Neuropharmacological Properties of Withania ...

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Neuropharmacological Properties of Withania somnifera – Indian Ginseng: An Overview on Experimental Evidence with Emphasis on Clinical Trials and Patents Sarat C. Yenesetti1, M. J. Manjunath2 and Muralidhara1,* 1

Drosophila Neurobiology Laboratory, Department of Zoology, Nagaland University (Central), Lumami, 798627, Nagaland, India; 2Department of Biochemistry & Nutrition, CSIR-Central Food Technological Research Institute, Mysore, 570 020 Karnataka, India Abstract: Background: Owing to the increasing aged population globally, disorders and diseases of the CNS are anticipated to increase and profoundly impact the health care. As these neurodegenerative diseases (NDD) are complex, multifactorial and do not have identified etiological factors, unfortunately, drugs developed for the purpose have not met with the expected success. Hence, there has been a constant demand for the development of natural therapeutic adjuvants which are safe and possess the potential to attenuate multiple pathways.

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Methods: Numerous herbal/natural products have been used as therapeutics in Ayurvedic system of medicine to treat NDD and other memory-related disorders. Withania somnifera (Ashwagandha, WS), popularly called as “Indian Ginseng” is one such plant which possesses a variety of beneficial neuropharmacological properties. In this review, we have attempted to review critically the existing literature and patents related to the neuroprotective efficacy of WS roots and the underlying mechanism/s. Results: Standardized extracts of Withania somnifera (WS) have been demonstrated to possess multidimensional neuromodulatory effects both in vitro and animal models. The spectrum of effects evidenced comprises of attenuation of oxidative damage by enhancing the antioxidant defense system with concomitant enhancement of the expression of marker proteins responsible for growth, differentiation and communication of neural cells. Specific effects of WS are attributable to its potential to modulate neurotrophic factors, cytoskeletal elements, cell adhesion molecules and synaptic proteins. Conclusion: Generation of new data by employing systematic contemporary approaches such as bioinformatics, molecular docking studies, identification of specific gene targets and epigenetic regulation would provide the necessary impetus to validate fully the neurotherapeutic potential of the phytochemicals derived from WS. More importantly, well-designed clinical trials are required to exploit the neuromodulatory propensity of WS extract/bioactives in specific neurodegenerative diseases such as AD and PD.

Keywords: Withania somnifera, neuroprotection, animal models, clinical trials, patents. Received: February 4, 2015

Revised: April 7, 2016

1. INTRODUCTION The World Health Organization (WHO) has estimated that the proportion of elderly people (over 60 years) will increase from 11% to 22% during the next 40 years throughout the world. Consequently, morbidity and mortality rates of age-related diseases such as neurodegenerative diseases (NDD) are sure to increase [1, 2]. Globally, central nervous system (CNS) disorders /diseases will have a greater impact on the healthcare concerns of the human population. Various neurological and psychiatric disorders are generally associated with loss of memory, cognitive deficits, impaired mental function etc. Drugs based on modern medicine have met *Address correspondence to this author at the DBT-Visiting Research Professor, Drosophila Neurobiology laboratory, Department of Zoology, Nagaland University (Central), Lumami, 798627, Nagaland, India; Tel:/Fax: ??????????????????????????; E-mails: [email protected]; [email protected] 1574-8898/15 $100.00+.00

Accepted: June 9, 2016

with limited success owing to the multi-factorial nature of these diseases. Accordingly, there exists a constant growing demand for developing novel therapeutic products which can target multiple pathways either independently or in combination with conventional drugs. In this regard, currently usage of herbal products based on traditional knowledge are on the increase both in developed and developing countries. “Ayurveda,” the renowned Indian traditional system of medicine which is more than 5000 years old has described several herbs and natural products for a variety of CNS diseases and therapy. It is well accepted and appreciated that the “Ayurveda-based drug discovery is promising and comparatively less time consuming since the drug candidates are first identified based on large-scale use in the population and then validated in clinical trials” [3-5]. Ayurveda has classified selected plants as “Medha rasayanas” (Medha means intellect and Rasayanas mean herbal preparation which promotes a youthful state of physical/mental health) that are specifi© 2015 Bentham Science Publishers

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cally beneficial in boosting memory, preventing cognitive deficits and improving brain function [4, 5]. Large number plant products have been employed in herbal/ traditional medicine since they offer significant therapeutic benefits in CNS disorders compared to pharmaceutical drugs. Withania somnifera (WS) Dunal belongs to the family, Solanaceae, Common name- Ashwagandha is one of the most reputed medicinal plants of Ayurveda. It is a traditional Indian medical plant extensively used for over 5000 years [6]. The medical practitioners of the traditional system of medicine in India regard WS as the “Indian Ginseng” [7]. In India, it is extensively cultivated in the regions of Madhya Pradesh, Uttar Pradesh, Gujarat, Rajasthan, and plains of Punjab [8]. 2. GENERAL PHARMACOLOGICAL PROPERTIES OF WS In recent times, the biological activities of WS and its bioactives have been the subject of several reviews [9-12]. WS products have been extensively used as an antioxidant, liver tonic, adaptogen, anti-inflammatory agent, astringent and also to treat ulcers, bacterial infections, venom toxins, senile dementia etc. Additionally, evidences obtained both in animal models and clinical trials have strongly supported the use of WS for anxiety, cognitive/neurological disorders, inflammation, hyperlipidemia and neurodegenerative diseases such as AD and PD. Various parts of the plant (flowers, leaves and roots) have been in use for centuries to treat a variety of human ailments and constantly a variety of newer biological activities are being ascribed. The roots of the plant categorized as

Medha Rasayanas are believed to promote general health and extend longevity by augmenting defense against various diseases, alleviate the process of aging, revitalize the body in debilitating situations, enhance the capacity of individual by rendering them less susceptible to environmental agents. 3. MAJOR BIOACTIVES OF WS The major biologically active constituents of WS are alkaloids (isopelletierine and anaferine), saponins (sitoindosides) and the C28 steroidal lactones - withanolides (steroidal lactones with ergostane skeleton) [13-14]. Withanolides are a group of naturally occurring C28 steroids comprised of an ergostane skeleton in which C-26 and C-22, or C-26 and C-23 which are oxidized to form a - or -lactone. Various alkaloids reported in WS include withanine, somniferine, somnine, somniferinine, withananine, pseudowithanine, tropine, pseudotropine, 3--gloyloxytropane, choline, cuscohygrine, isopelletierine, anaferine and anhydride. Two acyl stearyl glucoside viz. sitoindoside VII and sitoindoside VIII, two glycowithanoloids viz. sitoindoside IX or sitoindoside X have been isolated from the root [15] (Fig. 1). 4. SAFETY OF WS: EVIDENCE IN ANIMAL MODELS A significant amount of data regarding WS properties has come from established traditional knowledge that existed for thousands of years. Hence, there is inherent safety built into the usage of WS in humans. WS is generally considered to be safe and the acute LD50 value of ashwagandholine (total alkaloids from WS roots) reported in rats and mice respec-

Fig. (1). Some of the bioactive molecules and their chemical structures isolated from Withania somnifera. (A) Withaferin A, (B) Withanone, (C) Withanolide A, (D) Withanolide D, (E) Withanolide E, (F) Withanolide G, (G) Withanolide Q, (H) Withanolide R, (I) Withanolide P.

Neuropharmacological Properties of Withania Somnifera

Recent Patents on CNS Drug Discovery, 2015, Vol. 10, No. 2 3

Fig. (2). Schematic diagram showing speculated mechanisms responsible for the Therapeutic efficacy of Withania sommifera under various Neurological disorders.

tively are: 465mg/kg b.w (confidential limits: 332-651mg/kg b.w) and 432mg/kg (confidential limits, 229-626mg/kg) [16]. In a sub-acute study in rats, WS had no effect on various parameters such as food consumption, body weight and it caused no significant perturbations in various hematological, biochemical and histopathological parameters in vital organs [17]. 5. WS AND BRAIN HEALTH Traditionally, WS has been largely used as an adaptogenic, anti-stress and nervine tonic for the treatment of general debility, nervous exhaustion, insomnia and memory dysfunctions. Further, it is also recommended as a natural drug for neurological disorders like traumatic brain injuries, brain tumors and neurodegenerative pathologies [18-22]. 5.1. Protective efficacy of WS in Neurodegenerative Disease (NDD) Models A variety of WS preparations have been extensively evaluated for their potential therapeutic effects in several CNS related disorders. Experimental evidence clearly suggests that WS possess significant modulatory effect on GABAergic, cholinergic and oxidative systems. The spectrums of neuroprotection evidenced in various NDD models are briefly described below: 5.1.1. Anxiety WS has been extensively used to reduce symptoms of anxiety and stress [23]. Administration of WS (20-50 mg/kgbw; oral, once daily for 5d) was reported to cause significant anxiolytic effect [24]. A polyherbal formulation containing WS (NR-ANX-C) significantly reversed ethanolwithdrawal induced anxiety behavior in elevated plus maze (increased open arm time and/or open arm entries) and bright/dark arena paradigm tests [25].

5.1.2. Epilepsy and Related Models The anticonvulsant properties of WS have been demonstrated in both acute and chronic models of epilepsy [26]. The root extract of WS exhibited significant antiepileptic activity against pentylenetetrazol (PTZ)-induced kindling in mice, amygdaloid kindling in rats and in status epilepticus in rats [27]. The protective efficacies of WS in mice were speculated to be mediated through specific interaction with GABA- gated chloride ionophore [28]. A recent study has provided direct evidence for GABAergic activity of WS on mammalian ionotropic GABAA and GABA receptors [29]. 5.1.3. Nootropic and Behavioral Effects Withanolides were shown to significantly inhibit the activities of both acetylcholinesterase (AChE) and butryl cholinesterase (BChE) enzymes by possible modulation of muscarinic acetylcholine receptors thus regulating the cognitive functions [30]. Mice provided with supplements of methanolic extract of WS exhibited improved avoidance activity and spontaneous locomotor response in a‘T-Maze’ test [31]. Supplementation of WS also showed protective effects in attenuating diabetes-induced memory. Repeated oral administration of WS root extract (100-200 mg/kg, 14 d) also improved the 3-NPA induced cognitive deficits and restored the activity levels of AChE in cortex, hippocampal and striatum [32]. Further, WS supplements mitigated the memory impairment by prolonging the acquisition as well as retention transfer latency in propoxur-treated rats [33]. WS root extract exhibited therapeutic efficacy against protracted social isolation-induced behavior, orofacial dyskinesia and cognitive dysfunction induced by reserpine in rats [34]. 5.1.4. Cerebral Ischemia Employing a mouse model of permanent distal middle cerebral artery occlusion (pMCAO), recently, the prophylactic, as well as a therapeutic efficacy of WS extract, was dem-

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onstrated wherein WS (200 mg/kg) improved the functional recovery and significantly reduced the infarct volume in mice[35]. WS significantly up-regulated the expression of hemeoxygenase 1 (HO1), and attenuated the expression of the pro-apoptotic protein poly (ADP-ribose) polymerase-1 (PARP1) via the PARP1-AIF pathway, thus preventing the nuclear translocation of apoptosis-inducing factor (AIF) and subsequent apoptosis [35]. Pre-supplementation with WS (300 mg/kgbw, 30 d) to MCAO animals was also effective in restoring the AChE activity, lipid peroxidation, thiols and behavioural deficits [36]. WS reduced the cerebral infarct volume and ameliorated histopathological alterations. 5.1.5. Neuronal Outgrowth Bioactives of WS possess a wide range of actions in CNS, as evidenced by increased neural survival, extension of neurite growth and rescuing neurons from death due to various insults. In animal models, treatment with Withanoside IV rescued neuronal death by promoting axonal growth, increased peripheral myelin level and facilitated locomotion in spinal cord injured mice [37]. Significant regeneration of both axons and dendrites, in addition to the reconstruction of pre- and postsynapses in the neurons was demonstrated with withanolide A treatment [38]. Withanolide-A induced significant regeneration of both axons and dendrites in addition to the reconstruction of presynaptic densities and postsynaptic densities in the neurons. In human neuroblastoma SK-N-SH cells, methanolic extract of WS promoted neurite elongation [39]. Withanosides significantly improved memory deficits in amyloid-injected mice and prevented loss of axons, dendrites and synapses in the cortex and hippocampal brain regions [40]. In a recent study, sominone (an aglycone moiety of withanoside IV) was shown to reinforce the plasticity of neurons in axons/dendrites by activation of the RET pathway and enhance spatial memory in rats [41]. 5.1.6. Anti-Stress and Adaptogenic Action WS is extensively used as an anti-stress medication. In a chronic fatigue syndrome model (swim stress model), pretreatment of mice with WS (100 mg/kg bw, oral) caused a significant decrease in immobility time as compared to control stressed animals [42]. Interestingly, WS significantly reversed the extent of lipid peroxidation induced by chronic fatigue and the antistress activity was ascribed to the antioxidant property [43, 44]. Pretreatment with WS glycowithanolides produced a dose-related reversal of the stress effects and chronic foot shock-induced stress model [45]. 6. MECHANISM-BASED APPROACHES WITH WS In vitro studies: The anticonvulsant activities of WS extract have been attributed to be mediated through its specific effects on GABA receptor system [46]. WS extract inhibited the hydrogen peroxide (H2O2) -induced cytotoxicity and DNA damage in human non-immortalized fibroblasts [47]. The potential of WS extracts / Withanolides to promote dendrite formation has been adequately demonstrated in neuroblastoma cells [48]. Withanolides obtained from WS leaves have shown inhibition of cyclooxygenases and proliferation of tumor cells in vitro [49]. Withanoside IV facilitated axonal and dendritic outgrowth in cultures of rat cortical neurons [50]. Further, studies using PC 12 cells have demon-

strated that MAK-5 (a herbal formulation containing WS) enhanced the NGF-induced neural differentiation [51]. Withanolides exhibited calcium antagonistic properties and inhibited the activities of both AChE and BChE in a concentration-dependent fashion [30]. Recent studies have shown that an aqueous extract of WS offsets H2O2 and A (1-42) induced cytotoxicity in PC12 cells in a concentrationdependent manner [52-53]. In vivo evidences: The active principles of WS such as, sitoindosides VII–X and Withaferin A, have been extensively tested for antioxidant activity in vivo. Active glycowithanolides of WS (10-20 mg/kg b.w, i.p.) administered to rats once daily for 21 days, enhanced the activity levels of various antioxidant enzymes in frontal cortex and striatum [54]. Withanolides also increased the activity levels of catalase, glutathione peroxidase activities in rat cortex and striatum. The propensity of WS to inhibit cholinesterase activity is speculated to be chiefly responsible for its clinical use in AD and associated problems [55]. Evidence of their capacity to modulate dopaminergic system in the brain have been suggested to be responsible for the efficacy of WS in treating PD in humans [56]. Employing an immobilization stress model of rat pretreatment with root extract of WS (Stresscom® capsules) was shown to significantly reduce the number of degenerating cells in CA(2) and CA(3) subareas of hippocampal region [57]. 7. NEUROPROTECTIVE PROPERTIES IN EXPERIMENTAL AD AND PD MODELS 7.1. AD Models Substantial evidence exists on the neuroregenerative efficacy of WS root extracts in A-induced in vitro and mouse model of AD [40, 49, 58, 59]. The potential of WS extracts as well as the bioactives viz., Withanolide A, Withanoside IV and Withanoside VI to stimulate axonal growth, reconstruction of synapses, synaptic connectivity have been adequately demonstrated both in cell models and in vivo (Table 1). Interestingly, a single injection of sominone increased axonal density and improved object recognition memory in AD mice, but also stimulated neurite growth and enhanced memory function in normal mice [61]. In a mice AD model, chronic oral treatment with WS extract resulted in reduced cortical and hippocampal levels of A. A recent study speculated that the specific effect on A clearance may be due to upregulation of low-density lipoprotein receptor related protein (LRP) in liver and soluble form of LRP (sLRP) in plasma [62]. The other molecular effects of Ws extracts in AD models are presented in Table 2. 7.2. PD Models WS root extracts have been shown to protect significantly neuronal injury in vitro cell models and animal models of PD [64]. The beneficial effects of WS extract were adequately demonstrated in two neurotoxin models viz., 6OHDA and MPTP (Table 2). Studies from our laboratory have shown the efficacy of a standardized WS extract – enrichment to attenuate locomotor phenotype, enhance survival and mitigate rotenone-induced oxidative stress, mitochondrial dysfunction and neurotoxicity in the Drosophila


Table 1.


Effect of WS bioactives in various in vitro AD cell models (A 25-35 induced).

Withanolide A, Withanoside IV and Withanoside VI – Model -Human neuroblastoma cells Stimulated axonal growth) [48]. Withanolide A, Withanoside IV and Withanoside VI : Model Primary rat cortical neurons Resconstruction of Synapses [49]. Withanolide A: Model - Primary rat cortical neurons Reduced the expression of BACE1; b-secretase ; elevates disintegrin and ;Metalloproteinase domain-containing protein 10 ; Increased expression of IDE levels [60]. Sominone,: Model -Primary rat cortical neurons: Activates GDNF and stimulated axonal growth [41]. Methanolic root extract of WS (Model -human neuroblastoma SK-N-MC cells) internalization of Ab1-42 was markedly attenuated[58]. Aqueous root extract of WS (Model -PC12 cells): Protected against H2O2 & Ab91-42) induced cytotoxicity [53].

Table 2.

Neuroprotective effect of WS bioactives in AD nd PD models of neurodegeneration.

AD model Oral administration of withanolide A, withanoside IV or VI increased axonal growth and synaptic connectivity in cerebral cortex of A25e35 induced AD mice [40-41]. Single injection of Sominone increased axonal density and improved object recognition memory in 5XFAD mice; stimulated neurite growth and enhanced memory function in normal mice [41]. Prolonged oral administration of the methanolic extract for 30 days restored memory impairment assessed by radial arm maze test in transgenic mice; reduced cortical and hippocampal levels of Ab but increased the same in blood plasma. PD model WS extract (6-OHDA rodent model): prevented alterations in antioxidant enzyme activities, catecholamine content, dopaminergic D2 receptor binding and TH expression [56]. Ethanolic root extract (model: Maneb and Paraquat Mice): Reduced the level of dopamine in the substantia nigra and attenuated oxidative damage; reduced the expression of iNOS,; reduced the expression of pro-apoptotic markers Bax and Bcl- 2, and pro-inflammatory marker of astrocyte activation, GFAP [63]. Ethanolic root extract (MPTP mice model): increased DA, DOPAC and HVA levels ; normalized TBARS levels in the corpus striatum; showed improved motor function ; increased the level of GSH and GPx in the striatum; catecholamines levels, oxidative damage and physiological abnormalities [63].

system [65]. Interestingly, our recent findings in a rotenone model of PD revealed that oral supplements of standardized WS extract, besides modulating the endogenous levels of oxidative markers in the brain regions of mice, rendered the brain less susceptible to neurotoxicants as evidenced by significant attenuation of toxicant -mediated oxidative stress, mitochondrial dysfunction and neurotoxicity [66]. 7.3. Spinal Cord Injury Oral administration of withanoside IV for several weeks induced axonal growth and recovered motor function in spinal cord injury mouse model [67]. A synthetic compound, denosomin derived from sominone also facilitated axonal growth in spinal cord injured mice. Densomin was found to induce secretion of vimentin, an intermediate filament protein, from astrocytes in cell models clearly suggesting that

denosomin increased the vimentin-secreting astrocytes in the injured region, leading to axonal growth and recovery of motor function in spinal cord-injured mice [68]. 7.4. Hypoxia WS root extracts orally administered to hypobaric hypoxic rats exhibited neuroprotection and markedly ameliorated memory impairment. The recovery potential was associated with a decrease in the level of nitric oxide, corticosterone, oxidative stress and AChE activity in rat hippocampus [69]. Further studies suggested that Withanolide A contributed during recovery against hypoxia-induced memory impairment. Supplementation of Withanolide A during hypoxic exposure increased glutathione levels, augmented GSH dependent free radicals scavenging system and decreased the number of apoptotic cells in the hippocampus.

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Moreover, Withanolide A increased glutathione biosynthesis in neuronal cells by up regulating GCLC level through Nrf2 pathway in a corticosterone-dependent manner [70]. 8. EXISTING FORMULATIONS OF WS Several monoherbal, as well as polyherbal formulations, are available over the counter from reputed pharmaceutical companies in India. These are in the form of capsules and contain WS actives with various health claims such as nerve tonic, sedative, stress management, anti-anxiety, memory improvement, etc. A list of selected products sold in the market with their stated health claims is presented in Table 3. These formulations have also been evaluated in animal models. A polyherbal preparation, BR-16A (Mentat®), which contained WS as one of the main components exhibited marked protective effective against haloperidol or reserpineinduced catalepsy in mice [70]. Interestingly, a formulation containing WS and Mucuna pruriens seeds (as a concoction containing cow's milk) was shown to be effective in patients clinically diagnosed as PD [71]. 9. OTHER NEUROPROTECTIVE EFFECT OF WS 9.1. Scopolamine Toxicity The efficacy of WS leaf extract to improve memory was demonstrated in a recent study which examined the potential of alcoholic extract to protect against scopolamine-induced amnesia in a mice model [12]. Interestingly, Scopolamine induced down regulation of brain BDNF, and GFAP expression was markedly attenuated among WS treated mice. A similar mechanistic activity was also evident in various cell models (IMR neuronal cells, C6 glioma cells, and neuroblasTable 3.

toma cells) clearly suggesting that the WS extract and withanone may serve as a potential preventive as well as the therapeutic agent in memory disorders. Additional evidence was also reported in terms of significant improvement in phenotype in behavioral tests [71]. 9.2. Glutamate Toxicity Excitotoxicity which occurs by overstimulation of glutamate receptors which causes calcium overload is a common phenomenon in various neurodegenerative diseases. In a C6 glioma cell model, an aqueous extract of WS leaves markedly inhibited glutamate –induced cell death and HSP70 expression levels [72]. 9.4. Metal Induced Toxicity Owing to its demonstrated antioxidant activity and its ability to chelate metal ions, various researchers have examined the propensity of WS in modulating metal-induced toxicity in animal models. Studies have focused on metals such as cadmium, iron, and lead [11]. Water extract of WS leaves showed significant neuroprotection against lead exposure [12]. In a glioma cell model, WS extract normalized the expression of various molecular markers such as GFAP, mortalin and NCAM [73]. 9.3. Other Protective Effects Since WS is being extensively used as medha rasayana, root extracts have been tested in several experimental models to ascertain other biological activities. Some of the other significant biological activities ascribed to WS are presented in Table 4.

List of Withania somnifera registered products and their claimed therapeutic benefits

Registered Product

Manufacturer

Benefits claimed

Arshadi Pills

M/s Dehlvi Remedies

Depression, cardiac tonic Stress

Ashvagandha

M/s Morpheme Remedies

Combats stress

Ashwagandha

M/s Ayurceutics

Stress reliever

Ashwagandha pills

M/s Herbal Hills

Stress and revitalizer

Brento

M/s Zandu Pharmaceutical Works Ltd

Combats stress

Ashwagandharista

M/s Baidynath Ayurved Bhawan

Nerve tonic, memory /cognition improvement, improves concentration, relieves mental tension, natural sleep induction; recovery from nervous/ general debility

Geriforte (tablet and syrup)

M/s The Himalaya Drug Co

Stress relief and relief from insomnia

Mentat and Mentat DS


Memory and learning disorders, behavioural disorders, ADHD, anxiety and stress-related disorders, mental fatigue and an adjuvant in AD and PD

Ashvagandha (anti-stress)

M/s The Himalaya Drug Co.

Relieves Chronic stress-related physiological abnormalities

MindCare and MindCare Jr


Mental alertness, mental fatigue and occasional irritability

StressCare


Supports cortisol levels

Stresscom

M/s Dabur India Ltd

Relieves anxiety neurosis, physical / mental stress, and general depression

Stresswin

M/s Baidynath Ayurved Bhawan

Combats exertion, reduces anxiety, strain and stress


Table 4.


Other major biological activities ascribed to Withania somnifera.

Antiarthritic effect: Root powder (500-1000 mg/kg bw) suppressed experimentally induced gouty arthritis in rats [74]. Antibacterial effect: Aqueous/methanolic extracts of leaf / showed strong antibacterial activity against pathogenic S. typhimurium, E. coli and S. aureus [75]. Anticancer effects: Methanolic root extract (20 mg/kg bw) reduced DMBA induced papilloma; enhanced activity of antioxidant enzymes and inhibited lipid peroxides in skin/liver of mice; Withaferin A (2-10 μM) induced early reactive species formation and mitochondrial apoptosis by over-expressing TNF-1in Human myeloid leukemia cells; Ethanolic extract of root powder (400 mg/kg bw) along with paclitaxel inhibited B(a)P- induced free radical mediated cellular membrane in mice [76-78]. Anti-inflammatory and immunomodulatory activity: Aqueous suspension exhibited anti-inflammatory response against Complete Freund's Adjuvant (CFA) induced arthritis in rats [74]. Antiulcer effect: Reduced the volume of gastric secretion, ulcer index, acidity in gastric ulcer models induced by indomethacin and water immersion stress [10]. Cardioprotective effects: Hydro-alcoholic extract (25, 50 and 100 mg/kg) pretreatment for 30d attenuated isoproterenol induced myocardial ischemia in rats; [79]; Oral administration of extract (300mg/kg; 1.5% withanoloids) protected against doxorubicin induced cardiotoxicity in rats [80].

10. CLINICAL TRIALS Despite evidence on the broad spectrum of neuroprotective properties of WS, the number of published research articles highlighting its therapeutic effects is very limited. In a double-blind, placebo-controlled study patients with ICD-10 anxiety disorders were given an ethanolic extract of WS for 2-6 weeks. The drug was well tolerated, did not induce any adverse effects, and showed significant anxiolytic potential [81]. In another clinical study, a standardized WS root and leaf extract were evaluated for their anti-stress efficacy among chronically stressed subjects [82]. This study assessed the stress levels at 30-60 days using a modified Hamilton anxiety scale, and measurement of biochemical/ clinical variables such as serum cortisol, serum C-reactive protein, pulse rate and blood pressure. In a double-blind placebo-controlled trial, geriatric patients received a polyherbal formulation (Geriforte ) or a placebo at a dose of 2 tablets twice daily for a period of 6 months [83]. Significant rejuvenation of the anti-oxidant property was evidenced among the patients who received WS containing formulation as determined by the enzymatic and non-enzymatic anti-oxidants, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, reduced glutathione and malondialdehyde compared to patients treated with placebo and control group. In another prospective, double-blind, multi-dose, placebo-controlled, crossover study [84], 20 healthy male participants were randomized to receive two capsules twice daily of an encapsulated dried aqueous extract of roots and leaves of WS or a matching placebo for a period of 14 days. The cognitive and psychomotor performance was assessed pre-dose (day 1) and at 3 hrs post-dose on day 15 using a battery of computerized psychometric tests. Significant improvements were observed in reaction times with simple reaction, choice discrimination, digit symbol substitution, digit vigilance, and card sorting tests with WS extract compared to placebo. In a recent clinical trial, the standardized extract of WS (Sensorial) was assessed for its precognitive potential in 53

euthymic DSM-IV bipolar subjects [85]. In this study, compared to placebo, WS extract provided significant benefits for three cognitive tasks such as digit span backward, flanker neutral response time, and Social recognition response rating. 11. PATENTS There are several novel studies resulting in new numerous patents on the composition and application of the WS extracts which describe their efficacy for various purposes. Nevertheless patents on specific neurodegenerative diseases/disorders are limited. In this review we have selected patents based on criteria such as their neuro-beneficial claims and the recentness (years 2003-2014). Only a few patents have been briefly described below with their claims [86-96], while additional ones [97-102] with titles and inventor/s are presented in Tables 5 and 6. The majority of the patents actually deal with novel or improved herbal preparations either alone or as a combination with other plant products for application in human subjects with different ailments. A patent by Solaki et al., 2003 [86] describes a pharmaceutical or medicinal preparation which comprises of a mixture of seven herbs viz., WS, Tinospora cordifolia, Aloa vera, Curcuma longa, Achyranthus aspera, Ocimum sanctum and Picorrhiza kurroa. This invention describes a herbal formulation found to be effective for the treatment of cancer, more specifically haematological malignancies. The inventors claim that the formulation is specifically effective for the treatment of all acute leukaemias, including acute lymphoblastic leukaemias (L1—L3) and acute myeloblastic leukaemias. Another invention, by Patwardhan and Kapadi, 2004 [87] describes the compositions useful for ameliorating immune-suppression or side effects of myelosuppressive or immunosuppresive drug therapy. This invention describes a process for manufacturing the bioactives derived from WS which possess immunostimulating activity in animals and have anti-tumor activity in animals, in particular, when used in combination with other anti-tumor drugs. The authors claim that the formulation is useful for treating physiological conditions associated with myelosuppression, stimulating immune response, or for

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Table 5.

List of Selected Patents Relevant to Withania somnifera.

No

Patent number

Details

Ref

1

US6649185;

Pharmaceutical or medicinal preparation which comprises of a mixture of the following herbs: Withania somnifera, Tinospora cordifolia, Aloa vera, Curcuma longa, Achyranthus aspera, Ocimum sanctum and Picorrhiza kurroa

[86]

2

US 0033273

Composition obtained from a plant that has Withania somnifera fractions and mixtures of plant fractions having immune stimulating/anti tumor activity

[87]

3

US 0237786

Diet supplement for causing rapid weight loss, controlling appetite, managing stress, supporting relaxation, combating fatigue and supporting mental well-being

[88]

4

US 7250181

Pharmaceutical nutritional compositions of polyherbal extracts useful as anti-viral or immune-supporting agents viz., Withania somnifera, Mangifera indica and purified Shilajit

[89]

5

US 7241461

Antioxidant-promoting composition comprising of ashwagandha powder yielding overall decrease in oxidative damage and inflammation in mammals

[90]

6

US 7429397

Herbal formulation comprising root extract of Withania somnifera useful as brain tonic, memory enhancer and helps in recalling of thoughts

[91]

7

US 0213401

Nutritional supplement containing ashwaganda extract optimizes mental energy, decreases instances of forgetfulness

[92]

8

US 7550163

Herbal soft drink comprising decoction of Withania somnifera with Vitis vinifera, Boerhaavia diffusa and Tinospora cordifolia for antioxidant, immunoenhancing, nervine relaxation

[93]

9

US 7927633

Tea containing ashwagandha and other adaptogenic botanicals for providing energy, promoting stress resistance, promoting cellular activity and discouraging free radical generation

[94]

10

US 0177543

Natural compounds for modulation of stem cell function. combinations of a) Ashwagandha b) Milk Thistle c)Vitamin D3 d)Fucoidan; and e) Garcinia Indica

[95]

11

US 8636985

Nutraceutical gum or functional food compositions comprise ashwagandha (Withania somnifera) and Ltheanine in a sub lingual formulation

[96]

12

WO 067796

Herbal formulation as memory enhancer in alzheimer’s condition

[97]

13

WO 050015

Withania somnifera composition, method for obtaining same and pharmaceutical, nutritional and personal care formulations thereof

[98]

14

WO 014334

Method of treatment or management of stress

[99]

15

WO 042132

A novel herbal formulation for the modulation of immune system of HIV infected patients and a process of preparation thereof

[100]

16

WO 020422

A novel herbal formulation advocated for the prevention and

[101]

management of coronary heart disease 17

WO 010992

A synergistic herbal composition for treatment of rheumatic and musculo-skeletal disorders (rmsds)

[102]

18

US 7378112

Herbal composition to improve psychological functions as an anxiolytic, tranquilizer, and non-narcotic sedative, as well as other physiological functions

[103]

19

WO 056475

Composition for improving sexual health

[104]

20

US 0271944

Compositions for Alleviating Inflammation and Oxidative Stress in a Mammal

[105]

use alone or as an adjunct to anti-cell proliferative (e.g., anticancer) therapy. Several inventors have exploited the adaptogenic potential of Withania somnifera. One such patent by Heuer et al., 2007 [88] describes a method of manufacturing the diet supplement to be provided in a caplet form consumable several times per day. This formulation was claimed cause rapid weight loss, control appetite, manage stress, support relaxa-

tion, and mental well-being. The formulation is based on an extract of Rhodiola Rosea, Theanine, N-acetyl Tyrosine and a root extract of WS. The invention also describes a method of administering a diet supplement that comprises theanine and an extract of Russian Tarragon. Further, the invention also describes a method of administering to humans/animals a diet supplement that comprises an extract of Rhodiola rosea, Theanine, Astaxanthin extract of Algae, Vinpocetine, Nacetyl Tyrosine and an extract of WS.


In another patent, Ghosal et al., 2007 [89] provide a pharmaceutical, nutritional composition useful for the treatment of viral infections, or use as an adjuvant to an existing or conventional anti-viral therapy. The composition comprises of Withania somnifera and Mangifera indica and an extract of purified Shilajit. The authors claim that this cost effective immune-supporting agent is ideal for use during the maintenance phase of the treatment, following an initial viral load reduction phase in which it is used as an adjuvant to conventional anti-viral drug therapy. Several polyherbal formulations have been patented in which WS has been a vital component along with other wellknown botanicals. One such invention formulated by Myhill et al., 2007 [90], describes a polyherbal formulation which possess the potential to enhance antioxidant defenses. This formulation consists of Bacopa monniera extract, milk thistle extract, ashwagandha powder, green tea extract, Gotu kola powder, Ginko biloba leaf extract, Aloe vera powder, turmeric extract, and N-acetyl cystein and is claimed to alleviate inflammation and oxidative stress. The inventors claim that this composition significantly induces cellular antioxidant potential to achieve an overall net decrease in oxidative stress without the undesirable side-effects. This patent also describes a method for reducing the undesirable side-effects of free radicals in a subject by administering to a subject in need of such antioxidants an effective amount of antioxidantpromoting composition of the invention. Many formulations of WS with a variety of other medicinal plant extracts have been described which aim to specifically serve as a brain tonic and enhance memory and recall thoughts. One such invention by Pushpangadan et al., 2008 [91] describes a novel herbal formulation used to improve the memory and in treatment of senile and presenile dementia. This formulation(s) comprises of alcoholic extract of Tinospora cordifolia, Centella asiatica, WS, Mucuna pruriens and Curcuma longa. This product is formulated in various forms (tablets, capsules) can be used as a brain tonic to improve cognition and recalling of thoughts. In a conceptually identical patent, Smith, 2008 [92] describe a composition of a nutritional supplement containing WS extract and other botanical extracts which optimizes mental energy, decreases instances of forgetfulness. Another patent by Pushpangadan et al., 2009 [93] describes the preparation of a novel herbal soft drink comprising decoction of plants selected from Sida sps. Vitis vinifera, WS, Boerhaavia diffusa and Tinospora cordifolia for the protection and prevention of health and in particular, but not exclusively with antioxidant, immunoenhancing, hepatoprotective, cardiotonic, diuretic, digestive, choleretic, nervine relaxant properties. This formulation claims to possess immuno-enhancing, anti-oxidant, hepatoprotective, antifatigue, anti-stress herbal soft drink. In a recent patent, Swilling et al., 2011 [94], describe the preparation of a composition for brewing a natural tea to improve a general health. The composition contains adaptogenic botanicals claims to counter adverse physical, chemical, or biological stressors. The inventors claim that the formulation is useful as a natural tea for providing energy, promoting stress resistance, promoting t-cell activity, promoting k-cell activity, and discouraging free radicals. The composi-


tion claims to improve liver performance, resulting in improved toxin removal and improved nutrient absorption and improves capillary dilation, thereby providing better oxygenation to the brain. Further, a recent patent by Patel and Marleau, 2013 [95], describes the use of a formulation of natural compounds for modulation of stem cell function. This formulation is a combinations of WS, Milk Thistle, Vitamin D3, Fucoidan and Garcinia Indica. The authors claim the formulation significantly augments circulating stem cell numbers which has beneficial effect in degenerative conditions such as stroke, ischemic cardiomyopathy, critical limb ischemia, and myocardial infarction. Another recent patent by Barron, 2014 [96] describes a method of making a nutraceutical gum or functional food composition comprising Ashwagandha (Withania somnifera) and L-theanine. The formulation includes ingredients balanced in a synergistic manner to elicit complementary effects and claims to provide reduction of stress and alleviation of fatigue, enhancement of energy and a boost in mental clarity and concentration. 12. SUMMARY AND FUTURE PROSPECTS Standardized extracts of Withania somnifera have been demonstrated to possess multidimensional neuromodulatory effects in a variety of in vitro and experimental animal models. The spectrum of effects evidenced comprises of attenuation of oxidative damage by enhancing the antioxidant defense system with concomitant enhancement of the expression of marker proteins responsible for growth, differentiation and communication of neural cells. Further, the wide range of effects demonstrated with WS on neurotrophic factors, cytoskeletal elements, cell adhesion molecules and synaptic proteins clearly suggesting its multipotent attributes. As numerous researchers are deciphering the newer molecular targets of WS in animal models, it would be interesting to understand further how precisely the various signaling pathways converge resulting in neuroprotective effects. Moreover, there is an urgent need to exploit the neuromodulatory propensity of WS extract and its bioactives in specific neurodegenerative diseases such as AD and PD by conducting well-designed clinical trials. These can be achieved by obtaining fundamental insights on the cellular/molecular targets of the bioactives of WS by employing contemporary approaches such as bioinformatics, molecular docking studies, and identification of specific gene targets as well as understanding the post-translational/epigenetic regulation of the various effects. LIST OF ABBREVIATIONS NDD

=

Neurodegenerative diseases

AD

=

Alzheimer’s disease

PD

=

Parkinsons’s disease

AChE

=

Acetylcholinesterase

BChE

=

Butrylcholinesterase

BDNF

=

Brain derived neurotrpic factor

NGF

=

Neural growth factor

GFAP

=

Glial fibrillary acidic protein

Yenisettiet al.


NCAM

=

Neural cell adhesion molecule

GABA

=

Gammaminobutyric acid

PTZ

=

Pentylenetetrazol

CA (2) and CA (3) =

Cornu Ammonis areas

6-OHDA

=

6-hydroxydopamine

MPTP

=

1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine

GSH

=

Glutathione

GCLC

=

Glutamate—cysteine ligase

[14]

[15] [16]

[17]

[18]

CONFLICT OF INTEREST [19]

The authors confirm that this article content has no conflict of interest.

[20]

ACKNOWLEDGEMENTS This work is partly supported by the Department of Biotechnology (DBT), Ministry of Science and Technology, India (R&D grant no. BT/249/NE/TBP/2011, 25-4-2012) to the first author. The corresponding author is a recipient of a Visiting Research Professorship awarded by the Department of Biotechnology (DBT), Government of India under the North–East scheme and works at the Department of Zoology, Nagaland University (Central), Lumami, 798627, Nagaland.

[21]

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