Brine Shrimp Lethality Assay Research Articles

Biological Activities and Phytochemical Study of Pedicularis wilhelmsiana Fisch Ex. From Iran.

This research paper presents findings of an investigation about antibacterial effect of methanol/water fractions as well as cytotoxic activity of the extracts obtained from Pedicularis wilhelmsiana (Scrophulariaceae) which grows in Azarbaijan/Iran by agar well diffusion method and brine shrimp lethality test successively. Phytochemical study of this plant was determined as well. A combination of solid-phase extraction (SPE), preparative reversed-phase HPLC analysis and spectroscopic means were applied for fractionation, purification, and identification of ingredients respectively. Antimicrobial test demonstrated that 40% and 60% methanol/water fractions were more active than methanolic extract and other SPE fractions. No cytotoxic effect was detected from the extracts of this plant by brine shrimp lethality assay. Phytochemical study of aerial parts of Pedicularis wilhelmsiana (P. wilhelmsiana) afforded two phenylethanoids (verbascoside and martynoside), three iridoids (Aucubin, ipolamiid, 5-hydroxy-8-epi-loganin) and two flavonoids (luteolin, luteolin-7-glucoside) along with mannitol on the basis of spectral evidences (UV, 1H and 13CNMR) as well as comparison with literature data. The findings of this research supported further studies related to antibiotic potential of methanolic extract of P. wilhelmsiana.

Inhibitory effects of fermented soybean tempeh on the anti-adhesive properties of Actinomyces viscosus and plaque growth in vitro

Background: Tempeh, the Southeast Asian traditional food, has garnered great attention for its antibacterial property against Gram-positive and Gram-negative pathogens, and antidiarrheal effect. We have previously reported the potentiality of tempeh hexane fraction (HXF) in ceasing Actinomyces viscosus biofilm formation in vitro. Objective: Here, we investigated the efficacy of tempeh HXF on other cariogenic virulence traits of A. viscosus such as adhesive properties, acid production, and plaque growth. Materials and Methods: Anti adhesion of HXF was assessed based on its effects on the number of cells adhering to the surface of tooth in sucrose-dependent (SD) and sucrose-independent (SI) medium. The potential of HXF to inhibit the capability of A. viscosus to generate acids was investigated by pH drop assay. The HXF at different concentrations were used to determine the LC50based on brine shrimp lethality assay. Finally, the prospect of HXF as an inhibitor of plaque formation was investigated using artificial saliva-coated denture as an in vitro batch model. Results: HXF significantly decreased colony-forming unit of SD (1.07 log reduction) and SI (0.56 log reduction)-mediated adsorption of bacterial cells onto the tooth surface over 4- and 12-h incubation, respectively. Acid production was reduced after treated with HXF in a dose-dependent manner. Finally, a substantial reduction in plaque coverage area >55% was found on the HXF treated-denture. Conclusion: The anti-biofilm effect of HXF was associated with the suppression of A. viscosus adhesion to tooth surfaces and reduction in acid production. Furthermore, in vitro anti-plaque potential of HXF was demonstrated.

Phytochemical analysis, antioxidant and cytotoxic activities of Dryopteris ramosa.

Objective: The aim of present study was to evaluate phytochemical, cytotoxic and antioxidant potential of dichloromethane and methanol extracts of Dryopteris ramosa (Hope) C. Chr. belongs to family Dryopteridaceae. The plant is traditionally used as anthelminthic, diuretic, antibacterial, astringent and as tonic for various stomach and intestinal problems. Method: The phytochemical potential, total phenol, flavonoid and alkaloid contents of Dryopteris ramosa were determined for the first time. Plant was subjected to Brine shrimp lethality and DPPH inhibition assay was done. Identification and quantification of various phenolic content by was done HPLC. Results: Phytochemical analysis revealed that flavonoids, terpenoids, cardiac glycosides, alkaloids and saponins were present in the plant while tannins were absent. The results depicted high in vitro cytotoxic effect by dichloromethane extract with a LD50 0.6903 μl/ml as standardized with Etoposide with LD50 of 7.4625 μl/ml. The methanol extract showed the strong DPPH scavenging activity with 72.76 ± 1.52% inhibition as compared to the dichloromethane extract with 55.37 ± 3.055% inhibition. The high phenol content was found in methanol extract having 199.2 ± 4.50(mg of GA/g of extract). HPLC analysis showed the presence of various phenol compounds (Gallic acid, quercetin, caffeic aid, vanillic acid, cinnamic acid etc.) with their relevant concentration, in the plant. The dichloromethane extract exhibited high flavonoid content 73.02 ± 1.00 (mg of RE/g of extract). Alkaloidal content was highest in dichloromethane extract with 29.73 ± 4.04 (mg of AE/g of extract).

Lipophilic halogen-free ionic liquid with antibacterial and anti-biofilm activities against Pseudomonas aeruginosa

A halogen-free ionic liquid (IL) designed with long alkyl chain anion is reported. 1-methylimidazolium stearate (MIM stearate) synthesized through Bronsted acid-base reaction has shown improved lipophilic character and is able to penetrate bacterial cell walls. Antimicrobial activities against Gram-negative bacteria, Escherichia coli, and Pseudomonas aeruginosa were observed. Anti-biofilm assays showed effectivity against the biofilm of Pseudomonas aeruginosa. At 50 µg/mL the %biofilm inhibition of MIM stearate towards P. aeruginosa biofilm formation is comparable to the Bromofuran positive control. Brine shrimp lethality assay showed weak toxicity indicating the IL to be safe and benign. The synthesized MIM stearate showed good promise as an antimicrobial and anti-biofilm agent.

Characteristics of Kombucha Fermentation from Different Substrates and Cytotoxicity of Tea Broth

Kombucha is made from sweetened tea fermented by a symbiotic culture of bacteria and yeast consumed worldwide because of its potentially beneficial effects on health. However, there are only few studies on the safety of kombucha consumption that will establish it as a functional beverage. The present study compared, pH, temperature and sugar content of different tea mixtures of black or green tea as nitrogen sources and white and brown sugar as carbon sources in a 30-day fermentation period. A marked decrease in pH was observed throughout fermentation with Green tea-White (GW) sugar mixture showing the lowest recorded pH value of 2.37 on the 14th day of fermentation. Temperature is essential in the fermentation process and thus maintained at ambient 29±1°C. Black and green teas with white sugar (7°Brix) showed to have higher sugar level compared to tea mixtures with brown sugar (5°Brix). Brine Shrimp Lethality Assay was carried out to determine cytotoxicity of kombucha. The four substrate combinations have very low LC50 values with Black tea-Brown sugar (BB) mixture showing to have the lowest in both acute and chronic effects (0.073 ppm and 0.101 ppm, respectively). This indicates safety of kombucha for consumption.

Bioactivity-guided isolation of alkamides from a cytotoxic fraction of the ethyl acetate extract of Anacyclus pyrethrum (L.) DC. roots.

Abstract Introduction. The alcohol extract of Pellitory (Anacyclus pyrethrum) roots has been previously shown to exert anticancer activities on the Human Colorectal Cancer Cell Line (HCT) by targeting apoptosis, metastasis and cell cycle arrest. However, the nature of the cytotoxic molecules associated with this activity remains unexplored. Aims. This study aims to reinvestigate Pellitory root extract as regard to its cytotoxic activity and to proceed to a bioguided fractionation to explore its active fraction and to give new insight in their phytochemical constituents. Methods. Powdered roots were subjected to repeated extraction with Petroleum ether (Pe), Chloroform (Ch), Ethyl acetate (Ea) and Methanol (Me). Pellitory extracts were then screened for cytotoxic activity using the Brine Shrimp Lethality (BSL) bioassay. Results. Ea extract exhibited a marked cytotoxic activity, with LC50 of 249.26 μg/mL in the BSL bioassay. The remaining extracts (Pe,Ch,Me) treated groups exhibited no or low mortality in the range of tested concentrations (1-1000 µg/mL). BSL assay-guided chromatographic fractionation of Ea active Extract revealed a highly cytotoxic fraction (F11) with LC50 of 42.5 µg/mL. Multistep purifications of the active F11 fraction afforded four alkamides, namely N-isobutyldeca-2,4-dienamide or Pellitorine (I), N-propyldodeca- -2,8-dienamide (II), N-isobutyltetradeca-2,4-dienamide (III) and N-propylnona-2,5- -dienamide (IV). Conclusions. This study suggests that cytotoxic activity is localized mainly in the ethyl acetate extract (Ea) of pellitory roots. BSL assay fractionation of this active extract leads to the isolation of four alkamides, including pellitorine (I). While this isobutyl alkamide has previously shown strong cytotoxic activities against human cancer cell lines, the other compounds (II to IV) were not previously reported as cytotoxic. Subsequently, the isolated alkamides will be considered in future study as candidates for in depth in-vitro evaluation of their cytotoxicity against cancer and normal cell lines. Finally, through this study, BSL assay demonstrate again its usefulness as bench-top assay in exploring plant extracts for cytotoxic compounds.

Design and Synthesis of Novel 1,3,4-Oxadiazole Derivatives Bearing Azo Moiety as Biologically Significant Scaffolds

Introduction: Oxadiazoles having azo-functionality represent a new and an exciting class of physiologically active heterocyclic compounds. A number of molecules with these moieties have significant role in medicinal chemistry due to their diverse biological activities. Methodology: A series of 1,3,4-oxadiazole-2-thiol derivatives bearing azo-moiety have been designed and synthesized by multistep reaction sequences staring from p-aminobenzoic acid by employing literature know procedures. These compounds were investigated for their antimicrobial, cytotoxic, α-amylase and protein kinase inhibitory and antileishmanial potential. Results and Discussion: The newly synthesized compounds were characterized by spectroscopic techniques (IR, UV-Vis, NMR and MS). Antibacterial activity was investigated against four bacterial strains i.e. E. coli, P. aeruginosa, S. aureusand B. subtilis and good level of microbial inhibition was exhibited by most of the compounds with MIC 6.25 µg/mL. Brine Shrimp lethality assay revealed that the compound 31 is an excellent cytotoxic agent. (E)-5-(4-((4-Butoxyphenyl) diazenyl)phenyl)-1, 3,4-oxadiazole-2-thiol (21) showed good α-amylase inhibition (46.3± 0.45%) and (E)-2-(Decyloxythio)-5-(4-((propoxyphenyl)diazenyl)phenyl)-1,3,4-oxadiazole (30) exhibited excellent antileishmanial activity (88.4± 0.34%) at 10 µg/mL concentration. In case of protein kinase inhibition, remarkably, the oxadiazole-2-thiols (20-25) were found more active than the standard surfactinat tested concentration (25 µg/disc). Conclusion: We have designed, synthesized and characterized an interesting series of biologically active oxadiazole bearing diazene moiety derivatives. Noteworthy, the presence of diazene functionality, polar heterocyclic moiety as well as hydrophobic alkyl chain is special feature of these compounds. These studies provide a basic idea to further explore these types of scaffolds as biologically potent molecules in drug design and development.

EGFR Tyrosine Kinase Targeted In Silico Design and Synthesis of Novel Quinazoline Derivatives

Quinazolines are medicinally important as anti-convulsant, anti-cancer, anti-microbial, and anti-tubercular properties etc. They target epidermal growth factor receptors on tumor cells. This work is aimed to synthesize a new series of quinazoline derivatives which could deliver drug specifically to the EGFR over expressing tumors. A series of novel Schiff’s base analogues were developed by in silico screening methods. The drug likeness of the analogues was analyzed by using Molinspiration software. Biological activities of these analogues were evaluated by using PASS software. The candidates which obeyed Lipinski rule of five and having suitable anti-cancer and anti-microbial activity were taken for docking studies using Schrodinger software. All the proposed derivatives were docked with various protein targets obtained from PDB, using GLIDE software and satisfactory docking energy scores were obtained. Selected 10 derivatives of quinazoline have been synthesized by anthranilamide and benzaldehyde as starting materials. These analogues were purified by analyzing its melting point, Rf value. These were further characterized by FT-IR, 1H NMR and MASS spectral studies. The anti-cancer activity of these derivatives was done by MTT assay against HeLa cell lines, LD50 values were calculated by Brine Shrimp Lethality Assay. From these experiments it is clearly revealed that these analogues possess good anti-cancer activity and are good lead compounds against tumors.

Bumblebee death associated with Tilia x europaea L

For more than six decades, bumblebee death, which mainly occurs in August, has been assumed to be associated with intake of presumed toxic nectar from linden trees (Tilia spp.), a hypothesis which has been sustained by observations of a significant number of dead bumblebees under these trees during their flowering season. Several theories exist in current literature to account for these observations. The nectar has been assumed to contain compound(s) toxic to the bumblebees including the monosaccharide mannose, which cannot be metabolized by bumblebees. The presence of toxic compounds such as the alkaloid nicotine or pesticides of anthropogenic origin has also been indicated. However, none of the above suggested compounds have hitherto been properly characterized from the nectar. In the current paper we report on characterization of the composition of nectar of linden trees, under which a significant number of dead bumblebees were observed. The structure determinations were performed by selective 1D TOCSY NMR and extensive applications of 2D NMR spectroscopy. The nectar of the linden flowers was readily available in significant amounts during the entire period when dead bumblebees were observed under these trees. The nectar only contained non-toxic sugars such as α-glucopyranose, β-glucopyranose, sucrose, fructose and minor amounts of xylose. The nectar did not contain mannose, nor alkaloids or pesticides. Initial toxicity studies using brine shrimp lethality assay showed that the nectar did not exhibit any toxic effects even at concentrations higher than 1 mg/ml dry weight, providing disproving evidence against the assumption of the nectar's toxic character.

In vitro neuroprotective effect of charantin from Momordica charantia against neurotoxin and endoplasmic reticulum stress-induced cell death in SH-SY5Y cells

Aim: The study deals with the evaluation of neuroprotective effect of charantin. Charantin was isolated from Momordica charantia (MC) L. (Bitter gourd) and characterized by performing infrared (IR), nuclear magnetic resonance (NMR). As it was earlier proved that amyloid beta deposition and cognition are inter-related, neuroblastoma cell lines SH-SY5Y were utilized in this study to prove the effectiveness of charantin on the against neurotoxin and endoplasmic reticulum (ER) stress-induced cell death. Materials and Methods: Charantin was isolated from MC and characterized using IR, NMR spectroscopy studies. The cell viability analysis and IC50 determination for charantin in SH-SY5Y neuroblastoma cell line were performed by MTT assay and neuronal red uptake assay. The neuroprotective effect in SH-SY5Y cells against the MPP+ and ER stress damage was evaluated. The cell viability assays were performed at various concentration ranges from 0.5 to 1 mg/ml. Charantin was added at 0 h, 8 h, and 16 h post the neurotoxin treatment. Brine shrimp lethality assay and MTT assay were also performed to observe the cytotoxicity of charantin at different doses. Results and Discussion: Charantin was found to have a significant IC50 value in SH-SY5Y cells. IN case of MPP+ treatment 0.5 mg, 1 mg/ml concentration of charantin showed significant neuroprotection and improvement in cell viability. In tunicamycin-induced damage, the protection was seen at 0 h and not at 8 h and 16 h post-treatment. At the same time, it has less/no toxicity at therapeutic doses; very high doses may pronounce cytotoxicity. Conclusion: The present investigation shows the neuroprotective effect of MC and charantin obtained from the plant can be employed as a memory enhancer in Alzheimer’s related dementias; further to be evaluated clinically by formulating charantin.

Investigation of in vitro Cytotoxic Effects of Perchloroetylene by Using Brine Shrimp Lethality Assay

Perkloroetilen (PERC), kuru temizleme endüstrisinde çözücü, yağ giderici ve temizleyici olarak yaygın bir şekilde kullanılmaktadır. Perkloroetilen, güçlü ve etkili bir temizleyici olup giysilerin buruşmasını ve renginin solmasını engeller. Kuru temizleme dükkanlarının en az üçte ikisi kuru temizleme işlemlerinde perkloroetilen’i kullanmaktadır. Uluslararası Kanser Araştırmaları Ajansı (IARC, 1995) Perkloroetileni insan karsinojeni olarak Grup2A içerisine almıştır ve kurutemizleme endüstrisinde çalışan insanlar için kanserojen olabileceğini belirtmiştir. PERC’in kuru temizlemede çalışan işçilerde cilt lezyonlarının yanı sıra, lenfosarkom, lösemi, kolon, akciğer ve ürogenital kanserlere neden olduğuna dair çalışmalar bulunmaktadır. Bu çalışmada saf Perkloroetilen’in Artemia salina larvaları üzerindeki potansiyel sitotoksik etkisi Brine Shrimp Letalite Testi ile araştırılmıştır. Denemelerde negatif kontrol olarak tuzlu su, çözücü kontrol olarak etanol ve pozitif kontrol olarak da Mitomisin-C (MMC) kullanılmıştır. Çalışmanın sonuçları, LC50 değerlerine göre, in vitro ortamda PERC’e 24 saat maruz bırakılan A.salina larvaları üzerindeki sitotoksik etkinin sadece 1000 ppm’lik PERC uygulanması sonucunda ortaya çıktığını, diğer konsantrasyon aralıklarında (10 ve 100 ppm) ise sitotoksik aktivitesinin olmadığını göstermiştir.

Cytotoxicity of selected medicinal plants extracts using the brine shrimp lethality assay from Samburu county, Kenya

Cytotoxicity of selected medicinal plants extracts using the brine shrimp lethality assay from Samburu county, Kenya

Biosynthesis and characterisation of antimicrobial silver nanoparticles from a selection of fever-reducing medicinal plants of South Africa

Most developing countries, including South Africa, depend strongly on traditional medicine for a therapeutic outcome and have therefore employed numerous medicinal plants to treat fevers. Therefore, it is imperative that fever-reducing medicinal plants are investigated to establish their efficacy and to determine their potential as sources of new antimicrobials. The incorporation of nanotechnology in antimicrobial research with reference to medicinal plants is a growing domain. The interest in silver nanoparticles (AgNPs) encompasses the tested hypothesis that the chemical combination of silver with medicinal plant extracts results in nanoparticles with enhanced antimicrobial properties in comparison with plant extracts alone. This study investigated the antimicrobial properties from 10 medicinal plants of commercial significance used traditionally for the treatment of fever in South Africa and their potential for enhanced antimicrobial efficacy when incorporated within AgNPs. Plant extracts and AgNPs were tested against fever-related pathogens, i.e., two Gram-positive pathogens; Listeria monocytogenes (ATCC 19111) and Enterococcus faecalis (ATCC 29212) as well as two Gram-negative pathogens; Klebsiella pneumoniae (ATCC 13883) and Acinetobacter baumannii (ATCC 19606) using the broth microdilution method. Chemical characterisation of AgNPs included Ultraviolet–visible (UV–Vis) spectroscopy, dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM). The toxicity profiles of the AgNPs were evaluated using the brine-shrimp lethality assay (BSLA). Silver nanoparticles of both Eucomis autumnalis and Sclerocarya birrea display a dramatic increase in antimicrobial activity against the four test pathogens compared to the respective aqueous plant extracts. The greatest difference in antimicrobial activity was observed against E. faecalis where an increase in antimicrobial activity of at least 50-fold when E. autumnalis aqueous sample were compared with the AgNP-counterparts. Cytotoxicity of both AgNP samples from the BSLA emerged at less than 50% mortality, the results obtained in this study justify the use of selected fever-reducing plant extracts with the biosynthesis of AgNPs as promising antibacterial agents with low toxicity.

Antibacterial, cytotoxic and hemolytic activity of Holothuria parva sea cucumber from north Persian Gulf

In the present report, we focused on the screening of antibacterial, hemolytic and cytotoxic activities of Holothuria parva sea cucumber from the Persian Gulf for potential application in nutraceutical and pharmaceutical products. Ethanol, methanol and aqueous extracts of H. parva were prepared by maceration of milled body wall pieces. Cytotoxicity of extracts was tested using brine shrimp lethality assay. Antibacterial activity of extracts was tested against Vibrio alginolyticus, Vibrio parahaemolyticus, Shigella flexneri Salmonella group D, Escherichia coli and Staphylococcus aureus by serial dilution method. Hemolytic activity of extracts was performed using human erythrocytes. Results showed bacterial inhibition of ethanolic and methanolic extracts against E. coli, V. parahaemolyticus and S. aureus at concentrations higher than 20 µg/ml and minimum bactericidal concentration was observed for ethanolic and methanolic extracts at 500 mg/µl against E. coli. The median lethal concentration for brine shrimp was lowest (61.25 ± 8.53) for methanolic extracts, which shows this extract is more toxic against tested organisms. The methanolic and ethanolic extracts of H. parva showed a high level of hemolytic activity (0.62 ± 0.12 and 0.54 ± 0.9) to tested erythrocytes and were comparable to standard pure saponin, but no such activity was found in aqueous extracts.

Brine shrimp lethality and acute toxicity tests of different hydro-methanol extracts of Anacardium occidentale using in vitro and in vivo models: a preliminary study

The antimicrobial, antifungal, antiprotozoal, anthelminthic, and antiviral activities of various part of Anacardium occidentale have been reported in vitro. However, there are no records or documentation on the potential cytotoxicity and acute toxicity effects of the extracts using in vivo study. The present study was conducted to evaluate the cytotoxicity and acute toxicity effects of stem bark, root, stem, and leave extracts using in vivo and in vitro models. Plant materials were extracted using cold maceration in 70% hydro-methanol, and the extracts were concentrated under hot air oven at 40 °C. In brine shrimp lethality assay, five concentrations of each extract (10, 100, 250, 500, and 1000 ppm) were used. Each of the concentration was tested in triplicate and mortality of Artemia salina was monitored for 24 h post exposure. Sea water was used in control group. Acute toxicity testing was conducted according to the method of Organisation for Economic Co-operation and Development (OECD) using 2-week-old cockerels. Dose-probing tests were used to determine the median lethal dose (LD50). Four measures of acute toxicity tests were used, 3000, 1500, 750 and 375 mg/kg in 2-week-old cockerels. The LC50 was estimated using Fumey probit analysis. The LC50 of the four extracts were 336.34, 338.72, 486.36, and 657.62 ppm for stem bark, roots, stem, and leave, respectively. Acute toxicity tests did not show any sign of toxicity as no mortality was recorded in the four extracts. The treatment with all the plant extracts did not cause any morphological changes in the internal organs of the chickens grossly when compared to the control group. The study showed that the extracts of A. occidentale contain bioactive compounds which may be cytotoxic in vitro and non-toxic in vivo and can be used in ethnomedicine.

Physicochemical properties and toxicity of cocoa powder-like product from roasted seeds of fermented rambutan (Nephelium lappaceum L.) fruit

A novel way to fully utilize rambutan fruit and seed is to ferment peeled fruits followed by drying and roasting, and use the seeds to produce seed powder similar to that of cocoa powder. Hence, the objective of this study was to optimize the roasting time and temperature of rambutan fruit post-fermentation and drying, and to produce a cocoa-like powder product from the seeds. Parameters monitored during roasting were colour and total phenolic content, while seed powder obtained using optimized roasting conditions was analyzed for its physicochemical properties and toxicity. The latter was examined using the brine shrimp lethality assay. Results showed that the roasted seed powder possessed colour and key volatile compounds similar to that of cocoa powder. Besides, the brine shrimp lethality assay indicated that the roasted seed powder was non-toxic. Thus, the fruit, including its seed could be fully utilized and subsequently, wastage could be reduced.

Sponge chemical defenses are a possible mechanism for increasing sponge abundance on reefs in Zanzibar.

Coral reefs are experiencing increasing anthropogenic impacts that result in substantial declines of reef-building corals and a change of community structure towards other benthic invertebrates or macroalgae. Reefs around Zanzibar are exposed to untreated sewage and runoff from the main city Stonetown. At many of these sites, sponge cover has increased over the last years. Sponges are one of the top spatial competitors on reefs worldwide. Their success is, in part, dependent on their strong chemical defenses against predators, microbial attacks and other sessile benthic competitors. This is the first study that investigates the bioactive properties of sponge species in the Western Indian Ocean region. Crude extracts of the ten most dominant sponge species were assessed for their chemical defenses against 35 bacterial strains (nine known as marine pathogens) using disc diffusion assays and general cytotoxic activities were assessed with brine shrimp lethality assays. The three chemically most active sponge species were additionally tested for their allelopathic properties against the scleractinian coral competitor Porites sp.. The antimicrobial assays revealed that all tested sponge extracts had strong antimicrobial properties and that the majority (80%) of the tested sponges were equally defended against pathogenic and environmental bacterial strains. Additionally, seven out of ten sponge species exhibited cytotoxic activities in the brine shrimp assay. Moreover, we could also show that the three most bioactive sponge species were able to decrease the photosynthetic performance of the coral symbionts and thus were likely to impair the coral physiology.

Toxicity of Mid-Michigan plant extracts in the brine shrimp lethality assay and the effect of assay methodology on sensitivity

The Brine Shrimp Lethality Assay (BSLA) is a rapid and economical assay that has been used to screen plant extracts for potential anti-tumor compounds. The objective of this study was to screen Mid-Michigan plant extracts for activity in the BSLA to identify candidates for further pharmacological study. Based on the knowledge that many plants found in the region have been used in Native American traditional medicine for a variety of conditions, including cancer, we hypothesized that some plants would demonstrate activity in the BSLA. In addition, we aimed to determine any difference in sensitivity between the traditional test tube format and the more recently developed microwell format of the assay. After a preliminary screening of 38 plant extracts, eight extracts were active in the BSLA, including Achillea millefolium, Nepeta cataria, and Podophyllum peltatum. Both A. millefolium and N. cataria have been used as cold remedies in Native American traditional medicine, while P. peltatum derivatives etoposide and teniposide are current anti-tumor drugs. We also demonstrated that the sensitivity of the assay is dependent on the assay format. Percent lethality was higher in the 96-well plate format when compared to the 5 mL glass test tube format. These results suggest that several plants from the Mid-Michigan region may be worthy of further investigation to characterize their biological activity as it relates to potential anti-cancer properties. Furthermore, we suggest that researchers use the 96-well BSLA format to increase sensitivity and inter-laboratory consistency of the assay.

Toxicity Assessment of Nephrolepis exaltata (L.) Schott, Fam. Nephrolepidaceae

Abstract The fern Nephrolepis exaltata (L.) Schott, fam. Nephrolepidaceae, has little traditional medicinal use. In order to estimate its potential safety, in the present study we have investigated the phytotoxicity (on Triticum aestivum L.) and toxicity on brine shrimp of extracts from plants grown hydroponically. The species identity was confirmed by macroscopic and microscopic examinations on rhizomes, rachises, pinnae and runners, using bright field and fluorescent microscopy. Biological assays were performed on aqueous and ethanol solutions of the fronds. The brine shrimp lethality assay was performed on Artemia franciscana Kellog and a phytobiological bioassay on Triticum aestivum L. Lethality, root elongation and karyokinetic film modifications were evaluated, and LC50 and IC50 values were calculated. The microscopic analysis revealed the main histo-anatomic elements: polystelic structure and hypodermis (rhizome, rachis, runners), trichomes (rachis), homogenous structure, trichomes and diacytic/anisocytic stomata (leaves). The ethanol and aqueous extracts showed low cytotoxic effects on both Triticum aestivum roots and Artemia franciscana nauplii.

Chemical Composition, Toxicity, and Acaricidal Activity of Eucalyptus Globulus Essential Oil from Algeria

Abstract The study was aimed at determining the chemical composition, toxicity effect and field varroacidal efficacy of the essential oil distilled from the leaf of Eucalyptus globulus (Eg) grown in Algeria. Brine shrimp lethality (BSL) assay and bee hives infected by Varroa destructor were used to assess the toxicity and acaricidal effect, respectively. Steam distillation of leaves yielded 0.93 % (v/w) of essential oil (EO). GC/MS Analysis revealed 39 compounds, essentially oxygenated monoterpenes (86.01%). The main constituents of the oil were 1,8-cineole (78.45 %), o-cymene (2.18 %), isopinocarveole (1.74 %), α-pinene (1.69 %), pinocarvone (1.34%) and veridiflorol (1.31%). The BSL assay revealed a highly toxic value of LC50 (67.55 μg/mL). Furthermore, field efficacy testing on bee hives infected with Varroa destructor has confirmed the effectiveness of Eucalyptus globulus essential oil (EgEO) or thymol as varroacidal agents. Moreover, a EgEO + thymol association was more effective than EgEO or thymol alone. Finally, the use of EgEO + thymol may constitute a viable alternative to the thymol-based commercial treatment