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    The effect of crude water extracts of Tulbaghia violacea Harv. on scaffolds with cardiovascular applications
    (Vaal University of Technology, 2020-02) Madike, Lerato Nellvecia; Popat, K. C., Prof.; Pillay, M., Prof.
    Tulbaghia violacea Harv. has found extensive uses in traditional medicine for the treatment of numerous ailments among which are tuberculosis, oesophageal cancer, diabetes and cardiovascular diseases. Current reports show that cardiovascular diseases are now the primary cause of mortality worldwide. Thus, the potential of T. violacea plant extracts against cardiovascular diseases should be explored. The objectives of this study were, (i) to conduct qualitative and quantitative preliminary phytochemical screening of T. violacea aqueous leaf extracts, (ii) to conduct Gas chromatography–mass spectrometry (GC-MS) analysis for screening of compounds present in the plant extract, (iii) to evaluate the antioxidant activity of the T. violacea crude extracts using the DPPH:1.1-diphenyl-2-picrylhydrazyl and ABTS: 2,2-azino-bis 3-ethylebenzthiazoline-6-sulfonic acid assays, (iv) to evaluate the antimicrobial activity of the T. violacea crude extracts using disk diffusion and Minimum inhibitory concentration/Minimum bactericidal concentration (MIC/MBC), (v) to evaluate the antithrombogenic properties of T. violacea crude extracts on polystyrene, (vi) to fabricate polycaprolactone (PCL) and PCL-T. violacea incorporated scaffolds, (vii) to evaluate the antithrombogenic properties of T. violacea crude extracts on the fabricated PCL and PCL-T. violacea fabricated scaffolds and, (viii) to evaluate the growth and differentiation of adipose derived stem cells (ADSCs) on the fabricated scaffolds. The qualitative and quantitative phytochemical screening was conducted using standard procedures. Folin-Ciocalteu method was used to evaluate both total phenolic content (TPC) and total tannin content (TTC), the Aluminium chloride method was used for total flavonoid content (TFC) and GC-MS was used to screen for compounds present in the plant extract. The antioxidant activity was evaluated using DPPH and ABTS and the antimicrobial activity was evaluated using disc diffusion and MIC/MBC assays. The antithrombogenic properties of the T. violacea aqueous leaf extracts was then evaluated using platelet activation and whole blood clotting kinetics on polystyrene discs which have been reported to induce platelet activation. The experiment was performed in the absence and presence of 100 and 1000 μg/ml T. violacea plant extracts for both the platelet activation study which used blood plasma and the whole blood clotting kinetics assay which used fresh whole blood. Platelet adhesion was evaluated using fluorescence microscopy and a scanning electron microscope (SEM) was used to evaluate their morphology. Three scaffolds designated as PCL, 10% Tvio and 15% Tvio were fabricated which consisted of a 10% PCL powder and 10% as well as 15% T. violacea aqueous plant extract with respect to the PCL powder weight. The scaffolds were then characterized using Fourier-transform infrared spectroscopy (FTIR) and Energy-dispersive x-ray spectroscopy (EDS). The scaffolds were then evaluated for their antithrombogenic properties in the presence and absence of 100 and 1000 μg/ml T. violacea plant extracts. Platelet adhesion was evaluated using a fluorescent microscope and the morphology was evaluated using SEM. For the cell study, adipose derived stem cells (ADSCs) were cultured on the designed scaffolds and evaluated for their toxicity, viability, adhesion, proliferation, morphology and differentiation into osteoblasts over a period of 3 weeks. Lactate dehydrogenase (LDH) assay was used for toxicity studies, alamar blue assay was used for viability, fluorescence microscopy was used to evaluate cellular adhesion and proliferation while the alkaline phosphate (ALP) assay was used to evaluate differentiation of the cells into osteoblasts. Cell morphology was evaluated using SEM. Phytochemical screening of the prepared T. violacea aqueous extract revealed the presence of terpenoids, flavonoids, cardiac glycosides, saponins, protein, phenols, tannins, carbohydrates and amino acids. This is the first study that has identified the presence of carbohydrates and amino acids in T. violacea aqueous leaf extracts. Different concentrations of 0.1, 1.0 and 10 mg/ml of plant extract were used to conduct the quantitative phytochemical screening assays. There was a concentration dependent increase in the amount of phenols, tannins and flavonoids as the concentration of the plant extracts increased. This was the first study that evaluated the total tannic content of T. violacea plant extracts. The amount of total phenols was higher than that of flavonoids and tannins at every concentration range studied followed by the total flavonoids and lastly total tannins. The GC-MS analysis showed the presence of 33 compounds among which were 2,4 – Dithiapentate - 2,2-dioxide, Cannabidiol, 2,4,5,7 –Tetrathiaoctane and 2,4,5,7 - Tetrathiaoctane 2-dioxide. The presence of sulphur compounds support the characteristic garlic-like smell as well as some of the biological activities of T. violacea plant extracts. The antioxidant activities based on DPPH (0.49 mg/ml) and ABTS (0.24 mg/ml) suggest that T. violacea can be used as potential antioxidant agents. For the antimicrobial activity using disc diffusion, the extracts exhibited appreciable antibacterial activities against Bacillus subtilis, Serratia marcescens, Staphylococcus aureus and S. epidermidis. The highest zone of inhibition was observed for S. epidermidis at 19.50 ± 0.87 mm. The MIC results revealed that the plant extract of T. violacea was moderately active against B. subtilis, S. aureus, S. epidermidis, E. coli, and S. marcescens with MIC value of 2.5 mg/ml. However, the antimicrobial effect of the extract on S. epidermidis was bactericidal when compared to the bacteriostatic effect on the other active microorganisms. The antithrombogenic results on the polystyrene discs showed a significant reduction in the number of platelets that adhered on the polystyrene surfaces treated with plasma mixed with 100 μg/ml of plant extract when compared to the untreated control and the 1000 μg/ml treatment. For the 1000 μg/ml treatment, there was a significant increase in the number of platelets that adhered to polystyrene surfaces. These results were confirmed by the fluorescence and SEM results which showed a higher platelet count for the 1000 μg/ml treatment when compared to the other groups. The whole blood clotting kinetics study showed delayed blood clotting with the 100 μg/ml treatment over a period of 60 min when compared to the untreated control and the 1000 μg/ml treatment. These results correspond with the lower platelet adhesion observation and thus confirm the anticlotting properties of T. violacea aqueous leaf extracts at lower concentrations. The mean diameter of the scaffolds was recorded on the SEM as 275.60 ± 60.65 nm, 193 ± 30 nm and 537 ± 138 nm for the PCL, 10% Tvio and 15% Tvio scaffolds, respectively. The FTIR spectrum revealed the presence of amide groups as well hydroxyl O–H stretching groups which were the characteristic groups for the presence of T. violacea plant extracts in the polycaprolactone. The EDS results showed the presence of potassium, chlorine and sulphur compounds which were only present in the T. violacea scaffolds in addition to the carbon, oxygen and silicon observed in the PCL scaffold. The fabricated scaffolds were then used to evaluate platelet adhesion and activation on blood plasma in the absence and presence of 100 and 1000 μg/ml T. violacea aqueous leaf extracts. The results showed that the 10% Tvio scaffold was more effective in inhibiting platelet adhesion and activation at every treatment group especially when plasma was used in the absence of T. violacea plant extracts. A similar observation to the polystyrene study was observed were addition of 1000 μg/ml of plant extract resulted in the highest number of activated platelets. The study suggests the potential of the 10% Tvio scaffold in the prevention of platelet adhesion and aggregation. The in vitro cell adhesion, proliferation and differentiation of adipose derived stem cells (ADSCs) on the fabricated T. violacea loaded PCL nanofibers was then evaluated. The LDH assay illustrated less activity on the 10% Tvio scaffold when compared to PCL and 15% Tvio scaffolds however, none of the scaffolds were considered as toxic. The alamar blue assay was used for viability after 4 and 7 days of culture. The results showed a significant increase in cell viability for all scaffolds from day 4 to day 7 with the 10% Tvio scaffold having the highest overall cell viability for both day 4 and day 7 of cell cultures. Immunofluorescence staining was then used to count the number of cells using DAPI (4′,6-diamidino-2-phenylindole) stained images and illustrated that the T. violacea incorporated scaffolds supported better cell growth compared to the PCL scaffold. Cell morphology on the T. violacea scaffolds was denser and spread out into cellular extensions when compared to the PCL scaffold after 7 days of cell culture, supporting the higher number of adhered cells from the fluorescence results. For the long term cell study after week 1 and 3, the ALP results showed a significant difference in ALP activity between week 1 and week 3 for all scaffolds. The highest ALP activity was observed for the 15% Tvio scaffolds which is a marker for initial phase of bone matrix deposition. The designed T. violacea scaffolds supported better cell growth compared to the PCL scaffold and their morphology was more spread out and covered the entire surface of the scaffolds after week 3. Lastly, the cell count and osteocalcin differentiation was more prominent on 10% Tvio scaffold indicating higher levels of the protein marker for bone formation. Thus, supporting the use of the 10% Tvio scaffold for long-term cell studies. In conclusion, the results of this study indicated that the aqueous extract of T. violacea is rich is phytochemicals and also possess a broad range of pharmaceutically important compounds which may be attributed to the high antioxidant and antimicrobial activities identified. The results from this study suggest that T. violacea aqueous extracts have antithrombogenic properties at lower concentrations. Scaffolds fabricated with the incorporation of T. violacea plant extract also confirm the potential antiplatelet activity of the fabricated 10% Tvio scaffold. The results also suggest the potential of the fabricated 10% Tvio scaffold to enhance cell adhesion, proliferation and differentiation over long-term cell studies. It can thus be recommended that T. violacea may be useful for tissue engineering applications and bone repair with prospects of preventing cardiovascular diseases associated with bone defects. This research study has provided the foundation for clinical evaluation and outlined the potential effects of T. violacea aqueous leaf extracts as a clinical drug.