Collection and preparation of extracts
Undaria pinnatifida samples were collected seasonally at Belvedere Beach (45° 57'41, 8" S, 67° 33' 20.9" W), south of Comodoro Rivadavia city, in the central region of the Golfo San Jorge. During the collections, the temperature, pH and salinity of the water were determined. The algal material was washed with sea water to remove remains of sand and accompanying flora and fauna. One specimen from each collection was preserved in the Herbarium Regional Patagónico of the Universidad Nacional de la Patagonia San Juan Bosco under Nº HRP 7649, 7651, 7648, 7652, corresponding to winter, spring, summer and autumn, respectively. The rest of the material was dried indoors, protected from light, at room temperature. Once dry, the fronds (including the sporophyll), stripped of the holdfast, were reduced to powder in a paddle mill fitted with a 20 mesh screen and weighed (Fig. 1).
The extraction was performed exhaustively and sequentially with n-hexane, chloroform and methanol. To this end, dry material was extracted for nine days with each solvent, renewing it every three days, using a ratio of 1 kg of powder / 2 l of solvent in the first instance, and 1 kg / 1.5 l in the second and third renewals. The extracts obtained were separated by refrigerated centrifugation, combined according to the solvent, concentrated under reduced pressure on a rotary evaporator, and brought to dryness in a vacuum oven. Finally, they were weighed to calculate the extraction yield (Fig. 2).
This extraction method generated 12 extracts (three per season) named: EUpWHex, EUpWChlorof, EUpWMeOH, EUpSPHex, EUpSPChlorof, EUpSPMeOH, EUpSHex, EUpSChlorof, EUpSMeOH, EUpAHex, EUpAChlorof and EUpAMeOH (W, SP, S, A, corresponding to winter, spring, summer and autumn, respectively). For all of them, the extraction yield was calculated, and extracts were analyzed by i) chemical screening, ii) determination of planar chromatographic profiles oriented to the most important metabolites detected in the screening, and iii) in vitro biological activity assays to determine antioxidant capacity by the DPPH inhibition method and cytotoxicity by the Artemia salina test (Fig. 3).
Chemical screening
The chemical screening was performed by usual qualitative chemical reactions (Uhrich 2016).
Carbohydrates (Molisch reaction): 2 mL of the extract previously brought to dryness and redissolved in ethanol was added with two drops of Molisch's reagent (α-naphthol in ethanol at 10 % w/v). Then, 1 mL of concentrated H2SO4 was added through the walls of the tube. The formation of a purple ring at the interface between the recovered extract and sulfuric acid indicated a positive result.
Flavonoids (Shinoda reaction): 5 mL of the extract previously brought to dryness and redissolved in ethanol was added with zinc granules and 0.2 mL of analytical grade HCl. The development of a pink coloration indicated a positive result.
Lipids: 3 drops of the extract previously brought to dryness and redissolved in ethanol were placed on a filter paper, allowing each to dry before adding the next one; the paper was placed in a jar saturated with iodine vapors. The appearance of brown coloration maintained over time indicated a positive result.
Polyphenols and/or tannins: 0.5 mL of the extract previously brought to dryness and redissolved in ethanol was added with 0.5 mL of a 5 % aqueous solution of K2Cr2O7. The formation of a yellow or orange precipitate indicated a positive result.
Phenolic hydroxyls: 0.5 mL of the extract previously brought to dryness and redissolved in ethanol was added with drops of an aqueous solution of 2 % FeCl3. The appearance of a yellow, grayish-green or blackish-blue coloration, corresponding to the presence of isolated phenolic hydroxyls, or two or three adjacent phenolic hydroxyls, respectively, indicated a positive result.
Quinones (Bornträger reaction): 3 mL of the extract previously brought to dryness and redissolved in the same solvent in which it was extracted was added with 5 mL of the Bornträger’s reagent (5 % NaOH aqueous solution). The appearance of a reddish, orange or yellowish coloration in the alkaline aqueous phase indicated a positive result.
Steroids and triterpenes (Liebermann-Burchard reaction): 2 mL of the extract previously brought to dryness and redissolved in the same solvent in which it was extracted was added with 2 mL of a cooled mixture of acetic anhydride and chloroform (1:1) in an ice bath. Subsequently, drops of concentrated H2SO4, also cooled in an ice bath, were added through the walls of the tube, and the reaction was also made in cold bath. The appearance of a green-to-petroleum blue coloration if the sample contained steroids or the appearance of an orange or yellow-orange to brown coloration if the sample contained triterpenes, indicated a positive result.
Cardenolides (Kedde reaction): 3 drops of the extract previously brought to dryness and redissolved in ethanol were placed on a filter paper, allowing each to dry before adding the next one. Then 0.1 mL of Kedde’s reagent I (2 % 3,5-dinitrobenzoic acid in methanol) and 0.1 mL of Kedde’s reagent II (5.7 % aqueous KOH solution) were added. The appearance of pink coloration indicated a positive result
Alkaloids and related substances (Dragendorff reaction): 0.2 mL of the extract previously brought to dryness was redissolved in 2 mL of 1 % HCl. Then 2 drops of Dragendorff´s reagent was added. The appearance of an orange precipitate indicated a positive result.
Leukoanthocyanins (Rosenheim reaction): 2 mL of the extract previously brought to dryness and redissolved in 2 mL of an aqueous solution of 1 % HCl was added with 1 mL of concentrated HCl, mixed and placed on a water bath at 100 °C for 10 min. After the mixture cooled, it was partitioned with the same volume of amylic alcohol. The appearance of red color into the amylic alcohol phase indicated a positive result.
Direct reactions to the dry and powdered algae
Proteins and amino groups (Ninhydrin reaction): 1 g of the dried and powdered algae was heated with 100 mL of distilled water, in a water bath at 100 °C, for 30 min, and then filtered while still hot and concentrated to 5 mL. Three superimposed drops from this aqueous extract were placed on a filter paper, allowing each to dry before adding the next; then, three drops of 2 % ninhydrin solution in ethanol were added. The paper was heated in an oven at 110-120 °C for 5 min. The appearance of a purplish color indicated a positive result; the intensity was compared with the color produced by the positive control (amino acid) and by the negative control, corresponding to the reagent (Escobar Daza 2019).
Saponins (afrogenic power): 0.25 g of the dried algae for each season reduced to powder was heated with 4 mL of distilled water, in a water bath at 100 °C, for 30 min, and then filtered while still hot. After that, 1 mL of this aqueous hot extract was taken, placed in a hemolysis tube, covered and vigorously shaken for 15 sec. The foam column was measured at 0, 5 and 15 min. The result was considered positive when the foam height persisted for 15 min with a minimum height of 0.5 cm (Escobar Daza 2019).
Cyanoglycosides (Guignard reaction): 0.25 g of the dried algae reduced to powder was placed in a test tube and a few drops of chloroform were added. A plug was placed in the mouth of the tube, and a strip of dry filter paper previously soaked in a sodium picrate solution (prepared by mixing 10 mL of a 10 % aqueous NaOH solution with 0.5 g of picric acid and dried under light protection) was placed with the caution that the paper should not be in contact with the tube walls or the sample. The tube was then placed in an oven at 35 °C. The result is considered positive if there is a change in the color of the paper, from yellow to red. Before reporting a negative result, the tube must be allowed to remain in the oven for at least 24 hours (Becerra 2016).
Anthraquinones (Bornträger reaction modified): 0.4 g of the dried algae reduced to powder was placed in a test tube, added with 5 mL of an aqueous solution of 2 % NaOH, and then heated in a water bath at 100 °C, for 20 min, stirring with a rod. Once cold, the mixture was vacuum filtered and the supernatant was divided into three portions (Escobar Daza 2019), which were used as follows:
1. The first portion was placed in a test tube, acidified with 1 mL of a concentrated HCl and heated in a water bath at 100 °C, for 30 min; then it was partitioned with benzene. The organic phase was transferred to another tube and added with 0.25 mL of a 5 % NaOH aqueous solution. The appearance of a reddish coloration in the aqueous phase, which indicates the presence of anthraquinones released after the acid hydrolysis, was considered a positive result.
2. The second portion was placed in another test tube and added with drops of fuming nitric acid and then drops of ammonia, allowing them to mix gradually. The appearance of a reddish or purplish coloration, which indicates the presence of anthraquinones generated by oxidation from reduced total or partial forms, was considered a positive result.
3. The third portion was placed in another test tube, acidified with 1 mL of a concentrated HCl, added with 1 mL of a 1 % FeCl3 aqueous solution, and heated in a water bath at 100 °C for 30 min; then, it was partitioned with benzene. The organic phase was transferred to another tube and added with 0.25 mL of a 5 % NaOH aqueous solution. The appearance of a reddish coloration in the aqueous phase, which indicates the presence of anthraquinones released after oxidative degradation or oxidative hydrolysis treatment from dimers or C-glycosides, respectively, was considered a positive result.
Determination of planar chromatographic profiles
Chromatographic system for flavonoids (Wagner and Bladt 1996): Thin layer chromatography (TLC) plates containing Silicagel 60 F254 as stationary phase were used; the mobile phase was ethyl acetate-formic acid-glacial acetic acid-water (100:11:11:26). The extracts redissolved in the same extraction solvent were applied in 2.5 mm bands. Digoxin solutions (obtained from commercial tablets in a proportion of 5 mg of digoxin in 2 mL of methanol, in which the tablets are brought to powder in a mortar, and then added with methanol at 60 °C and filtered) was used as reference extract, and cholesterol (1 mg.mL-1 in chloroform) was used as standard. The plates were revealed in visible light, UV light (at 254 and 365 nm) and with NP-PEG (Natural Products – Polyethylene glycol reagent). The Rf were calculated and the results were compared with the literature.
Chromatographic system for steroids (Wagner and Bladt 1996): TLC plates containing Silicagel 60 F254 as stationary phase were used; the mobile phase was ethyl acetate-methanol-water (81:11:8). The extracts redissolved in the same extraction solvent were applied in 2.5 mm bands. Digoxin solutions (obtained from commercial tablets, in a proportion of 5 mg of digoxin in 2 mL of methanol, in which the tablets are brought to powder in a mortar, and then added with methanol at 60 °C and filtered) was used as reference extract, and cholesterol (1 mg.mL-1 in chloroform) was used as standards. The plates were revealed in visible light, UV light (at 254 and 365 nm), and with 20 % SbCl3 in chloroform and subsequent observation at 365 nm. The Rf were calculated and the results were compared with the literature.
Chromatographic system for pigments (Harborne 1984): Chromatography was carried out for the samples corresponding to the winter season (EUpWHex, EUpWChlorof, EUpWMeOH), redissolved in the same extraction solvent and applied in 2.5 mm bands. TLC plates containing Silicagel 60 F254 as stationary phase were used; the mobile phase used was dichloromethane-ethyl acetate (4:1). Reference extracts of carrot and chard were carried out as well. For this, 4 g of fresh tissue was grounded and extracted with 2 mL of acetone, with agitation for 20 minutes, at room temperature and sheltered from the light. After, was centrifuged at 4000 rpm for 15 minutes, using the supernatant at the TLC, under the same procedure than samples. The plates were revealed in visible light and UV light (at 254 and 365 nm). The Rf were calculated and the results were compared with the literature.
In vitro biological activity studies
Antioxidant capacity: The antioxidant capacity of the extracts was evaluated by the method of inhibition of DPPH (2,2-diphenyl-1-picrylhydracil radical), using absolute ethanol as solvent and butylated hydroxytoluene (BHT) as positive control, as described by Koleva et al. 2002. The DPPH solution was prepared at the moment to be used, with a concentration of 30 mM in absolute ethanol, and conserved in a flask covered with aluminum foil in a dark place at 4 ºC until the following measurement. For the preparation of the reaction blank, 1.5 mL of absolute ethanol and 1 mL of DPPH solution were added into a tube, whereas for the samples and positive control, similar proportions were used, being extract mass-DPPH (5.5:1) and BHT-DPPH (0.5:1). The tubes were incubated for 15 minutes in a dark place at 30 ºC, and absorbance was measured at 517 nm in a spectrophotometer. The measurements were made in triplicate and the antioxidant activity of the samples tested was expressed as percentage of DPPH inhibition, according to the following formula (Abs: absorbance).
Quantification of antioxidant capacity: This was realized by the determination of SC50 of each sample (SC50 is defined as the concentration sufficient to obtain 50 % of a maximum scavenging capacity), used the method described by Choi et al. 2002. To this end, solutions of 10, 50, 100, 250 and 500 µg.mL-1 of the samples to be tested were made, and 1.5 mL of each solution was mixed with 1 mL of DPPH solution (30 mM), and after 30 minutes at room temperature, absorbance was measured at 518 nm in a spectrophotometer. The measurements were made in triplicate, and the SC50 of each sample was determined by a graphic of percentage of inhibition against concentration.
Cytotoxicity: This was done realized by the Artemia salina test (or Brine Shrimp Test, BST). This test was carried out by the method described by McLaughlin et al. 1993. To this end, eggs of Artemia salina were incubated in artificial sea water (made by dissolving 38 g of commercial marine salt in 1 liter of tap water), in the presence of artificial light, at 27 °C for 48 h. With the samples to be tested, solutions of 10 mg.mL-1 were made, and after this, aliquots were taken, in such a way that after being dried and dissolved in 5 mL of artificial sea water, dilutions of 10, 100, 250, 500 and 1000 µg.mL-1 were obtained. Each tube was added with 10 nauplii and incubated for 24 h at 27 ºC in the presence of artificial light. A negative control was prepared in the same way, but using only artificial sea water. In all the cases, the procedure was made in triplicate. After the 24 h of incubation, the tubes were examined, and the number of dead organisms in each tube (individuals without movement) was determined. The 50 lethal dose (LD50) and the 95 % confidence intervals were calculated by the statistical method of Finney 1978.