Introduction

The Red Sea is a hub of marine biodiversity, hosting a significant number of species that are unique to the region. Approximately 40% of the 180 soft coral species discovered worldwide are indigenous to the Red Sea. A recent review focused on biomedical leads derived from marine crustaceans found in the Red Sea1. Sarcophyton is a widely distributed genus of corals that can be found from Eastern Africa and the Red Sea in the west to Polynesia in the east2,3. Out of the 36 recognized species of Sarcophyton, around 14 species can be found in the Red Sea4. Sarcophyton, a genus of soft corals in the family Alcyoniidae, is known for its prolific presence in coral reefs and its high levels of cembranoids5 and steroids6. Cembranoids are a type of 14-membered carbocyclic diterpenes that contain one isopropyl group and three methyl groups. They are frequently oxidized to generate hydroxymethyls or carboxylic acids and can also participate in the creation of lactone rings7. Without a doubt, S. trocheliophorum contains a high concentration of terpenes and cembranoids, which are the most commonly found structural types. These diterpenes are considered to have defensive, competitive, reproductive, or pheromonal properties, which have a functional purpose in the survival of the organisms that produce them8,9. From a medical standpoint, cembrane-type compounds exhibit a diverse array of biological actions, such as neuroprotective, antibacterial, and anticancer characteristics including neuroprotective, antimicrobial, and antitumor properties10,11.

Cancer is characterized by the aberrant proliferation of cells that have the capacity to metastasize or infiltrate other anatomical regions. It is regarded as a significant disease that poses a huge threat to humanity and continues to be the second leading cause of death globally12. Annually, over 10 million individuals are diagnosed with cancer, resulting in approximately 6 million deaths worldwide. It is projected that by 2025, the number of new cancer cases will surpass 20 million, and by 2030, the number of new cancer patients may reach 21.7 million, with approximately 13 million cancer-related deaths13.Currently, there is growing interest in the use of natural products derived from marine animals for maintaining human health and treating various ailments, as well as for discovering new drugs14,15.

Alternatively, pest control is an essential aspect of agricultural growth that has sparked substantial research. Extensive use of chemicals, such as pyrethroids and pyridoxandes, in control of different pests led to resistance of these pests to repetitive chemically synthesized pesticides16. Marine habitat is considered a source for massive biological resources compared to terrestrial habitat because of the special environmental conditions and huge territory. Various marine biological species offer important sources for getting a wide range of naturally occurring bioactive compounds with varied structure and diverse activities. Moreover, the number of compounds obtained from marine natural products with insecticidal properties, have been gradually increased in recent years17.

Culicidae and Muscidae are the most common families of insects, which are responsible for transmitting of huge number of diseases to human either biologically or mechanically18.Mosquitoes are a widely recognized group of insects due to their role in transmitting a range of diseases, including malaria, filariasis, and dengue, which result in millions of fatalities each19.The mosquito species Culex pipiens is prevalent in both urban and suburban regions of Africa, serving as the primary vector. However, the house fly, scientifically known as Musca domestica (Diptera: Muscidae), is also a notorious pest that affects livestock and has significant medical implications. Dipterous flies, including the house fly, are considered prime carriers and transmitters of bacteria, viruses, protozoan cysts, and various infections that affect both humans and animals. This is due to their unique biology and ecological characteristics20.

As part of our ongoing study on the bioactive compounds found in S. trocheliophorum21,22, a species collected from the Red Sea near the Hurghada beaches in Egypt, we have successfully isolated two compounds: sarcotrocheliol (1) and cholesterol (2). The chemical structures of compounds 1 and 2 (Fig. 1) were determined by the techniques of mass spectrometry and NMR spectroscopy. Compounds 1 and 2 were examined for their anticancer, antibacterial, and larvicidal characteristics. The biological activity of the isolated compounds was further assessed by the utilization of DFT calculations and molecular docking of compounds 1 and 2.

Fig. 1
figure 1

Chemical structures of compounds 1 and 2.

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Materials and methods

Electron Spray Ionization Mass Spectrometry ran on the Finnigan LCQ ion trap mass spectrometer. A Flux Instruments Rheos 4000 quaternary pump and HP 1100 HPLC equipment connected to the mass spectrometer. HPLC used a Nucleosil column with EC 125/2, 100-5, C 18 parameters. The experiment used a Jasco 851-AS autosampler and a Finnigan Surveyor LC System diode array detector. An Apex IV 7 Tesla Fourier-Transform Ion Cyclotron Resonance Mass Spectrometer (Bruker Daltonics, Billerica, MA, USA) generated the HRMS using ESI MS. The column chromatography was conducted utilizing Silica gel 60 (with a pore size of 60 Å, particle size ranging from 70 to 230 mesh, and measuring 63 to 200 μm in size, sourced from Fluka, Sigma-Aldrich Chemicals-Germany). The determination of Rf values was performed using Polygram SIL G/UV254 plates produced by Macherey & Nagel in Düren, Germany. Sephadex LH-20, a hydrophobic variant of Sephadex, was employed for size exclusion chromatography. The Sephadex LH-20 was obtained from Sigma-Aldrich Chemie, located in Steinheim, Germany, and produced by Amersham Biosciences, Ltd.

Animal material

The soft coral S. trocheliophorum (1.8 kg wet weight) was collected in September 2014 near Mahmieat of the Red Sea about ∼ 1 km off the coast of Hurghada, east Egypt, at a depth of ∼ 30 m (geographical coordinates: Latitude 27° 15’ 26 N, Longitude 33° 48’ 46 E) using SCUBA method. The collected organism was stored immediately in a freezer until working up and extraction23. The S. trocheliophorum was morphologically characterized by Dr. Mohamed A. Ghani, Hurghada, Egypt, and a voucher specimen (ST-92014) was deposited at Red Sea Marine Parks, P. O. Box 363, Hurghada, Red Sea, Egypt.

DNA extraction and sequencing

The DNA was extracted from tissue samples, stored in ethanol, and then the mitochondrial COI + igr1 gene was amplified and sequenced using the primer COII8068F and following the techniques described by McFadden et al.24. The forward and reverse sequencing results obtained from the PCR were edited and aligned using MEGA V14.0. The obtained S. trocheliophorum sequence was analyzed by comparing it to existing sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) available at http://blast.ncbi.nlm.nih.gov/Blast.cgi.

Extraction, isolation, and purification

The S. trocheliophorum was homogenized in a blender, macerated with 6 L of chloroform-methanol (8: 2) and kept at ∼ 5 celsius for 8 days. The solid material was filtered off, and the chloroform layer was evaporated in vacuo. The remaining aqueous methanol solution was reextracted with n-butanol. This butanol extract was similarly evaporated to dryness. Both extracts (chloroform and n-butanol) were combined according to TLC similarity and dried under vacuum, affording 62.8 g of a dark-green crude extract. The extract was subjected to column chromatography on silica gel (70 cm × 10 cm) and eluted with a cyclohexaneDCM-MeOH gradient (cyclohexane 1 L, cyclohexane-20% DCM 1 L, cyclohexane-40% DCM 1 L, cyclohexane-50% DCM 1 L, cyclohexane-80% DCM 1 L, DCM 1 L, DCM2% MeOH 1 L, DCM-5% MeOH 1 L, DCM-10% MeOH 1 L, DCM-20% MeOH 1 L, DCM-50% MeOH 1 L, MeOH 1 L) where 10 fractions were obtained. A subsequent continual purification of the remaining middle and polar fraction (VII) of the coral extract, into a series of subsequent chromatographic techniques, starting with silica gel columns of different dimensions eluted with n-hexane followed by a gradual increase of the polarity using a subsequent addition of DCM followed by a gradual increase of the polarity using a subsequent addition of methanol, followed by Sphadex LH-20 (DCM/40% MeOH) led to re-isolation of sarcotrocheliol (1, 16 mg) and cholesterol (2, 15 mg) as colourless solids.

Sarcotrocheliol (1)

Colorless needles; [α]D20= + 11.8 (CHCl3; c = 0.1);1H NMR (CDCl3, 500 MHz): δ = 4.51 (1 H, dd, J = 10.2, 4.8 Hz, H-1), 1.28 (1 H, m, H-2), 2.36 (1 H, m, H-3a), 1.22 (1 H, m, H-3b), 1.60 (1 H, m, H-4a), 1.40 (1 H, m, H-4b), 3.83 (1 H, d, J = 9.6 Hz, H-6), 1.72 (1 H, m, H-7a), 1.24 (1 H, m, H-7b), 2.07 (1 H, m, H-8a), 1.97 (1 H, m, H-8b), 4.96 (1 H, dd, J = 10.2, 4.8 Hz, H-10), 2.31 (1 H, m, H-11a), 2.08 (1 H, m, H-11b), 2.15 (1 H, m, H-12a), 1.96 (1 H, m, H-12b), 5.24 (1 H, d, J = 10.2 Hz, H-14), 1.16 (1 H, m, H-15), 0.70 (3 H, d, J = 6.6 Hz, H3-16), 0.85 (3 H, d, J = 6.6 Hz, H3-17), 0.99 (3 H, s, H3-18), 1.58 (3 H, s, H3-19), 1.62 (3 H, s, H3-20); 13C NMR (CDCl3, 125 MHz): δ = 138.5 (C, C-13), 135.9 (C, C-9), 125.4 (CH, C-14), 123.9 (CH, C-10), 75.0 (C, C-5), 71.0 (CH, C-1), 69.9 (CH, C-6), 46.5 (CH, C-2), 39.9 (CH2, C-12), 35.2 (CH2, C-8), 33.6 (CH2, C-3), 31.6 (CH2, C-7), 28.9 (CH, C-15), 25.1 (CH2, C-11), 24.2 (CH3, C-18), 20.7 (CH3, C-17), 20.2 (CH3, C-16), 18.7 (CH2, C-4), 17.6 (CH3, C-19), 15.0 (CH3, C-20); HRESIMS data m/z 307.2639 [M + H]+ (calculated for C20H34O2H+ 307.2631).

Cholesterol (2)

Colorless solid;  1H NMR (CDCl3, 500 MHz): δ = 1.31 (1 H, m, H-1a), 1.06 (1 H, m, H-1b), 1.52 (1 H, m, H-2a), 1.27 (1 H, m, H-2b), 3.53 (1 H, td, J = 6.5, 3.3 Hz, H-3), 2.21 (1 H, m, H-4a), 1.96 (1 H, m, H-4b), 5.35 (1 H, dt, J = 5.2, 2.1 Hz, H-6), 2.19 (1 H, m, H-7a), 1.94 (1 H, m, H-7b), 1.27 (1 H, m, H-8), 1.17 (1 H, m, H-9), 1.63 (1 H, m, H-11a), 1.38 (1 H, m, H-11b), 1.56 (1 H, m, H-12a), 1.31 (1 H, m, H-12b), 1.04 (1 H, m, H-14), 1.90 (1 H, m, H-15a), 1.65 (1 H, m, H-15b), 1.90 (1 H, m, H-16a), 1.65 (1 H, m, H-16b), 1.11 (1 H, m, H-17), 0.66 (3 H, s, H3-18), 1.00 (3 H, s, H3-19), 1.30 (1 H, m, H-20), 0.85 (3 H, d, J = 6.6 Hz, H3-21), 1.19 (2 H, m, H2-22), 1.25 (2 H, m, H2-23), 1.19 (2 H, m, H2-24), 1.62 (1 H, m, H-25), 0.94 (3 H, d, J = 6.5 Hz, H3-26), 0.94 (3 H, d, J = 6.5 Hz, H3-27); 1C NMR (CDCl3, 75 MHz): δ = 140.8 (C, C-5),121.8 (CH, C-6), 71.6 (CH, C-3), 56.5 (CH, C-14), 56.2 (CH, C-17), 50.8 (CH, C-9), 42.7 (C, C-13), 41.8 (CH2, C-4), 39.9 (CH2, C-24), 39.8 (CH2, C-12), 37.7 (C, C-10), 37.2 (CH2, C-1), 36.1 (CH2, C-22), 35.8 (CH, C-20), 32.0 (CH2, C-7), 31.8 (CH, C-8), 31.7 (CH2, C-2), 28.1 (CH, C-25), 26.3 (CH2, C-15),25.9 (CH2, C-16), 24.6 (CH2, C-23), 23.2 (CH3, C-26), 23.2 (CH3, C-27), 21.1 (CH2, C-11), 19.4 (CH3, C-21), 19.3 (CH3, C-19), 12.0 (CH3, C-18); (+)-ESIMS m/z 387.269 [M + H]+, 409.333 [M + Na]+.

In vitro cytotoxic activity

Cell culture

The HCT-116 and HepG2 cancer cell lines were purchased from the American Type Culture Collection (VACSERA Co., Cairo, Egypt). The cancer cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) and acquired from Corning Thomas Scientific, located in Swedesboro, NJ, USA. DMSO was acquired from Sigma-Aldrich in St. Louis, USA, whereas FBS was bought from Hyclone in Pittsburgh, PA, USA, and PSA was got from Mediatech Inc. in Herndon, VA, USA. The cancer cells were cultured in DMEM media with 5% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphotericin B (PSA) at a temperature of 37 °C in a 5% CO2 incubator. Similarly, HFB-4 cells were grown in RPMI-1640 media (Thermofisher Scientific Co., Waltham, MA, USA) with the addition of 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. The cells were incubated at a temperature of 37 °C in a controlled environment with a specific humidity level and an atmosphere containing 5% carbon dioxide25.

2.4.2. Assessment of cytotoxicity using MTT assay

The cytotoxicity of each chemical was assessed against Wi38 normal cell line, HCT-116 and HepG2 cancererous cells in accordance with the manufacturer’s instructions using the Cell Proliferation Kit I,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA). Precisely, 5000 cells were seeded per well into 96-well culture plates, followed by an incubation period of 24 h. Following that, the cells were exposed to varying concentrations of each drug (0, 6.25, 12.5, 25, 50, and 100 µg/ml) for a duration of 24 h in a humidified incubator containing 5% CO2 and 37 °C. On the day of detection, MTT powder was applied to both the treated and untreated cells for a duration of two hours. Violet crystals of varying colors began to form during this time period; these hues corresponded to the metabolic activity of the cells. Utilizing a Synergy™ 2 Multi-Mode Microplate Reader produced by BioTek Inc. in Vermont, USA, the colorimetric absorbance at 620 nm (A620) and 570 nm (A570) was quantified. The quantification of viable cells was performed utilizing the following equation26:

$$\:\text{P}\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\text{a}\text{g}\text{e}\:\text{o}\text{f}\:\text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}=\:\frac{\text{A}570-\text{A}620\:\text{o}\text{f}\:\text{U}\text{A}\:}{\text{A}570-\text{A}620\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\left(0{\upmu\:}\text{M}\right)\:}\times\:100$$

The IC50 value of each chemical was determined using the IC50 calculator provided by AAT Bioquest, Inc., CA, USA, using the methodology described by Luparello et al.27.  Treatment and analysis protocols were carried out in triplicate.

Antimicrobial activity

The antibacterial activity of compounds 1 and 2 was assessed against Pseudomonas aeruginosa ATCC 27,853, Escherichia coli ATCC 25,922, Bacillus subtilis ATCC 6051, Micrococcus luteus ATCC 10,240, and Staphylococcus aureus ATCC 25,923 using the agar well diffusion method as described in document M51-A2 of the Clinical and Laboratory Standards Institute (CLSI)28 with minor modifications. Mueller–Hinton agar (MHA) plates were seeded with bacterial suspensions of 1.5 × 107 (CFU/mL) individually. Subsequently, 100 µl of compounds 1 and 2 at a concentration of 3 µg/mL were put to the wells. The plates were refrigerated for 2 h and then incubated at 37 °C for 24 h. Subsequently, the diameter of the inhibitory zones was determined. Various concentrations of compounds 1 and 2, ranging from 3 to 0.187 µg/mL, were utilized for the detection of MIC29.

The antifungal activity was assessed against Candida albicans ATCC 90,028 and Cryptococcus neoformans ATCC 14,116. Potato dextrose agar (PDA) plates were cultured with fungi. Afterwards, 100 µL of solutions 1 and 2 at concentration of 3 µg/mL, were separately added to agar wells. The plates were incubated at a temperature of 30 °C for a period of 48 h. After the period of incubation, the dimensions of the inhibitory zones were assessed.

Insecticidal activity

Insects

The Culex pipiensmosquito used in this study was obtained from the established colony at the Animal house, Faculty of Science, Al-Azhar University, Cairo, Egypt. The third instar larvae were reared in 40-centimeter white enamel bowls containing 1000 ml of distilled water and kept in controlled laboratory conditions. The larvae were provided with an unlimited quantity of fish food as their dietary source30. The adult female house flies, scientifically identified as Musca domestica (L.), were acquired from the Research Institute of Medical Entomology situated in Dokki, Giza. The adults were provided with sustenance in the form of a sugar solution and a milk powder solution (10% w/v) that were dissolved in water. The insects that were examined were maintained and subjected to experiments in a controlled environment with a temperature of 27 ± 2 oC, a relative humidity of 75 ± 5%, and a day/night cycle of 14 –10 h31. For tested compounds, stock solutions of (1 mg/mL) were prepared for further investigations.

Toxicity on Culex pipiens larvae

The larvicidal assay was carried out according to the World Health Organization protocol32 with slight adjustments. Briefly, twenty-five 3rd instar larvae were exposed to serial concentrations (2, 4, 6, 8, and 10 ppm) of tested compounds diluted in dimethyl sulfoxide (DMSO), and each concentration was replicated five times. Each replicate was maintained in polyethylene plastic cup containing 25 larvae/250 mL. Larvae in the control group were reared in distilled water with the same amount of DMSO. Larval mortality was recorded 24 h post treatment to calculate lethal concentrations.

Toxicity on Musca domestica adults

Adulticidal activity of test compounds was estimated according to the method of Wright33 using topical application. Briefly, ten adult females were anesthetized with diethyl ether for 3 min then 1 µl of DMSO containing the compounds (doses: 4, 8, 16, 32 and 64 µg/adult) was applied by Hamilton microliter syringe 701-N (Sigma-Aldrich) on the dorsal thorax of 3–5 days old adult females. The control group was treated with DMSO without the compounds. After treatment, adults were returned to standard cages for recovery, where they were left for 24 h. Each dose was tested in five replicates, and adult mortality was recorded 24 h post-treatment.

Statistical analysis

Descriptive analysis was performed to determine the average and standard error (SE) for each treatment. A probit analysis was conducted to examine the mortality data for both larval and adult stages. The aim was to identify the lethal concentrations or doses, along with their 95% confidence intervals. The statistical analysis involved doing One-Way ANOVA, calculating lower and upper confidence limits, and determining Chi-square values using SPSS (version 25). The Tukey HSD post hoc test was used to do pairwise comparisons. The data is presented as the average value with the standard error (SE) indicated as the margin of error. A p-value below 0.05 was deemed to have statistical significance.

DFT (Density Functional Theory)

All computations in this investigation were done with the Gaussian 09 software and its default parameters34. DFT techniques were used to fully optimize the structures at the B3LYP level of theory utilizing 6-31G(d, p) basis sets. To determine the compounds’ lowest energy structures, a conformational study was done. The electronic characteristics, which included HOMO, LUMO, and HOMO-LUMO band gaps, were calculated as the difference between the energies of the highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMO). Gauss-View was used to view the compounds’ output structures.

Molecular docking study

For molecular modelling studies, the Molecular Operating Environment (MOE)version 2015.10 software was employed. Chem draws 18.0 was used to draw the studied compounds, which were then saved as MDL mol files. The crystal structures of Casp3 protein (PDB ID: 3KJF), M. tuberculosis (PDB ID:5UHF) and AChE [PDB ID: 1ACJ) were downloaded from the protein data bank (http://www.rcsb.org.pdb).

The crystal structures of B92 with Casp3 protein (PDB ID: 3KJF), 88D with M. tuberculosis (PDB ID: 5UHF), and THA with AChE [PDB ID: 1ACJ) were retrieved from the protein data bank. 3D protonation, in which hydrogen atoms are added to the enzyme’s conventional shape, was used to prepare it for docking. The compounds’ structures were aligned with the protein receptors using MOE-Dock, employing the triangle matcher placement approach. The London dG scoring function was used to evaluate the compatibility, and force field refinement was performed on the top 5 poses for each drug. In order to verify the docking procedure, B92, 88D, and THA were re-docked with the active sites of 3KJF, 5UHF, and 1ACJ, respectively. Tables 6, 7, and 8 depicted the relationships between amino acids, bond strength measured in Kcal/mol, docking energy score, and the lengths of hydrogen bonds measured in Angstroms (Ao).

Results and discussion

Structural identification of compounds 1, 2

Structures ofsarcotrocheliol (1) and cholesterol (2) were fully assigned based on 1D1H13, C) and 2D1H1, H COSY, HMQC and HMBC) NMR spectroscopy and ESI (positive/negative modes) mass spectrometry.

Sarcotrocheliol (1)

Compound 1 was isolated as an optically active colorless solid. It was assigned the molecular formula C20H34O2 indicated by HRESIMS m/z307.2639 [M + H]+ (calculated for C20H34O2H+ 307.2631), representing four degrees of unsaturation. The 13C NMR spectrum showed resonances for 20 carbons categorized by DEPT experiments into five methyls, six methylenes, six methines, and three quaternary carbons. Resonances for four olefinic carbons (δC138.5, 135.9, 125.4 and 123.9 ppm) in the 13C NMR spectrum accounted for two double bond equivalents, confirming that 1 has a bicyclic skeleton.

The 1H and 13C NMR spectra has shown resonances for five methyl groups: three tertiary methyl groups (δH/δC 0.99/24.2, 1.58/17.6, and 1.62/15.0) and isopropyl methyls (δH/δC 0.70/20.2 and 0.85/20.7, 3 H each, d, J = 6.6 Hz). Also, two oxygenated methines resonating at δH/δC3.83/69.9 and 4.51/71.0 and an oxygenated quaternary carbon at δC 75.0 were indicated from HMQC spectrum. Further studies of H, H COSY and HMBC correlations (Fig. 2) established the planar structure of compound 1 as a pyrane-based cembranoid which was further confirmed as sarcotrocheliol by comparing it with our previously published research article335.

Fig. 2
figure 2

COSY and key HMBC correlations of compounds 1 and 2.

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Cholesterol (2)

Compound 2 was obtained as a colorless solid. The molecular weight of 2 was determined by ESIMS as 386 Dalton with a corresponding molecular formula C27H46O requiring five degrees of unsaturation. 1H NMR spectrum showed a typical pattern for steroidal compounds36 in which resonances for five methyl groups were exhibited at δH 0.66 (s, H3-18), 1.00 (s, H3-19), 0.85 (d, J = 6.6 Hz, H3-21), 0.94 (d, J = 6.5 Hz, H3-26), 0.94 (d, J = 6.5 Hz, H3-27), in addition to one olefinic methine proton at δH 5.35 (dt, J = 5.2, 2.1 Hz, H-6), one oxymethine at δH 3.53 (td, J = 6.5, 3.3 Hz, H-3) and further signals integrated for twenty-eight protons being for eleven methylenes, and six non-oxygenated sp3-methines. The 13C NMR along with HMQC and DEPT spectra revealed resonances for 27 carbons classified into: three quaternary carbons including one olefinic carbon, eight methines among them one olefinic and one oxygenated carbon, eleven methylenes, and five methyls (in which two of them for isopropyl group). Further confirmation for structure of compound 2 was established on the basis of 2D NMR connectivities (H, H COSY and HMBC, Fig. 2). Consequently, the chemical structure of compound 2 was confirmed as cholesterol37.

Biological activities

Cytotoxic activity and induced apoptosis

To check biosafety of any compound, the evaluation the cytotoxicity of it toward normal cell line is requited. In the current study, both compounds 1&2 were assessed toward Wi38 normal cell line as shown in Table, 1, 2. Results revealed that, IC50 of compounds 1&2 toward Wi38 cell line was 112.5 & 97.2 µg/ml respectively. According to recent studies, it has been illustrated those bioactive compounds of soft coral genus Sarcophyton showed potential anti-cancer38 and cytotoxic activities39,40. Sarcophytol A, a cembrane-type diterpene, was extracted from Sarcophytonglaucum and has been scientifically demonstrated to possess anticancer properties and strong inhibitory effects against several types of tumor promoters41. Our investigation found that compounds 1 and 2 effectively suppressed the growth of HCT-116 and HepG2 cancer cell lines in a dose-dependent manner. The decrease in cell viability and rise in cytotoxicity were particularly significant, comparable to the effects observed with doxorubicin, a positive control. The IC50 values of compound 1 were 8.8 and 11.8 µg/mL for HepG2 and HCT-116, respectively, while compound 2 exhibited activity with IC50 10.4 and 12.0 µg/mL against both HCT-116 and HepG2 cancer cell lines, respectively (Table 1) and those for doxorubicin were 7.9 and 9.8 µg/mL against HCT-116 and HepG2 cancer cells, respectively. To confirm the safety of these compounds, the selectivity index (SI) was performed, where results showed that SI for both compounds 1&2 toward HCT-116 was 11.8 & 10.4 respectively. Likewise, SI for both compounds 1&2 toward HepG2 was 8.8 & 12 respectivty. These results confirmed the safety of the both compounds, compounds possessing SI values greater than two are considered acceptable selectivity toward cancer cells.

Table 1 Cytotoxic activities of compounds 1 and 2 on HCT-116 and HepG2 cancer cell lines.
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The caspase-3 inhibitory effects of compounds 1, 2 towards HCT-116 and HepG2 cancer cells were 252.97- 294.15 ng/mL and 233.11–265.21 ng/mL, respectively. These findings demonstrated that both compounds significantly induced apoptosis and antiproliferative activities of HepG2 and HCT-116 cancer cells through the mitochondrial mechanism (Table 2). The cytotoxicity performed herein for both compounds against HepG2 and HCT-116 cancer cell lines is being reported for the first time so far.

Table 2 Effect of compounds 1 &2 on caspase-3 in HCT-116 and HepG2 cancererous cell lines.
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Antimicrobial activity

The antimicrobial activity of sarcotrocheliol (1) and cholesterol (2) was evaluated herein for the first time against a set of bacterial and fungal strains as illustrated in Table 3. In accordance, sarcotrocheliol displayed antibacterial activity against P. aeruginosa, B. subtilis and M. luteus with inhibition zones of 15, 15 and 14 mm, respectively (Fig. 3). Moreover, sarcotrocheliol exhibited antifungal activity (15 mm) against C. albicans. Also, the MIC of sarcotrocheliol against P. aeruginosa, B. subtilis, M. Luteus, and C. albicans was recognized at concentration of 1.5 µg/mL. In contrast, sarcotrocheliol exhibited no activity against E. coli, S. aureus and C. neoformans (Fig. 3).

Table 3 Antimicrobial activity of compounds 1 and 2.
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On the other hand, cholesterol (2) has shown little antibacterial action against P. aeruginosa, B. subtilis, S. aureus, and C. albicans, resulting in inhibition zones measuring 10, 14, 12, and 10 mm correspondingly. Moreover, MICs of cholesterol against the reported test microorganisms (P. aeruginosa, B. subtilis, S. aureus and C. albicans) were at concentrations of 3.0, 1.5, 1.5 and 3.0 µg/mL, respectively. Prenner, Lewis 1reported the antibacterial activity of cholesterol played an important role in the attenuation of the interaction of gramicidin S with phospholipid bilayer membranes42. Also, conjugation of cholesterol with polyethylenimine-containing nano-agents led to increased antibacterial activity through photodynamic therapy43.

Fig. 3
figure 3

Antimicrobial activity of compounds 1 and 2.

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Insecticidal activity

Larvicidal activity

Table 4 shows the larval mortality percentages of Culex pipiens larvae treated with various concentrations of compounds 1 and 2 and their LC50 and LC90 values. The obtained results revealed that larval mortality was directly proportional to tested concentration. Tested compounds induced remarkable toxicity against the third instar larvae even at the lowest applied concentration of 2 ppm. Compound 2 induced much more larval toxicity than 1 with LC50 values of 3.928 and 6.39 ppm for 2 and 1, respectively (Fig. 4). The obtained Chi-square values, which are considered significant at P < 0.05, indicate the heterogeneity of the test population.

Table 4 Larvicidal activity of 1 and 2 against the third larval instar of the mosquito vector, Culex pipiens.
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Fig. 4
figure 4

Larvicidal activity of 1 and 2 against the third larval instar of the mosquito vector, Culex pipiens.

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Our current knowledge regarding the insecticidal properties of soft coral is limited. Various marine creatures have been identified as valuable reservoirs of unique bioactive chemicals or secondary metabolites with insecticidal capabilities. Kalimuthu et al.44 used the seaweed Gracilaria firma and the copepod Megacyclops formosanus for controlling Aedes aegypti larvae. It was discovered that the doses needed to eradicate the larvae exceeded the amounts documented in this study; Hasaballah and El-Naggar45 conducted a study to examine the effectiveness of the sea sponges Negombata magnifica and Callyspongia siphonella in eradicating mosquito larvae belonging to the Culex pipiens species. The researchers discovered that the LC50 values varied from 47.6 ppm to 610.3 ppm, which is eight times greater than the levels documented in this study. Recently, Elbahnasawy et al. (2023) found that sea cucumber Holothuria impatiens-ZnO-NPs induced larvicidal activity with LC50 of 2.756 ppm and LC90 of 9.294 ppm against the mosquito, C. pipiens which are considered similar to those reported here. However, the promising larvicidal activity reported here for the tested compounds may be attributed to a novel mode of action that possibly interferes with physiological processes and lead to quick mortality of targeted larvae.

Adulticidal activity

Adulticidal activity of various doses of 1 and 2 against the housefly, Musca domestica and their LD50 and LD90 valuesare given in Table 5. Obtained data revealed that tested compounds are distinctly potent toxicants against M. domestica adult females, and their mortality was directly proportional to tested doses. Compound 2 induced more pronounced adulticidal activity than 1 with LD50 values of 16.007 and 39.14 µg/adult for 2 and 1, respectively (Fig. 5). The obtained Chi-square values, which are considered significant at P < 0.05 indicate the heterogeneity of the test population.

Table 5 Adulticidal activity of 1 and 2 against the housefly, Musca domestica adult females.
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Fig. 5
figure 5

Adulticidal activity of 1 and 2 against the housefly, Musca domestica adult females.

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In harmony with these results, potent insecticidal activity (LC50 of 22.595 ppm) of the soft coral, Ovabunda macrospiculataZnO-nanoparticles against M. domestica was reported46. Additionally, Kilic et al. screened forty-seven macroalgae mostly from the Mediterranean Sea, for their insecticidal activity and found that selected extracts showed a potent adulticidal activity ranging from 80% to100% mortality when 5 µg/mosquito was applied47. However, although very rare studies on this model are present, marine habitat is still a promising and undiscovered source for novel compounds and secondary metabolites to be discovered.

DFT calculations

Frontier molecular orbital analysis

Frontier molecular orbitals (FMOs) are a highly efficient method for investigating intramolecular interactions. The electron donors are linked to the highest occupied molecular orbitals (HOMO). Conversely, the electron acceptors are linked to the LUMO, which corresponds to the molecular orbitals with the lowest energy level that do not contain any electrons. As the energy diminishes, the molecule undergoes greater polarization, exhibits heightened activity, and experiences a decline in kinetic bioactivity48.

Figure 6 exhibits the HOMO-LUMO orbitals and energy values of the isolated compounds. Figure 6 illustrates that compounds 1 and 2 have a very small energy gap of 0.211 and 0.201 eV, respectively. Therefore, they should possess the ability to unveil a more effective biological process. Table 6 presents the dipole moments of the compounds analyzed, revealing that compound 2 demonstrates higher dipole moments in comparison to compound 1. Compound 2 has the ability to enhance interactions with species that possess a significant dipole moment, particularly in biological systems49.

Table 6 DFT parameters calculated for compounds 1 and 2.
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Molecular electrostatic potential (MEP)

MEP is a useful technique for evaluating the comparative polarity and reactivity of a molecule. MEP can also be utilized for the anticipation and examination of molecular interactions, including hydrogen bonding, drug-receptor, and enzyme-substrate interactions. The Molecular Electrostatic Potentials (MEPs) at the B3LYP/6-31G (d, p) level of theory are generated using the optimized structures of compounds 1 and 2, as depicted in Fig. 7. The presence of the red color signifies the area with the most negativity, indicating a good position for electrophilic attacks. The blue tint signifies the most advantageous area, denoting a propitious ___location for nucleophilic assaults. Areas with zero potential are represented by the color green.

Fig. 6
figure 6

Molecular orbital distribution plots of HOMO and LUMO of compounds 1 and 2.

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Fig. 7
figure 7

MEP map of compounds 1 and 2.

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Molecular docking simulation

Docking evaluation against Caspase 3 (PDB ID: 3KJF)

Caspase 3 is the key executioner caspase that orchestrates the controlled dismantling of the cell during apoptosis or programmed cell death. It cleaves and activates other caspases and apoptotic factors, degrades cytoskeletal proteins, condenses chromatin, fragments DNA, packages cellular contents into apoptotic bodies, and signals phagocytes - all hallmark features of cells undergoing programmed suicide. The activation of caspase 3 commits the cell to apoptosis and inhibits inflammatory responses that would otherwise occur from necrosis. As shown in Table 7, the co-crystallized B92 protein exhibited the occurrence of two H- bonds with Arg 207, and Asn 208. The docking energy score of compounds 2 is greater (-6.24 Kcal mol[-1) than that of compound 1 (−4.88 Kcal mol[−1) and nearer to that of the co-crystallized B92 (docking score: −6.29 kcal/mol). Compound 1 revealed the presence of four H-bonds, with Ser 205 and Cys 163. In addition, compound 1 showed H-arene interactions with Tyr 204 residue. Compound 2, with the best docking score energy (S)= −6.24 kcal/mol, displayed three hydrogen bonds and one H−arene interaction (Fig. 8). The more negative the energy score, the stronger the interaction. So, the interaction followed the following order: 2 (−6.24) > 1 (−4.88). These results are consistent with the experimental findings about the anticancer activity mentioned earlier.

Table 7 Docking interaction data calculations of compounds 1 and 2 (compared with B92) with the active sites of the receptor of Casp3 protein (PDB ID:3KJF).
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Fig. 8
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3D molecular docking simulation studies of the interactions between the (a) compound 1, (b) compound 2, and (c) co-crystallized B92, with the active site of the receptor of Casp3 protein (PDB ID: 3KJF).

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Docking evaluation against PDB ID: 5UHF

The ECF sigma factor sigma H and 7nt short RNAs are crucial in governing gene expression in bacteria in response to stress. Sigma H is an ECF sigma factor that controls the expression of genes involved in the stress response of mycobacteria like oxidation, heat shock and cationic antibiotics exposure. It regulates nearly 200 genes by binding with the RNA polymerase holoenzyme to initiate transcription. From the data of Table 8, The co-crystallized 88D showed one arene-H contact with Gly 314 and four H-bonds with Asp 342, Pro 420, and Arg 364 amino acids. The docking energy scores of compounds 1 (−6.47 Kcal/mol) and 2 (−6.81 Kcal/mol) showed high similarity to those of the co-crystallized 88D (docking score: −6.47 kcal/mol). Compound 1 revealed two H-bonds with Glu 419 and Thr 365. Compound 2 produced one hydrogen bond with Met 315 amino acid. Moreover, compounds 1 and 2 showed similar binding modes with the receptor of 5UHF and this in agreement with the experimental results of the antimicrobial activity (Fig. 9).

Table 8 Docking interaction data calculations of compounds 1, 2, and 88D with the active sites of the receptor of M. Tuberculosis (PDB ID:5UHF).
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Fig. 9
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3D molecular docking simulation studies of the interactions between the (a) 1, (b) 2, and (c) 88D compounds with the active site of the receptor of M. tuberculosis (PDB ID: 5UHF).

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Docking evaluation against acetylcholinesterase (PDB ID: 1ACJ)

Acetylcholinesterase is the critical enzyme that hydrolyzes the neurotransmitter acetylcholine and terminates its action at cholinergic synapses in the nervous system. By rapidly splitting acetylcholine into choline and acetate, it prevents constant neuronal firing, regulates post-synaptic receptor sensitivity, and modulates key functions like muscle contractions, cognition, and wakefulness. Acetylcholinesterase controls the intensity and duration of cholinergic neurotransmission and is the target of neurotoxins and insecticides that inhibit its activity, leading to acetylcholine accumulation. According to the shown data in Table 9, the co-crystallized THA with an energy score S = −5.46 kcal/mol created one hydrogen bond with His 440. The docking score of compound 1 (S = −6.55 kcal/mol) is greater than that of compound 2 (S = −5.03 kcal/mol). Compound 1 showed H-arene interaction with Trp 84. On the other hand, compound 2 had good binding to 1ACJ active sites through two H-bonds with Tyr 130 and His 440, also two H-arene interactions with Phe 330 and Trp 84 (Fig. 10).

Table 9 Docking interaction data calculations of compounds 1, 2, and THA with the active sites of the receptor of AChE [PDB ID: 1ACJ).
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Fig. 10
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3D molecular docking simulation studies of the interactions between the (a) 1, (b) 2, and (c) THA compounds with the active site of the receptor of 1ACJ.

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Conclusion

The study demonstrates that the compounds sarcotrocheliol (1) and cholesterol (2) obtained from the soft coral S. trocheliophorum possess significant anticancer, antimicrobial, and insecticidal activities. Both compounds exhibited potent cytotoxicity against human cancer cell lines, induced apoptosis through caspase-3 upregulation, and displayed antimicrobial activity, with sarcotrocheliol (1) being more effective against a range of microorganisms. Notably, both compounds exhibited remarkable larvicidal and adulticidal effects against mosquitoes and houseflies, with cholesterol (2) showing stronger insecticidal properties. Computational analysis using Density Functional Theory (DFT) and molecular docking studies provided insights into the chemical reactivity and potential binding interactions of these compounds, suggesting their promise as drug candidates with broad bioactivities. These findings highlight the importance of exploring natural marine resources as a source of biologically active compounds with diverse therapeutic and insecticidal applications, warranting further investigation and potential development of these compounds as novel therapeutic agents or insecticides.