Effects of Kaempferol and Myricetin on Inducible Nitric OxideSynthase Expression and Nitric Oxide Production in Rats1

Evita Rostoka1, Larisa Baumane1, Sergejs Isajevs2, Aija Line3, Maija Dzintare1, Darja Svirina2, Jelena Sharipova1, Karina Silina3,

Ivars Kalvinsh1 and Nikolajs Sjakste1,2

1Latvian Institute of Organic Synthesis, Riga, Latvia, 2Faculty of Medicine, University of Latvia, Riga, Latvia, 3Latvian Biomedical Research

and Study Centre, Riga, Latvia

(Received 10 July 2009; Accepted 18 October 2009)

Abstract: When administered as drugs or consumed as food components, polyphenolic compounds synthesized in plants

interfere with intracellular signal transduction pathways, including pathways of nitric oxide synthase expression. However,

effects of these compounds in vivo do not always correlate with nitric oxide synthase-inhibiting activities revealed in experi-

ments with cultured cells. The initial goal of this work was to compare effects of flavonoids kaempferol and myricetin on

inducible nitric oxide synthase mRNA and protein expression monitored by real-time RT-PCR and immunohistochemistry

and to evaluate the impact of these effects on nitric oxide production in rat organs measured by means of electron paramag-

netic resonance spectroscopy. Kaempferol and myricetin attenuated the lipopolysaccharide-induced outburst of inducible nitric

oxide synthase gene expression; kaempferol also significantly decreased the lipopolysaccharide-induced outburst of inducible

nitric oxide synthase protein expression in the liver. Myricetin decreased nitric oxide production in intact rat liver. Kaempferol

did not decrease nitric oxide production neither in intact rats nor in the lipopolysaccharide-treated animals. Kaempferol even

enhanced the lipopolysaccharide-induced increase of nitric oxide production in blood. Myricetin did not interfere with lipo-

polysaccharide effects. As both kaempferol and myricetin are known as inhibitors of inducible nitric oxide synthase expres-

sion, our results suggest that modifications of nitric oxide level in tissues by these compounds cannot be predicted from data

about its effects on nitric oxide synthase expression or activity.

Flavonoid intake influences mortality from nitric oxide-

dependent processes: ischemic heart disease, stroke, diabetes

mellitus and cancer [1]. This implies significance of flavonoid

and other natural compound uptake for functions of cardio-

vascular, immune and nervous systems. Impact of a given

compound on nitric oxide production is usually deduced

from in vitro nitric oxide synthase expression and nitrite pro-

duction. However, modification of the nitric oxide synthesis

by drugs in animals and humans appears to be complicated

and dependent on numerous factors. Modifications of nitric

oxide synthesis is often organ-specific; data of in vitro and in

vivo experiments happen to be contradictory [2,3]. In our

opinion, only direct measurements of nitric oxide production

in vivo can reveal nitric oxide-dependent effects of a given

drug. The goal of this work was to monitor modification of

nitric oxide production in rat organs by the flavonoids,

kaempferol and myricetin. Chemical structures of the com-

pounds are given in fig. 1. According to published data, ka-

empferol produces inhibition of both inducible nitric oxide

synthase mRNA and protein in several cell cultures [4–7].

Myricetin inhibits nitric oxide release and inducible nitric

oxide synthase expression in cultured macrophages [8–10].

Taken together, these data indicate that the two compounds

should decrease lipopolysaccharide-induced nitric oxide pro-

duction in vivo. No data about modification of nitric oxide

production by these compounds are accessible. Thus, our

study was aimed to fill a significant gap in knowledge about

biological activities of natural compounds.

Experimental Procedures

Natural compounds. Kaempferol and myricetin were purchased

from Dayang Chemical Co., LTD (Taiwan 2).

Chemicals. Lipopolysascharide, diethylthiocarbamate, ferrous sul-

fate, sodium citrate and all other chemicals were from Sigma-Aldrich

Chemie GmbH (Taufkirchen, Germany).

Experimental design and drug administration. Animals were

purchased from the laboratory animal suppliers ‘Gailezers’ (Riga,

Latvia). All manipulations with animals were performed in accor-

dance with Republic of Latvia regulations, being in agreement with

European Union rules; permission from the Ethics Commission of

the Latvian Council for Science was obtained to perform this study.

Male Wistar rats weighing 200–300 g were used in the experi-

ments. The rats were assigned to the following experimental groups

(table 1). Group 1 (n = 24) served as a control for nitric oxide detec-

tion experiments. 30 min. after spin trap injection, the rats were

decapitated under slight ether narcosis. In Groups 2–4, correspond-

ing substances were administered per os in concentrations indicated

in table 1. After 3.5 hr of substance administration, spin trap was

injected, after 30 min., the rats were decapitated under slight ether

narcosis. In Group 5 (n = 28), the rats were intraperitoneally injected

lipopolysascharide (10 mg ⁄ kg), spin traps were administered 3.5 hr

B C P T 5 2 6 B Dispatch: 17.12.09 Journal: BCPT CE: Balaji prasad

Journal Name Manuscript No. Author Received: No. of pages: 6 PE: Bhagyalakshmi

Author for correspondence: Nikolajs Sjakste, Latvian Institute of

Organic Synthesis, Aizkraukles Street 21, Riga, LV1006, Latvia (fax

+371-7553142, e-mail nikolajs.sjakste@lu.lv).

� 2009 The Authors Doi: 10.1111/j.1742-7843.2009.00526.x

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later, 30 min. after spin trap injection, the rats were decapitated

under slight ether narcosis. In Groups 5–8, the rats were intraperito-

neally injected lipopolysascharide (10 mg ⁄kg), kaempferol or myrice-

tin (substances and doses are listed in table 1) were administered per

os at the same time, spin traps were administered 3.5 hr later,

30 min. after spin trap injection, rats were decapitated under slight

ether narcosis. Group 9 animals (n = 20) served as controls for real-

time PCR, the rats were decapitated under slight ether narcosis and

liver tissue was taken for RNA extraction. Group 10 (n = 3) rats

received kaempferol (50 mg ⁄ kg) per os and Group 11 (n = 3) ani-

mals were treated with myricetin (50 mg ⁄ kg per os). Four hours later,

the animals were killed and liver was taken for ribonucleic acid

extraction and histochemical examination. Group 12 (n = 21) ani-

mals received lipopolysaccharide (10 mg ⁄ kg) intraperitoneally, liver

tissue was taken for examination and ribonucleic acid extraction 4 hr

later. In Groups 13 and 14 (n = 3 for all), lipopolysaccharide

(10 mg ⁄ kg) was injected intraperitoneally at the same time as natural

substance was administered per os: kaempferol (50 mg ⁄ kg) and

myricetin (50 mg ⁄ kg). Correspondingly, 4 hr later, animals were

killed and liver was taken for RNA extraction and histochemical

examination.

Ribonucleic acid extraction and copy deoxyribonucleic acid prepara-

tion. Total ribonucleic acid was isolated from liver and brain cortex

using TRI reagent (Sigma Aldrich, USA3 ). Deoxyribonucleic acid

contaminations were removed with ribonuclease-free kit (Ambion,

USA4 ). The resulting ribonucleic acid quantity and purity were deter-

mined by spectrophotometry. Ribonucleic acid (2 lg) was reverse-

transcribed using a random hexamer primer (RevertAid� First

Strand cDNA Synthesis Kit, Fermentas, Lithuania) to obtain copy

deoxyribonucleic acid.

Real time RT-PCR. The mRNA expression rates of brain cortex,

liver-inducible nitric oxide synthase and reference gene were deter-

mined using the Applied Biosystems SYBR� Green PCR Master

Mix (USA5 ) according to the instructions of the manufacturer.

Amplification and detection of specific products were performed on

a StepOne� Real-Time PCR System (Applied Biosystems) using the

following temperature-time profile: one cycle of 95�C for 10.00 min.,

40 cycles of 95�C for 0.15 min. and 60�C for 1.00 min. To check

specificity of amplification products, the dissociation curve mode

was used (one cycle at 95�C for 0.15 min., 60�C for 1 min. and 95�C

for 0.15 min.). To evaluate suitability of candidates as reference

genes, we applied the GeNorm programme (http://medgen.ugent.be/

genorm/). Primers were designed using Primer3 software. The prim-

ers were ordered from Metabion international AG, Germany 6. The

2)DDCT method was applied for analysis of the results. Primer

sequences for inducible nitric oxide synthase gene were 5¢-GCTA

CACTTCCAACGCAACA-3¢ for forward and 5¢-CATGGTGAAC

ACGTTCTTGG for reverse primer, expected size of the product was

116 bp. Ribonucleic acid polymerase II was chosen as reference gene

(5¢-GCCAGAGTCTCCCATGTGTT-3¢and 5¢-GTCGGTGGGACT

CTGTTTGT-3¢, 135 bp [11].

Histological and immunohistochemical examination. Paraffin-embed-

ded tissue was cut in 4-micron-thick sections and stained with hae-

matoxylin and eosin for morphological examination.

Tissue sections were stained for visualization of inducible nitric

oxide synthase positive cells by immunohistochemical approach as

described [12]. Briefly, antigen retrieval was achieved by treatment in

microwave oven for 20 min. at 300 W in citrate buffer, pH = 6.0.

Endogenous peroxidase activity was blocked by 0.5% hydrogen per-

oxide for 10 min. Non-specific primary antibody binding was

blocked by serum-free protein block for 10 min. Rabbit polyclonal

active inducible nitric oxide synthase antibody (AbCam, UK 7) was

applied in 1:200 dilution and incubated for 1 hr at room temperature

in humidified chamber. Detection of primary antibody binding was

performed using specific peroxidise-conjugated polyclonal goat anti-

rabbit IgG (1:100 for 30 min.) and subsequently peroxidize-conju-

gated polyclonal rabbit anti-goat IgG (1:100 for 30 min.). The imm-

unoperoxidase colour reaction was developed by incubation with

diaminobenzidine (7 min.). A negative control without primary anti-

body was included in each staining run. Inducible nitric oxide syn-

thase positive cells were counted in twenty high-powered fields at

magnification ·400. All cell counts were expressed as cells per square

millimetre.

For morphological examination, at least three replicate measure-

ments of inducible nitric oxide synthase positive cells were performed

by the same observer in 10 randomly selected slides, and the intraob-

server reproducibility was assessed with the coefficient of variation

and with the interclass correlation coefficient. The intraobserver

coefficient of variation was 7% and the intraobserver correlation

coefficient was 0.90.

Administration of spin trap agents. To determine the nitric oxide

content in the tissues, we used the protocol originally elaborated by

A.L. Kleschyov and A. F. Vanin (reviewed in [13]). Spin traps were

administered 30 min. before killing the rats. Rats were administered

Table 1.

Effect of kaempferol and myricetine on nitric oxide production (ng ⁄ g tissue in 30 min.) in different rat organs and tissues in intact rats and

under lipopolysaccharide-induced inflammation.

Groups\organs Cortex Liver Kidney Blood Lungs

Control, n = 24 57.0 € 3.8 57.5 € 7.6 8.4 € 1.5 59.8 € 6.1 68.5 € 5.4

Kaempferol (50 mg ⁄ kg), n = 5 49.8 € 10.6 78.6 € 23.9 6.1 € 2.9 68.0 € 19.2 85.9 € 20.3

Kaempferol (350 mg ⁄ kg), n = 7 49.7 € 5.9 75.7 € 10.8 5.0 € 2.0 43.0 € 9.1 80.4 € 9.6

Myricetine (50 mg ⁄ kg), n = 10 56.7 € 4.1 31.4 € 3.9* 7.7 € 1.2 48.9 € 6.3 53.4 € 6.7

Lipopolysaccharide (10 mg ⁄kg), n = 28 172.2 € 13.3 1434.3 € 104.7 356.5 € 29.9 380.4 € 33.4 919.6 € 64.7

Kaempferol (50 mg ⁄ kg) + lipopolysaccharide

(10 mg ⁄ kg), n = 6

209.5 € 39.4 1488.3 € 296.5 387.3 € 73.2 509.7 € 112.5# 695.0 € 124.2

Kaempferol (350 mg ⁄ kg) + lipopolysaccharide

(10 mg ⁄ kg), n = 6

169.6 € 21.7 1449.6 € 105.7 369.4 € 47.4 566.2 € 65.8 1133.2 € 157.0

Myricetine (50 mg ⁄ kg) + lipopolysaccharide

(10 mg ⁄ kg), n = 10

206.5 € 49.2 1008.3 € 209.0 337.0 € 46.3 542.8 € 84.4 824.8 € 170.1

*p < 0.05 versus control group; #p < 0.05 versus lipopolysaccharide group.

10Fig. 1. Chemical structures of kaempferol and myricetine.

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400 mg ⁄kg of the nitric oxide scavenger diethylthiocarbamate via

intraperitoneal injection and ferrous citrate subcutaneously

(40 mg ⁄ kg ferrous sulphate + 200 mg ⁄kg sodium citrate). Diet-

hylthiocarbamate and ferrous citrate form a complex that traps nitric

oxide and forms a paramagnetic iron-diethylthiocarbamate – nitric

oxide complex that is easily detected by electron paramagnetic reso-

nance spectroscopy.

Sacrifice, organ dissection and sample preparation for electron

paramagnetic resonance spectroscopy. Following the drug and spin

trap administration, rats were decapitated under slight ether anaes-

thesia, and samples of brain cortex, liver, kidney, blood and lungs

were compacted in a glass tube 30 mm in length with inner diameter

of 4 mm and immediately frozen in liquid nitrogen. Before recording

the ESR spectra, the specimen was placed in a quartz finger Dewar

flask ER 167 FDS-Q (Bruker, Karlsruhe, Germany) filled with liquid

nitrogen.

Electron paramagnetic resonance spectroscopy. Electron paramag-

netic resonance spectra were recorded in liquid nitrogen using an

electron paramagnetic resonance spectrometer ‘Radiopan’

SE ⁄X2544 (Radiopan, Poznan, Poland). Conditions of electron

paramagnetic resonance measurements were: operation at X-band,

25 mW microwave power, 100 kHz modulation frequency, 5 G mod-

ulation amplitude, receiver gain 0.5 · 104, and time constant 1 sec.

Spectra were recorded for 4 min. The nitric oxide content in the sam-

ples was evaluated from the height of the third component of the

nitric oxide signal at g = 2.031.

The nitric oxide production (ng ⁄ g of tissue during 30 min.) was

calculated on the basis of calibration curves as described previously.

Briefly, different quantities of sodium nitrite (final concentrations 10,

20, 30, 40, 60, 100 lM) were mixed with diethylthiocarbamate

(33 mg ⁄ml) and ferrous sulphate (3.3 mM), an excess of sodium

hydrosulphite (2 M) was added to the mixture. The electron para-

magnetic resonance spectra were taken as described above. Further

details are given in our previous publications [2,3].

Statistical analysis. Results were expressed as mean € standard

deviation. Significance of differences in nitric oxide concentration

and inducible nitric oxide synthase expression between groups was

evaluated according to Student’s unpaired t-test. Mann-Whitney’s U

test was used for quantification of immunohistochemical experi-

ments.

Results

Effects of kaempferol and myricetin on inducible nitric oxide

synthase gene messenger ribonucleic acid and protein

expression in intact and lipopolysaccharide-treated rats.

Both flavonoids did not modify significantly the level of

inducible nitric oxide synthase gene expression in the liver of

intact animals. However, the lipopolysaccharide-induced out-

burst of inducible nitric oxide synthase gene expression was

attenuated, differences were statistically significant (fig. 2).

The substances did not modify much inducible nitric oxide

synthase protein expression with one exception: kaempferol

significantly decreased the lipopolysaccharide-induced out-

burst of inducible nitric oxide synthase protein expression in

the liver (fig. 3). Fig. 4 demonstrates that in the control and

kaempferol groups, there was little or no inducible nitric

oxide synthase expression in liver tissue, whereas lipopolysac-

charide administration significantly increased inducible nitric

oxide synthase expression. Kaempferol co-administered with

lipopolysaccharide decreased inducible nitric oxide synthase

over-expression.

Effects of kaempferol and myricetin on nitric oxide production

in intact and lipopolysaccharide-treated rats.

In order to test potential impact of ability of kaempferol and

myricetin to modify inducible nitric oxide synthase gene

11Fig. 2. Effects of kaempferol and myricetine on inducible nitric

oxide synthase gene expression in liver of intact and lipopolysaccha-

ride-treated animals. Results are presented as percentage against

average of the control. (A) intact animals; (B) lipopolysaccharide-

treated animals. All compounds were administered in a dose of

50 mg ⁄ kg. *p < 0.05 versus control group, #p < 0.05 versus lipopoly-

saccharide group. iNOS, inducible nitric oxide synthase; LPS, lipo-

polysaccharide. 8

12Fig. 3. Effects of kaempferol and myricetine on a number of induc-

ible nitric oxide synthase-positive cells in rat liver. Results are pre-

sented as percentage versus average of the control. (A) intact

animals; (B) lipopolysaccharide-treated animals. All compounds were

administered in a dose of 50 mg ⁄ kg. *p < 0.05 versus control group,

#p < 0.05 versus lipopolysaccharide group. iNOS, inducible nitric

oxide synthase; LPS, lipopolysaccharide. 9

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EFFECTS OF KAEMPFEROL AND MYRICETIN ON NITRIC OXIDE AND INDUCIBLE NITRIC OXIDE SYNTHASE 3

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expression on nitric oxide production in rat organs, the radi-

cal production was monitored in several rat organs and tis-

sues. Data are summarized in table 1. To determine the

background nitric oxide production levels in rat organs, rats

were injected with diethylthiocarbamate and ferrous citrate

and killed 30 min. later. Electron paramagnetic resonance

spectra of the different organs had a typical copper-diet-

hylthiocarbamate spectrum shape with a superposed iron-

diethythiocarbamate-nitric oxide peak; spectrawere published

previously (Sjakste et al. [3]). The nitric oxide production was

determined in brain cortex, liver, kidneys, blood and lungs

with the highest nitric oxide production levels in the brain cor-

tex, liver, lungs and blood (table 1). Nitric oxide production in

kidneyswas an order ofmagnitude lower.

When the control group of animals was compared to ani-

mals treated with kaempferol and myricetine, it turned out

that nitric oxide production was not greatly modified by

kaempferol; myricetin down-regulated nitric oxide production

in the liver only (table 1).

In the following set of experiments, the eventual activity of

the compounds as modifiers of nitric oxide production was

tested against the background of the inducible nitric oxide

synthase induction. Intraperitoneal injection of lipopolysac-

charide to the animals caused a drastic increase of nitric

oxide production levels in all tissues studied (table 1). The

highest accumulation of nitric oxide was detected in liver,

whereas very strong increases in nitric oxide accumulation

(50- to 100-fold compared to control) were observed in

blood and kidneys. The effects of lipopolysaccharide were

less pronounced in brain cortex tissue where nitric oxide

increased four times only.

The anticipated decrease of nitric oxide production by ka-

empferol and myricetine deduced from their impact on induc-

ible nitric oxide synthase gene expression was not observed

(table 1). Kaempferol even enhanced the lipopolysaccharide-

induced increase of nitric oxide production in blood.

Discussion

In our experiments, kaempferol decreased both inducible

nitric oxide synthase messenger ribonucleic acid and protein

expression enhanced by lipopolysaccharide. The messenger

ribonucleic acid and protein expression data confirm each

other; moreover, these are in good agreement with literature

data. Kaempferol produces inhibition of both inducible

nitric oxide synthase messenger ribonucleic acid and protein

in Chung liver cells; the mechanisms are likely to involve

nuclear factor-jB blockade [4]. It also decreases inducible

nitric oxide synthase, messenger ribonucleic acid, protein

and nitric oxide production in lipopolysaccharide-stimulated

macrophages [5,6] and glioma cells [7]. Probably, suppression

of inducible nitric oxide synthase induction by kaempferol is

produced via binding of the flavonoid to peroxisome prolifer-

ator-activated receptors gamma [14] or haemoxygenase acti-

vation [15].

However, inhibition of inducible nitric oxide synthase gene

and protein expression by kaempferol was not followed by

decrease in nitric oxide production. Lipopolysaccharide

effect was even enhanced in blood. Ability of the compound

to increase the nitric oxide production in some organs could

rather be ascribed to antioxidant activity of the substance

[16]. Scavenging of reactive oxygen species prevents involve-

ment of nitric oxide in interaction with these radicals and

thereby increasing its bioavailability. This effect is produced

by several natural compounds including cocoa polyphenols

[17] and resveratrol [18].

A B

C D

Fig. 4. Inducible nitric oxide synthase expression in liver tissue assessed by immunohistochemical methods. (A) control; (B) kaempferol; (C)

lipopolysaccharide; (D) lipopolysaccharide + kaempferol. Arrows indicate positively stained cells, magnification at ·200.

Colouronline,B&W

inprint

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Decrease of the inducible nitric oxide synthase gene

expression in liver of lipopolysaccharide-treated animals and

nitric oxide production in the liver of intact animals were the

only effects of myricetin observed in our study. These results

are in good correlation with literature data. Myricetin inhib-

its nitric oxide release in murine macrophages [10]. It also

inhibits nitric oxide production, inducible nitric oxide syn-

thase messenger ribonucleic expression, in part through

attenuating the nuclear factor-kappaB signalling pathway in

endotoxin-stimulated murine macrophages [8,9]. There are

also data on nitric oxide-enhancing effects of the substance:

it appeared to be capable of enhancing the endothelial nitric

oxide release of isolated porcine coronary arteries by direct

real-time measurement of the luminal surface nitric oxide

concentration with an amperometric microsensor [19]. Our

electron paramagnetic resonance spectroscopy data do not

reflect myricetine impact on inducible nitric oxide synthase

expression in lipopolysaccharide-treated animals. This might

be due to interference of several pathways of regulation of

nitric oxide production by the substance.

In our study, we have for the first time studied modifica-

tions of nitric oxide production by kaempferol and myricetin

in vivo; data were compared to nitric oxide synthase expres-

sion on gene and protein level. Analysing the results in gen-

eral, we can conclude that results of all the three methods

applied are not coherent. The observed decrease of nitric

oxide production in liver of intact animals by myricetin

could be attributed to its ability to inhibit inducible nitric

oxide expression, as the latter enzyme is expressed in liver

Kupfer cells and its activity is not much influenced by regu-

latory factors. However, we could not spot any significant

changes in nitric oxide synthase gene or protein expression

by myricetin. Down-regulation of the lipopolysaccharide-

triggered increase of the inducible nitric oxide synthase gene

expression achieved by administration of both the flavonoids

and decrease of protein expression by kaempferol did not

modify much nitric oxide production. Thus, the tested com-

pounds, being inhibitors of the inducible nitric oxide syn-

thase expression, can hardly be recommended for treatment

of septic shock. Inhibitors of the nitric oxide synthase enzy-

matic activity are also ineffective in this case; effective reme-

dies should be sought among compounds decreasing radical

production (discussed in [20]). Taken together, our results

suggest that modifications of nitric level in tissues by myrice-

tin and kaempferol cannot be predicted from data about

effects of these compounds on nitric oxide synthase expres-

sion. Moreover, it seems that inducible nitric oxide synthase

expression is not a limiting factor for nitric oxide production

in the LPS model used in this study. This stresses the impor-

tance of direct measurements of nitric oxide in the tissues.

Acknowledgements

This study was supported in part by the National Pro-

gramme ‘Novel drugs and biocorrection remedies: construc-

tion, transport forms and mechanisms of action’ sub-project

‘Study of synergism and resistance phenomena of anti-cancer

substances and creation of novel anti-cancer drugs’ managed

by I. Kalvinsh and the grant 04.1317 ‘Pathological produc-

tion of nitric oxide, possibilities of its pharmacological cor-

rection’ awarded to N. Sjakste by the Latvian Council of

Science. We thank L. Lauberte for technical assistance. Par-

ticipation of D. Meirena in experimental design and discus-

sion is greatly appreciated.

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by flavonoids in mouse macrophages. FEBS Lett 2001;496:12–8.

EFFECTS OF KAEMPFEROL AND MYRICETIN ON NITRIC OXIDE AND INDUCIBLE NITRIC OXIDE SYNTHASE 5

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Author Query Form

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USING E-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

Required Software

Adobe Acrobat Professional or Acrobat Reader (version 7.0 or above) is required to e-annotate PDFs. Acrobat 8 Reader is a free download: http://www.adobe.com/products/acrobat/readstep2.html

Once you have Acrobat Reader 8 on your PC and open the proof, you will see the Commenting Toolbar (if it does not appear automatically go to Tools>Commenting>Commenting Toolbar). The Commenting Toolbar looks like this:

If you experience problems annotating files in Adobe Acrobat Reader 9 then you may need to change a preference setting in order to edit.

In the “Documents” category under “Edit – Preferences”, please select the category ‘Documents’ and change the setting “PDF/A mode:” to “Never”.

Note Tool — For making notes at specific points in the text

Marks a point on the paper where a note or question needs to be addressed.

Replacement text tool — For deleting one word/section of text and replacing it

Strikes red line through text and opens up a replacement text box.

Cross out text tool — For deleting text when there is nothing to replace selection

Strikes through text in a red line.

How to use it:

1. Right click into area of either inserted text or relevance to note

2. Select Add Note and a yellow speech bubble symbol and text box will appear

3. Type comment into the text box

4. Click the X in the top right hand corner of the note box to close.

How to use it:

1. Select cursor from toolbar

2. Highlight word or sentence

3. Right click

4. Select Replace Text (Comment) option

5. Type replacement text in blue box

6. Click outside of the blue box to close

How to use it:

1. Select cursor from toolbar

2. Highlight word or sentence

3. Right click

4. Select Cross Out Text

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Approved tool — For approving a proof and that no corrections at all are required.

Highlight tool — For highlighting selection that should be changed to bold or italic.

Highlights text in yellow and opens up a text box.

Attach File Tool — For inserting large amounts of text or replacement figures as a files.

Inserts symbol and speech bubble where a file has been inserted.

Pencil tool — For circling parts of figures or making freeform marks

Creates freeform shapes with a pencil tool. Particularly with graphics within the proof it may be useful to use the Drawing Markups toolbar. These tools allow you to draw circles, lines and comment on these marks.

How to use it:

1. Click on the Stamp Tool in the toolbar

2. Select the Approved rubber stamp from the ‘standard business’ selection

3. Click on the text where you want to rubber stamp to appear (usually first page)

How to use it:

1. Select Highlighter Tool from the commenting toolbar

2. Highlight the desired text

3. Add a note detailing the required change

How to use it:

1. Select Tools > Drawing Markups > Pencil Tool

2. Draw with the cursor

3. Multiple pieces of pencil annotation can be grouped together

4. Once finished, move the cursor over the shape until an arrowhead appears and right click

5. Select Open Pop-Up Note and type in a details of required change

6. Click the X in the top right hand corner of the note box to close.

How to use it:

1. Click on paperclip icon in the commenting toolbar

2. Click where you want to insert the attachment

3. Select the saved file from your PC/network

4. Select appearance of icon (paperclip, graph, attachment or tag) and close