In-Vitro bioactivity testing of Medicago sativa L. leaf for anti-microbial, and cytotoxicity screening against Vero cells
Makhele Thapelo Simon1, Makhoahle Pakiso Moses1*, Mashele Sitheni Samson1
1 Faculty of Health and Environmental Sciences, Central University of Technology, Free State Province, Bloemfontein/Thabure 9300, Central South Africa.
Correspondence: Makhoahle Pakiso Moses, Faculty of Health and Environmental Sciences, Central University of Technology, Free State Province, Bloemfontein/Thabure 9300, Central South Africa. [email protected]
ABSTRACT
Anti-Microbial Drug Resistance (AMR) in pathogenic microbial organisms is a major threat to global public health. A certain study. isolated and tested a phenyl-propanol derivative from Tabernaemontana incospicua Stapf. (Apocynaceae) that not only displayed significant antimicrobial effects against the infectious Haemophilus influenzae 9435337A with the Minimum Inhibitory Concentration (MIC) of 62.5 μg/mL but also provides potential proof-of-ability towards combating the AMR pandemic using medicinal plants. In-vitro anti-microbial activity testing of five Alfalfa leaf extracts was evaluated using the Broth Micro-Dilution Assay and the colorimetric Microplate Alamar Blue Assay (MABA). Clinical antimicrobials Gentamicin, Vancomycin, and Fluconazole were used as controls. Cytotoxicity assays were used to determine whether cells continue to proliferate after exposure to a test compound for a specific time. The 3,4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to assess Vero cell viability following exposure to five Medicago sativa L. extracts. Vero cells treated with hexane, diethyl ether, and water extracts induced no decrease in the total number of cells compared to the untreated control. However, an increase of dead cells was observed after methanol, and butanol extracts treatment at 200 µg/mL. The global impact of AMR is wide and adverse, causing extended hospitalizations that amount to higher medical bills and high mortality rates.
Keywords: Anti-microbial drug resistance (AMR), Minimum inhibitory concentration (MIC), Microplate alamar blue assay (MABA), 3,4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT)
Introduction
Pharmaceutical research and development continue to struggle to meet the clinical needs of novel and effective anti-microbial agents during the ever-evolving anti-microbial drug resistance pandemic [1, 2]. The last two decades have seen the development of two novel antibiotic classes against Gram-positive bacteria: lipopeptides and oxazolidinones [3]. But the deficient existing library of newly approved anti-microbial compounds motivates an urgent need for testing natural medicines with anti-microbial activity against pathogenic and drug-resistant micro-organisms.
While superbugs (Antimicrobial Drug-Resistant Pathogens) kill over 700 000 people a year [4], the insect world also possesses potential bioactivity against biofilms as in the use of the degrading enzymes derived from the rat-tailed maggot of the drone fly Eristalis tenax [5]. The production of harmonine by the harlequin ladybird (Harmonia axyridis) is another example of a potential nature-derived antimicrobial agent used against malaria and tuberculosis as demonstrated by Christian Rohrich and others [6].
The Gram-negative organisms are notoriously implicated in mostly fatal intra-abdominal infections, urinary tract infections, pneumonia, and bacteraemia. The strong outer membrane of Gram-negative pathogens is responsible for the globally observed antibiotic resistance [7]. The innovation and development by Zurich University and Polyphor; the so-called Outer Membrane Protein Targeting Antibiotics (OMPTA), is a novel class of chimeric, cationic, and peptidomimetic antibiotics that have since demonstrated therapeutic activity against antibiotic-resistant Gram-negative pathogens [7].
The Anti-microbial resistance pandemic is a global concern affecting both developed and developing countries. There is no alternative but to control the use of antimicrobial drugs, to interpret the mechanisms of resistance, and thus to engineer novel therapeutics against drug-resistant pathogens [8]. The need for effective and cost-friendly therapeutics is urgent and requires a multi-pronged approach that includes looking back into our past (Sankofa) for clues and solutions [8]. The use of plants and plant-derived agents for medicinal purposes by humans and animals is old, diverse, and advanced. For humans and animals, plants play a central role in their health and wellness, vitality, and survival as plants continue to supply a large share of beneficial compounds and molecules to the food and Agri-pharmaceutical industries.
The most common bacterial Urinary Tract Infection (UTI) in women is caused by Escherichia coli [9]. The infection can occur anywhere in the urinary tract but frequently affects the bladder, urethra, or kidneys [9]. Antibiotic drugs are invaluable in the management and treatment of infections but also in the decrease of deaths associated with infections [10]. At the same time, Enterococcus faecalis is responsible for several nosocomial infections of the bloodstream, urinary tract, and of surgical sites [11]. Bacterial Meningitis (caused by Streptococcus pneumoniae) is a life-threatening condition that can lead to brain damage with sensual and cognitive impairment [12]. The species of Candida are notorious in the bloodstreams of hospitalized patients, as well as generally infecting our mouths, skins, and vagina [13]. S. epidermidis is also well-known as a frequent cause of prosthetic joint infections, prosthetic valve endocarditis, and neonatal sepsis [14].
Anti-biotic susceptibility testing is an effective tool in the treatment and management of infections; it allows clinicians to select the appropriate anti-microbial agent and to prescribe with precise accuracy, the bactericidal/bacteriostatic dosage [15]. The prescribed anti-biotic needs to be effective without causing significant or severe negative side effects to the person or animal or plant. The application of anti-microbial susceptibility testing for the evaluation of the potential bioactivity of natural products is one of the effective interventions instigated to curb the current surge of global AMR [16].
Cytotoxicity assays are tools used to determine whether cells continue to proliferate after exposure to a test compound for a specific time. Cytotoxicity is usually assessed by methods that compare either the cellular (enzymatic) function and/or cell integrity of control cells to those exposed to a test compound [17]. We used a quantitative colorimetric assay based on the metabolic activity of cells to evaluate cell viability after exposure to Alfalfa extracts (MTT assay).
This paper aimed to evaluate the anti-microbial bioactivity in-vitro and to report the MIC of Alfalfa (Medicago sativa L.) leaf extracts against a panel of five clinical pathogenic strains: E. coli, E. faecalis, S. epidermidis, S. pneumoniae, and C. albicans using the Broth Micro-dilution method and Microplate Alamar Blue assay.
Materials and Methods
The test (plant material), the extraction, and the preparation for in-vitro bio-analysis
The plant material (dried M. sativa L. leaves) was purchased from a nursery and medicinal plant farm North of Pretoria (South Africa) with Batch Number MH 71(10kg) as shown in Figure 1. The plant material was cultivated by Zizameleni Farming based in Mamogalieskraal, Northwest Province of South Africa. The test material was cultivated using regenerative natural farming principles and the fertilizers used were all natural and certified organics. The material was air-dried and stored in a cool dry area away from light and heat. The dry, grassy, and pale green leaves were extracted using five solvents: Butanol, Diethyl-ether, Hexane, Methanol, and Water. Sixty (60) grams of powdered plant material were extracted in 1 litre (1000 mL) of every solvent. The extracts were filtered using the Buchner funnel and Whatman no.1 filter paper. The extracts were frozen at -40 °C and freeze-dried for 48 hours at a yield of 9 g of dried extracts. The dried extracts were stored at -4 °C until analysis.
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Figure 1. M. sativa L. leaves |
The in-vitro bioactivity testing of five Alfalfa leaf extracts against pathogenic micro-organisms
There are countless approaches and techniques employed for the screening of biological extracts for therapeutic potential use as antimicrobial agents. The Broth Micro-dilution susceptibility method in a 96-well microtiter plate has become the preferred method for drug susceptibility testing due to its small sample requirements and high-throughput rate [18]. Microplate Alamar Blue assay (MABA) is a colorimetric assay that can be utilized for drug-susceptibility testing based on oxidation/reduction reactions [19]. This assay uses an oxidation/reduction indicator; Alamar Blue (CellTiter-Blue®), that changes color from blue to pink, indicating bacterial growth. The color change can be read visually and quantified fluorometrically by excitation at 530 nm and detecting emission at 590 nm [19].
Materials for the anti-microbial bio-activity testing of Alfalfa extracts
Enterococcus faecalis, Staphylococcus epidermidis, and Escherichia coli (clinical strains) were grown in Mueller-Hinton (MH) broth (Merck, USA). Candida albicans were maintained in Malt Extract broth (Merck, USA). S. pneumoniae was grown in Brain-Heart Infusion broth (Merck, USA). One microorganism colony, from an overnight streak plate, was inoculated in broth (10 mL) and allowed to grow for 16 hours (log growth phase) at 37 °C. Control drugs used: Gentamicin sulfate and Vancomycin hydrochloride (Sigma, USA) were used as positive controls against Gram-negative and Gram-positive bacteria, respectively. Fluconazole (Sigma, USA) was used as a positive control against C. albicans. Antibiotics were dissolved in ddH2O at stock concentrations of 2 mg/mL and filter sterilized (0.2 µM filter). Working concentrations were prepared in broth, depending on the antibiotics’ MIC values. Test Extracts were dissolved in Dimethyl Sulfoxide (DMSO) at stock concentrations of 100 mg/mL. Working concentrations of 4 mg/mL were prepared in MH broth/Malt Extract/Brain-Heart infusion broth.
The Anti-microbial bio-activity testing procedure (broth micro-dilution assay)
50 µL of MH broth/Malt Extract/Brain-Heart infusion broth was added to all test wells (i.e., plant extracts and antibiotics), except for the highest plant extract and antibiotic concentration wells to which 100 µL of the working concentrations were added. Serial dilutions were prepared for the plant extracts: 2 mg/mL to 125 µg/mL and for antibiotics: 64 to 0.25 µg/mL. The cultures were assessed and adjusted to a 0.5 McFarland standard (absorbance at 600 nm = 0.08-0.1; equivalent to ~1.5x108 cells/mL) and 50 µL was added to each test well.
Preparation of Controls:
Antibiotic/medium control (50 µL broth + 50 µL of highest antibiotic concentration). Extract color control (50 µL broth + 50 µL of highest plant extract concentration). 4% DMSO control (50 µL broth + 50 µL 8% DMSO). Microorganism control (50 µL broth + 50 µL microorganism). Plates were sealed with microplate sealing tape and incubated at 37°C for 24 h.
CellTiter-Blue® assay
After treatment, 20 μL of CellTiter-Blue® Reagent (Promega) was added to each well and incubated for 1 h. Wells were observed for the color change, where a blue color represented no growth (non-viable cells) and a pink color represented growth (viable cells). The fluorescence was read at excitation and emission wavelengths of 535 and 590 nm, respectively, using a BioTek® SYNERGY Mx fluorometer (Winooski, VT, USA).
The in-vitro cell viability testing of five Alfalfa leaf extracts against vero cells
The 3,4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay is commonly used to assess cell viability. The MTT assay is a quantitative colorimetric assay based on the metabolic activity of cells. The yellow water-soluble tetrazolium dye is reduced to a dark blue-to-purple insoluble formazan product by mitochondrial enzymes of viable cells. The insoluble formazan product is dissolved in dimethyl sulfoxide (DMSO) and can be measured spectrophotometrically at 540 nm. The amount of product produced is proportional to the number of viable cells [20, 21]. The limitation of this method is that other factors can influence the metabolic activity of cells and therefore we cannot accurately measure cell death [20].
Measuring the integrity of cell membranes is an alternative reliable method that can be used to assess cell viability [20] Dual staining with Hoechst and Propidium iodide (PI) has been used to assess viable and nonviable cells, respectively. Hoechst 33342 is a membrane-permeant dye that binds to the minor grooves of double-stranded DNA. Hoechst 33342 is excited at a wavelength of 350 nm and emits a blue fluorescence at 461 nm. Because Hoechst is a cell-permeant dye it stains all nucleic acids and therefore gives a total cell count, i.e., live plus dead cells [22]. PI is a red fluorescent molecule that binds to DNA and is excited at 536 nm and emits light at 623 nm. PI uptake is dependent on the integrity of the cell membrane and cannot enter cells with intact membranes. The uptake and exclusion of this dye can therefore be used to discriminate between viable and nonviable cells [23, 24].
Materials and methods
Vero cells were purchased from Cellonex, South Africa. DMEM low glucose cell culture medium and FBS were purchased from GE Healthcare Life Sciences (Logan, UT, USA). PBS with and without Ca2+ and Mg2+ and trypsin-EDTA was purchased from Lonza (Walkersville, MD, USA). Bis-benzamide H 33342 trihydrochloride (Hoechst) and propidium iodide (PI) was purchased from Sigma (St. Louis, MO, USA).
Image acquisition and analysis
Images were acquired using the ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices). A plate acquisition setup for a 96-well plate at a magnification of 10 times was used. Spatial distribution for the acquisition was set at 9 sites per well (3 by 3). The 9 images per site cover roughly 70% of the total well area. Filters used included DAPI (for Hoechst) and Texas Red (for PI). Analysis of acquired images was performed using the Multi-Wavelength Cell Scoring analysis module of the MetaXpress version 6.1 High-Content Image Acquisition and Analysis Software.
Cell seeding and treatment
Vero cells were seeded at a density of 5000 and 3000 cells/100 µL/well and incubated overnight at 37°C for attachment. Treatment for cytotoxicity assay was done by adding 100 µL of extract to achieve final concentrations of 50, 100, and 200 μg/mL. Cells were incubated at 37°C for 48 h. Melphalan at 6.25, 12.5, and 25 μg/mL served as the positive control as shown below in Table 1.
Cytotoxicity testing of Alfalfa leaf extracts procedure
After 48 h treatment medium was removed. Hoechst 33342 was diluted in 10 mL DPBS with Ca2+ and Mg2+ to a final concentration of 5 ug/mL and added to wells using 100 μL aliquots. Cells were incubated at 37°C for 30 min. PI was diluted in 1 mL PBS to a concentration of 110 μg/mL and added to wells in 10 μL aliquots before image acquisition to achieve a final concentration of 10 μg/mL.
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Table 1. The Plate Layout |
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1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
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A |
[50] rep1 |
[100] rep1 |
[200] rep1 |
[50] rep1 |
[100] rep1 |
[200] rep1 |
[50] rep1 |
[100] rep1 |
[200] rep1 |
[50] rep1 |
[100] rep1 |
[200] rep1 |
|
B |
[50] rep2 |
[100] rep2 |
[200] rep2 |
[50] rep2 |
[100] rep2 |
[200] rep2 |
[50] rep2 |
[100] rep2 |
[200] rep2 |
[50] rep2 |
[100] rep2 |
[200] rep2 |
|
C |
[50] rep3 |
[100] rep3 |
[200] rep3 |
[50] rep3 |
[100] rep3 |
[200] rep3 |
[50] rep3 |
[100] rep3 |
[200] rep3 |
[50] rep3 |
[100] rep3 |
[200] rep3 |
|
D |
[50] rep4 |
[100] rep4 |
[200] rep4 |
[50] rep4 |
[100] rep4 |
[200] rep4 |
[50] rep4 |
[100] rep4 |
[200] rep4 |
[50] rep4 |
[100] rep4 |
[200] rep4 |
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E |
[50] rep1 |
[100] rep1 |
[200] rep1 |
UNTREATED CONTROL |
POSITIVE CONTROL: Melphalan): 6.25, 12.5, 25 µM |
UNTREATED CONTROL |
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F |
[50] rep2 |
[100] rep2 |
[200] rep2 |
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G |
[50] rep3 |
[100] rep3 |
[200] rep3 |
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H |
[50] rep4 |
[100] rep4 |
[200] rep4 |
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A |
Hexane (SUNTB 1) LABEL: 1 |
Diethylether (SUNTB 4) LABEL: 2 |
Methanol (SUNTB 6) LABEL: 3 |
Butanol (SUNTB 7) LABEL: 4 |
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B |
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C |
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D |
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E |
Water (SUNTB 10) LABEL: 5 |
UNTREATED CONTROL |
POSITIVE CONTROL: Melphalan): 6.25, 12.5, 25 µM |
UNTREATED CONTROL |
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F |
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G |
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H |
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Results and Discussion
Results of the screening of five extracts of Medicago sativa L. leaves for antimicrobial activity against a panel of five microorganisms (E. coli, E. faecalis, S. epidermidis, S. pneumoniae, and C. albicans), using the Broth Micro-dilution method and Microplate Alamar Blue assay
Anti-microbial drug resistance (AMR) in pathogenic bacteria is a global concern that is threatening the effectiveness of conventional clinical care systems and treatments. The World Health Organization (WHO) considers AMR “an urgent threat that requires a multisectoral action to achieve the Sustainable Development Goals (SDGs)” [25]. ARM also imposes a pressurizing force on major medical interventions such as surgery and cancer chemotherapy [26]. The poor service delivery to disadvantaged communities and villages in developing countries means a lack of water and poor sanitation which can affect infection control and prevention.
The Broth Micro-Dilution Assay was used to evaluate the anti-microbial activity of Medicago sativa L. leaves against five clinical strains (E. coli, E. faecalis, S. epidermidis, S. pneumoniae, and C. albicans). The method is favored for its highly accurate results, availability of testing plates and standard reagents, and the possibility of reading results both qualitatively (color changes) and quantitatively by Spectrophotometric techniques [27].
The twofold dilutions of the candidate anti-microbial agent (plant leaf extracts) were incubated in broth with the 0.5 McFarland standard adjusted organism suspension. The clinical antimicrobial agent (Vancomycin/Gentamicin/ Fluconazole). Following the 24-hour incubation period, 20 μL of CellTiter-Blue® Reagent (Promega) was added to each well and incubated for 1 h. Wells were observed for the color change, where a blue color represented no growth (non-viable cells) and a pink color represented growth (viable cells). The fluorescence was read at excitation and emission wavelengths of 535 and 590 nm, respectively, using a BioTek® SYNERGY Mx fluorometer (Winooski, VT, USA). Fluorescence readings of 96-well plates were measured at excitation and emission wavelengths of 535 and 590 nm after the addition of CellTiter blue. Percentage inhibition for the extracts was calculated for some extracts showing inhibition of growth, but not for those that produced slight pink coloration.
All the Medicago sativa L. Leaves Extracts did not show inhibitory activity against E. coli. Medicago sativa L. Leaves Extracts (Hexane, Diethyl ether, Butanol) exhibited growth inhibition against E. faecalis with MIC (Minimum Inhibitory Concentration) of < 0.125 mg/mL. This experimental result confirms the ethnobotanical indication that Medicago sativa L. leaves possess anti-microbial activity against pathogenic organisms [28].
Medicago sativa L. Leaves Extracts (Hexane, Diethyl ether, Butanol) demonstrated inhibitory activity against S. pneumoniae with MIC (Minimum Inhibitory Concentration) of < 0.125 mg/mL. All the Medicago sativa L. Leaves Extracts did not show any inhibitory activity against C. albicans and S. epidermidis. The lack of inhibitory effect against one or more organisms is not enough to discourage further testing and deeper experimentation of M. sativa L.’s Anti-microbial activity against other pathogens. The experimental findings establish a scientific fact that plants can inhibit the growth and multiplication of disease-causing micro-organisms. This finding gives the religious habit of drinking herbal tea for health and wellness more value and potentiates the use of Medicago sativa L. as a possible alternative non-drug anti-microbial agent.
Results of cytotoxicity screening of Alfalfa Extracts in vero cells
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Figure 3. Total number of cells (live + dead) after treatment with extracts and positive control (melphalan). |
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Figure 3. Number of live cells after treatment with extracts and positive control (melphalan). |
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Figure 4. Number of cells with PI positive staining (dead cell number) after treatment with extracts and positive control (melphalan). |
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Figure 5. Percentage PI positive after treatment with extracts and positive control (melphalan). |
Cytotoxicity assays are tools used to determine whether cells continue to proliferate after exposure to a test compound for a specific time. Cytotoxicity results are shown and discussed for a cell density of 5 000 cells/100 µL/well. The Total number of cells (live + Dead): Vero cells treated with hexane (1), methanol (3), and water (5) extracts did not show a decrease in the total number of cells compared to the untreated control (Figure 2). A decrease in the total number of cells was observed for diethyl ether (2) and butanol (4) extracts at 200 µg/mL (Figure 2). The Number of Live cells: Vero cells treated with hexane (1) and water (5) extracts did not show a decrease in the number of live cells compared to the untreated control (Figure 3). A decrease in the number of live cells was observed for diethyl ether (2), methanol (3), and butanol (4) at a concentration of 200 µg/mL compared to the untreated control (Figure 3). The Number of PI-positive Cells (dead cells): Vero cells treated with hexane (1) and water (5) did not show an increase in the number of PI-positive cells (Figure 4). Diethyl ether (2), methanol (3), and butanol (4) showed an increase in the number of PI-positive cells (Figure 4). The Percentage PI positive: Vero cells treated with hexane (1) and water (5) did not show an increase in the percentage of PI-positive cells (6. Diethyl ether (2), methanol (3), and butanol (4) showed a dose-dependent increase in the percentage of PI-positive cells (Figure 5). We report therefore report that the hexane and water extracts demonstrated no evidence of toxicity or cytostatic activity. While the diethyl ether, methanol, and butanol extracts demonstrated toxicity at 200 µg/ml but not lower concentrations.
Plants and other natural sources continue to provide a diverse variety of compounds, analogues, and derivatives that can be used to formulate and develop the much-needed anti-microbial drugs that can combat antibiotic-resistant pathogens [29]. Anti-microbial susceptibility testing of medicinal plants is not only used to confirm the ethnobotanical literature but also to evaluate the plant as a prospective anti-microbial agent against drug-resistant clinical pathogens. The use of alfac-facah for medicinal purposes includes use as a neuroprotective, anti-microbial, anti-cancer, and cholesterol-lowering agent [28]. Newly isolated triterpenoid saponins from M. sativa L. demonstrated neuroprotective potential against hydrogen peroxide-induced SH-SY5Y (Human Neuroblastoma) cell death compared to the hydrogen peroxide-treated group [28]. The toluene and methyl tert-butyl ether (MTBE) extracts of M. sativa L. demonstrated in-vitro cytotoxic effects on two murine leukemia cell lines P388, whence the extracts also induced programmed cell death by activation of the caspase-3, leading to poly ADP ribose polymerase cleavage (PARP) [29].
Conclusion
Despite the good recording of negative clinical-drug-to-herbal-drug interactions, medicinal plants continue to play a critical role in disease prevention and drug development [30]. The regular and consistent consumption of herbal infusions was a key action that our ancestors used to flavor water and also to siphon nutrients and secondary metabolites (phytochemicals) from plants and herbs availed by nature. The native approach to processing roots and tree barks included preparation of decoctions, some fomentations for the skin, and soaking or irrigating a specific body area. The primitive hunter was also proficient in dealing with poisonous/venomous snake and insect bites, thereby using poultice or cataplasm of macerated plant material, and wrapping it against the skin or an area of much swelling probably using a leaf or flexible bark as a bandage.
A large library of the medicinal plants used for medicinal purposes still resides mostly within our traditional healers, herbalists, and medicine men/women (li-Ngaka, Lingaka-Tjhitja, Pepeduma, Makherenkhoa, Makhehla, le Baloi). The conventional medical care system still needs to integrate traditional or native medicinal approaches into its standard operating procedures. Advanced native Health care systems such as Ayurveda, homeopathy, Yunani Medicine, Phytotherapy, and Herbal Medicine are still largely isolated and not included in the scope of conventional medical care.
We rely on the results of our experiments reported here, to suggest that the perpetual drinking of a herbal beverage (tea) made from Medicago Sativa/Alfalfa leaf infusion for health and wellness purposes is hereby scientifically confirmed. Although we understand that laboratory-based conditions are far from a true reflection of the conditions in-vivo, we are confident to recommend the further testing of Medicago sativa-derived phytochemicals, as our experiments and experiments of others, demonstrate a significant therapeutic potential induced by the reliable agricultural fodder: Medica Sativa/Lucerne/Alfac-facah.
Acknowledgments: We extend our deepest gratitude to God for all. We thank the Central University of Technology for employment, grants, and supportive structures. We thank the scientist and facilities of the CSIR (Council for Scientific and Industrial Research), the University of Stellenbosch, and Nelson Mandela University. We also extend our gratitude to the botanists and horticulturists of the South African Biodiversity Institute (SANBI) for their teachings. We cannot do anything without our families, friends, and enemies, so we thank them for their upliftment, motivation, and support. I am grateful for the financial support from (CUT) the Central University of Technology, Free State, and the (DHET) Department of Higher Education and Training UCDP GRANT DHET and CUT are not liable for any opinion, finding and conclusion, or recommendation addressed by the authors of the article.
Conflict of interest: None
Financial support: Central University of Technology, Free State, and (DHET) Department of Higher Education and Training UCDP GRANT.
Ethics statement: None
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