**1. Introduction**

Seaweed extracts have been used in agricultural activities due to their content of macroelements (alginate, agar, carrageenan, etc.), which activate the synthesis of endogenous hormones in plants [1,2] and contribute microelements (N, Ca, Mg, Mn, B, Br, I, Zn, Cu, and Co), amino acids, and vitamins that enrich soil in plant crops [3]. Besides, seaweed extracts contain biochemical compounds such as chlorophylls, carotenoids, and phenolics that confer antioxidant protection [4,5]. The antioxidant properties of algae extracts have been widely evaluated and attributed to sulfated polysaccharides, pigments, and phenolic compounds [4–8], which provide desirable characteristics for their potential use in crops, since, in addition to conferring antioxidant protection, compounds such as polysaccharides have been linked to growth promoting activities [8].

Some environmentally-friendly extraction methods generally include boiling or soaking with distilled water, which have been used as biostimulants for plant growth [9]. Phytohormone-like Plant-Growth Regulators (PGRs) have been identified in algal extracts, such as abscisic acid, auxins, cytokinins, gibberellins, jasmonates, or salicylates, all of which regulate plant cell metabolism and boost production and growth [10–12]. For this reason, marine algae have been used in agriculture as organic fertilizers to achieve sustainable crop production [13,14] and counter the excessive use of fertilizers and synthetic hormones (e.g., 2.4-dichlorophenoxyacetic acid and naphthaleneacetic acid) that may potentially affect both the environment and humans [12,15].

Several authors have reported that algal extracts induce physiological processes in treated plants, such as germination, emergence, root growth, nutrient mobilization, maturation, tolerance to stress, and disease resistance; these responses are similar to those observed in plant crops treated with synthetic hormones [3,10,16–18]. Some seaweed extracts are marketed as liquid biofertilizers or biostimulants [3,16,19], mostly enriched with biomass of *Ascophyllum nodosum*, *Sargassum* spp., and *Macrocystis pyrifera* [3,20,21]. However, the different species of algae show variations regarding PGR biosynthesis [22]; thus, algal extracts exert variable physiological effects on different crops.

In Latin America, algal extracts have recently been used as biostimulants. Some reports demonstrate the benefits of the application of seaweed extracts harvested in coastal areas on various crops. The macroalgae *M*. *pyrifera*, *Gelidium robustum*, *Chondracanthus canaliculatus*, *Sargassum* spp., *Ulva lactuca,* and *Padina gymnospora*, have been used as biostimulants, fertilizers, and root promoters, as well as to stimulate growth and increase antifungal protection in tomato plants (*Solanum lycopersicum*) [20,23]. However, the exploitation of marine algae, mainly those involved in massive arrivals, is still incipient; moreover, it is not well known if improvements in yield and production of crops fertilized with algal extracts are due to the presence of PGRs in algal organic matter and/or if a possible contribution of other metabolites contribute to the biostimulant effects. Therefore, it is necessary to study the chemical and bioactive composition of a new algal extract when it is prepared to consider its potential use as plant grow stimulant.

In addition, PGRs in seaweeds have been insufficiently studied. Therefore, information is needed to support the use of marine sources, such as algae, to achieve sustainable agriculture practices in the future. This will reduce the environmental impact associated with the excessive use of chemical fertilizers, and also the potential risks to consumers resulting from the indiscriminate application of synthetic PGRs, along with the fact that algae provide other bioactive compounds that enhance protection against stress oxidative, improving plant health.

The objective of the present investigation was to characterize the chemical and bioactive composition (antioxidant activity, PGR identification and content) of aqueous extracts of the macroalgae *Ulva lactuca* and *Padina durvillaei*, and evaluate the use of such aqueous extracts as a potential biofertilizer.

#### **2. Material and Methods**

#### *2.1. Seaweed Collection and Reagents*

Specimens of the seaweeds *Padina durvillaei* (Bory Saint-Vicent, 1957) and *Ulva lactuca* (Linnaeus 1753) were collected in Mazatlan Bay, Sinaloa, Mexico (23◦1 2 9.1 ' LN, 106◦25 29.7 LW), in March 2017. Fresh samples were rinsed with distilled water, lyophilized, ground with a commercial grinder, and stored at −20 ◦C until used. All chemicals used in this research were analytical grade and supplied from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA), unless otherwise specified.

#### *2.2. Seaweed Extracts*

Seaweed extracts were obtained using distilled water according to Tierney et al. [24], modified as follows: dried algal material was mixed with water at 21 ◦C (1:10, *w*:*v*) with stirring for 3 h; then, the extract was filtered through a fiber glass filter (1.2 μm pore size) and the algal residue extracted again

(twice). Filtrates were pooled and centrifuged at 12,000× *g* and 4 ◦C for 20 min; then, the supernatant was collected. Finally, the aqueous extract was lyophilized and stored at −20 ◦C until analyzed.

The extraction yield was calculated according to Equation (1):

$$\text{Extraction yield} \left( \% \right) = \left( \text{grams of dry aqueous extract} \text{/grams of dry seawater} \right) \times 100 \tag{1}$$

### *2.3. Chemical Composition*

Carbohydrate content was measured using the phenol–sulfuric acid method [25] using D-glucose as standard. Soluble protein content was determined with Bradford's method using Bovine Serum Albumin (BSA) as standard [26].

Sulfate content was measured with the barium chloride-gelatin assay using potassium sulfate as standard [27]. The uronic acid content was determined with the sulfuric acid-carbazole colorimetric method using D-glucuronic acid as standard [28].

#### 2.3.1. Total Phenolic Content (TPC)

Total soluble phenolic content was determined using the Folin–Ciocalteu method [29]. Dry samples were reconstituted with acetone (1 mg/mL); then, a 100 mL of each sample was mixed with 150 mL of Folin solution (previously diluted 1:1 with deionized water) followed by the addition of 1 mL of 2% sodium carbonate in 0.4% sodium hydroxide. The mixture was incubated in the dark at room temperature for 20 min. The resulting blue complex was read in a spectrophotometer at 750 nm. Phenolic content was expressed as mg of gallic acid equivalent (GAE) per g of sample (dry weight). A gallic acid standard curve was constructed at the concentration range of 0–0.25 mg/mL.

#### 2.3.2. Total Flavonoids Content (TFC)

Total flavonoid content was assessed according to Luximon-Ramma et al. [30]. Samples of solutions (1 mL) were diluted in equal volumes of a 2% aluminum chloride solution (2 g of AlCl3·6H2O in 100 mL of methanol). The mixture was incubated at room temperature for 10 min. Absorbance was read at 367 nm. The results were expressed in mg of quercetin equivalents (QE) per gram of sample (dry weight). A quercetin standard curve was constructed at the concentration range of 0–0.5 mg/mL.
