neurotrophic activity of the carrageenophyte kappaphycus

Research ArticleNeurotrophic Activity of the CarrageenophyteKappaphycus alvarezii Cultivated at Different Depths and forDifferent Growth Periods in Various Areas of Indonesia

Gabriel Tirtawijaya1 Maria Dyah NurMeinita2 BintangMarhaeni2

Md Nazmul Haque3 Il SooMoon3 and Yong-Ki Hong 1

1Department of Biotechnology Pukyong National University Nam-gu Busan 48513 Republic of Korea2Faculty of Fisheries and Marine Science Jenderal Soedirman University Purwokerto 53123 Indonesia3Department of Anatomy College of Medicine Dongguk University Gyeongju 38066 Republic of Korea

Correspondence should be addressed to Yong-Ki Hong ykhongpknuackr

Received 1 June 2018 Revised 17 September 2018 Accepted 24 September 2018 Published 18 October 2018

Academic Editor Ching-Liang Hsieh

Copyright copy 2018 Gabriel Tirtawijaya 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 carrageenophyteKappaphycus alvarezii (Rhodophyta) hasneurotrophic activity in primaryhippocampal neuronsThis seaweedis abundant and easily cultivated in tropical coastal areas To determine the best growth conditions for neurotrophic activity thalliwere grown at different depths and for different periods in various areas of Indonesia Neurotrophic activity was measured basedon the number of primary neurites the total length of the primary neurites and the length of the longest neurite K alvarezii hadhigher neurotrophic activity than carrageenophytes K striatum and Eucheuma denticulatum cultured under the same conditionsK alvarezii grown at the surface for 45 days had higher (14- to 18-fold) neurotrophic activity than thalli grown at depth (2 m) orharvested sooner (15 days) (P lt 005) Relatively high activities were detected in thalli cultured at Ternate and Garut IndonesiaTherefore from a commercial perspective the culture conditions at the surface for 45 days were optimal for the production ofboth neurotrophic compounds and carrageenan K alvarezii produced neurotrophic compounds under various environmentalconditions although some conditions were optimal

1 Introduction

Neurodegenerative disorders are a crucial threat to humanhealth especially in the elderly These disorders are charac-terized by gradual degeneration of the structure and functionof the central or peripheral nervous system [1] The mostcommon neurodegenerative disorder is Alzheimerrsquos diseasePeople with Alzheimerrsquos disease have memory dysfunctionand hippocampal atrophy [2 3] Neurotrophic activity canprevent cell death in neurodegenerative processes and playkey roles in the survival differentiation synaptogenesis andmaturation of affected neurons in Alzheimerrsquos disease [4]

Macroalgae are used in health foods diet supplementsand other useful compounds The edible rhodophyte Kappa-phycus alvarezii produces a hydrocolloid carrageenan that isused as a food additive and a gelling emulsifying and sta-bilizing agent in nutraceutical and pharmaceutical products

[5] Recently this seaweed was reported to have beneficialeffects in preventing diet-induced metabolic syndrome [6]Other research has shown that K alvarezii is cardioprotective[7] and has wound healing [8] antioxidant [9] antimicrobial[10] and anti-inflammatory [11] properties Pangestuti andKim [12] reported that K alvarezii has antioxidant anti-neuroinflammatory and anti-cholinesterase activities Pre-viously we showed that K alvarezii exerts its neurotrophicactivity by accelerating the initial neuronal maturation andstimulating axodendritic arborization in primary cultures ofhippocampal neurons [13 14]

In Indonesia K alvarezii is abundant and is cultivated inmany localities Generally the bioactive compound contentof macroalgae depends on physical and biological factorssuch as climate water quality reproductive state blade agethallus section locality and seasonality [15]The biochemical

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 1098076 7 pageshttpsdoiorg10115520181098076

2 Evidence-Based Complementary and Alternative Medicine

Buoys

Bamboo frame (5 m x 5 m)

Nylon rope

Sandbag

Adjustable depth

Figure 1 The floating bamboo frame system used in this study The bamboo frame measured 5 m times 5 m Six culture nets were fixed to theframeThe depth of the culture nets was adjusted to 0 1 or 2 m Six algal thalli were grown on each culture line (5 m)

composition of K alvarezii can vary with the cultivationperiod and planting density [16] depth [17] season [18] andspatiotemporal conditions [19] It is important to evaluatethe growth conditions and bioactivity when macroalgae areexploited commercially Studies of the depth growth periodand locality are essential when characterizing the bioactivitiesof algae

Therefore we cultivated K alvarezii thalli at differentdepths and for different growth periods in various areas ofIndonesia and compared the neurite outgrowth-promotingactivity on hippocampal neuron cells

2 Materials and Methods

21MacroalgalMaterials andAquaculture Thefloating bam-boo method was used for aquaculture with the cages placed200 meters from the shore The bamboo frames (5 times 5 m2)were equipped with polyethylene plastic buoys for floatationand sandbags for anchors to prevent the influence of tides(Figure 1) Nylon lines were connected to the bamboo framevertically and horizontally The depth of nylon culture netswas adjusted to 0 1 or 2 meters At each depth a culturenet was used formacroalgae cultivation Approximately 200 g(wet weight) of fresh K alvarezii thalli was tied to the cultureropes with plastic twine Tissue samples were collected after0 15 30 or 45 days rinsed twice with seawater to removedebris and rinsed again with freshwater to remove salt Thenthe tissues were dried in the shade for 2sim3 days The driedsamples were pulverized into a powder in a grinder (HMF-340 Hanil Seoul Korea) and kept in the dark at ndash20∘C untilused

Thalli of the carrageenophytes K alvarezii K stria-tum and Eucheuma denticulatum were cultured in BulelengRegency (8∘11101584001510158401015840 S 114∘48101584039210158401015840 E) western Bali Indone-sia during the dry seasons (AprilndashJune) from 2015 to 2017The three carrageenophytes were cultured on nets at the

surface (0 m depth) for 45 days For the experiments atdifferent depths and for growing periods K alvarezii wascultivated at 0 1 and 2m for 0 15 30 and 45 days at the samesite in Buleleng Regency For the experiments comparinglocation K alvarezii thalli were cultured at the surface (0m depth) in cages of the same size for 45 days at 14 local-ities in Indonesia Bali Banten Batam Garut KalimantanKarimunjawa Kendari Lampung Lombok Morotai PapuaSumbawa Takalar and Ternate

22 Culture and Treatment of Primary Hippocampal NeuronsCultures of hippocampal neurons were prepared in 24-wellpolystyrene plates All cell culture reagents were purchasedfrom Invitrogen (Carlsbad CA USA) unless otherwisestated All animal care and use were in agreement withinstitutional guidelines and approved by the InstitutionalAnimal Care and Use Committee of the College of MedicineDongguk University Korea Primary hippocampal neuronswere prepared as described previously [20 21] BrieflySprague-Dawley rats on day 19 of pregnancy were euthanizedwith isoflurane and the fetuses were collected The neuronalcells dissociated from the fetal hippocampiwere countedwitha hemocytometer and plated at a density of approximately 1ndash2times 104 cellscm2 onto poly-DL-lysine-coated glass coverslips in24-well culture plates Cultures were maintained in serum-free neurobasal media supplemented with B27 and incubatedat 37∘C under 5 CO

2and 95 air for 2 days in vitro (DIV

2)Fine powder of K alvarezii was processed to obtain an

extract using 95 ethanol according to Tirtawijaya et al[13] The ethanol extract was dissolved in dimethyl sulfoxide(DMSO) to 8 mgml The extract with an optimal concentra-tion of 1 120583gml or vehicle control (DMSO final concentrationle 05) was added to the culture media prior to cellplating

Evidence-Based Complementary and Alternative Medicine 3

A B C

NPN

()

0

50

100

150

200

aa ax

x x

1 microgml10 microgml

(a)

A B C

TLPN

()

0

50

100

150

200 a

x xb

aby


(b)

A B C

LLN

()

0

50

100

150

200


ax

b

x

aby

(c)

Figure 2 Neurotrophic activity of Kappaphycus alvarezii (A) Kappaphycus striatum (B) and Eucheuma denticulatum (C) on primaryhippocampal neurons at day in vitro (DIV) 2 The seaweeds were grown at the surface for 45 days in west Bali The morphometry of theneurons (ge 50 cells) was measured based on (a) the number of primary neurites (NPN) (b) the total length of the primary neurites (TLPN)and (c) the length of the longest neurite (LLN) Activity () is expressed as the mean plusmn SE (n ge 3) relative to the control Extracts of 1 120583gml(black) and 10 120583gml (grey) were added to the neuron cultures Different letters at the 1 120583gml (a b) and 10 120583gml (x y) concentrations indicatesignificant differences (P lt 005)

23 ImageAcquisition Images (1388times 1039 pixels) were cap-tured using a Leica DM IRE2 research microscope equippedwith I3 S N21S and Y5 filter systems (Leica MicrosystemsAG Wetzlar Germany) and a high-resolution CoolSNAPCCD camera (Photometrics Munich Germany) under thecontrol of a computer using the Leica FW4000 programThedigital images were processed using Adobe Photoshop 70software (Adobe San Jose CA USA)

24 Image Analysis and Quantification Morphometric anal-yses and quantification were performed using ImageJ soft-ware version 148 (National Institutes of Health BethesdaMD USA httpimagejnihgovij) with the simple neuritetracer plug-inMorphometric parameters such as the numberof primary neurites (NPN neurites that originated directlyfrom the soma) the total length of primary neurites (TLPNthe sum of primary neurite lengths) and the length of thelongest neurite (LLN) were measured Neurons (a minimumof 50 cells) that were not intermingled with the processesof adjacent neurons were selected for analysis We alwayscompared the extract-treated cultureswith the vehicle control(cultures with DMSO)

25 Statistical Analysis Results are shown as the means plusmnstandard error (SE) of at least three independent experimentsData were checked for the normality of the distribution bythe Kolmogorov-Smirnov test and normally distributed datawere used for further analysis We conducted a two-wayanalysis of variance (ANOVA) on data of different depths andculture times while data from different species and localitieswere analyzed by a one-way ANOVA Duncanrsquos post hocmultiple comparison was used to determine significant (P lt005) differences among ge 3 treatment means Correlationsbetween cultivation conditions and NPN TLPN and LLNwere examined using Pearsonrsquos correlation coefficients with120572 lt 005 All calculations were carried out using SPSSstatistical software for Windows version 170 (SPSS IncChicago IL USA)

3 Results

Neurotrophic activity was quantified based onmorphometricanalyses of hippocampal neurons using NPN TLPN andLLN Adding the K alvarezii extract to the neuron culturesenhanced the growth of neurites First we compared threeabundant carrageenophytes (K alvarezii K striatum andE denticulatum) that are aquaculturable in Indonesia toselect the one with the highest neurotrophic activity Wegrew the plants at the surface (0 m depth) for 45 dayson bamboo frame nets in Buleleng Regency and comparedtheir neurotrophic activities (Figure 2) The three species didnot differ significantly in terms of NPN but they differedsignificantly in terms of TLPN and LLN (P lt 005) Kalvarezii had higher neurotrophic activity in terms of TLPNand LLN than K striatum or E denticulatum so K alvareziiwas selected as the best species for further neurotrophicexperiments

Next to determine the effective growth conditions topromote the neurotrophic activity of K alvarezii thalli weregrown at different depths and for different periods The Kalvarezii grown at 0 m for 45 days and at 1 m for 30 days hada significantly higher NPN increasing by approximately 14-and 14-fold respectively compared with the vehicle control(Table 1) The initial seeds at 0 d harvested from thalli grownfor 45 days had anNPN 12-fold higher than the control Aftergrowth for 15 days at all depths ie the young growing stageneurotrophic activity was similar to that of the controls iethere was no neurotrophic activity NPN tended to be lowerin cultures at a depth of 2 m for all harvest times

In terms of TLPN the seaweed thalli grown at 0 m for 45days and at 1 m for 30 days had significantly higher activitythan the control increasing by approximately 18- and 18-fold respectively (Table 2) Generally thalli grown at less than1 m and for longer than 30 days had high activity The initialseeds at 0 day from seaweed previously grown for 45 daysalso had a high TLPN No promotional activity was seen aftergrowth for 15 days at all depths TLPN tended to be lower forall cultures at a depth of 2 m for all harvest times


Table 1 Comparison of the number of primary neurites (NPN) in hippocampal neurons exposed to extracts of K alvarezii grown at differentdepths and for different culture periods

Depth (m) Culture period (days)0 15 30 45

0 121 plusmn 3abx 107 plusmn 4ax 126 plusmn 5bxy 135 plusmn 9bx

1 121 plusmn 3abx 105 plusmn 2bx 140 plusmn 2cx 122 plusmn 8ax

2 121 plusmn 3abx 110 plusmn 3ax 122 plusmn 5by 118 plusmn 3abx

Activity () is expressed as the mean plusmn SE (n ge 3) relative to the control Day 0 initial seeding day Means within each column with different letters (x y) differsignificantly (P lt 005) Means within each row with different letters (andashc) differ significantly (P lt 005)

Table 2 Comparison of the total length of primary neurites (TLPN) in hippocampal neurons exposed to extracts of K alvarezii grown atdifferent depths and for different culture periods


0 158 plusmn 5ax 115 plusmn 6bx 160 plusmn 5ax 180 plusmn 10ax

1 158 plusmn 5ax 100 plusmn 1bx 177 plusmn 6ax 164 plusmn 17axy

2 158 plusmn 5ax 100 plusmn 7bx 123 plusmn 6cy 131 plusmn 2cy


Table 3 Comparison of the length of the longest neurites (LLN) in hippocampal neurons exposed to extracts ofK alvarezii grown at differentdepths and for different culture periods


0 153 plusmn 7ax 105 plusmn 7bxy 154 plusmn 5ax 172 plusmn 18ax


2 153 plusmn 7ax 93 plusmn 5by 106 plusmn 6bcy 122 plusmn 2cy


With regard to the promotion of LLN thalli grown atdepth 0 m for 45 days had the highest activity increasingby approximately 17-fold higher than the control (Table 3)Generally thalli grown at less than 1 m and for longer than 30days had high activityThe initial seeds at 0 day also had a highLLNAt a culture time of 15 days at all depths no promotionalactivities were observed LLN tended to decrease at a depth of2 m for all harvest times

To investigate the influence of location thalli of Kalvarezii were cultured at the surface for 45 days in 14localities in Indonesia Extracts were prepared from the thallifrom the 14 localities and their neurotrophic activities weretested The NPN TLPN and LLN activities in extracts fromdifferent areas differed to varying degrees (Table 4) Relativelyhigh activities were detected in thalli cultured at TernateNorth Maluku and Garut West Java Thalli from Ternatepromoted NPN TLPN and LLN by 18- 21- and 19-foldrespectively compared with the control while thalli fromGarut promoted them by 17- 20- and 20-fold The highestNPNwas obtained with the extract fromTernate and the low-est with that from Takalar (11-fold the control) The highestTLPN was obtained with the extract from Ternate and thelowest from Batam (12-fold) The highest LLN was obtainedfromLombok (20-fold) and the lowest fromBatam (11-fold)

The neurotrophic activity of the K alvarezii extracts variedsignificantly with location (P lt 005)These results confirmedthat different environmental conditions affect the productionof neurotrophic compounds in K alvarezii tissues Figure 3shows representative neurons grown in vehicle and extractsof K alvarezii cultivated in Ternate and Garut on DIV 2

4 Discussion

Neurotrophic factors are molecules that are critical for neu-rite outgrowth and affect neuron survival synaptic plasticityand the formation of long-lasting memories [22] Previ-ously we found that the rhodophyte K alvarezii was thebest candidate for the production of neurotrophic factorsfrom among 34 common Indonesian macroalgae tested[13] This macroalga contains several lipophilic compoundsthat contribute to its neurotrophic activity [14] There ishigh demand for K alvarezii aquaculture in Indonesia forthe production of hydrocolloid carrageenan K striatumand E denticulatum two other carrageenan producers arealso widely cultivated in Indonesia The genus Kappaphycusproduces kappa-carrageenan which has greater gel strengthand 36-anhydrogalactose levels and fewer sulfate radicalsthan iota-carrageenan [23] The genus Eucheuma produces


Table 4 Neurotrophic activities of extracts of K alvarezii thalli grown in various areas in Indonesia

Culture area Activity ()NPN TLPN LLN

West Bali Bali 135 plusmn 9ab 180 plusmn 10a 172 plusmn 18ab

Banten Banten 149 plusmn 4bc 200 plusmn 11ab 184 plusmn 4ab

Batam Riau Islands 135 plusmn 8ab 118 plusmn 7c 106 plusmn 6c

Garut West Java 169 plusmn 3cd 196 plusmn 10ab 198 plusmn 12b

Bunyu Island North Kalimantan 164 plusmn 4cd 183 plusmn 3ab 189 plusmn 10ab

Karimunjawa Central Java 138 plusmn 2ab 144 plusmn 10cde 141 plusmn 12de

Kendari Southeast Sulawesi 152 plusmn 12bc 186 plusmn 6ab 162 plusmn 9ae

Lampung Lampung 149 plusmn 9bc 175 plusmn 10a 177 plusmn 7ab

Lombok West Nusa Tenggara 154 plusmn 4bc 187 plusmn 4ab 199 plusmn 8b

Morotai North Maluku 171 plusmn 9cd 185 plusmn 5e 152 plusmn 13ab

Papua Papua 120 plusmn 1ae 132 plusmn 5cde 118 plusmn 5cd

Sumbawa West Nusa Tenggara 135 plusmn 4ab 148 plusmn 9de 140 plusmn 6de

Takalar South Sulawesi 111 plusmn 2e 121 plusmn 2cd 136 plusmn 7de

Ternate North Maluku 181 plusmn 13d 209 plusmn 11b 189 plusmn 11ab

Thalli were cultured at the surface for 45 days in 14 different locations Activity () is expressed as the mean plusmn SE (n ge 3) relative to the control Means withineach column with different letters (andashe) differ significantly (P lt 005)

40 microm

20 microm

(a)

40 microm

20 microm

(b)

40 microm

20 microm

(c)

Figure 3 Representative images of neurons grown in vehicle (a) and extracts of Kappaphycus alvarezii grown at Ternate (b) and Garut (c)Primary neurites (arrows) and the longest neurite (arrowheads) were observed using ImageJ software to determine the NPN TLPN andLLN

iota-carrageenan Comparison of these three common car-rageenophytes revealed that K alvarezii had greater neu-rotrophic activity than the others The high neurotrophicactivity of K alvarezii did not appear to be related to thegel strength of carrageenan or the amount of sulfate oranhydrogalactose moieties in carrageenan Some researchhas indicated that the antioxidant activity and total phenoland carotene contents differ between E cottonii (now knownas K alvarezii) and E spinosum (now known as E den-ticulatum) [24] but Rosni et al [25] reported that severalvarieties of K alvarezii K striatum and E denticulatum hadinsignificant protein and total phenolic contents Thereforethe neurotrophic activity is not directly related to the levelsof antioxidants phenolics carotene or proteins

Two common methods are used for carrageenophytesaquaculture longlines and floating cages A floating cageresults in higher biomass production than a longlinebecause the macroalgae can be cultivated vertically in morespace and protected from herbivores [26] Given that lightdecreases exponentially as depth increases the chlorophyll

and carotenoid pigments increase with depth [17] We foundthat the neurotrophic activity was highest in K alvareziigrown at the surface Therefore the neurotrophic activityis also not related to pigment production The culture con-ditions that yield the best carrageenan from a commercialperspective were 45 days growth at the surface [16] Theseculture conditions were optimal for the production of bothcarrageenan and neurotrophic compounds in K alvareziiWhen grown for longer than 45 days thalli are prone tobreaking and are lost [16]

The values of NPN TLPN and LLN varied from 11119to 18109 11751 to 20849 and 10580 to 19906 respec-tively at all sites studied Significant spatial differences wereobserved in NPN TLPN and LLN (one-way ANOVA all Ple 005) indicating that different environmental conditionsaffected the neurotrophic activity The concentrations of sec-ondary metabolites in macroalgae may vary with changes inenvironmental conditions [15 27] Overall the neurotrophicactivity of K alvarezii from all localities studied was betterthan the vehicle control Therefore the carrageenophyte K


alvarezii produces neurotrophic compounds under variousenvironmental conditions although some conditions aremore optimal The macroalgae studied came from all regionsof Indonesia suggesting that K alvarezii can be cultivatedin various localities Future work should examine the rela-tionship between culture conditions in different localities andthe composition of neurotrophic compounds in K alvareziitissues

5 Conclusions

Neurotrophic activities from the carrageenophyte K alvarezii(Rhodophyta) were measured in primary cultures of rathippocampal neuronsThis seaweed had higher neurotrophicactivity than the other carrageenophytes K striatum andEucheuma denticulatum cultured under the same conditionsK alvarezii grown at the surface for 45 days had higherneurotrophic activity than thalli grown at a depth of 2 m orharvested sooner (15 days) Relatively high activity levels weredetected in thalli cultured at Ternate and Garut IndonesiaThus from a commercial perspective the culture conditionsat the surface for 45 days were optimal for the production ofboth neurotrophic compounds and carrageenan

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Gabriel Tirtawijaya andMariaDyahNurMeinita contributedequally to this work

Acknowledgments

We thank Mr Gigih Nur Akhmad and other farmers whoworked for the seaweed aquaculture This research wassupported by the International Research Collaboration andInternational Publication Grant Ministry of Research Tech-nology and Higher Education Republic of Indonesia

References

[1] HM Gao and J S Hong ldquoWhy neurodegenerative diseases areprogressive uncontrolled inflammation drives disease progres-sionrdquo Trends in Immunology vol 29 no 8 pp 357ndash365 2008

[2] J Barnes J W Bartlett L A van de Pol et al ldquoA meta-analysis of hippocampal atrophy rates in Alzheimerrsquos diseaserdquoNeurobiology of Aging vol 30 no 11 pp 1711ndash1723 2009

[3] H Jahn ldquoMemory loss in alzheimerrsquos diseaserdquo Dialogues inClinical Neuroscience vol 15 no 4 pp 445ndash454 2013

[4] T B Sampaio A S Savall M E Z Gutierrez and S PintonldquoNeurotrophic factors in Alzheimerrsquos and parkinsonrsquos diseasesImplications for pathogenesis and therapyrdquoNeural RegenerationResearch vol 12 no 4 pp 549ndash557 2017

[5] T D Pickering P Skelton and R J Sulu ldquoIntentional intro-ductions of commercially harvested alien seaweedsrdquo BotanicaMarina vol 50 no 5 pp 338ndash350 2007

[6] S Wanyonyi R Du Preez L Brown N A Paul and S KPanchal ldquoKappaphycus alvarezii as a food supplement preventsdiet-induced metabolic syndrome in ratsrdquo Nutrients vol 9 no11 pp 1261ndash1276 2017

[7] P Matanjun S Mohamed K Muhammad and N MMustapha ldquoComparison of cardiovascular protective effects oftropical seaweeds kappaphycus alvarezii caulerpa lentilliferaand sargassum polycystum on high-cholesterolhigh-fatdiet inratsrdquo Journal of Medicinal Food vol 13 no 4 pp 792ndash800 2010

[8] S G Fard R T R Tan AAMohammed et al ldquoWoundhealingproperties of Eucheuma cottonii extracts in Sprague-Dawleyratsrdquo Journal ofMedicinal Plant Research vol 5 no 27 pp 6373ndash6380 2011

[9] N Nagarani and A K Kumaraguru ldquoInvestigation of the effectof K alvarezii on antioxidant enzymes cell viability and DNAdamage inmale ratsrdquo Frontiers in Life Science vol 6 no 3-4 pp97ndash105 2012

[10] V Prabha D J Prakash and P N Sudha ldquoAnalysis of bioactivecompounds and antimicrobial activity of marine alga Kap-paphycus alvarezii using three solvent extractsrdquo InternationalJournal of Pharmaceutical Sciences and Research vol 4 no 1pp 306ndash310 2013

[11] P Ranganayaki S Susmitha and R Vijayaraghavan ldquoStudy onmetabolic compounds of Kappaphycus alvarezii and its in vitroanalysis of anti-inflammatory activityrdquo International Journal ofCurrent Research and Academy Review vol 2 no 10 pp 157ndash166 2014

[12] R Pangestuti and S-K Kim ldquoNeuroprotective effects of marinealgaerdquoMarine Drugs vol 9 no 5 pp 803ndash818 2011

[13] G Tirtawijaya M Mohibbullah M D N Meinita I S Moonand Y-K Hong ldquoThe ethanol extract of the rhodophyte Kap-paphycus alvarezii promotes neurite outgrowth in hippocampalneuronsrdquo Journal of Applied Phycology vol 28 no 4 pp 2515ndash2522 2016

[14] G Tirtawijaya M Mohibbullah M D N Meinita I S Moonand Y-K Hong ldquoThe tropical carrageenophyte Kappaphycusalvarezii extract promotes axodendritic maturation of hip-pocampal neurons in primary culturerdquo Journal of AppliedPhycology pp 1ndash9 2018

[15] J T Hafting J S Craigie D B Stengel et al ldquoProspects andchallenges for industrial production of seaweed bioactivesrdquoJournal of Phycology vol 51 no 5 pp 821ndash837 2015

[16] L Hayashi E C Oliveira G Bleicher-Lhonneur et al ldquoTheeffects of selected cultivation conditions on the carrageenancharacteristics of Kappaphycus alvarezii (Rhodophyta Solieri-aceae) in Ubatuba Bay Sao Paulo Brazilrdquo Journal of AppliedPhycology vol 19 no 5 pp 505ndash511 2007

[17] Indriatmoko Heriyanto L Limantara and T H P Brotosu-darmo ldquoComposition of photosynthetic pigments in a red algaKappaphycus alvarezii cultivated in different depthsrdquo ProcediaChemistry vol 14 pp 193ndash201 2015

[18] K Suresh Kumar K Ganesan and P V Subba Rao ldquoSeasonalvariation in nutritional composition of Kappaphycus alvarezii(Doty) Doty - an edible seaweedrdquo Journal of Food Science andTechnology vol 52 no 5 pp 2751ndash2760 2015


[19] C Periyasamy P V S Rao and P Anantharaman ldquoSpatialand temporal variation in carrageenan yield and gel strengthof cultivated Kappaphycus alvarezii (Doty) Doty in relationto environmental parameters in Palk Bay waters Tamil NaduSoutheast coast of Indiardquo Journal of Applied Phycology vol 28no 1 pp 525ndash532 2016

[20] K Goslin H Asmussen and G Banker ldquoRat hippocampalneurons in low-density culturerdquo in Culturing Nerve Cells GBanker and K Goslin Eds vol 58 pp 339ndash370 MIT PressMassachusetts MA USA 2nd edition 1998

[21] M A Hannan J-Y Kang Y-K Hong et al ldquoThe marine algaGelidiumamansii promotes the development and complexity ofneuronal cytoarchitecturerdquoPhytotherapy Research vol 27 no 1pp 21ndash29 2013

[22] C Deister and C E Schmidt ldquoOptimizing neurotrophic factorcombinations for neurite outgrowthrdquo Journal of Neural Engi-neering vol 3 no 2 pp 172ndash179 2006

[23] G A Santos ldquoCarrageenans of species of Eucheuma J Agardhand Kappaphycus Doty (Solieriaceae Rhodophyta)rdquo AquaticBotany vol 36 no 1 pp 55ndash67 1989

[24] L J Damongilala S B Widjanarko E Zubaidah and M R JRuntuwene ldquoAntioxidant activity against methanol extractionof Eucheuma cottonii and E spinosum collected from NorthSulawesi Waters Indonesiardquo Food Science and Quality Manage-ment vol 17 pp 7ndash13 2013

[25] S Mohd Rosni A Fisal A Azwan F Y Chye and P MatanjunldquoCrude proteins total soluble proteins total phenolic contentsand SDS-PAGE profile of fifteen varieties of seaweed from Sem-porna Sabah Malaysiardquo International Food Research Journalvol 22 no 4 pp 1483ndash1493 2015

[26] M Kasim and A Mustafa ldquoComparison growth of Kappaphy-cus alvarezii (Rhodophyta Solieriaceae) cultivation in floatingcage and longline in Indonesiardquo Aquaculture Reports vol 6 no1 pp 49ndash55 2017

[27] E A Shalaby ldquoInfluence of abiotic stress on biosynthesis of alga-chemicals and its relation to biological activitiesrdquo Indian Journalof Geo Marine Sciences vol 46 no 1 pp 23ndash32 2017

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Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


Buoys

Bamboo frame (5 m x 5 m)

Nylon rope

Sandbag

Adjustable depth

Figure 1 The floating bamboo frame system used in this study The bamboo frame measured 5 m times 5 m Six culture nets were fixed to theframeThe depth of the culture nets was adjusted to 0 1 or 2 m Six algal thalli were grown on each culture line (5 m)

composition of K alvarezii can vary with the cultivationperiod and planting density [16] depth [17] season [18] andspatiotemporal conditions [19] It is important to evaluatethe growth conditions and bioactivity when macroalgae areexploited commercially Studies of the depth growth periodand locality are essential when characterizing the bioactivitiesof algae

Therefore we cultivated K alvarezii thalli at differentdepths and for different growth periods in various areas ofIndonesia and compared the neurite outgrowth-promotingactivity on hippocampal neuron cells

2 Materials and Methods

21MacroalgalMaterials andAquaculture Thefloating bam-boo method was used for aquaculture with the cages placed200 meters from the shore The bamboo frames (5 times 5 m2)were equipped with polyethylene plastic buoys for floatationand sandbags for anchors to prevent the influence of tides(Figure 1) Nylon lines were connected to the bamboo framevertically and horizontally The depth of nylon culture netswas adjusted to 0 1 or 2 meters At each depth a culturenet was used formacroalgae cultivation Approximately 200 g(wet weight) of fresh K alvarezii thalli was tied to the cultureropes with plastic twine Tissue samples were collected after0 15 30 or 45 days rinsed twice with seawater to removedebris and rinsed again with freshwater to remove salt Thenthe tissues were dried in the shade for 2sim3 days The driedsamples were pulverized into a powder in a grinder (HMF-340 Hanil Seoul Korea) and kept in the dark at ndash20∘C untilused

Thalli of the carrageenophytes K alvarezii K stria-tum and Eucheuma denticulatum were cultured in BulelengRegency (8∘11101584001510158401015840 S 114∘48101584039210158401015840 E) western Bali Indone-sia during the dry seasons (AprilndashJune) from 2015 to 2017The three carrageenophytes were cultured on nets at the

surface (0 m depth) for 45 days For the experiments atdifferent depths and for growing periods K alvarezii wascultivated at 0 1 and 2m for 0 15 30 and 45 days at the samesite in Buleleng Regency For the experiments comparinglocation K alvarezii thalli were cultured at the surface (0m depth) in cages of the same size for 45 days at 14 local-ities in Indonesia Bali Banten Batam Garut KalimantanKarimunjawa Kendari Lampung Lombok Morotai PapuaSumbawa Takalar and Ternate

22 Culture and Treatment of Primary Hippocampal NeuronsCultures of hippocampal neurons were prepared in 24-wellpolystyrene plates All cell culture reagents were purchasedfrom Invitrogen (Carlsbad CA USA) unless otherwisestated All animal care and use were in agreement withinstitutional guidelines and approved by the InstitutionalAnimal Care and Use Committee of the College of MedicineDongguk University Korea Primary hippocampal neuronswere prepared as described previously [20 21] BrieflySprague-Dawley rats on day 19 of pregnancy were euthanizedwith isoflurane and the fetuses were collected The neuronalcells dissociated from the fetal hippocampiwere countedwitha hemocytometer and plated at a density of approximately 1ndash2times 104 cellscm2 onto poly-DL-lysine-coated glass coverslips in24-well culture plates Cultures were maintained in serum-free neurobasal media supplemented with B27 and incubatedat 37∘C under 5 CO

2and 95 air for 2 days in vitro (DIV

2)Fine powder of K alvarezii was processed to obtain an

extract using 95 ethanol according to Tirtawijaya et al[13] The ethanol extract was dissolved in dimethyl sulfoxide(DMSO) to 8 mgml The extract with an optimal concentra-tion of 1 120583gml or vehicle control (DMSO final concentrationle 05) was added to the culture media prior to cellplating


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5 Conclusions


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Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





































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20 microm

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(b)

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5 Conclusions


Data Availability






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of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















5 Conclusions


Data Availability






Acknowledgments


References

































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















neurotrophic activity of the carrageenophyte kappaphycus

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