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Evaluation of antioxidant activity of the extract n-Butanol and ethyl acetate of Catha Edulis from Yemen.

Introduction

khat platoon of natural steroids whose scientific name (Catha edulis) has other names by region located where [1]. He perennial tree greenish always grow at high altitudes, which extends from eastern to southern Africa, Afghanistan and Yemen. [2] Catha edulis belongs to the family flora Celastraceae [3]. Be this family in nearly 88 type and 1,300 species of plants [4]. High rate tree qat in Yemen from 1 meter to 5 meters while in Ethiopia Faisal along the tree khat to more than 18 meters [1]. Catha edulis is a major source of income for a number of people very involved in the marketing, production and distributors is also an important source of income for the government from its own tax [2,5,6]. Catha edulis leaves contain several chemical groups. It contains alkaloids, which are responsible for its stimulant effects especially vinyl alkyl compounds Secretary [7]. As studies have confirmed the existence of a complex set of alkaloids called Kathdulinat [8]. Tananse and flavonoids [9]. Terpenes and Sterolse [10]. Essential oils and amino acids [11,12]. And proteins, beta-carotene, calcium, vitamin C, riboflavin [13,14].

The main purpose of chewing khat leaves is to get an orgasm and activity and satisfy the mood. However, there are areas used khat leaves for many purposes, for example, in some countries in South Africa leaves and roots used to treat influenza, cough and gonorrhea, and asthma and other stomach problems, chest [15,16]. France has been working pharmaceuticals in 1910 of Catha edulis extract used to treat nerve disorder, especially when women [17].

Free radicals are generated by a process known as redox cycling and they are catalysed by transition metals, to cause DNA and RNA damage, thiol oxidation and lipid peroxidation [18,19]. The great potential of free radicals to react with various compounds by electron transfer, proton transfer, Hatom abstraction or addition reaction may involved in the pathological of various diseases [20,21].Many plant compounds can scavenge reactive oxygen species (ROS) and thereby directly reduce-oxidative stress [22].Among these, flavonoids seem to be potent candidates because they show broad pharmacological activities and widely distributed in many edible plants [23].

This study aims to search for the antioxidant activity of the extracts Butanol and ethyl acetate at the level of leaf Catha edulis for two different age groups.

Materials And Methods

Plant material:

The leaves of Catha edulis were collected from City Hajja--Yemen in September 2009. A voucher specimen of the plant material has been deposited at the department of biology (Sana'a University).

Preparation of the extracts:

Were collected leaves of the Catha edulis plant (soft twigs) from Hajja city of argument in mid-September 2009.Soak plant parts dried individual species in a mixture of ethanol_Water (7-3) and then left 24 hours, repeated the process four times filtered output and focus--and then eases with distilled water and add PB (CH3COO) 4 to get rid of resins and dust suspended and then begin the process of liquid_liquid extraction with ethyl acetate_butanol and the final outcome was as follows

plant\extrats   n-Butanol   Ethyl
                            acetate

3 age(600g)     36.87(g)    4.89(g)
50 age (317g)   33.74(g)    6.07(g)


Chemicals:

1,1-Diphenyl-2-picrylhydrazyl (DPPH[degrees]), potassium ferricyanide, gallic acid, ethylenediamine tetra acetic acid (EDTA), ferrozine, Folin-Ciocalteus's phenol reagent, quercetin, ascorbic acid, ferric chloride and sodiumcarbonate were from sigma, sigma Aldrich. All the chemicals used including the solvents, were of analytical grade.

Determination of antioxidant activity:

Determination of DPPH radical scavenging activity: The ability to scavenge the stable free radical l,l-diphenyl-2-picrylhydrazyl (DPPH[degrees]) was determined based on the method of Ohinishi et al. [24].with minor modifications. A solution of 0.2 mM DPPH in methanol was prepared and 1 ml of this solution was mixed with 1 ml of extract in methanol (5 to 150 ([micro]g/ml). The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. A control sample containing the same volume of solvent in place of extract was used to measure the maximum DPPH absorbance. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid and quercetin were used as references. Results were expressed as percentage of inhibition of the DPPH radical according to the following equation:

% Inhibition of DPPH = [(Absorbance of control - Absorbance of sample)/Absorbance of control] x 100

Determination of total phenolic contents:

Total phenolic content was determined using Folin-Ciocalteu reagent as adapted from Singleton and Rossi [25], with slight modifications. 100 [micro]l of extract was mixed with 250 [micro]l of Folin-Ciocalteu reagent (IN) and allowed to stand at room temperature for 2 min. 1250 [micro]l of sodium carbonate (20%) was added, and the mixture was mixed and allowed to stand at room temperature in the dark for 2 h. The absorbance was read at 765 nm, and the total polyphenols concentration was calculated from a calibration curve, using gallic acid as standard (50-1000 mg/L). The results were expressed as gallic acid equivalents (GAE)/g extract.

Determination of flavonoids:

Total flavonoid content was determined using the method of Ordon Ez et al. [26]. A volume of 0.5 ml of 2 % A1C13 ethanol solution was added to 0.5 ml of sample solution. After one hour at room temperature, the absorbance was measured at 420 nm. A yellow color indicated the presence of flavonoids. Extract samples were evaluated at a final concentration of 0.1 mg/ml. Total flavonoids were calculated as quercetin (mg/g) using the calibration curve. Results were expressed as mg quercetin equivalents (QE)/g extract.

Statistical analysis:

All assays were carried in triplicates and results expressed as means [+ or -] standard deviation. IC50-value ([micro]g extract/ml) is the effective concentration which proves 50% of activity, was calculated for each assay. Statistical comparisons were done with Student's test. Differences were considered to be higly significant at P < 0.01 and significant at P< 0.05.

Results And Discussion

Determination of antioxidant activity:

Scavenging effect on DPPH radical:

The antioxidants react with DPPH[degrees], a stable purple colored free radical and convert it into colorless a-[alpha]-diphenyl-[beta]-picryl hydrazine. The extent discoloration indicates the amount of DPPH scavenged [24]. As shown in Figure 1, the DPPH radical scavenging activities of various investigated extract n-Butanol and Ethyl acetate of Catha Edulis with age 3 years were in order of AcOEt extract (95.92%) at concentration 20 [micro]g/ml > n-BuOH extract (90.00%) at concentration 50 [micro]g/ml. have a remarkable ability to scavenge radicals with IC50 respectively) (Table 1).

In Figure 2, the DPPH radical scavenging activities of various investigated extract n-Butanol and Ethyl acetate of Catha Edulis with age 50 years were in order of n-BuOH extract (63.50%) at concentration 10 [micro]g/ml To be this dose responsiveness (96.95%) at concentration 20 [micro]g/ml >. AcOEt extract (95.55%) at concentration 150 [micro]g/ml have a remarkable ability to scavenge radicals with IC50 respectively) (Table 2).

Conclusion:

The results obtained in this study clearly showed that both AcOEt and n-BuOH extracts from the leaves of Catha edulis possess antioxidant activity. However, they differ from one age to another type in the plant is a little old AcOEt showed the effectiveness of anti-oxidant largest of n-BuOH developed. While at the plant with big old n-BuOH showed the effectiveness of anti-oxidant, even in lower concentration 10 [micro]g/ml and delayed reaction AcOEt where the response at a concentration 150 [micro]g/ml expressed.

References

[1.] Peters, D.W.A., 1952. Khat: its history, botany, chemistry and toxicology. Pharm J 169: 17-18: 36-37.

[2.] Kikorian, A.D. and A. Getahun, 1973. Chat: Coffee's Rival for Harrar: Ethiopoia II. Chemical Composition. Economic Botany, 27: 378-389.

[3.] Kennedy, J.G., 1987a. The Flower of Paradise The Institutionalized Useof the Drug Qat In North Yemen. p 176-188. D. Reidel: Dordrecht

[4.] Mabberley, D.J., 1997. The Plant-book. A portable dictionary of the vascular plants, Cambridge University Press, Cambridge, 2nd edn.

[5.] Kennedy, J.G., 1987b. The agriculture and economics of qat. In: Ibid, The flower of paradise: the institutionalized use of the drug qat in North Yemen. pp: 133-175. Dordrecht: D. Reidel.

[6.] Lemessa, D., 2001. Khat (Catha edulis): botany, distribution, cultivation, usage and economics in Ethiopia. Addis Ababa: UNEmergencies Unit for Ethiopia.

[7.] Kalix, P., 1992. Cathinone, a natural amphetamine. Pharmacol Toxicol., 70: 77-86.

[8.] Baxter, R.L., L. Crombie, D.J. Simmonds, D.A. Whiting, O.J. Braenden, K. Szendrei, 1979. Alkaloids of Catha edulis (khat). Part 1.Isolation and characterisation of eleven new alkaloids with sesquiterpene cores (cathedulins); identification of the quinone-methide root pigments. J Chem Soc Perkin Trans., 1: 2965-2971.

[9.] Kalix, P., O. Braenden, 1985. Pharmacological aspects of the chewing of khat leaves. Pharmacol Rev., 37: 149-164.

[10.] Crombie, L., 1980. The cathedulin alkaloids. Bull Narc., 32: 37-50.

[11.] Qedan, S., 1972. Catha edulis, Eine Wening Bekannte Rauch- und Genussdroge, Planta Medica, 21: 410-415.

[12.] Winterfeld, K, and G. Bernsmann, 1960. Zur Kenntnis der Inhaltstofe von Catha edulis. Arch.Pharm.(Weinheim) 63: P 991-1000.

[13.] Mustard, M., 1952. Ascorbic acid content of some miscellaneous tropical and subtropical plants and plant products. Food Research, 17: 31-35.

[14.] Alles, G.A., D. Fairchild and M. Jensen, 1961. Chemical pharmacology of Catha edulis. J. Med. Pharm. Chem. 3: 323-352.

[15.] Simmons, F.J., 1960. Northwest Ethiopia. Peoples and Economy. Madison University of Wisconsin press Xvii+250.

[16.] Githens, T.S., 1948. Drug Plants of Africa, African Handbooks 8. Committee on African Studies, The University of Pennsylvania Press.

[17.] Azais, R.P. and R. Chambard, 1931. Cinq Annees de recherches Archeologiques en Ethipie province du Harar et Ethiopie Meridionale. Paris: Librairie Orientaliste Paul Geuthner.

[18.] Halliwell, B. and J. Guttridge, 1999. The Free radicals in biology and medicine, Oxford University Press, Oxford.

[19.] Halliwell, B., 1994. Free radicals, antioxidants and human disease: curiosity, cause, or consequence? Lancet 344: 721-724.

[20.] Halliwell, H.B., 1993. The role of oxygen radicals in human disease, with particular reference to the vascular System. Haemostasis 23 (Suppl 1): 118-126.

[21.] Havsteen., B. 1983. Flavonoids, a class of natural products of high pharmacological potency. Biochem. Pharmacol. 32: 1141-1148.

[22.] Walgren, R.A., J.T. Lin., R.K. Kinne, T. Walle. 2000 b. Cellular uptake of dietary flavonoid quercetin 4'-beta-glucoside by sodiumdependent glucose transporter SGLT1. J. Pharmacol. Exp. Ther. 294: 837-843.

[23.] Rice-Evans, C.A., N.J. Miller, G. Paganga, 1996. Structure antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med., 20: 933-956.

[24.] Ohinishi, M., H. Morishita, H. Iwahashi, T. Shizuo, S. Yoshiaki, M. Kimura, R. Kido, 1994. Inhibitory effects of chlorogenic cids on linoleic acid peroxidation and haemolysis. Phytochemistry, 36: 579-583.

[25.] Singleton, V.L. & J.A. Rossi, 1965. Colorimetry of total phenolics with phosphor-molybdic-phosphotungstic acid reagent. Amer. J. Enol. Viticul., 16(3): 144-158.

[26.] Ordon Ez, A.A.L., J.D., Gomez, M.A Vattuone, M.I. Isla, 2006. Antioxidant activities of Sechium edule (Jacq.) Swart extracts. Food Chem., 97: 452-458.

(1) Methaq. Nasser. Algabr, (2) S. Ameddah, (2) A. Menad, (1) R. Mekkiou, (3) S. Benayache and (1) F. Benayache

(1) Laboratoire de Phytochimie et Analyses Physico-Chimiques et Biologiques, Universite Mentouri, Route de Ain El Bey, 25 000 Constantine, Algeria.

(2) Laboratoire de biologie et environnement, Universite Mentouri, Route de Ain El Bey, 25 000 Constantine, Algeria.

(3) Laboratoire de Valorisation des Ressources Naturelles et Synthese de Substances Bioactives, UniversiteMentouri, Route de Ain El Bey, 25 000 Constantine, Algeria.

Corresponding Author

Methaq. Nasser. Algabr, Laboratoire de Phytochimie et Analyses Physico-Chimiques et Biologiques, Universite Mentouri, Route de Ain El Bey, 25 000 Constantine, Algeria.

Table 1: shows the effectiveness of the phase of Ethyl acetate
and n-Butanol plant is 3 years old in DPPH root families

Ascorbic acid         n-BuOH                AcOEt

17,25 [+ or -] 2,52   13,55 [+ or -] 3,11   19,10 [+ or -] 8.21
51,11 [+ or -] 2,30   34,62 [+ or -] 7,66   68,18 [+ or -] 4.84
85,00 [+ or -] 3,77   45,64 [+ or -] 1,78   95,92 [+ or -] 0.30
96,00 [+ or -] 0,64   90,00 [+ or -] 2,55   95,43 [+ or -] 0.46
                      96,19 [+ or -] 0,43   95,09 [+ or -] 0.29
                      96,75 [+ or -] 0,40   95,51 [+ or -] 0.26

Ascorbic acid         ([micro]g/ml)
                      Concentration

17,25 [+ or -] 2,52   5
51,11 [+ or -] 2,30   10
85,00 [+ or -] 3,77   20
96,00 [+ or -] 0,64   50
                      100
                      150

Table 2: shows the effectiveness of the phase of Ethyl acetate
and n- Butanol plant is 50 years old in DPPH root families

Ascorbic acid         n-BuOH                AcOEt

17,25 [+ or -] 2,52   17,67 [+ or -] 3,56   2,44 [+ or -] 1,99
51,11 [+ or -] 2,30   63,50 [+ or -] 5,33   4,17 [+ or -] 3,84
85,00 [+ or -] 3,77   96,49 [+ or -] 0,39   6,05 [+ or -] 2,50
96,00 [+ or -] 0,64   96,07 [+ or -] 0,13   33,48 [+ or -] 4,91
                      95,73 [+ or -] 0,56   42,43 [+ or -] 9,61
                      95,55 [+ or -] 0,56   95,34 [+ or -] 0,17

Ascorbic acid         Concentration
                      ([micro]g/m)

17,25 [+ or -] 2,52   5
51,11 [+ or -] 2,30   10
85,00 [+ or -] 3,77   20
96,00 [+ or -] 0,64   50
                      100
                      150
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Article Details
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Title Annotation:Original Article
Author:Algabr, Methaq. Nasser.; Ameddah, S.; Menad, A.; Mekkiou, R.; Benayache, S.; Benayache, F.
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7YEME
Date:Sep 1, 2013
Words:2223
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