Detection and partial characterization of antimutagenic compounds from white shrimp (Litopenaeus vannamei) cephalothorax

ABSTRACT

Mutations are the result of effects produced by mutagenic agents, which may be responsible for the generation of chronic-degenerative diseases such as cancer. The aim of this work was to detect and characterize compounds with antimutagenic activity in white shrimp (Litopenaeus vannamei) cephalothorax. A serial extraction was carried out where different organic solvents, and its biological activity was evaluated. The antioxidant capacity was evaluated by DPPH and ABTS assay, where the methanol and water-soluble extracts had the highest antioxidant capacity. Antimutagenicity was evaluated by the Ames test, where the methanol-soluble extract showed the highest antimutagenic activity. The methanolic extract was fractionated by open column chromatography, the fractions obtained were determined antioxidant and antimutagenic activity and the ability to protect DNA from oxidative damage. Fraction H27 provided DNA the highest protection against the damage caused by H₂O₂. Partial chemical-characterization of fraction H27 suggests the presence of aromatic, hydroxylated, unsaturated compounds.

KEYWORDS:

• Antioxidant
• antimutagenic
• bioactivity
• by-product
• shrimp

Introduction

Cancer is a process by which cells grow and spread without control. Cancer is placed as the second cause of death in the world (World Health Organization, 2020). Oxidative stress is responsible for various diseases such as diabetes, atherosclerosis, metabolic disorders, vascular diseases, and cancer (Guerra 2001). When imbalances occur in the production of reactive oxygen species, free radicals are derived, which cause damage at the cellular level (García Romo et al., 2020) and harmful effects in molecules such as proteins, lipids, and nucleic acids take place (Pizzino et al., 2017).

The generation of changes in the DNA sequence of an organism is known as mutagenicity, which also is associated with metabolic problems in cellular systems causing morbidity and mortality in organisms. For this reason, it is important to search for components that can protect DNA from damage caused by mutagenic agents (Uribe-Yunda et al., 2012).

Chemoprevention is a strategy that has been considered for the benefits they present when treating diseases such as cancer. These compounds can be found naturally and synthetically, being able to prevent the different processes that damage cells, avoiding the early appearance of chronic-degenerative diseases (Steward et al., 2013).

The use of antioxidants of natural origin has been gaining attention lately, since they represent a healthy alternative to synthetic antioxidants; the latter have been reported as either toxicological or carcinogenic agents (Mitterer et al., 2021). Several studies have focused on the antioxidant effect present in terrestrial organisms, extracts from plants such as Secamone afzelii (Sinan et al., 2023), fruits such as pepper where their nutraceutical potential is highlighted (Della Valle et al., 2020) and peptides present in spinach species with anti-inflammatory and antinociceptive antioxidant activity (Maricianno Lorenza et al., 2023). However, the products of marine origin have been of great interest to scientists, due to the enormous number of biological activities they may exert (García Romo et al., 2020).

Previous studies have shown that white shrimp has antiproliferative, antioxidant and antimutagenic activity (Lopez-Saiz et al., 2016). However, studies that have reported antimutagenic activity in white shrimp (Litopenaeus vannamei) have been carried out in the compounds found in the muscle (Wilson-Sanchez et al., 2010). Antioxidant compounds can intervene in mutagenic processes, mainly preventing the oxidation of nitrogenous bases, therefore antioxidants have the potential to be antimutagenic (Renner et al., 1988). Based on the above, the aim of this study was to detect and characterize compounds with antimutagenic activity in white shrimp (Litopenaeus vannamei) cephalothorax.

Materials and methods

Raw material

White shrimp (Litopenaeus vannamei) specimens were obtained from a local shrimp processing company in Hermosillo, Sonora, Mexico, from which the cephalothorax was obtained and stored in polyethylene bags at −20°C until used.

Extraction

The extraction of bioactive compounds was carried out according to Osuna Ruiz et al. (2016). A serial extraction using hexane, acetone, methanol, and water soluble. Firstly, 50 g of cephalothorax were homogenized in a food processor with 150 mL of hexane (1:3 w/v), kept under constant stirring for 24 h and protected from light, at a maximum temperature of 25°C; samples were filtered under vacuum with a Buchner funnel using filter paper (Whatman No. 1). After the solvent was recovered by evaporation, the hexane-soluble extract (HE) was obtained, and the solid was macerated in the same way, with the different solvents serially obtaining the acetone (AE), methanol (ME) and water- (WE) soluble extracts. Extracts were concentrated using a rotary evaporator (Yamato, RE300) under negative pressure at 40°C and carried to dryness under a N₂ stream.

The extracts were re-suspended in the corresponding extraction solvent, bringing them to known concentrations and stored at 4°C until further use.

Antioxidant activity

DPPH 2,2-difenil-1-picrilhidracilo

The methodology reported by Brand Williams and Cuvelier (1995) was followed with a few modifications. Briefly, the extracts were prepared in seral dilutions (500, 250, 125, 62.5, 31.25 µg/mL). A 100 µL sample was mixed with 900 µL of DPPH solution (1.52 mM), incubated for 30 min and then the absorbance read at 540 nm in a spectrophotometer (BIOMATE 3S, UV-Visible Spectrophotometer). The inhibition percentage was calculated according to the formula 1.

(1) $\mathrm{\%}\mathit{Inh}=\left(1-\frac{\left(\mathit{AbsX}-\mathit{AbsControl}\right)}{\left(\mathit{C}-\right)}\right)\times 100$(1)

Where:

Abs X: Absorbance treatment

Abs Control: Absorbance control

C-: Negative control

The inhibition percentage was transformed into trolox equivalents, based on the absorbance data obtained for a curve of different concentrations of this commercial antioxidant, using the formula 2.

(2) $\mathit{y}=0.242\mathit{x}+1.4304$(2)

Where:

x: Trolox equivalents

y: Percentage of inhibition

ABTS 2,2´-azinobis (3-etilbenzotiazolinal 6-sulfonate)

The methodology described by Rea et al. (1999) was used with a few modifications. The extracts were prepared in serial dilutions (500, 250, 125, 62.5, 31.25 µg/mL). A 100 µL aliquot of the sample was mixed with 900 µL of ABTS (7 mM), incubated for 30 min and the absorbance read at 734 nm in a spectrophotometer (BIOMATE 3S, UV-Visible Spectrophotometer). The percentage of inhibition was calculated according to the formula 1.

The inhibition percentage was transformed into trolox equivalents, based on the absorbance data obtained for a curve of different concentrations of this commercial antioxidant, using the formula 3.

(3) $\mathit{y}=0.1031\mathit{x}+0.9362$(3)

Where:

x: Trolox equivalents

y: Percentage of inhibition

Antimutagenic activity

To evaluate the antimutagenic activity of shrimp cephalothorax extracts, the method reported by Maron and Ames (1983) was used. Top agar (2 mL) supplemented with a biotin and histidine solution, 100 µL of Salmonella typhimurium TA100 strain culture, 100 µL of shrimp cephalothorax extract solution (500, 250, 125, 62.5, or 31.25 µg/mL) and 0.1 mg/plate of sodium azide mutagen (AzS) as control mutagen, were combined in a test tube and poured onto minimal glucose agar plates. Negative (spontaneous revertants) and positive (AzS without stimulus) controls were used. Plates were incubated at 37°C for 48 h, and the number of revertant colonies per plate was counted. The inhibition percentage was calculated using the formula 4.

(4) $\mathit{Percentage}\mathit{\hspace{0.17em}}\mathit{of}\mathit{\hspace{0.17em}}\mathit{inhibition}\mathit{\hspace{0.17em}}\left(\mathrm{\%}\right)=\left(1-\frac{\left(\mathit{Treatments}-\mathit{SR}\right)}{\left(\mathit{Positive}\mathit{\hspace{0.17em}}\mathrm{Control}-SR\right)}\right)\times \mathit{\hspace{0.17em}}100$(4)

Where:

SR: Spontaneous Revertants

Open column chromatography

To isolate bioactive compounds from the extract with the highest antimutagenic activity, a fractionation process using open a silica gel (2.5 cm × 74 cm using 70–230 mesh Sigma-Adrich) column chromatography was carried. The elution was performed using different polarities solvents (acetone, methanol, and water). The eluates obtained were subjected to thin-layer chromatography (TLC) and revealed using UV light to identify those fractions sharing similar signals were combined into a single fraction. The mobile phase for TLC was that used for the open column chromatography.

Comet assay (single-cell gel electrophoresis)

Single gel electrophoresis or comet assay is a standard technique for the detection of DNA damage at the single-cell level and can be used to evaluate the protective effect that bioactive fractions may provide on DNA against oxidative damage. This assay was performed as described by Olive et al. (1990), Singh et al. (1998), Yañez et al. (2003) with modifications made for the analysis of antigenotoxic activity.

Vials containing 940 µL of DMEM medium (Dulbecco’s Modified Eagle Medium) were prepared to place 20 µL of whole blood. A 20 µL aliquot of the extract, at different concentrations (100, 50 or 25 µg/mL) was added to the vials, mixed and allowed to incubate for 1 h at 37°C. Subsequently, 20 µL of 10 mM H₂O₂ solution was added, mixed and incubated at room temperature for 10 min. The mixture was centrifuged at 2500 rpm for 30 s (Galaxy 13 microcentrifuge, VWR; max vel 10,000 rpm/7,200× g) and the supernatant was removed. A 225 µL aliquot of low melting point agarose solution (LMPA) (2.57 mg/mL) was added and mixed. A 75 µL volume of the LMPA-blood mixture was taken and placed in the center of a pre-coated slide with a layer of 0.5% regular agarose. A coverslip was placed over the mixture and left in the refrigerator for 10 min. After this, the coverslip was removed and 75 µL of LMPA were added, a coverslip was placed and left refrigerated for 10 min. Slides were removed and placed in Coplin cups containing cold lysis solution (10 mMTris-HCl, 2.5 M NaCl, and 0.1 M Na EDTA, pH 10). Coplin cups were kept refrigerated until electrophoresis.

The samples were then placed in a dark electrophoresis chamber which contained a cold alkaline buffer (300 mM NaOH, 1 nM EDTA, pH 13). The samples were allowed to stand for 20 min. Electrophoresis conditions were 25 mV, 300 mA, for 20 min. Once the electrophoresis was finished, the slides were removed and washed three times with neutralization buffer (0.4 mM Tris buffer at pH 7.5) for 5 min per wash. Finally, the slides were washed twice with anhydrous absolute ethyl alcohol for 5 min per wash. Excess of alcohol was removed, and slides were left to dry, stained with 60 µL of ethidium bromide and a coverslip was placed. An epifluorescence microscope equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm was used to visualize the comets. The comet image magnification was 200× . Olive tail moment was measured using the Image Analysis System (IAS-Delta Sistemi, Roma, Italy) software. Cell viability was determined by trypan blue dye exclusion and was always above 95%. The DNA damage produced was reported as the Olive Tail Moment (Olive et al., 1990).

Spectroscopic methods

UV-Visible spectroscopy

The sample was dissolved in DMSO and placed in a quartz cell where it was scanned using a spectrophotometer (BIOMATE 3S, UV-Visible Spectrophotometer) in the wavelength range 190–1100 nm.

¹³C and ¹H-NMR analysis

The chemical-structural characterization of the compounds with antimutagenic activity was carried out by means of carbon and proton magnetic resonance (¹³C- and¹H-NMR), using an Agilent Technologies equipment at 400 MHz. The sample was dissolved in CDCl₃ (Sigma-Aldrich, Saint Louis, MI, U.S.A.) at 20 mg/mL and tetramethylsilane (TMS) was used as internal standard; samples were placed in 5 mm diameter ultraprecision NMR tubes. Chemical changes were recorded in ppm.

Results and discussion

Extraction

From the serial extraction, 4 crude extracts were obtained from the head of the white shrimp (L. vannamei): hexanic (HE) with an intense orange color, acetonic (AE) with an orange color, methanolic (ME) with a yellow color and the water-soluble (WE) that presented an opaque white color. From the serial extraction with hexane, acetone, methanol and water-soluble, yields were obtained 6.3% ± 1.8 to (hexane), 4.0 ± 1.6 (acetone) 5.8% ± 3.5 (methanol) 4.9% ± 0.9 (water-soluble), obtaining a higher yield for the hexane extract. The physical characteristics represent the difference in the composition of the compounds present in each of the extracts.

Antioxidant activity DPPH

The extracts showed significant differences with respect to their capability of donating hydrogens (Table 1), being the methanolic extract, at test concentrations of 500, 250 and 125 µg/mL and the aqueous extract, at test concentration of 500 µg/µL, those where greater amounts of trolox equivalents were recorded, obtaining the highest value for the aqueous extract (304.84 µL TE/g). A dose-dependent reduction of the DPPH radical was observed for the methanolic extract, even at low concentrations. DPPH is a free radical that is inhibited by the donation of hydrogen atoms by an antioxidant; therefore, these results suggest that these extracts exhibit the capability of donating protons by which may stabilize free radicals.

Table 1. Antioxidant activity of crude extracts of white shrimp cephalothorax by DPPH and ABTS assay.

Concentration (µg/mL)	Hexane	Acetone	Methanol	Water-soluble
DPPH
31.2	−1.8 ± 1.8^g	−2.6 ± 2.5^g	101.2 ± 12.5^def	38.5 ± 7.9^fg
62.5	64.9 ± 3.9^efg	−2.2 ± 0.5^g	147.3 ± 4.2^cde	55.9 ± 4.4^efg
125	44.6 ± 1.3^fg	−1.9 ± 1.1^g	205.7 ± 4.2^abc	57.1 ± 36.6^efg
250	92.8 ± 2^.defg	0.6 ± 1.5^g	266.9 ± 5.4^ab	78.8 ± 55.5^efg
500	179.2 ± 5.7^bcd	5.4 ± 0.6^fg	288.6 ± 20.7ª	304.9 ± 24.3^a
ABTS
31.2	289.9 ± 81.5^gh	90.0 ± 45.2^h	371.6 ± 21.7^gh	839.4 ± 76.1^ef
62.5	553.4 ± 117.5^fg	139.3 ± 24.3^h	705.6 ± 72.9^ef	1219.8 ± 180.6^cd
125	925.2 ± 337.1^de	372.6 ± 232.1^gh	1298.4 ± 15.4^c	1836.4 ± 13.1ª
250	1398.0 ± 304.5^bc	522.3 ± 36.0^fg	1649.0 ± 218.2^ab	1938.2 ± 13.1^a
500	1872.7 ± 47.6^a	918.6 ± 33.4^de	1943.5 ± 8.6^a	1962.9 ± 1.8^a

The values are represented as means ± S.D. of three independent assays with five replicates each. Different letters within the same section indicate significant difference (p ≤ 0.05).

Antioxidant activity ABTS

The extracts with different polarity showed significant differences (p ≤ 0.05) with respect to their capability to donate electrons, as assessed by the ABTS method (Table 1). The aqueous (500, 250 and 125 µg/mL), the methanolic (500, 250 µg/mL), and the hexanic (500 µg/mL) extracts had the greatest amounts of trolox equivalents, obtaining the highest value for the aqueous extract (1962.97 µL TE/g).

These results suggest that the compounds present in the extracts obtained from the cephalothorax of white shrimp (L. vannamei) can donate electrons which may be transferred to radicals.

Since many mutagens may act through the generation of reactive oxygen species (ROS), and ROS are also able to oxidate nitrogenous bases triggering mutations (Słoczyńska et al., 2014), the data obtained from the antioxidant tests gives important information regarding the possible action mechanism by which shrimp compounds might exert the antimutagenic activity.

Antimutagenic activity

Crude extracts soluble in hexane, acetone, methanol and water-soluble were evaluated for their antimutagenic activity at different concentrations (500, 50, 5 and 0.5 μg/mL), using positive mutagenicity controls. The crude extracts did not show significant differences (p ≤ 0.05) at the highest concentrations and in the treatments, it was possible to observe a dose-response effect (Table 2). The inhibition percentages were established as strong (>60%), moderate (40–60%), weak (20–40%) and negligible (<20%) (Ikken et al., 1999).

Table 2. Antimutagenic activity of crude extracts and fractions of white shrimp cephalotorax obtained after open column chromatography tested on Salmonella typhimurium TA100 strain at different concentrations (0.5–500 μg/mL). Mutagen sodium azide (0.1 mg/mL) was used as a positive control.

Dose (µg/mL)	Crude extracts				Fractions
	Hexane	Acetone	Methanol	Water-Soluble	H8	H25	H27
500	56.0 ± 5.7^ab	53.9 ± 5.7^abc	70.4 ± 7.6^a	69.2 ± 3.9^a	76.4 ± 3.1^ab	75.3 ± 3.1^ab	84.2 ± 4.1^a
50	19.2 ± 4.0^efg	36.0 ± 7.2^bcde	53.6 ± 4.1^abc	52.3 ± 5.7^abcd	56.7 ± 2.1^cde	65.7 ± 3.9^bc	75.1 ± 2.2^ab
5	4.3 ± 7.5^fgh	27.0 ± 2.7^def	37.2 ± 8.6^bcde	32.3 ± 6.1^cde	48.8 ± 4.0^de	60.9 ± 1.5^cd	66.4 ± 4.6^bc
0.5	−1.5 ± 2.6^hi	−4.5 ± 18.7^gh	28.2 ± 8.9^cdef	20.5 ± 8.7^efg	45.6 ± 1.4^e	44.0 ± 10.0^e	61.5 ± 5.3^cd

Results are presented as the percentage of inhibition of AzS mutation. The values are represented as means ± S.D. of three independent assays with three replicates each. Different letters in each section indicates significant difference (p ≤ 0.05).

The HE only showed an inhibition percentage against sodium azide (AzS) of 56% at 500 μg/mL, which is considered a moderate inhibition; lower concentrations exerted no effect on AzS mutation. AE exerted a moderate inhibition of AzS with 54%, decreasing to 36% (50 μg/mL) and 27% (5 μg/mL), considering these last two as a weak effect. The ME inhibited up to 70% of AzS effect, which can be considered a strong effect. ME maintained higher inhibition values than AE and HE, at all the concentrations tested. Finally, the WE presented inhibition percentages similar to those described for ME, however, these were slightly lower. Based on the above, the effect of ME was the most promising as an antimutagenic compound against sodium azide, therefore, this extract was selected for further tests.

Open column chromatography

After the biological activity was studied, ME was subjected to an open column chromatography to separate the compounds according to their polarity, obtaining 45 eluates. The 45 eluates were subjected to thin-layer chromatography to combine those that showed similar patterns, thus obtaining 29 fractions in total (H1–H29); they were dried under a N₂ stream and resuspended in methanol for further analysis.

Antioxidant activity of fractions

The DPPH test was performed in all 29 fractions. Fraction H8 was the one that presented the highest value (307.25 μM ET) (Table 3). On the other hand, when the ABTS method was performed, fraction H23 presented the highest activity (956.3 μM eT) (Table 3), with values similar to those of fractions H8, H22, H24, H25, H26 and H27. Based on the results, antioxidant activity was determined using lower concentrations and the half-maximal inhibitory concentration (IC₅₀) for these fractions were determined (Table 4). Fractions with the lowest IC₅₀ were H8, H25 and H27, therefore, these fractions were selected to evaluate their capability of protecting DNA against oxidative damage.

Table 3. Antioxidant activity of fractions of Litopenaeus vannamei cephalotorax by DPPH (500 μg/mL) and ABTS (100 μg/mL) assays.

Fraction	DPPH μM eT	ABTS μM eT
H1	65.5 ± 3.2^fgh	181.6 ± 39.3^i
H2	71.6 ± 1.2^fgh	243.4 ± 22.6^ghi
H3	62.2 ± 2.9^gh	158.9 ± 7.4^ij
H4	93.6 ± 0.7^efgh	228.9 ± 7.9^ghi
H5	59.8 ± 1.7^gh	164.4 ± 20.6^ij
H6	57.4 ± 0.8^gh	65.3 ± 7.9^j
H7	63.4 ± 3.3^gh	207.9 ± 14.4^hi
H8	307.3 ± 1.5^a	950.9 ± 6.4^abc
H9	242.8 ± 3.9^abc	751.9 ± 8.8^d
H10	208.7 ± 0.8^abcd	324.2 ± 12.4^g
H11	123.4 ± 3.6^defgh	191.6 ± 17.5^i
H12	136.7 ± 4.1^cdefg	213.4 ± 4.7^ghi
H13	264.8 ± 3.5^ab	781.0 ± 8.4^d
H14	217.4 ± 3.4^abcd	464.1 ± 7.7^f
H15	243.7 ± 1.7^abc	590.3 ± 1.2^d
H16	258.4 ± 2.4^ab	634.8 ± 11.3^e
H17	247.6 ± 0.1^ab	536.7 ± 14.1^ef
H18	234.6 ± 1.4^abc	808.3 ± 36.2^d
H19	174.6 ± 12.5^bcdef	316.1 ± 6.7^gh
H20	23.9 ± 5.9^g	139.9 ± 23.5^ij
H21	212.0 ± 0.9^abcd	840.9 ± 20.1^cd
H22	198.1 ± 1.5^abcde	961.8 ± 0.7^a
H23	52.6 ± 31.9^gh	956.3 ± 2.4^ab
H24	79.7 ± 1.6^fgh	958.1 ± 1.2^a
H25	55.3 ± 0.8^gh	939.0 ± 9.5^abc
H26	48.9 ± 1.3^gh	954.5 ± 2.7^ab
H27	61.6 ± 0.5^gh	939.9 ± 7.1^abc
H28	55.3 ± 6.9^gh	843.7 ± 8.4^bcd
H29	83.0 ± 25.5^fgh	840.1 ± 10.1^cd

The values are represented as means ± S.D. of three independent assays with five replicates each. Different letters indicate significant difference in each column (p  ≤ 0.05).

Table 4. ABTS half-maximal inhibitory concentration (IC₅₀) of Litopenaeus vannamei cephalotorax methanolic fractions.

Fracción	IC50 (mg/mL)
H8	0.025
H22	0.019
H23	0.026
H24	0.014
H25	0.005
H26	0.024
H27	0.004

Antimutagenic activity of fractions

Fractions showing the highest antioxidant activity were analyzed to determine their antimutagenic potential. Fractions H8, H25 and H27 were tested against sodium azide as mutagen (Table 2).

Fractions analyzed showed a greater antimutagenic activity than crude fractions; this may be since bioactive compounds in fractions are more isolated, therefore, compounds are able to inhibit the mutagenic effect of AzS. H27 fraction was the one that showed the highest antimutagenicity against AzS when tested on Salmonella typhimurium TA100 tester strain, which is prone to base pair substitutions (Maron & Ames, 1983), therefore, the compounds present in the fractions obtained from the methanolic extract might be capable of reversing this type of mutations.

Antigenotoxicity test of fractions

Cell viability by trypan blue staining

Carrying out this technique allowed us to know if blood cells used as the basis for the study with the Comet Assay were in good condition. Values above 95% viability were obtained on each test.

Determination of the fraction with the highest protective activity against oxidative damage to DNA

The methanolic extract and the fractions obtained from it (H8, H25 and H27) were evaluated (100, 50 and 25 μg/mL) by the Comet Assay. Blood samples were incubated for 1 h and, subsequently, oxidative damage was caused (100 mM H₂O₂) and residual genotoxic damage was evaluated. To calculate this, 100 comets per slide were counted and scored, and damage was expressed as Olive Tail Moment (OTM) (Figure 1).

Figure 1. Protective effect (olive time moment) to different concentrations (25 to 100 μg/mL) of methanolic extract and fractions H8, H25, H27 on blood cells exposed to H₂O₂. The value is expressed as means ± S.D. from two independent experiments. Different letters represent statistical difference (p ≤ 0.05).

Figure 1 shows that, in the negative control (C-), a value of 21.41 OTM was obtained. The positive control (C+) presented a high genotoxic damage, with values of 45.3 OTM; this is due to the use of hydrogen peroxide, which can cause genotoxic damage to blood cells. It has been reported that H₂O₂ can cause the DNA helix to break due to its interaction with the most oxidizable sites; in fact, the reaction occurs widely at guanine (8-oxo-7,8-dihydroguanine) and, possibly, with thymine (glycol gymine) (Tayeb, 2020).

Significant differences were observed after treatments; the ME presented similar values to the positive control, suggesting that this extract did not provide protection to the DNA. On the other hand, when fractions were analyzed (100 and 50 μg/mL), they were able to reduce the OTM. Fraction H27, at a concentration of 100 μg/mL, showed lower values than those of the negative control; therefore, this extract, in addition to providing protection against H₂O₂ damage, may also have a repair effect on DNA, thus reversing the basal damage found in cells (Figure 2).

Figure 2. Comets observed in the DNA oxidative damage protection assay. (a) control + (b) control – H₂O_2, (c) H₂O₂ + methanolic extract, (d) H₂O₂ + H8 fraction, (e) H₂O₂ + H25 fraction, and (f) H₂O₂ + fraction H 27.

The lowest values obtained for each fraction were H27 with an OTM of 16.41, followed by H25 and H8 with OTM of 22.58, and 27.8, respectively. As for ME the OTM was 39.52. When presenting values lower than those of the negative control, the compounds in fraction H27 should continue to be studied. To our knowledge, this is the first report of antigenotoxic activity by compounds extracted from shrimp cephalothorax.

Chemical-structural characterization

Based on the results obtained from biological activity, fraction H27 was the one that could exert the highest antigenotoxic activity, therefore, spectrophotometric studies were carried out to determine its complexity and determine the presence of functional groups.

Spectrometry UV-Vis

Regarding the physical characteristics, fraction H27 was obtained as a purple liquid. A scan was performed to determine the absorbance signals in the wavelength range from 190 to 1100 nm (Figure 3). Signals between 200 and 220 nm have been reported as characteristic of aromatic and phenolic compounds (Boulet et al., 2017). On the other hand, the signals between 450 and 500 nm, characteristic of carotenoids (Lichtenthaler & Buschmann, 2001) present in white shrimp such as astaxanthin, β-carotene, echinone and canthaxanthin (Arredondo Figueroa et al., 2003), were not observed, this could be due to the affinity of these pigments for non-polar or low polarity solvents such as hexane and acetone (Rivera & Canela, 2012).

Figure 3. Spectra of H27 fraction obtained by (a) UV-Vis, (b) ¹³C NMR (dissolved in CDCl₃) and (c) ¹H NMR (dissolved in CDCl₃).

¹H-NMR analysis

On the other hand, a study was carried out by means of proton magnetic nuclear resonance and, according to the signals that could be observed in the spectrum (Figure 3), it can be affirmed that the fraction is still complex and that it is made up of several chemical structures, therefore, there is a great variety of signals resulting from the mixture of compounds present.

In the proton spectrum it can be seen, at high field, signals with chemical shifts between δ = 0.8–2.1 ppm due to the presence of protons adjacent to aliphatic carbons linked in simple bonds (H-C); these signals are difficult to attribute to specific compounds when there is a mixture of compounds, since there is an overlapping of signals; however, they are characteristics of single links. As for the signals found with chemical shifts between δ = 2.1and 3.0 ppm, the signals can be attributed to aliphatic protons attached to carbon atoms adjacent to carbonyl groups (HC-C=) (Zghari et al., 2017). Moving to a low field, there were signals with chemical shifts between δ = 3.0–4.0 ppm, which are characteristic of protons attached to carbons that are single bonded to oxygen (H-C-O) (Zghari et al., 2017). The signals with chemical shifts between δ = 5.0–6.0 are attributed to the presence of protons that are attached to carbons linked by double bonds, also known as vinyl protons (Rivero et al., 2017). Regarding the signals found in the low field, with chemical shifts between δ = 6.6–8.0, these can be attributed to protons that are attached to carbons of aromatic rings adjacent to hydroxyl groups (Charisiadis et al., 2014).

13C-NMR analysis

A carbon magnetic nuclear resonance study was also performed to determine chemical shifts; however, due to the yields obtained, the concentration of the sample does not allow to appreciate all the signals (Figure 3), however, those that were detected can be attributed as described in the following lines. At low field, with chemical shifts between δ = 0–50 ppm, the signals attributed to carbons linked in single bonds can be observed; moving to higher field, signals between δ = 50–70 ppm are attributed to carbons adjacent to a hydroxyl group; the signal seen at a shift δ = 100 ppm is attributed to aromatic-type compounds (Zghari et al., 2017).

The different compounds that compose fraction H27 seems to be responsible for the biological activities in the cephalothorax fraction of L. vannamei, for example, hydroxyl groups, aromatic rings and/or carbons that have double bonds they have characteristics that could help stabilize ROS, this being also a mechanism of antimutagenic activity (Słoczyńska et al., 2014). The mechanisms of action of the antioxidant activity can be by electron transfer (SET) or proton transfer (HAT), mechanisms which depend on the chemical structure of the compounds present in our extracts and fractions.

Conclusion

The methanolic and water-soluble extracts were able to exert antioxidant and inhibit the mutations of a known mutagen, therefore, they could be a possible source of chemopreventive compounds. Fraction H27, isolated from the methanolic extract, was able to protect DNA of cells against oxidative damage; spectroscopic studies suggest the presence of double bonds, aromatic rings and hydroxyl groups in the compounds of the fraction, nevertheless further studies are needed to identify the chemical structure for the compound responsible of the biological activity.

Acknowledgments

The authors acknowledge the National Council for Science and Technology (CONACyT) of Mexico for financing grant proposals A1-S-9762 and the graduated scholarship granted to Héctor-Enrique Trujillo-Ruiz. CVU 1078709.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Consejo Nacional de Humanidades, Ciencias y Tecnología (CONAHCYT) [A1-S-9762].