MATERIALS AND METHODS

Selection of four plant species: The choice of 4 species namely C. ternatum, C. transvaalensis, E. axillare and L. caffer implicated by Bapedi THs for RIs and RSs as candidates for further laboratory screening was based on their highest ethnobotanical indexes; (i) Fidelity Level, (ii) Use value and (iii) Preference ranking, compared to other species.

Plant collection and extracts preparation: Healthy anatomical part/s from C. ternatum (entire plant), C. transvaalensis (bark), E. axillare (entire plant) and L. caffer (root) as traditionally used by Bapedi THs to treat RIs and RSs (Table 1) were collected in the wilderness of Limpopo Province during May, 2018 and subsequently air dried at room temperature. Dried materials were deliberately utilised, firstly because they were used by THs and secondly due to the fact that there are fewer problems associated with their extraction compared to fresh plant materials³⁵. Desiccated plant parts was cut into smaller pieces and then pulverized to a fine powder using an electric grinder at the Limpopo Agro-Food Technology Station (LATS) (University of Limpopo) and stored in an air-tight container which was kept at room temperature in the dark until extraction procedure.

Extraction procedures: Small scale extraction was done using several solvents, from non-polar to polar. The 10 g of pounded part/s of the 4 selected plant species for laboratory study were extracted thrice in 200 mL of various solvents of distinct polarities (ranging from no-polar to more polar), namely; hexane, dichloromethane (DCM), acetone and methanol in 150 mL Erlenmeyer flask using IKA Magnetic Stirrers^® (RH digital) overnight and plant residues were allowed to settle.

Table 1:	Traditional use of Clerodendrum ternatum, Cryptocarya transvaalensis, Enicostema axillare and Lasiosiphon caffer by Bapedi healers to treat respiratory infections and related symptoms

Table 2:	Known therapeutic scientific assessments of Clerodendrum ternatum, Cryptocarya transvaalensis, Enicostema axillare and Lasiosiphon caffer
-: Not found, +: Pathogens known to cause RIs and RSs

With regards to the water extracts, it was done in 200 mL of distilled water in 250 mL round bottom flask using reflux apparatus for 30 min at 100°C and plant residues were allowed to settle. Generally, the extracts were filtered and solvents evaporated using a rotary evaporator (BUCHi Labotec rotavapor model R-205, Germany) and transferred into pre-weighed labelled glass beakers. Importantly, all the plant extracts were reconstituted with acetone to a final concentration of 10 mg mL^–1.

Phytochemical analysis: Chemical profile of the extracts from C. ternatum, C. transvaalensis, E. axillare and L. caffer materials were analysed by thin layer chromatography (TLC) using aluminium-backed TLC plates which included merck and silica gel 60 F254. The plant extracts were re-dissolved in acetone to give a final concentration of 10 mg mL^–1. Subsequently, 10 μL of the extracts was spotted onto a TLC plate. As described by Kotzé et al.³⁶, the TLC plates were developed in separation systems of varying polarities, namely, benzene/-ethanol/ammonium hydroxide (90:10:1): [BEA] (non-polar/basic), chloroform/ethyl acetate/formic acid (5:4:1): [CEF] (intermediate polarity/acidic) and ethyl acetate/ methanol/water (40:5:4:5): [EMW] (polar/neutral). The plates were then removed from the systems (tanks) and air dried under a fume-hood cabinet. Thereafter, the separated components were observed under visible and ultra-violet light at wavelengths of 254 and 365 nm. Separated compounds which were not visible under this light were detected using a method outlined by Wagner and Bladt³⁷. For instance, TLC plates were sprayed with vanillin sulphuric acid reagent and carefully heated for about five minutes at 110°C for optimal colour development.

Phytochemical constitutes: The mentioned morphological parts of C. ternatum, E. axillare (whole plant), C. transvaalensis (bark) and L. caffer (root) as used by Bapedi THs were examined for the present of the following components and appropriate colour change were used for interpretation.

Alkaloids: The Dragendorff’s reagent technique described by Harborne³⁸ was used. Milled parts (0.2 g) from the aforesaid species were extracted with 95% ethanol in a soxhlet extractor for 6 h. The ethanol extracts were evaporated to dryness using a vacuum evaporator at 45°C. Subsequently, the plant residues were dissolved in 5 mL of 1% hydrochloric acid and 5 drops of Dragendorff’s reagent was added. The samples were observed for the formation of colour (orange to orange red precipitate) to draw inference.

Anthraquinones: The presence of these secondary metabolites in studied plant extracts was tested by weighing 0.5 g of ethanol extracts and boiled with 10 mL of 97% sulphuric acid and carefully filtered while hot. The filtrate was shaken with 5 mL of chloroform. The chloroform layer was pipetted into another test tube to which 1 mL of dilute ammonia was added. The resulting solution was observed for pink colour³⁹.

Cardiac glycosides: For cardiac glycosides test, the Keller-Killiani experiment was used, where 0.5 g of plant extracts was added to 5 mL of water. The mixture of 2 mL of glacial acetic acid containing one drop of 0.1% ferric chloride solution was added to the diluted extracts. This was underplayed with 1 mL of concentrated sulphuric acid. A brown ring (with a violet ring beneath) at the interface indicates the presence of a deoxysugar characteristic of cardenolides while in the acetic acid layer, a greenish ring may also form gradually throughout the layer⁴⁰.

Flavonoids: Diluted ammonia solution (5 mL) was added to a portion of the aqueous filtrate of each plant extracts, followed by the addition of concentrated sulphuric acid (1 mL). A yellow colouration that disappears on standing represented the presence of flavonoids⁴⁰.

Phlobatannin: Powdered plant part samples (0.2 g) of each investigated species used by Bapedi THs were dissolved in 10 mL of distilled water and filtered. The filtrate was boiled with 2% hydrochloric acid solution. The deposition of a red precipitate confirmed the presence of phlobatannins⁴⁰.

Saponins: Odebiyi and Sofowora⁴¹’s persistent frothing test for saponins was used. One gram of powdered materials from four selected plant species was mixed with 30 mL of tap water. The mixture was vigorously shaken and heated at 100°C. The sample was observed for the formation of a persistent froth to indicate the presence of saponins.

Steroids: This was tested by adding 2 mL of acetic anhydride to 0.5 g of each plant extract, followed by addition of 2 mL of sulphuric acid. Colour changes from violet to blue or green in some plants indicates the presence of steroids⁴⁰.

Tannins: The tannins were tested by gently boiling 0.5 g of powdered materials from the selected plant species, in 5 mL of distilled water in a test tube, then cooled and filtered. Three drops of 0.1% ferric chloride was added to 1 mL of solution in a test tube and observed for green coloured precipitate⁴².

Terpenes: The Salkowski test was used to evaluate the presence of terpenes. Powdered plant part (0.5 g) of C. ternatum, E. axillare, C. transvaalensis and L. caffer were dissolved in 2 mL chloroform, followed by cautiously adding 3 mL of concentrated sulphuric acid, to form a layer. A reddish brown or greyish colouration of the interface was considered indicative of positive results⁴⁰.

Evaluation of the antioxidant activities: The antioxidant activities within the plant extracts were determined by using qualitative 2, 2-diphenyl-1-picrylhydrazyl (DPPH) on TLC and quantitative 2, 2-diphenyl-1-picrylhydrazyl assays.

Qualitative antioxidant assay using 2,2-diphenyl-1-picrylhydrazyl: As described earlier under phytochemical analysis section TLC plates were used to separate crude extracts of different polarities. To detect any antioxidant activities presence in the separated plant extracts, the chromatograms were firstly air-dried and afterwards subjected to 0.2% (w/v) DPPH solution in methanol sprayed (until saturated) by using a spray gun⁴³. Subsequently, yellow zones or spots (against purple background) appearing on the chromatograms indicated the presence of antioxidant.

Quantitative 2, 2-diphenyl-1-picrylhydrazyl assay: This assay was done on the studied medicinal plant extracts by adding 100 μL of distilled water into each well of 96-welled plate. These extracts were prepared at a concentration of 10 mg mL^–1. Subsequently, 100 μL of plant extracts were transferred into the first row of 96-welled plate and serially diluted 50%. Ascorbic acid (Vitamin C) at a concentration of 2 mg mL^–1 was used as the antioxidant standard or positive control. Fifteen micro litres of 0.2% of 2, 2-diphenyl-1-picrylhydrazyl (sigma^®) in methanol was added into each well of microtiter plate and allowed to react at room temperature in the dark for 30 min. The absorbance was measured at 540 nm using a microtiter plate reader (DTX 880 multimode detector; Beckman coulter, Inc.^®) as previously used by Ayoola et al.³⁹. Percentage antioxidant activities (AA %) values were calculated from the absorbance values using the equation:

Antibacterial assay
Used microorganisms: Mycobacterium smegmatis ATCC 1441 (a potential M. tuberculosis surrogate), Klebsiella pneumoniae, Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 were chosen to test the antimicrobial activities of the 4 studied plants extracts. These are microorganisms that are known to cause RIs and RSs or responsible for the majority of their cases. The test organisms were sub cultured on nutrient broth and incubated at 37°C for 24 h and subsequently preserved at 4°C in the refrigerator as stock strains prior to screening testing.

Qualitative antibacterial activities assay by bioautography: Thin layer chromatography plates were cut (10×10 cm) and loaded with 20 μL of each extract plant extract (10 mg mL^–1) dissolved in acetone in line with the method of Begue and Kline⁴⁴. The plates were prepared, developed in the different solvent systems as stipulated earlier and were allowed to dried at room temperature under a stream of air for 2-3 days, in order to remove residual solvent, which might inhibit bacterial growth. The 4 bacterial species used in microplate serial dilution assay were used the test organism. The developed plates were sprayed with the concentrated suspension containing actively growing bacteria until they were entirely wet and thereafter incubated at 37°C for 24 h in 100% relative humidity in the dark. After incubation, the plates were sprayed with a 2 mg mL^–1 solution of INT (Sigma^®) and incubated for a further 2-3 h. The bioautograms were subsequently observed for bacterial growth. The white areas (clear zones) indicated where reduction of INT to the coloured formazan did not take place due to the presence of compounds that inhibited the growth of the test bacteria.

Quantitative antibacterial activities assay by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC): The microplate serial dilution method of Eloff³⁵ was employed to determine the minimum inhibitory concentration (values of the plant extracts against M. smegmatis, K. pneumoniae, S. aureus and P. aeruginosa). Residues of plant extracts were dissolved to a concentration of 10 mg mL^–1 using the extracting solvent acetone. Consequently, the dissolved plant extracts were serially diluted (50%) with water in 96 well microtiter plates. Bacterial cultures were sub-cultured and thereafter transferred into fresh nutrient broth and 100 μL of the cultures were transferred into each well and acetone was included blanks. The microtiter plate (cultures) was incubated for 24 h at 37°C. After incubation, 20 μL of 2 mg mL^–1 p-iodonitrotetrazolium violet (Sigma^®) (INT) dissolved in water was added to each microplate wells as an indicator of growth. The covered microtiter plates were incubated for 30 min at 35°C and 100% relative humidity and all determinations were carried out in triplicate. Microorganism growth was indicated by a purple-red colour resulting from the reduction of INT into formazan. Clear wells indicated the presence of compound in the extracts that inhibited the growth of the microorganisms tested. The MIC value of each plant extracts was determined as the lowest concentration that led to an inhibition of bacterial growth after 24 h of incubation at 37°C. The total activities of the plant extract in mL g^–1 was calculated using a standard formula developed by Eloff³⁵. For instance, the MIC values were divided with the quantity extracted from 10 g of plant material. The resultant value indicates the volume to which the extract can be diluted and still inhibits the growth of the bacterial isolate.

The MBC were determined by further incubating for another 24 h, the same 96 well microtiter plates used for MIC. The lowest concentration with no visible growth of M. smegmatis, K. pneumoniae, S. aureus and P. aeruginosa was defined as the MBC, indicating 99.9% reduction in colony forming units’ number in the initial inoculum⁴⁵.

RESULTS AND DISCUSSION

Quantity of plant materials extracted: Table 3 depicts the percentage mass extracted from 10 g of C. ternatum, C. transvaalensis, E. axillare and L. caffer materials extracted in 5 different solvents namely acetone, dichloromethane, hexane, methanol and water. None of these solvents yielded the highest mass from these plant materials. In this regard, a wide range of quantity among extracts was observed depending on the extraction solvent and plant material used. Such disparity might be due to the variations in both particle sizes of grounded material and their phytochemical constituents, coupled with the ability of used solvents to solubilise compounds with similar polarity⁴⁶. Generally, different mass yields obtained using various solvents in our study indicate that the investigated plant species possess diverse chemical compounds.

An analysis of our findings showed that water and methanol respectively was the best extracting solvents, followed by acetone and dichloromethane. Most of the hexane materials from the investigated species gave much lower extraction yields. In generally polar solvents (acetone, methanol and water) extracted more materials than non-polar, thus suggesting that the studied plant species mainly contain polar compounds. The strongest extractability of water and methanol as observed in this study might indicate that both these solvents have great ability to extract higher concentration of compounds from the investigated species compared to other solvents. Therefore, these solvents can be considered the best as far as the biochemical extraction of C. ternatum, C. transvaalensis, E. axillare and L. caffer is concerned. We perceive the highest extraction yields of water in this study, as an interesting finding since it is a standard method of extract preparation by Bapedi THs. Therefore, it can be said that water as used by questioned THs have great potential to recoup numerous phytochemicals including those that are effective against RIs and RSs.

To the best of our knowledge, with the exclusion of E. axillare, there is presently no study reporting on either the application of acetone, dichloromethane, hexane, methanol and water as extracting solvents for the rest of the species included in this study, hence there is no literature to compare and contrast our findings with. The present study in this regard provides a baseline data. Analogously to the outcomes of our investigation, methanol (45.8%) and water (43.6%) extracts of E. axillare also yielded more materials in an Indian study⁴⁷. A comparable finding was reported by Gite et al.²⁶ in the same country. Findings from our research therefore, further buttress the significant of both water and methanol as the most efficient solvents for extraction of phytochemical constituents from E. axillare materials.

Table 3:	Percentage mass extracted (g) from powdered material species using different solvents

Fig. 1(a-b):	Thin layer chromatographic profiles, (a) Sprayed with vanillin-sulphuric acid reagent and heated in the oven to show compounds and (b) Prior spraying with this reagent and heating of investigated plants
	Species 1: E. axillare, Species 2: C. ternatum, Species 3: C. transvaalensis, Species 4: L. caffer, crude extracts A: Acetone, D: Dichloromethane, H: Hexane, M: Methanol, W: Water separated with 4 solvent systems namely: BEA, CEF and EMW and sprayed with vanillin-sulphuric acid reagent, to show compounds

Thin layer chromatography finger print profile of the plants: The separation of compounds from crude extracts of 4 studied medicinal plant species was determined using the thin-layer chromatography (TLC) and accordingly, TLC revealed complex mixture of compounds which exhibited different coloured reactions with and without the vanillin spray reagent (Fig. 1).

Generally, BEA (a non-polar), followed by EMW (polar/neutral) and CEF (intermediate polarity/acidic) respectively, proved to be a better solvent system for extraction of wide range of bioactive principles from all/most plants. Therefore, future studies further investigating the pharmacological activities of specific secondary metabolites from the 4 studied medicinal plants should utilize BEA and EMW TLC elution systems for column chromatography. However, the ability of the specific solvent used for each species to better isolate some compounds should not be overlooked by these studies. It worth mentioning that all water extracts from the studied plants displayed the presence of bioactive components, suggesting that the crude herbal remedies prescribed by Bapedi THs might be of help in the treatment and management of RIs and RSs.

Preliminary phytochemical analysis of crude extracts: Phytochemical constituents of 4 studied plant species exhibited the presence of alkaloids, anthraquinones, cardiac glycosides, flavonoids, phlobatannin, saponins, steroids, tannins and terpenes. With the exclusion of flavonoids, saponins, steroids, tannins and terpenes, which were found in all four plants, the rest of these bioactive compounds was present in certain species. Generally, the referred secondary metabolites were not detected in all 4 investigated species and their concentration (based on colour intensity) within some of these plants were either in low, moderate or high concentrations (Table 4). The latter might be attributed to variation in genetic makeup and chemical composition of plant specimen used, ascribed to the dissimilar geographical areas (i.e., altitude, climate and rainfall) were they were collected.

With the exception of E. axillare, to date there is no work done on the rest of the species namely C. ternatum, C. transvaalensis and L. caffer as far as phytochemical constituents are concerned. The presence of alkaloids, flavonoids, glycosides, saponins, steroids and tannins within E. axillare extracts was previously reported in India^14,29. Therefore, our findings further buttress this.

Fig. 2:	Chromatograms of investigated species
	Species 1: E. axillare, Species 2: C. ternatum, Species 3: C. transvaalensis, Species 4: L. caffer crude extracts (A, D, H, M and W) developed in four solvents systems (BEA, CEF and EMW) from top to bottom and sprayed with 0.2% DPPH in methanol as an indicator, with yellow zones indicating antioxidant activities exhibited by compounds

Table 4:	Preliminary phytochemical analysis of screened therapeutic plant species
+: Presence of phytochemicals, ++: Low concentration, +++: Moderate concentration, ++++: High concentration, A: Alkaloids, AN: Anthraquinones, CA: Cardiac glycosides, F: Flavonoids, PH: Phlobatannin, SA: Saponins, ST: Steroids, TA: Tannins, TE: Terpenes

Generally, the presence of phytochemicals within the investigated 4 crude plant extracts were mainly in high concentrations and none of the species had all the nine investigated secondary metabolites. Enicostema axillare and L. caffer were endured with more of these metabolites (n = 8) while C. ternatum and C. transvaalensis had six. Traces of several diverse biologically active phytochemicals reported in Table 3 within these species, justifies their use as treatment of various RIs and Rss. These phytochemicals serve as natural antibiotics, which help the body to fight infections and microbial invasion⁴⁸. Alkaloids, terpenes and steroids are reported to have anti-inflammatory and anti-bacterial activities^49-51, thus are helpful in the treatment of asthma and other respiratory infections (i.e., pneumonia and sore throat) which are caused by bacterial infections. Similarly, glycosides have been found to decrease inflammatory symptoms in different animal models of acute and chronic inflammation⁵². Tannin is known to hasten the healing of inflamed mucous membranes⁵³. Amutha and Doss⁵⁴ found that saponin fraction possess significant activities against K. pneumonia and S. aureus. Flavonoid supplementation decreased upper respiratory tract infections incidence by 33% (95% CI: 31-36%) compared with control in healthy adults, with no apparent adverse effects⁵⁵. Therefore, it can be said that the bioactive principles present in 4 investigated therapeutic plants prescribe by Bapedi THs might be of benefit for respiratory sicknesses patients.

Antioxidant activities of plant extracts: Qualitative antioxidant compounds activities of the extracts from studied plants were examined using the DPPH assay on TLC plates. The presence of these compounds with scavenging activities is indicated with yellowish bands on a purple background on TLC plates (Fig. 2).

Fig. 3(a-d):	Reducing power potential of 4 studied plant extracts, (a) L. caffer, (b) E. axillare, (c) C. ternatum and (d) C. transvaalensis
	A: Acetone, D: Dichloromethane, H: Hexane, M: Methanol, W: Water, ASC: Ascorbic acid with absorbance increasing with increasing concentration

The presence of antioxidant compounds activities of the studied plant extracts per solvent used on 3 separation systems (BEA, CEF and EMW) was also determined. Extracts from certain species alienated by these systems had antioxidant compounds. On the CEF chromatogram, C. ternatum water extract, acetone, dichloromethane, methanol and water extracts from L. caffer showed traces of these compounds. Same species extracted with these solvents but using EMW separation systems also showed some antioxidants activities. The mobile phase system; BEA exhibited antioxidant activities of compounds from C. ternatum (acetone and water), C. transvaalensis (acetone) and L. caffer (acetone, methanol and water) extracted with the mentioned solvents. Therefore, it can be said that C. ternatum and L. caffer are good candidates to isolate antioxidant compounds from. No yellow bands were observed when the plates were resolved on the BEA, CEF and EMW using all extracts from E. axillare and C. transvaalensis on CEF and EMW. Overall, with the exception of E. axillare which were previously reported by Krishnaveni and Mohandass¹⁴ as having antioxidant activities, the antioxidant capacity of the rest of the species investigated are mentioned for the first time in the present study.

Antioxidant activities (expressed as percentage) of C. ternatum, C. transvaalensis, E. axillare and L. caffer was quantified using quantitative 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activities assay, by comparing it to the activities of standard antioxidant drug, namely ascorbic acid (Fig. 3). Surprisingly, all plants including those that did not displayed any antioxidant activity using qualitative DPPH assay on TLC (Fig. 2) showed a certain level of scavenging activities when quantified via quantitative DPPH radical scavenging activities assay. Indeed, as reported by various researchers^56,57, one assay cannot reveal the presence of antioxidant compound in all plant extracts because the sensitivity of some methods depends on various factors, such as media pH, the presence of lipophilic and/or hydrophilic compounds and amongst the others. Therefore, antioxidant capacity of plant extracts should be evaluated by more than one method.

The mentioned extracts from E. axillare (water and hexane), L. caffer (water and acetone) and C. ternatum (acetone) showed excellent antioxidant activities almost comparable to ascorbic acid. Therefore, enquiries in to the possibilities of developing natural antioxidant drugs from these species should be a subject for future study. All C. transvaalensis extracts were less effective as opposed to ascorbic acid. Generally, the present of certain traces of anti-oxidants activities in all studied plants is very fundamental from the therapeutic stand-point pertinent to RIs and RSs.

Fig. 4:	Chromatograms of crude extracts of investigated species
	Species 1: E. axillare, Species 2: C. ternatum, Species 3: C. transvaalensis, Species 4: L. caffer, crude extracts A: Acetone, D: Dichloromethane, H: Hexane, M: Methanol, W: Water developed in three solvents systems (BEA, CEF and EMW) sprayed with S. aureus, M. smegmatis, P. aeruginosa and K. pneumonia, respectively (starting from right)

This is because the crucial role of this activities in preventing or slowing oxidative damage linked to asthma, infant and adult respiratory distress syndromes and chronic obstructive pulmonary disease were scientifically demonstrated⁵⁸.

Antibacterial activities of plant species
Qualitative antibacterial activities assay by bioautography: Different extracts from C. ternatum, C. transvaalensis, E. axillare and L. caffer were examined for antibacterial activities using the bioautography method. The appearances of a clear zones/white spots against the purple-red background on the bioautograms are areas of inhibition of the growth of used bacteria by certain compounds within the plant extracts. The bioautography results displayed a broad range of antibacterial activity compounds present in these extracts (Fig. 4). It worth mentioning that the position of most of these compounds (retention factor values of compounds was calculated but not presented) tallies with those observed during phytochemical profiling of the investigated plant species (Fig. 1). Thus, corroborating the assertion by the Bapedi THs that they prefer C. ternatum, C. transvaalensis, E. axillare and L. caffer due to their efficacy in the treatment and management of RIs and RSs.

There was a differential activity of various extracts against used bacterial species across the 3 mobile phases (BEA, EMW and CEF). This perhaps can be explained by the dissimilar sensitivity of used micro-organism towards the potential anti-bacterial phytochemicals present in the crude extracts on TLC mobile phases (Fig. 1). However, all bioautogram of S. aureus had more clear zone inhibition that corresponded with those noted in Fig. 1, thus indicating high level of antibacterial activities compared to mobile phases saturated with other micro-organisms. Analysis of the efficacy of the studied plant species extracts per three mobile phases used in this study showed a clear zones of inhibition by most extracts, that can be compared with bands displayed in Fig. 1. It should be stated that antibacterial activities observed within the plant extracts (at the top of the TLC plates eluted in some mobile phases especially EMW, might be due to synergy of various plant bioactive molecules which did not separate well, hence there are traces of chlorophyll. Importantly, a number of clear zones of inhibition (particularly those that correspond with bands in Fig. 1 exhibited by numerous studied therapeutic extracts against M. smegmatis, K. pneumoniae, S. aureus and P. aeruginosa across the different TLC elution systems (Fig. 4), show that the investigated plant species proffer beneficial effects towards alleviation or treatment of bacterial RIs and RSs.

Quantitative antibacterial activities assay by minimum inhibitory concentration: The bioautography results of the different extracts from C. ternatum, C. transvaalensis, E. axillare and L. caffer against tested bacteria strains was complemented by measuring the MIC values (Table 5).

Table 5:	Minimal inhibitory concentration (MIC) values and total activity values of four studied plant against four bacterial test organisms
A: Acetone, DCM: Dichloromethane, H: Hexane, M: Methanol, W: Water, MIC values>2.5 were included as >2.5 for the calculation purposes

These values were considered as the lowest concentration of the extract/s that completely inhibits the bacteria growth. In this regards, the lower MIC values depict greater antibacterial effectiveness of the crude extract/s and the opposite of this is true for high values. Extracts with MIC values <0.1 mg mL^–1 were considered to have significant activities; moderate activities were between 0.1 and 0.63 mg mL^–1 and weak or poor activities with MICs⁵⁹ >0.63 mg mL^–1. Generally, the MIC values of plants in the present study ranged between 0.63-2.5 mg mL^–1 after 24 h of incubation and there was no substantial difference in values observed between most plant extracts against tested strains, thus indicating that these extracts have the same inhibitory potential. With the exception of L. caffer, no other record, except our observations amongst the Bapedi, could be located regarding the MIC of the investigated plants. Pavithra et al.²⁹ found that hexane and methanol extracts of E. axillare harvested in India are ineffective against S. aureus, K. pneumoniae and P. aeruginosa. Likewise, in the present study these extracts did show activities against S. aureus and P. aeruginosa, but were effective against K. pneumoniae with MIC values of 1.25 and 0.625 mg mL^–1, respectively. This disjuncture may be due to the absence of antibacterial constituents present in the crude extracts of E. axillare collected in Indian. This is because the number of E. axillare phytochemical constituents in our study were higher compared to Pavithra et al.²⁹ study.

Table 6:	Minimum bactericidal concentration (MBC)
A: Acetone, D: Dichloromethane, H: Hexane, M: Methanol, W: Water

With the exception of all C. transvaalensis extracts which did not show any activities against four used bacteria species perhaps due to the insensitivity of this species to substances contained in the extracts, some extracts from the rest of the plants exhibited certain degrees of antibacterial activities against a particular bacteria strain/s tested (Table 5). None of the extract displayed noteworthy antibacterial activities. The moderate MIC value of 0.63 against the bacteria species were obtained with the stated extracts of E. axillare (acetone, dichloromethane, methanol and water against K. pneumonia) and L. caffer (hexane and water against K. pneumonia and acetone against S. aureus, P. aeruginosa and M. smegmatis). This finding confirms that E. axillare and L. caffer extracts contains secondary metabolites with potent antibacterial activities. However, it should be reported that the resistance of used bacteria in this study to some of the plant extracts using MIC assay may be due to insufficient amount of active ingredients. This speculation is based on the fact that same extracts portrayed antibacterial activities on bioautography assay.

The total activities values of four medicinal plant extracts investigated are also given in Table 5. Generally, these activities indicate the volume to which the bioactive compounds extracted from 10 g of plant material can be diluted and still inhibits growth of bacteria. The highest total activities were found in water extract of L. caffer (696 mL g^–1) against M. smegmatis and K. pneumonia compared with the other extracts, followed by same extract from this species (5125 mL g^–1) against S. aureus and P. aeruginosa. Other extracts which showed greater total activities includes E. axillare methanol (492 mL g^–1) and water (333 mL g^–1) against K. pneumonia. This finding came as no shock as both species had high percentage extract yield (Table 3). According to Eloff⁶⁰, total activities is dependent on the quantity of material extracted from the dried plant parts, meaning that the higher the total activities, the greater is the potential for application of the specific plant extract. Therefore, it is acceptable to state that all plant species extracts which yielded the moderate or lowest total activities against one or more bacteria is due to the lowest number of antibacterial compounds extracted. Overall, the plant extracts with higher total activities values against the tested bacterial strains should be selected for further fractionation.

Quantitative antibacterial activities assay by minimum bactericidal concentration: Minimum bactericidal concentration (MBC = lowest concentration that killed the bacteria) of all C. ternatum, C. transvaalensis, E. axillare and L. caffer extracts were determined at various concentrations tested over a 42 h period (Table 6), to further assess the therapeutic value of the plants implicated by Bapedi THs in the treatment of RIs and RSs. Minimum bactericidal concentration of these extracts ranged from 0.019-2.5 mg mL^–1.

Unexpectedly, the MBC values were greater than the MIC values and all extracts which depicted poor or no activities on the MIC were bactericidal. Although not specifically for the investigated species, this finding was previously reported by other researchers^61-63. Nevertheless, the observed disjunction between MBC and MIC values in the present study might be due to the strains high resistance, attributed to thick outer membrane in their cell wall acting as an obstacle for the plant extracts permeability over 24 h of exposure via MIC assay. Therefore, as these extracts perhaps fully came in contact with the test organisms over a longer duration (42 h period), they were able to completely eliminate organisms at various concentration. Generally, C. ternatum hexane and water extracts showed strongest bactericidal activities with MBC values of 0.078 and 0.019 mg mL^–1 against M. smegmatis and P. aeruginosa, respectively. All extracts from C. transvaalensis and L. caffer were also more effective (0.156 mg mL^–1) in killing P. aeruginosa. This suggests that these plants, when prescribed orally by Bapedi THs as antibacterials for RIs and RSs, might inhibit bacterial growth and ultimately kill it.

CONCLUSION

Findings of the present study indicated that crude extracts from four medicinal plants commonly used by Bapedi THs to cure and manage RIs and RSs contain diverse secondary metabolites active against bacterial strains causing these diseases. These findings therefore, provide support for the use of these plants in traditional medicine for RIs and RSs and could potentially be used as alternative therapeutic agents. Studies focusing on isolation, identification and characterization of the pure compounds responsible for antibacterial activities of C. ternatum, C. transvaalensis and L. caffer extracts and cytotoxicity of these compounds to determine any potential toxicity are in progress.

SIGNIFICANCE STATEMENT

This study is the first to report on the phytochemical constituents and biological activities of C. ternatum, C. transvaalensis and L. caffer. Overall, the study showed that the extracts from all the investigated four species can be beneficial in the treatment of RIs and RSs. A better understanding of these species’ phytochemical constitutes and biological activities will help the researchers to further investigate the possibilities of developing new herbal drugs that can be utilized against RIs and RSs.

ACKNOWLEDGMENTS

The authors would like to express their gratitude to the National Research Foundation, South Africa (Grant number T398) and Govan Mbeki Research and Development Centre (Grant number C169), University of Fort Hare for financial support to conduct this study.