Year : 2009 | Volume
: 5 | Issue : 20 | Page : 419--424
Antioxidant activity of aqueous and ethanolic extracts of Crocus sativus L. stigma and its bioactive constituents, crocin and safranal
Hossein Hosseinzadeh1, Fahimeh Shamsaie2, Soghra Mehri2,
1 Pharmaceutical Research Center, Pharmacodynamy and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, I.R, Iran
2 Pharmacodynamy and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, I.R, Iran
Pharmaceutical Research Center, Pharmacodynamy and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, I.R
The antioxidant activity of aqueous and ethanol extracts of saffron (Crocus sativus L.) stigma and its constituents, crocin and safranal were evaluated using deoxyribose, erythrocyte membrane lipid peroxidation and liver microsomal non-enzymatic lipid peroxidation methods in vitro. The aqueous and ethanolic extracts in different concentrations (10, 100, 250, 500 and 1000 µg/ml), crocin and safranal at 0.1, 0.5, 1 and 2 mM were tested. Results showed that test compounds could diminish the extent of MDA generation in three methods (P<0.01 VS control). The effect of crocin at 1,2 mM and ethanolic extract at 500 and 1000 µg/ml in liver microsomal lipid peroxidation were similar to BHT 100 µM in liver microsomal lipid peroxidation (P>0.05). Crocin the main carotenoid in saffron extracts probably and relatively safranal may have an important role in antioxidant properties of the extracts.
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Hosseinzadeh H, Shamsaie F, Mehri S. Antioxidant activity of aqueous and ethanolic extracts of Crocus sativus L. stigma and its bioactive constituents, crocin and safranal.Phcog Mag 2009;5:419-424
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Hosseinzadeh H, Shamsaie F, Mehri S. Antioxidant activity of aqueous and ethanolic extracts of Crocus sativus L. stigma and its bioactive constituents, crocin and safranal. Phcog Mag [serial online] 2009 [cited 2022 Sep 27 ];5:419-424
Available from: http://www.phcog.com/text.asp?2009/5/20/419/59768
Free radicals are involved in the etiology of different human disease such as vasculitis, rheumatoid arthritis, stroke  , congestive heart failure  , Parkinson, Alzheimer , and cancer  . Antioxidants are substances that can delay or inhibit oxidative stress, therefore they can help in prevention and therapy of disease caused by oxidative damage  .
Crocus sativus L. commonly known as saffron is a stemless herb of the Iridaceae family. Its constituents that are considered pharmacologically active and important are safranal, crocin, picrocrocin and crocetin .
Saffron and its constituents are widely evaluated for their pharmacological activities such as treatment of memory impairment  , antidepressant  , anticonvulsant  , antitussive  , aphrodisiac activity  , antianxiety  and hypnotic  and especially for their antitumor effect  .
Saffron or its constituents inhibited lipid peroxidation in renal  , hippocampal  and muscle skeletal  homogenates during ischemia-reperfusion-induced oxidative damage in rats. Radical scavenging activity of C. sativus L. extract and its bioactive constituents, safranal and crocin have been shown using DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging test  .
In this study we evaluated the antioxidant activity of ethanolic and aqueous extracts of saffron and its active constituents, crocin and safranal, in a range of lipid peroxidation using three experimental approaches i.e. deoxyribose assay, erythrocyte membrane peroxidation and rat liver microsomal lipid peroxidation induced by Fe2+/ascorbate.
Materials and Methods
Crocin, safranal, sodium azid and deoxyribose obtained from Fluka.
Male Wistar rats, 200-250 g were housed in colony rooms with 12/12 h light/dark cycle at 21±2°C and had free access to food and water. All animal experiments were carried out in accordance with Mashhad University of Medical Sciences, Ethical committee Acts.
Preparation of extracts
In the maceration method, 0.5g of sigma powder was macerated in 60 ml solvent (ethanol (80 v/v) and water) for 48 h. The mixture of plant and solvent was subsequently centrifuged (5 min, 3000 rpm) and the supernatants were evaporated to dryness under reduced pressure at 40°C. The yield of the extracts was 55% (w/w) for ethanolic extracts and 20% (w/w) for aqueous extract.
In this method all solutions were prepared freshly. 1.0 ml of the reaction mixture contained 100 µl of 28 mM 2-deoxy-2-ribose 500 µL solution of various concentrations of the material test (aqueous and ethanolic extracts of saffron, crocin and safranal in buffer), 200 µL of 200 µM FeCl3 and 1.04 mM EDTA (1:1 v/v), 100 µL H2O2 (1 mM) and 100 µl ascorbic acid (1 mM). After an incubation period of 1 h at 37°C, the extent of deoxyribose degradation was measured by the TBA reaction. 1 ml of TBA (1%in 50 mM NaOH) and 1 ml of TCA were added to the reaction mixture and the tubes were heated at 100 °C for 20 min. Absorbance was read at 532 nm  . The percentage of inhibition of deoxyribose degradation was calculated using the following equation
% inhibition = (A0-A1)/A0 × 100
A0: Absorbance of the control in the absence of samples
A1: The absorbance in the presence of samples.
DMSO was used as a positive control. 
Assay for site-specific reactions
This assay was done in the following three ways.
The assay was done as mentioned before except that 100 ml of FeCl3 instead of 200 ml of Fe 3+ -EDTA solution was added.The assessment was performed without ascorbic acid as a starter for oxidation.Deoxyribose itself was omitted from the assay  .
Erythrocyte membrane peroxidation
The protective effects of saffron extracts, crocin and safranal were determined according to the method that was described before , with modification. Wistar rats (200-250 g) were anesthized with chloroform, whole blood was drawn via a cardiac puncture and collected in to heparinized tubes. The RBC was separated from plasma by centrifugation at 1500 g for 15 min. Packed RBC was washed twice with NaCl 0.15 M, and preincubated with phosphate buffer (pH 7.4) containing sodium azid (1 mM) to inhibit catalase. Peroxidation was initiated by adding H2O2 (10 mM). 100 µl solution of various concentrations of the material test was added and the mixture was incubated at 37°C for 60 min. The reaction was terminated by addition of 28% (w/v) trichloroacetic acid. Lipid peroxidation was measured using thiobarbitoric method. The quantity of MDA was determined by measuring the absorbance at 532 nm ,. BHT was used as a positive control  .
Liver microsomal preparation
Liver microsomes were prepared according to the method that was described previously , with minor changes. Briefly, male Wistar rats (200-250) were sacrificed. The liver was perfused with ice-cold saline through the portal vein until uniformly pale, immediately removed. After being minced, the pieces of liver were homogenized with 4 volume of ice-cold 0.1 M potassium phosphate buffer (pH 7.4) containing 1.15% (w/v) KCl. The homogenate was centrifuged at 10000 g for 60 min. The supernatant was used for the study.
Microsomal lipid peroxidation induced by Fe2+/ascorbat
To measure the antioxidant activity of the various material test, lipid peroxidation of rat liver microsomes was carried out in vitro according to the method that was described previously  with minor changes. Briefly the reaction mixture containing 0.2 ml of rat liver microsome (2mg/ml) 0.1 ml of FeSO4 (final concentration 26% mM), 0.1 ml of ascorbate (final concentration 0.13 mM), 0.1 ml of the sample in 150 mM KCl/Tris-HCl buffer solution (pH 7.4). The mixture was incubated at 37 °C for 60 min in a water bath; the reaction was stopped by addition of 0.75 ml of 2M trichloroacetic acid /1.7 MHCl.
After centrifugation (4000 rpm, 10 min) 0.5 ml of the supernatant was mixed with 0.15 ml TBA and the mixture was heated at 95 °C for 10 min.
After cooling the quantity of MDA was determined by measuring the absorbance at 532 nm. Inhibition (I) of lipid peroxidation in percent was calculated by following equation. %I = (A0-A1/A0) × 100
A0: Absorbance of the control reaction
A1: The absorbance in the presence of the agents.
BHT was used as a positive control  .
Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. The P value less than 5% 0.05 were considered to be statistically significant. PCS software was used to calculate IC50 value.
Deoxyribose degradation assay
Absorption spectra of crocin aqueous and ethanolic extracts of saffron showed absorbance at 532 nm, therefor we prepared blank for each sample.
In deoxyribose degradation method, saffron aqueous and ethanolic extracts in various concentrations (10, 100, 250, 500, 1000 µg/ml) and its constituents crocin and safranal at (01, 0.25, 0.5, 1, 2 mM) were evaluated.
All of tested compound showed OH radical scavenging effect (P50 values for aqueous, ethanolic extract, crocin and safranal were 324 µg/ml, 338 µg/ml, 0.47 mM and 0.31 mM respectively. The results are shown in [Table 1] and [Table 2].
Assay for site specific actions
To assay site specific reaction when EDTA omitted from the system test Fe 3+ instead of Fe 3+ EDTA bound to deoxyribose and stimulated MDA generation. All of tested agents inhibit deoxyribose degradation in this condition and showed metal chelating effect.
Saffron aqueous and ethanolic extracts in concentration 500, 100 µg/ml, crocin at 1.2 mM stimulated peroxidation under test condition.
In the absence of deoxyribose, non of the tested compounds were able to react with TBA to produce colored products. Results are shown in [Table 3].
RBC Lipid Peroxidation
Both extracts in concentration 250, 500 and 1000 µg/ml inhibited RBC lipid peroxidation and MDA generation as compared with control (P50 value for crocin was 1.14 mM. Because extracts and safranal showed antioxidant effect less than 50%, IC50 for these agents were not calculated. Results are shown in [Table 4], [Table 5].
Liver microsomal lipid peroxidation
Treatment of liver microsomal with saffron extracts are (10, 100, 250, 500 and 1000 µg/ml) produced a significant decrease of MDA generation as compared with control treatment (P 0.05).
IC50 values for aqueous, ethanolic extract and crocin were 372.58 µg/ml, 44.8 µg/ml and 0.34 mM respectively. Results are shown in [Table 6], [Table 7]
In this study we evaluated the antioxidant activity of ethanolic and aqueous extracts of saffron and its major bioactive constituents, crocin and safranal, under in vitro methods.
In deoxyribose assay all agents showed hydroxyl radical scavenging activity in a dose-dependent manner. In site specific assay, in the absence of EDTA, crocin, safranal and extracts exhibited ferrous ion chelating activity. In the absence of ascorbic acid crocin (1.2 mM) and ethanolic extract (500 and 100 µg/mL) stimulated deoxyribose degradation and showed oxidant activity.
Metal ion chelation activity of an antioxidant compound prevents oxyradical generation and the consequent oxidative damage  . The inhibition of deoxyribose degradation in the absence of EDTA indicates the ability of agent to trap OH radicals and also to chelating iron  . The presence of carotenoids in saffron extracts may be responsible for the antioxidant properties ,, but carotenoids do not necessarily act as antioxidant and it was demonstrated that carotenoids could increase the total yield of free radicals depending on the oxidation potential of the carotenoids and the nature of the radicals  .
Membrane lipids are particularly susceptible to oxidative damage. This is not only because of their high polyunsaturated fatty acid content, but also because of their association in the cell membrane with enzymatic and non-enzymatic system able to generate free radical species  . Lipid peroxidation is important process in many pathological events and is one of the reactions that induced by oxidative stress ,, .
In this study all agents decrease MDA generation in RBC lipid peroxidation induced by H2O2 and liver microsomal non-enzymatic lipid peroxidation.
The antioxidant activity of saffron and its bioactive constituents may be responsible for its various pharmacological effects  such as inhibition of human cancer cells proliferation ,,, , protection of hepatocytes  and prevention muscle skeletal  , renal and cerebral ischemia-reperfusion induced oxidative injury , . Crocin a major carotenoid pigment of saffron inhibited PC12 lipid peroxidation and restored SOD activity  and also protected RBC against AAPH induced heamolysis  .
Safranal is monoterpene aldehyde which is the major constituent of the essential oil of saffron and is obtained by picrocrocin degradation , showed radical scavenging activity  and amelorated ischemia-reperfusion injury induced oxidative damage in rat hippocampus  . In this study safranal also exhibited antioxidant activity in three methods, thus the significant antioxidant activity of the saffron extracts should probably be attributed to an action of the main bioactive constituent mainly crocin but also safranal. The role of crocin in antioxidant activity of saffron extracts is more important because safranal is usually present in less that 1% in saffron extracts  .
The present study showed that ethanolic and aqueous extracts of saffron have antioxidant activity in different in vitro methods. Crocin, a major carotenoid of saffron may be have more role for antioxidant properties of saffron. Safranal a monoterpene aldehyde which is the major constituent essential oil of saffron also showed antioxidant activity.
We would like to thank School of Pharmacy, Mashhad University of Medical Sciences for financial support.
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