A new havanensin-type limonoid from Chisocheton macrophyllus

A new havanensin-type limonoid, 16β-hydroxydysobinin (1), along with four known limonoids (2–5), have been isolated from the seeds of Chisocheton macrophyllus. The chemical structure of the new compound was determined by referencing spectroscopic data, and by comparison to those related spectra previously reported. Each compound was evaluated for their cytotoxic effects against Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and display no significant activity.


Introduction
Limonoids, known as degraded triterpenes, are derived from a precursor with a 4,4,8-trimethyl-17-furanylsteroid four-ring skeleton labelled as A, B, C and D rings [1]. Limonoids are a class of secondary metabolites found in the order Rutales and the Meliaceae and Rutaceae family [2]. Meliaceae is a family of timber trees that are a rich source for limonoids and are widely distributed in tropical and subtropical regions with 50 genera and more than 1400 species [3,4]. Limonoids isolated from species of the family Meliaceae have been reported to have biologically activity as antifeedant, antimicrobial, antimalarial, and cytotoxins [5][6][7][8]. Genus Chisocheton is the second largest in the family Meliaceae, consisting of more than 50 species distributed across Nepal, India, Bhutan, Myanmar, South China, Thailand, Indonesia, Malaysia, and Papua New Guinea [7,9].
Chisocheton macrophyllus is a species distributed in the Nicobar Islands, peninsular Thailand, peninsular Malaysia, Singapore, Sumatera, Anambas Islands, Java and Borneo Islands [10]. Its seeds have been reported to yield bioactive limonoids such as dysobinol, 7α-hydroxyneotricilenone, dysobinin and nimonol with cytotoxic activity against P-388 murine leukemia cells [11], whereas the leaves to yield Epstein-Barr virus activation of Triterpenoids [12]. After further investigations for cytototoxic limonoids from the seeds of C. macrophyllus, we found and structural elucidation of a new havenensin-type limonoids (1) and four known limonoids (2)(3)(4)(5), along with their cytotoxic activity against MCF-7 breast cancer cells. Herein, the isolation, structural elucidation and cytotoxic activity against MCF-7 breast cancer cells are discussed.

Plant materials
Seeds of C. macrophyllus were collected from Bogor Botanical Garden, Bogor, West Java Province, Indonesia. The plant was identified by Mr. Harto, the staff of Bogoriense Herbarium, Research Center for Biology, Indonesia Science Institute, Bogor, Indonesia and a voucher specimen (No. Bo-1295453) was deposited at the Herbarium.

Instruments and reagents
Optical rotations were measured on a Perkin Elmer 341 Polarimeter (Waltham, MA, USA). UV spectra Nurlelasari et al. Appl Biol Chem (2021) 64:35 was measured using a TECAN Infinite M200 pro with MeOH. Furthermore, the IR spectra and mass spectra were recorded on a One PerkinElmer spectrum-100 FT-IR in KBr and Waters Xevo QTOF MS, respectively. NMR spectra were obtained with Bruker Topspin at 500 MHz for 1 H and 125 MHz for 13 C (compound 1) and for compounds 2-5 using JEOL JNM-ECZ500R/ S1 at 500 MHz for 1 H and 125 MHz for 13 C, using tetra methylsilane (TMS) as the internal standard. Chromatographic separations were carried out on the silica gel 60 (70-230 and 230-400 mesh, Merck). Thin layer chromatography (TLC) analysis was carried out on 60 GF 254 (Merck, 0.25 mm) using various solvent systems, and measured by irradiation under ultraviolet-visible light Vilber Lourmat (λ 254 nm dan 365 nm) followed by heating of silica gel plates, sprayed with 10% H 2 SO 4 in ethanol and Ehrlich's reagent (p-Dimethylaminobenzaldehyde in ethanol).

Cytotoxic activity test
The cytotoxicity of compounds 1-5 was determined with a cell viability test using PrestoBlue ® assay. The cells were maintained in a Roswell Park Memorial Institute (RPMI) medium with 10% (v/v) Fetal Bovine Serum (FBS) and 1 µL/1 mL antibiotics (1% Penicillin-Streptomycin). Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO 2 . MCF-7 cells plated in 96 multiwell culture plates at a density of 1.7 × 10 4 cells/well. After twenty-four hours, the medium was discarded and fresh medium containing sample with different concentrations 7.81, 15.63, 31.25, 62.50, 125.00, 250.00, 500.00, 1000.00 μg/mL and control was added. After incubation with the sample for 24 h, PrestoBlue ® reagent (resazurin dye) was added. The PrestoBlue ® assay results were read using a multimode reader at 570 nm. The IC 50 values were determined by linier regression method using Microsoft Excel software. The IC 50 value corresponds to the concentration of compounds that decreases by 50% the number of viable cells and the absorbance in control corresponds to 100% viability.

Results and discussion
The n-hexane fraction from the seeds of C. macrophyllus was subjected to vacuum-liquid chromatography (VLC) column packed with silica gel 60 by gradient elution. The VLC fractions were repeatedly subjected to normal phase column chromatography on silica gel to yield compounds 1-5 (Fig. 1).

Cytotoxic activity
All isolated compounds were evaluated for the cytotoxic activity against MCF-7 breast cancer cell line and cisplatin is used as a positive control according to the method previously described [16,22] and the results are shown in Table 2. Compound 1 showed the strongest activity against MCF-7 breast cancer cell with IC 50 (inhibitory concentration, 50%) values of 45.91 µ M, suggesting that the presence of hydroxyl at C-16 can increase the cytotoxic activity. In addition, the presence of epoxy ring and ketone group, like in compound 2-4, showed weak activity, indicated the presence of epoxy ring and ketone group can decrease activity.

Competing interests
There is so conflict of interests.