International Journal of Marine Science, 2017, Vol.7, No.31, 297-307
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2.3 Isolation of secondary metabolites compounds of
H.gracilllis
ethyl acetate
The crude extract of ethyl acetate as much as 2 g after gravity column chromatography vial vial-column results
were analyzed using TLC. TLC analysis of the results obtained 6 column fractions F1-F6. F1 0.94; F2 0.23; F3
0.11; F4 0.16; F5 0.20; and F6 0.09 g. Fraction F1 suggest the potential of secondary metabolites in the presence
of a white powder, TLC analysis results indicate that the compound is not pure then carried back to the field of
gravity separation obtained 3 fractions F1.1, F1.2, and F1.3. F1.2 fraction as much as 0.75 g after analyzed by
TLC using cerium sulfate indicates that the compound was pure (Figure 3).
Figure 2 Results TLC pure compound H.gracilis fraction of
n-hexane at cerium sulfate with the eluent n-hexane: EtOAc
(6:4)
Figure 3 Results TLC pure compound H.gracilis fraction of
n-hexane at cerium sulfate with the eluent n-hexane: EtOAc
(8:2)
2.4 Identification of isolated compounds results
2.4.1 Identification with 1H-NMR spectrum
1H-NMR spectrum of the compound showed signals typical isolation for a group of steroid/triterpenoid
compounds. This can be seen from the signal that accumulated in the area δH 0 - 3 ppm. 1H-NMR spectrum
showed characteristic absorption for the proton attached to the carbon sp2 (proton vinilik) in the area δH 5.34 ppm
(1H, t, J = 5.0 Hz), suspected of protons in this area is metin proton (CH) which cleaved by two neighboring
protons. 1H-NMR spectrum also showed uptake of protons bound to heteroatoms in the δH 3.51 ppm (1H, m)
which is suspected as metin proton divided by four neighboring protons. These protons on the steroid/triterpenoids
compound usually bound to third carbon atom (C3).
Fragment of 1H-NMR spectrum showed signals for two methylene protons that each is in the area δH 2.23 ppm –
δH 2.34 and δH 1.94 ppm ppm - δH 2.02 ppm (2H, m). Signal in the area δH 1.79 ppm - δH 1.85 ppm (3H, m) is
suspected as one of the metin protons and two methylene protons. Proton signals with the integration of the three
was also seen in the area δH 1.67 ppm (3H, m) and the signal is thought to also represent the methyl protons and
two methylene protons. 1H-NMR spectrum shows that the proton in the area δH 1.46 ppm – δH 1.50 ppm (6H, m)
allegedly representing three methylene protons, then the signal that appears in the δH 1.33 ppm (5H, m) is
suspected as four methylene protons and the protons metin. Methylene proton signals were also seen in the above
areas δH 1.01 ppm with four integration protons alleged methylene protons. 1H-NMR spectrum also showed the
presence of 4 pieces of signals for methyl protons. Fourth proton signals appear in the δH 1.00 ppm (3H, s); δH
0.82 ppm – δH 0.84 ppm (6H, m); δH 0.79 – δH 0.81 ppm (6H, d) and in the area δH 0.67 ppm (3H, s).
2.4.2 Identification with 13C-NMR spectrum
13C-NMR spectrum of isolated compound showed 29 carbon signals, two signals in the form of C sp2 namely in
the area δc 140.9 ppm and 121.9 ppm δc area and 27 signal in the form of C sp3. Signal sp3 carbon C located in
area, respectively: δc 71.9; 56.9; 56.2; 50.3; 46.2; 42.5; 42.4; 39.9; 37.4; 36.7; 36.4; 34.1; 32.1; 32.0; 31.8; 29.1;
28.4; 26.5; 24.5; 23.2;; 21.3; 19.8; 19.6; 19.2; 19.0; 12.5, and 12.0 ppm.
