REVIEW TRIBULUS TERRESTRIS L

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REVIEW TRIBULUS TERRESTRIS L. (ZYGOPHYLLACEAE): SAFETY AND EFFECTIVENESS OF STEROIDAL METABOLITES Thaís Omena Assunção1, Carlos Magno de Marce Rodrigues Barros1, Hércules Rezende Freitas*2, Thiago Huaytalla Silva3. Address: R.D., M.Sc. Hércules Rezende Freitas 1

Federal University of the State of Rio de Janeiro, Center for Biological and Health Sciences, Gaffreè & Guinle University Hospital, Rua Mariz e Barros,

775, 20270-004, Rio de Janeiro, Rio de Janeiro, +55 (21) 2264-5844. 2

Federal University of Rio de Janeiro, Carlos Chagas Filho Institute of Biophysics, Laboratory of Neurochemistry, Avenida Carlos Chagas Filho, s/n, 21941-

590, Rio de Janeiro, Rio de Janeiro, +55 (21) 3938-6452. 3

Federal University of the State of Rio de Janeiro, Center for Biological and Health Sciences, School of Nutrition, Avenida Pasteur, 296, 22290-240, Rio de

Janeiro, Rio de Janeiro, +55 (21) 2542-7262.

*Corresponding author: [email protected] / [email protected] ABSTRACT Plant extracts containing saponins and other phytosterols are regularly used as a medicinal resource for treating several symptoms and diseases, it may be provided in simple preparations (e.g. infusions) or into highly controlled pharmaceutical formulations. Investigations have recently been focused on the role of Tribulus terrestris as an ergogenic aid for physical activity practitioners, possibly due to its androgenic potential and capacity to reduce symptoms of erectile dysfunction. We have performed a systematized review of literature according to Freitas et al. (2015) and Batista and Freitas (2016), in which a judicious search methodology were applied to exclude non-related works. Data on the use of T. terrestris are heterogeneous, and analytical studies strongly suggest the need for better standardization of active compounds in commercial supplements, however, clinical trials and experimental data indicate that its effects occur in a dose-dependent manner, acting weakly on the production of androgen/estrogen hormones, but stimulating pathways related to libido and sexual behavior, especially in female subjects. T. terrestris extracts have failed to promote significant improvements in physical activity performance. Keywords: Plant extracts; Phytosterols; Protodioscin; Steroidal saponins; Tribulus terrestris.

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INTRODUCTION Tribulus terrestris (T. terrestris) belongs to the Zygophyllaceae family, also known as Puncturevine, abrolhos, cabeça-de-cabra, vidoeira-de-fumo and Gokshura. The plant is widely used in traditional Chinese medicine, usually prepared as infusion and/or dried extract. T. terrestris is native from temperate climate regions, its consume is popularized and stimulated due to health claims, such as improvements in symptoms of erectile dysfunction, increase in libido and supposedly promotion of muscle mass gains (Gauthaman et al., 2008; Moghaddam et al., 2013). There are, in scientific literature, case reports in which T. terrestris was successfully used to treat renal lithiasis, menorrhagia and rheumatic pain, however, mechanisms of action and phytopharmacological features of T. terrestris are poorly understood (Moghaddam et al., 2013). Ingestion of T. terrestris by physical activity practitioners is stimulated by allegations that plant metabolites are capable of raising plasma testosterone (TST), consequently inducing greater increases in skeletal muscle mass through its androgenic effects (Neychev and Mitev, 2005). This mechanism was initially proposed using in vivo experiments with animal models for sexual behavior correlated to variations in plasmatic levels of TST, dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA) sulfate (Gauthaman et al., 2002; 2008). In fact, a study from Gauthaman et al. (2003) have shown that mature male Sprague Dawley rats benefit from variable doses of a T. terrestris extract (2.5, 5 or 10 mg/Kg body weight). Rats receiving daily doses of 5-10 mg/Kg body weight for 8 weeks gained significantly more weight, improved sexual behavior parameters and increased intracavernous pressure (ICP) measurements. In humans, however, the effects in behavior and libido promoted by T. terrestris are apparently not related to serum values for androgenic steroidal hormones. Neychev and Mitev (2005) found no relationships between supplementation with T. terrestris (10-20 mg/Kg body weight) and TST, androstenedione (AND)

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or luteinizing hormone (LH) serum levels. Authors also explore the role of T. terrestris in optimizing performance gain and body composition. Although current data is conflicting, some results suggest that the plant may increase endurance to intense exercise and modulate beneficial blood markers (Milasius et al., 2009; 2010). The present review aimed to systematically acquire novel data from recently published documents using a judicious search methodology, focusing our work in analyzing chemical, absorptive, metabolic and toxic aspects of T. terrestris supplementation. We have also been able to propose new recommendations and restrictions to the ingestion of saponin-rich T. terrestris extracts. SURVEY METHODOLOGY Search parameters To ensure methodological and typographic quality of the collected studies, search procedures were applied according to Freitas et al. (2015) and Batista and Freitas (2016). Search for scientific articles was limited to indexed databases (e.g. PubMed) and high impact journals. The following keywords were Tribulus terrestris

Plant extracts

Phytosterols

Protodioscin

Steroidal saponins

Performance

Physical activity

Androgenic hormones . Terms of lexical proximity were also applied during collection of data. Experimental works, clinical studies and invited reviews were selected preferentially. References from acquired documents were also considered for analysis and subsequent use in this work. Excluded items were stored and reevaluated according to the pre-established search procedures. References from non-selected works were also read and additional documents were collected for verification. Experimental studies without explicit presentation of a statement of approval from a responsible ethics committee, or performing inadequate procedures that may impose indiscriminate risk to human and/or animal life, were not considered for the completion of this review. Documents published in non-indexed bases, entertainment magazine articles, blogs, social networks, documents containing unreferenced data and abstracts were automatically excluded. After initial selection, papers were carefully read and classified according to the items and sub-

Chemical composition e extraction

were have included works related to chemical analysis of T. terrestris samples, as well as those elucidating processes of extraction and preparation of the plant for use in supplements or pharmaceutical grade drugs

Absorption, metabolism and interaction with nutrients

how the body and cellular systems deal with T. terrestris, discussing the process of absorption, transformation and also interaction with other nutrients. In Toxicity

we discuss many of the chemical features of T. terrestris capable of inducing cell membrane lysis and death, and the impact of saponins in

epithelial absorptive tissues.

Therapeutic applications

T. terrestris, focusing on physical activity

and performance, once historical and popular applications indicate that the plant is a well-established ergogenic resource. Selection criteria Articles were excluded if not met the following inclusion criteria: chemical analysis and composition of T. terrestris OR T. terrestris supplementation OR metabolic processing of saponins OR nutritional aspects of T. terrestris AND physical activity OR therapeutic applications of plant extracts, including studies using non-human in vivo and in vitro experimental models, which were also considered for the purposes of this review. This study followed three major stages of document analysis: 1.

Search for keywords in indexed databases and printed issues (n > 3000 documents);

2.

Analysis title and summary, to exclude unrelated studies (n = 522 documents);

3.

Careful selection, followed by individual articles reading (n = 7 documents).

Due to the large number of initial documents, we used software (Zotero Standalone 4.0) to organize and select references. Considering the methodological limitations of this narrative review, we have adapted our search procedures from Freitas et al. (2015) and from the PRISMA (i.e. Preferring Reporting Items for Systematic Reviews and Meta-Analyses) norms and standards, allowing for systematic search, optimization of analysis and discussion of the data obtained. Figure 1 illustrates the procedures followed for document acquisition, selection and evaluation.

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Figure 1. General procedures for document collection and analysis.

*(STEP I) after raising a hypothesis, search parameters were determined and (STEP II) scientific documents searched within indexed databases. (STEP III) Obtained documents (STEP IV) were read in full and nonrelated/low quality works were excluded. (STEP V) A final group of documents were obtained and (STEP VI) the process repeated throughout the period for data analysis to ensure the inclusion of newly published works. Adapted from Batista and Freitas (2016). RESULTS AND DISCUSSION Several works explored the effects of T. terrestris and saponin-rich extracts, however, few of them are original experimental works using human models of physical activity, muscular strength, variations in serum androgenic/estrogenic hormones and saponin metabolites. Table 1 summarizes the main methodological procedures and results from selected articles. Table 1. Main features of collected studies regarding T. terrestris, physical activity and sexual function. Authors Aims Methodology Fifteen (n = 15) men ageing 18-35 y.o. Determine the effects of a underwent 8 weeks of supplementation (3,21 preparation containing T. mg of a T. terrestris extract/Kg/day) or Antonio et al., terrestris on body composition placebo + periodized training. Body mass, 2000 and physical performance of body composition, trained males. maximum strength, dietary intake, and humor were evaluated. Thirty (n = 30) active young men ageing 1929 y.o. underwent 8 weeks of resistance Evaluate the effects of training under the following androgen precursors and plant supplementation protocol: 300 mg Brown et al., extracts in the production of androstenedione; 150 mg 2000 testosterone and conversion of dehydroepiandrosterone (DHEA); 750 mg T. androgen hormones to terrestris; 625 mg chrysin; 300 mg indole-3estrogen. carbinol and 540mg saw palmetto. Serum levels of androstenedione and testosterone were analyzed. Twenty-two (n = 22) elite Rugby athletes (19.8 ± 2.9 years) underwent 5 weeks of Determine the effects of T. intense training and supplementation (or terrestris on strength, lean Rogerson et al., placebo) with 450 mg of a T. terrestris muscle mass, and the 2007 extract. Muscular strength, body testosterone/epitestosterone composition and urinary ratio. testosterone/epitestosterone ratio were evaluated.

Results There were no changes in body weight, fat percentage, body water, dietary intake, and humor. Results for muscular endurance and performance were changed for both groups without any additional benefits from T. terrestris supplementation. Androstenedione levels were higher at certain points of the experiment (weeks 2, 5 and 8), however, the supplement able to attenuate the estrogenic effect of precursors or raise testosterone levels (free or total). Moreover, supplementation of precursors and extracts was ineffective in inducing adaptation to resistance training. Supplementation was unable to optimize parameters of muscle strength and body composition and had no influence over urinary testosterone/epitestosterone ratio, which also makes the supplement unable to produce positive results in anti-doping tests.

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T. terrestris supplementation showed no effects on erythrocyte, hemoglobin and thrombocytes levels. Authors suggest negative effect of T. terrestris extract on the number of granulocytes (increase) and leukocytes (reduction). Creatine kinase levels were elevated, such as testosterone in the first 10 days of supplementation. Physical capacity and aerobic capacity were optimized with extract supplementation. Supplementation of T. terrestris was able to raise alactic-glycolytic anaerobic physical capacity and aerobic endurance through short-term exercise bouts (up to 30 seconds), while reducing the concentration of lactate in the blood. Authors also suggest positive influence of the extract on plasma testosterone levels.

Milasius et al., 2009

Establish the influence of a T. terrestris extract supplementation on athlete's physical development, physical capacity, functional aerobic capacity, circulation and respiratory systems.

Thirty-two (n = 32) trained men ageing 2022 y.o. underwent 20 days of supplementation with 1875mg of a T. terrestris extract (or placebo). The following parameters were assessed: physical development, physical capacity, functional aerobic capacity, blood circulation and respiratory system.

Milasius et al., 2010

To investigate the influence of T. terrestris supplementation on physical development, physical capacity, functional aerobic capacity and other biochemical parameters.

Thirty-two (n = 32) trained men ageing 2022 y.o. were subjected to 20 days of supplementation with 1875 mg of a T. terrestris extract (or placebo). Physical development, physical capacity and other biochemical parameters.

Ma et al., 2015

To investigate the effects of Tribulus terrestris (TT) extracts on muscle mass, muscle damage, and anaerobic performances of trained male boxers and its mechanism.

Fifteen (n = 15) male boxers undertook 3week high intensity and 3-week high volume trainings separated by a 4-week rest. T. terrestris extracts (1250 mg/day) were orally administered by boxers. T. terrestris extract compositions were detected by UHPLC QTOF/MS. Muscle mass, anaerobic performance, and blood indicators were explored.

T. terrestris extracts did not change muscle mass and plasma levels of testosterone (TST), DHT, and IGF-1 but significantly alleviated muscle damage and promoted anaerobic performance of trained male boxers, which may be related to the decrease of plasma IGFBP-3 rather than androgen in plasma.

Roaiah et al, 2015

Establish the effect T. terrestris on serum testosterone (free and total) and luteinizing hormone (LH), as well as the impact of supplementation on erectile function

Thirty (n = 30) aging male subjects with partial androgen deficiency were supplemented with 750 mg/day of a T. terrestris extract in 3 individual doses of 250 mg a day for 3 months.

Results showed a statistically significant difference in testosterone levels (total and free) and International Index of Erectile Function-5, but no statistically significant differences in the level of LH before and after treatment.

Chemical composition e extraction T. terrestris is essentially composed of oligosaccharides, in fact, Hammoda et al. (2013) highlight that several saccharides, especially di-p-coumaroylquinic acid derivatives, present in T. terrestris have potent antioxidant activity. However, detailed analysis reveals the presence of phenolic compounds, alkaloids, lignanamides, flavonoids and steroidal saponins, proposed to be responsible for increasing levels of androgens in the plasma of in vivo models (Su et al., 2009). There are several structural similarities between steroidal saponins found in T. terrestris extracts and the hormones TST and DHT, which derive from cholesterol metabolism in humans and has strong androgenic activity (Bartke et al., 1973). One of the most common strategies to extract the saponin content from T. terrestris is using chromatographic methods, and steroidal saponin concentrates are among the most consumed T. terrestris supplements (Mohammed et al., 2014). These methods can be used to identify, purify and isolate known substances from complex mixtures, such as in plant samples (Degani et al, 1998). Ganzera et al. (2001) have described the first analytical method for determination of steroidal saponins in T. terrestris. Separation were achieved through high performance liquid chromatography (HPLC) using a reversedphase (RP-18) column, evaporative light scattering (ELS) and water/acetonitrile as mobile phase. Several samples of T. terrestris from Bulgaria, India and China were analyzed, and quantification data indicates large variations in the saponin profile among plants. While an elevated concentration of steroidal saponins were identified in some products, in others the same saponin is virtually absent. Authors, therefore, highlight the need for standardization of plant samples used in supplements to ensure the presence of pharmacologically active compounds. Phytochemical screenings of a methanolic extract from a Nigerian variety of T. terrestris revealed the presence of alkaloids, tannins, saponins and cardiac glycosides. In fact, examination of antimicrobial activity has shown that such extract may be used as an effective drug for stomachic and urinary tract infections, once low minimum inhibitory concentrations (MIC) were active against an array of microorganisms, such as Salmonella typhi and Candida albicans (Usman et al., 2007). Liquid chromatography/mass spectrometry (LC/MS) analysis of geographically distinct samples of T. terrestris from Bulgaria, Greece, Serbia, Macedonia, Turkey, Georgia, Iran, Vietnam and India has shown that, as previously suggested by Ganzera et al. (2001), sample metabolites vary drastically from region to region. However, authors suggest the presence of common chemotypes within East South European and West Asian samples. Also, data indicate that there are no clear correlations between burrs morphology and chemical composition in the analyzed groups (Dinchev et al., 2008). Currently, structural elucidation of individual saponins from T. terrestris is achieved through a combination of infrared (IR), mass spectral (MS) and 1D/2D nuclear magnetic resonance (NMR). In an extensive study by Kostova and Dinchev (2005), authors explore the wide structural and geographical variety of saponins present in T. terrestris samples. Also, as discussed by Dinchev et al. (2008), there are chemotypes present exclusively in samples from a specific

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geographic region. Saponins with a cis A/B-rings juncture, for example, are exclusive from Chinese plants; tigogenin, gitogenin and hecogenin saponins are also predominant in Chinese samples. Diosgenin type spiro saponins, however, are widely present in Bulgarian, Moldovian, Romanian, Turkish and South African T. terrestris extracts, but absent in samples from China. As previously shown by Ganzera et al. (2001), Bulgarian samples of T. terrestris appear to contain higher levels of the furostanol saponin protodioscin (PTN), whereas Chinese and Indian plants appear to contain small or no amounts of PTN (see Kostova and Dinchev, 2005). Absorption, metabolism and interaction with nutrients Absorption mechanisms and metabolic pathways used by steroidal saponins are weakly elucidated, it is known, however, that glucosides containing polycyclic non-glycidic (aglycones) fractions, such as those present in T. terrestris saponins, have low rate of absorption and may induce attenuation in growth rate, weight loss, anorexia and gastroenteritis in in vivo models. This effect may be mediated by the surfactant and amphiphilic activity of saponins, which are capable of lysing the membrane of intestinal cells and increase the mucosal permeability in vitro. In addition, it is proposed that gastric acids, intestinal enzymes and enteric bacteria may play a role in processing and inactivating these substances (Kumar, 1992; Khokhar and Apenten, 2003). Furostanol-derived steroidal saponins, mainly PTN, protogracilin and prototribestin fractions are the main responsible for the biological effects of T. terrestris on infertility and libido, both in human and animal models (Kostova and Dinchev, 2005). Gauthaman et al. (2003) postulate that PTN is absorbed and directly metabolized to DHEA, which possess intense central nervous system effects, acting as antagonist for gamma-aminobutyric acid (GABA) receptors and thus favoring sexual behavior through previously known mechanisms. It is also proposed that PTN is capable of mediating sex behavior through elevations of TST, DHT and nitric oxide (NO) levels, which acts on diverse central stimulation pathways. Plants used in phytotherapy contain a wide range of metabolites with biological activity, which may have either positive or negative effects in the body. A negative example are glucosides with antinutritional properties, reducing the absorption of several essential nutrients. Saponins may behavior as antinutritional factors and are largely present in T. terrestris (Kumar, 1992). Saponins are considered toxic when intravenously administered, due to its ability to promote lysis of erythrocytes. The ingestion of products containing saponins has being associated to morphological alterations in cell membranes, although it has also been shown to reduce cholesterol levels in human subjects (Johnson et al., 1986). When provided orally, saponins are weakly absorbed, excreted and metabolized into the intestines. The mechanisms involved in this process are yet to be described, although it is believed that bacteria and enteric enzymes are the main agents of degradation. In a study with saponins from quinoa plant (Chenopodium quinoa), it was demonstrated that these substances could promote lysis in small intestine cells, thus increasing intestinal mucosal permeability (Kumar, 1992; Khokhar and Apenten, 2003). In ruminants, saponins were associated to promotion of swelling, symptoms of apathy, weight loss and gastroenteritis. Other investigators, however, indicate that large amounts of saponins (94 g/day) are necessary to promote toxicity (Kumar, 1992), and such concentrations are far from those supplemented in human studies (approximately 0.75 to 2 g/day). Toxicity There are few works exploring the side effects of T. terrestris extracts in humans. In a case report from Iran, a young man whom continuously ingested large doses of T. terrestris during two days with the aim to treat renal lithiasis was hospitalized for presenting symptoms of hepatitis, neurological disorders and renal lesions. After 22 days of suspending the use of T. terrestris and starting specific treatments, the patient has recovered hepatic and kidney functions, also, there were no observable neurological damage (Talasaz et al., 2010). Therefore, even though data related to toxicity and T. terrestris is scarce, the case strongly suggests the possibility of acute toxicity from supplementing the plant. Methodological procedures, such as dose or period of supplementation vary significantly between studies, however, in two selected works authors have provided young subjects (i.e. 20-22 y.o.) with 1875mg/day of a T. terrestris extract during 20 days, showing no side effects during supplementation protocol (Milasius et al., 2009; 2010). It is, however, important to highlight that small doses of T. terrestris extracts should allow for excretion and metabolic degradation of saponins, while large doses may cause plasmatic accumulation and induction of membrane lysis and apoptosis of erythrocytes. In an in vivo study, rats supplemented with 813mg/Kg/day presented several adverse effects in heart, kidney and liver tissues. Another study with sheep and goat models observed similar effects in addition to observable deposition of saponin crystals in biliary ducts (McDonough et al., 1994; Sangeeta et al., 1994). Proposed therapeutic applications of T. terrestris lack consistent evidences mainly due to the small number of well controlled studies and clinical trials supporting these claims. A restricted number of studies have been investigating T. terrestris as a pro-androgen substance, raising levels of plasma testosterone or derivatives and promoting lean mass accumulation. Also, it is also possible that products supplemented during these experiments may contain non-declared illegal steroids, thus comprising the effectivity and safety of these products. Therapeutic applications

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T. terrestris is a widespread medical plant used by physical activity practitioners mainly due to commercial claims (Pokrywka et al., 2014). Early studies showing beneficial effects of the use of T. terrestris were performed by a pharmaceutical company in Bulgaria. Preliminary results have shown that the supplementation raised plasma TST and, consequently, would promote gains in lean mass and strength. Authors proposed that weightlifting athletes would improve performance with T. terrestris supplementation and a protein-rich diet, suggesting an important effect caused by the presence of naturally occurring saponins in the plant, which are related to traditional mechanisms and/or androgenic pathways such as the release of LH and TST (Antonio et al., 2000; Pokrywka et al., 2014). One of the most common therapeutic allegations for T. terrestris supplementation is the possibility of improvements in libido and erectile dysfunction symptoms, however, a majority of studies with adult male individuals show no significant results (Santos et al., 2014). Data from T. terrestris supplementation for female groups, however, indicate a considerable improvement of libido after supplementation protocol. In an Iranian study, T. terrestris (60% saponins) were provided as a supplement for 30 female individuals during 4 weeks to evaluate hypoactive sexual desire disorder (HSDD) symptoms, as well as global desire, excitation, lubrication and pain levels. Results indicate significant improvements in all evaluated parameters and attenuation of HSDD signals (Akhtari et al., 2014). As shown in table 1, only male individuals (n = 161) were supplemented with T. terrestris, also, it is important to highlight that majority of participants in these studies were young adult (18-35 y.o.) individuals, therefore excluding other groups, which would benefit from extracts containing steroidal saponins. In one study from Roaiah et al, 2015, aging male subjects with partial androgen deficiency supplemented with 750 mg/day of a T. terrestris extract for 3 months showed increased levels of total/free testosterone and improvements in the International Index of Erectile Function-5. Recent investigations, however, show that DHEA or TST reposition have no beneficial effects to older subjects, which partially contradicts the idea that T. terrestris extracts are somehow effective for aging individuals through raising of androgen blood levels (Nair et al., 2006). Despite being proposed as an androgenic substance, elevating levels of plasma testosterone or precursors, these effects are controversial. Neychev and Mitev (2005) did not find significantly higher levels of TST and LH when T. terrestris were administered for 4 weeks to 21 healthy young (20-36 y.o.) men. Data from a selected study has shown that supplementation of 150mg dehydroepiandrosterone (DHEA), 300mg androstenedione (AND) and 750mg plant extracts (including T. terrestris) is capable of significantly raising plasma levels of AND without affecting the estrogenic effect of precursors or increasing free/total TST in a group of 30 young (19-29) participants. Also, the supplement did not improve adaptation to endurance training (Brown et al., 2000). T. terrestris extracts were shown to promote low to moderate improvements in parameters of sexual behavior in castrated rats, suggesting that the extract acts as an aphrodisiac substance through bioconversion of saponins, specially PTN, potentially converted into DHEA (Gauthaman et al., 2002). Data from Do et al. (2013) indicate that T. terrestris promoted increase in cyclic monophosphate adenosine (cAMP) and cyclic monophosphate guanosine (cGMP) levels in corpus cavernosum of in vivo models, suggesting that T. terrestris is a potential stimulator of nitric oxide (NO) synthesis in the tissue. Results from Milasius et al. (2009; 2010) indicate that T. terrestris supplementation showed no effects on erythrocyte, hemoglobin and thrombocytes levels and that supplementation had negative impact on the number of granulocytes (increase) and leukocytes (reduction). Creatine kinase levels were elevated, such as TST in the first 10 days of supplementation. Physical capacity and aerobic capacity were optimized with supplementation. T. terrestris was also able to raise alactic-glycolytic anaerobic physical capacity and aerobic endurance through short-term exercise bouts (up to 30 seconds), while reducing the concentration of lactate in the blood. Unlike other studies, authors also suggest positive influence of the extract on plasma TST levels. Although being suggested that T. terrestris increases levels of blood TST, commercially available extracts did not show significant influence on such parameters. In a study with elite rugby athletes, strength and muscle mass were not altered by 5 weeks of T. terrestris supplementation. Label information from T. terrestris supplements used in this study contained claims indicating that the product would allow for a 4,5Kg lean mass gain within a 5-week supplementation protocol (Rogerson et al., 2007). Ma et al. (2015) administered T. terrestris extracts (1250 mg/day) to male boxers participating in 3-week high intensity and 3-week high volume trainings separated by a 4-week rest. Before supplementation, extract compositions were detected by UHPLC QTOF/MS. T. terrestris extracts did not change muscle mass and plasma levels of TST, DHT and IGF-1 but significantly alleviated muscle damage and promoted anaerobic performance of trained male boxers. Authors suggest that these effects may be related to the decrease of plasma insulin-like growth factor binding protein-3 (IGFBP-3) rather than changes in androgen levels. Collectively, studies highlight the need for further detailed chemical analysis in T. terrestris supplements and extracts, once they may contain substances considered to be doping by international committees (e.g. androgenic anabolic steroids and androgenic pro-hormones). These substances would act as endogen precursors after hepatic biotransformation, therefore, authors argue if the higher levels of androgenic hormones found in urine samples are derived from the saponin metabolism or due to the presence of implicitly contained synthetic steroids (Brown et al., 2000; Pokrywka et al., 2014). important that future investigations use, as in Ma et al. (2015), plants in which the chemotype profiling is clearly known, allowing for standardization of dose and concentration of potential active substances, such as PTN. Figure 2 indicates possible biological pathways for PTN, one of the main saponins in T. terrestris extracts, possibly responsible for the effects on libido and erectile dysfunction.

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Figure 2. Possible biological pathways for protodioscin.

A. Protodioscin (PTN), a common saponin from T. terrestris, may stimulate nitric oxide (NO) and other mediators to modulate central dopaminergic activity and libido. PTN may also be converted to DHEA, which is capable of inhibiting GABA signaling, therefore promoting improvements in sexual function. B. The relationship between PTN and testosterone (TST), of stimulation or direct conversion, is still unknown, however, in vivo models indicates that T. terrestris may influence concentrations of TST and C. dihydrotestosterone (DHT). PubChem compound database codes: CID=441891 (PTN); CID=6013 (TST) and CID=10635 (DHT). Adapted from Gauthaman et al., 2003. CONCLUSIONS Several authors have investigated the participation of phytosterols present in T. terrestris extract, however, interventions in human models, especially those related to ascertain their participation in physical activity is scarce and poorly standardized, preventing these results to be properly replicated. A common problem encountered in studies using T. terrestris extracts is the dose range, with concentrations between 300 and 1875mg, indeed, the infrequently reported total content of steroidal saponins present in the tested products makes it difficult to reproduce supplementation protocols. Considering such limitations, comparative analyses and data compilation allows for the following conclusions: I.

necessary to standardize dosage, chemotype profiling and evaluation methods, making future investigations reproducible for all ages and genders.

II.

Optimization of aerobic and non-aerobic performance was demonstrated only when T. terrestris extract is provided in higher doses (1875mg/day) for a prolonged period (20 days), however, independent research groups try to replicate these data, thus reinforcing the validity of such outcomes.

III.

Supplementation of T. terrestris is regularly incapable of increasing levels of androgenic/estrogenic hormones in blood or urine samples, however, synergic actions over NO synthesis, GABAergic signaling inhibition (through DHEA), dopaminergic signaling and potential for bioconversion to androgens (1875mg doses), indicate a possible application in the treatment of erectile dysfunctions and HSDD.

IV.

Health claims, label statements and chemical composition of sports supplements should be constantly verified to avoid the presence of illegal drugs (e.g. synthetic steroids) and/or other substances considered unsafe by the national health agencies where such products are commercialized.

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