Anti-infective Properties and Time-Kill Kinetics of Phyllanthus muellerianus and its Major Constituent, Geraniin

Microbial resistance to existing antimicrobial agents remains a global challenge. In recent years, there has been a significant upsurge in the search for newer antimicrobial agents from nature with plants becoming the major focus in most parts of the world due to the vast availability of plants, which have not been screened for their antimicrobial activity. Hence, the study investigates the antimicrobial properties of aqueous aerial part extract of Phyllanthus muellerianus (PLE) and its major constituent, geraniin. The agar well diffusion and micro-dilution methods as well as time-kill kinetic studies were used to determine the antimicrobial activity of PLE and geraniin against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Bacillus subtilis NCTC 10073, Streptococcus pyogenes (clinical isolate) and Candida albicans (clinical isolate). The mean zones of growth inhibition for PLE and geraniin were in the range of 12.0 ± 0.0 to 22.7 ± 0.3 and 12.0 ± 0.0 to 21.6 ± 0.3 mm, respectively. MIC of both PLE and geraniin ranged from 0.31 to 5 and 0.08 to 1.25 mg/mL (90 to 1310 μM), respectively whiles the minimum cidal concentrations were 5.0 to 50.0 and 2.5 to 10 mg/mL (2.62 to 10.5 mM), respectively. The time-kill kinetics study showed that PLE and geraniin may act as microbiostatic agents. Preliminary phytochemical screening of PLE showed the presence of alkaloids, glycosides, saponins, tannins, flavonoids and terpenoids. The observed antimicrobial activity of the extract, PLE, may be due in large proportion to its major constituent, geraniin.


Introduction
Antibiotics are one of the most vital tools used in fighting bacterial infections and they have greatly improved the quality of health since their introduction in the fight against infectious disease. However, over the past few decades, these health benefits are under threat as many commonly used antibiotics have become less effective against known susceptible microbes due to emergence of antibiotic resistant bacteria [1][2][3][4].
Infectious diseases are ranked among wars and famines as one of the most serious factors that negatively influence the survival of man worldwide with developing countries facing the greatest impact of this menace [4][5][6]. In the developed nations, despite the tremendous advances made in the understanding of microorganisms and their control, incidence of epidemics due to drug resistant microorganisms and the emergence of new disease-causing microorganisms, pose huge public health concerns [3]. In addition, treatment option for some infections has become limited due to the emergence of multidrug resistant strains [7,8]. Along with bacterial infections, the fungal infections are also a significant cause of morbidity and mortality despite advances in medicine and the discovery of new antifungal agents is increasingly becoming scarce [9,10].
In recent years, the search for newer antimicrobial agents from nature to combat the microbial menace has increased significantly [2,11,12]. Plants have been shown in several studies to be one of the most promising sources for obtaining natural compounds that can act as anti-infective agents. Several plants including Pupalia lappaceae (L) Juss. [13], Strophanthus hispidus DC [14], Ocimum sanctum L., Eugenia caryophyllata (L.) Baill., Achyranthes bidentata Blume and Azadirachta indica A. Juss. [15], Centrosema pubescens Benth. [16] Myrianthus arboreus P. Beauv [17] have been shown to possess antimicrobial activity in in vitro models. Again natural products obtained from plants such as hyperforin from Hypericum perforatum L. [18], hyperenone A and hypercalin B from Hypericum acmosepalum N. Robson [19] as well as essential oils from plants such as Origanum vulgare L. and Thymus vulgaris L., as well as their components, carvacrol and thymol have been shown to possess antimicrobial activity [20]. Other compounds such as sterols from Laurencia papillosa C. Agardh [21], Ganoderma applanatum (Pers.) Pat. [22] and Curcubita maximus Duch [23] have been identified to be responsible for the anti-infective property of these plants. However, antimicrobial activity of many medicinal plants has not been studied. This has necessitated the continuous search for medicinal plants with anti-infective property.
Phyllanthus muellerianus Kuntze Exell which belongs to the family Euphorbiaceae is locally used in Ghana and other parts of West Africa in treating microbial infections. Biologically, the methanol and ethyl acetate stem bark extracts [24] as well as oil extracted via hydro-distillation [25] from P. muellerianus have been reported for antibacterial activity. The aqueous extract of P. muellerianus has been identified to possess antidiarrhoeal activity [26], antiplasmodial activity [27] and wound healing properties [28]. Geraniin is also reported to possess antiviral activity [29], inhibits TNF-α activity [30], antinociceptive activity [31] and antihypertensive effeet [32]. To confirm its ethnobotanical uses in the treatment of infections, we investigated the antimicrobial properties of aqueous aerial parts extract of P. muellerianus and its major constituent geraniin.

Plant collection
Aerial parts including the leaves of P. muellerianus were collected from Kuntunase (longitude 1.0° 28'18"W and latitude 6.0° 32'23"N), Ashanti Region, Ghana in February 2010. The plant was authenticated by Dr. Alex Asase of the Department of Botany, University of Ghana,

Preliminary phytochemical screening of aqueous extract of P. muellerianus (PLE)
Qualitative phytochemical screening were performed on PLE using standard methods of analysis to determine the presence of secondary metabolites such as tannins, glycosides, saponins, anthraquinones, alkaloids, flavonoids, steroids and terpenoids [34,35].

HPLC finger-printing of PLE
HPLC profile of PLE was determined according to the method described by Agyare et al. [33]. The test was performed using an HPLC with a UV detector set at a wavelength of 280 nm. The chromatographic conditions included a flow rate of 3 mL/min and a pressure of 15 MPa. Chromolith  performance RP-18e Merck (100 x 4.6 mm) was used as stationary phase.

Test organisms
Gram-positive, Gram-negative bacteria and a fungus were used and these include our (4)

Susceptibility testing (Agar diffusion method)
Susceptibility of test organisms to PLE and geraniin was determined using the agar diffusion method [36,37]. Nutrient agar and potato dextrose agar were used for the determination of the antibacterial and antifungal activities, respectively. Twenty millilitres of freshly prepared sterile nutrient agar and potato dextrose agar were seeded with 100 µL (1×10 6 CFU/mL) of test bacteria and fungus, respectively and transferred aseptically into sterile petri dishes. In each of these petri dishes, six equidistant wells (10 mm) were cut out using sterile cork borer (number 5) and wells filled with 100 µL of 100, 50, 25 and 12.5 mg/mL of aqueous extract dissolved in sterile distilled water and allowed to diffuse for 1 h at room temperature (25°C). The zones of growth inhibition (including diameter of well) were measured after 24 h of incubation at 37°C for bacteria and 72 h post incubation at 28°C for the fungus. The procedure was performed in independent triplicates and the mean zones of growth inhibition determined. Ciprofloxacin and ketoconazole (Sigma-Aldrich, Michigan, USA) were used as reference antimicrobial agents against test bacteria and fungus, respectively. The same procedure was repeated for geraniin at concentrations of 10, 5, 2.5, 1.25 mg/mL.

Minimum inhibitory concentration (MIC) determination
Minimum inhibitory concentrations of the PLE and geraniin were determined by the microdilution method using the method described by Salie et al. [38] and Wiegand et al. [39]. Micro-titre plates (96-well) were filled with 100 µL of double strength nutrient broth. PLE-containing test solutions at differfent concentrations within the range of 0.1 to 10 mg/mL were prepared and microbial inoculum size of 20 µL (1.0 x 10 5 CFU/mL) was added to each well. Activity of the PLE was determined against test microbes after incubating at 37°C. After 24 h post incubation, the MIC was determined as the lowest concentration of extract that inhibited growth which was indicated by the absence of purple colouration upon the addition of 20 µL of 1.25 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to the medium in the wells and incubated at 37°C for 30 min (14). Ciprofloxacin at concentration range of 0.01 to 1.0 µg/ mL and ketoconazole at concentration ranging from 1.0 to 10.0 µg/mL were used as standards. All tests were performed in triplicates (three independent experiments) to validate the results. The procedure was repeated for geraniin at concentration range of 0.02 to 5.0 mg/mL.

Determination of minimum bactericidal concentration and fungicidal concentration
Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of PLE and geraniin were determined according to the method described by Pfaller et al. [40]. Micro-titre plates (96-wells) were each filled with 100 µL of sterile double strength nutrient broth. A specified volume of the stock was added to each well to obtain a serial two-fold dilution of PLE in each well with concentrations within the range 1.0 to 100.0 mg/mL. An inoculum size of 20 µL (1.0×10 6 CFU/mL) of test organisms were added to the appropriately labeled wells and incubated at 37°C. After 24 h post incubation, aliquots (100 µL) were taken from the each well and inoculated into freshly prepared 10 mL nutrient broth. The inoculated nutrient broths were incubated at 37°C for 24 h. The MBC or MFC was determined as the least concentration of PLE at which no purple colouration was observed upon the addition of 20 µL of MTT (1.25 mg/mL) followed by incubation at 37°C for 30 min. The procedure was performed in triplicate. The same procedure was employed in determining the MBC and MFC of geraniin using concentrations in the range of 0.3 to 20.0 mg/mL.

Time-kill kinetics studies of PLE and geraniin
Time-kill kinetics of PLE and geraniin were determined using the method described by Petersen et al. [41]. Concentrations equivalent to the MIC, twice the MIC and four times the MIC of PLE were prepared. Inoculum size of 1.0 x 10 5 CFU/mL was used. Concentrations of the extracts were prepared in test tubes containing sterile nutrient broth and inoculated with test microorganisms. Aliquots of 1.0 mL of the medium taken at time intervals of 0, 1, 2, 3, 4, 5, 6, 12 and 24 h were inoculated into nutrient agar and incubated at 37°C for 24 h. A control test (negative) was the test organisms alone without the extracts or reference antibiotic. The colony forming units (CFU) after incubating for 24 h was determined. The procedure was performed in triplicates (three independent experiments) and a graph of the log CFU/mL was then plotted against time. The same procedure was repeated for geraniin as described above.

Statistical analysis
Data obtained from study were analysed with Graph Pad Prism Version 5.0 for Windows (Graph Pad Software Inc, San Diego, CA, USA) statistical package programme by using using One-way ANOVA followed by Dunnett's post hoc test.

HPLC finger-printing of PLE
For characterisation of PLE, HPLC profile on reversed phase RP18 stationary phase was performed. The main peak in the chromatogram was identified as Geraniin (2 isomers) by spiking and co-injection with the respective reference compound, geraniin (HPLC purity, 96 %). The retention time (R t ) of geraniin was 6.4 min ( Figure 1).

Antimicrobial activity of PLE and geraniin
Susceptibility testing (agar well diffusion): The mean zones of growth inhibition of PLE ranged from 12.0 ± 0.0 to 22.67 ± 0.3 mm against the test bacteria. In addition, the mean zones of growth inhibition for PLE against Gram-positive and Gram-negative bacteria were in the range of 12.0 ± 0.0 to 22.7 ± 0.3 mm and 13.0 ± 0.0 to 22.0 ± 0.33 mm, respectively. The mean zone of inhibition of PLE against C. albicans was between 12.0 ± 0.0 to 18.3 ± 0.3 mm (Table 1).
Furthermore, the mean zones of growth inhibition of geraniin against the test bacteria were from 12.0 ± 0.0 to 21.7 ± 0.33 mm. The mean zones of growth inhibition of geraniin against Gram-positive and Gram-negative bacteria were in the range of 12.0 ± 0.0 to 21.7 ± 0.3 mm and 12.3 ± 0.3 to 21.3 ± 0.3 mm, respectively. Against C. albicans, the mean zones of growth inhibition were 13.0 ± 0.6 to 18.3 ± 0.33 mm (Table 2).

MIC, MBC and MFC of PLE and geraniin:
MIC of PLE against the test Gram-negative and Gram-positive bacteria was within the range of 0.31 to 5 mg/mL. The MIC of PLE against C. albicans was 0.5 mg/ mL. The highest activity was observed against S. aureus and the lowest activity against E. coli. The MBC of PLE against test Gram-negative bacteria was found to be in the range of 10.0 to 50.0 mg/mL whiles the MBC of PLE against Gram-positive bacteria was in the range of 5.0 to 20.0 mg/mL. The MBC of PLE against C. albicans was 5.0 mg/mL ( Table 3).
MICs of geraniin against test Gram-negative and Gram-positive bacteria were in the range of 0.08 to 0.31 mg/mL (90 to 330 μM) and 0.08 to 1.25 mg/mL (90 to 1310 μM), respectively. The MIC of geraniin against C. albicans was 0.16 mg/mL (170 μM). The highest activity was observed against P. aeruginosa and S. pyogenes whiles the lowest activity was observed against E. coli. The MBC of geraniin against the test Gram-negative and Gram-positive bacteria were between the ranges of 2.  (Table 4).       (Figure 3).

44
]. Phytochemical screening of aqueous leaf extract of P. muellerianus (PLE) for secondary metabolites revealed the presence of alkaloids, glycosides, saponins, tannins, flavonoids and terpenoids. Steroids and anthraquinones were found to be absent in PLE. Bamisaye et al. [26] reported the presence of tannins, flavonoids, terpenoids, saponins and glycosides in the aqueous leaves and aerial parts extract of P. muellerianus. Doughari and Sunday [45] however, reported the presence of anthraquinones in the aqueous leaf extract in addition to alkaloids, tannins and flavonoids. The absence of anthraquinones in PLE may be due geographical location of the plant, the season and time of collection which are factors known to contribute to variations in the phytochemical constituents of same species of plants [46,47].
Although phytochemical screening for secondary metabolites can aid in the identification of plants, HPLC is preferred because it is more specific. HPLC profiling of extract aids in easy identification and confirmation of the plant based on qualitative and quantitative analysis of specific phytochemicals. This provides adequate identification parameters to prevent alterations of formulated herbal products containing the extract. The profile also represents the complex chemical composition of the extract [48]. The chromatogram revealed two peaks which were identified to represent the two isomers of geraniiin (a and b) which is the major composition of PLE at a retention time R t, of 6.44 min (Figure 3).
Agar diffusion method has been employed frequently and is recommended as a good method for determining the relative potency of complex extracts and for establishing their antimicrobial spectrum [49,50]. PLE and geraniin were active against both Gram-positive and Gram-negative bacteria as well as antifungal activity (Tables 1 and  2). The demonstration of antibacterial activity against both Grampositive and Gram-negative bacteria may be indication of the presence of broad-spectrum antimicrobial agent(s) or compound(s) [51]. The antimicrobial activity exhibited by PLE may likely be due to one or more of phytochemical constituents present in the plant [52,53] because phytochemical constituents such as tannins, flavonoids, alkaloids and glycosides, serve as defense mechanisms against predation by many microorganisms, insects and herbivores [52]. Again, recent studies have   indicated that tannins [54], alkaloids [55], saponins [56], glycosides [57], flavonoids [58] and terpenoids [59] possess antimicrobial activity and exert their effects by affecting the cell membrane integrity of the bacteria [60]. Furthermore, Assob et al. [24] and Doughari and Sunday [45] have shown that the methanol and ethyl acetate stem bark and aqueous leaf extract of P. muellerianus, respectively possess antibacterial activity and these support the findings of this study. Polyphenolic compounds have also been reported to possess antimicrobial activity which might be due to their ability to form complexes with bacterial cell wall which leads to inhibition of microbial growth [61]. Hence, this may account for the observed antimicrobial activity of the ellagitannin, geraniin which is a polyphenol. Gohar et al. [62] also reported that geraniin isolated from Erodium glaucophyllum was active against E. coli, S. aureus and C. albicans which is in agreement with our findings.
Studies have shown that plant extracts with MIC values between 2.5 and 8 mg/mL have led to the isolation of potent antimicrobial compounds [63][64][65][66]. This therefore suggests that PLE can be a source of potentially active antimicrobial compound. In addition, constituents or agents isolated from plants are routinely classified as potential antimicrobials on the basis of susceptibility tests that produce MICs of 100 to 1000 µg/mL [67]. This may suggest that geraniin can be classified as a potential antimicrobial agent.
Antimicrobials are usually regarded as bactericidal if the MBC/ MIC or MFC/MIC ratio is ≤ 4 and bacteriostatic if >4 [68]. The ratios obtained for all the test organisms were above 4 which indicated that both PLE and geraniin were bacteriostatic and fungistatic in action against test organisms ( Table 4). The bacteriostatic action of PLE and geraniin was also confirmed by the time-kill kinetic studies ( Figures  2,3). Bacteriostatic or fungistatic antimicrobial agents only inhibit the growth or multiplication of microbes giving the immune system of the host time to clear the microbes from the system [69].
The observed antimicrobial activity of the PLE and geraniin against S. aureus, S. pyogenes, P. aeruginosa, E. coli and C. albicans suggest that that PLE and geraniin may play a significant role in the management and treatment of infected wounds. In most infected wounds, Gram-positive organisms especially S. aureus and S. pyogenes are implicated due to their ability to produce enzymes that destroy the extracellular matrix in wound bed [70]. Also, the most prevalent Gram-negative bacteria found in wound infections is P. aeruginosa. Wounds contaminated with P. aeruginosa are in most cases difficult to manage due to high intrinsic resistant factors and the ability of these organisms to form biofilms [71,72]. Although, E. coli is not a common pathogen of wounds, some reports have implicated E. coli as the third most prevalent microbe in wounds after S. aureus and S. pyogenes [73]. It is also interesting to note that fungi such as C. albicans are also believed to contaminate and colonize wounds [74]. This may suggest that PLE and geraniin may be useful in the management of infected wounds.

Conclusion
The aqueous leaf extract of P. muellerianus (PLE) was found to possess antimicrobial activity that may confirms its traditional use as anti-infective agent. The antimicrobial activity of P. muellerianus may largely be due to its major isolate, geraniin and they act by static means.