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Fatty Acid Composition and Acute Toxicity Study on Moringa peregrina Fixed Oil in Albino Rats

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Vol. 5, No. 5 (2015): 1282-1288

Research Article Open Access

I

ISSSSNN:: 22332200--66881100

Fatty Acid Composition and Acute Toxicity Study on

Moringa peregrina

Fixed Oil in Albino Rats

Khaled A. kahilo

1

, Tarek Kamal

1

, Nasr Elsayed

1

, Mustafa Shukry

2

, Doaa Dishesh

1

and

Mohammed Abdalla Hussein

3

*

1

Biochemistry Department, Faculty of Veterinary Medicine, Kafr-Elsheikh University 2

Physiology Department, Faculty of Veterinary Medicine, Kafr-Elsheikh University 3

Biochemistry Department, Faculty of Pharmacy, October 6th University, 6th of October city, Egypt.

* Corresponding author: Mohammed Abdalla Hussein, e-mail: Prof.husseinma@o6u.edu.eg

ABSTRACT

One of crucial issues that are faced by global community is hunger and food insecurity. Moringa peregrina is a promising candidate for future crop, especially in arid regions where food insecurity prevalence is high.The GC-MS analyses of Moringa peregrina seeds oil revealed the presence of 12 components. 9-octadecenoic acid was the major constituent of Moringa peregrina seeds fixed oil representing (65.2%) followed by hexadecanoic acid methyl ester (10.6%). The objective of the second step was to evaluate the toxic effects of this oil. The results are given in shows that oral administration of Moringa peregrina seeds oil in doses of 3000, 6000, 9000, 12000, 15000 and 18000mg/kg. resulted in mortalities of 0, 1, 2, 7, 9 and 10 respectively. The dose of Moringa peregrina seeds oilthat killed half of the rats (LD50) was 11450mg/kg b.w. The results of liver and renal histopathology confirmed that the

death of rats. Based on these results it can be concluded that the Moringa peregrina fixed oil is safe and had low toxicity effect when given in concentrated doses for short period of time.

Keywords:

Moringa peregrina, Fatty acids, fixed oil, median lethal dose.

1. INTRODUCTION

Moringa peregrina (Forssk.) Fiori. (Moringaceae) occurs in the Middle East to India. In Iran it is a desert tree growing in Sistan and Baluchestan province and is locally called Gas-e-rowghan or Gaz Rokh [1,2]. Several reports on antioxidant activity of leaves and seeds of another Moringa species exist [3, 4]. Leaves of M. oleifera contain flavonoid pigments such as kaempferol, rhamnetin, isoquercitrin, and kaempferitrin [5]. Plants are rich sources of natural antioxidants, the best known are tocopherols, carotenoids, vitamin C, flavonoids, and different other phenolic compounds [5]. Recently, among natural antioxidants, flavonoids have received increasing attention. As compared with vitamin C and E, dietary flavonoids are considered to be more powerful antioxidants [6]. Recently, Moringa peregrina

seeds oil was tested for its antitumor activity [7]. As an extension of my interested research program in the extraction and therapeutic evaluation of rare medicinal plants [7, 8-10], we report herein, a facile route to

evaluate Moringa peregrina fixed oil composition as well as its toxicity in rats.

2. MATERIALS AND METHODS

2.1 Plant Material

Moringa peregrina seeds were obtained from Ankit Agrowal Co., India. The plant material was identified, authenticated taxonomically by Dr. Heba El-Gezawy, Pharmacognosy department, faculty of Pharmacy, October 6 University. The seed were cleaned, dried under direct sunlight and powdered by a mechanical grinder.

2.2 Extraction of Fixed oil:

After being cleaned by hand carefully to remove the foreign materials such as other seeds, stones and small stalks, Moringa peregrina seed were dried at 50°C for 12h in an oven, and then crushed into powder in a grinder with a size range of 0.55-1.0mm. The resulted powder was kept in a vacuum dryer until use. Moringa peregrina ground samples were mixed with hexane

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(1:10, m/V) at (60-80°C) using a Soxhlet apparatus. This process of extraction was repeated for 6h, the hexane distilled out by distillation assembly, then concentrated by hot plate drying and air-drying at temperature of 40±2 °C.

2.3 Materials:

Boron fluoride, methanol, pentane and hexane were obtained from Sigma-Aldrich, USA.

2.3.1 Preparation of BF3- Methanol Reagent:

One liter of reagent grade methanol, in a 2-liter flask, is cooled in an ice Bath. With the flask still in the bath, 125 grams of BF3 is bubbled through a glass tube into

the methanol in fume hood, and the gas should not flow so fast that white fumes emerge from the flask (the BF3

must be flowing through the glass tube before it is placed in and until it is removed from the methanol or the liquid may be drawn into the gas cylinder valve system) This reagent has an excellent shelf life and has been used up to 4months after preparation [11].

2.3.2 Preparation of FAME (fatty acid methyl ester):

Seeds (400 mg) were dried overnight at 50oC and

ground into powder with a mortar and pastle, after which 0.6ml of dichloromethane and 4.0ml of 0.5N sodium methoxide were added. Acidic catalysed esterification using the boron triflouride-methanol complex (14%w/ v) was added according to the method described by [12, 13]. The tube was shaken and heated for 30 min. at 50 0C. The reaction was stopped

by adding 5.0 ml of water containing 0.2 ml of glacial acetic acid. The esterified fatty acids were extracted with 3.0ml petroleum ether (40-600C). The clear

fraction was kept at -20 0C until further analysis.

2.4 Separation condition of Fatty Acids on GC/MS: Instrument

HP 6890 Series Gas Chromatograph System with an HP 5973 Mass Selective Detector.

The FAME in hexane (1μL) was injected into the column with a split ratio of 100:1. The injector and detector temperature were set at 200 and 250℃, respectively. He2 was used as the carrier gas at a flow

rate of 1.5mL/min. Separation was carried out on a TR-FAME (Thermo 260 M142 P) (30mm x 0.25mm ID) with a film thickness of 0.25υm film) (70% Cyanopropyl –Polysilphphenylene siloxane) capillary column. The column temperature was programmed from 100 to 160°C at 2°C/min and then to 250°C at 4°C/min and finally held at 250 ℃ for 20 min. The weights of the individual FAME were calculated on the basis of their relative peak area compared with that of internal standard, and then they were corrected using the corresponding GC response factors for each fatty acid.

2.5 Determination LD50 of Moringa peregrina seeds

oil:

2.5.1 Animals:

A 60 albino rats weighing around 180±10gms were divided into 6 groups, 10 rats in each. They were acclimatized to animal house conditions. Animals were provided with standard diet and water ad-libtum. Animals were kept under constant environmental condition and observed daily throughout the experimental work.

After administration of the tested Moringa peregrina

seeds oil, animals were observed individually every hour during the first day and every day for 21days. Behavior and clinical symptoms of animals are noted throughout the duration of the experiment.

Preliminary experiments were carried out on groups of 4 rats. Moringa peregrina seeds oil was administrated orally in different doses to find out the range of doses which cause zero and 100% mortality of animals. A range doses was determined for each compound.

LD50 was determined by oral administration of Moringa

peregrina seeds oilin different doses 3000, 6000, 9000, 12000, 15000 and 18000mg/kg. resulted in mortalities of 0, 1, 2, 7, 9 and 10 respectively, according to the method of Spearman and Karber [14].

The LD50 was then calculated by the application of the

following formula

LD50 = Dm - ∑ (Z . d)

--- n

Dm= The dose by which killed all the rats in the

group.

Z = Half the sum of the dead rats from 2 successive groups.

d = The difference between 2 successive doses. n = number of animals in each group.

2.6 Histological assessment:

Liver and kidney from rats of different groups were fixed in 10% neutral formalin solution, dehydrated in graded alcohol and embedded in paraffin. Fine sections obtained were mounted on glass slides and counter-stained with Hematoxylin Eosin (H&E) for light microscopic analyses according to the method of Bancroft and Steven [15]. The slides were coded and were examined by a histopathologist who was ignorant about the treatment groups after which photographs were taken.

3. RESULTS AND DISCUSSION

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Table 1: GC-MS analysis of the fixed oil constituents of Moringa peregrina seeds.

Peak

No. Compound Relative Retention time * Percentage (%)

1 Hexadecanoic acid, methyl ester 27.44 10.2 2 9-Hexadecenoic acid, methyl ester 28.04 2.54 3 Heptadecanoic acid, 16-methyl ester 30.89 0.49

4 Methyl stearate 31.22 8.20

5 9-Octadecenoic acid methyl ester 33.77 65.2 6 10-Octadecenoic acid methyl ester 31.82 2.96

7 Ethyl Oleate 32.15 1.68

8 9,12-Octadecadienoic acid 32.51 0.55 9 Methyl 18-methylnonadecanoate 34.28 3.00 10 cis-11-Eicosenoic acid 34.74 2.04 11 Docosanoic acid methyl ester 37.30 2.40 12 9,12-Octadecadienoic acid 40.12 0.28

Figure 1: Total Ion Chromatogram of the GC-MS analysis of the fixed oil of Moringa peregrina seeds.

3.1 LD50 of Moringa peregrina seeds oil

The results are given in table (2) shows that oral administration of Moringa peregrina seeds oil in doses of 3000, 6000, 9000, 12000, 15000 and 18000mg/kg. resulted in mortalities of 0, 1, 2, 7, 9 and 10 respectively. The dose of Moringa peregrina seeds oil that killed half of the rats (LD50) was 11450mg/kg b.w.

3.2 Toxic symptoms:

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Table 2: Determination of LD50 of Moringa peregrina seeds oil given orally in adult rats.

Group Number Dose

(mg/kg)

No. of

animals/group

No. of dead animals

(Z) (d) (Z.d)

1 3000 10 0 0.5 3000 1500

2 6000 10 1 1.5 3000 4500

3 9000 10 2 4.5 3000 13500

4 12000 10 7 8.0 3000 24000

5 15000 10 9 8.5 3000 25550

6 18000 10 10 0 00 00

3.3 Effect of Moringa peregrina seeds oil on

histological structure of liver tissue in groups (1 and 6) of rats:

Liver isolated from the group (1) rats received Moringa peregrina seeds oil (3000mg/kg.b.w.) showed normal

hepatic cells and free from inflammation or fibrosis (M.T X400) (graph 1).

Liver sections of group (6) rats received Moringa peregrina seeds oil (15000mg/kg.b.w.) revealed portal inflammation, hydropic degeneration with steatosis (fatty changes) (H&E X400) (graph 2).

Group(1): Rats received Moringa peregrina seeds oil (3000mg/kg.b.w.)

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.

Group (6): Rats received Moringa peregrina seeds oil (15000mg/kg.b.w.)

Graph 2: Mild lobular inflammation in hepatocytes (P.B X400).

3.4 Histopathology examination of the kidney tissues:

Graph 3 and 4 showed reprehensive histological

section in the kidney of groups (1 and 2). According to microscopic examinations no pathological lesions elicited in the kidney of group (1) rats received

Moringa peregrina seeds oil (3000mg/kg.b.w.) (Graph 3).

Kidney sections of the Moringa peregrina seeds oil (15000mg/kg.b.w.) group (6) of rats showed remarkable changes in the renal cortex versus. These changes include shrinkage of the glomeruli, tubular epithelial cells degeneration and tubular swelling were observed (Graph 4).

Group(1): Rats received Moringa peregrina seeds oil (3000mg/kg.b.w.)

Graph 3: Section in kidney showed normal (H&E X200)

Group(1): Rats received Moringa peregrina seeds oil (15000mg/kg.b.w.)

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Moringa peregrina is one of plant species that potentially become important in developing countries where hunger and undernourishment is a major concern. Traditionally, young seeds of the plant are eaten in India and the mature one are fried or roasted in Malawi [16]. Due to its valuable nutrient content and tolerant to severe drought, the plant could become an important future crop in arid and semi-arid regions. In this paper, the current status of M. peregrina in Saudi Arabia will be reviewed. Along with Yemen and Oman, Saudi Arabia is one of native distribution areas of the tree in the Middle East [17]. In the last decades the study on M. peregrina in Saudi Arabia has increased, leading to better understanding on its distribution and ecology, nutrient content from various parts of the plant, medicinal properties, threats and conservation status, and conservation action needed for protecting and utilizing the plant sustainably.

The LD50 of 11450 mg/ kg of the M. peregrina fixed oil

is an indication that the oil is safe. However, LD50 has

not been regarded as a biological constant because many variables such as animals’ species and strain, age, gender, diet, bedding, ambient temperature, caging conditions and time of the day can all affect the LD50

value obtained; hence there are considerable uncertainties in extrapolating LD50 value obtained for

specie to other species. Consequently, recognizing LD50

test as providing, at best, only a ball park estimate of human lethality has been advocated [18]. In the acute toxicity in male rats M. peregrina fixed oil orally at dose 3000mg/kg, there were no changes in animal behavior, but the body weight gains were significantly different in the treated rats. Since, the changes in animal behavior have been used as an indicator of adverse effects of drugs and chemicals [19]. The present results suggest that at the oral dose, the M. peregrina fixed oil is safe. The present study was designed to evaluate the effects of M. peregrina fixed oil on the liver and kidney histology of male rats. Liver and kidney are two important organs that perform vital function for the healthy survival of the body. The liver is known to be a key organ in the metabolism and detoxification of xenobiotic, is vulnerable to damage induced by a huge variety of chemicals as reported by Udem et al.,[20]. Liver isolated from the group (1) rats received Moringa peregrina seeds oil (3000mg/kg.b.w.) showed normal hepatic cells and free from inflammation or fibrosis (M.T X400). Liver sections of group (6) rats received

Moringa peregrina seeds oil (15000mg/kg.b.w.) revealed portal inflammation, hydropic degeneration with steatosis (fatty changes) (H&E X400). Histological section in the kidney of groups (1 and 2). According to microscopic examinations no pathological lesions elicited in the kidney of group (1) rats received

Moringa peregrina seeds oil (3000mg/kg.b.w.). Kidney sections of the Moringa peregrina seeds oil (15000mg/kg.b.w.) group (6) of rats showed remarkable changes in the renal cortex versus. These changes include shrinkage of the glomeruli, tubular epithelial cells degeneration and tubular swelling were observed. Histological examination indicated that, at

the oral dose, the M. peregrina fixed oil (3000mg/kg.b.w.) is safe.

4. CONCLUSION

The GC-MS analyses of Moringa peregrina seeds oil revealed the presence of 12 components. 9-octadecenoic acid was the major constituent of Moringa peregrina seeds fixed oil representing (65.2%) followed by hexadecanoic acid methyl ester (10.6%). It can be concluded that peregrina seeds fixed oil is hepatotoxic and has a toxic effect on the kidney at 15000mg/kg.b.w. and more safe at 3000mg/kg.b.w.

5. REFERENCES

1. Ghahreman, A. (2001). Flora of Iran. Tehran: Institute of Forests and Rangelands publications;. No. 2578.

2. Mozaffarian, V. (1996). A Dictionary of Iranian Plant Names. Tehran: Farhang mo’aser; p. 219.

3. Siddiq, A., Anwar, F., Manzoor, M., et al., (2005). Antioxidant activity of different solvent extracts of Moringa oleifera leaves under accelerated storage of sunflower oil. Asian J Plant Sci. 4:630–635.

4. Lalas, S., Tsaknis, J. (2002). Extraction and identification of natural antioxidant from the seeds of the Moringa oleifera tree variety of Malawi. J Am Oil Chem Soc.79:677–683. 5. Iqbal, S., Bhanger, M. (2006). Effect of season and production

location on antioxidant activity of Moringa oleiferaleaves grown in Pakistan. J Food Comp Anal.19:544–551.

6. Sultana, B., Anwar, F. (2008). Flavonols (kaempeferol, quercetin, myricetin) contents of selected fruits, vegetables and medicinal plants. Food Chem. 2008; 108:879–884. 7. Abdel-Maksoud, HA., Mohammed A. Hussein, M.A., et al.,

(2015). Antioxidant and Lung Protective Effects of Moringa peregrina seeds oil against benzene and/or Fluorouracil induced leukopenia in Rats. International Journal of Pharma Sciences. 5: 1108-1116.

8. Hussein, MA.(2008). Antidiabetic and antioxidant activity of Jasonia montana extract in Streptozotocin – induced diabetic rats. JSP., 16:214-221.

9. Hussein, MA. (2010). Purslane Extract Effects on ObesityInduced Diabetic Rats Fed a High- at Diet. Mal J Nut., 3:419-429.

10.Hussein, MA., Abdelgwad SM. (2010). In vivo Hepatoprotective Properties of Purslane Extracts on ParacetamolInduced Liver Damage. Mal J Nutr., 1: 161–170. 11.Metcalfe, LD., Schmitz, AA. (1961). The Rapid Preparation of

Fatty Acid Esters for Gas Chromatographic Analysis, Armour Industrial Chemical Co., McCook, March 33: 111.

12.AOAC, (1894). Official Methods of Analysis, Association of Analytical Chemists, Arlington, 14th ed., Official Method 963.22

13.Rezanka T, Rezankova, H. (1999). Characterization of fatty acids and triacylglycerols in vegetable oils by gas chromatography and statistical analysis. Anal Chim Acta. 398: 253.

14.Finney, D.J., 1964. Statistical Method in Biological Assay. Hafner, New York. Finney, D.J., 1971.

15.Bancroft, GD., Steven, A. (1983). In. Theory and practice of histological technique 4th Ed. London; Churchill Livingstone. Pp. 99 – 112.

16.Boulos, L. (1999). Flora of Egypt. Vol. 1 (Azollaceae Oxalidaceae). Nordic. J. Bot. 19:328.

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Rome.FAO, IFAD and WFP. (2013). The State of Food Insecurity in the World 2013. The multiple dimensions of food security. Rome, FAO.

18.Zbinden, G., Flury-Roversi, M. (1981). Significance of the LD50 test for the toxicological evaluation of chemical substances. Arch. Toxicol. 4792: 77-81.

19.El Hilaly, J., Israili, Z.H., Lyoussi, B. (2004). Acute and chronic toxicological studies of Ajugaivanin experimental animals. J. Ethnopharmacol. 91: 43-50.

20.Udem, SC., Obidoa, O., Asuzu, IU. (2009). Acute and chronic toxicity studies of Erythrina Senegalensis DC stem bark extract in mice. Comparative Clinical Pathology.19: 275-282

*****

© 2015; AIZEON Publishers; All Rights Reserved

Figure

Table 1: GC-MS analysis of the fixed oil constituents of Moringa peregrina seeds.
Table 2: Determination of LD50 of Moringa peregrina seeds oil given orally in adult rats

References

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