Antioxidant and prebiotic activity of five peonidin-based anthocyanins extracted from purple sweet potato (Ipomoea batatas (L.) Lam.)

Twelve kinds of anthocyanins from the Chinese purple sweet potato cultivar (Ipomoea batatas (L.) Lam.) were extracted and identified using LC-MS/MS, which had a high content of peonidin-based anthocyanins. Five peonidin-based anthocyanin monomers (P1, P2, P3, P4 and P5) were isolated by preparative liquid chromatography with structural analyses using an Impact II Q-TOF MS/MS. Then, the functional properties of the anthocyanin monomers, such as the antioxidant activities, proliferative effects on probiotics, and their inhibition on harmful bacteria in vitro, were investigated. The peonidin-based components in purple sweet potato anthocyanins (PSPAs) showed good properties regarding scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and superoxide anions, and had good potential in reducing the total power activity and Fe2+ chelating ability. While the order of the antioxidant abilities was as follows: P4 > P5 > P3 > P2 > P1 > PSPAs. Microbial cultivations showed that P1, P2, P3, P4, P5 and PSPAs could induce the proliferation of Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium infantis and Lactobacillus acidophilus, and they inhibited the growth of Staphylococcus aureus and Salmonella typhimurium, suggesting the anthocyanins might have prebiotic-like activity through the modulation of the intestinal microbiota. Our results indicate that peonidin-based anthocyanins could be further utilized in health foods and pharmaceutical developments.

Then, the main peaks were further purified using the preparative HPLC ( Fig. 1(b)), and the purities for peaks 2, 6, 10, 11 and 12 proved to be more than 98% by analytical HPLC, except for peak 4, which had impure peaks during the purification process. Thus, the anthocyanin monomer peaks 2, 6, 10, 11 and 12 were abbreviated as P1, P2, P3, P4 and P5, respectively, for further structural identifications by the tandem MS (MS/MS) spectral analysis.
In vitro antioxidant activity. The DPPH· scavenging capacities of PSPAs and PSP peonidin-based anthocyanin monomers are shown in Fig. 3(a). Compared with vitamin C (Vc) control, a significant increase of DPPH radical scavenging acidity was observably obtained with the increase in the concentration of anthocyanins (both the PSPAs and anthocyanin monomers), which suggested that the % DPPH· quenched value was depended on the concentration of the peonidin-based anthocyanins at a certain reaction time. The IC 50 values of the PSPAs, P1, P2, P3, P4 and P5 were 57.58, 61.07, 47.22, 37.52, 26.71 and 28.76 μg/mL, respectively, indicating that peonidin-based anthocyanin monomers had a stronger DPPH· scavenging activity.
The active oxygen (O 2− ) scavenging activities of the PSPAs and PSP peonidin-based anthocyanin monomers have been presented in Fig. 3(b). Although the scavenging activities of anthocyanins were weaker than that of Vc at 20 μg/mL, excellent superoxide anion-scavenging activities were shown with increased concentrations of anthocyanins, and the IC 50 values of the PSPAs, P1, P2, P3, P4 and P5 were obtained at 87.08, 57.88, 45.14, 37.08, 29.05 and 30.62 μg/mL, respectively.  The total reducing power of the peonidin-based anthocyanins was measured by the increase in the absorbance at 700 nm and is shown in Fig. 3(c). It was obviously found that the TRP of anthocyanins increased with an increase in the concentrations. Good TRP values from 0.11 to 0.28 were observed in the peonidin-based anthocyanin concentration range between 20 and 100 μg/mL, indicating that the antioxidant concentration might determine the electron donating ability, which could reduce the ferric ion complex to the ferrous form. Meanwhile, the highest absorbance was obtained in the P4 group, which had the same activity rank as the superoxide anion radical scavenging activity.
As presented in Fig. 3(d), the metal chelating activity increased depending on the increase in the concentration of both the PSPAs and anthocyanin monomers. Although the ferrous ion-chelating activities of anthocyanins were significantly lower than that of EDTA, the acylated anthocyanins expect P1 (IC 50 value of 353.23 μg/mL) exhibited stronger chelating effects, as the IC 50 values of P4, P5, P3, P2 reached 116.95, 133.06, 167.27 and 213.74 μg/mL, respectively, which were lower than the IC 50 of PSPAs (287.85 μg/mL).

Effects of peonidin-based anthocyanins on the probiotic bacteria proliferation. The numbers of
Bifidobacterial strains (B. infantis, B. adolescentis, and B. bifidum) and Lactobacillus acidophilus (L. acidophilus) were represented as log cfu/mL in Table 2   Meanwhile, as shown in Table 3, the significant decreases of the pH values in the media were obtained in the proliferation processes of B. bifidum, B. adolescentis, B. infantis and Lactobacillus acidophilus with the supplementation of peonidin-based anthocyanins at 0.5 mg/mL, and the changes were similar to the variations in the bacterial counts.

Effects of peonidin-based anthocyanins on the growth of harmful bacteria. The antibacterial
properties of five peonidin-based anthocyanins on Staphylococcus aureus and Salmonella typhimurium were evaluated by the filter paper method (Fig. 4). The sigmoidal curves for the diameter of the inhibition zone were observed over a concentration range of 1-5 mg/mL, and each anthocyanin fraction demonstrated a significant inhibitory effect on the growth of Staphylococcus aureus and Salmonella typhimurium, respectively, compared with the control without anthocyanin (0 mm). The sizes of the inhibition zones increased with the increase of the concentration of the samples, and the inhibition activity on the growth of Staphylococcus aureus and Salmonella typhimurium was in the order of P4 > P5 > P3 > P2 > PSPAs > P1. In addition, compared to Staphylococcus aureus, the antibacterial activity of peonidin-based anthocyanins on Salmonella typhimurium was more effective.
The minimum inhibitory concentration (MIC) was established in Table 4 as the lowest concentration of anthocyanin that inhibited the visible growth of the pathogenic bacteria from the intestinal tract. Staphylococcus aureus and Salmonella typhimurium were inhibited by the anthocyanins with MIC values ranging from 0.25 to 0.50 mg/mL, and high inhibitory effects of the anthocyanins against Gram-positive and Gram-negative strains are presented in the P4 and P5 groups at 0.25 mg/mL, followed by P3 (0.50 and 0.25 mg/L, respectively).

Discussion
Various anthocyanins contents have been found in different PSP cultivars. The total anthocyanin content from Ipomoea batatas L. was calculated to be 13.73 ± 0.13 mg/100 g PSP, which was similar to the anthocyanin content of 12.40 mg/100 g of the Japanese 'Purple Sweet' variety peonidin-type 15 , and it confirmed the effectiveness of the extraction method. Since few qualitative and quantitative studies on the anthocyanins from PSP cultivars in China have been reported, the anthocyanin profile analysis based on UV and MS is important.
The abundance differences of each peak between HPLC ( Fig. 1(a)) and preparative HPLC ( Fig. 1(b)) resulted from the variability in the chromatographic conditions. As shown in Fig. 1(a) and Table 1, ten anthocyanins were acylated with p-hydroxybenzoic acid, caffeic acid and/or ferulic acid, except the anthocyanins of peaks 1 and 2. Meanwhile, two kinds of glycosides, sophoroside and glucoside, were found to be connected with all twelve anthocyanins. And the twelve anthocyanin peaks were tentatively identified as two major kinds of compounds, cyanidin-based anthocyanins and peonidin-based anthocyanins, due to the characteristic MS-MS fragments at  13 . Interestingly, all the main peaks (2, 4, 6, 10, 11 and 12) were peonidin-types (Figs 2 and S1-S4), and the total content of the peonidin-based anthocyanins was approximately four-fold higher than the cyanidin-based anthocyanins, which was similar to the peonidin/cyanidin ratio of 4.52 calculated from the new Japanese cultivar, 'Purple Sweet' , and the new American variety, 'Stokes Purple' 16 .
A number of researchers have shown a higher correlation between individual anthocyanin and biological activities because of the type and content 5 . To our knowledge, a series of cyanidin-based anthocyanin in vitro antioxidant activities have been investigated according to the structural variations 17 . However, few studies have been published regarding to antioxidant and prebiotic properties of the most abundant peonidin-based anthocyanins that exist in PSP. Furthermore, the higher acylated ratio in the current study was also a potential suggestion for high activity 5 . As expected, the five peonidin-based anthocyanins were ranked as P4 (peak 11) > P5 (peak 12) > P3 (peak 10) > P2 (peak 6) > P1 (peak 2) in the free radical-scavenging activity analysis at a low concentration (60 μg/mL) (Fig. 3(a)), which corresponded with the structure characteristics. The scavenging activity of anthocyanins could be explained by the donation of hydrogen, and it has been well known that di-acylated anthocyanins possess higher antioxidant acidity than the mono-acylated and non-acylated anthocyanins 2 .
Recently, much attention has been focused on the potential scavenging activity of anthocyanins against reactive oxygen and nitrogen species, which might prevent cell damage. Similar to the results of the DPPH scavenging activity analysis, the superoxide anion radical scavenging activity of five anthocyanin compounds was in the order of P4 > P5 > P3 > P2 > P1 (Fig. 3(b)), which was attributed to the chemical structure-activity relationship. The  presence of a hydroxyl group at C-3 and the higher phenolic acids content, such as p-hydroxybenzoic acid, caffeic acid and ferulic acid, enhanced the O 2− scavenging effect 18 . As indicated in Fig. 3(c), although the reducing power of Vc was significantly higher, all the tested anthocyanin samples showed more higher potent activities at low concentrations in the microgram range. And the variations in the ability to quench radicals by electron donation might be due to the total phenolic acids content 19 .
Because of the catalysis of transition metals in lipid peroxidation, hydroxyl radicals (OH·) would be generated due to oxidative damage. Therefore, the chelating activity of ferrous ion is important to form chelates with the transition metal ion 20 . The rank of the metal chelating activities of the tested anthocyanins (Fig. 3(d)) was similar to the antioxidant activities in the superoxide anion radical scavenging activity and total reducing power activity. Although few reports on the metal chelating activity have previously been published, our results suggest that the spatial conformation, position and number of electron-donating ligating groups in anthocyanin compounds could result in a higher metal chelating activity 21 .
A concentration-dependent analysis was carried out to evaluate the effect of five peonidin-based anthocyanins and PSPAs on the proliferation of probiotic bacteria. Interestingly, our results ( Table 2) did not follow the stronger dose-response correlation for the cyanidin-based anthocyanins as previously reported 22 . According to the published results, the catabolism of peonidin-based anthocyanins would be favorable for bacterial growth, while cytoplasm pyknosis and disintegration could also be induced by high phenolic acid content through the reaction with the mycoproteins and enzymes of the strains 23 .
As shown in Table 3, the decrease in the pH at an anthocyanin concentration of 0.5 mg/mL was accompanied by the proliferation of probiotic bacteria, which indicated that anthocyanins would be deglycosylated by β-glucosidases that were secreted through probiotics, and the resulting glucose could be utilized by the bacteria. Meanwhile, phenolic/organic acid could be generated by the degradation of the de-glycosylated anthocyanins which could adjust the environmental pH for bacterial growth 24 . Our results also showed that the higher concentrations of peonidin-based anthocyanins might inhibit the nutrition intake of the tested probiotic bacteria, and anthocyanins could be utilized by varying degrees depending on the bacterial species.
Furthermore, the acylated anthocyanins exhibited a higher inhibition sensitivity on harmful bacteria (Fig. 4, Table 4). According to the previous reports, the propagation and spread as well as the nutrition intake of harmful bacteria could be limited by anthocyanins, and the cellular metabolism of pathogens could be disturbed by anthocyanin metabolites 25 .

Extraction of anthocyanins.
Previous procedures with minor modifications were applied to extract anthocyanins from PSP 26 . One hundred grams of PSP were cleaned and cut into small strips (3 mm × 1 mm), and then ground in a colloid mill (JM, Shanghai Aisijie Co., Ltd., China) with 1.5 L extracting solvent, which contained 3.5% citric acid and 79 U/mL cellulose. The samples were subsequently incubated at 55 °C for 2 h, and followed by centrifugation at 4000 rpm for 20 min. The extraction process was repeated three times, and all the extracts were filtered and concentrated by separation membranes with 3 and 0.2 kDa molecular weight cut-offs, respectively. Then, a macroporous resin X-5 column was applied for the further purification at a flow rate of 2.0 mL/min. The column was washed with three column volumes (3 CV) of deionized water and eluted with 4 CV of ethanol (80:20, V/V). The eluent was dialyzed and freeze-dried till the water content was less than 5%. Then, the PSPAs were obtained and stored at −20 °C for further experiments.
Determination of total anthocyanin content. A modified pH differential method was utilized to determine the total anthocyanin content 4 . Briefly, 1.0 mg/mL PSPAs was prepared and aliquoted in duplicate. One duplicate was diluted with hydrochloric acid-potassium chloride buffer (0.2 M, pH 1.0), and the other was diluted with sodium acetate buffer (0.2 M, pH 4.5). After equilibration for 15 min, the absorbance of each sample was measured at wavelengths of 522 nm and 700 nm, respectively, by a UV-Vis spectrophotometer (Precision Scientific Instrument Co., Ltd., Shanghai, China) with pure water as a blank. The absorbance was calculated as follows: pH pH 522 700 1 0 522 700 4 5 The total anthocyanin content was calculated and expressed as cyanidin-3-glucoside equivalents using the following equation: Where C was the total anthocyanin content calculated as cyanidin-3-glucoside (mg/100 g PSP), V is the collection volume prior to freeze drying, MW was the molecular weight of cyanidin-3-glucoside (449.2 g/mol), DF was the dilution factor, ε was the molar absorbance of cyanidin-3-glucoside (26900 L/cm/mol), and L was the cell path length (1 cm).

HPLC analysis and LC-MS/MS identification.
PSPAs were dissolved in pure water containing 0.05% hydrochloric acid at a concentration of 1 mg/mL, and passed through a 0.20 µm filter membrane (Jinteng Experiment Equipment Co., Ltd., Tianjin, China) prior to HPLC analysis using a Waters apparatus (2410, Waters Co., Ltd., USA) equipped with a 2598 UV-Vis detector and connected to a 4.6 mm × 250 mm C18 column (Waters Co., Ltd., USA). The temperature of the column was set as 30 °C, and the flow rate and injection volume were 0.8 mL/min and 20 µL, respectively. The anthocyanins were detected at 520 nm and eluent A was 0.05% trifluoroacetic acid in water (V/V), while eluent B was acetonitrile. The elution process was 10% B for 0-5 min, 10-12% B for 5-10 min Purification of five peonidin-based anthocyanin monomers from PSP. Five peonidin-based anthocyanin monomers from PSP were purified using preparative high-performance liquid chromatography (Preparative HPLC) (Lisui Technology Co., Ltd., Suzhou, China) connected with a Galaksil EP-C18 column (20 mm × 250 mm, 10 μm). Five milliliters of PSPAs were injected at a concentration of 20 mg/mL. The temperature of the column was set at 30 °C and the flow rate was 12 mL/min. The anthocyanins were detected at 520 nm using eluent A (0.05% trifluoroacetic acid) and B (acetonitrile) as the mobile phase. The solvent gradient was 10% B for 8 min, 10-16% B for 8 min, 16-17% B for 8 min, 17-19% B for 8 min, 19-20% B for 10 min, 20-30% B for 15 min, and 30-40% B for 10 min. The elution peaks were collected by an automatic collector, and then freeze-dried till the water content was less than 5%. The purity of the five peonidins was characterized by the HPLC method.
DPPH radical (DPPH·) scavenging capacity analysis. The DPPH· scavenging capacity was determined using the method described by Brand-Williams with some modifications 27 . Two milliliters of test samples were mixed with 2 mL of 20 mM DPPH· solution in ethanol. Then, the mixtures were shaken and placed at room Scientific RePoRts | (2018) 8:5018 | DOI:10.1038/s41598-018-23397-0 temperature for 30 min before measuring the absorbance at 517 nm (A 1 ). Distilled water was used to replace the DPPH· solution and test samples, which were blank one and two, respectively. The DPPH· scavenging capacity was calculated as follows: Where A 2 and A 0 were the absorbance of blank one and two, respectively.
Superoxide anion (O 2− ·) scavenging capacity analysis. The pyrogallol autoxidation method was applied to determine the O 2− · scavenging capacity. Two milliliters of the test samples and 75 μL of 45 mM pyrogallol were successively mixed with 2.25 mL of 50 mM Tris-HCl buffer solution (pH 8.2). Then, 20 μL of 10 M HCl were added for termination after a 4-min reaction, and the absorbance of the mixture was measured at 320 nm (A 1 ). Distilled water was used to replace the pyrogallol and test samples as blank one and two, respectively. The O 2− · scavenging capacity was calculated as follows: Where A 0 and A 2 were the absorbance of blank one and two, respectively.
Total reducing power (TRP) analysis. A ferricyanide/Prussian blue method was performed to analyze the total reducing power activity. The phosphate buffer solution (2.5 mL, 0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide solution were successively mixed with 1 mL of the test samples. Then, the mixtures were incubated at 50 °C for 20 min. Upon cooling to room temperature, 2.5 mL of the mixture was taken out and mixed with 2.5 mL of 10% trichloroacetic acid, 0.5 mL of 0.1% ferric trichloride solution and 2.5 mL water. Reducing the power activity (A) was represented by the absorbance of the mixture measured at 700 nm after a 10-min incubation at room temperature.
Ferrous ion-chelating activity analysis. Ferrous ion-chelating activity was evaluated according to the method of Dinis with some modification 28 . A sample (2 mL) was mixed with 0.05 mL 2 mM FeCl 2 solution, and 0.2 mL 5 mM Ferric ion solution was added. After a 10-min incubation at room temperature, the absorbance of the mixture was measured at 562 nm (A 1 ), and the distilled water sample was set as the blank without metal chelating activity. The ferrous ion-chelating activity (F) was calculated as follows: Where A 0 was the absorbance of the blank.

Effects of five peonidin-based anthocyanins on the probiotic bacteria proliferation. The
Bifidobacterial strains (B. infantis, B. adolescentis, and B. bifidum) and Lactobacillus acidophilus were used to investigate the prebiotic activity of the peonidin-based anthocyanins. The Bifidobacterial strains and Lactobacillus acidophilus were activated with BS and MRS medium, respectively, according to the method established by Huebner 29 . The samples were added to the culture mediums at concentrations of 0, 0.5, 1.0, 1.5, 2.0, or 2.5 mg/ mL, respectively, with fructooligosaccharide (FOS) as the control. After recording the initial total bacterial count, the media was incubated in an anaerobic incubator (YQX-III, Shanghai Wanrui Laboratory Equipment Co., Ltd. Shanghai, China) at 37 °C for 36 h with a culture condition of 85% N 2 , 10% H 2 and 5% CO 2 . Then, the count of the probiotics and the pH of each sample were measured. For each sample, serial dilutions of the fluid medium were prepared with sterile 1% (W/V) peptone solution and duplicated 100 μL aliquots were spread on MRS agar plates. After anaerobic incubation at 37 °C for 48 h, the bacteria count was rescored as log cfu/mL.
Effects of five peonidin-based anthocyanins on the growth of harmful bacteria. The antibacterial activity of the peonidin-based anthocyanins on the growth of Staphylococcus aureus and Salmonella typhimurium were detected by the filter paper method. The pathogenic bacteria were activated with TSB medium in a constant temperature shaker (HASUC, Shanghai union instrument manufacturing Co., Ltd. Shanghai, China) at 37 °C and 180 r/min till the OD value was 0.5, and 100 μL of the bacteria suspension was added to the petri dishes and coated evenly under aseptic conditions. The filter papers disks (Whatman No. 1, 6 mm diameter) containing 15 μL of each sample at different concentrations (1, 2, 3, 4 and 5 mg/mL, respectively) were put in the center of the petri dishes. The diameter of the resulting zone of inhibition was measured after a 12 h incubation at 37 °C. The minimum inhibitory concentration (MIC) of the peonidin based anthocyanins against the growth of Staphylococcus aureus and Salmonella typhimurium was determined. Two-fold serial dilutions of anthocyanins were mixed with culture media to get a final concentration ranging from 25 to 3000 μg/mL. The bacterial suspension was inoculated onto each plate and incubated at 37 °C for 12 h, including on a growth control without anthocyanins and a sterility control without bacterial suspension. The MIC was defined as the lowest concentration of anthocyanins that prevented the visible growth of the bacteria 30 .

Statistical analysis.
Each experiment was carried out in triplicate. One-way analysis of variance and Tukey's comparison of the means were performed using IBM SPSS Statistical software (version 20, Chicago, IL, USA), and a difference of P < 0.05 was considered significant.

Conclusions
The main anthocyanins from the Chinese cultivar PSP (Ipomoea batatas (L.) Lam.) were identified as the peonidin types. Five peonidin-based anthocyanin monomers exerted stronger in vitro antioxidant activities. And the Scientific RePoRts | (2018) 8:5018 | DOI:10.1038/s41598-018-23397-0 peonidin-based anthocyanin monomers might be potential natural probiotic sources that could increase the proliferation of Bifidobacterial strains (B. infantis, B. adolescentis, and B. bifidum) and Lactobacillus acidophilus, as well as inhibit the growth of the intestinal pathogens of Staphylococcus aureus and Salmonella typhimurium. The results revealed the potential benefits for the consumption of the Chinese cultivar PSP in the human diet, and the characteristics of anthocyanin monomers will be useful for functional foods and pharmaceutical developments.