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Research ArticleEvaluationof thePhysicochemical Antioxidant andAntibacterialProperties of Tunichrome Released from Phallusia nigra PersianGulf Marine Tunicate

Zohreh Marhamati 1 Mohammad Hossein Marhamatizadeh 1

and Gholamhossein Mohebbi 2

1Department of Food Hygiene Faculty of Veterinary Medicine Kazerun Branch Islamic Azad University Kazerun Iran2e Persian Gulf Marine Biotechnology Research Center e Persian Gulf Biomedical Sciences Research InstituteBushehr University of Medical Sciences Bushehr Iran

Correspondence should be addressed to Mohammad Hossein Marhamatizadeh drmarhamatigmailcom

Received 11 February 2021 Revised 26 March 2021 Accepted 23 April 2021 Published 28 April 2021

Academic Editor Laura Arru

Copyright copy 2021 Zohreh Marhamati et al (is 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 isproperly cited

(e aim of this study was to evaluate the physicochemical nutraceutical antioxidant and antibacterial properties of tunichromereleased from Persian Gulf tunicate (Phallusia nigra) For this purpose molecular weight (SDS-PAGE) amino acid profilechemical composition (GC-MS) mineral composition functional groups (FTIR) total phenol content (TPC) total flavonoidcontent (TFC) antioxidant activity and antimicrobial properties were investigated (e results showed that tunichrome con-tained a high amount of essential amino acids (ie Lys 3224mg100 g) and essential minerals According to GC-MS resultstunichrome had different antioxidant and antimicrobial components (e TPC and TFC of tunichrome were 055mgGAg and021mg quercetin100 g respectively Tunichrome showed higher antioxidant activity than ascorbic acid and its radical scav-enging activity values were increased from 3028 to 8208 by increasing concentration from 50 to 200 ppm Inhibition zones ofStaphylococcus aureus Bacillus cereus Salmonella enterica and Escherichia coliO157H7 were 14 18 17 and 15mm respectivelyMoreover the minimum inhibitory concentration values of tunichrome for S aureus Bacillus cereus S enterica and E coliO157H7 were 117 059 059 and 117mgml respectively (e minimum bacterial concentrations were 234 117 117 and 234mgml for S aureus Bacillus cereus S enterica and E coli O157H7 respectively (ese results showed that tunichrome of Phallusianigra has excellent biological effects as a bioactive compound for food fortification

1 Introduction

Since the synthetic antioxidants and antimicrobial com-ponents could exert several side effects the interest ofconsumers and producers for safe and natural ingredients isgrowing [1] Bioactive compounds are the most attractiveingredients in the design and development of functionalfoods (e tunicates are a wide marine animal group whosebodies are covered by the cellulose-containing tunic (eyare marine filter invertebrates that exhibit the properties ofthe vertebrates (e accumulation of a high level of metals(ie either vanadium or iron) in seawater can be possible by

intracellular polymer matrices [2 3](us in the presence ofmetal chelators such as catechol and pyrogallol groupstunichromes are associated with metal reduction Moreovertunichrome can form covalent crosslinking interactions orcomplexes with different types of multivalent ions in sea-water (is ability can affect its functional properties such asswelling solubilization coagulation and precipitationbehavior

(e ascidians thaliaceans and appendicularians are thethree main groups of tunicates [4] (ese animals areconsumed in Asia Chile and the Mediterranean in the past(ese products come from sea wild and cultured

HindawiJournal of Food QualityVolume 2021 Article ID 5513717 11 pageshttpsdoiorg10115520215513717

populations when the demand is high especially for Hal-ocynthia and Styela species Edible species are usually fromsolitary stolidobranchs Halocynthia aurantium H roretziMicrocosmus hartmeyeri M sabatieriM vulgaris Polycarpapomaria Pyura chilensis Styela clava and S plicata are theimportant species eaten(ese groups are presented in freshand dried forms in the markets High-quality processes ofH aurantium H roretzi P chilensis S clava and S plicataare the main seafood exports to Europe and America [5]

Generally tunicates have a high nutritional value due tothe presence of a high level of bioactive components andprotein and low calories [6ndash10] Moreover some of themcontain different vitamins (ie vitamin E vitamin B12 andvitamin C) minerals (ie Na K Ca Mg P Fe Zn and Cu)amino acids folic acid fatty acid and pantothenic acid [11]Some groups of tunicates have different low molecularweight peptides in their bodies such as styelins plicatamidehalocyamines lamellarins and ferreascidin from Styelaclava Styela plicata Halocynthia roretzi Didemnum char-taceum and Pyura stolonifera respectively as well astunichromes from different tunicates species [5 12 13]

Some tunicates species can accumulate low molecularweight oligopeptides in their blood cell which are known astunichromes Tunichromes could play an important role indefense mechanism mainly due to the phenoloxidase whichcan attack easily tunichrome It is considered as a keycomponent for both tunic formation and tunic woundhealing which use the same primary biochemical mecha-nisms [13] Tunichromes have different colors such as pinkred and blue [14] (ese pigments are ingredients in theblood cells of tunicates Tunichromes were identified in 11groups of different tunicates [5] (e broad biological ac-tivity of tunichromes remains mainly unknown Howeversome researches offer unique functionalities for tunichromein terms of antioxidant [15ndash17] antimicrobial [5 18] andanticancer activities [19]

To the best of our knowledge there is no information onthe biological activities of the amazing tunichrome releasedfrom the Phallusia nigra Persian Gulf marine tunicate(erefore this research is a first attempt to evaluate thephysicochemical nutraceutical antioxidant and antibac-terial properties of tunichrome released from Phallusia nigraPersian Gulf marine tunicate

2 Materials and Methods

21 Materials Phallusia nigra tunicate was randomly col-lected from Nayband Bay situated in the north (27deg 30primeS 52deg35primeE) of Bushehr Iran using SCUBA diving at the depthranging from 05 to 15m in spring of 2019 Media culturesand all other chemical materials were prepared in analyticalgrade from Merck and Sigma-Aldrich

22 Methods

221 Sampling and Sample Preparation 50 samples ofPhallusia nigra tunicates were washed by double distilledwater (DDW) to remove contaminants (ey were imme-diately transported to the laboratory in a cool box (in ice-

cold condition) and kept at room temperature for 30min torelease a pink color solution (tunichrome) (e obtainedsuspension was centrifuged (4000times g for 5min) and thenfiltrated by Whatman No 1 filter paper (e final solutionwas freeze-dried and powdered for further studies

222 Total Protein Content Total protein content wasmeasured based on the AOAC method by the Kjeldahldevice (Buchi Switzerland)

223 Molecular Weight Measurement (SDS-PAGE) For themolecular weight determination the tunichrome (5mgmL)was dissolved in 1 SDS solution prepared by phosphatebuffer (pH 70) and stirred for 24 h followed by centrifu-gation at 5000times g for 6min at 20degC [20] (e supernatant(20 μl) was added to 10 μL Tris-HCl buffer (10mM Tris-HCl10 (wv) SDS 01 (wv) bromophenol blue and 10 (vv) glycerol) with or without 2 (vv) β-mercaptoethanol(e suspension was then heated at 95degC for 11min and20 μL of the suspension was developed on the gel slab whichwas contained resolving gel (125 pH 88) and stacking gel(6 pH 68) PowerPac 1000 (Bio-Rad USA) was used forrunning the electrophoresis (e running buffer was madeby diluting 100mL 10 X TrisGlycineSDS buffer with theDDW at the constant voltage 220V(e Coomassie BrilliantBlue was used for staining the gel for 60min and water wasused for destaining for 24 h (e molecular weights wereevaluated by comparison to Sinaclon markers (PR901641CinnaGen Co Tehran Iran) at the ranges of 245ndash11 kDa

224 Amino Acidsrsquo Profile To evaluate amino acid profile intunichrome peptide bonds of protein were broken by hy-drolysis (e hydrolysis process was conducted by heatingthe sample in an oxygen-free condition containing 6M HCland 01 phenolphthalein at 110degC overnight Amino acidswere measured by a Varian chromatographic system con-taining a 1525 pump a 9100 autoinjector and a UV-vis de-tector (e hydrolyze suspension was injected into anautomatic precolumn reaction by 01mL of derivatizing re-agent (is chromatographic process contained a solventmixture (PBS buffer (10mM pH 4) acetonitrile (25 75) at aflow rate of 1mLmin In this system a C18Waters Nova-Packreverse phase column (particle size 5μm 250times 46mm internaldiameter) was applied All the chromatographic data wereprocessed in a V 45 Star workstation supplied by Varian [21]

225 Na Ca K Mg Mn Cu Fe and Zn Measurements(e concentrations of Na Ca K Mg Mn Cu Fe and Zn oftunichrome were analyzed by a Selectra 2 auto-analyzer(Vital Scientific Spankeren Netherlands) To this end 1 g oflyophilized powder was dissolved in the 5 g of DDW for 24 hand then centrifuged

226 Heavy Metal (Cd As Pb and Hg) Measurements(e lyophilized powder of tunichrome (02 g) was weighedand mixed with concentrated HNO3 (7mL 65 vv) and

2 Journal of Food Quality

H2O2 (1mL 30 vv) in the polytetrafluoroethylene vessel(e vessel temperature was raised to 160degC for 21min bymixing at 1500W in the magnetron (ETHOS One Mile-stone Italy) After this digestion process the sample wascooled for 120min and then the final volume of the samplereached 50mL with DDW (e determination of heavymetals was conducted by an atomic absorption spectrometry(Atomic Absorption Spectrophotometer Varian AA240 FS)system which contained an electrode discharge lamp forevaluating volatile and nonvolatile toxic heavy metals byusing argon gas A graphite furnace was applied to deter-mine nonvolatile compounds (Pb and Cd) and a flow in-jection-mercury hydride system was applied to calculatevolatile (As and Hg) compounds

227 Fluoride Chloride Bromide Nitrite Nitrate Phos-phate and Sulfate Content Measurement (is measure-ment was done by an ion chromatograph 761 Compact IC(Metrohm Herisau Switzerland) with anion self-regener-ating suppressor Metrohm Suppressor Module MSM andconductivity detector Anion separation was performed by aldquoStar-Ion-A300rdquo column (100mmtimes 460mm PhenomenexTorrance USA) A ldquoMetrosep A PCC 1 HCrdquo column(125mmtimes 40mm Metrohm Herisau Switzerland) wasapplied for preconcentration (e volume sample loop was20 μl Sample injection to the ion chromatography was doneby a 5ml Becton Dickinson syringe (Fraga Spain) [22]

228 FTIR Spectroscopy FTIR spectroscopy (WQF-510FTIR Rayleigh Beijing Rayleigh China) was used for theevaluation of the peak absorbance intensity of the functionalgroups in the structure of tunichrome To this end driedpowder of tunichrome was mixed with KBr to form acompact plate (e FTIR spectra were recorded in thewavenumber range of 4000ndash400 cmminus1

229 Determination of Chemical Composition by GC-MS(e chemical composition of tunichrome was investigatedby a GC (7890B Agilent Technologies Santa Clara CAUnited States) system containing MS (5977ANetworkAgilent Technologies) Briefly 200mg of tunichrome washomogenized with 200ml of methanol chloroform hexane(1 1 1) for 24 h in 200 rpm and then centrifuged for 20minat 4000 rpm (e final supernatant was used for evaluatingthe chemical composition (e GC system was equippedwith an HP5 MS column (nonpolar column AgilentTechnologies internal diameter 30mtimes 250 μM filmthickness 025 μm) (e flow rate of carrier gas (Helium)was 1mLmin (e temperature of the injector was set at120degC (e oven temperature program included three steps(1) 50degC for 1min (2) temperature raising to 300degC at a rateof 15degCmin and (3) holding at 300degC for 20min (e totalprocessing time was 3766min (e scanning range of massspectra was 50ndash550mz in the EI mode at 70 eV Compo-nents were identified based on mass spectra in comparisonwith those deposited in the database of NIST11 (US

Department of Commerce Gaithersburg MD UnitedStates) and literature data [23]

2210 Antioxidant Properties

Antioxidant Activity (e radical scavenging activity (RSA)() was determined by the DPPHdeg radical scavengingmethod according to the technique of Ruengdech andSiripatrawan [24] First 200mg of tunichrome with differentconcentrations within the range of 50 100 150 and 200 μgmL was mixed with 800 μL methanol (en 400 μL of eachdiluted tunichrome was homogenized with 16mL of DPPHdeg

solution (01mM) (e suspension was kept at 25degC for 1 hunder dark conditions and finally the absorbance of thesuspension was recorded at 517 nm A sample withouttunichrome was used as a control (e RSA () was de-termined based on the following equation

RSA() Ac minus As( 1113857

Ac

1113890 1113891 times 100 (1)

where Ac and As express the absorbances of control and testsamples respectively

Total Phenol Content (TPC) (e TPC was determined byFolin-Ciocalteu reagent assay based on the method ofMajzoobi et al [25] In brief samples were mixed with750 μL of 10 ww Folin-Ciocalteu (diluted in DDW) andthen hold at 20degC for 15min Afterward 750 μL of 02sodium carbonate was mixed with the suspension and theabsorbance of the sample was determined at 765 nm after 1 hincubation in a dark place (e TPC was calculated via acalibration curve of different concentrations of a gallic acidsolution (0 to 150 μgmL) and reported as mg gallic acidg oftunichrome weight

Total Flavonoid Content (TFC) (e method developed byBagheri et al [26] was used to measure TFC For thispurpose 05 g of samples and 500 μL of methanol werehomogenized with 100 μL of AlCl3 (10 wv) 100 μL ofCH3CO2K (1M) and 2800 μL of DDW Absorbance at415 nm was measured after incubation at 23degC for 35min(e standard calibration curve was plotted at the samewavelength for various quercetin concentrations (5 to 30 μgmL methanol) (e TFC was reported as mg querceting ofthe sample weight

2211 Antimicrobial Properties

Well Diffusion Agar Method (e tunichrome sample anti-microbial activity was measured against selected Gram-positive and Gram-negative bacteria including Bacillus ce-reus (ATCC 11778) Staphylococcus aureus (ATCC 6538)Salmonella enterica (ATCC14028) and Escherichia coli(ATCC 35218) by well diffusion agar method Muller Hintonagar plates were cultured using 01mL of bacterial sus-pension with a cell density of 05 Mc-Farland standard(asymp15times108 CFUml) Wells with 4mm diameter were

Journal of Food Quality 3

created by a sterile punch and filled with 50 μl of the sample(150mgml) After 24 h incubation at 37degC the diameterinhibition zone (DIZ) was determined in mm

Minimum Inhibitory Concentration (MIC) and MinimumBactericidal Concentration (MBC) Method (e brothmicrodilution technique was used for the evaluation of MICFirst broth subcultures were produced by inoculating onecolony of each bacteria grown 24 h in the 50ml flask with20ml Mueller Hinton Broth in a shaker incubator (JalTehran Iran) at 150 rpm (en the final concentration ofbacterium was set at 15times106 CFUml followed by sepa-rating the cells using centrifugation at 6000times g for 5min(en the pellet was dispersed in sterile saline (09 NaCl)and was applied for inoculation by 96-well microplates withtunichrome serial dilutions from 0 to 150mgml (emicroplates were held at 37degC overnight After incubationbacterial growth was determined by turbidity methods (econcentration around MIC was cultured on the MRS agarfor determining the MBC

2212 Statistical Analysis All data were determined intriplicate Analysis of variance (ANOVA) and the Duncanmultiple range tests were done to evaluate the significantdifferences (Plt 005) among the average values (SAS ver 912002ndash2003 by SAS Institute Inc Cary NC USA)

3 Results and Discussion

31 Molecular Weight Measurement SDS-PAGE of tuni-chrome released from Persian Gulf marine tunicate is shownin Figure 1 (e bands at 245 180 135 100 and 75 between75 and 63 between 63 and 48 48 35 between 35 and 25 2520 between 20 and 17 17 between 17 and 11 and 11 kDwere identified in the sample Twomain bands were between63 and 48 and 20 kD (is result showed that there weredifferent proteins and peptides with antioxidant and anti-microbial properties in the tunichrome

32AminoAcidsrsquo Profile (e protein content of tunichromereleased from Phallusia nigra Persian Gulf marine tunicatewas 07plusmn 002 Amino acidsrsquo profile of tunichrome byHPLC is reported in Table 1 (e main amino acids werelysine arginine glycine aspartic acid and proline withconcentrations of 3224 1434 1244 1062 and 947mg100 g respectively (erefore tunichrome can be consideredas a suitable source of lysine essential amino acid (EAA)Kumaran and Bragadeeswaran [27] reported that the releaseof E viride is containing leucine (5823 μgg) arginine(3654 μgg) lysine (3445 μgg) threonine (2956 μgg) andisoleucine (2312 μgg) and the release of D psammathodescontains leucine (5409 μgg) arginine (4012 μgg) lysine(3854 μgg) threonine (3125 μgg) and isoleucine(2541 μgg) Karthikeyan et al [28] evaluated the amino acidprofile of solitary ascidian Microcosmus exasperatus andreported the presence of a total of seventeen essential andnonessential amino acids (nEAA) Among them ten

essential and seven nonessential amino acids were reportedin ascidians mussel (e maximum recorded level of theessential amino acid (5673mg) was leucine and the leastlevel of nonessential amino acids (0212mg) was asparticacid Tabakaeva and Tabakaev [29] also evaluated the dif-ferent amino acids of the ascidianHalocynthia aurantium inthe Japan Sea Among eighteen identified amino acids eightof them were essential and the others were nonessentialInternal organs had the highest essential amino acid(5061) content and tunic had the lowest (3501) content(e common nonessential amino acid in all parts(584ndash1016) was aspartic acid

(e contents of EAA semiessential amino acids (SEAA)and nEAA of tunichrome released from Persian Gulf marinetunicate are reported in Figure 2 It contained 444 213 and34 of EAA SEAA and NEAA respectively Cho et al [7]and Kang et al [10] reported that the protein content andamino acid type are related to the age and type of tunicateenvironmental condition pH of water and the salt type andconcentration

33 Mineral Content Table 2 shows the Na K Mg Ca MnZn Fe and Cu contents of tunichrome released fromPhallusia nigra Persian Gulf marine tunicate (e maincation ions of tunichrome were sodium potassium andmagnesium with concentrations of 8584 7785 and947mg100 g respectively Based on these results thissample is a good source of essential minerals for the humanbody Lee et al [30] also reported that the internal part oftunicate from Korea was containing Na K Mg Ca Mn ZnFe and Cu in the range of 14711ndash12579 39ndash983 428ndash781291ndash2733 52ndash09 02ndash04 25ndash09 and 0ndash02mg100 grespectively Papadopoulou and Kanias [31] reported thatZn and Fe of tunic ofCiona intestinaliswere 110 and 610mgkg Cho et al [7] reported that the mineral type is related tothe age and type of tunicate environmental condition andpollution

Table 3 shows the heavy metals of tunichrome releasedfrom Phallusia nigra Persian Gulf marine tunicate (issource contained high amounts of heavy metals such as Pband Hg with concentrations of 3635 and 8 μgg respectively(e main idea is culturing this tunicate in the pure waterinstead of the sea Lee et al [30] evaluated the internal part oftunicate from Korea and reported a trace content of chro-mium lead silver and arsenic heavy metals Papadopoulouand Kanias [31] reported 1 19 0011 0021 0041 37 044and 015 ppm of Se Cr Ag Cs Sc Rb Co and Sb for thetunic of Ciona intestinalis respectively

Table 4 shows the concentrations of fluoride chloridebromide nitrite nitrate phosphate and sulfate of tuni-chrome released from Phallusia nigra Persian Gulf marinetunicate Chloride (1750 μgg) nitrate (850 μgg) andfluoride (1750 μgg) were the main anions of tunichromeSince a high amount of nitrite and nitrate in the processedfood can lead to different cancers the low content of nitriteand nitrate in tunichrome showed its beneficial health effects[32]


245180135100

75

63

48

35

25

20

17

11

kDa

Tris-Glycine4-20

(a) (b)

Figure 1 SDS-PAGE (a) sinaclon marker (Tris-Glycine) and (b) tunichrome released from Phallusia nigra Persian Gulf marine tunicate

Table 1 Amino acidsrsquo profile of tunichrome released from Phallusia nigra Persian Gulf marine tunicate by HPLC

Amino acid Retention time (min) Concentration (mg100 g) Content ()1 Asp 556 1062plusmn 0004 92plusmn 0042 Glu 663 655plusmn 0003 67plusmn 0033 H-Pro 1079 029plusmn 0002 05plusmn 0024 Ser 1440 279plusmn 0003 77plusmn 0035 Gly 1555 1244plusmn 0003 97plusmn 0026 His 1719 087plusmn 0002 32plusmn 0027 Arg 1911 1434plusmn 0002 78plusmn 0028 (r 1962 445plusmn 0002 46plusmn 0029 Ala 1983 583plusmn 0004 54plusmn 00410 Pro 2038 947plusmn 0002 66plusmn 00211 Tyr 2600 072plusmn 0003 22plusmn 00312 Val 2722 197plusmn 0002 35plusmn 00213 Met 2843 532plusmn 0003 56plusmn 00314 Ile 3117 402plusmn 0035 23plusmn 00415 Leu 3155 772plusmn 0002 51plusmn 00216 Phe 3392 587plusmn 0002 31plusmn 00217 Lys 3472 3224plusmn 0003 170plusmn 003Data represent meanplusmn standard deviation of three independent repeats


34 FTIR Infrared spectroscopy is a valuable analyticaltechnique for obtaining rapid information about thestructure and chemical changes of compounds during thedifferent processes(e FTIR spectra of tunichrome releasedfrom Phallusia nigra Persian Gulf marine tunicate arepresented in Figure 3 (e spectrum had two low-intensitypeaks at 1624 and 1728 cmminus1 (e signal around 1624 cmminus1

was due to the symmetric deformation vibration of adsorbedwater molecules [33] (e peak at 1728 cmminus1 was attributedto the CO stretching vibration of formate ester moieties(e low intensity of this band suggested that only smallamounts of this specific band are formed [34] Tunichromeshowed a strong OndashH stretching absorption band around3300 cmminus1 and CndashH stretching absorption band around2962 cmminus1 (Table 5) Similar results were reported by Nuntildeez-Pons et al [35] and Song et al [36] Moreover these results

were in agreement with those obtained by GC-MS for thedetection of carbocyclic acidic and steric groups

35 Chemical Composition (e chemical composition oftunichrome released from Phallusia nigra Persian Gulfmarine tunicate by GC-MS is reported in Table 6 Octa-decanoic acid methyl ester (2574) nonanal (1408)hydrazinecarbothioamide (1238) octadecanoic acid(967) and hexadecanoic acid (906) were the maincomponents extracted by methanol hexane chloroform (1 1 1) Similar components in tunichrome of a marine as-cidian Lissoclinum bistratum were reported by Karthi et al[37] after analysis by the GC-MS method

(is important analysis showed that tunichrome re-leased from Phallusia nigra Persian Gulf marine tunicatecontained a large number of bioactive compounds which areknown as antioxidant and antimicrobial natural productsSeveral amazing medicinal and pharmacological activitieswere also reported for tunichrome According to the pre-vious studies some identified components such as 3-methyl-2-[4-(3-methyl-butoxy)-benzoylamino]-butyric acid [38]hexadecanoic acid [39] and phenol 24-bis(11-dimethy-lethyl)- [40] exhibit high antioxidant activity

(e antimicrobial activities of oxazine [41] 13-oxazine[42] nonanal [43] hydrazinecarbothioamide [44] triazene[45] butyric acid [46 47] (+)-trans-34-Dimethyl-2-phe-nyltetrahydro-14-thiazine [48ndash50] hexadecanoic acid [47]octadecanoic acid [51] spirost-8-en-11-one3-hydroxy- (3β5α 14β20β 22β 25R)- [52 53] and phenol 24-bis(11-dimethylethyl)- [40] were also well documented

In addition other pharmaceutical properties such assedative analgesic antipyretic anticonvulsant antitubercularantitumor and antimalarial properties of 4-phenyltetrahydro-13-oxazine-2-thione [41] antitumor and anti-inflammatoryproperties of (+)-trans-34-dimethyl-2-phenyltetrahydro-14-thiazine [54] antitrypanosomal properties of hydrazi-necarbothioamide 2-[1-(4-nitrophenyl)ethylidene]- [55]and anticancer antiproliferative and anti-inflammatoryproperties of spirost-8-en-11-one3-hydroxy-(3β 5α 14β20β22β 25R)- [56 57] were reported previously

36 Antioxidant Properties DPPH is a stable radical with anabsorbance at 517 nm which can react with any antioxidant(is interaction can decrease the absorbance due to the color

444

213

34

05

EAASEAA

NEAAMisc

Figure 2 Essential amino acids (EAA) semiessential amino acids(SEAA) and nonessential amino acids (nEAA) of tunichromereleased from Phallusia nigra Persian Gulf marine tunicate

Table 2 Na K Mg Ca Mn Zn Fe and Cu contents of tunichromereleased from Phallusia nigra Persian Gulf marine tunicate

Name Concentration (mg100 g)Na 8584plusmn 002K 7785plusmn 003Mg 947plusmn 002Ca 21plusmn 002Mn 03plusmn 003Zn 06plusmn 002Fe 03plusmn 003Cu 01plusmn 004Data represent meanplusmn standard deviation of three independent repeats

Table 3 Heavy metals of tunichrome released from Phallusia nigraPersian Gulf marine tunicate

Name LOQ Concentration (μgg)Cd 03 0013plusmn 0003As 0025 0013plusmn 0002Pb 42 3635plusmn 0010Hg 3 800plusmn 0001Data represent meanplusmn standard deviation of three independent repeats

Table 4 Fluoride chloride bromide nitrite nitrate phosphateand sulfate contents of tunichrome released from Phallusia nigraPersian Gulf marine tunicate

Name RT (min) Concentration (μgg)Fluoride 375 1750plusmn 02Chloride 568 1750plusmn 03Bromide 859 15plusmn 02Nitrite 733 5plusmn 01Nitrate 1039 850plusmn 02Phosphate 1172 5plusmn 01Sulfate 1441 15plusmn 01Data represent meanplusmn standard deviation of three independent repeats


change from purple to white or yellow [58 59] TPC andTFC of the sample were 055mgGAg and 021mg quer-cetin100 g respectively (e RSA () values of tunichromereleased from Phallusia nigra Persian Gulf marine tunicateand ascorbic acid are reported in Table 7 Tunichromeshowed higher antioxidant activity than ascorbic acid andRSA values were increased from 3028 to 8208 by in-creasing the concentration from 50 to 200 ppm indicatingits dose-dependent activity (ese results showed thattunichrome released from Phallusia nigra Persian Gulfmarine tunicate can be an appropriate source of naturalantioxidants for food and pharmaceutical purposes

(e antioxidant activities of 3-methyl-2-[4-(3-methyl-butoxy)-benzoylamino]-butyric acid [38] and hexadecanoicacid [39] and phenol 24-bis(11-dimethylethyl)- [40] werealso reported previously Lee et al [15] evaluated the anti-oxidant activity of extracts of Stalked sea squirt Styela clavatunic (ey reported that this extract showed high dose-dependent antioxidant activity and water extract had0192mgml Also Lee et al [60] showed that the RSA of thewater and ethanol Styela clava tunic extracts was 31 and486 at 10mgml respectively Lee et al [61] also reported

high scavenging activities (50) of starfish Acanthasterplanci extracts at concentrations of 162mgml gt10mgmland 403mgml for the ethanol ethyl acetate and n-butanolextracts respectively Kim [14] reported that hydrolysatesof solitary tunicate (Styela clava) had high antioxidantactivity

37 Antimicrobial Content

371 Well Diffusion Agar Antimicrobial activity of tuni-chrome was reported by Sugumaran and Robinson [62]previously Inhibition zones (mm) of tunichrome releasedfrom Phallusia nigra Persian Gulf marine tunicate (150 μgml) and gentamicin (10 μgml) are reported in Table 8Inhibition zones of Staphylococcus aureus Staphylococcusaureus Salmonella enterica and Escherichia coli O157H7were 14 18 17 and 15mm respectively(is sample had thehighest antimicrobial activity against Bacillus cereus Inde-pendent of bacterial strain the antimicrobial activity oftunichrome was lower than gentamicin as shown in Table 8(ese results showed the significant antimicrobial activity oftunichrome released from Phallusia nigra Persian Gulfmarine tunicate as a natural compound in food application

(e antimicrobial activities of oxazine and its derivativeswere reported by Sindhu et al [41] against S aureus andE coli (e antimicrobial activities of 13-oxazine againstEnterococcus faecalis and Listeria monocytogenes were alsoreported by Hamza et al [42] (e antimicrobial activity ofnonanal was reported by Zhang et al [43] (e antimicrobialpotential of hydrazinecarbothioamide against Bacillus sub-tilis E coli Staphylococcus aureus Pseudomonas aeruginosaPaenibacillus macerans and Salmonella typhimurium wasreported by Shim et al [44] Mohammadi [45] studied theantimicrobial activities of triazene against Bacillus subtilisPseudomonas aeruginosa Salmonella enterica and Micro-coccus luteus (e butyric acid activities against Salmonellaenterica E coli and Campylobacter jejuni [46] Candidaalbicans Streptococcus mutans and Streptococcus sanguinis

Table 5 FTIR of tunichrome released from Phallusia nigra PersianGulf marine tunicate

Wavenumber (cmminus1)1 2962 C-H stretching2 2573 O-H stretch vibration3 1728 CO stretch vibration4 1624 CC symmetric deformation vibration5 1608 N-O stretch vibration6 13887 12288 10769 89910 85211 65212 536 C-Br stretch vibration

2962

2573

652 536

852899

107612281388

160816241728

0

20

40

60

80

100

Tran

smitt

ance

()

2800 2400 2000 1600 1200 800 4003200Wave number (cmndash1)

Figure 3 FTIR spectrum of tunichrome released from Phallusia nigra Persian Gulf marine tunicate


[47] were also documented Moreover the antimicrobialactivities of (+)-trans-34-dimethyl-2-phenyltetrahydro-14-thiazine against Mycobacterium tuberculosis [48] Staphy-lococcus aureus and E coli [49] Staphylococcus aureusE coli Bacillus subtilis and Pseudomonas aeruginosa werereported previously [50]

(e antimicrobial activities of hexadecanoic acid againstCandida albicans Streptococcus mutans and Streptococcussanguinis were shown by Huang et al [47] According to daSilva et al [51] octadecanoic acid had significant antimi-crobial activities against Bacillus subtilis and Bacillus cereus(e antimicrobial activities of spirost-8-en-11-one3-hy-droxy-(3β 5α 14β20β 22β 25R)- against E coli Bacillussubtilis Staphylococcus aureus and Pseudomonas aeruginosawere reported in [52 53] (e antimicrobial activities ofphenol 24-bis(11-dimethylethyl)- against Aspergillus nigerFusarium oxysporum and Penicillium chrysogenum werereported by Varsha et al [40]

Galinier et al [18] evaluated the two antimicrobialpeptides from hemocytes of the Halocynthia papillosa tu-nicate (ey reported that these components had high an-timicrobial activities against S aureus and E coli Cai et al[5] reported that tunichromes showed antimicrobial activityagainst E coli and Photobacterium phosphoreum

372 MIC and MBC Table 9 shows the MIC and MBC oftunichrome released from Phallusia nigra Persian Gulfmarine tunicate MIC values of tunichrome for Staphylo-coccus aureus Staphylococcus aureus Salmonella entericaand Escherichia coli O157H7 were 117 059 059 and117mgml respectively Moreover the values of MBC forthe Staphylococcus aureus Staphylococcus aureus Salmo-nella enterica and Escherichia coli O157H7 were 234 117117 and 234mgml respectively It was reported thatstyelins clavanins halocyamine and plicatamide of tuni-chrome separated from ascidian blood cells exhibit anti-microbial properties [62 63] Cai et al [5] also reported thatthere are different components in the tunichrome with invitro antibiotic properties

4 Conclusion

(e aim of this study was to evaluate the physicochemicalnutraceutical antioxidant and antibacterial properties oftunichrome released from Phallusia nigra Persian Gulfmarine tunicate (e results showed that this release con-tained a high amount of essential amino acids especially

Table 6 Chemical composition of tunichrome released from Phallusia nigra Persian Gulf marine tunicate by GC-MS

RT (min) Name Formula MW (gmol) Content ()1 322 4-Phenyltetrahydro-13-oxazine-2-thione C10H11NOS 19327 0712 592 Nonanal C9H18O 14224 14083 839 33-Dimethyl-1-(2-carboxyphenyl)triazene C9H11N3O2 19320 0874 848 3-Methyl-2-[4-(3-methyl-butoxy)-benzoylamino]-butyric acid C17H25NO4 30740 0875 868 p-Cynophenyl p-(2-propoxyethoxy)benzoat C19H19NO4 32540 0876 888 (+)-trans-34-Dimethyl-2-phenyltetrahydro-14-thiazine C12H17NS 20730 0877 1706 Phenol 24-bis(11-dimethylethyl)- C14H22O 20632 0738 1768 Hexadecanoic acid methyl ester C17H34O2 27050 9069 1877 n-Hexadecanoic acid C16H32O2 25642 58410 2196 Hydrazinecarbothioamide 2-[1-(4-nitrophenyl)ethylidene]- C9 H10N4O2S 23827 123811 2302 Octadecanoic acid methyl ester C19H38O2 29850 257412 2402 Octadecanoic acid C18H36O2 28450 96713 2732 Spirost-8-en-11-one3-hydroxy-(3β 5α 14β20β 22β 25R)- C27H40O4 42800 08714 2937 Hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester C19H38O4 33050 1743

Table 7 RSA () of tunichrome released from Phallusia nigraPersian Gulf marine tunicate and ascorbic acid

Concentration (ppm) Tunichrome Ascorbic acid50 3028plusmn 02Ca 1786plusmn 01Db100 4759plusmn 01Ca 2204plusmn 02Cb150 6628plusmn 02Ba 4247plusmn 01Bb200 8208plusmn 01Aa 5383plusmn 01AbData represent meanplusmn standard deviation of three independent repeatsDifferent capital letters in each column and lowercase ones in each rowindicate significant differences (Plt 005)

Table 8 Inhibition zones (mm) of tunichrome released fromPhallusia nigra Persian Gulf marine tunicate (150 μgml) andgentamicin (10 μgml)

Bacteria Tunichrome (mm) Gentamicin (mm)Staphylococcus aureus 14plusmn 03Db 16plusmn 02DaBacillus cereus 18plusmn 03Ab 19plusmn 02AaSalmonella enterica 17plusmn 02Bb 18plusmn 03BaEscherichia coli O157H7 15plusmn 02Cb 17plusmn 03CaData represent meanplusmn standard deviation of three independent repeatsDifferent capital letters in each column and lowercase ones in each rowindicate significant differences (Plt 005)

Table 9 MIC and MBC values of tunichrome released fromPhallusia nigra Persian Gulf marine tunicate

Bacteria MIC (mgml) MBC (mgml)Staphylococcus aureus 117plusmn 000Ab 234plusmn 000AaBacillus cereus 059plusmn 000Bb 117plusmn 000BaSalmonella enterica 059plusmn 000Bb 117plusmn 000BaEscherichia coli O157H7 117plusmn 000Ab 234plusmn 000AaData represent meanplusmn standard deviation of three independent repeatsDifferent capital letters in each column and lowercase ones in each rowindicate significant differences (Plt 005)


lysine Also the GC-MS profile showed that this compoundincluded the functional antioxidant and antimicrobialcomponents Tunichrome had high antioxidant activityTPC and TFC Also the strong antimicrobial activitiesagainst Staphylococcus aureus Staphylococcus aureus Sal-monella enterica and Escherichia coli O157H7 were ob-served (erefore tunichrome can be considered as a goodsource of natural antioxidants and antimicrobials for futurefood and pharmaceutical applications

Data Availability

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

Conflicts of Interest

(e authors declare that they have no conflicts of interest

References

[1] V Silva G Igrejas V Falco et al ldquoChemical compositionantioxidant and antimicrobial activity of phenolic compoundsextracted from wine industry by-productsrdquo Food Controlvol 92 pp 516ndash52 2018

[2] A D Dwivedi R Permana J P Singh et al ldquoTunichrome-inspired gold-enrichment dispersion matrix and its applica-tion in water treatment a proof-of-concept investigationrdquoACS Applied Materials amp Interfaces vol 9 no 23pp 19815ndash19824 2017

[3] Q F Kuang A Abebe J Evans and M Sugumaran ldquoOxi-dative transformation of tunichromes-model studies with 12-dehydro-N-acetyldopamine and N-acetylcysteinerdquo Bio-organic Chemistry vol 73 pp 53ndash62 2017

[4] P Lemaire ldquoEvolutionary crossroads in developmental bi-ology the tunicatesrdquo Development vol 138 no 11pp 2143ndash2152 2011

[5] M Cai M Sugumaran and W E Robinson ldquo(e cross-linking and antimicrobial properties of tunichromerdquo Com-parative Biochemistry and Physiology Part B Biochemistry andMolecular Biology vol 151 no 1 pp 110ndash117 2008

[6] G Ananthan M Karthikeyan P A Selva andC Raghunathan ldquoStudies on the seasonal variations in theproximate composition of ascidians from the Palk Baysoutheast coast of Indiardquo Asian Pacific Journal of TropicalBiomedicine vol 2 no 10 pp 793ndash797 2012

[7] N-D Cho J Zeng B-D Choi and H-S Ryu ldquoShelf life ofbottled sea squirt Halocynthia roretzi meat packed in vege-table oil (BSMO)rdquo Fisheries and Aquatic Sciences vol 17pp 37ndash46 2014

[8] R Roje-Busatto and I Ujevic ldquoPSP toxins profile in ascidianMicrocosmus vulgaris (Heller 1877) after human poisoningin Croatia (Adriatic Sea)rdquo Toxicon vol 79 pp 28ndash36 2014

[9] M Tamilselvi V Sivakumar H Abdul Jaffar Ali andR (ilaga ldquoPreparation of pickle from Herdmania pallidasimple ascidianrdquo World Journal of Dairy amp Food Sciencesvol 5 pp 88ndash92 2010

[10] C-K Kang E J Choy W C Lee N J Kim H-J Park andK-S Choi ldquoPhysiological energetics and gross biochemicalcomposition of the ascidian Styela clava cultured in sus-pension in a temperate bay of Koreardquo Aquaculture vol 319no 1-2 pp 168ndash177 2011

[11] G Lambert R Karney W Rhee and M Carman ldquoWild andcultured edible tunicates a reviewrdquoManagement of BiologicalInvasions vol 7 no 1 pp 59ndash66 2016

[12] N D Sanandiya S Lee S Rho H Lee I S Kim andD S Hwang ldquoTunichrome-inspired pyrogallol functionalizedchitosan for tissue adhesion and hemostasisrdquo CarbohydratePolymers vol 208 pp 77ndash85 2019

[13] A Abebe Q F Kuang J Evans W E Robinson andM Sugumaran ldquoOxidative transformation of a tunichromemodel compound provides new insight into the crosslinkingand defense reaction of tunichromesrdquo Bioorganic Chemistryvol 71 pp 219ndash229 2017

[14] A D Dwivedi R Permana J P Singh et al ldquoTunichromemimetic matrix its perspective in abatement for carcinogenichexavalent chromium and specific coordination behaviorrdquoChemical Engineering Journal vol 328 pp 629ndash638 2017

[15] S-M Lee Y-R Lee K-S Cho et al ldquoStalked sea squirt (Styelaclava) tunic waste as a valuable bioresource cosmetic andantioxidant activitiesrdquo Process Biochemistry vol 50 no 11pp 1977ndash1984 2015

[16] A Sato T Shindo N Kasanuki and K Hasegawa ldquoAnti-oxidant metabolites from the tunicate Amaroucium multi-plicatumrdquo Journal of Natural Products vol 52 no 5pp 975ndash981 1989

[17] H-J Lee S-J Chae P S Saravana and B-S Chun ldquoPhysicaland functional properties of tunicate (Styela clava) hydroly-sate obtained from pressurized hydrothermal processrdquoFisheries and Aquatic Sciences vol 20 pp 1ndash8 2017

[18] R Galinier E Roger P-E Sautiere A Aumelas B Banaigsand G Mitta ldquoHalocyntin and papillosin two new antimi-crobial peptides isolated from hemocytes of the solitarytunicateHalocynthia papillosardquo Journal of Peptide Sciencevol 15 no 1 pp 48ndash55 2009

[19] S M Kim ldquoAntioxidant and anticancer activities of enzy-matic hydrolysates of solitary tunicate (Styela clava)rdquo FoodScience and Biotechnology vol 20 p 1075 2011

[20] Y Shen X Tang and Y Li ldquoDrying methods affect physi-cochemical and functional properties of quinoa protein iso-laterdquo Food Chemistry vol 339 p 127823 2020

[21] P B Zamudio-Flores M H Gonzalez and V G G CanoldquoFood supplements from a Grasshopper a developmentalstage-wise evaluation of amino acid profile protein and vi-tamins in Brachystola magna (Girard)rdquo Emirates Journal ofFood and Agriculture vol 31 pp 561ndash568 2019

[22] E Kapinus I Revelsky V Ulogov and Y A LyalikovldquoAmino acids and related compounds of the ascidian Hal-ocynthia aurantium from the sea of Japanrdquo Journal ofChromatography B vol 800 pp 321ndash323 2004

[23] M Mollah M Imam M C Roy et al ldquoVariations of indolemetabolites and NRPS-PKS loci in two different virulentstrains of Xenorhabdus hominickiirdquo Frontiers in Microbiol-ogy vol 11 p 2870 2020

[24] A Ruengdech and U Siripatrawan ldquoApplication of catechinnanoencapsulation with enhanced antioxidant activity in highpressure processed catechin-fortified coconut milkrdquo LWTvol 140 p 110594 2021

[25] M Majzoobi F Ghiasi and A Farahnaky ldquoPhysicochemicalassessment of fresh chilled dairy dessert supplemented withwheat germrdquo International Journal of Food Science amp Tech-nology vol 51 no 1 pp 78ndash86 2016

[26] N Bagheri H A Al Lawati and J Hassanzadeh ldquoSimulta-neous determination of total phenolic acids and total flavo-noids in tea and honey samples using an integrated lab on achip devicerdquo Food Chemistry vol 342 2021


[27] N S Kumaran and S Bragadeeswaran ldquoNutritional com-position of the colonial ascidian eudistoma viride andDidemnum psammathodesrdquo Biosciences Biotechnology Re-search Asia vol 11 no SE pp 331ndash338 2014

[28] M Karthikeyan G Anathan and A J Ali ldquoNutritional valuesof solitary ascidian Microcosmus exasperatus heller 1878(ascidiacea pyuridae)rdquo Global Veterinaria vol 4 pp 255ndash259 2010

[29] O V Tabakaeva and A V Tabakaev ldquoAmino acids and re-lated compounds of the ascidian Halocynthia aurantium fromthe sea of Japanrdquo Chemistry of Natural Compounds vol 53no 4 pp 722ndash725 2017

[30] J-S Lee S-J Kang and B-D Choi ldquoSeasonal variation in thenutritional content of Mideodeok Styela clavardquo Fisheries andAquatic Sciences vol 9 pp 49ndash56 2006

[31] C Papadopoulou and G D Kanias ldquoTunicate species asmarine pollution indicatorsrdquoMarine Pollution Bulletin vol 8no 10 pp 229ndash231 1977

[32] F Hosseini M Majdi S Naghshi F SheikhhosseinK Djafarian and S Shab-Bidar ldquoNitrate-nitrite exposurethrough drinking water and diet and risk of colorectal cancera systematic review and meta-analysis of observationalstudiesrdquo Clinical Nutrition vol 20 2020

[33] Q G Fan D M Lewis and K N Tapley ldquoCharacterization ofcellulose aldehyde using Fourier transform infrared spec-troscopyrdquo Journal of Applied Polymer Science vol 82 no 5pp 1195ndash1202 2001

[34] H Yuan Y Nishiyama M Wada and S Kuga ldquoSurfaceacylation of cellulose whiskers by drying aqueous emulsionrdquoBiomacromolecules vol 7 no 3 pp 696ndash700 2006

[35] L Nuntildeez-Pons M Carbone J Vazquez et al ldquoNaturalproducts from Antarctic colonial ascidians of the generaAplidium and Synoicum variability and defensive rolerdquoMarine Drugs vol 10 no 12 pp 1741ndash1764 2012

[36] G Song J Delroisse D Schoenaers et al ldquoStructure andcomposition of the tunic in the sea pineapple Halocynthiaroretzi a complex cellulosic composite biomaterialrdquo ActaBiomaterialia vol 111 pp 290ndash301 2020

[37] S Karthi B Somanath and A Abdul Jaffar ldquoEfficacy ofmethanolic extract of a marine ascidian Lissoclinum bis-tratum for antimicrobial activityrdquo Journal Of ChemicalBiological and Physical Sciences B vol 5 pp 4119ndash41252015

[38] A P Kumar M Chougala C D Nandini and P V SalimathldquoEffect of butyric acid supplementation on serum and renalantioxidant enzyme activities in streptozotocin-induced di-abetic ratsrdquo Journal of Food Biochemistry vol 34 pp 15ndash302010

[39] G Belakhdar A Benjouad and E Abdennebi ldquoEfficacy ofmethanolic extract of a marine ascidian Lissoclinum bis-tratum for antimicrobial activityrdquo Journal of Materials andEnvironmental Science vol 6 pp 2778ndash2783 2015

[40] K K Varsha L Devendra G Shilpa S Priya A Pandey andK M Nampoothiri ldquo24-Di-tert-butyl phenol as the anti-fungal antioxidant bioactive purified from a newly isolatedLactococcus sprdquo International Journal of Food Microbiologyvol 211 pp 44ndash50 2015

[41] T Sindhu S Arikkatt G Vincent M Chandran A Bhat andK Krishnakumar ldquoBiological activities of oxazine and itsderivatives a reviewrdquo International Journal of Pharma Sci-ences and Research vol 4 pp 134ndash143 2013

[42] A Hamza H A El-Sayed M G Assy N H Ouf andM E Farhan ldquoSynthesis and antimicrobial activity of somenew triazine 1 3-oxazine fused pyridine and pyrimidine

derivativesrdquo World Applied Sciences Journal vol 36pp 637ndash645 2018

[43] J Zhang L Zeng H Sun S Chen T Wang and S Ma ldquoUseof nonanal-wax as postharvest fungicide of tomato againstbotrytis cinereardquo Journal of Food and Nutrition Researchvol 5 no 7 pp 458ndash466 2017

[44] J Shim N R Jyothi and N A M Farook ldquoBiological ap-plications of thiosemicarbazones and their metal complexesrdquoAsian Journal of Chemistry vol 25 no 10 pp 5838ndash58402013

[45] A Mohammadi ldquoNovel triazene dyes based on N-phenyl-piperazine synthesis anti-bacterial activity and sol-vatochromic propertiesrdquo Journal of Molecular Liquidsvol 193 pp 69ndash73 2014

[46] L Kovanda W Zhang X Wei et al ldquoIn vitro antimicrobialactivities of organic acids and their derivatives on severalspecies of gram-negative and gram-positive bacteriardquo Mole-cules vol 24 no 20 p 3770 2019

[47] C B Huang Y Alimova T M Myers and J L EbersoleldquoShort- and medium-chain fatty acids exhibit antimicrobialactivity for oral microorganismsrdquo Archives of Oral Biologyvol 56 no 7 pp 650ndash654 2011

[48] S Indumathi S Perumal D Banerjee P Yogeeswari andD Sriram ldquol-Proline-catalysed facile green protocol for thesynthesis and antimycobacterial evaluation of [14]-thiazinesrdquoEuropean Journal of Medicinal Chemistry vol 44 no 12pp 4978ndash4984 2009

[49] S Badshah and A Naeem ldquoBioactive thiazine and benzo-thiazine derivatives green synthesis methods and their me-dicinal importancerdquo Molecules vol 21 no 8 p 1054 2016

[50] S S Didwagh and P B Piste ldquoSynthesis and antimicrobialactivity of thiazine derivativesrdquo Journal of Chemical andPharmaceutical Research vol 55 pp 171ndash174 2013

[51] L L D da Silva M Nascimento D H S Silva M Furlan andV da Silva Bolzani ldquoAntibacterial activity of a stearic acidderivative from Stemodia foliosardquo Planta Medica vol 68pp 1137ndash1139 2002

[52] A Dogan S Otlu O Ccedilelebi and P A Kilicle ldquoAn investi-gation of antibacterial effects of steroidsrdquo Turkish Journal ofVeterinary and Animal Sciences vol 41 pp 302ndash305 2017

[53] J H Hussein Y H Mohammed and H H Imad ldquoStudy ofchemical composition of Foeniculum vulgare using Fouriertransform infrared spectrophotometer and gas chromatog-raphy-mass spectrometryrdquo Journal of Pharmacognosy andPhytotherapy vol 8 no 3 pp 60ndash89 2016

[54] P K Sharma and R Makkar ldquoA review thiazines derivativestreated as potential antimicrobial agentsrdquo Asian Journal ofPharmaceutical and Clinical Research vol 10 pp 43ndash46 2017

[55] A Perez-Rebolledo L R Teixeira A A Batista et al ldquo4-Nitroacetophenone-derived thiosemicarbazones and theircopper(II) complexes with significant in vitro anti-trypano-somal activityrdquo European Journal of Medicinal Chemistryvol 43 no 5 pp 939ndash948 2008

[56] J Raju and R Mehta ldquoCancer chemopreventive and thera-peutic effects of diosgenin a food saponinrdquo Nutrition andCancer vol 61 pp 27ndash35 2009

[57] L Xiang X Yi Y Wang and X He ldquoAntiproliferative andanti-inflammatory polyhydroxylated spirostanol saponinsfrom Tupistra chinensisrdquo Scientific Reports vol 6 pp 1ndash132016

[58] Q Liu B Kong Y L Xiong and X Xia ldquoAntioxidant activityand functional properties of porcine plasma protein hydro-lysate as influenced by the degree of hydrolysisrdquo FoodChemistry vol 118 no 2 pp 403ndash410 2010


[59] X Peng Y L Xiong and B Kong ldquoAntioxidant activity ofpeptide fractions fromwhey protein hydrolysates as measuredby electron spin resonancerdquo Food Chemistry vol 113 no 1pp 196ndash201 2009

[60] D-W Lee D-H You E-K Yang et al ldquoAntioxidant andACE inhibitory activities of Styela clava according to har-vesting timerdquo Journal of the Korean Society of Food Scienceand Nutrition vol 39 no 3 pp 331ndash336 2010

[61] C-C Lee H J Hsieh C-H Hsieh and D-F HwangldquoAntioxidative and anticancer activities of various ethanolicextract fractions from crown-of-thorns starfish (Acanthasterplanci)rdquo Environmental Toxicology and Pharmacology vol 38no 3 pp 761ndash773 2014

[62] M Sugumaran and W E Robinson ldquoStructure biosynthesisand possible function of tunichromes and related com-poundsrdquo Comparative Biochemistry and Physiology Part BBiochemistry and Molecular Biology vol 163 no 1 pp 1ndash252012

[63] J A Tincu L P Menzel R Azimov et al ldquoPlicatamide anantimicrobial octapeptide from Styela plicata hemocytesrdquoJournal of Biological Chemistry vol 278 no 15 pp 13546ndash13553 2003


populations when the demand is high especially for Hal-ocynthia and Styela species Edible species are usually fromsolitary stolidobranchs Halocynthia aurantium H roretziMicrocosmus hartmeyeri M sabatieriM vulgaris Polycarpapomaria Pyura chilensis Styela clava and S plicata are theimportant species eaten(ese groups are presented in freshand dried forms in the markets High-quality processes ofH aurantium H roretzi P chilensis S clava and S plicataare the main seafood exports to Europe and America [5]

Generally tunicates have a high nutritional value due tothe presence of a high level of bioactive components andprotein and low calories [6ndash10] Moreover some of themcontain different vitamins (ie vitamin E vitamin B12 andvitamin C) minerals (ie Na K Ca Mg P Fe Zn and Cu)amino acids folic acid fatty acid and pantothenic acid [11]Some groups of tunicates have different low molecularweight peptides in their bodies such as styelins plicatamidehalocyamines lamellarins and ferreascidin from Styelaclava Styela plicata Halocynthia roretzi Didemnum char-taceum and Pyura stolonifera respectively as well astunichromes from different tunicates species [5 12 13]

Some tunicates species can accumulate low molecularweight oligopeptides in their blood cell which are known astunichromes Tunichromes could play an important role indefense mechanism mainly due to the phenoloxidase whichcan attack easily tunichrome It is considered as a keycomponent for both tunic formation and tunic woundhealing which use the same primary biochemical mecha-nisms [13] Tunichromes have different colors such as pinkred and blue [14] (ese pigments are ingredients in theblood cells of tunicates Tunichromes were identified in 11groups of different tunicates [5] (e broad biological ac-tivity of tunichromes remains mainly unknown Howeversome researches offer unique functionalities for tunichromein terms of antioxidant [15ndash17] antimicrobial [5 18] andanticancer activities [19]

To the best of our knowledge there is no information onthe biological activities of the amazing tunichrome releasedfrom the Phallusia nigra Persian Gulf marine tunicate(erefore this research is a first attempt to evaluate thephysicochemical nutraceutical antioxidant and antibac-terial properties of tunichrome released from Phallusia nigraPersian Gulf marine tunicate

2 Materials and Methods

21 Materials Phallusia nigra tunicate was randomly col-lected from Nayband Bay situated in the north (27deg 30primeS 52deg35primeE) of Bushehr Iran using SCUBA diving at the depthranging from 05 to 15m in spring of 2019 Media culturesand all other chemical materials were prepared in analyticalgrade from Merck and Sigma-Aldrich

22 Methods

221 Sampling and Sample Preparation 50 samples ofPhallusia nigra tunicates were washed by double distilledwater (DDW) to remove contaminants (ey were imme-diately transported to the laboratory in a cool box (in ice-

cold condition) and kept at room temperature for 30min torelease a pink color solution (tunichrome) (e obtainedsuspension was centrifuged (4000times g for 5min) and thenfiltrated by Whatman No 1 filter paper (e final solutionwas freeze-dried and powdered for further studies

222 Total Protein Content Total protein content wasmeasured based on the AOAC method by the Kjeldahldevice (Buchi Switzerland)

223 Molecular Weight Measurement (SDS-PAGE) For themolecular weight determination the tunichrome (5mgmL)was dissolved in 1 SDS solution prepared by phosphatebuffer (pH 70) and stirred for 24 h followed by centrifu-gation at 5000times g for 6min at 20degC [20] (e supernatant(20 μl) was added to 10 μL Tris-HCl buffer (10mM Tris-HCl10 (wv) SDS 01 (wv) bromophenol blue and 10 (vv) glycerol) with or without 2 (vv) β-mercaptoethanol(e suspension was then heated at 95degC for 11min and20 μL of the suspension was developed on the gel slab whichwas contained resolving gel (125 pH 88) and stacking gel(6 pH 68) PowerPac 1000 (Bio-Rad USA) was used forrunning the electrophoresis (e running buffer was madeby diluting 100mL 10 X TrisGlycineSDS buffer with theDDW at the constant voltage 220V(e Coomassie BrilliantBlue was used for staining the gel for 60min and water wasused for destaining for 24 h (e molecular weights wereevaluated by comparison to Sinaclon markers (PR901641CinnaGen Co Tehran Iran) at the ranges of 245ndash11 kDa

224 Amino Acidsrsquo Profile To evaluate amino acid profile intunichrome peptide bonds of protein were broken by hy-drolysis (e hydrolysis process was conducted by heatingthe sample in an oxygen-free condition containing 6M HCland 01 phenolphthalein at 110degC overnight Amino acidswere measured by a Varian chromatographic system con-taining a 1525 pump a 9100 autoinjector and a UV-vis de-tector (e hydrolyze suspension was injected into anautomatic precolumn reaction by 01mL of derivatizing re-agent (is chromatographic process contained a solventmixture (PBS buffer (10mM pH 4) acetonitrile (25 75) at aflow rate of 1mLmin In this system a C18Waters Nova-Packreverse phase column (particle size 5μm 250times 46mm internaldiameter) was applied All the chromatographic data wereprocessed in a V 45 Star workstation supplied by Varian [21]

225 Na Ca K Mg Mn Cu Fe and Zn Measurements(e concentrations of Na Ca K Mg Mn Cu Fe and Zn oftunichrome were analyzed by a Selectra 2 auto-analyzer(Vital Scientific Spankeren Netherlands) To this end 1 g oflyophilized powder was dissolved in the 5 g of DDW for 24 hand then centrifuged

226 Heavy Metal (Cd As Pb and Hg) Measurements(e lyophilized powder of tunichrome (02 g) was weighedand mixed with concentrated HNO3 (7mL 65 vv) and











Ac

1113890 1113891 times 100 (1)



















245180135100

75

63

48

35

25

20

17

11

kDa

Tris-Glycine4-20

(a) (b)













444

213

34

05

EAASEAA

NEAAMisc

















2962

2573

652 536

852899

107612281388

160816241728

0

20

40

60

80

100

Tran

smitt

ance

()








4 Conclusion












Data Availability




References













































































Ac

1113890 1113891 times 100 (1)



















245180135100

75

63

48

35

25

20

17

11

kDa

Tris-Glycine4-20

(a) (b)













444

213

34

05

EAASEAA

NEAAMisc

















2962

2573

652 536

852899

107612281388

160816241728

0

20

40

60

80

100

Tran

smitt

ance

()








4 Conclusion












Data Availability




References
















































































245180135100

75

63

48

35

25

20

17

11

kDa

Tris-Glycine4-20

(a) (b)













444

213

34

05

EAASEAA

NEAAMisc

















2962

2573

652 536

852899

107612281388

160816241728

0

20

40

60

80

100

Tran

smitt

ance

()








4 Conclusion












Data Availability




References












































































444

213

34

05

EAASEAA

NEAAMisc

















2962

2573

652 536

852899

107612281388

160816241728

0

20

40

60

80

100

Tran

smitt

ance

()








4 Conclusion












Data Availability




References












































































2962

2573

652 536

852899

107612281388

160816241728

0

20

40

60

80

100

Tran

smitt

ance

()








4 Conclusion












Data Availability




References








































































4 Conclusion












Data Availability




References





































































Data Availability




References




































































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Transcript of 5513717.pdf - Hindawi.com