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International Food Research Journal 23(5): 1953-1959 (2016)
Anti-cholinesterase inhibitory activities of different varieties of chili peppers
Nantakornsuttanan, N., Thuphairo, K., Kukreja, R.K., Charoenkiatkul, S. and *Suttisansanee, U.
Institute of Nutrition, Mahidol University, Phutthamonthon 4 Rd.,Salaya, Phutthamonthon, Received: 1 July 2015 Alzheimer's disease (AD) is one of the leading neurological disorders that degrade learning, Received in revised form: memory and cognitive functions of a nervous system, eventually leaving a person with an 28 January 2016 inability to perform any function on his/her own. One of several AD causes involves loss of Accepted: 10 February 2016 presynaptic markers of a cholinergic system due to cholinesterase enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which degrade neurotransmitters, acetylcholine (ACh). Currently, the investigation in natural products that can act as functional foods is of an interesting matter to promote health benefits. Chili pepper is previously reported to contain various bioactive compounds that can promote several health benefits. Nevertheless, limited information regarding the control of AD through inhibition of AChE and BChE are available. Alzheimer's disease Therefore, the aim of this experiment was to investigate the cholinesterase inhibitory activities Chili peppers of Thai local chili peppers including Yellow pepper, Bird pepper, Green pepper, Cayenne Pepper, Jinda-green pepper, Jinda-red pepper, Young pepper, Chili Spur pepper and Sweet pepper using a colorimetric high throughput screening methodology. As results, all chili peppers (in exception of Yellow pepper) exhibited AChE inhibitory activities under the range of 5-26% inhibition with Young pepper extract exhibiting the highest inhibition. Interestingly, all chili peppers (in exception of Young pepper) exhibited the BChE inhibitory activities under the range of 2-24% inhibition with Jinda-green pepper extract exhibiting the highest inhibition. Both AChE and BChE inhibitory activities were in dose-dependent manners. These results possibly that the bioactive compounds such as capsaicin, myricetin, quercetin and luteolin in chili pepper might function as anti-cholinesterase agents, since these compounds have been previously reported to be capable of effectively inhibiting cholinesterase enzymes in vitro. The information received from this study would support further investigation on potential natural bioactive compounds from chili pepper with anti-AD property through inhibition of All Rights Reserved There are currently two cholinesterase enzymes in focus; acetylcholinesterase (AChE), an enzyme that Alzheimer's disease (AD), the most common degenerates neurotransmitter acetylcholine (ACh) type of dementia, is an age-related neurodegenerative as its specific substrate (Vengaiah et al., 2007), disorder. The causes of AD could possibly come and butyrylcholinesterase (BChE), an enzyme from brain cells damaging/injury and complicate that degenerates neurotransmitters; butyrylcholine with neuron interaction, which induce brain cells to (BCh) and ACh (Park et al., 2012). Degeneration be inoperable, dysfunctional and eventually death. of the neurotransmitters results in decline of these In the early stage, AD patients are incapable of neurons. It has been found that the cholinergic controlling their performances and might be partially markers, the enzymes responsible for ACh synthesis unconscious during daily activity. In the long term, and degradation, were significantly impaired in AD they may lose cognitive abilities necessary for patients in comparison to elderly control subjects (Sun maintaining an independent living style.
et al., 2007). As well, inhibition of AChE resulted in As of present, there is no certain pathway the increase of non-plaque forming amyloids in the for treatment of AD. However, inhibition of cortex of rat brain (Deepa et al., 2007).
cholinesterase has so far provided the most The β–amyloid plaque formation along with promising results in terms of AD treatment (Luke plaque deposit is one of the main hypotheses or and Wildman, 2006; Al Othman et al., 2011). histological associated AD. The β-amyloid plaques *Corresponding author. Tel: 0-2800-2380 ext. 422; Fax: 0-2441-9344 Nantakornsuttanan et al./IFRJ 23(5): 1953-1959 are aggregated protein fragments, which are degraded Table 1. The general information of chili peppers used in from amyloid precursor protein (APP) in brain cell membrane. Hydrolysis of APP by β- and γ-secretases can lead to development of amyloid plaques (amyloidogenic pathway). These β-amyloid plaques or senial plaques can disturb brain cell communication and stimulate innate immune response, leading to cell inflammatory, cell abnormality, and eventually cell death (Checler, 1995).
Many medical treatments for AD have been investigated, in which 6 major groups of anti- AD drugs, including AChE inhibitors (AChE-I), N-methyl-D-aspartate (NMDA), receptor antagonists, monoamine oxidase (MAO) inhibitors, antioxidants, metal chelators and anti-inflammatory drugs, have been developed. Originally, anti-AD drug Materials and Methods
was discovered as AChE inhibitors, which cause an increase in acetylcholine levels, leading to reduced Plant material and extraction AD symptoms such as memory loss, abnormality Chili peppers (Yellow pepper, Bird pepper, Green of thinking and language. These drugs have been pepper, Cayenne Pepper, Jinda-green pepper, Jinda- approved by Food and Drug Administration (FDA), red pepper, Young pepper, Chili Spur pepper and including donepezil, galantamine, rivastigmine and Sweet pepper) (Table 1) were purchased during June, tacrine. However, these drugs have side effects such 2014 from local market in Nakhon Pathom province, as usually diarrhea, tiredness, dizziness, confusion, Thailand. The samples were clean with deionized headache, vomiting, nausea, fatigue, insomnia, water and cut into small piece (approx. 0.5 x 0.5 cm). heart attack and stroke (Kannappan et al., 2011). The samples were then freeze-dried (Heto Power Thus, prevention and treatment of AD from natural Dry PL9000, Thermo Fisher Scientific, Waltham, products such as fruits and vegetables that can be MA, USA) before being ground into a fine powder consumed daily are of interest due to no/less side by a cyclotec sample mill (series 1903 with 200– effect or toxicity to be concerned.
240V and 50/60 Hz; FOSS, Höganäs, Sweden). The Chili pepper, a significant ingredient in many moisture content after freeze drying was determined traditional cuisines in various countries, has been using Association of Official Analytical Chemists widely investigated regarding their biological (AOAC) method 930.15 (AOAC, 2005). All samples properties toward health benefits (Pakaski et al., were kept in vacuum bags and stored at –20°C.
2009). Chili pepper is an important source of Dry chili powder (0.2 g dry weight) was extracted bioactive compounds with antioxidant activities with 70% (v/v) aqueous ethanol (8 mL). The mixture such as vitamin C, vitamin E, carotenoids, phenolic was vortexed for 5 min and then sonicated in a water compounds and alkaloids (Kulisic-Bilusic et al., bath sonicator (model B1510, 40 KHz; Bransonic 2008; Stefano, 2013). Interestingly, some of these Bransonic® Ultrasonic, Danbury, CT, USA) for 10 antioxidants exhibit biological functions against min before shaking in a water bath shaker (Memmert AChE and BChE (multi-functional compounds), GmbH, Wisconsin, USA) at 60ºC for 1 hour. The suggesting the relationship among these biological mixture was then centrifuged at 1190 x g for 10 min, functions. Even through chili pepper has been reported and the supernatant was collected for further analysis.
as a rich source of antioxidants, its function against AD is limited. Therefore, the aim of this research was Cholinesterase inhibitory activity to investigate the biochemical properties of customly The enzymatic assay for cholinesterase activity consumed chili peppers against some key enzymes was performed utilizing a well established protocol (beta-site APP-cleaving enzyme 1 (BACE-1) and (Jung et al., 2009) with some changes as follows. The cholinesterases) that control AD. This research will inhibitory enzymatic assay consisted of cholinesterase be useful for promoting chili pepper consumption (5–20 ng Electrophorus electricus AChE (1,000 units/ for health benefits and developing functional food, mg) or 10–50 ng equine serum BChE (≥10 units/mg nutraceutical or dietary supplement with biological protein)), thiocholine (0.08 mM acetylthiocholine property against AD. (ATCh) or 0.1 mM butyrylthiocholine (BTCh)), 5,5'–dithiobis(2–nitro benzoic acid) (DTNB, 0.8 Nantakornsuttanan et al./IFRJ 23(5): 1953-1959 mM) and chili extracts (5 mg/mL) in a 96–well plate. All chemicals were received from Sigma–Aldrich (St. Louis, MO, USA). Enzyme inhibitory activity was measured at a wavelength of 412 nm using a 96-well microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) with a Gen5 data analysis software. The initial rate was fitted by the Michaelis– Menten equation with least squares fit parameter using a GraphPad Prism software version 5.00 (GraphPad Software, Inc., La Jolla, CA, USA). The enzymatic inhibitory activity was calculated as % inhibition using the following equation; % inhibition = 100 x (1 – ((B–b)/(A–a))), Figure 1. The AChE inhibitory activity from various types of chili peppers (5 mg/mL) including Young pepper, Chili where A was an initial velocity of the control reaction spur pepper, Green pepper, Jinda-green pepper, Sweet (without plant extract) with the enzyme, a was pepper, Jinda-red pepper, Bird pepper and Cayenne an initial velocity of the control reaction without pepper. The different letters showed statistically significant enzyme, B was an initial velocity of the enzyme difference at p value < 0.05using one-way ANOVA and reaction with chili extract and b was an initial Duncan's Multiple Range Test velocity of the reaction with chilli extract but without enzyme. Eserine (Sigma–Aldrich, St. Louis, MO), of presynaptic markers of a cholinergic system, a reversible anti–cholinesterase drug, was used as a accumulation of β-amyloid plaque in the brain, standard inhibitor for both AChE and BChE assays.
neurofibrillary tangles or abnormal tau protein and oxidative stress induction (Jung et al., 2009). Beta-secretase inhibitory activity Most researches have been focused on cholinergic Beta-secretase (BACE1) inhibitory activity hypothesis and β-amyloid formation, which are two was analyzed using β-secretase (BACE1) FRET important platforms for development of anti-AD (fluorescence resonance energy transfer) Assay Kit drugs. Nevertheless, the adverse effects of these (Sigma–Aldrich, St. Louis, MO, USA). The assay synthesized drugs were recently reported, and green consisted of BACE1 enzyme (0.006 U/µL), BACE1 medicines have been in the center of current interest.
substrate (Rh–EVNLDAEFK–Quencher in 50 mM Chili with different degree of spicy favor is an ammonium bicarbonate), fluorescent assay buffer (50 important ingredient that is customly consumed in nM sodium acetate) and chili extract (5 mg/mL) in the various quantities in many dishes around the world. 96–well plate. The reactionwas mixed and incubated Being involved in many traditional cuisines, it is of at 37°C for 2 hours. The reaction was monitored at interest to investigate the effect of different types of an excitation wavelength of 545 nm and an emission chili regarding anti-AD properties via inhibition of wavelength of 585 nm using the 96-well microplate some key enzymes that control the disease. Chili, a reader. The inhibitory activity will be reported as plant in the Capsicum genus and Solanaceae family, percentage of inhibition as above.
can be divided into five domesticated species, including C. annuum, C. frutescens, C. chinense, C. baccatum and C. pubescensa. In Thailand, only C. All data were expressed as mean ± standard annuum (Green pepper, Young pepper, Chili Spur deviation (SD) of triplicate assays. One way analysis pepper and Sweet pepper) and C. fretescens (Yellow of variance (ANOVA) and Duncan test were pepper, Bird pepper, Jinda-red pepper, Jinda-green performed to determine the significant differences pepper and Cayenne Pepper) are agriculturally and between values with p<0.05. All statistical analyses commercially available. Thus, variation between were performed using IBM SPSS Statistics version these species that affects anti-AD properties is of 19.0 (IBM Corp, Armonk, NY).
interest for health maintaining purpose.
As a result, all chili peppers, in exceptionof Results and Discussion
Yellow pepper, exhibited AChE inhibitory activities under the range of 5-26% inhibition with Young Scientific researches proposed four main pepper extract exhibiting the highest AChE inhibitory hypotheses of AD development, including a loss activity (Figure 1). Young pepper (C. annuum) that Nantakornsuttanan et al./IFRJ 23(5): 1953-1959 was used in this experiment is a hot pepper in its young-green (premature) stage. It was previously found that hot pepper fruits in C. annuum L. var. acuminatum species contain different bioactive compounds, depending on ripening stage (Conforti et al., 2007; Menichini et al., 2009). The first stage of maturation (small green premature stage) exhibited the highest antioxidant activity (IC of 129 µg/ mL) as being detected by DPPH (1,1–diphenyl–2– picrylhydrazyl)–radical scavenging assay and total phenolic contents (76 mg/g) as being detected by Folin–Ciocalteu method (Conforti et al., 2007). Besides, the investigation on the effect of ripening stage of methanolic extract of C. annuum L. var. acuminatum regarding its AChE inhibitory activity Figure 2. The BChE inhibitory activity from various types was suggested that the half maximal inhibitory of chili peppers (5 mg/mL) including Jinda-green pepper, concentration (IC ) of the premature green pepper Bird pepper, Chili spur pepper, Green pepper, Yellow extract exhibited the highest AChE inhibitory activity pepper, Cayenne pepper, Jinda-red pepper and Sweet (IC of 84.30 µg/mL) (Loizzo et al., 2008). This pepper.The different letters showed statistically significant inhibitory activity was decreased during ripening difference at p value < 0.05using one-way ANOVA and stage of mature green and red peppers (IC of Duncan's Multiple Range Test 96.69 and 130.03 µg/mL, respectively). However, pepper fruits in other ripening stages (Conforti et when comparing to the IC of physostigmine (0.07 al., 2007). Myricetin is a strong antioxidant (DPPH µg/mL), the commercial anti-cholinesterase drug, value of 16.2 µM) (Khanduja and Bhardwaj, 2003) it was suggested that the chili peppers under this with anti-AChE activity (K of 37.8 µM) (Katalinic et investigated extraction condition might be insufficient al., 2010). Similarly, sterol content was found to be for treatment of AD. Nevertheless, when comparing decreased during ripening stage of C. annuum L. var. to other fruits and vegetables such as ginkgo (Mitra acuminatum (Conforti et al., 2007). Some sterols were et al., 2013), pomegranate (Choi et al., 2011), proven to be AChE inhibitors (Hopia and Heinonen, mulberry (Shih et al., 2010), lemon juice (Girones- 1999). However, when comparing to eserine (K of Vilaplana et al., 2012), black chokeberry (Girones- 0.144 µM as being measured in our laboratory), a Vilaplana et al., 2012), turmeric (Kannappan et al., reversible anti–AChE drug, it was found that these 2011), garlic (Kannappan et al., 2011), black pepper compounds might not be the effective anti-AChE (Kannappan et al., 2011), ginger (Kannappan et al., agents for AD treatment but might be useful for 2011), and cinnamon (Kannappan et al., 2011), these chemoprevention of AD.
chili peppers might be the potential food source for On the other hand, all chili peppers, in exception prevention of AD.
of Young pepper, exhibited the BChE inhibitory The chemical composition analysis of C. annuum activities under the range of 2-24% inhibition L. var. acuminatum suggested that some polyphenolic with Jinda-green pepper and Bird pepper extracts compounds with antioxidant properties such as exhibiting the highest BChE inhibitory activities luteolin, myricetin and sterol might be responsible (Figure 2). Jinda-green pepper and Bird red-pepper for high antioxidant and anti-AChE activities as (C. frutescens) that were used in this experiment are being observed in the experiments (Helmja et al., hot peppers in their mature stage. This species (C. 2007). It was found that premature green pepper frutescens) is commonly used to make flavoring in exhibited the highest quantity of luteolin (76.0 food due to their hot spicy taste (hotter than chili mg/g), followed by mature green pepper (73.8 mg/g) peppers in C. annuum species). Among all five and matures red pepper (43.2 mg/g), respectively domesticated species, C. frutescens chili peppers (Conforti et al., 2007). Luteolin possessed the DPPH possess the highest quantity of capsaicinoid (1560 radical-scavenging activity (IC ) of 2.051 µg/mL mg/100 g), a phenolic compound that causes chili to (Conforti et al., 2007) and AChE inhibitory activity have spicy and pungent favors (Orhana et al., 2007). with reversible inhibition constant (K ) of 65.8 µM Capsaicin (8-methyl-N-vanillyl-6-nonenamide), (Katalinic et al., 2010). Likewise, it was found a main capsaicinoid in chili peppers, under the that green pepper exhibited the highest quantity of concentration of 1 mg/mL was significantly inhibited myricetin (658.2 µg/g), comparing to other chili BChE reaction with 75.3% inhibition (Orhana et al., Nantakornsuttanan et al./IFRJ 23(5): 1953-1959 2007). Comparing to galantamine, an anti-AD drug suggested that (1) consumption of chili peppers might for mild to moderate AD patients, under the same possibly prevent AD occurrence through inhibition concentration with 80.3% BChE inhibition, capsaicin of some key enzymes that control the disease, (2) could be a future potential anti-AD agent (Katalinic some antioxidants could exhibit multi-functional et al., 2010). Other bioactive compounds, including properties, (3) AD hypotheses could be related to each myricetin, quercetin and luteolin could also reversibly other, and (4) different types of chili peppers could inhibit human plasma BChE with K of 71.0, 68.0 influent different degree of anti-AD properties. The and 166.1 µM, respectively (Orhana et al., 2007; first suggestion was also depended on bioavailability Katalinic et al., 2010). However, when comparing to and absorption of bioactive compounds in chili eserine (K of 0.305 µM as being measured in our peppers. These bioactive compounds were also laboratory), it was found that these compounds might found to possess multi-biological functions i.e. not be the effective anti-BChE agents for AD patients being antioxidants and anti-cholinesterase agents, but might be useful for AD prevention.
thus leading to correlation between AD hypotheses Interestingly, it was previously reported that (i.e. cholinergic hypothesis and oxidative stress capsaicin could promote amyloidogenic route of induction).
brain amyloid precursor protein processing (Pakaski Interestingly, different types of chili peppers et al., 2009). However, no inhibitory activity in could interact with AChE and BChE with different β-secretase assay was observed in all chili peppers degrees of inhibition. It has been previously found extracted under investigated conditions (final that BChE has less substrate specificity than AChE concentration of extracts in the assay was 5 mg/mL). and has a wider range of substrates acting on both It was possible that low concentration of the extract ACh and BCh substrates (Orhan et al., 2007). The acyl could not inactivate β-secretase. The experiment (functional moiety on the substrate) binding region of was then repeated using Sweet pepper with higher AChE and BChE are different in terms of catalytic concentration (30 mg/mL) as a study case and was residues. AChE consists of larger Phe residues in the found that the extract could inhibit β-secretase with active site, leading preferable small substrates such 31% inhibition. Thus, it could be concluded that as ACh. This smaller substrate would fit better in the chili peppers at the concentration of 5 mg/mL could catalytic pocket than the larger molecule. On the other effectively inactivate cholinesterase reactions but not hand, the active site of BChE consists of Leu and Val β-secretase. Since effective inhibitors are normally residues, which are smaller amino acids, allowing designed to mimic natural substrate of individual it to accept larger substrates such as BCh (Orhan et enzyme, these results might be correlated to the al., 2007). Hence, AChE is able to hydrolyze ACh particular characteristics of the substrates (such as efficiently but has low ability in cleaving esters with structure, size, and interaction(s) between enzyme bulkier acyl moieties such as BCh. BChE, on the other and substrate) for each enzyme. Cholinesterase hand, is an esterase with less substrate specificity due hydrolyzes small organic compounds, ACh and to its structure that allows cleavage of various esters BCh, while β-secretase hydrolyzes APP, the integral including ones with bulkier acyl moieties (Katalinic membrane protein. The inhibitors for β-secretase et al., 2010). Not only that these two enzymes can be divided into two main groups, pseudopeptide are also found to work differently, but also may β-secretase inhibitors and non-peptidomimetic contribute to the reason as to why BChE inhibitory β-secretase inhibitors (Ghosh et al., 2012; Ghosh and activity was higher in comparison to AChE. BChE Osswald, 2014). The former are generally substrate acts even when the substrate is in excess, but under analogues, while the later are organic compounds i.e., the same condition, feed-back inhibition is observed macrocyclic inhibitors, hydroxyethylamine-based for AChE (Tougu, 2001). In this experiment, all chili inhibitors and carbinamine-based inhibitors (Ghosh peppers, in exception of Jinda-green pepper, Bird et al., 2012; Ghosh and Osswald, 2014). These non- pepper and Yellow pepper, exhibited higher AChE peptidomimetic β-secretase inhibitors possess large inhibitory activities than BChE inhibitions, which molecular size with unique functional groups in their possibly suggested that anti-cholinesterase agents in structures, which can form specific interactions with these peppers might be more specific towards AChE β-secretase. Thus, smaller anti-cholinesterase agents active site than that of BChE. On the other hand, anti- found in chili peppers might not be able to form proper cholinesterase agents in Jinda-green pepper, Bird interaction(s) or fit properly in the catalytic pocket of pepper and Yellow pepper might be less specific to β-secretase, leading to no inhibitory activity that was cholinesterases. For example, these peppers might observed at low concentration of chili extracts.
contain various types of large molecules that could From these experimental results, it could be fit into the active site of BChE but could not enter Nantakornsuttanan et al./IFRJ 23(5): 1953-1959 the catalytic pocket of AChE, thus resulting in higher pepper (Capsicum annuum L.) genotypes during BChE inhibition than AChE inhibition.
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