Effects of Ashwagandha (Withania Somnifera) on Physical J

Journal of Functional Morphology and Kinesiology

Review Review EffectsEffects of Ashwagandha of Ashwagandha (Withania (Withania somnifera somnifera) on Physical) on Physical Performance:Performance: Systematic Systematic Review Review and Bayesian and Bayesian Meta-Analysis Meta-Analysis

Diego A.Diego Bonilla A.1,2,3,4, Bonilla* ,1,2,3,4, Yurany*, Yurany Moreno Moreno1,2, Camila 1,2, Camila Gho 1Gho, Jorge 1, Jorge L. PetroL. Petro1,3 1,3, Adrián Odriozola-Martínez 4,5,6 Adrián Odriozola-Martand Richard B.ínez Kreider4,5,6 7 and Richard B. Kreider 7

1 Research1 Division, Research Dynamical Division, Dynamical Business & Business ScienceSociety—DBSS & Science Society—DBSS International International SAS, SAS, Bogotá 110861,Bogotá Colombia; 110861, Colombia; [email protected] [email protected] (Y.M.); [email protected] (Y.M.); [email protected] (C.G.); (C.G.); [email protected]@dbss.pro (J.L.P.) (J.L.P.) 2 Research2 Group Research in Biochemistry Group in Biochemistry and Molecular and Biology,Molecular Universidad Biology, Universidad Distrital Francisco Distrital Jos Franciscoé de Caldas, José de Caldas, Bogotá 110311,Bogotá Colombia 110311, Colombia 3 Research3 Group Research in Physical Group in Activity, Physical Sports Activity, and Sports Health and Sciences Health (GICAFS), Sciences Universidad(GICAFS), Universidad de Córdoba, de Córdoba, Montería 230002,Montería Colombia 230002, Colombia 4 kDNA Genomics,4 kDNA Genomics, Joxe Mari KortaJoxe Mari Research Korta Center, Research University Center, University of the Basque of the Country Basque UPV/EHU, Country UPV/EHU, 20018 Donostia,20018 SanDonostia, Sebasti Sanán, Spain;Sebastián, [email protected] Spain; [email protected] 5 Sport Genomics5 Sport ResearchGenomics Group, Research Department Group, Department of Genetics, of Physical Genetics, Anthropology Physical Anthropology and Animal and Physiology, Animal Physiology, Faculty of ScienceFaculty andof Science Technology, and Technology, University ofUniversity the Basque of the Country Basque (UPV/EHU), Country (UPV/EHU), 48940 Leioa, 48940 Spain Leioa, Spain 6 Phymo Lab,6 Phymo Physiology Lab, Physiology and Molecular and Laboratory,Molecular Laboratory, 08028 Barcelona, 08028 SpainBarcelona, Spain 7 Exercise7 & Exercise Sport Nutrition & SportLaboratory, Nutrition Labo Humanratory, Clinical Human Research Clinical Facility, Research Texas Facility, A&M Texas University, A&M University, College Station,College TX Station, 77843, USA;TX 77843, [email protected] USA; [email protected] * Correspondence:* Correspondence: [email protected]; [email protected]; Tel.: +57-320-335-2050 Tel.: +57-320-335-2050

Abstract: AshwagandhaAbstract: Ashwagandha (Withania somnifera(Withania) issomnifera considered) is considered a potent adaptogen a potent adaptogen and anti-stress and anti-stress agent agent that couldthat have could some have potential some potential to improve to improve physical physical performance. performance. This preferred This preferred reporting reporting items items for Citation: Bonilla, D.A.; Moreno,for Y.; systematic systematic reviews reviews and meta-analyses and meta-analyses (PRISMA)-based (PRISMA)-based comprehensive comprehensive systematic systematic review and review and Citation: Bonilla,Gho, D.A.;C.; Petro, Moreno, J.L.; Y.;Odriozola-Bayesian meta-analysisBayesian meta-analysis aimed to aimed evaluate to evaluate clinical trials clinical up trials to 2020 up to from 2020 PubMed, from PubMed, ScienceDirect, ScienceDirect, and Gho, C.; Petro,Martínez, J.L.; A.; Kreider, R.B. Effectsand of Google Google Scholar Scholar databases databases regarding regarding the effect the ofeffect Ashwagandha of Ashwagandha supplementation supplementation on physical on physical per- Odriozola-Martínez, A.; Kreider, R.B. Ashwagandha (Withania somniferaperformance) on formance in healthy in healthy individuals. individuals. Besides Besides implementing implementing estimation estimation statistics statistics analysis, analysis, we devel- we developed Effects of Ashwagandha (Withania Physical Performance: Systematicoped BayesianBayesian hierarchical hierarchical models models for a for pre-specified a pre-specified subgroup subgroup meta-analysis meta-analysis on strength/power, on strength/power, cardi- somnifera) on Physical Performance: Review and Bayesian Meta-Analysis.cardiorespiratory J. orespiratory fitness fitness and fatigue/recoveryand fatigue/recovery variables. variable As. totalA total of 13of 13 studies studies met met the the require- requirements of Systematic ReviewFunct. andMorphol. Bayesian Kinesiol. 2021, 6, 20. J. Funct. Morphol. ments of thisthissystematic systematic review,review, althoughalthough only 12 were were included included in in the the quan quantitativetitative analysis. analysis. A low-to-mod- A Meta-Analysis.https://doi.org/10.3390/jfmk6010020 Kinesiol. 2021, 6, 20. https:// low-to-moderateerate overall overall risk risk of of bias bias of of the the trials trials included included in in this this study study was was detected. detected. All All Bayesian Bayesian hierarchical doi.org/10.3390/jfmk6010020 hierarchicalmodels models converged converged to to a a target target distribution distribution ( Ȓ = 1) for both meta-analytic effecteffect sizesize( (µμ)) and and between- Received: 1 January 2021 study standard deviationτ (τ). The meta-analytic approaches of the included studies revealed that Accepted: 9 February 2021 between-study standard deviation ( ). The meta-analytic approaches of the included studies revealed Received: 1 January 2021 Published: 11 February 2021 that AshwagandhaAshwagandha supplementation supplementation was morewas more efficacious efficaciou thans than placebo placebo for improvingfor improving variables variables related Accepted: 9 February 2021 related to physicalto physical performance performance in healthy in health meny men and and female. female. In fact, In fact, the Bayesianthe Bayesian models models showed showed that that future Published: 11 February 2021 Publisher’s Note: MDPI staysfuture interventionsinterventions might might be at be least at least in some in some way beneficialway bene onficial the on analyzed the analyzed outcomes outcomes considering considering the neutral with regard to jurisdictionalthe 95% credible95% credible intervals intervals for the meta-analyticfor the meta-analytic effect size. effect Several size. practical Several applicationspractical applications and future and future Publisher’s Note:claimsMDPI in published stays neutral maps anddirections directions are discussed, are discussed, although morealthough comparable more comparab studies arele studies needed are in needed exercise in training, exercise and training, and with regard toinstitutional jurisdictional affiliations. claims in athletic populationsathletic populations are needed are to deriveneeded a to more derive stable a more estimate stable of estimate the true underlyingof the true underlying effect. effect. published maps and institutional affiliations. Keywords:Keywords:herbal supplements; herbal supplements; muscle strength; muscle cardiorespiratory strength; cardiorespiratory fitness; exercise fitness; tolerance; exercise quality tolerance; qual-

of life; sleepity latency of life; sleep latency Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: ©This 2021 article by is the an authors.open access article Licensee MDPI,distributed Basel, under Switzerland. the terms and1. Introduction1. Introduction This article isconditions an open accessof the Creative article Commons Traditional botanical knowledge is characteristic of cultural contexts with long ex- distributed underAttribution the (CC terms BY) andlicense Traditional botanical knowledge is characteristic of cultural contexts with long expe- conditions of(http://creativecommons.org/licenses the Creative Commons perience inrience the naturalin the natural environment; environment; however, however, as it as is it transmitted is transmitted from from generation generation to to gener- Attribution (CC/by/4.0/). BY) license (https:// generation,ation, it adapts it adapts and and transforms transforms through through scientiﬁc scientific experimentation. experimentation. Within Within the tradi-the traditional creativecommons.org/licenses/by/ tional medicine of India (Ayurveda), the Rasavatam¯ provides nutritional and behavioral 4.0/). recommendations for all phases of life, which may delay the onset of aging processes [1].

J. Funct. Morphol. Kinesiol. 2021, 6, 20. https://doi.org/10.3390/jfmk6010020 www.mdpi.com/journal/jfmk J. Funct. Morphol. Kinesiol. 2021, 6, 20. https://doi.org/10.3390/jfmk6010020 https://www.mdpi.com/journal/jfmk J. Funct. Morphol. Kinesiol. 2021, 6, 20 2 of 31

Thus, there is a wide range of Rasavatam¯ preventive or therapeutic strategies with various health objectives (e.g., improvement of the libido). One of the most popular plants in Ayurveda is Ashwagandha (Withania somnifera), which is considered a potent adaptogen and anti-stress agent [2]. Brekhman and Dardymov [3] proposed in 1969 the term adaptogen as an innocuous agent that increases in a nonspecific way the resistance against harmful factors or physical, chemical, biological and psychological “stressors”, normalizing the homeostasis of individuals. Ashwagandha, commonly known as Indian ginseng or poison gooseberry, belongs to the Solanaceae family (NCBI:txid126910). In addition to its adaptogenic properties, several authors have attributed multiple medicinal benefits including antitumor, anti-inflammatory, hypoglycemic and antioxidant effects [4,5]. These characteristics have generated a great scientific interest in the study of the chemical composition of this plant. Several bioactive compounds, including flavonoids, tannins, alkaloids, glycosides and steroid lactones have been identified in leaves, stems and roots [6]. Within the steroid lactones are the withanolides, which have been described as the main secondary metabolites responsible for the beneficial properties of this plant. Withanolides are polyoxygenated steroids consisting of a 28C ergostane backbone. The structural diversity of withanolides generally depends on the nature and number of oxygenated substitutes and the degree of saturation of the rings. These compounds can be classified into two main groups based on the C17 side chain, those containing an δ-lactone or δ-lactol between C22 and C26, and those with an γ-lactone that usually forms between C23 and C26. The first group can be further classified into withanolides with intact (withaferin A) and modified (fisalin C) backbone. Currently, the presence of withanolides with unmodified skeletons has been commonly identified in nature, of which approximately 580 have been reported in the Solanaceae family. The wide structural variety of withanolides may be related to the multiple biological functions that have been described for Ashwagandha: antimicrobial, anti-inflammatory, immunomodulatory, neuroprotective, cytotoxic, and antioxidant, among others [5]. However, several experimental strategies have been developed for the extraction and purification of these compounds. Recently, Nile et al. (2019) compared different methods of withanolide and withanoside extraction from Ashwagandha leaf and root extracts and the effects on their biological activity. The authors demonstrated that subcritical water extraction allowed higher yield in solubilization of these compounds mainly in root extracts, maintaining their biological properties, compared to other methods such as maceration, Soxhlet extraction, and microwave-assisted extraction, which require long periods of time and the use of organic solvents [7]. Trivedi et al. (2017) identified a total of 43 withanolides in the hydroalchoholic extract of Ashwagandha root using LC/MS, GC/MS and NMR, including withaferin A, withanolide A, withanoside IV and VI, withanolide D, dihydrowithanolide D and withanolide sulfoxide [8]. Of these, withaferin A and withanolide A possess therapeutic properties for the treatment of cancer and neurodegenerative diseases, such as Parkinson’s and Alzheimer’s [9–11]. Although it has been used therapeutically for a number of reasons, Ashwagandha supplementation lacks sufficient information to be considered effective for all proposed conditions [4]. However, several recent systematic reviews and meta-analyses have shown its potential and safety for controlling anxiety [12], fighting male infertility [13], improving the function of the reproductive system [14], serving as an adjuvant to the treatment of diabetes [15] and avoiding the deterioration of cognitive function [16]. However, it seems that some of the secondary metabolites of Ashwagandha could have some potential at the level of physical performance improvement, being responsible for various effects at the metabolic and physiological level through the regulation of certain anti-inflammatory and anti-oxidative pathways [4,17]. Considering that nutrition is one of the fundamental pillars to optimize sports performance, some have suggested that Ashwagandha may provide ergogenic benefits for active individuals and athletes. In fact, Pérez-Gómez et al. [18] recently reported that consumption of this herbal extract significantly increases maximum oxygen uptake (VO2max), although this meta-analysis has some limitations that warrants caution when interpreting results. On the other hand, some previous work has shown that J. Funct. Morphol. Kinesiol. 2021, 6, 20 3 of 31

the administration of Ashwagandha during a program of resistance training improves the strength and muscular power of the upper and lower limbs [19,20]. Despite the above, no meta-analytic study has been conducted to evaluate the effect of Ashwagandha supplementation on physical performance. Moreover, no comprehensive systematic review with Bayesian meta-analysis has been carried out on clinical trials that have investigated the effect of the aqueous extract of the root of this plant on the different physical capacities and variables related to physical performance in healthy adult subjects. Thus, this systematic review and meta-analysis evaluated clinical trials up to 2020 that have assessed the effect of Ashwagandha (Withania somnifera) supplementation on physical performance (considering as main variables the muscle strength, VO2max, muscle fatigue, tiredness, and physical recovery (i.e., pain, inﬂammatory status, and sleep quality)) in healthy individuals.

2. Methods 2.1. Protocol and Registration The present systematic review was conducted and reported according to the estab- lished guidelines of the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines [21]. As this review was not eligible to be registered on PROSPERO, the summary information was uploaded to Figshare to make it publicly accessible in order to avoid unnecessary duplication (doi:10.6084/m9.ﬁgshare.12725081).

2.2. Eligibility Criteria The inclusion criteria for this systematic review were as follows: (1) clinical trials (randomized or not) in healthy females and males; (2) articles that were published from 2010 onwards; (3) studies written in English, German and Spanish; (4) trials that assessed the effect of Ashwagandha supplementation compared to a control group or with repeated measures; and (5) trials that reported the effects on variables related to physical performance (e.g., muscle strength, VO2max, muscle fatigue, tiredness, and physical recovery). Studies that did not correspond to original research (e.g., editorials, notes, reviews, dissertations, etc.) or include adults (e.g., children, elderly, etc.) were excluded.

2.3. Information Sources The following academic and free research databases were selected to examine the literature: PubMed/Medline, ScienceDirect, and Google Scholar. Further papers were sought by hand-searching.

2.4. Search Methods The following Boolean algorithms were used to perform the search: PubMed/MEDLINE (ashwagandha OR withania somnifera OR indian ginseng OR winter cherry OR poison gooseberry) AND (performance OR sports OR strength OR recovery OR sleep) NOT a disease; and ScienceDirect (ashwagandha OR withania somnifera) AND (performance OR sports OR strength OR recovery). The identiﬁcation of potential studies was enriched by performing a hand-search in Google Scholar with free language terms related to Ashwa- gandha supplementation and physical performance.

2.5. Study Selection After the search of published articles, the ﬁlter options of the databases were used to meet the inclusion criteria 1 to 3. After this searching process, the remaining references were ﬁltered by screening the title, abstract, or full-text publication. A network graph was built using the Connected Papers tool (www.connectedpapers.com accessed on 23 June 2020) to ensure the inclusion of recent publications and to visually discover relevant studies. The study selection took place during May and June of 2020, although an updated search was conducted in December 2020 prior to manuscript submission. J. Funct. Morphol. Kinesiol. 2021, 6, 20 4 of 31

2.6. Data Collection Process and Items The full-text articles of the selected studies were evaluated for meeting the inclusion criteria. The following data were obtained and analyzed from the selected studies: (i) descriptive statistics of the study population; (ii) study length; (iii) characteristics of the Ashwagandha supplementation protocol; (iv) magnitude and units of the analyzed variables; (v) the percentage of change according to the formula: ((postpre)/pre) × 100, and signiﬁcant difference in comparison to placebo or control group (if existed); and (vi) study conclusions.

2.7. Risk of Bias Two authors (D.A.B. and C.G.) independently evaluated the risk of bias of all included clinical trials using the Cochrane risk of bias tool RoB 2.0 [22], including bias due to randomization, bias due to deviations from intended intervention, bias due to missing data, bias due to outcome measurement, and bias due to selection of reported results. All randomized participants in the analysis were included, as it was the least-biased way to analyze clinical effects. Discrepancies were identiﬁed and resolved through discussion (with a third author where necessary). The ﬁgures summarizing the results of the risk of bias assessment were developed using the risk-of-bias VISualization package (robvis)[23].

2.8. Summary Measures The primary outcome was changes in physical performance variables (muscle strength, VO2max, muscle fatigue, tiredness, and physical recovery) after Ashwagandha supplementation. Selected publications that met all the requirements went on to the next phase of data analysis and synthesis, where a table of their results and ﬁndings comparison was developed and complemented by the review authors considering the items mentioned before (see data items).

2.9. Data Analysis and Synthesis All studies included in this meta-analysis compared an experimental group (i.e., a group that received Ashwagandha supplementation) against a control group (i.e., a group of participants that received a placebo) in regards to physical performance variables. A pre-specified subgroup analysis (strength/power, cardiorespiratory fitness and fatigue/recovery) was conducted to evaluate interaction and effect modification. In detail, we extracted the changes from baseline (mean change and SDs of the changes) in the experimental interventions (Ashwagandha) and the comparator interventions (placebo). Following the Cochrane Handbook for Systematic Review of Interven- tions [24], the correlation coefficients (Corr) from studies reported in considerable detail were calculated for each variable in order to impute a change-from-baseline SD in studies √ 2 for which these values were missing, according to the expression SD change = (SD baseline 2 + SD final − (2 x Corr × SDbaseline × SDfinal)) [25]. We also had e-mail communication with some authors to obtain missing information. Subsequently, the effect sizes, as standardized mean differences, were calculated as unbiased Cohen’s d (dunb), also known as Hedges’ g [26]. Positive scores of standardized mean differences indicated a higher physical performance in each subgroup; however, it should be noted that this method did not correct for differences in the direction of the scale, so to ensure that all the scales point in the same direction, we multiplied some variables by −1 (mainly fatigue/recovery variables), before standardization [24]. There were some complex data structures within the analyzed studies that required attention before computing a summary effect across studies. All solutions to these potential issues were based on the recommendations by Borenstein et al. [27]. On one hand, several studies reported multiple outcomes from the same participants, which may be problematic due to the fact that treating each outcome as a separate unit in the analysis will assign more weight to studies with two outcomes than those reporting one, which leads to a wrong estimate of the precision of the summary effect. To address this problem, we computed the mean of the outcomes for correlated variables in each study

Review Effects of Ashwagandha (Withania somnifera) on Physical J. Funct. Morphol. Kinesiol. 2021, 6, 20 Performance: Systematic Review and Bayesian5 of 31 Meta-Analysis

Diego A. Bonilla 1,2,3,4,*, Yurany Moreno 1,2, Camila Gho 1, Jorge L. Petro 1,3, Adrián Odriozola-Martínez 4,5,6 and usedand Richard this synthetic B. Kreider score 7 as the unit of analyses per study. This was different for non-correlated variables given that setting the correlation among the outcomes at 0.00 will treat the comparison as independent of each other and yields the same precision as a 1 separate unit in the meta-analysis. On Research the other Division, hand, Dynamical some studies Business used & Science a single Society—DBSS placebo International SAS, Bogotá 110861, Colombia; [email protected] (Y.M.); [email protected] (C.G.); group and several treatment [email protected] which represent (J.L.P.) certain problems when we want to incorporate both treatment groups2 Research in the Group same in analysis. Biochemistry To and combine Molecular these Biology, multiple Universidad Distrital Francisco José de Caldas, comparisons within the studies, we setBogotá the correlation110311, Colombia among comparisons at 0.50. Finally, to compute effects in3 each Research pre-specified Group in Physical subgroup, Activity, the Sports meta-analysis and Health Sciences was (GICAFS), Universidad de Córdoba, Montería 230002, Colombia based on dunb between two independent group change-from-baseline means by employing 4 a single pooled standard deviation. kDNA The heterogeneity Genomics, Joxe Mari was Korta evaluated Research byCenter means, University of the of the Basque Country UPV/EHU, 20018 Donostia, San Sebastián, Spain; [email protected] diamond ratio with values approaching or exceeding 2.0, implying considerable hetero- 5 Sport Genomics Research Group, Department of Genetics, Physical Anthropology and Animal Physiology, geneity. To address any substantial heterogeneity,Faculty of Science we and checked Technology, outlier University effects of and the Basque explored Country (UPV/EHU), 48940 Leioa, Spain the impact of excluding studies with6 Phymo a standardized Lab, Physiology mean and differenceMolecular Laboratory, higher than 08028 5Barcelona, (see Spain AppendixA), considering such big effect7 Exercise sizes & highly Sport Nutrition influence Labo theratory, summary Human effect.Clinical TakingResearch Facility, Texas A&M University, into account the assumption of exchangeability,College Station, a random-effect TX 77843, USA; [email protected] was used. This first meta-analytic approach was performed* Correspondence: within the [email protected]; Exploratory Software Tel.: +57-320-335-2050 for Confidence Intervals (ESCI) [28]. Additionally, to directly modelAbstract: the uncertainty Ashwagandha (which (Withania has been somnifera found) tois considered be superior a potent adaptogen and anti-stress agent in estimating the between-study heterogeneitythat could have and some pooled potential effect) to improve and to produce physical fullperformance. pos- This preferred reporting items for teriorCitation: distributions Bonilla, D.A.; (which Moreno, allows Y.; calculatingsystematic actualreviews probabilities), and meta-analyses a Bayesian (PRISMA)-based hierarchical comprehensive systematic review and modelGho, was C.; Petro, used J.L.; to Odriozola- pool effects in aBayesian Bayesian meta-analysis meta-analysis aimed using to evaluate “Stan” clinical (brms )trials package up to 2020 from PubMed, ScienceDirect, and v2.13.0Martínez, [29], A.; within Kreider, the R.B. R Effects statistical of Google computing Scholar environment databases regarding v4.0.2 [ 30the]. effect Importantly, of Ashwagandha the supplementation on physical per- effectAshwagandha sizes used (Withania in the somnifera Bayesian) on meta-analysisformance in healthy (and displayedindividuals. in Besides the forest implementing plot) do estimation not statistics analysis, we developed Physical Performance: Systematic correspond to the calculated effectBayesian sizes in hierarchical the original models studies for (da pre-specifiedunb), but the subgroup estimate meta-analysis on strength/power, cardi- Review and Bayesian Meta-Analysis. J. of the “true” effect size (θk) of eachorespiratory study (obtained fitness from and fatigue/recovery the model with variable the mentioneds. A total of 13 studies met the requirements of Funct. Morphol. Kinesiol. 2021, 6, 20. function (brms). The convergence assessment,this systematic that review, is, if although the Hamiltonian only 12 were Markov included chain in the quantitative analysis. A low-to-mod- https://doi.org/10.3390/jfmk6010020 Monte Carlo algorithm found an optimalerate overall solution, risk wasof bias conducted of the trials based included on posterior in this study pre- was detected. All Bayesian hierarchical dictive checks and with the inspectionmodels of potential converged scale to a target reduction distribution factor ( Ȓ )=values 1) for both of the meta-analytic effect size (μ) and between- Received: 1 January 2021 parameter estimates (meta-analyticstudy effect standard size [µ] anddeviation tau [ τ(]).τ). TheThe meta-analytic posterior distributions approaches of the included studies revealed that Accepted: 9 February 2021 of thePublished:θk for each 11 February study, 2021 the posteriorAshwagandha mean, and the supplementation 95% credible was intervals more efficaciou (95% CrI)s than were placebo for improving variables related reported. This last is an interval in whichto physical there performance is a 95% certainty in healthy that men the and “true” female. value In fact, of the Bayesian models showed that future 2 the parameterPublisher’s Note: lies. MDPI The staysτ value andinterventions its squared numbermight be (atτ least) were in calculatedsome way bene to reportficial on the the analyzed outcomes considering the estimatedneutral standardwith regard to deviation jurisdictional and variance95% credible of underlying intervals for effects the meta-analytic across studies effect with size. this Several practical applications and future meta-analyticclaims in published approach, maps and respectively. directions Based on are the discussed, obtained although Bayesian more model, comparab we calculatedle studies are needed in exercise training, and the exactinstitutional probabilities affiliations. that µ will beathletic smaller/larger populations than are a givenneeded effect to derive size valuea more by stable looking estimate of the true underlying effect. at the empirical cumulative distribution function (ECDF) of the posterior distribution for the pooled effect size. The function forest()Keywords:, which herbal is includedsupplements; in the musclebrmstools strength;package, cardiorespiratory and fitness; exercise tolerance; qual-

the ggplot2 functions were used toity draw of life; forest sleep and latency density plots within R. Copyright: © 2021 by the authors. 3. ResultsLicensee MDPI, Basel, Switzerland. 3.1. StudyThis article Selection is an open access article Afterdistributed running under the the terms search and algorithms1. Introduction with Boolean operators and free language terms, 1310conditions references of the were Creative obtained. Commons After the screening process of the publications found Attribution (CC BY) license Traditional botanical knowledge is characteristic of cultural contexts with long expe- (filtering by date, type of article and availability of full text) resulted in 484 potentially (http://creativecommons.org/licenses rience in the natural environment; however, as it is transmitted from generation to gener- eligible studies. However, after evaluating the abstracts and full text of these studies /by/4.0/). ation, it adapts and transforms through scientific experimentation. Within the traditional to filter out duplicates and analyze strict compliance with the other inclusion criteria, 471 articles were excluded. A total of 13 studies met the requirements of this systematic review. Figure1 shows a flowchart of the literature search.

J. Funct. Morphol. Kinesiol. 2021, 6, 20. https://doi.org/10.3390/jfmk6010020 www.mdpi.com/journal/jfmk J. Funct.J. Morphol. Funct. Morphol. Kinesiol. Kinesiol. 2021,2021 6, 20, 6 , 20 6 of 316 of 31

Figure 1. Preferred reporting items for systematic reviews and meta-analyses (PRISMA) flow diagram. Figure 1. Preferred reporting items for systematic reviews and meta-analyses (PRISMA) flow diagram. The verification of relevant publications in the field of supplementation with Ash- wagandha,The verification and a visualization of relevant of publications the trends, popular in the field works ofand supplementation dynamics of the with field, Ashwa- is gandha,shown and in Figure a visualization2 through a graphof the that trends, shows popular each node works as an and academic dynamics product of relatedthe field, is shownto a sourcein Figure document, 2 through which a graph was selected that shows according each tonode the as number an academic of citations product and the related to ahigh source degree document, of relationship which with was the selected objective according of this systematic to the number review. Consideringof citations theand the highabove, degree the of work relationship selected was wi thatth the of Wankhedeobjective etof al.,this 2015 systematic [12]. review. Considering the above,3.2. Riskthe ofwork Bias selected within Studies was that of Wankhede et al., 2015 [12]. The methodological quality of the trials included in this systematic review and meta- analysis is presented in Figure3.

3.3. Results of Individual Studies With a total of 615 healthy adults participating in the studies analyzed in this systematic review, there is direct evidence suggesting that Ashwagandha supplementation has a positive effect on different variables related to physical performance when compared to a placebo or control group. The most relevant aspects of the selected publications are summarized in Table1. J. Funct. Morphol. Kinesiol. 2021, 6, 20 7 of 31

J. Funct. Morphol. Kinesiol. 2021, 6, 20 7 of 31 J. Funct. Morphol. Kinesiol. 2021, 6, 20 7 of 31

Figure 2. Network graph of the studies related to Ashwagandha supplementation. Node size is proportional to the number of citations, and the color is the publishing year. This graph was developed within www.connectedpapers.com accessed on 23 June 2020.

FigureFigure 2. Network 2. Network graph3.2. of the graphRisk studies of of Bias relatedthe within studies to Ashwagandha Studies related to supplementation. Ashwagandha Node supplementation. size is proportional Node to the size number is of citations,proportional and the color to the is the numberThe publishing methodological of ci year.tations, This graph and quality wasthe color developedof the is trials the within publishing included www.connectedpapers.com in year. this Thissystematic graph accessed wasreview devel- on and meta- 23 June 2020. oped within www.connectedpapers.canalysis is presented inom Figure accessed 3. on 23 June 2020.

3.2. Risk of Bias within Studies The methodological quality of the trials included in this systematic review and meta- analysis is presented in Figure 3.

FigureFigure 3. Risk 3. Risk of bias of bias summary summary for for included studies. studies. Weighted Weighted bar-chart bar-chart of the of distribution the distribution of risk-of-bias of risk-of-bias judgments. judgments. These Thesegraphics graphics were were obtained obtained using using the robvisthe robvispackage package within within the R statistical the R statistical computing computing environment. environment.

3.3. Results of Individual Studies With a total of 615 healthy adults participating in the studies analyzed in this systematic review, there is direct evidence suggesting that Ashwagandha supplementation has a Figure 3. Risk of bias summary for included studies. Weighted bar-chart of the distribution of risk-of-bias judgments. positive effect on different variables related to physical performance when compared to a These graphics were obtained using the robvis package within the R statistical computing environment. placebo or control group. The most relevant aspects of the selected publications are summarized in Table 1. 3.3. Results of Individual Studies With a total of 615 healthy adults participating in the studies analyzed in this systematic review, there is direct evidence suggesting that Ashwagandha supplementation has a positive effect on different variables related to physical performance when compared to a placebo or control group. The most relevant aspects of the selected publications are summarized in Table 1. J. Funct. Morphol. Kinesiol. 2021, 6, 20 8 of 31

Table 1. Evidence of the effects of Ashwagandha supplementation on physical performance.

Ashwagandha (WS) Supplementation Sample (F:M) Age/ Analyzed Change Reference Design/ Dosage Conclusions BMI Groups (Withanolides) Variables Length (mg) WS may be useful • · −1 a against physical WS (n = 10) VMax (m s ) b +2.9% †* exhaustion and to TA (n = 10) • Wabsolute (W) Sandhu et al., 40 20.6 (2.5) RCT-SB/ 500 +8.8% †* improve speed, WS+TA (n = 10) ARE (ND) • Wrelative 2010 [31] (18:22) 21.9 (2.2) 8 weeks (o.d. qAM) − +10.1% †* lower limb muscle Placebo (W·kg 1) b +6.8% †* strength and (ﬂour, n = 10) • VO c 2máx neuromuscular coordination. 750 per day × Muscle strength (kg): WS supplementation 10 days, 1000 • +8.0% NS Handgrip is not only safe Raut et al. 18 24.3 (2.1) CT-O/ per day × 10 • +21.5% † WS (n = 18) ARE 8:1 (ND) Quadriceps but has positive 2012 [32] (6:12) 24.2 (2.7) 30 days days and 1250 • +15.4% † Back extensors effects on per day × 10 • −26.9% NS RPE-Borg (VAS) muscle strength. days (b.i.d.) First study to c report signiﬁcant WS (n = 18) • VO2máx +13% †* Shenoy et al., 40 19.6 (1.4) RCT/ 1000 improvements in Placebo ARE (ND) • TTE (min) +7.2% †* 2012 [33] (20:17) 19.6 (1.9) 8 weeks (500 b.i.d.) c the sports (starch, n = 19) • RER +1.8% NS performance of elite athletes. WS supplementation WS (n = 16) d Malik et al., 32 17.4 (1.7) RCT-SB/ 500 • VO2máx +6.67% †* for eight weeks Placebo ARE (ND) 2013 [34] (0:32) 20.9 (2.9) 8 weeks (q.h.s.) • [Hb] +5.14% †* improves VO (sugar, n = 16) 2max and [Hb] in hockey players. J. Funct. Morphol. Kinesiol. 2021, 6, 20 9 of 31

Table 1. Cont.

Ashwagandha (WS) Supplementation Sample (F:M) Age/ Analyzed Change Reference Design/ Dosage Conclusions BMI Groups (Withanolides) Variables Length (mg)

WS contributes to • [Hb] +6.29% † 3 the increase of WS (n = 11) • MCV (µm ) +8.12% † [Hb] and Kuchewar 30 18 to 45 years Guduchi (n = 10) RCT-DB/ 1000 • MCHC (%) +4.45% † ARE (ND) hematological et al., 2014 [35] (20:10) old Placebo 6 meses (500 b.i.d.) • MCH (pg) +8.15% † markers while (CaCO , n = 9) • MDA −18.2% † 3 preventing • SOD +18.1% † oxidative stress.

Fourteen days of WS ARLE Reaction time: supplementation RCT-DB WS (n = 10) Sensoril® • −6.2% †* decreases reaction Crossver/ SRT Pingali et al., 20 24.9 (4.18) Placebo 1000 ≥10% • −3.39% †* time compared 14 days CDT 2014 [36] (0:20) 22.38 (1.1) (carbohydrates (500 b.i.d.) withanolides • −8.14% †* to placebo, (14 days DSST mixture, n = 10) and ≤0.5% • −3.16% †* indicating a wash-out) DVT withaferin-A positive effect on cognitive and psychomotor function.

e WS root extract • VO2máx increases 20 to WS (n = 25) ARE Quality of life test: +6.2% †* cardiorespiratory Choudhary 49 45 years old/ Placebo RCT-DB/ 600 KSM-66® 5% • +14.7% †* endurance and et al., 2015 [37] (ND) 18.5 to (saccharose, 3 months (300 b.i.d.) Physical health withanolides • +19.6% †* improves quality 24.9 kg·m−2 n = 24) Psychological health of life in healthy athletic adults. J. Funct. Morphol. Kinesiol. 2021, 6, 20 10 of 31

Table 1. Cont.

Ashwagandha (WS) Supplementation Sample (F:M) Age/ Analyzed Change Reference Design/ Dosage Conclusions BMI Groups (Withanolides) Variables Length (mg) WS • PB 1-RM supplementation +138% †* • KE 1-RM during a +51.9% †* • 2 resistance training Arm (cm ) +17.1% †* WS (n = 25) ARE • program increases Wankhede 50 28.0 (8.0) RCT-DB/ 600 Chest (cm) +3.32% †* Placebo (starch, KSM-66® 5% 2 muscle strength et al., 2015 [19] (0:50) /NA 8 weeks (300 b.i.d.) • Thigh (cm ) +8.0% † n = 25) withanolides and size while • % BF f +16.0% †* accelerating • [T] +15.2% †* post-exercise •↑ [CK] 24/48 −98.9% †* recovery in untrained subjects. WS330: +15.7% †* +15.9 †* WS • Max Dist g +9.3% NS supplementation WS330 (n = 10) • g · −1 +4.95% † for 28 days 330 and 500 per Vav (km h ) Tripathi et al., 30 27.3 (2.48) WS500 (n = 10) RCT-O/ −1 g +5.40% † increases muscle group (o.d. ARE (ND) • Vmax (km·h ) 2016 [38] (0:30) 22.9 (1.44) Placebo (starch, 28 days WS500 strength and qAM) • Handgrip (kg): n = 10) +15.9% †* cardiorespiratory • VO h 2máx +16.2% †* capacity in +12.0% † healthy men. +4.93% † +6.09% † J. Funct. Morphol. Kinesiol. 2021, 6, 20 11 of 31

J. Funct. Morphol. Kinesiol. 2021, 6, 20 10 of 31 Table 1. Cont.

Ashwagandha (WS) Supplementation Sample (F:M) Age/ Analyzed Change • HAM-A −40.8% †* Reference Design/ Dosage Conclusions WS reduces anxiety levels, [cor- BMI Groups (Withanolides) Variables WS (n = 30) ARLE • DASS-21 −30.0 † Length (mg) Lopestri et al., 60 42.2 (2.44) RCT-DB/ 240 tisol] and [DHEA-S], with a Placebo (toasted Shoden® ≥35% with- • [Cortisol] −23.3% †* 2019 [40] (23:37) 24.6 (0.60) 60 days (o.d. qPM) non-significant tendency to in- rice, n = 30) anolide glycosides • [DHEA-S] −8.15% †* • SQ 1-RM crease [testosterone] in men. • [T] +11.4 † • SQ peak • RSQ-W power (W) i +72% †* WS • SOL (min) k 120 mg of WS extract increases • SQ mean −27.2% †* supplementation ARLE • WASO k the overall quality of sleep i WS+18.0% (n = 75)†* −14.7% †* Deshpande et peak150 (W)36.8 (10.9) RCT-DB/(500 mg·day120−1 ) Shoden® ≥35% with- • TTS (min) k (time and efficiency) and in- • Placebo+8.45% (rice †* +4.8% †* al., 2020 [41] (78:72)SQ Reps 25.0 (3.9) 6 weeksfor twelve (o.d. weeks qPM) anolide glycosides (42 mg • SE (%) k creases the quality of life at the powder,+4.64% n = 75) † +4.6% †* ARLE 65%-RM of a resistance HPLC) Quality of life test: physical and psychological lev- +22.8% † +13.1 † Sensoril® • BP 1-RM training program • Physical health els. WS (n = 19) +13.6% † +11.8 † Ziegenfuss 38 24.4 (4.2) RCT-DB/ 500 ≥10% • BP peak improves upper • Psychological health Placebo i +11.3% †* et al., 2018 [20] (0:38) 26.2 (3.4) 3 months (o.d. qAM) withanolidesData are expressedpower (W) as mean (SD). The change valuesand lower are expresse limb d as the percentage change of the group supplemented with Ashwagandha according to the (flour, n = 19) +11.6% †* and ≤0.5%formula:• ((postpre)/pre)BP mean × 100. ↑[CK] 24/48: increasemuscle in serum strength creatine kinase concentration (U/L) from 24 h to 48 h post-exercise; [cortisol]: serum cortisol concen- i +28.1% † withaferin-A peak−1 (W) and power −1 −1 tration (μg·dL ); [DHEA-S]: serum−21.2% DHEA † sulfate concentration (μg·dL ); [Hb]: blood hemoglobin concentration; [T]: serum testosterone concentration (ng·dL );% • BP Reps and perceived BF: body fat percentage; 1-RM: one-repetition+14.4% † maximum (kg); ARE: aqueous root extract; ARLE: aqueous root and leaves extract; b.i.d.: bis in die—twice a day; BMI: body mass 65%-RMindex (kg·m−2); BP: bench press; CDT: choicerecovery discrimination in test; CT-O: open-label clinical trial; DASS-21: depression anxiety stress scales-21; DSST: digit • −1.93% † symbol substitutionTime-trial test; DVT: digit vigilance test; recreationallyF: female; HAM-A: Hamilton anxiety rating scale; KE: knee extension; M: male; MCH: mean corpuscular hemo- j globin; MCHC:7.5 km mean (s) cell hemoglobin concentration;active MCV: subjects. mean corpuscular volume; MDA: malondialdehyde (nmol·mL−1); Max Dist: maximum distance (km); • NA: not available;PRS (VAS) o.d.: omne in die—once a day; PRS: perceived recovery scale; PSS-10: perceived stress scale; qAM: quaque ante meridiem—every morning; q.h.s.: • [Hb] quaque hora somni—every night at bedtime; QoS: quality of sleep; qPM: quaque post meridiem—every afternoon or evening; RCT-O: open-label randomized clinical trial; RCT-SB: single-blinded randomized clinical trial; RCT-DB: double-blinded randomized clinical trial; Reps 65%-RM: as many repetitions as possible Eight weeks of with a load of 65% RM; RER: respiratoryWS250: exchange ratio; RPE-Borg: rating of perceived exertion; RSQ-W: restorative sleep questionnaire-weekly version (0–100); supplementation−1 SE: sleep efficiency; SOD: superoxide−33.7% dismutase †* (U·g Hb); SQ: squat; SOL: sleep onset latency; SRT: simple reaction time; TA: Terminalia arjuna; TTE: time to of aqueous WS exhaustion; TTS: total time of sleep;− VAS:16.5 †* visual analog scale; VMax: maximum velocity; Vav: average velocity; Wabsolute: absolute power; Wrelative: relative power; WASO: root extract was a b c −1 −1 d WS250 (n = 19) wake •after PSS-10sleep onset (min); WS:− 13.0%Withania NS somnifera (Ashwagandha). Photoelectric sensor; contact mat; ergospirometry (mL·kg ·min ); Cooper’s 12 min run 250 and ARE −1 −1 e −1 f associated with a g h Salve et al., 58 31.1 (7.5) WS600 (n = 20) RCT-DB/ test (mL·kg• [cortisol]·min ); Léger’s test− (L·min41.0% †*); tetrapolar bioelectrical impedance analysis; six-minute cycle ergometer exercise test; YMCA cycle ergometer sub- 600 per group KSM-66® 5% significant 2019 [39] (ND) /NA Placebo (starch, 8 weeks maximal• testHAM-A (mL·kg−1·min−1); i linearWS600 position transducer system TENDO; j cycle ergometer; k ActiSleep®; † significant difference in comparison to baseline; * signif- (o.d. qAM) withanolides reduction of stress n = 19) icant difference• QoS in comparison to− placebo;38.3% †* ⁑ significant difference in comparison to the other Ashwagandha group; NS no significant change. levels and −32.6% †* improved the −16.3% †* overall quality −46.0% †* of life. J. Funct. Morphol. Kinesiol. 2021, 6, 20 12 of 31

Table 1. Cont.

Ashwagandha (WS) Supplementation J. Funct. Morphol. Kinesiol. 2021, 6, 20 10 of 31 Sample (F:M) Age/ Analyzed Change Reference Design/ Dosage Conclusions BMI Groups (Withanolides) Variables Length (mg)

WS reduces • HAM-A −40.8% †* • HAM-A − WS reduces anxiety levels, [cor- ARLE WS (n = 30) 40.8% †* anxietyARLE levels, • DASS-21 −30.0 † Lopestri et al., 60 42.2 (2.44)® • DASS-21 RCT-DB/ − 240 tisol] and [DHEA-S], with a WS (n = 30) Shoden Placebo (toasted 30.0 † Shoden[cortisol]® ≥35% and with- • [Cortisol] −23.3% †* Lopestri et al., 60 42.2 (2.44) RCT-DB/ 2019240 [40] (23:37) 24.6 (0.60) • [Cortisol] 60 days (o.d. qPM) non-significant tendency to in- Placebo (toasted ≥35% rice, n = 30) −23.3% †* [DHEA-S],anolide glycosides with a • [DHEA-S] −8.15% †* 2019 [40] (23:37) 24.6 (0.60) 60 days (o.d. qPM) • [DHEA-S] crease [testosterone] in men. rice, n = 30) withanolide −8.15% †* non-significant • [T] +11.4 † • [T] glycosides +11.4 † tendency to increase • RSQ-W +72% †* [testosterone] in men. • SOL (min) k 120 mg of WS extract increases −27.2% †* ARLE • WASO k the overall quality of sleep • WS RSQ-W(n = 75) −14.7% †* Deshpande et 150 36.8 (10.9) RCT-DB/ 120 Shoden® ≥35% with- • TTS (min) k (time and efficiency) and in- Placebo (rice k 120 mg of WS +4.8% †* al., 2020 [41] (78:72) 25.0 (3.9) • SOL (min) 6 weeks (o.d. qPM) anolide glycosides (42 mg • SE (%) k creases the quality of life at the powder, n = 75)k +72% †* extract increases +4.6% †* ARLE • WASO HPLC) Quality of life test: physical and psychological lev- −27.2% †* the overall quality +13.1 † Shoden® • TTS (min) k • Physical health els. WS (n = 75) −14.7% †* of sleep (time and +11.8 † Deshpande 150 36.8 (10.9) RCT-DB/ 120 ≥35% k • Psychological health Placebo (rice • SE (%) +4.8% †* efficiency) and et al., 2020 [41] (78:72) 25.0 (3.9) 6 weeks (o.d.Data qPM) are expressedwithanolide as mean (SD). The change values are expressed as the percentage change of the group supplemented with Ashwagandha according to the powder, n = 75) Quality of life test: +4.6% †* increases the glycosides ↑ formula: ((postpre)/pre) × 100. [CK] 24/48: increase in serum+13.1 creatine † kinasequality concentration of life (U/L) from 24 h to 48 h post-exercise; [cortisol]: serum cortisol concentration (μg·dL(42−1 mg); [DHEA-S]: HPLC) serum• Physical DHEA sulfate health concentration (μg·dL−1); [Hb]: blood hemoglobin concentration; [T]: serum testosterone concentration (ng·dL−1);% • Psychological +11.8 † at the physical and BF: body fat percentage; 1-RM: one-repetition maximum (kg); ARE: aqueouspsychological root extract; levels ARLE:. aqueous root and leaves extract; b.i.d.: bis in die—twice a day; BMI: body mass index (kg·m−2); BP: benchhealth press; CDT: choice discrimination test; CT-O: open-label clinical trial; DASS-21: depression anxiety stress scales-21; DSST: digit symbol substitution test; DVT: digit vigilance test; F: female; HAM-A: Hamilton anxiety rating scale; KE: knee extension; M: male; MCH: mean corpuscular hemo- Data are expressed as mean (SD). The change values are expressed as the percentage change of the group supplementedglobin; MCHC: with mean Ashwagandha cell hemoglobin according concentration; to the formula: ((postpre)/pre)MCV: mean corpuscular× 100. ↑[CK] volume; 24/48: MDA: malondialdehyde (nmol·mL−1); Max Dist: maximum distance (km); −1 −1 increase in serum creatine kinase concentration (U/L) from 24 h to 48 h post-exercise; [cortisol]: serum cortisolNA: concentration not available; (µg·dL o.d.:); omne [DHEA-S]: in die—once serum DHEA a day; sulfate PRS: concentration perceived recovery (µg·dL scale;); [Hb]: PSS-10: blood perceived stress scale; qAM: quaque ante meridiem—every morning; q.h.s.: hemoglobin concentration; [T]: serum testosterone concentration (ng·dL−1);% BF: body fat percentage; 1-RM: one-repetition maximum (kg); ARE: aqueous root extract; ARLE: aqueous root and leaves extract; b.i.d.: bis in die—twice a day; BMI: body mass index (kg·m−2); BP: bench press; CDT: choice discrimination test;quaque CT-O: open-labelhora somni—every clinical trial; night DASS-21: at bedtime; depression QoS: anxiety quality stress scales-21;of sleep; DSST:qPM: digit quaque symbol post meridiem—every afternoon or evening; RCT-O: open-label randomized substitution test; DVT: digit vigilance test; F: female; HAM-A: Hamilton anxiety rating scale; KE: knee extension;clinical M: male; trial; MCH: RCT-SB: mean single-blinded corpuscular hemoglobin; randomized MCHC: clinical mean trial; cell hemoglobin RCT-DB: double-blinde concentration; d randomized clinical trial; Reps 65%-RM: as many repetitions as possible MCV: mean corpuscular volume; MDA: malondialdehyde (nmol·mL−1); Max Dist: maximum distance (km); NA:with not a available;load of 65% o.d.: RM; omne RER: in die—once respiratory a day; exchange PRS: perceived ratio; recovery RPE-Bo scale;rg: rating PSS-10: of perceived perceived exertion; RSQ-W: restorative sleep questionnaire-weekly version (0–100); stress scale; qAM: quaque ante meridiem—every morning; q.h.s.: quaque hora somni—every night at bedtime; QoS:SE: sleep quality efficiency; of sleep; qPM: SOD: quaque superoxide post meridiem—every dismutase (U·g afternoon−1 Hb); or SQ: evening; squat; RCT-O: SOL: open-label sleep onset latency; SRT: simple reaction time; TA: Terminalia arjuna; TTE: time to randomized clinical trial; RCT-SB: single-blinded randomized clinical trial; RCT-DB: double-blinded randomized clinical trial; Reps 65%-RM: as many repetitions as possible with a load of 65% RM; RER: exhaustion; TTS: total time of sleep; VAS: visual analog scale; VMax: maximum velocity; Vav: average velocity; Wabsolute: absolute power; Wrelative: relative power; WASO: respiratory exchange ratio; RPE-Borg: rating of perceived exertion; RSQ-W: restorative sleep questionnaire-weekly version (0–100); SE: sleep efficiency; SOD: superoxide dismutase (U·g−1 Hb); SQ: squat; SOL: wake after sleep onset (min); WS: Withania somnifera (Ashwagandha). a Photoelectric sensor; b contact mat; c ergospirometry (mL·kg−1·min−1); d Cooper’s 12 min run sleep onset latency; SRT: simple reaction time; TA: Terminalia arjuna; TTE: time to exhaustion; TTS: total time of sleep; VAS: visual analog scale; VMax: maximum velocity; Vav: average velocity; Wabsolute: absolute a −1 −1b e c −1 f −1 −1 d g h power; Wrelative: relative power; WASO: wake after sleep onset (min); WS: Withania somnifera (Ashwagandha). testPhotoelectric (mL·kg sensor;·min ); contact Léger’s mat; testergospirometry (L·min ); tetrapolar (mL·kg · minbioelectrical); Cooper’s impedance 12 min run analysis; test six-minute cycle ergometer exercise test; YMCA cycle ergometer sub- (mL·kg−1·min−1); e Léger’s test (L·min−1); f tetrapolar bioelectrical impedance analysis; g six-minute cycle ergometermaximal exercise test (mL·kg test; h YMCA−1·min cycle−1); i linear ergometer position submaximal transducer test (mL system·kg−1 ·TENDO;min−1); i linear j cycle position ergometer; k ActiSleep®; † significant difference in comparison to baseline; * signif- transducer system TENDO; j cycle ergometer; k ActiSleep®; † significant difference in comparison to baseline; *icant significant difference difference in comparison in comparison to to placebo; placebo; ⁑ significantsignificant difference difference incomparison in comparison to the to other the other Ashwagandha group; NS no significant change. Ashwagandha group; NS no significant change.

Review Effects of Ashwagandha (Withania somnifera) on Physical Performance: Systematic Review and Bayesian Meta-Analysis

Diego A. Bonilla 1,2,3,4,*, Yurany Moreno 1,2, Camila Gho 1, Jorge L. Petro 1,3, Adrián Odriozola-Martínez 4,5,6 and Richard B. Kreider 7

1 Research Division, Dynamical Business & Science Society—DBSS International SAS, Bogotá 110861, Colombia; [email protected] (Y.M.); [email protected] (C.G.); [email protected] (J.L.P.) 2 Research Group in Biochemistry and Molecular Biology, Universidad Distrital Francisco José de Caldas, Bogotá 110311, Colombia 3 Research Group in Physical Activity, Sports and Health Sciences (GICAFS), Universidad de Córdoba, Montería 230002, Colombia 4 kDNA Genomics, Joxe Mari Korta Research Center, University of the Basque Country UPV/EHU, 20018 Donostia, San Sebastián, Spain; [email protected] 5 Sport Genomics Research Group, Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain 6 Phymo Lab, Physiology and Molecular Laboratory, 08028 Barcelona, Spain 7 Exercise & Sport Nutrition Laboratory, Human Clinical Research Facility, Texas A&M University, College Station, TX 77843, USA; [email protected] J. Funct. Morphol. Kinesiol. 2021, 6, 20 * Correspondence: [email protected]; Tel.: +57-320-335-205013 of 31

Abstract: Ashwagandha (Withania somnifera) is considered a potent adaptogen and anti-stress agent 3.4. Synthesis of Results that could have some potential to improve physical performance. This preferred reporting items for All studiesCitation: included Bonilla, D.A.; in the Moreno, quantitative Y.; systematic analysis werereviews randomized and meta-analyses placebo-controlled (PRISMA)-based comprehensive systematic review and trials; hence,Gho, only C.; onePetro, repeated-measures J.L.; Odriozola- studyBayesian was meta-analysis excluded since aimed it to did evaluate not include clinical the trials up to 2020 from PubMed, ScienceDirect, and placebo groupMartínez, [32]. A.; In Kreider, addition, R.B. Effects both of meta-analyticGoogle Scholar approaches databases wereregarding based the on effect changes of Ashwagandha supplementation on physical per- from baseline,Ashwagandha which according (Withania somnifera to current) on literature,formance in might healthy be individuals. more efficient Besides and implementing powerful estimation statistics analysis, we developed than a comparisonPhysical Performance: of post-intervention Systematic valuesBayesian solely, hierarchical as it removes models thefor a between-person pre-specified subgroup meta-analysis on strength/power, cardi- variability [24Review]. Very and Bayesian extreme Meta-Analysis. effect sizes J. wereorespiratory excluded fitness from and the fatigue/recovery meta-analysis since variable theys. A total of 13 studies met the requirements of Funct. Morphol. Kinesiol. 2021, 6, 20. may affect the overall effect and/or increasethis heterogeneity; systematic review, notwithstanding, although only 12 we were reported included in the quantitative analysis. A low-to-mod- https://doi.org/10.3390/jfmk6010020 all results (including outliers) in AppendixerateA to demonstrateoverall risk of howbias of the the overall trials included effect would in this study was detected. All Bayesian hierarchical have been. In this sense, after excluding outliers,models allconverged Bayesian to a models target distribution resulted in (Ȓ =values 1) for both meta-analytic effect size (μ) and between- Received: 1 January 2021 equal to one for both parameters (µ and τ),study which standard signified deviation convergence. (τ). The Additionally,meta-analytic approaches all of the included studies revealed that Accepted: 9 February 2021 our BayesianPublished: explorations 11 February to quantify2021 posteriorAshwagandha distributions supplementation with the Hamiltonian was more efficaciou Monte s than placebo for improving variables related Carlo algorithm were performed by runningto physical fourMarkov performance chains, in health whichy men implemented and female. In fact, the Bayesian models showed that future 1000 warmupPublisher’s iterations Note: and MDPI atotal stays of 4000 postinterventions warmup might sampling be at iterations. least in some The way following beneficial on the analyzed outcomes considering the subsections showneutral with the regard pre-specified to jurisdictional subgroup 95% analysis. credible intervals for the meta-analytic effect size. Several practical applications and future claims in published maps and directions are discussed, although more comparable studies are needed in exercise training, and 3.4.1. Strength and Power institutional affiliations. athletic populations are needed to derive a more stable estimate of the true underlying effect. The quantitative selection of this sub-group meta-analysis included five trials. Three articles [19,20,31] provided multiple outcomes,Keywords: while herbal one supplements; [38] reported muscle both strength; multiple cardiorespiratory fitness; exercise tolerance; qual-

outcomes and multiple treatment arms. Inity total, of life; seven sleep effectlatency sizes were estimated from different strength-Copyright: and © 2021 power-related by the authors. variables (muscle strength of upper and lower limbs, handgrip, maximumLicensee MDPI, velocity, Basel, Switzerland. relative power and testosterone concentration) using data from 198 participants,This article is an ofopen which access article 104 were in Ashwagandha groups and 94 in placebo groups. Thedistributed results of under the the random-effects terms and meta-analysis1. Introduction of standardized mean differences for strength/powerconditions of variables the Creative Commons is shown in Figure4. Compared to placebo, the pooled Attribution (CC BY) license Traditional botanical knowledge is characteristic of cultural contexts with long expe- treatment effect of Ashwagandha supplementation was medium (dunb: 0.68; 95% CI: 0.40 to 0.95). There(http://creativecommons.org/licenses was little heterogeneity amongrience the in studies the natural (diamond environment; ratio = 1.11). however, as it is transmitted from generation to gener- Our Bayesian/by/4.0/). model (at convergence,ation,= 1) it revealed adapts and an estimate transforms of the through pooled scientific effect experimentation. Within the traditional size of µ = 0.67 with the 95% CrI ranging from 0.28 to 1.04. Therefore, there was a medium overall effect size of the Ashwagandha interventions studied in this subgroup meta-analysis. Relatively small between-study standard deviation was estimated (τ = 0.31 (95% CrI 0.01– 0.96)) with an absolute value of the true variance of τ2 = 0.09 (low heterogeneity). The ECDF showedJ. Funct. that Morphol. the probability Kinesiol. 2021 of, the 6, 20. pooled https://doi.org/10.3390/jf effect being greatermk6010020 than 0.20 is very high www.mdpi.com/journal/jfmk (98.3%). Therefore, the effects of the Ashwagandha supplementation are very likely to be meaningful on strength/power-related variables. The results of the Bayesian random- effects meta-analysis conducted on the evaluated studies are shown in Figure5.

3.4.2. Cardiorespiratory Fitness This sub-group meta-analysis included seven studies. One article [34] provided multiple outcomes, while one study [38] reported on multiple comparisons. A total of eight effect sizes were estimated from research designs that compared Ashwagandha administration (total n = 119) with placebo groups (total n = 107) on cardiorespiratory fitness-related variables (VO2max and [Hb]). However, this first pooled treatment effect (dunb: 1.37; 95% CI: 0.26 to 2.48) was considered as merely exploratory since there was a very high degree of heterogeneity (diamond ratio = 3.74; see Figure A1). Thus, we proceed to the identification of potential outliers and found that the upper bound of the 95% CI of one study [20] was lower than the lower bound o0f the pooled effect CI (extremely small effect size). After exploring the impact of excluding that study, the new heterogeneity was considerably reduced to a medium-to-high-level (diamond ratio = 1.41). The results of this second random-effects meta-analysis of standardized mean differences for VO2max and [Hb] is shown in Figure6. Compared to placebo, the pooled treatment effect of Ashwagandha supplementation on cardiorespiratory fitness was very large (dunb: 1.85; 95% CI: 1.40 to 2.31). J. Funct. Morphol. Kinesiol. 2021, 6, 20 12 of 31

J. Funct. Morphol. Kinesiol. 2021, 6, 20 14 of 31

FigureFigure 4.4. ForestForest plotplot depictingdepicting thethe standardizedstandardized meanmean differencesdifferences fromfrom thethe meta-analysis meta-analysisof of studies studies comparing comparing groups groups with with Ashwagandha Ashwagandha or or placebo placebo on on strength/power. strength/power. ColoredColored

squaressquares representrepresent the the d dunbunb for individual trials, while colored and green polygons depictdepict thethe meta-analytic ddunbunb forfor the the indicated indicated subgroups subgroups and and for for the the overall overall (total) (total) results, results, respectively. respectively. −1 HorizontalHorizontal lineslines representrepresent thethe 95%95% conﬁdence confidence intervals intervals for for the the data. data. LL: LL: lower lower limbs; limbs; RM: RM: one-repetition one-repetition maximum maximum (kg); (kg); UL: UL: upper upper limbs; limbs; Vmax: Vmax: maximum maximum velocity velocity (m (m·s·s−1); Wrelative: −1 relativerelative powerpower (W(W·kg·kg−1);); WS330: WS330: WithaniaWithania somnifera somnifera 330330 mg; mg; WS500: WS500: WithaniaWithania somnifera somnifera 500500 mg. mg. J. Funct. Morphol. Kinesiol. 2021, 6, 20 15 of 31 J. Funct. Morphol. Kinesiol. 2021, 6, 20 13 of 31

Figure 5. ForestFigure plot of 5. aForest random plot effect of meta-analysis a random effect on stre meta-analysisngth/power variables on strength/power after Ashwagandha variables supplementation. after Ashwa- (A) The plot shows the names of the studies with their respective variable or multiple comparison/multiple outcomes on gandha supplementation. (A) The plot shows the names of the studies with their respective variable the left. On the right are the θk and 95% credible interval (CrI). The value of the overall weighted mean of effect sizes is drawn as a verticalor multiple line in comparison/multiplethe middle of the plot, which outcomes represents on the the left. reference On the line right to test are the the nullθk hypothesisand 95% crediblein each study. The posteriorinterval distributions (CrI). The value of the ofestimated the overall effect weighted sizes for each mean study of effect are shown sizes as is drawncyan densities. as a vertical The black line circle in the represents themiddle posterior of themean, plot, and which the horizontal represents line the extend referenceing from line the to testpoint the is nullthe 95% hypothesis CrI. The inbottom each row study. is the The meta-analytic effect size (μ). (B) Multivariate kernel density estimation plot of the posterior distribution of μ (x-axis) and τ (y-axis) withposterior the darker distributions zone indicating of theincreased estimated plausibility effect sizesof values. for each study are shown as cyan densities. The black circle represents the posterior mean, and the horizontal line extending from the point is the 95% CrI. The bottom row is the meta-analytic effect size (µ). (B) Multivariate kernel density estimation plot of the posterior distribution of µ (x-axis) and τ (y-axis) with the darker zone indicating increased plausibility of values. J. Funct. Morphol. Kinesiol. 2021, 6, 20 15 of 31

J. Funct. Morphol. Kinesiol. 2021, 6, 20 16 of 31

FigureFigure 6. Forest 6. Forest plot plot depicting depicting the the standardized standardized meanmean differences differences from from the th meta-analysise meta-analysis of studies of studies comparing compar groupsing groups with Ashwagandha with Ashwagandha or placebo or on placebo cardiorespiratory on cardiorespiratory ﬁtness. Colored fitness.

Coloredsquares squares represent represent the dunb thefor d individualunb for individual trials, while trials, colored while and colored green and polygons green depictpolygons the meta-analyticdepict the meta-analytic dunb for the indicateddunb for the subgroups indicated and subgroups for the overall and (total)for the results, overall respectively. (total) results, respectively.Horizontal Horizontal lines represent lines the represent 95% conﬁdence the 95% intervals confidence for the intervals data. Hb: for blood the data. hemoglobin Hb: blood concentration; hemoglobin VO concentration;2max: maximum VO oxygen2max: maximum uptake; WS330: oxygenWithania uptake; somnifera WS330:330 Withania mg; WS500: somnifera 330 Withaniamg; WS500: somnifera Withania500 mg.somnifera 500 mg.

Review Effects of Ashwagandha (Withania somnifera) on Physical Performance: Systematic Review and Bayesian Meta-Analysis

Diego A. Bonilla 1,2,3,4,*, Yurany Moreno 1,2, Camila Gho 1, Jorge L. Petro 1,3, Adrián Odriozola-Martínez 4,5,6 and Richard B. Kreider 7

1 Research Division, Dynamical Business & Science Society—DBSS International SAS, Bogotá 110861, Colombia; [email protected] (Y.M.); [email protected] (C.G.); [email protected] (J.L.P.) 2 Research Group in Biochemistry and Molecular Biology, Universidad Distrital Francisco José de Caldas, Bogotá 110311, Colombia 3 Research Group in Physical Activity, Sports and Health Sciences (GICAFS), Universidad de Córdoba, Montería 230002, Colombia 4 kDNA Genomics, Joxe Mari Korta Research Center, University of the Basque Country UPV/EHU, 20018 Donostia, San Sebastián, Spain; [email protected] 5 Sport Genomics Research Group, Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain 6 Phymo Lab, Physiology and Molecular Laboratory, 08028 Barcelona, Spain 7 Exercise & Sport Nutrition Laboratory, Human Clinical Research Facility, Texas A&M University, College Station, TX 77843, USA; [email protected] * Correspondence: [email protected]; Tel.: +57-320-335-2050

Abstract: Ashwagandha (Withania somnifera) is considered a potent adaptogen and anti-stress agent that could have some potential to improve physical performance. This preferred reporting items for Citation: Bonilla, D.A.; Moreno, Y.; systematic reviews and meta-analyses (PRISMA)-based comprehensive systematic review and Gho, C.; Petro, J.L.; Odriozola- Bayesian meta-analysis aimed to evaluate clinical trials up to 2020 from PubMed, ScienceDirect, and Martínez, A.; Kreider, R.B. Effects of Google Scholar databases regarding the effect of Ashwagandha supplementation on physical per- Ashwagandha (Withania somnifera) on formance in healthy individuals. Besides implementing estimation statistics analysis, we developed Physical Performance: Systematic J. Funct. Morphol. Kinesiol. 2021, 6, 20Bayesian hierarchical models for a pre-specified subgroup meta-analysis on strength/power, cardi-17 of 31 Review and Bayesian Meta-Analysis. J. orespiratory fitness and fatigue/recovery variables. A total of 13 studies met the requirements of Funct. Morphol. Kinesiol. 2021, 6, 20. this systematic review, although only 12 were included in the quantitative analysis. A low-to-mod- https://doi.org/10.3390/jfmk6010020 erate overall risk of bias of the trials included in this study was detected. All Bayesian hierarchical modelsAfter converged conﬁrming to a target convergence distribution ( Ȓ == 1) 1), for the both Bayesian meta-analytic model effect indicated size (μ) and a very between- large, Received: 1 January 2021 studypooled standard effect deviation size that ( favoredτ). The meta-analytic the Ashwagandha approaches supplementation of the included (studiesµ = 1.89 revealed (95% CrI that− Accepted: 9 February 2021 Published: 11 February 2021 Ashwagandha1.30 to 2.51)). supplementation There was a low-to-medium was more efficaciou studys than heterogeneity placebo for improving in terms of variables between-study related tostandard physical performance deviation ( inτ = health 0.55y (95% men and CrI female. 0.03–1.44)). In fact, However, the Bayesian the models inclusion showed of that the future outlier Publisher’s Note: MDPI stays interventionseffect size withinmight be the at Bayesianleast in some model way reducedbeneficial the on pooledthe analyzed effect outcomes size (µ =considering 1.38 (95% the CrI neutral with regard to jurisdictional 95%− 0.03credible to 2.81)) intervals and for increased the meta-analytic the heterogeneity effect size. substantiallySeveral practical (τ =applications 1.91 (95% and CrI future− 1.03 claims in published maps and directionsto 3.54)). are The discussed, posterior although predictive more distribution comparable studies for a future are needed observation in exercise showed training, that and the institutional affiliations. athleticprobability populations of the are effect needed size to is derive greater a more than 0.20stable is estimate absolutely of the high true (100%), underlying which effect. suggests positive effects of Ashwagandha supplementation on cardiorespiratory ﬁtness (i.e., VO2max J. Funct. Morphol. Kinesiol.Keywords:and 2021 [Hb])., 6, 20 herbal The supplements; results of the muscle Bayesian strength; random-effects cardiorespiratory meta-analysis fitness;16 of exercise 31 conducted tolerance; onqual- the

ityevaluated of life; sleep studies latency of this subgroup are shown in Figure7. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license Traditional botanical knowledge is characteristic of cultural contexts with long expe- (http://creativecommons.org/licenses rience in the natural environment; however, as it is transmitted from generation to gener- /by/4.0/). ation, it adapts and transforms through scientific experimentation. Within the traditional

J. Funct. Morphol. Kinesiol. 2021, 6, 20. https://doi.org/10.3390/jfmk6010020 www.mdpi.com/journal/jfmk

Figure 7. Forest plot of a random effect meta-analysis on cardiorespiratory fitness variables after Ashwagandha supplementation. (AFigure) The plot 7.showsForest the names plot of the of studies a random with their effect respective meta-analysis variable or multiple on comparison/multiple cardiorespiratory ﬁtness variables after outcomes on the left. On the right are the θk and 95% CrI. The value of the overall weighted mean of effect sizes is drawn as a vertical lineAshwagandha in the middle of the supplementation.plot, which represents the reference (A) The lineplot to test showsthe null hypothesis the names in each of study. the Th studiese with their respective posterior distributions of the estimated effect sizes for each study are shown as cyan densities. The black circle represents variable or multiple comparison/multiple outcomes on the left. On the right are the θk and 95% CrI. the posterior mean, and the horizontal line extending from the point is the 95% CrI. The bottom row is the meta-analytic effect size (μ).The (B) multivariate value of kernel the density overall estimation weighted plot of the mean posterior of distribution effect sizes of μ (x-axis) is drawn and τ (y-axis) as a with vertical line in the middle of the darker zonethe indicating plot, increased which plausibility represents of values. the reference line to test the null hypothesis in each study. The posterior distributions of the estimated effect sizes for each study are shown as cyan densities. The black circle represents the posterior mean, and the horizontal line extending from the point is the 95% CrI. The bottom row is the meta-analytic effect size (µ). (B) multivariate kernel density estimation plot of the posterior distribution of µ (x-axis) and τ (y-axis) with the darker zone indicating increased plausibility of values.

Review Effects of Ashwagandha (Withania somnifera) on Physical Performance: Systematic Review and Bayesian Meta-Analysis

Diego A. Bonilla 1,2,3,4,*, Yurany Moreno 1,2, Camila Gho 1, Jorge L. Petro 1,3, Adrián Odriozola-Martínez 4,5,6 and Richard B. Kreider 7 J. Funct. Morphol. Kinesiol. 2021, 6, 20 18 of 31

1 Research Division, Dynamical Business & Science Society—DBSS International SAS, Bogotá 110861, Colombia; [email protected] (Y.M.); [email protected] (C.G.); 3.4.3. Fatigue and Recovery [email protected] (J.L.P.) 2 Research Group in Biochemistry and Molecular Biology, Universidad Distrital Francisco José de Caldas, Taking into account the presenceBogotá of extremely 110311, Colombia small effects (the upper limit of the 95% CI was lower than the lower bound3 Research of the pooledGroup in effectPhysical CI), Activity, two Sports outliers and were Health identified Sciences (GICAFS), Universidad de Córdoba, and were removed from the analysis,Montería leaving 230002, a total Colombia of nine effect sizes from eight studies. 4 Salve et al. (2019) reported both multiple kDNA Genomics, outcomes Joxe Mari and Korta multiple Research treatment Center, University arms [ 39of ].the Basque Country UPV/EHU, 20018 Donostia, San Sebastián, Spain; [email protected] The analyzed outcomes encompassed variables of physical health, muscle fatigue, muscle 5 Sport Genomics Research Group, Department of Genetics, Physical Anthropology and Animal Physiology, damage/soreness, sleep recovery, andFaculty cortisol of Science from and 403 Technology, participants, University of which of the 203Basque were Country in (UPV/EHU), 48940 Leioa, Spain Ashwagandha groups and 200 in placebo6 Phymo groups. Lab, Physiology To ensure and Molecular that all the Laboratory, scales pointed 08028 Barcelona, in the Spain same direction in this subgroup meta-analysis7 Exercise & Sport of Nutrition fatigue/recovery-related Laboratory, Human Clinical outcomes, Research we Facility, Texas A&M University, multiplied several mean differencesCollege by − 1Station, (i.e., physicalTX 77843, USA; health [email protected] component of the quality of life test, time to exhaustion, perceived* Correspondence: recovery [email protected]; status scale, superoxide Tel.: +57-320-335-2050 dismutase and sleep efficiency); therefore, negative values can be interpreted as counteracting fatigue and favored physical recovery effect.Abstract: In comparison Ashwagandha to the(Withania placebo, somnifera the pooled) is considered treatment a potent adaptogen and anti-stress agent that could have some potential to improve physical performance. This preferred reporting items for effect of Ashwagandha supplementation was very large (dunb: −1.17; 95% CI: −3.01 to −1.049)Citation: with Bonilla, a medium-high D.A.; Moreno, Y.; heterogeneitysystematic amongreviews the and studies meta-analyses after removal (PRISMA)-based of the outliers comprehensive systematic review and (diamondGho, C.; Petro, ratio J.L.; = Odriozola- 1.89). The resultsBayesian of this meta-analysis random-effects aimed meta-analysis to evaluate clinical of standardized trials up to 2020 from PubMed, ScienceDirect, and meanMartínez, differences A.; Kreider, for R.B. fatigue/recoveryEffects of Google variablesScholar databases is shown regarding in Figure the8 effect. The of preliminary Ashwagandha supplementation on physical per- meta-analysisAshwagandha (Withania with thesomnifera inclusion) on offormance outliers in (extremely healthy individuals. high heterogeneity: Besides implementing diamond estimation ratio statistics analysis, we developed = 5.11)Physical is Performance: shown in Systematic the Appendix ABayesian Figure hierarchical A2. models for a pre-specified subgroup meta-analysis on strength/power, cardi- ReviewFinally, and Bayesian after Meta-Analysis. the exclusion J. oforespiratory outliers, the fitness pooled and effectfatigue/recovery size in our variable Bayesians. A modeltotal of 13 studies met the requirements of Funct. Morphol. Kinesiol. 2021, 6, 20. was −1.18 (95% CrI from −1.70 tothis−0.63). systematic Thus, review, there was although a large only overall 12 were effect included size that in the fa- quantitative analysis. A low-to-mod- https://doi.org/10.3390/jfmk6010020 vored the Ashwagandha interventionserate inoverall this subgrouprisk of bias meta-analysisof the trials included on fatigue/recovery- in this study was detected. All Bayesian hierarchical related variables. Our Bayesian modelmodels converged converged to to a a target target distribution distribution ( Ȓ = 1) 1). for A both medium meta-analytic effect size (μ) and between- Received: 1 January 2021 between-study standard deviationstudy was standard identified deviation (τ = 0.66 (τ). (95% The CrImeta-analytic−0.25–1.31)) approaches with an of the included studies revealed that Accepted: 9 February 2021 τ2 absolutePublished: value 11 February of the 2021 true varianceAshwagandha of = 0.43 (medium supplementation heterogeneity). was more The efficaciou ECDFs showed than placebo for improving variables related that the probability of the pooledto effect physical being performance greater than in health 0.20y in men a future and female. observation In fact, the is Bayesian models showed that future veryPublisher’s high (99.9%), Note: MDPI which stays suggestsinterventions the effects might of the be Ashwagandha at least in some supplementation way beneficial on arethe analyzed outcomes considering the veryneutral probable with regard to beto jurisdictional meaningful to95% reduce credible fatigue intervals and tofor improve the meta-analytic recovery. effect The resultssize. Several of practical applications and future thisclaims Bayesian in published random-effects maps and meta-analysisdirections are is showndiscussed, in Figurealthough9. more comparable studies are needed in exercise training, and institutional affiliations. athletic populations are needed to derive a more stable estimate of the true underlying effect.

Keywords: herbal supplements; muscle strength; cardiorespiratory fitness; exercise tolerance; qual-

ity of life; sleep latency Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license Traditional botanical knowledge is characteristic of cultural contexts with long expe- (http://creativecommons.org/licenses rience in the natural environment; however, as it is transmitted from generation to gener- /by/4.0/). ation, it adapts and transforms through scientific experimentation. Within the traditional

J. Funct. Morphol. Kinesiol. 2021, 6, 20. https://doi.org/10.3390/jfmk6010020 www.mdpi.com/journal/jfmk J. Funct. Morphol. Kinesiol. 2021, 6, 20 18 of 31

J. Funct. Morphol. Kinesiol. 2021, 6, 20 19 of 31

Figure 8. Forest plot depictingdepicting the standardizedstandardized meanmean differencesdifferences fromfrom thethe meta-analysismeta-analysis ofof studiesstudies comparingcomparing groupsgroups withwith AshwagandhaAshwagandha oror placeboplacebo onon fatigue/recovery.fatigue/recovery. Colored squares represent the dunb for individual trials, while colored and green polygons depict the meta-analytic dunb for the indicated subgroups and for the overall (total) results, respectively. squares represent the dunb for individual trials, while colored and green polygons depict the meta-analytic dunb for the indicated subgroups and for the overall (total) results, respectively. Horizontal lineslines representrepresent thethe 95% 95% conﬁdence confidence intervals intervals for for the the data. data. SRT: SRT: simple simple reaction reaction time; time; PH-QoL: PH-QoL: physical physical health health component component of the of the quality quality of life of test;life test; TTE: TTE: time time to exhaustion; to exhaustion; PRS: PRS: perceived recovery scale; CK: creatine kinase; SOD: superoxide dismutase; SE: sleep efficiency; QoS: quality of sleep; WS250: Withania somnifera 250 mg; WS600: Withania somnifera perceived recovery scale; CK: creatine kinase; SOD: superoxide dismutase; SE: sleep efﬁciency; QoS: quality of sleep; WS250: Withania somnifera 250 mg; WS600: Withania somnifera 600 mg. 600 mg. J. Funct. Morphol. Kinesiol. 2021, 6, 20 20 of 31 J. Funct. Morphol. Kinesiol. 2021, 6, 20 19 of 31

Figure 9. ForestFigure plot of 9. a Forestrandom plot effect of meta-analysis a random effect on fati meta-analysisgue/recovery variables on fatigue/recovery after Ashwagandha variables supplementation. after Ashwa- (A) The plot shows the names of the studies with their respective variable or multiple comparison/multiple outcomes on gandha supplementation. (A) The plot shows the names of the studies with their respective variable the left. On the right are the θk and 95% CrI. The value of the overall weighted mean of effect sizes is drawn as a vertical line in the middleor multiple of the plot, comparison/multiple which represents the reference outcomes line on tothe test left.the null On hypothesis the right arein each the study.θk and The 95% posterior CrI. The distributions ofvalue the estimated of the overall effect sizes weighted for each mean study of are effect shown sizes as cyan is drawn densities. as a The vertical black linecircle in represents the middle the pos- of the terior mean, andplot, the which horizontal represents line extending the reference from the line poin tot is test the the95% null CrI. hypothesisThe bottom row in each is the study. meta-analytic The posterior effect size (μ). (B) Multivariate kernel density estimation plot of the posterior distribution of μ (x-axis) and τ (y-axis) with the distributions of the estimated effect sizes for each study are shown as cyan densities. The black darker zone indicating increased plausibility of values. circle represents the posterior mean, and the horizontal line extending from the point is the 95% CrI. The bottom row is the meta-analytic effect size (µ). (B) Multivariate kernel density estimation plot of the posterior distribution of µ (x-axis) and τ (y-axis) with the darker zone indicating increased plausibility of values.

Review Effects of Ashwagandha (Withania somnifera) on Physical Performance: Systematic Review and Bayesian Meta-Analysis

Diego A. Bonilla 1,2,3,4,*, Yurany Moreno 1,2, Camila Gho 1, Jorge L. Petro 1,3, Adrián Odriozola-Martínez 4,5,6 and Richard B. Kreider 7

1 Research Division, Dynamical Business & Science Society—DBSS International SAS, J. Funct. Morphol. Kinesiol. 2021, 6, 20 Bogotá 110861, Colombia; [email protected] (Y.M.); [email protected] (C.G.);21 of 31 [email protected] (J.L.P.) 2 Research Group in Biochemistry and Molecular Biology, Universidad Distrital Francisco José de Caldas, Bogotá 110311, Colombia 3 Research Group in Physical Activity, Sports and Health Sciences (GICAFS), Universidad de Córdoba, 4. DiscussionMontería 230002, Colombia 4.1. Summary4 kDNA of Evidence Genomics, Joxe Mari Korta Research Center, University of the Basque Country UPV/EHU, 20018 Donostia, San Sebastián, Spain; [email protected] The aim of this systematic review and meta-analysis was to evaluate the effect of 5 Sport Genomics Research Group, Department of Genetics, Physical Anthropology and Animal Physiology, AshwagandhaFaculty supplementation of Science and Technology, on physical University performance. of the Basque A Country comprehensive (UPV/EHU), 48940 systematic Leioa, Spain review was6 Phymo performed Lab, Physiology to report and the Molecular different Laboratory, experimental 08028 Barcelona, designs Spain and findings of the available7body Exercise of evidence.& Sport Nutrition Furthermore, Laboratory, usingHuman estimation Clinical Research statistics Facility, analysis Texas A&M and University, devel- oping BayesianCollege hierarchical Station, TX 77843, models, USA; [email protected] the meta-analysis of the included studies on three components* Correspondence: of physical performance [email protected]; (strength/power, Tel.: +57-320-335-2050 cardiorespiratory fitness and fatigue/recovery) revealed that comparing Ashwagandha administration and placebo resulted inAbstract: a pooled Ashwagandha effect size estimate (Withania substantially somnifera) is considered different froma potent zero adaptogen that favored and anti-stress the agent treatmentthat effect. could Collectively, have some potential the findings to improve of this physical study demonstratedperformance. This that preferred Ashwagandha reporting items for Citation: Bonilla, D.A.; Moreno,supplementation Y.; systematic was reviews more efficaciousand meta-analyses than placebo (PRISMA)-based for improving comprehensive physical performance. systematic review and Gho, C.; Petro, J.L.; Odriozola-In addition,Bayesian the implementation meta-analysis aimed of a to Bayesian evaluate clinical approach, trials theup to assessment 2020 from PubMed, of the models’ ScienceDirect, and Martínez, A.; Kreider, R.B. Effectsintegrity of andGoogle the Scholar medium-to-low databases heterogeneityregarding the effect among of theAshwagandha studies (both supplementation diamond ratio on and physical per- Ashwagandha (Withania somniferaτ2) on provide formance certain in confidence healthy individuals. in the results Besides that implementing have been obtained.estimation Instatistics fact, according analysis, we to developed Physical Performance: SystematicKruschke Bayesian and Liddell hierarchical (2017), “Bayesian models for methodsa pre-specified are well subgroup suited formeta-analysis meta-analytic on strength/power, modeling cardi- Review and Bayesian Meta-Analysis.because J. meta-analyticorespiratory fitness models and are fatigue/recovery a kind of hierarchical variables. model A total and of 13 Bayesian studies met methods the requirements are of Funct. Morphol. Kinesiol. 2021, 6, 20. exceptionallythis systematic useful for review, hierarchical although models” only 12 [ 42were]. included in the quantitative analysis. A low-to-mod- https://doi.org/10.3390/jfmk6010020 Basederate on ouroverall results, risk of we bias must of the highlight trials included the following in this facts:study (i)was all detected. input Markov All Bayesian chains hierarchical had similarmodels behavior converged for the to a studied target distribution variables ( Ȓ = 1) 1), for which both meta-analytic is sufficiently effect indicative size (μ) and of between- Received: 1 January 2021 no problemsstudy in standard the model deviation fit; (ii) ( althoughτ). The meta-analytic often hard approaches to interpret, of the the included estimation studies ofthe revealed that Accepted: 9 February 2021 τ τ2 Published: 11 February 2021 between-studyAshwagandha standard supplementation deviation and was variance more efficaciou ( and s than, respectively) placebo for improving offered certain variables related advantagesto tophysical the meta-analysis performance in if wehealth considery men and they female. are insensitive In fact, the to Bayesian the number models of showed studies that future Publisher’s Note: MDPI staysand the precision;interventions and might (iii) we be wereat least able in some to make way reasonablebeneficial on inferences the analyzed of probabilityoutcomes considering on the neutral with regard to jurisdictionalfuture observations95% credible based intervals on thefor the present meta-analytic evidence effect in the size. meta-analysis. Several practical With applications regard to and future claims in published maps andAshwagandha directions supplementation, are discussed, although we can bemore quite comparab confidentle studies that future are needed interventions in exercise may training, and institutional affiliations. be at leastathletic in some populations way beneficial are needed on the to derive analyzed a more components stable estimate of physical of the true performance underlying effect. since the 95% credible intervals for the meta-analytic effect size lay completely on the “favoring”Keywords: side of the herbal intervention. supplements; In spite muscle of thestrength; above, cardiorespiratory due to the low fitness; number exercise of studies tolerance; qual-

in each subgroupity of life; and sleep the latency inclusion of multiple effect sizes from single studies (i.e., multiple Copyright: © 2021 by the authors.outcomes), the pooled treatment effects should be interpreted with caution, and further Licensee MDPI, Basel, Switzerland.research is needed to confirm the ergogenic effects of Ashwagandha. This article is an open access article It has been reported that Ashwagandha supplementation (between 240 and 600 mg distributed under the terms anddaily) might1. Introduction enhance strength/power-related variables in healthy untrained individu- conditions of the Creative Commonsals that are not frequently involved in exercise training and physical conditioning pro- Attribution (CC BY) license grams [19,31,38Traditional,40]. Only botanical one study knowledge [20] has demonstrated is characteristic that of exercisecultural contexts adaptations with to long expe- (http://creativecommons.org/licensesresistancerience training in the can natural increase environment; after twelve however, weeks of asAshwagandha it is transmitted supplementation from generation to gener- /by/4.0/). (500 mg o.d.ation, qAM, it adapts Sensoril and ®transforms≥10% withanolides through scientific & ≤0.5% experimentation. withaferin-A) Within in strength- the traditional trained individuals. Similarly, the study of Raut et al. (2012), which was excluded from the meta-analysis since it was not placebo-controlled, concluded that Ashwagandha is not only safe at increasing doses of 750 to 1250 mg·day−1 but also has positive effects on muscle strength in physically active healthy individuals [32]. The changes reported J. Funct. Morphol. Kinesiol.in 2021 the, 6, analyzed20. https://doi.org/10.3390/jf studies includemk6010020 variables such as muscle strength, musclewww.mdpi.com/journal/jfmk size, serum testosterone, maximum velocity, absolute and relative power. Although more research in exercise-trained individuals is needed, this Bayesian meta-analysis showed that the probability of having a clinically significant increase in strength/power compared to placebo after the Ashwagandha supplementation is very high. In regards to cardiorespiratory fitness, Pérez-Gómez and colleagues (2020) reported in a recent meta-analysis that Ashwagandha supplementation significantly increases VO2max in both untrained and exercise-trained participants [18]. However, unlike their methodology, we did not exclude data from the meta-analysis because we based our analysis on changes from baseline, which might be more efficient and powerful than the comparison of post-intervention values solely according to the current version of the Cochrane Handbook for Systematic Reviews of Interventions [24]. Furthermore, the authors of this recent meta- J. Funct. Morphol. Kinesiol. 2021, 6, 20 22 of 31

analysis conducted a wrong extraction process of the data for their analysis. For example, Shenoy et al. [33] reported that three male subjects dropped out due to inconsistent atten- dance in training sessions (one from the placebo and two from the Ashwagandha group with a final sample size of n = 19 and n = 18, respectively); notwithstanding, Pérez-Gómez et al. performed the meta-analysis with the initial number of participants in each group (n = 20 each one). Moreover, Choudhary et al. [37] reported the following values of VO2max: 41.74 (5.21), 46.65 (6.29) and 47.41 (6.63) at baseline, week 8 and week 12, respectively; however, Pérez-Gómez et al. performed the meta-analysis with 46.65 (6.29) as value for the week 12. These flaws can be easily identified in Figure 3 and Table 2 of their published article. The results of our subgroup meta-analysis (six studies, seven effect sizes) showed a large, pooled treatment effect on cardiorespiratory fitness. Additionally, VO2max and [Hb] increased significantly and were statistically different from placebo across the studies after 8–24 weeks of daily supplementation with 330–1000 mg of Ashwagandha. There is a high probability of having clinical significance in future observations, and it seems that the higher the dosage, the better the outcomes and more suitable for athletes and trained individuals, but further investigations are necessary. Finally, the consumption of 120–1000 mg Ashwagandha per day has been shown to reduce fatigue and optimize recovery in healthy individuals. The selected studies that were included in this systematic review showed improvements in physical health (a component of the quality-of-life test), muscle fatigue (increasing both times to exhaustion and perceived recovery), muscle damage/soreness (less CK levels and increase in SOD), sleep recovery (better overall quality of sleep) and stress levels (considerable reductions in cortisol levels). Very high heterogeneity was obtained among the studies (diamond ratio = 5.11) after the first exploratory meta-analysis. This was due to the inclusion of two outliers: (i) an extremely positive impact on the physical health component of the quality-of-life test after Ashwagandha supplementation, as reported by Deshpande et al. [41] (very high effect size); and (ii) a very large reduction on cortisol concentrations in the Ashwagandha group compared to placebo (−23.3% vs. +0.5%, respectively) observed by Lopestri et al. [40]. Al- beit these reported changes were in agreement with other references [37,39], their inclusion in the meta-analysis resulted in severe heterogeneity; however, we should bear in mind that this high between-study variance is itself informative as may be seen as opportunities to explore ways to improve the current status of literature. After the removal of these outliers, the Bayesian meta-analysis model revealed that, in comparison to the placebo, the pooled effect of Ashwagandha supplementation was very large (−1.18 (95% CrI from −1.70 to −0.63)) and that future observations are very probable to be meaningful on the reduction of fatigue and the improvement of recovery.

4.2. Potential Mechanisms of Action Currently, there are no studies describing the molecular mechanisms of Ashwagandha extracts associated with improving physical performance and reducing muscle fatigue in humans. However, the positive effects of the aqueous root extract of Ashwagandha on the muscle strength/power, cardiorespiratory fitness and fatigue/recovery that were described by most of the studies included in this systematic review might be due, in part, to the antioxidant properties of this plant. It is well known that adequate physiological levels of reactive oxygen species (ROS) are required for the processes of physiological adaptation to exercise training; notwithstanding, overtraining, low energy availability or inadequate sleep hygiene might increase the ROS production, which in turn can negatively affect the time course of exercise-induced adaptations [43–45]. For instance, these molecules have been shown to induce structural modifications of myofibrillar proteins, which affect their function (e.g., low sensibility to intracellular Ca2+). In this sense, several studies have demonstrated that antioxidant supplementation (such as N-acetylcysteine) may delay muscle fatigue during prolonged exercise in humans [46–48], but high doses have also been associated with reductions in exercise benefits. Thus, a common rationale for the increase in strength and power has been the possible optimization in the process of muscle J. Funct. Morphol. Kinesiol. 2021, 6, 20 23 of 31

adaptation and recovery after a physical effort, which has a practical implication on the frequency, and therefore the volume, of the training. Even though animal and in vitro studies cannot be directly extrapolated to human effects and their results have to be interpreted with attention [49], several animal and cell studies have identified potential molecular targets of the secondary metabolites of this herbal extract that could be involved in the regulation of oxidative stress at the cellular level. Palliyaguru et al. (2016) revealed that withaferin A is a potent inducer of Nrf2, a transcription factor that regulates the expression of antioxidant enzymes in response to oxidative stress, through the activation of the PTEN/PI3K/Akt pathway, in a murine model with induced liver toxicity [50]. Likewise, Yan et al. (2018) demonstrated that treatment with withaferin A of myoblast cells exposed to simulated ischemia/reperfusion or treated with H2O2 increased cell survival by inducing the expression of proteins with antioxidant activity HO-1, SOD2, SOD3 and Prdx-1, dependent on the activation of Akt [51]. The cytoprotective effect of Ashwagandha through the activation of Nrf2 has been corroborated in different cellular contexts and in different combinations of herbal extracts [52–54]. On the other hand, several anabolic and catabolic signaling pathways regulating muscle protein synthesis and energy metabolism might be affected after the administration of Ashwa- gandha. Of these, the NF-κB signaling pathway has been involved in the regulation of myogenesis in skeletal muscle in humans and in animal models. Although several authors have described a promyogenic function after the activation of this pathway, others have demonstrated the potent inhibitory effect of myogenesis and muscle regeneration, suggesting a complex regulation of this signaling pathway during muscle formation [55]. Several studies have shown the effect of withanolides on the regulation of NF-κB transcriptional activity in different in vivo and in vitro experimental models [10,56,57]. Ichikawa et al. (2006) reported that withaferin A and viscosalactone, along with their acetylated derivatives, block this signaling pathway by inhibiting the phosphorylation and degradation of IB, blocking the expression of NF-κB-related genes [56]. Kaileh et al. (2017) demonstrated that withaferin A directly inhibits IKK kinase activity, which phosphorylates IκBα prior to its ubiquitination and degradation, through a redox mechanism sensitive to thiol alkylation in a MEK1/ERK1 dependent manner [10]. Likewise, withaferin A and withanolide D blocked the angiogenic activity of endothelial cells by inhibiting the degradation of IB and inducing the expression of the antioxidant enzyme HO-1 [57]. Thus, although there is no evidence describing the mechanisms associated with increased muscle strength/power after supplementation with Ashwagandha root extract, withaferin A, among other secondary metabolites, have been identified as regulators of different cellular contexts that may favor myogenesis and oxidative metabolism in humans. In terms of the cardiorespiratory fitness and endurance performance, it has been reported that Ashwagandha supplementation contributes to the increase of [Hb] and hematological markers (mean corpuscular hemoglobin concentration [MCH], mean cell hemoglobin concentration [MCHC], and mean corpuscular volume [MCV]) while preventing oxidative stress [34,35]. These physiological effects might be crucial mechanisms to explain the increase in VO2max. Schumacher et al. [58] reported higher MCV and MCH in the competition phase compared to the off-season period in the elite cyclists. Borges et al. [59] also showed a substantial increase in MCV and MCH in elite kayakers after a period of high-intensity training. While Rietjens et al. (2002) reported a low correlation between [Hb] and VO2max obtained in cycle ergometer and treadmill in triathletes [60], some research has shown a correlation between these variables in athletes trained at altitude [61] and between the [Hb] and the ability to exercise [62,63]. Additionally, some authors highlight the importance of monitoring hematological status and possible iron supplementation since, throughout the year, several athletes show values of [Hb], hematocrit and ferritin below the normal range [60]. Interestingly, Shenoy et al. (2012) reported a higher increase in time to exhaustion in men in comparison to women (10.7% vs. 4.3%, respectively), which resulted in higher outcomes of VO2max for men (16.1% vs. 9.0%, respectively). This im- plies that men responded more to supplementation than women, perhaps mediated by an J. Funct. Morphol. Kinesiol. 2021, 6, 20 24 of 31

Ashwagandha effect on the endocrine system [33]. It should also be noted that: (i) athletes and physically active women are more susceptible to iron deficiency, which can contribute directly and indirectly to energy deficiency and changes in performance due to changes at the hematological level [64]; and (ii) supplementation with Ashwagandha extracts (600 mg per day delivering 21 mg withanolide glycoside) could not only counteract the production of ROS during exercise but also increase the levels of testosterone in healthy males [65] (probably through metabolic pathways of steroid hormone biosynthesis involving the conversion of saponins or sterane derivatives found in Ashwagandha [6] to testosterone and derivatives such as DHEA), which would justify the differences in physical performance between men and women if we consider the well-documented erythrogenic effects of testosterone [66]. Finally, considering the importance of rest and sleep in the processes of adaptation to physical effort, some of the reviewed references support the fact that supplementation with Ashwagandha might optimize the quality of sleep. In fact, Ashwagandha root extract (300 mg twice daily for 10 weeks) has been found to have the potential to induce sleep and improve sleep quality in patients with insomnia without presenting side effects [67]. This could not only favor the processes of adaptation to exercise but also accelerate rehabilitation and physical readaptation after a musculoskeletal injury. In this sense, a randomized, double-blind, placebo-controlled clinical trial showed that supplementation with 125 mg and 250 mg of Ashwagandha, both taken bis in die in patients with knee joint pain over a 12 week period, had a significant reduction in treatment efficacy and tolerability outcomes compared to baseline and placebo (with better results when taking 250 mg twice per day) while any significant gastrointestinal distress was detected [68]. Based on a model of predictive regulation [69], Ashwagandha might help overcome the allostatic overload and accelerate exercise-induced adaptations by the activation of actuator variables that modify the allostatic state (e.g., allostatic response to physical exercise) [45], albeit further standardized research is needed to have a better comprehension of its effects under certain bio-psycho-environmental situations. Figure 10 represents the potential mechanisms of action with feedback control of the Ashwagandha effects on physical performance variables.

4.3. Practical Applications Our comprehensive systematic review and Bayesian meta-analysis showed that Ash- wagandha supplementation protocols between 120 mg and 1250 mg per day might enhance physical performance. In regards to timing, seven studies asked participants to supplement with Ashwagandha once a day as follows: four studies every morning, two studies every afternoon or evening, and one study implemented every night at bedtime supplementation. On the other hand, six studies have implemented doses bis in die (i.e., twice per day—early in the morning and at night). Interestingly, higher doses were preferred for trained individuals and athletes; therefore, it seems that 300–500 mg twice per day (morning and before sleep) might be a safe and effective supplementation protocol for both female and male undergoing strenuous resistance or endurance training, but we are aware that further research is needed to individualize accordingly. Lower doses (≤300 mg once a day) might be considered for non-physically active individuals, those with no training experience and/or those engaging in exercise programs. J. Funct. Morphol. Kinesiol. 2021, 6, 20 25 of 31 J. Funct. Morphol. Kinesiol. 2021, 6, 20 24 of 31

FigureFigure 10. Potential 10. Potential mechanisms mechanisms of actionof action with with feedback feedback control controlof of thethe AshwagandhaAshwagandha effects effects on on physical physical performance performance variables. 3β-HSD: 3β-hydroxysteroid dehydrogenase; 17β-HSD: 17β-hydroxysteroid dehydrogenase; DHEA: dehydroe- β β β β variables.piandrosterone; 3 -HSD: 3SNP:-hydroxysteroid single nucleotide dehydrogenase; polymorphism. 17 -HSD: 17 -hydroxysteroid dehydrogenase; DHEA: dehy- droepiandrosterone; SNP: single nucleotide polymorphism. 4.3. Practical Applications The concentrations of withanolides were between 5% and 35% (only one study re- Our comprehensive systematic review and Bayesian meta-analysis showed that ported 42 mg by HPLC quantiﬁcation) and less than 0.5% of withaferin A. The most Ashwagandha supplementation protocols between 120 mg and 1250® mg per® day might ® frequentenhance commercial physical performance. products used In regards in the studies to timing, were seven KSM-66 studies, asked Shoden participantsand Sensoril to . Wesupplement would like with to highlight Ashwagandha the common once a day phytochemical as follows: four variability studies every that morning, can be found two in all herbalstudies extracts,every afternoon including or evening, Ashwagandha. and one study In fact, implemented Sangwan etevery al. [night70] concluded at bedtime that frequentsupplementation. oscillations On might the other be due hand, to factors six studies such have as (i) implemented heterogeneous doses bioresources bis in die (i.e., (wild and/ortwice harvested); per day—early (ii) physiologicalin the morning andand at ecological night). variations in plantations; (iii) harvest and postharvest collection operations; (iv) biomass processing and product manufacturing process; and (v) unregulated, contaminated and undescribed supplements. Recently, new analytical methods (e.g., high-performance thin-layer chromatography, HPTLC) have been reported to rapidly and selectively detect and quantify certain withanolides and phenolic J. Funct. Morphol. Kinesiol. 2021, 6, 20 26 of 31

compounds present in different components of Ashwagandha (root, stem and leaf) [71], which will ensure the quality and reliability of the products used in future studies. Although we did not perform a rigorous analysis of discontinuation rates and patient satisfaction, it should be noted that none of the clinical trials have found serious adverse effects from the consumption of Ashwagandha in the doses and length administered. Actually, Raut et al. (2012) demonstrated that the aqueous root extract of Ashwagandha seems to be safe on hematological and biochemical organ function tests. More recently, Salve et al. (2019) noted that 600 mg of Ashwagandha per day was well tolerated with no adverse events reported by the participants during eight weeks.

4.4. Future Directions According to the results obtained in the present systematic review, and considering recent results that suggest that the Ashwagandha root extract can be used to control body mass in adults under chronic stress [72], future studies may evaluate the effect of Ashwagandha supplementation during nutritional and exercise intervention programs to improve body composition for body esthetics, health or sports performance. Studies are also needed at different altitudes and environmental conditions (e.g., heat and humidity) since it may be a strategy to reduce environmental impact and maximize adaptations to the exercise. A registered clinical trial will allow us to know more about the effects on endurance performance (NCT03596307), but further research is still needed in other sports. More research comparing sex differences is needed, considering that it seems the production of testosterone derivatives (e.g., DHEA) from withanolides may lead to important differences. In this sense, the positive benefits on the hormonal profile (DHEA-S and testosterone) in overweight men over 50 s [65], on the immediate and general memory, executive function, attention and speed of information processing in subjects aged 50 with mild cognitive impairment [73], and on the quality of life, mental alertness and quality of sleep in healthy men and women over 70 s [74], makes the Ashwagandha root extract a nutritional alternative to optimize physical condition, bone and muscle health while preventing loss of muscle mass and strength in elderly. Further research is required to evaluate and identify the most important secondary metabolites of Ashwagandha in order to generate enriched supplements while optimiz- ing and standardizing the methods of quantitative analysis in broad-spectrum products (e.g., KSM-66), which will increase the reproducibility of studies in terms of dose and concentration of withanolides. Finally, this is an open field for new studies on different exercise- and sports-related variables, but considering our experience dealing with missing data, low-quality biased studies or data misinterpretation, researchers are encouraged: (i) to register their protocols in any of the current organizations or collaboration networks (e.g., US Clinical Trials, EU Clinical Trials Register, OSF International, Campbell Collaboration, among others); and (ii) to follow robust reporting guidelines to increase quality and transparency of clinical research (visit https://www.equator-network.org/ accessed on 28 February 2021). Far from being a meaningless value judgment, this is a call to action to report in detail relevant information within the current paradigm of open science.

4.5. Limitations This systematic review with meta-analysis has certain limitations: (i) the low number of studies published to date; (ii) we limited the databases for data extraction to PubMed/Medline, ScienceDirect, and Google Scholar; (iii) there were considerable differences in the exercise training experience among the participants included in the analyzed studies; (iv) although τ2 is insensitive to the number and the precision of the included studies, further analytical approaches are necessary with other types of heterogeneity measures when more placebo-controlled randomized-clinical trials are available. In spite of the above, the Bayesian method has tremendous advantages compared to the frequentist J. Funct. Morphol. Kinesiol. 2021, 6, 20 27 of 31

method if we consider that the estimates allow us to discuss probabilities, which makes it more intuitive, direct, coherent, accurate, and ﬂexible than frequentist approaches [42].

5. Conclusions The findings of this comprehensive systematic review and Bayesian meta-analysis revealed that Ashwagandha supplementation was more efficacious than placebo for improving variables related to strength/power, cardiorespiratory fitness and fatigue/recovery in healthy men and female. In fact, the probability of at least a small effect size on physical performance favoring subjects supplemented with Ashwagandha is very high (>95%). However, more comparable studies in exercisers/athletes are needed to derive a more stable estimate of the true underlying effect of the consumption of this aqueous herbal extract in trained individuals.

Author Contributions: Conceptualization, D.A.B.; methodology, D.A.B.; search and risk of bias assessment; D.A.B. and C.G., Formal analysis and visualization, D.A.B.; writing—original draft preparation, D.A.B., C.G. and Y.M.; writing—review and editing, D.A.B., Y.M., A.O.-M., J.L.P. and R.B.K.; project administration, D.A.B. All authors have read and agreed to the published version of the manuscript. Funding: The APC was funded by DBSS International. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data supporting this systematic review and meta-analysis are from previously reported studies and datasets, which have been cited. Acknowledgments: The authors would like to thank Tripathi, Petare and Raut for providing com- plementary data for our meta-analysis (e-mail communication). Moreover, we would like to thank the practicum student of Universidad Santo Tomás (Brayan Pulido), who collaborated as a research assistant at DBSS International with the first version of the drafted manuscript. Conflicts of Interest: D.A.B. serves as Science Product Manager for MTX Corporation® in Europe, a company that sells a dietary product with Ashwagandha, has acted as a scientific consultant for MET-Rx in Colombia and is a current scientific affiliate to the “Creatine in Health” scientific advisory board for AlzChem (Tostberg, GmbH). R.B.K. has conducted a number of industry-sponsored studies on sport nutrition-related nutrients that include Ashwagandha, has served as a paid consultant and received honorariums to speak at conferences by industry, and currently serves as chair of the “Creatine in Health” scientific advisory board for AlzChem. The other authors declare no conflicts of interest.

Appendix A The exploratory subgroup meta-analysis of the seven initial studies (including the outlier study) on the evaluation of cardiorespiratory ﬁtness after Ashwagandha supplementation is shown in Figure A1. J. Funct. Morphol. Kinesiol. 2021, 6, 20 27 of 31

of the “Creatine in Health” scientific advisory board for AlzChem. The other authors declare no conflicts of interest.

Appendix A

J. Funct. Morphol. Kinesiol. 2021, 6, 20 The exploratory subgroup meta-analysis of the seven initial studies (including28 ofthe 31 outlier study) on the evaluation of cardiorespiratory fitness after Ashwagandha supplementation is shown in Figure A1.

Figure A1. Forest plot depicting the standardized mean differences from a meta-analysis of all studies (including outliers) Figure A1. Forest plot depicting the standardized mean differences from a meta-analysis of all studies (including outliers) comparing groups with Ashwagandha or placebo on cardiorespiratory fitness. Colored squares represent the dunb for in- comparing groups with Ashwagandha or placebo on cardiorespiratory ﬁtness. Colored squares represent the d for dividual trials, while colored and green polygons depict the meta-analytic dunb for the indicated subgroups and unbfor the individualoverall (total) trials, results, while respectively. colored and Hori greenzontal polygons lines represent depict the the meta-analytic 95% confidence dunb intervalsfor the for indicated the data. subgroups Hb: blood and hemo- for theglobin overall concentration; (total) results, VO2max respectively.: maximum Horizontaloxygen uptake; lines WS330: represent Withania the 95% somnifera conﬁdence 330 mg; intervals WS500: for Withania the data. somnifera Hb: blood 500 hemoglobinmg. concentration; VO2max: maximum oxygen uptake; WS330: Withania somnifera 330 mg; WS500: Withania somnifera 500 mg. The exploratory subgroup meta-analysis of the initial studies (including two outliers) on theThe evaluation exploratory of subgroupfatigue/recovery meta-analysis after Ashwagandha of the initial studies supplementation (including twois shown outliers) in J. Funct. Morphol. Kinesiol. 2021Figureon, 6, 20 the evaluationA2. of fatigue/recovery after Ashwagandha supplementation28 of is 31 shown in Figure A2.

Figure A2.FigureForest A2. plotForest depicting plot depicting the standardizedthe standardized mean mean differencesdifferences from from a meta-analysis a meta-analysis of all ofstudies all studies (including (including outliers) outliers) comparing groups with Ashwagandha or placebo on fatigue/recovery. Colored squares represent the dunb for individual comparing groups with Ashwagandha or placebo on fatigue/recovery. Colored squares represent the d for individual trials, while colored and green polygons depict the meta-analytic dunb for the indicated subgroups and for theunb overall trials, while(total) colored results, andrespectively. green polygonsHorizontal depictlines represent the meta-analytic the 95% confidence dunb intervalsfor the for indicated the data. SRT: subgroups simple reaction and for time; the overall (total) results,PH-QoL: respectively. physical health Horizontal component lines of the represent quality of the life 95%test; TTE: conﬁdence time to exhaustion; intervals PRS: for the perceived data. SRT:recovery simple scale; reaction CK: time; creatine kinase; SOD: superoxide dismutase; SE: sleep efficiency; QoS: quality of sleep; WS250: Withania somnifera 250 mg; PH-QoL:WS600: physical Withania health somnifera component 600 mg. of the quality of life test; TTE: time to exhaustion; PRS: perceived recovery scale; CK: creatine kinase; SOD: superoxide dismutase; SE: sleep efﬁciency; QoS: quality of sleep; WS250: Withania somnifera 250 mg; WS600:ReferencesWithania somnifera 600 mg. 1. Keßler, C.; Gupta, H. Rasayanas: Ayurvedische Behandlung von altersbedingten Erkrankungen. Z. Komplementärmedizin 2015, 7, 22–25, doi:10.1055/s-0035-1570133. 2. Mishra, L.C.; Singh, B.B.; Dagenais, S. Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): A review. Altern. Med. Rev. 2000, 5, 334–346. 3. Brekhman, I.I.; Dardymov, I.V. New Substances of Plant Origin which Increase Nonspecific Resistance. Annu. Rev. Pharmacol. 1969, 9, 419–430, doi:10.1146/annurev.pa.09.040169.002223. 4. Singh, N.; Bhalla, M.; De Jager, P.; Gilca, M. An Overview on Ashwagandha: A Rasayana (Rejuvenator) of Ayurveda. Afr. J. Tradit. Complement. Altern. Med. 2011, 8, doi:10.4314/ajtcam.v8i5S.9. 5. Mirjalili, M.H.; Moyano, E.; Bonfill, M.; Cusido, R.M.; Palazon, J. Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 2009, 14, 2373–2393, doi:10.3390/molecules14072373. 6. Tripathi, S.; Sangwan, R.S.; Mishra, B.; Jadaun, J.S.; Sangwan, N.S. Berry transcriptome: Insights into a novel resource to understand development dependent secondary metabolism in Withania somnifera (Ashwagandha). Physiol. Plant. 2019, 168, 148–173, doi:10.1111/ppl.12943. 7. Nile, S.H.; Nile, A.; Gansukh, E.; Baskar, V.; Kai, G. Subcritical water extraction of withanosides and withanolides from ashwagandha (Withania somnifera L) and their biological activities. Food Chem. Toxicol. 2019, 132, 110659, doi:10.1016/j.fct.2019.110659. 8. Trivedi, M.K.; Panda, P.; Sethi, K.K.; Jana, S. Metabolite Profiling in Withania somnifera Roots Hydroalcoholic Extract Using LC/MS, GC/MS and NMR Spectroscopy. Chem. Biodivers. 2017, 14, e1600280, doi:10.1002/cbdv.201600280. 9. Jayaprakasam, B.; Zhang, Y.; Seeram, N.P.; Nair, M.G. Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves. Life Sci. 2003, 74, 125–132, doi:10.1016/j.lfs.2003.07.007. 10. Kaileh, M.; Vanden Berghe, W.; Heyerick, A.; Horion, J.; Piette, J.; Libert, C.; De Keukeleire, D.; Essawi, T.; Haegeman, G. Withaferin a strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity. J. Biol. Chem. 2007, 282, 4253–4264, doi:10.1074/jbc.M606728200.

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