Food Chemistry 161 (2014) 22–26

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Isolation and identification of colourless caffeoyl compounds in purple sweet potato by HPLC-DAD–ESI/MS and their antioxidant activities Jin-Ge Zhao a, Qian-Qian Yan a, Ren-Yu Xue a, Jian Zhang b,⇑, Yu-Qing Zhang a,⇑ a Silk Biotechnology Laboratory, School of Biology and Basic Medical Sciences, Soochow University, RM702-2303, No. 199, Renai Road, Dushuhu Higher Edu. Town, Suzhou 215123, PR China b College of Pharmaceutical Science, Soochow University, No. 199, Renai Road, Dushuhu Higher Edu. Town, Suzhou 215123, PR China

a r t i c l e

i n f o

Article history: Received 19 December 2012 Received in revised form 12 March 2014 Accepted 15 March 2014 Available online 26 March 2014 Keywords: Caffeoyl Purple sweet potato Semi-preparative HPLC ESI/MS NMR Antioxidant activity

a b s t r a c t More than 10 red anthocyanins and related glucosides have been isolated and identified from purple sweet potato (Ipomoea batatas, Ayamurasaki) in the recent decades. This paper reports the isolation of colourless caffeoyl compounds from purple sweet potato using AB-8 macroresin absorption and semipreparative HPLC-DAD. The structures of the five isolated monomers were identified as: 5-caffeoylquinic acid (1), 6-O-caffeoyl-b-D-fructofuranosyl-(2-1)-a-D-glucopyranoside (2) and trans-4,5-dicaffeoylquinic acid (3), 3,5-dicaffeoylquinic acid (4), 4,5-dicaffeoylquinic acid (5), and by ESI/MS and NMR. Compounds 1, 4 and 5 were reported previously in combination with anthocyanins in purple sweet potato, whereas 2 and 3 were found for the first time. In vitro antioxidant assay showed trans-4,5-dicaffeoylquinic acid has significant antioxidant activities. These results should lay the groundwork for further work identifying purple sweet potato as a healthy food. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Anthocyanins, a group of plant pigments that are widely distributed in fruits (Rommel, Heatherbell, & Wrolstad, 1990), vegetables (Gusti & Wrolstad, 1996; McDougall, Fyffe, Dobson, & Stewart, 2007; Murai, & Wilkins, 1990) and some flowers (Markakis, 1982), are flavonoids that commonly produce blue, red or purple colours and are members of a class of water-soluble glycosides and acylglycosides of anthocyanidins (Bridle & Timberlake, 1997). Anthocyanins possess two benzene rings joined by a linear three carbon chain (C2–C4), represented as the C6–C3–C6 system. This particular chemical structure makes them highly reactive towards the free radicals and powerful natural antioxidants (Galvano, 2005). Anthocyanins have been found to down-regulate pro-inflammatory cytokines in a murine asthma model (Park, Shin, Seo, & Kim, 2007). Thus, anthocyanins are notable not only as natural food colourants, but consumption may also reduce the risk of age-related diseases. It has been shown that some phenolics act as antioxidants through a number of mechanisms that involve free radical scavenging and metal ion chelation (Meyer, Donovan, Pearson, Waterhouse, & Frankel, 1998; Rice-Evans, Miller, Bolwell, Bramley ⇑ Corresponding authors. Tel./fax: +86 0512 65880181 (Y.-Q. Zhang). E-mail addresses: [email protected] (J. Zhang), [email protected] (Y.-Q. Zhang). http://dx.doi.org/10.1016/j.foodchem.2014.03.079 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

& Pridham, 1995; Terao, Karasawa, Arai, Nagao, Suzuki, & Takama, 1993). Caffeoylquinic acids are formed by esterification quinic acid and caffeic acid. The best known and recognized caffeoylquinic acid is a monocaffeoyl derivative, chlorogenic acid (5-caffeoylquinic acids), which occurs in many plants, fruits and vegetables (Azuma, Ippoushi, Nakayama, Ito, Higashio, & Terao, 2000). There are several advantageous health properties associated with this class of compounds, such as antioxidant capacity and anti-inflammatory and antibacterial properties (Farah, Monteiro, Donangelo, & Lafay, 2008). The dicaffeoylquinic acid isomers are known to possess higher antioxidant activity as well as tyrosinase inhibition and anti-proliferation activities than monocaffeoylquinic acid (Iwai, Kishimoto, Kakino, Mochida, & Fujita, 2004). However, as for antioxidant activity in humans, their poor bioavailability and easy metabolism raise serious doubts about the benefits of the parent caffeoylquinic acids. Sweet potato (Ipomoea batatas L.) contains high levels of phenolic compounds, including chlorogenic acids (Clifford, Johnston, Knight, & Kuhnert, 2003), three isomers of mono-caffeoylquinic acid: 3-caffeoylquinic acid (neochlorogenic acid), 4-caffeoylquinic acids (cryptochlorogenic acid) and 5-caffeoylquinic acid (chlorogenic acid), and three dicaffeoylquinic acids: 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid (isochlorogenic acids A–C, respectively) (Ishiguro, Yahara, & Yoshimoto, 2007). Jung, Lee, Kozukue, Levin, and Friedman (2011) studied the distribution of phenolic compounds between different parts

J.-G. Zhao et al. / Food Chemistry 161 (2014) 22–26

of the sweet potato plant. The results showed that the stem end of the root had significantly more phenolics and the predominant chlorogenic acids present were 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acids. Anthocyanins in purple-fleshed sweet potatoes (Ayamurasaki) are acylated by caffeoylquinic acid, p-hydroxybenzoic acid or ferulic acid, which are also responsible for their radical-scavenging properties (Suda, Oki, Masuda, Kobayashi, Nishiba, & Furuta, 2003). Various pigments in purple sweet potato (PSP) have been analyzed and most of these pigments have been found to be anthocyanins. Montilla, Hillebrand, Butschbach, Baldermann, Watanabe, and Winterhalter (2010) investigated the anthocyanin composition of PSP using HPLC-DAD and ESI–MS, and identified two major pigments, cyanidin and peonidin derivatives, both of which are linked to caffeoyl compounds (Montilla et al., 2010). Similar results reported by Yukihiro et al. showed that the 3-O-(6-O-trans-caffeyt-2-O-b-glucopyranosyl-/3-glucopyranoside)-5-O-fl-glucoside of cyanidin and peonidin could be isolated from the storage roots of sweet potato (Yukihiro, Takashige, Yoshiaki, Nakamura, Maitani, & Yamada, 1997). Eight acylated anthocyanins were isolated from the roots of the PSP by Terahara, Shimazu, and Kato (1999). They were identified as cyanidin and peonidin, in which each acyl substituent was a p-hydroxybenzoyl, caffeyl or ferulyl residue. Qiu, Luo, Yao, Ma, and Kong (2009) applied high-speed counter-current chromatography to isolate and purify peonidin glucosides and cyanidin glucopyranosides with caffeoyls from PSP. These were identified as 1H NMR, 13C NMR and ESI-MSn (Qiu et al.,, 2009). Previous studies on PSP roots focused on anthocyanins that combined with caffeoyl compounds. There have been no reports of caffeoyl compounds in PSP where they are not combined with other compounds. This study isolated colourless caffeoyl compounds from PSP roots, which had strong antioxidant characteristic. This paper describes the isolation, purification and identification of these caffeoyl compounds using pre-preparative HPLC and their characterization using ESI–MS and NMR and their specific bioactivities. 2. Materials and methods 2.1. Chemicals and materials The purple sweet potato (I. batatas, Ayamurasaki) was provided by Jinshu Co., Ltd., Shandong, China. Solvents used for HPLC were of chromatogram grade (Fair Lawn, New Jersey 07410). All other solvents were of analytical grade. 2.2. Sample preparation of red and non-red compounds There were slight modifications to the extraction methods for PSP active compounds described previously (Lu & Zhang, 2010). PSPs were heated in a microwave oven for 15 min. The cooked potatoes (1 kg approximately) were crushed and suspended in acidic electrolyzed water (2 L, pH = 3.0). The suspension was cooled to 0 °C for 12 h without stirring. To remove the solid residue, the suspension was centrifuged at 12,000 rpm and 4 °C for 20 min prior to analysis using an Amberlite AB-8 column. The column was rinsed with water and the extracts were eluted with a solution of water/ethanol (7:3 v/v). The elute was concentrated in vacuo, freeze-dried and referred to as the crude PSP extracts. Caffeoyl compounds were then isolated and purified from the crude PSP extracts by semi-preparative high-performance liquid chromatography (HPLC). HPLC was performed using a Semi-Preparative Shimadzu LC-6A equipped with a DAD detector (SPDM20A). The column and mobile phase consisted of a Shim-pack Prep-ODS (H) column (250  20 mm) fitted with a guard module (Shim-pack G-ODS). The mobile phase was composed of 10% acetic

23

acid in water (A) and 100% acetonitrile (B). The gradient conditions were as follows: for 0–30 min, the content of mobile phase B was increased linearly from 7% to 30%, and remained at 30% for the next 30 min. The flow rate was 5 ml min1 and injection volume was 5 mL. All solvents and samples were filtered through a 0.22 lm Millipore filter. Fractions were collected using a FRC-10A fraction collector (SHIMADZU). 2.3. Electrospray ionization multiple mass spectrometry (ESI–MS) For ESI–MS experiments, pure pigments were dissolved in a mixture of water/acetic acid (9:1, v/v) and were directly delivered to the ESI source (Agilent LC/MSD SL) for mass spectrometry analysis. The mass spectrometry parameters were as follows: negative ion mode; scan range, m/z 110–1000 and 1000–2000; dry temperature, 350 °C; MSD detection voltage, 3650 V and Fragmentor chipped voltage, 110 V. 2.4. Proton and carbon nuclear magnetic resonance spectroscopy (NMR) 1 H and 13C NMR measurements were performed using a Bruker AV 500 spectrometer (Bruker Biospin, Rheinstetten, Germany) at 125.77 and 500.13 MHz, respectively. Pure pigments were dissolved in DMSO-d6 and MeOD-d4 and the data were processed by WIN-NMR software version 6.1.0.0.

2.5. Assay of DPPH radical scavenging activity DPPH radical scavenging activities were measured using the method of Rao, Tiwari, Kumar, Reddy, Ali, and Rao (2003) with some modifications. In brief, a solution of DPPH was prepared by dissolving DPPH in ethanol and the solution was kept in the dark at room temperature. Various concentrations of the test samples were mixed with DPPH in ethanol and the absorbance measured at 517 nm. Percentage scavenging activity was determined by comparison with the untreated control group. The antioxidant activity of the test compounds was expressed as an IC50, which is defined as the concentration of compounds required to inhibit DPPH radicals by half. 2.6. Reducing power assay The reducing power of PSP extracts was measured using the method of Wang, Xing, Hu, Zhu, & Wang (1994). The absorbance was measured spectrophotometrically at 700 nm. Higher absorbance indicated increased reducing activity. 2.7. Assay of total antioxidant capacity The total antioxidant capacity of the samples was determined using a modification of the FRAP assay (Benzie & Strain, 1996). Briefly, a 100 lL aliquot of the extracts was added to 900 lL of FRAP reagent. Thereafter, the procedure was followed as described (Benzie & Strain, 1996). The results were compared with a standard curve using FeSO4 comprised of concentrations in the range 100–1000 lmol L1. The results were expressed as lmoles of ferric reducing/antioxidant power per 1 g of extracting power (lmol FeSO4 g1 dw). 3. Results and discussion 3.1. Isolation and preparation of colourless fractions Five components: C1–C5, were isolated based on different retention times in a semi-preparative reversed-phased chromatogram

24

J.-G. Zhao et al. / Food Chemistry 161 (2014) 22–26

column. Fig. 1 shows the HPLC chromatogram for the double wavelengths. C1–C5 were strongly detected at 324 nm and five red compounds (A1–A5) were detected at 524 nm. According to the mass spectrum data (Table 1) and previous studies (Montilla et al., 2010), the five red compounds were acylated cyanidin or peonidin glucoside compounds, specifically: cy-3-(600 ,000 -dicaffeoylsoph)-5-glcc, cy-3-(600 -caffeoyl-6000 -feruloylsoph)-5-glcc, pn-3-(600 ,6000 -dicaffeoylsoph)-5-glcc, pn-3-(600 -caffeoyl-6000 -p-hydroxybenzoylsoph)-5-glcc and pn-3-(600 -caffeoyl-6000 -feruloylsoph)-5-glcc.

Table 1 Mass spectrometric properties of anthocyanins A1–A5.

3.3. Structure identification of colourless compounds

34.48533

C1

A4

36.768

31.73333

35.70133

18.14

A1

15.91

ABS AU

0.2

A5

A2 A3

C2

0.3

C5

38.784

0.4

C4

46.24

324nm 524nm

C3

41.48

0.5

40.00

The results showed that the five colourless compounds were caffeoyl compounds (Table 2, Fig. 3). Their molecular ions [M+H+] were: 355, 504, 517, 517 and 517, respectively, and it is likely the last three are isomers. The 1H and 13C NMR spectroscopic data are detailed below: C1: 5-O-caffeoy lquinic acid (C16H18O9). Data from 1H-NMR (CD3OD, 500 MHz) using methanol as the solvent are shown in Supplementary data 1. The spectroscopic data was the same as the known substance, 5-O-caffeoylquinic acid (Xu, Zhang, & Gong, 2012). Data from 1H-NMR (DMSO-d6, 500 MHz) using DMSO as the solvent are shown in Supplementary data 2. The spectroscopic data was same as the known substance, 5-O-caffeoylquinic acid. C2: 6-O-caffeoyl-b-D-fructofuranosyl-(2-1)-a-D-glucopyranoside (C21H28O14). Data from 1H-NMR (DMSO-d6, 500 MHz) using DMSO as the solvent are shown in Supplementary data 3. The spectroscopic data was the same as the known substance, 6-O-caffeoyl-b-D-fructofuranosyl-(2-1)-a-D-glucopyranoside, which structure was showed in Fig. 3. C3: trans-4,5-dicaffeoylquinic acid (C25H24O12). Data from 1H-NMR (MeOD, 500 MHz) using methanol as the solvent are shown in Supplementary data 4. Data from 1H-NMR

Fragments (m/z)

31.73

1097

cy-3-(6 -caffeoyl-6 -feruloylsoph)5-glcc pn-3-(600 ,6000 -dicaffeoylsoph)-5-glcc

34.48

1111

35.70

1111

pn-3-(600 -caffeoyl-6000 -phydroxybenzoylsoph)-5-glcc pn-3-(600 -caffeoyl-6000 -feruloylsoph)5-glcc

36.77

1069

38.78

1125

935, 287 949, 287 949, 301 907, 301 963, 301

A1

cy-3-(600 ,000 -dicaffeoylsoph)-5-glcc

A2 A3

A5

The UV–Vis spectra of the five PSP caffeoyl fractions had similar characteristics (Fig. 2). They all had strong absorption peaks at 330 nm and characteristic spectral absorption peaks at 295 nm. This suggested that the five pigments had similar chemical structures (Table 2).

[M]+ (m/z)

Compound

A4

3.2. UV–Vis spectra of colourless fractions

Rt (min)

Peak

00

000

449, 449, 463, 463, 463,

(DMSO-d6, 500 MHz) using DMSO as the solvent are shown in Supplementary data 5. C4: 3,5-dicaffeoylquinic acid (C25H24O12). Data from 1H-NMR (MeOD, 500 MHz) using methanol as the solvent are shown in Supplementary data 6. The spectroscopic data was same as 3,5-dicaffeoylquinic acid from the honeysuckle leaf (Wang, Shao, Li, & Zhu, 1999). Data from 1H-NMR (DMSO-d6, 500 MHz) using DMSO as the solvent are shown in Supplementary data 7. The spectroscopic data was the same as 3,5-dicaffeoylquinic acid from the honeysuckle leaf (Wang, Wang, Yao, & Susumu, 2006). C5: 4,5-dicaffeoylquinic acid (C25H24O12). Data from 1H-NMR (MeOD, 500 MHz) using methanol as the solvent are shown in Supplementary data 8. The spectroscopic data was the same as 4,5-dicaffeoylquinic acid from Floret ghost needle grass (Wang et al., 2006). Data from 1H-NMR (DMSO-d6, 500 MHz) using DMSO as the solvent are shown in Supplementary data 9. The spectroscopic data was the same as 4,5-dicaffeoylquinic acid from the honeysuckle leaf (Liu & Chen, 2010). 3.4. Total antioxidant capacity The total antioxidant capacity of the five caffeoyl compounds is shown in Fig. 4. The results showed that the total antioxidant capacity increased as the concentration of the caffeoyl compounds rose and the trends for all five compounds was similar to vitamin C. Nevertheless, C4 and C5, which had longer column retention times, had much greater antioxidant capacity, almost twice the values determined for the other compounds, over a 0.05–0.5 mg ml1 range. Their total antioxidant capacities reached 70–80% of vitamin C. C3 and C5 had similar structures and the only difference was spatial arrangement at the 40 -quinic site. However, the total antioxidant activity of C3 was two thirds that of C5. The results suggested that quinic acid with di-caffeoyl has stronger antioxidant capacity than mono-caffeoyl. Furthermore, with regards to di-caffeoyl quinic acid, cis-dicaffeoyl quinic acid has a much stronger antioxidant capacity than trans-dicaffeoyl quinic acid. In addition, the antioxidant activity of 6-O-caffeoylb-D-fructofuranosyl-(2-1)-a-D-glucopyranoside was higher than that of 5-O-caffeoyl quinic acid. 3.5. Reducing power of caffeoyl compounds

0.1

0.0 15

20

25

30

35

40

45

Retention Time (min) Fig. 1. HPLC-PDA detection of PSP caffeoyl compounds.

50

Fig. 5 shows the reducing power of the five caffeoyl compounds. As with total antioxidant capacity, C3–C5 had much greater reducing power than C2 and C1. Quinic acid combined with dicaffeoyl had a much greater reducing power than when combined with mono-caffeoyl or when diglucoside was combined with mono-caffeoyl. However, their reducing power was only 15–50% of vitamin C.

25

J.-G. Zhao et al. / Food Chemistry 161 (2014) 22–26

4.0

1.0 0.9

C1 C2 C3 C4 C5

0.7

ABS (AU)

0.6 0.5 0.4 0.3 0.2 0.1 0.0 250

300

350

400

450

500

C1 C2 C3 C4 C5 Vc

3.5

FeSO4 equivalently(mmol)

0.8

3.0 2.5 2.0 1.5 1.0 0.5 0.0

550

0.0

0.1

0.2

Fig. 2. UV–Vis spectra of PSP caffeoyl compounds.

5

OH

2

6

C1 C2 C3 C4 C5 Vc

2.5

3

OR3

2.0

OH

4

OR4 OR5 Quinic acid

Caffeoyl

Peak

Compound

R1

R3

R4

R5

C1 C3 C4 C5

5-Caffeoylquinic acid trans-4,5-Dicaffeoylquinic acid 3,5-Dicaffeoylquinic acid 4,5-Dicaffeoylquinic acid

H H H H

H H Caffeoyl H

H Caffeoyl H Caffeoyl

Caffeoyl Caffeoyl Caffeoyl Caffeoyl

ABS(AU)

1

HO

0.5

3.0

O

OR1

0.4

Fig. 4. Total antioxidant status of PSP caffeoyl compounds.

Table 2 The chemical structures of caffeoyl compounds. O

0.3

Concentrate(mg/ml)

Wavelength (nm)

1.5

1.0

0.5

0.0

0.20

0.25

3.6. DPPH free radical scavenging activities of caffeoyl compounds

0.35

0.40

Fig. 5. Scavenging effect of PSP caffeoyl compounds on reducing power.

80

C1 C2 C3 C4 C5 Vc

70 60

Inhibition rate(%)

As shown in Fig. 6, the five caffeoyl compounds from purple sweet potato had strong DPPH free radical scavenging activities compared with vitamin C. The scavenging activities increased as the concentration of the caffeoyl compounds rose. Their inhibition rates (IC50) were 7.6–12.4 lg ml1, and the value for vitamin C was 4.7 lg ml1. The difference in scavenging activities between the five pigments was not as great as the total antioxidant capacities and reducing power. It made no significant difference whether quinic acid was combined with caffeoyl quinic acid, caffeoyl diglucoside, mono-caffeoyl or di-caffeoyl, as these combinarions all had strong DPPH free radical scavenging activities suggesting the scavenging activity was related to the caffeoyl groups. So, it does seem that scavenging activity is related to the caffeoyl groups. But, caffeoyl has no electrons or H-atoms to donate meaning the

0.30

Concentrate(mg/ml)

50 40 30 20 10 0

HO O HO HO

OH OH O 6 5

O 2

-10

1

O O OH OH 2 4 OH OH 3

Fig. 3. Structure of caffeoyl glucoside (C2) in PSP.

0

5

10

15

20

25

Concentrate (µg/ml) Fig. 6. The scavenging effect of PSP caffeoyl compounds on DPPH free radicals.

mechanism(s) accounting for the scavenging activity are unknown; the radical and the compound may be forming a stable complex.

26

J.-G. Zhao et al. / Food Chemistry 161 (2014) 22–26

4. Conclusions Caffeoyl quinic acid compounds are widely distributed in plants. Most of them are combined with other molecules and have significant antioxidant and pharmacological activities. In this study, using LC–MS, 1H-NMR and 13C-NMR, five caffeoyl compounds were identified, which contained caffeoyl quinic acid and caffeoyl diglucoside. According to previous reports, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid are combined with anthocyanins in PSP and do not occur separately. This study found 6-O-caffeoyl-b-D-fructofuranosyl-(2-1)a-D-glucopyranoside and trans-4,5-dicaffeoylquinic acid in purple sweet potato for the first time. The antioxidant properties of the five caffeoyl compounds in PSP were evaluated and the results showed that quinic acids when combined with di-caffeoyl had much stronger antioxidative activities than when combined with mono-caffeoyl. This suggests the antioxidant activities are closely related to the number of caffeoyl compounds present. Furthermore, cis-dicaffeoyl quinic acid had a much greater antioxidant capacity than trans-dicaffeoyl quinic acid. These studies form a basis for future research into caffeoyl quinic acid compounds, their consumption in the human diet and potential health benefits. In summary, five caffeoyl compounds were identified, further purified and separated and they showed strong antioxidant and DPPH radical scavenging activities. The results obtained in this study aim to lay the foundation for further progress in developing purple sweet potato or its extracts as a health foods and as environmentally friendly food colourings and active compounds. It could also provide the basis for a new functional breeding program for purple sweet potato. Acknowledgements The authors are very grateful to Professor Jian Zhang at Medical College of Soochow University for her instruction of structure analysis. The authors gratefully acknowledge the earmarked fund (CARS-22-ZJ0504) for China Agriculture Research System (CARS) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, P.R. China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 03.079. References Azuma, K., Ippoushi, K., Nakayama, M., Ito, H., Higashio, H., & Terao, J. (2000). Absorption of chlorogenic acid and caffeic acid in rats after oral administration. Journal of Agricultural and Food Chemistry, 48, 5496–5500. Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: The FRAP assay. Analytical Biochemistry, 239, 70–76. Bridle, P., & Timberlake, C. F. (1997). Anthocyanins as natural food colours—Selected aspects. Food Chemistry, 58, 103–109. Clifford, M. N., Johnston, K. L., Knight, S., & Kuhnert, N. (2003). Hierarchical scheme for LC–MSn identification of chlorogenic acids. Journal of Agricultural and Food Chemistry, 51, 2900–2911. Farah, A., Monteiro, M., Donangelo, C. M., & Lafay, S. (2008). Chlorogenic acids from green coffee extract are highly bioavailable in humans. The Journal of Nutrition, 138, 2309–2315.

Galvano, F. (2005). The chemistry of anthocyanins. Functional Ingredients, July 1, 2005. Available from http://newhope360.com/managing-your-business/ chemistry-anthocyanins. Gusti, M. M., & Wrolstad, R. E. (1996). Radish anthocyanin extract as a natural red colorant from maraschino cherries. Journal of Food Science, 61, 688–694. Ishiguro, K., Yahara, S., & Yoshimoto, M. (2007). Changes in polyphenols content and radical-scavenging activity of sweetpotato (Ipomoea batatas L.) during storage at optimal and low temperatures. Journal of Agricultural and Food Chemistry, 55, 10773–10778. Iwai, K., Kishimoto, N., Kakino, Y., Mochida, K., & Fujita, T. (2004). In vitro antioxidative effects and tyrosinase inhibitory activities of seven hydroxycinnamoyl derivatives in green coffee beans. Journal of Agricultural and Food Chemistry, 52, 4893–4898. Jung, J. K., Lee, S. U., Kozukue, N., Levin, C. E., & Friedman, M. (2011). Distribution of phenolic compounds and antioxidative activities in parts of sweet potato (Ipomoea batatas L.) plants and in home processed roots. Journal of Food Composition and Analysis, 24, 29–37. Liu, C. J., & Chen, S. P. (2010). Studies on chemical constituents of leaves of Lonicera japonica. Chinese Journal of Experimental Traditional Medical Formulae, 16, 90–92. Lu, L. Z., & Zhang, Y. Q. (2010). Anthocyanin extracts from purple sweet potato by means of microwave baking and acidified electrolysed water and their antioxidation in vitro. International Journal of Food Science and Technology, 45, 1378–1385. Markakis, P. (1982). Anthocyanins as food colors food science and technology—A series of monographs. New York: Academic press. McDougall, G. J., Fyffe, S., Dobson, P., & Stewart, D. (2007). Anthocyanins from red cabbage-stability to simulated gastrointestinal digestion. Phytochemistry, 68, 1285–1294. Meyer, A. S., Donovan, J. L., Pearson, D. A., Waterhouse, A. L., & Frankel, E. N. (1998). Fruit hydroxycinnamic acids inhibit human low-density lipoprotein oxidation in vitro. Journal of Agricultural and Food Chemistry, 46, 1783–1787. Montilla, E. C., Hillebrand, S., Butschbach, D., Baldermann, S., Watanabe, N., & Winterhalter, P. (2010). Preparative isolation of anthocyanins from Japanese purple sweet potato (Ipomoea batatas L.) varieties by high-speed countercurrent chromatography. Journal of Agricultural and Food Chemistry, 58, 9899–9904. Murai, K., & Wilkins, D. (1990). Natural red color derived from red cabbage. Food Technology, 44, 31. Park, S. J., Shin, W. H., Seo, J. W., & Kim, E. J. (2007). Anthocyanins inhibit airway inflammation and hyperresponsiveness in a murine asthma model. Food and Chemical Toxicology, 45, 1459–1467. Qiu, F., Luo, J. G., Yao, S., Ma, L., & Kong, L. (2009). Preparative isolation and purification of anthocyanins from purple sweet potato by high-speed countercurrent chromatography. Journal of Separation Science, 32, 2146–2151. Rao, R. J., Tiwari, A. K., Kumar, U. S., Reddy, S. V., Ali, A. Z., & Rao, J. M. (2003). Novel 3-O-Acyl mesquitol analogues as free radical scavengers and enzyme inhibitors: Synthesis, biological evaluation and structure activity relationship. Bioorganic & Medicinal Chemistry Letters, 13, 2777–2780. Rice-Evans, C. A., Miller, N. J., Bolwell, P. G., Bramley, P. M., & Pridham, J. B. (1995). The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radical Research, 22, 375–383. Rommel, A., Heatherbell, D. A., & Wrolstad, R. E. (1990). Red raspberry juice and wine: Effect of processing and storage on anthocyanin pigment composition, color appearance. Journal of Food Science, 55, 1011–1017. Suda, I., Oki, T., Masuda, M., Kobayashi, M., Nishiba, Y., & Furuta, S. (2003). Physiological functionality of purple-fleshed sweet potatoes containing anthocyanins and their utilization in foods. JARQ, 37, 167–173. Terahara, N., Shimazu, T., Kato, Y., et al. (1999). Six diacylated anthocyanins from the storage roots of purple sweet potato, Ipomoea batatas. Bioscience, Biotechnology, and Biochemistry, 63, 1420–1424. Terao, J., Karasawa, H., Arai, H., Nagao, A., Suzuki, T., & Takama, K. (1993). Peroxyl radical scavenging activity of caffeic acid and its related phenolic compounds in solution. Bioscience, Biotechnology, and Biochemistry, 57, 1204–1205. Wang, J., Wang, N. L., Yao, X. S., & Susumu, K. (2006). Caffeoylquinic acid derivatives from Bidens parviflora and their antihistamine release activites. Chinese Traditional and Herbal Drugs, 37, 966–970. Wang, J. C., Xing, G. S., Hu, W. D., Zhu, T., & Wang, Q. (1994). Effects of Ge-132 on oxygen free radicals and the lipid peroxidation induced by hydroxyl free radical in vitro. Chinese Pharmaceutical Journal, 29, 23–25. Wang, M. F., Shao, Y., Li, J. G., & Zhu, N. (1999). Antioxidative phenolic glycosides from sage (Salvia officinalis). Journal of Natural Production, 62, 454–456. Xu, J., Zhang, T. J., & Gong, S. X. (2012). Chemical compounds research of hemostatic active site of Cirsium setosum. Chinese Traditional and Herbal Drugs, 41, 542–544. Yukihiro, G., Takashige, S., Yoshiaki, K., Nakamura, M., Maitani, T., Yamada, T., et al. (1997). Two acyleted anthocyanins from purple sweet potato. Phytochemistry, 44, 183–186.