Preliminary in vitro antimicrobial potential and phytochemicals study of some medical plants

Introduction

The ethnobotanical use of medicinal plants and their derivatives (essential oil, resins and soluble extracts) in Africa dates back to early civilization (Sofowora, 1993). Herbal formulated medicine and traditional healthcare practice are globally perceived as comparatively cheaper and more widely accessible to most rural and less-privileged populations around the world than synthetic drugs and orthodox medicine respectively (Lawal et al., 2012). Statistics 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 was estimated that about 39% of drugs developed since 1980 have been from natural plants, their derivatives or analogs (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 natural plants, a number largely composed of analgesics (e.g. morphine), cardiotonic, chemotherapeutics and antimalarials (e.g. quinine and artemisinin). In recent decades, there have been growing global concerns on the affordability, the rising cost of producing synthetic drugs and accessibility by end users. Furthermore, the complexity involved in assessing their toxicological profile, eliminating their periodic side-effects and episodic efficacy contributed to undermining humans’ clinical dependency on synthetic drugs and scale-up their cost of production (Gupta et al., 2016). Other synthetic and semi-synthetic medicines use that has generated concerns in modern healthcare delivery are associated with possible side-effects and growing emergence of spontaneous or build-up biotic resistance (Langdon et al., 2016). Fair & Tor (2014), and Langdon et al. (2016) hypothesized that induced microbial resistance to synthetic antibiotics or their analogs involves various ecophysiological mechanisms such as neutralization of antibiosis effect, modulation of binding receptor site, mutation or acquisition of genes coded for antibiotic resistance, and regurgitation of antibiotic chemicals. These have caused a renascence of herbal screening for chronopharmacologically safer alternative remedies from natural plants and other biogenetic resources (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 (Bandow et al., 2003). This has redirected the focus of pharmacological research into the exploit of natural plants and their allies (ferns, fungi, algae) for safer generic bio-equivalents with parallel or better therapeutic capacity (Rojas et al., 2003; Savoia, 2012). Synthetic drugs use in animal farming also have implications for the global development of organic foods production.

Plant-derived medicine accounted 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 improves human health is constantly 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 botanical data in capturing the global representation of medicinal plants and ethnobotanical knowledge. This was due to possible inaccessibility 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 variable use as herbal remedies for treating diverse ailments may predates civilization (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 acuminata (P. Beauv.) Schott and Endl. (Kolanuts), and Moringa oleifera Lam. (Moringa) were listed among over 5,000 species of documented medicinal plants reported by Cunningham (1993) and Mahomoodally (2013). These plants except Chrysophyllum albidum, based on ethnobotanical data, were found to be common in many Nigerian cultures and traditional ceremonies (marriage, coronation, invocation) where they feature as masticatory and spiritual materials (George et al., 2018; Idu et al., 2007; Pasewu et al., 2008). They are also ethnopharmacologically valuable in maintaining, preventing and improving health, and in the traditional treatment of 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 biologically 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 interactive or counteractive modulation of bioactive compounds in many extracts responsible for inducing therapeutic effectiveness is still a mystery even though ethnomedicine hypothesized that herbal remedies are stable, and safe. This present study aims to cursorily compare the in vitro antimicrobial potential, phytochemical profile and solubility of the phytometabolome of selected medicinal plants in aqueous and ethanol solvents.

Methods

Results

Antibacterial activity

The plant extracts investigated showed various degrees of antibacterial activity. 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 investigated showed mild to average inhibition capacity which is inferior compared to the positive control (Chloramphenicol). Unlike the target bacteria, the fungus (Apergillus fumigatus) is insensitive to the extracts compared to what was observed in the griseofulvin control treatment(Figure 1 & Figure 2). Proteus mirabilis was more sensitive to the aqueous extract of Moringa seeds and Chrysophyllum albidum respectively fruits compared to their ethanol extracts. The negative control wells showed no inhibitory activity. Plant extracts derived from C. nitida, C. acuminata, A. melegueta and moringa pods using ethanol solvents showed better inhibitory activity than the aqueous extracts against Pseudomonas aeruginosa. In addition, Pseudomonas aeruginosa was the most susceptible target bacterium to the plant extracts screened (Figure 2). Conversely, the aqueous extracts of C. albidum and moringa seeds showed better inhibitory performance against the target bacteria than the ethanol extracts. A susceptibility contrast was, however, observed for Proteus mirabilis treated with the aqueous extracts of Cola nitida. Similarly, the aqueous extracts of Chrysophyllum albidum had the best inhibition activity against Pseudomonas aeruginosa (11.3mm) and Proteus mirabilis (9.4 mm) respectively. The highest inhibitory activity 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.

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

Figure 2. Comparative inhibition performance (mm ± SD) of plant extracts on Pseudonomas aeruginosa.

Plants aqueous extracts inhibition performance on Pseudomonas aeruginosa; Plants ethanol extracts inhibition performance on Pseudomonas aeruginosa.

Discussion

Herbal healthcare practice is an age long tradition that is now assuming a global dimension, attracting interest primarily in the improvement of the techniques for standardizing herbal prescriptions and production (Venkatasubramanian et al., 2018). In many African traditional healthcare services, herbal remedies prescribed in the form of decoction or infusion in tincture of ethanol or water are without accurate information to end users on expiring dates, dosage, composition, and usage. While this is presently improving among traditional medicine practitioners and local herbs vendors, the issues of proper approach to their scientific quality control, legislation and regulation of herbal drugs use is still a concern to many African nations. The use of herbal knowledge data in the development of pharmaceutical industries and primary public healthcare in many nations of the world is rapidly growing amidst emerging clinical health and therapeutic concerns associated with synthetic drugs use. Pharmacopeias derived from natural herbs is now rife in the global pharmaceutical market and has become a huge investment (Mahomoodally, 2013). The plants selected for this study were indigenous to Nigeria and were all observed to show promise as antibacterial. This observation corroborated already existing ethnomedicinal data that validated their utility in traditional healthcare provisions.

While further studies may be required to ascertain the in vivo toxicologically safety, dosage and microbial resistance responses to different herbal medicines, reports have more frequently traced positive pharmacological, biological and toxicological reaction by humans to the compositional dynamics of bioactive secondary metabolites (Ramawa et al., 2009). Results from the study inferred no correlation in antimicrobial activity and phytochemical yields. This may hypothetically suggest that the yields may not be the true reflection of the requisite composites of the extracts nor the phytochemical composition relevant to microbial antibiosis. Consequently, the positively interactive or synergistic and counter-interactive mechanisms responsible for the optimal microbial inhibitory capacity of plants’ extracts vary with the biological nature of the target microbes and are not yet fully understood. It is also philosophical to correlate the inhibitory action of the extracts of the five medicinal plants investigated to the structural modulation of signal transduction and functional groups reaction (Vinoth et al., 2012; Wink, 2015). While the mechanism underlying the solubility preference of secondary metabolites in aqueous or ethanol is not fully understood, it is logical to infer that the aqueous extract selectively solvated more of the non-cytotoxic or bioactively weak secondary metabolites contrary to the ethanol (95%) that theoretical had lower phytochemical composition. Hypothetically, ethanol showed the capacity to selectively solvate a consortium of phytochemicals that are potently interactive for optimal biologically activities or that afforded synergic reactions with the best bactriocin action. This compared to the aqueous could have also accounted for the superior inhibitory performance of ethanol despite the low yield percentage values obtained (Nascimento et al., 2000). While the variation observed in the concentration and distribution of phytochemicals may be linked to the part of plant, nature of solvent and method used, it has implication for the plants' bioactivity. It is infrequently noted in literature that the indiscriminate use of herbal tinctures of ethanol and aqueous infusions or decoctions could have histopathological consequence in humans (Fair & Tor, 2014; Langdon et al., 2016; Osemwegie et al., 2017). Conversely, their oral administrations in healthcare practice in this part of the world remained poorly standardized and without scientific therapeutic protocol. Although every plant materials used for this study were perceived by most local populations to have multitherapeutic uses in the treatment of numerous ailments (cancer, fever, infections, inflammations, hypertension, diabetics, obesity, dementia, etc.) with no recorded cases of side effect(s) in humans, they showed varying level of systemic potency against the target bacteria. This variation may be linked to the intrinsic or acquired immunity resulting from concomitant exposure to rapidly changing ecological pressures (Soares et al., 2012).

The target 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 may be attributed to a number of factors including their chronopharmacology, growth history of the plant, physiochemical nature of the solvent extractants used and selection of bioactive metabolites in the extracts (Wikaningtyas & Sukandar, 2015). The choice of ethanol and water for this study is consistent with several other antimicrobial studies involving medicinal plants (Ezeifeka et al., 2004). The aqueous extractant was observed to solvate more constituents that did not impact on the inhibitory performance of the extracts of investigated plant materials compared to ethanol (Ahmad et al., 1998; Abu-Shanab et al., 2004; Bacon et al., 2017; Cowan, 1999; Mothana et al., 2010). Phenols and flavonoids ware present in most of the extracts phytochemically assayed, they may have influenced positively the observed antimicrobial activity as demonstrated by the results of the ethanolic extracts of A. melegueta seeds. The inconsistent pattern of distribution of the phytochemicals may theoretically conflicted such inference. Phenols were detected in all the plant extracts except Moringa oleiferaseeds. This may have accounted for the observed antimicrobial capacity of the screened extracts (Bukar et al., 2010; Manisha & Vibsha, 2004). The cellular proteins disruption potential and interspecific interactions of phenols or other phytochemicals (flavonoids, tannins, polyketides) may be assumed to be responsible for the 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 seeds and pods of moringa plant. The difference in the extraction and susceptibility test protocols may have accounted for the contradiction and detection of more of the assayed phytochemicals in the fruit extracts of Cola nitida. Furthermore, the fruits of C. nitida and C. acuminata extracts screened in this study showed low antibacterial activity relative to the other plants’ parts. 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 antibacterial activity that is comparable with the seeds of A. melegueta, Moringa oleifera seeds and pods. This observation concurred with the study by George et al. (2018) and validates Chrysophyllum albidum fruits as a health food and an equally valuable pharmacological resource for the development of antibiotics.

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 (cytotoxic and non-cytotoxic) that may compromise the resistance of the target bacteria. The potency and efficacy of the extracts may be the product of the interaction of multiple phytochemicals. Mild sensitivity by Proteus mirabilis could hypothetically be as results of induced resistance (Venkatasubramanian et al., 2018; Wink, 2015). Further studies on the isolation and identification of the phytochemical agent(s) responsible for the observed antibacterial activity using High-Performance Liquid Chromatography and Gas Chromatography may be required for better understanding of their mode of action. Target bacteria growth inhibition may also be attributed to a disruption of cell protein biochemistry such as ion channels and pumps, enzyme actions, cytoskeleton function and membrane biochemical modulation (Ekpendu, 1995; Idowu et al., 2006; Wink, 2015). The insensitivity of Aspergillus fumigatus may be due to its biological complexity coupled with its innate defensive enzyme mechanism (Osemwegie et al., 2017). While the method adopted for the sensitive test of the fungus may influence the result, the result may be assumed to be derived from fungi long lasting symbiotic relationship with terrestrial plants.

This preliminary study attested to the inherent antibacterial property in all the plants investigated irrespective of the parts used corroborating their prevalence in the Nigeria herbal healthcare practice. Similarly, ethanol as an extraction solvent selectively optimized more effectively the expression of antimicrobial principles in the plants compared to the aqueous solvent. This observation supported the common practice of herbal tincture and offered a scientific basis for the continuous use of ethanol tincture in most local herbal preparations. It also validated the antiseptic nature of ethanol and preferred use of normal saline in the negative control set-up of the experiment (Senguttuyan et al., 2014). It is interesting to uncover the potential antibiotic property of C. albidum fruits which is hitherto neglected as an ethnobotanical and was one of the least popular of the five medicinal plants screened. The therapeutic usage of Moringa oleifera plants’leaf predominates in ethnomedicine and predates the utility of the other plant parts (e.g. roots, barks, pods, fruits) in this part of the world. In addition, the result opened up a potential utility trajectory for ethanol use in optimizing bioactive composites from natural plants for the production of safe, cheap and alternative antimicrobials, and other pharmaceuticals (Abdul et al., 2010; Abu-Shanab et al., 2004). Further investigation of these medicinal plants’ effect on a larger spectrum of target plant, human and animal pathogenic microorganisms is required to properly evince their pharmacognostic potentials. The observed effect demonstrated by the five medicinal plants on target bacteria used in this study encourages their possible use as alternative pharmacopeia to leverage global consciousness for safer antimicrobial drugs of organic origins (Trease & Evans, 2002).

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).