Chemical Composition and Antimicrobial Activities of Essential Oil from the Leaves of Acalypha wilkesiana on Pathogenic Microorganisms

Acalypha wilkesiana is a tropical plant used to treat a wide range of medical conditions. The leaves of A. wilkesiana were tested for phytochemicals with microbicide activity against a variety of microorganisms known to cause severe human infections. Antimicrobial efficacy of essential oil extracts from pulverised dried leaves of A. wilkesiana was examined. Phytochemical and proximate assessments of the leaves were carried out using standard techniques. The essential oil was extracted using hydro-distillation and collected using two processes. The first form was extraction for four hours straight, and the second mode involve hourly collection for four times. A total of five fractions of the essential oil were extracted. The chemical constituent of the oil was separated with Gas chromatography–mass spectrometry (GC-MS) in accordance with standard procedures. The proximate composition of leaves showed low moisture and high carbohydrate (61.39%). Tannins, flavonoids, and alkaloids were among the secondary metabolites found in the leaves. Six bioactive compounds including n-Hexadecanoic acid and 6-Benzamido-4-benzoyl-1, 2, 4-triazine-3, 5 were identified in the GC–MS analysis. The oils from A. wilkesiana leaves have excellent broad-spectrum antimicrobial activity at various concentrations on Staphylococcus species, Bacillus coagulans, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Candida albicans. It could not, however, inhibit the growth of Pseudomonas aeruginosa and Gardnerella vaginalis. The findings justify the usage of A. wilkesiana leaves in ethnomedicine for possible management of skin conditions, gastrointestinal problems as well as other related microbial infections.


INTRODUCTION
Globally, plants are used primarily in health care, either in crude or refined forms (Cordell, 2009;Sofowora et al., 2013). They have contributed significantly to making the earth habitable for all living things, most especially man. Due to their ability to convert solar energy into metabolites, which are then turned into food and pharmaceutics for animal and human use; and have also become a valuable source of income and medications. Principally, plants contain bioactive substances such as flavonoids, alkaloids, tannins and other chemical compounds that have medicinal properties.
Acalypha wilkesiana is a tropical plant used to treat a wide range of medical conditions. The leaves of A. wilkesiana were tested for phytochemicals with microbicide activity against a variety of microorganisms known to cause severe human infections. Antimicrobial efficacy of essential oil extracts from pulverised dried leaves of A. wilkesiana was examined. Phytochemical and proximate assessments of the leaves were carried out using standard techniques. The essential oil was extracted using hydro-distillation and collected using two processes. The first form was extraction for four hours straight, and the second mode involve hourly collection for four times. A total of five fractions of the essential oil were extracted. The chemical constituent of the oil was separated with Gas chromatography-mass spectrometry (GC-MS) in accordance with standard procedures. The proximate composition of leaves showed low moisture and high carbohydrate (61.39%). Tannins, flavonoids, and alkaloids were among the secondary metabolites found in the leaves. Six bioactive compounds including n-Hexadecanoic acid and 6-Benzamido-4-benzoyl-1, 2, 4-triazine-3, 5 were identified in the GC-MS analysis. The oils from A. wilkesiana leaves have excellent broad-spectrum antimicrobial activity at various concentrations on Staphylococcus species, Bacillus coagulans, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Candida albicans. It could not, however, inhibit the growth of Pseudomonas aeruginosa and Gardnerella vaginalis. The findings justify the usage of A. wilkesiana leaves in ethnomedicine for possible management of skin conditions, gastrointestinal problems as well as other related microbial infections.
These plant-derived chemicals have prompted a shift in medical practices from restorative to preventative approaches, particularly as pathogens become more resistant to synthetic drugs.
Phytochemicals such as alkaloids and tannins have been extensively employed in the management of serious microbial infections. Vernonia amygdalina (bitter leaves), for example, has been shown in animal studies to have the ability to suppress the occurrence of diarrhea (Gudeta et al., 2020) and high antibacterial properties against clinical isolates of Enterobacter aerogenes, Escherichia coli and Salmonella typhi, (Ohwofasa and Pondei, 2018). Several studies have also shown that essential oils derived from a variety of plants could be interesting alternatives for the treatment of infectious diseases. Essential oils are volatile and aromatic compounds which may be obtained from various plant parts, particularly leaves, flowers and seeds. Lemon grass (Cymbopogon citratus) essential oils have been discovered to have antibiofilm and antibacterial properties against Candida species, Staphylococcus aureus, and Klebsiella pneumoniae (Naik et al., 2018;Gao et al., 2020). Ghavam and colleagues reported that essential oils derived from Salvia hydrangea flowers and leaves are effective in treating fungal and bacterial infections. (Ghavam et al., 2020).
The plants of the Acalypha genus belong to the family Euphorbiaceae (Elkhouly et al., 2017) and are usually cultivated as house plants because of their attractive colours. In addition, they are traditionally known to have remarkable medicinal values and nearly every component of the plant, such as the stems, roots, and leaves, is used as a therapeutic agent to treat and control a variety of diseases owing to their broad phytochemical constituents (Seebaluck et al., 2015;Asekunowo et al., 2019). Acalypha hispida and A. indica, for instance, possess interesting pharmacological active constituents such as proteins, crude fats, cyanogenic glucosides, acalyphine, triacetoneamine as well as alkaloids with therapeutic potentials and as such could be used in ethnomedicine to treat bacterial infections and other diseases. The screening of phyto-constituents of the leaves of A. wilkesiana (copper leaf) revealed the presence of flavonoid, carotenoids and alkaloids. These substances are thought to exert a significant role in managing the risk factors associated with cardiac infections. Likewise, the boiled decoction of the leaves has been suggested for the management of intestinal diseases, dermatitis caused by fungi including Tinea pedis, Tinea corporis, Candida intetrigo and Pityriasis versicolar (Ogundaini, 2005) and diabetes. Whilst most studies have focussed on the aqueous and methanolic extracts of A. wilkesiana leaves (Omage et al., 2018;Okoye and Amadi, 2019;Osibote et al., 2020), research on the potential benefits of its essential oil has been very limited. As a result, the current study is aimed at evaluating the antimicrobial activities of essential oil extracts from A. wilkesiana on pathogenic organisms and to identify potential bioactive constituents.

MATERIALS AND METHODS Plant Materials:
Fresh A. wilkesiana leaves were collected from a facility within the premises of University of Lagos in Nigeria. The plant was classified as well as verified at the Department of Botany of the University (Voucher No: LUH 6497) (Osibote et al., 2020). The leaves were air dried before processing into rough powdery texture.

Test Microorganisms:
Eleven strains of microorganisms which were maintained in glycerol-peptone broth at 4 o C were obtained from the bacteriology laboratory of the Microbiology Department, Faculty of Science of the University.
The While preliminary evidence of the proximate and phytochemical findings was given in a previous article (Osibote et al., 2020). The total carbohydrate, fiber, fat, crude protein and moisture content in addition to the fat and ash content of A. wilkesiana leaves were determined in triplicate using standard methods (AOAC, 2006;Iniaghe et al., 2009). Besides, the nutritional contents of the leaves of three different species of Acalypha (A. indica, A. marginata and A. hispida) were also determined. The phytochemicals of A. wilkesiana were identified using conventional procedures (Sofowora, 2008;Osibote et al., 2020).

Extraction of Essential Oil:
One hundred (100) gram of the powdered leaves was mixed with 3 L of distilled water and hydro-distillation was used to recover the essential oil, which was collected in hexane (Ogunlesi et al., 2010). The extraction was carried out in twofold: one for four hours at a time, with the extract retrieved every four hours, and another for four hours at a time, with the extract obtained every hour. Evaporation at room temperature separated the hexane from the essential oil and any remaining water in the extract was removed using anhydrous sodium sulphate. Essential Oil Antimicrobial Activity: The antibacterial and antifungal activities of the essential oil from the leaves of A. wilkesiana was analyzed using agar well diffusion method as previously described (Ochei and Kolharkar, 2008;Osibote et al., 2009). Using a colorimeter set to 540 nm, a suspension of each organism was prepared and calibrated to a concentration equivalent to 0.5 McFarland standards. 100µl the suspension was introduced into the sterile Muller Hinton agar in duplicate and spread evenly with sterile glass rod. The essential oils were poured into the wells drilled into the Mueller Hinton using an 8 mm cork borer. The inoculated plates were left standing for some minutes to allow the extracts to thoroughly dissipate. After 24 hours of incubation at 37 o C, the inoculated plates were examined for inhibition zones. For the bacterial organisms, imipenem (10µg) was used for quality control to determine the effectiveness of the oil against the isolates. The percentage sensitivity was calculated as follows: Diameter of zone of inhibition of the standard antibiotic disc

Gas chromatography-mass spectrometry (GC-MS) Analysis:
Profiling of the components of the essential oil was conducted as described previously Osibote et al., 2009;Osibote et al., 2020). The MS library identified the observed molecular ions (NIST MS search 2.0).

Minimum Inhibition Concentration (MIC):
Using the agar well dilution method, the minimum inhibitory concentration (MIC) of essential oil of A. wilkesiana was determined. Different concentrations of the essential oils were added to sterile Mueller Hintion agar plates inoculated with test organisms (Cheesbrough, 2013). The stock solution of the essential oil (extract E) was dissolved in hexane at varying concentrations ranging from 50 to 250 mg/ml. 100µl of each concentration was applied to the inoculated plates and incubated for 24 hrs. at 37 o C. Hexane was used as negative control. After an overnight incubation, the plates were examined for the lowest concentration that inhibited observable growth of the microorganisms, establishing the MIC.

Data Analysis:
The results of the phytochemical and proximate measurements were presented as the mean standard deviation. Microsoft Excel spreadsheet (version 2010) was used to create graphical representations of the results.

Phytochemical and Proximate Composition:
The basic secondary metabolites discovered were tannins, cardiac glycosides, anthraquinones, flavonoids, saponins and alkaloids. These are shown in Table 1. The results of the proximate study revealed that A. wilkesiana leaves possessed high carbohydrate (61.39%) and fiber (17.00%) but low moisture content (8.43%). The standard deviation for crude fiber ranges from 0.02 to 2.791 for crude fat. The percentage composition is depicted on Figure 1.

GC-MS Analysis of The Essential Oil:
All the extracts (A-E) of the essential oil of A. wilkesiana leaves had sharp herbal odour and light yellow colouration. The GC/MS analysis showed the existence of six bioactive compounds, including pyrrole, and 1,3,7-Octatriene with retention time of 23.57 and 23.92 mins. respectively (Table 2). n-Hexadecanoic acid was the prominent compound.

Antimicrobial Susceptibility Testing:
The antimicrobial susceptibility testing of the essential oil against some bacterial and fungal organisms revealed that the plant was very effective against S. aureus, S. typhimurium, B. coagulans and methicillin resistant S. aureus (MRSA) ( Table 3). The essential oil that was extracted for four hours stretch (extract E) was the most effective of all the extracts, more than some of the hourly extracts (A, B, C, D) (Table 3). However, it showed limited inhibitory activities against P. aeruginosa and G. vaginalis. Table 3: Antimicrobial Activity of essential oil from Acalypha wilkesiana leaves +: degree of inhibition (inhibition zone measured in millimeter); 1+: 5-9mm, 2+: 10-19 mm, 3+: > 20mm3+: indicates a very good antimicrobial activity, 2+: good antimicrobial activity and 1+: fair antimicrobial activity. -ND: not detected.

Minimum Inhibition Concentrations (MICs):
The essential oil showed high antimicrobial potential against S. aureus compared to the control. This was followed by S. typhimurium, B. coagulans and MRSA.
The MICs of most of the isolates were at the following concentrations: 20µg/ml (B. coagulans; K. pneumoniae), 40µg/ml (S. aureus) and 60µg/ml (MRSA; B. subtilis). All the essential oil (extracts A-E) were able to exhibit good inhibitory activities but extract E was the most effective since it had all the components that were present in extracts A-D (Table 4). Thus, the order of sensitivity of the essential oil extracts was E > A > B > C > D.  (Ikewuchi et al., 2010). Generally, the chemical composition of plants, regardless of the parts, depends on the species, geographical region, seasonal variation, and extraction process. The phytochemicals identified in this study have been shown to have health-promoting properties (Basu et al., 2007). Tannins, for example, have been shown to significantly aid in the repair of underlying tissue during wound healing (de Sousa et al., 2015). Flavonoids, another significant plant constituent, have antiinflammatory, anti-oxidative, anticarcinogenic and anti-mutagenic effects, as well as their ability to modify essential cellular enzyme activity (Beking and Vieira, 2010). In general, antioxidants protect against radiation damage and boost the immune system.
The present investigation also revealed that the leaves of A. wilkesiana contain high carbohydrate (61.39%) and low moisture (8.43%). The low moisture content can be beneficial in restricting the growth of microorganisms, shelf life and quality of the leaves. This is in agreement with the results of Adeyeye and Ayejuyo (1994). In addition, we found that the leaves of A. wilkesiana have very high crude fiber when compared with those of A. indica, A. hispida and A. marginata leaves. Studies have suggested that fiber intake can help to reduce the occurrence of certain diseases such as diabetes, obesity, hypertension and gastrointestinal problems (SACN, 2008). Fiber consumption could also protect the gut by increasing faecal mass, thus diluting elevated bile concentrations associated with high fat consumption (Dillard and German, 2000). Accordingly, the importance of A. wilkesiana in disease management is thereby acknowledged.
Our current work supported previous studies by confirming the antimicrobial activity of A. wilkesiana essential oil on the pathogenic organisms. The essential oils derived from the leaves of A. wilkesiana have potent antifungal properties and significant inhibitory effects on C. albicans, S. epidermidis, B. coagulans, S. typhimurium, K. pneumoniae and E. coli. Significantly, the inhibitory activity was more pronounced against S. aureus, S. typhimurium, B. coagulans and MRSA. These organisms have been implicated in cases of skin infections, sepsis, nosocomial infections (Ayub et al., 2015). Previous study of the leaves extract had indicated that the plant has significant antibacterial effects that correspond with its phytochemical properties (Akharaiyi et al., 2019). Also, Okoye and Amadi (2019) discovered that the aqueous extracts of A. wilkesiana leaves had significant inhibitory activities against some fungal species. The essential oil extracted for four hours (extract E) was the most effective in this study because it contained all of the components of the hourly extracts. Regrettably, it has minimal impact on the growth of P. aeruginosa and G. vaginalis.
The GC-MS analysis showed the existence of specific medicinal compounds that have been linked to the powerful therapeutic potential of essential oil. Compounds like pyrrole have demonstrated positive anti-malarial, antifungal, antibacterial, and anticancer properties. 2, 5 -Pyrrolidinedione, 1-(benzoyloxy) has been recognised to have antibacterial and antifungal effects. Bacteriostatic, antioxidative, and antimicrobial abilities are also possessed by 6-Benzamido-4-benzoyl-1, 2, 4-triazine-3,5 (Karrouchi et al., 2018). Another bioactive component of the oil is 4-Hexen-2-one, 3-methyl, which is a wellknown antioxidant, anti-inflammatory, antitumor, and antimicrobial phytochemical that has been shown to have anti-inflammatory, lipolytic, mucolytic, anti-coagulant and stimulatory qualities. Gyawali and Kyong-Su (2012) attributed these qualities the mild electronegativity and high polarity of its ketones.

CONCLUSION
The findings of this study indicate that the different constituents of the essential oil derived from A. wilkesiana have effective therapeutic activities. These properties, as well as the constituents from the oil promote its use as possible treatment option for skin conditions, salmonellosis, respiratory, and candida infections.