disease (AD) is one of the most widespread neurodegenerative diseases that
involve dementia and mainly afflict people over 65 years of age. The therapy of
early and moderate stages of AD is mainly based on acetylcholine esterase
inhibitors such as synthetic donepezil and galanthamine. However, these licensed medicines
have drawbacks of inducing severe peripheral and central side effects,
including gastrointestinal disturbances, insomnia, fatigue or depression. On
the other hand, since the butyrylcholinesterase (BuChE) and
acetylcholinesterase (AChE) activity in AD increases progressively as the
severity of dementia progresses, researchers have investigated selective BuChE
and AChE inhibitors in the treatment of AD as well . The serious side
effects caused by licensed drugs used to treat AD have forced researchers to
investigate safer AChE inhibitors from natural sources. One of the best sources of new substances to treat AD are natural
products and their derivatives.
Traditionally, plants have been used to enhance memory and to alleviate other symptoms associated with AD .
Liquidambar styraciflua (L.) from
Hamamelidaceae family, is known as sweet gum tree. It is native to
southeastern, east-central, and south-central United States, southern Mexico
and central America . The bark is grayish brown, deeply furrowed into
narrow, somewhat rounded ridges. The leaves are alternate, simple,
dark green and lustrous above. Flowers are monoecious consisting of 2-beaker
ovaries subtended by minute scales. The fruit is a dangling brown, woody spiny
tipped “gum ball” with seeds brownish and winged [4, 5]. In traditional
medicine, L. styraciflua is used in treatment of wounds, the roots were
boiled into a strong tea to treat skin sores, diarrhea and dysentery . The
bark was used to make an infusion that was used as a sedative for nervous
patients and for patients who were well in the day but sick during the night.
The plant was used to treat colic, internal diseases and to “comfort the
heart.”. One report about cancer chemopreventive activity from
triterpenoids from the cones of the plant . The main aim of this present
study is to evaluate
acetylcholinesterase inhibitory activity of L. styraciflua aerial parts
methanol extract and also investigate phytochemical content of the plant
UV/VIS: Shimadzu UV-visible recording spectrophotometer model-UV 240
(NRC, Egypt). 1H-NMR and 13C-NMR (Varian Unity Inova). MS
(Finnigan MAT SSQ 7000, 70 ev). (Silica gel (0.063-0.200 mm for column
chromatography) and Sephadex LH-20 (Pharmacia Fine Chemicals). Thin layer
chromatography (TLC) F254 plates. Solvent mixtures, BAW (n-butanol: acetic acid: water 4:1:5
upper phase, 15% acetic acid: water: glacial acetic acid: 85:15). Paper
Chromatography (PC) Whatman No.1 (Whatman Led.Maid Stone, Kent, England) sheets
for qualitative detection of flavonoids and sugars were used in this study.
Plant identification and collection
Liquidambar styraciflua aerial parts were collected
garden, Giza, Egypt in May 2011. The plant was identified by Dr. Mohammed El-Gebaly, department of botany, national research centre
(NRC) and by Mrs. Tereeza Labib consultant of plant taxonomy at the ministry of
agriculture and director of Orman botanical garden, Giza, Egypt. A voucher specimen was
deposited in the herbarium of
Al-Zohiriya garden, Giza, Egypt.
Air dried aerial parts of L. styraciflua (760 g) were extracted with
methanol: distilled water 80:20 (v/v) several times at room temperature by
maceration method. The extract was concentrated under reduced pressure to give 37 g of methanol extract.
The extract was phytochemically screened according to that described by Yadav
and Agarwala .
Isolation of the
bioactive components of methanol extract of L. styraciflua
styraciflua (35 g)
of aerial parts methanol extract
was subjected to silica gel column chromatography eluting with hexane, dichloromethane,
ethyl acetate and methanol gradually.
One hundred and fifty fractions
of 100 ml conical flask were collected. The fractions that showed similar Paper
Chromatography (PC) in two solvent systems, butanol–acetic acid–water (BAW)
4:1:5 and 15% acetic acid were combined to give 4 fractions (I, II, III and
IV). Fraction I (2.1 g)
was subjected to sub–column of silica gel eluted with n-hexane: dichloromethane
(50:50) gave compound 1 and elution with dichloromethane: n-hexane (80:20) gave
Fraction II (1.45 mg) was
subjected to sub–column of silica gel eluted with dichloromethane: ethyl
acetate (90:10) yielded compound 3 and elution with dichloromethane: ethyl
acetate (95:5) gave compound 4. Fraction III (1.85 g) was subjected to
sub–column of silica gel eluted with ethyl acetate : methanol (95: 5) to give
compound 5 while compound 6 was obtained
from elution with ethyl acetate: methanol (90:10) and also compound 7 was
obtained by elution with ethyl acetate: methanol (80:20). Fraction IV (1.25 g) was subjected to
sub–column of silica gel eluted with ethyl acetate: methanol (70:30) gave
compound 8 while compound 9 was obtained by elution with ethyl acetate:
methanol (50:50). All the isolated compounds were purified on sephadex LH–20
column using different systems of methanol and distilled water.
General method for acid
hydrolysis of flavonoid glycosides
mg of each flavonoid glycoside 6, 7, 8 and 9 in 5 ml 10% HCl was heated for 5 h. The
aglycones were extracted with ethyl acetate and identified by co-TLC with
authentic standards. The sugars in the aqueous layer were identified by
co-paper chromatography (co-PC) with authentic markers on Whatman No. 1 sheets
in solvent system (n-BuOH-AcOH-H2O 4:1:5 upper layer).
Acetylcholinesterase inhibition assay
methanol extract of L. styraciflua was dissolved in methanol to prepare
solution of 10 mg/mL. Then, 1.5 µL of the extract was spotted on silica gel TLC
plate and developed with chloroform: methanol 9:1 after which the enzyme
inhibitory activity was detected using Ellman’s method “in situ” on the plate
[9, 10]. The developed plate was sprayed with 1 mM Ellman's reagent
[5,5'-dithiobis-(2-nitrobenzoic acid)] (DTNB) and 1 mM acetylthiocholine iodide (ATCI) in buffer A. It dried
for 3-5 minutes, then an enzyme solution of AChE from an electric eel (type
VI-s lyophilized, 261 U/mg solid, 386 U/mg protein) dissolved in buffer A (500
U/mL stock solution) was diluted with buffer A to obtain 5 U/mL enzyme and was
then sprayed on the plate . Yellow background with white spot for
inhibiting extract was visible after about 5 minutes. The observation must be
recorded within 15 minutes because they fade after 20-30 minutes. To observe
whether the positive results of the extract in TLC or the microplate assay are
due to enzyme inhibition or to the inhibition of the chemical reaction between
DTNB and thiocholine, (the product of the enzyme reaction), 5 units/mL of AChE
was premixed with 1 mM
ATCI in buffer A and incubated for 15 minutes at 37oC.
This enzyme-substrate mixture was used as thiocholine spray . The extract
was spotted on the silica gel TLC plate developed as described above and
sprayed with 1 mM
solution DTNB followed by the thiocholine spray. White spot on a yellow
background was observed for false positive extract.
The inhibitory effect quantitative of methanol extract of L.
styraciflua on acetylcholinesterase activity is evaluated using and
adaptation of the spectrophometric method of Ellman et al.  modified by Rhee
et al. . Five different concentrations were prepared in triplicate,
starting from the methanol extract of L. styraciflua (1 mg/mL; 0.5 mg/ml;
0.25 mg/mL; 0.125 mg/mL and 0.0625 mg/mL). The reaction was monitored at 412 nm
for 5 min in spectrophotometer.
In test tube is placed 100 µL of the extract (concentration 0.1%
solution in 50 mM
Tris-HCl pH 8, and methanol 10%) was
mixed with 100 µL of AChE 0.22 U / ml (22 U of enzyme diluted in 100 mL of 50 mM Tris-HCl pH 8, 0.1%
BSA) and 200 µL of buffer (50
mM Tris-HCl, pH 8, BSA 0.1%). Incubating the mixture for
5 min at 30°C.
Subsequently add, 500 µL of DTNB (concentration of the 3
mM in Tris-HCl pH 8, 0.1 M NaCl, 0.02 M MgCl2) and
100 µL of ATCI (4 mM
in water). A blank should also be prepared by substituting AChE with 100 µL of
buffer (50 mM
Tris-HCl buffer pH 8, 0.1% BSA). The reaction is monitored for 5 min at 412 nm
and initial velocity (V0) recorded. Anticholinesterase activity (%) was
I (%) = (1 –
Vo sample) x 100/ Vo white
Vo and Vo represents the initial rates blank samples and white.
concentration 50% (IC50) values ??so obtained by plotting
Log-Probit. Neostigmine (or other commercial acetylcholinesterase inhibitor) is
used as positive control at the same.
The present study was focused
on the evaluation of
acetylcholinesterase activity of
L. styraciflua aerial parts methanol extract and
the extract showed a significant inhibition for acetylcholinesterase. enzyme with (IC50 = 0.070 mg/mL). We investigated the presence of phytochemicals and
bioactive constituents in L. styraciflua methanol extract. Phytoconstituents are shown in table 1. The
major bioactive components of L. styraciflua aerial parts methanol extract are ß-sitosterol,
oleanolic acid, ursolic acid, luteolin, luteolin
luteolin 6-C-ß-glucoside (isoorientin),
kaempferol 3-O-a-rhamnoside and kaempferol 3-O-ß-glucoside. The
chemical structures of the bio-active components were elucidated by different
spectroscopic analyses and shown in Figure 1.