Features and Advantages of Supercritical CO2 Extraction of Sea Cucumber Cucumaria frondosa japonica Semper, 1868

Extraction process of Cucumaria frondosa japonica Semper, 1868, which are subspecies of Cucumaria frondosa (Gunnerus, 1767), were studied. It was shown that supercritical carbon dioxide extraction of holothuria was more effective than conventional solvent extraction. Step-by-step extraction with carbon dioxide followed by supercritical extraction with the addition of a co-solvent of ethanol can almost double the yields of extracts of triterpene glycosides, styrenes and carotenoids. Moreover, the fraction of triterpene glycosides practically does not contain colored impurities, in contrast to traditional ethanol extraction. The obtained extracts by HPLC in combination with tandem mass spectrometry (HPLC-MS/MS) identified 15 triterpene glycosides, 18 styrene compounds and 14 carotenoids. Supercritical extraction made it possible to obtain extracts with yields superior to conventional hexane and alcohol extracts. Moreover, such an approach with the use of supercritical fluid extraction (SFE) and subsequent profiling of metabolites can help with the study of holothuria species that are not as well studied.


Introduction
Oceans are the least studied part of the Earth. Every year, dozens of new compounds are isolated and described from marine hydrobionts around the world. Many of them have different biological activities and many have been used in drug development. Secondary metabolites of the representatives of the class of echinoderms are of great practical and fundamental interest. New secondary metabolites isolated from echinoderms are discovered annually. Many articles have been published describing the

Carotenoids Identified in Supercritical CO 2 Extract
During the first step, SFE fatty fraction was isolated. Total yield was 1.1 g. Fourteen carotenoids were identified with a total mass of 71.1 mg ( Table 2). The major component of carotenoid fraction was Cucumariaxanthin yielding 33.6 mg, that corresponded to 56.1 mg per 100 g of dry weight. The second main carotenoid was Canthaxanthin, 25.4 mg/100 g. The third main component was Cucumariaxanthin B. Lutein was detected in the smallest amount 0.5 mg/100 g.

Triterpene Glycosides Identified in Supercritical CO 2 Extract
In the second step, SFE extracts were analyzed by HPLC in combination with tandem mass spectrometry (HPLC ESI MS/MS). A total of 15 different triterpene glycosides were identified (Table 3). All of the detected triterpene glycosides were found earlier in this type of holothuria, but this is the first study when it was possible to detect all of these glycosides at the beginning of one complex experiment. The structural formulas of the detected glycosides are shown in Figures 1 and 2 All of the detected triterpene glycosides were found earlier in this type of holothuria, but this is the first study when it was possible to detect all of these glycosides at the beginning of one complex experiment. The structural formulas of the detected glycosides are shown in Figures 1 and 2       Total compounds chromatogram of detected triterpene glycosides in negative ion mode shown in Figure 3. The figure shows that the total fraction without preliminary separation has poorly resolved peaks, and although the full qualitative composition can be determined due to the mass spectrometer detector, this does not allow quantitative analysis. Total compounds chromatogram of detected triterpene glycosides in negative ion mode shown in Figure 3. The figure shows that the total fraction without preliminary separation has poorly resolved peaks, and although the full qualitative composition can be determined due to the mass spectrometer detector, this does not allow quantitative analysis.

Discussion
Cucumaria frondosa japonica Semper, 1868 is a subspecies of Cucumaria frondosa (Gunnerus, 1767). Cucumaria frondosa japonica's habitat is in the Pacific Ocean but does not occur in the Atlantic Ocean. These two species have very similar metabolites but are slightly modified. Both of these are commercial species of holothuria, are widely used in food and are a source of concentrates for the pharmaceutical industry [9,10]. All this has led to the fact that over the past 40 years, the biochemical composition of this species has been fairly well studied. One of the first works on the biochemistry of this holothurian dates back to 1980. The triterpene glycoside of these holothurians is most well studied, since they are promising substances for medicine [11][12][13]. Use of supercritical extraction allowed us to increase the yield of the total fraction with triterpene glycosides and for the first time in one complex experiment we showed all 15 triterpene glycosides present in Cucumaria frondosa japonica. For simultaneous qualitative characterization of the present triterpene glycosides, we used the metabolite profiling approach used earlier by other groups for the analysis of triterpene glycosides [14,15]. This approach allows reliable identification of known molecules, even if they are

Discussion
Cucumaria frondosa japonica Semper, 1868 is a subspecies of Cucumaria frondosa (Gunnerus, 1767). Cucumaria frondosa japonica's habitat is in the Pacific Ocean but does not occur in the Atlantic Ocean. These two species have very similar metabolites but are slightly modified. Both of these are commercial species of holothuria, are widely used in food and are a source of concentrates for the pharmaceutical industry [9,10]. All this has led to the fact that over the past 40 years, the biochemical composition of this species has been fairly well studied. One of the first works on the biochemistry of this holothurian dates back to 1980. The triterpene glycoside of these holothurians is most well studied, since they are promising substances for medicine [11][12][13]. Use of supercritical extraction allowed us to increase the yield of the total fraction with triterpene glycosides and for the first time in one complex experiment we showed all 15 triterpene glycosides present in Cucumaria frondosa japonica. For simultaneous qualitative characterization of the present triterpene glycosides, we used the metabolite profiling approach used earlier by other groups for the analysis of triterpene glycosides [14,15]. This approach allows reliable identification of known molecules, even if they are not well separated during HPLC.
The first stage of extraction was carried out at a pressure of 2500 psi, which allows the extraction of almost only hydrophobic nonpolar and weakly polar molecules, such as carotenoids, fats and sterols. This was used to analyze the carotenoids and sterols contained in Cucumaria frondosa japonica. Supercritical extraction made it possible to obtain extracts with yields superior to conventional hexane and alcohol extracts. Moreover, such an approach with the use of SFE and subsequent profiling of metabolites can help with the study of holothuria species that are not as well studied as Cucumaria frondosa japonica. This approach helps to better study the carotenoid composition and minor components of other types of Echinodermata. Supercritical extraction is a very promising approach and since it avoids unnecessary oxidation of substances, prevents unpleasant odors, improves product quality and yield, and is more profitable to operate.

Sea Cucumber
The live specimens of sea cucumber Cucumaria frondosa japonica Semper, 1868, were collected in Peter the Great Gulf (Russia, Sea of Japan) in 2019. The body wall of the sea cucumbers, total weight 1450 g, was dissected free of intestine and detritus with scissors, cut into small pieces (about 2 cm long), and stored in a polyethylene bag at −70 • C until used. Later, sea cucumbers with the total weight of 1450 g, were freeze-dried to 120 g, then the sample was separated into two equal parts, for ethanol extraction and supercritical fluid extraction (SFE).

Supercritical Fluid Extraction
Frozen sea cucumbers were freeze dried and minced. Dry weight was 120 g. Then 60 g was extracted in supercritical CO 2 pressure extraction apparatus (Thar SFC 500, San Diego, CA, USA). Extraction was carried out in two steps, first to get carotenoids and sterols fraction with CO 2 under the pressure of 2500 psi, at a temperature of 60 • C, and flow rate was 50 mL/min for liquid CO 2 . flow of 20 g/min. Total yield was 1.1 g or 1.8% from dry weight. The second step was with a flow rate 50 mL/min for liquid CO2 and 1.00 mL/min for EtOH under the pressure of 7000 psi, at a temperature of 60 • C, and a flow of 20 g/min. Yield was 3.4 g and approximately 1.1 g was triterpene glycosides. Another 60 g of dried sea cucumbers were used for usual ethanol extraction.

Ethanol Extraction
The second part of freeze-dried body with the weight of 60 g was extracted with ethanol. The extraction was carried out at a ratio of 1:2 in a dark container at a temperature of 40 • C, with constant alternation on a stirrer for 3 h. Then the extract was filtered off and another 60 mL of ethanol was poured into the precipitate for re-extraction within 3 h. The precipitates were combined and concentrated in a rotary evaporator under vacuum at a temperature of 40 • C and then evaporated under vacuum until a constant mass was yielded. Total yield was 2.7 g.
ESI-MS and ESI-MSn spectra were recorded using an amaZon SL ion trap (BRUKER DALTONIKS, Bremen, Germany) equipped with an electrospray ion source. ESI MS ionization parameters were optimized as follows: a capillary voltage of 4500 V, end plate bend voltage: 1500V, collision energy: 60 eV, nebulization with nitrogen at 29 psi, dry gas flow of 10 L/min at a temperature of 160 • C. The mass spectra were recorded within m/z mass range of 500-2000 in negative and positive ion mode. Product ion mass spectra were recorded in auto MS/MS mode. Identification of compounds was carried out according to the Bruker library and literature data [11,16,17].

Conclusions
It was shown that supercritical carbon dioxide extraction of holothuria Cucumaria frondosa japonica Semper, 1868 was more effective than conventional solvent extraction. Supercritical extraction is a very promising approach since it avoids unnecessary oxidation of substances, prevents unpleasant odors, improves product quality and yield, and is more profitable to operate.
Step-by-step extraction with carbon dioxide followed by supercritical extraction with the addition of a co-solvent of ethanol can almost double the yields of extracts of triterpene glycosides, styrenes and carotenoids. Moreover, the fraction of triterpene glycosides practically does not contain colored impurities, in contrast to traditional ethanol extraction. The obtained extracts by HPLC in combination with tandem mass spectrometry (HPLC-MS/MS) identified 15 triterpene glycosides, 18 styrene compounds and 14 carotenoids. Separation of the triterpene glycoside fraction into sub-fractions by hydrophobic chromatography on a Polychrome-I carrier made it possible to additionally identify 3 minor triterpene glycosides cucumarioside A 7-1 , cucumarioside A 7-2 and cucumarioside E. We managed to isolate all triterpene glycosides, including minor ones, but no new triterpene glycosides were found. This publication is primarily of methodological and technological value. These data will be useful for organizing production facilities for the commercial isolation of valuable products from mountainuria and for studying the composition of triterpene glycosides of not so widespread and well-studied species in order to discover new triterpene glycosides.

Conflicts of Interest:
The authors declare no conflict of interest.