Investigations on the effects of Typha capensis on male reproductive functions
Introduction: Typha capensis, commonly referred to as bulrush also called "love reed'' growing in Southern Africa's wetlands, is one of South Africa indigenous medicinal plants that are traditionally used to treat male fertility problems and various other ailments. Previous studies revealed that T. capensis has indeed a beneficial effect on male reproductive functions and aging male symptoms. The T.capensis rhizomes are used in traditional medicine during pregnancy to ensure easy delivery, for venereal diseases, dysmenorrhea, diarrhoea, dysentery, and to enhance the male potency and libido. Typha genuses contain flavones and other phenolic compounds, which exhibit anti-oxidative capacity. Materials and Methods: This study encompasses three parts (part 1: Exposure of different cell lines to crude aqueous extracts of T. capensis rhizomes; part 2: HPLC analysis of Typha capensis crude rhizome extract and exposure of different cell lines to the F1 fraction of the summer season; part 3: Compound identification by means of NMR spectrometric analysis and exposure of different cell lines to bioactive compounds (Quercetin and Naringenin) isolated from T. capensis rhizomes. Part 1: TM3-Leydig cells and LNCaP cells incubated with different concentrations of crude aqueous extract of T. capensis rhizomes (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) and control (without extract) for 24 and 96 hours, after incubation. The following parameters were evaluated: cell morphology and viability (determined by means of MTT assay). Part 2: The crude extract HPLC profiles were obtained by preparing the extracts for different seasons (Autumn, Winter, Spring, Summer). TM3-Leydig cells, LNCaP cells and PWR-1E cells incubated with different concentrations T. capensis rhizomes extract F1 fraction of the summer season (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) and control (without extract) for 24 and 96 hours, after incubation. The following parameters were evaluated: cell morphology was observed and recorded, viability (determined by means of MTT assay), testosterone production (testosterone ELISA test), cell early apoptosis (determined by means of Annexin V-Cy3 binding), DNA fragmentation (determined by means of the TUNEL assay). Part 3: NMR spectrometric analysis was performed on a 13C spectra were recorded at 400 MHz. TM3-Leydig cells and LNCaP cells incubated with different concentrations of bioactive compounds (Quercetin and Naringenin) isolated from T. capensis rhizomes, for acute exposure (24, 96 hours) and chronic exposure (96 hours), after incubation, the following parameters were evaluated: cell morphology and viability (determined by means of MTT assay), testosterone production (testosterone ELISA test), cell early apoptosis (determined by means of Annexin V-Cy3 binding) and DNA fragmentation (determined by means of the TUNEL assay). Results: Part 1: for TM3-Leydig cells the results reveal no observable morphological changes and no significant influence on cell viability except at highest concentration indicating cellular stress. However, LNCaP cells showed a decline in cell viability at the incubation period 96 hours (-82.4%) more than 24 hours (-64.7%) indicating more cell death. Part 2: HPLC data showed that the most effective fraction was the F1 fraction from the summer harvest. Results revealed that the T. capensis rhizome extract F1 fraction of the summer season significantly enhanced testosterone production in TM3 cells and was more toxic towards cancer cells (LNCaP cells ) compared to the normal cell lines (TM3-Leydig, PWR-1E cells). Part 3: NMR data showed 2 bioactive compounds which were identified as Quercetin and Naringenin. The assays showed that LNCaP cells are more sensitive to the cytotoxic effects and apoptosis induction of both compounds, whereas, the assays resulted in weak effects toward TM3-Leydig cells. However, testosterone production in TM3-Leydig cells was significantly enhanced at low concentrations of Quercetin and Naringenin at all exposure types (acute and chronic) testosterone beak significantly at around 0.100 and 0.125 μg/ml (P<0.0001), stimulatory activity in a dose-dependent manner. Conclusion: Typha capensis enhanced the production of testosterone and might be useful to treat male infertility and aging male problems. Results further reveal that the F1 fraction from the summer harvest had highest biological activity. This study, for the first time, investigated the effects of bioactive compounds (Quercetin and Naringenin) yeilded from aqueous extraction of Typha capensis rhizomes in cell lines investigating male reproductive functions. Active compounds present in the rhizomes have caused an increased production of testosterone level in TM3-Leydig cells. Furthermore, the active compounds of Typha capensis rhizomes in the high dose had a negative effect on the percentage of DNA fragmentation in LNCaP cells. When compared to the effect of the low dose, the two compounds induced significant apoptosis in cancer cell line (LNCaP) compared with the normal cell line (TM3-Leydig). The isolated compounds are significantly selective towards the cancer cells than the normal cell compared with the exposure of bioactive compounds used in this study.