Comparative study of the in vitro phytochemicals and antimicrobial potential of six medicinal plants

Introduction

The ethnobotanical use of medicinal plants and/or their derivatives (essential oil, resins and soluble extracts) in Africa dates back to early civilization (Sofowora, 1993). This practice is globally perceived as comparatively cheaper and more widely accessible to most rural or less-privileged economies of the world than modern synthetic drugs (Lawal et al., 2012). Statistics as presented by Fabricant & Farnsworth (2001) in the bulletin of the World Health Organization showed that close to 65% of the world population relied on medicinal plants for their primary healthcare drugs (Eddouks & Ghanimi, 2013). Consequently, it is estimated that about 39% of drugs developed since 1980 have being from natural plants, their derivatives or analogues (Newman & Cragg, 2007; Verpoorte et al. (2010). In addition, Ramawa et al. (2009) and Verpoorte et al., (2010) noted that approximately 25% of the currently used modern drugs are derived from plants, a number largely composed of analgesics (e.g. morphine), cardiotonics, chemotherapeutics and antimalarials (e.g. quinine and artemisinin). In recent decades, there have been growing global concerns on the rising cost of buying synthetic drugs, assessing their toxicological profile, and redressing their periodic side-effects and unstable efficacy (Gupta et al., 2016). These undermine their continuous use in modern healthcare delivery and cause a renaissance of herbal screening as well as the chronopharmacological process (Westh et al., 2004). The clinical and pharmacokinetics sustainability of the efficacy of many synthetic antibiotics, prophylactics and curative drugs is threatened by the growing emergence of multi-drug resistant pathogenic strains and cost of production (Bandow et al., 2003). These concerns caused researchers to shift focus and exploit more intensely natural plants and their allies (ferns, fungi and algae) for safer generic bioequivalents to synthetic drugs or substitutions with stable therapeutic values (Rojas et al., 2003; Savoia, 2012). Synthetic drugs use in animal farming also have implications for the global development of organic foods.

Plant-derived medicine accounts for more than a quarter of today’s pharmacopoeia and over US$3.5 billion in annual export value of pharmaceutical (Eddouks & Ghanimi, 2013). While the global inventory of ethnobotanicals is growing, the catalogue of their bioactive compounds that can improve human health is constantly being updated. Over 12,000 bioactive metabolites (primary and secondary) and pigments of plant origin with a wide range of biological activities as well as therapeutic values were documented. Osemwegie et al. (2014) noted the inadequacy of current data in capturing the global totality of medicinal plants due to either neglect of plants in remote ecozones or bias against other related plant biota. While the prehistoric and historic knowledge of numerous health-beneficial plants in Africa seems threatened in recent times, their use in primary healthcare delivery predates modern medicine (Pasewu et al., 2008). This has facilitated the hybridization of both traditional and modern primary healthcare systems in some continents of the world (Eddouks & Ghanimi, 2013).

Aframomum melegueta (Roscoe) K. Schumann (Alligator pepper), Chrysophyllum albidum G. Don (Cherry), Cola nitida (Vent.) Schott and Endl., Cola acuminate (P. Beauv.) Schott and Endl. (Kolanuts), and Moringa oleifera Lam. (Moringa) were listed among over 5,000 species of documented medicinal plants reported by Mahomoodally (2013). These plants, from ethnobotanical surveys, were found to be common in many Nigerian culture and traditional festivities, featuring as masticatory and spiritual materials at traditional marriage, coronation and invocation ceremonies (Anwar et al., 2007; Idu et al., 2007; Pasewu et al., 2008). They are also ethnopharmacologically valuable in maintaining, preventing and improving health, and in treating different forms of illness in many African nations (Duraipandiyan et al., 2006; Idu et al., 2007; Mahomoodally, 2013). While the data on West African medicinal plants is hardly current, reports are inconsistent on the use of these plants as insecticides, antimicrobial, molluscicides and nematocides across cultures (Odugbemi, 2006). Similarly, the therapeutic scope and potency of biological active constituents of plants are to a large extent improved by ecosystem factors, methods of extraction of biometabolites, polarity of extraction solvent(s) used and part of plant assayed (Saini et al., 2016; Shitan, 2016). The emerging human allergic response or side effects from synthetic drugs usage, explosion of multi-drug resistant microorganisms, and global paradigm for more efficient stable biopharmacopoeia have increased toxicological, mechanism of action, ethnobotanical and phytometabolomic investigations of medicinal plants. This present study therefore aims to compare the antimicrobial potential, phytochemical profile and solubility of the phytometabolome of selected medicinal plants in aqueous and ethanol media.

Methods

Results

Antibacterial activity

The plant extracts investigated showed various degrees of antimicrobial activity against the target bacterial organisms. The observed zone of inhibition from the edge of each well differed from one target organism to the other. Raw data for zones of inhibition are available on OSF (Nwonuma, 2019). All the plant extracts assayed showed mild inhibition activity compared to the positive control (Chloramphenicol), to which the target bacteria were radically susceptible to (Figure 1). The negative control wells, containing normal saline solutions, showed no inhibitory activity. Plant extracts derived using ethanol solvents showed better inhibitory capacity than the aqueous extracts against Pseudomonas aeruginosa. In addition, Pseudomonas aeruginosa was the most susceptible target bacterium to the plant extracts used for the study. A susceptibility contrast was, however, observed for Proteus mirabilis treated with the aqueous extracts of Cola nitida. Similarly, the aqueous extracts of Chrysophyllum albidium at 400 mg in 1 ml of distilled water had the best inhibition activity against Pseudomonas aeruginosa and Proteus mirabilis (11.3 mm and 9.4 mm) respectively. The highest inhibitory activities were observed for the ethanol extracts of Cola nitida (11.8 mm), Aframomum melegueta (15.8 mm) and moringa pod (13.4mm) against Pseudomonas aeruginosa. The susceptibility results obtained for both aqueous and ethanol assays of investigated plants on Proteus mirabilis vary and were consistently low.

Figure 1. Comparative inhibitory performance (mm ± SD) of plant extracts on Pseudonomas aeruginosa and Proteus mirabilis.

Plants aqueous extracts inhibition performance on Pseudomonas aeruginosa (A) and Proteus mirabilis (C). Ethanol extract of plants inhibitory performance against Pseudomonas aeruginosa (B) and Proteus mirabilis (D).

Discussion

Herbal practice is now assuming a global dimension, attracting interest primarily in the standardization of the production chain, quality control and indigenous knowledge inventorying (Venkatasubramanian et al., 2018). The use of herbal knowledge in the development of pharmaceutical industries and primary healthcare in many nations of the world is rapidly growing amidst rising clinical health and unstable therapeutic concerns associated with synthetic drugs. Pharmacopeias derived from natural herbs is now rife in the global pharmaceutical market and has become a huge investment (Mahomoodally, 2013). Furthermore, all the plants selected for this study were indigenous, with ethnomedicinal implications at lower minimum inhibitory concentrations (MICs); they elicit pharmacological, biological or toxicological effects on humans (Ramawa et al., 2009). Conversely, their administrations in healthcare practice in this part of the world remained poorly standardized and without clear therapy specificity. Although every one of the plant materials used for this study are misconceived as having multifunctional bioactive compounds for curing numerous ailments (cancer, fever, infections, inflammations, hypertension, diabetics, obesity, dementia, etc.) and zero side effect in humans, they showed varying level of antimicrobial potential against two prevalent clinical Gram-negative target bacteria.

The tested microorganisms were observed to be most susceptible to the fruit, seed and pod extracts (ethanol and aqueous) of Cola nitida, Aframomum melegueta and Moringa oleifera respectively. This comparative variation maybe attributed to a number of factors involving the chronopharmacology, storage concentration and separation of electric charges (polarity) of the solvent used and composition of bioactive metabolites in the extracts (Wikaningtyas & Sukandar, 2015). The structural and induced resistance of the target microbes coupled with the diversity and richness of relevant solvated metabolites could have equally accounted for the observed variations of inhibitory capacity in the medicinal plants investigated. The choice of ethanol and water for this study is consistent with several other antimicrobial studies involving medicinal plants that used distilled water or ethanol as preliminary extractants of bioactive metabolites of (Ezeifeka et al., 2004). The aqueous extractant was observed to have solvated more constituents as supported by the percentage yield results across the investigated plant materials than ethanol. This finding is inconsistent with the mean zones of inhibition results obtained for the ethanol extract, which suggests better antimicrobial activity by ethanol derived plant extracts (Ahmad et al., 1998; Abu-Shanab et al., 2004; Bacon et al., 2017; Cowan, 1999; Mothana, et al., 2010).

While the mechanism underlying the solubility preference of secondary metabolites in aqueous or ethanol is not fully understood, it is logical to assume that the aqueous extract selectively solvated more of the non-cytotoxic or biologically weak secondary metabolites contrary to the ethanol (95%) that extracted lower but optimized consortium of potent biologically active phytochemicals (Nascimento et al., 2000). It is also philosophical to correlate the superior inhibition action of the ethanol extracts of the plants investigated to the capacity of the extractant to solvate phytochemicals that structurally modulate through reactive functional groups for optimal antimicrobial activity (Vinoth et al., 2012; Wink, 2015). This may therefore suggest that the full representation of phytochemicals assayed in the various plant extracts studied was selective or limited and accounted for the huge variations in the mean zones of inhibition obtained for the experimental extracts and positive control (chloramphenicol) tested. Phenols was detected in all the plant extracts except Moringa oleifera. This may have accounted for the observed antimicrobial capacity of tested extracts and phytotherapeutic popularity the medicinal plants screened (Bukar et al., 2010; Manisha & Vibsha, 2004). The cellular proteins disruption potential and interspecific interactions of phenols or other phytochemicals (flavonoids, tannins and polyketides) may be assumed to be responsible for multi-therapeutic uses of the investigated plants. This finding is, however, inconsistent with the report of Saini et al. (2016) and Fahal et al. (2018), who both observed higher deposits of flavonoids than phenols in the plant seeds and pods. Difference in the extraction and susceptibility test protocols may have accounted for the contradiction in experimental observations and the detection of more of the assayed phytochemicals in the fruit extracts of Colanitida.

Although this study showed that Pseudomonas aeruginosa is relatively more sensitive to both crude aqueous and ethanol-based plant extracts than Proteus mirabilis, the mechanism underlying the sensitivity reaction remains unclear. In theory, the plant extracts are made up of biochemicals with specific and non-specific modes of actions that compromise the resistance of the target bacteria. The potency and efficacy of the extracts are measured by collective modes of action of the phytochemicals and the corresponding reaction(s) of the target organisms (structural and inductive resistance). Mild resistance posed by Proteus mirabilis could have induced by chemical signaling mechanism (Venkatasubramanian et al., 2018). Further study on the isolation and identification of the phytochemical agent(s) responsible for the observed antimicrobial activity using modern techniques involving High Performance Liquid Chromatography and gas chromatography may be required for better understanding of the mode of action. The inhibition of growth of target bacteria is also attributed to the destruction of cell protein machinery such as ion channels and pumps, enzyme actions, cytoskeleton function and membrane biochemical modulation (Ekpendu, 1995; Idowu et al., 2006; Wink, 2015). The structural complexity of the test fungus Aspergillus fumigatus may have been responsible for its non-susceptibility to all the investigated extracts compared to the positive control of commercial griseofulvin.

This preliminary study confirmed all the plants investigated as having antibacterial property and corroborating their prevalent use in Nigeria herbal healthcare services. Similarly, ethanol as an extraction solvent optimized more effectively the antimicrobial agents released in the plant extracts compared to the aqueous solvent, supporting the common practice as well as offering the scientific basis to employing tincture for most local herbal preparations. The therapeutic usage of Moringa oleifera leaf however predominates over other plant parts (e.g. roots, barks, pods and fruits) in this part of the world in herbal healthcare predisposing it to possible exploitation for pharmacopeia. It was observed from this study that its seeds had phytochemical efflux with low antimicrobial activity relative to the other plants screened. While Chrysophyllum albidum was the least ethnobotanically popular of the plant studied (Adewoye et al., 2011; Idu et al., 2007; Okoli & Okere, 2010), its fruit extracts proved to have antimicrobial activity that is comparable with the fruits of kola nuts and the alligator pepper. Qualitative assays for flavonoids, terpenoides, phenolics and saponin in all the extracts showed the prevalence of phenolics and inconsistent distribution of the other phytochemicals. Consequently, this by no means accounted for the total phytochemical representations of the plant extracts studied due to non-detection of some unsolvated ones but proved that they have the potential for use as safe, cheap and alternative sources of antimicrobials, and other pharmaceuticals (Abdul et al., 2010; Abu-Shanab et al., 2004).

Data availability

Complete raw data containing the zones of inhibition for each extract are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/5APVE (Nwonuma, 2019).

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).