Phytochemical screening, gas chromatograph/mass spectrometer (GCMS) analysis and molecular toxicological potential of Hunteria umbellata aqueous fruit extract against Staphylococcus aureus in accessory gene regulators (AGRs)

Abstract

Background

Fruits of Hunteria umbellata (HU) have been utilized in folk medicine as potent against Staphylococcus aureus (SA) infections, particularly skin and nasal related conditions. However, there is scarcity of literature concerning toxicological evaluation of graded doses of HU fruit, especially at molecular level, specifically targeting the accessory gene regulator (AGR) system to prevent abuse of doses in the treatment of bacterial infections. This research evaluated molecular toxicological property of SA exposed to varied concentrations of aqueous HU fruit extract ranging from 500 to 3.90625 µg/µL using broth microdilution method and quantification of AGR I and II genes’ expression employing two-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). We first identified quality and quantity of chemical compounds in aqueous fruit of HU using phytochemistry and hybrid gas chromatograph–mass spectrometer (GC–MS) technique. Additionally, preliminary bactericidal potential of HU was assessed before molecular toxicology.

Results

Results revealed six phytochemicals and twenty analytical grade compounds from a standard library were identified from chromatograms of HU fruit extract. Some main compounds detected are n-Hexadecanoic acid (25.24%), 2-Pentadecanone, 6,10,14-trimethyl (16.08%), Cuparene (16.63%), Tetradecanoic acid (6.21%) and 9-Octadecenoic acid, (E)- (5.70%). Bactericidal activity shows significant (p < 0.05) toxicity in the tested (8) concentrations of HU fruits in a dose-response relationship compared to the controls (positive and negative). The quantified expression of AGR I and II genes in SA was most significantly increased (p < 0.05) at both 250 and 500 µg/µL of HU fruit extract while least significant increase (p < 0.05) was recorded at 125 µg/µL compared to control.

Conclusions

Notably, the study highlighted a potential risk of augmented bacterial infection especially with higher doses of HU extracts during boils’ treatment and other epidermal infections instigated by Staph. Expression of both AGR genes at higher doses (250 and 500 µg/µL) is indicative of further expression of several other genes responsible for virulence factors in a variety of human bacterial infections. Thus, consumption of HU fruit to treat boils and blisters should be with great caution especially at high doses.

Background

The emerging integration of modern molecular technology in toxicological evaluation of medicinal plants has become an integral part of decoding progression of infectious diseases and possible development of promising drugs and vaccines. This is because quantitative molecular technique such as RT-qPCR used for gene expression profiling from targeted bacterial will create an intrinsic understanding of the roles of virulence genes in disease pathogenicity [31]. Staphylococcus aureus (SA) emerges as one out of five leading gram-negative antibiotic-resistant bacteria causing death globally with the highest prevalence in sub-Saharan Africa super-region as recently documented by antimicrobial resistance collaborators [25]. Antibiotic-resistance of S. aureus may be associated with acquisition of novel virulency via one lateral gene transfer event or more, and subsequently modifies an earlier nonpathogenic strain to a hypervirulent and/or multidrug resistant [23]. Currently, there is a focus on development of preventive alternate strategies (vaccination and phage therapy) through AGR regulatory genes to forestall occurrence of staphylococcal infections [8]. The regulation of AGR has been described to be imperative in the development of staphylococcal infections involving formation of biofilm [29]. Recurrent Staph. infection has become an important source of health loss through colonization of the skin and nostrils of human host [27]. The frequency of infections and persistent evasion of most antimicrobials of SA colonizing strain has led to alternative treatment in traditional medicine by means of various parts of medicinal plants’ parts, especially in Africa. Most communities in Africa including Nigeria still rely on indigenous herbs as most sourced health treatments since these plants are easily accessible and affordable [38]. Hunteria umbellata (HU) fruit has been popularly employed in folk medicines without satisfactory doses for the management and treatment of many disease conditions caused by pathogenic bacterial in humans.

Although, several therapeutic potentials of many parts have been highlighted in the literature [37, 39]. Hunteria umbellata have been extensively harnessed in traditional herbal medicine as anti-diabetic potent agents with hyperlipidaemic activities [6], natural erectogenic and antioxidant extracts [35]. However, under exploitation of the in vitro molecular toxicity of varying doses from low to high of HU fruit in SA still persists. Hence, this study design was necessitated to precisely offer evidence based scientific findings on in vitro molecular toxicology in virulent genes’ expression of SA as the causative agent of boils when treated with HU fruit extracts. Additionally, to establish basis for possible mechanisms of response underlying medicinal plant-resistance as implicated in noxious exhibition in human staphylococcal communicable diseases.

Methods

Preparation of fruit extract

The HU fruit samples which were purchased in the second month (February) of the year 2023 from a local resident marketplace in Dopemu, Lagos State, Nigeria (6° 07′ N to 7° 00′ N latitude and 3° 43′ E to 4° 00′ E longitude) had been identified, authenticated and assigned voucher LUH 8996 as earlier illustrated by Salisu et al. [43]. Extraction employed was modified using the method of maceration [2]. The process involved preparing the fresh fruits by washing, slicing, oven-drying at 40 °C and grinding into a powder using electronic milling machine grinder, Lab. Mill, Serial No. 4745, Christy and Norris Ltd. The ground fruit (254 g) was macerated in 1.4 L of absolute sterile water within 72 h. A thin cloth and cotton wool were used to filter homogenate to get rid of every possible residue. The filtrate was placed in a fan assisted oven at 20 °C to dry. The aqueous extract (paste like form) yielded 3.95 g was stored in glassware and used for further analysis.

Qualitative phytochemical examination

Crude aqueous extract (20 mg) from HU fruit was mixed gently with 6 ml of 1% HCl, warmed, filtered, then tested for availability of alkaloids, anthraquinone and cardiac glycosides, saponins, phenol, flavonoids, tannins, phyloba-tannins, terpenoids as well as steroids. The presence (+) and absence (−) of tested phytochemicals were observed and recorded.

Test for alkaloids

Wagner’s assay

Dilute HCl and Wagner’s reagent (1 ml) were added to fruit filtrate (3 ml) and shook well. Afterward, colored precipitate (reddish–brown) was formed as the presence of alkaloids [40].

Tests for glycosides

Borntrager’s test of anthraquinone glycoside

Addition of 5% H₂SO₄ (1 ml) to fruit extract solution (1 ml) made a mixture that was boiled, filtered, shaken with the same volume of chloroform and kept to still for 5 min. Then the lower layer of chloroform was shaken with half of its volume with dilute ammonia. Nonappearance of bright red coloration of ammoniacal layer indicates absence of anthraquinone glycosides in the fruit [40].

Keller–Killiani test of cardiac glycoside

A small amount of fruit extract (5 mg) were treated with anhydrous acetic acid (1 ml) and few drops of ferric chloride solution in a test tube. Then, concentrated sulphuric acid (2 ml) was added cautiously to prepared mixture. An observed reddish–brown ring at the junction of two layers and development of aqua at the lower layer indicated cardiac glycosides existence [45].

Test for saponins

A mixture of fruit sample (1 g) and distilled water (10 ml) was prepared, boiled for 5 min and shaken vigorously and observed in a test tube. Absence of froths indicates lack of saponins [15].

Test for phenol

A few fruits sample (1 g), ethanol (10 ml) and three drops of phenol solution were mixed in a labeled test tube. Nonappearance of a deep green coloration specifies absence of phenol [16].

Test for flavonoids

A mixture of HU fruit extract (1 g), 10 ml of ethanol and 2 drops of FeCl₃ was prepared. Observed dark green color specifies flavonoid [15].

Test for tannins

Decoction of fruit extract (1 g) with 10 ml of distilled water was prepared by boiling for 10 min, filtered, cooled and mixed with 0.1% ferric chloride reagent. An observed blue–black precipitate implies tannins availability as delineated in a modified method of Banso and Adeyemo [10].

Test for phyloba-tannins

Aqueous hydrochloric acid (1 ml) was stirred with fruit extract (2 ml), boiled and allowed to cool down. Availability of phyloba-tannins was implicated in precipitate (red) observed. [10].

Test for terpenoids

Some concentrated sulphuric acids (5 ml) were gently added to fruit extract sample (0.5 g) in a test tube. Observation revealed reddish brown coloration indicating terponoids’ presence [9].

Salkowski test for steroids

The crude fruit extracts (2 mg each) were dissolved with chloroform (2 ml) and mixed gently by shaking to form a mixture. Oil of vitriol (2 ml) was poured in prepared mixture to observe red topmost layer in the test tube. This indicates existence of terpenoid [9].

Gas chromatograph/mass spectrometer (GCMS) analysis of bioactive components of Hunteria umbellata fruits

Gas Chromatograph/Mass Spectrometer (GCMS) analysis as reported earlier [42] was carried out in Central Research Laboratory, University of Lagos Akoka, using 7890A Gas chromatography system attached to VL/MSD 5975C mass spectrometer (GC–MS Agilent Technologies, Santa Clara, USA) instrument employing the following settings: Column HP5MS fused silica capillary column [30 m (length) × 0.32 mm (diameter) × 0.25 μm (film thickness)] composed of 100% dimethyl polysiloxane). 1 g of ground fruit samples was dissolved in 10 ml chromatographic grade methanol, filtered by using syringe filter and the clear sample was later injected in GCMS column. Helium gas (99.9999%) was used as the carrier gas at constant flow rate of 1 ml/min and an injection volume of 1 μl was employed with injector temperature at 250 °C and pressure at 8.802 psi. The oven temperature was programmed originally from 80 °C (held for 2 min) with an increase of 5–120 °C/min., then 10–240 °C/min., to hold for 6 min. The total GC running time for the fruit sample was 24 min. The area under a peak accurately represents the quantity of the component present in the sample. Software adopted to handle mass spectra and chromatogram was a ChemStation. The interpretation on mass spectrum of GCMS was done by means of the database of National Institute of Standard and Technology (NIST) version 2, year 2015 library. The mass spectrum of the unknown components was compared with the spectrum of the known components stored in the NIST library to establish the name, molecular weight and structure of the components of the fruit extract.

Collection of bacterial isolates and antibacterial assay

A gram-positive organism, S. aureus strain MBSA2208, was used for this study. The bacteria isolate was obtained from the Department of microbiology, Nigerian Institute of Medical Research (NIMR), plated on salt agar (Mannitol) and then brooded (at 40 °C for 24 h) to obtain pure culture. Antibacterial activity of HU fruit sample was tested by method of broth micro-dilution. A sterilized well (96) microplate was prepared for the experiment. Each well was filled with 90 µL of nutrient broth. In the standard wells, 10 µL of 1 mg/ml vancomycin (a susceptible antibiotic) was added, while the control well, received 10 µL of sterile broth as a sterility control [44]. Minimal inhibitory concentration (MIC) was evaluated by preparation of 0.4 µL of a stock concentration HU extract (250 mg/ml) to arrive at a working solution of 500 µg/µL. This working solution was then serially diluted to obtain two-fold dilutions. The resulting concentrations in column 3 to 11 were 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, and 3.90625 µg/µL. Each well, except for the blank well in column 12, was filled up to 200 µL with nutrient broth that had contained bacteria already inoculated at a standard density of 0.5 McFarland, equivalent to 1.5 × 10⁸ (CFU/mL). This setup allowed for the estimation of the bacterial activity of fruit extract by observing a noticeable growth or inhibition of the bacteria in each well of the microplate. After setting up the microplate with the appropriate concentrations of the extract, standard antibiotic and control. The incubation of the plate was done at 37 °C on a shaker in not more than 24 h. After incubation period, measurements of absorption values in each well with microplate reader at 600 nm absorbance were recorded. This measurement allowed for the determination of the growth difference between the wells before and after incubation, indicating the effectiveness of the extract in inhibiting bacterial growth.

To determine the point of inhibition for each extract, preparation of resazurin dye (0.8 mg/ml) was done. After the 24-h incubation period, A small volume of prepared resazurin solution (30 µL) was dropped in individual well of microplate. Resazurin is a dye that changes color from blue to pink in the presence of oxygen. The microplate was then incubated for an additional 3 h to allow the color change to occur. This color change visually indicated microbes’ growth inhibition due to smallest amount of HU fruit extract in each well.

RNA extraction and purification

For the extraction of single-stranded RNA from the aqueous extracts of H. umbellata, three concentrations were chosen: the highest (500 µg/µL), a moderate (250 µg/µL) and the lowest (125 µg/µL). The extraction was performed via the NIMR Biotech extraction kit according to manufacturer’s description.

The extraction process involved several steps. First, the cells in the extracts were lysed to disrupt cell membranes and make RNA available freely. Lysed samples were then incubated to facilitate the extraction process. After incubation, RNA precipitation was carried out to isolate the RNA from other cellular components. The cell debris and impurities were removed through a washing step to obtain a purified RNA sample. The elution step utilized a spin column-based method, where the purified RNA was eluted from the column, leaving behind any contaminants.

Finally, the extracted single-stranded RNA was stored at − 20 °C until further use. This freezing temperature helps maintain the stability and integrity of the RNA for future analysis or experiments.

Dual-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)

A separate reverse transcription coupled with qPCR was carried out. First, extracted RNA was converted to complementary DNA (cDNA). Synthesis mix of FIREScript RT cDNA (Solis Biodyne, Estonia) was used, alongside random primers and Oligo (DT). Prepared reaction mixture consisted of 6.8 µl of double-distilled, molecular-grade water, 2 µl of mastermix, 0.1 µl each of forward and reverse primers in Table 1 as manufactured by the NIMR-MTN Oligo Synthesis Laboratory and 1 µl of the RNA template. These components were combined to create a 10 µl reaction mixture as followed by manufacturer’s manual. Transcription (reverse) process took place for a duration of 30 min at a temperature of 50 °C. After 5 min, transcriptase enzyme was deactivated at a lesser temperature of 85 °C using a BioRad thermal cycler.

Table 1 List of Nucleotide Primers designed for Virulence Genes in Real-Time PCR Technique

The second step was the quantitative Polymerase chain reaction (PCR). This reaction was executed to quantify amplifications of expressed genes. The number of amplifications of virulence genes (AGR I and II genes) and the house keeping gene (SA442) of S. aureus used in this study was determined using a real-time system (BioRad CFX96 Deepwell), in accordance with the manufacturer’s description. A 10 µl reaction mixture was prepared containing 5.8 µl double distilled, nuclease free water, 2 µl Solis Biodyne Eva green mastermix, 0.1 µl each of both forward and reverse primers and 2 µl template coding deoxyribonucleic acid (cDNA) for the two primers separately. The processes involved are; initial and final denaturation, annealing, initial and final extension. First denaturation (DNA unwinding) occurred at 95 °C for 2 min, final unwinding took place for just 30secs at the same temperature as the first melting of DNA, annealing was set for 30secs at 54 °C and repeated for 40cycles followed by extension step with temperature range of 55–80 °C with 0.5 °C increment for 5secs.

Ct values (cycle threshold) were generated at the completion of the reaction and used to calculate relative quantification 2^{−(∆∆ct)}.

Data analysis

The cycle threshold (ct value) generated from the melt curves plotted by the thermal cycler was used to calculate relative expression using Microsoft Excel (2016 version). Relative expression values were further subjected to IBM SPSS statistics version 22, to determine the increment or decrease in expression of the virulence genes (AGR I and II). All data entries were given as descriptive average alongside standard error means (mean ± SEM), mean difference was compared using a one-way ANOVA analysis and a post-hoc test of LSD (least significance difference). A confidence interval of 95% was used as the criterion for significance to show variations between compared sets.

Results

Phytochemical analysis of chemical components of Hunteria umbellata fruits

The screening of phytochemicals in HU fruit, aqueous extracts revealed six (6) phytochemicals which include specific secondary metabolites like alkaloids, cardiac glycosides, flavonoids, tannins, phyloba-tannins and terpenoids. Four compounds (anthraquinone glycosides, saponins and steroids) were absent in the fruit (see Table 2.)

Table 2 Presence (+ ) & absence (−) of Phytochemicals in Hunteria umbellata Fruit

Bioactive components of H. umbellata fruit from chromatograms of GC–MS

Twenty (20) non-polar volatile compounds appeared in HU fruit (methanol extract) as analyzed from GCMS chromatograms in Fig. 1 below. Individual peak denotes a compound with dissimilar quality and quantity based on percentage by ChemStation's calibration mode (Software). All compounds identified with their molecular formula, molecular weight (MW), retention time (RT), area percentage composition (quantity), quality (matching factor > 80) and chemical structure of fruit extract are presented in Table 3. The presence of compounds that combine both the matching factor (> 80) and percentage composition (> 5%) were classified as main compounds while others below both quality (matching factor > 80) and quantity (> 5%) were regarded as minor compounds. The five major compounds (Table 4) detected in HU fruit are n-Hexadecanoic acid (99; 25.2%), 2-Pentadecanone, 6,10,14-trimethyl (96;16.1%), Cuparene (95; 6.6%), Tetradecanoic acid (95; 6.2%) and 9-Octadecenoic acid, (E)- (83; 5.7%). The 15 minor compounds comprise 1-Methylenespiro [2.4] heptan-4-one (43; 1.1%), 2-Butynedioic acid, di-2-propenyl ester (49; 2.1%), Phthalic acid, butyl oct-3-yl ester (86: 2.2%), 7-Hexadecene, (Z)- (97; 2.6%), camphor (42: 1.6%), Methyl 14-methylpentadecanoate (97: 3.3%), 1-Octadecene (97: 4.7%), Methyl linoleate (95: 1.1%), Methyl elaidate (99: 1.4%), Bromoacetic acid, octadecyl ester (49: 1.76%), Cyclohexaneethanol, 4-methyl-.beta.-methylene-, trans-(45: 5.8%), Octadecanoic acid (70: 2.31%) and 1-Octadecene (97: 2.5%).

Fig. 1

Total Ion GCMS Chromatogram of methanol fruit extract of Hunteria umbellata

Table 3 Compounds recognized in methanol fruit extract of H. umbellatta

Table 4 Nature and reported bioactivity of the major phyto-compounds in methanol fruit extract of H. umbellatta

Bactericidal property of aqueous Hunteria umbellata fruit extract on Staphylococcus aureus

Result obtained from the antibacterial assay showed that Vancomycin, a standard drug for Staphylococcus infection greatly displayed repressive properties on proliferation of SA. None of eight tested concentrations of H. umbellata with 500 µg/µL being the highest stalled the growth of S. aureus in comparison to control as shown below (Fig. 2). This was confirmed in all the wells containing resazurin that changed from blue to pink. The change of resazurin from blue indicates the presence of viable organisms (producing oxygen).

Fig. 2

Graphical representation of Microbial growth in Hunteria umbellata fruit extract before and after 24 h of incubation

Amplification of AGR I and II genes’ expression in SA predisposed to three selected doses of HU fruit extracts

The amplification and melt curves of the two AGR genes in SA exposed to aqueous HU fruit extracts were provided in Figs. 3 and 4 respectively. The amplification curves are indicative of the binding of DNA to SYBR green fluorescent dye present in the master mix which started after the 20th cycles in AGR I and 30th cycle in AGR II of real time PCR. The melt curves began after 60 °C for AGR I and 75 °C for AGR II.

Fig. 3

Amplification curves of AGR (I and II) genes of SA treated with HU fruit extracts. RFU represent relative fluorescence units

Fig. 4

Melt curves of AGR (I and II) genes of SA treated with HU fruit extracts. RFU represent relative fluorescence units

Quantification of AGR I and II gene in Staphylococcus aureus after exposure to HU aqueous fruit extract

The result obtained after the quantitative real time PCR showed increased expression of AGR I and II genes at 500 and 125 µg/µL compared to control. However, AGR I and II genes were more significantly (p < 0.05) expressed at 250 µg/µL of H. umbellata aqueous fruit extract relative to the other two doses and control. (see Fig. 5).

Fig. 5

Graphical representation of AGR I gene expression of Staphylococcus aureus exposed to Hunteria umbellata aqueous fruit extract

Discussion

The safety profile of certain plants, their potential therapeutic benefits and considerations of toxicity effect have been criticized as a result of ambiguity in treatment prescriptions by indigenous traditional healers. This is due to ancestral believe in guaranteed safety with no side effect in any consumption of most parts of medicinal plants without adequate doses [36]. Regrettably, this supposition has translated to numerous cases of health complications and in some cases demise of the users [21]. This often results to patients overdosing of herbs and herbal products, as regulatory bodies are totally not in control over the usage of herbal medicines. Despite the goodness of herbal medication, certain plant species have been documented to exhibit cytotoxic effects at elevated doses. The implication is that a whole plant or its parts considered highly safe can become toxic at elevated doses. Serious concerns related to potential toxicity-related to phytomedicine or phytotherapy including issues like carcer-inducing effect, liver toxicity, mutation inducement and genetic damage have been brought to the forefront [30]. Therefore, prevailing perception among users of herbal medicines that these remedies are inherently safe without adverse effects due to their natural origins in the plant kingdom calls for serious concern [32].

Secondary metabolites of different parts of HU have consistently been documented in therapeutic intervention or management of numerous ailments. The results of phytochemical screening of HU fruit revealed certain specialized metabolites including alkaloids, cardiac glycosides, flavonoids, tannins, phyloba-tannins and terpenoids. This result is in accordance with the compounds found in HU methanol seed as well as their known bioactivities as reported by Salami and Ladokun [41], Aderele et al. [5] and Akinrotoye et al. [7]. Another study reported 21 different compounds from chromatograms of both seed and leave methanol extracts [28]. The GCMS analysis from this study resolved 20 compounds from HU fruit, comprising five major compounds and 15 minor compounds based on quality and quantity. The major compounds are palmitic acid, aldehyde, sesquiterpene (terpene), myristic acid and stearic acid. Palmitic acid (n-hexadecanoic acid) constituted the most abundant with almost 100% matching factor (Table 4). Notably, maximum concentration of 50 µg/ml, n-hexadecanoic acid has established only modest antagonistic bacterial activity against S. aureus, as specified by Ganesan et al. [22]. This means overdose of plant rich in palmitic acid in the treatment of bacterial infection could be lethal. Void activity of fatty aldehydes against clinical pathogenic bacteria has been documented by Xie et al. [49] while literature has described terpenes as promising antibacterial especially in multiple drug resistance (MDR) bacterial strains [20, 33]. Though the precise molecular machinery responsible for the antibacterial effects of terpenes are yet to be fully understood. Most plant parts possess fatty acids (FA) as good sources of carbon with structural functions and bactericidal properties [14]. However, sublethal concentrations of some of these fatty acids serve as signaling molecules in modulating bacterial virulence as explained by Cortes-López et al. [17]. Despite the antibacterial activity of most fatty acids, palmitic, myristic and stearic acids have been associated with increase in pathogenicity of bacteria [26]. Although their probable impact on the effectiveness of anti-virulence treatments coupled with participation in intricate network of pathogenicity regulation are increasingly recognized, representing a promising strategy to manage antibiotic-resistant bacterial diseases.

Several reports have shown that Hunteria umbellata, possesses antagonistic bactericidal efficacy against the two major representative of bacteria (gram positive and negative), S. aureus and E. coli bacteria respectively, at doses less than 150 mg/ml [5, 7]. Contrarily to these reports, our findings revealed negative antibacterial activity of H. umbellata aqueous fruit extract against S. aureus tested. Increase in growth was recorded in S. aureus, the causative agent of these staph infections, when it was exposed to H. umbellata aqueous fruit extract at doses ranging from 3.9 to 500 µg/µL. The increment could be indicative that the extract supports the growth of Staph infections in the body. This difference could also be attributed to the growing evolution and resistance of pathogenic bacteria [18, 34]. Nevertheless, our study corroborates minimal and inconsequential inhibitory effects of the fruit pulp (water extract) noted against SA in old fact-findings of Igbe et al. [24] as reviewed by Fadahunsi et al. [19].

The expression levels of gene have been generally regarded as an excellent match for protein expression and thus be harnessed to deduce expression of either protein and/or virulence susceptibility. Equivalent data will offer more enlightened strategy for the choice of medicinal plants for the development of drugs to manage persistent evaded pathogenic processes. Our previous study has established increase in the amount of Laf A and ExsE genes’ expressions in, as a main causative agent of gastroenteritis, when exposed to high dosages of both H. umbellata seed and stem extracts [43]. This present study further evaluated the AGR system of S. aureus which is a vital component underlying numerous genes’ expression that code for virulence tendencies, formation of biofilms during the pathogenicity of bacterium and inducement of both type 2 and interleukin-17-dependent epidermal inflammation in host cell [47]. Significant increase in expression of both AGR genes of SA exposed to the three tested doses (500, 250 and 125 µg/µL) is in accordance with expression of AGR 1 and II which has been ascribed to invasive infection like bacteraemia [12].

Every pathogen has virulence genes associated with them, these genes are critical for triggering diseases when the host has been colonized successfully to cause bacterial infections [48]. Our studies revealed persistent increase of AGR I gene followed by AGR II gene in the tested doses as compared to control, in agreement with other study that both genes are the most common allelic groups of AGR system [1]. The AGR I system is often connected with strains of S. aureus which produces the Panton-Valentine leukocidin (PVL-SA) toxin, commonly implicated in necrotic skin infection and persistent cellulitis [13]. AGR II is frequently found in antibiotic (methicillin) resilient S. aureus strains, linked with hospital—acquired infections [46]. Variation in amplification peaks during quantification of the AGR genes revealed difference in expression of AGR genes in the three tested concentrations of the extract (500, 250 and 125 µg/µL) compared to normal control. This further indicates the presence of the genes in the S. aureus. AGR I and II genes were expressed significantly (p < 0.05) at 250 µg/µL and 500 µg/µL doses while dose 125 µg/µL significantly decreased (p < 0.05) genes’ expression. This suggests that AGR I and II genes would be expressed as doses of H. umbellata fruit extract increases. However, this molecular toxicity at genetic level is scarce in the literature, as most studies have been focused on the antibacterial activity of H. umbellata aqueous fruit extract. Until this present study, there isn’t any recorded side effects of H. umbellata at molecular level. Adeneye et al. [4] indicated in his study that HU exhibits a notably low level of oral toxicity. That is, consumption of the HU fruit is not dangerous or toxic. Yet, this study confirms the expression of both types of AGR genes (I and II) as the colonization factor for S. aureus in the development of staphylococcal related infections. These findings affirmed the importance of molecular toxicity especially at gene and protein levels to validate conventional and modern therapeutics in either prevention or treatment of microbial infectious diseases globally. This will further elucidate the toxicity pathways underlying antimicrobial resistance associated with virulence.

Conclusion

Consumption of indiscriminate dosages of H. umbellata extracts may aggravate the staphylococcal diseases such as boils, abscess and staph food poisoning. Hence, H. umbellata fruit extract maybe be dangerous to health when consumed in high quantities.

Recommendation

Extensive in vitro studies should be conducted on different herbal plants and concoctions to verify their antibacterial activities. Most especially, molecular toxicity tests must be incorporated in the toxicology of medicinal plants to fully ascertain its safety and safe dosage. In addition, enhancing awareness about potential health risks linked to the utilization or excessive use of medicinal plant parts and quality control guidelines should be encouraged and enforced on traditional use of herbal plants in any disease management.