Isolation and characterization of compounds from Calodendrum capense (Rutaceae) and Lydenburgia cassinoides (Celastraceae) for treatment of fungal and bacterial infections in immunocompromised patients

Abstract

Infectious diseases are a serious concern worldwide especially in immune-compromised patients. Some of these diseases are considered to be contagious and are spread via airborne transmission, while others are not contagious, i.e. non-communicable diseases. The problem is compounded by the emergence of pathogens resistant to currently used antimicrobial drugs. A wide range of microbes including bacteria, fungi, parasites, viruses and protozoans are implicated as causative agents of various diseases. Many patients without ready access to Western medical facilities rely on medicinal plants for the cure of various ailments including infectious diseases. Two plant species used in South African traditional medicine for treating infectious diseases, namely Calodendrum capense Thunb. (Rutaceae) and Lydenburgia cassinoides N. Robson (syn. Catha transvaalensis, Celastraceae) were screened for antimicrobial activities against a range of fungi, bacteria and mycobacteria. The test organisms included Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Mycobacterium smegmatis and M. bovis BCG. The L. cassinoides acetone extract generally had good activity, with MIC values ranging from 0.04 to 0.15 mg/ml while the C. capense extract MIC values ranged from 0.31 to 0.62 mg/ml against the fungi. The hexane extract of L. cassinoides had good activity (MIC = 0.04 mg/ml) against M. smegmatis and the methanol extract had MIC = 0.16 mg/ml against M. bovis BCG. The two plant species had reasonable antibacterial activity against S. aureus and P. aeruginosa, with MIC values ranging from 0.16 to 0.32 mg/ml. Antibacterial activity against Escherichia coli and Enterococcus faecalis resulted in MIC = 0.63 mg/ml for both plants. However in the bioautography assay, the hexane extracts of C. capense and L. cassinoides had good activity against S. aureus, showing active zones of bacterial growth inhibition. The aim of this study was to isolate and characterize the active compounds, with emphasis on antifungal activity, from Calodendrum capense and Lydenburgia cassinoides that may be useful in treating opportunistic infections in immunocompromised patients. Bioassay-guided evaluation of the antimicrobial active components of both hexane fractions using C. neoformans and C. albicans as test organisms led to the isolation and characterization of lupeol from C. capense and ß-amyrin from L. cassinoides. The MIC values of lupeol and ß-amyrin ranged from 1.5 to 6.2 µg/ml against all the tested organisms. Both compounds were also tested against a resistant strain of Candida albicans which resulted in MICs of 3.2 and 6.2 µg/ml respectively. Crude extracts and compounds were also tested for cytotoxicity against human liver (C3A) cells. The crude plant extracts had a low cytotoxicity with average LC50 values of 205.8 ± 8.38 µg/ml for L. cassinoides and 83.07 ± 44.66 µg/ml for C. capense. LC50 values for the isolated compounds were greater than 200 µg/ml, the highest concentration tested. Selectivity index (SI) values were calculated using the formula SI = LC50/MIC. The SI values of the crude extracts of the two plant species ranged between 0.18 and 0.91, showing that these extracts were relatively toxic compared to the antimicrobial activity as the SI values were less than 1. However, the compounds ß-amyrin and lupeol had good activity and low toxicity with SI values greater than 10. In conclusion both plant species showed broad-based antimicrobial activity against the standard ATCC strains of bacterial and laboratory isolates of fungal pathogens. Purified compounds with very good antifungal activity and negligible detectable cytotoxicity, namely lupeol from C. capense and ß-amyrin from L. cassinoides (both pentacyclic triterpenoids) were isolated. This is apparently the first report of these two compounds from these two plant species. In our research group, lupeol has been isolated from various other plant species and it is known to have antimicrobial and anti-inflammatory activities. The second compound, ß-amyrin, reportedly has anti-inflammatory, antitumor, antibacterial, gastroprotective and hepatoprotective effects. The findings from this study show that the two isolated compounds were highly active against fungal and bacterial pathogens, with the lowest MIC value of 0.015 mg/ml. Both compounds showed much better selectivity index values with regard to antifungal activity compared to those of the crude extracts. The compounds also had good activity against the two Mycobacterium strains tested, indicating potential application in antimycobacterial therapy. The results may validate to an extent the use of these two plants as anti-infectious agents in traditional medicine. The compounds have potential for development into therapeutic agents, but various factors will need to be investigated further, including in vivo efficacy and safety, as well as other aspects such as mode of administration.