An Antisalmonellal Agent and a New Dihydroanthracenone from Cassia petersiana
Pierre C. Djemgou,
Abiodun H. Adebayo,
Bonaventure T. Ngadjui,
Godwin I. Adoga
Phytochemical and biological investigation of the leaves of Cassia petersiana afforded four compounds including a new dihydroanthracenone (1), two known chromones (2,3), in addition to stigmasterol glucoside (4). The work was guided by the antisalmonellal activity of the extract and fractions. Compound 4 was found to be the active principle. The structures of the compounds were determined by combination of spectroscopic techniques, including 1H, 13C, DEPT, COSY, HMQC, HMBC, MS and IR.
|| Structures of the isolated compounds
Typhoid fever is caused by Salmonella typhi, whereas paratyphoid fevers
are caused by Salmonella paratyphi A and Salmonella paratyphi
B (Cheesbrough, 1991). Typhoid fever continues to be a marked public health
problem in developing countries in general and in Sub-Saharan Africa in particular,
where it is endemic (Gatsing et al., 2003). The greater prevalence of
resistance to all three first line antimicrobial (ampicillin, chloramphenicol
and co-trimoxazole) has been established (WHO, 1981; Gatsing et al.,
2003). Previous phytochemical investigation on this plant species led to the
isolation of diterpene (Msonti, 1984) and flavonoids (Coetze et al.,
1999). As part of our contribution to the phytochemical and chemotaxonomic survey
of the genus Cassia and in a continuation of our search for therapeutic
agents from natural sources with potential for the treatment of typhoid and
paratyphoid fevers (Gatsing et al., 2003; Gatsing et al., 2006),
we carried out the investigation of the CH2Cl2-MeOH (1:1)
extract of the leaves of C. petersiana, a species from Cameroon. We report
herein the isolation and structural elucidation of four compounds including
one new and three known (Fig. 1). The extract and the pure
compounds were assessed for their antisalmonellal activity. The compounds were
characterised from comprehensive 1D and 2D NMR interpretation and comparison
with literature data.
Materials and Methods
1H, 13C-NMR and 2D spectra were recorded at 400 MHZ
using a Bruker DPX 400 spectrometer; trimethylsilane (TMS) was used as the internal
standard. EIMS spectra were recorded on TSQ-70-Triple Stage Quadruple mass spectrometer
(70 ev). The IR spectra (CHCl3) were recorded on a Perkin-Elmer FT-IR-spectrometer.
The leaves of Cassia petersiana Bolle were collected in Bafia, Centre
province of Cameroon in July 2003. Plant material was identified by Dr. Onana
at the Cameroon National Herbarium, Yaoundé, where a voucher specimen
(N° 6494/SFR/Cam) was deposited.
Test Bacteria and Culture Media
The test microorganisms, Salmonella typhi, Salmonella paratyphi
A and Salmonella paratyphi B, were obtained from the Medical Bacteriology
Laboratory of the Pasteur Centre, Yaoundé, Cameroon. The culture media
used namely Salmonella-Shigella agar (SS agar) and Selenite Broth, were supplied
by International Diagnostics Group PLC, Topley House, 52 Wash Lane, Bury, Lancashire
Extraction and Isolation
The air-dried and pulverized leaves of C. petersiana (800 g) were
extracted by maceration with CH2Cl2-MeOH (1:1), (11 L,
72 h) and evaporated under reduced pressure to give 80 g of crude extract. Part
of this extract (30 g) was fractionated by silica gel column chromatography
(CC) eluted successively with n-hexane-EtOAc and EtOAc-MeOH in a step gradient
by using different ratios. Three fractions A (8 g), B (11 g) and C (3 g) were
recorded. Fraction A was purified on Sephadex LH-20 column (n-hexane-CH2Cl2-MeOH,
7:4:1) and preparative TLC to give 4-Acetyl-3,4-dihydro-3,8-dimethyl-3-hydroxy-6-methoxyanthracen-1(2H)-one
(1, 9 mg). Fraction B was purified on silica gel column (n-hexane-EtOAc, 9:1)
and Sephadex LH-20 to give 5-acetonyl-7-hydroxy-2-hydroxymethylchromone (2,
4 mg) and 5-acetonyl-7-hydroxy-2-methylchromone (3, 11 mg). Stigmasterol-3-O-β-D-glucoside
(4, 500 mg) was recrystallised with acetone from fraction C.
(1): Yellow powder, mp 291.3 C (uncorrected), IR 3320, 3480, 2814, 1624, 1597, 1430, 1300, 970; 1H NMR (CD3OD, 400 MHZ) and 13C NMR (CD3OD, 100 MHZ), Table 1, EIMS [M+] m/z 312, [M-H]+ m/z 311, [M-CH3]+ m/z 297, [M-OH]+ m/z 295, [M-Ac]+ m/z 270, [M-OH-Ac]+ m/z 254, HREIMS 312.3737 [calcd. for C19H20O4, 312.3630].
The antibacterial activity was determined using both agar diffusion and
broth dilution techniques as previously described (Cheesbrough, 1991; Gatsing
et al., 2006).
Agar diffusion susceptibility testing was done using the method of wells. On
each plate containing Salmonella-Shigella agar (SS agar) meduim already inoculated
with the test organism (100 μL of the bacteria suspension in Selenite broth,
at the concentration of 5x105 cfu mL-1) wells (of 6 mm
diameter) were bored using a cork borer. The bottom of each well was sealed
with a drop of molten agar. The compounds and the extract were dissolved in
dimethysulfoxide (DMSO). The wells were filled with 150 μL of the solution
(of known concentration) of various compounds and extracts to be tested.
|| 13C and 1H NMR data of compound 1 at
100 and 400 MHZ in CD3OD
Chloramphenicol (Sigma) was used as the standard drug. The extract, compounds and chloramphenicol were tested at the concentration of 50, 0.5 and 0.1 mg mL-1, respectively. The petridishes were left at room temperature for 45 min to allow the compounds and extract to diffuse from the wells into the medium. They were then incubated at 37°C for 24 h, after which the zones of no growth were noted and their diameters recorded as the zones of inhibition.
For the broth dilution susceptibility testing, the solution (maximum concentration) of the active compound (ie the compound that induced a zone of inhibition; compound 4) was dissolved in DMSO, serially (2-fold) diluted and 0.5 mL of each dilution was introduced into a test tube containing 4.4 mL of Selenite broth; then 0.1 mL of bacteria suspension (5x105 cfu mL-1) was added and the mixture was homogenised. The total volume of the mixture was 5 mL, with the test compound concentrations in the tube ranging from 180 to 5.625 μg mL-1 and those of chloramphenicol ranging from 40 to 0.625 μg mL-1. After 24 h of incubation at 37°C, the Minimum Inhibitory Concentration (MIC) was reported as the lowest concentration of antimicrobial that prevented visible growth. The Minimum Bactericidal Concentration (MBC) was determined by subculturing the last tube to show visible growth and all the tubes in which there was no growth on already prepared plates containing SS agar medium. The plates were then incubated at 37°C for 24 h and the lowest concentration showing no growth was taken as the MBC.
Results and Discussion
The air-dried and pulverized leaves of C. petersiana were extracted
by maceration with CH2Cl2/MeOH (1/1). Part of the resulting
extract was fractionated by silica gel column chromatography (CC) eluted successively
with n-hexane-EtOAc and EtOAc-MeOH in a step gradient by using different ratios.
Three fractions A (8 g), B (11 g) and C (3 g) were recorded and purified to
(1, 9 mg), 5-acetonyl-7-hydroxy-2-hydroxymethylenechromone (2, 4 mg), 5-acetonyl-7-hydroxy-2-methylchromone
(3, 11 mg) and stigmasterol-3β-D-glucoside (4, 500 mg).
Compound 1 was isolated as a yellow powder, mp 291.3°C (uncorrected); its EIMS gave a molecular ion peak at m/z 312. The cross formula was deduced to be C19H20O4 from combination of HREIMS, EIMS and RMN data. Moreover, interesting peaks were observed, each characteristic of a precise moiety, m/z 270, [M-Ac]+; 297, [M-H2O]+; 295, [M-CH3]+. A peak at m/z 311 (M-1) suggested the presence of hydroxyl group in the structure. The 1H NMR (CD3OD, 400 MHZ (Table 1) revealed eleven different hydrogens in the molecule, which could be classified as seven singlets, three doublets and a doublet of doublet. The methoxyl appeared at δ 4.18 (3H, s, OMe). The carbone13 NMR (CD3OD, 100 MHZ) spectra reveals 19 carbon atoms in the molecule (Table 1). Analyses of 13C, DEPT (90 and 135) and HMQC led to their classification as two carbonyls, three methyls, a methoxyl at δ 50.9 (-OMe), one saturated methine carbon at δ 64.5 (C-4), one methylene carbon at δ 49.5 (C-2), one saturated quaternary carbon at δ 72.9 (C-3), six aromatic quaternary carbons and four aromatic
methine carbons. The assignement of the structure was based on the following
observations on the spectra: (i) The position of the hydroxyl (OH) was based
on the chemical shift of C-3 and confirmed by the HMBC (Fig. 1).
(ii) The acetyl group was deduced from an intense correlation on HMBC between
the methyl at δ 2.40 (H-12) and the carbonyl at δ 209.9 (C-11). (iii)
This was confirmed by the chemical shift of the methyl carbon at δ 33.9
(C-12), downfield, compared to the methyl C-14 (δ 20.3) for example. The
other substituents were attributed from interpretation of the HMBC spectra (Fig.
2). (iv) H-9, δ 6.55 and H-10, δ 6.00 were placed according to
the HMBC where they showed a cross correlation with the carbonyl at δ 203.3
(C-1) and the methine carbon at δ 64.5 (C-4) respectively. H-5, δ
6.22 and H-7, δ 6.19 were meta oriented based on their coupling
constant 2.20 Hz.
|| Some remarquable HMBC correlations of compound 1
On proton COSY, H-5, δ 6.22 showed a weak 4J correlation with
H-10, δ 6.00. (v) As H-2b was a doublet of doublet, this suggest a weak
coupling with H-4, confirmed from the 1H-1H COSY spectra,
where a W type coupling was observed between the protons at δ 2.50 (H-2b)
and 42.0 (H-4). Their chemical shift, downfield compared to a normal proton
in their position, could be due to the effect of the neighbouring carbonyl.
(vi) The stereochemistry was not determined. Taking into account the above comprehensive
NMR interpretation and the comparison of these data to those of related compounds
(Ingkaninan et al., 2000; Lee et al., 2001; Dagne et al.,
1996), structure 1 was attributed to the compound which is a new dihydroanthracenone
qualified as 4-acetyl-3,4-dihydro-3,8-dimethyl-3-hydroxy-6-methoxyanthracen-1(2H)-one,
trivially named petersone A.
The 1H, 13C, IR and mass spectral data of 2 and 3 were similar to those previously reported from the phytochemical investigation of C. siamea (Ingkaninan et al., 2000). These compounds were then identified as 5-acetonyl-7-hydroxy-2-hydroxymethylenechromone (2) and 5-acetonyl-7-hydroxy-2-methylchromone (3).
Compound 4 was isolated as a white powder hardly soluble in most of the organic solvents. 1H and 13C NMR are characteristic of stigmasterol-3-O-β-D-glucoside (Neera and Wichtl, 1987), common in the plant kingdom.
The crude CH2Cl2/MeOH (1:1) leaf extract of Cassia petersiana showed antibacterial activity against all the three bacteria species used and the diameter of inhibition were 14-16 mm against S. typhi, 15-18 mm against S. paratyphi A and 17-18 mm against S. paratyphi B. Four compounds namely 4-acetyl-3,4-dihydro-3,8-dimethyl-3-hydroxy-6-methoxyanthracen-1(2H)-one, 5-acetonyl-7-hydroxy-2-hydroxymethylene-chromone, 5-acetonyl-7-hydroxy-2-methylchromone and stigmasterol-3-O-β-D-glucoside were isolated from the above crude extract and were tested for their antisalmonellal activities. The results obtained showed that compound 4 (stigmasterol-3-O-β-D-glucoside) was the only active compound with the following diameters of inhibition: 15-18 mm against S. typhi, 17-19 mm against S. paratyphi A and 19-21 mm against S. paratyphi B. Chloramphenicol, used as the standard, showed the diameters of 27-28, 25-27 and 26-28 mm against S. typhi, S. paratyphi A and S. paratyphi B, respectively (Table 2).
Compound 4, which showed antibacterial activity against all the three bacteria
species used, was further studied using broth dilution technique and the following
results were obtained: the MIC and MBC values were 22.5 and 90 μg mL-1,
respectively, against all the three bacteria tested. For chloramphenicol the
MIC and MBC values were 2 and 16 μg mL-1, respectively, against
the same bacteria species (Table 3).
||Diameters of inhibition of S. typhi, S.
paratyphi A and S. paratyphi B by the extract and compounds
isolated from the leaves of Cassia petersiana
|NA: Not Active; 1: 4-Acetyl-3-hydroxy-6-methoxy-3,8-dimethyl-dihydroanthracenone;
2: 5-Acetonyl-7-hydroxy-2-hydroxymethylene-chromone; 3: 5-Acetonyl-7-hydroxy-2-methylchromone
and 4: Stigmasterol-3-O-β-D-glucoside
|| Inhibition parameters (MIC, MBC) of compound 4, isolated
from the leaves of C. petersiana
|Compound 4: Stigmasterol-3β-D-glucoside; MIC: Minimum
Inhibitory Concentration; MBC: Minimum Bactericidal Concentration
Antimicrobial substances are considered as bactericidal agents when the ratio MBC/MIC ≤4 and bacteriostatic agents when the ratio MBC/MIC>4 (Carbonnelle et al., 1987). For compound 4, the ratio MBC/MIC = 4, suggesting that it may be classified as bactericidal agent. Based on the MIC values, compound 4 was about eleven times less active than chloramphenicol.
Chromones were previously isolated from various species of the genus Cassia (Lee et al., 2001; Lu et al., 2001). The appearance of chromones derivatives in our results is of great interest for the investigation of members of this genus where they constitute a possible chemotaxonomic marker. In addition, the antisalmonellal activities of the CH2Cl2/MeOH (1:1) extract of the leaves of Cassia petersiana and the isolated compounds are here described for the first time and attest the traditional used of this plant against typhoid fever.
Authors are grateful to the Third World Academy of Science (TWAS) for the fellowship given to one of us (D.P.C.). Agence Universitaire de la Francophonie (AUF) is acknowledged for financial support of the work. We will like to thank Dr. Onana for plant identification." class="btn btn-success" target="_blank">View Fulltext