Background: In our earlier study, we have shown the memory enhancing and scopolamine-induced amnesia recovery properties of Ashwagandha leaf extract using behavioral paradigm and expression analysis of synaptic plasticity genes. Purpose: However, the exact mechanism through which Ashwagandha demonstrates these effects is still unknown. Methods: In the present study, we hypothesized that the alcoholic extract of Ashwagandha leaves (i-Extract) possesses cholinergic properties, which in turn inhibit the anti-cholinergic nature of scopolamine. Therefore, the potential of i-Extract to recover from the scopolamine-induced cholinergic deficits was assessed by measuring acetylcholine (neurotransmitter) and Arc (synaptic activity-related gene) expression level in the mouse brain. Results: The enzymatic activity of acetyl cholinesterase and choline acetyltransferase was assessed through colorimetric assays, and expression level of Arc protein was examined by Western blotting. Furthermore, mRNA level of these genes was examined by semi-quantitative reverse-transcriptase PCR. We observed that the treatment of i-Extract in scopolamine-induced amnesic mouse attenuates scopolamine-induced detrimental alterations in the cholinergic system. Conclusion: Thus, our study provided biochemical and molecular evidence of cholinergic properties of Ashwagandha leaf extract during brain disorders associated with cholinergic dysfunction.
Ashwagandha (Withania somnifera (L.) Dunal) is a member of Solanaceae family, commonly referred to as ‘Indian Ginseng'. It is one of the widely used herbs in traditional system of home medicine, Ayurveda [1,2,3]. It is also known as ‘Queen of Ayurveda' because it exhibits adaptogenic, analgesic, antioxidant, anti-stress, immunomodulatory and immunostimulant properties . The extracts from different parts of the plant including roots, shoot and leaves have been shown to possess a large variety of health promoting effects [1,5,6]. However, the exact mechanism of these properties of Ashwagandha is still not clear. We have earlier reported the pharmacological characterization of alcoholic leaf extract of Ashwagandha (i-Extract) and standardization of its dose and duration along with scopolamine administration [7,8,9,10]. In the present study, we have used scopolamine hydrobromide, a gold standard for inducing amnesia in rodents, for disrupting the normal cholinergic processes in the mouse brain and to focus on the possible cholinergic properties of i-Extract.
Scopolamine hydrobromide, a tropane alkaloid, is a non-selective antagonist of muscarinic receptor which blocks the effects of acetylcholine neurotransmitter . It impairs long-term potentiation and induces amnesia in mammals . The use of scopolamine as a pharmacological model of amnesia became very popular after the cholinergic hypothesis of memory dysfunction was postulated. This hypothesis assumed that the decline in memory and cognitive functions during aging and disorders was predominantly related to a decrease in the integrity of cholinergic neurotransmission. Therefore, the enzymatic assay of acetyl cholinesterase (AChE), an enzyme that hydrolyzes acetylcholine, and choline acetyltransferase (ChAT), an enzyme that synthesizes acetylcholine, was undertaken. Since interference in cholinergic signaling modulates the expression of activity-regulated cytoskeletal-associated protein (Arc) , a neuronal immediate early gene critical for synaptic plasticity, we also examined the expression of Arc at mRNA and protein level in the brain of control, scopolamine-treated and i-Extract-treated mice.
Chemicals and Reagents
Acetyl CoA, acetyl thiocholine iodide, Arc antibody (sc-17839), DTNB, neostigmine bromide, TRI reagent and monoclonal anti-β-actin-peroxidase (A3854) were purchased from Sigma-Aldrich (USA); enhanced chemiluminescence (ECL) reagents, random hexanucleotides, RNase inhibitor, dNTPs, Taq polymerase and reverse transcriptase enzymes were obtained from the New England Biolabs (USA); peroxidase conjugated secondary antibodies were purchased from Bangalore Genei (India) and polyvinyl difluoride (PVDF) membrane was procured from Millipore (Germany); i-Extract was synthesized and characterized at AIST (Japan). All other biochemicals were purchased from Merck (Germany).
Plant Material and Extraction
Alcoholic extract of Ashwagandha leaves (i-Extract) was prepared by air drying of Ashwagandha leaves, crushing it to a fine powder and subjecting to extraction with methyl alcohol in a Soxhlet apparatus for 4-5 days at 60°C. This extract was further extracted with n-hexane to get rid of chlorophyll and other pigments and then with ethoxyethane that was evaporated to obtain the ether extract. This extract was stored at 4°C and freshly prepared by suspending in 0.5% DMSO for oral administration to mice. The fingerprint analysis of i-Extract is shown in online supplementary figure 1 (for all online suppl. material, see www.karger.com/doi/10.1159/000443573).
Animals and Drugs Treatment
Young (12 ± 2 weeks) male Swiss albino strain mice were inbred in the animal house of Department of Zoology, Banaras Hindu University, India. They were maintained at 24 ± 2°C under 12:12-hour light and dark schedule with ad libitum standard mice feed and drinking water. All mice were handled and used according to guidelines of the institutional animal ethical committee. Mice were administered normal saline intraperitoneally in vehicle control group and 3 mg/kg bw scopolamine hydrobromide dissolved in normal saline in experimental groups. To examine the neuroprotective, therapeutic as well as per se effect of W. somnifera leaves, 200 mg/kg bw i-Extract dissolved in 0.05% DMSO was administered orally 1 h before or after scopolamine treatment and alone, respectively.
Mice were divided into 5 groups: (1) C-mice administered with normal saline, (2) S-mice injected with scopolamine, (3) SA-mice injected with scopolamine followed by i-Extract after 1 h, (4) A-mice treated with i-Extract alone and (5) AS-mice treated with i-Extract followed by scopolamine after 1 h. Drugs were treated at 9 a.m. every day for 7 days. On the seventh day, mice were sacrificed and the whole brain was removed quickly on ice.
Acetyl Cholinesterase Assay
Acetyl cholinesterase assay was done based on the principle and protocol as described by Ellman et al.  with some modifications. Briefly, the whole brain from each mouse was weighed and 20% homogenate was prepared for each sample in 0.1 M phosphate buffer (pH 8). Then 2.6 ml phosphate buffer (0.1 M, pH 8), 100 μl DTNB and aliquot of 0.4 ml homogenate was added in a glass cuvette and mixed thoroughly. A412 was measured in a UV-VIS spectrophotometer till the absorbance reached a constant value (basal reading). Finally, 20 μl of acetyl thiocholine iodide was added as substrate and change in absorbance at the interval of 2 min was noted down to calculate the change in absorbance per minute. The AChE activity was determined by formula: r = 5.74 × 10-4 × ∆A/C, where r = rate of enzymatic activity (in moles of acetylthiocholine hydrolyzed/min/g tissue), ∆A = change in absorbance/min and C = concentration of the tissue in homogenate (mg/ml).
Choline Acetyltransferase Assay
Choline acetyltransferase assay was done based on the principle and protocol as described earlier by Chao and Wolfgram  with few modifications. Briefly, the mixture of 20 μl 0.05 M Trisbuffer, pH 7.2, 3 mM sodium chloride, 1 M choline chloride, 6.5 mM dithioerythritol, 1.1 mM EDTA, 0.76 mM neostigmine bromide along with 40 μl 6.2 mM acetyl CoA and 20 μl 0.1 M creatine was pre-incubated at 37°C for 5 min. Thereafter, 100 μg of protein homogenate from brain was added to this mixture and incubated at 37°C for 20 min. After boiling it for 2 min, 800 μl 2.5 mM sodium arsenite was added. Finally, all the tubes were centrifuged at 12,000 g for 5 min to collect the supernatant. The tubes were allowed to stand for 15 min after adding 10 μl 1 mM 4-dithiopyrimidine in each and A342 was measured in a UV-VIS spectrophotometer.
The protein homogenate of whole brain was prepared in RIPA buffer and centrifuged at 10,000 g for 15 min. The supernatant was separated and concentration of protein was determined by Bradford method . Thereafter, 30 µg protein was mixed with 6 × loading dye and loaded on 10% SDS-PAGE for resolving the bands. All protein bands were transferred onto PVDF membrane through semi-dry electroblotting apparatus. This membrane was blocked in 5% fat-free skimmed milk, incubated overnight at 4°C with 1,000 × diluted anti-Arc antibody, washed in 1 × PBS, incubated for 2 h at room temperature with 1,000 × diluted HRP-conjugated goat anti-mouse IgG, washed in 1 × PBS and signal was detected with ECL reagent on a X-ray film. For loading control, the same membrane was reprobed for β-actin with HRP-conjugated anti-β-actin antibody (1:10,000) for 3 h at room temperature.
The whole brain was homogenized in TRI reagent and RNA was isolated from control and experimental mice as instructed by the company. The quantity of RNA obtained was estimated by taking absorbance at 260 nm, whereas purity was checked by calculating absorbance ratio (A260/280). Only RNA samples with A260/280 ≥1.8 were taken further to resolve on 1% agarose formaldehyde gel for checking the quality of 18S and 28S rRNA using UV transilluminator. Five micrograms RNA from each group of mice was used for first strand synthesis by means of reverse transcriptase. The resulting cDNA was used as a template for PCR amplification using specific primers for AChE , ChAT  and Arc  with GAPDH  as internal control in each case. Following primer sequences for PCR amplification were used: AChE: 5′-TCTTTGCTCAGCGACTTA-3′ (upstream), 5′-GTCACAGGTCTGAGCA-3′ (downstream); ChAT: 5′-GTCTTGGATGTTGTCATTAATTTC-3′ (upstream), 5′-TCTCTGGTAAAGCCTGTAGTAAGC-3′ (downstream); Arc: 5′-GGCGACCAGATGGAGCTGGACCATA-3′ (upstream), 5′-CTGGCCCCTCTATTCAGGCTGGGTC-3′ (downstream); GAPDH: 5′-GTCTCCTGCGACTTCAG-3′ (upstream), 5′-TCATTGTCATACCAGGAAATGAGC-3′ (downstream). Amplifications were performed as follows: AChE: 40 cycles at 57°C for 45 s, ChAT: 35 cycles at 59°C for 45 s, Arc: 35 cycles at 59°C for 90 s, GAPDH: 26 cycles at 52°C for 30 s. Finally, the PCR products were analyzed on 2% agarose gel after staining with ethidium bromide under UV light.
All sets of experiment were repeated thrice (n = 3 × 3 mice/group). Results are expressed as mean ± SEM and statistical analysis of the data was done by applying one way analysis of variance, followed by post hoc tests of LSD method through PASW Statistics for Windows (version 18) for all parameters. The p value <0.05 was considered statistically significant. In each experiment, scopolamine-injected (second group) and only i-Extract per se administered group (fourth group) have been compared with vehicle control (first group), whereas both the treatment groups, that is, scopolamine followed by i-Extract (third group) and i-Extract followed by scopolamine (fifth group) have been compared with scopolamine-injected amnesic group (second group).
For AChE and ChAT assays, results are expressed as enzymatic activity in terms of micromoles of substrate hydrolyzed per minute per gram of tissue weight. For both Western blotting and reverse transcriptase PCR, the bands were documented by AlphaImager system consisting of 10 bit CCD camera and their intensity was analyzed densitometrically using Alpha-EaseFC software (Alpha Innotech Corp., USA). Signal intensity (IDV) for every candidate gene or protein was normalized against signal intensity (IDV) of respective loading control to obtain the final relative density value of signal intensity of candidate gene or protein and analyzed by calculating the fold change with respect to control group.
Effect of i-Extract on AChE in Scopolamine-Injected Mouse
Scopolamine hydrobromide increased the activity of AChE by 2-fold, whereas per se treatment of i-Extract decreased this by 1.7-fold. With the post- and pre-treatment of i-Extract, we found a significant decrease (1.25 and 1.4-fold, respectively) in the AChE activity as compared to scopolamine-injected group (fig. 1a). Likewise, AChE mRNA level showed corresponding changes in the expression level of all the experimental groups. There was about 3-fold increase of AChE mRNA level in scopolamine-injected group, whereas i-Extract alone treated group showed a downregulation with 2.4-fold change when compared to control group. Both post- and pre-treatments of i-Extract in scopolamine-injected group illustrated significant downregulation (2.6 and 1.9-fold) in AChE mRNA. This downregulation brings back AChE mRNA level to that of the control group level (fig. 1b). The correlation coefficient between the enzymatic activity and mRNA level of AChE was calculated as +0.8666 (fig. 1c).
Effect of i-Extract on ChAT in Scopolamine-Injected Mouse
Contrary to the above results, scopolamine hydrobromide caused decrease in the ChAT activity by 2.4-fold; while administration of i-Extract as such augmented its activity by same fold. Both, post- and pre-treatment of i-Extract in scopolamine-injected group showed significant increase of about 1.3-fold in the activity of ChAT each as compared to the scopolamine-injected group (fig. 2a). In the same way, mRNA level of ChAT showed related changes in the expression level of all the experimental groups. Though scopolamine hydrobromide downregulated ChAT mRNA by 2.3-fold as compared to control group, the administration of i-Extract alone upregulated it by 1.5-fold. The post- as well as pre-treatment of i-Extract in scopolamine-injected group showed significant upregulation of 1.7 and 1.8-fold in ChAT mRNA (fig. 2b). The correlation coefficient between the enzymatic activity and mRNA level of ChAT was calculated as +0.95 (fig. 2c).
Effect of i-Extract on Arc in Scopolamine-Injected Mouse
As compared to control, scopolamine treatment resulted in significant downregulation (1.9-fold) of Arc protein in the whole brain, whereas i-Extract extensively upregulated (∼1.3-fold) the Arc protein. Most conspicuously, treatment with i-Extract in scopolamine-injected mice showed recovery of Arc protein and this revival of Arc protein level was significant in both post- and pre-treatment (∼1.5-fold in each) when compared to scopolamine treated group (fig. 3a). Inconsistent with the Arc protein results, both the post- and pre-treatment with i-Extract did not show any significant change in the Arc mRNA level when compared to scopolamine alone injected group. Though scopolamine and i-Extract per se administration significantly downregulated (0.65-fold) and upregulated (1.5-fold) the expression of Arc mRNA respectively in the whole brain as compared to the control saline treated mice (fig. 3b). A significant correlation between the protein and mRNA level of Arc was obtained as r = +0.916 (fig. 3c).
As described in Ayurveda, Ashwagandha provides a natural resource for safe and effective remedies against stress, sexual weakness, low stamina, rheumatism, arthritis, leprosy, cancer and several other fatal disorders [21,22]. Leaves of Ashwagandha are the major site and source of biosynthesis and are indeed rich in withanolides (widely identified as active components). Although there are reports to support the efficacy of Ashwagandha in animal models, its exact mechanism is still elusive. For identifying the probable involvement of cholinergic pathway during neuroprotection by i-Extract treatment, we recruited integrative approaches including chemical analysis of the extract and generation of mice models for toxicity and neurodegenerative diseases. We first performed the chemical analysis of alcoholic extract of Ashwagandha leaves to support its functionality and found that it is rich in withanolides including withanone and withaferin A (online suppl. fig. 1). We then used scopolamine-induced amnesia in mouse as a model to investigate its cholinergic properties. The cholinergic mechanism of such effect was determined by analysis of major regulators of cognitive response, acetylcholine (neurotransmitter) and Arc (synaptic activity-related gene).
Scopolamine is a potent psychoactive drug and is regarded as a gold standard for inducing amnesia in animals and humans. It is a non-selective muscarinic receptor antagonist that inhibits central cholinergic neuronal activity and impairs memory and is one of the well-established pharmacological models of memory impairment that mimics those occurring during aging, neurodegenerative, neuropsychiatric pathologies and traumatic brain injuries . One of the advantages of this model is that it provides a simple and quick way for screening anti-amnesic and cognition enhancing drugs. The use of scopolamine as a pharmacological model of amnesia became very popular after the cholinergic hypothesis of memory dysfunction was postulated, according to which the decline in memory and cognitive functions during aging and disorders was predominantly related to a decrease in the integrity of cholinergic neurotransmission .
In consistency with these reports, we observed significant decrease in the level of acetylcholine neurotransmitter due to scopolamine injection as evident by the increased AChE activity and decreased ChAT activity, which in fact implies that there is rapid degradation of available acetylcholine and lesser synthesis of new acetylcholine as compared to normal conditions. The mRNA level of AChE and ChAT enzymes revealed that there was a significant positive correlation between their activity and mRNA level suggesting correlation between the enzymatic activity and the available level of translated protein, that is, functional enzyme. It has also been reported from human studies on Alzheimer's disease patients and mild cognitive impairment cases . Interestingly, treatment of scopolamine-challenged mice with i-Extract reverses the effects in the activity as well as mRNA level of both AChE and ChAT. This change denoted the recovery of diminished level of acetylcholine neurotransmitter in the brain (fig. 4).
Arc (or Arg3.1) is an effector immediate-early gene, whose mRNA is localized in an NMDA receptor-dependent manner to activate synaptic sites, where the newly translated proteins are likely to play a critical role in synaptic plasticity and memory [26,27,28]. In our study, we found that Arc protein and mRNA level decreased with the scopolamine injection. This result was similar to that of the earlier study  and expected as the activation of muscarinic acetylcholine receptor (mAChR) causes increase in expression of Arc , and scopolamine acts as a blocker of muscarinic receptors. On the other hand, the post- and pre-treatment with i-Extract in scopolamine-injected mice led to the upregulation of Arc protein. However, this increase in protein level was not due to increase in Arc mRNA level. Hence, we hypothesize that the increase in Arc protein may be due to the synaptic activation of mAChR by i-Extract, which generally leads to the increase in dendritic transport as well as translation of Arc mRNA at synapses [30,31].
To the best of our knowledge, the present study is the primary instance of indicating cholinergic consequences of Ashwagandha leaf-derived i-Extract, which is rich in withanone, on scopolamine-induced cholinergic deficits. The fact that i-Extract treatment caused remarkable changes in the acetylcholine and Arc levels in control and amnesic mice suggested that i-Extract has cholinergic properties for serving as a defensive therapy for amnesia and neuroplasticity disorders that are constantly affected with age and stress. The study also proposes the use of Ashwagandha leaves for protection and enhancement of brain functions involved in cholinergic nervous system-associated cognitive responses.
A.G. conceived, designed and performed the experiments, analyzed the data and wrote the manuscript. R.W. prepared and analyzed i-Extract and performed concise review of the manuscript. M.K.T. contributed in reagents, materials, analysis tools and preparation of the manuscript.
The authors declare no conflict of interest.
A.G. was a recipient of Senior Research Fellowship from the Council of Scientific and Industrial Research, India. The work was supported by grants from the Department of Biotechnology (BT/PR3996/MED/97/57/2011), Government of India to M.K.T.