Invasive Alien Species as a Potential Source of Phytopharmaceuticals: Phenolic Composition and Antimicrobial and Cytotoxic Activity of Robinia pseudoacacia L. Leaf and Flower Extracts.

Black locust (Robinia pseudoacacia L.), an invasive tree in Europe, commonly known for its negative impact on biodiversity, is a rich source of phenolic compounds recognized in traditional medicine. Since the metabolite profile depends on the environment and climate, this study aimed to provide the first LC-MS phytochemical screening of the black locust from the Istria region (Croatia). The compounds were extracted from leaves and flowers with 70% ethanol and 80% methanol. Total phenolics (TP) and flavonoids (TF), as well as antioxidant capacity (AC) measured by ABTS (17.49-146.41 mg TE/g DW), DPPH (24.67-118.49 mg TE/g DW), and FRAP (7.38-77.53 mg TE/g DW) assays, were higher in leaf than in flower extracts. Higher TP and total non-flavonoid (TNF) values were displayed in ethanolic than in methanolic extracts. In total, 64 compounds were identified, of which flavonols (20) and hydroxycinnamic acid derivatives (15) were the most represented. Flavanols such as catechin dominated in leaf extracts, followed by flavonols, with kaempferol glucuronyl rhamnosyl hexosides as the main compound, respectively. Flower extracts had the highest share of flavones, followed by ellagitannins, with luteolin dirhamnosyl hexosides and vescalagin, respectively, being predominant. The extracts had good quorum sensing, biofilm formation prevention, and eradicating capacity. The results provided new insights into the phytochemical properties of R. pseudoacacia as the first step toward its potential pharmaceutical use.

LC-DAD-MS Phenolic Characterisation of Six Invasive Plant Species in Croatia and Determination of Their Antimicrobial and Cytotoxic Activity.

Invasive plants' phytochemicals are important for their invasiveness, enabling them to spread in new environments. However, these chemicals could offer many pharmaceutical compounds or active ingredients for herbal preparations. This study provides the first LC-MS phytochemical screening of six invasive alien plant species (IAPS) in the Istria region (Croatia): Ailanthus altissima, Ambrosia artemisiifolia, Conyza canadensis, Dittrichia viscosa, Erigeron annuus, and Xanthium strumarium. The study aims to identify and quantify the phenolic content of their leaf extracts and assess their antimicrobial and cytotoxic potential. A total of 32 species-specific compounds were recorded. Neochlorogenic, chlorogenic, and 5-p-coumaroylquinic acids, quercetin-3-glucoside, and kaempferol hexoside were detected in all the tested IAPS. Hydroxycinnamic acid derivatives were the main components in all the tested IAPS, except in E. annuus, where flavanones dominated with a share of 70%. X. strumarium extract had the best activity against the tested bacteria, with an average MIC value of 0.11 mg/mL, while A. altissima and X. strumarium extracts had the best activity against the tested fungi, with an average MIC value of 0.21 mg/mL in both cases. All the plant extracts studied, except X. strumarium, were less cytotoxic than the positive control. The results provided additional information on the phytochemical properties of IAPS and their potential for use as antimicrobial agents.

Genotoxic effects of Dukhan: A smoke bath from the wood of Acacia seyal used traditionally by Sudanese women.

ETHNOBOTANICAL RELEVANCE: Smoke from the wood of Acacia seyal Delile has been used by Sudanese women for making a smoke bath locally called Dukhan. The ritual is performed to relieve rheumatic pain, smooth skin, heal wounds and achieve general body relaxation. AIM OF THE STUDY: The present study was designed to investigate the in vitro anti-inflammatory effect of the smoke condensate using cyclooxygenase -1 (COX-1) and -2 (COX-2) as well as its potential genotoxic effects using the bacterial-based Ames test and the mammalian cells-based micronucleus/cytome and comet assays. MATERIAL AND METHODS: The smoke was prepared in a similar way to that commonly used traditionally by Sudanese women then condensed using a funnel. Cyclooxygenase assay was used to evaluate its in vitro anti-inflammatory activity. The neutral red uptake assay was conducted to determine the range of concentrations in the mammalian cells-based assays. The Ames, cytome and comet assays were used to assess its potential adverse (long-term) effects. RESULTS: The smoke condensate did not inhibit the cyclooxygenases at the highest concentration tested. All smoke condensate concentrations tested in the Salmonella/microsome assay induced mutation in both TA98 and TA100 in a dose dependent manner. A significant increase in the frequency of micronucleated cells, nucleoplasmic bridges and nuclear buds was observed in the cytome assay as well as in the % DNA damage in the comet assay. CONCLUSIONS: The findings indicated a dose dependent genotoxic potential of the smoke condensate in the bacterial and human C3A cells and may pose a health risk to women since the smoke bath is frequently practised. The study highlighted the need for further rigorous assessment of the risks associated with the smoke bath practice.

Corrigendum: Anthelmintic activity of acetone extracts from South African plants used on egg hatching of Haemonchus contortus.

No abstract available.

Corrigendum: Investigation of the acaricidal activity of the acetone and ethanol extracts of 12 South African plants against the adult ticks of Rhipicephalus turanicus.

No abstract available.

Anti-influenza A virus activity of two Newtonia species and the isolated compound myricetin-3-o-rhamnoside.

BACKGROUND: Some viruses play a key role in the disturbance of the digestive system. The common viruses which cause infectious diarrhoea (gastroenteritis) include astrovirus, caliciviruses, coronavirus and torovirus which are single-stranded RNA viruses. Influenza A virus (H1N1) also causes diarrhoea in addition to being associated with respiratory symptoms. In preliminary studies, Newtonia hildebrandtii and N. buchananii leaf extracts had good antibacterial activity against some bacteria implicated in causing diarrhoea. The aim of this study was to evaluate the anti-influenza activity of two Newtonia species extracts and the isolated compound (myricitrin). METHODS: N. hildebrandtii and N. buchananii acetone, and MeOH: DCM (methanol-dichloromethane) leaf and stem extracts, and an antibacterial compound myricetin-3-o-rhamnoside (myricitrin), isolated from N. buchananii, were evaluated for their antiviral efficacy against influenza A virus (IAV) PR8/34/H1N1 as a model organism. The MTT and hemagglutination assays were used to assess the extracts and compound interference with cell viability and viral surface HA glycoprotein. The quantitative real-time PCR was performed to assess the viral load. RESULTS: Plant extracts of N. hildebrandtii and N. buchananii were effective against IAV. The extracts in combination with H1N1 showed highly significant antiviral activity (P < 0.01) and maintained cell viabilities (P < 0.05). Myricitrin was non-cytotoxic at concentration 104 µg/ml. Myricitrin was most effective against IAV in a co-penetration combined treatment, thereby confirming the inhibitory effect of this compound in the viral attachment and entry stages. Myricitrin treatment also resulted in the highest viability of the cells in co-penetration treatment. The activity of myricitrin indicates the potential of the extracts in controlling viral infection at the attachment stage. The antiviral effect of myricitrin on IAV load in MDCK cell culture was confirmed using quantitative real-time PCR. CONCLUSION: Data from this study support further research and development on Newtonia hildebrandtii, Newtonia buchananii and myricitrin to address diarrhoea and related conditions caused by viruses in both human and veterinary medicine. Further work needs to be conducted on the activity of the extracts and the purified compound on other viruses of importance which have similar symptoms to influenza virus such as the coronavirus which led to a recent global pandemic.

Inhibitory effect of Newtonia extracts and myricetin-3-o-rhamnoside (myricitrin) on bacterial biofilm formation.

BACKGROUND: Diarrhoea is a major health issue in both humans and animals and may be caused by bacterial, viral and fungal infections. Previous studies highlighted excellent activity of Newtonia buchananii and N. hildebrandtii leaf extracts against bacterial and fungal organisms related to diarrhoea-causing pathogens. The aim of this study was to isolate the compound(s) responsible for antimicrobial activity and to investigate efficacy of the extracts and purified compound against bacterial biofilms. METHODS: The acetone extract of N. buchananii leaf powder was separated by solvent-solvent partitioning into eight fractions, followed by bioassay-guided fractionation for isolation of antimicrobial compounds. Antibacterial activity testing was performed using a broth microdilution assay. The cytotoxicity was evaluated against Vero cells using a colorimetric MTT assay. A crystal violet method was employed to test the inhibitory effect of acetone, methanol: dichloromethane and water (cold and hot) extracts of N. buchananii and N. hildebrandtii leaves and the purified compound on biofilm formation of Pseudomonas aeruginosa, Escherichia coli, Salmonella Typhimurium, Enterococcus faecalis, Staphylococcus aureus and Bacillus cereus. RESULTS: Myricetin-3-o-rhamnoside (myricitrin) was isolated for the first time from N. buchananii. Myricitrin was active against B. cereus, E. coli and S. aureus (MIC = 62.5 µg/ml in all cases). Additionally, myricitrin had relatively low cytotoxicity with IC50 = 104 µg/ml. Extracts of both plant species had stronger biofilm inhibitory activity against Gram-positive than Gram-negative bacteria. The most sensitive bacterial strains were E. faecalis and S. aureus. The cold and hot water leaf extracts of N. buchananii had antibacterial activity and were relatively non-cytotoxic with selectivity index values of 1.98-11.44. CONCLUSIONS: The purified compound, myricitrin, contributed to the activity of N. buchananii but it is likely that synergistic effects play a role in the antibacterial and antibiofilm efficacy of the plant extract. The cold and hot water leaf extracts of N. buchananii may be developed as potential antibacterial and antibiofilm agents in the natural treatment of gastrointestinal disorders including diarrhoea in both human and veterinary medicine.

The ultrastructural damage caused by Eugenia zeyheri and Syzygium legatii acetone leaf extracts on pathogenic Escherichia coli.

BACKGROUND: Antibiotics are commonly added to livestock feeds in sub-therapeutic doses as growth promoters and for prophylaxis against pathogenic microbes, especially those implicated in diarrhoea. While this practice has improved livestock production, it is a major cause of antimicrobial resistance in microbes affecting livestock and humans. This has led to the banning of prophylactic antibiotic use in animals in many countries. To compensate for this, alternatives have been sought from natural sources such as plants. While many studies have reported the antimicrobial activity of medicinal plants with potential for use as phytogenic/botanical feed additives, little information exists on their mode of action. This study is based on our earlier work and describes ultrastructural damage induced by acetone crude leaf extracts of Syzygium legatii and Eugenia zeyheri (Myrtaceae) active against diarrhoeagenic E. coli of swine origin using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescent microscopy (FM). Gas chromatography/mass spectrometry (GC-MS) was used to investigate the chemical composition of plant extracts. RESULTS: The extracts damaged the internal and external anatomy of the cytoplasmic membrane and inner structure at a concentration of 0.04 mg/mL. Extracts also led to an increased influx of propidium iodide into treated bacterial cells suggesting compromised cellular integrity and cellular damage. Non-polar compounds such as α-amyrin, friedelan-3-one, lupeol, and ß-sitosterol were abundant in the extracts. CONCLUSIONS: The extracts of S. legatii and E. zeyheri caused ultrastructural damage to E. coli cells characterized by altered external and internal morphology. These observations may assist in elucidating the mode of action of the extracts.

Experimental validation and computational modeling of anti-influenza effects of quercetin-3-O-α-L-rhamnopyranoside from indigenous south African medicinal plant Rapanea melanophloeos.

BACKGROUND: Influenza A virus (IAV) is still a major health threat. The clinical manifestations of this infection are related to immune dysregulation, which causes morbidity and mortality. The usage of traditional medication with immunomodulatory properties against influenza infection has been increased recently. Our previous study showed antiviral activity of quercetin-3-O-α-L-rhamnopyranoside (Q3R) isolated from Rapanea melanophloeos (RM) (L.) Mez (family Myrsinaceae) against H1N1 (A/PR/8/34) infection. This study aimed to confirm the wider range of immunomodulatory effect of Q3R on selective pro- and anti-inflammatory cytokines against IAV in vitro, to evaluate the effect of Q3R on apoptosis pathway in combination with H1N1, also to assess the physical interaction of Q3R with virus glycoproteins and RhoA protein using computational docking. METHODS: MDCK cells were exposed to Q3R and 100CCID50/100 µl of H1N1 in combined treatments (co-, pre- and post-penetration treatments). The treatments were tested for the cytokines evaluation at RNA and protein levels by qPCR and ELISA, respectively. In another set of treatment, apoptosis was examined by detecting RhoA GTPase protein and caspase-3 activity. Molecular docking was used as a tool for evaluation of the potential anti-influenza activity of Q3R. RESULTS: The expressions of cytokines in both genome and protein levels were significantly affected by Q3R treatment. It was shown that Q3R was much more effective against influenza when it was applied in co-penetration treatment. Q3R in combination with H1N1 increased caspase-3 activity while decreasing RhoA activation. The molecular docking results showed strong binding ability of Q3R with M2 transmembrane, Neuraminidase of 2009 pandemic H1N1, N1 and H1 of PR/8/1934 and Human RhoA proteins, with docking energy of - 10.81, - 10.47, - 9.52, - 9.24 and - 8.78 Kcal/mol, respectively. CONCLUSIONS: Quercetin-3-O-α-L-rhamnopyranoside from RM was significantly effective against influenza infection by immunomodulatory properties, affecting the apoptosis pathway and binding ability to viral receptors M2 transmembrane and Neuraminidase of 2009 pandemic H1N1 and human RhoA cellular protein. Further research will focus on detecting the detailed specific mechanism of Q3R in virus-host interactions.

Addition of a surfactant to water increases the acaricidal activity of extracts of some plant species used to control ticks by Zimbabwean smallholder farmers.

BACKGROUND: Many studies have revealed that bioactive compounds for different indications are not extracted from plants with water, the only extractant practically available to rural communities. We compared the acaricidal activity of acetone extracts of 13 species used traditionally to protect cattle against ticks. We also investigated if the extraction of biologically active compounds against Rhipicephalus (Boophilus) decoloratus ticks could be enhanced by adding a liquid soap that is locally available to smallholder farmers. METHODS: A total of 13 plant species selected based on reported traditional use in Zimbabwe, were dried and finely ground before extraction with water, or water plus a surfactant, or acetone. The adapted Shaw Larval Immersion Test (SLIT) method was used to determine the activity of acetone and crude water extracts with or without liquid soap against the tick larvae. The activity of four fractions of crude acetone extracts (extracted using solvents of different polarity), of the most active plant species, Maerua edulis (tuber and leaf) was also compared to identify the most active fraction. RESULTS: Aqueous plant extracts were not toxic to ticks, but the addition of 1% liquid soap as a surfactant increased mortality of the R. (B) decoloratus larvae significantly. With the Maerua edulis tuber extract, the efficacy of the 1% liquid soap was comparable to that of the amitraz based commercial synthetic acaricide. The use of acetone as an extractant, also increased the mortality of the tick larvae in all the plant species. With M. edulis (tuber and leaf), Monadenium lugardae and Kleinia sp. acetone extracts, the activity was comparable to that of the positive control (a commercially available amitraz-based synthetic acaricide). The non-polar fractions of the acetone extract of leaf and tuber of M. edulis caused up to 100% mortality. This indicates that non-polar to intermediate polarity compounds are responsible for the acaricidal activity. CONCLUSION: Organic solvents such as acetone extracted active compounds but water did not. By adding commonly available dishwashing soap to water active compounds were extracted leading to a high acaricidal activity of the plant extracts. In some cases, it was as active as non-polar extracts and a synthetic commercial acaricide (positive control). This approach makes it possible for the smallholder farmers and traditional healers to extract biologically active compounds from plants by using water.

Acaricidal activity of the aqueous and hydroethanolic extracts of 15 South African plants against Rhipicephalus turanicus and their toxicity on human liver and kidney cells.

Hot water and hydroethanolic (70:30) extracts were prepared from 15 plant species, which were investigated to discover eco-friendly and less expensive tick control methods as an alternative to synthetic acaricides. A contact bioassay was used to determine the acaricidal activity of these extracts against the cattle tick, Rhipicephalus turanicus (Acari: Ixodidae) at a concentration of 20% (200 mg/mL). The hydroethanolic extracts had better activity than the hot water extracts against R. turanicus. The hydroethanolic extract from Tabernaemontana elegans (leaves) had the best mortality (87.0%). This was followed by Calpurnia aurea (stems) with a mortality of 75.0%, Schkuhria pinnata (whole plant) with a mortality of 67.0% and Aloe rupestris (leaves) with a mortality of 66.6%. The toxicity of the plant extracts was also investigated and it was found that most of the hydroethanolic and hot water extracts were either safe or very safe on human Vero kidney and liver HepG2 cells. From this study, it was evident that botanicals have the potential to be developed as environmentally benign natural acaricides against R. turanicus.

Antibacterial and antibiofilm activity of acetone leaf extracts of nine under-investigated south African Eugenia and Syzygium (Myrtaceae) species and their selectivity indices.

BACKGROUND: Antimicrobial resistance (AMR) remains an important global health issue but the gap between AMR and development of new antimicrobials is increasing. Plant extracts may have good activity per se or may be sources of effective antimicrobial compounds which can act against planktonic and/or biofilms of pathogens. We determined the antimicrobial efficacy and cytotoxicity of some under-investigated plants from the Myrtaceae family endemic to South Africa. The ability of the plant extracts to inhibit or destroy pre-formed bacterial biofilms was also determined. METHODS: Based on previous preliminary in vitro screening and on chemotaxonomy, nine species from the Myrtaceae family were selected. The antimicrobial activity of the crude acetone leaf extracts was determined against six common nosocomial pathogens, namely: Gram-positive bacteria (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium) using a two-fold serial microdilution assay with p-iodonitrotetrazolium violet as growth indicator. The number of antimicrobial compounds present in extracts was determined by bioautography. Cytotoxicity of extracts was determined against Vero kidney cells using a colorimetric tetrazolium-based assay. The total antibacterial activity (TAA) in ml/g and selectivity index (LC50/MIC) of the plant extracts were calculated. A modified crystal violet assay was used to determine the antibiofilm activity of the extracts. RESULTS: Syzygium legatii, Syzygium masukuense, and Syzygium species A had the best activities against Gram-negative and Gram-positive bacteria (MIC) values ranging from 0.04-0.08 mg/ml. Eugenia erythrophylla had the best MIC (0.02 mg/ml) against Bacillus cereus. Many extracts had relatively low cytotoxicity (LC50 > 20 µg/ml) leading to reasonable selectivity indices. Three leaf extracts (Syzygium masukuense, Syzygium species A, and Eugenia natalitia) were moderately cytotoxic (20 µg/ml < LC50 < 100 µg/ml). The plant extracts had a good capacity to reduce biofilm formation and good to poor potential to destroy pre-formed biofilms. CONCLUSIONS: The plant species examined in this study had varying degrees of antibacterial activity against bacterial planktonic and biofilm forms with some having good activity against both forms. Several of these selected species may be potential candidates for further investigation to isolate antimicrobial compounds and to determine the mechanism of activity.

Fractions and isolated compounds from Oxyanthus speciosus subsp. stenocarpus (Rubiaceae) have promising antimycobacterial and intracellular activity.

BACKGROUND: Tuberculosis is a deadly disease caused by Mycobacterium species. The use of medicinal plants is an ancient global practice for the treatment and prevention of diverse ailments including tuberculosis. The aim of this study was to isolate and characterize antimycobacterial compounds by bioassay-guided fractionation of the acetone leaf extract of Oxyanthus speciosus. METHODS: A two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against mycobacteria. Cytotoxicity and nitric oxide inhibitory activity of the isolated compounds was determined to evaluate in vitro safety and potential anti-inflammatory activity. Intracellular efficacy of the crude extract against Mycobacterium-infected macrophages was also determined. RESULTS: Two compounds were isolated and identified as lutein (1) and rotundic acid (2). These had good antimycobacterial activity against the four mycobacteria tested with MIC values ranging from 0.013 to 0.1 mg/mL. Rotundic acid had some cytotoxicity against C3A human liver cells. Lutein was not cytotoxic at the highest tested concentration (200 µg/mL) and inhibited nitric oxide production in RAW 264.7 macrophages by 94% at a concentration of 25 µg/mL. The acetone crude extract (120 µg/mL) of O. speciosus had intracellular antimycobacterial activity, reducing colony forming units by more than 90%, displaying bactericidal efficacy in a dose and time-dependent manner. CONCLUSION: This study provides good proof of the presence of synergism between different compounds in extracts and fractions. It is also the first report of the antimycobacterial activity of lutein and rotundic acid isolated from Oxyanthus speciosus. The promising activity of the crude extract of O. speciosus both in vitro and intracellularly in an in vitro macrophage model suggests its potential for development as an anti- tuberculosis (TB) herbal medicine.

Antibacterial activity and mode of action of acetone crude leaf extracts of under-investigated Syzygium and Eugenia (Myrtaceae) species on multidrug resistant porcine diarrhoeagenic Escherichia coli.

BACKGROUND: Diarrhoea, a global economically important disease burden affecting swine and, especially piglets, is commonly caused by infection with entero-toxigenic E. coli (ETEC). Adherence of ETEC to porcine intestinal epithelial cells following infection, is necessary for its pathogenesis. While antimicrobials are commonly given as therapy or as feed additives for prophylaxis against microbial infections, the concern over increased levels of antimicrobial resistance necessitate the search for safe and effective alternatives in livestock feed. Attention is shifting to natural products including plants as suitable alternatives to antimicrobials. The activity of acetone crude leaf extracts of nine under-explored South African endemic plants from the Myrtaceae family with good antimicrobial activity were tested against pathogenic E. coli of porcine origin using a microplate serial dilution method. Bioautography, also with p-iodonitrotetrazolium violet as growth indicator was used to view the number of bioactive compounds in each extract. In vitro toxicity of extracts was determined against Caco-2 cells using the 3-(4,5-dimethythiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay. The antimicrobial susceptibility of E. coli isolates was tested on a panel of antimicrobials using the Kirby-Bauer agar diffusion method while the anti-adherence mechanism was evaluated using a Caco-2 cell enterocyte anti-adhesion model. RESULTS: The MIC of the extracts ranged from 0.07-0.14 mg/mL with S. legatii having the best mean MIC (0.05 mg/mL). Bioautography revealed at least two active bands in each plant extract. The 50% lethal concentration (LC50) values ranged between 0.03-0.66 mg/mL. Eugenia zeyheri least cytotoxic (LC50 = 0.66 mg/ml) while E. natalitia had the highest cytotoxicity (LC50 = 0.03 mg/mL). All the bacteria were completely resistant to doxycycline and colistin sulphate and many of the plant extracts significantly reduced adhesion of E. coli to Caco-2 cells. CONCLUSIONS: The extracts of the plants had good antibacterial activity as well as a protective role on intestinal epithelial cells against enterotoxigenic E. coli bacterial adhesion. This supports the potential use of these species in limiting infection causes by E. coli. Some of these plants or extracts may be useful as phytogenic feed additives but it has to be investigated by animal feed trials.

Bioassay-guided isolation and identification of gametocytocidal compounds from Artemisia afra (Asteraceae).

BACKGROUND: Optimal adoption of the malaria transmission-blocking strategy is currently limited by lack of safe and efficacious drugs. This has sparked the exploration of different sources of drugs in search of transmission-blocking agents. While plant species have been extensively investigated in search of malaria chemotherapeutic agents, comparatively less effort has been channelled towards exploring them in search of transmission-blocking drugs. Artemisia afra (Asteraceae), a prominent feature of South African folk medicine, is used for the treatment of a number of diseases, including malaria. In search of transmission-blocking compounds aimed against Plasmodium parasites, the current study endeavoured to isolate and identify gametocytocidal compounds from A. afra. METHODS: A bioassay-guided isolation approach was adopted wherein a combination of solvent-solvent partitioning and gravity column chromatography was used. Collected fractions were continuously screened in vitro for their ability to inhibit the viability of primarily late-stage gametocytes of Plasmodium falciparum (NF54 strain), using a parasite lactate dehydrogenase assay. Chemical structures of isolated compounds were elucidated using UPLC-MS/MS and NMR data analysis. RESULTS: Two guaianolide sesquiterpene lactones, 1α,4α-dihydroxybishopsolicepolide and yomogiartemin, were isolated and shown to be active (IC50 < 10 µg/ml; ~ 10 µM) against both gametocytes and intra-erythrocytic asexual P. falciparum parasites. Interestingly, 1α,4α-dihydroxybishopsolicepolide was significantly more potent against late-stage gametocytes than to early-stage gametocytes and intra-erythrocytic asexual P. falciparum parasites. Additionally, both isolated compounds were not overly cytotoxic against HepG2 cells in vitro. CONCLUSION: This study provides the first instance of isolated compounds from A. afra against P. falciparum gametocytes as a starting point for further investigations on more plant species in search of transmission-blocking compounds.

Croton gratissimus leaf extracts inhibit cancer cell growth by inducing caspase 3/7 activation with additional anti-inflammatory and antioxidant activities.

BACKGROUND: Croton species (Euphorbiaceae) are distributed in different parts of the world, and are used in traditional medicine to treat various ailments including cancer, inflammation, parasitic infections and oxidative stress related diseases. The present study aimed to evaluate the antioxidant, anti-inflammatory and cytotoxic properties of different extracts from three Croton species. METHODS: Acetone, ethanol and water leaf extracts from C. gratissimus, C. pseudopulchellus, and C. sylvaticus were tested for their free radical scavenging activity. Anti-inflammatory activity was determined via the nitric oxide (NO) inhibitory assay on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and the 15-lipoxygenase inhibitory assay using the ferrous oxidation-xylenol orange assay. The cytotoxicity of the extracts was determined on four cancerous cell lines (A549, Caco-2, HeLa, MCF-7), and a non-cancerous African green monkey (Vero) kidney cells using the tetrazolium-based colorimetric (MTT) assay. The potential mechanism of action of the active extracts was explored by quantifying the caspase-3/- 7 activity with the Caspase-Glo® 3/7 assay kit (Promega). RESULTS: The acetone and ethanol leaf extracts of C. pseudopulchellus and C. sylvaticus were highly cytotoxic to the non-cancerous cells with LC50 varying between 7.86 and 48.19 µg/mL. In contrast, the acetone and ethanol extracts of C. gratissimus were less cytotoxic to non-cancerous cells and more selective with LC50 varying between 152.30 and 462.88 µg/mL, and selectivity index (SI) ranging between 1.56 and 11.64. Regarding the anti-inflammatory activity, the acetone leaf extract of C. pseudopulchellus had the highest NO inhibitory potency with an IC50 of 34.64 µg/mL, while the ethanol leaf extract of the same plant was very active against 15-lipoxygenase with an IC50 of 0.57 µg/mL. A linear correlation (r<0.5) was found between phytochemical contents, antioxidant, anti-inflammatory and cytotoxic activities of active extracts. These extracts induced differentially the activation of caspases - 3 and - 7 enzymes in all the four cancerous cells with the highest induction (1.83-fold change) obtained on HeLa cells with the acetone leaf extract of C. gratissimus. CONCLUSION: Based on their selective toxicity, good antioxidant and anti-inflammatory activities, the acetone and ethanol leaf extracts of C. gratissimus represent promising alternative sources of compounds against cancer and other oxidative stress related diseases.

Immunomodulatory properties of quercetin-3-O-α-L-rhamnopyranoside from Rapanea melanophloeos against influenza a virus.

BACKGROUND: Influenza infection is a major public health threat. The role of influenza A virus-induced inflammatory response in severe cases of this disease is widely recognized. Drug resistance and side effects of chemical treatments have been observed, resulting in increased interest in alternative use of herbal medications for prophylaxis against this infection. The South African medicinal plant, Rapanea melanophloeos (RM) (L.) Mez of the family Myrsinaceae was selected owing to its traditional use for the treatment of several diseases such as respiratory ailments and also previous preliminary studies of anti-influenza activity of its methanolic extract. The aim of this study was to investigate the immunomodulatory properties of a glycoside flavone isolated from RM against influenza A virus. METHODS: The non-cytotoxic concentration of the quercetin-3-O-α-L-rhamnopyranoside (Q3R) was determined by MTT assay and tested for activity against influenza A virus (IAV) in simultaneous, pre-penetration and post-penetration combination treatments over 1 h incubation on MDCK cells. The virus titer and viral load targeting NP and M2 viral genes were determined using HA and qPCR, respectively. TNF-α and IL-27 as pro- and anti-inflammatory cytokines were measured at RNA and protein levels by qPCR and ELISA, respectively. RESULTS: Quercetin-3-O-α-L-rhamnopyranoside at 150 µg/ml decreased the viral titer by 6 logs (p < 0.01) in the simultaneous procedure. The NP and M2 genes copy numbers as viral target genes, calculated based on the Ct values and standard formula, significantly decreased in simultaneous treatment (p < 0.01). The expression of cytokines was also considerably affected by the compound treatment. CONCLUSIONS: This is the first report of quercetin-3-O-α-L-rhamnopyranoside from RM and its immunomodulatory properties against influenza A virus. Further research will focus on detecting the specific mechanism of virus-host interactions.

Isolation and characterization of two acaricidal compounds from Calpurnia aurea subsp. aurea (Fabaceae) leaf extract.

The menace caused by ticks and tick-borne diseases is a major limitation to the livestock industry in Africa. The high costs and non-availability of synthetic, chemical acaricides to resource-limited farmers, resistance of ticks to available acaricides and residue problems in meat and milk consumed by humans further complicate matters. The use of plant extracts as a possible source of new acaricides has received much interest in the last decade. In our endeavour to discover natural acaricidal compounds, tick toxicant bioassays were conducted and the chloroform fraction of Calpurnia aurea ethanol leaf extract had good acaricidal activity. Further purification of the fraction revealed two flavonoids, isolated from C. aurea for the first time. These flavonoids were characterized as apigenin-7-O-ß-D-glycoside and isorhoifolin by means of NMR spectroscopic and mass spectrometry analysis. Isorhoifolin was the most potent compound (LC50 = 0.65 mg/ml), was not cytotoxic and should be further investigated for its potential as an acaricidal agent.

South African medicinal plant extracts active against influenza A virus.

BACKGROUND: Influenza infection remains a major health threat for animals and humans which crucially requires effective antiviral remedies. The usage of herbal medications as readily available alternatives for their compatibility with the body and fewer side effects compared to synthetic chemical treatments has become popular globally. The aim of this study was to investigate and screen in vitro anti-influenza activity of extracts of five South African medicinal plants, namely Tabernaemontana ventricosa, Cussonia spicata, Rapanea melanophloeos, Pittosporum viridiflorum and Clerodendrum glabrum, species which are used traditionally for the treatment of several diseases such as inflammatory and respiratory diseases. METHODS: Methanol, ethanol (100% and 30%), acetone, hot and cold water extracts of the powdered plants leaves were obtained by standard methods. The cytotoxicity was determined by the MTT colorimetric assay on MDCK cells. The concentrations below CC50 values were tested for antiviral activity against influenza A virus (IAV) in different combination treatments. The effect of extracts on viral surface glycoproteins and viral titer were tested by HI and HA virological assays, respectively. RESULTS: Based on the applied methods, the most effective results against IAV were obtained from Rapanea melanophloeos methanol leaf extract (EC50 = 113.3 µg/ml) and Pittosporum viridiflorum methanol, 100% and 30% ethanol and acetone leaf extracts (EC50 values = 3.6, 3.4, 19.2, 82.3 µg/ml, respectively) in all types of combined treatments especially in pre- and post-penetration combined treatments with highly significant effects against viral titer (P ≤ 0.01). CONCLUSION: The outcomes offer for the first time a scientific basis for the use of extracts of Rapanea melanophloeos and Pittosporum viridiflorum against IAV. It is worth focusing on the isolation and identification of effective active compounds and elucidating the mechanism of action from these species. However, Tabernaemontana ventricosa, Cussonia spicata and Clerodendrum glabrum leaf extracts were ineffective in vitro in this study.

Investigation of the acaricidal activity of the acetone and ethanol extracts of 12 South African plants against the adult ticks of Rhipicephalus turanicus.

The acaricidal activity of acetone and ethanol extracts of 12 plant species was evaluated using the contact method on Rhipicephalus turanicus (Acari: Ixodidae) ticks at an initial concentration of 20% (200 mg/mL). Eight of the 12 plants had mortality greater than 50% and the acetone extracts had better acaricidal activity than the ethanol extracts. The acetone extract of Calpurnia aurea (leaves and flowers) had the highest corrected mortality (CM) of 92.2% followed by Schkuhria pinnata (whole plant) with a CM of 88.9%, Ficus sycomorus (bark and stems) 86.7% and Senna italica subsp. arachoides (roots, leaves and fruits) 83.3%. Selected extracts were tested at five different concentrations using the adult immersion test. From dose-response assays, EC₅₀ values of 61.82 mg/mL, 115.21 mg/mL and 161.02 mg/mL were obtained for the acetone extracts of S. pinnata (whole plant), S. italica subsp. arachoides (roots, leaves and fruits) and C. aurea (leaves and flowers) respectively. The ethanol extract of Monsonia angustifolia (whole plant) had the highest CM of 97.8% followed by S. pinnata (whole plant) with a CM of 86.7%, C. aurea (leaves and flowers) 81.1% and Cleome gynandra (leaves) 77.8%. There is potential for the development of environmentally benign botanicals as natural acaricides against R. turanicus.