Antileishmanial anthraquinones from the rhyzomes of Rumex abyssinicus Jacq (Polygonaceae)

Abstract Phytochemical investigation of the rhyzomes of Rumex abyssinicus (Polygonaceae) afforded six anthraquinones viz chrysophanol (1), physcion (2), emodin (3), mixture of physcion-8-O-β,D-glucopyranoside (4) and chrypsophanol-8-O-β,D-glucopyranoside (5), and emodin-8-O-β,D-glucopyranoside (6). All the compounds were characterised and identified by comparison of their MS and NMR data with available literature data. The isolated compounds were evaluated for their antileishmanial activity. Emodin (3) was the most active compounds with IC50 13.82 and 0.26 µg/mL against Leishmania donovani amastigotes and promastigotes, respectively. Emodin-8-O-β,D-glucopyranoside (6) also showed a moderate activity with IC50 27.53 and 37.08 µg/mL. This is the first report of antileishmanial compounds from R. abyssinicus and the antileishmanial activities of compounds 2, 4, 5 and 6 are here reported for the first time. Graphical Abstract


Introduction
Rumex abyssinicus Jacq. Commonly called spinach rhubarb, is a flowering perennial herb of up to 4 m height largely distributed in central and eastern Africa belonging to the Polygonaceae (Worku et al. 2013). It is used in traditional medicine in several African countries for the treatment of several diseases. The rhizomes are used for the treatment of constipation, hepatitis, gonorrhoea, malaria, mild diabetes, poisoning and hypertension (Mekonnen et al. 2010). The water decoction of leaves is used as vermifuge and in the treatment of haemorrhoids, stomach distention, thypus and wound (Worku et al. 2013). Previous studies have demonstrated the analgesic, anticancer, anti-inflammatory, antimalarial, antimicrobial, antioxidant, cholinesterase inhibitor and wound healing potential of the plant (Muganga et al. 2010;Mulisa et al. 2015;Ntemafack et al. 2020). Anthraquinones, flavonoids and terpenoids have been reported in the plants (Fassil et al. 1985;Tala et al. 2018;Ntemafack et al. 2020).
The search of antileishmanial compounds from natural sources such as plants represent an alternative for the discovery of potent antileishmanial agents. In fact, the continuous search of treatment against leishmaniasis is much needed due to the limitation of available antileishmanial drugs with the risk of relapse in immunosuppressive patient and the absence of vaccine (WHO 2021). Moreover, plants represent a rich source of diverse chemical entities with application in medicine (Newman and Cragg 2020). Indeed, several compounds isolated from plants such as racemoside A from Asparagus racemosus with IC 50 ¼ 1.31 mg/mL against Leishmania donovani promastigotes, have demonstrated significant antileishmanial activity (Lal and Singh 2016). Therefore, in the present study, we have investigated the antileishmanial activity of secondary metabolites isolated from R. abyssinicus.
The in vitro antileishmanial potential of the isolated compounds (1-3, 4-6) from R. abyssinicus against Leishmania donovani amastigotes and promastigotes was measured by direct counting of live amastigotes and promastigotes after exposure to various concentrations of the compounds ranging from 0.16 to 100 mg/mL. The results are showed in Table S1. The isolated compounds exhibited a moderate inhibitory potential against L. donovani amastigotes (IC 50 ranging from 13.82 to 27.53 mg/mL) and promastigotes (IC 50 ranging from 0.26 to 49.56 mg/mL). Emodin (3) was the most active against L. donovani amastigotes IC 50 ¼ 13.82 ± 0.05 mg/mL followed by emodin-8-Ob,D-glucopyranoside (6) IC 50 ¼ 27.53 ± 0.03 mg/mL. Chrysophanol (1) and physcion (2) were not active at the concentration tested. Coming to L. donovani promastigotes, emodin (3) was also the most active IC 50 ¼ 0.26 ± 0.57 mg/mL followed by emodin-8-Ob,D-glucopyranoside (6) IC 50 ¼ 37.08 ± 0.01 mg/mL while chrysophanol (1) and physcion (2) showed a weak activity. This strong activity on promastigotes forms which is the insect vector-based forms of Leishmania parasites by comparison on amastigotes forms, could be due to the non-ability of emodin to cross the various membrane cells to attend intracellular amastigotes or could be due to the rapid intracellular degradation of the compound (St George et al. 2006). Moreover, it has been demonstrated that parasites form express stage-specific molecules that can serve as virulence factor. Those molecules located on the surface of the parasite form act differently and therefore their interaction with a potential antileishmanial agent should be different (Ilgoutz andMcConville 2001, Kima 2007). In addition, promastigotes forms are more sensitive to potential drugs than the amastigotes forms (De Muylder et al. 2011). It was noticed that emodin (3) was more active than emodin-8-O-b,D-glucopyranoside (6). This reduction in activity could be due to the presence of the 8-O-b,D-glucopyranoside moiety in 6. In addition, the weak antileishmanial activity of compounds 1 and 2 compared to those of 3 and 6 could be due to the absence or blockage of the free hydroxyl group in position C-6 in emodin (3) and emodin-8-O-b,D-glucopyranoside (6). Leishmanicidal activity of anthraquinones could be explained by their ability to produce Reactive Oxygen Species (ROS) (Tewabe et al. 2019). It has been demonstrated that aromatic compounds with free hydroxyl group are electron donating group and therefore produce ROS (Lee et al. 2015). Malaria and Leishmaniasis parasites belong to the same family of protozoa and share some common therapeutic targets (Ferede et al. 2017). Anthraquinones with hydroxyl substituents in meta-or ortho-arrangement have demonstrated strong antiplasmodial activity (Osman et al. 2019). Therefore, there is no doubt that compound 3 and 6 showed good antileishmanial activity than the other isolated compounds. In addition, it should be noted that activity increase along with hydroxylation (Winter et al. 1995). This is the first report of the antileishmanial activity of physcion (2)  promastigotes with IC 50 ¼ 14.3 mg/mL and a good activity for emodin (3) L. donovani amastigotes with IC 50 ¼ 2 mg/mL (Mbwambo et al. 2004;Ghoneim et al. 2013).
The cytotoxicity of the isolated compounds was evaluated against RAW cell lines macrophages using resazurin assay. Globally, compounds 2 and 3 and the mixture of 4 and 5 were found to have a moderate inhibition with IC 50 ranging from 32.41 to 42.85 mg/mL. Compound 3 been the most save molecule with SI of 122.53 and 2.34 on promastigote and amastigote form, respectively. Compared to other compounds, compounds 2 and 3 are more active on amastigote than on promastigote with SP < 1. The remaining compounds were not cytotoxic at the tested concentrations.

Experimental
The experimental section is available online in supplementary material.

Conclusions
This is the first report of the evaluation of the antileishmanial activity of isolated compounds from R. abyssinicus. Emodin and emodin-8-O-b,D-glucopyranoside were found to be the most active compounds among the six compounds isolated. The finding regarding emodin is in agreement with previous studies, which have demonstrated that emodin isolated from Vismia orientalis possessed good antileishmanial activity.

Disclosure statement
No potential conflict of interest was reported by the authors.