Abstract: Phytochemical investigation of the leaves of C. petersiana afforded two new compounds (4 α-Acetyl-3,7-dihydroxy-3,6-dimethyldihydronaphtalenone and 5-Hydroxy-7-acetonyl-2-methylenechromone), in addition to common terpenes as sitosterol, stigmasterol glucoside. The crude extract and the new compounds were tested for antisalmonellal activity. The structures of the compounds were determined from comprehensive NMR studies, including 1H, 13C, DEPT, COSY, HMQC, HMBC, MS, IR and comparison with literature data.
INTRODUCTION
Cassia is the major genus of the family Caesalpiniaceae and possesses about 600 species distributed worldwide (Viega et al., 2004). This genus constitutes a possible lead for the search of Aromatic compounds (Rao et al., 1999; Ingkaninan et al., 2000; Moriyama et al., 2003; Li et al., 2001), meanwhile terpenes and alkaloids could also be found (Msonti, 1984; Ingkaninan et al., 2000). Significant biological activities have been reported from member of this genus (Moriyama et al., 2003; Villasenor et al., 2002). Cassia petersiana Bolle is a tree generally distributed in equatorial countries from Sierra Leone to D. R. Congo. This plants species is commonly growing on sandy soils up to 12 m high and at altitude of up to 1050 m above sea level. It has pinnate leaves and yellow flowers. The compressed, hairy pods are eaten either raw or cooked as gruel. The roots of the plant are used as a treatment for coughs, colds, syphilis and stomachache. It is also used as an anthelmentic. The roots are mixed with those of Fagara nitens (Rutaceae) and Stegmanotaenia araliacea (Umbellifereae) and burnt to charcoal, which is pulverized and rubbed on incisions cut in the ankles and between the forefinger against snake bite. In Southern Africa, the leaf is used as a febrifuge and as a cure for skin diseases (Msonti, 1984). Previous phytochemical investigation on this plant species led to the isolation of diterpenes (Msonti, 1984) and flavonoids (Coetzee 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, 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 structure elucidation of two new compounds from this natural source. The extract and the pure compounds were assessed for their antisalmonellal activity. The compounds were characterised from comprehensive NMR interpretation including 1D and 2D.
MATERIALS AND METHODS
General
1H, 13C NMR and 2D spectra were recorded on JEOL 500
MHZ, Lambda spectrometer with TMS as an 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.
Plant Material
The leaves of Cassia petersiana Bolle were collected in Bafia, Centre
province of Cameroon in July 2003. Plant material was identified by Dr. Onana,
National Herbarium, Yaoundé, Cameroon, 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, Yaounde, 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
BL96AU, UK.
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 on 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, B and C were recorded.
Fraction A was purified on a Sephadex LH-20 column (n-hexane-CH2Cl2-MeOH,
7:4:1) and preparative TLC to give 4α-Acetyl-3,7-dihydroxy-3,6-dimethyldihydro-naphtalenone
(1, 11 mg). Fraction B was purified on a silica gel column (n-hexane-EtOAc
9:1) and Sephadex LH-20 to give 5-Hydroxy-7-acetonyl-2-methylenechromone (2,
6 mg). Sitosterol and stigmasterol-3-O-β-D-glucoside were identified from
cochromatography of fraction C with authentic sample available from our laboratory.
4α-Acetyl-3,7-dihydroxy-3,6-dimethyldihydronaphtalenone (1): Yellow powder, mp 117.1°C (uncorrected), IR 3370, 3100, 1710, 1290, 980; 1H NMR (CDCl3, 500 MHZ), (Table 1), 13C NMR (CDCl3, 125 MHZ), (Table 1) EIMS, m/z 248 (10), 247 (30) 233 (56), 231 (40), 206 (50), 190 (100), HREIMS 248.2753 [calcd. for C14H16O4, 248.2764].
5-Hydroxy-7-acetonyl-2-hydroxymethylenechromone (2): Yellow powder, mp more than 300°C, IR 3300, 1740, 1631, 926. 1H NMR (CDCl3, 500 MHZ), (Table 1), 13C NMR (CDCl3, 125 MHZ), (Table 1) EIMS, m/z 248 (15), 220 (60), 217 (35), 212 (100), HREIMS 248.2431 [calcd. for C13H12O5, 248.2428].
| Table 1: | 1H and 13C NMR data of compound 1 and 2 in CDCl3, at 500 and 125 MHZ |
Antimicrobial Assay
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) medium already inoculated with the test organism (100 μL of the bacteria suspension in Selenite broth, at the concentration of 5.105 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 dimethylsulfoxide (DMSO). The wells were filled with 150 μL of the solution (of known concentration) of various compounds and extract to be tested. 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 extract (that induced a zone of inhibition) was prepared 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 (5.105 cfu mL-1) was added and the mixture was homogenised. The total volume of the mixture was 5 mL, with the extract concentrations in the tube ranging from 48 to 0.75 mg 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 Bolle were extracted with CH2Cl2/MeOH (1/1), to give an organic extract. Part of this 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, B and C were recorded and purified to give two new compounds, 1 and 2 (Fig. 1).
| Fig. 1: | Structures of the isolated compounds |
Compound 1, was isolated as a yellow powder, mp 117.1°C (uncorrected). The IR spectrum showed bands at 3370 (hydroxyl group), 1710 (carbonyl), 1290 (hydroxyl bond) and 980 (substituted aromatic ring) cm-1; the cross formula was deduced to be C14H16O4 on the basis of a molecular ion peak at m/z 248.2753 on HRMS. On EIMS a fragment was observed at m/z 247 [M-H]+, suggesting the structure to possess hydroxyl group. Further fragments could also be observed at, m/z 233 [M-CH3]+, 231 [M-OH]+, 206 [M-Ac]+, 190 [M-OAc]+. The 1H NMR and the 13C NMR data are listed in Table 1. The structure was established from the following observations on the spectra. (I) From the 1H NMR we could observed three singlets each integrating for three protons attributed to the methyl’s H-10, H-11 and H-12. (ii) From the 13C the structure was suggested to possess two carbonyls (C-1 and C-9) and two carbons link to hydroxyl group (C-3 and C-7). (iii) H-5 and H-8 were placed para to each other, as they were both singlet. (iv) The positions of H-5 and H-8 were distinguished from a correlation on HMBC between H-8 (δ 7.40) and the carbonyl at (δ 203.1, C-1). (v) Me-12 was placed at C-6 based on the HMBC where H-5 (δ 6.31) was correlated to C-12 (37.0). (iv) The acetyl moiety was deduced from an intense correlation of H-10 to C-9 on HMBC. Used of HMQC help to assign carbon multiplicities. The stereochemistry was deduced from comparison of our data to those of a closely related compound (Ingkaninan et al., 2000). Based on our data here described and compared to those of similar compound (Ingkaninan et al., 2000), structure 1 was attributed to the molecule which is a new derivative trivially named petersone A.
Compound 2 was isolated as a yellow powder, which did not melt before 300°C. The HREIMS spectrum showed a molecular ion peak at m/z 248.2431 [M]+, compatible with the molecular formula C13H12O5. The chromone nature was attributed to the structure from a peak at m/z 220 (M-CO) on EIMS, characteristic for γ-pyrone system (Wagner et al., 1978). Fragments could also be observed at m/z 247 [M-OH]+, 220 [M-CO]+, 217 [M-OCH3]+ and 202 [217-CH3]+. The IR spectrum showed absorption bands at 3300 (OH), 1740 (C = O), 1631 (dienone) and 926 (substituted aromatic ring) cm-1. The 1H and 13C NMR data are listed in Table 1. The structure was established from deep studies of the spectra, thus the following items could be made: (I) A 2,5,7-trisubstituted chromone skeleton was deduced from the 1H spectra. (ii) The location of the hydroxyl group at C-5 was determined from the downfield signal at δ 12.44, hydrogen bond with the ketone at C-4. From the 13C NMR it was evident that there are two carbonyls (C-4, C-2’) and four carbon connected to oxygen atom (C-2, C-5, C-8a and C-1). Our data were very close and even identical to those previously reported by Ingkaninan et al. (2000); the difference came to be the 5-OH. From comparison of our data to those of related compounds previously reported (Ingkaninan et al., 2000; Wagner et al., 1978), structure 2 was attributed to the molecule characterised as 7-Acetonyl-5-hydroxy-2-hydroxymethylenchromone which is a new chromone derivative trivially named petersone B.
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. Two compounds, namely 4α-Acetyl-3,7-dihydroxy-3,6-dimethyldihydronaphtal-enone and 5-Hydroxy-7-acetonyl-2-hydroxymethylenechromone, were isolated from the above crude extract and were tested for their antisalmonellal activities.
| Table 2: | Diameters of inhibition of S. typhi, S. paratyphi A and S. paratyphi B by the CH2Cl2/MeOH (1:1) extract and compounds isolated from the leaves of Cassia petersiana |
| Key: NA: Not Active, 1: 4α-Acetyl-3,7-dihydroxy-3,6-dimethyldihydronaphtalenone, 2: 5-Hydroxy-7-acetonyl-2-hydroxymethylenechromone | |
| Table 3: | MIC and MBC values of the CH2Cl2/MeOH (1:1) extract of the leaves of Cassia petersiana |
| Key: MIC: Minimum inhibitory concentration, MBC: Minimum bactericidal concentration | |
The results obtained showed that none of these compounds was active against the bacteria strains used. 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).
The crude extract, 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 1.5 and 12.0 mg mL-1, respectively, against all the three bacteria tested. For chloramphenicol the MIC and MBC values were 2.0 and 16.0 μg mL-1, respectively, against the same bacteria species (Table 3).
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 the crude extract, the ratio MBC/MIC > 4, suggesting that it may be classified as bacteriostatic agent.
CONCLUSION
Chromones are regularly isolated from various species of the genus Cassia (Lee et al., 2001; Lu et al., 2001; Biswas and Malik, 1986; Kuo et al., 2002). Petersone B is a new derivative here reported for the first time. Its presence in our results is of great interest for the investigation of members of this genus where it constitutes a possible chemotaxonomic marker.
ACKNOWLEDGMENT
We are grateful to TWAS (Third World Academy of Sciences) for the fellowship given to one of us (D.P.C.); AUF (Agence Universitaire de la Francophonie) is also acknowledged for partial financial support of the work. Authors will like to thank Dr. Onana for plant identification.