FLAVONOLS FROM THE LEAVES Lygodium microphyllum ( Lygodiaceae )

Flavonol compounds, quercetin (1) and quercetin-3-O-β-D-glucopyranoside (2) have been isolated from the ethyl acetate extract of Lygodium microphyllum leaves. The chemical structures of flavonol compounds were identified based on spectroscopic data and by comparison of spectral data obtained previously. The discovery of flavonol compounds in Lygodium microphyllum was shown in this study for the first time.


INTRODUCTION
Susu A fern has survived since Paleozoic era and can adapt to variety environmental changes (Wallace et al., 1991), thus fern contain a lot of useful secondary metabolites, including flavonoids, steroids, alkaloids, phenols, triterpenoids, various kinds of amino acids and fatty acids (Zeng-fu et al., 2008).One from thousands of ferns species that have interesting pharmacological benefits is Lygodiaceae family.The genus belong to Lygodiaceae family only Lygodium (Guo-gang et al., 2012).Generally, Lygodium genus is a group of ferns that spread and always propagate in other plants.Lygodium genus are different from other kinds of ferns because it has roots that crawl on the ground rhizomes and fleshy and can only live in the open because they like the sunlight.Some plants from Lygodium genus are invasive and has become a problem in a number of forest areas.It's growing fast and lack of predator makes these plants dominate, displacing wildlife, threatens biodiversity, and enhance the human-animal conflict (Zheng and Xing, 2009).One of the invasive species from Lygodium genus is L. microphyllum.Plants of this genus have a variety of properties that have been widely recognized, thus the utilization of plants from genus is quite expected.Some herbs of Lygodium genus widely used by people one of them as traditional medicine as hepatitis medicine (Zheng and Xing, 2009), back pain, rheumatism and treatment for kidney stones (Lee et al., 2008), expectorant, scabies, eczema and liver treatment (Upreti et al., 2009), diuretics, antiplasmodial, and treatment for lung and kidney (Upreti et al., 2009), laxative, headache and digestive disorders (Zheng and Xing, 2009).Phytochemical studies on the Lygodium genus had reported to contain a compound with unique structure and had diverse biological activities such as flavonoids (Zhang et al., 2006), phenolic glycoside (Ye et al., 2007), naphthoquinone (Chen et al., 2010), ecdysteroids (Zhu et al., 2009) and phenylpropanoid glycoside (Duan et al., 2012).
L. microphyllum based on ethnobotany and ethnopharmacological information provides many benefits in the field of reproductive health as well as simple patterns make the plant is classified as herbs are easily available so that benefit of these plants is required primarily to determine the chemical constituents of this plant.In our ongoing research to find new biologically active compounds from Indonesia Lygodium plants, we isolated and describe steroid compounds from the leaves of L. microphyllum (Kuncoro et al., 2015).In the further search for biologically active dompounds from the polar fraction, the ethyl acetate extract showed flavonoid constituents.In this paper we decribe isolation and structural elucidation of flavonols from the leaves of L. microphyllum.

General
Melting point measured on electrothermal melting point apparatus and not corrected.IR spectra were measured on Perkin-Elmer 1760X spectrophotometer, FT-IR on KBr.Mass spectra recorded with a mass spectrometer Water, Qtof HR-MS XEV otm . 1 H and 13 C NMR spectra are obtained by JEOL NMR 500 MHz, JEOL NMR ECZR 600 MHz used TMS as an internal standard.Chromatographic separation is carried out on silica gel 60 (Merck), octa desyl silane (ODS, Fuji silysia).TLC Plate filled with silica gel GF254 (Merck, 0.25 mm) and detection was obtained with the appearance of 10% H2SO4 in ethanol followed by heating and under ultraviolet-visible light at wavelength of 257 and 364 nm.

Plant Material
The leaves of L. microphyllum was collected from forest areas in Samarinda, East Kalimantan in June 2014.The plant was identified by staff at the Faculty of Forestry, University of Mulawarman, Samarinda and sample specimens stored at the Faculty of Forestry, University of Mulawarman, Samarinda.

Extraction and Isolation
Dried leaves of L. microphyllum (3.54 kg) was extracted with methanol at room temperature.The methanol extract obtained was concentrated at low pressure to produce a concentrated methanol extract (526 g).The concentrated methanol extract was dissolved in water (4: 1) and partitioned successively with n-hexane, ethyl acetate, and nbutanol.Evaporation of the solvents resulted nhexane (59 g), ethyl acetate (72 g) and n-butanol extract (54 g).The ethyl acetate extract (50 g) was separated using column chromatography with silica gel eluted with n-hexanel-ethyl acetatemethanol as a gradient solvent yield 22 fractions (A01-22).Fraction A10-15 were combined (920 mg) and separated using column chromatography on silica gel eluted with ethyl acetate: methanol (7:3) to obtained 22 fractions (B01-22).Fraction B02-05 were combined (420 mg) and further separated using flash column chromatography on silica gel eluted with a gradient ratio of solvent, ethyl acetate-methanol to obtained 20 fractions (C01-22).Fraction C05 (190 mg) was separated using column chromatography on ODS eluted with a gradient solvent ratio methanol-water to obtain compound 1 (12.5 mg) and 2 (5.4 mg) as a yellowish powder.

RESEARCH AND DISCUSSION
The ethyl acetate extract of L. microphyllum leaves was fractionated by a classical column chromatography on silica gel and octa desyl silane adsorbens to yield quercetin (1) and 3-O--Dglucopyranoside (2).

Conclusion
Two flavonol compounds, quercetin (1) and quercetin-3-O--glucopyranoside (2) were isolated from the leaves of L. microphyllum.The phytocehmical studies of flavonol compounds from this species was shown for first time reported in this study.

Figure 1 .
Figure 1.Chemical structure of compounds 1 and 2

Table 1 .
NMR data for compounds 1 and 2