Abstract: A new antibacterial ester (1), 2-O-butyl-1-O-(2’-ethylhexyl) benzene-1,8-dicarboxylate and a ketone (2), 1-phenyl-1,4-pentanedione was isolated from Vitellaria paradoxa (G.Don) heper by directing the fractionation of an ethanol extract of the air dried root bark with bacterial sensitivity assay. The structures of 1 and 2 were determined from 13C-NMR, 1H-NMR, DEPT, IR and GC-MS spectral data. The two compounds were found to be active against gram positive Bacillus subtilis and Staphylococcus aureus and gram negative Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa. The isolation, structural elucidation, NMR spectral assignment and bioactivities of compounds 1 and 2 are reported.
INTRODUCTION
Vitellaria paradoxa (G.Don) Heper. Commonly called ‘Shea butter tree’ in English, belongs to the family Sapotaceae. It is a plant that grows naturally in the wild in the dry savannah belt of West Africa from Senegal in the west to Sudan in the east and is locally abundant in Nigeria (NRC, 2006; FAO, 2007). It is a small to medium size tree which grows up to a height of 25 m. The tree is much branched, dense and spread in form of hemispherical crown. It consist of one of the most affordable and widely used vegetable fats in the Sahel and the nuts are important internationally where they are being sold to European and Japanese food industries. (Akhter et al., 2008).
Vitellaria paradoxa is reported to have a great medicinal value particularly in the preparation of skin ointment. The shea butter which is the fat extracted from the kernel is reported to contain a high level of UV-absorbing triterpenes ester (Wiesman et al., 2003; Brucken et al., 2008). Its anti-oxidant properties have led to its use in the protection of skin from sun burn, eczema and as a skin rejuvenator (Badifu, 1989; Alender, 2004; Akhter et al., 2008). Analysis of the kernel revealed the presence of phenolic compounds such as gallic acid, catechin, epicachin, epigallocachin gallete as well as quercetin and transcinnamic acid (Steven and Nissim, 2003; Maranz et al., 2004). The plant is used to treat inflammation, rashes in children, dermatitis, chapping, irritation, ulcer and rheumatism (Hong et al., 1996). Leaf decoctions are used for the treatment of stomach ache, head ache and as an eye lotion. The paste of the root bark is taken orally to cure jaundice as well as diarrhea and stomach ache in humans and applied topically to treat chronic sores and girth sores in horses (Mallogo, 1989). Its stem bark decoction is used in a bath to facilitate child birth, encourage lactation after delivery, treatment of leprosy, neutralization of venom of spitting cobra and for gastric problems as well as for diarrhea and dysentery (Vining, 1992; Von Maydell, 1986). The immense applications of Vitellaria paradoxa to the treatments of many bacterial diseases motivated us to investigate the plant for antibacterial components.
MATERIALS AND METHODS
Plant collection: The roots of Vitellaria paradoxa (G.Don)
heper were collected in August 2006 at Nigerian Defence Academy, Afaka, Kaduna
state, Nigeria. The plant was authenticated at the herbarium section of Department
of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria. A specimen
of the root is preserved at the Herbarium.
Extraction and isolation: The air dried root bark of Vitellaria paradoxa (500 g) was milled and extracted at room temperature by percolation with ethanol (2.5 L) for two weeks. The extract was concentrated using rotary evaporator at 40°C to give a residue (150 g). Twenty gram of the ethanolic residue was chromatographed on silica gel column (silica gel, 235 g i.d. 4.5 cm). The column was eluted successively with solvents of increasing polarity to give fractions and residue as shown in parenthesis: petroleum ether (0.5 g); CHCl3 (3.9 g); hexane:EtOAc, 1:1, (0.8 g); CHCl3-EtOAc, 1:1, (0.9 g); EtOAc (0.7 g); EtOAc-MeOH, 1:3, (12.9 g) and EtOAc-MeOH, 1:1, (2.9 g). The CHCl3 fraction a light brown oil (3.5 g) which was found to be highly active against both the shrimp larvae and the targeted bacteria was further chromatographed on silica gel column (silica gel, 100 g, i.d. 2.5 cm) and eluted with a solvent mixture of CHCl3-EtOAc, 1:1, to give 6 fractions. The first fraction after elution with CHCl3-EtOAc, was found be very active and was therefore further chromatographed and eluted with CHCl3 to give the ester (1) (0.98 g) with Rf value of 0.75. The refractive index, viscosity and color of the oil were determined using standard method (AOAC, 1997) and are summerized in Table 4. Fraction (F18) eluted with EtOAc-MeOH, 1:3, (4.3 g) was further chromatographed and eluted with a solvent mixture of CHCl3-EtOAc-MeOH (1:2:1) to give fractions F1 to F15. Fraction 5 which was found to be highly active was coated on a preparatory TLC plate and developed in EtOAc-MeOH (3:1) to obtained three bands; the top band gave the ketone 2 (400 mg) with Rf value of 0.65 and melting point of 23°C.
Brine shrimp lethality test (BST): Fractions were evaluated for lethality to brine shrimp using standard methods (Meyer et al., 1982; McLaughlin, 1991; Solis et al., 1993). In this test a drop of DMSO was added to vials of the test and control substances to enhance the solubility of test materials.
Antibacterial assay: The antibacterial activity of the crude/isolated fractions were determined by paper disc diffusion method (Navarro et al., 1996; Pelczar et al., 1993; Okeke et al., 2001; Ayandele and Adebiyi, 2007) using nutrient agar. A stock solution of each extract was prepared by dissolving 20 mg in 5 cm3 of the respective solvents of extraction to give 4000 μg cm-3 of the stock solution. Using a micropipette, 0.35, 0.3, 0.25 and 0.2 cm3 of the solution were separately drawn into vial and the volumes adjusted to 2 cm3 to give approximate concentrations of 7x102, 6x102, 5x102 and 4x102 μg cm-3, respectively. Filter paper was carefully labeled and cut into sizes of 0.5 cm diameter and separately introduced in each vial containing the prepared solution. They were dried at 40°C. A control was similarly set up using distilled water and ethanol.
Nutrient agar was used as the growth medium for the microbes. Each medium was prepared by dissolving 38 g of the agar in 1000 cm3 of distilled water, heated to dissolve and autoclaved at 120°C for 15 min. It was then cooled and poured into a petri dish to solidify. Isolates of S. aureus, S. typhi, E. coli, P. aeruginosa and B. subtilis were separately cultured on each nutrient agar plate; sterile paper disc incorporated with the extract, were placed on each agar and incubated at 37°C 18 to 24 h. All tests were performed in triplicate and the zones of inhibition diameter were measured with the aid of a plastic ruler and the Minimum Inhibitory Concentrations (MIC) determined.
RESULTS AND DISCUSSION
The chromatograhed chloroform fraction which was found to be lethal to the shrimp larvae (BST LC50 = 23.20 μg cm-3) was also active against S. aureus, S. typhi, E. coli, P. aeruginosa and B. subtilis at 700 μg cm-3. The fraction after been further chromatographed yielded six fractions. Three of the fractions viz: F2 (0.49 g), F3 (0.31 g) and F4 (0.13 g) were similar and pure (Rf value 0.75 and BST LC50 = 23.7814 μg cm-3) and gave the yellow-orange ester. The refractive index of the isolated ester does not differ much from the refractive index of palm oil while the viscosity obtained fall within category of most fluid (AOAC, 1997). They were highly active against all the test bacteria. They recorded zones of inhibition diameter of 28 and 26 mm against the gram positive bacteria B. subtilis and S. aureus respectively and 25 mm against the gram negative bacteria E. coli, S. typhi and P. aeruginosa (Table 1-3).
The preparatory TLC carried out on fraction 5 of F18, gave three pure components (F5:1 (0.40 g), F5:2 (0.04 g) and F5:3 (0.03 g) which have Rf values of 0.65, 0.75 and 0.85 g, respectively. Activity tests showed that TLC fraction F18:5:1 (BST LC50 = 100 μg cm-3) was active against B. subtilis, P. aeruginosa, E. coli, S. aureus and S. typhi (zones of inhibition diameter range of 20-28 mm) and was elucidated as a ketone (2).
The isolated compounds (1 and 2) showed high activities (zones of inhibition diameter range of 25-28 mm) against the gram positive S. aureus and B. subtilis at the concentration of 7x102 μg cm-3. Similarly the activities of compounds 1 and 2 against gram negative E. coli, S. typhi and P. aeruginosa were high, (zone of inhibition range from 25-28 mm) at the concentration of 7x102 μg cm-3.
Comparisons of the antimicrobial efficacies of the compounds with those of reference standard (Amoxicillin, Erythromycin, Chloramphenicol and Gentamycin) indicated that the activities of the compounds were slightly lower than those of the reference standards (Table 1 and 2). For instance, Amoxicillin and compound 2 exhibited zones of inhibition diameter of 30 and 28 mm, respectively against B. subtilis at the concentration of 7x102 μ cm-3. Similarly, gentamycin and compound 2 recorded zones of inhibition of 30 and 28 mm, respectively against P. aeruginosa at the same concentration. This is expected in view of the higher activities exhibited by synthetic drugs compared to their counterparts obtained from natural sources (Bhavanani and Ballow, 2000; Chung et al., 2004; Nair and Chanda, 2004; Borris, 1996; Essawi and Srour, 2005).
| Table 1: | Activity of isolated compounds against gram positive bacteria |
| NI: No inhibition, C: Control | |
| Table 2: | Activity of isolated compounds against gram negative bacteria |
| NI: No inhibition, C: Control | |
| Table 3: | 13CNMR for compounds 1 and 2 |
| Table 4: | Physical properties of the isolated compounds |
The 1HNMR, 13CNMR, APT and DEPT of compound 1 and 2 were obtained by analyzing the samples on Mercury BB 200 MHZ. The 1HNMR of compound 1 recorded signals at d 7.65 (1H, d, H-3 and H-6), 7.55 (1H, d, H-4 and H-5) which are aromatic protons, 4.55 (2H, t, H-1’ and 7’), 4.00 (2H, d, H-8’) are methelene protons, 1.75 (1H, d, H-2’) a methine proton, 1.55 (3H, q, H-6’) and 0.85 (1H, s, H-10’) are methyl protons.
The 13CNMR spectrum had signals recorded at d 167.81 (C-1) and 167.75 (C-8) which are carbonyl carbons, d 132.70 (C-2 and C-7) are aromatic quaternary carbons (Table 3) as well as 131.15 (C-3 and C-6) and 129.08 (C-4 and C-5) which are aromatic carbons. The signals at d 68.40 (C-1’ and C-7’), 66.45 (C-8’), 64.59 (C-9’), 35.67 (C-3’), 30.62 (C-4’), 29.19 (C-5’) and 24.02 (C-1’=) are methylene groups. Those at d 19.86 (C-10’), 14.30 (C-2’=) and 11.23 (C-6’) are methyl groups. The signal at d 39.00 (C-2’) is a methine group (Fig. 1). The IR spectrum showed a strong absorption at 1729.31 cm-1 which is due to carbonyl stretching (C=O), 1127.53 cm-1 due to the presence of C-O-Cstr of an ester, 1588.28 cm-1 due to the presence of C=C of aromatic ring, 1460.31 cm-1 due to the presence of methyl and methylene groups and the presence of methyl group was further confirmed by the presence of a peak at 1379.36 (Fig. 2) (Robert and Robert, 1992). The GC/MS gave the molecular weight of the compound as 334, the signal at m/z 267 correspond to the loss of C5H7+ from the molecular ion, the signal at m/z 223 corresponds to the loss of C3H7+ and the signal at m/z 205 corresponds to the loss of H2O molecule from the ester molecule.
| Fig. 1: | 13Cnmr of compound 1 |
| Fig. 2: | IR spectra of compound 1 |
The signal at m/z 160 is due to the loss of CH3CH2O. radical, the signal at 149 corresponds to the base peak and is due to the loss C8H4O3+. The signal at m/z 76 corresponds to the loss of C6H4+ a substituted benzene ring from the molecule. Taking all the above information into consideration the ester isolated from the CHCl3 fractions is interpreted as 2-O-butyl- 1-O-(2’-ethylhexyl) benzene-1,8-dicarboxylate (1).
Fractions F18:5:1 had 1HNMR signals recorded at d 8.60 (1H, s, H-2’ and H-6’), 8.20 (1H, d, H-3’ and H-5’) and 7.60 (1H, d, H-4’), are aromatic protons. The one at d 3.60 (2H, t, H-2), 1.20 (2H, d, H-3), are methylene protons. The signal at 2.0 (1H, s, H-5) a methyl group proton. The 13CNMR spectrum showed signals at d 98.0 (C-1) and 190.0 (C-4) which are carbonyl carbons. Those at d 130.08 (C-1’), 128.60 (C-2’ and C-6’), 115.18 (C-3’ and C-5’), 102.50 (C-4’) are aromatic carbons. The signals at d 29.58 (C-2), 22.55 (C-3) are methylene groups and the one at 13.28 (C-5) is a methyl group (Fig. 3).
| Fig. 3: | 13Cnmr of compound 2 |
| Fig. 4: | IR spectra of compound 2 |
The IR spectrum showed no OH absorption indicating the absence of OH group. The peak at 1729.31 cm-1 is due to the presence of carbonyl carbon. The peak at 1557.93 cm-1 is due to aromatic C=C ring stretching vibration and those at 828.81 and 722.09 cm-1 are due to aromatic out of plane C=C bending vibrations (Fig. 4).
The GC/MS gave the molecular weight of the compound as 176. The signal at m/z 161 is due to the loss of CH3 group from the molecular ion, the signal at m/z 133 corresponds to the loss of carbonyl group (C=O) from the molecule. The signal at m/z 105 is due to the loss of benzene ring which is the base peak and the signal at m/z 77 is due to the loss of C2H3+ from the molecule. Taking all the information’s into consideration fraction F18:5:1 is interpreted as 1-phenyl-1,4-pentanedione (2).
CONCLUSIONS
2-O-butyl-1-O-(2’-ethylhexyl)benzene-1,8-dicarboxylate and 1-phenyl-1,4-pentanedione isolated from Vitellaria paradoxa (root) showed high activity against S. aureus, S. typhi, P. aeruginosa, B. subtilis and E. coli, suggesting that the two compounds may be employed as broad spectrum antibiotics against these organisms.
ACKNOWLEDGMENT
The researchers wish to acknowledge the contribution of Mr. A. Okolo of the Microbiology Laboratory, Department of Biological Sciences, N.D.A. Kaduna.