AIP Conference Proceedings 2092, 030016 (2019); https://doi.org/10.1063/1.5096720 2092, 030016
© 2019 Author(s).
Phytochemical analysis and antioxidant activity of marine algae Eucheuma Sp.
Cite as: AIP Conference Proceedings 2092, 030016 (2019); https://doi.org/10.1063/1.5096720 Published Online: 09 April 2019
Trivani Putri, Ade Arsianti, Priscilla Aya Maheswari Subroto, and Elvira Lesmana
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Phytochemical Analysis and Antioxidant Activity of Marine Algae Eucheuma Sp.
Trivani Putri
1,a), Ade Arsianti
2,3,b), Priscilla Aya Maheswari Subroto
1,c), and Elvira Lesmana
1,d)1Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
2Department of Medical Chemistry, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
3Drug Development Research Cluster, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
Corresponding author: a)[email protected], b)[email protected], c)[email protected],
Abstract. Marine algal is one of marine resources which has a potent antioxidant activity. This research aims to explore two species of Indonesia’s marine algal, Eucheuma cottonii and Eucheuma spinosum, as a natural antioxidant. In this work, Seaweed Eucheuma Sp. originated from Labuan Aji beach, Nusa Tenggara Barat, Indonesia, were extracted into two different polarity of organic solvents, that is, semipolar solvent of ethyl acetate and polar solvent of ethanol. The extracts were used for phytochemical test and analyzed by Thin Layer Chromatography (TLC). Afterwards, antioxidant activity evaluation of ethyl acetate, and ethanol extracts Eucheuma cottonii and Eucheuma spinosum were conducted by DPPH method. Phytochemical analysis of those two Eucheuma Sp. extracts are positive for a secondary metabolite of flavonoid, triterpenoid, alkaloid and tannin. TLC analysis revealed that the extracts contain one until four chemical components.
Among Four concentrated extracts of Eucheuma Sp., the best antioxidant activity has shown by polar ethanolic extract of Eucheuma cottonii with IC50 value of 35.51 ppm, suggesting that ethanol extract of Eucheuma Sp. is potential to be developed as a promising naturally obtained antioxidant.
Keywords: antioxidant, DPPH method, Eucheuma Sp., marine algal, phytochemistry
INTRODUCTION
Reactive oxygen species (ROS) are naturally produced by cellular enzymatic reaction in our body. Besides the highly reactive activity of this molecule, ROS are required to maintain cell homeostasis [1]. Antioxidant are compound that can inhibit reactive oxygen and free radical in the body by giving one or more its electron to free radical so that it becomes a normal molecular shape again and stop the various damage [1]. Our body has its own antioxidant defense system to fight the harmful effect caused by increased level of ROS. Unfortunately, human body does not have enough antioxidant stores if there is exposure to excess free radical. In that case, we need exogenous antioxidant [2].
Growing knowledge of ROS, triggered the development of antioxidant compound research. Since the use of synthetic antioxidant, such as butylated hydroxytoluen (BHT) and butylated hydroxyanisole (BHA) are strictly restricted due to their carcinogenic effect, researcher start using natural ingredients as antioxidant. Phenolic compound such as flavonoids, cinnamic acid, benzoic acid, gallic acid, phlorotanninns and quercetin are antioxidant compounds produced by plants. Among marine organism, seaweed represent one of the richest source of antioxidant. The absence of oxidative damage in the structural component of seaweeds besides exposure to a combination of stressful factor, including light and oxygen, suggest their cell have protective antioxidative defense system [2].
Seaweeds are marine organism that are easily found throughout the world. Until today, more than 10.000 marine algae species have been known. Based on their pigmentation, there are three main groups of seaweed, brown seaweed (phylum ochrophyta), red seaweed (phylum Rhodophyta), and green seaweed (phylum chlorophyta). Seaweed has been extensively used as traditional medicine in Asian country. The use of plants as traditional medicine has been known by Indonesian people for long time before synthetic drugs are exist. 90% of herbal plants in Asia are located in Indonesia, which making Indonesia as one of the country that has various traditional medicines [3].
Seaweed has several bioactive substances like polysaccharide, protein, and polyphenol, with potential medicinal use as anticancer, inflammation, and degenerative diseases [4]. Chemical and nutritional composition of seaweed varies with individual, species, and depend on geographical origin, seasonal, physiological variations, dan water temperature. Antioxidant activity has been reported in numerous marine algae, including Gracilaria, Turbinaria, Colpomenia, Halymenia [5]. Based on previous study on red algae, the in vitro antioxidant activities of methanol extract and different organic fractions of Hypnea musciformis, Hypnea valentiae, and Janua rubens, exhibited dose dependency, and increase with increasing concentratrion. Ethyl acetate fractions of red seaweed were found to be more effective than n-hexane, dichloromethane, and crude methanol extract [5].
Antioxidant properties of seaweed have been evaluated by numerous studies, yet there is still limited information about antioxidant potential of seaweed species. Based on this background, present research were aim to evaluate the phytochemical and antioxidant activities of Euchema sp. as one of red algae. This research consists of several stages.
First, sample Eucheuma sp. are collected and identified. The second stage is extraction on sample Eucheuma sp. into four different polarity of organic solvent, which are n-hexane, chloroform, ethyl acetate, and ethanol. The third stage is phytochemical test and analyzed by TLC. Last stage is evaluation of antioxidant activity using DPPH method.
DPPH method is an easy, fast and sensitive method for testing antioxidant activity in certain plant extract.
METHODS
Extraction and Fractionation of Seaweed Samples
Powder sample of Eucheuma cottonii and Eucheuma spinosum are macerated using two different solvents, that are ethyl acetate, and ethanol. This procedure is done in stages, which is carried out by immersing macroalgae powder in 500 mL ethyl acetate for 24 hours twice, then filtered. The liquid filtrate is collected, while the solid filtrate will be dissolved in ethanol solvent. The filtrate solution from each solution is then concentrated using a rotary evaporator, producing extract of seaweed in ethyl acetate and ethanol. These extracts were then analyzed using thin layer chromatography to determine the number of compounds contained in the fraction.
Thin Layer Chromatography
Thin layer chromatography is a method used to separate sample components based on their polarity. This method is performed by dripping the sample at approximately 1.5 cm from the bottom of the plate. TLC plate usually coated with an adsorbent material such as silica gel or cellulose, known as stationary phase. In this research, the mixture of chloroform (CHCl3)- methanol(CH3OH) in ratio 3:1 was used as a mobile phase, and silica gel as a stationary phase.
The TLC plate then placed into a glass that has been filled with a suitable eluent. Different compound in the sample will move at different rates due to its different polarity against stationary phase and solubility in eluent. This procedure will show the amount of metabolite contents. Rf (retention factor) value is defined by the distance taken by sample compared to solvent.
Rf (Retention factor) = distance traveled by sample / distance traveled by solvent
Phytochemical Analysis
Organic compounds inside sample of ethyl acetate and ethanol extracts of Eucheuma sp. can be identified through phytochemical test, which consist of few stages.
Alkaloid Test
About 2 mL of sample solutions in the form of paste were evaporated on a porcelain cup. The residue formed will be diluted with 5 ml of HCL 2N and then divided into 3 tubes. The first tube will be added with HCl 2N and works as blanko. Second tube will be added with 3 drops of dragendorff reagent and 3 drops of meyer reagent will be added to the last tube. Positive result will show orange precipitate in the second tube or yellow precipitate in the third tube.
Saponin Test
10 ml of solution is added to the tube and vertical shuffle is done for about 10 second. After another 10 second, if there is presence of stabile foam around 1-10 cm in height in less than 10 minutes indicates positive result.
Confirmation test can be done by adding 1 drop of HCl 2N, the foam is supposed to stay for positive result.
Flavonoid Test
Flavonoid screening is done with evaporating 1 ml solution until it dry. Left solution will be mixed with acetone and smooth powder boric acid and axalate acid. After being heated with water bath, residue that is formed will be mixed with 10 ml of ether. Presence of yellow fluorescence after using UV light with a wavelength of 365 nm, indicates positive result.
Triterpenoid and Steroid Test
This test is done with Liebemann-Burcahrd reaction. About 2 ml of solution is evaporated and the residue is dissolved in 0.5 ml of chloroform and 0.5 ml of anhydrate acetate acid. 2 ml of concentrated sulfuric acid is added through the tube wall. Formation of blue-green ring, indicates positive result for triterpenoid and steroid.
Tannin Test
1 ml of solution is dissolved in iron (III) chloride 10%. Dark blue or black greenish color suggest the presence of tannin.
Glycoside Test
Evaporation of 0.1 ml solution is done on water bath. The remaining solution is dilutes into 5 ml of acetic acid anhydrate and 10 drops of concentrated sulfuric acid will be added. Positive result for glycoside is indicated by the formation of blue or green product.
Antioxidant Analysis
Antioxidant test is done using DPPH (2,2-diphenyl-1-picrylhydrazyl) method. 100 L of the extracted filtrate will be taken and placed into a tube. About 2,9 ml DPPH with 0,004% concentration in methanol is added into the tube and then incubated in room temperature and lightless condition for 120 minutes. Absorbance was then measured using 515 nm wave. The percentage of free radical inhibition is calculated by the formula:
DPPH inhibition (%) = (absorbence of blank – absorbence of sample) / absorbance of blanko) x 100%
Information:
Absorbance of blank = DPPH absorbance without sample Absorbance of sample = absorption of DPPH by given sample
The standard curve employed is y = 3.93x + 7.82 (r2 = 0.907) and potency of antioxidant is expressed as equivalent to mg of ascorbic acid per gram of sample dry weight.
RESULTS AND DISCUSSION Phytochemical Composition
Phytochemical composition of Eucheuma cottoni is summarized in Table 1 and for Eucheuma spinosum is displayed in Table 2.
Eucheuma cottonii
TABLE 1. Phytochemical Composition of Eucheuma cottonii
Metabolites Solvent
Ethyl acetate Ethanol
Saponin - -
Flavonoid + +
Triterpenoid + +
Steroid - -
Alkaloid + -
Tannin - +
Glycoside - -
Eucheuma spinosum
TABLE 2. Phytochemical Composition of Eucheuma spinosum
Metabolites Solvent
Ethyl acetate Ethanol
Saponin - -
Flavonoid + +
Triterpenoid + -
Steroid - +
Alkaloid + -
Tannin - +
Glycoside - -
From all metabolites that are being tested, only flavonoid, triterpenoid, alkaloid, and tannin show positive result in E. cottonii extracts. Both flavonoid and triterpenoid are present in all E. cottonii extracts, while alkaloid only show positive result in ethyl acetate extract and tannin in ethanol extract. As for Eucheuma spinosum, flavonoid presents in all extract. Besides flavonoid, triterpenoid, and alkaloid also show positive results in ethyl acetate extract of E.
spinosum. Flavonoids are a group of natural low-molecular-weight phenolic compounds with variable structures [6].
In plant, flavonoid are involves in so many processes, which are plant-pathogen interaction, pollination, light screening, and seed development [7]. Their biosynthesis gene will be induced by biotic and abiotic stress [7]. Few reports tent to show that secondary metabolites of phenolic nature including flavonoids are responsible for many pharmacological activities, including protective effects against many infectious and degenerative disease (cardiovascular, cancer, and other age-related diseases), and anti-inflammation activity [8].
Different seaweed species and solvents will give a difference in the number of total phenolic compounds [9].
External factors such as radiation, depth, and nutrition can give variation in total phenolic compound of macroalgae.
Besides that, variation of total phenolic compounds also influenced by intrinsic factors such as morphology of seaweed, age, and stage of reproduction [10]. It is well known that solvent polarity will play an important role in determining phenolic solubility. Non-polar solvent such as n-hexane usually used to extract lipophilic compounds, including terpenoids and alkaloids [11]. Methanol, ethanol, and acetone are polar solvents which mainly used to extract polar compounds, including flavonols, alkaloids, polyphenol, and saponins [11]. Meanwhile, ethyl acetate is usually used to extract some alkaloid, flavonoids, and terpenoid [11]. Based on previous study, Tomsone et al., states that ethanol is the best solvent for phenolic extraction horseradish roots [12]. Similar result are found in study that was conducted by Do et al, in which extract of Limnophila aromatica in 100% ethanol fraction showed the highest amount of total phenolic content and total flavonoid content than the other solvents system (water, methanol, acetone) [13].
Thin Layer Chromatography
The results of TLC analysis of E. cottonii and E. spinosum are shown in Table 3 and Table 4.
TABLE 3. TLC analysis and Retention factor (Rf) of E. cottonii extracts.
TABLE 4. TLC analysis and Retention factor(Rf) of E. spinosum extracts.
Extract Rf value
1 2 3
Ethyl Acetate 0.281 0.625 0.906
Ethanol 0.218 0.281 0.656
*Rf = Retention factor
(a)
(b)
FIGURE 1. (a) TLC Result from n-hexane, ethanol, chloroform, and ethyl acetate extract of E. cottonii. (b) TLC result from chloroform, n-hexane, ethyl acetate, and ethanol of E. spinosum.
Thin layer chromatography (TLC) separates different compound based on their polarity. TLC analysis on Eucheuma cottonii extract shows that ethyl acetate extract contains only one spot of chemical compound with Rf at 0.906, whereas ethanol extract has two spots of chemical compounds. TLC analysis of ethyl acetate and ethanol extracts of Eucheuma spinosum revealed that both of extracts have three spots of chemical components, which are similar because they have one spots with the same Rf at 0.281.
Antioxidant Activity
In present study, antioxidant activity was evaluated using DPPH method. DPPH (2,2-diphenyl-1-picrylhydrazyl) is a free radical that is stable in room temperature and can be reduced when antioxidant molecule is present [14]. When DPPH solution is mixed with antioxidant, which act as hydrogen donor, there will be a loss of DPPH violet colour [15]. The degree of discoloration indicates the scavenging potential of antioxidant extract [15]. This method is widely used to measure the ability of compound to act as free radical scavengers or hydrogen donor because it is one of the easy, rapid, simple, and inexpensive method [16].
Eucheuma cottonii
Parameter that is used for the interpretation of DPPH method is IC50 value. This defines as concentration of
Extract Rf value
1 2 3
Ethyl Acetate 0.906 - -
Ethanol 0.187 0.968 -
the substrate [17]. In present study, the test was performed on ethanol extract of Eucheuma cottonii with duplo system.
Test result is shown in Table 5.
TABLE 5. Antioxidant Activity of Ethanol Extract of Eucheuma cottonii Concentrations of Ethanol extract
of E. cottonii %Inhibition SD
3.75 -2.0 0.0242
6.25 6.7 0.0159
12.5 13.2 0.0662
25 34.7 0.0548
50 72.1 0.0728
IC50 value from ethanol extract of Eucheuma cottonii obtained from the calculation of linear regression equation, which is y = 1.5647x – 5.5685 and R2 = 0.99605. Coefficient y represents IC50 value, whereas coefficient x represent the amount of concentration required to absorb 50% of DPPH radical activity.
Based on the calculation using equation above, IC50 value of ethanol extract of Eucheuma cottonii is 35.51 ppm.
Highest concentration of ethanol extract tested, which is 50 g/mL, showed inhibition of 72.1% of DPPH activity.
This indicates the promising antioxidant activity from Eucheuma cottonii.
TABLE 6. Antioxidant Activity of Ethyl Acetate Extract of Eucheuma cottonii Concentrations of Ethyl Acetate Extract
of E. cottonii %Inhibition SD
3.75 -4.8 0.0257
6.25 -3.8 0.0470
12.5 -3.6 0.0304
25 -1.7 0.0122
50 1.3 0.0391
Antioxidant test result using ethyl acetate extract of Eucheuma cottonii is shown in Table 6. IC50 value from ethyl acetate extract of Eucheuma cottonii obtained from the calculation of linear regression equation, which is y = 0.1263x – 4.9864 and R2 = 0.98825. IC50 extract value required to inhibit 50% of DPPH radical activity is 435.36 ppm. This value is outside the curve. The highest concentration tested, which was 50 g/mL, can only inhibit 1.2981% of DPPH activity. These results indicate the sample has better inhibitory activity on its ethanol extract than its ethyl acetate extract.
Previous study stated that antioxidant activity can be divided into 3 classes depend on its 1C50 values. Substances with IC50 < 50 ppm, said to have very strong antioxidant activity, IC50 = 50-100 ppm said to be a potential antioxidant, and IC50 > 150 ppm classified as weak antioxidant [18]. IC50 value for ethanol extract of E. cottonii is 35.51 ppm, this suggest that ethanol extract has very strong antioxidant activity. On the other hand, IC50 for ethyl acetate extract is 435.36 ppm, which means this extract has weak antioxidant activity.
Eucheuma spinosum
TABLE 7. Antioxidant Activity of Ethanol Extract of Eucheuma spinosum Concentrations of Ethanol Extract of E. spinosum %Inhibition SD
4.687 -16.3 0.0009
18.75 -0.2 0.0010
37.5 0.7 0.0010
62.5 3.1 0.0001
125 3.1 0.0007
250 8.8 0.0000
500 5.3 0.0005
Same method as before, applied for antioxidant test in ethanol extract of Eucheuma spinosum. IC50 value is obtained from equation, y = 4.4543ln(x) – 17.671 and R2 = 0.77637. Using this equation, concentration needed to inhibit 50% of DPPH activity is 1386,79 ppm. As we can see from the Table 7, 500g/mL of ethanol extract of Eucheuma spinosum can only inhibit 5.3% of DPPH activity. This result suggested that ethanol extract of Eucheuma spinosum has weak antioxidant activity compare to ethanol extract of Eucheuma cottonii.
TABLE 8. Antioxidant Activity of Ethyl Acetate Extract of Eucheuma spinosum Concentrations of Ethyl Acetate
Extract of E. spinosum %Inhibition SD
18.75 44.8 0.0004
125 48.3 0.0001
500 50.4 0.0004
After took percent inhibition and log. concentration of ethyl acetate extract into count, we got the regression linear equation, y = 0.01x + 45.695 and R2 = 0.81184. From this equation, we calculate the concentration needed to inhibit 50% of DPPH activity and the result is 430.50 ppm, which indicate this ectract has weak antioxidant activity. Whereas, 500 g/mL of ethyl acetate extract of Eucheuma spinosum shows 50% inhibition activity of DPPH. This result suggest sample has better inhibitory activity on its ethyl acetate extract than its ethanol extract.
TABLE 9. Summarizes Antioxidant Activity (IC50 value in ppm) of Eucheuma cottonii and Eucheuma spinosum Extracts
Extract IC50 value (μg/mL)
Eucheuma spinosum Eucheuma cottonii
Ethanol 1386.79 35.51
Ethyl acetate 430.50 435.36
Based on those results above, if we compare ethanol extract of Eucheuma cottonii and Eucheuma spinosum, it shows that ethanol extract of Eucheuma cottonii has better inhibitory activity than Eucheuma spinosum. This is proved by the data, in which IC50 value in ethanol extract of Eucheuma cottonii is 35.51 ppm, whereas IC50 value in ethanol extract of Eucheuma spinosum is 1386,79 ppm. The smaller the value of IC50, the greater free radical inhibition activity.
As for IC50 value from ethyl acetate extract of Eucheuma cottonii dan Eucheuma spinosum, the results do not show any significant differences.
Significant correlation between the phenolic content and antioxidant activity in seaweed extracts have been proved by several studies. Chakraborty et al., observed a higher percentage of total phenolic contents in the polar solvent fraction, which is ethyl acetate, of Turbinaria spp. (brown seaweed) indicate their high antioxidant activity [19]. This research findings were in agreement with the result of Farasat et al., which reported a positive and significant correlation between DPPH radical scavenging activity and phenolic and flavonoid contents of Ulva species [20]. In their study, flavonoid is the main contributor of antioxidant activity from Ulva species [20]. Jaminez-escrig et al., also proved that there were positive correlation between DPPH-free radical scavenging and the total polyphenol content of brown and red algal extracts [21]. Basically, phenolic compounds act as electron donors. This reaction will neutralize free radicals in the body [22]. Unfortunately, in the current study, we do not analyze total phenolic contents and flavonoids from each extracts quantitatively. Besides its promising function as natural antioxidant, we still not sure whether is there any correlation between total phenolic contents and flavonoids from this extracts and its antioxidant activity.
CONCLUSION
Seaweed Eucheuma cottonii and Eucheuma spinosum showed antioxidant activity. In this study, ethanol extract of Eucheuma cottonii with IC50 value of 35.51 ppm has the strongest antioxidant activity.
ACKNOWLEDGMENTS
We wish to express our gratitude to Directorate of research and Public Service University of Indonesia for the research grant PITTA (Publikasi Internasional Terindeks Scopus Untuk Tugas Akhir Mahasiswa) for fiscal year 2018 (contract number : 2040/UN2.R3.1/HKP.05.00/2018).
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