SUPPLEMENTARY MATERIAL
Phytochemical study of Caulerpa racemosa (Forsk.) J. Agarth, an invading alga
in the habitat of La Maddalena Archipelago
Luigi Ornanoac, Yuri Donnob, Cinzia Sannac, Mauro Balleroc, Mauro Serafinidc, Armandodoriano
Biancoac.
a
Dipartimento di Chimica, Università di Roma “La Sapienza”, P.le Aldo Moro, 5, 00185 Roma.
b
Parco Nazionale, Arcipelago della Maddalena, Via D. Millelire, , 07024 La Maddalena (OT).
c
Consorzio CoSMeSe, Dipartimento di Scienze della Vita e dell’Ambiente, Università di Cagliari,
Via S.Ignazio da Laconi 11, 09123 Cagliari.
a
Dipartimento di Biologia Ambientale, Università di Roma “La Sapienza”, P.le Aldo Moro, 5,
00185 Roma.
<luigi.ornano@uniroma1.it>
Abstract
Caulerpa racemosa is a marine Chlorophyta, widely distributed in tropical areas,
introduced into the Mediterranean Sea since 1990. It has been invading the
Mediterranean Sea, causing ecological problems. This invasive event can be
considered as one of the most serious in the history of species introduced into the
Mediterranean Sea, even if C. racemosa has not triggered as much attention as the
famous ‘‘killer alga’’ Caulerpa taxifolia. The aim of this work is the
phytochemical analysis of C. racemosa for the first time investigated in the
Northern Sardinia area for secondary metabolites. Marine algae shows the
molecular pattern of bis-indole alkaloids, sesquiterpenes, diterpenes and sterols.
The intention to expand phytochemical analysis in order to understand just how
significant the anti-tumor, anti-inflammatory and antinociceptive actions can be.
Key
words:
sesquiterpenoids
Caulerpa
racemosa,
caulepin,
racemosin,
caulerprenyol,
1. Experimental
1.1 Plant material
Caulerpa racemosa were collected at the end of November 2013 from marine
protected area, in the La Maddalena archipelago, at south of Budelli island. (GPS
coordinates: N 41°16' 47.58" E9° 21' 35.67"). The samples were identified by
Dr.Yuri Donno (National Pak of La Maddalena Archipelago) and Dr.ssa Cinzia
Sanna (Co.S.Me.Se.).
1.2 Extraction and isolation
The fresh marine alga (dry weight,166.6g) was removed unwanted impurities,
then was extracted three times with 96% aqueous EtOH (1L for each) for four
days each times at room temperature. The obtained extracts were concentrated
under reduced pressure to eliminate the ethanol and resulting water suspension
was first frozen to -20°C and lyophilized to recover 1.6 g of crude extract. A
portion of 1.6 g was partitioned on silica gel column using chloroform/methanol
9:1.
1.3 Spectrometric identification
NMR spectra were recorder on Varian Mercury 300 MHz instrument using
CDCl3, CD3OD and D2O as deuterated solvents, the chemical shift was expressed
in ppm from TMS (the signal of HDO at 4.78 ppm is used as reference for spectra
in D2O). MS spectra were performed on a Q-TOFMICRO spectrometer
(MIcromass, now Waters, Manchester UK) equipped with an ESI source, that was
operated in the negative and/or positive ion mode. The flow rate of sample
injection was 10µL/min. With 100 acquisition per spectrum. Data were analyzed
using Masslynx software developed by Waters.
Caulerpin (1): (dimethyl(6E,13E)-5,12-dihydrocycloocta[1,2-b:5,6-b']diindole6,13-dicarboxylate), (Ciavatta ML et al. 2006).
caulerpic acid (2): (6E,13E)-5,12-dihydrocycloocta[1,2-b:5,6-b']diindole-6,13dicarboxylic caulerpic acid (Ciavatta MLet al. 2006).
Compound 1: 1H-NMR, CD3OD, 300 MHz, : 3.88 (H-11, 3H, s), : 8.04 (H-9,
1H, s), : 7.17 (H-6, 1H, dt, J=1.4, 7.8 Hz), : 7.07 (H-5, 1H, dt J=1.4, 7.8Hz), :
7.41 (H-4, 1H, dt, J=7.6Hz), : 9.02 (NH-1, 1H, s).13C-NMR, (CD3OD) 300
MHz, : 67.74 (-OMe), 106.647 (C-3), 113.35 (C-7), 114.82 (C-4), 122.98 (C-6),
123.35 (C-5), 123.37 (C-3a), 124.25 (C-8), 134.27 (C-2), 137.52 (C-7a), 148.74
(C-9’), 168.70 (C-10’). ESI -MS: m/z[M-H]- =397.1191 .
Compound 2:1H-NMR, CD3OD, 300 MHz, : 6.85 (m, 8H-Ar), : 8.15 (s, 2H,
CH=C),
13
C-NMR, CD3OD, 300 MHz, 112.9), 118.6, 123.71273.0, 128.2, 138.9
134.2,143.1,167.36 ESI-MS: m/z[M-H]- = 369.1685.
Racemosin A (3): (methyl-2-((Z)-3-oxoindolin-2-ylidene)-3-((Z)-2-oxoindolin-3ylidene)propanoate) (Liu DQ et al 2013).
Compound 3: 1H-NMR, CD3OD, 300 MHz, : 3.68 (H-10, s), : 7.17 (H-6, 1H, dt,
J=1.4, 7.8Hz), : 6.83 (3’a,5’’, d J=7.8 Hz), : 7.06 (3’a,7’a, t, J= 7.6Hz), : 7.24
(4’,7’a, t, J= 7.8Hz), : 7.62 (6’,7’a, d, J= 7.8Hz), : 7.72 (br s), : 9.30 (br s).13CNMR, CD3OD, 300 MHz, : 53.7(-OMe), 166.6(C-10’), 129.3(C-9’), 127.72(C8’), 140.2 (C-7’a), 109.3 (C-7’), 129.3 (C-6’), 122.2 (C-5’), 120.1(C-4’), 124.2(C3’a), 108.7 (C-3’), 167.70(C-2’), 152.1 (C-7’), 111.5 (C-7), 122.1 (C-5’), 152.1
(C-7’), 112.8 (C-7), 137.1 (C-6), 121.1 (C-5), 125.0 (C-4), 120.6 (C-3a), 180.0
(C-3), 143.1 (C-2).
ESI-MS: m/z[M-Na]+ =369.1385.
5,11-dihydroindolo[3,2-b]carbazole, (4) (Liu DQ et al 2013)
Compound 4: 1H-NMR, CD3OD, 300 MHz : 8.22 (H-3,H-3a,H-6,H-7a) (1H, d,
J=7.8Hz), : 7.31(H-3a,H-6,H-7) (1H, t, J=7.8Hz), : 7.40 (H-7a,H-4) (1H, t,
J=7.8Hz), : 7.42 (H-3a,H-5) (1H, d, J=7.8Hz), : 9.02 (NH-1, 1H, s),13C-NMR
CD3OD, 300 MHz :120.4 (C-H,3,3a,6,7a), :120.1 (C-H, 3a,6,7a), :125.2 (CH, 4,7a), :111,4 (C-H, 3a,5).
ESI-MS: m/z[M-Na]+ =279.2564
Caulerpal A, (5): (1S)-4-formyl-1-hydroxy-1-methyl-7-(2-methylprop-1-en-1-yl)2,3-dihydro-1H-inden-2-yl acetate (Liu AH: et al 2013) (Mao SC. et al 2006).
Compound 5: 1H-NMR, CD3OD, 300 MHz : 6.80 (bs,H-11) : 10.01(s, H-15,
CHO), : 7.40 (d, t, J=7.8Hz,H-6), : 7.65 (d, t, J=7.8Hz,H-7), : 1.85 (s, H-13),
: 3.02 (dd, J=17.4Hz, 8.9Hz, Ha1), : 3.90 (dd, J=17.4Hz, 8.9Hz, Ha1), : 5.27
(dd, J=17,4Hz, 8,9Hz, H-2), : 1.30 (s, H3-10),
C-NMR CD3OD, 300 MHz :121.5 (C-11), :138.1 (C-12), :25.5 (C-14),
13
:19,5 (C-13), :141,5 (C-5), :143,0 (C-6,C-4), :130,6 (C-5, C-8).
ESI-MS: m/z[M-Na]+ =311.2831
Caulerpal B, (6): ((1S)-2-hydroxy-1-methoxy-1-methyl-7-(2-methylprop-1-en-1yl)-2,3-dihydro-1H-indene-4-carbaldehyde) (Mao SC et al 2006).
Compound 6: 1H-NMR, CD3OD, 300 MHz : 2.83 (s, H-10, O-CH3) : 3.83 (s, H2), : 5.70-5.29 (m, H-2 -OH). 13C-NMR CD3OD, 300 MHz :33.5 (C-1), :74.1
(C-2), :140.5 (C-9), :141,5 (C-43).
ESI-MS: m/z[M-H]- . =258.9013
Occidol (7). ((R)-2-(5,8-dimethyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-2-ol),
(Liu AH et al 2006)
Compound 7: 1H-NMR, CD3OD, 300 MHz : 2.15-2.22 (m, 2H, -CH2), : 2.26(s,
3H, H-2), : 2.81-3.03 (m, 4H), : 3.05 (m, 3H), : 5.15-5.28 (m, 1H), : 6.85 (bs,
3H), : 6.92 (bs, 1H).13C-NMR CD3OD, 300 MHz :136.5 (C-10), :135.5 (C-9),
:130.5 (C-8), :129,5 (C-5), :128,7 (C-6), :78,5 (C-2), :39,7 (C-13), :33,5
(C-3), :28,8 (C-1), :26,4 (C-4), :20,5 (C-12), :19,3 (C-11).
ESI-MS: m/z[M-Na]+ =240.9992
((E)-2-(2-oxoethyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-2-en-1-yl
acetate)
(8), (Paul VJ et al 1987).
Compound 8:1H-NMR, (CDCl3) 300 MHz, : 1.98 (s, OAc), : 0.92 (s H-15), :
0.88 (s, H-14), : 1,65 (s, H-13), : 4.45 (s, H-12), : 1.15 (s, H-10), : 1.45 (m,
H-12), : 5.35 (m, H-8), : 1.60 (m, H-6), : 2.16 (mm, H-5), : 5.81 (t, J=7Hz, H4), : 3.11 (t, J=2Hz, H -2), : 9.61 (t, J=2Hz, H-1)13C-NMR, (CDCl3) 300 MHz,
:196.3 (C-1), :44.3 (C-2), :125.3 (C-3), :136.2 (C-4), :29.2 (C-5), :49.9
(C-67),
:135.5
(C-7),
:121.5
(C-8),:31.6(C-9),:23.0(C-10),:32.5(C-
11),:69.5(C-12), :27.0(C-13), :28.6(C-15), :165.6(OAc).
ESI-MS: m/z[M+Na]+= 301.2386
(1Z,4E)-1,5-diphenylpenta-1,4-diene (9), ( Anjaneyulu ASR et al 1992)
Compound 9:1H-NMR, (CD3OD)300 MHz, : 2.81 (2H (-CH2-), t, J=5.6Hz), :
5.95-6.5 (4H (=CH-), m), : 7.0-7.3 (10H (H-Ar). 13C-NMR, (CD3OD) 300 MHz,
:120.4 (C-H,3,3a,6,7a), :120.1 (C-H, 3a,6,7a), :125.2 (C-H, 4,7a), :111,4 (CH, 3a,5).
ESI-MS: m/z [M-H]- =218.8975
Trans-phitol (10): ((E)-3,7,11,15-tetramethylhexadec-2-en-1-ol, (Wailed et.al.
2011,. Khalid MN et.al. 2011).
Compound 10: 1H-NMR, (CDCl3) 300 MHz , : 0.88-0.90 (6H, d, J=6.6 Hz, CH3), : 0.91 (3H,d, J=6.6 Hz, -CH3), : 0.65 (3H, s, -CH3), : 1.1 (3H, s, -CH3),
: 1.58 (3H, d, J=6.6 Hz, -CH3), : 3.53 (3H, m, H-3), : 5.50 (3H, m, H-6), :
2.28 (m, H-25).13C-NMR, (CDCl3) 300 MHz, :39.3 (C-1), :36.2 (C-2), :71.3
(C-3), :42.5 (C-4), :140.6 (C-5), :120.9 (C-6), :36.5 (C-7), :35.5 (C-8),
:50.1(C-9), :39.1(C-10), :27.5(C-14), :31.5 (C-15), :56.71 (C-17),  :11.6
(C-18), :19.6 (C-19), :39.6 (C-20), :25.6 (C-21), :37.2 (C-22), :31.9 (C-23),
:145.6 (C-24), :115.4 (C-28), :18.8 (C-29).
ESI-MS: m/z [M+Na]+=310.8564
Ergosterol (11): (24-methyl-cholesta,7,22-dien-3β-ol); ((3S,10S,13R)-17-((E)-5,6dimethylhept-3-en-2-yl)-10,13-dimethyl-2,3,4,5,6,9,10,11,12,13,14,15,16,17tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol (Tao R et al, 2013).
Compound 11 1H-NMR, (CDCl3) 300MHz , : 3.58-3.75 (1H, m, H-3), : 5.135.20 (1H, dd, H-22), : 5.20-5.27 (1H, dd, H-23), : 5,37-5,39 (1H, ddd, H-8), :
5.56-5.58 (1H, dd, H-6),
13
C-NMR, (CDCl3) 300MHz, :12.3 (C-18), :16.3 (C-
28), :17.3 (C-17), :19.5 (C-26), :19.6 (C-27), :19.9 (C-27), :21.5 (C-21),
:22.5 (C-15), :28.5 (C-16),:31.0 (C-2), :33.5 (C-25), :39.5 (C-1), :37.0 (C10), :39.6 (C-12), :39.8 (C-20), :40.8 (C-4), :42.8 (C-24), :42.8 (C-13),
:46.8 (C-9), :54.8 (C-14), :55.8 (C-17), :55.8 (C-17), :70.4(C-3), :116.3 (C7), :119.8 (C-6), :132.0 (C-23), :135.0 (C-22), :140.0 (C-5), :142.0 (C-8).
ESI-MS: m/z [M-H]+=397.2564
β-tocopherol
(12):
(R)-2,5,8-trimethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)
chroman-6-ol (Chi-Rong C et al 2007)
β-tocopherol 12: 1H-NMR, (CDCl3) 300MHz, : 0.83-0.88 (m, H-4’, H-8’, H-12’,
-13’), : 1.30 (s, H-2a), : 2.10-2.12 (s, H-5a,H-8b), : 2.65 (2H, m, H-4), : 4.12
(1H, br s, OH-6), : 6.35 (1H, s, H-7).
ESI-MS: m/z [M-H]+=415.3664
palmitic acid (13)
Compound 13:1H-NMR, (CD3OD) 300 MHz, : 0.84-0.93 (3H terminal methyl,
m), : 1,25-1-33 (-CH2 (C-3), m), : 2.17-2.31 (-CH2 (C-2), s)
13
C-NMR,
(CD3OD) 300 MHz, :14.3 (CH3), :22.5 (CH2-αCH3), :29.2 (CH2)-12), :32.2
(CH2-βCH3), :173.7 (COO).
ES-MS: m/z [M+Na]+=279.2564