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Assessment of anti-inflammatory effect of5�-hydroxypalisadin B isolated from red seaweedLaurencia snackeyi in zebrafish embryo in vivomodel

W.A.J.P. Wijesinghea,1, Eun-A Kima, Min-Cheol Kanga, Won-Woo Leea,Hyi-Seung Leeb, Charles S. Vairappanc,∗, You-Jin Jeona,∗∗

a Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Koreab Marine Natural Products Laboratory, Korea Ocean Research & Development Institute, P.O. Box 29, Ansan 426-744,Republic of Koreac Laboratory of Natural Products Chemistry, Institute for Tropical Biology and Conservation, University MalaysiaSabah, Kota Kinabalu 88440, Sabah, Malaysia

a r t i c l e i n f o

Article history:

Received 31 July 2013

Received in revised form

31 October 2013

Accepted 3 November 2013

Available online 9 November 2013

Keywords:

5�-hydroxypalisadin B

a b s t r a c t

5�-Hydroxypalisadin B, a halogenated secondary metabolite isolated from red seaweed

Laurencia snackeyi was evaluated for its anti-inflammatory activity in lipopolysaccharide

(LPS)-induced zebrafish embryo. Preliminary studies suggested the effective concentrations

of the compound as 0.25, 0.5, 1 �g/mL for further in vivo experiments. 5�-Hydroxypalisadin

B, exhibited profound protective effect in the zebrafish embryo as confirmed by survival

rate, heart beat rate, and yolk sac edema size. The compound acts as an effective agent

against reactive oxygen species (ROS) formation induced by LPS and tail cut. Moreover, 5�-

hydroxypalisadin B effectively inhibited the LPS-induced nitric oxide (NO) production in

zebrafish embryo. All the tested protective effects of 5�-hydroxypalisadin B were compa-

Anti-inflammatory

Functional ingredient

Laurencia snackeyi

Zebrafish embryo

rable to the well-known anti-inflammatory agent dexamethasone. According to the results

obtained, 5�-hydroxypalisadin B isolated from red seaweed L. snackeyi could be considered

as an effective anti-inflammatory agent which might be further developed as a functional

ingredient.

and nitric oxide, which can originate locally or from cells

1. Introduction

Inflammation is a highly regulated biological process thatenables the immune system to efficiently remove the injuriousstimuli and initiate the healing process (Masresha et al., 2012).

∗ Corresponding author. Tel.: +6088 320000x2397; fax: +6088 320291.∗∗ Corresponding author at: Jeju National University, Marine Life Scien

fax: +82 64 756 3493.E-mail addresses: csv@ums.edu.my (C.S. Vairappan), youjinj@jejunu

1 Present address: Food Research Unit, Department of Agriculture, Pe1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.etap.2013.11.006

© 2013 Elsevier B.V. All rights reserved.

The inflammatory symptoms result from the action of inflam-matory agents such as bradykinin, histamine, prostaglandins,

ce, Ara 1dong, Jeju, Republic of Korea. Tel.: +82 10 4572 3624;

.ac.kr (Y.-J. Jeon).radeniya, Sri Lanka.

that infiltrate in the sight of insult (Mequanint et al., 2011).Although inflammation is a defense mechanism, the complexevents and mediators involved in the prolonged inflammation

p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 110–117 111

cmti

imbmoh2

i(ooccefafHpa

vnImfctupm

2

2L

RaKotrsPfawotg(U

Fig. 1 – Chemical structure of 5�-hydroxypalisadin B

e n v i r o n m e n t a l t o x i c o l o g y a n d

an induce various diseases and disorders. Hence, the employ-ent of anti-inflammatory agents may be useful in the

herapeutic treatment of those pathologies associated withnflammatory reactions (Yonathan et al., 2006).

Secondary metabolites play an important ecological rolen marine organisms. Isolation and characterization of such

arine natural products may facilitate the investigation ofioactive metabolites in order to development of the phar-aceutical agents. In contrast, the development of marine

rganisms-derived compounds into functional ingredientsas been attracted attention over the years (Thomas & Kim,011).

Seaweeds have been one of the richest and most promis-ng sources of bioactive primary and secondary metabolitesde Almeida et al., 2011). Many researchers have focusedn red algae as a potential source of bioactive compoundsver the past few years. The red algal genus Laurencia typi-ally produces halogenated secondary metabolites, and theseompounds have been exhibited antimicrobial and cytotoxicffects (Kamada and Vairappan, 2012). Once the structures andunctional properties of these biologically active compoundsre understood, they may serve as potential active ingredientsor the development of nutraceuticals or pharmaceuticals.ence, understanding of the action pathways of such com-ounds is critical in the development of effective therapeuticgents for a particular disease.

Zebrafish (Danio rerio) is becoming increasingly popular inivo model organism for examining the biological properties ofatural products with biomedical relevance (Pyati et al., 2007).

n contrast, over the past few years the zebrafish has beenanipulated in a large number of studies as a model organism

or exploration of biological functions of the natural bioactiveomponents (Rinkwitz et al., 2011). Therefore, in this study,he zebrafish, a common aquatic vertebrate embryo has beensed as a tool for the assessment of in vivo anti-inflammatoryotential of 5�-hydroxypalisadin B, a brominated secondaryetabolite isolated from red seaweed L. snackeyi.

. Materials and methods

.1. Isolation of 5ˇ-hydroxypalisadin B from red algaaurencia snackeyi.

ed alga, L. snackeyi (Weber-van Bosse) Masuda was collectedt the depth of 5 m by scuba divers at PulauSulug Island, Kotainabalu, Sabah, Malaysia. Collected specimens were cleanedff epiphytes, sand and organic debris, brought to the labora-ory under 4 ◦C in a chiller. In the laboratory the algae wereinsed in three exchanges of double distilled water (DDW) andubjected to air-drying under 24 ◦C away from direct sunlight.artially dried algal thallus (220 g) was extracted with MeOHor 5 days. The MeOH solution was concentrated in vacuond partitioned between Et2O and H2O. The Et2O solution wasashed with water, dried over anhydrous Na2SO4, and evap-rated to leave a dark green oil (1.9 g). Chemical profiling of

he crude extract was done by spotting crude extract on SiO2

el F254 thin layer chromatography and developed in toluene100%) and hexane:EtOAc (3:1) solvent systems, visualized byV light (254 nm) and molybdophosphoric acid and heated.

isolated from red seaweed L. snackeyi.

The crude extract (1.0 g) was then fractionated by Si gel col-umn chromatography with a step gradient (hexane/EtOAc inthe ratio of 9:1, 8:2, 7:3, 5:5, and 100% EtOAc). Fraction elutedwith hexane-EtOAc (5:5) was subjected to preparative TLCwith CHCl3 to give 5�-hydroxypalisadin B (Fig. 1). Independentstructural elucidation and comparison of spectroscopic datato publish data lead to the identity of the isolated metaboliteas 5�-hydroxypalisadin B (de Nys et al., 1993; Tan et al., 2011).The final confirmation of the compound was made based onthe HRMS and optical rotation values and was similar to theones in the published documents. In addition, the purity ofthe compound was determined via HPLC and 1H-NMR to be>99%.

2.2. Origin and maintenance of parental zebrafish

Adult zebrafishes were obtained from a commercial dealer(Seoul aquarium, Korea), and 10 fishes were kept in 3 Lacrylic tank with the following conditions; 28.5 ◦C, with a14/10 h light/dark cycle. They were fed three times a day, 6d/week, with tetramin flake food supplemented with live brineshrimps (Artemiasalina). Embryos were obtained from naturalspawning that was induced in the morning by turning on thelight. Collection of embryos was completed within 30 min.

2.3. Measurement of the toxicity of the isolatedcompound

Sample toxicity was determined by means of survival rateand the heart beat rate of the zebrafish embryos. Briefly, theembryos (n = 15) were transferred to individual wells of 12-wellplates containing 950 �L embryo media from approximately 3to 4 h post-fertilization (3–4 hpf), 5�-hydroxypalisadin B wasintroduced to the embryos up to 24 hpf. The survival rate wasmeasured every day. Then, the zebrafish embryos were rinsedin fresh embryo media. The heart-beating rate of both atriumand ventricle was measured at 24 hpf to determine the sam-ple toxicity. Counting and recording of atrial and ventricularcontraction were performed for 3 min under the microscope,and the results were presented as the average heart-beating

rate per min (Cha et al., 2011). The survival rate of zebrafishembryos exposed to 5�-hydroxypalisadin B was determineduntil 5 day post-fertilization (5 dpf).

d p h

112 e n v i r o n m e n t a l t o x i c o l o g y a n

2.4. Measurement of the toxicity of the isolatedcompound by means of the cell death

Cell death was detected in live embryos using acridine orangestaining, a nucleic acid selective metachromatic dye thatinteracts with DNA and RNA by intercalation or electrostaticattractions. Acridine orange stains cells with disturbed plasmamembrane permeability, so it preferentially stains necrotic orvery late apoptotic cells. At 3 dpf, a zebrafish larvae weretransferred to 96-well plates, treated with acridine orangesolution (7 �g/mL) and incubated for 30 min under the dark at28.5 ± 1 ◦C. After, incubation, the zebrafish larvae were rinsedby fresh embryo media and anaesthetized by 2-phenoxyethanol (1/500 dilution sigma) before observation. Then pho-tographed under the microscope CoolSNAP-Pro color digitalcamera (Olympus, Japan). Individual zebrafish larvae fluores-cence intensity was quantified using an image J program.

2.5. Estimation of oxidative stress-induced ROSgeneration and image analysis

At 3 dpf, zebrafish larvae were transferred to 96-well plate,treated with DCF-DA solution (20 �g/mL) and incubated for 1 hin the dark at 28.5 ± 1 ◦C. After incubation, the zebrafish larvaewere rinsed by fresh embryo media and anaesthetized using2-phenoxy ethanol (1/500 dilution sigma) before observationand then photographed under the microscope CoolSNAP-Procolor digital camera (Olympus, Japan). Individual zebrafish lar-vae fluorescence intensity was quantified using an image Jprogram.

2.6. Measurement of in vivo NO production

At 3 dpf, zebrafish larvae were transferred to 96-well plates,treated with DAF-DM-DA solution (5 �M) and incubated for1 h in the dark at 28.5 ± 1 ◦C. After, incubation, the zebrafishlarvae were rinsed in fresh embryo media and anaesthetizedby 2-phenoxy ethanol (1/500 dilution sigma) before observa-tion. Then photographed under the microscope CoolSNAP-Procolor digital camera (Olympus, Japan). Individual zebrafish lar-vae fluorescence intensity was quantified using an image Jprogram.

2.7. Statistical analysis

The data are expressed as the mean ± standard error (S.E),and one-way ANOVA test (using SPSS 11.5 statistical software)was used to compare the mean values of each treatment. Sig-nificant differences between the means of parameters weredetermined by the student’s t-test (p < 0.05, p < 0.01).

3. Results

3.1. Effect of 5ˇ-hydroxypalisadin B on survival rate,heart beat rate and morphological changes in zebrafish

embryo

In order to determine the toxicity of the isolated compound5�-hydroxypalisadin B, in this study, we observed the survival

a r m a c o l o g y 3 7 ( 2 0 1 4 ) 110–117

rate, heart beat rate, and morphological changes in zebrafishembryos. According to the results obtained, there is no signifi-cant change in survival rate compared to the control indicatingthat there is no toxicity at the tested concentrations (Fig. 2A).In addition, almost the same trend was observed in the heartbeat rates at the sample concentrations tested. However, aslight increase in the heart beat rate was observed at the con-centration of 5 �g/mL (Fig. 2B). Moreover, some mormpologicalchange in zebrafish embryos was observed at the concentra-tion of 5 �g/mL (Fig. 2C). But, the lower concentrations did notshow any toxic effects on the developmental stages of thezebrafish embryo.

3.2. Effect of 5ˇ-hydroxypalisadin B on cell death inzebrafish embryo

Toxicity of 5�-hydroxypalisadin B was evaluated by meansof cell death in zebrafish embryos. Significantly highercell death was observed at the concentration of 5 �g/mL(Fig. 3). In contrast, a significantly increased intensity ofacridine orange was observed in the microscopic pictureof the zebrafish embryos treated with 5�-hydroxypalisadinB at the concentration of 5 �g/mL (Fig. 3A). With theresults of the preliminary studies, concentrations wereselected for the further experiments. Due to the toxici-ties at the higher concentrations, we adjusted the sampleconcentrations as 0.25, 0.5, and 1 �g/mL for the further exper-iments.

3.3. In vivo protective effect of 5ˇ-hydroxypalisadin Bon LPS-induced stress in zebrafish embryo

With the treatment of LPS, the survival rate of zebrafishembryo was slightly decreased. However, with the treatmentsof 5�-hydroxypalisadin B, the survival rates were observedas the control (Fig. 4A). In addition, LPS treatment signif-icantly increased the heart beat rate of the embryos. But,the sample effectively reduced the heart beat rate to thenormal level (Fig. 4B). Moreover, significantly increased yolksac edema size was observed with the LPS treatment. How-ever, 5�-hydroxypalisadin B dose-dependently reduced thesize of yolk sac edema up to the normal level (Fig. 4C).In this study, dexamethasone was used as a standardanti-inflammatory agent. 5�-hydroxypalisadin B showed acomparable protective effect compared to the dexameha-sone.

3.4. In vivo effect of 5ˇ-hydroxypalisadin B onstress-induced ROS generation and image analysis

In this study, the protective effect of 5�-hydroxypalisadin B onstress-induced ROS generation was determined in two differ-ent stress conditions. Fig. 5 shows the in vivo protective effectof 5�-hydroxypalisadin B on ROS generation in LPS-inducedzebrafish embryo. The control, which contained no LPS or5�-hydroxypalisadin B, generated clear image, whereas the

positive control, which got treated only with LPS, generatedfluorescence image, which suggests that generation of ROShas taken place in the presence of LPS in the zebrafish embryo.However, when the zebrafish embryos were treated with

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Fig. 2 – Effects of 5�-hydroxypalisadin B on (A) survival rate (B) heart beat rate, and (C) morphological changes of zebrafishembryo. Survival rate after treated with different concentrations of the compound was measured at 5 dpf. The average heartbeat rate per minute of 5�-hydroxypalisadin B treated embryos was recorded at 3 dpf. Representative photographs of themorphological changes of the embryos treated with different concentrations of the compound were taken. Experimentswere performed in triplicate and the data are expressed as mean ± SE. Values with different alphabets are significantlydifferent at p < 0.05 as analyzed by DMRT.

Fig. 3 – Effect of 5�-hydroxypalisadin B on cell death in zebrafish embryo. (A) The cell death levels were measured afteracridine orange staining by image analysis and fluorescence microscope. (B) The cell death was quantified using an image Jprogram. Experiments were performed in triplicate and the data are expressed as mean ± SE. Values with differentalphabets are significantly different at p < 0.05 as analyzed by DMRT.

114 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 110–117

Fig. 4 – Effects of 5�-hydroxypalisadin B or Dexamethasone on (A) survival rate (B) heart beat rate and (C) yolk sac edemasize of LPS treated zebrafish embryo. Experiments were performed in triplicate and the data were expressed as mean ± SE.Values with different alphabets are significantly different at p < 0.05 as analyzed by DMRT.

Fig. 5 – Protective effect of 5�-hydroxypalisadin B against LPS-induced ROS production in zebrafish larvae. (A) The ROSlevels were measured by image analysis and fluorescence microscope. (B) Individual zebrafish fluorescence intensity wasquantified using an image J program. Experiments were performed in triplicate and the data are expressed as mean ± SE.Values with different alphabets are significantly different at p < 0.05 as analyzed by DMRT.

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Fig. 6 – Protective effect of 5�-hydroxypalisadin B against tail cutting-induced ROS production in zebrafish larvae. (A) TheROS levels were measured by image analysis and fluorescence microscope. (B) Individual zebrafish fluorescence intensitywas quantified using an image J program. Experiments were performed in triplicate and the data are expressed asmean ± SE. Values with different alphabets are significantly different at p < 0.05 as analyzed by DMRT.

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�-hydroxypalisadin B prior to LPS treatment; a dose-ependent reduction in the generation of ROS was observed.

In addition, a similar trend was observed in the tail cut-nduced ROS production in the zebrafish embryo (Fig. 6).ail cut induced the generation of ROS according to theuorescence image and however, with the sample treat-ent dose-dependent reduction in the ROS generation was

bserved.

.5. In vivo effect of 5ˇ-hydroxypalisadin B onPS-induced NO production

he effect of 5�-hydroxypalisadin B on LPS-induced NO pro-uction was shown in Fig. 7. Stimulation of the zebrafishmbryo with LPS resulted in an enhancement of NO pro-uction. However, pretreatment of zebrafish embryo with theompound decreased the NO production in a dose-dependentanner.

. Discussion

t is well-known that naturally originated agents with min-mum side effects are desirable to substitute chemicalherapeutics (Conforti et al., 2008). Therefore, the use ofioactive natural ingredients is widespread and plants still

represent a large source of secondary metabolites that mightserve as leads for the development of novel therapeuticagents. Thus, natural products continue to be a major sourceof pharmaceuticals and for the discovery of new molecularstructures (McCloud, 2010). Hence, the aim of the presentinvestigation was to evaluate the anti-inflammatory potentialof a marine-derived halogenated secondary metabolite in thezebrafish embryo in vivo model. In contrast, our report pro-vides the first evidence of in vivo anti-inflammatory effect of5�-hydroxypalisadin B isolated from red seaweed L. snackeyi.

Inflammation is a disorder involving localized increases inthe number of leukocytes and a variety of complex media-tors (Mosquera et al., 2011). The mechanism of inflammationis attributed, in part, to release of ROS from activated neu-trophils and macrophages (Conforti et al., 2008). ROS, whenpresent in a high enough concentration, can attack biologi-cal molecules and lead to cell or tissue injury associated withdegenerative diseases including inflammation (Choi et al.,2002; Shibata et al., 2008). In addition, ROS induces inflamma-tion by stimulating release of cytokines such as interleukin-1�,interleukin-6, and tumor necrosis factor- ̨ which stimulaterecruitment of additional neutrophils and macrophages. As

free radicals are important mediators that provoke inflamma-tory processes, the suppression of such free radicals might bean effective approach toward the attenuation of inflammation(Geronikaki and Gavalas, 2006).

116 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 110–117

Fig. 7 – Protective effect of 5�-hydroxypalisadin B against LPS-induced NO production in zebrafish larvae. (A) The NO levelswere measured by image analysis and fluorescence microscope. (B) Individual zebrafish fluorescence intensity wasquantified using an image J program. Experiments were performed in triplicate and the data are expressed as mean ± SE.

p < 0

r

Values with different alphabets are significantly different at

5. Conclusion

In the present work, the isolated compound was evaluatedfor its anti-inflammatory activity in zebrafish embryo in vivomodel compared to the standard drug dexamethasone, awell-known anti-inflammatory agent. 5�-hydroxypalisadin Bshowed a profound protective effect against stress-inducedROS formation in zebrafish embroyos. In addition, accord-ing to the obtained results, 5�-hydroxypalisadin B can actas an effective agent for inhibiting the LPS-induced NOproduction. Taken together, 5�-hydroxypalisadin B can beconsidered as an effective anti-inflammatory candidate frommarine. In addition, it could be suggested that in vivoanti-inflammatory potential of the halogenated secondarymetabolite 5�-hydroxypalisadin B isolated from red seaweedL. snackeyi may facilitate the development of novel functionalingredient.

Conflict of interest

Nothing Declared

Acknowledgement

This work was supported by a grant from the Ministry ofLand, Transport and Maritime Affairs, Korea (PM57121) andCSV would like to express his appreciation to KORDI for par-tially funding this project. The permit to collect this seaweed

.05 as analyzed by DMRT.

was obtained by CSV from Sabah Parks, Malaysia, and it ishighly appreciated.

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