Phytochemical screening and pharmacological evaluation of herbal concoctions sold at Ga Maja Limpopo Province

South African Journal of Botany 117 (2018) 1–10

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Phytochemical screening and pharmacological evaluation of herbal concoctions sold at Ga Maja Limpopo Province M.M. Matotoka, P. Masoko ⁎ Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa

a r t i c l e

i n f o

Article history: Received 22 February 2018 Received in revised form 6 April 2018 Accepted 20 April 2018 Available online xxxx Edited by S Van Vuuren Keywords: Concoction Toxicology Antioxidant Antibacterial

a b s t r a c t Informal street merchants and traditional health practitioners at Ga Maja (Limpopo Province) primarily offer consumers semi-processed herbal preparations that are indicated to have blood cleansing, detoxifying, antidiarrheal and pain relieving properties. The focus of this study was to evaluate the phytochemical composition of the concoctions and substantiate the pharmacological effects and safety indicated by the traders. Five herbal concoctions and plant material used in their preparation were purchased from five independent traders and a laboratory concoction was prepared according to the traders' instructions. Possible bacterial and fungal contaminants were isolated and identified using Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Qualitative phytochemical analysis was determined using standard chemical tests and thin layer chromatography. Total polyphenol content was quantified. Antioxidant activity was quantified using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay and ferric reducing power. Antimicrobial activities were determined using a broth micro-dilution assay and bioautography. Cell viability assay was used to determine the cytotoxicity of the concoctions. Pathogenic bacteria, Enterobacter cloacae, Enterobacter aerogenes, Escherichia coli and Klebsiella pneumoniae were identified as bacterial contaminants. The commercial concoctions and the laboratory standard had similar phyto-constituents and phytochemical fingerprint profiles. The antimicrobial properties of the concoctions were a result of synergistic effects of the compounds because no single compound was observed to have antimicrobial activities on the bioautograms. The phenolic content, antioxidant and antimicrobial activities varied substantially amongst the concoctions. The lack of standardisation methods reduces the pharmacological potential of the products. This study concludes that while plants with biological activities were used by the traders to prepare the concoctions, the efficacious concentrations to produce a therapeutic response were not adequately measured and adhered to. Furthermore, although the concoctions did not exhibit cyctotoxic effects, toxicities may arise from endotoxins produced by the microbial contaminants. Hygienic processing and packaging are essential to ensure that consumers receive quality products that are safe to consume. © 2018 The Authors. Published by Elsevier B.V. on behalf of SAAB. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

contribute to the short expiry period. Unfortunately, the production of such remedies leads to the depletion and wastage of plant material (Nwankwo et al., 2012). The plant parts commonly used as ingredients in preparation of the herbal concoctions include leaves, stems, barks, roots, rhizomes, bulbs and/or seeds. The complexity of the formulations is dependent on the severity of the ailment. Simple home remedies can be prepared for trivial ailments such as diarrhoea, coughs, pains and gastrointestinal disorder. However, more elaborate procedures of preparations are required for life-threatening conditions (Cano and Volpato, 2004). A single plant species can produce numerous bioactive compounds that are neither stringently required for metabolic processes nor do they form part of nutrition. The production of the compounds is subject to the interaction of the plant with the environment in which it is supposed to thrive (Okem et al., 2015). The biological activity of these compounds is attributed to their role in the plants' survival. Some of these compounds are synthesised to effectively shield the plant from

1. Background Informal street merchants and traditional health practitioners primarily offer consumers semi-processed herbal preparations that are commonly prepared in small batches. In preparing the herbal concoctions, fresh or dry plant material can be used; the plant material can either be macerated in water for several days or generally boiled in hot water (Ndhlala and Van Staden, 2012). In South Africa, herbal products that are sold by informal traders are usually indicated to be immune and energy boosters, blood cleansers, detoxifiers and aphrodisiacs (Ndhlala et al., 2009). Some of these formulations are unstable, vary in strength and generally have short shelf lives. The poor physical conditions employed in preparation, such as unsterile working environment and storage, ⁎ Corresponding author. E-mail address: [email protected] (P. Masoko). https://doi.org/10.1016/j.sajb.2018.04.013 0254-6299/© 2018 The Authors. Published by (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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bacterial, fungal, parasitic and viral attacks (Kennedy and Wightman, 2011). Therefore, in preparing herbal remedies by using various plant parts from different sources, one can realise the extent of the number of compounds which are present and the possibility for the constituents to chemically interact. There has been increasing interest in the use of herbal products. This is witnessed by the frequent use of herbal products by people not only living in the rural locations, but also urban areas. This demonstrates that even though impoverished rural communities use herbal products due to a lack of healthcare infrastructure and the cost of modern pharmaceuticals (Marsland, 2007), people in urban locations use traditional medicine due to the lack of trust in the ability of western medicine to treat not only the diseases itself but rather also the mental aspect of ill-health. The broad use of traditional herbal remedies has encouraged manufacturers, private traders and street merchants to capitalise on this upsurge by increasing the availability of herbal remedies to those who desire them (Ndhlala and Van Staden, 2012). The allure in the use of traditional herbal medicines is the holistic approach which is used to treat an individual. The mode of treatment does not only involve the use of herbal remedies, but includes performing incarnations that are believed to give the remedy more strength. Herbal concoctions here in this study were purchased from traders established at Ga Maja (Limpopo Province). The traders sell herbal concoctions indicated to have aphrodisiac, antidiarrheal, blood cleansing and pain relieving properties. The traders at these locations use 500 mL to 5 L recycled plastic bottles. The bottles are not labelled with product information and only the word of the trader has to be taken into account regarding the ingredients. This may compromise the products due to more prone possible microbial contamination, whilst the lack of labelling may permit adulteration regarding materials used. The proper labelling of herbal products is of utmost importance. Labels on herbal products provide the consumer with information about its contents, that is; the list and quantity of the active ingredients, the mode and frequency of administration and potential side effects. Moreover, the labels should include details about the expiry date, any additives such as preservatives and appropriate methods for storing and maintaining the product. The degree of chemical purity of phyto-medicines can be assessed using an array of analytical methods. In order to analyse the phytochemical profile of a complex mixture, techniques such a thin layer chromatography (TLC) become useful and powerful. It is time efficient and a quick resolution towards challenges involved with being able to discriminate and develop fingerprints for major chemical classes that are present in mixtures (Zeng et al., 2008). In order for the herbal concoctions to be reputable, maintain quality, reliability and be marketable, their efficacy and safety status must meet quality health standards. However, investigating herbal products is accompanied by the challenge that some herbalists, traditional healers

and/or traders are reluctant to divulge the ingredients and formulae to some of their products. This underscores research efforts aimed at standardising and providing evidence-based pharmacological effects of the remedies (Matotoka and Masoko, 2017). This study was undertaken to evaluate and compare the phytochemical profiles of different commercial concoctions as well as to substantiate the pharmacological effects indicated by the traders. Safety regarding the consumption on these concoctions was evaluated by assessing microbial contamination and cytotoxic effects. 2. Methods and materials 2.1. Sample procurement and processing Five herbal concoctions were randomly purchased from five independent traders from Ga Maja; this included the plant material used in their preparation. Table 1 shows the list of the collected plant material. The tables also include the vernacular names of the plants and the concoction samples in which they are added as ingredients. The voucher specimens for the collected plant species were deposited in the Larry Leach herbarium of the University of Limpopo. The plant material was left to dry at room temperature in a well ventilated room away from sunlight. The corms were cut into smaller pieces to increase the surface area of the parts to allow for a quicker drying period. The dried material was ground to fine powder using a commercial blender and stored in airtight glass containers. The samples were stored at room temperature in the dark. 2.2. Isolation and identification of microbial contaminants from concoctions The traders prepared the herbal concoctions by boiling the plant material (Table 1) in water and packaged them in recycled plastic bottles. The five products were cultured on nutrient agar (NA) (Fluka, Switzerland) and Potato dextrose agar (PDA) (Fluka, Switzerland) plates immediately on the day of collection, using the spread-plate technique. Under aseptic conditions, 100 μL of each concoction were spread over the surface of separate and labelled plates using a sterile bent glass rod. The PDA plates were incubated at 25 °C for 48 h and the NA plates at 37 °C for 24 h. Viable colonies were purified using light microscopy and Gram staining procedures. The identification of the pure cultures of bacteria was carried out using Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). This technology is rapid and primarily relies on the detection of a great range of proteins which enables it to differentiate and classify even closely related organisms (Biswas and Rolain, 2013). 2.3. Sample preparation In preparation, the traders rely on visual observation to try and keep the amount of plant material (leaves and roots) added constant.

Table 1 Plant material used by traders to prepare herbal concoctions. Plant material

Voucher number

Vernacular name

HC1

HC2

HC3

HC4

HC5

Kirkia wilmsii (leaves) Kirkia wilmsii (corm) Kirkia wilmsii (twigs) Hypoxis hemerocallidea (corm) Monsonia angustifolia (leaves) Drimia elata (corm) Sarcostemma viminale Vahlia capensis “Tšhikwana/Morotwa tšhwene” (powder)a

SS 94 SS 94 SS 94 SS 115 121393 S 18 121404 121394

Legaba/modumela Legaba/modumela Legaba/modumela Monna maledu Tee ya thaba Sekanama Moema Makgonatsohle

+ + − + + + − + −

+ + − + + + − + +

+ + − + + + + + +

+ + + + + − + + +

− + + + + − + + +

Key: (+): Included, (−): Not included, HC1-sample 1; HC2- sample 2; HC3-Sample 3; HC4-Sample 4; HC5-Sample5; LC- Lab Concoction. a To the best of our knowledge, Tšhikwana/Morotwa tšhwene is a powdered plant mixture consisting D. elata, S. viminale, V. capensis and roots of unspecified plant species. The traditional healers were reluctant to divulge all the components of this mixture citing that secrecy contributed efficacy of these powders because intervension bytheir ancestral gods enhanced the therapeutic action of the treatment.

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Regarding corms, the traders have a designated number that they add to their preparation. A standard laboratory herbal concoction was prepared in a similar procedure followed by the traders. One gram of each of the plants indicated to have been used in preparing the commercial concoctions was added to 140 mL of distilled water and boiled for 5 min. The standard and the purchased samples were separately filtered through a Whatman No.1 filter paper and freeze-dried. All the samples were reconstituted to a concentration of 10 mg/mL for use in subsequent analytical methods. 2.4. Phytochemical fingerprint Thin layer chromatography (TLC) was used to establish the phytochemical fingerprint of the samples. Ten microliters of 10 mg/mL of each sample were loaded onto aluminium-backed TLC plates (Merck, Silica gel F254) coated with silica. Butanol/acetic acid/water (BAW) (3:2:2) (Merk, technical grade) was used to separate the chemical species that constitute the concoctions. Once the mobile phase reached the solvent front, the chromatograms were air-dried. To detect separated compounds, ultra-violet (UV) light (254 and 365 nm) was used to visualise compounds which have the ability to fluoresce under that electromagnetic spectrum. 2.5. Screening of phyto-constituents Screening of phyto-constituents following TLC phytochemical analyses was performed; terpenoids (Salkowski test), flavonoids, cardiac glycosides (Keller- Killiani test), phlobatannin, steroids and tannins (Borokini and Omotayo, 2012). Procedures reported by Odebiyi and Sofowara (1978) were adapted for detecting saponins (froth test) and alkaloids (Drangendoff's reagent test). 2.6. Total phenolic content The quantity of phenolics present in each concoction was determined by using the Folin-Ciocaltleu reagent method as (Humadi and Istudor, 2008) with minor modifications. The concoction (10 μL) was diluted with 490 μL of distilled water, followed by the addition of 0.25 mL of folin-ciocaltleu reagent. To stop the reaction, Sodium carbonate (1.25 mL) was added and the mixture was incubated in the dark at room temperature for 30 min. An ultraviolet/visible (UV/VIS) spectrophotometer was used to determine the absorbance of the mixtures at 725 nm. A blank and the standard curve were prepared in a similar manner, except that the plant extracts were replaced by distilled water and various concentration of tannic acid (1.25, 0. 63, 0.31, 0.16, 0.08 mg/mL), respectively. The results obtained from the linear regression formula of the tannic acid standard curve were expressed as milligram tannic acid equivalence/gram of extract (mg of TAE/g extract). The experiment was conducted in triplicates and independently repeated three times. 2.7. Total tannin content The folin–ciocalteu method described by Tambe and Bhambar (2014) was used to determine the tannin content in the concoctions. Briefly, 100 μL of 10 mg/mL extract was added to a clean test tube containing 7.5 mL of distilled water. The Folin–Ciocalteu reagent (0.5 mL) was added to the mixture and vortexed. Ten millilitre of a 35% solution of sodium carbonate (Na2CO3) was added to mixture. The mixture in the tube was transferred to a 10 mL volumetric flask and the volume of the mixture was made up to 10 mL by distilled water. The mixture was shaken and kept at room temperature for 30 min in the dark. Gallic acid was used as a standard and reference standard solutions (1–0.625 mg/mL) were prepared. The absorbance for the solutions was measured against a blank that was prepared in the same manner as the test solutions without adding any extract. A UV/VIS spectrophotometer was used to measure

3

the absorbance at 725 nm. Tannin content was expressed as milligram gallic acid equivalence/gram of extract (mg GAE/g extract). The experiment was conducted in triplicates and independently repeated three times.

2.8. Total flavonoid content Total flavonoid content was determined by the aluminium chloride colorimetric assay described by Tambe and Bhambar (2014). Briefly, 100 μL of 10 mg/mL extract was added to 4.9 mL of distilled water in a clean test tube. To this reaction mixture, 300 μL of 5% sodium nitrite (NaNO2) dissolved in distilled water was added and the mixture was left at room temperature for 5 min. After the 5 min, 300 μL of 10% aluminium chloride (AlCl3) (dissolved in distilled water) was added to the reaction mixture. The reaction was allowed to stand for 5 min at room temperature, after which 2 mL of sodium hydroxide (NaOH) was added to the solution. The mixture in the test tube was then made up to 10 mL with distilled water. Quercetin was used as a standard. Different concentrations (500–31.5 μg/mL) of the quercetin were prepared in the same method as the extracts. The absorbance of the experimental samples and the standard were determined using a UV/VIS spectrophotometer at a wavelength of 510 nm. The blank was prepared in the same manner as the experimental and standard samples, however, 100 μL of distilled water was added instead of the concoctions. The total flavonoid content of the samples was expressed as milligram quercetin equivalence/gram of extract (mg QE/g extract).

2.9. Qualitative DPPH assay on TLC Thin layer chromatography coupled with 2,2-Diphenyl-1picrylhydrazyl (DPPH) (Sigma) was used to screen for possible antioxidant activity of the concoctions. The chromatograms were prepared and developed identically to phytochemical fingerprinting. DPPH solution (0.2% w/v) (prepared by dissolving 0.2 g of the DPPH free radical in 100 mL of methanol) was sprayed unto the air-dried chromatograms. The presence of antioxidant activity was indicated by the development of yellow spots against a purple background (Deby and Margotteaux, 1970).

2.10. Quantitative DPPH assay Free radical scavenging activity of the concoctions was quantified and compared using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (Sigma) method reported by Chigayo et al. (2016) with modifications. Briefly, different concentrations of the concoctions (250–15.63 μg/mL) were prepared to a volume of 1 mL of the solution. L-Ascorbic acid was used as standard by preparing the same concentration range as the concoctions. To this 1 mL solutions, 2 mL of 0.2 mmol/L DPPH solution dissolved in methanol was added and vortexed thoroughly. All the prepared mixtures were left to stand in the dark for 30 min. The control solution was prepared by adding 2 mL of 0.2 mmol/L DPPH to 1 mL of distilled water. After the elapsed time, the solutions were analysed with a UV/VIS spectrophotometer. The absorbance of the solutions was read at 517 nm and the percentage antioxidant potential was calculated using the formula:

%Inhibition ¼

Ac−As  100 Ac

where Ac is absorbance of the control solution, As is the absorbance of the concoctions. The experiment was run in duplicate and repeated three times.

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2.11. Ferric reducing power The ferric reducing power of the concoctions was determined using the methods of Vijayalakshmi and Ruckmani (2016) and Ahmed et al. (2012). Five different concentrations of the concoctions (625–39 μg/mL) were prepared by serially diluting a stock solution of 1250 μg/mL. The different concentrations (2.5 mL) were mixed with 2.5 mL of sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1% w/v in distilled water) respectively, in a test tube. The mixtures were vortexed after addition of solutions. The mixtures were incubated at 50 °C for 20 min. Two mL of trichloroacetic acid (10% w/v in distilled water) was added to the test tubes after incubation. The mixtures were centrifuged at 3000 rpm for 10 min and 5 mL of the resulting supernatant was transferred to a clean test tube. To this solution, 5 mL of distilled water and 1 mL ferric chloride (0, 1% w/v in distilled water) were added consecutively with thorough vortexing after each addition. A UV/VIS spectrophotometer was used to read the absorbance of solutions at 700 nm wavelength. The blank for this procedure was prepared in the same manner, however, the concoctions were replaced by an equal amount of distilled water. L-Ascorbic acid (625–39 μg/mL) was used as a positive control and was prepared similar to the concoctions. The experiments were performed in duplicates and repeated three times. 2.12. Micro-dilution assay for antimicrobial activity The antibacterial activity of the concoctions was determined using the micro-dilution methods described by Eloff (1998) and Masoko et al. (2005). Sterile distilled water (100 μL) was added to each well of a 96 well microtitre plate using a multi-channel micropipette. The plant extracts (100 μL) were separately serially diluted to 50% with the distilled water in the wells of the 96 well microtitre plates. The culture (100 μL) was aseptically added to each well. The antibiotic ampicillin was used as a positive control for bacteria and cycloheximide was used for fungi. Sterile distilled water served as a negative control. The microtitre plates were covered with laboratory plastic wrap and incubated for 24 h at 37 °C for bacteria and at 25 °C for fungi. After incubation, 40 μL of 0.2 mg/mL of p-iodonitrotetrazolium chloride (INT) (Sigma) dissolved in distilled water was added to each well of the microtitre plates and further incubated for 30 min (bacteria) and 2–3 h (fungi). INT, served as a growth indicator, whereby the growth of the microorganism reduced the tetrazolium salt to a purple formazan. MIC was determined as the lowest concentration of the plant extract that was able to inhibit bacterial growth i.e. MIC values were recorded as the concentrations of the lowest clear wells of each extract. Microbial growth in the wells was indicated by a violet-purple colour, whereas clear wells indicated growth inhibition. The assay was repeated three times in duplicate. 2.13. Qualitative antimicrobial activity The antibacterial activities of the concoctions were qualitatively determined using bioautography on TLC as described by Begue and Kline (1972) with modifications by Masoko and Eloff (2005). The chromatograms of the concoctions were prepared and developed in a butanol/ acetic acid/water (3:2:2) as described for phytochemical fingerprinting. The chromatograms were air-dried for 2 weeks to completely remove any possible lurking residue of the mobile system. The chromatograms were sprayed with overnight cultures of bacterial and fungal species using a spray-gun, until the plates were moistened. The moist chromatograms were incubated for 24 h at 37 °C (bacteria) and at 25 °C (fungi) in 100% relative humidity. After incubation, 0.2 mg/mL of piodonitrotetrazolium chloride (INT) (Sigma) dissolved in water was sprayed onto the chromatograms. The chromatograms were incubated at 37 °C (bacteria) and at 25 °C (fungi) in 100% relative humidity for 30 min (bacteria) and 2–3 h (fungi). Clear zones against a purple

background would serve as an indication of the presence of compounds in the extract that inhibited the growth of the microorganisms used. 2.14. Cytotoxicity assay In order to determine the toxicological outcomes of the consumption of the concoctions, their effect on cell viability of normal primary fibroblast Kmst-6 cell line (PC-201-012) was assessed. The MTT calorimetric assay described by Mosmann (1983) was performed with modifications. The cell culture was maintained in a flask with Dulbeco minimal essential medium (DMEM, Whitehead scientific) supplemented with 10% foetal bovine serum (FBS) (Adcock-Ingram). Prior to seeding 96 well microtitre plates, the medium discarded and the cells washed with 5 mL phosphate buffered saline (PBS). The PBS was discarded and 2 mL of trypsin was used to detach the cells from the surface. Trypan blue was used to dye the cells and an automatic cell counter (model) was used to quantify viable cells. The cells were diluted with DMEM to obtain 1 × 105 cells/mL cell suspension. One hundred microliter of the cell suspension was added into each of the wells of the 96 well microtitre plate using a multi-channel micropipette. The plates were incubated at 37 °C in a 5% carbon dioxide (CO2) incubator for 24 h to allow the cells to attach to the bottom surface of the wells. The stock solutions of the extracts were prepared to a concentration of 200 mg/mL dissolved in (6:4) dimethyl sulfoxide: distilled water (DMSO: dH2O). The stock solutions were diluted to 10 mg/mL with DMEM. The 10 mg/mL dilutions were then filter-sterilised into sterile 25 mL centrifuge tubes. The 10 mg/mL dilutions were further diluted to 1 mg/mL with DMEM supplemented with 10% FBS. In a separate sterile 96 well microtitre plate, the 1 mg/mL extracts were serially diluted 50% with in DMEM with FBS to obtain a concentration range of (1000–31.25 μg/mL). Prior to treating the cells with the prepared concentrations, the DMEM was aspirated from the cells and the cells were washed with 100 μL of 1X PBS. The PBS was then discarded. One hundred microliters of the extracts (1000–31.25 μg/mL) prepared in a separate 95 well plate were transferred to the plate containing the cell cultures. Thus, the different concentrations were used to evaluate their effect of cell viability. The microtitre plates were incubated at 37 °C in a 5% carbon dioxide incubator for 24 h. Following incubation, 50 μL of 1 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) dissolved in 1X PBS was added to each well and the plates were further incubated for 3 h. After incubation, the media was removed from the plates and 100 μL of DMSO was added to each well. The plates were carefully swirled to dissolve the purple formazan crystals. Purple formazan crystals are formed when MTT is reduced by metabolically active cells. Thus, the amount of formed formazan products produced provides an indication of the amount of viable cells. A microtitre plate reader (promega) was used to measure the absorbance of the purple colour at 560 nm. Cells treated with the extracts were compared with untreated cells (positive control) and the cells treated with actinomycin (negative control). 3. Results and discussion In the Limpopo Province, South Africa, the largest ethnic group occupying the land is the Bapedi and they constitute 57% of the regional populace (Limpopo Provincial Government, 2013). The Limpopo Province has a high distribution of Bapedi traditional health practitioners (THPs) and they are distributed mainly in the Capricorn, Sekhukhune and Waterberg districts, which collectively include more than 17 municipalities in which they are operational (Semenya and Potgieters, 2015). The traders at this marketplace utilise previously used bottles from sodas, water and energy drinks to store their herbal concoctions. In this study, safety was investigated by evaluating microbial contamination because during the stages of production of the herbal concoctions, there were no informed attempts by the handlers to decontaminate

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Table 2 Bacterial species isolated from herbal concoctions and identified with MALDI-TOF. SIM agar medium was used to evaluate motility and Hydrogen sulphide and Indole production. Identified bacterial species H2S production Indole production Motility Infection

Reference

Enterobacter cloacae Enterobacter aerogenes Escherichia coli Leclercia adecarboxylata Citrobacter braakii Klebsiella pneumoniae

− − − − + −

− + + + − −

+ + + + + −

Nosocomial pathogen: intra-abdominal, endocarditis and septic arthritis Respiratory, bacteremia and urinary tract Diarrheal diseases and urinary tract infections Blood, wounds, urinary tract infections in immunocompromised hosts Bacteremia Urinary tract infections, septicemia, pneumonia, bacteremia and meningitis

Pantoea agglomerans Bacillus subtilis

− −

− −

− −

Uncommon pathogen −

Fata et al., 1996 Philippe et al., 2015 Wanke, 2001 Tam and Nayak, 2012 Hirai et al., 2016 Cheng et al., 1991; Ko et al., 2002 Cruz et al., 2007 −

Key: (+): positive; (−): Negative.

the bottles and the plant parts used. Some of the plant parts used are underground parts [Kirkia wilmsii (corm), Hypoxis hemerocallidea (corm), Drimia elata (corm), “Tšhikwana/Moroto wa tšhwene” (powdered root mixture)] (Table 1). The large number and varieties of microorganisms that are associated with the soil are consequently carried onto the plant parts. The use of water is the only means by which the traders and traditional healers clean the plant material and bottles. This approach of sterilisation is inefficient to decontaminate the bottles, because water alone does not have the strength to remove all microbes. Table 2 represents the identified bacterial contaminants of the concoctions using MALDI-TOF. From these results, it was observed that all the isolated bacteria except for Bacillus subtilis, belonged to the Enterobacteriaceae family. This family of bacteria are Gram negative rods and some members are able to secrete endotoxins (lipopolysaccharides) that occur on the outer membrane of the bacterial cell. The secretion of these chemical substances can lead to sepsis and haemolysis to mention a few (Ramachandran, 2014). Motility is a beneficial characteristic, and motile bacteria are more advantaged in that they are able to adjust to vast environmental conditions. These bacteria are capable of propagating towards favourable conditions or move away from threats and this gives them the advantage over non-motile bacteria (Duan et al., 2011). This could explain the larger number of viable motile bacteria in the concoctions. Majority of the bacteria; Enterobacter cloacae, Enterobacter aerogenes, Escherichia coli, Leclercia adecarboxylata and Citrobacter braakii were found to be motile. Motility was observed as cloudy growth or protrusions moving away from the stab line in the tube. Motility was further validated by visualising wet mounts of the bacteria using a light microscope at 100× objective. Motile bacteria are more damaging, owing to their capability to colonise cells and propagate through vast host cells, tissues and vital organs (Duan et al., 2011). The intake of these concoctions suggests that consumers would be predisposed to bacterial infections that would effectively deteriorate their health status and may be more debilitating to immune compromised consumers. It is therefore appropriate and essential that more effective sterilisation methods be used to ensure the purity of the product. The contamination of the herbal concoctions by commonly occurring fungal species (Table 3) presents a health hazard to consumers. Fungi produce low-molecular weight secondary metabolites known as mycotoxins and exposure to these toxic metabolites leads to the onset of diseases in animals and humans. Dietary exposure remains the largest rick factor leading to mycotoxicoses (Bennett, 1987).

Thin layer chromatography (TLC) was used to separate the chemical constituents to establish phytochemical fingerprints of the samples. Fig. 1 demonstrates that compounds with structural diversity react differently and fluoresce at different wavelengths. The different colours observed on the chromatograms are indicative of the different chemical species present in the concoctions. Concoction 1, 2 and 3 seemed to share a closely related phytochemical profile. This suggests that they were prepared using at most similar plant constituents. An analogous observation was found in the phytochemical profiles of concoction 4, 5 and standard, in that these three concoctions also demonstrated a similar profile. Therefore, one is also tempted to postulate that these samples were prepared with more similar plant constituents. This indicates that the traders may share common knowledge regarding the pharmacological effects of the plants around their environment. Medicinal plants owe their pharmacological properties from bioactive phytochemicals that are involved not only in the preventive but also the curative aspect of diseases (Karunyadevi et al., 2009). The constituents of the concoctions are medicinal plants, therefore, to better understand the phytochemical profile and subsequent pharmacological effects of the concoctions, various phyto-constituents were screened. Table 4 shows the different phyto-constituents present in the concoctions. Phlobatannins were absent in all the concoctions and this suggests that either these class of phytochemicals was not present on the individual plants used or they were destroyed during preparation of the concoctions due to exposure to high temperatures. Cardiac glycosides have been used in the treatment of ulcers and diabetic disorders (Karunyadevi et al., 2009). Terpenoids were detected in all the concoctions and have been reported to have been applied in the treatment of microbial infections (Harborne and Williams, 2000). Flavonoids and tannins were also detected in all the concoctions, these phytochemicals are known as polyphenols and possess antioxidant, anti-allergic, antiinflammatory, antimicrobial and anticancer properties (Balasundram et al., 2006). The biological activities associated with saponins include anti-inflammatory, antimicrobial and cytotoxic effects (Sarikurkcu and Tepe, 2015). The presence of these different phyto-constituents in the concoctions specifies a host of potential beneficial ways they can be used to improve health. Compounds that can reduce oxidative stress in cellular metabolism are increasingly becoming popular in the food industry. This has led to the production of synthetic antioxidants, however, they have been met with a concern regarding their use. Phenolic compounds are a class of phytochemicals that display a number of biological activities which include, but not limited to anti-allergenic, anti-inflammatory,

Table 3 Yeast isolates identified from the concoction with Vitek 2. Identified species

Characteristics

Percentage identification score (%)

Infections

References

Stephanoascus ciferrii

Coccus budding yeast

98

De Gentile et al., 1991

Cryptococcus laurentii

Coccus budding yeast

96

Ear diseases, non-insulin heart disease and most especially with cases of onychomycosis Fungemia

Johnson et al., 1998

6

M.M. Matotoka, P. Masoko / South African Journal of Botany 117 (2018) 1–10

BAW

A

Table 4 Phyto-constituents present in the plants and plant mixtures used to manufacture herbal concoctions. Phyto-constituents

HC1

HC2

HC3

HC4

HC5

LC

Terpenoids Alkaloids Saponins Flavonoids Phlabatannins Cardiac glycosides Tannins Steroids

+ + + + − + + +

+ + + + − + + +

+ + + + − + + +

+ + + + − + + +

+ + + + − + + +

+ + + + − + + +

Key: (+): positive; (−): Negative, HC1-sample 1; HC2- sample 2; HC3-Sample 3; HC4Sample 4; HC5-Sample 5; LC- Lab Concoction.

HC1 HC2 HC3

HC4

B

HC1 HC2 HC3

HC5 LC BAW

HC4

HC5 LC

Fig. 1. Chromatograms showing separated compounds in the herbal concoctions. The chromatograms were developed in the BAW solvent system. The Fluorescing compounds were visualised with UV light at 365 (A) and 245 nm (B). Key: HC1-sample 1; HC2- sample 2; HC3-Sample 3; HC4-Sample 4; HC5-Sample 5; LC- Lab Concoction.

antimicrobial, antioxidant, anti-thrombotic and cardio-protective effects (Balasundram et al., 2006). The concoctions were indicated by the traders to have blood cleansing and pain relieving properties, henceforth, the total phenolic, flavonoid and tannin content of the concoctions was determined. The quantification of these phyto-constituents served as a swift approach to the investigation of the claimed therapeutic effects. From Fig. 1, it was observed that to a large extent the concoctions had similarities in their chemical profiles. Therefore, the inconsistencies in the polyphenol content (Table 5) of the herbal concoctions may indicate that the ratios of plants used by the traders could be compromised by the lack of adequate weighing and measuring systems. Moreover, some traders may add an easily accessible plant in larger quantities than those that are otherwise more challenging to obtain in order to meet the increasing demand of these herbal products. The high phenolic

content in the products was the basis for screening antioxidant activity because they are natural harbours of antioxidants. The herbal concoctions generally had high content of total phenolics. This could be because each plant species used in the preparation of the concoctions, additively deposited a certain amount of its constituents. However, the tannin content (Table 5) of the concoctions maybe less because the folin–ciocalteu reagent used to detect the tannins may have also detected other present phenolics. The standard, herbal concoction 1 and 2 exhibited compounds with moderate antioxidant activity visualised by the low intensity yellow colour on the chromatograms (Fig. 2). Results of the free radical scavenging activity of the concoctions are shown in Fig. 3. Herbal concoction 1, 2 and the lab standard had higher antioxidant activity than herbal concoction 3, 4 and 5. This observation indicated that the antioxidant activity of the concoctions is not dependent on single compounds but the collective interaction of the compounds in the crude extract. Ferric reducing power (FRP) measures the formation of Pearl's Prussian blue complex as a result of the reduction of Fe3+ to Fe2 + in the presence of antioxidant compounds (Fig. 4). Except for concoction 3, the concoctions generally showed good reducing activity compared to L-ascorbic acid. The antioxidant activity observed on the DPPH assay and ferric reducing power demonstrate that the mode of antioxidant activity of the concoctions is different i.e. they can donate both protons (DPPH assay) and electrons (FRP assay). Furthermore, these results demonstrate that single herbal concoctions can have different strengths pertaining to various mechanisms it uses to prevent oxidative damage. The combined effect of the different mechanisms may have the effect to amplify the therapeutic effect of the concoctions. Therefore, there could be basis for the use of the concoctions as detoxifiers and blood cleansing remedies. Furthermore, antioxidants have been implicated in the reduction of inflammatory responses (Govindappa et al., 2011) and thus their consumption could be basis for the reduction and management of pain mediators. Some of the selected plant materials used to prepare the concoctions have been reported to possess antibacterial activities against a vast number of microorganisms. The corm of H. hemerocalledia was reported by Katerere and Eloff (2008) to have a range of antibacterial activities Table 5 Total phenol content in herbal concoctions. Concentrations are expressed as milligram tannic acid equivalence per gram plant extract (mg TAE/g).a Herbal concoctions

Phenolic content (mg TAE/g extract)a

Tannin content (mg GAE/g extract)a

Flavonoid content (mg QE/g extract)a

Concoction 1 Concoction 2 Concoction 3 Concoction 4 Concoction 5 Lab concoction

65,551 ± 0,164 61,677 ± 0,064 52,636 ± 0,031 64,257 ± 0,187 55,761 ± 0,026 60,970 ± 0,103

7.754 ± 0.094 9.567 ± 0.142 4.505 ± 0.217 3.120 ± 0.062 7.175 ± 0.155 28.858 ± 0.471

1.561 ± 0.036 2.402 ± 0.076 0.536 ± 0.024 1.841 ± 0.002 1.754 ± 0.024 1.416 ± 0.036

Key: HC1-sample 1; HC2- sample 2; HC3-Sample 3; HC4-Sample 4; HC5-Sample 5; LC- Lab Concoction. a Values are mean ± standard deviation (SD) (n = 3).

M.M. Matotoka, P. Masoko / South African Journal of Botany 117 (2018) 1–10

BEA

HC1

HC2

HC3

HC4

HC5

LC CEF

HC1

HC1

HC2

HC3

HC4

HC2

HC3

HC4

HC5

HC5

LC BAW

LC

Fig. 2. Chromatogram of herbal concoctions developed in BEA, CEF and BAW solvent systems. The Chromatograms were sprayed with 0.2% DPPH solution. Yellow colouration against a purple background is indicative of antioxidant activity. Key: HC1-sample 1; HC2- sample 2; HC3-Sample 3; HC4-Sample 4; HC5-Sample5; LC- Lab Concoction.

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against both Gram negative and positive species. Eloff et al. (2010) demonstrated both the antibacterial and antifungal activities of K. wilmsii leaves. In the Limpopo Province, Drimia elata was reported by Semenya et al. (2013) to be mixed with several plants, and the mixtures used in treatment of HIV/AIDS. Therefore, although microbial contaminants were present as described above, the concoctions had to have some potential antibacterial activities against the contaminants due to the present plant constituents. Table 6 demonstrates the antibacterial activity against the bacterial contaminants. The results indicated that indeed the concoctions contained compounds with antibacterial activities, especially concoction 4 which has an average MIC of 0.63 mg/mL against the various bacterial contaminants. Results from Table 6 also highlight that although the concoctions consisted of antimicrobial compounds, the concentrations of the components may have been dilute in the final product to enforce an inhibitory response towards the bacterial contaminants. Therefore, the concentrations of the commercial herbal concoctions were not prepared to efficacious concentrations. As mentioned above, the lack of adequate weighing and measuring systems are overlooked challenges the informal traders face during the production of their products. The concoctions were screened for antibacterial and antifungal activities against causative pathogens. Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans (yeast) are economically important causative agents of diarrhoea (Ahmed et al., 2012). E. coli exhibited the most susceptibility towards all the concoctions, more especially with herbal concoction 1 (Table 6). The latter concoction also showed good antibacterial activity against Gram positive bacterial test species. The other concoctions exhibited low antibacterial activity towards Gram positive bacteria. The determined antibacterial activities of the concoctions suggest that they could be more effective in combating infections caused by Gram negative bacteria. Although the concoctions generally showed moderate antibacterial activity, there still lies potential antidiarrheal activity. This is because the mechanisms of diarrhoea and urinary tract infections are multidimensional, therefore, therapeutic actions may be a result of concoctions enhancing other biological activities such as antioxidant activity through synergy

120

100

% Inhibition

80

60

40

20

0 0

50

100

150

200

250

300

Concentration (µg/mL) Concoction1 Concoction5

Concoction 2 Lab concoction

Concoction3 Ascorbic acid

Concoction4

Fig. 3. Percentage free radical (DPPH) inhibition of the herbal concoctions. Ascorbic acid was used a standard to which the experimental samples are compared. Values used are means of triplicates ± standard deviation (n = 3).

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M.M. Matotoka, P. Masoko / South African Journal of Botany 117 (2018) 1–10

6

Absorbance (700nm)

5

4

3

2

1

0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Concentration of concoctions (mg/mL) Ascobic acid

Concoction 1

Concoction 2

Concoction 4

Concoction 5

Lab concoction

Concoction 3

Fig. 4. The ferric reducing power of herbal concoctions at varying concentrations expressed as Absorbance at 700 nm. Ascorbic acid was used as a standard to which the concoctions were compared. Values used are means of triplicates ± standard deviation (n = 3).

of the diverse phyto-constituents (Ahmed et al., 2012). At the highest tested concentration (2 mg/mL), the herbal concoctions showed no antifungal activity against C. albicans. Cell viability assay was conducted to determine the cytotoxic effect of the samples against normal primary fibroblast Kmst-6 cell line (PC201-012™) (human skin cells). In vitro toxicological studies employ vast assays to determine cell viability and cytotoxicity that results from the exposure of chemical substances. In turn the determinations from these in vitro cytotoxicity assays can be used to predict possible human toxicities (Fotakis and Timbrell, 2006). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a tetrazolium salt that is converted to an insoluble purple formazan by metabolically active cell. The tetrazolium ring is cleaved by succinate dehydrogenase present in the mitochondria. Due to the impermeability of the resulting formazan, this purple product becomes collected inside healthy cells. Therefore, the amount of absorbance (purple colour) gives an

estimation of the number of viable cells. The percentage viability of the concoctions is represented in Fig. 5(A and B). The lowest percentage cell viabilities observed were that of the lab concoction and sample 5 which were below 60% at 1000 μg/mL (Fig. 5B). A general trend that was observed after the 24 h incubation of the treated cell lines was that at higher concentrations of the concoctions, less cells were viable and this viability increased as the concentrations decreased. At the highest tested concentration (1000 μg/mL) percentage cell viability for samples (Begue and Kline, 1972; Balasundram et al., 2006; Ahmed et al., 2012) was above 60% and at the lowest tested concentrations (78 μg/mL), it was above 90% (Fig. 5A). Despite these differences, the cytotoxic concentration (CC50) values of all the concoctions were above the highest concentration used (1000 μg/mL); comparatively to the negative control, Actinomycin D (CC50 of 0.6 μg/mL). Acute toxicity seems less probable upon the consumption of the concoctions.

Table 6 Antibacterial activity (MIC mg/mL) of the concoctions against isolated bacterial contaminants and nosocomial infections.

4. Conclusion

Microorganism

HC1

HC2

HC3

HC4

HC5

LC

Amp (μg/mL)

MIC (mg/mL) Herbal concoctions against isolated bacterial contaminants E. coli 2.5 1.25 N2.5 0.63 1.25 1.25 K. pneumoniae 2.5 1.25 N2.5 0.31 0.63 0.63 P. agglomerans 1.25 0.31 N2.5 0.63 2.5 1.25 E. aerogenes 2.5 2.5 N2.5 0.63 0.31 2.5 E. cloacae 1.25 0.31 N2.5 1.25 1.25 1.25 L. adecarboxylata 1.25 2.5 N2.5 0.63 0.63 1.25 C. braakii 2.5 1.25 N2.5 0.63 1.25 1.25 B. subtilis 1.25 0.31 2.5 0.31 1.25 0.63 Average 1.88 1.21 N2.5 0.63 1.13 1.25

0.17 0.13 0.1 0.16 0.12 0.15 0.13 0.14 0.14

Herbal concoctions against pathogenic ATCC bacterial strains 0.63 2.5 2.5 2.5 2.5 1.25 0.63 2.5 2.5 2.5 2.5 2.5 0.63 2.5 2.5 0.63 1.25 1.25 0.31 0.63 0.63 0.63 0.63 0.31 0.55 2.03 2.03 1.56 1.72 1.33

0.08 0.16 0.13 0.16 0.13

S. aureus E. faecalis P. aeruginosa E. coli Average:

Key: HC1-sample 1; HC2- sample 2; HC3-Sample 3; HC4-Sample 4; HC5-Sample5; LC- Lab Concoction; Amp-Ampicillin.

The preliminary toxicological results from this study suggest that the concoctions may be non-toxic to human cells. The aspect of safety still needs to be addressed in that, toxicities can emanate from the microbial contaminants due to their ability to produce endotoxins and/or induce infections when in contact with the host's internal organs. Information, regulations and strategies of maintaining sterility need to be adhered to during preparation, handling and storage in order to eliminate microbial contamination. The lack of standardisation methods reduces the pharmacological potential of the products. Although plants with biological activities were used by the traders, the efficacious concentrations to produce a therapeutic response were not adequately measured and adhered to. This has a negative impact towards the expected pharmacological effects of their products. This study highlights the importance of standardisation to ensure the consumers receive quality products that have efficacy and safe to consume.

Declaration of interest The authors declare that there is no conflict of interest.

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Fig. 5. A: Percentage cell viability of the herbal concoctions. Untreated cells were used as a positive control and actinomycin was used as a negative control. B: Cytotoxic effects of the herbal concoctions expressed as percentage cell viability. Untreated cells were used as a positive control and actinomycin was used as a negative control.

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