LAUSR

Tetradenia (T.) riparia (Hochst.) Codd (Lamiaceae), popularly known as umuravumba, is frequently used in traditional Rwandese medicine. Using a bioassay-guided isolation (with Staphylococcus aureus as the test organism), three active compounds: umuravumbolide, deacetylumuravumbolide, and 8(14),15-sandaracopimaradiene-7α,18-diol were identified by a combination of NMR and mass spectrometry. Candida auris has become a global threat, causing nosocomial infections that are often resistant to multiple antifungals. As T. riparia is traditionally used for the treatment of infectious diseases including those of the skin, and C. auris is reported to colonize the skin, we tested the aforementioned three bioactive compounds against C. auris (planktonic and biofilm). The diterpenediol 8(14),15-sandaracopimaradiene-7alpha, 18-diol inhibited the growth of C. auris (IC50 21.45 μg/ml; 95% confidence interval: 13.36–34.48 μg/ml), as well as its biofilm formation (IC50 29.14 μg/ml; 95% confidence interval: 18.5–45.91 μg/ml). Interestingly, the α-pyrone: umuravumbolide was also active against C. auris (IC50: 131.2; 95% confidence interval: 83.72–205.6 μg/ml), but not against Saccharomyces cerevisiae. Based on the predicted toxicity profiles using ProTox, 8(14),15-sandaracopimaradiene-7α,18-diol (class V) seems comparatively a safer candidate than the standard antifungal agent miconazole (class IV). T. riparia is a widespread shrub throughout oriental intertropical Africa reputed to possess substantial antimicrobial activity against both bacterial and fungal pathogens (Boily & Van Puyvelde, 1986; Fernandez et al., 2017; Van Puyvelde et al., 1986). Recent reports document its traditional use in several skin infections, possibly caused by fungi (wound infections, itching, rashes etc.) (de Melo et al., 2015; Kakande et al., 2019). The yeast Candida (C.) auris is an emerging pathogen that has become a public health threat worldwide over the past decade. Amongst its pathogenic traits is its capability to adhere strongly to and persist on surfaces, making it difficult to eliminate once it establishes itself in healthcare environments. This fungus can colonize, for example, skin, and cause invasive infections in vulnerable patients that are very difficult to treat with the most commercially available antifungal agents. Thus, systemic infections can cause death in up to 70% of humans. C. auris often show resistance to the major classes of antifungal drugs; most are resistant against at least one class of antifungal drugs, one-third against two classes, and some even to three classes (Nishikawa et al., 2016). Therefore, scientists world-wide have focused efforts on understanding its biology as well as finding effective treatment strategies to combat this fungus. As there is an urgent need to discover drugs against C. auris, we tested the activity of three antimicrobial compounds from T. riparia against this emerging pathogen. Furthermore, we predicted some of their toxicity parameters using the software tool ProTox. All chemicals, reagents, and solvents (HPLC grade) were purchased from Acros Organics (Geel, Belgium) or Sigma-Aldrich (St. Louis, MO, USA). Leaves of cultivated T. riparia were harvested in October 2014 in Mukoni, Huye, Rwanda, and identified by an expert botanist (Dr. Vedaste Minani). A voucher specimen (No. 86) was deposited in the National Herbarium at the National Industrial Research and Development Agency (NIRDA), Huye, Rwanda. The extraction procedures (liquid–liquid separation, silica gel chromatography) were essentially as described before (Van Puyvelde et al., 2021). Briefly, the active dichloromethane extract (4.8 g). was chromatographed over a silica gel (230–400 mesh) column (Ø 5 cm, h: 50 cm), and eluted with a hexane-ethyl acetate-methanol step-gradient (40 ml/min; 400 ml per solvent system; fractions of 40 ml). Active fractions (pooled based on TLC analysis), were further purified on a small silica gel column (Ø 2 cm, h: 35 cm) and offered three compounds after crystallization in hexane. These compounds were subjected to H and C NMR (Bruker Avance II + 600 spectrometer, Fallanden, Switzerland) for identification (Van Puyvelde et al., 2021; for details, see Supplementary Material 1). The in vitro antifungal activity was evaluated against Candida auris (OS299) and Saccharomyces cerevisiae (ATCC 7754) using a 96-well microplate broth dilution method essentially as described before (Hu et al., 2021). The (frozen) storage, maintenance, and preparation of working culture suspensions were carried out following established procedures described in a previous study (Hu et al., 2021). Miconazole (50 μg/ml) was used as a positive control, while 2% dimethyl sulfoxide (DMSO) served as a negative (solvent) control. The percentage inhibition of the test sample was determined as 100% [{(ODtest sample-ODsample control) 100%}/ OD solvent control]. Biofilm activity was determined in duplicate after 24 h of incubation in RPMI 1640 plus MOPS medium using a resazurin reduction assay. The antifungal concentration that caused ≥50% reduction in the metabolic activity of the biofilm compared with the control (incubated in the absence of the antifungal agent) was determined, and the concentration resulting in a 50% reduction in the metabolic activity was considered the biofilm IC50 (BIC50). In addition, hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, Received: 4 June 2022 Revised: 2 September 2022 Accepted: 12 September 2022