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Browsing Faculty of Applied and Computer Science by Subject "2³ Factorial design"

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    An investigation of the immunomodulatory effects of crude extracts from Carpobrotus edulis on macrophages in vitro
    (Vaal University of Technology, 2024-12) Laloo, Neelan; Ssemakalu, S. S., Prof.; Pillay, M., Prof.
    Carpobrotus edulis is an edible halophyte which is used widely in traditional medicine in South Africa for a variety of ailments. The plant has antioxidant, anti-inflammatory, antibacterial, antifungal and anticancer properties and is used in the treatment of skin and wound infections, insect bites and mouth and throat infections. Despite its wide use as a medicinal plant, empirical research focusing specifically on its immunomodulatory effects remains sparse. This study aimed to bridge this knowledge gap by rigorously investigating the immunomodulatory effects of C. edulis on RAW264.7 macrophage cells using aqueous and methanol extracts. Two 2³ factorial designs were used to determine the optimal parameters to maximize the yield of crude leaf extracts using the maceration technique. The variables included solvent (water or methanol), pH (5 or 9), extraction temperature (25 or 40℃) and extraction duration (72 or 168 h). Design Expert 11 software was used to determine which extraction parameters produced the highest yield and phytochemical content. Quantitative phytochemical analysis was conducted using the Folin-Ciocalteau and Aluminium Chloride methods to determine total phenolic concentration (TPC) and total flavonoid concentration (TFC), respectively. The highest extraction yield (31.03 ± 0.58%) with water obtained at pH 9, for 72 hours at 40℃, had a TPC of 6.42 ± 0.03 mg Gallic Acid Equivalent per gram of extract. The highest yield with methanol (64.21 ± 2.12%) was obtained at pH 9, 168 hours and 40℃. The highest TPC (7.44 ± 0.50 mg GAE/g of extract) with methanol was obtained at pH 9, 72 hours of extraction, and 25℃. The radical scavenging activity of the extracts was determined using the ABTS and DPPH assays. For the aqueous extracts, the conditions which produced the highest yield and TPC (pH 9, 72 h and 40℃) also produced the lowest IC50 values for both assays – 140.77 μg/mL for ABTS and 298.28 μg/mL for DPPH. However, with methanol different extraction parameters produced differing results. The lowest IC50 value for the ABTS assay (26.79 μg/mL) was obtained at pH 5, 168 hours of extraction and 25℃; whereas the lowest IC50 value for the DPPH assay (109.84 μg/mL) was from the extract obtained at pH 9, 72 hours extraction time and at 25℃. All the IC50 values were higher than those for the positive control, ascorbic acid. Strong correlation was found between the TPC and radical scavenging activity for the aqueous and methanol extracts – as the TPC increased, the IC50 decreased. Using the DPPH results, r values were -0.952 and -0.759 for the aqueous and methanol extracts, respectively. For the ABTS assay, the correlation coefficients were -0.879 and -0.320 for the aqueous and methanol extracts, respectively. The effect of the extracts on the metabolic activity and protein content of the RAW264.7 cells was determined using the CellTiter Blue® (CTB) and Sulforhodamine B (SRB) assays, respectively. The cells were metabolically active even after treatment with 3000 μg/mL of the aqueous extract. However, protein content decreased as the aqueous extract concentration exceeded 1000 μg/mL. The aqueous extract with the highest metabolic activity, using the CTB assay, was obtained at pH 5, 168 hours and 40℃. However, the SRB assay showed the highest protein content in an extract obtained using the same time and temperature, but at pH 9. RAW264.7 cells treated with the methanol extracts showed increases in metabolic activity of greater than 1200% than the untreated control cells. These extracts were both obtained at 40℃ and after 72 hours of extraction except that one was at pH 5 and the other at pH 9. The latter parameters also produced the highest protein content. The potential pro- or anti-inflammatory effect of the extracts was determined using the Griess assay for nitric oxide production. RAW264.7 cells stimulated with lipopolysaccharide (LPS) at 100 ng/mL were treated with extract at concentrations from 1 μg/mL to 1500 μg/mL. In the cells treated with three of the eight aqueous extracts, the nitric oxide production increased as the extract concentration increased. Cells treated with an aqueous extract obtained at pH 9, and 72 hours of extraction at 25℃. produced the highest NO. Two treatments did not produce NO for concentrations of the aqueous extract above 10 μg/mL; these extracts were produced at 40℃ and 168 hours at either pH value. None of the cells treated with methanol extracts (at concentrations above 100 μg/mL) produced NO. The aqueous extract obtained at pH 9, 40℃ and 168 hours and the one for methanol obtained at pH 9, 40℃ and 72 h were selected for further study based on their viability, proliferative potential, and low nitric oxide production. Different concentrations of these were used to treat LPS-stimulated RAW264.7 cells to obtain RNA at 24 and 48 hours. The RNA was converted to cDNA, which was amplified using qPCR to determine the expression levels of three genes associated with the M1 macrophage phenotype (Stat1, NF-κB, Nos2) and three associated with the M2 phenotype (Stat6, Irf4, Arg1). The aqueous extract did not trigger an inflammatory response, as the expression levels (calculated as fold change 2-(ΔΔCt)) were close to zero for NF-κB and Nos2 suggesting that the expression was significantly downregulated (p = 0.0009). The aqueous extract at 1500 μg/mL did significantly (p = 0.0002) increase in the expression of Stat1 after 24 hours. The aqueous extract did not increase the expression of the M2 genes either. Only a slight increase in Irf4 expression was observed in comparison with the untreated control cells (p > 0.05). The response of the M1 genes to the methanol and aqueous extract was similar. A significant (p < 0.0001) increase in Stat6 and a slight increase in Arg1 (p = 0.0179) were detected after 24 hours in response to the methanol extract at 250 μg/mL. To further investigate the response of the cells to the two C. edulis extracts, a pre-mixed Luminex MAP® cytokine kit was used to detect the expression of cytokines and chemokines related to the M1 and M2 macrophages. At concentrations up to 1000 μg/mL and 48 hours of treatment, the aqueous extract significantly increased the expression of the M1 molecules CCL2 and GM-CSF (p < 0.0001) and at up to 500 μg/mL, CXCL10 (p = 0.0003). Molecules associated with the M2 phenotype only increased in concentration after 48 hours at extract concentrations up to 1000 μg/mL. TNF-α and IL-6, which are expressed by both M1 and M2 macrophages increased in concentration after treatment with the aqueous extract up to 1000 μg/mL (p < 0.0001 for both). A similar response was observed for cells treated with the methanol extract up to 100 μg/mL, but onlythe IL-6 concentration was significantly different from the control (p < 0.0001). The M1 molecules CCL2, GM-CSF and CXCL10 increased significantly in comparison with the untreated cells (p < 0.0001) when treated with up to 100 μg/mL of the methanol extract. These results suggest that in the absence of a strong anti-inflammatory response, the cells are being polarized towards an M1 phenotype. High concentrations of GM-CSF attract monocytes and macrophages to the site of injury. This suggests that the two C. edulis extracts used here are nontoxic and have an immunostimulatory effect on the RAW264.7 cells. This study showed that the therapeutic effects of C. edulis appear to be dependent on extraction conditions and the concentration of the extracts.