Goji berry species are deciduous woody perennial plants which are grown in the northwestern part of China, primarily in the Ningxia Hui Autonomous Region. Goji berry is prized for its versatility of color and nut-like taste in common meals, snacks, beverages, and medicinal applications. It has been widely used as a functional ingredient in nutraceuticals since excessive studies have been carried out and demonstrated that goji berry act as a crucial role in improving of vision, prevention of aging and age-related diseases, inhibition of cancer development and boosting immune system [1
]. These healthy contributions have been proved and associated with the presence of various functional components including betaine, phenolics, β-carotenes and polysaccharides.
Beside the dry fruit of goji berry sold in the market, the most notable products are the goji beverage and goji wine (goji marinated in grain liquor) which have been recognized as functional drinks. Based on the principle of Chinese medicine, the grain liquor can act as organic solvent to enhance the medicinal function of herbal medicine and food materials due to the solubility of active compounds [3
]. Therefore, the functional compounds of goji berry can be extracted by grain liquor.
Betaine, one of the most notable components in goji berry, known as trimethylglycine (TMG), is a metabolic product of choline. Betaine from goji berries helps to relieve anxiety, increases memory, promotes muscle growth and protects against fatty liver illness [4
]. Many important biochemical processes rely on methylation, including the metabolism of lipids, neurotransmitters, and DNA [5
]. It is well known that methylation ability of human body declines with age, therefore the decline of methylation contributes to the aging process. Betaine supplementation, therefore, has an interesting potential prevention benefit against the aging process [6
Extensive researches have indicated that the antioxidant activity of goji berry is related to β-carotene and phenolic compounds [7
]. The attractive red-yellow color of goji wine is mainly associated with a group of lipophilic compounds, known as carotenoids [9
]. The carotenoids have been identified to be effective in preventing chronic diseases, such as cardiovascular diseases and skin cancer [10
]. The β-carotene which is chemically a terpenoid accounts for the major portion of carotenoid in goji berry [12
]. Other antioxidants existing naturally in goji berry are polyphenols, including tannins, lignans, flavonoids, and some other simple phenolic compounds. External stimuli (microbial infections, ultraviolet radiation, and chemical stressors) induce their synthesis to defense against herbivores and pathogens [14
]. It is widely accepted that significant antioxidant activity of food is related to high total phenolics content [15
]. Therefore, the use of flavonoid or other phenolics in diets and medicine has been intensively studied, particularly as treatments for neurodegenerative diseases and aging-related diseases [16
]. Recent researches have strengthened the importance of flavonoids because of their imperative roles in antioxidant activities and other biological activities [17
]. Several methods have been intensively employed to measure the antioxidant capacity, such as DPPH, or ferric reducing antioxidant power (FRAP).
This study is aimed at determining the content of these functional components and to assess the diffusion profiles of these components and investigate how antioxidant capacity influences by alcohol concentration and steeping time.
2. Materials and Methods
2.1. Food Materials and Chemicals
Ningxia goji berries were purchased from a local supermarket in Zhuhai, China. 2-Diphenyl-1-picryhydrazyl (DPPH), betaine, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich Co. (Munich, Germany). Folin-Ciocalteu reagent was supplied by Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Catechin, Reinecke’s salt and triphenyltetrazolium chloride (TPTZ) were supplied by Yuanye Biotechnical Company (Shanghai, China). Gallic acid was purchased from Damao Chemical Reagent Company (Tianjin, China). Other chemicals were purchased from Guangzhou Chemical Reagent Company (Guangzhou, China). Unless otherwise stated, all the chemicals were of analytical grade.
2.2. Sample Preparation
Due to grain liquor in market can be categorized into three main groups, including low, medium, and high concentration alcohol. Therefore one concentration of alcohol was selected as low concentration group and different concentrations of alcohol were selected in both medial and high concentration groups. Therefore 30 treatments were obtained by combining five different concentrations (15%, 25%, 38%, 55%, and 65%) of alcohol and six different steeping times (1 day, 3 days, 5 days, 7 days, 10 days and 14 days). Each group was steeped in triplicate. Therefore total 90 samples were prepared. Goji berry (5 g) and 45 mL of alcohol solution were added into individual Erlenmeyer flask (the ratio of goji berry and alcohol is referred to the ratio of making commercial goji wine). The Erlenmeyer flasks were sealed with parafilm and tinfoil. The solid and liquid phases were separated by filtration with funnel and filter paper after finishing steeping process at room temperature. All liquid samples were stored in refrigerator at 40 °C for further analysis.
2.3. Determination of Betaine
According to the report of Zhu [6
], trimethylglycine (betaine) can form aubergine color precipitate with Reineche’s salt after hydrolyzation. Then this color can be measured by UV-spectrophotometer and the results could be used for the calculation of betaine content.
Therefore 20 mg of betaine standard weighed accurately and dissolved in 15.4 mL of distilled water to make 1.3 mg/mL stock standard solution. Then 0.8 mL, 1.6 mL, 2.4 mL, 3.2 mL, and 4 mL stock standard solution was transferred using pipettes to centrifugal tubes, respectively. The stock standard solutions were diluted to the 12 mL mark line with distilled water. Hydrochloric acid (2 M) was used to adjust the pH of standard solution to 1 and the samples were cooled down to 0–4 °C. After cooling, 8 mL of 3% Reinecke’s salt solution (made fresh) was added in each sample tube and the aging process of samples were carried out for 3–4 h in 0–4 °C. By using glass sand funnel, the aubergine color precipitate was filtered and washed by diethyl ether until the filtrate was colorless. Then the precipitates on filter paper were dissolved with 70% acetone solution and be transferred to 10 mL volumetric flask and diluted to mark line with distilled water. The absorbance of standard solution was measured and recorded using a UV-Visible spectrophotometer at 525 nm against 70% acetone solution as blank. All absorbance number was used to build up a standard curve correlated to known concentration of standard solution, which would be used to read and calculate the betaine content of samples.
Certain amount of sample (14 mL) of each tube was sucked into test tubes and the pH of samples was adjusted to 1 using 2 M hydrochloric acid. Then the samples were followed the procedures of standard solution to measure the absorbance of sample and to determine the betaine content in goji wine samples.
2.4. Determination of β-Carotene
The quantitative analysis of β-carotene was conducted as the following procedures: 4 mL of goji wine sample was added in cuvette and then measured with the UV-Visible spectrophotometer at both 405 nm and 503 nm. The absorbance reading was used to calculate β-carotene content in sample according to the empirical equation [18
] as below: Cβ-carotene
= 4.642 × A450
− 3.091 × A503
. Where C is the concentration of carotenoid expressed in μg/mL, and A450 and A503 represent the absorbance at 450 nm and 503 nm, respectively.
2.5. Determination of Phenolic Compounds
2.5.1. Determination of Total Phenolic Content (TPC)
The TPC was determined by a Folin-Ciocalteu assay [19
] with slight modifications [14
] using gallic acid (GA) as the standard. The absorbance was measured with the UV-Visible spectrophotometer at 765 nm. The TPC was expressed as milligrams GA equivalents per gram goji berry (mg GAE/g) on a dry weight basis for solid sample and milligrams GA equivalents per milliliter goji wine (mg GAE/mL) for liquid sample through the calibration curve of GA. The linearity range of the calibration curve was 50–1000 μg/mL (r
2.5.2. Determination of Total Flavonoid Content (TFC)
The TFC was determined using a colorimetric method described previously [14
]. The absorbance of standard solution, blank and samples were measured against the blank at 510 nm using the UV-Visible spectrophotometer. The TFCs were expressed as milligrams catechin equivalents per gram goji berry (mg CAE/g) on a dry weight basis or milligrams catechin equivalents per milliliter goji wine using the calibration curve of (+)-catechin. The linearity range of the calibration curve was 400–10000 μg/mL (r
2.6. Antioxidant Capacity Measurement
2.6.1. In Vitro DPPH Free Radical Scavenging Capacity Analysis
The DPPH free radical scavenging capacity of goji wine was evaluated according to our previous communication [14
]. The absorbance of all samples, blank, and standards were measured against the blank at 517 nm using the spectrophotometer. The scavenging rate was calculated as the equation: The scavenging rate (%) = (Ablank
× 100% (A represent absorbance). The DPPH antioxidant values were also expressed as micromoles of Trolox equivalents per gram goji berry (μmol TE/g) on a dry weight basis using the calibration curve of Trolox. The linearity range of the calibration cure was 20–1000 μM (r
2.6.2. The Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP was performed as described previously [14
]. The absorbance of all samples, blank, and standard solutions were measured against the blank at 593 nm by the spectrophotometer under condition of 37 °C water bath. The FRAP value was expressed as millimoles of Fe2+
equivalent (FE) per gram goji berry (mmol FE/g) on a dry weight basis using the calibration curve of Fe2+
. The linearity range of the calibration curve was 0.1–1.0 mM (r
2.7. Statistical Analysis
All steeping treatments were carried out in triplicates. Analyses were done based on triplicate samples. Means, standard deviations were done using Microsoft Excel 2003. Significant differences of the results for part of the experiment were statistically analyzed by SPSS 14.0. Statistical significance was accepted at a level of p < 0.05.
Goji wines have already widely accepted by most elder people and are recognized as one of the functional wine against aging and aging-related disease. Nevertheless, the concentration of alcohol added and the steeping time are not explicated by most manufacturers. The present investigation shows that low alcohol concentration can promote the diffusion of functional components in large extent, except the total flavonoid content which is boosted by high alcohol concentration. However, the steeping time has no significant effect on the diffusion of functional components and basically the diffusion profile is fluctuated during the soaking process. The antioxidant activity may influenced by steeping time and combined action of β-carotene, TPC, TFC, and other active compounds. The high content of β-carotene, TPC and TFC can develop moderate antioxidant capacity. Therefore, some indications could be summarized for goji wine industries. Firstly, the manufacturers could treat goji berry with 25% of alcohol and separate the goji berry from goji wine after 14 days to obtain goji wine containing high betaine. Secondly, the goji berry should be treated with low concentration alcohol and relative short marinating time (less than 7 days) to obtain high betaine and antioxidant components. Thirdly, if the target customers demand vision beneficial goji wine, manufactures could employ 55% of alcohol and soak for at least 14 days to gain high β-carotene content.