Antimicrobial evaluation of various leaves extracted samples of nettle desert ( Forsskaolea tenacissima L . )

The present study evaluates the anti-microbial activity of three different crude extracts (ethanol, aqueous and n-hexane) of Forsskaolea tenacissima L. leaves against gram negative and gram positive bacteria and fungi using well diffusion method. N-hexane extract showed tremendous inhibition of 12mm (80% ZI) and 10mm (71.42% ZI) against Staphylococcus aureus and Bacillus subtillis at 1000 μg/ml concentration. Similarly, aqueous extract at the concentration of 1000 μg/ml reduced the growth of Xanthomonas maltophilia and Escherichia coli as 11mm (68.75% ZI) and 9mm (60.00%), respectively. However, ethanol extract showed good activity of 12mm (75.00%) against Clavibacter michiganense Aqueous extract showed 9mm (75.00%) against Acromonium alternatum at 1000 μg/ml. Rhizopus stolinifer and Trichoderma reesei both were found sensitive to aqueous extract, which showed 11 and 8mm (68.75 and 57.14% ZI) at 1000 μg/ml concentration, respectively. The growth of Aspergillus niger was inhibited by ethanol extract through 9mm (56.25% ZI) at the concentration of 1000 μg/ml. The above study determined the medicinal importance of Forsskaolea tenacissima.


Introduction
In developed countries, about 80% of plants are used as traditional medicines that serve as excellent sources of compounds (drugs).The plants are collected for the properties they exhibit such as the synthesis of secondary product [1], and inhibitory effect against various growing human [2].Traditional medicines are being used for practices, knowledge and also need for making of better plants and animal-based approaches [1, 3].The use of natural medicine and local practices are common in the treatment of various diseases [4].An increase in number of infectious agents with strong resistance to commercial antimicrobial compounds has been noticed [5].Due to this reason, the secondary product produced by medicinal plant is of excellent value due to its antimicrobial constituents [6].These antimicrobial agents mark the most important discovery of 20 century in the field of medicine [7].Also, traditional medicines system is now well popular and adopted at global level as a primary health care system [8].Main resources for modern drugs are from Mother Nature.According to World Health Organization (WHO) reports, the use of traditional medicine in the 1 st world countries is at peak.Failure of conventional medicine that can cure chronic diseases, emergence of multi-drug resistance pathogens and parasites, adverse effects of chemical drugs and increasing cost are some of the reasons that made traditional medicines using again by people [9].

Materials and methods
Experiments regarding this study were conducted in research Laboratory of Department of Botany, Islamia College University Peshawar, Pakistan.

Plant materials
Forsskaolea tenacissima leaves were collected from Jarjorey F-R Peshawar Tribal area.The leaves were subjected to room temperature and shade for a period of 3 month to completely dry.An ordinary grinder was used to grind the leaves.Preparation of crude extract About 70g of ground plant powder was taken three times in three round bottom flasks, first round bottom flask ethanol to the second nhexane and to 3 rd round bottom flask distal water was added.After 24 hours, the filtrate was filtered with cotton and the process was repeated 3 times.The concentration of the extracts was performed by rotary evaporator at 60°C.The material was again dried through water bath at 55°C and was stored in bottles.

Well diffusion susceptibility method
Well diffusion method was used to study antimicrobial activity of different plant extract.Fungal culture was grown on PDA while bacterial cultural were grown on agar media as mentioned by [27].For the purpose to know antimicrobial activity of the species, petri plates impregnated with microbes were added with the extracts obtained from the plant with dissimilar concentration such as 500 µg and 1000 µg into holes present in media.The bacterial culture incubated at 37°C for 24 hours and fungal cultural were incubated at 37°C for 3 Days.

Antimicrobial activity bioassay
To determine antibacterial activity, specific amount of nutrient agar was mixed in sterilized distal water contained in bottle.All the experimental apparatus such as petri plates, borer and liquid media were sterilized at the pressure of 1.5 lbs and 121ºC for fifteen to twenty minutes.Then the agar was poured in petri plates in sterilized environment and the agar was allowed to solidify.A sterilized borer was used to make holes in the media.For antifungal activity, the same procedure was used except the nutrient agar media.In antifungal activity, PDA media was used instead of nutrient agar.But the procedure was same as antibacterial activity.

Applying antifungal test
For antifungal activity require amount of Potato Dextrose Agar (PDA) media (14.25 gm in 400 ml of distal water) was prepared for 12 petri plates.The media was prepared in bottle.The media and all the apparatus used in this experiment were sterilized in autoclave for 20 minutes at 1.5 lbs of pressure and 121 º C temperatures.Later, the sterilized media was poured in sterilized petri plates in laminar flow hood and allow it to solidify in petri plates and made 3 holes in each petri plates through sterilized borer.To avoid contamination all the procedure was attempted inside the laminar flow hood.After formation of holes the fungus was applied by streaking with sterile inoculation loop on the PDA media plates in a laminar flow.When streaking was completed, plant extract was then added and antibiotic at different concentration.An in first hole 1000 µg/ml of plant extract was added and in second hole 500 µg/ml of plant extract was added while in last hole standard antifungal against fungi was added.After that the petri plates were closed and shifted from laminar flow hood to incubator and kept in incubator at 37ºC for 3 days.Zone of inhibition around each hole for antifungal potential was recorded after three days.Positive control used against fungal strains 0.05% Flumetazole was used as a positive control.

Statistics
Simple statistics was used as the mean values were converted in percent via the following formula.

Results
The present work was conducted on three different crude extracts (ethanol, n-hexane and aqueous) of Forsskaolea tenacissima (leaves) against gram positive and gram negative bacteria.The result revealed that ethanol extract of Forsskaolea tenacissima against Staphylococcus aureus showed 8 (53.35%) and 10 (66.66%) mm zone of inhibition (ZI) in concentration of 500 and 1000 µg/ml.The n-hexane extract showed 10mm and 12mm zone of inhibition at concentration of 500 µg/ml and 1000 µg/ml, (66.66 and 80%) respectively.Against Bacillus subtilis the ethanol extract reduced the growth by 6mm (42.75% ZI) at concentration of 500 µg/ml and 8mm (57.14% ZI) at concentration of 1000 µg/ml, respectively.The n-hexane extract inhibited the growth of Bacillus subtilis by 7mm (50% ZI) at concentration of 500 µg/ml and 10mm (71.42 ZI) at concentration of 1000 µg/ml, respectively.Against Xanthomonas maltophilia, highest zone of inhibition was recorded by aqueous extract at the concentration of 500 and 1000 µg/ml, which showed 9 and 11mm (56.25 and 68.75% ZI), respectively.The ethanol extracted sample showed 7mm (43.75% ZI) at concentration of 500 µg/ml and 9mm (56.25% ZI) at concentration of 1000 µg/ml, respectively.Against Escherichia coli highest zone of inhibition was recorded by aqueous extract, as 9mm (60% ZI) at the concentration of 1000 µg/ml, while lowest zone of inhibition was recorded by N-hexane as 5mm (33.33%ZI), at the concentration of 500 µg/ml.Ethanol extracted sample showed 6 and 7mm (40 and 46.66% ZI) at the concentration of 500 and 1000 µg/ml, respectively.
Against Clavibacter michiganense all samples were effective and showed reliable results.The highest zone was recorded ethanol extract which was 8 and 12mm (50 and 75% ZI) at the concentration of 500 µg/ml and 1000 µg/ml, respectively.While n-hexane extracted sample displayed lowest ZI of 7 mm and 9 mm (43.75 and 56.25% ZI) at the concentration of 500 and 1000 µg/ml, respectively.The antifungal activity of ethanol, n-hexane and aqueous extracts of Forsskaolea tenacissima leaves against Acromonium alternatum have been shown in table 1.The aqueous extract showed 7 and 9mm (58.33 and 75% ZI) at the concentration of 500 and 1000 µg/ml, respectively.The lowest ZI was recorded by n-hexane extract as 5mm (41.66%ZI) at the concentration of 500 µg/ml.Against Rhizopus stolinifer the aqueous extract inhibited the growth by 9 and 11mm (56.25 and 68.75% ZI) at the concentration of 500 and 1000 µg/ml, respectively.The ethanol and n-hexane extracts showed 8 and 9mm (50 and 56.25 % ZI) at higher concentration.Data regarding antifungal activity is shown in table 1. Results revealed that Trichoderma reesei was highly inhibited by ethanol and aqueous at higher concentration.The data was recorded as 8mm (57.14% ZI) at the concentration of 1000 µg/ml.The antifungal activity of ethanol, n-hexane and aqueous extracted sample from Forsskaolea tenacissima leaves against Aspergillus niger is shown in table 1. Aspergillus niger was highly sensitive to ethanol extract, which Per cent inhibition= Zone of inhibition of extract (mm) X 100 Zone of inhibition of standard (mm) showed 9 and 7mm (43.75 and 56.25% ZI) at the concentration of 500 and 1000 µg/ml, respectively.Our data also revealed that Aspergillus niger was susceptible to the n-hexane extract, which showed shown 5 and 7mm (31.25 43.75% ZI) at the concentration of 500 and 1000 µg/ml, respectively.N-hexane extracted sample of Forsskaolea tenacissima (leaves) also showed maximum zone of inhibition against all tested bacteria.Ethanol extract showed best result against Clavibacter michiganense As, 12mm (75% ZI) at higher concentration, while against Acromonium alternatum aqueous extract showed the highest inhibition of 9mm (75% ZI).In this study the authors investigated the antimicrobial activity of n-hexane extracted sample of Urtica dioica, which showed maximum zone of inhibition against gram positive bacteria [31].Another study was conducted to observe the antimicrobial activity of leaf, stem and root extracts of ethyl acetate against Xanthomonas malvacearum bacteria.The ethyl acetate showed activity with 500 µg/ml more than the pure antibiotic [31].Against Rhizopus stolinifer aqueous extract showed the highest ZI at both the concentration, while against Trichoderma reesei, highest ZI was recorded by Ethanol and aqueous extracts, which were recorded as 8mm (57.14% ZI) at the concentration of 1000 µg/ml.Ethanol extract showed highest ZI against Aspergillus niger at both the concentration as compared to other extracts.Conclusion N-hexane extract showed tremendous activity of 12mm (80.00%ZI) against Staphylococcus aureus, while ethanol extract showed 12mm (75.00%ZI) against Clavibacter michiganense at higher concentration 1000 µg/ml, respectively.