Antioxidant and Cytotoxic Properties of Soft Coral Nepthea sp

Soft coral Nepthea sp. grows in the seas of South-East Sulawesi, Indonesia. However, information on the chemical and pharmaceutical aspects of this genus is still limited. Therefore, this research aims to explore the chemical contents and biological activities of Nepthea sp. The sample was collected from the waters of Saponda Island by SCUBA diving. It was extracted by ethyl acetate and fractionated using vacuum liquid chromatography. The chemical content was analyzed by phytochemical screening, LC-MS/MS analysis, Total Phenolics Content and Total Flavonoids


Introduction
Southeast Sulawesi is an archipelagic province, has 651 islands, 74.25% of its territory is the ocean (Bangwilsultra, 2016). One of the important and abundant marine natural resources is soft coral. In Waworaha Waters, Konawe, with a monitoring area of 700 m2, eight types of soft coral were found (Wanda et al., 2018). The chemical content and efficacy in the pharmaceutical field have not been reported yet. Even though there are many new compounds from soft corals that have very interesting properties, such as sarcophine from S. glaucum growing in the Red Sea is active as a modulator of glycine receptors in the nervous system (Saleh et al., 2020), S. solidum produces diterpene compounds (Zhu et al., 2015), and several terpenoid compounds are also obtained from Lobophytum crasum and oxygenated steroids from S. pauciplicatum, can increase the immune system to fight infection of cancer patients (Florean et al., 2020).
In continuing our study on chemical and pharmaceutical aspects of marine natural resources, soft coral especially Nepthea sp. was chosen as the research sample. Before research on soft corals, we had worked with sponges and through the process of isolation and structure determination, we obtained a new compound from Chlatria sp, namely Clathruohate, and has been published in MJAS, 2018 (Sahidin et al., 2018), and chemical screening of some sponges . Biological activities of sponges include antihyperlipidemic , Anti-Inflammatory , antioxidant properties and acute toxicity (Fristiohady et al., 2020).
Based on the literature review (searching) in Science Direct and Springer Link, no information has been found regarding the study of chemical and pharmaceutical aspects of Nepthea sp. from South East Sulawesi (Indonesia). Soft corals from Indonesia whose chemical and pharmaceutical aspects have been reported include Sinularia depresa from the South China Sea which produces sinulasterol A-C which plays a role in cancer prevention through antiinflammatory effects  and ethyl acetate and butanol extracts of Lobophytum sp. from Selayar, South Sulawesi, is active as an anti-bacterial and antioxidant (Putra et al., 2016). Meanwhile, the study of soft corals from Southeast Sulawesi is still in the mapping stage (Pedoja et al., 2018). This article explains the study of antioxidant and toxicity against breast cancer cells lines (MCF-7) as well as the study of the chemical content using LC-MS/MS of the nonpolar fraction of soft coral Nepthea sp. that grows in Southeast Sulawesi.

General procedures
A Waters Acquity UPLC I-Class was used in conjunction with a Xevo G2-X2 Quadrupole Time-of-Flight (QToF) mass spectrometer to perform the LC-MS/MS study. Methanol, ethyl acetate, and n-hexane are among the chemicals used, and the aquades are of analytical quality.Thin-layer chromatography plate:

Nepthea sp. collection
The sample was taken on the reef slopes of the Saponda Islands in Indonesia's Province of Southeast Sulawesi. SCUBA diving at a depth of 4-10 m was used to collect the sample. The material was collected and put in separate ice containers before being returned to the laboratory for further analysis.

Extraction and fractionation
Fresh Nepthea sp. (3 kg) samples were chopped into small pieces and extracted in ethyl acetate (3x10 L, 24 h each time) at room temperature. Each dried soft coral extract was collected under reduced pressure and stored at 4°C for future analysis in an amber bottle. The ethyl acetate extract (160 g) was fractionated using Vacuum Liquid Chromatography (VLC) with silica gel as an adsorbent and a combination of n-hexane: ethyl acetate (100:0 up to 0:100%) and methanol 100 per cent as eluent, yielding six fractions (A-F).

Phytochemical screening
The presence of alkaloids, flavonoids, tannins, terpenoids, steroids, and saponins in the selected sponges was determined using phytochemical screening methods (Harborne, 1973).

Total Phenolics Content (TPC) and Total Flavonoids Content (TFC)
The TPC of the samples were determined using the Folin and Ciocalteu reagent, following Singleton and Rossi's method with minor adjustments and the total flavonoids of the samples were determined using Chang et al's (Chandra et al., 2014). The nonpolar fraction of the soft coral Nepthea sp. was prepared for LC-MS/MS analysis using the standard operating procedure of this instrument . Antioxidant assay of the samples was assessed using the DPPH radical . The modified Moniruzzaman method was used for ABTS Assay (Wahyuni et al., 2021). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess cytotoxicity in MCF-7 cells in vitro (Asasutjarit et al., 2021).

Result and Discussion
Vacuum Liquid Chromatography with silica gel as an adsorbent and a solvent mixture of n-hexane: ethyl acetate (100%:0 % up to 0%:100%) followed by methanol was used to fractionate ethyl acetate extract, to give six fractions (A-F). Fraction A and B have interesting TLC chromatogram patterns after being developed by n-hexane : ethyl acetate=8:2 as eluent (Figure 1.), and also have sufficient weight for further experiments such as biological activity tests or isolation of nonpolar compounds, 35.5 g of A and B= 4.3 g, respectively (Tabel 1.). Table 1 shows that the ethyl acetate extract contains saponins, tannins, alkaloids and terpenoids, while fractions A and B only contain terpenoids. Because of fractions, A and B are the non-polar fraction, so that the compounds in the two fractions are non-polar. This phenomenon can also be observed from the Retardation factor (Rf) values of fractions A and B which are higher than the other fractions and also become the place where the top (non-polar) part of the ethyl acetate extract (T) TLC chromatogram is concentrated. The absence of polar compounds such as flavonoids, saponins, tannins, alkaloids and other phenolics in Fraction A and B can be seen from the value of TPC and TFC, where TPC and TFC of fractions A and B are 0.0 mgGAE.g Ex -1 and 0.0 mgQE. g Ex -1 respectively. This means that the polar portion of the ethyl acetate extract is stored in the other fraction. The presence of non-polar compounds from the non-polar fraction of Nepthea sp. is relevant with the compounds previously found from Nepthea sp. (Coll et al., 1985).
An in-depth study of the compounds of the non-polar fraction of Nepthea sp. ethyl acetate extract can be clearly explained based on the data from the LC-MS/MS analysis in Table 2 and Figure 2. Ethyl acetate extract has ten compounds and five of them has been identified, Fraction A has five compounds and seven compounds of Fraction B. Generally, all of the identified compounds are terpenoids consisting of monoterpenes (compound 3), sesquiterpenes (1,2,4,5,6,7,8,10) and steroids (9), which matches the previous invention (Almeida et al., 2014;Florean et al., 2020;Saleh et al., 2020).
An interesting phenomenon is the presence of nitrogenous compounds in the non-polar fraction. Usually, nitrogenous compounds in secondary metabolites are alkaloids which are semi-polar up to polar compounds. It is presumed that the alkaloids are found in the form of free nitrogen and not in the form of nitrogen salts. The use of this fractionation method can describe the diversity of compounds in the non-polar fraction of Nepthea sp., it will also make it easier to carry out the next step, namely the isolation and elucidation of non-polar compounds from Nepthea sp. The biological activity of all samples will be clearer and more interesting when presented in Table  3 and graphical form in Figure 3. Antioxidant activity is represented by the IC50 value of DPPH and ABTS. Cytotoxicity of samples was evaluated against breast cancer cell lines MCF-7.
Based on the IC50 value of DPPH and ABTS of all samples in the Table 3, the IC50 value of all samples is >150 ppm (mgL -1 ) or in other words the antioxidant properties of all samples are in the weak category (Tristantini et al., 2016). The study for each sample shows that fractions A and B have antioxidant potential better than ethyl acetate extract. Although the ethyl acetate extract contains phenolic and flavonoid compounds which are indicated by the TPC and TFC values as listed in Table 3, the diversity and solubility of the compounds, as well as the mole fraction of non-TPC and non-TFC compounds, greatly affect the antioxidant power of the ethyl acetate extract. Furthermore, the antioxidant properties of fraction B were slightly better than fraction A, both based on the IC50 DPPH and ABTS values. This fact is supported by differences in compound content. Fraction B has compounds that have been identified LC-MS/MS consisting of 1,9 and 10 while fraction A contains 6,7 and 8. Compounds 1,9 and 10 contain hydroxyl units, so it is easier to donor hydrogen radicals than compounds 6,7 and 8. In other words, the compounds in Fraction B are easier to donate proton radicals or are easier to neutralize radicals or have stronger antioxidant properties.  Ethylacetate extract and Fraction A have cytotoxic potential against breast cancer cells lines (MCF-7), although their activities are weaker than that positive control. Based on the level of cytotoxicity (Weerapreeyakul et al., 2012) that the extract is categorized as very active if it has IC50 <10 mg.mL -1 , active (10-100 mg.mL -1 ), moderately active (100-500 mg.mL -1 ), then ethylacetate extract and fraction A were categorized as moderately active. For the samples, the cytotoxic properties of Fraction A had better cytotoxic properties than the ethylacetate extract and Fraction B. Furthermore, the ethyl acetate extract was stronger than the fraction B. This phenomenon is still quite difficult to explain. However, when looking at the structure of the standard compound (positive control) in this study, namely doxorubicin, and the active compound used in the treatment of breast cancer (tamoxifen) (Thayyib et al., 2020), both compounds are alkaloids. Doxorubicin and tamoxifen have structural formulas C27H29NO11 and C26H29NO, respectively.

Conclusion
The non-polar fraction of Nepthea sp. ethyl acetate extract has interesting potential for both the diversity of compounds and their biological activities. The compounds in the non-polar fraction based on LC-MS/MS data include achillin, atractylenolide II, buthyl isobuthyl phthalate, rengyolester, 2aacetoxycostic acid, ocotillol acetate, petasitolone and some unidentified compounds. Furthermore, the nonpolar fraction can be further developed as raw material for the discovery of new compounds, antioxidant and anticancer agents, especially breast cancer.