Acridone alkaloids from Vepris verdoorniana (Excell & Mendonça) Mziray (Rutaceae)

Albert Fouda Atanganaa, Flavien Aristide Alfred Tozea, Moses K. Langatb, Emmanuel

Ngeufa Happia, Luc Leonard Meva’a Mbazea, Dulcie A. Mulhollandb, Alain François

Kamdem Waffoa, Norbert Sewaldc, Jean Duplex Wansia,*

Affiliation

a. Department of Chemistry, University of Douala, Faculty of Sciences, 24157 Douala,

Cameroon.

b. Natural Products Research Group, Department of Chemistry, Faculty of Engineering

and Physical Sciences, University of Surrey, Guildford, GU2 7XH, United Kingdom.

c. Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University,

33501 Bielefeld, Germany.

*Corresponding author. Tel. +237 674 725 425. E-mail address: jdwansi@yahoo.fr (J.D.

Wansi).

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Abstract

Two new acridone alkaloids, verdoocridone A (1) and B (4), together with fifteen known

compounds were isolated from methanol extracts of the roots and leaves of Vepris

verdoorniana. The structures of all compounds were determined by comprehensive

spectroscopic analyses (1D and 2D NMR, EI- and ESI-MS). The 13C-NMR values of 1,2,3,5-

tetramethoxy-N-methylacridone (2) and 5-methoxyaborinine (3) are also reported. The crude

extracts and compounds (1-6) were tested for their antimicrobial activity. The test delivered

moderate activities for crude extracts and compounds 1, 5 and 6 against the bacterium

Staphylococcus aureus and the fungi Mucor meihei and Candida albicans with MIC values

between 115 and 180 µg/mL for extracts and between 21.3 and 29.4 µM for compounds,

compared to gentamycin with 0.2 µM and nystatin with 5.2 µM against both fungi. The

determination of the radical scanvenging activity using 1,1-dephenyl-2-picrylhydrazyl

(DPPH) assay gave moderate antioxidant values for all tested compounds, with IC50 between

0.29 and 0.41 µM, compared to the standard 3-t-butyl-4-hydroxyanisole (BHA) displaying

0.03 µM.

Keywords: Vepris verdoorniana; Rutaceae; acridone alkaloids, antimicrobial and antioxidant

activities

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1. Introduction

Vepris verdoorniana (Excell & Mendonça) Mziray (Rutaceae), synonym Teclea

verdoorniana Excell & Mendonça, is a shrub or small tree up to 22 m tall, the trunk is up to

1.2 m in girth. The species is endemic to Western and Central Africa, stretching along the

coast line from Liberia over Nigeria to Cameroon and Gabon and then westwards to the

Central African Republic, Eastern Democratic Republic of Congo and Western Uganda.

Various plant parts are used in traditional African medicine to treat fever, malaria, colds,

conjunctivitis, arterial hypertension, tape-worm and cough (Nordeng et al., 2013; Rasoanaivo

et al., 1999; Chhabra et al., 1991; Gurib-Fakim et al., 1993). In Uganda, the wood is used to

make walking sticks, spear shafts and bark-cloth mallets. The leafy twigs, containing an

inflammable resin, are bundled to serve as torches. To the best of our knowledge, no

phytochemical studies had yet been carried out on this species, while other more than fifteen

species of the genus revealed the presence of acridones, furoquinolines, quinolones, amides,

azole, coumarins, fatty acids, flavonoids, indoloquinazolines, limonoids, lignans, phenolic

compounds and terpenoids (Ayafor et al., 1980; 1982a-c; Khalid and Waterman, 1982;

Brader et al., 1996; Rasoanaivo et al., 1999; Chaturvedula et al., 2003; Cheplogoi et al., 2008;

Kiplimo et al., 2012; Kiplimo and Koorbanally, 2012). Interestingly, some compounds

isolated from above chemical classes exhibit potent antibacterial, antioxidant, antimalarial

and cytotoxic activities (Rasoanaivo et al., 1999; Chaturvedula et al., 2004; Cheplogoi et al.,

2008; Kiplimo et al., 2012; Kiplimo and Koorbanally, 2012). In this article, we report the

isolation and structural elucidation of two new acridone alkaloids, verdoocridone A (1) and B

(4), together with the 13C-NMR values of 1,2,3,5-tetramethoxy-N-methylacridone (2) and 5-

methoxyaborinine (3).

2. Results and discussion

The roots and the leaves of V. verdoorniana were separately extracted with MeOH and

subjected to vacuum liquid chromatography (VLC), column chromatography carried out on

silica gel and preparative thin layer chromatography (pTLC) to afford two new acridone

alkaloids and fifteen known compound (Fig. 1). By comparison with the reported data, the

known compounds were identified as 1,2,3,5-tetramethoxy-N-methylacridone (2), 1-hydroxy-

2,3,5-trimethoxy-N-methylacridone (3), 6-methoxytecleanthine (5), tecleanthine (6),

citracridone II (7), 5-methoxynoracronycine (8), citropsine A (9), scopoletin (10), limonin

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(11), 24R-ethyl-5α-cholestane-3β,6α-diol (12), β-sitosterol, stigmasterol, stigmasterol-3-O-β-

D-glucopyranoside, β-sitosterol-3-O-β-D-glucopyranoside and lupeol (Rosaoanaivo et al.,

1999; Wijerantne et al., 1992; Bowen et al., 1980; Mbaze et al., 2010; Happi et al., 2011;

Fomani et al., 2016; Chaurasia and Wichti, 1987), while the new acridone alkaloids were

identified by comprehensive spectroscopic analyses.

Compound 1 was obtained as a yellow amorphous powder. The molecular ion was found to

be C19H21O6N by HR-EIMS ([M]+ at m/z 359.1360, calcd. 359.1369). The IR spectrum

exhibited vibration bands due to a conjugated carbonyl group (max1633 cm-1). The UV

absorptions at 222, 265, 338 and 409 nm indicated 1 to be a 9-acridone derivative (Wansi et

al., 2006).

The 1H NMR spectrum (Table 1) exhibited five methoxy groups at δH 4.01 (s, OCH3-1), 4.00

(s, OCH3-3), 3.98 (s, OCH3-6), 3.89 (s, OCH3-2), and 3.78 (s, OCH3-5), and a N-methyl

group at δH 3.94 (s). In the aromatic region, a singlet at δH 6.59 (H-4) and an AB system of

two aromatic protons at δH 8.18 (d, J = 7.2 Hz); 6.93 (d, J = 7.2 Hz) could be attributed to H-8

and H-7, respectively. These assignments were determined through the lower-field signal of

H-8, which was deshielded by the adjacent carbonyl group of the acridone moiety (Wansi et

al., 2006). This inference was confirmed by the 13C NMR data (Table 1) and DEPT,

displaying five methoxy groups at δC 61.9 (C-1), 61.3 (C-5), 61.2 (C-2), 56.4 (C-6) and 56.1

(C-1), an N-methyl group at δC 41.5 and a carbonyl at δC 176.6 (Naidoo et al., 2005). The

positions of the five methoxy groups were determined by HMBC and NOESY spectra. The

HMBC spectrum showed correlation between N-CH3 (δH 3.94) and carbon signals at C-10a

(δC 138.6); C-4a (δC 144.9) and in addition an important 4J correlation with C-4 (δC 94.0)

confirming the singlet proton at H-4 (δH 6.59). Furthermore, the proton H-4 (δH 6.59)

displayed correlation with the carbon signals at C-9a (δC 112.4), C-2 (δC 138.0), C-4a (δC

144.9) and C-3 (δC 157.8) affirming the position C-2 and C-3 for two methoxy groups. The

HMBC correlations between H-8 (δH 8.18) and carbon signals at C-7 (δC 107.4), C-8a (δC

121.4), C-10a (δC 138.6) and C-6 (δC 158.6) as well as between H-7 (δC 6.93) and carbon

signals at C-8a (δC 121.4), C-8 (δC 123.6), C-5 (δC 137.1) and C-6 (δC 158.6) verified the

position C-5 and C-6 for further two methoxy groups. The position of MeO-1 was reassured

by the NOESY correlations indicating cross-peaks between N-CH3 and H-4, H-4 and MeO-3,

MeO-3 and MeO-2 as well as MeO-2 and MeO-1 and between N-CH3 and MeO-5 and in

addition between MeO-5 and MeO-6, as shown in Fig. 2. In addition, cross-peaks confirming

the position C-5 and C-6 were observed as well between H-7 and MeO-6 and in addition

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between MeO-6 and MeO-5. From the above spectroscopic data, the structure of compound 1

was determined as 1,2,3,5,6-pentamethoxy-N-methylacridone and was named verdoocridone

A.

Compound 4 was obtained as a yellow amorphous powder. The molecular composition was

found to be C17H15NO6 by HR-EIMS ([M]+, m/z 329.0890, calcd. 329.0899). This value was

30 mass units less than that of compound 1 suggesting the absence of two CH3 in compound

4. The UV absorption bands (220, 267, 340 and 425nm) and the IR spectrum (1220, 1596,

1635, 1720, 3310, 3390 cm-1) also suggested an acridone skeleton for compound 4 as well

(Wansi et al., 2006).

The 1H NMR, 13C NMR, DEPT, COSY, HMQC and HMBC spectra showed the presence of

the same aromatic spin systems as in compound 1 (Table 1). While the 1H NMR spectrum

revealed the presence of the free hydroxyl function at δH 6.28 (brs), which was exchangeable

with D2O and a singlet at δH 6.03 corresponding to a methylenedioxy group, the 13C NMR and

DEPT spectra confirmed the presence of the free hydroxy group at C 153.6, and

methylenedioxy at C 101.9. The methylenedioxy revealed to be fixed to C-2 and C-3 by the

HMBC correlation from H-4 (H 6.65) to C-9a (C 112.5), C-2 (C 134.3), C-4a (C 144.7) and

to C-3 (C 153.6), while the free hydroxy group was shown to be at C-6 position by the

HMBC correlations between H-8 (δH 8.13) and carbon signals at C-7 (δC 111.2), C-8a (δC

121.1), C-10a (δC 137.4) and C-6 (δC 153.6) and further correlations between H-7 (δC 6.93)

and carbon signals at C-8a (δC 121.1), C-8 (δC 124.5), C-5 (δC 134.3) and C-6 (δC 153.6). The

position of MeO-1 was confirmed by the NOESY cross-peaks between N-CH3 and H-4, H-4

and methylenedioxy as well as between methylenedioxy and MeO-1. Furthermore, cross-

peaks confirming the position C-5 for the methoxy and C-6 for the hydroxy were observed

between H-7 and OH-6 as well as between OH-6 and MeO-5. From the above spectroscopic

data, the structure of compound 4 was determined as 6-hydroxy-1,5-dimethoxy-2,3-

methylenedioxy-N-methylacridone (6-hydroxytecleathine) and named verdoocridone B.

Mimimum inhibitory (MIC) assay with compounds and crude extracts was carried out

against the bacteria Bacillus subtilis (BS), Escherichia coli (EC), Staphylococcus aureus

(SA), Streptomyces viridochromogenes (SV) and the fungi Mucor miehei (MM) and Candida

albicans (CA). Best results were received against the bacterium Staphylococcus aureus and

the fungi Mucor meihei and Candida albicans with MIC values between 21.3 and 29.4 µM

for compounds 1, 5 and 6 compared to gentamycin with 0.2 µM and nystatin with 5.2 µM,

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respectively. Results correspond well the low MIC values of 115-180 µg/mL received for

extracts (table 2).

Samples were also screened for radical scavenging activity by 1,1-diphenyl-2-

picrylhydrazyl (DPPH) assay showing moderate antioxidant values for all tested compounds,

with IC50 between 0.29 and 0.41 µM compared to the standard 3-t-butyl-4-hydroxyanisole

(BHA) displaying 0.03 µM. Results correspond well to the low IC50 values of 160.51 and

150.32 µg/mL measured for leaf and root extract, respectively (table 3).

3. Experimental

3.1. General experimental procedures

Ultraviolet spectra were recorded on a Hitachi UV 3200 spectrophotometer in MeOH and

Infrared spectra on a JASCO 302-A spectrophotometer, while ESI-HR mass spectra were

measured on a Bruker FTICR 4.7 T mass spectrometer and EI-MS on a Finnigan MAT 95

spectrometer (70 eV) with perfluorokerosene as reference substance for EI-HR-MS. The 1H-

and 13C-NMR spectra were recorded at 500 MHz and 125 MHz respectively on Bruker DRX

500 NMR spectrometers in CDCl3. Methyl, methylene and methine carbons were

distinguished by DEPT experiments. Homonuclear 1H connectivities were determined by

using the COSY experiment. One-bond 1H-13C connectivities were determined with HMQC

gradient pulse factor selection, and two- and three-bond 1H-13C connectivities by HMBC

experiments. Chemical shifts are reported in δ (ppm) using TMS as internal standard, while

coupling constants (J) were measured in Hz. Column chromatography was carried out on

silica gel (70-230 mesh, Merck). Thin layer chromatography was performed on Merck

precoated silica gel 60 F254 aluminium foil, and colour spots were detected using ceric

sulphate spray reagent after heating. The degree of purity of the positive control compounds

was ≥ 98%, while that of the isolated compound was > 95%. The purity of isolated

compounds was investigated by means of EI-HR-MS. Nystatin was purchased from Maneesh

Pharmaceutic Pvt. Ltd., and gentamicin from Jinling Pharmaceutic (Group) Corp. All

reagents used were of analytical grade.

3.2. Plant material

The roots and leaves of Vepris verdoorniana were collected at the lake site BAROMBI,

KUMBA locality, South-West region of Cameroon in March 2015. The material was

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identified by the botanist Mr. Nana Victor, National Herbarium of Cameroon, Yaoundé,

where a voucher specimen (ref. 5659HNC) has been deposited.

3.3. Extraction and isolation

The air-dried and powdered roots (2.0 kg) and leaves (3.5 kg) of Vepris verdoorniana were

extracted with methanol at room temperature for 72 h. Evaporation under reduced pressure

resulted in 63.2 g of dried crude extract from the roots and 158.2 g from the leaves. Extracts

were then fractionated by Vacuum Liquid Chromatography (VLC) to eliminate chlorophyll

residues using the silica gel 60 (10-40 µm) with a hexane/AcOEt gradient of 1/9; 2/8; 2/3;

1/1; 3/2 and pur AcOEt. The roots sub-fractions were combined into four main fractions A-D

and those of the leaves into four main fractions A’-D’, on the basis of TLC analysis.

Fraction A (5.4 g) was chromatographed over silica gel 60C column with a hexane/AcOEt

gradient. A total of 24 fractions of around 100 mL each were collected and combined on the

basis of TLC. While the combined fractions 10-15 precipitated to yield lupeol (7.5 mg),

fractions 16-24 were combined and chromatographed over a silica gel 60H column with

hexane/AcOEt (7/3) to yield a mixture of β-sitosterol and stigmasterol (22.5 mg) and 24R-

ethyl-5α-cholestane-3β,6α-diol (15.0 mg).

Fraction A’ (9.8 g) was also studied using the same technique to yield lupeol (17.5 mg) in the

combined fractions 1-12, a mixture of β-sitosterol and stigmasterol (51.5 mg) in the

combined fractions 13-25 and a yellow amorphous powder identified as scopoletin (37.0 mg)

in the combined fractions 25-34.

Fraction B (11.3 g) was chromatographed over silica gel 60C in a column with a

hexane/AcOEt gradient. A total of 23 fractions of ca. 100 mL each were collected and

combined on the basis of TLC as well. The combined fractions, 1-19 were further

chromatographed over a silica gel 60H column with hexane/AcOEt (1/1) to yield yellow

fibres identified as 1,2,3,5-tetramethoxy-N-methylacridone (23.5 mg) and yellow pellets

identified as 1-hydroxy-2,3,5-trimethoxy-N-methylacridone (26.0 mg).

In addition, Fraction B’ (15.2 mg) was also studied using the same method to

yieldverdoocridone A (19.0 mg) and verdoocridone B (53.2 mg) as a yellow powder in

hexane/AcOEt (3/7).

Fraction C (17.8 g) was chromatographed over a silica gel 60C in a column with a

hexane/AcOEt gradient. A total of 30 fractions of around 100 mL each were collected and

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combined on the basis of TLC. Fractions 2-15 were further chromatographed over a silica gel

60H column with a mixture of hexane/AcOEt (1/2) to yield citracridone II (13.1 mg), 5-

methoxynoracronycine (10.5 mg) and citropsine A (7.0 mg).

Fraction C’ (20.0 mg)was also chromatographed over a silica gel 60C column with

hexane/AcOEt (1/2) to yield 6-methoxytecleanthine (14.0 mg) and tecleanthine (16.6 mg).

Finally, fraction D and D’ were combined on the basis of the TLC (32.5 g) and

chromatographed over silica gel 60C column with a hexane/AcOEt gradient. A total of 25

fractions of around 100 mL each were collected and combined on the basis of TLC. Sub-

fractions 15-25 precipitated as a white powder to yield a mixture of stigmasterol-3-O-β-D-

glucopyranoside and β-sitosterol-3-O-β-D-glucopyranoside (20.0 mg).

3.4. Verdoocridone A (1)

Yellow amorphous powder (CHCl3); Rf = 0.48, silica gel 60 F254, Hex/CH2Cl2 (1/4);

UV (MeOH) max (log ) 222 (2.40), 265 (2.14), 277 (1.80), 290 (1.60), 303 (1.50), 338

(1.40), 409 (2.00) nm; IR (KBr) max 3350, 2980, 1700, 1633, 1510, 1394, 1280, 1170 cm1; 1H and 13C NMR data, see Table 1; EI-MS (%) m/z 359.1 (C19H21NO6, 32), 328 (98), 298

(28), 266 (74), 251 (45), 216 (18), 181 (85), 149 (40); HR-EIMS [M] +m/z359.1360 (calcd for

C19H21NO6,359.1369).

3.5. Verdoocridone B (4)

Yellow amorphous powder (CHCl3); Rf = 0.44, silica gel 60 F254, Hex/CH2Cl2 (1/4);

UV (MeOH) max (log ) 220 (3.62), 267 (3.51), 290 (3.72), 326 (2.54), 340 (3.45), 366

(3.60), 425 (3.50) nm; IR (KBr) max 3390, 3310, 2835, 1720, 1664, 1635, 1596, 1391, 1220,

1012, 755 cm1; 1H and 13C NMR data, see Table 1; EI-MS (%) m/z 329.2 ([M]+, 70), 298

(48), 286 (25), 257 (45), 242 (75), 157 (35), 148 (33), 123(25), 81 (41); HR-EIMS [M] +m/z

329.0890 (calcd for C17H15NO6,315.0899).

3.6. Biological activities

3.6.1. Determination of minimum inhibition concentrations against bacteria and fungi

The minimum inhibition concentrations (MICs) of test samples and reference drugs were

measured by the microdilution broth susceptibility assay (CLSI/NCCLS, 2008) against the

bacteria Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Streptomyces

viridochromogenes and the fungi Mucor miehei and Candida albicans originating from the

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strain collections Göttingen / Tübingen, Germany and the Centre Pasteur du Cameroun,

Yaoundé, Cameroon. The inocula of bacterial and fungal strains were prepared from 12-h

broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. The

samples were dissolved in 10% DMSO and serial twofold diluted in sterile 96-well microtiter

plates in duplicate, using BHI broth for bacterial and Sabouraud dextrose broth for fungal test

lines. Standardized inocula of test strains were added, and after incubation at 37°C for 24 h

on a rotary shaker at 200 rpm, MICs were read as the lowest concentration with inhibition of

the growth of the test organisms. Nystatin was used as positive control for fungi and

gentamycin for bacteria, while BHI and Sabouraud dextrose broth containing 10% DMSO

was used for negative control.

3.6.3. Determination of the radical scavenging activity

DMSO (5 μL) containing the test sample and 95 μL of 1,1-diphenyl-2-picrylhydrazyl

(DPPH) (Sigma, 300 μM) in ethanol was given into a well of a 96-well microtiter plate

(Molecular Devices, Germany) and incubated at 37°C for 30 min. The absorbance was

measured at 515 nm and the percentage of radical scavenging activity determined by

comparison with a DMSO containing control. IC50 values represent concentrations of

compounds to scavenge 50 % of DPPH radicals. 3-t-butyl-4-hydroxyanisole (BHA) was used

as a positive control. All the chemicals used were of analytical grade (Sigma, USA).

Acknowledgement.

The authors wish to thank the Alexander von Humboldt Foundation, Germany for Research

Group Linkage funding 2015/2018 of the Norbert Sewald/Jean Duplex Wansi research group

cooperation and the Alexander von Humboldt Foundation for the generous support with

laboratory equipment.

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Table 1. 1H (500 MHz) and 13C (125 MHz) NMR assignments for (1-4) in CDCl3

Attribution 1 2 3 41H 13C 13C 13C 1H 13C

1 - 154.3 153.9 155.1 - 143.22 - 138.0 138.0 138.0 - 134.33 - 157.8 157.9 157.8 - 153.64 6.59 (s) 94.0 93.9 93.2 6.65 (s) 90.94a - 144.9 145.0 145.2 - 144.95 - 137.1 149.7 149.5 - 134.36 - 156.8 114.8 114.9 - 153.67 6.93 (d, J = 7.2) 107.4 122.1 122.5 6.93 (d, J = 7.0) 111.28 8.18 (d, J = 7.2) 123.6 119.0 120.0 8.13 (d, J = 7.0) 124.58a - 121.4 127.4 127.5 - 121.19 - 176.6 176.9 176.7 - 176.79a - 112.4 112.7 112.3 - 112.510a - 138.6 135.0 135.2 - 137.4

OCH3-1 4.01 (s) 61.9 61.9 - 4.15 (s) 61.1OCH3-2 3.89 (s) 61.2 61.6 61.5 - -OCH3-3 4.00 (s) 56.1 56.1 55.9 - -OCH3-5 3.78 (s) 61.3 56.5 56.4 3.76 (s) 61.2OCH3-6 3.98 (s) 56.4 - - - -N-CH3 3.94 (s) 41.5 42.4 42.1 3.91 (s) 40.5

O-CH2-O - - - - 6.03 (s) 101.9Assignments were based on HMQC, HMBC and NOESY experiments

12

Table 2. Minimum inhibition concentrations of MeOH extracts of leaves (VVL), roots (VVR) and compounds 1-6 from Vepris verdoorniana

MicroorganismSample BSb SVc SAd ECe MMf CAg

VVL 240.5 291.2 250.3 210.0 150.1 145.2VVR 200.3 321.2 180.1 280.5 120.3 115.0

1 85.0 66.0 70.0 54.3 39.1 26.12 75.1 49.3 42.2 49.5 41.5 40.53 82.2 39.4 34.1 42.3 42.5 43.54 66.5 48.3 32.4 39.5 43.2 31.25 51.0 30.2 29.4 35.1 27.0 21.36 41.1 40.5 40.5 34.2 26.0 25.5

RA 0.2 0.5 0.2 0.2 5.2 5.2aMIC for extracts in µg/mL and for compounds in µM;bBacillus subtilis; cStreptomyces viridochromogenes; dStaphylococcus aureus; eEscherichia coli; fMucor meihei; gCandida albicans. Reference Antibiotics (RA):gentamycin for bacteria and nystatin for fungi

Table 3. Free radical scavenging activity of MeOH extracts of leaves (VVL), of roots (VVR) and compounds 1-6from Vepris verdoorniana against DPPH (1,1-diphenyl-2-picrylhydrazyl)

Sample aIC50 ± S.E.M.VVL 160.51 ± 1.43VVR 150.32 ± 2.21

1 0.41 ± 0.032 0.40 ± 0.073 0.29 ± 0.014 0.32 ± 0.035 0.38 ± 0.056 0.40 ± 0.03

BHAb 0.03 ± 0.01 aIC50 for extracts in µg/mL and for compounds in µM; bBHA = 3-t-butyl-4-hydroxyanisole.

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Figure 1. Structures of some isolated compounds.

N

O

CH3

OCH3OCH3

OCH3OCH3

H3CO

H

H

H

Figure 2. Selected NOESY correlations of compound 1.

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