Propidium monoazide PCR, a method to determine OsHV-1 undamaged capsids and to estimate virus Lethal Dose 50.

Ostreid herpes virus 1 (OsHV-1) has been classified within the Malacoherpesviridae family from the Herpesvirales order. OsHV-1 is the etiological agent of a contagious viral disease of Pacific oysters, C. gigas, affecting also other bivalve species. Mortality rates reported associated with the viral infection vary considerably between sites and countries and depend on the age of affected stocks. A variant called µVar has been reported since 2008 in Europe and other variants in Australia and in New Zealand last decade. These variants are considered as the main causative agents of mass mortality events affecting C. gigas. Presently there is no established cell line that allows for the detection of infectious OsHV-1. In this context, a technique of propidium monoazide (PMA) PCR was developed in order to quantify "undamaged" capsids. This methodology is of interest to explore the virus infectivity. Being able to quantify viral particles getting an undamaged capsid (not only an amount of viral DNA) in tissue homogenates prepared from infected oysters or in seawater samples can assist in the definition of a Lethal Dose (LD) 50 and gain information in the experiments conducted to reproduce the viral infection. The main objectives of the present study were (i) the development/optimization of a PMA PCR technique for OsHV-1 detection using the best quantity of PMA and verifying its effectiveness through heat treatment, (ii) the definition of the percentage of undamaged capsids in four different tissue homogenates prepared from infected Pacific oysters and (iii) the approach of a LD50 during experimental viral infection assays on the basis of a number of undamaged capsids. Although the developped PMA PCR technique was unable to determine OsHV-1 infectivity in viral supensions, it could greatly improve interpretation of virus positive results obtained by qPCR. This technique is not intended to replace the quantification of viral DNA by qPCR, but it does make it possible to give a form of biological meaning to the detection of this DNA.

Nutritional Composition, Fatty Acids Profile, Mineral Content, Antioxidant Activity and Acute Toxicity of the Flesh of Helix aspesra Müller.

Humans consume snail flesh as part of their diet. To assess its nutritional value and toxicity, chemical analyses were conducted to confirm the presence of protein, total and reduced carbohydrates, fat, fatty acid composition and mineral components. Furthermore, an acute toxicity study was carried out to determine the safety of Helix aspersa Müller snail flesh. H. aspersa Müller snail flesh exhibits a high nutritional content, a good ω3/ω6 ratio and higher levels of unsaturated fatty acids. Various minerals have been found in the flesh of H. aspersa Müller. Around 76.91 kcal, or 3.84% of the energy of a daily meal of 2000 kcal, are present in 100 g of this flesh. The evaluation of the antioxidant capacity indicated that the flesh's extracts contained a large quantity of antioxidant biomolecules. Administration of the aqueous extract of H. aspersa Müller flesh didn't cause death in laboratory rats, indicating that the lethal dose 50 is greater than 2000 mg·kg-1 body weight. The consumption of the flesh of H. aspersa Müller is highly recommended for human consumption due to its high concentration of nutrients and essential elements, as well as unsaturated fats, and due to its safety.

Virulence network of interacting domains of influenza a and mouse proteins.

There exist several databases that provide virus-host protein interactions. While most provide curated records of interacting virus-host protein pairs, information on the strain-specific virulence factors or protein domains involved, is lacking. Some databases offer incomplete coverage of influenza strains because of the need to sift through vast amounts of literature (including those of major viruses including HIV and Dengue, besides others). None have offered complete, strain specific protein-protein interaction records for the influenza A group of viruses. In this paper, we present a comprehensive network of predicted domain-domain interaction(s) (DDI) between influenza A virus (IAV) and mouse host proteins, that will allow the systematic study of disease factors by taking the virulence information (lethal dose) into account. From a previously published dataset of lethal dose studies of IAV infection in mice, we constructed an interacting domain network of mouse and viral protein domains as nodes with weighted edges. The edges were scored with the Domain Interaction Statistical Potential (DISPOT) to indicate putative DDI. The virulence network can be easily navigated via a web browser, with the associated virulence information (LD50 values) prominently displayed. The network will aid influenza A disease modeling by providing strain-specific virulence levels with interacting protein domains. It can possibly contribute to computational methods for uncovering influenza infection mechanisms mediated through protein domain interactions between viral and host proteins. It is available at https://iav-ppi.onrender.com/home.

Acute and subchronic toxicity studies of rhein in immature and d-galactose-induced aged mice and its potential hepatotoxicity mechanisms.

Rhein is a key ingredient in many herbal remedies and is widely used. However, herbs containing rhein are frequently associated with poisoning incidents, especially in elderly subjects. Acute and subchronic toxicity of rhein in Kunming mice (KM) was investigated in this experiment. Acute toxicity tests showed a 40% lethality at a given rhein dose of 4000 mg/kg, and the LD50 of rhein was calculated by the bliss method to be greater than 2185.6 mg/kg. In subchronic toxicity, d-gal-induced aged and immature animals were randomized into three groups that were exposed to rhein of 0, 175, and 375 mg/kg/d for 75 days, respectively. No mortality was observed in immature mice group, whereas 55.5% (5/9) subjects in aged mice groups died in the high dosage group. AST, ALT, IL-6, TNF-α levels and typical histopathological changes indicate that rhein causes liver injury. In addition, our investigation explored possible hepatotoxic mechanisms of rhein and experimental results showed increased ROS production, NRF-2 and MDA levels and decreased SOD levels, demonstrating that rhein causes oxidative stress. MMP and mitochondrial swelling levels were able to assess the impact of rhein on mitochondrial function. Furthermore, the effect of rhein on apoptosis can be detected by flow cytometry. Our studies suggested that rhein induces oxidative stress leading to mitochondria dysfunction and apoptotic activation. Multidrug resistance protein (MRP) is an efflux transporter protein and is capable of transporting cellular oxidative stress-related substances. To further clarify the role of MRP in rhein induced oxidative stress, we examined MRP expression in the liver. However, the expression of MRP has no statistical significance.

Preliminary evaluation of the efficacy and safety of brimonidine for general anesthesia.

BACKGROUND: To determine the hypnotic and analgesic effects of brimonidine, and evaluate its efficacy and safety for general anesthesia. Potentiation of pentobarbital sleeping time following brimonidine administration was observed in mice, as was the analgesic activity of brimonidine. METHODS: The median effective dose (ED50) and lethal dose (LD50) of intraperitoneally injected brimonidine were determined in hypnotized mice. In addition, the LD50 of intravenously injected brimonidine, and ED50 of intravenously, intramuscularly, and intrarectally injected brimonidine in hypnotized rabbits were determined. Finally, the synergistic anesthetic effect of brimonidine and chloral hydrate was evaluated in rabbits. RESULTS: Intraperitoneal injection of 10 mg/kg brimonidine enhanced the hypnotic effect of a threshold dose of pentobarbital. Intraperitoneally injected brimonidine produced dose-related analgesic effects in mice. The ED50 of intraperitoneally administered brimonidine in hypnotized mice was 75.7 mg/kg and the LD50 was 379 mg/kg. ED50 values of intravenous, intramuscular, and intrarectal brimonidine for hypnosis in rabbits were 5.2 mg/kg, 8.8 mg/kg, and 8.7 mg/kg, respectively; the LD50 of intravenous brimonidine was 146 mg/kg. Combined intravenous administration of 0.6 mg/kg brimonidine and 0.03 g/kg chloral hydrate had a synergistic anesthetic effect. CONCLUSIONS: Brimonidine elicited hypnotic and analgesic effects after systemic administration and exhibited safety. Moreover, brimonidine enhanced the effects of other types of narcotics when combined.

Toxicity and protein composition of venoms of Hottentotta saulcyi, Hottentotta schach and Androctonus crassicauda, three scorpion species collected in Iran.

BACKGROUND: Scorpion stings comprise a serious problem throughout the globe, especially in regions where they are more frequent. Despite a recent upsurge of interest in scorpion venoms by various research groups, there remain many challenges. OBJECTIVE: Therefore, in this study, we aimed to study the toxicity and protein composition of venoms of Hottentotta saulcyi, Hottentotta schach and Androctonus crassicauda, three scorpion species collected in Iran. MATERIALS AND METHODS: Scorpion species were collected from Esfahan farm scorpion company and maintained in the laboratory in containers that mimic their natural habitat. Venom was extracted from A. crassicauda, H. schach and H. saulcyi by electrical stimulation of 8 and 10 V. The toxicity of each venom was established by using four groups of male Swiss albino mice aged 2 months (weighting 18-20 g) for testing each dose of venom. One group was used as a control. Venom was injected into mice by subcutaneous route. Then, animals were monitored for 24 h and LD50 was estimated by the graphic method of Miller and Tainter. Thus, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was used to determine amino acids in the venom, and protein concentrations were determined by the Biuret method. RESULTS: LD50 of scorpion venoms by subcutaneous route was found to be 1.70 mg/kg b.w (A. crassicauda), 1.47 mg/kg b.w (H. saulcyi) and 0.85 mg/kg b.w (H. schach). A. crassicauda, H. saulcyi and H. schach contain 26, 30, and 31 amino acids, respectively. A. crassicauda contains low concentrations of alpha-aminoadipic acid, beta-aminoisobutyric acid, beta-alanine and citrulline. H. saulcyi contains a concentration of hydroxylysine, whereas H. schach has no such concentration. A. crassicauda also had the highest levels of tyrosine and threonine. Only A. crassicauda venom contains a low proportion of proteins (14.80%) compared with those of H. schach (16.26%) and H. saulcyi (16.20%). Albumin content in the venoms was 11.7% (H. saulcyi), 5.4% (H. schach) and 4.4% (A. crassicauda). CONCLUSION: Scorpions venoms have a variable toxicity and an interesting composition in amino acids and proteins. Work on the development of anti-venom is fundamental.

Subchronic neurotoxicity of diazinon in albino mice: Impact of oxidative stress, AChE activity, and gene expression disturbances in the cerebral cortex and hippocampus on mood, spatial learning, and memory function.

Diazinon (DZN) with prominent neurotoxic effects perturbs CNS function via multiple mechanisms. This investigation intends to explore mood, spatial learning, and memory dysfunction, acetylcholine esterase (AChE) activity, and neurodegeneration-related gene expression in the cortex and hippocampus regions of mice exposed to DZN for 63 consecutive days (subchronic exposure). Adult male albino mice were orally given sublethal DZN (DZNL = 0.1 mg/kg, DZNM = 1 mg/kg and DZNH = 10 mg/kg). All mice in the DZNH group died within 3 weeks postexposure. DZNL and DZNM caused body and brain weight loss (p < 0.05). Completing 9 weeks of DZN exposure, a marked decline in AChE activity and oxidative stress level was indicated in both brain regions (p < 0.05). Also, synaptophysin, vesicular acetylcholine transferase, and glutamate decarboxylase gene expressions were affected in both brain regions (p < 0.05). Furthermore, the present study revealed that DZN administration increased anxiety and depressive-like behaviors (p < 0.0001). Spatial learning and short- and long-memory were severely affected by DZNL and DZNM treatments (p < 0.0001). Taken together, subchronic exposure to low and medium doses of DZN can cause AChE inhibition, oxidative damage, and neurotransmitter disturbances in brain cells and induce neurodegeneration. These changes would impair mood, spatial learning, and memory function.

Lipophilic and hydrophilic leaf extracts of Portulaca oleracea (Purslane) disrupts female sex hormones in albino rats (Rattus norvegicus).

BACKGROUND AND AIM: Decoctions and infusions from the aerial parts of Portulaca oleracea Linn., especially the leaves and stems, are used by traditional medicine practitioners in Nigeria to enhance fertility in humans. The scarcity of literature on the use of this plant for the said purpose as well as its efficacy prompted this research. Study investigated effect of lipophilic and hydrophilic leaf extracts of Portulaca oleracea on oestrous cycle, female sex hormones at various phases of oestrous cycle and ovarian and uterine histomorphology in albino rats. EXPERIMENTAL PROCEDURE: Experimental animals were randomly divided into 7 groups of 5 rats each. Group A (control) received 0.5 ml 20% Tween 80 (vehicle), groups B, C & D received 125, 250 & 500 mg/kg of the lipophilic extract respectively and E, F & G received 125, 250 & 500 mg/kg of the hydrophilic extract respectively for 21 days. Oestrous cycle was assessed daily. At the end, blood samples (for hormones) and ovarian &uterine sections (histoarchitecture) were collected. RESULTS AND CONCLUSION: Both extracts had no significant (p > 0.05) effect on oestrous cycle, ovarian & uterine histoarchitecture and female sex hormones except at proestrus phase where significant (p < 0.05) decrease in LH and FSH was recorded. P.oleracea as used in this study may have deleterious effect on female reproductive system as shown by the disruption of the hormones at proestrus phase. This can form a basis to refute the use of P.oleracea leaf extracts in enhancing fertility as it has been shown to affect the gonadotropins involved in folliculogenesis.

Anti-Proliferative Properties, Biocompatibility, and Chemical Composition of Different Extracts of Plantago major Medicinal Plant.

Background: To study the anticancer activity of Plantago major, we assessed the effect of ethanolic, methanolic and acetonic extracts of this plant on HCT-116, SW-480, and HEK-293 cell lines as control. Methods: The cytotoxic activity, biocompatibility, and toxicity were evaluated by MTT assay, hemolysis, and Artemia salina-LD50 (on mice) tests, respectively. The analysis of the extracts was performed by GC-MS analysis. Results: The results showed that all the extracts had the most antiproliferative properties on the HCT-116 cell line. The P. major root extract was more effective than the aerial parts, and IC50 values for ethanolic, methanolic and acetonic root extracts were 405.59, 470.16, and 82.26 µg/mL, respectively on HCT-116 cell line at 72 h. Hemolysis degree of the ethanolic extract of aerial and root parts were approximately 1% at 400 µg/mL.. Using the ethanolic extracts, the Artemia survived every concentration, and no toxicity was observed. One week after the oral administration of different parts of P. major extracts, none of the mice died, even those were administered 2000 mg/kg. The results of GC/MS analysis showed that P. major extracts contain potential anticancer compounds, such as stearic acid (8.61%) in aerial parts of methanolic extract and 1,2- Benzenedicarboxylic acid, mono(2-ethylhexyl)ester (88.07% and 40.63%) in aerial and root parts of acetonic extract of P. major. Conclusion: Our findings suggest that the P. major is a source of potential compounds with antiproliferative properties.

Computational screening of phytochemicals from three medicinal plants as inhibitors of transmembrane protease serine 2 implicated in SARS-CoV-2 infection.

Background: SARS-CoV-2 infection or COVID-19 is a major global public health issue that requires urgent attention in terms of drug development. Transmembrane Protease Serine 2 (TMPRSS2) is a good drug target against SARS-CoV-2 because of the role it plays during the viral entry into the cell. Virtual screening of phytochemicals as potential inhibitors of TMPRSS2 can lead to the discovery of drug candidates for the treatment of COVID-19. Purpose: The study was designed to screen 132 phytochemicals from three medicinal plants traditionally used as antivirals; Zingiber officinalis Roscoe (Zingiberaceae), Artemisia annua L. (Asteraceae), and Moringa oleifera Lam. (Moringaceae), as potential inhibitors of TMPRSS2 for the purpose of finding therapeutic options to treat COVID-19. Methods: Homology model of TMPRSS2 was built using the ProMod3 3.1.1 program of the SWISS-MODEL. Binding affinities and interaction between compounds and TMPRSS2 model was examined using molecular docking and molecular dynamics simulation. The drug-likeness and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of potential inhibitors of TMPRSS2 were also assessed using admetSAR web tool. Results: Three compounds, namely, niazirin, quercetin, and moringyne from M. oleifera demonstrated better molecular interactions with binding affinities ranging from -7.1 to -8.0 kcal/mol compared to -7.0 kcal/mol obtained for camostat mesylate (a known TMPRSS2 inhibitor), which served as a control. All the three compounds exhibited good drug-like properties by not violating the Lipinski rule of 5. Niazirin and moringyne possessed good ADMET properties and were stable in their interactions with the TMPRSS2 based on the molecular dynamics simulation. However, the ADMET tool predicted the potential hepatotoxic and mutagenic effects of quercetin. Conclusion: This study demonstrated the potentials of niazirin, quercetin, and moringyne from M. oleifera, to inhibit the activities of human TMPRSS2, thus probably being good candidates for further development as new drugs for the treatment or management of COVID-19.

Eficacia protectora de una vacuna inactivada contra el virus de la enfermedad hemorrágica del conejo tipo 2 preparada a partir de un aislado local en Egipto / Protective efficacy of an inactivated vaccine against Rabbit hemorrhagic disease virus 2 prepared from a local isolate in Egypt

Rabbit hemorrhagic disease is a contagious viral disease of rabbits controlled by vaccination. The present study was aimed to diagnose rabbit hemorrhagic disease from 11 infected farms from Qalubia governorate during 2019 and to prepare homologous vaccine against rabbit hemorrhagic disease virus 2. For this purpose, 11 liver samples were collected from suspected cases and subjected to detection and identification of circulating rabbit hemorrhagic disease virus. Ten samples were confirmed to be rabbit hemorrhagic disease virus using hemagglutination test, animal inoculation and reverse transcriptase polymerase chain reaction. Sequencing and phylogenetic analysis of two isolates (R5&R6) revealed the presence of rabbit hemorrhagic disease virus 2 (A/Qalubia/2019 and B/Qalubia/2019) under accession number MT07629 and MT067630 respectively. The inactivated rabbit hemorrhagic disease virus vaccines were prepared using Montanide ISA 206 oil or aluminum hydroxide gel adjuvants. Prepared vaccines were inoculated subcutaneously in susceptible rabbits and submitted to sterility, safety and potency tests. Obtained results showed that mean hemagglutination inhibition titer for aluminum hydroxide gel vaccine was 6,7.7,8.9 and 9.1 log2 while, Montanide vaccine reached to 6.7,8.7,9.2 and 9.5 log2 at 1st, 2nd, 3rd, and 4th weeks post vaccination, respectively. Immunized rabbits with Montanide vaccine showed better protection reach to 70 percent, 90 percent percent, 100 percent and 100 percent when compared to aluminum hydroxide gel vaccine 60 percent, 70 percent, 90 percent and 90 percent at 1st, 2nd, 3rd and 4th weeks post vaccination respectively. It was concluded that newly emerged rabbit hemorrhagic disease virus 2 was isolated from suspected cases. The two prepared vaccines were sterile, safe and potent. The oily adjuvanted rabbit hemorrhagic disease virus 2 vaccine stimulated an earlier and higher humoral immune response than the aluminum hydroxide gel adjuvanted vaccine. This humoral immune response achieved significant level of protection(AU)

La enfermedad hemorrágica del conejo es una enfermedad viral contagiosa de los conejos que se controla mediante vacunación. El presente estudio tuvo como objetivo diagnosticar la enfermedad hemorrágica del conejo en 11 granjas infectadas de la provincia de Qalubia, durante 2019 y preparar una vacuna homóloga contra el virus de la enfermedad hemorrágica del conejo tipo 2. Para este propósito, se recolectaron 11 muestras de hígado de casos sospechosos y se sometieron a detección e identificación de virus circulante de la enfermedad hemorrágica del conejo. Se confirmó que diez muestras eran positivas al virus de la enfermedad hemorrágica del conejo, utilizando para ello la prueba de hemaglutinación, inoculación en animales y Reacción en cadena de la polimerasa con transcriptasa inversa. La secuenciación y el análisis filogenético de dos aislamientos (R5 y R6) revelaron la presencia del virus de la enfermedad hemorrágica del conejo tipo 2 (A/Qalubia/2019 y B/Qalubia/2019) con los números de acceso MT07629 y MT067630 respectivamente. Las vacunas inactivadas del virus de la enfermedad hemorrágica del conejo se prepararon usando adyuvantes de gel de hidróxido de aluminio o aceite Montanide ISA 206. Las vacunas preparadas se inocularon por vía subcutánea en conejos susceptibles y se sometieron a pruebas de esterilidad, seguridad y potencia. Los resultados obtenidos mostraron que el título medio de inhibición de la hemaglutinación para la vacuna en gel de hidróxido de aluminio fue de 6; 7,7; 8,9 y 9,1 log2, mientras que la vacuna de Montanide alcanzó 6,7; 8,7; 9,2 y 9,5 log2 en la 1ª, 2ª, 3ª y 4ª semanas después de la vacunación, respectivamente. Los conejos inmunizados con la vacuna Montanide tuvieron una mejor protección, alcanzándose niveles de 70 por ciento, 90 por ciento, 100 por ciento y 100 por ciento en comparación con la vacuna en gel de hidróxido de aluminio 60 por ciento, 70 por ciento, 90 por ciento y 90 por ciento en la 1ª, 2ª, 3ª y 4ª semanas después de la vacunación, respectivamente. Se concluyó que el virus de la enfermedad hemorrágica del conejo tipo 2 de reciente aparición se aisló de los casos sospechosos. Las dos vacunas preparadas fueron estériles, seguras y potentes. La vacuna contra el virus de la enfermedad hemorrágica del conejo tipo 2 con adyuvante oleoso estimuló una respuesta inmune humoral más temprana y mayor que la vacuna con adyuvante en gel de hidróxido de aluminio. Esta respuesta inmune humoral confirió un nivel significativo de protección(AU)

Neutralización de la actividad letal del veneno de serpiente Bothrops atrox por suero hiperinmune de llama (Lama glama) / Neutralization of the lethal activity from Bothrops atrox venom by hyperimune llama serum (Lama glama)

RESUMEN Objetivos: Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio. Materiales y métodos: Se calculó la dosis letal media (DL50) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE50) durante el periodo de inmunización. Resultados: La DL50 del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE50 de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones. Conclusiones: El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

ABSTRACT Objectives: To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice. Materials and methods: Mean lethal dose (LD50) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED50) during the immunization period. Results: The venom's LD50 was 3.96 μg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED50 of 3.30 μL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations. Conclusions: The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

Acute and Subchronic Oral Toxicity of Oil Palm Puree in Sprague-Dawley Rats.

Palm puree is rich in antioxidants and is produced via blending various proportions of mesocarp fibre and crude palm oil. The aim of this study was to assess the acute and subchronic toxicity of palm puree in male and female Sprague-Dawley rats. For the acute toxicity study, animals administered single palm-puree doses (2000 mg kg-1) by gavage were observed daily for 14 d. For the subchronic toxicity study, the rats were administered 500, 1000, or 2000 mg kg-1 palm puree daily for 28 d. We evaluated body and organ weights; performed haematological, biochemical, and histopathological analyses of blood and organ samples during and after treatment; and calculated the oral no-observed-adverse-effect level (NOAEL). The toxicity studies showed no signs of toxicity or mortality. The haematological, biochemical, and histopathological analyses and body and organ weights indicated no evidence of substantial toxicity at any dose of palm puree. The oral lethal dose and NOAEL for the palm puree were greater than 2000 mg kg-1 d-1 over 28 d. To the best of our knowledge, the present study is the first to confirm the safety of palm puree as a novel functional food. These encouraging results warrant further studies to elucidate its potential for pharmaceutical formulations.

Acute toxicity "six-pack" studies supporting approved new drug applications in the U.S., 2015-2018.

The acute toxicity "six-pack" is a battery of animal tests used to evaluate acute systemic toxicity by three routes of exposure, skin and eye irritation/corrosion, and skin sensitization. A perception exists that these tests are not required for pharmaceuticals. For the four years from 2015 through 2018, we tallied the number of corresponding tests submitted by sponsors in approved, original new drug applications, as reported by the U.S. Food and Drug Administration (FDA) in publicly available reviews. In 125 reviews, we identified 228 single dose acute toxicity studies, 62 in vivo local tolerance studies, and 32 in vivo skin sensitization studies, as well as 37 in vitro or ex vivo local tolerance studies. A total of 4798 animals were used in these studies; however, FDA's reporting was inconsistent, and we estimate the actual number of animals used to be 8998. For the evaluation of single dose acute toxicity, we accessed two guidance documents with conflicting recommendations regarding routes of administration and number of species to be used. For the evaluation of local tolerance and skin sensitization, most studies examined were conducted by routes other than that intended for human administration. Non-animal methods used to evaluate skin sensitization were not reported.

Toxicity of Vipera palaestinae venom and antagonistic effects of methanolic leaf extract of Eryngium creticum lam.

Vipera palaestinae is responsible for many venomous incidents in the Middle East. However, this species is not included in the antigenic pool of venoms for the production of the regionally available polyvalent antivenoms. In an attempt to develop a potential complementary alternative therapy for snakebite patients, this study is investigating the antagonistic effect of Eryngium creticum against V. palaestinae venom. In this context, the concentration of the venom as well as the electrophoretic profile, and the venom LD50 were determined by intraperitoneal injection (ip). The methanolic leaf extract was prepared, and its safety on rats was examined. Adult male Sprague-Dawley rats were divided into 8 groups (n = 6); G1-G3 were injected subplantar in the right hind paws with 2.5, 3.125, and 3.75 mg kg-1 then 200 mg kg-1 extract ip. G4-G6 were given the same venom dose with no extract, respectively. Controls were G7 that only had the extract ip, and G8 that was injected subplantar with PBS. The swollen paws were measured at Hour 0 (before injection), Hour 1, Hour 6, and Hour 24. IL-6 and TNF-α were measured in serum using ELISA. Histopathological changes were examined in paw sections. The pooled venom concentration was 176.93 ±â€¯35.81 mg ml-1, revealed 10 protein bands (5-80 kDa), and the LD50 via ip rout was 6.56 mg kg-1. Paw edema peaked at Hour 1. At Hour 6, edema in G1 was significantly reduced (p < 0.05) compared to G6, while at Hour 24 there was no significant difference between all groups including the controls. Treated animals in G1-G3 expressed IL-6 significantly lower (p < 0.001) than untreated G4-G6, respectively. Levels of TNF-α in G1 and G2 were significantly (p < 0.001) lower than G3-G6, while G5 and G6 were significantly (p < 0.001) higher than G1-G4. Histopathological changes showed intensifying edema, hemorrhage, and inflammation with incrementing venom doses. Sections from treated animals expressed less adverse changes compared to untreated animals. Together, the outcomes are encouraging future utilization of E. creticum as a supportive remedy for snakebite cases.

Foeniculum vulgare as Valuable Plant in Management of Women's Health.

This review paper evaluates use of Foeniculum vulgare extracts as a popular female plant in management of different ailments of women. Information in this paper was gathered from accessible sources (PubMed, Science Direct, Springer, Wiley, and Google), and traditional books (Persian or English modern traditional books), unpublished data (R&D reports, thesis and dissertation) by keywords based on the words F. vulgare or fennel and women. Efficacy of oral fennel oil in management of dysmenorrhea, premenstrual syndrome, amenorrhea, menopause, lactation, and polycystic ovary syndrome were confirmed according to results of clinical studies. Results of clinical efficacy of fennel oil on menstrual bleeding is complicated, but results of one meta-analysis study revealed that fennel oil significantly increased means of bleeding in the first menstrual periodic cycle (P = 0.001), while fennel oil had no significant effect on bleeding in the second menstrual cycle (P = 0.67). Topical and vaginal fennel extract (5%) exhibited good efficacy in treatment of sexual function, vaginal atrophy, and hirsutism. Fennel had no effect on bone density, or body mass index of menopause women. Results of clinical studies introduce fennel as a valuable medicinal plant in management of women's ailments, but understanding the mechanism of action could be the subject of future studies.

Foeniculum vulgare as Valuable Plant in Management of Women's Health

This review paper evaluates use of Foeniculum vulgare extracts as a popular female plant in management of different ailments of women. Information in this paper was gathered from accessible sources (PubMed, Science Direct, Springer, Wiley, and Google), and traditional books (Persian or English modern traditional books), unpublished data (R&D reports, thesis and dissertation) by keywords based on the words F. vulgare or fennel and women. Efficacy of oral fennel oil in management of dysmenorrhea, premenstrual syndrome, amenorrhea, menopause, lactation, and polycystic ovary syndrome were confirmed according to results of clinical studies. Results of clinical efficacy of fennel oil on menstrual bleeding is complicated, but results of one meta-analysis study revealed that fennel oil significantly increased means of bleeding in the first menstrual periodic cycle (P = 0.001), while fennel oil had no significant effect on bleeding in the second menstrual cycle (P = 0.67). Topical and vaginal fennel extract (5%) exhibited good efficacy in treatment of sexual function, vaginal atrophy, and hirsutism. Fennel had no effect on bone density, or body mass index of menopause women. Results of clinical studies introduce fennel as a valuable medicinal plant in management of women's ailments, but understanding the mechanism of action could be the subject of future studies.

Toxicological assessment of Xanthigen® nutraceutical extract combination: Mutagenicity, genotoxicity and oral toxicity.

Xanthigen® is a nutraceutical combination for weight management capable of increasing energy expenditure via uncoupling protein 1 (UCP-1) in white adipose tissue. It consists of brown seaweed Undaria pinnatifida extract, rich in the carotenoid fucoxanthin (FX) and pomegranate seed oil (PSO), rich in punicic acid. Xanthigen was screened to determine its genotoxicity and 90-days repeated oral toxicity. Genotoxicity was assessed with the Ames test (TA89, TA100, TA1535, TA1537, WP2), chromosomal aberration assay (Chinese hamster ovary cells) and mammalian micronucleus test (in mice). Xanthigen did not exhibit genotoxicity in any tested strain. Sub-chronic toxicity was evaluated with daily oral administration of 250, 500 and 1000 mg/kg/day doses of Xanthigen® to Sprague-Dawley rats over 90 days. No deaths and no deleterious effects were observed during the 90-day treatment, indicating an absence of sub-chronic toxicity and a no observed adverse effect level greater than 1000 mg/kg/day. A statistically significant decrease in bodyweight and food intake in Xanthigen® treated groups was attributed to the weight loss property of Xanthigen®. Overall, Xanthigen® shows no significant mutagenic or toxic effects.

Ameliorating effect of Alstonia scholaris L. bark extract on histopathological changes following viper envenomation in animal models.

OBJECTIVES: The primary symptoms associated with snake envenomation are both systemic and local. The local symptoms are characterized by pain, swelling, haemorrhage and myonecrosis at the site of bite. The present study investigates the ameliorating effect of the aqueous bark extract of Alstonia scholaris bark on viper venom induced histopathological and biochemical changes in liver and kidney of swiss albino mice models. METHODS & MATERIALS: Swiss albino mice (20 ± 2)g were treated with sublethal doses(0.5 µg and 1 µg) of Vipera russelli venom(VRV) intraperitonially The following groups were assigned in the study-Group I(saline control); Group II & III(Venom treated-0.5 µg ie » LD50 and 1 µg ie 1/2 LD50) and Group IV &V(Venom-0.5 µg and 1 µg respectively incubated with Aqueous Alstonia scholaris (AAS) extract; 200 mg/kg bw) and Group VI (Antivenom serum (AVS) (2 mg/ml) followed by 1 µg Vipera russelli venom (VRV). The animals were sacrificed and their organs were immersed in Bouin's fixative for 24 h and stained with haematoxylin/eosin and observed under the microscope. The serum samples were collected from the animals and tested for serum Alanine transaminase (ALT) and Aspartate transaminases (AST) following the method of Reitman & Frankel(1957) and serum creatinine. RESULTS: The histological alterations observed in Group II and III liver sections were mainly pyknosis, karyorrhexis, cytoplasmic vacuolation, necrosis, fatty changes and hepatocytes atrophy. Sinusoidal dilatation, amyloidosis, portal vein thrombosis which was significantly reduced by AAS extract in Groups IV and V. A venom dose of 1 µg induced tubular cell acidophilia indicating cell damage, peritubular congestion, degenerating changes in the proximal tubules in the form of cytoplasmic vacuolations, partially destroyed bowman's capillaries with dilated Bowman's space in Group III that was significantly reduced by Aqueous Alstonia scholaris (AAS) (200 mg/kg bw) extract in Groups IV and Group V.AVS gave significant protection against venom induced action in Group VI. CONCLUSION: The present paper thus highlights the histopathological changes associated with Vipera russelli venom and systemic venom neutralization potential of Aqueous Alstonia scholaris (AAS) in animal models.

Parenteral nanosuspensions: a brief review from solubility enhancement to more novel and specific applications.

Advancements in in silico techniques of lead molecule selection have resulted in the failure of around 70% of new chemical entities (NCEs). Some of these molecules are getting rejected at final developmental stage resulting in wastage of money and resources. Unfavourable physicochemical properties affect ADME profile of any efficacious and potent molecule, which may ultimately lead to killing of NCE at final stage. Numerous techniques are being explored including nanocrystals for solubility enhancement purposes. Nanocrystals are the most successful and the ones which had a shorter gap between invention and subsequent commercialization of the first marketed product. Several nanocrystal-based products are commercially available and there is a paradigm shift in using approach from simply being solubility enhancement technique to more novel and specific applications. Some other aspects in relation to parenteral nanosuspensions are concentrations of surfactant to be used, scalability and in vivo fate. At present, there exists a wide gap due to poor understanding of these critical factors, which we have tried to address in this review. This review will focus on parenteral nanosuspensions, covering varied aspects especially stabilizers used, GRAS (Generally Recognized as Safe) status of stabilizers, scalability challenges, issues of physical and chemical stability, solidification techniques to combat stability problems and in vivo fate.