Brevipedicelones D and E, Two C–O–C Flavonoid Dimmers from the Leaves of Garcinia brevipedicellata and Anti-onchocercal Activity

A novel isoflavone–chromone flavonoid C–O–C dimmer, brevipedicelone D (1), along with one new C–O–C biflavonoid derivative, brevipedicelone E (2), were isolated from the ethyl acetate extract of the leaves of Garcinia brevipedicellata, a medicinal plant used in folk medicine in parts of Cameroon. Their structures were elucidated by extensive spectroscopic techniques, including 1D- and 2D- NMR, MS experiments, as well as comparing their spectral data with those of known analogues. Anti-onchocercal screening of 1 showed moderate inhibition of adult worm motility of Onchocerca ochengi by 60% at the highest concentration (20 µg/mL) and inhibited motility of both the juvenile worms of O. ochengi and Loa loa by 90% at this same concentration.


Introduction
Garcinia is the most represented genus of the (Clusiaceae) family. Long known by the name Guttifreae, the Clusiaceae family is the most represented of this family, widely distributed from temperate to tropical regions and are represented by six (06) sub families which are: Kielmeyeroidae, Calophyloidae, Moronoboidae, Lorostermonoidae, Hypericoidae and Clusioidae [1]. The family at times regroups trees, shrubs, herbs and rarely lignans. Members are generally unbeared and the plants are easily recognized by their yellow or orange resinous latex which usually flows slowly when the stems, flowers and fruits are wounded while the leaves hardly produce latex [2]. Largely represented in Africa and Asia this family is made up of about 1350 species regrouped into 47 genera in low altitudes humid dense forests and are composed of six genera in Africa (i.e. Allanblackia, Calophyllum, Garcinia, Pentadesma, Symphonia and Mammea) [3]. Garcina brevipedicellata Oliv. is an African species that is abundant in forest regions of Cameroon and is a deciduous shrub that attains a height of about 5-9 m and about 30 cm in diameter. Previous phytochemical investigation of the leaves and stem bark of G. brevipedicellata have shown the presence of sterols, depsidones and tannins [4][5][6]. In a previous report from our lab., eight biflavonoids were isolated and characterized from the methanol extract of the stem heartwood of this plant, with three of them namely; brevipedicelones A, B, and C, being new flavonoid C-O-C dimmers [7]. This prompted further investigation of the plant.
Further investigation of the ethyl acetate extract of the leaves of this plant resulted in the isolation of a novel isoflavone-chromone flavonoid C-O-C dimmer, brevipedicelone D (1) and a new C-O-C biflavonoid derivative, brevipedicelone E (2), along with three known biflavonoids, robustaflavone (3), O-methylrobustaflavone (4) brevipediclone C (5) and one flavonoid, luteoline (6) (Fig. 1). We now report the isolation, structure elucidation of compounds 1 and 2 and the anti-onchocercal activity of 1 on the adult and juvenile worms (microfilariae) of Onchocerca ochengi and Loa loa, parasites responsible for human onchocerciasis (river blindness).

Results and Discussion
The air-dried and powdered leaves of garcinia brevipedicellata were extracted with methanol to obtain a gum which was re-extracted with warm ethyl acetate to give a crude extract which was first fractionated by exclusion column chromatography on Sephadex LH-20. The fractions obtained were subjected to a combination of column and preparative thin layer chromatography over silica gel that led to the isolation of compounds: 1 (52 mg), 2 (10 mg), 3 (11 mg), 4 (32 mg), 5 (8 mg) and 6 (12 mg  22 Hz, H-6 0 ) and 6.65 (1H, brd, 8.46, H-5 0 ) were attributed to carbons C-2 0 , C-6 0 and C-5 0 respectively of a trisubstituted benzynic ring (ring B). A singlet observed at d H 6.35 (1H, s, H-6 00 ) was attributed to the proton on carbon C-6 00 of a penta-substituted benzynic ring (ring A 0 ).
In addition, a highly deshielded proton observed at d H 9.10 (1H, s, H-2 00 ) suggested a chromone building block. The DEPT spectrum of 1 displayed signals for six hydroxylated carbons, six aromatic carbons and two ethylenic carbons bearing an oxygen bridge to the pyran ring of the benzopyran moieties. We also noticed in its HMBC spectrum a correlation between H-2 0 and C-8 00 and the axis was assigned to be located at C-3 0 /C8 00 by a typical downfield shift of C-8 00 (119.0 ppm), along with an upfield shift at C-2 0 (114.5 ppm) which further confirmed the ether linkage between the two derivatives via C-3 0 and C-8 00 (Table 1). This ether linkage was confirmed by selected HMBC, COSY and NOESY interactions (Fig. 2). From the above spectroscopic data leading to the structure shown for compound 1, it is characterized as a novel isoflavonechromone flavonoid C-O-C dimmer described for the first time and named brevipedicelone D. Compound 2 was obtained as yellow powder in CH 2 Cl 2 / MeOH (10:1) mixture. The molecular formula was C 31 H 20 O 10 as deduced from HRTOFMS.
On its 1 H NMR spectrum, two tetra-substituted aromatic rings with two protons exhibiting meta coupling signals at d On the DEPT spectrum, it was deduced that apart from the C-atom of the CH 3 O group which is saturated and whose chemical shift appears at d  Table 1).
The HMBC spectrum of 2 showed correlations between the two protons carried by ring B (H-2 0 and H-6 0 at d C 7.59) and the carbon atom at d C 164.5, (C-3, ring C). A first flavone sub-structure in 2 was deduced from correlations between proton H-3 (d H 6.82) and the C-4 (d C 181.9) carbonyl. Correlations were also observed on one hand between proton H-2 000 (ring B 0 ) at d H 8.04 and H-3 00 (ring C 0 ) at d H 6.89 and on the other hand between the carbonyl carbon (C-4 00 ring C 0 ) at d C 182.0 and the proton H-3 00 at d H 6.89 (ring C 0 ). These led to the suggestion of a second flavone sub-structure in 2.
On the NOESY spectrum of 2, it was observed that there exist a correlation between H-3 0 and H-5 0 (d H 6.71, ring B) with H-6 00 and H-8 00 (d H 6.81, ring A 0 ). This suggested that the linkage between the two flavonoid units in 2 is between C-4 (ring C) and C-7 00 (ring A 0 ). From the above spectroscopic data leading to the structure shown for compound 2, it is characterized as a new ether biflavonoid derivative also described for the first time and named brevipedicelone E.

General
The mass spectra (HRTOFMS) were measured in a time of flight mode spectrometers. The 1 H NMR spectra were registered on a 600 MHz NMR spectrometer with tetramethylsilane (TMS) as an internal standard; while 13 C NMR spectra were recorded on a 150 MHz NMR spectrometer using DMSO as solvent. Methyl, methylene and methine carbons were distinguished by DEPT experiments. Homonuclear 1 H connectivities were determined by using the COSY experiment. One-bond 1 H-13 C connectivities were determined with HMQC gradient pulse factor selection. Two and three bonds 1 H-13 C connectivities were determined by HMBC experiment. Chemical shifts were reported in d (ppm) and coupling constants (J) were measured in Hz.

Extraction and Isolation
Air dried and ground leaves of Garcinia brevipedicellata (1.5 kg) were extracted using methanol. After evaporating the solvent the methanolic residue obtained was exhaustively fractionated into three, by extraction with three solvents in increasing polarity. First with ethyl acetate, followed with acetone and lastly with methanol. Only the ethyl acetate fraction (62 g) was investigated. This extract was divided into four equal portions A-D and each portion fractionated by size exclusion chromatography on Sephadex gel LH-20 and eluted with methanol. Sub-fractions were pooled together to obtain five main fractions. N 1 (6.1 g), N 2 (4.3 g), N 3 (34 g), N 4 (5.5 g) and N 5 (3.2 g). Fraction N 4 was purified twice by chromatography on a silica gel column with CH 2 Cl 2 /MeOH (10:1) to afford Compound 1 (52 mg). N 3 and N 2 were repeatedly purified on an open silica gel column with CH 2 Cl 2 /MeOH (10:1) followed by preparative thin layer chromatography to give Compounds 2 (10 mg), 3 (11 mg) and 4 (32 mg), 5 (8 mg), 6 (12 mg) and more of compound 3 (8 mg) respectively.

Extraction of Onchocerca ochengi Adult Worms
Onchocerca ochengi adult worm masses were extracted from cattle skin by the method employed in [32]. Briefly, fresh pieces of umbilical cattle skin containing palpable nodules were obtained from local slaughterhouse in Buea, Cameroon. The piece of skin was immediately transported to laboratory. The skin was thoroughly washed with soap and distilled water, drained, dried by blotting with a piece of cloth and then transferred to a sterile laminar flow hood. It was then entirely covered with 70% ethanol and allowed to evaporate completely on its own. The nodules were carefully dissected using a sterile razor blade and the pale orange-yellow worms (in appearance) were immediately submerged in sterile 12-well culture plates (NUNC, USA) containing 2 ml of complete culture medium [RPMI-1640 supplemented with 25 mM HEPES, 2 g/L sodium bicarbonate, 20 mM L-glutamine, 10% new born calf serum (SIGMA, USA), 2 9 antibiotic-antimycotic (Sigma, USA)], pH 7.4]. After overnight culture, 1 mL of medium was added before addition of drug making a total volume of 3 mL. Adult worm cultures were carried out at 37°C under an atmosphere of 5% CO 2 in humidified air in an incubator (HERACell 150, Haraeus, Germany).

Extraction of Onchocerca ochengi Microfilariae
The microfilariae of O. ochengi were extracted by the method of [33], with some slight modifications. Cattle skin got from the slaughterhouse was thoroughly cleaned and sterilized as above. The skin was then firmly attached onto a sterilized flat wooden board using autoclaved thumbnails. The outer surface was carefully shaved with a sterile razor blade, and then rinsed twice with distilled water. A clean dry adsorbent cloth was used to remove excess moisture from the skin. The entire skin was covered with 70% ethanol and allowed to evaporate in a laminar flow hood. This sterilization process was done twice. Once the alcohol had completely evaporated from the skin, skin snips were obtained from different locations of the skin. These sleeves were carefully scrapped and the snips submerged in 15 mL of complete culture medium. The assemblage was incubated at room temperature for 2 h to allow for emergence of microfilariae. The highly motile microfilariae that emerged were concentrated by centrifugation at 4009g for 10 min. The supernatant was decanted, and the pelleted mfs were re-suspended in fresh complete culture medium. The highly motile microfilariae were quantified using an inverted microscope (Euromex, Holland). One hundred microlitres of culture medium containing microfilariae were distributed into 96 well culture plate containing LLC-MK2 cell layers to obtain an average of 12-15 mfs per well. Culture conditions were the same as that of adult worms above.

Isolation and Culture of Loa loa Microfilariae
Blood was collected from Loa loa infected individuals at the Edea Health District after confirmation by Giemsa stain. The blood was rapidly transferred to the University laboratory. The microfilariae were isolated by the method of [32]. Freshly collected L. loa -infected blood was diluted (1:2) with culture media used above but without sera. The diluted blood was carefully layered on 4 mL of Ficollpacque (TM) in a 15 mL centrifuge tube. The tube was spun in a swing bucket centrifuge at 4009g for 15 min. The recovered mfs were washed three times with culture media (without sera) and then re-suspended in media containing sera. The mfs were then distributed in wells of a 96-well microtiter plate containing LLC-MK2 cell layers. Each well contained 12-15 mfs in 100 of media.

Preparation of Compound and Assessment of Activity
Compound 1 was dissolved in C 99.9% sterile dimethyl sulfoxide (SIGMA, USA) giving a stock concentration of 5 lg/ml. The compound was prepared at 29 the final concentration and distributed to wells containing parasites. For the microfilariae, 100 lL was added while 1 mL was added to wells containing adult worms to give a final volume of 200 lL and 4 mL for microfilariae and adult worms respectively. All the cultures were conducted for 120 h post addition of the compound. Auranofin [34], served as positive control for adult worm assay while ivermectin and amocarzine were used as positive control drugs for O. ochengi and L. loa mfs respectively. The diluent (dimethyl sulfoxide) was added to the negative control wells. Inhibition of microfilariae motility was assessed using an inverted microscope. Effect of compound on adult worm viability was assessed using the MTT-formazan assay following procedures employed by [32,33].

Ethical Issues
We got ethical clearance (2013/11/371/L/CNERSH/SP) and administrative authorization (631-06.14) from respectively, the Cameroon National Ethical Committee and the Ministry of Public Health, Cameroon. Local administrative authorization was also obtained from the District Medical Officer of the Edea Health District. Informed consent was obtained freely from individuals who harbored high L. loa mf load.