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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



+ 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



+ 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



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



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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