Abstract Neurodegenerative diseases commonly induce irreversible destruction of central nervous system (CNS) neuronal networks, resulting in permanent functional impairments. Effective medications against neurodegenerative diseases are currently lacking. Ashwagandha (roots of Withania somnifera Dunal) is used in traditional Indian medicine (Ayurveda) for general debility, consumption, nervous exhaustion, insomnia, and loss of memory. In this review, we summarize various effects and mechanisms of Ashwagandha extracts and related compounds on in vitro and in vivo models of neurodegenerative diseases such as Alzheimer’s disease and spinal cord injury.

1. INTRODUCTION In neurodegenerative diseases such as Alzheimer’s disease, spinal cord injury, Parkinson’s disease, and Huntington’s disease, destruction of neuronal networks is a critical cause of the functional impairment. In neurodegenerative diseases, neuronal networks are believed not spontaneously regenerated by distinct causes; for example, amyloid β (Aβ) induces axonal atrophy in Alzheimer’s disease, and inhibitory proteoglycans inhibit regeneration of axons in spinal cord injury.1–10) Although several drugs are clinically used for the treatment of neurodegenerative diseases, most of these drugs are for symptomatic treatment; drugs for fundamental cure of neurodegenerative diseases are not clinically available and greatly needed. Ashwagandha (roots of Withania somnifera Dunal) is one of the most valuable herbal drugs used in Indian traditional medicine (Ayurveda) as a rasayana drug that is capable of imparting long life, youthful vigor, and good intellectual powers.11) Ashwagandha is clinically used for the treatment of general debility, consumption, nervous exhaustion, insomnia, loss of memory, and so on.12,13) These traditional uses imply that Ashwagandha may possibly be useful at improving neurodegenerative diseases. Indeed, this herbal drug has been reported to exert various pharmacological effects such as anti-inflammatory, anti-tumor, anti-oxidant, immunomodulatory, and anti-neuropsychiatric disease effects.14,15) In this review, we describe effects of Ashwagandha extracts, constituents of Ashwagandha (mainly withanolides: steroidal lactones with ergostane skeleton), and its derivatives in the context of neurodegenerative diseases.

2. ALZHEIMER’S DISEASE 2.1. Axon Regeneration and Synaptic Reconstruction In Alzheimer’s disease, the formation of Aβ deposits in the brain is widely accepted to be a critical cause of axonal atrophy and synaptic degeneration.1,2,16) We propose that promotion of axonal and synaptic regeneration may lead to reconstruction of neuronal networks and fundamental recovery from Alzheimer’s disease. Therefore the effects of methanol extracts of Ashwagandha on neurite outgrowth using an in vitro culture system were investigated; methanol extract of Ashwagandha showed neurite outgrowth-promoting activity in human neuroblastoma SK-N-SH cells.17) Withanolide A, withanoside IV, and withanoside VI (Fig. 1) were identified as active constituents from the methanol extract that induced neurite outgrowth in human neuroblastoma SH-SY5Y cells and rat cortical neurons.18,19) An in vitro axonal atrophy model was established using an active partial fragment of Aβ such as Aβ25–35.20) Aβ25–35 induced axonal atrophy as potently as full-length Aβ1–42.21) Withanolide A, withanoside IV, and withanoside VI were individually treated to the neurons displaying axonal atrophy. Each of these 3 compounds induced axonal growth even in the presence of Aβ25–35.21–23) Subsequently, an in vitro synaptic degeneration model was established. Rat cortical neurons formed synapses in vitro during 21-d culture. Thereafter, Aβ25–35 was treated to the neurons, resulting in losses of densities of presynapses and postsynapses.21) Nonetheless, post treatment with withanolide A, withanoside IV, or withanoside VI increased the synaptic densities. These 3 compounds were then tested in vivo. Aβ25–35 was intracerebroventricularly (i.c.v.) injected to mice brains. Densities of axons and synapses in the parietal cortex were reduced, and spatial memory of the mice was diminished by i.c.v. injection of Aβ25–35. Consecutive oral administration of withanolide A, withanoside IV, or withanoside VI for 12 d increased the densities of axons and synapses in the parietal cortex and improved spatial memory deficit. Fig. 1. Structures of Constituents of Ashwagandha and Derivatives from Withanoside IV Withanoside IV conjugates 2 glucoses at position C3. It was reported that, after oral administration, several glycosides of natural products were deglycosylated by human intestinal bacteria and subsequently absorbed in the blood.24,25) Therefore orally administered withanoside IV was speculated deglycosylated at position C3 by the intestinal bacteria. After oral administration of withanoside IV to mice, serum was collected and analyzed by liquid chromatography/mass spectrometry.22) As a result, withanoside IV itself was not detected in the serum whereas sominone, an aglycon of withanoside IV, was detected (Fig. 1). Sominone induced axonal regeneration and synaptic reconstruction in Aβ25–35-treated cortical neurons in vitro. In Alzheimer’s disease model 5XFAD mice,26) a single intraperitoneal (i.p.) administration of sominone increased axonal density in the brain and improved object recognition memory impairment.27) These results suggest that withanoside IV is metabolized to its active principle sominone after oral administration, which subsequently induces marked recovery of neurites, synapses, and memory. These data suggest that withanoside IV and its metabolite sominone are potential drugs against Alzheimer’s disease. In normal adult mice, a single i.p. administration of sominone increased axonal density in the brain and enhanced object location memory.28) Rearranged during transfection (RET) phosphorylation was increased in the brain by sominone-treatment. RET is a part of the receptor complex for glial cell-line-derived neurotrophic factor (GDNF) and is phosphorylated by GDNF stimulation.29,30) Experimental knockdown of RET inhibited sominone-induced axonal growth in cultured cortical neurons.28) GDNF secretion was not influenced by treatment with sominone in cultured cortical neurons. These results indicate that sominone activates RET and induces axonal growth, possibly leading to memory enhancement. Other groups have reported effects of withanolide A on Alzheimer’s disease experimental models. Patil et al.31) reported that withanolide A decreased beta-site amyloid precursor protein cleaving enzyme 1 (BACE1; known as β-secretase) expression and increased a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10; known as α-secretase) expression in cultured normal rat cortical neurons. Aβ is produced from amyloid precursor protein (APP) by processing with BACE1 and presenilin 1 (PS1; known as γ-secretase).32) When APP is processed by ADAM10, Aβ production is attenuated whereas non-toxic soluble APPα is alternatively produced. As a result, withanolide A increased soluble APPα production in the cultured neurons. It was also shown that withanolide A increased expression levels of insulin-degrading enzyme (IDE), a major proteolytic enzyme involved in Aβ degradation. These results suggest that withanolide A possibly reduces Aβ by increasing soluble APPα production and Aβ clearance. Considering these reports, withanolide A is an important candidate as a multifunctional drug against Alzheimer’s disease. 2.2. Neuroprotective Effects Kurapati et al.33) investigated effects of methanol–chloroform (3 : 1) extract of Ashwagandha against Aβ1–42-induced toxicity in cultured human neuroblastoma SK-N-MC cells. Aβ1–42 induced cell death and internalization of Aβ1–42. Simultaneous treatment with the extract and Aβ1–42 inhibited the above phenomena induced by Aβ1–42. Expression level of peroxisome proliferator-activated receptor-γ (PPARγ) was decreased by Aβ1–42 and reversed by simultaneous treatment with the extract. The authors indicated that upregulation of PPARγ by the extract supports neuroprotective effects against Aβ, but a causal relation between PPARγ expression and neuroprotective effects was not clarified. Effects of the alcoholic extract of Ashwagandha leaves (i-Extract)34) on scopolamine-induced cell damages were investigated by Konar et al.35) Scopolamine is a muscarinic receptor antagonist that induces amnesia in rodents.36) Scopolamine also influences expression of genes related to muscarinic receptor signaling pathways, apoptosis, and cell differentiation in the rat brain.37) Konar et al. showed that scopolamine-treatment induced cell deaths in cultured human neuroblastoma IMR32 cells and cultured rat glioma C6 cells, whereas i-Extract-treatment before scopolamine-treatment protected cells from death.35) Scopolamine treatment induced DNA damage and oxidative stress in C6 cells, whereas i-Extract treatment prevented those. Withaferin A and withanone (Fig. 1) were focused as major constituents in i-Extract. Although withanone showed preventive effects comparable to i-Extract, withaferin A did not. The authors concluded that withanone is a predominant active constituent in i-Extract that protects cells against scopolamine-induced damage probably via its anti-oxidative effects. 2.3. Clearance of Aβ Sehgal et al.38) reported the effects of an authenticated Ashwagandha product serially extracted with chloroform-methanol (Arya Vaidya Sala, Kottakkal, India). In APP/PS1 Alzheimer’s disease model transgenic mice,39) consecutive oral administration of the Ashwagandha extract for 30 d reversed behavioral memory deficits in the radial arm task and decreased Aβ level in the cerebral cortex and hippocampus but increased that in the blood plasma. Low-density lipoprotein receptor-related protein (LRP) in the liver and soluble form of LRP (sLRP) in plasma were upregulated by administration of the Ashwagandha extract. LRP mediates efflux of Aβ from the brain into the periphery.40,41) LRP in the liver is cleaved and released into the blood, where it remains present as sLRP.42) Liver-specific knockdown of LRP blocked both sLRP and Aβ increment in plasma and Aβ reduction in the brain after administration of the extract.38) Therefore the Ashwagandha extract promotes Aβ clearance in the brain via upregulation of liver LRP.

3. SPINAL CORD INJURY In the injured region of the spinal cord, astrocytes are increased and form a glial scar where inhibitory proteoglycans are secreted from the astrocytes. Such deposit of inhibitory proteoglycans interferes with axonal regeneration.5,8–10) Methylprednisolone sodium succinate is the only available medication for acute spinal cord injury; however, this drug is now less widely used because of very moderate efficacy coupled with recognized side effects. We attempted to induce axonal growth in injured spinal cord, which could lead to functional recovery. In our study, withanoside IV showed activity to induce axonal growth in the degenerated central nervous system.22) Therefore the effects of withanoside IV on spinal cord-injured mice were investigated.43) One hour after contusion injury at the L1 spinal cord, consecutive oral administration of withanoside IV was started. Administration of withanoside IV for 21 d induced axonal growth in the spinal cord and recovered hindlimb motor function. After oral administration, withanoside IV is metabolized into sominone in the intestine as described above, and sominone is thought an active principle for axonal growth activity.22) We synthesized a novel compound “denosomin” that is an analogous derivative of sominone44) (Fig. 1). In cultured cortical neurons, denosomin showed axonal growth activity comparably to sominone. Synthesis of denosomin is easier and more efficient than that of sominone. Therefore we investigated the effects of denosomin on spinal cord-injured mice.45) One hour after contusion injury at the L1 spinal cord, consecutive oral administration of denosomin was started. Administration of denosomin for 14 d recovered hindlimb motor function. At that time, axonal growth was facilitated by administration in the injured center; additionally glial fibrillary acidic protein-positive reactive astrocytes also increased in the injured center. In astrocyte pure culture, denosomin increased astrocytic proliferation and inhibited H 2 O 2 -induced astrocytic death. Conditioned medium isolated from denosomin-treated astrocytes induced axonal growth in cultured cortical cells; nonetheless the conditioned medium did not contain denosomin. We identified that vimentin, an intermediate filament protein, was increased in cultured astrocytes after denosomin-treatment, and denosomin increased secretion of vimentin from cultured astrocytes. Although functions of the secreted vimentin on neurons had not been identified, an effect of extracellular vimentin on axonal growth was confirmed in cultured cortical cells. Also in vivo, denosomin-administration increased vimentin-positive astrocytes but not inhibitory proteoglycan-positive ones in the injured region of the spinal cord-injured mice. These results indicate that denosomin increases vimentin-secreting astrocytes in the injured region, leading to axonal growth and recovery of motor function in spinal cord-injured mice. In spinal cord injury, astrocytes in the glial scar were thought a major impediment to axonal regrowth.5,8–10) However, upregulation and secretion of vimentin converts this burden into a force for functional recovery from spinal cord injury.

4. SAFETY OF ASHWAGANDHA AND ITS CONSTITUENTS Considering activities against neurodegenerative diseases, clinical application of Ashwagandha and its constituents is expected to improve such diseases. Some groups have previously reported safety of Ashwagandha and its constituents. Administration of water extract of Ashwagandha (100 mg/kg/d) with drinking water for 8 months did not show any toxicity in rats.46) Consecutive oral administration of 80% methanol extract of Ashwagandha (2000 mg/kg/d for 28 d) also showed no toxicity.47) However, oral administration of the alcoholic extract from defatted seeds of Ashwagandha induced acute toxicity in mice: LD 50 was 1750±41 mg/kg.48) Following i.p. administration of an ethanol extract of Ashwagandha, LD 50 was 1259 mg/kg in mice.49) Although withanolide A, withanoside IV, and withanoside VI showed neurite outgrowth activity at 1 µM, withaferin A and withanolide D (Fig. 1) showed neurotoxicity at 1 µM in cultured SH-SY5Y cells18,19) and normal cortical neurons.23) Withaferin A and withanolide D are known to elicit anti-tumor effects.50–52) Other reports also showed that withaferin A revealed cytotoxic activities in normal and cancer cells53) and did not show anti-Alzheimer’s disease effects.18,19,23,35) When i.p. administered, LD 50 of withaferin A in mice was approximately 80 mg/kg.54) Considering that Ashwagandha has been used in Ayurveda since ancient times, traditional use of Ashwagandha including some detoxification methods might show no severe toxicity. However, considering the toxicity of several of its constituents, toxic compounds contents in Ashwagandha should be taken into account particularly when used in high doses.

5. CONCLUSION Ashwagandha and its constituents showed various activities against models of Alzheimer’s disease and spinal cord injury. Ashwagandha extracts also showed ameliorative effects against other neurodegenerative disease models such as Parkinson’s disease and Huntington’s disease,55–60) suggesting that Ashwagandha may be useful against various neurodegenerative diseases (Table 1). Additionally, a novel drug candidate for spinal cord injury was synthesized as a derivative of constituents of Ashwagandha. These results suggest that Ashwagandha is a potential basis for novel drugs against neurodegenerative diseases. In addition, pharmacological analyses of Ashwagandha extracts and related compounds gave us novel insights for the treatment of neurodegenerative diseases; for instance, modulation of astrocyte properties can lead to recovery form functional impairment in spinal cord injury, and peripheral organ-targeted effects can lead to clearance of Aβ in the brain. However, direct target molecules of Ashwagandha-related compounds have not been identified as yet. Clarification of these issues may lead to identification of novel targets for therapy of neurodegenerative diseases. Further studies of Ashwagandha will probably contribute to resolving an urgent unmet medical need: efficacious treatments that may offer cure of neurodegenerative diseases. Disease models Materials Functions Alzheimer’s disease/Dementia Extract Neurite outgrowth in vitro17) Memory improvement38) Neuroprotective effects in vitro33,35) Aβ clearance in vivo38) Withanolide A Neurite outgrowth in vitro18,19) Axonal regeneration and synaptic reconstruction in vitro and in vivo21) Memory improvement21) Upregulation of BACE1, ADAM10 and IDE in vitro31) Withanoside IV Neurite outgrowth in vitro18,19) Axonal regeneration and synaptic reconstruction in vitro and in vivo22) Memory improvement22) Withanoside VI Neurite outgrowth in vitro18,19) Axonal regeneration and synaptic reconstruction in vitro and in vivo23) Memory improvement23) Sominone Axonal regeneration and synaptic reconstruction in vitro22) Axonal growth in vivo28) Memory improvement27) and memory enhancement28) Denosomin Axonal regeneration and neuroprotective effect in vitro45) Withanone Neuroprotective effects in vitro35) Spinal cord injury Withanoside IV Axonal growth in vivo and functional recovery43) Denosomin Axonal growth in vivo and functional recovery45) Parkinson’s disease Extract Anti-oxidant effect in vivo55–59) Neuroprotective effect in vivo55,59) Functional recovery55–59) Huntington’s disease Extract Anti-oxidant effect in vivo60) Functional recovery60) Table 1. Effects of Ashwagandha Extracts, Constituents, and Derivatives on Neurodegenerative Diseases

Acknowledgment We thank Dr. M. Hattori, Dr. K. Zou, Dr. M. R. Meselhy, Dr. N. Nakamura, Dr. J. Zhao, Ms. N. Nakayama, Ms. E. Joyashiki, Dr. K. Teshigawara, Ms. M. Shigyo, and colleagues of the Institute of Natural Medicine, University of Toyama and Dr. Y. Matsuya, Dr. H. Nemoto and colleagues of Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama for their extensive contributions to this study. Author’s works were partially funded by the Grants-in-Aid for Scientific Research (C) (23500439, C.T.), Young Scientists (B) (23700376, T.K.) and the Expansion Program, Regional Innovation Cluster Program, Global Type (II) (C.T. and K.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Natural Medicine and Biotechnology Research and Toyama prefecture, Japan (T.K., C.T.).

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