A new gnidiflavanone-flavonol dimer and other constituents from Gnidia apiculata

Abstract A new 3,8′′-flavanone-flavonol dimer gnidiflavanone-flavonol (1) and 10 known compounds (2–11), including four rare primula-type flavones 2–5, were isolated from the roots of Gnidia apiculata. Compounds 2–5 and 7 were reported for the first time from the plant family Thymelaeceae. Structures of the isolated compounds were established by spectroscopic data, including 1D and 2D NMR (COSY, HMBC, HSQC and ROESY) and mass spectrometry, as well as by the comparison with literature data. The crude roots extract and isolated compounds were evaluated for antimicrobial and antiplasmodial activities. Among isolated compounds, 6-hydroxyflavone (4) and 6-O-acetylflavone (4a) showed antiplasmodial activity against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Graphical Abstract


Results and discussion
Compound 1 (Figure 1) was isolated as yellow powder and found to be dextrorotatory ([a] 26 D þ 30.3 (c 0.33, MeOH). Its ESI-HRMS ( Figure S1) showed a protonated molecular ion peak at m/z 589.0979 [M þ H] þ , attributed to molecular formula C 30 H 20 O 13 . The UV spectrum of 1 showed absorption bands at k max 280 and 380 nm, suggesting a flavone chromophore, and the FTIR spectrum ( Figure S2) displayed bands at max 3275 and 1601 cm À1 , attributed to hydroxy and conjugated carbonyl groups, respectively. The 13 C NMR data (Table S1 and Figure S3) of Compound 1 revealed 30 signals between d C 50.4 and 198.1, which were assigned to 2 carbonyls (d C 198.1 and 177.2), 2 methines (d C 50.4, C-3 and 82.8, C-2) typical of a flavanone moiety, and 26 sp 2 hybridized carbon atoms (9 hydrogenated, 12 oxygenated and 5 quaternary carbons). The 1 H NMR spectrum (Table S1 and Figure S5 (Ito et al. 1999). In addition, two deshielded intramolecular hydrogen bonded peaks were observed at d H 12.2 (C-5-OH) and 12.6 (C-5 00 -OH), characteristic for hydroxyl groups. These data so far suggested a dimer, comprising of a flavanone unit linked to a flavonol moiety.
The crude roots extract of G. apiculata, Compounds 1-11 and 4a were tested for in vitro antimicrobial and antiplasmodial activities against selected bacteria, fungi and Plasmodium falciparum. All the isolated compounds were found inactive towards bacteria and fungi. However, 6-hydroxyflavone (4) and its acetylated derivative 4a showed activities against chloroquine-sensitive (D6) and -resistant (W2) strains of P. falciparum. Compound 4 exhibited IC 50 values of 4.6 and >4.7 mg/mL against D6 and W2 strains, respectively, while 4a had IC 50 values of 3.4 and 4.6 mg/mL against D6 and W2, compared to reference standards chloroquine and artemisinin (IC 50 0.02, 0.16, and IC 50 0.005 and 0.003 mg/mL, respectively).

General experimental procedures
Optical rotation was determined by AUTOPOLV R IV polarimeter. The UV spectra were recorded using Shimadzu UV-VIS spectrophotometer and the IR spectra were measured by Shimadzu (IRAfinity-1S) FTIR spectrophotometer. The 1 H-and 13 C NMR spectra were recorded on a Bruker Avance 400 or 500 MHz spectrometers. HMBC, HSQC and ROESY were measured on an Agilent DD2-500 NMR spectrometer. The ESI-HRMS spectral data were obtained by utilizing an Agilent 6545 LC/Q-ToF system. LC/MS data was measured using Agilent 1290 Infinity series UHPLC instrument, coupled to an Agilent 6120 quadrupole mass spectrometer with a dual ESI and APCI interface. TLC analysis was done using analytical silica gel 60 PF 254 þ 366 pre-coated alumina plates (Merck, 0.25 mm thick). CC was done on Silica Gel (Loba Chemie, 70-230 mesh) or (CDH, 60-120 mesh) and sephadex LH-20 (Merck). Solvents for extraction and CC were distilled before use.

Plant material
The roots of G. apiculata were collected from Machakos County, about 40 km from Nairobi city, Kenya, and a voucher specimen (RWK 2017/03) was deposited in the University herbarium in the Department of Botany, University of Nairobi.

O-Acetylation of 6-hydroxyflavone (4)
The acetylation of Compound 4 was performed using acetic anhydride (Ranu et al. 2003). A neat mixture of Compound 4 (10 mg) and acetic anhydride (12 mL) was placed in a 100 mL round bottomed flask and heated at 100 C with constant stirring for 2 hours. Progress of the reaction was monitored by TLC analysis. The product was extracted with CH 2 Cl 2 followed by crystallization, to yield a white solid of 6-O-acetylflavone (4a) (

Antimicrobial assay
The in vitro antimicrobial activity of crude extract and pure compounds was determined against selected standard ATCC organisms (bacteria and fungi) from ATCC, using protocol as described in literature (Kumarihamy et al. 2021). The crude extract was tested at 200 mg/mL, while the pure compounds were dissolved to make a stock solution of 2 mg/mL. The pure compounds were tested at concentrations of 20, 4 and 0.8 mg/mL and the respective IC 50 's determined. The IC 50 values were calculated using XLFit software. Amphotericin B (92% pure, MP Biomedical, CA, USA) and vancomycin (90% pure, from Sigma-Aldrich, MO, USA) were used as positive controls against fungi and bacteria, respectively.

Antiplasmodial assay
The in vitro antiplasmodial activity of the crude extract and pure compounds was done by parasite Lactate Dehydrogenase Assay (Nogueira and Ros ario 2010) on chloroquine -resistant W2 and -sensitive D6 strains of Plasmodium falciparum (Kumarihamy et al. 2021). The compounds were dissolved in dimethyl sulfoxide and pre-diluted in RPMI 1640 medium to make concentrations between 528.9 to 4760 ng/mL. Chloroquine and artemisinin were used as positive controls. The percent inhibition of the parasites by the phytochemicals was determined from the absorbance at 650 nm, shown by SpectraMAX ELISA Reader. The XLFit software was used to calculate IC 50 ; the concentration of the sample that caused inhibition of 50% of the tested parasites.