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1 Chapter 1 : Foods Free Full-Text Phenolic Profiles and Antioxidant Activity of Germinated Legumes HT Nonflavonoid Phenolic Compoundsâ Structure and Their Dietary Occurrence Overview of Flavonoid and Phenolic Biosynthetic Pathways Optimization of the Flavonoid and Phenolic Profiles in Crop Plants. 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 Response surface methodology was applied to optimization of the conditions for reflux extraction of Pandan Pandanus amaryllifolius Roxb. Response surface plots showed that increasing these operation parameters induced the responses significantly. The optimized model was validated by a comparison of the predicted and experimental values. The experimental values were found to be in agreement with the predicted values, indicating the suitability of the model for optimizing the conditions for the reflux extraction of Pandan. Introduction Phytochemicals are important compounds found in medicinal plants that exert positive effects on human health or in amelioration of diseases. Though many phytochemicals have been identified, a great many are yet to be identified [ 1 ]. Extraction and analysis of plant matrices are primary and important processes for the quality control, modernization, and development of herbal formulations [ 3 ]. In general, the first step of complete extraction is the selection of plant parts and careful preparation of plant extracts and a thorough review of the existing literature to determine the most suitable protocols for a specific group of phytochemicals or plant species. Traditionally, the extraction of phenolic acids and flavonoid compounds is accomplished by reflux or Soxhlet extraction [ 4 ]. However, prolonged extraction at high temperature may degrade flavonoid and phenolic acid compounds [ 5 ] and involves a high energy cost. A model for optimizing the most relevant operational parameters is required in order to achieve higher extraction yield. Response surface methodology RSM is a collection of statistical and mathematical techniques that is used to optimize the range of variables in various experimental processes with reducing the number of experimental runs, cost, and time compared to other methods [ 6, 7 ]. Pandan Pandanus amaryllifolius Roxb. Pandan has narrow and strap-shaped green leaves with a spiral arrangement [ 8 ]. Pandan is characterized by a sweet and delightful flavor and is widely used as a natural flavor in East Asian countries including Indonesia, Thailand, India, and Malaysia. In Malaysia, Pandan leaves are also used in the production of coconut jam, sweets, desserts, and ice cream. A number of studies have demonstrated that Pandan leaves are a valuable source of phenolic compounds [ 9 â 11 ]. However, far too little attention has been paid to optimization of Pandan extract in folk medicine. To the best of our knowledge, there are no studies and reports undertaken to optimize the flavonoid and phenolic extraction from Pandan leaf, following that improving of antioxidant activity using RSM. The aim of this study is to optimize the extraction conditions of a Malaysian Pandanus amaryllifolius variety to achieve high TF and TP content and thus to enhance the AC based on the use of RSM method with a central composite design CCD for optimization of the reflux extraction conditions. Material and Methods 2. Plant Material Plant samples P. The leaves were shade dried and were powdered using a mechanical grinder. This powered material was used for further analysis. After reflux, the solutions were cooled at room temperature and were filtered with a Whatman number 1 filter paper and used for future analysis. Independent variables and their coded and actual values used for optimization. Determination of Total Flavonoids After extraction, 1 ml of extracts was diluted with distilled water 4 ml. Absorbance of the solutions was read at nm [ 12 ]. Solutions were allowed to stand for 5 min. The absorbance of the mixtures was read at nm [ 13 ]. Determination of Antioxidant Capacity 2. DPPH was dissolved in methanol to give final concentration of 2 mm. For the control, methanol was used as a blank. The absorbance of the samples was read at nm using spectrophotometer. BHT butylhydroxytoluene and -tocopherol were used as positive controls. The scavenging activity was calculated using the following formula: In this system two mobile phases including 0. The range of detecting wavelength was between and nm. Gradient elution was performed as follows: To prepare the standard solution all flavonoid and phenolic acid standards were dissolved in HPLC grade methanol. The Page 1

2 linear regression equation was calculated with, where was concentration of flavonoid and was the peak area of flavonoids obtained from UHPLC [ 15 ]. Compounds were tentatively identified by comparison of retention times of standards. All flavonoids and phenolic acids standards were purchased from Sigma-Aldrich Malaysia. The central composite experimental design with 3 levels and 3 factors was used to examine the extraction variables. Design-Expert software Version 7. Analysis of variance and response surface analysis were employed to determine the regression coefficients and statistical significance of the model terms and to fit the mathematical models of the experimental data that aimed to optimize the overall region for both response variables. A model was applied to predict the response variables as given below: The relationship between the independent variables MC: Table 1 shows information about extraction temperature, MC, and LS ratio of the 20 experiments. Result and Discussion 3. In addition, the Maillard reaction may occur at high temperatures, resulting in undesired compounds [ 17 ]. The results of the experiment and the extraction conditions are shown in Table 2. Significant regression relationships between the response and independent variables were observed. High TF and TP contents of 1. The predicted TF and TP contents for treatment 5 were 1. Increasing the LS ratio resulted in an increment of the TF content of the extracts. In addition, a lower TF content was recorded at lower LS ratio In the analysis of the TF content, a good coefficient of determination was obtained, where the model explained most of the observed variations Table 3. The model value of 0. The lack of fit test for the model describes the variation in the data around the fitted model. If the model does not fit the data well, the lack of fit value will be significant; consequently, proceeding with investigation and optimization of the fitted response surface is likely to give misleading results. However, no interactive effect of the independent variables was observed. The predicted model obtained for TF was as follows: The data in Table 2 demonstrate that when the LS ratio increased from It is plausible that these results are due to the fact that more solvent could enter the cells while more phenolic compounds could permeate into the solvent at higher LS ratios [ 18 ]. The highest content of phenolics was reportedly obtained from fruits of E. In current study, the predicted model obtained for TP extraction was as follows: There is only a 0. The effect of the variables and the interaction of these variables on the responses can be seen in Figures 1 and 2. Moreover, the results indicated that the MC was the most significant factor affecting the responses at the level. As shown in Figure 2, the TP content increased significantly to ca. It is difficult to explain this result, but this trend might be related to the increased solubility of the flavonoid compounds in the mixture of methanol and water [ 21 ]. The findings of the current study are consistent with those of Liyana-Pathirana and Shahidi [ 22 ], who found that the TF content of wheat increased with increasing ethanol concentration. Predicted models and statistical parameters calculated after implementation of three-factor central composite design. In different plants and organs this temperature will be variable and according to the previous studies this changes in phenolic acids and flavonoids at high temperature could be related to PAL or CHS enzymes activity at high or low temperature [ 25 ]. At high temperature, the flavonoid and phenolic content can be increased as a result of enhancement of their solubility, extraction rate, diffusion rate, and the reduced surface tension and solvent viscosity [ 26 ]. However, further increment of the ET may degrade flavonoids and phenolics due to destabilization of the compounds by reaction with other plant components or enzymatic and chemical degradation, thus reducing the extraction efficiency [ 27 ]. Page 2

3 Chapter 2 : Antioxidant Capacity and Phenolic Contents of Three Quercus Species Response surface methodology was applied to optimization of the conditions for reflux extraction of Pandan (Pandanus amaryllifolius Roxb.) in order to achieve a high content of total flavonoids (TF), total phenolics (TP), and high antioxidant capacity (AC) in the extracts. Joe Vinson and Christopher J. Bioactive compounds, which are naturally produced in plants, have been concerned with the food and pharmaceutical industries because of the pharmacological effects on humans. In this study, the individual phenolics of six legumes during germination and antioxidant capacity from sprout extracts were determined. It was found that the phenolic content significantly increased during germination in all legumes. A total of 13 phenolic acids were detected and quantified. There were 11 phenolic constituents identified in adzuki beans; 10 in soybeans; 9 in black beans, mung beans, and white cowpeas; and 7 compounds in peanuts. Sinapic acid and cinnamic acid were detected in all six legume sprouts, and their quantities in germinated peanuts were the highest The study reveals that, among the investigated legumes, germinated peanuts and soybeans obtained maximum phenolics and antioxidant capacity. Introduction Consumption of legumes potentially reduces the risk of chronic diseases [ 1 ] such as stroke, type II diabetes [ 2 ], cardiovascular [ 3 ], and gastrointestinal cancer [ 4 ]. In addition to fiber, legume grains also contain many substances to improve health such as vitamins, minerals, and other substances, including phenolic compounds [ 2 ]. Phenolic compounds are resistant to oxidation and protect cell damage to prevent the risk of degenerative diseases thanks to antioxidative, anti-inflammatory, antiallergic and anticarcinogenic activities [ 5, 6 ]. Various studies have proved the dramatic change in beneficial substances that are heavily linked to germination of grains. Total phenolics and antioxidant activities of grains and germinated grains have been widely investigated. Germination produced high phenolic content and consequently increased the antioxidant activities of lupin seeds Lupinus angustifolius L. Zapaton [ 8 ]. Lin and Lai [ 9 ] obtained similar results when they compared the phenolic content and reducing powers of 19 domestic legumes in Taiwan, including soybeans, black soybeans, adzuki beans, and mung beans. However, the identification of individual phenolics and the relationship among phenolic components in germinated legumes are rarely reported. The aim of this study was to investigate the changes in phenolics and antioxidant capacity of six popular legumes during germination to evaluate their potential uses in foods and pharmaceuticals. Total Phenolic Content The change in total phenolic content in legumes during germination is illustrated in Table 1. In ungerminated grains, the content of total phenolics ranged from 5. In particular, peanuts had the highest concentration, which was Identification and Quantification of Phenolic Components Phenolic profiles of legume sprouts are shown in Table 2. Different concentrations of 13 out of 15 standard phenolics Figure 1 ranged from 2. There were 11 compounds found in adzuki beans, 10 in soybeans, 9 in black beans, mung beans and white cowpeas, and 7 in peanuts. Sinapic acid and cinnamic acid were detected in all legume sprouts. Germinated peanuts contained the highest concentrations in the two phenolic acids, which constituted Sinapic acid was also the dominant compound in adzuki bean sprouts with Benzoic acid was the most abundant in germinated mung beans It was found that the six legume extracts had different antioxidant activity levels at a 0. In particular, peanuts had the highest antioxidant activity at The reducing power of germinated peanuts obtained maximum activity Correlation between Phenolics and Antioxidant Activity It was observed that the levels of antioxidant activity depend on both concentrations and types of phenolics, and the correlations between antioxidant activities and phenolics are shown in Table 4, Figure 2 and Figure 3. Ellagic acid, cinnamic acid and ferulic acid showed maximum correlations with reducing power, as the R2 values were 0. Total Phenolic Content Total phenolics are naturally produced during the growth and development of plants to protect themselves from biotic stresses such as diseases, insects and environmental stresses [ 5, 10 ]. The dramatic change in phytochemicals in the germination process has been considered as a natural phenomenon of plants. This research supports the theory that phenolic content positively increases with germination, as shown by the six legumes. The rise in Page 3

4 polyphenol content after germination has been broadly reported in chickpeas [ 11 ], lupin seeds [ 12 ], beans, lentils and peas [ 13 ], oats [ 6 ], and Ceiba pentandra seeds [ 14 ]. Because the metabolism initiates in the presence of water, the particles inside the grains change, and accordingly generate a great amount of energy and new compounds, including phenolics [ 6, 13 ]. During the germination of peanuts, resveratrol, a typical phenolic in peanuts, was highly synthesized [ 15 ]. In addition, flavonoids and epicatechins were other two dominant phenolics in this legume [ 16 ]. In soybeans, the primary phenolics were flavonoids, while those in mung beans, adzuki beans, black beans and white cowpeas were phenolic acids [ 17 ]. Some studies have shown that levels of phenolics depend on extracting solvents. According to Nepote et al. For phenolic acids, to achieve the high levels of extraction, ethanoic acids are solvents that are commonly used [ 17 ]. For these reasons, in this study, the total phenolic content of peanuts was reasonably higher than that of other legumes. Identification of Phenolic Profiles in Germinated Legumes Phenolic compounds consist of phenolic acids, tannins, and flavonoids, which are generally produced in plants, and the abundance of these chemicals depends on species and stage of growth [ 6, 13, 19 ]. Sinapic acid and cinnamic acid are common phenolics found in plants [ 20 ], so these could be the dominant phenolics in all six legumes. In addition, p-hydroxybenzoic acid and chlorogenic acid were also detected in both peanut kernels and skin [ 15 ]. In this study, ferulic acid, sinapic acid and ellagic acid highly contributed to the total phenolic content of peanuts because these phenolic acids had the highest concentrations. Adzuki beans and black beans are dark skin seeds that contain anthocyanidin pigments consisting of delphinidin, cyanidin, pelargonidin, malvidin, and petunidin [ 22 ]. According to Fidrianny et al. The main phenolic compounds in soybeans were isoflavones, which were identified as aglycones daidzein, genistein, glycitein, glucosides daidzin, genistin, glycitin, malonylglucosides, malonyldaidzin, malonylgenistin, malonylglycitin, and acetyl glucosides acetyl daidzin, acetyl genistin, acetyl glycitin [ 9 ]. The Antioxidant Capacity of Germinated Legume Extracts Antioxidant activity is closely related to phenolic content [ 6, 24, 25 ]. In this study, peanuts contained a maximum concentration of phenolics, which may result in the strongest antioxidant activity of the legume. Furthermore, according to Corral-Aguayo et al. In addition, the main substances in soybean phenolics were flavonoids, whereas, in other legumes chickpeas and black, red, and white cowpeas, the major components were phenolic acids [ 17 ]. Correlation between Phenolics and Antioxidant Activities It is believed that phenolic structures play a crucial role in bioactive activities [ 23 ]. Particularly, the number and location of hydroxyl groups in phenolic structures inextricably link to antioxidant activity [ 27 ]. If an additional hydroxyl group is added to ortho or para positions of the benzen ring, the antioxidant activity will significantly increase. Principally, the ortho position can create the intramolecular hydrogen bond, so such a position is proposed to be more effective than a para or a meta one [ 28 ]. Similarly, the antioxidant capacity is higher if there is one or more methoxy groups introduced. Therefore, the inhibiting effectiveness of cinnamic acid derivatives was stronger than that of benzoic acid derivatives [ 29 ]. As a result, sinapic acid was the most correlative with the antioxidant activity of legume phenolics because sinapic acid contains both 3,5-dimethoxyl and 4-hydroxyl groups [ 30 ]. Moreover, biological activities such as anxiolytic, anti-inflammatory and peroxinitrite scavenging were successfully evaluated [ 30 ]. Ellagic acid is highly contained in berries raspberries and strawberries, walnuts, tea, red wine, longan seed, mango, kernel, pomegranate, and herbal plants [ 31, 32 ]. Ellagic acid has positive effects on anti-inflammation, anti-proliferation, anti-angiogenesis, anticarcinogenesis, antimutagenensis, anti-cancer, and antiradical [ 32 ]. Materials and Methods 4. Samples Commercial legume grains, including mung beans Vigna radiata, white cowpeas Vigna unguiculata, black beans Vigna cylindrica, adzuki beans Vigna angularis, soybeans Glycine max and peanuts Arachis hypogaea, were purchased from the Coopmark supermarket, Can Tho city, Vietnam. The supernatant was taken and filtered through the filter membrane with a size of 0. The pellet was extracted twice, and the solutions were mixed together. Methanol was used to dissolve the dried extracts. The absorbance was measured at nm after 30 min using a spectrophotometer DU Beckman Coulter, Germany. The 5-day germinated legumes were taken for evaluating the antioxidant activity and phenolic identification. Evaluation of Antioxidant Activity 4. Reducing Power Page 4

5 Assay For the assay of reducing power, the protocol of Singhal et al. One milliliter of the filtrate was mixed with 2. The reaction was then stopped by adding 2. In the reducing power assay, the more the absorbance of the reaction increased, the more reducing power was obtained. The percentage of the reducing power was calculated based on the following formula: The absorbance was recorded at nm after 30 min. The capacity of radical scavenging was calculated with the following formula: Quantification of Phenolic Components in Germinated Legumes Phenolic compounds found in plants usually exist in conjugated forms with sugars of esters. Therefore, to determine the phenolics in germinated legumes, an acidic fraction of the samples was obtained via the extraction method described in Xuan et al. The two solvents used were absolute methanol A and 0. The peaks of the samples were identified and calculated based on the retention times and peak areas of the phenolic standards. Conclusions In this study, 13 phenolic compounds were detected in germinated grains of black beans, mung beans, soybeans, peanuts, adzuki beans and white cowpeas with high concentrations. Among these identified phenolic acids, sinapic acid, ellagic acid, ferulic acid and cinnamic acid showed high correlations with antioxidant activities. The findings showed that germinated legumes are a promising source of phenolic compounds with beneficial effects for human health. Acknowledgments We are extremely grateful to the Australian Development Awards Alumni Program who provided funding for the purchase of reagents for this study. All authors read and approved the final manuscript. Conflicts of Interest The authors declare no conflict of interest. Abbreviations The following abbreviations are used in this manuscript: Page 5