محتویات کیفی و کمی ساپونین در پنج خیار دریایی از اقیانوس هند Qualitative and Quantitative Saponin Contents in Five Sea Cucumbers from the Indian Ocean

۱٫ Introduction Holothuroids, also known as sea cucumbers, are marine animals that are characterized by a slow motion and the absence of prominent structural defenses. As a direct consequence, they are vulnerable to predation. As a mean of defense, most sea cucumbers contain, in their body wall and viscera, secondary metabolites named saponins [1–۴]. Their structures are based on a lanosterol-type triterpene with a distinctive D-ring with fused γ-lactone skeleton named holostane and a carbohydrate chain containing up to six sugar residues such as glucose (Glc), 3-O-methylglucose (MeGlc), quinovose (Qui), and xylose (Xyl) [5,6]. In addition, in some species, sulfate groups may be present at certain positions of the sugar units [7]. Because these compounds possess a large spectrum of pharmacological activities [8], numerous studies are being currently conducted to identify new congeners and new natural sources of saponins. On the other hand, the biological roles of saponins in holothuroids are still very speculative [9,10]. They are abundant in the body wall which, in addition to its role as a physical barrier protecting the animal, is also the largest organ [3,11,12]. They also appear to be particularly concentrated in the Cuvierian tubules, a specialized defense system developed by some sea cucumber species, all belonging to the family Holothuriidae [3,4,11]. This organ, located in the posterior part of the animal, consists of multiple tubules that can be expelled by the individual after stimulation [13,14]. Expelled tubules lengthen into sticky white threads that may entangle potential predators. To date, however, only a few authors have investigated the qualitative and quantitative differences between body wall and Cuvierian tubule saponins (see e.g., Kobayashi et al. [11]). Recent studies have demonstrated that mass spectrometry (MS) procedures represent very valuable techniques for the detection and identification of saponins [15–۱۷]. Indeed, using MS methods such as MALDI-MS (Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry) and LC-MS (Liquid Chromatography-Mass Spectrometry) techniques, we were able to highlight remarkable differences between the saponin mixtures from the body wall and the Cuvierian tubules of Holothuria forskali [18]. In this species, which was first studied by Rodrigez et al. [19], only five saponins were described, with no indication on their organ of origin. Using MS, we detected 12 different saponins in the body wall and 26 in the Cuvierian tubules and highlighted the occurrence of many isomer congeners [18]. In continuation of our study on triterpene glycosides from sea cucumbers of the family Holothuriidae, we carried out a large-scale comparative investigation on saponins from five tropical species from the Indian Ocean. MS methods were used to detect and analyze saponins and a semi-quantitative study was also performed to compare total saponin contents. A peculiar attention was paid to the differences that could occur between the body wall and the Cuvierian tubules in a given species. ۲٫ Results ۲٫۱٫ Qualitative study Saponins of the five species have been extracted and analyzed using MALDI-MS/MS and LC-MS/MS. MALDI-MS technique was used for direct detection and analysis of saponin mixtures, while the LC-MS technique was performed to achieve chromatographic separation of potential isomers (see Supplementary Figure 1 for a typical example). Indeed, in a previous work, we highlighted the  presence of isomers in such saponin mixtures [18]. The entire extraction and purification procedures and the mass spectrometric analyses were the same as those used in Van Dyck et al. [18]. Figure 1. Comparison between the collision-induced fragmentation patterns of holothurin A (A) and holothurinoside A (B). Full and dotted arrows are two possible fragmentation patterns (see Figure 2 for molecular structures of these saponins and their respective fragments). As a characteristic example, Figure 1 presents the tandem MS spectra of holothurin A (1) and of holothurinoside A (10) ions. It is noteworthy that the observed ions arise from cationization (Na+ attachment) of the neutral molecules upon ESI or MALDI. Based on MS/MS spectra and as described in detail in Van Dyck et al. [18], the molecular structures of the saponins can be obtained by the identification of the mass transitions between the successive collision-induced fragmentation peaks. For instance, in Figure 1A, two competitive sequences of decompositions are represented by arrows showing the consecutive losses of sodium monohydrogenosulfate (NaHSO4), of the aglycone, of xylose, and of quinovose, glucose and 3-O-methylglucose from the mass-selected holothurin A (1) ions (m/z 1243.5). This sequence of fragmentation is exemplified in Figure 2A. Similarly, Figure 1B is characterized by the consecutive losses of the aglycone, glucose and xylose, and, competitively, of quinovose, glucose and 3-O-methylglucose (see also Figure 2B). The detection of this sequence of fragmentations allows the identification of the m/z 1303.3 ions as cationized holothurinoside A (10).