Method for producing a sea urchin extract enriched with 1,4-polyhydroxylated naphthoquinones with antimicrobial and antioxidant activity

Abstract

The invention refers to a method for obtaining polyhydroxy 1,4-naphthoquinones from live sea urchins, comprising the following steps: a) injecting a KCl saline solution into the perivisceral coeloma of female sea urchins to induce spawning; b) collecting the eggs, and grinding them into a fine powder; c) mixing said fine powder with an alcoholic solvent; d) separating the alcohol supernatant comprising the polyhydroxy 1,4-naphthoquinones in solution.
Where the polyhydroxy 1,4-naphthoquinones are selected from: echinochrome A (6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone), spinochrome A (2-acetyl-3,5,6,8-tetrahydroxy-1,4-naphthoquinone), spinochrome B (2,3,5,7-tetrahydroxy-1,4-naphthoquinone), spinochrome C (2-acetyl-3,5,6,7,8-pentahydroxy-1,4-naphthoquinone), spinochrome D (2,3,5,6,8-pentahydroxy-1,4-naphthoquinone), spinochrome E (hexahydroxy-1,4-naphthoquinone) or mixtures thereof.

Claims

1. A method for obtaining polyhydroxy 1,4-naphthoquinones from live sea urchins comprising: a) injecting a potassium chloride saline solution into the perivisceral coeloma of female sea urchins to induce spawning which produces eggs; b) collecting the eggs, and grinding them into a fine powder; c) mixing said fine powder with ethanol or methanol to form an ethanol or methanol supernatant; d) separating out the ethanol or methanol supernatant which comprises the polyhydroxy 1,4-naphthoquinones in solution, wherein the potassium chloride is used at a concentration range between 0.1 M to 2 M and in a volume of solution between 0.1 ml to 2 ml, the method is performed in the absence of acid and base and wherein the sea urchins are kept alive.

2. The method of claim 1, wherein the polyhydroxy 1,4-naphthoquinones are concentrated, separated or purified from the ethanolic or methanolic supernatant obtained in step d).

3. The method of claim 2, wherein the ethanolic or methanolic supernatant is precipitated using a different organic solvent or subjected to evaporation wherein the resulting crystals are resuspended in ethanol to obtain polyhydroxylated 1,4-naphthoquinones with a high degree of purity.

4. The method of claim 2, wherein the methanolic or ethanolic supernatant is concentrated by evaporation and purified by High performance liquid chromatography.

5. The method of claim 1, wherein the polyhydroxy 1,4-naphthoquinones are selected from the group consisting of echinochrome A (6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone), spinochrome A (2-acetyl-3,5,6,8-tetrahydroxy-1, 4-naphthoquinone), spinochrome B (2,3,5,7-tetrahydroxy-1,4-naphthoquinone), spinochrome C (2-acetyl-3,5,6,7,8-pentahydroxy -1,4-naphthoquinone), spinochrome D (2,3,5,6,8-pentahydroxy-1,4-naphthoquinone), spinochrome E (hexahydroxy-1,4-naphthoquinone) and mixtures thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. HPLC chromatography of the organic extract obtained from sea urchin eggs (Tetrapygus niger), using extraction with ethanol (A) or ethanol and hydrochloric acid (pH 2) (B). The presence of EqA was monitored at 520 nm. The mobile phase is isocratic with formic acid 0.1% (50%), acetonitrile (32%) and methanol (18%) and a constant flow of 0.5 ml/min was used. The insert in the figure illustrates the absorbance spectrum of the main compound, which is similar to that reported by Kuwahara et al. (2010).

(2) FIG. 2. Nuclear Magnetic Resonance Analysis of the main compound present in the organic extract of sea urchin (Tetrapygus niger). H.sup.1 NMR (A), C.sup.13 NMR (B), two-dimensional heteronuclear NMR HMBC (Acronym in English: Heteronuclear Multiple Bond Correlation) and HMQC (Acronym in English: Heteronuclear Multiple Quantum Correlation). The analysis that correlates the NMR signals with the 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone structure (equinochrome A) is included.

DESCRIPTION OF THE INVENTION

(3) The invention relates to a new method for obtaining high purity polyhydroxylated 1,4-naphthoquinones, so that it can be used directly in the pharmaceutical and food industries.

(4) A first innovation of the method of the invention with respect to what is known in the state of the art is the raw material used to obtain the polyhydroxylated 1,4-naphthoquinones. Currently, polyhydroxylated 1,4-naphthoquinones such as EqA are extracted mainly from the shells and spines of urchins. The invention proposes to obtain the polyhydroxy 1,4-naphthoquinones from urchin eggs, the first step of the method being then to chemically induce spawning. Since the raw material is ova and not shells or spines, the chemical conditions necessary for the extraction and purification of the EqA pigment are much milder, resulting in a lower expense and contamination associated with the process, given that acids or strong bases are not used.

(5) In addition, the manipulation to induce the spawning process of the sea urchin eggs, which is used in this invention, allows the urchins to remain alive, which enables the production of more eggs by these urchins. In this way the procedure is renewable and sustainable.

(6) Once the eggs are obtained, the process for obtaining the extract enriched in polyhydroxylated 1,4-naphthoquinones comprises the following additional steps: grinding the eggs until a fine powder is obtained, an extraction process with an alcoholic solvent, such as ethanol or methanol in absence of acids or bases, optionally a separation and/or concentration of the alcoholic extract and, if necessary, a final purification by liquid chromatography with a reversed phase column. If all these steps are carried out, the yield of the product EqA is ˜0.2% by dry weight of raw material, with a purity of ˜98%.

(7) In general terms, the invention relates to a method for obtaining polyhydroxylated 1,4-naphthoquinones from live sea urchins, comprising the following steps:

(8) a) injecting a saline solution into the perivisceral coelom of female sea urchins to induce spawning; b) collecting the eggs, and grinding them to obtain a fine powder; c) mixing said fine powder with an alcoholic solvent; d) separating the alcohol supernatant comprising the polyhydroxylated 1,4-naphthoquinones in solution.

(9) Optionally comprising concentrating, separating and/or purifying from the alcoholic supernatant obtained in step d) the polyhydroxylated 1,4-naphthoquinone molecules. In one embodiment the alcohol supernatant is precipitated using another organic solvent, or subjected to evaporation, for example under vacuum; and the crystals are resuspended in ethanol to obtain polyhydroxylated 1,4-naphthoquinones with high degree of purity. In one embodiment, the alcohol supernatant is concentrated by evaporation and purified by HPLC. In another embodiment, the alcohol supernatant is concentrated by evaporation in vacuo, the obtained crystals are resuspended in ethanol and purified by HPLC.

(10) Where the KCl salt used in step a) is in a concentration range between 0.1 M to 2 M, and in a volume between 0.1 to 2 mL.

(11) To grind the eggs, as indicated in step b) these are optionally frozen or dried. The alcohol solvent of step c) is preferably selected from methanol, ethanol or mixtures thereof.

(12) The polyhydroxylated 1,4-naphthoquinones are purified equinochrome A (6-ethyl-2,3,5,7,8-pentahydroxy-1, 4-naphthoquinone), spinochrome A (2-acetyl-3,5,6,8-tetrahydroxy-1,4-naphthoquinone), spinochrome B (2,3,5,7-tetrahydroxy-1,4-naphthoquinone), spinochrome C (2-acetyl-3,5,6,7,8-pentahydroxy-1, 4-naphthoquinone), spinochrome D (2,3,5,6,8-pentahydroxy-1, 4-naphthoquinone), spinochrome E (hexahydroxy-1,4-naphthoquinone) or mixtures thereof.

(13) The process of the invention begins with spawning, which is induced by injection of KCl (0.5 M) into the perivisceral coelom. The induced urchin females are deposited on a smaller diameter vessel, which contains filtered and sterile seawater, where the eggs are received. The collected eggs are filtered, preferably using a fine sieve of pore size 1/32 inches (0.079375 centimeters) to remove impurities. Afterwards it is washed with filtered and sterile seawater. The excess water is subsequently removed by decanting the eggs. Then the eggs are dried at 60-80° C. for 24-72 hours, and are ground to a fine powder, which is cleaned by passing through a sieve of pore size 1/120 inches (0.021 centimeters). The product is then extracted by mixing the powder with methanol or ethanol at a proportion of 1:30 v/v under constant agitation. It is further filtered to remove the egg debris and evaporated under vacuum at 55° C. to 1/10 of the initial volume. The resulting extract is purified by chromatography with a C18 RP column, elution with 30-40% methanol to extract mainly spinochrome E, and finally an elution with 100% ethanol for the extraction of EqA. The resulting extract possesses equinochrome A with a purity of 99%. Finally the product is stored at −20° C. where it maintains a high stability for months.

(14) The present invention in a preferred embodiment allows 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone (echinochrome A) to be obtained from sea urchin eggs, especially from black sea urchin (Tetrapygus niger) spawned by chemical induction, followed by extraction with ethanol or methanol without acids or bases. Given its antimicrobial and antioxidant properties, the obtained EqA can be used in aquaculture, fish processing, the food and medical industry, veterinary medicine and cosmetology. In addition, it can be applied on inert surfaces, to provide antimicrobial properties.

(15) In order to test the antibiotic activity of these compounds, the inventors studied the antibacterial activity, measured as minimum inhibitory concentration (MIC), of the black sea urchin (Tetrapygus niger) extract enriched in equinochrome A (85% purity) and the purified EqA. (99% purity) against various pathogenic bacterial strains of clinical origin, whose results are shown in Table 1.

(16) The results show that the extract, which comprises a mixture of polyhydroxylated 1,4-naphthoquinones, has better antibiotic activity than pure EqA, although both the extract and EqA show good results against strains resistant to conventional antibiotics.

(17) TABLE-US-00001 TABLE 1 Antimicrobial activity of sea urchin extract Tetrapygus niger enriched in equinochrome A (85% purity) and EqA (99% purity) against various pathogenic strains MIC of MIC of Origin of the EqA Extract Bacteria of clinical (μg/ml) (μg/ml) clinical origin isolate Antibiotic resistance pH 5 pH 7 pH 5 pH 7 GRAM POSITIVE S. aureus tracheal penicillin 8 32 4 16 aspirate hemocultivo penicillin 8 32 4 16 hemocultivo penicillin 8 32 4 16 hemocultivo penicillin 8 32 4 16 hemocultivo penicillin, oxacillin, 32 32 16 16 erythromycin, clindamycin, ciprofloxacin GRAM NEGATIVA E. coli hemocultivo ampicillin, chloramphenicol, 32 >64 16 64 cotrimoxazole hemocultivo — 32 >64 16 64 urine ampicillin, aztreonam, 32 64 16 32 cefepime, cefotaxime, ceftazidime, ciprofloxacin Klebsiella hemocultivo ampicillin 64 >64 16 32 oxytica Klebsiella urine ampicillin, aztreonam, 64 >64 16 32 pneumoniae cephalothin, cefepime, cefotaxime, ceftazidime, ertapenem, nitrofurantoin urine ampicillin, aztreonam, 64 >64 16 32 cephalothin, cefepime, cefotaxime, ceftazidime, nitrofurantoin hemocultivo ampicillin, aztreonam, 64 >64 16 32 cephalothin, cefepime, cefotaxime, ceftazidime, ciprofloxacin, chloramphenicol, colistin, cotrimoxazole, gentamicin. Pseudomona hemocultivo cefepime, ceftazidime, 32 >64 16 32 aeruginosa ciprofloxacin, gentamicin cateter ampicillin, aztreonam, 32 >64 16 32 cefepime, ceftazidime, imipenem, meropenem aspirado ampicillin, aztreonam, 32 >64 16 32 traqueal cefepime, ceftazidime, imipenem, meropenem aspirado ampicillin, ciprofloxacin, 32 >64 16 32 traqueal imipenem, meropenem aspirado ampicillin, aztreonam, 64 >64 16 32 traqueal ceftazidime, imipenem, meropenem MIC, minimum inhibitory concentration.

(18) As we have indicated, the polyhydroxylated 1,4-naphthoquinones of sea urchin, such as EqA, possess a high antioxidant activity, which was demonstrated by the DPPH radical capture assay (2,2-diphenyl-1-picrylhydrazyl). The antioxidant activity of EqA, which was observed in the DPPH assay carried out by the authors of this invention, was greater than the antioxidant activities of α-tocopherol and ascorbic acid, both compounds recognized for their antioxidant activity. The antioxidant activity of EqA is dependent on the concentration of the pigment, the pH and the concentration of calcium. In the absence of calcium, the antioxidant activity is observed only at alkaline pH, but in the presence of calcium, the antioxidant activity is observed at alkaline pH and neutral pH, by the formation of stable semiquinone-calcium complexes (Levedeb et al., Archives of Biochemistry and Biophysics (2003) 413: 191-198). The invention is illustrated in the following examples:

EXAMPLE 1

(19) In the present method, female sea urchins (black urchin Tetrapygus niger) are induced to spawn by injecting 0.1-1.0 ml of KCl (0.5 M) between the teeth and the hard outer shell. For the release of the red eggs, the urchin is placed face up on a precipitated glass or some other container of smaller diameter than the urchin, which contains filtered and sterile seawater. The eggs collected at the bottom of the container are washed of solid impurities with filtered and sterile seawater, and by filtration through a metallic sieve of pore size 1/32 inch (0.079375 cm) until all the solid impurities have been removed. The eggs are decanted to remove excess water, prior to processing or storage at −20° C. The eggs are dried at 60-80° C., milled and filtered through a sieve of pore size 1/120 inch (0.021 cm). Fifty ml of eggs are poured onto 1500 ml of ethanol or methanol and incubated at room temperature under constant agitation for 24-48 hours. The extract is collected and the raw material is re-extracted in methanol until no more red color is observed.

(20) The extracts are mixed, concentrated at 55° C. in vacuo to obtain 1/10 of the initial volume. The purification is performed by HPLC with a C18 RP column, washed with 30%-40% methanol to elute spinochrome E or other pigments, and finally elute the EqA with 100% ethanol. The spectrum is shown in FIG. 1A.

EXAMPLE 2

(21) 108 grams (wet weight) of sea urchin eggs (black urchin Tetrapygus niger) are extracted and incubated with 3 liters of methanol. 6 extractions are made until the extract shows no red color. The extracts are concentrated with a rotary evaporator to a volume of 150 ml. Water is added to a final volume of 400 ml and then chromatography of the extract is carried out by a C18 column, previously calibrated with water. Elutions are performed with 10% methanol, 30% methanol and 40% methanol, eluting spinochrome E or other pigments with a lower degree of hydrophobicity than EqA. Then, the EqA retained in the column is eluted with ethanol or methanol (100%), presenting a high purity (>90%).

EXAMPLE 3

(22) Fifty ml of fresh or frozen and washed eggs are poured onto 1500 ml of methanol, and then processed and extracted as described in Example 1.

EXAMPLE 4

(23) The product obtained from Example 1 or Example 3 is precipitated using another organic solvent, or subjected to evaporation. Finally the crystals are resuspended in ethanol to obtain equinochrome A with a high purity (>99%).

(24) The concentration of EqA in ethanol was quantified at 341 nm, considering its molar extinction coefficient of 10650 (M.sup.−1×cm.sup.−1). The samples were diluted to readings between 0.2 and 1.0 absorbance units. The readings were made in triplicate. The purity of the product was determined by high performance liquid chromatography (HPLC) with diode array detector (DAD). A C18 RP column (250 mm×4.6 mm, 3.5 μm) was used. La presencia de EqA se monitoreó a una longitud de onda de 520 nm. Se utilizó un flujo de 0.5 mL/min, con una temperatura de horno de 25° C. The presence of EqA was monitored at a wavelength of 520 nm. A flow of 0.5 mL/min was used, with an oven temperature of 25° C. The mobile phase consisted of a mixture of 50% formic acid 0.1% (solution A), 32% acetonitrile (solution B) and 18% methanol (solution C). Under these conditions the EqA had a retention time of 10.6 minutes. This product was identified by its absorbance spectrum with 2 absorption maximums at 341 and 468 nm, as described by Kuwahara et al. (2010). Alternatively, at a constant flow of 0.5 ml/min the following gradient was used: 0-3 min, solution A (95%), solution C (5%); 3-7 min, solution A (75%), solution B (10%), solution C (15%); 7-15 min solution B (30%), solution C (70%); 15-18 min, solution C (100%); 20 min, solution A (95%), solution C (5%). Under these conditions the EqA had a retention time of 14.8 min.

(25) The mass of the purified EqA was determined by MALDI-TOF by analyzing the positive ion having a mass m/z 267 [M−H+], deducting a mass of 266 u.m.a. This value corresponds to that reported by Kuwahara et al. (2010). Nuclear magnetic resonance analysis of protons, carbon 13, and two-dimensional analysis of heteronuclear correlation HMBC (Acronym in English: Heteronuclear Multiple Bond Correlation) and HMQC (Acronym in English: Heteronuclear Multiple Quantum Correlation) confirm the structure of Echinochrome A as 6-ethyl-2,3,5,7,8-pentahydroxy-1, 4-naphthoquinone (FIG. 2).

(26) In the extract which contains EqA (85-90%) and another compound (10%) that has a retention time of 6 min in the HPLC chromatogram (FIG. 1A). This compound has an absorbance spectrum with two maximums at 353 nm and 478 nm, which correspond to the values reported for spinochrome E (hexahydroxy-1-4-naphthoquinone). The mass spectrometric analysis by MALDI-TOF of this compound indicates a positive ion of mass m/z 255 [M−H+], which allows the deduction of a mass of 254 a.m.u., which corresponds to the molecular mass of spinochrome E. Consequently, it is concluded that the extract of sea urchin eggs of the invention contains spinochrome E.

(27) The black sea urchin ova extract (Tetrapygus niger) possesses antimicrobial activity against various pathogenic bacterial strains, including some multiresistant strains of antibiotics, with a minimum inhibitory concentration in a range of 4-64 μg/ml (Table 1), where the maximum activity is at acidic pH. The purified EqA of this extract containing EqA (85%) possesses an antibiotic activity with a minimum inhibitory concentration between 8 and 64 μg/ml (Table 1) on pathogenic bacteria which are multiresistant to antibiotics. This result suggests that the antibiotic activity of the extract is mainly due to the presence of EqA.

(28) The antioxidant activity of the purified extract and equinochrome A was evaluated by the DPPH radical capture assay (2, 2-diphenyl-1-picryihydracil) (Shankarlal et al., (2011) Am-Euras, J. Sci. Res. 6:178-181). The EC50 of equinochrome A extract (85% EqA) for this assay was 18 μM and the EC50 of pure equinochrome A (99% purity) was 13 μM. Under these conditions the EC.sub.50 of α-tocoferol is 100 μM and of ascorbic acid is 45 μM, indicating a higher antioxidant activity of pure EqA against these known antioxidants. These results also suggest that the EqA possesses the main antioxidant activity of the extract of the invention.

EXAMPLE 5

(29) In order to compare the method of the invention with respect to the traditional method, an extraction was made from the eggs adjusting the pH to 2 with hydrochloric acid.

(30) For which HCl was added to 50 ml of eggs until it reached a pH of 2, later the acidified eggs are poured on 1500 ml of ethanol or methanol and incubated at room temperature in constant agitation for 24-48 hours. The extract is collected and the raw material is re-extracted in methanol until no more red color is observed.

(31) The extracts are mixed, concentrated at 55° C. in vacuo to obtain 1/10 of the initial volume. The purification is performed by HPLC with a C18 RP column, washed with 30%-40% methanol to elute spinochrome E or other pigments, and finally elute the EqA with 100% ethanol. The spectrum of this comparative example is shown in FIG. 1B.

(32) The extraction with ethanol and hydrochloric acid and the extraction with ethanol without hydrochloric acid, make it possible to obtain the same final product (FIGS. 1A and 1B), which corresponds to equinochrome A. The absorbance spectrum and the mass spectrum of the product correspond to the absorbance and mass spectra of EqA (Kuwahara et al., LWT-Food Science and Technology (2010) 43:1185-1190). This is expected since in the traditional method hydrochloric acid is used to dissolve the calcareous structure of shells and spines of urchin, which are the raw material of said traditional method. However, because the eggs of urchins do not have a calcareous structure, when comparing extraction with HCl, or traditional, with the method of the invention, we verify that there are no differences in the profile of molecules eluted with both methods.

(33) In this way, the invention makes it possible to obtain polyhydroxylated 1,4-naphthoquinones without residues of hydrochloric acid, or of any other strong acid or base, since it is not used in its extraction.