Phytochemical screening, antioxidant and anticholinesterase effects of Alangium salvifolium (L.F) Wang root extracts

Alangium salvifolium wang is a medicinal plant of the Alanginaceae family which was used as a traditional medicine to cure or prevent a variety of ailments. The aim of the study was to investigate and compare the phytochemical profiles, antioxidant and anticholinesterase effects of ethanol (EASR), dichloromethane (DASR), chloroform (CASR) and aqueous (AASR) extracts of A. salvifolium root. Phytochemical screening was done by using qualitative methods whereas total phenol content (TPC), total flavonoid content (TFC) and total flavonol content (TFlC) were determined by Folin-Ciocalteau reagent, aluminium trichloride and sodium acetate solution methods, respectively. Antioxidant activities were assessed by DPPH radical scavenging, ferric reducing antioxidant power (FRAP) and total antioxidant content (TAC) assay. Ellman's assay was applied to investigate acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme inhibitory effect. Preliminary phytochemical screening revealed the presence of valuable phytochemicals with significantly (P*<0.05, P**<0.01, P***<0.001) different content of TPC, TFC and TFlC. CASR, among the extracts, had shown the highest TPC (492.38±22.34 mg/g gallic acid), followed by TFC (276.25±17.23 mg/g quercetin) and TFlC (332.92±7.07 mg/g quercetin). Moreover, maximum antioxidant potential, including DPPH radical scavenging (IC50: 11.26±1.29 μg/ml), FRAP (EC50: 26.64±2.17 μg/ml) and TAC (639.55±10.51 mg/g ascorbic acid) was found in the CASR. Donepezil, a standard drug, showed maximum inhibitory effect of AChE (IC50: 7.94±1.12 μg/ml) and BChE (IC50:12.58±2.15 μg/ml). CASR followed by DASR had potent inhibitory effects while AASR had mild and EASR practically had no inhibitory effects of the enzymes. The present study has demonstrated that the root extracts of the A. salvifolium have moderate to potent antioxidant and enzyme inhibitory effects.


INTRODUCTION
Free radical damage and oxidative stress are considered as important causative factors for generation as well as exacerbation of various ailments like cancer, diabetes, asthma, and the pathogenesis of alzheimer's disease (AD) (Asmat et al., 2015). Oxidative stress, a potential source of damage to DNA, lipids, sugars and proteins, causes an imbalance between the intracellular production of free radicals/reactive oxygen species (ROS) and antioxidant defense mechanisms, resulting in cellular injury (Gjumrakch et al., 2008). The brain consumes a large proportion of the inhaled oxygen, and therefore produces a comparatively large quantity of free radical by-products (Yongxin et al., 2013). However, less quantity of the reactive oxygen (ROS) species are the precondition to keep the integrity of the neuronal cells and subsequently their normal functioning, since the elevated level of the radicals can lead to neuronal cell death (Yongxin et al., 2013). In contrast, antioxidants, being the defensive agents against the oxidative stress, have multiple functions in biological systems, including maintenance of cell integrity and cell signaling pathways (Kumar et al., 2008). One principal cellular function of antioxidants is to prevent damage caused by the ROS. Various studies have proved that an antioxidant may scavenge a highly reactive free radical or may inactivate it by donating a proton atom or by accepting an electron from the radical, and eventually prevents the free radicalinduced diseases (Jiaojiao et al., 2012).
Alzheimer, the most common among the neurodegenerative disorders and dementia, is a major challenge of the modern era, and is a slowly progressive disease of the brain that is characterized by the impairment of memory (Rahmat et al., 2012). For normal functioning of brain, sufficient level of acetylcholine (Ach) is necessary which is essential for proper neurotransmission. Acetylcholinesterase (AChE) enzyme catalyzes hydrolysis reaction of the Ach and butyrylcholinesterase (BChE) potentiates the catalyzing activity of the AChE, resulting in a decreased level of Ach in the brain (Zeb et al., 2014). This condition leads to neurodegeneration and subsequently cognition. So, inhibition of AChE and BChE may be the most effective way of protecting the Ach to prevent or to improve dementia.
Alangium salvifolium wang belongs to the family of Alanginaceae. Ankola and Alangi are its common name in India, and Stone Mango in English. It is a small deciduous thorny tree or shrub (Uthiraselvam et al., 2012) which is distributed in tropical and subtropical region such as Bangladesh, India, China Phillipines, Africa, Srilanka and Indochina (Ronok et al., 2013). An array of ailments including diabetes, jaundice, gastric disorders, protozoal diseases, rheumatic pain, burning sensation, haemorrhages, lung cancer, poisonings, leprosy and many inflammatory patches have been treated by using various parts of the plant (Meera et al., 2013). Many bioactive phytochemicals such as several flavanoids, phenolic compounds, irridoid glycosides and oxyoglucosides have been isolated by phytochemical screening of it (Gopinath, 2013). Literature review of the plant indicates the presence of coumarins, triterpenoids, and some potent alkaloids in it (Savithramma et al., 2012). The aim of the present study was to evaluate antioxidant and anticholineesterase effects of various extracts of the A. salvifolium root.

Plant
For the investigation, A. salvifolium wang root was collected from Rajshahi, Bangladesh between January and June, 2013 and identified by an expert of the Bangladesh National Herbarium, Dhaka, where a voucher specimen number was retained with an accession no. 40214. The collected plant part was cleaned, dried for one week and pulverized into a coarse powder using a suitable grinder. Powdered material was stored in an airtight container and kept in a cool, dark, and dry place until further analysis was taken.

Extract preparation
Approximately 500 g of powdered root was placed separately in four clean and flat-bottomed glass containers and soaked in ethanol, dichloromethane, chloroform and distilled water. All the containers with their contents were sealed and kept for 7 days. Then extraction was carried out using ultrasonic sound bath accompanied by sonication (40 min). The entire mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then the extract was filtered through Whatman filter paper and concentrated by using a rotatory evaporator at reduced pressure. The gummy extracts were then dried by using an electric oven, and finally obtained EASR (12.25 g), DASR (9.5 g), CASR (7.5 g) and AASR (14.17 g). The dried extracts were separately stored in air tight containers until completion of the analysis.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Germay. All other reagents and solvents used for the study were of highest purity grade and commercially available.

Phytochemical screening
Phytochemical screening of the extracts was done by applying some previously established methods. Alkaloids, saponins, terpenoids and steroids were detected by applying Harborne (Harborne, 1973) method. Flavonoids and tannins were examined by applying methods of Sofowara (Sofowara, 1993). Reducing sugar and resins were evaluated by following methods of Dipali (Dipali et al., 2013). Coumarins, anthraquinones, cardiac glycosides and phlobatannins were detected by applying the methods of Trease and Evans (Trease and Evans., 1989).

Determination of total phenolic content (TPC)
TPC of the extracts was determined by using the Folin-Ciocalteau method with slight modification (Gao et al., 2000). Briefly, the extracts and standard gallic acid solution (1 ml) was mixed with 2.58 ml of Folin-Ciocalteu's phenol reagent. After 3 min, 0.3 ml of saturated sodium carbonate solution was added to the mixture and incubated at room temperature (25°C) for 20 min. Then, absorbance of each sample was measured at 760 nm with a spectrophotometer. TPC of the extracts was calculated from the regression equation (r 2 = 0.958) of the standard gallic acid and the results were expressed as milligram per gram of gallic acid equivalent of the dried extracts.

Determination of total flavonoid content (TFC)
1 ml extract in methanol (200 mg/ml) was mixed with 1 ml aluminium trichloride in ethanol (20 mg/ml, and a drop of acetic acid), and then the mixture was diluted by the addition of ethanol up to its 25 ml volume. Blank samples were prepared by adding all the reagents with equal volume used in the sample, except the extract. The absorbance of the solution was read at 415 nm after 40 min of incubation at room temperature. Using the same procedure for absorbance of quercetin, standard compound of flavonoid was read and TFC of the extracts was calculated from the standard curve (r 2 = 0.902) of the quercetin (12.5 to 200 mg/ml). Total flavonoid content was expressed as mg/g of quercetin equivalent (Kumaran and Karunakaran, 2007).

Estimation of total flavonol content (TFlC)
TFlC was determined by applying a method previously described by Mbaebie et al. with slight modification (Mbaebie et al., 2012). According to the method, 1 ml of the extracts (200 µg/ml) was taken separately in different test tubes. 2 ml ethanol solution of AlCl 3 and 3 ml of (50 g/l) sodium acetate solution were added in the test tubes. After gently mixing, all the test tubes were allowed to stand for 2.5 h at 20°C temperature. Then, absorbance was determined by using a spectrophotometer at a wavelength of 440 nm. Quercetin was used as standard flavonol compound. Following the aforementioned procedure, absorbance of the quercetin was taken at various concentrations (25 to 400 μg/ml) of series dilution. TFlC of the extracts was calculated from regression equation (r 2 = 0.951) of the standard quercetin and the results were expressed as milligram per gram of quercetin equivalent of the dried extracts.

Determination of total antioxidant content (TAC)
TAC of the extracts was evaluated by phosphomolybdenum complex method with slight modification, which was described by Prieto et al. (1999). Briefly, a reagent solution was prepared having 0.6M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate in distilled water. 1 ml of each extract was combined with the reagent solution in separate test tubes. After shaking gently, the test tubes were incubated for 90 min at 95°C temperature. Then after cooling at room temperature, absorbance was measured at 695 nm wavelength using a spectrophotometer. Similarly, ascorbic acid, a standard antioxidant, was run through the process at different concentration gradient (25 to 400 μg/ml). Using this absorbance value, a standard calibration curve and a regression equation (r 2 = 0.964) was derived, from which TAC of each of the extracts was determined and expressed as mg/g of ascorbic acid equivalent of the dried extracts.

Determination of 1, 1-dipheny-l-2-picrylhydrazyl (DPPH) radical scavenging activity
The DPPH free radical scavenging activity was measured by an established method described by Braca et al. (2002). Briefly, 0.004% w/v of DPPH radical solution was prepared in methanol and then 900 μl of this solution was mixed with 100 μl of extract or standard ascorbic acid solution (12.5 to 200 μg/ml) and kept in a dark place for thirty minutes. Then, absorbance was measured at 517 nm. Scavenging capacity of DPPH radicals (% Inhibition) was measured by the following formula and finally the 50% inhibition concentration (IC 50 ) was calculated using MS-Excell software.
Where A 0 = Absorbance of control group, A s = Absorbance of sample.

Ferric reducing antioxidant power (FRAP) assay
The Fe 3+ reducing power was determined by the method of Oyaizu (1986) with slight modifications. Shortly, 1 ml of extract or standard ascorbic acid solution was taken in a test tube and mixed with 2.5 ml of phosphate buffer solution (0.2 M, pH 6.6). Then 2.5 ml of potassium ferricyanide (1%) was added and incubated at 50°C for 30 min. After that, 2.5 ml of trichloroacetic acid (10%) was added and centrifuged at 4000 rpm for 10 min. Finally, 2.5 ml of the supernatant was mixed with 2.5 ml of distilled water and 0.1 ml of FeCl 3 (0.1%) solution followed by incubation at 35°C for 10 min. The absorbance was measured at 700 nm and the reducing power of the extracts was compared with the standard ascorbic acid. From standard calibration curve, median effective concentration (EC 50 ) was calculated. The EC 50 value (µg/ml) is the effective concentration giving an absorbance of 0.5.

Anticholinesterase (AChE and BChE) assays
AChE from Electric eel and BChE from equine serum were used to explore the enzymes inhibitory potential of A. salvifolium root extracts by using Ellman's assay (Classics et al., 1961). The assay is based on the hydrolysis of acetylthiocholine iodide or butyrylthiocholine iodide by the respective enzymes and the formation of 5-thio-2-nitrobenzoate anion followed by complexation with DTNB to give a yellow colour compound which is detected with spectrophotometer beside the reaction time.

Preparation of solutions
A phosphate buffer solution (0.1 M and 8.0 ± 0.1 pH) was prepared by adding K 2 HPO 4 (17.4 g/L) and KH 2 PO 4 (13.6 g/L) in distilled water. Various concentrations (25,50,100,200,400, 800 μg/ml) of the extracts and standard drug Donepezil were prepared by series dilution. AChE (518 U/mg solid) and BChE (7 to 16 U/mg) were diluted by adding the freshly prepared buffer solution up to obtain 0.03 and 0.01 U/ml concentration of the enzymes, respectively. Solutions of DTNB (0.0002273 M), ATChI and BTChI (0.0005 M) were prepared in distilled water and were kept in eppendorp caps in the refrigerator at 8°C temperature.

Spectroscopic analysis
For these assays, 5 μl of AChE/BChE enzymes were taken in different cuvette followed by addition of 205 μl sample (extracts/standard solution) and 5 μl DTNB reagent solutions. The solution mixture in each cuvette was mixed gently and maintained at 30°C for 15 min using water bath with subsequent addition of 5 μl substrate solution (ATChI in AChE containing cuvettee and BTChI in BChE containing cuvettee). Absorbance was read against a blank solution by using a UV-Visible spectrophotometer. The absorbance of each solution along with the reaction time was taken for four minutes at 30°C. The enzyme activity and enzyme inhibition by control and tested samples were calculated from the rate of absorbance change with time (V = ΔAbs / Δt) as follows: Enzyme inhibition (%) = 100 -percent enzyme activity. Enzyme activity (%) = 100 × V/V max . Where, V is the enzyme activity in the presence of standard drug or extracts and V max is the enzyme activity in the absence of extracts or standard drug. 50% inhibition concentration (IC 50 ) values were calculated by using MS-Excel software.

Determination of correlation (r 2 ) between antioxidant activities and phytochemical assay
MS-excel program was used to determine the correlations between antioxidant activities and phytochemical contents. IC 50 values of DPPH, EC 50 values of FRAP and TAC were put against TPC, TFC and TFlC values of the extracts. In each set, pearson correlation (r 2 value) was determined from the regression equation.

Statistical analysis
All the data were presented as the mean value of triplicate experiment (n=3) along with standard deviation (Mean±SD). P* < 0.05, P** < 0.01 and P*** < 0.001 were considered as significance level. ANOVA, followed by dunnett's test was done in SPSS version 15.0 and 95% confidence of interval was calculated from it. IC 50 and EC 50 values were calculated by using the MS-excel program.TPC, TFC and TFlC were calculated from regression equation of each standard sample by using the program (MS-excel). All the figures were prepared by using Graph Pad Prism software, version 5.0.

Preliminary phytochemical screening
Preliminary phytochemical screening of the extracts revealed the important bioactive metabolites which are presented in Table 1.

Total phenol content (TPC)
All the extracts showed phenolic content with significant (P**<0.01, P***<0.001) difference among them which are summarized in Figure 1A. DASR, among the extracts, showed the highest phenolic content followed by CASR.

DPPH free radical scavenging activity
All the extracts inhibited DPPH radicals at concentration gradient manner (more concentration more inhibition).  (Table 2).

Ferric reducing power assay
Reducing power of all the extracts and the standard compound ascorbic acid was increased with the gradual increase of concentration.  (Table 2).

Total antioxidant content (TAC)
The phosphomolybdate method, another quantitative method of antioxidant effect measurement, is based on the reduction of molybdenum (VI) to molybdenum (V) which takes place for the presence of antioxidant compound in the extracts. In the present study, all experimented samples had good TAC but in significantly (P* < 0.05, P** < 0.01 and P*** < 0.001) different extent. CASR had the highest (639.55 ± 10.51) while EASR had the lowest TAC (114.11 ± 12.83). The order of TAC among the extracts was CASR > DASR > AASR > EASR ( Figure 1D).  (Table 3).

Correlation between antioxidant effects and phytochemicals
The correlation analysis was performed to investigate the Data are expressed as mean ± standard deviation (n = 3). P*< 0.05, P**<0.01 and P***<0.001 are considered as significant difference of IC 50 /EC 50 value compared with the highest value.
relationship between the phytochemicals and antioxidant activity of the extracts. Among the phytochemicals, TFlC showed strong positive correlation with DPPH (r 2 = 0.913), FRAP (r 2 = 0.803) and TAC (r 2 = 0.782). TFC had well positive correlation with TAC (r 2 = 0.764) while weak correlation with DPPH and FRAP effects. TPC showed weak correlation with FRAP and TAC but moderate correlation with the DPPH test (Table 4).

DISCUSSION
Oxidative stress plays a vital role for generation and progression of AD, where nerve cells or cellular components are oxidized by some free radicals that are considered as powerful oxidizing agents. Among these, the ROS ( • O 2 -, • OH, H 2 O 2 , O 3 ) are very potential to induce lipid peroxidation and subsequently cell death. These are generated mostly by mitochondrial oxidation and moderately by the influence of environmental pollutants, smoking and harmful radiations (Lobo et al., 2010). We have a self protective mechanism against the radicals, namely antioxidant defense system, composed of some enzymatic antioxidants, main function of which is to protect our body from the oxidative stress. Here, antioxidants, enzymatic or non enzymatic, show their Data are expressed as mean ± standard deviation (n = 3). P * < 0.05, P ** <0.01 and P *** <0.001 are considered as significant difference of IC 50 value compared with the highest value.  (Laura et al., 2012). The coordinate action of antioxidant system is very critical for the detoxification of the radicals. Superoxide dismutase acts on highly reactive superoxide radical ( • O 2 -) and converts it to less reactive H 2 O 2 radical. Then catalase and glutathione peroxidase converts the H 2 O 2 to water, and thus brain tissues are protected from the reactive radicals (Lin et al., 2008). However, in the case of stress condition, defensive power of the natural antioxidant system declines sharply, since the brain cells consume large proportion of the inhaled oxygen which consequently generates increased number of free radicals due to high metabolic rate in it. Moreover, ascorbate and transient metals, largely present in the nerve tissues, acts as prooxidant and potentiates the oxidative damage of the nerve cells due to their high content of polyunsaturated fatty acids (Laura et al., 2012). So, when free radicals exceeds their normal threshold level, oxidative stress proceeds abundantly, and the cells fail to function effectively, and consequently cellular degeneration takes place which is a way of the AD progression. In this condition, antioxidant supplement is essential to combat with the radicals, and to protect the brain from the cell degeneration (Varcin et al., 2012).
AD is developed by numerous pathogenic factors such as formation of abnormal compound, namely amyloid-β peptide (Aβ) and intracellular neurofibrillary tangles (NFTs), reduction of acetylcholine level and exacerbation of oxidative stress (Iwaki and Namoto, 2014).
Acetylcholine, an organic molecule, acts as a neurotransmitter, and is associated with neuronal networking in central and peripheral nervous systems. Naturally, it is produced in some of our brain cells which are called cholinergic neurons. After a specific life span, ACh goes to break down by the AChE and BChE enzymes. In case of normal healthy people, the rate of synthesis and cleavage of the ACh remain steady to maintain its normal level. In this case, the AChE is 1.5-fold to 60-fold more active than that of BChE. But, in the case of AD, enzyme performance shifts towards the BChE, where its activity increases up to 120%. In contrast, AChE loses its effectiveness by 10 to 15% (Faiyaz et al., 2013). This abnormality, increased break down rate of Ach, leads to decrease the availability of the ACh than its normal physiological scale. Furthermore, the reduced level of ACh adversely affects the physiological functions of the brain. In addition, AChE and BChE potentiates neuronal degeneration by forming some protein complexes such as: neurofibrillary tangles (NFT) and neuritic plaques (NP) which are aggregates of hyperphosphorylated tau protein and extracellular neurotoxic deposits of Aβ, respectively (Dominik and Kamila, 2012). AChE/BChE bind with Aβ and a protein called ApoE protein, resulting in the formation of a highly stable complex (AChE/BChE-Ab-ApoE complex) in cerebrospinal fluid (CSF) of the brain. This stable complex directly interacts with ACh receptors and therefore, interferes with their signal transductions and potentiates ultrafast hydrolysis of ACh (Swetha et al., 2013). Researchers, for example, from postmorterm studies of AD patients, have found strongly reduced number of ACh receptors and loss of basal forebrain and cortical cholinergic neurons (Taiwo et al., 2010). Therefore, inhibition of AChE and BChE is the most effective therapeutic approach to treat the symptoms of AD. Consequently, cholinesterase inhibitors are the only approved drugs for treating patients with mild to moderately severe Alzheimer's disease (Faiyaz et al., 2013).
Many phytochemicals have been reported to have satisfactory antioxidant and anticholinesterase effects. Among these phenolics and flavonoids, potent antioxidative compounds act as free radical scavengers (Fadwa et al., 2012). Majority of the phytochemicals having potent AChE and BChE inhibitory effects, are alkaloids followed by terpinoids, steroids, flavonoids, glycosides, saponins and essential oils (Seyed et al., 2014). Since most of the natural or synthetic products, having enzyme inhibitory effects are known to contain nitrogen atom, the promising effect of the medicinal plants could be due to their high alkaloidal contents (Seyed et al., 2014).
Alangium salvifolium is rich with biologically active phytochemicals where various types of alkaloids have been isolated and identified. Among these alangimaridine, meyhyl-1H pyrimidine-2, 4-dione, alangine A and B, alangicine, markindine, lamarckinine and emetine are important. Besides, phytochemical screening of it revealed the presence of flavonoids, phenolics, glycosides etc (Ashalatha and Gopinath, 2013;Ronok et al., 2013;Savithramma et al., 2013). So, these compounds may be considered for the antioxidant effect and enzyme (AChE and BChE) inhibitory activities of the extracts.

Conclusion
A. salvifolium wang is extensively used as folk medicine. The present study showed that the plant is important for its phytochemical constituents. It has significant amount of phenolics, flavonoid and flavonol. Root extracts of the plant have shown moderate to potent antioxidant potential. These are also effective to inhibit AChE and BChE enzymes. So the plant is effective to protect from alzheimer disease. However, further analysis is necessary to isolate the key compounds and to find out the actual mechanism of action.