Abstract: This approach intends to add new data on the helminthes parasites of laboratory mice. It has been investigated the anthelmintic activity of Solanum lycocarpum (Solanaceae) extracts against Vampirolepis nana and Aspiculuris tetraptera in mice naturally infected. The extracts were applied for oral saw (intragastric), into the volume of 0.04 mL g-1, with the employing of a dead and bend probe during three consecutive days. The fecal material, collected 24 h after each application, performing a total of four fecal collection, have been softened previously, transferred about to sieve of network of 125 µm and tested under microscope stereoscope, with the objective of behave the identification and counting from the worms eliminated of the second to the fifth day of the experimental. Tukey-Kramer Multiple Comparisons Test was applied to compare the results. According to the analysis of the results it was observed that there were no differences in the % of elimination to the concentration of 10% between TM and UR (from 28.4±3.55 to 29.2±2.92) and statistical differences between TM and C (from 28.4±3.55 to 15.0±1.5) and UR and C (from 29.2±2.92 to 15.0±1.5). To the concentration of was observed that there were differences in the of eliminating between TM and UR (from 30.9±3.43 to 7.7±1.1) and C (from 30.9±3.43 to 15±1.5) related to the experimental which the animal was infecting with Vampirolepis nana. To the concentration of 5% it was observed that there were no differences in the % of elimination between TM and UR (from 4.18±3.33 to 3.70±3.16), TM and C (from 4.18±3.33 to 1.56±3.16) and UR and C (from 3.70±3.16 to 1.56±3.16) related to the experimental which the animal was infected with Aspiculuris tetraptera. It was described that in the chemical analysis of the studied extract there is the presence of glycoalkaloids, a finding that represents a reason for concern since many of these substances are generally toxic and maybe anthelmintic. It was published that medicinal plants which were reported as useful in the treatment of diabetes the S. lycocarpum was the sixth most frequently mentioned. According to the results obtained in the present study, we can speculate that the anthelmintic effect of S. lycocarpum would be related to the action of steroidal alkaloid oligoglycosides as well as the presence of short-chain fatty acids as well as with the concentration of the referred compounds in the extracts.
INTRODUCTION
The Brazilian flora is one the world richest sources of bioactive material due to its biodiversity. Several plants are currently used in Brazilian traditional medicine to treat diabetes. Solanum lycocarpum St. Hill, Solanaceae has been widely used and commercialized as a hypoglycemic agent in Brazil. Recently, it was carried out a chemical analysis of the starch and tried to correlate its supposed hypoglycemic activity with the polysaccharide content. However, these investigators did not conduct any experimental test to directly demonstrate the hypoglycemic effect attributed to the starch. As far as we know, no studies have evaluated the potential hypoglycemic effect of the starch of S. lycocarpum in experimental animals or the pattern of its use by a group of diabetic patients (Dall'Agnol and von Poser, 2000).
S. lycocarpum is a plant which is shrubs ranging in height from 1.2 to 3 m. The fruit is yellow in color and resembles a medium sized tomato. Parts of the plant are poisonous if it gets in your system. When it is in bloom, it is medium blue. It blooms in the late winter, early spring, late fall, early winter and mid winter. It is velvety or fuzzy. It needs water regularly. It is found in the Brazilian savannah but has been said to grow in San Antonio, Texas. S. lycocarpum is commonly used in Brazilian folk medicine. Solanaceae or Lobeira is a plant used as a hypoglycemic agent. A study reported that the extract reduces glycemia in alloxan induced diabetic rats. It was reported that the potential of S. lycocarpum as antioxidant was capable reduce in 27% nitrate generation in diabetic animals. In literature has been demonstrated that S. lycocarpum is not ulcerogenic and restored haemoglobin and haematocrit to normal values in diabetic animals (Perez et al., 2006).
It is called the wolf apple because maned wolves eat them. It is not actually an apple but instead it is similar to a big green tomato. It has leaves covered with white hairs that make them look gray. The flowers are shaped like a star and are blue with yellow stamens. Scientific research is being made for medicinal purposes. Yoshikawa et al. (2007) described that steroidal alkaloid oligoglycosides as solamargine, solasonine and 12-hydroxysolasonine, inhibited the increase of rat serum glucose levels by suppressing the transfer of sucrose from the stomach to the small intestine.
It grows in wet, red clay. It needs water regularly. It doesn’t need as much water in the winter because it needs full sunlight and mild temperatures. They are edible by humans. This plant contains steroidal glycoalkaloids that can be transformed into an intermediate for steroidal drug production. In this way, it is very possible that these glycoalkaloids and its aglycone, once in the body by ingestion of S. lycocarpum fruits, may act by disrupting the endocrine system. Because its fruits may be consumed by pregnant animals in the fields, various studies determined the possible toxic effects of exposure to S. lycocarpum fruit from gestation. The unripe fruits contained 0.6% of solamargine and 0.9% of solasonine. It was related that S. lycocarpum, during gestation and the beginning of lactation reduces intrauterine growth. It is known that during adulthood, female offspring showed impaired sexual behavior and male offspring showed prominent degeneration of testis germinative cells, characterized by a reduced number of germ cells and vacuolation. It has been documented that the exposed offspring showed reduced hypothalamic norepinephrine (NOR), vanillylmandelic acid (VMA), 3-methoxy-4-hydrophenylglycol (MHPG) and homovanillic acid (HVA) levels and reduced striatum NOR, HVA, VMA, MHPG, dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) levels. It is suggest that the fruit may act as an estrogen, with a long-term effect, impairing the receptive lordosis behavior of female offspring and promoting testis abnormalities in male offspring at adulthood. It appears to disrupt brain organization since important central monoamine level alterations were also related (Schwarz et al., 2005a).
It was described by Vieira et al. (2003) the anti-inflammatory effects of the crude ethanol extract and its alkaloid fraction from Solanum lycocarpum fruits. Due to the referred study the alkaloid fraction induced a dose-dependent reduction in ear oedema formation and leukocyte migration, suggesting that S. lycocarpum fruits may contain steroidal alkaloids accounting for the anti-inflammatory effect of the crude ethanol extract.
Maruo et al. (2003) demonstrated the embryotoxic effects of S. lycocarpum fruit ingestion during preimplantation and during organogenesis in rats. In this study few differences were observed in food and water consumption without biological importance. It was observed that the placental weight in the group that received the plant during the organogenesis period was decreased. An increase in sternebra abnormalities was observed in animals treated with the plant during organogenesis. Olfactory bulb hemorrhage was increased in the group that received the plant during preimplantation when compared to the control group. These results indicate that consumption of S. lycocarpum at 3% in diet during pregnancy cause slight toxicological effects. Chang et al. (2002) evaluating the toxic effects of lobeira during the fetogenesis period, related that no clinical signs of maternal toxicity were observed. The placenta weights of the treated rats were lower than those of the control. Lungs and kidneys of the fetuses treated with lobeira were also significantly reduced, suggesting a fetotoxic effect of this plant.
Rodents, as mice and rats are the most common laboratory animals used in research and testing. They are seldom investigated for autochthonous ecto- and endoparasites prior their utilization in the experiments. Pinworms commonly infecting laboratory rodents include mainly the mice pinworms Syphacia obvelata and Aspiculuris tetraptera and in rats Syphacia muris (Perec-Matysiak et al., 2006). Fecal specimens obtained from rats and mice in general are infected with one or more helminth species. Syphacia muris and Syphacia obvelata are more frequently in rats and Aspiculuris tetraptera, S. obvelata and Hymenolepis nana, respectively, in mice (Senlik et al., 2005).
Some plant extracts are efficient due to their anthelmintic activity. It was related that ethanolic and aqueous extracts obtained from nine plant species from seven families selected depending on their use in Turkish folk medicine, including Citrillus lanatus (Thunb.) Matsum. (seed), Jasminum fruticans L. (branches), Juniperus drupacea Labill. (fruits), Juniperus nana L. (fruit and leaves), Juniperus oxcycedrus L. (fruit and leaves), Mentha longifolia L. (herba), Pinus nigra ssp. pallasiana (Lamb.) Richt. (fruits), Plantago lanceolata L. (leaves) and Zea mays L. (seed) were evaluated for their in vivo anthelmintic activity. Among the plant extracts studied, both ethanolic and aqueous extracts of Jasminum fruticans, Mentha longifolia and Pinus nigra ssp. pallasiana, the aqueous extracts of Zea mays, the ethanolic extracts of Citrillus lanatus, Juniperus drupacea (fruit), Juniperus oxcycedrus and Plantago lanceolata displayed significant anthelmintic activity against pinworms, Syphacia obvelata and Aspiculuris tetraptera, in mice. Rest of the extracts from plants did not show any remarkable anthelmintic activity (Kozan et al., 2006).
Some plant extract may act differently due to its action against the parasite. In a study the anthelmintic activity of the extracts obtained from Luxemburgia octandra was evaluated naturally infected mice with Aspiculuris tetraptera and Vampirolepis nana. The leaves extracts were given to the animals during three days. The ethanolic and ethyl acetate extracts presented significant increase of the V. nana elimination, but did not present the nematicide effect against A. tetraptera (Silva et al., 2005).
In the present study, we evaluated the anthelmintic activity of Solanum lycocarpum extracts in a concentration of 5% and 10% against Vampirolepis nana and Aspiculuris tetraptera in naturally infected mice.
MATERIALS AND METHODS
Vegetal Extracts
Dried leaves of units of had been used in the anthelmintic tests Solanum
lycocarpum collected in the City of Três Marias, State of Minas Gerais
and in the City of Seropédica, State of Rio de Janeiro. The botanical
identification was carried through in the Department of Botany of the Rural
Federal University of Rio de Janeiro, having been the exsiccates deposited under
numbers RBR 28010 and RBR 14071. For the execution of the tests, the extracts
had been gotten by infusion (tea), submitted to the filtration in nylon and
the express concentrations in g/100 mL (p/v).
Animals and Anthelmintic Tests
For anthelmintc test have been used lots of albinos mice, male and females
weighted in media of 25 g and naturally infecting for Vampirolepis nana
and Aspiculuris tetraptera originated from Oswaldo Cruz Foundation-FIOCRUZ
and held into the Institute of Biology from Rural Federal University from Rio
de Janeiro. The animals have been held into bird cages individual of polypropylene
(30x20x13 cm), it has at the bottom road of screen stark and stiff (network
of 7x7 mm) upon a sheet of absorbent paper with the aim to facilitate the collection
diary of excrement (Steward, 1955; Amorim et al., 1987; Amorim and Borba,
1990).
The extracts were applied for oral saw (intragastric), into the volume of 0.04 mL g-1, with the employing of a dead and bend probe during three consecutive days. The faecal material, collected 24 h after each application, performing a total of four fecal collection, have been softened previously, transferred about to tames of network of 125 μm and evaluated under microscope stereoscope, with the objective of behave the identification from the worm eliminated of the second to the fifth day of the experimental. Into the fifth and last days from the tests, the mice have been sacrificing for inhalation of vapors of ether ethyl, examining humid weight of the contents of the small intestine, in order to access the number of the proglotes collected of V. nana remnants examining in the colon the number of the A. tetratptera remnants (Amorim et al., 1999). On the tests have been used the extracts of Solanum lycocarpum (leaves dried from Três Marias in the concentration of 5 and 10%) and (leaves dried from UFRRJ in the concentration of 5 and 10%). Additional lots of mice have been used with standard, they receiving doses of 20 mg kg-1 day-1 of mebendazol and 100 mg kg-1 day-1 of nitroscanato and they were submitted to the identical assessment anthelmintic description about to the animals treated with the plant extracts. A batch control, without a treatment served about to appraise the elimination spontaneous from the helminthes studied. The outcome anticestode was denominated in terms of percentile average of elimination of proglotes, through the humid weight of the segments eliminated on the faecal material where upon treatment in relation to on the dough total of segments. To the Aspiculuris tetraptera the outcome antinematode also was denominated in terms percentile average of roundworm eliminated, considering the number of roundworm eliminated in the faecal material in relation to the total number. Statistical analysis were performed and Tukey-Kramer Multiple Comparisons Test was applied to compare the results.
RESULTS
According to the analysis of the results it was observed that there were differences (p<0.001) in the % of elimination between TM and C (from 28.4±3.55 to 15±1.5) and to UR versus C (from 29.2±2.92 to 15±1.5). There was observed no difference (p>0.05) in the comparison between TM and UR (from 28.4±3.55 to 29.2±2.92) (Table 1).
| Table 1: | Anthelmintic activity of the extracts obtained of Solanum lycocarpum in the elimination of Vampirolepis nana in mice naturally infected |
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| The extracts were applied for oral saw (intragastric), into the volume of 0.04 mL g-1, with the employing of a dead and bend probe during three consecutive days. The excrements, collected 24 h after each application, performing a total of four fecal collection, have been softened previously, transferred about to tames of network of 125 μm and evaluated under microscope stereoscope, with the objective of behave the identification of the worm eliminated of the second to the fifth day of the experimental. Tukey-Kramer Multiple Comparisons Test was applied to compare the results | |
| Table 2: | Anthelmintic activity of the extracts obtained of Solanum lycocarpum in the elimination of Aspiculuris tetraptera in mice naturally infected |
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| The extracts were applied for oral saw (intragastric), into the volume of 0.04 mL g-1, with the employing of a dead and bend probe during three consecutive days. The excrements, collected 24 h after each application, performing a total of four fecal collection, have been softened previously, transferred about to tames of network of 125 μm and evaluated under microscope stereoscope, with the objective of behave the identification of the worm eliminated of the second to the fifth day of the experimental. Tukey-Kramer Multiple Comparisons Test was applied to compare the results | |
| Table 3: | Anthelmintic activity of the extracts obtained of Solanum lycocarpum in the elimination of Vampirolepis nana in mice naturally infected |
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|
| The extracts were applied for oral saw (intragastric), into the volume of 0.04 mL g-1, with the employing of a dead and bend probe during three consecutive days. The excrements, collected 24 h after each application, performing a total of four fecal collection, have been softened previously, transferred about to tames of network of 125 μm and evaluated under microscope stereoscope, with the objective of behave the identification of the worm eliminated of the second to the fifth day of the experimental. Tukey-Kramer Multiple Comparisons Test was applied to compare the results | |
According to the analysis of the results it was observed that there were no differences (p>0.05) in the % of elimination between TM and C (from 4.18±3.33 to 1.56±3.16), UR and C (from 3.70±3.16 to 1.56±3.16) and TM and UR (from 4.18±3.33 to 3.70±3.16) (Table 2).
According to the analysis of the results it was observed that there were no differences (p<0.001) in the % of elimination between TM and UR (from 30.9±3.43 to 7.7±1.1), TM and C (from 30.9±3.43 to 15±1.5) and (p<0.05) but not relevant to UR versus C (from 7.7±1.1 to 15±1.5) (Table 3).
DISCUSSION
Animal models have been exhaustively investigated regarding aspects related to their suitability for the development of experimental protocols under laboratory conditions. Nevertheless, in most of the adopted procedures, the prior detection of their ecto and endo parasites are generally overlooked related to the really effects of natural extracts in their biological cycle.
In the Brazilian cerate, a preparation obtained from the fruits of Solanum lycocarpum St.-Hill. (Solanaceae), popularly known as fruta-de-lobo (wolf-fruit), have been widely employed for diabetes management, obesity and to decrease cholesterol levels. The medicinal preparation consists of the green fruits which are ground in aqueous solution and filtered. The white 'gum' deposited is decanted and slowly dried providing a powder which is commercialized in capsules with the name of polvilho-de-lobeira. Through phytochemical analysis of this phytomedicine and the fruit of S. lycocarpum were found polysaccharides as the main component. Some polysaccharides slow gastric emptying and act on the endocrinous system affecting the liberation of gastrointestinal hormones, lowering blood glucose levels. According to Schwarz et al. (2005b) it is well known that this plant contain steroidal glycoalkaloids that can be transformed into an intermediate for steroidal drugs production, like oral contraceptives. In this way, it is very possible that these glycoalkaloids and its aglycone, once in the body by ingestion of S. lycocarpum fruits, may act disrupting to the endocrine system as well as it may probably affect the reproductive system of helminthes. The hypocholesterolemic activity could be due to the increased fecal bile acid excretion as well as to the action of the short-chain fatty acids, coming from fermentation, on the synthesis of delta-aminolevulinate and by the increase of the cholesterol 7-alpha-hydroxylase and 3-hydroxy-3-methylglutaryl CoA reductase synthesis (Dall' Agnol and Von Poser, 2000). Due to the effect related to the extracts in the concentration of 10% to the animals infected with Vampirolepis nana it may be possible that these fatty acids could act as an anthelmintic, once that the extract TM and UR increased the % of elimination in comparison to the control. Related to the action of the TM extract it was observed that its effect was effectively as anthelmintic as well as UR extract in comparison to Mebendazol. In the studied concentration the extracts showed a similar effect which may be resulted from the biochemistry compounds in the equivalents proportions in spite of different conditions as soil composition, light and water availability.
A possible explanation of the anthelmintic of the extracts may be related to a toxic effect of S. lycocarpum on the male reproductive system of the Swiss mouse, with possible antiandrogenic activity, which in spite of no present apparent antifertility activity in rats may be toxic to the helminthes (de Cassia da SeSa et al., 2000).
Related to the similar effect found in comparison to the action of the extracts, it may be support by possible modifications in ribosomal DNA spacer region suggesting that it could result in genetic and geographical variability as well as different bioactivity which may not be effective depend on the concentration of the extract (de Arruda et al., 2003). Due to the effect related to the concentration of 5% of the extracts to the animals infected with Aspiculuris tetraptera it may be possible that these fatty acids could act as an anthelmintic, although in he present study the TM and UR extracts did not be effective related to % of elimination in comparison to the control. Related to the obtained results due to the action of the TM and UR extracts it may be explained by their low concentration which were not effectively as anthelmintic in comparison to Mebendazol and nitroscanato. In the studied concentration, the extracts showed a similar no effect which may be resulted from the biochemistry compounds in the equivalents proportions in spite of different conditions as soil composition, light and water availability. Related to the obtained results due to the action of the TM extract it may be explained by their concentration as well as originated region which may explain the effect due to the biochemistry compounds in the equivalents proportions in spite of different conditions as soil composition, light and water availability.
We can speculate that the other effect would be related to the steroidal alkaloid oligoglycosides suppressing the transfer of sucrose from the stomach to the small intestine which could diminish the support of glucose to helminthes together with its antioxidant effect which is capable of reducing the nitrate generation which can be used in the protein synthesis. This fact is of extreme relevance, taking into account that cestode infections induce very high levels of immunogenic responses in the parasitized animals and consequently a great consumption of glucose.
CONCLUSION
Based on the results we can suggested that the anthelmintic effect of Solanum lycocarpum extracts, would be related to the action of steroidal alkaloid oligoglycosides as well as the presence of short-chain fatty acids. The similar action of the extracts may be explained by adaptation mechanisms related to the genetic and geographical variability which can be notary related to the concentration of the extracts.
ACKNOWLEDGMENT
We thanks to Thiago de Azevedo Amorin, Technician of Herbarium of the Botanical Department of Rural Federal University from Rio de Janeiro, by the identification of the studied species.