Assessment of Acute and Subacute Toxicity of the Total Dichloromethane-Ethanol Extract of Morinda morindoides ( Baker )

Context: ETDE shown good antihypertensive and antioxidant activities in rats made hypertensive. This present study aims to assess its toxicity. Aims: This study was designed to study the toxicity of dichloromethaneethanol extract of Morinda morindoides. Settings and Design: Toxicological activity in vivo. Methods and Material: Alkaloids were characterized from reagents of Bouchardat, flavonoids by reacting the cyanidrine, tannins by the reagent Stiasny, polyphenols by reacting ferric chloride, quinones by the reagent Bornstraëgen, sterols and polyterpenes by the reaction of Libermann and saponins by observing the foam after agitation of the extract. Acute and subacute toxicity were studied using respectively 423 and 407 OECD guidelines for testing of chemicals. Statistical analysis used: The graphical representation of the data was performed using the Graph Pad Prism 5.0. The mean value is accompanied by the standard error of the mean (Mean ± SEM). The difference between the two values is considered significant when P<0.001. Statistical analysis of results was performed using analysis of variance (ANOVA). Results: The phytochemical screening showed the presence in the ETDE of polyphenols, alkaloids, flavonoids, sterols and polyterpenes. The toxicological study shows that ETDE has a LD50 between 2000 and 5000 mg/kg bw therefore classified in the hazard category 5. The administration of ETDE at repeated dose for 28 days did not significantly affect the weight gain, hematological and biochemical parameters of rats. Conclusion: ETDE toxicity is relatively low with LD50 between 2000 and 5000 mg/kg bw. It does not cause damage to the heart, liver and kidney. ETDE can be used without risk of intoxication.


INTRODUCTION
The use of plants especially for therapeutic purposes is growing around the world, mainly in countries like Côte d'Ivoire, which is known for its richness in plants.Several factors explain the greater use often irrational, anarchic and uncontrolled.Therefore, the share of poisoning related to the use of plants is not negligible.Plants, because they are natural, are mistakenly considered non-hazardous and the population uses there in very varied and numerous contexts.Natural products are of great importance in the development of new pharmaceutical products or plant protection products, 1,2 but many studies have shown that these products contain potential toxic effects. 3,4It is therefore essential, before use as a therapeutic drug form said plant extract in the traditional environment, to conduct its toxicological study to assess its safety for use without risk of poisoning.Morinda morindoides is a woody plant used by traditional practioner in Ivory Coast for the treatment of various illnesses.The work of Boga et al 5 showed that the total dichloromethane-ethanol extract of this plant has a very good antioxidant activity in rats made hypertensive by adrenaline.Our previous study had shown significant antihypertensive activity of the dichloromethane-ethanol extract of M. morindoides in rabbit. 6The present study aims to study the acute and subacute toxicity of the dichloromethane-ethanol extract of Morinda morindoides.

SUBJECTS
White albino male and female rats, Wistar strain aged 2 to 3 months are used for the study of oxidative stress of the extract.The rats were kept in plastic cages with stainless steel covers containing a bed of wood chips renewed every two days.The animals are fed regularly with rat pellets and received standard tap water as the drinking water in stainless steel cylinders.The plant material is constituted by leaves of Morinda morindoides (Baker) Milne-Redh., collected to Gbahiri in the municipality of Lakota, South-western city of Côte d'Ivoire, between January and February 2012.These leaves were dried and powdered for the preparation of the extract.

Preparation of the extract
The total dichloromethane-ethanol extract of Morinda morindoides (Baker) Milne-Redh.(ETDE) was prepared according to the method described by Zirihi et al. 7 According to this method, one hundred and fifty grams (150 g) of powder of Morinda morindoides (Baker) Milne-Redh.were dissolved in eight hundred milliliters (800 mL) of a mixture of dichloromethane-ethanol solvent (1/3 (V/V)).The mixture is homogenized for 24 hours at laboratory temperature (25-30°C) using a magnetic stirrer IKAMAG RCT.The homogenate is filtered twice on cotton wool and once on Whatman 3 mm paper filter.The recovered filtrate is evaporated using a BÜCHI rotary evaporator.We got a greenish paste that forms the total dichloromethane-ethanol extract of Morinda morindoides (Baker) Milne-redh.(ETDE).Different doses for testing were prepared from this extract.

Quantitative Analysis of the ETDE
The different groups of ETDE were characterized using the techniques described in the work of Békro et al. 8

Search of total polyphenols
The total polyphenols were characterized by the reaction with ferric chloride.These compounds form with ferric chloride (FeCl 3 ), a colorful blue-black or green precipitate.The assessment of this coloration is made with respect to a control test with a phenolic compound reference.At 2 mL of plant extract are added a few drops of alcoholic solution of ferric chloride 2%.The appearance of a blue-black color more or less dark green indicates the presence of polyphenol derivatives.

Search of alkaloids
Alkaloids were characterized from Buchardat reagent (reagent iodoiodide) and Dragendorff (reagent potassium iodo-bismuthate).Six (6) mL of ETDE were evaporated to dryness and the residue taken up in 6 ml of alcohol at 60°C.The addition of 2 drops of Dragendorff reagent on the alcoholic solution caused an orange precipitate.The appearance of a reddish brown precipitate following the addition of 2 drops of reagent Buchardat indicates a positive reaction.

Search of flavonoids
Flavonoids were detected by reaction with cyanidin.Two (2) mL of ETDE were evaporated to dryness and the residue taken up in 5 mL of hydrochloric alcohol diluted 2 times.2-3 magnesium chips are added which caused a release of heat and a purplish color.The addition of 3 drops of isoamyl alcohol has intensified this coloration, which confirmed the presence of flavonoids.

Search of sterols and polyterpenes
Sterols and polyterpenes have been characterized by the reaction of Libermann.Five (5) mL of ETDE were evaporated on a sand bath.The residue obtained was dissolved in hot 1 acetic mL d'anhydride then 0.5 mL of concentrated sulfuric acid were added.There was appearing at the interface, a purple purplish ring, which turned blue to green.This indicated a positive reaction.

Search of tannins
Catechin tannins were sought from Stiasny reagent.Five (5) mL of ETDE were evaporated to dryness.The residue obtained are added 15 ml of Stiasny reagent and the mixture was kept in a water bath at 80°C for 30 minutes.The observation of a precipitate in large flakes characterizes catechin tannins.For gallic tannins, the mixture is filtered and the collected filtrate is saturated with sodium acetate.The appearance of a deep blue-black color, after adding 3 drops of FeCl 3 sign the presence of gallic tannins.

Search of quinones
Quinone substances have been characterized from Bornstraëgen reagent.Two (2) mL of ETDE were evaporated to dryness.The residue obtained was triturated in 5 ml of hydrochloric acid 1/5.The triturate is poured into a test tube and heated in a water bath for 30 minutes.After cooling, it is extracted with 20 mL of chloroform.The appearance of a red or violet color after addition of 0.5 ml of dilute ammonia twice to the chloroform solution, is the sign of the presence of quinones.

Search of saponins
Research saponins, 10 mL of ETDE were poured into a test tube, the tube is shaken for 15 seconds and allowed to stand for 15 minutes.Persistent foam height greater than 1 cm indicates the presence of saponins.

Assessment of acute toxicity of ETDE
The acute toxicity was investigated using the OECD Guideline 423 9 for testing of chemicals.This method is a sequential process using three animals of one sex by step.Depending on the mortality and/or moribund state of animals, two to four steps are needed to assess the average acute toxicity of the test substance.Aged female rats 3 to 4 months and weighing between 136.56 and 196.86 grams on average were used for experimentation.We did not have information on the toxicity of ETDE so we started the test with a dose of 300 mg/kg bw.Three rats deprived of food during the night but no water, are weighed and then were given orally 1 ml of ETDE at a dose of 300 mg/kg bw as a single dose using a cannula intubation.After administration of ETDE, the three are again fasted for three hours before giving them food.They are observed individually the first thirty minutes, periodically during the first 24 hours after treatment.They were then observed daily for up to 14 days.24 hours after administration of the ETDE at a dose of 300 mg/kg bw, no mortality and no moribund were observed, we have gone to the dose of 2000 mg/kg bw.After this dose, there was observed no mortality and no moribund.We then moved at a dose of 5000 mg /kg bw.Then three rats are treated in the same way.

Evaluation of subacute toxicity
Guideline 407 OECD 10 for testing of chemicals, with some modifications, was used for this study.The test substance is administered daily oral (gavage) at different dose levels of several batches of animals at the rate of one dose level per group, for a period of 28 days.20 rats aged 2 to 3 months and weighing between 158.25 and 257.57grams on average were used.The rats were divided into 5 groups of 4 rats each according to their weight.Rats in the control group (group 1) received individually by gavage daily 1 ml of distilled water for 28 days.The four lots (Lots 2, 3, 4 and 5) received by gavage 50 respectively; 100; 200 and 500 mg/kg bw ETDE daily for 28 days.The volume of extract administered daily in a single dose was 2 ml per 100 g body weight.The experiment was conducted in accordance with the principles of good laboratory practice.During the 28 days of treatment, the animals were observed daily for clinical signs and symptoms of toxicity and death, before, immediately and 3 hours after administration of the extract.The 29 th day or the day after the last day of treatment, blood is taken from rat's tail docking for hematological and biochemical analyzes.Rats were weighed every three to four days to determine the effect of the extract on the weight gain of animals.

Assays of haematological parameters
The hematological analysis was carried out using an automatic hematology analyzer (Sysmex KX-21).The parameters such as: the counting of Red blood cells (RBC), the number of White blood cells (WBC), Hemoglobin (Hb), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), platelet count and the percentage of cells were determined.

Assays of biochemical parameters
The blood contained in the dry tubes was centrifuged using a centrifuge at 3000 rpm/min for 5 minutes.The resulting serum was removed and stored at -20° for analysis of serum parameters of living kidney, liver and heart with the analyzer COBAS integral 400 plus.The protocol for each assay was preset and then incorporated into the device during the assays.We assayed Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Blood glucose, Blood urea, Creatinine, Creatine kinase (CK), Total protein (TP), Total cholesterol (Chol-T), HDL cholesterol (HDL-C), LDL-cholesterol (LDL-c) and the blood electrolytes: K + , Ca 2+ , Mg 2+ and Na + .

Chemical composition of ETDE
The phytochemical screening of ETDE revealed the presence of large chemical groups such as alkaloids, flavonoids, polyphenols, sterols and polyterpenes.It is clear from these results that the ETDE contains very high proportion of polyphenols, flavonoids and alkaloids but does not contain saponins, tannins and quinones (Table 1).

Acute toxicity of ETDE
After administration of ETDE at doses ranging from 300 to 2000 mg/kg bw to rats, no signs of toxicity were observed during the 14 days of observation.As against the dose of 5000 mg/kg bw resulted dyspnea, anorexia, bleeding and death of animals.

Subacute toxicity of ETDE Effect of ETDE on body weight parameters of rats
The results in Table 2 show that the control group had a daily weight gain of 0.74 g (percentage of daily weight gain of 0.26%) and an average daily weight gain of 0.27 g (percentage of average daily weight gain of 0.11%).The oral administration of ETDE at doses of 50, 100, 200 and 500 mg/ kg bw did not significantly affect these parameters.And at doses of 50, 100, 200 and 500 mg / kg bw, the rats had respective daily weight gains of 0.85; 0.79; 0.64 and 0.79 g (percentage of daily weight gain of 0.27; 0.26; 0.22 and 0.21%).As for average daily weight gains, they varied from 0.30 respectively; 0.27; 0.26 and 0.28 g (percentage of average daily weight gain of 0.12; 0.11; 0.12 and 0.12%).It is clear from these results that the ETDE had no significant effect on weight gain in rats.During the first 6 days of feeding, the weight of rats decreased for all other doses except 500 mg/kg bw, the weight decline continued until the ninth day.After those days, growth resumed and developed normally from the control group until the last day of treatment (Figure 1).

Effect of ETDE on hematological parameters in rats
Hematology rats were measured after 28 days of treatment with ETDE.Rats received the ETDE by gavage at doses of 50, 100, 200, 500 mg/kg bw for lots 2, 3, 4, 5 and tap water for the control group.Blood is then collected in EDTA tubes by amputation of tail and analyzed using an automatic hematology analyzer (Sysmex KX-21).The effect of ETDE on hematological parameters in rats treated is presented in Table 3.The results show that all hematologic parameters have not evolved significantly compared to the control group.At doses used in this experiment, the ETDE caused no significant change in blood parameters that are the White blood cells (WBC), Red blood cells (RBC), Hemoglobin (Hb), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean cor-  puscular hemoglobin concentration (MCHC), Platelets (PLT) and Lymphocytes (Lym) compared to the control group.The values of GR (6.647 ± 0.94 x 10 6 /µL), Hb (11 ± 1.77 g/dL), hematocrit (36.2 ± 6.36%) are in the range of usual values.We note that the percentage of lymphocytes nearly doubled, but this increase was not significant effect.This is also true for the platelets.

Effect of ETDE on biochemical parameters of rats
Table 4 shows the effect of ETDE on biochemical parameters in rats.The results show that of the 16 biochemical parameters measured during the treatment period (28 days), no parameters have changed significantly.At doses of 50, 100, 200, 500 mg/kg bw, ETDE resulted in no significant change settings.

DISCUSSION
The results of the phytochemical study of the extract of ETDE show the presence of alkaloids, flavonoids, polyphenols, sterols and polyterpenes.These results disagree with those of the methanol extract of Morinda morindoides witch contained saponins but not alkaloids 11 and those of various extracts (aqueous, ethanol, ethyl acetate and hexane extracts) of Morinda morindoides. 12fter administration of ETDE at doses ranging from 300 to 2000 mg/kg bw to rats, no signs of toxicity were observed during the 14 days of observation.As against the dose of 5000 mg/kg bw resulted dyspnea, anorexia, bleeding and death of animals.According to OECD Guideline 423 line for testing chemicals, ETDE has lethal dose 50 (LD 50 ) between 2000 and Data are expressed as mean ± SEM (n=4).; ns (not significant); ALT: Aspartate aminotransferase (U/l); ALT: Alanine aminotransferase (U/l); Urea (mg/l); Creatinine (mg/l), LDH: Lactate dehydrogenase (U/l); CK: Creatine kinase (U/l); PAL: Alkaline Phosphatase (U/L); Total proteins (g/l); Cholesterol (g/l), Triglycerides (g/l); HDL: High-density lipoprotein; LDL: Low-density lipoprotein (g/l).5000 mg/kg bw.In principle, this process is not intended to determine a value of the precise LD 50 , but serves as a suggestion for the classification of the crude extract based on the prediction of the dose at which animals have to survive. 13The ETDE can be classified in the hazard category 5, acute toxicity is relatively low but may, under certain conditions, be dangerous for vulnerable populations.The administration of ETDE at doses of 50, 100, 200 and 500 mg/kg bw had no significant effect (p<0.05) on weight parameters of animals compared to control group, which means that ETDE has a negligible effect on the growth of animals.In general; body weight gain and mouse internal organs changes reflect the toxicity after exposure to toxic substances. 14ody weight changes are indicators of adverse effects of chemical drugs and products, and it will be important if the loss of body weight is more than 10% from the initial weight. 15,16However, it is reported that increases in body weights of the animals are more closely related to the accumulation of fat rather than toxic effects of drugs or chemicals 17 The reductions in body weights observed the first days of treatment may be associated with normal physiological responses of adaptation of animals to the extract 18 but also to stress caused by treatment. 19reatment of rats with ETDE at repeated doses for 28 days, caused no significant change (p<0.001) in hematological and biochemical parameters in rats treated compared to the control group.Red blood cells (RBC), White blood cells (WBC), Platelets (PLT), Lymphocytes (LYM), Hemoglobin (Hb) and other hepatocytes are the most sensitive target of toxic compounds and is an important index of pathological and physiological state of human and animal. 20In our study, there were no significant changes in these parameters.Or a modification of these parameters provides information on the human toxicity when these data are derived from studies in animals. 21Changing these parameters also determine the safety of a substance subject to a toxicological study.RBC and WBC rates have not significantly different compared to the control group.Platelets and lymphocytes have seen their value almost doubled; however, these values are within the ranges of normal values. 22The ETDE therefore did not affect the immune system; lymphocytes are the main effector cells of the immune system. 23ur results show that the activities of enzymes such as the ALT, AST and ALP were not disturbed by ETDE.These enzymes are liver markers whose activities increase in liver toxicity. 24,25Their concentration in serum informs about a hepatocyte injury. 26LT is an enzyme specific to the liver in dogs, rats, rabbits, cats and primates. 27It can provide a quantitative assessment of the degree of damage to the liver. 28Our results show that ETDE caused no damage to the liver as shown Sucheta et al. 29 Urea and creatinine are markers of renal function 25 have not seen their rates varied by ETDE.No significant change in the CK and LDH activities was observed.These enzymes are markers of cardiac function. 30Our results show that ETDE, at the doses used, did not provoked significantly change (p<0.001) of serum biochemical values of these parameters compared with rats in the control group, suggesting that the ETDE did not affect kidney, or heart, or the liver.Blood sugar has not changed significantly during treatment, suggesting that ETDE did not affect the blood sugar control system.ETDE was also not significantly changed (p<0.001),serum values of HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), total cholesterol and triglycerides.However, a slight decrease in LDL-c rates and a slight increase in HDL-c rates are observed compared to the control group.The ETDE could therefore prevent cardiovascular complications.Blood electrolytes, such as calcium (Ca 2+ ), potassium (K + ), magnesium (Mg 2+ ) and sodium (Na + ), no significant variation (p<0.001) were noted with respect to the control group.That suggests that ETDE did not influence the hemodynamic balance.It is clear from our study that at doses used, ETDE is not toxic and could be used without causing damage to the body.Our results are consistent with those of Balogun and Akinloye 11 which showed that the administration of the methanol extract of Morinda morindoides is not harmful to rats at the same doses.The study of subacute toxicity showed that ETDE preserves the integrity of vital organs.

CONCLUSION
ETDE is an extract whose toxicity is relatively low with LD 50 between 2000 and 5000 mg/kg bw.This extract does not cause damage to the toxic target organs such as the heart, liver and kidney.The ETDE can be used without risk of intoxication.

Figure 1 :
Figure 1: Effect of EDTE on the weight of rats in function with the time

Table 2 : Effect of ETDE on change in body weight parameters of rats
Data are expressed in mean ± SEM; (n=4); ns, p<0.05: no significant difference compared with rats of control group.