Background
The nutrition and health of the world population are the main upcoming challenges particularly in developing countries. In Sub Saharan Africa, there are more than 45,000 plant species of which about 1000 can be consumed as leafy vegetables which happen to be the mainstay of traditional African diets [
1]. Leafy vegetables are plant species whose leaves are used as a vegetable in the sauce preparation. They play a very important role in our diet and are the most readily available sources of carbohydrates, fats, proteins, vitamins, minerals, essential amino acids [
2,
3]. Many leafy vegetables are mainly consumed for their nutritional values since immemorial times without much consideration for their medicinal importance. Apart from their nutritional intake, they have the ability to synthesize several secondary metabolites of relatively complex structures possessing antioxidants [
4]. These metabolites produce specific effects on the physiology of human being and other organisms. Recent reports indicate that there is an inverse relationship between the dietary intake of antioxidant rich foods and the incidence of human diseases [
5]. The interest on these leafy vegetable has increased as a result of epidemiological studies linking eating habits and prevalence of certain diseases. Previous research indicated antimicrobial [
6,
7], antidiabetic [
8], anti-histaminic [
9], anti-carcinogenic and hypolipidemic [
10,
11] properties of leafy vegetables. Leafy vegetables are popular in Benin but there is no scientific data available on their medicinal properties and toxicity.
Acmella uliginosa (Sw.) Cass. (Asteracea) is a species of flowering plant which is indigenous and widely distributed in the tropics and sub-tropics especially in the West Indies, Venezuela, Brazil, Africa, Indonesia and Malaysia [
12]. It is found in the North-west of Benin specifically in Atacora region [
13].
Acmella uliginosa is commonly used by the Malay community in Malaysia to relieve pain often associated with mouth ulcers, toothache, sore throat, and stomach ache [
14]. The methanolic flowers extract was reported to display potent antinociceptive property [
15]. In Benin it is a traditional leafy vegetable which has been domesticated in rural areas and whose sauce is a good dewormer and antibiotic [
13,
14]. Aside these reports, and to the best of our knowledge, no other pharmacological effects of this plant related to its traditional use as antibiotic have been reported. Therefore, the objectives of the present study were (1) to evaluate the antimicrobial and antioxidant properties of the extracts of
Acmella uliginosa leaves; (2) to determine the toxicity of aqueous extract of
A. uliginosa using an acute oral toxicity test in animal models.
Methods
Plant collection
The leaves of A. uliginosa were collected in September 2012 from the airport garden of Cotonou, department of Littoral, Southern Benin. Identification of the specie was carried out by botanists from the University of Abomey-Calavi and a voucher specimen (AA6624/HNB) was deposited at the National Herbarium of Bénin. After identification, the collected leaves were ground using an electric grinder (MARLEX Electroline Excella).
The leaves of A. uliginosa (620 g) were successively extracted by maceration with 1000 ml of dichloromethane (DCM) and 750 ml of methanol (MeOH) for 72 h stirring while a second extraction (decoction) with 1000 ml of sterile distilled water (H2O) was carried out with five hundred grams (500 g) of plant material. Each extraction was repeated three times. The filtrates of each extraction were desiccated under vacuum and the obtained extracts were stored at 4 ° C until biological assay.
Phytochemical
Phytochemical screening of the plant was carried out according to the standards methods for the detection of plant secondary metabolites [
16,
17]. Alkaloids, flavonoids, steroids, coumarins, saponins, naphthoquinones, triterpenes, lignans, pigments, anthracene derivatives were investigated using TLC method [
16] while tannins were characterized using iron-III-chloride reagent [
17].
Antibacterial activity
Bacterial strains
Dichloromethane, methanol and aqueous extracts of A. uliginosa were individually tested against a panel of bacteria including four Gram-positive: Staphylococcus aureus (ATCC 6538), S. epidermidis (CIP8039), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus Methicillin Resistant (SARM) and two Gram-negative: Escherichia coli (CIP 53126) and Pseudomonas aeruginosa (CIP82118) obtained from Laboratoire de Biophotonique et Pharmacologie, University of Strasbourg, France.
Growth inhibition effect of extracts at 10 mg/ml
Sensivity of different bacterial strains to various extracts was determined using 96-well microplate. The aim of this method was to eliminate the extracts, which at 10 mg/ml do not inhibit the growth of bacteria [
18]. The extracts were reconstituted to a concentration of 20 mg/ml in acetone/Muller Hinton broth culture. A volume of 100 μl of each extract (20 mg/ml) was introduced in triplicate microplate already seeded with 100 μl of the Muller Hinton broth culture inoculums (10
6 CFU/ml) of the tested bacteria. The microplate was incubated at 37 °C. After 18 h of incubation, 40 μl of 0.2 mg/ml solution of
p-iodonitrotetrazolium (Sigma Aldrich) were added to each well and microplate was incubated at 37 °C. Finally, after 30 min, the color change (extract color to red) of mix in each well was examined to select actives extracts. Active extract do not change color.
Minimum inhibitory concentration and total activity
The Minimum inhibitory concentrations (MIC) of selected extracts were determined by the method of broth microdilution using
p-iodonitrotetrazolium (INT) as an indicator of bacterial viability [
19]. Briefly, 100 μl of Mueller Hinton broth (DIFCO) were added to each well of a 96-well microplate and 100 μl of extracts (20 mg/ml) were added to the first well (A) of the plate. A two-fold dilution was carried to make 8 concentrations. Then, 100 μl of bacterial broth at 10
6 CFU/ml were finally added into all the wells. After 18 h incubation at 37 °C, 40 μl of
p-iodonitrotétrazolium (0.2 %) were added to each well and the incubated was continued at 37 °C. After 1 h incubation, the MIC values were recorded. Gentamicin was used as positive control. Each assay was run in triplicate. The total activity of each extract was calculated by dividing the MICs with the amount of extract obtained from 1 g of plant material [
20]. This value indicates the volume in which the active extract obtained from 1 g of dry plant material can be diluted to always have inhibitory activity against organisms [
21].
Antifungal assay
Test organisms
Fungi strains used in this study included
Aspergillus flavus CMBB75,
A. parasiticus CMBB20,
A. ochraceus CMBB91,
A. nidulans CMBB90,
A. clavatus NCPT24 and
A. fumigatus CMBB89. They were obtained from the laboratory of biochemistry and molecular biology at the University of Abomey. These microorganisms are the most common fungal pathogens of vegetables, animals and humans. They play an important role in opportunistic infections in immunocompromised patients [
22].
Antifungal test
The in vitro antifungal activity of extracts was evaluated on mycelia development and sporulation as described previously by Dohou et al. [
23] with minor modifications. 10 ml of the mixture of potato dextrose agar-extract at 1 mg/ml were poured into sterile petri dishes. Fungi suspension were prepared in tween (5 %) and 100 spores were dropping in the center of petri dishes. After 5 days incubation at 25 °C, the diameter of mycelia was measured and the number of spores was counted microscopically using Malassez cell. Each test was performed in triplicate. Three petri dishes containing potato dextrose agar without extract were used as negative control and Fluconazol (100 μg/ml) was used as positive control. The percentage of inhibition (PI) of extracts was determined according to the formula below:
$$ \mathrm{PI}\ \left(\%\right) = \kern0.5em \frac{{\mathrm{A}}_{\mathrm{v}\ \mathrm{control}}\hbox{-}\ {\mathrm{A}}_{\mathrm{v}\ \mathrm{tested}\ \mathrm{extract}}}{{\mathrm{A}}_{\mathrm{v}\ \mathrm{control}}}\kern0.5em \mathrm{x}\ 100 $$
In which Av control = average diameter of the mycelia or estimated number of spores of control (n = 3), Av tested extract average diameter of the mycelia or estimated number of spores of tested extracts (n = 3).
DPPH radical-scavenging activity
The ability of the extracts to scavenge the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical was evaluated. The antioxidant activity was determined according to the method previously described by Velazquez et al. [
24]. The stock solution of the extracts was prepared at 1 mg/ml. Then, a two-fold serial dilution was carried to make 8 concentrations (1 - 0.007 mg/ml). Briefly, 1.5 ml of a freshly prepared methanolic solution of DPPH (2 %) was mixed with 0.75 ml of extract solution. After 15 min incubation in the dark, at room temperature, the absorbance of the mixture was read at 517 nm using a spectrophotometer (Jenway, Genova). The blank consisting of a mixture of 1.5 ml of methanol and 0.75 ml of extract solution. Quercetin was used as positive control. All assays were performed in triplicate. The percentage of inhibition of DPPH radical was calculated according to the following formula:
$$ \%\ \mathrm{inhibition} = \kern0.5em \frac{{\mathrm{A}}_{\mathrm{blank}}\hbox{--}\ {\mathrm{A}}_{\mathrm{sample}}}{{\mathrm{A}}_{\mathrm{blank}}}\kern0.5em \mathrm{x}\ 100 $$
Ablank = absorbance of blank, Asample = absorbance of tested extract
Oral acute toxicity
Experimental animals
Six female wistar rats with body weight ranged from 180-200 g were obtained from the Laboratoire de Biologie Humaine, Faculty of Medicine, University of Abomey-Calavi. The used animals were nulliparous and non-pregnant. The rats were fed with standard laboratory diets, given water ad libitum and maintained under laboratory conditions (22 ± 3 °C), a relative humidity between 30-70 % and a constant light-dark schedule (12 h light/dark cycle).
Oral acute toxicity testing
The toxicity of the aqueous extract of
A. uliginosa was evaluated according to the Organization for Economic Co-operation and Development guidelines n° 423 [
25]. The protocol related to acute toxicity test was approved by the scientific committee of research protocols (VPMAS/PFCR-2/UAC), University of Abomey-Calavi, Bénin. A total of six females rats were divided into two groups with three animals each and kept in different cages for easy observation during experiment. Distilled water (10 ml/kg body weight) was given to control group (group I). The group II was given a single dose of the aqueous extract solution (2000 mg/kg body weight). Following administration of extract, rats were closely monitored for 30 min and 2, 4, 8 and 24 h. Mortality, food and water consumption and general acute toxicity or clinical symptoms were recorded. Body weight was also recorded on days 1, 7 and 14.
Haematological and biochemical parameters
At the end of the experiment, all the rats were anaesthetized using thiopental at 0.5 ml/Kg body weight. Animals were then sacrificed and the blood for biochemical and hematological analysis were collected through cardiac puncture into ethylenediaminetetraacetic acid (EDTA) tubes. Each blood sample was analyzed for haematological parameters such as hematocrit (Ht), red blood cells (Rbc), hemoglobin concentration (Hc), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular hemoglobin levels (MCH), white blood cells (Wbc), basophils (B), lymphocytes (L), monocytes (M)using an automatic hematological analyzer (Sysmex, XP-300, Japan). Biochemical parameters such as Glucose (GLU), Creatinine (CREA), cholesterol (CHOL), alanine aminotransferase (ALT), aspartate transaminase (AST) were determined using an autoanalyzer (ErbaChem 7, Germany). The liver and kidneys of rats (group I and II) were collected, weighed immediately and transferred to a saline solution. These organs were fixed in 10 % buffered formalin for histological examination. The samples were then treated with increasing concentrations of ethanol and infiltrated with paraffin. Then, the thin cuts were made and stained with hematoxylin and eosin stains.
Statistical analysis
The t-student test was used for statistical analysis of data on body weight, hematological and biochemical parameters. The difference was considered statistically significant when the p value was 0.05 or less (p < 0.05). All data were expressed as mean ± SD. The graphical representation was performed using the Graph Pad Prism 5.0 software (Microsoft, USA).
Discussion
The therapeutic effects of plant materials generally result from the combination of secondary metabolites. These secondary metabolites are not only essential in the cell structure, but often are involved in the protection of plants against biotic and abiotic stresses. Natural products, as pure compounds or standardized extracts, provide unlimited opportunities for the drug discovery because of the unmatched availability of chemical diversity inside the plants. Phytochemicals are bioactive compound present in leafy-vegetables which could be responsible for their bioactivity linked to the reduced risk of major chronic diseases. It has indeed been estimated that a healthy diet could prevent approximately 30 % of all cancers [
28]. The phytochemical screening showed that leaves of
Acmella uliginosa contain coumarin, flavonoid, naphtoquinone, anthracene derivative, saponin, lignan, triterpene, and tannin. Bioactive compounds are normally accumulated as secondary metabolites in all plant cells but their concentration varies according to the plant parts, seasons, climates, extracting solvent in plant and particular growth phases [
29]. Leaves are one of the highest sources of accumulation and are highly beneficial [
30].
In this study, dichloromethane extract showed interesting antibacterial (0.625-1.25 mg/ml) and/or antifungal (PI up to 99.77 %) activity by inhibiting one or more microorganisms. These results confirm a statement that the intermediate polarity compounds usually have the highest antimicrobial activity found with many different plant species [
31]. The interesting antimicrobial activity of dichloromethane extract against the tested microorganisms could be due to the presence of flavonoids, naphtoquinone and triterpene as reported previously [
32‐
34]. Flavonoids are hydroxylated phenolic substances known to be synthesized by plants in response to microbial infection and they have been found to be antimicrobial substances against wide array of microorganisms in vitro [
35]. Flavonoids are known antimicrobial agents throught various mechanisms like inhibition of nucleic acid synthesis, inhibition of cytoplasmic membrane function and energy metabolism [
36]. Naphthoquinone and triterpenes have been also reported to possess antibacterial activity [
37,
38]. In our study, coumarin and tannin were detected in methanol and aqueous extracts. Coumarins represent a large group of compounds that have been reported to possess a wide range of biological activities including antimicrobial [
39] and antioxidant [
40]. Tannins are known antimicrobial agents that could inhibit the growth of microorganisms by precipitating the microbial protein and thus depriving them of nutritional proteins needed for their growth and development [
41]. This could explain the antimicrobial activity of methanol and aqueous extracts.
DPPH is a stable free radical which accepts an electron or hydrogen radical to become a stable diamagnetic molecule. In the presence of hydrogen doner, DPPH is reduced. It has been showed that the scavenging effects on the DPPH radical increased with the increasing concentration of the samples to a certain extent. In the present study, methanol and aqueous extracts showed appreciable free radical scavenging activity than dichloromethane extract at 500 and 1000 μg/ml. This may be due to the different polarities of antioxidant compounds present in the extracts [
42]. The difference in the DPPH radical scavenging activity implies that the extracting solvent would affect the presence of secondary metabolites of extract and then the radical scavenging potency. The antioxidant capacity of methanol and aqueous extracts could be due to the presence of phenolic compounds such as coumarins and tannins. Several researches reported the antioxidant activity of these chemical compounds [
43‐
45].
The oral acute toxicity of aqueous extract at 2000 mg/kg body weight resulted in no mortality and no signs of acute toxicity throughout the 14 days. This suggests that the LD50 is greater than 2000 mg/kg body weight. The increase in body weight observed between day 1 and 14 in the treated groups could be attributed to the increase in food consumption. This could be explained by an excitation of appetite of animal by aqueous extract. In this case, aqueous extract could have hypoglycemia effect. A significant increase (
P < 0.05) observed in the relative kidney weight of treated animals compare to control could also correlate with the growth of animals or to a dysfunction of kidney cells. Previous studies have shown that a reduction in body weight gain and organ weights is an internal simple and sensitive index of toxicity after-exposure to toxic substances [
46,
47]. Contrary to this, the results of our studies showed an increase in body weight and organs of treated rats. Then, it could be concluded that a change in body weight and organs is an index of toxicity.
Hematological assessment is useful to determine the extent of toxic effects of plant extracts on the blood constituents of an animal [
48]. In this study, we found a significant difference in hematological parameters such as Rbc, Ht, MCH, MCV, WBC, Nc, L and M. Increase in the production of WBC and it's differentials is generally considered to be a marker of stress and a defense mechanism triggered by immune system against various inflammatory conditions (Polymyalgia rheumatica, bacterial infections, hemorrhage, leukemia etc.) [
49]. The significant changes in the level of WBC suggest the toxic effect of the leaves of
A. uliginosa. Creatinin is known as an effective indicator of kidney function and any significant increase in creatinin levels induces functional nephron damage [
50]. The significant rise of creatinine concentration in plasma indicates the implicit effect of the plant on renal filtration mechanism. It was noted a significant reduction of AST and ALT in animals treated compared to the control. This could mean that the aqueous extract of
A. uliginosa had a harmful effect on the liver. Histological analysis showed lesions in the liver and kidneys. The cellular architecture of these organs confirms the significant changes in hematological and biochemical parameters of treated animals. Coumarins found in
A. uliginosa extracts are known for their hepatotoxicity and have been reported to be toxic to rats and mice [
51]. Thus, this class of compounds may be involved in the changes caused by the aqueous extract of
A. uliginosa. The lesions observed in the organs could be due to the presence of pesticide residues and/or heavy metal in the leaves of
A. uliginosa, since the gardener use pesticides and the harvest was made in the garden near Cotonou International Airport. Previous study showed that presence of pesticides in leaf-vegetable could justify the increased kidney weight and increased incidence of chronic nephrosis [
52].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LL designed the study, followed the implementation, wrote the manuscript, AMA Collected plant, prepared the extracts and carried out the study, participated to write the manuscript, RA carried out antibacterial and phytochemical test, AL carried out toxicity and histological test, AS coordination and helped to revise the manuscript. All authors read and approved the final manuscript.