Free Radical Scavenging Properties and LC/MS Analysis of Bulgarian Crataegus Oxycantha Fruits Ethanol Extract
V Hadjimitova, T Traykov, P Hadjieva, P Traldi
crataegus oxycantha, free radicals, lcms, lipid peroxidation, procyanidin
V Hadjimitova, T Traykov, P Hadjieva, P Traldi. Free Radical Scavenging Properties and LC/MS Analysis of Bulgarian Crataegus Oxycantha Fruits Ethanol Extract. The Internet Journal of Alternative Medicine. 2008 Volume 6 Number 2.
Investigations on procyanidin profile on Bulgarian
The antioxidant properties of
We identified poliphenols in this extract and we proved the antioxidant effect in two biologically relevant systems.
Crataegus oxycantha (Cr. oxycantha) is used for treatment of cardiovascular diseases such as hypertension, hyperlipidemia, and in particular congestive heart failure, cancer, diabetes and sexual weakness in Arabic traditional medicine. Hawthorn may induce anti-ischemia/reperfusion-injury and anti-arrhythmic effects 1,2,3,4 .
The observed effects may be due to inhibition of free radicals generation as well as suppression growth of free radicals damage of biologically important molecules. There are investigations on antioxidant properties of hawthorn on in vitro systems. Investigations on medical effects of leaves, fruits, green fruits and flowers of many Crataegus species extracted by different methods showed antioxidant effects. It was established that Cr. monogynya, Cr. pinnatifida and Cr. aronia inhibits metal-induced lipid peroxidation 5,6,7,8,9 . These beneficial effects may in part be due to the presence of antioxidant flavonoid components 2 .
In the last decade new and interesting information on the content of polar components of natural extracts have been gained by the coupling of LC and MS technique mainly based on APC and electro spray ionization (ESI) systems. The recent availability of these ionization methods coupled with simple and effective MS/MS systems has suggested the possibility of direct infusion of the natural extract and the characterization of the different components by MS/MS. Of course this approach fails in the presence of isometric compounds and in general exhibits a specificity lower than that of LC-MS and LC-MS/MS systems. However, considering the time saving aspect of this approach, it can be thought of interest for a fast and preliminary description of natural extract mixtures.
The present study is devoted to the identification of flavonoid and procyanidin profile from Bulgarian Crataegus oxycantha fruit ethanol extract and the antioxidant properties of this extract on iron-dependant LP and on UV irradiation induced oxidation. Both LC- and direct injection – MS/MS approaches were employed in order to evaluate the power of the former in this specific frame.
Material And Methods
Preparation of the ethanol extract
Fresh fruits from Bulgarian Crataegus oxycantha (Rosaceae) were extracted with 95 % ethanol and were distilled at vacuum rotating evaporator. The yielded viscous residues were analyzed.
Instrumentation: LCQ DECA Thermoquest
ESI Parameters: Negative ion mode; Mass range: 50 – 1500; Spray voltage: 4 kV; Capillary temperature: 270 ºC; Capillary voltage: -8 V
Column: Luna C18 150 x 4.5 mm; guard column: C18; injection volume: 20 µl; solvents
A = H2O + 0.05 % acetic acid (HAC) and B = MeOH.
Gradient profile of solvent B: 10 % for 5 min, 10 – 100% in 55 min; flow rate: 0.5 ml/min.
5 µl/min; solvent H2O : MeOH 50%
Registration of tiobarbituric acid reactive species (TBA-RS) induced by Fe 2+ .
The TBA-RS of LP was measured in lipisomal suspension obtained from phospholipids of egg yolk extracted according to Folch et al. 10 . Each sample was prepared in PBS: 1 mg lipid/ml and diluted substances or a buffer for the controls. After addition of 0.1 mmol/l FeCl2 samples was incubated at 37 °C for 30 min. The 0.5 ml of 2.8% trichloroacetic acid and 0.5 ml of 0.5 % TBA were added. The solution was heated at 100 °C for 20 min. The absorption was measured at 532 nm.
The deoxyribose assay was carried out as Halliwell et al. 11 with small modifications. One ml samples of PBS. pH 7.4 containing: 0.3 mmol/l 2-deoxy-D-ribose and tested drug at concentrations between 1 and 100 µmol/l. or a buffer for the controls. After 25 min of UV irradiation (UV 220-400 nm) 0.5 ml of 2.8% trichloroacetic acid and 0.5 ml of 1 % TBA ware added. The solution was heated at 100 C for 20 min. The chromophore absorption was measured at 532 nm.
The antioxidant activity (AOA) was calculated by:
Calculation of C-50
The value concentration that provide AOA = 50 % was termed C-50. C-50 was calculated by:
The calculations used fitting of the data to the “sigmoid” model, where
Results and Discussion
The TIC LC-MS chromatograms of the natural extract is reported in Fig. 1. The system has been set to direct negative ions and in these conditions some of the most abundant peaks are due to procyanidins and to anthocyanidin with [M-H] - at m/z 463. This has been verifying by reconstructed ion chromatograms of components related to [M-H] - species (peaks
Interestingly the same fragments are observed by MS/MS of the [M-H] - species. The most abundant peak detected in the LC run (peak
With the same molecular and fragment ions, m/z 463 and 301, could be a glucoside flavonoid of quercitin type, but it is easy to distinguish from isobaric anthocyanidins by their IR spectrum 13 .
The natural extract has been injected by direct infusion leading the TIC MS in Fig. 2. Worth nothing is the fact that, aside the ion at m/z 191, which do not belong to the antocyanidin class, the most abundant anions are due to the glucoside of peonidine and to procyanidins (monomer, dimmer, trimer and, tetramer). Their nature has been confirmed by comparing their MS/MS spectra with those of the [M-H] - species of the peaks detected in LC-MS conditions. Interestingly the same trend of relative abundance is observed by measuring the relative abundance of the three mass spectrometric peaks (Fig. 2) and the relative areas of the three chromatographic peaks (see Table 1). This result is not surprising considering that both the data originate from the same ionization mechanism, i.e. procyanidin deprotonation, and put in evidence the validity of direct infusion spectrum for a semiquantitative evaluation of the main procyanidin components present in the natural extract. In the same table the times requested for the two analytical approaches are reported, making evident the advantage the direct infusion.
It is consider the presence of these anthocyanidins and procyanidins leads to the elucidation of cardiotonic action of Cr. oxycantha ethanol extract used in the medicine. In the present study we investigated the antioxidant properties of the analyzing extract in habitual systems.
Our research on antioxidant properties of this extract includes two methods. They investigate the influence of the extract on Fe 2+ -induced LP and · OH scavenging and UV protective effects. The choice of the methods is based on the healing Cr. oxycantha effect, which successfully influences cardiac performance, high blood pressure and protection against ischemia/ reperfusion brain damage; against chronic diseases such as atherosclerosis and coronary heart disease 14 . All these medical conditions are accompanied with heightened radical production and an increased LP level.
The ability of the extract to affect LP processes induced by catalytically active iron and hence to protect cell membranes from oxidative damages can be evaluated by a system of Fe 2+ induced LP (Fig 3).
Antioxidant activity values between 20% and 90% were found within the tested concentration range of 0.1 to 10 mg/ml. The last case means that only 10% of membrane lipids have been oxidized (and 90% are not) which permits us to classify the extract as a good antioxidant for the system tested. Those values are obtained as a result of three independent experiments for each of shown in Fig. 3 concentrations. On the basis of data shown in Fig. 3 C-50 value was calculated. In the tested model system we found a value of C-50 = 0.70 ± 0.02 mg/ml.
The absorbances measured for the same model system but in the presence of one of the main biologically active Crataegus fruits extract component proved by GC/MS analysis are shown in Table 2 15 . The tested concentration of 0.3 mg/ml is the same for all substances. Six among eight investigated components showed antioxidant properties. Surprisingly the nicotinic acid showed prooxidant activity. Asiatic acid did not show significant difference from the controls.
In the UV irradiation oxidation of deoxyribose system mainly hydroxyl radicals are generated and they are mostly responsible for the DNA damages 11
In our experiments the Crataegus fruits extract was tested within the concentration range of 0.01 to 1 mg/ml. Absorbance values found were calculated as an average of absorbance of 3 independent experiments for each of shown in Fig. 4 concentrations. On the basis of these data C-50 value (which produces twofold decrease of deoxyribose oxidation) was calculated. In the tested here system we found a value of C-50 = 0.26 ± 0.01 mg/ml.
The comparison of C-50 of the Crataegus extract for the both tested systems showed that its antioxidant activity is about 2.5 fold stronger in the systems of UV irradiation generated hydroxyl radicals.
Hawthorn polyphenol prepared by ethyl acetate treatment of the ethanol extract of Chinese hawthorn fruit were detected 15 polyphenols comprise flavonoids and procyanidin (monomer, dimmer, trimer, tetramer and pentamer) (2) 16 .Similar components were found in ethanol fruit extract of Bulgarian Crataegus oxicantha (Fig. 1 and 2). Those polyphenols manifested a strong superoxide and hydroxyl radical scavenging effect with C-50 values of 6.3 µg/ml and 1.1 µg/ml respectively
We obtained · OH scavenging effect lower then Chinese hawthorn but we used whole Crataegus oxycantha extract no polyphenols only.
It has been shown a positive correlation between antioxidant activity and polyphenol contents in different Bulgarian herbs water extract 17,18 . In this paper we identified poliphenols in Cr. oxycantha fruits ethanol extract and we proved the antioxidant effect in two biologically relevant and complementary systems.
The application of direct infusion – ESI – MS for the procyanidin content analysis, coupled with an evaluation of free radicals scavenging properties of the plant extract are important approaches for the phytotherapy.