A New Ceramide (Rumexamide) and Other Chemical Constituents from Rumex abyssinicus Jacq (Polygonaceae): Isolation, Characterization, Antibacterial Activities and Chemophenetic Significance

The chemical study of Rumex abyssinicus Jacp (Polygonaceae) led to the isolation of a new ceramide named (R)-2'-hydroxy-N-[(2S,3S,4R,16E)-1,3,4-trihy-droxyhexacos-16-en-2-yl]hexadecanamide (rumexamide) (1) together with sixteen known compounds: bis-(2-ethylhexyl) phthalate ( 2), chrysophanol (3), physcion bacterial activities of extracts (MeOH, n-BuOH and EtOAc), as well as compounds 9, 11, 12, 15 + 16 and 17 against pathogenic bacteria (Staphylococcus aureus ATCC 43300, Shigella flexneri NR 518, Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922), were performed using the broth microdilution method and the results show that, extracts were not active (MIC > 1000 µg/mL) while compounds were weakly or not active (MIC ≥ 500 µg/mL) against all bacteria strains. Furthermore, the chemophenetic relationships of the isolated compounds and their significances were discussed.


Introduction
Since their discoveries, antibiotics have been essential in the treatment of diseases caused by pathogens (especially bacterial infections) which have helped in extending the average life expectancy. However, their overexploitation has caused the appearance and increase in the rate of resistance of microorganisms to said antibiotics [1]. It is well established that this resistance is nowadays responsible for nearly 7 million deaths per year and it is estimated that it will reach to 10 million deaths per year by 2050 [2]. Thus, in order to overcome this state of affairs, efforts are being made to find new antimicrobial agents such as medicinal plants [3]. Rumex abyssinicus is a large annual herbaceous plant with generally sagittate leaves, highly branched inflorescence and light brown hazelnuts and locally named in Amharic "mekmako" [4]. It is a medicinal plant widely distributed in the highlands of tropical Africa and distributed throughout North Africa and Ethiopia [5]. Indeed, Rumex abyssinicus is used in traditional medicine to treat several diseases like rheumatism, malaria, typhoid and hepatitis [5] [6]. Members of the genus Rumex have been reported to produce a wide range of secondary metabolites such as anthraquinones [7], flavonoids [8], stilbenoids [9] [10], tannins [11], triterpenoids [12], steroids and saponins [13]. Some of them possess several pharmacological activities such as antioxidant, antitumour, antimicrobial, antiparasitic, antiviral [14]. Previous pharmacological studies carried out on R. abyssinicus revealed its antimicrobial activities against Salmonella typhimurium, Listeria monocytogenes (ATCC 29211), Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC 4944) [15] and antiplasmodial activity against chloroquine-sensitive Plasmodium falciparum strain (3D7) [16]. In our continuous search for bioactive secondary metabolites from Cameroonian medicinal plants [17] [18] [19] [20], we report in this paper the isolation, characterization, antibacterial activities and Chemophe-

General Experimental Procedures
Bruker IR-Alpha spectrometer apparatus was used for scanning IR (Infrared) spectroscopy using KBr pellets. Column chromatography was carried out on silica gel 230 -400 mesh, Merck (Merck, Darmstadt, Germany), 70 -230 mesh (Merck) and/or gel permeation on Sephadex LH-20 (Sigma-Aldrich, Munich, Germany). High resolution mass spectra were obtained with QTOF (Quadrupole Time of Flight) Compact Spectrometer (Bruker, Germany) equipped with a HRESI source. The spectrometer was operated in positive and negative modes (mass range: 50 -1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 0.4 ppm deviation using Na formate as calibrant. The following parameters were used for experiments: spray voltage of 4.5 kV, capillary temperature of 200˚C. Nitrogen was used as sheath gas (4 L/min). The 1 H and 13 C Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker DRX 500 MHz and 600 MHz NMR spectrometers and on a Bruker Avance III 600 and 500 spectrometers equipped with a cryo-platform. 2D NMR experiments were performed using standard Bruker microprograms (Xwin-NMR version 2.1 software) in deuterated solvents. Chemical shifts (δ) are reported in parts per million (ppm) using the residual solvent signals as secondary reference relatively to TMS (Tetramethylsilane) (δ = 0), while the coupling constants (J values) are given in Hertz (Hz). Thin-layer chromatography (TLC) was carried out on Merck pre-coated silica gel (60 F 254 ) aluminium foil (Merck) with detection accomplished by spraying with diluted sulfuric acid (50% H 2 SO 4 , Riedel-de Haen AG, 95% -97%) followed by heating at 100˚C, or by visual inspection under Ultraviolet (UV) lamp at 254 and 365 nm.

Plant Material
In the present study the whole plant of Rumex abyssinicus was collected in February 2018 in Dschang Western Region of Cameroon, with GPS data of: latitude: N 5˚27'5.94828'' and longitude: E 10˚3'17.39556''. The botanical identification was carried out by Victor Nana, a botanist of the National Herbarium of Cameroon, where a specimen was deposited under the voucher number N˚ 50551/HNC.

Methanolysis of Compound 1
Compound 1 (5.0 mg, 7.3421 μMol) was dissolved in 5% HCl-MeOH (3 mL) (HCl, 37% produce by Fisher Scientific and MeOH obtained from SOLEVO-Cameroon) and refluxed for 14 h at 70˚C. The reaction was monitored using TLC analysis. At the end of the reaction, the mixture obtained was extracted with methylene chloride (CH 2 Cl 2 obtained from SOLEVO-Cameroon) after neutralization with dilute potassium carbonate (K 2 CO 3 , produce by Fisher Scientific with 99.9% purity). The organic phase was separated and concentrated to yield 2.1 mg.

Bacteria Strains and Sample Preparation
The microorganisms used in this study were consisted of four bacterial strains namely: Staphylococcus aureus NR 43300, Shighella flexineri NR 518, Klebsiella, pneumonia ATCC 700603, Escherichia coli ATCC 25922 obtained from our local stocks. They were cultivated in petri dishes containing Mueller Hinton Agar (MHA) followed by an incubation period of 24 hours at 37˚C. Each microorganism was sub-cultured in a new agar plate and incubated in the same above mentioned conditions prior each experiment. Extracts and compounds were weighed and dissolved in different solvents (pyridine, DMSO) for a final of 100 mg/mL and 20 mg/mL for extracts and compounds respectively. Positive control Ci-profloxacin was prepared at 1 mg/mL. Extracts, compounds and the reference drugs were screened at 1000 µg/mL, 500 µg/mL and 119 µg/mL, respectively.

Determination of Minimal Inhibitory Concentration (MIC)
The antimicrobial activity of each product was done as previously described by the CLSI, (2012) using the broth microdilution method in 96-wells microplates.
The absolute configuration of the stereocenters C-2, C-3, C-4 and C-2', were assigned to be 2R, 3S, 4R, 2'R on the basis of their carbon chemical shifts which are very close to those of vernoguinamide [37].
The length of the acid chain (ALC) was determined using the ESI (

Spectroscopic Data of Compounds 1-17
Bis-(2-ethylhexyl) phthalate (2)  Citreorosein (5) nepalensis [44], compound 8 from R. obtusifolius [45] and compounds 7 and 8 from R. acetosa [46]. Base to the fact that, anthraquinones represent one of the main classes of secondary metabolites isolated from the genus Rumex [14] [ 47], their presence in all of the above species allowed us to conclude that R. abyssinicus and the other species mentioned exhibit very close chemotaxonomic relationships. The isolation of a new ceramide (1)  (2) isolated for the first time from Rumex genus has already been reported from Polygonaceae family especially from Polygonum runcinatum [52].

Conclusion
In conclusion, the chemical study of the whole plant of R. abyssinicus led to the isolation of one previously undescribed ceramide (1) and sixteen known (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) compounds which enriched the chemical diversities of the plant, genus Rumex and Polygonaceae family. Compounds 10-12 may demonstrate here the relationship between F. aubertii and other species belonging to other genera herbs of Polygonaceae. Unfortunately, the extracts showed no antibacterial activity against the strains tested while compounds were either not active or weakly active. It will be interesting in future studies to test these extracts as well as the compounds isolated on other microbial strains.