Exploring The Potential Of Icelandic Seaweeds Extracts Produced By Aqueous Pulsed Electric Fields-Assisted Extraction For Cosmetic Applications Part 2

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2.4. Antioxidant Capacities of Icelandic Seaweeds Extracts

A.esculenta had the strongest DPPH scavenging activity among the crude extracts of the three algae species (p<0.05), with scavenging effects higher than 90%(Table 3). Compared with the different standard solutions, A.esculenta showed comparable scavenging activity as 100ug/mL of ascorbic acid (87.9%), gallic acid (91.0%), and α-tocopherol (87.9%). Our results were in agreement with recent studies [50], which also reported a positive antioxidant activity of A.esculenta extracts. Surprisingly, no significant differences in antioxidant activity were observed between the different extraction methods tested (p>0.05). It was expected that PEF extracts would show better antioxidant values than the extracts produced with the hot traditional extraction since other studies have shown that green techniques(such as microwave-assisted extraction or enzymatic extraction) could effectively avoid the decomposition of bioactive compounds, exhibiting higher antioxidant activities [59,60].

The ability of seaweed extracts to reduce ferric (Fe8#) to ferrous (Fe2+)ion and the ability to scavenge the radical ABTS was also studied, by the FRAP and ABTS method, respectively. FRAP results showed similar trends to DPPH, showing A. esculenta had the strongest ability to reduce ferric (Fe3+) to ferrous (Fe2+) ions among the crude extracts of the three algae species (p<0.05). However, a different behavior was found for the ABTS. All seaweed extracts showed a similar ability to scavenge the radical ABTS (p>0.05), indicating that these species probably contain some efficient compounds which are responsible for their scavenging activity.

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In general, brown algae are known to present higher antioxidant potential in comparison to red and green families [61]. Our results also showed that aqueous extracts from A. esculenta exhibited effective antioxidant activities with regard to the scavenging of free radicals and reducing power, suggesting that A. esculenta could potentially be a resource for natural antioxidants. The high antioxidant activity observed for A.esculenta extracts could be linked to the high content of phenolic compounds determined in the brown algae extracts. In many studies, the antioxidant activity of algae extracts has been ascribed to the phenolic compounds, showing positive correlations between phenolic content and scavenging capacity mostly with DPPH[62,63]. Similar correlation results were found in the current study for A.esculenta extracts (see a better discussion in Section 2.6. Correlations between chemical compounds and bioactive properties).

2.5.Enzumatic Inhibitory Activities of Icelandic Seaweed Extracts

Icelandic seaweed extracts exhibited positive inhibitory effects towards all enzymes tested(Table 4), opening new avenues for the exploitation of natural enzymatic inhibitors from algae resources. To the best of our knowledge, this is the first time that enzymatic inhibitory activities of Icelandic seaweed extracts produced by PEF have been tested.

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2.5.1. Collagenase Inhibition Activity

A.esculenta extracts showed positive collagenase inhibition ranging from 68 to 91%, while P. palmaria and U. Lactuca extracts exhibited insignificant inhibition activities against collagenase (Table 4).A.esculenta hot water extract exhibited 71.1% collagenase inhibition activity, which was higher than epigallocatechin-3-gallate(EGCG)standard solution(63.2%)and comparable with positive standard providing by the commercial enzymatic kit (74.9%). An important finding was that the A.esculenta extracts produced by the PEF showed a collagenase inhibition of 91%, exhibiting even higher activity than the inhibitor provided by the commercial kit. It should be highlighted that this activity was only observed in the water extracts produced by PEF and not by the combination of PEF+HW. This behavior can be explained by the possibility that the hot water process could have a negative effect on the compounds responsible for inhibiting collagenase activity. However, additional studies are needed to explain these results due to the complexity of crude algal extracts. The aforementioned research group is currently working on the identification of the inhibition molecules in A.esculenta extracts to better understand these positive effects produced by the PEF.

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Results regarding the inhibition of collagenase by A.esculenta extracts are in accordance with previous data, in which A.esculenta is being used in commercial extracts due to its antiaging effect. The degradation of collagen occurs with aging due to collagenase activity, resulting in wrinkles on the skin. The inhibition of collagenase by naturally occurring compounds is an interesting opportunity for anti-aging products. For instance, SEPPIC, a supplier of ingredients for the cosmetics industry, is offering a lipophilic extract of A.esculenta(Kalpariane AD) [64].

2.5.2. Elastase Inhibition Activity

Only the crude extracts of A.esculenta inhibited elastase, exhibiting activities higher than 70% of inhibition (Table 4). However, the anti-elastase activities of A.esculenta extracts did not statistically differ among extraction methods (p>0.05). Compared with quercetin solutions, a well-known elastase inhibitor that showed 100% inhibition at 1 mM and 58.7%at 0.5 mM, the performance of extracts from A. esculenta was high.

Elastase is a proteinase enzyme that can reduce elastin by breaking specific peptide bonds. Consequently, the inhibition of elastase activity in the dermis layer can be used to maintain skin elasticity [65]. Many plant extracts have been identified as elastase inhibitors [17]; however, few investigations have been carried out on the elastase inhibition from algae resources. According to literature data, polyphenols extracted from plants are known to be strong elastase and hyaluronidase inhibitors [66]. A recent study reported that the phlorotannins, the type of tannin in brown algae, extracts of sea kelp Eisenia bicycles, and brown alga Ecklonia cava, benefit the skin by reducing the elastase activity significantly [67]. The A.esculenta extracts produced in this study showed the highest TPC and TFCvalues in comparison to the other species studied (Table 4), so this could be the reason why the aqueous extracts from P. palmaria and U. Lactuca did not show anti-elastase activities. To confirm this hypothesis, a Pearson correlation analysis was conducted, suggesting that the anti-enzymatic activities positively correlate with the content of phenolic substances (see a further discussion in Section 2.6.Correlations between chemical compounds and bioactive properties).

2.5.3. Tyrosinase Inhibition Activity

A.esculenta extracts showed positive tyrosinase inhibition higher than 90% for all the extraction methods used, while P.palmaria and U. Lactuca extracts did not exhibit tyrosinase inhibitory effects (Table 4). However, the anti-tyrosinase activities of A.esculenta extracts did not differ (p<0.05) with extraction methods. Comparing the effect of A.esculenta extracts with the quercetin solutions tested, the crude extracts of the brown algae showed better inhibitory activities than these solutions(88 and75% for the 0.5 and 1 mM quercetin solutions, respectively). Based on the literature,anti-tyrosinase activities of plants, bacteria, and fungi have been reported by several researchers I68I. However, though different studies suggest that bioactive compounds derived from marine algae have a good potential to be utilized as skin whitening agents [13], this is still an unexplored domain and only a few studies have been carried out. Most of the studies performed in this area have been focused on brown algae, agreeing with the results of the present study in which A.esculenta extracts exhibited the best anti-tyrosinase activities. For instance, phloroglucinol derivatives and phlorotannins, common secondary metabolites found in brown algae, have shown inhibitory activity against tyrosinase due to their ability to chelate copper [69]. In a recent study, the extract of the brown algae Lessonia trabeculate produced by microwave-assisted extraction inhibited a tyrosinase activity of 33.73%[60].In another study, the extract of the brown algae Turbinaria conoides showed activity as an antioxidant and tyrosinase inhibitor, however, in this case, ethanol was used as solvent [70]. A significant correlation between the inhibitory potency of polyphenols extracted from plants on mushroom tyrosinase has been reported in previous studies [68]. Likewise, the results of this study suggest that the inhibitory activity towards tyrosinase was positively correlated with flavonoid and phenolic content (see Section 2.6. Correlations between chemical compounds and bioactive properties).

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Tyrosinase plays an important role in the biosynthesis of melanin pigment in the skin. Melanin is responsible for the protection against harmful ultraviolet irradiation, which can cause several pathological conditions [71]. In addition, it can create aesthetic problems when melanin is accumulated as hyperpigmented spots [72]. Thus, incorporating tyrosinase inhibitors in cosmetic products can be attractive due to whitening and or lightening effects.

2.5.4. Hyaluronidase Inhibition Activity

All the seaweed extracts exhibited significantly high anti-hyaluronidase activity (Table 4), showing comparable results to the tannic acid solutions (a well-known inhibitor of hyaluronidase). Specifically, A.esculenta extracts showed 100% of inhibition for all the methods tested. Moreover, U. Lactuca extracts exhibited activities higher than 90% of inhibition, where the inhibition of the extracts produced by PEF(96.8%o) and the combination of PEF+ HW (97.3%) was higher than the inhibition produced by the traditional hot water method 93.4%)(p<0.05). All P.palmaria extracts exhibited similar activities(p<0.05), the inhibition of the extracts produced by PEF was (91.9 %)and the combination of PEF+HW (89.5%) and the traditional hot water method (91.8%).

Other authors also described the anti-hyaluronidase activity of different seaweed extracts, especially extracts rich in phlorotannins from brown algae [73,74]. However, to the best of our knowledge, this is the first time that hyaluronidase inhibitory activities of P.palmata and U. Lactuca extracts produced by PEF have been reported.

Hyaluronic acid is a major component of the dermis, where it is involved in tissue repair, it breaks down with aging, causing wrinkles and loss of skin firmness. In this sense, hyaluronidase inhibitors increase the hyaluronic acid level of the dermal extracellular matrix for the improvement of the appearance of aging facial skin [13]. cistanch Therefore, the results of this study might open new avenues for the exploitation of natural hyaluronidase inhibitors from algae resources with potential use in cosmetic products.

In summary, the data gathered allowed us to conclude that A.esculenta extracts exhibited overall better inhibitory activities than P.palmaria and U.lactuca towards the enzymes tested. Thus, being the most promising seaweed specie with excellent anti-enzymatic activities and therefore it was selected for further studies in our laboratory. Although crude extracts from A. esculenta appear to be good candidates for in vitro experiments, further studies need to be carried out to elucidate the identity of the metabolites responsible for these biological effects.

2.6.Correlations between Chemical Compounds and Bioactive Properties

The results from principal component analysis (PCA), showed that the main separation of the groups was defined by PC1 and PC2, which accounted for71.9% of and 14.5% of the variance in the data, respectively (Figure 2). The A.esculenta extracts were characterized by higher contents of flavonoids and phenolic compounds, inhibitory effects on enzymes (collagenase, tyrosinase, and elastase), and DPPH and FRAP values, than the other species, P. palmata, and U.lactuca. On the other hand, A.esculenta had lower carbohydrate content, especially compared to P. palmitate (which was located at the opposite side of the PC1). The variation in data along the PC2 was mainly related to ABTS and hyaluronidase inhibition. As indicated by the location on the plot, P. palmitate had a stronger correlation to ABTS whereas U.lactuca was more related to hyaluronidase inhibition effects, in comparison to these two species.

A high and significant positive correlation between TPC, TFC, DPPH, FRAP, and inhibitory effects on collagenase, elastase, and tyrosinase was demonstrated by Pearson correlation analysis (Table 5).

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This was in an agreement with previous studies, reporting that phenolic compounds (including flavonoids) are the main contributors to the antioxidant activity of various seaweeds [75-77]. The high antioxidant activity of extracts from brown macroalgae has been related to a specific group of polyphenols, phlorotannins, and their unique molecular structure. Phlorotannis from brown algae is reported to have up to eight interconnected phenol rings that act as electron traps [78,79]. It was expected that ABTs would correlate with TPC and other antioxidant parameters. Possible reasons might be that the methods are based on different reaction conditions and that reactivity differs both with regard to time and the range of components. For example, the ABTSreagent reacts with a broader range of antioxidants than the DPPH radical [80]. On the other hand, one of the limitations mentioned for ABTS is a long reaction and the general reaction time may not allow reaching an endpoint.

The results indicate that there is a high positive correlation between TPC and TFC to the inhibitory activity of collagenase, elastase, and tyrosinase (0.93-0.99), whereas the relationship to inhibition of hyaluronidase was not as strong (r=0.42 and 0.54, respectively). This indicates that other components may have contributed to the inhibitory effect of the extracts. Other studies have reported that polysaccharides have hyaluronidase-inhibitory activity, for instance, alginic acid in brown algae [81,82]. Further studies on the chemical composition of the macroalgae species for the effects of isolated compounds on the enzyme are needed to evaluate the contribution of each chemical component as in this study the focus was on crude extracts. The findings were in harmony with previous studies, stating that the chemical composition and levels of bioactivity of the extracts vary significantly between the three lineages (red, green, and brown algae) and between different species belonging to the same phylum and are influenced by age and tissue type. Furthermore, the composition and characteristics depend on many environmental factors affecting the distribution and growth of macroalgae. For example, light (UV-radiation), temperature, nutrient availability, exposure to air, water motion, wave exposure, and salinity. The temperature has been described as the factor having the strongest effects on pigment formation and nutrient concentration, salinity, and UV radiation as the factors influencing the concentration of TPC [83].

The distribution of different macroalgae species varies with water depth. Positions higher up the shore in the intertidal or littoral zone are more stressful as the species growing there, must withstand multiple changes in abiotic factors due to tidal changes. For instance, the drying effect of air, high solar irradiances (at low tide), changes in salinity and temperature, and, under conditions of low air temperatures, including freezing. Below the low water mark, increasing depth results in a very rapid decrease in light intensity and less exposure to irradiance.

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Algae growing in the tidal range have lower sensitivity to UV Radiation and recover more rapidly from solar stress. Whereas algae growing in the sublittoral zone are more sensitive to UV radiation and have lower recovery from solar stress [84]. At the same time, the water column provides protection. In the present study, the exposure to sunlight was presumably stronger for P. palmitate, compared to the other species. Other studies have shown that the formation of MAAs is directly related to sunlight [85], protecting the organisms against UV-A and UV-B radiation. Moreover, it was shown that the specific amount of MAAs decreased with increasing collecting depth. Kelps such as A.esculenta, are known to grow in the upper sublittoral zone but also extend into the lowest intertidal just above the low water mark. Meaning the water column provided stronger protection than for P. palmitate. In addition, the morphological characteristics are different, the blades of A.esculenta are thicker compared to the other two species. UI. lactuca, growing mainly in the intertidal and sublittoral is able to photosynthesize and grow under very low irradiances. Exposure to UVB light has been stated to accelerate the recovery of photosynthetic parameters of U.lactuca from the negative effects of UVA light. It is smaller, simpler in structure, and shorter-lived (3 months) than both A.esculenta (5-7 years) and P. palmata which has a new growth every year.

In summary, the assumptions can be drawn that the main differences in the properties of the extracts are the variation in life span, morphological characteristics, and growth conditions of the algae species.

3. Materials and Methods

3.1. Materials

Icelandic seaweeds U.lactuca(green algae), A.esculenta (brown algae), and P. palmitate (red algae)were provided by Icelandic Blue mussels and Seaweed, which harvested sea-weeds in Breidafjordur (West-Iceland).After harvesting the seaweeds were dried(to approximately 90% dry material), milled, and delivered vacuum packed. Samples were kept in a dry and dark place at room temperature until used.

Tyrosinase from mushroom, L-3,4-dihydroxyphenylalanine (L-DOPA), elastase from porcine pancreas, ascorbic acid, N-Succinyl-Ala-Ala-Ala-p-nitroanilide(AAPVN), hyaluronidase from bovine testes, quercetin, a-tocopherol, tannic acid,2,2-diphenyl-1-picrylhydrazyl(DPPH),2.4,6-Tripyridyl-s-Triazine(TPTZ), Trolox, Folin-Ciocalteu reagent, gallic acid, and a collagenase activity colorimetric assay kit (MAK293) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Hyaluronic acid sodium salt was purchased from MakingCosmetics (Redmond, WA, USA). All other chemicals and reagents used were analytical grade and obtained from VWR International, LLC. Deionized water (Elix Essential, Merck, Darmstadt, Germany) was used for the extraction and preparation of water-based solutions.

3.2.Experimental Design

A factorial design was used for evaluating the effects of Icelandic seaweed species (U. Lactuca, A. esculenta, P. palmitate) and extraction treatment (hot water extraction (HW,95 °C), PEF-assisted extraction (PEF) and the combination of both techniques (PEF + HW), on extract composition and bioactivity (Table 6). The extraction was carried out in triplicate for each group and every extract replicate was analyzed in triplicate.

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3.3. The Extraction of Bioactives from the Icelandic Seaweeds

The exploitation of macroalgal biomass at different levels has motivated scientists to explore more eco-friendly, efficient, and cost-effective extraction techniques, based on green extraction approaches. In this work, PEF-assisted extraction was evaluated as a novel and green method to produce functional extracts, while traditional hot water extraction was used for comparison. Moreover, the effect of the combination of both techniques, PEF treatment of macroalgae followed by the traditional hot water extraction, on the bioactive recovery was studied. Due to the expected electroporation produced in the cell membranes after the physical treatment, the following extraction with hot water could further facilitate the release of the intracellular material [86], increasing the extraction field. A time after treatment is needed for the materials to diffuse out of the cells [87,88], and in this experiment, the suspensions waited overnight until the separation of the liquid (extract)from the pulp.

Regarding extraction medium, distilled water was used to produce the seaweed extracts to overcome limitations concerning the use of toxics and organics solvents. Water proved to be a good solvent for the extraction of several bioactive compounds from seaweeds [46,89-91], and is environmentally friendly. In addition, water is commonly used for PEF-assisted extraction as it is a good conductor for electricity.

3.3.1. Extraction Procedures

For every replicate in each group, seaweeds(15 g) were soaked overnight at room temperature(22°C) in deionized water(300 mL). Then, the suspension was treated with PEF (PEF), heated (HW), or both PEF-treated and heated (PEF+HW). The suspensions were kept overnight in a refrigerator followed by filtration with a coarse (20 um) filter paper. Then the filtrates (extracts) were stored at 4°C until their analyses.

The pulsed electric field-assisted extraction was carried out by using a pulse generator built in-house. It had a F.u.G.HCK-200-2000 capacitor(Fu.G.Elektronik GmbH, Rosenheim, Germany) and spark gap (18.5 kV OG75, Perkin-Elmer Optoelectronics, GMBH, Wiese-baden, Germany). The PEF equipment generated exponential decay pulses with a width of 0.96 us and an amplitude of 18 kV. A plexiglass treatment chamber with the dimensions (L× H× W) 20×8×2.5 cm, with the shortest distance being between the plate electrodes were used to treat the suspensions with an 8 kV/cm electric field at 1.2 Hz for 10 min. The HW extracts were prepared by heating the suspension in a beaker in a thermostatic water bath and kept at 95°Cfor 45 min. For the combined pulsed electric field and heating treatment, the suspensions were PEF treated and then placed in a beaker, heated in a water bath, and kept at 95 °C for 45 min.

3.3.2. Conductivity, pH, and Temperature Measurements

The electrical conductivity and pH of seaweeds suspensions were measured after soaking and after the extraction treatments, at room temperature, using a pH meter (Orion StarTM A215 pH/Conductivity Benchtop Meter, Thermo Scientific, Waltham, MA, USA)equipped with a conductivity sensor and pH/ARC triode combination electrode. Furthermore, temperature changes due to treatments were recorded.

3.4. Spectral Profiles of the Seaweed Extracts

The UV-VIS absorption spectra of the different seaweed extracts were measured in the range of 200 to 450 nm using a double beam Thermo Scientific Evolution 350 UV-Vis Spectrophotometer(Thermo Fisher Scientific, Waltham, MA, USA) with 1 cm quartz cuvettes. Three scans were performed for each seaweed extract.

3.5. Determination of Total Polyphenolic Content

The total phenolic content (TPC) in seaweed extracts was determined by using the Folin-Ciocalteu reagent following a slightly modified method described by Zhang [92] using a Multiskan Sky Microplate Spectrophotometer(Thermo Fisher Scientific, Waltham, MA USA）. A volume of 20 μL of seaweed extract or serial standard solution was mixed with 100 μL of Folin-Ciocalteu reagent （10% in distilled water）. After 5 min， 80 μL of 7.5%(/w)sodium carbonate solution was added. The reaction mixture was incubated at room temperature and darkness for 30 min. Absorbance was measured at the wavelength of 760 nm. Distilled water was used as blank. A standard curve of gallic acid was used to determine the total phenolic content and expressed as μg of gallic acid equivalents（GAE）per gram of dry material （μg GAE/g do）.

3.6. Determination of Total Flavonoid Content

The total flavonoid content (TFC) in seaweed extracts was determined by the method described by Kamtekar 【93】 and adapted to 96-well microplates. Briefly， a volume of 25 μL of seaweed extract or serial standard solution was mixed with 100 μL of sodium nitrite （0.375%w/o）. After5 min，25 μLof aluminum chloride （3%w/o）was added to the mixture and incubated for 6 min at room temperature. Then， 100 μL of sodium hydroxide （2%w/ø）was added to the mixture and mixed. Immediately， absorbance was measured at the wavelength of 510 nm. Distilled water and ethanol were used as blanks. A standard curve of quercetin(dissolved in ethanol) was used to determine the total phenolic content and expressed as μg of quercetin equivalents（QE） per gram of dry material （μg QE/g do）. 3.7.Determination of Carbohydrate Content

The free sugar content was measured according to the method described by [94], with slight modifications. A 50 μL of phenol solution （4%） and 250 μL of sulfuric acid （96%）were added to 100 μL of sample or standard solution. After 10 min of incubation at room temperature, the absorbance of the mixture was read at 490 nm. A standard curve of glucose was used to determine the total carbohydrate content and expressed as mg of glucose equivalents (GluE) per gram of dry material (mg GluE/g do).

3.8. Antioxidant Properties of Seaweeds Extracts

3.8.1.2,2 Diphenyl-1-picrylhydrazyl (DPPH) Free Radical Scavenging Assay

The antioxidant activity (DPPH) of seaweed extracts was determined following the previously described methodology 【94】 with some modifications. Briefly，200 μL of 10.825 × 10-5 M DPPH solution was added to 100 uL of sample (1:1 in methanol) in a 96-well plate. The same volume of DPPH was mixed with 50μL standard +50μL methanol. Then the samples and standard were incubated in a dark place at room temperature for 30 min. The absorbance was measured at the wavelength of 517 nm. Distilled water was used as a blank. The ability to scavenge the DPPH radical was calculated using the following equation: where control is the absorbance of the control (DPH solution without sample), the A sample is the absorbance of the test sample (DPPH solution plus test sample), and the A sample blank is the absorbance of the sample only (sample without DPPH solution) and Amethanol blank is the absorbance of methanol only. Commercial antioxidants (ascorbic acid, gallic acid, and α-tocopherol) were used as positive controls.

3.8.2.Ferric Ion Reducing Antioxidant Power (FRAP) Assay

FRAP activity was measured according to the method of Benzie and Strain [95]. Briefly, acetate buffer (300mM,pH3.6),2.4,6-tripyridyl-s-triazine(TPTZ)10mM in 40mMHCl, and FeCl; 6H-O (20 mM) were mixed in the ratio of 10:1 to obtain the working FRAP reagent. The reaction mixture was incubated at 37℃for10min. A50 μL sample from every extract was mixed with 150 μL of working FRAP solution for 8 min at room temperature. The absorbance of the colored product, Ferrous-TPTZ was measured at the wavelength of 593 nm. FRAP values of seaweed extracts were expressed as uM of Trolox equivalents (TE)per gram of dry material.

3.8.3.2,2 Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid)(ABTS) Assay

The analysis was performed using the ABTS decolorization protocol [76] with some modifications. An ABTS radical cation(ABTS+) was produced by reacting ABTS(66mg)with 10 mL of potassium persulphate solution (2.45 mM). The mixture was left in the dark at room temperature for 12-16 h before use. The ABTS+ solution was diluted with water to an absorbance of 0.700 at 734 nm. The reaction mixture(200 ul) was transferred to a microplate，50 μL of the sample was added and then 150 μL of the reagent solution. The plate was shaken for 10 s at medium speed, and the absorbance was measured at734 nm after 5 min of incubation at room temperature. A standard curve was prepared by plotting the inhibition of A734nm of Trolox standards as a function of their concentrations. The Trolox equivalent antioxidant capacity (TEAC) value of the samples was calculated using the equation obtained from the linear regression of the standard curve substituted A734nm values for each sample:

3.9. Anti-Enzymatic Activities of Seaweeds Extracts

3.9.1. Collagenase Inhibition Assay

A collagenase activity colorimetric assay kit (MAK293), purchased from Sigma-Aldrich, was used to determine the collagenase inhibition of seaweed extracts. The kit measured collagenase activity using a synthetic peptide (FALGPA)that mimics collagen's structure. The procedure was performed according to the kit instructions.

3.9.2. Elastase Inhibition Assay

The elastase inhibition of seaweed extracts was investigated in TRIS buffer solution with the modified method as described earlier 【96】. Briefly，100 μL of 0.1 M TRISbuffer solution （pH8.0）， 25 μL of elastase（1 U/mL in TRIS buffer）and 25μL sample extracts were mixed and incubated for 15 min at 30 C before adding the substrate to begin the reaction. After incubation time， 50μL of 2 mM AAAPVN solution was added. Then， the absorbance at 420 nm was monitored for 20 min using a microplate reader under a constant temperature of 30 C. Finally, elastase inhibition was calculated in percentage using the equation: where Abs control is the absorbance of the assay using the buffer instead of inhibitor(sample)and Absmpleis the absorbance of the sample extracts. Quercetin was used as a positive control. Tris buffer was used as blank.

3.9.3. Tyrosinase Inhibition Assay

Tyrosinase inhibitory assay was performed according to the method previously described by 【66】using L-DOPA as substrate. A 20 μL of the sample， 10 μL of mushroom tyrosinase solution （50 U/mL in phosphate buffer）, and 80 μL of phosphate buffer （pH=6.8）were mixed in a microplate and pre-incubated at 37Cfor 5 min. Then, 90 uL of L-DOPA (2 mg/mL)was added. The formation of dopachrome was immediately monitored for 20 min at 475 nm in a microplate reader under a constant temperature of 37°C. The percent inhibition of tyrosinase enzyme was calculated using the equation: where Abs control is the absorbance of the assay using the buffer instead of inhibitor(sample)and A sample is the absorbance of the sample extracts. Quercetin was used as a positive control. Phosphate buffer was used as blank.

3.9.4. Hyaluronidase Inhibition Assay

Hyaluronidase inhibitory activity was measured as previously described by [66] with few modifications. A volume of 100 μlof type-1-Sbovine testes hyaluronidase（2100 U/mL）dissolved in 0.1 M acetate buffer （pH 3.5）was mixed with 100 μL of extract and incubated at 37°C for 20 min. A volume of 200 μL of 6mM of calcium chloride was added to the reaction mixture, and then the mixture was incubated at 37 ℃C for 20 min. This Ca2+activated hyaluronidase was treated with 250 uL of sodium hyaluronate(1.2 mg/mL)dissolved in 0.1 M acetate buffer (pH 3.5) and then incubated in a water bath at 37°C for 40 min. A 50 μL of 0.9 M sodium hydroxide and 100 μL of 0.2 M sodium borate were added to the reaction mixture and then incubated in a boiling water bath for 5 min. After cooling to room temperature, 250 uL of p-dimethylaminobenzaldehyde (DAMB) solution was added to the reaction mixture. The DAMB solution was prepared by dissolving 0.25 g of DAMB in 21.88 mL of 100% acetic acid and 3.12 ml of 10N hydrochloric acid. The control group was treated with 100 μL of 5% of water instead of extract. The absorbance was measured at the wavelength of 585 nm after 45 min. The percentage enzyme inhibition was calculated using the following equation: where Abs control is the absorbance of the assay using the buffer instead of inhibitor (sample)and Abs sample is the absorbance of the sample extracts. Tannic acid is used as a reference standard.

3.10.Statistical Analysis

The average of the triplicate analysis of every extract was calculated and used to find the mean values and standard deviations for each group (n = 3). General linear models (GLM)for fixed factors were applied to evaluate the main effects and two-way interactions of the experimental factors (species and extraction methods)on measured variables. Furthermore, ANOVA and the Tukey-Kramer test were used to identify significant (p<0.05)differences between the groups. Pearson correlation was used to evaluate linear relationships between the variables. Principal component analysis (PCA) was used to detect structure in the relationship between measured variables and experimental factors. The PCA reduces voluminous data to a small set of linear combinations of related variables(i.e., factors) based on patterns of correlation among the original variables. The resulting linear attribute combinations can be used for profiling specific product characteristics based on the variables studied. All statistical analyses were performed using NCSS 2020 Statistical Software (2020) (NCSS, LLC., Kaysville, UT, USA).

4. Conclusions

The outcomes of this first screening experiment showed the potential of three Icelandic seaweed species by providing effective beneficial effects via several pathways. The green approach developed using aqueous pulsed electric fields exhibited similar results to the traditional hot water extraction, showing several advantages such as its non-thermal nature and shorter extraction time(10 min vs. 45 min). Amongst the three algal species, the brown macroalgae A.esculenta showed the highest content of TPC and TFC also exhibiting the greatest antioxidant capacities Moreover, A.esculenta water extracts exhibited better inhibitory activities than P. palmaria and U. Lactuca towards collagenase, elastase, tyrosinase, and hyaluronidase being the most promising seaweed specie with excellent anti-enzymatic activities for their use in skin whitening, anti-aging and skin health. Interestingly, the A.esculenta extracts produced by the PEF method showed a collagenase inhibition of 91%, higher than the inhibition activity shown by the traditional hot water extraction and even higher than the inhibitor provided by the commercial kit. In conclusion, our preliminary study suggests that Icelandic seaweed-based extracts, especially the extracts from the brown macroalgae A. esculenta, produced by aqueous pulsed electric fields-assisted extraction are potential functional ingredients that could be used as active compounds for cosmetic and cosmeceutical formulations in the near future.

This article is extracted from Mar. Drugs 2021, 19, 662. https://doi.org/10.3390/md19120662 https://www.mdpi.com/journal/marinedrugs