*2.2. NMR Spectroscopy*

### 2.2.1. NMR Analysis of Female Gonads Aqueous Extracts

The 1H NMR spectrum of the aqueous extract of *R. pulmo* ovaries was characterized by free amino acids, organic acids, and derivatives (Figure 2). Many signals due to different compounds, such as betaine (δ 3.27 and 3.90), taurine (δ 3.27 and 3.41), homarine (δ 4.37, 7.97, 8.04, 8.55, 8.71), lactate (δ 1.33 and 4.16), succinate (δ 2.41), acetate (δ 1.92), and formate (δ 8.46), were identified. High signals at δ 3.57 and the doublet at δ 1.48 were also assigned to glycine and alanine, respectively. The multiplet of proline appeared at δ 2.1–2.0, 2.32–2.36, 3.30–3.40, 4.10–4.14, while the multiplet at δ 2.07 and 2.36 were assigned to glutamate. Other amino acids such as leucine (δ 0.96, 1.70), isoleucine (δ 1.01, 1.97), valine (δ 1.05, 2.29), threonine (overlapping doublets at δ 1.33 and multiplets at δ 3.68 and 4.29) were detected. Using 2D NMR experiments (Figure 3), and by comparison with literature data [22–24], two other osmolytes were identified: β-alanine (triplets at δ 2.56 and 3.20) and hypotaurine (triplets at δ 2.65 and 3.37). In the aromatic region, low-intensity signals at δ 6.90 and 7.19 were assigned to tyrosine, while δ 8.08, 8.84, and 9.13 signals were also identified for trigonelline (*N*-methylpicolinic acid). Quantitative analysis [25] showed that taurine, betaine, and glycine (concentrations >10 mM) were the most abundant free metabolites. Homarine, β-alanine, and alanine contents ranged from 5 to 3 mM. Finally, trigonelline, acetate, valine, formate, succinate, and hypotaurine were present in very low concentrations (≤1 mM).

**Figure 2.** Typical 1H NMR spectrum obtained at 600 MHz of *R. pulmo* ovaries aqueous extract.

**Figure 3.** Expansions of COSY spectrum of *Rhizostoma pulmo* aqueous extract. Colored boxed regions correlate with the various resonances of homarine, trigonelline, betaine, taurine, hypotaurine, and glycine.

### 2.2.2. NMR Analysis of Female Gonads Lipid Extracts

The lipid extracts of the examined jellyfish female gonads were characterized by the presence of triglycerides (TG), polyunsaturated fatty acids (PUFAs), diunsaturated fatty acids (DUFAs), monounsaturated fatty acids (MUFAs), saturated fatty acids (SFAs), and minor components such as sterols (cholesterol) and phospholipids. The main signals, marked in the spectrum (Figure 4), corresponded to −CH2 in α and β-position to the carboxylic acid esters (COOCH2CH2), unsaturations (CH=CH-CH2-CH=CH), and monounsaturated fatty acids (docosahexaenoic, DHA C22:6, and eicosapentaenoic acids, EPA C20:5, ω-3) or other PUFAs (two and more than two double bonds) of long fatty acids alkyl chain, including PUFA CH3s and terminal CH3s of phospholipids.

**Figure 4.** Typical 1H NMR spectrum obtained at 600MHz of CD3OD/CDCl3 *R. pulmo* female gonad lipid extract (**a**) high-, (**b**,**<sup>c</sup>**) middle-, and (**d**) low-frequency regions, (**e**) full spectrum.

The COSY cross peaks correlated with the multiplets from the glycerol moiety of TG appeared at δ 4.14 and 4.11 (sn 1,3) and δ 5.24 (sn 2), with very low intensities. The signals in the range of δ 2.32–2.27 and δ 1.66–1.57 were assigned to protons of COOCH2 and COOCH2CH2, respectively, for all the fatty acids chains, except for DHA (signal at δ 2.38, COOCH2CH2,) and EPA (signal at δ 1.70 COOCH2CH2).

The presence of ω-3 PUFAs is confirmed by the appearance of a triplet at δ 0.98 related to the terminal methyl group. This terminal methyl group is clearly separated from other methyl groups at δ 0.80 and 0.91, ascribable to all other non-ω-3 fatty acids, such as DUFAs, MUFAs, and SFAs. The spectra also indicated intense signals in the range δ 2.88–2.75 for the presence of bis-allylic (CH=CH-CH2-CH=CH) protons of long alkyl chain fatty acids components. In particular, the multiplet at δH 2.85–2.80 were assigned to bis-allylic protons (CH=CH-CH2-CH=CH) of PUFAs (such as DHA and EPA), while bis-allylic protons of other PUFAs, such as α-linolenic fatty acid and DUFAs, appeared at δH 2.77. The presence of partially overlapping singlets at δH 3.22 are due to the polar head group (N(CH3)3) of phosphatidylcholine (PC), while the signal at δH 3.03 is attributed to the CH2N group of phosphatidylethanolamine (PE). The presence of phospholipids in the extracts was confirmed by 31P NMR analysis of few samples (data not shown). Furthermore, signals at δH 0.68–0.69, 0.92, and 1.01, due to characteristic resonances of cholesterol (CHO) and multiplets in ranges of δH 5.26–5.13 and 4.28–4.12, assigned to 1,2-diacylglycerols (DAGs), were also observed. Finally, homarine signals appeared at δH 8.61, 8.44, 8.09, 7.85. The assignments were confirmed by 2D experiments (Figure 5) and literature data [26–29].

**Figure 5.** Expansion of the COSY spectrum of *R. pulmo* lipid extract. Colored boxed regions correlate with the various resonances of homarine.

### *2.3. Lysozyme-like Activity in R. pulmo Oocyte Lysate*

Oocyte lysate of *R. pulmo* showed a natural lysozyme-like activity. By the standard assay on Petri dishes, a diameter of lysis of 9.33 ± 0.32 mm corresponding to 1.21 mg/mL of hen egg-white lysozyme was observed (Figure 6A). The lysozyme activity of the egg lysate was significantly affected by temperature (*p* = 0.0002), ionic strength (*p* = 0.0028), and pH (*p* = 0.0002) of the incubation (Figure 6B–D; Table 1). Post hoc analyses clarified better responses at different experimental conditions tested (Table 2). Increasing the temperature improved proportionally lysozyme-like activity (Figure 6B), showing significant differences among different conditions. The lytic activity increased significantly after dialysis against PB at I = 0.175 (Figure 6C, Table 2). Among all experiments, the maximum

diameter of lysis was reported at pH 4.0, although there were no significant differences among the measured diameters at pH 4 or 6 (Figure 6D, Table 2). A dose-response correlation was obtained when increasing amounts of oocyte lysate were plotted against the respective lysis area diameters (Figure 7). The diameter of the lysis area was positively correlated with the sample volume.

**Figure 6.** (**A**) Lysozyme-like activity of *R. pulmo* oocyte lysate measured on Petri dish. The arrow indicates the diameter of lysis around each well (6.3 mm in diameter) in which the oocyte lysate (30 μL) was loaded. All the wells were loaded with 30 μL of oocyte lysate and represent replicates; (**B**) effect of the temperature (5, 15, 22, and 37 ◦C) on lysozyme-like activity measured at ionic strength (I) = 0.175 and pH 6.0; (**C**) the effect of ionic strength (I = 0.0175, 0.175, 1.75) on lysozyme-like activity measured at temperature 37 ◦C and pH 6.0; (**D**) the effect of the pH (4, 5, 6, 7, 8) on lysozyme-like activity measured at 37 ◦C and I = 0.175. Data are reported as mean value ± standard error.

**Table 1.** Results from the multivariate permutational analysis (PERMANOVA) showing differences in lysozyme activity among different tested conditions.


df = degree of freedom; MS = mean sum of squares; F = F value by permutation; *p* = *p*-value by permutation. \*\* = *p* < 0.01; \*\*\* = *p* < 0.001.

**Table 2.** Results of the pairwise tests showing differences in lysozyme activity among various levels in different laboratory conditions (temperature, ionic strength, pH).


T = T value; P(MC) = probability level after Monte Carlo simulations. \* = *p* < 0.05; \*\* = *p* < 0.01; \*\*\* = *p* < 0.001; ns = not significant.

**Figure 7.** Dose-response curve of lysozyme-like activity of *R. pulmo* oocyte lysate.
