Experimental research procedures will be applied in identifying lichen species and acids including the quantification of compounds such as DPPH scavenging, total phenolic, ascorbic acid, and in finding the antibacterial potential of isolated mycobiont from the lichen apothecia.
The study will be conducted at the Center for Natural Sciences Research Laboratory of Saint Mary’s University, Bayombong, Nueva Vizcaya from February 2018 to May 2018.
Materials and Methods
Collection of Lichens
The lichen species will be collected in the forest of Nueva Vizcaya (Imugan in Sta. Fe) through purposive sampling. The collected lichens will be placed into paper bag or newspaper.
Identification of Lichens
The lichen species will be identified through characteristics of their morphoanatomical parts such as the thallus and reproductive structures. Thalline spot test using K (potassium hydroxide), P (paraphenylenediamine) and C (sodium hypochlorite) will be performed. Color changes in a cut of the thallus and unto the exposed medulla were observed and recorded for taxonomic identification purposes.
Extraction of Lichens
The lichen thalli will be pulverized using mortar and pestle and will be placed in a beaker with acetone as a solvent. Afterwards, incubate for 24 hours at room temperature then decant using whatman filter paper. The extracted liquid will be positioned into water bath until it settles similar to paste texture.
Isolation of Mycobiont
The isolation of mycobiont will be performed using Mcdonald et al. (2013) method. The apothecia of lichens will be washed with 1mL of water for 4 hours, blotted and affixed to the top of an inverted petri dish with double-stick tape or petroleum jelly and maintain at 20 °C or room temperature allow the spores to germinate into potato-carrot agar (Potato-20g, Carrots-20g, Agar-15g and distilled water-1L) as media. After 5 days of continued monitoring spore germination and mycelial growth will be observed.
Extraction of Mycobiont
The isolated mycobiont is cut into pieces and place in a beaker with acetone as solvent. The set up will be incubated for 24 hours at room temperature. After 24 hours, pour the solvent into another beaker using Whatman filter paper and place the sample in the water bath and let it dry until the texture appearance is like a paste.
Determination of Lichen Acids: Thin Layer Chromatography Screening
Thin layer chromatography (TLC) will be completed (Lumbsch, 2002) to identify the specific lichen acids present in the mycobiont and lichen extracts (10 mg/ml) which initially determine in the thalline spot test. Lines will be measured 2cm above the base and 2cm below the top edge of a silicate plate are drawn using a pencil. The same measurements are applied at both left and right edges of the plate. Starting from the left edge point margin, points measuring 1 cm apart will be drawn until the point margin at the right edge is met. Along these points, the extracts are spotted with the use of a capillary tube. A filter paper is placed back of a TLC tank and saturated with toluene – acetic acid (170:30 ml) solvent to achieve uniform vapor saturation throughout the tank. The tank is filled with the solvent system up to around 1 cm height and left to stabilize. The spotted TLC (silica) plates will be placed in the TLC tank with the silica side facing the filter paper to start elution. After elution, the TLC plate is removed from the tank and the front of the solvent will be marked and air dry for 30 minutes in a fume cupboard. The TLC plates are examined for pigments that appear as colored spots initially in daylight and under 360 nm UV. Spots are marked using a pencil. The TLC plate is directly brushed with 10% sulfuric acid and left until dry before heating it at 30 ?C in an oven for 5 – 10 minutes to develop spots. Color is recorded at once and will be examined under 360 nm UV light. Identification of chemical constituents is done by using Rf value classes as presented in Culberson (1972), Culberson & Kristinsson (1970) and Culberson et al. (1981).
Determination and Quantification of Compounds
Antioxidant activity assay
Antioxidant property of mycobiont lichen extracts is determined using the thiocyanate method adapted from Rankovic et al. (2010). The sample (1 mg) in 1 mL solvent is mixed with 5 mL linoleic acid emulsion (0.02 M, pH 7.0) and 5 mL phosphate buffer (0.2 M, pH 7.0). A Linoleic acid emulsion is prepared by mixing 0.5608 g of linoleic acid with 0.5608 g of Tween 20 as the emulsifier, and 100 mL phosphate buffer and the mixture is regulated. The reaction mixture is incubated at 37°C. Aliquots of 0.1 mL are noted at different intervals during incubation. The degree of oxidation is measured according to the thiocyanate method by sequentially adding 4.7 ml ethanol (75%), 01 mL ammonium thiocyanate (30%), 0.1 mL sample solution, and 0.1 mL ferrous chloride (0.02 M, in 3.5% HCl), The mixture stood for 3 minutes and the peroxide value is then determined by reading the absorbance at 500 nm using a UV visible spectrophotometer. A control is performed with linoleic acid without the extract. Ascorbic acid solutions are prepared as a positive control. All experiments must be carried out in triplicate. The inhibition percent is computed using the following formula:
1% = 1 – (absorbance of sample at 500 nm/absorbance of control at 500 nm) × 100.
1 = absorbance of control (blank) at 500 nm
The method of Oyiazu (1986) will be followed to determine the total reducing power of lichen thalli and mycobiont extracts. The stock solution will be prepared by mixing 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium fericyanide K3Fe(CN)6 (1%). The prepared stock solution will be mixed in 1 ml of the sample extracts and will be followed by incubation at 50?C in a water bath for 20 minutes before being cooled rapidly, spiked with 2.5 ml of 10% trichloroacetic acid and centrifuge (3000 rpm for 10 min). The supernatant (5 ml) is then mixed with an equal amount of distilled water and 1 ml of 0.1% ferric chloride (FeCl3). After 10 minutes, the absorbance of 700 nm will be measured using a spectrophotometer. The higher the absorbance indicates the stronger reducing power. The assay is carried out in triplicate and the results are expressed as mean values ± standard deviations. Ascorbic acid is used as positive control.
Determination of Total Phenolic contents
Total phenolic content will be determined with Folin – Ciocalteu reagent according to the method of Rankovic et al. (2010) using gallic acid as a standard. In order to determine the total phenol content in the lichens by Folin – Ciocalteu assay and gallic acid are used as usual standards. The gallic acid is selected since it is structurally more similar to the lichen acids. The extract of 1 mL (contains 5 mg of mycobiont extracts) is mixed with 1 mL of Folin – Ciocalteu reagent. The mixture is blended vigorously and allow to stand at room temperature for 5 minutes before the addition of 2 mL of 20% Na2CO3 is added and allow to stand for 2 hours with occasional shaking. The absorbance is measured at 760 nm. All experiments are carried out in triplicate. The concentration of total phenolic compounds in the extract is determined as milligrams of gallic acid equivalent per gram of the dry extract by using an equation that is obtained from standard rutin graph as:
Absorbance = 0.0144 x total flavonoid + 0.0556
Antibacterial Assay: Paper disc diffusion method
The antibacterial assay will be performed as presented in Guevarra et al. (2005) and Benson (2002). The extract lichen mycobionts are tested for their activities against the selected bacteria species. The bacterial cell suspension is prepared from cultures and adjusted to 0.5 McFarland standards, and swab on Petri plates pre-filled with Nutrient Agar (NA). Control disks (Streptomycin) for positive control; acetone for negative control) and lichen disks measuring 6 mm in diameter are placed into the inoculate NA plates (five disks per plate). Each disk is impregnated with lichens mycobiont crude extracts, positive and negative control. This will incubate at 37 ?C for 24 hours. Following incubation, zones of inhibition including paper disks are measured with digital caliper and record. Assays are in 3 replicates.
Minimum Inhibitory Concentration
One gram of each lichen species crude extracts with antibacterial activities is prepared using 10 mg/ mL acetone as solvent. The Sterile well plates and pipette tip are used for the MIC assay. The pure extract is introduced to the first well of the plate and then dilutes using two-fold serial dilution. Streptomycin as positive control and acetone as a negative control is used for selected microorganisms to determine the minimum inhibitory concentration of the different lichen extracts. MTT 3-(4, 5-dimethylthiazol-2, 5-diphenyl tetrazolium bromide with 0.2 % concentration is used as an indicator of bacterial growth. A formation of violet formazan in wells indicates microbial growth while yellow solution in wells indicates no microbial growth.
Minimum Bactericidal Concentration (MBC)
The different lichens mycobiont species extracts with no microbial growth in MIC are selected for MBC testing. The contents of the wells with no microbial growth are subculture by streaking them into prepared agar plates using a sterile cotton swab. The agar plate will be numbered according to the number from which the subculture originated. The plates are incubating for 24 hours at 37 °C and plates are observed for growth of colonies after the incubation period.
Treatment of Data
The zones of inhibition will be measured and means are computed. These measure zones of inhibition between and among the lichen mycobionts extracts and with the standard (Streptomycin) are analyzed for significant differences using Analysis of Variance and also Independent Samples t-test. To determine where the significant differences are, a post hoc test using Scheffe’s test is employed and also Levene’s Test for Equality of Means. It is noted that there are no zones of inhibition measure for the negative control (acetone), thus, data for this is not included anymore in the analysis. All analyses are done at 0.05 level of significant and run in the SPSS software.
Rankovic, B. Rankovic, D. & Maric, D. (2010, December). Antioxidant and Antimicrobial Activity of Some Lichen Species. ResearchGate, Vol. 79, pp. 809–815. Retrieved from https://www.researchgate.net/publication/227050778
McDonald, T.R. Gaya, E. & Lutzoni, F. (2013, March 24). Twenty-five cultures lichenizing fungi available for experimental studies on a symbiotic system. Springer Science+Business Media Dordrecht 2013, Volume 59(Number 3), 59:165-171.
Oyaizu, M. (1986) Studies on products of browning reaction prepared from glucoseamine. Jpn J Nutr, 44:307-314.