Abstract: Background: Plants of the genus Jacaranda and Kigelia (Bignoniaceae) are traditionally used to relieve ear infection, abscesses, ulcers, diarrhea, dysentery, boils, fungal infections, psoriasis and eczema. The main objective was to undergone the phytochemical investigation and antibacterial activity of the stem barks of Jacaranda mimosifolia and Kigelia africana. Materials and Methods: Plants were extracted by solvents of various polarities. Compounds isolation was carried out using chromatography methods (medium and high-pressure liquid chromatography, open column and thin-layer chromatography). The isolated compounds were identified and characterized by using 1D and 2D NMR spectroscopy. The antimicrobial activity was assayed against some Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi) and Gram-positive bacteria (Staphylococcus aureus) by determining the Minimum Inhibitory Concentration (MIC). Results: The phytochemical investigation of Jacaranda mimosifolia and Kigelia africana led to the isolation of seventeen known compounds, seven terpenoid, four quinone and seven order compounds. The extracts and pure compounds exhibited antibacterial activities with inhibition zone diameters ranging from 0.0±0.0 to 11.0±0.2 mm. Selected crude extracts, fractions and compounds showed Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) ranging from 0.1±0.0 to >0.83±0.00 and 0.83±0.00 to >0.83±0.00 mg mL–1, respectively, against the tested microorganisms. Compound 17showed the best MIC against the four strains. Conclusion: The results obtained indicate that these plants could be a possible source of an effective antibacterial and justify the traditional uses.
INTRODUCTION
Microbial infections represent the world’s leading cause of pre-mature death and our well-being depends on the production of new clinically useful antibiotics to curtail and/or eradicate pathogens in our communities1. For over a decade, the pace of development of new antimicrobial agents has slowed down while the prevalence of resistance has been growing at an astronomical rate. Today, multiple antibiotic resistances among bacterial pathogens are a major public health concern worldwide2. Bacterial infections due to Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi and Eschericha coli have continued to be a major source of morbidity and mortality in hospitals and these organisms are now exhibiting multi-drug resistance to commonly used antibiotics, hence a significant cause for concern among physicians3. The presence of efflux pumps and multidrug resistance (MDR) proteins in antibiotic resistant organisms contribute significantly to the intrinsic and acquired resistance in these pathogens4. The increase in prevalence of multiple-drug resistance has slowed down the development of new synthetic antimicrobial drugs and has necessitated the search for new antimicrobials from alternative sources. Recent progress to discover drugs from natural sources has resulted in compounds that treat cancer, resistant bacteria, viruses and immunosuppressive disorders5.
Phytochemicals from medicinal plants with demonstrated antimicrobial activities have the potential of satisfying these needs, because their structures are different from those previously studies and therefore their mode of action are likely to differ. There is growing interest in correlating the phytochemical constituents of a medicinal plant with its pharmacological activity6, 7. Screening the active compounds from plants has led to the discovery of new and efficient drugs against various diseases8.
The genus Jacaranda and Kigela (Bignoniaceae) are widely distributed in the tropical and subtropical areas of the world. Some species are used in traditional medicine of different countries to cure wounds, ulcers and as an astringent in diarrhoea and dysentery9. Jacaranda mimosifolia (D.), Don is an ornamental tree with attractive foliage and owers. The leaves, owers and seeds are used in Africa to treat hypertension and amoebic infections10. Kigelia africana (Lam.) Benth is widely distributed in south, central and west Africa11. It is a plant with medicinal properties not only because of its perceived characteristics such as bitterness, astringent taste or smell but also because of forces that it seems to emit in connection with its location, orientation and association with other plants12. Most commonly traditional healers used it to treat a wide range of skin ailments like, fungal infections, boils, psoriasis and eczema13. It also has interval application including the treatment of dysentery, malaria, diabetes and pneumonia14. Previous phytochemical studies revealed that iridoids, flavonoids, naphthoquinones, monoterpenoid naphthoquinones, limonoids steroids, isocoumarins and lignans are the main secondary metabolites isolated from K. africana15-16,18-19, while triterpenes, flavonoids, acetoside, quinones, phenylpropanoidderivatives, fatty acid and anthocyanins have been reported from J. mimosifolia 20-25. The present study aims at evaluating the antibacterial activity of crude extracts, fractions and isolated compounds from the stem bark of J. mimosifolia and K. africana.
MATERIALS AND METHODS
Plant material: The stem barks of J. mimosifolia and K. africana were collected from Melen, Yaounde-Cameroon (February, 2010) and Dschang, Cameroon (November, 2012), respectively. These two plants were identified by Mr. Victor NANA of the Cameroon National Herbarium (HNC) where voucher specimens for J. mimosifolia (N°50081/HNC) and K. africana (N° 157/HNC) have been deposited
Extraction and isolation: The air-dried stem bark of J. mimosifolia was pulverised and 2 kg was exhaustively macerated with 12 L of Methylene Chloride-Methanol (Cl2CH2-MeOH) (1:1v/v) at room temperature for 72 h. The macerate was filtered and evaporated under reduced pressure to obtain 300 g of a crude extract labeled JMB. Two hundred grams of this extract was solubilized with ethyl acetate (AcOET) filtered and evaporated under reduce pressure to obtain 75 g of AcOEt extract fraction labeled JMB1. Seven gram of this AcOEt extract fraction was then subjected to silica gel 60 (0.063-0.200 mm) column chromatography using n-hexane, n-hexane-ethyl acetate gradient and ethyl acetate. One hundred and fifty fractions of 150 mL, each were collected and concentrated under vacuum. The JMB1 AcOEt extract fraction yielded four compounds: namely benzoic acid 1 (25 mg)26, 1-naphthaleneacetic acid, 5-carboxy-1, 2, 3, 4, 4a, 7, 8, 8a- octahydro-1,2,4a-trimethyl-,[1S-(1α, 2β, 4aβ, 8aα)] 2 (23 mg)27, betulinic acid 3 (15 mg)28, lupeol 4 (15 mg)22 and ursolic acid 5 (3 g)28, obtained, respectively in n-Hex-AcOEt, 93:7 (frs. 18-21), n-Hex-AcOEt, 90:10 (frs. 33-35), n-Hex-AcOEt, 75:25 (frs. 65-69), n-Hex-AcOEt, 70:30 (frs. 80-85) and n-Hex-AcOEt, 60:40 (frs. 110-117).
The air-dried and powdered stem bark (1.5 kg) of K. africana was extracted with MeOH using the soxhlet apparatus. The solvent was then concentrated under reduced vacuum pressure to obtain 160 g of a crude extract labeled KAB. One hundred forty grams of this extract was solubilized successively with n-Hex-AcOEt, (1:1v/v), AcOEt and H2O followed by filtration to give three main fractions labelled KAB1 (45 g), KAB2 (40 g) and KAB3 (40 g), respectively. Thirty gram g of the n-Hex-AcOEt fraction (KAB1) was subjected to silica gel 60 (0.063-0.200 mm) column chromatography using n-hexane, n-hexane-ethyl acetate gradient and ethyl acetate. One hundred and fifty fractions of 150 mL each were collected and concentrated under vacuum to yield four compounds namely lapachol 6 (65 mg)29, dehydro-α-lapachone 7 (15 mg)29, 2-acetylfuro-1,4-naphthoquinone 8 (20 mg), p-coumaric acid 9 (30 mg)30 and caffeic acid 10 (29 mg)31 obtained, respectively in n-Hex-AcOEt, 99:1 (frs. 26-33), n-Hex-AcOEt 98:2 (frs. 45-48), n-Hex-AcOEt 90:10 (frs. 70-74), n-Hex- AcOEt 55:45 (frs. 80-85) and n-Hex-AcOEt 55:45 (frs. 90-93).
Twenty five grams of the ethyl acetate fraction (KAB2) was subjected to 40 g silica gel 60 (0.063-0.200 mm) column chromatography using n-Hexane, n-Hexane-ethyl acetate gradient, ethyl acetate and ethyl acetate-methanol (AcOEt-MeOH) gradient. One hundred and ninty eight fractions of 150 mL each were collected and concentrated under vacuum to yield six compounds namely, 2-(4-hydroxyphenyl)ethyl ester 11 (25 mg)30, β-sitosterol 12 (35 mg)32, kigelinol 13 (25 mg)33, oleanolic acid 14 (35 mg)34, β-friedelinol 15 (20 mg)35, pomolic acid 16 (15 mg)36 obtained, respectively in n-Hex-AcOEt 95:5 (frs. 26-33), n-Hex-AcOEt 90:10 (frs. 45-50), n-Hex-AcOEt 80:20 (frs. 64-67), n-Hex-AcOEt 80:20 (frs. 73-77), n-Hex-AcOEt 75:25 (frs. 96-100), AcOEt-MeOH 95: 5 (frs. 115-119).
Twenty five gram of the aqueous fraction (KAB3) was subjected to 40 g silica gel 60 (0.063 - 0.200 mm) column chromatography (40 g) using ethyl acetate and ethyl acetate-methanol gradient. Sixty fractions of 150 mL each were collected and concentrated under vacuum to yield one compound namely kojic acid 17 (100 mg)37 obtained in AcOEt-MeOH 90:10 (frs. 30-35).
General experimental technique: Fractions were monitored by TLC and performed on precoated silica gel 60 F254 plates (Merck, Dramstadt, Germany). The spots were revealed using both ultra-violet light (254 and 366 nm) and 10% H2SO4 spray reagent. The structures of isolated compounds were elucidated by means of spectroscopic experiments mainly 1D and 2D NMR performed, on a 600 MHz Bruker Avance III-600 spectrometer equipped with a 5 mm BBFO+probe at 300 K and ESIMS/HRESIMS analyses recorded on a SYNAPT G2 HDMS (Waters) mass spectrometer and by comparison with literature data.
Microbial strains: Four bacterial strains were used as test microorganisms in this study. One Gram-positive bacteria, Staphylococcus aureus CIP 7625 and three Gram-negative, Pseudomonas aeruginosa CIP 76110, Salmonella typhi and Escherichia coli ATCC 25922, were obtained from the Essos Central Hospital, Yaounde-Cameroon. Bacteria were grown and maintained in culture on Muller Hinton Agar slants at 35°C until used. They were then stored under aerobic conditions.
Inoculum preparation: Before any test, bacteria were subcultured on Mueller Hinton agar slants at 35°C for 18 h. Mature colonies were collected with inoculating loop and introduced into a tube with 5 mL of sterile saline (0.9% NaCl) and homogenized. The turbidity of the solution was adjusted at 0.5 McFarland standards.
Antibacterial assays
Antibacterial screening of plants extracts and compounds by disk diffusion method: In vitro antibacterial activity was screened by disc diffusion method using Mueller Hinton Agar (MHA) obtained from Mast Group Ltd. The MHA plates were prepared by pouring 15 mL of molten media into sterile petriplates (90 mm). The plates were allowed to solidify for 5 min and 0.1 mL of inoculum suspension was swabbed uniformly and the inoculum allowed to dry for 5 min.
The different extracts at 20 mg discs–1 and compounds at 2 mg discs–1 were loaded on 5 mm sterile individual discs. The loaded discs were placed on the surface of the medium in which the extracts and compounds were allowed to diffuse for 5 min. The plates were incubated at 35°C for 24 h. Negative control was prepared using 10% DMSO as solvent while Amoxicillin (2 mg disc–1) was used as positive control. At the end of incubation, inhibition zones formed around the disc were measured with a vernier calliper in millimeter. Each experiment was performed in triplicate38.
MIC and MBC determination: The Minimum Inhibitory Concentration (MIC) was determined according to National Committee for Clinical Laboratory Standards (NCCLS) M38, a microdilution method using (12×8 wells) microtitre plates. This method was applied on extracts and compounds that demonstrated high efficacy against microorganisms using the disc diffusion method. In the well of the first line (line 1), 100 μL of culture medium Mueller Hinton Broth (Mast Group Ltd.) was introduced and 100 μL in the remaining well of the plates. Later on, 100 μL of stock solution of crude extracts and compounds at 20 and 2 mg mL–1، respectively was added to the first well. The medium and sample in the first well were mixed thoroughly before transferring 100 μL of the resultant mixture to the well of the second line. Then two-fold serial dilutions of the test samples were made from line 1 until line 11 and 20 μL of inoculum standardized at 0.5 McFarland standards were introduced in the entire well containing the test substances except the columns of blank (column C and F) which constitute the sterility control. The concentration range was 0.0081-8.3000 mg mL–1 for crude extracts and fractions and 0.83000-0.00081 mg mL–1 for compounds. In each microtiter plate, a column with broad-spectrum antibiotic (Amoxicillin) with the concentration range from 0.00195-2 mg mL–1 was used as positive control. After incubating at 35°C for 24 h, turbidity was observed as indication of growth. Thus the lowest concentration inhibiting the growth of bacteria was considered as the Minimum Inhibitory Concentration (MIC).
The MBC was determined by transferring 25 μL aliquots of the clear wells into 100 μL of freshly prepared Muller Hinton Broth medium and incubating at 35°C for 24 h. The MBC is the lowest concentration of test sample which did not produce turbidity as above, indicating no microbial growth. All tests were performed in triplicate. Amoxicillin was used as positive control.
Statistical analysis: The different results were statistically analyzed using the software SPSS 17.0 for windows and variance analysis by ANOVA coupled with turkey test where p<0.05 was considered as statistically significant.
RESULTS AND DISCUSSION
Fractionation and isolation of compounds: Fractionation of J. mimosifolia and K. africana stem bark extracts by column chromatography lead to the isolation and purification of five (1-5) and twelve compounds (6-17), respectively. The structures of the isolated compounds were determined by spectroscopic analysis, especially, 1H and 13C NMR spectra in conjunction with 2D experiments, COSY, HSQC, HMBC and direct comparison with reference data from available literature. The compounds isolated from stem bark of J. mimosifolia and K. africana are shown in Fig. 1.
They are the terpenoids betulinic acid (3), lupeol (4), ursolic acid (5), oleanolic acid (14), β-friedelinol (15), pomolic acid (16), quinones identified as lapachol (6), dehydro-α-lapachone (7), 2-acetylfuro-1,4-naphtoquinone (8), kigelinol (13), steroid β-sitosterol (12) and phenolic acids identified as benzoic acid (1), 1-naphthaleneacetic acid, 5-carboxy-1, 2, 3, 4, 4a, 7, 8, 8a-octahydro-1, 2, 4 a-trimethyl-[1S-(1α, 2β, 4aβ, 8aα)] (2), p-coumaric acid (9), caffeic acid (10), nonacosanoic acid, 2-(4-hydroxyphenyl)ethyl ester (11) and kojic acid (17).
The bacterial inhibition zone diameters of crude extracts, fractions and compounds are summarized in Table 1 and 3. The results of the MIC and MBC are represented in Table 2, 4, 5 and 6. The results in Tables 1 and 3 below indicate that the inhibition zone diameters vary from 0.0±0.0 to 11.00±0.02 mm on both bacteria strains and are dependent on the concentration of extracts and compounds tested on the microorganisms. The fraction JMB1 and the compounds p-coumaric acid (9), kojic acid (17), betulinic acid (3) in low concentration have a broad spectrum of action on both strains. Kojic acid (17) and betulinic acid (3) displayed moderate inhibition diameters on S. aureus and E. coli ATCC 25922 with inhibition zones of 11.0±0.0 and 10.0±0.0 mm, respectively. S. typhi was not susceptible to any extracts and isolated compounds except p-coumaric acid (9) and kojic acid (17). In general, the inhibition zone diameter shown by amoxicillin is less than those of extracts and compounds.
The extracts, fractions and compounds that had broad spectrum of action on bacteria strains were selected for the determination of the Minimum Inhibitory Concentration and Minimum Bactericidal Concentration. The results of the Minimum Inhibitory Concentration ranged from 0.1±0.0 to >0.83±0.00 mg mL–1 on all the tested microorganisms (Table 2). These results showed that the two plant extracts were active against both the Gram-positive and Gram-negative organisms with better activity against the Gram negative organisms. This implies that the use of these extraction procedure releases some active ingredients which have high activity against some enteric organisms. In fact, kojic acid (17) isolated from K. africana is the most active on the four strains with the MIC of 0.83±0.00, 0.2±0.0, 0.1±0.0 and 0.2±0.0 mg mL–1, respectively on S. aureus, P. aeruginosa, S. typhi and E. coli with a significant difference at p<0.05. These results showed an important difference in inhibitory activity of this compound between Gram-positive and Gram-negative organisms.
With other compounds there appears to be a slight antibacterial activity at the first concentration of study (0.83±0.00 mg mL–1). Amoxicillin, the standard antibiotic used as controls had much lower MIC values than the tested compounds.
DISCUSSION
The search for antimicrobials from natural sources has received much attention and efforts have been made to identify compounds that can act as suitable antimicrobial agents to replace synthetic drugs. Phytochemicals derived from plant products serve as a prototype to develop less toxic and more effective medicines in controlling the growth of microorganism39. These compounds have significant therapeutic application against human pathogens including bacteria, fungi or virus.
In the present investigation, the extracts of two medicinal plants were tested against the pathogenic microbes; Escherichia coli, the most common bacteria whose virulent strains cause gastroenteritis, urinary tract infections, neonatal meningitis; Staphylococcus aureus, a wound infecting pathogen which can cause septicemia, endocarditis and toxic shock syndrome; Pseudomonas aeruginosa which infects the pulmonary tract, urinary tract, burns and wounds and Salmonella typhi which causes typhoid fever. The wide spread of these infectious pathogens raises the need for new, cheap, effective and safe drugs. One of the best candidates to address this need appears to be the natural resources40. Varying level of potency of the crude extracts, fractions and compounds from stem bark of J. mimosifolia and K. africana were observed against different bacteria strains. The MIC values of the stem bark extracts and isolated compounds from the two plants against different tested strains vary. The relationship between zone of inhibition and MIC value may not be related. In fact, kojic acid (17) that gives the high zone of inhibition against S. aureus (11.0±0.0 mm) give the MIC value (0.83±0.00 mg mL–1) less than those obtain against the three other strains. This is similar to previous observations that some compounds or the crude extracts have the weak diffusion power that may influence the formation of significant zone of inhibition on the agar medium8. On the other hand, these tested strains may have different level of intrinsic tolerance to antimicrobials and thus the MIC values differ from one isolate to the other.
The result of this study showed that K. africana and J. mimosifolia extracts have varied antibacterial activities against the tested organisms. This correlates with the observation of previous research which showed that these plants contain substances with antimicrobial activity13, 41. In fact, kojic acid isolated from K. africana showed good antibacterial activity against the four bacterial strains with MIC range from 0.1±0.0 to 0.83±0.00 mg mL–1. Kojic acid is classified as a multifunctional, reactive γ-pyrone with weakly acidic properties. It is reactive at every position on the ring. At carbon 5 position, the hydroxyl group acts as a weak acid, which is capable of forming salts with few metals such as sodium, zinc, copper, calcium, nickel and cadmium42. These properties of kojic acid related to his structure could explain the fact that it possesses antibiotic properties against gram-negative as well as gram-positive microorganisms43. Beelik44 previously reportedthat the bacterial growth is generally inhibited in the presence of more than 0.5 % (w/v) of kojic acid.
This observation is justified by the values of MIC on the four strains obtained above. Some compounds isolated in this study have been reported to possess antibacterial activity. Betulinic acid (3) from stem bark of J. mimosifolia showed weak activity against the four bacterial strains (MIC>0.83±0.00 mg mL–1). Chandramu et al.45 reported that the same compound isolated from the leaves of Vitex negundo exhibited antimicrobial activity against Bacillus subtilis and Staphylococcus epidermidis (MIC of 128 μg mL–1) but no activity against the Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli. P-coumaric acid (9) isolated in this study from stem barks of K. africana present a weak antibacterial activity against the four strains with zone of inhibition range from 6.0±0.0 to 7.0±0.0 mm with the best activity on Salmonella typhi (MIC = 0.4±0.0 mg mL–1). The antibacterial activity of p-coumaric acid (9) observed in the present study is in agreement with the findings of Lawrence et al.46 who isolated the same compounds from Aloe vera. The present study shows that ursolic acid (5) has moderate antibacterial activity (MICs in the range 0.83±0.00 to 0.83±0.00 mg mL–1) on the four bacterial strains. These results is different from that of Moodley et al.28 who isolated ursolic acid from the fruits of Carissa macrocarpa which showed moderate antibacterial activity (MICs in the range 0.12 to 1.00 mg mL–1) against S. aureus (ATCC 25923 and ATCC 43300), E. coli (ATCC 25922) and P. aeruginosa (ATCC 35032). The difference in MIC values could be due to the difference between the strains species.
Accordingly, the discovery of natural, effective and cheap drug against these resistant bacteria might be considered as a breakthrough in the solution of this problem all around the world39. Therefore, the demonstrated antimicrobial activities of these plant extract and isolated compounds in this study, confirms its traditional use in treating antimicrobial infections like dysentery, wound infection, sore, ear infection and abscesses.
CONCLUSION
The results provide justification for the use of these plants in folk medicine to treat various infectious diseases. This study might be considered as a prelude to discover new antibacterial agents to the problematic pathogenic bacteria. Moreover, the broad spectrum activity of isolated compounds as kojic acid gives the opportunity for possible discovery of new, effective components for downstream clinical development.
ACKNOWLEDGMENTS
The authors are grateful to the Institute of Medical Research and Medicinal Plants Studies (IMPM) of Cameroon for their laboratory facilities.