INTRODUCTION
Statistical data in 2015 estimated that 46.8 million people in the world have dementia. The amount is predicted to increase almost two times higher so that in 2,030 and 2,050 will reach 74.7 and 131.5 million people (Prince et al., 2015). Generally, people with dementia are from the elderly, and Alzheimer’s is a disease characterized by dementia. Alzheimer's disease is a syndrome of brain cell damage due to the presence of small bodies of protein (called: senile plaque), resulting in decrease neurotransmitters such as acetylcholine. Acetylcholine is a conducting compound of nerve excitatory (neurotransmitter), while acetylcholinesterase (AChE) is an enzyme that can hydrolyze the acetylcholine into choline and acetic acid. This reaction is needed so that the nerves can return to take a rest after the activation process, but it can cause damage to the cells in the brain.
Dementia prevalence in people aged 60 years or above range from 4.6% in Central Europe to 8.7% in North Africa and the Middle East. The estimated number is higher in East Asia and Africa (Prince et al., 2015). The cause of the disease is not fully known, but there is a possibility caused by an inadequate diet. In contrast, Kausler et al. (2007) write out that the disease is less prevalent in China and perhaps other Eastern countries compared to the United States. Some experts believed that one of the ways to avoid Alzheimer's disease is consuming vegetables (Kausler et al., 2007), but this still needs to be proven through scientific research. In Indonesia, Sundanese and Javanese ethnics have a habit of consuming vegetables for daily food. Various kinds of foods that contain vegetables are pecel, lotek, urap, and gado-gado, in the local name.
For centuries, plants have been used as food sources and also as traditional medicines to improve cognitive function and memory due to the elderly (Mukherjee and Houghton, 2009). The consumption of fruit and vegetable every day can reduce the risk of dementia (Barberger-Gateau et al., 2007). Some plants, fruits, vegetables, and spices show the AChE inhibitory activity, thereby potentially increasing memory in people with Alzheimer's. These plants include pomegranates (Mathew and Subramanian, 2014), strawberry forest, apples, potato tubers, celery, parsley leaves (Szwajgier and Borowiec, 2012), cinnamon, rosehip, red cabbage (BoÄŸa et al., 2011), and kale leaves (Dhanasekaran et al., 2015).
There are several synthetic drugs for the treatment of Alzheimer's whose chemical structure comes from plants, which are physostigmine (Physostigma venenosa), galantamine (Galanthus nivalis), and huperzin (Huperzia serrata) (Bhadra et al., 2015). Many research studies showed that phenolic and flavonoid compounds played a role in neurodegenerative diseases. Consumption of foods containing higher levels of flavonoids, especially flavonol, was associated with lower dementia rates in populations in European countries, New Zealand, Australia, America, and Canada (Beking and Vieira, 2010). Previous research studies described the benefits of flavonol quercetin and its glycosylated derivatives in the cellular and animal model of Alzheimer's disease. In vitro experiments showed that quercetin could inhibit the formation of amyloid fibrils (Jiménez-Aliaga et al., 2011). Metabolite quercetin-3-O-glucuronide can reduce the generation of amyloid peptides from the Tg2576 Alzheimer’s disease mouse model. (Ho et al., 2013). Clinical trials with multivariate models showed that food intake with high total polyphenol was associated with better language and verbal memory. High intake of flavonoids and phenolic acids can help to maintain verbal memory, which is the most vulnerable important factor in brain aging (Kesse-Guyot et al., 2012).
This study aims to investigate AChE inhibitory activity from 13 different vegetables that are widely used by Sundanese and Javanese ethnics (Table 1) and also to determine the phenolic and flavonoid content of the extracts. Potential plants as an AChE inhibitor were fractionated by a liquid–liquid partition with n-hexane and water.
MATERIALS AND METHODS
Chemicals
AChE from electric eel (product number C3389, 500 U/mg solid), acetylthiocholine iodide (ATCI), 5,5´-dithiobis-2-nitrobenzoic acid (DTNB), eserine (pyridostigmine), quercetin, and gallic acid was purchased from Sigma-Aldrich. Buffer Tris HCl, NaCl, MgCl2, aluminum chloride, potassium acetate, Folin–Ciocalteu, sodium carbonate, and other chemicals used were analytical grade.
Plant material
Vegetable materials were obtained from various regions in West Java and Central Java, Indonesia. Fresh vegetables were harvested from uncultivated or cultivated fields near Bandung, West Java, such as Lembang, Rancaekek, and Cimahi. Kenikir was obtained from Bogor, West Java, while Turi was obtained from Semarang, Central Java, Indonesia. All of the test materials were determined at Herbarium Bandungense Institut Teknologi Bandung.
Extraction
Fresh materials were dried at temperature 40°C–50°C and powdered. Part of the plant used is presented in Table 2. The dried samples were extracted by maceration method with 95% ethanol . The process was done for 3 days and every day was repeated using a new solvent. The mixture was filtered so that a liquid extract is obtained. The liquid extract was dried using a rotary evaporator (Heidolph). The yield of dry extract was calculated to the starting material (Table 2). The dry extract was stored in a desiccator to avoid humid influence.
Determination of total phenolic content in vegetable extracts
Analysis of total phenolic content in vegetable extracts was carried out spectrophotometrically according to the Folin–Ciocalteu method (Chun et al., 2003) with slight modification. A standard curve was obtained by preparing gallic acid solutions with a concentration of 50, 100, 150, 200, 250, and 300 μg/ml. The standard solution (200 μl) was added to 0.4 ml Folin–Ciocalteu reagent, shaken, allowed to stand for 5 minutes, then it was added 4.0 ml of 7% sodium carbonate solution. The final mixture was shaken until homogeneous and then incubated for 120 minutes at room temperature. The absorbance was measured at 763.0 nm. The solution of ethanol extracts (200 μl) was treated the same as a standard solution. The total phenolic content was expressed as milligrams of gallic acid equivalent (GAE) per gram dry extract.
 | Table 1. List of vegetables including chemical contents. [Click here to view] |
Determination of total flavonoid content in vegetable extracts
Quantitative analysis of the total flavonoid content was carried out by the colorimetric method using aluminum chloride reagent, according to Chang et al. (2002) with slight modification. Quercetin was chosen as a standard with the concentration series of 2, 4, 6, 8, 10, and 12 μg/ml. The standard solution (1.0 ml) was mixed with 1.5 ml of 95% ethanol, 0.2 ml of 10% aluminum chloride, 0.2 ml of 1 M potassium acetate, and 2.1 ml of deionized water. After incubation for 30 minutes at room temperature, the absorbance of the reaction mixture was measured at a wavelength of 422.9 nm. Similarly, each vegetable extracts (1.0 ml) in a solvent were reacted with aluminum chloride for the determination of the total flavonoid content as described above. The total flavonoid content was expressed as milligrams quercetin equivalent (QE) per gram of dry extract.
Fractionation
Ocimum americanum L. and Cosmos caudatus Kunth ethanol extract were continued to fractionation. The ethanol extract was mixed with water (ratio 1:10). The mixture was placed into a separating funnel then added with n-hexane (ratio 1:1). The mixture was then vigorously shaken until interaction occurs between the two phases. After the shaking process, the mixture was allowed to stand for two separate phases. The n-hexane phase was evaporated with a temperature of 45°C until it was dry. The water phase was dried using a freeze dryer.
 | Table 2. Parts used and the yield of dry extract from several plants tested. [Click here to view] |
Determination of AChE inhibitory activity using Ellman’s colorimetric method in 96-well plate
AChE activity was measured using a modified 96-well plate assay as described by Mathew and Subramanian (2014) with minor modifications based on Ellman et al. (1961). Briefly, AChE was from electric eel (product number C3389, 500 U/mg solid). The further enzyme-dilution was done in the Tris-HCl buffer and kept at −20°C. In the 96-well plates, 100 μl of 3 mM DTNB, 20 μl of 0.26 U/ml of AChE, and 60 μl of each extract in various concentrations (500, 750, 1,000, 1,250, and 1,500 μg/ml) dissolved in buffer containing 10% methanol and they were filled in to the wells. Meanwhile, the concentration of the fractions was 200, 400, 600, 800, and 1,000 μg/ml. After mixing, the plate was incubated at 25°C for 15 minutes, and the absorbance value was measured at 412 nm in Tecan infinite 200 pro microplate reader and reading was used as a blank. The enzymatic reaction was initiated by the addition of 20 μl of 15 mM ATCI, and 5-thio-2-nitrobenzoate product was measured by reading the absorbance value every 5 minutes for 30 minutes. Pyridostigmine was used as the standard. The enzyme inhibition (%) was calculated as follows:
Enzyme inhibition (%) = [(E − S)/E] × 100
which E is enzyme activity without extract and S is enzyme activity with the extract. Enzyme activity was calculated from the rate of absorbance change with time (V = Abs/Δt). The IC50 value was determined by linear regression analysis between the inhibition percentage versus the extract concentrations using the Microsoft Excel program. The data were expressed as mean ± standard deviation for each group for the determination in triplicates.
RESULTS AND DISCUSSION
Total phenolic and flavonoid contents
This study determined the chemical characteristic of the extracts that are total phenolic and flavonoid contents (Table 3). Total phenolic contents were calculated based on the equation of the gallic acid standard curve y = 0.00207 x + 0.13513, R2 = 0.99879 and expressed as GAE (mg GAE/g extract). Based on the standard curve quercetin y = 0.04942 x+ 0.14529, R2 = 0.99828, the total flavonoid contents were calculated and expressed in term QE (mg QE/g extract).
 | Table 3. Total phenolic and flavonoid contents of vegetable extracts. [Click here to view] |
Table 3 showed that the extract of C. caudatus, Nasturtium officinale, Nothopanax fruticosus, and O. americanum had higher total phenolic and flavonoid contents compared to other extracts. The total phenolic content of C. caudatus ethanol extract in this study (313.70 ± 27.86 mg GAE/g extract) was almost the same as the results of Siregar and Kristanti, (2019) study that was 312.075 ± 4.772 mg GAE/g extract. Meanwhile, the total phenolic content of N. officinale extract in this study (315.74 ± 1.60 mg GAE/g extract) was lower than the results obtained by Fenton-Navarro et al. (2018), which was 552.5 ± 39.12 mg GAE/g extract. This can be caused by biotic or abiotic factors. Biotic factors are the interaction of plants with microorganism around their environment or plant physiological aspects. Abiotic factors include all physical factors that affect plant habitat including light or ultraviolet (UV)-Vis radiation, water availability, temperature, and soil composition. Some biosynthetic and bioactivity of phenolic compounds are affected by exposure to UV or light (Pavarini et al., 2012).
Ethanol extract from 13 types of vegetables which were commonly consumed by Sundanese and Javanese ethnics had been tested for its AChE inhibitory activity by using Ellman’s method in 96-well plate. The results were shown in Table 4, which represented the percentage of AChE inhibition from various types of extracts. In this study, pyridostigmine was used as a standard of AChE inhibitor, and the IC50 was 0.344 ± 0.005 μg/ml.
Four types of vegetables that could not inhibit the activity of AChE were Cucumis sativus, Sechium edule, Sesbania grandiflora, and Vigna unguiculata (L.) Walp. The four materials showed absorbance values that tend to increase during observation time when compared to treatment without the addition of extracts. The result of AChE inhibitory activity from cucumber ethanol extract was different from water extract that has been reported by Oboh et al. (2017) The study reported that aqueous extract of cucumber could inhibit AChE enzymes, and the extract contained quercetin, caffeic acid, and gallic acid (Oboh et al., 2017). Other studies related to cucumber were carried out by in vivo experiments. Cucumber paste could increase cognition in rodents. Biochemical analysis of AChE activity in brain tissue showed that 9 g/kg of cucumber paste significantly (p < 0.001) reduced brain AChE activity (Kumar and Parle, 2014). The difference in the type of extracts (ethanol extract, water extract, and paste) as the test material caused diversity in the phytochemical content in each extract. Based on this review, the cucumber had AChE inhibitory activity in their water extract or paste form so that it could attract almost all the polar chemical content such as phenolic and flavonoid compounds.
The ethanol extract of S. grandiflora was not able to inhibit AChE. The other studies also reported that flower infusions and tincture of S. grandiflora had the same profile with ethanol extract, but the exception occurs in decoction flower that could inhibit AChE (Baessa et al., 2018). The results of the experiment can be different due to the various type of solvents that were used for extraction. V. unguiculata had various chemical constituents such as cycloartenol, stigmasterol, sitosterol 3-β-ÆŠ-glucoside (Noorwala et al., 1995), anthocyanin delphinidin, cyanidin 3-O-glucoside, quercetin glycosides, and kaempferol glycosides (Ojwang et al., 2012). Legume V. unguiculata was known to have several bioactivities such as antioxidant and antihypertensive but its neuroprotective effect had never been reported (Shakir et al., 2013). This study reported that V. unguiculata could not inhibit AChE activity, so that it might occur because of the adverse effect between the chemical compounds.
Meanwhile, seven types of vegetables (Amaranthus cruentus, Carica papaya, Etlingera hemisphaerica, Leucaena leucocephala, N. officinale, N. fruticosus, and Phaseolus vulgaris) had IC50 values more than 1,000 μg/ml. These seven types of ethanol extracts had never been done before for their AChE inhibitory activity. The smallest AChE inhibitory activity was from P. vulgaris, which produced 8.9% ± 0.3% inhibition at 1,000 μg/ml. Common beans contained phenolic compounds, tocopherols, and unsaturated fatty acids, among other constituents (Los et al., 2018). The methanol extract of common beans contained alkaloid and steroid compounds, while the water extract contained saponins (Doss and Pugalenthi, 2012). Even though common beans have divergence chemical contents, the inhibitory activity of AChE was the lowest.
 | Table 4. Percentage of inhibition and IC50 value of vegetable extract for AChE inhibition assays. [Click here to view] |
The ethanol extract of L. leucocephala seeds only produced 12.5% ± 1.1% inhibition to AChE at 1,000 μg/ml concentration, but other studies reported that acetone extract of L. leucochepala leaves had IC50 values 118.23 ± 4.40 μg/ml (Dzoyem and Eloff, 2015). This showed that L. leucochepala leaves had better inhibitory activity against AChE than its seeds.
The ethanol extract of N. officinale and C. papaya produced 15.6%–16.7% AChE inhibition at 1,000 μg/ml concentration. Both of these extracts had a similar activity for AChE inhibition. Five percents papaya fruit in citric acid solution had AChE inhibitory activity with IC50 20.47 mg/ml (Gironés-Vilaplana et al., 2015). This indicated that the experiment gave relatively similar IC50 value among papaya leaves ethanol extract and papaya fruit juice, which were above 1,000 μg/ml. N. officinale contained flavonoid glycosides (Martinez-Sanchez et al., 2008) and also thiocarbamate group compounds (Breme et al., 2007). Carbamate moiety was a pharmacophore for AChE inhibitory activity. Various carbamates had been proposed as AChE inhibitors (Krátký et al., 2016).
The ethanol extract of A. cruentus had 22.6% ± 0.7% AChE inhibitory activity at 1,000 μg/ml concentration. Phytochemical composition of methanolic extract of A. cruentus revealed the presence of polyphenols, tannins, flavonoids, steroids, and carotenoids (Nana et al., 2012). Ethanol and methanol solvents had almost the same polarity, so they probably had the same active compounds that were extracted in both solvents. The ethanol extract of N. fruticosus leaves also had AChE inhibitor activity with 22.8% ± 0.3% inhibition at 1,000 μg/ml. Three bisdesmosidic saponins were isolated from the methanol extract of N. fruticosus leaves (Hanh et al., 2016). Other studies in vivo reported that saponin compounds which were extracted from Fenugreek seeds (Trigonella foenum-graecum) increased the AChE inhibitor in Alzheimer's-induced rats (Khalil et al., 2016). Therefore, the active compounds in N. fruticosus, which were thought to contribute to AChE inhibition, are saponins.
The ethanol extract of Etlingera hemisphaerica had 45.8% ± 1.3% AChE inhibitory activity at 1,000 μg/ml. This result had never been reported before, but the antioxidant activity of the flower extract and inflorescent extract E. hemisphaerica had existed in previous studies (Maimulyanti and Prihadi, 2015; Sungthong and Srichaikul, 2018). The content of chemical compounds such as tannins, flavonoids, steroids, and phenolics in E. hemisphaerica was believed to play a role in the antioxidant activity, as well as AChE inhibition.
Only two types of vegetables had IC50 values less than 1,000 μg/ml, which were ethanol extract of O. americanum and C. caudatus, with IC50 values 519.4 ± 35.9 and 790.2 ± 20.7 μg/ml, respectively. The ethanol extracts of O. americanum leaves had been studied before and provide 20% inhibition against AChE, while the ethanol extract of the stems did not show any inhibition (Khattak et al., 2005). The methanol extract of O. americanum leaves showed AChE inhibitory activity with IC50 was 2.571 ± 0.199 mg/ml (Farag et al., 2016a). If all of these results were compared, the O. americanum leaves ethanol extract reported in this study showed the higher activity than methanol extract. The ethanol extract of C. caudatus had a slightly lower AChE inhibitory activity compared to O. americanum based on its IC50 value. C. caudatus contained phenolic compounds such as quercitrin, catechin, and rutin (Seyedreihani et al., 2017).
Out of the 13 plants that were screened for their activity toward AChE, there were two plants that were considered potential to be developed further because they had IC50 less than 1,000 μg/ml, which were ethanol extract of O. americanum and C. caudatus. To simplify the chemical compound contained in the extract and obtained greater AChE inhibitory activity, both extracts were fractionated. The fractionation process was using a liquid–liquid partition with n-hexane and water.
The results showed that the water fraction of C. caudatus and the n-hexane fraction of O. americanum could inhibit AChE with the IC50 values less than 1,000 μg/ml. Meanwhile, the n-hexane fraction of C. caudatus and the water fraction of O. americanum could not inhibit AChE activity (Table 5). The fractionation process of these two types of extracts could produce a more active fraction than the extract. This could be seen from the smaller IC50 value from the fraction (on average around 325–374 μg/ml) compared to IC50 value from the extract (on average around 519–790 μg/ml). The chemical content that could be dissolved in n-hexane would be very different from water because these solvents had different polarity. The n-hexane fraction would more easily attract nonpolar compounds, while the water fraction would attract polar compounds.
 | Table 5. The AChE inhibition and IC50 value of the fraction. [Click here to view] |
The water fraction of C. caudatus could extract polar substances such as phenolic and flavonoid compounds. Phytochemical screening of C. caudatus extract contained phenolic and flavonoid compounds, with total phenolic and flavonoid content of 313.70 ± 27.86 mg GAE/g extract and 4.38 ± 0.08 mg QE/g extract (Table 3). C. caudatus contained many chemical compounds, such as chlorogenic acid, catechin, rutin, quercetin, quercetin 3-O-rhamnoside, quercetin 3-O-β-arabinofuranoside, and quercetin 3-O-β-glucoside (Mediani et al., 2012). All of these compounds could be dissolved into the water fraction and were certainly contributed to their AChE inhibitory activity.
On the other hand, the n-hexane fraction of O. americanum was believed to be able to extract essential oils in its leaves because of their similar polarity. Phytochemical screening of O. americanum extract contained terpenoid and flavonoid compounds. Other research reported that essential oils prepared from O. americanum inhibited AChE activity with IC50 value was 570 μg/ml, whereas camphor (terpenoid group) was the most abundant compound found in O. americanum (Farag et al., 2016b). The n-hexane fraction of O. americanum could also extract other non-polar substances like flavonoid group. O. americanum contains flavone class such as luteolin, apigenin, pilosin, and salvigenin (Vieira et al., 2003). Interactions that occurred between all these compounds (terpenoid and flavonoid) could produce a greater inhibitory effect so that the inhibitory activity against AChE was greater. This was shown from the IC50 value of the n-hexane fraction, which was smaller than the essential oil of O. americanum.
CONCLUSION
Thirteen ethanol extracts from various vegetables, which were widely consumed by Javanese and Sundanese people in Indonesia, had been screened for its AChE inhibitory activity, their total phenolic and flavonoid content in the extracts were also determined. Ethanol extracts of C. caudatus and O. americanum were expected to have a promising AChE inhibitory activity, their total phenolic and flavonoid content were higher than other extracts. The active compounds such as phenolic, flavonoid, and terpenoid that contain in water fraction of C. caudatus and n-hexane fraction of O. americanum were a source of natural products as a potent AChE inhibitor.
FINANCIAL SUPPORT AND SPONSORSHIP
Nil.
CONFLICT OF INTEREST
The author’s declare no conflict of interest.
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