2.1. Influence of the Cultivar and Harvest Season on Phenolic Compounds Contents
The TPC and TFC quantification in rabbiteye blueberry leaves from different harvest seasons and cultivars is shown in Table 1
. The leaves collected in March presented the highest TPC (ranging from 154 ± 1 to 222 ± 1 mg GAE/g) and TFC contents (ranged to 49.8 ± 0.8 to 38.3 ± 0.8 µg rutin/g) (Table 1
) confirming that the leaves’ phytochemical composition varies according to the stages of the plant growth [15
] and the plant maturity status is reflected the physiological, biochemical and structural processes of the plant tissue [5
There are many reports about TPC and TFC of blueberry leaves and fruits, what makes it possible to notice that the Vaccinium
genotype and the harvest season are factors that exert a great influence on the content of these antioxidant compounds. Zhu et al., observed that for the aqueous extract of blueberry leaves (Vaccinium ashei
) cultivated in China, in different seasons, the highest TFC (114.21 ± 0.03 mg rutin equivalent/g extract) was reported in May and the highest TPC (425.2 ± 0.2 mg gallic acid equivalent/g extract) in November [8
]. Similarly, Li et al., reported that rabbiteye blueberry (Vaccinium ashei
; cv. Brightwell) from Nanjing (China), harvested in July had the highest TPC (339 ± 3 mg GAE/g DW) and TFC (198 ± 2 mg of quercetin/g DW) in the extract of leaves in comparison with fruits and pomace [6
]. Routray and Orsat observed that highbush blueberry (Vaccinium corymbosum
) leaves had a high amount of total phenolics in October (for Nelson 152 ± 3 and for Elliot 156 ± 2 mg GAE/g dry matter) [15
According to Venskutonis and co-workers, during seasonal development, blueberry plants first concentrate the secondary metabolites in the fruits, but later those metabolites are concentrated in the leaves vegetative portion [7
]. This observation corroborates the results obtained in this study. On the other hand, these data are not in agreement with Percival and Mackenzie’s report. In that study during harvest, blueberry (Vaccinium angustifolium
) leaves presented a higher total phenolic content in green leaf tissues at harvest than those observed two weeks after the collection period [22
In Brazil, the flowering of blueberry fruits starts in August and ends between early September and the end of October. The blueberry fruit maturation starts during the second half of December until January and the harvest period lasts 37 days [21
]. Leaves from December (late spring and early summer) are from the end of fruit ripening and leaves from March (late summer and early autumn) correspond to the phenological stage of the plants when pruning occurs.
Considering the effect of cultivars on TPC and TFC values, the Clímax and Aliceblue cultivars showed higher TPC levels for both collections (133.6 ± 0.4 to 222 ± 1 mg GAE/g). Regarding the TFC quantification, the Clímax from December showed (32.3 ± 0.2 µg/g) and Bluegem (45.2 ± 0.5 µg/g), Powderblue (48.7 ± 0.7 µg/g). The Clímax (49.8 ± 0.8 µg/g) from March presented the highest compound contents. Ehlenfeldt and Prior previously reported TPC values from fruits and leaves of 87 Vaccinium corymbosum
cultivars from late July [9
]. In the leaves, phenolics values were higher than in the fruit, ranging from 23.6 GAE/g of fresh weight (cv. Reka) to 77.4 GAE/g of fresh weight (cv. Little Giant), with a mean of 44.80 GAE/g of fresh weight. In dried blueberry leaves (Vaccinium corymbosum
; cv. Bluecrop), collected at the beginning of August, Skupien et al., reported a TPC of 111.5 mg% [23
2.2. Phenolic Compounds Identification by HPLC
A typical HPLC chromatogram of solution and percentages of phenolic compounds in blueberry leaves extracts from different harvest months and cultivars are shown in Figure 1
and Figure 2
, respectively. All blueberry leaves extracts had similar phenolic composition, however quantitative differences were observed depending on the cultivar and collection month (Table 2
Three phenolic compounds were identified: chlorogenic acid (retention time - tR = 19.3 min, peak 1), rutin (tR = 30 min, peak 2) and quercetin (tR = 38 min, peak 3), comparing the retention time and UV spectra with 11 commercial standards (Figure 1
). The quantification of chlorogenic acid, rutin and quercetin by HPLC-UV/DAD was based on reference standard calibration curves. Calibration curve for chlorogenic acid: y = 168508x − 52225 (r = 0.9980); rutin: y = 72823x − 1900.7 (r = 0.9982); and quercetin: y = 187893x − 163671 (r = 0.9984).
Chlorogenic acids (3-O
-caffeoylquinic acids) were the most prevalent phenolic compound in blueberry leaf extracts (2.03 ± 0.03 to 21.28 ± 0.05 mg/g DW; corresponding to 32.2 and 87%, respectively) (Table 2
and Figure 2
), which was consistent with Ferlemi et al. [12
] who reported that the leaves are one the richest sources of chlorogenic acids and previous leaves analysis from 38 rabbiteye blueberry, 37 northern highbush blueberry and 29 southern highbush blueberry leaves collected in China in October showed substantial differences from each other. The eight chlorogenic acids were detected and this compound was the most abundant phenolic compounds in leaves of all cultivars [4
In present study, the content of rutin ranged from 2.59 ± 0.04 to 15.8 ± 0.1 mg/g DW (corresponding to 13 and 64.8%, respectively). Quercetin was not detected in the chromatograms of some cultivars (0.83 ± 0.02 to 11.9 ± 0.2 mg/g DW)
In a study performed by Ferlemi et al. (2015) Vaccinium corymbosum
leaf extract (cv. Bluecrop and Patriot) demonstrated by LC-ESI/MS and HPLC-DAD five major polyphenols (chlorogenic acid, rutin, hyperoside, isoquercetin and quercetin aglycone) which were able to protect the affected tissues (cortex, liver, from the overdose of selenite) and enhanced the antioxidant state of the least perturbed tissues [24
Chlorogenic acid is an ester of caffeic acid and quinic acid, while rutin (quercetin-3-O
-rutinoside) is a quercetin glycoside. These compounds have a well-established antioxidant activity [25
]. The chlorogenic acid antioxidant activity is attributed to the catechol phenyl ring and the double bond together with the catechol group serving as a site for the attack of free radicals [27
]. Besides, chlorogenic acids have activity against hepatocellular carcinomas and fibroblastic sarcomas, as well as anti-inflammatory, cardioprotective, cardioprotective, and neuroprotective properties [4
]. In turn, rutin has demonstrated a number of pharmacological properties, including cytoprotective, vasoprotective, anticarcinogenic, neuroprotective and cardioprotective activities [28
] suggesting the pharmacological potential of blueberry leaves.
shows the effect of harvest season on chlorogenic acid values. It is possible to observe that the Powderblue and Aliceblue cultivars from March showed higher chlorogenic acid content (21.28 ± 0.05 and 18.21 ± 0.05 mg/g of DW, respectively). Besides, FloridaM, Climax and Aliceblue cultivars from December showed higher chlorogenic acid content (17.34 ± 0.05, 15.87 ± 0.03 and 15.58 ± 0.05 mg/g of DW, respectively). The Bluegem cultivar from March showed higher rutin content (15.84 ± 0.13 mg/g of DW). Quercetin was identified in Aliceblue, Powderblue and FloridaM cultivars and the highest concentrations were observed in the December collection.
This variation was also observed by Zhu et al. where the presence of the chlorogenic acid (34 ± 2, 8.8 ± 0.2 and 14.07 ± 0.02 mg/g), caffeic acid (0.5 ± 0.1, 0.09 ± 0.01 and 0.13 + 0.01 mg/g), rutin (7 ± 1, 2.30 ± 0.03 and 2.6 ± 0.4 mg/g), hyperoside (3 ± 1, 1.1 ± 0.2 and 4.8 ± 0.7 mg/g), galuteolin (27 ± 2 ) and quercitrin (4.2 ± 0.4, 1.33 ± 0.03 and 2.10 ± 0.07 mg/g) was detected in aqueous extracts of blueberry leaves from different seasons, specifically for the samples from the months of May, September and November, respectively [8
Grace and co-workers showed that UV light stimulates the production of foliar chlorogenic acid content in plants [29
]. Fully exposed leaves produced higher levels of chlorogenic acid, whereas in shaded leaves it similar chlorogenic acid contents were found between seasons. Besides, one of the abiotic stresses which affects temperate plants is the low temperature, so in autumn, an increase in the content of a range of cryoprotective substances with the purpose of maximize their cold tolerance it can be seen [30
]. Considering this, it is possible to suggest that the plant from March used in this study was already accumulating the metabolites to be prepared for the winter. After this period, the rutin, quercetin and chlorogenic acid concentration in the leaves decreases the development of the flowers in spring and resource allocation shifting from defense to reproduction [19
2.3. Influence of Cultivar and Harvest Season on Extracts Antioxidant Activity
The antioxidant properties of the blueberry leaves extracts from different harvest months and cultivars by the DPPH (2,2-diphenyl-2-picrylhydrazyl) free radical capture method and the oxygen radicals removal ability method (ORAC) are shown in Table 3
. The DPPH and ORAC methods are common analyses used to evaluate the antioxidant activities of medicinal plants. To the best of our knowledge, no report on the antioxidant activity of rabbiteye blueberry as a result of different harvesting months and cultivars from Brazil exists.
According to the results expressed in Table 3
, the values ranged from 5.80 ± 0.04 to 105 ± 2 µg/mL (DPPH) and 178 ± 5 to 431 ± 8 mmol Trolox/100 g (ORAC). In general, the cultivars from March showed higher average antioxidant properties than the ones from the December collection, regardless of the test employed. Concerning the influence of the cultivar type, the Bluegem from March presented the highest antioxidant properties in both assays (5.80 ± 0.04 µg/mL DPPH and 431 ± 8 mmol Trolox/100 g ORAC).
The results demonstrated that there was a variation in the antioxidant activity depending on the cultivar and the collection period, regardless the method used in the evaluation. As previously exposed, changes in environmental conditions during the different seasons and genetic predisposition can explain these variations [15
]. For the species Vaccinium corymbosum
, Ehlenfeldt and Prior reported values of about 490.4 µmol Trolox/g (ORAC) for hydroalcoholic extracts of leaves from different cultivars [9
] and Pervin, Hasnat and Lim reported values of about 0.12 ± 0.003 mg/mL by DPPH assay [20
Moreover, in studies with blueberry fruits, the total antioxidants activities in six different varieties varied about 2.6 times according to the ORAC assay, and 2 times by the peroxyl radical scavenging capacity (PSC) assay [31
]. The same was observed by Cardeñosa and co-workers, where the genotype influenced the antioxidant capacity and the content of the three groups of phenolics in blueberry fruits [32
]. In accordance of Sarkar and co-workers, the genotype versus environment interactions are most critical in the in vitro anti-diabetic-relevant functionalities of blueberry bioactives [33
The correlation between the phenolic compounds (total phenolic, flavonoid content, chlorogenic acid and rutin contents) with antioxidant activity (DPPH and ORAC) of blueberry leaves from December and March is shown in Table 4
According to Table 4
, rabbiteye blueberry leaves from December showed the highest correlation coefficient for interaction between DPPH and chlorogenic acid (0.99), which describes a strong positive correlation (0.8 < r < 1). On the other hand, interactions between DPPH and total phenolics (0.76) and the interaction between ORAC and total flavonoids (0.69) presented a moderate positive correlation (0.5 < r < 0.8). Despite the high overall phenolics and flavonoids total content, no correlation was found for interactions between phenolics, flavonoids total content and chlorogenic acid versus DPPH or ORAC to blueberry leaves from March (Table 4
In contrast, rabbiteye blueberry leaves from March showed a strong positive correlation (0.8 < r < 1) between rutin and DPPH (0.83) and between rutin and ORAC (0.80). The same was observed between DPPH and chlorogenic acid (0.98) to rabbiteye blueberry leaves from December. This correlation helps to understand the contribution of rutin to the antioxidant capacity of rabbiteye blueberry leaves. In accordance with Yang and co-workers [34
], rutin play an important role in terms of antioxidant capacity against numerous in vitro antioxidant systems and this capacity depends on its concentration.
In conclusion, in view of the different phenolic compounds contents and the antioxidant properties identified in the leaves, this study contributes to better understand the influence of different cultivars and harvest seasons, as well as, it extends the blueberry aplications not limiting them only to its fruits. The results demonstrated that the Bluegem variety harvested in March is the most promising, and considering the high cost associated with growing blueberry fruits, the use of their leaves can be considered advantageous in this aspect. The byproducts derived from leaves could be used as infusions being a coadjuvant treatment for many conditions where oxidative stress is involved. Moreover, these byproducts can be viewed as intermediate products to produce final pharmaceutical and nutraceutical dosage forms.