Li-Yu Chung

Larvicidal activities of ginger (Zingiber officinale) against Angiostrongylus cantonensis.

In this study, we investigated the anthelmintic activity of [6]-gingerol, [10]-shogaol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin, a constituent isolate from the roots of ginger (Zingiber officinale), for the parasite Angiostrongylus cantonensis. This study found that the above constituents killed A. cantonensis larvae or reduced their spontaneous movements in a time- and dose-dependent manner. The larvicidal effect or ability to halt spontaneous parasite movement of [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin at various concentrations was reached from 24 to 72 h, respectively. Further investigation to determine minimal effective doses of [10]-gingerol and hexahydrocurcumin revealed [10]-gingerol to have a greater maximum larvicidal effect and loss of spontaneous movements than hexahydrocurcumin, mebendazole and albendazole. These constituents of ginger showed effects against DPPH and peroxyl radical under larvicidal effect. Together, these findings suggest that these constituents of ginger might be used as larvicidal agents against A. cantonensis.

Larvicidal Constituents of Zingiber officinale (Ginger) against Anisakis simplex

In this study, we investigated the anthelmintic activity of [10]-shogaol, [6]-shogaol, [10]-gingerol and [6]-gingerol, compounds isolated from the roots of Zingiber officinale L., Zingiberaceae (ginger), against Anisakis simplex. The above compounds kill or reduce spontaneous movement in A. simplex larvae. The maximum lethal efficacy of [10]-shogaol and [10]-gingerol was approximately 80% and 100%, respectively. We further examined the time course of compound-induced loss of mobility in A. simplex. The results showed that various concentrations of [10]-shogaol, [6]-shogaol, [10]-gingerol and [6]-gingerol have maximum effects on loss of spontaneous movement from 24 to 72 h. In addition, the time course of mortality and the percentage of loss of spontaneous movements were ascertained to determine the minimum effective doses of [10]-gingerol and [10]-shogaol. [10]-Gingerol exhibited a larger maximum larvicidal effect and greater loss of spontaneous movement than [10]-shogaol and albendazole. In addition, these constituents of Zingiber officinale showed effects against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and peroxyl radicals. These constituents of Zingiber officinale are responsible for its larvicidal activity against A. simplex.

Anthelmintic constituents from ginger (Zingiber officinale) against Hymenolepis nana

This study investigated the anthelmintic activity of gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, hexahydrocurcumin, 3R,5S-[6]-gingerdiol and 3S,5S-[6]-gingerdiol, a constituent isolate from the roots of ginger, for the parasite Hymenolepis nana. The cestocidal activity or ability to halt spontaneous parasite movement (oscillation/peristalsis) in H. nana of above constituents was reached from 24 to 72h in a time- and dose-dependent manner, respectively. The [10]-shogaol and [10]-gingero1 have maximum lethal efficacy and loss of spontaneous movement than the others at 24-72h. In addition, worms treated with 1 and 10μM [10]-gingero1, more than 30% had spontaneous movement of oscillation at 72h but [10]-shogaol at 72h only about 15-20% of oscillation. This showing that [10]-gingero1 had less loss of spontaneous movement efficacy than [10]-shogaol. After exposure to 200μM [10]-shogaol, 100% of H. nana had died at 12h rather than died at 24h for [10]-gingerol, showing that [10]-gingero1 had less lethal efficacy than [10]-shogaol. In addition, these constituents of ginger showed effects against peroxyl radical under cestocidal activity. In order to evaluate the cestocidal activity and cytokine production caused by gingers extract R0 in the H. nana infected mice, we carried out in vivo examination about H. nana infected mice BALB/c mice were inoculated orally with 500 eggs. After post-inoculation, R0 (1g/kg/day) was administered orally for 10 days. The R0 exhibited cestocidal activity in vivo of significantly reduced worms number and cytokines production by in vitro Con A-stimulated spleen cells showed that INF-γ and IL-2 were significantly increases by R0. IL-4, IL-5, IL-6, IL-10 and IL-13 were significantly decreases and Murine KC and IL-12 were not significantly changes by R0. Together, these findings first suggest that these constituents of ginger might be used as cestocidal agents against H. nana.

Anthelmintic Activities of Aporphine from Nelumbo nucifera Gaertn. cv. Rosa-plena against Hymenolepis nana

Nelumbo nucifera Gaertn. cv. Rosa-plena (Nelumbonaceae), commonly known as lotus, is a perennial aquatic plant grown and consumed throughout Asia. All parts of N. nucifera have been used for various medicinal purposes in oriental medicine. From the leaves of Nelumbo nucifera Gaertn. cv. Rosa-plena (an aquatic plant), liriodenine (1), lysicamine (2), (−)-anonaine (3), (-)-asimilobine (4), (-)-caaverine (5), (-)-N-methylasimilobine (6), (-)-nuciferine (7), (-)-nornuciferine (8), (-)-roemerine (9), 7-hydroxydehydronuciferine (10) and cepharadione B (11) were isolated and identification and anthelmintic activities of aporphine was evaluated against Anisakis simplex and Hymenolepis nana. This study found that the above constituents killed H. nana or reduced their spontaneous movements (oscillation/peristalsis). However, the above constituents at various concentrations demonstrated no larvicidal effect or ability to halt spontaneous parasite movement for 72 h against A. simplex, respectively. In addition, according to an assay of cestocidal activity against H. nana and nematocidal activity against A. simplex, we found that the above compounds showed greater lethal efficacy on H. nana than against A. simplex. Further investigation showed that these above constituents have effects against peroxyl radicals under cestocidal effect. Together, these findings suggest that these constituents of Nelumbo nucifera Gaertn. cv. Rosa-plena might be used as anthelmintic agents against H. nana.

Sequence analysis in partial genes of five isolates of Angiostrongylus cantonensis from Taiwan and biological comparison in infectivity and pathogenicity between two strains.

Angiostrongylus cantonensis is the most common infectious agent causing eosinophilic meningitis and is present in Taiwan, Thailand and the Pacific islands. Clinical symptoms vary within different endemic regions, and their severity is probably dependent on the number of ingested parasites and the diversity among strains. The experimentally definitive host is the rat, and non-permissive hosts are certain mammals such as humans and mice. In this study, the partial gene sequences of two A. cantonensis strains isolated from five different regions in Taiwan were selected and molecularly analyzed. The internal transcribed spacer gene and cytochrome-c oxidase subunit I gene sequences of the Hualien (H) strain of A. cantonensis differed from those of the Pingtung (P) strain and the other three strains by 19% and 11%, respectively. We analyzed the infectivity, fecundity, and development of the H and P strain in rats and host pathogenicity in mice inoculated with both strains. The number of the emerged first-stage larvae, adult recovery, and average length of adults in Sprague-Dawley rats significantly differed between rats inoculated with the H and P strain. Young adult recovery, average length of young adults, eosinophil counts in the cerebrospinal fluid (CSF), glutathione peroxidase concentration, levels of reactive oxygen species as well as malondialdehyde concentration in the CSF, and the survival of mice significantly differed between BALB/c mice inoculated with the H and P strain. The H strain of A. cantonensis had lower infectivity, delayed fecundity, and poor development in rats, and caused milder pathology and lower mortality in mice than the P strain. These data clearly indicate that the H strain of A. cantonensis is a pathogenically distinct strain with lower infectivity to its definitive host, and causing mild pathogenic symptoms to its non-permissive host.

Kinetic change of oxidative stress in cerebrospinal fluid of mice infected with Angiostrongylus cantonensis

Abstract The cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis was examined for kinetic changes in oxidative stress parameters, including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase, malondialdehyde (MDA), 8-isoprostane, and 8-hydroxy-2′-deoxyguanosine (8-OHdG). The ROS increased gradually in the early stage of infection. During days 12–30 post-infection, the infected mice revealed ROS levels significantly higher than that in uninfected controls (P < 0.001). The ROS levels peaked at day 24 and then returned to that observed in uninfected controls at day 45 post-infection. The kinetics of MDA, 8-isoprostane, and 8-OHdG concentration changes observed in the CSF of the infected mice corresponded with kinetic changes in ROS levels. Thus, the excess ROS caused lipid peroxidation and DNA damage to cells in the central nervous system (CNS) of mice infected with A. cantonensis despite the increased antioxidant SOD and catalase enzyme activities during post-infection days 12–30. The oxidative stress in the CNS of C57BL/6 mice was apparently increased by diseases associated with A. cantonensis infection.

Angiostrongylus cantonensis (Nematode: Metastrongiloidea): In Vitro Cultivation of Infective Third-Stage Larvae to Fourth-Stage Larvae

The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.

Eosinophilic meningitis risk associated with raw Ampullarium canaliculatus snails consumption

In Taiwan, Angiostrongylus cantonensis infection has been reported in foreign laborers who had consumed raw Ampullarium canaliculatus snails. This study analyzed three foreign laborers who had contracted enzyme‐linked immunosorbent assay‐confirmed A cantonensis infection while working in Taiwan. All three workers had consumed either roasted snails or raw snails flavored with seasoning while drinking wine. This study investigated possible risk factors for A cantonensis, including naturally occurring A cantonensis in A canaliculatus snails, viability of third‐stage A cantonensis larvae in raw seasoned snails and in roasted snails, infectivity of larvae, and effects of alcohol while consuming snails. Positive infection rates in snails from five different irrigation canals in south Taiwan ranged from 12.3% to 29.4% and the average number of motile larvae per infected snail ranged from 36 to 65. The number of motile and coiled larvae in snail meat after 120 minutes seasoning was 93 (27.7%) and 233 (69.3%), respectively. After 20 minutes of roasting, most larvae in the snail meat were dead. The infectivities of motile and coiled larvae from snail meat after 60 minutes seasoning were 53.2% and 33.2%, respectively, and those from snail meat after 5 minutes roasting were 33.2% and 7.0%, respectively. Eating Taiwan A canaliculatus snails raw is extremely risky given their high infection rates and infection intensities. Even after 120 minutes seasoning or after 20 minutes roasting, snail meat should be considered unsafe for human consumption. Finally, experimental rodent studies indicated that consuming alcohol while ingesting larvae does not significantly reduced infectivity.

Excretory/secretory proteases and mechanical movement of Anisakis pegreffii infective larvae in the penetration of BALB/c mice gastrointestine

Anisakiasis is a human parasitic disease caused by infection with the infective larvae of Anisakis. Accidental infection in humans causes the gastrointestinal pathophysiological effects of mechanical tissue damage by migrating larvae. The mechanism of the infective larval invasion and migration is suspected to involve larval excretory/secretory proteases and motility. This study demonstrates the penetration rate of the infective larvae of Anisakis pegreffii in mouse gastrointestine depends on the time after infection, and that only 15% of larvae remain in the gastrointestinal tract 3 h after infection. Strong activities of matrix metalloproteinases (MMPs) and serine proteases, especially plasmin, were found in the excretory/secretory products of A. pegreffii; these can be inhibited by ONO‐4817 and phenylmethylsulfonyl fluoride, respectively. The protease activity was also significantly decreased in another 1 h of cultivation of larvae in fresh 0.9% normal saline (NS) after previous cultivation for 48 h in NS. The motility scores of larvae were significantly lower after 48 h of cultivation in NS. The penetration rate of A. pegreffii larvae in the gastrointestine of infected mice sequentially were 90% in the freshly prepared, 68% in serine protease inhibited, 55% in MMPs inhibited larvae, and 16% in larvae cultivated in NS for 48 h. Therefore, this study demonstrates that MMPs and serine proteases excreted and secreted by A. pegreffii and the mechanical movement of infective larvae participate in the penetration of the gastrointestine of mice after infection.