Abstract: Background: The use of antibiotics over the years has led to increased bacterial resistance. With the current menace of microbial resistance to already existing antibiotics, there is an urgent need to discover new antimicrobial agents with different and novel mechanism of actions for new and re-emerging infectious diseases. Pupalia lappacea (L.) Juss is an annual or perennial herb (Family Amaranthaceae) which is used for the treatment of boils, wounds and skin infections. Objective: To determine the antimicrobial potential and antibiotic resistance modulation of the chloroform (PLC), petroleum ether (PLP) and ethanol (PLE) extracts and evaluate their possible cytotoxic effects. Methods: These include the determination of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) Minimum Fungicidal Concentration (MFC) and microbial time-kill kinetics of the extracts against Pseudomonas aeruginosa ATCC 4853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis NTCC 10073, Proteus vulgaris ATCC 4175 and clinical strains of Streptococcus pyogenes, Klebsiella pneumonia and Candida albicans. The ability of the extracts at sub-MIC concentrations to modulate the activity of some selected antibiotics including amoxicillin, cloxacillin, tetracycline, erythromycin and ampicillin was evaluated. Influence of the extracts on viability, proliferation and Lactate Dehydrogenase (LDH) release of HaCaT keratinocyte cells were evaluated at various selected concentrations. Results: All the extracts of P. lappacea exhibited broad-spectrum antimicrobial activity. The PLC demonstrated the highest antimicrobial activity with MIC of 2.5 to 4.0 mg mL-1 and MBC/MFC value ranging from 4.0-8.0 mg mL-1. Time-kill kinetics generally revealed a concentration and time-dependent rate of microbiocidal activity of the extracts and MBC/MIC ratio <4. The PLC and PLP at sub-MIC concentrations of 0.5 mg mL-1 caused a 20% reduction in the MICs of amoxicillin, erythromycin, ampicillin and cloxacillin against K. pneumonia. There was also a 96% reduction in the MIC of amoxicillin against E. coli and P. vulgaris. None of the extracts exhibited cytotoxic activity against HaCaT keratinocytes within the concentrations used. Conclusion: All extracts of P. lappacea exhibited broad-spectrum antimicrobial activity and sub-MIC concentrations of the extracts showed microbial resistance modulation potential among the selected antibiotics.
INTRODUCTION
Many infectious diseases have been known to be treated with herbal remedies throughout the history of mankind. Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. It is estimated that about 80% of world’s population depend on medicines of plant origin which may consist of the additives aside the active ingredients1. Antibiotic bacterial resistance is the ability of bacteria to survive at the exposure to a Minimum Inhibitory Concentration (MIC) of an antibiotic. The use of antibiotics over the years has led to increased bacterial resistance. With the current menace of microbial resistance to already existing antibiotics, there is an urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases2. Considering the diversity of nature and the enormous potential of plants, research into plants as crude antimicrobial agents or sources of lead compounds is on the increase. Methanol fruit extract of Aframomum melegueta and other Ghanaian medicinal plants have been found to possess antimicrobial activity3.
Pupalia lappacea (L.) Juss is an annual or perennial herb (Family Amaranthaceae) found growing in the tropics and sub-tropical regions including Ghana, Nigeria, Congo DR etc. It is used for treatment of boils, wounds and skin infections. P. lappacea has been found to possess antioxidant and anticancer4,5, wound healing6,7, antinociceptive and antipyretic8 properties. In previous study, the influence of chloroform, petroleum ether and ethanol leaf extracts of P. lappacea on the rate of wound closure, histology of treated and untreated wound tissues in rats has been investigated and reported as well as the antioxidant properties of these extracts. The HPLC profiles and secondary metabolites present in extracts have also been determined6. In this current study, the anti-infective, antibiotic resistance modulation and cytotoxic activities of chloroform, petroleum ether and ethanol leaf extracts of P. lappacea have been reported.
MATERIALS AND METHODS
Collection of plant material: Leaves of P. lappacea were collected in May, 2012 at Kuntenase in the Bosomtwi District, Ashanti Region of Ghana. It was authenticated and a voucher specimen (KNUST/HMI/2013/L006) has been deposited at the Department of Herbal Medicine Herbarium, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
Extraction of plant material: The plant sample was cleaned of foreign material and dried at room temperature (25-28°C). It was then milled into coarse powder in a lab mill machine (Christy and Norris, United Kingdom). A quantity of 600 g was weighed and divided into 200 g portions of three. The various portions were extracted separately by cold maceration using 1.0 L of chloroform (BDH, England), petroleum ether (BDH, England) and 70% ethanol (Scharlau, United Kingdom) for 72 h. After 72 h, the extracts with solvents were centrifuged (3000 g for 10 min). The extract obtained was concentrated using rotary evaporator (Buchi, Disendorf, Germany) at 40°C and lyophilized. The dried powdered extracts were stored at 4°C.
Test organisms: The test organisms were Pseudomonas aeruginosa ATCC 4853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis NTCC 10073, Proteus vulgaris ATCC 4175 and clinical strains of Streptococcus pyogenes, Candida albicans and Klebsiella pneumonia.
Determination of antimicrobial activity: The antimicrobial activities of the extracts were screened using the agar-well diffusion method9. Concentrations of 12.5, 50, 100 and 200 mg mL-1 of extracts were prepared with 50% v/v methanol as solvent for the chloroform (PLC) and petroleum ether (PLP) extracts and sterile distilled water as solvent for the ethanol extract (PLE). Twenty millilitres of nutrient agar (Oxoid, United Kingdom) was melted, stabilized at 45°C and seeded with 100 μL of 1.0x105 colony forming unit (CFU)/mL of test organisms and four equidistant wells (diameter of 6 mm) made in the seeded agar. One hundred microliters of different concentrations of the extracts were introduced into the wells and allowed to diffuse adequately for 1 h. The nutrient agar plates were then incubated at 37°C for 24 h. Zones of growth inhibition were determined. The activity of 50% v/v methanol was determined and it was found to exhibit no activity against the all the test organisms. The above procedure was repeated for C. albicans using Sabouraud dextrose agar (Oxoid, United Kingdom) as the medium and incubated at 30°C for 48 h. Ciprofloxacin (10 μg mL-1) and ketoconazole (400 μg mL-1) (Sigma-Aldrich, Damstadt, Germany) were used as reference antibiotics for the bacteria and C. albicans respectively. The experiment was carried out in triplicates.
Determination of Minimum Inhibitory Concentration (MIC): The minimum inhibitory concentrations of the extracts were determined by micro-dilution method10. The micro-titre plates were filled with 100 μL of double strength nutrient broth and the appropriate concentrations of the extracts were aseptically introduced into the wells and 10 μL of 1.0x105 CFU mL-1 of test microorganisms added and then incubated at 37°C for 24 h. The MIC was detected as the lowest concentration of extract that inhibited growth which was indicated by the absence of purple coloration upon the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazoliumbromide (MTT) (Sigma-Aldrich, Damstadt, Germany) after incubating at 37°C for 24 h. The above procedure was repeated for C. albicans using Sabouraud dextrose broth as the medium and incubated at 30°C for 5 days. Ciprofloxacin and ketoconazole were used as reference antibiotics for test bacteria and C. albicans, respectively. The experiments were carried out in triplicates.
Determination of minimum bactericidal and fungicidal concentration: The minimum bactericidal (MBC) and fungicidal concentrations (MFC) were determined according to the method described by Pfaller et al.11. Appropriate volumes of different concentrations of the extracts were added to micro-titre plates containing test bacteria and nutrient broth and incubated at 37°C for 24 h. Aliquots were taken from the various concentrations after incubation and inoculated onto nutrient agar plates. The inoculated nutrient agar plates were incubated at 37°C for 24 h. The MBC was determined as the minimum or least concentration of extract which showed no visible growth of microorganisms on the surface of the nutrient agar. The procedure was repeated for C. albicans using Sabouraud dextrose agar medium and incubated the plates at 30°C for 3 days. The experiments were carried out in triplicates.
Determination of the time-kill kinetics of extracts: The time kill kinetics test was performed according to the modified method described by Wiegand et al.10. Concentrations of the MIC, 2X MIC and 4X MIC of each extract were prepared in test tubes containing 10 mL nutrient broth and inoculated with 100 μL of 1.0x105 CFU mL-1 of the test organisms and incubated at 37°C. Aliquots of 1.0 mL were taken at time intervals of 0, 1, 2, 3, 4, 5, 6 and 24 h onto 20 mL nutrient agar and incubated at 37°C for 24 h. A control test was performed with the test organisms in the medium without the extracts. The CFU was determined after incubating for 24 h. A graph of the log CFU mL-1 was then plotted against time. The experiment was carried out in triplicates.
Determination of microbial resistance modulation of extracts: The ability of the extracts at sub-MIC concentrations to modulate the resistance of some microorganisms to some antibiotics was also evaluated. This involved the determination of the MIC of the antibiotic against the microbes without the extract and determination of MIC of the antibiotic in the presence of sub-MIC concentration of the extract. The microbial resistance modulation tests were performed according to a modified method described by Wiegand et al.10. The wells of the 96-well micro-titre plates were filled with 100 μL of double strength nutrient broth. Various concentrations of antibiotics including cloxacillin, amoxicillin, ampicillin, tetracycline and erythromycin with their HPLC purities of >98% (Sigma-Aldrich, Damstadt, Germany) were used in the determination of MICs. The reference antibiotics were tested against P. aeruginosa, E. coli, S. aureus, S. pyogenes, P. vulgaris and K. pneumonia. The MICs of the antibiotics were determined after incubation for 24 h at 37°C and upon the addition of MTT. Sub-inhibitory concentrations of 0.50 and 1.0 mg mL-1 of extracts and various concentrations of the antibiotics (cloxacillin, amoxicillin, ampicillin, tetracycline and erythromycin) plus the same inoculum size were incubated at 37°C for 24 h and MICs of the antibiotics in presence of the extracts were determined according to the method described above.
Influence of extracts on cell viability of HaCaT keratinocytes: The influence of the extracts on HaCaT keratinocyte cells was evaluated according to the method described by Agyare et al.12. Extract concentrations of 0.1, 1.0, 10.0, 50.0 and 100.0 μg mL-1 were added to the cells (6x103 cells mL-1) in a 96 well Micro-Titre Plate (MTP) containing HaCaT keratinocyte medium (comprising of Dulbecco’s Modified Eagle Medium (DMEM) high glucose with glutamine, Foetal Calf Serum (FCS), non-essential amino acids and penicillin/streptomycin) and incubated at 35°C with 5% CO2. The medium was then washed with 100 μL Phosphate Buffer Solution (PBS) per well and filled with 50 μL of MTT solution. This was then incubated at 35°C for 12 h. Five percent (5%) Foetal Calf Serum (FCS) was used as a positive control while untreated HaCaT keratinocytes served as negative control. The MTT solution was then removed and dissolved with 50 μL DMSO. The intensity of the violet colouration was measured with MTP reader (Corona Electric Co. Ltd, Japan) at 595 nm against 690 nm.
Influence of extracts on proliferation of HaCaT keratinocytes: The influence of the extracts on the proliferation of HaCaT keratinocytes was performed using ELISA cell proliferation test kit13. The HaCaT keratinocyte cells (6x103 cells mL-1) were cultured in micro-titre plates containing concentrations of 0.1, 1.0, 10.0, 50.0 and 100.0 μg mL-1 of extracts constituted in HaCaT keratinocyte medium and incubated at 35°C for 48 h. After incubation, 10 μL labelling solution was added to the cells in 96-well tissue culture plates and incubated at 35°C with 5% CO2 for 24 h. The medium was later removed and the cells treated with 100 μL fixation-denaturation solution for 30 min at 20 to 25°C. The fixation-denaturation solution was later removed and the cells incubated with 50 μL antibody solution BrdU-POD (peroxidase-marked-mouse anti-BrdU-antibody) at 20 to 25°C for 90 min with constant shaking. The antibody solution was later washed out three times and 50 μL substrate solution was added to the wells. It was later incubated at room temperature between 20 to 25°C until the formation of a clear blue solution. The peroxidase reaction was terminated upon the addition of 10 μL 1M H2SO4 solution. The termination of the reaction resulted in a change of the solution from blue to yellow. The absorbance of the yellow solution was determined with MTP reader at a wavelength of 450 nm against 690 nm.
Influence of extracts on lactic dehydrogenase (LDH) release from HaCaT keratinocytes: This was determined according to the method described by Decker and Lohmann-Matthes14 . HaCaT keratinocytes (6x103 cells mL-1) were grown in 96-well micro-titre plates containing concentrations of 0.1, 1.0, 10.0, 50.0 and 100.0 μg mL-1 of extracts constituted in HaCaT keratinocyte medium and incubated at 35°C with 5% CO2 for 24 h. After incubation, 25 μL of each test medium (supernatant) was transferred into a 96-well MTP. To the remaining medium, 25 μL cell lysis solution (10% Triton X-100 in 5% FCS) was added. This was incubated for 1 h 30 min at room temperature with constant shaking. Twenty-five microlitres reaction mixture were added to both the supernatant and lysed cell medium and incubated in the dark at 20°C for 30 min. The reaction was terminated by the addition of 10 μL HCl 1M solution to each well. The absorbance of the resultant solution was measured with MTP reader at 490 nm against 690 nm.
Statistical analysis: GraphPad Prism Version 5.0 for Windows (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. Data are presented as Mean±SEM (N = 5) and analysed by one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05, **p<0.01 and ***p<0.001.
RESULTS
The antimicrobial screening was performed by the agar-well diffusion method against the different strains of Gram-negative and Gram-positive bacteria and a fungus. The extracts exhibited broad spectrum antimicrobial activity against the selected test organisms (Table 1). The antimicrobial activity of the extracts generally increased with increasing concentration of extract but not for all the test organisms.
Minimum inhibitory concentration (MIC) of extracts and reference antibiotics: PLC demonstrated the highest antimicrobial activity with lowest MIC range of 2.5 to 4.0 mg mL-1 against the test organisms. PLE had high MIC of 20 to 40 mg mL-1 against the test organisms and this indicates low antimicrobial activity (Table 2).
Minimum-bactericidal (MBC) and fungicidal concentration (MFC) of extracts: The MBC/MIC value was determined for the extracts against the various test organisms. This indicates the static or cidal activity against the test organism. PLC and PLE showed bactericidal and fungicidal activities whiles PLP displayed static activity against E. coli. PLC exhibited the lowest MBC/MFC ratio indicative of good microbial killing effect. PLE also had high MBC and MFC values, indicating poor microbial killing effects (Table 3 and 4).
Microbial time kill kinetics of ethanol leaf extract (PLE) of P. lappacea: PLE exhibited antibacterial and antifungal activities against the test organisms with reduction of initial viable cells and subsequent gradual increment after the sixth hour (Fig. 1a-f).
Microbial time-kill kinetics of pet ether leaf extract of P. lappacea (PLP): PLP demonstrated antibacterial and antifungal activities at the various concentrations and times tested. The microbial time killing effect was generally concentration-dependent with increasing concentration resulting in increased microbial destruction (Fig. 2a-f).
Microbial time-kill kinetics of chloroform leaf extract of P. lappacea (PLC): PLC demonstrated concentration dependent antibacterial and antifungal activities against the test bacteria and fungus. There was a gradual reduction in the initial inoculum size of the test organisms with increasing concentration of extract (Fig. 3a-f).
Antibiotic resistance modulation: The ability of the extracts at sub-MIC concentrations to modulate the activity of some antibiotics to which some microbes have developed resistance was evaluated. PLC and PLE extracts modulated the resistance of some microorganisms to the antimicrobial effects of some antibiotics. The PLC at sub-MIC concentration of 0.5 mg mL-1 reduced the MIC of tetracycline and amoxicillin from >100 μg mL-1 to <1 μg mL-1 against S. pyogenes, K. pneumonia, P. aeruginosa, P. vulgaris and S. aureus.
It also caused a reduction in the MIC of cloxacillin on E. coli from >100 μg mL-1 to 2 μg mL-1 (98% reduction) resulting in an improved antibacterial activity (Table 5-6).
PLC further reduced the MICs of cloxacillin on P. vulgaris and S. aureus from >100 to 80 μg mL-1 (20% reduction in the MIC). The PLP extract at sub-MIC concentration of 0.5 mg mL-1 demonstrated resistance modulation effects on amoxicillin, cloxacillin, ampicillin and erythromycin against the test organisms.
It caused a reduction in the MICs of amoxicillin, erythromycin, ampicillin and cloxacillin against K. pneumonia, P. aeruginosa, P. vulgaris and S. aureus, respectively from >100 to 80 μg mL-1 (20% reduction in the MIC) and hence improving the antibacterial activities of these antibiotics (Tables 5 and 6).
Influence of extracts on viability, proliferation and LDH release of HaCaT keratinocytes: The influence of the PLC extract on the viability of cells was not statistically significant (p>0.05). The PLC extract had no cytotoxic effects at the concentrations tested (Fig. 4).
The PLE caused a reduction in cell proliferation of HaCaT keratinocytes at higher concentrations (50 to 100 μg mL-1); however, there was increment in the cell viability with no cytotoxicity at the concentrations tested (Fig. 5). keratinocytes at higher concentrations (50 to 100 μg mL-1); however, there was increment in the cell viability with no cytotoxicity at the concentrations tested (Fig. 5). The PLP (within the concentrations tested) caused statistically insignificant (p>0.05) reduction in cell proliferation and cell viability of the HaCaT keratinocytes (Fig. 6). No cytotoxic effect was observed for the various extracts used.
DISCUSSION
The leaf extracts of P. lappacea exhibited some level of biological activity. With respect to antimicrobial activity, the PLC and PLP extracts exhibited antimicrobial activity against Gram-positive bacteria (B. subtilis, S. aureus and S. pyogenes) and Gram-negative bacteria (E. coli and P. aeruginosa) and fungus (C. albicans) using the agar-well diffusion method. PLE however exhibited antimicrobial activity only against C. albicans and the Gram-positive organisms (Table 1). PLC exhibited low MIC values ranging from 2.5 to 4.0 mg mL-1 against the test organisms, PLP exhibited MIC values ranging from 4.0 to 12 mg mL-1 (Table 2).
The antimicrobial activity of the extracts may be attributed to the presence of polyphenolic compounds and tannins15 found in the different leaf extracts of P. lappacea6. The presence of other secondary metabolites such as saponins and alkaloids in the extracts may also contribute their antimicrobial activities6,16. The low MICs of PLC (<8 mg mL-1) indicate strong antimicrobial activity against the test organisms17. In folkloric medicine, P. lappacea is used for the management of infections and wounds18 and hence the activities of the extracts against some common pathogenic microbes (S. aureus, S. pyogenes, P. aeruginosa) that colonize wounds and cause infections leading to chronic, unhealed wounds or delay wound healing are very significant and of important observation. This may justify the use of the plant in folklore for the treatment of wounds and other infections. It may also give an indication of the presence of potent antimicrobials against organisms which can be isolated and purified for therapeutic use. The broad-spectrum antimicrobial activity exhibited by the extracts is also demonstrated in the MBC and MFC determinations (Table 3). According to Haas et al.19, the type of antimicrobial action exhibited by an antimicrobial agent can be determined by calculating the ratio of MBC to MIC (MBC/MIC). MBC/MIC≥4 indicates bacteriostatic or fungistatic activity while MBC/MIC ratio 4 represents bactericidal or fungicidal activity.
The PLC and PLE exhibited bactericidal and fungicidal activities against all the tested organisms since MBC/MIC ratios were less than 4. PLP showed bacteriostatic activity against E. coli and bactericidal and fungicidal activities against the other test organism (Table 4).
The time-kill kinetics of the extract of P. lappacea was performed to determine the rate of kill of the extracts and the extent of bactericidal or fungicidal action over 24 h. PLE exhibited time and concentration-dependent rate of kill against S. aureus, P. aeruginosa and E. coli with the 4 times the MIC concentration causing the highest reduction in the viable organisms. This situation was not similar with B. subtilis and S. pyogenes as the effect of the extract was not concentration dependent but only time dependent. PLP also exhibited concentration-dependent time kill kinetics with the 4X MIC and 2X MIC concentrations causing the most significant reduction in inoculum size against all the test organisms. This result revealed that extracts of P. lappacea exhibited a time and concentration dependent rate of microbial destruction with maximum reduction of the viable organisms within the first five hours and these finding were similar to other reports involving plant materials3,20,21.
In most developing countries, most people who patronize herbal or traditional medicine may combine herbal remedies with orthodox medicine in order to enhance the activity and efficacy of these herbal preparations or orthodox medications especially the use of antibiotics for the treatment of skin infections12,22. The ability of the extracts of P. lappacea at sub-MIC concentrations to modulate the resistance of some microorganisms to certain antibiotics was also evaluated. According to Adu et al.20, bacteria resistance modifying agents have the ability to reduce the MIC of an antibiotic to which resistance has occurred. Kamatou et al.21 reported that antimicrobials from plants have been found to be synergistic enhancers; thus at sub-inhibitory concentrations, they can enhance the antimicrobial effects of standard antibiotics. Plants are also known to produce multi-drug resistance inhibitors (MDRIs) which enhance the activities of antimicrobial compounds23. The PLP extract at sub-MIC concentration of 0.5 mg mL-1 exhibited resistance modulation effects on amoxicillin, cloxacillin, ampicillin and erythromycin against the test organisms. It caused a reduction in the MICs of amoxicillin, erythromycin, ampicillin and cloxacillin against K. pneumonia, P. aeruginosa, P. vulgaris and S. aureus, respectively from 100 to 80 μg mL-1. The MICs of amoxicillin against P. vulgaris and E. coli were also reduced by the extract from 100 to 4 μg mL-1 (96% reduction in the MIC). There was also a reduction in the MICs of ampicillin against S. pyogenes, P. vulgaris, S. aureus, E. coli by 0.5 mg mL-1 PLP extract from to 1 μg mL-1 (99% reduction in the MIC). These results show that the 0.5 mg mL-1 sub-MIC extract concentration of PLP and PLC modulated the resistance of the antibiotics better than the 1 mg mL-1 sub-MIC extract concentration. The ability of the extracts to modulate the resistance of the various organisms to the effects of the antibiotics may be due to a breaching of the various resistance mechanisms of the microbes which include efflux pump systems, the production of inactivating agents such as β-lactamases particularly to the penicillin antibiotic or altering antimicrobial binding sites24. The inhibition of efflux pumps or mechanism in a bacterial cell according to Adu et al.20 could be a means of reversing resistance. The ability of these extracts to cause resistance modulation can be attributed to the phytochemicals in the plant particularly flavonoids which have been reported to reverse S. aureus resistance to many antibiotics22. These agents might have caused a reversal in the resistance mechanisms of these organisms hence causing the antibiotics to exert their effects. The extracts might have also enhanced the production of MDRIs resulting in the reduction in resistance. The screening of these resistance modulation agents in plants serves as a first step for the isolating and purification of compounds which can be combined with standard antibiotics to improve their activity.
Considering the extent of folkloric use of P. lappacea in the management of various ailments including wounds, skin infections, etc. and the various biological activities4,5,18, reported and the possible cytotoxic effects of the plant extracts skin cells, the influence of the extracts on the viability, proliferation and LDH release on HaCaT keratinocyte cells ware evaluated. PLC increased the proliferation of the HaCaT keratinocytes at lower concentrations of 0.1 to10.0 μg mL-1 compared to the untreated cells. This observation was similar to other reports on the cytotoxicity studies of plant materials used for treatment of wound and other skin infections12,25.
The effect of the extracts on LDH release from the cytoplasm of cells into extracellular fluids which indicates cell necrosis or apoptosis may be an indication of cytotoxic effects. The extracts (PLC, PLP and PLE) of P. lappacea did not exhibited significant (p>0.05) release of LDH from the HaCaT cells compared to the untreated cells and relative amount of LDH release less than 0.5 is considered as non-cytotoxic or necrotic25 and hence no necrotic or toxic effects was observed for the extracts at the concentrations tested (Fig. 4-6). There is a need to isolate and characterize the bioactive compounds or principles responsible for the various biological activities exhibited by these extracts of P. lappacea.
CONCLUSION
The chloroform, ethanol and petroleum ether leaf extracts of P. lappacea exhibited broad-spectrum antimicrobial activity. The extracts showed concentration and time-dependent rate of microbial destruction against the test organisms. The petroleum ether and chloroform extracts at sub-MIC concentrations showed microbial resistance modulation potential among the selected antibiotics. The extracts (PLC, PLP and PLE) did not show cytotoxic effects within the concentrations used.
ACKNOWLEDGMENTS
Special thanks go to the technical staff of the Department of Pharmaceutics (Microbiology Section), Kwame Nkrumah University of Science and Technology, Kumasi, Ghana for their assistance, Mr. Eric Gyebi of Jachie, Ashanti Region, Ghana for the collection of plant material and Dr. Alex Asase, Department of Botany University of Ghana, for the identification of the plant.