Ergosterol Peroxide and Stigmasterol from The Stembark of Aglaia simplicifolia (Meliaceae) and Their Cytotoxic against HeLa Cervical Cancer Cell Lines

Two steroid compounds, ergosterol peroxide (1) and stigmasterol (2) have been isolated from the stembark of Aglaia simplicifolia belong to Meliaceae family. The chemical structures of 1 and 2 were identified based on spectroscopic evidence including UV, IR, 1D NMR, 2D NMR as well as mass spectra and by comparison with those previously reported spectra data. Both compounds were evaluated for their cytotoxic effects against cervical cancer HeLa cells in vitro. Compounds 1 and 2 showed cytotoxicity activity against HeLa cervical cancer cells with IC50 values of 0.80 and 26.42 μM, respectively.


INTRODUCTION
Meliaceae is the important plant families that have utilized and generally grow in tropical countries. Meliaceae plant is known for the presence of the various secondary metabolite compounds that exhibit interesting biological activity such as hypoglycemia, anticancer, anti-inflammation, antifeedant, antitumor Leong et al., 2016;Su et al., 2006) and insecticidal activity (Nugroho et al., 1999).
The Aglaia genus is a plant of the tropical rain forest in the Indomalesiana region and mainly distributed in tropical countries including India, Indonesia, Malaysia and parts of the Western Pacific. Aglaia is the largest genus belongs to the Meliaceae family contains more than 150 species (Hidayat et al., 2017a;Hidayat et al., 2017b;Awang et al., 2012) and about 65 species grown in Indonesia (Wood, et al., 1970;Heyne 1982). Phytochemical studies on Aglaia species have led to the identification of main compounds such as sesquiterpenoid, diterpenoid, triterpenoid, limonoid, steroid, lignan, and alkaloid groups (Harneti & Supratman, 2021).
Aglaia simplicifolia is found in Sumatra and Kalimantan, Indonesia. So far, reports on the content of secondary metabolite compounds from this plant are the only senecracidiol isolated from the bark of the stem (Kurniasih et al., 2019). Although steroids of other Aglaia species have been investigated previously, the ergosterol peroxide of A. simplicifolia is yet to be reported.

Cytotoxic Activity (Resazurin assay)
Cell viability was assessed by resazurin assay following the previously reported procedures (Sittampalam et al., 2004). Cells were seeded into a 96-well plates at a density of 17,000 cells/well and stabilized at 37 °C in 5% CO 2 for 24 h. Cells were incubated for 24 h with compounds 1 and 2. Ten cells were treated with 10 µL of Presto Blue™ Cell Viability Reagent for another 1-2 hours. Cell viability assessed by measuring the absorbance at 570 nm with a reference wavelength of 600 nm using an EMax Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). For the positive control, cells were incubated for 24h with 100 µL of Cisplatin.

Structure Elucidation
In our phytochemical research on Aglaia simplicifolia, two steroids, ergosterol peroxide (1) and stigmasterol (2) (Figure 1) were isolated from the nonpolar fractions. Their structures were determined by a detailed analysis of their spectroscopic data.   (Table 1), which could be classified with the help of HSQC data as six Me, seven CH 2 , and eleven CH groups (two oxygenated), and four Cq -atoms. The presence of two disubstituted olefins (δ 130.78 (C-7), 132.33 (C-23), 135.41 (C-6), 135.19 (C-22)), indicating that the sterol fragment of compound 1 is an ergosterol derivative. Besides, two oxygenated quaternary carbons of δ 82.15 (C-5) and 79.42 (C-8) suggested the presence of a peroxide structure.
The signals at δ H 6.22 and 6.51 (d, J = 8 Hz, 2H, H-6, H-7) in the 1 H-NMR spectrum revealed the presence of a disubstituted double bond which were correlated with carbon signals of 135.41 (C-6) and 130.78 (C-7) in the HMBC spectrum. The 1 H-NMR showed also signals for six methyl groups, two singlets at 0.81 and 0.83, and four doublets at 0.84 (J = 6.8 Hz), 0.87 (J = 6.6 Hz), 0.93 (J = 6.8 Hz) and 1.17 (J = 6.7 Hz). Moreover, a multiplet at 3.95, characteristic of a steroid oxymethine signal located at C-3, was observed. The 2D-NMR experiments confirmed that compound 1 is a steroid, containing a peroxy function at C-5/C-8 and two double bonds in the side chain and at C-6/C-7.
In the HMBC correlations (Figure 2), these three methylene proton signals were correlated to the methine carbon signal at δ C 66.84 (C-3), and the methylene carbon signal at δ C 35.06 (C-1) was correlated with the methyl proton signal at δ H 0.89 (H-19). The methylene proton signals at δ H 1.94 and 2.11 (H-4) were clearly correlated to two carbon signals at δ C 83.10 (C-5) and 135.80 (C-6) in 5α,8α-epidioxy system. The methyl proton signal at δ H 0.89 (H-19) was long-rangecorrelated to the methyne carbon signal at δ C 83.10 (C-5) to which the proton signals at δ H 1.94 and 2.11 (H-4) were correlated, but not to the signal at δ C 79.82 (C-8). The methyl proton signal was also correlated to the methine carbon signal at δ C 51.43 (C-9), but not to the signal at 52.05 (C-14) which correlated with the methyl proton signal at δ H 0.83 (H-18). Also an olefinic proton signal in 5α,8αepidioxy system at (5 6.25 (H-7) was correlated to the carbon signals at δ C 51.43 (C-9) and 52.05 (C-14), while the other olefinic proton signal at δ H 6.51 (H-6) was correlated to carbon signals at δ C 37.29 (C-4) and 37.33 (C-10). Therefore, the structure of ergosterol peroxide (5,8-epidioxy-5 ,8 -ergosta-6,22E-dien-3 -ol) was thus elucidated to be 1. In this paper ergosterol peroxide was isolated from A. simplicifolia first time, so it is a new compound for this species. The known compounds stigmasterol (2), were confirmed by comparison and biogenetic analysis of these compounds with values reported by Cayme & Ragasa (2004).

Cytotoxic Activity
The cytotoxic effects of compounds 1 and 2 against HeLa cervical cancer cells were conducted according to the resazurin assay method (Sittampalam et al., 2004) and were used cisplatin (IC 50 0.67 µM) as a positive control. As shown in Figure 3, treatment with both compounds resulted in the dosedependent inhibition of HeLa cervical cancer cell growth when assessed at 24 hours posttreatment. Higher concentrations of both compounds were required to inhibit cell growth.
Ergosterol peroxide (1) has a much stronger activity with an IC 50 value of 0.80 µM compared to stigmasterol (2) which has an IC 50 value of 26.42 µM. This shows that the value of cytotoxic activity against HeLa cervical cancer cells is influenced by the presence of peroxide groups bound to C-5 and C-8. In fact, the ergosterol peroxide compound from the stem bark of Aglaia simplicifolia is much stronger to inhibit the growth of cervical cancer cells HeLa than those isolated from marine fungus Phoma sp with an IC 50 value of 29.20 µM (Wu et al., 2018).
Ergosterol peroxide was sensitive to cancer cells, while less sensitive or nontoxic to normal cells. Wu et al., (2018)

CONCLUSIONS
Two steroid compounds, ergosterol peroxide (1) and stigmasterol (2) have been isolated from the stembark of Aglaia simplicifolia and were shown for the first time in this species. The presence of peroxide in steroid structure plays an important role in cytotoxic activity against HeLa cervical cancer cells.