Antibacterial constituents of Rumex nepalensis spreng and its emodin derivatives

Abstract The CH2Cl2−MeOH (1:1) extract of roots of Rumex nepalensis (Polygonaceae) displayed significant antibacterial activity against five bacterial strains with MICs (62.5–31.2 μg.mL−1). The EtOAc soluble fraction displayed a significant activity against the same strains with MICs (31.2–3.9 μg.mL−1). The purification of the EtOAc fraction yielded one new phenylisobenzofuranone derivative, berquaertiide (1), along with 19 known compounds (2–20). Their structures were elucidated based on the analysis of their NMR and MS data. All the isolated compounds were assessed for their antibacterial activity. Compound 2 was the most active against all the tested strains (15.7 to 1.9 μg.mL−1), while compounds 3–7 displayed good activities on at least one of the tested strains. In addition, seven analogues (21–27) of compound 2 were prepared and further assessed for their antibacterial activity. Compounds 26 and 27 were most active than 2 against Salmonella enterica and Klebsiella pneumoniae with MIC (125 and 15.6 μg.mL−1, respectively). Graphical Abstract


Introduction
Bacterial infections kill over 7 million people annually, and it may kill up to 10 million people by the year 2050 if appropriate measures are not taken (Yadav and Kapley 2021).The progress of bacterial-resistance to available antibiotics is alarming and makes the treatment of even simple bacterial infections difficult (Aslam et al. 2018).This concern requires a continuous search for new and efficient lead antibacterial agents.In this context, plants can play a key role in the discovery of new lead drugs since in most African countries, people have been relying mainly on medicinal plants to treat themselves from bacterial diseases and other infections .Plants have been reported as an important source of bioactive molecules (Stafford 2002).The genus Rumex is the largest one of the family Polygonaceae with more than 250 species distributed worldwide (Rao et al. 2011).Plants of the genus Rumex are commonly used in the treatment of skin infections (Getie et al. 2003), inflammation of the gastrointestinal tract, and against digestive disorders (Vazquez et al. 1997).It is also used as laxative or as antidiarrheal agents (Loi et al. 2004), and as a treatment for upper respiratory tract diseases (nasal sinuses and throat) (S€ uleyman et al. 1999).Previous chemical investigations of plants of the Rumex genus led to the isolation of anthraquinones (Ntemafack et al. 2020), flavonoids (Desta et al. 2015), triterpenoids (Jang et al. 2005), stilbenoids (Kerem et al. 2003), tannins (Bicker et al. 2009), steroids, and saponins (Shahnaz et al. 2017).Among these secondary metabolites, some exhibited antibacterial properties such as polyphenols (Vaquero et al. 2007) and tannins (Amarowicz et al. 2008).Also known as Rumex berquaertii De Wild, the previous chemical studies of R. nepalensis Spreng led to the isolation of a wide range of secondary metabolites including anthraquinones, naphthalenes, stilbenoids, flavonoids, tannins, and terpenoids (Gonfa et al. 2021).In our continuing search for new bioactive agents from Cameroonian medicinal plants, we report herein the bioguided isolation of secondary metabolites from the CH 2 Cl 2 -MeOH (1:1) root extract of R. nepalensis, their activities against seven strains including Salmonella typhi CPC, Staphylococcus aureus ATCC43300, S. aureus ATCC25923, Pseudomona aeruginosa HM801, and Klebsiella Pneumoniae (clinical isolate).In addition, some structural modifications were performed on the most active isolated compound 2 and the prepared derivatives assessed for their antibacterial activities.
All these isolated compounds were assessed in vitro for their antibacterial activity on the above mentioned strains and compounds 2-7 exhibited strong to moderate antibacterial activities ranging from 1.9 to 62.5 lg.mLÀ1 against S. typhi, S. aureus ATCC25923, K. Pneumoniae NR41388 and P. aeruginosa HM801 (Table S2).Emodin (2) showed the best antibacterial activity, particularly against S. typhi CPC, P. aeruginosa HM801 and K. Pneumoniae Clinical isolate with MICs value of 1.9 lg.mL À1 , while compounds 3-7 displayed good activities on at least one of the tested strains.The result obtained with the anthraquinone, emodin (2), corroborate with those reported by Chukwujekwu and collaborators (2006) against S. aureus with an MIC value of 3.9 lg.mL À1 .1,8-dihydroxyanthraquinones are known to possess good antibacterial activity.Their antibacterial activity is due to their interaction with the cell wall and cell membrane by which it increases the permeability of the cell envelope and leads to the leakage of cytoplasm and the deconstruction of cell (Wei et al. 2015).
While comparing the activity of the isolates, the crude extract and the EtOAc fraction, it is evident that apart from S. enterica strain, the partition led to an increase in the activity of the EtOAc against six strains of bacteria and a decrease in the activity of n-hexane and n-BuOH soluble fractions.The increase or the decrease of activity may be due to the synergistic and antagonistic effects of the constituents of the fractions, respectively.
Apart from compound 2 that was more active than all the extracts and fractions, compounds 3-7 were more active than the EtOAc soluble fraction on at least one of the assayed bacterial strains.In addition, the good activity of the crude extract, the EtOAc soluble fraction and that of compounds 1-7 against S. aureus, K. Pneumoniae, S. typhi and Salmonella enterica may partially justify the use of this plant in traditional medicine in the treatment of typhoid fever, skin diseases and respiratory tract infections.
Owing to the antibacterial potential of emodin ( 2) and the fact that the methylation of its hydroxy groups led to inactive analogue towards methicillin-resistant S. aureus at 200 mg/mL (Chalothorn et al. 2019), some structural modifications were carried out to evaluate the effect of allylation and acetylation on its antibacterial potency.The first series of analogues were synthesized by using Williamson ether synthesis method for allylation and acetylation (Scheme 1).In fact, the allylation was achieved by treatment with allyl bromide in the presence of K 2 CO 3 to yield monoallylated emodin [6-allyloxyemodin ( 21)], diallylated emodin [6,8-diallyloxyemodin ( 22) and 1,6-diallyloxyemodin ( 23)] and fully allylated emodin, 1,6,8-triallyloxyemodin (24).The acetylation was done with acetic anhydride in the presence of 4-(dimethylamino) pyridine to give 1,6,8-triacetylemodin (25) (Scheme 1).Analogues 21-25 were assessed in vitro for their antibacterial activity against the aforementioned bacteria strains and all the derivatives were inactive (Table S3).These results confirm the hydroxy groups are important for the antibacterial activity of emodin, and this is in accordance with previous findings (Chalothorn et al. 2019).Thus, the absence of the hydroxy group at C-6 or C-8 may justified the inactivity of questin (5), chrysophanol (8), physcion (9), questinol ( 13) and emodin-6-O-b-D-glucoside (20) (Omosa et al. 2016) on some strains.In addition, the absence of the hydroxy group at the 8-position in questinol (13) could justify the fact that this compound was also inactive against all the tested bacteria strains except for the S. typhi CPC strain with a MIC value of 31.2 lg mL À1 .Despite the absence of the hydroxy groups at C-8 and C-6 in the mixture of physcionin (6) and chrysophanein (7), Scheme 1. Allylation and acetylation of hydroxy groups of emodin (2) it exhibited moderate activity against S. typhi, P. aeruginosa HM801 and K. Pneumoniae Clinical isolate and this is probably due to the synergistic effect of these two compounds.

General experiment procedures
The 1 H NMR spectra were recorded on Bruker AM Avance DRX 500 ( 1 H NMR, 500 MHz and 13 C NMR, 125 MHz) and Bruker Avance DRX 600 ( 1 H NMR, 600 MHz and 13 C NMR, 150 MHz) using deuterated solvent.Infrared (IR) spectra (KBr tablet or film) were recorded on Bruker Tensor 27 FTIR-spectrometer equipped with a diamond ATR.Highresolution mass spectra were recorded on Bruker QTOF Compact Spectrometer equipped with an ESI source.Column chromatography were carried out on 230-400 mesh silica gel (Merck, Darmstadt, Germany), 70-230 mesh silica gel (Merck, Darmstadt, Germany) and Sephadex LH-20 (Sigma-Aldrich, Munich, Germany).MPLC (B € UCHI Reveleris X2 Flash Chromatography System) normal phase with pre-packed silica-gel columns as stationary phase was used for the purifications.Precoated plates of silica gel 60 F254 (Merck; Darmstadt, Germany) were used for analytical purposes and the spots were detected with a UV lamp at 254 and 366 nm and by spraying with diluted sulfuric acid before.L-rhamnose (Sigma-Aldrich, Munich, Germany) was used for comparison of sugar units.

Plant material
The roots of R. nepalensis were harvested in May 2016 in Bamenda, Northwest Region of Cameroon, and identified at the Cameroon National Herbarium, Yaound e, with voucher specimen number N o 7665/SRFCam.

Antibacterial assay
The minimum inhibitory concentration (MIC) of samples was evaluated following the broth microdilution method as described by Eloff, with little modifications (Eloff 1998).Extracts, compounds, and reference drug were dissolved in DMSO-MHB.The strain inocula were prepared and their turbidity adjusted to 0.5 McFarland standard to give an approximate 1.5 Â 10 8 CFU/mL.Ciprofloxacin was used as positive control.Briefly, one hundred microliters of Mueller Hinton Broth were added into all wells of the 96well plate, and 100 mL of the compounds/extracts were introduced to the wells in the first row (A) and mixed thoroughly.The sample mixture (100 mL) was removed from well from row A to perform a two-fold serial dilution down the rows (B-H).The last 100 mL was discarded.Then, 100 mL of the inoculum was introduced into the corresponding wells.The final volume in each well was 200 mL.Each extract concentration was assayed in triplicate and each test realized twice.After the incubation period of 18 h at 37 C, 20 mL of Alamar Blue were added to each well.The plates were then reincubated for 30 mn at 37 C. A blue color in the well was scored as 'no bacterial growth', while a pink color was scored as 'growth occurrence'.MIC values were read as those concentrations where a pronounced change of color formation was noticed (from blue to pink).

Acid hydrolysis
Compound 1 (2 mg) was refluxed in 2 M HCl (5 mL) at 100 C for 4 h.The reaction mixture was poured into water and extracted with EtOAc (5 mL three times).The aqueous layer was concentrated and used for the sugar identification by TLC and comparison of the optical rotation with hose of L-rhamnose.

Conclusions
Besides providing the knowledge on the chemistry of R. nepalensis, this study highlighted the antibacterial potency of the CH 2 Cl 2 -MeOH (1:1) extract of the roots of R. nepalensis, its EtOAc soluble fraction and constituents.Among the isolated compounds, the phenylisobenzofuranone derivative 1 was found to be a new compound and the furanones 17 and 18 isolated for the first time from Polygonaceae family.These results support the uses of this plant in traditional medicine and also confirm the antibacterial potential of anthraquinones known as biomarkers of the Polygonaceae family.Moreover, the study also confirmed the role of the hydroxy group on the activity of emodin, a secondary metabolite that could be a lead in the search of new antibacterial agents.