# Castanea sativa Mill. Flowers amongst the Most Powerful Antioxidant Matrices: A Phytochemical Approach in Decoctions and Infusions

Authors: Márcio Carocho; Lillian Barros; Albino Bento; Celestino Santos-Buelga; Patricia Morales; Isabel C. F. R. Ferreira
Journal: BioMed Research International
 (2014)

---

## Abstract

Infusions and decoction of chestnut tree flowers have been used for different medical purposes, but their phytochemical profile and antioxidant activity are still mostly unknown. Herein, decoctions and infusions of flowers from the two most appreciated chestnut cultivars (longal and judia) in Trás-os-Montes, Portugal, were prepared and characterized with regard to their content in free sugars, organic acids, and phenolic compounds, such as flavonoids and hydrolyzable tannins, and their antioxidant activity. Overall, the decoction of the cultivar judia was the sample with both the highest quantity of flavonoids and antioxidant activity. The phenolic compound with the highest abundance in all samples was trigalloyl-HHDP-glucoside, followed by pentagalloyl glucoside. The sample with the highest quantity of total phenolic compounds was judia infusion, closely followed by longal decoction, which also gave the highest quantities of ellagitannins. Regarding sugars and organic acids, the profiles were more similar. These results corroborate ancestral claims of the health benefits of infusions and decoctions of chestnut flowers.


---

## Body

## 1. Introduction

In the Trás-os-Montes region of Portugal and across a good part of the Mediterranean countries, chestnut trees are a considerable part of the landscape. These trees and their respective nuts have been important in the past and are still a source of income for those regions. In Portugal, chestnuts are almost totally exported, translating into a revenue of 32 million euros in 2012 [1, 2]. The chestnut tree has a variety of applications; the nuts are used for human and animal feed, being widely appreciated and even transformed into many typical dishes and desserts. The wood is used for high class furniture. The leaves are used in many ethnobotanic formulations against colds, coughs, diarrhea, and even high blood cholesterol [3]. Furthermore, some patents indicate the use of chestnut flowers in beverages like teas and refreshments [4–6].The nutritional and bioactive properties of the fruits and flowers of chestnuts have been reported [7–9]. Barros et al. [8, 9] described the high antioxidant potential and phenolic compounds profile of methanolic extracts obtained from the flowers. In fact, some of the most antioxidant molecules are not always found in fruits, but in richer polyphenolic matrices like flowers. Other antioxidant molecules include some organic acids that among many other beneficial activities are known to act against free radicals [8, 10].The characterization of antioxidant molecules present in flowers is fundamental to draw conclusions concerning their antioxidant potential, which could be interesting for the food industry, by adding antioxidant extracts to foodstuffs or using them in coatings, in order to extend their shelf-life and reduce consumption of chemical additives [11].In this report, the decoctions and infusions of flowers from the two most appreciated chestnut cultivars (longal andjudia) from Trás-os-Montes, Portugal, were characterized regarding their content of hydrophilic antioxidant molecules (free sugars, organic acids, and phenolic compounds), reducing power, free radicals scavenging activity, and lipid peroxidation inhibition.


## 2. Materials and Methods

### 2.1. Standards and Reagents

Acetonitrile 99.9%, of high performance liquid chromatography (HPLC) grade, and sulphuric acid were acquired from Fisher Scientific (Lisbon, Portugal). Formic acid was acquired from Panreac (Barcelona, Spain). Sugar standards [D(−)-fructose, D(+)-glucose anhydrous and D(+)-sucrose], organic acid standards (malic acid, shikinic acid; oxalic acid and quinic acid), and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were acquired from Sigma Chemical Co. (Saint Louis, MO, USA). Phenolic compound standards (catechin, gallic acid, isorhamnetin 3-O-glucoside, kaempferol 3-O-glucoside, kaempferol 3-O-rutinoside, myricetin, quercetin 3-O-glucoside and quercetin 3-O-rutinoside) were purchased from Extrasynthese (Genay, France). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). Water was treated by means of a Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).


### 2.2. Flower Samples

Castanea sativaMill. flowers of the cultivarsjudia andlongal were collected in June 2013 in Oleiros, Bragança (north-eastern Portugal) (41°51′02′′N, 6°49′54′′W). The specimens were lyophilized (FreeZone 4.5, Labconco, Kansas, USA), milled down to a fine powder, and finally stored at −5°C until analysis.


### 2.3. Preparation of the Decoctions and Infusions

For the infusions preparation, the lyophilized flowers (1 g) were added to 200 mL of boiling distilled water, left to stand for 5 min, and finally filtered through a Whatman filter paper. The obtained infusions were frozen and lyophilized.For the decoctions preparation, the lyophilized flowers (1 g) were added to 200 mL of boiling distilled water, boiled for 5 min, and then left to stand at room temperature for 5 more minutes. After filtration through a Whatman filter paper, the obtained decoctions were frozen and lyophilized.


### 2.4. Analysis of Free Sugars

Free sugars were determined by HPLC coupled to a refraction index (RI) detector as described previously [8]. The equipment consisted of a pump (Knauer, Smartline System 1000, Berlin, Germany), a degasser (Smartline Manager 5000), an autosampler (AS-2057 Jasco, Easton, MD, USA), and a RI detector (Knauer Smartline 2300). The identification was achieved by comparing the relative retention times of sample peaks with standards. Quantification was made by the internal standard method, and the results are expressed in mg per g of lyophilized decoction or infusion.


### 2.5.  Analysis of Organic Acids

Organic acids were determined following a procedure previously optimized and described by the authors [10]. Analysis was performed on a Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu Cooperation, Kyoto, Japan) coupled to photodiode array detector (PDA, Shimadzu), using 215 nm and 245 nm as the preferred wavelengths. Separation was achieved on a SphereClone (Phenomenex, Torrance, CA, USA) reverse phase C18 column (5 μm, 250 mm × 4.6 mm i.d) thermostatted at 35°C. Analytes were eluted with 3.6 mM sulphuric acid at a flow-rate of 0.8 mL/min. The organic acids found were quantified by comparison of the area of their peaks recorded at 215 nm or 245 nm (for ascorbic acid) with calibration curves obtained from commercial standards of each compound: oxalic acid (
y
=
1
x
107
x
+
96178; R
2
=
0.999
); quinic acid (
y
=
601768
x
+
8853.2; R
2
=
1
); malic acid (
y
=
952269
x
+
17803; R
2
=
1
); shikimic acid (
y
=
8
x
107
+
55079; R
2
=
0.999
). The results were expressed in mg per g of lyophilized decoction or infusion.


### 2.6. Analysis of Phenolic Compounds

Phenolic compounds were determined by HPLC (Hewlett-Packard 1100, Agilent Technologies, Santa Clara, CA, USA) as previously described [9]. Double online detection was carried out with a PDA using 280 nm and 370 nm as the preferred wavelengths and a mass spectrometer (MS) connected to the HPLC system via the PDA cell outlet. Mass spectrometric detection was performed by means of an API 3200 (Applied Biosystems, Darmstadt, Germany) triple quadrupole-ion trap analyser equipped with an ESI source. Spectra were recorded in negative ion mode betweenm/z 100 and 1700. The phenolic compounds were characterized according to their UV, mass spectra, retention times, and comparison with authentic standards when available. For the quantitative analysis of phenolic compounds, a baseline to valley integration with baseline projection mode was used to calculate peak areas. For quantification, calibration curves were generated by injection of known concentration (2.5–100 μg/mL) of standard compounds: catechin (y
=
132.76
x
-
59.658; R
2
=
1
); gallic acid (
y
=
556.94
x
-
738.37; R
2
=
0.999
); isorhamnetin-3-O-glucoside (
y
=
262.31
x
-
9.8958; R
2
=
1
); kaempferol-3-O-glucoside (
y
=
190.75
x
-
36.158; R
2
=
1
); kaempferol-3-O-rutinoside (
y
=
175.02
x
-
43.877; R
2
=
0.999
); myricetin (
y
=
778
x
-
1454.3; R
2
=
0.999
); quercetin-3-O-glucoside (
y
=
316.48
x
-
2.9142; R
2
=
1.000
); and quercetin-3-O-rutinoside (
y
=
222.79
x
-
243.11; R
2
=
0.999
). The results were expressed in mg per g of lyophilized decoction or infusion.


### 2.7. Evaluation of Antioxidant Activity

Thein vitro antioxidant activity assays were performed following the previously described methodology of Barros et al. [8]. The lyophilized infusions and decoctions were dissolved in water (final concentration 10 mg/mL); the final solution was further diluted to different concentrations to be used in the following assays. DPPH radical-scavenging activity was evaluated using an ELX800 microplate Reader (Bio-Tek Instruments, Inc.; Winooski, VT, USA) and calculated as a percentage of DPPH discolouration after 1 hour of incubation with the antioxidant extract, using the formula: [(
A
DPPH
-
A
S
)
/
A
DPPH]  × 100, where A
S is the absorbance of the solution containing the sample at 515 nm, and A
DPPH is the absorbance of the DPPH solution. Reducing power was evaluated by the capacity to reduce Fe3+ into Fe2+, measuring the absorbance at 690 nm in the microplate reader mentioned. Inhibition of β-carotene bleaching was evaluated through the β-carotene/linoleate assay; the neutralization of linoleate free radicals avoids β-carotene bleaching, which is measured by the formula: (β-carotene absorbance after 2 h of assay/initial absorbance) × 100. Lipid peroxidation inhibition in porcine (Sus scrofa) brain homogenates was evaluated by the decrease in thiobarbituric acid reactive substances (TBARS); the colour intensity of the malondialdehyde-thiobarbituric acid (MDA-TBA) was measured by its absorbance at 532 nm; the inhibition ratio (%) was calculated using the following formula: [
(
A
-
B
)
/
A
] × 100%, where A and B were the absorbance of the control and the sample solution, respectively. The results of the antioxidant activity were expressed in EC50 value (sample concentration providing 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay).


### 2.8. Statistical Analysis

All the assays were carried out in triplicate for both decoctions and infusions of each cultivar. The results are expressed as mean values ± standard deviations (SD). The statistical differences represented by letters were obtained through one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test withα
=
0.05. Statistical analyses were carried out using the SPSS v. 18.0 program.


## 2.1. Standards and Reagents

Acetonitrile 99.9%, of high performance liquid chromatography (HPLC) grade, and sulphuric acid were acquired from Fisher Scientific (Lisbon, Portugal). Formic acid was acquired from Panreac (Barcelona, Spain). Sugar standards [D(−)-fructose, D(+)-glucose anhydrous and D(+)-sucrose], organic acid standards (malic acid, shikinic acid; oxalic acid and quinic acid), and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were acquired from Sigma Chemical Co. (Saint Louis, MO, USA). Phenolic compound standards (catechin, gallic acid, isorhamnetin 3-O-glucoside, kaempferol 3-O-glucoside, kaempferol 3-O-rutinoside, myricetin, quercetin 3-O-glucoside and quercetin 3-O-rutinoside) were purchased from Extrasynthese (Genay, France). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). Water was treated by means of a Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).


## 2.2. Flower Samples

Castanea sativaMill. flowers of the cultivarsjudia andlongal were collected in June 2013 in Oleiros, Bragança (north-eastern Portugal) (41°51′02′′N, 6°49′54′′W). The specimens were lyophilized (FreeZone 4.5, Labconco, Kansas, USA), milled down to a fine powder, and finally stored at −5°C until analysis.


## 2.3. Preparation of the Decoctions and Infusions

For the infusions preparation, the lyophilized flowers (1 g) were added to 200 mL of boiling distilled water, left to stand for 5 min, and finally filtered through a Whatman filter paper. The obtained infusions were frozen and lyophilized.For the decoctions preparation, the lyophilized flowers (1 g) were added to 200 mL of boiling distilled water, boiled for 5 min, and then left to stand at room temperature for 5 more minutes. After filtration through a Whatman filter paper, the obtained decoctions were frozen and lyophilized.


## 2.4. Analysis of Free Sugars

Free sugars were determined by HPLC coupled to a refraction index (RI) detector as described previously [8]. The equipment consisted of a pump (Knauer, Smartline System 1000, Berlin, Germany), a degasser (Smartline Manager 5000), an autosampler (AS-2057 Jasco, Easton, MD, USA), and a RI detector (Knauer Smartline 2300). The identification was achieved by comparing the relative retention times of sample peaks with standards. Quantification was made by the internal standard method, and the results are expressed in mg per g of lyophilized decoction or infusion.


## 2.5.  Analysis of Organic Acids

Organic acids were determined following a procedure previously optimized and described by the authors [10]. Analysis was performed on a Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu Cooperation, Kyoto, Japan) coupled to photodiode array detector (PDA, Shimadzu), using 215 nm and 245 nm as the preferred wavelengths. Separation was achieved on a SphereClone (Phenomenex, Torrance, CA, USA) reverse phase C18 column (5 μm, 250 mm × 4.6 mm i.d) thermostatted at 35°C. Analytes were eluted with 3.6 mM sulphuric acid at a flow-rate of 0.8 mL/min. The organic acids found were quantified by comparison of the area of their peaks recorded at 215 nm or 245 nm (for ascorbic acid) with calibration curves obtained from commercial standards of each compound: oxalic acid (
y
=
1
x
107
x
+
96178; R
2
=
0.999
); quinic acid (
y
=
601768
x
+
8853.2; R
2
=
1
); malic acid (
y
=
952269
x
+
17803; R
2
=
1
); shikimic acid (
y
=
8
x
107
+
55079; R
2
=
0.999
). The results were expressed in mg per g of lyophilized decoction or infusion.


## 2.6. Analysis of Phenolic Compounds

Phenolic compounds were determined by HPLC (Hewlett-Packard 1100, Agilent Technologies, Santa Clara, CA, USA) as previously described [9]. Double online detection was carried out with a PDA using 280 nm and 370 nm as the preferred wavelengths and a mass spectrometer (MS) connected to the HPLC system via the PDA cell outlet. Mass spectrometric detection was performed by means of an API 3200 (Applied Biosystems, Darmstadt, Germany) triple quadrupole-ion trap analyser equipped with an ESI source. Spectra were recorded in negative ion mode betweenm/z 100 and 1700. The phenolic compounds were characterized according to their UV, mass spectra, retention times, and comparison with authentic standards when available. For the quantitative analysis of phenolic compounds, a baseline to valley integration with baseline projection mode was used to calculate peak areas. For quantification, calibration curves were generated by injection of known concentration (2.5–100 μg/mL) of standard compounds: catechin (y
=
132.76
x
-
59.658; R
2
=
1
); gallic acid (
y
=
556.94
x
-
738.37; R
2
=
0.999
); isorhamnetin-3-O-glucoside (
y
=
262.31
x
-
9.8958; R
2
=
1
); kaempferol-3-O-glucoside (
y
=
190.75
x
-
36.158; R
2
=
1
); kaempferol-3-O-rutinoside (
y
=
175.02
x
-
43.877; R
2
=
0.999
); myricetin (
y
=
778
x
-
1454.3; R
2
=
0.999
); quercetin-3-O-glucoside (
y
=
316.48
x
-
2.9142; R
2
=
1.000
); and quercetin-3-O-rutinoside (
y
=
222.79
x
-
243.11; R
2
=
0.999
). The results were expressed in mg per g of lyophilized decoction or infusion.


## 2.7. Evaluation of Antioxidant Activity

Thein vitro antioxidant activity assays were performed following the previously described methodology of Barros et al. [8]. The lyophilized infusions and decoctions were dissolved in water (final concentration 10 mg/mL); the final solution was further diluted to different concentrations to be used in the following assays. DPPH radical-scavenging activity was evaluated using an ELX800 microplate Reader (Bio-Tek Instruments, Inc.; Winooski, VT, USA) and calculated as a percentage of DPPH discolouration after 1 hour of incubation with the antioxidant extract, using the formula: [(
A
DPPH
-
A
S
)
/
A
DPPH]  × 100, where A
S is the absorbance of the solution containing the sample at 515 nm, and A
DPPH is the absorbance of the DPPH solution. Reducing power was evaluated by the capacity to reduce Fe3+ into Fe2+, measuring the absorbance at 690 nm in the microplate reader mentioned. Inhibition of β-carotene bleaching was evaluated through the β-carotene/linoleate assay; the neutralization of linoleate free radicals avoids β-carotene bleaching, which is measured by the formula: (β-carotene absorbance after 2 h of assay/initial absorbance) × 100. Lipid peroxidation inhibition in porcine (Sus scrofa) brain homogenates was evaluated by the decrease in thiobarbituric acid reactive substances (TBARS); the colour intensity of the malondialdehyde-thiobarbituric acid (MDA-TBA) was measured by its absorbance at 532 nm; the inhibition ratio (%) was calculated using the following formula: [
(
A
-
B
)
/
A
] × 100%, where A and B were the absorbance of the control and the sample solution, respectively. The results of the antioxidant activity were expressed in EC50 value (sample concentration providing 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay).


## 2.8. Statistical Analysis

All the assays were carried out in triplicate for both decoctions and infusions of each cultivar. The results are expressed as mean values ± standard deviations (SD). The statistical differences represented by letters were obtained through one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test withα
=
0.05. Statistical analyses were carried out using the SPSS v. 18.0 program.


## 3. Results and Discussion

### 3.1. Analysis of Free Sugars and Organic Acids

Three free sugars, namely, fructose, glucose, and sucrose, were detected in all the samples (Table1). Sucrose was the least abundant sugar, while fructose and glucose were the most abundant in the cultivarsjudia andlongal, respectively. This had been also reported for methanolic extracts ofC. sativa flowers [9]. Glucose and fructose were present at higher levels in decoctions than infusions, probably due to the longer extraction time, albeit significant differences were only found in the case of the cultivarlongal. By contrast, higher concentrations of sucrose were extracted in the infusions, although the increase was only statistically significant in the case ofjudia.No significant differences were observed among samples.Table 1
Content of free sugars and organic acids in mg/g of lyophilized decoction/infusion of the flowers of twoCastanea sativa cultivars.
Decoctionjudia
Decoctionlongal
Infusionjudia
Infusionlongal
Sugars
Fructose
160.41 ± 0.01a
152.08 ± 6.52a
148.94 ± 4.60a
123.58 ± 1.76b
Glucose
149.09 ± 0.04b
191.91 ± 7.35a
145.71 ± 5.63b
164.07 ± 2.31b
Sucrose
27.01 ± 0.45b
25.69 ± 0.99b
35.68 ± 1.45a
26.67 ± 0.50b
Total
336.51 ± 0.48a
369.69 ± 14.85a
330.32 ± 11.69a
314.32 ± 4.57a
Organic Acids
Oxalic acid
72.91 ± 1.82a
71.85 ± 0.52ab
43.42 ± 0.75c
55.84 ± 1.82bc
Quinic acid
84.61 ± 0.64a
52.59 ± 3.14b
69.04 ± 2.81ab
61.63 ± 4.40b
Malic acid
27.17 ± 1.36a
22.94 ± 0.10a
25.03 ± 0.52a
20.82 ± 0.70a
Shikimic acid
1.83 ± 0.02a
1.26 ± 0.08a
1.35 ± 0.02a
1.35 ± 0.07a
Total
186.52 ± 3.84a
148.65 ± 2.79ab
138.83 ± 2.61b
139.64 ± 6.99b
In each row different letters mean significant differences with aP
<
0.05. Letters refer to Tukey’s post hoc test; therefore, significant different values were classified using letters in alphabetic order.Oxalic, quinic, malic, and shikimic acids were quantified in all the samples, with quinic acid being the most abundant acid (Table1). Decoctions generally yielded higher quantities of oxalic acid in comparison to infusions, and statistical differences for this compound were only noted for the infusion oflongal. Thelongal cultivar did not display statistical differences for quinic acid in both extraction methods, but forjudia statistical differences were found between extraction methods. Malic acid and shikimic acid displayed similar concentrations in all the samples, whereas some differences were found among samples regarding quinic acid. The decoction ofjudia displayed the highest concentrations of total organic acids of the samples analysed.


### 3.2. Analysis of Phenolic Compounds

An exemplary phenolic profile of thejudia cultivar ofC. sativa recorded at 280 nm and 370 nm is shown in Figure 1. Twenty-seven phenolic compounds were identified in bothjudia andlongal cultivars (infusion and decoction preparations). Peak characteristics and tentative identities are presented in Table 2.Table 2
Retention time (Rt), wavelength of maximum absorption (λ
max
⁡), mass spectral data, and tentative identification of phenolic compounds in decoctions and infusions of Castanea sativa flowers.
Peak
Rt (min)
λ
max
⁡ (nm)
Pseudomolecular ion [M-H]− (m/z)
MS2 (m/z) (% of base peak)
Tentative identification
1
4.96
276
783
481 (10), 301 (41)
Pedunculagin isomer (bis-HHDP-glucose)
2
5.79
268
783
481 (6), 301 (45)
Pedunculagin isomer (bis-HHDP-glucose)
3
7.06
272
633
463 (17), 301 (100)
Galloyl-HHDP-glucose
4
7.67
274
937
637 (15), 467 (2), 301 (4)
Trigalloyl-HHDP-glucose
5
8.02
278
289
245 (91), 203 (60), 137 (38)
(+)-Catechin
6
13.77
276
939
631 (31), 469 (66), 169 (100)
Pentagalloyl glucose
7
14.92
276
935
633 (15), 301 (18)
Galloyl-bis-HHDP-glucose
8
15.25
274
933
915 (5), 633 (8), 451 (24), 301 (7)
Castalagin/vescalagin
9
15.46
278
907
767 (3), 607 (24), 467 (35), 169 (5)
Galloyl-HHDP derivative
10
16.13
274
937
767 (2), 637 (8), 467 (68), 301 (10)
Trigalloyl-HHDP-glucoside
11
16.65
358
493
317 (100)
MyricetinO-glucuronide
12
17.07
350
479
317 (100)
Myricetin 3-O-glucoside
13
17.30
274
937
767 (2), 637 (5), 467 (58), 301 (7)
Trigalloyl-HHDP-glucoside
14
19.89
356
609
301 (100)
Quecetin 3-O-rutinoside
15
20.64
356
477
301 (100)
Quercetin 3-O-glucuronide
16
20.86
356
463
301 (100)
Quercetin 3-O-glucoside
17
21.30
356
463
301 (100)
QuercetinO-hexoside
18
23.54
350
593
285 (100)
Kaempferol 3-O-rutinoside
19
24.05
354
433
301 (100)
QuercetinO-pentoside
20
24.46
268
907
767 (3), 607 (23), 467 (67), 169 (7)
Galloyl-HHDP derivative
21
25.11
348
477
285 (100)
Kaempferol 3-O-glucoside
22
26.00
354
477
315 (100)
IsorhamnetinO-hexoside
23
26.25
354
491
315 (100)
IsorhamnetinO-glucuronide
24
27.71
274
907
767 (2), 607 (27), 467 (76), 169 (8)
Galloyl-HHDP derivative
25
29.87
250/368
343
328 (97), 313 (100), 298 (36)
Tri-O-methylellagic acid
26
32.38
358
609
463 (76), 301 (40)
QuercetinO-rhamnosyl hexoside
27
33.13
356
519
477 (5), 315 (77)
IsorhamnetinO-acetylhexosidePhenolic profile of the infusion ofCastanea sativa flowers (judia cultivar) recorded at 370 nm (a) and 280 nm (b).
(a)
(b)Peaks 1–4, 6–10, 13, 20, 24, and 25 showed UV spectra coherent with galloyl and hexahydroxydiphenoyl (HHDP) derivatives [12–14]. According to the literature, the main characteristic in the mass spectra of these compounds is the deprotonated molecule [M-H]− and the loss of one or more ellagic acid (302 mu), gallic acid (170 mu), and/or galloyl groups (152 mu) [12, 15, 16]. Peaks 1 and 2 presented a singly charged pseudomolecular ion [M-H]− atm/z 783 and daughter ions atm/z 481 and 301, which together with their early elution allowed their identification as pedunculagin (i.e., bis-HHDP-glucose) isomers [17]. Mass characteristics of peak 3 ([M-H]− atm/z 633; fragment ions atm/z 463 and 301) coincided with a galloyl-HHDP-glucose isomer [16, 17], whereas peaks 4, 10, and 13 ([M-H]− atm/z 937; fragment ions atm/z 767, 637, 467 and 301) were coherent with trigalloyl-HHDP-glucose isomers [13], already reported inC. sativa heartwood [18] and flowers [9]. Peak 6 presented a pseudomolecular ion [M-H]− atm/z 939, yielding MS2 fragment ions atm/z 631 [M-2galloyl-3H]−, 469 [M-2galloyl-3H-glu]−, being identified as pentagalloylglucose. Peak 7 with a pseudomolecular ion [M-H]− atm/z 935 and MS2 product ions atm/z 633 and 301, likely due to the loss of HHDP and galloyl-glucose moieties, was consistent with a galloyl-bis-HHDP-glucose isomer [15, 17, 19]. Pseudomolecular ion ([M-H]− atm/z 933) and fragmentation pattern (ions atm/z 915, 631, 451 and 301) of peak 8 were in agreement with those attributed to castalagin or vescalagin isomers [17, 20], already reported inC. sativa heartwood [18]. Peaks 9, 20, and 24 are most likely galloyl-HHDP derivatives, with an unusual parent ion [M-H]− atm/z 907, but with characteristic fragments of this type of compounds (m/z at 767, 467 and 169). No structure could be assigned to these peaks that remain unidentified. A fragment ion withm/z at 907 was reported as released from the cleavage of a di(HHDP-galloylglucose)-pentose found in pomegranate juice [21] and from unknown ellagitannins present in blackberries [17]. Peak 25 presented an ellagic acid-like UV spectrum (λ
max
⁡ around 250 and 368 nm) and a pseudo molecular ion [M-H]− atm/z 343, releasing three fragments atm/z 328, 313 and 298 mu, corresponding to the successive losses of three methyl groups (−15 mu), which allowed its tentative identification as a tri-O-methylellagic acid.Regarding flavonoids, flavonol derivatives were the main compounds found in the analyzed samples (Table2). Catechin (peak 5), myricetin 3-O-glucoside (peak 12), quercetin 3-O-rutinoside (peak 14), quercetin 3-O-glucoside (peak 16), kaempferol 3-O-rutinoside (peak 18), and kaempferol 3-O-glucoside (peak 21) were positively identified according to their retention, mass, and UV-vis characteristics by comparison with commercial standards. Peak 11 was assigned to a myricetinO-glucuronide, according to the pseudomolecular ion [M-H]− atm/z 497 and MS2 fragment released atm/z 317 ([M-H-176]−, loss of glucuronyl moiety). Peaks 15, 17, 19, and 26 presented UV spectra with λ
max
⁡ around 350 nm and an MS2 product ion atm/z 301 indicating that they correspond to quercetin derivatives. According to their pseudomolecular ions, they were identified as quercetin 3-O-glucuronide (peak 15; [M-H]− atm/z 477), which was confirmed by comparison with a standard obtained in our laboratory [22], quercetinO-hexoside (peak 17; [M-H]− atm/z 463), quercetinO-pentoside (peak 19; [M-H]− atm/z 433), and quercetinO-rhamnosyl hexoside (peak 26; [M-H]− atm/z 609). Similarly reasoning also allowed assigning peaks 22 and 23 as isorhamnetinO-hexoside and isorhamnetinO-glucuronide, respectively. Peak 27 should correspond to isorhamnetinO-acetylhexoside according to the pseudomolecular ion [M-H]− atm/z 519 and MS2 fragment released atm/z 315 ([M-H-42-162]−, loss of an acetylhexoside moiety). The individual polyphenol with the highest concentration in all samples was a trigalloyl-HHDP-glucoside (peak 10), followed by pentagalloyl glucose (peak 6), whereas quercetin 3-O-glucuronide (peak 15) and a quercetin hexoside (peak 17) were the most abundant flavonoids injudia andlongal cultivars, respectively (Table 3). The sample with the highest concentration of total polyphenols was the infusion ofjudia, closely followed by the decoction oflongal. The preparations of the cultivarjudiapresented higher flavonoid levels, while those oflongal displayed higher concentrations of hydrolyzable tannins (Table 3). The compounds present in the samples were, to some extent, different from those found in aC. sativa hydromethanolic extract [9]. Nevertheless, the main ellagitannins found in this study were in accordance with the mentioned study.Table 3
Quantification of phenolic compounds in infusions and decoctions ofCastanea sativa flowers expressed in mg/g of lyophilized decoction/infusion.
Compounds
Decoctionjudia
Decoctionlongal
Infusionjudia
Infusionlongal
Pedunculagin isomer (bis-HHDP-glucose)
5.21 ± 0.11
8.48 ± 0.51
5.98 ± 0.16
7.68 ± 0.11
Pedunculagin isomer (bis-HHDP-glucose)
1.12 ± 0.09
1.44 ± 0.19
0.92 ± 0.92
1.21 ± 0.04
Galloyl-HHDP-glucose
3.07 ± 0.14
3.18 ± 0.00
3.18 ± 0.07
3.05 ± 0.09
Trigalloyl-HHDP-glucose
2.81 ± 0.25
5.51 ± 0.06
2.60 ± 0.06
4.30 ± 0.47
(+)-Catechin
1.14 ± 0.12
1.58 ± 0.14
1.11 ± 0.03
1.10 ± 0.05
Pentagalloyl glucose
5.61 ± 0.08
6.04 ± 0.15
5.73 ± 0.14
6.47 ± 0.15
Galloyl-bis-HHDP-glucose
0.45 ± 0.04
0.29 ± 0.07
0.66 ± 0.01
0.30 ± 0.01
Castalagin/vescalagin
0.75 ± 0.06
0.73 ± 0.11
1.06 ± 0.09
1.61 ± 0.04
Galloyl-HHDP derivative
0.38 ± 0.02
0.55 ± 0.07
3.60 ± 0.16
3.72 ± 0.01
Trigalloyl-HHDP-glucoside
26.72 ± 0.15
28.16 ± 0.11
30.70 ± 0.40
28.73 ± 1.34
MyricetinO-glucuronide
1.13 ± 0.02
0.09 ± 0.00
0.96 ± 0.01
0.08 ± 0.00
Myricetin 3-O-glucoside
0.66 ± 0.01
0.09 ± 0.01
0.60 ± 0.01
0.09 ± 0.00
Trigalloyl-HHDP-glucoside
1.33 ± 0.00
0.54 ± 0.01
1.61 ± 0.05
0.70 ± 0.04
Quecetin 3-O-rutinoside
1.57 ± 0.01
0.52 ± 0.03
1.31 ± 0.10
0.48 ± 0.05
Quercetin 3-O-glucuronide
3.55 ± 0.10
1.56 ± 0.04
3.38 ± 0.01
1.49 ± 0.07
Quercetin 3-O-glucoside*
1.80 ± 0.01
0.89 ± 0.02
1.60 ± 0.04
0.91 ± 0.10
QuercetinO-hexoside
2.21 ± 0.07
1.82 ± 0.05
2.11 ± 0.02
1.85 ± 0.06
Kaempferol 3-O-rutinoside
0.21 ± 0.01
0.21 ± 0.03
0.18 ± 0.01
0.21 ± 0.01
QuercetinO-pentoside
0.43 ± 0.02
0.20 ± 0.01
0.34 ± 0.00
0.22 ± 0.02
Galloyl-HHDP derivative
4.18 ± 0.10
4.54 ± 0.14
2.41 ± 0.14
1.96 ± 0.12
Kaempferol 3-O-glucoside
0.53 ± 0.00
0.49 ± 0.01
0.41 ± 0.00
0.46 ± 0.04
IsorhamnetinO-hexoside
0.33 ± 0.04
0.27 ± 0.01
0.27 ± 0.00
0.30 ± 0.01
IsorhamnetinO-glucuronide
0.35 ± 0.03
0.50 ± 0.02
0.33 ± 0.01
0.46 ± 0.04
Galloyl-HHDP derivative
2.04 ± 0.06
2.02 ± 0.03
0.72 ± 0.01
0.59 ± 0.01
Tri-O-methylellagic acid
0.10 ± 0.01
tr
0.08 ± 0.01
tr
QuercetinO-rhamnosyl hexoside
0.23 ± 0.00
0.10 ± 0.01
0.23 ± 0.03
0.09 ± 0.00
IsorhamnetinO-acetylhexoside
0.11 ± 0.00
0.08 ± 0.01
0.12 ± 0.01
0.04 ± 0.00
Total flavonoids
14.26 ± 0.14
a
8.38 ± 0.01
c
12.94 ± 0.17
b
7.79 ± 0.35
d
Total hydrolyzable tannins
53.78 ± 0.31
c
61.49 ± 0.80
a
59.25 ± 0.03
b
60.32 ± 1.87
ab
Total phenolic compounds
68.04 ± 0.18
c
69.88 ± 0.81
bc
72.20 ± 0.14
a
68.10 ± 1.52
c
In each row different letters mean significant differences with aP
<
0.05. They refer to Tukey’s post hoc test; therefore, significant different values were classified using letters in alphabetic order. tr: compound detected in trace amount. *Estimation due to its low resolution.


### 3.3. Antioxidant Activity

The antioxidant activity of theC. sativa samples was determined through various assays, namely, DPPH scavenging activity, reducing power through Prussian-blue assay, inhibition of β-carotene bleaching, and finally inhibition of TBARS formation in brain cell homogenates (Table 4). Decoctions showed greater antioxidant activity than infusions; this might be explained by the longer time at boiling point that decoctions were subjected to during extraction. In terms of scavenging of DPPH radicals, statistically significant differences were found between decoctions and infusions. Reducing power, β-carotene bleaching inhibition and TBARS assays gave statistically significant different results among samples, but the lowest EC50 values were always obtained in the TBARS assay, this could be due to its specificity, sensibility, and low quantity of interferences.Table 4
Antioxidant activity of decoctions and infusions of the flowers of twoCastanea sativa cultivars.
Antioxidant activity (EC50 values, μg/mL)
Decoctionjudia
Decoctionlongal
Infusionjudia
Infusionlongal
DPPH scavenging activity
99.47 ± 0.006b
100.04 ± 0.01b
126.61 ± 0.005a
133.56 ± 0.005a
Reducing power
68.51 ± 0.001d
76.07 ± 0.001c
90.65 ± 0.001b
98.79 ± 0.001a
β-carotene bleaching inhibition
47.89 ± 0.002d
184.92 ± 0.001b
177.23 ± 0.004c
195.10 ± 0.01a
TBARS inhibition
38.73 ± 0.001b
48.63 ± 0.000a
15.24 ± 0.002d
19.79 ± 0.003c
EC50 values correspond to the sample concentration achieving 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay. In each row different letters mean significant differences with a P
<
0.05. They refer to Tukey’s post hoc test; therefore, significant different values were classified using letters in alphabetic order.This assay was the only one that reported lower EC50 values for infusions, which can be related to the low heat resistance of antioxidants that inhibit lipid peroxidation, like tocopherols and other vitamins [23].Among the four samples, the decoction ofjudia proved to be the most antioxidant among the samples analysed. This could be explained by antioxidant variability of cultivars, translating into a higher antioxidant potential ofjudia when compared tolongal. Further research should be carried out to determine what antioxidants apart from phenolic compounds are present in these flowers that could help clarify this antioxidant variability between cultivars. Nevertheless, in general, all the studied samples proved to be powerful antioxidants when compared to other herbal matrices previously studied by the authors [8, 9].


## 3.1. Analysis of Free Sugars and Organic Acids

Three free sugars, namely, fructose, glucose, and sucrose, were detected in all the samples (Table1). Sucrose was the least abundant sugar, while fructose and glucose were the most abundant in the cultivarsjudia andlongal, respectively. This had been also reported for methanolic extracts ofC. sativa flowers [9]. Glucose and fructose were present at higher levels in decoctions than infusions, probably due to the longer extraction time, albeit significant differences were only found in the case of the cultivarlongal. By contrast, higher concentrations of sucrose were extracted in the infusions, although the increase was only statistically significant in the case ofjudia.No significant differences were observed among samples.Table 1
Content of free sugars and organic acids in mg/g of lyophilized decoction/infusion of the flowers of twoCastanea sativa cultivars.
Decoctionjudia
Decoctionlongal
Infusionjudia
Infusionlongal
Sugars
Fructose
160.41 ± 0.01a
152.08 ± 6.52a
148.94 ± 4.60a
123.58 ± 1.76b
Glucose
149.09 ± 0.04b
191.91 ± 7.35a
145.71 ± 5.63b
164.07 ± 2.31b
Sucrose
27.01 ± 0.45b
25.69 ± 0.99b
35.68 ± 1.45a
26.67 ± 0.50b
Total
336.51 ± 0.48a
369.69 ± 14.85a
330.32 ± 11.69a
314.32 ± 4.57a
Organic Acids
Oxalic acid
72.91 ± 1.82a
71.85 ± 0.52ab
43.42 ± 0.75c
55.84 ± 1.82bc
Quinic acid
84.61 ± 0.64a
52.59 ± 3.14b
69.04 ± 2.81ab
61.63 ± 4.40b
Malic acid
27.17 ± 1.36a
22.94 ± 0.10a
25.03 ± 0.52a
20.82 ± 0.70a
Shikimic acid
1.83 ± 0.02a
1.26 ± 0.08a
1.35 ± 0.02a
1.35 ± 0.07a
Total
186.52 ± 3.84a
148.65 ± 2.79ab
138.83 ± 2.61b
139.64 ± 6.99b
In each row different letters mean significant differences with aP
<
0.05. Letters refer to Tukey’s post hoc test; therefore, significant different values were classified using letters in alphabetic order.Oxalic, quinic, malic, and shikimic acids were quantified in all the samples, with quinic acid being the most abundant acid (Table1). Decoctions generally yielded higher quantities of oxalic acid in comparison to infusions, and statistical differences for this compound were only noted for the infusion oflongal. Thelongal cultivar did not display statistical differences for quinic acid in both extraction methods, but forjudia statistical differences were found between extraction methods. Malic acid and shikimic acid displayed similar concentrations in all the samples, whereas some differences were found among samples regarding quinic acid. The decoction ofjudia displayed the highest concentrations of total organic acids of the samples analysed.


## 3.2. Analysis of Phenolic Compounds

An exemplary phenolic profile of thejudia cultivar ofC. sativa recorded at 280 nm and 370 nm is shown in Figure 1. Twenty-seven phenolic compounds were identified in bothjudia andlongal cultivars (infusion and decoction preparations). Peak characteristics and tentative identities are presented in Table 2.Table 2
Retention time (Rt), wavelength of maximum absorption (λ
max
⁡), mass spectral data, and tentative identification of phenolic compounds in decoctions and infusions of Castanea sativa flowers.
Peak
Rt (min)
λ
max
⁡ (nm)
Pseudomolecular ion [M-H]− (m/z)
MS2 (m/z) (% of base peak)
Tentative identification
1
4.96
276
783
481 (10), 301 (41)
Pedunculagin isomer (bis-HHDP-glucose)
2
5.79
268
783
481 (6), 301 (45)
Pedunculagin isomer (bis-HHDP-glucose)
3
7.06
272
633
463 (17), 301 (100)
Galloyl-HHDP-glucose
4
7.67
274
937
637 (15), 467 (2), 301 (4)
Trigalloyl-HHDP-glucose
5
8.02
278
289
245 (91), 203 (60), 137 (38)
(+)-Catechin
6
13.77
276
939
631 (31), 469 (66), 169 (100)
Pentagalloyl glucose
7
14.92
276
935
633 (15), 301 (18)
Galloyl-bis-HHDP-glucose
8
15.25
274
933
915 (5), 633 (8), 451 (24), 301 (7)
Castalagin/vescalagin
9
15.46
278
907
767 (3), 607 (24), 467 (35), 169 (5)
Galloyl-HHDP derivative
10
16.13
274
937
767 (2), 637 (8), 467 (68), 301 (10)
Trigalloyl-HHDP-glucoside
11
16.65
358
493
317 (100)
MyricetinO-glucuronide
12
17.07
350
479
317 (100)
Myricetin 3-O-glucoside
13
17.30
274
937
767 (2), 637 (5), 467 (58), 301 (7)
Trigalloyl-HHDP-glucoside
14
19.89
356
609
301 (100)
Quecetin 3-O-rutinoside
15
20.64
356
477
301 (100)
Quercetin 3-O-glucuronide
16
20.86
356
463
301 (100)
Quercetin 3-O-glucoside
17
21.30
356
463
301 (100)
QuercetinO-hexoside
18
23.54
350
593
285 (100)
Kaempferol 3-O-rutinoside
19
24.05
354
433
301 (100)
QuercetinO-pentoside
20
24.46
268
907
767 (3), 607 (23), 467 (67), 169 (7)
Galloyl-HHDP derivative
21
25.11
348
477
285 (100)
Kaempferol 3-O-glucoside
22
26.00
354
477
315 (100)
IsorhamnetinO-hexoside
23
26.25
354
491
315 (100)
IsorhamnetinO-glucuronide
24
27.71
274
907
767 (2), 607 (27), 467 (76), 169 (8)
Galloyl-HHDP derivative
25
29.87
250/368
343
328 (97), 313 (100), 298 (36)
Tri-O-methylellagic acid
26
32.38
358
609
463 (76), 301 (40)
QuercetinO-rhamnosyl hexoside
27
33.13
356
519
477 (5), 315 (77)
IsorhamnetinO-acetylhexosidePhenolic profile of the infusion ofCastanea sativa flowers (judia cultivar) recorded at 370 nm (a) and 280 nm (b).
(a)
(b)Peaks 1–4, 6–10, 13, 20, 24, and 25 showed UV spectra coherent with galloyl and hexahydroxydiphenoyl (HHDP) derivatives [12–14]. According to the literature, the main characteristic in the mass spectra of these compounds is the deprotonated molecule [M-H]− and the loss of one or more ellagic acid (302 mu), gallic acid (170 mu), and/or galloyl groups (152 mu) [12, 15, 16]. Peaks 1 and 2 presented a singly charged pseudomolecular ion [M-H]− atm/z 783 and daughter ions atm/z 481 and 301, which together with their early elution allowed their identification as pedunculagin (i.e., bis-HHDP-glucose) isomers [17]. Mass characteristics of peak 3 ([M-H]− atm/z 633; fragment ions atm/z 463 and 301) coincided with a galloyl-HHDP-glucose isomer [16, 17], whereas peaks 4, 10, and 13 ([M-H]− atm/z 937; fragment ions atm/z 767, 637, 467 and 301) were coherent with trigalloyl-HHDP-glucose isomers [13], already reported inC. sativa heartwood [18] and flowers [9]. Peak 6 presented a pseudomolecular ion [M-H]− atm/z 939, yielding MS2 fragment ions atm/z 631 [M-2galloyl-3H]−, 469 [M-2galloyl-3H-glu]−, being identified as pentagalloylglucose. Peak 7 with a pseudomolecular ion [M-H]− atm/z 935 and MS2 product ions atm/z 633 and 301, likely due to the loss of HHDP and galloyl-glucose moieties, was consistent with a galloyl-bis-HHDP-glucose isomer [15, 17, 19]. Pseudomolecular ion ([M-H]− atm/z 933) and fragmentation pattern (ions atm/z 915, 631, 451 and 301) of peak 8 were in agreement with those attributed to castalagin or vescalagin isomers [17, 20], already reported inC. sativa heartwood [18]. Peaks 9, 20, and 24 are most likely galloyl-HHDP derivatives, with an unusual parent ion [M-H]− atm/z 907, but with characteristic fragments of this type of compounds (m/z at 767, 467 and 169). No structure could be assigned to these peaks that remain unidentified. A fragment ion withm/z at 907 was reported as released from the cleavage of a di(HHDP-galloylglucose)-pentose found in pomegranate juice [21] and from unknown ellagitannins present in blackberries [17]. Peak 25 presented an ellagic acid-like UV spectrum (λ
max
⁡ around 250 and 368 nm) and a pseudo molecular ion [M-H]− atm/z 343, releasing three fragments atm/z 328, 313 and 298 mu, corresponding to the successive losses of three methyl groups (−15 mu), which allowed its tentative identification as a tri-O-methylellagic acid.Regarding flavonoids, flavonol derivatives were the main compounds found in the analyzed samples (Table2). Catechin (peak 5), myricetin 3-O-glucoside (peak 12), quercetin 3-O-rutinoside (peak 14), quercetin 3-O-glucoside (peak 16), kaempferol 3-O-rutinoside (peak 18), and kaempferol 3-O-glucoside (peak 21) were positively identified according to their retention, mass, and UV-vis characteristics by comparison with commercial standards. Peak 11 was assigned to a myricetinO-glucuronide, according to the pseudomolecular ion [M-H]− atm/z 497 and MS2 fragment released atm/z 317 ([M-H-176]−, loss of glucuronyl moiety). Peaks 15, 17, 19, and 26 presented UV spectra with λ
max
⁡ around 350 nm and an MS2 product ion atm/z 301 indicating that they correspond to quercetin derivatives. According to their pseudomolecular ions, they were identified as quercetin 3-O-glucuronide (peak 15; [M-H]− atm/z 477), which was confirmed by comparison with a standard obtained in our laboratory [22], quercetinO-hexoside (peak 17; [M-H]− atm/z 463), quercetinO-pentoside (peak 19; [M-H]− atm/z 433), and quercetinO-rhamnosyl hexoside (peak 26; [M-H]− atm/z 609). Similarly reasoning also allowed assigning peaks 22 and 23 as isorhamnetinO-hexoside and isorhamnetinO-glucuronide, respectively. Peak 27 should correspond to isorhamnetinO-acetylhexoside according to the pseudomolecular ion [M-H]− atm/z 519 and MS2 fragment released atm/z 315 ([M-H-42-162]−, loss of an acetylhexoside moiety). The individual polyphenol with the highest concentration in all samples was a trigalloyl-HHDP-glucoside (peak 10), followed by pentagalloyl glucose (peak 6), whereas quercetin 3-O-glucuronide (peak 15) and a quercetin hexoside (peak 17) were the most abundant flavonoids injudia andlongal cultivars, respectively (Table 3). The sample with the highest concentration of total polyphenols was the infusion ofjudia, closely followed by the decoction oflongal. The preparations of the cultivarjudiapresented higher flavonoid levels, while those oflongal displayed higher concentrations of hydrolyzable tannins (Table 3). The compounds present in the samples were, to some extent, different from those found in aC. sativa hydromethanolic extract [9]. Nevertheless, the main ellagitannins found in this study were in accordance with the mentioned study.Table 3
Quantification of phenolic compounds in infusions and decoctions ofCastanea sativa flowers expressed in mg/g of lyophilized decoction/infusion.
Compounds
Decoctionjudia
Decoctionlongal
Infusionjudia
Infusionlongal
Pedunculagin isomer (bis-HHDP-glucose)
5.21 ± 0.11
8.48 ± 0.51
5.98 ± 0.16
7.68 ± 0.11
Pedunculagin isomer (bis-HHDP-glucose)
1.12 ± 0.09
1.44 ± 0.19
0.92 ± 0.92
1.21 ± 0.04
Galloyl-HHDP-glucose
3.07 ± 0.14
3.18 ± 0.00
3.18 ± 0.07
3.05 ± 0.09
Trigalloyl-HHDP-glucose
2.81 ± 0.25
5.51 ± 0.06
2.60 ± 0.06
4.30 ± 0.47
(+)-Catechin
1.14 ± 0.12
1.58 ± 0.14
1.11 ± 0.03
1.10 ± 0.05
Pentagalloyl glucose
5.61 ± 0.08
6.04 ± 0.15
5.73 ± 0.14
6.47 ± 0.15
Galloyl-bis-HHDP-glucose
0.45 ± 0.04
0.29 ± 0.07
0.66 ± 0.01
0.30 ± 0.01
Castalagin/vescalagin
0.75 ± 0.06
0.73 ± 0.11
1.06 ± 0.09
1.61 ± 0.04
Galloyl-HHDP derivative
0.38 ± 0.02
0.55 ± 0.07
3.60 ± 0.16
3.72 ± 0.01
Trigalloyl-HHDP-glucoside
26.72 ± 0.15
28.16 ± 0.11
30.70 ± 0.40
28.73 ± 1.34
MyricetinO-glucuronide
1.13 ± 0.02
0.09 ± 0.00
0.96 ± 0.01
0.08 ± 0.00
Myricetin 3-O-glucoside
0.66 ± 0.01
0.09 ± 0.01
0.60 ± 0.01
0.09 ± 0.00
Trigalloyl-HHDP-glucoside
1.33 ± 0.00
0.54 ± 0.01
1.61 ± 0.05
0.70 ± 0.04
Quecetin 3-O-rutinoside
1.57 ± 0.01
0.52 ± 0.03
1.31 ± 0.10
0.48 ± 0.05
Quercetin 3-O-glucuronide
3.55 ± 0.10
1.56 ± 0.04
3.38 ± 0.01
1.49 ± 0.07
Quercetin 3-O-glucoside*
1.80 ± 0.01
0.89 ± 0.02
1.60 ± 0.04
0.91 ± 0.10
QuercetinO-hexoside
2.21 ± 0.07
1.82 ± 0.05
2.11 ± 0.02
1.85 ± 0.06
Kaempferol 3-O-rutinoside
0.21 ± 0.01
0.21 ± 0.03
0.18 ± 0.01
0.21 ± 0.01
QuercetinO-pentoside
0.43 ± 0.02
0.20 ± 0.01
0.34 ± 0.00
0.22 ± 0.02
Galloyl-HHDP derivative
4.18 ± 0.10
4.54 ± 0.14
2.41 ± 0.14
1.96 ± 0.12
Kaempferol 3-O-glucoside
0.53 ± 0.00
0.49 ± 0.01
0.41 ± 0.00
0.46 ± 0.04
IsorhamnetinO-hexoside
0.33 ± 0.04
0.27 ± 0.01
0.27 ± 0.00
0.30 ± 0.01
IsorhamnetinO-glucuronide
0.35 ± 0.03
0.50 ± 0.02
0.33 ± 0.01
0.46 ± 0.04
Galloyl-HHDP derivative
2.04 ± 0.06
2.02 ± 0.03
0.72 ± 0.01
0.59 ± 0.01
Tri-O-methylellagic acid
0.10 ± 0.01
tr
0.08 ± 0.01
tr
QuercetinO-rhamnosyl hexoside
0.23 ± 0.00
0.10 ± 0.01
0.23 ± 0.03
0.09 ± 0.00
IsorhamnetinO-acetylhexoside
0.11 ± 0.00
0.08 ± 0.01
0.12 ± 0.01
0.04 ± 0.00
Total flavonoids
14.26 ± 0.14
a
8.38 ± 0.01
c
12.94 ± 0.17
b
7.79 ± 0.35
d
Total hydrolyzable tannins
53.78 ± 0.31
c
61.49 ± 0.80
a
59.25 ± 0.03
b
60.32 ± 1.87
ab
Total phenolic compounds
68.04 ± 0.18
c
69.88 ± 0.81
bc
72.20 ± 0.14
a
68.10 ± 1.52
c
In each row different letters mean significant differences with aP
<
0.05. They refer to Tukey’s post hoc test; therefore, significant different values were classified using letters in alphabetic order. tr: compound detected in trace amount. *Estimation due to its low resolution.


## 3.3. Antioxidant Activity

The antioxidant activity of theC. sativa samples was determined through various assays, namely, DPPH scavenging activity, reducing power through Prussian-blue assay, inhibition of β-carotene bleaching, and finally inhibition of TBARS formation in brain cell homogenates (Table 4). Decoctions showed greater antioxidant activity than infusions; this might be explained by the longer time at boiling point that decoctions were subjected to during extraction. In terms of scavenging of DPPH radicals, statistically significant differences were found between decoctions and infusions. Reducing power, β-carotene bleaching inhibition and TBARS assays gave statistically significant different results among samples, but the lowest EC50 values were always obtained in the TBARS assay, this could be due to its specificity, sensibility, and low quantity of interferences.Table 4
Antioxidant activity of decoctions and infusions of the flowers of twoCastanea sativa cultivars.
Antioxidant activity (EC50 values, μg/mL)
Decoctionjudia
Decoctionlongal
Infusionjudia
Infusionlongal
DPPH scavenging activity
99.47 ± 0.006b
100.04 ± 0.01b
126.61 ± 0.005a
133.56 ± 0.005a
Reducing power
68.51 ± 0.001d
76.07 ± 0.001c
90.65 ± 0.001b
98.79 ± 0.001a
β-carotene bleaching inhibition
47.89 ± 0.002d
184.92 ± 0.001b
177.23 ± 0.004c
195.10 ± 0.01a
TBARS inhibition
38.73 ± 0.001b
48.63 ± 0.000a
15.24 ± 0.002d
19.79 ± 0.003c
EC50 values correspond to the sample concentration achieving 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay. In each row different letters mean significant differences with a P
<
0.05. They refer to Tukey’s post hoc test; therefore, significant different values were classified using letters in alphabetic order.This assay was the only one that reported lower EC50 values for infusions, which can be related to the low heat resistance of antioxidants that inhibit lipid peroxidation, like tocopherols and other vitamins [23].Among the four samples, the decoction ofjudia proved to be the most antioxidant among the samples analysed. This could be explained by antioxidant variability of cultivars, translating into a higher antioxidant potential ofjudia when compared tolongal. Further research should be carried out to determine what antioxidants apart from phenolic compounds are present in these flowers that could help clarify this antioxidant variability between cultivars. Nevertheless, in general, all the studied samples proved to be powerful antioxidants when compared to other herbal matrices previously studied by the authors [8, 9].


## 4. Conclusion

The ancestral claims of health benefits of chestnuts flowers were partially corroborated in this paper. The organic acids could be responsible for some antioxidant potential, but further research can be carried out in terms on tocopherols and other vitamins and minerals that could provide more insight into the total amount of antioxidants. Further research should be carried out on the potentiality of these flowers in the pharmaceutical and food industries, among others. Pharmaceutical industries could use the flowers as excipients for dietary supplements, benefiting from their natural content in polyphenols for health purposes. The food industry, which has recently started to intensify its search for natural conservatives and additives, to add to transformed foodstuffs, meeting the consumers habits, can use the high antioxidant power and natural high abundance of tannins in the flowers to preserve food and inhibit lipid deterioration and microorganism development. Although quite beneficial, the flowers used for these purposes should not be picked from the tree, but rather from the ground to avoid interference of pollination. Methanolic extractions have also been performed for chestnut flowers and yielded good results [8, 9], although the usage of methanol is not recommended in the food industry; but on the other hand, extractions with this solvent could carry hydrophobic antioxidant compounds.



---

*Source: 232956-2014-04-14.xml*