Ashwagandha

Last Updated: September 28 2022

Ashwagandha has been called the king of Ayurvedic herbs. It’s best known for reducing stress and anxiety. It may also modestly enhance various aspects of physical performance, increase testosterone levels, and improve reproductive health, but more research is needed to confirm these effects.

Ashwagandha is most often used for.



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    1.

    Sources and Composition

    1.1

    Sources and Usage

    Withania somnifera (of the family solanaceae) is a highly esteemed medicinal herb in Ayurveda and most popularized as Ashwagandha although other common names include the King of Ayurveda,[20][21] Indian Ginseng (not at all related to panax ginseng), and Wintercherry.[22] The herb is classified as rasayana in ayurvedic medicine due to being a general tonic and in modern terms it is called an adaptogen for similar reasons,[23] and is also classified as bhalya (Increases strength) and vajikara (aphrodisiac).[24]

    The name Ashwagandha comes from the translation 'Smell of Horse', which is thought to be due to two main reasons; the root itself smells like a horse, and the root is supposed to imbibe you with the strength and virility of a horse.[25]

    Beyond those uses, it has been traditional used as an analgesic, astringent, antispasmodic, and immunostimulant while being used to treat inflammation, cancer, stress, fatigue, diabetes, and cardiovascular complications[26][27] while its adaptogenic usage is emphazied for persons with stress related insomnia, debility, and nervous exhaustion.[28] Ashwagandha has also been reported to be an immunostimulant compound, with particular emphasis for a stress-related suppression in immunity.[28]

    Ashwagandha is a highly esteemed medicinal plant in traditional Indian medicine for a wide variety of ailments but usually focused on stress, immune support (with regards to stress), anxiety and depression (again in regards to stress) and the treatment of cancer and inflammation; historically, it boasts low toxicity when consumed as the root extract alongside food

    1.2

    Composition

    Ashwagandha (roots unless otherwise specified) tends to include:

    • The steroidal lactones Withanone (Dry weight of the roots at 5.54+/-0.4mg/g and 18.42+/-0.8mg/g leaves[29]), 27-deoxywithanone (1.63+/-0.2mg/g in the leaves and 3.94+/-0.4mg/g in the roots[29]), and 27-hydroxywithanone (0.50+/-0.1mg/g dry weight leaf and root[29])
    • The 5,6-epoxy steroidal lactones Withaferin A[30] (22.31+/-1mg/g dry weight of leaves and 0.92+/-0.4mg/g in roots[29]) and 17-hydroxy-27-deoxy-Withaferin A (3.61+/-0.5mg/g dry weight of leaf and 0.66+/-0.2mg/g root[29])
    • The Withanolide series of 6,7-epoxy steroidal lactones mostly looking at Withanolide A[30] (root at 3.88+/-0.7mg/g, leaf at 2.11+/-0.5mg/g[29]) but also B-D;[31] there are some variants such as 27-hydroxy Withanolide B (0.55+/-0.2mg/g root and 2.78+/-0.5mg/g dry leaf weight[29])
    • The Withanoside series of steroidal lactones, usually Withanoside IV (0.44+/-0.1mg/g in the root dry weight and 1.60+/-0.2 in the leaves[29]) and VI (1.90+/-0.2mg/g in the leaves and 3.74+/-0.2mg/g in the roots[29]) although up to 19 exist[31]
    • Diepoxy variants of the withanolides such as 5β,6β,14α,15α-diepoxy-4β,27-dihydroxy-1-oxowitha-2,24-dienolide[32]
    • Chloronated withanolide variants such as 27-acetoxy-4β,6α-dihydroxy-5β-chloro-1-oxowitha-2,24-dienolide[32][33] and Withanolide Z[34]
    • Withanolide glycosides, which are referred to as Sitoindosides (or glycowithanolides)[35][36]
    • 12-deoxywithastromonolide at 2.15+/-0.5mg/g in the leaves and 1.90+/-0.5mg/g in the roots[29]
    • Physagulin (3.46 ± 0.4mg/g in the leaves; not detectable in the root[29]) with variants (4,16-dihydroxy-5β,6β-epoxyphysagulin D[37]) and glycosides (27-O-β-d-glucopyranosylphysagulin D[37])
    • Ashwagandhanolide (Withaferin A dimer bound by a sulfur bridge which breaks the epoxide site, or 'thiowithanolide'),[38][39] the same molecule but with a sulfoxide rather than a sulfur bridge (Withanolide sulfoxide),[39]
    • Viscosa lactone B[31]
    • Other sulfated steroidal lactones[40]
    • Kaempferol at 0.06mg/g (fruit dry weight, none in roots nor leaves)[41]
    • Naringenin at 0.50mg/g in the fruit by dry weight (none in roots nore leaves)[41]
    • (+)-Catechin at 12.82mg/g (roots), 19.48mg/g (fruits), and 28.38mg/g dry weight (leaves)[41]
    • Gallic acid at 0.18mg/g dry weight of the leaves (none in root nor fruit)[41]
    • Phenolic acids such as Syringic acid (0.30mg/g in the leaves), p-coumaric acid (0.80mg/g in the leaves), vanillic acid (0.15mg/g leaf dry weight), and benzoic acid (0.80mg/g in the leaves)[41]
    • A 1,4-dioxane derivative (2,5-Dioxo-3-tetratriacont-3′-enyl-1,4-dioxane)[42]
    • β-sitosterol and stigmaterol, as well as each molecule's glucoside.[43][29]
    • Trigonelline (1.33+/-0.3mg/g in the leaves[29])
    • Palmitic acid in the leaves (3.55+/-0.5mg/g dry weight[29]) and root (1.18+/-0.2mg/g dry weight[29])
    • Oleic acid in the leaves (0.71+/-0.1mg/g dry weight[29]) and root (0.39+/-0.1mg/g dry weight[29])
    • Linoleic acid in the leaves (1.52+/-0.2mg/g dry weight[29]) and roots (1.31+/-0.2mg/g dry weight[29])
    • Linolenic acid in the leaves (4.38+/-0.5mg/g dry weight[29]) and roots (0.15+/-0.1mg/g dry weight[29])

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    There is also a polysaccharide content in the roots (196mg per 20g dry root), which are 65% sugars (52% arabinose, 22% galactose, 18% glucose, 6% rhamnose, and 2% fucose) with 22% protein and 9% uronic acid.[44][45] An acidic 28kDa glycoprotein also exists in the roots of Ashwagandha[46] which has inhibitory effects on hyaluronidase.[47]

    Ashwagandha is a source of withanolide structures, which are either steroidal lactones (the basic four ring steroid structure with the five carbon lactone group on the top right of the structures) or the glycosides thereof. These appear to be the main components (and the ones unique to this plant) while there may be bioactive polysaccharides as well

    Withanolides are present in all plants in the Solanaceae family of plants, of which Withania Somnifera(Ashwagandha) is the highest in concentrations.[48]

    There is a phenolic content of ashwagandha reaching 17.8-32.6mg/g dry weight[41] which is comparable to the detected flavonoid content 15.49-31.58mg/g dry weight; in both instances, leaves were the highest and roots lowest (with the fruits intermediate).[41] In 80% ethanolic extracts, the content of flavonoids in the roots are around 530+/-80mg/100g (quercetin equivalents) and 520+/-60mg/100g in the leaves.[49]

    There has been reported to be high variability in the amount of active withanolides in common nutritional supplements,[50] which may be due either to lack of standardization of root powder.

    Unless otherwise standardized to a certain percentage, the amount of active Withanolide A (seen as the primary ingredient) and Withaferin-A are 1% of dry weight of the leaves (with negligible content in the roots) of Withania Somnifera.[51]

    A 50% ethanolic extract of the roots has been noted to contain Withaferin A (17+/-4mg/100g), Withanoside VI (24+/-3mg/100g), Withanoside IV (79+/-5mg/100g), physagulin (103+/-3mg/100g), 27-hydroxywithanone (22+/-2mg/100g), Withanolide A (1,340+/-6mg/100g), Withanone (315+/-5), 12-deoxywithastramonolide (23+/-3mg/100g), and withastramonolide (17+/-2mg/100g) and no detectable Withanolide D.[52]

    1.3

    Physicochemical Properties

    Withaferin A appears to be more soluble in ethanol than water, and when stored in standard conditions in 90% ethanol Withaferin A appears to be 90% stable after 6 months and 80% stable after a year.[53]

    1.4

    Variants and Formulations

    There is a compound formula called Mamsyadi Kwatha which includes Ashwagandha alongside Jatamansi (Nardostachys jatamansi) and Parasika Yanavi (Hyocymus niger) in an 4:8:1 ratio; it is used for the treatment of psychiatric disorders.[54]

    2.

    Molecular Targets

    2.1

    Vimentin

    It was initially noted that Withaferin A was able to irreversibly degrade a 56kDa protein in HUVEC cells[55] and it was later discovered that this protein was Vimentin, an intermediate filament protein;[56] a protein involved in wound healing, angiogenesis, and cancer growth and metastasis.[57][58] Withaferin A docks to the amino acids Gln324, Cys328, and Asp331 (initially thought to need to bind to Cys328[56] but this is not required[59]) and while this bonding does not per se block aggregation of vimetin into a tetramer (involved in its mechanisms of action[60]) it alters its binding and induces its fragmentation or depolymerization.[61][56] Withaferin A also appears to phosphorylate serine 56 on vimentin (250-500nM concentration)[61] which is a site phosphorylated prior to dissasembly,[62] and the C3 carbon on the A-ring of Withaferin (two carbons between the epoxide and ketone groups) is critical for this phosphorylation;[61] This phosphorylation has been noted in vivo when 4mg/kg is injected into mice bearing breast cancer tumors.[61]

    The decrease in vimentin is not associated with reduced total cell protein levels until chronic incubation[53] and can occur at low nanomolar concentrations, reducing vimentin content in a concentration and time dependent manner, suggesting selectivity.[63] Withaferin A may also reduce the TGF-β induced increase in Vimentin (500-1,000nM range)[53] although it does not appear to prevent TGF-β from increasing Vimentin mRNA levels[53] nor does it inherently reduce Vimentin mRNA levels.[53][61]

    Withaferin A appears to directly bind to Vimentin and induces its degradation. The reduction of vimentin is thought to be one of the main mechanisms of action for Withaferin A, since it underlies the proteasomal inhibition (which itself underlies a lot of the anti-cancer mechanism) and underlies both the suppression of metastasis and angiogenesis

    It has been noted that the binding to vimentin is not unique as many intermediate filament proteins are also affected in a similar manner with Withaferin A, although they are less sensitive (keratin heteropolymer IFs or KIFs needing 4µM to induce dissasembly; peripherin (PF) and neurofilament triplet protein (NIF) needing 1µM)[59] and the inhibitory effects on vimentin (despite being irreversible when coincubated) are reversible after three hours of removal of Withaferin A from medium.[59]

    Due to influencing all four intermediate filament proteins (KIFs, PFs, NIFs, and VIF), there is an apparent disruption of microtubules and microfilament formation in the cell's cytoskeleton and an increase in actin stress fibers at 2µM Withaferin A.[59]

    It appears that all intermediate filament proteins are affected in a similar manner as Vimentin (although vimentin is more sensitive), and high levels of Withaferin A adversely affect cellular structure and integrity; it may be prudent to not exceed nanomolar concentrations of Withaferin A since low concentrations (100-500nM) are selective for Vimentin and not other IFPs

    2.2

    NF-kB

    NF-kB is a signalling locus for inflammation and cellular survival which is kept inactive by its inhibitor IκB (directly forms a complex to prevent NF-kB from signalling). IκB can be phosphorylated by IKK (IκB kinase) to release NF-kB, which means that IKK positively influences NF-kB activity.[64]

    IKK itself is a complex with two subunits, IKKα and IKKβ, and a regulatory subunit known as NEMO (NF-kB essential modulator) which is sometimes called IKKc.[65] IKKβ seems to be enough on its own to stimulate IκB phosphorylation, and inhibiting the formation of IKKβ and NEMO is thought to be a novel mechanism for suppressing NF-kB.

    Withanolide A has shown direct docking to NEMO in its binding pocket with a binding energy of -9.44kcal/mol primarily at Glu 89[66] which is critical for binding of NEMO to the Ser 733 of IKKβ,[67] and withanolide A has also bound to Glu 99 at the other end while being involved in binding to Phe 92, Leu 93, Phe 97 and Ala 100 (all of which are involved in NEMO binding to IKKβ, with only Arg 101 being involved yet not influenced by Withanolide A)[66][67] although it was somewhat unstable in MD simulations.[66]

    Withanolide A has been noted to directly bind to NEMO and interfere with the NEMO:IKKβ interaction, which would lead to less activation of NF-kB. Subsequently, less activation of NF-kB will result in less cellular survival of tumor cells and an augmentation of other apoptosis inducing agents

    Withaferin A is also able to inhibit NF-kB activation secondary to inhibiting the degradation of IκBα (an inhibitor whose degradation is required to release active NF-kB), which is by blocking IKKβ (acts to degrade IκBα via phosphorylation) secondary to the MEK1/ERK pathway with an IC50 of 250nM reaching up to 95% inhibition.[68] This potent inhibition via MEK1/ERK is hindered by reducing agents, which is thought to be due to a thioalkylation reaction between the lactone group and cysteine groups on proteins (theorized to occur with steroidal lactones[55] as it occurs with lactone groups[69]).

    However, at least one study has noted that the NF-kB inhibition (as well as the suppression in Akt signalling) are both partially abrogated when vimentin is removed in a cell.[63]

    Withaferin A potently inhibits NF-kB via a different mechanism (increasing signalling from MEK1/ERK, which suppresses IKKβ and prevents IKKβ from releasing NF-kB from its inhibitor (IκBα); this is thought to be due to modification of the MEK1/ERK proteins via direct thioalkylation, although Vimentin is implicated

    2.3

    20S Proteasome

    Withaferin A has been found to inhibit the chymotrypsin like activity of the rabbit 20s proteasome (IC50 of 4.5μM) and isolated prostate cancer cells (5-10μM).[70] It appears that the ketone structure of Withaferin A is mandatory (90% inhibition at 10μM was reduced to 30% when the ketone group was reduced) similar to how celastrol (from Thunder God Vine) works on proteasome inhibition;[71][70] and it been noted that inhibition at maximal concentrations (10μM) is fairly weak when directly inhibiting the catalytic activity (the catalytic inhibition being measured at 340+/-80 at 0.5–10μM for Withaferin A; by comparison, the direct proteosome inhibitor epoxomicin reached 44,510+/-7,000 at 10–75nM).[56] Withaferin A is known to bind to the specific catalytic β subunit of the 20S-proteasome at Thr1,[72] resulting in its inhibition with three hours of incubation and showing maximum inhibition of 30-60% after 6 hours; the concentration of Withaferin A which this occurred at was 10nM and was comparable to bortezomib.[73] Since there was an activity attributed to proteasomal inhibition that did not occur at 100pM or 1µM, these doses were thought to be ineffective.[73]

    Withaferin A is known to inhibit proteasomal activity in vitro and shows direct binding to the 20S proteasome as well; however, the direct binding of Withaferin A does not lead to the potent inhibitory effects on proteasomal activity since the overall activity (despite occurring at low concentrations) is minimal

    Intermediate filament protein aggregation is known to impair the functions of the proteasome[74] and it appears that the ability of Withaferin A to inhibit the proteasome is greatly decreased in cells that do not express Vimentin;[59] this suggests that the biologically relevant mechanism for proteasomal inhibition is secondary to Vimentin degradation.

    The degradation of Vimentin is thought to explain the proteasomal inhibitory actions of Withaferin A

    Inhibition of proteasomal activity is known to cause accumulation of its target proteins (which it normally degrades) including Bax, IκB-α, and p27Kip1.[70]

    One study (using malignant pleural mesothelioma or MPM cells) noted that the inhibition of the proteasome from 10µM Withaferin A was accompanied (expectedly) by a downregulation of a vast amount of antiapoptotic proteins, but there were notable upregulations including Thioredoxin redutase 1 (3.46-fold), TGFβ induced protein of 68kDa (2.37-fold), TIMP2 (2.2-fold), and CARP-1;[75] CARP-1, a protein involved in suppressing growth of cells,[76] appeared to be critical to the anti-growth properties of Withaferin A.[75]

    Proteasome inhibition has been confirmed in vivo when 4-8mg/kg of Withaferin A was intraperitoneally injected to mice (associated with 54-70% inhibition of tumor growth).[70]

    Proteasomal inhibition will suppress the levels of many proteins, but it appears to be associated with a few increases in proteins; one of these proteins, CARP-1, appears to be associated with the antigrowth properties of Withaferin A in cancer cells. This proteasomal inhibition has been confirmed to be relevant in vivo following injections of Withaferin A

    2.4

    Antiapoptotic Proteins

    Withanone has been noted to have strong (-19.1088kJ/mol) affinity towards the protein known as survivin, particularly the BIR5 domain;[77] as survivin is an anti-apoptotic protein in cancer cells, its inhibition will allow apoptosis in cancer cells.

    There is a related protein called mortalin (a heat shock protein in the Hsp70 family regulating proliferation and stress responses[78] and is enriched in cancer cells[79][80]) which complexes with p53 to sequester it in the nucleus[81] which can increase lifespan in normal cells[82] but also cause cancerous cells to become more robust against chemotherapy.[80] Withanone also seems to bind to mortalin like it does to survivin,[83] binding to the segments of mortalin that MKT-077 (known ligand of mortalin[84]) binds to, at the Phe 272 and Asn 139 (ring lactone group of withanone), with some other bonds at Asp 277 and Arg 284 with binding energies in the range of -5.99 to -6.60kcal/mol.[83]

    Withanone may be a direct inhibitor of both survivin and mortalin by directly binding to them, and since in cancer cells both of these small signalling proteins are involved in making cancer cells more robust their inhibition will allow cancer cells to be killed more readily

    2.5

    Aurora A

    TPX2-Aurora A is a complex formed between the protein Aurora A and the spindle protein TPX2 (after TPX2 is released from importins α and β by the GTPase Ran) and since this complex prevents PP1 from negatively regulating Aurora A, genomic signalling from Aurora A can occur;[85][86] Aurora A is an oncogene[87] that tends to be overactive in several cancers such as cervical,[88] breast,[89] and pancreatic[90] so the inhibition of Aurora A[91] (or the inhibiton of TPX2 activity, causing indirect inhibition of Aurora A[92]) is thought to be theapeutic to various cancers.

    Ashwagandha has had its apoptotic effects in cancer cells attenuated when TPX2 is abolished with siRNAs[20] and withanone has shown direct semiflexible docking (binding energy of −7.18kcal/mol) via hydrogen bonding to His 280 on Aurora A[93] which is a residue required to bind to TPX2[94] and the other end of withanone binding to Arg 180 and Thr 288 on Aurora A;[93] ultimately this (and some possible interactions directly with TPX2 at Phe 35 and Lys 83) inhibit the complex formation and reduce the activity of TPX2-Aurora A, and this was confirmed in vitro with Withanone (15µg/mL) via less histone H3 activation (target of Aurora A) and complex formation via immunoprecipitation.[93]

    Withanone prevents the complex of Aurora A and TPX2 from forming via physically blocking their interaction, and since these two proteins cannot bind they cannot influence the genome together; ultimately, this results in less of their activity and since Aurora A is a tumor promoting oncogene less of their activity is seen as therapeutic for cancer

    2.6

    PKC

    PKC has been noted to have docking from both withanone (binding energy of -22.57kcal/mol) and Withaferin A (binding energy of -28.47kcal/mol) which results in its inhibition; the two dock in a similar site on PKC near the catalytic site[95] and this inhibition has been thought to occur in skin cells.[96]

    2.7

    Hsp90

    Heat shock proteins (HSPs) are small intracellular signalling proteins which are known as chaperons, and are involved in aiding the folding and configuration of other protein structures.[97][98] Of these, Hsp90 is one of the more important and abundant heat shock proteins (1-2% of total proteins in a cell under nonstressed conditions[99]) and beyond folding it can support the structure of 'client' proteins with those that are clients of Hsp90 include the androgen receptor, p53, Raf-1, and Akt amongst more than 100 others.[100]

    Many of Hsp90's clients are overexpressed during cancers, so inhibiting Hsp90 activity is thought to be therapeutic under cancerous conditions.[101][102][103] It can be inhibited by suppressing or blocking its co-chaperones (other chaperone proteins required to form the Hsp90 active 'superchaperone' complex[104]) and cell division cycle protein 37 (Cdc37) is a major co-chaperone.[105]

    Withaferin A has been noted to inhibit Hsp90 signalling in pancreatic cancer cells previously,[106] and Withaferin A has been noted to dock to the binding pocket on Hsp90 (binding energy of -9.10 Kcal/mol with an inhibition constant of 214.73nM) associated with hydrogen bonding primarily to Asp102 and partially with Asp54, with Van der Waals forces between a variety of amines (Leu48, Asn51, Asp54, Ala55, Leu107, Ala111, Val136 and Phe138) which are not the binding sites for Cdc37.[107] That being said, it appeared that binding of Withaferin A structurally disrupted the active site where Cdc37 binds to Hsp90 which caused inhibition of complex formation.[107]

    Hsp90 is a heat shock protein that is induced in response to stress and aids in the formation and maintenance of other proteins in the cell; it require a co-chaperone to function well and is overactive in cancer cells, and Withaferin A appears to noncompetitively inhibit the binding of Hsp90 to its co-chaperones which inhibits its function

    3.

    Pharmacology

    3.1

    Serum

    In mice given 10mg/kg of isolated Withaferin A orally, a Cmax of 8.41+/-1.4μg/mL was reached after 3 hours with a half-life of 7.1+/-1.2 hours and an overall AUC of 55.01±8.4 μg/h/mL.[73]

    A water extract of ashwagandha (0.046% Withaferin A and 0.048% Withanolide A) fed orally to mice at 1,000mg/kg resulted in rapid Cmax values of 16.69+/-4.02ng/mL (Withaferin A) and 26.59+/-4.47ng/mL (Withanolide A) at the Tmax of 20 and 10 minutes, respectively.[108] Their respective half lives were 60 and 45 minutes, conferring AUC values of 1673.10+/-54.53ng/h/mL and 2516.41+/-212.10ng/h/mL.[108]

    Very limited pharmacokinetic data on Ashwagandha ingestion, but it appears that oral supplementation of ashwagandha basic water extracts tends to result in a blood concentration of the main bioactives in the low nanomolar range; there is no data on ethanolic extracts

    3.2

    Distribution

    The volume of distribution of Withaferin A was noted to be 0.043L and the Mean Residence Time (MRT) was 6.52 hours.[73]

    3.3

    Mineral Bioaccumulation

    Ashwagandha has once been noted to reduce cadmium bioaccumulation in the body when dosed at 0.1% of the diet in chickens given in a rehabilitative manner after 100ppm cadium in the diet (28 days) noted that supplementation was able to reduce cadmium bioaccumulation by 81% (liver) and 55% (kidneys) within two weeks;[109] the potency of Ashwagandha was comparable to ocimum sanctum (Holy Basil) and nonsignificantly greater than all other adaptogens tested and these two also normalize the changes in oxidative stress to a degree that correlates with cadmium removal.[109][110]

    Ashwagandha has demonstrated protection against lead nitrate (where an 80% methanolic root extract at 200-500mg/kg taken alongside the lead dose dependently reduced hematological and liver toxicity).[111]

    Ashwagandha appears to be capable of reducing mineral bioaccumulation in the body following oral administration, and the potency (among adaptogens) seems to be comparable with Holy Basil and greater than others

    3.4

    Phase II Enzyme Interactions

    Heme oxygenase 1 (HO-1) is a redox sensitive stress inducible antioxidant protein that works via releasing the gasotransmitter carbon monoxide.[112][113] In liver tissue, it has been noted that 100mg/kg Ashwagandha (root extract) has failed to alter basal HO-1 concentrations although the increase in HO-1 expression in response to gamma irradiation was enhanced 45.6% above the irradiated control;[114] this enhanced reactivity was met with fully abrogation in oxidative changes such as MDA, glutathione, SOD, and catalase and a significant attenuation of DNA damage.[114]

    It has also been noted that ashwagandha (as well as bacopa monnieri and green tea catechins) have failed to induce HO-1 in isolated cells (neuroblastoma and pancreatic), but potentiated the HO-1 induction by curcumin and/or silymarin (from milk thistle).[115]

    Ashwagandha appears to potentiate the ability of prooxidants (including hormetic supplements as well as just environmental oxidative damage) in causing an induction of HO-1 via the Nrf2/ARE pathway, but it does not appear to actually influence this pathway by itself. In practical situations, due to environmental oxidative damage, it would exert effects similar to HO-1 induction

    4.

    Longevity

    4.1

    Rationale

    Withanone is able to downregulate P21WAF1 in normal fibroblast cells (TIG-1, MRC5, and WI38 cells) due to downregulating P53[116] despite the apparent upregulation of p53 in cancerous cells;[117] due to P21WAF1 positively influencing the rate of cellular senesence in normal cells,[118] 2.5ug/mL withanone induced downregulation of P21WAF1 caused 10-12 more population doublings which correlated to a 20% increase in cellular lifespan[116] and not only was there a relative decrease in accumulated molecular damage associated with withanone the increase of P21WAF1 seen with Withaferin-A was abolished with withanone.[116]

    Withanone appears to downregulate P21WAF1 in normal cells despite an upregulation in cancer cells, and this appears to delay the rate of cellular aging at a reasonably low concentrations

    5.

    Neurology

    5.1

    Mechanisms

    One study in rats using using pentylenetetrazole to increase brain inhibition MAO-A (109.1%) and MAO-B (70.6%) activity noted that Ashwagandha glycowithanolides (1.13% yield from the root) at 20-50mg/kg was able to prevent said inhibitory activity;[119] this was also seen with the reference drug lorazepam (500µg/kg), suggesting that GABAergic signalling may be associated.[119]

    Appears to attenuate the inhibition of MAO enzymes, which may serve a beneficial role in preventing excessive MAO inhibition from combining supplements (as MAO inhibition in high levels tends to predispose organisms to side-effects); still imited evidence otherwise, but it at least appears to be confirmed in vitro

    5.2

    Cholinergic Neurotransmission

    In regards to the acetylcholinesterase enzyme, Withanolide A appears to directly influence acetylcholinesterase in an inhibitory manner (molecular docking on Thr78, Trp81, Ser120 and His442[120]) which has been noted in vitro with an IC50 of 84.0+/-1.5µM (stronger than 5β,6β-epoxy-4β,17α,27-trihydroxy-1-oxowitha-2,24-dienolide and 5β,6β-epoxy-4β-hydroxy-1-oxowitha-2,14,24-trienolide at 161.5µM and 124.0µM respectively, but weaker than 6α,7α-epoxy-5α,20β-dihydroxy-1-oxowitha-2,24-dienolide at 50µM).[121]

    An injection of 40mg/kg of an alkaloid mixture (half Withanolide A and half mixed sitoindosides) has been noted to influence acetylcholinesterase noted a slight enhancement of activity in the lateral septum and globus pallidus regions of the brain while a decrease in activity (indicative of inhibition) was noted in the basal forebrain nuclei.[122]

    When 100mg/kg of the water root extract is fed to mice, there is a mild decrease in acetylcholinesterase activity relative to control (approximately 10%).[123]

    Withanolide A has direct molecular docking onto acetylcholinesterase where it can inhibit its function, but the concentration required to inhibit half of the enzyme activity is very high and may not apply to oral supplementation of these molecules; despite that, the basic water root extract has shown mild inhibitory activity in rodents

    100mg/kg of the Ashwagandha water root extract over a month alongside a neurological oxidative toxin (Propoxur, a pesticide[123]) was able to significantly attenuate the impairments to memory, but this was not associated with practically significant alterations in acetylcholinesterase (which Propoxur is known to decrease in activity).[124]

    Ashwagandha injections (40mg/kg of the alkaloids; half of which were Withanolide A) has been noted to enhance M1 receptor binding in some brain regions (lateral and medial septum) while enhancing M2 receptor binding in other brain regions (cingulate, piriform, parietal and retrosplenial cortices) while the frontal cortex experienced enhancements in both.[122]

    There may be positive modulation of cholinergic signalling at the receptor level associated with ashagandha bioactives, but the practical relevance of this information to oral supplementation is not currently known

    5.3

    Glutaminergic Neurotransmission

    A relatively small concentration of the ethanolic extract of ashwagandha (400ng/mL) was able to induce neuronal depolarization secondary to enhancing NMDA receptor signalling, in part through the glycine-binding site of the NMDA receptors as it is partially inhibited by blocking this site.[125]

    The NMDA or AMPA glutamate receptors do not appear to be altered with systemic administration of ashwagandha bioactives (Withaferin A and a series of sitoindosides),[122] although in epileptic rats both ashwagandha (100mg/kg) and isolated withanolide A (100µM/kg) can reduce the abnormal elevation of glutamate (potency similar to carbamazepine) and acted to partially normalize the adverse changes in AMPA receptor affinity and content.[126] This protective effect extends to NMDA receptors.[127]

    The ethanolic extract appears to potentiate NMDA signalling in part via interacting with the glycine binding site, and while it does not inherently appear to modify glutaminergic receptors there may be a preserving effect secondary to general neuroprotection

    Glioma and neuronal cell models (RA differentiated C6[128] and IMR-32[129]) subject to 0.01% of the culture as a water extract of ashwagandha and glutamate excitotoxicity note protective effects of ashwagandha on cell morphology and biomarkers of cell death,[130] which may be related to a previously noted increase in glutathione concentrations in cells exposed to ashwagandha[131] or due to preventing oxidation-induced changes to the NMDA receptors (which predispose cells to glutamate-induced oxidative stress),[127] although preventing oxidation-induced changes in Hsp70 appear to be vital.[130]

    Appears to have neuroprotective properties against glutamate-induced neurotoxicity, although it is unclear what mechanisms are at play and what molecules in ashwagandha mediate these effects

    5.4

    GABAergic Neurotransmission

    GABAA receptors are the subclass of GABA receptors that cause chloride influx into a neuron[132][133] similar to glycinergic signalling (via glycine receptors), which acts to suppress the ability of neurons to subsequently fire. GABAB receptors are G-protein coupled receptors.

    Ashwagandha appears to be involved in signalling through the GABAA receptor, as its beneficial influences on sleep are abolished with GABAA antagonists and potentiated by GABAA agonists[134] and the ability of ashwagandha to enhance GABAA signalling via diazepam has been noted elsewhere with 5µg of the methanolic extract[135] and 100-200mg/kg oral ashwagandha (mice).[136] Acting through the GABAA receptors also underlies the ability of 400ng/mL of the methanolic extract to release GnRH.[137]

    This enhancement of GABAA signalling is similar to that seen with scutellaria baicalensis, and while ashwagandha has been noted to hinder GABA binding to the receptor (5mcg causing 20% inhibition, 1mg causing 100% inhibition) it enhances flunitrazepam binding (to the benzodiazepine binding site).[135]

    Ashwagandha appears to enhance signalling through GABAA receptors in a similar manner as scutellaria baicalensis, which seem to underlie the sleep enhancement and possibly anxiolytic effects of ashwagandha

    5.5

    Dopaminergic Neurotransmission

    Despite there being a partial preservation of dopaminergic neuroamines in the brains of rats given a dopaminegic toxin (6-OHDA) by 25-60%, 100-300mg/kg of oral Ashwagandha root extract daily for three weeks does not inherently modify these neurotransmitter levels.[138]

    In regards to spiperone binding to D2 receptors which are increased after the administration of 6-OHDA, 100-300mg/kg Ashwagandha can attenuate the increased binding seen alongside toxicity without inhernetly modifying the binding affinity of these receptors.[138]

    5.6

    Adrenergic Neurotransmission

    The antidepressant effects of Ashwagandha appear to be blocked by the pretreatment of prazosin (general α-adrenergic receptor blocker)[139] while the depressive symptoms induced by clonidine (α2 adrenergic and imidazoline agonist) and reserpine (catecholamine depleter) were abrogated with pretreatment of Ashwagandha at the antidepressive dose, while haloperidol (dopamine antagonist) was not involved in the antidepressive effects.[139] These effects are similar to yohimbine which can block the depressive effects of clonidine directly and both Ashwagandha[139] and yohimbine[140] potentiate the effects of SSRIs.

    Adrenergic signalling appears to be involved in the antidepressant effects of Ashwagandha, and there are some parallels to the antidepressive effects of yohimbine as well. It is not clear how Ashwagandha mediates these effects though

    5.7

    Serotonergic Neurotransmission

    Supplementation of 100mg/kg ashwagandha root to normal rats for up to eight weeks has been noted to reduce 5-HT1A serotonin signalling in response to agonists while increase sensitivity of 5-HT2 signalling.[141]

    One study using ashwagandha has noted increases in plasma serotonin in stressed rats alongside the antidepressive effects, but it was confounded with other herbs (Clitoria ternatea, bacopa monnieri, and asparagus racemosus).[142] Elsewhere, one study in depressed mice noted that ashwagandha root in isolation was effective in preventing serotonin losses (although not completely) which was attributed to reducing corticosterone[143]

    Ashwagandha seems to have some evidence to suggest that it can increase 5-HT2 receptor signalling while reducing 5-HT1A receptor signalling, thus repartitioning serotonin signalling

    The 5-HT2 receptors appears to be involved in suppressingly nNOS activity in neurons (nNOS being localized with glutaminergic NMDA receptors[144] and is involved in NMDA dependent learning and excitotoxicity[145]) as inhibition of 5-HT2 enhances nNOS activity[146] and ashwagandha has been noted to enhance signalling of this receptor subset[141] and reduce nNOS immunostaining following stress.[143]

    It is possible that this enhanced signalling via the 5-HT2 subset underlies some of the neuroprotective effects of ashwagandha

    5.8

    Neuroprotection

    Withanolides and sitoindosides VII–X have been noted to enhance glutathione peroxidase, superoxide dismutase, and catalase in the frontal cortex and striatum of rats following oral ingestion[147][148] with 10-20mg/kg having a potency comparable to 2mg/kg deprenyl.[35]

    Appears to induce antioxidant enzymes in the brain following oral administration, which may underlie some neuroprotective effects of ashwagandha

    There is a chain of events that occur after the switching of serotonergic signalling from 5-HT1A towards 5-HT2, as this modification is implicated in suppressing nNOS (and reducing nitric oxide formation), this enzyme being what causes an increase of corticosterone and subsequent memory loss[143] which ashwagandha is known to abrogate and is circumvented with additional nitric oxide.[149]

    Whatever causes the enhanced serotonergic signalling via 5-HT2 receptors ultimately prevents an excessive increase in nNOS and nitric oxide, which prevents excessive levels of corticosterone and thus exerts a neuroprotective and adaptogenic effect

    Ashwagandha has been demonstrated to, after reserpine-induced toxicity resulting in tardive dyskinesia in mice, to dose-dependently reduce symptoms (orofacial) of tardive dyskinesia.[150] This reversal of symptoms has also been seen with haloperidol-induced dyskinesia[151] and both are suspected to benefit symptoms secondary to increased anti-oxidant enzyme expression.[150][151] Dopaminergic neurons are also protected via Ashwagandha during periods of morphine withdrawal;[152] which tend to be associated with significant localized atrophy in dopaminergic neurons.[152][153]

    It is possible that these inductions of anti-oxidant enzymes are secondary to induction of Heme-Oxygenase 1, which is from Ashwagandha acting on KEAP-1 to induce activation of Nrf2; however, this lead is one in vitro study with industry funding[115] and a PLoS entry[154] demonstrating that Ashwagandha (specifically, the isolated withanone) was able to inhibit premature oxidant-induced senescence of cells via inducing Nrf2 and the Anti-oxidant response element and keep levels of anti-oxidant enzymes fairly stable near control levels at 10uM.[154] This induction was greater than that recorded by Genistein, a soy isoflavone.

    An underlying mechanism(s) related to the anti-oxidant enzymes appears to mediate protection from a variety of cognitive diseases associated with oxidative stress. This may be Nrf2 induction, which would suggest that the effects are similar to many other polyphenolic compounds

    5.9

    Neurogenesis

    One aspect of the neuroprotective effects of ashwagandha is the ability of ashwagandha to induce neurogenesis, which is thought to play a rehabilitative role in cognitive decline.[155] A few isolated molecules have shown this property, including Withanolide A at concentrations as low as 1µM[156][157][158] and the Withaonsides IV and VI[159] and the aglycone of Withanoside IV known as Sominone.[160] Withanoside IV (and its aglycone sominone) have been noted to increase neurogenesis and axonal length of neurons when coincuated with Alzheimer's fibrils (Aβ25-35)[161] perhaps in part due to its protective effects against these fibrils.[162][161]

    When incubated in glial cells, ashwagandha leaf extract (800ng/mL) and withanone (5µg/mL) but not withaferin A (200ng/mL) have been noted to promote astrocyte differentiation.[163]

    On a cellular level, ashwagandha components can induce neurogenesis and prevent select neurotoxins (Aβ25-35) from suppressing neurogenesis. The concentration which this occurs at is low enough that it may occur following oral ingestion of the supplements

    Sominone appears to be able to induce axon extension (maximal efficacy at 100nM[160]) and extend dendrites (maximal efficacy at 1µM[160]) which is thought to be due to a direct phosphorylation of the RET receptor (to 124.3% of control with 1µM sominone),[160] the molecular target for the glial derived neurotrophic factor GDNF;[164][165] this was confirmed with intraperitoneal injections of sominone (10µM/kg; maximal effective dose) into mice where RET was phosphorylated within an hour.[160] The receptor expression does not appear significantly altered, just its phosphorylation, and sominone did not induce secretion of GDNF.[160]

    When looking at BDNF (a neurological growth factor), ashwagandha leaf extracts at 200mg/kg appears to upregulate brain mature BDNF to near 130% of control levels in mice over the course of a week.[163]

    When looking at the mechanisms of ashwagandha induced neurogenesis, sominone appears to be a direct agonist for the RET receptor and some other components in ashwagandha appear to stimulate the production of BDNF (another neurotrophic growth agent, which acts upon a different set of receptors)

    In mice given ashwagandha (100-300mg/kg) orally before scopolamine induced amnesia, the reductions seen in BDNF and GFAP with scopolamine was halved with the lowest dose and fully reversed with 200-300mg/kg (equipotent),[163] with mature BDNF being increased to double of saline control despite the presence of scopolamine and GFAP just being normalized.[163]

    Oral intake of 200mg/kg ashwagandha (leaf extract; has a higher level of steroidal lactones than the root extract dose) has once been confirmed to fully overcome and reverse the scopolamine induced reduction in BDNF

    5.10

    Stroke and Oxygenation

    In rats pretreated with Ashwagandha (unspecificed hydroalcoholic at 1,000mg/kg oral ingestion) for 15-30 days prior to an MCAO induced stroke, supplementation was able to time-dependently preserve motor function after stroke as assessed by a foot fault test (40-68%), grip test (50% attentuation to 33% reversal), and rotarod test (54-70%) with statistical significance only occurring at 30 days[166] and alongside improvements in physical function there was less lipid peroxidation and evidence of neurological damage.[166]

    5.11

    Stress and Anxiety

    Ashwagandha is known as an adaptogen compound, which may be due to the withanolide glycosides (ie. Withanoside IV rather than Withaferin A).[167][168][169] Adaptogens tend to reduce the perception of stress, and while their mechanisms are not well known in the case of Ashwagandha it may be related to preventing a stress-induced increase in NADPH diaphosphorase[143] (ie. nNOS[170][171]) which may be related to preserving the decrease in its negative regulators (serotonin) while preventing its positively regulators (corticosterone, glutamate) from increasing during stress.[143]

    Ashwagandha is highly antistress, and this antistress effect seems to be related to corticosterone signalling as well as suppressing neuronal excitation (nNOS and glutamate) in response to stress. There are also anxiety reducing effects secondary to the anti-stress properties, but perhaps also some inherent ones (serotonergic and GABAergic signalling)

    When looking at the roots, a 70% ethanolic extract of the roots (9.23% yield) that is subfractionated into an aqueous fraction (1.43% total yield) appears to contain the main bioactive, where 12.5-100mg/kg noted that all doses increased swimming time (35.03-93.68%) and appeared to reduce stress as assessed by stomach ulceration (12-58% protection from swimming and immobility stress).[167] Oxidation in organs such as the liver is also significantly reduced[168] and when compared against panax ginseng (100mg/kg) it was noted that in a model of chronic stress that 25-50mg/kg of an ashwagandha extract (aforementioned withanolide glycoside) was nonsignificantly more effective at reducing stress biomarkers.[172] The basic water extract of the root is also effective acutely, albeit at the dose of 360mg/kg.[169]

    There is also a significant physical antifatigue effect as assessed by the rotarod test (balance tested immediately after stressed in a swimming test), where 100mg/kg of the extract immediatley prevented the fatigue from stress while after 30 minutes all doses between 25-100mg/kg were fully effective.[167]

    Ashwagandha appears to have an anti-stress component which underlies its claim as an adaptogen, and it appears to be more related to a decrease in circulating cortisol (see the hormone section on corticosteroids) and improves physical functioning under psychological stress

    20-50mg/kg of the withanolide glycosides (1.13% yield of the root) for five days appears to increase social interaction in rats at a dose that does not impair locomotion.[119] The standard dose of the basic root extract (100-500mg/kg) also appears to be effective in rats subject to social isolation inherently[173] while subeffective doses potentiated the effects of diazepam.[173]

    In stressed humans given 300mg of ashwagandha daily for 60 days, there have been reported improvements in social functioning as assessed by the General health questionnaire-28[174] which reached a 68.1% reduction in 'social dysfunction' (placebo noted a 3.7% increase).[25] Interestingly, usage of Ashwagandha as adjuvant (2,000mg thrice daily) in cancer patients who did not self-identify as being stressed also noted improvements in social and romantic functionality and well being.[175]

    In regards to social interaction (a mechanism related to serotonin neurotransmission and anxiety), Ashwagandha appears to both inhernetly promote social interaction as well as attenuate the negative effects that prolonged isolation has on social function

    In rats, 20-50mg/kg of oral withanolide glycosides (1.13% dry weight of the root; taken daily for five days prior to an elevated plus maze test) is comparable to 500µg/kg lorazepam (a benzodiazepine) for reducing anxiety.[119] Similar to the potentiation of diazepam seen with low dose ashwagandha,[173] it also appears to synergistically enhance the anxiolytic effects of alcohol[176] which is known to work via GABAergic signalling.

    Standard doses of Ashwagandha are known to possess anxiolytic effects secondary to GABAergic signalling, and low doses of Ashwagandha appear to potentiate the effects of any GABAergic anxiolytic; this includes drinking alcohol

    In humans, persons with chronic mental stress given 300mg ashwagandha have noted significantly greater reductions in stress and anxiety as assessed by the percieved stress scale (44%; placebo at 5.5%) and the General health questionnaire-28 (all tested domains in the range of 58-89% more improvement than placebo).[25] Elsewhere, stressed persons given 125-250mg of ashwagandha (11.90% withanolide glycosides, 1.05% withaferin A, and 40.25% oligosaccharides with 3.44% polysaccharides) in two divided doses for the same time period have also note significant reductions in anxiety and its comorbidities (forgetfulness, lack of sleep, etc.),[177] 300mg twice daily (1.5% withanolide) alongside counselling and learning breathing techniques was associated with a 56.5% improvement of symptoms (placebo 30.5%),[178] and 250mg twice daily ethanolic root extract for six weeks in persons with diagnosed anxiety (mostly generalized anxiety disorder) outperformed placebo in reducing symptoms on the HAMA rating scale.[179]

    Ashwagandha's anxiety reducing properties appear to extend to humans, although the efficacy of ashwagandha as an anxiolytic appears to be weak as monotherapy (used by itself to treat anxiety) and more beneficial in instances where the subject has anxiety secondary to stress, where it appears to have quite a respectable potency

    5.12

    Depression

    In animals, ashwagandha has repeatedly shown anti-depressive effects over the course of a few weeks[141] and 20-50mg/kg withanolide glycosides (1.13% of the dry root weight) is nonsignificnatly lesser in potency than the reference 10mg/kg imipramine for reducing depressive effects, reducing the immobility in a forced swim test by 30.4-44.7% relative to control.[119] One study has noted that while haloperidol failed to block the antidepressant effects of Ashwagandha (suggesting no dopaminergic mechanism) that prazosin was able to block the antidepressant effects, suggesting adrenergic signalling.[139]

    50-150mg/kg Ashwagandha basic root extract for 14 days prior to testing was able to cause dose-dependent antidepressive effects in rats with a potency statistically comparable to 32-64mg/kg Imipramine (trending to be less effective) while the combination of the lowest doses of 50mg/kg Ashwagandha with Imipramine (16mg/kg) was more effective than either monotherapy in a learned helplessness test and forced swim test.[180] This study also noted that Bacopa monnieri was synergistic with imipramine, but weakly effective on its own and ultimately less synergistic than Ashwagandha[180] while subeffective dsoes of Ashwaganhda (50mg/kg) have also been noted to be synergistic with Diazepam in reducing depression[173] and synergistic again with Imipramine (replicated) and the SSRI fluoxetine elsewhere.[139]

    Appears to exert antidepressive effects on its own with a potency comparable to Imipramine (although requiring a slightly higher dose), but seems to be highly synergistic with other antidepressants such as imipramine and fluoxetine. This potency is currently only seen in animal research

    In chronically stressed persons, symptoms of depression (assessed by GHQ-28 and DASS) have been noted to be reduced with 300mg of ashwagandha daily over the course of 60 days by 77-79.2%.[25]

    The lone study measuring depressive symptoms from Ashwagandha measured stressed persons, and it noted a remarkable reduction in depressive symptoms alongside improvements in stress; there is currently no research in depressed persons who are not also stressed

    5.13

    Memory and Learning

    In a study assessing the anti-amnesiac effects of Ashwagandha, giving Ashwagandha to an otherwise healthy control group (when compared to the drug-free control) failed to increase memory formation when 100mg/kg of the water root extract was ingested over one month.[124]

    Limited evidence in otherwise healthy rodents suggest that there is no otherwise inherent nootropic effect of ashwagandha on learning and memory formation

    In rats given scopolamine to induce amnesia, the 50% ethanolic leaf extract (high withanone and withaferin A content) is known to attenuate the amnesia associated with decreased Arc (Activity-regulated cytoskeletal-associated protein) expression in the hippocampus and frontal cortex.[181]

    The amnesiac effects of beta-amyloid proteins appears to be effectively reversed with 10umol/kg of oral withanolide A over the course of 13 days[157] and the basic root extract has been shown to reduce the amnesiac effects of hypoxia by preventing the excessive production of nitric oxide (via nNOS, which the increases the levels of corticosterone and neuronal cell loss).[149] Anti-amnesiac effects have also been noted against streptozotocin,[182] of which intracerebrovascular injections are also a model for Alzheimer's.[183]

    There appear to be an anti-amnesiac effect in neurological toxins that are associated with inducing Alzheimer's disease, and this appears to be due to withanolide A and withanone (although other steroidal lactones may also be active)

    The damage to the hippocampus seen with immobilization stress in mice appears to be partially attenuated with Ashwagandha at 20mg/kg of the hydroalcoholic root extract for one month prior to stress, as assessed by histologicla examination of the CA2 and CA3 regions.[184]

    5.14

    Sedation and Sleep

    Ashwagandha (100-200mg/kg) appears to be similar in potency to 500mcg diazepam in reducing sleep latency and improving sleep quality in mice[185][134] and the GABAA receptors appear to be implicated as the effects of ashwagandha are inhibited by GABAA antagonists (picrotoxin) and potentiated by GABAA agonists (muscimol).[134]

    The oxidative stress seen with disturbed sleep in mice is also reversed with supplementation of ashwagandha root at the 100-200mg/kg range over the course of five days.[185]

    Ashwagandha is thought to promote sleep due to having a signalling effect via GABAA receptors, and due to this being potentiated by direct GABAA agonists it seems to be a potentiating effect

    In rat studies not investigating sleep quality, 3,000mg/kg of ashwagandha (a higher than normal dosage) appears to induce sedation in rats despite lower doses promoting libido[186] and similar effects have been noted with 100mg/kg ashwagandha in mice with obsessive compulsive disorder (symptom reduction, but also a reduction in motor unit recruitment indicating sedation).[187]

    Rat studies which use high acute doses of Ashwagandha to note sedation as a side-effect of treatment

    Studies in humans include two where an 'ayurveda' control group consisting of a few herbs (10g total, of which 2,000mg was ashwagandha root and other major components were 1,000mg Emblica officinalis and 250mg of both Sida cordifolia and Terminalia Arjuna) was paired against yoga, and while herbal supplementation showed a nonsignificant trend to improve sleep conclusions cannot be drawn due to nutrient confounds.[188][189] One study using 750-1,250mg of the water root extract (6-10g root equivalent) in otherwise healthy humans has noted that 6/17 subjects reported better sleep with supplementation; the study was not blinded.[190]

    Human studies where ashwagandha is used throughout the day (even if it is not inherently treating stress) tend to note sporadic reports of improved sleep quality

    5.15

    Obsession and Addiction

    A study has suggested that Withania Somnifera (Ashwagandha) can help with Obsessive-Compulsive Disorder.[187] Based on the assumption that Ashwagandha has traditionally been used to cure 'mood disturbances', a study was conducted on mouse marble-burying behaviour (an established research model for OCD[191]) and found that 10-100mg/kg bodyweight ethanolic extract of Withania Somnifera was able to reduce OCD-like symptoms; with 25 and 50mg/kg being seen as best, as 10mg/kg was statistically ineffective and 100mg/kg associated with sedation (the anti-OCD effects still held true, but sedation was suggestive of impairment and lowered internal validity somewhat).[187] Ashwagandha was about as effective at 10mg/kg as Fluoxetine at 5mg/kg bodyweight (both dosed too low to be significantly different than control) yet the combination of the two abolished OCD-like behaviour while pairing Ashwagandha with ritanserin (a serotonergic antagonist) negated the benefits of both; suggesting Ashwagandha benefits OCD via serotonergic mechanisms.[187]

    Ashwagandha may reduce obsessive behaviours, and appears to be synergistic with the tested SSRI (fluoxetine)

    In animals given an alcohol addiction with continual exposure and then given ashwagandha root (200-500mg/kg) upon cessation, it was noted that the increase susceptability to convulsions in rats undergoing alcohol withdrawal were abolished with the higher dose of ashwagandha (200mg/kg not significantly effective) and 500mg/kg had antidepressive and anxiolytic effects in these rats to a level similar to 1mg/kg diazepam.[192]

    6.

    Cardiovascular Health

    6.1

    Absorption

    The combination of Ashwagandha roots alongside other herbs (Glycyrrhiza glabra, Chlorophytum borivilianum, Asparagus racemosus, and sesamum indicum) has been noted to increase fecal cholesterol (11-19%), sterols (12-23%), and bile acids (18-34%) when in varying ratios with the other plants totalling 5% of the diet;[193] Ashwagandha roots by itself in hypercholesteromic rats has also been noted to increase fecal cholesterol (14.21-17.68%), neutral sterols (12.4-18.85%), and bile acids (22.43-28.52%) at 0.75-1.5% of the rat diet[194] which also applied to rats without high cholesterol but to a lesser degree.[194]

    Ashwagandha has been noted to inhibit cholesterol absorption when consumed at moderately high levels in the rat diet (alongside high cholesterol intake); the influence on lipid absorption is not known and the practical significance of this data is also not known

    6.2

    Cardiac Tissue

    Oral supplementation of Ashwagandha (25-100mg/kg of a hydroalcoholic extract) for a month prior to cardiotoxic stimuli (such as isoproterenol induced toxicity or ischemia-reperfusion) noted that the middle dose of 50mg/kg was most effective and significantly preserved all measured antioxidant enzymes (catalase, SOD, glutathione peroxidase) to levels greater than 100mg/kg Vitamin E and normalized lipid peroxidation assessed by MDA concentrations.[195] When examining the cardiac tissue via histology, ashwagandha at 50mg/kg appears to be highly protective of necrosis[195] and this same dose elsewhere has been noted to reduce cellular apoptosis rates following ischemia-reperfusion alongside the betterment of the antioxidant profile.[196]

    This protective effect has also been noted against doxorubicin induced cardiotoxicity, where an ashwagandha extract containing 1.5% total withanolides given at the dose of 300mg/kg to rats for a week prior to and after doxorubicin is able to fully prevent the increase in TUNEL positive cells (apoptotic) seen with the drug as well as other markers of oxidation (MDA, MPO, and protein carbonyls).[197]

    Oral ashwagandha supplementation at relatively low doses of the hydroalcoholic extract (50mg/kg in rats correlating to 8mg/kg in humans) appears to be cardioprotective against a variety of insults to a fairly respectable degree against ischemia and strong protection against doxorubicin

    Supplementation of ashwagandha by itself (500mg of an aqueous root extract) has been implicated in improving velocity (2.9%), power (average by 8.8% and relative by 10%) and VO2 max (6.8%) in otherwise untrained persons subject to sprint training.[198] These benefits were additive with Terminalia arjuna.[198]

    Oral supplementation of ashwagandha in otherwise healthy persons appears to improve cardiorespiratory parameters during exercise, notably VO2 max; possibly indicative of the neural (power output) and lung interactions more than cardiac tissue though

    6.3

    Red Blood Cells

    High concentrations of withaferin A (5-10µM) are able to cause calcium influx and ceramide production in erythrocytes, resulting in cellular death; 2.5µM Withaferin A or less were not toxic.[199]

    Higher than normal concentrations of Withaferin A (normal concentrations are 50-500nM or 0.05-0.5µM) appear to be cytotoxic to red blood cells, indicative of toxicity with high doses of this molecule

    6.4

    Blood Pressure

    Ashwagandha given to otherwise healthy persons subject to exercise training has failed to modify blood pressure.[198]

    Very limited evidence in Ashwagandha and blood pressure, with no significant influence in otherwise healthy persons being noted

    6.5

    Cholesterol

    In rats with normal cholesterol levels fed a standard chow diet, supplementation of ashwagandha at 0.75-1.5% of the diet for four weeks seems to be able to reduce total cholesterol (10.5-16.6%) and LDL-C (29.3-48.4%) while causing a minor increase in HDL-C (3.1-7.0%) relative to baseline, significnatly greater than the control group.[194]

    In diet-induced hypercholesterolemic rats given 0.75-1.5% ashwagandha root in the diet for four weeks, there is a reduction in total cholesterol (41.5-53%) and LDL-C (49.2-62.7%) with a significant increase in HDL-C (15.1-17.7%).[194]

    In diabetic rats, the abnormalities seen in cholesterol (total, LDL-C, and HDL-C) appears to be fully normalized with ingestion of 1,000mg/kg of the fruit extract (60% ethyl acetate) relative to nondiabetic control; a potency comparable to atorvastatin.[200]

    In rodent studies, Ashwagandha appears to better the lipoprotein profile with a mild increase in HDL-C and more notable reductions in LDL-C and total cholesterol. The reduction in LDL cholesterol actually appears to be a per se mechanism of action rather than just fixing a metabolic abnormality, and occurs in normal rats as well as those with metabolic ailments

    One study in otherwise healthy human subjects given 750-1,250mg of the water extract (6-10g root equivalent) in a scaling dose over 30 days noted that at the end of the study there was a significant reduction in LDL-C by 9.7% (175.9mg/dL down to 159.6mg/dL);[190] the changes in HDL-C and total cholesterol were not statistically significant.[190]

    The reduction in LDL cholesterol has been confirmed in otherwise healthy persons who did not have elevated LDL cholesterol at baseline

    6.6

    Triglycerides

    In rats given 0.75-1.5% of the diet as ashwagandha root, after four weeks there is a reduction in triglycerides in the normal rat control (18.1-34.2%) as well as the rats with high cholesterol (31.2-44.8%).[194]

    In alloxan induced diabetic rats Ashwagandha (100-200mg/kg of an 80% ethanolic extract) attenuates the increase in triglycerdes to 23-35% of diabetic control, a potency comparable to 600µg/kg of the reference drug glibenclamide (28%).[49] The fruits (1000mg/kg of the 60% ethyl acetate extract) has elsewhere been shown to be comparable to atorvastatin in fully normalizing triglycerides in streptozotocin induced diabetic rats, and the two were not additive since they were both able to normalize triglycerides relative to control.[200]

    There is a mild reduction in triglycerides associated with oral ingestion of Ashwagandha

    In persons with metabolic syndrome, supplementation of 400mg ashwagandha extract thrice daily over the course of 30 days is able to reduce triglycerides by approximately 12% relative to baseline (placebo had no benefit).[201] Otherwise healthy persons given 750-1,250mg of the water extract (6-10g root equivalent) have failed to experience changes in triglycerides over 30 days.[190]

    The reduction in triglycerides seen in persons with metabolic syndrome appears to be mild, and there is no influence of Ashwagandha on the triglycerides of otherwise healthy persons

    7.

    Interactions with Glucose Metabolism

    7.1

    Blood glucose

    In a model of type I diabetes (mice injected with streptocozin), Withania Somnifera at 6.25% of the diet may raise fasting blood glucose slightly but significantly.[202]

    7.2

    Type II Diabetes

    200-400mg/kg of the aqueous root extract (3.9% withaolides) has been noted to normalize blood glucose in streptozotocin induced diabetic rats after five weeks by 76-89%, which correlated with improvements in oxidative biomarkers and pancreatic β-cell structure.[203]

    Doses of Ashwagandha (100-200mg/kg) have been shown to reduce adverse changes to glucose metabolism after oral ingestion in rats.[49] The higher dose of 200mg/kg was as effective as 0.6mg/kg glibenclamide (anti-diabetic drug) at reducing blood glucose and HbA1c (to near control levels, although not completely) and increasing hepatic liver glycogen and hemoglobin levels.[49] A decrease in glucose-6-phosphatase activity is also seen in the livers of diabetic animals treated with Ashwagandha, to a similar potency to glibenclamide.[49]

    1000mg/kg of the dried fruits (60% ethyl acetate extract) is also effective in reducing blood glucose in streptozotocin induced diabetic mice with a potency comparable with 1mg/kg glipizide over four weeks, and the combination appeared to be additive in reducing blood glucose.[200]

    Appears to be able to reduce blood glucose in rodent models of diabetes when given at the standard oral doses, with a potency comparable to reference drugs (and additive with glipizide, an insulin secretagogue)

    One study in rats noted that 6.25% Ashwagandha (on a food weight basis) could attenuate the adverse changes in lutenizing hormone and follicle-stimulating hormone associated with diabetes.[204] No effects were seen on estrogen and progresterone (despite the latter dropping in the experimental group of diabetes) and testosterone changes were not related to the state of diabetes.[204]

    May also normalize other parameters associated with diabetes, thought to be vicariously through reducing blood glucose

    In persons with metabolic syndrome, supplementation of 400mg ashwagandha root extract thrice daily over the course of 30 days was able to reduce fasting blood glucose by approximately 13%; there was no reference drug used in this study, but it outperformed placebo.In persons with metabolic syndrome, supplementation of 400mg ashwagandha extract thrice daily over the course of 30 days is able to reduce triglycerides by approximately 12% relative to baseline (placebo had no benefit).[201]

    There appears to be a mild reduction in blood glucose when supplemented in persons with metabolic syndrome (insulin resistance)

    8.

    Obesity and Fat Mass

    8.1

    Adipogenesis

    Withaferin A appears to induce apoptosis in 3T3-L1 adipocytes when incubated with 1-25µM Withaferin A for 24 hours, although the most practically relevant concentration (1µM) was noted to suppress viability of preconfluent preadipocytes (16.73%) while enhancing viability of postconfluent preadipocytes (22.5%) and had no significant influence on mature adipocytes.[205]

    Has the potential to induce apoptotic cell death in fat cells, but the concentrations that this occurs at are impractically high for oral supplementation of Ashwagandha

    8.2

    Weight and Body Fat

    Healthy, untrained men put on a periodized strength training program for 8 weeks while also taking 300 mg of a commmercial ashwagandha extract (KSM-66, standardized to 5% withanolides) twice daily lost 2% more body fat than the placebo group as measured by bioelectrical impedance (1.5% versus 3.5% for placebo versus ashwagandha, respectively).[19]

    9.

    Skeletal Muscle and Physical Performance

    9.1

    Power Output

    One study in otherwise healthy sedentary persons has noted that supplementation of the water extract of ashwagandha at 750-1,250mg daily (6-10g dry root equivalent) over 30 days, despite no exercise program, increased force production from the lower back (15.4%) and quadriceps (21.5%) but not grip test;[190] there was no influence on the rate of perceived exertion, and results were seen after 30 days but not 10-20.[190]

    Another study in healthy, untrained men who underwent a periodized strength training regimen for 8 weeks while taking 300 mg of ashwagandha extract (standardized to 5% withanolides) or placebo twice daily found that the ashwagandha's 1RM for the bench press increased by 20 kg (44 lbs) more than the placebo group. The 1RM gains for the leg extension exercise of the treatment group was about 4.5 kg (10 lbs).[19]

    Preliminary evidence suggests that ashwagandha can increase power output in sedentary people who start resistance training and also those who undergo no training.

    9.2

    Aerobic Exercise

    In elite cyclists given 500mg of the aqueous root extract twice daily over the course of eight weeks, there was a statistically significant increase in VO2 max (12.5%) and time to fatigue on the VO2 test (7.2% or 1.14m) although fat oxidation as assessed by the respiratory exchange ratio was unaffected.[206]

    Ashwagandha has once been implicated in increasing VO2 max in otherwise trained cyclists at the standard oral dose

    9.3

    Lean Mass

    Aswagandha at 750-1,250mg daily (6-10g dry root equivalent) for a month in otherwise healthy sedentary persons has been noted to cause a trend to increase lean mass and decrease fat mass, although the changes were not statistically significant.[190]

    A trend to increase lean mass has been noted in otherwise sedentary persons which failed to reach statistical significance; no studies currently exist in trained individuals

    10.

    Bone and Joint Health

    10.1

    Osteoblasts

    Inhibition of proteasomal activity appears to promote osteoblastic cell proliferation[207][208] and Withaferin A appears to directly bind to the specific catalytic β subunit of the 20S-proteasome in an inhibitory manner;[73] secondary to this, Withaferin A (10nM; both 100pM and 1µM ineffective) increased ALP secretion three-fold and increased bone mineralization rates to a level greater than the reference proteasome inhibitor (bortezomib) at the same concentration.[73] The reason proteasomal inhibition increased bone mineralization was said to be an induction of BMP2 resposive genes and preserves Smad proteins; Smad proteins will then influence Smurf2 (suppress) causing an increase in RunX2 expression which then increases bone mineralization.[73][209]

    It appears that the proteasomal inhibition associated with ashwagandha bioactives (Withaferin A) can also promote osteoblastic differentiation and growth, which would lead to increased mineral accumulation; this appears to occur at a low enough concentration that it may be relevant following oral ingestion

    10.2

    Osteoclasts

    In osteoclasts, 10nM of Withaferin A has been noted to reduce TRAP positive osteoclasts by 30-40% after nine days of incubation in a manner that was replicated with bortezomib and thus said to be secondary to proteasomal inhibition.[73] This indicates a reduction in osteoclast differentiation, and Withaferin A was further able to prevent TNF-α from increasing the activity of NF-kB in vitro[73] and serum concentrations of TNF-α in ovarectomized mice given 10mg/kg Withaferin A orally was reduced to control levels (mimicked by bortezomib).[73]

    The same proteasomal inhibition that causes osteoblastic differentiation may also suppress osteoclastic differentiation, which would also be a mechanisms associated with increased bone mass

    10.3

    Rheumatism

    Acute gouty arthritis (rheumatism associated with monosodium urate crystal deposition[210]) treated with 500-1,000mg/kg of the basic root extract of Ashwagandha in rats is able to abrogate inflammation to around half of control (similar potency at both doses, and similar to 3mg/kg indomethacin) with similar partial efficacy for analgesia.[211]

    Limited evidence suggests a protective effect against experimentally induced rheumatism in rodents

    10.4

    Osteoarthritis

    In vitro, a water extract of Ashwagandha root has shown antiinflammatory properties in cartilage removed from chronic osteoarthritic patients (50µg/mL over eight days) secondary to mildly suppressing nitric oxide release; this was no associated with an inherent chondroprotective effect (assessed via GAG release), and some persons were deemed nonresponders;[212] this study is duplicated in Medline.[213]

    Natural therapies from Ayurveda in particular seem to be investigated for their benefits against osteoarthritis[214] and there has been a trial using ayurvedic combination treatment for osteoarthritis (combining Ashwagandha alongside Boswellia serrata, ginger, and Turmeric) which has outperformed placebo over 32 weeks.[215]

    While ashwagandha is thought to confer protective effects towards osteoarthritis, there is currently not a lot of good evidence to suggest a protective effect and the evidence that does exist is either confounded or suggests that Ashwagandha may not be overly potent

    10.5

    Bone Mass

    Oral ingestion of 5-10mg/kg of Withaferin A daily to female mice (ovarectomized) who experienced a bone injury noted that both doses increased bone remineralization in a dose dependent manner, while in osteopenic mice 10mg/kg was required for benefits to bone structure, resulting in a greater compressive energy required to break bones.[73]

    A 50% ethanolic extract of the roots (65mg/kg) fed to a rat model of menopause for 16 weeks alongside a diet deficient in calcium was able to decrease urinary excretion of calcium and phosphorus[216] and increased the amount of force required to snap the tibia, suggesting a bone preserving effect.[216]

    May promote bone mass in ovarectomized rodents (models of menopause) with feasible oral doses

    11.

    Inflammation and Immunology

    11.1

    Immunostimulation

    When incubated in splenocytes, Ashwagandha is able to augment LPS-induced spleen cell proliferation by 6-fold relative to LPS alone[217] and the T-cell lymphocyte proliferation response (from Concavalin A) is increased with isolated Withanolide A.[218]

    Ashwagandha appears to potentiate the stimulatory effect of mitogens on splenic cell proliferation, and this appears to be due to Withanolide A (other withanolides not necessarily excluded)

    Ashwagandha is known to possess an anti-inflammatory potential (ie. in adjuvant induced arthritis[219]) and in rats with chronic inflammation via adjuvant, a model for arthritis, 1,000mg/kg of the basic root extract daily for one week noted that the proliferation of lymphocytes in response to a mitogen (PHA) seen in arthritic control was attenuated with Ashwagandha.[220]

    The proliferation of lymphocytes may only occur under otherwise normal scenarios, as in instances where there is a high level of inflammation there actually appears to be a mild suppression (or 'attenuation of increase') seen with ashwagandha

    11.2

    Immunosuppression

    Dexamethasone induced suppression of Th1 cell activity is recovered in vitro with 0.1-10ng/mL of withanolide A when coincubated with a mitogen (PHA), although the IL-4 suppression (seen with withanolide A by itself) does not occur in this concentration[52] In stressed rats (high corticosteroid concentrations in serum), the water extract of the root at 25-200mg/kg is able to somewhat preserve the reduction in total T-cell count associated with a reduction in circulating cortisol to near control levels,[221] and isolated Withanolide A is able to reduce corticosterone concentrations by itself (0.25-1mg/kg oral ingestion) to near control levels in stressed mice associated with preservation of T-cell function.[218]

    Withaferin A appears to suppress the IL-10 secretion from myeloid-derived suppressor cells (MDSCs) also also reduced ROS formation, the latter being a STAT3 dependent mechanism;[222] secondary to the suppressive effects on MDSCs, Withaferin A preserved the tumor-orientated cytotoxicity of CD4+ and CD8+ T-cells and reduced tumor weight in mice.[222] The IL-10 secretion from MDSCs, as mentioned, is STAT3 dependent[223] and Ashwagandha (100-400mg/kg oral intake of 50% ethanolic extract) has been confirmed to suppress STAT3 activity in vivo over nine days in mice.[224]

    Withaferin A and Withanolide A appears to abrogate the corticosteroid-induced suppression of Th1 cell activity. This is in part due to the antistress aspects of Ashwagandha (reducing cortisol) but may also be related to STAT3 inhibition (can reduce suppression of T-cells independent of cortisol)

    Expression of CD4+ and CD8+ receptors on T-cells after cyclosporin (immunosuppressant) are minorly preserved, but this may be secondary to Ashwagandha (100-200mg/kg oral intake of the 50% methanolic extract for 14 days) inherently increasing the levels of these receptors.[225]

    The neutropenia (reduction in neutrophil count) seen with paclitaxel appears to be significantly attenuated with oral ingestion of 200mg/kg of Ashwagandha extract daily for four days prior to paclitaxel injection and for another subsequent eight days.[226] The reduction in neutrophils was fully abrogated, with an increase in neutrophil count relative to baseline seen in the Ashwagandha group despite injections of paclitaxel, and was comparbale to 25µg/kg injections of Granulocyte-Monocyte Colony Stimulating Factor (GM-CSF).[226]

    There appear to also be immunosupportive actions of Ashwagandha against other immunosuppressants such as cyclosporin or paclitaxel, and in regards to neutropenia it appears to be quite effective

    11.3

    Interferons and Immunoglobulins

    In mice fed 30mg/kg of a 50% ethanolic extract of ashwagandha for 15 days with an antigen presented on day 9 (SRBC), it was noted that the antibody titer of immunoglobulin M (IgM) by 128%, and while the immunoglobulin G (IgG) profile overall experienced an increase this was due to a large increase in IgG2a (around a four-fold induction) since the IgG1 isotope decline mildly by 40% in all oral doses (10-100mg/kg) which correlated with IL-4 concentrations in serum.[52]

    An increase in IgM has been noted with Ashwagandha, and IFNγ is known to also be increased fairly reliable associated with T-cell stimulation (see T-cell section)

    11.4

    Macrophages

    In mice subject to stress (in this study, cold stress), Ashwagandha at 512.5mcg daily (20.5-25.625mg/kg) was able to increase phagocytosis by macrophages to near control levels although it was outperformed by beta-glucans from Maitake (at 30.75μg; 1.25-1.5mg/kg).[227]

    The reduction in macrophage activity seen during stress appears to be significantly reduced with oral ingestion of Ashwagandha at relatively low oral doses

    This increase in phagocytosis has been noted elsewhere with the 70% methanolic extract of ashwagandha at 20mg/kg (intraperitoneal injections) by 142% when tested ex vivo.[228]

    Ashwagandha 50% ethanolic extract (30mg/kg to mice for 15 days) noted an increase in IL-12 and TNF-α production with no influence on IL-10, and this increase in macrophage activity (assessed by nitrite) inhibited by dexamethasone.[52]

    Ashwagandha can stimulate macrophage activity (nitrite production) in a manner that is inhibited by exogenous corticosteroids, and can also stimulated phagocytosis even when the subject is otherwise normal

    11.5

    Natural Killer Cells

    NK cells are known to be stimulated in mice fed Ashwagandha whether they bear tumors or not either alone[217] or as a combination formula alongside Guduchi, Holy Basil, and Amla.[229] This occurs in tumor bearing mice as well, where oral ingestion of 100-400mg/kg of a 50% ethanolic root extract increases NK cell population by 20-40% over nine days.[224]

    In rodents, it appears that Ashwgandha is able to increase NK cell activity and population in both otherwise healthy animals and those bearing tumors

    In humans, a medicinal tea containing Ashwagandha (0.5%) amongst other medicinal herbs (totalling 4.0%) has been noted to, when given to adults with low NK cell activity at the dose of 2.06g for two months, increase NK cell activity only after the two month point but to a degree reaching 60% more than placebo tea;[230] this was confirmed in a larger trial where it took two months to increase NK cell activity and all subjects experienced a reduction after two and a half months of tea cessation.[230] Elsewhere, a root extract of Ashwagandha (3:1 ethanolic extract taken at 6mL; dry weight equivalent unknown) twice daily for four days alongside whole milk caused an increase in NK cell CD56+ receptors although it was noted there was interindividual differences.[231]

    The human evidence at this moment in time is not well structured and uses atypical dosing strategies (with the lone study not using confounded herbs not stating the dry root equivalent), but it seems to suggest that the NK cell activation in rodents also applies to humans

    11.6

    T Cells

    A 50% ethanolic extract of Ashwagandha fed to mice for 15 days (with SRBC antigen given on day 9) noted that 30mg/kg (the dose more effective than 10mg/kg and 100mg/kg) was able to promote proliferation of T cells and both CD3+ and CD4+/CD8+ associated with a cytokine profile more reflective of Th1 cells (IFNγ and IL-2 increased by around 60%);[52] this was thought to be due to Withanolide A, which suppressed IL-4 with no dose-dependence in the range of 0.1-100ng/mL and increase IFNγ and IL-1 with peak efficacy at 1ng/mL.[52] This occurs in tumor bearing mice as well, where oral intake of 100-400mg/kg of the 50% ethanolic extract, again with most potency at 200mg/kg.[224]

    Elsewhere, 25-200mg/kg of an Ashwagandha extract (50% methanolic) was able to increase IFNγ secretion in cells while 400mg/kg was less effective and the potency of Ashwagandha exceeded the reference of 2.5mg/kg Levamisole;[225] there was a stimulatory effect on IL-2 at 100-200mg/kg comparable to Levamisole, and both CD4+ and CD8+ receptors were increased[225] and these effects are seen with comparable potency with the basic water extract of the root at 100-200mg/kg[221] and in vitro are maximal at 100-1,000ng/mL of the 50% ethanolic extract.[224]

    Ashwagandha appears to have a dose-dependent stimulatory effect on Th1 cells, and increases their receptor levels and the cytokines that they secrete (IFNγ and IL-2). This occurs in a dose dependent manner up to 200mg/kg (human equivalent being 32mg/kg) of the basic root extract and appears to apply to the standard oral doses of Ashwagandha

    IL-4 has been noted to steadily decrease in the range of 15-30% with the 50% ethanolic extract, and with 0.1-100ng/mL of isolated Withanolide A[52] and elsewhere IL-4 is unaffected at an oral dose (100-200mg/kg) that influences IFNγ and IL-2[225] or nonsignificantly attenuated at the concentration (100-1,000ng/mL) that most effectively activate Th1 cells.[224]

    There is either a mild suppressive effect on Th2 cells seen with Ashwagandha or there is no significant influence on their function, usually assessed by the cytokine IL-4

    11.7

    B Cells

    A 50% ethanolic extract of Ashwagandha fed to mice for 15 days (with SRBC antigen given on day 9) noted that 30mg/kg was able to promote proliferation of B cells to a level greater than 10mg/kg and 100mg/kg, and this was accompanied by an increase in CD19+ cells.[52]

    In mice given SRBC either in a normal state or in an immunosuppressed state (cyclophosphamide), oral ingestion of 100-200mg/kg of the 50% methanolic root extract in mice appears to increase the overall antibody titre (11-16% and 14-30% in normal and immunosuppressed, respectively);[225] the potency was lesser than the reference drug of levamisole (2.5mg/kg).[225] An increase in antibody titre has been noted per se in mice given 20mg/kg injections of the 70% methanolic extract, which is though to be due to proliferating the type of cells that produce antibodies in the spleen.[228]

    While there may be an indirect stimulatory effect on B-cells, it is not a highly researched topic in regards to Ashwagandha supplementation

    11.8

    Bacterial Interactions

    Ashwagandha appears to be moderately active in inhibiting Trichophyton mentagrophytes (MIC value of 3.125mg/mL), Staphylococcus aureus (MIC value of 6.25mg/mL) and its methicilin resistant strain known as MRSA (MIC value of 12.5mg/mL).[232]

    In pulmonary tuberculosis which has been known to respond benficially to ayurvedic combination therapy,[233] 500mg of ashwagandha twice daily over the course of 28 days given alongside standard anti-tuberculosis drugs (Rifampicin, Pyrazinamide, Ethambutol, Isoniazid; given as needed) noted a significant increase in IgG and IgM as well as total white blood cell counts (monocyte and eosinophyll mostly, no influence on neutrophil nor lymphocytes) and significantly reduced bacterial load relative to drug control.[234]

    12.

    Interactions with Hormones

    12.1

    Testosterone

    A slight increase in testosterone was seen in non-diabetic rats used as a control, alongside a much larger spike in progesterone.[204]

    There is some evidence for usage of Ashwagandha as a testosterone-boosting compound in infertile men.[235] This aforementioned study used 5g of basic Ashwagandha root powder daily for 6 months and found increases in testosterone in the three tested infertile groups (Asthenozoospermic rose to 121% of baseline, Oligozoospermic to 140% of baseline, and Normozoospermic to 114% of baseline) yet no group surpassed the fertile control groups testosterone level, although normozoospermic was insignificantly different after the end of the trial.[235] Elsewhere, three groups of men who were infertile and normozoospermic (smokers, stressed, neither) given the same dose of ashwagandha for three months noted improvement in testosterone in the range of 10-22% with a larger spike in the stressed men.[236]

    A rise in serum testosterone was also seen in healthy, untrained men who began a periodization strength training protocol while taking 300 mg twice daily of ashwagandha root extract; the placebo group's testosterone (measured before workouts) remained unchanged, while the ashwagandha group's rose about 15%.[19]

    Preliminary evidence that ashwagandha can normalize reduced testosterone levels in infertile men, and may also boost testosterone levels in men who are also undergoing resistance training.

    12.2

    Estrogen

    Withaferin-A has been noted to suppress the expression of the alpha subset of the estrogen receptor (ERα) in breast cancer cells (MCF-7 and T47D) at 1.25-2.5μM concentrations to a variable 50-90% reduction, with a concomitant 20-30% increase in the expression of ERβ.[237]

    Although there is some evidence to suggest that ashwagandha can alter the expression of the estrogen receptors, these studies are currently conducted in tumor cells and may not apply to normal cells

    12.3

    Luteinizing Hormone

    One study to note LH levels in infertile men noted an increase and a trend towards normalization when compared to fertile men[235] and 5g of the root extract daily for three months has noted an increase in LH levels in serum by 11-21% with more efficacy in men who self-identified as being stressed.[236]

    A study in diabetic rats who had two control groups (non-intervention, Ashwagandha intervention) found LH spikes about three-fold higher with 6.25% Ashwagandha (as assessed on a weight basis of food intake) from 0.2mIU/mL to 0.6mIU/mL after four weeks.[204]

    12.4

    Follicle-Stimulating Hormone

    The one study to note FSH levels noted a decline in tested infertile men, signalling a trend to normalization when compared to the fertile control[235] which has been replicated with 5g of the basic root extract.[236]

    These effects have been noted in diabetic mice, and a slight decrease is also seen in control mice with no health complications.[204]

    12.5

    Thyroid Hormones

    In animals, Ashwagandha has been shown to increase circulating T4 levels with no influence on T3 (at 1.4g/kg root extract daily in mice for 20 days[238]) and another study noted both hormones (T3 and T4) increased under the same protocol.[239] The latter study was conducted in male mice, and tested females showed different results; when female mice were tested in isolation,[238] the same increase in hepatic anti-oxidant enzymes (catalase up by 12%) and decrease in lipid peroxidation (34%) was seen, and increased T4 (lesser active thyroid hormone) by approximately 60% without significantly influencing T3 (more active hormone).[238] Ashwagandha has also been investigated as a combination with Guggulsterone gum and Bauhinia Bark (from Bauhinia Purpurea)[240] and appears to be well-tolerated; and a rise in T3 was recorded with this blend[240] possibly due to the Bauhinia.[238]

    Beyond that, Ashwagandha possibly has benefit as an adjunct therapy. While Metformin administration can alleviate many effects of experimentally induced type II diabetes, it further reduces circulating T4 levels; Ashwagandha administration at 1.4g/kg ameliorated these adverse changes.[241]

    At least one human case study has noted a medically relevant case of hyperthyroidism after usage of a supplement containing Ashwagandha.[242]

    Seems to have the ability to stimulate T4 production at the level of the thyroid, but there may be a gender difference in regards to T3 where men can produce more in response to Ashwagandha than women; more studies would be needed for conclusison

    12.6

    Corticosteroids

    In rats, the increase in serum cortisol seen with stress is fully abrogated at an oral intake of 100-200mg/kg of the water root extract with partial attenuation at 50mg/kg[221] while lower doses (25-50mg/kg) of the withanolide glycosides (ie. Withanoside IV) appears to fully abrogate the increase in corticosteroids seen with stress;[172] a potency greater than 100mg/kg panax ginseng (57% attenuation).[172] This is also noted with Withanolide A at 0.25-1mg/kg (1-2mg/kg both causing full normalization of serum corticosterone in stressed mice, thus maximal efficacy at 1mg/kg), despite not being a withanolide glycoside.[218]

    Ashwagandha appears to reduce the stress related increase in corticosteroids in serum, and at the higher doses of supplementation (still within the doses humans use) this appears to fully normalize cortisol to control levels

    One case study exists where a woman with non-classical adrenal hyperplasia was treated with ashwagandha root for six months and experienced a therapeutic decrease in corticosteroids (18-OH-hydroxycorticoserone by 31%, 17-OH-pregnenolone by 66%, corticosterone by 69%, and 11-deoxycortisol by 55%) alongside a decrease in clinical side-effects associated with excessive corticosteroid levels such as hair loss.[243]

    Other human studies have noted that cortisol is reduced by at least 14.5% in persons who report that they are chronically stressed (250-500mg in two divided doses for sixty days)[177] and elsewhere has been noted to be reduced by up to 27.9% (sixty days of 300mg of a supplement with at least 5% total withanolides)[25] and in the range of 36-48% (measured at a controlled timepoint) with 5g of the dry root daily for three months with more potency in those who reported being highly stressed.[236]

    The reductions in cortisol seen with Ashwagandha in humans given the lower end of the standard supplemental doses (300-500mg of extracts or 5,000mg of the root itself) tends to be in the range of 15-50%, with more potency in those who self-report as being significantly more stressed

    13.

    Interactions with Oxidation

    13.1

    Lipid Peroxidation

    In rats fed 0.75-1.5% of the diet as ashwagandha root for four weeks, lipid peroxidation (MDA) in the liver is reduced 12.4-18.2% and reached a more significant reduction in the rats with high cholesterol (36.3-36.5%).[194]

    13.2

    Hydrogen Peroxide

    In rats fed 0.75-1.5% of the diet as ashwagandha root for four weeks, there is an increase in catalase activity in the liver are increased in otherwise normal rats (11-15.2%) as well as those with diet-induced hypercholesterolemia (37.1-43.3%).[194]

    13.3

    Superoxide

    In rats fed 0.75-1.5% of the diet as ashwagandha root for four weeks, superoxide dismutase levels in the liver are increased in otherwise normal rats (2.9-3%) as well as those with diet-induced hypercholesterolemia (20-31%).[194]

    13.4

    Irradiation

    Withanone has been demonstrated to increase cell viability (survival) in normal human fibroblasts when cells are insulted by either UV rays or by H2O2, although not to control levels.[116] Overall reductive capacity of a cell also appears to be increased (as evidenced by up to 40% increases in Glucose-6-phosphate dehydrogenase activity).[116]

    Withaferin A at a concentration too small to induce cytotoxicity alone (500nM) in U937 (lymphoma) cells is able to enhance the cytotoxicity experienced from radiation associated with increasing both superoxide (O2-) and hydrogen peroxide (H2O2) concentrations in the cell to higher levels with the combination.[244]

    14.

    Peripheral Organ Systems

    14.1

    Pancreas

    Withaferin A (1.25mg/kg injections) given for a week after islet cell transplantation into mice appeared to facilitate the transplantation by reducing pancreatic cell apoptosis associated with reducing NF-kB translocation and reducing IκB degradation.[245] This was noted in vitro in isolated pancreatic β-cells, where 500ng/mL of Withaferin A fully prevented an increase in NF-kB from cytokines and 1µg/mL actually caused lower levels than control despite the presence of cytokines;[245] 1µg/mL, however, is high enough to inherently reduce cell viability.[245]

    Oral administration of Ashwagandha root (water extract of 3.9% withanolides) has been noted to significantly preserve β-cell viability in diabetic rats when given at 200-400mg/kg for five weeks after diabetes induction.[203]

    14.2

    Liver

    In normal rats given 0.75-1.5% ashwagandha in the diet for four weeks, there appears to be a reduction in liver weight in the range of 6.1-9.77% relative to baseline associated with reductions in hepatic triglycerides and cholesterol as well as a relative increase in bile acids.[194]

    There appears to be a minor decrease in liver weight associated with Ashwagandha in otherwise normal rats whihc is not associated with any apparent toxicity

    In diabetic animals, both the root and leaf of Ashwagandha is also able to decrease circulating liver enzymes at 200mg/kg by 45% (AST and ALT), 31% (ACP) and 16% (ALP).[49] The increases in liver enzymes as a result from radiotherapy are also fully abrogated[114] and the increase in ALT seen with chronic psychological stress in mice is mildly attenuated with supplementation of isolated Withanolide A (able to reduce corticosterone) at 0.25-1mg/kg.[218]

    Radiation induced damage to the liver appears to be fully abrogated with oral ingestion of 100mg/kg ashwagandha root daily for a week prior to radiotherapy, as assessed by alterations in MDA and antioxidant enzymes;[114] this was thought to be due to augmenting the HO-1 induction from radiotherapy (an additional 45.6% increase) without much of an inherent effect.[114] There is also a protective effect against iron overload (mechanism not stated and Iron concentrations of the liver not measured) where Ashwagandha root extract at 50mg/kg is as effective as 20mg/kg silymarin from Milk thistle in normalizing liver enzymes and lipid peroxidation.[246]

    Ashwagandha may enhance the induction of HO-1 in response to stressors, although it does not appear to have an inherent effect on HO-1 protein levels in the liver

    14.3

    Intestines

    In intestinal epithelial cells (Capable of secreting IL-7[232] which can stimulate CD8+ T cell antigen responsiveness[247] as well as CD4+ T cells[248]) incubated with Ashwagandha extract, IL-7 mRNA is increased two-fold at 100µg/mL Ashwagandha and was concentration dependent up until that point (which was peak efficacy).[232]

    Ashwagandha may stimulate intestinal cells in such a way that they can prime T cell function

    14.4

    Lung

    The polysaccharide component of Ashwagandha appears to have antitussive effects against chemical induced coughs when fed to guinea pigs at 50mg/kg and measured 30-300 minutes after oral intake with the potency trending to be more effective than 10mg/kg codeine phosphate, but less effective than glycyrrhiza glabra polysaccharide;[44] this has been replicated elsewhere with the same dose and methodology.[45]

    The polysaccharide fragment of ashwagandha appears to have antitussive properties which may reduce chemical or irritant induced coughing. The 50mg/kg dose in guinea pigs is 11mg/kg in humans (750mg for a 150lb human) and due to this polysaccharide being at 196mg per 20g of the dry weight, unless it is concentrated it is a very large dose of ashwagandha to get the above antitussive properties

    14.5

    Kidneys

    500mg/kg of ashwagandha (root extract) given to rats for two weeks prior to and alongside gentamicin induced nephrotoxicity was able to significantly attenuate the increase in kidney weight and biomarkers of kidney damage (urinary protein and glucose, serum creatinine), although the protection was not absolute;[249] 500mg/kg outperformed 250mg/kg and 750mg/kg.[249]

    14.6

    Bladder

    Some other plants in the withania genera have diuretic activity such as withania aristata[250][251] which may be related to it Withaferin A content since Withaferin A by itself at 5-10mg/kg in rats is able to increase 6-hour diuresis by 76-147%, a potency less than the reference drug of 10mg/kg hydrochlorothiazide.[251]

    15.

    Cancer Metabolism

    15.1

    Immunotherapy

    The neutropenia induced by paclitaxel (a chemotherapeutic drug) appears to be fully abrogated and partially reversed with oral ingestion of 200mg/kg Ashwagandha root extract for four days prior to paclitaxel injections (and continued for another eight days),[226] and beyond that it has shown protective effects from paclitaxel induced oxidative damage in normal cells[252][253] while enhancing its cytotoxic properties against tumors[252][254] in animal models of benzo(a)pyrene induced lung cancer.

    Appears to be quite synergistic with paclitaxel therapy, in part due to abrogating the immunosuppression seen with paclitaxel and (possibly secondary to preserving neutrophil function) enhancing tumor cytotoxicity

    Withaferin A appears to suppress the IL-10 secretion from myeloid-derived suppressor cells (MDSCs) also also reduced ROS formation, the latter being a STAT3 dependent mechanism;[222] secondary to the suppressive effects on MDSCs, Withaferin A preserved the tumor-orientated cytotoxicity of CD4+ and CD8+ T-cells and reduced tumor weight in mice.[222] The IL-10 secretion from MDSCs, as mentioned, is STAT3 dependent[223] and Ashwagandha (100-400mg/kg oral intake of 50% ethanolic extract) has been confirmed to suppress STAT3 activity in vivo over nine days in mice.[224]

    May cause a T-cell profile that is more favorable to causing death of tumor cells, which is thought to be secondary to suppressing STAT3 (which results in less IL-10 to be secreted; IL-10 normally suppresses the cytotoxicity of T-cells, so its reduction is met with an increase in T-cell mediated cytotoxicity)

    TRAIL mediated apoptosis is a mechanism of apoptosis mediated by 'death receptors' on cancer cells, which are activated by immune cell cytokines such as TRAIL or TNF-α; this mechanism of apoptosis tends to boast good selectivity since there is minimal toxicity towards non cancerous cells.[255][256]

    A protein known as cellular FADD-like IL-1β-converting enzyme inhibitory protein (c-FLIP) is suppressed from Withaferin A in Caki cancer cells secondary to NF-kB inhibition[257][68] and downregulation of c-FLIP tends to potentiate death receptor signalling (TRAIL);[258] 1.2μM of Withaferin A in TRAIL resistant cells (Caki, SK-Hep1, Hep3B, and Huh-7) by increasing levels of the death receptor (DR5) in a manner mediated by CHOP and reactive oxygen species[259] (a mechanism similar to curcumin[260] or sulforaphane[261]) and an observed downregulation of c-FLIP observed was thought to contribute[259] since this downregulation is associated with both NF-kB inhibition and sensitization to TRAIL mediated apoptosis.[262]

    Elsewhere, Ashwagandha (50% ethanolic root extract at 20µg/mL) in HL-60 cells has been noted to increase death signalling via the TNF-α receptor (TNF-R1) and it also increased the Death receptor 4 (other receptor for TRAIL).[224]

    The inhibition of NF-kB (which then signals through reactive oxygen species and CHOP) upregulates the death receptor 5 (DR5), which mediates the ability of some immune cells to destroy cancer cells; this mechanism sensitizes cancer cells to death from the immune system without necessarily influencing immune cells, and both DR4 and TNF-R1 have also been implicated in being increased

    15.2

    Adjuvant Therapy

    In an open-label trial of women with breast cancer given either ashwagandha (2,000mg of the root thrice daily) or used as control over the course of six cycles of standard chemotherapy patient fatigue as assessed by PFS, SCFS-6, and the fatigue domain of the EORTC QLQ-C30 noted significant reductions in fatigue in the study groups;[175] the other domains of the EORTC QLQ-C30 noted improved physical, emotional, role, and social functioning with a reduction in insomnia and pain.[175]

    Ashwagandha has once been used to reduce fatigue and improve well being and general function in persons undergoing chemotherapy

    15.3

    Proliferation and Angiogenesis

    Withaferin A has been noted to suppress proliferation of HUVECs with an IC50 of 12nM while to suppressed NF-kB activation in these cells (IC50 of 500nM) and required up to 5µM to induce cytotoxicity (associated with AP-1 signalling);[263] these potent effects were confirmed in vivo with injections of 7µg/kg Withaferin A injected into mice (reducing the angiogenic index to near a quarter of control).[263] This is mostly secondary to inducing degradation of Vimentin, since a dose that reduces angiogenesis by 73% was reduced to 29% in mice lacking vimentin.[56]

    Withaferin A appears to be a very potent angiogenesis inhibitor in otherwise normal endothelial cells, and at the doses it is effective it does not appear to be cytotoxic

    15.4

    Metastasis and Invasion

    Withaferin A has been noted to suppress MMP9 expression in invasive cancer cells (Caski and SK-Hep1) associated with preventing TGF‑β from activating Akt signalling.[264]

    A phenomena known as epithelial-to-mesenchymal transition (EMT) occurs to a cell before it becomes migratory and invasive[265] and EMT is characterized by changes at the level of the membrane with a loss of E-cadrin and increase in vimentin and fibronectin;[266] Withaferin A directly binds to Vimentin at Cys328 in an inhibitory manner[56][59] and Withaferin A or extracts with a high (70%) Withaferin A content can inhibit vimentin activity at 500-1,000nM and EMT of breast cancer cells with efficacy in the nanomolar range (100-1,000nM);[53] mice fed an extract (pure withanolides; 79% Withaferin A) by gavage at 1-8mg/kg thrice weekly significantly reduced metastasis by more than half.[53]

    It should be noted that the anti-invasive properties of Withaferin A are weaker in cells that lack vimentin, as assessed by a BEAS-2B cell line (lacks vimentin yet is invasive) having an IC50 in the range of 1.9-2.9µM[53] while it usually shows efficacy at 100nM in cells that express Vimentin.[53]

    Withaferin A appears to be anti-metastatic as well as anti-angiogenetic, suggesting that at concentrations below its cytotoxic dose it can suppress the proliferation of cancer cells

    15.5

    Breast Cancer

    Isolated withaferin A and withanone are able to induce ROS formation in MCF-7 and MDA-MB-231 cells resulting in apoptosis via the mitochondrial dependent pathway.[20][267] Withaferin A is able to induce cytotoxicity in both normal and cancer cells and cause DNA damage to both (assessed by γH2AX levels) while Withanone is not,[20] although one study noted that the presence of withanone attenuates the cytotoxicity of Withaferin A in noncancerous cells.[116] This ROS is also associated with autophagy, although autophagy does not appear to be involved in the cytotoxicity seen with Withaferin A[268] and is associated with an induction in Bax/Bak pro-apoptotic proteins.[267]

    Ashwagandhanolide (thiowithanolide) has been found to have a 50% growth inhibitory concentration of 1.45µg/mL in MCF-7 estrogen responsive tumor cells[38] which is similar to the sulfoxide variant of this dimer, having a GI50 of 1.25µg/mL.[39]

    Withaferin A and Withanone are both able to induce apoptosis in breast cancer cells associated with reactive oxygen species production and apoptosis via the mitochondrial pathway

    Withaferin A has been noted to inhibit both inducible and IL-6 induced STAT3 activation (STAT3 positively mediating cancer cell survival[269][270]) at 2-4μM in both estrogen responsive (MCF-7) and nonresponsive (MDA-MB-231) breast cancer cells up to 80% or greater;[271] the protein levels of STAT3 and an upstream regulator (JAK2[272]) were both concentration dependently reduced, suggesting an initial effect upstream of JAK signalling.[271] IC50 values for cell apoptosis over 24 hours have been noted to be lesser than 2μM elsewhere,[273] and associated with an increase in both the apoptotic (Bak, Bax, and Bim) and antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1) proteins, but mostly Bim secondary to activation of FOXO3a signalling.[274][273]

    STAT3 inhibition and FOXO3a signalling have both been partially implicated in the apoptotic effects of ashwagandha on breast cancer cells

    The Notch signalling pathway regulates a set of genes involved in cellular proliferation and differentiation[275] and its increased activity is involved in breast cancer tumorigenesis;[276][277] while it was once noted that withaferin A inhibited Notch-1 in colon cancer cells[278] it was later found to induce the activity of Notch2 and Notch4 (although it still managed to inhibit the activity of Notch1) in a manner not associated with estrogenic signalling; Withanone and Withanolide A were not effective.[279] It was thought that induction of presenilin1 and nicastrin mediated these effects on Notch proteins, and inhibiting Notch2 and Notch4 augmented the apoptotic and anti-proliferative properties of Withaferin A.[279]

    In MCF-7 and SUM159 breast cancer cells, Withaferin A appears to induce phosphorylation of the MAPKs (ERK, JNK, p38) with ERK being partially reliant on superoxide; despite that, further inhibiting ERK and p38 augments the apoptosis induced by Withaferin A while inhibiting JNK suppresses it and inducing myeloid cell leukemia-1 (Mcl-1) attenuates apoptosis from Withaferin A as well.[280]

    Despite its ability to induce apoptosis in cancer cells, Withaferin A appears to cause an increase in Notch signalling and in MAPK signalling; both of which slightly circumvent the apoptotic effects of Withaferin A and their inhibition (Notch2 and Notch4; ERK and p38) augments the apoptotic effects of Withaferin A

    It is thought that activation of CDKN1A-p21 is a critical mediator of apoptotic effects of an Ashwagandha ethanolic extract in MCF-7 cancer cells, since silencing four target genes of Ashwagandha (TPX2, ING1, TFAP2A and LHX3) all seem to reduce the activation of CDKN1A-p21 from Ashwagandha;[20] furthermore, p21 deficient cells are resistant to ashwagandha (whereas alterations in p53 do not influence its effects much).[20]

    The p21 pathway appears to be involved in the antiapoptotic effects of ashwagandha, and it seems to integrate signalling from various upstream anticancer molecular targets (TPX2, ING1, TFAP2A and LHX3)

    p53 induction from Withaferin A (2.5μM) appears to confer partial protection in estrogen responsive cancer cells via suppressing the expression of ERα.[237]

    The p53 pathway appears to be relevant in the anti-apoptotic effects of ashwagandha on cancer cells since it can suppress excessive estrogen signalling in cancer cells (Withaferin A) and Withanone can possible prevent nuclear accumulation of p53, increasing its anticancer signalling properties

    Injections of Withaferin A to female mice bearing MDA-MB-231 tumors (4mg/kg Withaferin A five times weekly) over 10 weeks noted a reduction in tumor size to nearly half that of control mice.[273]

    Oral ingestion of Ashwagandha root at 150mg/kg over the course of 155 days in female rats given a mammary carcinogen (metylnitrosourea) appears to be able to reduce tumor occurrence (23%) and size (21%).[281]

    Injections of pure Withaferin A appear to confer anti-tumor properties in mice, and this has been confirmed in rats given the basic root extract as an oral supplement as well

    15.6

    Neurological Cancers

    Ashwagandhanolide (thiowithanolide) has been found to have a 50% growth inhibitory concentration of 840ng/mL in SF-268 in glioblastoma tumor cells[38] while its sulfoxide dimer (rather than a basic sulfur bridge) is less effective with a GI50 of 1.78µg/mL.[39]

    15.7

    Stomach Cancer

    Ashwagandhanolide (thiowithanolide) has been found to have a 50% growth inhibitory concentration of 430ng/mL in AGS gastric tumor cells[38] and replacing the sulfur bridge with a sulfoxide group slightly attenuates the 50% inhibitory concentration to 730ng/mL.[39]

    15.8

    Prostate Cancer

    When testing various components of Ashwagandha, it was noted that a 50% ethanolic extract of the leaves was able to induce up to 88% toxicity in PC-3 prostatic tumor cells and 70% in DU-145 cells at a concentration of 100µg/mL; significantly more cytotoxic than the root or stem extracts.[282]

    In mice bearing PC-3 prostate tumors, injections of 4-8mg/kg Withaferin A is able to reduce tumor size by 54-70% associated with proteasome inhibition.[70]

    Injections of pure Withaferin A appear to reduce prostatic tumors in male mice

    15.9

    Lung Cancer

    In isolated lung cancer cells (NCI-H460), Withaferin A appears to inhibit growth with an GI50 (50% growth inhibition) of 140nM and induce cytotoxicity with a median lethal concentration (LC50) of 450nM;[32] the values being comparable to doxorubicin (GI50 of 70nM and LC50 of 860nM)[32] and elsewhere Ashwagandhanolide (thiowithanolide) has been found to have a 50% growth inhibitory concentration of 1.48µg/mL in these NCI-H460 lung cancer cells.[38]

    15.10

    Colon Cancer

    Ashwagandha leaf extract (50% ethanolic) has been noted to induce 27-98% inhibition of HCT-15 colonic cancer cells at 100µg/mL in vitro, a potency greater than that seen in the root or stem.[282] Ashwagandhanolide (thiowithanolide) has been found to have a 50% growth inhibitory concentration of 1.25µg/mL in HCT-116 colon tumor cells[38] hwile its sulfoxide variant is significantly less effective (3.59µg/mL).[39]

    15.11

    Renal Cancer

    Withaferin A (2-6μM) has been noted to induce oxidative stress in renal cancer cells secondary to the endoplasmic reticulum, causing an upregulation of REDD1 and CHOP resulting in mitchondrial-dependent apoptosis.[283]

    Withaferin A has been noted to inhibit STAT3 phosphorylation induced by IL-6 as well as constituatively active STAT3 in renal cancer cells (Caki) at 4μM Withaferin A, with no influence on constituative or induced STAT1 activity.[284] This is thought to be downstream of JAK2 inhibition (positively influences STAT3 activity at Tyr705 via phosphorylation[285]) which was also inhibited at the constituative and inducible level by Withaferin A.[284] This also occurred in breast cancer cells at the same concentration[284][271] and explained why IL-6 failed to promote growth of cancer cells in the presence of Withaferin A[271] and why STAT3 overexpression attenuates the apoptotic effects of Withaferin A.[284]

    Target downstream genes of STAT3 are those that promote survival of cancer cells (survivin, Bcl-xL, and Bcl-2),[286][287] and their reduction induces apoptosis in renal Caki cells.[284]

    The inhibition of the JAK-STAT pathway appears to be involved in the apoptotic effects of Withaferin A on renal cancer cells in vitro

    15.12

    Skin Cancer

    In isolated JB6 P+ epidermal cells, 625nM of Withaferin A was able to suppress the cancerous cell transformation induced by the tumor promoters 12-O-tetradecanoylphorbol 13-acetate (TPA) or UVC by around 84-90% in vitro.[288] This was associated with enhanced isocitrate dehydrogenase 1 (IDH1) activity at 1.25-5µM and a preservation of mitrochondrial dysfunction seen with TPA control;[288] mechanisms known to suppress the actions of tumor promoters in these cells.[289][290]

    The above was thought to apply when withaferin A was added topically (20µg) before TPA, since there was significantly less lactic acid production within cells.[288]

    Neoplasms from tumor promoters appear to be significantly suppressed with Withaferin A due to preserving mitochondrial function

    Withaferin A has been noted to cause apoptosis in melanoma cell lines (Lu1205, M14, Mel501 and SK28) with IC50 values of 1.8µM, 3.2µM, 6.1µM, and 5.9µM respectively; this apoptosis was associated with DNA fragmentation and reactive oxygen species formation causing apoptosis via the mitochondrial pathway, and it appeared to be secondary to downregulating Bcl-2.[291]

    15.13

    Pancreatic Cancer

    Withaferin A is able to exert antiproliferative effects in pancreatic cell lines Panc-1 (IC50 of 1.24µM), MiaPaCa2 (2.93µM), and BxPc3 (2.78µM) and induced apoptosis in PANC-1 cells with 1-10μM Withaferin A;[106] this was due to disrupting signalling from Hsp90 and causing its client proteins (the glucocorticoid receptor, Akt, and Cdk4) to degrade,[106] later found to be due to noncompetitive inhibition of Hsp90 with its co-chaperones.[107]

    Withaferin A inhibits Hsp90 function, and due to this it cannot support its client proteins of which one of them is Akt (a pro survival protein); loss of function of Hsp90 results in less Akt signalling amongst other things, and reduces the viability of cancer cells

    15.14

    Cervical Cancer

    Human papilloma virus (HPV) is known to express the oncoproteins E6 and E7, which reduce the activity of the tumor suppressor proteins p53 and pRb[292][293] and with less p53 to act upon p21CIP1/WAF1 there is less apoptosis induced secondary to less Bax induction.[294][295]

    Ashwagandha is able to induce apoptosis in cervical cancer cell lines CaSki (IC50 of 200nM), HeLa (IC50 of 450nM), SiHa (IC50 of 1.0μM) and C33a (IC50 of 1.2μM) associated with suppressing the aforementioned oncoproteins (and causing a relative increase in p53 activity and reduction in STAT3 phosphorylation).[296]

    Injections of 8mg/kg Withaferin A into mice bearing cervical cancer tumors for 6 weeks has resulted in a 70% reduction in tumor size relative to cancerous control.[296]

    15.15

    Immunological Cancers

    The 50% ethanolic extract of Ashwagandha root has shown apoptotic effects in isolated HL-60 cells with an IC50 value of 20μg/mL, with inhibitory actions on normal cells (hGF) with an IC50 of 600μg/mL; this apoptosis was associated with increases in reactive oxygen species acting upon the mitochondria to release caspases.[224]

    When fed to mice at the dose of 350mg/kg, the 50% ethanolic extract has been noted to inhibit 62% of the growth seen in Ehrlich Ascites Carcinoma cells over the next nine days of measurement. [224]

    15.16

    Sarcoma

    Sarcoma cells appear to be sensitive to Withaferin A, with various cell lines (PLS-1, STS26T, HT1080, and SKLMS1) having suppressed growth within two hours of incubation with IC50 values of 370-500nM associated with reduced anchorage of cells, while concentrations above 2.5µM were required for noncancerous cells.[63]

    Injections of Ashwagandha (50% ethanolic extract of the root) have been noted to reduce sarcoma tumor growth by 52% relative to tumor bearing control over nine days in mice.[224]

    16.

    Sexuality and Pregnancy

    16.1

    Libido

    Ashwagandha is sometimes used as an aphrodisiac, which may be vicariously through its 'adaptogenic' stress reduction (as chronic stress induces sexual dysfunction). One study using a moderate dose in mice (25-50mg/kg bodyweight for 21 days) noted both reductions in stress and decreases in the reductions in sexual activity induced by chronic stress; in a relatively dose-dependent manner.[172] Another study using 3g/kg in mice daily did note reductions in libido, hypothesized to be secondary to sedative effects of Ashwagandha and the larger dose used.[186]

    16.2

    Seminal Interactions

    When 5g of Withania Somnifera root powder is supplemented by otherwise infertile men, all measured seminal parameters (motility, anti-oxidation status, cell count, concentration, and volume) increase alongside biomarkers of nutritional status of semen (Vitamin C and fructose), although not to the levels of the fertile control group.[235] A later study with the same dose found universal improvements in all biomarkers of semen (lactate, citrate, glycerophosphocholine, etc.) associated with supplementation in infertile men, and confirmed an increase in seminal parameters such as motility and count.[297]

    This protective effect is currently thought to be secondary to promoting antioxidant enzymes and reducing oxidant stress,[298] as reactive oxygen species generation in sperm cells has been confirmed to be reduced with ashwagandha at this dosage.[235][298]

    One study investigating normozoospermic who were infertile, infertile and heavy smokers, or infertile and psychologically stressed noted improvements with 5g of the root powder daily for 3 months in regards to sperm count (17, 20 and 36%) and motility (9, 10 and 13%) alongside a decrease in semen liquefaction time (19, 20 and 34%);[236] there was no correlation with stress in regards to SOD or lipid peroxidation, but there was with catalase.[236]

    When supplemented by infertile men, ashwagandha appears to enhance all seminal parameters and is thought to enhance fertility secondary to this. This is currently thought to be due to enhancing the antioxidant status of the testicles and sperm cells

    16.3

    Erections

    One human study has been conducted on 'psychogenic erectile dysfunction' (lack of erections due to anxiety and fear of failure) in persons with DSM-IV confirmed Male Erectile Disorder Psychogenic type, and 2g of Ashwagandha root extract taken with meals for 60 days was ineffective in treating this condition.[299] Ashwagandha was associated with significant benefit on all measured parameters (International Index of Erectile Dysfunction), but placebo group matched this benefit.[299]

    17.

    Interactions with Aesthetics

    17.1

    Skin

    The pigmentation induced by Endothelin-1 in isolated melanocytes is blocked with Ashwagandha (10µg/mL) when incubated over 14 days in a manner that is independent of directly inhibiting tyrosinase but is upstream, inhibition of PKC activity which prevented tyrosinase upregulation and activation of MEK and Raf-1;[96] the calcium efflux from endothelin-1 was not prevented (suggesting that the mechanism was not upstream of PKC), and 10-50µM Withaferin A was able to replicate the observed effects[96] while elsewhere being shown to directly inhibit PKC by docking onto it (binding energy of -28.47kcal/mol) as well as withanone (binding energy of -22.57kcal/mol) with similar affinity to near the active site of PKC.[95]

    There may also be inhibitory effects on ERK phosphorylation in melanocytes stimulated with stem cell factor, which acts on its receptor (c-KIT) to induce melanogenesis via another pathway, the MAPK pathway;[300] ashwagandha (50% ethanolic extract at 10µg/mL) inhibits ERK phosphorylation from SCF in melanocytes[301]

    May possess a depigmentation property in skin cells, and both Withanone and Withaferin A have shown direct inhibitory properties on one of the proteins in the pigmentation signalling cascade (PKC) while ashwagandha itself inhibits ERK phosphorylation from SCF.

    17.2

    Hair

    It was mentioned in the discussion of one study[116] that a trial of older men in India (50-59 years) given 3g Ashwagandha daily for a year enabled a greater amount of hair melanin content to be preserved, which should theoretically preserve hair color to a degree. Mentioned in the text entitled Clinical applications of Ayurvedic and Chinese herbs: monographs for the western herbal practitioner, the study (Kuppurajan, K, et al; 1980) cannot be located online.

    18.

    Other Medical Conditions

    18.1

    Alzheimer's Disease

    Ashwagandha has failed to suppress amyloid precursor protein (APP) mRNA in mice, and β-secretase (which is an enzyme in the process of forming Aβ42 fibrils) is also unaffected following oral intake of an ashwagandha extract (75% withanolides and 20% withanosides) at 1,000mg/kg for 7-30 days.[302]

    Ashwagandha (water extract of the roots) at 6.25-50µg/mL appears to inhibit Aβ1-42 fibril formation, and this reduced fibril formation has been noted to reach maximal potency (50% inhibition) at 25µg/mL.[303]

    When looking at the formation of protein aggregates (fibrils), it appears to ashwagandha can slightly reduce their formation

    In PC12 cells incubated with preformed Aβ1-42 a concentration of 50-100µg/mL Ashwagandha water extract was required to reduce neuronal damage from the fibrils, but was sufficient to preserve neurons to 80% of control (Aβ1-42 being reduced to 30%);[304] this has been noted with isolated Withanoside IV and its metabolite sominone[162] and has been noted with oral ingestion of 10µmol/kg Withanoside IV in rats.[161]

    When looking at already formed fibrils, the damage they exert onto neurons appears to be attenuated in the presence of Askwagandha. This appears to be relevant to oral ingestion of isolated Withanoside IV

    When given to mice genetically induced with Alzhiemer's at the oral dose 1,000mg/kg of a highly purified extract (75% withanolides and 20% withanosides) for 7-30 days it can reduce Aβ42 buildup in the cortex (49-77%) and hippocampus (52-78%) with more efficacy in middle aged than older mice.[302] This study also noted reduction in brain Aβ40 and the plasma ratio of Aβ42/40 after 30 days, but since there was a spike in plasma Aβ42/40 within 7-14 days it signified Aβ42 efflux from the brain.[302]

    When looking at proteins involved in efflux, LDL-related protein (LRP) was increased in the endothelium but not brain while RAGE and clusterin were reduced; ApoE was unaffected.[302] The changes that were of highest magnitude and coincided with the Aβ42 efflux were hepatic levels of LRP (57-239%), soluble LRP in plasma (46-274%), and neprilysin (NEP; 16-45%) all of which showed time-dependent effects and occurred in normal mice without genetic predisposition to Alzheimer's or Aβ42 buildup;[302] sLRP normally yends to be decreased in Alzheimer's disease[305] and its reduction (and a relative increase in RAGE) is associated with progression of Alzheimer's disease,[306] but hepatic LRP seemed to be the major target since knockdown of this protein inhibited the effects of Ashwagandha.[302][307]

    Ashwagandha appears to induce the expression of LDL related protein (LRP), which then promotes efflux of the Aβ42 fibrils from the brain into plasma; this reduces Aβ42 buildup in the brain and preserves cognition in the mice with Alzheimer's, and appears to occur as a per se mechanism in mice

    18.2

    Parkinson's Disease

    In a paraquat induced rat model of Parkinson's disease (causes neurotoxicity with a similar histological and clinical profile as Parkinson's[308][309]) where ashwagandha at 100mg/kg was given alongside the paraquat for nine weeks, ashwagandha appears to partially attenuate the alterations in motor performance and neurological inflammatory biomarkers relative to control in a time-dependent manner.[310] Similar effects are also seen against rotenone, where 100-400mg/kg of ashwagandha is able to reduce the oxidative stress in the brain, although there was effectively a normalization in some parameters (ie. nitric oxide) with the higher dose,[311] and a third toxin thought to replicate Parkinson's (6-OHDA) in rats pretreated with 100-300mg/kg Ashwagandha root for 21 days was able to attenuate subsequent changes in motor performance, with 200mg/kg and 300mg/kg performing equally and to about half preservation.[138]

    When assessing the effects of ashwagandha (100mg/kg for 7-28 days) against MPTP toxicity in mice, there was a slight preservation in dopamine concentrations in the mouse striatum after a week which did not increase in potency after a month; DOPAC and homovanillic acid were unaffected, and this minor buffering effect correlated with the preservation in glutathione concentrations.[312] Elsewhere, MPTP toxicity followed up by a month of 100mg/kg ashwagandha root extract noted that supplementation was able to significantly preserve functional tests such as hang time and balance on a rotorod test which again correlated with improvements in oxidative status in the brain.[313] A preload of the leaf extract (100mg/kg) for a week prior to injections of MPTP has confirmed that this plant part also has partial attenuation of oxidative changes in the striatum and cortex and physical performance.[314]

    Ashwagandha (either pretreatment or rehabilitative treatment) appears to benefit rodent models of Parkinson's, although it appears that the maximal protective effect is around half preservation or rehabilitation; this suggests mild protective effects at doses that are relevant to oral supplementation

    18.3

    Huntington's Disease

    In a mouse model of Huntington's Disease (3-nitropropionic acid induced) supplementation of Ashwagandha at 100-200mg/kg for two weeks alongside the 3-NP toxin was able to dose-dependently attenuate alterations in motor function and body weight seen with 3-NP control, this was associated with reductions in lipid peroxidation and (at the higher dose) mild preservation of mitochondrial function.[315]

    Also appears to have protective effects against Huntington's disease, but the effects here are also apparently mild

    18.4

    Schizophrenia

    In a double blind pilot study, supplementation of ashwagandha (400mg thrice daily for one month) in schizophrenic patients who also suffered from metabolic syndrome noted that supplementation (alongside their medication) was able to reduce serum glucose and triglycerides without causing any adverse effects;[201] this study did not give a breakdown of the medications used.[201]

    18.5

    Systemic Lupus Erythematosus

    A water extract of the roots of Ashwagandha in a pristane-induced mouse model of systemic lupus erythematosus (SLE) at 500-1,000mg/kg for a month prior to induction of SLE and then continued for six months afterwards fully abolished inflammation in the peritoneal cavity (assessed by ascites nitrites and histological examination) and significantly attenuated serum biomarkers of inflammation.[316] A later study starting ashwagandha intervention a month after SLE induction and continuing for five months noted a significant attenuation of inflammation in ascites fluid as assessed by nitrite (93.7-96%) and IL-6 (81.5-90%) which outperformed the reference drug of Indomethacin (3mg/kg; 93% and 82% reductions) at the higher dose of ashwagandha.[317]

    There appears to be a significant reduction in inflammation in the rodent models of lupus (pristane induced), with notable potency towards inflammation in ascites and the peritoneal cavity. There are currently no other animal models or human studies assessing lupus, but this potential usage seems very promising

    19.

    Nutrient-Nutrient Interactions

    19.1

    Perment

    Perment is a poly-Ayurvedic formula consisting of equal parts (125mg each) of four herbs; Ashwagandha, Clitoria Ternatea, Bacopa Monnieri, and Asparagus Racemosus. It is apparently used clinically for its anti-depressive and anxiolytic effects, and is effective in rat models of chronic unpredictable manageable stress.[142] The combination of the four herbs shows synergism in this regard, but whether synergism exists between all four or just 2-3 herbs is not demonstrated.[142]

    20.

    Safety and Toxicity

    20.1

    General

    The acute LD50 for Ashwagandha extract (2% pure alkaloids) was found to be 465mg/kg in rats and 432mg/kg in mice.[318][36] Whereas other studies using alcohol extracts of Ashwagandha found LD50 values around 1750+/-41mg/kg, 1076 +/- 78 mg/kg and 1564 +/- 92 mg/kg for Ashwagandha, sitoindosides VII and VIII, and withaferin-A respectively[36][319] while a basic water extract of Ashwagandha at 2,000mg/kg to rats failed to exert any clinical or biochemical toxicity over the course of 28 days.[320]

    In vitro results suggest no toxicity to human blood cells (erythrocytes) with standard doses of the extract[321] although the dose of Withaferin A that is known to be toxic to healthy cells also appears to cause erythrocytic cell death.[199]

    In a subchronic toxicity study using a 10:1 ratio of Panax Ginseng to Ashwagandha taken at 8.50mg/kg, 12.75mg/kg, and 17.00mg/kg bodyweight in rats (4, 6, and 8 times the therapeutic dose) noted that despite increased food intake (70-80% in experimental, 41% in control) there was no abnormal serum parameter indicative of toxicological signs; liver weight increased, but it was uncertain as to whether this was due to the intervention per se or the increased food intake observed. An increase in haematopoesis was seen 1.7-1.9 gm% which led to an increase of 2-2.1million/cumm in RBC count.[322]

    20.2

    Case studies

    One confirmed case has established causation with ashwagandha at 5g oral ingestion daily for 10 days (a treatment dosage for libido) caused a burning/itching sensation on the penis mucous membrane, and slight discoloration and reddening of the head and prepuce.[323]

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