Sapium ellipticum (Hochst.) Pax Leaf Extract: In-vitro Antioxidant Activities and Lethal Dose (LD 50 ) Determination in Wistar Rats

Prior to this investigation, the LD 50 and antioxidant activities of Sapium ellipticum leaf extract have not been reported, hence; the focus of this study. The LD 50 of the extract was determined through three routes of administration using the method of Lorke. The Intraperitoneal (i.p) and intramuscular (i.m) LD 50 values were determined as 979.80 and 1,341.60 mg/kg body weight (BW) respectively. Oral administration of the extract (to a dose of 45,000 mg/kg BW) did not cause any negative behavioral changes in the animals, and no mortality was recorded within and after 24h of the experiment. The antioxidant properties of the extract was assessed in vitro in terms of its free radical scavenging, metal chelating and reducing power activities as well as its ability to inhibit the formation of malondialdehyde (MDA), an index of lipid peroxidation. The total phenolic content of the extract was determined as 74.23±3.12 mg GAE/g. Data obtained indicate that the extract exhibited appreciable free radical scavenging activity (IC 50 = 0.128 mgmL -1 ) and strong reducing Article power in comparison with butylated hydroxyl toluene (BHT, IC 50 = 0.118 mgmL -1 ) and ascorbic acid (IC 50 = 0.120 mgmL -1 ) used as reference antioxidants. The inhibition of linoleic acid induced-lipid peroxidation by the extract was comparable to that of BHT and greater than that elicited by ascorbic acid. In terms of metal chelating activity, ethylene diamine tetraacetic acid (EDTA) used as positive control elicited a significantly (p ≤ 0.05) higher activity than the extract at all concentrations used. Findings from this study credit Sapium ellipticum ethanol leaf extract with significant antioxidant properties. The plant material may therefore be of immense relevance in combating oxidative stress and its related ailments.


INTRODUCTION
Free radicals or reactive oxygen species (ROS) induced-oxidative stress has been widely implicated in the aetiology and progression of several disease conditions [1][2][3][4]. Normally, reactive species exist in all aerobic cells in balance with cellular antioxidants [5]. Oxidative stress occurs when the production of ROS exceeds the level the body's natural antioxidant defense mechanisms can cope with; causing damage to cell membranes and molecules such as DNA, proteins and lipids [6][7][8]. Interestingly, many plants known to possess antioxidant properties have been proposed in the prophylactic and curative treatments of different pathologies induced by oxidative stress. This is probably consequent upon their ability to scavenge or mop up free radicals amidst other protective properties such as metal chelating and reducing power activities.
The antioxidant properties of Sapium ellipticum stem bark extract has earlier been reported by Adesegun et al. [9]. Cytotoxicity screening of selected Nigerian plants used in traditional cancer treatment on HT29 (colon cancer) and MCF-7 (breast cancer) cell lines indicated that S. ellipticum leaf extract expressed the highest cytotoxic activity among other plants with anticancer potential [10]. S. ellipticum (Hochst.) pax (family Euphorbiaceae) is commonly referred to as jumping seed tree. The plant is common on the outskirts of evergreen forest and in wooded ravines. It is widely distributed in Eastern Africa and tropical Africa. In Western part of Nigeria, particularly among the Ilorin indigenes, the plant is popularly known as aloko-ạgbọ. According to Burkill [11][12][13], a number of pharmacological effects have been traditionally associated with the plant: A preparation of dried leaves of S. ellipticum is applied to wounds in Tanzania, the leaf preparation is also used for sore-eyes and abdominal swelling. In Central Africa, the decoction of the stem bark is used for scurvy and stomatitis, as purgative in Congo and for treatment of eczema. Interestingly, it is believed to be a cure for stammering in Zaire. A rootconcoction is prepared as a fomentation in East Africa for enlarged spleen in babies and is taken by adults for malaria. In Tanganyika, a leafpreparation is used to relieve pains in the head, chest, shoulders and back. Surprisingly, extremely few investigations have been conducted on the plant. The focus of this present study was to evaluate the antioxidant status of the plant's leaf extract and prescribe a safe dose range for its use through the knowledge of its lethal dose vis-a-viz different routes of administration.

Collection of Sapium ellipticum and Preparation of Leaf Extracts
Fresh S. ellipcitum leaves were harvested in the month of December 2012 from a forest in a suburb of Ibadan, Southwest of Nigeria. The taxonomical authentication was done by a botanist (Mr.Odewo, T.K) at the Lagos University Herbarium (LUH), Nigeria, where herbarium specimen with voucher number LUH 5423 was deposited. The plant material was freed of extraneous materials; air dried at room temperature and was milled into a fine powder with a milling machine. Fifty grams of the dried powdery sample was macerated in 500mL of the extracting solvent (absolute ethanol) at room temperature. The mixture was allowed to stand for 72 h and stirred intermittently to facilitate extraction. Sieving of the mixture was achieved with a muslin cloth of mesh size, 42. The resulting volume on sieving was reduced with a rotary evaporator. Final solvent elimination and drying was done using a water bath at 40°C. The crude extracts were stored in sterile screwed (airtight) bottles and aliquots were taken when required.

The Animals-collection and Management
Male and female albino rats of the Wister strain (100 to 120 g) were used for lethal dose determination. They were purchased from the animal breeding unit of Institute for Advance Medical Research and Training (IMRAT), at the University College Hospital (UCH), Ibadan. Ethical approval number (LCUEC 107) was obtained from the local institution ethical committee. All procedures for maintenance and sacrifice (care and use) of animals were carried out according to the criteria outlined by the National Academy of Science and published by the National Institute of Health [14]. The animals were handled humanely, kept in a plastic suspended cage placed in a well ventilated and hygienic rat house under suitable conditions of temperature and humidity. They were provided rat pellets and served water ad libitum and subjected to natural photoperiod of 12 h light and 12 h dark cycle. The animals were allowed two weeks of acclimatization before the commencement of the study.

Lethal Dose Determination (Acute Toxicity Test)
Lethality studies to determine the 50% lethal dose (LD 50 ) of the extract was performed according to the procedure described by Lorke [15]. It was assessed through three routes of administration {Intraperitoneal (i.p), intramuscular (i.m) and oral (p.o)}. For each of i.p and i.m determinations, forty rats (both sexes) were randomly assigned to ten (10) groups, with each group having four (4) animals. They were respectively treated with 200, 400, 600, 800, 1000, 1,200, 1,400, 1,600, 1,800 and 2000 mg/kg body weight of the extract in saline. The animals were then returned to their respective cages, allowed free access to food and drinking water. They were thereafter monitored for clinical signs, symptoms and mortality within 24h of the experiment.
In the oral LD 50 determination, three different sets of ten (10) animals (60 in number) were used. The first set of ten animals was randomized into five groups, each containing 4 rats. They were treated with 1000, 2000, 3000, 4000 and 5000 mg/kg respectively with no mortality recorded after 24 hours. In the second phase, doses of 6000, 8000, 10,000, 12,000 and 14,000 mg/kgBW were respectively administered to another set of animals. When no mortality was recorded, a third set of ten animals equally assigned to 5 groups were respectively treated with doses of 15,000, 20,000, 30,000, 40,000 and 45,000 mg/kg BW of the extract in saline. They were closely observed for negative behavioral changes and mortality within 24h of the experiment. The lethal dose of the extract through the different routes was calculated using the formula: Where D o = Maximum dose that produce 0% mortality, D 100 = Minimum dose that produce 100% mortality.

DPPH radical scavenging activity
The free radical scavenging activity of extract was measured from the bleaching of the purplecoloured methanol solution of 1,1-diphenyl-2picrylhydrazyl (DPPH) using the method of Burits and Bucar [16]. One milliliter of various concentrations (0.025-0.25 mgmL -1 ) of the extract in ethanol was added to 4 mL of 0.004% DPPH solution. The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 30 min. The absorbance was then read at against a blank at 520 nm using spectrophotometer. Ascorbic acid and BHT were used as positive controls and deionized water in place of extract in addition to other reagents was used as blank. The decrease in absorbance of the reaction mixture indicated higher free radical scavenging activity. Percentage inhibition of free radical was calculated using the formula % inhibition = (A blank -A sample /A blank ) x 100, where A blank is the absorbance of the control reaction (containing all reagents except the test extract) and A sample is the absorbance of the test material. Extract concentration providing 50% inhibition (IC 50 ) was calculated from the plot of percentage inhibition against extract concentration. Tests were carried out in triplicate.

Fe 2+ -chelating ability
The Fe 2+ -chelating ability of the extract with EDTA used as positive control was assessed using the method described by Decker and Welch [17]. The extract (1 mL, 0.1-10.0 mg mL -1 ) was diluted to 20% of the original concentration with water, mixed with FeCl 2 (0.1 mL, 2.0 mM) and after 30 min, ferrozine (0.2 mL, 5 mM) was added. The resulting mixture was shaken vigorously and left to stand for 10 min at room temperature. The absorbance of the resulting solution was measured at 562 nm. Decreased absorbance of the reaction mixture indicates higher Fe 2+ -chelating ability. The percentage of inhibition of ferrozine-Fe 2+ complex formation was calculated from the absorbance ratio to that of the blank without any sample. All determinations were carried out in triplicate.

Total phenol estimation
The total phenol content of the extract was estimated according to the method of Singleton et al. [18] using Folin-Ciocalteau reagent. The extract (100 mg mL-1, 1.0 mL) was mixed thoroughly with 5 mL Folin-Ciocalteau reagent (diluted ten-fold) and after 5 min., 4.0 mL of sodium carbonate (0.7 M) was added and the mixture was allowed to stand for 1h with intermittent shaking. The absorbance was measured at 765 nm in a spectrophotometer. Gallic acid was used as a standard. Serial dilution of 10 mg/mL of the standard was made to obtain a calibration curve. Total phenol was determined from the calibration concentration curve as gallic acid equivalent (GAE). All determinations were carried out in triplicate.

Anti-peroxidant assay
The method of Chang et al. [19] was used to measure the anti-peroxidant activity of the extract, indexed by its ability to inhibit linoleic acid emulsion induced-lipid peroxidation. The extract (0.5 mL, 1.0-8.0 mg mL -1 ) was mixed with phosphate buffer (2 mL, 0.2 M, pH 7.0) and linoleic acid emulsion (2.5 mL, 0.56% w/v, pH 7.0). The mixture was then incubated at 60°C in the dark for 12 h to accelerate oxidation. Ethanol (4.5 mL, 75%), ammonium thiocyanate solution (0.2 mL, 4 M), sample solution (0.1 mL) and ferrous chloride (0.2 mL, 20 mM in HCl) were mixed in sequence and after 3 min the absorbance for the red colour was measured at 500 nm. The level of lipid peroxidation inhibition (%) by the extract was calculated from the absorbance ratio to that of the blank without any sample. BHT and Ascorbic acid were used as standards. All determinations were carried out in triplicate.

Ferric reducing/antioxidant power assay (FRAP)
The method of Lai et al. [20] was used to measure the reducing power of the extract. One milliliter of various concentrations (0.2-2.0 mg mL -1 ) was added to 2.5 mL phosphate buffer (0.2 M, pH 6.6). This was mixed with potassium ferricyanide (2.5 mL, 1%). The mixture was incubated at 50°C for 20 min. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the mixture and centrifuged at 1000 g for 10 min. The upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl 3 (0.5 mL, 0.1%) and the absorbance was measured at 700 nm against a blank in the spectrophotometer. Ascorbic acid and BHT were used as positive controls. Increased absorbance of the reaction mixture indicated increased reducing power. All determinations were carried out in triplicate.

Statistical Analysis
Data were analyzed by SPPS version 19.0 using one way ANOVA and subjected to Fisher LSD post hoc test. Values are presented as mean ± SEM. Differences between means were accepted to be significant at p < 0.05.

Lethal Dose Determination
The LD 50 values of the investigated S. ellipticum leaf extract through i.p and i.m routes were determined as 979.80 and 1,341.60 mg/kg BW respectively. The values are respectively about and above 1000 mg/kg BW. These figures connote substantial degree of safety for the use of S. ellipticum leaf extract in terms of toxicity level assessment through these routes. Interestingly, p.o administration of the extract (to a dose of 45,000 mg/kg BW) did not cause any negative behavioral changes in the animals, and no mortality was recorded within and after 24 h of the experiment, rather; increased appetite was observed in the animals. Possibly, the plant material was poorly absorbed through this route of administration (p.o) or biotransformation of the active component of the extract into non or less toxic metabolites occurred in the gastrointestinal tract (GIT) of the animals by the action of certain modifying enzymes. Increase in food intake by the animals as observed in this study suggests the presence of a digestive or appetizing agent in SE leaf extract.

In vitro Antioxidant Assessments
Polyphenolic compounds are strongly associated with antioxidant capabilities. Compounds with high phenol content have been found to be good antioxidants [21,22]. This is probably explained by the fact that polyphenols generally have the ability to readily donate electron or hydrogen atom to highly unstable molecules with unpaired electron such as free radicals or reactive oxygen species (ROS). 1, 1-diphenyl-2-picrylhydrazyl (DPPH) model system is often employed as a source of free radicals or unpaired electrons to assess the ability of a test material to function as an antioxidant in vitro [16,19,23]. Findings from this present investigation showed that S. ellipticum leaf extract possesses a total phenolic content of 74.23±3.12 mg GAE/g and elicited appreciable free radical scavenging activity of IC 50 value (0.128 ± 0.01 mgmL -1 ) comparable to that of BHT (0.118 ± 0.00 mgmL -1 ) and ascorbic acid (0.120 ± 0.01 mgmL -1 ) used as reference antioxidants. Fig. 1 shows the DPPH radical scavenging effects of the extract, BHT and LAA. The radical scavenging effects increased with increasing concentration in each case. At all concentration studied, there was no significant difference (P≤0.05) in the scavenging activities of the tested materials. The extract amazingly showed significantly greater inhibition of linoleic acid induced-lipid per oxidation in a dose dependent manner, particularly at 5.0 and 7.5 mg/mL than BHT and LAA Fig. 2. The reducing activity on fe 3+ /ferricyanide complex exercised by the leaf extract followed the same pattern Fig. 3.
In terms of metal chelating activity, ethylene diamine tetraacetic acid (EDTA) used as positive control elicited a significantly (p≤0.05) higher activity than the extract at all concentrations used. The chelating capacity at a concentration of 0.4 mgmL -1 was 28.7% compared to 74.2% expressed by EDTA at the same concentration Fig. 4. Higher concentration (0.8 mg/mL) did not cause any significant increase in the chelating ability (31.4%) of the extract. As noted by Adesegun et al. [9] in a previous study on the stem bark extract of the same plant, metal chelation probably contribute very little to the antioxidant relevance of S. ellipticum.
Free radicals or reactive species (RS) effect damage to cell membranes and molecules such as DNA, proteins and lipids through processes involving degradation of fatty acids (per oxidation), removal of electrons (oxidation) and combining with target molecules (formation of adducts) [7,8]. This usually occurs when the endogenous antioxidants (enzymes and molecules) are overwhelmed by excess amount of oxidants or free radicals, a phenomenon describes as oxidative stress. Antioxidants when present even in small concentration but in the right proportion with RS are capable of muffling their actions. Donation of electron or hydrogen atom (reducing activity), scavenging or mopping of free radicals (acceptance of electron), breaking of free radical chains (chelating action) are some of the mechanisms by which antioxidants elicit their protective properties. Data obtained from this study credit S. ellipticum leaf extract with substantial free radical scavenging and reducing activities, as well as lipid per oxidation inhibitory effect comparable to those of BHT and L-Ascorbic acid, well known synthetic and endogenous antioxidants respectively. These chemicals have proven abilities to protect the human body against free radicals and oxidative stress associated injuries. In this regard, findings from this study afford S. ellipticum leaf extract significant antioxidant usefulness which may be adduced to arrays of secondary metabolites such as flavonoids and other polyphenols present in the plant. The plant material may therefore be of immense relevance in combating oxidative stress relatedderangements. Moreover, comparing the findings of this investigation with the study of Adesegun et al. [9] on the stem bark extract of S. ellipticum, suggests that the leaf extract of the plant possess relatively better antioxidant values.

CONCLUSION
The investigated extract performed creditably well against renown synthetic and endogenous antioxidants (BHT & L-Ascorbic acid) in most of the In vitro antioxidant evaluation model systems employed. Collectively, the results of this study indicate that ethanol leaf extract of S. ellipticum is a good source of natural antioxidants and extremely safe for oral consumption. These observations affirm the use of the plant in alternative or traditional medicine across Africa. On the basis of available scientific documentations, this report is apparently the first on the antioxidant properties and LD 50 status of the leaf extract of S. ellipticum. In vivo assessment of the extract is currently ongoing in our laboratory. This is necessary because some plant materials with impressive In vitro antioxidant relevance fail to replicate the same In vivo.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All authors hereby declare that "Principles of laboratory animal care" (NIH publication 2010) were followed, as well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee.