Isolation and characterization of antifungal compounds from Clerodendron glabrum var glabrum (Verbenaceae) used traditionally to treat candidiasis in Venda, South Africa

Abstract

The aim of this study was to isolate and characterize antifungal compounds from the most active medicinal plant species that could be used to address secondary infection problems in immunocompromised patients. An ethnobotanical study was conducted and 45 medicinal plant species used traditionally to treat candidiasis and related infections in HIV/AIDS patients were identified and documented. The most popular plant species used included Acacia caffra, Clerodendrum glabrum, Croton gratissimus, Elaeodendron transvaalense, Faurea saligna, Hippocratea longipetiolata, Osyris lanceolata, Richardia brasiliensis, Schkuhria pinnata, Schotia brachypetala, Spilanthes acmella, Strychnos potatorum, Vangueria infausta subsp. infausta and Withania somnifera. The plant parts used in the therapeutic preparations were roots (26.7%), bark (22.2%), and a combination of roots and bark (17.7%). Decoctions (44.4%), infusions (20%) and macerations (17.7%) were used. Most of the herbal remedies were administered orally. Chemical profiles of the plant species were established by using thin layer chromatography. Leaf extracts of these plant species were tested for antimicrobial activity against two common pathogenic fungal species in humans (Candida albicans and Cryptococcus neoformans) and four nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) using a two-fold serial microdilution method and bioautography. All plant species investigated had some degree of antimicrobial activity against the test microorganisms. The hexane and the acetone extracts of Clerodendrum glabrum, Hippocratea longipetiolata, Schkuhria pinnata and Withania somnifera were the most active with MIC values ranging from 0.06 to 0.08 mg/ml. The most susceptible pathogen to the test samples was C. neoformans while C. albicans was resistant to most of the plant extracts. The water extracts of Withania somnifera and Hippocratea longipetiolata (14%) had MIC < 1 mg/ml against C. albicans. C. neoformans was susceptible to nine water plant extracts (64%) with MIC < 1 mg/ml and the promising activity was observed in Hippocratea longipetiolata and Faurea saligna extracts with MIC values of 0.16 and 0.31 mg/ml respectively. The hexane extract of C. glabrum was the most active against C. albicans with an MIC value of 0.06 mg/ml and total activity of 550 ml/g. In the bioautography, most plant extracts tested had few active compounds, others had no active components at all and this may be attributed to the disruption of synergism by the thin layer chromatography. C. glabrum had eight active antifungal compounds on bioautograms and most of these components were observed in the EMW solvent system. Based on this and its wide distribution in rural areas, C. glabrum was chosen for further study. The antioxidant activity and possible immune boosting potential of the species were determined using 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), 2, 2’ azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. In the DPPH qualitative assay, the aqueous plant extracts had several prominent antioxidant components than the organic plant extracts. The aqueous plant extracts which had the most prominent antioxidant activity were F. saligna with 8 compounds, followed by E. transvaalense, H. longipetiolata O. lanceolata, R. brasiliensis and S.brachypetala, with five compounds each and their Rf values ranged from 0;06 to 0.94. This appears to validate the ethnomedicinal use of the plant species to some extent because decoction is the most common method used in the preparation of the remedy by the traditional healers. With regard to the organic plant extracts, only one plant extract, F. saligna had two prominent antioxidant components at Rf values 0.81 and 0.88. A third of the plant species had a high level of free radical scavenging activities in the DPPH, ABTS and FRAP assays. However, all plant extracts had lower antioxidant activity than the positive control (Trolox) used. The selected plant species were also evaluated for their in vitro toxicity against the Vero monkey kidney cell line using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The acetone plant extracts of O. lanceolata, S. acmella, S. pinnata and S. brachypetala had high cytotoxic activity against Vero cells with IC50 values of 13.7±0, 19.9±0.001, 21.6±0.001 and 28.34±0.001 μg/ml respectively. However, their IC50 values were higher than that of the positive control, doxorubicin (IC50 = 9.9±0 μg/ml). The rest of the acetone plant extracts (64%) had moderate cytotoxic activity (30 < IC50<100 μg/ml). The aqueous plant extracts were relatively non-toxic to the Vero cells with IC50 values ranging from 137 to > 500 µg/ml. This supports the use of aqueous extracts in the traditional medicine. However, their low selectivity index values ranging from 0.26 to 1.68 suggest that the plant extracts are probably suitable for external use only. Fractionation of the hexane extract of the leaves of C. glabrum by chromatographic techniques yielded six fractions of which fractions C and D had significant antifungal activity (average MIC value = 0.1 mg/ml) against C. albicans and C. neoformans. From these fractions, one new triterpenoid, 3-(1-oxobutyl)-11α-hydroxytaraxast-20(30)-ene-24,28-dioic acid (clerodendrumic acid) (1) was isolated along with known heptadecanoic acid (2). C. albicans was relatively insensitive to clerodendrumic acid (1) (MIC value = 125 µg/mL) and was resistant to heptadecanoic acid (2) (MIC value = 188 µg/ml). Compounds 1 and 2 were non-toxic against monkey kidney Vero cells in vitro with IC50 values of 202.6 and 108.4 µg/ml respectively. Due to its low antifungal activity, the novel compound clerodendrumic acid (1) is not a viable candidate for drug development which could be used to combat candidiasis and related fungal infections. However, due to its relative safety, it may possibly be used as a lead compound to produce new chemically modified active derivatives or could be used together with known antibiotics to mitigate their undesirable side effects. To the best of our knowledge, the isolation of a novel, clerodendrumic acid (1) and a known heptadecanoic acid (2) compounds from leaf extracts of C. glabrum is reported herein for the first time. The results obtained from this study generally substantiate the rationale behind the use of the selected plant species in the traditional medicine to treat candidiasis and related infections to some extent. This study showed the potential of studying traditional medicine in the search for effective plant extracts or new lead compounds that could be developed into drugs for combating microbial infections among the rural poor people.