In vitro assessment of selected Korean plants for antioxidant and antiacetylcholinesterase activities

Abstract Context: Antiacetylcholinesterase (AChE) drugs have been a main therapeutic treatment for Alzheimer’s disease because increased AChE levels play a key role in reducing neurotransmission. Objectives: Extracts from 35 Korean plants were selected and screened for antioxidant and anti-cholinesterase activity to explore new sources derived from Korean natural resources that could be used as AD therapeutic agents. Materials and methods: The antioxidant effect of extracts from 35 selected Korean plants was determined using two most common free radical scavenging assays using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS). Additionally, the effect of extracts, identified as antioxidants, on acetylcholinesterase inhibition was assessed by an acetylcholinesterase assay kit. Results: Out of 36 extracts of 35 plants tested, Oenothera biennis L. (9.09 μg/mL), Saururus chinensis (Lour.) Baill. (9.52 μg/mL) and Betula platyphylla var. japonica (9.85 μg/mL) showed strong DPPH scavenging activity. Twelve other extracts also exerted moderate free radical scavenging activities with IC50 values ranging from 10 to 50 μg/mL. Antioxidant capacity detected by ABTS assay was only significant in O. biennis (23.40 μg/mL), while the other extracts were weak or unable to reduce the production of ABTS. Based on the antioxidant activities of these plant extracts, 19 extracts with IC50 values less than 100 μg/mL in DPPH assay were selected for further AChE inhibition assay. Among the extracts tested, the IC50 value for Prunella vulgaris var. lilacina NAKAI (18.83 μg/mL) in AChE inhibitory activity was the lowest, followed by O. biennis (20.09 μg/mL) and Pharbitis nil Chosy (22.79 μg/mL). Conclusions: Considering complex multifactorial etiology of AD, the extracts of P. vulgaris var. lilacina (aerial part), O. biennis (seed) and P. nil (seed) may be safe and ideal candidates for future AD modifying therapies.


Introduction
Alzheimer's disease (AD), a progressive age-related disease of the central nervous system (CNS), is characterized by deterioration in neurological function (Bartzokis 2004;Adewusi et al. 2011). AD is the most common type of dementia; 50-60% of dementia cases in the aging population are reported to be AD (Nordin et al. 1995). Although its exact cause remains uncertain, previous research has shown that lack of cholinergic neurotransmission and deposition of misfolded extracellular b-amyloid (Ab) plaques and neurofibrillary tangles in the CNS are hallmarks of this disease (Ali et al. 2015). As the main pathological feature of AD, Ab deposition prevents neurons from acquiring sufficient nutrition and causes increased levels of reactive oxygen metabolites (Ali et al. 2015;Haque and Nazir 2016;Wu et al. 2017).
Oxidative stress is known to be a key factor in the aging process. Since this is associated with Ab plaque deposition which causes neuronal oxidative stress in AD patients, it is considered as a main pathogenesis cause of AD (Adewusi et al. 2011;Zhao et al. 2013). It was previously reported that reactive oxygen species (ROS) play an important role in neurodegenerative diseases (Zhao et al. 2013), and eventually contribute to neuronal death, ultimately causing impaired memory, cognitive ability and behavioral problems (Ali et al. 2015;Haque and Nazir 2016;Wu et al. 2017).
Recent studies have also elucidated the involvement of acetylcholinesterase (AChE) in AD cognitive deficits (Haque and Nazir 2016). The enzyme AChE hydrolyzes and breaks down acetylcholine (ACh) in the synaptic cleft. ACh is the neurotransmitter responsible for cholinergic transmission in the brain, and deposited within neurofibrillary tangles and Ab plaques in the CNS (Dhanasekaran et al. 2015). The resulting lack of cholinergic neurotransmission due to reduced ACh levels eventually leads to cognitive deficits and in the worst cases, death (Adewusi et al. 2011;Ali et al. 2015). Thus, therapies that inhibit AChE and thereby increase ACh levels are promising temporary treatments for AD (Sallam et al. 2016).
AD's multifactorial nature suggests that a multitargeted therapeutic approach might be more advantageous than single-target drugs and combination therapies. This has led to sustained searches by many research groups for natural drug candidates with antiamyloidogenic and antioxidant properties in addition to cholinesterase inhibitory activity (Mathew and Subramanian 2014). Natural products have been proven as antioxidant sources as well as AChE inhibitors (Adewusi et al. 2011;Mathew and Subramanian, 2014). The discovery of the naturally derived AChE inhibitors galantamine and rivastigmine, which are approved by the US Food and Drug Administration (FDA), has encouraged the search for other novel AD therapeutic agents from natural products (Mehta et al. 2012;Wang et al. 2016). Even though these AChE inhibitors are unable to prevent disease progression, they can improve cognitive dysfunction in mild to moderate AD cases (Wilcock et al. 2000;Schulz 2003). However, these inhibitors, along with other currently available inhibitors, are reported to have adverse side effects including gastrointestinal disturbances (Schulz 2003;Mukherjee et al. 2007;Adewusi et al. 2011). Thus, there is a need to discover new potent anti-AD agents with minimal side effects (Ali et al. 2015).
In this study, extracts from various plants originating in Korea were screened for antioxidant and AChE inhibitory activity. In the search for new anti-AD agents, these plants are appropriate to screen since the plants themselves and health supplement formulations derived from them are well tolerated with few side effects. They also possess definite beneficial effects such as anti-inflammatory and antioxidant activity, which are related to brain function. In this study, 35 different plants traditionally used in Korea for rejuvenation, anti-inflammation and/ or improving memory and cognitive function were selected (Table 1) (Zee 2009). The current study is the first attempt to identify and compare potential antioxidant and AChE inhibition candidates from these plants.

Plant materials
All plant materials were procured from the National Institute of Horticultural and Herbal Science (NIHHS), Eumseong-gun, Chungcheongbuk-do, Korea, and authenticated by Dr Seung-Eun Lee, Department of Herbal Crop Research, NIHHS, Eumseonggun, Chungcheongbuk-do, Korea. Voucher specimens of the   Table 1.

Plant extract preparation
Freshly collected plant materials were dried in a hot air oven at 55 C and then pulverized. Plant parts used are presented in Table 2. To prepare the extracts, 5 g of each powdered plant was extracted using the indicated solvent under different conditions (Table 2) and each filtered extract was concentrated by complete evaporation in a vacuum centrifuge. The dried extract was then stored at À20 C. The entire study was conducted using a single batch of each plant extract to avoid batch-to-batch variation and maximize product consistency.
Determination of antioxidant activity by scavenging effect on 2,2'-diphenyl-1-picryl hydrazyl radical (DPPH) In each well of 96-well microplate, 100 lL aqueous solution from the sample (control: 100 lL of distilled water) was added to an ethanolic solution of DPPH (100 lL, 60 lM) based on a previously reported method with minor modifications (Eom et al. 2016). The absorbance at 540 nm was measured using a microplate reader (Tecan SPECTRAFluor; Tecan UK, Goring-on-Thames, UK) after mixed gently and allowed to stand at room temperature for 30 min. Ascorbic acid was used as a DPPH scavenging positive control. Determination of antioxidant activity by scavenging effect on 2,2 0 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) The ABTS activity was measured according to the previously reported method (Moreno-Montoro et al. 2017). After adding 0.5 mL of sample to 3 mL of the diluted ABTS solution, the absorbance was measured at 415 nm using a spectrophotometer. The ABTS radical scavenging (%) was calculated as (1ÀA/ A 0 ) Â 100, where A 0 is the absorbance of the control, and A is the absorbance of the samples. The IC 50 value was calculated as the concentration of sample required to scavenge 50% of free radicals. Ascorbic acid was used as an ABTS scavenging positive control.

AChE enzyme activity assay
Acetylcholinesterase inhibition activity was determined using an acetylcholinesterase assay kit according to the manufacturer's protocol (BioVision, Milpitas, CA). In brief, 50 mL sample and assay buffer was added to each well of a 96-well plate, followed by the addition of 50 mL of the reaction mixture. After incubation at 37 C for 20 min, the absorbance at 570 nm was measured using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT).

Statistical analysis
Statistical significance was determined with analysis of variance (ANOVA) followed by a multiple comparison test with Bonferroni's adjustment. p Values less than 0.05 were considered statistically significant. Analyses were performed using SPSS ver. 19.0 (SPSS Inc., Chicago, IL).

Results
Free radical scavenging activity of the extracts using DPPH and ABTS assay Multiple factors are involved in the development of neurodegenerative disease. Considering AD's complex multifactorial etiology, phytochemicals that have antioxidant as well as AChE inhibitory activity have been considered to be safer and better therapeutic candidates for treating AD (Parodi et al. 2015). Antioxidant therapy has proven successful for improving cognitive function and behavioral deficits in patients with mild to moderate AD (Gutzmann and Hadler 1998).
The antioxidant activity of extracts from 35 selected Korean plants (Table 1) was determined using the free radical DPPH. IC 50 values for DPPH radical scavenging were determined based on the concentration of the extract required for approximately 50% of the original activity. IC 50 values for all extracts are presented in Table 2. Strong IC 50 values were obtained for O. biennis L. (9.09 lg/mL), S. chinensis (Lour.) Baill. (9.52 lg/mL) and B. platyphylla var. japonica (9.85 lg/mL), whereas the value for ascorbic acid was 4.97 lg/mL. F. japonica (Houtt.) Ronse Decr., A. capillaris Thunb., S. buergeriana Miq., S. tenuifolia (Benth.) Briq., A. koraiensis NAKAI, C. pilosula (FR.) NANNF, I. tinctoria var. yezoensis OHWI, A. tartaricus L., H. dulcis Thunb, P. vulgaris var. lilacina NAKAI, P. nil Chosy and C. setidens NAKAI also exerted moderate free radical scavenging activities with IC 50 values ranging from 10 to 50 lg/mL (Table 2). Interestingly, the MeOH extract from the roots of S. buergeriana Miq. exhibited good DPPH scavenging activity with a corresponding IC 50 value of 48.10 lg/mL, whereas the MeOH extract from its aerial parts was not active (IC 50 > 200 lg/mL). In addition, the extracts of 35 selected Korean plants were investigated for their antioxidant properties using ABTS radical scavenging capacity assay (Table  2). Antioxidant capacity detected by ABTS assay was only significant in O. biennis (23.40 lg/mL), while the other extracts were weak or unable to reduce the production of ABTS (Table 2).

Acetylcholinesterase (AChE) inhibitory activity of the selected extracts
Based on plant extract antioxidant activities, 19 extracts with IC 50 values less than 100 lg/mL in DPPH assay were selected for further AChE inhibition assays. The main pathological feature that characterizes AD is reduction in cholinergic acetylcholine (ACh) neurotransmission (Bae and Lee 2015;Li et al. 2016;Xu et al. 2016). Acetylcholinesterase (AChE) is responsible for ACh hydrolysis. Reduced levels of ACh eventually lead to cognitive dysfunction and even death. Thus, AChE inhibition is considered as the most valuable therapy for AD (Sim et al. 2014). Although this disease cannot be prevented from progressing, there are various AChE inhibitors currently available that can improve symptoms in mild to moderate AD patients (Schulz 2003;Mehta et al. 2012). However, intensive research is still needed to discover new candidates against AD since the current AChE inhibitors carry adverse effects (Wilcock et al. 2000;Mehta et al. 2012). 19 extracts selected based on their antioxidant activity were tested for AChE inhibitory activity and the results are shown in Table 3 represent % inhibition at 100 mg/mL and the IC 50 for tested extracts. Donepezil hydrochloride was used as the standard AChE inhibitor in this study which showed an IC 50 of 0.03 lg/ mL. The IC 50 value for AChE inhibitory activity was the lowest for P. vulgaris var. lilacina (18.83 lg/mL) followed by O. biennis (20.09 lg/mL) and P. nil (22.79 lg/mL). These plant extracts also showed very high antioxidant activity in the DPPH assay. In addition, O. biennis exhibited significant activity in reducing the production of ABTS (Table 2). 92.42 ± 3.72 0.03 a Tested extracts were the same as those used for the antioxidant assay. b IC 50 values were determined by curve-fitting the data points using nonlinear regression. c Donepezil hydrochloride was used as a positive control for the AChE inhibition activity.

Discussion
Out of 35 Korean plants screened, the extracts of dry plant parts from P. vulgaris var. lilacina (aerial part), O. biennis (seed) and P. nil (seed) were selected as candidate sources for potent AChE inhibitors as well as antioxidants. Due to AD's multifactorial pathogenesis, multi-targeted drugs are preferred as an effective therapeutic strategy. These selected Korean plants exhibiting in vitro AChE inhibition and antioxidant activity act on multiple therapeutic AD targets and can be consumed daily in our diet to provide their neuroprotective effects.
Oenothera biennis (Onagraceae) is commonly known as evening primrose and its seeds are known for their high antioxidant activities (Budin cevi c et al. 1995). The seeds of O. biennis are also believed to have medicinal value mainly due to the presence of c-linolenic acid, which is known to be an essential dietary supplementation for humans. c-Linolenic acid improves many pathological conditions including dermatitis, platelet aggregation and high blood pressure (Corrigan et al. 1998;Barre 2001). c-Linolenic acid is an omega-6 (n-6) fatty acid, which is known to have anti-inflammatory activity along with omega-3 (n-3) fatty acids and affects the pathogenesis of many diseases where inflammation plays a critical role including cancer, diabetes, heart disease and AD (Kapoor and Huang 2006). In one study, n-6 fatty acids were reported to contribute to the improvement in learning tasks and recovery from neurotoxins, which suggested the potential for O. biennis seeds to be utilized as a candidate therapeutic agent for AD (Yehuda et al. 1996). The crude extract from O. biennis seeds was also reported to have antioxidant activities due to the involvement of phenolic constituents (Wettasinghe et al. 2002). In this work, the MeOH extract from O. biennis seeds showed AChE inhibitory effects with an IC 50 value of 20.09 lg/ mL, which was the highest antioxidant activity among the tested extracts.
The aerial parts of P. vulgaris var. lilacina (Labiatae), which have been used in Chinese folk medicine to calm irritated skin and heal wounds, are rich in phenolic acids such as rosmarinic acid, caffeic acid and kaempferol. Rosmarinic acid is the main component in the aerial parts of P. vulgaris var. lilacina. It has been shown to exhibit antioxidant effects by suppressing lipoperoxidation and scavenging superoxide radicals ( Skottov a et al. 2004). In addition, it also inhibits the formation of b-amyloid (Ab) plaques. Deposition of misfolded extracellular Ab plaques is one of the main possible causes of AD. The inhibitory activity of rosmarinic acid eventually protects against memory impairment (Alkam et al. 2007). Caffeic acid, one of the main components of the aerial parts of P. vulgaris var. lilacina, is well-known for its antioxidant and anti-inflammatory activities and was also found to possess a significant protective effect against b-amyloid-induced neurotoxicity by inhibiting calcium influx and tau phosphorylation (Sul et al. 2009). Kaempferol, another major component of P. vulgaris var. lilacina, has been found to protect PC12 and T47D cells from b-amyloid-induced toxicity (Roth et al. 1999). In this regard, the aerial parts of P. vulgaris var. lilacina, with these phytochemicals as the major content, should be further assessed as an adjuvant therapeutic agent for AD. In this study, the MeOH extract from the aerial parts of P. vulgaris var. lilacina was tested for AChE inhibition as well as antioxidant activity and it showed the highest AChE inhibition with an IC 50 value of 18.83 lg/mL along with high antioxidant activity.
Pharbitis nil (Convolvulaceae) is known as morning glory and the seeds of P. nil (Pharbitidis Semen) have been used as a purgative drug in folkloric medicine in Asian countries ). The seeds of P. nil are reported to be a rich source of diverse phytochemicals, including resin glycosides, gibberellins, flavonoids, anthocyanins, diterpenoids, lignans, triterpene saponins and phenolic compounds (Kim et al. 2008(Kim et al. , 2010(Kim et al. , 2011(Kim et al. , 2013Park et al. 2016). The bioactivity of the constituents of P. nil is still underexplored; however, polysaccharides from P. nil seeds were recently reported to possess antioxidant activities (Wang et al. 2014). Although study on the possible medicinal usage of P. nil seeds is still limited, the properties of phytochemicals such as polysaccharides, flavonoids, diterpenoids and phenolic compounds suggest the potential use of P. nil seeds as a treatment for AD. Flavonoids are known to possess neuroprotective properties that improve cognitive function, and show protective effects against memory deficits associated with normal aging (Macready et al. 2009). There has also been a report on the potential AChE inhibitory activity of diterpenoids, which provides a theoretical basis for further research and utilization of P. nil seeds for cholinesterase inhibitory activity (Hung et al. 2011). In addition, some of the diterpenes, lignans and phenolic compounds isolated from P. nil seeds were found to show anti-neuroinflammatory activity by inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 microglia cells (Kim et al. 2011(Kim et al. , 2013. Under pathological conditions, microglia cells, which are the immune resident cells of the brain, are over-activated and produce a variety of pro-inflammatory mediators including NO, which consequently leads to various neurodegenerative conditions of the CNS including Parkinson's and Alzheimer's disease (Kim et al. 2015;Suh et al. 2016). In the current study, the MeOH extract from the seeds of P. nil was tested for antioxidant activity and showed high activity in scavenging reactive oxygen species, which led to a subsequent assay to observe its AChE inhibitory effects. The extract showed AChE inhibition with an IC 50 value of 22.79 lg/mL.

Conclusions
The extracts from plants originating in Korea, which have been used in Korea for rejuvenation and anti-inflammation and/or improving memory and cognitive function, were screened for AChE inhibition and antioxidant activity for the first time.
Of the 35 plant materials tested, the extracts from the aerial part of P. vulgaris var. lilacina, the seeds of O. biennis and the seeds of P. nil were selected as promising candidates for sources of potent AChE inhibitors as well as antioxidants. Considering the complex multifactorial etiology of AD, these selected plant extracts may be safe and ideal candidates as therapies against AD. Further evaluation to identify active ingredients and assess safety and bioavailability using in vivo animal models is required.