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ANTI-ACETYL CHOLINESTERASE COMPOUNDS ISOLATED FROM 1
THE LEAVES OF KIGELIA AFRICANA (LAM) BENTH 2
(BIGNONIACEAE) 3
ABSTRACT 4
Acetyl cholinesterase (AChE) is an enzyme that is involved in the breakdown of some 5
neurotransmiiters. Its inhibition is one of the treatment strategies employed in the management 6
Alzemhier diseases. Flavonoids isolated from the leaves of Kigelia africana were investigated 7
for their comparative AChE inhibition. 8
Powdered leaves of the plant was extracted with 80% methanol for 72 h and concentrated on a 9
rotary evaporator at 35 °C to obtain the crude extract The extract was subjected to vacuum 10
liquid chromatography (VLC) to obtain four fractions using n-hexane (n-hex, 100 %), n-11
hexane/dichloromethane (hex/DCM, 1:1), dichloromethane/ethyl acetate (DCM/EtOAc, 1:1) 12
and ethyl acetate/methanol (EtOAc/MeOH, 1:1). The four fractions were subjected to AChE 13
inhibitory study with DCM/EtOAc (1:1) fraction showing highest inhibitory activity. Three 14
flavonoids were isolated from this fraction and their structures were elucidated and 15
characterised using 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy and mass 16
spectrometry (MS) techniques. Their spectroscopic data compared well with literature. 17
The compounds demonstrated considerable inhibition of AChE activity with luteolin (1), rutin 18
(2) and quercetin (3) that showed IC50 of 945.0, 282.1, 254.8 μg/ml respectively as against the 19
IC50 of 38.93 μg/ml for rivastigmine, a well-known acetylcholinesterase agent. Compound 3 20
showed 17.89 ± 0.57 and 7.70 ± 0.64 μ/l/mg protein at 200 and 400 μg/ml respectively, for 21
AChE activity as against 10.37 ± 0.99 and 6.24 ± 1.24 μ/l/mg protein showed by rivastigmine 22
at 200 and 400 μg/ml respectively. 23
This study showed that the phytochemical responsible for the AChE inhibition in the crude 24
extract as reported by Falode et al., 2017 resided in the DCM/EtOAc (1:1) fraction. The 25
structural-activity relationship of the flavonoids revolves round substitution on position 3 of 26
the compounds. 27
28
Keyword: Kigelia africana, flavonoids, structural elucidation, dementia, acetyl cholinesterase 29
inhibition. 30
31
INTRODUCTION 32
Alzheimier disease is the main cause of dementia accounting for about 75% of all dementia 33
cases. Dementia refers to a very large group of brain diseases that bring about a long term and 34
frequently gradual decline in the ability to think and remember things which in turn affect the 35
individual’s daily performance, but oftentimes, consciousness is not affected [1]. Dementia is 36
one of the most common causes of disability among the old [2], which has been estimated to 37
result in economic costs of 604 billion United States dollar (USD) a year (WHO, 2014). 38
People with dementia are often physically or chemically restrained and undemonstrative. 39
Globally, dementia affected about 46 million people in 2015 [3]. About 10% of people 40
develop the disorder at some point in their lives [4]; it becomes more common with age [5]. 41
A dementia diagnosis requires a change from a person's usual mental functioning and a 42
greater decline than one would expect due to aging [1]. The most common type of dementia 43
is Alzheimer's disease, which makes up 50% to 70% of cases; the most common symptoms 44
of Alzheimer's disease are short-term memory loss and word-finding difficulties. The part of 45
the brain most affected by Alzheimer's is the hippocampus; other parts of the brain that show 46
shrinking (atrophy) include the temporal and parietal lobes [6]. Other frequent types include 47
vascular dementia (25%), Lewy body dementia (15%), and front temporal dementia [1,2]. 48
More than one type of dementia may exist in the same person [1]. Cholinesterase inhibitors 49
such as rivastigmine, donepezil and galantamine are often used and may be beneficial in mild 50
to moderate disorder [7,8,9]; overall benefit, nevertheless, may be minor [9], this necessitated 51
the need for alternative medicines. 52
Despite the evolvement of four pharmacotherapy used in the management of dementia, 53
galanthamine is the only natural occurring drugs in the treatment of dementia. The use of 54
alternative medicine has been encouraged in the management of disease due to the multi- 55
target potentials they have Kigelia aficana is used in the southwest Nigeria in the 56
management of dementia. It is even one of the ingredients used in the treatment of mentally 57
retarded patients. It is referred to in Yoruba as ‘ewe isoye’. Kigelia africana (Lam) Benth 58
(family Bignoniaceae) is commonly called sausage tree in English and “Pandoro” in South-59
Western Nigeria [10]. The Bignoniaceae family is noted for the occurrence of iridoids, 60
naphthoquinones, flavonoids, terpenes, tannins, steroids, saponins and caffeic acid in the 61
fruits, stem, leaves and roots [11,12,13,14,15]. Several pharmacological properties have been 62
attributed to the fruits of K. africana but there is a relative paucity of data on the leaves [16]. 63
The aqueous extract of the leaves was reported to possess central nervous system (CNS) 64
stimulatory effect [17]. In addition, the antioxidant, antiulcerogenic, arginase inhibitory, 65
hepatoprotective and antibacterial properties of the aqueous leaf extract have been reported 66
[18,19,20,21]. Previous studies have shown that among compounds isolated from the 67
Bignoniaceae, naphthoquinones in general and furanonaphthoquinones in particular, exhibit a 68
broad spectrum of biological activities [22-25,26]. Falode et al., 2016 evaluated and 69
compared the antioxidant activity of the fruit and leaf extracts of the plant. The sausage tree 70
flavonoid extract has been reported to be neuroprotective in AlCl3-induced Alzheimer’s 71
disease model [28]. Previous phytochemical studies on Kigelia africana extract showed 72
central nervous system stimulant properties [16]. However, there has been no report about 73
neuropharmacological studies on its isolated compounds. 74
The present study assessed the acetyl cholinesterase inhibitory capacity of some flavonoids 75
(quercetin, luteolin and rutin) isolated from the leaf extract of Africana kigelia. 76
77
MATERIALS AND METHODS 78
Chemicals 79
Aluminium chloride (AlCl3), rivastigmine, acetylcholine iodide, 5′, 5
′-dithiobis-2-80
nitrobenzoate (DTNB), thiobarbituric acid (TBA) and sucrose were obtained from Sigma-81
Aldrich Co. (Munich, Germany). n-Hexane, Dichloromethane (DCM), Ethylacetate (EtOAc), 82
Methanol (MeOH), Vanillin/Sulphuric acid (H2SO4) was used as TLC spray reagent. 83
Visualization was done by observing under UV at 254 and 366 nm prior to detection with 84
chemical spray. Other chemicals and reagents used were of analytical grade and obtained 85
from standard suppliers. 86
87
Materials 88
Spatula, Masting tape, aluminium foil, sample vials, ruler, pencil, silica gel (100-200 mesh), 89
volumetric flasks of various volumes, TLC Silica gel 60 F254 (Aluminium sheet 20 x 20 cm), 90
rotary evaporator (Buchi), Vacuum-Liquid Chromatography (VLC)-Setup, open columns 91
(48x4 cm, 50x2.3 cm). 92
93
Plant material and extraction 94
Leaves of K. Africana were obtained from Ikere-Ekiti, South-Western Nigeria and 95
authenticated at the Botany Department, University of Ibadan, Ibadan, Nigeria (voucher 96
number KA06). The leaves were cleaned with distilled water, air dried and powdered. The 97
pulverized sample (1.2 kg) was extracted by maceration in 80% methanol for 72 h and then 98
filtered. The filtrate was concentrated in a rotary evaporator and freeze-dried to obtain the 99
crude extract (55 g, KAE). 100
101
Bioassays on the fractions obtained 102
Acetyl cholinesterase activity was carried out on the four fractions obtained and the isolated 103
compounds according to standard procedure [29]. 104
105
Isolation and characterization 106
The crude extract (50 g) was dissolved in minimum volume of methanol and adsorbed on 107
silica gel (150 g). The adsorbed silica gel was air-dried and then packed into a sintered glass 108
funnel. This was eluted gradiently by using n-Hex., DCM, EtOAc and MeOH. Four fractions 109
eluting with n-hex (600 ml), n-hex: DCM (1:1, 1000 ml), DCM: EtOAc (1:1, 2600 ml), 110
EtOAc: MeOH (1:1, 1800 ml) were collected that yielded 1.12 g, 0.17 g, 6.00 g and 38.25 g 111
of their respective fractions. 112
Fraction, DCM: EtOAc(1:1) was subjected to column chromatography based on the results of 113
the bio-assay. About 5.7 g of the fraction was dissolved in 5 ml MeOH and adsorbed on silica 114
gel. The adsorbed silica gel was air dried and then packed into a column (4:48 cm) that 115
contained plain silica gel in ratio 1:5. Then gradient elution followed from n-hex. through 116
EtOAc to MeOH and 157 (20 ml each) fractions were collected which was bulked into 10 117
fractions according to their TLC profile: K1 (0.10 g), K2 (0.08 g), K3 (0.93 g), K4 (0.81 g), 118
K5 (0.68 g), K6 (0.32 g), K7 (0.12 g), K8 (0.42 g), K9 (0.23 g) and K10 (0.09 g). 119
Bulked fraction K5 (0.68 g) was further subjected to repeated column chromatography based 120
on the colour (yellow) and fewer number of spots on the TLC plate. The fraction K5 was 121
dissolved in methanol (2 ml) and the solution was adsorbed on silica gel. The adsorbed silica 122
gel was allowed to air-dry before packing into the column. The column was eluted gradiently 123
using n-hex through EtOAc to MeOH mixtures. Eluants were collected in 15 ml test tubes 124
and a total of 88 fractions were collected which was bulked into 6 fractions according to their 125
TLC profile: K5a (10 mg), K5b (200 mg), K5c (100 mg), K5d (300 mg), K5e (5 mg) and K5f 126
(10 mg). K5c (light yellow) was eluted with 40 % n-hex. in EtOAc and it gave a single spot 127
on analytical TLC plate. This was labelled compound 3 (100 mg). 128
Bulked fraction K5b (200 mg) that gave two spots on TLC which were coded K5b1 and 129
K5b2 from the top of the plate and were separated by PTLC. K5b2 (59 mg) gave a light 130
yellow single spot on TLC which was labelled compound 2 (59 mg) while K5b1 (125 mg), an 131
impure solid, was further purified on column chromatography by dry packing followed with 132
gradient elution of n-hex/EtAOc/MeOH mixture. At 40 % n-hex.:EtOAc mixture K5b1 133
yielded a light yellow single on TLC that was labelled compound 1 (20 mg). 134
135
3.3.1. 2-(3,4-Dihydroxyphenyl)-5,7-dihydroxychromen-4-one (compound 1) 136
Yellow powder, 1H and
13C NMR: Table 1. HRMS-ESI (negative mode): m/z [M-H]
+ calcd 137
for C15H9O6, 285.0399; found 285.0393. 138
139
3.3.2. 2-(3,4-Dihydroxyphenyl)-3-O-glucosyl-6-O-rhamnosyl-5,7-dihydroxychromen-4-one 140
(compound 2) 141
Yellow powder, 1H and
13C NMR: Table 1. HRMS-ESI (negative mode): m/z [M-H]
+ calcd 142
for C27H29O16, 609.1455; found 609.1455. 143
144
3.3.3. 2-(3.4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one (compound 3) 145
Yellow powder, 1H and
13C NMR: Table 1. HRMS-ESI (negative mode): m/z [M-H]
+ calcd 146
for C15H9O7, 301.0348; found 301.0339. 147
148
RESULTS AND DISCUSSION 149
Effect of fractions on AChE activity 150
The effect of n-Hex (100 %), Hex:DCM (1:1), DCM:EtOAc (1:1) and EtOAc:MeOH (1:1) on 151
cerebral activity of AChE is illustrated in Fig. 1. The result showed that fraction DCM:EtOAc 152
(1:1) had the highest inhibitory effect on the activity of acetyl cholinesterase. This result also 153
showed that the inhibitory activity on the AChE by the fraction is significantly comparable to 154
the reference sample, rivastigmine and this suggested that the phytochemical(s) responsible 155
for the inhibitory activities observed in the crude extract as reported by Falode et al., 2017 156
reside in this fraction. 157
158
159
Fig. 1: Effects of various solvent fractions on AChE activity 160
161
Effect of isolated compounds on AChE activity 162
The effect of the isolated compounds from the DCM:EtOAc (1:1) fraction on the AChE 163
activity were shown in figures 2. The result showed dose-dependent activity with compound 164
3, quercetin, demonstrating highest inhibitory activity in the assay and the inhibitory activity 165
of compound 3 was significantly comparable especially at 400 μg/ml to the reference sample, 166
rivastigmine. This suggested that the compound responsible for the inhibitory activity of 167
AChE was compound 3. The structural-activity relationship of the compounds was noted to 168
0 5
10 15 20 25 30 35 40 45
ace
tyl c
ho
line
ste
rase
act
ivit
y (μ
mo
l/m
in/m
g/p
rote
in)
AChE
AChE
revolve round position 3. It was noted that the more the electronegative the atom or group of 169
atoms in position 3 of the compounds are the more the inhibitory activity of the compound. 170
171
172
Figure 2: Effect of isolated compounds on AChE activity 173
174
Structural elucidation of the isolated compounds 175
Compound 1 176
The HRESIMS (negative mode) of the compound showed a pseudo-molecular ion peak at 177
m/z 285.0393 of the molecular ion peak calculated to be 286.0477 which is consistent with a 178
molecular formula C15H10O6. The 1H NMR of the compound showed resonances for aromatic 179
protons at δ 6.20 (d, J = 3), δ 6.43(d, J = 3), δ 6.56 (s), δ 6.79 (d, J = 3), 6.81 (d, J = 3) and δ 180
6.93 (dd, J = 3, 8) each integrated to be one proton suggesting two protons on positions 6 and 181
8 on ring A. And three protons on positions 2, 5 and 6 on ring B of a flavone. The 13
C NMR 182
showed very distinct signals for fifteen carbon atoms at δ 93.6, 98.7, 101.9, 102.4, 112.7, 183
114.5, 121.8, 125.9, 144.9, 145.0, 158.0, 161.3, 161.8, 166.8 and 182.5. The NMR data 184
compared well with Luteolin in literature [30]. Hence the compound was identified as 185
Luteolin (Fig. 3). 186
Compound 2 187
0
5
10
15
20
25
30
35
Compound 1 Compound 2 Compound 3 Rivastigmine
Ace
tylc
ho
line
ste
rase
act
ivit
y
(μ/L
/mg
pro
tein
)
400
200
The HRESIMS (negative mode) of the compound showed a pseudo-molecular ion peak at 188
m/z 609.1455 of the molecular ion peak calculated to be 610.1534 which is consistent with a 189
molecular formula C27H30O16. The 1H NMR of the compound showed resonances for 190
aromatic protons at δ 6.19 (s), δ 6.37(s), δ 6.88 (d, J = 8), δ 7.64 (d, J = 8) and δ 7.67 (s) each 191
integrated to be one proton suggesting two protons on positions 6 and 8 on ring A. And three 192
protons on positions 2, 5 and 6 on ring B of a flavonol. The 13
C NMR showed very distinct 193
signals for twenty seven carbon atoms at δ 178.0, 164.7, 161.5, 157.9, 157.1, 148.4, 144.4, 194
134.3, 122.2, 121.7, 116.3, 114.7, 104.2, 103.4, 101.0, 98.6, 93.5, 76.8, 75.8, 74.3, 72.6, 70.9, 195
70.7, 70.0, 68.3, 67.2 and 16.5. The 1H and
13C data indicated a glycoside with two sugar 196
moiety and aglycone to be C6-C3-C6 compound which was easily identified as quercetin. 197
The HMBC spectrum showed correlations between the anomeric proton of glucose at δH 5.11 198
(1H, d) with carbon atom at δc 134.3 which indicated point of attachment of one of the sugars 199
at position 3 of the aglycone. The protons on C-6 of the glucose at δH 3.83 and 3.38 (2H) 200
showed HMBC correlation with the anomeric carbon atom of rhamnose at 101.0 which 201
indicated the attachment of the second sugar, rhamnose, at position 6 of the first sugar, 202
glucose. The NMR data compared well with Rutin in literature [31]. Hence the compound 203
was identified as Rutin (Fig. 3). 204
Compound 3 205
The HRESIMS (negative mode) of the compound showed a pseudo-molecular ion peak at 206
m/z 301.0339 of the molecular ion peak calculated to be 302.0427 which is consistent with a 207
molecular formula C15H10O7. The 1H NMR of the compound showed resonances for aromatic 208
protons at δ 6.28 (d, J = 4), δ 6.54 (d, J = 4), δ 7.01 (d, J = 8), δ 7.72 (dd, J = 0, 8) and δ 7.83 209
(d, J = 0) each integrated to be one proton suggesting one proton each on positions 6 and 8 on 210
ring A. And one proton each on positions 2, 5 and 6 on ring B of a flavonol. The 13
C NMR 211
showed very distinct signals for fifteen carbon atoms at δ 175.6, 164.1, 162.2, 156.9, 147.4, 212
146.0, 144.9, 135.8, 122.9, 120.6, 115.3, 114.8, 103.2, 98.2 and 93.6, The only correlation in 213
the COSY spectrum is between protons at δ 7.72 (dd, J = 0, 8) and δ 7.01 (d, J = 8) which 214
agreed with the coupling constant from the 1H NMR spectrum. The HMQC spectrum showed 215
correlations between these carbon atoms at δc 120.6, 115.3, 114.8, 98.2 and 93.6 with protons 216
at δH 7.72, 7.01, 7.83, 6.28 and 6.54 respectively. The HMBC spectrum showed correlations 217
between proton at δH 7.01 with carbon atoms at δc 147.4, 146.0 and 122.9. Correlation was 218
also observed with a proton at 6.54 and a carbon atom at 156.9. The NMR data compared 219
well with Quercetin in literature [31]. Hence the compound was identified as Quercetin (Fig. 220
3). 221
222
O
R
OOH
HO
OH
OH
2
35
7
24
6
223
Compound R
1
2
3
H
O-glucose-rhamnose
OH
224
Fig. 3: Structures of flavonoids isolated from the leaves of Kigelia africana. 225
Table 1: NMR data for compounds 1, 2 and 3 in CD3OD (multiplicities and J values are given in Hz in parenthesis). 226
Position 1 2 3
δH (600 MHz) δC (150 MHz) δH (600 MHz) δC (150 MHz) δH (600 MHz) δC (150 MHz)
2
3
4
4a
5
6
7
8
8a
1'
2'
3'
4'
5'
6'
1''
2''
3''
4''
5''
6''
1'''
2'''
3'''
4'''
5'''
6'''
-
6.93
-
-
-
6.20 (s)
-
6.43 (s)
-
-
6.81 (s)
6.56 (s)
-
-
6.79 (s)
-
-
-
-
-
-
-
-
-
-
-
-
168.8
101.9
182.5
102.4
161.3
93.6
161.8
98.7
158.0
121.8
125.9
114.5
144.9
145.0
112.7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6.19
-
6.37
-
-
7.64
6.88
-
-
7.67
5.11
-
-
-
-
3.83, 3.38
4.53
-
-
-
-
1.12
157.9
134.3
178.0
104.2
161.5
98.6
164.7
93.5
157.1
122.2
121.7
114.7
144.4
148.4
116.3
103.4
74.3
75.8
70.9
76.8
67.2
101.0
70.7
70.0
72.6
68.3
16.5
-
-
-
-
-
6.28 (s)
-
6.54 (s)
-
-
7.72 (s)
7.01 (s)
-
-
7.83 (s)
-
-
-
-
-
-
-
-
-
-
-
-
144.9
135.8
175.6
103.2
162.2
98.2
164.1
93.6
156.9
122.9
120.6
115.3
147.4
146.0
114.8
-
-
-
-
-
-
-
-
-
-
-
-
227
CONCLUSION 228
This work showed the isolation of luteolin, rutin and quercetin from the leaves of Kigelia 229
africana and their comparative cholinesterase inhibitory capacities on AChE. It showed 230
quercetin as the major inhibitor of acetyl cholinesterase in the leaves of the plant. Though the 231
cholinesterase inhibitory capacity of luteolin, rutin and quercetin have been reported but their 232
activity had not been correlated in this manner. 233
Conclusively, this research has established the phytoconstituents responsible for potential 234
neuroprotection in the extract, it has also elucidated the mechanism of action of the extract. 235
This report could be built upon in further researches leading to drug discovery. 236
237
CONFLICT OF INTEREST 238
The authors declare that they have no conflict of interest. 239
240
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