Anthelmintic flavonoids and other compounds from Combretum glutinosum Perr. ex DC (Combretaceae) leaves

Nine known compounds have been isolated from Combretum glutinosum leaves, including flavonoids and triterpenes. The crystal structures of umuhengerin and (20S,24R)-ocotillone are reported for the first time; the latter structure confirms the absolute configuration. The crude extracts and the isolated compounds were tested for their anthelmintic activity on the larvae and adult worms of Haemonchus contortus. The best activity on both stages of the parasite was obtained with the flavonoids.


Introduction
Combretaceae are trees, shrubs or often lianas widely distributed in subtropical to tropical regions. This family consists of 18 genera, including 370 species of Combretum (Malgras, 1992;McGaw et al., 2001, Amadou, 2004. These species are widely used in traditional medicine for their numerous pharmacological properties (Komlan, 2002). C. glutinosum is a tree of the genus Combretum belonging to the family Combretaceae. This plant is most often present in tree savannas, normally on shallow soils (Akoè gninou et al., 2006). It is distributed in tropical Africa from Mauritania to Uganda, passing through, for example, Senegal, Cameroon and Chad. In Bé nin, the plant is spread in the North in Kandi, Ké tou, Toukountouna, south of Malanville, Bessassi and Porga, and in the Pendrari Park (Akoè gninou et al., 2006). This species is among the most widely used of the medicinal plants in West Africa (Kerharo & Adam, 1974). It has been reported by Toklo et al. (2021) that it is used in the treatment of malaria, ISSN 2053ISSN -2296 dysentery, diarrhea, bronchitis and hypertension. The traditional uses of this plant have led to numerous pharmacological studies, including antibacterial, antifungal, anthelmintic, antimalarial and antidrepanosite properties (Baba-Moussa et al., 1999;Ouattara et al., 2006;Usman et al., 2017;Sall et al., 2017;Alowanou et al., 2019). Previous phytochemical studies of the genus Combretum led to the isolation of tannins, flavonoids, triterpenoids and steroids (Jossang et al., 1994;Dawe et al., 2013;Roy et al., 2014, Amako et al., 2016Sene et al., 2018;N'Diaye et al., 2017;Balde et al., 2019). In the search for a new active ingredient effective against increasing biological resistance to synthetic anthelmintics, the study reported here was undertaken on the leaves of C. glutinosum, which were obtained from plants in Bé nin. The search for bioactive secondary metabolites from the leaves revealed nine known compounds (Scheme 1), of which the crystal structures of two, one flavonoid and one triterpene, have been determined for the first time. The biological activity of these compounds on the larvae and adult worms of H. contortus, a hematophage that causes parasitic disorders in small ruminants, has also been investigated.

Chromatographic and spectroscopic analysis
Column chromatography was performed using 230-400 mesh silica gel (Merck, Darmstadt, Germany), 70-230 mesh silica gel (Merck) and sephadex LH-20 (Sigma-Aldrich). Thin-layer chromatography (TLC) was performed on a precoated aluminium sheet of silica gel 60 F254 (Merck). The spots of compounds were detected using UV lamps at two wavelengths (254 and 365 nm) and then fixed using a 10% sulfuric acid spray reagent, followed by heating to 373 K. The high-resolution mass spectra were recorded in positive mode using a QTOF mass spectrometer (Bruker, Germany) equipped with an HESI source. The spectrometer operates in positive mode (mass range 100-1500, with a scan rate of 1.00 Hz), with automatic gain control to provide high accuracy mass measurements within the mass range. NMR spectra were recorded in deuterated chloroform (CDCl 3 ) and/or deuterated methanol (MeOD) using a Bruker DRX 500 NMR spectrometer (Bruker, Rheinstetten, Germany); the chemical shifts () are given in ppm relative to tetramethylsilane (TMS) (Sigma-Aldrich, Germany) as the internal standard.

Collection of plant material, extraction and isolation of compounds
The leaves of C. glutinosum were collected in April 2018 in Kandi (in northern Bé nin) and identified at the national herbarium of the University of Abomey-Calavi. A reference specimen was stored under the accession number YH 241/ HNB after authentication.
Based on the TLC profiles, the FCG2-2 subfraction was combined with the FCG3 fraction and subjected to silica-gel column chromatography using a gradient elution of hex/ EtOAc with increasing polarity to obtain the compounds 5-demethylsinensetin [(1); 17 mg] and umuhengerin [(2) research papers 22 mg]. The FCG4 fraction was also eluted with a mixture of ethyl acetate and 5% methanol to give eight subfractions (FCG4 1-8), which all contained an impure compound (CCG20). The FCG4-2 fraction was passed through a Sephadex LH-20 column and eluted with methanol to give solely pure CCG20, which was identified as -sitosterol glucoside [(9); 48 mg].
Colourless needle-like crystals of (2) and colourless platelike crystals of (3) were obtained by slow diffusion of dichloromethane into their solutions in methanol. Selected crystals were mounted on cryo loops.

Aqueous extract
An aqueous extract was obtained by boiling 100 g of C. glutinosum leaf powder in 1000 ml of distilled water brought to the boil for 30 min. After decantation, the mixture was filtered on Whatman paper and the filtrate obtained was evaporated under vacuum to obtain the dry extract.

Anthelmintic tests
2.4.1. Test for inhibition of larval migration and motility of adult worms. The test of larval migration and motility of adult worms in the presence of the samples was evaluated following the procedure of Hounzangbe-Adote et al. (2005). The observation of the worms in the presence of the extracts was done every 6 h and every 3 h in the presence of the com-pounds. The concentration of the tested compounds was 150 mg ml À1 in phosphate buffer solution (PBS, pH 7 and 0.15 M), analogous to that used by Brunet & Hoste (2006). Levamisole and PBS were used as positive and negative reference controls, respectively.
2.4.2. Statistical analysis. The different values were included in a two-criteria repeated measures analysis of variance model. The comparison of means for the different tests was done using the SNK procedure, which runs the Student-Newman-Keuls test in the R software. Differences were considered significant at the 5% level.

Refinement
Crystal data, data collection and structure refinement details for (2) and (3) are summarized in Table 1. For both structures, the hydroxy H atoms were located in a difference Fourier map and their positions were refined freely along with individual isotropic displacement parameters. The methyl H atoms were constrained to an ideal geometry (C-H = 0.98 Å ), with U iso (H) = 1.5U eq (C), but were allowed to rotate freely about the C-C bonds. All other H atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms, with C-H distances of 0.95 (aromatic), 0.99 (methylene) or 1.00 Å (methine) and with U iso (H) = 1.2U eq (C). The absolute configuration of (3) was determined confidently from the diffraction experiment by refinement of  Table 1 Experimental details.
For both structures: Z = 4. Experiments were carried out at 160 K with Cu K radiation. H atoms were treated by a mixture of independent and constrained refinement. The absorption correction was numerical based on Gaussian integration over a multifaceted crystal model (Coppens et al., 1965) plus empirical (using intensity measurements) using spherical harmonics (CrysAlis PRO; Rigaku Oxford Diffraction, 2021). (2) ( 3) the absolute structure parameter using the intensity quotients method (Parsons et al., 2013). For (2), one reflection was omitted from the final cycles of refinement because its observed intensity was much lower than the calculated value as a result of being partially obscured by the beam stop; a correction for secondary extinction was also applied.

The crystal structures of
The hydroxy group in each independent flavonoid molecule forms an intramolecular hydrogen bond with the adjacent carbonyl O atom (Table 2). In the crystal packing, the molecules form stacks, each of which consists of repeats of just one of the independent molecules. The molecules containing atom O1 lie tilted within an otherwise uniform column that runs parallel to the [100] direction. The molecular plane is tilted by approximately 45 with respect to the stacking direction. Nonetheless, there are no significantinteractions, because the ring offsets resulting from the tilting preclude significant overlap of the ring systems. The molecules containing atom O21 also stack parallel to the [100] direction in a similar 45tilted fashion, but the orientation of the tilted planes differs from that in the O1-containing stacks (Fig. 2); the normals to the molecular planes in the two independent stacks point in  Table 2 Hydrogen-bond geometry (Å , ) for (2).  Figure 1 Separate views of the two symmetry-independent molecules of (2), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level. H atoms are represented by spheres of arbitrary size. different directions. Each type of stack runs parallel to another stack of the same kind related by a centre of inversion to give a centrosymmetric double-stack pair. As the planes of the molecules in the two independent types of pairs of stacks are oriented differently,interactions between the stacks are precluded and the stacks are not intertwined with one another. The Cambridge Structural Database (CSD, Version 2020.3.0 with May 2021 update; Groom et al., 2016) contains data for six closely related flavones with hydroxy or methoxy substituents at least at the 5-, 6-, 7-, 3 0 -and 4 0 -positions. The ring systems in four of these structures are planar, with perhaps a tendency towards a slight bowing along the axis of the threering system, similar to that observed in (2), as seen solely from visual inspection. These structures are 5,7,4 0 -trihydroxy-6,3 0 ,5 0trimethoxyflavone ethyl acetate solvate (Martinez-Vazquez et al., 1993), 5,3 0 -dihydroxy-6,7,4 0 -trimethoxyflavone (Parvez et al., 2001), 5,7-dihydroxy-6,3 0 ,4 0 -trimethoxyflavone (Suleimenov et al., 2005) and 5,7,3 0 -trihydroxy-6,4 0 ,5 0 -trimethoxyflavone (Adizov et al., 2013;Turdybekov et al., 2014). In the structure of 5,6,7,2 0 ,3 0 ,4 0 -hexamethoxyflavone (Butler et al., 2018), the bowing within the fused rings appears to be more prominent. In the structure of 5,3 0 -dihydroxy-6,7,2 0 ,4 0 ,5 0pentamethoxyflavone (Al-Yahya et al., 1987), the individual planes of the phenyl and fused rings are significantly tilted from one another, with a dihedral angle of 12.23 (14) ; this is the only example with four substituents on the phenyl ring (three methoxy and one hydroxy).
The crystal structure of the triterpene (20S,24R)-ocotillone, (3), has one molecule in the asymmetric unit (Fig. 3). In the chosen crystal, the compound is enantiomerically pure and the absolute configuration of the molecule was determined independently by the diffraction experiment; the value of the absolute structure parameter (Parsons et al., 2013) was À0.07 (4). According to the numbering of the atoms used in the refinement model, the absolute configuration of the stereogenic C atoms of the molecule is established as follows: 5R,8R,9R,10R,13R,14R,17S,20S,24R. The isolation and identification of 20S-and 20R-ocotillones have been reported on several occasions (Bisset et al., 1966(Bisset et al., , 1967Betancor et al., 1983;Aalbersberg et al., 1991). The isolation of the corresponding alcohol, ocotillol, appears to be mentioned for the first time by Warnhoff & Halls (1965). The absolute configuration of (20S,24R)-ocotillone was deduced from an X-ray crystal structure of a bromobenzoyl derivative of the corresponding ocotillol (Yamauchi et al., 1969). The crystal structure determination of (3) is the first time the absolute configuration has been confirmed crystallographically for the native (20S,24R)-ocotillone.

Anthelmintic activity
3.3.1. About the extracts. The crude extracts obtained by aqueous decoction and hydro-ethanolic maceration, as well as the nine isolated compounds, were tested for their anthelmintic activity on the larvae and adult worms of H. contortus. The larval migration inhibition technique applied is based on the measurement of the migration rate of parasite larvae through a membrane after contact with the tested extract. At different doses, aqueous and hydro-ethanol extracts of C. glutinosum significantly inhibited in vitro larval migration of H. contortus (p < 0.001) (Fig. 5). This effect is independent of the dose and does not vary with the extraction solvent (p > 0.05). However, the aqueous extract appeared to be more effective than the hydro-ethanolic extract (Fig. 5). Similarly, both extracts significantly reduced the motility of adult H. contortus worms (p < 0.001). Although the inhibition effect did not vary with dose and extraction solvent (p > 0.05), it did vary with time (p < 0.001) and, paradoxically, the hydro-ethanolic extract appeared to inhibit adult worm motility more (Table 4). In order to know the chemical composition of these two extracts for the identification of the active principle, the present work was continued with the hydro-ethanolic extract and the compounds isolated therefrom were tested on H. contortus larvae and worms.
3.3.2. On the compounds. In vitro, the effect of the compounds was evaluated on H. contortus larvae and adult worms. All the compounds inhibited the migration of H. contortus larvae (Fig. 6) and the three isolated flavonoids seem to present the best results with inhibition percentages of 75.37, 53.26 and 47.73%, respectively, for compounds (1), (2) and (8), although they are all less active than the reference drug levamisol (95.97%). For the adult worms observed every 3 h with a magnifying glass after their contact with the tested compounds, the total inhibition of their motility was observed with the positive reference control (levamisol) after just 3 h of exposure. This inhibition was total at 12 h with compounds (1), (2), (4), (5) and (8). On the other hand, in phosphate buffer solution (PBS), 75% of adult worms were still mobile after 18 h (

Figure 4
The crystal packing of (3) adult worms within the same time as levamisole, compared with the negative control (p < 0.001). On adult worms, the inhibitory effect varied with time (p < 0.001) and flavonoids; in particular, 5-demethylsinensetin, (1), would be responsible for the known anthelmintic activity of the plant. Indeed, the class of polyphenols is strongly suspected as being the active agent in the anthelmintic effect of plants (Ayers et al., 2008). Condensed tannins are frequently reported as being responsible for such effects, for example, in the report by Hoste et al. (2018). Nonetheless, other reports do link anthelmintic properties to flavonoids (Paolini et al., 2003;Barrau et al., 2005). Given the results of the in vivo tests, the known anthelmintic activity of C. glutinosum appears to be related to the presence of the flavonoids isolated from this plant. Thus, following the report that C. glutinosum is an anthelmintic plant (Alowanou et al., 2019), the present study has allowed the anthelmintic capacity of the different compounds isolated from this plant to be ranked and highlighted. It appears that these compounds, although less active than the positive reference control, have a larvicidal and vermicidal effect on H. contortus, with 5-demethylsinensetin, (1), being the most active. The decrease in the migration of infesting larvae and the reduction of the motility of adult worms could disrupt their settlement in the mucosal wall of the digestive tract and thus ensure their progressive elimination from the infested animal (Dedehou et al., 2014). These results could serve as a basis for a conformational analysis leading to the proposal of a new compound with a broader spectrum of activity than current commercially available anthelmintics.

Conclusion
The phytochemical investigation of the leaves of C. glutinosum led to the isolation of nine known compounds, which were characterized using spectroscopic analyses and by comparison with literature data. The crystal structures of two compounds were described for the first time in the present work and four compounds have been isolated for the first time from the genus Combretum. The flavonoids isolated from the plant presented the best in vitro activity on H. contortus. The results of this study could be verified in vivo on sheep in order to gain further insight into and enhance the status of this plant. ants'. MPT thanks the YaBiNaPA project coordination team, in particular, Professor Bruno N. Lenta, Dr Billy T. Tchegnitegni and Dr Joseph Tchamgoue for their diverse contributions to the realization of this work. Dr Olivier Blacque of the Department of Chemistry, University of Zurich, is thanked for assistance with one diffraction data collection.  Table 5 The motility (%) of adult H. contortus worms in the presence of the isolated compounds (150 mg ml À1 ), as determined by an adult worm motility inhibition assay (AMIA). The effect on H. contortus larval migration caused by C. glutinosum extracts.