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Evidence-Based Complementary and Alternative Medicine
Volume 2015, Article ID 349235, 8 pages
http://dx.doi.org/10.1155/2015/349235
Research Article

Anti-Inflammatory Activity and Changes in Antioxidant Properties of Leaf and Stem Extracts from Vitex mollis Kunth during In Vitro Digestion

1División de Desarrollo Biotecnológico, Centro Universitario de la Ciénega (CUCI), Universidad de Guadalajara (UdeG), Avenida Universidad 1115, Colonia Lindavista, 47820 Ocotlán, JAL, Mexico
2Coordinación 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
3Unidad de Tecnología Alimentaria, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ) AC, Avenida Normalistas 800, Colonia Colinas de la Normal, 44270 Guadalajara, JAL, Mexico
4Departamento de Biotecnología y Ciencias Alimentarias, Instituto Tecnológico de Sonora (ITSON), 5 de Febrero 818 Sur, Colonia Centro, 85000 Ciudad Obregón, SON, Mexico
5Departamento de Investigación y Posgrado en Alimentos, Universidad de Sonora, Rosales y Niños Héroes S/N, 83000 Hermosillo, SON, Mexico

Received 7 June 2015; Accepted 1 September 2015

Academic Editor: Filippo Maggi

Copyright © 2015 Juan Alfredo Morales-Del-Rio et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Vitex mollis is used in traditional Mexican medicine for the treatment of some ailments. However, there are no studies on what happens to the anti-inflammatory activity or antioxidant properties and total phenolic content of leaves and stem extracts of Vitex mollis during the digestion process; hence, this is the aim of this work. Methanolic, acetonic, and hexanic extracts were obtained from both parts of the plant. Extract yields and anti-inflammatory activity (elastase inhibition) were measured. Additionally, changes in antioxidant activity (DPPH and ABTS) and total phenols content of plant extracts before and after in vitro digestion were determined. The highest elastase inhibition to prevent inflammation was presented by hexanic extracts (leaf = 94.63% and stem = 98.30%). On the other hand, the major extract yield (16.14%), antioxidant properties (ABTS = 98.51% and DPPH = 94.47% of inhibition), and total phenols (33.70 mg GAE/g of dried sample) were showed by leaf methanolic extract. Finally, leaf and stem methanolic extracts presented an antioxidant activity increase of 35.25% and 27.22%, respectively, in comparison to their initial values after in vitro digestion process. All samples showed a decrease in total phenols at the end of the digestion. These results could be the basis to search for new therapeutic agents from Vitex mollis.

1. Introduction

Inflammation is a complex process and is a protective reaction of cells/tissues of the body to allergic or chemical irritation, injury, and/or infections [1]. This process has many ways to act; one of them is by elastase enzyme [EC: 3.4.4.7]. This enzyme is a serine protease associated with the granular fraction of polymorphonuclear leukocytes, which hydrolyzes multiple bonds of connective tissue matrix protein substrates such as elastin, collagen, proteoglycans, and keratins [2]. UV irradiation stimulates the activity of fibroblast elastases in skin [3]. Additionally, elastase can display chronic obstructive pulmonary disease-like features including widespread lung inflammation, goblet cell metaplasia, increased lung volume, emphysema, and decreased elastic recoil [4]. Therefore, elastase plays a critical role in inflammatory processes. Plants with potent biological effect and fewer side effects have attracted great interest and have been evaluated for their use in treating inflammation [2, 57].

The use of plants is becoming more important not only for their anti-inflammatory properties but also for their ability to prevent cellular damage induced by free radicals. The excess of free radicals is produced during oxidative metabolism causing damage in cellular lipids, proteins, or DNA, inhibiting their normal function [8]. For these reasons, the importance of free radicals neutralization by antioxidants is a very important aspect of living organisms. Phenolic compounds are commonly found in plants exhibiting antioxidant properties that are attributed to their intrinsic reducing properties [9]. However, when the plants or their extracts are consumed, the digestive process can affect their antioxidant properties due to their hydrophobicity, molecular mass, degree of polymerization, environment, kind of sugar present in the molecule, and pH [10]. Furthermore, several studies have shown that the effectiveness of phenols absorption through the intestine is influenced by the action of digestive enzymes such as pepsin and pancreatin [11, 12]. Therefore, the importance of the antioxidant properties of plant materials in the maintenance of health and protection against several diseases has raised interest among scientists [13]. In this context, Vitex mollis is a medicinal plant widely used in traditional Mexican medicine for the treatment of diarrhea and dysentery [14] that possesses antispasmodic [14], antiprotozoal [15], and antioxidant [16] activities. The aim of the present work was to investigate the anti-inflammatory properties of leaves and stem extracts of Vitex mollis, as well as changes in their total phenol contents and antioxidant properties that might occur during the digestion process.

2. Methodology

The material and reagents such as porcine pancreatic elastase (PPE, type IV), succinyl-Ala-Ala-Ala-p-nitroanilide (ESIV, elastase substrate IV), DPPH [2,2-diphenyl-1-picrylhydrazyl], ABTS [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], gallic acid [3,4,5-trihydroxybenzoic acid], 2 N Folin-Ciocalteu phenol reagent solution, tris-HCl buffer (T-3253), pepsin (P-7012), pancreatin (P-1750), phosphate buffered saline (PBS-3813), and dialysis tubing (D-9527) were purchased from Sigma-Aldrich Co. (USA). All other chemicals and solvents were of the highest commercial grade.

2.1. Extraction

Leaves and stems of Vitex mollis were collected from the Ciénega region of Jalisco, Mexico (102°48′W, 20°21.6′N, 1800 m of altitude), in May 2014. Taxonomic identification of the plant was made by the Botanic Institute of the University of Guadalajara (IBUG) with the register number 192156. The extractions of compounds were made according to Del-Toro-Sánchez et al. [17] with some modifications. Three grams of each dried and ground sample was mixed with 20 mL of solvent (methanol, acetone, or hexane). After homogenization for 1 min (Ultraturrax, T 25 DS1 digital homogenizer) and sonication at 4°C for 15 min (Bransonic, 151-DTH), the samples were centrifuged at 4,000 ×g for 15 min at 4°C. The supernatants were collected and the residues were used for a second extraction employing the same procedure. The supernatants were combined and evaporated to dryness using a rotavapor (Heidolph Rotavapor, 4003 VAC Senso T) at 45–50°C. The dry extracts were weighed to obtain the yield of the extract and solubilized in aqueous 1% Tween 20 to a final concentration of 750 μg/mL. Leaves samples were identified as methanolic, acetonic, and hexanic extracts (LM, LA, and LH, resp.). The same designation procedure was used for the stem extracts (SM, SA, and SH).

2.2. Anti-Inflammatory Activity

The anti-inflammatory activity was evaluated by the method of Lee et al. [2]. This method consists in the spectrophotometric measurement at 410 nm of the amount of p-nitroaniline released from the substrate [N-Succ-(Ala)3-p-nitroanilide] by porcine pancreatic elastase (PPE). The reaction mixture (final volume of 150 μL) contained 0.2 M Tris-HCl buffer (pH 8.0), 1 μg/mL elastase (0.046 U/mL), 0.8 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide as substrate, and leaves and stem extracts as inhibitors. Aliquots (10 μL) of the extracts, previously incubated for 20 min at 25°C, were added at different concentrations (0, 150, 300, 450, 600, and 750 μg/mL). The reaction was started by adding the substrate. Proper blanks were prepared and contained all the components except the enzyme. The rate of the reaction was determined as the slope of the line recorded and was proportional to elastase activity. A control curve was prepared with elastase in the absence of inhibitor. One unit of elastolytic activity was defined as the amount of enzyme required to produce 1 mM of p-nitroaniline/min under the conditions of the assay. For p-nitroaniline, an ε 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 (IC50) were also obtained.

2.3. Antioxidant Activity by DDPH Assay

The antioxidant properties of the plant samples using the DPPH assay were measured by the method described by Molyneux [18]. Briefly, an aliquot of 0.1 mL of the sample solutions was mixed with 3.9 mL of a free radical DPPH solution (6 × 10−5 mol/L). The reaction mixtures were incubated for 30 min in the darkness and their absorbance was measured at 515 nm against a blank prepared with aqueous 1% Tween 20. All determinations were carried out in triplicate. The results were converted into percentage of antioxidant activity using the following equation:

2.4. Antioxidant Activity by ABTS Assay

The antioxidant activity of the plant samples was determined by the ABTS cation radical method described by Re et al. [19]. A volume of 2.97 mL of the cation radical solution was combined with 0.03 mL of each plant extract. The absorbance was measured at 734 nm after 20 min of incubation at room temperature. A control was prepared containing the cation radical solution with no plant extracts. Aqueous 1% Tween 20 was used as the blank. Antioxidant activity, expressed as % of inhibition, was calculated using the formula mentioned in the DPPH assay.

2.5. Total Phenols

The total phenolic content was assayed by a spectrophotometric method using the Folin-Ciocalteu reagent in a 96-well microplate format [20, 21]. Briefly, 30 μL of each extract solution was combined with 150 μL of 0.1 M Folin-Ciocalteu reagent. After 10 min of incubation at room temperature, 120 μL of a 7.5% sodium carbonate solution was added and the absorbance was read at 760 nm. A gallic acid standard calibration curve (0–100 mg/L) was prepared and the results were expressed as mg of gallic acid equivalents (GAE) per gram of dry sample.

2.6. In Vitro Digestion

The method described by Gil-Izquierdo et al. [22] with some modifications was utilized to perform the in vitro digestion. The method consists in simulating the stomach and small intestine conditions to evaluate the in vitro bioavailability of the antioxidant properties of Vitex mollis extracts. The hexanic extracts (LH and SH) were not considered for this assay due to their low antioxidant activity. The rest of the extracts (300 μg/mL) were adjusted to pH 2.0 with 1 M HCl. A volume of 0.5 mL from each acidified extract was combined with 0.75 mL of pepsin (315 U/mL) and 1.75 mL of deionized water. The mixture was incubated at 37°C in a shaking water bath (WiseBath, DAIHAN Scientific, WSB-18) at 80 rpm for 2 h. After incubation, the samples were neutralized with 1.25 M NaHCO3 and 0.375 mL of pancreatin solution (4 mg/mL) was added. This mixture was transferred to dialysis tubes, placed in an Erlenmeyer flask containing 35 mL of phosphate buffer (PBS), and placed again under incubation in the shaking water bath (4 h, 80 rpm, 37°C). The antioxidant activity and total phenols were determined before (initial) and after (dialysate) digestion. The dialysate was the PBS buffer + compounds that passed through the membrane as equivalent to intestinal absorption after digestion. Since the lipid content of the extracts analyzed in this process was very low, there was no necessity to add bile salts.

2.7. Statistical Analysis

Analysis of data was carried out by analysis of variance (ANOVA) using a single-factor experimental design. Comparison of means was performed by Fisher’s least significance test () using the Statgraphics Centurion XV v.15.2.06 software package. The experiments were run in triplicate.

3. Results and Discussion

3.1. Extract Yields

Different extract yields were obtained from leaves and stems of Vitex mollis with the different solvents used in this study (Table 1). LM extract presented the highest yield. This extract was approximately five times higher than the rest of the leaf extracts. On the other hand, stem extracts did not show significant differences among them. Hence, extract yields progressively decreased according to the following order: LM > SH, SM, SA > LA, LH. Our results suggest that the more represented compounds of Vitex mollis leaves were polar fractions, whereas in stem extracts there were similar proportions of polar and nonpolar fractions.

Table 1: Extract yields from leaf and stem of Vitex mollis.

Within the same genus Vitex, the extract yields may vary. For example, methanolic leaf extracts from Vitex trifolia and Vitex glabrata showed extract yields of 21.22% [23] and 18.86% [24], respectively. These values were higher in comparison with the methanolic leaf extract from Vitex mollis in our study obtaining 16.4% of extract yield. However, the different result obtained in this study may be due to the different polarity of compounds or to the extraction procedure employed. In the studies of the other authors, the solvent was in contact with the samples for several days (in this case 6 to 9). In contrast, in our extraction procedure, the samples were mixed with the solvent for approximately 30 min. Similar experimental condition to ours was used by Thenmozhi et al. [25] to obtain leaf extracts from Vitex trifolia. They obtained a yield of 8.62%, a value lower than ours in Vitex mollis. Therefore, the method used in the extraction of Vitex mollis provides good yields in a short period of time.

On the other hand, there are very few studies about the stem extract yields from Vitex genus. Tijjani et al. [26] obtained a yield of 14.8% from stem of Vitex doniana by five-day maceration in ethanol. Using this same variety, James et al. [27] obtained approximately 9.9, 9.2, and 7.5% extract yields from two-day methanolic, ethanolic, and acetonic maceration, respectively. These results are similar to the extract yields of our study. However, ethanolic extract yields in these studies [26, 27] were obtained with the same method, solvent, and plant; hence, the time of maceration or the maturity of the plant affects extract yields.

3.2. Anti-Inflammatory Activity

The anti-inflammatory activities evaluated in this study were higher in stems than leaves extracts (Figure 1(b)). However, the hexanic extracts of both parts of the plant (LH and SH) presented the highest elastase inhibition; consequently, they have the major anti-inflammatory activity (Figures 1(a) and 1(b)). SH extract requires a minor amount to reach the 50% of elastase inhibition (IC50 = 197.86 μg/mL) compared to the rest of the samples. Hence, these results suggest that nonpolar compounds are responsible for this activity.

Figure 1: Anti-inflammatory activity evaluated by inhibition of elastase activity (expressed in %). The IC50 values of control (C, deionized water + 1% of Tween 20), (a) leaf (L), and (b) stem (S) extracts of Vitex mollis from the different solvents, methanol (M), acetone (A), and hexane (H), are reported beside each graph. ND: not detected.

Some authors mentioned that nonpolar compounds have more anti-inflammatory activity in the genus Vitex such as Vitex altissima [28], Vitex rotundifolia [2931], Vitex trifolia [32], Vitex negundo [33], and Vitex agnus-castus [34]. Different anti-inflammatory mechanisms are proposed for nonpolar compounds from Vitex including the inhibition of lymphocyte proliferation [29] and nuclear factor inhibition throughout reactive oxygen species (ROS) [30] or vascular-endothelial-growth-factor (VEGF) [31] among others. In our study, the inhibition of elastase enzyme was the mechanism used. This is the first study in leaf and stem extracts from Vitex mollis about inhibition of elastase enzyme to prevent inflammation. Hence, this plant could be a good candidate to obtain and identify anti-inflammatory compounds.

3.3. Antioxidant Activity and Total Phenols

The antioxidant activities were measured using the DPPH and ABTS assays. In general, all the extract samples showed a higher antioxidant activity by the ABTS than the DPPH assay, with the higher values recorded in LM (98.51%) and SA (96.79%), respectively (Figure 2(a)), whereas the higher values by using the DPPH assay were recorded in both the methanolic extracts (LM = 94.47% and SM = 77.22%). Similarly, LM and SM (33.70 and 25.54 mg GAE/g of dry sample, resp.) presented also the major phenolic content (Figure 2(b)). In the study of Gacche et al. [35], polar extracts of Vitex negundo showed higher DPPH radical scavenging activity, while the nonpolar extract did not react with this radical. Similar to our results, in hexanic extracts of Vitex mollis, DPPH radical inhibition was very low in both parts of the plant. But, as in the present study, the methanolic leaf extracts from Vitex doniana presented the highest antioxidant activity and total phenols in the work of James et al. [27]. However, correlations are useful to indicate the relationship between the factors in study. For example, the correlation between DPPH and total phenols (Figure 3(a)) was very high (), whereas ABTS and total phenols (Figure 3(b)) showed a low correlation (). Furthermore, the correlation value of ABTS with DPPH (Figure 3(c)) was low (). These differences can be attributed to the idea that when radical is formed, indeed, it reacts with any hydroxylated aromatic compounds independently of their real antioxidative potential in a wide pH range. Additionally, in the absence of phenolics, is rather stable, but it reacts energetically with a H-atom donor; therefore, is much faster than the DPPH assay [36, 37]. DPPH is likely more selective than in the reaction with H-donors at a neutral or alkaline pH [38], but it does not react with aromatic acids containing only one OH-group [39] nor with phenols that contain no OH-groups in B-ring [40]. Therefore, it is probable that compounds different from the phenols contribute to determination of the antioxidant activity of extracts in the ABTS assay, that is, ascorbate, glutathione, urate, tocopherols, or carotenoids [41]. Conversely, in the DPPH assay, the higher antioxidant responses elevated are probably due to the phenolic components of the studied extracts.

Figure 2: Antioxidant activities and content of total phenols in extracts of Vitex mollis obtained with leaf (L) and stem (S) from methanol (M), acetone (A), and hexane (H) solvents.
Figure 3: Linear correlation between (a) DPPH and (b) ABTS assays with total phenols and (c) DPPH with ABTS assays.
3.4. In Vitro Digestion

Changes in the antioxidant properties and content of total phenols were determined before (initial) and after (dialysate) the simulated digestion of leaf and stem extracts from Vitex mollis (Figure 4). In the dialysed samples, the methanolic extracts showed the highest value of antioxidant activity.

Figure 4: Antioxidant activity evaluated by (a) ABTS and (b) DPPH assays and (c) content of total phenols of initial and dialysate extracts obtained in the in vitro digestion of leaf (L) and stem (S) extracts of Vitex mollis from methanol (M) and acetone (A) solvents.

Moreover, the same samples gave the higher increase of their antioxidant properties (expressed as % of inhibition) after the in vitro digestion, both in the ABTS and DPPH assays. In the ABTS assay, LM and SM showed an increase of 35.25% and 27.22%, respectively, compared to the initial amounts (Figure 4(a)). A similar behavior was observed in the DPPH test results (LM = 30.34% and SM = 26.43% of increase) (Figure 4(b)). Unfortunately, the rest of the samples decreased their antiradical potential after digestion, especially the acetonic stem extracts. Therefore, these results suggest that polar compounds could increase their antioxidant capacity after influence of pH and enzyme digestion. From the chemical point of view, it is possible that at lower pH the compounds such as phenolics will have less susceptibility towards oxidation because hydroxyl groups will be shielded by protonation. Hence, the pH definitely affects the antioxidant activity. However, the effect is either way depending on type of these compounds.

On the other hand, all samples showed a decrease in total phenols (from 36 to 81.4%) at the end of the digestion, particularly the acetonic extracts (Figure 4(c)). The effectiveness of phenols absorption could be influenced by many factors such as hydrophobicity, molecular mass, degree of polymerization, environment, kind of sugar present in the molecule, and pH [10]. In our study, two pH values were tested (pH 2 and pH 7) to simulate digestion and probably the pH change could have negatively affected the phenols adsorption. However, the antioxidant activities of some of the dialysed samples increased anyway. Bermúdez-Soto et al. [11] demonstrated that polyphenols are highly sensitive to the mild alkaline conditions in the small intestine and a proportion of these compounds can be transformed before absorption. However, various studies have reported that the absorption of polyphenols that can reach the small intestine is very low, changing between 5 and 10% [12, 42]. Furthermore, it is possible that some polyphenols, which cannot pass through the intestinal barrier, can be fermented by colonic microflora exerting their antioxidant activity after colonic fermentation [12]. In our experiment, the model did not take into account the interactions with the microflora. Moreover, active transport mechanisms that may occur in the small intestine cannot be mimicked in the in vitro study, and partition of the compounds into the dialysis tubing is solely dependent on the diffusion rates and their stability [43]. Therefore, these factors require further attention.

4. Conclusions

The nonpolar compounds of leaf and stem obtained from Vitex mollis by hexanic extraction could be responsible for the higher anti-inflammatory activity registered in the elastase inhibition test. Hence, according to the results of the present study, Vitex mollis is a good candidate as a potential source of anti-inflammatory compounds. On the other hand, from leaves of this plant it is possible to obtain high methanolic extract yields and high antioxidant properties. Additionally, the in vitro digestion process appeared to be an important factor for potentiating the antioxidant activity of methanolic extracts from leaves and stems of Vitex mollis. Therefore, the in vitro digestion could be suggested as a rapid and simple method to assess the stability of phytochemical compounds obtained in phytoextracts.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

This work was supported by the Grant 05-2010-1-947 from Consejo Estatal de Ciencia y Tecnología de Jalisco (COECyTJAL).

References

  1. E. O. Iwalewa, L. J. McGaw, V. Naidoo, and J. N. Eloff, “Inflammation: the foundation of diseases and disorders. A review of phytomedicines of South African origin used to treat pain and inflammatory conditions,” African Journal of Biotechnology, vol. 6, no. 25, pp. 2868–2885, 2007. View at Google Scholar · View at Scopus
  2. K.-K. Lee, J.-H. Kim, J.-J. Cho, and J.-D. Choi, “Inhibitory effects of 150 plant extracts on elastase activity, and their anti-inflammatory effects,” International Journal of Cosmetic Science, vol. 21, no. 2, pp. 71–82, 1999. View at Publisher · View at Google Scholar · View at Scopus
  3. G. Imokawa, “Recent advances in characterizing biological mechanisms underlying UV-induced wrinkles: a pivotal role of fibrobrast-derived elastase,” Archives of Dermatological Research, vol. 300, no. 1, pp. S7–S20, 2008. View at Publisher · View at Google Scholar · View at Scopus
  4. U. Sajjan, S. Ganesan, A. T. Comstock et al., “Elastase- and LPS-exposed mice display altered responses to rhinovirus infection,” American Journal of Physiology—Lung Cellular and Molecular Physiology, vol. 297, no. 5, pp. L931–L944, 2009. View at Publisher · View at Google Scholar · View at Scopus
  5. S.-J. Kim, S. A. Sancheti, S. S. Sancheti, B.-H. Um, S.-M. Yu, and S.-Y. Seo, “Effect of 1,2,3,4,6-penta-O-galloyl-β-D-glucose on elastase and hyaluronidase activities and its type II collagen expression,” Acta Poloniae Pharmaceutica - Drug Research, vol. 67, no. 2, pp. 145–150, 2010. View at Google Scholar · View at Scopus
  6. N. Maity, N. K. Nema, M. K. Abedy, B. K. Sarkar, and P. K. Mukherjee, “Exploring Tagetes erecta linn flower for the elastase, hyaluronidase and MMP-1 inhibitory activity,” Journal of Ethnopharmacology, vol. 137, no. 3, pp. 1300–1305, 2011. View at Publisher · View at Google Scholar · View at Scopus
  7. S. J. Hwang and H.-J. Lee, “Phenyl-β-d-Glucopyranoside exhibits anti-inflammatory activity in lipopolysaccharide-activated RAW 264.7 cells,” Inflammation, vol. 38, no. 3, pp. 1071–1079, 2015. View at Publisher · View at Google Scholar · View at Scopus
  8. M. Valko, D. Leibfritz, J. Moncol, M. T. D. Cronin, M. Mazur, and J. Telser, “Free radicals and antioxidants in normal physiological functions and human disease,” International Journal of Biochemistry and Cell Biology, vol. 39, no. 1, pp. 44–84, 2007. View at Publisher · View at Google Scholar · View at Scopus
  9. Z. Hodzic, H. Pasalic, A. Memisevic, M. Srabovic, M. Saletovic, and M. Poljakovic, “The influence of total phenols content on antioxidant capacity in the whole grain extracts,” European Journal of Scientific Research, vol. 28, no. 3, pp. 471–477, 2009. View at Google Scholar · View at Scopus
  10. T. Tarko, A. Duda-Chodak, P. Sroka, P. Satora, and J. Michalik, “Transformations of phenolic compounds in an in vitro model simulating the human alimentary tract,” Food Technology and Biotechnology, vol. 47, no. 4, pp. 456–463, 2009. View at Google Scholar · View at Scopus
  11. M.-J. Bermúdez-Soto, F.-A. Tomás-Barberán, and M.-T. García-Conesa, “Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to in vitro gastric and pancreatic digestion,” Food Chemistry, vol. 102, no. 3, pp. 865–874, 2007. View at Publisher · View at Google Scholar · View at Scopus
  12. H. G. Akillioglu and S. Karakaya, “Changes in total phenols, total flavonoids, and antioxidant activities of common beans and pinto beans after soaking, cooking, and in vitro digestion process,” Food Science and Biotechnology, vol. 19, no. 3, pp. 633–639, 2010. View at Publisher · View at Google Scholar · View at Scopus
  13. M. P. Kähkönen, A. I. Hopia, H. J. Vuorela et al., “Antioxidant activity of plant extracts containing phenolic compounds,” Journal of Agricultural and Food Chemistry, vol. 47, no. 10, pp. 3954–3962, 1999. View at Publisher · View at Google Scholar · View at Scopus
  14. L. Osuna, M. E. Tapia-Pérez, J. E. Jiménez-Ferrer, B. A. Carrillo-Quiróz, and J. Silva-Sánchez, “Screening of Alternanthera repens , Boerhavia coccinea , Flaveria trinervia, Tournefortia densiflora , and Vitex mollis extracts to evaluate their antibacterial activity and effect on smooth muscle. I,” Pharmaceutical Biology, vol. 43, no. 1, pp. 749–753, 2005. View at Publisher · View at Google Scholar · View at Scopus
  15. M. E. Tapia-Pérez, A. Tapia-Contreras, R. Cedillo-Rivera, L. Osuna, and M. Meckes, “Screening of Mexican medicinal plants for antiprotozoal activity—part II,” Pharmaceutical Biology, vol. 41, no. 3, pp. 180–183, 2003. View at Publisher · View at Google Scholar · View at Scopus
  16. F. Ruiz-Terán, A. Medrano-Martínez, and A. Navarro-Ocaña, “Antioxidant and free radical scavenging activities of plant extracts used in traditional medicine in Mexico,” African Journal of Biotechnology, vol. 7, no. 12, pp. 1886–1893, 2008. View at Google Scholar · View at Scopus
  17. C. L. Del-Toro-Sánchez, N. Bautista-Bautista, J. L. Blasco-Cabal, M. Gonzalez-Ávila, M. Gutiérrez-Lomelí, and M. Arriaga-Alba, “Antimutagenicity of methanolic extracts from Anemopsis californica in relation to their antioxidant activity,” Evidence-Based Complementary and Alternative Medicine, vol. 2014, Article ID 273878, 8 pages, 2014. View at Publisher · View at Google Scholar
  18. P. Molyneux, “The use of the stable radical Diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity,” Songklanakarin Journal of Science and Technology, vol. 26, no. 2, pp. 211–219, 2004. View at Google Scholar
  19. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, and C. Rice-Evans, “Antioxidant activity applying an improved ABTS radical cation decolorization assay,” Free Radical Biology and Medicine, vol. 26, no. 9-10, pp. 1231–1237, 1999. View at Publisher · View at Google Scholar · View at Scopus
  20. V. L. Singleton, R. Orthofer, and R. M. Lamuela-Raventós, “Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent,” Methods in Enzymology, vol. 299, no. 1, pp. 152–178, 1998. View at Publisher · View at Google Scholar · View at Scopus
  21. L. M. Magalhães, F. Santos, M. A. Segundo, S. Reis, and J. L. F. C. Lima, “Rapid microplate high-throughput methodology for assessment of Folin-Ciocalteu reducing capacity,” Talanta, vol. 83, no. 2, pp. 441–447, 2010. View at Publisher · View at Google Scholar · View at Scopus
  22. A. Gil-Izquierdo, P. Zafrilla, and F. A. Tomás-Barberán, “An in vitro method to simulate phenolic compound release from the food matrix in the gastrointestinal tract,” European Food Research and Technology, vol. 214, no. 2, pp. 155–159, 2002. View at Publisher · View at Google Scholar · View at Scopus
  23. T. Masuda, S. Yonemori, Y. Oyama et al., “Evaluation of the antioxidant activity of environmental plants: activity of the leaf extracts from Seashore plants,” Journal of Agricultural and Food Chemistry, vol. 47, no. 4, pp. 1749–1754, 1999. View at Publisher · View at Google Scholar · View at Scopus
  24. P. Luecha, K. Umehara, T. Miyase, and H. Noguchi, “Antiestrogenic constituents of the Thai medicinal plants Capparis flavicans and Vitex glabrata,” Journal of Natural Products, vol. 72, no. 11, pp. 1954–1959, 2009. View at Publisher · View at Google Scholar · View at Scopus
  25. S. Thenmozhi, R. Sundaram, J. Kumar, and C. G. Bihari, “Pharmacognostical and phytochemical investigation on leaves of Vitex trifolia Linn,” Journal of Pharmacy Research, vol. 4, no. 4, pp. 1259–1262, 2011. View at Google Scholar
  26. M. A. Tijjani, F. I. Abdurahaman, I. Z. Khan, and U. K. Sandabe, “The effect of ethanolic extract of Vitex doniana stem bark on peripheral and central nervous system of laborotory animals,” Journal of Applied Pharmaceutical Science, vol. 2, no. 3, pp. 74–79, 2012. View at Google Scholar
  27. D. B. James, V. D. Sheneni, A. Kadejo, and K. B. Yatai, “Phytochemical screening, and in-vitro antioxidant activities in different solvent extracts of Vitex doniana leaves, stem bark and root bark,” American Journal of Biomedical and Life Sciences, vol. 2, no. 1, pp. 22–27, 2014. View at Publisher · View at Google Scholar
  28. C. Sridhar, K. V. Rao, and G. V. Subbaraju, “Flavonoids, triterpenoids and a lignan from Vitex altissima,” Phytochemistry, vol. 66, no. 14, pp. 1707–1712, 2005. View at Publisher · View at Google Scholar · View at Scopus
  29. K. M. You, K. H. Son, H. W. Chang, S. S. Kang, and H. P. Kim, “Vitexicarpin, a flavonoid from the fruits of Vitex rotundifolia, inhibits mouse lymphocyte proliferation and growth of cell lines in vitro,” Planta Medica, vol. 64, no. 6, pp. 546–550, 1998. View at Publisher · View at Google Scholar · View at Scopus
  30. S. M. Lee, Y. J. Lee, Y. C. Kim, J. S. Kim, D. G. Kang, and H. S. Lee, “Vascular protective role of vitexicarpin isolated from Vitex rotundifolia in human umbilical vein endothelial cells,” Inflammation, vol. 35, no. 2, pp. 584–593, 2012. View at Publisher · View at Google Scholar · View at Scopus
  31. B. Zhang, L. Liu, S. Zhao, X. Wang, L. Liu, and S. Li, “Vitexicarpin acts as a novel angiogenesis inhibitor and its target network,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 278405, 13 pages, 2013. View at Publisher · View at Google Scholar · View at Scopus
  32. G. Alam, S. Wahyuono, I. G. Ganjar, L. Hakim, H. Timmerman, and R. Verpoorte, “Tracheospasmolytic activity of viteosin-A and vitexicarpin isolated from Vitex trifolia,” Planta Medica, vol. 68, no. 11, pp. 1047–1049, 2002. View at Publisher · View at Google Scholar · View at Scopus
  33. F. Díaz, D. Chávez, D. Lee et al., “Cytotoxic flavone analogues of vitexicarpin, a constituent of the leaves of Vitex negundo,” Journal of Natural Products, vol. 66, no. 6, pp. 865–867, 2003. View at Publisher · View at Google Scholar · View at Scopus
  34. M. I. Choudhary, Azizuddin, S. Jalil et al., “Antiinflammatory and lipoxygenase inhibitory compounds from Vitex agnus-castus,” Phytotherapy Research, vol. 23, no. 9, pp. 1336–1339, 2009. View at Publisher · View at Google Scholar · View at Scopus
  35. R. Gacche, R. Shaikh, M. Pund, and R. Deshmukh, “Cyclooxygenase inhibitory, cytotoxicity and free radical scavenging activities of selected medicinal plants used in indian traditional medicine,” Pharmacognosy Journal, vol. 3, no. 19, pp. 57–64, 2011. View at Publisher · View at Google Scholar · View at Scopus
  36. R. L. Prior, X. Wu, and K. Schaich, “Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements,” Journal of Agricultural and Food Chemistry, vol. 53, no. 10, pp. 4290–4302, 2005. View at Publisher · View at Google Scholar · View at Scopus
  37. V. Roginsky and E. A. Lissi, “Review of methods to determine chain-breaking antioxidant activity in food,” Food Chemistry, vol. 92, no. 2, pp. 235–254, 2005. View at Publisher · View at Google Scholar · View at Scopus
  38. M. S. Bhoyar, G. P. Mishra, P. K. Naik, and R. B. Srivastava, “Estimation of antioxidant activity and total phenolics among natural populations of Caper (Capparis spinosa) leaves collected from cold arid desert of trans-Himalayas,” Australian Journal of Crop Science, vol. 5, no. 7, pp. 912–919, 2011. View at Google Scholar · View at Scopus
  39. A. von Gadov, E. Joubert, and C. F. Hansmann, “Comparison of the antioxidant activity of aspalathin with that of other plant phenols of Roobies tea (Aspalathus linearis), α-tocopherol, BHT, and BHA,” Journal of Agricultural and Food Chemistry, vol. 45, no. 1, pp. 632–638, 1997. View at Publisher · View at Google Scholar
  40. T. Yokozawa, C. P. Chen, E. Dong, T. Tanaka, G.-I. Nonaka, and I. Nishioka, “Study on the inhibitory effect of tannins and flavonoids against the 1,1-diphenyl-2-picrylhydrazyl radical,” Biochemical Pharmacology, vol. 56, no. 2, pp. 213–222, 1998. View at Publisher · View at Google Scholar · View at Scopus
  41. M. A. Eastwood, “Interaction of dietary antioxidants in vivo: how fruit and vegetables prevent disease?” Quarterly Journal of Medicine, vol. 92, no. 9, pp. 527–530, 1999. View at Publisher · View at Google Scholar · View at Scopus
  42. M. N. Clifford, “Diet-derived phenols in plasma and tissues and their implications for health,” Planta Medica, vol. 70, no. 12, pp. 1103–1114, 2004. View at Publisher · View at Google Scholar · View at Scopus
  43. G. J. McDougall, P. Dobson, P. Smith, A. Blake, and D. Stewart, “Assessing potential bioavailability of raspberry anthocyanins using an in vitro digestion system,” Journal of Agricultural and Food Chemistry, vol. 53, no. 15, pp. 5896–5904, 2005. View at Publisher · View at Google Scholar · View at Scopus