In vitro time kill assessment of crude methanol extract of Helichrysum pedunculatum leaves

The in vitro antibacterial activities and time kill regimes of crude methanol extract of Helichrysum pedunculatum was assessed using standard microbiological procedures. The experiment was conducted against a panel of bacterial species made up of clinical, environmental and reference strains. The extract was active against eleven of the twenty-one bacteria tested at a concentration of 10 mg/ml. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.1 – 5.0 mg/ml. The average log reduction in viable cell count in time kill assay ranged between 0.17 Log10 to 6.37 Log10 cfu/ml after 6 h of interaction, and between 0.14 Log10 and 6.99 Log10 cfu/ml after 12 h interaction in 1×MIC and 2×MIC of the extract. The extract was bactericidal against 8 of the test bacteria at 1×MIC and against 9 of the test bacteria at 2×MIC from 12 h interaction period. At both MIC levels, the extract was bactericidal to all the reference strains and four of the six environmental strains at both MIC levels after 12 h of interaction. Also the extract was bactericidal to four of the six environmental strains at both MIC levels after 12 h of interaction and bacteriostatic during the first 6 h of interaction. Inhibitory levels of crude methanol extract of H. pedunculatum could be bacteriostatic or bactericidal independent of Gram’s characteristic.


INTRODUCTION
The Genus Helichrysum, from the family Asteraceae, is large, comprising of about 500 species with 246 growing in South Africa (Afolayan and Meyer, 1997).Some members of the family are: Helichrysum pedunculatum, Helichrysum longifolium, Helichrysum stoechas, Helichrysum apendunculatum, Helichrysum kraussii and Helichrysum nudifolium.Members of the genus are usually aromatic, perennial shrubs, having dense-woolly leaves with hardy inflorescence parts that are usually brightly coloured (Mathekga et al., 2000).
Extracts of various species have been used to treat topical infections, respiratory ailments and as dressing of circumcision wounds (Stafford et al., 2005).The antimicrobial activities of extracts from Helichrysum species have been widely reported (Bougatsos et al., 2004;Eloff, *Corresponding author.Email: aokoh@ufh.ac.za.1999; Grierson and Afolayan, 1999;Mathekga et al., 2000), and in East Africa, they are used for menstrual and abdominal pains.While the first therapeutic uses of the plant were based on folk medicine, in vivo and in vitro studies also proved its choleric and hepatoprotective properties (Czinner et al., 2001).In Europe, infusions are prepared from inflorescences of Helichrysum because of their bile regulatory and diuretic effects (Cosar and Cubucku, 1990).In South Africa, Helichrysum species are used extensively for stress-related ailments and as dressings for wounds normally encountered in circumcision rites, bruises, cuts and sores (Grierson and Afolayan, 1999;Lourens et al., 2004).
H. pedunculatum (Hilliard and Burtt) is a perennial herb with a wide distribution, it is found within the range of boarders of Southern Lesotho to the Eastern Cape Province of South Africa (Meyer and Dilika, 1996).The 'Xhosas' of the Eastern Cape Province of South Africa commonly use the plant to dress wound acquired after circumcision rite.However, there is paucity of information on the nature of bacterial inhibition of the plant.Hence, in this paper, we report the antibacterial activity of the methanol extract of H. pedunculatum and the nature of their inhibition by in vitro time-kill assay.

Plant material
Leaves of H. pedunculatum were collected from the vicinity of the Research Farm of the University of Fort Hare, Alice, Eastern Cape Province of South Africa, during September 2007.The plant materials were compared with the voucher specimen earlier collected from the same spot and deposited at the Griffin's Herbarium of the Plant Science building of the University of Fort Hare in Alice.The Plant materials were later confirmed by the curator of the Herbarium to be H. pedunculatum.The leaf was picked and washed with water to remove all unwanted plant materials and sand, air-dried, pulverized in a mill (Christy Lab Mill, Christy and Norris Ltd; Process Engineers, Chelmsford, England) and stored in an air-tight container for further use.

Preparation of extract
Exactly 135 g of the pulverized leaf of the plant was cold extracted using methanol with occasional shaking (Okeke et al., 2001).The mixture was then filtered (using Whatmann's no 1 filter paper); the filtrate was concentrated to dryness in vacuo using a rotary evaporator.This gave a yield of about 12 g of the crude extract.

Antibacterial susceptibility test
Screening of the methanol extract of the plant for antibacterial activity was done in accordance with the method of Afolayan and Meyer (1997).Stock solution of the extract was prepared by reconstituting the dried extract in the extracting solvent.This was used to prepare dilution of the extract in molten Mueller Hinton agar maintained in a water bath at 50 o C to attain a concentration of 10 mg/ml and final methanol concentration of 5%.The inoculum size of each test strain was standardized at 5 × 10 5 cfu/ml using McFarland Nephelometer standard according to the National Committee for Clinical Laboratory Standards (NCCLS, 1993) (now Clinical and Laboratory Standards Institute (CLSI)).The inocula were streaked Aiyegoro et al. 1685 in radial patterns on the agar plates (Afolayan and Meyer, 1997).
Plates were incubated under aerobic conditions at 37 o C for 24 h.Two blank plates containing nutrient agar and 5% methanol without the extract, served as controls.Each test was done in triplicate.

Determination of minimum inhibitory concentration (MIC)
The MIC was determined using the agar dilution method (EUCAST, 2000).The extract was diluted such that the highest concen-tration of the solvent in agar was 5% (Predetermined to have no inhibitory effect on the test organism).Plates were inoculated with overnight broth cultures of the test organisms diluted 1:100 with fresh sterile nutrient broth and incubated for 18 h at 37 o C. The MIC was taken as the lowest dilution (least concentration) of extract showing no visible growth of the test organism.

Time-kill assay
Determination of the rate of kill of the crude extract was done following the procedure described by Okoli and Iroegbu (2005).Inocula were prepared following the described guidelines of EUCAST (2003).The resultant suspension was diluted 1:100 with fresh sterile broth and used to inoculate 50 ml volumes of Mueller Hinton broth incorporated with extract at MIC and 2 × MIC to a final cell density of approximately 5 × 10 5 cfu/ml.The flasks were incubated at 37 o C on an orbital shaker at 120 rpm.A 500 µl sample was removed from cultures at 0, 6 and 12 h, diluted serially and 100 µl of the diluted samples were plated on Mueller Hinton agar plates and incubated at 37 o C for 24 h.Controls included extract free Mueller Hinton broth seeded with the test inoculum.

RESULTS AND DISCUSSION
Twenty-one bacteria species made up of seven Gram positive and fourteen Gram negative bacteria were screened for susceptibility to crude methanol extract of the H. pedunculatum (Table 1).Two of test organisms were clinical isolates; ten were environmental strains and nine were reference strains.Eleven of the test bacteria were susceptible at the test concentration (10 mg/ml) (Table 1), of which five were reference strains and the remaining six were environmental strains.The 2 clinical isolates S. aureus OKOH 2B and S. aureus OKOH 3 were not susceptible to the extract.The minimum inhibitory concentrations (MICs) of the extract against the susceptible bacteria generally ranged between 0.1-5.0mg/ml.Specifically, MICs for the reference and environmental strains ranged between 0.5-5.0 mg/ml and 0.1-5.0 mg/ml respectively (Table 1).
The results of time-kill studies are presented in Table 2. Data are presented in terms of the log 10 cfu/ml change and are based on the conventional bactericidal activity standard, that is, a 3Log 10 cfu/ml or greater reduction in the viable colony number.Average log reduction in viable cell count in time kill assay ranged between 0.17 Log 10 to 6.37 Log 10 cfu/ml after 6 h of interaction, and between 0.14 Log 10 and 6.99 Log 10 cfu/ml after 12 h interaction in 1×MIC and 2×MIC of the extract.The extract produced a range of 0.41 Log 10 -1.19 Log 10 and 0.54 Log 10 -6.37 Log 10 reduction after 6 h at concentration equal to 1×MIC and 2×MIC respectively and a range of 5.98 Log 10 -6.99 Log 10 reduction after 12 h at both 1×MIC and 2×MIC respectively on the reference strains.On the other hand, the range for environmental strains is between 0.17 Log 10 -5.44 Log 10 and 0.65 Log 10 -5.44 Log 10 reductions after 6 h at concentration equal to 1×MIC and 2×MIC respectively and a range between 0.99 Log 10 -6.44 Log 10 after 12 h at 1×MIC and 2×MIC respectively.Log reduction on B. subtilis (environmental strain) was 5.44 Log 10 cfu/ml after 6 h of interaction at 1×MIC, the log reduction was constant until after 12 h even at 2×MIC, thus suggesting that the total population of the bacteria have been wiped off by the sixth hour at both 1×MIC and 2×MIC.There is an increase in inoculum size for M. kristinae (environ-mental strain), the increase in log range from 0.05 Log 10 -0.44 Log 10 after 12 h at both MIC levels and this indicate resistance to the extract.The greatest reductions achieved within the reference strains are in P. aeruginosa ATCC 19582 and P. aeruginosa ATCC 7700.The value is on the average of 6.99 Log 10, after 12 h at 2×MIC, while the greatest reduction with the environmental strains is achieved with M. luteus which is 6.44 Log 10 reduction after 12 h at 2×MIC.Generally the reference strains were more susceptible to the extract than the environmental strains.
The extract was bactericidal against 8 of the test bacteria at 1×MIC and against 9 of the test bacteria at 2×MIC for 12 h interaction period.At both MIC levels, the extract was bactericidal to all the reference strains after 12 h and generally bacteriostatic during the first 6 h of interaction.Also the extract was bactericidal to four of the six environmental strains at both MIC levels after 12 h of interaction and bacteriostatic during the first 6 h of interaction.The extract exhibited bacteriostatic effect on K. pneumoniae (environmental strain) after 12 h interaction at both the 1×MIC and 2×MIC, but the effect was more pronounced at 2×MIC.It was not able to inhibit the growth of M. kristinae (environmental strain) at both 1×MIC and 2×MIC, but after 12 h of interaction at 2×MIC, it only showed about 0.14 Log 10 cfu/ml reduction in inoculum size (a weak activity).Inhibitory levels of crude methanol extract of H. pedunculatum could therefore be bacteriostatic or bactericidal independent of Gram's staining characteristic.
Results obtained from this study suggest the possible reason why H. pedunculatum is being used in folkloric medicines for the treatment of various human topical infections.The effectiveness of an antibacterial agent is measured by its ability to inhibit and kill bacteria (Nostro et al., 2001).In vitro time-kill assays are expressed as the rate of killing by a fixed concentration of an antimicrobial agent and are one of the most reliable methods for determining tolerance (Nostro et al., 2001).Generally, the effect of the crude methanol extract of H. pedunculatum on the test bacteria in this experiment is time and concentration dependent, as it is evident from the data presented.At higher concentration (2×MIC) and longer duration of interaction (12 h), more bacteria were killed.The in vitro data corroborates the reported efficacies of the several different crude extracts of H. pedunculatum on a wide range of microorganisms and this support the folkloric uses of this plant in treatment of different topical ailments among the traditional people.

Conclusion
Crude methanol extract of H. pedunculatum has a broad spectrum antibacterial activity against many bacteria species.The nature of bacterial inhibition of extract could either be bacteriostatic or bactericidal depending on the Aiyegoro et al. 1687 concentration of the extract and the exposure time irrespective of Gram's staining characteristic of the test organism.The resistance of the clinical isolates to the extract is worrisome and this could be one reason why, there is an incidence of high death rate resulting from circumcision wounds infection even after treating such wounds with H. pedunculatum leaf.Perhaps the plant could be of more relevance in combination therapy and a source of resistance modifying principles which is the subject of on going studies in our group.

Table 1 .
Antibacterial activities of crude methanol extract of H. pedunculatum leaves on bacterial isolates.
Key: are clinical isolates; § are environmental strains; -represents no antibacterial activity; + represent presence of antibacterial activity; MIC represents minimum inhibitory concentration; ND represents not determined.

Table 2 .
Nature of inhibition of crude methanol extracts of H. pedunculatum leaves against bacterial isolates.