Ultrastructure for the diagnosis of primary ciliary dyskinesia in South Africa, a resource-limited setting

Introduction: International guidelines recommend a multi-faceted approach for successful diagnoses of primary ciliary dyskinesia (PCD). In the absence of a gold standard test, a combination of genetic testing/microscopic analysis of structure and function/nasal nitric oxide measurement is used. In resourcelimited settings, often none of the above tests are available, and in South Africa, only transmission electron microscopy (TEM) is available in central anatomical pathology departments. The aim of this study was to describe the clinical and ultrastructural findings of suspected PCD cases managed by pediatric pulmonologists at a tertiary-level state funded hospital in Johannesburg. Methods: Nasal brushings were taken from 14 children with chronic respiratory symptoms in keeping with a PCD phenotype. Ultrastructural analysis in accordance with the international consensus guidelines for TEM-PCD diagnostic reporting was undertaken. Results: TEM observations confirmed 43% (6) of the clinically-suspected cases (hallmark ultrastructural defects in the dynein arms of the outer doublets), whilst 57% (8) required another PCD testing modality to support ultrastructural observations. Of these, 25% (2) had neither ultrastructural defects nor did they present with bronchiectasis. Of the remaining cases, 83% (5) had very few ciliated cells (all of which were sparsely ciliated), together with goblet cell hyperplasia. There was the apparent absence of ciliary rootlets in 17% (1) case. Discussion: In resource-limited settings in which TEM is the only available testing modality, confirmatory and probable diagnoses of PCD can be made to facilitate early initiation of treatment of children with chronic respiratory symptoms

In Vitro Studies on Antioxidant and AntiParasitic Activities of Compounds Isolated from Rauvolfia caffra Sond

As part of an ongoing study of natural products from local medicinal plants, the methanol extract of stem bark of Rauvolfia caffra Sond was investigated for biological activity. Column chromatography and preparative thin-layer chromatography were used to isolate lupeol (1), raucaffricine (2), N-methylsarpagine (3), and spegatrine (4). The crude extract, fractions and isolated compounds were tested for anti-oxidant, antitrypanosomal and anti-proliferation activities. Two fractions displayed high DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity and reducing power with IC50 (The half maximal inhibitory concentration) and IC0.5 values of 0.022 ± 0.003 mg/mL and 0.036 ± 0.007 mg/mL, and 0.518 ± 0.044 mg/mL and 1.076 ± 0.136 mg/mL, respectively. Spegatrine (4) was identified as the main antioxidant compound in R. caffra with IC50 and IC0.5 values of 0.119 ± 0.067 mg/mL and 0.712 ± 0 mg/mL, respectively. One fraction displayed high antitrypanosomal activity with an IC50 value of 18.50 μg/mL. However, the major constituent of this fraction, raucaffricine (2), was not active. The crude extract, fractions and pure compounds did not display any cytotoxic effect at a concentration of 50 μg/mL against HeLa cells. This study shows directions for further in vitro studies on the antioxidant and antitrypanosomal activities of Rauvolfia caffra Sond

Isolation and structure elucidation of bioctive compounds from Rauvolfia Caffra Sond

MSc (Chemistry)Department of ChemistryRauvolfia caffra Sond, a species of evergreen trees and shrubs in the dogbane family, (Apocynaceae), is used as a medicinal plant among traditional communities in many countries for the treatment of malaria, diabetes, coughs, gastrointestinal disturbances, skin infections, impotence, insomnia, diarrhoea, dysentery, scabies, worm infections, and both parasitic and microbial infections. Phytochemical studies have revealed that indole alkaloids are the major constituents of the stem bark. However, there are limited studies linking the compounds with the ethnomedicinal uses. The aim of this study is to isolate and characterize bioactive compounds from Rauvolfia caffra Sond. The highest phenolic content found in a fraction was 16.06±0.125 mg GAE/g, while the highest flavonoid content measured was 9.453±0.081 mg QE/g. In the DPPH free radical scavenging activity and reducing power tests, a lowest IC50 value of 0.022±0.003 μg/mL and IC0.5 value 0.518±0.044 μg/mL, respectively, was found. Six compounds were isolated from the stem bark, including lupeol, a pentacyclic tri-terpenoid isolated for the first time from the genus Rauvolfia; raucaffricine, a rare glycoalkaloid of the monoterpenoid indole class; N-methylsarpagine, an indole alkaloid isolated for the time from R. caffra and spegatrine, an indole alkaloid isolated for the first time from R. caffra, respectively. Concerning antimicrobial activity, the highest activity of a fraction was against B. cereus with MIC values as low as 12.5 mg/mL. One fraction at the tested concentration (250 μg/mL) decreased the viability of Plasmodium falciparum (4.149±6.979 %) with an IC50 value of 6.533 μg/mL. The crude extract and some fractions affected the viability of the Trypanosomes at the tested concentration (250 μg/mL), giving -0.133 ± 0.206 %, 11.334 ± 2.692 %, 1.026 ± 0.143 % and 20.769 ± 9.054 % with IC50 values of 18.50 μg/mL, 14.15 μg/mL, 15.58 μg/mL and 34.71 μg/mL, respectively. Furthermore, the fractions did not show significant cytotoxic effects at a concentration of 50 μg/mL.NR

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

Molecular Networking-Based Metabolome, In Vitro Antidiabetic and Anti-Inflammatory Effects of <i>Breonadia salicina</i> (Vahl) Hepper & J.R.I. Wood

Breonadia salicina (Vahl) Hepper & J.R.I. Wood is widely distributed throughout Africa. It is used ethnobotanically to treat various diseases. However, the metabolic profile of the Breonadia species is not well characterized and the metabolites that are responsible for the bioactivity of this plant remain unknown. Therefore, there is a need to determine the phytochemical and bioactivity profile to identify metabolites that contribute to the antidiabetic, anti-inflammatory and antiproliferation activity, including the genotoxicity and cytotoxic effects, of Breonadia salicina. The study is aimed at exploring the metabolomic profile antidiabetic, anti-inflammatory and antiproliferation activity, as well as the genotoxicity and cytotoxicity effects, of constituents of B. salicina. The compounds in the B. salicina extract were analyzed by ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and the resultant data were further analyzed using a molecular networking approach. The crude stem bark and root extracts showed the highest antidiabetic activity against α-amylase at the lowest test concentration of 62.5 µg/mL, with 74.53 ± 0.74% and 79.1 ± 1.5% inhibition, respectively. However, the crude stem bark and root extracts showed the highest antidiabetic activity against α-glucosidase at the lowest test concentration of 31.3 µg/mL, with 98.20 ± 0.15% and 97.98 ± 0.22% inhibition, respectively. The crude methanol leaf extract showed a decrease in the nitrite concentration at the highest concentration of 200 µg/mL, with cell viability of 90.34 ± 2.21%, thus showing anti-inflammatory activity. No samples showed significant cytotoxic effects at a concentration of 10 µg/mL against HeLa cells. Furthermore, a molecular network of Breonadia species using UPLC-QTOF-MS with negative mode electrospray ionization showed the presence of organic oxygen compounds, lipids, benzenoids, phenylpropanoids and polyketides. These compound classes were differentially distributed in the three different plant parts, indicating the chemical differences between the stem bark, root and leaf extracts of B. salicina. Therefore, the identified compounds may contribute to the antidiabetic and anti-inflammatory activity of Breonadia salicina. The stem bark, root and leaf extracts of B. salicina yielded thirteen compounds identified for the first time in this plant, offering a promising avenue for the discovery of new lead drugs for the treatment of diabetes and inflammation. The use of molecular networking produced a detailed phytochemical overview of this Breonadia species. The results reported in this study show the importance of searching for bioactive compounds from Breonadia salicina and provide new insights into the phytochemical characterization and bioactivity of different plant parts of Breonadia salicina

Metabolomic Profiling and Antioxidant Activities of Breonadia salicina Using 1H-NMR and UPLC-QTOF-MS Analysis

Breonadia salicina (Vahl) Hepper and J.R.I. Wood is widely used in South Africa and some other African countries for treatment of various infectious diseases such as diarrhea, fevers, cancer, diabetes and malaria. However, little is known about the active constituents associated with the biological activities. This study is aimed at exploring the metabolomics profile and antioxidant constituents of B. salicina. The chemical profiles of the leaf, stem bark and root of B. salicina were comprehensively characterized using proton nuclear magnetic resonance (1H-NMR) spectroscopy and ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The antioxidant activities of the crude extracts, fractions and pure compounds were determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging and reducing power assays. A total of 25 compounds were tentatively identified using the UPLC-QTOF-MS. Furthermore, the 1H-NMR fingerprint revealed that the different parts of plant had differences and similarities among the different crude extracts and fractions. The crude extracts and fractions of the root, stem bark and leaf showed the presence of α-glucose, β-glucose, glucose and fructose. However, catechin was not found in the stem bark crude extracts but was found in the fractions of the stem bark. Lupeol was present only in the root crude extract and fractions of the stem bark. Furthermore, 5-O-caffeoylquinic acid was identified in the methanol leaf extract and its respective fractions, while the crude extracts and fractions from the root and dichloromethane leaf revealed the presence of hexadecane. Column chromatography and preparative thin-layer chromatography were used to isolate kaempferol 3-O-(2″-O-galloyl)-glucuronide, lupeol, d-galactopyranose, bodinioside Q, 5-O-caffeoylquinic acid, sucrose, hexadecane and palmitic acid. The crude methanol stem bark showed the highest antioxidant activity in the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity with an IC50 value of 41.7263 ± 7.6401 μg/mL, whereas the root crude extract had the highest reducing power activity with an IC0.5 value of 0.1481 ± 0.1441 μg/mL. Furthermore, the 1H-NMR and UPLC-QTOF-MS profiles showed the presence of hydroxycinnamic acids, polyphenols and flavonoids. According to a literature survey, these phytochemicals have been reported to display antioxidant activities. Therefore, the identified hydroxycinnamic acid (caffeic acid), polyphenol (ellagic acid) and flavonoids (catechin and (epi) gallocatechin) significantly contribute to the antioxidant activity of the different parts of plant of B. salicina. The results obtained in this study provides information about the phytochemistry and phytochemical compositions of Breonadia salicina, confirming that the species is promising in obtaining constituents with medicinal potential primarily antioxidant potential

In Vitro Studies on Antioxidant and Anti-Parasitic Activities of Compounds Isolated from Rauvolfia caffra Sond

As part of an ongoing study of natural products from local medicinal plants, the methanol extract of stem bark of Rauvolfia caffra Sond was investigated for biological activity. Column chromatography and preparative thin-layer chromatography were used to isolate lupeol (1), raucaffricine (2), N-methylsarpagine (3), and spegatrine (4). The crude extract, fractions and isolated compounds were tested for anti-oxidant, antitrypanosomal and anti-proliferation activities. Two fractions displayed high DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity and reducing power with IC50 (The half maximal inhibitory concentration) and IC0.5 values of 0.022 &plusmn; 0.003 mg/mL and 0.036 &plusmn; 0.007 mg/mL, and 0.518 &plusmn; 0.044 mg/mL and 1.076 &plusmn; 0.136 mg/mL, respectively. Spegatrine (4) was identified as the main antioxidant compound in R. caffra with IC50 and IC0.5 values of 0.119 &plusmn; 0.067 mg/mL and 0.712 &plusmn; 0 mg/mL, respectively. One fraction displayed high antitrypanosomal activity with an IC50 value of 18.50 &mu;g/mL. However, the major constituent of this fraction, raucaffricine (2), was not active. The crude extract, fractions and pure compounds did not display any cytotoxic effect at a concentration of 50 &mu;g/mL against HeLa cells. This study shows directions for further in vitro studies on the antioxidant and antitrypanosomal activities of Rauvolfia caffra Sond

Isolation, Chemical Profile and Antimalarial Activities of Bioactive Compounds from Rauvolfia caffra Sond

In this study, the chemical profile of a crude methanol extract of Rauvolfia caffra Sond was determined by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Column chromatography and preparative thin layer chromatography were used to isolate three indole alkaloids (raucaffricine, N-methylsarpagine and spegatrine) and one triterpenoid (lupeol). The antiplasmodial activity was determined using the parasite lactate dehydrogenase (pLDH) assay. The UPLC-MS profile of the crude extract reveals that the major constituents of R. caffra are raucaffricine (m/z 513.2) and spegatrine (m/z 352.2). Fraction 3 displayed the highest antiplasmodial activity with an IC50 of 6.533 μg/mL. However, raucaffricine, isolated from the active fraction did not display any activity. The study identifies the major constituents of R. caffra and also demonstrates that the major constituents do not contribute to the antiplasmodial activity of R. caffra