Alzheimer’s disease (AD) is a progressive neurodegenerative disorder associated with the loss of cognitive functions. It is pathologically characterized by accumulation of extracellular beta amyloid plaques and intracellular neurofibrillary tangles of tau protein.1 Deterioration of cholinergic neurons in the basal forebrain and subsequent loss of cholinergic neurotransmission in the cerebral cortex are major neurochemical abnormalities linked with AD.2 Inhibition of the acetylcholinesterase enzyme in order to uphold the levels of acetylcholine is the ‘only’ approved therapy for treatment of AD.3 However, there are several side effects associated with these drugs and they have only been able to provide symptomatic relief.4 Plants are a rich source of numerous phytoconstituents and hence are constantly being explored for anti-Alzheimer potential.5 In an attempt to search plants with potential for management of AD, the in vitro acetylcholinesterase inhibition, antioxidant and cytotoxic potential of two culinary plant species, Allium cepa (Amaryllidaceae) and Ocimum basilicum (Lamiaceae) has been examined. These plants have neuroprotective effect.6,7,8,9 However, their anti-Alzheimer potential is not explored comprehensively.
MATERIALS AND METHODS
The bulbs of Allium cepa var. NHRDF-Red were collected in July 2013 from National Horticulture Research and Development Foundation (NHRDF), Bathinda, Punjab, India. The collected bulbs were authenticated by Mr. H.K. Sharma, Director, NHRDF, Bathinda (NHRDF/SC/BTI/2013-14/458). The leaves of Ocimum basilicum were procured in November 2013 from Indian Institute of Integrative Medicine, Jammu and were authenticated by Dr. Sunita Garg, Chief Scientist, Raw Material Herbarium and Museum, Delhi, CSIR-NISCAIR (NISCAIR/RHMD/Consult/2013/2337-117-1).
Acetylthiocholine iodide (ATCI), acetylcholinesterase (AChE), 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB) and 3-[4, 5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma–Aldrich. Donepezil was procured from Ind-Swift Ltd Jawahrpur, Punjab. All other chemicals used were of analytical grade.
6 Glioma cell lines
Rat C6 glioma cell line was obtained from the National Centre for Cell Sciences, Pune, India and maintained on Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with streptomycin (100 U/ml), gentamycin (100 μg/ml), 10% fetal bovine serum at 37°C and humid environment containing 5% CO2.
The outer scales of A. cepa bulbs and leaves of O. basilicum were shade dried and coarsely powdered. The powdered plant material was defatted with petroleum ether by maceration. The marc was extracted with methanol: distilled water (70: 30) mixture by maceration in a shaking incubator (r.p.m. = 100; Temperature = 37°C). The extracts so obtained were dried under vacuum and the percentage yields were calculated on dry weight basis.
Phytochemical screening and standardization of plant extracts
The prepared hydro-methanol (HM) extracts were subjected to phytochemical screening.10,11 Total phenolic content (TPC) was estimated by Folin-Ciocalteu procedure.12 and stated as milligram gallic acid equivalents (mg GAE)/g of extract. Aluminum chloride colorimetric method was used to establish total flavonoid content (TFC).13 and expressed as milligram quercetin equivalent (mg QE)/g of extract.
In vitro antioxidant activity
DPPH (2, 2- diphenyl-1-picryl hydrazyl) test
Inhibition of DPPH free radical test was carried out by method of Blois.14 DPPH radical is a compound having a proton free radical with a characteristic absorption which decreases considerably on exposure to proton radical scavengers. The method is based on capability of antioxidant samples to scavenge DPPH, decreasing its primary concentration and changing the color of solution from purple to yellow. The absorbance was read after 30 min incubation period at room temperature against a blank at 517 nm. Ascorbic acid was used as positive control. All readings were taken in triplicate. The percent of inhibition was calculated using following formula:
Sample concentration exhibiting 50% inhibition (IC50) was calculated from the graph between inhibition percentage and extract concentration.
FRAP (ferric reducing antioxidant power) test
The method reported by Yen and Chen.15 was used to determine the reducing power of extracts. Due to reductive capability of antioxidant compounds, they cause the reduction of ferric (Fe3+) form to the ferrous (Fe2+) form. Addition of FeCl3 to the ferrous (Fe2+) form lead to the formation of Prussian blue-colored complex. Reducing power is determined by measuring the formation of Perls’ Prussian blue at 700 nm. Different concentrations of HM extracts were mixed with sodium phosphate buffer and potassium ferricyanide. The mixture was incubated at 50°C for 20 min. After that trichloroacetic acid was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Supernatant was mixed with distilled water and ferric chloride solution, and absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.
Acetylcholinesterase inhibition assay
The acetylcholinesterase inhibition assay was determined by Ellman.16 colorimetric method with some modifications. It is a colorimetric assay based on enzymatic hydrolysis of acetylthiocholine iodide by the enzyme. The basic reaction involved in the process is as follows:
The anticholinesterase compounds hinder the action of acetylcholinesterase enzyme thus preventing the breakdown of acetylthiocholine. Reduction in the yellow color of reaction mixture indicates preventive effect by the test compounds.
In a total volume of 2 ml, 1500 μl of phosphate buffer 0.1 M (pH 8), 100 μl solution of HM extracts at different concentrations and 100 μl of enzyme solution containing 0.1 U/ml were incubated for 15 min at room temperature. After that, 100 μl solution of acetylthiocholine iodide and 200 μl of DTNB (4 mM) were added and the final mixture was incubated for 30 min at room temperature. Absorbance of the mixture was measured at 405 nm in a UV-Visible spectrophotometer. Donepezil was used as positive control. The percentage inhibition of enzyme activity was calculated by using the following formula:
Where, Asample = absorbance of the test extracts; Acontrol = absorbance of the control
The concentration of extract providing 50% inhibition (IC50) was obtained by plotting the percentage inhibition against extract concentration.
Stock solutions (200 μg/ml) of HM extracts were prepared by reconstituting the dried extracts in 70% methanol and then calculating the percentage yield. The cytotoxic effect of each plant extract was evaluated by tetrazolium- dye, MTT, assay.17 This colorimetric assay is based on the breakdown of tetrazolium ring of MTT by dehydrogenases in active mitochondria of living cells to estimate the number of viable cells.
In this assay, after adjusting the viable cell count (using trypan blue dye and dilution by DMEM), the C6 glioma cells were seeded in 96-well plates at a density of 40×103 cells/well in 100 μl DMEM. Following 24 h of incubation and attachment at 37°C in 5% CO2, the cells were treated with different concentrations of extracts (200, 100, 50, 25, 12.5, 6.2, 3.1 μg/ml) for a further 24 h under the same conditions. After treatment, the media was replaced with MTT solution (100 μl of 0.5 mg/ml per well) prepared in medium and incubated for 4 h at 37°C in a humidified incubator with 5% CO2. After removal of the MTT solution by gentle aspiration, the purple formazan crystals formed upon reduction of MTT by succinate dehydrogenase in the mitochondria of viable cells were dissolved by adding 100µl of DMSO per well. The plates were gently shaken for 1 min and absorbance was measured at 490 nm by microtiter plate reader (LabSystems, Finland).
The concentrations at which the cell viability was equivalent to the control cells were considered as non-toxic concentrations. All concentrations were tested in triplicate and the cell viability was calculated by the formula given below.
The percentage yields of HM extracts on dry weight basis for A. cepa var. NHRDF-Red and O. basilicum were 5.8 % w/w and 26.7% w/w respectively. The test extracts showed the presence of phenols, flavonoids and triterpenoids; hence were standardized with respect to TPC and TFC. A. cepa contains 456.1±11.7 mg GAE/g of extract and 278.4±21.6 mg QE/g of extract whereas O. basilicum extract contains 112.1±15.8mg GAE/g of extract and 20.41±8.7mg QE/g of extract.
In vitro antioxidant activity
The prepared HM extracts were evaluated for in vitro antioxidant activity using DPPH and FRAP assay. Both the plant extracts exhibited antioxidant activity in both in vitro assays.
In DPPH free radical scavenging assay IC50 value was calculated for both the plant extracts and compared with the standard (ascorbic acid). The IC50 values of ascorbic acid, A. cepa extract and O. basilicum extract were 6.32±0.27, 30.10±6.96, 38.67±1.72 µg/ml respectively. The present investigation confirmed that HM extracts of both the plants are significant radical scavengers.
In the FRAP assay, a higher absorbance indicates a higher ferric reducing power. With an increase in the concentration of the extracts, there is an increase in absorbance (Figure 1). HM extract of A. cepa showed more marked reducing capacity with maximum absorbance at concentration of 320 μg/ml.
Acetylcholinesterase inhibition assay
The summary of acetylcholinesterase inhibition by plant extracts used in this study is shown in Table 1. With an increase in concentration of extracts, the percentage inhibition increased. Of both the extracts, A. cepa extract displayed noteworthy inhibition with IC50 value of 51.78±1.05 µg/ml. A. cepa extract was found to be an effective cholinesterase inhibitor in comparison to the standard drug.
Plant extracts at different concentrations were evaluated for cytotoxicity against C6 glioma cells using MTT assay. The percentage cell viability was calculated for both the extracts. Treatment with the extracts considerably inhibited the proliferation of C6 cells in a concentration-dependent manner as shown in Figure 2 (a and b) MTT assay revealed affected cell viability at higher concentration in comparison to control. Significant toxic effects were observed at concentrations greater than 100µg/ml for both the plant extracts. These results revealed that concentrations below 100µg/ml for each extract are safe to C6 glioma cells and therefore can be selected for further evaluation of neuroprotective experiments.
Plants can be an excellent reservoir for drugs for the management of Alzheimer’s disease owing to the presence of variety of phytochemicals.18 Due to multifactorial nature of the disease, different plant constituents present in an extract may act alone on one pathological factor or synergistically to curb a pathway. This may prove beneficial in preventing various pathological events linked to the disease. The loss of cholinergic neurons in cerebral cortex is accepted as the main feature of Alzheimer’s disease.19 The use of acetylcholinesterase inhibitors to enhance the levels of acetylcholine is an important and solitary preventive approach for Alzheimer patients.20 Free radical production is also a major factor contributing to Alzheimer’s disease. Brain of AD patients witnesses changes in the balance of redox transition metals, like Fe and Cu, increase in lipid perxoxidation, increase in protein and DNA oxidation. Studies have also reported that the formation of senile plaques enhance free radical production.21,22 Reserachers have evaluated A. sativum for the management of Alzheimer’s disease.23,24 However, A. cepa has not been explored for controlling pathogenic mechanisms related to the disease. Hydro-methanol extract of O. basilicum has been reported as acetylcholinesterase inhibitor from our laboratory.9,25 whereas cytotoxic activity of the extract has not been explored.
In our study both plant extracts are potent antioxidants. A. cepa extract was found to be a good acetylcholinesterase inhibitor. High phenolic and flavonoid content of A. cepa might be responsible for the activity.26 Cytotoxicity studies on plant extracts revealed that both the extracts were found to be non-toxic to C6 glioma cells at concentrations below 100 µg/ml. This assay is helpful in testing the safety of plant extracts in different cell culture experiments. Both the activities are vital for a drug candidate for the management of Alzheimer’s disease hence A.cepa is an excellent candidate for drug development.
In the present investigation anticholinesterase, antioxidant and cytotoxic potential of the two culinary plants- Allium cepa and Ocimum basilicum was investigated. Both the plant extracts displayed significant antioxidant activity and were found to be non-toxic in MTT assay below the concentration of 100µg/ml. The A. cepa extract had strong antioxidant and acetylcholinesterase inhibitory effect probably due to higher total phenol and flavonoid content. This can be considered an excellent candidate for developing as a drug for management of AD.