Evaluation of health benefits of sea lamprey (Petromyzon marinus) isolates using in vitro antiinflammatory and antioxidant assays

Sea lamprey (Petromyzon marinus), a parasitic fish which survives on blood of other fishes, is consumed as a delicacy in many countries. Our earlier studies on sea lamprey compounds that showed potential to deter adult sea lampreys yielded several sterols, glycerides, free fatty acids, amino acids, organic acids and nitrogenous compounds. Therefore, this study was to assess the health-benefits of these compounds including additional isolates from HPLC fractions that kept aside due to lack of activity in sea lamprey deterrent assays. In vitro cyclooxygenase enzymes (COX-1 and -2) and lipid peroxidation (LPO) inhibitory assays, respectively, were used to determine antiinflammatory and antioxidant activities. Among the tested sterols, cholesteryl eicosapentaenoate and cholesteryl arachidonate exhibited IC50 values of 14.6 and 17.7 μg/mL for COX-1 and 17.3 and 20.8 μg/mL for COX-2, respectively. Cholesteryl palmitate and cholesteryl oleate showed moderate COX-1 and COX-2 enzyme inhibition at 25 μg/mL. Amino acids arginine, tyrosine, glutamic acid, tryptophan and asparagine also showed moderate COX-1 and COX-2 inhibition at the same concentration. Among the twelve new isolates from fractions that we did not investigate earlier, a novel uracil derivative petromyzonacil showed COX-1 and COX-2 inhibition at 25 μg/mL by 35 and 15%, respectively. Cholesterol esters tested at 25 μg/mL exhibited LPO inhibition between 38 and 82 percent. Amino acids cysteine, methionine, aspartic acid, threonine, tryptophan, histidine, glutamic acid, phenylalanine and tyrosine at 25 μg/mL showed LPO inhibition between 37 and 58% and petromyzonacil by 32%. These assay results indicate that consumption of sea lamprey offer health-benefits in addition to nutritional benefits.


Introduction
The sea lamprey (Petromyzon marinus) is native to the northeast Atlantic coasts of Norway, Iceland and the Barents Sea, and south along the Atlantic shore to northern Africa [1]. It is an eel-like fish with an anadromous lifestyle in the native range, with a land-locked population in the Laurentian Great Lakes [2]. Historically, it was consumed during lent by the Romans. It is sold in European supermarkets as a highly priced seasonal delicacy and consumed by incorporating it in pies and blood sausages [3,4]. There are many studies on the composition of lipophilic and nitrogenous constituents in muscles and other organs of sea lamprey [5][6][7][8]. We have also studied sea lamprey extracts for its adult sea lamprey deterrent activity in field and raceway assays. This resulted in the identification of several lipophilic and nitrogenous compounds from the deterrent fractions of sea lamprey extract [9,10]. Some of the subfractions in the past research, minor in quantities and not deterrent, were kept aside [10]. Since this study was to evaluate the health-benefits of the constituents in the sea lamprey, we isolated and identified pure compounds from those subfractions and conducted bioassays of new and earlier isolates as a block by in vitro antiinflammatory and antioxidant activities [11][12][13][14][15].

Isolation and identification of compounds in fractions I-IV
Sea lampreys were collected according to the process approved by the Michigan State University institutional animal care and use committee (permit # AUF 01/14-007-00) [9,10] (see S1 File for experimental details). Experimental details of sea lamprey skin extraction, solvent-solvent partition and MPLC fractionation are provided in the S1 File. Previously, the purification of the water-soluble fraction of the sea lamprey skin extract afforded many subfractions by HPLC. Among these, subfractions I-IV were not investigated due to lack of sea lamprey deterrent activity in the field assays [10]. However, human health-effects are independent of adult sea lamprey deterrent activity. Therefore, isolation of compounds from all fractions including subfractions (I-IV) was important to assess the health-beneficial effects of sea lamprey isolates (S1 File).

Results
Fractionation and purification of the uninvestigated fractions earlier from water-soluble fraction of sea lamprey extract used in the sea lamprey deterrent studies were carried out with reverse phase MPLC and preparative HPLC methods [10]. The chemical identity of all pure isolates was determined by 1 H (500 MHz), 13 C-NMR (125 MHz) and HRESIMS experiments. The 1 H and 13 C chemical shift values were expressed in parts per million (ppm), and residual solvent signals for D 2 O at 3.79 ppm, and for DMSO-d 6 at 2.50 and 39.9 ppm were used as internal standards, respectively.

Discussion
The aqueous ethanolic Soxhlet extraction of 1 kg of whole adult sea lamprey yielded 83.5 g of lipophilic and 37.6 g of water-soluble constituents. Similarly, separate extraction of 1 kg of skin gave 12.3 g of lipophilic and 10.3 g of water-soluble constituents [9,10]. Previous work by Araujo et al reported about 30-35% of adult sea lamprey body contained dry matter [6]. Further analysis of the dry matter revealed that about 38-51% and 42-54% of it was composed of lipids and proteins, respectively [5][6][7][8]. We have reported that the lipophilic fraction of the sea lamprey skin consisted of 18.4% sterols, 34.5% of glycerides and 40.5% of free fatty acids and its lipophilic fraction of the whole sea lamprey is consisted of 4.2% sterols, 57.6% of glycerides and 38.2% of free fatty acids, respectively [9]. The steroidal composition was primarily composed of free cholesterol followed by cholesterol fatty acid esters such as cholesteryl palmitate, cholesteryl oleate, cholesteryl eicosapentaenoate and cholesteryl arachidonate [9]. The glycerides fraction was composed of 90% triglycerides and 10% di-glycerides (1,3 and 1,2 di- Table 2 substituted glycerides). Based on GCMS analysis, combined free fatty acids and glycerides fractions from sea lamprey skin extract constituted 36.1% saturated, 46.6% monounsaturated (MUFAs) and 17.3% polyunsaturated (PUFAs) fatty acids [9]. Studies revealed that fatty acids and sterols are essential for the body due to their crucial roles in metabolism and signal mediation as well as source of energy [22]. Among the PUFAs we consume ɷ-3, ɷ-6 and ɷ-9 PUFAs plays a vital role in our body and C18:3ɷ3 (α-linolenic acid), C20:5ɷ3 (eicosapentaenoic acid), C22:6ɷ3 (docosahexaenoic acid), C18:2ɷ6 (linoleic acid) and C2O:4ɷ6 (arachidonic acid) are considered as essential fatty acids. In addition, the protective roles of ɷ-3 PUFAs such as docosahexaenoic acid (DHA, C22:6ɷ3) and eicosapentaenoic acid (EPA, C20:5ɷ3)) have been reported in the prevention of many diseases including cancers, inflammatory and autoimmune diseases, cardiovascular diseases as well as psychiatric and mental illnesses [23][24][25]. Analysis of amino acids composition in liver, muscles and plasma collected from sea lamprey have been reported. Among the free amino acids from liver and the muscles, about 20-25% of it comprised of essential amino acids [5][6][7]. Our studies showed that the water-soluble fraction contained 19.2% of amino acids, 80.1% of nitrogenous compounds and 0.72% of organic acids [10]. Even though the previous work on liver and muscle tissues and plasma reported amino acids alanine and lysine, we did not detect those in our studies with se lamprey extract [10,11].

. Percent antiinflammatory (COX-1 and -2) and antioxidant (LPO) inhibitory activities of compounds isolated from sea lamprey lipophilic and water-soluble fractions tested at 25
Cyclooxygenase enzymes (COX-1 and -2) catalyze the conversion of arachidonic acid to prostaglandin and other signaling intermediates responsible for the onset of inflammation in the body [11][12][13][14][15]21]. Foods with ability to inhibit COX enzymes therefore could relieve the symptoms of inflammation and pain by functioning as modulators of inflammation signaling pathways. The cholesterol esters from sea lamprey showed moderate COX-1 enzyme inhibition by 37-74%, and COX-2 by 17-66% at 25 μg/mL concentration (Table 2) (Fig M in S1  File). As reported earlier, an increase in double bonds in fatty acid chain enhanced the inhibition of COX enzymes and a similar trend in activity was evident in the case of cholesterol esters [21]. Our data indicated that cholesteryl eicosapentaenoate and cholesteryl arachidonate exhibited IC 50 values of 14.6 and 17.7 μg/mL for COX-1 enzyme and 17.3 and 20.8 μg/mL for COX-2 enzyme, respectively (Fig Q in S1 File). Increasing the concentrations of cholesteryl palmitate and cholesteryl oleate to 50 μg/mL in the assay did not affect the inhibition of COX enzymes. The COX enzymes inhibitory activity profiles of cholesteryl palmitate and cholesteryl oleate were similar to the activity profiles of aspirin and ibuprofen and cholesteryl eicosapentaenoate and cholesteryl arachidonate to that of naproxen. The water-soluble fraction composed of 19.2% of free amino acids and among them about 50% were essential amino acids [10]. The COX enzyme inhibitory activities of the isolated amino acids at 25 μg/mL showed that arginine, tyrosine, glutamic acid, tryptophan and asparagine inhibited COX-1 enzyme moderately by 30, 37, 32, 37 and 28%, and COX-2 by 12, 22, 14, 20 and 17%, respectively. Increasing the concentration to 50 μg/mL of these amino acids did not impact the inhibitory activities of both COX enzymes. Rest of the amino acid isolates from sea lamprey did not inhibit COX enzymes at 25 μg/mL (Table 2) (Fig N in S1 File). Petromyzonacil and inosine inhibited COX-1 enzyme by 25 and 35%, and COX-2 by 15 and 26%, respectively at 25 μg/mL (Fig O in S1 File). Dose response of petromyzonacil against COX-1 and COX-2 enzymes were carried out at 12.5, 25, 50, 100 and 200 μg/mL concentrations and showed an IC 50 value of >200 μg/mL (Fig R in S1 File). Other nitrogenous compounds spermine, putrescine, adenosine, hypoxanthine, xanthine, adenine and creatine and organic acids α-ketovaleric, α-ketoisovaleric, α-ketobutyric, β-hydroxybutyric, pyruvic and 3-phenyllactic acids did not inhibit COX enzymes at 25 μg/mL ( Table 2) (Figs O and P in S1 File).
The antioxidant assay, the lipid peroxidation inhibitory (LPO) assay, used in this study measures free radical scavenging capability of pure compounds and extracts [11][12][13][14][15]21]. In this assay, peroxidation of the lipid was initiated by the addition of Fe 2+ resulting in the generation of radicals, which cause the peroxidation of LUV and in turn the fluorescent probes embedded in it. The loss of fluorescent intensity in the assay indicates the oxidative damage to the lipid component of the LUV [11][12][13][14][15]21] (S1 File). At 25 μg/mL concentration, cholesteryl palmitate, cholesteryl oleate, cholesteryl eicosapentaenoate and cholesteryl arachidonate inhibited LPO by 38, 60, 72 and 82%, respectively (Table 2) (Fig S in S1 File). The most abundant steroidal compound in sea lamprey was cholesterol and it lacked LPO inhibitory activity. Antioxidant activity profiles of the cholesterol esters were like its antiinflammatory profiles. That is, an increase in chain length and unsaturation increased the LPO activity. Majority of the lipophilic fraction of the sea lamprey extract consisted of glycerides and free fatty acids [9]. We have reported antioxidant activities of glycerides and free fatty acids and hence did not investigate those compounds again in this study [12,14,21]. Among the amino acids tested at 25 μg/ mL, aspartic acid, cysteine, methionine, threonine, tryptophan, histidine, glutamic acid, phenylalanine and tyrosine showed moderate LPO inhibition by 37, 47,59, 45, 58, 40, 46, 44 and 55%, respectively. Other amino acid isolates from sea lamprey did not exhibit significant LPO inhibition at the same concentration (Table 2) (Fig T in S1 File). Petromyzonacil showed moderate LPO inhibition by 32% at 25 μg/mL (Fig U in S1 File). Nitrogenous isolates spermine, putrescine, adenosine, hypoxanthine, xanthine, adenine, inosine and creatine and organic acids (α-ketovaleric, α-ketoisovaleric, α-ketobutyric, β-hydroxybutyric, pyruvic and 3-phenyllactic acid) did not inhibit LPO at 25 μg/mL ( Table 2) (Figs U and V in S1 File).

Conclusions
The whole adult sea lamprey yielded 8.35% of lipophilic and 3.76% of water-soluble extracts and its skin yielded 1.23% of lipophilic and 1.03% of water-soluble extracts, respectively. We have characterized most of the isolable compounds in sea lamprey extract as sterols, glycerides and free fatty acids, and water-soluble extract contained amino acids, organic acids and nitrogenous compounds. Isolation of petromyzonacil and keto acids from adult sea lamprey in this study is for the first time. This is also the first report on the evaluation of in vitro antiinflammatory and antioxidant activities of sea lamprey isolates. Among the tested compounds, cholesteryl eicosapentaenoate and cholesteryl arachidonate showed antiinflammatory activity profiles similar to NSAID naproxen at about the same concentration. Our data suggests that the total fatty acids and glycerides content in sea lamprey is similar to anchovy and mackerel. The monounsaturated and polyunsaturated fatty acids (EPA and DHA) in sea lamprey are strong antiinflammatory and antioxidant agents. Bioassays clearly indicated moderate to good antiinflammatory and antioxidant activities for the most compounds present in sea lamprey and the activity profiles were comparable to over the counter or prescription antiinflammatory agents and approved antioxidants employed in food industry. It implies that consumption of sea lamprey provides health-benefits in addition to nutritional benefits like other functional foods. This study also encourages to view sea lamprey as a potential food source rather than just an invasive parasite to fishes and an economic opportunity for the Great Lakes fishing industry.