Seagrasses are flowering plants (Angiospermae) that live inundated in shallow sea water2 and have been used for the treatment of indigestion, muscle pain, skin diseases, wounds and fever as traditional medicine.1 One of seagrasses commonly found spread throughout Indonesian coast among 13 species recorded is Thalassia hemprichii (Ehrenb.) Asch, belong to the family of Hydrocharitaceae.2
T. hemprichii has been reported to contain polyphenolic compounds such as phenolic acids (chicoric acid, ferulic acid, p-coumaric acid, caffeic acid, gallic acid, and p-hydroxybenzoic acid)3-4 and flavonoids (thalassiolin A-B4 and thalassiolin D).5 Its potency as a source of antioxidant has also been reported.1,3,6 Several studies reported that polyphenolic content in a plant contributes to their antioxidant properties.1,3,7-8 Phenolic compounds of T. hemprichii showed wide range of pharmacological properties as antiviral HCV protease,5 antibiotic9 and skin-regenerating activities.10 The ethanol extract of T. hemprichii has also been reported its potency as an anti-wrinkle cosmetics.11
Extraction of phytochemicals from plant materials is impacted by many factors such as solvent, solvent concentration, extraction time, temperature and pH.8,12 Significant effect on the antioxidant activity and phenolic content from different pH and temperature of extraction conditions have been studied by Inggrid et al.13 and Wong et al.14 Moreover, studies on the evaluation of antioxidant activity from T. hemprichii generally have been reported from the methanol, ethyl acetate, and n-hexane extract, while the ethanol extract is still limited.1,3,6 Hence in this study, ethanol will be chosen and the extraction factors from various ethanol concentration, the addition of HCl, and temperature will be optimized using factorial design to obtain higher total phenolic content and antioxidant activity from seagrass T. hemprichii.
MATERIALS AND METHODS
Ascorbic acid, gallic acid, quercetin, and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich. Sodium carbonate, sodium acetate, and aluminum chloride were obtained from Merck. Folin-Ciocalteu, methanol, ethanol, and other solvents used were analytical grade purchased from Merck.
T. hemprichii seagrass collected in August 2017 from Pasauran Beach, located in Serang, Banten, Indonesia. The identification of sample was determined by Center for Marine Studies, Biology Department, University of Indonesia. After harvested, seagrass directly rinsed to remove salt, sand, and foreign particles attached. All parts of samples were then dried at room temperature, shattered into powder and kept in a tight container.
Optimization of extraction
The extraction was carried out using factorial design with three factors namely ethanol concentration (50%, 70%, and 100%), the addition of HCl 1 N (Yes-No), temperature (600C, 300C); and with two responses, the IC50 value of antioxidant activity and total phenolic content. The significant factors on responses will be analyzed using ANOVA.
Method of extraction refers to Jeyapragash et al.1 with modifications. About five grams of sample was soaked in 100 mL solvent for 24 h at room temperature under dark condition, followed by acidification of 0.5 mL HCl 1 N and kept for 24 h at a temperature based on the matrix described in Table 1. The mixture solution was then filtered and centrifuged at 3000 rpm for 5 min. The filtrate was then evaporated with vacuum rotary evaporator and dried on water bath at 500C.
% Yield of extract
The percentage yield of all samples was calculated by comparing the weight of dry extracts obtained to the initial weight of the sample.
DPPH Scavenging Activity
The antioxidant activity was evaluated using radical scavenging DPPH method refers to Bobo-Garcia et al.15 with slight modifications. About 20 μL sample solution was added with 180 μL DPPH solution (150 μmol/L) under dark condition. After stored for 40 min, the absorbance was read at wavelength 519 nm using Versamax Microplate Reader (USA). A control solution was applied by adding 20 μL of 80% methanol instead of sample. Ascorbic acid used as positive control and 80% methanol as blank. The scavenging activity was obtained from the equation follows:
The sample concentration capable of providing DPPH radical inhibition by 50% (IC50) was then calculated using the linear regression y = a + bx, where x is the sample concentration (μg/mL) and y is the percentage of the inhibition.
Total Phenolic Content (TPC)
Folin-Ciocalteu method was applied in total phenolic content (TPC) with reference to Bobo-Garcia et al.15 Under dark condition, 20 μL of extract solution was added with 100 μL of 20% diluted Folin - Ciocalteu and homogenized for 1 min. After 4 min stand, then added with 75 μL 10% sodium carbonate and shaken for 1 min. The mixture was stored for 120 min and the absorbance was measured at wavelength 750 nm using microplate reader. TPC of each sample was calculated based on the calibration curves of 20-100 μg/mL gallic acid standard solution and stated in gallic acid equivalent per gram sample (mg GAE per gram).
% Yield of extract
Higher percentage yield of extract was generally obtained from the sample of 50% ethanol compared to other concentrations of ethanol. The highest yield was shown in sample R1 (22%), while the lowest was in R11 (4%) as shown in Figure 1.
The antioxidant activity of seagrass T. hemprichii in this experiment is expressed in IC50 value. Higher antioxidant properties of a compound in an extract was indicated by the lower IC50. Refers to Molyneux,16 strong antioxidant activity was shown in R1 and R9 samples with IC50 under 100 μg/mL; intermediate activity was shown in R2-R8 with IC50 below 150 μg/mL, whereas the R10-R12 had a weak activity with IC50 above 200 μg/mL as presented in Figure 2. The highest antioxidant activity was shown in R1 with IC50 83.48 μg/mL and the lowest activity was shown from R12 with IC50 499.24 μg/mL.
Total Phenolic Content (TPC)
TPC of all sample were calculated using the equation of gallic acid calibration curve obtained, y = 0.0688x + 0.005 (r=0.9996). All samples showed total phenolic content in a range of 9.92 – 41.63 mg GAE per gram (Figure 3). Sample R9 had shown the highest total phenolic content (41.63 mg GAE per gram), while the lowest was in sample R12 (9.92 mg GAE per gram).
Statistical analysis of the results
The experimental design used in this study was factorial design with variable factors of ethanol concentration, the addition of HCl, and temperature. The ethanol concentration was divided in three treatment levels, namely 50%, 70%, and 100%. The HCl factor was divided into two levels, namely added (Yes), and not added (No), while the temperature factor was divided into two levels of 300C and 600C. The response variable was antioxidant activity and total phenolic content. From the above treatment factors analyzed by ANOVA, the HCl factor gave the significant influence compared with other factors, in the response of antioxidant activity with a p-value of 0.048, and in the response of total phenolic content with a p-value of 0.034 (p-value <0.05). This suggests that the addition of HCl has the most significant effect on the antioxidant activity and total phenolic content of T. hemprichii extract.
T. hemprichii is one of the scattered seagrasses on the coast of Indonesia and yet has not been utilized. Several studies have shown their potency as an antioxidant, therefore, the authors are interested to explore it. Phenolic acids and flavonoids have been reported in this seagrass.3,4,5 The phenolics were reported capable of scavenging free radical chain by donating the hydrogen,17 and flavonoids also contribute to the antioxidant activity because of their hydroxyl functional group at the site of the structure.18
Extraction played important role in isolating phytochemicals from the plant materials. One of the factors that could affect the extraction process is a selection of solvent. The ethanol was chosen in this study because its considered safer, and environmentally friendly compared to other solvents. Moreover, several studies have demonstrated higher antioxidant activity as well as total phenolic content when extracted with ethanol in Caesalpinia bonduc,19 seagrass T. testidunum,10 palm kernel-by product14 and in black tea and mate tea.20
Another significant influence in obtaining the phenolic content and antioxidant activity is the proportion of water in ethanol.14 According to Markham,21 the best solvent for extracting phytochemicals compounds is a mixture of alcohol and water, rather than water or alcohol alone. The possible reason is due to the plant swelling by the water through the increase of surface contact area as also reported by Xiao et al.22
In this study, other than the solvent, an addition of HCl and temperature have also shown significant influence on antioxidant activity and total phenolic content of seagrass T. hemprichii as seen in the results (Figure 2 and 3). The used of HCl and high temperature in the extraction will make the plant cell wall becomes more fragile so that the intracellular compounds can be extracted out into the solvent. A similar trend was also reported by Inggrid et al.6 in obtaining the antioxidant activity and phenolic content from strawberry fruits. According to Inggrid et al,6 the stability of some phenolic compounds e.g anthocyanin strongly affected with pH and temperature. Higher temperature from 50C to 600C could increase diffusion rate and solubility of phenolic content, moreover, lower pH could also affect the result of phenolic content.6
The used of factorial design in this study was to investigate the effect of all experimental factors on responses and finding the most significant factors through ANOVA test. The results of ANOVA showed that the significant influence on antioxidant activity and total phenolic content was from the addition of HCl. The correlation test between the result of antioxidant activity (IC50) and total phenolic content of seagrass showed a strong linear correlation (R2 = 0.8768, r= -0,936) and similar with the one reported by Jeyapragash et al.1 (R2 = 0.864).
The percentage yield of extracts showed in Figure 1 was generally increased by the increasing portion of water in ethanol. Samples extracted in 50% ethanol solvent showed a higher yield of extract compared to samples in 70% and 100% ethanol. This is probably because 50% ethanol has the same ratio of ethanol and water so the phenolic compounds that soluble in organic solvents or in water can be extracted into that solvent.
The extraction condition used in this study has been able to obtain higher antioxidant activity and total phenolic content from the seagrass T. hemprichii. Higher antioxidant activity (IC50) 83.48 μg/mL was shown in sample R1 (extracted with 50% ethanol, added with HCl 1 N, and 600C). The sample showed total phenolic content 41.03 mg GAE per gram and 22% yield extract.