Research progress of bacterial endotoxin detection methods / 中国生物制品学杂志

@#As a powerful pyrogen substance,bacterial endotoxin in small amounts can cause many serious effects on human health and would cause fever,microcirculation disorders,endotoxemia,endotoxin shock,diffuse intravascular coagulation and even death.Therefore,it is very important to detect endotoxin in pharmaceutical products.In recent years,due to overfishing of horseshoe crab and environmental deterioration,the number of horseshoe crab in China is decreasing rapidly.It has been listed as the second-class protected animal in China,and the traditional endotoxin detection methods of limulus amoebocyte lysate will be replaced gradually.With the deepening of research,a series of rapid,sensitive and accurate methods for endotoxin detection have been developed.This paper reviews various endotoxin detection methods,focusing on their innovations such as recombinant factor C method and biosensor method,and elaborates their advantages,disadvantages and development trends with the hope that new detection technologies will be more widely developed and applied.

Effect of myrrh and thyme on Trichinella spiralisenteral and parenteral phases with inducible nitric oxide expression in mice

Trichinellosis is a serious disease with no satisfactory treatment. We aimed to assess the effect of myrrh (Commiphora molmol) and, for the first time, thyme (Thymus vulgaris L.) against enteral and encysted (parenteral) phases of Trichinella spiralis in mice compared with albendazole, and detect their effect on inducible nitric oxide synthase (iNOS) expression. Oral administration of 500 mg/kg of myrrh and thyme led to adult reduction (90.9%, 79.4%), while 1,000 mg/kg led to larvae reduction (79.6%, 71.3%), respectively. Administration of 50 mg/kg of albendazole resulted in adult and larvae reduction (94.2%, 90.9%). Positive immunostaining of inflammatory cells infiltrating intestinal mucosa and submucosa of all treated groups was detected. Myrrh-treated mice showed the highest iNOS expression followed by albendazole, then thyme. On the other hand, both myrrh and thyme-treated groups showed stronger iNOS expression of inflammatory cells infiltrating and surrounding encapsulated T. spiralis larvae than albendazole treated group. In conclusion, myrrh and thyme extracts are highly effective against both phases of T. spiralis and showed strong iNOS expressions, especially myrrh which could be a promising alternative drug. This experiment provides a basis for further exploration of this plant by isolation and retesting the active principles of both extracts against different stages of T. spiralis.

Comparative evaluation of pyrogens tests in pharmaceutical products

A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.