Storage Effect on Phenols and on the Antioxidant Activity of Extracts from Anemopsis californica and Inhibition of Elastase Enzyme

1División de Desarrollo Biotecnológico, Centro Universitario de la Ciénega (CUCI), Universidad de Guadalajara (UdeG), Avenida Universidad 1115, Colonia Lindavista, 47820 Ocotlán, JAL, Mexico 2Unidad de Tecnologı́a Alimentaria, Centro de Investigación y Asistencia en Tecnologı́a y Diseño del Estado de Jalisco (CIATEJ) AC, Avenida Normalistas 800, Colonia Colinas de la Normal, 44270 Guadalajara, JAL, Mexico 3Departamento de Biotecnologı́a y Ciencias Alimentarias, Instituto Tecnológico de Sonora (ITSON), 5 de Febrero 818 Sur, Colonia Centro, 85000 Ciudad Obregón, SON, Mexico 4Coordinación de Genómica Alimentaria, Universidad de la Ciénega del Estado de Michoacán de Ocampo (UCM), Avenida Universidad 3000, Colonia Lomas de la Universidad, 59103 Sahuayo, MICH, Mexico


Introduction
Phenolics are found in all plants as their secondary metabolites and are synthesized during normal development and in response to conditions of stress.These compounds comprise one of the most widely occurring groups of phytochemicals and have many industrial applications in fields such as medicine and cosmetics and in the food industry.To date, less attention has been paid to the stability of polyphenolic compounds and their degradation under different conditions.However, these aspects can substantially influence their potential applications [1].
Inflammation may be defined as a degenerative process that is sufficiently intense to cause local accumulation of lowmolecular-weight (LMW) catabolic products, which in turn elevates osmotic pressure in tissue that, in turn, attracts extra fluid [5].Elastase plays a critical role in inflammation and is the sole enzyme that is capable of degrading elastin.Elastin is the main component of the elastic fibers of the connective tissue and tendons.However, elastase is capable of hydrolyzing nearly all proteins, including the supporting structural proteins of the connective tissues, increasing inflammation [6,7].Therefore, the enzyme has received great attention, primarily because of its reactivity and nonspecificity.An alternative to inhibit elastase is the use of extracts from food plants, because some of them are a good source of antiinflammatory compounds [6,8,9].
Anemopsis californica, commonly known in Spanish as yerba mansa or hierba mansa, is distributed in the southeastern US and in northern and central Mexico [10,11].The plant has been found in the state of Querétaro (a central state in Mexico) and is employed by local healers to treat a variety of ailments.Fresh-or dry-leaf, rhizome, or root samples are prepared as tinctures, decoctions, and teas for internal use; wilted leaves or root powders are employed externally [12,13].Traditionally, this plant is utilized to prevent and treat venereal diseases [14], rheumatism [15], and cancer [16,17].Furthermore, A. californica possesses important antimicrobial [18], antimutagenic [19], and antioxidant [20] activities.Due to its properties, A. californica has a great potential in addition to other foods or in pharmaceutical and herbal medicine industries.However, to date and to our knowledge, no antioxidant stability and anti-inflammatory evaluations have been conducted.Therefore, in the present work, the amount of total phenols and flavonoids and the antioxidant activity of leaf, stem, and rhizome methanolic extracts from Anemopsis californica under different storage conditions were investigated.The inhibition of elastase enzyme was also evaluated.

Methanolic Extraction.
Leaves, stems, and rhizomes of Anemopsis californica were collected in Juriquilla, Querétaro (Mexico), in March 2013; they were cut, dried, and ground separately.Three g of each sample was mixed with 15 mL of methanol and homogenized in an Ultraturrax (T 25 DS1 digital homogenizer) for 1 min.Afterward, the sample was ultrasonicated (Bransonic, 151-DTH) at 4 ∘ C for 15 min.The supernatant was collected after centrifugation at 4,000 rpm for 15 min at 4 ∘ C. The extraction procedure was repeated for a second time.The combined supernatants were evaporated under vacuum conditions at 45-50 ∘ C (Heidolph Rotavapor, 4003 VAC Senso T) to dryness.The dry extract was weighed to obtain the yield of the extract.After that, it was solubilized in methanol to a final concentration of 14 mg/mL [19].

Storage Conditions.
Ten mL of leaf, stem, and rhizome extracts was introduced into glass bottles protected (amber) and not protected (no amber) from light.The samples were stored at 50, 25, 4, and −20 ∘ C for 180 days.Extract stability was measured every 30 days by determining total phenols, flavonoids, and antioxidant activity (DPPH and ABTS methods).All samples were carried out in triplicate.
2.4.Total Phenols.Determination of total phenolic content was performed by Folin-Ciocalteu method in a 96-well microplate format [21,22].Leaf, stem, and rhizome extracts were diluted in methanol at a ratio of 1 : 300, 1 : 200, and 1 : 100, respectively.Gallic acid,prepared in six concentrations ranging from 4 to 20 mg/l, was used as standard.Thirty L of each extract or standard solution, except in a blank probe in which only the solvent was used, was added to 150 L of 0.1 mol/l Folin-Ciocalteu reagent and mixed with 120 L of sodium carbonate (7.5%) after 10 min.Absorbance at 760 nm was read after 2 h.Phenolic concentration was determined by comparison with the standard calibration curve of gallic acid, and the results are presented as a mean value of tests conducted in triplicate.Total phenol value was expressed as mg of gallic acid equivalents (GAE) per g of dry weight.

Total Flavonoids.
Total flavonoids were determined by utilizing the aluminum chelating method [23].Fifty L aliquots of methanol extract were transferred into the test tubes and their volumes were completed up to 750 L with deionized water.After the addition of 250 L of AlCl 3 reagent, the tubes were vortexed and maintained at room temperature for 30 min to allow for the complete reaction between the reagent and the flavonoids.Absorbance of the yellow colored solution was recorded at 405 nm against blank containing 50 L of methanol.Total flavonoid content was calculated as quercetin equivalents (QE) employing calibration curves prepared with quercetin standard solutions with a concentration range between 10 and 80 g/mL.Total flavonoid value was expressed as mg of QE per g of dry weight.

DPPH Assay.
A free-radical (F-R) DPPH solution was prepared at 6 × 10 −5 mol/l [19,24].A 0.1 mL volume of leaf, stem, and rhizome extracts was added to 3.9 mL of DPPH solution.The decrease in absorbance was determined at 515 nm at 0 and 30 min under conditions of darkness.The control sample was prepared containing the same volume without any extract.Methanol was used as the blank.All determinations were carried out in triplicate.The results are reported as % of inhibition with the following equation:
(1)   the line recorded and is proportional to elastase activity.A control curve was prepared with elastase in the absence of inhibitor.One unit of elastolytic activity is defined as the amount of enzyme releasing 1 mM of p-nitroaniline/min.For p-nitroaniline, an  of 8,800 at 410 nm was employed.Percentage of inhibition was calculated as follows: where  is the enzyme activity without inhibitor and  is the activity in the presence of inhibitor.Concentrations of extracts required for half maximal inhibitory concentration (IC 50 ) were also obtained.

Statistical Analysis.
Data was analyzed by analysis of variance (ANOVA), and the Fisher least significant difference (LSD) test was utilized to identify differences among the means.Changes and significant differences were considered at  < 0.05.All assays were performed in triplicate.

Yields of Methanolic Extracts.
Quantitative results of leaf, stem, and rhizome methanolic extracts from A. californica are summarized in Table 1.Leaf extracts exhibited the highest yield, that is, approximately 5 times more than stem and rhizome extracts.The three samples exhibited significant differences.Therefore, the order was leaf > rhizome > stem extracts.

Changes in Total Phenols and Flavonoids.
Highest amounts of total phenols (Table 2) and flavonoids (Table 3) were identified in extracts from rhizome.During storage, high temperature and exposure to light (not using amber bottles) comprised the main factors for degradation of these compounds from A. californica.They were stable during at least 60 days at 4 ∘ C and at −20 ∘ C, maintaining 97 and 98% stability, respectively, in amber bottles.After this time period, total phenols and flavonoids decreased drastically.Final extract degradations among each of the temperatures were different; however, on comparing the same temperature with leaf, stem, and rhizome extracts, similar behavior was observed.At the end of storage (180 days), the best condition in the three extracts was −20 ∘ C, with amber bottle, the rhizome extract was the most stable, conserving approximately 79% of total phenols and 73% of total flavonoid.

Antioxidant Activity.
High temperature and exposure to light were the main factors that decreased antioxidant activity.At the beginning of the experiment using the DPPH method, stem and rhizome extracts presented highest antioxidant activity (Figure 1).However, during storage, the stem extract exhibited best stability in each treatment.A contrasting situation occurred with the rhizome extract with lowest F-R scavenging activity, despite the fact that it possesses the greatest content of phenols and flavonoids.Only at −20 ∘ C did all extracts demonstrate up to 95% stability at 60 days of storage using amber bottles.However, at 180 storage days, the stem extract maintained 70% of DPPH inhibition, the highest during this time period (Figure 1(d)).
Otherwise, high inhibition of the ABTS radical was observed in the extracts in all of the treatments (Figure 2).The best conditions were found at 4 ∘ C (95%) and −20 ∘ C (98%) with protection from light from stem extracts (Figures 2(c) and 2(d)).Antioxidant activity using the ABTS radical did not correlate with phenolic content and its behavior was different from that of the DPPH method.

Discussion
Differences in the yields obtained in leaf, stem, and rhizome extracts could be caused by several factors, that is, the composition of each particular plant part and differences in solubility and polarity [26].It is, however, difficult to provide a definite explanation for the results obtained within the scope of the present study.Despite the fact that the leaf extract had the greatest yield, this extract did not present best amount of total phenols and flavonoids, nor of antioxidant activity, in comparison with stem and rhizome extracts.However, it inhibited elastase activity at the highest proportion, suggesting the presence of important anti-inflammatory compounds, which could be polyphenols, in the extract [27].
Polyphenol stability under different conditions is a very important aspect that must be taken into account to ensure that phenolic compounds possess the desired properties and maintain their activity under different storage conditions, which can involve high temperatures and light [1].Within this context, when extracts were exposure to 50 ∘ C and to light, degradation was faster, presenting a maximum of 60% degradation at the end of the experiment.The three extracts were very similarly susceptible to polyphenol degradation under these conditions.High temperature and light could change or degrade the structure of the polyphenols, resulting in marked changes in their affinity [28,29].However, this effect was not observed at low temperatures, under which they maintained high stability (up of 97%) during 60 days.
On the other hand, flavonoids are phenolic compounds; hence, the amount of total phenols includes flavonoids, both of which were correlated.Consequently, flavonoids exhibited the same behavior as total phenols.
Radical scavenging activity of leaf, stem, and rhizome methanolic extracts from A. californica was evaluated by DPPH and ABTS assays.In DPPH, the antioxidant activity of the plant extracts could be related with their phenolic content [1].Contrariwise, in ABTS, the results indicated that antioxidant activity was not correlated with these compounds, suggesting that nonphenolic compounds might engage in major F-R scavenging activity in the plant materials studied [30].In this latter assay, antioxidant activity was higher than in the DPPH method.At 180 days of storage, the stem extract demonstrated best antioxidant stability in each treatment and radical assay.On the other hand, the rhizome extract was more sensitive to antioxidant degradation.It is probable that antioxidant activity depends not only on phenolic concentration, but also on polarity and the specific chemical structure of each phenolic compound (degree of hydroxylation and extent of conjugation); some works in the literature report examples of hierarchies for phenol antioxidant activity and reduction potential [28,31].This matter could also explain the contrast with high antioxidant activity by means of ABTS assay.Similar results were obtained under similar storage conditions in extracts from sour cherry [2], pomegranate peel [32], cereals [33], mango [34], thuja fruit, and peach [35].
The inhibitory effects of leaf, stem, and rhizome extracts from A. californica on elastase activity were investigated.Leaf and rhizome extracts showed highest inhibition.However, inhibition of rhizome was more rapid than that of other extracts; therefore, its IC 50 value was lower.This may be due to the different composition of the extracts.Perhaps rhizome extracts contain more compounds related with the enzyme.Within this context, the amount of phenolic compounds was highest in this extract and presented antioxidant activity.These could exert an influence on inhibition of the enzyme, because some investigations demonstrated that antioxidants such as quercetin prevent the elastase activity [27].On the other hand, our work suggested that the IC 50 values of Anemopsis californica extracts are similar to those of antiinflammatory plants, such as Curcuma longa, Alpinia katsumadai, and Areca catechu [6].However, numerous studies have been conducted in relation to medicinal plants or their active compounds as anti-inflammatory remedies [6][7][8][9]36].
In this study, we demonstrated that leaf, stem, and rhizome methanolic extracts from Anemopsis californica maintain high stability in phenolic content and antioxidant activity during 60 days of storage at low temperatures in the dark.

Conclusions
To our knowledge, there is no previous report on the stability of extracts from Anemopsis californica; thus, this information could provide the basis for further applications in different industries, such as those of the food and pharmaceutical sectors.On the other hand, these extracts, mainly the leaf and rhizome extracts, have an elastase inhibitory effect; nevertheless, more studies are necessaries to deepen on the compounds that act as anti-inflammatory agents through the inhibition of this enzyme.Finally, Anemopsis californica could comprise a good alternative of use as antioxidant in foods.
* Mean ± standard deviation (SD) of three independent experiments.Different letters in each line indicate significant difference at  < 0.05.

Table 2 :
Changes in total phenols during storage of extracts from Anemopsis californica.
[6]ata are reported as mg gallic acid equivalents (GAE)/g of dry weight.PL: protect light using amber bottle; NP: not using amber bottle.*Mean±standarddeviation (SD) of three independent experiments.Different letters in each line indicate significant difference at  < 0.05.2.6.3.Assay for Elastase Activity.The effect of extracts on elastase activity was assayed spectrophotometrically by the method of Lee et al.[6].Porcine IV, Sigma) as substrate.Leaf, stem, and rhizome extracts were added to the reaction mixture at different concentrations (0, 250, 500, 750, and 100 g/mL).Each inhibitor was preincubated for 20 min at 25 ∘ C and the reaction was started by adding substrate.Blanks contained all of the components except for the enzyme.The reaction rate is the slope of

Table 3 :
Changes in total flavonoids during storage of extracts from Anemopsis californica.
dData are reported as mg of quercetin equivalents (QE)/g of dry weight.PL: protected from light using amber bottle; NP: not using amber bottle.* Mean ± standard deviation (SD) of three independent experiments.Different letters in each line indicate significant difference at  < 0.05.