Role of Artemisia macrocephala as enzymes inhibitor in dementia and nociception ! A pharmacological study

This study was conducted for investigating potentials of methanolic extract (Am. CME) and subsequent fractions of Artemisia macrocephala (A. macrocephala) for the inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and cyclo-oxygenase (COX-1 and COX-2). Ellman's assay was followed for the investigation of AChE and BChE inhibitory potentials of Am. CME and subsequent fractions of A. macrocephala while cyclo-oxygenase inhibition was investigated using a COX inhibition assay. For AChE inhibitory assay, chloroform (Am.CHF), ethyl acetate (Am.EtOAc) fractions and Am.CME showed 85.76 ± 1.49, 77.50 ± 0.86 and 71.00 ± 1.15 % inhibition of the enzyme respectively at 1000μg/mL concentration. Similarly, for BChE inhibition assay, Am.CHF, Am.EtOAc and Am. CME showed 82.56±0.68, 73.56 ± 1.56 and 68.55±0.81% inhibition of the enzyme respectively at 1000μg/mL concentration. Am.CHF and Am.CME were found to be most active against COX-1 and COX-2. Against COX-1, AmCHF and Am.CME showed 75.23 ± 0.98 and 73.33 ± 0.46% inhibition respectively at 1000μg/mL concentration. Similarly against COX-2, they showed 81.23 ± 1.23 and 79.40 ± 0.46% respectively at 1000μg/mL concentration. It can be concluded from results that Artemisia macrocephala can be used in the treatment of cognitive disorders, along with neurodegenerative ailments, various neuro-pharmacological ailments and certain painful conditions.


Introduction
Neurodegenerative disease is a general terminology used for various diseased conditions developing from persistent breakdown and corrosion of neurons in central nervous system.Amongst several types of dementia, Alzheimer's disease (AD) has been common that affect about 20 million people in the world.Owing to its multi-factorial nature, the single therapeutic loom is based on the cholinergic hypothesis of cognitive dysfunction [1,2].AD is a progressive, neurodegenerative ailment affecting mainly elders and about 50-60 % of dementia patients have age above 65 years.The main symptoms associated with the later stages of AD engross cognitive dysfunction, principally memory loss leading to sever loss in the abilities of mental level.It affects language, thought and memory control centers of the brain.It is associated with loss in neurons, abnormal tangles and plaques in neurons [3,4].In mammalian brain, there are present two main types of cholinesterases, namely, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) [5].The common notable biochemical change that AD patients are facing is diminution of acetylcholine (ACh) level in the brain.AChE is present in excitable tissues (nerves, muscles, erythrocytes and placenta), while BuChE mostly in peripheral, central nervous system, plasma and liver.Therefore, AChE and BChE inhibition, the enzymes accountable in Ach hydrolysis in cholinergic synapse, is presently the most recognized approach in treatment of AD [6].Till

Extraction
Drying of plant material was carried out at room temperature in shade, followed by crushing and mechanical grinding to get fine powder.Soaked the powder (5.5 kg) for 22 days in commercial grade (90 %) methanol at room temperature with occasional shaking.Filtration of the materials was carried out.The materials filtered off 3 times.With the help of rotary evaporator at reduced pressure, the combined filtrate was evaporated till obtaining a dark greenish color crude Am.CME.yielding 555 gram (10.09 %).Fractionation Am.CME (500 g) of A. macrocephala suspended in distilled water (400 ml) was sequentially fractionated with n-hexane (400 mL).For thorough fractionation, process was repeated three times.The same method was followed for obtaining ethyl acetate and chloroform fractions which successively gave 40 g (8%) of n-hexane fraction (Am.NHX), 61.7 g (12.34 %) of chloroform fraction Am.CHF, 39.4 g (7.88%) of ethyl acetate Am.EtOAc and 40.7 g (8.14 %) of remaining aqueous fraction (Am.Aq).

Anticholinestrase assays
For enzyme inhibitory potentials investigation of the Am.CME and its consequent fractions AChE and BChE were used following previously reported Ellman's assay [24].The test samples were dissolved in DMSO (few drops) and further dilution (125-1000 μg/mL) was done in buffer (phosphate, 0.1 M).AChE (518 U/mg) and BChE (7-16 U/mg) were further diluted with 0.1 M buffer (phosphate, pH 8.0) till 0.03 U/mL (AChE) and 0.01 U/mL (BChE).Prepared DTNB (0.2273 mM), BTchI (0.5 mM) and ATchI (0.5 mM) solutions in distilled water and stored at 8 °C.Thereafter, added 5 μL enzyme solution in cuvette before the addition of test sample (205 μL) and 5 μL reagent (DTNB) in each essay.Kept this whole mixture for 15 min at 30°C in water bath and substrate solution (5 μL) was subsequently added.The absorbance was measured spectrophotometrically (Thermo electron corporation, USA) at 412 nm.Galanthamine served as reference standard.Absorbance with the time of reaction was noted at 30°Cfor four minutes.The experiment was performed in triplicate.Percent activity of enzyme and inhibition of enzyme by test samples and control were calculated from absorption rate with time change (V = ΔAbs/Δt) as: Enzyme inhibition (%) = 100 -% enzyme activity Enzyme activity (%) = 100 × V/Vmax (Vmax =Enzyme activity without inhibitor drug).

Cyclo-oxygenase inhibition assays
Cyclo-oxygenase enzymes (COX-1, COX-2) inhibition assays were conducted as previously described [25].The controls contained solvent blank and background correction reaction for enzymes inactivation with HCl on ice prior to arachidonic acid (16 Ci/mol; 30 M) addition.Indomethacin was used as a reference drug (100 mg/mL).Crude extract and subsequent fractions were tested at 62.5-1000 mg/mL concentrations.COX inhibition was calculated via comparing radioactivity quantity of sample to blank using through the formula as: where DPM is disintegrations per min of extract, background and solvent blank.Results are given as means of three experiments and values are given as percentage mean ± standard error mean.

Results and discussion
The acetylcholinesterase inhibitory potentials of Am.CME and subsequent fractions of A. macrocephala are summarized in Figure 1.Among all the tested samples, Am.CHF showed the highest percent acetylcholinesterase inhibition, causing 85.76 ± 1.49 percent inhibition at 1000 µg/mL concentration.This was followed by Am.EtOAc and Am.CME with 77.50 ± 0.86 and 71.00 ± 1.15 percent inhibition, respectively at 1000 µg/mL concentration.Galanthamine showed 94.45 ± 2.37 percent inhibition at 1000 µg/mL concentration.All samples showed good to moderate percent enzyme inhibition in a concentration dependent manner.Butyrylcholinesterase inhibition results of all the samples of A. macrocephala are summarized in Figure 2.Among all the samples, Am.CHF caused maximum percent butyrylcholinesterase inhibition which was 82.56 ± 0.68 % at 1000 µg/mL concentration.Similarly EtOAc, Am.CME and Am.Aq showed 73.56 ± 1.56, 68.55 ± 0.81 and 67.57± 0.82 percent enzyme inhibition respectively at 1000 µg/mL concentration.All samples showed enzyme inhibition in concentration dependent manner and were from moderate to good.Standard galanthamine showed 94.45 ± 2.37 percent inhibition at 1000 µg/mL.Central cholinergic system is considered to be very important in cognitive functions regulation.Cholinergic neuronal loss is the main characteristic of AD and augmentation of central cholinergic action by use of anticholinesterase, is currently the basis of pharmacotherapy of senile dementia of Alzheimer type [26].AChE is among the fastest identified enzymes that catalyzes the breakage of acetylcholine after depolarization in synaptic cleft.AChE inhibitors such as galanthamine, are frequently used in AD pharmacotherapy.
The BChE, less specific, has freshly been a center of research, since its concentration remains the same, or is even up-regulated, whereas, AChE is down-regulated dramatically in AD patients [27].Thus, regulating Ach level via AChE and BChE inhibition is considered to be a useful therapeutic approach in treatment of AD and several other types of dementia.Prostanoids are bioactive constituents composed of prostaglandins (PGs), thromboxane and prostacyclin.They are originated from arachidonic acid, which come out from plasma membrane phospholipids intracellularly due to damage of tissue and inflammation.This arachidonic acid converted to the precursors of PGs.PGs, the last metabolites of arachidonic acid, are responsible for the generation of pain.Enzymes like LOX and COX are involved in the formation of these PGs [32].Before going to test the samples for analgesic activities, their in-vitro screening against COX enzyme is imperative as these types study can predict the in-vivo fate of the test samples.Novel, clinically effective and economically cheap anti-inflamatory and antinociceptive drugs from medicinal plants are the best alternative to the hazardous synthetic drugs.The in vitro cyclooxygenase assays, the crude extract and subsequent fraction of Artemisia macrocephala showed COX-1 and COX-2 inhibition in a dose dependent manner.Among all tested samples, Am.CHF and Am.CCME showed the enhanced inhibition activity against both COX-1 and COX-2.While other fractions showed moderate percent inhibition of the enzymes.Am.CHF and Am.CCME caused 75.23 ± 0.98 (IC50 122 µg/mL) and 73.33 ± 0.46% (IC50 241 µg/mL) COX-1 inhibition respectively at 1000µg/mL concentration.Similarly against COX-2, they showed 81.23±1.23 (IC50 60.25 µg/mL) and 79.40 ± 0.46% (IC50 112.97 µg/mL) respectively at 1000µg/mL concentration as shown in Table 1.Am.Aq showed the lowest inhibitory activity for both the enzymes even the highest concentrations.The in-vitro enzyme inhibition activity of the plant can be attributed to the presence of bioactive compounds like flavonoids and terpenes in the plant as reported earlier [20].The inhibition of COX exhibited by the crude extract and subsequent fractions of Artemisia macrocephala make it as alternative candidate for further work in search for new anti-nociceptive and antiinflammatory compounds.

Conclusion
The results of the present study show that the plant is rich in bioactive compounds which are responsible for enzymes inhibition activity.From the results of this study, it can be concluded that the plant can be used to treat cognitive disorders, including neurodegenerative diseases and different neuro-pharmacological disorders.This primary screening will further open new channels for the isolation, structural characterization and in-vivo evaluation of the bioactive compounds for enzymes inhibition potentials and then ultimately will lead the molecular level investigation for enzymes inhibition responsible for AD, other forms of dementia and nociception.

Figure 2 .
Figure 2. The butyrylcholinesterase inhibitory potentials of Am.CME and subsequent fractions of A. macrocephalaProstanoids are bioactive constituents composed of prostaglandins (PGs), thromboxane and prostacyclin.They are originated from arachidonic acid, which come out from plasma membrane phospholipids intracellularly due to damage of tissue and inflammation.This arachidonic acid converted to the precursors of PGs.PGs, the last metabolites of arachidonic acid, are responsible for the generation of pain.Enzymes like LOX and COX are involved in the formation of these PGs[32].Before going to test the samples for analgesic activities, their in-vitro screening against COX enzyme is imperative as these types study can predict the in-vivo fate of the test samples.Novel, clinically effective and Conceived and designed the experiments: I Shah, Performed the experiments: I Shah, M Shoaib, N Ali & SWA Shah, Analyzed the data: I Shah & M Shoaib, Contributed

and asthma [15, 16]. In
Aerial fresh parts of A. macrocephala were collected in August, 2014in hills near Badwan Chowk, Dir Lower, Pakistan.It was identified by Dr. Nasur Ullah, University of Malakand Chakdara, Dir Lower, KPK, Pakistan.Voucher sample "AM-01-2014" has been submitted in University of Malakand herbarium.
macrocephala.Antioxidant activity was reported by evaluation of its different fractions [22].We have reported its essential oil for AChE and BuChE inhibition assays with remarkable results [23].