attributed to the baicalein - induced inhibition of the nonphysiological degeneration of muscle , a finding reported in literature describing the anti - inflammatory and antifibrotic effects of baicalein .
as reported previously , estrogen has an angioproliferative effect on skeletal muscle [ 51 , 52 ] and a loss of capillaries by 25% could be observed in sarcopenia because of aging [ 21 , 53 ] .
notably , we could show that baicalein leads to a significant gain in the capillary to muscle fibre ratio in all baicalein - treated groups . in m. soleus
, baicalein treatment showed a dose - dependent increase in the number of capillaries per muscle fibre .
similar results could also be seen in m. gastrocnemius and in m. longissimus , where the 10 mg / kg bw concentration evinced the greatest effect on capillarization .
the expected loss of capillaries under estrogen deficiency could not be observed when treated with baicalein .
the hypothesis that baicalein , applied subcutaneously , stimulates angiogenesis in skeletal muscle despite estrogen deficiency was proven .
nonetheless , there were no significant differences in capillary to muscle fibre ratio after ovx , which might be due to insufficient trial duration , which was too short to reveal the decapillarization in sarcopenic muscle , as seen in other rat models .
we purported that decreased muscle diameter due to estrogen loss , as described in rats [ 5356 ] and humans , could be restored by baicalein .
indeed , the baicalein c2 and c3 groups had significantly increased diameters of m. soleus , a finding similar to that for body weight .
these findings support our hypothesis that baicalein counteracts muscle atrophy at high concentrations . in all three fibre types
( i , iia , and iib ) in m. gastrocnemius we saw a tendency of all baicalein - treated groups to have increased fibre diameters .
interestingly , the group treated with baicalein c2 concentration showed the greatest fibre diameters . when analysed in detail , the diameter to body weight ratio showed that types i , iia , and iib fibres in almost all baicalein groups were slightly increased .
we also found a tendency for greater diameter per body weight values in the non - ovx group when compared with the ovx group .
overall , baicalein appears to induce hypertrophy in the estrogen - deficient skeletal muscle . in m. longissimus
, the development of sarcopenia after ovariectomy , resulting in estrogen deficiency , which leads to reduced muscle fibre diameter is supported .
altogether , the expected atrophy of skeletal muscle fibres , indicated by the decline in fibre diameter , appears to be rescued by baicalein .
the gain of iia fibre diameter is noteworthy , considering that the diameters are typically affected in sarcopenia .
the fact that lipoxygenase inhibitors could counteract muscle atrophy has already been shown in clinical trials . in m. longissimus
, the increase in types i and iia fibre areas suggests that baicalein can counteract the estrogen - dependent atrophy . in m. gastrocnemius , the increase of type
nonetheless , this gain in area and diameter can also be attributed to the storage of fat and water .
moreover , estrogen deficiency can lead to an increase in noncontractile muscle fibres that can hardly be differentiated from normal muscle tissue . whether this is
microscopically , no greater fat deposits or lipid content could be observed . in affecting muscle fibre types i , iia , and iib
, baicalein appears to react with oxidative- and glycolytic - working muscle groups . in m. longissimus , significantly increased proportions of fast iia but not type i fibres were observed in the baicalein c2 group .
this finding disagrees with the decline in fast fibres , the described  fast - to - slow fibre type shift ,  which is characteristic for muscle atrophy and wasting . in conclusion , these results suggest a positive , though not fibre type - specific , effect of baicalein on estrogen - deficient muscles in rats .
whereas in m. gastrocnemius and m. soleus the number of capillaries in muscle was proportional to the dose of baicalein , in m. longissimus , the medium baicalein concentration ( 10  mg / kg bw ) showed the greatest effect .
the results of muscle diameter and area also showed a positive baicalein effect in comparison with the control groups .
moreover , regarding fibre ratios , a gain in iia fibres was seen in m. longissimus , which contradicts the  fast - to - slow fibre type shift . 
as observed in human studies , there was no difference between sarcopenic and control groups regarding calcium and magnesium levels [ 62 , 63 ] .
serum concentration of creatine kinase , a clinical marker of muscle state [ 32 , 64 ] , in baicalein c3 group was found to be significantly decreased .
investigating molecular mechanisms of baicalein treatment was not within the scope of this descriptive study , but bhattacharya et al . showed that skeletal muscle atrophy ( during aging and neurodegenerative diseases ) can be diminished with inhibition of 12-lox and 15-lox , while the activation of this pathway led to increased nadph oxidase activity , protein ubiquitination , and proteolytic degradation .
they also demonstrated that baicalein reduces denervation - induced muscle atrophy in wild type but not in 12-lox and 15-lox knockout mice .
therefore , baicalein chiefly operates through this pathway and is not arbitrated through the reduction of inflammatory response .
possibly the main effect of baicalein in this study could be observed because of the above - mentioned 12-lox and 15-lox pathway inhibiting nadph oxidase activity and protein ubiquitination as well as 
another study by wang et al .   demonstrates the multiple signaling pathways in which baicalein is involved ( e.g. , akt / mtor , erk 1/2 , nf-b , and calcineurin ) .
the aim of this study was to elucidate whether baicalein , in different doses , applied subcutaneously , could neutralize the estrogen - induced loss of muscle and atrophy in the skeletal muscle of rats .
we found that baicalein has a positive effect on estrogen - dependent atrophy of skeletal muscle .
this inflammatory effect has not been reported so far but did not affect overall health , as was seen by the analyses of body weight , food intake , and general conditions ( clean coat , absence of porphyrin around eyes , etc . ) .
the inflammatory processes were not part of this study and we could not confirm an anti - inflammatory effect after s.c .
therefore , it is advisable to use an alternative route of administration , such as orally [ 50 , 68 ] or intravenously .
to emphasize differences between baicalein and ovx groups , trial duration could be elongated and more groups could provide better conditions to find the ideal dose and route of baicalein administration . moreover , a healthy non - ovx group should be included . as we only investigated skeletal muscle , we can not exclude systemic effects . no fever or severe infections occurred throughout the experiments .
however , the development of the necrotic lesions at the injection sites should be taken into consideration .
smooth muscles and cardiomyocytes as well as infection parameters , such as crp , il-6 , and procalcitonin , could be further examined to rule out effects on these tissues . to exclude the fact that gains in area and diameter are attributed to fat and water retention , biochemical analyses of muscle fibres
additionally , there could be a possible effect of osteotomy on muscle tissue , so these experiments could be repeated without this operative procedure ; however , this effect would exist in all rats .
different models of muscle inflammation could also be used to further investigate the anti - inflammatory effect of baicalein .
in addition , the bone of rats and its biomechanical and histomorphometric composition could also be evaluated , as recently done by li et al . , but were not part of this study .
detection of the molecular mechanism underlying the effects of baicalein in rats was not part of this descriptive study .
this discrepancy could be explained by baicalein - induced inhibition of the nonphysiological degeneration of muscle due to the anti - inflammatory , antifibrotic [ 50 , 72 ] , and antioxidant [ 66 , 73 ] effects of baicalein but should be further examined .
 sarcopenia , a loss of muscle mass accompanying osteoporosis , leads to falls and fall - related injuries . 
 baicalein , as a phytochemical agent , has an antioxidative and anti - inflammatory effect in muscle . in this study , 
 sixty - one female sprague dawley rats were divided into five groups : four groups were ovariectomized ( ovx ) and one control group was nonovariectomized ( non - ovx ) . 
 eight weeks after ovariectomy , three disparate concentrations ( 1  mg / kg body weight ( bw ) , 10  mg / kg  bw , and 100  mg / kg  bw ) of baicalein were applied subcutaneously daily in three ovx groups . 
 mm . 
 soleus , gastrocnemius , and longissimus were extracted ; their diameter , area , relation to body , and muscle weights as well as number of capillaries per fibre were recorded . in mm . 
 soleus and gastrocnemius , the baicalein effect ( increasing number of capillaries per fibre ) was proportional to the dose applied . 
 the fibre diameters and area under baicalein treatment were significantly greater compared to ovx and non - ovx groups . in m. longissimus , we observed a shift to type iia fibres . 
 serum creatine kinase levels were significantly lower in highest baicalein concentration group . 
 we conclude that baicalein can stimulate angiogenesis , though not fibre type - specific , in skeletal muscle and reduce the estrogen - related loss of fibre diameter and area in the skeletal muscle in rats . 
 therefore , a protective effect of baicalein on muscle cells can be assumed . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Limitations