The Effect of Different Extraction Conditions on Acetylcholinesterese Inhibitory Activities
of Gynura procumbens Leaves
Triwoot Phanyotha11,*, Chalat Santivarangkna22, Nattapol Tangsuphoom33, Warangkana
Srichamnong44, Uthaiwan Suttisansanee55,#
1
M.Sc. Food Science for Nutrition, Institute of Nutrition, Mahidol University, Phutthamonthon 4
Rd., Salaya, Phutthamonthon, NakhonPathom 73170), Thailand
2
Institute of Nutrition, Mahidol University, Phutthamonthon 4 Rd., Salaya, Phutthamonthon,
NakhonPathom 73170), Thailand
*
e-mail: triwootpolo1@hotmail.com, #e-mail: uthaiwan.sut@mahidol.ac.th
Abstract
Gynura genus, especially G. procumbens, is a local medicinal plant, which can be
customarily consumed as side dish vegetable. It contains high bioactive compounds with wellknown biological functions against oxidative stress related diseases. Some of these compounds
have been reported to possess multi-functional properties such as being anti-oxidative agents as
well as being inhibitors against some key enzymes that control some non-communicable diseases
i.e., lipase inhibitors (control obesity) and cholinesterase inhibitors (control Alzheimer’s
disease). Nevertheless, little information on the acetylcholinesterese (AChE) inhibitory activity
of G. procumbens leaves regarding its extraction conditions is currently available. Thus, the
present study was aimed to investigate the effect of different extraction conditions on AChE
inhibitory activities of G. procumbens leaves. Freeze-dried G. procumbens leaves (0.2 g of
sample)were extracted using designed experiments including extraction time (15-240 minutes),
concentration of aqueous ethanol (0-100% v/v), liquid to solid ratio (20-50% v/w) and extraction
temperature (30-90 ºC). As results, the optimized extraction conditions of G. procumbens leaves
were 60 minutes of extraction time, 40% (v/v) aqueous ethanol, 40% (v/w) of liquid-to-solid
ratio and 30 °C of extraction temperature. The information received from this research would
support further investigation on optimized extraction conditions by using response surface
methodology and isolation of bioactive compounds from leaves of G. procumbens.
Keywords: Gynura procumbens, Alzheimer’s disease, Acetylcholinesterese Inhibitor, Extraction
condition
Introduction
Alzheimer’s disease (AD) is the most common form of dementia, which is diagnosed in
aged people over 65 years (1). The disease is characterized as impaired ability to retain own skill
(2) as well as non-cognitive function that are associated with behavior and emotional disability
(3, 4). AD occurrences have been suggested as genetic inherence and environmental induction
(5). These matters could happen through several hypotheses such as oxidative stress induced
tissue damage, formation of β–amyloid plaques, abnormality of Tau protein and decreased
cholinergic synapses. Currently, the medicinal treatment of AD has been focused on the
termination of physiological role of cholinergic synapses, which is related to cholinesterase
enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The cholinergic
function depends on the rapid hydrolysis of the neurotransmitter acetylcholine (ACh) by
cholinesterases (6). Hence, inhibition of these enzymes is one of the pathways currently
employed to support the prevention and treatment of AD. Interestingly, AChE or primary
cholinesterase are located on the surface of synaptic cleft and erythrocytes, while BChE or
pseudocholinesterase can be found in general body such as liver, brain, skin, gastrointestinal
smooth muscle and plasma. Besides, AChE is more specific to its own substrate, the
neurotransmitter ACh, than BChE, which degrades other esters such as benzoylcholine, procaine,
succinylcholine and propanidid as well. Thus, AChE-targeted inhibition is more specific for
preventing AD occurrence than BChE.
Currently, anti-AD drugs are synthetic medicines i.e., tacrine, donepezil, and the natural
product-based rivastigmine (7). These drugs are employed to reduce the rate of broken down
acetylcholine (8). However, AChE inhibitors that used for AD treatment have most common side
effects such as are nausea and vomiting, both of which are linked to cholinergic excess (9).
Therefore, the substituted compound from natural produce that has high potential for prevention
of AChE without severe side effects is currently of interest.
Gynura genus, especially Gynura procumbens, is a folk medicinal plant, which can be
customarily consumed as a side dish vegetable. This plant could be grown and populated around
the world, especially in tropical regions such as Thailand and other Southeast Asian countries. It
contains high bioactive compounds with well-known biological functions against oxidative stress
related diseases. However, limited information is available regarding the pharmacologic
properties against AD through AChE inhibition. Thus, the aim of this study was to determine
AChE inhibitory activities of G. procumbens leaves under selected extraction conditions.
Materials and Methods
Fresh leaves of G. procumbenswere freeze-dried and then ground to fine powder by a
cyclotex sample mill (series 1903 with 200–240 V and 50/60 Hz from FOSS, Höganäs, Sweden)
before being stored at –20 °C. Sample powder was extracted under designed extraction
parameters including extraction time (X1), concentration of ethanol (X2), liquid-to-solid ratio
(X3), and extraction temperature (X4). The limited ranges in each factor were designed as 15-240
minute in X1, 0-100% (v/v) aqueous ethanol in X2, 20-50% (v/w) in X3, and 30-90 °C in X4. The
mixture after extraction was centrifuged at 2,810 g for 10 minutes. The supernatant was collected
and filtered through Whatman No.1 filter paper. The filtrate was then kept at -20 °C for further
analysis.
The cholinesterase activity was determined utilizing a well-established protocol (10) with
some changes as follows. The inhibitory enzyme assay consisted of AChE (5-20 µg),
acetylthiocholine (0.08mM), DTNB (0.8 mM), and G. procumbens extracts. The reaction was
then monitored at a wavelength of 412 nm using a microplate reader (BioTek Instrumental, Inc.,
Winooski, VT) and Gen5 data analysis software. The inhibitory activity was determined as %
inhibition. The percentage of inhibition is 100 x (1 – ((B–b)/(A–a))), where A is an initial
velocity of the control reaction with enzyme, a is an initial velocity of the control reaction
without enzyme, B is an initial velocity of the enzyme reaction with extract and b is an initial
velocity of the reaction with extract but without enzyme. One way analysis of variance
(ANOVA) and Tukey’s multiple comparison tests were performed to determine significant
differences between values (p<0.05). All statistical analyses were carried out using SPSS
software for window (version 16.0, SPSS Inc., IL, USA).
Results
Despite being widely investigated on its biological functions, the AChE inhibitory
activity of G. procumbens leaves has never been previously reported. These studies were, thus,
aimed to investigate the AChE inhibitory activity of G. procumbens leaves under designed
extraction conditions. As results (Table 1), the AChE inhibitory activities of G. procumbens
extracts (0.2 g of sample) ranged from 12-60% inhibition as being analyzed by varying
extraction times, concentrations of ethanol, liquid-to-solid ratios and extraction temperatures.
The optimized extraction conditions were determined as the highest AChE inhibitory activities of
G. procumbens leaves that were extracted under designed experiments. It was suggested that 60
minutes of extraction time, 40% (v/v) of aqueous ethanol, 40% (v/w) of liquid-to-solid ratio and
30 °C of extraction temperature provided statistically significant inhibitory activities, which were
higher than others under the particular set of experiments.
Table 1: The AChE inhibitory activities of G.procumbens leaves extracted with various extraction conditions.
Extraction condition of G. procumbens leaves
% inhibition of Acetylcholinesterase#
% Aqueous
liquid-to-solid
Temperature
Time (min)
ethanol (v/v)
ratio (v/w)
(°C)
70
59.44 ± 3.57ab
61.88 ± 0.91ab
62.73 ± 2.98b
55.54 ± 2.67a
57.57 ± 1.94ab
70
15.31 ± 2.84a
21.25 ± 0.09b
34.23 ± 0.99c
15.46 ± 0.52a
14.20 ± 2.21a
12.26 ± 0.96a
20
30
40
50
70
42.06 ± 5.01a
45.35 ± 2.21ab
54.56 ± 5.89b
49.92 ± 3.62ab
50
30
50
70
90
49.47 ± 1.50b
47.91 ± 1.61b
47.77 ± 2.95b
42.09 ± 1.99a
15
30
60
120
240
40
60
0
20
40
60
80
100
40
40
60
60
40
40
#
Each value was represent as mean ± SD (n = 3). Mean within a column in each tested condition was shown with
difference superscript letters, which was significantly different (p<0.05).
Discussion and Conclusion
All the factors used for the extraction were found to be statistically significant (p<0.05).
Under investigated extraction solvents, the AChE inhibitory activity of G. procumbens extracts
significantly increased from 15.31 to 34.23% inhibition when the concentration of ethanol
increased from 0 to 40% v/v. Low ethanol concentration could reduce enzyme activity as a result
of solubility of anti-AChE agents that are likely dissolved in a solvent with lower polarity than
water. However, as the concentration of ethanol continued to rise, the AChE inhibitory activity
declined to 12.26% inhibition, suggesting that ethanol could decrease enzyme activity by
interfering stability of AChE.
Interestingly, it was found that extraction time was associated with thermal factor. It was
observed that when fixing the extraction temperature at 70 °C and varying the extraction time
from 30 to 60 minutes, the AChE inhibitory activity was significantly raised from 59.44 to
62.73% inhibition. Short extraction time could lessen enzyme activities since low quantity of
anti-AChE agents was extracted. However, when extraction time was increased to 240 minutes,
the AChE inhibitory activity was dropped to 57.57% inhibition. Thus, the optimized extraction
time was 60 minutes. Since the reaction proceeded at high temperature (70 °C), enzyme activity
could decline with longer reaction time as a result of decreased AChE stability at high
temperature. This hypothesis was confirmed by varying extraction temperatures from 30 to 90
°C (while fixing extraction time at 60 minutes), where the AChE inhibitory activities were
significantly decreased from 49.47 to 42.09% inhibition. High temperature could, as well,
destroy anti-AChE agents and demote solvent extraction.
Lastly, under investigated extraction liquid-to-solid ratio, the optimized AChE inhibitory
activity was found at 40% (v/w) ratios. The inhibitory activity was significantly elevated from
42.06 to 54.56% inhibition as increased liquid-to-solid ratio (20–40% v/w). However, decreased
inhibitory activity was observed with increased liquid-to-solid ratio, which might be a result of
the solubility of anti-AChE agents in G. procumbens leaves. Larger extraction volume likely
provided higher anti-AChE activities than lower volume. This matter could be explained in term
of the increase in driving force for mass transfer or increased solvent-sample interface area.
Thus, the highest AChE inhibitory activities of G. procumbens extracts were found to be
under 60 minutes of extraction time, 40% (v/v) aqueous ethanol, 40% (v/w) of liquid-to-solid
ratio, and 30 °C of extraction temperature. The information received from this research would
support further investigation on the optimization of extraction conditions using response surface
methodology and the isolation of bioactive compounds from leaves of G. procumbens.
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Acknowledgements: This research was performed at the Institute of Nutrition, Mahidol
University. Financial support is received from the Nutrition Association of Thailand through
Amway for Nutrition Research Grant 2013.