Antibacterial Activity and Identification of Metabolites from the Semi-Purified Fraction of Chrysophyllum albidum Leaf (African Star Apple)

Traditional medicine which rely on plant resources to treat various diseases and ailment remains an important (in some case, the only) form of treatment in many developing countries and its use is increasing worldwide. 1 According to the world Health Organization (WHO), 75% of world population still rely on plant based traditional medicines for their primary healthcare of which 85% is based on the use of plant extracts. 2 Similarly, it is estimated that 39% of 520 new approved drugs (between 1983 and 1994), 5080% of antibacterial and anticancer drugs were derived from natural product. 3 The increased interest in the use of natural products is for a number of reasons, the perception that such products are safe, ecofriendly, low cost, readily available and of little or no side effects. The use of natural products has led in many drug discoveries as they provided chemical building blocks used in their synthesis or used as new drugs in their unmodified state. 4


Introduction
Traditional medicine which rely on plant resources to treat various diseases and ailment remains an important (in some case, the only) form of treatment in many developing countries and its use is increasing worldwide. 1 According to the world Health Organization (WHO), 75% of world population still rely on plant based traditional medicines for their primary healthcare of which 85% is based on the use of plant extracts. 2 Similarly, it is estimated that 39% of 520 new approved drugs (between 1983 and 1994), 50-80% of antibacterial and anticancer drugs were derived from natural product. 3 The increased interest in the use of natural products is for a number of reasons, the perception that such products are safe, ecofriendly, low cost, readily available and of little or no side effects. The use of natural products has led in many drug discoveries as they provided chemical building blocks used in their synthesis or used as new drugs in their unmodified state. 4 Many natural products are produced as part of chemical defense. Examples of this include synthesis of antimicrobial or antifeedant substances systems to protect the producing organism from attack. This inherent biological activity associated with natural products is the reason for the use of plants in traditional medicines as remedies for various infections. [5][6][7] Chrysophyllum albidum G. known as African star apple, belonging to the family Sapotaceae is primarily a forest tree with its natural occurrences in diverse eco-zones of Africa In Nigeria, C. albidum is known as "agbalumo" in the South West and "udara" in the South East. The fruit usually available during the dry season (December-April) has been enjoyed over the years by Nigerians. The fleshy pulp of the fruits is eaten especially as snack and its fruit has been found to have higher contents of ascorbic acid than oranges and guava. 9,10 Extracts from different parts, including the bark, leaves, roots and seeds of C. albidum have been used for the treatment of different ailments, such as yellow fever, malaria, certain skin diseases, stomach ache, and diarrhoea, infertility problems as well as dermatological and urinary related infections. [11][12][13][14] The extracts have also found use as liniments and in stopping microbial growth in open wounds. [15][16][17] The split stems and wigs are used as chewing sticks in many parts of Africa and have been commercialized in many cities for years offering natural dental care. The extracts of the leaves and fruits have shown antibacterial and antioxidant properties in vitro and in vivo. [18][19][20][21][22][23][24][25] Despite numerous studies on phytochemical composition, medicinal and antimicrobial activities of plants, reports on African star apple are limited especially report on the particular component responsible for the biological activities. Moreover reports on the phytochemical composition and activity of the metabolite are also scanty. This study therefore focuses on the antibacterial activity of the metabolite from the ethanol extract of C. albidum with a view to identify the most active fraction.

Sample collection and preparation
Fresh leaves of C. albidum were collected within Owerri municipal in Imo State, Nigeria in July 2018. The plant material was identified at the crop science department in the school of Agricultural Technology, Federal University of Technology Owerri. The leaves were dried at room temperature to constant weight, ground to fine powder and then stored in an air-tight container. The powdered sample (80 g) was extracted with 250 mL of ethanol for 12 h using a soxhlet extractor. The ethanol extract after extraction was allowed to evaporate

Preparation of basic metabolite
The filtrate of Chrysophyllum albidum extract was treated with dilute HCl and extracted with chloroform in a separatory funnel. The lower chloroform layer was removed and reserved for the preparationof the neutral metabolite. The HCl layer was treated with NaHCO3 until the mixture became basic. The precipitate from the resultant solution was filtered out and washed well with distilled water. The precipitate was dissolved in 40 mL of 95% ethanol and filtered. The filtrate was used for the antibacterial screening without further purification.

Preparation of neutral metabolite
The chloroform layer obtained from basic metabolites was placed in a separatory funnel and treated with NaOH solution (2 M). After equilibrating, the aqueous NaOH solution layer was removed and reserved for preparation of the acidic metabolite. The chloroform layer was removed and allowed to evaporate completely at room temperature to form a gel which was dissolved in 95% ethanol and filtered. The filtrate was used without further purification for antibacterial screening.

Preparation of acidic metabolite
The aqueous alkaline layer obtained from neutral metabolite was treated with dilute HCL until the solution became acidic. The precipitate of the mixture was filtered out and washed well with distilled water. The precipitate was dissolved in 40 mL of 95% ethanol and filtered and the filtrate was used for the antibacterial screening without further purification.

Phytochemical analysis
The phytochemical screenings of the extracts were carried out using the methods described by Harborne 27 and Evans. 28 The substances that were tested for include tannins, saponins, and flavonoids, cardiac glycosides, alkaloids, anthraquinones and steroids.

Chromatographic purification
Preparative thin layer chromatography was carried out on the neutral metabolite using a chromatographic plate coated with silica gel and cellulose. The sample was spotted on the plate and then developed in a chromatographic tank for 1 hour using ethanol (95%) and chloroform mixture (2:1) as the eluting solvent. After separation, three distinct fractions were obtained and labeled N1, N2 and N3. The fractions were scrapped into different conical flasks re-dissolved with ethanol (95%) and filtered. The filtrates in the different flasks were allowed to evaporate to dryness at room temperature and each fraction was screened to determine their anti-microbial activity against some test microbes.

Antimicrobial Tests Collection of test organisms
Clinical isolates of Staphylococcus aureus, Streptococcus spp, Escherichia coli, Salmonella typhi and the coliform bacilli: Enterobacter cloacae were collected from the Department of microbiology Federal Medical Centre, Owerri, Nigeria.

Sterility test of the extracts
Sterility of the extracts was determined by streak inoculation onto freshly prepared Nutrient, Blood and MacConkey agar plates. Plates were incubated at 37 o C for 24 hours. At the end of incubation period, plates were examined for growth.

Antibacterial screening
The Disc Diffusion Method was used as earlier described. An inoculating loop or needle was touched on five isolated colonies of the pathogens growing on agar and then used to inoculate a tube of culture broth. The culture was incubated for a few hours at 35-37 o C until it becomes slightly turbid and was diluted to match a turbidity standard. A sterile cotton swab was dipped into the standardized bacterial test suspension and used to evenly inoculate the entire surface of an agar plate. After the agar surface had dried for about 5 min, the appropriate antibiotic test disc of each fraction was placed on it and the agar plate was immediately placed in the incubator maintained at 35-37 o C. After 16-18 h of incubation, the minimum inhibition zone diameters were measured and recorded to the nearest mm. [29][30][31] The fraction with the highest inhibition zone diameter was selected for spectrometric analyses using Infrared Spectroscopy (IR) and GC-MS.
Spectroscopic analysis IR spectroscopic analysis of selected antibacterial active fractions was carried out at the Central Research Laboratory University of Ibadan, using the Perken-Elmer IR spectrophotometer while the GC-MS analysis was carried out at the Obafemi Awolowo University Ile-Ife.

GC-MS analysis
GC-MS analysis was carried out using the method of Dhivya and Manimegalai. 32 Identification of the compounds based on their massto-charge ratio were determined by the mass spectrometer. 33 Chemical analysis was by triple quadruple gas chromatography mass spectrometry. Analysis was performed in TIC scan mode. Oven temperature was set at 50 o C for 2 min and programmed at 50 o C to 230 o C at a rate of 4 o C/min and at hold 230 o C for 2 min resulting in the complete elution of peaks analysed. Table 1 shows the result of the phytochemical screening carried out on the extract and various metabolites. Table 2 compares the antibacterial activity of the crude extract and of the various fractions (metabolites) at same concentration of 100 mg/mL. Table 3 compares the antibacterial activity of the three fractions (N1, N2, N3) obtained from the chromatographic separation with that of the control drugs at same concentrations. The result of the phytochemical screening showed the presence of tannins, saponins, flavonoids, phenols, cardio-active glycosides and alkaloids. It has been reported that plant that contained tannins have astringent, hemostatic, antiseptic and toning properties. 33 Tannin containing herbs have been reportedly used in stopping minor hemorrhage, sore mouth, bronchitis, burns, scars of the skin, wound diarrhea and other diseases. 34 Saponin have been reported to be active in fighting cancer and also in lowering cholesterol level and blood sugar. 35 It has been used in the treatment of hypercalciuria in human and as antidote against lead poising. 35 Flavonoid on the other hand have been reported to have wide range of biological and pharmacological activities in in vitro studies which include antiallergic, anti-inflammatory, antioxidant, antimicrobial, anticancer and anti-diarrheal activities. 36 Natural phenolic compounds have been reported to play an important role in cancer prevention and treatment. 37 Phenolic compounds from medicinal herbs and dietary plants include phenolic acids, flavonoids, tannins, stilbenes, curcuminoids, coumarins, lignans, quinones, and others. Various bioactivities of phenolic compounds are responsible for their chemopreventive properties (e.g., antioxidant, anticarcinogenic, or antimutagenic and anti-inflammatory effects) and also contribute to their inducing apoptosis by arresting cell cycle, regulating carcinogen metabolism and ontogenesis expression, inhibiting DNA binding and cell adhesion, migration, proliferation or differentiation, and blocking signaling pathways. 37 Cardaic active glycoside is valuable in the treatment of congestive heart failure. 38 The presence of these phytochemical scientifically proves the leaves of C. albidum as a medicinal plant. The results of the preliminary antibacterial screening of the crude extract and various metabolites of C. albidum are shown in Tables 2, from which it was observed that the crude extract, acidic, basic and  These differences in bioactivity arose probably from the differences in the phytochemical composition of the various metabolites. The result of the control experiment with the following drugs Cefuroxime, ciprofloxacin and gentamycin against the test micro-organisms are presented in Table 3.

Results and Discussion
Hence the observed antibacterial activity of the crude extract, acidic, basic and neutral metabolites against the test microorganisms may be due to the presence of tannins or other phenolics, saponins and flavonoids found to be present in the plant. The neutral metabolites due to its high activity were chosen for purification and further study. Fraction N3 obtained from the chromatographic separation of the neutral metabolites exhibited high activity against Salmonella and least activity against Streptococcus which implies that the neutral metabolite may be used to treat diseases caused by Salmonella spp. The neutral metabolite was purified using preparative thin layer chromatography and three fractions were obtained which were labeled N1, N2 and N3. Antibacterial analysis was carried out on the three fractions and it was observed that fraction N3 was most active against the test microbes because it gave the highest inhibition zone diameter of 18 mm for Streptococcus spp., 20 mm for S. aureus, 25 mm for Salmonella typhi, 20 mm for E.coli and no inhibition for Enterobacter cloacae. Due to its high activity against the test organisms, fraction N3 was chosen for IR and GC-MS analyses.

IR Spectral data of Fraction N3
The IR Spectrum of fraction N3 is presented in Figure 1 and showed the following absorptions.

Conclusion
This study has demonstrated the antibacterial activity of the metabolites of the leaves of Chrysophyllum albidum against some human pathogens, and it has been established scientifically that the leaves is medicinal as shown by the antibacterial activity and the presence of potentially bioactive phytochemicals.