Shatavari Supplementation in Postmenopausal Women Alters the Skeletal Muscle Proteome and Upregulates Proteins and Pathways Involved in Training Adaptation

doi:10.21203/rs.3.rs-2369451/v1

Purpose

Shatavari is an understudied but widely available herbal supplement. It contains steroidal saponins and phytoestrogenic compounds. We previously showed that 6 weeks of shatavari supplementation improved handgrip strength and increased markers of myosin contractile function. Mechanistic insights into shatavari’s actions are limited. Therefore, we performed global proteomics on vastus lateralis (VL) samples that remained from our original study.

Methods

In a randomised double-blind trial, women (68.5 ± 6 years) ingested either placebo or shatavari (equivalent to 26,500 mg/d fresh weight) for 6 weeks. Tandem mass tag global proteomic analysis of VL samples was conducted (participants - N = 7 shatavari, N = 5 placebo). Data were normalised to total peptides and scaled using a reference sample across experiments. Data were filtered using a 5% FDR. Log2 transformed fold change (week 6 vs baseline) was calculated and Welch’s t-test performed. Over-representation (ORA) and pathway enrichment analyses (PADOG) were conducted in Reactome (v79).

Results

76 VL proteins were differentially expressed between placebo and shatavari. ORA demonstrated that proteins in pathways related to metabolism of proteins, amino acids and RNA were downregulated by shatavari. Proteins related to the pentose phosphate pathway were upregulated. PADOG showed that proteins in pathways related to integrin/MAPK signalling, cell growth, metabolism, apoptosis, elastic fibre formation, the neuronal system and chemical synapse transmission were significantly upregulated.

Conclusion

Our analyses indicate that shatavari may support muscle adaptation responses to exercise. These data provide useful signposts for future investigation of shatavari’s utility in conserving and enhancing musculoskeletal function. Trial registration NCT05025917 30/08/21, retrospectively registered.

asparagus racemosus

skeletal muscle

nutrition

proteomics

Shatavari (Asparagus Racemosus Willd.) is a herb that has been widely used in Ayurvedia, but without much empirical evidence for its effectiveness. Recently, evidence has emerged that shatavari supplementation may increase skeletal muscle strength. We showed that 6 weeks of shatavari supplementation significantly improved handgrip strength in postmenopausal women [1]. Further, myosin regulatory light chain phosphorylation, which is a known marker of improved myosin contractile function, was increased in the vastus lateralis following shatavari supplementation. Shatavari also increased the phosphorylation of Akt^ser473, but not other traditional molecular markers of muscle protein synthesis e.g. phosphorylation of p70S6k^Thr389, 4EBP1^Thr37/46 or S6^Ser240/244. An earlier study showed that shatavari promoted strength gains in young men during eight weeks of bench press training [2]. However, this study did not attempt to elucidate possible mechanisms of shatavari action. There is therefore a need for greater mechanistic insight into the actions of shatavari in skeletal muscle. This will provide molecular corroboration of its functional effects, guide further research questions and ensure that these questions are targeted at the most appropriate athletic and patient populations.

Estradiol receptor activation by phytoestrogens could improve muscle strength, particularly in the estradiol deficient postmenopausal population that we studied previously. Indeed, this was the rationale for our original work [1]. Estradiol is known to improve myosin binding function and muscle force production [3]. Hormone replacement therapy improves muscle strength in postmenopausal women [4]. However, the work of Anders et al. in young men suggests that the mechanism of shatavari action in skeletal muscle is likely more complicated than estradiol-mimicking effects alone [2].

The bioactive constituents of shatavari are manifold. They comprise steroidal saponins (shatavarins I–IV), known antioxidants such as racemosides, racemosol, racemofuran and asparagamine A and phytoestrogens e.g. rutin, kaempferol, genistein, daidzein and quercetin [5]. Identifying and quantifying the bioactive constituents of shatavari that are responsible for its functional effects remains a distinct challenge. However, the complex bioactive constituents of shatavari may evoke skeletal muscle adaptations that are relevant to endurance performance and metabolic health. For example, kaempferol can increase mitochondrial complex IV activity and adenosine triphosphate (ATP) production in hypoxic C2C12 myotubes [6]. Rutin supplementation of rats with diet-induced obesity increases mitochondrial size, mitochondrial DNA content and the expression of genes implicated in mitochondrial biogenesis [7]. However, the evidence surrounding the effects of individual compounds in these scenarios is disparate. Such evidence also remains centred around animal and in vitro work that is of variable quality, and it is unclear whether such effects would translate to specific human scenarios. Bioactive supplements and compounds that are known to have anti-inflammatory and anti-oxidant effects are understudied in relation to long term human muscle function and adaptations to resistance training. The dearth of evidence in this area may stem from observations that high doses of some dietary supplements e.g. anti-oxidant vitamins (e.g. vitamin C and vitamin E) adversely affect resistance training adaptations, although the balance of evidence is equivocal [8–10]. We have recently shown that a polyphenol-rich tart cherry supplement is unlikely to act as a direct anti-oxidant, rather 8 days of supplementation upregulates endogenous skeletal muscle glutathione peroxidase expression [11]. This suggests that the effects of complex bioactive supplements might differ from effects that have been observed when high doses of exogenous antioxidant vitamins have been administered. Resistance training in young men increases the skeletal muscle expression of antioxidant gene terms [12]. Further, resistance training in older men increases skeletal muscle gene expression of superoxide dismutase 1 (SOD1) and 2 (SOD2), catalase (CAT) and glutathione peroxidases (GPXs), although protein levels were unchanged and enzyme activities varied. The enzymatic activity of CAT increased, whereas GPX activity decreased [13]. This limited evidence suggests that complex bioactive supplements may support or at the very least not impair, resistance training adaptations. In the case of shatavari, this remains speculative.

Therefore, to provide mechanistic insights and signposts for future work, we performed tandem mass tagged global proteomics on skeletal muscle samples that remained from our original shatavari supplementation study in postmenopausal women. Given our previous work and the work of Anders et al. [2] we hypothesised that shatavari supplementation might increase the levels of proteins associated with muscle protein synthesis and myosin function.

Study Design and Supplementation Protocol

The skeletal muscle samples that were used for these analyses were obtained as part of a previously published study [1]. This double-blind, placebo-controlled parallel design study was approved by the University of Exeter’s Sport and Health Sciences Research Ethics Committee (190206/B/01) and an amendment to this approval was secured to allow for the analyses presented in this paper. All participants had consented to the use of their samples in future work.

Briefly, participants attended a screening and consent visit during which eligibility was again confirmed and informed consent was obtained. Participants returned for visit two having fasted overnight. They completed tests of leg strength. These consisted of a warm-up, three sets of three maximal isokinetic repetitions (concentric and eccentric effort) and three maximum voluntary contractions with the knee joint fixed at 90 degrees of flexion. All sets were separated by one minute of rest.

A vastus lateralis skeletal muscle biopsy was performed [1]. A researcher who was not involved in any other aspect of the study randomised participants to receive either placebo or shatavari supplementation for six weeks using a random number generator. Participants consumed two capsules per day containing either placebo (N=10; magnesium stearate, 1000 mg total) or shatavari (N= 10; 1000 mg powder equivalent to 26,500 mg fresh weight shatavari root). The capsules were visually identical and did not have an odour.

Compliance was assessed by providing an excess of capsules and counting the number that remained at the end of the study period. No participant missed more than three doses over the six-week supplementation period. Visit three took place at the conclusion of this supplementation period and was identical to visit two.

Proteomic analysis

We powered our analyses in the original study (using standard deviations derived from unpublished internal immunoblotting data from older adults) to be able to detect a 1.6-fold change in myogenin protein expression with 10 participants per group. For this follow-up work, a complete set of skeletal muscle samples were available for analysis from N = 7 shatavari supplemented individuals and N = 5 placebo supplemented individuals. Skeletal muscle (c. 15 mg) was placed in microcentrifuge tubes with 250 μL of radioimmunoprecipitation assay (RIPA) buffer (Pierce 89900 RIPA buffer, ThermoFisher Scientific) containing protease and phosphatase inhibitors (Pierce A32961 Protease and Phosphatase Inhibitor EDTA-free mini tablet, ThermoFisher Scientific). Samples were homogenised for 1 min using a bead homogeniser (Speedmill Plus, Analytik Jena AG). Samples were removed to clean microcentrifuge tubes and vortexed thoroughly before incubation on ice for 30 min, with occasional vortexing. Samples were centrifuged for 10 min at 8000 g at 4°C. The supernatant was removed to a clean microcentrifuge tube and the pellet was discarded. Protein concentrations were determined by bicinchoninic acid (BCA) assay (Pierce 23225 BCA Protein Assay Kit, ThermoFisher Scientific) according to the manufacturer’s instructions.

TMT Labelling, High pH reversed-phase chromatography and Phospho-peptide enrichment.

Aliquots of 50µg of each sample were digested with trypsin (1.25µg trypsin; 37°C, overnight), labelled with Tandem Mass Tag (TMTpro) sixteen plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, LE11 5RG, UK) and the labelled samples pooled.

An aliquot of 200ug of the pooled sample was desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130Å, 3.5 µm, 2.1 mm X 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM Ammonium Hydroxide in acetonitrile, pH 10) from 0-95% over 60 minutes. The resulting fractions (20 in total) were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific).

Nano-LC Mass Spectrometry

High pH RP fractions were further fractionated using an Ultimate 3000 nano-LC system in line with an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). In brief, peptides in 1% (vol/vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Scientific). After washing with 0.5% (vol/vol) acetonitrile 0.1% (vol/vol) formic acid peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column (Thermo Scientific) over a 150 min organic gradient, using 7 gradient segments (1-6% solvent B over 1min., 6-15% B over 58min., 15-32%B over 58min., 32-40%B over 5min., 40-90%B over 1min., held at 90%B for 6min and then reduced to 1%B over 1min.) with a flow rate of 300 nl min−1. Solvent A was 0.1% formic acid and Solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.0kV using a stainless-steel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary temperature of 300°C.

All spectra were acquired using an Orbitrap Fusion Lumos mass spectrometer controlled by Xcalibur 3.0 software (Thermo Scientific) and operated in data-dependent acquisition mode using an SPS-MS3 workflow. FTMS1 spectra were collected at a resolution of 120 000, with an automatic gain control (AGC) target of 200 000 and a max injection time of 50ms. Precursors were filtered with an intensity threshold of 5000, according to charge state (to include charge states 2-7) and with monoisotopic peak determination set to Peptide. Previously interrogated precursors were excluded using a dynamic window (60s +/-10ppm). The MS2 precursors were isolated with a quadrupole isolation window of 0.7m/z. ITMS2 spectra were collected with an AGC target of 10 000, max injection time of 70ms and CID collision energy of 35%.

For FTMS3 analysis, the Orbitrap was operated at 50 000 resolution with an AGC target of 50 000 and a max injection time of 105ms. Precursors were fragmented by high energy collision dissociation (HCD) at a normalised collision energy of 60% to ensure maximal TMT reporter ion yield. Synchronous Precursor Selection (SPS) was enabled to include up to 10 MS2 fragment ions in the FTMS3 scan.

Data Analysis

The raw data files were processed and quantified using Proteome Discoverer software v2.4 (Thermo Scientific) and searched against the UniProt Human database (downloaded January 2022: 178486 entries) using the SEQUEST HT algorithm. Peptide precursor mass tolerance was set at 10ppm, and MS/MS tolerance was set at 0.6Da. Search criteria included oxidation of methionine (+15.995Da), acetylation of the protein N-terminus (+42.011Da) and Methionine loss plus acetylation of the protein N-terminus (-89.03Da) as variable modifications and carbamidomethylation of cysteine (+57.0214) and the addition of the TMTpro mass tag (+304.207) to peptide N-termini and lysine as fixed modifications. Searches were performed with full tryptic digestion and a maximum of 2 missed cleavages were allowed. The reverse database search option was enabled and all data was filtered to satisfy false discovery rate (FDR) of 5%.

Bioinformatics

Overrepresentation analysis

For each protein, the log2 fold change between the pre and post supplementation biopsies was calculated for each patient. A Welch’s t-test was then performed to compare treatment groups. Further analyses were performed using Reactome Version 82. Overrepresentation analyses were performed separately on lists of proteins that were found to be significantly 1) downregulated and 2) upregulated.

Overrepresentation pathway analyses spotlight pathways in which proteins that appear in many molecular pathways are present. We therefore performed differential gene expression analysis using Reactome’s ‘Pathway Analysis with Down-weighting of Overlapping Genes’ (PADOG) algorithm. PADOG uses log2 transformed quantitative proteomics data and calculates a gene set score and down-weights genes/proteins that appear in many molecular pathways. Where a protein was not present in at least half of the samples in a treatment group, it was removed from the analysis. We report p values and false discovery rates for overrepresentation and PADOG analyses.

Proteome Changes

4411 proteins were detected at quantifiable levels, with 3,121 of these proteins being detected in all samples. Of these, 76 proteins were differentially expressed between placebo and shatavari supplementation conditions (Fig. 1). 30 were downregulated by shatavari supplementation and 46 were upregulated. Table 1 shows the 10 most prominently upregulated and downregulated proteins (detected in all samples, ranked by lowest p value for the Welch’s t test comparison).

Using Reactome, we performed separate overrepresentation analyses of the upregulated and downregulated protein lists. Pathways related to metabolism of proteins, amino acids and RNA dominate the pathways that were found to be downregulated using this analysis approach (Table 2). Only the pentose phosphate pathway was found to be upregulated (p = 3.13E-5, FDR = 6.07E-3).

Differential expression analysis found that pathways related to integrin/MAPK signalling, cell growth (BRAF/RAF/RAS signalling), metabolism, apoptosis, elastic fibre formation, the neuronal system and chemical synapse transmission were significantly upregulated (Fig. 2). The corresponding fold change, p values and false discovery rates are presented in Online Resource 1.

Here, we performed tandem mass tagged global proteomics on skeletal muscle samples that remained from a previous 6-week shatavari supplementation study in postmenopausal women. That study showed that 6 weeks of shatavari supplementation improved handgrip strength and increased the phosphorylation of the myosin regulatory light chain, a molecular indicator of improved myosin function. Further the work of Anders et al. demonstrated that shatavari supplementation during a resistance training programme improved bench press strength [2]. Therefore, we hypothesised that shatavari supplementation might increase the levels of proteins associated with muscle protein synthesis and myosin function.

Over-representation analysis of significantly downregulated proteins indicated that pathways related to protein translation, metabolism of RNA and metabolism of proteins (particularly pathways related to stress and starvation conditions) were downregulated. It is possible that shatavari supplementation may induce a state of reduced protein turnover within skeletal muscle. Skeletal muscle mass may be gained or lost through an imbalance between muscle protein synthesis (MPS) and breakdown (MPB). In the context of age-related loss of skeletal muscle mass (sarcopenia), a blunted muscle protein synthetic response to nutritional and exercise stimuli is thought to be one major aetiological factor. Further, nutritional strategies to counteract this age-related imbalance have traditionally been found to succeed via ‘correcting’ this blunted MPS [14]. However, this does not preclude reduced MPB contributing to any effects of shatavari supplementation on muscle protein turnover. The existence of a change in skeletal muscle protein turnover with skeletal muscle supplementation should be investigated further. This could be confirmed via specific investigation of shatavari’s effects on MPS and MPB in the different skeletal muscle protein subfractions including myofibrillar, sarcoplasmic, mitochondrial, extracellular/collagenous using minimally invasive stable isotope tracer techniques [15]. . Interestingly, differential expression analysis of these data demonstrated an upregulation of skeletal muscle apoptosis processes (Fig. 2, Online Resource 1). This suggests that any reduction in protein turnover is not necessarily accompanied by a lack of skeletal muscle maintenance or ‘quality control’. We note that overrepresentation analysis suggested an increase in proteins associated with the pentose phosphate pathway. This pathway is a branch of the main glycolytic pathway, producing NADPH and ribose 5-phosphate which are central to nucleic acid, fatty acid nucleotide and non-essential amino acid synthesis. NADPH is also important in facilitating the conversion of glutathione to its reduced form; this is a critical component of cellular antioxidant defence [16]. The functional and adaptive value of these observations remain to be explored.

It is notable that we demonstrated a shatavari-induced upregulation of pathways involved in integrin and MAPK signalling. Integrins act as force sensors at the sarcolemma – the muscle cell membrane – connecting the extracellular matrix to the skeletal muscle contractile apparatus. Integrins are implicated in skeletal muscle hypertrophic responses to exercise and generation of external forces [17]. The mechanism/s that underpin this remain opaque, although it appears that this occurs independently of mammalian target of rapamycin complex 1 (mTORC1) [18]. Evidence is emerging that an integrin–integrin-linked kinase–RICTOR–Akt (Rapamycin-insensitive companion of mTOR = RICTOR; protein kinase B = Akt) pathway may mediate muscle adaptations to exercise [18, 19]. The results of our original targeted immunoblotting analyses of these samples found a significant increase in Akt phosphorylation, with no changes in the phosphorylation status of effectors of muscle protein synthesis downstream of mTORC1 (Ribosomal protein S6, Ribosomal protein S6 kinase beta-1, Eukaryotic translation initiation factor 4E-binding protein 1). Notably, these biopsies were taken approximately 30 min after a moderate bout of resistance exercise, suggesting that shatavari supplementation may indeed enhance signalling in pathways other than mTORC1 that are responsive to resistance exercise. Shatavari also enhanced the expression of proteins in pathways involved in mitogen-activated protein kinases (MAPK) signalling. The MAPK pathway is responsible for transmitting diverse extracellular signals to allow for a co-ordinated cellular response. Extracellular signal-regulated kinase 1/2 (ERK), c-Jun N-terminal kinase (JNK) and p38 are the ‘big three’ effector kinases in human skeletal muscle. ERK and JNK have long been known to have central roles and complex roles in co-ordinating skeletal muscle repair and myogenesis [20–22]. Recent work has identified a key role for JNK in the adaptive response to resistance exercise, via Mothers against decapentaplegic homolog 2 (SMAD2) inhibition of myostatin [23]. ERK is activated by endurance [24] and resistance [25] exercise. We note the enrichment of proteins that are mapped to pathways involving Ras and Raf (Fig. 2). Upstream of ERK 1/2, the GTPase Ras activates the c-Raf protein kinases which activates Mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) and in turn, ERK 1/2 [26].

Proteins associated with the neuronal system, axon guidance and chemical synapse transmission pathways were also observed to be increased following shatavari supplementation (Fig. 2). The human neuromuscular junction remains structurally stable across the lifespan, despite animal models suggesting the contrary [27]. However, the link between anatomical and functional stability is unclear. Intramuscular electromyogram (EMG) data suggest that synaptic transmission becomes compromised in older age, although this may be somewhat modifiable by exercise [28–31]. A prominent feature of neuromuscular ageing is denervation of muscle fibres and the growth of new axons to re-innervate and rescue these fibres. This is known as axonal sprouting. This process is imperfect and leads to larger motor units, with reduced discharge rates, increased recruitment thresholds and impaired muscle performance. There is some evidence to suggest that older athletes do display enhanced axonal sprouting and re-innervation of muscle fibres [32, 33]. Our data suggest that shatavari may have the potential to improve neuromuscular function in older populations and support such adaptations to resistance or endurance training. This is speculative and requires further study.

We note with interest the increase in proteins associated with metabolic pathways, including several which are implicated in carbohydrate metabolism, insulin secretion and the interconversion of nucleotide di- and triphosphates. The implications of these observations for skeletal muscle ATP availability, carbohydrate metabolism and whole-body glycaemic control under resting and exercise conditions are unclear. However, the value of these data in providing signposts for future work that might not have otherwise been considered is evident.

We did not analyse the bioactive constituents of our shatavari root supplement. High quality quantitative characterisations of such supplements are lacking, with many investigations and reviews providing qualitative, rather than quantitative information [5, 34]. One recent high-quality study exists and notably, they identified 33 different steroidal saponins [35]. These saponins were particularly enriched within shatavari root compared to the leaf and the fruit. They also identified 16 triterpene saponins that had previously not been described in shatavari. Additionally, 31 flavonoids were described. The discovery of new compounds that had previously not been described in the literature demonstrates the importance of further detailed characterisations of shatavari root. Characterisations of whole food dietary supplements are typically incomplete and provide only a snapshot of prominent, known compounds that may not necessarily be responsible for the biological effects of the supplement. For this reason, we believe that it is important to first do work to prove/refute efficacy for such supplements that are already being sold over the counter and used by consumers. This is what we have done in our recent work. Identification of the individual compound/s within shatavari root that exert biological effects then becomes a worthy research question requiring complex analytical work. This should now be a priority in the shatavari supplementation research field.

These analyses were conducted using samples that remained from previously published work. The original study was designed to detect 1.6-fold changes in the expression of proteins analysed by immunoblotting, a technique that is well-recognised as having quantitative limitations [36, 37]. Insufficient VL sample remained for proteomics analysis for several participants. Therefore, in this follow-up study, the participant numbers are smaller. However, pathway analyses improve statistical power by aggregating changes in all proteins involved in a pathway/process [38]. Indeed, these sample numbers have proved sufficient to identify pathway-level proteomic differences between the supplementation groups. We acknowledge that a risk of false and non-generalisable conclusions does exist, although the ‘dual’ controls of both a placebo condition and pre-post supplementation changes in each group minimise this. Critically in this regard, the proteomic differences between these supplementation groups are consistent with the limited published evidence of the effects of shatavari on skeletal muscle function. Specifically, increased muscle strength is accompanied by downregulation of proteins in pathways related to metabolism of proteins, amino acids and RNA and upregulation of proteins in pathways related to integrin/MAPK signalling, cell growth, metabolism, apoptosis, elastic fibre formation, the neuronal system and chemical synapse transmission. Therefore, we consider these data to be a novel, valuable and timely contribution to this emerging area of bioactives research. Most importantly, these data will influence the design and direction of future shatavari investigations.

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Author Contributions

M.F.O. and J.L.B. conceived and planned the experiments. M.F.O., J.L.B., S.R.J., carried out the experiments. M.F.O. and J.L.B. contributed to the interpretation of the results. M.F.O. took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research, analysis and final manuscript. All authors have approved the submitted version. All authors have agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated and resolved, and that the resolution documented in the literature. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Pukka Herbs Ltd. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the University of Exeter’s Sport and Health Sciences Research Ethics Committee (190206/B/01).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Acknowledgments

We thank the participants in this study for their time and commitment. We thank Dr Kate Heesom and Dr Phil Lewis (University of Bristol) for their technical support of the proteomics work presented here. We thank Matthew Campbell and Vlad Sabou for their assistance with muscle biopsy procedures.

Conflicts of Interest

The authors declare no conflict of interest

Data Availability Statement

The datasets used and/or analysed during the current study will be available from Open Research Exeter once the article has been accepted for publication. Upon approval into ORE, the dataset will be allocated a DOI, which can then be included in this data access statement prior to publication. These datasets have been uploaded with this manuscript for review purposes.

Table 1. Most prominently upregulated and downregulated proteins (shatavari supplementation vs placebo)
UniProt ID	Gene	Protein Name	Log Fold Change		p-value	Know Function/s

Upregulated
B3KNZ3	LIMS2	LIM and senescent cell antigen-like-containing domain protein		0.99	6.01E-04	Interacts with integrin-linked kinase), alters protein-protein interactions with the extraceullar matrix
Q07812	BAX	Apoptosis regulator BAX (Bcl-2-like protein 4)		0.74	7.48E-03	Triggers apoptosis under stress conditions
Q9H4A5	GOLPH3L	Golgi phosphoprotein 3-like (GPP34-related protein)		0.47	8.20E-03	Regulation of Golgi trafficking
P00488	F13A1	Coagulation factor XIIIa		0.94	1.07E-02	Element of blood coagulation cascade. Control of bleeding and wound healing. Alpha9 beta1 integrin signalling
P05106	ITGB3	Integrin beta-3		0.84	1.14E-02	Facilitates platelet aggregation Myogenic precursor adhesion, motility; skeletal muscle regeneration
Q9H299	SH3BGRL3	SH3 domain-binding protein 1		0.39	1.29E-02	Not well characterised. Thought to modulate glutaredoxin activity; possible involvement in redox signaling and glucose metabolism
O95816	BAG2	BAG family molecular chaperone regulator 2 (BAG-2)		0.34	1.35E-02	Co-chaperone for heat shock chaperone proteins; involved in protein folding and degradation
P21980	TGM2	Protein-glutamine gamma-glutamyltransferase 2		0.67	1.46E-02	Diverse, incl. bone development,angiogenesis, wound healing, cellular differentiation, chromatin modification and apoptosis
O43847	NRDC	Nardilysin		0.56	1.71E-02	Not well characterised. May regulate body temperature through the modulation of PGC-1α
A0A3B3ISX9	TNXB	Tenascin-X		0.53	2.16E-02	Regulates stability of elastic fibers organizes extra-cellular matrix collagen
Downregulated
Q8NCA5	FAM98A	Protein FAM98A		-0.65	2.85E-03	Skeletal homeostasis, increased expression in low protein diets
Q8IVN3	MUSTN1	Musculoskeletal embryonic nuclear protein 1		-0.84	4.88E-03	Development and regeneration of the musculoskeletal system. Increased expression in diabetes, Duchenne's Muscular Dystrophy
Q00688	FKBP3	Peptidyl-prolyl cis-trans isomerase FKBP3		-0.39	1.42E-02	Skeletal homeostasis
A8E631	KIAA1881	KIAA1881 protein (Fragment)		-0.88	1.43E-02	Perilipin family; triacylglycerol packaging in adipocytes
O14558	HSPB6	Heat shock protein beta-6 (HspB6)		-0.29	1.62E-02	Maintains denatured proteins in a folding-competent state. May be protective against atrophy, ischemia, metabolic dysfunction. Inhibits platelet aggregation
Q4VC31	MIX23	Protein MIX23		-0.28	1.67E-02	Up-regulated during mitoprotein-induced stress conditions
P42766	RPL35	60S ribosomal protein L35		-0.73	1.74E-02	Ribosomal protein synthesis
Q9Y2Q9	MRPS28	28S ribosomal protein S28, mitochondrial		-0.39	2.01E-02	Mitochondrial protein synthesis
Q96GD7		CSDA protein		-0.58	2.33E-02	Not well studied
P46776	RPL27A	60S ribosomal protein L27a		-0.53	2.40E-02	Ribosomal protein synthesis

Table 2. Overrepresentation Analysis – Downregulated Pathways
Notable Downregulated Pathways	p-value	FDR
Metabolism of proteins	7.11E-03	1.42E-02
Eukaryotic Translation Termination	1.41E-07	1.76E-06
Mitochondrial translation	1.00E-04	2.98E-04
Eukaryotic Translation Initiation	5.84E-07	2.92E-06
Peptide chain elongation	1.10E-07	1.76E-06
SRP-dependent co-translational protein targeting to membrane	4.12E-07	2.47E-06
Metabolism of RNA	3.70E-05	1.48E-04
rRNA processing in the nucleus and cytosol	8.88E-06	3.55E-05
rRNA processing	1.18E-05	4.72E-05
Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC)	1.75E-08	6.13E-07
mRNA 3'-end processing	1.02E-02	2.04E-02
Metabolism of amino acids and derivatives	3.45E-04	1.03E-03
Cellular responses to stress	5.57E-04	1.67E-03
Response of EIF2AK4 (GCN2) to amino acid deficiency	2.27E-07	1.82E-06
Cellular response to starvation	1.28E-07	1.76E-06
Parent pathways presented in bold.

Shatavari Supplementation in Postmenopausal Women Alters the Skeletal Muscle Proteome and Upregulates Proteins and Pathways Involved in Training Adaptation

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