The effectiveness of many antimicrobial agents is currently decreasing; therefore, it is important to search for alternative therapeutics. Our study was carried out to assess the in vitro antibiofilm activity using microtiter plate assay, to characterize the bioactive compounds using Ultra Performance Liquid Chromatography-Diode Array Detection and Liquid Chromatography-Mass Spectrometry and to test the oral acute toxicity on Sprague Dawley rats of extract derived from a novel bacterial species of Paenibacillus strain 139SI. Our results indicate that the crude extract and its three identified compounds exhibit strong antibiofilm activity against a broad range of clinically important pathogens. Three potential compounds were identified including an amino acid antibiotic C8H20N3O4P (MW 253.237), phospholipase A2 inhibitor C21H36O5 (MW 368.512), and an antibacterial agent C14H11N3O2 (MW 253.260). The acute toxicity test indicates that the mortality rate among all rats was low and that the biochemical parameters, hematological profile, and histopathology examination of liver and kidneys showed no significant differences between experimental groups (P > 0.05). Overall, our findings suggest that the extract and its purified compounds derived from novel Paenibacillus sp. are nontoxic exhibiting strong antibiofilm activity against Gram-positive and Gram-negative pathogens that can be useful towards new therapeutic management of biofilm-associated infections.
Aminoglycoside antibiotics are widely employed clinically due to their powerful bactericidal activities, less bacterial resistance compared to beta lactam group and low cost. However, their use has been limited in recent years due to their potential induction of nephrotoxicity. Here we investigate the possibility of reversing nephrotoxicity caused by gentamicin in rat models by using ethanolic crude extract of the medicinal plant Jatropha Mollissima. Nephrotoxic male Wistar rats was obtained by gentamicin antibiotic, which then treated with two doses of J. mollissima crude extract for 3 weeks with monitoring their parameter in weekly base. Our results indicate that J. mollissima crude extract at both doses has strong protection ability against gentamicin nephrotoxicity, most of tested parameters backed to normal values after few days from the administration of the crude extract, which could be due to the antagonized the biochemical action of gentamicin on the proximal tubules of the kidney. The results of histopathologic analysis showed observable improvement in J. mollissima treated groups compared with untreated groups. Our findings suggests the J. mollissima has exceptional nephron protection potentials able to reverse the nephrotoxicity caused by gentamicin antibiotic.
The epidermal mucus of fish contains antimicrobial agents that act as biological defence against disease. This study aims to identify antibacterial activity and protein concentration of epidermal mucus of Barbodes everetti, a Bornean endemic freshwater fish. The epidermal mucus was extracted with 3% acetic acid, 0.85% sodium chloride and crude solvents. The mucus activity against eight strains of human pathogenic bacteria, including Bacillus cereus ATCC 33019, Escherichia coli O157:H7, Listeria monocytogenes ATCC 7644, Pseudomonas aeruginosa ATCC 27853, Salmonella braenderup ATCC BAA 664, Salmonella typhimurium, Staphylococcus aureus ATCC 25933, and Vibrio cholerae, were tested. The acetic acid mucus extract of B. everetti was able to inhibit five strains of bacteria and show no activity toward E. coli O157:H7, B. cereus ATCC 33019 and L. monocytogenes ATCC 7644. Moreover, the highest protein concentration was quantified in crude extract, followed by aqueous and acetic acid extracts. This study provides a preliminary knowledge on the activity of epidermal mucus of B. everetti towards five out of the eight human pathogens tested, therefore it may contain potential sources of novel antibacterial components which could be further extracted for the production of natural antibiotics towards human-related pathogenic bacteria.
Three species of the lichen Usnea (U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr. and U. pectinata Stirt.) and nine associated endolichenic fungi (ELF) were evaluated using a metabolomics approach. All investigated lichen crude extracts afforded antibacterial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC): 0.0625 mg/mL), but none was observed against Escherichia coli, while the ELF extract Xylaria venustula was found to be the most active against S. aureus (MIC: 2.5 mg/mL) and E. coli (MIC: 5 mg/mL). X. venustula was fractionated and tested for to determine its antibacterial activity. Fractions XvFr1 to 5 displayed bioactivities against both test bacteria. Selected crude extracts and fractions were subjected to metabolomics analyses using high-resolution LC-MS. Multivariate analyses showed the presence of five secondary metabolites unique to bioactive fractions XvFr1 to 3, which were identified as responsible for the antibacterial activity of X. venustula. The p-values of these metabolites were at the margin of significance level, with methyl xylariate C (P_60) being the most significant. However, their high variable importance of projection (VIP) scores (>5) suggest these metabolites are potential diagnostic metabolites for X. venustula for "dual" bioactivity against S. aureus and E. coli. The statistical models also showed the distinctiveness of metabolites produced by lichens and ELF, thus supporting our hypotheses of ELF functionality similar to plant endophytes.
The performance of a compost biofilter inoculated with mixed microbial consortium was optimized for treating a gas-phase mixture of benzene and toluene. The biofilter was acclimated to these VOCs for a period of ∼18d. The effects of concentration and flow rate on the removal efficiency (RE) and elimination capacity (EC) were investigated by varying the inlet concentration of benzene (0.12-0.95g/m(3)), toluene (0.14-1.48g/m(3)) and gas-flow rate (0.024-0.072m(3)/h). At comparable loading rates, benzene removal in the mixture was reduced in the range of 6.6-41% in comparison with the individual benzene degradation. Toluene removal in mixture was even more affected as observed from the reductions in REs, ranging from 18.4% to 76%. The results were statistically interpreted by performing an analysis of variance (ANOVA) to elucidate the main and interaction effects.
Liquid biphasic flotation (LBF), an integrated process of liquid biphasic system (LBS) and adsorptive bubbles flotation, was used for the purification of C-phycocyanin from S. platensis microalgae. Various experimental parameters such as type of phase forming polymer and salt, concentration of phase forming components, system pH, volume ratio, air flotation time and crude extract concentration were evaluated to maximise the C-phycocyanin recovery yield and purity. The optimal conditions for the LBF system achieving C-phycocyanin purification fold of 3.49 compared to 2.43 from the initial LBF conditions was in polyethylene glycol (PEG) 4000 and potassium phosphate combination, with 250 g/L of polymer and salt concentration each, volume ratio of 1:0.85, system pH of 7.0, air flotation duration of 7 min and phycocyanin crude extract concentration of 0.625 %w/w. The LBF has effectively enhanced the purification of C-phycocyanin in a cost effective and simple processing.
A binary mixture of starch-starch or starch with other biopolymers such as protein and non-starch polysaccharides could provide a new approach in producing starch-based food products. In the context of food processing, a specific adjustment in the rheological properties plays an important role in regulating production processing and optimizing the applicability, stability, and sensory of the final food products. This review examines various biopolymer mixtures based on starch and the influence of their interaction on physicochemical and rheological properties of the starch-based foods. It is evident that the physicochemical and rheological characteristics of the biopolymers mixture are highly dependent on the type of starch and other biopolymers that make them up mixing ratios, mixing procedure and presence of other food ingredients in the mixture. Understanding these properties will lead to improve the formulation of starch-based foods and minimize the need to resort to chemically modified starch.
An endophytic fungus isolated from the plant Cinnamomum mollissimum was investigated for the bioactivity of its metabolites. The fungus, similar to a Phoma sp., was cultured in potato dextrose broth for two weeks, followed by extraction with ethyl acetate. The crude extract obtained was fractionated by high-performance liquid chromatography. Both crude extract and fractions were assayed for cytotoxicity against P388 murine leukemic cells and inhibition of bacterial and fungal pathogens. The bioactive extract fraction was purified further and characterized by nuclear magnetic resonance, mass spectral and X-ray crystallography analysis. A polyketide compound, 5-hydroxyramulosin, was identified as the constituent of the bioactive fungal extract fraction. This compound inhibited the fungal pathogen Aspergillus niger (IC(50) 1.56 μg/mL) and was cytotoxic against murine leukemia cells (IC(50) 2.10 μg/mL). 5-Hydroxyramulosin was the major compound produced by the endophytic fungus. This research suggests that fungal endophytes are a good source of bioactive metabolites which have potential applications in medicine.
Elephantopus scaber and Clinacanthus nutans are traditionally used as wound healing herb. The objective of the present study is to develop a new polyherbal formulation, by comparison, the herbal combination of Elephantopus scaber and Clinacanthus nutans as an in vitro antioxidant activity with their individual herbal activity followed by fractionation of polyherbal formulation for in vivo wound healing activities and identification of bioactive compounds from their active fractions. Antioxidant activity was performed in vitro by DPPH scavenging antioxidant activity followed by in vivo wound healing activities using excision wound model, incision wound model, and burn wound model. Toxicity of the fractions of the polyherbal formulation was performed by a dermal toxicity test. The result showed that Elephantopus scaber crude extract on the basis of EC50 performs a much faster action (15.67 μg/mL) but with less % inhibition (87.66%) as compared to the combination of the new polyherbal formulation of crude extract (30 μg/mL). The polyherbal formulation has the highest % inhibition (89.49%) at the same dose as compared to Elephantopus scaber (87.66%). In comparison among all crude and fractions of new polyherbal formulation, it was found that the ethyl acetate fraction of polyherbal formulation has the fastest activity (EC50 14.83 μg/mL) with % inhibition (89.28%). Furthermore, during evaluation of wound contraction on excision and incision wound model, ethyl acetate fraction possesses the highest activity with (P < 0.001) and (P < 0.0001), respectively. During burn wound model, aqueous fraction (P < 0.001) possesses the highest activity followed by an ethyl acetate fraction (P < 0.0001). LC-MS analysis discovered the presence of several flavonoid-based compounds that work synergistically with sesquiterpene lactone and other bioactive compounds. In conclusion, flavonoid increases the antioxidant activity that surges the rate of wound contraction and works synergistically with other bioactive compounds.
We present a model case study of the activity of aqueous extract of Hericium erinaceus fresh fruit bodies in promoting functional recovery following crush injury to the peroneal nerve in adult female Sprague-Dawley rats. The aim was to explore the possible use of this mushroom in nerve repair. The activities of aqueous extract were compared to activities exhibited by mecobalamin (vitamin B12), which has been widely used in the treatment of peripheral nerve disorders. Analysis of walking track indicated that return of hind limb function and normal toe spreading occurred earlier in treated groups than in the negative control (non-treated) group. Regeneration of axons and reinnervation of motor endplates/neuromuscular junction in extensor digitorum longus muscle of rats in treated groups developed better than in the negative control group. Further, immunofluorescence studies also showed that dorsal root ganglia neurons ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for Akt and MAPK signaling pathways as well as c-Jun and c-Fos genes compared to the negative control group. Akt cascade plays a major role in mediating neurotrophin-promoted cell survival, while MAPK cascade is involved in mediating neurite outgrowth. Immediate early gene expression was also involved in the cascade of events leading to regeneration. Local axonal protein synthetic machinery was also enhanced in the distal segments of crushed nerves in treated groups. Therefore, daily oral administration of H. erinaceus could promote the regeneration of injured rat peroneal nerve in the early stage of recovery.
This study aimed to determine anthocyanins and their antioxidative and cardioprotective properties in defatted dabai parts. Anthocyanins in crude extracts and extract fractions of defatted dabai peel and pericarp were quantified using UHPLC, while their antioxidant capacity and oxidative stress inhibition ability were evaluated by using DPPH and CUPRAC assays as well as linoleic acid oxidation system, hemoglobin oxidation, and PARP-1 inhibition ELISA. Cardioprotective effect of the defatted dabai peel extract was evaluated using hypercholesterolemic-induced New Zealand white rabbits. Six anthocyanins were detected in the defatted dabai peel, with the highest antioxidant capacities and oxidative stress inhibition effect compared to the other part. The defatted dabai peel extract has also inhibited lipid peroxidation (plasma MDA) and elevated cellular antioxidant enzymes (SOD and GPx) in the tested animal model. Major anthocyanin (cyanidin-3-glucoside) and other anthocyanins (pelargonidin-3-glucoside, malvidin-3-glucoside, cyanidin-3-galactoside, cyanidin-3-arabinoside, and peonidin-3-glucoside) detected in the defatted dabai peel are potential future nutraceuticals with promising medicinal properties.
The aim of this study was to develop and validate a rapid and simple high performance thin layer chromatographic (HPTLC) method to screen for antioxidant activity in algal samples. 16 algal species were collected from local Victorian beaches. Fucoxanthin, one of the most abundant marine carotenoids was quantified directly from the HPTLC plates before derivatization, while derivatization either with 2,2-diphenyl-1-picrylhydrazyl (DPPH) or ferric chloride (FeCl3) was used to analyze antioxidants in marine algae, based on their ability to scavenge non biological stable free radical (DPPH) or to chelate iron ions. Principal component analysis of obtained HPTLC fingerprints has classified algae species into 5 groups according to their chemical/antioxidant profiles. The investigated brown algae samples were found to be rich in non-and moderate-polar compounds and phenolic compounds with antioxidant activity. Most of the phenolic iron chelators also have shown free radical scavenging activity. Strong positive and significant correlations between total phenolic content and DPPH radical scavenging activity showed that, phenolic compounds, including flavonoids are the main contributors of antioxidant activity in these species. The results suggest that certain brown algae possess significantly higher antioxidant potential when compared to red or green algae and could be considered for future applications in medicine, dietary supplements, cosmetics or food industries. Cystophora monilifera extract was found to have the highest antioxidant concentration, followed by Zonaria angustata, Cystophora pectinate, Codium fragile, and Cystophora pectinata. Fucoxanthin was found mainly in the brown algae species. The proposed methods provide an edge in terms of screening for antioxidants and quantification of antioxidant constituents in complex mixtures. The current application also demonstrates flexibility and versatility of a standard HPTLC system in the drug discovery. Proposed methods could be used for the bioassay-guided isolation of unknown natural antioxidants and subsequent identification if combined with spectroscopic identification.
The aim of this study was to evaluate the genotoxic effects of a crude extract of khat (Catha edulis, Forsk) leaves in rats. Two groups were fed khat crude extract, 1000 and 2000mg/kg body weight, for 90 days and were compared with a control group. The alkaline (pH>13) version of comet assay was used in this study. However, no previous published work has been undertaken and showed the effect of khat on DNA migration in the comet assay. To compare the comet assay results with another genetic endpoint, blood samples were analyzed for chromosomal aberrations. These results showed no DNA damage detected using comet assay in both the khat treated groups, while the results of chromosomal aberrations assay showed a significant increase (P<0.05) in the 2000mg/kg body weight treated group compared to the control group.
Protease was extracted from two maturity stages of noni fruits (Morinda citrifolia L.), unripe (stage 1) and ripe (stage 5). The crude extract was partially purified by acetone precipitation method followed by dialysis, gel filtration chromatography and freeze drying. Protein concentrations, proteolytic activity, molecular weight distribution, pH stability, temperature stability and storage efficiency of the resulting protease were evaluated. The unripe and ripe noni fruit contains 0.65 and 0.35% protein, respectively. Molecular weight of the proteases from both stages ranged approximately between 3 to 28 kDa based on the SDS-PAGE results. The optimum activity were at pH 7s and 6, temperatures of 40 and 50°C, respectively for proteases obtained from the unripe and ripe fruit. Analysis from the freeze dried protease indicated that protease from ripe noni fruits had higher protein concentration and specific activity compared to those from unripe fruit. However, it is more sensitive to pH and temperature and less stable during storage as it shows lower proteolytic activity compared to protease from unripe fruit. Based on its high proteolytic activity reaching up to 70.31 U/mg and storage stability (30% lost of activity), noni fruit could be an alternative source of plant protease.
Tinospora crispa and Tabernaemontana corymbosa have been used traditionally to treat fever, diabetes, rheumatism and sinusitis. The objective of this study was to evaluate the antioxidant activity of Tinospora crispa and Tabernaemontana corymbosa. The presence of apigenin and magnoflorine was detected using LCMS/MS in Tinospora crispa (Patawali) whereas appararicine, voafinine, conodusarine, conodurine, voacamine and voacangine were detected in Tabernaemontana corymbosa (Susur kelapa) methanol extract. The stem extract of Tinospora crispa showed high antioxidant activity in the following order: DPPH radical scavenging, reducing power and metal chelating assay (98.8%, 0.957, 81.97%) than Tabernaemontana corymbosa of leaves (90.04%, 0.652, 69.64%), stem (82.78%, 0.819, 36.70%) and root extracts (63.25%, 0.469, 51.56%), respectively. The high antioxidant activity in the stem extract of Tinospora crispa is due to the presence of apigenin and magnoflorine. The high antioxidant activity in Tabernaemontana corymbosa extract is due to its high phenol contents. There were significant linear positive correlation (r=0.788, p<0.001, r2=0.621) between the total phenolic content and DPPH free radical scavenging assay in the crude extracts of Tinospora crispa and Tabernaemontana corymbosa. Meanwhile, a significant moderate positive correlation was observed between the total phenolic content and ferric reducing power assay (r= 0.556, p<0.05, r2= 0.309). However, there was no significant difference in the correlation coefficient of total phenolic content and metal chelating assay.
Kaffir lime or “limau purut” (Citrus hystrix) is a popular traditional herbal plant with aromatic leaves. It produces fruits which are commonly used in Asian cuisines as a flavouring agent. Essential oil has been a popular and valuable product applicable in various industries, but its availability is scarce. Essential oil from the leaves of Citrus hystrix is a complex mixture of volatile and semi-volatile compounds. It is rich in bioactive molecules that act synergistically to improve the well-being of an individual. Essential oil is extracted from Citrus hystrix leaves using a few extraction methods; however, the most excellent extraction methods have yet to be ascertained. This review paper highlights several extraction methods that determined the final yield of Citrus hystrix leaves’ essential oil and the comparison of advantages and disadvantages between each method. Two types of methods are discussed, which are conventional and modern methods. Conventional methods include hydro-distillation, steam distillation, Soxhlet distillation and solvent extraction, while the modern method includes pressurized liquid extraction (PLE). In addition, pre- treatment processes are also included as they can significantly affect the performance of other important processes and production yield. This paper also found that the highest yield of 56.16% was obtained using PLE method. Essentially, each of the extraction and pre-treatment methods has its own pros and cons; hence choosing a suitable method depends heavily on the producer's demand and requirement.
Threadfin bream (Nemipterus japonicas) muscle was hydrolysed using protease extracted from
bilimbi (Averrhoa bilimbi L.) fruit. This study was performed in order to compare the efficiency of bilimbi protease in producing threadfin bream protein hydrolysate with the commercial protease; alcalase 2.4 L. Initially, protease was extracted and then purified using 40% ammonium sulfate precipitation method. The proteolytic activity of the crude extract and purified protease was determined. Precipitation using 40% ammonium sulfate resulted in bilimbi protease specific activity of 2.36 U/mg and 23.13% recovery. Threadfin bream hydrolysate was prepared based on the pH-stat method by hydrolysis for 2 hrs. Hydrolysis using bilimbi protease produced 34.76% degree of hydrolysis (DH) and 3.75% yield while hydrolysis using alcalase resulted in 86.6% DH with 22.78% yield. Alcalase hydrolysate showed higher solubility than bilimbi protease hydrolysate at pH 7 with 70.87 and 32.16% solubility, respectively. Results also showed that protein content of threadfin bream hydrolysate produced using alcalase was higher (86.86%) than those produced using bilimbi protease (22.12%). However, both hydrolysates showed low moisture content between 3.93 to 7.00%. The molecular weight distribution analysis using SDS–PAGE indicated the distribution of smaller peptides especially in alcalase hydrolysate. Overall, the results showed that alcalase is more efficient enzyme choice than bilimbi protease for preparing threadfin bream hydrolysates. However, both hydrolysates could play an important role thus contribute to the food industry.
Methanol extract of the Gracilaria changii has been screened for antimicrobial activity against Pseudomonas aeruginosa. Antimicrobial activities were carried out using disc diffusion assay and broth dilution method against P. aeruginosa. The methanol extract of G. changii showed a good antimicrobial activity against P. aeruginosa with MIC (Minimum Inhibitory Concentration) value of 6.25mg/ml. Exposure of P. aeruginosa cells to 6.25mg/ml of methanol extract of G. changii resulted in complete inhibition of the bacterial cells. The main abnormalities noted via SEM and TEM studies were the alterations in morphology and cytology of the bacterial cells. The main reason for this deterioration was discussed. The effect of the methanol extract on the growth profile for the bacteria was also done and confirmed the bactericidal effect of the G. changii methanol extract on P. aeruginosa by changing the normal growth profile of P. aeruginosa. In an acute toxicity study using mice, the median lethal dose (LD(50)) of the extract was greater than 2000 mg/kg, and we found no pathological changes in macroscopic examination by necropsy of mice treated with extract. We conclude that G. changii might be safely used as an antimicrobial agent.
Antimicrobial activities of plants have long been evaluated for their promising use as antimicrobial agent and in minimizing the unwanted resistance effects of microorganisms. The study was conducted to evaluate the antibacterial activity of Quercus infectoria gall crude extracts against multidrug resistant (MDR) bacteria in vitro. The screening test was determined by disc diffusion technique using sterile filter paper discs impregnated with 1 mg/ disc (50 mg/ml) aqueous and ethanol extracts of Q. infectoria galls tested on five selected MDR bacterial strains. The minimum inhibitory concentration (MIC) was determined using the twofold serial micro dilution technique at concentration ranging from 5.00 mg/ml to 0.01 mg/ml. The minimum bactericidal concentration (MBC) was determined by sub culturing the microtitre wells showing no turbidity on the agar plate to obtain the MBC value. Both extracts showed substantial inhibitory effects against methicillin resistant coagulase negative Staphylococcus (MRCoNS) and methicillin resistant Staphylococcus aureus (MRSA). A slightly reduced inhibitory zone diameter was observed with MDR Acinetobacter sp. while no inhibitory effect was displayed among the extended spectrum beta lactamases (ESBL) K. pneumoniae and ESBL E. coli isolates. A significant difference in the zone sizes between both extracts was only observed in MRSA (p < 0.05). The MIC values ranged from 0.08 mg/ml to 0.63 mg/ml for aqueous and ethanol extracts against MRSA, MRCoNS and MDR Acinetobacter sp. while their MBC to MIC ratio values were 2 and less. The Q. infectoria gall extracts have shown very promising in vitro antibacterial activities and may be considered as a potentially good source of antimicrobial agent especially against MDR Gram positive bacteria.