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Effects of pine bark extract administered to immunosuppressed adult mice infected with Cryptosporidium parvum

American Journal of Chinese Medicine, Summer-Fall, 2001 by Hyeon Cheol Kim, Jeffrey M. Healey

Abstract: The treatment of cryptosporidiosis using pine bark extract (Pycnogenol[R]) in immunosuppressed adult C57BL/6N mice infected with Cryptosporidium parvum was investigated. Five groups of 10 mice/group were used. Groups 1, 2, 3, and 5 served as normal, toxicity, placebo, and positive controls, respectively. Mice in groups 2-5 were immunosuppressed with dexamethasone phosphate administered ad libitum in drinking water at a dosage level of 12 [micro]g/ml. Mice in groups 3-5 were inoculated per os with [10.sup.6] C. parvum oocysts on the day immunosuppression was started. Mice in groups 2 and 4 were treated by administering Pycnogenol[R] orally at 30 mg/kg/day. In group 4, Pycnogenol[R] was first administered on day 3 postinoculation. Of the four groups of mice immunosuppressed with DEXp (groups 2-5), the two groups treated with Pycnogenol[R] (groups 2 and 4) had no premature deaths. The other two groups (groups 3 and 5) had 3 and 4 mice die, respectively, before the experiment ended. Consequently, Pycnogenol[R] was judged to be non-toxic at the dosage level used and even afforded mice some positive health benefits. Fecal oocyst shedding in groups 3-5 was initially detected on day 3 postinoculation. These mice continued to shed oocysts throughout the duration of the 28-day experiment. Oocyst shedding intensities were greater in group 3 and 5 than in group 4. However, histological examination of infected intestinal tissues in groups 3-5 revealed no significant difference with regard to parasite colonization and villus/crypt (V/C) length ratios. As a result, Pycnogenol[R] was determined to be therapeutically effective against C. parvum at 30 mg/kg/day only when measured by fecal oocyst shedding intensity. There was no effect on parasite tissue colonization and V/C ratios in infected mice. We conclude that Pycnogenol[R] is a useful dietary supplement for C. parvum-infected patients by affording some positive health benefits, significantly reduces fecal oocyst shedding, but does not decrease parasite colonization of intestinal tissue.

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Cryptosporidium parvum is an intracellular protozoan parasite that colonizes epithelial cells of the respiratory and digestive tracts of humans and other animals (Fayer and Ungar, 1986; Moore and Frenkel, 1991; O'Donoghue, 1995). The infection is usually mild and self-limiting in hosts with a normal immune system, but can be chronic and life-threatening in immunocompromised individuals (Navin and Juranek, 1984; Casemore et al., 1985). Despite progress, prevention and treatment of the disease remains limited by the absence of approved vaccines or immunotherapies and by the lack of safe and effective parasite-specific drugs (Blagburn and Soave, 1997; Riggs, 1997). The antimicrobials paromomycin and azithromycin have limited efficacy against cryptosporidiosis in patients with AIDS (Vakil et al., 1996; White et al., 1994). Smith et al. (1998) reported that the treatment of cryptosporidiosis with combined paromomycin and azithromycin was associated with a significant reduction in oocyst excretion and some clinical improvement. Fortunately, patients with reversible immunodeficiencies usually recover from cryptosporidiosis (Fayer and Ungar, 1986).

There is growing interest in the biologic activities of plant extracts in the treatment of disease (Packer et al., 1999; Park et al., 2000). Extracts from the bark of pine have been used in different parts of the world as traditional medicines and are believed to affect various pathologies, ranging from vascular disease to arthritis (Virgili et al., 1998). Pycnogenol[R] (Horphag Research, Geneva, Switzerland), the extract from the bark of the French maritime pine tree (Pinus maritima), is a concentrate of bioflavonoids, mainly phenolic acids and procyanidins, as well as other components (Packer et al., 1999). This combination is a very useful antioxidative substance (Rohdewald, 1998). Although data are lacking regarding the use of pine bark extract in the treatment of cryptosporidiosis, Molan et al., (1999) reported that proanthocyanidines may disrupt the life cycle of nematodes by reducing egg viability and larval development. The purpose of the present study was to observe the therapeutic effect of Pycnogenol[R] in mice infected with C. parvum.

Materials and Methods

Animals and Parasites

Female C57BL/6N mice (Simonson Laboratories, Gilroy, California), age 6 to 8 weeks and weighing 15 to 20 g each, were used. The mice were immunosuppressed with dexamethasone phosphate (DEXp) (Sigma Chemical Co., St. Louis, Missouri) administered ad libitum in drinking water (12 [micro]g/ml) (Yang and Healey, 1993). They were maintained in isolation during the course of the study and were housed in wire-floored cages. The cages were placed on trays containing 1.8% potassium dichromate solution to prevent the feces from drying out.

Mice were inoculated with the Iowa isolate of C. parvum. Oocysts were maintained by passage in experimentally infected mice and purified from feces using discontinuous sucrose gradients (Arrowood and Sterling, 1987). Purified oocysts were stored in 2.5% potassium dichromate solution at 4 [degrees] C for less than 4 months prior to use. Oocyst inocula were prepared by washing purified oocysts with distilled water 3 times to remove the potassium dichromate. Washed oocysts were enumerated on a hemocytometer using microscopy and then administered to mice by orogastric intubation ([10.sup.6] C. parvum oocysts/mouse) as reported previously (Yang and Healey, 1994).

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Effects of pine bark extract administered to immunosuppressed adult mice infected with Cryptosporidium parvum

American Journal of Chinese Medicine, Summer-Fall, 2001 by Hyeon Cheol Kim, Jeffrey M. Healey

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