Evaluation of cholinesterase inhibitory and antioxidant potential of Wedelia chinensis: possible implications in alleviating Alzheimer’s disease

Wedelia including Although the the of in present study to evaluate and The extract and fractions of the plant were evaluated for acetylcholinesterase and butyrylcholinesterase inhibitory activity by modied Ellman method. The antioxidant activity was assessed in several in vitro models/assays such as reducing power, total antioxidant capacity, total phenolic and avonoid content, scavenging of 2,2’-diphenyl-1-picrylhydrazyl (DPPH) free radical and hydroxyl radical, and inhibition of brain lipid peroxidation. Chromatographic and spectroscopic methods were used to isolate and identify the active compound from the extract. phenolic content and DPPH radical scavenging activity, and lipid peroxidation inhibition and a moderate association was found between total phenolic content with hydroxyl radical scavenging & BChE inhibitory activity. These results indicated strong association of avonoids with both the cholinesterase inhibitory and antioxidant activities.

were procured from Sigma-Aldrich, Germany. Catechin, ascorbic acid and gallic acid were obtained from Wako Pure Chemical Company Ltd., Japan. Methanol, ethylacetate, chloroform and petroleum ether were purchased from Active Fine Chemicals Limited, Dhaka, Bangladesh. All other chemicals, unless speci ed, were of analytical grade.

Animals and collection of brain
Mice were used only for collection of brain as a source of crude acetylcholinesterase enzyme. Swiss Albino Mice having 5-6 weeks were purchased from the Animal House, Jahangirnagar University, Savar, Dhaka and were caged in the experimental room. A standard diet and water ad libitum were given to the mice. The study was carried out in compliance with the ARRIVE guidelines. The international ethical guidelines were followed to deal with the laboratory animals. The procedures were approved by the by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) of the University of Rajshahi, Bangladesh (Ethical clearance number: 102).
To collect brain from mice, mice were anesthetized with sodium pentobarbital (30 mg/kg; intraperitoneal injection; Taj Pharmaceuticals Ltd., India) and then sacri ced by cervical dislocation. Cervical dislocation was performed by a trained person. Brains were taken quickly; washed in ice cold saline and homogenized in 50 mM Tris buffer, PH 7.4 to yield the crude acetylcholinesterase enzyme [17]. Crude methnol extract (CME, 18.5 g) was prepared from plant powder (500 g) of W. chinensis by hot extraction with methanol in a soxhlet apparatus. The extract (10 g) was then concentrated in vacuo with a rotary evaporator and suspended in water. It was then sequentially partitioned with solvents of increasing polarity such as petroleum ether, chloroform, ethylacetate and water by the method as described earlier [17] to yield the corresponding petroleum ether (PEF, 3.2 g), chloroform (CLF, 2.5 g), ethylacetate (EAF, 1.4 g) and aqueous (AQF, 2.9 g) fractions. All the fractions were preserved in a refrigerator at 4°C until further use.

Phytochemical analysis
Phytochemical screening of the plant extract Qualitative tests were performed to identify the classes of phytochemicals such as avonoids, glycosides, alkaloids, tannins, saponins, and steroids in the different fractions by the methods as described earlier [18].
Quantitation of total phenolic content (TPC) Folin-Ciocalteu method was used to determine the the total phenolic content of the extractives of W. chinensis as described [19]. To a mixure of 2.5 ml of 10% Folin-Ciocalteu reagent and 2.5 ml of 7.5% sodium carbonate solution, 0.5 ml sample was added and left in the dark for 20 minutes at 25 0 C. The absorbance of the reaction mixure was recorded by a spectrophotometer at 760 nm. A standard curve was obtained for gallic acid and the phenolic content was determined from extrapolation of this curve.
Quantitation of total avonoid content (TFC) Aluminum chloride colorimetric method was used to measure the total avonoid content of the extracts of W. chinensis as described earlier [20]. To a mixure of methanol (3.0 ml), 10% AlCl 3 (0.2 ml), 1M potassium acetate (0.2 ml) and 5.6 ml of distilled water, plant extract (1.0 ml) was added and left at room temperature for 30 minutes. The absorbance of the reaction mixture was recorded by a spectrophotometer at 420 nm. A standard curve was obtained for catechin and the avonoid content was determined from extrapolation of this curve.

Antioxidant activity
Reducing power assay The reducing ability of the extracts of W. chinensis was determined by the method of Oyazu et al. (1986) [21]. To a mixure of 0.2 M potassium buffer (2.5 ml) and 1% potassium ferricyanide (2.5 ml), plant extract (1 ml) was added and incubated at 50°C for 20 minutes. Then 10% TCA solution (2.5 ml) was added to the reaction mixture and centrifuged (3000 rpm) for 10 minutes. Finally, 2.5 ml of solution was mixed with 2.5 ml of ultrapure water and 0.5 ml of 0.1% ferric chloride solution. The absorbance of the reaction mixture was recorded at 700 nm. A reference standard catechin was used for comparison.

Total antioxidant capacity assay
The antioxidant capacity of the extracts of W. chinensis was assessed by the method as described earlier [18]. To a mixture of sulphuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4 mM), plant extract was added and heated in a water bath at 95 0 C for 90 min. After cooling to room temperature, the absorbance of the mixture was recorded at 695 nm against blank. A reference compound catechin was used for comparison.

DPPH radical scavenging assay
The ability of the extracts of W. chinensis to scavenge DPPH radical was measured by the modi ed method of Choi et al. (2000) [22]. A reference compound catechin was used for comparison. Methanolic solution of plant extract or reference compound was mixed with 0.135 mM of methanolic DPPH and left in dark for 30 minutes. The absorbance of the reaction mixture was recorded at 517 nm. The percent scavenging was calculated using the equation: Where, A control is the absorbance of control and A sample is the absorbance of extract or reference compound. The percentage inhibition was plotted against the compound concentration in order to calculate the IC 50 values.

Determination of hydroxyl radical scavenging activity
The capacity of the extracts of W. chinensis to scavenge hydroxyl radical was assessed by the modi ed method of Elizabeth et al. (1990) [23]. A reference compound catechin was used for comparison. Plant extract or reference compound was added to a 1 ml reaction mixture containing 2.8 mM 2-deoxy-2-ribose, 20 mM phosphate buffer (pH 7.4), 100 μM FeCl 3 , 100 μM EDTA, 1 mM H 2 O 2 and 100 μM ascorbic acid and then incubated at 37°C for 60 minutes. 0.5 ml of the reaction mixture was mixed with 1 ml of TCA (2.8%) and 1 ml of TBA (1%) and heated in a water bath at 90°C for 15 minutes. After cooling to room tempaerature, the absorbance of the mixture was recorded at 532 nm in a spectrophotometer against an appropriate blank solution. The percent scavenging of hydroxyl radical was calculated as in DPPH radical scavenging assay.

Determination of lipid peroxidation inhibition activity
The ability of the extracts of W. chinensis to inhibit the peroxidation of lipid was assesses by the method as described [18]. Brain homogenate was employed for in vitro lipid peroxidation assay. Mice brain homogenates were prepared by homogenizing brain in 50 mM phosphate buffer (pH 7.4) containing 0.15 M KCl using a homogenizer and centrifuged at 10000 g at 4 o C for 20 min. To a mixture of brain homogenates (0.5 ml), 0.15 M KCl (1 ml) and 10 µM hydrogen peroxide (100 µl), plant extract was added and incubated at 37 o C for 30 minutes. A solution of 2 ml of HCl (0.25 N) containging TCA (15%), TBA (0.38%), and BHT (5%) was added to the reaction mixture and heated in a water bath at 80 o C for 60 minutes. After cooling to room temperature, the mixture was centrifuged to separate the supernatant and then the absorbance was measured at 532 nm by spectrophotometer. The percent inhibition of lipid peroxidation was determined as in DPPH radical scavenging assay. A reference standard catechin was used for comparison.

Cholinesterase inhibitory activities
The assessment of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibiting activities were performed by the colorimetric method of Ellman et al. (1961) [24]. Crude AChE enzyme was prepared from mice brain as mentioned above and BChE enzyme was prepared from human blood according to the method as described earlier [18]. The acetylthiocholine iodide and butyrylthiochoilne iodide were used as substrates for investigation of AChE and BChE assays, respectively. The hydrolysis of acetylthiocholine and butyrylthiocholine were determined spectrophotometrically. Plant extract or reference compound was added in an enzyme solution and incubated at 37°C for 15 min for interaction. This was followed by the addition of a 50 mM sodium phosphate buffer (pH 8.0) containing 0.5 mM acetylthiocholine and 1 mM DTNB and immediately the absorbance of the solution was recorded against a blank solution. All the experiments were taken in triplicate. For comparison, a reference compound donepezil was used for AChE activity and galantamine was used for BChE activity. The percent inhibition of cholinesterase activity was computed using the equation: Where, A control is the absorbance of control and A sample is the absorbance of extract or reference compound. IC 50 value could be calculated from the dose response curve obtained by plotting the percent inhibition values against test concentrations of each extract or compound.
Isolation and characterization of an active compound from the bioactive extract The EAF (5.6 g) of W. chinensis was subjected to column chromatography using silica gel 60 (Merck, Germany) as a stationery phase in an open column and sequentially eluted with n-hexane, dichloromethane and methanol stepwise gradient to yield ve major subfractions (F1 to F5). Fraction F2 with potent AChE and BChE inhibitory activity was puri ed on silica gel GF 254 by preparative thin layer chromatography with n-hexane-acetone (6:4) as the mobile phase to obtain the pure compound 1 (18 mg). 1 H-and 13 C-NMR spectra of the compound 1 was recorded in DMSO-d 6 on a Jeol-Ex 400 MHz and FT-NMR 100 MHz spectrometers. The chemical structure of the compound 1 was con rmed by comparing its spectral data with the reported values in the literature [27].

Statistical Analysis
All experiments were carried out in triplicate. The results were expressed as mean ± SD. Graph Pad Prism (version 8.0.1) and Microsoft Excel 2010 were used for the statistical and graphical evaluations. T-test was employed to estimate the statistical signi cance (P-value < 0.05) between the average values. IC 50 values of different fractions/extractives were calculated using non-linear regression (Dose-Response --Inhibition equation; log 10 (inhibitor) vs. normalized response --variable slope) in Graph Pad Prism -8.0.1.
Correlation study was performed using Pearson correlation test.

Phytochemical analysis
A preliminary phytochemical analysis conducted on the CME revealed that the plant contains tannins, phenolics and avonoids, alkaloids, phytosterols and saponins. Qualitative analysis of the four fractions showed that they all contained phenolics and avonoids, but higher amounts were found in the EAF and AQF (Supplementary Table S1).

Cholinesterase inhibitory activity
The CME and its fractions were evaluated for AChE inhibition at different concentration (0-200 µg/ml) using the widely used Ellman method. The percent inhibition of AChE by the extractives has been presented in Fig. 1A. Donepezil was used as the reference AChE inhibitor in this study that showed an IC 50 of 9.21±0.45 µg/ml. All the test extract and fractions exerted dose dependent inhibition of AChE

Antioxidant activity
The antioxidant activity of the extractives of W. chinensis were assessed by using several in vitro models such as DPPH and hydroxyl free radicals scavenging, reducing power and total antioxidant activity.
DPPH is a stable free radical which is widely used for evaluation of scavenging activity of the antioxidant. The percent scavenging of DPPH free radical by different concentration of extract has been shown in Fig. 2A scavenging activity followed by AQF. EAF was found to be more potent than that of the standard catechin whose IC 50 was 5.14±0.10 µg/ml. The CLF and PEF had relatively lower DPPH radical scavenging activity.
Hydroxyl radical is the most harmful radical among the radicals generated in the biological system.
Hydroxyl radicals were generated in vitro in Fenton reaction and the ability of the extractives to scavenge the radicals was determined (Fig. 2B) Reducing power assay was used for assessing the reducing ability of the CME and its fractions and the result has been shown in the Fig. 3A. All the extract and fractions were found to possess the reducing activity and the activity was increased with the increase of the concentration of the extract. At high concentration of 80 mg/ml, the absorbance of CME, AQF, EAF, CLF, PEF and catechin were 1.783±0.027, 2.542±0.05, 2.245±0.06, 1.383±0.02, 0.942±0.01 and 2.451±0.07, indicating that AQF has the highest activity followed by CAT, EAF, CME, CLF and PEF. Notably, the activity of AQF was found to be higher than that of the standard antioxidant catechin.
The total antioxidant activity of the extractives was assessed based on their capacity to reduce Mo (VI) to Mo (V) and the result has been shown in the Fig. 3B. Similar to reducing power, the AQF and EAF exhibited high total antioxidant activity among the extractives ( Figure 3B). At high concentration of 80 mg/ml, CME, AQF, EAF, CLF, PEF and catechin gave an absorbance of 0.456±0.008, 0.556±0.008, 0.529±0.007, 0.365±0.005, 0.347±0.005 and 0.489±0.004, respectively. Interestingly, the total antioxidant activity of AQF and EAF were found to be greater than that of the standard antioxidant catechin.
Oxidation of lipid by free radicals results in lipid peroxidation. In this study, lipid peroxidation of the mouse brain homogenate was induced by hydrogen peroxide and the effect of the different extractives of W. chinensis in the inhibition of lipid peroxidation were assessed via thiobarbituric acid reactive species (TBARS). As shown in the Fig. 4

Correlation between total phenolic and avonoid content and the acetylcholinesterase inhibitory and antioxidant activities
Phenolics and avonoids have been reported to be associated with the antioxidant activity [25,26]. Since AQF and EAF contained a large amount of phenolics and avonoids and exhibited high cholinesterase inhibitory and antioxidant activities, we therefore tested their correlations by Pearson's correlation analysis and the result has been given in the  Activity guided isolation of an active compound Due to potential bioactivity, the EAF was investigated further to isolate and identify the active compounds. Activity guided chromatographic separation resulted in the isolation of a major compound 1 from the EAF and the structure of the compound 1 was established as apigenin by direct comparison of its 1 H-and 13 C-NMR spectral data with previously reported values (Table 3, Fig. 5) [27]. The compound exhibited inhibition of AChE and BChE enzymes as well as antioxidant activity, which are in accordance with the previous results [28,29]. Further studies are required to isolate and identify the active compounds present in the AQF.

Discussion
Alzheimer's disease is a progressively developing neurodegenerative disorder of the elderly people and the most common cause of dementia. Until now there is no effective treatment for AD. Approximately 50 million people are affected worldwide in AD which will triple by 2050 [30]. The problem is much more pronounced in the developing countries [31]. Due to the growing population and extended lifespan, AD has become a serious heath concern in the elderly people. To date, only three cholinesterase inhibitors namely donepezil, rivastigmine, galantamine and one partial NMDA receptor antagonist memantine are the only approved drugs by the Food and Drug Administration (FDA) to treat AD [32]. These drugs offer the symptomatic relief of the disease and only slow the progression modestly, but does not stop the progression of AD [33]. The handful of drugs and the limitations of their use led us to develop new potent drugs. Plant has already proved to be an important source of different classes of drugs and new candidate drugs for AD has been developed. W. chinensis is a potential medicinal herb with multiple biological activities [1,2,3]. Herein, we report for the rst time the cholinesterase inhibitory and antioxidant activities of the extractives of W. chinensis in vitro and apigenin as the major compound contributing to the activity.
The crude methanol extract (CME) of W. chinensis was fractionated with petroleum ether, chloroform, ethylacetate and water for investigating their phytochemical pro le and biological activity. Qualitative analysis of the CME showed the different classes of phytochemicals present in the extract which were differentially distributed in the different solvent fractions. Interestingly, phenolics and avonoids were found to be present in each fraction, but high concentration was found in the ethylacetate and AQF (Supplementary Table S-1). Phenolics and avonoids are an important class of secondary metabolites of plants that display important biological activities. They are known as natural antioxidants due to the ability to scavenge free radical by donating electron or hydrogen. Quantitative analysis of phenolic and avonoid content revealed that EAF contained the highest content of phenolics (97.28 ± 0.49 mg GAE/g dried extract) followed by CME (80.00 ± 0.62 mg GAE/g dried extract) and AQF (61.05 ± 0.21 mg GAE/g dried extract). Whereas AQF contained the highest content of avonoids (175.78 ± 0.69 mg CE/g dried extract) followed by CME (174.02 ± 1.01 mg CE/g dried extract) and EAF (144.35 ± 0.51 mg AE/g dried extract). PEF and CLF had lower content of phenolics and avonoids ( Table 1). The presence of large amount of phenolics and avonoids in the EAF and AQF indicated that they might have potential biological activity.
AChE is still a therapeutic target of choice among other targets for the development of AD drug. Inhibitors of AChE increase the concentration of acetylcholine at the synapse, augment the cholinergic neurotransmission and improve the memory and cognition in animal. Medicinal plants contain a diverse chemical compounds having cholinesterase inhibiting properties and currently used in the treatment of AD [34]. Interest in natural AChE inhibitor has increased due to less toxic effects of the natural compounds. In this study, we report for the rst time a substantial AChE inhibitory activity of the CME of W. chinensis and its fractions (Fig. 1A). IC 50 values were calculated for quantitative evaluation of the inhibitory potency of the different fractions. The IC 50 value of CME was found to be 93.64 ± 0.28 µg/ml. A large number of plants used in traditional medicine use to enhance memory have been investigated, but only a few of them have been found to possess an acceptable level of AChE inhibitory capacity [35]. In comparison with these plants, W. chinensis extract appeared to be a potential AChE inhibitor. When the fractions were evaluated, highest inhibition was found in the AQF and EAF with the IC 50 values of 40.02 ± 0.16 µg/ml and 57.76 ± 0.37 µg/ml, respectively, indicating the polarity of the active compounds. In contrast, CLF and PEF had less activity and their IC 50 were 121.97 ± 0.74 µg/ml and 152.60 ± 1.14 µg/ml, respectively. Pearson's correlation showed a signi cant association of total avonoid content with the AChE inhibitory activity (R 2 = 0.7748, p < 0.05) ( Table 2). These results suggest that the extract of W. chinensis and its fractions possess an appreciable AChE inhibitory activity which might be attributed to avonoid compounds.
After AChE, BChE is another drug target of choice for AD. BChE is a cholinesterase involved in the catalysis of acetylcholine and plays a co-regulatory role in the cholinergic neurotransmission that accounts for 20% of cholinesterase activity [36]. The increased activity of BChE in the late stage of pathogenesis suggests it as a target for AD drug. There are reports that dual inhibition of AChE and BChE have been found a better outcome in AD [37]. Of the three approved AChE inhibitor, rivastigmine has dual activity. In this investigation, the crude methanol extract of W. chinensis was found to exert inhibition of BChE with an IC 50 value of 69.09 ± 0.44 µg/ml (Fig. 1B).  (Table 2). Taken together, these results suggest a dual role of W. chinensis and hold promise as a source of drug for dual activity.
Oxidative stress plays a crucial role in the development of AD. Oxidative stress results from the excessive production of free radicals by amyloid beta-protein, a major culprit according to amyloid cascade hypothesis [12,13]. Although a number of biomolecules are affected due to oxidative stress, lipids in neuronal membrane are most vulnerable that lead neuronal dysfunction and death. Lipid peroxidation is thus considered as an important marker for oxidative stress in AD [17,18,38]. In this study, we found the potential antioxidant activity of the crude methanol extract and its fractions in all antioxidant assays when compared with the other medicinally important plant. In DPPH radical scavenging, which is a stable synthetic radical, EAF showed the highest activity followed by AQF and CME with IC 50 values of 4.78 ± 0.01, 8.60 ± 0.04, 10.13 ± 0.44 µg/ml, respectively ( Fig. 2A). The IC 50 value of catechin was found to be 5.14 ± 0.10 µg/ ml. In hydroxyl radical scavenging, which is relevant to biological system, AQF showed the highest activity followed by CAT, EAF and CME with IC 50 values of 9.65 ± 0.25, 14.89 ± 0.25, 28.21 ± 0.39 and 42.49 ± 0.51 µg/ml, respectively (Fig. 2B). These results suggest that EAF and AQF are potential radical scavenger due to hydrogen donating abilities. Similarly, in reducing power assay, AQF showed the highest activity followed by CAT, EAF and CME (Fig. 3A) and in total antioxidant capacity test, AQF showed the highest activity followed by EAF, CAT and CME (Fig. 3B). At high concentration of 80 µg/ml, the absorbance of AQF, CAT, EAF and CME were 2.542 ± 0.05, 2.452 ± 0.07, 2.245 ± 0.06 and 1.783 ± 0.03 in reducing power assay and 0.556 ± 0.01, 0.488 ± 0.00, 0.529 ± 0.01 and 0.456 ± 0.01 in total antioxidant assay, respectively (Table 1). Both the assays indicated the potential ability of the EAF and AQF to donate electron or proton to free radicals in terminating the chain reaction. It is noteworthy that the activity of AQF was superior to the standard antioxidant catechin; whereas the activity of EAF was very close that of catechin. The antioxidant activity of the crude extracts was re ected in the inhibition of peroxidation of brain lipid from mouse. Among the extractives, the AQF showed the highest inhibition followed by CAT, EAF and CME with IC 50 values of 25.53 ± 0.04, 30.91 ± 0.20, 45.20 ± 0.10 and 47.18 ± 1.07 µg/ml, respectively (Fig. 4). Pearson's correlation showed a strong association of total avonoid content with DPPH radical scavenging, reducing power, total antioxidant activity, hydroxyl radical scavenging, lipid peroxidation inhibition, AChE and BChE inhibitory activity (Table 2). While a signi cant correlation was observed between total phenolic content and DPPH radical scavenging activity, and lipid peroxidation inhibition and a moderate association was found between total phenolic content with hydroxyl radical scavenging & BChE inhibitory activity. These results indicated strong association of avonoids with both the cholinesterase inhibitory and antioxidant activities.
Because the crude methanol extract of W. chinensis and its fractions were found to possess potential cholinesterase inhibitory and antioxidant activity, we explored the bioactive compounds from EAF by bioassay guided chromatography. A major active compound was isolated from EAF and its structure was established as apigenin by 1 H-and 13 C-NMR spectral studies (Table-3, Fig. 5) [27]. The compound exerted substantial inhibition of AChE and BChE enzymes and exhibited appreciable antioxidant activity.
Apigenin is one of the most naturally occurring avonoids that are found in edible and medicinal plants.
The compound has been reported earlier to inhibit Aβ aggregation, and neuroin ammation involved in AD [39]. Apigenin can be absorbed in the intestine and cross the blood brain barrier, suggesting its potential as a therapeutic agent in the neurodegenerative pathologies including AD. In the present study, the isolation of apigenin from W. chinensis having both cholinesterase inhibitory and antioxidant properties, which according to the previous report, can be useful to prevent or slow down the progression of AD.
Taken together, all these evidences suggest that apigenin has neuroprotective potential to prevent or slow down the progression of AD.

Conclusion
Our results demonstrated that Wedelia chinensis exerts substantial inhibition of cholinesterase activity and antioxidant properties, which could be useful for the management of Alzheimer's disease. Apigenin was identi ed as a major compound that contributes to the inhibitory activities. To the best our Declarations MAI, KB, MKH carried out the collection and extraction of plant materials, isolation of compound and the assays for antioxidant and acetylcholinesterase inhibitory activities; MYA, AHMKA performed the statistical analysis, interpretation of data and drafted the manuscript; GS, SZ, TT has made substantial contribution to conception and design, participated in general coordination of the study, and revising the manuscript critically for important intellectual content. All authors read and approved the nal manuscript.

Funding
The research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
International ethical guidelines were followed to deal with the animals and the procedures were approved by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC), Institute of Biological Sciences, University of Rajshahi, Bangladesh (Ethical clearance number: 102).

Consent for publication
Not applicable.