Phenolic, flavonoid contents, anticholinesterase and antioxidant evaluation of Iris germanica var; florentina

Abstract This study was designed to investigate antioxidant and anticholinesterase potential of Iris germanica var; florentina. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potential of plant samples were investigated by Ellman’s assay. Antioxidant activity was performed using DPPH, H2O2 and ABTS free radical scavenging assays. Total phenolics and flavonoids contents were expressed in mg GAE/g dry weight and mg RTE/g, respectively. In AChE inhibition assay, Ig.Fl, Ig.Sp and Ig.Cf fractions exhibited highest activity with IC50 values of < 0.1, 5.64 and 19 μg/mL, respectively. In BChE inhibitory assay, Ig.Fl, Ig.Sp, Ig.Cf and Ig.Cr were most active with IC50 of < 0.1, < 0.1, 31 and 78 μg/mL, respectively. In DPPH assay, Ig.Fl and Ig.Cf exhibited highest inhibition of free radicals, 80.52% (IC50 = 9 μg/mL) and 78.30% (IC50 = 8 μg/mL), respectively. In ABTS assay Ig.Cr, Ig.Cf, Ig.Fl and Ig.Sp exhibited IC50 values of < 0.1, 2, 2 and 3 μg/mL, respectively. Graphical abstract

cognitive functions including learning, memory, arousal and excitement processes (Gold 2003). Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the key enzymes involved in the breakdown of ACh, is the most useful strategy in the treatment of various neurological disorders including Alzheimer's disease (AD), Parkinson's disease, senile dementia, myasthenia gravis and ataxia (Mukherjee et al. 2007). Several cholinesterase inhibitors such as galanthamine, tacrine, rivastigmine and donepezil are used in the treatment of above-mentioned neurological disorders, but their use is limited due to the major side effects (Knapp et al. 1994;Schulz 2003) and less effectiveness (Schneider 2001). Therefore, a constant investigation for the discovery of new, safe and effective drug candidates is the interest of current researchers.
Oxygen is vital for aerobic respiration and animal's life on earth. The oxygen consumption leads to the production of a chain of reactive oxygen species (ROS), which play an important role in energy production, regulation of cell growth, intercellular signalling and phagocytosis (Verma et al. 2009). However, ROS may also be very injurious as they can readily attack biomolecules such as DNA, lipids, proteins, leading to genotoxicity, cancer, arthritis, arteriosclerosis, inflammation, diabetes and neurological disorders such as AD (Shah et al. 2013). Plants and their products, rich in antioxidant compounds, are known to retard progression of AD and neuronal degeneration ). Owing to their capacity of scavenging free radicals, these antioxidants attenuate inflammation pathways, decrease cancer progression, heart diseases and other degenerative problems associated with ageing (Henry et al. 2002;Ferreira et al. 2006). Nowadays, the use of antioxidants in the management of some disease states and as food supplements has tremendously increased. The health hazards associated with the toxicity of synthetic AChE and BChE inhibitors, and antioxidants have led to the continuous search for naturally occurring drugs especially of plant origin.
Amongst the antioxidant compounds, significant attention has been given to flavonoids and phenolic compounds. Flavonoids are widely distributed in plant kingdom with more than 10,000 known compounds (Xiao, Mao, et al. 2011). Flavonoids are of major interest due to their bioactivities including anti-inflammatory, anti-genotoxic, anti-cancer, neuroprotective, anti-Alzheimer's and antiglycative properties (Chidambara Murthy et al. 2012). All these properties are basically attributed to the antioxidant nature of flavonoids (Xiao, Chen, et al. 2011).
IGF belongs to the family Iridaceae. Iris is a rhizomatous or bulbous herb. Iris genus is rich in isoflavonoids which have a wide range of biological activities including antioxidant, anti-inflammatory and cancer chemopreventive properties (Rahman et al. 2003). Iris rhizomes are also popular in cosmetic preparations, and its oil is regarded as one of the most precious ingredients in perfume industries (Schütz et al. 2011). Iris suaveolens, another specie of iridaceae family, has been reported for antioxidant and anticholinesterase activities and potential usefulness in neurological disorders (Hacıbekirog and Kolak 2011). In an effort to discover new sources which can potentially be used in the treatment of AD, this study was designed to evaluate the AChE, BChE inhibitory and diverse antioxidant potentials of IGF.

Results and discussion
Development of novel-and mechanism-based cholinesterase inhibitors is amongst the most valuable approaches for the treatment of neurodegenerative diseases, due to their role in the breakdown of neurotransmitter ACh (Ayaz et al. 2014). Neurodegenerative disorders such as AD are characterised by reduced cholinergic activity in brain (Nunes-Tavares et al. 2012).
The enzymes AchE and BchE cause hydrolysis of ACh; therefore, inhibition of these enzymes plays a key role in enhancing cholinergic activity. Besides reduced cholinergic activity, oxidative stress has also been recognised as a key factor in the pathogenesis of AD (Lee et al. 2012). Oxidation is an integral process in the cellular metabolism. Generally, the free radicals generated during metabolic processes have unpaired electrons (Pietta 2000), are highly reactive and produce cellular injury by causing membrane lipid peroxidation and damage to enzymes and DNA (Rafat Husain et al. 1987). The cell injury and multiple hazardous effects of free radicals may lead to several nervous disorders and eventually may cause neuronal death. In this context, huge investigational studies demonstrate the efficacy of phenolic antioxidants from plant origin to reduce or block neuronal death occurring in the pathophysiology of AD. Flavonoids being a group of polyphenolic compounds have been reported to be responsible for antioxidant activity, as they can effectively scavenge the free radicals and protects the body cells especially the neuronal cells from injurious effect of free radicals (Valko et al. 2006). Furthermore, the hydrogen donating substituent (hydroxyl groups) attached to the aromatic ring structures of flavonoids enable them to undergo a redox reaction, which in turn, helps them scavenge free radicals (Robards et al. 1999). Similarly, another group of secondary metabolite, i.e. the saponins have also been reported to possess antioxidant and anticholinesterase potentials (Gülçin et al. 2004). Bacopa monniera and Ginkgo biloba have been traditionally used in dementia and cognitive disorders and as reported earlier that these plants are rich in saponins and possess strong anticholinesterase potentials (Das et al. 2002).
Regarding antioxidant activities Ig.Chf, Ig.Fl and Ig.Sp were found most potent in DPPH free radical scavenging assay exhibiting IC 50 values of 8, 9 and 30 μg/mL, respectively. In ABTS free radical scavenging assay, Ig.Cr, Ig.Chf, Ig.Fl and Ig.Sp were found most potent amongst all fractions with IC 50 values of <0.1, 2, 2 and 3 μg/mL, respectively. In H 2 O 2 radicals scavenging activity, Ig.Fl and Ig.Chf exhibited IC 50 values of 11 and 12 μg/mL, respectively ( Figure S4). Concentrations of flavonoids, phenolics, saponins correlated well to their antioxidant and anticholinesterase activities ( Figure S4 A-E). Antioxidant and enzyme inhibition activities of flavonoids, phenolics, saponins were comparable to standard drugs. Antioxidant potential of the Iris germanica coupled with promising anticholinesterase activity can be a better remedy for the treatment of neurological disorders such as AD, parkinsonism, amnesia and dementias. Activity-guided isolation can give novel and more useful compounds.

Conclusions
Based on the importance of natural medicine, the gap between traditional remedies and scientific validation should be bridged. From this study, it may be concluded that the phenolics, flavonoids and saponins of I. germanica are potential remedies for the treatment of oxidative stresses which leads to AD. It may also be inferred that the Ig.Chf of Ig.Fl are rich in active principles responsible for the antioxidant and anticholinesterase activities. The targeted isolation of bioactive compounds is required to develop novel drugs which may alleviate AD and other neurological disorders.