The current study shows the biochemical, biomechanical and morphological alterations in Achilles tendon of rats concurrently submitted to endurance training and H. aphrodisiaca treatment.
The implications of endurance training [[29–32]] and AAS treatment [[10, 13, 14]] on tendon properties have been studied by several investigators. The use of AAS has resulted in a stiffer tendon that failed when submitted to less elongation than was achieved by the control Wistar rats. Exercise did not significantly alter tendon elongation in athlete animals. However, the combination of exercise and steroids significantly increased stiffness and decreased elongation, as well as the energy that the tendon could absorb at tendon failure . In all cases the maximum load that the tendon could withstand did not seem to be affected. However, the use of steroids in the presence of exercise increased the cross-sectional area and reduced the flexibility of the tendon . Wood et al.  in similar study, reported alterations in collagen content and the toe-limit strain in rats that had been treated with anabolic steroids and exercise.
In the present study, the maximum stress, modulus of elasticity and stiffness were higher in tendons of trained and treated rats, which also exhibited higher hydroxyproline content and increased cross-sectional area. These data showed that HT animals have more resistant tendons, differing from other studies in which the combination of AAS and exercise did not improve the tendon's biomechanical properties [10, 13]. Moreover, the interaction between strenuous exercise and H. aphrodisiaca promoted significant increase in the material properties (maximum stress and modulus of elasticity) and of collagen content, resulting in stronger tendons able to support intense muscular contraction. Tendons may show a faster response to the number of loading cycles, rather than to the magnitude of the load . Simonsen et al.  found that a strength-training regimen (high force with few loading cycles) did not stimulate increase in strength of the Achilles tendon of rats; however, low-force endurance training (e.g. swimming) resulted in stronger tendons. They suggested that the tendons may respond better to the number of muscle contractions that occur during training rather than the absolute tension exerted by the muscle. In this case, the increased tendon mechanical resistance observed during endurance training and H. aphrodisiaca treatment may represent a mechanism to prevent tendon damage due to mechanical fatigue. This biomechanical behavior could be due to the increase of the collagen content, to the fiber orientation and to the interaction between collagen and ground substance . There is a relationship between mechanical properties and collagen content , since more resistant tissues have either more collagen per area or collagen fibers with larger diameters .
The biomechanical results corroborate the results obtained by polarized microscopy. The organizational aspect of the fibers is better understood when slides are analyzed under polarized microscopy, due to the birefringence properties of collagen bundles. This observation is important because it shows micro-morphological details hidden within these bundles. In the present study, this technique revealed high birefringent brightness due to the condensation and highly tidy fiber array in the trained group. Besides, in the trained group that also received the plant infusion (HT) the results were even more prominent, showing brighter collagen fibers, possibly indicating highly compacted bundles. The lower birefringence found in sedentary animals reflects less organized collagen bundles in these groups.
Some observations in the compression region showed that, in HT animals, there was an increase in the round cell population (stereological data not shown), as well as in the metachromasy intensity, which indicates a greater proteoglycan accumulation due to the increased compressive forces during endurance exercise. However, this result was not confirmed by the GAG dosage, which used the whole tendon. It is important to say that microscopical analysis of sections stained with toluidine blue specifically evaluate a region of the tendon where there is greater accumulation of proteoglycans due to the presence of localized compressive forces, as has been observed in tendons of rats  and pigs . Nevertheless, proteoglycans, mainly the low weight ones, are distributed all over the tendon, associated to the collagen fibers , probably regulating collagen fibrillogenesis .
The degradation of collagen, as well as of a great number of other extracellular matrix compounds, is initiated by metalloproteinases (MMPs). An increase in net MMP activity is likely to indicate matrix degradation and accelerated remodeling [39, 40]. Increased MMPs activity was verified in human tendons after acute running exercise  and in exercised rats (jumping in water) . In the present study, zymography showed that MMP-2 activity in CT animals was similar in the sedentary groups, differing from other studies in which exercise increased the MMPs levels in tendons [14, 39]. It is noteworthy that the MMP activity in the above studies was analyzed after only 3 days and 6 weeks of training, respectively.
In the present study, the animals were killed after 11 weeks of training, which means that the MMP activity was evaluated after a long training period. The biomechanical and morphological data demonstrated that the tendons of treated and trained animals had undergone adaptation to the increased demand. Thus, we suggest that there was a period of increased MMP activity to permit tissue remodeling, followed by a period of reduced activity when the tendons were already adapted to the load required. In this case, the mechanical load did not represent a stimulus for the synthesis of pro-MMPs and their eventual activation. Also, the MMP-9 activity did not alter in the exercised animals, which confirms the tendons' adaptation, since high activity of this protein is associated to the presence of immune cells during the initial inflammatory process , which occurs in response to tissue damage caused by exercise. In human tendons, protein synthesis and degradation were chronically elevated 4 weeks after the beginning of the training period, whereas protein synthesis remained high throughout a 12-week training cycle, while the degradation was slowly reduced. This suggests that there was probably an early period in the exercise program when collagen turnover in tendons was increased in order to restructure and readapt the tendon to the increased loading pattern .
Marqueti et al.  showed that the MMPs activity strongly decreased in AAS-treated animals. The inhibition could be due to a decrease in MMP synthesis or inhibition of activation of latent pro-MMPs. Also, that exercise by itself was not enough to compensate the inhibition of MMP activity induced by AAS treatment. In the present investigation the plant infusion without exercise did not inhibit MMP activity. The data obtained suggests that the infusion associated to exercise could have increased the MMPs' activity in the initial training period (1-3 weeks) when the highest mechanical loading occurred, considering that in the HT group the MMP-2 activity was reduced compared to all other groups, and that in this group the tendons were more resistant, according to the biomechanical results, in relation to the CT group. Therefore, we suggest that remodeling was more efficient in the HT group. However, further research is necessary to evaluate the effect of the plant infusion on MMP activity in exercised animals.
The present study faced some limitations regarding to the applied methodology, such as the lack of specific phytochemical data considering the active compounds present in H. aphrodisiaca infusion. Infusion preparation was based on previous studies that have been performed by our group using the same plant species [[3, 4, 42]], attempting to mimic the infusion traditionally used by folk medicine. Results of administering H. aphrodisiaca without exercise are not significantly different from the control, which would suggest a mandatory exercise program for efficacy of the plant extract altering rat tendons.
Common side effects due to long term endurance exercise together with synthetic AAS intake have been described as: collagen dysplasia, greater stiffness, reduction of strain, impaired tissue remodeling and others, none of which were noticed in this study after the training protocol and plant infusion administration. Scarce but important data lead us believe that there are no deleterious side effects related to H. aphrodisiaca intake, which was proved by evaluating blood biochemical parameters as well as kidney and liver morphology after long term administration of the plant infusion (data not published). Based on the promising data presented so far, future experiments crossing the variables of infusion dosages and time could be performed in order to potentiate H. aphrodisiaca effects along with endurance exercise. Additionally, phytochemical studies using the same infusion concentration are being carried out to reveal which active components could be related to the anabolic effects shown in this study.