Phytochemistry and biological studies of constituents from Breonadia Salicina (VAHL) Hepper and J. R. I. Wood

PhD (Chemistry)Department of ChemistryBreonadia salicina (Vahl) Hepper and J.R.I. Wood is a tree used widely to treat numerous infectious diseases in South Africa and other African countries, and ethnopharmacological studies have shown a number of biological activities of the crude extracts. Furthermore, phytochemical studies have indicated that the stem bark is rich in tannins, and alkaloids have been isolated from the twigs and leaves. However, few studies have correlated the phytochemistry to the physiological activties. This study aimed to explore the phytochemistry of B. salicina using a metabolomic approach and correlating the phytochemistry to the biological activities for possible drug development. Samples of B. salicina were collected at Fondwe, Limpopo Province, South Africa. Phytochemical studies followed a metabolomics approach, with repeated column chromatography and preparative thin-layer chromatography yielding a number of pure compounds. Antimalarial and antitrypanosomal activities of the crude extracts, pure compounds, fractions, and seasonal samples were evaluated using the parasite lactate dehydrogenase (pLDH) and Trypanosoma brucei assays, respectively. Furthermore, the antioxidant activities of the crude extracts, fractions and pure compounds were evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) and reducing power assays. The antimycobacterial activities of the crude extracts and fractions were determined against Mycobacterium tuberculosis (H37RvMA strain), and anti-diabetic activities of the crude extracts were determined using α-amylase and α-glucosidase inhibition assays. The anti-inflammatory activities of the crude extracts were assessed using the Griess assay, while the in vitro toxicology of the crude extracts was evaluated using cell toxicity, NucRed nuclei dye, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Hoechst 33342/Propidium iodide (PI) dual staining assays. Eight compounds (bodinioside Q, 5-O-caffeoylquinic acid, D-galactopyranose, hexadecane, kaempferol 3-O-(2’’-O-galloyl)-glucuronide, lupeol, palmitic acid, and sucrose) were isolated for the first time from the root, stem bark, and leaf extracts of B. salicina, while 51 compounds were tentatively identified from the crude extracts, fractions and seasonal samples by UPLC-QTOF-MS and 1H-NMR spectroscopy. The aligned UPLC-QTOF-MS data were analysed chemometrically to determine the chemical variability of the crude extracts of the roots, stem bark, and leaves collected during four different consecutive seasons. The principal component analysis (PCA) model, hierarchical cluster analysis (HCA) and partial least square discriminant analysis (PLS-DA) were constructed. These indicated the presence of two main clusters related to the different parts of the plant (root, stem bark, and leaf). In the antiplasmodial activity, three fractions collected in the first year affected the viability of Plasmodium falciparum, with viabilities of 16.16 ± 8.63 %, 27.01 ± 4.47 % and 31.07 ± 6.71 %; and IC50 values of 2.19 ± 0.09 μg/mL, 1.91 ± 0.05 μg/mL and 3.02 ± 0.08 μg/mL, respectively, at a concentration of 10 μg/mL. However, all the tested crude extracts and fractions collected in the first year contained potent antiplasmodial activities at a concentration of 50 μg/mL. Furthermore, the dichloromethane leaf extract collected in the second year in autumn, winter, spring and summer displayed high activities, with viabilities of 18.57 ± 1.99 %, 32.07 ± 4.91 %, 38.11 ± 5.07 % and 20.21 ± 5.19 % at a concentration of of 50 μg/mL with IC50 values of 7.90 ± 0.06 μg/mL, 18.15 ± 0.07 μg/mL, 19.40 ± 0.06 μg/mL and 15.26 ± 0.05 μg/mL at a concentration of 300 μg/mL, respectively. The pure compounds, including kaempferol 3-O-(2"-O-galloyl)-glucuronide (1) and palmitic acid (8), caused a significant decrease in parasite viability at a concentration of 50 μg/mL, with viabilities of 29.37 ± 1.29 % and 24.97 ± 5.21 %; and IC50 values of 9.06 ± 0.036 μg/mL and 6.792 ± 0.046 μg/mL at a concentration of 200 μg/mL. In the antitrypanosomal test, the crude methanol leaf extract, dichloromethane leaf extract, and two fractions attained in the first year strongly affected the viability of trypanosomes at the tested concentration of (50 μg/mL), with a viability of 6.74 ± 0.06 %, 6.38 ± 2.15 %, 7.78 ± 0.08 % and 5.05 ± 0.35 %; and IC50 values of 11.4 ± 0.42 μg/mL, 10.6 ± 0.07 μg/mL, 2.0 ± 0.09 μg/mL and 7.1 ± 0.14 μg/mL at a concentration of 300 μg/mL, respectively. Furthermore, the crude methanol leaf extract collected in the second year in autumn, spring and summer displayed higher activities, with viabilities of 5.84 ± 0.38 %, 26.66 ± 3.91 % and 9.05 ± 0.80 % at a concentration of 50 μg/mL; and IC50 values of 12.0 ± 0.36 μg/mL, 5.2 ± 0.74 μg/mL and 10.6 ± 0.07 μg/mL at a concentration of 300 μg/mL, respectively. However, the crude dichloromethane leaf extract collected in the second year in autumn, winter, spring and summer displayed higher activities, with viabilities of 3.50 ± 0.59 %, 4.13 ± 0.06 %, 29.47 ± 1.25 % and 3.85 ± 0.10 % at a concentration of 50 μg/mL; and IC50 values of 4.6 ± 1.82 μg/mL, 5.1 ± 0.30 μg/mL, 5.1 ± 0.72 μg/mL and 4.0 ± 0.08 μg/mL at a concentration of 300 μg/mL, respectively. The isolated compounds, including bodinioside Q (4), kaempferol 3-O-(2"-O-galloyl)-glucuronide (1), lupeol (2), and palmitic acid (8), exhibited antitrypanosomal activity with viabilities of 12.99 ± 0.53 %, 20.38 ± 2.35 %, 5.46 ± 0.04 %, and 5.83 ± 0.28 % at a concentration of 20 μg/mL; and IC50 values of 4.0 ± 0.09 μg/mL, 1.1 ± 0.22 μg/mL, 4.2 ± 0.27 μg/mL and 5.7 ± 0.09 μg/mL, respectively; On the other hand, the reference drug pentamidine showed an IC50 of 10.2 ± 0.07 μg/mL. The anti-oxidant assays revealed that the crude stem bark extract had the highest DPPH free radical scavenging activity, with an IC50 of 41.7263 ± 7.6401 μg/mL. Furthermore, the crude root extract had the highest reducing power with an IC0.5 of 0.1481 ± 0.1441 μg/mL. In the antimycobacterial activity test, none of the tested plant samples produced significant antimycobacterial activity at a concentration of 90 μg/mL. All the samples produced a MIC value of >62.5 μg/mL against 7H9_ADC_GLU_TW, 7H9_ADC_GLU_N_TW and 7H9_ADC_GLY_TW media. Furthermore, the crude stem bark and root extracts showed very strong antidiabetic activity at the lowest tested concentration of 62.5 μg/mL, with an inhibition of 74.53 ± 0.737 % and 79.1 ± 1.494 % against α-amylase enzyme. However, for the α-glucosidase inhibition assay, the crude stem bark and root extracts showed complete inhibition at the lowest tested concentration of 31.3 μg/mL at 98.20 ± 0.15 % and 97.98 ± 0.22 %. The crude dichloromethane leaf extract showed a decrease in nitrite concentration at the highest concentration of 200 μg/mL, with a cell viability of 79.06 ± 1.88 %, indicating anti-inflammatory activity. The crude stem bark, root and methanol leaf extracts were not cytotoxic against Vero cells at the concentrations of 15.125 μg/mL, 31.25 μg/mL, 125 μg/mL and 250 μg/mL. Furthermore, none of the extracts were cytotoxic at the following concentrations: 50 μg/mL, 100 μg/mL and 200 μg/mL, against RAW 264.7 macrophages. However, the crude stem bark and root extracts showed cytotoxic effects against Vero cells at 250 μg/mL.National Research Foundation (NRF