Cholestan Steroids from The Stem Bark of Aglaia angustifolia Miq and Their Cytotoxic Activity against MCF-7 Breast Cancer Cell Lines.

. With about 120 species, Aglaia is one of the largest genera of the plant family Meliaceae (the mahogany plants). Various Aglaia species have been investigated since the 1960s for their phytochemical constituents and biological properties. This research objective was to find secondary metabolites that have activity as anti-breast cancer compounds from endemic Indonesian Aglaia, such as Aglaia angustifolia Miq. Two cholestan type steroids, stigmast-5en-3 α -acetat (1), as a new steroid with α -sterochemistry of acetyl moiety at C-3 and 23a-homostigmast-5en-3 β -ol (2), with unusual side chain were isolated for the first time from the stem bark of Aglaia angustifolia Miq or known as segara tree in Kalimantan. The chemical structures of two steroids were identified with spectroscopic data, including IR, NMR (1H, 13C, DEPT 135°, HMQC, HMBC, NOESY, 1 H-1 H COSY) and HRTOF-MS, as well as by comparing with previously reported spectral data. These two steroids were isolated for the first time from this genus. Steroids 1 and 2 were evaluated for cytotoxic activity against MCF-7 breast cancer cells and showed weak activity with IC 50 values of 829.0 and 903.0 µg/mL, respectively.


INTRODUCTION
Steroids are a structurally diverse group of natural products that exist in a variety of organisms exhibiting extensive biological activities (Li et al., 2021;Ratnaweera et al., 2015;Kikuchi et al., 2017;Gu et al., 2018;Ren et al., 2019). To date, more than 100 steroidal agents have been approved as pharmaceuticals by the FDA for the treatment of cancer, heart failure, inflammation, pain, traumatic brain injury etc (Kim et al., 2018). Continuous efforts in searching for structurally novel and active steroids may thus provide structural lead for drug development, which will also make the prosperity of the steroid chemistry. Aglaia lour., the largest genus of subtropical and tropical angiosperm family Meliaceae (the mahagony plants), consists of 130 species. Aglaia is native to the tropical rain forests of the Indo-Australian region, ranging from India and Srilanka eastward to Polynesia and Micronesia. Various Aglaia species have been investigated since 1960s for their phytochemical constituents and biological proterties (Agarwal et al., 2021;Farabi et al., 2018). Aglaia angustifolia mainly distributed in Indonesia (Sumatera, Kalimantan) and declared almost extinct, until today only a few phytochemical studies have been carried out to identify its active metabolites (Hutagaol et al., 2021); Hutagaol et al., 2020). Previous phytochemical studies on some species Aglaia plants reported the presence of a series of secondary metabolite such as triterpenoids, lignans, rocaglate derivatives, sesquiterpenoids, tetraterpenoids and steroids. Phytochemical research of the species A. angustifolia has not been widely carried out, steroids that have been found and published previously from this species are stigma-4-en-3on (Hutagaol et al., 2022). Previous phytochemical studies on some species Aglaia plants reported the presence of a series of secondary metabolite such as triterpenoids, lignans, rocaglate derivatives, sesquiterpenoids, tetraterpenoids and steroids. Those which have been shown to posses antifungal, insecticidal and anticancer properties (Zhang et al., 2012;Awang et al., 2012;Harneti et al., 2014;. Fractionation of the n-hexane and ethyl acetate extracts from the stembark of this Aglaia angustifolia led to the isolation of one steroid compound 1, as a new steroid with α-sterochemistry of acetyl moety at C-3 along with one knew steroid compound 2, with unusual side chain (Figure 1). Based on the literature that the two steroids 1 and 2 from the results of this study are the first steroid structures found in Aglaia. The Isolation, structure elucidation of compounds based on spectroscopic data are described herein. Breast cancer is one of a kind cancer by prevalence highest in the world. According to WHO in 2020, as many as 2.1 million womans has been diagnosed have breast cancer every year. In vitro testing using modeling culture being a method dependable for find a cure for cancer. MCF-7 cells have used for more than 40 years in research breast cancer. Cell culture was isolated from the patient breast cancer which metastasize to pleura. This study used MCF-7 cells because MCF-7 cells are many cells used for in vitro tests and has the best form. MCF-7 cells are included in breast cancer subtype luminal A which has ER+ molecular characteristics, PR+ and HER2- (Lailatul & Nurrachma, 2020). As part of our studies on anti breast cancer cells candidate compounds from Indonesia Aglaia plants. The obtained steroid compounds were evaluated biological activity against MCF-7 human breast cancer cell line and showed weak activity with IC50 829.0 µg/mL (1) and 903.0 µg/mL (2), with cisplatin as standard 38.1 µg/mL. The biological activity test method used in this study used Presto Blue method with cisplatin as a standard compound, but different in the compounds tested, which in this study were tested against steroids 1 and 2, while tested limonoids (Supriatno et al., 2018).

EXPERIMENTAL SECTION General Experimental Procedure
Optical rotations were measured on a Perkin Elmer 341 Polarimeter (Waltham, MA, USA). The IR Spectra were recorded on a Perkin Elmer 1760 X FT-IR in KBr (Waltham, MA, USA). Mass spectra were obtained with a Water Qtof. HR-MS XEV otm mass spectrometer (Waters, Milford, MA, USA). 1D NMR spectra ( 1 H, 13 C and DEPT) and 2D spectra (COSY, HSQC, HMBC and NOESY) spectra were recorded with a Bruker 600 MHz/Topspin 3.5P17 spectrometer for 1 and JEOL JNM ECZ-600 spectrometer for 2, (at 600 MHz for 1 H and 125 MHz for 13 C-NMR, with CDCl3 as a solvent, chemical shift were given on a δ (ppm) scale and both using tetra methyl silane (TMS) as the internal standard. Technical solvents were distilled prior to use for maceration, isolation and spectral grade solvents were employed for spectroscopic measurements.
Chromatographic separation were carried out on silica gel 60 (70-230 mesh and 230-400 mesh; Merck, Darmstadt, Germany) and Octa Dodecyl Silane (ODS Fuji Sylisia, Japan). TLC plates were precoated with silica gel GF254 (Merck, 0.25 mm). Spots were visualized under UV light of 254 nm and 365 nm simultaneously and by spraying with 10% H2SO4 in ethanol or vanillin reagent followed by heating.
In the 13 C-NMR and DEPT 135 spectra, two other olefinic carbons at C 125-140 ppm were also seen.
However on the HMBC spectrum, the above signals have no correlation with other signals, so that those signals were assumed to be impurities found in compound 1.
The relative configuration of compound 1 was identified by a NOESY experiment (Figure 3), which showed the NOESY correlations between H-19β (Me-19β at H 1.01) with H-4 (H 2.31) and H-1 (H 1.87).  Correlations also shown by H-3 (H 4.60) H-4 and H-1. These correlations indicated that the acetyl group at C-3 is α-oriented. Similar to those observations from NOESY, showed the cross peak between H-18β (Me-18β at H 0.68) with H-20 (H 1.33), and cross peak between H-19β with H-20. Correlation also shown by H-20 with H-24 (H 0.92), these NOESY correlations indicated that H-20 and H-24 was βoriented. Compound 1 was similar to a compound that have been found previously from the stem bark of Chisocheton cumingianus (Meliaceae) and from the aerial parts of Polygonum bellardii, β-sitosterol-3-Oacetate but with difference stereochemistry of acetyl moety at C-3 (Katja et al., 2017); (El-kader et al., 2012). Therefore, compound 1 was elucidated to be a new derivative cholestan-type steroid with α-oriented relative configuration of acetyl group at chiral carbon C-3 and was named 24S, 24-ethylcholest-5en-3αacetate.
Furthermore, the olefinic proton, H 5.34 (1H, br d, J = 4.8 Hz, H-6) was correlated to methylene carbon at C-4 (C 38.3) and C-7 (C 32.1), quaternary carbon at C-10 (C 36.8) indicating an olefinic moiety was located at C-5 and C-6 ( 5,6 ). Correlations from methylene protons, H-4 H 2.25(2H, m) and H-1 H 1.84 (1H, m) with C-3 (C 71.9), were used to assign an hydroxyl group which was located on C-3. The HMBC spectrum of 2 exhibited interaction of C-23a at δC 26.0 with H-28 at δH 1.25 and 1.21 and with H-23 at δH 1.23. Correlations also shown from methylene protons, H-23a H 1.13 (2H, m) with methine carbon at δC 45.9 (C-24), than from methylene protons H-23 at H 1.23 (2H, m) with methylene carbon at δC 26.0 (C-23a). These HMBC correlation with The 13 C NMR, DEPT and COSY spectrum defined the presence of sp 3 methylene carbon C-23 at δC 29.8 and C-23a at δC 26.0. The 1 H NMR and 13 C NMR spectral data were compared with β-sitosterol, these data suggested the presence of Δ 5 steroid-type like stigmas-5-en-3β-ol with had one double bond previously published (Cayme & Ragasa, 2004). The similar signals except for marked difference in the addition of one methylene carbon at C-23, so named C-23a-Homo after C-23 at [δC 26.0, δH 1.13, (2H, m, H-23a)], so compound 2 steroid-type cholestan named 23a-Homostigmast-5en-3β-ol. A detailed comparison of the 1 H NMR and 13 C NMR spectral data of compound 2 were similar to those of the 23a-Homostigmast-5-en-3β-ol which were previously isolated from n-hexane fraction of the roots of Fumaria parviflora (Naz et al., 2013). The structures of steroid compounds that have been isolated from the genus Aglaia compared to steroids 1 and 2 have differences in the basic structure and in their side groups. Steroid compounds that have been found from the genus Aglaia include, pregnantype steroids, pregnasetal from A. silvestris (Pointinger et al., 2008). (Weber et al., 2000) have succeeded in isolating androstan-type steroid compounds, as well as cholestan-type steroid compounds, from the leaves of A. rubiginosa. (Harneti et al., 2014), had succeeded in finding steroid compound from the stem bark of A. eximia and showed cytotoxic activity against murine leukemia P-388 cells with an IC50 value of 11.42 µg/mL.  have succeeded in finding a steroid (β-sitosterol) from the stem bark of A. argentea. (Farabi et al., 2018), have also succeeded in finding pregnane-type steroids from the stem bark of A. elliptica. Steroids 1 and 2 showed the weak activity against MCF-7.

CONCLUSIONS
Two cholestan type steroid compounds were investigated from ethyl acetate and n-hexane extract of Aglaia angustifolia Miq stem bark and identified as a Stigmast-5en-3α-acetat (1), as a new steroid with αsterochemistry of acetyl moety at C-3 and 23a-Homostigmast-5en-3β-ol (2), a steroid with unique side chain moiety. The isolated compounds showed weak activity against MCF-7 breast cancer cell lines.