Study on Inflammation and the Nervous system of Ethanol extract of Jatropha Curcas seed

Introduction: Jatropha curcas L. seeds are used in traditional medicine to treat a variety of diseases or conditions. The aim of this study is to evaluate effects on inflammation and the nervous system of ethanol extract of J. curcas seeds. Materials and methods: It was used 64 mice divided in 8 groups; respectively, 4 groups received 400, 600, 800 and 1000 mg/kg of ethanol extract of J. curcas seed; and the rest intake Diclofenac, Diazepam, Caffeine and a control group not received any substance. The effects on inflammation was evaluated by Carrageenan-Induced paw oedema test and by Paw skin temperature. Neurological symptoms of toxicity were evaluated using the Irwin test. For the analysis of quantitative variables were used the following tests: one-way ANOVA, Tukey, Shapiro-Wilk and Pearson correlation; for qualitative variables Chi square was used. Results: According to the paw oedema, it was showed a trend on an inflammatory effect of the seeds of J. curcas; this activity was statistically significant in doses of 1000 mg/kg. Also, the skin temperature measurements outcomes reveal a positive dose response manner. Regard to neurological manifestations, Straub tail was founded in doses of 400 mg/kg. Stereotypies were founded in doses of 400, 600, 800 and 1000 mg/kg throughout the evaluation. Conclusion: J. curcas seeds were showed an inflammatory effect. In addition, effects on the nervous system were founded as stereotypes and Straub tail.


INTRODUCTION
Jatropha curcas (Linnaeus) is a plant which belongs to family Euphorbiaceae and genus Jatropha.This plant is native of America and is widely distributed, is also located in Africa, Asia and Europe where it grows in arid, tropical and sub-tropical environments.Its name derives from the Greek words "Jatros" (doctor) and "trophe" (food) which translates its medicinal traditional use. 1 Such medicinal use is reported in diverse consumption of different parts of J. curcas, some of them through infusions for various conditions such as fever, burn, rheumatism, allergies, diarrhea, intestinal cramping, healing, dysentery, parasitic infections, etc. 2,3 Thus J. curcas seeds have acquired importance for its extra-medicinal properties 4 and its use in native communities for some conditions as arthritis, gout, jaundice, convulsions, constipation, diarrhea, fever and inflammation. 3,5,6n Peru, this plant is located in departments of Lima, Piura, Cajamarca, San Martin, Cusco, Ucayali and Loreto where its traditional practices were also recognised. 3,7,8reclinical studies have demonstrated the biological activity of the different parts of J. curcas, so, antifungal and acaricide activity of seeds; [9][10][11][12][13] antidiarrheal, anti inflammatory and antimicrobial effect of the root, stem, bark and leaf; 3,14-17 abortifacient activity of fruit 18 and activity on the blood coagulation of latex. 6][26][27][28] Considering the increase of interest for the value of this medicinal plant, 29 this study focused on assessment of effects on inflammation and the nervous system of ethanol extract of J. curcas seeds in escalating doses.

Plant material and extraction
It was used Jatropha curcas seeds which was collected in the Department of Loreto (Iquitos) under the criteria of the Cerrate method E.1969: 30 1.-First, the vascular plant was collected at 08:00 hrs 2.-Then, it was put immediately into a field press 3.-After that, it was dried Berta Loja, who is biologist of The Research Centre of Traditional Medicine and Pharmacology, FMH-USMP, authenticated the taxonomy of the plant.For mark the specimens it was crossover the information with the herbaria of the Universidad Nacional Mayor de San Marcos (USM) and the Missouri Botanical Garden (MO).][36][37] The extract was prepared from dried and ground seeds of J. curcas, this was macerated in 70% ethanol for a week, then the mixture was filtered and the obtained residue was dried in an oven for a period of 7 days.The dried sample was ground in a mortar to obtain a fine powder which was stored in airtight containers and refrigerated until its experimental use.

Animals and study design
64 Mus musculus male albino mice were obtained from National Institute of Health (Instituto Nacional de Salud, INS-Bioterio, Chorrillos, Lima-Perú), whose weight average were between 25 and 30 g. Mice were undergone to an acclimatization process in FMH-USMP; they were housed 4 per cage and were maintained at the following conditions: temperature 22°C (± 2), humidity between 30 and 70%, 12 hr light/dark cycles and noise levels less than 70 dB.Mice were provided with balanced food and water ad libitum and then they were deprived of food 12 hrs before the development of experiment.Animals were divided in 8 groups of 8 mice each group.4 experimental groups which orally intake ethanol extracts of J. curcas seed in escalating doses of 400, 600, 800 and 1000 mg/kg.4 control groups which three of them received orally caffeine 32 mg/kg, diazepam 32 mg/kg, diclofenac 8 mg/kg, and a control group who not received any substance.For randomized distribution and allocation of groups, the simple randomization by raffle method was used.Each procedure with all mice during the experiment respected the principles and guidelines for researches in laboratory animals, cited in International Guiding Principles for Biomedical Research Involving Animal (1985). 38

Irwin Test in mice
The test was conducted as an adaptation of the previously described, 39,40 The substance was administered to mice in a double-blind manner.Subsequently, the animals were observed during a one hour in time ranges of 15, 30, 45 and 60 min.The end points analyzed were presence or absence of the following: lethality, convulsions, straub tail, sedation, agitation, abnormal gait (rolling or toes), jumps, motor incoordination, piloerection, abdominal cramps, stereotypies (sniffing, chewing or head movements), head shaking, itching, and abnormal breathing. 24,25

Carrageenin-induced paw oedema in mice
This was performed as previously indicated. 41,42The carrageenan 0.2% was injected subcutaneously into the mice plantar aponeurosis.The volume of the injected paw was measured before and after each injection with a LETICA digital plethysmometer (LE-750).Observations were made on 6 periods: pre-test (before the time of administration), 1, 2, 3, 4 and 5 hrs after administration.The inflammatory activity was calculated using the formula: Oedema plantar (ml) = Vt-Vb Vt= volume of the foot at measurement point.Vb= paw volume before carrageenan administration (pre-test)

Irwin Test
The ethanol extract of the seeds of J. curcas induced neurotoxicity effects during the first hour assessment.The mainly evidenced effects were sedation, stereotypies and Straub tail in groups at doses of 400, 600, 800 and 1000 mg/kg of J. curcas seeds (See Table 1).

Carrageenan induced paw edema
The direct correlation between increased paw edema and administration dose was evidenced.The experimental groups 400, 600, 800 and 1000 mg/kg of J. curcas seeds showed statistical difference versus control groups only during the fifth hour of evaluation.Moreover, the group 1000 mg/kg of J. curcas seeds presented an increasing of plantar edema during all hours of evaluation (See Table 2).

Paw skin temperature
In a dose-response profile, the experimental groups showed increased of the skin temperature in a positive way.Particularly, the group at doses of 1000 mg/kg presented the highest temperature throughout the evaluation (See Table 3).

DISCUSIÓN
The presence of stereotype and Straub tail, would mean a stimulant effect in the the central nervous system level, which may be due to a possible induction of the serotonergic, dopaminergic, and opioidergic systems. 24 is recognized the neurotoxicity and fatality affects by the secondary metabolites phorbol esters from the seeds of J. curcas, 43 in contrast, other studies in some varieties of this plant showed poor toxicity. 45,46her secondary metabolites present in the seed of J. curcas are alkaloids, flavonoids, tannins and saponins among others, [20][21][22][23] could be responsible for the effects observed in this study.
Considering the presence of stereotype and Straub tail, it is possible that the ethanolic extract of J. curcas seed have a lipophilic chemical constitution, which would make it possible to cross the blood-brain barrier, and therefore, it should alert the potential risk during pregnancy and others.
In this study, doses of 400, 600, 800 and 1000 mg/kg of ethanol extract of J. curcas seeds showed throughout early hours of evaluation an inflammatory effect similar to control group.Contrary, a research showed an anti-inflammatory effect of methanol extract of J. curcas root at dose of 100 and 200 mg/kg. 48Similar results were showed in other study with the alcohol extracts of root, stem and leaf, at doses of 200 mg/kg showed a decrease of edema, 49 and with aqueous extract of J. curcas leaves at doses of 300 mg/kg reduced edema significantly. 50e contradictory inflammatory effects presented by the seeds of the J. curcas in front of the others parts of the plant might have explanation in the presence or absence of their secondary metabolites, inclusively in the concentration or others, 28,47,51 these variables are need to study in future studies.
In addition, the outcomes of the skin temperature are according to the inflammation cascade produced by carrageenan 26,28,47,52,54 and in this case it is possible that the ethanolic extract of J. curcas induced elevation of the skin temperature by release autacoids like serotonin or prostaglandins, nonetheless, which should be clarified in future studies

For
quantitative variables the following statistics were applied: 1 tail ANOVA, Tukey and Pearson Correlation Coefficient tests.For qualitative variables, Fisher's statistic was applied.It was considered statistical significance for p<0.05 value and a confidence interval of 95%.It was used like informatics support Microsoft Office Excel 2013 and the Graph Pad Prism Software version 5.01.

Figure 1 :
Figure 1: Correlation between dose of J. curcas and paw skin temperature.