Isolation and Structure Elucidation of Quercetin-Derivative (amino-flavone) from Centaurea perrottetii DC (Asteraceae)

The plant, Centaurea perrottetii DC (Asteraceae) is a herbaceous thistle-like shrub used in traditional medicine for the treatment of malaria, fever, syphilis, ulcer and skin infections. The aim of the study is to isolate and elucidate the structure of compound(s) from C. perrottetii . Quercetin-derivative (an amino-flavone) was isolated from the n-hexane fraction of the methanol extract of the whole plant of Centaurea perrottetii using a combination of silica gel column and gel filtration chromatography. The structure of the compound as 2-(3,4-dihydroxyphenyl)-3-((1-ethylcyclopropyl)amino)-5,7-dihydroxy-4Hchromen-4-one was established via chemical tests, Ultraviolet (UV), Infrared (IR), 1D- and 2D-dimensional NMR spectroscopy. This is the first report of isolation of this compound from Centaurea perrottetii .


Introduction
Centaurea perrottetii DC ( Figure 1) is a species of the genus Centaurea of the family Asteraceae (Burkil,2000). It is a herbaceous thistle-like shrub which is usually prostrate or erect growing up to 50 cm high; it is covered with rough hairs, undulate, spiny margins (and flowers heads) with deeply lobed leaves (Burkil,2000). The plant is widely distributed from Mauritania to Niger, northern Nigeria, Western Cameroon and through the Sahara (Burkil, 2000;Alebiosu et al., 2019). It is commonly known as star thistle and Danyii or Danar Maguzawo in Hausa (Alebiosu et al., 2019).
The whole plant is used traditionally in Nigeria for the treatment of various ailments such as malaria, ear aches, syphilis, fevers, skin infections and ulcer without scientific validation (Burkil,2000). Thus, the antimalarial activity of the methanol extract of the plant and its fractions have been reported (Alebiosu et al., 2019). We report herein the isolation of a quercetinderivative (amino flavone) from the n-hexane soluble fraction of the methanol extract of the whole plant of Centaurea perrottetii for the first time. The study was limited to isolation and characterization of compound(s) from the plant.

General Procedures
Nuclear Magnetic Radiation (NMR) data were recorded on a Bruker AVANCE III spectrophotometer (600 MHz) with residual solvent as internal standard. Thin Layer Chromatography (TLC) was carried out using silica gel 60 GF254 pre-coated Aluminium sheets by Sigma Aldrich, Germany. Column chromatography was conducted using LONA cheme silical gel (60 -200 mesh), while gel filtration chromatography was performed using sephadex LH -20 (Sigma, Space street, St. Louis, MO, USA). Spots on TLC plates were visualized using 10 % H2SO4 followed by heating at 105 º C for ten minutes.

Collection and Identification of Plant Material
Whole plant of C. perrottetii was collected in November, 2014 from Ilella, Gwadabawa Local Government Area, Sokoto State, Nigeria and was identified and authenticated by a taxonomist, Mal. M. A. Salihu. Voucher specimens were prepared and deposited at the Herbarium of the Taxonomy Unit, Department of Botany, Usmanu Danfodiyo University, Sokoto, Nigeria, and voucher specimen number (UDUH/ANS/0034) was given.

Preparation of the Extract Partitioning
The plant material was shade dried, pulverize to powder (2.1 kg) and it was extracted with 70 % methanol (25 L) using maceration method for 7 days with constant agitation. The extract obtained was filtered and evaporated in-vacuo using a rotary evaporator at 40 º C to yield a dark green residue (147.84 g) subsequently referred to as the crude methanol extract (CME). Some part of the CME (120 g) was suspended in distilled water (500 mL) and partitioned successively with n-hexane (1.5 L), chloroform (500 mL), ethylacetate (1.0 L) and n-butanol (1.5 L) to obtain hexane fraction (HF), chloroform fraction (CF), ethyl-acetate fraction (EF), nbutanol fraction (BF) an d the residual aqueous fraction (AQF), respectively.

Isolation and Characterization of Compound SHA
The n-hexane fraction (5.0 g) was subjected to column chromatography on a silica gel (60-120 mesh Merck) column and it was gradiently eluted with n-hexane (100 %), n-hexane : ethyl acetate (10:90), ethyl acetate (100 %), ethyl acetate : methanol (5: 95) and methanol (100 %). A total of 125 fractions of 20 mL each, were collected and combined based on their TLC profiles to obtain 17 major fractions coded H1 -H17. Fraction H-4 was subjected to further purification using Sephadex LH-20 and methanol (100 %) as mobile phase; 5 mL each of a total of 100 collections were made and combined based on the TLC profile to afford 7 major fractions coded HA -HG. Repeated gel filtration of fraction HC and HF led to the isolation of a compound labelled as SHA which was characterized using chemical tests, melting point and spectroscopic analysis.

Discussion
Compound SHA was isolated from the n-hexane soluble fraction of C. perrottetii as a yellow solid substance (5 mg); it reacted positively to Shinoda and Wagner's tests, suggesting the presence of phenolic and alkaloidal nucleus (Silva et al., 1998). The uncorrected melting point of compound SHA was 258 -260 º C. TLC analysis of SHA using chloroform and ethylacetate (1:6) gave a single homogenous spot with Rf value of 0.76.
The UV spectrum (in MeOH) of SHA indicates 307 nm as the wavelength of maximum absorption, other absorptions observed were 209 and 256 nm. The IR spectrum (in KBr) values at 3420 cm -1 which is due to OH stretching, 2923 cm -1 (CH bending), 1650 and 1034 cm -1 due to N-H bending and C-OH vibration.