The Peperomias constitute a large genus of herbs widely distributed in tropical and sub-tropical regions [21
]. The two species used in this study are found around Mount Cameroon in the South West and in parts of the North West of Cameroon [22
]. Scleria striatinux is also found in the North West of Cameroon. Crude extracts of the three plants, P. vulcanica, P. fernandopoioana and S. striatinux investigated in this study all demonstrated antibacterial activity against some of the bacterial organisms except for the methanol extract of P. fernandopoioana. From the flow charts (Figures and ), one could observe clear activity in the fractions of moderate polarity. Both Gram-negative (E. coli, S. typhimurium and P. aeruginosa) and -positive bacteria (S. aureus) were selected based on their frequent occurrence in wounds and implication in common bacterial diseases such as diarrhoea, urinary tract infections and respiratory tract infections associated with coughing. Four clinical isolates were sensitive to standard antibiotics and one was a resistant strain of S. aureus that showed total resistance to vancomycin with a zero diameter of inhibition and intermediate resistance to erythromycin and ampicillin. Isolation of a resistant strain in the study area is not unexpected given the abusive use of antibiotics which favours the emergence of resistance as earlier described [23
Six of the seven crude extracts from the three plants inhibited two of four bacterial species tested. The methylene chloride extracts of the Peperomia species and the methylene chloride:methanol (1:1) extract of Scleria striatinux were the most active (Table ). Antibacterial activity had been demonstrated in the crude methanolic extracts of the whole plant of these two Peperomia species [5
]. The earlier work constituted a basis for this study and our results confirm their findings and provide further evidence to support the use of these plants in Cameroonian folk medicine to treat bacterial infections [22
]. The methanol extract of P. fernandopoioana showed no activity against all 6 bacterial test organisms, probably due to absence of compounds active against the bacteria following the sequential extraction performed on the plant powder.
The crude hexane extract of P. vulcanica showed no activity against the clinical isolate of S. aureus (Table ). Interestingly, 3 of the 8 fractions obtained from the hexane extract showed moderate activity against this same sample with DZI
mm suggesting antagonism between compounds in the crude extract which could have been eliminated following fractionation. These fractions obtained with moderately polar solvents were the most active with clear zones of inhibition; this suggests that the bio-active principles in these fractions are moderately polar. The hexane and methylene chloride extracts of P. fernandopoioana showed moderate activity against E. coli and S. aureus (Table ). However, the activity of fractions from these extracts was not certain given the unclear zones observed. The latter observation suggests that the antibacterial action of the crude extracts of this plant may be due to the combined effect of the compounds present in them. Upon separation by fractionation, the antibacterial action was weakened or the active principles in the fractions were too small to be isolated in sufficiently high amounts to show activity.
Several plants have been reported to display antibacterial activity on the bacterial species used in this study [24
]. The current study employed quantities of crude extract similar to those used in an earlier study and similar zones of inhibition were recorded, i.e. ranging from 7–15
mm as against 7–13
mm in this study. Also, much lower MICs were recorded (32–512
μg/mL against 4 - 8
mg/mL in this study). This higher activity is likely due to the secondary metabolites in the plant which was rich in alkaloids, flavonoids, phenol, quinines, and terpenoids [24
]. Another study [25
] also observed higher activity for six Phyllantus species having a similar phytochemical profile. The Peperomias tested in this study contained lignans, steroids, flavonoids and fatty acids. The difference in phytochemical composition may largely account for the difference in the reported activity while other factors such as experimental method may contribute to a smaller extent. This explanation is supported by yet similar findings on a plant with a similar secondary metabolite composition [26
]. One of the investigations [27
] reported much higher inhibition zones (14 - 36
mm) for the same bacteria species alongside higher MICs (4–64
mg/mL) and MBCs (8 - 128
mg/mL); these higher zones may be due to the high amount of extract (25
Previous studies have shown that most of the common phytochemical constituents of Peperomia species are phenylpropanoid, benzopyran, chromone, prenylated quinone, secolignan, and acylcyclohexane-1, 3-dione [21
]. Some of these compounds in the crude extracts and fractions of P. vulcanica and P. fernandopoioana may be responsible for the antibacterial activities observed.
The isolation of a large number of compounds (27 in total) from the two Peperomia species is not surprising. Nineteen compounds were isolated from P. sui of the same family [21
] suggesting that the Piperaceae may be quite rich in secondary metabolites. Hence, sequential extraction and bioassay-guided fractionation were done to narrow down the number of metabolites and increase our chances of obtaining active compounds. The residual methanol extracts turned out to be weak or inactive, suggesting that sequential extraction actually localized the active compounds in the hexane and methylene chloride extracts (containing moderately polar compounds). However, none of the compounds from the Peperomias showed antibacterial activity, suggesting that the observed antibacterial activity for some of the crude extracts and fractions is likely due to the synergistic or additive interaction of some of these compounds; but this remains to be demonstrated experimentally.
One of the compounds, Okundoperoxide (OKP), from S. striatinux showed considerably high activity against a resistant and sensitive strain of S. aureus (Table ). Considering the multi-drug resistance that has developed in S. aureus [2
], a bacterium with potentially serious pathology in humans [28
], this activity is therefore highly significant. This finding provides a strong basis for explorative structure-activity relationship studies which may yield new potent antibacterial lead(s) with enhanced activity. Okundoperoxide had been isolated, its structure determined (Figure ) and antiplasmodial activity demonstrated, justifying the use of the roots of the plant as herbal tea for fevers in Cameroon [7
]. Menthol, a terpenoid isolated from Mentha longifolia L. leaves showed much higher activity (25
mm zone and MIC of 15.6
μg/mL) against S. aureus [29
]. A higher activity (MIC
μg/mL) was also recorded against S. aureus for three plant-derived triterpenoids which showed a high degree of synergism with standard antibiotics [30
]. The effect of OKP combined with standard antibiotics should also be investigated to discover useful combinations with enhanced antibacterial action to combat resistant bacteria.
The lack of susceptibility in P. aeruginosa and S. typhimurium to the crude extracts, fractions or pure compounds may be attributed to several factors including multi-drug efflux pumps common in P. aeruginosa and S. typhimurium and the low permeability of the bacterial envelopes [31
]. Efflux pumps extrude the drug from the cell before they attain an adequate concentration at the site of action [32
]. Some studies have reported no inhibition zone for 24 plant extracts against S. typhimurim [33
] and menthol against P. aeruginosa [29
]. Whereas no antibacterial activity was also reported for 40 plant extracts against S. typhimurium and S. aureus using the disc diffusion method, considerable inhibition of these organisms was recorded using the tube dilution method [5
]. This suggests the tube dilution method may permit a more rapid accumulation and concentration of active principles than the diffusion method. The higher concentration of active secondary metabolites obtained with the tube dilution method would translate into more efficient inhibition of bacterial growth.
Based on some classifications of antibacterial activity using the disc method [14
], the most active extracts [the methylene chloride extracts of P. vulcanica, P. fernandopoioana, and the methylene chloride:methanol (1:1) extract of S. striatinux] exhibited moderate activity (DZI between 11–16
mm). On the same basis, the pure compound (Okundoperoxide) showed high activity against S. aureus with a DZI ranging from 10–19
mm. Based on the MICs recorded for E. coli and S. aureus (4–8
mg/mL) and the apparently high MBC (> 10
mg/mL), these plants can be classified as possessing broad-spectrum bacteriostatic activity. Similarly Okundoperoxide with a high MIC of 1
mg/mL may only be bacteriostatic against S. aureus.
of Okundoperoxide on monkey kidney cells (LLC-MK2
) was 46.88
μg/mL whereas the active dose was 500
μg/disc i.e. about 10 times the LC50.
This suggests OKP may be toxic to mammalian cells. However, use of different formulations, disc for antibacterial activity and solution for cytotoxicity makes comparison of the results difficult as the rate of distribution of the compound in both experimental systems is likely to be different. An LC50
μg/mL has been suggested to indicate moderate cytotoxicity [35
], while a CC50
μg/mL has been categorized as non-cytotoxic [36
]. Thus OKP with a higher LC50
can be considered as having a very low risk of cytotoxicity on mammalian cells. For P. vulcanica, no mortality was recorded in the acute toxicity study, suggesting that despite containing a large number of compounds this plant may be non-toxic to humans. The changes in body weight of the animals could have resulted from corresponding changes in feeding due to alteration of appetite or effects on the metabolism of the animals.
In conclusion, the observed antibacterial activity of the two Peperomia species is likely due to the combined effect of the moderately polar compounds present in them. Furthermore, the apparent lack of acute toxicity in P. vulcanica supports its use in traditional medicine. In view of its potency and relatively low cytotoxicty, Okundoperoxide may serve as a template for the development of new antibiotics. Moreover, given the structural similarity that is found among the secondary metabolites of a given plant, the isolation of other compounds from S. striatinux may result in the identification of other interesting antibacterial agents.