ABSTRACT
RESUMEN Introducción: Los productos naturales con actividad farmacológica requieren de evaluaciones preclínicas que justifiquen su empleo sobre una base científica. El ensayo de pirógenos es una prueba dentro de la Farmacología de Seguridad que se realiza para determinar la presencia de endotoxinas y constituye un método valioso, para demostrar la seguridad de bioderivados con potencial prebiótico en el campo de la inmunonutrición. Objetivo: Evaluar la pirogenicidad de bioproductos fúngicos de Pleurotus ostreatus (extractos acuosos del micelio y cuerpos fructíferos) y un biopreparado de levadura Kluyveromyces marxianus, empleando el ensayo de pirógenos en conejos Nueva Zelanda. Método: Se ensayaron concentraciones de 1,0 y 10,0 mg/mL de cada muestra por vía endovenosa en dosis de 0,5 y 5,0 mg/kg de peso. El diseño experimental cumplió las buenas prácticas de laboratorio según lo establecido por el International Council for Laboratory Animals Science y se realizó de acuerdo a los procedimientos normalizados de trabajo del Centro de Toxicología y Biomedicina, Santiago de Cuba. Resultados: Los extractos de Pleurotus ostreatus y el biopreparado de levadura (0,5 mg/kg) no mostraron signos de pirogenicidad. En los resultados del biopreparado (5,0 mg/kg), los valores de temperatura caen en un rango de incertidumbre, según la Farmacopea de Estados Unidos (USP) y se sugirió repetir el estudio. Conclusiones: Los extractos de Pleurotus ostreatus y el biopreparado de Kluyveromyces marxianus (0,5 mg/kg) no indujeron un aumento de temperatura significativo en los animales, lo cual sugiere que en estos bioproductos no existen niveles de endotoxinas que puedan provocar pirogenicidad.
ABSTRACT Introduction: Natural products with pharmacological activity require preclinical evaluations to justify their uses scientifically. The pyrogen assay is a safety pharmacology test performed to determine the presence of endotoxins and it is a valuable method to demonstrate the bio-derivative products safety and their prebiotic potential in the field of immunonutrition. Objective: To evaluate the pyrogenicity of fungal bioproducts from Pleurotus ostreatus (aqueous extracts from mycelium and fruiting bodies) and a biopreparation from Kluyveromyces marxianus yeast, using a pyrogen assay in New Zealand rabbits. Method: Concentrations of 1.0 and 10.0 mg/mL of each sample were tested intravenously at doses of 0.5 and 5.0 mg/kg body weight. The experimental design complied with good laboratory practices as established by the International Council for Laboratory Animal Science and was carried out according to the standard work procedures of the Centro de Toxicología y Biomedicina, Santiago de Cuba. Results: Pleurotus ostreatus extracts and the yeast biopreparation (0.5 mg/kg) showed no signs of pyrogenicity. In the biopreparation results (5.0 mg/kg), temperature values fall in the uncertainty range according to the United States Pharmacopoeia (USP), and therefore it was suggested to repeat the study. Conclusions: Pleurotus ostreatus extracts and Kluyveromyces marxianus biopreparation (0.5 mg/kg) did not induce a significant temperature increase in the animals, thereby suggesting that there are no endotoxin levels in such bioproducts that could cause pyrogenicity.
RESUMO Introdução: Produtos naturais com atividade farmacológica requerem avaliações pré-clínicas que justifiquem seu uso em bases científicas. O ensaio de pirogênio é um teste dentro da Farmacologia de Segurança que é realizado para determinar a presença de endotoxinas e é um método valioso para demonstrar a segurança de bioderivados com potencial prebiótico no campo da imunonutrição. Objetivo: Avaliar a pirogenicidade de bioprodutos fúngicos de Pleurotus ostreatus (extratos aquosos do micélio e corpos de frutificação) e de uma biopreparação da levedura Kluyveromyces marxianus, utilizando o ensaio pirogênico de coelho da Nova Zelândia. Método: Concentrações de 1,0 e 10,0 mg/mL de cada amostra foram testadas por via intravenosa nas doses de 0,5 e 5,0 mg/kg de peso. O desenho experimental obedeceu às boas práticas laboratoriais estabelecidas pelo Conselho Internacional para a Ciência dos Animais de Laboratório e foi realizado de acordo com os procedimentos de trabalho padrão do Centro de Toxicologia e Biomedicina, Santiago de Cuba. Resultados: Os extratos de Pleurotus ostreatus e a biopreparação de leveduras (0,5 mg/kg) não apresentaram sinais de pirogenicidade. Nos resultados da biopreparação (5,0 mg/kg), os valores de temperatura estão dentro de uma faixa de incerteza, segundo a Farmacopeia dos Estados Unidos (USP) e foi sugerido repetir o estudo. Conclusões: Os extratos de Pleurotus ostreatus e a biopreparação de Kluyveromyces marxianus (0,5 mg/kg) não induziram um aumento significativo da temperatura nos animais, o que sugere que não há níveis de endotoxinas nesses bioprodutos que possam causar pirogenicidade.
ABSTRACT
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.
ABSTRACT
Objective: To study the effect of eliminating pyrogen with ultrafiltration on the quatity of Chuanshentong Injection. Methods: The sample solution was filtered with the ultrafiltration membranes of molecular weight 6000 and 20000. Then,the results of eliminating pyrogens and the examination results of quality of sample solution before and after ultrafiltration were compared.Results: The results of elminating pyrogens is satisfactory. But there are larger changes in many items of quality markers after ultrafiltration. Conclusion: Therefore, the ultrafiltration should be used with caution in eliminating pyrogens of injection of Chinese medicinals.
ABSTRACT
Fever model was made by intravenously injected leucocytic pyrogen(LP,EP). Chai Ge Jie Ji Tang CGJJT fluid was perfused into rabbits stomach by mouth. 65 New Zealand white rabbits were divided into four groups: (1) control group; (2) LP group; (3) CGJJT fluid group; (4) LP+ CGJJT fluid group. Effects of CGJJT fluid on LP fever and cAMP concentration in cerebrospinal fluid (CSF)were observed. The results obtained from our experiments showed that: (1) The rectal temperature of normal rabbits markedly affected by CGJJT fluid. (2) Febrile response of LP was significantly inhibited by CGJJT fluid (P
ABSTRACT
78 rabbits in this study were divided into two parts and each part had 39 rabbits. In part Ⅰ and Ⅱ the effects of rhubarb injection on the febrile response and the change of cAMP contents in CSF in these rabbits were observed respectively. The results showed that LP+rhubarb group: △T (0.70?0.12); TPI_4 (3.37?0.07) and LP group:△T (1.11?0.11);TRI_4 (5.56?0.10) comparing of two groups, had markable statistical significance (P
ABSTRACT
In order to analyze the mechanism of febrifugal effect of electric needling "Baihui (Hundreds Converge)" point of febrile rabbits, 108 rabbits were divided into 5 groups, serveing as experimental animals. The results obtained showed: 1. In febrile rabbits induced by monoeytic pyrogen, electric needling "Bai hui" points has significant febrifugal effect, i. e. inhibiting the rise of body temperature, in the meantime the cAMP content rose in c. s. f. was markedly inhibited. 2. On febrile rabbits, electric needling blocking "Bai hui" point and Non-acupoint exhibited no marked febrifugal effect, i. e. showing apparently fever response, and at the same time cAMP contents in c. s. f. was strikingly increased. 3. Blocking of "Bai hui" point itself did not produce any effect on pyrogen-induced fever and cAMP content increased.The author inferred that the mechanism of inhibitory effect of electric needling "Bai hui" points in febrile rabbits might be closele assocated with feeling of local sensible tissue (e. g. free nerve endings) in "Bai hui" point, and with producing nerve impuls which transmitted through some mode to brain. As a result, increased cAMP was inhibited and setpoint to move up was inhibited.
ABSTRACT
The experiments were divided into two parts. The first part: inhibitory effect of Bupleurum Chinense injection on fever was observed. The second part: while Bupleurum Chinense injection inhibited fever, the cAMP concentration in cerebrospinal fluid was observed. The experimental fever models were induced by endogenous pyrogen from monocytes of rabbits' blood. The results obtained demonstrated that the Bupleurum Chinense injection (250mg/kg) had marked inhibitory effect on the endogenous pyrogen fever and the cerebrospinal fluid cAMP levels of the experimental group was obviously lower than in control. The authors inferred that inhibitory effect of Bupleurum Chinense injection on endogenous pyrogen fever may be obtained through some-link to reduced central cAMP content.
ABSTRACT
Crude monocytic endogenous pyrogens from rabbits' blood were further purified by means of ultrafiltration membranes. The sterile pyrogen obtained was passed through gel filtration on a 1.5 by 100cm G-75 Sephadex column, and subje- cted to a 1.5 by 60cm ion-exchange chromatography on QAE Sephadex or subjected to isoelectric focusing. Under our experimental conditions high concentration of monocyte pyrogen could be obtained. In above described method used two pyrogen peak whose specific activity was 0.5—3.0ug, caused fever up to 0.6℃ in rabbits. In addition, isoelectric focusing column employed indicated isoeletric points of specific activity protein that induced fever were PI 5.1 and 3.6 respectively.
ABSTRACT
Staphylococcus albus (St albus) given intravenously to conscious rabbits produced a dose-dependent fever. When the St albus doses reached a certain limit, hyperthermic ceiling was approached. The pyrogenicity of plasma increased progressively along with the increase of St albus doses. When hyperthermic ceiling was approached, pyrogenicity of plasma stopped raising any further. Fever induced by St albus was associated with a dose related increase in the concentration of cAMP in csf as well as that in the plasma. The increase of cAMP in plasma and in csf was limited at the occurrence of upper limit of fever. It is concluded that cAMP may be an important central mediator in the fever induced by staphylococci, whilst the limitation of production and release of EP and the limitation of increase of cAMP in central nervous system may be two important factors in "staphylococcus hyperthermic ceiling".
ABSTRACT
In order to study the mechanisms of ET and EP hyperthermic ceiling the concentration of cAMP in samples of c. s. f. and plasma were measured using the competitive protein binding assay after intraveneous injections of ET and EP in doses of nonhyperthermic ceiling, hyperthermic ceiling and hyperthermic ceiling plus the other pyrogen load. The results showed: (1) The concentration of cAMP in c. s. f. was elevated markdly with increase of fever reaction induced by EP, and was limited when EP hyperthermic ceiling was reached. But there was no analogous change of cAMP in plasma. (2) The contents of cAMP both in c. s. f. and in plasma were apparently correlated simultaneously to fever reaction induced by ET, and were limited when ET hyperthermic ceiling was reached. (3) Although the febrele level of ET hyperthermic ceiling was evidently hegher than that of EP and the altitude of fever exceed EP hyyerthermic ceiling when EP hyperthermic ceiling was added the load of ET. Contents of cAMP in c. s. f. had no significant differences among these groups. The results above indicate that the inhibition of central cAMP synthesis is closely related to the formation of EP hyperthermic ceiling and also is one of an important factors that induce. ET hyperthermic ceiling, which, however, is involved in other mediator except cAMP.
ABSTRACT
Since the endotoxic fever is mediated essentially by endogenous pyrogen (EP), we have examined the possibility that the central cAMP is involved in the mechanism of biphasic fever induced by endotoxin, as in the case of biphasic fever induced by EP. The results showed that administration of endotoxin in rabbits elicited typical biphasic fever. The fluctuation of concentration of cAMP in cerebrospinal fluid (CSF) paralleled with the change of the rectal temperature, similar to that of the bipbasic fever caused by EP. Raised level of cAMP in plasma was observed only in the first phase. The cAMP concentration in CSF correlated positively with the febrile response. The correlation of cAMP levels in CSF and plasma was observed only in the first phase but not in all the febrilic periods. These findings indicated that the mechanism of endotoxic biphasic fever is associated with the Change of central cAMP. Content. The raised level of cAMP in CSF during fever is likely to represent the biosynthsis in brain regions. However, the possibility that the raised cAMP in plasma enters CSF in the first phase is not excluded.
ABSTRACT
In 100 rabbits, the febrifugal effect of needling on the endogenous pyrogen fever was studied. These experiments were divided into two parts:Part Ⅰ: Influence of acupoint block on the needling febrifugal effect of rabbit. Eighty rabbits were redivided into 4 groups. Group Ⅰ was with blocking of acupoint "Bai Hui (Hundreds Converge)". In this group, the febrile rabbits were needled after blocking "Bai hui" point with lidocaine (hydrochloride). Group Ⅱ was a group of needling "Bai hui". GroupⅢ was a control group of febrile rabbits. In all the above three groups, febrile effect was induced by intravenous injection of 10ml supernatant containing endogenous pyrogen (Ep). Group Ⅳ was a blank control.Part Ⅱ: Influence of acupoint block on the febrile effect of rabbit. Twenty rabbits were divided into experimental (acupoint block) and control (no block) groups.It was found that needling "Bai hui" point of febrile rabbits would produce marked febrifugal effect, while little febrifugal effect could be observed by needling blocked "Bai hui" point of febrile rabbits. The difference was significant (P
ABSTRACT
In order to determine whether the central mediator participates in the mechanism of the EP biphasic fever, the following experiments were carried out: 1. Effect of EP in different doses on the fever pattern; 2. Changes of the cAMP content in CSF and in plasma during the EP biphasic fever. The results obtained demonstrated: 1. After EP were given intravenously in different doses, different fever patterns were produced. 2. The cAMP contents in CSF were increased markedly in every phase of the EP biphasic fever compared with the normal control. These changes of the cAMP content were paralleled and correlated apparently with the body temperature. However, there Were no obvious changes of the cAMP content in plasma during the EP biphasic fever. It demonstrated that cAMP increased in CSF was synthesized and released by CNS for oneself. The authors deduced from above: The wave of body temperature may be related to the changes of cAMP in center during the EP biphasic fever. The fluctuation of cAMP content in the center caused the set-point to move up and down, and further caused the fluctuation of the body temperature and formed the biphasic fever.
ABSTRACT
In order to study mechanisms of endotoxin (ET) and endogenous pyrogen (EP) byperthermic ceilings, the febrile responses induced in rabbits by intravenous injection of varying doses of EP were observed, and the doses in which the febrile reactions of rabbits to ET and EP are limited by hyperthermic ceiling were identified. Furthermore, the febrile reactions of typerttermic ceiling plus different pyrogen load were observed. T, e experiment stowed: 1. The level of ET hypert ermic ceiling is notably higher in comparison to that of EP hypertiermic ceiling. The findings indicated that the mechanisms of ET fever and ET Lyperthermic ceiling are different from those of EP fever and EP hyperthermic ceiling. 2. After EP hyperthermic ceiling was added the load of ET. the altitude of fever exceeded the EP hyperthermic ceiling; but after ET hypertthermic ceiling was added the load of EP, the altitude couldn't exceed ET hyperthermic ceiling. The results suggested that ET hyperthermic ceiling contained EP hyperttermic ceiling. The latter was an important component of ET hyperttermic ceiling, but there are other factors participating in the mechanisms of ET fever and ET hyperthermic ceiling except EP.
ABSTRACT
Fever model induced by endogenous pyrogen was used to study the effect of needling "Bai hui" point on fever. 126 rabbits served as experimental animal. The present experiment was divided into three parts: Ⅰ. Ⅱ. (injection of pyrogen)and Ⅲ. (no injection), and each part was redivided into experimental (needling "Bai hui" point or "non-channel point) and control (no needling) groups.The findings were: (1) In febrile rabbits induced by endogenous pyrogen, needling "Bai hui" points had significant febrifugal effect, i.e, inhibiting the rise of body temperature, increasing the amplitude of defervescence and accelerating the febrifugal rate. (2) In febrile rabbits, needling "non-channel points" had no marked febrifugal effect, i. e. showing the rise of body temperature, after that, accelerating the febrifugal rate. (3) On body temperature of healthy rabbits, needling "Bai hui" points hnd no hignificant effect.