Sarcophyton species are a group of soft corals belonging to family Alcyoniidae,
subclass Octocorallia, Class Anthozoa and phylum Cnidaria.
These soft corals attracted great attention because they yield a group of cembranoids
with wide spectrum of biological activities. Sarcophine is an abundant cembranolide
diterpene isolated from the Red Sea soft coral, Sarcophyton glaucum (Fig.
1) (Bernstein et al., 1974). Sarcophine constitutes the corals chemical
defense mechanism against the natural predators. It has toxic effects on fish,
mice and rats (Neeman et al., 1974). It acts as an inhibitor of
a number of vital enzymes including cholinesterase and phosphofructokinase by
reacting with their thiol groups (Neeman et al., 1974; Erman and
Neeman, 1977). Sarcophine was also found to have a potential to inhibit
tumorigensis assessed by its ability to inhibit 12-O-tetradecanoylphorbol-13-acetate
(TPA)-induced transformation, a relevant model for investigating neoplastic
transformation (El Sayed et al., 1998).
|| Molecular structure of sarcophine isolated from Sarcophyton
The microbial transformation of sarcophine and its chemical oxidation with
selenium dioxide yielded a group of compounds with cancer chemopreventive activity
higher than sarcophine (El Sayed et al., 1998; Katsuyama et al.,
2002 ; Fahmy et al., 2004).
In addition to sarcophine, the Red Sea Sarcophyton glaucum was found to produce a lactone cembrane diterpene, sarcophytolide. It displayed a strong cytoprotective effect against glutamate- induced neurotoxicity and antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Saccharomyces cerevisiae (Badria et al., 1998).
An important issue that tends to be overlooked in the search for marine natural products and its associated research is that of fundamental taxonomy, that is accurate identification of the species. The identification of Sarcophyton species, as all octocorals, depends on the colony morphology and sclerite architecture, but these characters exhibit an extra-ordinary high degree of variability (Lohuis et al., 1990). The phylogenetic relationships within the Anthozoa were re-evaluated based on the 41 morphological characters and the nuclear sequences of the 18S ribosomal DNA. The trees from the morphological data did not coincide closely with the molecular data (Won et al., 2001). Furthermore, the phylogenetic analysis of the nuclear 18S rDNA from the octocorallians did not support either the historical or the current classification systems. Thus, the morphological characters used to derive the current classification system are not reflected consistently with the results revealed by the 18S rDNA performed (Brenston et al., 2001).
The accurate identification is of enormous importance once the marine organism becomes a source for large scale production of a potential pharmaceutical drug. Hence, for the industrial purposes, the proper species, location and environmental conditions need to be predetermined to ensure a sufficient and steady supply of the drug. Consequently, this study focused on testing the reliability of the morphological and sclerite based taxonomy against the chemical fingerprint and the protein profile for the different species of the genus Sarcophyton from the Red Sea and hence offer new insights for a more precise classification of the genus. Also, the study aimed at determining whether the chemical diversity within the genus is due to the geographical location and/or is species specific.
Materials and Methods
The chemical fingerprint was performed using HPTLC plates 10x10 cm silica
gel 60 F 254 (Merck). Fully automatic TLC sampler, horizontal developing chamber
10x10 cm and chromatogram immersion device, equipped with 20x20 cm dip tank
HPLC Shimadzu instrument, equipped with a model series LC-10 ADVP pump, SCL-10 AVP system controller, DGU-12 A Degasser, Rheodyne 7725 injector with a 20 μL loop and a SPD-10AVP UV-VIS detector and with a HPLC column TSK-Gel ODS-80 TM column, 5 μm, with dimensions 150x4.6 mm were used to determine the quantity of sarcophine in the organic extract. The UV spectral analysis of sarcophine was measured in double-beam Shimadzu UV-Visible spectrophotometer, model UV-1601 PC equipped with 1 cm quartz cells. The bundled software was UVPC personal spectroscopy software version 3.7. EM science silica gel 60/230-400 mesh (flash) was used for column chromatography.
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed in the SE 600 vertical slab gel casting unit (Amersham, Pharmacia, Sweden).
We collected 26 samples of the soft coral Sarcophyton species by
hand using scuba diving from different locations in the Red Sea: Sharm El- Sheikh
(SH), Ras Mohammed (RM), ElGouna (GN), Hurghada (HU), Safaga (SA) and
Marsa Alam (MA). Each sample was preserved in two portions. The first one of
each individual was frozen immediately after collection in liquid nitrogen then
at -80°C till manipulation. The second portion was preserved in 70% ethanol
for morphological identification.
The stalk and capitulum sclerites were prepared from the alcohol preserved
samples following the method described by Fabricus and Aderslade (2001). Different
Sarcophyton species were identified by the sclerite examination according
to the keys described by Versevldt (1982).
In order to obtain the organic extract, the frozen samples were freeze dried
and then extracted with methanol, dichloromethane mixture (1:1) for five days.
The solvent mixture was freshly changed daily to ensure extraction efficiency.
The extract was dried under vacuum, dissolved in chloroform and partitioned
against water to remove the sodium chloride traces from the extract then dried
Pure sarcophine standard was prepared by the fractionation of a portion of the organic extract of one sample on silica gel column using 30% ethyl acetate in hexane as an eluent. The fraction containing sarcophine was further purified by crystallization out of the cold hexane. The identity of sarcophine was confirmed by comparing the spectral data with the published one (Bernstein et al., 1974).
HPTLC technique was applied in order to obtain a chemical fingerprint for the organic extract using a mobile phase made up of 30% ethyl acetate in n- Hexane. Samples were applied on HPTLC plates in the form of bands of 7 mm width leaving 5 mm in between. The sample volume and concentration was 1 μL and 1 μg/30 μL methanol, respectively. The separated bands were detected using the anisaldehyde spray reagent after heating to 120° for 5 min.
HPLC was carried out to quantify the amount of sarcophine present in the organic extract of each Sarcophyton sample. The best separation was obtained using a mobile phase of 70% acetonitrile, 30% deionized water and pH adjusted to 3.5 by phosphoric acid. The detector was set to 220 nm and the flow rate was 1.5 mL min-1 and the injection volume was 20 μL. All determinations were performed at ambient temperature. Six standard solutions were prepared by diluting the stock with methanol to reach a concentration range of 10 to 80 μg mL-1. The used concentrations were 10, 20, 40, 50, 60 and 80 μg mL-1. Triplicate of 20 μL injections were chromatographed under the conditions described above. The peak area of each concentration was plotted against the corresponding concentration to obtain the calibration graph. The samples were prepared by dissolving a specified weight of the extract in methanol so that the final concentration was 20 mg% and filtered through 0.45 μm disposable filters and 20 μL injection of each sample was assayed.
This is the first time to attempt isolating the total cellular proteins
from the genus Sarcophyton. Therefore, different optimization steps were
made so as to obtain maximal total cellular protein yield with minimal proteolysis
and contamination. Protein were extracted from the capitulum tissue, where equal
amounts of the capitulum sample and the protein extraction buffer were manually
homogenized over ice. The protein extraction buffer was composed of 50 mM Tris-HCl
(pH 8), 1 mM Dithiothreitol, 50 mM EDTA, 0.5 M NaCl, 1% v/v Tritonx100, 1 mM
Benzamidine and 1 mM Phenyl methyl sulfonyl fluoride. Proteins were separated
by centrifugation for 15 min at 4°C. The extracted proteins were boiled
for 3 min with the sample buffer. Equal protein concentration of each sample
was loaded on 10% discontinuous SDS-PAGE prepared as described by Laemmli in
1970 (Walker, 2002). Protein bands were visualized by Coomassie blue R 250 staining.
A volume of 20 μL of each sample was loaded per well. Molecular weight
estimate was done by comparison to a full range marker of molecular weight from
205 to 14 KDa (Amersham Pharmacia Biotech). The molecular weight of the protein
bands was determned by comparison to the marker using the software Gel Documentation,
Advanced Software Version 2.0. The absence of contamination from the symbiotic
algae Zooxanthellae proteins was verified by isolating the algal cells
from the Sarcophyton tissue as described by Lin et al. (2000).
The sclerite examination indicated the presence of seven Sarcophyton
species in the study, in addition to two unidentified samples from Safaga
and Sharm El Sheikh that could not be identified by sclerite key. The similarities
of sclerite shapes among the different species of the genus Sarcophyton made
the unambiguous identification, a difficult task. In some instances, the variation
in the shape of sclerites does not necessarily mean different species, instead
it can reflect the age of the colony (Versevldt, 1982). Species identification
by gross morphology of the Sarcophyton colony was complicated by the
fact that it harbors symbiotic algae that often show different colors within
the same species. Also all the colonies capitulae showed different extension
and contraction phases according to the water currents and the environmental
conditions at the collection site. This further complicated the gross morphology
based identification. According to sclerite examination, the identified species
in our study were S. auritum, S. digitatum, S. ehrenbergi, S. glaucum,
S. gemmatum, S. pauciplactum and S. trocheliophorum. Different
sclerite shapes are shown in Fig. 2-4.
High Performance Thin Layer Chromatography (HPTLC)
HPTLC analysis was used to obtain a chemical fingerprint of the organic
extract obtained from different Sarcophyton species at different locations.
Figures 5-9 show HPTLC plates with good
separation of the sarcophine band in the extract. The plates revealed that all
Sarcophyton species, identified according to the sclerite architecture,
contained sarcophine; but in different concentrations.
Sarcophyton auritum produced a distinctive fingerprint that was consistent
in almost all locations (Fig. 6-8) except
for the one from Ras Mohamed (Fig. 5) that showed different
pattern. S. trocheliophorum (Fig. 8) showed another
pattern that was not found in any other species. The chemical fingerprints of
S. ehrenbergi collected from Marsa Alam and from El-Gouna were not identical.
Furthermore, S. digitatum collected from two different locations, Hurghada
and Sharm El-Sheikh (Fig. 8 and 9, respectively),
had different pattern. S. glaucum, S.gemmatum and S. Pauciplactum collected from
different locations (Fig. 5-9) and the unidentified
species collected from Safaga (Fig. 6) showed similar chemical
||Different shapes of the capitulum sclerites common in all
Sarcophyton species. (100x magnification)
||The key sclerite of the interior stalk of Sarcophyton glaucum
||The key sclerite of the interior stalk of Sarcophyton trocheliophorum.
||HPTLC fingerprint for Sarcophyton species from El Gouna
(GN), Ras Mohamed (RM) and Sharm El Sheikh (SH). Lane 1: Sarcophine standard.
Lanes 2 and 3: S. glaucum from GN and 6 from RM. Lane 4: S. auritum
from RM. Lane 5: S. pauciplactum from RM. Lane 7: S. gemmatum
||HPTLC fingerprint for Sarcophyton species from Safaga
(SA) and Sharm El Sheikh (SH). Lane 1: Sarcophine standard. Lane 2 and 5:
S. pauciplactum from SH and SA, respectively. Lane 3: S. gemmatum
from SH. Lane 4: unidentified sample from SA. Lane 6: S. auritum
from SA. Lane 7: S. glaucum from SA
Another species collected from Sharm El Sheikh (Fig. 9),
that we were not able to identify it by sclerite architecture, showed a distinctive
chemical profile that was not found in any other species. The heterogeneity
in the chemical fingerprints that was obtained from the same species collected
at different locations might be attributed to the variation of the type and
concentration of the complementary metabolites with the environmental conditions
at the site of collection (Fabricius and Alderslade, 2001). Using HPTLC, it
was possible to identify the sarcophine band using short UV when compared to
sarcophine standard. Interestingly, it was found that all the Sarcophyton
species studied contained sarcophine except for S. auritum and S.
||HPTLC fingerprint for Sarcophyton species from Marsa
Alam (MA) and El-Gouna (GN). Lane 1: Sarcophine standard. Lane 2: S.
auritum from MA. Lanes 3 and 4: S. glancum from MA. Lane 5: S.
ehrenberg from MA. 6 and 7: S. ehrenbergi from GN
||HPTLC fingerprint for Sarcophyton species from Hurghada
(HU). Lane 1: Sarcophine standard. Lane 2: S. glaucum. Lane
3: S. digitatum. Lane 4: S. trocheliophorum. Lane 5: S.
High performance liquid chromatography (HPLC)
The developed reversed phase high performance liquid chromatography (RP
HPLC) method was applied to determine the sarcophine concentration in the organic
extract. The chromatographic conditions were studied and optimized as a function
of the pH and the acetonitrile concentration in the mobile phase. Figure
10 shows the chromatogram of 40 μg mL-1 of sarcophine standard.
The average retention time±standard deviation (SD) was found to be 4.6±0.012
min. The HPLC method was specific to sarcophine, hence we used short UV (220
nm) at which only sarcophine has maximum absorbance with minimum interference
from other compounds in the extract. The specificity of the RP HPLC is shown
in Fig. 11 where there is a good separation of sarcophine
from the rest of the analyzed matrix. This validated HPLC method provides a
simple, accurate and reproducible quantitative analysis for the determination
of sarcophine in Sarcophyton natural extracts without interference from
other compounds in the extract (unpublished data).
||HPTLC fingerprint for Sarcophyton species from Sharm
El-Sheikh. Lane 1: Sarcophine standard. Lanes: 2, 3 and 6: S. glaucum.
Lane 5: S. digitatum. Lane 4: unidentified sample
|| A typical chromatogram of 20 μL injection of 40 μg
mL-1 of sarcophine standard
The HPLC results indicated that all Sarcophyton species contained sarcophine but with variable quantities that ranged from 0.05 and 0.1% for S. auritum and S. trocheliophorum, respectively and increased from 0.2 to 8.2% for the S. digitatum, S. ehrenbergi, S. gemmatum S. pauciplactum and S. glaucum (Table 1). This may explain the low rate of mortality in Sarcophyton colonies and their long term stability despite having slow growth rates and being poor colonizers (Cornish and Didonato, 2004). Of the Red Sea species and locations tested using the described HPLC method; Sarcophyton glaucum from Hurghada location produced the highest concentrations of sarcophine (Table 1). The HPLC results revealed that the sarcophine concentration is variable among the different species and within the individual species collected from different locations. This finding agrees with another report (Koh et al., 2000) on the distribution of sarcophytol A in the genus Sarcophyton. Therefore, sarcophine concentration is influenced by both, genetic factors and environmental conditions.
||Percentage of sarcophine in the dry weight of the Red Sea
soft coral Sarcophyton species from different locations. *SH: Sharm
El sheikh,HU: Hurghada, SA: Safaga, MA: Marsa Alam, GN: Gouna and RM: Ras
||Chromatogram showing the chromatographic separation of sarcophine
from other organic compounds of S. glaucum from Marsa Alam. The sarcophine
concentration was 2.3% of the dry weight. CRS is a closely related substance
The HPLC and HPTLC analyses showed the presence of a compound that elutes very close to sarcophine. It appears that this compound is closely related to sarcophine (designated CRS). It was shown by HPLC (Fig. 11) that the concentration of sarcophine is inversely proportional to the concentration of CRS.
||10% SDS-PAGE of different Sarcophyton species proteins
from Hurghada (HU) and Safaga (SA) in the presence of Sinularia as
an out group, the arrows indicate the common bands in Sarcophyton
species. Lane 1: Sinulariaspecies as an out group collected from
HU. Lane 2 and 10: S. glaucum from HU and SA, respectively. Lane
3: S. digitatum from HU. Lane 4: S. trocheliophorum from HU.
Lane 5 and 9: S. auritum from HU and SA, respectively. Lane 6: molecular
weight marker. Lane 7: unidentified sample collected from SA. Lane 8: S.
pauciplactum collected from SA
||10% SDS-PAGE of different Sarcophyton species proteins
from Marsa Alam (MA) and El Gouna (GN). Lane 1: S. auritum collected
from MA. Lanes 2 and 3: S. glaucum obtained from MA and 8 and 9 from
GN. Lanes 4: S. ehrenbergi from MA and 6 and 7 from GN. Lane 5: Molecular
The SDS-PAGE of proteins indicated that there was distinctive protein pattern
for the Sarcophyton species, when compared to other members of the same
family Alcyoniidea, Sinularia, (Fig. 12, lane 1).
This was apparent in the high molecular weight proteins of molecular weight
characteristic for Sarcophyton. The results also revealed that a 60 and
45 KDa protein bands are common in all Sarcophyton species. Figure
12- 14 show the 10% SDS-PAGE of different Sarcophyton
species collected from different locations.
|| 10% SDS-PAGE of different Sarcophyton species proteins
from Sharm El Sheikh (SH) and Ras Mohammed (RM). Lanes 1, 2, 5: S. glaucum
from SH and 9 from RM. Lane 3: unidentified species from SH. Lane 4: S.
digitatum from SH. Lane 6: Molecular weight marker. Lane 7: S. auritum
from RM. Lanes 8 and 11: S. pauciplactum from RM and SH. Lanes 10
and 12: S. gemmatum from SH
In all locations S. glaucum can be characterized by the presence of two consecutive protein bands of 27 and 29 KDa, in addition to the common protein bands. The protein profile coincides with high sarcophine content in almost all samples, except for the S. glaucum collected from El- Gouna and Ras Mohammed that showed low sarcophine concentration, 0.6 and 0.35%, respectively along with high closely related substance (CRS) concentration (Fig. 11). The highest sarcophine concentration was found in S. glaucum from Hurghada (8.2%). This particular sample had an additional protein band of molecular weight of 26 KDa. Whether this band is characteristic to the S. glaucum with the highest sarcophine concentration requires further investigation.
The slight individual differences in the low molecular weight proteins of S. glaucum collected from different locations might be due to environmental conditions that may induce the production of heat-shock protein reported in other soft corals (Tom et al., 1999). Heat-shock proteins are induced by stressful factors such as extremes of temperature, UV radiation, the presence of heavy metals and amino acids analogs (Tom et al., 1999). Moreover, the difference in the low molecular weight proteins might be attributed to the growth state of the colony and its sexual maturity. Sexual maturity is after six to seven years for male colonies and ten years for female colonies (Benayahu and Loya, 1986). In addition, the variation in the low molecular weight proteins may also be due to the altered gene expression as well as posttranslational modification of polypeptides, which may be due to environmental stress and/or aging (Soti and Csermely, 2003).
S. auritum showed a unique pattern of a 35 KDa protein band in addition to the characteristic protein bands found in the genus. This protein can be used to characterize the auritum species. Interestingly, the protein profile was similar in the three S. auritum collected from Hurghada, Safaga and Marsa Alam samples. These three specimens contained small amount of sarcophine between 0.04 and 0.05%. However, the S. auritum that was identified by sclerite architecture and collected from Ras Mohammed had a different protein profile of 27 and 29 KDa proteins without the unique 35 KDa band. In addition, the HPLC analysis revealed that it contained high sarcophine content (4%) and the HPTLC indicated that it has a different chemical fingerprint not identical to the other S. auritum samples. Taking together the protein profile and the chemical fingerprint along with the high concentration of sarcophine found in the Ras Mohammed sample, it could be suggested that this sample might be S. glaucum due to the resemblance between the two.
S. ehrenbergi collected from El Gouna, Fig. 13, lane 6 had a relatively high sarcophine concentration of 1% and the protein bands of 27 and 29 KDa characteristic to S. glaucum and a chemical fingerprint as S. glaucum. Therefore, these findings suggest that this species may be better identified as S. glaucum. Moreover, the S. Pauciplactum collected from Ras Mohammed, Fig. 14, lane 8 had the same pattern of high sarcophine concentration of 4.6% as well as the protein bands and the chemical fingerprint characteristic for S. glaucum which also suggest that it might be S. glaucum.
Furthermore, the S. ehrenbergi from El-Gouna, Fig. 13, lane 7, S. Pauciplactum and S. gemmatum collected from Sharm El-Sheikh, Fig. 14 lanes 11 and 12, respectively , all have the protein bands characteristic to S. glaucum, but the sarcophine concentration was low (0.07, 0.25 and 0.29%, respectively). Present findings also indicate the presence of an inverse relation between the concentration of sarcophine and its CRS (Fig. 11). Therefore, these samples appear to belong to S. glaucum which biosynthesized more of the CRS at the expense of sarcophine.
S. ehrenbergi collected from Marsa Alam, as was identified by sclerites, showed protein bands of molecular weight of 34 , 26 and 24 KDa. Furthermore, the S. trocheliophorum sample collected from Hurghada showed an interesting protein profile with protein bands of molecular weight of 25 and 27 KDa. These protein profiles can be used to characterize the S. ehrenbergi and the S. trocheliophorum samples.
S. digitatum samples were collected from two locations, Hurghada and
Sharm El Sheikh (Fig. 12 and 14, lanes
3 and 4, respectively). The Hurghada sample followed the sclerite keys described
by Verseveldt (1982) for the identification of S. digitatum. It had a
characteristic protein profile of the common bands of the genus Sarcophyton
in addition to a 30 KDa protein band and with high sarcophine concentration
of 5.7%. On the other hand, the Sharm El Sheikh sample was not well identified
according to the sclerite keys. It had also the common bands of the genus Sarcophyton
in addition to a band of molecular weight of 28 KDa and not of 30 KDa band,
its sarcophine concentration was 0.2% along with high CRS concentration. Furthermore,
the HPTLC chemical fingerprints of the two samples were not identical. This
might indicate that the two samples are different and the Hurghada sample is
the only S. digitatum included in the study.
The protein profile of the S. digitatum from Sharm El Sheikh is identical to the protein profile of the one of the S. gemmatum collected from Sharm El Sheikh as well as the unidentified sample and S. pauciplactum collected from Safaga (Fig. 14, lanes 4 and 10 and Fig. 12, lanes 7 and 8, respectively). Because the current taxonomy is based on the colony morphology and the sclerite architecture and these characters exhibit an extra-ordinary high degree of variability and were proven to be inaccurate tools for identification when compared to genetic based taxonomy (Won et al., 2001; Brenston et al., 2001), the above samples were misidentified. Moreover, the sarcophine concentration was variable among these samples, ranging from 0.189 to 5.52%. Therefore, further investigation is needed to correctly identify these samples.
The unidentified species collected from Sharm El Sheikh (Fig. 14, lane 3) had a unique protein pattern and chemical fingerprint on HPTLC that was different from the other species studied along with high sarcophine concentration (4.4%). Further studies are needed before this sample can be accurately and correctly identified.
As far as we are aware, this is the first study of the genus Sarcophyton that has correlated morphological, chemical and protein profiling for each sample. We hope that it will stimulate a broader approach to the delimitation of species identification in the Red Sea.
The results of this work indicate that the chemical fingerprint and the protein profile were in agreement but in some instances did not support the morphological or the sclerite based identification. Based on sclerite architecture, seven morphologically different Sarcophyton species were identified in the study, in addition to two unidentified species collected from Safaga and Sharm El Sheikh. The chemical fingerprint obtained by HPTLC and the protein profiles obtained by 10% SDS-PAGE supported the presence of five species only. These species are S. auritum, S. digitatum, S. ehrenbergi, S. glaucum and S. trocheliophorum. The chemical and protein fingerprints were instrumental in confirming the identification of the collected species. They were also important tools in correcting the sclerite based misidentification in some species . The identity of S. gemmatum and S. pauciplactum was uncertain because the samples collected had chemical and protein fingerprints similar to either S. glaucum or to the unidentified sample from Safaga indicating problems with the sclerite identification. Furthermore, the unidentified Sharm El Sheikh sample showed a unique protein profile and chemical fingerprint that was not found in any other species included in this study.
It is clear from the current study that depending solely on the sclerite based taxonomy, which is the widely used technique for all soft corals identification, is unreliable. Therefore, additional techniques such as the chemical and protein fingerprints should be used to support and ensure the reliability of identification. The accurate identification is of enormous importance once the organism becomes a source for large-scale production of a potential pharmaceutical drug. Hence, for the industrial purposes, the proper species, location and environmental conditions need to be predetermined to ensure a sufficient and steady supply of the drug.
The authors wish to thank Mr. Tarek Temraz of the Marine Science Department, Suez Canal University for helping with the sclerite based taxonomy of the different Sarcophyton species. The authors would also like to acknowledge the Egyptian Academy of Scientific Research for financial assistance under the Program of National Strategy for Biotechnology and Genetic Engineering, project title: The Red Sea: A natural source of biologically active compounds, number: 34.