Comparative chemical profiling, cholinesterase inhibitions and anti-radicals properties of essential oils from Polygonum hydropiper L: A Preliminary anti- Alzheimer’s study

Background Cholinesterase inhibition is a vital target for the development of novel and mechanism based inhibitors, owing to their role in the breakdown of acetylcholine (ACh) neurotransmitter to treat various neurological disorders including Alzheimer’s disease (AD). Similarly, free radicals are implicated in the progression of various diseases like neurodegenerative disorders. Due to lipid solubility and potential to easily cross blood brain barrier, this study was designed to investigate the anticholinesterase and antioxidant potentials of the standardized essential oils from the leaves and flowers of Polygonum hydropiper. Methods Essential oils from the leaves (Ph.LO) and flowers (Ph.FO) of P. hdropiper were isolated using Clevenger apparatus. Oil samples were analyzed by GC-MS to identify major components and to attribute the antioxidant and anticholinesterase activity to specific components. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potentials of the samples were determined following Ellman’s assay. Antioxidant assays were performed using 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) and hydrogen peroxide (H2O2) free radical scavenging assays. Results In the GC-MS analysis 141 and 122 compounds were indentified in Ph.LO and Ph.FO respectively. Caryophylene oxide (41.42 %) was the major component in Ph.FO while decahydronaphthalene (38.29 %) was prominent in Ph.LO. In AChE inhibition, Ph.LO and Ph.FO exhibited 87.00** and 79.66***% inhibitions at 1000 μg/ml with IC50 of 120 and 220 μg/ml respectively. The IC50 value for galanthamine was 15 μg/ml. In BChE inhibitory assay, Ph.LO and Ph.FO caused 82.66*** (IC50 130 μg/ml) and 77.50***% (IC50 225 μg/ml) inhibitions respectively at 1000 μg/ml concentration. In DPPH free radical scavenging assay, Ph.LO and Ph.FO exhibited IC50 of 20 and 200 μg/ml respectively. The calculated IC50s were 180 & 60 μg/ml for Ph.LO, and 45 & 50 μg/ml for Ph.FO in scavenging of ABTS and H2O2 free radicals respectively. Conclusions In the current study, essential oils from leaves and flowers of P. hydropiper exhibited dose dependent anticholinesterase and antioxidant activities. Leaves essential oil were more effective and can be subjected to further in-vitro and in-vivo anti-Alzheimer’s studies. Electronic supplementary material The online version of this article (doi:10.1186/s12944-015-0145-8) contains supplementary material, which is available to authorized users.


Background
The cholinergic concept of Alzheimer's disease (AD) was initially resulted from postmortem studies of the brain [1,2], which ultimately led to the development of new drugs based on the inhibition of the key enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [3,4]. Therapy with such drugs resulted in a significant improvement in cognitive functions and also hampered the progression of the disease [5][6][7]. Two cholinesterases, AChE encoded by gene on chromosome 7 and BChE encoded by gene on chromosome 3 occur in the human central nervous system (CNS) [8,9]. These enzymes share about 65 % amino acid sequence homology even though coded on different genes [10]. In the human brain BChE mostly appears to have a neuroglial distribution, while AChE is principally located within cholinergic axons and in the neuronal cell bodies. Both enzymes are also present in neuritic plaques and tangles in AD patients [11,12]. The ratio of cholinesterases in the human brain varies during the course of AD. A decline of 10-15 % in the activity of AChE in the hippocampus and cerebral cortex has been reported in advanced stages of the disease, whereas BChE activity increases by 40-90 % [11,13]. These changes in the ratio of cholinesterases and variation in the level of the neurotransmitters in dementia must be considered in order to optimize the therapeutic balance between AChE and BChE inhibitions. This balance may be sustained via the selective or non-selective inhibition of the enzymes. A significant correlation between the inhibition of BChE activity in the cerebrospinal fluid (CSF), but not AChE, with an enhancement in cognitive performance in patients with mild to moderate AD after treatment with rivastigmine (non-specific inhibitor of cholinesterases) has been reported [14]. Experimental data also revealed that BChE specific inhibitors not only raise the levels of acetylcholine (ACh) in rats but also improve memory in elderly rats [9,15]. These findings also signify that inhibition of BChE in addition to AChE may be vital in the treatment of Alzheimer's type dementia.
A currently available drug like tacrine is observed to have severe side effects like liver transaminase elevations and gastrointestinal complainsts [16], and are only useful in mild type of AD [17]. Therefore, it is required to search new, safe and effective drug candidates. Natural products are potential sources of novel bioactive compounds and have an extensive history of therapeutic utility since the establishment of human era. Galanthamine, an anticholinestrase alkaloid was isolated from snowdrop, and is approved for the therapy of AD [18]. Research has been directed to study the biological effects of plants traditionally used as cholinesterase inhibitors [18,19].
Free radicals including reactive oxygen species (ROS) are implicated in a variety of disorders including neuroinflammation, gastritis, ischemic heart diseases, reperfusion injury of tissues and atherosclerosis [20,21]. Free radicals generated during oxidation process are converted to non-radical forms by catalase and hydroperoxidase enzymes in living systems. But in case of excessive radical generation or depletion of human immune system natural antioxidants as free radical scavengers may be required [22]. In Alzheimer's patients and aging brain, dysfunctional mitochondria generate free radicals, thus lead to oxidative stress followed by oxidative damage and pathological changes. β-amyloid (Aβ) is a powerful originator of reactive oxygen and nitrogen species which are primary initiators of oxidative harm thus effecting neural, microglial, cerebrovascular cells and tissues [23]. Currently, available synthetic antioxidants including gallic acid esters, tertiary butylated hydroquinone and butylated hydroxy toluene (BHT) are associated with adverse health consequences [24]. Numerous natural bioactive compounds have been shown to possess strong antioxidant potential which reveals that these compounds have the ability to scavenge free radicals inside the body and provide very low chances of adverse effects [25,26].
Among plants which have been investigated for the treatment of neurodegenerative disorders, Polygonum hydropiper is one of the most numerous genuses in the family Polygonaceae which is abundant in North West of Pakistan. This plant has a long history of use in folk medicine as remedy for the treatment of a multiplicity of disorders including inflammation, rheumatoid arthritis, epilepsy, headache, colic pain, fever, chill, joint pain, oedema and infectious diseases [27][28][29]. It is also used as diuretic, CNS stimulant, anthelmintic, to treat insomnia, kidney diseases, hemorrhoids, hypertension and angina [30]. Other species of Polygonaceae family have been reported for their effectiveness in Parkinson's disease [31], cerebral ischemia [32] and neuroprotective agents [33]. We recently reported the solvent extracts of P. hydropiper for antioxidant, anticholinestrase activities and its potential effectiveness to treat neurodegenerative disorders [29]. Volatile constituents of the essential oils from P. hydropiper are expected to readily cross the blood-brain barrier owing to their small molecular size and lipophilic nature. Their volatile nature may also facilitate their administration in the form of inhalation avoiding the alimentary canal with its attendant denaturing of active molecular species.

Cholinesterase inhibition assays
The acetyl and butyrylcholinesterase inhibitions potentials of essential oils isolated from leaves and flowers of P. hydropiper are shown in Table 2.

Antioxidant assays
The antioxidant potentials of essential oils from leaves and flowers of P. hydropiper were determined using DPPH, ABTS and H 2 O 2 free radicals. The results are summarized in Table 3.

Discussion
Steam distillation, subsequently GC/MS and GC/FID analysis were used to determine the chemical compositions of essential oils from the leaves and flowers of P. hydropiper. Chromatograms with the identified peaks ( Fig. 1) as well as the chemical structures of the major identified compounds from leaves and flowers oils are shown in Figs. 2 and 3 respectively. In GC, GC-MS analysis of Ph.LO, 141 compounds were identified among which decahydronaphthalene (38.29 %) was in highest concentration (Fig. 2, Table 1(A)). Likewise, in analysis of Ph.FO, caryophylene oxide (41.42 %) was present in highest concentration as given in Fig. 3, Table 1(B). The number of identified compounds in Ph.LO were greater than Ph.FO and both anticholinesterase and antioxidant potentials of Ph.LO were observed to be higher than Ph.FO. This is not astonishing that the chemical composition of essential oils greatly depends upon the genetics, age, season and varies with environmental conditions of the plant [34]. Up to the best of our knowledge this is the most detailed report on the chemical composition of essential oils from P. hydropiper. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are the key enzymes catalyzing the breakdown of the important neurotransmitter acetylcholine (ACh) in the nervous system to form acetate and choline [25]. ACh insufficiency in the cerebral cortex of humans is among the vital pathophysiologies observed in AD patients [35,36]. An important tool for treating AD is to boost the level of ACh in the brain by the administration of safe and effective AChE inhibitors [37]. Among the clinically approved drugs, four are cholinesterase inhibitors including tacrine, donepezil, rivastigmine and galantamine, whereas, the fifth one is glutamatergic system modifier called memantine (Fig. 4). Among these four drugs, the use of tacrine is limited due to hepatotoxic effects associated with it [38,39]. Further, studies during clinical trials revealed that cholinesterase inhibitors may help AD patients to sustain their ability to perform routine activities with less frequent behavioral changes [40]. Other studies suggest that cholinesterase inhibitors may improve the cognitive performance of the AD patients even in the advanced stages of the disease [41]. Consequently, cholinesterase inhibitors may improve cognitive decline and thus reduce the emergence of new behavioral turbulence.
In the current study, we observed that Ph.LO were most effective against AChE causing 87.00 ± 1.15 % inhibition followed by Ph.FO with 79.66 ± 0.88 % enzyme inhibition at 1000 μg/ml. Among both oils, Ph.LO was more potent with IC 50 of 120 μg/ml, while the IC 50 for Ph.FO was 220 μg/ml. The IC 50 value for galanthamine was 15 μg/ml. Both oils exhibited concentration dependent activity as shown in Table 2. In BChE inhibitory assay, again Ph.LO was more active causing 82.66 ± 1.20 % inhibition at 1000 μg/ml and IC 50 of 130 μg/ml. Moreover, Ph.FO revealed 77.50 ± 0.44 % inhibition at the same concentration with IC 50 of 225 μg/ml. Positive control inhibition was 96.00 ± 1.52 % at the same concentration and IC 50 was 10 μg/ml. Presently, there is no complete preventative or curative drug therapy available for AD, leaving the symptomatic relief presented by AChE/BChE inhibitors as the single approved therapeutic choice. Recently, galanthamine from Amaryllidaceae family is approved for clinical use and has become a vital therapeutic option effective to retard the process of neurological degeneration in AD. Galanthamine provides an efficient symptomatic therapy for AD patients and also delay the progression of the disease. Another isoquinoline alkaloid berberine, isolated from Rhizoma coptidis and Cortex phellodendri is reported as an effective neuroprotective agent in diseases like cerebral ischemia, schizophrenia, AD, depression and anxiety [42,43]. Berberine is reported to reduce extracellular Aβ fabrication and BACE activity without affecting the release of LDH in H4 neuroglioma (APPNL-H4) cells [8]. Berberine therapy also reduces cognitive dysfunction as indicated by decrease in errors using MWM task in comparison to usual reference memory and memory retention (probe trial) in APP transgenic mice. [44]. The essential oils in the current study exhibited comparative percent inhibitions with the standard drug in both assays. However, the IC 50 of essential oils were higher than that of an orally administered standard drug galanthamine. We speculate that the essential oils administered in the form of vapors (aerosol) will have better availability than orally administered drugs due to high lipid solubility and bypassing presystemic metabolism. However, further in-vivo studies on genetically modified animals' models are required to confirm its in-vivo bioavailability and potential efficacy in neurological disorders.
Modern research revealed that beta secretase enzyme (BACE1) catalyze the breakdown of amyloid precursor protein (APP) to form ß-amyloid peptides in AD brain, which provoke inflammatory process with consequent release of free radicals oxygen species causing neuronal damage [45][46][47]. Antioxidant drugs may contribute to AD chemotherapy by attenuation of the inflammatory pathways via scavenging of free radicals [37]. In recent times, natural products has got more attention as antioxidants as they are safer and these substances could be supplied as food components or in the form of pharmaceuticals for human use [48]. Among those, essential oils from aromatic and medicinal plants are well known to reveal antioxidant and cholinesterase inhibitory properties and thus can be very helpful in the treatment of AD [49]. In DPPH assay, Ph.LO was most effective causing 85.00 ± 1.15, 79.50 ± 0.28 and 72.00 ± 1.04 % free radicals scavenging at concentrations of 1000, 800 and 400 μg/ml respectively. DPPH free radicals

Conclusions
Essential oils from P. hydropiper were investigated for the first time for anticholinesterase and antioxidant potentials. All samples exhibited concentration dependent enzyme inhibitions and anti-radical activities with Ph.LO most affective. In GC, GC-MS analysis 144 and 122 compounds were identified in Ph.LO and Ph.FO respectively. Further in-vivo studies are required for possible use of these samples in neurodegenerative disorders.

Collection of plant material
Fresh leaves of P. hydropiper were collected from Talash Valley Dir (L) Pakistan in the month of September 2014.

Isolation of the essential oils
Fresh leaves of P. hydropiper were macerated and hydrodistilled using a Clevenger type apparatus supplied with condenser. Distillation process was continued for 3 days at 100°C, and the volatile oils (yellowish in color) were collected in glass bottles. Anhydrous sodium sulfate was used to remove water after extraction [50]. Flowers of P. hydropiper were hydro-distilled with a Likens-Nickerson-type apparatus using diethyl ether for 3 h. White to yellow color obtained which was dried over anhydrous sodium sulphate. Finally, the oils were properly sealed in glass vials and stored in refrigerator at −30°C before further analysis.

Gas chromatography (GC) analysis
Essential oils samples were analyzed by means of an Agilent USB-393752 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with HHP-5MS 5 % phenylmethylsiloxane capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Restek, Bellefonte, PA) equipped with an FID detector. The temperature of Oven was maintained at 70°C for 1 min at first, and then increased at the rate of 6°C/min to 180°C for 5 min and lastly at the rate of 5°C/min to 280°C for 20 min. Injector and detector temperatures were set at 220°C and 290°C, correspondingly. Helium was used as carrier gas at a flow rate of 1 ml/min, and diluted samples (1/1000 in n-pentane, v/v) of 1.0 μl were injected manually in the split-less mode.  Fig. 4 Clinically available drugs for Alzheimer's therapy the fragmentation pattern of the mass spectra with data published in the literature [51,52] or with mass spectra from literature.

Anticholinesterase assays
AChE and BChE inhibitory potentials of the samples were carried out following Ellman's assay [53,54]. Using this procedure, acetylthiocholine iodide or butyrylthiocholine iodide are hydrolyzed by the respective enzymes to form 5-thio-2-nitrobenzoate anion which then form complex with DTNB and give UV detectable yellow color compound.

Spectroscopic analysis
In each experiment, 5 μl enzyme solutions were added to the cuvette and oil samples were added at the above mentioned concentrations. Finally, DTNB reagent (5 μl) was added to the cuvette and the resultant mixture was incubated at 30°C for 15 min using water bath. A substrate solution (5 μl) was added at the end and absorbance was measured at 412 nm using a double beam spectrophotometer (Thermo electron corporation USA). Negative control contained all components except oil samples, while positive control galanthamine (10 μg/ ml) was used in the assay as standard cholinesterase inhibitor. Change in absorbance along with the reaction time was recorded for 4 h at 30°C. The experiments were performed in triplicate. Enzymes activity and enzyme inhibition by control and tested samples were determined from the rate of absorption with change in time (V = ΔAbs /Δt) as; Enzyme inhibition (%) = 100 -percent enzyme activity; Where (V max ) is enzyme activity in the absence of inhibitor drug.

Antioxidant assays DPPH free radicals scavenging assay
Free radicals scavenging ability of the essential oil was determined following well established procedures [29,55]. Different dilutions (12.5, 50, 100, 200, 400, 800 and 1000 μg/ml) of essential oils (0.1 ml) were added to 0.004 % methanolic solution of DPPH. After 30 min, absorbance was measured at 517 nm using UV spectrophotometer (Thermo electron corporation, USA). Percent DPPH scavenging activity was calculated as; Ascorbic acid was used as positive control. Where A 0 characterize absorbance of control and A 1 is the absorbance of the essential oils. All experiments were performed in triplicate and inhibition graphs were made with the help of GraphPad prism program (GraphPAD, San Diego, California, USA). Median inhibitory concentrations IC 50 values were calculated using Microsoft Excel programme.

ABTS free radicals scavenging assay
The ABTS free radical scavenging potential of samples were evaluated using previously reported procedure [56]. The test is based on the ability of antioxidants present in the sample to scavenge ABTS radicals leading to reduction. Using this procedure, solutions of ABTS 7 mM and potassium persulphate (K 2 S 2 O 4 ) 2.45 mM were mixed and stored in dark place at room temperature for 12-16 h to obtain a dark colored solution. This solution was diluted using Phosphate buffer (0.01 M) pH 7.4 and absorbance value was adjusted to 0.70 at 734 nm. Finally, 300 μl solution of test sample was added to 3.0 ml of ABTS solution in cuvette and was analyzed spectrophotometerically at 734 nm. The decline in absorbance was determined after one minute of mixing the solutions and analysis was continued for 6 min. Ascorbic acid was used as positive control. The assay was repeated in triplicate and percentage inhibition was calculated using formula: % scavenging effect ¼ control absorbancesample absorbance =control absorbance Â 100 Hydrogen peroxide free radicals scavenging assay The hydrogen peroxide scavenging activity of extracts was determined using methods described previously [57]. Using this method 2 mM hydrogen peroxide solution was prepared in 50 mM phosphate buffer having pH of 7.4. Oil samples (0.1 ml) were taken in test tubes and their volumes were increased to 0.4 ml using 50 mM phosphate buffer solution. Hydrogen peroxide (0.6 ml) was added to the tubes and was vertexed. Absorbance of each sample was measured at 230 nm against the blank after 10 min. Hydrogen peroxide scavenging activity was calculated using equation; 1−absorbance of sample Absorbance of control Â 100

Estimation of IC 50 values
Concentrations of the oils which inhibited substrate hydrolysis (AChE and BChE) by 50 % (IC 50 ) were calculated from dose response ratio using Microsoft Excel program. In DPPH, ABTS and H 2 O 2 the IC 50 values were calculated using the same procedure.

Statistical data analysis
All the assays were repeated in triplicate and values were expressed as mean ± S.E.M. One way ANOVA followed by Dunnett's multiple comparison test was applied for the comparison of positive control with the test group at 95 % confidence interval using GraphPad prism Software USA. The P values less than 0.05 were considered as statistically significant.