Bioactive compounds, minerals and antioxidants of edible flowers of peach and apple

ABSTRACT Peach and apple flowers were evaluated for their human nutritional value by obtaining baseline information on their contents of bioactive compounds, minerals and antioxidant capacity. We used simple random sampling and nested statistical analysis to examine flowers of three peach cultivars (“July Flame,” “Cary Mac” and “Fair Time”) and two crab apple cultivars (“Manchurian” and “John Downie”). Flowers of “John Downie” stood out for their high contents of ash, protein and crude fat. Carbohydrate contents were highest for “Fair Time” and “Cary Mac.” Peach and apple differed in their contents of total phenols (73.23 ± 1.94 mg GAE/g peach and 86.65 ± 1.18 mg GAE/g, apple) and total flavonoids (TFl) (5.29 ± 0.10 mg QE/100 g peach and 7.99 ± 0.3 mg QE/100 g apple). The peach cultivar “Fair Time” and the crab apple cultivars “Manchurian” and “John Downie” showed the highest values of K+ and Mg2+. Micronutrient concentrations differed between species, however, Zn2+ and Na+ values were similar among species but differed between cultivars. Antioxidant capacity showed inter-species variations with the methods ABTS and FRAP. The results indicate a good potential for both peach and apple flowers to be incorporated in the human diet, for fresh consumption as functional foods.


Introduction
Since the beginning, most human civilizations and cultures have used plants as their primary food source.They have also used plant-derived products in other ways including in their medicines and cosmetics and, their flowers and foliage in particular, have been used for aesthetic and religious purposes. [1]A plant's various organs -fruits, leaves, stems, roots, seeds and flowers -each contains its own unique profile of nutritional compounds (including sugars, essential oils, carotenoids and vitamins), of bioactive compounds (including flavonoids, phenols and anthocyanins) and of minerals (including N, P, K + , Ca 2+ , Mg 2+ , Fe 2+ , Cu 2+ , Mn 2+ and Zn 2+ ). [2]However, our consumption of foods of low nutritional value but high energy has contributed to an increase in the occurrence of obesity and Likewise, inflorescences were also collected from crab apple trees (5 April) with (cvs."Manchurian crab apple" Malus baccata and "John Downie crab apple" Malus domestica) established in "Campo C," Cuauhtemoc, Chihuahua, Mexico (28°38'0'"N;106°59'53"'W; 2050 masl).Collections were made early in the day between 06:00 and 09:00 h to reducing mechanical damage.
Throughout the experiment, the peach and apple orchards were managed according to standard commercial practice for phytosanitary control (weeds, pests and diseases), mineral nutrition and irrigation scheduling.All inflorescences were quickly transported in paper bags in a container of dry ice to the Chemistry Laboratory of the Faculty of Agrotechnological Sciences (UACH), where they were separated into individual flowers which were then freeze-dried in a BK-F10PT freeze dryer (BIOBASE®, USA).Briefly, 50 g of petals were placed in trays (180 mm x 20 mm) for freeze-drying with a capacity of 0.9 L for 72 h (constant weight) in the equipment previously stabilized at -56°C and 0.1 mBar.Sampling was random with three replicates per peach cv. and five per apple cv.Trees were chosen to be of similar size (peach 40 ± 1 cm diam.And 2.5 m height, apple 30 ± 1 cm diam.And 3 m height).

Proximate composition
To determine moisture, dry matter, crude protein and raw fat we used the method indicated by Tomasik. [15]The extract free nitrogen (EFN), also known as total carbohydrate, represents sugars and starches, and was calculated by difference.The results are expressed as percentages.The total energy of one serving (100 g fresh weight) was calculated according to the equation: Total energy = (energy content of 1 g protein × g protein of sample) + (energy content of 1 g fat × g fat of sample) + energy content of 1 g carbohydrate × g carbohydrate of sample). [16]he titratable acidity was determined according to Tomasik. [15]Briefly, a 0.5 g sample was homogenized with 10 mL of distilled water in an Ultra Turrax T25 homogenizer (IKA®, USA) for 2 min, then filtered and titrated with NaOH (0.1N) in which 2% phenolphthalein was added as indicator.The results are expressed as % citric acid.The total soluble solids (TSS) content was determined by placing a drop of the filtrate on the prism of a portable digital refractometer PAL-1 (ATAGO®, Japan).The results are expressed in °Brix.The color coordinates (L = brilliance, C = chrome and h = hue angle) of the flowers were also determined using a portable SP62 sphere colorimeter (X-Rite®, USA).

Bioactive compounds
The determination of the total anthocyanin (Tan) content was carried out according to the method described by Lee et al. [17] with slight modifications.Briefly, two buffer solutions, pH 1.0 and 4.5, were prepared.Then, 0.1 g of lyophilized tissue was taken, homogenized with 10 mL of water + ethanol (v:v) and centrifuged at 10,000 rpm for 10 min at 4°C.Last, 1 mL of the supernatant was taken to obtain absorbance readings at 520 and 700 nm in the buffer solutions, pH 1.0 and 4.5, respectively.The results are expressed in mg cyanidin-3-glucoside 100 g −1 .The determination of total phenols (TP) was carried out according to the Folin-Ciocalteu method described by Waterman & Mole, [18] for which 0.1 g of tissue was taken and homogenized in 400 µL of distilled water from which an aliquot of 500 µL was taken to form a mixture with 2.5 mL of Folin-Ciocalteu reagent (2N) and 2.0 mL of 20% Na 2 CO 3 (w:v) which was left to stand for 2 h at room temperature (22 ± 1°C) in the dark.After this, the absorbance was determined at 760 nm and a standard curve for gallic acid was constructed for different concentrations (10-100 mg L −1 ).The results are expressed as mg GAE g −1 DW (dry weight).We determined total flavonoids by using the methodology by Arvouet-Grand et al. [19] Briefly, 0.2 g of tissue was homogenized with 1.8 mL of methanol in an Ultra Turrax (IKA®, USA) and centrifuged for 15 min at 10,000 g at 4°C; 2 mL of sample were taken from the supernatant, that reacted with 2 mL of 2% p/v AlCl₃, left to rest for 15 min in the dark and the absorbance was determined at 415 nm.Results are expressed in mg QE 100 g −1 .

Antioxidant capacity
The ABTS assay was carried out as described by Re et al., [20] for which the ABTS solution was prepared by mixing equal volumes of 7 mM of the ABTS reagent [(2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid)] and potassium persulfate (K 2 S 2 O 8 ) at 2.45 mM (w:v), which was left to stand for 116 h at room temperature.Subsequently, 20% ethanol was diluted to obtain an absorbance value between 0.24 and 0.67 at 734 nm and then 10 µL of the sample diluted in ethanol +90 µL of distilled water +3 mL of ABTS applied every 30 s were added and left to react for 15 min.The absorbance at 734 nm was obtained and the results are expressed as µmol TE (Trolox equivalents) g −1 .The FRAP (Ferric Reducing Antioxidant Power Assay) method was used to determine the antioxidant capacity (AC) as described by Benzie et al. [21] Briefly, 0.5 g of tissue was homogenized in 15 mL of deionized water and placed in a flat-bottomed tube.This mixture was then filtered with Whatman® 40 qualitative filter paper (Sigma-Aldrich, USA) and centrifuged at 11,500 rpm for 20 min.Subsequently, 10 µL of the sample +90 µL of distilled water +1900 μL of FRAP reagent were mixed and placed in a Heratherm VCA 230® (Thermo Scientific, USA) oven for 30 min at 37°C.The absorbance value was obtained at 593 nm and the results are expressed as µmol Fe 2+ g −1 .The DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay was carried out according to Brand-Williams et al. [22] with modifications.Briefly, 0.1 g of sample was homogenized in 90 μL of distilled water.Next 3 mL of DPPH reagent was added and the mixture allowed to stand for 15 min at room temperature before obtaining the absorbance value at 517 nm, where 20% (v:v) methanol was used as a blank.The results are expressed as % inhibition of DPPH.

Statistical analyses
Data were analyzed using a nested model with the upper hierarchy factor being the species and the lower hierarchy factor being the cultivar or crab apples.To validate the analysis of variance (ANOVA), Levene's test (homogeneity of variances) and Shapiro-Wilk's test (normality) were applied. [23]The multiple separation of means for peach and apple employed a Tukey's test and a t-test (p ≤ .05),respectively.A Pearson's correlation matrix was also constructed between the nutritional and petal quality variables.In all procedures, SAS version 9.0 (SAS Institute, 2002) software was used.

Results
The values obtained from the proximate analysis are shown in Table 1.Significant differences (p ≤ .05)were found between species and cultivars.The values found for moisture fluctuated between 83.9 ± 0.08 and 88.6 ± 0.03%, where the extreme values, i.e. the lowest or highest values corresponded to apple pollinators.In the case of ash (5.1 ± 0.06 and 6.3 ± 0.03%), protein (16.2 ± 0.01 and 16.9 ± 0.05%) Mean values ± standard deviation (n = 3 and 5, respectively); values with the same letter within each column for peach cultivars and crab apples do not differ significantly (P ≤ .05;Tukey's test and t-test, respectively).ns -not significant; *, ** significant at 5 and 1%, respectively.Data are expressed on a dry weight basis.P-C; peach cultivars; C-A; crab apples; M: Moisture; A: Ash; P: protein; FC: Fat crude; TC: total carbohydrates; TEOS; total energy per 100 g fresh weight portion; JF; "July Flame;" CM; "Cary Mac;" Ftime; "Fair Time;" Mn; "Manchurian;" JD; "John Downie;" C -Cultivars and crude fat (1.7 ± 0.10 and 1.9 ± 0.02%), the highest values were found in the apple pollinator "John Downie."The total carbohydrate content was higher for the peach varieties "Fair Time" and "Cary Mac" with values of 65.7 ± 0.05 and 64.5 ± 0.13%, respectively.In general, peach and apple flowers offer nutritional benefits as fresh foods, due to their significant mineral, protein and carbohydrate contents but low calorific content.Flowers and inflorescences have brightly colored petals that are very attractive (chrome and hue angle) to pollinating organisms, and also to the eye of the human consumer.Both components were significant (p ≤ .05)only for fruit trees, however, chromaticity also showed variation between peach and apple varieties or pollinators, respectively (Table 2).Likewise, peach and apple blossoms did not show statistical variation with respect to TSS (0.61 ± 0.01 and 0.62 ± 0.02 °Brix) and TA (2.97 ± 0.18 and 3.27 ± 0.04% citric acid) values, where the lowest value was observed for "John Downie." The bioactive compounds total anthocyanins (TA), total phenols (TP) and total flavonoids (TFl) showed significant differences between peach and apple flowers, respectively (Table 3), where values were: TP 73.23 ± 1.94 and 86.65 ± 1.18 mg GAE/g and TFl 5.29 ± 0.10 and 7.99 ± 0.3 mg QE/100 g.Also, TFl was significant among the cultivars and crab apples analyzed.

Discussion
Flowers generally have high moisture contents (≥88%), a condition favoring short shelf life.They also support an active microbial population of both fungi and bacteria. [3]Moisture contents of petals of different colors of wild and cultivated dahlia species report values ranging from 88 to 92% [(wild: D. australis, D. appiculata, D. brevis, D. coccinea, D. campanulata and D. pinnata [24] and cultivated: D. x hortorum]. [8]However, lower moisture contents are reported for rose (Rosa x grandiflora, 84.56 ± 0.122%) and sunflower (Helianthus annuus L., 86.45 ± 0.377%) by de Lima-Franzen et al. [25] The range of results is likely associated with morphological variations in size and structure and the content of other components (fibers and minerals).There appear to be no clear standards for edible flower quality, with turgor, aroma and color being much affected by production system, management, harvest timing and postharvest handling. [6,13]hese peach and apple flowers would seem to offer a healthy option as a fresh food as they are a source of carbohydrates and low calorific content.Flowers, including those of peach and apple, are considered a healthy food because of their minimal fat content [7] and high water content.In contrast, "Gala" apple flowers [13] contain carbohydrates ranging from 20.63 ± 0.03 to 24.39 ± 0.01 g 100 g −1 .Similar values are reported for cactus flowers from Hidalgo in Mexico [3] including from Cylindropuntia rosea (67.44 ± 0.45%, 1456.22 ± 1.45 kJ), Opuntia oligacantha, (57.31 ± 0.86%, 1338.97 ± 2.59 kJ), O. matudae (61.20 ± 0.63%, 1356.63 ± 1.54 kJ) and Echinocereus cinerascens (55.83 ± 0.11%, 1341.01 ± 9.06 kJ).Other flowers with similar carbohydrate contents are cultivated dahlia (D. x hortorum) (56.21 to 64.09%), [4] colorin (Erythrina americana) (56.64 ± 1.23%), sabila (Aloe vera) (68.98 ± 1.11%) and garambullo (Myrtillocactus geometrizans) (68.13 ± 0.80%). [7]onsumption of flowers, including peach and apple, also provides some essential and non-essential amino acids, necessary for formation of proteins involved in cell structure and function. [9,13]The protein percentages obtained in our study are similar to those reported in flowers of aloe vera (Aloe vera L.), [7] cultivated dahlias (Dahlia x hortorum) (cherry, yellow, white, purple and red). [6]However, in this same dahlia species, Martínez-Damián et al. [8] report values of 18.54 and 19.25% for pink and purple petals, respectively.Such variation can be related to the degree of domestication, flowering stage, management, extraction method, conservation of samples etc.
Edible flowers can be a complementary source of mineral nutrients, often determined as ash content. [3]The ash content we measure for "John Downie" flowers (6.25 ± 0.11 g 100 g −1 ) is similar to that reported for flowers of "Gala" apple (6.47 ± 0.15 g 100 g −1 ), [13] everlasting flower (Helichrysum italicum) (6.31 ± 0.67 g 100 g −1 ), [26] but higher than that reported for yellow cosmos (Cosmos sulphureus cav.) (0.4 g 100 g −1 ) and Rose "Purple Fragrance" (0.6 g 100 g −1 ). [27]However, none of the flowers so far analyzed exceed the values reported for Japanese pagoda tree (Sophora japonica L.) (10.38 ± 0.19 g 100 g-1) and black locust (Robinia pseudoacacia L.) (21.30 ± 0.93 g 100 g −1 ), [28] these differences relate mainly to the size of the flower or inflorescence, as well as to the drying process, where most reports consider drying with dry air and not by freeze-drying, which significantly reduces the mineral components, including ash. [10]here is increasing interest in flower consumption associated with improved eating habits and lifestyles.Globally, cookbooks, TV cooking programmes and culinary magazines all contribute to raising the standard of food presentation which includes use of the edible flowers of cultivated plants (ornamental, vegetable and medicinal) and wild plants.Flowers are presented fresh, dehydrated, and minimally or substantially processed (candied, preserved) as simple decorations or in dishes such as tamales and atoles. [5,29,30]The colors of peach and apple flowers range from white, through pink, to purple and are a property of their absorption and reflectance of incident (sun)light.While their coloring has primarily to do with their attractiveness to pollinators their subtle shades also explain their aesthetic attraction to us. [31]Interspecific variations in the degrees of flower pigmentation can be discrete or continuous, depending on the synthesis and accumulation in the petal's epidermal cells of pigments that may include flavonoids, anthocyanins, carotenoids and betacyanins. [9,16]In general, their bright colors have a positive visual impact, however, the consumer also associates different colors with different flavors, e.g., red (sweet cherry or strawberry flavors), yellow (citrus or acid flavors) and blue/purple with sugary foods. [32]However, it is well known that flavor originates from specific concentrations of various compounds, including carbohydrates, organic acids, proteins, fatty acids and minerals, where the most abundant compounds are usually the sugars and organic acids. [33] study on "Idared" apple flowers [34] reports the presence of organic acids (mg g −1 ) including citric (4.0), malic (0.05), shikimic (0.0024) and fumaric (0.0008).Meanwhile, Pereira et al. [35] indicate that the organoleptic properties in plant foods (including flowers) are associated with the balance between sugars and organic acids, where the latter contribute to the release of protons and while each of their different anions is responsible for a distinctive flavor. [36]The metabolism and concentrations of these compounds can be affected by complex networks of environmental factors and agronomic managements, [1] however, in the case of leaves and flowers, as they do not function as reserve structures, these contribute to their accelerated degradation, where organic acids are the main respiratory substrate for chemical energy generation. [6]ne of the main factors that has stimulated interest in the consumption of flowers and inflorescences is their content of a range of secondary metabolites such as anthocyanins, phenols and flavonoids.These compounds are characterized by a high biological activity but they are also recognized their involvement in factors such as color, flavor, astringency and nutritional value. [4,11]owever, none of the flowers evaluated in this study are considered in the catalog of novel foods, [37] which is why it is worthwhile generating baseline information on them to assess their nutritional potential as products for fresh consumption.Previous studies on flowers of some apricot cultivars (Prunus armeniaca L.) by Göttingerová et al. [2] report a wide range of values for the content of total phenols (between 404.08 ± 0.28 and 768.45 ± 0.25 mg GAE 100/g) and for total flavonoids (between 198.76 ± 0.06 and 538.11 ± 0.04 mg EC/100 g), however, for total anthocyanins, the values obtained in our study exceeded those of most of the cultivars evaluated by these authors, with the exception of "Blenheim" (13.40 ± 0.1 mg/100 g) and "Vestar" (28.41 ± 0.07 mg/100 g), both with white petals.It has also been reported that the yield of secondary metabolites can be affected by extraction methodresults varying with different solvents and drying methods (freeze-drying vs. air-drying at 25°C) -as observed for flowers of wood violet (Viola odorata L.) [38] and Korean mint (Agastache rugosa). [10]hile it has been shown the purple hue is due to a mixture of phenolic compounds, anthocyanins, flavonoids and carotenoids, the results for total anthocyanins did not show much variation for either species with values ranging between 10.001 and 10.0002 mg/100 g.
Previous studies have demonstrated that edible flowers contain numerous chemicals with intense biological activities or antioxidant capacities, these include organic acids, ascorbic acid, flavonoids, polyphenols, anthocyanins and carotenoids. [44,45]However, in most flowers, AC is defined by the presence of polyphenols, carotenoids and ascorbic acid, [46] compounds that are widely recognized as important in human nutrition. [37]The determination of antioxidant activity in extracts of flowers, fruits or leaves cannot be analyzed by a single, simple assay, due to the different properties of the compounds involved. [47]In our study, ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid, ABTS .+), FRAP (ferric 2,4,6-tripyridyl-s-triazine complex [Fe 3+ -(TPTZ)2] 3+ ) and DPPH (2,2-diphenyl-1-picrylhydrazyl, DPPH.),where DPPH is a free radical, stable, commercially available and the ability to scavenge it constitutes one of the most widely used methods to evaluate the antioxidant properties of fruits and vegetables, because the samples do not require special treatment. [48]Similar results have been reported (DPPH % inhibition) for edible flowers of fruit trees such as pomegranate (Punica granatum L.) (65.0 ± 0.7). [47]However, our study results exceed those for papaya (Carica papaya L.) (50.5 ± 0.3), banana (Musa spp) (44.2 ± 4.6) and mahua (Madhuca longifolia) (81.9 ± 0.3). [47]On the other hand, CA values determined with FRAP (between 64.65 ± 1.8 and 75.03 ± 1.75 µmol Fe 2+ g −1 ) and ABTS (between 126.82 ± 1.7 and 153.21 ± 2. 54 µmol TE (Trolox equivalents)/g) did not exceed those reported with these methods (µmol Fe 2+ g −1 and µmol TE (Trolox equivalents) g −1 , respectively) in edible flowers of fruit tree species such as almond (Amygdalus persica) (311 and 192), apricot (Armeniaca mume) (499 and 220), apple (Malus pumila var.domestica) (184 and 231), pomegranate (Punica granatum) (1275 and 460) and sour orange (Citrus x aurantium) (155 and 126). [49]Oxidation of biological systems involves the transfer of electrons between molecules to a highly reactive oxidizing agent, hence the need to evaluate compounds (antioxidants) that eliminate or minimize this process.Edible flowers have different ways of preventing oxidation, including oxygen scavenging, binding pro-oxidants (primary antioxidants) and phenolic compounds (secondary antioxidants). [2]In general, the presence of AC compounds in flowers helps minimize the processes of senescence and lipid peroxidation at the cell membrane level, which is linked to free radical activity with the consequent loss of ions and changes in osmotic pressure. [10]Edible flowers, including those of peach and apple, are a potentially important source of a large group of non-essential nutrients which include: polyphenols, flavonoids, phenylpropanoids, stilbenoids, benzoic acid derivatives.All these offer benefits for human health due to their high biological and antioxidant activities. [1]ost of the compounds reported in edible flowers are considered secondary metabolites that allow the flower to interact with the environment for its fitness and survival.These serve as signaling agents for pollinating organisms, for seed dispersal and for defense against predators. [37,50]Thus, secondary metabolites and micronutrients participate in redox reactions, electron transfer, structural components of enzymes and play important roles as precursors in the synthesis and accumulation of a great diversity of compounds, including polyphenolic compounds (phenylpropanoids and flavonoids), terpenes or isoprenoids, nitrogenous compounds (alkaloids, cyanogenic glycosides, glycosides and glucosinolates). [51]

Conclusion
Our baseline analysis allows some understanding of the different nutritional values of peach and apple flowers.The crab apple "John Downie" showed the highest values of ash, protein and crude fat.Meanwhile, carbohydrate contents were higher in the peach cultivars "Fair Time" and "Cary Mac."On the other hand, differences between species were found for total phenols and total flavonoids, however, only flavonoids were significant between cultivars and crab apples.Among the macronutrients, statistical variation between species was found for K + and Mg 2+ with "Fair Time," "Manchurian" and "John Downie" standing out.The concentration of Fe 2+ , Cu 2+ , Mn 2+ , Zn 2+ and Na + was different between species, however, the values of Zn 2+ and Na + were similar between varieties.Likewise, with the exception of Fe 2+ , apple flowers were characterized by high concentrations of Cu 2+ , Mn 2+ , Zn 2+ and Na + .Finally, the antioxidant capacity only showed inter-species variation with methods ABTS and FRAP.These findings allow us to elucidate peach and apple flowers as novel ingredients that can be considered as functional foods for fresh consumption.However, further research is needed for the flowers of these fruit trees, as none of them are considered in the catalog of novel foods.

Table 1 .
Nutritional value of edible flowers of peach and apple.

Table 2 .
Colour, total soluble solids and titratable acidity in edible flowers of some peach varieties and apple pollinators.

Table 3 .
Bioactive compounds in edible flowers of some peach varieties and apple pollinators.
Mean values ± standard deviation (n = 3 and 5, respectively); values with the same letter within each column for peach varieties and apple pollinators do not differ statistically (P ≤ .05;Tukey's test and t-test, respectively).ns -not significant; *, ** significant at 5 and 1%, respectively.Data are expressed on a dry weight basis.TA: total anthocyanins; TP: total phenols; TFl: total flavonoids; C-A; Crab apples; C; Cultivars

Table 4 .
Mineral composition in edible flowers of peach and apple.
Mean values ± standard deviation (n = 3 and 5, respectively); values with the same letter within each column for peach varieties and apple pollinators do not differ statistically (P ≤ .05;Tukey's test and t-test, respectively).ns -not significant; *, ** significant at 5% and 1%, respectively.Data are expressed on a dry weight basis.C-A; Crab apples; C; cultivars

Table 5 .
Antioxidant capacity in edible flowers of some peach varieties and apple pollinators.

Table 6 .
Pearson's correlation coefficient for mineral composition, total soluble solids, titratable acidity, bioactive compounds and antioxidant capacity in flowers of some peach cultivars and crab apples.