Plant material
The aerial parts (leafy branches) of Helichrysum faradifani (Asteraceae) Scott-Elliot were harvested in the Marisoa commune, Fianarantsoa region, Madagascar, on May 20th, 2020. A reference herbarium specimen under the code RLL1826 was deposited at the Herbarium of the National Center for Pharmaceutical Research Application (CNARP). The harvested aerial parts were dried in a ventilated dryer at a temperature of 40°C for 2 weeks and then ground to obtain a powder.
Preparation of Helichrysum faradifani leafy twig extract
An amount of 400 g of powdered aerial parts of H. faradifani was decocted in 4 liters of water for two minutes at 100°C. After that, it was divided into liquid-liquid phases using 480 milliliters of hexane, ethyl acetate, and butanol in order of increasing solvent polarity. A rotary evaporator operating at 56°C was used to evaporate the solvants. After extracting the dry materials, the yields were calculated.
Phytochemical screening
Phytochemical screening is a method for gathering processes aimed at detecting principal families of chemicals found in plants, such as alkaloids, terpenoids, phenolic compounds, and polysaccharides. The presence or absence of major classes of compounds was characterized by color reactions and/or precipitations according to the methods described by Daira et al., 2016 and Norman, 1966.
Total flavonoid determination
The total flavonoid content of the aqueous extract of H.faradifani was determined by UV spectrophotometry using the method of colorimetry with 2% (w/v) aluminum trichloride (AlCl3) by the method of Nickavar and Esbati, (2012) and Aslan et al., (2007). The calibration curve for quercetin was prepared by mixing 3 ml of quercetin solution in methanol at concentrations ranging from 0.1 to 0.006 mg/ml with 3 ml of AlCl3 solution. Then, 3 ml of the sample (at concentrations ranging from 0.1 to 0.05 mg/ml) was mixed with 3 ml of the AlCl3 solution. After 40 minutes of incubation in the dark, the absorbance was measured at 415 nm, starting with the blank, followed by the least concentrated standard, and finally the sample to be assayed. The total flavonoid content of the Helichrysum faradifani aqueous extract was expressed in μg of quercetin equivalents per mg of extract (μg EQ/mg of extract).
DPPH• free radical scavenging activity assays
The DPPH• scavenging capacity was evaluated as follows, with slight modifications according to the methods of Ahmad et al., (2010) and Awika et al., (2003) : A quantity of 3800 μl of 4.5% DPPH prepared in methanol was combined with 200 μl of various concentrations of H. faradifani extract and the ascorbic acid standards (0.003125 - 1 mg/ml). The mixtures were vortexed and incubated in the dark for 30 minutes. The absorbance was then measured at 517 nm using a UV spectrophotometer. The antioxidant activity, which expresses the ability to scavenge the free radical, is estimated by the percentage of DPPH discoloration in solution in methanol. It is given by the following formula:
The 50% inhibitory concentration of free radicals, or IC50, is then obtained using the equation of the linear regression curve resulting from the percentage of inhibition as a function of concentration. Extracts with an IC50 < 30 μg/ml are considered strong antioxidant activity, 30 μg/ml < IC50 < 100 μg/ml are considered moderate activity, and an IC50 > 100 μg/ml are considered inactive according to the scale of Ahmad et al., (2010).
Antimicrobial activity
Eight microorganisms, including five Gram-positive bacteria: Bacillus cereus (LMG6910), Bacillus anthracis (ATCC22151), Clostridium perfringens (ATCC13124), Streptococcus pneumoniae (ATCC6301), Enterococcus faecalis (ATCC15922), three Gram-negative bacteria: Escherichia coli (ATCC8739), Pseudomonas aeruginosa (ATCC10145), Enterobacter cloacae (ATCC13047) and one yeast Candida albicans (ATCC10231). These strains are part of the collection of the National Center for Pharmaceutical Research Application (CNARP). Thery were used for antibacterial testing extracts from H. faradifani.
The antimicrobial screening was conducted using the antibiogram method as described by Moreira et al., 2005 ; Ponce et al., 2003. The determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was evaluated according to the methods of [20] and Kuete et al., 2011 with some modifications. Specifically, 90 µL of Mueller-Hinton broth was placed inside the wells. A quantity of 100 µL of each extract, tested at 1 mg/mL, was placed in wells A1 and A2 of the microplate, forming the stock solution. Then, 100 µL of this stock solution was serially diluted twofold from the second to the twelfth column of the microplate. Finally, 10 µL of the inoculum at 10⁶ CFU/mL was added to all wells except those used as bacterial growth control and medium sterility control. The microplates were incubated at 37°C for 24 h. After 24 hours of incubation, each well received 40 µL of a Thiazolyl Blue Tetrazolium solution (10% w/v) to evaluate microbial growth. The MIC was measured visually as the lowest concentration of each sample that produced no apparent growth. Next, 5 µL of each well was plated on Mueller-Hinton agar. The MBC was defined as the lowest concentration at which no colonies grew on the plates after 24 hours of incubation at 37°C.
Statistical analysis
All experiments were performed in triplicate, and the results were presented as mean values ± standard deviation. The data were analyzed using ANOVA. Significant differences between mean values were determined by Tukey's HSD test at a significance level of p < 0.05.