<i>Launaea taraxacifolia </i> is a leafy vegetable of the family of Asteraceae (Compositae) found in several countries in West Africa including Ghana, Benin and Nigeria. The plant leaves are eaten either fresh as salad or cooked as sauces. They are also consumed as infusion to fight against several diseases including non-communicable diseases such as diabetes and hypertension. Several studies have been conducted in Ghana, Nigeria on the nutritional and medicinal values of this plant but no study has yet been conducted in Benin on the virtues of this plant. In this work we have achieved the phytochemical characterization and evaluated the cytotoxicity as well as hypolipidemic and anti-oxidant effects of the ethanol-aqueous extracts of <i>Launaea taraxacifolia </i> leaves. Cytotoxicity and hypolipidemic activities have been performed on HepG2 cells; the antioxidant effect has been performed on the PLB985 cells. The results showed that the ethanol-aqueous extracts of <i>Launaea taraxacifolia </i> leaves contained the following metabolites: catechic tannin, flavonoids, phenolic acids, mucilage and leucoanthocyanins. Only very high concentrations (>20 mg/ml) of leaves extracts are toxic for HepG2 cells. <i>Launaea taraxacifolia </i> leaves have significant antioxidant and hypolipidemic activities.
Cardiovascular diseases and diabetes are the most common non-communicable diseases in the world. One third of the world population suffers from hypertension and one tenth suffers from diabetes; worldwide, three million and six million people die from hypertension and diabetes respectively a year. 80% of these deaths occur in low-income countries (Global Status Report on Non-Communicable Diseases, 2010). These diseases are caused by unbalanced diet and lack of physical exercise and affect more and more poor countries. The majority of patients in poor countries use medicinal plants to treat these diseases because modern medicine is too expensive. Cardiovascular diseases and diabetes could come from oxidative stress associated with hyperlipidemia. Indeed it has been shown that hyperlipidemia induces accumulation of fatty acids and triglycerides in the liver which causes liver steatosis and oxidative stress [
Launaea taraxacifolia (L. taraxacifolia) is a leafy vegetable of the family Asteraceae (Compositae) that is present in several African countries including Ghana, Senegal, Benin and Nigeria where it is more known and domesticated [
In this work we have achieved the phytochemical characterization of ethanol-aqueous extracts of Benin species of L. taraxacifolia leaves and evaluated its cytotoxicity, hypolipidemic and antioxidant properties in HepG2 and PLB985 cells lines.
L. taraxacifolia plants were collected in the month of May, 2014 from sakete in southern of Benin. A specimen was deposited in the National Herbarium of the Department of Botany, Abomey-Calavi University. Samples were dried in the shade at room temperature (25˚C) until stabilization of their mass and then pulverized into coarse powder.
HepG2 cells were obtained from ATCC and PLB985 cells were obtained from Dr. Marie-José Stasia (University Hospital, Grenoble, France).
Gallic acid, Butyl Hydroxy Anisole (BHA), quercetin and catechin were purchased from Sigma Chemical Co. (St. Louis, MO) while 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu reagent were obtained from Acros Organics (Morris Plains, NJ). All solvents used are analytical grad.
DMEM medium, oleic acids, oil red O, Phorbol Myristate Acetate, luminol were purchased from Sigma (France), RPMI 1640 was obtained from BioWhittaker, (Walkersville, MD, USA), MTS-PMS reagent was obtained from Promega (France).
All samples were ground in a commercial coffee grinder for extraction. The mixture ethanol-water 50% (v/v) was used as extraction solvent. The extract was concentrated in vacuo using a rota vapor and the yield (Y) was calculated by the formula below:
We explored the chemical potential of the leaves of L. taraxacifolia by a series of coloring techniques. This phytochemical screening was based on standard coloring reactions and/or the precipitation reactions of the chemical compounds in plants according to the published methods and routinely used in our laboratory.
1) Alkaloids
Three various properties based on the capacity of alkaloids to combine with heavy metals or iodine (Dragendoff’s reagent, Mayer’s reagent and iodoplatinate test) were implemented [
2) Coumarins
The characterization of coumarins was made according to the method described by Rizk [
3) Saponosides or saponins: Index foam
Two grams of dry and ground Sorghum caudatum were used to prepare a decoction with 100 mL of distilled water and submitted to boiling for 30 min, then the resulting solution was divided in 10 tubes: 1 mL, 2 mL, 3 mL, …, 10 mL of decoction. The content of each tube was adjusted to 10 mL with distilled water. Each tube was shaken vigorously in a horizontal position for 15 seconds. After 15 min in vertical position, persistent foam measurement was obtained. If it was close to 1 cm in the 10th tube, the foam index was calculated by the following formula:
The presence of saponins was confirmed by an index exceeding 100 [
A qualitative approach on methanolic extracts, prepared according to the method described for research of alkaloids, was done by TLC with AcOEt/MeOH/H2O (100:13.5:4) as solvent of migration, and was visualized with sulfuric vanillin.
4) Sterols and terpenes
The Sterol and terpenes were identified by Liebermann-Buchard reaction [
5) Carotenoids and quinones
We carried out the characterization of:
・ carotenoids by the reaction of Carr and Price;
・ free anthraquinones by the reaction of Bornträger [
・ combined quinones (O-heteroside and C-heteroside) by methods of characterization usually used in our laboratory.
6) Polyphenols
The determination of phenolic compounds was made by the reaction of ferric chloride [
7) Flavonoids
Flavonoids identification was carried out by the test of cyanidin [
8) Anthocyanins
To an infusion, we added 5 ml of 10% H2SO4 and 5ml of 50% NH4OH. The appearance of a red color that turned purplish blue in basic medium indicates the presence of anthocyanins [
9) Leuco-anthocyanins
0.5 ml of 12 N HCl was poured into 3 ml of hydro-alcoholic extract. The acidified solution was brought to boiling water bath for 30 minutes. After cooling, the appearance of a purplish red color indicated the presence of leuco-anthocyanins [
10) Mucilages
1 ml of decoction 10% and 5 ml of ethyl ether were introduced in a test tube. After ten minutes, obtaining a flocculent precipitate indicated the presence of mucilages [
Total polyphenols: The method of determination of total polyphenols consisted to use a mixture of phosphotungstic and phosphomolybdic acid which was reduced during the oxidation of phenols in the mixture of tungsten blue oxide and molybden [
Total flavonoids: The method of aluminum trichloride (AlCl3) was used to quantify the total flavonoids. This technique was based on the formation of the aluminum-flavonoids complex that had a maximum absorption at 500 nm [
Condensed tannins: condensed tannins dosing was achieved by using the method of vanillin sulfuric [
HepG2 cells (hepatocellular carcinoma cell line) were cultured in 75 cm2 polystyrene flasks with DMEM culture medium to which were added 10% FBS and 1% penicillin streptomycin at 37˚ under athmosphere of 5% CO2.
PLB985 cells are human leukemic cells that are capable of differentiating into neutrophils [
Cytotoxicity was performed by the MTS assay [
1) Induction of lipid accumulation by oleic acid
105 HepG2 cells were plated on slides for 24 h in normal culture medium. After 24 hours the culture medium was replaced with either normal culture medium (negative control cells), culture medium without FBS containing 1 mM of oleic acid (positive control) or culture medium without FBS containing 1 mM of oleic acid and L. taraxacifolia leaves extracts at concentration of 20 mg/ml. The cells were again incubated for 24 h before lipid staining.
2) Stained with Oil Red O (ORO)
The ORO is a lipid-soluble molecule that can color the lipids [
Phorbol myristate acetate (PMA) induces the activity of NADPH oxidase and the production of free radicals in human neutrophils [
As we can see in
The extraction yield of the secondary metabolites from L. taraxacifolia leaves by the mixture ethanol-water is “19.851%”. The total phenol content is expressed as Gallic Acid Equivalent (GAE); the flavonoids content is expressed in mg of Quercetin Equivalent (QE) per gram of dry matter and the tannin content is expressed as mg Catechin Equivalent (CE) per gram of dry matter. The results show that L. taraxacifolia extracts have a high total phenolic and flavonoids contents but a low tannin content: respectively “32.275 ± 1.11” mg GAE/g of dry matter; “56.959 ± 0.385” mg QE/g of dry matter and “3.212 ± 0.036” mg CE/g of dry matter (
Phenolic acids can be distinguished by derivatives of benzoic acid and derivatives of cinnamic acid. The gallic acid (benzoic acid) content of edible plants is generally very low, with the exception of certain red fruits, black radish, onions and tea [
Flavonoids include severals derivatives: flavonols, flavones, isoflavones, flavanones, flavanols, anthocyanidins. Flavonols are the most ubiquitous flavonoids in foods and the main representatives are quercetin and kaempfero. The high dose of quercetin in L. taraxacifolia leaves can be explained by the fact that flavonols accumulate in the outer and aerial tissues of plants (skin and leaves) because their biosynthesis is stimulated by light [
The MTS test is used to assess cell viability. The enzymes involved in cellular redox activity of NADPH are able to reduce MTS to formazan, and then reflect cell viability. The reduction of MTS depends on the cellular metabolic activity and the flow of NADPH. The cells that divide rapidly have a high metabolism and greatly reduce the MTS [
Secondary metabolites | L. taraxacifolia | |
---|---|---|
Tannin | Gallic | − |
Catechic | + | |
Flavonoids | + | |
Mucilage | + | |
Anthocyanin | − | |
Leucoanthocyanin | + | |
Reducing sugar | − | |
Anthraquinones | − | |
Alkaloids | − | |
Saponosids | − | |
Quinone derivatives | − |
+: presence; −: absence.
radiolabeled polyphenols are given to rats, radioactivity is recovered in blood and in several tissues including brain, endothelial cells, heat, kidney, uterus, mammary gland at the concentrations ranged from 30 ng to 3 μg/g of tissue depending on the dose administered and the tissue considered [
The antioxidant activity of ethanol-aqueous L. taraxacifolia leaves extracts is demonstrated by measuring the production of free radicals by the PLB985 cells in the presence of 100 nM PMA alone or “100 nM” PMA with different concentrations of extracts. It’s known that PMA induces the activity of NADPH oxidase in human neutrophils [
The experiment was repeated with lower concentrations of extracts (“0.05 - 0.5 μg/μl”). Under these conditions ROS production induced by “100 nM” PMA decreased and slowed considerably depending on the dose and was zero in the presence of “0.5 µg/µl” extracts (
In response to growth factors and cytokines, and during normal metabolic events such as respiration and phagocytosis, eukaryotic cells produce oxidants. To compensate for this, the cells have evolved both enzymatic and nonenzymatic mechanisms to protect against oxidants’ toxic effects. The enzymatic mechanisms include the actions of enzymes such as catalase and glutathione peroxidase. The non-enzymatic antioxidants include glutathione, ascorbate. However, in pathophysiologic circumstances, an excess of oxidants can overwhelm the scavenging capacity of cellular antioxidant systems. The subsequent oxidative stress damages the cell’s lipids, membranes,
proteins, and DNA [
The hypolipidemic activity of L. taraxacifolia leaves extract was investigated by measuring it effect on lipid accumulation induced by oleic acid in HepG2 cells. “1 mM” of oleic acid induced lipid accumulation (steatosis) in HepG2 cells [
cells. The recent works demonstrated that Oleic acid induced steatosis significantly increased TNF-α production and secretion in HepG2 cells [
Our results showed that the ethanol-aqueous extracts of L. taraxacifolia leaves were toxic only at very high concentrations. These leaves can be consumed both in food and as an infusion without risk of toxicity. However as a bioactive substance the dosage must be controlled since the very high doses are toxic. The phytochemical characterization we achieved showed that the leaves extracts contained a high proportion of polyphenols which may account for the reported medicinal properties of the plant. L. taraxacifolia leaves extracts could help to prevent or fight against non-communicable diseases such as cardiovascular diseases, diabetes and cancer. We therefore recommend the domestication of this plant in Benin since it can be used as a health food.
OmédineKoukoui,PascalAgbangnan,SylvianeBoucherie,MahudroYovo,OliverNusse,LaurentCombettes,DominiqueSohounhloué, (2015) Phytochemical Study and Evaluation of Cytotoxicity, Antioxidant and Hypolipidemic Properties of Launaea taraxacifolia Leaves Extracts on Cell Lines HepG2 and PLB985. American Journal of Plant Sciences,06,1768-1779. doi: 10.4236/ajps.2015.611177