Article pubs.acs.org/JAFC
Flavonols, Alkaloids, and Antioxidant Capacity of Edible Wild Berberis Species from Patagonia Antonieta Ruiz,†,§ Moises Zapata,† Constanza Sabando,† Luis Bustamante,† Dietrich von Baer,† Carola Vergara,† and Claudia Mardones*,† †
Department of Instrumental Analysis, Faculty of Pharmacy, University of Concepción, P.O. Box 160-C, Concepción, Chile Department of Chemical Science and Natural Resources, University of La Frontera, Casilla 54-D, Temuco, Chile
§
S Supporting Information *
ABSTRACT: There are 20 species of the Berberidaceae family described in Chile, whose fruits are edible and show high anthocyanin and hydroxycinnamic acid levels. Berberis microphylla G. Forst, commonly known as calafate, is the most extensively distributed. Flavonols and alkaloids in seed, pulp, skin, and whole calafate berry extracts and other Berberis were studied using HPLC-DAD-ESI-MS/MS and HPLC with fluorescence detector. Berry samples from different locations in Chilean Patagonia, including different phenological stages, were systematically addressed. Results were compared with other organs of the plant and with other Berberis species. Total flavonol concentration in calafate (n = 65) was 1.33 ± 0.54 μmol/g. Glycosyl metabolites of quercetin and isorhamnetin were the most abundant. Similar profiles were observed in calafate from distinct locations, but important differences were observed for the other edible Berberis species. Calafate pulp and skin have higher flavonol concentrations than seeds, and the maturation process reduced its levels. TEACCUPRAC and TEACABTS of whole calafate extracts and fractions are also explored. Finally, only berberine was detected in the fruit (0.001%), mainly in seeds. Results contribute to the promotion of this berry as a superfruit from Patagonia. KEYWORDS: flavonols, alkaloids, HPLC-DAD-ESI-MS/MS, core−shell columns, berberine, Berberis microphylla G. Forst, antioxidant capacity
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species from Asia, Europe, and South America,5 in which high levels of berberine and jatrorrhizine were described. Berberine was also detected at high concentration in calafate roots from Patagonia;6 however, there is no scientific information about its presence in calafate berries, which constitute the only edible part of the plant. Acute toxicity of berberine has been studied in rats;7 however, this compound has also been described as antidiabetic, hepatoprotective, anticarcinogenic, and neuroprotective, among other effects.8 Considering these facts, the main aim of this research is to increase the knowledge of Berberis species, now from the perspective of their flavonol and alkaloid compositions and antioxidant capacity, in the context of nutraceutical potential and safety of Berberis berries, with emphasis on calafate. This research includes the study of an important number of calafate samples obtained from different locations around Patagonia in two successive collection years by using HPLC-DAD-ESI-MS/ MS and HPLC with fluorescence detection. A study of flavonol and alkaloid profiles and concentrations in calafate fruit with different phenological stages was also accomplished. The distribution of these compounds in the different parts of the fruit and plant and their comparison to other Berberis species are also discussed in this work. The antioxidant capacity of whole extracts and flavonol/hydroxycinnamic acids fractions
INTRODUCTION Berberis is the only genus of the Berberidaceae family found in South America. In Chile, 20 species of this genus have been described, with the most extensively distributed being Berberis microphylla G. Forst (commonly known as calafate), Berberis darwinii, and Berberis empetrifolia.1 Calafate is an endemic wildgrowing plant that is not cultivated and grows extensively in southern Patagonia in Chile and Argentina. Very high concentrations of anthocyanins and hydroxycinnamic acid derivatives (17.81 μmol/g fresh weight (FW) and 2.62 μmol/ g (FW) as mean, respectively) have been described in this fruit, making it a very promising berry from a nutraceutical perspective.2−4 It is necessary to study other bioactive compounds in this fruit and others of its genus to better understand its potential competence as a functional fruit. In a previous study carried out with eight mature calafate samples obtained in Patagonia, a range between 0.11 and 0.21 μmol/g (FW) of flavonols was reported, with the main compounds being myricetin and quercetin derivatives.2 There is no information about the flavonol distribution in the fruit (skin, pulp, and seed), the effect of the maturation process on their profiles and concentrations, or the effect of fruit-growing latitudes. All of this is important to understand the effect of these parameters on their potential as functional food. On the other hand, considering the presence of alkaloids reported in different species of this genus, their determination in calafate is also necessary to demonstrate its safety. In this context, isoquinolinic alkaloids have been detected in different structures (branches, leaves, roots, and berries) of Berberis © XXXX American Chemical Society
Received: July 3, 2014 Revised: November 28, 2014 Accepted: November 29, 2014
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Shimadzu (Kyoto, Japan). The data collection was done using an interactive software graphics version 6.2 Varian Inc. (Palo Alto, CA, USA). Sample Pretreatments. Flavonols. Whole extract preparation and cleanup were carried out on the basis of the methods described by Ruiz et al. and Castillo-Muñoz et al.2,11 with minor modifications. Five grams of whole fruit was ground in a stainless steel mixer with 10 mL of methanol/water (93:7% v/v); the extract was treated for 45 s with the ultrasonic bar, followed by 16 h of mechanical shaking under darkness. After centrifugation, the separated supernatant was transferred, and the residual solid was washed three times with 5 mL of the same solvent by mechanical shaking for 15 min. Cleanup of the combined extracts (25 mL) was made by using Oasis MCX cartridges, to eliminate the interfering anthocyanins. One milliliter of crude extract was dried in a rotary evaporator and reconstituted in 1.0 mL of 0.1 N hydrochloric acid. This solution was loaded on a MCX cartridge previously conditioned with 5.0 mL of methanol and 5.0 mL of water. A washing step, using 5.0 mL of 0.1 N hydrochloric acid and 5.0 mL of water, was necessary to remove sugars. The flavonol fraction was eluted with 3 × 5.0 mL of methanol and, finally, the solvent was distilled off by vacuum evaporation and the residue was redissolved in 1 mL of the mobile phase (0.1% formic acid in water/acetonitrile 85:15%v/v) for its chromatographic analysis. This fraction also contained the hydroxycinnamic acids. In the study of flavonol distribution in different parts of the fruits (seeds, pulp, and skin), frozen berries were peeled, the pulp was separated from the seed, and all were carefully dried with paper toweling. Each was separately ground before the same extraction procedure as described above was applied. Alkaloids. The method was adapted from a procedure described for alkaloid extraction of lupine seeds and plants,9 which was based on the method proposed by Wink,10 with modifications: 1 g of calafate sample (fruit, seeds, leaves, branches, or roots) was extracted with 8 mL of 0.1 N HCl with the assistance of an ultrasonic bar for 45 s and mechanical shaking for 10 min. After centrifugation, the supernatant was transferred to a 25 mL volumetric flask, and the solid residues were washed three times with 5 mL of the same solvent (in the case of fruits, seeds, and leaves) and 8 mL (in the case of roots and branches). The whole extract was neutralized using NH4OH and completed to 25 mL. Twenty milliliters of extract was transferred to an Extrelut NT-20 column, to carry out the liquid−liquid extraction, using dichloromethane as the elution solvent (4 × 25 mL). After that, the solvent was evaporated at reduced pressure (30 °C) and finally redissolved in 1 mL of mobile phase (0.1% of formic acid in water/acetonitrile 20:80% v/v) and filtered before its chromatographic analysis. Flavonol Analyses. HPLC-DAD. Three C18 columns were evaluated for flavonol HPLC separation: (A) C18 column (Kromasil C18 100 A, 5 μm, 250 × 4.6 mm) with a C18 precolumn (Nova-Pak Waters, 4 μm, 22 × 3.9 mm); (B) C18 core−shell column (Kinetex C18 100 A, 2.6 μm, 150 × 4.6 mm) with a C18 precolumn for UHPLC columns (Phenomenex AJ0-9000, 2.2 μm, 22 × 3.9 mm); (C) C18 UHPLC column (Shim-pack XR-ODS III, 2.2 μm, 150 × 2.0 mm) without a precolumn. The chromatographic separation conditions were optimized for each column and were carried out at 30 °C using 0.1% of formic acid in water (A) and acetonitrile (B) as mobile phase. The optimized separation using column A was carried out at a flow rate of 1 mL/min, with a gradient starting with 80% A for 6.5 min, from 80 to 76% in 6 min, from 76 to 60% in 2.5 min, and from 60 to 0% in 0.5 min, maintaining the proportion for 6.5 min, and a final stabilization step of 10 min at 80% A. The injection volume was 10 μL. The separation using column B was carried out also at 1 mL/min with a gradient starting with 85% A for 0.1 min, from 85 to 80% in 5 min, from 80 to 70% in 2.5 min, from 70 to 0% in 0.5 min, maintaining the proportion for 2 min, and with a final stabilization step of 5 min at 85% A. The injection volume was 5 μL. Finally, the separation in column C was carried out at a flow rate of 0.5 mL/min, with a gradient starting with 85% A for 12 min, from 85 to 60% in 3 min, from 60 to 0% in 0.5 min, maintaining the proportion for 5 min, and with a final stabilization step of 7 min at 85% A. The injection volume in this case was also 5 μL.
are also explored in relation with their concentrations to evalute their contribution to the antioxidant capacity of calafate. This research is a contribution to promote, from a scientific perspective, the potential of this underexploited fruit, complementing previous work about anthocyanins and hydroxycinnamic acids now with flavonol and alkaloid levels and antioxidant capacity.
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MATERIALS AND METHODS
Reagents, Standards, and Extraction Materials. Commercial standards of quercetin-3-rutinoside (97%), quercetin-3-glucoside (>90%), quercetin-3-rhamnoside (85%), quercetin-3-O-(6″malonyl)-D-β-glucoside (85%), berbamine dihydrochloride, and berberine hydrochloride (95% of the flavonols were extracted, which is a good compromise between extraction efficiency and extraction time. The internal precision of flavonol determination in calafate samples, considering the whole method including liquid−solid E
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Figure 3. Individual flavonol concentrations in 65 samples of B. microphylla G. Forst obtained in Magallanes, Aysen, and Los Lagos and Araucania.
between samples obtained in 2011 and 2012 (1.34 ± 0.55 and 1.32 ± 0.49 μmol/g FW, respectively). Samples from more southerly latitudes (Magallanes 51−54° S) showed higher concentrations than those from the northern ones (Aysen 43− 47° S) and Chiloe Main Island (42° S). This trend is similar to that observed for hydroxycinnamic acid derivates,3 in which a decrease in their concentrations was observed from south to north. However, in the flavonol study, an exception was observed for four samples obtained further north, in Gorbea (39° 06′ S; 72° 40′ W in the Araucaniá region), which showed similar mean concentrations to the Magallanes samples. The quantitative results by region are summarized in Table 2. The profile and concentration of flavonols in mature samples of calafate (B. microphylla) were compared with those for mature berry samples of other endemic Berberis species obtained in the same study zone (between Bio-Bio and Magallanes regions). The respective flavonol chromatograms for the Berberis species are presented in Figure 4. The results showed different profiles for the species, especially for B. empetrifolia, B. ilicifolia, and B. darwinii, in comparison with B. microphylla. In the first one, only three flavonols (rutinoside, galactoside, and glucoside derivatives of quercetin) predominated, only traces of other flavonols being observed (isorhamnetin-3-rutinoside, isorhamnetin-3-galactoside, and isorhamnetin-3 malonylglucoside). In B. ilicifolia, isorhamnetin-3-rutinoside predominated, whereas quercetin-3rhamnoside, quercetin-malonyl derivatives, and the other isorhamnetin malonyl derivatives were absent. In B. darwinii, the absence of isorhamnetin-3-rutinoside and isorhamnetin-3galactoside was noted, with a similar proportion of the other flavonols to B. microphylla. B. montana and B. vulgaris presented flavonol profiles similar to that of calafate, with similar proportions of them. This observation is interesting because
extraction, SPE cleanup steps, and chromatographic separation, was 7.9%, and the extracts were stable for 7 months at −20 °C. The analytical response and other analytical parameters for the main flavonols are summarized in the Supporting Information. Total flavonols were determined by the sum of individual flavonols detected over the limit of quantitation of the method. Flavonol Quantitation in Calafate and Other Berberis Berries. Sixty-five samples of calafate berries obtained at different latitudes in Patagonia were analyzed. In all of them, 16 flavonol derivates were detected. The most abundant was quercetin-3-rutinoside, showing a mean concentration of 0.32 ± 0.15 μmol/g FW, with a range from traces to 0.74 μmol/g FW. Quercetin-3-galactoside, quercetin-3-rhamnoside, and isorhamnetin-3-rutinoside also showed interesting concentrations of 0.24 ± 0.16, 0.25 ± 0.12, and 0.24 ± 0.14 μmol/g FW, respectively. For the other quercetin and isorhamnetin derivates, their mean concentrations were 60% of the total flavonols in seed). It is important to note that this flavonol is one of the predominant flavonols in calafate. This distribution within the different structures of the fruit is very interesting and must be explained by the calafate plant metabolism; however, this has not been studied until now. The profile and concentrations of flavonols in different phenological stages was evaluated using berry samples obtained from the same plant in the stages of fruit setting, green fruit, and full-colored fruit. The samples were taken on December 15, 2011; January 15, 2012; and February 15, 2012, respectively. Results showed that the flavonol profile changed in this process. Quercetin-3-malonylglucoside decreased, whereas quercetin-3rhamnoside increased, and the overall flavonol concentration decreased to 75% of the initial level. These results are presented in Figure 5. These observations are similar to those previously obtained for hydroxycinnamic acid derivatives in calafate3 and are correlated with those observed by Arena et al.16 for total polyphenol and flavonoid contents in calafate. The phenolic content decrease has also been observed for other berries, including different cultivars of the blueberry,17 in which a change in the pool of total phenolics toward anthocyanin synthesis, with an overall decline in the content of other phenolic components, is observed during ripening. These findings about flavonols in calafate are very relevant because of the enhancement of calafate and other Berberis from a nutraceutical point of view. They also contributed to finding the optimal growing time and conditions. Reducing Capacity of Whole and SPE Fractions Calafate Extracts. Four previously quantitated fractions obtained by SPE were subjected to in vitro antioxidant capacity assays (TEACCUPRAC and TEACABTS). This was carried out to evaluate the contribution of these anthocyanin free fractions on the antioxidant capacity of calafate. These fractions contained mainly flavonols and hydroxycinnamic acids. Two of these samples (C1 and C2) had low concentrations of these compounds, whereas the other two were included due to its high concentrations (C3 and C4). In Table 3 are summarized these results. High TEACCUPRAC and TEACABTS values were observed for whole extracts with high total phenolics (C1 and C4). This is explained mainly due to the high anthocyanin concentrations present in these extracts. Also, it is possible to observe a trend of higher TEACCUPRAC and TEACABTS values for SPE fractions with high flavonol−hydroxycinnamic acid concentrations (C3 and C4), compared with the fraction with minor concentrations of this compounds (C1 and C2); however, this trend is less clear than those observed for whole extracts. The antioxidant capacities obtained for flavonol plus hydroxycinnamic acid fractions represent 15 ± 5 and 5 ± 4% of TEACCUPRAC and TEACABTS values, respectively, in comparison with the values obtained for the whole extracts. This shows that flavonols and hydroxycinnamic acids have a notable incidence in the antioxidant capacity of calafate, despite its lower concentration in comparison with anthocyanins. Alkaloid Identification and Quantitation in Calafate and Other Berberis Berries. Alkaloid determination was done with the objective of guaranteeing the innocuousness of Berberis. The identification of alkaloids in calafate and other
0.91−1.16
0.97−1.32 1.75−2.22 2.19−2.63
0.94−1.33 1.61−1.80 1.25−2.09 1.37−1.90 1.64−1.78
4 2 4 3 2 1 1
0.71 0.87 0.83 0.92 1.04 0.94 0.77
± 0.33 ± 0.51 ± 0.10 ± 0.71 ± 0.29 ± 0.00 ± 0.00
0.27−0.97 0.51−1.20 0.74−0.94 0.71−1.24 0.83−1.24
4
1.52 ± 0.10
1.38−1.58
B. montana and B. microphylla are taxonomically similar,1 which could explain their similar profiles. However, B. vulgaris is an introduced species, which is used as an ornamental plant in gardens and is very different from B. microphylla. The quantitative results of the 16 samples of other Berberis species were compared with the results for calafate samples summarized in Table 2. The total flavonol concentration of B. microphylla was less than that observed for B. montana samples (n = 4), showing 1.33 ± 0.54 and 1.61 ± 0.64 μmol/g FW, respectively, and for B. empetrifolia (2.01 μmol/g FW, n = 1), but was higher than that observed for B. ilicifolia (0.71 ± 0.18 μmol/g FW, n = 1), B. darwinii (1.04 ± 0.31 μmol/g FW, n = 6), and the only available sample of B. vulgaris (0.72 μmol/g FW), which was obtained from University of Concepción gardens. The flavonol concentration in different parts of the calafate fruit (skin, pulp, and seeds) was determined in two composed samples, one of them from Magallanes and the other from the Aysen region. The results were similar for samples from both G
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Figure 4. Chromatographic profiles of flavonols extract of different Berberis species, by HPLC-DAD using core−shell column: (A) B. empetrifolia (sample from Parque Pinguino Rey, Provenir, Tierra del Fuego); (B) B. vulgaris (sample from gardens of University of Concepción, Concepción); (C) B. darwinii (sample from Gorbea, Temuco); (D) B. ilicifolia (sample from Lago Deseado, Porvenir, Magallanes); (E) B. montana (sample from Valle Las Trancas, Chillan); ( (F) B. microphylla 2012 (sample from Lago Fagnano, Magallanes); (G) B. microphylla 2011 (sample from Seno Otway, Magallanes).
Berberis species was done by comparison of tR with commercial standards. The identity confirmation was based on their MS/ MS and UV−vis spectra. Table 4 presents spectroscopic characteristics of alkaloids detected in different Berberis structures. In all calafate fruits analyzed in this part of the research (n = 62), only berberine was detected. However, jatrorrhizine and palmatine were detected at trace levels in calafate leaves. The alkaloid coridaline was assigned to a signal
found in fruits of B. ilicifolia based on the MS/MS spectra, in accordance with Wang et al.18 Quantitative analysis of berberine was carried out by HPLC with fluorescence detection using an external calibration curve, which was performed by triplicate injection at six levels of berberine standard concentration. The calibration curve showed a low level of dispersion, with a regression coefficient of 0.9993 and an equation of (y = 560704.27x − 80514.59). Fluorescence detection showed a high sensitivity and low H
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Figure 5. Flavonols profile and concentration during the maturation process of B. microphylla G. Forst (sample from Sector Barranco Amarillo, Magallanes).
Table 3. In Vitro Antioxidant Capacity (TEACCUPRAC and TEACABTS) of SPE Fractions and Whole Extracts of B. microphylla G. Forsta whole calafate extracts total phenolic concn (μmol/g FW) calafate calafate calafate calafate
1 2 3 4
32.63 3.06 11.56 37.68
± ± ± ±
flavonols concn (μmol/g FW) calafate calafate calafate calafate
1 2 3 4
0.26 1.56 0.97 1.44
± ± ± ±
0.01 0.04 0.01 0.03
0.14 0.04 0.03 0.54
TEACCUPRAC (μmol TE/g FW) 405.95 179.61 224.68 577.73 SPE fractions (flavonols
HCADs concn (μmol/g FW) 0.68 0.89 4.21 4.88
± ± ± ±
0.00 0.00 0.02 0.00
± 33.01 ± 12.12 ± 12.19 ± 35,.5 + HCADs)
105.48 71.56 106.92 133.47
TEACCUPRAC (μmol TE/g FW) 34.87 35.79 42.88 66.85
± ± ± ±
TEACABTS (μmol TE/g FW)
7.44 0.69 11.39 11.27
± ± ± ±
4.43 0.12 15.92 3.17
TEACABTS (μmol TE/g FW) 4.69 5.29 5.68 11.45
± ± ± ±
0.14 0.14 0.18 0.44
a The identifications of each sample are, respectively, calafate 1, Puerto tranquilo, Aysen; calafate 2, Gorbea, Temuco; calafate 3, Rio San Luis, Magallanes; calafate 4, Camino Provenir. Km 25, Magallanes. HCADs, hydroxycinnamic acids.
Berberine levels were evaluated in fruits of different Berberis species, showing low concentrations; however, some variations between the different samples were observed. The highest concentration of berberine was detected in B. ilicifolia (n = 3) with 160.0 nmol/g FW. In calafate fruits (B. microphylla) collected in the year 2011 (n = 45) levels of berberine were between 1.8 and 126.9 nmol/g FW, whereas in samples collected in 2012 (n = 18) concentrations between 3.3 and 152.8 nmol/g FW were determined. Higher contents of berberine were found in B. ilicifolia (55.9−160.5 nmol/g FW)
detection and quantitation limits (0.6 and 1.5 nmol/g, respectively). Repeatability and intermediate precision were also evaluated, giving RSD values of 8.9 and 10.9%, respectively. The quantitative method validation included the efficiency of the extraction, and it was evaluated in the same manner as that of flavonols. The results showed that with three extractions, a recovery of >90% of berberine was obtained, reaching 94.3% when four steps were carried out. Three extractions were considered optimal as a good compromise between accuracy and time/cost of analysis. I
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Table 4. Spectroscopic Characteristic of Alkaloidd Found in Berberis Genera from Patagony by HPLC-DAD-MS/MS tR (min)
λmax (nm)
[M + H]+
6.7
274
609
coridaline berberine
11.99 12.88
345 347
370 337
jatrorrhizine palmatine
11.96 12.66
344 344
338 352
berbamine
a
a
product ions 566, 305, 535, 552 322, 192, 307 321, 292, 278, 306 323, 294 337, 308
detected in
B. ilicifolia (fruits) B. microphylla (fruits, leaves, seeds, branches, and roots), B. montana, B. ilicifolia, B. empetrifolia, B. vulgaris B. microphylla (only in leaves) B. microphylla (only in leaves)
Not detected in the Berberis samples studied. Data were obtained for a standard solution.
and B. darwinii (57.7−156.1 nmol/g FW), whereas the upper concentrations are in berries of B. microphylla. In two samples of B. vulgaris collected from the University of Concepcion gardens, lower levels of berberine (1.8−3.3 nmol/g FW) were observed. The only B. montana analyzed contained 8.3 nmol/g FW berberine. The observed variations could be explained by different factors that can contribute to the biosynthesis of alkaloids in plants, such as the Berberis species, the place of collection, climate conditions, and number of seeds per fruit, as well as insect and disease attack.19 The distribution of berberine in calafate plants and fruit was evaluated using four samples. The results showed that berberine levels decreased with the distance of the analyzed structure from the roots. The highest levels were detected in the roots (4655.7 ± 1067.3 nmol/g FW), followed by the branches (1343.8 ± 981.1 nmol/g FW), and much lower levels in their fruits (32.7 ± 11.9 nmol/g FW) and leaves (35.7 ± 8.9 nmol/g FW). On the other hand, in two of these fruit samples, it was possible to analyze the berberine distribution, showing a higher level in the seeds (between 55.0 and 121.3 nmol/g FW) than in a sample of pulp mixed with the skin (between 3.6 and 6.2 nmol/g FW). Seeds are principally responsible of berberine content in the edible part of calafate fruits. This implies that in calafate products such as juices or jams, which are produced without seeds, berberine levels will be even lower than in whole fresh berries. Berberine levels were also studied in fruits with different phenological stages, using the same methods as in the flavonol study. A low concentration of berberine in the first stage (fruit setting) was observed (6.8 nmol/g FW), which increased toward maturation, reaching concentrations of 47.0 and 41.9 nmol/g FW in green berry and colored berry, respectively. This is consistent with the maturation process, if one of the functions of the seeds is to provide chemical protection to the plant during germination. LD50 of berberine has been previously reported in mice.20 According to these data and assuming a 7 times lower metabolic rate in humans,8,21 a consumption of 139 kg of calafate fruits, assuming the highest level of berberine detected in calafate berries in the present study, is necessary to reach the LD50 in humans. The berberine concentrations in calafate fruits suggest that they are innocuous for human consumption. On the other hand, considering the beneficial biological effect attributed by several authors to this molecule,22,6 its low presence in calafate fruit would not be relevant from a nutraceutical point of view; however, calafate roots and branches constitute an interesting source of this compound. It mediates the chemical defense of plants against viruses, bacteria, fungi, other plants, insects, and vertebrates,23 protecting the vegetative part of the plant and the seeds, but
not the edible part of the berry, facilitating the dissemination of the species, that is, by birds. Finally, the distribution of these phytochemicals in the calafate berries, the effect of the maturity process, the different growing latitudes, and the antioxidant capacity of the extract have influence on the nutraceutical potential of this wild and underexploited fruit.
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ASSOCIATED CONTENT
* Supporting Information S
Analytical parameters for flavonol quantification. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(C.M.) Phone: 56-41-2204598. Fax: 56-41-2226382. E-mail: [email protected]. Funding
We thank FONDECYT (Grant 1100944) and CONICYT, Chile (doctoral fellowship). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We thank Prof. Dr. Roberto Rodriguez, Director of Herbario CONC, and the Department of Botany, University of Concepción, for botanical classification of the Berberis samples.
(1) Marticorena, C.; Rodríguez, R. Flora de Chile Vol. 2: Berberidaceae−Betulaceae, 1st ed.; Universidad de Concepción: Concepción, Chile, 2003. (2) Ruiz, A.; Hermosin-Gutierrez, I.; Mardones, C.; Vergara, C.; Herlitz, E.; Vega, M.; Dorau, C.; Winterhalter, P.; von Baer, D. Polyphenols and antioxidant activity of calafate (Berberis microphylla) fruits and other native berries from southern Chile. J. Agric. Food Chem. 2010, 58, 6081−6089. (3) Ruiz, A.; Mardones, C.; Vergara, C.; Hermosín-Gutiérrez, I.; von Baer, D.; Hinrichsen, P.; Rodríguez, R. Analysis of hydroxycinnamic acids derivatives in calafate (Berberis microphylla G. Forst) berries by liquid chromatography with photodiode array and mass spectrometry detection. J. Chromatogr., A 2013, 1281, 38−45. (4) Ruiz, A.; Mardones, C.; Vergara, C.; von Baer, D.; GómezAlonso, S.; Gómez, M. V.; Hermosín-Gutiérrez, I. Isolation and structural elucidation of anthocyanidin 3,7-β-O-diglucosides and caffeoyl-glucaric acids from calafate berries. J. Agric. Food Chem. 2014, 62, 6918−6925. (5) Mokhber-Dezfuli, N.; Saeidnia, S.; Reza-Gohari, A.; KurepazMahmoodabadi, M. Phytochemistry and pharmacology of Berberis species. Pharmacogn. Rev. 2014, 8, 8−15.
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(6) Fajardo, V. Alcaloides en Especies del Género Berberis en Chile, ́ Quimica de la Flora de Chile, 1st ed.; Universidad de Chile: Santiago, Chile, 1992. (7) Kheir, M.; Wang, Y.; Hua, L.; Hu, J.; Li, L.; Lei, F.; Dua, L. J. Acute toxicity of berberine and its correlation with the blood concentration in mice. Food Chem. Toxicol. 2010, 48, 1105−1110. (8) Tillhon, M.; Ortiz, L. M. G.; Lombardi, P.; Scovassi, A. I. Berberine: new perpective for old remedies. Biochem. Pharmacol. 2012, 84, 1260−1267. (9) Marivil, P. Variación del perfil de alcaloides durante el crecimiento de ́ L. albus y en semillas de L. angustifolius. Tesis para optar al titulo de ́ Bioquimico, Universidad de Concepción, Concepción, Chile, 1995. (10) Wink, M.; Meißner, C.; Witte, L. Patterns of quinolizidine alkaloids in 56 species of the genus Lupinus. Phytochemistry 1995, 38, 139−153. (11) Castillo-Muñoz, N.; Gomez-Alonso, S.; Garcia-Romero, E.; Hermosin-Gutierrez, I. Flavonol profiles of Vitis vinifera red grapes and their single-cultivar wines. J. Agric. Food Chem. 2007, 55, 992−1002. (12) Hungria, M.; Joseph, C.; Phillips, D. Anthocyanidins and flavonols, major nod gene inducers from seeds of a black-seeded common bean (Phaseolus vulgaris L.). Plant Physiol. 1991, 97, 751− 758. (13) Meyer, V. Practical High-Performance Liquid Chromatography, 4th ed.; Wiley: Hoboken, NJ, USA, 2005. (14) Guiochon, G.; Gritti, F. Shell particles, trials, tribulations and triumphs. J. Chromatogr., A 2011, 1218, 1915−1938. (15) Cho, M. J.; Howard, L.; Prior, R.; Clark, J. Flavonol glycosides and antioxidant capacity of various blackberry and blueberry genotypes determined by high-performance liquid chromatography/mass spectrometry. J. Sci. Food Agric. 2005, 85, 2149−2158. (16) Arena, M. E.; Postemsky, P.; Curvetto, N. R. Flavonol glycosides and antioxidant capacity of various blackberry and blueberry genotypes determined by high-performance liquid chromatography/mass spectrometry. N. Z. J. Bot. 2002, 50, 15−28. (17) Rodarte Castrejon, A. D.; Eichholz, I.; Rohn, S.; Kroh, L. W.; Huyskens-Keil, S. Phenolic profile and antioxidant activity of highbush blueberry Vaccinium corymbosum L. during fruit maturation and ripening. Food Chem. 2008, 109, 564−572. (18) Wang, Y.; Huang, Y.; Yiming, L.; Aimin, X. Berberine prevents hyperglycemia-induced endothelial injury and enhances vasodilatation via adenosine monophosphate-activated protein kinase and endothelial nitric oxide synthase. Cardiovasc. Res. 2009, 82, 484−492. (19) Roberts, M. F.; Wink, M. Alkaloids: Biochemistry, Ecology, and Medical Applications, 1st ed.; Plenum Press: New York, 1998. (20) Yi, J.; Ye, X.; Wang, D.; He, K.; Yang, Y.; Liu, X.; Li, X. Safety evaluation of main alkaloids from Rhizoma coptidis. J. Ethnopharmacol. 2012, 145, 303−310. (21) Terpstra, A. H. M. Differences between humans and mice in efficacy of the body fat lowering effect of conjugated linoleic acid: role of metabolic rate. J. Nutr. 2001, 131, 2067−2068. (22) Chen, C.; Xu, X. M.; Chen, Y.; Xu, M. Y.; Wen, F. Y.; Zhang, H. Identification, quantitation and antioxidant activity of acylated flavonol glycosides from sea buckthorn (Hippophae rhamnoides ssp. sinensis). Food Chem. 2013, 141, 1573−1579. (23) Schmeller, T.; Latz-Brüning, B.; Wink, M. Biochemical activities of berberine, palmatine and sanguinarine mediating chemical deference against microorganisms and herbivores. Phytochemistry 1997, 44, 257−266.
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Flavonols, Alkaloids, and Antioxidant Capacity of Edible Wild Berberis Species from Patagonia Antonieta Ruiz,†,§ Moises Zapata,† Constanza Sabando,† Luis Bustamante,† Dietrich von Baer,† Carola Vergara,† and Claudia Mardones*,† †
Department of Instrumental Analysis, Faculty of Pharmacy, University of Concepción, P.O. Box 160-C, Concepción, Chile Department of Chemical Science and Natural Resources, University of La Frontera, Casilla 54-D, Temuco, Chile
§
S Supporting Information *
ABSTRACT: There are 20 species of the Berberidaceae family described in Chile, whose fruits are edible and show high anthocyanin and hydroxycinnamic acid levels. Berberis microphylla G. Forst, commonly known as calafate, is the most extensively distributed. Flavonols and alkaloids in seed, pulp, skin, and whole calafate berry extracts and other Berberis were studied using HPLC-DAD-ESI-MS/MS and HPLC with fluorescence detector. Berry samples from different locations in Chilean Patagonia, including different phenological stages, were systematically addressed. Results were compared with other organs of the plant and with other Berberis species. Total flavonol concentration in calafate (n = 65) was 1.33 ± 0.54 μmol/g. Glycosyl metabolites of quercetin and isorhamnetin were the most abundant. Similar profiles were observed in calafate from distinct locations, but important differences were observed for the other edible Berberis species. Calafate pulp and skin have higher flavonol concentrations than seeds, and the maturation process reduced its levels. TEACCUPRAC and TEACABTS of whole calafate extracts and fractions are also explored. Finally, only berberine was detected in the fruit (0.001%), mainly in seeds. Results contribute to the promotion of this berry as a superfruit from Patagonia. KEYWORDS: flavonols, alkaloids, HPLC-DAD-ESI-MS/MS, core−shell columns, berberine, Berberis microphylla G. Forst, antioxidant capacity
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species from Asia, Europe, and South America,5 in which high levels of berberine and jatrorrhizine were described. Berberine was also detected at high concentration in calafate roots from Patagonia;6 however, there is no scientific information about its presence in calafate berries, which constitute the only edible part of the plant. Acute toxicity of berberine has been studied in rats;7 however, this compound has also been described as antidiabetic, hepatoprotective, anticarcinogenic, and neuroprotective, among other effects.8 Considering these facts, the main aim of this research is to increase the knowledge of Berberis species, now from the perspective of their flavonol and alkaloid compositions and antioxidant capacity, in the context of nutraceutical potential and safety of Berberis berries, with emphasis on calafate. This research includes the study of an important number of calafate samples obtained from different locations around Patagonia in two successive collection years by using HPLC-DAD-ESI-MS/ MS and HPLC with fluorescence detection. A study of flavonol and alkaloid profiles and concentrations in calafate fruit with different phenological stages was also accomplished. The distribution of these compounds in the different parts of the fruit and plant and their comparison to other Berberis species are also discussed in this work. The antioxidant capacity of whole extracts and flavonol/hydroxycinnamic acids fractions
INTRODUCTION Berberis is the only genus of the Berberidaceae family found in South America. In Chile, 20 species of this genus have been described, with the most extensively distributed being Berberis microphylla G. Forst (commonly known as calafate), Berberis darwinii, and Berberis empetrifolia.1 Calafate is an endemic wildgrowing plant that is not cultivated and grows extensively in southern Patagonia in Chile and Argentina. Very high concentrations of anthocyanins and hydroxycinnamic acid derivatives (17.81 μmol/g fresh weight (FW) and 2.62 μmol/ g (FW) as mean, respectively) have been described in this fruit, making it a very promising berry from a nutraceutical perspective.2−4 It is necessary to study other bioactive compounds in this fruit and others of its genus to better understand its potential competence as a functional fruit. In a previous study carried out with eight mature calafate samples obtained in Patagonia, a range between 0.11 and 0.21 μmol/g (FW) of flavonols was reported, with the main compounds being myricetin and quercetin derivatives.2 There is no information about the flavonol distribution in the fruit (skin, pulp, and seed), the effect of the maturation process on their profiles and concentrations, or the effect of fruit-growing latitudes. All of this is important to understand the effect of these parameters on their potential as functional food. On the other hand, considering the presence of alkaloids reported in different species of this genus, their determination in calafate is also necessary to demonstrate its safety. In this context, isoquinolinic alkaloids have been detected in different structures (branches, leaves, roots, and berries) of Berberis © XXXX American Chemical Society
Received: July 3, 2014 Revised: November 28, 2014 Accepted: November 29, 2014
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Shimadzu (Kyoto, Japan). The data collection was done using an interactive software graphics version 6.2 Varian Inc. (Palo Alto, CA, USA). Sample Pretreatments. Flavonols. Whole extract preparation and cleanup were carried out on the basis of the methods described by Ruiz et al. and Castillo-Muñoz et al.2,11 with minor modifications. Five grams of whole fruit was ground in a stainless steel mixer with 10 mL of methanol/water (93:7% v/v); the extract was treated for 45 s with the ultrasonic bar, followed by 16 h of mechanical shaking under darkness. After centrifugation, the separated supernatant was transferred, and the residual solid was washed three times with 5 mL of the same solvent by mechanical shaking for 15 min. Cleanup of the combined extracts (25 mL) was made by using Oasis MCX cartridges, to eliminate the interfering anthocyanins. One milliliter of crude extract was dried in a rotary evaporator and reconstituted in 1.0 mL of 0.1 N hydrochloric acid. This solution was loaded on a MCX cartridge previously conditioned with 5.0 mL of methanol and 5.0 mL of water. A washing step, using 5.0 mL of 0.1 N hydrochloric acid and 5.0 mL of water, was necessary to remove sugars. The flavonol fraction was eluted with 3 × 5.0 mL of methanol and, finally, the solvent was distilled off by vacuum evaporation and the residue was redissolved in 1 mL of the mobile phase (0.1% formic acid in water/acetonitrile 85:15%v/v) for its chromatographic analysis. This fraction also contained the hydroxycinnamic acids. In the study of flavonol distribution in different parts of the fruits (seeds, pulp, and skin), frozen berries were peeled, the pulp was separated from the seed, and all were carefully dried with paper toweling. Each was separately ground before the same extraction procedure as described above was applied. Alkaloids. The method was adapted from a procedure described for alkaloid extraction of lupine seeds and plants,9 which was based on the method proposed by Wink,10 with modifications: 1 g of calafate sample (fruit, seeds, leaves, branches, or roots) was extracted with 8 mL of 0.1 N HCl with the assistance of an ultrasonic bar for 45 s and mechanical shaking for 10 min. After centrifugation, the supernatant was transferred to a 25 mL volumetric flask, and the solid residues were washed three times with 5 mL of the same solvent (in the case of fruits, seeds, and leaves) and 8 mL (in the case of roots and branches). The whole extract was neutralized using NH4OH and completed to 25 mL. Twenty milliliters of extract was transferred to an Extrelut NT-20 column, to carry out the liquid−liquid extraction, using dichloromethane as the elution solvent (4 × 25 mL). After that, the solvent was evaporated at reduced pressure (30 °C) and finally redissolved in 1 mL of mobile phase (0.1% of formic acid in water/acetonitrile 20:80% v/v) and filtered before its chromatographic analysis. Flavonol Analyses. HPLC-DAD. Three C18 columns were evaluated for flavonol HPLC separation: (A) C18 column (Kromasil C18 100 A, 5 μm, 250 × 4.6 mm) with a C18 precolumn (Nova-Pak Waters, 4 μm, 22 × 3.9 mm); (B) C18 core−shell column (Kinetex C18 100 A, 2.6 μm, 150 × 4.6 mm) with a C18 precolumn for UHPLC columns (Phenomenex AJ0-9000, 2.2 μm, 22 × 3.9 mm); (C) C18 UHPLC column (Shim-pack XR-ODS III, 2.2 μm, 150 × 2.0 mm) without a precolumn. The chromatographic separation conditions were optimized for each column and were carried out at 30 °C using 0.1% of formic acid in water (A) and acetonitrile (B) as mobile phase. The optimized separation using column A was carried out at a flow rate of 1 mL/min, with a gradient starting with 80% A for 6.5 min, from 80 to 76% in 6 min, from 76 to 60% in 2.5 min, and from 60 to 0% in 0.5 min, maintaining the proportion for 6.5 min, and a final stabilization step of 10 min at 80% A. The injection volume was 10 μL. The separation using column B was carried out also at 1 mL/min with a gradient starting with 85% A for 0.1 min, from 85 to 80% in 5 min, from 80 to 70% in 2.5 min, from 70 to 0% in 0.5 min, maintaining the proportion for 2 min, and with a final stabilization step of 5 min at 85% A. The injection volume was 5 μL. Finally, the separation in column C was carried out at a flow rate of 0.5 mL/min, with a gradient starting with 85% A for 12 min, from 85 to 60% in 3 min, from 60 to 0% in 0.5 min, maintaining the proportion for 5 min, and with a final stabilization step of 7 min at 85% A. The injection volume in this case was also 5 μL.
are also explored in relation with their concentrations to evalute their contribution to the antioxidant capacity of calafate. This research is a contribution to promote, from a scientific perspective, the potential of this underexploited fruit, complementing previous work about anthocyanins and hydroxycinnamic acids now with flavonol and alkaloid levels and antioxidant capacity.
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MATERIALS AND METHODS
Reagents, Standards, and Extraction Materials. Commercial standards of quercetin-3-rutinoside (97%), quercetin-3-glucoside (>90%), quercetin-3-rhamnoside (85%), quercetin-3-O-(6″malonyl)-D-β-glucoside (85%), berbamine dihydrochloride, and berberine hydrochloride (95% of the flavonols were extracted, which is a good compromise between extraction efficiency and extraction time. The internal precision of flavonol determination in calafate samples, considering the whole method including liquid−solid E
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Figure 3. Individual flavonol concentrations in 65 samples of B. microphylla G. Forst obtained in Magallanes, Aysen, and Los Lagos and Araucania.
between samples obtained in 2011 and 2012 (1.34 ± 0.55 and 1.32 ± 0.49 μmol/g FW, respectively). Samples from more southerly latitudes (Magallanes 51−54° S) showed higher concentrations than those from the northern ones (Aysen 43− 47° S) and Chiloe Main Island (42° S). This trend is similar to that observed for hydroxycinnamic acid derivates,3 in which a decrease in their concentrations was observed from south to north. However, in the flavonol study, an exception was observed for four samples obtained further north, in Gorbea (39° 06′ S; 72° 40′ W in the Araucaniá region), which showed similar mean concentrations to the Magallanes samples. The quantitative results by region are summarized in Table 2. The profile and concentration of flavonols in mature samples of calafate (B. microphylla) were compared with those for mature berry samples of other endemic Berberis species obtained in the same study zone (between Bio-Bio and Magallanes regions). The respective flavonol chromatograms for the Berberis species are presented in Figure 4. The results showed different profiles for the species, especially for B. empetrifolia, B. ilicifolia, and B. darwinii, in comparison with B. microphylla. In the first one, only three flavonols (rutinoside, galactoside, and glucoside derivatives of quercetin) predominated, only traces of other flavonols being observed (isorhamnetin-3-rutinoside, isorhamnetin-3-galactoside, and isorhamnetin-3 malonylglucoside). In B. ilicifolia, isorhamnetin-3-rutinoside predominated, whereas quercetin-3rhamnoside, quercetin-malonyl derivatives, and the other isorhamnetin malonyl derivatives were absent. In B. darwinii, the absence of isorhamnetin-3-rutinoside and isorhamnetin-3galactoside was noted, with a similar proportion of the other flavonols to B. microphylla. B. montana and B. vulgaris presented flavonol profiles similar to that of calafate, with similar proportions of them. This observation is interesting because
extraction, SPE cleanup steps, and chromatographic separation, was 7.9%, and the extracts were stable for 7 months at −20 °C. The analytical response and other analytical parameters for the main flavonols are summarized in the Supporting Information. Total flavonols were determined by the sum of individual flavonols detected over the limit of quantitation of the method. Flavonol Quantitation in Calafate and Other Berberis Berries. Sixty-five samples of calafate berries obtained at different latitudes in Patagonia were analyzed. In all of them, 16 flavonol derivates were detected. The most abundant was quercetin-3-rutinoside, showing a mean concentration of 0.32 ± 0.15 μmol/g FW, with a range from traces to 0.74 μmol/g FW. Quercetin-3-galactoside, quercetin-3-rhamnoside, and isorhamnetin-3-rutinoside also showed interesting concentrations of 0.24 ± 0.16, 0.25 ± 0.12, and 0.24 ± 0.14 μmol/g FW, respectively. For the other quercetin and isorhamnetin derivates, their mean concentrations were 60% of the total flavonols in seed). It is important to note that this flavonol is one of the predominant flavonols in calafate. This distribution within the different structures of the fruit is very interesting and must be explained by the calafate plant metabolism; however, this has not been studied until now. The profile and concentrations of flavonols in different phenological stages was evaluated using berry samples obtained from the same plant in the stages of fruit setting, green fruit, and full-colored fruit. The samples were taken on December 15, 2011; January 15, 2012; and February 15, 2012, respectively. Results showed that the flavonol profile changed in this process. Quercetin-3-malonylglucoside decreased, whereas quercetin-3rhamnoside increased, and the overall flavonol concentration decreased to 75% of the initial level. These results are presented in Figure 5. These observations are similar to those previously obtained for hydroxycinnamic acid derivatives in calafate3 and are correlated with those observed by Arena et al.16 for total polyphenol and flavonoid contents in calafate. The phenolic content decrease has also been observed for other berries, including different cultivars of the blueberry,17 in which a change in the pool of total phenolics toward anthocyanin synthesis, with an overall decline in the content of other phenolic components, is observed during ripening. These findings about flavonols in calafate are very relevant because of the enhancement of calafate and other Berberis from a nutraceutical point of view. They also contributed to finding the optimal growing time and conditions. Reducing Capacity of Whole and SPE Fractions Calafate Extracts. Four previously quantitated fractions obtained by SPE were subjected to in vitro antioxidant capacity assays (TEACCUPRAC and TEACABTS). This was carried out to evaluate the contribution of these anthocyanin free fractions on the antioxidant capacity of calafate. These fractions contained mainly flavonols and hydroxycinnamic acids. Two of these samples (C1 and C2) had low concentrations of these compounds, whereas the other two were included due to its high concentrations (C3 and C4). In Table 3 are summarized these results. High TEACCUPRAC and TEACABTS values were observed for whole extracts with high total phenolics (C1 and C4). This is explained mainly due to the high anthocyanin concentrations present in these extracts. Also, it is possible to observe a trend of higher TEACCUPRAC and TEACABTS values for SPE fractions with high flavonol−hydroxycinnamic acid concentrations (C3 and C4), compared with the fraction with minor concentrations of this compounds (C1 and C2); however, this trend is less clear than those observed for whole extracts. The antioxidant capacities obtained for flavonol plus hydroxycinnamic acid fractions represent 15 ± 5 and 5 ± 4% of TEACCUPRAC and TEACABTS values, respectively, in comparison with the values obtained for the whole extracts. This shows that flavonols and hydroxycinnamic acids have a notable incidence in the antioxidant capacity of calafate, despite its lower concentration in comparison with anthocyanins. Alkaloid Identification and Quantitation in Calafate and Other Berberis Berries. Alkaloid determination was done with the objective of guaranteeing the innocuousness of Berberis. The identification of alkaloids in calafate and other
0.91−1.16
0.97−1.32 1.75−2.22 2.19−2.63
0.94−1.33 1.61−1.80 1.25−2.09 1.37−1.90 1.64−1.78
4 2 4 3 2 1 1
0.71 0.87 0.83 0.92 1.04 0.94 0.77
± 0.33 ± 0.51 ± 0.10 ± 0.71 ± 0.29 ± 0.00 ± 0.00
0.27−0.97 0.51−1.20 0.74−0.94 0.71−1.24 0.83−1.24
4
1.52 ± 0.10
1.38−1.58
B. montana and B. microphylla are taxonomically similar,1 which could explain their similar profiles. However, B. vulgaris is an introduced species, which is used as an ornamental plant in gardens and is very different from B. microphylla. The quantitative results of the 16 samples of other Berberis species were compared with the results for calafate samples summarized in Table 2. The total flavonol concentration of B. microphylla was less than that observed for B. montana samples (n = 4), showing 1.33 ± 0.54 and 1.61 ± 0.64 μmol/g FW, respectively, and for B. empetrifolia (2.01 μmol/g FW, n = 1), but was higher than that observed for B. ilicifolia (0.71 ± 0.18 μmol/g FW, n = 1), B. darwinii (1.04 ± 0.31 μmol/g FW, n = 6), and the only available sample of B. vulgaris (0.72 μmol/g FW), which was obtained from University of Concepción gardens. The flavonol concentration in different parts of the calafate fruit (skin, pulp, and seeds) was determined in two composed samples, one of them from Magallanes and the other from the Aysen region. The results were similar for samples from both G
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Figure 4. Chromatographic profiles of flavonols extract of different Berberis species, by HPLC-DAD using core−shell column: (A) B. empetrifolia (sample from Parque Pinguino Rey, Provenir, Tierra del Fuego); (B) B. vulgaris (sample from gardens of University of Concepción, Concepción); (C) B. darwinii (sample from Gorbea, Temuco); (D) B. ilicifolia (sample from Lago Deseado, Porvenir, Magallanes); (E) B. montana (sample from Valle Las Trancas, Chillan); ( (F) B. microphylla 2012 (sample from Lago Fagnano, Magallanes); (G) B. microphylla 2011 (sample from Seno Otway, Magallanes).
Berberis species was done by comparison of tR with commercial standards. The identity confirmation was based on their MS/ MS and UV−vis spectra. Table 4 presents spectroscopic characteristics of alkaloids detected in different Berberis structures. In all calafate fruits analyzed in this part of the research (n = 62), only berberine was detected. However, jatrorrhizine and palmatine were detected at trace levels in calafate leaves. The alkaloid coridaline was assigned to a signal
found in fruits of B. ilicifolia based on the MS/MS spectra, in accordance with Wang et al.18 Quantitative analysis of berberine was carried out by HPLC with fluorescence detection using an external calibration curve, which was performed by triplicate injection at six levels of berberine standard concentration. The calibration curve showed a low level of dispersion, with a regression coefficient of 0.9993 and an equation of (y = 560704.27x − 80514.59). Fluorescence detection showed a high sensitivity and low H
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Figure 5. Flavonols profile and concentration during the maturation process of B. microphylla G. Forst (sample from Sector Barranco Amarillo, Magallanes).
Table 3. In Vitro Antioxidant Capacity (TEACCUPRAC and TEACABTS) of SPE Fractions and Whole Extracts of B. microphylla G. Forsta whole calafate extracts total phenolic concn (μmol/g FW) calafate calafate calafate calafate
1 2 3 4
32.63 3.06 11.56 37.68
± ± ± ±
flavonols concn (μmol/g FW) calafate calafate calafate calafate
1 2 3 4
0.26 1.56 0.97 1.44
± ± ± ±
0.01 0.04 0.01 0.03
0.14 0.04 0.03 0.54
TEACCUPRAC (μmol TE/g FW) 405.95 179.61 224.68 577.73 SPE fractions (flavonols
HCADs concn (μmol/g FW) 0.68 0.89 4.21 4.88
± ± ± ±
0.00 0.00 0.02 0.00
± 33.01 ± 12.12 ± 12.19 ± 35,.5 + HCADs)
105.48 71.56 106.92 133.47
TEACCUPRAC (μmol TE/g FW) 34.87 35.79 42.88 66.85
± ± ± ±
TEACABTS (μmol TE/g FW)
7.44 0.69 11.39 11.27
± ± ± ±
4.43 0.12 15.92 3.17
TEACABTS (μmol TE/g FW) 4.69 5.29 5.68 11.45
± ± ± ±
0.14 0.14 0.18 0.44
a The identifications of each sample are, respectively, calafate 1, Puerto tranquilo, Aysen; calafate 2, Gorbea, Temuco; calafate 3, Rio San Luis, Magallanes; calafate 4, Camino Provenir. Km 25, Magallanes. HCADs, hydroxycinnamic acids.
Berberine levels were evaluated in fruits of different Berberis species, showing low concentrations; however, some variations between the different samples were observed. The highest concentration of berberine was detected in B. ilicifolia (n = 3) with 160.0 nmol/g FW. In calafate fruits (B. microphylla) collected in the year 2011 (n = 45) levels of berberine were between 1.8 and 126.9 nmol/g FW, whereas in samples collected in 2012 (n = 18) concentrations between 3.3 and 152.8 nmol/g FW were determined. Higher contents of berberine were found in B. ilicifolia (55.9−160.5 nmol/g FW)
detection and quantitation limits (0.6 and 1.5 nmol/g, respectively). Repeatability and intermediate precision were also evaluated, giving RSD values of 8.9 and 10.9%, respectively. The quantitative method validation included the efficiency of the extraction, and it was evaluated in the same manner as that of flavonols. The results showed that with three extractions, a recovery of >90% of berberine was obtained, reaching 94.3% when four steps were carried out. Three extractions were considered optimal as a good compromise between accuracy and time/cost of analysis. I
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Table 4. Spectroscopic Characteristic of Alkaloidd Found in Berberis Genera from Patagony by HPLC-DAD-MS/MS tR (min)
λmax (nm)
[M + H]+
6.7
274
609
coridaline berberine
11.99 12.88
345 347
370 337
jatrorrhizine palmatine
11.96 12.66
344 344
338 352
berbamine
a
a
product ions 566, 305, 535, 552 322, 192, 307 321, 292, 278, 306 323, 294 337, 308
detected in
B. ilicifolia (fruits) B. microphylla (fruits, leaves, seeds, branches, and roots), B. montana, B. ilicifolia, B. empetrifolia, B. vulgaris B. microphylla (only in leaves) B. microphylla (only in leaves)
Not detected in the Berberis samples studied. Data were obtained for a standard solution.
and B. darwinii (57.7−156.1 nmol/g FW), whereas the upper concentrations are in berries of B. microphylla. In two samples of B. vulgaris collected from the University of Concepcion gardens, lower levels of berberine (1.8−3.3 nmol/g FW) were observed. The only B. montana analyzed contained 8.3 nmol/g FW berberine. The observed variations could be explained by different factors that can contribute to the biosynthesis of alkaloids in plants, such as the Berberis species, the place of collection, climate conditions, and number of seeds per fruit, as well as insect and disease attack.19 The distribution of berberine in calafate plants and fruit was evaluated using four samples. The results showed that berberine levels decreased with the distance of the analyzed structure from the roots. The highest levels were detected in the roots (4655.7 ± 1067.3 nmol/g FW), followed by the branches (1343.8 ± 981.1 nmol/g FW), and much lower levels in their fruits (32.7 ± 11.9 nmol/g FW) and leaves (35.7 ± 8.9 nmol/g FW). On the other hand, in two of these fruit samples, it was possible to analyze the berberine distribution, showing a higher level in the seeds (between 55.0 and 121.3 nmol/g FW) than in a sample of pulp mixed with the skin (between 3.6 and 6.2 nmol/g FW). Seeds are principally responsible of berberine content in the edible part of calafate fruits. This implies that in calafate products such as juices or jams, which are produced without seeds, berberine levels will be even lower than in whole fresh berries. Berberine levels were also studied in fruits with different phenological stages, using the same methods as in the flavonol study. A low concentration of berberine in the first stage (fruit setting) was observed (6.8 nmol/g FW), which increased toward maturation, reaching concentrations of 47.0 and 41.9 nmol/g FW in green berry and colored berry, respectively. This is consistent with the maturation process, if one of the functions of the seeds is to provide chemical protection to the plant during germination. LD50 of berberine has been previously reported in mice.20 According to these data and assuming a 7 times lower metabolic rate in humans,8,21 a consumption of 139 kg of calafate fruits, assuming the highest level of berberine detected in calafate berries in the present study, is necessary to reach the LD50 in humans. The berberine concentrations in calafate fruits suggest that they are innocuous for human consumption. On the other hand, considering the beneficial biological effect attributed by several authors to this molecule,22,6 its low presence in calafate fruit would not be relevant from a nutraceutical point of view; however, calafate roots and branches constitute an interesting source of this compound. It mediates the chemical defense of plants against viruses, bacteria, fungi, other plants, insects, and vertebrates,23 protecting the vegetative part of the plant and the seeds, but
not the edible part of the berry, facilitating the dissemination of the species, that is, by birds. Finally, the distribution of these phytochemicals in the calafate berries, the effect of the maturity process, the different growing latitudes, and the antioxidant capacity of the extract have influence on the nutraceutical potential of this wild and underexploited fruit.
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ASSOCIATED CONTENT
* Supporting Information S
Analytical parameters for flavonol quantification. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(C.M.) Phone: 56-41-2204598. Fax: 56-41-2226382. E-mail: [email protected]. Funding
We thank FONDECYT (Grant 1100944) and CONICYT, Chile (doctoral fellowship). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We thank Prof. Dr. Roberto Rodriguez, Director of Herbario CONC, and the Department of Botany, University of Concepción, for botanical classification of the Berberis samples.
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