Anti-Microbial and Antioxidant Activities of Ziziphus spina Christi, Sudanese Medicinal Plant

This work is an anti-Microbial and Antioxidant activity of Ziziphus spina Christi based on four selected bacteria: Escherichia coli, Psedumonus aeruginosa, Bacillussubtilis and Staphylococcus aureus. These species were collected and prepared in Sudan. The concentrations were carried out and compared with standard control sample, the suspension of anti-microbial activities have shown different results in Fagonia cretica. The antioxidant activity is the ability of bioactive compounds to prevent, delay and protect against oxidant of virus substrates such as DNA and lipid materials, both in living organisms (e. g. human) and in food products. The result showed an antioxidant activity based on standard liquid reagent [1].


Introduction
Anti-Microbial is an agent that kills microorganisms such as bacterial or stops their growth. Anti-microbial medicines can be grouped according to the microorganisms they act primarily against. For example: antibiotics were used against bacteria and antifungals too [2]. Anti-Microbial can be further subdivided into bactericidal agent; instead, certain types of porous media have been developed to kill microbes on contact [3]. On the other hand, antioxidants are compounds that inhibit oxidation. This is a chemical reaction that can produce free radicals. Thereby, leading to chain reactions that may damage the cells of organisms. For example: thiols or ascorbic acid terminates these chain reactions. Mostly, antioxidants have two entirely different groups of substances: industrial chemical reactions which can be added to products and natural chemicals which can be presented in foods and tissues for resistant of cancer [4].Plant and animals maintain complex system of overlapping antioxidant [5].

Study area
The batch was found in west Om-Dorman region, Sudan. This area is mostly populated and features a hot arid climate routinely exceeded to 40 °C in midsummer, the region lay between 15.6476° N, 32.4807°E. This region considered as heliostat city of Sudan. The soil of this area is partly divided into two types: slab stone and muddy.

Population
According to ethno-botanical Omdurman area includes several people such as Arabs and Africans. Meanwhile, most of these people are officers beside other traditional works such as agriculture and animal husbandry.

Preparation of crude extract
One hundred gm. of whole plant material (Sedir -local name) was extracted by ethyl acetate for (24) hours at room temperature.
The extracts were filtered using filter paper and air-dried. The final volume was adjusted to give the specific concentration. Yield of all extract was respected to dry starting materials.

Anti-microbial activity (preparation of culture)
Twenty-eight gm. of nutrient agar media was added to (1000) ml. of distilled water. Seven gm. was added to (250) ml. distilled water on glass bottle volume (250) ml. (4 bottles), the four bottles were shacked very well to dilute the powder media and (32.5) gm. of sabouraud dextrose agar was added to (500) ml. distilled water.

Preparation of sabouraud
Sixty two gm. of powdered sabouraud dextrose agar was weighted and dispersed in (1) liter of distilled water and allowed to soak for (10) minutes swirl to mixed and sterilized by autoclave for (15) minutes at (121°C) and cooled to (47 °C). Lastly, mixed well and poured into petri dishes.

Preparation of organism's suspension
One ml. aliquots of a (24) hours broth culture of the testing organisms were aseptically distributed on nutrient agar slopes and incubated at (37 °C) for (24) hours, the bacteria growth was then harvested and washed off with (100) ml. sterile normal saline to produce a suspension containing a bout (108 -109) C.F.U/ml., the suspension was then stored in a refrigerator at (4 °C). The average number of organisms per ml. of the stock suspension was determined by the surface counting technique. (Miles and Misra, 1938).Serial dilutions of the stock suspension were made in sterile normal saline solution and (0.02) ml. volumes of the appropriate dilutions were transferred by micro-pipette onto the surface of dried nutrient agar plates, the plates were allowed to stand for (2) hours at room temperature with drops to dry and incubated at (37 °C) for (2) hours, after incubation the number of developed colonies in each drop was counted. The average number of the colonies per drop of (0.02) ml. was multiplied by (50) and by dilution factor to give viable count of the stock suspension which expressed as a number of colony of the stock suspension which expressed as a number of colony forming unit (C.F.U). Therefore, each time of fresh stock suspension was prepared.

Testing for anti-microbial activity
The cup-plate agar diffusion method was adopted with some minor modifications to assess the anti-bacterial activity of the prepared extract [6]. One ml. of standardized bacterial stock suspension 108 -109 C.F.U/ml was thoroughly mixed with (100) ml. of molten sterile nutrient agar which was maintained at (45 °C).
Twenty ml. aliquots of inoculated nutrient agar were distributed into sterile petri-dishes, the agar was left to set in each of this four cup-plates (10 mm. in diameter) were cut using a sterile cork borer and agar was removed. Alternate cups were filtered with (0.1) ml.

DPPH radical scavenging assay
The DPPH assay scavenging was determined according to the method of Shimada [1] with some modification. In 96-well plate, the test samples were allowed to react with 2,2-Di (4-tert-octyl phenyl)-1-picryl-hydrazyl stable free radical (DPPH) for half an hour at 37 °C. The concentration of DPPH was kept as (300μM).
The test samples were dissolved in DMSO while DPPH was prepared in ethanol. After incubation, decrease in absorbance was measured at (517nm) using multiplate reader spectrophotometer.
Percentage radical scavenging activity by samples was determined in comparison with a DMSO treated control group. All tests and analysis were run in triplicate.

Anti-Microbial
Ethyl acetate solvent had been detected a precise output. The numbers of bacteria were infected by concentrations Table 1

Antioxidant
Propyl gallate was used as standard and showed the result of (31±0.03) in Table 2   Also, propyl gallate exhibits antimicrobial activity and has reported to an effective antioxidant based hepatoprotector. Finally, this reagent used as anti-fade in fluorescence microscopy to reduce photobleaching of fluorescent probes such as rhodamine and fluorescein [1]. A balance between free radical and antioxidant is necessary for proper physiological function. If free radical overwhelms the body's ability to regulate them a condition known as oxidative stress ensues. Application of external source of antioxidants can assist in coping oxidative stress [9][10][11].

Conclusion
Anti-microbial • Anti-biotics had the ability to inhibit the growth of bacteria; therefore, we must detect the suitable one for treatment Table 1.
• Anti-bacterial activity differs with applied extractive method.
• Anti-bacterial activities support further studies to discover the new chemical structures that can contribute to alleviate or cure some illnesses.

•
Antioxidants counteract by products called free radical.
• Antioxidant may possible the development of cancer.
• Some food sources of antioxidant such as Beta-Carotene, vitamin (A), (C) and (E) must be available within the meals.