Study In-vitro and in-silico of ethylacetate extract and fractions of soft coral Lobophytum sp. towards Artemia salina Brine Shrim (BSLT)

. The article aims to describe the findings of chemical and pharmaceutical aspects of Lobophytum sp. from Southeast Sulawesi, Indonesia. Ethylacetate extract was fractionated by Vacuum liquid chromatography (VLC). Toxicity was evaluated by BSLT test, and the phytochemical screening and LCMSMS method were used to determine the chemical composition and molecular docking for in-silico study. The results showed that the ethylacetate extract was produced seven fractions namely Fraction A-G. The weight of each fraction was A (12.8% w/w), B (9.7%), C (10.1%), D (2.0%), E (7.0%), F (25, 3%) and G (11.5%). The toxicity potency of Fraction B is the most toxic with LC 50 (mg/L) 26.70 ± 0.58. LCMSMS data indicated that the fraction B contains 19 b -glukocyl-14- deoxy-11,12-didehydrographoside, 3-isoazmalicine, abietraticine, arachidonic acid, neociwujiaphenol, oxyphyliacinol, saurufuran B and some unidentified compounds with molecular formulas C 37 H 46 O 7 , C 35 H 44 O 5 , and C 20 H 26 O 2 . Based on computational simulations, Ar-Abietatriene and 3-Iso-ajmalicine have the potential to inhibit CDK-6. These compounds hinder the progression of the cell cycle and the proliferation of cancer cells by forming molecular interactions with residues Ile19, Val27, Ala41, Val77, Phe98, Val101, Leu152


Introduction
In continuing our study on the chemical and pharmaceutical aspects of marine natural resources, we selected soft coral, particularly Lobophytum sp., as a sample.Before studying soft corals, we have isolated and identified the structure of a novel molecule from a sponge Chlatria sp., called Clathruohate [1], and chemically screened a few sponges [2].Antihyperlipidemia [3], anti-inflammatory [4], antioxidant capabilities and acute toxicity [5], as well as chemical and medicinal characteristics of soft coral Nepthea sp.[6] are just a few of the biological activities of sponges that have been noted.Southeast Sulawesi's waters are home to soft corals, one of them is the Lobophytum sp.[7].A few studies on the chemical and pharmaceutical aspects of this genus have been reported, including three cembrene diterpenoids named Lobophylins F-H from L. crassum growing in Taiwan [8] and lobocrasols A-D were identified from Vietnamese's L. crassum which have potential as anti-inflammatory [9], and Chinese's L. crassum produced polyoxygenated cembranoids namely lobophycrasins A-D and hum ulisin A [10].Some Vietnamese's soft corals also produce interesting compounds including laevigatol A-D which was active as anti-inflammatory and anticancer agents from L. laevigatum [11], lobocompactol A-B and sterols from L. compactum is active as antioxidant and cytotoxic against HL-60 (leukaemia) and A549 (lung) cell lines [12].The South China Sea-dwelling Lobophytum sp. has also been investigated for its chemical and pharmacological properties, including the production of Lobophytrols A-C [13], the antiinflammatory and antibacterial activity of durumolides A to E from L. durum [14], and the activity of lobophysterols A-D against the cell lines HT-29, SNU-398, and Capan-1 [15].The cembranolide chemicals generated by an Okinawan-based Lobophytum sp. were cytotoxic to the HeLa, A459, B16-F10, and RAW246.7 cell lines [16].
The study of soft corals from Eastern Indonesia is still in the mapping stage [17,18].However, some research reports on marine organisms from the area are interesting, for example, Lobophytum sp. from Selayar (South Sulawesi) has potential as an anti-bacterial and antioxidant [19].Lobozoathamine, an alkaloid from Lobophytum sp, growing in Manado, North Sulawesi, has been successfully isolated and identified [20].This paper will present the Antioxidant, toxicity and secondary metabolites of ethyl acetate fraction from soft coral Lobophytum sp.growing in South East Sulawesi.

Soft Coral Collection, Preparation and Extraction
Southeast Sulawesi's Hoga Island, located at 5 o 27' 54' 36" LS and 123 o 45' 20" LE, was the site of a soft coral collection of Lobophytum sp.3.0 kg of dried soft coral powder was macerated in 10 L of ethyl acetate (EtOAc) for 3 x 24 hours and concentrated in a vacuum rotary evaporator.Furthermore, the ethyl acetate extract was fractionated using liquid vacuum chromatography (VLC) with Si-gel adsorbent and a mixture of n-hexane: ethyl acetate and methanol as the eluent.[6].

Phytochemical Screening
To identify the secondary metabolite of the ethyl acetate fractions of the soft coral Lobophytum sp., phytochemical screening was done.The Harborne Method was used to assess the secondary metabolites, which included flavonoids, saponins, tannins/polyphenols, alkaloids, and terpenoids [6].Additionally, utilizing this instrument's regular operating method, LC-MS/MS analysis was used to determine the precise chemical composition of Lobophytum sp.[2].

Toxicity Assays
Brine Shrimp Lethality Test (BSLT) assay was used for toxicity evaluation of the sample [21].

Protein and Ligand Preparation
We chose the three-dimensional structures of CDK-6 (PDB ID: 5L2I) [22] from the Protein Data Bank (https://www.rcsb.org/).The compounds identified from the LC-MS/MS results were gathered from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).AutoDockTool v1.5.6 was utilized to prepare the protein and ligands.Water molecules were removed for the protein, and protonations and the Kollman charges were added [23].The ligand was configured to rotate freely, and the Gasteiger charges were incorporated [24].

Molecular Docking Simulation
The docking process was run with the assistance of AutoDock Vina v.1.1.2[25].The binding site is set following the position of Palbociclib in CDK-6 with a grid area of 20 x 20 x 20 Å and 0,375 Å point spacing.The validated procedures were identified with a root mean square deviation (RMSD) of after redocking process of Palbociclib was below 2 Å [26].Other docking procedures are set by default.Lastly, the protein and compounds' interactions were analyzed and visualized with help of Discovery Studio Visualizer software v17.2.0.16349.

Result and Discussion
Ethyl acetate was used to extract the Lobophytum sp.powder (3 kg), producing a 75 g concentrated extract (2.50%).In fractionation, the ethyl acetate extract was produced in seven fractions namely Fraction A-G.The weight of each extract was A (12.8% w/w), B (9.7%), C (10.1%), D (2.0%), E (7.0%), F (25.3%) and G (11.5%).Fraction F is the largest fraction in terms of weight so it was chosen for further study.The phytochemical screening of the fraction F gave information which showed in Table 1 The LC-MS/MS data informs that there are 10 main compounds contained in the Fraction B of ethyl acetate extract of Lobophytum sp., of which 7 compounds have been identified and the rest have not been identified.The seven compounds that have been identified are 19β-Glucosyl-14-deoxy-11,12-didehydroand-rographoside, Neociwujiaphenol, Oxyphyllacinol, Saurufuran B, 3-Iso-ajmalicine, ar-Abietatriene, Arachidonic acid and some unidentified compounds with molecular formulas C37H46O7, C35H44O5, and C20H26O2.The structure of the seven compounds that have been identified is shown in Fig. 1.  3.  3 shows that the toxicity of an extract and fraction of Lobophytum sp. using BSLT was evaluated LC50 (mg/mL) value.If the LC50 value is less than 1000 mg/mL, it is categorized as active [22].Thus, all samples have high toxic properties and Fraction B of the ethyl acetate extract is more active than ethylacetate extract of Lobophytum sp.In general, extracts or compounds that have high toxicity have the opportunity to be developed as anticancer drugs.Anticancer potential testing was carried out in-silico using molecular docking analysis.

Mole cular Docking Analysis
Cyclin-Dependent Kinase 6 (CDK-6) plays a significant role in cancer by regulating the cell cycle and promoting cell proliferation.Hyperactivation of CDK-6 is commonly observed in various types of cancers [35].This hyperactivation can result from mutations, gene amplification, or dysregulation of pathways that control CDK-6 expression [36].High CDK-6 activity can lead to uncontrolled cell division and tumor formation.Given its critical role in cell cycle progression and proliferation, CDK-6 has become a target for cancer therapy [35].CDK-6 inhibitors, such as palbociclib, have been developed to block the activity of CDK-6 selectively.These inhibitors aim to slow down or halt the proliferation of cancer cells, particularly in hormone receptor-positive breast cancers [37].
This research aims to present insights into the potential of compounds derived from the soft coral Lobophytum sp. as molecular inhibitors of CDK-6.To validate the accuracy of our simulations, we conducted a redocking procedure using the palbociclib crystal structure back into CDK-6 and evaluated the Root Mean Square Deviation (RMSD).A lower RMSD value signifies that the docking parameters successfully replicated the conformation of the cocrystal ligand, as observed through x-ray crystallography.The most optimal palbociclib conformation within CDK-6 is depicted in Figure 1.The re-docking analysis exhibited a pose similar to the reported x-ray crystallography, showing an RMSD of 1.821 Å.Based on the docking simulation results, palbociclib exhibited the highest affinity towards CDK-6 with a binding energy of -9.3 kcal/mol.In this regard, this compound's carboxyl and pyrimidine groups formed hydrogen bonds with the residues Val101, Asp163, Ile19, and Glu99 at the active site of CDK-6 (Figure 1).Additionally, the benzene ring of the pyrimidine group contributed to the formation of hydrophobic interactions with the residues Ala41, Val77, Phe98, His100, Asp102, Leu152, and Ala162.Meanwhile, a total of 7 compounds from fraction B were found to have affinities within the binding energy range of -8.9 to -6.4 kcal/mol (Table 1).Two compounds from the B fraction of Lobophytum sp., Ar-Abietatriene, and 3-Iso-ajmalicine, exhibited the best affinities among the other compounds, with binding energies of -8.9 kcal/mol and -8.8 kcal/mol, respectively.
-glucosyl-14-deoxy-11,12dedihydroandrogaphoside -7.8 Gln149, Asn150, Asp163 Ile19, Val27, Ala41, Lys43, Leu152, Ala162 Arachidonic Acid -6.4 -Tyr24, Val27 The molecular interactions of Ar-Abietatriene were observed with the methyl group and its benzene ring, forming hydrophobic contacts with Ile19, Val27, Ala41, Val77, Phe98, Leu152, and Ala162 (Figure 2A).Interestingly, although this compound did not exhibit hydrogen bond, its affinity was superior to other compounds.We speculate that interactions with the residues Ala41, Val77, and Phe98 are crucial in their affinity [22].On the other hand, compound 3-Iso-ajmalicine revealed hydrogen bonding with the residues His100, Val101, and Glu99, facilitated by this compound's carbonyl and pyrrole groups.Additionally, its quinolizine ring contributed to hydrophobic interactions with the residues Ile19, Val27, Asp102, and Leu152 at the CDK-6 active site (Figure 3B).Overall, all three compounds were able to interact hydrophobically with residues in the active site of ERα, such as Ile19, Val27, Ala41, Leu152, and Ala162.Additionally, they exhibited varying hydrogen bonding compared to palbociclib and the two best compounds (Figure 3).These residues in CDK-6 play crucial roles in its structure and function.Ile19 and Ala47 are located near the active site of CDK-6.It is involved in forming hydrophobic interactions with ligands that bind to CDK-6.These residues contribute to stabilizing ligand binding and help maintain the structural integrity of the active site.In addition, Ala162, situated near the ATP binding site of CDK-6, plays a crucial role in establishing a cavity within the active site of CDK-6, facilitating its interaction with the ligand.

Palbociclib
Ar-Abietatriene Through this computational study, two potential compounds from Fraction B have been identified: Ar-Abietatriene and 3-Iso-ajmalicine.These compounds are projected to have a crucial role in anticancer activity.Their promising characteristics suggest they hold significant potential as candidates for cancer treatment.The substantial evidence supporting this potential lies in the remarkable affinity exhibited by these three compounds towards CDK-6.CDK-6, which is critical in driving cell cycle progression and proliferation, is frequently deregulated and overly active in various cancer types.Targeting CDK-6 with specific inhibitors has emerged as a promising strategy to curtail tumor growth and enhance the overall outcomes of cancer treatments.The strong interaction of these compounds with CDK-6 highlights their potential, offering a new avenue to improve cancer treatments.

Conclusion
Fractionation of ethylacetate extract of Lobophytum sp.produced seven fractions (A-G).The Fraction B (9.7% w/w) is the most toxic toward Artemia salina brine shrimp.LC-MS/MS data indicated that the fraction contains 19b-glukocyl-14-deoxy-11,12-didehydrographoside, 3-isoazmalicine, abietraticine, arachidonic acid, neociwujiaphenol, oxyphyliacinol, saurufuran B and some unidentified compounds with molecular formulas C37H46O7, C35H44O5, and C20H26O2.Further studies on the potential of fractions and compounds that have an important role are carried out in in-silico cancer cells.Ar-Abietatriene and 3-Isoajmalicine play vital roles in the anti-cancer attributes of Lobophytum sp.'s fraction B. Their interactions with important cancer-related receptors show their potential to inhibit cell cycle progression and cell proliferation.Additional investigations and studies are needed to comprehensively delve into these compounds' therapeutic capabilities in cancer treatment.

Table 1 .
. Weight and chemical composition of fraction F ethyl acetate extract

Table 1
reveals that the content of secondary metabolites in soft corals of Lobophytum sp, especially those which are the polar part of the ethyl acetate extract, called fraction B as much as 12.93 g (9.7% w/w).Its composition includes saponins, tannins/phenolic and alkaloids, while terpenoids and flavonoids are not found in the fraction F of Lobophytum sp.The composition of secondary metabolites in more detail was carried out by translating the LC-MS/MS data as shown in Table2.

Table 2 .
Compounds of Fraction B of Lobophytum sp based on LC-MS/MS data

Table 3 .
Toxicity of Ethylacetate extract and Fraction B ethyl acetate extract of Lobophytum sp.

Table 4 .
Summary of the docking result and interactions of all identified compounds in Lobophytum sp.'s fraction B against CDK-6