Background

Halimeda sp lives in shallow waters. This biota can grow well in protected water types such as bays and sheltered island. Maspari Island is the only island located in South Sumatra Province. The island has a large natural resource potential, one of which is seaweed. There are at least 19 types of seaweed in Maspari Island. One of them comes from the genus Halimeda. Seaweed type Halimeda micronesica dominate the waters, while the seaweed type Halimeda macroloba does not dominate.

Antioxidants are compounds that can counteract free radicals and reduce the negative effects of oxidants in the body (Julyasih et al., 2009). Antioxidants have molecular structures that can give electrons to free radical molecules without disturbing their function and can break the chain reaction of free radicals (Ramadan, 2015).

This research is expected to provide information about the antioxidant activity of seaweed extract of H. micronesica and H. macroloba as input for further development and increase the added value of seaweed type H. micronesica and H. macroloba as the natural source of antioxidant.

1 Materials and Methods

1.1 Time and place

The study was conducted on March-October 2019. Seaweed samples were taken in the waters of Maspari Island, South Sumatra. The analysis of antioxidant test was conducted at Marine Biophysics Laboratory, Marine Oceanography and Instrumentation Laboratory, Marine Science and Basic Chemistry Laboratory, Chemistry Department, Faculty of Mathematics and Natural Sciences, Sriwijaya University, Inderalaya. The sampling location is located at coordinates 106º13’0,743”BT and 3º12’57,151”LS. The location of the study is presented in Figure 1.

Figure 1 Research site

1.2 Sampling and handling samples

Seaweed is cleaned by using seawater to prevent dirt and epiphytes from sticking out. Then the sample is taken to Marine Biotech Laboratory, Marine Science Study Program, Faculty of Mathematics and Natural Sciences, Sriwijaya University, Inderalaya for further extraction.

1.3 Seaweed extraction

Seaweed extraction process is very important to get the rough extract of seaweed to be tested as an antioxidant. Here's how to extract seaweed.

1.3.1 Mixed maceration

The sample uses fresh seaweed because according to Suryaningrum et al. (2006), fresh samples yield more extracts than dry samples. Samples are macerated by using 2 solvents with different polar properties namely ethyl acetate (semi polar) and methanol (polar). Samples of 100 grams are dissolved in 400 ml of ethyl acetate (1: 4 b/v) solvent for 2x24 hours and then filtered with Whatman 40 filter paper to obtain macerate and pulp. The dregs are again macerated with methanol solvent and then filtered by the same treatment (Septiana and Asnani, 2013). The process of maceration is done in 1 repetition.

1.3.2 Evaporation of macerate

The produced macerates are evaporated using a Rotary Evaporator. The temperature used in the evaporation process is 40℃ so that the compound contained in it is not damaged (Pramesti, 2013).

1.4 Potential test of antioxidant activity

Seaweed extract is weighed as much as 0.03 gr and 30 mL of methanol is added which resulted in a stock solution with a concentration of 1 000 ppm. DPPH solution is prepared by dissolving 0.003 gr of DPPH crystals into 10 mL of methanol and obtaining a solution with a concentration of 300 ppm. The concentration series is made by dilution of the main liquor to 750, 500, 250 and 50 ppm (Suhendra and Arnata, 2009).

Sample extract solution, sample control, negative control, blank and positive control are homogenized and incubated in dark spaces at room temperature for 30 minutes. Each solution is introduced into quvet (v=4 mL) of 3.75 mL. Each concentration is done in 3 times repetition. The active extracts are re-assessed with concentrations of 40, 80, 120, 160 and 200 ppm to determine the antioxidantlamic activity. The active extracts are tested at concentrations of 40, 80, 120, 160 and 200 ppm.

According to Sari et al. (2015), Husni et al. (2014), Pramesti (2013), Nurjanah et al. (2011) and Salamah et al. (2008), DPPH uptake is measured using a UV-Vis spectrophotometer at λ_maks 517 nm. The antioxidant activity of an extract is based on the value of percentage calculation of DPPH uptake inhibition and the ability of an extract to reduce 50% of DPPH radical through IC₅₀ value.

1.5 Phytochemical test

1.5.1 Steroid and triterpenoid test

Extract and chloroform are dropped on the drop plate to be set aside for a few minutes to dry. After it got dried, 10 drops of anhydrous acetic acid and stirred until homogeneous are added. After that, 3 drops of 96% sulfuric acid are added. Positive samples contain steroids if they are blue or green and positively contain triterpenoids when they are red or violet.

1.5.2 Flavonoid test

Positive samples contain flavonoids when yellow to orange are formed. Flavonoid test is done by adding 5 mL 70% of ethanol to the extract and heating it. After that, the extract is filtered and the filtrate is taken. 2 drops of 10% NaOH are added.

1.5.3 Saponin test

The extract is added with distilled water and it is boiled for 2 minutes. After it gets cold, 1 drop of HCl 2N is added and shake until homogeneous. The positive extract contains saponins if the foam is formed.

1.5.4 Alkaloid test

The extract is added with 10 mL of chloroform ammonia solution and homogenized for 1 min. The filtrate is taken by using a spotting pipette and then filtered. After that, the filtrate is added with H₂SO₄ 96% amounted to 5 drops. Water and chloroform are separated and meyer, dragondorf, and wagner reagents are added. Observe the changes in the extract formed by the deposit.

1.5.5 Tanin test

2 mL extract solution was added to a test tube with 4 drops of 1% NaCl. Then, 3 drops of gelatin solution are also added to be homogenized. The positive extract contains tannin if there is deposit at the bottom of the test tube.

1.6 Data analysis

1.6.1 Percentage calculation of antioxidant inhibition

According to Fithriani et al. (2015), the inhibition percentage of DPPH absorbance by the extract to determine antioxidant activity is calculated using the following formula:

Where, A=Abs Negative control; B=Abs blank; C=Abs extract; D=Abs control extract

According to Mega and Swastini (2010), a compound has antioxidant activity when its percentage is more than or equal to 50%. If the percentage of resistance 0-<50% means no potential as an antioxidant and 100% indicates the need for dilution of the sample to know the limits of activity concentration.

1.6.2 IC₅₀ Value calculation

According to Sugiat et al. (2010), IC₅₀ is the value of extract concentration to inhibit free radicals by 50%. The equation of quadratic line formed from percentage data of inhibition and concentration is used to determine IC₅₀ value. The equation of the squared line can be expressed as:

Y=a+bX

According to Jun et al. (2003) in Samin et al. (2013), the antioxidant power criterion is IC₅₀<50 ppm=strong, IC₅₀ 50-100 ppm=active, IC₅₀ 101-250 ppm=medium, IC₅₀ 251-500=weak and IC₅₀>500 ppm=inactive.

2 Results

H. macroloba lives on a sandy substrate and has a long thallus of 76.65 mm and a width of 30.42 mm. H. macroloba has holdfast with a smooth fiber containing sand. Branching H. macroloba has a dichotomous type and has a flat oval thallus. Measurements of H. macroloba are presented in Figure 2.

Figure 2 Halimeda macroloba

Halimeda micronesica has a lush, small, curved and green thallus. Thallus will turn whitish when H. micronesica dies. The seaweed has a single, hard holdfast. The branching of thallus has a trichotomous type. The thallus segment of H. micronesica has a width of 66.15 mm and a segment length of 93.60 mm. The thallus segment forms a semicircle like a kidney shape. The results of H. micronesica measurements are presented in Figure 3.

Figure 3 Halimeda micronesica

The result of measurement of waters quality in Maspari Island obtained average temperature value of 29.33°C, DO (Dissolved oxygen) content of 4.25 mg/L, salinity 32.67‰, and pH value of 7 and density of 1 024.67 kg/m³. The value of the measurement of the parameters of the waters is presented in Table 1.

Table 1 Average Measurement of Seawater Parameters on Maspari Island Note: Source: Research Results, 2019

The waters of Maspari Island can be categorized as the suitable place for the growth of seaweed. This is in accordance with the statement of Marianingsih et al. (2013) that the macroalgae can grow optimum at temperatures ranging from 25℃~31℃ and pH ranges from 7~8. Meanwhile, according to BSNI (2010), water quality requirements for seaweed production with temperatures ranging from 26℃~32℃, salinity ranges from 28~34 mg/L and the pH ranges from 7~8.5. Ladunta et al. (2015) state that good water quality for marine algae growth with temperatures ranging from 29.8℃~31℃, DO content 4.2~5.4 mg/L, salinity ranges from 31~35 ppt and pH ranges from 7.5 to 8.1.

The multilevel maceration performed in this study resulted in a crude extract of H. micronesica with an ethyl acetate solvent of 0.73 gr and a methanol of 1.05 gr. H. macroloba rough extract with ethyl acetate solvent of 0.98 gr and methanol 1.22 gr. The yield of H. micronesica extract was 1.78% while H. macroloba was 2.2%, as presented in Table 2.

Table 2 Extraction Results and Yield Extracts of H. micronesica and H. macroloba Using Ethyl Acetate and Methanol Solvents Note: Source: Research Data Result, 2019

H. macroloba sample extracts have more quantities than H. micronesica extracts. This is caused by the filtering process at the time of maceration leaving the extract on the filter. In addition, the maceration time and evaporation process also affect the amount of the produced cruide extract. The longer the maceration time the more dissolved extract compound will be. Too long evaporation process will cause the extract to be too dry. Thus, it will be difficult to move from the flask to the vial tube. If it is too fast then the extract will be difficult to dry.

Based on Table 3 the lowest IC₅₀ values are found in H. micronesica samples with ethyl acetate solvent which is worth 52.81 ppm whereas the highest IC₅₀ value is found in H. micronesica sample with methanol solvent amounted to 2 142 442.09 ppm. Samples that potentially have antioxidant activity are samples of H. micronesica with an ethyl acetate solvent. The other three samples are not potential antioxidants because they have too high IC50 values. This may occur due to a multilevel maceration process that causes compounds that have the ability as an antioxidant already dissolved in an ethyl acetate solvent. So when the H. micronesica sample is dissolved using methanol, the antioxidant compound is gone.

Table 3 Results of linear regression calculations and IC50 extract of H. micronesica and H. Macroloba Note: Source: (Data Research Result, 2017); Information: Hmi EA= H. micronesica Ethyl Acetate; Hma EA= H. macroloba Ethyl Acetate; Hmi M= H. micronesica Methanol; Hma M= H. macroloba Methanol

Phytochemical test results of seaweed extract of H. micronesica ethyl acetate containing alkaloid, steroid and flavonoid. H. micronesica methanol extract contains only alkaloid compounds. Phytochemical test results are presented in Table 4. Based on Table 3 ethyl acetate extract H. micronesica has active antioxidant activity. It can be caused by the content of the compounds contained in the extract of flavonoid compounds that function as antioxidants (Winarno, 1996 in Salamah et al., 2008; Redha, 2010; Firdiyani et al., 2015). H. micronesica methanol extract has weak antioxidant activity because based on phytochemical test results only contain alkaloid compounds that function as antibacterial as proposed by Robinson (1995) in Darsana et al. (2012).

Table 4 Phytochemical Test Results of Halimeda micronesica Ethyl Acetate and Methanol Extracts Note: Source: (Research Result Data, 2019)

3 Conclusion

Based on the result of the research, the conclusion is as follows:

I. micronesica extract in an ethyl acetate solvent (Hmi-EA) has a percentage of free radical inhibition ranging from 43%~89.2%. The percentage of free radical inhibition on H. micronesica extract in methanol solvent (Hmi-M) ranging from 11.14%~21.61%. H. macroloba extract in ethyl acetate (Hma-EA) solvent has inhibition percentage ranging between 16.57%~29.01% and H. macroloba extract in methanol solvent (Hma-M) ranging from 10.68%~21.79%.

Hmi-EA extract has potential antioxidant activity with IC₅₀ value of 52.81 ppm. All three other extracts have no antioxidant activity potential.

The results of phytochemical test of the Hmi-EA extract contain alkaloid compound, steroid and flavonoid. Flavonoid can function as antioxidants.

Authors’ contributions

Muhammad Hendri participated in the design of the study and performed the statistical analysis, conceived of the study, participated in its design and coordination and helped to draft the manuscript, writing and correspondence. Rozirwan participated in the sequence alignment. All authors read and approved the final manuscript.

Acknowledgements

The authors wish to thank Universitas Sriwijaya for support throughout the research, in particular the Faculty of Mathematics and Natural Sciences, especially the Department of Marine Sciences and the faculty staff. Special thanks go to Yoga Pebrian Firmansyah, S. Kel, Adi Murdianto, S. Kel, Tri Rizky Oktariansyah, Mas Ebiet and Mas Kacong (Pahawang) for their assistance during the research.  

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