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.