Antifungal, Antibacterial, Phytotoxic and Insecticidal Activities of Nigella sativa Seeds

Plants have been recognized for their therapeutic and medicinal potential since ages. A medicinal plant is one that contains substances which possess some therapeutic property or have the potential to be used as precursor for the synthesis of chemo-pharmaceutical products. Though plants are bestowed with large number of therapeutic chemicals but unfortunately most of these needs to be explored. Pakistan has a wide floral diversity consisting of about 6000 different genus and 700 species are being used for medicinal and aromatic purposes, but still the real commercial potential of these plants needs to be explored. The restorations of natural drugs started in last two decades mainly because of the broad and extended belief that green medicine is better than artificial products. Now-a-day, there are various medicinal plants based industries due to increase in the awareness of use of medicinal plants, all over the world, which are growing at a rate of 7-15 % yearly. About 60 % of the world’s populations depend on the use of traditional plant extracts for their major health-care needs. Antifungal, Antibacterial, Phytotoxic and Insecticidal Activities of Nigella sativa Seeds

At present, there are growing resistance against existing antibiotics and therefore a number of infectious diseases cannot be cured effectively 7,8 .
World Health Organization (WHO) reported that about 80 % of the world population relies on the traditional medicine for the treatment of different diseases. Since 1980, WHO has been encouraging countries to identify and develop traditional medicine. Therefore, the assessment of rich heritage traditional medicine is indispensable.
In this regard, one such plant is N. sativa (Ranunculaceae), which is a small herb distributed and cultivated all over the world and regarded as one of the best healing medicine available. In Arabic known as habbat al barakah and commonly called "black seed" black cumin" or "fennel flower" in English, N. sativa is a very old food and medicinal harvest 9,10 .
Our Prophet Muhammad (PBUH) told that the N. sativa has the healing power except death. Avicenna said that N. sativa is Canon of Medicine, restore the energy and recover from fatigue. It is found in the "Tibb-e-Nabavi and also in the Unani Tibb for its benefits 5,11 .
The seeds of N. sativa have been used in pharmacological investigations. The studies have showed a broad spectrum of actions 10,12,13 ; the plant also showed antitumor activity 14 . The seeds of N. sativa are used for buffalo reproductive efficiency and for the treatment of Helicobacter pylori disease in human [15][16][17] . The seeds are used as diuretic, jaundice, intermittent fever, dyspepsia, paralysis, skin diseases and pile 5 . Seed oil is used as condiment, carminative, food additive and painkiller in different parts of the world 18,19 . The world's biggest food company, Nestle is in search of rights on the use of N. sativa to stop food allergies 9 .Therefore keeping in view the importance of seeds of N. sativa the current study was aimed at screening it for antifungal, antibacterial, phytotoxic and insecticidal activities.

EXPERIMENTAL
The seeds of N. sativa were purchased from the local herbal market in Peshawar and identified by Prof. Dr. Farrukh Hussain, Department of Botany, University of Peshawar, Pakistan.
Extraction: Dried seeds of N. sativa (10 kg) were grinded to powder form and soaked in methanol for fifteen days at room temperature, with occasional shaking. The material was filtered and concentrated at 40 °C under vacuum by rotary evaporator. About 850 g of crude methanolic extract was obtained.
Antibacterial activity: The methanolic extract and different fractions of N. sativa were tested for antibacterial activity against E. coli, P. mirabilis, B. cereus and X. oryzae. Stock solutions were prepared in dimethyl sulfoxide (DMSO, < 1 %) at concentration of 3 mg/mL of the test samples. The microorganisms were inoculated in nutrients broth (Sigma-Aldrich, Germany) and incubated at 37 °C. After incubation, 18-24 h culture of each test organism was transferred to sterile Nutrient agar plates for preparation of bacterial lawn and 6 mm wells were dug in plates at equal distance. 100 µL from the stock solutions of the test samples were loaded to their respective wells. For the positive and negative controls, amoxicillin and DMSO were used as controls respectively. The petri plates were left for 2-3 h undisturbed inside the laminar air flow to get the best diffusion of the test samples to media. The petri plates were then incubated at 37 °C for 24 h. The inhibition zones (mm) were calculated after incubation in comparison with positive control 20,21 .
Phytotoxic bioassay: The test samples were checked against Lemna minor L. as per reported procedure 22 . Stock solutions of the test samples were prepared in methanol at concentration of 20 mg/mL and E-medium was prepared for the L. minor growth. 1000, 100 and 10 µg/mL from the stock solution were poured to three flasks, one for each sample and left at room temperature till the organic solvent was dried.
The E-medium and twenty healthy plants with a rosette of three fronds were added to all flasks and incubated at 28 ± 1°C for seven days. Paraquat was used as standard growth inhibitor. Results were noted after seven days of incubation by counting the number of damaged plants 21,22 .
Insecticidal activity: For this activity, direct contact toxicity was used 21 . Describing the procedure, the test sample (200 mg/mL) was dissolved in 3 mL of methanol and applied to filter papers (90 mm diameter). The dried filter paper was placed in the separate Petri dish along with 10 adults of T. castaneum. Permethrin (235.71 µg/cm 2 ) was used as reference insecticide. These were kept without food for the interval of 24 h after which mortality was calculated.
Antifungal activity: Antifungal activity of the test samples were evaluated as per reported procedure 21 against P. oxalicum, A. strictum, V. lecanii and T. harzianum. Sabouraud dextrose agar (Sigma-Aldrich, Germany) was prepared, autoclaved and 4 mL was dispensed into sterile test tubes. The (non-solidified) SDA media was mixed with test solution 66.6 µL. The tubes allowed for solidification in the slanted position at room temperature. An agar surface streak of the selected fungi was employed for non-mycelial growth. DMSO and standard antifungal drugs served as negative and positive control, respectively. After 7 days of incubation at 28 ± 1 °C linear inhibition was calculated.
Statistical analysis: In SPSS (two way anova) calculation showed highly significant difference between the different fractions (P = .000) and between the different concentrations 1000, 100 and 10 mg/mL it was insignificant (P = .076).

RESULTS AND DISCUSSION
Antibacterial activity: The demand of medicinal plants has increased with the advancement of science and increase in infectious diseases particularly caused by E. coli, P. mirabilis, B. cereus and X. oryzae, all becoming resistant to many antibiotics 23 . This problem can be solved by the plant origin active ingredients; we have selected N. sativa which is locally available and traditionally used for different purposes. The previous research was conducted on different plant extracts e.g., Zizyphus jujuba 20 , Tylophora hirsute 22 and on Myrsine africana 21 on different strains of bacteria 10,13,24 . In our research methanolic extract possess significant antibacterial activity against E. coli (100 %), P. mirabilis (100 %), B. cereus (100 %) and X. oryzae (100 %). The n-hexane fraction possess significant activity against P. mirabilis (100 %), B. cereus (100 %) and X. oryzae (100 %) while low activity against E. coli (2 %). Chloroform fraction possess significant antibacterial activity against P. mirabilis (100 %), X. oryzae (100 %) and E. coli (100 %) while low activity against B. cereus (5 %). Aqueous fraction showed significant activity against P. mirabilis (100 %), B. cereus (100 %) and X. oryzae (100 %) while low activity against E. coli (30 %). The SPSS (two way anova) showed (P > 0.05) insignificant difference for different bacterial strains but significant difference for (P < 0.05) different fractions as shown in Fig. 1.