Chemical Composition and Antimicrobial Activities of Fruit Extracts of Ceratonia siliqua

Materials

Plant Material: Carob pods (Ceratonia siliqua L) were randomly harvested from various parts of several trees grown in different locations in the plant garden in Kadogli (South of Kordofan); the samples were collected in the morning in December 2018. The plant was authenticated by a plant taxonomist at Elobeid agricultural researches station to be Ceratonia siliqua, same physiological maturity (dark brown) and of uniform shape and size. The fruits were shade-dried, cleaned and grinded by a mechanical grinder. The grounded samples were stored at room temperature to be ready for further extraction.

Methods

Preparation of Plant Extracts: Hundred grams of the dried fruits powder were macerated exhaustively for three days and five hours at room temperature with 1000 ml of ethanol 99% and with 1000ml of distill water separately and respectively. 250g of dried fruits powder were macerated exhaustively for three days at room temperature with 2500 ml hexane. The extracts were filtered and the solvents were left to evaporate, after evaporation of the solvents, solid products with beige color for water extract, dark orange color for ethanol extract and viscous liquid product with dark green color for hexane extract was obtained. Preparation of Bacterial Suspensions: One ml aliquots of a 24 hours broth culture of the test organisms were aseptically distributed onto nutrient agar slopes and incubated at 37ºC for 24 hours. The bacterial growth was harvested and washed off with 100 ml sterile normal saline, to produce a suspension containing about 108- 109 C.F.U/ ml. The suspension was stored in the refrigerator at 4°C till used. The average number of viable organisms per ml of the stock suspension was determined by means of the surface viable counting technique. Serial dilutions of the stock suspension were made in sterile normal saline solution and 0.02 ml volumes of the appropriate dilution were transferred by micro pipette onto the surface of dried nutrient agar plates. The plates were allowed to stand for two hours at room temperature for the drops to dry and then incubated at 37°C for 24 hours. After incubation, the number of developed colonies in each drop was counted. The average number of colonies per drop (0.02 ml) was multiplied by 50 and by the dilution factor to give the viable count of the stock suspension, expressed as the number of colony forming units per ml suspension. Each time a fresh stock suspension was prepared. All the above experimental conditions were maintained constant so that suspensions with very close viable counts would be obtained [12].

Preparation of Fungus Suspension: The fungus culture was maintained on sabouraud dextrose agar, incubated at 25°C for 4 days. The fungus growth was harvested and washed with sterile normal saline and finally suspended in 100ml of sterile normal saline, and the suspension were stored in the refrigerator until used [12].

• Bacterial Microorganisms
• NCTC 8236 (Gram + ve bacteria)-Bacillus subtilis
• Staphylococcus aureus-ATCC 25923(Gram + ve bacteria)
• Escherichia coli-ATCC 25922(Gram -ve bacteria)
• ATCC 27853 (Gram -ve bacteria) Pseudomonas aeruginosa
• National Collection of Type Culture (NCTC), Colindale, England.
• American Type Culture Collection (ATCC) Rockville, Maryland, USA.

Fungus Microorganism
Candida albicans-ATCC7596

GC-MS Analysis Conditions: The qualitative and quantitative analysis of the samples was carried out by using GC/MS technique model (GC/MS-QP2010-Ultra) from Japans 'Simadzu Company, with serial number 020525101565SA and capillary column (Rtx-5ms-30 m × 0.25 mm × 0.25 µm). The sample was injected by using split mode, helium helium as the carrier gas passed with flow rate 1.61 ml/min, the temperature program was started from 60°C with rate 10°C /min to 300°C as final temperature degree with 3 minutes hold time, the injection port temperature was 300°C, the ion source temperature was 200°C and the interface temperature was 250°C. The sample was analyzed by using scan mode in the range of m/z 40-500 charges to ratio and the total run time was 27 minutes. Identification of components for the sample was achieved by comparing their retention index and mass fragmentation patents with those available in the library, the National Institute of Standards and Technology (NIST).

Assessment of Antimicrobial Activities of the Extracts

Assessment of antimicrobial activities of ceratonia siliqua extracts with different concentrations (100, 80, 60, 40, 20,10,5 and 2.5 mg/ml) were carried out against four types of bacteria (two gram positive: Bacillus Subtilis and staphylococcus and two gram negative: Escherichia coli and Pseudomonas) and one type of fungus (Candida albicans). Tetracycline and ultra griseo fulavin were used with different concentrations (100, 80, 60 and 40 mg/ml) as positive control for bacteria and fungus respectively. The assessment of antimicrobial activities of the plant extracts and tetracycline and ultra griseo fulavin against bacteria and fungus were shown in Tables 1-4 respectively. Hexane extract showed no antibacterial activity against Escherichia coli with concentration 40 mg/ml and below. Ethanol extract exhibited no antibacterial activity against Escherichia coli with concentrations (60, 40, 20, 10, 5and 2.5mg/ml) and Pseudomonas aeruginosa with all concentrations. The water extract showed no antibacterial activity against the four tested bacteria with the different concentrations. The fruit extracted by hexane and ethanol exhibited partial activity against the tested fungus (Candida albicans) with the different concentrations. The minimum inhibition concentrations (MIC) of ethanol extract was 40mg/ml against staphylococcus and Bacillus Subtilis (with inhibition zones 14mm and 15mm respectively). The MIC of hexane extract was 40mg/ml against Pseudomonas aeruginosa, staphylococcus and Bacillus Subtilis (all have inhibition zones 12mm). The MIC of ethanol and hexane extracts was 40mg/ml against the Candida albicans (with inhibition zones 12mm and 10mm respectively).

Table 4: Antifungal activity of ultra griseo fulavin (antifungal) against candida albican (inhibition zone in mm). E.c = Escherichia coli, Ps.a = Pseudomonas aeruginosa, S.a = Staphylococcus aureus, B.s = Bacillus subtilis, C.a = Candida albicans, Mic= Minimum inhibition concentration. 18 mm and above = very active, 13-18 mm = active, 9-12 mm = partially active, 9 mm and below = Resistant.

Chemical Compounds of Different Extracts

Analysis of water fruit extract by using GC-MS showed the presence of 21 compounds and listed in Table 5. Ethanol fruit extract was analyzed by using GC-MS leading to the identification of 20 compounds. The compounds were shown in Table 6. Analysis by GC-MS identified 28 compounds in hexane fruit extract which are presented in Table 7. The chemical compounds identified in the crude extracts were listed in tables according to their retention time.