Phytochemical Evaluation of Terminalia canescens DC. Radlk. Extracts with Antibacterial and Antibiotic Potentiation Activities against Selected ß-Lactam Drug-Resistant Bacteria.

Terminalia canescens DC. Radlk. (family: Combretaceae) is native to northern Australia. Species of the genus Terminalia are widely used as traditional medicines to treat diverse ailments, including bacterial infections. However, we were unable to find any studies that had examined the antimicrobial activity of T. canescens. In this study, T. canescens was screened against a panel of bacterial pathogens, including multi-antibiotic-resistant strains. Solvents with different polarities were used to extract different complements of phytochemicals from T. canescens leaves. Methanolic and aqueous extracts exhibited substantial antimicrobial activity against various pathogens, including those that are multidrug-resistant strains. When combined with some selected clinical antibiotics, some extracts potentiated the antibacterial inhibitory activity. This study identified two synergistic, eleven additive, eleven non-interactive and eight antagonistic interactions. The toxicities of the plant extracts were examined in the Artemia franciscana nauplii assay and were found to be non-toxic, except the aqueous extract, which showed toxicity. Metabolomic liquid chromatography-mass spectrometry (LC-MS) analyses highlighted and identified several flavonoids, including vitexin, quercetin, orientin and kaempferol, as well as the tannins ellagic acid and pyrogallol, which may contribute to the antibacterial activities observed herein. The possible mechanism of action of these extracts was further explored in this study.

Selected Australian Terminalia Species Extracts Inhibit ß-Lactam Drug-Resistant Bacteria Growth and Potentiate the Activity of Conventional Antibiotics: Bioactivities and Phytochemistry.

Terminalia ferdinandiana Exell, Terminalia grandiflora Benth., Terminalia microcarpa Decne., and Terminalia muelleri Benth. (family: Combretaceae) belong to the genus Terminalia. Plants of this genus have been extensively used as traditional medicines to treat a variety of illnesses, including pathogen infections. However, we were unable to find any studies that have investigated the antibacterial activity of T. microcarpa. Similarly, whilst some preliminary studies have examined the antimicrobial properties of T. muelleri and T. grandiflora, they did not test the extracts against antibiotic-resistant pathogens. This study screens the antimicrobial activity of T. grandiflora, T. microcarpa, and T. muelleri and compares it to that of T. ferdinandiana extracts prepared from both the fruit and leaves against a range of pathogens, including multi-antibiotic-resistant strains. Solvents with varying polarities were used to extract different phytochemical constituents from the leaves of T. grandiflora, T. microcarpa, and T. muelleri and from the fruit and leaves of T. ferdinandiana. The aqueous and methanolic extracts each displayed significant antimicrobial activity when tested against the bacterial pathogens, including against the multidrug-resistant strains. When these extracts were tested in combination with selected antibiotics, some extracts potentiated the antimicrobial activity. This study identifies twelve synergistic, fifty-eight additive, and sixty non-interactive combinations, as well as thirty antagonistic effects. The extracts were evaluated for toxicity using the Artemia franciscana nauplii lethality assay (ALA) and were each classified as non-toxic, with the exception of the methanolic and aqueous T. ferdinandiana fruit extracts and the aqueous and ethyl acetate T. ferdinandiana leaf extracts. Metabolomic analysis using liquid chromatography-mass spectrometry (LC-MS) highlighted several flavonoids and tannins that may contribute to the antimicrobial activities reported herein. The potential antibacterial mechanism(s) of the T. ferdinandiana extracts are discussed in this study.

Terminalia petiolaris A.Cunn ex Benth. Extracts Have Antibacterial Activity and Potentiate Conventional Antibiotics against ß-Lactam-Drug-Resistant Bacteria.

Terminalia petiolaris A. Cunn. Ex Benth. (genus: Terminalia, family: Combretaceae) is native to Australia. Terminalia spp. have traditionally been used to treat various ailments, including bacterial infections. Solvents of varying polarity were used to extract compounds from leaves of this species, and the extracts were tested against a panel of bacteria, including antibiotic-resistant strains. The methanolic and water extracts showed substantial inhibitory activity against several bacteria, including antibiotic-resistant strains in both disc diffusion and liquid dilution assays. Combining these extracts with selected conventional antibiotics enhanced the inhibition of bacterial growth for some combinations, while others showed no significant interaction. In total, two synergistic, twenty-five additive, twenty-three non-interactive and one antagonistic interaction were observed. The methanolic and ethyl acetate plant extracts were found to be non-toxic in Artemia franciscana nauplii toxicity assays. A liquid chromatography-mass spectrometry metabolomics analysis identified several flavonoid compounds, including miquelianin, trifolin and orientin, which might contribute to the observed activities. The potential modes of these active extracts are further discussed in this study.

Bacterial Foodborne Illness in Malaysia: Terminalia spp. as a Potential Resource for Treating Infections and Countering Antibiotic Resistance.

Acute diarrhoea is becoming a major public health problem in Malaysia, with more than 13.5 million cases reported annually. Foodborne bacterial pathogens are a predominant cause of diarrhoea, with infections causing prolonged illness durations and higher patient mortality rates, placing a tremendous burden on the Malaysian economy. Due to increasing incidences of diarrhoea in Malaysia caused by foodborne pathogens and the increasing levels of resistance towards antibiotics from many different classes, new drugs and/or therapies are urgently required. The evidence for plants as new sources of antibiotics has increased dramatically in recent years and there has been a substantial increase in interest in traditional and herbal medicines. Several Terminalia spp. are native to Malaysia, with previous research demonstrating that Terminalia spp. are rich in therapeutic phytochemicals and possess antibacterial properties. However, limited research has been conducted on the native Malaysian Terminalia spp. for their potential as new antibacterial therapies. The current review discusses the types of bacteria, including antibiotic-resistant strains, that cause food poisoning in Malaysia, and reports the phytochemical content and antibacterial properties of eight of these useful plant species. Future directions pertaining to drug discovery pathways are also suggested.

Phytochemistry, Medicinal Properties, Bioactive Compounds, and Therapeutic Potential of the Genus Eremophila (Scrophulariaceae).

The genus Eremophila (family Scrophulariaceae) consists of approximately 200 species that are widely distributed in the semi-arid and arid regions of Australia. Multiple Eremophila spp. are used as traditional medicines by the First Australians in the areas in which they grow. They are used for their antibacterial, antifungal, antiviral, antioxidant, anti-diabetic, anti-inflammatory, and cardiac properties. Many species of this genus are beneficial against several diseases and ailments. The antibacterial properties of the genus have been relatively well studied, with several important compounds identified and their mechanisms studied. In particular, Eremophila spp. are rich in terpenoids, and the antimicrobial bioactivities of many of these compounds have already been confirmed. The therapeutic properties of Eremophila spp. preparations and purified compounds have received substantially less attention, and much study is required to validate the traditional uses and to highlight species that warrant further investigation as drug leads. The aim of this study is to review and summarise the research into the medicinal properties, therapeutic mechanisms, and phytochemistry of Eremophila spp., with the aim of focussing future studies into the therapeutic potential of this important genus.

Comparison of the antibacterial activity of Australian Terminalia spp. extracts against Klebsiella pneumoniae: a potential treatment for ankylosing spondylitis.

Traditional medicines prepared using Terminalia species have been used globally to treat inflammation and pathogenic infections. Recent studies have demonstrated that multiple Asian and African Terminalia spp. inhibit bacterial triggers of some autoimmune inflammatory diseases, including ankylosing spondylitis. Despite this, the effects of Australian Terminalia spp. on a bacterial trigger of ankylosing spondylitis (K. pneumoniae) remain unexplored. Fifty-five extracts from five Australian Terminalia spp. were investigated for K. pneumoniae growth inhibitory activity. Methanolic, aqueous and ethyl acetate extracts of most species and plant parts inhibited K. pneumoniae growth, with varying potencies. Methanolic leaf extracts were generally the most potent bacterial growth inhibitors, with minimum inhibitory concentration (MIC) values of 66 µg/mL (T. ferdinandiana), 128 µg/mL (T. carpenteriae) and 83 µg/mL (T. petiolares). However, the aqueous leaf extract was the most potent T. grandiflora extract (MIC = 87 µg/mL). All T. catappa extracts displayed low growth inhibitory activity. The Terminalia spp. methanolic leaf extracts were examined by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). All contained a relative abundance of simple gallotannins (particularly gallic and chebulic acids), the flavonoid luteolin, as well as the monoterpenoids cineole and terpineol. Notably, all Terminalia spp. were non-toxic or of low toxicity in ALA and HDF toxicity assays, highlighting their potential for preventing the onset of ankylosing spondylitis and treating its symptoms once the disease is established, although this needs to be verified in in vivo systems.

Chemical Composition, Antibacterial Activity, and Antibiotic Potentiation of Boswellia sacra Flueck. Oleoresin Extracts from the Dhofar Region of Oman.

The emergence of MDR bacterial pathogens has directed antibiotic discovery research towards alternative therapies and traditional medicines. Boswellia sacra oleoresin (frankincense) was used to treat bacterial infections in traditional Arabian and Asian healing systems for at least 1000 years. Despite this, B. sacra extracts have not been rigorously tested for inhibitory activity against gastrointestinal pathogens or bacterial triggers of autoimmune diseases. Solvent extracts were prepared from Boswellia sacra oleoresins obtained from three regions near Salalah, Oman. MIC values were quantified against gastrointestinal pathogens and bacterial triggers of selected autoimmune diseases by disc diffusion and broth dilution methods. The antibacterial activity was also evaluated in combination with conventional antibiotics, and the class of interaction was determined by ΣFIC analysis. Isobolograms were used to determine the optimal ratios for synergistic combinations. Toxicity was evaluated by ALA and HDF cell viability bioassays. The phytochemical composition of the volatile components of all extracts was identified by nontargeted GC-MS headspace analysis. All methanolic extracts inhibited the growth of all of the bacteria tested, although the extracts prepared using Najdi oleoresin were generally more potent than the Sahli and Houjari extracts. Combinations of the methanolic B. sacra extracts and conventional antibiotics were significantly more effective in inhibiting the growth of several bacterial pathogens. In total, there were 38 synergistic and 166 additive combinations. Approximately half of the synergistic combinations contained tetracycline. All B. sacra extracts were nontoxic in the ALA and HDF cell viability assays. Nonbiased GC-MS headspace analysis of the methanolic extracts putatively identified a high diversity of monoterpenoids, with particularly high abundances of α-pinene. The antibacterial activity and lack of toxicity of the B. sacra extracts indicate their potential in the treatment and prevention of gastrointestinal and autoimmune diseases. Furthermore, the extracts potentiated the activity of several conventional antibiotics, indicating that they may contain resistance-modifying compounds.

The Therapeutic Properties of Plants Used Traditionally to Treat Gastrointestinal Disorders on Groote Eylandt, Australia.

The First Australians had well-developed healing systems. Groote Eylandt inhabitants used a variety of plant species to treat diarrhoea and other gastrointestinal illnesses. This study was undertaken to test, identify, and evaluate traditional medicines to treat these conditions against gastrointestinal bacterial, protozoal, and viral pathogens, as well as against cancer cell proliferation. Six plant species (Buchanania obovata Engl., Casuarina equisetifolia L., Eucalyptus tetrodonta F. Muell., Planchonia careya (F. Muell.) R. Knuth, Terminalia carpentariae C. T. White, and Vigna vexillata (L.) A. Rich.) were selected from a survey of a panel of elders from the Warnindhilyagwa tribe and compared with the published literature. Decoctions prepared according to traditional methods were screened for growth inhibitory activity of a panel of diarrhoea-causing bacterial pathogens by disc diffusion and liquid dilution MIC assays. Inhibitory activity against the gastrointestinal protozoal parasite Giardia duodenalis and antiproliferative activity against human colorectal (Caco2) and cervical (HeLa) cancer cell lines were evaluated using MTS-based colorimetric cell proliferation assays. Preliminary antiviral screening was accomplished using an MS2 bacteriophage plaque reduction assay. Toxicity was evaluated using Artemia franciscana nauplii mortality and HDF cell viability bioassays. All traditional medicines tested inhibited bacterial growth, often with MIC values substantially <1000 µg/mL. T. carpentariae was particularly noteworthy, with MIC values of 230-350 µg/mL against Citrobacter freundii, Salmonella newport, Shigella sonnei, Staphylococcus aureus, and Staphylococcus epidermidis. This species also had MICs 450-950 µg/mL against all other bacterial pathogens. B. obovata Engl. and E. tetrodonta were also good inhibitors of bacterial growth, albeit with substantially higher MIC values than determined for T. carpentariae. The T. carpentariae decoction was also the best inhibitor of MS2 phage replication (IC50 = 427 µg/mL) and Caco2 and HeLa proliferation (IC50 values of 885 and 85 µg/mL, respectively). None of the extracts were particularly strong inhibitors of Giardia duodenalis growth. All decoctions were nontoxic in the Artemia nauplii and HDF cell viability bioassays, indicating their suitability for therapeutic use.

Inertial Microfluidic Purification of Floating Cancer Cells for Drug Screening and Three-Dimensional Tumor Models.

Floating cancer cells can survive the programmed death anoikis process after detaching from the extracellular matrix for the anchorage-dependent cells. Purification of viable floating cancer cells is essential for many biomedical studies, such as drug screening and cancer model development. However, the floating cancer cells are mixed with dead cells and debris in the medium supernatant. In this paper, we developed an inertial microfluidic device with sinusoidal microchannels to continuously remove dead cells and debris from viable cells. First, we characterized the differential inertial focusing properties of polystyrene beads in the devices. Then, we investigated the effects of flow rate on inertial focusing of floating MDA-MB-231 cells. At an optimal flow condition, purification of viable cells was performed and the purity of live cells was increased significantly from 19.9% to 76.6%, with a recovery rate of 69.7%. After separation, we studied and compared the floating and adherent MDA-MB-231 cells in terms of cell proliferation, protrusive cellular structure, and the expression of cyclooxygenase (Cox-2) which is related to epithelial-mesenchymal transition (EMT) changes. Meanwhile, drug screening of both floating and adherent cancer cells was conducted using a chemotherapeutic drug, doxorubicin (Dox). The results revealed that the floating cancer cells possess 30-fold acquired chemoresistance as compared to the adherent cancer cells. Furthermore, a three-dimensional (3D) double-cellular coculture model of human mammary fibroblasts (HMF) spheroid and cancer cells using the floating liquid marble technique was developed.

Lapatinib inhibits doxorubicin induced migration of HER2-positive breast cancer cells.

Inflammatory breast cancer (IBC) is an uncommon and highly aggressive form of breast cancer. The disease is characterized by rapid progression with approximately 50% of IBC patients to have human epidermal growth factor receptor 2 (HER2) amplification. HER2-positive IBC is associated with unfavourable prognosis and increased risk of brain metastasis. Ironically, HER2-positive metastatic breast cancer is still prevalent where therapeutic targeting of HER2-receptor is well developed. In addition, the ability to accurately predict the risk of metastatic potential in these cells poses a substantial challenge. Lapatinib (Lap), a dual kinase inhibitor of HER2 and epidermal growth factor receptor is used in the treatment of advanced HER-2 positive breast cancers and is currently being evaluated in the adjuvant setting. In this study, we report the effectiveness of Lap in the suppression of low-dose response to doxorubicin (Dox) in HER2-positive SKBR3 cells. Upon treatment of SKBR3 cells with 0.1 µM of Dox, the cell viability was significantly increased as compared to the human mammary fibroblasts, and triple-negative human breast cancer MDA-MB-231 cells. Interestingly, the effect of 0.1 µM Dox revealed morphological changes consistent with a significant increase in the formation of prominent F-actin filaments and mitochondrial spread compared with the control SKBR3 cells. Furthermore, an enhanced migration was also evident in these cells. However, a combinational dose of 0.1 µM Dox + 5 µM Lap suppressed the observed phenotypic changes in the 0.1 µM Dox treated SKBR3 cells. There was a significant difference in the prominent F-actin filaments and the mitochondrial spread compared with the 0.1 µM Dox versus combination regimen of 0.1 µM Dox + 5 µM Lap. In addition, the combinational therapy showed a decrease in the percentage of wound closure when compared to the control. Hence, the combinational therapy in which Lap suppresses the low-dose effect of Dox in SKBR3 cells may provide an effective intervention strategy for reducing the risk of metastasis in HER2-positive breast cancers.

Terminalia ferdinandiana Exell: Extracts inhibit Shewanella spp. growth and prevent fish spoilage.

Shewanella spp. are major causes of fish spoilage. Terminalia ferdinandiana (Kakadu plum) extracts were investigated for their ability to inhibit Shewanella spp. growth. Leaf and fruit extracts displayed potent growth inhibitory properties against all Shewanella spp. The methanolic leaf extract was a particularly potent inhibitor of S. putrefaciens (DD MIC 93; LD MIC 73 µg/mL), S. baltica (DD MIC 104 µg/mL; LD MIC 85 µg/mL), S. frigidimarina (DD MIC 466 µg/mL; LD MIC 391 µg/mL) and S. loihica (DD MIC 95 µg/mL; LD MIC 55 µg/mL) growth. The aqueous and ethyl acetate leaf extracts were also potent growth inhibitors, with MIC values generally substantially <1000 µg/mL. Treatment of Acanthopagrus butcheri Munro fillets with methanolic Kakadu plum extracts significantly inhibited bacterial growth for 15 days at 4 °C. All Kakadu plum extracts were nontoxic in the Artemia franciscana bioassay. LC-MS analysis identified several compounds which may contribute to the inhibition of Shewanella spp. growth.

Terminalia chebula Retz. Fruit Extracts Inhibit Bacterial Triggers of Some Autoimmune Diseases and Potentiate the Activity of Tetracycline.

Terminalia chebula Retz. is a northern Indian plant species known for its anti-inflammatory and antimicrobial properties. T. chebula fruit powder was extracted with solvents of varying polarity and screened for bacterial growth inhibition by disc diffusion assay. The minimum inhibitory concentration (MIC) was quantified by both liquid dilution and disc diffusion techniques. To screen for combinatorial effects, the T. chebula fruit extracts were combined with a range of conventional antibiotics and tested against each bacteria using a liquid dilution assay. Where synergy was detected, the optimal ratios were determined using isobologram analysis. Toxicity was examined using Artemia nauplii and HDF bioassays. T. chebula fruit methanolic, aqueous and ethyl acetate extracts displayed strong antimicrobial activity against the bacterial triggers of all autoimmune inflammatory diseases except K. pneumoniae, for which only moderate inhibition was observed. Indeed, MIC values as low as 195 µg/mL were measured for the aqueous extract against a resistant strain of P. aeruginosa. Of further note, both the aqueous and ethyl acetate extracts interacted synergistically in combination with tetracycline against K. pneumoniae (Σ FIC 0.38 and 0.25 respectively). All extracts were nontoxic in the Artemia and HDF toxicity assays, further indicating their potential for medicinal use.

Is the pharmaceutical industry's preoccupation with the monotherapy drug model stifling the development of effective new drug therapies?

Drug discovery and development is heavily biased towards the development of monotherapies. Screening, testing, and evaluation of mono-entity drugs are generally much simpler than drug combinations, and are generally easier to get approval from the regulatory authorities for their clinical use. However, monotherapy drugs may not have optimal activity, may have associated toxicities, or may lose activity over time as their target develops resistance. Drug combinations, often developed from existing monotherapies, may have improved efficacy and/or be less toxic. Furthermore, the existing drugs which have lost efficacy due to the development of resistance can often be re-activated by combining them with other chemical entities. Thus, whilst the current climate for drug approval, registration, and clinical use drives the majority of drug development research towards the development of monotherapies, combinations are often a substantial improvement on the original drug. This commentary examines monotherapy and combinational therapy models and discusses the benefits and limitations of each model.

Inhibition of Shewanella spp. growth by Syzygium australe and Syzygium luehmannii extracts: natural methods for the prevention of fish spoilage.

Syzygium australe and Syzygium luehmannii fruit and leaf were investigated for their ability to inhibit Shewanella spp. growth. Extracts of both Syzygium spp. displayed potent growth inhibitory properties against all Shewanella spp. tested in disc diffusion and liquid diffusion assays. In general, S. australe extracts were more potent inhibitors of Shewanella spp. growth, and the fruit extracts were generally better than the corresponding leaf extracts. The methanolic S. australe fruit extract was a particularly potent inhibitor of all Shewanella spp. growth, with MIC values as low as 87 µg/mL. The aqueous and ethyl acetate S. australe fruit extracts were similarly potent inhibitors of Shewanella spp. growth, albeit with slightly higher MIC values. Several other Syzygium spp. extracts also were potent bacterial growth inhibitors, albeit with MIC values generally >1000 µg/mL. The most potent S. australe fruit extracts were nontoxic in the Artemia franciscana bioassay, with LC50 values substantially >1000 µg/mL. The potent bacterial growth inhibitory activity and lack of toxicity of the S. australe fruit extracts indicate their potential as natural fish and seafood preservatives.

High Performance Liquid Chromatography-mass Spectrometry Analysis of High Antioxidant Australian Fruits with Antiproliferative Activity Against Cancer Cells.

BACKGROUND: High antioxidant capacities have been linked to the treatment and prevention of several cancers. Recent reports have identified several native Australian fruits with high antioxidant capacities. Despite this, several of these species are yet to be tested for anticancer activity. MATERIALS AND METHODS: Solvent extracts prepared from high antioxidant native Australian fruits were analyzed for antioxidant capacity by the di (phenyl)-(2,4,6-trinitrophenyl) iminoazanium free radical scavenging assay. Antiproliferative activities against CaCo2 and HeLa cancer cells were determined by a multicellular tumor spheroid-based cell proliferation assay. Toxicity was determined by Artemia franciscana bioassay. RESULTS: Methanolic extracts of all plant species displayed high antioxidant contents (equivalent to approximately 7-16 mg of vitamin C per gram of fruit extracted). Most aqueous extracts also contained relatively high antioxidant capacities. In contrast, the ethyl acetate, chloroform, and hexane extracts of most species (except lemon aspen and bush tomato) had lower antioxidant contents (below 1.5 mg of vitamin C equivalents per gram of plant material extracted). The antioxidant contents correlated with the ability of the extracts to inhibit proliferation of CaCo2 and HeLa cancer cell lines. The high antioxidant methanolic extracts of all species were potent inhibitors of cell proliferation. The methanolic lemon aspen extract was particularly effective, with IC50 values of 480 and 769 µg/mL against HeLa and CaCo2 cells, respectively. In contrast, the lower antioxidant ethyl acetate and hexane extracts (except the lemon aspen ethyl acetate extract) generally did not inhibit cancer cell proliferation or inhibited to only a minor degree. Indeed, most of the ethyl acetate and hexane extracts induced potent cell proliferation. The native tamarind ethyl acetate extract displayed low-moderate toxicity in the A. franciscana bioassay (LC50 values below 1000 µg/mL). All other extracts were nontoxic. A total of 145 unique mass signals were detected in the lemon aspen methanolic and aqueous extracts by nonbiased high-performance liquid chromatography-mass spectrometry analysis. Of these, 20 compounds were identified as being of particular interest due to their reported antioxidant and/or anticancer activities. CONCLUSIONS: The lack of toxicity and antiproliferative activity of the high antioxidant plant extracts against HeLa and CaCo2 cancer cell lines indicates their potential in the treatment and prevention of some cancers. SUMMARY: Australian fruit extracts with high antioxidant contents were potent inhibitors of CaCo2 and HeLa carcinoma cell proliferationMethanolic lemon aspen extract was particularly potent, with IC50 values of 480 µg/mL (HeLa) and 769 µg/mL (CaCo2)High-performance liquid chromatography-mass spectrometry-quadrupole time-of-flight analysis highlighted and putatively identified 20 compounds in the antiproliferative lemon aspen extractsIn contrast, lower antioxidant content extracts stimulated carcinoma cell proliferationAll extracts with antiproliferative activity were nontoxic in the Artemia nauplii assay. Abbreviations used: DPPH: di (phenyl)- (2,4,6-trinitrophenyl) iminoazanium, HPLC: High-performance liquid chromatography, IC50: The concentration required to inhibit by 50%, LC50: The concentration required to achieve 50% mortality, MS: Mass spectrometry.