Chemical composition and antibacterial activity of Xeromphis nilotica bark extracts

This present study was carried out in Khartoum state-Sudan, during March 2019. The dried bark of Xeromphis nilotica was extracted successively with (Ethyl acetate, Ethanol) and (Methanol, Water). The phytochemical screening carried out on four different extracts of Xeromphis nilotica bark and it contain amount of secondary metabolites such as (Alkaloids, Carbohydrates, Tannins, Phenols, Sterols & Triterpenes, Saponins, Flavonoids, Amino acids and Proteins were studied. The antibacterial activity of extracts were evaluated against four standard bacteria (Gram positive; Bacillus subtilis, Staphylococcus aureus) and (Gram negative; Escherichia coli, Pseudomonas aeruginosa) and the results showed high inhibition zone (21mm) in methanol extract against Escherichia coli and low inhibition zone in aqueous extract against all bacterial strains tested.


Introduction
Xeromphis nilotica belong to Rubiaceae family, is the medicine plant that distributed in tropical and subtropical regions [1], the genus Xeromphis is represented in Sudan by one species, namely Xeromphis nilotica(Stapf) Keay [2], which is widespread in Central and East Africa as well as inCameroon and Nigeria [3]. Locally it is known as Shagart-Elmarfaein [4].It grows as a medium height shrub (usually less than 3 m) with grey globose drupes, stiff spines, and deciduous leaves clustered below the spines [5], it is use in different traditional medicines systems of Sudan for antispasmodic, anti-dysenteric, anti-inflammatory, immunomodulatory and anti-fertility properties [6][7][8][9][10]. Many species of plants grow abundantly in the Sudan and other African countries and are used by the village populations for treatment of various disorders [11] [12], and are the main medicinal source to treat infectious diseases [13], in many rural area, the medicinal plants hadbeen an important methods in the treatment of diseases. The medicinal plants contain a number of secondary metabolites compounds such as alkaloids‚ flavonoids‚ tannins‚ saponins‚ amino acids, proteins, carbohydrates, Phenol, Sterols &Triterpenes [12], the traditional medicinal plants are increase in both developing and industrialized countries. [14]reported thatboth literate and illiterate people still use local plants as drugs in many conditions [15].
The aim of the present study was to determine the phytochemical screening and antibacterial activity of Xiromphis nilotica bark.

Materials and methods
All the chemicals and reagents used in this study were of analytical grade such as chloroform, distilled water, ethanol, methanol, acetic anhydride, sulphuric acid, gelatine salt, ferric chloride, reagents (Wagner, Hager, and Dragendorffs), aluminum chloride and potassium hydroxide.

Plant material, collection and identification
Xiromphis nilotica bark was collected from Elsalihen area, locality of algoze, South kordofan State-Sudanand identified in herbarium of natural research Centre and compared with herbarium of Faculty of Science University of Khartoum

Preparation of Crude Extracts
50g of the dried plant was weighted and extracted successively with (ethyl acetate, ethanol) and (methanol aqueous)by shaker apparatus for four hours at room temperature, each extract was filtrated through Whatman No 1 filter paper, followed by concentrated under vacuum room. The crude extracts were then kept at -20 ºC in sterile universal bottles.

Phytochemical screening of different crude extracts
General phytochemical screening for the active constituents was carried out for extracts using the methods of [16].

Preparation of media
28g of powdered nutrient agar was weighted, dispersed in 1 liter of distilled water and allowed to soak for 10 minutes, swirl to mix then sterilized by autoclaving for 15 minutes at 121ºC, cooled to 47c, mixed well then poured into Petridishes [17].

Testing of organisms:
The two (gram positive and negative bacteria were tested in table (2). The bacterial strains tests used for screening were Bacillus subtiles (NCTC 8236 Gram positive bacteria).Staphylococcus aureus (ATCC 25923 Gram positive bacteria).Escherichia coli (ATCC 25922 Gram negative bacteria) and Pseudomonas aeruginosa (ATCC 27853 Gram negative bacteria).

Testing for antibacterial Activity
The cup-plate agar diffusion method [17], was adopted with some minor modifications to assess the antibacterial of the prepared extracts. One ml of the standardized bacterial stock suspension 108-109 C.F.U/ ml were thoroughly mixed with 100 ml of molten sterile nutrient agar which was maintained at 45 ºC. 20 ml aliquots of the inoculated nutrient agar were distributed into sterile Petri-dishes. The agars was left to set and in each of these plates 4 cups (10 mm in diameter) were cut using a sterile cork borer (No. 4) and agar discs were removed. Alternate cups were filled with 0.1 ml sample of the extracts using automatic micro-liter pipette, and allowed to diffuse at room temperature for two hours. The plates were then incubated in the upright position at 37 ºC for 18 hours. Two replicates were carried out for the extracts against each of the tested organisms. After incubation the diameters of the resultant growth inhibition zones were measured.  Phytochemical screening for major constituents was undertaken using standard qualitative methods of [16]. The plant extracts were screened for the presence of (Alkaloids, Carbohydrates, Tannins, Phenols, Sterols &Triterpenes, Saponins, Flavonoids and Amino acids). Aqueous Key:Concentrations of extracts (100, 50, 25, 12.5mg/ml) .Zone of inhibition in (mm), -no inhibition, <9mm inactive, 9-12mm partially active, 13-18mm active, >18mm very active.The methanol and ethanol extracts showed high inhibition zone between (21-18)against four tested microorganisms (E.c, S.a, B.s, and P.s),

Results and discussion
The Aqueous extract was recorded low inhibition zone between  comparing with other extracts.

Discussion
Phytochemical analysis provided significant ideas for the development of new herbicides and drugs against deadly diseases. Natural products of wild plants still play a central role in the biological activities and healthcare system of large proportions of the world's population [18,19].The total of four extracts of Xeromphis nilotica bark (Methanol, Ethanol, Ethyl acetate and aqueous) were used to evaluate the antibacterial profile and phytochemical screening of the plant. The Xeromphis nilotica bark found to be good source of (Alkaloids, Flavonoids, Carbohydrates, Tannins, Phenols, Sterols &Triterpenes, Saponins and Amino acids).The antibacterial assay shows that all of the extracts were found to be active against the tested pathogens, but the aqueous extract of the plant shows minimum activity against staphyllococcus aureus at concentration 12.5 when compared with other extracts. The presence of different phytochemicals in the plant is the possible answer for its active antibacterial profile.

Conclusion
It was concluded from the present study that the crude extracts of Xeromphis nilotica bark has significant inhibitory effect against both gram positive and gram negative bacterial culture. Among different extracts methanolic extract was very effective. The extraction bark of Xeromphis nilotica used in this study showed the significant phytochemicals and the bark contained high amounts of secondary metabolites. In general the bark extracts has better chemical characteristics.