Brine Shrimp Lethality Assay Research Articles

Chemical profiling of Justicia vahlii Roth. (Acanthaceae) using UPLC-QTOF-MS and GC-MS analysis and evaluation of acute oral toxicity, antineuropathic and antioxidant activities

Ethnopharmacological relevanceJusticia vahlii Roth. (Acanthaceae), also called as kodasoori and bhekkar is an annual therophyte erect or decumbent herb used traditionally in toothache, skin diseases (itching, topical inflammation) and for the treatment of various respiratory disorders. Aim of the studyThe current study aimed at exploring pain cessation potential of J. vahlii Roth. via murine model of neuropathic pain and its phytochemical, toxicological and antioxidant profiles. Materials and methodsThe hydro-alcoholic extract of J. vahlii (HAEJv) prepared by maceration technique was subjected to preliminary phytochemical screening, total bioactive content determination, UPLC-QTOF-MS and GC-MS analysis. Toxicity assessment was carried out by using brine shrimp lethality assay and acute oral toxicity test. Murine model of neuropathic pain was applied to assess the antineuropathic potential of the species. Furthermore effect of the extract on catalase, superoxide oxide dismutase (SOD), Glutathione (GSH), interleukin-1beta (IL-1β) and total necrosis factor-alpha (TNF-α) was also studied. In vitro antioxidant profile was explored by using four methods; 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis(3-ethylbenothiazoline)-6-sulfonic acid (ABTS), CUPric reducing antioxidant capacity (CUPRAC) and Ferric reducing antioxidant power (FRAP) assay. ResultsThe phytochemical screening revealed the presence of phenols, flavonoids, coumarins, alkaloids and lignans as the major classes of secondary metabolites. The extract was found rich in total phenolics content (TPC) and total flavonoids content (TFC) with identification of total 59 bioactives in UPLC-QTOF-MS and 40 compounds in GC-MS analysis. The extract was found nontoxic up to 4000 mg/kg (p.o.) in mice and no mortality observed in brine shrimp lethality assay. The HAEJv significantly reduced number of acetic acid induced abdominal constrictions at 100 mg/kg (p < 0.01) and 200 mg/kg (p < 0.001) and increased paw withdrawal threshold p < 0.05 at 100 mg/kg and p < 0.001 at 200 mg/kg, and an increase in tail withdrawal latency time p < 0.001 at 200 mg/kg was observed. The extract significantly increased levels of catalase, SOD and GSH while decreased IL-1β and TNF-α levels in sciatic nerve tissue of mice. HAEJv showed highest antioxidant activity through CUPRAC method 121.32 ± 1.22 mg trolox equivalent per gram of dry extract (mg TE/g DE) followed by DPPH 81.334 ± 4.35 mg TE/g DE, FRAP 69.89 ± 3.05 mg TE/g DE and ABTS 38.17 ± 2.12 mg TE/g DE. ConclusionThe current study back the traditional use of J. vahlii in pain cessation through antioxidant based antineuropathic pain activity and revealed the extract non-toxic with number of functional phytoconstituents and warrants further research on isolation of the compounds and sub-acute toxicity studies.

Brine shrimp lethality assay with selected medicinal plants extracts

Brine shrimp lethality assay with selected medicinal plants extracts

Assessment of Cytotoxic, Antioxidant, Thrombolytic, Anti Inflammatory and Antimicrobial Activity of Curcuma longa Linn, Cissus quadrangularis and Boerhaavia diffusa Herbal Mixture - An In vitro Study

Background and Aim: Plants play an important role in drug research, and the pharmaceutical industry is heavily reliant on natural products for new drug development. Curcumin, a natural compound contained in the rhizomes of the plant Curcuma longa Linn., has been shown to have anti-inflammatory properties in scientific studies. Cissus quadrangularis L. is a fleshy plant that can be found all over the world, especially in Asia, Africa, and a few other warm tropical areas. Boerhaavia diffusa L. (Nyctaginaceae) is present in the tropical regions of India, South America, and Africa. B. diffusa roots are commonly used in Ayurveda to treat various ailments. The aim of the study is to assess the cytotoxic, antioxidant and thrombolytic, antiinflammatory and antimicrobial properties of the aqua-alcoholic extract of the herbal mixture (Curcuma longa Linn +Cissus quadrangularis +Boerhaavia diffusa )&#x0D; Methods: Aqua-alcoholic extract of mixture of Curcuma longa Linn, Cissus quadrangularis and Boerhaavia diffusa was prepared. Phytochemical in-vitro studies were done using the CCB mixture cytotoxicity by brine shrimp lethality assay, antioxidant activity by DPPH assay, anti inflammatory activity by albumin denaturation assay, antimicrobial activity against Staphylococcus aureus, Streptococcus mutans, Escherichia coli and Candida albicans by agar well diffusion method, thrombolytic activity by clot lysis assay.&#x0D; Results: The CCB mixture showed good cytotoxic activity at varying concentration in brine shrimp lethality assay, increasing antioxidant activity with DPPH assay, increasing thrombolytic activity, increasing anti inflammatory activity with increasing concentration, the anti microbial activity was not efficient as positive controls.&#x0D; Conclusion: In this in-vitro study the CCB mixture shows improved cytotoxic, antioxidant, thrombolytic, anti-inflammatory and antimicrobial activity. Further studies on invivo animal and human clinical trials needs to be done.

HPLC-DAD Based Polyphenolic Profiling and Evaluation of Pharmacological Attributes of Putranjiva roxburghii Wall.

The current study was intended to explore the phytochemical profiling and therapeutic activities of Putranjiva roxburghii Wall. Crude extracts of different plant parts were subjected to the determination of antioxidant, antimicrobial, antidiabetic, cytotoxic, and protein kinase inhibitory potential by using solvents of varying polarity ranges. Maximum phenolic content was notified in distilled water extracts of the stem (DW-S) and leaf (DW-L) while the highest flavonoid content was obtained in ethyl acetate leaf (EA-L) extract. HPLC-DAD analysis confirmed the presence of various polyphenols, quantified in the range of 0.02 ± 0.36 to 2.05 ± 0.18 μg/mg extract. Maximum DPPH scavenging activity was expressed by methanolic extract of the stem (MeOH-S). The highest antioxidant capacity and reducing power was shown by MeOH-S and leaf methanolic extract (MeOH-L), respectively. Proficient antibacterial activity was shown by EA-L extract against Bacillus subtilis and Escherichia coli. Remarkable α-amylase and α-glucosidase inhibition potential was expressed by ethyl acetate fruit (EA-F) and n-Hexane leaf (nH-L) extracts, respectively. In case of brine shrimp lethality assay, 41.67% of the extracts (LC50 < 50 µg/mL) were considered as extremely cytotoxic. The test extracts also showed mild antifungal and protein kinase inhibition activities. The present study explores the therapeutic potential of P. roxburghii and calls for subsequent studies to isolate new bioactive leads through bioactivity-guided isolation.

Antitumor Potential of Annona muricata Linn. An Edible and Medicinal Plant in Mexico: In Vitro, In Vivo, and Toxicological Studies.

Annona muricata (Am) is a plant used in traditional Mexican medicine to treat cancer. In this study, ethanol extracts of Am collected in Acapulco and Tecpan from Guerrero state were evaluated orally on Balb/c mice inoculated with 4T1 cells, for cytotoxic activity (CA) on 4T1 cells, in brine shrimp lethality assay (BSLA), and for acute oral toxicity in mice. In addition, ethanol extracts were subjected to high-performance liquid chromatography (HPLC) with diode array detection. Results showed that the extracts collected in December in Acapulco (AcDe) and Tecpan (TeDe) exhibited the most significant antitumor and cytotoxic activity. In the BSLA, the most important effect was observed in the extracts from Acapulco and Tecpan collected in June (AcJu) and August (TeAg), respectively. The samples from Acapulco (AcJu, and AcAg) and Tecpan (TeJu and TeAg) showed the highest toxicity. The analysis of the extracts, AcDe and TeDe, by HPLC revealed that flavonoids, rutin, narcissin, and nicotinflorin were the major components. These findings suggest that extracts from Am collected in Acapulco and Tecpan in the month of December may be an important source to obtain flavonoid glycosides with anticancer potential specifically against breast cancer. This also supports the use of Am to treat cancer in Mexican traditional medicine.

Anticancer and Apoptotic Potentials of Gliricidia sepium Leaf Extract on Breast HCC1395, Prostate DU145 and Colorectal CT26 Cell Lines

Background: Plants have become a household name in the quest for effective and safe cancer chemotherapy in the Pharmaceutical industry. We studied the Brine shrimps lethality, antiproliferative and apoptotic potentials of Gliricidia sepium leaf extract against some selected cancer cell lines.&#x0D; Methodology: Plant leaves extraction was done with 70% ethanol and petroleum ether. Twenty four hours old shrimp’s larvae exposure to different concentrations (1 mg/ mL, 100 µg/ mL, 10 µg/ mL and 1 µg/ mL) of the extracts were used to evaluate the cytotoxicity in Brine shrimps lethality assay (BSLA). Three cancer cell lines: Breast (HCC1395); Prostate (DU145); Colorectal (CT26) and one normal cell line (Vero E6) were used for the in-vitro cytotoxicity testing using MTT assay. IC50, CC50 and Selectivity Index (SI) were determined respectively, using standard methods. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out on HCC1395 and DU145 cell lines. Primers for the genes p53 and BAX were generated and amplified for apoptotic evaluation. The fold change relative to GADPH, the house­keeping gene, was calculated using double delta Ct analysis [2ˉΔΔCT].&#x0D; Results: The results of BSLA showed that both extracts of G. sepium are strongly toxic (LC50 &lt; 100 µg/ml). However, petroleum ether extract (PGS) exhibited the highest toxicity to the shrimps, with LC50 of 11.95 µg/ml. This is about 8 times more cytotoxic than cyclophosphamide (LC50 of 98.76 µg/ml). On cancer cells, PGS exhibited varying antiproliferative activities; it was high on prostate (IC50 = 12.76 µg/ml), antiproliferative (IC50 = 23.55 µg/ml) and moderately antiproliferative (IC50 = 77.58 µg/ml) on breast and colorectal cancer respectively. PGS CC50 value was greater than 100 µg/ml. Ethanol extract (EGS) showed very high toxicity to all the tested cancer cells (IC50 &lt;20µg/ml) with low cytotoxicity (CC50 = 41.81 µg/ml) to the normal cells. It exhibited a significant difference and high selectivity index across all the cell lines used in this study. EGS also upregulated both p53 and BAX biomarkers in the qRT-PCR apoptotic study.&#x0D; Conclusion: The leaf extracts of G. sepium is a potential anticancer agent. The 70% ethanol extract selectively induced antiproliferative activities on cancer cells and upregulated apoptotic genes. The Petroleum ether extract also showed very low cytotoxicity to the normal cells. The plant should be considered a novel candidate for further studies.

Essential Oil Blends: The Potential of Combined Use for Respiratory Tract Infections.

This study investigated the potential efficacy of 369 commercial essential oil combinations for antimicrobial, anti-toxic and anti-inflammatory activity with the aim of identifying synergy among essential oils commonly used in combination by aromatherapists for respiratory purposes. Essential oil combinations were assessed for their antimicrobial activities using a panel of Gram-positive, Gram-negative, and yeast strains associated with respiratory tract infections. The antimicrobial activity was measured by determining the minimal inhibitory concentration (MIC) of microbial growth. The fractional inhibitory concentration index (ΣFIC) was calculated to determine the antimicrobial interactions between the essential oils in the combination. The toxicity of the essential oil combinations was tested in vitro using the brine shrimp lethality assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on RAW 264.7 mouse macrophage cells and A549 lung cancer cell lines. In addition, an inflammatory response was evaluated measuring nitric oxide production. The essential oils, when in combination, demonstrated an increased antimicrobial effect, a reduction in toxicity and provided improved anti-inflammatory outcomes. Five distinct combinations [Cupressus sempervirens (cypress) in combination with Melaleuca alternifolia (tea tree), Hyssopus officinalis (hyssop) in combination with Rosmarinus officinalis (rosemary), Origanum marjorana (marjoram) in combination with M. alternifolia, Myrtus communis (myrtle) in combination with M. alternifolia and Origanum vulgare (origanum) in combination with M. alternifolia] were found to be the most promising, demonstrating antimicrobial activity, reduced cytotoxicity and improved anti-inflammatory effects. With the increased prevalence of respiratory tract infections and the growing antimicrobial resistance development associated with antimicrobial treatments, this study provides a promising complementary alternative for the appropriate use of a selection of essential oil combinations for use in the respiratory tract.

Comparative toxicity, phytochemistry, and use of 53 Philippine medicinal plants

The study compares the toxicity of 53 selected medicinal plants commonly used in the Philippines to treat various diseases. It uses as a benchmark Vitex negundo L., which was approved by the Philippine Food and Drug Administration as an herbal drug for cough and asthma after passing clinical trials for safety and efficacy. The methods were chosen for their simplicity and accessibility even for resource-limited laboratories. Extracts (95 % ethanol) of the medicinal parts of the plants were (1) chemically profiled using qualitative phytochemical tests that detect the presence of key classes of bioactive compounds; and (2) evaluated for toxicity using the brine shrimp (Artemia sp.) lethality assay (BSLA). General phytochemical screening revealed the presence of tannins in 50 plant extracts, alkaloids in 43, glycosides in 33, flavonoids in 31, steroids in 21, triterpenoids in 20, anthraquinones in 10, and saponins in 8. Extracts from eight plants had LC50 values lower than the potassium dichromate control (approximately 12 μg/mL) and were considered highly toxic; extracts from 21 plants had LC50 values between 12 μg/mL and 100 μg/mL and were considered moderately toxic; extracts from 19 plant extracts, including Vitex negundo and some common vegetables, had LC50 values between 100 μg/mL and 500 μg/mL, and were considered mildly toxic and likely to have reasonable safety margins; five plant extracts, including common vegetables, had LC50 values above 500 μg/mL and were considered essentially nontoxic. No apparent correlation could be found between toxicity and chemical diversity or a specific class of phytochemicals present. Our findings may serve as a guide for herbal drug and nutraceutical development, especially in prioritizing plants for more detailed safety studies.

Fluconazole and biogenic silver nanoparticles-based nano-fungicidal system for highly efficient elimination of multi-drug resistant Candida biofilms

HypothesisSecretion of extracellular hydrolytic enzymes such as secreted aspartyl proteinases (SAP) and biofilm formation are considered as important virulence determinants in resistant strains of Candida sp. which facilitate the pathogenicity towards host cells. The resistance in fungal pathogens results in use of high dosage of antifungal drug, therefore in order to study the effect of reduced amount of antifungal, combination therapy was utilized. Currently, no data related to combined effect of mycogenic silver nanoparticles and fluconazole, in SAP inhibition by resistant Candida spp. has been described. In this study, synergistic combination along with anti-virulence was used as a way for discovering new antifungal potential of mycogenic silver nanoparticles (Ag-NPmyc). ExperimentsThis study utilized Ag-NPmyc, synthesized using Aspergillus oryzae SZ1 and their combined effect with fluconazole on planktonic and 24 h (h) biofilm forms of clinical isolates of six Candida species (C. albicans, C. galabrata, C. parapsilosis, C. krusie, C. tropicalis, C. albicans ATCC 24433). The cytotoxic effects of Ag-NPmyc mycogenic using brine shrimp lethality assay was also determined. Ag-NPmyc and fluconazole (FLC) suspensions were used to treat Candida sp. biofilms grown on polystyrene surfaces for 24 h (h) and their efficacy was determined by MIC, time-kill growth curve analysis, cell viability analysis (XTT reduction assay), inhibition of virulence factors exopolymeric substances (EPS) and SAP activity. Nanoparticles were characterized using Ultraviolet–Visible (UV–Vis) spectroscopy, X-Ray Diffraction (XRD) and transmission electron microscopy (TEM). FindingsThe surface plasmon resonance band of Ag-NPmyc was observed at 430 nm by Ultraviolet–Visible spectroscopy, X-Ray Diffraction and TEM confirmed with uniform, spherical shaped and monodispersed with size range of 1–50 nm. Maximum cytotoxicity was achieved after 0.3 mg/ml. The results showed that combination use of Ag-NPmyc with FLC exhibit strong in vitro antifungal synergy (FICI values ranged from 0.2812 to 0.375) against the majority of the Candida strains tested with gradual decrease in CFU till 12 h, and after 15 h fungistatic growth was observed. Comparable effects on biofilms of Candida sp. were obtained. Overall, >60% reduction of metabolic activity was observed in different Candida sp. at 16 μg/ml FLC in combination with Ag-NPmyc. The EPS inhibition (%) in planktonic cells ranged from 2 to 73, 3–82 and 1–19 by 15 ppm Ag-NPmyc/FLC, 25 ppm Ag-NPmyc/FLC and FLC alone, respectively. Whereas in case of biofilm forms, EPS inhibition (%) ranged from 1 to 84, 1–93 and 1–32 by 15 ppm Ag-NPmyc/FLC, 25 ppm Ag-NPmyc/FLC and FLC alone, respectively. Highest EPS inhibition in planktonic cells was observed with Ag-NPmyc/FLC, 25 ppm which ranged 3–82%. Whereas in case of biofilm forms, EPS inhibition ranged from 1 to 93% with the same combination. Less inhibition seen in case of FLC treated Candida spp. planktonic (1–15%) and biofilm (1–14%) cells. The results obtained, showed that combinations of 15 ppm and 25 ppm Ag-NPmyc with FLC effectively inhibited the SAP in Candida spp. with an inhibition range of 1–82% and 1–79%, respectively in case of planktonic cells. Whereas 1–78% and 2–92% in case of biofilm cells as compared to less inhibition (<20%) seen in case of FLC treated Candida spp. planktonic and biofilm cells. ConclusionAg-NPmyc in combination with fluconazole promoted significant reduction in virulence factors of resistant forms of Candida sp. Ag-NPmyc at different concentrations were not found cytotoxic to brine shrimps. The results highlight the improved antifungal effect of Ag-NPmyc in combination with FLC against planktonic and biofilm forms of different species of Candida while inhibiting (>70%) in SAP production. The effect of azole-NP combination on EPS inhibition was also significant. Synergistic effect showed inhibition in SAP production both in planktonic and biofilm forms of Candida spp.

In vivo Toxicity Evaluation of Ethanolic Extract From Two Mangrove Plants

Background: Mangrove plants, often possessing a unique collection of metabolites, have extensively been used for the primary healthcare of coastal residents. Objectives: To determine the safety level and enrich the scientific data, the present study aimed to investigate the toxicity of Avicennia officinalis and Excoecaria agallocha. Methods: Ethanolic leaf and stem extracts were evaluated for cytotoxicity by brine shrimp lethality assay. The obtained extracts were administered to Swiss albino in a single dose (200, 400, 800, 1600, &amp; 3200 mg/kg body weight) by oral-gavage for acute toxicity assay. Furthermore, systematic observation was performed by close monitoring for any toxic manifestations and mortality after dosing for the first 4 h, at 24 h and twice daily for 6 days. Evaluating the adverse effects were estimated by comparing the test groups with the controls. After sacrificing all group animals, relative organ weight was measured and histopathological analysis was conducted. Results: Having Lethal Concentration (LC50) of 44.66 µg/mL, E. agallocha leaf was found with the highest toxicity against brine shrimp nauplii. The toxicological study data demonstrated no death and noticeable change in behavioral patterns in the test mice groups, compared with the control group. Moreover, no significant (P&gt;0.05) differences were found in body weight and relative organ weights, compared to the controls. The histological structures of the liver in the treated mice displayed regular tissue configurations similar to the control group. Conclusion: In this study, the mice model exhibited no harmful effects; thus, the reported results indicated that the ethanolic extracts of leaf and stem of these two mangrove plants are safe for therapeutic use. Further long-term toxicological impact of the extracts should be determined for well-founded confirmation.

Eucalyptus gunnii and Eucalyptus pulverulenta 'Baby Blue' Essential Oils as Potential Natural Herbicides.

The phytotoxicity and eco-compatibility of essential oils (EOs) from Eucalyptus gunnii (EG) and E. pulverulenta ‘Baby Blue’ (EP), cultivated in Italy for their cut foliage, were investigated. Leaf micromorphology, EOs phytochemical characterization, and phytotoxicity were analysed. EP revealed a significantly higher oil gland density and a higher EO yield with respect to EG. In both EOs, 1,8-cineole was the major compound (~75%), followed by α-pinene in EG (13.1%) and eugenol in EP (7.5%). EO phytotoxicity was tested on both weeds (Lolium multiflorum, Portulaca oleracea) and crops (Raphanus sativus, Lactuca sativa, Lepidium sativum, Solanum lycopersicum, Pisum sativum, Cucumis sativus). EG EO inhibited germination of P. oleracea, R. sativus, and S. lycopersicum seeds (ranging from 61.5 to 94.6% for the higher dose used), while affecting only radical elongation in S. lycopersicum (ranging from 66.7 to 82.6%). EP EO inhibited germination of P. oleracea and R. sativus (ranging from 41.3 to 74.7%) and affected radical elongation of L. sativum and L. multiflorum (ranging from 57.4 to 76.0%). None of the EOs affected the germination and radical growing of L. sativa, P. sativum, and C. sativus. Moreover, EP EO was more active than EG EO in inhibiting α-amylase, a key enzyme for seed growth regulation. Brine shrimp lethality assay showed that both EOs are safe for aquatic organisms, suggesting their high eco-compatibility. The data collected provide useful information for future applications of these EOs in agriculture as safe and selective bioherbicides.

Preparation of Boerhaavia diffusa Mediated Selenium Based Mouthwash-A Comparative Microbial and Cytotoxic Effect

Introduction: The extract of Boerhaavia diffusa root, a plant used in Indian traditional medicine, has significant immunomodulatory Potential. Selenium, a nutrient element that has a massive function in biological systems, is one of the interesting compounds to integrate with antibacterial agents. Recently several studies have pointed out the ability of selenium nanoparticles to exhibit anticancer, antioxidant, antibacterial and antibiofilm properties.&#x0D; Aim: To analyze the antimicrobial and cytotoxic activity of Boerhaavia diffusa mediated selenium nanoparticles based mouthwash.&#x0D; Materials and Methods: 1 g of Boerhavia diffusa was added in 100 ml of distilled water. It was boiled; the plant extract was filtered using Whatman’s no.1 filter paper. In 250 ml conical flask, 60 ml of 20 millimolar sodium selenite was prepared and 40 ml of the filtered plant extract was mixed. This flask was kept in a magnetic stirrer. The nanoparticle solution was centrifuged at 8000rpm to prepare nanoparticle pellets. The nanoparticle pellet was dried in a hot air oven at 80 degree celsius. The dried powder was sent for Characterization. A mouthwash is prepared.&#x0D; The prepared mouthwash was tested for cytotoxic activity by brine shrimp lethality assay and antimicrobial activity evaluated the zone of inhibition of agar well diffusion method.&#x0D; Results and Discussion: It was proved that cytotoxic activity of selenium bound mouthwash was less compared to the commercial mouthwash. The antibacterial activity of the selenium bound mouthwash against C. albicans and S. mutans was significant when compared to the standard antimicrobial agent.&#x0D; Conclusion: Based on the results, this mouthwash has the required qualities to be commercially be used. Therefore further studies can be done to prove that this mouthwash can be used commercially.

Anxiolytic effects of silibinin and naringenin on zebrafish model: A preclinical study.

OBJECTIVE:Stress exacerbates the pathophysiology of major neurodegenerative disorders. In this study, the zebrafish (Danio rerio), the frequently used model for experimental studies of stress and other central nervous system disorders, was used to evaluate the anxiolytic potential of flavonoids, namely silibinin and naringenin on alleviating acute stress-induced anxiety.MATERIALS AND METHODS:A molecular docking study with Molegro Virtual Docker software was done to assess the binding potential of flavonoids on serotonin and dopamine receptors. To determine the bioactivity and investigate the toxicity of the flavonoids, silibinin, and naringenin, brine shrimp lethality assay, and an acute toxicity study was conducted according to Organization for Economic Co-operation and Development (OECD) Test Guideline 203. The effect of silibinin and naringenin was assessed using behavioral tasks such as the novel tank assay and the light-dark test on the zebrafish model of acute stress.RESULTS:Molecular docking studies showed a higher affinity of silibinin and naringenin for the serotonin and dopamine receptors. In comparison to the LC50 value, 13.15 μg/ml of the reference standard potassium dichromate, silibinin, and naringenin yielded higher LC50 values, 34.10 μg/ml and 91.33 μg/ml, respectively. The LC50 value of silibinin and naringenin was observed to be >100 mg/l from the acute toxicity study on adult zebrafish. After transferring to a novel tank, silibinin and naringenin-treated zebrafish groups were found to explore the upper level of the tank, similar to standard drugs, and spent a long time in the upper level of the tank compared to the control group (p < 0.01). Both silibinin and naringenin treatment group spent increased amounts of time in the tank's illuminated part in contrast to that of the dark side as evidenced by the number of zebrafish entering or remaining in the illuminated part of the tank through the light-dark test. Silibinin and naringenin treated groups were found to spend increased time in the light side significantly on the day 15th of evaluation as compared to the control group (p < 0.05 and p < 0.001, respectively).CONCLUSION:The flavonoids, silibinin, and naringenin were found to mitigate acute stress-induced anxiety, owing to their anxiolytic properties in the zebrafish model and may be explored as the potential therapeutic agents for treating anxiety.

Evaluation of acute oral toxicity, cytotoxicity, antidepressant and antioxidant activities of Japanese mint (Mentha arvensis L.) oil

Menthol-rich essential oil of Mentha arvensis L . (Japanese mint) have been identified as a valuable and new source of natural bioactive compounds with applications not only in industry but also in our daily life. The present study aimed to assess the cytotoxicity, acute toxicity, antidepressant and antioxidant activities of Japanese mint oil (JMO). Brine shrimp lethality assay was performed to investigate cytotoxicity of JMO. The acute toxicity was evaluated by Lorke's method. Forced swimming test (FST) was performed for determining antidepressant property. The antioxidant potentials were performed by DPPH scavenging assay, ferric reducing antioxidant potential (FRAP) and Fe 2+ ion chelating ability methods. In acute toxicity study, JMO showed toxic effects within 5 minutes and estimated LD 50 value was 2070.4 µl/kg body weight after 10 days oral treatment. The brine shrimp lethality assay exhibited concentration–dependent approach and the LC 50 value was determined to be 139.73 l/ml, with lower and upper limits of 82.2428 l/ml and 202.7082 l/ml, respectively. In the FST method, intraperitoneal injections of JMO emulsion at doses of 78l/kg, 156l/kg, and 225l/kg of body weight resulted in a significant (p<0.005) increase in struggling time and a reduction in immobility. This observation had a nearly identical effect to the reference drug. Besides, JMO showed weak antioxidant activity in DPPH assay with an IC 50 of 72.58±6.16387 μl/ml. But in other two methods, it exhibited strong antioxidant abilities whereas 1.4010 μl/ml reducing power for FRAP assay and 66.66% chelating rate for Fe2+ ion chelating ability test at 100 μl/ml concentration. The investigation clearly showed that JMO have significant antidepressant and cytotoxic properties whereas acute toxicity was moderate. Besides, JMO also possess considerable antioxidant activities.

TOKSISITAS EKSTRAK KARANG LUNAK Cespitularia sp. DARI TELUK MANADO PROVINSI SULAWESI UTARA

Secondary metabolites produced by marine organisms commonly have unique chemical structures and very interesting pharmacological activities. Marine organisms that have the most chemical conent are produced by marine invertebrates, followed by marine plants. Groups that include marine invertebrates include: sponges, bryozoans, cnidarians and tunicates. Soft coral, which is one of the marine invertebrates, has biological activities, such as antifungal, cytotoxic, antineoplastic, antimicrobial, HIV inhibitor and anti-inflammatory. This study aims to determine the value of the soft coral extract Cespitularia sp. taken from Manado Bay by the Brine Shrimp Lethality Assay (BSLA) method. This toxicity test was carried out quantitatively by analyzing data based on the number of deaths caused by the test extract of Artemia sp. The results obtained based on data analysis namely the value of the soft coral extract Cespitularia sp. is 53.27 ppm, this means that the extract can be developed as an anticancer drug candidate due to below below to 1000 ppm. Soft coral Cespitularia sp. categorized as toxic based on the Meyer classification or moderately toxic based on the Tanamatayarat classification and very toxic based on the Clarkson classification.Keywords: Soft coral; Cespitularia sp.; Toxicity;

Screening of anti-microbial, anti-biofilm activity, and cytotoxicity analysis of a designed polyherbal formulation against shigellosis

BackgroundShigellosis is an infectious intestinal disease common in rural communities. In developing countries, shigellosis is caused predominantly by Shigella flexneri and has been determined as a major cause of morbidity and mortality. ObjectiveThe study investigates the anti-biofilm, anti-microbial, and anti-shigellosis activity of a designed formulation. Materials and methodsThe potential of the formulation against S. flexneri (MTCC 1457) was investigated using a well-diffusion assay. Further, the effect of the designed formulation on bacterial growth and biofilm formation was analyzed by the spectrophotometry method. Anti-quorum sensing activity was analyzed using Chromobacterium violaceum CV026, a bacterial strain. Finally, the cytotoxicity of the formulation was examined by using cell line and brine shrimp lethality assay. ResultsThe MIC value of the aqueous extract of the formulation was 2.4 mg ml−1 and an inhibitory zone of 23 mm was observed. On the other side, the formulation significantly inhibited the bacterial growth, biofilm formation (23.78%), violacein inhibition (27.68%) at 0.6 mg ml−1 concentration (did not significantly affect the growth curves) and was found non-toxic in cell assay and brine shrimp lethality assay. ConclusionAccording to the result obtained, the designed formulation was found effective and non-toxic, so it can be used to treat shigellosis and Shigella-related infections.

Evaluation of chemical profiles and biological properties of Gliricidia sepium (Jacq.) Walp. volatile oils from Nigeria

Volatile oils obtained by hydrodistillation from Gliricidia sepium (Jacq.) Walp. leaf and stem were examined for their chemical composition and biological activity. The oils were analyzed by gas chromatographic-mass spectrometric (GC-MS) techniques. Toxicity, antimicrobial and antioxidant activities were evaluated via brine shrimp lethality assay, agar-diffusion, and DPPH radical-scavenging methods, respectively. A total of 43 and 44 constituents were identified in the leaf and stem oils, correspondingly. The major components recognized in the leaf oil were (E)-hexadecatrienal (16.9%) and pentadecanal (16.0%) while humulene epoxide II (17.5%) and caryophyllene oxide (10.6%) dominated the stem oil. There was no significant activity against the bacteria but moderate inhibition zones (IZ) between 1.8±0.3 and 8.1±0.1 mm were observed against the fungi. The stem oil showed better antifungal activity than the leaf oil but not as active as the standard drug ketoconazole which inhibited the test fungi with IZ range of 10.4±0.4-21.0±1.4 mm at 200 μg. Both oils were toxic to brine shrimp (Artemia salina) giving LC50 of 79.7 μg/mL (leaf) and 38.7 μg/mL (stem). The leaf and stem oils had IC50 of 84.3 μg/mL and 142.2μg/mL, respectively, in the DPPH radical-scavenging assay, indicating moderate antioxidant activity relative to positive controls, butylated hydroxylanisole (IC50=45.1 μg/mL) and α-tocopherol (IC50 =81.6 μg/mL). The results suggest that G. sepium volatile oil may find potential use as a natural antioxidant and antifungal agent.

Ethnobotanical Documentation, Phytochemical Screening, and Cytotoxicity Evaluation of Medicinal Plants Used to Manage Snakebite Envenomation in Mwingi West Subcounty, Kenya.

Snakebite envenomation (SBE) is a life-threatening global public health problem affecting over 2.7 million persons annually, with a bigger burden lying in the developing world. Despite the successful management of SBE by antivenom therapy in conventional medicine, it is of low efficacy due to the diverse venom composition across snake types, which limits its usefulness. As a result, inhabitants of the sub-Sahara region, where SBE incidence is high, utilise medicinal plants as an alternative remedy for SBE. However, most plants have not been ethnobotanically documented and validated empirically and hence this study is needed. An ethnobotanical survey to document medicinal plants used to manage SBE in Migwani ward, Mwingi West Subcounty, Kitui County, was conducted between January and February, 2021. Ethnobotanical data were collected from 45 purposefully sampled respondents from Migwani ward using semistructured questionnaires, field walks, and oral interviews. In this study, 14 medicinal plants which are used to manage SBE were documented. Four plants with the highest Relative Frequency of Citation (RFC) (Entada leptostachya Harms-stem bark (0.58), Senna singueana-roots (0.53), Securidaca longipendunculata-roots (0.36), and Strychnos henningsii-stem bark (0.46)) were selected and extracted using water, methanol, and dichloromethane according to the standard procedures. Qualitative phytochemical analysis of the plant extracts and their cytotoxic effects on brine shrimp nauplii (brine shrimp lethality assay) was conducted according to the standard techniques. Qualitative phytochemical screening revealed the presence of anti-SBE-associated phytochemicals, such as alkaloids, saponins, tannins, phenols, and flavonoids, in the aqueous and methanolic extracts of the studied plant extracts. However, the tested phytochemicals were not detected in dichloromethane extracts of all the studied extracts. The anti-SBE effects of the documented plants could be attributable to these associated bioactive phytocompounds, which are synthesized by the studied plants and transfered to humans when consumed. Furthermore, the aqueous and methanolic extracts of Entada leptostachya and Senna singueana had high LC50 of >1000 µg/ml and were considered noncytotoxic. However, Securidaca longipendunculata had an LC50 of <1000 µg/ml and was considered slightly cytotoxic. Further empirical investigations to characterise the bioactive phytochemicals and their safety should be done.

Antibacterial and Cytotoxic Potential of Two Steroids from the Indonesian Soft Coral Sinularia polydactila

Background: Soft corals of the genus Sinularia are well recognized as a rich source of steroidal compounds. These constituents have been reported as possessing antitumor, antimicrobial, and antiviral activities. Objectives: This study was designed to isolate and elucidate antibacterial and cytotoxic compounds from the soft coral Sinularia polydactila. Methods: Structure elucidation of steroids was determined based on spectroscopic data through 1D and 2D NMR analyses and mass spectrometry, with the results compared to data in the literature. Antibacterial activity was determined using four human bacterial pathogens, namely B. subtilis (ATCC 6633), P. aeruginosa (ATCC 27853), S. aureus (ATCC 25923), and E. coli (ATCC 25922). Cytotoxic activity was evaluated using the human colon cancer cell HCT 116 and brine shrimp lethality assay (BSLA). Results: Two steroids (Compounds 1 - 2) were isolated from the Indonesian soft coral Sinularia polydactila. (22R,23R,24R)-22,23-methylene-24-methylcholest-6-en-5α,8α-epidioxy-3β-ol (1) and 5α,8α-Epidioxy-24(R)-methylcholesta-6,22-dien-3α-ol (2) showed moderate activity against colon carcinoma cancer HCT 116 at the concentrations of 24.8 and 27.3 μg/mL, respectively. Furthermore, compounds 1 and 2 showed cytotoxic activity using the brine shrimp lethality assay with the concentrations of 57.1 and 121.3 3 μg/mL, respectively. Compound 2 showed moderate activity against S. aureus and B. subtilis at the 250 μg/mL concentration. Conclusions: Two steroids isolated from soft coral Sinularia polydactila were found to possess moderate cytotoxic and antibacterial activities.

Antimicrobial Efficacy, Cytotoxicity, Acute Oral Toxicity, and Phytochemical Investigation of the Aqueous and Methanolic Stem Bark Extracts of Bridellia micrantha (Hochst.) Baill

Introduction: Microbial infections cause high morbidity and mortality in humans globally. Antimicrobial resistance, emergence of new strains, high costs of antibiotics, inaccessibility-especially in remote areas, and adverse effects, impede successful eradications of pathogens, hence the need for novel strategies. Bridellia micrantha is used in traditional medicine to treat microbial infections; however, it has not been empirically validated. Methods: Antimicrobial activity of the aqueous and methanolic stem bark extracts of Bridellia micrantha was investigated using the disk diffusion and broth microdilution techniques, described by the Clinical Laboratory Standards Institute (CLSI) guidelines. The brine shrimp lethality assay technique was used to investigate the cytotoxic effects of the studied plant extracts in exposed nauplii. Acute oral toxicity effects of the studied plant extracts in Winstar rats were investigated following the up-and-down procedure described by the Organisation for Economic Development and Co-operation (OECD). Qualitative phytochemical screening was performed following standard procedures. Results: The aqueous and methanolic extract of B. micrantha indicated varied antimicrobial activities against E. coli, S. typhimurium, S. aureus, and C. albicans, with inhibition zones ranging from 6.00mm to 19.00mm. Furthermore, the studied plant extracts exhibited low MIC values (≤100 μg/ml) on selected microbes. Since the MIC values were much lower than 1000μg/ml (the cutoff for antimicrobial efficacy appraisal), it is anticipated that, the studied plant extracts can be strong antibiotics. The aqueous and methanolic stem bark extracts of B. micrantha were cytotoxic to brine shrimp nauplii, with LC50 values of 486.67±3.15 μg/ml and 458.33±2.87 μg/ml, respectively; however, these extracts did not elicit any observable signs of toxicity in rat models. Pharmacologically active phytochemicals, including flavonoids, alkaloids, saponins, tannins, phenols, and anthraquinones were detected in the two studied extracts. Conclusions: The aqueous and methanolic stem bark extracts of B. micrantha have appreciable antimicrobial activity against E. coli, S. typhimurium, S. aureus and C. albicans. Besides, the studied plant extracts are cytotoxic to brine shrimp nauplii; but they do not cause acute oral toxicity effects in rat models. Additionally, the studied plant extracts contain bioactive phytochemicals, with antimicrobial activity.