Liqing Du, Yixiao Shen, Xiumei Zhang, Witoon Prinyawiwatkul, Zhimin Xu
Miracle berry is known for its unique characteristic of modifying sour flavours to sweet. Twelve phenolics were identified and quantified in the miracle berry flesh at a level from 0.3 for kaempferol to 17.8 mg/100 g FW for epicatechin. Lutein and a-tocopherol were also quantified at a level of 0.4 and 5.8 mg/100 g FW, respectively. The TP and TF contents were 1448.3 GA and 9.9 QR mg Equiv/100 g FW for the flesh, respectively, compared with 306.7 GA and 3.8 mg QR mg Equiv/100 g FW of the seeds. The free radical scavenging and reducing percentage of the flesh extract was 96.3% and 32.5% in DPPH and ABTS assays, respectively. Additionally, the flesh extract had a high FRAP of 22.9 mmol/100 g. It significantly inhibited the oxidation of PUFA in fish oil as well. Thus, miracle berry could also serve as an antioxidant-rich fruit to provide health promoting function.
Introduction
Miracle berry (Synsepalum dulcifificum), also called miracle fruit or red berry, is an indigenous tropical plant growing in West Africa. Generally, miracle berry is about an inch in length with a bright red colour. It has a big seed surrounded by a thin layer of berry flesh with a faint cherry-like flavour. It has a unique ability to convert sour tasting foods to sweet. The glycoprotein miraculin in miracle berry was reported to be responsible for this unique function by binding to the sweet receptor cells of the tongue, thus suppressing the response of a sour taste in the central nervous system.
This effect would last until the miraculin was diluted and eliminated by saliva. With the taste modification function, miracle berry has a great potential in food application as an alternative sweetener or taste modifier to mask undesirable sour tastes in food products. Miracle berry could also be an abundant source of antioxidant-rich phytochemicals. These phytochemical antioxidants have been confirmed to possess health promoting functions in preventing various chronic diseases, such as cardiovascular diseases, obesity, diabetes and certain cancers. In this study, the hydrophilic and lipophilic phytochemicals in miracle berry were identified and quantified.
The high antioxidant activity of miracle berry could provide health promoting function in its food application, in addition to the taste modifying function. In general, the results obtained from this study would be helpful to explore the mechanism of the health promoting functions of miracle berry and provide a potential utilisation of miracle berry extract as a food ingredient with both antioxidant and taste modification functions.
Materials and methods
2.1. Chemicals and materials
HPLC grade acetonitrile, acetic acid, methanol and hexane, Acetone, Ethyl acetate, Tween 20, 2,2-diphenyl-L picrylhydrazyl (DPPH), 2,4,6-trip yridyl-s-triazine (TPTZ), Folin–Ciocalteau reagent, Trolox, menhaden fish oil, heptadecanoic acid (C17:0), EPA, DHA, a-tocopherol, a-tocotrienol, c-tocopherol, c-tocotrienol, cyanidin chloride, epicatechin, rutin, myricetin, quercetin, kaempferol, gallic, ellagic, syringic and ferulic acid standards, Fresh miracle berry (S. dulcifificum).
2.2. Extraction of phytochemicals, tocopherols and carotenoids in miracle berry
After miracle berry seeds were separated from the flesh, the seeds and flesh were separately ground using a kitchen blender. Twenty grams of the ground flesh or seed sample was homogeneously mixed with 50 ml of methanol to extract phytochemicals at 60 C for 30 min. After 10 min of centrifugation, the methanol layer was transferred to a clean tube. The residue was mixed with 50 ml of methanol to repeat the extraction. The methanol layer was combined with the previously obtained methanol layer. Miracle berry flesh or seed extract was obtained after the methanol was evaporated by a vacuum centrifuge evaporator (Labconco, Kansas City, MO, USA). After the extracts were weighed, each extract was used to prepare a stock solution (100 mg/ml) with its corresponding extraction solvent. For quantifying tocopherols and carotenoids in the flesh, hexane and acetone instead of methanol were used to perform the extraction, respectively, with the same extraction procedure as the phytochemicals extraction.
2.3. Determination of total phenolic and flavonoid contents in the flesh and seed extracts
2.3.1. Total phenolics
The method used to determine the total phenolic content includes: Folin–Ciocalteu reagent (0.75 ml) was diluted 10 times and mixed with 0.1 ml of diluted extract solution (1 mg/ml). The reaction was carried out for 5 min in dark. Then, sodium bicarbonate (60 g/l, 0.75 ml) was added. The reaction mixture was incubated at 25 C for 90 min. The absorbance was measured by a UV–Vis double beam spectrometer (1600 Shimadzu, Kyoto, Japan) at 750 nm. Gallic acid was used to plot the calibration curve for calculation. The total phenolics content of the extract was calculated and expressed as mg gallic acid equivalent (GA Equiv)/100 g FW.
2.3.2. Total flavonoids Total flavonoid content was determined. One milliliter of diluted extract solution (1 mg/ml) was mixed with 0.3 ml of 5% NaNO2 and 4 ml of distilled water. An aliquot (0.3 ml) of 10% AlCl3 was added to the mixture followed by adding 2 ml of 1 M NaOH. The solution was immediately diluted to 10 ml using distilled water. The absorbance of the solution was measured at 506 nm. The total flavonoid content was calculated by using a calibration curve obtained from a quercetin standard and expressed as mg quercetin equivalent (QR Equiv)/100 g FW.
2.4. Identification and quantification of phytochemicals, ascorbic acid and tocols in the extracts
Phytochemicals such as anthocyanins, phenolic acids and ascorbic acid, as well as carotenoids, were determined. The mobile phase was a mixture of A: 1% acetic acid in water and B: acetonitrile, with the percentage of B ramped from 0% to 100% in 100 min and then changed back to 0% at 101 min for 9 min with a constant flow rate of 0.8 ml/min. The detector was set at 520 nm for monitoring anthocyanins. The wavelength for monitoring each phenolic or ascorbic acid was based on the maximum absorption of its standard. Each anthocyanin was identified by comparison of an elution order. The concentration of each anthocyanin was calculated by the calibration curve of cyanidin chloride in molar concentration and converted to lg/g of sample based on its molecular weight. The concentrations of other phenolics were calculated by the external calibration curves of their corresponding standards.
2.5. Determination of antioxidant activities of the extracts by using DPPH, ABTS & FRAP methods
2.5.1. DPPH
The DPPH assay was performed. One milliliter of 0.135 mM DPPH methanolic solution was mixed with 1 ml of the extract (1000 lg/ml), kamperferol, catechin, gallic acid solution (100 lg/ml), or methanol as a blank. The mixture was then vortexed vigorously and left for 30 min at room temperature in the dark before its absorbance (Abs) was measured at 517 nm. The DPPH free radical scavenging activity was calculated by the equation below and expressed as the percentage of inhibition rate compared with the blank: DPPH free radical scavenging activityð%Þ
¼ ð1 Abssample=AbsblankÞ 100 where Abssample was the absorbance of the mixture of the test sample and DPPH reagent after reaction; Absblank was the absorbance the mixture of methanol and DPPH reagent after reaction.
2.5.2. ABTS
The solution consisting of 7 mM of ABTS and 2.4 mM potassium persulfate (1:1 v/v) was reacted in the dark for twelve hours at room temperature. Then, it was mixed with methanol to obtain an absorbance value 0.700 at 734 nm. One milliliter of the diluted solution was mixed with 1 ml of the extract (1000 lg/ml), kamperferol, catechin, gallic acid solution (100 lg/ml), or methanol as a blank. After a 7 min reaction, the absorbance (Abs) was measured at 734 nm. The free radical scavenging capability was calculated by the equation below and expressed as the percentage of inhibition rate of free radical scavenging compared with the blank. ABTS radical scavenging activityð%Þ ¼ ð1 Abssample=AbsblankÞ 100 where Absblank and Abssample were the absorbance of the mixtures of blank and test samples, respectively.
2.5.3. FRAP
The FRAP reagent contained 25 ml of sodium acetate (300 mM in acetic acid, pH 3.6), 2.5 ml of TPTZ solution (10 mM in 40 mM HCl) and 2.5 ml of FeCl36H2O solution (20 mM). Ten microliters of the extract (1000 lg/ml), kamperferol, catechin, gallic acid solution (100 lg/ml), or FeSO4 (1.0 mmol/l) as a reference was mixed with 1 ml distilled water and 1.8 ml of the FRAP solution. Then the mixture reacted at 37 C for 10 min. The absorbance of the reaction solution was recorded at 593 nm. The ferric reducing capability was calculated by comparing the absorbance of the reaction solution to the absorbance of the FeSO4 reference and converting to mmol/100 g extract or standard.
2.6. Determination of antioxidant activity of the berry flesh extract in stabilizing fish oil
An emulsion consisted of 1% menhaden fish oil and 1% Tween 20 in phosphate buffer (pH 7.0). Then, 3.0 or 6.0 mg/ml of the berry flesh hydrophilic extract in the emulsion was prepared in the same way as the treatments. Based on the results obtained from the determination of the total phenolic content, the total phenolic content of 3.0 mg of the extract was equivalent to 0.5 mg of gallic acid. Thus, 0.5 mg/ml of gallic acid in the emulsion was also prepared as a reference. The emulsion without the extract or gallic acid was used as a blank. Each treatment or blank emulsion (20 ml) was added to a 40 ml test vial. The vials were incubated at 37 C with continuous agitation by a multiple magnetic stirrer (Multistirrer, VELP Company, Italy) until the experiment was completed. The levels of EPA and DHA in each fish oil emulsion were determined at 0, 24, 48 and 72 h. One milliliter of each emulsion sample was extracted with 2 ml of hexane which contained C17:0 as the internal standard (100 lg/ml). The hexane layer was separated and evaporated to obtain dry oil extracts in a clean test tube. Then, 2 ml of BCl3 was added to the dried oil to perform esterification at 60 C in a water bath for 30 min. Then, the reaction solution was mixed and vortexed with 1 ml hexane and 1 ml water. The upper hexane layer was dehydrated by anhydrous sodium sulfate and transferred to a GC vial. The retained EPA and DHA in the emulsion were calculated with the following formula: Retained rateð%Þ¼ðCt=C0Þ 100 where C0 was the concentration of EPA or DHA at 0 h; Ct was the concentration of EPA or DHA at 0, 24, 48 or 72 h in the same emulsion.
2.7. Data analysis
Each determination was repeated in triplicate. The results of the total phenolic and flavonoid content, DPPH, ABTS and FRAP assays, and identified components in the extracts were expressed as means ± standard deviation. The significant differences among treatments were conducted by one-way ANOVA at P < 0.05 (SAS, 9.1.3, Cary, NY, US).
3. Results and discussions
3.1. Extraction yields, total phenolic and flavonoid contents and phytochemicals in miracle berry flesh and seeds
The total flavonoid content, the berry flesh contained 9.9 ± 0.5 mg of QR Equiv/100 g FW, which was approximately three times higher than in the berry seeds (3.8 ± 0.4 mg of QR Equiv/ 100 g FW) Based on these results, the overall antioxidant capability of miracle berry flesh was much higher than that of the berry seeds. In other words, more antioxidant-rich phytochemicals in miracle berry are located in the flesh rather than the seeds. Anthocyanins in the berry flesh may be responsible for the red colour of miracle berry. Also, lutein was the only carotenoid detected in the miracle berry flesh. Compared with other antioxidant-rich berries, the miracle berry had higher levels of important hydrophilic phenolics and lipophilic tocols and carotenoid, which may contribute to the antioxidant capability and health promoting functions of miracle berry.
3.2. The antioxidant capabilities of miracle berry flesh and seed extract in DPPH, ABTS, and FRAP assays
Three antioxidant activity assays, DPPH, ABTS and FRAP were applied to assess the antioxidant capabilities of both miracle berry flesh and seed extracts. The antioxidant activity determined by DPPH assay is the activity of quenching free radicals or H-donor capability of the antioxidant. The DPPH free radicals react with a hydrogen donated from the antioxidant and form their corresponding hydrazine. The results of this study indicated that the free radical scavenging percentage of miracle berry flesh extract was 96.3%, and not significantly different from that of catechin (94.4%) or gallic acid (92.9%) at 100 lg/ml.
The results obtained from the traditional antioxidant activity assays indicate that the miracle berry flesh extract had significantly higher antioxidant capability than the seed extract. This capability was also equivalent to common phenolic acid and flavonoid antioxidants at the same concentration.
Miracle berry exhibited significant higher ABTS scavenging activity than grapes. In the FRAP assay, the miracle berry flesh extract maintained stronger ferric-reducing power (22.9 mmol/100 g) than that of myrtle berry extract (0.7–8.4 mmol/100 g). Therefore, the antioxidant capability of miracle berry was superior to many other recognised antioxidant-rich fruits in scavenging free radicals.
3.3. The antioxidant capability of miracle berry flesh in stabilizing fish oil
Due to the high degree of unsaturation, the incorporation of EPA and DHA in food could increase the tendency for lipid oxidation. In order to retain the omega-3 fatty acids and stabilizing fish oil against oxidation during food processing and storage, usually a synthetic antioxidant is added in the food.
However, the safety of long term consumption of synthetic antioxidants is a concern, as the antioxidants could accumulate in the liver or even cause carcinogenesis.
In this study, the capability of stabilizing EPA and DHA against oxidation was evaluated. Also, the fish oil was homogenised with tween in phosphorous buffers (pH = 7.2) and incubated at 37 C to simulate the environment of vulnerable lipids in the human serum. The retention of EPA or DHA in the emulsion reflected the status of lipid oxidation in fish oil. It also indicated that the traditional antioxidant activity assays could not directly reflect the capability of an antioxidant in preventing lipid oxidation in an oil-water emulsion. Actually, lipid oxidation or the related oxidised products in the human body is the initiator of tissue cell inflammatory and could result in the risk of developing various chronic diseases. The effectiveness of an antioxidant in preventing lipid oxidation indicates the higher capability in reducing toxic lipid oxidation production and potentially preventing the risk of tissue inflammatory and chronic diseases.
Thus, the miracle berry extract could be a food antioxidant to effectively stabilize lipids in food products and prolong their shelf life. It could also provide effective health promoting functions due to its greater potential in inhibiting lipid oxidation and toxic oxidised products in human serum.
Conclusion
Epicatechin, rutin, quercetin, myricetin, kaempferol, gallic, ferulic, syringic acid, three anthocyanins (delphinidin glucoside, cyanidin galactoside and malvidin galactoside), three tocopherols (a-tocotrienol, a- and c-tocopherol) and lutein were identified and quantified in the miracle berry flesh. Some of the important antioxidant-rich phenolics and ascorbic acid in the miracle berry had much higher levels than those in well recognised antioxidant-rich berries, such as blueberry and blackberry. In the ABTS and DPPH assays, the free radical scavenging activities of the flesh extract was similar to other antioxidant standards. However, in the FRAP assay, the activity of the flesh extract was significantly higher than other antioxidant standards.
Furthermore, the miracle berry extract exhibited greater capability in preventing lipid oxidation in the fish oil emulsion than gallic acid in this study. Thus, it could potentially be used as a food ingredient not only to replace synthetic food antioxidants, but also to provide health promoting functions in reducing the risk of chronic diseases associated with lipid metabolism problem.
Reference:
Full journal research copy available here
,
Comments