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.

Hymenolepis nana is a common opportunitistic cestode parasite and is found worldwide. H. nana infections are typically asymptomatic but heavy infections also cause anorexia, headaches, weakness, diarrhea, and abdominal pain [4]. H. nana infection is more dangerous for small children than adults, especially in regions with inadequate sanitation and hygiene. H. nana is the only cestode without any intermediate hosts in its life cycle [5]. H. nana infection is typically acquired from eggs in contaminated food. Eggs are ingested by an arthropod intermediate host and hatch in the duodenum, releasing oncospheres, and develop into cysticercoid larvae. Upon rupture of the villus, the cysticercoids return to the intestinal lumen, evaginate their scoleces, attach to the intestinal mucosa, and mature into adults that reside in the ileal portion of the small intestine, producing gravid proglottids. The eggs are then passed in stools when released from the proglottids or disintegration of proglottids in the small intestine. An alternate mode of infection consists of internal autoinfection without passing through the external environment. The short life span and rapid course of development also facilitates the spread and ready availability of this worm, but internal autoinfection allows the infection to continue for years [5].
Anisakis nematodes are marine mammal parasites and have fish and crustaceans as intermediate hosts. Anisakiasis is a widely distributed zoonosis associated with fish consumption. In humans they carry out an incomplete cycle as the larvae do not have the adequate environment to reach the adult stage [6]. Humans act as accidental hosts by consuming undercooked and/or raw second intermediate hosts that contain A. simplex third-stage larvae (AsL3). A. simplex rarely develops further within the human gastrointestinal tract, instead, by means of proteolytic enzymes, they typically become embedded in the gastric or intestinal mucosa and die, or invade the muscular layers of the stomach and intestine to induce allergic reactions and a variety of abdominal symptoms that are characterized as anisakiasis or anisakidosis [7]. Infection by the A. simplex [8][9][10] depends on both the viability of the larvae and on the activity of their somatic and/or secretory/excretory antigens. Four major clinical symptoms in human anisakiasis are gastric, intestinal, ectopic (extra-gastrointestinal) and allergic disease. Anisakidosis is globally recognized as a public health problem. It is relative to Asia and Europe [7]. The prevalence of anisakidosis has increased unusually because of the increasing popularity of Japanese cuisine, such as sushi and sashimi. The availability of an anthelmintic compound against A. simplex has the potential to shorten the clinical course and prevent invasive intervention from endoscopic procedures.
Free radical scavenging activities have been implicated in some inflammatory diseases. Some agents also have free radical scavenging activity and antiprotozoan activity [11]. However, free radical scavenging activity failed to reduce their larvicidal activity [12]. Other studies have suggested that free radical scavenging may reduce larvicidal activity by permitting larvae survival. Therefore, exactly how free radical scavenging affects the cestocidal activity of certain anthelminthic agents still remains unclear. Whether or not a correlation exists between the possible scavenger activity of aporphine and their anthelminthic activity is determined using oxygen radical absorbance capacity (ORAC) assays.

Preparation of H. nana Adult Worms
H. nana adult worms were obtained from each part of the intestines of wild type mice, purchased from Lin's farm in Fengshan, Kaohsiung, Taiwan. These parts of the duodenum, jejunum, ileum, colon and rectum were used. The H. nana adult worms ranged in length from 5-50 mm, and were collected using a needle with a blunt tip, before being placed in Petri dishes (Gibco BRL Life Technologies, Grand Island, NY, USA) with 0.9% NaCl and gentamycin (10 mg/mL). They were then washed several times. The adult worms were individually observed under an inverted microscope and those that exhibited any kind of internal or external damage were discarded. The adult worms were then identified by their morphological features, divided into groups and placed in 24-well plates contained cultivated media RPMI-1640 (Gibco BRL Life Technologies) plus 20% FBS, pH 7.4, in an atmosphere of 95% O 2 /5% CO 2 , 37 °C. These culture conditions have been shown to maximize the development and survival of H. nana [18].

A. simplex Larvae Preparation
The AsL3 were obtained from the muscle and peritoneum of fresh Trichiurus lepturus (largehead hairtail, Pacific cutlassfish) that were purchased from the fish market of Kaohsiung,Taiwan. The AsL3 had an average length of 20-22 mm, and were collected using a needle with a blunt tip, placed in Petri dishes with 0.9% NaCl and washed several times. The majority of the larvae were encysted, but they quickly became excysted upon washing in NaCl solution. They were individually observed under an inverted microscope and those that showed any internal or external damage were discarded. The larvae were then identified by morphological features, divided into groups and placed in 24-well plates containing cultivated media RPMI-1640 (Gibco BRL Life Technologies) plus 20% FBS, pH 4.0, in an atmosphere of 95% O 2 /5% CO 2 , 37 °C. These culture conditions have been shown to maximize the development and survival of A. simplex [19][20][21].

Evaluation of Oxygen Radical Absorbing Capacity (ORAC)
The automated ORAC assay was carried out on a FLUOstar Galaxy plate reader (Roche Diagnostic System Inc., Branchburg, NJ, USA) as described in a previous report by Gillespie and co-workers [22]. The experiment was conducted at 37 °C under pH 7.0 condition with a blank sample in parallel. Briefly, AAPH was used as a peroxyl generator, and Trolox (1 μM), a water-soluble analogue of vitamin E, was used as a control standard. The final reaction mixture for each black microplate in a 96-well microplate assay contained fluorescent (0.06 μM), AAPH (18.75 mM) and appropriate test substance (1 μM) in phosphate buffer (75 mM). The analyzer was programmed to record the fluorescence of FL (FLUOstar Galaxy plate reader, Vienna, VA, USA) every minute after the addition of AAPH. All fluorescent measurements are expressed relative to the initial reading (excitation/emission at 495/530 nm). Parameters of assay for the plate reader were as follows: