Ital. J. Food Sci., vol. 30, 2018 - 428 PAPER BIOACTIVE AND PHARMACOKINETIC CHARACTERISTICS OF PRE-MATURED BLACK RASPBERRY, RUBUS OCCIDENTALIS D. SHIN1, K.S. CHAE1, H.R. CHOI1, S.J. LEE1, S.W. GIM1, G.T. KWON1, H.T. LEE3, Y.C. SONG3, K.J. KIM3, H.S. KONG*2 and J.W. KWON*1 1Berry & Biofood Research Institute, Gochanggun, Jeonbuk, Republic of Korea 2College of Animal Biotechnology & Resource, Sahmyook University, Seoul, Republic of Korea 3College of Pharmacy, Sahmyook University, Seoul, Republic of Korea *Corresponding author: Tel.: +82 635605190 *E-mail address: hskong0813@syu.ac.kr, kjwung@hanmail.net ABSTRACT Black raspberry (BR, Rubus occidentalis) is a berry originating from North America. It has a high anthocyanin and flavonoid content that seems to be dependent on ripening. Therefore, the bioactive and pharmacokinetic effects of BR were evaluated using pre- matured BR(PBR) collected from May to June. The total polyphenol, flavonoid and vitamin C content in PBR decreased while anthocyanin increased from 0.13 to 10.70 mg/g at 35 days after fruit set. The antioxidant activities due to DPPH and ABTS determination decreased as BRs were matured. Moreover, it is clear that the content of many phenolic compounds including gallic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin, myricetin, luteolin and kaempferol was diminished during maturing. Both ferulic acid and rutin of BR generated the highest decrease as compared to other phenolic compounds. The pharmacokinetic Tmax and Cmax of PBR were 0.6 h and 0.264 µg/mL, respectively. Based on these results, PBR can generate qualified functionality as food and/or as a raw medicinal material. Keywords: black raspberry, anthocyanin, flavonoid, polyphenol, pharmacokinetic Ital. J. Food Sci., vol. 30, 2018 - 429 1. INTRODUCTION Due to concerns for wellbeing and lifestyles of health and sustainability (LOHAS) resulting from a sharply increasing income, the consumption for functional foods is also growing very fast. Many studies have been conducted to develop functional food products containing natural, bioactive compounds to meet consumer demand. Berries – such as blueberry, strawberry, and black raspberry – are used as common functional food additives to meet demand from health-conscious consumers. Black raspberry (BR, Rubus occidentalis), which originated from North America, is one of the main functional food additives due to its pharmacological effects that were written about in Donguibogam, an ancient Korean medicinal textbook written by J. Hur (CHO et al., 2005). Most of the BR cultivated in Gochang, Korea belongs to Rubus occidentalis. However, the BR written of in Donguibogam, Korea is Rubus coreanus Miquel. BR is normally cultivated in soil with pH 6.0–7.2, and it shows medium water usage (OZGEN et al., 2008). Since BR is generally cultivated in moderate to low incident light on coarsely textured soil with average to low levels of organic matter and inorganic nitrogen, BRs form in June in Korea (BAJCZ, 2014). A morphological study indicates BR is a multi-stemmed shrub that forms broad colonies. BR contains thorns with three (ternate) or five palmately arranged leaflets (LEE et al., 2014). BR seems to generate a bright red flower in May, and it tends to breed a floral leaf that is shorter than its sepal. It has semi-spherical shape with naps. BR berry will turn to wine in color when the berry has matured. Normally, edible BR berries are harvested at the early stage of summer. However, some pre-matured BR berries are also gathered as medicines to improve sexual dysfunction of males, as mentioned in Donguibogam (ZHAO et al., 2011). Approximately 1.7% of anthocyanin in BR tends to be responsible for most BR functions. The main anthocyanins of BR are cyanidin 3-glucoside, cyanidin 3-rutinoside, cyanidin 3- sambubioside, and cyanidin 3-xylosylrutinoside (TIAN et al., 2006; TULIO et al., 2008; LEE et al., 2013). TORRE and BARRITT (1977) have indicated that both cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside of BR anthocyanin seem to contribute to the red or black color and antioxidant effect. In addition, as mentioned above, J. Hur during the Joseon Dynasty indicated that BR (Rubus coreanus Miquel) has anti-biotic, anti-cancer, and anaphylaxis effects (BAEK et al., 2005). Such effects have been confirmed by many current scientists (BAEK et al., 2005; LIU et al., 2005; DUNCAN et al., 2009; JEONG et al., 2010; LEE et al., 2011). A study conducted by CHO et al. (2005) showed that BR can increase antioxidant activities, and it was confirmed through ABTS, DPPH and TBARS when 1 g BR is added to 100 mL of 10 or 100% ethanol. The study also showed that protocatecuic acid is the most abundant phenolic compound in BR when analyzed via HPLC (CHO et al., 2005). CHAE et al. (2014) and BOBINAITE et al. (2012) demonstrated that BR contains a high amount of ellagic acid. They also reported that ellagic acid of BR possesses strong antioxidant effects (BOBINAITE et al., 2012; CHAE et al., 2014). Moreover, since ellagic acid is not easily metabolized by microflora and/or broken down by stomach acids, it is commonly selected as standard index material for berries. To increase the use of BR as a functional food additive in Korea, BR was collected and its individual phenolic content was determined in this study. Based on the phenolic content of BR, its antioxidant effects were examined for BR depending on the stage of maturation. In addition, to evaluate the pre-matured BR (PBR, <28 days after fruit set) value and its quality as a functional food additive, the ellagic acid content, which is the standard material index for berries, was evaluated to determine the pharmacokinetic index of PBR. Ital. J. Food Sci., vol. 30, 2018 - 430 2. MATERIALS AND METHODS 2.1. Sample preparation According to research conducted by CHUNG et al. (2008), BR planting soil in Gochang, Korea mostly consisted of silt loam (64%), loam (35%) and clay (1%). BRs were cultivated in an open field with soil at pH 5.3∼6.7 and harvested from 4-year-old BR plants. All BRs were sampled from May to June of 2014 with an interval of 3-4 days (Fig. 1). The average temperature and humidity of Gochang, Korea during harvest, was 17.4∼21.7°C and 72.2∼81.0 %, respectively (KMA, 2017). All BRs collected were similar to the Shuttleworth, which was confirmed by the random amplified polymorphic DNA (RAPD). After harvest of the BR, they were dried at 60°C for 36 h and then powered for storage. 10 g of BR powder were sampled and mixed with 90 mL double distilled water (DDW). The mixture was distilled twice at 80°C for 2 h. All BR extracts were filtered, freeze dried, and stored at -70°C until use. 15 days after fruit set 22 days after fruit set 25 days after fruit set 28 days after fruit set 32 days after fruit set 38 days after fruit set 41 days after fruit set Figure 1. Pre-matured black raspberry (PBR, Rubus occidentalis) and matured black raspberry fruits harvested from May to June with an interval of 2-3 days. 2.2. Determination of the total phenolics, flavonoids, anthocyanins, and ascorbic acid content The total phenolics of BR were measured using a modified Folin-ciocalteu colorimetric method (SINGLETON and ROSSI, 1965). 20 µL of filtered BR (1 mg/mL) was mixed with water and Folin-ciocalteu reagent. After 3 min of incubation at room temperature, 300 µL of 20% Na2CO3 were added. The mixture was then incubated in the dark for 30 min at Ital. J. Food Sci., vol. 30, 2018 - 431 40°C. The absorbance was measured at a wavelength of 725 nm (Beckman Du 730, Beckman Coulter Inc., Fullerton, CA, USA) and expressed as its gallic acid equivalent. The flavonoid content was determined using the method described by MEDA et al. (2005) with slight modifications. Briefly, 0.25 mL of BR (1 mg/mL) were mixed with 1 mL DDW, 0.075 mL of 5% NaNO2, 0.075 mL of 10% AlCl3, and 0.5 mL of 1 M NaOH. The final volume was then adjusted to 2.5 mL with DDW. The absorbance of each sample was then measured at a wavelength of 410 nm (Beckman Du 730, Beckman Coulter Inc., Fullerton, CA, USA). Quercetin was used as a standard. All readings were expressed as micrograms of quercetin equivalent. Monomeric anthocyanin was evaluated using the modified pH differential method (CHAOVANALIKIT and WROLSTAD, 2004). Optimum dilution for BR filtrate was defined using 0.025 M of potassium chloride buffer. Individual BR was diluted with either potassium chloride buffer or sodium acetate buffer to reach an optimum state. After 15 min of incubation, the absorbance of each diluted BR was measured at a wavelength of 700 nm. The amount of anthocyanin was calculated as follows: Anthocyanin (mg/g) = (A × MW × DF × 1000)/(ε × 1) A: absorbance of diluted BR = (Aλvis-max – A700nm) pH1.0 – (Aλvis-max – A700nm) pH 4.5; MW: expressed as cyanidin 3-glucoside (450); DF: dilution factor; ε: molar absorptivity (26,900). The ascorbic acid content was determined using the modified method described by KAMPFERNKEL et al. (1995). Briefly, 0.8 mL of 10 % (w/v) trichloroacetic acid were added to 200 µL of BR filtrate (1 mg/mL). Each mixture was then pre-cooled and centrifuged at 3,000 rpm (4°C) for 5 min. Then, 1.5 mL water and 0.2 mL 0.2 N Folin- ciocalteu were added to 0.5 mL of BR supernatant. After 10 min of incubation at room temperature, the absorbance was recorded at a wavelength of 765 nm. L-ascorbic acid was used as a standard. 2.3. DPPH and ABTS radical scavenging activities The 1,1-dipenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity of each extract sample was determined using the method described by BRAND-WILLIAMS et al. (1995). Briefly, freeze-dried BRs were diluted with methanol to obtain five different concentrations. Each diluted BR (10 µL from 1 mg/mL of BR) was mixed with 0.2 mM of DPPH in dimethyl sulfoxide. After 30 min of incubation at room temperature in the dark, the absorbance was measured at a wavelength of 517 nm using a spectrophotometer. The inhibition percentage was calculated from the equation below: DPPH radical scavenging activity (%) = [(Control absorbance – BR absorbance)/Control absorbance] × 100 The 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging activities of the BR extract were determined using the method described by RE et al. (1999). 2.45 mM of potassium persulfate was added to 7 mM of ABTS and the mixture was kept in the dark at room temperature for 12-16 h. The ABTS radical cation solution was diluted with phosphate buffer saline to obtain an absorbance value of less than 0.70 at a wavelength of 734 nm before the analysis. After adding 2.0 mL of diluted ABTS radical cation solution to 20 µL of BR sample (1 mg/mL), the reaction mixture was incubated in a cuvette at 30°C for 6 min. Trolox, an analog of vitamin E antioxidant, was used as control. Ital. J. Food Sci., vol. 30, 2018 - 432 The ABTS radical scavenging capacity of the BR was calculated using the following equation: ABTS free radical scavenging activity (%) = [(Control absorbance – BR absorbance)/Control absorbance] × 100 All DPPH and ABTS values calculated using Graphpad Prism 5.0 (Graphpad Prism Version 5.0, Graphpad Software Inc., San Diego, CA, USA), were generated as the 50% inhibition concentration (IC50). 2.4. Liquid Chromatography analysis for the phenolic quantification and ellagic acid identification Five g of BR were homogenized in 20 mL of 80 % acetone containing 0.2 % formic acid for 1 min. BR was then concentrated, mixed with 10 mL of acidified water, and passed through an activated C18 Sep-Pak cartridge (Waters Corp., Milford, MA). All adsorbed phenolics onto the C18 column were then recovered with 2 mL of acidified methanol containing 3% formic acid. Individually recovered BR in methanol was filtered, and 10 µL of BR methanol extract was used to analyze the phenolics using liquid chromatography (LC) (Acquity H-class, Waters) equipped with an autosampler/injector and photodiode Array (PDA) detector. A Shiseido Capcellpak C18 UG (5 um, 4.6 × 250 mm) was used for separation. Both mobile phase A (0.2 M ortho-phosphoric acid, pH1.57) and B (20% 50 mM ammonium dihydrogen phosphate, pH2.6 in 80% acetonitrile) were used for elution. The flow rate was 1.0 mL/min, and detection was performed at 280 nm (gallic acid), 320 nm (caffeic acid, p-coumaric acid, ferulic acid) and 360 nm (rutin, myricetin, luteolin, kaempferol). A gradient was employed as follows: 0-10 min, 80% A; 10-15 min, 70% A; 15- 20 min, 60% A; 20-25 min, 10% A; 25-30 min, 10 % A; 30-40 min, 95% A. All phenolics were identified and quantified using external standards (gallic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin, myricetin, luteolin, and kaempferol). Data scanned at 280–360 nm were collected. All LC handling steps were followed as mentioned in a previous study (CHAE et al. 2014). 2.5. Animal model and blood sample preparation Male Sprague Dawley rats (7 wks, 220 g) were obtained from KOSA Bio, Korea. A total of six rats were acclimated in an environmentally controlled breeding room at a temperature of 22±2°C and relative humidity of 50±10 % with a 12 h dark/light cycle for one week before being used for the experiments. They were provided ad libitum access to commercial chow and water. All rats were handled in accordance with the recommendation of the Regulations for the Administration of Affairs Concerning Experimental Animals (BBRI- IACUC-16001). PBR at a dose of 150 mg/kg was orally administered to each rat. A serial of blood samples was collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post oral administration. All blood samples were immediately transferred to a BD vacutainer lithium heparin tube and centrifuged at 3,000 rpm for 10 min at 4°C. The pH of rat plasma (100 µL) was adjusted to pH 2.5 using 1 M potassium dihydrogen phosphate solution. Serial addition of 50% phosphoric acid and acetonitrile was then performed. The plasma samples were centrifuged at 12,000 rpm for 8 min under refrigerated conditions. The supernatant was taken, dried, and re-dissolved in 0.1 mL methanol. Each supernatant sample was then used for ellagic acid analysis under the LC/MS/MS conditions summarized in Tables 1 and 2. Ital. J. Food Sci., vol. 30, 2018 - 433 Table 1. Liquid chromatography (LC)/MS/MS1 analytical conditions used for analysis of ellagic acid in Sprague Dawley rat serum. Parameter Condition HPLC system Agilent 1290 Infinity Binary HPLC system with 6420 triple quadrupole LC/MS system Column Shiseido Capcellpak C18 UG (5 μm, 4.6×250 mm) Column Temperature 40oC Flow rate 1.0 mL/min Injection volume 10 μL Mobile phase A : 0.1% Formic acid B : Methanol Gradient step Time (min) A (%) B (%) 0 70 30 7.0 50 50 13.0 10 90 15.0 70 30 18.0 70 30 1Ionization Type: ESI (Electospray ionization) Negative; Gas temperature (°C): 320; Gas flow (L/min): 9; Nebulizer (psi): 15; Capillary (v): 4,000; Scan mode: Multiple reaction monitoring (MRM). Table 2. Liquid chromatography (LC)/MS analytical conditions used for analysis of ellagic acid in pre- matured black raspberry (PBR, Rubus occidentalis) extract. Compound RT 1 (min) MW 2 Precursor Ion (MS m/z) Product Ion (MS, m/z) Confirm ion Frag (V) Collision Energy (V) Ellagic acid 12.1∼12.3 302 30[M-H]- 229 257 185 170 20/16/15 1RT: Retention time; 2MW: Molecular Weight. 2.6. Pharmacokinetic analysis 5 µL of different concentrations (0.625, 1.25, 2.5, 5.0, and 10.0 µg/mL) of ellagic acid in plasma were used to obtain the ellagic acid calibration curve (final concentrations of 31.25, 62.50, 125, 250, 500 ng/mL). As a result of ellagic acid calibration (R2 = 0.998), a linear regression equation was obtained. Each plasma sample was then injected to LC/MS/MS. The pharmacokinetic parameters of ellagic acid were calculated using WinNonlin software version 6.3 on non-compartmental analysis (Pharsight Cor., Mountain View, USA). 2.7. Statistical analysis All experiments were carried out in triplicates or quadruplicates and expressed as mean ± standard deviation. Statistical analyses were performed using SPSS program (SPSS version 12.0, SPSS Chicago, IL, USA). Unpaired t-tests or one-way repeated measures ANOVA were performed when appropriate. If significant in the ANOVA test, differences in the Ital. J. Food Sci., vol. 30, 2018 - 434 means were determined using Duncan’s multiple range tests. Statistical significance was considered when the p-value was less than 0.05. 3. RESULTS 3.1. Quality properties and phenolic compound of Rubus occidentalis The anthocyanin content in BR increased from 0.13 to 10.70 mg/g, although the total polyphenols, total flavonoids, and vitamin C content in BR decreased during maturing (Table 3). The DPPH and ABTS radical scavenging activities of BR collected on June 19th were 8.44 and 4.53 times higher, respectively, compared to those of BR collected on May 23. This indicates that antioxidant activities decreased while maturing. Gallic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin, myricetin, luteolin, and kaempferol were detected in BR (Table 4). Both gallic acid and ferulic acid were found to be its major phenolics. Depending on the BR harvest day, the amount of eight different phenolics decreased (P < 0.05) when collected on June 19th, the last day of harvest. Five phenolics (caffeic acid, p- coumaric acid, rutin, myricetin, and kaempferol) had the maximum contents on May 26 or May 30. However, the luteolin content was the highest on June 9th. For this reason, luteolin generated the least disappearance, and only 42.5% of luteolin disappeared when BR was collected on June 19. However, only 1% rutin was left at the end of harvest. The rutin and ferulic acid contents at the end of the harvest were 1 and 3%, respectively. The content decreases for both were the highest compared to the other six phenolics. 3.2. Pharmacokinetic and kinetic analysis PBR was orally administrated to male Sprague Dawley rats. The plasma concentration- time profile of ellagic acid in Sprague Dawley rats (n = 5) is shown in Fig. 2. Ellagic acid was confirmed and quantified when plasma was sampled at 0.25, 0.5, 1, 2, and 4 h after oral administration of PBR. Figure 2. The mean plasma concentration-time profile of ellagic acid in rats after oral administration of pre- matured black raspberry (PBR, Rubus occidentalis) at 150 mg/kg (Mean±SD, n = 5). Time (h) 0 2 4 6 8 10 Co nc en tra tio n (u g/ m L) 0.0 0.1 0.2 0.3 0.4 0.5 Ital. J. Food Sci., vol. 30, 2018 - 435 Table 3. Chemical quality properties of pre-matured black raspberry (PBR, Rubus occidentalis)1 harvested from late May to early June. Days After Fruit Set (Date) Total polyphenol (mg/g) Total flavonoid (mg/g) Anthocyanin (mg/g) Ascorbic acid (mg/100g) DPPH2 IC50 (ug/mL) ABTS3 IC50 (ug/mL) 15 (May 23) 170.75±4.65a 1.21±0.02a 0.13±0.01e 24.30±0.38a 95.9±4.52e 261.87±10.25d 18 (May 28) 162.16±4.06a 1.10±0.02b 0.18±0.01e 21.86±0.51b 114.57±4.85de 278.13±15.39d 22 (May 30) 148.64±7.78b 1.07±0.04b 0.08±0.01e 20.34±0.14c 126.57±5.69de 266.23±9.58d 25 (June 2) 126.52±8.37c 0.80±0.04c 0.12±0.01e 17.03±0.12d 160.67±5.91de 326.60±25.54d 28 (June 5) 111.41±2.94d 0.68±0.01d 0.05±0.01e 15.25±0.03e 193.97±1.36d 406.13±31.42d 32 (June 9) 79.04±2.87e 0.51±0.05f 0.81±0.05d 16.79±0.94f 418.83±18.75c 677.73±25.28c 35 (June 13) 60.44±2.62f 0.60±0.01e 3.75±0.12c 10.78±0.03f 824.93±59.99a 912.50±4.90b 38 (June 16) 61.16±7.37f 0.45±0.01f 6.08±0.05b 14.42±0.03g 511.13±57.92b 771.53±99.54bc 41 (June 19) 45.15±0.71g 0.52±0.02g 10.70±0.1a 10.62±0.03h 809.77±99.82a 1186.17±294.46a 1Values are the Mean±SD (n = 3) based on dry basis. 22,2-diphenyl-1-picrylhydrazyl. 32,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid. a-hValues with different superscripts within the same columns are significantly different (p < 0.05). Table 4. Phenolic compositions of pre-matured black raspberry (PBR, Rubus occidentalis)1 harvested from late May to early June. Days After Fruit Set (date) Phenolic compound (μg/g) Gallic acid Caffeic acid p-Coumaric acid Ferulic acid Rutin Myricetin Luteolin Kaempfrol 15 (May23) 3321.72±1.25a 381.71±52.17ab 48.38±46.51ab 1733.63±53.32a 326.11±131.49a 63.66±5.18ab 2.52±0.13b 11.40±0.31b 18 (May26) 2638.79±6.67b 390.88±72.29a 52.40±22.98a 1260.60±19.55b 398.79±100.58a 60.59±4.34b 2.73±0.05b 13.56±0.38a 22 (May30) 3040.08±0.01c 325.25±65.51ab 31.54±13.53abc 1241.73±15.96b 324.48±126.41a 70.12±5.70a 2.33±0.23b 11.30±2.26b 25 (June2) 1826.81±0.14d 330.65±53.15ab 52.44±5.79a 703.88±6.22c 106.96±77.03b 23.05±5.90d 3.08±0.12b 9.55±0.60cd 28 (June5) 1225.55±2.72e 310.03±33.78b 23.17±14.66abc 414.35±1.26d 37.89±45.01b 30.16±1.10c 3.44±0.07b 10.18±0.10bc 32 (June9) 489.50±1.06f 147.24±9.90c 18.05±0.49abc 119.31±5.28e 38.89±14.71b 12.63±2.46e 6.35±3.39a 8.24±0.17d 35 (June13) 373.67±1.23g 21.21±1.50d 15.34±3.63bc 77.24±0.32f 46.04±53.14b 13.19±4.02e 3.35±0.15b 4.93±0.43e 38 (June16) 303.98±0.19h 46.84±11.87d 12.64±1.32bc 53.15±3.08f 73.47±63.69b 8.79±0.24e 3.22±0.03b 4.47±0.10e 41 (June19) 278.71±3.39i 26.74±1.17d 5.98±0.20c 48.30±0.44f 3.68±3.19b 8.66±0.69e 1.45±1.17b 2.08±0.35f 1Values are the Means ± SD (n = 3) based on dry basis. a-iValues with different superscripts within the same columns are significantly different (p < 0.05). Ital. J. Food Sci., vol. 30, 2018 - 436 Ellagic acid was below the detectable level at 8 h after oral administration of PBR. It had the maximum plasma concentration (250 ng/mL) at 0.5 h post oral administration of PBR. The plasma level of ellagic acid decreased as time went by, and its half life (t1/2) was 1.018 h. The results of other pharmacokinetic parameters of ellagic acid are summarized in Table 5. Table 5. Main pharmacokinetic parameters1 of ellagic acid in rats plasma after oral administration of pre- matured black raspberry (PBR, Rubus occidentalis) at 150 mg/kg (Mean ± SD, n = 5). Unit Mean2 AUC0-t ug h mL -1 0.330±0.109 AUC0-∞ ug h mL -1 0.363±0.118 t1/2 h 1.018±0.195 Tmax H 0.600±0.223 Cmax ug mL -1 0.264±0.109 MRT h 1.494±0.309 1AUC0-t: Area under the curve, AUC0-∞: Area under the curve, t1/2: half life time, Tmax: maximum time, Cmax: maximum concentration, MRT: Mean residence time. 2Each value represents the Mean±SD. 4. DISCUSSION BR was collected during its period of maturation at an interval of 2-3 days. The results for the amount of anthocyanin indicated that anthocyanin may not be a major component in BR contributing to its antioxidant effects before its complete maturation (Table 3). FLOEGEL et al. (2011) reported that high-pigmented and hydrophilic BR antioxidants seem to have both DPPH and ABTS scavenging activities, with higher activities toward ABTS than that for DPPH. In a study conducted by LEE et al. (2013), seven different anthocyanins were detectable in BR. Only cyanidin 3-glucoside, which has a lower amount compared to cyanidin 3-rutinoside and/or cyanidin 3-xylosylrutinoside, is found to have stronger antioxidant effect (DPPH scavenging activity) than ascorbic acid, but not Trolox (KÄHKÖNEN and HEINONEN, 2003). However, BORGES et al. (2010) suggested that the antioxidant activity of raspberries is influenced by ascorbic acid content rather than cyanidin 3-glucoside in this case, they showed 10.5 and 8.1 % antioxidant activity, respectively. In this study, PBR polyphenol and flavonoid contents were found to have significantly diminished with increase in maturation days. The study also indicated that the amount of ascorbic acid also showed a similar trend as evidenced by both polyphenol and flavonoid content. Therefore, in PBR, both DPPH and ABTS seem to be influenced by the amount of polyphenol, flavonoid and ascorbic acid rather than anthocyanin. This is similar to results obtained in a study conducted by OGAWA et al. (2008), which suggests that ascorbic acid and flavonols contribute to the antioxidant properties of berries. In PBR, eight major phenolic compounds were detected. Each phenolic compound showed the least amount when BR was harvested on June 19th (Table 4). A study conducted by KANG et al. (2015), showed that whole black raspberry did not contain luteolin and myricetin but catechin, epicatechin, rutin, gallic acid and quercetin were detectable. Although our study did not trace any catechin, epicatechin and quercetin due to different analysis conditions, the amount of rutin was similar to that of fully matured black raspberry. In addition to that, many phenolic compounds of PBR were noted to decrease Ital. J. Food Sci., vol. 30, 2018 - 437 during maturing periods. CHIRINOS et al. (2007) indicated that the contents of both phenolic compounds and flavan 3-ols during maturity stage of 10 mashua cultivars were different, and diminution of both phenolic compound and flavan 3-ols were attributed due to genotypic difference of mashua. Therefore, among BRs, the Shuttleworth at Gochang, Korea is a cultivar that may decrease some phenolic compounds in maturing stag, therefore, ascorbic acid and flavonols may be major factors that contributed to the DPPH and ABTS scavenging activities of BR at the early stage of the harvest. To determine the LC protocol accuracy (recovery) and repeatability (precision) for ellagic acid determination in both PBR and rat plasma, ellagic acid was used. The recovery (103.01–104.23 %) and relative standard deviation (RSD, ≤ 3.35%) implied that the protocol had satisfactory accuracy and high recovery for ellagic acid. In addition, the RSD of intra- (1.92 %) and inter-day (0.61–2.14 %) differences indicated that the LC protocol was precise. Hence, the LC method seemed to be accurate and precise to quantify ellagic acid in PBR (32.13 ± 0.62 mg/g) (data are not shown). Based on the accuracy and repeatability of LC, ellagic acid plasma concentration-time profile in rats and six major pharmacokinetic parameters were analyzed. The pharmacokinetic results of PBR ellagic acid indicated that PBR ellagic acid in rats seemed to increase rapidly (Table 5), and the Cmax reached 0.264 µg/mL. The half time of ellagic acid was found to be 1.018 h after oral administration. Similar results were reported in a pharmacokinetic study by LEI et al. (2003), in which the maximum concentration of pomegranate leaf ellagic acid in rat plasma is found to be 213 ng/mL after 0.55 h of oral administration. The pharmacokinetic characteristics revealed that ellagic acid seems to have poor absorption and rapid elimination after oral administration (SEERAM et al., 2004; BALA et al., 2006). The rapid elimination of ellagic acid has been confirmed via urine and feces analysis by SMART et al. (1986), with up to 70 % ellagic acid being detected in urine and feces. 5. CONCLUSIONS This is the first study to characterize pre-matured black raspberry (PBR, Rubus occidentalis). PBRs are pre-matured black raspberry when BRs are collected from 15 to 28 days after fruit set. The results showed that PBR under 28 days after the fruit set seemed to produce a high content of phenolic compounds, flavonoids, and vitamin C, thereby possessing excellent antioxidant activities. Based on the PBR pharmacokinetic results and chemical analysis along with antioxidant results, PBR can be used as a functional food or medicinal material derived from the nature. Our data could be used us a baseline to develop nutraceuticals, natural colorants, and other related products. 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