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Natural Product ResearchFormerly Natural Product LettersPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713398545
Assessing the antioxidative properties and chemicalcomposition of Linaria vulgaris infusionV. Vrchovská a; J. Spilková a; P. Valentão b; C. Sousa b; P. B. Andrade b; R. M.Seabra ba Faculty of Pharmacy, Department of Pharmacognosy, Charles University, HradecKr lov , Czech Republicb Faculdade de Farm cia Universidade do Porto, REQUIMTE/Servi o deFarmacognosia, Porto, Portugal
Online Publication Date: 01 June 2008
To cite this Article: Vrchovská, V., Spilková, J., Valentão, P., Sousa, C., Andrade, P. B. and Seabra, R. M. (2008)'Assessing the antioxidative properties and chemical composition of Linaria vulgaris infusion', Natural ProductResearch, 22:9, 735 — 746
To link to this article: DOI: 10.1080/14786410601132360URL: http://dx.doi.org/10.1080/14786410601132360
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Natural Product Research, Vol. 22, No. 9, 15 June 2008, 735–746
Assessing the antioxidative properties and chemical
composition of Linaria vulgaris infusion
V. VRCHOVSKAy, J. SPILKOVAy, P. VALENTAOz, C. SOUSAz,P. B. ANDRADEz and R. M. SEABRA*z
yFaculty of Pharmacy, Department of Pharmacognosy, Charles University,Hradec Kralove, Czech Republic
zFaculdade de Farmacia Universidade do Porto,REQUIMTE/Servico de Farmacognosia, Porto, Portugal
(Received 19 June 2006; in final form 18 October 2006)
The ability of Linaria vulgaris (Scrophulariaceae) infusion to act as a scavenger of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, reactive oxygen species (superoxide radical, hydroxyl radical,hypochlorous acid (HOCl)) and nitric oxide was investigated. The obtained data indicate thatthe infusion has a good scavenging activity against superoxide radical and is a very potent nitricoxide and DPPH scavenger. In hydroxyl radical assay a pro-oxidant capacity was noticed,especially for concentrations higher than 31.25 mgmL�1. No effect was found against HOCl.A phytochemical study of this extract was also performed. The HPLC/UV analysis allowed theidentification and quantification of eight organic acids (oxalic, aconitic, citric, ketoglutaric,ascorbic, malic, shikimic and fumaric acids). The phenolic composition of the lyophilisedinfusion was also determined by HPLC/DAD and four compounds were quantified, but,despite its high content, only linarin was managed to be identified.
Keywords: Linaria vulgaris; DPPH; reactive oxygen species; nitric oxide; phenolic compounds;organic acids
1. Introduction
Linaria vulgaris Mill., known as yellow toadflax, is a perennial herb fromScrophulariaceae family widely dispersed in all parts of Europe, being extensivelyconsumed as an infusion [1]. In traditional folk medicine, fresh or dried flowering herbis used internally to aid digestion problems and urinary disorders. Externally, the plantis applied in the treatment of hemorrhoids, ulcus cruris, for ablution of festeringwounds, and skin rashes. It is also reported to have anti-inflammatory effect [1] and totreat coughs and asthma [2]. L. vulgaris is reported to contain alkaloids, iridoidglucosides, flavonoids, and aurones [1–5]. As far as we know, nothing has been reportedabout organic acids composition of this species.
*Corresponding author. Email: [email protected]
Natural Product Research
ISSN 1478-6419 print/ISSN 1029-2349 online � 2008 Taylor & Francis
http://www.tandf.co.uk/journals
DOI: 10.1080/14786410601132360
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In recent years, there has been an increasing interest in antioxidants derived from
fruits, vegetables, herbs and beverages. Antioxidants are of the interest to the food
industry because they prevent rancidity. Natural antioxidants seem to be promising
substitutes of conventional synthetic antioxidants, such as butylated hydroxyanisole
(BHA) or butylated hydroxytoluene (BHT), whose application have been restricted
because of the possible existence of reactive intermediates [6]. Antioxidants are also of
interest to biologist and clinicians, because they may help to protect the human body
against damage by reactive oxygen and nitrogen species (ROS and RNS, respectively)
[7]. These reactive species are implicated in the development of numerous chronic
diseases and also in the ageing process [8–10].However, despite its wide use, there is no available information concerning the
antioxidant activity of L. vulgaris infusion or about its main phytochemicals. The aim
of the present study was to evaluate the antioxidant potential of L. vulgaris lyophilised
infusion, since this is the most common form of using the species. Therefore, its capacity
to act as scavenger of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide
radical, hydroxyl radical, hypochlorous acid (HOCl) and nitric oxide was investigated.
The polar phenolic compounds and organic acids were determined by HPLC/DAD and
HPLC/UV, respectively, since both classes are reported to have antioxidant effect [11].
A possible relation between the chemical composition and the antioxidant potential
is discussed.
2. Results and discussion
2.1. Phenolic compounds
The analysis of the phenolic compounds in L. vulgaris lyophilised infusion revealed the
presence of four compounds, from which only linarin (acacetin-7-O-rutinoside) was
managed to be identified (table 1). This compound was already described to occur in
this species (PDR for Herbal Medicines, 1998). The other three detected compounds
(compounds 1, 2 and 3) exhibited a UV spectra identical to that of linarin, with two
maxima at 273 and 330 nm. Linariin, an acetylated derivative exhibiting an additional
shoulder at around 230 nm and previously reported in other Linaria species [12], was
not noticed.Quantification of the detected compounds revealed a high content of phenolics
(ca 65 g kg�1), from which compound 1 was the major one (ca 61.3% of total
Table 1. Phenolic composition of L. vulgaris lyophilised infusion (mgkg�1)a.
Compound Mean SD
Linarin (RT 23.2min) 3840.9 5.0a (RT 23.8min) 39576.1 430.2b (RT 29.7min) 3825.0 60.7c (RT 31.6min) 17340.1 201.8R 64582.1
aResults are expressed as mean of three determinations; SD, standard deviation; �, sum of thedetermined phenolic compounds.
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compounds) (table 1). Linarin and compound 3 were present in minor amounts,
representing ca 6.0% of total phenolic compounds (table 1).
2.2. Organic acids
The HPLC/UV analysis of L. vulgaris infusion allowed the identification and
quantification of eight organic acids: oxalic, aconitic, citric, ketoglutaric, ascorbic,
malic, shikimic and fumaric acids (figure 1). All these compounds are reported for the
first time in L. vulgaris. The total organic acids content was found to be ca 36 g kg�1,
with ascorbic acid as the major compound (ca 65.5% of total acids) (table 2), followed
by malic acid, which represented ca 25.3% of total acids (table 2). Oxalic, aconitic
and fumaric acids were the compounds present in minor amounts, lower than 0.2% of
total organic acids (table 2).
7
10.000.00 20.00 30.00 40.00 50.00 60.00 70.0
0
20
40
60
80
100
MP
8 6
5
4
3
2 1
Figure 1. HPLC organic acid profile of L. vulgaris aerial parts. Detection at 214 nm. Peaks: (MP) mobilephase; (1) oxalic acid; (2) aconitic acid; (3) citric acid; (4) ketoglutaric acid; (5) ascorbic acid; (6) malic acid;(7) shikimic acid; (8) fumaric acid.
Table 2. Organic acids composition of L. vulgaris infusion (mgkg�1)a.
Compound Mean SD
Oxalic acid (RT 19.6min) 62.5 14.0Aconitic acid (RT 25.1min) 30.1 2.2Citric acid (RT 27.9min) 2167.0 78.1Ketoglutaric acid (RT 30.04min) 651.0 24.3Ascorbic acid (RT 29.6min) 23450.0 310.0Malic acid (RT 33.9min) 9050.4 129.3Shikimic acid (RT 43.2min) 395.0 0.7Fumaric acid (RT 58.4min) 26.0 0.2R 35801.2
aResults are expressed as means of three determinations; SD, standard deviation; �, sum of thedetermined organic acids.
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2.3. Antioxidant activity
The DPPH test provides basic information on the ability of extracts to scavenge freeradicals. L. vulgaris lyophilised infusion notably reduced the concentration of DPPHfree radical in a concentration-dependent way (IC50¼ 353.85 mgmL�1) (figure 2).
Superoxide radical is known to be very harmful to cellular components as a precursorof more reactive oxygen species [7]. The extract exhibited potent superoxide radicalscavenging activity using the xanthine/xanthine oxidase (X/XO) system (figure 3a) andthe observed effect was concentration dependent (IC50¼ 48.36 mgmL�1). To evaluatethe effect of the infusion on the enzyme itself (Valentao et al., 2002), we also performeda control experiment monitoring the conversion of xanthine to uric acid (figure 3b).The results showed that at the tested concentrations, the infusion has a weak inhibitoryeffect on XO (IC50¼ 418.06 mgmL�1), so it was not possible to show a clear-cutscavenging effect on superoxide radical. To confirm the scavenging capacity of thelyophilised infusion against superoxide radical, we performed an assay using a chemicalsystem composed of phenazine methosulfate (PMS), �-nicotinamide adenine dinucleo-tide (NADH) and oxygen for production of superoxide radical. The extract stronglyinhibited the formation of blue formazan and the percentage of inhibition wasproportional to the concentration, with an IC50 value of 14.11mgmL�1 (figure 3c).
The hydroxyl radical scavenging activity was investigated using the Fenton reaction, inwhich hydroxyl radicals are generated by a mixture of Fe3þ-EDTA, H2O2 and ascorbicacid and are assessed by monitoring the degradation of deoxyribose throughmalonaldehyde (MDA) formation [7]. This experiment can be done under two differentconditions to find out (i) hydroxyl trapping and (ii) metal chelation activity. In the firstcase, the assay is performed in the presence of ethylenediaminetetraacetic acid disodiumsalt (EDTA), constituting a non-site-specific assay. EDTA forms a complex with ironand hydroxyl radicals are generated in solution. In the site-specific assay, where noEDTA is available, omission of the chelator allows iron ions to bind directly to the sugarand produce hydroxyl radicals at this site itself. Under such conditions, compoundsinhibit deoxyribose degradation not only by reacting with hydroxyl radicals, but alsobecause they present ion-binding capacity and can withdraw the iron ions and render
Concentration (mgmL−1)
0 250 500 750 1000
0
25
50
75
100
DP
PH
in
hib
itio
n (
%)
Figure 2. Effect of L. vulgaris infusion on DPPH. reduction. Values show mean� SE from threeexperiments performed in triplicate.
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them inactive or poorly active in Fenton reactions [13]. As shown in table 3, in both the
conditions, a weak activity was observed.If we omit ascorbate in the reaction mixture and if some pro-oxidant compounds are
present, they will be able to redox cycle the metal ion required for hydroxyl generation,
increasing the radical production [14]. Interesting results were obtained in the assay
Concentration (mgmL−1)
0 100 200 300 400 500 600
Concentration (mgmL−1)
0 100 200 300 400 500 600
Concentration (mgmL−1)
0 100 200 300 400 500
0
25
50
75
100(a)
(b)
(c)
Inh
ibit
ion
of
NB
Tre
du
ctio
n (
%)
0
25
50
75
Inh
ibit
ion
of
xan
thin
ere
du
ctio
n (
%)
0
25
50
75
100
Inh
ibit
ion
of
NB
Tre
du
ctio
n (
%)
Figure 3. Effect of L. vulgaris infusion on: (a) NBT reduction induced by superoxide radical generated in anX/XO system; (b) on XO activity; (c) NBT reduction induced by superoxide radical generated in a NADH/PMS system. Values show mean� SE from three experiments performed in triplicate.
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undertaken in the absence of ascorbic acid. At concentrations higher than31.25 mgmL�1, L. vulgaris lyophilised infusion exerted a very potent pro-oxidantactivity (table 3). The results obtained in the hydroxyl radical assay can be of great
relevance, since hydroxyl radical is the most reactive radical known in chemistry thatcan attack and damage almost every molecule found in living cells [7]. Hydroxylradicals are produced in vivo by Fenton-type reactions, in which transition metals areinvolved. In addition, reducing agents, such as ascorbic acid, accelerate hydroxylradical formation [15]. It seems that L. vulgaris infusion stimulates the generation ofhydroxyl radical without ascorbic acid and these findings can be important in thecase of pathological situations like Wilson’s disease or hemochromatosis, in which highnon-quelated transition metals contents, namely copper and iron, respectively, are
involved [16,17].HOCl is the most powerful oxidant produced by human neutrophils, contributing to
the damage caused by these inflammatory cells. It is produced from H2O2 and chlorideby neutrophil-derived enzyme myeloperoxidase [18]. It is a non-specific oxidising andchlorinating agent that reacts promptly with a variety of biological important moleculesbearing thiol groups [19]. Thus, HOCl damages and induces target cell lysis, caused bysulfhydryl oxidation in plasma membrane proteins [20], inactivates �1-antiprotease,activates collagenase and gelatinase, depletes antioxidant vitamins such as ascorbic acidand inactivates antioxidants enzymes like catalase [13,21]. In the present assay anHOCl scavenger inhibits the oxidation of 5-thio-2-nitrobenzoic acid (TNB) into5,50-dithiobis(2-nitrobenzoic acid) (DNTB) [22]. L. vulgaris lyophilised infusion
exhibited no activity against HOCl (figure 4a). Lipoic acid was used as a referencecompound in this assay and effectively scavenged HOCl in a concentration-dependentmanner, presenting a protective effect of 80% at 500 mgmL�1 (figure 4b). Thus, thereported traditional use of L. vulgaris as anti-inflammatory agent [1] cannot beattributed to the scavenging of HOCl.
Nitric oxide is a highly diffusible, moderately reactive, and unstable free radicalproduced in vivo by both constitutive and inducible isoforms of nitric oxide synthase[23]. Over-production of NO has been suggested to contribute to the pathology ofseveral diseases, including neurodegenerative disease and chronic inflammation [24].In fact, NO is a very important mediator in the inflammatory process: it is produced
at the inflammatory sites by an inducible nitric oxide synthase [24]. In the present workL. vulgaris extract exhibited NO radical scavenging activity, in a concentration-dependent manner, with an IC25 at 68.95 mgmL�1 (figure 5). These findings could beassociated with its anti-inflammatory efficiency [1].
Table 3. Absorbance and scavenging effect obtained in the deoxyribose assay in the presence and absence ofascorbic acid (-AA) or EDTA (-EDTA).
L. vulgaris lyophilisedinfusion (mgmL�1) ABS Scavenging ratio (%) ABS (-AA) ABS (-EDTA)
0.00 0.480 – 0.370 0.3517.81 0.410 14.5 0.356 0.32715.63 0.403 16.1 0.357 0.32731.25 0.433 9.2 0.398 0.32362.50 0.447 6.7 0.465 0.327
125.00 0.499 – 0.547 0.328
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As far as we know, this is the first report concerning the antioxidant activity ofL. vulgaris. The antioxidant capacity is supposed to be the resulting sum of effects of theseveral hydrophilic compounds present in the infusion. So, the antioxidative propertiesof L. vulgaris infusion observed in this study may be due, at least to some extent, to thepresence of flavonoids, since they are described as having significant antioxidantcapacity [7,25]. In fact, linarin and its aglycone, acacetin, have already been evaluatedfor their antioxidant ability [26,27]. Organic acids may also contribute to the scavengingproperties observed, since they are reported to have antioxidant capacity [11].In addition, antioxidant activity of other compounds should also be taken intoaccount. The strong pro-oxidant effect observed in the Fenton assay performedwithout ascorbic acid can be attributed to the presence of high amounts of this reducingagent in L. vulgaris infusion.
In conclusion, a potent antioxidant activity of L. vulgaris infusion was shown,considering the scavenging abilities displayed against superoxide radical, DPPH. andnitric oxide. This capacity can be mainly ascribed to the flavonoid derivatives
50
60
70
80
90
Concentration (mgmL−1)
TN
B r
emai
nin
g (
%)
0 100 200 300 400 500 600
Concentration (mgmL−1)
0 100 200 300 400 500 600
40
50
60
70
80
90(a)
(b)
TN
B r
emai
nin
g (
%)
Figure 4. Effect of L. vulgaris infusion (a) and lipoic acid (b) on the oxidation of TNB by HOCl. Valuesshow mean� SE from three experiments performed in triplicate.
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present in the infusion. Plant-based dietary antioxidants are hypothesised to have an
important role in maintaining human health and L. vulgaris infusion seems to be
a good source of bioactive compounds. Thus, L. vulgaris can become an interesting
object for both the food and pharmaceutical industry. However, further investigation
of individual compounds is necessary.
3. Experimental
3.1. Standards and reagents
Oxalic, citric, ketoglutaric, malic, shikimic, and fumaric acids were purchased from
Sigma (St. Louis, MO, USA). Aconitic acid and linarin were purchased from
Extrasynthese (Genay, France). Ascorbic acid, methanol, and formic acids were
obtained from Merck (Darmstadt, Germany) and sulphuric acid from Pronalab
(Lisboa, Portugal). The water was treated in a Milli-Q water purification system
(Millipore, Bedford, MA, USA). DPPH, xanthine, XO grade I from buttermilk
(EC 1.1.3.22), NADH, PMS, nitroblue tetrazolium chloride (NBT), ferric chloride
anhydrous (FeCl3), EDTA, trichloroacetic acid, thiobarbituric acid, deoxyribose,
sodium hypochlorite solution with 4% available chlorine (NaOCl), DTNB and
sulphanilamide were obtained from Sigma Chemical Co. (St. Louis, USA). Sodium
nitroprussiate dehydrate (SNP) was obtained from Riedel-de-Haen and N-(1-naphtyl)-ethylene-diamin dihydrochloride from Merck (Darmstadt, Germany).
3.2. Plant material and sampling
Sample was purchased from Natura (Decın, Czech Republic). A voucher specimen was
deposited at Department of Pharmacognosy, Faculty of Pharmacy, Charles University,
Hradec Kralove, Czech Republic.
Concentration (mgmL−1)
0 1000 2000 3000 40000
25
50
75
100
NO
sca
ven
gin
g (
%)
Figure 5. Effect of L. vulgaris infusion on the reduction of NO radical. Values mean�SE from threeexperiments performed in triplicate.
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3.3. Sample preparation
Linaria vulgaris infusion was prepared by pouring 500mL of boiling water on 5 g of
plant material. The mixture was left to stand for 15min and then filtered over a Buchner
funnel. The resulting infusion was then lyophilised (Labconco 4.5, Kansas City, MO,
USA). The yield of the lyophilised extract was 1.37 g. The lyophilised extract was kept
in an exsicator, in the dark.
3.4. Organic acids extract
The lyophilised infusion was redissolved in acid water (pH 2 with HCl) and the aqueous
solution was passed through a solid-phase extraction C18 (45 mm particle size, 60 A
porosity; 0.5 g sorbent mass/6mL reservoir volume) column (Chromabond), previously
conditioned with 2mL of methanol and 5mL of acid water (pH 2 with HCl). Organic
acids and other polar compounds were eluted with the aqueous solution. This aqueous
extract was evaporated to dryness under reduced pressure (40�C), redissolved in 0.01N
sulphuric acid (1mL) and 20 mL were analysed by HPLC/UV. A mixture of standards
was prepared by dissolving the compounds in 0.01N sulphuric acid.
3.5. HPLC analysis of organic acids
The separation was carried out according to a described procedure [28] with an
analytical HPLC unit (Gilson), using an ion exclusion Nucleogel� Ion 300 OA
(300� 7.7mm) column in conjunction with a column-heating device set at 30�C.
Elution was carried out isocratically with sulphuric acid 0.01N, at a flow rate of
0.2mLmin�1. Detection was performed with a UV detector set at 214 nm. Organic
acids quantification was achieved by the absorbance recorded in the chromatograms
relative to external standards.
3.6. HPLC analysis of phenolics
The lyophilised infusion was redissolved in water and 20 mL were subjected to HPLC
analysis. This was performed using an HPLC unit (Gilson) and a Spherisorb ODS2
reversed-phase (Waters, Milford, USA) column (250� 4.6mm, 5 mm particle size).
The solvent system was a mixture of formic acid 5% (A) and methanol (B), with a flow
rate of 1mLmin�1, and the gradient was as follows: 0min – 50% B, 20min – 60% B,
35min – 60% B. Detection was achieved with a Gilson diode array detector. Linarin was
identified on the basis of its chromatographic behaviour, by comparison of its retention
time and UV–VIS spectrum in the range of 200–400 nm, with that of authentic standard.
Peak purity was checked by means of the software contrast facilities. The data were
processed with Unipoint system Software (Gilson Medical Electronics, Villiers le Bel,
France). Phenolic compounds quantification was achieved by the absorbance recorded in
the chromatograms relative to external standard at 320 nm. All the detected compounds
were quantified as linarin.
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3.7. DPPH. scavenging activity
The antiradical activity of the extracts was determined spectrophotometrically in anELX808 IU Ultra Microplate Reader (Bio-Tek Instruments, Inc.), by monitoring thedisappearance of DPPH. at 515 nm, according to a described procedure [11]. For eachextract, a dilution series (five different concentrations) was prepared in a 96-well plate.The reaction mixtures in the sample wells consisted of 25 mL of lyophilised infusion and200 mL of DPPH. dissolved in methanol. Three experiments were performed in triplicate.
3.8. Evaluation of superoxide (O��2 ) radical scavenging activity
Antiradical activity was determined spectrophotometrically in an ELX808 IU UltraMicroplate Reader (Bio-Tek Instruments, Inc.), by monitoring the effect of thelyophilised infusion on the O��2 -induced reduction of NBT at 562 nm.
3.8.1. Non-enzymatic assay. Superoxide radicals were generated by the NADH/PMSsystem according to a described procedure [29]. All components were dissolved inphosphate buffer 19mM, pH 7.4. Three experiments were performed in triplicate.
3.8.2. Enzymatic assay. Superoxide radicals were generated by the X/XO systemfollowing a described procedure [29]. Xanthine was dissolved in NaOH 1 mM andsubsequently diluted in phosphate buffer 50mM with EDTA 0.1mM, pH 7.8, XO inEDTA 0.1mM and the other components in phosphate buffer 50mM with EDTA0.1mM, pH 7.8. Three experiments were performed in triplicate.
3.8.3. Effect on XO activity. The effect of the lyophilised infusion on XO activity wasevaluated by measuring the formation of uric acid from xanthine in a double beamspectrophotometer (He�ios �, Unicam), at room temperature. The reaction mixturescontained the same proportion of components as in the enzymatic assay for superoxideradical scavenging activity, except NBT, in a final volume of 600 mL. The absorbancewas measured at 295 nm for 2min. Three experiments were performed in triplicate.
3.9. Hydroxyl radical (.OH) assay
The deoxyribose method for determining the effect of the infusion on hydroxyl radicalswas performed according to a described procedure [14]. Reaction mixtures containedascorbic acid, FeCl3, EDTA, H2O2, deoxyribose and lyophilised extract. Allcomponents were dissolved in KH2PO4-KOH buffer 10mM, pH 7.4. This assay wasalso performed without either ascorbic acid or EDTA, in order to evaluate the extractspro-oxidant and metal chelation potential, respectively. In each case three experimentswere performed in triplicate.
3.10. Hypochlorous acid (HOCL) scavenging activity
The inhibition of HOCl-induced TNB oxidation to 5,50-dithiobis(2-nitrobenzoic acid)was performed according to a described procedure [14], in a double beam
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spectrophotometer (He�ios �, Unicam). HOCl and TNB were preparedimmediately before use. Scavenging of HOCl was ascertained by using lipoic acid asa reference scavenger. The amount of TNB unchanged after incubation was calculatedand expressed as percentage of the initial value. Three experiments were performed intriplicate.
3.11. Nitric oxide (.NO) scavenging activity
The ability of the lyophilised infusion to scavenge nitric oxide radical was determinedspectrophotometrically in an ELX808 IU Ultra Microplate Reader (Bio-TekInstruments, Inc.) according to a described procedure [30] with some modifications.A dilution series (five different concentrations) was prepared in a 96-well plate.The reaction mixtures in the sample wells consisted of extract and SNP dissolved insaline phosphate buffer pH 7.4. The plates were incubated at 25�C for 60min underlight. Afterwards Griess reagent (1% sulphanilamide and 0.1% naphthyletylenediaminedihydrochloride in 2% H3PO4) was added and the absorbance of the chromophoreformed during the diazotisation of nitrite with sulphanilamide and subsequentcoupling with naphthylethylenediamine was read at 562 nm. Three experiments wereperformed in triplicate.
Acknowledgements
V. Vrchovska is grateful to Mobility Fund of Charles University and Zentiva a.s. forfinancial support (MSM 0021620822).
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