In Vitro Sensitivity of Some Mycolic Acid-containing Actinomycetes to Nigella Sativa Extracts

Nigella sativa L. (b lack seed or cumin) is an herbaceous plant, used for centuries for the treatment of various ailments, including infectious diseases. This study investigates the in vitro effect of extract of N. sativa against representative of mycolic acid containing actinomycetes notably members of the genera Gordonia, Mycobacterium, Nocardia and Rhodococcus. Methanolic and petroleum ether ext racts of N. sativa at different concentrations (0.3 mg/ mL, 0.6 mg/ mL, 1.25 mg/ mL, 2.5 mg/ mL and 5 mg/ mL) were used to impregnate filter paper disks. The disks were incorporated onto Diagnostic Sensitivity Test agar, plates inoculated with test strains and incubated aerobically for up to seven days at 37 °C. Inhibition zones around discs were measured in millimetres. At 0.6 mg/ mL all test strains showed inhibition zones of 1.5 cm except Mycobacterium phlei and R. rhodochrous. Petroleum ether ext ract was found to be more effective than the methanolic extracts since relat ively h igher concentrations from the latter were needed to achieve similar inhibit ions zones produced by petroleum ether ext racts. It be concluded that extracts of N. sativa is a promising candidate for the in vivo application since it is a natural food additive with no reported harmfu l effect.


Introduction
Many cultures around the world have strong beliefs in the use of many different plants as medicine which has rendered some cultures, up to date, almost co mpletely dependent on the use of plants in medicine rather than co mmon commercial medical products. Nigella sativa L., belongs to the family Ranunculaceae, is a s mall genus of annual herb found in many regions including the African countries. Its seeds are angular, dark gray in co lor and are locally known as "black cu min". Its oil is generally regarded as safe and are considered carminative, stimu lant, diuretic, emmenagogue, galactagogue and other healing effects [1,6]. Different crude extracts of N. sativa have been tested for their antimicrobial effectiveness and found to have promising effects against some organis ms [3,9,12].
The aim of this study was to investigate the in vitro effect of petroleum ether and methanolic extract of N. sativa on representative mycolic acid containing actino mycetes notably Nocardia africana which has been recently described in Sudan [8]. Also, strains fro m the genera: Gordonia, Mycobacterium, Nocardia and Rhodococcus were tested.

Methodology
Fifty grams of air dried and coarsely powdered of clean seeds of N. sativa were extracted in So xh let apparatus to obtain methanolic and petroleum ether extracts [5]. The extracts were filtered, and the filtrates were vaporized to dryness, and weighed in order to determine the % yield of the extracts, follo wing the formula: % yield = (weight of extract/ weight of ground plant material) × 100. The stock solutions of the crude methanolic and petroleum ether extracts were prepared by diluting the dried extracts with 50% methanol to obtain the desired final concentrations of 0.3 mg/ mL, 0.6 mg/ mL, 1.25 mg/ mL, 2.5 mg/ mL and 5 mg/ mL. These concentrations were used to impregnate filter paper disks (10 mm d iameters). Disk imp regnated into 50% ethanol were used as control.
Antibiotic susceptibility testing was performed following the Clin ical and Laboratory Standards Institute (CLSI) guidelines as described previously [13]. The dried disks containing different concentrations and the controls were incorporated onto Diagnostic Sensitivity Test agar (Difco) onto which bacteria was inoculated, then incubated aerobically for up to seven days at 37 o C. Inhib ition zones around discs were measured in millimetres. In vitro sensitivity of selected mycolic acid containing actinomycetes to petroleum ether extract of Nigella sativa using disc method on DST agar. Arrows indicate the direction of increasing concentration: 312.5μg/ml, 650μg/ml, 1,250μg/ml, 2,500μg/ml, 5,000μg/ml, and 10,000μg/m The effect of N. sativa in terms of inhibition zones is shown in Figure 1 and Table 1. At 312.5 µg / ml, 0.5 cm inhibit ion zone noticed in all test strains except Rhodococcus rhodochrous. At 625 µg/ ml all test strains showed inhibition zones of 1.5 cm (+++) except Mycobacterium phlei and R. rhodochrous. Petroleum ether extract was found to be more effective than the methanolic ext racts since relatively h igher concentrations from the latter were needed to achieve similar inhibit ions zoned produced by petroleum ether extracts.

Discussion
Mycolic acid containing actinomycetes (MACA) are a wide group of bacteria belonging to the genera Mycobacterium, Nocardia, Gordonia and Rhodococcus [7]. It includes some species that cause diseases such as tuberculosis, mastitis, and pulmonary infections [10]. MACA are known to be ext remely tough and resistant to many therapeutic agents, both in man and animals [2,4]. Mycobacterium is the main strong acid fast organisms, Gordonia and Nocardia are week acid fast whereas Rhodococcus is a non acid fast [11].
Throughout this study, the effects of the petroleum ether extract of N. sativa on the selected organisms were found good (Table 1). Organisms were inhib ited at different concentrations. The most susceptible organisms were G. bronchialis and N. otidiscaviarum, and the inhibitory zones of 0.5 cm have been noticed on disk imp regnated with the 312.5 µg/ ml concentration.
On the other hand M. phlei showed 0.5 cm inhibit ion zone at 625µg/ ml, wh ile in case of R. rhodochrous is was 1250 µg/ml. These organisms can therefore be considered the most resistant to the anti microbial activity of N. sativa petroleum ether extract among tested strains.
Fro m these comparisons, it is clear that the petroleum ether extract o f N. sativa, as an antimicrobial agent, is mo re effective than the methanolic extract. Furthermore, it should be noted that the effect of the petroleum ether extract on N. otidiscaviarum and G. bronchialis was stronger than on M. phlei and R. rhodochrous ( Table 1).
The study concluded that extract of N. sativa is a promising candidate for the in vivo applicat ion since it is a natural food additive with no reported harmful effect. The study needed to be supported by a wider in vitro application, not against MACA, but perhaps against a range of gram positive and gram negative organisms.