Research ArticlePurification and Characterization of Bioactive Compoundsfrom Styela clava

Bao Ju1 Bin Chen2 Xingrong Zhang1 Chunling Han1 and Aili Jiang1

1 Department of Bioengineering Yantai University Yantai Shandong 264005 China2 Yantai Institute of Coastal Zone Research Chinese Academy of Sciences Yantai Shandong 264003 China

Correspondence should be addressed to Aili Jiang jal9035livecn

Received 17 March 2014 Accepted 1 April 2014 Published 30 April 2014

Academic Editor Qiusheng Zheng

Copyright copy 2014 Bao Ju et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The immunomodulatory activity of extract from Styela clava was studied systematically based on activity tracking in vitro inorder to find out novel-structured secondary metabolite The proliferation rates of mouse splenic lymphocytes and peritonealmacrophages were used as screening index as well as NO release promoting activities The crude extract (CE) and its differentpolar fractions from S clava all exhibited proliferative activity of splenolymphocytes andmousemacrophages as well as NO releasepromoting activities among which petroleum ether fraction (PE) showed the strongest effect The antioxidant experiment in vitroshowed that CE demonstrated antioxidant ability in 11-diphenyl-2-picrylhydrazyl (DPPH) system and the beta carotene-linoleicacid system the activity of ethyl acetate fraction (ET) was much stronger than that of the others Further isolated by silica gelcolumn chromatography ET was classified into seven sub-components (E1simE7) listed in the order of activity as E5 gt E6 gt E4 gtE3 gt E7 gt E2 gt E1 Five compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol(4) batilol and (5) ceramide among which (1) (2) and (4) were isolated for the first time from S clava

1 Introduction

The ascidians are commonly found in waters all over theworld along the coasts and deep to the bottoms which isthe most important marine source for active agents exceptsponge In the early 1970s it had been found that theextract from ascidians had a variety of bioactivities such ascytotoxicity antitumor in vivo and immune regulation Since1980s a greater variety of bioactive substances were extractedfrom the ascidians with the activities of antitumor antivirusantimicrobial immune regulation and biocatalysis Furtherstudies showed that those active chemical compounds insea squirt mainly included peptides alkaloids polyethersmacrolides terpenes and polysulfides [1ndash9] among whichsome have been tested at the clinical phase as anticancerreagents [10ndash13]

By strengthening organismsrsquo defense and host immunesystem against tumor cells cell immunity can improve itsoverall antitumor ability to inhibit tumor and thus play

an important role in immune antitumor treatment Alco-hol from Styela clava (51205728120572-cyclicobioxygen-24-bimethyl-6-vinyl-3120573-cholesterol SC) a lead compound against a varietyof viruses has a wide application to immune regulation asa supplement to maintain normal immune response for theremoval of viruses and recovery [14]

The ascidian has become the hot topic for research anddevelopment both at home and abroad for its numerousactive agents with high activity Styela clava the dominantpopulation in the Yellow Sea and the Bohai Sea has a negativeimpact on marine culture for its wide distribution and largequantity while the changes in its population may be regardedas an index for environment contamination If fed in seawaterin proper conditions S clava may grow and reproduce veryfast with low cost therefore with rational exploitation Sclava can become treasures instead of waste to benefit botheconomy and environment protection

S clava remains neglected as a potential marine chemicalresource and there are few reports on its components

Hindawi Publishing CorporationJournal of ChemistryVolume 2014 Article ID 525141 9 pageshttpdxdoiorg1011552014525141

2 Journal of Chemistry

worldwide besides few individual components without in-depth investigation Up to now there have been no reports ofthe systemic in-depth study on S clavarsquos compounds and theiractivity Integrated exploitation of its active agents may ononehandprovide theoretical basis for resource exploitation toachievemaximumeconomic results and on the other improveour lives and medical care to achieve direct social results

This paper is a study on S clava in terms of its chemicalstructure and bioactivity to obtain novel-structured activeagents as a theoretical basis for new drugs or lead compoundfor new drugs These studies may be used as references forthe research and development of marine functional food anddrugs

2 Materials and Methods

21 Materials Styela clava was collected from Yantai Sishili-wan Bay Shandong Province China in August 2010 adher-ents were removed and frozen

Kunming mice of clean grade were purchased fromShandong Animal Experiment Center with weight being 18ndash22 g for each

ConA LPS MTT peptone 11-DPPH 120573-carotene andlinoleic acid were purchased from Sigma Chemical Co(USA) RPMI1640 from Gibco fetal bovine serum fromHyClone DMSO from AMRESCO and penicillin and strep-tomycin from Shandong Lukang Pharmaceutical Group CoLtd Other analytical reagents were purchased from homemarkets

22 Methods

221 Extraction of Active Agents from S clava Tissue ofS clava (15 kg) was minced centrifuged and then soakedin 95 ethanol three times in volume for 1 week at roomtemperature The extraction was repeated three times Thecombined extract was concentrated under vacuum to give acrude extract (CE) as brown extractum of 205 g The crudeextract was suspended in distilled water of 2000mL and thenfurther extracted with petroleum ether chloroform ethylacetate and butanol (800mL times3) respectively resulting inpetroleum ether extract (PE) 787 g ethyl acetate extract (ET)186 g butyl alcohol extract (BU) 402 g and the remainder(AR) 647 g

222 Isolation Purification and Structural Identification ofthe Chemical Components fromS clava After activity screen-ing normal phase silica gel column chromatography (CC)and ODS reversed phase silica gel column chromatographywere adopted respectively for the most active ET whilepetroleum ether and acetone chloroform and carbinol wereused for silica gel column chromatography gradient elutionSeven components (E1ndashE7) were isolated among whichcomponents E2 E3 E4 and E5 for their large quantity werefurther purified by Sephadex LH-20 PTLC Amberlite XAD-2 and recrystallization and 5 compounds were obtained andtheir structures were identified by IR (Nicolet impact 400)

MS (HP 5988AGCMS) and 1DNMRand 2DNMR (Bruker-500MHz-FT-NMR)

223 Proliferation of Spleen Lymphocyte Ten days aftervaccinated S180 Kunming mice of clean grade were killedby cervical dislocation and spleens were taken out underasepsis for the preparation of lymphocyte suspension whichwas incubated in the 96-well plates (Coastar USA) at theconcentration of 1 times 107 cellsmL with 100120583L for each welland six repeats for each treatingMediumof 100120583Lwas addedto the control group 10 120583gmL ConA to the positive groupand the sample solutions of different concentration to theexperiment groups resulting in the concentrations of 125 2550 100 and 200 120583gmL cultured at 37∘C in 5CO

2incubators

(by SHEL LAB) for 44 h and afterwards 5mgmL of MTTwas added with 20120583L for each well and further culturedfor 4 h After the media were removed 015mL of DMSOwas added to each well and shaken for 10min for completemixture at room temperature for 10min before ELX-800(BioTek Instruments Inc) was used to measure absorbanceat 570 nm Consider

Proliferation ratio of lymphocytes ()

= [

119860 sample minus 119860control

119860control] times 100

(1)

where 119860 sample is the absorbance of tested sample and 119860controlis the absorbance of control group

224 Proliferation ofMacrophages 2 (wv) peptone of 2mLwas injected into the micersquos peritoneal cavity every day forthree days After cervical dislocation precooled Hankrsquos of5mL was injected into the peritoneal cavity then abdominalfluid was sucked and the supernatant was removed bycentrifugation at 2000 rpm the precipitate cells were washedtwice1 times 10

6mL of cell suspension was made with 10 ofcalf serum in RPMI 1640 incubated in the tissue cultureplate with 100 120583L for each well and cultured at 37∘C in 5CO2incubators for 2 h The upper medium was removed

and the wells were washed with PBS twice to remove thenonadherent cells somacrophagemonolayers were obtainedFresh medium was put into the 96-well plates following withfresh medium of 100 120583L to the control group 100120583L LPSto the positive group (with final concentration of 2 120583gmL)and 100 120583L different samples of different concentrationsto the other groups (the sample concentrations are as inSection 223) and cultured at 37∘C in 5 CO

2incubators

for 24 h Afterwards upper medium was removed and newmedium was added with 100 120583L for each hole as well as5mgmL of MTT with 20120583L further cultured for 4 h thenthe supernatant was carefully sucked and 150120583L DMSO wasadded before shaking for 10min for complete mixture After

Journal of Chemistry 3

placing at room temperature for 10min ELX-800 was used tomeasure absorbance at 570 nm Consider

Proliferation ratio of macrophages ()

= [

119860 sample minus 119860control

119860control] times 100

(2)

where 119860 sample is the absorbance of tested sample and 119860controlis the absorbance of control group

225 NO Release Activities of Macrophages Macrophageswere prepared with the same method as in Section 22425times10

5mL of cell suspension was made with 10 of calfserum in RPMI 1640 in sterile conditions inoculated on thetissue culture plate with 100 120583L for each well and three wellsfor each sample were cultured at 37∘C in 5 CO

2incubators

for 2 h Then LPS solution (with final concentration of2 120583gmL) of 100 120583L was put into the positive group medium1640 of 100 120583L was added to the control group and 100 120583Lwith different samples of different concentrations was addedto the other groups and further cultured for 48 h Afterwardsupper medium of 50 120583L from each well was removed andtransferred to a new 96-well plate and measured accordingto the following method

Standard Curve Standard solution nitrite of 01molL wasdiluted in 1 1000 with medium A1simH3 on the tissue cultureplate were used for standard curve Standard solution nitriteof 100 120583L was put into A1simA3 respectively and medium of50120583Lwas put into B1-H3 respectively Doubling dilution wasconducted to AndashG in turn and the final fluid from G wasremoved and nitrite concentration coefficient (100 50 25125 625 313 156 and 0 120583molL) was obtained Standardcurve was necessary before each test with the same mediumas for the samples

Measurement NED solution and sulfanilamide solution werekept at room temperature for 15 to 30min Each of testedsamples of 100 120583L was placed on the tissue culture plate 2 to3 repeated wells were used for every sample Sulfanilamidesolution of 50120583L was put into all standard curve wells andsample wells were kept in dark place at room temperature for5 to 10min and then the same was done with NED solutionof 50120583L Absorbance at 540 nm was measured within 30minand the nitrite concentration in the samples was calculatedaccording to standard curve

226 In Vitro Antioxidant Activity

(1) DPPH Radical Scavenging Assay Modified Binsanrsquosmethod [15] was adopted 15mL sample in different concen-trations was added to 15mL of 02mM DPPH in ethanol

15mL DPPH of 02mmol in anhydrous ethanol wasadded to all the samples of 15mL after mixing vigorouslyand kept at room temperature in dark for 30min Theabsorbance of the resulting solution was measured at 517 nmwithwater as the control and the ability to scavenge theDPPH

radical was expressed by clearance rate (119864) in the followingformula

119864 () = [1 minus119860 sample minus 119860 sample blank

119860control] times 100 (3)

where 119860 sample means the absorbance of test sample solution(DPPH + sample) 119860 sample blank means the absorbance of thesample only (ethanol + sample) and 119860control means theabsorbance of control (DPPH + ethanol)

The half clean concentration (EC50) is calculated by

regression equation of E and sample concentration

(2) 120573-Carotene-Linoleic Acid System Modified Jayaprakasharsquosmethod [16] was adopted 120573-carotene of 02mg was dissolvedin 02mL trichloromethane into which linoleic acid of 20mgand Tween-40 of 200mg were added and mixed completelyAfter chloroform was removed under vacuum at 40∘Cdistilled water of 50mL (oxygenated for 2-3min) was addedThe reaction emulsion was prepared after vigorous agitationThe control was prepared as above except for the addition of120573-carotene Sample solution of 50120583L and reaction mediumof 200120583L were added into 96-well plates respectively andthe absorbance was measured at 450 nm immediately (119905 =0) and remeasured every 20min until the color of 120573-carotene faded in the control (about 240min) Three parallelmeasurements were conducted and the antioxidant activity(AA) was expressed in the following formula

AA = [1 minus119860

0minus 119860

119905

119860

1015840

0minus 119860

1015840

119905

] times 100 (4)

where 1198600and 119860

119905were the absorbance of the sample while

119905 = 0 and 119905 = 240min respectively and 11986010158400and 1198601015840

119905were

the absorbance of the control while 119905 = 0 and 119905 = 240minrespectively

(3) Reducing Power The reducing power was determinedaccording to Yen and Duhrsquos method [17] All samples weredissolved with phosphate buffer solution (02molL pH 66)of 25mL and 1 potassium ferricyanide (wv) of 25mLwas added before reacting at 50∘C for 20min then 10trichloroacetic acid (wv) of 25mL was added to stopreaction Reaction mixture was centrifuged at 650timesg for10min supernatant of 25mL was mixed with distilled waterof 25mL and 01 FeCl

3(wv) of 05mLThe absorbance was

measured at 700 nm and the higher absorbance means thestronger reducing power The reducing power is convertedin ascorbic acid and expressed as ascorbic acid equivalent(AscAE) in milligrammes of ascorbic acid per gramme ofsample (AscAE mgg AscA)

23 Statistical Analysis All data were expressed by mean plusmnstandard deviation All statistical analyses were carried outusing SPSS 130 for Windows Tukeyrsquos multiple comparisontests were used to detect differences between groups and aprobability of 119875 lt 005 was taken as an acceptable level ofsignificance

4 Journal of Chemistry

0

10

20

30

40

50

60

Prol

ifera

tion

rate

()

BU PE CE ConAET

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmLPositive control

lowast

lowast

minus10

minus20

lowastlowast

Figure 1 Effects of extracts on the proliferation of the mousesplenocytes lowast119875 lt 005 and lowastlowast119875 lt 001 compared with the control

3 Results and Discussion

31 Proliferation of Spleen Lymphocyte As organismrsquos firstline defending tumor immune system plays an importantrole in the recognition and removal of malignant cells Theimmune status evaluation of the body bearing neoplasm isimportant for mechanism study of antitumor drugs

As shown in Figure 1 four extracts of S clava demonstratesignificant activity on splenocytes proliferation (comparedwith the control 119875 lt 005 or 119875 lt 001) amongwhich the proliferation effect of PE is the most activewith proliferation rate being near 50 at a concentration of25 120583gmL close to the positive control group ConA (5671)However its proliferation rate reduces with the increasingconcentration and the proliferation rate is only 476 whenthe concentration reaches 400 120583gmL which shows that PEinhibits the proliferation of the mouse splenocytes at highconcentration CE is less active than PE on the proliferationof the mouse splenocytes demonstrating a rising trend atfirst and then a falling one The effects of ET and BUare relatively weak and ET shows distinctive inhibition at50120583gmL and 100120583gmL with the inhibition rate of 1262at 100 120583gmL but on the contrary BU reaches the strongestlevel at this concentration with the proliferation rate of1452 while distinctive inhibition was shown below thispoint

32 Proliferation of Macrophages Figure 2 shows the effectsof S clavarsquos extracts on the proliferation of the mouseperitonealmacrophagesThe four extracts all show significanteffect on the proliferation of the mouse macrophages at alldesigned concentrations (compared with the control 119875 lt005 or 119875 lt 001) but it is weaker than that of thepositive control LPS as proliferation rate of 6319 PE is themost active extract on the proliferation at concentration of

05

101520253035404550556065

Prol

ifera

tion

rate

()

ET BU PE CE LPS

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmLPositive control

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

Figure 2 Effects of extracts on the proliferation of the mouseperitoneal macrophages lowast119875 lt 005 and lowastlowast119875 lt 001 compared withthe control

25 120583gmL with proliferation rate 4070 but afterwards theproliferation rate dropswith the increasing concentration andthe proliferation rate is only 1085 when the concentrationreaches 400120583gmL showing similar trend as on the lympho-cytes

ET and CE are close on the effect of proliferation ETreaches the lowest point at 25120583gmL with the proliferationrate of 194 and the highest point at 200120583gmL with theproliferation rate of 2597 CE shows similar trend asET reaching the highest and lowest points at 100 120583gmLand 400120583gmL with the proliferation rate of 2519 and1017 respectively BU shows the weakest effect with thehighest proliferation rate of only 1008 at 200120583gmL andthe same effect as the control at 25120583gmL the prolifer-ation rates at all the other concentrations are all below10

33 NO Release Activities of Macrophages Macrophages arethe important components in the body immune systemfunctioning as immunization effect regulating the immunesystem and NO is one of the active agents produced bymacrophages after they are activated [18] Figure 3 showseffects of extract from S clava on the NO release activitiesof mouse macrophages The effects of ET PE and CE are allin positive correlation with concentration in promoting therelease of NO PE shows the strongest effect among themreaching the highest point of 1614 120583molmL at 400 120583gmLCE is less effective with NO releasing the highest point of1476 120583molmL at 400 120583gmL The effects of BU and ET arerather weak only 870120583molmL for ET at 400120583gmL as thehighest point while at the other concentrations the NOreleasing abilities for ET and BU are close to and approaching6 120583molmL

Journal of Chemistry 5

Table 1 DPPH scavenging activity of different components isolated from S clava

Component DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

CE 192 plusmn 037 3012 plusmn 060 379 plusmn 064 426 plusmn 088

PE 483 plusmn 011 1011 plusmn 022 1424 plusmn 038 1850 plusmn 037

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

BU 838 plusmn 021 1929 plusmn 037 2532 plusmn 049 3077 plusmn 062

AR 210 plusmn 005 305 plusmn 011 426 plusmn 008 489 plusmn 010

Table 2 DPPH scavenging activity of subcomponents E1ndashE7 isolated from ET of S clava

Subcomponent DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

E1 824 plusmn 026 1417 plusmn 040 1864 plusmn 059 2080 plusmn 065

E2 1137 plusmn 038 1819 plusmn 088 2430 plusmn 162 2918 plusmn 125

E3 1846 plusmn 064 2530 plusmn 124 3428 plusmn 122 4580 plusmn 099

E4 2280 plusmn 032 3820 plusmn 102 4937 plusmn 157 5678 plusmn 101

E5 3026 plusmn 088 4620 plusmn 092 5916 plusmn 111 6731 plusmn 165

E6 2492 plusmn 077 4200 plusmn 106 5340 plusmn 070 6442 plusmn 162

E7 1400 plusmn 031 2029 plusmn 066 2713 plusmn 048 3250 plusmn 101

0

2

4

6

8

10

12

14

16

18

ET BU PE CE

NO

(120583m

olm

L)

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Figure 3 Effects of extracts on NO production lowast119875 lt 005 andlowastlowast119875 lt 001 compared with the control

34 In Vitro Antioxidant Activity

341 DPPH Radical Scavenging Assay The DPPH free rad-icals scavenging method is a colorimetric assay and can beused to evaluate the radical scavenging activity in a shorttime DPPH is an organic free radical with maximal absorp-tion at 517 nmWhenwith free radical scavengerDPPHrsquos loneelectronwill be paired and its absorbancewill decrease whichcan be used to evaluate its antioxidant activity As shown

in Table 1 there was a positive correlation between DPPHscavenging activity of CE and its concentration Among thefour extracted components PE and BU demonstrate poorscavenging activity while AR shows no scavenging activityThe scavenging activity of ET reaches 5064 at 200 120583gmLmuch higher than CE (119875 lt 005) indicating that compoundswithDPPH scavenging activity in S clava aremostlymediumpolar

E1ndashE7 subcomponents are isolated from ETwith silica gelcolumn chromatography whose DPPH scavenging activity isshown in Table 2 At all experiment concentrations E5 showsthe strongest scavenging activity followed by E6 E4 E3 E7and E2 with E1 the weakest E5 and E6 show much strongerscavenging activity than ET (119875 lt 005) while E4 is close toET in scavenging activity EC

50of E5 and E6 are 12206120583gmL

and 13968 120583gmL respectively

342 Antioxidant Activity in 120573-Carotene-Linoleic Acid Sys-tem 120573-carotene is a polyene pigment apt to be oxidized tofade In the reaction medium the superoxide produced bythe oxidized linoleic acid results in 120573-carotenersquos fading andas time goes on the absorbance decreases The degree of120573-carotenersquos fading depends on the antioxidant activity inthe system Table 3 shows antioxidant activities of differentcomponents isolated from S clava in the 120573-carotene-linoleicacid system CE has some antioxidation activities positivelycorrelated with its concentration ET is much higher in itsantioxidant activity than other components reaching 50 at200120583gmL and is even higher than the positive control AscAat all experimental concentrations which further indicatesthat compounds with higher antioxidant activity in the120573-carotene-linoleic acid system are mostly medium-polarcomponents

6 Journal of Chemistry

Table 3 Antioxidant activity of different components isolated from S clava in the 120573-carotene-linoleic acid system

Component Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

CE 1062 plusmn 038 2010 plusmn 112 2948 plusmn 130 4035 plusmn 112

PE 460 plusmn 021 1022 plusmn 044 1650 plusmn 049 2240 plusmn 068

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

BU 720 plusmn 048 1439 plusmn 062 2260 plusmn 077 2600 plusmn 089

AR 215 plusmn 005 690 plusmn 014 820 plusmn 018 980 plusmn 011

AscA 1050 plusmn 012 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

Table 4 Antioxidant activity of subcomponents E1ndashE7 isolated from ET in the 120573-carotene-linoleic acid system

Subcomponent Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

E1 646 plusmn 050 1380 plusmn 046 2820 plusmn 064 2219 plusmn 097

E2 742 plusmn 031 1540 plusmn 074 2474 plusmn 107 3090 plusmn 121

E3 930 plusmn 037 1778 plusmn 087 2741 plusmn 103 3277 plusmn 127

E4 1238 plusmn 067 2472 plusmn 118 3270 plusmn 079 4365 plusmn 198

E5 2024 plusmn 144 4060 plusmn 125 5410 plusmn 157 6892 plusmn 167

E6 1520 plusmn 079 3440 plusmn 085 4852 plusmn 171 6236 plusmn 168

E7 1070 plusmn 047 2010 plusmn 027 2842 plusmn 080 3680 plusmn 098

AscA 1050 plusmn 072 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

GA 2180 plusmn 041 4280 plusmn 188 5861 plusmn 120 7010 plusmn 146

Table 5 Reducing power of extract and subcomponents E1ndashE7isolated from ET

Component AscAE (mggAscA)CE 4020 plusmn 110

PE 1432 plusmn 042

ET 6260 plusmn 155

BU 2660 plusmn 051

WT 572 plusmn 026

E1 2040 plusmn 077

E2 2820 plusmn 122

E3 4620 plusmn 118

E4 6160 plusmn 111

E5 8440 plusmn 210

E6 7520 plusmn 150

E7 4160 plusmn 095

Theantioxidant activity of subcomponents E1ndashE7 isolatedfrom ET in the 120573-carotene-linoleic acid system is shownin Table 4 At all experiment concentrations E5 shows thestrongest antioxidant activity followed by E6 E4 E7 E3 andE2 with E1 being the weakest Among the seven subcom-ponents E5 and E6 show much stronger antioxidant activitythan ET (119875 lt 005) while the antioxidant activity of E5 is6892at 200120583gmL close to the synthesized antioxidantGA

343 Reducing Power Table 5 shows the reducing power ofthe extract from S clava and subcomponents E1ndashE7 isolated

from ET The reducing powers of all the samples demon-strate a pattern similar to DPPH scavenging activity andantioxidant activity in the 120573-carotene-linoleic acid systemAmong all the extracted components ET is the highest inthe reducing power (119875 lt 005) and the reducing power ofthe seven subcomponents isolated fromET is in the followingorder E5 gt E6 gt E4 gt E3 gt E7 gt E2 gt E1

35 Structural Identification of Compound Compound 1colorless needle crystal is identified as C

27H46O with EI-MS

and NMR 120575347 in 1H NMR 120575718 in 13C NMR and DEPTshow that there is hydroxyl in themolecule 120575531 in 1HNMR12057514018 and 1205751217 in 13C NMR and DEPT further confirmthe double bond in the molecule mz 255 and 147 in MScombinedwith 120575531 (1H H-7) 120575356 (1HmH-3) 120575101 (3Hs Me-19) and 120575088 (3H s Me-18) in 1HMMR 1205751408 (s C-8) 1205751217 (d C-7) and 120575718 (d C-3) in 13CNMR and DEPTreferred to the physical constant and spectral data reportedby Tsuda and Schroepfer Jr [19] compound 1 was identifiedas cholesteric-7-en-3120573-ol whichwas isolated from S clava forthe first time

Compound 2 colorless needle crystal mp 244sim246∘C isidentified as C

27H46O2with EI-MS andNMRThe two signals

of methyl group connected with quaternary carbon (120575126 s120575071 s) and three signals of methyl group connected withtertiary carbon (120575091 d 120575087 d 120575085 d) show that thiscompound is sterol The signal of 19-Me at 120575126 shifts toa lower field in contrast to cholesterol which proves theexistence of 6120573-OH Meanwhile in contrast to cholesterolsignal (120575418) of 3120572-H also shifts to a lower field and so is

Journal of Chemistry 7

HO

HO

HO

OH

OH

OH

OH

OH

H2C

H2C (CH2)17CH3

C

O

O

(CH2)16CH3

CH3(CH2)14

NH

HC

Compound 1 Compound 2

Compound 3 Compound 4

Compound 5

Figure 4 Structures of compounds

proved of 45-double bond considering single peak 120575555Compound 2 is identified as cholesteric-4-en-3120573 6120573-diolreferring to the physical constant and spectral data reportedby Wahidullah et al [20] which is isolated from S clava forthe first time

Compound 3 is a white needle crystal mp 148sim149∘Cpurplish red at 10 sulfuric acid-ethanol solution The mul-tiple peaks at 120575353 in 1H NMR is the characteristic signal of3120572-H in sterol while the multiple peaks at 120575535 is signal ofH-6 In addition five methyl group signals appear in highfield of 1H NMR among which the 120575101 s and 120575068 sbelong to 19-Me and 18-Me respectively 120575091 d is the signalof 21-Me and 120575084 d and 120575082 d belong to 26-Me and27-Me respectively Contrasting the physical constant andspectral data with Su et al [21] compound 3 is identified ascholesterol

Compound 4 is white powder mp 68sim69∘C purplish redat 10 sulfuric acid-ethanol solution The molecular weight

is 345 according to EI-MS As shown in 13C NMR spectrum120575641 (t C-1) 120575319 (t C2) 120575296 (t C3-C15) 120575260 (t C16)120575226 (t C17) and 120575141 (t C18) are characteristic signalsof long chain fatty acid and 120575722 (C-1) 120575706 (C-2) and120575718 (C-3) belong to glycerol so this compound should bea long chain triglyceride However there is no signal of estercarbonyl therefore this compound is identified as glycidylether agreeing with Yang et al [22] andWang et al [23] andis confirmed as batilol which is isolated from S clava for thefirst time

Compound 5 molecular formula is C36H71NO3accord-

ing to EI-MS andNMR 120575637 (1H) in 1HNMR spectrum and1205751739 (C=O) in 13C NMR spectrum proves the existence ofacylamino and 120575545 in 13C NMR spectrum indicates thatwhat is connected with nitrogen is methyne 120575202 (2H) in1HNMR indicates that there are two ndashOH in the compoundand 1205757474 and 1205756240 in 13C NMR indicate that ndashCHndash andndashCH2ndash connect with ndashOH respectively Meantime a strong

8 Journal of Chemistry

hydrogen signal of 120575126 in 1H NMR spectrum and twosignals ndashCH

3ndash of 120575084 (6H) determine two long chain alkyl

groups This compound is identified as ceramide referring tothe physical constant and spectral data reported by Yu andYang [24]

The structures of these compounds are shown in Figure 4As reported by Cai et al [25] the number and positionof hydroxyl groups largely determined radical scavengingactivity of phenolic compounds Compounds 1 2 and 3 allhave only one phenolic hydroxyl group and there is nofunctional groups in orthoposition little steric hindranceeffect increased their contact with free radicals

4 Conclusion

The immunomodulatory and antioxidant activity of crudeand fractionated extracts of the ascidian Styela clava weredetermined by in vitro screening All these extracts demon-strate immunomodulatory activity through increasing theproliferation rate of spleen lymphocyte and macrophagesas well as the NO release activities of macrophages Amongthem the petroleum ether fraction shows the strongestimmune active in vitro The ethyl acetate fraction (ET) wasmuch higher in its antioxidant activity in DPPH systemreducing power assay and 120573-carotene-linoleic acid systemcompared with the other fractions and its subcomponentE5 demonstrated the strongest antioxidant activity as well asreducing power higher than the positive control AscA andclose to the synthesized GA ET was isolated systemically andfive compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol (4) batiloland (5) ceramide among which (1) (2) and (4) were isolatedfor the first time from S clava

Conflict of Interests

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

Authorsrsquo Contribution

Ju Bao and Chen Bin contributed equally to this project

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (no 31070368) and the Sci-ence and Technology Department of Shandong Province(BS2010HZ022 and ZR2011DL008)

References

[1] T M Zabriskie and C M Ireland ldquoThe isolation and structureofmodified bioactive nucleosides from Jaspis johnstonirdquo Journalof Natural Products vol 52 no 6 pp 1353ndash1356 1989

[2] J Kobayashi M Tsuda M Ishibashi et al ldquoAmphidinolideF a new cytotoxic macrolide from the marine dinoflagellateAmphidinium sprdquo Journal of Antibiotics vol 44 no 11 pp 1259ndash1261 1991

[3] B S Davidson and C M Ireland ldquoLissoclinolide the first non-nitrogenous metabolite from a Lissoclinum tunicaterdquo Journal ofNatural Products vol 53 no 4 pp 1036ndash1038 1990

[4] T N Makarieva V A Stonik A S Dmitrenok et al ldquoVaracinand three new marine antimicrobial polysulfides from the far-Eastern ascidian Polycitor sprdquo Journal of Natural Products vol58 no 2 pp 254ndash258 1995

[5] P W Ford and B S Davidson ldquoPlakinidine D a newpyrroloacridine alkaloid from the ascidianDidemnum rubeumrdquoJournal of Natural Products vol 60 no 10 pp 1051ndash1053 1997

[6] C Ireland and P J Scheuer ldquoUlicyclamine and ulithiacy-clamide two new small peptides from a marine tunicaterdquoJournal of the American Chemical Society vol 102 no 17 pp5688ndash5691 1980

[7] H G Chun B Davies and D Hoth ldquoDidemnin B the firstmarine compound entering clinical trials as an antineoplasticagentrdquo Investigational New Drugs vol 4 no 3 pp 279ndash2841986

[8] M A Baker D R Grubb and A Lawen ldquoDidemnin B inducesapoptosis in proliferating but not resting peripheral bloodmononuclear cellsrdquo Apoptosis vol 7 no 5 pp 407ndash412 2002

[9] M D Vera and M M Joullie ldquoNatural products as probes ofcell biology 20 Years of didemnin researchrdquoMedicinal ResearchReviews vol 22 no 2 pp 102ndash145 2002

[10] S Jin B Gorfajn G Faircloth and K W Scotto ldquoEcteinascidin743 a transcription-targeted chemotherapeutic that inhibitsMDR1 activationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 12 pp 6775ndash6779 2000

[11] C van Kesteren E Cvitkovic A Taamma et al ldquoPharma-cokinetics and pharmacodynamics of the novel marine-derivedanticancer agent ecteinascidin 743 in a phase I dose-findingstudyrdquo Clinical Cancer Research vol 6 no 12 pp 4725ndash47322000

[12] E G C Brain ldquoSafety and efficacy of ET-743 the Frenchexperiencerdquo Anti-Cancer Drugs vol 13 supplement 1 pp S11ndashS14 2002

[13] J C M Waterval J C Bloks R W Sparidans et al ldquoDegra-dation kinetics of aplidine a new marine antitumoural cyclicpeptide in aqueous solutionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 754 no 1 pp 161ndash1682001

[14] C K Cai Research of anti-HBV activity and pharmacodynamicaction and toxicity of SC [PhD thesis] Beijing University ofChinese Medicine Beijing China 2006

[15] W Binsan S Benjakul W Visessanguan S Roytrakul MTanaka and H Kishimura ldquoAntioxidative activity of Mun-goong an extract paste from the cephalothorax ofwhite shrimp(Litopenaeus vannamei)rdquo Food Chemistry vol 106 no 1 pp185ndash193 2008

[16] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[17] G C Yen and P D Duh ldquoAntioxidative properties of methano-lic extracts from peanut hullsrdquo Journal of the American OilChemistsrsquo Society vol 70 no 4 pp 383ndash386 1993

[18] R N Albrecht Immunologie Tolerance and Macrophage Func-tion Aeademie New York NY USA 1 edition 1979

[19] M Tsuda and G J Schroepfer Jr ldquoCarbon-13 nuclear magneticresonance studies of C27 sterol precursors of cholesterolrdquoJournal of Organic Chemistry vol 44 no 8 pp 1290ndash1293 1979

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

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Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

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Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

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CatalystsJournal of

2 Journal of Chemistry

worldwide besides few individual components without in-depth investigation Up to now there have been no reports ofthe systemic in-depth study on S clavarsquos compounds and theiractivity Integrated exploitation of its active agents may ononehandprovide theoretical basis for resource exploitation toachievemaximumeconomic results and on the other improveour lives and medical care to achieve direct social results

This paper is a study on S clava in terms of its chemicalstructure and bioactivity to obtain novel-structured activeagents as a theoretical basis for new drugs or lead compoundfor new drugs These studies may be used as references forthe research and development of marine functional food anddrugs

2 Materials and Methods

21 Materials Styela clava was collected from Yantai Sishili-wan Bay Shandong Province China in August 2010 adher-ents were removed and frozen

Kunming mice of clean grade were purchased fromShandong Animal Experiment Center with weight being 18ndash22 g for each

ConA LPS MTT peptone 11-DPPH 120573-carotene andlinoleic acid were purchased from Sigma Chemical Co(USA) RPMI1640 from Gibco fetal bovine serum fromHyClone DMSO from AMRESCO and penicillin and strep-tomycin from Shandong Lukang Pharmaceutical Group CoLtd Other analytical reagents were purchased from homemarkets

22 Methods

221 Extraction of Active Agents from S clava Tissue ofS clava (15 kg) was minced centrifuged and then soakedin 95 ethanol three times in volume for 1 week at roomtemperature The extraction was repeated three times Thecombined extract was concentrated under vacuum to give acrude extract (CE) as brown extractum of 205 g The crudeextract was suspended in distilled water of 2000mL and thenfurther extracted with petroleum ether chloroform ethylacetate and butanol (800mL times3) respectively resulting inpetroleum ether extract (PE) 787 g ethyl acetate extract (ET)186 g butyl alcohol extract (BU) 402 g and the remainder(AR) 647 g

222 Isolation Purification and Structural Identification ofthe Chemical Components fromS clava After activity screen-ing normal phase silica gel column chromatography (CC)and ODS reversed phase silica gel column chromatographywere adopted respectively for the most active ET whilepetroleum ether and acetone chloroform and carbinol wereused for silica gel column chromatography gradient elutionSeven components (E1ndashE7) were isolated among whichcomponents E2 E3 E4 and E5 for their large quantity werefurther purified by Sephadex LH-20 PTLC Amberlite XAD-2 and recrystallization and 5 compounds were obtained andtheir structures were identified by IR (Nicolet impact 400)

MS (HP 5988AGCMS) and 1DNMRand 2DNMR (Bruker-500MHz-FT-NMR)

223 Proliferation of Spleen Lymphocyte Ten days aftervaccinated S180 Kunming mice of clean grade were killedby cervical dislocation and spleens were taken out underasepsis for the preparation of lymphocyte suspension whichwas incubated in the 96-well plates (Coastar USA) at theconcentration of 1 times 107 cellsmL with 100120583L for each welland six repeats for each treatingMediumof 100120583Lwas addedto the control group 10 120583gmL ConA to the positive groupand the sample solutions of different concentration to theexperiment groups resulting in the concentrations of 125 2550 100 and 200 120583gmL cultured at 37∘C in 5CO

2incubators

(by SHEL LAB) for 44 h and afterwards 5mgmL of MTTwas added with 20120583L for each well and further culturedfor 4 h After the media were removed 015mL of DMSOwas added to each well and shaken for 10min for completemixture at room temperature for 10min before ELX-800(BioTek Instruments Inc) was used to measure absorbanceat 570 nm Consider

Proliferation ratio of lymphocytes ()

= [

119860 sample minus 119860control

119860control] times 100

(1)

where 119860 sample is the absorbance of tested sample and 119860controlis the absorbance of control group

224 Proliferation ofMacrophages 2 (wv) peptone of 2mLwas injected into the micersquos peritoneal cavity every day forthree days After cervical dislocation precooled Hankrsquos of5mL was injected into the peritoneal cavity then abdominalfluid was sucked and the supernatant was removed bycentrifugation at 2000 rpm the precipitate cells were washedtwice1 times 10

6mL of cell suspension was made with 10 ofcalf serum in RPMI 1640 incubated in the tissue cultureplate with 100 120583L for each well and cultured at 37∘C in 5CO2incubators for 2 h The upper medium was removed

and the wells were washed with PBS twice to remove thenonadherent cells somacrophagemonolayers were obtainedFresh medium was put into the 96-well plates following withfresh medium of 100 120583L to the control group 100120583L LPSto the positive group (with final concentration of 2 120583gmL)and 100 120583L different samples of different concentrationsto the other groups (the sample concentrations are as inSection 223) and cultured at 37∘C in 5 CO

2incubators

for 24 h Afterwards upper medium was removed and newmedium was added with 100 120583L for each hole as well as5mgmL of MTT with 20120583L further cultured for 4 h thenthe supernatant was carefully sucked and 150120583L DMSO wasadded before shaking for 10min for complete mixture After

Journal of Chemistry 3

placing at room temperature for 10min ELX-800 was used tomeasure absorbance at 570 nm Consider

Proliferation ratio of macrophages ()

= [

119860 sample minus 119860control

119860control] times 100

(2)

where 119860 sample is the absorbance of tested sample and 119860controlis the absorbance of control group

225 NO Release Activities of Macrophages Macrophageswere prepared with the same method as in Section 22425times10

5mL of cell suspension was made with 10 of calfserum in RPMI 1640 in sterile conditions inoculated on thetissue culture plate with 100 120583L for each well and three wellsfor each sample were cultured at 37∘C in 5 CO

2incubators

for 2 h Then LPS solution (with final concentration of2 120583gmL) of 100 120583L was put into the positive group medium1640 of 100 120583L was added to the control group and 100 120583Lwith different samples of different concentrations was addedto the other groups and further cultured for 48 h Afterwardsupper medium of 50 120583L from each well was removed andtransferred to a new 96-well plate and measured accordingto the following method

Standard Curve Standard solution nitrite of 01molL wasdiluted in 1 1000 with medium A1simH3 on the tissue cultureplate were used for standard curve Standard solution nitriteof 100 120583L was put into A1simA3 respectively and medium of50120583Lwas put into B1-H3 respectively Doubling dilution wasconducted to AndashG in turn and the final fluid from G wasremoved and nitrite concentration coefficient (100 50 25125 625 313 156 and 0 120583molL) was obtained Standardcurve was necessary before each test with the same mediumas for the samples

Measurement NED solution and sulfanilamide solution werekept at room temperature for 15 to 30min Each of testedsamples of 100 120583L was placed on the tissue culture plate 2 to3 repeated wells were used for every sample Sulfanilamidesolution of 50120583L was put into all standard curve wells andsample wells were kept in dark place at room temperature for5 to 10min and then the same was done with NED solutionof 50120583L Absorbance at 540 nm was measured within 30minand the nitrite concentration in the samples was calculatedaccording to standard curve

226 In Vitro Antioxidant Activity

(1) DPPH Radical Scavenging Assay Modified Binsanrsquosmethod [15] was adopted 15mL sample in different concen-trations was added to 15mL of 02mM DPPH in ethanol

15mL DPPH of 02mmol in anhydrous ethanol wasadded to all the samples of 15mL after mixing vigorouslyand kept at room temperature in dark for 30min Theabsorbance of the resulting solution was measured at 517 nmwithwater as the control and the ability to scavenge theDPPH

radical was expressed by clearance rate (119864) in the followingformula

119864 () = [1 minus119860 sample minus 119860 sample blank

119860control] times 100 (3)

where 119860 sample means the absorbance of test sample solution(DPPH + sample) 119860 sample blank means the absorbance of thesample only (ethanol + sample) and 119860control means theabsorbance of control (DPPH + ethanol)

The half clean concentration (EC50) is calculated by

regression equation of E and sample concentration

(2) 120573-Carotene-Linoleic Acid System Modified Jayaprakasharsquosmethod [16] was adopted 120573-carotene of 02mg was dissolvedin 02mL trichloromethane into which linoleic acid of 20mgand Tween-40 of 200mg were added and mixed completelyAfter chloroform was removed under vacuum at 40∘Cdistilled water of 50mL (oxygenated for 2-3min) was addedThe reaction emulsion was prepared after vigorous agitationThe control was prepared as above except for the addition of120573-carotene Sample solution of 50120583L and reaction mediumof 200120583L were added into 96-well plates respectively andthe absorbance was measured at 450 nm immediately (119905 =0) and remeasured every 20min until the color of 120573-carotene faded in the control (about 240min) Three parallelmeasurements were conducted and the antioxidant activity(AA) was expressed in the following formula

AA = [1 minus119860

0minus 119860

119905

119860

1015840

0minus 119860

1015840

119905

] times 100 (4)

where 1198600and 119860

119905were the absorbance of the sample while

119905 = 0 and 119905 = 240min respectively and 11986010158400and 1198601015840

119905were

the absorbance of the control while 119905 = 0 and 119905 = 240minrespectively

(3) Reducing Power The reducing power was determinedaccording to Yen and Duhrsquos method [17] All samples weredissolved with phosphate buffer solution (02molL pH 66)of 25mL and 1 potassium ferricyanide (wv) of 25mLwas added before reacting at 50∘C for 20min then 10trichloroacetic acid (wv) of 25mL was added to stopreaction Reaction mixture was centrifuged at 650timesg for10min supernatant of 25mL was mixed with distilled waterof 25mL and 01 FeCl

3(wv) of 05mLThe absorbance was

measured at 700 nm and the higher absorbance means thestronger reducing power The reducing power is convertedin ascorbic acid and expressed as ascorbic acid equivalent(AscAE) in milligrammes of ascorbic acid per gramme ofsample (AscAE mgg AscA)

23 Statistical Analysis All data were expressed by mean plusmnstandard deviation All statistical analyses were carried outusing SPSS 130 for Windows Tukeyrsquos multiple comparisontests were used to detect differences between groups and aprobability of 119875 lt 005 was taken as an acceptable level ofsignificance

4 Journal of Chemistry

0

10

20

30

40

50

60

Prol

ifera

tion

rate

()

BU PE CE ConAET

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmLPositive control

lowast

lowast

minus10

minus20

lowastlowast

Figure 1 Effects of extracts on the proliferation of the mousesplenocytes lowast119875 lt 005 and lowastlowast119875 lt 001 compared with the control

3 Results and Discussion

31 Proliferation of Spleen Lymphocyte As organismrsquos firstline defending tumor immune system plays an importantrole in the recognition and removal of malignant cells Theimmune status evaluation of the body bearing neoplasm isimportant for mechanism study of antitumor drugs

As shown in Figure 1 four extracts of S clava demonstratesignificant activity on splenocytes proliferation (comparedwith the control 119875 lt 005 or 119875 lt 001) amongwhich the proliferation effect of PE is the most activewith proliferation rate being near 50 at a concentration of25 120583gmL close to the positive control group ConA (5671)However its proliferation rate reduces with the increasingconcentration and the proliferation rate is only 476 whenthe concentration reaches 400 120583gmL which shows that PEinhibits the proliferation of the mouse splenocytes at highconcentration CE is less active than PE on the proliferationof the mouse splenocytes demonstrating a rising trend atfirst and then a falling one The effects of ET and BUare relatively weak and ET shows distinctive inhibition at50120583gmL and 100120583gmL with the inhibition rate of 1262at 100 120583gmL but on the contrary BU reaches the strongestlevel at this concentration with the proliferation rate of1452 while distinctive inhibition was shown below thispoint

32 Proliferation of Macrophages Figure 2 shows the effectsof S clavarsquos extracts on the proliferation of the mouseperitonealmacrophagesThe four extracts all show significanteffect on the proliferation of the mouse macrophages at alldesigned concentrations (compared with the control 119875 lt005 or 119875 lt 001) but it is weaker than that of thepositive control LPS as proliferation rate of 6319 PE is themost active extract on the proliferation at concentration of

05

101520253035404550556065

Prol

ifera

tion

rate

()

ET BU PE CE LPS

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmLPositive control

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

Figure 2 Effects of extracts on the proliferation of the mouseperitoneal macrophages lowast119875 lt 005 and lowastlowast119875 lt 001 compared withthe control

25 120583gmL with proliferation rate 4070 but afterwards theproliferation rate dropswith the increasing concentration andthe proliferation rate is only 1085 when the concentrationreaches 400120583gmL showing similar trend as on the lympho-cytes

ET and CE are close on the effect of proliferation ETreaches the lowest point at 25120583gmL with the proliferationrate of 194 and the highest point at 200120583gmL with theproliferation rate of 2597 CE shows similar trend asET reaching the highest and lowest points at 100 120583gmLand 400120583gmL with the proliferation rate of 2519 and1017 respectively BU shows the weakest effect with thehighest proliferation rate of only 1008 at 200120583gmL andthe same effect as the control at 25120583gmL the prolifer-ation rates at all the other concentrations are all below10

33 NO Release Activities of Macrophages Macrophages arethe important components in the body immune systemfunctioning as immunization effect regulating the immunesystem and NO is one of the active agents produced bymacrophages after they are activated [18] Figure 3 showseffects of extract from S clava on the NO release activitiesof mouse macrophages The effects of ET PE and CE are allin positive correlation with concentration in promoting therelease of NO PE shows the strongest effect among themreaching the highest point of 1614 120583molmL at 400 120583gmLCE is less effective with NO releasing the highest point of1476 120583molmL at 400 120583gmL The effects of BU and ET arerather weak only 870120583molmL for ET at 400120583gmL as thehighest point while at the other concentrations the NOreleasing abilities for ET and BU are close to and approaching6 120583molmL

Journal of Chemistry 5

Table 1 DPPH scavenging activity of different components isolated from S clava

Component DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

CE 192 plusmn 037 3012 plusmn 060 379 plusmn 064 426 plusmn 088

PE 483 plusmn 011 1011 plusmn 022 1424 plusmn 038 1850 plusmn 037

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

BU 838 plusmn 021 1929 plusmn 037 2532 plusmn 049 3077 plusmn 062

AR 210 plusmn 005 305 plusmn 011 426 plusmn 008 489 plusmn 010

Table 2 DPPH scavenging activity of subcomponents E1ndashE7 isolated from ET of S clava

Subcomponent DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

E1 824 plusmn 026 1417 plusmn 040 1864 plusmn 059 2080 plusmn 065

E2 1137 plusmn 038 1819 plusmn 088 2430 plusmn 162 2918 plusmn 125

E3 1846 plusmn 064 2530 plusmn 124 3428 plusmn 122 4580 plusmn 099

E4 2280 plusmn 032 3820 plusmn 102 4937 plusmn 157 5678 plusmn 101

E5 3026 plusmn 088 4620 plusmn 092 5916 plusmn 111 6731 plusmn 165

E6 2492 plusmn 077 4200 plusmn 106 5340 plusmn 070 6442 plusmn 162

E7 1400 plusmn 031 2029 plusmn 066 2713 plusmn 048 3250 plusmn 101

0

2

4

6

8

10

12

14

16

18

ET BU PE CE

NO

(120583m

olm

L)

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Figure 3 Effects of extracts on NO production lowast119875 lt 005 andlowastlowast119875 lt 001 compared with the control

34 In Vitro Antioxidant Activity

341 DPPH Radical Scavenging Assay The DPPH free rad-icals scavenging method is a colorimetric assay and can beused to evaluate the radical scavenging activity in a shorttime DPPH is an organic free radical with maximal absorp-tion at 517 nmWhenwith free radical scavengerDPPHrsquos loneelectronwill be paired and its absorbancewill decrease whichcan be used to evaluate its antioxidant activity As shown

in Table 1 there was a positive correlation between DPPHscavenging activity of CE and its concentration Among thefour extracted components PE and BU demonstrate poorscavenging activity while AR shows no scavenging activityThe scavenging activity of ET reaches 5064 at 200 120583gmLmuch higher than CE (119875 lt 005) indicating that compoundswithDPPH scavenging activity in S clava aremostlymediumpolar

E1ndashE7 subcomponents are isolated from ETwith silica gelcolumn chromatography whose DPPH scavenging activity isshown in Table 2 At all experiment concentrations E5 showsthe strongest scavenging activity followed by E6 E4 E3 E7and E2 with E1 the weakest E5 and E6 show much strongerscavenging activity than ET (119875 lt 005) while E4 is close toET in scavenging activity EC

50of E5 and E6 are 12206120583gmL

and 13968 120583gmL respectively

342 Antioxidant Activity in 120573-Carotene-Linoleic Acid Sys-tem 120573-carotene is a polyene pigment apt to be oxidized tofade In the reaction medium the superoxide produced bythe oxidized linoleic acid results in 120573-carotenersquos fading andas time goes on the absorbance decreases The degree of120573-carotenersquos fading depends on the antioxidant activity inthe system Table 3 shows antioxidant activities of differentcomponents isolated from S clava in the 120573-carotene-linoleicacid system CE has some antioxidation activities positivelycorrelated with its concentration ET is much higher in itsantioxidant activity than other components reaching 50 at200120583gmL and is even higher than the positive control AscAat all experimental concentrations which further indicatesthat compounds with higher antioxidant activity in the120573-carotene-linoleic acid system are mostly medium-polarcomponents

6 Journal of Chemistry

Table 3 Antioxidant activity of different components isolated from S clava in the 120573-carotene-linoleic acid system

Component Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

CE 1062 plusmn 038 2010 plusmn 112 2948 plusmn 130 4035 plusmn 112

PE 460 plusmn 021 1022 plusmn 044 1650 plusmn 049 2240 plusmn 068

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

BU 720 plusmn 048 1439 plusmn 062 2260 plusmn 077 2600 plusmn 089

AR 215 plusmn 005 690 plusmn 014 820 plusmn 018 980 plusmn 011

AscA 1050 plusmn 012 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

Table 4 Antioxidant activity of subcomponents E1ndashE7 isolated from ET in the 120573-carotene-linoleic acid system

Subcomponent Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

E1 646 plusmn 050 1380 plusmn 046 2820 plusmn 064 2219 plusmn 097

E2 742 plusmn 031 1540 plusmn 074 2474 plusmn 107 3090 plusmn 121

E3 930 plusmn 037 1778 plusmn 087 2741 plusmn 103 3277 plusmn 127

E4 1238 plusmn 067 2472 plusmn 118 3270 plusmn 079 4365 plusmn 198

E5 2024 plusmn 144 4060 plusmn 125 5410 plusmn 157 6892 plusmn 167

E6 1520 plusmn 079 3440 plusmn 085 4852 plusmn 171 6236 plusmn 168

E7 1070 plusmn 047 2010 plusmn 027 2842 plusmn 080 3680 plusmn 098

AscA 1050 plusmn 072 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

GA 2180 plusmn 041 4280 plusmn 188 5861 plusmn 120 7010 plusmn 146

Table 5 Reducing power of extract and subcomponents E1ndashE7isolated from ET

Component AscAE (mggAscA)CE 4020 plusmn 110

PE 1432 plusmn 042

ET 6260 plusmn 155

BU 2660 plusmn 051

WT 572 plusmn 026

E1 2040 plusmn 077

E2 2820 plusmn 122

E3 4620 plusmn 118

E4 6160 plusmn 111

E5 8440 plusmn 210

E6 7520 plusmn 150

E7 4160 plusmn 095

Theantioxidant activity of subcomponents E1ndashE7 isolatedfrom ET in the 120573-carotene-linoleic acid system is shownin Table 4 At all experiment concentrations E5 shows thestrongest antioxidant activity followed by E6 E4 E7 E3 andE2 with E1 being the weakest Among the seven subcom-ponents E5 and E6 show much stronger antioxidant activitythan ET (119875 lt 005) while the antioxidant activity of E5 is6892at 200120583gmL close to the synthesized antioxidantGA

343 Reducing Power Table 5 shows the reducing power ofthe extract from S clava and subcomponents E1ndashE7 isolated

from ET The reducing powers of all the samples demon-strate a pattern similar to DPPH scavenging activity andantioxidant activity in the 120573-carotene-linoleic acid systemAmong all the extracted components ET is the highest inthe reducing power (119875 lt 005) and the reducing power ofthe seven subcomponents isolated fromET is in the followingorder E5 gt E6 gt E4 gt E3 gt E7 gt E2 gt E1

35 Structural Identification of Compound Compound 1colorless needle crystal is identified as C

27H46O with EI-MS

and NMR 120575347 in 1H NMR 120575718 in 13C NMR and DEPTshow that there is hydroxyl in themolecule 120575531 in 1HNMR12057514018 and 1205751217 in 13C NMR and DEPT further confirmthe double bond in the molecule mz 255 and 147 in MScombinedwith 120575531 (1H H-7) 120575356 (1HmH-3) 120575101 (3Hs Me-19) and 120575088 (3H s Me-18) in 1HMMR 1205751408 (s C-8) 1205751217 (d C-7) and 120575718 (d C-3) in 13CNMR and DEPTreferred to the physical constant and spectral data reportedby Tsuda and Schroepfer Jr [19] compound 1 was identifiedas cholesteric-7-en-3120573-ol whichwas isolated from S clava forthe first time

Compound 2 colorless needle crystal mp 244sim246∘C isidentified as C

27H46O2with EI-MS andNMRThe two signals

of methyl group connected with quaternary carbon (120575126 s120575071 s) and three signals of methyl group connected withtertiary carbon (120575091 d 120575087 d 120575085 d) show that thiscompound is sterol The signal of 19-Me at 120575126 shifts toa lower field in contrast to cholesterol which proves theexistence of 6120573-OH Meanwhile in contrast to cholesterolsignal (120575418) of 3120572-H also shifts to a lower field and so is

Journal of Chemistry 7

HO

HO

HO

OH

OH

OH

OH

OH

H2C

H2C (CH2)17CH3

C

O

O

(CH2)16CH3

CH3(CH2)14

NH

HC

Compound 1 Compound 2

Compound 3 Compound 4

Compound 5

Figure 4 Structures of compounds

proved of 45-double bond considering single peak 120575555Compound 2 is identified as cholesteric-4-en-3120573 6120573-diolreferring to the physical constant and spectral data reportedby Wahidullah et al [20] which is isolated from S clava forthe first time

Compound 3 is a white needle crystal mp 148sim149∘Cpurplish red at 10 sulfuric acid-ethanol solution The mul-tiple peaks at 120575353 in 1H NMR is the characteristic signal of3120572-H in sterol while the multiple peaks at 120575535 is signal ofH-6 In addition five methyl group signals appear in highfield of 1H NMR among which the 120575101 s and 120575068 sbelong to 19-Me and 18-Me respectively 120575091 d is the signalof 21-Me and 120575084 d and 120575082 d belong to 26-Me and27-Me respectively Contrasting the physical constant andspectral data with Su et al [21] compound 3 is identified ascholesterol

Compound 4 is white powder mp 68sim69∘C purplish redat 10 sulfuric acid-ethanol solution The molecular weight

is 345 according to EI-MS As shown in 13C NMR spectrum120575641 (t C-1) 120575319 (t C2) 120575296 (t C3-C15) 120575260 (t C16)120575226 (t C17) and 120575141 (t C18) are characteristic signalsof long chain fatty acid and 120575722 (C-1) 120575706 (C-2) and120575718 (C-3) belong to glycerol so this compound should bea long chain triglyceride However there is no signal of estercarbonyl therefore this compound is identified as glycidylether agreeing with Yang et al [22] andWang et al [23] andis confirmed as batilol which is isolated from S clava for thefirst time

Compound 5 molecular formula is C36H71NO3accord-

ing to EI-MS andNMR 120575637 (1H) in 1HNMR spectrum and1205751739 (C=O) in 13C NMR spectrum proves the existence ofacylamino and 120575545 in 13C NMR spectrum indicates thatwhat is connected with nitrogen is methyne 120575202 (2H) in1HNMR indicates that there are two ndashOH in the compoundand 1205757474 and 1205756240 in 13C NMR indicate that ndashCHndash andndashCH2ndash connect with ndashOH respectively Meantime a strong

8 Journal of Chemistry

hydrogen signal of 120575126 in 1H NMR spectrum and twosignals ndashCH

3ndash of 120575084 (6H) determine two long chain alkyl

groups This compound is identified as ceramide referring tothe physical constant and spectral data reported by Yu andYang [24]

The structures of these compounds are shown in Figure 4As reported by Cai et al [25] the number and positionof hydroxyl groups largely determined radical scavengingactivity of phenolic compounds Compounds 1 2 and 3 allhave only one phenolic hydroxyl group and there is nofunctional groups in orthoposition little steric hindranceeffect increased their contact with free radicals

4 Conclusion

The immunomodulatory and antioxidant activity of crudeand fractionated extracts of the ascidian Styela clava weredetermined by in vitro screening All these extracts demon-strate immunomodulatory activity through increasing theproliferation rate of spleen lymphocyte and macrophagesas well as the NO release activities of macrophages Amongthem the petroleum ether fraction shows the strongestimmune active in vitro The ethyl acetate fraction (ET) wasmuch higher in its antioxidant activity in DPPH systemreducing power assay and 120573-carotene-linoleic acid systemcompared with the other fractions and its subcomponentE5 demonstrated the strongest antioxidant activity as well asreducing power higher than the positive control AscA andclose to the synthesized GA ET was isolated systemically andfive compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol (4) batiloland (5) ceramide among which (1) (2) and (4) were isolatedfor the first time from S clava

Conflict of Interests

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

Authorsrsquo Contribution

Ju Bao and Chen Bin contributed equally to this project

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (no 31070368) and the Sci-ence and Technology Department of Shandong Province(BS2010HZ022 and ZR2011DL008)

References

[1] T M Zabriskie and C M Ireland ldquoThe isolation and structureofmodified bioactive nucleosides from Jaspis johnstonirdquo Journalof Natural Products vol 52 no 6 pp 1353ndash1356 1989

[2] J Kobayashi M Tsuda M Ishibashi et al ldquoAmphidinolideF a new cytotoxic macrolide from the marine dinoflagellateAmphidinium sprdquo Journal of Antibiotics vol 44 no 11 pp 1259ndash1261 1991

[3] B S Davidson and C M Ireland ldquoLissoclinolide the first non-nitrogenous metabolite from a Lissoclinum tunicaterdquo Journal ofNatural Products vol 53 no 4 pp 1036ndash1038 1990

[4] T N Makarieva V A Stonik A S Dmitrenok et al ldquoVaracinand three new marine antimicrobial polysulfides from the far-Eastern ascidian Polycitor sprdquo Journal of Natural Products vol58 no 2 pp 254ndash258 1995

[5] P W Ford and B S Davidson ldquoPlakinidine D a newpyrroloacridine alkaloid from the ascidianDidemnum rubeumrdquoJournal of Natural Products vol 60 no 10 pp 1051ndash1053 1997

[6] C Ireland and P J Scheuer ldquoUlicyclamine and ulithiacy-clamide two new small peptides from a marine tunicaterdquoJournal of the American Chemical Society vol 102 no 17 pp5688ndash5691 1980

[7] H G Chun B Davies and D Hoth ldquoDidemnin B the firstmarine compound entering clinical trials as an antineoplasticagentrdquo Investigational New Drugs vol 4 no 3 pp 279ndash2841986

[8] M A Baker D R Grubb and A Lawen ldquoDidemnin B inducesapoptosis in proliferating but not resting peripheral bloodmononuclear cellsrdquo Apoptosis vol 7 no 5 pp 407ndash412 2002

[9] M D Vera and M M Joullie ldquoNatural products as probes ofcell biology 20 Years of didemnin researchrdquoMedicinal ResearchReviews vol 22 no 2 pp 102ndash145 2002

[10] S Jin B Gorfajn G Faircloth and K W Scotto ldquoEcteinascidin743 a transcription-targeted chemotherapeutic that inhibitsMDR1 activationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 12 pp 6775ndash6779 2000

[11] C van Kesteren E Cvitkovic A Taamma et al ldquoPharma-cokinetics and pharmacodynamics of the novel marine-derivedanticancer agent ecteinascidin 743 in a phase I dose-findingstudyrdquo Clinical Cancer Research vol 6 no 12 pp 4725ndash47322000

[12] E G C Brain ldquoSafety and efficacy of ET-743 the Frenchexperiencerdquo Anti-Cancer Drugs vol 13 supplement 1 pp S11ndashS14 2002

[13] J C M Waterval J C Bloks R W Sparidans et al ldquoDegra-dation kinetics of aplidine a new marine antitumoural cyclicpeptide in aqueous solutionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 754 no 1 pp 161ndash1682001

[14] C K Cai Research of anti-HBV activity and pharmacodynamicaction and toxicity of SC [PhD thesis] Beijing University ofChinese Medicine Beijing China 2006

[15] W Binsan S Benjakul W Visessanguan S Roytrakul MTanaka and H Kishimura ldquoAntioxidative activity of Mun-goong an extract paste from the cephalothorax ofwhite shrimp(Litopenaeus vannamei)rdquo Food Chemistry vol 106 no 1 pp185ndash193 2008

[16] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[17] G C Yen and P D Duh ldquoAntioxidative properties of methano-lic extracts from peanut hullsrdquo Journal of the American OilChemistsrsquo Society vol 70 no 4 pp 383ndash386 1993

[18] R N Albrecht Immunologie Tolerance and Macrophage Func-tion Aeademie New York NY USA 1 edition 1979

[19] M Tsuda and G J Schroepfer Jr ldquoCarbon-13 nuclear magneticresonance studies of C27 sterol precursors of cholesterolrdquoJournal of Organic Chemistry vol 44 no 8 pp 1290ndash1293 1979

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 3

placing at room temperature for 10min ELX-800 was used tomeasure absorbance at 570 nm Consider

Proliferation ratio of macrophages ()

= [

119860 sample minus 119860control

119860control] times 100

(2)

where 119860 sample is the absorbance of tested sample and 119860controlis the absorbance of control group

225 NO Release Activities of Macrophages Macrophageswere prepared with the same method as in Section 22425times10

5mL of cell suspension was made with 10 of calfserum in RPMI 1640 in sterile conditions inoculated on thetissue culture plate with 100 120583L for each well and three wellsfor each sample were cultured at 37∘C in 5 CO

2incubators

for 2 h Then LPS solution (with final concentration of2 120583gmL) of 100 120583L was put into the positive group medium1640 of 100 120583L was added to the control group and 100 120583Lwith different samples of different concentrations was addedto the other groups and further cultured for 48 h Afterwardsupper medium of 50 120583L from each well was removed andtransferred to a new 96-well plate and measured accordingto the following method

Standard Curve Standard solution nitrite of 01molL wasdiluted in 1 1000 with medium A1simH3 on the tissue cultureplate were used for standard curve Standard solution nitriteof 100 120583L was put into A1simA3 respectively and medium of50120583Lwas put into B1-H3 respectively Doubling dilution wasconducted to AndashG in turn and the final fluid from G wasremoved and nitrite concentration coefficient (100 50 25125 625 313 156 and 0 120583molL) was obtained Standardcurve was necessary before each test with the same mediumas for the samples

Measurement NED solution and sulfanilamide solution werekept at room temperature for 15 to 30min Each of testedsamples of 100 120583L was placed on the tissue culture plate 2 to3 repeated wells were used for every sample Sulfanilamidesolution of 50120583L was put into all standard curve wells andsample wells were kept in dark place at room temperature for5 to 10min and then the same was done with NED solutionof 50120583L Absorbance at 540 nm was measured within 30minand the nitrite concentration in the samples was calculatedaccording to standard curve

226 In Vitro Antioxidant Activity

(1) DPPH Radical Scavenging Assay Modified Binsanrsquosmethod [15] was adopted 15mL sample in different concen-trations was added to 15mL of 02mM DPPH in ethanol

15mL DPPH of 02mmol in anhydrous ethanol wasadded to all the samples of 15mL after mixing vigorouslyand kept at room temperature in dark for 30min Theabsorbance of the resulting solution was measured at 517 nmwithwater as the control and the ability to scavenge theDPPH

radical was expressed by clearance rate (119864) in the followingformula

119864 () = [1 minus119860 sample minus 119860 sample blank

119860control] times 100 (3)

where 119860 sample means the absorbance of test sample solution(DPPH + sample) 119860 sample blank means the absorbance of thesample only (ethanol + sample) and 119860control means theabsorbance of control (DPPH + ethanol)

The half clean concentration (EC50) is calculated by

regression equation of E and sample concentration

(2) 120573-Carotene-Linoleic Acid System Modified Jayaprakasharsquosmethod [16] was adopted 120573-carotene of 02mg was dissolvedin 02mL trichloromethane into which linoleic acid of 20mgand Tween-40 of 200mg were added and mixed completelyAfter chloroform was removed under vacuum at 40∘Cdistilled water of 50mL (oxygenated for 2-3min) was addedThe reaction emulsion was prepared after vigorous agitationThe control was prepared as above except for the addition of120573-carotene Sample solution of 50120583L and reaction mediumof 200120583L were added into 96-well plates respectively andthe absorbance was measured at 450 nm immediately (119905 =0) and remeasured every 20min until the color of 120573-carotene faded in the control (about 240min) Three parallelmeasurements were conducted and the antioxidant activity(AA) was expressed in the following formula

AA = [1 minus119860

0minus 119860

119905

119860

1015840

0minus 119860

1015840

119905

] times 100 (4)

where 1198600and 119860

119905were the absorbance of the sample while

119905 = 0 and 119905 = 240min respectively and 11986010158400and 1198601015840

119905were

the absorbance of the control while 119905 = 0 and 119905 = 240minrespectively

(3) Reducing Power The reducing power was determinedaccording to Yen and Duhrsquos method [17] All samples weredissolved with phosphate buffer solution (02molL pH 66)of 25mL and 1 potassium ferricyanide (wv) of 25mLwas added before reacting at 50∘C for 20min then 10trichloroacetic acid (wv) of 25mL was added to stopreaction Reaction mixture was centrifuged at 650timesg for10min supernatant of 25mL was mixed with distilled waterof 25mL and 01 FeCl

3(wv) of 05mLThe absorbance was

measured at 700 nm and the higher absorbance means thestronger reducing power The reducing power is convertedin ascorbic acid and expressed as ascorbic acid equivalent(AscAE) in milligrammes of ascorbic acid per gramme ofsample (AscAE mgg AscA)

23 Statistical Analysis All data were expressed by mean plusmnstandard deviation All statistical analyses were carried outusing SPSS 130 for Windows Tukeyrsquos multiple comparisontests were used to detect differences between groups and aprobability of 119875 lt 005 was taken as an acceptable level ofsignificance

4 Journal of Chemistry

0

10

20

30

40

50

60

Prol

ifera

tion

rate

()

BU PE CE ConAET

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmLPositive control

lowast

lowast

minus10

minus20

lowastlowast

Figure 1 Effects of extracts on the proliferation of the mousesplenocytes lowast119875 lt 005 and lowastlowast119875 lt 001 compared with the control

3 Results and Discussion

31 Proliferation of Spleen Lymphocyte As organismrsquos firstline defending tumor immune system plays an importantrole in the recognition and removal of malignant cells Theimmune status evaluation of the body bearing neoplasm isimportant for mechanism study of antitumor drugs

As shown in Figure 1 four extracts of S clava demonstratesignificant activity on splenocytes proliferation (comparedwith the control 119875 lt 005 or 119875 lt 001) amongwhich the proliferation effect of PE is the most activewith proliferation rate being near 50 at a concentration of25 120583gmL close to the positive control group ConA (5671)However its proliferation rate reduces with the increasingconcentration and the proliferation rate is only 476 whenthe concentration reaches 400 120583gmL which shows that PEinhibits the proliferation of the mouse splenocytes at highconcentration CE is less active than PE on the proliferationof the mouse splenocytes demonstrating a rising trend atfirst and then a falling one The effects of ET and BUare relatively weak and ET shows distinctive inhibition at50120583gmL and 100120583gmL with the inhibition rate of 1262at 100 120583gmL but on the contrary BU reaches the strongestlevel at this concentration with the proliferation rate of1452 while distinctive inhibition was shown below thispoint

32 Proliferation of Macrophages Figure 2 shows the effectsof S clavarsquos extracts on the proliferation of the mouseperitonealmacrophagesThe four extracts all show significanteffect on the proliferation of the mouse macrophages at alldesigned concentrations (compared with the control 119875 lt005 or 119875 lt 001) but it is weaker than that of thepositive control LPS as proliferation rate of 6319 PE is themost active extract on the proliferation at concentration of

05

101520253035404550556065

Prol

ifera

tion

rate

()

ET BU PE CE LPS

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmLPositive control

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

Figure 2 Effects of extracts on the proliferation of the mouseperitoneal macrophages lowast119875 lt 005 and lowastlowast119875 lt 001 compared withthe control

25 120583gmL with proliferation rate 4070 but afterwards theproliferation rate dropswith the increasing concentration andthe proliferation rate is only 1085 when the concentrationreaches 400120583gmL showing similar trend as on the lympho-cytes

ET and CE are close on the effect of proliferation ETreaches the lowest point at 25120583gmL with the proliferationrate of 194 and the highest point at 200120583gmL with theproliferation rate of 2597 CE shows similar trend asET reaching the highest and lowest points at 100 120583gmLand 400120583gmL with the proliferation rate of 2519 and1017 respectively BU shows the weakest effect with thehighest proliferation rate of only 1008 at 200120583gmL andthe same effect as the control at 25120583gmL the prolifer-ation rates at all the other concentrations are all below10

33 NO Release Activities of Macrophages Macrophages arethe important components in the body immune systemfunctioning as immunization effect regulating the immunesystem and NO is one of the active agents produced bymacrophages after they are activated [18] Figure 3 showseffects of extract from S clava on the NO release activitiesof mouse macrophages The effects of ET PE and CE are allin positive correlation with concentration in promoting therelease of NO PE shows the strongest effect among themreaching the highest point of 1614 120583molmL at 400 120583gmLCE is less effective with NO releasing the highest point of1476 120583molmL at 400 120583gmL The effects of BU and ET arerather weak only 870120583molmL for ET at 400120583gmL as thehighest point while at the other concentrations the NOreleasing abilities for ET and BU are close to and approaching6 120583molmL

Journal of Chemistry 5

Table 1 DPPH scavenging activity of different components isolated from S clava

Component DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

CE 192 plusmn 037 3012 plusmn 060 379 plusmn 064 426 plusmn 088

PE 483 plusmn 011 1011 plusmn 022 1424 plusmn 038 1850 plusmn 037

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

BU 838 plusmn 021 1929 plusmn 037 2532 plusmn 049 3077 plusmn 062

AR 210 plusmn 005 305 plusmn 011 426 plusmn 008 489 plusmn 010

Table 2 DPPH scavenging activity of subcomponents E1ndashE7 isolated from ET of S clava

Subcomponent DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

E1 824 plusmn 026 1417 plusmn 040 1864 plusmn 059 2080 plusmn 065

E2 1137 plusmn 038 1819 plusmn 088 2430 plusmn 162 2918 plusmn 125

E3 1846 plusmn 064 2530 plusmn 124 3428 plusmn 122 4580 plusmn 099

E4 2280 plusmn 032 3820 plusmn 102 4937 plusmn 157 5678 plusmn 101

E5 3026 plusmn 088 4620 plusmn 092 5916 plusmn 111 6731 plusmn 165

E6 2492 plusmn 077 4200 plusmn 106 5340 plusmn 070 6442 plusmn 162

E7 1400 plusmn 031 2029 plusmn 066 2713 plusmn 048 3250 plusmn 101

0

2

4

6

8

10

12

14

16

18

ET BU PE CE

NO

(120583m

olm

L)

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Figure 3 Effects of extracts on NO production lowast119875 lt 005 andlowastlowast119875 lt 001 compared with the control

34 In Vitro Antioxidant Activity

341 DPPH Radical Scavenging Assay The DPPH free rad-icals scavenging method is a colorimetric assay and can beused to evaluate the radical scavenging activity in a shorttime DPPH is an organic free radical with maximal absorp-tion at 517 nmWhenwith free radical scavengerDPPHrsquos loneelectronwill be paired and its absorbancewill decrease whichcan be used to evaluate its antioxidant activity As shown

in Table 1 there was a positive correlation between DPPHscavenging activity of CE and its concentration Among thefour extracted components PE and BU demonstrate poorscavenging activity while AR shows no scavenging activityThe scavenging activity of ET reaches 5064 at 200 120583gmLmuch higher than CE (119875 lt 005) indicating that compoundswithDPPH scavenging activity in S clava aremostlymediumpolar

E1ndashE7 subcomponents are isolated from ETwith silica gelcolumn chromatography whose DPPH scavenging activity isshown in Table 2 At all experiment concentrations E5 showsthe strongest scavenging activity followed by E6 E4 E3 E7and E2 with E1 the weakest E5 and E6 show much strongerscavenging activity than ET (119875 lt 005) while E4 is close toET in scavenging activity EC

50of E5 and E6 are 12206120583gmL

and 13968 120583gmL respectively

342 Antioxidant Activity in 120573-Carotene-Linoleic Acid Sys-tem 120573-carotene is a polyene pigment apt to be oxidized tofade In the reaction medium the superoxide produced bythe oxidized linoleic acid results in 120573-carotenersquos fading andas time goes on the absorbance decreases The degree of120573-carotenersquos fading depends on the antioxidant activity inthe system Table 3 shows antioxidant activities of differentcomponents isolated from S clava in the 120573-carotene-linoleicacid system CE has some antioxidation activities positivelycorrelated with its concentration ET is much higher in itsantioxidant activity than other components reaching 50 at200120583gmL and is even higher than the positive control AscAat all experimental concentrations which further indicatesthat compounds with higher antioxidant activity in the120573-carotene-linoleic acid system are mostly medium-polarcomponents

6 Journal of Chemistry

Table 3 Antioxidant activity of different components isolated from S clava in the 120573-carotene-linoleic acid system

Component Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

CE 1062 plusmn 038 2010 plusmn 112 2948 plusmn 130 4035 plusmn 112

PE 460 plusmn 021 1022 plusmn 044 1650 plusmn 049 2240 plusmn 068

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

BU 720 plusmn 048 1439 plusmn 062 2260 plusmn 077 2600 plusmn 089

AR 215 plusmn 005 690 plusmn 014 820 plusmn 018 980 plusmn 011

AscA 1050 plusmn 012 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

Table 4 Antioxidant activity of subcomponents E1ndashE7 isolated from ET in the 120573-carotene-linoleic acid system

Subcomponent Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

E1 646 plusmn 050 1380 plusmn 046 2820 plusmn 064 2219 plusmn 097

E2 742 plusmn 031 1540 plusmn 074 2474 plusmn 107 3090 plusmn 121

E3 930 plusmn 037 1778 plusmn 087 2741 plusmn 103 3277 plusmn 127

E4 1238 plusmn 067 2472 plusmn 118 3270 plusmn 079 4365 plusmn 198

E5 2024 plusmn 144 4060 plusmn 125 5410 plusmn 157 6892 plusmn 167

E6 1520 plusmn 079 3440 plusmn 085 4852 plusmn 171 6236 plusmn 168

E7 1070 plusmn 047 2010 plusmn 027 2842 plusmn 080 3680 plusmn 098

AscA 1050 plusmn 072 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

GA 2180 plusmn 041 4280 plusmn 188 5861 plusmn 120 7010 plusmn 146

Table 5 Reducing power of extract and subcomponents E1ndashE7isolated from ET

Component AscAE (mggAscA)CE 4020 plusmn 110

PE 1432 plusmn 042

ET 6260 plusmn 155

BU 2660 plusmn 051

WT 572 plusmn 026

E1 2040 plusmn 077

E2 2820 plusmn 122

E3 4620 plusmn 118

E4 6160 plusmn 111

E5 8440 plusmn 210

E6 7520 plusmn 150

E7 4160 plusmn 095

Theantioxidant activity of subcomponents E1ndashE7 isolatedfrom ET in the 120573-carotene-linoleic acid system is shownin Table 4 At all experiment concentrations E5 shows thestrongest antioxidant activity followed by E6 E4 E7 E3 andE2 with E1 being the weakest Among the seven subcom-ponents E5 and E6 show much stronger antioxidant activitythan ET (119875 lt 005) while the antioxidant activity of E5 is6892at 200120583gmL close to the synthesized antioxidantGA

343 Reducing Power Table 5 shows the reducing power ofthe extract from S clava and subcomponents E1ndashE7 isolated

from ET The reducing powers of all the samples demon-strate a pattern similar to DPPH scavenging activity andantioxidant activity in the 120573-carotene-linoleic acid systemAmong all the extracted components ET is the highest inthe reducing power (119875 lt 005) and the reducing power ofthe seven subcomponents isolated fromET is in the followingorder E5 gt E6 gt E4 gt E3 gt E7 gt E2 gt E1

35 Structural Identification of Compound Compound 1colorless needle crystal is identified as C

27H46O with EI-MS

and NMR 120575347 in 1H NMR 120575718 in 13C NMR and DEPTshow that there is hydroxyl in themolecule 120575531 in 1HNMR12057514018 and 1205751217 in 13C NMR and DEPT further confirmthe double bond in the molecule mz 255 and 147 in MScombinedwith 120575531 (1H H-7) 120575356 (1HmH-3) 120575101 (3Hs Me-19) and 120575088 (3H s Me-18) in 1HMMR 1205751408 (s C-8) 1205751217 (d C-7) and 120575718 (d C-3) in 13CNMR and DEPTreferred to the physical constant and spectral data reportedby Tsuda and Schroepfer Jr [19] compound 1 was identifiedas cholesteric-7-en-3120573-ol whichwas isolated from S clava forthe first time

Compound 2 colorless needle crystal mp 244sim246∘C isidentified as C

27H46O2with EI-MS andNMRThe two signals

of methyl group connected with quaternary carbon (120575126 s120575071 s) and three signals of methyl group connected withtertiary carbon (120575091 d 120575087 d 120575085 d) show that thiscompound is sterol The signal of 19-Me at 120575126 shifts toa lower field in contrast to cholesterol which proves theexistence of 6120573-OH Meanwhile in contrast to cholesterolsignal (120575418) of 3120572-H also shifts to a lower field and so is

Journal of Chemistry 7

HO

HO

HO

OH

OH

OH

OH

OH

H2C

H2C (CH2)17CH3

C

O

O

(CH2)16CH3

CH3(CH2)14

NH

HC

Compound 1 Compound 2

Compound 3 Compound 4

Compound 5

Figure 4 Structures of compounds

proved of 45-double bond considering single peak 120575555Compound 2 is identified as cholesteric-4-en-3120573 6120573-diolreferring to the physical constant and spectral data reportedby Wahidullah et al [20] which is isolated from S clava forthe first time

Compound 3 is a white needle crystal mp 148sim149∘Cpurplish red at 10 sulfuric acid-ethanol solution The mul-tiple peaks at 120575353 in 1H NMR is the characteristic signal of3120572-H in sterol while the multiple peaks at 120575535 is signal ofH-6 In addition five methyl group signals appear in highfield of 1H NMR among which the 120575101 s and 120575068 sbelong to 19-Me and 18-Me respectively 120575091 d is the signalof 21-Me and 120575084 d and 120575082 d belong to 26-Me and27-Me respectively Contrasting the physical constant andspectral data with Su et al [21] compound 3 is identified ascholesterol

Compound 4 is white powder mp 68sim69∘C purplish redat 10 sulfuric acid-ethanol solution The molecular weight

is 345 according to EI-MS As shown in 13C NMR spectrum120575641 (t C-1) 120575319 (t C2) 120575296 (t C3-C15) 120575260 (t C16)120575226 (t C17) and 120575141 (t C18) are characteristic signalsof long chain fatty acid and 120575722 (C-1) 120575706 (C-2) and120575718 (C-3) belong to glycerol so this compound should bea long chain triglyceride However there is no signal of estercarbonyl therefore this compound is identified as glycidylether agreeing with Yang et al [22] andWang et al [23] andis confirmed as batilol which is isolated from S clava for thefirst time

Compound 5 molecular formula is C36H71NO3accord-

ing to EI-MS andNMR 120575637 (1H) in 1HNMR spectrum and1205751739 (C=O) in 13C NMR spectrum proves the existence ofacylamino and 120575545 in 13C NMR spectrum indicates thatwhat is connected with nitrogen is methyne 120575202 (2H) in1HNMR indicates that there are two ndashOH in the compoundand 1205757474 and 1205756240 in 13C NMR indicate that ndashCHndash andndashCH2ndash connect with ndashOH respectively Meantime a strong

8 Journal of Chemistry

hydrogen signal of 120575126 in 1H NMR spectrum and twosignals ndashCH

3ndash of 120575084 (6H) determine two long chain alkyl

groups This compound is identified as ceramide referring tothe physical constant and spectral data reported by Yu andYang [24]

The structures of these compounds are shown in Figure 4As reported by Cai et al [25] the number and positionof hydroxyl groups largely determined radical scavengingactivity of phenolic compounds Compounds 1 2 and 3 allhave only one phenolic hydroxyl group and there is nofunctional groups in orthoposition little steric hindranceeffect increased their contact with free radicals

4 Conclusion

The immunomodulatory and antioxidant activity of crudeand fractionated extracts of the ascidian Styela clava weredetermined by in vitro screening All these extracts demon-strate immunomodulatory activity through increasing theproliferation rate of spleen lymphocyte and macrophagesas well as the NO release activities of macrophages Amongthem the petroleum ether fraction shows the strongestimmune active in vitro The ethyl acetate fraction (ET) wasmuch higher in its antioxidant activity in DPPH systemreducing power assay and 120573-carotene-linoleic acid systemcompared with the other fractions and its subcomponentE5 demonstrated the strongest antioxidant activity as well asreducing power higher than the positive control AscA andclose to the synthesized GA ET was isolated systemically andfive compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol (4) batiloland (5) ceramide among which (1) (2) and (4) were isolatedfor the first time from S clava

Conflict of Interests

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

Authorsrsquo Contribution

Ju Bao and Chen Bin contributed equally to this project

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (no 31070368) and the Sci-ence and Technology Department of Shandong Province(BS2010HZ022 and ZR2011DL008)

References

[1] T M Zabriskie and C M Ireland ldquoThe isolation and structureofmodified bioactive nucleosides from Jaspis johnstonirdquo Journalof Natural Products vol 52 no 6 pp 1353ndash1356 1989

[2] J Kobayashi M Tsuda M Ishibashi et al ldquoAmphidinolideF a new cytotoxic macrolide from the marine dinoflagellateAmphidinium sprdquo Journal of Antibiotics vol 44 no 11 pp 1259ndash1261 1991

[3] B S Davidson and C M Ireland ldquoLissoclinolide the first non-nitrogenous metabolite from a Lissoclinum tunicaterdquo Journal ofNatural Products vol 53 no 4 pp 1036ndash1038 1990

[4] T N Makarieva V A Stonik A S Dmitrenok et al ldquoVaracinand three new marine antimicrobial polysulfides from the far-Eastern ascidian Polycitor sprdquo Journal of Natural Products vol58 no 2 pp 254ndash258 1995

[5] P W Ford and B S Davidson ldquoPlakinidine D a newpyrroloacridine alkaloid from the ascidianDidemnum rubeumrdquoJournal of Natural Products vol 60 no 10 pp 1051ndash1053 1997

[6] C Ireland and P J Scheuer ldquoUlicyclamine and ulithiacy-clamide two new small peptides from a marine tunicaterdquoJournal of the American Chemical Society vol 102 no 17 pp5688ndash5691 1980

[7] H G Chun B Davies and D Hoth ldquoDidemnin B the firstmarine compound entering clinical trials as an antineoplasticagentrdquo Investigational New Drugs vol 4 no 3 pp 279ndash2841986

[8] M A Baker D R Grubb and A Lawen ldquoDidemnin B inducesapoptosis in proliferating but not resting peripheral bloodmononuclear cellsrdquo Apoptosis vol 7 no 5 pp 407ndash412 2002

[9] M D Vera and M M Joullie ldquoNatural products as probes ofcell biology 20 Years of didemnin researchrdquoMedicinal ResearchReviews vol 22 no 2 pp 102ndash145 2002

[10] S Jin B Gorfajn G Faircloth and K W Scotto ldquoEcteinascidin743 a transcription-targeted chemotherapeutic that inhibitsMDR1 activationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 12 pp 6775ndash6779 2000

[11] C van Kesteren E Cvitkovic A Taamma et al ldquoPharma-cokinetics and pharmacodynamics of the novel marine-derivedanticancer agent ecteinascidin 743 in a phase I dose-findingstudyrdquo Clinical Cancer Research vol 6 no 12 pp 4725ndash47322000

[12] E G C Brain ldquoSafety and efficacy of ET-743 the Frenchexperiencerdquo Anti-Cancer Drugs vol 13 supplement 1 pp S11ndashS14 2002

[13] J C M Waterval J C Bloks R W Sparidans et al ldquoDegra-dation kinetics of aplidine a new marine antitumoural cyclicpeptide in aqueous solutionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 754 no 1 pp 161ndash1682001

[14] C K Cai Research of anti-HBV activity and pharmacodynamicaction and toxicity of SC [PhD thesis] Beijing University ofChinese Medicine Beijing China 2006

[15] W Binsan S Benjakul W Visessanguan S Roytrakul MTanaka and H Kishimura ldquoAntioxidative activity of Mun-goong an extract paste from the cephalothorax ofwhite shrimp(Litopenaeus vannamei)rdquo Food Chemistry vol 106 no 1 pp185ndash193 2008

[16] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[17] G C Yen and P D Duh ldquoAntioxidative properties of methano-lic extracts from peanut hullsrdquo Journal of the American OilChemistsrsquo Society vol 70 no 4 pp 383ndash386 1993

[18] R N Albrecht Immunologie Tolerance and Macrophage Func-tion Aeademie New York NY USA 1 edition 1979

[19] M Tsuda and G J Schroepfer Jr ldquoCarbon-13 nuclear magneticresonance studies of C27 sterol precursors of cholesterolrdquoJournal of Organic Chemistry vol 44 no 8 pp 1290ndash1293 1979

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Chemistry

0

10

20

30

40

50

60

Prol

ifera

tion

rate

()

BU PE CE ConAET

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmLPositive control

lowast

lowast

minus10

minus20

lowastlowast

Figure 1 Effects of extracts on the proliferation of the mousesplenocytes lowast119875 lt 005 and lowastlowast119875 lt 001 compared with the control

3 Results and Discussion

31 Proliferation of Spleen Lymphocyte As organismrsquos firstline defending tumor immune system plays an importantrole in the recognition and removal of malignant cells Theimmune status evaluation of the body bearing neoplasm isimportant for mechanism study of antitumor drugs

As shown in Figure 1 four extracts of S clava demonstratesignificant activity on splenocytes proliferation (comparedwith the control 119875 lt 005 or 119875 lt 001) amongwhich the proliferation effect of PE is the most activewith proliferation rate being near 50 at a concentration of25 120583gmL close to the positive control group ConA (5671)However its proliferation rate reduces with the increasingconcentration and the proliferation rate is only 476 whenthe concentration reaches 400 120583gmL which shows that PEinhibits the proliferation of the mouse splenocytes at highconcentration CE is less active than PE on the proliferationof the mouse splenocytes demonstrating a rising trend atfirst and then a falling one The effects of ET and BUare relatively weak and ET shows distinctive inhibition at50120583gmL and 100120583gmL with the inhibition rate of 1262at 100 120583gmL but on the contrary BU reaches the strongestlevel at this concentration with the proliferation rate of1452 while distinctive inhibition was shown below thispoint

32 Proliferation of Macrophages Figure 2 shows the effectsof S clavarsquos extracts on the proliferation of the mouseperitonealmacrophagesThe four extracts all show significanteffect on the proliferation of the mouse macrophages at alldesigned concentrations (compared with the control 119875 lt005 or 119875 lt 001) but it is weaker than that of thepositive control LPS as proliferation rate of 6319 PE is themost active extract on the proliferation at concentration of

05

101520253035404550556065

Prol

ifera

tion

rate

()

ET BU PE CE LPS

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmLPositive control

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

Figure 2 Effects of extracts on the proliferation of the mouseperitoneal macrophages lowast119875 lt 005 and lowastlowast119875 lt 001 compared withthe control

25 120583gmL with proliferation rate 4070 but afterwards theproliferation rate dropswith the increasing concentration andthe proliferation rate is only 1085 when the concentrationreaches 400120583gmL showing similar trend as on the lympho-cytes

ET and CE are close on the effect of proliferation ETreaches the lowest point at 25120583gmL with the proliferationrate of 194 and the highest point at 200120583gmL with theproliferation rate of 2597 CE shows similar trend asET reaching the highest and lowest points at 100 120583gmLand 400120583gmL with the proliferation rate of 2519 and1017 respectively BU shows the weakest effect with thehighest proliferation rate of only 1008 at 200120583gmL andthe same effect as the control at 25120583gmL the prolifer-ation rates at all the other concentrations are all below10

33 NO Release Activities of Macrophages Macrophages arethe important components in the body immune systemfunctioning as immunization effect regulating the immunesystem and NO is one of the active agents produced bymacrophages after they are activated [18] Figure 3 showseffects of extract from S clava on the NO release activitiesof mouse macrophages The effects of ET PE and CE are allin positive correlation with concentration in promoting therelease of NO PE shows the strongest effect among themreaching the highest point of 1614 120583molmL at 400 120583gmLCE is less effective with NO releasing the highest point of1476 120583molmL at 400 120583gmL The effects of BU and ET arerather weak only 870120583molmL for ET at 400120583gmL as thehighest point while at the other concentrations the NOreleasing abilities for ET and BU are close to and approaching6 120583molmL

Journal of Chemistry 5

Table 1 DPPH scavenging activity of different components isolated from S clava

Component DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

CE 192 plusmn 037 3012 plusmn 060 379 plusmn 064 426 plusmn 088

PE 483 plusmn 011 1011 plusmn 022 1424 plusmn 038 1850 plusmn 037

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

BU 838 plusmn 021 1929 plusmn 037 2532 plusmn 049 3077 plusmn 062

AR 210 plusmn 005 305 plusmn 011 426 plusmn 008 489 plusmn 010

Table 2 DPPH scavenging activity of subcomponents E1ndashE7 isolated from ET of S clava

Subcomponent DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

E1 824 plusmn 026 1417 plusmn 040 1864 plusmn 059 2080 plusmn 065

E2 1137 plusmn 038 1819 plusmn 088 2430 plusmn 162 2918 plusmn 125

E3 1846 plusmn 064 2530 plusmn 124 3428 plusmn 122 4580 plusmn 099

E4 2280 plusmn 032 3820 plusmn 102 4937 plusmn 157 5678 plusmn 101

E5 3026 plusmn 088 4620 plusmn 092 5916 plusmn 111 6731 plusmn 165

E6 2492 plusmn 077 4200 plusmn 106 5340 plusmn 070 6442 plusmn 162

E7 1400 plusmn 031 2029 plusmn 066 2713 plusmn 048 3250 plusmn 101

0

2

4

6

8

10

12

14

16

18

ET BU PE CE

NO

(120583m

olm

L)

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Figure 3 Effects of extracts on NO production lowast119875 lt 005 andlowastlowast119875 lt 001 compared with the control

34 In Vitro Antioxidant Activity

341 DPPH Radical Scavenging Assay The DPPH free rad-icals scavenging method is a colorimetric assay and can beused to evaluate the radical scavenging activity in a shorttime DPPH is an organic free radical with maximal absorp-tion at 517 nmWhenwith free radical scavengerDPPHrsquos loneelectronwill be paired and its absorbancewill decrease whichcan be used to evaluate its antioxidant activity As shown

in Table 1 there was a positive correlation between DPPHscavenging activity of CE and its concentration Among thefour extracted components PE and BU demonstrate poorscavenging activity while AR shows no scavenging activityThe scavenging activity of ET reaches 5064 at 200 120583gmLmuch higher than CE (119875 lt 005) indicating that compoundswithDPPH scavenging activity in S clava aremostlymediumpolar

E1ndashE7 subcomponents are isolated from ETwith silica gelcolumn chromatography whose DPPH scavenging activity isshown in Table 2 At all experiment concentrations E5 showsthe strongest scavenging activity followed by E6 E4 E3 E7and E2 with E1 the weakest E5 and E6 show much strongerscavenging activity than ET (119875 lt 005) while E4 is close toET in scavenging activity EC

50of E5 and E6 are 12206120583gmL

and 13968 120583gmL respectively

342 Antioxidant Activity in 120573-Carotene-Linoleic Acid Sys-tem 120573-carotene is a polyene pigment apt to be oxidized tofade In the reaction medium the superoxide produced bythe oxidized linoleic acid results in 120573-carotenersquos fading andas time goes on the absorbance decreases The degree of120573-carotenersquos fading depends on the antioxidant activity inthe system Table 3 shows antioxidant activities of differentcomponents isolated from S clava in the 120573-carotene-linoleicacid system CE has some antioxidation activities positivelycorrelated with its concentration ET is much higher in itsantioxidant activity than other components reaching 50 at200120583gmL and is even higher than the positive control AscAat all experimental concentrations which further indicatesthat compounds with higher antioxidant activity in the120573-carotene-linoleic acid system are mostly medium-polarcomponents

6 Journal of Chemistry

Table 3 Antioxidant activity of different components isolated from S clava in the 120573-carotene-linoleic acid system

Component Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

CE 1062 plusmn 038 2010 plusmn 112 2948 plusmn 130 4035 plusmn 112

PE 460 plusmn 021 1022 plusmn 044 1650 plusmn 049 2240 plusmn 068

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

BU 720 plusmn 048 1439 plusmn 062 2260 plusmn 077 2600 plusmn 089

AR 215 plusmn 005 690 plusmn 014 820 plusmn 018 980 plusmn 011

AscA 1050 plusmn 012 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

Table 4 Antioxidant activity of subcomponents E1ndashE7 isolated from ET in the 120573-carotene-linoleic acid system

Subcomponent Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

E1 646 plusmn 050 1380 plusmn 046 2820 plusmn 064 2219 plusmn 097

E2 742 plusmn 031 1540 plusmn 074 2474 plusmn 107 3090 plusmn 121

E3 930 plusmn 037 1778 plusmn 087 2741 plusmn 103 3277 plusmn 127

E4 1238 plusmn 067 2472 plusmn 118 3270 plusmn 079 4365 plusmn 198

E5 2024 plusmn 144 4060 plusmn 125 5410 plusmn 157 6892 plusmn 167

E6 1520 plusmn 079 3440 plusmn 085 4852 plusmn 171 6236 plusmn 168

E7 1070 plusmn 047 2010 plusmn 027 2842 plusmn 080 3680 plusmn 098

AscA 1050 plusmn 072 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

GA 2180 plusmn 041 4280 plusmn 188 5861 plusmn 120 7010 plusmn 146

Table 5 Reducing power of extract and subcomponents E1ndashE7isolated from ET

Component AscAE (mggAscA)CE 4020 plusmn 110

PE 1432 plusmn 042

ET 6260 plusmn 155

BU 2660 plusmn 051

WT 572 plusmn 026

E1 2040 plusmn 077

E2 2820 plusmn 122

E3 4620 plusmn 118

E4 6160 plusmn 111

E5 8440 plusmn 210

E6 7520 plusmn 150

E7 4160 plusmn 095

Theantioxidant activity of subcomponents E1ndashE7 isolatedfrom ET in the 120573-carotene-linoleic acid system is shownin Table 4 At all experiment concentrations E5 shows thestrongest antioxidant activity followed by E6 E4 E7 E3 andE2 with E1 being the weakest Among the seven subcom-ponents E5 and E6 show much stronger antioxidant activitythan ET (119875 lt 005) while the antioxidant activity of E5 is6892at 200120583gmL close to the synthesized antioxidantGA

343 Reducing Power Table 5 shows the reducing power ofthe extract from S clava and subcomponents E1ndashE7 isolated

from ET The reducing powers of all the samples demon-strate a pattern similar to DPPH scavenging activity andantioxidant activity in the 120573-carotene-linoleic acid systemAmong all the extracted components ET is the highest inthe reducing power (119875 lt 005) and the reducing power ofthe seven subcomponents isolated fromET is in the followingorder E5 gt E6 gt E4 gt E3 gt E7 gt E2 gt E1

35 Structural Identification of Compound Compound 1colorless needle crystal is identified as C

27H46O with EI-MS

and NMR 120575347 in 1H NMR 120575718 in 13C NMR and DEPTshow that there is hydroxyl in themolecule 120575531 in 1HNMR12057514018 and 1205751217 in 13C NMR and DEPT further confirmthe double bond in the molecule mz 255 and 147 in MScombinedwith 120575531 (1H H-7) 120575356 (1HmH-3) 120575101 (3Hs Me-19) and 120575088 (3H s Me-18) in 1HMMR 1205751408 (s C-8) 1205751217 (d C-7) and 120575718 (d C-3) in 13CNMR and DEPTreferred to the physical constant and spectral data reportedby Tsuda and Schroepfer Jr [19] compound 1 was identifiedas cholesteric-7-en-3120573-ol whichwas isolated from S clava forthe first time

Compound 2 colorless needle crystal mp 244sim246∘C isidentified as C

27H46O2with EI-MS andNMRThe two signals

of methyl group connected with quaternary carbon (120575126 s120575071 s) and three signals of methyl group connected withtertiary carbon (120575091 d 120575087 d 120575085 d) show that thiscompound is sterol The signal of 19-Me at 120575126 shifts toa lower field in contrast to cholesterol which proves theexistence of 6120573-OH Meanwhile in contrast to cholesterolsignal (120575418) of 3120572-H also shifts to a lower field and so is

Journal of Chemistry 7

HO

HO

HO

OH

OH

OH

OH

OH

H2C

H2C (CH2)17CH3

C

O

O

(CH2)16CH3

CH3(CH2)14

NH

HC

Compound 1 Compound 2

Compound 3 Compound 4

Compound 5

Figure 4 Structures of compounds

proved of 45-double bond considering single peak 120575555Compound 2 is identified as cholesteric-4-en-3120573 6120573-diolreferring to the physical constant and spectral data reportedby Wahidullah et al [20] which is isolated from S clava forthe first time

Compound 3 is a white needle crystal mp 148sim149∘Cpurplish red at 10 sulfuric acid-ethanol solution The mul-tiple peaks at 120575353 in 1H NMR is the characteristic signal of3120572-H in sterol while the multiple peaks at 120575535 is signal ofH-6 In addition five methyl group signals appear in highfield of 1H NMR among which the 120575101 s and 120575068 sbelong to 19-Me and 18-Me respectively 120575091 d is the signalof 21-Me and 120575084 d and 120575082 d belong to 26-Me and27-Me respectively Contrasting the physical constant andspectral data with Su et al [21] compound 3 is identified ascholesterol

Compound 4 is white powder mp 68sim69∘C purplish redat 10 sulfuric acid-ethanol solution The molecular weight

is 345 according to EI-MS As shown in 13C NMR spectrum120575641 (t C-1) 120575319 (t C2) 120575296 (t C3-C15) 120575260 (t C16)120575226 (t C17) and 120575141 (t C18) are characteristic signalsof long chain fatty acid and 120575722 (C-1) 120575706 (C-2) and120575718 (C-3) belong to glycerol so this compound should bea long chain triglyceride However there is no signal of estercarbonyl therefore this compound is identified as glycidylether agreeing with Yang et al [22] andWang et al [23] andis confirmed as batilol which is isolated from S clava for thefirst time

Compound 5 molecular formula is C36H71NO3accord-

ing to EI-MS andNMR 120575637 (1H) in 1HNMR spectrum and1205751739 (C=O) in 13C NMR spectrum proves the existence ofacylamino and 120575545 in 13C NMR spectrum indicates thatwhat is connected with nitrogen is methyne 120575202 (2H) in1HNMR indicates that there are two ndashOH in the compoundand 1205757474 and 1205756240 in 13C NMR indicate that ndashCHndash andndashCH2ndash connect with ndashOH respectively Meantime a strong

8 Journal of Chemistry

hydrogen signal of 120575126 in 1H NMR spectrum and twosignals ndashCH

3ndash of 120575084 (6H) determine two long chain alkyl

groups This compound is identified as ceramide referring tothe physical constant and spectral data reported by Yu andYang [24]

The structures of these compounds are shown in Figure 4As reported by Cai et al [25] the number and positionof hydroxyl groups largely determined radical scavengingactivity of phenolic compounds Compounds 1 2 and 3 allhave only one phenolic hydroxyl group and there is nofunctional groups in orthoposition little steric hindranceeffect increased their contact with free radicals

4 Conclusion

The immunomodulatory and antioxidant activity of crudeand fractionated extracts of the ascidian Styela clava weredetermined by in vitro screening All these extracts demon-strate immunomodulatory activity through increasing theproliferation rate of spleen lymphocyte and macrophagesas well as the NO release activities of macrophages Amongthem the petroleum ether fraction shows the strongestimmune active in vitro The ethyl acetate fraction (ET) wasmuch higher in its antioxidant activity in DPPH systemreducing power assay and 120573-carotene-linoleic acid systemcompared with the other fractions and its subcomponentE5 demonstrated the strongest antioxidant activity as well asreducing power higher than the positive control AscA andclose to the synthesized GA ET was isolated systemically andfive compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol (4) batiloland (5) ceramide among which (1) (2) and (4) were isolatedfor the first time from S clava

Conflict of Interests

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

Authorsrsquo Contribution

Ju Bao and Chen Bin contributed equally to this project

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (no 31070368) and the Sci-ence and Technology Department of Shandong Province(BS2010HZ022 and ZR2011DL008)

References

[1] T M Zabriskie and C M Ireland ldquoThe isolation and structureofmodified bioactive nucleosides from Jaspis johnstonirdquo Journalof Natural Products vol 52 no 6 pp 1353ndash1356 1989

[2] J Kobayashi M Tsuda M Ishibashi et al ldquoAmphidinolideF a new cytotoxic macrolide from the marine dinoflagellateAmphidinium sprdquo Journal of Antibiotics vol 44 no 11 pp 1259ndash1261 1991

[3] B S Davidson and C M Ireland ldquoLissoclinolide the first non-nitrogenous metabolite from a Lissoclinum tunicaterdquo Journal ofNatural Products vol 53 no 4 pp 1036ndash1038 1990

[4] T N Makarieva V A Stonik A S Dmitrenok et al ldquoVaracinand three new marine antimicrobial polysulfides from the far-Eastern ascidian Polycitor sprdquo Journal of Natural Products vol58 no 2 pp 254ndash258 1995

[5] P W Ford and B S Davidson ldquoPlakinidine D a newpyrroloacridine alkaloid from the ascidianDidemnum rubeumrdquoJournal of Natural Products vol 60 no 10 pp 1051ndash1053 1997

[6] C Ireland and P J Scheuer ldquoUlicyclamine and ulithiacy-clamide two new small peptides from a marine tunicaterdquoJournal of the American Chemical Society vol 102 no 17 pp5688ndash5691 1980

[7] H G Chun B Davies and D Hoth ldquoDidemnin B the firstmarine compound entering clinical trials as an antineoplasticagentrdquo Investigational New Drugs vol 4 no 3 pp 279ndash2841986

[8] M A Baker D R Grubb and A Lawen ldquoDidemnin B inducesapoptosis in proliferating but not resting peripheral bloodmononuclear cellsrdquo Apoptosis vol 7 no 5 pp 407ndash412 2002

[9] M D Vera and M M Joullie ldquoNatural products as probes ofcell biology 20 Years of didemnin researchrdquoMedicinal ResearchReviews vol 22 no 2 pp 102ndash145 2002

[10] S Jin B Gorfajn G Faircloth and K W Scotto ldquoEcteinascidin743 a transcription-targeted chemotherapeutic that inhibitsMDR1 activationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 12 pp 6775ndash6779 2000

[11] C van Kesteren E Cvitkovic A Taamma et al ldquoPharma-cokinetics and pharmacodynamics of the novel marine-derivedanticancer agent ecteinascidin 743 in a phase I dose-findingstudyrdquo Clinical Cancer Research vol 6 no 12 pp 4725ndash47322000

[12] E G C Brain ldquoSafety and efficacy of ET-743 the Frenchexperiencerdquo Anti-Cancer Drugs vol 13 supplement 1 pp S11ndashS14 2002

[13] J C M Waterval J C Bloks R W Sparidans et al ldquoDegra-dation kinetics of aplidine a new marine antitumoural cyclicpeptide in aqueous solutionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 754 no 1 pp 161ndash1682001

[14] C K Cai Research of anti-HBV activity and pharmacodynamicaction and toxicity of SC [PhD thesis] Beijing University ofChinese Medicine Beijing China 2006

[15] W Binsan S Benjakul W Visessanguan S Roytrakul MTanaka and H Kishimura ldquoAntioxidative activity of Mun-goong an extract paste from the cephalothorax ofwhite shrimp(Litopenaeus vannamei)rdquo Food Chemistry vol 106 no 1 pp185ndash193 2008

[16] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[17] G C Yen and P D Duh ldquoAntioxidative properties of methano-lic extracts from peanut hullsrdquo Journal of the American OilChemistsrsquo Society vol 70 no 4 pp 383ndash386 1993

[18] R N Albrecht Immunologie Tolerance and Macrophage Func-tion Aeademie New York NY USA 1 edition 1979

[19] M Tsuda and G J Schroepfer Jr ldquoCarbon-13 nuclear magneticresonance studies of C27 sterol precursors of cholesterolrdquoJournal of Organic Chemistry vol 44 no 8 pp 1290ndash1293 1979

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 5

Table 1 DPPH scavenging activity of different components isolated from S clava

Component DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

CE 192 plusmn 037 3012 plusmn 060 379 plusmn 064 426 plusmn 088

PE 483 plusmn 011 1011 plusmn 022 1424 plusmn 038 1850 plusmn 037

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

BU 838 plusmn 021 1929 plusmn 037 2532 plusmn 049 3077 plusmn 062

AR 210 plusmn 005 305 plusmn 011 426 plusmn 008 489 plusmn 010

Table 2 DPPH scavenging activity of subcomponents E1ndashE7 isolated from ET of S clava

Subcomponent DPPH scavenging rate ()50 (120583gmL) 100 (120583gmL) 150 (120583gmL) 200 (120583gmL)

ET 2290 plusmn 070 3810 plusmn 114 4748 plusmn 130 5064 plusmn 112

E1 824 plusmn 026 1417 plusmn 040 1864 plusmn 059 2080 plusmn 065

E2 1137 plusmn 038 1819 plusmn 088 2430 plusmn 162 2918 plusmn 125

E3 1846 plusmn 064 2530 plusmn 124 3428 plusmn 122 4580 plusmn 099

E4 2280 plusmn 032 3820 plusmn 102 4937 plusmn 157 5678 plusmn 101

E5 3026 plusmn 088 4620 plusmn 092 5916 plusmn 111 6731 plusmn 165

E6 2492 plusmn 077 4200 plusmn 106 5340 plusmn 070 6442 plusmn 162

E7 1400 plusmn 031 2029 plusmn 066 2713 plusmn 048 3250 plusmn 101

0

2

4

6

8

10

12

14

16

18

ET BU PE CE

NO

(120583m

olm

L)

25120583gmL50120583gmL100120583gmL

200120583gmL400120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Figure 3 Effects of extracts on NO production lowast119875 lt 005 andlowastlowast119875 lt 001 compared with the control

34 In Vitro Antioxidant Activity

341 DPPH Radical Scavenging Assay The DPPH free rad-icals scavenging method is a colorimetric assay and can beused to evaluate the radical scavenging activity in a shorttime DPPH is an organic free radical with maximal absorp-tion at 517 nmWhenwith free radical scavengerDPPHrsquos loneelectronwill be paired and its absorbancewill decrease whichcan be used to evaluate its antioxidant activity As shown

in Table 1 there was a positive correlation between DPPHscavenging activity of CE and its concentration Among thefour extracted components PE and BU demonstrate poorscavenging activity while AR shows no scavenging activityThe scavenging activity of ET reaches 5064 at 200 120583gmLmuch higher than CE (119875 lt 005) indicating that compoundswithDPPH scavenging activity in S clava aremostlymediumpolar

E1ndashE7 subcomponents are isolated from ETwith silica gelcolumn chromatography whose DPPH scavenging activity isshown in Table 2 At all experiment concentrations E5 showsthe strongest scavenging activity followed by E6 E4 E3 E7and E2 with E1 the weakest E5 and E6 show much strongerscavenging activity than ET (119875 lt 005) while E4 is close toET in scavenging activity EC

50of E5 and E6 are 12206120583gmL

and 13968 120583gmL respectively

342 Antioxidant Activity in 120573-Carotene-Linoleic Acid Sys-tem 120573-carotene is a polyene pigment apt to be oxidized tofade In the reaction medium the superoxide produced bythe oxidized linoleic acid results in 120573-carotenersquos fading andas time goes on the absorbance decreases The degree of120573-carotenersquos fading depends on the antioxidant activity inthe system Table 3 shows antioxidant activities of differentcomponents isolated from S clava in the 120573-carotene-linoleicacid system CE has some antioxidation activities positivelycorrelated with its concentration ET is much higher in itsantioxidant activity than other components reaching 50 at200120583gmL and is even higher than the positive control AscAat all experimental concentrations which further indicatesthat compounds with higher antioxidant activity in the120573-carotene-linoleic acid system are mostly medium-polarcomponents

6 Journal of Chemistry

Table 3 Antioxidant activity of different components isolated from S clava in the 120573-carotene-linoleic acid system

Component Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

CE 1062 plusmn 038 2010 plusmn 112 2948 plusmn 130 4035 plusmn 112

PE 460 plusmn 021 1022 plusmn 044 1650 plusmn 049 2240 plusmn 068

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

BU 720 plusmn 048 1439 plusmn 062 2260 plusmn 077 2600 plusmn 089

AR 215 plusmn 005 690 plusmn 014 820 plusmn 018 980 plusmn 011

AscA 1050 plusmn 012 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

Table 4 Antioxidant activity of subcomponents E1ndashE7 isolated from ET in the 120573-carotene-linoleic acid system

Subcomponent Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

E1 646 plusmn 050 1380 plusmn 046 2820 plusmn 064 2219 plusmn 097

E2 742 plusmn 031 1540 plusmn 074 2474 plusmn 107 3090 plusmn 121

E3 930 plusmn 037 1778 plusmn 087 2741 plusmn 103 3277 plusmn 127

E4 1238 plusmn 067 2472 plusmn 118 3270 plusmn 079 4365 plusmn 198

E5 2024 plusmn 144 4060 plusmn 125 5410 plusmn 157 6892 plusmn 167

E6 1520 plusmn 079 3440 plusmn 085 4852 plusmn 171 6236 plusmn 168

E7 1070 plusmn 047 2010 plusmn 027 2842 plusmn 080 3680 plusmn 098

AscA 1050 plusmn 072 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

GA 2180 plusmn 041 4280 plusmn 188 5861 plusmn 120 7010 plusmn 146

Table 5 Reducing power of extract and subcomponents E1ndashE7isolated from ET

Component AscAE (mggAscA)CE 4020 plusmn 110

PE 1432 plusmn 042

ET 6260 plusmn 155

BU 2660 plusmn 051

WT 572 plusmn 026

E1 2040 plusmn 077

E2 2820 plusmn 122

E3 4620 plusmn 118

E4 6160 plusmn 111

E5 8440 plusmn 210

E6 7520 plusmn 150

E7 4160 plusmn 095

Theantioxidant activity of subcomponents E1ndashE7 isolatedfrom ET in the 120573-carotene-linoleic acid system is shownin Table 4 At all experiment concentrations E5 shows thestrongest antioxidant activity followed by E6 E4 E7 E3 andE2 with E1 being the weakest Among the seven subcom-ponents E5 and E6 show much stronger antioxidant activitythan ET (119875 lt 005) while the antioxidant activity of E5 is6892at 200120583gmL close to the synthesized antioxidantGA

343 Reducing Power Table 5 shows the reducing power ofthe extract from S clava and subcomponents E1ndashE7 isolated

from ET The reducing powers of all the samples demon-strate a pattern similar to DPPH scavenging activity andantioxidant activity in the 120573-carotene-linoleic acid systemAmong all the extracted components ET is the highest inthe reducing power (119875 lt 005) and the reducing power ofthe seven subcomponents isolated fromET is in the followingorder E5 gt E6 gt E4 gt E3 gt E7 gt E2 gt E1

35 Structural Identification of Compound Compound 1colorless needle crystal is identified as C

27H46O with EI-MS

and NMR 120575347 in 1H NMR 120575718 in 13C NMR and DEPTshow that there is hydroxyl in themolecule 120575531 in 1HNMR12057514018 and 1205751217 in 13C NMR and DEPT further confirmthe double bond in the molecule mz 255 and 147 in MScombinedwith 120575531 (1H H-7) 120575356 (1HmH-3) 120575101 (3Hs Me-19) and 120575088 (3H s Me-18) in 1HMMR 1205751408 (s C-8) 1205751217 (d C-7) and 120575718 (d C-3) in 13CNMR and DEPTreferred to the physical constant and spectral data reportedby Tsuda and Schroepfer Jr [19] compound 1 was identifiedas cholesteric-7-en-3120573-ol whichwas isolated from S clava forthe first time

Compound 2 colorless needle crystal mp 244sim246∘C isidentified as C

27H46O2with EI-MS andNMRThe two signals

of methyl group connected with quaternary carbon (120575126 s120575071 s) and three signals of methyl group connected withtertiary carbon (120575091 d 120575087 d 120575085 d) show that thiscompound is sterol The signal of 19-Me at 120575126 shifts toa lower field in contrast to cholesterol which proves theexistence of 6120573-OH Meanwhile in contrast to cholesterolsignal (120575418) of 3120572-H also shifts to a lower field and so is

Journal of Chemistry 7

HO

HO

HO

OH

OH

OH

OH

OH

H2C

H2C (CH2)17CH3

C

O

O

(CH2)16CH3

CH3(CH2)14

NH

HC

Compound 1 Compound 2

Compound 3 Compound 4

Compound 5

Figure 4 Structures of compounds

proved of 45-double bond considering single peak 120575555Compound 2 is identified as cholesteric-4-en-3120573 6120573-diolreferring to the physical constant and spectral data reportedby Wahidullah et al [20] which is isolated from S clava forthe first time

Compound 3 is a white needle crystal mp 148sim149∘Cpurplish red at 10 sulfuric acid-ethanol solution The mul-tiple peaks at 120575353 in 1H NMR is the characteristic signal of3120572-H in sterol while the multiple peaks at 120575535 is signal ofH-6 In addition five methyl group signals appear in highfield of 1H NMR among which the 120575101 s and 120575068 sbelong to 19-Me and 18-Me respectively 120575091 d is the signalof 21-Me and 120575084 d and 120575082 d belong to 26-Me and27-Me respectively Contrasting the physical constant andspectral data with Su et al [21] compound 3 is identified ascholesterol

Compound 4 is white powder mp 68sim69∘C purplish redat 10 sulfuric acid-ethanol solution The molecular weight

is 345 according to EI-MS As shown in 13C NMR spectrum120575641 (t C-1) 120575319 (t C2) 120575296 (t C3-C15) 120575260 (t C16)120575226 (t C17) and 120575141 (t C18) are characteristic signalsof long chain fatty acid and 120575722 (C-1) 120575706 (C-2) and120575718 (C-3) belong to glycerol so this compound should bea long chain triglyceride However there is no signal of estercarbonyl therefore this compound is identified as glycidylether agreeing with Yang et al [22] andWang et al [23] andis confirmed as batilol which is isolated from S clava for thefirst time

Compound 5 molecular formula is C36H71NO3accord-

ing to EI-MS andNMR 120575637 (1H) in 1HNMR spectrum and1205751739 (C=O) in 13C NMR spectrum proves the existence ofacylamino and 120575545 in 13C NMR spectrum indicates thatwhat is connected with nitrogen is methyne 120575202 (2H) in1HNMR indicates that there are two ndashOH in the compoundand 1205757474 and 1205756240 in 13C NMR indicate that ndashCHndash andndashCH2ndash connect with ndashOH respectively Meantime a strong

8 Journal of Chemistry

hydrogen signal of 120575126 in 1H NMR spectrum and twosignals ndashCH

3ndash of 120575084 (6H) determine two long chain alkyl

groups This compound is identified as ceramide referring tothe physical constant and spectral data reported by Yu andYang [24]

The structures of these compounds are shown in Figure 4As reported by Cai et al [25] the number and positionof hydroxyl groups largely determined radical scavengingactivity of phenolic compounds Compounds 1 2 and 3 allhave only one phenolic hydroxyl group and there is nofunctional groups in orthoposition little steric hindranceeffect increased their contact with free radicals

4 Conclusion

The immunomodulatory and antioxidant activity of crudeand fractionated extracts of the ascidian Styela clava weredetermined by in vitro screening All these extracts demon-strate immunomodulatory activity through increasing theproliferation rate of spleen lymphocyte and macrophagesas well as the NO release activities of macrophages Amongthem the petroleum ether fraction shows the strongestimmune active in vitro The ethyl acetate fraction (ET) wasmuch higher in its antioxidant activity in DPPH systemreducing power assay and 120573-carotene-linoleic acid systemcompared with the other fractions and its subcomponentE5 demonstrated the strongest antioxidant activity as well asreducing power higher than the positive control AscA andclose to the synthesized GA ET was isolated systemically andfive compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol (4) batiloland (5) ceramide among which (1) (2) and (4) were isolatedfor the first time from S clava

Conflict of Interests

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

Authorsrsquo Contribution

Ju Bao and Chen Bin contributed equally to this project

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (no 31070368) and the Sci-ence and Technology Department of Shandong Province(BS2010HZ022 and ZR2011DL008)

References

[1] T M Zabriskie and C M Ireland ldquoThe isolation and structureofmodified bioactive nucleosides from Jaspis johnstonirdquo Journalof Natural Products vol 52 no 6 pp 1353ndash1356 1989

[2] J Kobayashi M Tsuda M Ishibashi et al ldquoAmphidinolideF a new cytotoxic macrolide from the marine dinoflagellateAmphidinium sprdquo Journal of Antibiotics vol 44 no 11 pp 1259ndash1261 1991

[3] B S Davidson and C M Ireland ldquoLissoclinolide the first non-nitrogenous metabolite from a Lissoclinum tunicaterdquo Journal ofNatural Products vol 53 no 4 pp 1036ndash1038 1990

[4] T N Makarieva V A Stonik A S Dmitrenok et al ldquoVaracinand three new marine antimicrobial polysulfides from the far-Eastern ascidian Polycitor sprdquo Journal of Natural Products vol58 no 2 pp 254ndash258 1995

[5] P W Ford and B S Davidson ldquoPlakinidine D a newpyrroloacridine alkaloid from the ascidianDidemnum rubeumrdquoJournal of Natural Products vol 60 no 10 pp 1051ndash1053 1997

[6] C Ireland and P J Scheuer ldquoUlicyclamine and ulithiacy-clamide two new small peptides from a marine tunicaterdquoJournal of the American Chemical Society vol 102 no 17 pp5688ndash5691 1980

[7] H G Chun B Davies and D Hoth ldquoDidemnin B the firstmarine compound entering clinical trials as an antineoplasticagentrdquo Investigational New Drugs vol 4 no 3 pp 279ndash2841986

[8] M A Baker D R Grubb and A Lawen ldquoDidemnin B inducesapoptosis in proliferating but not resting peripheral bloodmononuclear cellsrdquo Apoptosis vol 7 no 5 pp 407ndash412 2002

[9] M D Vera and M M Joullie ldquoNatural products as probes ofcell biology 20 Years of didemnin researchrdquoMedicinal ResearchReviews vol 22 no 2 pp 102ndash145 2002

[10] S Jin B Gorfajn G Faircloth and K W Scotto ldquoEcteinascidin743 a transcription-targeted chemotherapeutic that inhibitsMDR1 activationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 12 pp 6775ndash6779 2000

[11] C van Kesteren E Cvitkovic A Taamma et al ldquoPharma-cokinetics and pharmacodynamics of the novel marine-derivedanticancer agent ecteinascidin 743 in a phase I dose-findingstudyrdquo Clinical Cancer Research vol 6 no 12 pp 4725ndash47322000

[12] E G C Brain ldquoSafety and efficacy of ET-743 the Frenchexperiencerdquo Anti-Cancer Drugs vol 13 supplement 1 pp S11ndashS14 2002

[13] J C M Waterval J C Bloks R W Sparidans et al ldquoDegra-dation kinetics of aplidine a new marine antitumoural cyclicpeptide in aqueous solutionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 754 no 1 pp 161ndash1682001

[14] C K Cai Research of anti-HBV activity and pharmacodynamicaction and toxicity of SC [PhD thesis] Beijing University ofChinese Medicine Beijing China 2006

[15] W Binsan S Benjakul W Visessanguan S Roytrakul MTanaka and H Kishimura ldquoAntioxidative activity of Mun-goong an extract paste from the cephalothorax ofwhite shrimp(Litopenaeus vannamei)rdquo Food Chemistry vol 106 no 1 pp185ndash193 2008

[16] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[17] G C Yen and P D Duh ldquoAntioxidative properties of methano-lic extracts from peanut hullsrdquo Journal of the American OilChemistsrsquo Society vol 70 no 4 pp 383ndash386 1993

[18] R N Albrecht Immunologie Tolerance and Macrophage Func-tion Aeademie New York NY USA 1 edition 1979

[19] M Tsuda and G J Schroepfer Jr ldquoCarbon-13 nuclear magneticresonance studies of C27 sterol precursors of cholesterolrdquoJournal of Organic Chemistry vol 44 no 8 pp 1290ndash1293 1979

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Journal of Chemistry

Table 3 Antioxidant activity of different components isolated from S clava in the 120573-carotene-linoleic acid system

Component Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

CE 1062 plusmn 038 2010 plusmn 112 2948 plusmn 130 4035 plusmn 112

PE 460 plusmn 021 1022 plusmn 044 1650 plusmn 049 2240 plusmn 068

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

BU 720 plusmn 048 1439 plusmn 062 2260 plusmn 077 2600 plusmn 089

AR 215 plusmn 005 690 plusmn 014 820 plusmn 018 980 plusmn 011

AscA 1050 plusmn 012 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

Table 4 Antioxidant activity of subcomponents E1ndashE7 isolated from ET in the 120573-carotene-linoleic acid system

Subcomponent Antioxidant activity ()10 (120583gmL) 50 (120583gmL) 100 (120583gmL) 200 (120583gmL)

ET 1270 plusmn 065 2876 plusmn 112 3742 plusmn 088 4837 plusmn 155

E1 646 plusmn 050 1380 plusmn 046 2820 plusmn 064 2219 plusmn 097

E2 742 plusmn 031 1540 plusmn 074 2474 plusmn 107 3090 plusmn 121

E3 930 plusmn 037 1778 plusmn 087 2741 plusmn 103 3277 plusmn 127

E4 1238 plusmn 067 2472 plusmn 118 3270 plusmn 079 4365 plusmn 198

E5 2024 plusmn 144 4060 plusmn 125 5410 plusmn 157 6892 plusmn 167

E6 1520 plusmn 079 3440 plusmn 085 4852 plusmn 171 6236 plusmn 168

E7 1070 plusmn 047 2010 plusmn 027 2842 plusmn 080 3680 plusmn 098

AscA 1050 plusmn 072 2070 plusmn 031 3290 plusmn 018 4660 plusmn 032

GA 2180 plusmn 041 4280 plusmn 188 5861 plusmn 120 7010 plusmn 146

Table 5 Reducing power of extract and subcomponents E1ndashE7isolated from ET

Component AscAE (mggAscA)CE 4020 plusmn 110

PE 1432 plusmn 042

ET 6260 plusmn 155

BU 2660 plusmn 051

WT 572 plusmn 026

E1 2040 plusmn 077

E2 2820 plusmn 122

E3 4620 plusmn 118

E4 6160 plusmn 111

E5 8440 plusmn 210

E6 7520 plusmn 150

E7 4160 plusmn 095

Theantioxidant activity of subcomponents E1ndashE7 isolatedfrom ET in the 120573-carotene-linoleic acid system is shownin Table 4 At all experiment concentrations E5 shows thestrongest antioxidant activity followed by E6 E4 E7 E3 andE2 with E1 being the weakest Among the seven subcom-ponents E5 and E6 show much stronger antioxidant activitythan ET (119875 lt 005) while the antioxidant activity of E5 is6892at 200120583gmL close to the synthesized antioxidantGA

343 Reducing Power Table 5 shows the reducing power ofthe extract from S clava and subcomponents E1ndashE7 isolated

from ET The reducing powers of all the samples demon-strate a pattern similar to DPPH scavenging activity andantioxidant activity in the 120573-carotene-linoleic acid systemAmong all the extracted components ET is the highest inthe reducing power (119875 lt 005) and the reducing power ofthe seven subcomponents isolated fromET is in the followingorder E5 gt E6 gt E4 gt E3 gt E7 gt E2 gt E1

35 Structural Identification of Compound Compound 1colorless needle crystal is identified as C

27H46O with EI-MS

and NMR 120575347 in 1H NMR 120575718 in 13C NMR and DEPTshow that there is hydroxyl in themolecule 120575531 in 1HNMR12057514018 and 1205751217 in 13C NMR and DEPT further confirmthe double bond in the molecule mz 255 and 147 in MScombinedwith 120575531 (1H H-7) 120575356 (1HmH-3) 120575101 (3Hs Me-19) and 120575088 (3H s Me-18) in 1HMMR 1205751408 (s C-8) 1205751217 (d C-7) and 120575718 (d C-3) in 13CNMR and DEPTreferred to the physical constant and spectral data reportedby Tsuda and Schroepfer Jr [19] compound 1 was identifiedas cholesteric-7-en-3120573-ol whichwas isolated from S clava forthe first time

Compound 2 colorless needle crystal mp 244sim246∘C isidentified as C

27H46O2with EI-MS andNMRThe two signals

of methyl group connected with quaternary carbon (120575126 s120575071 s) and three signals of methyl group connected withtertiary carbon (120575091 d 120575087 d 120575085 d) show that thiscompound is sterol The signal of 19-Me at 120575126 shifts toa lower field in contrast to cholesterol which proves theexistence of 6120573-OH Meanwhile in contrast to cholesterolsignal (120575418) of 3120572-H also shifts to a lower field and so is

Journal of Chemistry 7

HO

HO

HO

OH

OH

OH

OH

OH

H2C

H2C (CH2)17CH3

C

O

O

(CH2)16CH3

CH3(CH2)14

NH

HC

Compound 1 Compound 2

Compound 3 Compound 4

Compound 5

Figure 4 Structures of compounds

proved of 45-double bond considering single peak 120575555Compound 2 is identified as cholesteric-4-en-3120573 6120573-diolreferring to the physical constant and spectral data reportedby Wahidullah et al [20] which is isolated from S clava forthe first time

Compound 3 is a white needle crystal mp 148sim149∘Cpurplish red at 10 sulfuric acid-ethanol solution The mul-tiple peaks at 120575353 in 1H NMR is the characteristic signal of3120572-H in sterol while the multiple peaks at 120575535 is signal ofH-6 In addition five methyl group signals appear in highfield of 1H NMR among which the 120575101 s and 120575068 sbelong to 19-Me and 18-Me respectively 120575091 d is the signalof 21-Me and 120575084 d and 120575082 d belong to 26-Me and27-Me respectively Contrasting the physical constant andspectral data with Su et al [21] compound 3 is identified ascholesterol

Compound 4 is white powder mp 68sim69∘C purplish redat 10 sulfuric acid-ethanol solution The molecular weight

is 345 according to EI-MS As shown in 13C NMR spectrum120575641 (t C-1) 120575319 (t C2) 120575296 (t C3-C15) 120575260 (t C16)120575226 (t C17) and 120575141 (t C18) are characteristic signalsof long chain fatty acid and 120575722 (C-1) 120575706 (C-2) and120575718 (C-3) belong to glycerol so this compound should bea long chain triglyceride However there is no signal of estercarbonyl therefore this compound is identified as glycidylether agreeing with Yang et al [22] andWang et al [23] andis confirmed as batilol which is isolated from S clava for thefirst time

Compound 5 molecular formula is C36H71NO3accord-

ing to EI-MS andNMR 120575637 (1H) in 1HNMR spectrum and1205751739 (C=O) in 13C NMR spectrum proves the existence ofacylamino and 120575545 in 13C NMR spectrum indicates thatwhat is connected with nitrogen is methyne 120575202 (2H) in1HNMR indicates that there are two ndashOH in the compoundand 1205757474 and 1205756240 in 13C NMR indicate that ndashCHndash andndashCH2ndash connect with ndashOH respectively Meantime a strong

8 Journal of Chemistry

hydrogen signal of 120575126 in 1H NMR spectrum and twosignals ndashCH

3ndash of 120575084 (6H) determine two long chain alkyl

groups This compound is identified as ceramide referring tothe physical constant and spectral data reported by Yu andYang [24]

The structures of these compounds are shown in Figure 4As reported by Cai et al [25] the number and positionof hydroxyl groups largely determined radical scavengingactivity of phenolic compounds Compounds 1 2 and 3 allhave only one phenolic hydroxyl group and there is nofunctional groups in orthoposition little steric hindranceeffect increased their contact with free radicals

4 Conclusion

The immunomodulatory and antioxidant activity of crudeand fractionated extracts of the ascidian Styela clava weredetermined by in vitro screening All these extracts demon-strate immunomodulatory activity through increasing theproliferation rate of spleen lymphocyte and macrophagesas well as the NO release activities of macrophages Amongthem the petroleum ether fraction shows the strongestimmune active in vitro The ethyl acetate fraction (ET) wasmuch higher in its antioxidant activity in DPPH systemreducing power assay and 120573-carotene-linoleic acid systemcompared with the other fractions and its subcomponentE5 demonstrated the strongest antioxidant activity as well asreducing power higher than the positive control AscA andclose to the synthesized GA ET was isolated systemically andfive compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol (4) batiloland (5) ceramide among which (1) (2) and (4) were isolatedfor the first time from S clava

Conflict of Interests

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

Authorsrsquo Contribution

Ju Bao and Chen Bin contributed equally to this project

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (no 31070368) and the Sci-ence and Technology Department of Shandong Province(BS2010HZ022 and ZR2011DL008)

References

[1] T M Zabriskie and C M Ireland ldquoThe isolation and structureofmodified bioactive nucleosides from Jaspis johnstonirdquo Journalof Natural Products vol 52 no 6 pp 1353ndash1356 1989

[2] J Kobayashi M Tsuda M Ishibashi et al ldquoAmphidinolideF a new cytotoxic macrolide from the marine dinoflagellateAmphidinium sprdquo Journal of Antibiotics vol 44 no 11 pp 1259ndash1261 1991

[3] B S Davidson and C M Ireland ldquoLissoclinolide the first non-nitrogenous metabolite from a Lissoclinum tunicaterdquo Journal ofNatural Products vol 53 no 4 pp 1036ndash1038 1990

[4] T N Makarieva V A Stonik A S Dmitrenok et al ldquoVaracinand three new marine antimicrobial polysulfides from the far-Eastern ascidian Polycitor sprdquo Journal of Natural Products vol58 no 2 pp 254ndash258 1995

[5] P W Ford and B S Davidson ldquoPlakinidine D a newpyrroloacridine alkaloid from the ascidianDidemnum rubeumrdquoJournal of Natural Products vol 60 no 10 pp 1051ndash1053 1997

[6] C Ireland and P J Scheuer ldquoUlicyclamine and ulithiacy-clamide two new small peptides from a marine tunicaterdquoJournal of the American Chemical Society vol 102 no 17 pp5688ndash5691 1980

[7] H G Chun B Davies and D Hoth ldquoDidemnin B the firstmarine compound entering clinical trials as an antineoplasticagentrdquo Investigational New Drugs vol 4 no 3 pp 279ndash2841986

[8] M A Baker D R Grubb and A Lawen ldquoDidemnin B inducesapoptosis in proliferating but not resting peripheral bloodmononuclear cellsrdquo Apoptosis vol 7 no 5 pp 407ndash412 2002

[9] M D Vera and M M Joullie ldquoNatural products as probes ofcell biology 20 Years of didemnin researchrdquoMedicinal ResearchReviews vol 22 no 2 pp 102ndash145 2002

[10] S Jin B Gorfajn G Faircloth and K W Scotto ldquoEcteinascidin743 a transcription-targeted chemotherapeutic that inhibitsMDR1 activationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 12 pp 6775ndash6779 2000

[11] C van Kesteren E Cvitkovic A Taamma et al ldquoPharma-cokinetics and pharmacodynamics of the novel marine-derivedanticancer agent ecteinascidin 743 in a phase I dose-findingstudyrdquo Clinical Cancer Research vol 6 no 12 pp 4725ndash47322000

[12] E G C Brain ldquoSafety and efficacy of ET-743 the Frenchexperiencerdquo Anti-Cancer Drugs vol 13 supplement 1 pp S11ndashS14 2002

[13] J C M Waterval J C Bloks R W Sparidans et al ldquoDegra-dation kinetics of aplidine a new marine antitumoural cyclicpeptide in aqueous solutionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 754 no 1 pp 161ndash1682001

[14] C K Cai Research of anti-HBV activity and pharmacodynamicaction and toxicity of SC [PhD thesis] Beijing University ofChinese Medicine Beijing China 2006

[15] W Binsan S Benjakul W Visessanguan S Roytrakul MTanaka and H Kishimura ldquoAntioxidative activity of Mun-goong an extract paste from the cephalothorax ofwhite shrimp(Litopenaeus vannamei)rdquo Food Chemistry vol 106 no 1 pp185ndash193 2008

[16] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[17] G C Yen and P D Duh ldquoAntioxidative properties of methano-lic extracts from peanut hullsrdquo Journal of the American OilChemistsrsquo Society vol 70 no 4 pp 383ndash386 1993

[18] R N Albrecht Immunologie Tolerance and Macrophage Func-tion Aeademie New York NY USA 1 edition 1979

[19] M Tsuda and G J Schroepfer Jr ldquoCarbon-13 nuclear magneticresonance studies of C27 sterol precursors of cholesterolrdquoJournal of Organic Chemistry vol 44 no 8 pp 1290ndash1293 1979

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 7

HO

HO

HO

OH

OH

OH

OH

OH

H2C

H2C (CH2)17CH3

C

O

O

(CH2)16CH3

CH3(CH2)14

NH

HC

Compound 1 Compound 2

Compound 3 Compound 4

Compound 5

Figure 4 Structures of compounds

proved of 45-double bond considering single peak 120575555Compound 2 is identified as cholesteric-4-en-3120573 6120573-diolreferring to the physical constant and spectral data reportedby Wahidullah et al [20] which is isolated from S clava forthe first time

Compound 3 is a white needle crystal mp 148sim149∘Cpurplish red at 10 sulfuric acid-ethanol solution The mul-tiple peaks at 120575353 in 1H NMR is the characteristic signal of3120572-H in sterol while the multiple peaks at 120575535 is signal ofH-6 In addition five methyl group signals appear in highfield of 1H NMR among which the 120575101 s and 120575068 sbelong to 19-Me and 18-Me respectively 120575091 d is the signalof 21-Me and 120575084 d and 120575082 d belong to 26-Me and27-Me respectively Contrasting the physical constant andspectral data with Su et al [21] compound 3 is identified ascholesterol

Compound 4 is white powder mp 68sim69∘C purplish redat 10 sulfuric acid-ethanol solution The molecular weight

is 345 according to EI-MS As shown in 13C NMR spectrum120575641 (t C-1) 120575319 (t C2) 120575296 (t C3-C15) 120575260 (t C16)120575226 (t C17) and 120575141 (t C18) are characteristic signalsof long chain fatty acid and 120575722 (C-1) 120575706 (C-2) and120575718 (C-3) belong to glycerol so this compound should bea long chain triglyceride However there is no signal of estercarbonyl therefore this compound is identified as glycidylether agreeing with Yang et al [22] andWang et al [23] andis confirmed as batilol which is isolated from S clava for thefirst time

Compound 5 molecular formula is C36H71NO3accord-

ing to EI-MS andNMR 120575637 (1H) in 1HNMR spectrum and1205751739 (C=O) in 13C NMR spectrum proves the existence ofacylamino and 120575545 in 13C NMR spectrum indicates thatwhat is connected with nitrogen is methyne 120575202 (2H) in1HNMR indicates that there are two ndashOH in the compoundand 1205757474 and 1205756240 in 13C NMR indicate that ndashCHndash andndashCH2ndash connect with ndashOH respectively Meantime a strong

8 Journal of Chemistry

hydrogen signal of 120575126 in 1H NMR spectrum and twosignals ndashCH

3ndash of 120575084 (6H) determine two long chain alkyl

groups This compound is identified as ceramide referring tothe physical constant and spectral data reported by Yu andYang [24]

The structures of these compounds are shown in Figure 4As reported by Cai et al [25] the number and positionof hydroxyl groups largely determined radical scavengingactivity of phenolic compounds Compounds 1 2 and 3 allhave only one phenolic hydroxyl group and there is nofunctional groups in orthoposition little steric hindranceeffect increased their contact with free radicals

4 Conclusion

The immunomodulatory and antioxidant activity of crudeand fractionated extracts of the ascidian Styela clava weredetermined by in vitro screening All these extracts demon-strate immunomodulatory activity through increasing theproliferation rate of spleen lymphocyte and macrophagesas well as the NO release activities of macrophages Amongthem the petroleum ether fraction shows the strongestimmune active in vitro The ethyl acetate fraction (ET) wasmuch higher in its antioxidant activity in DPPH systemreducing power assay and 120573-carotene-linoleic acid systemcompared with the other fractions and its subcomponentE5 demonstrated the strongest antioxidant activity as well asreducing power higher than the positive control AscA andclose to the synthesized GA ET was isolated systemically andfive compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol (4) batiloland (5) ceramide among which (1) (2) and (4) were isolatedfor the first time from S clava

Conflict of Interests

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

Authorsrsquo Contribution

Ju Bao and Chen Bin contributed equally to this project

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (no 31070368) and the Sci-ence and Technology Department of Shandong Province(BS2010HZ022 and ZR2011DL008)

References

[1] T M Zabriskie and C M Ireland ldquoThe isolation and structureofmodified bioactive nucleosides from Jaspis johnstonirdquo Journalof Natural Products vol 52 no 6 pp 1353ndash1356 1989

[2] J Kobayashi M Tsuda M Ishibashi et al ldquoAmphidinolideF a new cytotoxic macrolide from the marine dinoflagellateAmphidinium sprdquo Journal of Antibiotics vol 44 no 11 pp 1259ndash1261 1991

[3] B S Davidson and C M Ireland ldquoLissoclinolide the first non-nitrogenous metabolite from a Lissoclinum tunicaterdquo Journal ofNatural Products vol 53 no 4 pp 1036ndash1038 1990

[4] T N Makarieva V A Stonik A S Dmitrenok et al ldquoVaracinand three new marine antimicrobial polysulfides from the far-Eastern ascidian Polycitor sprdquo Journal of Natural Products vol58 no 2 pp 254ndash258 1995

[5] P W Ford and B S Davidson ldquoPlakinidine D a newpyrroloacridine alkaloid from the ascidianDidemnum rubeumrdquoJournal of Natural Products vol 60 no 10 pp 1051ndash1053 1997

[6] C Ireland and P J Scheuer ldquoUlicyclamine and ulithiacy-clamide two new small peptides from a marine tunicaterdquoJournal of the American Chemical Society vol 102 no 17 pp5688ndash5691 1980

[7] H G Chun B Davies and D Hoth ldquoDidemnin B the firstmarine compound entering clinical trials as an antineoplasticagentrdquo Investigational New Drugs vol 4 no 3 pp 279ndash2841986

[8] M A Baker D R Grubb and A Lawen ldquoDidemnin B inducesapoptosis in proliferating but not resting peripheral bloodmononuclear cellsrdquo Apoptosis vol 7 no 5 pp 407ndash412 2002

[9] M D Vera and M M Joullie ldquoNatural products as probes ofcell biology 20 Years of didemnin researchrdquoMedicinal ResearchReviews vol 22 no 2 pp 102ndash145 2002

[10] S Jin B Gorfajn G Faircloth and K W Scotto ldquoEcteinascidin743 a transcription-targeted chemotherapeutic that inhibitsMDR1 activationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 12 pp 6775ndash6779 2000

[11] C van Kesteren E Cvitkovic A Taamma et al ldquoPharma-cokinetics and pharmacodynamics of the novel marine-derivedanticancer agent ecteinascidin 743 in a phase I dose-findingstudyrdquo Clinical Cancer Research vol 6 no 12 pp 4725ndash47322000

[12] E G C Brain ldquoSafety and efficacy of ET-743 the Frenchexperiencerdquo Anti-Cancer Drugs vol 13 supplement 1 pp S11ndashS14 2002

[13] J C M Waterval J C Bloks R W Sparidans et al ldquoDegra-dation kinetics of aplidine a new marine antitumoural cyclicpeptide in aqueous solutionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 754 no 1 pp 161ndash1682001

[14] C K Cai Research of anti-HBV activity and pharmacodynamicaction and toxicity of SC [PhD thesis] Beijing University ofChinese Medicine Beijing China 2006

[15] W Binsan S Benjakul W Visessanguan S Roytrakul MTanaka and H Kishimura ldquoAntioxidative activity of Mun-goong an extract paste from the cephalothorax ofwhite shrimp(Litopenaeus vannamei)rdquo Food Chemistry vol 106 no 1 pp185ndash193 2008

[16] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[17] G C Yen and P D Duh ldquoAntioxidative properties of methano-lic extracts from peanut hullsrdquo Journal of the American OilChemistsrsquo Society vol 70 no 4 pp 383ndash386 1993

[18] R N Albrecht Immunologie Tolerance and Macrophage Func-tion Aeademie New York NY USA 1 edition 1979

[19] M Tsuda and G J Schroepfer Jr ldquoCarbon-13 nuclear magneticresonance studies of C27 sterol precursors of cholesterolrdquoJournal of Organic Chemistry vol 44 no 8 pp 1290ndash1293 1979

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 Journal of Chemistry

hydrogen signal of 120575126 in 1H NMR spectrum and twosignals ndashCH

3ndash of 120575084 (6H) determine two long chain alkyl

groups This compound is identified as ceramide referring tothe physical constant and spectral data reported by Yu andYang [24]

The structures of these compounds are shown in Figure 4As reported by Cai et al [25] the number and positionof hydroxyl groups largely determined radical scavengingactivity of phenolic compounds Compounds 1 2 and 3 allhave only one phenolic hydroxyl group and there is nofunctional groups in orthoposition little steric hindranceeffect increased their contact with free radicals

4 Conclusion

The immunomodulatory and antioxidant activity of crudeand fractionated extracts of the ascidian Styela clava weredetermined by in vitro screening All these extracts demon-strate immunomodulatory activity through increasing theproliferation rate of spleen lymphocyte and macrophagesas well as the NO release activities of macrophages Amongthem the petroleum ether fraction shows the strongestimmune active in vitro The ethyl acetate fraction (ET) wasmuch higher in its antioxidant activity in DPPH systemreducing power assay and 120573-carotene-linoleic acid systemcompared with the other fractions and its subcomponentE5 demonstrated the strongest antioxidant activity as well asreducing power higher than the positive control AscA andclose to the synthesized GA ET was isolated systemically andfive compounds were separated as (1) cholesteric-7-en-3120573-ol (2) cholesteric-4-en-31205736120573-diol (3) cholesterol (4) batiloland (5) ceramide among which (1) (2) and (4) were isolatedfor the first time from S clava

Conflict of Interests

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

Authorsrsquo Contribution

Ju Bao and Chen Bin contributed equally to this project

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (no 31070368) and the Sci-ence and Technology Department of Shandong Province(BS2010HZ022 and ZR2011DL008)

References

[1] T M Zabriskie and C M Ireland ldquoThe isolation and structureofmodified bioactive nucleosides from Jaspis johnstonirdquo Journalof Natural Products vol 52 no 6 pp 1353ndash1356 1989

[2] J Kobayashi M Tsuda M Ishibashi et al ldquoAmphidinolideF a new cytotoxic macrolide from the marine dinoflagellateAmphidinium sprdquo Journal of Antibiotics vol 44 no 11 pp 1259ndash1261 1991

[3] B S Davidson and C M Ireland ldquoLissoclinolide the first non-nitrogenous metabolite from a Lissoclinum tunicaterdquo Journal ofNatural Products vol 53 no 4 pp 1036ndash1038 1990

[4] T N Makarieva V A Stonik A S Dmitrenok et al ldquoVaracinand three new marine antimicrobial polysulfides from the far-Eastern ascidian Polycitor sprdquo Journal of Natural Products vol58 no 2 pp 254ndash258 1995

[5] P W Ford and B S Davidson ldquoPlakinidine D a newpyrroloacridine alkaloid from the ascidianDidemnum rubeumrdquoJournal of Natural Products vol 60 no 10 pp 1051ndash1053 1997

[6] C Ireland and P J Scheuer ldquoUlicyclamine and ulithiacy-clamide two new small peptides from a marine tunicaterdquoJournal of the American Chemical Society vol 102 no 17 pp5688ndash5691 1980

[7] H G Chun B Davies and D Hoth ldquoDidemnin B the firstmarine compound entering clinical trials as an antineoplasticagentrdquo Investigational New Drugs vol 4 no 3 pp 279ndash2841986

[8] M A Baker D R Grubb and A Lawen ldquoDidemnin B inducesapoptosis in proliferating but not resting peripheral bloodmononuclear cellsrdquo Apoptosis vol 7 no 5 pp 407ndash412 2002

[9] M D Vera and M M Joullie ldquoNatural products as probes ofcell biology 20 Years of didemnin researchrdquoMedicinal ResearchReviews vol 22 no 2 pp 102ndash145 2002

[10] S Jin B Gorfajn G Faircloth and K W Scotto ldquoEcteinascidin743 a transcription-targeted chemotherapeutic that inhibitsMDR1 activationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 12 pp 6775ndash6779 2000

[11] C van Kesteren E Cvitkovic A Taamma et al ldquoPharma-cokinetics and pharmacodynamics of the novel marine-derivedanticancer agent ecteinascidin 743 in a phase I dose-findingstudyrdquo Clinical Cancer Research vol 6 no 12 pp 4725ndash47322000

[12] E G C Brain ldquoSafety and efficacy of ET-743 the Frenchexperiencerdquo Anti-Cancer Drugs vol 13 supplement 1 pp S11ndashS14 2002

[13] J C M Waterval J C Bloks R W Sparidans et al ldquoDegra-dation kinetics of aplidine a new marine antitumoural cyclicpeptide in aqueous solutionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 754 no 1 pp 161ndash1682001

[14] C K Cai Research of anti-HBV activity and pharmacodynamicaction and toxicity of SC [PhD thesis] Beijing University ofChinese Medicine Beijing China 2006

[15] W Binsan S Benjakul W Visessanguan S Roytrakul MTanaka and H Kishimura ldquoAntioxidative activity of Mun-goong an extract paste from the cephalothorax ofwhite shrimp(Litopenaeus vannamei)rdquo Food Chemistry vol 106 no 1 pp185ndash193 2008

[16] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[17] G C Yen and P D Duh ldquoAntioxidative properties of methano-lic extracts from peanut hullsrdquo Journal of the American OilChemistsrsquo Society vol 70 no 4 pp 383ndash386 1993

[18] R N Albrecht Immunologie Tolerance and Macrophage Func-tion Aeademie New York NY USA 1 edition 1979

[19] M Tsuda and G J Schroepfer Jr ldquoCarbon-13 nuclear magneticresonance studies of C27 sterol precursors of cholesterolrdquoJournal of Organic Chemistry vol 44 no 8 pp 1290ndash1293 1979

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 9

[20] S Wahidulla L DrsquoSouza andM Govenker ldquoLipid constituentsof the red alga Acantophora spiciferardquo Phytochemistry vol 48no 7 pp 1203ndash1206 1998

[21] G C Su S Zhang and S H Qi ldquoChemical constituents ofAmtipathes dichotomardquo Chinese Traditional and Herbal Drugsvol 39 no 11 pp 1606ndash1609 2008

[22] J Yang S-H Qi S Zhang andQ-X Li ldquoChemical constituentsfrom the south China sea gorgonian coral Subergorgia reticu-latardquo Journal of Chinese medicinal materials vol 29 no 6 pp555ndash558 2006

[23] G S Wang Q Liu and L M Zeng ldquoThe chemical constituentsof soft Cladiella densa from South China Seardquo Acta ScientiarumNaturalium Universitatis Sunyatseni vol 34 no 1 pp 110ndash1121995

[24] D Q Yu and J S Yang Handbook of Analytical Chmuistry vol7 of 123 Chemical Industry Press Beijing China 2nd edition1999

[25] Y Z Cai M S M Sun J X J Xing Q Luo and H CorkeldquoStructure-radical scavenging activity relationships of phenoliccompounds from traditional Chinese medicinal plantsrdquo LifeSciences vol 78 no 25 pp 2872ndash2888 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of