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Keywords:

  • Andes berries;
  • anthocyanins;
  • antioxidant properties

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. References

ABSTRACT:  Anthocyanins, total phenolic content, ascorbic acid content, and the antioxidant activity were determined in extracts of Andes berry fruit (Rubus glaucus Benth). Anthocyanis (ACNs) were isolated and characterized by means of high-performance liquid chromatography (HPLC) with photodiode array detection and electro spray ionization/mass spectrometry (PDA-ESI/MS/MS) analysis. The anthocyanin (ACN) content was 45 mg/100 g FW. The isolated anthocyanins were characterized as cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylorutinoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, and pelargonidin 3-rutinoside. The ascorbic acid content was 10.1 mg/100 g FW. The total phenolic content as determined by the Folin–Ciocalteau method was 294 mg GAE/100 g FW while the antioxidant activity as measured by ABTS·+ radical scavenging capacity and ferric reducing antioxidant power (FRAP) was 2.01 and 4.50 mmol TE/100 g FW or 8.22 mmoles ferric iron reduced/100 g FW, respectively. The high phenolic content and antioxidant capacity of Andes berry suggest that this fruit could be a rich source of natural pigments, nutraceuticals, and natural antioxidants.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. References

Epidemiological studies have shown the importance of fruit consumption in the human diet for the prevention of coronary heart disease, cancer, diabetes, and stroke. The protective effects of fruits have been attributed to the presence of polyphenols and ascorbic acid (Wang and others 1996). Particular attention has been devoted to Rubus species because of their high antioxidant activity, anthocyanin pigment, and phenolic content (Deighton and others 2000; Moyer and others 2002; Sellappan and others 2002; Shahidi and Naczk 2003).

Rubus represents a diverse genus of plants distributed globally as wild and cultivated species and genotypes. Such a variety is reflected in a wide range of fruit types and pigmentation found within the genus. As a result, there is a significant variation in the anthocyanin content and antioxidant capacities of existing Rubus species (Deighton and others 2000).

Andes berry (Rubus glaucus Benth) is a climbing perennial shrub belonging to the Rosaceae family and native to the Andes in northern South America, where it grows year round. Andes berry fruit is appreciated for its attractive color (dark-red), tartness, juiciness, and superior flavor, and quality in comparison to most cultivated blackberries and raspberries (Popenoe and others 1989).

In Colombia, the yearly production of Andes berry fruit is around 80000 tons. A considerable portion of this crop is processed into natural juices, marmalades and jellies. However, around 30% of the harvested product is discarded due to difficulties in postharvest conservation and high perishability of the fruit (Agrios 2001). These byproducts may be a potential source of natural colorants, nutraceuticals, and natural antioxidants.

Although Rubus anthocyanins and antioxidant activity from several species have been well characterized (Sapers and others 1986; Fan-Chiang 1999; Moyer and others 2002; Shahidi and Naczk 2003) there are no reports documenting the ACN profile or antioxidant activity of Andes berry.

Because of its high availability and potential as a source of natural colorants and nutraceuticals, identification of ACNs, determination of total phenolics, antioxidant activity, and ascorbic acid content in Andes berry is of interest to researchers and may have commercial potential for applications in new products within Colombia and elsewhere. Accordingly, the 1st objective of this study was to use high-performance liquid chromatography (HPLC) with photodiode array detection and electrospray ionization/mass spectrometry (PDA-ESI/MS/MS) to evaluate the anthocyanin composition of Andes berry. Additional objectives were to measure the amounts of ascorbic acid, total phenolics, total monomeric anthocyanins, ABTS radical scavenging capacity, and ferric reducing antioxidant power (FRAP) of Andes berry extracts.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. References

Fruit

Fresh ripe Andes berry (Rubus glaucus Benth) was obtained from a local market in Bogota, Colombia. The fruit was classified according to color, size, soluble solids content (°Brix) using a digital refractrometer Abbe II (Reichert-Jung, Leica Inc., Buffalo, N.Y., U.S.A.), and total titratable acidity (TA). The TA was determined as grams citric acid per 100 g fruit by titrating a sample of fruit extract with 0.1 N NaOH to a final pH of 8.2. The measurement of the pH was done with a Schott Gerate® pH meter, model CG820 (Mainz, Germany).

Reagents

Cyanidin 3-glucoside was purchased from Polyphenols Laboratories (Hanaveien, Norway), formic acid and acetonitrile were purchased from Fisher Scientific (Fair Lawn, N.J., U.S.A.). Sodium carbonate, potassium persulfate, and Folin–Ciocalteau reagent were purchased from Merck® (Darmstadt, Germany). Trolox (6-hydroxy-2,5,7,8-tetramethychromane-2-carboxylic acid), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), TPTZ (2,4,6-tripyridyl-s-triazine), and gallic acid were from Chemical Co. (St. Louis, Mo., U.S.A.). L-ascorbic acid, DTT (1,4-dithiothreitol), and FeCl3·6H2O were purchased from Fisher Scientific Co.

Extraction of anthocyanins and polyphenolics

For extraction and isolation of anthocyanins and polyphenolics, the method described by Rodriguez and Wrolstad (2001) was followed. Plant material was powdered in a stainless steel Waring blender (Waring Commercial, Torrington, Conn., U.S.A.) after addition of liquid nitrogen. The powder was mixed with 2 L acetone/kg material and the extracted material was separated from the cake by filtration on a Buchner funnel. The filter cake remnant was reextracted with 70% (v/v) aqueous acetone twice. Filtrates were combined and partitioned with chloroform (1: 2 acetone: chloroform, v/v) and stored overnight at 1 °C. The aqueous portion containing the ACNs was recovered and separated from the residual acetone in a Buchi rotoevaporator at 40 °C and resolubilized in 0.01% HCl. Extractions were replicated 4 times. All extracts were stored at −70 °C until analyzed.

Monomeric anthocyanin content

Monomeric ACN pigment content was determined by the pH differential method described by Giusti and Wrolstad (2001). Absorbencies were read at 510 and 700 nm. For comparison purposes, pigment content was calculated as cyanidin 3-glucoside using an extinction coefficient (∈) of 26900 L/cm-mol and molecular weight of 449.2. Measurements were replicated 4 times with means being reported.

Solid-phase extraction with C18 resin

Cleaning of the aqueous extract containing the ACN and phenolic fractions was achieved with a C-18 cartridge (high load C-18 tube), 20 mL capacity (Alltech Assoc., Inc., Ill., U.S.A.). The minicolumn was activated with methanol, which was subsequently eluted with 0.01% HCl (v/v). After loading the extract, sugars, acids, and other water-soluble compounds were removed with acidified water. Polyphenolics other than ACNs were subsequently eluted with ethyl acetate and the ACNs were then recovered with methanol containing 0.01% HCl (v/v). Methanol was evaporated using a rotary evaporator at 40 °C and the remaining pigment was freeze dried until further analysis.

HPLC-MS/MS of anthocyanins

HPLC analysis was carried out on a Waters 2695 gradient HPLC separation module (Waters Corp., Milford, Mass., U.S.A.) equipped with an autoinjector and a 996-photodiode array detector (PDA). Chromatographic separations were performed on a C18 Symmetry column (75 mm × 4.6 mm i.d., 3.5 μm) (Waters Corp.). The mobile phase consisted of 5% (v/v) formic acid in water (solvent A) and 5% (v/v) formic acid in acetonitrile (solvent B) at a flow rate of 1.0 mL/min. A gradient from 0% B to 20% B over 8 min was applied. Absorbance spectra were recorded every 1.2 nm from 200 to 700 nm. The eluate was split 1: 10 before introducing into the mass spectrometer.

Mass spectrometry was performed on a quadrupole/time-of-flight mass spectrometer (QTof Premier, Micromass Limited, Manchester, U.K.) equipped with an electrospray ionization (ESI) source. Positive ion ESI conditions for anthocyanin analysis included a capillary voltage of 3.2 kV, cone voltage of 35 V, ion guide at 1 V, source temperature of 100 °C, and nitrogen desolvation gas temperature of 400 °C flowing at 600 L/h. During collisionally induced dissociation (CID) experiments, argon was held at a pressure of 3 mbar. Cyanidin 3-glucoside was used as a standard for identification. UV-visible spectral characteristics, molecular masses (m/z), and fragmentation pattern compared to previous work with black raspberry extracts using the same HPLC system (Tian and others 2005a) assisted identification of the compounds.

Total phenolics

Total phenolics were determined as gallic acid equivalents GAE/100 g FW using the method described by Waterhouse (2001). A 20-μL sample aliquot of extract or gallic acid standard (50 to 500 mg/L) was mixed with 1.58 mL water followed by 100 μL Folin–Ciocalteau's reagent. After vortexing and incubating at room temperature for 8 min, 300 μL of 20% aqueous sodium carbonate solution were added. Samples were vortexed and held at room temperature for 2 h. Absorbance of the blue-color solution was recorded at 765 nm on a Shimadzu UV visible spectrophotometer, model UV 160 U (Japan), using 1-cm disposable cells. All measurements were replicated 4 times.

Antioxidant activity

Antioxidant properties were determined by the ABTS and FRAP assays. ABTS·+ radical cation scavenging activity was determined according to the method described by Re and others (1999). ABTS·+ was produced by mixing ABTS stock solution (7 mm in water) with 2.45 mm potassium persulfate (final concentration). The solution was held at room temperature in the dark for 16 h before use. Once the radical was formed, the absorbance at 734 nm was adjusted to 0.7 by dilution with 95% ethanol. Fresh ABTS·+ solution was prepared for each analysis. ABTS·+ (1 mL) was added to 10 μL sample and the reaction mixture was allowed to stand at 30 °C for 6 min and the absorbance at 734 nm was immediately recorded. The percentage decrease of the absorbance at 734 nm was calculated by the formula I = [(ABAA)/AB]× 100; where: I = ABTS·+ inhibition, %; AB = absorbency of a blank sample (t = 0 min); AA = absorbency of a tested extract solution at the end of the reaction.

A standard curve was obtained by using Trolox standard solution at various concentrations (0.5, 1, 1.5, 2 mM) with ethanol. The absorbance of the reaction samples was compared to that of the Trolox standard and the results were expressed in terms of mmoles Trolox equivalents (TE)/100 g FW. All measurements were replicated 4 times.

The FRAP assay was performed according to the method of Benzie and Strain (1999). FRAP reagent was prepared by mixing 2.5 mL TPTZ solution (10 mM in 40 mM HCl), 25 mL acetate buffer (300 mM, pH 3.6), and 2.5 mL of FeCl3.6H2O solution (20 mM). After 4-min incubation at 37 °C, the reagent was used as a blank by determining the absorbance at 593 nm. FRAP solution (900 μL) was added to 90 μL of distilled water and 30 μL of standards or extracts. Standard curves using trolox (0 to 0.25 mM) and ferrous sulphate (0 to 0.5 mM) were run with each set of extracts. The reaction of the samples was followed until it reached the plateau. Final results were expressed as mmoles ferric iron reduced/100 g FW or as mmoles TE/100 g FW.

Ascorbic acid content

Ascorbic acid content was determined using the method described by Garzón and Wrolstad (2002) with modifications. A fruit sample (20 g) was processed in a Warring blender with 80 mL with deionized water. After centrifugation of the sample at 6000 RPM for 10 min, the extract was collected and its pH was adjusted to 5 to 5.2 with NaOH 0.1 N. Ascorbic acid was kept in the reduced form by adding 0.1% 1,4-dithiothreitol (DTT). After 2 h reaction with DTT, the sample was filtered through a 0.45-μm millipore filter (type HA) and analyzed by HPLC.

HPLC analysis of total ascorbic acid was carried out using Shimadzu LC-20AD/T HPLC equipped with a SPD-6AUV detector (Kyoto, Japan) and a LiChrosorb RP 18 column (5 micron) 250 × 4.6 mm (E Merch, Inc., Darmstadt, Germany). The mobile phase was 2% KH2PO4, pH 2.5 with 0.1% DTT and the running conditions were: isocratic program for 15 min, elution at 0.5 mL/min, injection volume 20 μL, detection at 254 nm.

For quantitation of ascorbic acid in the samples, a standard curve of ascorbic acid (10 to 50 ppm) was developed. Linear regression analysis was applied to determine the ascorbic acid concentration in the extract.

Statistical analysis

The total monomeric ACN content, ascorbic acid content, antioxidant activity, and total phenolic content were calculated as mean ± SD (n = 4).

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. References

Total anthocyanin content

The total monomeric ACN content in Andes berry was 45 ± 7.07 mg/100 g FW. In comparison to reports by other researchers, this anthocyanin concentration is lower than that of blackberries (80 to 230 mg/100 g FW) and black raspberries (464 to 627 mg/100 g FW) (Moyer and others 2002; Siriwoharn and Wrolstad 2004; Fan-Chiang and Wrolstad 2005), but close to that of some raspberry genotypes (Rotundo and others 1998; Moyer and others 2002; Benvenuti and others 2004).

Characterization of Andes berry anthocyanins

Figure 1A is the HPLC–PDA chromatogram (518 nm) of Andes berry extract. Although peaks 1 and 2 were unresolved by PDA due to the coelution of anthocyanins, mass spectrometry analysis indicated the presence of 6 anthocyanins with unique elution times, whose extracted ion chromatograms are presented in Figure 2A to 2F. Peaks 1, 5, and 6 were minor peaks representing 5% of the total area while the 3 major peaks (2, 3, and 4) accounted for 40%, 10%, and 45% of the total area, respectively. Percentages were calculated as the individual MS area divided by the total MS area for the 6 anthocyanins. Assuming similar extinction coefficients for the various species, we found that MS predicted the PDA profile in a reliable manner.

image

Figure 1—. HPLC–PDA chromatogram of Rubus glaucus Benth detected at 518 nm. Peak 1, cyanidin 3-sambubioside; peak 2, cyanidin 3-glucoside; peak 3, cyanidin 3-xylorutinoside; peak 4, cyanidin 3-rutinoside; peak 5, pelargonidin 3-glucoside; peak 6, pelargonidin 3-rutinoside.

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image

Figure 2—. Extracted ion chromatograms of anthocyanins present in Rubus glaucus Benth. (A) cyanidin 3-sambubioside, (B) cyanidin 3-glucoside, (C) cyanidin 3-xylorutinoside, (D) cyanidin 3-rutinoside, (E) pelargonidin 3-glucoside, (F) pelargonidin 3-rutinoside.

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Mass spectral data of the anthocyanins in Andes berry are shown in Table 1. This information along with UV-visible spectral characteristics of the compounds was used to confirm their identity. The Abs440/Absλmax ratios for all compounds ranged from 0.31 to 0.49. These values are typical of 3-glycosylated anthocyanidins (Hong and Wrolstad 1990). In addition, only weak UV absorption was detected in the 310 to 320 nm region, which indicates no acylation with hydroxycinnamic acids (Hong and Wrolstad 1990).

Table 1—.  Chromatographic and spectroscopic characteristics of anthocyanins detected in Andes berry (Rubus glaucus Benth).
Peaktr (min)Anthocyanin assignmentMS/MS
Parent ion M+(m/z)Fragment ions M+-X (m/z)
  1. Cy = cyanidin; Pg = pelargonidin; gluc = glucose; rham = rhamnose; rut = rutinose; sam = sambubiose; xylrut = xylorutinose.

16.58Cy 3-sambubioside581.15287.06 (M+-sam)
26.60Cy 3-glucoside449.11287.06 (M+-glu)
36.77Cy 3-xylorutinoside727.21287.06 (M+-xylrut), 581 (M+-rham), 433 (M+-rham-gluc)
46.94Cy 3-rutinoside595.16287.06 (M+-rut), 449 (M+-rham)
57.16Pg 3-glucoside433.10271.05 (M+-glu)
67.59Pg 3-rutinoside579.15271.05 (M+-rut)

Compound 1 was characterized by MS/MS analysis as cyanidin 3-sambubioside since it exhibited a parent ion of m/z 581 and a daughter ion at m/z 287 corresponding to the aglycon cyanidin. Cyanidin 3-sambubioside has been shown to fragment directly to the aglycone without evidence for intermediate fragments such as cyanidin 3-glucoside (m/z 449). This has been rationalized as due to high relative stability of the xylose–glucose glycosidic bond compared to the glucose to cyanidin bond, which is the preferred fragmentation site (Tian and others 2005b).

Peaks 2, 3, and 4 exhibited a λmax at 516, 521, and 517 nm, respectively, and were characterized as cyanidin derivatives. Peak 2, which was identified as cyanidin 3-glucoside exhibited a molecular cation of m/z 449 and a fragment ion at m/z 287 ([M-glucose]+). Compound 3 was characterized as cyanidin 3-xylorutinoside. It exhibited a parent ion of m/z a 727, a daughter ion of m/z 581 corresponding to the loss of rhamnose ([M- rhamnose]+), an aglycone at m/z 287 (cyanidin), and a fragment at m/z 433 corresponding to the loss of glucose and rhamnose ([M-rhamnose-glucose]+). This last fragment does not match any expected fragments after neutral loss of the constituent sugars as ([M-rhamnose]+) = 581 m/z, ([M-xylose]+) = 595 m/z, ([M-rhamnose-xylose]+) = 449 m/z, and ([M-rhamnose-xylose-glucose]+) = 287 m/z. It has been suggested that 433 m/z may represent cyanidin rhamnoside (Wu and Prior 2005), but considering the structure this fragment should not be possible unless the NMR analysis was in error. Recently, another report has emerged with the structural elucidation of cyanidin xylorutinoside in black raspberry by multidimensional NMR (Tulio and others 2008) and found consistent with previous reports that all 3 sugars are attached to one another with linkage to cyanidin via the glucose moiety. Based on this information, black raspberry can serve as a validated reference standard for cyanidin xylorutinoside and the other major cyanidin glycosides on our system.

Peak 4 was labeled as cyanidin 3-rutinoside. It exhibited a molecular cation of m/z 595, an aglycon of m/z 287 (cyanidin), and a daughter ion at m/z 449 corresponding to the loss of rhamnose ([M-rhamnose]+).

Peaks 5 and 6 were identified as pelargonidin 3-glucoside and pelargonidin 3-rutinoside. They had an absorbance spectrum with a λmax of 501 and 504 nm, respectively, and a pronounced shoulder in the 400 to 450 nm region typical of pelargonidin derivatives (Hong and Wrolstad 1990). Their mass spectrum contained parent ions of m/z 433 and 579, respectively, and both exhibited a fragment ion at m/z 271 (pelargonidin aglycon).

The major ACNs found for Rubus glaucus Benth (cyanidin 3-glucoside and cyanidin 3-rutinoside) are similar to those present in 18 different blackberry varieties from the United States, Mexico, Chile, France, and Macedonia as reported by Fan-Chiang and Wrolstad (2005). Cyanidin 3-glucoside and cyanidin 3-rutinoside were established as major anthocyanins in those varieties with cyanidin 3-glucoside representing between 43% and 95% of the total peak area and cyanidin 3-rutinoside ranging from trace to 53%. On the other hand, Tian and others (2005a) reported that Rubus occidentalis Jewel (black raspberry) ACNs were predominantly cyanidin rutinoside and cyanidin xylorutinoside while pelargonidin rutinoside was found and has been described as a minor anthocyanin in this commodity (Tian and others 2006).

Total phenolic content

The total phenolic content in the Andes berry extract was 294 ± 37.2 mg GAE/100g FW. This level is lower than that previously reported by Vasco and others (2008) (2167 mg GAE/100 g FW) for Andes berry harvested in the country of Ecuador. However, our results showed that Andes berry from Colombia had a similar level of phenolic content to those of Rubus idaeus (raspberry) and Rubus fruticosus (blackberry) (307 to 320 mg GAE/100 g FW) as reported by Costantino and others (1992), Rotundo and others (1998), and Benvenuti and others (2004). The variance in reported levels for Andes berry could be due to differing methods of extraction. We analyzed the total phenolic content in the extract obtained with pure acetone and acetone: water (70: 30) followed by partition with chloroform while Vasco and others (2008) analyzed the extract obtained first with methanol: water (50: 50, v/v) and then with acetone: water (70: 30, v/v). Our extraction method theoretically determines the maximal amount of ACNs, total phenolics, and antioxidant capacity present in a plant sample (Moyer and others 2002). However, methanol has been reported as better extracting solvent than acetone due to its polarity and good solubility for phenolic components from plant materials (Sobhy and others 2008). Another possible explanation for the difference in values is the variation in phenolic levels according to season and growing location (Connor and others 2005).

Antioxidant activity

The antioxidant activity of the fruit extract determined as mmol TE/100 g FW using the ABTS·+ and FRAP assays was 2.01 ± 0.12 and 4.50 ± 1.22, respectively. Vasco and others (2008) reported values of 5.5 and 6.2 mmol TE/100 g FW for these assays, which is consistent with the higher phenolic content obtained in their study.

Although the ABTS·+ radical scavenging activity of Andes berry in our study is lower than that reported by Vasco and others (2008), it is within the range for other Rubus species (0 to 2.53 mmol TE/100 g FW), which have been recommended for the improvement of nutritional value due to their high antioxidant activities (Deighton and others 2000). According to these researchers, the degree of pigmentation and the phenolic content appear to be important factors in determining the antioxidant capacity of berries. These facts agree with our results that show lower ACN content, phenolic content, and antioxidant activity of Colombian Andes berry as compared to Andes berry from Ecuador. Various factors such as variety, growing condition, maturity, season, geographic origin, fertilizer, soil type, storage conditions, and amount of sunlight received, among others, might be responsible for the observed differences (Al-Farsi and others 2005). The FRAP value expressed as mmoles ferric iron reduced/100 g FW was 8.22 ± 1.50, which is close to the overall mean (7.92 mmoles ferric iron reduced/100 g FW) for 37 Rubus species and cultivars harvested in the state of Oregon, U.S.A. (Moyer and others 2002).

The ABTS method measures the ability of the antioxidant to quench ABTS·+ radicals probably by an electron transfer reaction (Dejian and others 2005) while the FRAP assay measures the potential of an antioxidant to reduce the yellow ferric–TPTZ complex to a blue ferrous–TPTZ complex by electrodonating substances under acidic conditions (Nilsson and others 2005). According to the results, Andes berry is a good electron donor as its extract was able to quench ABTS·+ radicals and reduce the ferric complex to a ferrous complex.

Ascorbic acid content

The ascorbic acid content was 10.1 ± 1.42 mg/100 g FW. This content is comparable to that found by Vasco and others (2008) (10 to 11 mg/100 g FW) in Andes berry from Ecuador. Conversely, Deighton and others (2000) reported 68 to 242 mg/100 g FW for several Rubus species while Pantelidis and others (2007) determined ranges between 14.3 and 32.4 mg/100 g FW for Rubus ideaus, Rubus fructicosus cultivars, and Rubus hybrids.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. References

The ACN content and profile in Colombian Andes berry (Rubus glaucus Benth) were investigated. Anthocyanin content was 45 ± 7.07 mg/100 g FW. Cyanidin 3-rutinoside was the major pigment representing 45% of the total pigment followed closely by cyanidin 3-glucoside at 40%. Andes berry also contained important amounts of phenolics and ascorbic acid and exhibit high antioxidant activity as measured by ABTS and FRAP. The year-round availability of Andes berry along with the considerable percentage of discarded fruit makes this fruit a potential source of natural pigments, nutraceuticals, and natural antioxidants.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. References
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