Antimicrobial screening of the crude extract of Hannoa klaineana against some pathogens

Hannoa klaineana is an ever green plant belonging to the Simaroubaceae family commonly known as Oghuru in Igbo, itekiri in Edo and igi gun in Yoruba. It has been used in ethnomedicine for the treatment of malaria, cough, and stomach problems. The present study assessed the antimicrobial property of the extract of H. klaineana against Staphylococcus aureus, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Bacilus substilus, and Candida albicans using the agar diffusion method. Our preliminary phytochemical studies showed the presence of resins, steroids, terpenoids, glycosides, alkaloids, flavonoids, tannins, saponins, and phenols. Quantitative determination of the phytochemicals present revealed alkaloids (1.2%), tannins (11.2%), flavonoids (3%), saponins (29.3%), and phenolic compounds (45.7%). The extract of H. klaineana showed good antimicrobial activity against C. albicans and moderate active against S. aureus, S. typhi, E. coli, P. aeruginosa, B. substilus. These results further confirm the potentials of Nigerian plants as a source of bioactive compounds with pharmaceutical, agricultural or industrial applications.


Introduction
Medicinal plants are described by the World Health Organization (WHO) as plants that hold within compounds that can be utilized for therapeutic purposes or can be combined to create very able drugs [1]. Natural products are compounds created by microorganisms e.g. bacteria, fungi, etc., and plants to respond to changes. They are greatly utilized in industries e.g. pharmaceuticals, etc., for a range of pharmacological activities and striking structural diversity [2]. The rise in the population of the world, disease, health problems, etc. is of great concern. These have led to the use of these chemical molecules or compounds from these plants as pharmaceutical agents.
Phytochemicals are bioactive compounds found in plant diets such as fruits, vegetables, grains, beans that are responsible for disease protection [3]. Hannoa klaineana has been used several in ethnomedicine to treat cough, malaria, stomach problems. Some studies have proven its antimicrobial, anti-inflammatory, antiviral activity, anti feedant, and anti-tumor activities. It is the most known in Nigeria, among the other species of Simaroubaceae [4]. It is used in other African countries to take care of fevers, malaria, cough, colic diseases, and antitumor activity [5]. The general objective of this study was to evaluate the potential activity of the extract and fractions of the stem bark of H. klaineana on bacteria and to validate the medicinal values of the plant extract.

Plant collection and extraction
The stem bark of the plant was brought together from Orba, Nsukka, Enugu State Nigeria, and impurities were removed, air-dried in the Pharmacognosy laboratory.
The extraction and fractionation process was carried out according to the method by [6]. It was pulverized into a powder and extracted in methanol with shaking for 48 hours. The filtrates were collected and concentrated to dryness with a rotary evaporator at 40ºC. The crude methanol extract of H. klaineana was first absorbed on silica gel and sequentially extracted using n-hexane, chloroform, ethylacetate, methanol in increasing order of polarity. The fractions so obtained were filtered twice using Whatman no 1 filter paper. A rotary evaporator was used to concentrate the fractions at 45º±5ºC. The extract and fractions obtained were stored at 4ºC. The extracts of the plant were analyzed for the antimicrobial property.

Qualitative Phytochemical Screening
Phytochemical screening tests were carried out on the powdered stem bark and crude extract to determine phytochemical constituents using methods described by [7].

Quantitative phytochemical analysis
The coarse powder of the plant material was tested for quantitative estimation to determine the number of alkaloids, flavonoids, phenols, saponins, and tannins present.

Determination of total phenols by spectrophotometric method
5 g of sample was boiled with 50 ml of ether for the extraction of the phenolic component for 15 mins. 5 ml of the extract was placed in 50 ml flask, and then 10 ml of distilled water was added. 2 ml of ammonium hydroxide solution and 5 ml of concentrated amyl alcohol were also added. The samples were left to react for 30 mins for colour development [8]. This was measured at 505 nm. The standard curve was prepared using 0, 50, 100, 150, 200,250 mg/L solutions of gallic acid in methanol: water (50:50 v/v).

Alkaloid determination
5 g of the sample of powdered bark was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added and allowed to stand for 4 hrs. This was filtered and the extract was concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop-wise to the extract until the precipitation ceases. The precipitate was collected and washed with dilute ammonium hydroxide and then filtered, the residue is the alkaloid, which was dried and weighed [9].

Flavonoid determination
10 g of the plant sample was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman filter paper. The filtrate was later transferred into a crucible and evaporated into dryness over a water bath and weighed to a constant weight [10].

Saponin determination
This was done according to the method by Obdoni and Ochuko [11]. 20 g of sample powder in a conical flask was added 100 ml of 20% aqueous ethanol. The samples were heated over a hot water bath for 4 hrs with continuous stirring at about 55ºC. The mixture was filtered and the residue re-extracted with another 200 ml of 20% aqueous ethanol. The combined extracts were reduced to 40ml over a water bath at about 90ºC. The concentrated was transferred into a 250 ml separating funnel and 20 ml diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated with 60 ml of n-butanol. The combined n-butanol was washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the samples were dried in the oven to a constant weight and the saponin content was calculated as the percentage weight.

Determination of tannins content
Dried plant material (0.5 g) was extracted with 300 ml of ether for 20 hours at room temperature. The residue was boiled for 2 hrs with 100 ml of distilled water, and then allowed to cool and was filtered, the extract was adjusted to a volume of 100 ml in a volumetric flask. The content of the tannins in the extract was determined colorimetrically using Foling-Denis reagent and by measuring the absorbance of the blue complex at 760 nm using the tannic acid solution as a standard solution [12].

Antimicrobial Assay
The antimicrobial susceptibility testing was carried out using the agar well diffusion technique [13]. The stock solution of the extract was prepared by dissolving 4g of the extract in DMSO to get a final concentration of 400 mg/ml. 0.5 ml of the test bacterium and fungi isolates standardized to an inoculums concentration equivalent to 1 x 10 8 CFU (Mac Farland 0.5 standard) were introduced using Finn Pipette and spread into Mollen mullen Hinton agar (for bacterial isolates) and Sabourard agar (for fungi isolates) plates to achieve a confluent growth. The plates were allowed to dry and sterile cork borer of 6 mm diameter was used to bore wells in the agar plates. 20µl of the various concentration of the plant extract (400, 200, 100, 50, 25, 12.5, and 6.25 mg/ml) were introduced into the wells. The plates were allowed to stand for 1 hour or more for diffusion to take place and then incubated at 37ºC for 24 hrs. The zone of inhibition was recorded to the nearest millimeter; the MIC (minimum inhibitory concentration) of the extract that was able to produce a zone of inhibition on the test organisms. These analyses were performed in triplicate.

Results
The results were analyzed based on phytochemical parameters and antimicrobial activity obtained in the methanol crude extract. Preliminary qualitative phytochemical screening indicates the presence of alkaloids, glycosides, flavonoids, fats and oils, resins, steroids, terpenoids, tannins, acidic compounds, saponins, proteins, and starch. The quantitative estimation of phyto-constituents in the stem bark of H. klaineana is shown in Table 1; having phenols (45.7%), saponins (29.3%), and tannins (11.2%) as the highest phytochemical components in the stem bark of this plant. The results of the antimicrobial activity are presented in Tables 2 and 3. The methanol crude extract of the stem bark of H. klaineana has an inhibitory activity on all bacterial and fungal studied in this work. The inhibition zone diameter varied from 6.1 mm with S. aureus and P. aeruginosa to 4 mm with E. coli, S. Typhi, and B. substilis for bacterial tested with the highest concentration (400 mg/ml). This plant has antifungal activity ( Table 2).

Discussion
The results of this study showed that H. klaineana has biological activity. According to Odeghe et al. [14], who carried out a qualitative analysis on the selected medicinal plants (Achomanes difformis stem, Dioscorea bulbifera stem bark, Fadogia cienkowskii leaf, H. klaineana stem bark, and Vitex simplicifolia leaf); the study revealed the presence of essential phytochemicals such as alkaloid, glycoside, saponin, terpenoid, protein, carbohydrate, steroid, resin and flavonoid.
It was observed in the present study that the phytochemicals are present in these parts of the plant. The results of the phytochemicals analysis have shown the presence of alkaloids (1.2%), tannins (11.2%), flavonoids (3%), saponins (29.3%) and the highest percentage of phenolic compounds (45.7%) ( Table 1).
H. klaineana has antimicrobial activity. According to Danijela et al. [15], methanol and acetone extract of the leaf extracts of Ailanthus altissima (Simaroubaceae) has antibacterial and antifungal activity equivalent to standard antibiotics. The MIC of positive control gentamicin for antibacterial activity was 0.0 mg/mL in all cases, and positive control Bamphotericin for antifungal was 0.1 mg/mL. With P. aeruginosa as an exception, the acetone extract showed a higher or similar activity to that of methanol: dichloromethane extract. The results for the extracts against different microorganisms varied from 0.0 to 0.6 mg/mL. The study has presented that the acetone leaf extract had an excellent MIC of 0.04 mg/mL against E. coli. This value was as good as that of the generic drug gentamicin. This discrepancy with the results of other authors may be due to the extraction solvents or less sensitive bioassay methods used. The result against the fungus C. albicans was also very promising. The extracts had higher activity than amphotericin B, a gold standard in antifungal therapy.
It was observed in the present study that the antimicrobial activity of H. klaineana extract is more effective against fungus C. albicans (MIC= 12.5 mg/ml), and moderately against some bacteria strains (E. coli (MIC 100 mg/ml), P. Aeruginosa (MIC=50 mg/ml), S. aureus (MIC=50 mg/ml), S. typhi (MIC=100 mg/ml), and B. substilis (MIC=100 mg/ml) - Table 3. This antimicrobial activity can be attributed to the phytochemicals composition of this plant (alkaloids, saponins, tannins, flavonoids, etc. when compared with work done by Ajaiyeoba and Krebs [16] who worked on Quassia amara and Quassia undulate (Simaroubaceae). The extracts of the leaves and stem bark were effective against the six clinical strains and five fungi; the differences can be the different species used and/or differences in extraction and month of collection and even temperature.

Conclusion
From this study, it can be concluded that the antimicrobial screening of the crude extract of H. klaineana have very little effect on the anti-microbial activity, but showed more effect on the fungus, C. albicans. Further medicinal and toxicity tests can be carried out to show that the plant is safe for consumption as herbal medicine without plausible toxicity to body organs and tissues.