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Department of Food Engineering, State University of Ponta Grossa (UEPG), Av. Carlos Cavalcanti, 4748, 84030-900, Ponta Grossa, BrazilFood Processing and Quality, Innovative Food System, Production Systems Unit, Natural Resources Institute Finland (Luke), Tietotie 2, FI-02150 Espoo, Finland
This work aimed to characterize the phenolic composition and in vitro antioxidant and antiproliferative properties of lyophilized camu-camu (Myrciaria dubia) seed extract (LCE), and to assess the effects of LCE on the antioxidant and sensory traits of yogurt. The LCE contained 46.3% (wt/wt) total phenolic content; the main compounds quantified were vescalagin, castalagin, gallic acid, procyanidin A2, and (−)-epicatechin. The LCE had antioxidant activity, as measured by different chemical assays (2,2-diphenyl-1-picrylhydrazyl, Folin–Ciocalteu reducing capacity, total reducing capacity, ferric reducing antioxidant power, and Cu2+ chelating capacity), and inhibited the cell proliferation of HepG2 cells (human hepatoma carcinoma; IC50 = 1,116 µg/mL) and Caco-2 cells (human colorectal adenocarcinoma epithelial cells; IC50 = 608.5 µg/mL). In addition, LCE inhibited the in vitro activity of α-amylase, α-glucosidase, and angiotensin-converting enzyme, and protected DNA from peroxyl radical–induced scission. When added to yogurts, different concentrations of LCE (0, 0.25, 0.5, 0.75, and 1.0 g/100 g) increased the chemical antioxidant and reducing capacities. The camu-camu yogurt containing LCE at 0.25 g/100 g had an acceptance index of 84%, showing that camu-camu seed extract may be a potential ingredient for addition to yogurts.
Camu-camu [Myrciaria dubia (HBK) McVaugh] is a fruit from South America that is mainly consumed on its own or as frozen pulp. The frozen pulp is exported to many European and Asian countries because of its taste and versatility in household cooking. However, its peel and seeds are discarded by frozen pulp companies, generating a large quantity of byproduct. The consumption of camu-camu pulp is related to its unique taste and high ascorbic acid content, but recently chemical and functional analyses of camu-camu byproducts have indicated in vitro and in vivo functional properties, such as antioxidant, antihyperglycemic, antihyperlipidemic, antihypertensive, antimicrobial, and neuroprotective effects (
Evaluation of phenolic-linked bioactives of camu-camu (Myrciaria dubia McVaugh) for antihyperglycemia, antihypertension, antimicrobial properties and cellular rejuvenation.
In vitro antioxidant and antihypertensive compounds from camu-camu (Myrciaria dubia McVaugh, Myrtaceae) seed coat: A multivariate structure-activity study.
). Camu-camu byproducts have the potential to be used in developing new bioactive-rich foods and possibly eliminating the use of synthetic chemical preservatives in processed foods. No patents are registered with the Brazilian Industrial Property Institute (INPI; search on June 3, 2019, using the key words “camu-camu yogurt” and “camu-camu dairy food”), indicating that the dairy sector should explore the use of camu-camu pulp, seeds, and peel to develop new potentially functional dairy foods. To date, yogurts manufactured with pomegranate (Punica granatum) and jacaranda (Jacaranda mimosifolia) seed flours (0.5 g of flour/100 g of yogurt) have been shown to increase antioxidant activity, as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and the degree of liking of taste (
, 38% of US consumers are willing to pay a premium for foods that offer health benefits beyond basic nutrition, and roughly 54% of those consumers actively seek out functional foods. In 2018, 54% of US consumers sought out preservative-free (“natural”) foods, and within this context, dairy foods manufactured with natural extracts are suitable alternatives (
). Adding phenolic-rich extracts or matrices to dairy foods such as ice creams and yogurts is among a number of new approaches aimed at giving these foods a naturally healthy halo (
Enzymatic extract from flowers of Algerian spontaneous⨿Cynara cardunculus: Milk-clotting properties and use in the manufacture of a Camembert-type cheese.
added a lyophilized clove aqueous extract to fermented milk, finding that the total phenolic and antioxidant activity increased considerably, and that the sensory acceptance of the product was enhanced. Similarly,
added Fuzhuan brick tea and aqueous saffron extract, respectively, to yogurts and verified that increasing concentrations of the bioactive-rich aqueous extracts led to higher antioxidant activity in the yogurts.
In vitro antioxidant and antihypertensive compounds from camu-camu (Myrciaria dubia McVaugh, Myrtaceae) seed coat: A multivariate structure-activity study.
), we performed a statistical optimization of the extraction conditions of camu-camu seeds, and the most promising solvent system to extract the highest content of bioactive compounds was 40.7% water, 16% ethyl alcohol, and 43.3% propanone. Taking advantage of technological trends in the dairy sector, the present work aimed to characterize the lyophilized camu-camu seed extract (LCE) in terms of phenolic composition, antioxidant, and cytotoxic effects, and to assess the effects of different concentrations on the antioxidant activity of yogurt. We also conducted proximate analysis and a sensory evaluation of the yogurt with added LCE to characterize its potentially functional properties.
MATERIALS AND METHODS
Chemicals
We purchased gallic acid, DPPH, FeCl3·6H2O, Folin–Ciocalteu reagent, 2,4,6-tripyridyl-s-triazine, acetonitrile, analytical standards (HPLC, purity >97%), ellagic acid, caffeic acid, ferulic acid, (+)-catechin, (−)-epicatechin, quercetin, quercetin-3-rutinoside, chlorogenic acid, α-amylase (A3176-500KU from porcine pancreas type VI-B), 4-nitrophenyl-d-glucopyranoside, dinitrosalicylic reagent, acarbose, and α-glucosidase (G5003-100UN from Saccharomyces cerevisiae type I) from Sigma-Aldrich (São Paulo, Brazil), and acquired trans-resveratrol from Extrasynthese (Genay, France). Ethyl alcohol 99.8%, methyl alcohol 99.8%, and FeSO4 were purchased from Neon (São Paulo, Brazil). Hydrochloric acid, isobutanol, anhydrous CH3CO2Na, NaH2PO4, K4[Fe(CN)6], and ascorbic acid were purchased from Vetec (Rio de Janeiro, Brazil). Sodium hydroxide and propanone 99.5% were purchased from Synth (Diadema, Brazil). Formic acid was obtained from Reagen (Curitiba, Brazil). Aqueous solutions were prepared using ultrapure water (Millipore, São Paulo, Brazil). The HepG2, Caco-2, and IMR90 (normal lung fibroblast cells) cell lines were obtained from the Rio de Janeiro cell bank.
Camu-Camu Seeds
Samples of mature camu-camu fruit [Myrciaria dubia (HBK) McVaugh] were supplied by a producer from the municipality of Iguape (geographical coordinates: 24°41′51″S, 47°34′16″W at 6 m altitude) in April 2017. Fruits were sanitized (NaOCl at 100 mg/L for 15 min) and dried for 35 h at 35°C. Dried seeds were ground (model M20; Ika Werke, São Paulo, Brazil) and sieved using a 42 Tyler mesh (0.354 mm) to obtain a standardized camu-camu seed flour. This project was registered at the National Genetic Heritage Management System (SisGen) of the Brazilian Ministry of the Environment (www.sisgen.gov.br; A1848EB).
From previous work involving the extraction of phenolic compounds from camu-camu seeds (
In vitro antioxidant and antihypertensive compounds from camu-camu (Myrciaria dubia McVaugh, Myrtaceae) seed coat: A multivariate structure-activity study.
), we used 3 solvents for extraction: 40.7% water, 16% ethyl alcohol, and 43.3% propanone. Extractions were performed using a 1:20 ratio (mass/vol). For this purpose, camu-camu seed flour was mixed with the solvent mixture at a ratio of 1:20 (wt/vol). We used a technique of continuous agitation with temperature control, in which the water remained at 45 ± 1°C for 45 min in a thermostatically controlled glass cell (Dubnoff TE-0.53; Tecnal, Ourinhos, Brazil). The extract was filtered and the solvent fully eliminated under vacuum using a rotatory evaporator. The aqueous solution was freeze-dried under vacuum at 1,200 μL of Hg (LD 1500A, Terroni, São Carlos, Brazil) to obtain the LCE.
Lyophilized Camu-Camu Seed Extract
We analyzed total phenolic content [mg of gallic acid equivalent (GAE)/100 g], ortho-diphenols (mg of chlorogenic acid equivalent/100 g), and condensed tannins [mg of catechin equivalent (CE)/100 g] 3 times in the LCE according to a methodology previously described (
Optimized Camellia sinensis var. sinensis, Ilex paraguariensis, and Aspalathus linearis blend presents high antioxidant and antiproliferative activities in a beverage model.
). We determined individual phenolic compounds by liquid chromatography using a LC-20T Prominence chromatograph (Shimadzu, Tokyo, Japan) equipped with diode array and fluorescence detectors (LC-DAD). We conducted separation using a reverse phase column (C18, 150 mm × 4.6 mm, particle size 3.5 μm) and the experimental conditions described by
In vitro antioxidant and antihypertensive compounds from camu-camu (Myrciaria dubia McVaugh, Myrtaceae) seed coat: A multivariate structure-activity study.
We measured the chemical antioxidant activity of LCE using the following assays: DPPH free-radical scavenging activity [mg of ascorbic acid equivalent (AAE)/100 g]; Folin–Ciocalteu reducing capacity (FCRC; mg of GAE/100 g), ferric reducing antioxidant power (FRAP; mg of AAE/100 g), Cu2+ chelating capacity (% inhibition of pyrocatechol violet-Cu2+ complex formation), and total reducing capacity (mg of quercetin equivalent/100 g), following experimental conditions described by
In vitro antioxidant and antihypertensive compounds from camu-camu (Myrciaria dubia McVaugh, Myrtaceae) seed coat: A multivariate structure-activity study.
Optimized Camellia sinensis var. sinensis, Ilex paraguariensis, and Aspalathus linearis blend presents high antioxidant and antiproliferative activities in a beverage model.
We evaluated the cytotoxicity of the camu-camu seed extract to Caco-2 and HepG2 using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) as previously described (
). Briefly, cells were seeded into 96-well plates at densities of 2 × 104 cells/well (HepG2) and 1 × 104 cells/well (Caco-2). After adhesion, the cells were treated for 48 h with serial concentrations of LCE (100, 500, 1,000, and 2,000 µg/mL). We determined cell viability at the time of treatment and after 48 h, according to the following parameters: IC50 represented the concentration of the agent that inhibited cell growth by 50%, (T/C) × 100 = 50, where T was the number of cells at the time of treatment and C was the number of control cells at the time of treatment; GI50 represented the concentration of the agent that inhibited cell growth by 50% relative to untreated cells [(T − T0)/(C − T0)] × 100 = 50, where T0 was the number of cells at time zero; and LC50 represented the concentration of the agent that resulted in a net loss of 50% of cells relative to the number at the start of treatment ([T − T0]/T0) × 100 = −50 (
We measured cellular reactive oxygen species (ROS) levels with fluorescence probes using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; 25 mmol/L). The cell lines IMR90 (normal human lung fibroblast) and HepG2 (6 × 104 per well) were treated for 1 h with different concentrations (10–100 µg/mL) of LCE or 15 μmol/L H2O2 (positive control) or culture medium (negative control). Following treatment, H2O2 at 15 μmol/L was added to all wells, and fluorescence intensity was measured at 485 nm of excitation and 538 nm of emission (
. Supercoiled pBR 322 DNA (50 μg/mL) was dissolved in phosphate buffer (10 mmol/L, pH 7.4), and extracts (1 mg/mL) were dissolved in water. In a tube, LCE was diluted with phosphate buffer (catechin 10 μg/mL and extract 100 μg/mL). Then, test samples (2 μL), phosphate buffer (2 μL), DNA (2 μL), and 2,2′-azobis(2-methylpropanimidamide) dihydrochloride (11.25 mmol/L, 4 μL) were added to the Eppendorf tube. A control (DNA with peroxyl radical) and a blank (DNA only) were also prepared. To quantify the DNA scission, samples were electrophoresed using a 0.7% (wt/vol) agarose gel in Tris-acetic acid-EDTA buffer (40 mmol/L Tris acetate, 1 mmol/L EDTA, pH 8.5) at 60 V for 5 h using a B1A horizontal mini gel electrophoresis system (Owl Separation Systems Inc., Portsmouth, NH). The intensity of the bands in the photographs was analyzed using Alpha Ease standalone software (Alpha Innotech Co., San Leandro, CA). The protective effects of LCE were calculated according to the equation below and expressed the results as inhibitory percentage of DNA scission.
DNA retention (%) = (intensity of supercoiled DNA with the peroxyl radical/intensity of supercoiled DNA in control) × 100.
We also analyzed LCE for its ability to inhibit the activity of α-amylase, α-glucosidase, and angiotensin-converting enzyme (ACE). First, LCE (100 mg) was diluted with 1 mL of ultrapure water and stirred at 200 rpm (SL180/D, Solab, Piracicaba, SP, Brazil) for 1 h at room temperature. Then, the extract was filtered and analyzed. We assessed ACE inhibition according to
The evaluation of dipeptidyl peptidase (DPP)-IV, α-glucosidase and angiotensin converting enzyme (ace) inhibitory activities of whey proteins hydrolyzed with serine protease isolated from Asian pumpkin (Cucurbita ficifolia).
Int. J. Pept. Res. Ther.2014; 20 (25364320): 483-491
, and expressed the results as percentage of inhibition. All analyses were performed in triplicate and results are expressed as mean and standard deviation.
Processing and Characterization of Yogurts with Added Camu-Camu Seed Extract
Yogurts (∼1 L per formulation) were processed as described by
. Whole bovine milk (Lactobom, Toledo, Brazil; 2.5 g/100 mL of total lipids and 3.4 g/100 mL of total proteins), 11% sucrose (Union, São Paulo, Brazil), 3.5 g/100 g of whole bovine milk powder (Tyrol, São Paulo, Brazil; 27.7 g/100 g of total fat and 37 g/100 g of total carbohydrates) were mixed and pasteurized at 65°C/30 min. After pasteurization, 0.01 g/100 g of lyophilized bacterial cultures (Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus) were added when the temperature reached 42°C. The fermentation process was stopped at pH 4.6. The curd was cooled to 6°C for 8 h, and then the LCE was added at different concentrations: 0.25, 0.50, 0.75, and 1.00 g/100 g. We also prepared a negative control yogurt (0 g of LCE/100 g) for comparison. An orange flavoring agent (Mix, São Bernardo do Campo, Brazil) was added to all samples at a concentration of 0.25 g/100 g.
To evaluate the effect of the LCE on the antioxidant activity of the yogurts, we extracted 5 g of yogurt with 10 mL of methyl alcohol, under constant stirring (vortex) for 10 min. Then, the mixture was centrifuged at 700 × g for 30 min, and the supernatant was collected and immediately analyzed for FCRC (mg of GAE/100 g), FRAP (mg of AAE/100 g), and DPPH (mg of AAE/100 g) following the experimental conditions described by
In vitro antioxidant and antihypertensive compounds from camu-camu (Myrciaria dubia McVaugh, Myrtaceae) seed coat: A multivariate structure-activity study.
Optimized Camellia sinensis var. sinensis, Ilex paraguariensis, and Aspalathus linearis blend presents high antioxidant and antiproliferative activities in a beverage model.
In a preliminary sensory evaluation of the 5 yogurt formulations (data not shown), LCE concentrations higher than 0.25 g/100 g enhanced the bitterness and astringency of the yogurts, which was an undesirable effect of the polyphenols on yogurt acceptability. Therefore, we chose the yogurt formulation containing 0.25 g of LCE/100 g for further sensory evaluation. For this purpose, a total of 75 assessors (22 men and 53 women between 18 and 60 yr of age) signed the consent form and were informed about the sensory protocol, which had been previously approved by the ethics committee (CAAE: 65493717.9.0000.0105). The yogurt (25 g) was served in coded (3 digits) tumblers at 4°C, and assessors used a 9-point hedonic structured scale (1 = extreme dislike; 5 = neither like nor dislike; 9 = extreme like) to indicate the degree of liking of taste, odor, consistency, color, and overall impression. Assessors were also asked to indicate whether they would pay (R$0 to R$3.00; R$1 = US$0.25) for a 100-g yogurt source of natural antioxidants compared with a natural yogurt without antioxidants (R$1.00). As a supplementary analysis, we also asked assessors to indicate whether they considered yogurt to be a healthy food (yes/no) and whether they considered the consumption of yogurts with added natural extracts to be healthy (yes/no).
We determined the proximate composition of the yogurt with added 0.25 g of LCE/100 g and the negative control yogurt according to
: protein (N × 6.38), ash, moisture, and total lipids (Bligh-Dyer method) and results are expressed as grams per 100 g. We estimated total carbohydrates by difference, and calculated the total energy value using Atwater factors (4 kcal/g for proteins and carbohydrates and 9 kcal/g for lipids; results are expressed as kcal/100 g). The pH was also measured using a calibrated pH meter (model PHS-3B; Hanna Instruments, Barueri, Brazil), and we assessed total titratable acidity using a 0.11 mol/L NaOH solution (results are expressed as g ofg lactic acid/100 g of yogurt). We assessed syneresis at 5°C of the yogurt with added LCE by centrifugation (7,000 × g for 10 min); results are expressed as percentages.
In addition, the instrumental color of the yogurts was assessed by measuring lightness (L*), red/green color (a* coordinate), and yellow/blue color (b* coordinate) using a handheld MiniScan EZ 4500L colorimeter (Hunter Laboratory, Reston, VA) in reflectance mode. We assessed the instrumental texture profile of the yogurts at 5°C using a TA-XT Plus Texture Analyzer (Stable Micro Systems, Godalming, UK) to assess firmness (g) and consistency (g/s). For this purpose, we used a cylindrical stainless steel probe with an acrylic disk (35 mm diameter), a penetration depth of 30 mm, and a penetration rate of 1 mm/s.
Data Analysis
The experimental data are presented as means ± standard deviations (n = 3 replicates). When appropriate, we compared means between groups using 1-way ANOVA. For this purpose, we verified homoscedasticity using the Brown-Forsythe test and TIBCO Statistica (version 13.3; TIBCO Ltd., Palo Alto, CA). We identified differences between means using Fisher's least significant difference (LSD) test, considering P ≤ 0.05 to be significant. To compare sensory data between men and women and the physicochemical characteristics of the LCE and LCE-free yogurts, we used unpaired Student's t-tests, and calculated the 2-sided comparisons between acceptance indexes using the z-test (
Phenolic Composition, Antioxidant Activity, and Cytotoxicity/Antiproliferative Evaluation of LCE
The results for phenolic composition (individual and major phenolic classes), antioxidant activity, and cytotoxic and antiproliferative effects of LCE in relation to the different cell lines are presented in Table 1.
Table 1Phenolic composition, chemical antioxidant activity, and cytotoxicity against cancerous cell lines of lyophilized camu-camu seed extract
GAE = gallic acid equivalent; CE = catechin equivalent; CAE = chlorogenic acid equivalent; AAE = ascorbic acid equivalent; QE = quercetin equivalent; GI50 = concentration of the agent that inhibits growth by 50%, relative to untreated cells; IC50 = concentration of the agent that inhibits cell growth by 50%; LC50 = concentration of the agent that results in a net loss of 50% cells, relative to the number at the start of treatment.
Value
Phenolic class
Total phenolic content (mg of GAE/100 g)
43,598 ± 923
Total condensed tannins (mg of CE/100 g)
5,766 ± 478
Total ortho-diphenols (mg CAE/100 g)
13,193 ± 1,041
Phenolic composition (mg/100 g)
Gallic acid
473.18 ± 5.03
Syringic acid
35.34 ± 0.61
Caffeic acid
42.38 ± 0.19
Rosmarinic acid
17.26 ± 0.73
Chlorogenic acid
24.91 ± 0.23
2,4-Dihydroxybenzoic acid
73.73 ± 0.72
Ferulic acid
14.96 ± 0.15
2-Hydroxycinnamic
6.07 ± 0.07
p-Coumaric acid
20.14 ± 0.15
Castalagin
8,771 ± 278
Vescalagin
3,967 ± 75
Quercetin
6.39 ± 0.10
Procyanidin A2
200.16 ± 3.05
(+)-Catechin
130.00 ± 0.01
(−)-Epicatechin
144.29 ± 0.14
Quercetin-3-rutinoside
136.05 ± 0.45
trans-Resveratrol
20.75 ± 0.09
Ellagic acid
86.61 ± 0.31
Chemical antioxidant activity
Folin–Ciocalteu reducing capacity (mg of GAE/100 g)
21,324 ± 547
Total reducing capacity (mg of QE/100 g)
34,858 ± 1,223
Ferric reducing antioxidant power (mg of AAE/100 g)
48,436 ± 6,346
2,2-Diphenyl-1-picrylhydrazyl (mg of AAE/100 g)
49,798 ± 682
Cu2+ chelating capacity (% inhibition of complex formation)
17 ± 2
Cytotoxicity toward cancerous cells (μg/mL)
Caco-2 LC50
936.8
Caco-2 IC50
608.5
Caco-2 GI50
491.8
HepG2 LC50
1,912
HepG2 IC50
1,116
HepG2 GI50
538.1
Inhibition of enzymes (% inhibition)
α-Amylase
40.70 ± 0.40
α-Glucosidase
16.60 ± 0.46
Angiotensin-converting enzyme
34.40 ± 0.72
1 GAE = gallic acid equivalent; CE = catechin equivalent; CAE = chlorogenic acid equivalent; AAE = ascorbic acid equivalent; QE = quercetin equivalent; GI50 = concentration of the agent that inhibits growth by 50%, relative to untreated cells; IC50 = concentration of the agent that inhibits cell growth by 50%; LC50 = concentration of the agent that results in a net loss of 50% cells, relative to the number at the start of treatment.
The contents of total phenolics, ortho-diphenols, and condensed tannins in LCE were roughly 43.6 g of GAE/100 g of LCE, 13.2 g of CAE/100 g of LCE, and 5.8 g of CE/100 g LCE, respectively. These values were higher than those in camu-camu powder produced by spouted bed drying (8.16 g of GAE/100 g and 7.22 g of quebracho tannin equivalent/100 g;
Impact of spouted bed drying on bioactive compounds, antimicrobial and antioxidant activities of commercial frozen pulp of camu-camu (Myrciaria dubia McVaugh).
). Several phenolic acids, flavonoids, ellagitannins, and trans-resveratrol have already been identified in different parts of the camu-camu pulp, peel, or seed coat (
Ellagic acid derivatives, ellagitannins, proanthocyanidins and other phenolics, vitamin C and antioxidant capacity of two powder products from camu-camu fruit (Myrciaria dubia).
Evaluation of phenolic-linked bioactives of camu-camu (Myrciaria dubia McVaugh) for antihyperglycemia, antihypertension, antimicrobial properties and cellular rejuvenation.
Evaluation of phenolic-linked bioactives of camu-camu (Myrciaria dubia McVaugh) for antihyperglycemia, antihypertension, antimicrobial properties and cellular rejuvenation.
identified quercetin derivatives; ellagic, syringic, and gallic acids; myricetin; cyanidin-3-o-glucoside; castalagin; and vescalagin in camu-camu powder (peel and pulp).
Ellagic acid derivatives, ellagitannins, proanthocyanidins and other phenolics, vitamin C and antioxidant capacity of two powder products from camu-camu fruit (Myrciaria dubia).
also identified gallocatechin derivatives, procyanidin dimer B type, (+)-catechin, catechin gallate, and other glycosylated ellagic acid derivatives.
To determine the antioxidant activity of LCE, we used 4 chemical methods to measure the ability of LCE antioxidants to act as single-electron transfer agents: FRAP, FCRC, total reducing capacity, and DPPH. We also measured Cu2+ chelating capacity to determine the capacity of LCE to chelate a transition metal ion (Table 1). The results showed strong chemical antioxidant activity using all methods, especially metal chelating capacity: LCE was diluted 100-fold to render a 17% inhibition of the pyrocatechol violet-Cu2+ complex formation. These antioxidant activity values were associated with the high total phenolic content in LCE. Our data were in agreement with of
, who analyzed the antioxidant activity of camu-camu flour (pulp and peel) using DPPH, oxygen radical absorbance capacity, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods. Similarly,
Impact of spouted bed drying on bioactive compounds, antimicrobial and antioxidant activities of commercial frozen pulp of camu-camu (Myrciaria dubia McVaugh).
found high antioxidant activity as measured by FCRC (11 g of GAE/100 g), FRAP [65,000 mmol of Trolox equivalents (TE)/100 g], and DPPH (3,800 mmol of TE/100 g) in lyophilized camu-camu powder. The in vitro chemical antioxidant activity of camu-camu frozen pulp was associated with a decrease in total cholesterol, triacylglycerols, uric acid, and thiobarbituric acid reactive substances in the liver and blood of diabetic Wistar rats (
Using the MTT assay, we showed that LCE decreased cell viability in a dose-dependent manner in both Caco-2 and HepG2 cancer cells. According to Figure 1, LCE exhibited pro-carcinogenic behavior at low concentrations in Caco-2 cells (100 µg/mL) but antiproliferative activity at higher concentrations (500–2,000 µg/mL). Similarly,
pointed out that quercetin (a phenolic compound highly distributed in vegetables) could stimulate or inhibit cell proliferation depending on its concentration. Another study showed that Cichorium intybus L. extract, rich in anthocyanins and other phenolic compounds, also showed this biphasic effect (pro-carcinogenic and antiproliferative) at specific concentrations (
). Moreover, it is important to consider that individual bioactive compounds exist in different proportions in whole natural matrices. This explains why, in many cases, whole matrices present better bioactivity or behavior than isolated or single nutrients (
). The antiproliferative results showed that among the cell lines, HepG2 cells were more resistant to LCE, with a high GI50 value (538 μg/mL). In contrast, Caco-2 cells were more susceptible to LCE, with a lower GI50 value (491.8 μg/mL). Regarding cell death (cytotoxic effect), Caco-2 also showed a lower LC50 value (936.8 µg/mL) compared with HepG2 (LC50 = 1,912 µg/mL). Interestingly,
Camu-camu (Myrciaria dubia) from commercial cultivation has higher levels of bioactive compounds than native cultivation (Amazon Forest) and presents antimutagenic effects in vivo..
showed that camu-camu pulp extract exerted protection against 1,2-dimethylhydrazine dihydrochloride mutagenic effects on the colon in in vivo studies. Thus, according to our results, it may be that the potential bioactivity of camu-camu extracts depends on the cancer cell line and the type of study (in vivo versus in vitro).
Figure 1Cell viability and evaluation of the concentration-dependent effect after 48 h exposure to camu-camu extract in Caco-2 and HepG2 cancer cells. (A) IC50 (concentration of the agent that inhibits cell growth by 50%); (B) GI50 (concentration of the agent that inhibits growth by 50%, relative to untreated cells); and (C) LC50 (concentration of the agent that results in a net loss of 50% cells, relative to the number at the start of treatment).
We assessed the ROS generated in cell lines exposed to various concentrations of LCE using DCFH-DA. The levels of ROS induced by H2O2 was higher than in the negative control for both cells (Figure 2), indicating its influence on ROS generation. Our results revealed that LCE exhibited antioxidant activity at all concentrations against oxidative stress induced by H2O2 in HepG2 cancer cells and IMR90 normal cells. In the same way,
showed that tea polyphenols could attenuate H2O2-induced oxidative stress in vitro by adjusting ROS generation. We suggest that LCE induced no antiproliferative effects through ROS generation in HepG2 cells. The generation of ROS is one of the stimuli that can trigger an apoptosis cascade, leading to cell death. Thus, the activation of apoptosis may be independent of an extract's antioxidant capacity (
). In the present study, the cell death observed in the cell viability test could have occurred as a result of different types of physicochemical stress stimuli, such as cell membrane damage; mitochondrial dysfunction and destabilization (
). Moreover, plant extracts rich in polyphenols, as well as pure polyphenols alone or in combination, can exert many pro-apoptotic effects, often selectively targeting cancer cells, through extrinsic and intrinsic mechanisms of action, tested in vitro and at concentrations generally higher than those found in food matrices or in herbal medicine (
). For instance, trans-resveratrol, a phenolic compound in our camu-camu extract, was found to possess apoptotic effects due to the formation of acidic vesicular organelles, autophagic vacuoles, and DNA fragmentation in oral cancer cells (
Figure 2Results of intracellular reactive oxygen species measurement in (A) IMR90, and (B) HepG2 cell lines. Treatment = lyophilized camu-camu seed extract at concentrations of 10, 50, or 100 μg/mL. DCF = 2'-7'dichlorofluorescein. Quantitative data are mean ± SD. Bars with different letters (a, b) differ (P ≤ 0.05).
We hypothesize that the antioxidants contained in LCE neutralized additional ROS production, preventing cancer cell death by the ROS pathway. Because we added LCE to a food consumed by all age groups on all continents, animal-based experiments and clinical trials should be undertaken to assess the effects of yogurt with added camu-camu seed extract on the biomarkers of oxidative and nitrosative stress, such as lipid peroxidation, low-density lipoprotein oxidation, and induction of proinflammatory cytokines (
In addition to its high chemical antioxidant activity, LCE protected DNA against peroxyl radical–induced scission. Indeed, LCE (100 μg/mL) had an inhibition rate of 89.3 ± 2.7%, which was comparable (P = 0.713) to the protection offered by (+)-catechin (10 μg/mL; 88.6 ± 1.4%; Figure 3). These results show that both LCE and (+)-catechin protected DNA from oxidative reactions and scission. In fact, peroxyl radicals may cause oxidative stress in cells that can lead to DNA damage, causing mutagenesis and carcinogenesis in vivo (
). This is the first report indicating the DNA protection of camu-camu seed extract against ROS.
Figure 3Gel electrophoresis showing inhibition of lyophilized camu-camu seed extract (LCE at 100 µg/mL) against peroxyl radical-induced DNA scission compared with blank (DNA only), control (DNA with peroxyl radical), and (+)-catechin as a standard (10 µg/mL).
The effects of LCE [0 (negative control), 0.25, 0.50, 0.75, and 1.00 g/100 g] on the antioxidant activity of yogurt (FRAP, DPPH, and FCRC) are shown in Figure 4. All antioxidant assays showed dose-dependent effects: that is, the higher the concentration of LCE, the greater the in vitro antioxidant activity of the yogurts. Similar observations have been obtained for yogurts and fermented milks manufactured with added herbal and other natural extracts rich in bioactive compounds.
showed that the addition of moringa aqueous extract (Moringa oleifera; 0–0.20 g/100 g) to yogurts enhanced FCRC (6.5–12.5 mg of GAE/100 mL) and antioxidant activity as measured by the DPPH (10–72% radical inhibition) and ABTS (15–83% radical inhibition) assays in a dose-dependent manner. Similarly,
observed that when Fuzhuan brick tea leaves were added to yogurts (1–3 g/100 mL), FCRC ranged from 15 to 65 mg/100 mL, and antioxidant activity as measured by the ABTS assay ranged from 20 to 85% radical inhibition.
used a chia extract obtained with ethanol or water to prepare yogurts and observed dose-dependent total phenolic content and antioxidant activity as measured by the DPPH and ABTS assays when extracts were added at 0.05 to 0.10 g/100 g. These results emphasize the remarks made by
that dairy foods should be the target of research regarding the addition of natural bioactive-rich extracts, aimed at decreasing the use of synthetic compounds.
Figure 4Effects of different concentrations (0 to 1 g/100 g) of lyophilized camu-camu seed extract (LCE) on (A) Folin–Ciocalteu reducing capacity, (B) 2,2-diphenyl-1-picrylhydrazyl (DPPH), and (C) ferric reducing antioxidant power (FRAP) of yogurts. GAE = gallic acid equivalent; AAE = ascorbic acid equivalent. Data are expressed as means followed by SD (n = 3). Different letters for the same method represent statistically significant differences between samples (P < 0.05).
The proximate composition results of the yogurt with 0.25 g of LCE/100 g and the negative control yogurt were, respectively, as follows: moisture, 76.30 ± 5.91 and 72.61 ± 0.04 g/100 g; total carbohydrates 16.79 ± 6.16 and 18.20 ± 0.10 g/100 g; proteins 4.03 ± 0.31 and 4.20 ± 0.10 g/100 g; total lipids 1.97 ± 0.10 and 2.05 ± 0.10 g/100 g; ashes 0.91 ± 0.01 and 1.03 ± 0.01g/100 g; pH 4.02 ± 0.01 and 4.01 ± 0.01; total titratable acidity 0.99 ± 0.01 and 1.38 ± 0.01 g/100 g (as lactic acid); and mean caloric value 101 and 108 kcal/100 g. These values were in accordance with the proximate composition of yogurts with added extracts rich in bioactive compounds (
). In a study relating the effects of added grape peel flour and grape juice on the proximate composition of an organic yogurt with added grape pomace,
observed similar values for moisture (76.81 g/100 g), total carbohydrates (7.16 g/100 g), protein (3.39 g/100 g), total lipids (6.81 g/100 g), and ash (0.72 g/100 g).
Regarding instrumental color attributes, the yogurt with added LCE had a higher (P < 0.001) a* color (2.21 ± 0.07) value than the LCE-free yogurt (−1.26 ± 0.02). Conversely, the LCE-free yogurt had higher (P < 0.001) lightness (88.89 ± 0.15) value than the yogurt with added LCE (72.91 ± 0.48). We observed no significant difference (P = 0.053) for the b* coordinate between the LCE-free yogurt (13.11 ± 0.06) and the yogurt with added LCE (13.47 ± 0.22). The yogurt with added LCE had 28.4 ± 1.2% syneresis, a consistency of 1,451 ± 219 g/s, and a firmness of 59.33 ± 8.50 g.
Sensory analysis of the yogurt formulation containing 0.25 g of LCE/100 g was conducted with 75 untrained consumers (22 men and 53 women; 18 to 60 years of age) who consumed yogurt or fermented milk at least twice a week. The mean scores of liking for taste (7.6 ± 1.1), odor (7.5 ± 1.4), consistency (7.1 ± 1.5), color (7.7 ± 1.4), and overall acceptance (7.6 ± 0.1) were between “moderately liked” and “liked very much.” The global acceptance index of the yogurt formulation containing 0.25 g of LCE/100 g was 84.3%, indicating a high sensory acceptance of the yogurt. Comparing the sensory data between men and women we found no statistically significant difference in the degree of liking of taste (men vs. women: 7.73 ± 0.93 vs. 7.53 ± 1.22; P = 0.495), consistency (6.68 ± 1.72 vs. 7.28 ± 1.31; P = 0.104), odor (7.54 ± 1.37 vs. 7.41 ± 1.46; P = 0.721), color (7.45 ± 1.33 vs. 7.79 ± 1.47; P = 0.356), overall acceptance (7.50 ± 0.96 vs. 7.62 ± 0.92; P = 0.607), and acceptance index (83.3 vs. 84.4%; P = 0.906). These results showed consensus in the scores attributed to the sensorial parameters evaluated. Of the 75 assessors, 73 considered yogurt to be a healthy food, and 74 considered the consumption of yogurts with added natural antioxidants to be healthy. In addition, 56 assessors indicated that they would pay from R$2.00 to R$3.00 for yogurts (100 g) that were sources of natural antioxidants compared with a conventional antioxidant-free yogurt (R$1.00).
CONCLUSIONS
Camu-camu seed extract is a rich source of phenolic compounds, especially vescalagin, castalagin, gallic acid, and procyanidin A2. The bioactive compounds in the extract promoted high chemical antioxidant activity as measured by 5 different assays; showed antiproliferative and cytotoxic effects in cancerous cell lines (HepG2 and Caco-2); and decreased ROS generation in a normal human cell line (IMR90). When added to yogurts, different concentrations of camu-camu seed extract increased the yogurts' antioxidant capacity. The camu-camu yogurt containing 0.25 g of LCE/100 g was sensorially accepted, indicating that camu-camu seed extract may be a sustainable ingredient for addition to potentially functional yogurts. Future studies should assess the shelf life and the effects of simulated digestion on the bioactive compounds of yogurt with added camu-camu seed extract.
ACKNOWLEDGMENTS
The authors thank Coordination for the Improvement of Higher Education Personnel (CAPES), Minas Gerais Research Foundation (FAPEMIG), Fundação Araucária and Brazilian National Council for Scientific and Technological Development (CNPq; Processes 405890/2018-4 and 303188/2016-2) for the PhD scholarships and partial funding of this research. We also thank the Department of Chemistry (State University of Ponta Grossa, UEPG) and Complexo de Laboratórios Multiusuários (C-LABMU, UEPG) for the infrastructure used in the laboratory work. This work was also partially funded by Natural Resources Institute Finland (Luke project 41007-00173600). We also thank Renata D. S. Salem (Department of Food Engineering, State University of Ponta Grossa, Ponta Grossa, Brazil) for the support in the texture analysis. The authors declare no conflict of interest.
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