Chemical and biological characterization of antibacterial compounds present in Ochna pretoriensis (Ochnaceae) leaf extracts

In preliminary work done in a tree leaf screening project in the Phytomedicine Programme (www.up.ac.za/phyto) Ochna pretoriensis acetone leaf extracts had good antibacterial activity against several important bacterial pathogens. The main aim of this study was to isolate and characterize antibacterial compounds present in the acetone leaf extract of Ochna species growing in South Africa. In a preliminary screening, the minimum inhibitory concentration of acetone leaf extracts of Ochna natalitia, Ochna pretoriensis, Ochna pulchra, Ochna gamostigmata, and Ochna. Serullata, against Staphylococcus aureus, Escherichia coli, Enterococcus faecalisand Pseudomonas aeruginosa were determined by using a serial microplate dilution assay. The number of antibacterial compounds in the extracts was also determined by bioautography against the same bacteria. The MIC values of the five species ranged from 0.039 mg/ml to 1.25 mg/ml. The lowest average MIC values observed were for O. Pretoriensis especially against E. Faecalis and E. Coli. The most sensitive organism to all the plants was E. coli. O. Pretoriensis had the lowest average MIC value and the highest total activity value of 1538 ml/g. Based on bioautography some of the Ochna species had antibacterial compounds with similar Rf values. The thin layer chromatography chemical profiles of the five plant extracts may be useful in the taxonomy of the genus. O. Pretoriensis was chosen for fractionation and isolation of antibacterial compound because it has the lowest average MIC values and highest total activity especially against E. Faecalis and E. coli. The acetone extract of O. Pretoriensis was fractionated into seven fractions (hexane, carbon tetrachloride, chloroform, ethyl acetate, 35% water in methanol, 70% water in methanol and butanol) by solvent-solvent fractionation. Only three of the seven fractions (carbon tetrachloride, chloroform, ethyl acetate fractions) had clearly defined antibacterial spots/lines on bioautograms. The three fractions were further fractionated using column chromatography from which three compounds were successfully isolated. The chemical structures of the isolated compounds were determined using NMR spectroscopy as β-Sitosterol (SS), ochnaflavone (OF) and ochnaflavone 7-O- methyl ether (OFME). Compounds that are related to sitosterol have activity against neurodegenerative disorders as well as estrogenic, analgesic, anti-inflammatory, anthelminthic and antimutagenic activity. OF and OFME are biflavonoids which belong to the group ochnaflavones previously characterized from Ochna obtusata. These compounds have anti-atherosclerotic, anti-inflammatory, and anti-tumor activity. They also inhibit lymphocytes proliferation, archidonic acid release and phospholipase activity. Moreover, OFME was reported to inhibit HIV-1 activity as well as HIV-1 reverse transcriptase activity. The antibacterial activity, and potential cytotoxic, genotoxic and antigenotoxic effects of the isolated compounds were determined. The MIC values ranged from 31.3 to 250 μg/ml. SS was more active against P. Aeruginosa with an MIC of 62.5 μg/ml, OF against P. Aeruginosa and E. Faecalis with MICs of 0.03 mg/ml and OFME against P. Aeruginosa with an MIC of 31.3 μg/ml. The isolated compounds were much less active than the positive control gentamycin. The compounds had low cytotoxic activity, with LC50 values of 193.8 μg/ml for β-Sitosterol, 125.9 μg/ml for OF and 125.9 μg/ml for OFME against Vero cells. The therapeutic indexes of the crude extract and the isolated compounds varied between 0.77 and 3.27, which is an indication of non-specific antibacterial activity i.e. general toxicity, thus the crude plant extract and compounds isolated from O. Pretoriensis can only be recommended for external applications. e.g. topical treatments. None of the compounds tested had potential genotoxic and/or antigenotoxic effects. The number of revertants in the mutagenicity experiments was less than twice the number of revertants in the negative control. The percentage inhibition of 4NQO in the antimutagenicity experiments were less than 45%. The results obtained in this case may be principally associated with the general toxicity of the test samples to the bacteria used in this study. Comparison of the total activity of the crude extract and the fractions gave a clear indication of synergic interaction of compounds in the crude extract to successfully inhibit the growth of the test pathogens. Approximately 76% of activity was lost in the 34% of dry mass lost during fractionation. Twelve percent of activity was present in the chloroform fraction and 6% in the carbon tetrachloride fraction. Despite the evidence for synergistic activity, the crude extract was also relatively toxic to the Vero cells with a therapeutic index of 0.8. As far as could be established, the antibacterial activity of members of the Ochna genus and the cytotoxicity of ochnaflavones were determined for the first time in the current study. The two most active antibacterial compounds (ochnaflavone and ochnaflavone 7-O- methyl ether) are being reported from this species for the first time. The relative safety of the crude extract and the compounds isolated from this plant was relatively low. Preparations of O. Pretoriensis may be safe in a topical application but internal use cannot be recommended for treating antibacterial infections before animal toxicity studies have been carried out. Caution is also required in using the isolated compounds or crude extracts for other applications. CopyrightDissertation (MSc)--University of Pretoria, 2009.Paraclinical Sciencesunrestricte

Apoptosis in cancer cells is induced by alternative splicing of hnrnpa2/b1 through splicing of bcl-x, a mechanism that can be stimulated by an extract of the South African medicinal plant, cotyledon orbiculata

Alternative splicing is deregulated in cancer and alternatively spliced products can be linked to cancer hallmarks. Targeting alternative splicing could offer novel effective cancer treatments. We investigated the effects of the crude extract of a South African medicinal plant, Cotyledon orbiculata, on cell survival of colon (HCT116) and esophageal (OE33 and KYSE70) cancer cell lines. Using RNASeq, we discovered that the extract interfered with mRNA regulatory pathways. The extract caused hnRNPA2B1 to splice from the hnRNPB1 to the hnRNPA2 isoform, resulting in a switch in the BCL2L1 gene from Bcl-xL to Bcl-xS causing activation of caspase-3-cleavage and apoptosis. Similar splicing effects were induced by the known anti-cancer splicing modulator pladienolide B. Knockdown of hnRNPB1 using siRNA resulted in decreased cell viability and increased caspase-3-cleavage, and over-expression of hnRNPB1 prevented the effect of C

In vitro cytotoxicity and genotoxicity of five Ochna species (Ochnaceae) with excellent antibacterial activity

Extracts and fractions of some Ochna species had excellent antibacterial activity. Before considering the potential therapeutic use of these extracts it is important to determine the safety of extracts. The cytotoxicity of Ochna natalitia, Ochna pretoriensis, Ochna pulchra, Ochna gamostigmata, and Ochna serrulata (Ochnaceae) was determined in monkey kidney (Vero) cells, human hepatocellular carcinoma (C3A) cells and bovine dermis cells using the mitochondrial viability MTT assay. Their potential mutagenic effects were also determined using the Ames testwith strains Salmonella typhimuriumTA98 and TA100 with andwithoutmetabolic activation. The LC50 values (the lethal concentration at which 50% of the cells are killed) of the extracts on the various cell lines ranged from 26 to 99 μg/ml. None of the plant species was mutagenic (mutagenic index values ≤ 1.59 for TA98 and ≤0.92 for TA100). In a previous study, we determined the antibacterial activity of the five extracts against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa. From this we calculated the selectivity index (SI) values by dividing the LC50 value by the minimum inhibitory concentration (MIC) to obtain the ratio of toxicity to bioactivity of each extract. The plant extracts had low SI values ≤ 1.307. This is a clear indication of non-selective toxicity, i.e. extracts are almost equally toxic to the bacteria and mammalian cell lines used in the assays. As a result, the extracts may have limited application as ingestible or intravenous therapeutic agents based on the in vitro findings. However, it may be necessary to also evaluate in vivo toxicity of the extracts in animal models as in vitro toxicity does not always equate to in vivo toxicity because of the difference in physiological microenvironment in live animals and tissue culture. Additionally, if it is the case that the toxic compounds are not the same as the active compounds, it may be possible to potentiate the extracts by the removal of toxic compounds and concentration of active compounds. The extracts may then be useful for development into treatments for topical bacterial infections.National Research Foundation of South Africa, the Medical Research Council and the University of Pretoria.http://www.elsevier.com/locate/sajbhb201

Novel polycarbo-substituted alkyl (thieno[3,2-c]quinoline)-2- carboxylates : synthesis and cytotoxicity studies

Direct one-pot base-promoted conjugate addition–elimination of 6,8-dibromo-4- chloroquinoline-3-carbaldehyde with methyl mercaptoacetate and subsequent cyclization afforded methyl [(6,8-dibromothieno[3,2-c]quinoline)]-2-carboxylate. The latter undergoes Suzuki-Miyaura cross-coupling with arylboronic acids to yield exclusively the corresponding alkyl [(6,8-diarylthieno[3,2-c]quinoline)]-2-carboxylates,. The cytotoxicity of the prepared compounds was evaluated against the human breast cancer cell line MCF-7 using the MTT assay. The effects of compounds 2, 3c and 4d on cell kinetics were further determined using the xCELLigence Real Time Cell Analysis (RTCA) system. In both the MTT assay and Real Time Cell Analysis, the compounds inhibited cancer cell growth in a dose- and time-dependent manner. Furthermore, on the basis of the calculated LC50 values, the compounds compared favourably with nocodazole, a well-established anticancer drug.University of South Africa and the National Research Foundation in South Africa.http://www.mdpi.com/journal/hb201

Apoptosis in Cancer Cells Is Induced by Alternative Splicing of hnRNPA2/B1 Through Splicing of Bcl-x, a Mechanism that Can Be Stimulated by an Extract of the South African Medicinal Plant, Cotyledon orbiculata

Alternative splicing is deregulated in cancer and alternatively spliced products can be linked to cancer hallmarks. Targeting alternative splicing could offer novel effective cancer treatments. We investigated the effects of the crude extract of a South African medicinal plant, Cotyledon orbiculata, on cell survival of colon (HCT116) and esophageal (OE33 and KYSE70) cancer cell lines. Using RNASeq, we discovered that the extract interfered with mRNA regulatory pathways. The extract caused hnRNPA2B1 to splice from the hnRNPB1 to the hnRNPA2 isoform, resulting in a switch in the BCL2L1 gene from Bcl-xL to Bcl-xS causing activation of caspase-3-cleavage and apoptosis. Similar splicing effects were induced by the known anti-cancer splicing modulator pladienolide B. Knockdown of hnRNPB1 using siRNA resulted in decreased cell viability and increased caspase-3-cleavage, and over-expression of hnRNPB1 prevented the effect of C. orbiculata extract on apoptosis and cell survival. The effect of the hnRNPA2/B1 splicing switch by the C. orbiculata extract increased hnRNPA2B1 binding to Bcl-xl/s, BCL2, MDM2, cMYC, CD44, CDK6, and cJUN mRNA. These findings suggest that apoptosis in HCT116, OE33, and KYSE cancer cells is controlled by switched splicing of hnRNPA2B1 and BCL2L1, providing evidence that hnRNPB1 regulates apoptosis. Inhibiting this splicing could have therapeutic potential for colon and esophageal cancers. Targeting hnRNPA2B1 splicing in colon cancer regulates splicing of BCL2L1 to induce apoptosis. This approach could be a useful therapeutic strategy to induce apoptosis and restrain cancer cell proliferation and tumor progression. Here, we found that the extract of Cotyledon orbiculata, a South African medicinal plant, had an anti-proliferative effect in cancer cells, mediated by apoptosis induced by alternative splicing of hnRNPA2B1 and BCL2L1

Microbiomics in collusion with the nervous system in carcinogenesis : diagnosis, pathogenesis and treatment

The influence of the naturally occurring population of microbes on various human diseases has been a topic of much recent interest. Not surprisingly, continuously growing attention is devoted to the existence of a gut brain axis, where the microbiota present in the gut can affect the nervous system through the release of metabolites, stimulation of the immune system, changing the permeability of the blood–brain barrier or activating the vagus nerves. Many of the methods that stimulate the nervous system can also lead to the development of cancer by manipulating pathways associated with the hallmarks of cancer. Moreover, neurogenesis or the creation of new nervous tissue, is associated with the development and progression of cancer in a similar manner as the blood and lymphatic systems. Finally, microbes can secrete neurotransmitters, which can stimulate cancer growth and development. In this review we discuss the latest evidence that support the importance of microbiota and peripheral nerves in cancer development and dissemination.The South African Medical Research Council (SAMRC).https://www.mdpi.com/journal/microorganismsam2022Surger

Natural compounds isolated from African mistletoes (loranthaceae) exert anti-inflammatory and acetylcholinesterase inhibitory potentials : in vitro and in silico studies

DATA AVAILABILITY STATEMENT: All data generated or analysed during this study are included in this published article.Please read abstract in article.The Central University of Technology operational expenses and the National Research Foundation (NRF) of South Africa.https://www.mdpi.com/journal/applsciParaclinical Science

A multinational review : oesophageal cancer in low to middle-income countries

Oesophageal cancer (OC) is an aggressive neoplasm that manifests in the gastrointestinal tract and is the result of numerous factors that can contribute to the development of the disease. These may include old age, nutritional deficiencies, oesophageal obstruction and food ingestion difficulties. Environmental factors serve a large role in increasing the risk of developing OC. Two factors that serve an increasing risk of developing OC are the use of tobacco and the consumption of alcohol. Genetic factors also exhibit a large effect on the risk of developing OC, for example, the causative genes in Black Africans differ from other races. OC is 3‑4 times more common among men than women. OC has been previously reported in >450 000 individuals worldwide, and its incidence is increasing. The current review compares OC in low to middle‑income countries with developed countries. The incidence of OC, particularly squamous cell carcinoma (SCC) is high in low and middle‑income countries. In developed countries, the incidence of SCC is low compared with adenocarcinoma. The majority of OC cases are diagnosed in the late stages of the disease, leading to high mortality rates. The current review aimed to discuss factors that contribute to the development of this disease in different geographical areas and genetic mechanisms governing these findings. The current review also aims to discuss the preventative treatment options for the disease, and also discusses the diagnosis and surveillance in five LMICs, including South Africa, China, Tanzania, India and Brazil.The Medical Research Council of South Africahttp://www.spandidos-publications.com/olam2021Internal MedicineObstetrics and Gynaecolog

The in vitro inhibition of genotoxicity by plant extracts and the isolation and characterization of antimutagenic compounds from Combretum microphyllum (Combretaceae)

The possibility of moderating the response of cells to a particular mutagen by phytomedicines opens new horizons in cancer prevention. On this basis, the search for antimutagens presents many possibilities for the discovery of new anticarcinogenic compounds. Determination of the antimutagenic potential of plant extracts is an important step in the discovery of new effective cancer chemopreventive agents. The main aim of this study was to isolate and characterize antimutagenic compounds active against 4-nitroquinoline 1-oxide (4NQO), mitomycin-C (MMC) and ethyl methanesulfonate (EMS) in vitro. Antioxidant compounds play a preventive role against mutation related diseases and thus may have potential antimutagenic activity. It was for this reason that methanol leaf extracts of 120 plant species from the existing plant material collection of tree leaves in the Phytomedicine Programme of the University of Pretoria were assayed for qualitative antioxidant activity. Almost 98% of the extracts (117) had well defined antioxidant compounds. From these 117 species, 31 were selected for investigation of qualitative antioxidant activity, total phenolic content, mutagenic and antimutagenic activity. Methanol extracts of the selected 31 species effectively reduced the DPPH free radical with EC50 values ranging from 1.20 ± 0.22 to 19.07 ± 1.50 μg/ml and total phenolic content measured in gallic acid equivalents (GAE) ranged from 5.17 ± 0.97 to 18.65 ± 3.86 mgGAE/mg extract. In some instances, the plant extracts had better antioxidant activity than the positive control, L-ascorbic acid (vitamin C) with EC50 value of 2.28 ± 0.02 μg/ml. Only one plant (Halleria liucida) extract was mutagenic in the Ames test using Salmonella typhimurium TA98 and TA100. Upon investigating antimutagenicity, the percentage inhibition of 4-NQO in the Ames test ranged from 8.8 ± 2.4 to 76.7 ± 4.7% in S. typhimurium TA98 and from 0.8 ± 6.9 to 99.00 ± 2.9% in TA100. There was a direct correlation between the presence of antioxidant activity and antimutagenic activity of the plant extracts confirming the initial hypothesis of the study. Some of the plant extracts had a comutagenic effect as they potentiated the mutagenic effects of 4-NQO. From the 31 plant species investigated, 4 species (2 antimutagenic and 2 comutagenic) were selected for in-depth genotoxicity (mutagenicity and antimutagenicity) studies using the Ames test, cytokinesis block micronucleus/cytome assay and alkaline single-cell gel electrophoresis/comet assay. These species were: Combretum microphyllum Klotzsch (Combretaceae), Leucospermum erubescens Rourke (Proteaceae), Kirkia wilmsii Engl (Simaroubaceae) and Thespisia acutiloba (Baker f) Exell & Mendonça (Malvaceae). No plant extract was mutagenic in the Ames test and micronucleus/cytome assay. However, some extracts were slightly mutagenic in the comet assay and this may be attributed to cytotoxicity rather than genotoxic effects. The extracts of C. microphyllum and L. erubescens inhibited the mutagenic effects of 4-NQO (S. typhimurium TA98 and TA100) and MMC (S. typhimurium TA102) with values from 10% to more than 30% in the Ames test. However, extracts of K. wilmsii and T. acutiloba enhanced the mutagenic effects of 4-NQO and MMC in all tester strains. Extracts of C. microphyllum and L. erubescens prevented micronuclei induction by up to 65.9%, chromosomal rearrangements by 51.9% and gene amplification by 86.1% in the micronucleus/cytome assay. In the comet assay, there was a clear dose dependent decrease in comet tail length. Based on the preliminary screening results, in depth genotoxicity investigation results and availability of plant material, C. microphyllum was selected for the isolation of antimutagenic compounds. Bioassay-guided fractionation of the crude methanol leaf extract, using the Ames test (S. typhimurium TA98, TA100 and TA102) as an indicator of antimutagenicity was used for the isolation of antimutagenic compounds. To simplify the isolation of the antimutagenic compounds, the methanol leaf extract of C. microphyllum was first separated into four fractions based on polarity using a solvent-solvent fractionation procedure. The solvents used were: hexane, ethyl acetate, water and butanol. The fractions were subjected to antimutagenicity testing in the Ames test. The ethyl acetate fraction was the most active in all three tester strains i.e. S. typhimurium TA98, 100 and 102 with percentage antimutagenicity of up to 32.7 ± 2.1, 30.6 ± 3.8% and 21.4 ± 3.1% respectively at the highest concentration (5 mg/ml) assayed. The activity was dose dependent. Bioactivity-guided fractionation of the ethyl acetate fraction by open column chromatography led to the isolation of three compounds. The structures of the compounds were determined using NMR and were identified as n-tetracosanol (C1), eicosanoic acid (C2) and olean-12-ene-28-oic acid (arjunolic acid) (C3). The antimutagenic activity in the Ames test using S. typhimurium TA98, 100 and 102, and the cytotoxicity on C3A human hepatocarcinoma cell line of the isolated compounds were determined. In the Ames test, the compounds were assayed at concentrations 10 times lower than the concentrations used for the crude extract and fraction. Arjunolic acid was more active in all three tester strains with percentage antimutagenicity of up to 41.9 ± 9.6%, 35.8 ± 1.5% and 43.8 ± 0.18% in S. typhimurium TA98, 100 and 102 respectively, followed by eicosanoic acid and lastly n-tetracosanol. Overall, the compounds had much higher antimutagenic activity than the crude extract and the fractions. The quantities of the isolated compounds were not sufficient to allow testing in the micronucleus/cytome assay and comet assay. The compounds were not cytotoxic at the highest concentration tested i.e. 200 μg/ml. n-Tetracosanol and eicosanoic acid had LC50 values > 200 μg/ml (with percentage cell viability of 59.7 ± 7.2and 50.1 ± 6.2% at the highest concentration respectively) and arjunolic acid had LC50 value of 106.4 ± 5.1 μg/ml. Arjunolic acid was the only compound with pronounced antioxidant activity with an EC50 value of 6.3 ±0.3 μg/ml. This was moderate antioxidant activity compared to that of vitamin C. n-Tetracosanol and eicosanoic acid did not have antioxidant activity. The antimutagenic activity of arjunolic acid at least in part may be attributed to its antioxidant activity resulting in the detoxification of reactive oxygen species produced during mutagenesis, but other mechanisms were probably involved with the other compounds. These results also show that it would not have worked by just isolating antioxidant compounds and testing these for genotoxicity. Combretum microphyllum has potential antimutagenic activity and protective effects against cancer since the crude extract of this plant species effectively inhibits the genotoxic end-points induced by 4-nitroquinoline 1-oxide (4NQO), mitomycin-C (MMC) and ethyl methanesulfonate (EMS) in vitro. This conclusion is supported by the fact that chromosomal biomarkers of genomic instability are relevant to cancer and that genotoxicity involving gene mutations, chromosomal aberrations and rearrangements and DNA strand breakages play a major role in cancer initiation. Pure compounds isolated from the ethyl acetate fraction of this extract had antimutagenic effects in the Ames test using S. typhimurium TA98, 100 and 102. The compounds had better activity compared to the crude extract and fractions at concentrations 10 times lower. The compounds were not cytotoxic against C3A human hepatocarcinoma cells at the highest concentration tested of 200 μg/ml. Overall, these types of studies on plant extracts may provide leads to the discovery of chemopreventive agents that can be used to develop pharmacologically active agents for prevention of chronic degenerative diseases. The compounds isolated in this study have been previously isolated from other plant species and are known to possess numerous biological activities, including amongst others: antioxidant activity, antimicrobial activity, antitumor effects and anticholinesterase activity. Even when new chemical structures are not found in medicinal plant research studies and drug discovery, known compounds with new biological activities can provide important drug leads Some of the extracts, fractions or compounds may be used as elements in nutraceuticals, functional foods and other applications as antimutagens to limit the possibility of mutations. Antimutagens and anticarcinogens play a major role in the primary prevention of mutations and cancer development by lowering the frequency or rate of mutations. This is the first report of the isolation of n-tetracosanol, eicosanoic acid and arjunolic acid from C. microphyllum. We also report for the first time the potential antigenotoxic effects of the crude extract, fractions and the compounds isolated from C. microphyllum.Thesis (PhD)--University of Pretoria, 2014.tm2015Paraclinical SciencesPhDUnrestricte

Regulation of alternative splicing in obesity-induced hypertension

Obesity is the result of genetics which predisposes an individual to obesity and environmental factors, resulting in excessive weight gain. A well-established linear relationship exists between hypertension and obesity. The combined burden of hypertension and obesity poses significant health and economic challenges. Many environmental factors and genetic traits interact to contribute to obesity-linked hypertension. These include excess sodium re-absorption or secretion by the kidneys, a hypertensive shift of renal-pressure and activation of the sympathetic nervous system. Most individuals suffering from hypertension need drugs in order to treat their raised blood pressure, and while a number of antihypertensive therapeutic agents are currently available, 50% of cases remain uncontrolled. In order to develop new and effective therapeutic agents combating obesity-induced hypertension, a thorough understanding of the molecular events leading to adipogenesis is critical. With the advent of whole genome and exome sequencing techniques, new genes and variants which can be used as markers for obesity and hypertension are being identified. This review examines the role played by alternative splicing (AS) as a contributing factor to the metabolic regulation of obesity-induced hypertension. Splicing mutations constitute at least 14% of the disease-causing mutations, thus implicating polymorphisms that effect splicing as indicators of disease susceptibility. The unique transcripts resulting from the alternate splicing of mRNA encoding proteins that play a key role in contributing to obesity would be vital to gain a proper understanding of the genetic causes of obesity. A greater knowledge of the genetic basis for obesity-linked hypertension will assist in the development of appropriate diagnostic tests as well as the identification of new personalized therapeutic targets against obesity-induced hypertension.The South African Medical Research Council (SAMRC) and the National Research Foundation (NRF).https://www.dovepress.com/diabetes-metabolic-syndrome-and-obesity-targets-and-therapy-journalam2019Obstetrics and Gynaecolog