Antibacterial activity and in situ efficacy of Bidens pilosa Linn and Dichrostachys cinerea Wight et Arn extracts against common diarrhoea-causing waterborne bacteria

Antibacterial activity and in situ efficacy of Bidens pilosa Linn and Dichrostachys cinerea Wight... Background: Bidens pilosa and Dichrostachys cinerea extracts were investigated for the antibacterial properties against waterborne diarrhoeagenic bacteria. Methods: The plant materials were extracted using the direct and serial exhaustive methods using solvents of varying polarities, namely, hexane, dichloromethane, ethyl acetate, acetone and methanol. Qualitative phytochemical analysis and quantitative determination of total phenolic content of the leaf powders of the two plants were tested. The antioxidant activities of the plants were determined using the 2, 2-diphenyl-1-picrylhydrazyl method. The toxic effect of the extracts on C2C12 muscle cell line were assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method and the antibacterial activity was determined using the serial microbroth dilution. Results: Methanol leaf extracts both plants had the highest yield in both direct and serial exhaustive extraction methods. Phytochemical profiling of the extracts displayed the presence of various secondary metabolites. The Benzene: ethanol: ammonia hydroxide solvent system showed a good resolution of chemical compounds in plant extracts from both plants. Most antioxidant compounds observed were developed in chloroform: ethyl acetate: formic acid and ethyl acetate: methanol: water solvent systems. All the bacterial species tested were sensitive to the effect of different extracts of both plant species, with E. coli being less sensitive to the effect of the extracts from D. cinerea. Following the simulated gastric fluid (SGF) treatment, a decrease in the antibacterial potency of the extracts was observed. No extract was toxic to the C2C12 muscle cell line. Conclusion: The presence of the secondary metabolites and nontoxic effect of the two plants tested may affirm the medicinal value of these leaf extracts. Our results suggest that B. pilosa and D. cinerea contain constituents with antioxidant and antimicrobial activities, which could be used in the treatment of diarrhoea in a case where untreated surface water is used. Keywords: Diarrhoea, Simulated gastric fluid, Medicinal plants, Antibacterial activity, Probiotics * Correspondence: kgabo.moganedi@ul.ac.za Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 2 of 10 Background been reported to have antioxidant activity [14–16]and Many communities in South Africa still depend on un- anti-hypertensive and antibacterial effects [17, 18]. treated surface water for their domestic needs such as Bidens pilosa L. is a species of flowering plant of the drinking, cooking, laundry and for personal hygiene [1], Asteraceae family. In South Africa, B. pilosa is found mostly due to the shortage of potable water. Surface water is in the wild and homestead gardens in the KwaZulu-Natal not safe for human consumption and its consumption and Limpopo Provinces [19]. It is commonly known as often results in waterborne diseases such as gastroenter- Black-jack in South Africa and other countries [20]. Al- itis, which can be very severe in immunocompromised though B. pilosa is considered a weed in some parts of the individuals and children with underdeveloped immune world, in Africa it is commonly consumed as a rich source systems [2]. Water-borne diarrhoea and increased bacter- of food and medicine for human and animals [21, 22]. The ial drug-resistance remain a challenge for public health aerial parts of B. pilosa, i.e., the leaves, flowers, seeds, stems [3]. Diarrhoea constitutes a major cause of morbidity and and roots are used in folk medicine as dry powder, decoc- mortality in South Africa [4], due to widespread usage of tion, maceration or tincture [23]. The B. pilosa extracts non-potable water sources and unhygienic practices. The have been reported to possess antiviral [24], antifungal [25] importance of diarrhoea as a public health problem is and antibacterial properties [26–28]. marked by relatively reduced number of drugs for its The purpose of this study was to investigate the antibac- treatment due to their high costs [5]. The high costs of an- terial properties of D. cinerea and B. pilosa extracts against tibiotics [6] and shortage of healthcare facilities in some awidespectrumofcommonwaterborne diarrhoea-causing regions of South Africa [7] leave many affected people bacteria. Notably, B. pilosa has not been tested specif- with few options for the treatment of infections. ically for anti-diarrhoeal efficacy, while D. cinerea has Medicinal plants are commonly used in Africa in the been reported [29]. Little information is available on the treatment of many ailments and constitute the first health unintended effects that emanate from consumption of D. recourse for about 80% of the population [8]. Many rural cinerea preparations. It is deemed appropriate that when communities in South Africa still rely on traditional medi- testing for anti-diarrhoeal properties of medicinal plants, a cines for the treatment of gastric ailments and other dis- wide spectrum of appropriate microbiota, which are asso- eases [9]. Medicinal plants are rich in compounds that ciated with and are commonly implicated in diarrhoeal may potentially be natural drugs, which may serve as episodes in humans are used. It is important to broaden sources of alternative, affordable and safe antimicrobial the knowledge on the effectiveness of these selected plants treatment for common diseases. Considering the levels of as sources of anti-diarrhoeal agents against common diar- dependency on medicinal plant remedies, scientific re- rhoea causing bacteria; to support their traditional use search studies have largely reported on the antibacterial and to further shed light on the potential unintended ef- and other medicinal properties of the traditional medicinal fects associated with consumption of their herbal prepara- plants. However, there is less consideration for the investi- tions. The unintended effects of the plant extracts include gation of the safety and in situ efficacy of plant extracts their effect on probiotics and their retention of antibacterial used in the treatment of ailments such as diarrhoea. Prop- potency following exposure to gastric fluid. erties such as potency, possible presence of toxic com- pounds, spectrum range of the antimicrobial agents and Methods their effect on normal and beneficial flora should be con- Plants collection sidered with the use of plant preparations. These factors The selection of medicinal plants was based on their trad- form an integral part in the evaluation process of drug itional use as reported in Mabogo [10] and from personal leads. It is thus important to investigate the effectiveness communication with herbalists (Ramathavha R and of the plants used for the treatment of diarrhoea, and to Nemakwelengwe M, pers. com, 2014). Leaves of B. pilosa further study secondary complications, which may result Linn (Voucher specimen no: UNIN 12895) and D. cinerea from the usage of medicinal plants used traditionally to Chiov, (Voucher specimen no: UNIN 12894) were col- treat diarrhoea based on indigenous knowledge. lected from Venda (Miluwani village) and Mankweng area Dichrostachys cinerea Wight et Arn. and Bidens pilosa (University of Limpopo) in the Limpopo Province. The Linn were investigated in this study due to their wide Larry Leach Herbarium (UNIN) at the University of usage in the treatment of diarrhoea in rural communities. Limpopo was used as reference point for the identification These plant species are used by the vhaVenda people for of plants and as a repository for voucher specimens. the treatment of diarrhoea as prescribed by the trad- itional healers and the herbalists [10], Ramathavha R Plant extraction and Nemakwelengwe M (pers. com). The dried fruits Collected plant materials were air-dried in the dark at of D. cinerea are commonly used as condiments [11, 12], room temperature for several days, ground with an elec- for the management of fever and headache [13] and have tric grinder into fine powders and stored in airtight Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 3 of 10 containers. The powdered plant materials were subjected yellow spots against a purple background on the TLC to extraction using the direct and serial exhaustive methods plates. [30, 31]. Briefly, 2 and 5 g of powdered leaves were separ- ately mixed with 20 mL and 50 mL of hexane, dichloro- Quantitative determination of total phenolic content methane, ethyl acetate, acetone and methanol according to The amount of total phenolics in the extracts was quan- the solvents polarities from non-polar to the more polar titatively determined with the Folin-Ciocalteu method solvent in polyester centrifuge tubes. The tubes were [36]. Plant extracts were dissolved in methanol to shaken for 30 min at room temperature in a series 2 achieve a concentration of 2 mg/mL. A blank was pre- incubator shaker (New Brunswick Scientific Co., Inc) pared from all the reagents minus the plant extract. An at 200 rpm. The plant extract was filtered into pre-weighed aliquot of 0.5 mL for each sample was mixed with labelled vial through a Whatman No. 1 filter paper. The 2.5 mL of a 10-fold diluted Folin Ciocalteu reagent and process was repeated three times until the plant constitu- 2 mL of 7.5% sodium carbonate in a test tube. The tubes ents were fully collected and the extracts were combined. were covered with parafilm and allowed to stand for The solvents were removed under a stream of cold air 30 min at room temperature prior to taking the absorb- using a fan. Dried extracts were weighed, recorded and ance reading at 760 nm with the Glomax microtiter reconstituted in acetone to obtain stock solutions of plate reader (Promega, U.S.A). Gallic acid was used as a 100 mg/mL. Extracts were stored as aliquots at − 20 °C standard at concentrations of 0.01 to 0.05 mg/mL (m/v) until further tests for phytochemicals and antibacterial in methanol. The total phenolics were expressed as gallic activity. acid equivalent (GAE/mg of extracted material). Antibacterial activity of plant extracts Determination of phytoconstituents Bacterial cultures Qualitative phytochemical analyses of B. pilosa and D. The microbiota for the antimicrobial study included cinerea leaf powders were done for the following classes the surface water-borne isolate Klebsiella pneumoniae, of secondary metabolites: total phenols [32], terpenoids, commercial probiotics (combination of Lactobacillus steroids and flavonoids [33] and saponins [34]. acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lacto- Screening for phytochemical compounds bacillus bifidum, Lactobacillus breve, Lactobacillus lactis, The chemical constituents of the plant extracts were also and Lactobacillus thermophillus), E. coli ATCC25922™, analysed by thin layer chromatography (TLC) using Salmonella typhimurium ATCC13311™, Shigella boydii aluminium-backed TLC plates (Silica gel F254, Fluka) as ATCC9207™ and Vibrio parahaemolyticus ATCC17802™. reported by Kotze and Eloff [30]. Ten microliter of each The microorganisms were maintained on Nutrient agar extract (10 mg/mL) was loaded on TLC plates and de- slant at 4 °C and sub-cultured on fresh Nutrient agar veloped in saturated chambers using three mobile phases plates for 24 h at 37 °C prior to antimicrobial test. The of different polarities; namely, benzene/ethyl acetate/ammo- Nutrient broth (NB) was used for the Minimum Inhibition nia hydroxide (BEA) (non-polar/basic) (9:1:0.1), chloro- Concentration (MIC) assay. form/ethyl acetate/formic acid (CEF) (intermediate polarity/ acidic) (5:4:1) and ethyl acetate/methanol/water (EMW) Minimum inhibition concentration (polar/neutral) (10:5.4:4). The TLC plates were dried in the The microplate serial dilution method described by Eloff fume-hood. The developed compounds on the TLC plates [31] was used to determine the Minimum Inhibition Con- were examined under ultraviolet light (254 and 365 nm) centration (MIC) values of the plant extracts against bac- and sprayed with vanillin-sulphuric acid reagent [0.1 g terial pathogens in 96 well microtiter plates. MIC was vanillin (Sigma ®): 28 mL methanol: 1 mL concentrated defined as the lowest concentration of the crude plant ex- sulphuric acid] and heated at 110 °C for optimal colour tract that inhibits bacterial growth after incubation period. development. The plant extract was serially diluted with distilled water to a concentration range of 2.5–0.02 mg/mL as described Qualitative antioxidant activity by Eloff [31]. Hundred microliters each of fresh bacterial Antioxidant activity was determined using the 2, cultures of probiotics mixture (2.4 × 10 CFU/mL), S. 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scaven- boydii (2.4 × 10 CFU/mL) and S. typhimurium, E. coli, V. ging ability as reported by Deby and Margotteaux [35]. parahaemolyticus and K. pneumonia (1.10 × 10 CFU/mL) Thin Layer Chromatography (TLC) plates were devel- were added to the wells of the microtiter plates. Similar oped as described above. The plates were sprayed with serial dilutions were performed for Ampicillin (1 mg/mL) 0.2% (w/v) DPPH in methanol as an indicator. The as a positive control and acetone as a negative control. presence of antioxidant compounds was detected by Microtiter plates were covered and incubated at 37 °C for Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 4 of 10 24 h. After incubation, 2 mg/mL of p-iodonitrotetrazolium overnight at 37 °C in 5% CO . Cells were treated with vari- violet (INT) reagent was used as an indicator of bacterial ous concentrations of the extracts with decreasing dilutions growth. from 1000 to 200 μg/mL for 24 h. After 24 h of incubation, the treatment medium was aspirated and 100 μLofMTT Exposure of plant extracts to simulated gastric fluid at a concentration of 1 mg/mL was added and further incu- Simulated gastric fluid (SGF) was prepared according to bated for 3 h at 37 °C in the incubator. The formazan prod- the US Pharmacopeia. Two grams of NaCl, 3.2 g pepsin uct was solubilised in 100 μLofDMSOand was leftfor (Sigma) and 80 mL of 1 M HCl were mixed together to 30 min. Spectrophotometric analysis was performed using make 1 L with distilled water. Five hundred microliters the Glomax microtiter plate reader (Promega, U.S.A) at of each plant extract (10 mg/mL) that had an activity on 560 nm. Untreated cells with 1000 μg/mL of DMSO served MIC was incubated with 1 mL of SGF in a shaking incu- as a negative control and Actinomycin D at a concentration bator at 200 rpm for 2 h at 37 °C. Following incubation, of 40 μg/mL was used as a positive control. The percentage MIC was determined for residual antibacterial activities of cell viability was calculated using the [(A/B) × 100] for- of the plant extracts. mula, where A is the absorbance value for treated cells and B is the absorbance value for untreated cells. Synergistic activity The choice of leaf extracts for use in this assay was Results and discussion based on the lowest MIC values obtained in this study The plant extraction capabilities of the different solvents for the individual plant extracts. Following the investiga- of varying polarities using the direct and serial exhaust- tion of the independent MIC of the selected plants, the ive extraction approaches are indicated in Fig. 1. Extrac- synergistic or antagonistic interactions between the ex- tion is a very important first step in the analysis of tracts were investigated. Aliquots of 100 μL of bacterial medicinal plant properties because the choice of solvent cultures were grown in 100 mL of nutrient broth for influences the types of compounds that can be extracted 24 h at 37 °C. Different active plant extracts were com- and ultimately, the biological activities imparted by the bined at a ratio of 1:1 (v/v). The extract interactions extracted compounds. Two extraction methods were were achieved by determining the MIC of the combina- used to check which one will give better yield. After dry- tions exhibiting antibacterial activity to establish any ing the extracts, better yield was obtained when using interaction effect. The fractional inhibitory concentra- the direct extraction method than the serial exhaustive tion (FIC) was calculated for the 1:1 combinations of the method (results not shown). This is in agreement with plants. This was determined with the equation below, the study conducted by Masoko et al. [40]. During the where (i) and (ii) represented the different 1:1 plant com- phytochemical analysis, more non-polar compounds binations [37]. The FIC index was expressed as the sum of were reactive to vanillin sulphuric acid reagent than the FIC and FIC and this was used to classify the inter- polar compounds (Fig. 2). This infers that both plant (i) (ii) action as either synergistic (≤0.50), additive (0.50–1.00), species contain more of non-polar than polar com- indifferent (> 1.00–4.00) or antagonistic (> 4.00) [38]. pounds based on the phytochemical profiles. More compounds with antioxidant activity were ob- MIC of ðÞ a in combination with ðÞ b served in the intermediate and polar extractants in both FICðÞ i MIC of ðÞ a independently plant species, i.e., ethyl acetate, acetone and methanol, which separated well in the polar and intermediate polar MIC of ðÞ b in combination with ðÞ a FICðÞ ii mobile systems (Fig. 3). This observation is not uncom- MIC of ðÞ b independently mon based on the study by Masoko and Eloff [41] and Sudha and Srinivasan [42] who reported the predomin- Evaluation of the cytotoxic activity of the plant extracts on ant presence of antioxidant compounds in polar than in cell culture non-polar plants extracts. The toxic effect of the extracts on muscle cell line C2C12 The total phenolic content (Fig. 4) of the leaf extracts was assessed by the 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphe- of B. pilosa and D. cinerea was expressed using the gallic nyltetrazolium bromide (MTT) (Sigma®) method [39]. The acid equivalent. Ethyl acetate, acetone and methanol ex- amount of the insoluble formazan formed from reduction tracts were selected for determination of the total phen- of MTT is directly proportional to the amount of viable olic content because of the high antioxidant activities of cells. Cells were maintained in Dubleco’s Modified Essential the extracts observed in this study (Fig. 3). Furthermore, Medium (DMEM) supplemented with 10% of Foetal bovine phenolic compounds have been reported to exhibit anti- serum (FBS) and 1× Penicillin-Streptomycin-Neomycin oxidants activity [43]. The total activity increased with (PSN). The C2C12 cells were seeded at a density of 5 × 10 an increase in polarity of the solvent for D. cinerea while cells/well in a 96 well microtiter plates and incubated and the converse was observed for B. pilosa. The type of Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 5 of 10 H DCM EA A M H DCM EA A M Direct method Serial exhaustive method B. pilosa D. cinerea Fig. 1 The percentage yield of B. pilosa and D. cinerea leaf extracts following extraction using the direct and serial exhaustive extraction methods. H, hexane; DCM, dichloromethane; EA, ethyl acetate; A, acetone; M, methanol solvent used for extraction plays a role in the ability to the distribution of antioxidant compounds from D. cinerea extract phenolic components from plant materials [44]. was dominant in the polar to intermediate polar mo- Several studies have shown that methanol and acetone bile system and the phenolic activity was higher in can extract higher amount of phenolics [45–47] and the the polar extracts. On the contrary, the antioxidant presence of phenolic compounds might be responsible activity for B. pilosa was apparent in the intermediate for the bioactivity of these extracts. The positive associ- to non-polar mobile systems while the total phenolic ation between total phenolic content and antioxidant ac- activity was higher in the intermediate polar extract tivity of B. pilosa and D. cinerea was observed, because (Figs. 3 and 4). Fig. 2 TLC fingerprint profile of plants extracted using the direct extraction method eluted in three solvent systems of varying polarities that show reactive compounds detected by vanillin sulphuric-acid reagent. BEA, Benzene: ethanol: ammonia hydroxide; CEF, Chloroform: ethyl acetate: formic acid; EMW, Ethyl acetate: methanol: water; H, hexane; D, dichloromethane; EA, ethyl acetate; A, acetone; M, methanol Percentage yield (%) Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 6 of 10 Fig. 3 The presence of antioxidant activity depicted by the yellow bands against a purple background. DPPH (0.2%) in methanol was used as an indicator. BEA, Benzene: ethanol: ammonia hydroxide; CEF, Chloroform: ethyl acetate: formic acid; EMW, Ethyl acetate: methanol: water; H, hexane; D, dichloromethane; EA, ethyl acetate; A, acetone; M, methanol The phytochemical compounds assayed in B. pilosa type of solvent used for the extraction. The dichloro- showed a predominance of steroids whereas in D. cinerea methane extracts of both plant species had high anti- the phenols saponins were the most dominant (Table 1). bacterial activity against all the bacterial species tested The antibacterial activity of the medicinal plant ex- with an average MIC value of 0.56 mg/mL (Table 2). tracts varied according to the bacteria tested and the Nevertheless, K. pneumoniae was sensitive to B. pilosa Fig. 4 The total phenolic content of leaf extracts of B. pilosa and D. cinerea extracted by ethyl acetate, acetone and methanol using the direct extraction method Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 7 of 10 Table 1 Phytoconstituents of B. pilosa and D. cinerea leaf although no antioxidant activity was detected for this extracts solvent in this study. Shigella sp. was the most suscep- Phytochemicals tested B. pilosa D. cinerea tible of all the test bacteria with an MIC values of 0.04 mg/mL. The observed antibacterial activity against Phenols – +++ the probiotics must be considered by the consumers Steroids +++ + and traditional healers for continuous consumption of Flavonoids + ++ the traditional remedies as this may pose a significant Saponins – +++ health risk in sick patients and can further weaken their Terpenoids ++ + immune system. Destabilisation of probiotic activity in - Absent, + slightly present, ++ moderately present, +++ highly present the human gut often creates an imbalance of gut flora and this aggravates diarrhoeal episodes. The imbalance and D. cinerea extracts but resistant to hexane extracts of the intestinal flora may consequently lead to the of both plant species. Ethyl acetate extract of D. cinerea surge of opportunistic pathogens, and this occurrence showed no activity against most of the tested bacteria, may inadvertently reverse the beneficial effect of the plant including the probiotics. The dichloromethane extract extract, and eventually produce a detrimental effect. Some of both plant species specifically showed higher anti- of the extracts had high MIC values (1.25–2.5 mg/mL) bacterial activities against the test organisms, with the that is an indication of lack of activity against tested most being against the probiotics for B. pilosa.The microorganisms. The secondary metabolites, namely, lower antibacterial activities are generally biased to- terpenoids, steroids and flavonoids were detected in the ex- wards the non-polar solvents for B. pilosa,and this is tracts (Table 1) and these compounds are known to have congruent with the dominance of anti-oxidant activities antibacterial activities [48]. Previous studies has reported and phenolic content which separated more in the antibacterial activity of D. cinerea against Staphylococcus non-polar solvents as well. It is noteworthy to mention aureus, Bacillus subtilis, Escherichia coli, Pseudomonas that all the extracts were less potent against the test aeruginosa and Candida albicans [49]. da Silva et al. [50] bacteria due to the observed high MIC values and again also reported antibacterial activity of B. pilosa against the potency of ampicillin which was used as a positive Staphylococcus aureus. The findings in this study that polar control. Hence, these extracts may not have good can- solvents such as acetone and methanol produced better didates as drug leads for antibacterial activity because antioxidant and antibacterial activities, support the trad- of these high MIC values due to the importance of the itional use of water as an extractant for preparations of potency of the antibacterial agent in drug development, remedies. amongst other factors. Comparatively, the dichloro- All the plant extracts that had MIC value ≤1 mg/mL methane extracts of both plant species gave lower MIC against tested bacteria were exposed to the simulated values against all the test organisms and the probiotics, gastric fluid (SGF). Table 3 shows the results of the plant extracts after exposure to the SGF. B. pilosa and D. cinerea Table 2 The MIC values of B. pilosa and D. cinerea leaf extracts hexane extracts lost their antibacterial activityfollowing the against selected microorganisms Bacteria Plants MIC values (mg/mL) Table 3 The MIC values of B. pilosa and D. cinerea leaf extracts H D EA A M Ampicillin after exposure to simulated gastric fluid S. typhimurium B. pilosa 1.25 1.88 0.31 2.5 2.5 0.23 Bacteria Plants MIC values (mg/mL) D. cinerea 2.5 1.25 2.5 1.25 1.25 H D EA A M Ampicillin S. boydii B. pilosa 0.16 0.08 0.94 0.16 0.16 0.04 S. typhimurium B. pilosa 2.5 NA 2.5 NT NT 0.63 D. cinerea 1.25 0.04 0.31 0.31 0.16 D. cinerea NA NA NT 1.25 1.25 V. parahaemolyticus B. pilosa 0.31 0.31 0.47 0.47 0.94 0.12 S. boydii B. pilosa NA 1.25 2.5 1.25 1.25 0.31 D. cinerea 0.63 1.88 2.5 1.88 2.5 D. cinerea NA 0.63 1.25 0.63 1.25 E. coli B. pilosa 1.25 0.08 0.63 0.63 1.25 0.31 V. parahaemolyticus B. pilosa NA NA 1.25 1.25 1.25 0.31 D. cinerea 2.5 0.63 2.5 2.5 2.5 D. cinerea NA NA NA 2.5 NA K. pneumoniae B. pilosa 2.5 0.12 0.31 0.31 0.31 0.47 E. coli B. pilosa NA 1.25 1.25 2.5 NA 0.63 D. cinerea 2.5 0.31 0.63 0.16 0.16 D. cinerea NT NA NT NT NA Probiotics B. pilosa 0.31 0.01 0.31 0.16 0.16 0.47 K. pneumoniae B. pilosa NT 1.25 2.5 2.5 NA 0.63 D. cinerea 2.5 0.16 2.5 1.25 1.25 D. cinerea NT NA 1.25 1.25 1.25 Average 1.47 0.56 1.16 0.97 1.1 NT Not tested, NA No activity, H hexane, D dichloromethane, EA ethyl acetate, H hexane, D dichloromethane, EA ethyl acetate, A acetone, M methanol A acetone, M methanol Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 8 of 10 Table 4 MIC values of synergistic activity for B. pilosa and D. Cytotoxicity effect of the extracts of D. cinerea and cinerea leaf extracts B. pilosa leaves were investigated against C2C12 cell Bacteria B. pilosa + D. cinerea MIC values in FIC index line and the results are shown in Fig. 5. The effects (independent MIC, mg/mL of extracts were compared with the positive control mg/mL) (combination) after exposure for 24 and 48 h. None of the crude S. typhimurium EA (0.31) + D (1.25) 0.63 2.53 extracts at different concentrations were toxic to S. boydii D (0.08) + D (0.04) 0.08 3 C2C12 cell lines for both plants. Cytotoxicity of these V. parahaemolyticus H (0.31) + H (0.63) 0.08 0.38 two plants has been investigated for different cell line by other authors and the results showed no effect on E. coli D (0.08) + D (0.63) 0.16 2.25 the cell line after the treatment [53, 54]. The fact that K. pneumoniae D (0.12) + D (0.31) 0.31 3.58 there was no toxicity observed on the cell line makes EA ethyl acetate, D dichloromethane, H hexane the plants extracts beneficial to the rural community of South Africa. This is good especially in the case of treatment of SGF. Vermaak et al. [51] and Keating et al. B. pilosa which is used as a vegetable as part of everyday [52] reported that SGF can modify or degrade antibacterial food by the African people living in the rural villages. compounds in plants extracts and this can subsequently lead to the loss of initial antibacterial activity in the extracts. Conclusion In the present study, loss of activity was observed with Waterborne bacterial infections are a serious problem in polar extracts of D. cinerea against S. typhimurium as com- many developing countries and the decline in fresh pared with the plant extracts alone (Table 3). Interestingly, water resources aggravates these incidences. Africans the gastric fluid played a protective role towards the bac- preferentially use traditional medicine either as a choice teria by reducing their sensitivity to the extracts. The same or due to financial constraints wherein western medicine effect was observed with ampicillin, which served as the is not an option. While the biological activities of plant positive control. This protective phenomenon could explain extracts can be empirically validated, as was the case in their tenacious survival in the human gut during infections. this study, it remains important to investigate other un- The results obtained suggest that most of the activities of intended effects that may result in secondary complica- the orally consumed plant extracts are weakened because tions from consumption of these medicinal preparations. of the interaction with the SGF. This current study has shown that leaf extracts of B. All extracts with the lowest MIC values for all the bac- pilosa and D. cinerea are potential sources of antioxidant teria used were further subjected to synergistic analysis. and antimicrobial agents, which can find application in Table 4 shows the results of the MIC values of synergistic the treatment of bacterial diarrhoea conditions. Polar activity of B. pilosa and D. cinerea leaf extracts. Syn- solvents such as acetone and methanol showed good ergy was observed with an FID index of 0.38 for the antioxidant and antibacterial activities, and this is an ad- combination of extracts of B. pilosa and D. cinerea vantage to herbalist and traditional healers who use against V. parahaemolyticus, whereas the antibacterial water for preparations of remedies. However, the loss of activity of the other combinations did not improve from some antibacterial activity when plant extracts were ex- the independent MIC values of the individual B. pilosa posed to gastric fluid is concerning because this may and D. cinerea extracts. lead to continuous survival of the pathogens in the B. pilosa D. cinerea Concentration (µg/ml) Fig. 5 The cytotoxic effects of B. pilosa and D. cinerea extracts against C2C12 % Cell Viability Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 9 of 10 human gut and prolonged disease episodes. In addition, 5. Johnson J, Kuskowski M, Menard M, Gajewski A, Xercavins M, Garau J. Similarity between human and chicken Escherichia coli isolates in relation to the adverse effect of plant extracts on the probiotics can ciprofloxacin resistance status. J Infect Dis. 2006;194(1):71–8. be circumvented by re-dosing with probiotics food prod- 6. Debruyne D. 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The ethnobotany of the vhaVenda. Master of science MTT: (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PSN: Penicillin- dissertation. South Africa: University of Pretoria; 1990. Streptomycin-Neomycin; SGF: Simulated gastric fluid; TLC: Thin layer 11. Tchiegang C, Mbougueng PD. Chemical composition of spices used in the chromatography cooking of nah poh and nkui of western Cameroon. Tropicultura. 2005;23:193–200. 12. Kuate D, Etoundi BCO, Ngondi JL, Oben JE. Effects of Dichrostachys Acknowledgements glomerata spice on cardiovascular diseases risk factors in normoglycemic The authors are thankful to Mr. A Njanje for assistance with tissue culture and type 2 diabetic obese volunteers. Food Res Int. 2011;44(5):1197–202. assays and the University Herbarium for helping with the identification of 13. World Agroforestry Centre. Agroforestree Database. Nairobi: World the plants species. Agroforestry Centre; 2005. http://www.worldagroforestry.org/output/ agroforestree-database. (2017-08-04) Funding 14. Abdou Bouba A, Njintang YN, Scher J, Mbofung CMF. Phenolic compounds We would like to thank National Research Foundation for student financial and radical scavenging potential of twenty Cameroonian spices. Agric Biol J support under Scarce Skills Doctoral study programme, grant number 101384. North America. 2010;1:213–24. 15. Kuate D. Effects of some spices on glucose and lipid metabolism and Availability of data and materials oxidative stress. Cameroon: PhD Thesis. University of Yaounde. All data generated and analysed during this study are included in this article. 16. Kuate D, Etoundi BCO, Soukontoua YB, Ngondi JL, Oben JE. Antioxidant characteristics of Dichrostachys glomerata spice extracts. CyTA J Food. Authors’ contributions 2010;8:23–37. PDS carried out the experiments, analysed and interpreted the data on 17. Fankam AG, Kuete V, Voukeng IK, Kuiate JR, Pages JM. Antibacterial activities of efficacy of plant materials, influence of gastric juice and synergistic effects of selected Cameroonian spices and their synergistic effects with antibiotics against combinatorial treatments of bacteria. KMM, SCT contributed to the multidrug-resistant phenotypes. BMC Compl Altern Med. 2011;11:104–14. conception, the design of the study, the interpretation of the data. PM, 18. Fotie J, Nkengfack AE, Peter MG, Heydenreich M, Fomum ZT. Chemical analysed and interpreted the data. All authors prepared, proofread and constituents of the ethyl acetate extracts of the stem bark and fruits of approved the final manuscript. Dichrostachys cinerea and the roots of Parkia bicolor. Bull Chem Soc Ethiop. 2004;18(1):111–5. 19. Department of Agriculture, Forestry & Fisheries (DAFF). Most common Authors’ information Indigenous food crops of South Africa; 2013. p. 9. Ms. Pfarelo Daphney Shandukani, Doctoral Student; Dr. Shonisani Cathphonia 20. 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Antibacterial activity and in situ efficacy of Bidens pilosa Linn and Dichrostachys cinerea Wight et Arn extracts against common diarrhoea-causing waterborne bacteria

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Medicine & Public Health; Complementary & Alternative Medicine; Internal Medicine; Chiropractic Medicine
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Abstract

Background: Bidens pilosa and Dichrostachys cinerea extracts were investigated for the antibacterial properties against waterborne diarrhoeagenic bacteria. Methods: The plant materials were extracted using the direct and serial exhaustive methods using solvents of varying polarities, namely, hexane, dichloromethane, ethyl acetate, acetone and methanol. Qualitative phytochemical analysis and quantitative determination of total phenolic content of the leaf powders of the two plants were tested. The antioxidant activities of the plants were determined using the 2, 2-diphenyl-1-picrylhydrazyl method. The toxic effect of the extracts on C2C12 muscle cell line were assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method and the antibacterial activity was determined using the serial microbroth dilution. Results: Methanol leaf extracts both plants had the highest yield in both direct and serial exhaustive extraction methods. Phytochemical profiling of the extracts displayed the presence of various secondary metabolites. The Benzene: ethanol: ammonia hydroxide solvent system showed a good resolution of chemical compounds in plant extracts from both plants. Most antioxidant compounds observed were developed in chloroform: ethyl acetate: formic acid and ethyl acetate: methanol: water solvent systems. All the bacterial species tested were sensitive to the effect of different extracts of both plant species, with E. coli being less sensitive to the effect of the extracts from D. cinerea. Following the simulated gastric fluid (SGF) treatment, a decrease in the antibacterial potency of the extracts was observed. No extract was toxic to the C2C12 muscle cell line. Conclusion: The presence of the secondary metabolites and nontoxic effect of the two plants tested may affirm the medicinal value of these leaf extracts. Our results suggest that B. pilosa and D. cinerea contain constituents with antioxidant and antimicrobial activities, which could be used in the treatment of diarrhoea in a case where untreated surface water is used. Keywords: Diarrhoea, Simulated gastric fluid, Medicinal plants, Antibacterial activity, Probiotics * Correspondence: kgabo.moganedi@ul.ac.za Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 2 of 10 Background been reported to have antioxidant activity [14–16]and Many communities in South Africa still depend on un- anti-hypertensive and antibacterial effects [17, 18]. treated surface water for their domestic needs such as Bidens pilosa L. is a species of flowering plant of the drinking, cooking, laundry and for personal hygiene [1], Asteraceae family. In South Africa, B. pilosa is found mostly due to the shortage of potable water. Surface water is in the wild and homestead gardens in the KwaZulu-Natal not safe for human consumption and its consumption and Limpopo Provinces [19]. It is commonly known as often results in waterborne diseases such as gastroenter- Black-jack in South Africa and other countries [20]. Al- itis, which can be very severe in immunocompromised though B. pilosa is considered a weed in some parts of the individuals and children with underdeveloped immune world, in Africa it is commonly consumed as a rich source systems [2]. Water-borne diarrhoea and increased bacter- of food and medicine for human and animals [21, 22]. The ial drug-resistance remain a challenge for public health aerial parts of B. pilosa, i.e., the leaves, flowers, seeds, stems [3]. Diarrhoea constitutes a major cause of morbidity and and roots are used in folk medicine as dry powder, decoc- mortality in South Africa [4], due to widespread usage of tion, maceration or tincture [23]. The B. pilosa extracts non-potable water sources and unhygienic practices. The have been reported to possess antiviral [24], antifungal [25] importance of diarrhoea as a public health problem is and antibacterial properties [26–28]. marked by relatively reduced number of drugs for its The purpose of this study was to investigate the antibac- treatment due to their high costs [5]. The high costs of an- terial properties of D. cinerea and B. pilosa extracts against tibiotics [6] and shortage of healthcare facilities in some awidespectrumofcommonwaterborne diarrhoea-causing regions of South Africa [7] leave many affected people bacteria. Notably, B. pilosa has not been tested specif- with few options for the treatment of infections. ically for anti-diarrhoeal efficacy, while D. cinerea has Medicinal plants are commonly used in Africa in the been reported [29]. Little information is available on the treatment of many ailments and constitute the first health unintended effects that emanate from consumption of D. recourse for about 80% of the population [8]. Many rural cinerea preparations. It is deemed appropriate that when communities in South Africa still rely on traditional medi- testing for anti-diarrhoeal properties of medicinal plants, a cines for the treatment of gastric ailments and other dis- wide spectrum of appropriate microbiota, which are asso- eases [9]. Medicinal plants are rich in compounds that ciated with and are commonly implicated in diarrhoeal may potentially be natural drugs, which may serve as episodes in humans are used. It is important to broaden sources of alternative, affordable and safe antimicrobial the knowledge on the effectiveness of these selected plants treatment for common diseases. Considering the levels of as sources of anti-diarrhoeal agents against common diar- dependency on medicinal plant remedies, scientific re- rhoea causing bacteria; to support their traditional use search studies have largely reported on the antibacterial and to further shed light on the potential unintended ef- and other medicinal properties of the traditional medicinal fects associated with consumption of their herbal prepara- plants. However, there is less consideration for the investi- tions. The unintended effects of the plant extracts include gation of the safety and in situ efficacy of plant extracts their effect on probiotics and their retention of antibacterial used in the treatment of ailments such as diarrhoea. Prop- potency following exposure to gastric fluid. erties such as potency, possible presence of toxic com- pounds, spectrum range of the antimicrobial agents and Methods their effect on normal and beneficial flora should be con- Plants collection sidered with the use of plant preparations. These factors The selection of medicinal plants was based on their trad- form an integral part in the evaluation process of drug itional use as reported in Mabogo [10] and from personal leads. It is thus important to investigate the effectiveness communication with herbalists (Ramathavha R and of the plants used for the treatment of diarrhoea, and to Nemakwelengwe M, pers. com, 2014). Leaves of B. pilosa further study secondary complications, which may result Linn (Voucher specimen no: UNIN 12895) and D. cinerea from the usage of medicinal plants used traditionally to Chiov, (Voucher specimen no: UNIN 12894) were col- treat diarrhoea based on indigenous knowledge. lected from Venda (Miluwani village) and Mankweng area Dichrostachys cinerea Wight et Arn. and Bidens pilosa (University of Limpopo) in the Limpopo Province. The Linn were investigated in this study due to their wide Larry Leach Herbarium (UNIN) at the University of usage in the treatment of diarrhoea in rural communities. Limpopo was used as reference point for the identification These plant species are used by the vhaVenda people for of plants and as a repository for voucher specimens. the treatment of diarrhoea as prescribed by the trad- itional healers and the herbalists [10], Ramathavha R Plant extraction and Nemakwelengwe M (pers. com). The dried fruits Collected plant materials were air-dried in the dark at of D. cinerea are commonly used as condiments [11, 12], room temperature for several days, ground with an elec- for the management of fever and headache [13] and have tric grinder into fine powders and stored in airtight Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 3 of 10 containers. The powdered plant materials were subjected yellow spots against a purple background on the TLC to extraction using the direct and serial exhaustive methods plates. [30, 31]. Briefly, 2 and 5 g of powdered leaves were separ- ately mixed with 20 mL and 50 mL of hexane, dichloro- Quantitative determination of total phenolic content methane, ethyl acetate, acetone and methanol according to The amount of total phenolics in the extracts was quan- the solvents polarities from non-polar to the more polar titatively determined with the Folin-Ciocalteu method solvent in polyester centrifuge tubes. The tubes were [36]. Plant extracts were dissolved in methanol to shaken for 30 min at room temperature in a series 2 achieve a concentration of 2 mg/mL. A blank was pre- incubator shaker (New Brunswick Scientific Co., Inc) pared from all the reagents minus the plant extract. An at 200 rpm. The plant extract was filtered into pre-weighed aliquot of 0.5 mL for each sample was mixed with labelled vial through a Whatman No. 1 filter paper. The 2.5 mL of a 10-fold diluted Folin Ciocalteu reagent and process was repeated three times until the plant constitu- 2 mL of 7.5% sodium carbonate in a test tube. The tubes ents were fully collected and the extracts were combined. were covered with parafilm and allowed to stand for The solvents were removed under a stream of cold air 30 min at room temperature prior to taking the absorb- using a fan. Dried extracts were weighed, recorded and ance reading at 760 nm with the Glomax microtiter reconstituted in acetone to obtain stock solutions of plate reader (Promega, U.S.A). Gallic acid was used as a 100 mg/mL. Extracts were stored as aliquots at − 20 °C standard at concentrations of 0.01 to 0.05 mg/mL (m/v) until further tests for phytochemicals and antibacterial in methanol. The total phenolics were expressed as gallic activity. acid equivalent (GAE/mg of extracted material). Antibacterial activity of plant extracts Determination of phytoconstituents Bacterial cultures Qualitative phytochemical analyses of B. pilosa and D. The microbiota for the antimicrobial study included cinerea leaf powders were done for the following classes the surface water-borne isolate Klebsiella pneumoniae, of secondary metabolites: total phenols [32], terpenoids, commercial probiotics (combination of Lactobacillus steroids and flavonoids [33] and saponins [34]. acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lacto- Screening for phytochemical compounds bacillus bifidum, Lactobacillus breve, Lactobacillus lactis, The chemical constituents of the plant extracts were also and Lactobacillus thermophillus), E. coli ATCC25922™, analysed by thin layer chromatography (TLC) using Salmonella typhimurium ATCC13311™, Shigella boydii aluminium-backed TLC plates (Silica gel F254, Fluka) as ATCC9207™ and Vibrio parahaemolyticus ATCC17802™. reported by Kotze and Eloff [30]. Ten microliter of each The microorganisms were maintained on Nutrient agar extract (10 mg/mL) was loaded on TLC plates and de- slant at 4 °C and sub-cultured on fresh Nutrient agar veloped in saturated chambers using three mobile phases plates for 24 h at 37 °C prior to antimicrobial test. The of different polarities; namely, benzene/ethyl acetate/ammo- Nutrient broth (NB) was used for the Minimum Inhibition nia hydroxide (BEA) (non-polar/basic) (9:1:0.1), chloro- Concentration (MIC) assay. form/ethyl acetate/formic acid (CEF) (intermediate polarity/ acidic) (5:4:1) and ethyl acetate/methanol/water (EMW) Minimum inhibition concentration (polar/neutral) (10:5.4:4). The TLC plates were dried in the The microplate serial dilution method described by Eloff fume-hood. The developed compounds on the TLC plates [31] was used to determine the Minimum Inhibition Con- were examined under ultraviolet light (254 and 365 nm) centration (MIC) values of the plant extracts against bac- and sprayed with vanillin-sulphuric acid reagent [0.1 g terial pathogens in 96 well microtiter plates. MIC was vanillin (Sigma ®): 28 mL methanol: 1 mL concentrated defined as the lowest concentration of the crude plant ex- sulphuric acid] and heated at 110 °C for optimal colour tract that inhibits bacterial growth after incubation period. development. The plant extract was serially diluted with distilled water to a concentration range of 2.5–0.02 mg/mL as described Qualitative antioxidant activity by Eloff [31]. Hundred microliters each of fresh bacterial Antioxidant activity was determined using the 2, cultures of probiotics mixture (2.4 × 10 CFU/mL), S. 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scaven- boydii (2.4 × 10 CFU/mL) and S. typhimurium, E. coli, V. ging ability as reported by Deby and Margotteaux [35]. parahaemolyticus and K. pneumonia (1.10 × 10 CFU/mL) Thin Layer Chromatography (TLC) plates were devel- were added to the wells of the microtiter plates. Similar oped as described above. The plates were sprayed with serial dilutions were performed for Ampicillin (1 mg/mL) 0.2% (w/v) DPPH in methanol as an indicator. The as a positive control and acetone as a negative control. presence of antioxidant compounds was detected by Microtiter plates were covered and incubated at 37 °C for Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 4 of 10 24 h. After incubation, 2 mg/mL of p-iodonitrotetrazolium overnight at 37 °C in 5% CO . Cells were treated with vari- violet (INT) reagent was used as an indicator of bacterial ous concentrations of the extracts with decreasing dilutions growth. from 1000 to 200 μg/mL for 24 h. After 24 h of incubation, the treatment medium was aspirated and 100 μLofMTT Exposure of plant extracts to simulated gastric fluid at a concentration of 1 mg/mL was added and further incu- Simulated gastric fluid (SGF) was prepared according to bated for 3 h at 37 °C in the incubator. The formazan prod- the US Pharmacopeia. Two grams of NaCl, 3.2 g pepsin uct was solubilised in 100 μLofDMSOand was leftfor (Sigma) and 80 mL of 1 M HCl were mixed together to 30 min. Spectrophotometric analysis was performed using make 1 L with distilled water. Five hundred microliters the Glomax microtiter plate reader (Promega, U.S.A) at of each plant extract (10 mg/mL) that had an activity on 560 nm. Untreated cells with 1000 μg/mL of DMSO served MIC was incubated with 1 mL of SGF in a shaking incu- as a negative control and Actinomycin D at a concentration bator at 200 rpm for 2 h at 37 °C. Following incubation, of 40 μg/mL was used as a positive control. The percentage MIC was determined for residual antibacterial activities of cell viability was calculated using the [(A/B) × 100] for- of the plant extracts. mula, where A is the absorbance value for treated cells and B is the absorbance value for untreated cells. Synergistic activity The choice of leaf extracts for use in this assay was Results and discussion based on the lowest MIC values obtained in this study The plant extraction capabilities of the different solvents for the individual plant extracts. Following the investiga- of varying polarities using the direct and serial exhaust- tion of the independent MIC of the selected plants, the ive extraction approaches are indicated in Fig. 1. Extrac- synergistic or antagonistic interactions between the ex- tion is a very important first step in the analysis of tracts were investigated. Aliquots of 100 μL of bacterial medicinal plant properties because the choice of solvent cultures were grown in 100 mL of nutrient broth for influences the types of compounds that can be extracted 24 h at 37 °C. Different active plant extracts were com- and ultimately, the biological activities imparted by the bined at a ratio of 1:1 (v/v). The extract interactions extracted compounds. Two extraction methods were were achieved by determining the MIC of the combina- used to check which one will give better yield. After dry- tions exhibiting antibacterial activity to establish any ing the extracts, better yield was obtained when using interaction effect. The fractional inhibitory concentra- the direct extraction method than the serial exhaustive tion (FIC) was calculated for the 1:1 combinations of the method (results not shown). This is in agreement with plants. This was determined with the equation below, the study conducted by Masoko et al. [40]. During the where (i) and (ii) represented the different 1:1 plant com- phytochemical analysis, more non-polar compounds binations [37]. The FIC index was expressed as the sum of were reactive to vanillin sulphuric acid reagent than the FIC and FIC and this was used to classify the inter- polar compounds (Fig. 2). This infers that both plant (i) (ii) action as either synergistic (≤0.50), additive (0.50–1.00), species contain more of non-polar than polar com- indifferent (> 1.00–4.00) or antagonistic (> 4.00) [38]. pounds based on the phytochemical profiles. More compounds with antioxidant activity were ob- MIC of ðÞ a in combination with ðÞ b served in the intermediate and polar extractants in both FICðÞ i MIC of ðÞ a independently plant species, i.e., ethyl acetate, acetone and methanol, which separated well in the polar and intermediate polar MIC of ðÞ b in combination with ðÞ a FICðÞ ii mobile systems (Fig. 3). This observation is not uncom- MIC of ðÞ b independently mon based on the study by Masoko and Eloff [41] and Sudha and Srinivasan [42] who reported the predomin- Evaluation of the cytotoxic activity of the plant extracts on ant presence of antioxidant compounds in polar than in cell culture non-polar plants extracts. The toxic effect of the extracts on muscle cell line C2C12 The total phenolic content (Fig. 4) of the leaf extracts was assessed by the 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphe- of B. pilosa and D. cinerea was expressed using the gallic nyltetrazolium bromide (MTT) (Sigma®) method [39]. The acid equivalent. Ethyl acetate, acetone and methanol ex- amount of the insoluble formazan formed from reduction tracts were selected for determination of the total phen- of MTT is directly proportional to the amount of viable olic content because of the high antioxidant activities of cells. Cells were maintained in Dubleco’s Modified Essential the extracts observed in this study (Fig. 3). Furthermore, Medium (DMEM) supplemented with 10% of Foetal bovine phenolic compounds have been reported to exhibit anti- serum (FBS) and 1× Penicillin-Streptomycin-Neomycin oxidants activity [43]. The total activity increased with (PSN). The C2C12 cells were seeded at a density of 5 × 10 an increase in polarity of the solvent for D. cinerea while cells/well in a 96 well microtiter plates and incubated and the converse was observed for B. pilosa. The type of Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 5 of 10 H DCM EA A M H DCM EA A M Direct method Serial exhaustive method B. pilosa D. cinerea Fig. 1 The percentage yield of B. pilosa and D. cinerea leaf extracts following extraction using the direct and serial exhaustive extraction methods. H, hexane; DCM, dichloromethane; EA, ethyl acetate; A, acetone; M, methanol solvent used for extraction plays a role in the ability to the distribution of antioxidant compounds from D. cinerea extract phenolic components from plant materials [44]. was dominant in the polar to intermediate polar mo- Several studies have shown that methanol and acetone bile system and the phenolic activity was higher in can extract higher amount of phenolics [45–47] and the the polar extracts. On the contrary, the antioxidant presence of phenolic compounds might be responsible activity for B. pilosa was apparent in the intermediate for the bioactivity of these extracts. The positive associ- to non-polar mobile systems while the total phenolic ation between total phenolic content and antioxidant ac- activity was higher in the intermediate polar extract tivity of B. pilosa and D. cinerea was observed, because (Figs. 3 and 4). Fig. 2 TLC fingerprint profile of plants extracted using the direct extraction method eluted in three solvent systems of varying polarities that show reactive compounds detected by vanillin sulphuric-acid reagent. BEA, Benzene: ethanol: ammonia hydroxide; CEF, Chloroform: ethyl acetate: formic acid; EMW, Ethyl acetate: methanol: water; H, hexane; D, dichloromethane; EA, ethyl acetate; A, acetone; M, methanol Percentage yield (%) Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 6 of 10 Fig. 3 The presence of antioxidant activity depicted by the yellow bands against a purple background. DPPH (0.2%) in methanol was used as an indicator. BEA, Benzene: ethanol: ammonia hydroxide; CEF, Chloroform: ethyl acetate: formic acid; EMW, Ethyl acetate: methanol: water; H, hexane; D, dichloromethane; EA, ethyl acetate; A, acetone; M, methanol The phytochemical compounds assayed in B. pilosa type of solvent used for the extraction. The dichloro- showed a predominance of steroids whereas in D. cinerea methane extracts of both plant species had high anti- the phenols saponins were the most dominant (Table 1). bacterial activity against all the bacterial species tested The antibacterial activity of the medicinal plant ex- with an average MIC value of 0.56 mg/mL (Table 2). tracts varied according to the bacteria tested and the Nevertheless, K. pneumoniae was sensitive to B. pilosa Fig. 4 The total phenolic content of leaf extracts of B. pilosa and D. cinerea extracted by ethyl acetate, acetone and methanol using the direct extraction method Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 7 of 10 Table 1 Phytoconstituents of B. pilosa and D. cinerea leaf although no antioxidant activity was detected for this extracts solvent in this study. Shigella sp. was the most suscep- Phytochemicals tested B. pilosa D. cinerea tible of all the test bacteria with an MIC values of 0.04 mg/mL. The observed antibacterial activity against Phenols – +++ the probiotics must be considered by the consumers Steroids +++ + and traditional healers for continuous consumption of Flavonoids + ++ the traditional remedies as this may pose a significant Saponins – +++ health risk in sick patients and can further weaken their Terpenoids ++ + immune system. Destabilisation of probiotic activity in - Absent, + slightly present, ++ moderately present, +++ highly present the human gut often creates an imbalance of gut flora and this aggravates diarrhoeal episodes. The imbalance and D. cinerea extracts but resistant to hexane extracts of the intestinal flora may consequently lead to the of both plant species. Ethyl acetate extract of D. cinerea surge of opportunistic pathogens, and this occurrence showed no activity against most of the tested bacteria, may inadvertently reverse the beneficial effect of the plant including the probiotics. The dichloromethane extract extract, and eventually produce a detrimental effect. Some of both plant species specifically showed higher anti- of the extracts had high MIC values (1.25–2.5 mg/mL) bacterial activities against the test organisms, with the that is an indication of lack of activity against tested most being against the probiotics for B. pilosa.The microorganisms. The secondary metabolites, namely, lower antibacterial activities are generally biased to- terpenoids, steroids and flavonoids were detected in the ex- wards the non-polar solvents for B. pilosa,and this is tracts (Table 1) and these compounds are known to have congruent with the dominance of anti-oxidant activities antibacterial activities [48]. Previous studies has reported and phenolic content which separated more in the antibacterial activity of D. cinerea against Staphylococcus non-polar solvents as well. It is noteworthy to mention aureus, Bacillus subtilis, Escherichia coli, Pseudomonas that all the extracts were less potent against the test aeruginosa and Candida albicans [49]. da Silva et al. [50] bacteria due to the observed high MIC values and again also reported antibacterial activity of B. pilosa against the potency of ampicillin which was used as a positive Staphylococcus aureus. The findings in this study that polar control. Hence, these extracts may not have good can- solvents such as acetone and methanol produced better didates as drug leads for antibacterial activity because antioxidant and antibacterial activities, support the trad- of these high MIC values due to the importance of the itional use of water as an extractant for preparations of potency of the antibacterial agent in drug development, remedies. amongst other factors. Comparatively, the dichloro- All the plant extracts that had MIC value ≤1 mg/mL methane extracts of both plant species gave lower MIC against tested bacteria were exposed to the simulated values against all the test organisms and the probiotics, gastric fluid (SGF). Table 3 shows the results of the plant extracts after exposure to the SGF. B. pilosa and D. cinerea Table 2 The MIC values of B. pilosa and D. cinerea leaf extracts hexane extracts lost their antibacterial activityfollowing the against selected microorganisms Bacteria Plants MIC values (mg/mL) Table 3 The MIC values of B. pilosa and D. cinerea leaf extracts H D EA A M Ampicillin after exposure to simulated gastric fluid S. typhimurium B. pilosa 1.25 1.88 0.31 2.5 2.5 0.23 Bacteria Plants MIC values (mg/mL) D. cinerea 2.5 1.25 2.5 1.25 1.25 H D EA A M Ampicillin S. boydii B. pilosa 0.16 0.08 0.94 0.16 0.16 0.04 S. typhimurium B. pilosa 2.5 NA 2.5 NT NT 0.63 D. cinerea 1.25 0.04 0.31 0.31 0.16 D. cinerea NA NA NT 1.25 1.25 V. parahaemolyticus B. pilosa 0.31 0.31 0.47 0.47 0.94 0.12 S. boydii B. pilosa NA 1.25 2.5 1.25 1.25 0.31 D. cinerea 0.63 1.88 2.5 1.88 2.5 D. cinerea NA 0.63 1.25 0.63 1.25 E. coli B. pilosa 1.25 0.08 0.63 0.63 1.25 0.31 V. parahaemolyticus B. pilosa NA NA 1.25 1.25 1.25 0.31 D. cinerea 2.5 0.63 2.5 2.5 2.5 D. cinerea NA NA NA 2.5 NA K. pneumoniae B. pilosa 2.5 0.12 0.31 0.31 0.31 0.47 E. coli B. pilosa NA 1.25 1.25 2.5 NA 0.63 D. cinerea 2.5 0.31 0.63 0.16 0.16 D. cinerea NT NA NT NT NA Probiotics B. pilosa 0.31 0.01 0.31 0.16 0.16 0.47 K. pneumoniae B. pilosa NT 1.25 2.5 2.5 NA 0.63 D. cinerea 2.5 0.16 2.5 1.25 1.25 D. cinerea NT NA 1.25 1.25 1.25 Average 1.47 0.56 1.16 0.97 1.1 NT Not tested, NA No activity, H hexane, D dichloromethane, EA ethyl acetate, H hexane, D dichloromethane, EA ethyl acetate, A acetone, M methanol A acetone, M methanol Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 8 of 10 Table 4 MIC values of synergistic activity for B. pilosa and D. Cytotoxicity effect of the extracts of D. cinerea and cinerea leaf extracts B. pilosa leaves were investigated against C2C12 cell Bacteria B. pilosa + D. cinerea MIC values in FIC index line and the results are shown in Fig. 5. The effects (independent MIC, mg/mL of extracts were compared with the positive control mg/mL) (combination) after exposure for 24 and 48 h. None of the crude S. typhimurium EA (0.31) + D (1.25) 0.63 2.53 extracts at different concentrations were toxic to S. boydii D (0.08) + D (0.04) 0.08 3 C2C12 cell lines for both plants. Cytotoxicity of these V. parahaemolyticus H (0.31) + H (0.63) 0.08 0.38 two plants has been investigated for different cell line by other authors and the results showed no effect on E. coli D (0.08) + D (0.63) 0.16 2.25 the cell line after the treatment [53, 54]. The fact that K. pneumoniae D (0.12) + D (0.31) 0.31 3.58 there was no toxicity observed on the cell line makes EA ethyl acetate, D dichloromethane, H hexane the plants extracts beneficial to the rural community of South Africa. This is good especially in the case of treatment of SGF. Vermaak et al. [51] and Keating et al. B. pilosa which is used as a vegetable as part of everyday [52] reported that SGF can modify or degrade antibacterial food by the African people living in the rural villages. compounds in plants extracts and this can subsequently lead to the loss of initial antibacterial activity in the extracts. Conclusion In the present study, loss of activity was observed with Waterborne bacterial infections are a serious problem in polar extracts of D. cinerea against S. typhimurium as com- many developing countries and the decline in fresh pared with the plant extracts alone (Table 3). Interestingly, water resources aggravates these incidences. Africans the gastric fluid played a protective role towards the bac- preferentially use traditional medicine either as a choice teria by reducing their sensitivity to the extracts. The same or due to financial constraints wherein western medicine effect was observed with ampicillin, which served as the is not an option. While the biological activities of plant positive control. This protective phenomenon could explain extracts can be empirically validated, as was the case in their tenacious survival in the human gut during infections. this study, it remains important to investigate other un- The results obtained suggest that most of the activities of intended effects that may result in secondary complica- the orally consumed plant extracts are weakened because tions from consumption of these medicinal preparations. of the interaction with the SGF. This current study has shown that leaf extracts of B. All extracts with the lowest MIC values for all the bac- pilosa and D. cinerea are potential sources of antioxidant teria used were further subjected to synergistic analysis. and antimicrobial agents, which can find application in Table 4 shows the results of the MIC values of synergistic the treatment of bacterial diarrhoea conditions. Polar activity of B. pilosa and D. cinerea leaf extracts. Syn- solvents such as acetone and methanol showed good ergy was observed with an FID index of 0.38 for the antioxidant and antibacterial activities, and this is an ad- combination of extracts of B. pilosa and D. cinerea vantage to herbalist and traditional healers who use against V. parahaemolyticus, whereas the antibacterial water for preparations of remedies. However, the loss of activity of the other combinations did not improve from some antibacterial activity when plant extracts were ex- the independent MIC values of the individual B. pilosa posed to gastric fluid is concerning because this may and D. cinerea extracts. lead to continuous survival of the pathogens in the B. pilosa D. cinerea Concentration (µg/ml) Fig. 5 The cytotoxic effects of B. pilosa and D. cinerea extracts against C2C12 % Cell Viability Shandukani et al. BMC Complementary and Alternative Medicine (2018) 18:171 Page 9 of 10 human gut and prolonged disease episodes. In addition, 5. Johnson J, Kuskowski M, Menard M, Gajewski A, Xercavins M, Garau J. Similarity between human and chicken Escherichia coli isolates in relation to the adverse effect of plant extracts on the probiotics can ciprofloxacin resistance status. J Infect Dis. 2006;194(1):71–8. be circumvented by re-dosing with probiotics food prod- 6. Debruyne D. 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