PHYTOCHEMICAL SCREENING AND IN VITRO ANTIMICROBIAL ACTIVITY OF IRVINGIA GABONENSIS (AUBRY-LECOMTE EX O’RORKE) BAILL

Chinelo Anthonia Ezeabara¹*, Mary Chinenye Ihedimbu¹, Wisdom Chibuzo Anyanele¹

ABSTRACT

Irvingia gabonensis is an African deciduous tree species that bear edible mango-like fruits. The phytochemical screening and in vitro antimicrobial activity of the plant part extracts were evaluated. The methanol extracts of the plant were used for all the analyses. Both the qualitative and the quantitative analyses of the plant extracts were carried out using standard techniques. The susceptibility of the test organisms to the herbal extractswas done using the determination of the minimuminhibitory concentration (MIC). The significant difference was measured usingDuncan’s Multiple Range Test.Alkaloids, flavonoids, saponins, tannins, and terpenoids occurred in high levels in theleaf, stem bark, and ripe fruit peel of I. gabonensiswhereas low values of anthraquinones, phenols, and steroids were also found in all the parts. The plant extracts exhibited dose-dependent effects on the microorganisms tested. The methanol extracts of I. gabonensis parts effectively inhibited the growth of Staphylococcus aureus, Streptococcus viridians, Escherichia coli, Pseudomona saeruginosa, Salmonella enterica, Shigella sonnei, Aspergillus niger, Aspergillus flavus, Penicillium chrysogenum, Fusarium oxysporum, and Rhizopus stolonifer. These extracts, therefore, showedgood antibacterial and antifungal activities at different concentrations in vitro. Hence, the pharmaceutical application of these plant parts' antimicrobial propertiesissuggested.

Keywords: Plant chemicals, Antifungal agent, Antibacterial, Pant extracts, Anti-staphylococcal , Anti-streptococcal

Introduction

Irvingia is a dicot genus of the family, Irvingiaceae. This is a family of flowering plants, consisting of 13 species distributed in three genera, Allantospermum, Irvingia, and Klainedoxa [1]. The genus comprises seven species, Irvingia gabonensis (Aubry-Lecomte Ex O’rorke) Baill., I. grandifolia (Engl.) Engl., I.malayana Oliver ex. Bennett, I. robur Mildbr., I. smithii Hook.f., I. tenuinucleata Tiegh.and I. womboluVermoesen [2]. The botanic description of I. gabonensis was documented in Hutchinson and Dalziel [3]. The flowers are short, clustered, mostly axillary racemes, or subpaniculate. The pedicel is up to 10 mm long. The leaves are obovate-elliptic or, more or less cuneate or narrowly rounded at the base, shortly and broadly acuminate. The fruits are broadly, somewhat flattened, about 5‒6 cm long with smooth skin, fibrous exocarp, and hard endocarp.

Plants serve as medicine since ancient days. A wide range of phytochemicals that are traditionally classified as primary and secondary metabolites occur naturally in plants [4]. They dissolve in an array of solvents based on their nature [5]. Among the diverse uses ofsecondary metabolites is their function as a pharmacological active compound and they occur in various structural classes. The type and level of the biological active compounds in plants are responsible for their medicinal properties. Their concentrations in various plant parts vary [6, 7]. Their syntheses and accumulations are influenced by the environment and defense against herbivory [8].

Moreover, when bacteria form a parasitic association with other organisms, they are classified as pathogens. Pathogenic microorganisms cause human diseases and subsequent death. The plant extracts exhibit dose-dependent effects on the microorganisms [9, 10]. The level of inhibitory activity of a plant extract against pathogenic microorganisms determines the degree of its potency [11, 12]. Phytochemicals are the mechanisms that plants use to protect themselves against the effects of their pathogens [13]. Hence, constitutes the natural source of antimicrobial substances.

Furthermore, the tropical rainforest zone is rich in medicinal plants possessing a wide range of therapeutic potentials that are underutilized. Irvingia gabonensis is one of the medicinal plants that are underexploited. In addition, medicinal plants have been proven efficacious in the treatment of various diseases, and this has led to a boost in their search over the last two decades. The objectives of this study, therefore, were to screen the I. gabonensis leaf, stem bark, and ripe fruit peel for the presence of phytochemicals and as well as determine their antimicrobial activity.

Materials and Methods

Collection of Plant Material

The leaf, stem bark, and ripe fruit peel of I. gabonensis used in this work were collected in June from Ihioma, Imo State Nigeria. The samples were authenticated at the Herbarium of the Department of Botany, Nnamdi Azikiwe University, Awka, Nigeria, where the voucher specimen was deposited.

Preparation of Samples

The ripe fruits of I. gabonensis were peeled with a table knife. The leaf, stem bark, and ripe fruit peel were sliced with a table knife and then oven-dried (LDD906MF, Australia) at a temperature of 70^o C for 12 hours. The samples were then ground in a mortar with a pestle, and later into powdered form with an electric blender (Omega, USA). The powdered samples were then kept in an air-tight container before use.

Extraction of Plant Material

The methanol extracts of the plant were prepared by adding the powdered samples of the leaf, stem bark, and ripefruit peel in 100ml of methanol. The concentrations of the extracts were determined by adding 50g, 75g, 100g, and 150g to100ml of methanol. The whole setup was left for 24hours at room temperature and thereafter filtered using Whatman filter paper. The extract was then concentrated to 50ml, stored in an air-tight container, and kept in a refrigerator at 40^o C before use.

Qualitative Phytochemical Analysis

Qualitative tests were conducted using the standard methods described by Harborne [14], and their presence was denoted by a sign (+).

Quantitative Phytochemical Screening

The quantitative phytochemicaldeterminationsof the samples were carried out using standard procedures. Alkaloid, flavonoid, and steroid contents were determined by the gravimetric methods of Harborne [15]. The method of Ezeabara and Egwuoba [16] was used to determine the anthraquinone content. The tannin level of the samples was determined using the Folin-Dennis colorimetric method described by Kirk and Sawyer [17]. The method of AOAC [18] was used to determine the saponin content. Concentrations of phenols were determined using the Folin-cioCaltean colorimetric method [19]. The total terpenoid content of the plant specimen was determined by the method described by Ferguson [20].

Microbial Analysis

Preparation of Microorganisms for the Experiment

The pure culture of the microorganisms was obtained from the Pathology Department of the National Root Crop Research Institute, Umudike, Abia State, Nigeria. The bacteria isolates include Gram-positive: Staphylococcus aureus, and Streptococcus viridans, and the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, and Shigella sonnei. The fungi were Aspergillus niger, Aspergillus flavus, Penicillium chrysogenum, Fusarium oxysporum and Rhizopus stolonifer. The stock cultures of bacteria were sub-cultured in nutrient agar (NA) slants while mould on Sabour and Dextrose Agar (SDA) slants and stored at 4^oC.

Antimicrobial Test Procedures Preparation of Stock Solution

The initial concentration of each plant extract (5 g) was diluted with 50 ml of methanol to obtain the stock culture. Moreover, 100, 150, 200, and 250mg/ml concentrations were obtained from the stock culture and stored atroom temperature before use.

Antimicrobial Susceptibility Testing

The test organismswere checked for susceptibility to the herbal extracts by carrying out antimicrobial screeningusing the extracts and by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). A measured 20ml of sterile nutrient agar was poured into the sterile Petridish and allowed to gel. The surface was flooded with 2ml of 18 hours broth culture standardized according to the National Committee for Clinic Laboratory Standard [21] by gradually adding normal saline to compare its turbidity to the McFarland standard of 0.5 which is approximately1.0x10 cfu/ml. The surface was allowed to dry and a sterile No.4 Cork borer was used to bore six holes of 2.5cm equal in size on the surface. A measured 0.1ml of the extracts at different concentrations of 6.25%w/v, 12.5%w/v, 25%w/v, 50%w/v, and 100%w/v wasdropped into each hole and the plate was kept for an hour at room temperature and incubated at 37°C for 18hours. The diameter of zones of inhibition was measured after incubation to the nearest millimeter (mm). The experiment was repeated three times and the mean diameter was taken. The effects of the extracts on bacteria and fungi pathogens were compared with those of the standard antibiotics, amoxicillin, and fungabacter for bacteria and fungi respectively as standard controls.

Statistical Analysis

Analysis of Variance (ANOVA) using SPSS version 21 was used in analyzing the data collected from the study. Duncan’s Multiple Range Test (DMRT) was used to measure the test of significance, and the data were expressed as mean±standard deviation of triplicate determinations.

Results and Discussion

All the phytochemicals tested were present in the I. gabonensis leaf, stem bark, and ripe fruit peel (Table 1). Hence, the plant parts are filled with bioactive compounds that have powerful health benefits. Alkaloids, flavonoids, saponins, tannins, and terpenoids occurred athigh levels in all the plant parts while low concentrations of anthraquinones, phenols, and steroids were detected, especially in the ripe fruit peel. This finding allied with the report of the previous study, where relatively high levels of alkaloids, flavonoids, saponins, tannins, and terpenoidsas well as low values of phenols and steroids were also detected in the leaf and stem bark of this plant [22]. The highest percentage of saponins (1.67±0.03) and tannins (1.41±0.04) were found in the leaf. The alkaloid concentrations followed a pattern that the ripe fruit peel>leaf>stem bark. Alkaloids are found in approximately 20% of plant species [23]. Traditionally, the stem bark of this plant is used as a pain relieverin Sierra Leone [24]. This is probably a result of the analgesic property of alkaloids. Hence, the I.gabonensis stem bark extract could be regarded as an effective painkiller.

Table 1. Mean quantitative phytochemical composition of the leaf, stem bark, and fruit peel of Irvingiagabonensis

Results are in Mean±Std of triplicate determinations. Means with the same letter in a column are not significantly different (p>0.05)

On the antimicrobial activity investigation, the crude extracts showed varied levels of activityagainst the microorganisms tested (Table 2). The degree of the inhibitory activity of the leaf and ripe fruit peel wasintenser when compared with that of the stem bark against all the test microorganisms. The extent of the effectiveness was dependent on the level of the concentrations of the plant extracts. Escherichia coli, P. aeruginosa, Salmonella enterica, Shigella sonnei, Staphylococcusaureus, and Streptococcus viridiansare disease-causative agents. Pseudomonas and E. coli are among the most critical group of threatening multidrug-resistant bacteria in hospitals, nursing homes, and among patients whose care requires devices such as ventilators and blood catheters [25]. Besides, severe and often lethal infections such as bloodstream infections and pneumonia can be attributed to them. Traditionally, the decoction of the I. gabonesis stem bark is used in treating gonorrhea, liver, and gastrointestinal disorders, in Senegal [26]. Moreover, decreased gastrointestinal motility and protection against diarrhea were reported in animal studies administered with both aqueous and methanol leaf extracts of I. gabonesis [27, 28]. Therefore, the effectiveness of the leaf extract of this plant against E. coli (12.24±0.03 mm) and P. aeruginosa (12.56±0.03 mm) at the concentration of 250 Mg/mm could be the confirmation for the traditional use of the leaf for diarrhea and gastrointestinal disorder treatments. Furthermore, the antidiarrhoeal potentials of plant-based tannins have extensively been reported [29, 30]. Therefore, the high tannin level (1.41±0.04%) of the leaf extract could presumably be responsible for the antidiarrhoeal effect. The leaf extract of this plant was most effective against Salmonella enterica at 11.81±0.02 mm, Staphylococcusaureusat 13.22±0.02 mm, Streptococcus viridians at 13.63±0.01 mmwhen compared with the stem bark and ripe fruit peel extracts at 250Mg/ml concentration. Salmonella enterica is the cause of life-threatening systemic enteric fever [31]. Staphylococcus aureus was reported to be the second main pathogen for deaths associated with antimicrobial resistance in 2019 [32]. Besides, an estimated 20% to 30% of the human population are long-term carriers of S. aureus [33, 34]. Streptococcus viridians are associated with sepsis and pneumonia in the neutropenic host, and sepsis and meningitis in the neonate [35]. In addition, the I. gabonensis leaf extract showed the highest level of inhibition against Shigella sonnei at all the concentrations, except at150Mg/ml, where the highest value of the inhibition was 7.67±0.01mm in the ripe fruit peel extract. Alkaloids extracted from Sanguisorba officinalis L. also had antimicrobial qualities against P. aeruginosa and E. coli [36]. Moreover, the extracts of Trema guineensis (Schumach. & Thonn.) Ficalho, Phyllanthus discoideus (Baill) Mull.Arg. and Acalypha wikesiana Mull.Arg. that are traditionally used in South-West Nigeria also showed antimicrobial effects against E. coli and S. aureus [37]. Moreover, alkaloids, anthraquinones, flavonoids, saponins, and tannins were reported present in these plants. Similarly, the high activity of the I. gabonensis leaf extract against E. coli, P. aeruginosa, Salmonella enterica, Shigella sonnei, Streptococcus viridians, and Staphylococcus aureus may be attributed to the high concentrations of alkaloids (1.33±0.01%), flavonoids (1.62±0.01%), saponins (1.67±0.03%), tannins (1.41±0.04%) and terpenoids (1.61±0.02%). Hence, the effective antibacterial action of I.gabonensis leaf could be hugely due to the synergistic actions of these phytochemicals.

Table 2. Effects of methanol extracts of Irvingiagabonensis leaf, stem, and ripe fruit peel on bacterial pathogens

 Results are in Mean± Std of triplicate determinations. This means with the same letter in a column is not significantly different (p>0.05)

However, on the antifungal test, the effectiveness of I. gabonensis extracts against A. niger, P. chrysogenum, R. stolonifer, F. oxysporum, and A. favuswasdose-dependent (Table 3). All thesetest microorganisms are among the disease-causing fungi. The ripe fruit peel extract of I. gabonensis was the most active against A. niger at 12.32±0.00 mm, in comparison with the other parts. The aggressive nature of A. niger as a causative agent of pneumonia has been demonstrated [38]. In addition, the effectiveness of the extracts of the I. gabonensis parts against F. oxysporumfollowed a sequence that the leaf (12.27±0.01mm)>ripe fruit peel (12.18±0.00 mm)>stem bark (11.07±0.02 mm) at 250Mg/ml concentration. Fusarium species have long been associated withlocalized infections in immunocompetent individuals [39] and circulated infections among those who are severely immunocompromised [40]. Fusarium species infections regularly involve the skin, either as the primary or the metastatic site [41]. The inhibitory effect of the ripe fruit peel extract (14.60±0.03 mm) at 250Mg/ml concentration was most pronounced against P. chrysogenum while the least was the leaf extract (12.31±0.01mm). Penicillium chrysogenum is often identified in immunosuppressed patients, either due to human immunodeficiency virus or from immunosuppressant medications post-transplantation [42]. Therefore, it is a rare cause of infection in immunocompetent patients. The leaf and stembark extracts of I. gabonensis at 250Mg/ml, inhibited the growth of A. flavus (13.45±0.01 mm; 13.89±0.01 mm) and R. stolonifer (13.21±0.03 mm; 13.40±0.02 mm) respectively. The methanol leaf extract of I. gabonensis at 200mg/ml had a lesser inhibitory effect against A. flavus at 9.95±0.01 mm and R. stolonifer at 9.52±0.01 mm, when compared with the effect of the ethanol leaf extractof Dacryodes edulis (G. Don) H. J. Lam. in previous work [43]; where the antifungal activity against A. flavus and R. stolonifer were 13.58±0.0 mm and 13.60±0.00 mm respectively. Aspergillus flavus is the second most common (approximately 15‒20%) causative agent of invasive Aspergillus infections [44]. The test organisms exhibited different patterns of susceptibility to the I. gabonensis extracts at different concentrations. The methanol leaf, stem bark, and ripe fruit peel extracts of this plant could, therefore, be regarded as effective antifungals.

Table 3. Effects of methanol extracts of Irvingiagabonensis leaf, stem, and ripe fruit peel on fungal pathogens

Results are in Mean±Std of three different determinations. The same letter in a column is not significantly different (p>0.05).

Conclusion

The levels of alkaloids, flavonoids, saponins, tannins, and terpenoids were high in these parts of I. gabonensis; hence, this plant could be regarded as a rich source of them. The findings of this study suggested that all the compounds detected in the parts of I. gabonensis have antimicrobial effects. Hence, the extracts displayed various degrees of antibacterial and antifungal effects against all the test microorganisms, in vitro. Moreover, the effectiveness of theseplant extracts increases with the increase in concentrations. The anti-staphylococcal of the leaf extract and the anti-streptococcalactivities of the leaf and ripe fruit peel extracts of this plant can be further explored.

Acknowledgments: We heartly appreciate Mr Afam Ikpeama, National Root Crop Research Institute, Umudike, Abia State, Nigeria, for his technical assistance.

Conflict of interest: None

Financial support: None

Ethics statement: None

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