Two new flavone glycosides from the leaves of Ochna afzelii Oliv. (Ochnaceae)

Abstract Two new glycosylflavones, 6′′-O-acetyl-8-C-β-D-galactopyranosylchrysoeriol (1) and 8-C-β-D-galactopyranosylchrysoeriol (2) were isolated from the methanol extract of the leaves of Ochna afzelii Oliv., along with four known compounds namely 8-C-β-D-galactopyranosylapigenin (3), ochnaflavone (4), sitosterol-3-O-β-D-glucopyranoside (5) and D-mannitol (6). Isolation was performed chromatographically and the structures of the purified compounds were elucidated by analyzing their spectroscopic and mass spectrometric data. The antibacterial activity of extract, fractions, and compounds 1 − 4 was evaluated using broth microdilution method against Gram-positive and Gram-negative bacteria while the antioxidant capacity was performed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the ferric reducing antioxidant power (FRAP) methods. The new flavones (1 and 2) displayed moderate antibacterial activities (MIC = 32 − 64 µg/mL) and weak antioxidant properties. Graphical Abstract


Introduction
The genus Ochna (Ochnaceae) comprises 85 species distributed in tropical Asia, Africa and America (Bandi et al. 2012).Ochna afzelii Oliv. is a small tree, found in groups in forest areas.Its most important medicinal uses include the treatment of jaundice, toothache, female sterility, menstrual complaints, lumbago and dysentery (Pegnyemb et al. 2001(Pegnyemb et al. , 2003a(Pegnyemb et al. , 2003b)).Many secondary metabolites belonging mainly to flavonoids (bi-, tri-, tetra-, and pentaflavonoids), anthranoids, triterpenoids, steroids, fatty acids, and a few others have been reported from this genus (Pegnyemb et al. 2001;Bandi et al. 2012).Some of the reported compounds as well as their initial crude extracts exhibited an array of interesting biological activities, including analgesic, anti-inflammatory, anti-HIV-1, antimalarial, antimicrobial, and cytotoxic properties (Pegnyemb et al. 2003a;Bandi et al. 2012).In the course of our search for new bioactive compounds from Cameroonian medicinal plants (Mpetga et al. 2021;Nago et al. 2021;Nguetsa et al. 2021), we herein report the isolation, structure elucidation as well as the antibacterial and antioxidant activities of two new galactopyranosyl flavonoids from the leaves of O. afzelii.

Isolation and structure elucidation
The methanol extract from the leaves of O. afzelii was repeatedly fractionated using column chromatography on silica gel and Sephadex LH-20 to afford six compounds (Figure 1) including two new C-galactopyranosylflavones (1 and 2).Their structures were determined based on their spectroscopic and spectrometric data.
Compound 1 was obtained as a yellow powder from EtOAc.Its molecular formula C 24 H 24 O 12 was deduced from the positive-ion mode HRESI-MS, which revealed the pseudomolecular ion peak [M þ H] þ at m/z 505.1343 (calcd.505.1341 for C 24 H 25 O 12 þ ).
The IR spectrum exhibited vibrational bands for hydroxyl (3393 cm À1 ), conjugated carbonyl (1652 cm À1 ) and aromatic C ¼ C (1508 cm À1 ) functionalities.The 1 H NMR spectrum of 1 (Table SM1) showed a signal exchangeable with D 2 O at d H 13.35, attributed to the chelated hydroxyl group of flavonoids at C-5 position (Bitchagno et al. 2015(Bitchagno et al. , 2016)).Three aromatic protons were also evidenced by signals consisting of a double doublet at d H 8.26 (1H, J ¼ 8.5 and 2.2 Hz, H-6 0 ) and two doublets at d H 7.56 (1H, J ¼ 2.2 Hz, H-2 0 ) and 6.89 (1H, J ¼ 8.4 Hz, H-5 0 ) respectively, suggesting the presence of a 1,3,4-trisubstituted aromatic ring in 1 (Anuradha et al. 2006).Four signals were also observed as singlets at d H 6.99 (1H), 6.28 (1H), 3.89 (3H) and 1.98 (3H) and were respectively assigned to the olefinic H-3 proton of flavones (Bitchagno et al. 2015(Bitchagno et al. , 2016)), an aromatic proton, a methoxy group and an acetoxy methyl group.This spectrum revealed the glycosylated nature of the flavone by also exhibiting a set of resonances attributed to a sugar unit between d H 4.68 and 3.45.The large coupling constant of the anomeric proton at d H 4.68 (1H, d, J ¼ 7.8 Hz, H-1 00 ) suggested a b-configuration for the glycopyranosyl unit.The 13 C NMR and DEPT-135 spectra of 1 (Table SM1) resolved 24 carbon signals consisting of two carbonyl, twelve aromatic, two olefinic, six oxygenated aliphatic, one methoxy and one methyl carbons.The carbonyl  (1652 cm À1 ) and aromatic double bond (1506 cm À1 ) functionalities.The NMR data of 2 (Table SM1) also showed characteristic signals of a C-galactopyranosyl flavone and were similar to those of 1, except the upfield shift of signals at C-6 00 position (d H 3.91 and 3.59) and the lack of resonances attributable to an acetyl group in 2. Compound 2 thus appeared to be the deacetylated derivative of 1. Consequently, the structure of 2 was established as 8-C-b-D-galactopyranosylchrysoeriol.Likhitwitayawuid et al. (2005) highlighted that C-glycosylated flavones isolated from Ochna species are either C-6 or C-8-glucosides, structurally related to apigenin or luteolin.Compounds 1 and 2 described herein are 8-C-glycosyl derivatives of chrysoeriol, the 3 0 -O-methylated derivative of luteolin (Miranda et al. 2014).Meanwhile, as far as the nature of the glycosylation is concerned, the present study reports for the first time, C-galactopyranosyl flavones from the genus Ochna.The known compounds were identified as 8-C-b-D-galactopyranosylapigenin (3) (Chari et al. 1980), ochnaflavone (4) (Zintchem et al. 2007;Bahia et al. 2010), sitosterol-3-O-b-D-glucopyranoside (5) (Nguetsa et al. 2021) and D-mannitol (6) (Koagne et al. 2017) by comparison of their spectroscopic data with those reported in the literature.
Supporting spectra for compounds 1 and 2 are embedded as figures (Figures SM.2-SM.19,Supplementary material)

Antibacterial activity
The MeOH extract, derived fractions, as well as compounds 1 2 4 from O. afzelii showed selective effects on some human pathogenic bacteria (Table 1).According to the antimicrobial activity scale defined by Tamokou et al. (2017), the MeOH extract was highly active (MIC < 100 mg/mL) against E. coli, S. typhi and P. aeruginosa, and significantly active (100 MIC 512 mg/mL) against E. faecalis.Fractions A and B were highly active against all the tested bacteria.Fraction C was found to be highly active against S. typhi and significantly active against E. coli, E. faecalis and P. aeruginosa.Fraction D was highly active against E. coli and P. aeruginosa and significantly active against S. typhi and E. faecalis, whereas fraction E was significantly active against all the tested bacteria.The findings of the present investigation suggested that the fractionation improved the antibacterial activities of fractions A and B. Among the botanicals, the lowest MIC value of 16 lg/mL corresponding to the largest antibacterial activity, as well as the lowest MBC value of 32 lg/mL corresponding to the highest bactericidal effect were both recorded for fraction B against P. aeruginosa.Compounds 1 2 4 displayed moderate activities (10 < MIC 100 mg/mL) (Tamokou et al. 2017) against all the tested bacteria, with compounds 2 and 4 being the most active (MIC value of 32 mg/mL) respectively against 4/4 and 3/4 of the used microorganisms.The results also showed that the MBC values of extracts and compounds were at most four-fold higher than their MICs on the sensitive microorganisms, revealing the bactericidal effects of the tested samples.This is the first report demonstrating the antimicrobial property of extracts and compounds from O. afzelii and the results obtained from this study corroborate those from other Ochna species (Bandi et al. 2012).

Antioxidant capacity
The antioxidant capacity of the methanol extract and compounds 1, 2 and 4 was determined by two complementary methods, namely the 2,2-diphenyl-1-picrylhydrazyl (DPPH) in vitro assay for radical scavenging and the ferric reducing antioxidant power (FRAP) in vitro assay for assessing reducing power (Messi et al. 2016).The methanol extract showed a significant radical scavenging activity (EC 50 ¼ 4.62 ± 0.53 mg/mL) compared with that of vitamin C (EC 50 ¼ 2.29 ± 0.19 mg/mL) used as the reference antioxidant.However, the isolated compounds exhibited weak antioxidant capacity (EC 50 > 850 mg/mL) against DPPH radicals, presumably due to the absence in their chemical structures of the 3 0 ,4 0 -ortho-dihydroxy group which also plays an important role in the antioxidant capacity of flavonoids by favoring a high delocalization effect of electrons through intramolecular hydrogen bonds (Lin et al. 2014).The methanol extract and compounds 1, 2 and 4 were further assessed for their ferric reducing antioxidant power and the results supported the radical scavenging activity.The methanol extract exhibited a reducing power of 85.50 ± 0.16 mmol/g and was therefore the most potent compared to compounds 1 (82.10 ± 0.33 mmol/g), 2 (67.15 ± 0.65 mmol/g) and 4 (62.70 ± 0.28 mmol/g), respectively.

General experimental procedures
IR spectra were recorded using JASCO Fourier transform IR-420 spectrometer.Optical rotation was measured on a Perkin Elmer polarimeter model 241 using a 10 cm cell and a wavelength of 589 nm (Sodium lamp).The NMR spectra were recorded on a Bruker DRX-500 MHz (Bruker, Rheinstetten, Germany) spectrometer.Chemical shifts (d) were reported in parts per million (ppm) using tetramethylsilane (TMS) (Sigma-Aldrich, Munich, Germany) as internal standard, while coupling constants (J) were measured in Hz.ESI-MS spectra were recorded on Agilent 6220 TOF LCMS mass spectrometer with perfluorokerosene as reference substance for ESI-HR-MS.Column chromatography was performed on silica gel Merck 60 F 254 (0.200-0.500 mm and 0.063-0.200mm, resp.)(Darmstadt, Germany).Pre-coated silica gel 60 F 254 thin layer chromatography (TLC) plates (0.25 mm thick) (Merck, Darmstadt, Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm) then sprayed with 20% sulfuric acid (H 2 SO 4 ) followed by heating to 100 C. Solvents were distilled prior to use.The absorbance values and optical densities in the antioxidant assays were read on a spectrophotometer (FLUOstar Omega Microplate Reader).

Plant material
The leaves of Ochna afzelli were collected in Dschang, West Region of Cameroon in June 2018.The identification was done at the Cameroon National Herbarium (Yaound e) by Mr. Fulbert Tadjouteu, a botanist, by comparison with a voucher specimen kept under the number 27064/SRP/CAM.

Antibacterial assay
INT colorimetric assay was used to determine the minimal inhibitory concentrations (MICs) of extracts/compounds against Gram-positive (Enterococcus faecalis ATCC 29212) and Gram-negative (Escherichia coli ATCC 8739, Salmonella typhi ATCC 6539 and Pseudomonas aeruginosa ATCC 76110) bacteria as previously described (Yanda et al. 2022).The tested bacteria were taken from the Research Unit of Microbiology and Antimicrobial Substances, Department of Biochemistry, University of Dschang, Cameroon.They were grown at 37 C and maintained on nutrient agar (NA, Conda, Madrid, Spain).The MIC values of extract/compounds were determined by adding into each well 40 lL of a 0.2 mg/mL p-iodonitrotetrazolium violet solution followed by incubation at 35 C for 30 min.Viable bacteria reduced the colourless dye to pink.MIC represented the lowest concentration that prevented this change and displayed full inhibition of bacterial growth.The lowest concentrations of the tested samples which gave no growth of bacteria after sub-cultivation on Mueller Hinton Agar plates were taken as the minimum bactericidal concentration (MBC) values.Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) was tested as reference antibacterial.All assays were made in independent replicates.
3.4.2.Antioxidant assays 3.4.2.1.DPPH scavenging assay.The antioxidant ability of the methanol extract and some isolated compounds was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay as early described by Mensor et al. (2001).The optical densities were read on a spectrophotometer (FLUOstar Omega Microplate Reader) at 517 nm and converted to percentages of antioxidant capacity.Vitamin C (L-ascorbic acid) was used as the positive control.For each sample, three replicates were performed.The percentages of antioxidant capacity of each sample were calculated according to the following formula:each sample were calculated according to the following formula: 3.4.2.2.Ferric reducing antioxidant power (FRAP).The reducing power of the methanol extract and some isolated compounds was determined as previously described (Benzie and Strain 1996).The optical density was read on a spectrophotometer (FLUORstar Omega microplate reader) at 593 nm.Vitamin C was used as the positive control.The antioxidant power of samples was calculated from the calibration curve of the FeSO 4 solution (the number of moles of the FeSO 4 solution varying from 156.25 mmol to 10 000 mmol) and expressed in mmol FeSO 4 equivalents per gram of sample.

Conclusion
Six compounds among which four flavonoids including two new 8-C-galactopyranosyl flavones were isolated from the leaves of O. afzelii.These results are chemotaxonomically relevant as the isolated compounds include ochnaflavone, the characteristic biflavonoid of the Ochna genus.Moreover, this is the first report of C-galactopyranosyl flavones from this plant genus.The antibacterial and antioxidant activities of O. afzelii are also reported for the first time from this study.The significant activities of the extract suggest that this plant species could be a source of molecules with interesting antioxidant and antibacterial properties that could lead to the development of new drugs.It might therefore be useful to carry out a further, more detailed bio-guided reinvestigation of the subject botanical.
signal at d C 182.7 (C-4) and the olefinic carbon resonances at d C 103.0 (C-3) and 164.4 (C-2) were in good agreement with a flavone skeleton(Pinzon et al. 2011); while the signals at d C 74.3 (C-1 00 ), 68.3 (C-2 00 ), 75.6 (C-3 00 ), 69.9 (C-4 00 ), 77.3 (C-5 00 ) and 65.5 (C-6 00 ) supported the presence of a glycopyranosyl moiety in the molecule and were compatible with those of a galactosyl unit(Beier et al. 1980;Chari et al. 1980;Agrawal 1992;Stark and Hofmann 2006).The position of the methoxyl group (d C 56.6) on the flavone skeleton at C-3 0 was proved by a complete sequence of HMBC and NOE interactions (Figure SM.1, Supplementary material).Indeed, HMBC cross peaks were observed from H-2 0 (d H 7.56), H-5 0 (d H 6.89) and 3 0 -OCH 3 (d H 3.89) to C-3 0 (d C 148.3), while ROESY interactions arose between H-3 (d H 6.99) and H-2 0 (d H 7.56), and between H-2 0 and 3 0 -OCH 3 (d H 3.89).The 13 C NMR data of the aglycone moiety in 1 were superimposable to those of chrysoeriol(Bashyal et al. 2019), except the downfield shift by 10.3 ppm of C-8 resonance (d C 104.8) in 1 compared to C-8 (d C 94.5) in chrysoeriol (Bashyal et al. 2019).Additionally, the appearance in the NMR spectra of compound 1 of H-6 signal (d H 6.28) as a singlet instead of a meta-coupled doublet as in chrysoeriol, and the resonance of the anomeric carbon in 1 at d C 74.3 suggested the C-glycosylation of the flavone skeleton at C-8 position.This was further confirmed by the HMBC interactions from H-6 (d H 6.28) and the anomeric proton (d H 4.68) to C-8 (d C 104.8).Further longrange correlations from the oxymethylene protons of the galactosyl unit (d H 4.23 and 4.08) and the methyl protons at d H 1.98 to the carbonyl at d C 170.9 supported the location of the acetoxy group at C-6 00 .Based on all the above evidences, compound 1 was elucidated as 6 00 -O-acetyl-8-C-b-D-galactopyranosylchrysoeriol.Compound 2 was obtained as a yellow powder from EtOAc.Its molecular formula C 22 H 22 O 11 was deduced from the positive-ion mode ESI-MS which revealed a sodium adduct ion peak [M þ Na] þ at m/z 485.1056 (calcd.485.1054 for C 22 H 22 O 11 Na þ ).The IR spectrum exhibited vibrational bands for hydroxyl (3383 cm À1 ), conjugated carbonyl

Figure 1 .
Figure 1.Chemical structures of isolated compounds from the leaves of O. afzelii.

%
of antioxidant capacity ¼ ½absorbance of DPPHÀ absorbance of the test À absorbance of blank ð Þ absorbance of DPPH x 100 where, test ¼ sample þ methanolic solution of DPPH and blank ¼ sample þ methanol.The different percentages of antioxidant capacity were used for the determination of the EC 50 (the concentration of the sample that can trap 50% of DPPH) (Yassa et al. 2008).To achieve this, the regression lines were drawn using the values of the different percentages of antioxidant capacity and the decimal logarithm of the concentrations of the samples [% of antioxidant capacity ¼ f (log C)].The equation of the regression lines in the form of y ¼ ax þ b was used.Assuming each time y ¼ 50, we got EC 50 ¼ 10 x , where x ¼ (50b)/a.

Table 1 .
Antibacterial activity (MIC and MBC in mg/mL) of extracts and compounds as well as the reference antibiotic drug.