Endang Dwi Wulansari*), Yully Sri Mulyati*), Herry Pratikno**)
*)STIFAR “Yayasan Pharmasi”, Semarang, Indonesia;
**)Faculty of Animal Agriculture, Diponegoro University, Semarang, Indonesia
ABSTRACT
Antibacterial activity of Nigella sativa seeds against Streptococcus pyogenes was investigated.
The seed extracts were prepared by soxhlet extracted method using n-hexane, ethyl acetate, and ethanol. Several concentrations of Nigella sativa extract were tested by the well diffusion method. The inhibition zones of the Muller Hinton agar in different extract concentrations such as 20%, 40%, and 80%, showed that n-hexane extract was found to be inactive against Streptococcus pyogenes, while ethanolic extract at 80% showed the biggest inhibition zones. Bioautography assay of ethyl acetate extract and ethanolic extract showed there were flavonoid and saponin compounds that inhibited Streptococcus pyogenes growth.
Key words: Nigella sativa, antibacterial activity, bioautography, Streptococcus pyogenes
INTRODUCTION
Nigella sativa is commonly known as black seed which belongs to botanical family of Ranunculaceae. The seeds have been claimed to have several traditional medicinal properties (Roshan, et,al., 2010). The seed extracts and its essential oil have been reported to exhibit many pharmalogical effects including antidiabetic (Khanam, et.al, 2008), antiinflammatory (Elbandy, et.al, 2009), antibacterial (Zuridah, et.al., 2008), anti oxidant (Yoruk, et,al., 2010), and anti stress (Roshan, et,al., 2010).
Pharyngitis (strep throat) is common illness in Indonesia. It may happen because of bacteria, such as Streptococcus pyogenes. Beside the anti-inflammatory agent, it needs an antibacterial agent to cure the illness. Nigella sativa is becoming the alternative medicine for strep throat, because of it anti- inflammatory activity and antibacterial activity. The aim of this study is to determine the antibacterial activity of several seed extracts from Nigella sativa against Streptococcus pyogenes.
METHODOLOGY
Plant material and extraction procedure
The Nigella sativa seeds were purchased from a local herbal shop in Semarang, Indonesia. A modification of soxhlet extraction procedure by Ali, et.al (2007) was used. The seeds of Nigella sativa were powdered in a mixer. 30 g Nigella sativa seed powder placed in a soxhlet and extracted with 300 ml n-hexane at 70˚C. The plant residue in soxhlet, was then re extracted with 300 ml ethyl acetate at 80˚C. The lastly, the plant residue in soxhlet was extracted with 300 ml ethanol at 70˚C. All the extract, n-hexane, ethyl acetate, and ethanolic extracts were each evaporated after extraction using a vacuum rotary evaporator.
Bioautography assay
Volatile oil, flavonoid and saponin compound of n-heksan extract, etil acetate extract, and ethanolic ectract have been analyzed using thin layer chromatography (TLC) with silica gel GF 254 plates (Merck), according to the method of Wagner and Bladt (1996) and contact bioautography assay procedure by Gende, et,al. (2008). N-hexane extract was used for volatile oil identification using toluene-etil asetat (93:7) as the mobile phase. Etil acetate extract and ethanolic extract were used for flavonoid and saponin identification. Flavonoid identification used butanol-asam asetat-air (4:1:5) as the mobile phase. Saponin identification used kloroform : metanol : air (64 : 50 : 10) as the mobile phase.
In Vitro Antibacterial Activity…..
The stationary phase that used for identification, was placed on the inoculated plates within 30 minutes. The plates were incubated at 37˚C for 24 hours. Microbial growth inhibition zone was determined by measuring the area of inhibition zones. The inhibition zone was calculated as average of three measurements per TLC plate.
Determination of antibacterial activity
Nigella sativa seeds extract were tested against Streptococcus pyogenes. This strains were obtained from Health Laboratory Office, Semarang.
Antibacterial activity was determined by the well diffusion method. Muller Hinton agar plates were cultured with Streptococcus pyogenes for 24 hours. The inoculums size was adjusted to 1 MacFarland I turbidity standard (3x108 cfu/ml). Muller Hinton agar plates were inoculated with Streptococcus pyogenes suspentions using spread method. The dried plant extracts were dissolved in polyethylenglicol (PEG) to give concentrations of 20%, 40%, and 80% (w/v). Wells (1 cm diameter) were cut into the agar and filled with 50µl of the plant extract. The inoculated plates were incubated at 37˚C. The antibacterial activity was evaluated by measuring the diameter of the inhibition zone. The experiment was repeated five times for each concentration and the mean of diameter of the inhibition zones was calculated.
RESULTS AND DISCUSSIONS
The ability of an antibacterial agent to inhibit bacterial growth in vitro may be estimated by the diffusion method. This study investigated the antibacterial effect of n-hexsane, ethyl acetate, and ethanolic seed extract on Streptococcus pyogenes. The n-hexane extract did not show the antibacterial activity. TLC analysis of volatile oil in n-hexane extract showed there was one blue colour spot (Rf 0.28) using anisaldehid-sulphonic acid, but did not show inhibitory effect against Streptococcus pyogenes on bioautography assay.
TLC analysis of flavonoids in ethyl acetate extract and ethanolic extract showed one spot (Rf 0.67) in each extract. It is possible that flavonoid can be extracted with both ethyl acetate and ethanol.
Bioautography assay of flavonoids in ethyl acetate extract and ethanolic extract showed an inhibitory zones with 1.363 cm diameter for ethyl acetate extract and 1.281 cm for ethanolic extract. TLC analysis of saponins in ethyl acetate extract and ethanolic extract showed different spots with different values of Rf, 0.88 cm (ethyl acetate extract) and 0.77 cm (ethanolic extract). The diameters of the inhibition zones of saponin for ethyl acetate extract and ethanolic axtract were 1.296 cm and 1.371 cm (Figure. 1).
The bioautography technique was employed to define the active constituents (Iskan, et al., 2002).
There are two constituent in Nigella sativa seeds which are flavonoid and saponin can inhibited Streptococcus pyogenes growth. Saponin have shown activity against a broad range of microorganism including bacteria, filamentous fungi and yeast (Kredy, 2010).
Figure 1. Bioautography assay of flavonoids (A) and saponin compound (B) in ethyl acetate extract and ethanolic extract
Endang Dwi Wulansari, et al.
In this study, we investigated the antibacterial effects of n-hexane, ethyl acetate, and ethanolic seed extracts on Streptococcus pyogenes (Table 1.).
Table 1. Inhibition zones of ethyl acetate extract and ethanolic extract at various concentration against Streptococcus pyogenes.
Concentration
Zone of inhibition (cm) 20% 40% 80%
Ethyl acetate extract 1.358 1.608 2.177
Ethanolic extract 1.963 2.163 2.319
Positive control 2.292
There are significantly different inhibition zones from various kind of solvent and concentration extract. The ethanolic extract gave higher value compared to ethyl acetate extract. It is possible because of the polarity of the flavonoid and saponin are similar to ethanol. So, it can good extracted by ethanolic solvent. The best inhibition was seen at 80% ethanolic extract. The inhibition zones showed higher value on the higher concentration, due to the concentration of constituents of the extract.
The end-point of inhibition is judged by the naked eye at the edge where the growth starts, but there are exceptions like the heaped-up growth characteristics (Zuridah, et.al., 2008). Ciprofloxacin (50mg/L) and PEG were used on the MHA for positive control and negative control, respectively.
In this preliminary study, Minimum Inhibitory Concentration (MIC) of the extract was not carried out, however the diameters of Streptococcus pyogenes were reported here. This study showed that the ethanolic extract of Nigella sativa seed at 80% concentration had the best antibacterial activity.
CONCLUSION
In our study we have found that ethyl acetate and ethanolic extracts of Nigella sativa seed, which its compounds were flavonoid and saponin, could be used as antibacterial agent against Streptococcus pyogenes. More studies may be needed to make final conclusion regarding the use of Nigella sativa seed as a good medicinal plant for strep throat.
REFERENCES
Ali, O., Basbϋlbϋl, G., Aydin, T., 2007, Antimicotic and Antibacterial Effects of the Nigella sativa L. Seed, Caryologia, 60 (3) : 270-272
Elbandy, M., Kang, O.H., Kwon, D.Y., Rho, J.R., 2009, Two New Antiinflammatory Triterpen Saponins from the Egyptian Medicinal Food Black Cumin (Seeds of Nigella sativa), Bull. Korean Chem.Soc., 30 (8) : 1811-1815
Gende, L.B., Floris, I., Frits, R., Javier, M., 2008, Antimicrobial Activity of Cinnamon (Cinnamomum zeylanicum) Essensial Oil and Its Main Components Againts Renibacillus Larvae from Argentine, Bulletin of Insectologi, 61 (1) : 1-4
Iskan, G., Kirimer, N., Kϋrkcϋooglu, M., Hϋsnϋ, K., Demirci, F., 2002, Antimicrobial Screening of Mentha piperita Essensial Oil, Journal of Agricultural and Food Chemistry, 50 : 3943-3946
Khanam, M., Dewan, Z. F., 2008, Effects of the Crude and the n-hexane Extract of Nigella sativa Linn.
(kalajira) upon Diabetic Rats, Bangladesh J.Pharmacol, 4 : 17-20
Kredy, H.M., 2010, Antibacterial Activity of Saponin Extract from Sider (Zingiphus spina_christi), Journal of Thi-Qar University, 1(6) : 1-7
Roshan, S., Khan, A., Tazneem, B., Ali, S., 2010, To Study The Effect of Nigella sativa on Various Biochemical Parameter on Stress Induced in Albino Rats, International Journal of Pharmacy and Pharmaceutical Sciences, 2 (4) : 185-188
In Vitro Antibacterial Activity…..
Wagner, H., Bladt, S., 1996, Plant Drug Analysis in Thin Layer Chromatography Atlas, 2sd edition, Springer Verlag, Berlin
Yoruk, O., Gur, F.O., Uyanik, H., yasar, M., Mutlu, V., Atlas, E., Baysal, E., Taysi, S., 2010, Antioxidant Effects of Nigella sativa in The Treatment of Experimentally Induced Rhinosinusitis, Macedonian Journal of Medical Sciences, 3 (2) : 132-137
Zuridah, H., Fairuz, A.R.M., Zakri, Rahim, M.N.A., 2008, In vitro Antibacterial Activity of Nigella sativa Againts Staphylococcus aureus, Psedumonas aeruginosa, Klebsiella pneumonia, Escherichia coli, and Bacillus cereus, Asian Journal of Plant Sciences, 7 (3) : 331-333