*Corresponding author: Sahar, Bastawy
1 THE POSSIBLE PROPHYLACTIC EFFECT OF EGYPTIAN MORINGA OLEIFERA SEED OIL AS ANTI-ULCER ACTIVITY AND ANTI-INFLAMMATORY IN RATS. Sahar, B. A. 1 and Rania, A.H. Abd El-Aal 1 1 Biochemistry Department,
1 Developmental Pharmacology Department, National Organization for Drug Control and Research (NODCAR), Giza 12553, Egypt Received 11 October 2015; Accepted 14 November 2015 ABSTRACT Moringa oleifera Lam. is the multipurpose tree (therapeutic potential and nutritive value). In this work, authors focused on importance of Moringa oleifera seed oil (MSO) as antioxidant, anti-inflammatory and antiulcer. The search describes the lethal dose of 50 % (LD50), some Physico-chemical, antioxidant, antiulcer and anti-inflammatory of MSO.
The results illustrated that the LD50 of MSO is more than 62.5 g/kg. MSO treated animals (250 and 500 mg/kg) a significantly decreased in the gastric juice, free acidity(FA) and total acidity (TA), also occurs improvement in pH of gastric juice(PH). There was a significant anti-ulcer activity, in ethanol-induced gastric ulcer of the MSO treated groups compared to ranitidine (R)( 197 mg/kg )as standard. Also, MSO (20 mg/kg, 72%) inhibited the carrageenan-induced inflammation in the hind paw of rats compared with ibuprofen (20 mg/kg, 33%) and mefenamic acid (100 mg/kg, 39%) as standard. Keywords: lethal dose of 50 %, ibuprofen, Moringa oleifera Lam., pylorus ligation, antiulcer activity, antiinflammatory. INTRODUCTION Moringa species (Moringaceae) are one of the most useful trees in the tropics and subtropics of Asia and Africa. These species are the most widely cultivated and utilized by the ancient Egyptians, Romans and Greeks. Traditionally, Moringa flower, fruits and roots are edible and consumed as the vegetable. It used in folk Medicine for the treatment of many diseases such as rheumatic and anticular pain, abdominal tumors, hysteria, scurvy, paralytic attacks, helminthic bladder, prostate troubles, sores and skin infections .
Moringa oleifera has been reported as analgesic and antiinflammation  and hence the analgesic and antiinflammatory effects may be attributed due to flavonoids, steroids and tannins by its extract. However, its pharmacological actions and mechanisms have not been precisely documented in spite of its increasing usage recently. Moringa oleifera seed can be used in a variety of ways including as medicinal and herbal remedies, nutritional supplements, and for industrial and agricultural purposes. Its seeds contain 19 to 47 % oil , similar as olive oil and are rich in palmitic, stearic, behenic and oleic acids .
This oil contains about 75% oleic acid, a monounsaturated fatty acid that is less vulnerable to oxidative stress than polyunsaturated fats. The protective agents of oleic acid in reducing cardiovascular disease, breast and skin cancer levels may be attributed to its ability to reduce the inflammation .
It has been used for the treatment of inflammation, infectious diseases and gastrointestinal .
Moringa seed oil content and its properties show a wide variation depending mainly on the species and environmental conditions .
Inflammation is defined as the local response of living mammalian tissues to injury due to any agent. The body defense of inflammation reaction occurs by eliminating the spread of injurious agents as well as to remove the consequent necrosis cells and tissue and to manifest the body's response to tissue damage and infection.
The inflammatory reaction may be beneficial as the body defense against agents deranging its homeostasis or harmful as damage the surrounding tissues .
The inflammation associated chronic diseases can be possible ameliorate with the potent antiinflammatory activity of MO bioactive compounds .
The biphenols are abundant in MSO that have Sahar, B. A. and Rania, A.H. Abd El-Aal Journal of Biomedical and Pharmaceutical Research 4 (6) 2015, 01-09 © 2015 All Rights Reserved. CODEN (USA):
JBPRAU 2 anti-inflammatory, antioxidant , and immunomodulatory  properties suggest that they may have beneficial effects on inflammatory bowel diseases (IBD) which is characterized by chronic intestinal inflammation. Although its etiology is still unknown, immune dysfunction, inflammatory mediators, reactive oxygen species (ROS) and cytokines play crucial roles in its development [12-13].
There are two main approaches for treating peptic ulcer; first reducing the production of gastric acid and reinforcing gastric mucosal protection . Treat of gastric ulcer by commercially available anti-ulcer drugs is usually accompanied by various side effects  such as H2-receptor antagonists (e.g. cimetidine) may cause gynaecomastia in men and galactorrhoea in women , while proton pump inhibitors (e.g. omeprazole and lansoprazole) can cause nausea, abdominal pain, constipation and diarrhea . As a result of these problems, there is a need to find new antiulcer agents that are highly effective with potentially less or no side effects. Medicinal plants have always been the main source of new drugs for the handle of gastric ulcers .
The clout of research is to investigate some physico-chemical, antioxidant properties, anti-inflammatory and anti-ulcer of Egyptian Maringa oleifera seed oil. MATERIALS and METHODS Plant material Moringa oleifera seeds (MS) were collected from National Research Center, Dokki, Cairo, Egypt. The seeds coat and wings were removed manually and it has crushed into powder using a laboratory blender. Extraction of oil using n- hexane: 500 g of MS powder was added to 1 L of n-hexane and continuously shaken for 4 h and then allowed to stay overnight and the oil was decanted. The filtrate was concentrated using a rotary evaporator at 40˚C. The recovered oil was used for physicochemical and biochemical analysis . Physicochemical properties The physico-chemical properties of the oil as (density, refractive index, iodine value, peroxide value, acidity, saponification value, and unsaponifiable matter were carried out using the method described by A.O.A.C. . Biochemical Analysis: Median lethal dose (LD50):
For determination of acute toxicity, each animal was carefully given single orally dose of MSO and death was observed after 24h of treatment . Antioxidant Activity: Determination of Total phenolic content Total phenolic content of MSO was spectrophotometrically determined using FolinCiocalteu reagent .
Determination of total flavonoid content The total flavonoid content was assayed according to Wong, et al., . Determination of radical DPPH scavenging activity Free radical scavenging capacity of MSO was determined using the stable 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) according to Hwang, et al., . Experimental Animals Anti-inflammatory activity Carrageenan-induced paw oedema test Carrageenin-induced rat hind paw oedema model The method adopted resembles essentially [25, 26].
The animals were studied for toxicity of DMSO up to 10% v/v in saline, and 5% DMSO was selected as a vehicle to suspend the standard drugs and the test compounds. Albino rats weighing between 150 and 250 g of either sex were starved for 18 h prior to the experiment. The animals were weighed, marked for identification and divided into groups of six. The standard drugs, ibuprofen (20 mg/kg body weight), mefenamic acid (100 mg/kg body weight) and three graded doses (10, 20 and 40 mg/kg body weight) of the test compounds were given orally as a suspension using 5% DMSO as a vehicle.
One hour later foot paw oedema was induced by injecting 0.1 ml of 1% carrageenin subcutaneously into the planter portion of the right hind paw of each rat. Initial foot paw volume was measured immediately by mercury plethysmometer. Oedema was measured 3 h after carrageen in administration. The swelling in test group animals was used to calculate the % inhibition M ± SEM of oedema achieved by the compound at the test dose compared with the vehicle control group. The % protection of oedema Sahar, B. A. and Rania, A.H. Abd El-Aal Journal of Biomedical and Pharmaceutical Research 4 (6) 2015, 01-09 © 2015 All Rights Reserved. CODEN (USA):
JBPRAU 3 was calculated according to the formula, % antiinflammatory activity = 100 X (1 - Vt/Vc) where Vt and Vc are the volume of oedema in test compounds and control groups, respectively. Antiulcer activity Animal rats were fasted for 18 hours before pylorus ligation with water ad libitum and it has divided into four groups (each: n=6): Group I: Ulcer induced control; Group II: MSO extract (250 mg/kg body wt p.o.) in ulcer rats; Group III:
MSO extract (500 mg/kg body wt p.o.) in ulcer rats; Group IVRanitidine (179 mg/kg body wt p.o.) in ulcer rats. Pylorus Ligated (PL) - Induced Ulcers Rats weighing 150-200 g were used for Pylorus Ligated (PL) - Induced Ulcers using the method described by to Deshpande, et al.,  .
Scoring of ulcer will be made as follows . Normal colored stomach....... (0) Red coloration.......... (0.5) Spot ulcer............…. (1) Hemorrhagic streak... (1.5) Deep Ulcers....................… (2) Perforation.............… (3) Mean ulcer score for each animal will be expressed as ulcer index. The percentage of ulcer protection was determined as follows: Ulcer index (UI) was measured by using following formula: . UI = UN + US + UP Χ 10−1 Where, UI= Ulcer Index; UN = Average number of ulcers per animal; US = Average number of severity score; UP = Percentage of animals with ulcers Percentage inhibition of ulceration was calculated as below: % Inhibition of ulceration = (ulcer index controlulcer index Test) × 100 /ulcer index control. Ethanol-induced gastric ulcer in rats Rats were deprived from food but had ad libitum access to tap water for 24 h before ulcer induction. Gastric mucosal damage was induced in conscious rats by gavage of 5.0ml/kg b.wt. of absolute ethanol (99.5%). The standard drug (Ranitidine 179 mg/kg) or MSO (250 & 500 mg/kg) were taken one hour before ethanol administration. The animals were anaesthetized with ether. Stomachs were isolated and ulcer index was determined . Parameters measured: Ulcer index and % protection was measured. pH of the gastric juice was also measured. STATISTICAL ANALYSIS The statistical significance was assessed using oneway analysis of variance (ANOVA) followed by Dunnet comparison test. The values are expressed as mean ± S.E. .
RESULTS The results illustrated that the LD50 of MSO is more than 62.5g/kg b.w., as all doses below this incurred zero percentage of death among all groups of rats. Physicochemical property of MSO is illustrated in Table 1. As shown in Table 2, MSO exhibited higher phenolic content and good scavenging abilities against DPPH radicals. The total phenolic, total flavonoid and DPPH of MSO were 35.05 mg gallic acid / g extract, 2.29 mg quercetin /g extract and 75.75 % respectively. Carrageenan-induced edema in rat hind paw is the most widely used primary test for screening of anti-inflammatory agents. The percentage inhibition of edema at 1h in ibuprofen (20 mg/kg) and mefenamic acid (100 mg/kg) was 30.0% and 8.0% respectively, since the percentage inhibition of edema was 72.2% in hexane extract of MSO treated group at a dose 20 mg/kg were investigated in table 3. The percentage inhibition for hexane extracts of MSO was highest at the doses of 20 mg/kg (72%) (p<0.05) which was comparable to that of ibuprofen and mefenamic acid (30% and 8% respectively). Table 4 and 5 illustrate that MSO was effective in the all of tested models of gastric ulcers. Sahar, B. A. and Rania, A.H. Abd El-Aal Journal of Biomedical and Pharmaceutical Research 4 (6) 2015, 01-09 © 2015 All Rights Reserved. CODEN (USA): JBPRAU 4 Table 1: Physicochemical property of Moringa oleifera seed oil Properties Moringaoleifera oil Oil yelid % 38.23 Acidity (as oleic acid ) % 1.50 Peroxide value (meq kg-1 of oil) 24.70 ±0 .24 Iodine value (g /100 g of oil) 66.11± 0.30 Density (g/cm3 ) 25o C 0.928±0 .02 Refractive index (nD 25 °C) 1.454± 0.04 Saponification value (mg of KOH/g of oil) 183.4± 0.27 Unsaponifiable matter (%) 0.85±0.09 Table 2:
The total phenolic, total flavonoid contents and DPPH antioxidant test of Moringa oleifera seed oil extract. Parameters Total phenols (mg/g) Total flavonoids (mg/g) DPPH (%inhibition ) Oil extract 35.05±0.03 2.29±.04 75.75±0.1 Table 3: Anti-inflammatory activity carrageenin-induced rat hind paw oedema % protection. Tested Compounds Ibuprofen (20 mg/kg) Mefenamic acid (100 mg/kg) MSO (20mg/kg) % Protection 33 8 72 Table 4: demonstrated descriptive statistic of Moringa oleifera seed oil on Pylorus Ligation-Induced Gastric Ulcers Group Treatment Gastric juice (GJ) (ml) pH of gastric juice(PH) Free Acidity(FA) mEq/litre Total Acidity(TA) I Control ( C ) (pyloric ligation) 8.5 ± 0.21 3.21±0.04 120±0.25 95.1±1.5 II MSO (L) (250mg/kg) 4.6 ± 0.10*** 4.8±0.18** 70±0.30* 66±0.12* III MSO (H ) (500mg/kg) 4.2 ± 0.10*** 3.8±0.05* 65±0.28* 58 ±0.14** IV Ranitidine (R) (197mg/kg) 4.1 ± 0.10*** 5.35±0.15*** 3 ± 0.17*** 37±0.24*** All values are mean ± S.E. ,n = 6 , ***p