Cytotoxic Activity of Flavonols from Macaranga gigantea

Two flavonols, glyasperin A ( 1 ), and meliternatin ( 2 ) has been isolated from the leaves of Macaranga gigantea (Rchb.f. & Zoll.) Müll.Arg. Extraction and isolation of flavonols were used methanol with the maceration method. The process of fractionation and purification used column chromatography and radial chromatography. The structure of both flavonols was determined by spectroscopic methods, including UV-Vis, IR, HRESIMS, 1D NMR ( 1 H, and 13 C-NMR) and 2D NMR (HMBC and HMQC). The cytotoxic activity of glyasperin A ( 1 ), and meliternatin ( 2 ) toward P-388 leukemia murine cells by MTT method, showing IC 50 values 3.44 and 30.04 µg/mL, respectively .


Introduction
Macaranga (Euphorbiaceae) is one of the pioneer plants that are found in secondary forests, especially those that get lots of suns. The genus Macaranga consists of 310 species, and in Indonesia, around 140 species are found. The spread of this plant is quite extensive, covering Africa to the tropical regions of Asia to the Pacific region (Blattner et al., 2001). This plant is widely used by the community as traditional medicines, among others, as medicine for wounds, infections, diarrhea, and coughs (Heyne, 1987). Macaranga produces phenolic compounds, especially flavonoids (Agustina et al., 2012, Tanjung et al., 2018, 2014 and stilbenoids (Aldin et al., 2019;Beutler et al., 1998;Tjahjandarie et al., 2019). Flavonoids and stilbenoids of Macaranga have terpenyl side chains such as isoprenyl (C5), geranyl (C10), and farnesyl (C15), which are attached to the aromatic nucleus. Flavonoids and stilbenoids of Macaranga show bioactivity as antimalarial, antioxidant, antimicrobial, anti-inflammatory, and anticancer (Pailee et al., 2015;Peresse et al., 2017;Magadula et al., 2014). On this occasion, two flavonols will be reported, namely glyasperin A (1), and meliternatin (2) from the ethyl acetate extract of Macaranga gigantea (Rchb.f. & Zoll.) Müll.Arg. leaves. Phytochemical data on this plant is still very limited. It will also be reported to test the cytotoxic activity of the two flavonols toward P-388 murine leukemia cells using the MTT method.

General procedure
Cerium sulfate reagent is used as a stain to show flavonoids compounds. Silica gel is used as a stationary phase in gravity column chromatography and radial chromatography. Thin layer chromatography analysis (TLC) using T25 silica gel 60 GF254 0.25 mm (Merck) TLC plates. The UV spectrum was determined with a Shimadzu 1800 UV-Vis spectrophotometer. The IR spectrum was determined with the Shimadzu IR spectrophotometer. The mass spectrum was determined with the HRESIMS Merck Waters LCT XE ESI-TOF spectrometer, the NMR spectrum was determined by the NMR JEOL ECA 400 spectrophotometer operating at 400 MHz ( 1 H-NMR) and 100 MHz ( 13 C-NMR). Cytotoxic activity test against P-388 murine leukemia cells was determined using the MTT method.

Plant materials
Plant samples used in the study were M. gigantea leaves. Plant samples were obtained from the forest area of Jalan Samarinda-Sanga-Sanga, Palaran District, Samarinda, East Kalimantan. The identification of plant samples was carried out at the Bogoriensis Herbarium. Experiments

Extraction and isolation
Extraction of M. gigantea (2.5 kg) leaves using methanol at room temperature for 24 hours three times. Evaporation of the solvent using a low-pressure device produces crude methanol extract. The crude methanol extract, partitioned with nhexane to remove chlorophyll and nonpolar compounds. The methanol extract was added with 10% v/v H2O and partitioned with ethyl acetate. Evaporation of the solvent using a low-pressure device produces a crude EtOAc extract of 70 g. Separation of EtOAc extracts using gravity column chromatography using n-hexane: EtOAc (9: 1 to 3: 7) produces three main fractions, namely the A-C fraction. Fractions A and C show the presence of flavonoid compounds with cerium sulfate reagents characterized by brownish-yellow spots. The separation of fraction C by gravity column chromatography using a mixture of n-hexane: EtOAc (8: 2 and 1: 1) produced three subfractions, C1-C3. Separation of C2 subfraction from the Sephadex column using methanol results in C21-C23 subfraction. Purification of the C21 subfraction by radial chromatography with a mixture of n-hexane: acetone (9: 1 to 7: 3) resulted in 20 mg of meliternatin (2). Separation of fraction A by gravity column chromatography using hexane: EtOAc (9: 1 and 7: 3) produces three subfractions, namely A1-A3. Purification of the A1 subfraction by radial chromatography using nhexane: CHCl3 (7: 3 to 100% CHCl3) produced 45 mg glyasperin A (1) compounds.

Cytotoxic activity
Determination of the anticancer activity test of the flavonols (1-2) was determined using the MTT method in vitro. Cytotoxic activity of the isolated compounds 1-2 to P-388 murine leukemia cells was determined according to the MTT assay, as previously reported (Tanjung et al., 2018;Tjahjandarie et al., 2020).

Conclusion
Two flavonol derivates, glyasperin A and meliternatin, were isolated from the leaves of M. gigantea.
Glyasperin showed moderate activity to P-388 cells, meliternatin was inactive.