Atricephenols A and B, two phenolic compounds from Indigofera atriceps Hook.f. (Fabaceae)

Abstract The phytochemical investigation of a previously unstudied species of the genus Indigofera, I. atriceps Hook.f. was undertaken and two new phenolic compounds, atricephenols A (1) and B (2) were isolated, along with nine known secondary metabolites viz., (-)-melilotocarpan D (3), genistein (4), melilotocarpan A (5), maackiain (6), p-hydroxybenzaldehyde (7), bornesitol (8), β-sitosterol (9), sitosterol-3-O-β-D-glucopyranoside (10) and stigmasterol-3-O-β-D-glucopyranoside (11). Their structures were elucidated by extensive NMR spectroscopic analyses and HRESIMS, and by comparing their data with those reported in the literature. Compounds 1, 4, 7–11 were tested for their antibacterial efficacies and for their potential to inhibit the enzyme urease. Compounds 7 and 9 showed significant antibacterial activity against Salmonella typhi (ZOIs of 13 and 15 mm, respectively), while the best urease inhibition was measured for compound 9 with an IC50 value of 18.6 µM, which is higher than that of the potent inhibitor, thiourea (IC50 = 21.5 µM). Graphical Abstract


Introduction
Infectious diseases caused by the development of bacteria or yeasts in humans or animals, constitute the major causes of mortality and morbidity in humanity (WHO 2019a). They kill ca. 17 million people every year around the world and are now classified as the third cause of mortality due to the resistance of microbial strains to existing antimicrobial agents (Balkis et al. 2002;WHO 2019aWHO , 2019b. On the other hand, urease enzymes that hydrolyse urea into ammonia and carbon dioxide, have a considerable impact on bacteria, as they are potential causes and/or factors contributing to the persistence of some pathogen infections (Rego et al. 2018). Thus, compounds with antimicrobial and/or urease inhibitory properties are very advantageous for their dual interventions in the fight against infectious diseases. Although, plants of the genus Indigofera are well known to produce indigo dyes (Wu et al. 1999), species such I. gerardiana and I. atriceps are used in traditional medicine for the treatment of generic body pains and microbial infections (Nisar et al. 2009;Soladoye et al. 2010). In fact, Indigofera atriceps Hook.f. (Fabaceae) is a blossoming and herbaceous plant with legume type fruits and is distributed throughout the tropical and subtropical zones of Africa, Asia and America (Rahman et al. 2018). Previous biological studies of crude extracts and metabolites from various species have shown this genus to exhibit diverse properties such as anti-inflammatory, antiulcergenic, teratogenic, cytotoxic, insecticidal, phytotoxic, antimicrobial, urease inhibitory and hepatotoxic Pound 1968, 1970;Dahot 1999;Shahjahan et al. 2005;Lopes et al. 2011;Uddin et al. 2011aUddin et al. , 2011bMotamarri et al. 2012;Rahman et al. 2017;Halim et al. 2018;Mouafon et al. 2021). Chemical investigations of these species have led to the isolation of secondary metabolites belonging to different classes including benzofurans, flavonoids, lignins, triterpenoids, steroids, alkaloids, fatty acids containing amino groups, saponins, quinines and tannins (Rahman et al. 2017;Halim et al. 2018;Mouafon et al. 2021). As part of our continuing search for bioactive metabolites from Cameroonian medicinal plants (Mountessou et al. 2018;Kemayou et al. 2021;Mouafon et al. 2021), the chemical investigation of I. atriceps was undertaken. Two phenolic compounds named atricephenols A (1) and B (2) together with nine known secondary metabolites (3-11), as well as their antibacterial and enzyme inhibitory activities are herein reported.

Results and discussion
The CH 2 Cl 2 /MeOH extract of the whole plant of I. atriceps was subjected to repeated silica gel column chromatography and the resulting fractions were separately submitted to Sephadex LH-20 column chromatography, and preparative TLC to afford atricephenols A (1) and B (2) (Figure 1), and nine known secondary metabolites (3-11) (see Figure S1). Atricephenol A (1) was obtained as a white amorphous powder. It gave a purple colouration upon addition of aqueous ferric chloride, characteristic of phenolic compounds. Its molecular formula C 16 H 12 O 7 was deduced from the negative ion mode HR-ESI-MS, which showed a pseudo molecular ion peak [M⎯H] ⎯ at m/z 315.0506 (calcd. for C 16 H 11 O 7 ⎯ , 315.0510), corresponding to eleven degrees of unsaturation. The IR spectrum showed absorption bands for an unsaturated aldehyde and an ester carbonyl groups (1661 and 1725 cm À1 , respectively). The UV spectrum displayed absorption bands at k max 297, 262 and 230 nm, suggesting the presence of a benzoyl system (Harborne et al. 1975). The 13 C NMR (BB and DEPT) spectra displayed sixteen signals including those for one methoxyl, one methylene dioxy and six methine groups, as well as those for eight quaternary carbons. The 1 H NMR spectrum showed a downfield resonance for an aldehydic proton at d H 9.99 and a typical ABX spin system at d H 7.96 (d, J ¼ 8.0 Hz, H-6), 6.54 (d, J ¼ 1. 6 Hz, H-3) and 6.48 (dd, J ¼ 8.0, 1.6 Hz, H-5), assigned to a 1,2,4-trisubstituted benzene ring, which was attached to an ester carbonyl (d C 165.2) as inferred from the strong HMBC cross peak ( Figure S10) observed between this carbonyl signal and the proton signal at d H 7.96. Moreover, HMBC cross peaks were observed between the later signal (d H 7.96, H-6) and two carbon signals at d C 164.3 (C-2) and 165.1 (C-4), as well as between the proton signal at d H 6.54 (H-3) and the carbon signals at d C 164.3 (C-2) and 108.1 (C-1). Furthermore, the 1 H NMR spectrum displayed two singlets of aromatic protons at d H 7.27 and 6.83, indicating the presence of another benzene ring. The later was linked to the benzene ring based on 2 J and 3 J correlations observed on the HMBC spectrum ( Figure S10). In fact, the proton signal at d H 9.99 showed cross peaks to the carbon signals at d C 124.1 (C-2 0 ) and 107.1 (C-3 0 ). In addition, the presence of a methylenedioxy moiety could be deduced from the signal of a downfield singlet integrating for two protons at d H 6.12 (H-2 00 ) in the 1 H NMR spectrum, and a carbon signal at d C 104.1 (C-2 00 ) in the 13 C NMR spectrum. In the HMBC spectrum, cross peaks were observed between the signal at d H 6.12 and the carbon signals at d C 154.8 and d C 147.5, which suggested the methylenedioxy group to be attached to the two adjacent carbons C-4 0 and C-5 0 of the second benzene ring. The three-proton singlet signal at d H 3.86 in the 1 H NMR spectrum was assigned to a methoxyl group, which was located at C-2 based on the cross peak observed in the HMBC spectrum between the carbon signal at d C 164.3 (C-2) and the proton signal at d H 3.86. The allocated position of the methoxyl group was further confirmed by the observed correlation between the proton signals at d H 6.54 (H-3) and 3.86 (-OCH 3 ) in the NOESY spectrum ( Figure S11). Based on the molecular weight of compound 1, a hydroxyl group was attached to carbon C-4 (d C 165.1). All these data agreed with the assigned structure for atricephenol A (1). The proposed structure was further confirmed by its mass spectrum where characteristic peaks were observed at m/z 270 and 165 ( Figure S24). Atricephenol B (2) was also obtained as a white amorphous powder, which gave a purple colouration to the ferric chloride test indicating its phenolic nature. Its molecular formula C 17 H 16  corresponding to ten double bond equivalents. The IR spectrum showed absorption bands for a hydroxyl group (3366 cm À1 ) and conjugated carbonyl groups (1739 and 1696 cm À1 ). The UV spectrum displayed two minor absorption bands at k max 273 and 230 nm, suggesting the presence of a benzoyl system (Harborne et al. 1975). Although the LC-MS chromatogram displayed a single peak, indicating a pure compound, both the 1 H and 13 C NMR spectra exhibited doubled sets of signals in an intensity ratio of ca 1:3, suggesting the presence of two stereoisomers 2a and 2 b (Hu et al. 2012;Niesen et al. 2013). The 13 C NMR (BB and DEPT) spectra displayed 17 carbon signals including those for two methoxy methyls, one methylenedioxyl, six methines and eight quaternary carbons. In the 1 H NMR spectrum ( Figure S15), the major isomer 2a displayed an ABX system at d H 7.88 (d, J ¼ 8.4 Hz, H-6), 6.46 (dd, J ¼ 1. 8, 8.4 Hz, H-5) and 6.38 (d, J ¼ 1.8, H-3) assignable to a 1,2,4-trisubstituted benzene ring attached to a carbonyl group as deduced from its HMBC spectrum. In fact, correlations ( Figure S21) between the proton signal at d H 7.88 (H-6) and the carbon signals at d C 198.2 (C-7 carbonyl group), 166.8 (C-4) and 163.0 (C-2) were observed. Moreover, the 1 H and 13 C NMR spectra exhibited additional signals including those of two methoxyl groups at d H/C 3.70/55.7 and 2.92/51.8 and an oxymethine group at d H/C 5.96/73.2. In the HMBC spectrum, cross peaks were observed between the carbon signal at d C 163.0 (C-2) and the proton signal at d H 3.70 (methoxy group), which in turn showed a strong cross peak with the proton signal at d H 6.38 (H-3) in the NOESY spectrum indicating the proximity of this proton to the C-2 methoxyl group. Likewise, an important cross peak was observed between the oxymethine proton signal (d H 5.96, C-8) and two characteristic signals at d C 198.2 (C-7 carbonyl signal) and d C 116.8 (C-1) in the HMBC spectrum. Thus, the oxymethine group was attached and adjacent to the carbonyl group. Further investigation of the 1 H/ 13 C NMR spectrum revealed a set of signals at 186.9, 170.7, 143.7, 131.0, 100.5, 98.8 and 98.5 typical for those of a 4-methoxy-4,5-methylenedioxycyclohexa-2,5-dienone substructure (Buchi et al. 1978), further sustained by observed HMBC correlations between the methylenedioxy protons signal at d H 5.56 and the carbon signals at d C 170.7 (C-4 0 ) and 98.8 (C-5 0 ). The aforementioned substructure was attached to the oxymethine group (d H 5.96) at C-1ꞌ based on observed HMBC cross peaks between the proton signal at d H 5.96 and the carbon signals at d C 186.9 (C-2 0 ), 143.7 (C-1 0 ) and 131.0 (C-6 0 ). Further cross peaks were observed in the HMBC spectrum between the olefinic proton signal at d H 6.63 (H-6 0 ) and the carbon signals at d C 73.2 (oxymethine group) and 186.9 (C-2ꞌ, carbonyl group), corroborating these findings. The absolute configurations (R/S) of the two stereogenic centres at C-8 and C-5 0 could not be determined at this stage. Based on this evidence, the structure of atricephenol B was deduced as presented in Figure 1.
Compounds 1, 4 and 7 À 11 were assessed for their antibacterial properties and as inhibitors of the urease enzyme. The antibacterial activity was determined using the agar disk diffusion method with streptomycin as reference. The results, in terms of diameter of inhibition zone (ZOI) are presented in Table S1. p-Hydroxybenzaldehyde (7) and b-sitosterol (9) showed significant activity against S. typhi with ZOIs of 13 and 15 mm, respectively as compared to streptomycin (ZOI ¼ 23 mm). The obtained results are in line with previous findings on the antibacterial properties of these compounds (Friedman et al. 2003;Ododo et al. 2016). Atricephenol A (1) exhibited moderate antibacterial activity against Micrococcus sp. and S. typhi with ZOIs of 11 and 9 mm, respectively. According to the literature, the observed activity might be due to the methylenedioxy moiety in 1 (Sundararajan et al. 2014). Genistein (4), bornesitol (8), b-sitosterol-3-O-b-D-glucopyranoside (10), and stigmasterol-3-O-b-D-glucopyranoside (11) showed moderate activity with ZOIs ranging from 7 to 10 mm.
Selected isolated compounds (1, 4, 7 and 9 À 11) were also evaluated for their inhibitory activity against the urease enzyme and their IC 50 values are also summarised in Table S1. The highest urease inhibitory activity was recorded for b-sitosterol (9) (IC 50 ¼ 18.6 mM), which is higher than that of the standard, thiourea (IC 50 ¼ 21.5 mM). Compounds 4, 7, 10 and 11 showed significant activity with IC 50 values of 28.9, 39.6, 36.3 and 38.6 mM, respectively.

General experimental procedures
All details are provided in the supplementary material.

Plant material
The whole plant of I. atriceps (Fabaceae) was collected in March 2018 from the district of Koutaba, Western Region of Cameroon. It was identified by Mr. Victor Nana, a botanist at the National Herbarium of Cameroon, Yaounde, where a voucher specimen was deposited under the number 41126/HNC.

Extraction and isolation
All details are provided in the supplementary material.

Antibacterial and urease inhibitor assays
The detailed description is provided in the supplementary material.

Conclusion
The present study reports the isolation of two new phenolic compounds, viz. atricephenols A and B, and nine known secondary metabolites (3-11) from I. atriceps. The antibacterial efficacy and urease inhibitory potential of 1, 4, 7-11 were evaluated. Compounds 7 and 9 showed significant antibacterial activity against Salmonella typhi and the best urease inhibition was measured for compound 9. The observed activities substantiate the ethnomedicinal use of this plant and compounds 7 and 9 might be among those responsible for its therapeutic properties. The above results indicate that I. atriceps shares similar chemical characteristics with other species of Indigofera, with the presence of phenolic compounds (1-7), carbohydrate (8) and steroids (9-11). Moreover, all these compounds were reported for the first time from this plant and could serve as potential chemotaxonomic markers of this species which is mostly found in arid zones. The presence of phenolic compounds in this species is not surprising as it was collected in the District of Koutaba where it is used for crop shading.