The phytochemical constituent and antioxidant activity of Sea Weed Euchaema Cottonii

This study aimed to determine the phytochemical content and antioxidant activity of the red sea weed Euchaema cottonii. The phytochemical component of this seaweed can be used as an agent for enteric methane mitigation. The antioxidant can be used to reduce the reaction of free radicals when the animals are in heat stress conditions. The study presented used fresh and oven-dried E. cottonii. The methods of study started with the extracting process of two seaweed samples (fresh and sundried) using 3 types of solutions, namely water, hexane, and dichloromethane (DCM). The phytochemical content was analyzed using spektro-tanat, while antioxidant activity was determined using the DPPH test (1,1-diphenyl-2-picrilhidrazil). The result showed that the extract seaweed E. cottonii contained alkaloid, flavonoid, saponin, and steroid/triterpenoid. The seaweed did not contain tannin. The seaweed contains 0.249 to 2.430 mg TAE/gram of total phenolic. Based on the results of the antioxidant activity test using DPPH obtained extract seaweed E. cottonii is active as an antioxidant. The highest phytochemical and phenolic content resulted when the dried seaweed was extracted using the DCM. It is concluded that sea weed E. cottonii contains some phytochemicals and has an antioxidant activity.


Introduction
Seaweed is one of the plant's waters, which abundant production, has economic value and has many benefits.Seaweed uses for food, feed, cosmetics and even medicine, so the opportunities for using seaweed are very wide [1][2][3].Macroalgae bioactive compounds are one of the ingredients that can be applied in the health sector such as antioxidants, antibacterial, antifungals and antivirals [4].Abundant seaweed production makes Indonesia one of the largest exporting countries in the world.Total national fresh seaweed production in 2020 was 5.01 million tones, of which 4.66 million tones (93.01%) came from the sea and 351 thousand tones (6.99%) from ponds [5].
Red seaweed grows abundantly in tropical areas/Indonesian waters, containing the substance bromoform (CHBr3) which can manipulate microbial digestive enzymes responsible for methane 1312 (2024) 012006 IOP Publishing doi:10.1088/1755-1315/1312/1/012006 2 production.A wide range of bioactive substances are thought to exist in red sea weed, including polyphenolic compounds, carotenoids, minerals, vitamins, phlorotannins, peptides, tocotrienols, proteins, tocopherols, and carbohydrates (polysaccharides) [6][7][8].The red seaweed Eucheuma cottonii is a seaweed species that is widely cultivated in Indonesian waters because its cultivation techniques are relatively easy and cheap.Preliminary testing of the effectiveness of E. cottonii as feed additive, shows that this seaweed can reduce enteric methane production up to 16.74% [9].With the potential for other types of seaweed to be abundant in Indonesian waters, the use of seaweed as a livestock feed additive has the potential to reduce enteric methane gas emissions from ruminant livestock.
The E. cottonii has high nutritional value [10] and is rich in important nutrients, including enzymes, nucleic acids, amino acids, minerals, trace elements, especially iodine, and vitamins A, B, C, D, E and others [11].Apart from that, E. cottonii contains alkoloids, flavonoids, phenol hydroquinone, and triterpenoids/steroids, tannins, saponins and antioxidants.As photosynthetic organisms, seaweeds are subjected to a variety of stressful conditions, including light and oxygen, which can lead to the production of free radicals and other oxidative reagents.However, the lack of oxidative damage in seaweeds' structural elements shows that their cells have robust antioxidative defence mechanisms.Moreover, the potassium hydroxide-extracted E. cottonii shown strong antioxidant activity against the DPPH free radical, with an IC50 value of 39.94 ppm [12,13].The effective bioactive potential of seaweeds is determined by the quantity of particular secondary metabolites, with phenolic compounds being the most common type.However, since the nutrients content of the seaweed varies greatly depending on the area of growing, therefore this study aimed to determine the phytochemical content and antioxidant activity of the red sea weed E. cottonii growing on the coast of the western region of Indonesia.

Collection and preparation of Euchaema cottonii
The study used E. cottonii which was cultivated in the waters of North Banten and is directly harvested from the seaweed nursery of the "Sukadiri" famer group.Lontar village, Tirtayasa District.The seaweed was dried in the oven at a temperature of 45 o C until the water content reaches 20%.The dried E. cottonii was then ground and filtered using a 40-mesh sieve.The E. cottonii powder was stored in a closed container and kept in a cold room.While, the fresh E. cottonii is stored in the freezer (temperature -4 o C) for later use in the extraction process.

Extraction methods
The extraction process is carried out at the Laboratory of Feed Technology at the IRIAP, Ministry of Agriculture and at the Agrochemical Process Technology Laboratory, Center for Agro-Industrial Technology, BRIN.The extraction method used three solvents, including water, hexane, and dichloromethane (DCM).Two samples of E. cottonii (fresh and oven dried) was used in extraction process.The seaweed samples were weighed 10 grams each for 10 replications per each type of solvent.The extraction process was following method described by Praptiwi et al. [14].In an Erlenmeyer flask, a dried seaweed powder (10 grams) was weight out and a 50 mL extraction solvent each (water, hexane, and dichloromethane) was added; sonicated for 15 minutes and then follow for extraction in a vibrate vertical shaker for 20 hours at room temperature.After the mixture was left for overnight, the mixture was filtered using Whatman filter No. 41.The supernatant was collected, and the remaining residue was re-extracted twice with the same solvents and shake for 6-7 hours and left overnight.The supernatant from 3 times extraction and filtration were mixed.Supernatant from water extraction was kept frozen at -80 o C, the other supernatant from the hexane and DCM extracts were evaporated using a rotary vacuum evaporator under 40 o C to reach 1/3 of the volume of the initial solution.

Qualitative analysis of Phytochemical compounds of the extracts
Three replicates of supernatant generated from each type of solvents were qualitative analysed for the chemical constituents of extracts, including saponin, tannins, alkaloids, flavonoids, phenols, triterpenoids, and steroids were conducted following method described by Harborne [15].On a TLC plate, the antioxidative activity was carried out utilizing DPPH free radical scavenging.The Dot-Blot technique and elution with a development solvent were also used [16].The active chemicals in the extract were identified by the white band on the TLC plate.Serial microdilution concentration on a 96well microplate was also used to assess the IC50 value for antioxidative activity, which was then computed using a linear regression equation [17].
Data for total phenolic content and antioxidant activity were statistical analyzed using ANOVA for completed randomized design.

Results and Discussion
In several studies, the solvent used for extraction will produce different phytochemical contents.The current study found that fresh E. cottonii extracted using water produced qualitatively more phytochemicals, namely alkaloids, flavonoids and saponins than the solvents dichloromethane and hexane.In contrast, in dried E. cottonii the solvent dichloromethane produced qualitatively more types of phytochemicals than the solvents water and hexane (Table 1).The most positive components in fresh seaweed are produced from dichloromethane and hexane solvents, while in dried seaweed they are produced from dichloromethane solvents.Different results were reported by Syafitri et al. [12] that the most positive components were using n-hexane solvent, while methanol and distilled water obtained the same positive components.The difference in solvent polarity is one of the factors that causes saponin not to be detected in extracts with non-polar solvents such as n-hexane.
Alkaloids, a group of nitrogen-containing organic compounds, were analyzed using three different reagents: Dragendorff, Mayer, and Wagner.When fresh E. cottonii was extracted with Dichloromethane and Hexane, moderate levels of alkaloids were detected using Dragendorff and Wagner reagents.This indicates the presence of these alkaloids in both solvents.However, fresh seaweed extracted with water showed no presence of alkaloids using Dragendorff, while Hexane exhibited a moderate presence using Wagner's reagent.In contrast, oven-dried seaweed in water displayed a significant presence of alkaloids using the Wagner reagent.These results suggest that the choice of solvent and drying conditions significantly affect the detection of alkaloids in E. cottonii.Flavonoids, a class of polyphenolic compounds with various biological activities, were detected in fresh seaweed when extracted with water, albeit in small amounts.In contrast, Dichloromethane and Hexane extracts of fresh seaweed did not show the presence of flavonoids.Interestingly, oven-dried seaweed also displayed the presence of flavonoids in water extracts, albeit in small quantities.These findings suggest that the drying process may concentrate flavonoids in the seaweed, making them more detectable.
Saponins, glycosides with foaming properties and diverse biological activities, were detected in both fresh and oven-dried seaweed when extracted with water.The presence of saponins was also observed in Hexane extracts of fresh seaweed.These results imply that water is an effective solvent for extracting saponins from E. cottonii, regardless of its moisture content.
Steroids/Triterpenoids, compounds with diverse physiological roles, were primarily detected in the dichloromethane extract of both fresh and oven-dried seaweed.This suggests that dichloromethane is the solvent of choice for isolating steroids/triterpenoids from E. cottonii.Interestingly, fresh seaweed extracts showed no presence of these compounds, highlighting the impact of the drying process on their concentration.Tannins, polyphenolic compounds known for their astringent properties, were not detected in any of the extracts, whether from fresh or oven-dried seaweed, and regardless of the solvent used.This suggests that E. cottonii may not be a significant source of tannins.
The results of this analysis demonstrate the complex and solvent-dependent nature of compound extraction from E. cottonii.The choice of solvent and the moisture content of the seaweed can greatly influence the presence and concentration of specific compounds.These findings provide valuable insights for researchers and industries interested in harnessing the bioactive potential of E. cottonii, whether for pharmaceuticals, food products, or other applications, and emphasize the importance of tailored extraction methods based on the targeted compounds of interest.Further research is warranted to explore the specific properties and potential benefits of each compound class within this seaweed species.
Table 2. Total phenolic content and antioxidant activity from fresh and oven-dried E. cottonii extracted using water, dichloromethane and hexane as the solvents.
Fresh Note : Different superscript in the same row indicates a significant different among the treatment (P<0.05).

DCM = Dichloromethane
The table provides valuable insights into the total phenolic content of fresh and oven-dried E. cottonii extracts using different solvents.The results reveal significant variations in phenolic content (mgTAE/g): depending on the type of solvent used for extraction.Among the fresh seaweed extracts, the Hexane extract exhibited the highest total phenolic content with a value of 0.408 mgTAE/g.This suggests that non-polar solvents like Hexane are effective in extracting phenolic compounds from fresh seaweed.The Dichloromethane extract of fresh seaweed also displayed a substantial phenolic content of 0.288 mgTAE/g, while the water extract had the lowest phenolic content at 0.249 mgTAE/g.
In contrast, the oven-dried seaweed extracts showed a different pattern.The Dichloromethane extract of oven-dried seaweed had the highest total phenolic content at 2.430 mgTAE/g, indicating a significant increase compared to fresh seaweed extracts.This suggests that the drying process may concentrate phenolic compounds in the seaweed, making them more accessible for extraction with Dichloromethane.The Hexane extract of oven-dried seaweed also exhibited a notable phenolic content of 1.009 mgTAE/g, while the water extract had a total phenolic content of 1.275 mgTAE/g.These findings underscore the influence of both the solvent choice and drying conditions on the phenolic content of E. cottonii extracts.
In this study, it was discovered that the total phenolic content of fresh E. cottonii was lower than oven-dried seaweed.The solvent used on fresh E. cottonii did not significantly differ on the total phenolics produced.Meanwhile, in oven-dried E. cottonii, the solvent had a significant effect on the total phenolic content, where the dichloromethane solvent produced the highest total phenolics (Table 2).The total phenolic content in this study was considered low, this result was lower than the study reported by Foon et al. [18] and Chang and Teo [19].Low total phenolics according to Loho et al. [20] because the habitat of E. cottonii is in areas that do not receive enough sunlight.This is supported by Peinado et al. ( 2013) that there is a correlation between exposure to sunlight and high phenolic content.
The data on antioxidant activity in the table reflect the ability of the seaweed extracts to neutralize harmful free radicals.In the case of fresh seaweed, the Dichloromethane and Hexane extracts displayed the highest antioxidant activity, with values of 47.766 mg/L and 46.437 mg/L, respectively.This suggests that non-polar solvents are effective in extracting antioxidants from fresh seaweed, aligning with the trend observed in total phenolic content.
However, in the oven-dried seaweed extracts, a different pattern emerged.The Dichloromethane extract exhibited the highest antioxidant activity at 10.907 mg/L, while the Hexane extract also demonstrated notable antioxidant activity at 18.760 mg/L.Unlike the phenolic content, the water extract of oven-dried seaweed showed no detectable antioxidant activity.This disparity in antioxidant activity between fresh and oven-dried seaweed extracts may be attributed to changes in the concentration and composition of antioxidant compounds during the drying process.The antibiotic activity of E. cottonii in this study was lower (Table 2) when compared with the study by Maharany et al. [13] obtained an antioxidant activity value (IC50) of 106.021 ppm.The antioxidant activity of fresh E. cottonii was higher than dried.The use of water as a solvent did not detect any antibiotic activity in either fresh or dried E. cottonii.
The results emphasize the complex relationship between solvent choice, drying conditions, total phenolic content, and antioxidant activity in E. cottonii extracts.While non-polar solvents like Hexane and Dichloromethane are effective for extracting phenolic compounds and antioxidants, the drying process appears to significantly influence the concentration of these bioactive compounds.These findings are pertinent to industries interested in utilizing E. cottonii for its potential health benefits and underscore the need for tailored extraction methods based on the desired bioactive compounds.Further research is essential to identify the specific phenolic compounds responsible for the observed antioxidant activity and their potential applications.

Conclusion
It can be concluded that seaweed E. cottonii are rich on Phytochemical component and has antibiotic activity.There were different contain of the component when it was extracted using different solvents.The best results were when the fresh E. cottonii extracted using Hexane and using dichloromethane for dried E. cottonii.

Table 1 .
The content of alkaloids, flavanoids, saponins, steroids/triterpenoids, and tannins from fresh and oven-dried E. cottonii extracted using water, dichloromethane, and hexane as the solvents.