PHYTOCHEMICAL COMPOSITION, TOXICITY, ANTIOXIDANT AND LACTOGENIC ACTIVITIES OF EUPHORBIA HIRTA (L.).

was to investigate its on studies achieved to identify compounds, to assess and activity of the Oral acute and

Toxicity Studies:-Acute toxicity study The acute toxicity test was conducted according to OECD guideline (OECD, 2001). Adult nulliparous and no pregnant Wistar rats weighing between 146 and 184 g, aged ten (10) weeks were used. A single oral dose of 2000 mg/kg body weight (b.w.) of aqueous extract of E. hirta (AEEH) was administered to three rats. After treatment, the animals were observed individually during one (01) hour and daily fourteen (14) consecutive days. The mortality and the signs of toxicity such as the changes of skin, fur, and eyes were recorded.

Sub-acute toxicity study
The sub-acute toxicity test was carried out according to OECD guidelines (OECD, 2008) with some modifications. Twenty-four nulliparous and no pregnant Wistar rats weighing between 106 and 131 g, aged eight (08) weeks were used. The rats were randomized into four groups of six animals each. The group I served as control and received distilled water. The groups II, III, IV were treated with the aqueous extract of E. hirta (AEEH) at the doses of 50, 100, 200 mg/kg b.w., respectively. The extract was administered orally to all animals daily during twenty-eight (28) days. During the period of treatment all animals were weighed weekly. Toxicity signs, mortality and body weight changes were recorded. At the end of the treatment, the rats were fasted overnight. Twenty four (24) hours after, the animals were anesthetized with a mixture of ketamine and xylazine (1/0.7) by intraperitoneal injection and blood samples were obtained by cardiac puncture for haematological and biochemical analysis.
For biochemical analysis, blood samples were collected into the dry vacutainer tubes and centrifuged at 3500 rpm for 5 min. The sera obtained were used for the test. The methodology of spinreact were used for spectrophotometric determination of the different biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea, cholesterol and total proteins. After blood collection, internal organs such as liver, lung, spleen, heart, kidney, uterus, ovary and adrenal gland were removed and weighed.
Antioxidant Activity:-Diphenylpicrylhydrazyl (DPPH) radical assay Diphenylpicrylhydrazyl (DPPH) is a free radical with violet color. When it is mixed with a substance that can donate a hydrogen atom or an electron, it was reduced to a pale yellow color due to the acceptance of hydrogen or an electron.
The stable free radical 2, 2 diphenyl-1-picrylhydrazyl was used for in vitro determination of free radical scavenging activity of aqueous and hydroethanolic extracts of E. hirta. In a 96 microwells plate, 100µL of different concentrations (0.05-40µg/mL) of each extract was added to 200µL of DPPH solution (2mg/mL). The mixture was incubated in dark and at room temperature to complete the reaction for 15 min. Quercetin and ascorbic acid were used as reference compounds. The free radicals scavenging activity of extracts was evaluated using spectrophotometer at 517 nm. The pourcentage of discoloration was calculated by following formula: DPPH radical scavenging activity (%) = [(AC 517 -AE 517 )/AC 517 ]*100 AC 517 is the absorbance of a DPPH solution without extract AE 517 is the absorbance of the tested plant extract with DPPH IC 50 was determined for 50 % inhibition concentration using a graph. All tests were performed in triplicate.

Ferric reducing power (FRAP) assay
The reducing power of extracts was determined according to Hinneburg et al. (2006).

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This reduction concerns extract ability to reduce ferric ions (Fe 3+ ) to ferrous ions (Fe 2+ ). For this evaluation, 0.5 mL of extract of different concentrations (0.1-1 mg/mL) were mixed with 1.25 mL of phosphate buffer (0.2 M, pH 6.6) and 1.25 mL of 1% aqueous potassium hexacyanoferrate [K 3 F e (CN) 6 ]. The mixture was incubated at 50°C for 30 min. In the mixture, 1.25 mL of trichloroacetic acid (10%) was added to stop the reaction and the total mixture was centrifuged at 3000 rpm for 10 min. After, 125 µL of upper layer was mixed with 125 µL of distilled water and 25 µL of F e Cl 3 (0.1%) fresh solution in 96 microwells plate. Ascorbic acid was used to produce a calibration curve by reading absorbences at 700 nm (y=0.0749x + 0.1569, r 2 =0.992). Quercetin and gallic acid were used as positive controls. All tests were performed in triplicate.

Lipid peroxidation inhibition
Lipid peroxidation assay was carried out using the method of Ohkawa et al. (1979) adapted by Guenné et al. (2012). The lipid peroxidation was induced by FeSO 4 , 7H 2 O and H 2 O 2 in the normal rat liver and brain homogenate. Zero points two (0.2) millilitres of aqueous and hydroethanolic extracts of E. hirta (1,5 mg/mL) were mixed with 1 mL of rat liver or brain homogenate in 1 % of Tris-HCl buffer (50 mM; pH 7, 4) then, 50 µL FeSO 4 (0.5 mM) and 50 µL of H 2 O 2 (0.5 mM) were added. The mixture was incubated at 37 °C for 60 min. After incubation, 1 mL of trichloroacetic acid (15%) and 1 mL of thiobarbituric acid (0.67%) were added to the mixture and heated in boiling water for 15 min. The lipid peroxidation inhibition was measured at 532 nm using a spectrophotometer. Ascorbic acid was used as standard.

Effect of aqueous extract of E. hirta on milk production
The effect of E. hirta on milk production of rats was evaluated as described by Lompo- Ouédraogo et al. (2004). Fifteen (15) nulliparous female rats were used. The animals were mated with male in plastic cages in the standard conditions of breeding until pregnancy. Two (02) weeks after mating, all the pregnant rats were housed individually in plastic cages. Following birth, the litters were adjusted to nine pups per dam. At the beginning of lactation, the animals weighed 241.22±9.64 g and were divided into three experimental groups of five lactating dams each. The group I received NaCl (0.9%) and served as blank control, the group II treated with metoclopramide (5mg/kg b.w.) served as reference control and group III was given aqueous extract of E. hirta at the dose of 200 mg/kg b.w. The administration was done orally by gavage. All the animals were treated daily, starting from the evening (17 h 00) of day 3 of lactation to day 17. Milk production was measured from day 4 to day 17 of lactation. Milk yield, weight of pups, body weight gain of pups were determined each day. Milk production was estimated 18 h and 23 h after treatment. Each day during the period of experimentation, the pups were weighed at 7 h 00 (w1) and isolated from their mother for 4 h. At 11 h 00, the pups were weighed (w2) and returned to their mother and allowed to feed for 1 h. At 12 h 00 they were weighed again (w3). Milk yield 18 h after treatment was estimated as w3-w2 with a correction for weight loss due to metabolic processes (urination, defection and respiration) in the pup as (w2-w1)/4. For the estimation of milk yield 23 h after the treatment, the pups were subsequently isolated at 12 h 00 for 4 h. At 16 h 00 they were weighed (w4) and reunited with their mother for 1 h of feeding. Finally the pups were weighed (w5) again at 17 h 00 and left with their mother to the night. Milk yield 23 h after treatment was calculated as w5-w4 with a correction for weight loss due to metabolic processes in the pups as [(w2-w1) + (w4-w3)]/8.

Statistical analysis
Data are expressed as mean ± standard error of mean (Mean ±S.E.M.). The data obtained were analyzed using Graph Pad Prism version 5.03. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test were used to determine differences between the groups. P-value <0.05 was considered as statistically significant.

Phytochemical screening
The qualitative phytochemical screening of aqueous and hydroethanolic extracts of whole plant of E. hirta revealed the presence of compounds which are recorded in the table I.

Sub-acute toxicity
Oral administration of aqueous extract of E. hirta at the doses of 50, 100 and 200 mg/kg b.w. did not cause any mortality or morbidity in rat during 28 days of treatment. In addition, no signs of toxicity were observed in all the extract treated groups compared to control group.
During the period of treatment, the increase of body weight was noted in animals treated with the extract similarly to control group (p > 0.05) (Fig 1).
The relative organ weight of liver, kidney, lung, heart, spleen, uterus, ovary and adrenal gland are presented in Fig 2, 3 and 4. These results show that there was no significant difference (p > 0.05) in organ weight of extract treated groups compared to control group. Nevertheless, a slight dose-dependent decrease of relative weight of uterus was observed.

Effect of AEEH on haematological and biochemical parameters
There were no significant changes (p > 0.05) between control and the treated groups at the doses of 50 and 100 mg/kg. A significant decrease (p < 0.05) of red blood cells level at the dose of 200 mg/kg was noted compared to control (Table II). The results of biochemical analysis showed that at the doses of 100 and 200mg/kg b.w., no significant difference were observed compared to control. A significant decrease (p < 0.01) of creatinine level was observed at the dose of 50mg/kg b.w. compared to control (table III).  (table IV). Indeed, scavenging activity of free radicals of hydroethanolic extract of E. hirta is seven-fold lower than that of ascorbic acid whereas the aqueous was seventy four-fold less potent than ascorbic acid.

Ferric reducing power determination (FRAP)
The hydroethanolic extract exhibited the high reducing activity than the aqueous extract. Both extracts showed less reducing activity than the quercetin and gallic acid (tableV).

Lipid peroxidation inhibition in rat brain and liver
All extracts had the property to reduce lipid peroxidation. Nevertheless, the extracts showed a more potent capacity to decrease lipid peroxidation in liver homogenate than in brain (table VI).

Effect of aqueous extract of E. hirta on milk production in rats
The effect of E. hirta on milk production are presented in Fig 5. The extract induced a non-significant increase (p > 0.05) of milk production in rats comparatively to control groups (NaCl and metoclopramide).
The mean milk production was 0.84 ± 0.06; 0.95 ± 0.06 and 1.04 ± 0.07 g/pup/day respectively for the blank, reference controls and E. hirta extract group (Fig 6). The extract did not cause an increase of milk production 18 h after the treatment compared to the control. Whereas, 23h after the treatment a non-significant (p > 0.05) increase of milk production was recorded.   8). There were no significant (p > 0.05) changes in body weight of pups between all groups but a slight body weight gain was observed for reference and extract treated groups compared to blank control.

Toxicity
The acute and sub-acute toxicities of aqueous extract of E. hirta were evaluated on Wistar rats in order to detect the harmful effects on human health. Indeed, the single dose of 2000 mg/kg b.w. did not cause any death and no signs of toxicity.
The result of acute toxicity showed that the LD 50 was higher than 2000 mg/kg b.w. According to the scale of Hodge and sterner (1943), the extract is classified as a slight toxic drugs. Our results are similar to those of Ping et al. (2013) who showed that the extract of E. hirta does not cause an acute toxicity effect at the dose of 5000 mg/kg b.w. and then the DL 50 is more than 5000 mg/kg b.w.
The effect of the extract after a long-term treatment (28 days) was evaluated. The oral administration of aqueous extract of E. hirta at the doses of 50, 100, 200 mg/kg b.w. did not show any morbidity and mortality in rats. In addition no signs of toxicity were recorded. Moreover, the increase of body weight or body weight gain were observed for all animals (treated and control groups). Our results are in agreement with the findings of other authors who also observed the body weight gain in rats treated with E. hirta (Ping et al. 2013). According to these authors this weight gain is due to the food and water intake suggesting that the extract did not induce the loss of appetite during the period of the experiment. Futhermore, the extract did not adversely interfere with the nutritional benefits maintaining the appetite and led to the body weight gain. Subsequently, there was no significant changes in the relative weight of the internal organs of extract treated animals compared to control except the uterus where slight dose dependent decrease of relative weight was observed. These results are similar to those of Ping et al. (2013) who found no significant changes in relative weight of internal organs.
The haematological and biochemical analysis were carried out to determine the effect of the extract on the internal organs functions. For haematological parameters, no significant changes were noted at the dose of 50 and 100 mg/kg b.w. while at the dose of 200 mg/kg b.w. a significant decrease of red blood cells was observed. This decrease of red blood cells is an indicator of anemia which may be due to bone marrow dysfunction or suppression (Mohajeri et al., 2007). This result suggests that the extract at this dose and beyond may cause anemia. According to Kluwe (1981) and

Effect of aqueous extract of E. hirta on milk production in rat
Milk production is a complex physiological process involving physical and emotional factors and the interaction of several hormones especially prolactin (PRL) and growth hormone (GH). These factors modulate the milk synthesis in lactating mothers and their disturbance can cause some problems of breastfeeding such as the agalactia or the 333 hypogalactia (Baig and Bhagwat, 2009;Zuppa et al., 2010). Some galactogogues are used to initiate, increase and maintain breast milk production. Among these drugs, metoclopramide is widely used, it antagonizes the release of dopamine in the central nervous system and increase the prolactin levels. However, these synthetic drugs present some side effects in mother and infants (Zuppa et al., 2010).
Milk production was slightly higher in aqueous extract treated group than blank control group. In addition, increase of milk yield was marked 23 h after the administration. Milk production depends on the number of mammary epithelial cells in the gland and their secretory activity (Capuco et al. 2003;Boutinaud et al., 2004). The increase of number or activity of mammary secretory cells are regulated by the endocrine system and the physiological state (Akers, 1985;Boutinaud et al., 2004). In our study the increase of milk production in lactating rats may be explained by the proliferation of mammary epithelial cells and their activity. This proliferation of mammary epithelial cells is due to the action of extract on mammary glands and some hormones involving in lactation like PRL (Lompo-Ouedraogo et al., 2004). In addition, Flint and Gardner, 1994 showed that PRL and GH are the major regulators of milk production. Indeed, PRL maintains milk synthesis by inhibiting epithelial cells loss, maintaining cellular differentiation and have effect on biochemical processes involved in the synthesis of the major compounds of milk. Whereas GH acts directly on mammary gland to stimulate milk synthesis. Our results are similar to those of Sawadogo (1987) who showed that the extract of E. hirta boosts and maintains the lactation in rabbits. Thereafter he found that the extract is able to induce casein synthesis in rat and stimulate prolactin secretion in ewes.
There is a linear increase of body weight of suckling pups during the period of experimentation. The variation of body weight gain of pups was observed for all groups from day 4 to day 17 of lactation. The body weight gain of pups of extract treated lactating rats and treated metoclopramide rats was slightly higher than that of the blank control group. Thereby, the pup's weight gain per day was assumed to be approximately proportional to the production of milk during the lactation. Thus, it was used as an indicator of milk production in rats (Azizah et al., 2012).

Conclusion:-
E. hirta contains several phytochemical compounds including steroids and triterpenoids, tannins, flavonoids, coumarins, reducing sugars which may explain its utilization in traditional medicine. Toxicity studies in rats revealed that the aqueous extract of the plant is practically non-toxic and safe for use when it administered orally. Moreover the plant exhibited a strong antioxidant activity. The aqueous extract stimulated milk production in rats, this result confirms the traditional use of the plant in the lactation insufficiency. Subsequent studies must be carried out to evaluate the effects of the plant on mouse mammary cells lines.