Preliminary in vitro antimicrobial potential and phytochemicals study of some medical plants [ version 3 ; peer review : 2 approved with reservations , 3 not approved ]

Background: Plants in traditional healthcare services in West Africa were selected based on ethnobotanical data for this study. Aqueous and ethanol extracts from these plants’ parts were comparatively screened for phytochemicals and in vitro antimicrobial activity. Methods: The antimicrobial activity of five medicinal plants’ extracts (aqueous and ethanol) were evaluated against Proteus mirabilis (LHC201), Pseudomonas aeruginosa (LHC181) and Aspergillus fumigates (LUML56) using the agar-well diffusion protocol. Retailed chloramphenicol and griseofulvin were used as positive controls respectively. Phytochemicals and percentage yield were determined by modified standard methods. Results: The target bacteria showed varied degrees of susceptibility to both aqueous and ethanol extracts. A. fumigates was insensitive to the treatments. The ethanol extracts of the sampled plants’ parts showed better inhibitory performance against the target bacteria compared to aqueous extracts. Aqueous and ethanol extracts of Aframomum melegueta, Moringa oleifera and Cola nitida showed marginal difference in inhibitory activity with higher inhibition zones observed for the ethanol extracts of A. melegueta seed and M. oleifera pod against the target bacteria. Phytochemicals composition and density observed in extractants and plants’ parts also varied. Phenols were detected in both the aqueous and ethanolic extracts of C. nitida and C. acuminata, but appeared relatively richer in extracts of A. Open Peer Review


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) andVerpoorte 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), andLangdon 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).

Amendments from Version 2
The manuscript title has been changed to " Preliminary in vitro antimicrobial potential and phytochemicals study of some medical plants" The manuscript has been reviewed thoroughly and it was felt that the work is too preliminary and lacks the scientific rigour. There are some points which led to this conclusion: The phytochemical analysis has not been described clearly and satisfactorily for the interpretation and usage by the readers.
We have described more comprehensively the phytochemical results in the discussion (middle part of 3 rd paragraph) with the introduction of "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".
The new manuscript has updated by addressing the concerns raised by the reviewers.
"Material and methods section needs to be rearranged appropriately, where phytochemical analysis should be mentioned after preparation of culture media and standardization of test organisms". Has been rearranged "Conclusion of the study and its significance not drawn appropriately". Has been edited with an introduction of "Further investigation of these medicinal plants with a larger spectrum of clinical and pathologic microorganisms as targets is required to properly situate their pharmacognostic potentials".
"Why is saline used as negative control for antimicrobial screening of the ethanolic extracts and not 95% ethanol?"

REVISED
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. The seeds of plants used for this study were rid of their coat, air-dried for 14 days and then pulverized with a stainless-steel electric blender. This was then sieved with a 325 micron-mesh sieve before storage in labeled, air-tight, sterile universal bottles. The Moringa, seeds and pods were also air dried for 5 days before milling while the two species of kola nuts (Cola nitida and Cola acuminata) were also sorted, air-dried for 14 days, pounded with a mortar and pestle, air-dried again for another 4 days before milling. The powdered form was later stored in air-tight sterile jars and left on the laboratory bench.

Materials
In the same vein, the fruit pulp and the fruit apicarp of Chrysophyllum albidum were manually separated from the seeds, air dried together for 12 days before subjected to similar protocol as described for others.

Preparation of aqueous extracts
A total of 80 g of each of the pulverized plant materials was weighed, poured into a labeled 500-ml conical flask and then soaked with 400 ml distilled water prior to each being vortexed in linear motion for effective extraction using a bench-top reciprocal shaker for 18 h. The preparations were then filtered with a steam-sterilized white handkerchief affixed to a glass filter funnel that drained to labelled 250 ml sterilize conical flasks. The residue from each plant extraction was stored in labelled cylindrical jar and refrigerated while each filtrate while each filtrate was further processed using a rotary evaporator (Model R-205V) at 55°C until a thick concentrate was obtained. This was later transferred into 250 ml beakers and further concentrated on a Water bath at 50°C until a paste was formed (dry crude extract). The paste was later spatulated into freshly labelled sterile universal bottles and weighed (weight of the dry extracts = weight of the universal bottle containing extract -weight of empty universal bottle).

Preparation of ethanol extracts
A similar weight (80 g) of each pulverized plant material was weighed and soaked in 400 ml of 95% ethanol in labelled 500 ml conical flasks. These were vortexed with a benchtop reciprocal shaker (E5850) for a period of 18 h. The mixture was then filtered and processed using similar extraction protocol for aqueous. Aliquots were prepared from the paste for phytochemical and in vitro sensitivity assays.
The percentage extraction yield was estimated for both aqueous and ethanol extracts using the formula: Extraction yield (%) = Weight of dry extract (g) x 100/Weight of sample used for extraction (g) Where: weight of dry extract is the actual weight of the extracts and the weight of the sample used for the extraction (g) is the initial weight of the samples measured (80 g).
Preparation of culture media A total of 28 g of nutrient agar (NA) and 65 g of Sabouraud dextrose agar (SDA) were each weighed into a sterile conical flask containing 1000 ml of distilled water and mixed vigorously. Each flask was then corked with an absorbent cotton wool stopper after which it was autoclaved for 15 mins at 121°C.
This was later allowed to cool to 40°C and dispensed to labelled Petri dishes in a laminar flow chamber where they were allowed to solidify. The bottom of each of the plates was marked into 4 quadrants.

Standardization of test organisms
In this study, the McFarland turbidity standard method, as described by Forbes et al. (2007) was used. The 0.5 McFarland standard, which is equivalent to 1.5 X 10 8 bacteria/ml was prepared according to standard methods (Zapata & Ramirez-Arcos, 2015). Furthermore, normal saline suspensions of the pure culture of each target bacteria were prepared. Turbidity was comparable to the 0.5 McFarland standards by visual determination. Similarly, a serial dilution of Aspergillus fumigatus suspension of 10 -3 diluent containing 10 5 spores/ml was used.
Preparation of positive and negative controls A concentration of 0.5 mg/ml of chloramphenicol and griseofulvin was prepared, serving as positive controls for antibacterial and antifungal activities, respectively. A solution of 0.85% NaCl was prepared and used as negative control.

Assessment of antimicrobial activity
The agar well diffusion method was used for the antimicrobial assay (Balouiri et al., 2016;Karunanidhi et al., 2019;Murray et al., 2016;Ncube et al., 2008). Previously prepared nutrient agar plates were flooded evenly with 1 ml of 1.5 X 10 8 bacteria/ml of each bacterial strain and and 10 5 spores/ml for the fungus suspensionin triplicates. These were left for 5 min after which four 5 mm wells were bored aseptically with a sterile corkborer into each inoculated agar plate. Next, 80 mg from each of the prepared crude extract was dissolved in 2 ml of sterile distilled water or 0.1% ethanol, as appropriate. 100 μl of each plant extracts was then used to fill 4 equidistant wells using a micropipette. For the controls, 100 μl each of the commercial antimicrobials (positive control) and normal saline (negative control) solutions was used to fill the agar wells. The plates were all allowed to incubate at 36°C for 24 h for bacteria and room temperature for 48 h for the fungus. These were then observed for zones of inhibition around the wells. Diameters of zones of inhibition were measured in millimeters with a meter rule.

Phytochemical analysis
Phytochemical screening of both the aqueous and ethanol extracts of the different plant materials was done according to the method of Tiwari et al. (2011) and Gul et al. (2017). The extracts' concentrates were dissolved with distilled water and ethanol (0.1%) respectively to assay saponins, phenolics, flavonoids and terpenoides of each plant extract respectively. 0.10g of each of the plant crude extracts was weighed in a labelled sterile universal bottle, appropriately solubilized with 10 ml of either distilled water for aqueous crude extract or ethanol (0.1%) for the ethanol crude extract. The aliquote were then transferred into clean labelled test-tubes, heated over a Bunsen flame for 3 min, agitated, filtered with Whatmann filter paper (32 mm), cooled, agitated again continuously for 2 min, left to stand for 10 min and observed for froth. In another protocol, phenols, flavonoids and terpenoids were qualitatively investigated using previously described methods (Harborne, 1998). Visual colour intensity scale of mild (+), strong (++), and extreme (+++) was used determine the concentration of phytochemicals. Colour change was measured against a standard RGB Colour.

Yields
The ethanol and water used in this study elicited varying solubility capacities of plant phytochemicals. The water extract from the different plants' parts used had the highest yield range (2.13-22.88%) of soluble phytochemicals compared to 95% ethanol extracts' yield range (1.76-22.74%) ( Table 1). A marked contrast was observed between the aqueous and ethanol extract yields of Chrysophyllum albidum and Cola nitida respectively. Ethanol had the least phytochemical yield value of 1.76% for Moringa oleifera pod. This was apparently lower than the lowest yield value for the aqueous (2.13%) extract of the same plant material (Table 2).

Phytochemicals
Assessment of the phytochemical profile of each plant extract showed a marked variation in the phytochemical content of each plant material used in this study. Phenol was detected in all the plant materials evaluated, with the exception of Moringa oleifera seeds and pods. Saponin, flavonoids and terpenoids showed inconsistent distributions across the various plant extracts studied, with aqueous extracts of Moringa oleifera seeds richer in terpenoid compared to the pod extracts (Table 3). Conversely, Aframomum melegueta was negative for terpenoids in the two extractants used for this study. Levels of saponin were observed to be negligible except in extracts of Cola nitida and moringa seeds of both aqueous and ethanol respectively (Table 4).

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.

Antifungal activity
Aspergillus fumigatus showed no visible susceptibility to the various plant extracts investigated with no clear inhibition zone.

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).

References
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 nonspecific 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 are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

I confirm that I have read this submission and believe that I have an appropriate level of
different polarities. Non-polar compounds do not diffuse well into the aqueous agar matrix and this underestimates activity. MIC using serial dilution delivers reproducible results to compare results in different laboratories and only extracts with MICs less than 0.1 mg/ml are considered, as interesting ones. Using crude extracts of plant materials with concentrations above 1000 μg/ml in antimicrobial screening protocols should be avoided, because using high concentrations of plant crude extracts can bring to false positive results (Rios and Recio, 2005). During the current study authors used 400 mg/ml concertation of the extracts in antimicrobial tests, which is too high. The concentrations of positive controls are also too high (5 mg/ml) In the article, it is stated that bacterial test strains (Pseudomonas aeruginosa (ATCC27856) and Proteus mirabilis (ATCC21784)) were isolated from immune-compromised patients of the University Health Centre and available in Microbial Bank of the Microbiology Laboratory, Landmark University. But the ATCC reference numbers were given to them. Are they available in ATCC's microorganism collection? 6.
The discussion must be completely rearranged. 7.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? No
We sincerely thank you for the thorough analytics and comments raised. We have already initiated efforts at addressing the issues of comment and wish to respond to some of the issues we disagreed in your reviewer's comment.
There are no proper negative controls. Particularly sterile distilled water and ethanol should be used as negative controls at the concentrations they present in the final test solution. Taking into account that ethanol has antiseptic properties how the authors can distinguish detected antimicrobial activity as result of ethanol or tested phytochemicals action. 1.
The method adopted only used ethanol as an extractant of bioactive phytochemicals of the test medicinal plants and not meant for solubilizing the crude for antimicrobial assay. We regret such error and appreciate your pointing it out. Similarly, the extracts were only suspended as crude in distilled water. Therefore, the antiseptic reference of ethanol is not appropriate.
In figure 1 there are six columns regarding the inhibition zones of positive control chloramphenicol differing each other against the same bacterial strains. This is not clear: why? 1.
The method only affords one positive and one negative control per treatment. No inhibition was observed and recorded (suppressed column) for the negative control treatments hence the number of columns presented. Differing reaction by the same strain of bacterium to in vitro antibiotic treatment is a possibility possible linked to undetectable slow differing changes in the local micro-environmental conditions.  (Rios and Recio, 2005). During the current study authors used 400 mg/ml concertation of the extracts in antimicrobial tests, which is too high. The concentrations of positive controls are also too high (5 mg/ml). One 2019 (Arunkumar et al., 2019) reference has been used to reflect currency and spread of literature review. On the technical dept. of the methods, the preference for disc technique in susceptibility test expressed by the version 1 report is scientifically debatable and appreciated. Well diffusion method is also listed among the approved routine antimicrobial susceptibility testing methods in clinical laboratory practices. Selection of method is influenced by a range of factors that may include the nature of culture media, test material and target organism. Literature as recent as 2019 also exists that have employed this method (Mounyr et al., 2016 -Journal of Pharmaceutical Analysis; Arunkumar et al., 2019 -Molecules). Ethanol used on crude was 0.1% while the concentration of crude extract is 200mg/ml instead of 400mg/ml. The three (3) reviewers called fungal spore suspension concentration (10 5 spores/ml) which is provided in the Assessment of antimicrobial activity).

1.
We partially concur with you that well diffusion method is becoming antiquated but its ineffectiveness in investigating the antimicrobial property of plant extracts remains debatable and it is still receiving acceptance in reputable journal outlets (Balouiri et al., 2016 -http://dx.doi.org/10.1016/j.jpha.2015.11.005). We wish for you to know that the first intent of the study was to prove that all the test plants have antimicrobial activity. We refer you to the last paragraph of the discussion which clearly mentioned that the work is preliminary. The concentration used was set based on the outcome of ethnobotanical survey of the location of study and it still generated the expected result despite disagreeing with Rios and Recio, 2005. All other comments on the voucher number for the test plants, reconstruction of the abstract and the discussion, expressive narration of the results will be addressed. While we do not understand what you mean by the language is poor, we wish to appeal for your guidance in the use of the right language hence we would be glad you return to use the edited version of the article. Thank you.