)joebxj1vcmjtijoh$psqpsbujpo …

Research ArticleBioaccessibility In Vitro of Nutraceuticals fromBark of Selected Salix Species

Urszula Gawlik-Dziki1 Danuta Sugier2 Dariusz Dziki3 and Piotr Sugier4

1 Department of Biochemistry and Food Chemistry University of Life Sciences Skromna Street 8 20-704 Lublin Poland2Department of Industrial and Medicinal Plants University of Life Sciences in Lublin Akademicka 15 20-950 Lublin Poland3Department of Thermal Technology University of Life Sciences Doswiadczalna 44 20-280 Lublin Poland4Department of Ecology Faculty of Biology and Biotechnology Maria Curie-Skłodowska University Akademicka 1920-033 Lublin Poland

Correspondence should be addressed to Urszula Gawlik-Dziki urszulagawlikuplublinpl

Received 31 August 2013 Accepted 29 December 2013 Published 17 February 2014

Academic Editors P Cos and W Gelderblom

Copyright copy 2014 Urszula Gawlik-Dziki et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of this studywas to investigate and to compare the extractability bioaccessibility and bioavailability in vitro of antioxidativecompounds from bark of selected Salix species S alba (SA) S daphnoides (SD) S purpurea (SP) and S daphnoides x purpurea(SDP) hybrid willow clones originating from their natural habitats and cultivated on the sandy soilThe highest amount of phenolicglycosides was found in the bark of SDP and SD The best source of phenolics was bark of SDP The highest content of flavonoidswere found in SD bark samples whereas the highest concentration of bioaccessible and bioavailable phenolic acids was determinedin SDP bark Bark of all tested Salix species showed significant antiradical activity This properties are strongly dependent onextraction system and genetic factors Regardless of Salix genotypes the lowest chelating power was found for chemically-extractable compounds Bark of all Salix species contained ethanol-extractable compounds with reducing ability Besides this highbioaccessibility and bioavailability in vitro of Salix bark phytochemicals were found Obtained results indicate that extracts frombark tested Salix genotypes can provide health promoting benefits to the consumers however this problem requires further study

1 Introduction

The willow bark is a constituent of many herbal drugs andalso dietary supplements such as an analgesic antipyreticantiphlogistic and weight loss enhancement remedies Theterm Salicis Cortex [SC] is defined as whole or fragmenteddried bark of young branches or whole dried pieces of currentyear twigs of various species of the genus Salix [1] SC isstandardised based on the content of salicin a compoundwith analgesic and antiphlogistic properties However clini-cal trials suggest that other compounds also present in SalicisCortex can contribute to the pharmacological effects Theresults of clinical trials suggest that besides salicylic deriva-tives other substances like polyphenols (flavonoids flavan-3-ols) and simple phenols (phenolic acids) can contribute to thetherapeutic effects of SC [2 3]

Phenolic compounds possess strong antioxidant activitythus this effect probably results from synergistic interactionsbetween them and salicin Antioxidant activity is the fun-damental property of phenolic medicinal plant compoundsimportant for their health protecting including antimuta-genic anticarcinogenic and antiaging activity Reactive oxy-gen species (ROS) scavenging preserves the genomic stabilityof cells through elimination of carcinogens and interferencewith DNA adducts formation [4] Free radicals are constantlygenerated in the body as a result of oxidativemetabolismThecreation of ROS also is connected with lipoxygenase (LOX)and xanthine oxidase (XO) activity Subsequently oxidativestress occurs if the antioxidant defense in the organism is notadequateThe condition of in vivo ldquooxidative stressrdquo is definedas elevated levels of free radicals or other ROSwhich can eliciteither direct or indirect damage to the body [5]

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 782763 10 pageshttpdxdoiorg1011552014782763

2 The Scientific World Journal

Plant extracts containing secondary metabolites haveserved as antioxidants in phytotherapeutic medicines toprotect against various diseases for centuries Natural antiox-idants exhibit a wide range of pharmacological activitiesand have been shown to have anticancer anti-inflammatoryand antiaging properties [6] Accordingly phytochemicalscan directly interfere with signaling systems involved inthe regulation of inflammatory processes angiogenesis andcancer invasion in amanner dependent on their antioxidativeactivity and concomitant inhibitory effect on the enzymesinvolved with pathological condition [7 8]

S purpurea L S daphnoides Vill and S alba L are avery popular herbal species affirmed in the natural habitatsand field-cultivated in Poland [9 10]The phenolic glycosidescontained in willow bark of this species are known fortheir anti-inflammatory analgesic and fever-reducing effectsand have been shown to relieve rheumatic disturbancesinfections and headache [3 11] Additionally bark of thisspecies contains p-hydroxybenzoic vanillic cinnamic p-coumaric ferulic and caffeic acids phenolic acids and narin-genin [1] However little is as yet known about their synergiceffects A particularly important issue on the effectivenessof dietary supplements is bioavailability and bioavailabilityof active compounds Thus the aim of this study was toinvestigate and to compare the extractability bioaccessibilityand bioavailability in vitro of antioxidative compounds frombark of selected Salix species

2 Materials and Methods

21 Plant Material Selected S alba (SA) S daphnoides (SD)S purpurea (SP) and S daphnoides x purpurea (SDP) hybridwillow clones with the highest phenolic glycoside contents[9 10] originating from their natural habitats were cultivatedon the sandy soil (heavy loamy sand) The study was carriedout in 2009 and 2010 on a 5-6 year-old plantation Theexperiment was a completely randomized block design withthree replicates conducted in the area of experimental fieldsat theUniversity of Life Sciences in Lublin (51∘331015840N 22∘441015840E)The plantation was established at the spacing of 40 times 20 cmEach plot was 16m2 The sandy soil was characterized byan average content of humus (141) very low phosphorus(173mgsdotkgminus1) very low potassium (332mgsdotkgminus1) very lowmagnesium (150mgsdotkgminus1) and strong acid reaction (pHKClmdash41) Mineral fertilization was applied in spring beforethe beginning of vegetation in both soil types Nmdash20 kg Pmdash131 kg Kmdash498 kg calculated per 1 ha

Willow shoots were harvested in November every year(2009-2010) in three replicates The plant material was col-lected in the form of 60 annual shoots (20 entire shootsper plot) from every taxon The shoots were washed withdeionized water Bark was separated from the wood bypeeling and subsampled for chemical analysis

The bark material sampled for salicylate analysis wasdried at room temperature and intensivelymixed andhomog-enized After drying the phenolic glycosides content cal-culated on salicin was determined by means of the HPLC

technique [12] in the Laboratory of Labofarm in StarogardGdanski and expressed as mgmg dry mass (DM)

22 Chemicals Ferrozine (3-(2-pyridyl)-56-bis-(4-phenyl-sulfonic acid)-124-triazine) ABTS (221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) 120572-amylasepancreatin pepsin bile extract Folin-Ciocalteau reagentlinoleic acid ammonium thiocyanate and haemoglobinwere purchased from Sigma-Aldrich Company (PoznanPoland) All others chemicals were of analytical grade

23 Extracts Preparation

231 Chemical Extract (CE) Two grams of plant materialwas filled with 100mL of 70 ethanol and left in darknessby 2 weeks

232 Buffer Extract (BE) Powdered samples of willow bark(1 g) were extracted for 1 h with 20mL of PBS buffer (phos-phate buffered saline pH 74) The extracts were separated bydecantation and the residues were extracted again with 20mLof PBS buffer Extracts were combined and stored in darknessat minus20∘C

233 Digestion In Vitro (DE) In vitro digestion and absorp-tion were performed according to Gawlik-Dziki [13] Thebark samples (1 g) were homogenized in a stomacher lab-oratory blender for 1min to simulate mastication with thepresence of 15mL of simulated salivary fluid (prepared bydissolving 238 g Na

2HPO4 019 g KH

2PO4 and 8 g NaCl

100mg of mucin in 1 liter of distilled water The solutionwas adjusted to pH = 675 and 120572-amylase (EC 3211)was added to obtain 200U per mL of enzyme activity Forthe gastric digestion 300UmL of pepsin (from porcinestomach mucosa pepsin A EC 34231) in 003molL NaClpH = 12 was prepared Further simulated intestinal juicewas prepared by dissolving 005 g of pancreatin (activityequivalent 4 xUSP) and 03 g of bile extract in 35mL01molLNaHCO

3) and subsequently the samples were shaken for

10min at 37∘C The samples were adjusted to pH = 12 using5molL HCl and subsequently 15mL of simulated gastricfluid was added The samples were shaken for 60min at37∘C After digestion with the gastric fluid the samples wereadjusted to pH = 6 with 01molL of NaHCO

3and then

15mL of a mixture of bile extract and pancreatin was addedThe extracts were adjusted to pH = 7 with 1molL NaOHand finally 5mL of 120mmolL NaCl and 5mL of mmolLKCl were added to each sample The prepared samples weresubmitted for in vitro digestion for 120 minutes at 37∘Cin the darkness After that samples were centrifuged andsupernatants were used for further analysis

234 Absorption In Vitro (AE) Considering that antioxi-dants absorption takes placemainly at the intestinal digestionstage the resulting mixture (fluids obtained after in vitrodigestion) was transferred to the dialysis sacks (D9777-100FTSigma-Aldrich) placed in an Erlenmeyer flask containing50mL of PBS buffer and incubated in a rotary shaker (2 times

The Scientific World Journal 3

per 2 h 37∘C) The PBS buffer together with the compoundsthat passed through the membrane (dialysate) was treated asan equivalent of the raw material absorbed in the intestineafter digestion

24 Determination of Total Phenolics Content (TPC) Totalphenols were estimated according to the Folin-Ciocalteaumethod [14] A 05mL sample of the extract was mixedwith 05mL of H

2O 2mL of Folin reagent (1 5 H

2O) and

after 3min with 10mL of 10 Na2CO3 After 30min the

absorbance of mixed samples was measured at a wavelengthof 725 nm The amount of total phenolics was expressed asgallic acid equivalents (GAE) per gram of dry mass (DM)

25 Determination of Total Flavonoids (TFC) Total flavon-oids were estimated according to the method described byBahorun et al [15] One milliliter of sample was mixed with1mL 2 g100mL AlCl

3times 6H2O After 10min absorbance

at 430 nm was measured The total flavonoids content wasexpresses as quercetin equivalent (QE) in milligrams pergram of DM

26 Determination of Total Phenolic Acids (TPA) Total phe-nolic acids content was determined according to the Arnovmethod [16] One milliliter of sample was mixed with 5mLof distilled water 1mL 05molL HCl 1mL of Arnov reagent(10 g sodium molybdate and 10 g sodium nitrite dissolvedin 100mL of distilled water) and 1mL 1molL NaOH andcomplete to 10mL with distilled water Absorbance wasmeasured at 490 nm The total phenolic acids content wasexpressed as caffeic acid equivalent (CAE) inmicrograms pergram of DM

27 Free Radical Scavenging Assay (AA) The experimentswere performed using an improved ABTS decolorizationassay [17] ABTS+∙ was generated by the oxidation of ABTSwith potassiumpersulfateTheABTS radical cation (ABTS+∙)was produced by reacting 7mmolL stock solution of ABTSwith 245mmolL potassium persulphate (final concentra-tion) The ABTS+∙ solution was diluted (with distilled water)to an absorbance of 07plusmn005 at 734 nmThen 40120583Lof samplewas added to 18mL of ABTS+∙ solution and the absorbancewas measured at the end time of 5min The ability of theextracts to quench the ABTS free radical was determinedusing the following equation

scavenging = [(119860119862minus 119860119860)

119860119862

] times 100 (1)

where 119860119862 absorbance of control and 119860

119860 absorbance of

sampleAntioxidant activity was expressed as IC

50mdashextract con-

centration provided 50 of activity based on dose-dependentmode of action

28 Metal Chelating Activity (CHEL) Chelating power wasdetermined by the method of Guo et al [18] The extractsamples (5mL) were added to a 01mL of 2mM FeCl

2

solution and 02mL 5mM ferrozine and the mixture wasshaken vigorously and left standing at room temperature for10min Absorbance of the solution was then measured spec-trophotometrically at 562 nmThe percentage of inhibition offerrozine-Fe2+ complex formation was given below formula

inhibition = [1 minus (119860119875119860119862

)] times 100 (2)

where 119860119862 absorbance of the control and 119860

119875 absorbance in

the presence of the sampleAntioxidant activity was expressed as IC

50mdashextract con-


29 Ferric Reducing Power (FRAP) Reducing power wasdetermined using the method described by Oyaizu [19]Extracts (25mL) were mixed with phosphate buffer (25mL200mmolL pH 66) and 25mL of 1 g100mL aqueoussolution of potassium ferricyanide K

3[Fe(CN

6)]Themixture

was incubated at 50∘C for 20min A portion (05mL) of10 g100mL trichloroacetic acid was added to the mix-ture which was then centrifuged at 25timesg for 10min Theupper layer of solution (25mL) was mixed with distilledwater (25mL) and 05mL of 01 g100mL FeCl

3 and the

absorbance was measured at 700 nm IC50

value (mgmL) isthe effective concentration at which the absorbance was 05for reducing power and was obtained by interpolation fromlinear regression analysis

210 Inhibition of Lipoxygenase (LOXI) Lipoxygenase activ-ity was determined spectrophotometrically at a temperatureof 25∘C by measuring the increase of absorbance at 234 nmover a 2min period [20] The reaction mixture contained245mL 115molL phosphate buffer 002mL of lipoxygenasesolution (167UmL) and 005mL of inhibitor (vegetableextract) solution After preincubation of the mixture at 30∘Cfor 10min the reaction was initiated by adding 008mL25mmolL linoleic acid One unit of LOX activity wasdefined as an increase in absorbance of 0001 per minute at234 nm

Antioxidant activity was expressed as IC50mdashextract con-


211 Inhibition of Xanthine Oxidase (XOI) TheXOI activitieswith xanthine as a substrate were measured spectrophoto-metrically [21] with the following modification the assaymixture consisted of 05mL of test solution 13mL of115molL phosphate buffer (pH 75) and 02mL of enzymesolution (001UmL in M15 phosphate buffer) After prein-cubation of the mixture at 30∘C for 10min the reactionwas initiated by adding 15mL of 015mmolL xanthinesolution The assay mixture was incubated at 30∘C and theabsorbance (295 nm) was measured every minute for 10minXO inhibitory activity was expressed as the percentage


inhibition of XO in the above assay mixture system and wascalculated as follows

inhibition = (1 minusΔ119860mintestΔ119860minblank

) times 100 (3)

where Δ119860mintest is the linear change in absorbance perminute of test material and Δ119860minblank is the linear changein absorbance per minute of blank



212 Theoretical Approach The following factors were deter-mined to better evaluate the extractability of phenolic com-pounds

(i) the mastication efficiency factor (MEF) which is anindication extractability of the phytochemicals duringsimulated mastication

MEF = 119862BE119862CE (4)

(ii) the digestion efficiency factor (DEF) which is anindication extractability of the phytochemicals duringsimulated digestion

DEF =119862GE119862CE (5)

(iii) the absorption efficiency factor (AEF) which is anindication extractability of the phytochemicals duringsimulated absorption

AEF = 119862AE119862CE (6)

where 119862BE is the concentration of phenolics in rawextract (BE) 119862GE is the concentration of phenolicsin extract after simulated gastrointestinal digestion(GE) and 119862AE is the concentration of phenolics inextract after simulated intestinal absorption (AE)

The following factors were determined to better under-stand the relationships between biologically active com-pounds in the light of their bioaccessibility bioavailabilityand bioefficiency

(i) the antioxidant bioaccessibility index (BAC) which isan indication of the bioaccessibility of antioxidativecompounds

BAC = 119860BE119860GE (7)

(ii) the antioxidant bioavailability index (BAV) which isan indication of the bioavailability of antioxidativecompounds

BAV =119860GE119860AE (8)

c

a

b

a

0

20

40

60

80

100

120

S alba S daphnoides S purpurea S d x p

Salic

in co

nten

t (m

gg

DM

)

Figure 1 Comparison of phenolic glycosides content in Salix barkBars (means) followed by the different letters differ significantly(Tukey-test 119875 lt 005)

(iii) the antioxidant bioefficiency index (BEF) which is anindication of the bioactivity of bioavailable antioxi-dant compounds

BEF = 119860BE119860AE (9)

where 119860BE is EC50

of raw extract (BE) 119860GE is IC50

of extract after simulated gastrointestinal digestion(GE) and AAE is IC

50of extract after simulated

intestinal absorption (AE)

213 Statistical Analysis All experimental results are dis-played as plusmn SD of three parallel experiments (119899 = 9) anddata were evaluated by analysis of variance (one-way Anova)The statistical differences between the groups were estimatedusing the Tukey test 120572 = 005

3 Results and Discussion

31 Comparison of Phytochemicals Content Salix bark sam-ples were characterized by a diverse content of pheno-lic glycosides The highest amounts were found in thebark of SDP and SD whereas the lowestmdashin the bark ofSA (Figure 1) These results were in accordance with thoseobtained by Sugier et al [10] Several studies have focusedon phytochemical investigation of Salix species used forpreparing the final willow bark products and for exampleS daphnoides S pentandra S purpurea S alba and Sfragilis have been investigated for their content of phenolicglycosides Salicylates (calculated as salicin) are found in allmembers of Salix species but S daphnoides S fragilis andS purpurea contain the greatest yield [22]These constituentsare also reported to possess antirheumatic antipyretic hyper-glycemichypoglycemic uricosuricantiuricosuric activitiesincreases prothrombin time and plasma-albumin binding[23] In addition to salicylates flavonoids and condensed tan-nins constitute the major groups of secondary metabolites inSalix species and these compounds are believed to contribute


Table 1 Comparison of content and extractability factors of phenolic compounds

Plant material Extracts Extractability factorsChemical Buffer Digested Absorbed MEF DEF AEF

TPC

S alba 38984 plusmn 1532dA11464 plusmn 611

dB8220 plusmn 255

dC5188 plusmn 212

dD 029 021 013S daphnoides 46897 plusmn 1435

bA16250 plusmn 598

bB14117 plusmn 414

bC13561 plusmn 554

bC 035 030 029S purpurea 41493 plusmn 1725

cA13490 plusmn 512

cB11329 plusmn 329

cC10892 plusmn 438

cD 033 027 026S d x p 74012 plusmn 2021

aA24992 plusmn 721

aB19049 plusmn 922

aC18074 plusmn 542

aD 034 026 024

TFC

S alba 2193 plusmn 099dA

1051 plusmn 055dB

1548 plusmn 077dC

751 plusmn 033dD 048 071 034

S daphnoides 27595 plusmn 958aA

6173 plusmn 269aB

6972 plusmn 123aB

3153 plusmn 125aC 022 025 011

S purpurea 8605 plusmn 321cA

2747 plusmn 141cB

3279 plusmn 113cC

1704 plusmn 081cD 032 038 020

S d x p 17790 plusmn 789bA

5229 plusmn 254bB

5899 plusmn 225bB

2622 plusmn 084bC 029 033 015

TPA


bB13773 plusmn 655

dC14519 plusmn 345

cD 125 082 086S daphnoides 25281 plusmn 1011

bA26944 plusmn 913

aA35761 plusmn 1011

bB29774 plusmn 918


cA11776 plusmn 777

cB21022 plusmn 923

cC17520 plusmn 713

dD 058 104 087S d x p 32294 plusmn 948

aA21040 plusmn 1145

bB45487 plusmn 1225

aC43354 plusmn 1452

aD 065 141 134The values designated by the different small letters in the columns of the table (within individual extract and fraction) are significantly different (120572 = 005)The values designated by the different small letters in the lines of the table are significantly different (120572 = 005)

to the analgesic and anti-inflammatory effect of willow bark[24]

As it can be seen from Table 1 regardless of kind ofextract the best source of phenolic compounds (expressed asgallic acid equivalent) was bark of SDP However the highestcontents of flavonoids were found in samples obtained fromSD bark Taking into account alcohol and buffer extractsthe highest content of phenolic acids was determined insamples obtained from SD bark however the highest theirconcentration was determined in extracts obtained aftersimulated digestion and absorption of SDP bark Taking intoaccount data presented in Table 1 it can be concluded that theleast valuable source of phenolic compounds was SA bark

Taking into account extractability of phenolic com-pounds fractions the highest extractability of total phenolicsin all samples has been reached in the simulated masticationstage whereas simulated digestion efficiencywas significantlylower Importantly efficiency of simulated absorption (withthe exception of SA bark) was comparable to that determinedfor the simulated digestion systemThese resultsmay indicatehigh bioavailability of phenolic compounds from Salix barkTaking into account flavonoids and phenolic acids thehighest efficiency was found for simulated digestion system(except SA phenolic acids) which may indicate high bioac-cessibility of these compounds For flavonoids effectivenessof simulated absorption system was the lowest which mayindicate low bioavailability of these compounds whereas forphenolic acids AEF values were generally comparable toDEFvalues which may indicate high bioavailability of phenolicacids from tested sources (Table 1)

Results concerning total phenolics content obtained inour laboratory were significantly higher than those concern-ing S aegyptiaca [25] The total phenol content of 212 plusmn 4mgGAEg of DM was obtained in the ethanolic extract of Saegyptiaca bark while the lowest total phenol content of 4 plusmn1mg GAEg of dried sample was obtained in cyclohexaneextract of bark However in comparison with our studyethanolic extract of S aegyptiaca bark contained higher

amount of flavonoids 479 plusmn 63mg CEg of DM was observedin the ethanolic extract of bark This difference may be par-tially explained by the fact that in our study total flavonoidscontent was expressed by quercetin equivalent Accordingto our research bark of analysed Salix genotypes containedsignificant amount of phenolic acids The presence of p-hydroxybenzoic vanillic cinnamic p-coumaric ferulic andcaffeic acids was reported in the genus Salix [1 25] Accordingto work of Podłocka-Olech et al [1] the forms of glycosidesor ester derivatives are wider spread than free phenolic acidsFurthermore the revealed presence of pyrocatechol in thebark of some willow species raises a question regarding thesafety of the application of this herbal medicine mainly thetwigs [1] On the other hand Freischmidt et al [26] concluded(from the in vitro data) that not only flavonoids and salicinderivatives but also catechol can probably contribute to theanti-inflammatory activity of willow bark extracts

32 Extractability of Antioxidants Several phenolic acidsincluding salicylic and caffeic acids possess anti-inflamma-tory and analgesic activity which has been associated withtheir antioxidant activity Scavenging oxygen free radicalsdecides the anti-inflammatory activity of gallic and proto-catechuic acids [27] Antioxidant activity was also shownfor caffeic ferulic and chlorogenic acids [1] Bark of alltested Salix species showed significant antiradical activity(Figure 2)These properties strongly dependent of extractionsystem and genetic factors Taking into account extractionprocedure better results were obtained when using EtOHthan PBS buffer with the exception of SDP bark Digestionin vitro in all cases (except SDP bark) released compoundsable to quench ABTS radicals Antiradical compounds fromSA and SP were easily bioavailable in vitro whereas potentialbioavailability of antiradical compounds released during invitro digestion from bark of SD and SDP was difficultRegardless of this the highest antiradical activity was foundfor extracts obtained from SDP (Figure 2)


acd c

h

bb

j

i

c

fd

ghde d e

g

05

1015202530354045


IC

(mg

DM

mL)

50

Chemical extractBuffer extract

DigestedAbsorbed

Figure 2 Effect of extraction system on antiradical activity ofphytochemicals from bark of different Salix genotype Bars (means)followed by the different letters differ significantly (Tukey-test 119875 lt005)

Taking into account the chelating power of analyzedsamples the similar relationships have been received forall Salix varieties (Figure 3) Regardless of plant materialthe lowest activity was found for chemically extractablecompounds Significantly higher activity was observed inthe cases of potentially mastication-extractable compounds(buffer extracts) Digestion in vitro released compounds ableto metal ions chelate from all samples except SDP bark Mostimportantly active compounds were highly bioavailable invitro whereas significant differences between samples werenot found (Figure 3)

Bark of all Salix species contained ethanol-extractablecompounds with comparable reducing ability Significant dif-ferences were concerning potentially mastication-extractablephytochemicalsmdashthe highest activity was observed in thecase of SD and SDP bark whereas the lowest for bark ofSP Digestion in vitro unexpectedly did not release activecompounds-activity of these extracts and was the lowest inall cases Most importantly compounds with reducing abilityeasily permeated dialysis membrane which may indicatetheir bioavailability In all cases activity of extracts aftersimulated absorption was the highest (Figure 4)

Antioxidant activity of Salix bark was widely studied[24 26 27] but comparative analysis of results was difficultdue to different ways of its measure and expression And soantiradical activity of bark of S aegyptiaca (depending onthe extraction system) ranged from 10 to 105mg quercetinequivalentd DM [26]

The results from in vitro studies showed that methanolicextract from S nigra bark is an effective scavenger of bothsuperoxide and hydrogen peroxide radicals This may be dueto the phenolic content particularly flavonoids present inS nigra extract which are believed to be potent superoxideradical scavengersMoreover presence of such compounds inthe extract may help neutralize hydrogen peroxide into waterthrough electron donation The antioxidative properties ofmethanolic extract from S nigra on scavenging hydrogen

a

ef

ef

b b b b

cc c

b

d d d d

000

040

080

120

160

200



DigestedAbsorbed

IC50

(mg

DM

mL)

Figure 3 Effect of extraction system on chelating power of phy-tochemicals from bark of different Salix genotype Bars (means)followed by the different letters differ significantly (Tukey-test 119875 lt005)

a a a a

bc

d

c

ded

de

b

f

hg

i

0

1

2

3

4

5

6

7



DigestedAbsorbed

IC50

(mg

DM

mL)

Figure 4 Effect of extraction system on reducing power of phy-tochemicals from bark of different Salix genotype Bars (means)followed by the different letters differ significantly (Tukey-test 119875 lt005)

peroxide radicals were found to be superior (IC50

value was175ndash180 120583gmL) [23] while the ability to inhibit superoxideradicals was lower than that reported for S caprea flowerextract [28]This activity of Salix bark is attributed to the highantioxidant contents which could react with free radicals tostabilize and terminate radical chain reactions [29] Oxidativestress and inflammation have been reported to be closelyassociatedwith tumor promotion stage of carcinogenesisTheinactivation of enzymes by free radicals and the accumulationof oxidized proteins may play a critical role in the alterationof cellular function and cell death Some essential growthregulatory proteins lose their function when damaged by freeradicals [30]

In the light of data given in Figure 5 it may be concludedthat compounds able to inhibit LOX were easily ethanol-extractable Activity of all extracts was comparable andvery high in comparison with other samples obtained fromthe same plant material Significantly lower efficiency was


a a a a

cdc

dbc

f

bc

b

e e e

d

000

050

100

150

200

250

300



DigestedAbsorbed

IC50

(mg

DM

mL)

Figure 5 Effect of extraction system on LOX-inhibitory activity ofphytochemicals from bark of different Salix genotype Bars (means)followed by the different letters differ significantly (Tukey-test 119875 lt005)

observed after using PBS buffer Extracts containing poten-tially mastication extractable phytochemicals were charac-terized by comparable activity Significant differences wereobserved for extracts obtained after simulated digestionDigestion in vitro released active compounds fromall samplesexcept S alba bark Only in this case activity of simulateddigesta was lower than activity of buffer extract Unfortu-nately compounds able to inhibit LOXwith difficulty perme-ated dialysis membrane Activity of extracts after simulatedabsorption was the lowest in all cases except SA sample(Figure 5)

Most of the tumor promoters have been reported toincrease the activity of XO thus increasing superoxide radicalgeneration and enhancing hydrogen peroxide generation[30] Taking into account activity of XO inhibitors it maybe concluded that the best results were obtained after usingethanol for extraction Extractability in a buffer system wassignificantly lower Especially significant differences wereobserved for SA and SD active compounds whereas thelowestmdashfor phytochemicals from SDP Changes occurringduring simulated digestion caused decrease of activity in allsamples except SD This tendency is strongly visible in SDPand SP bark All the more interesting is the fact that XOinhibitors easily permeated dialysis membrane (Figure 6) Inall cases the highest activity was found for extracts aftersimulated absorption

Results presented in this study confirm the resultsobtained by Sultana and Saleem [30] Cited investigatorsstated that the pretreatment of S caprea was observed toreverse the chemical induction in XO activity and hydrogenperoxide content This suggests the antitumor promotingpotential of S caprea High antioxidant activity under in vitroconditions of methanol extractfrom S nigra was also con-firmed It has a great potential to ameliorate the progressionof collagen-induced arthritis by controlling inflammatoryproteins nitric oxide and antioxidant enzymes [23]

On the other hand is interesting to note that water extractfrom SD bark used as elicitor significantly increased LOX

a a a a

ef

d

c

f fg g

b b ba

0123456789

10



DigestedAbsorbed

IC50

(mg

DM

mL)

Figure 6 Effect of extraction system on XO-inhibitory activity ofphytochemicals from bark of different Salix genotype Bars (means)followed by the different letters differ significantly (Tukey-test 119875 lt005)

and XO-inhibitory activity in broccoli sprouts Treatmentwith 1 SD water extract also significantly improved SOD-like activity and increase of OH∙ radicals scavenging abilityprobably by stimulating phenolics overproduction in broccolisprouts [31 32]

33 Studies of Potential Bioaccessibility and BioavailabilityThe gastrointestinal tract may be considered as an extractorwhere both the mechanical action during mastication andthe chemical action during the digestive phase contributeto the extraction of phenolic compounds [33] It should betaken into consideration that high content and activity ofphytochemicals determined in chemical system not alwaysgo hand in hand in high activity in vivo For this reasonthe investigations of bioaccessibility and bioavailability ofbioactive compounds should be carried out Models basedon human physiology have been developed as simple cheapand reproducible for investigating the food components Invitro digestionmodels are widely used for studying structuralchanges digestibility and release of food components undersimulated gastrointestinal conditions [34]

Data given in Table 2 indicate that the best source ofbioaccessible antiradical compounds was bark of SP whilethe lowest BAC value was obtained for bark of SDP howeverhighly bioavailable in vitro were phytochemicals able toquench free radicals released during in vitro digestion frombark of SP and SA The highest bioefficiency was found forantiradical from SP Taking into account potential bioacces-sibility of compounds able to metal ions chelate samples maybe ordered as follows 119878119860 gt 119878119863 ge 119878119875 gt 119878119863119875 Unexpectedlythe highest bioavailability and bioefficiency in vitro werefound for SDP phytochemicals Similar relationship wasobserved comparing bioavailability and bioefficiency in vitroof reductive compoundsmdashthe highest BAV and BEF valueswere determined for SDP bark samples while their potentialbioaccessibility was relatively low Though relatively lowbioaccessibility in vitro (BAC values not exceed 1) LOXinhibitors released from tested Salix bark were strongly


Table 2 Comparison of antioxidants bioaccessibility (BAC) bioavailability (BAV) and bioefficiency (BEF) factors

Activity Plant material FactorsBAC BAV BEF

Antiradical activity

S alba 162 135 218S daphnoides 257 071 182S purpurea 316 123 390S d x p 044 087 039

Chelating power


Reducing power


LOX inhibitory activity


XO inhibitory activity


bioavailable and bioefficient in vitro The highest values ofthese parameters were obtained for S alba phytochemicalswhereas the lowestmdashfor SDP compounds Bark of SA con-tained also bioavailable and bioefficient compounds able toinhibit XO Although the highest BAC value was determinedin the case of SDP phytochemicals potential bioavailability ofthese compoundswas relatively lowThe highest bioefficiencywas determined for XO inhibitors derived from the bark ofSDP and SA

While white willow is the willow species most commonlyused formedicinal purposes purplewillow (S purpurea) andviolet willow (S daphnoides) are all salicin-rich and may besold under the label of willow bark The major metabolitesof salicin are gentisic acid salicylic acid and salicyluricacid with salicylic acid being the major component in theserum After oral ingestion of willow bark peak levels ofsalicylic acid were found in less than 2 hours A salicincontent of 240mg corresponds to approximately 87mg ofacetylsalicylate which is more cardioprotective than anal-gesic It is also found that the bioavailability of the salicin inthe formulation being evaluated was greater than that foundin other studies This suggests that different formulationsof willow bark extract result in different bioavailabilities aconcept that may be applicable to all herbal medications [35]

4 Conclusion

Unlike in the case of synthetic pharmaceuticals based on anactivity of single (chemical) active compounds numerousphytochemical compounds act in a beneficial manner by anadditive of synergistic activity in one or numerous target sites

connected to physiological processes This idea has foundan application in pharmacology during investigations oncombinations of few metabolites in multidirectional therapy[36] It has been proposed that although willow extracts havebeen traditionally used as anti-inflammatory compoundsfor their salicin content the presence of high amounts ofphenolic compounds can contribute to the beneficial effects[2 3] Our investigations support this thesis In the light ofpresented data it may be concluded that the best source ofpotentially mastication-extractable compounds with multi-directorial biological activity was SD and SDP bark Otherfactors often overlooked in studies of the biological activity ofphytochemicals are their bioaccessibility and bioavailabilityOur study shows high bioaccessibility and bioavailabilityin vitro of Salix bark phytochemicals which may indicatethat extracts from this species of plants may provide healthpromoting benefits to the consumers however this issuerequires further study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] L Pobłocka-Olech M Krauze-Baranowska D Głod A Kaw-iak and E Łojkowska ldquoChromatographic analysis of simplephenols in some species from the genus salixrdquo PhytochemicalAnalysis vol 21 no 5 pp 463ndash469 2010


[2] B Schmid R Ludtke H-K Selbmann et al ldquoEfficacy andtolerability of a standardized willow bark extract in patientswith osteoarthritis randomized placebo-controlled doubleblind clinical trialrdquo Phytotherapy Research vol 15 no 4 pp344ndash350 2001

[3] M T KhayyalMA El-Ghazaly DMAbdallah SNOkpanyiO Kelber and D Weiser ldquoMechanisms involved in the anti-inflammatory effect of a standardized willow bark extractrdquoArzneimittel-Forschung vol 55 no 11 pp 677ndash687 2005

[4] A R Collins ldquoInvestigating oxidative DNA damage and itsrepair using the comet assayrdquo Mutation Research vol 681 no1 pp 24ndash32 2009

[5] B Halliwell ldquoAntioxidants in human health and diseaserdquoAnnual Review of Nutrition vol 16 pp 33ndash50 1996

[6] C-H Chen H-C Chan Y-T Chu et al ldquoAntioxidant activityof some plant extracts towards xanthine oxidase lipoxygenaseand tyrosinaserdquoMolecules vol 14 no 8 pp 2947ndash2958 2009

[7] U Gawlik-Dziki M Jezyna M Swieca D Dziki B Baraniakand J Czyz ldquoEffect of bioaccessibility of phenolic compoundson in vitro anticancer activity of broccoli sproutsrdquoFoodResearchInternational vol 49 no 1 pp 469ndash476 2012

[8] U Gawlik-Dziki M Swieca M Sułkowski D Dziki B Bara-niak and J Czyz ldquoAntioxidant and anticancer activities ofChenopodium quinoa leaves extracts in vitro studyrdquo Food andChemical Toxicology vol 57 pp 154ndash160 2013

[9] D Sugier P Sugier M Pawełek and U Gawlik-Dziki ldquoSalixmyrsinifolia Salisb As a source of phenolic glycosides distribu-tion and characteristic of habitat conditions in the mid-easternPolandrdquoActa Scientiarum Polonorum HortorumCultus vol 10no 3 pp 75ndash88 2011

[10] D Sugier P Sugier A Banas and C Szewczuk ldquoThe contentof phenolic glycosides and macroelements (K Ca Mg) in thebark of herbal willowsrdquoActa ScientiarumPolonorumHortorumCultus vol 12 no 4 pp 31ndash41 2013

[11] M Krauze-Baranowska and E Szumowicz ldquoWillow the sourceof antiinflammatory and analgesic medicinal plantsrdquo PostepyFitoterapii vol 5 no 2 pp 77ndash86 2004

[12] ldquoEuropeanPharmacopoeia 6 0 Council of Europerdquo StrasbourgFrance 2004

[13] U Gawlik-Dziki ldquoChanges in the antioxidant activities ofvegetables as a consequence of interactions between activecompoundsrdquo Journal of Fumctional Foods vol 4 no 4 pp 872ndash882 2012

[14] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswitch phosphomolybdic-phosphotungstics acid reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 no 3 pp144ndash158 1965

[15] T Bahorun A Luximon-Ramma A Crozier andO I AruomaldquoTotal phenol flavonoid proanthocyanidin and vitamin C lev-els and antioxidant activities ofMauritian vegetablesrdquo Journal ofthe Science of Food and Agriculture vol 84 no 12 pp 1553ndash15612004

[16] M Szaufer-Hajdrych ldquoPhenolic acids in leaves of species of theAquilegia genusrdquoHerba Polonica vol 50 no 2 pp 10ndash14 2004

[17] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[18] J-T Guo H-L Lee S-H Chiang F-I Lin and C-Y ChangldquoAntioxidant properties of the extracts from different parts ofBroccoli in Taiwanrdquo Journal of Food and Drug Analysis vol 9no 2 pp 96ndash101 2001

[19] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activities of products of browning reaction preparedfrom glucosaminerdquo Japanese Journal of Nutrition vol 44 no6 pp 307ndash315 1986

[20] B Axelrod T M Cheesbrough and S Laakso ldquoLipoxygenasefrom soybeansrdquo Methods in Enzymology vol 71 pp 441ndash4511981

[21] A P Sweeney S G Wyllie R A Shalliker and J L MarkhamldquoXanthine oxidase inhibitory activity of selected Australiannative plantsrdquo Journal of Ethnopharmacology vol 75 no 2-3pp 273ndash277 2001

[22] B Meier O Sticher and R Julkunen-Tiitto ldquoPharmaceuticalaspects of the use of willows in herbal remediesrdquo PlantaMedicavol 54 no 6 pp 559ndash560 1988

[23] S Sharma D Sahu H R Das and D Sharma ldquoAmelioration ofcollagen-induced arthritis by Salix nigra bark extract via sup-pression of pro-inflammatory cytokines and oxidative stressrdquoFood and Chemical Toxicology vol 49 no 12 pp 3395ndash34062011

[24] S Agnolet S Wiese R Verpoorte and D Staerk ldquoCompre-hensive analysis of commercial willow bark extracts by newtechnology platform combined use of metabolomics high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy and high-resolutionradical scavenging assayrdquo Journal of Chromatography A vol1262 pp 130ndash137 2012

[25] S Enayat and S Banerjee ldquoComparative antioxidant activity ofextracts from leaves bark and catkins of Salix aegyptiaca sprdquoFood Chemistry vol 116 no 1 pp 23ndash28 2009

[26] A Freischmidt G Jurgenliemk B Kraus et al ldquoContribution offlavonoids and catechol to the reduction of ICAM-1 expressionin endothelial cells by a standardised Willow bark extractrdquoPhytomedicine vol 19 no 3-4 pp 245ndash252 2012

[27] B H Kroes A J J van den Berg H C Quarles Van UffordH Van Dijk and R P Labadie ldquoAnti-inflammatory activity ofgallic acidrdquo Planta Medica vol 58 no 6 pp 499ndash504 1992

[28] M S Alam G Kaur Z Jabbar K Javed and M AtharldquoEvaluation of antioxidant activity of Salix caprea flowersrdquoPhytotherapy Research vol 20 no 6 pp 479ndash483 2006

[29] V Roginsky and E A Lissi ldquoReview of methods to determinechain-breaking antioxidant activity in foodrdquo Food Chemistryvol 92 no 2 pp 235ndash254 2005

[30] S Sultana and M Saleem ldquoSalix caprea inhibits skin car-cinogenesis in murine skin inhibition of oxidative stressornithine decarboxylase activity and DNA synthesisrdquo Journalof Ethnopharmacology vol 91 no 2-3 pp 267ndash276 2004

[31] U Gawlik-Dziki M Swieca and D Sugier ldquoEnhancement ofantioxidant abilities and the lipoxygenase and xanthine oxidaseinhibitory activity of broccoli sprouts by biotic elicitorsrdquo ActaScientiarum Polonorum Hortorum Cultus vol 11 no 1 pp 13ndash25 2012

[32] U Gawlik-Dziki M Swieca and D Sugier ldquoImprovement ofnutraceuticals value of broccoli sprouts by natural elicitorsrdquoActa ScientiarumPolonorumHortorumCultus vol 12 no 1 pp129ndash140 2013

[33] M Pinelo A Arnous and A S Meyer ldquoUpgrading of grapeskins significance of plant cell-wall structural components andextraction techniques for phenol releaserdquoTrends in Food Scienceand Technology vol 17 no 11 pp 579ndash590 2006

[34] A G Oomen A Hack M Minekus et al ldquoComparison of fivein vitro digestion models to study the bioaccessibility of soil


contaminantsrdquo Environmental Science and Technology vol 36no 15 pp 3326ndash3334 2002

[35] A R Setty and LH Sigal ldquoHerbalmedications commonly usedin the practice of rheumatology mechanisms of action efficacyand side effectsrdquo Seminars in Arthritis and Rheumatism vol 34no 6 pp 773ndash784 2005

[36] D P Briskin ldquoMedicinal plants and phytomedicines linkingplant biochemistry and physiology to human healthrdquo PlantPhysiology vol 124 no 2 pp 507ndash514 2000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Plant extracts containing secondary metabolites haveserved as antioxidants in phytotherapeutic medicines toprotect against various diseases for centuries Natural antiox-idants exhibit a wide range of pharmacological activitiesand have been shown to have anticancer anti-inflammatoryand antiaging properties [6] Accordingly phytochemicalscan directly interfere with signaling systems involved inthe regulation of inflammatory processes angiogenesis andcancer invasion in amanner dependent on their antioxidativeactivity and concomitant inhibitory effect on the enzymesinvolved with pathological condition [7 8]

S purpurea L S daphnoides Vill and S alba L are avery popular herbal species affirmed in the natural habitatsand field-cultivated in Poland [9 10]The phenolic glycosidescontained in willow bark of this species are known fortheir anti-inflammatory analgesic and fever-reducing effectsand have been shown to relieve rheumatic disturbancesinfections and headache [3 11] Additionally bark of thisspecies contains p-hydroxybenzoic vanillic cinnamic p-coumaric ferulic and caffeic acids phenolic acids and narin-genin [1] However little is as yet known about their synergiceffects A particularly important issue on the effectivenessof dietary supplements is bioavailability and bioavailabilityof active compounds Thus the aim of this study was toinvestigate and to compare the extractability bioaccessibilityand bioavailability in vitro of antioxidative compounds frombark of selected Salix species

2 Materials and Methods

21 Plant Material Selected S alba (SA) S daphnoides (SD)S purpurea (SP) and S daphnoides x purpurea (SDP) hybridwillow clones with the highest phenolic glycoside contents[9 10] originating from their natural habitats were cultivatedon the sandy soil (heavy loamy sand) The study was carriedout in 2009 and 2010 on a 5-6 year-old plantation Theexperiment was a completely randomized block design withthree replicates conducted in the area of experimental fieldsat theUniversity of Life Sciences in Lublin (51∘331015840N 22∘441015840E)The plantation was established at the spacing of 40 times 20 cmEach plot was 16m2 The sandy soil was characterized byan average content of humus (141) very low phosphorus(173mgsdotkgminus1) very low potassium (332mgsdotkgminus1) very lowmagnesium (150mgsdotkgminus1) and strong acid reaction (pHKClmdash41) Mineral fertilization was applied in spring beforethe beginning of vegetation in both soil types Nmdash20 kg Pmdash131 kg Kmdash498 kg calculated per 1 ha

Willow shoots were harvested in November every year(2009-2010) in three replicates The plant material was col-lected in the form of 60 annual shoots (20 entire shootsper plot) from every taxon The shoots were washed withdeionized water Bark was separated from the wood bypeeling and subsampled for chemical analysis

The bark material sampled for salicylate analysis wasdried at room temperature and intensivelymixed andhomog-enized After drying the phenolic glycosides content cal-culated on salicin was determined by means of the HPLC

technique [12] in the Laboratory of Labofarm in StarogardGdanski and expressed as mgmg dry mass (DM)

22 Chemicals Ferrozine (3-(2-pyridyl)-56-bis-(4-phenyl-sulfonic acid)-124-triazine) ABTS (221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) 120572-amylasepancreatin pepsin bile extract Folin-Ciocalteau reagentlinoleic acid ammonium thiocyanate and haemoglobinwere purchased from Sigma-Aldrich Company (PoznanPoland) All others chemicals were of analytical grade

23 Extracts Preparation

231 Chemical Extract (CE) Two grams of plant materialwas filled with 100mL of 70 ethanol and left in darknessby 2 weeks

232 Buffer Extract (BE) Powdered samples of willow bark(1 g) were extracted for 1 h with 20mL of PBS buffer (phos-phate buffered saline pH 74) The extracts were separated bydecantation and the residues were extracted again with 20mLof PBS buffer Extracts were combined and stored in darknessat minus20∘C

233 Digestion In Vitro (DE) In vitro digestion and absorp-tion were performed according to Gawlik-Dziki [13] Thebark samples (1 g) were homogenized in a stomacher lab-oratory blender for 1min to simulate mastication with thepresence of 15mL of simulated salivary fluid (prepared bydissolving 238 g Na

2HPO4 019 g KH

2PO4 and 8 g NaCl

100mg of mucin in 1 liter of distilled water The solutionwas adjusted to pH = 675 and 120572-amylase (EC 3211)was added to obtain 200U per mL of enzyme activity Forthe gastric digestion 300UmL of pepsin (from porcinestomach mucosa pepsin A EC 34231) in 003molL NaClpH = 12 was prepared Further simulated intestinal juicewas prepared by dissolving 005 g of pancreatin (activityequivalent 4 xUSP) and 03 g of bile extract in 35mL01molLNaHCO

3) and subsequently the samples were shaken for

10min at 37∘C The samples were adjusted to pH = 12 using5molL HCl and subsequently 15mL of simulated gastricfluid was added The samples were shaken for 60min at37∘C After digestion with the gastric fluid the samples wereadjusted to pH = 6 with 01molL of NaHCO

3and then

15mL of a mixture of bile extract and pancreatin was addedThe extracts were adjusted to pH = 7 with 1molL NaOHand finally 5mL of 120mmolL NaCl and 5mL of mmolLKCl were added to each sample The prepared samples weresubmitted for in vitro digestion for 120 minutes at 37∘Cin the darkness After that samples were centrifuged andsupernatants were used for further analysis

234 Absorption In Vitro (AE) Considering that antioxi-dants absorption takes placemainly at the intestinal digestionstage the resulting mixture (fluids obtained after in vitrodigestion) was transferred to the dialysis sacks (D9777-100FTSigma-Aldrich) placed in an Erlenmeyer flask containing50mL of PBS buffer and incubated in a rotary shaker (2 times





2O) and









119860119862

] times 100 (1)




50mdashextract con-



2



)] times 100 (2)




50mdashextract con-



3[Fe(CN

6)]Themixture


3 and the










) times 100 (3)






MEF = 119862BE119862CE (4)


DEF =119862GE119862CE (5)


AEF = 119862AE119862CE (6)




BAC = 119860BE119860GE (7)


BAV =119860GE119860AE (8)

c

a

b

a

0

20

40

60

80

100

120


Salic

in co

nten

t (m

gg

DM

)



BEF = 119860BE119860AE (9)












TPC


dB8220 plusmn 255

dC5188 plusmn 212


bA16250 plusmn 598

bB14117 plusmn 414

bC13561 plusmn 554


cA13490 plusmn 512

cB11329 plusmn 329

cC10892 plusmn 438

cD 033 027 026S d x p 74012 plusmn 2021

aA24992 plusmn 721

aB19049 plusmn 922

aC18074 plusmn 542

aD 034 026 024

TFC


1051 plusmn 055dB

1548 plusmn 077dC

751 plusmn 033dD 048 071 034


6173 plusmn 269aB

6972 plusmn 123aB

3153 plusmn 125aC 022 025 011


2747 plusmn 141cB

3279 plusmn 113cC

1704 plusmn 081cD 032 038 020


5229 plusmn 254bB

5899 plusmn 225bB

2622 plusmn 084bC 029 033 015

TPA


bB13773 plusmn 655

dC14519 plusmn 345


bA26944 plusmn 913

aA35761 plusmn 1011

bB29774 plusmn 918


cA11776 plusmn 777

cB21022 plusmn 923

cC17520 plusmn 713

dD 058 104 087S d x p 32294 plusmn 948

aA21040 plusmn 1145

bB45487 plusmn 1225

aC43354 plusmn 1452









acd c

h

bb

j

i

c

fd

ghde d e

g

05

1015202530354045


IC

(mg

DM

mL)

50


DigestedAbsorbed






a

ef

ef

b b b b

cc c

b

d d d d

000

040

080

120

160

200



DigestedAbsorbed

IC50

(mg

DM

mL)


a a a a

bc

d

c

ded

de

b

f

hg

i

0

1

2

3

4

5

6

7



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

cdc

dbc

f

bc

b

e e e

d

000

050

100

150

200

250

300



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

ef

d

c

f fg g

b b ba

0123456789

10



DigestedAbsorbed

IC50

(mg

DM

mL)










Chelating power


Reducing power








4 Conclusion





References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2O) and









119860119862

] times 100 (1)




50mdashextract con-



2



)] times 100 (2)




50mdashextract con-



3[Fe(CN

6)]Themixture


3 and the










) times 100 (3)






MEF = 119862BE119862CE (4)


DEF =119862GE119862CE (5)


AEF = 119862AE119862CE (6)




BAC = 119860BE119860GE (7)


BAV =119860GE119860AE (8)

c

a

b

a

0

20

40

60

80

100

120


Salic

in co

nten

t (m

gg

DM

)



BEF = 119860BE119860AE (9)












TPC


dB8220 plusmn 255

dC5188 plusmn 212


bA16250 plusmn 598

bB14117 plusmn 414

bC13561 plusmn 554


cA13490 plusmn 512

cB11329 plusmn 329

cC10892 plusmn 438

cD 033 027 026S d x p 74012 plusmn 2021

aA24992 plusmn 721

aB19049 plusmn 922

aC18074 plusmn 542

aD 034 026 024

TFC


1051 plusmn 055dB

1548 plusmn 077dC

751 plusmn 033dD 048 071 034


6173 plusmn 269aB

6972 plusmn 123aB

3153 plusmn 125aC 022 025 011


2747 plusmn 141cB

3279 plusmn 113cC

1704 plusmn 081cD 032 038 020


5229 plusmn 254bB

5899 plusmn 225bB

2622 plusmn 084bC 029 033 015

TPA


bB13773 plusmn 655

dC14519 plusmn 345


bA26944 plusmn 913

aA35761 plusmn 1011

bB29774 plusmn 918


cA11776 plusmn 777

cB21022 plusmn 923

cC17520 plusmn 713

dD 058 104 087S d x p 32294 plusmn 948

aA21040 plusmn 1145

bB45487 plusmn 1225

aC43354 plusmn 1452









acd c

h

bb

j

i

c

fd

ghde d e

g

05

1015202530354045


IC

(mg

DM

mL)

50


DigestedAbsorbed






a

ef

ef

b b b b

cc c

b

d d d d

000

040

080

120

160

200



DigestedAbsorbed

IC50

(mg

DM

mL)


a a a a

bc

d

c

ded

de

b

f

hg

i

0

1

2

3

4

5

6

7



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

cdc

dbc

f

bc

b

e e e

d

000

050

100

150

200

250

300



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

ef

d

c

f fg g

b b ba

0123456789

10



DigestedAbsorbed

IC50

(mg

DM

mL)










Chelating power


Reducing power








4 Conclusion





References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




) times 100 (3)






MEF = 119862BE119862CE (4)


DEF =119862GE119862CE (5)


AEF = 119862AE119862CE (6)




BAC = 119860BE119860GE (7)


BAV =119860GE119860AE (8)

c

a

b

a

0

20

40

60

80

100

120


Salic

in co

nten

t (m

gg

DM

)



BEF = 119860BE119860AE (9)












TPC


dB8220 plusmn 255

dC5188 plusmn 212


bA16250 plusmn 598

bB14117 plusmn 414

bC13561 plusmn 554


cA13490 plusmn 512

cB11329 plusmn 329

cC10892 plusmn 438

cD 033 027 026S d x p 74012 plusmn 2021

aA24992 plusmn 721

aB19049 plusmn 922

aC18074 plusmn 542

aD 034 026 024

TFC


1051 plusmn 055dB

1548 plusmn 077dC

751 plusmn 033dD 048 071 034


6173 plusmn 269aB

6972 plusmn 123aB

3153 plusmn 125aC 022 025 011


2747 plusmn 141cB

3279 plusmn 113cC

1704 plusmn 081cD 032 038 020


5229 plusmn 254bB

5899 plusmn 225bB

2622 plusmn 084bC 029 033 015

TPA


bB13773 plusmn 655

dC14519 plusmn 345


bA26944 plusmn 913

aA35761 plusmn 1011

bB29774 plusmn 918


cA11776 plusmn 777

cB21022 plusmn 923

cC17520 plusmn 713

dD 058 104 087S d x p 32294 plusmn 948

aA21040 plusmn 1145

bB45487 plusmn 1225

aC43354 plusmn 1452









acd c

h

bb

j

i

c

fd

ghde d e

g

05

1015202530354045


IC

(mg

DM

mL)

50


DigestedAbsorbed






a

ef

ef

b b b b

cc c

b

d d d d

000

040

080

120

160

200



DigestedAbsorbed

IC50

(mg

DM

mL)


a a a a

bc

d

c

ded

de

b

f

hg

i

0

1

2

3

4

5

6

7



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

cdc

dbc

f

bc

b

e e e

d

000

050

100

150

200

250

300



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

ef

d

c

f fg g

b b ba

0123456789

10



DigestedAbsorbed

IC50

(mg

DM

mL)










Chelating power


Reducing power








4 Conclusion





References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




TPC


dB8220 plusmn 255

dC5188 plusmn 212


bA16250 plusmn 598

bB14117 plusmn 414

bC13561 plusmn 554


cA13490 plusmn 512

cB11329 plusmn 329

cC10892 plusmn 438

cD 033 027 026S d x p 74012 plusmn 2021

aA24992 plusmn 721

aB19049 plusmn 922

aC18074 plusmn 542

aD 034 026 024

TFC


1051 plusmn 055dB

1548 plusmn 077dC

751 plusmn 033dD 048 071 034


6173 plusmn 269aB

6972 plusmn 123aB

3153 plusmn 125aC 022 025 011


2747 plusmn 141cB

3279 plusmn 113cC

1704 plusmn 081cD 032 038 020


5229 plusmn 254bB

5899 plusmn 225bB

2622 plusmn 084bC 029 033 015

TPA


bB13773 plusmn 655

dC14519 plusmn 345


bA26944 plusmn 913

aA35761 plusmn 1011

bB29774 plusmn 918


cA11776 plusmn 777

cB21022 plusmn 923

cC17520 plusmn 713

dD 058 104 087S d x p 32294 plusmn 948

aA21040 plusmn 1145

bB45487 plusmn 1225

aC43354 plusmn 1452









acd c

h

bb

j

i

c

fd

ghde d e

g

05

1015202530354045


IC

(mg

DM

mL)

50


DigestedAbsorbed






a

ef

ef

b b b b

cc c

b

d d d d

000

040

080

120

160

200



DigestedAbsorbed

IC50

(mg

DM

mL)


a a a a

bc

d

c

ded

de

b

f

hg

i

0

1

2

3

4

5

6

7



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

cdc

dbc

f

bc

b

e e e

d

000

050

100

150

200

250

300



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

ef

d

c

f fg g

b b ba

0123456789

10



DigestedAbsorbed

IC50

(mg

DM

mL)










Chelating power


Reducing power








4 Conclusion





References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


acd c

h

bb

j

i

c

fd

ghde d e

g

05

1015202530354045


IC

(mg

DM

mL)

50


DigestedAbsorbed






a

ef

ef

b b b b

cc c

b

d d d d

000

040

080

120

160

200



DigestedAbsorbed

IC50

(mg

DM

mL)


a a a a

bc

d

c

ded

de

b

f

hg

i

0

1

2

3

4

5

6

7



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

cdc

dbc

f

bc

b

e e e

d

000

050

100

150

200

250

300



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

ef

d

c

f fg g

b b ba

0123456789

10



DigestedAbsorbed

IC50

(mg

DM

mL)










Chelating power


Reducing power








4 Conclusion





References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


a a a a

cdc

dbc

f

bc

b

e e e

d

000

050

100

150

200

250

300



DigestedAbsorbed

IC50

(mg

DM

mL)






a a a a

ef

d

c

f fg g

b b ba

0123456789

10



DigestedAbsorbed

IC50

(mg

DM

mL)










Chelating power


Reducing power








4 Conclusion





References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Chelating power


Reducing power








4 Conclusion





References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

)joebxj1vcmjtijoh$psqpsbujpo …

Documents