Reduction in the Number of Infective Trichinella spiralis

Nirmal C. Sukul Sudeshna Ghosh Santi P. Sinhababu
Department of Zoology, Visva-Bharati University, Santiniketan, West Bengal, India

Key Words
Trichinella spiralis · Homeopathic drugs · Cina · Podophyllum · Santonin
Summary
Background: Trichinellosis caused by the gastrointestinal nematode Trichinella spiralis occurs in humans, domestic animals and wild animals. It is difficult
to control the muscle phase of the parasite. Homeopathic drugs such as Cina and Santoninum have anthelmintic properties. We have observed that in material doses, the homeopathic drug Podophyllum also has nematotoxic properties. We have also observed that homeopathic potency can influence the water permeability of cells. Objective: The purpose of this study was to investigate whether potentized homeopathic drugs such as Cina 30, Santoninum
30 and Podophyllum mother tincture can affect the muscle phase of the parasite T. spiralis in mice. Another objective was to see whether trichinellosis and its treatment with the 3 named homeopathic drugs could alter the water content in the muscle tissue of mice. Materials and Methods: Cina 30 and Santoninum 30 were prepared from the mother tincture of the flowering tops of Artemisia nilagirica and its active principle santonin, in each case by successive dilution (1:100) with 90% ethanol and sonication in 30 steps following the single glass method (K30). Ethanol 30 was prepared by successive dilution of 90% ethanol with 90% ethanol (1:100) followed by sonication in 30 steps. In each step, the dilution was sonicated at 20 KHz for 30 s. We have observed before that sonication is a more uniform, measurable and effective process of mechanical agitation of a liquid than manual succussion. Experimentally infected mice were orally treated with an aqueous Podophyllum suspension at 60 mg/kg/day. Each potentized drug was diluted 1:20 with distilled water and administered orally at 0.05 ml/mouse/day. Each mouse was inoculated with T. spiralis larvae at a dose of 200 larvae/mouse by esophageal intubation. Treatment was started on day 7 post-infection and
continued for 120 days. After completion of treatment, the mice were sacrificed and the larvae were extracted from muscles by HCl-pepsin digestion.
The water content of the muscles was measured by determining the difference between fresh weight and dry weight of the tissue. Results: Podophyllum
θ, Cina 30 and Santoninum 30 reduced the larval population in the studied mice by 68.14%, 84.10% and 81.20%, respectively, as compared to the untreated
control group. Ethanol 30 achieved no significant reduction in the larval population compared to the untreated control group. The water content of
the muscle tissue in the untreated control group and the Podophyllum-treated groups was significantly higher than in the Ethanol 30-, Cina 30- and Santoninum
30-treated groups. Conclusions: (1) Podophyllum θ, Cina 30 and Santoninum 30 were effective in the muscle phase of T. spiralis infection and
significantly reduced the larval population in the treated mice. The potencies were more effective than the mother tincture, an effect which was not due
to the medium ethanol. (2) The potencies significantly reduced the water content of the muscle tissue which might have affected the larvae. The effect
of Podophyllum θ might be due to the direct toxic effect of the drug on the larvae.

Introduction

Trichinellosis is a cosmopolitan parasitic disease affecting humans, domestic animals and wild animals. In case of severe infection it causes lethality. New-born larvae of the parasite migrate to the heart and cause eosinophilic myocarditis in mice [1]. Adult worms, which are nematodes, live for some weeks in the intestine of the host and give birth to larvae which penetrate the intestine, enter the blood stream and encyst in muscle tissue. If infected muscles are eaten by a potential host, the digestive enzymes set free the larvae. In the intestine, the larvae develop into adults within 40-50 h. This zoonotic disease is re-emerging in several countries with pigs serving as the main intermediate host for disease transmission to humans [2]. It is difficult to eradicate the disease with available anthelmintic drugs. Albendazole, a derivative of benzimidazole, is effective against murine trichinellosis when given at the stage of maturation of adult worms and production of newborn larvae. However, the drug does not change the course of Trichinella infection when given at the stage of larval infectiveness [3]. In previous experiments, we observed potentized homeopathic drugs such as Cina 30, Cina 200 and Cina 1000 to have antifilarial activity against canine dirofilariasis [4, 5]. In these experiments, Cina potencies reduced microfilarial densities in the blood of dogs infected with Dirofilaria immitis. The results promoted us to study the effects of a potency of Cina and Santonin on trichinellosis. Cina potencies are produced from the ethanolic extract of the plant Artemisia sp. The active principle of Artemisia is santonin [6]. The crude extract of Artemisia and santonin are known to have anthelmintic properties [4, 7,8]. Therefore, the material doses of these two substances were not used in the present experiment. On the other hand, Podophyllum è has not yet been reported to have anthelmintic effect. Once this effect is established for Podophyllum è, as was done in this study, its potencies can be tested for the same effect. The purpose of the present study was to see whether the crude extracts of Podophyllum, Cina 30 and Santoninum 30 could reduce the larval population of T. spiralis in mice. We further wanted to see whether the homeopathic drugs could alter the water content in the muscles of mice. We previously observed that homeopathic potencies influenced the water permeability of cells [9]. If we are to establish the scientific basis for homeopathy, we have to conduct experiments in a rigorous way including sacrificing animals where it is necessary.

Materials and Methods

Drugs

Flowering tops of Artemisia nilagirica were collected from Shillong, dried in the shade and extracted with 90% ethanol in the laboratory. The extract, called mother tincture or Cina è, contained 1 mg of soluble solids/ml of the solvent. Cina è was diluted with 90% ethanol in the proportion of 1:100 and the mixture was sonicated at 20 KHz for 30 s to prepare the first centesimal potency of the drug Cina 1. This potency was successively diluted with 90% ethanol and sonicated in a similar manner in 30 steps to prepare the 30th potency (Cina 30) following the single glass method (K30) [10]. Santonin, purchased from Sigma Chemical Co. (St. Louis, MO, USA), was dissolved in distilled water in the proportion of 1 mg/ml. This solution was successively diluted with 90% ethanol and sonicated in 30 steps to prepare Santoninum 30 following the same procedure as with Cina 30. Ethanol 30 was prepared from 90% ethanol in a similar manner. Podophyllum è, which is prepared from the extracts of the rhizomes of Podophyllum hexandrum, was obtained from M. Bhattacharyya & Co., Kolkata, India. The extract was evaporated at room temperature in large petri dishes under continuous fanning for 4 days. The residue, desiccated over anhydrous calcium chloride, was mixed with distilled water in the proportion of 4 g / 50 ml and used for the treatment of infected mice. Experiments on animals were conducted without causing any stress to the animals, following the conventions and guidelines on animal experimentation in the University.

Animals

Swiss albino mice (20-25 g) were intramuscularly injected with hydrocortisone at a dose of 7.5 mg/kg/day for 15 days. Hydrocortisone decreases the immunity and helps establish a more or less uniform infection in the animals [11, 12]. T. spiralis, originally obtained from the Gifu University, Japan, had been maintained in mice at our laboratory. An infected mouse from the laboratory stock was sacrificed by decapitation. The skeletal muscles of the animal were cut up and digested with pepsin-HCl (0.8% pepsin and 0.8% HCl in 0.85% physiological saline) for 1 h at 37 °C [1]. The isolated larvae were washed three times in phosphate buffer solution (PBS) and then given to mice by esophageal intubation within 1 hour of digestion. Each mouse received a dose of 200 larvae in PBS suspension. A total of 32 mice were inoculated in this way. A further 16 mice were kept as uninoculated controls. All mice were kept in the animal house and received their usual diet and water as desired.

Treatment

While the infected mice were divided into 5 groups, the uninfected animals were divided into 2 groups. Of the infected mice, 4 groups were treated with Ethanol 30, Podophyllum è, Cina 30 and Santoninum 30. The remaining infected group served as the untreated control. Treatment was started on day 7 post-infection and was continued for 120 days. We had observed earlier that repeated doses of the 30th potencies are more effective than one single dose (unpublished). During treatment, 1 or 2 mice died in each group, which is why results were obtained from 6 mice of each group. Podophyllum suspension was administered orally at 60 mg/kg/day. Each of the 30th potencies were diluted with distilled water in the proportion of 1:20 and administered orally at 0.05 ml/mouse/day. All mice were sacrificed after the last day of treatment, and the larval population in each infected mouse was estimated by a standard method of pepsin-HCl digestion of chopped muscles. An aliquot of chopped muscles of each group of mice was weighed, kept in a BOD incubator at 90 °C for 12 h to remove water and then weighed again. Total water content in the muscles was assessed by measuring the difference between fresh weight and dry weight of the tissues [9, 13, 14]. The adult worms live in the intestine. Males die or are excreted within 30-35 h of infection. Females give birth to larvae and die within 9-16 weeks [15]. Obviously, no adults were found in the intestine of infected mice when they were sacrificed about 7 weeks after infection. Larvae are encapsulated in the muscles tissue, which is why the entire animal except for the skin and brain is chopped into pieces and examined.

Results

The mean larval population was determined with standard errors (SE) for each group of infected mice. Each treatment group was compared by the student t-test with the untreated but infected control group. Similarly, the mean water content of the muscle tissue of each infected and uninfected group was determined with SE’s and expressed in g/g of dry tissue. While one of the uninfected groups received the same dose of Cina 30 every day for 120 days, the other group was kept as uninoculated and untreated control. All the data is presented in table 1. Podophyllum è, Cina 30 and Santoninum 30 reduced the larval population in mice by 68.14, 84.10 and 81.20%, respectively, compared to the untreated control. Treatment with Ethanol 30 resulted in a 14.71% reduction in the larval population compared to the untreated control. However, the difference was of no statistic significance. The water content of the muscle tissue was significantly higher (p < 0.01) in the infected mice than in the uninfected animals. The water content in the muscle tissue of the uninfected and untreated mice did not show any significant difference to that of the uninfected but Cina 30treated mice. Treatment with Ethanol 30, Cina 30 and Santoninum 30 reduced the water content in the muscles significantly (p < 0.05), compared to the infected but untreated control group.

Discussion

Potentized drugs such as Cina 30 and Santoninum 30 were more effective in reducing the larval population in the muscles of mice than the crude extract of Podophyllum. The duration of action of hydrocortisone, which was administered to establish a more or less uniform Trichinella infection, is very short, with biological half-life of 8-12 h. After withdrawal, the treated animals exhibit a nearly normal immune response [16]. Thus, the mice exhibited a normal immune response at the   time of treatment with the homeopathic drugs. All potentized drugs significantly reduced the water content in the muscle tissue of the animals, compared to the infected and untreated control. However, this was not the case with Podophyllum è. It is possible that potentized drugs adversely affected the intracellular larvae by producing water stress. We had observed earlier that Nux vomica 30 and Mercury chloride 30 interfered with the transport of water through the plasma membrane of erythrocytes in fresh water cat fish. We proposed that homeopathic potencies acted on water channel proteins of the membrane and produced that effect [9]. After entering the muscle cell, T. spiralis induces significant phenotypic changes in the cell which then re-enter the cell cycle and replicate their DNA [17, 18]. Usually, differentiated skeletal muscle cells terminally withdraw from the cell cycle [19, 20]. The larva has a lag phase in growth of about 5 days post-infection after which it resumes growth and attains a 10-fold increase in volume by day 20 post-infection [21]. Homeopathic potencies may interfere with the dedifferentiation of the muscle cells, thereby arresting the growth of the larvae. Podophyllum è might have directly affected the larvae by its toxic effect on them. Homeopathic potencies higher than 12 consist of specifically structured H-bonded water molecules preserved by ethanol

molecules. The pattern of structural diffusion (formation and

breaking of H-bonds) appears to be specific to a particular

homeopathic potency. The pattern varies with different potentized

drugs [22]. If the potentized homeopathic drug is diluted

with water at a ratio of 1:1000 or more, it still remains effective

on patients or treated organisms [23]. This shows that a potentized

drug is capable of influencing bulk water in such a way

that the latter behaves as a potency. A homeopathic potency,

applied on the cell surface – in this case the oral mucosa of

mice – could modify the organization of water covering the

cell surface. This would affect all surface proteins of cells including

water channel proteins, thereby producing a cascade

of biochemical events inside the cells which come in contact

with the drug. The sensory neurons in the contact area – in

this case the oral mucosa – would transmit impulses to the

brain which would process the message and produce an appropriate

reaction through efferent nerves in the target cells,

i.e. the infected muscle cells. In a diseased state, the water

structure covering all cell surfaces as a continuous medium assumes

a different configuration, depending on the nature of

the disease [22]. The intracellular water of muscle fibers is

structured and aligned along the myofilaments. The state of

the intracellular water changes with physiological processes in

the muscle fibers. The structured water suppresses the diffusion

of solutes in an anisotropic fashion [24]. This shows that

structured water can alter diffusion of solutes in cells and play

an important role in cell physiology. An appropriate homeopathic

potency actually holds the complementary water structure

which induces change in the water structure of the diseased

cell, thereby restoring the normal state of the cellular

water. The change in water structure is brought about by a

change in the number and strength of hydrogen bonds [22].

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