A phylogeny-based comparative study of the phytochemical and pharmacological characteristics of Croton species occurring in KwaZulu-Natal.

Abstract

Ethnobotanical enquiries often lead to the discovery of phytocompounds with pharmacological activities. Against this background a comparative and quantitative evaluation of the phytochemical and antioxidant activity of extracts from six Croton species; C. gratissimus Burch., C. sylvaticus Hochst., C. menyhartii Pax, C. pseudopulchellus Pax, C. steenkampianus Gerstner and C. rivularis Müll.Arg., all collected from KwaZulu-Natal (KZN), South Africa, was conducted. The analysis included a comparison of the different plant organs to explore the possibility of using leaves rather than bark for medicinal purposes. The latter would result in less destructive harvesting and would contribute to sustainable use of these medicinal plant resources. Extraction of the different plants and their organs were done in water and, in different organics solvents, including methanol (MeOH), dichloromethane (DCM), and petroleum ether (PE). The extracts were screened for antibacterial and antifungal activities using the microdilution technique. All of the tested plant samples showed some notable antibacterial activity in one or two of their organs, except for C. rivularis, which was the only species in the list that had no record of medicinal use. The most potent antibacterial activity was exhibited by the dichloromethane (DCM) extracts of C. steenkampianus leaves and the petroleum ether (PE) extracts of C. pseudopulchellus stem bark, both sampled from the Durban Botanic Gardens, which yielded a minimum inhibition concentration (MIC) value of 0.04 mg/ml against Enterococcus faecalis (E. faecalis). The DCM stem bark and leaf extracts, as well as the PE twig extracts of C. pseudopulchellus, (Durban Botanic Gardens) also exhibited noteworthy activities against S. aureus (MIC value of 0.08 mg/ml). A broad spectrum of activity was observed in the DCM and PE twig extracts of C. sylvaticus collected from Umdoni Park, with a MIC ranging from 0.31-0.94 mg/ml. This activity was against E. faecalis, Staphylococcus aureus (S. aureus) and Klebsiella pneumoniae (K. pneunomiae). Noteworthy antifungal activity against Candida albicans was only observed in one extract, the MeOH leaf extract of C steenkampianus collected at Kosi Bay. The MIC of this extract was 0.6 mg/ml. Water extracts did not show any antimicrobial activity. The results of the pharmacological study suggested that the aerial plant organs, such as the leaves and the twigs, could replace bark as they exhibited significant antimicrobial activity when compared to the preferred bark. The study also revealed that the same species collected from different regions may not necessarily exhibit similar biological activities, as pharmacological activities are as a result of the phytochemicals present in the plant, which are triggered by environmental stimuli. The phenolic profiles of aqueous (50%) methanol extracts obtained from the plants were assessed using the Folin and Ciocalteu (Folin C), butanol-hydrochloric acid and aluminium chloride assays. Aqueous (50%) methanol extracts were also run on thin layer chromatography plates and the plates were later stained with the Dragendorff reagent to determine the possible presence of alkaloids. Antioxidant activity was determined with two different assays, β-carotene /Linoleic model system and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay. The overall EC50 values of the different Croton species displayed by the DPPH assay ranged from 1.76 to 5.35 μg/ml. The β-carotene /Linoleic model system displayed antioxidant activity that ranged between 48.66 to 81.97%. In the phytochemical study; the leaf extracts of C. pseudopulchellus from Mkuze exhibiting the highest phenolic content at 23.8±1.1 mg GAE/g Dry Weight. The highest condensed tannin content was from the leaf extracts of C. gratissimus from Southport at a concentration of 31.3± 0.1 mg CCE/g Dry Weight. The highest flavonoid content observed in the leaf extracts of C. gratissimus from Southport at a concentration of 31.2±0.7 mg CE/g Dry Weight. Higher phytochemical contents were also observed in the leaves and twigs. These phytochemicals are believed to be the reason for most of the notable antimicrobial activities exhibited by these plant organs. The mutagenic potential of the most biologically active Croton extracts was tested. An Ames with two Salmonella tester strains (TA98 and TA102) revealed that the species are not toxic as they did not produce His+ revertant colonies in Salmonella tester strains that were more than twice the number of His+ revertant produced by the positive control, Nitroquinoline-N-oxide (4-NQO). Standard DNA barcodes of the Croton species occurring in KZN were generated, since these are useful in plant identification and authentication. These were used to run a phylogenetic analysis in order to assess whether the phytochemical profile is clade-specific. The results showed that the quantity and quality of phytochemicals closely related species may vary. DNA barcoding may be a useful tool in medicinal plant identification, especially where fragments of the plants are traded and morphological identification is not possible. In this study, the biological activity of different parts of six Croton species occurring in KZN, was investigated with the aim of replacing bark as the source of local medicine with other plant parts (leave and twigs) that can be more sustainably harvested, as this will contribute to the conservation of these species.