O A RIGINAL RTICLE

2375
Advances in Environmental Biology, 7(9): 2375-2379, 2013
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Evaluation of antioxidant activity of the extract n- Butanol and Ethyl acetate of Catha
Edulis from Yemen
1
Methaq. Nasser. Algabr, 2S. Ameddah, 2A. Menad, 1R. Mekkiou, 3S. Benayache and
1
F. Benayache
1
Laboratoire de Phytochimie et Analyses Physico-Chimiques et Biologiques, Université Mentouri, Route de Ain
El Bey, 25 000 Constantine, Algeria.
2
Laboratoire de biologie et environnement, Université Mentouri, Route de Ain El Bey, 25 000
Constantine,Algeria.
3
Laboratoire de Valorisation des Ressources Naturelles et Synthèse de Substances Bioactives,
UniversitéMentouri, Route de Ain El Bey, 25 000 Constantine, Algeria.
Methaq. Nasser. Algabr, S. Ameddah, A. Menad, R. Mekkiou, S. Benayache and F. Benayache:
Evaluation of antioxidant activity of the extract n- Butanol and Ethyl acetate of Catha Edulis from
Yemen
ABSTRACT
Flavonoids play a very important role. The beneficial effect of flavonoids is mainly associated with the
different various antioxidative mechanisms which act as enzyme inhibitor, reducing agents, trapping free radical
and by acting as iron- In this study was to evaluate the Butanol (n-BuOH) extract and ethyl acetate (AcOEt) to
two different types at the age of Catha edulis, (AcOEt) extract of the age of 3 years, which contains a high
level of poly-phenols and flavonoids showed antioxidant capacity of the most effective look so high dose
independent method for the liquidation of the root activity (DPPH °) While (n-BuOH) extract age is 50 years
showed antioxidant capacity most efficiently.
Key words: Catha edulis, Polyphenols, flavonoids, antioxidant activity.
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
Corresponding Author
Methaq. Nasser. Algabr, Laboratoire de Phytochimie et Analyses Physico-Chimiques et
Biologiques, Université Mentouri, Route de Ain El Bey, 25 000 Constantine, Algeria.
2376
Adv. Environ. Biol., 7(9): 2375-2379, 2013
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 midSeptember 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
extrats
n- Butanol
Ethyl acetate
36.87(g)
4.89(g)
33.74(g)
6.07(g)
plant
3 age (600g)
50 age (317g)
Chemicals:
1,1-Diphenyl-2-picrylhydrazyl
(DPPH°),
potassium ferricyanide, gallic acid, ethylenediamine
tetra acetic acid (EDTA), ferrozine, FolinCiocalteus’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:
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 to150 μg/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:
The ability to scavenge the stable free radical
1,1-diphenyl-2-picrylhydrazyl
(DPPH°)
was
% Inhibition of DPPH = (Absorbance of control - Absorbance of sample) x 100
Absorbance of control
U
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 μl of
extract was mixed with 250 μl of Folin-Ciocalteu
reagent (1N) and allowed to stand at room
temperature for 2 min. 1250 μl 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:
U
Total flavonoid content was determined using
the method of Ordon Ez et al. [26]. A volume of 0.5
ml of 2 % AlCl3 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 ± standard deviation. IC50-value
(μg 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
2377
Adv. Environ. Biol., 7(9): 2375-2379, 2013
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°, a stable
purple colored free radical and convert it into
colorless α-α- diphenyl-β-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 μg/ml > n-BuOH
extract (90.00%) at concentration 50 μg/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 μg/ml To be this dose
responsiveness (96.95%) at concentration 20 μg/ml
>. AcOEt extract (95.55%) at concentration 150
μg/ml have a remarkable ability to scavenge radicals
with
IC50
respectively)
(Table
2).
Fig. 1: DPPH radical-scavenging activities of different extracts of Ethyl acetate and n- Butanol of Catha edulis
is 3 years and standards. Each value represents a mean ± SD (n=3), P<0.05
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
(µg/ml) Concentration
17,25±2,52
13,55±3,11
19,10±8.21
5
51,11±2,30
34,62±7,66
68,18±4.84
10
85,00±3,77
45,64±1,78
95,92±0.30
20
96,00±0,64
90,00±2,55
95,43±0.46
50
96,19±0,43
95,09±0.29
100
96,75±0,40
95,51±0.26
150
Fig. 2: DPPH radical-scavenging activities of different extracts of Ethyl acetate and n- Butanol of Catha edulis
is 50 years and standards. Each value represents a mean ± SD (n=3), P<0.05
2378
Adv. Environ. Biol., 7(9): 2375-2379, 2013
Table 2: shows the effectiveness of the phase of Ethyl acetate and n- Butanol plant is 50 years old in DPPH root families
Concentration
Ascorbic acid
n-BuOH
AcOEt
(µg/m)
17,25±2,52
17,67±3,56
2,44±1,99
5
51,11±2,30
63,50±5,33
4,17±3,84
10
85,00±3,77
96,49±0,39
6,05±2,50
20
96,00±0,64
96,07±0,13
33,48±4,91
50
95,73±0,56
42,43±9,61
100
95,55±0,56
95,34±0,17
150
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 μg/ml and delayed reaction
AcOEt where the response at a concentration150
μg/ml expressed.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Peters, D.W.A., 1952. Khat: its history, botany,
chemistry and toxicology. Pharm J 169: 17-18:
36-37.
Kikorian, A.D. and A. Getahun, 1973. Chat:
Coffee’s Rival for Harrar: Ethiopoia II.
Chemical Composition. Economic Botany, 27:
378-389.
Kennedy, J.G., 1987a. The Flower of Paradise –
The Institutionalized Useof the Drug Qat In
North Yemen.p 176 – 188. D. Reidel:Dordrecht
Mabberley, D.J., 1997. The Plant-book. A
portable dictionary of the vascular plants,
Cambridge University Press, Cambridge, 2nd
edn.
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.
Lemessa, D., 2001. Khat (Catha edulis): botany,
distribution, cultivation, usage and economics in
Ethiopia. Addis Ababa: UNEmergencies Unit
for Ethiopia.
Kalix, P., 1992. Cathinone, a natural
amphetamine. Pharmacol Toxicol., 70: 77-86.
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: 29652971.
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 acids on
2379
Adv. Environ. Biol., 7(9): 2375-2379, 2013
linoleic acid peroxidation and haemolysis.
Phytochemistry, 36: 579-583.
25. Singleton, V.L. & J.A. Rossi, 1965. Colorimetry
of total phenolics with phosphor-molybdicphosphotungstic 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.