Identification of anthocyanins isolated from black rice (Oryza sativa L.) and their degradation kinetics

Food Research International 50 (2013) 691–697

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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Identification of anthocyanins isolated from black rice (Oryza sativa L.) and their degradation kinetics Zhaohua Hou a, b, Peiyou Qin a, Yan Zhang a, Songhuan Cui b, Guixing Ren a,⁎ a

Institute of Crop Science, Chinese Academy of Agricultural Sciences, No. 80 South Xueyuan Road, Haidian District, Beijing 100081, PR China State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Science, Chinese Academy of Agricultural Sciences, No. 15 Luming Road, Jilin City, Jilin Province 132109, PR China

b

a r t i c l e

i n f o

Article history: Received 3 December 2010 Accepted 28 July 2011 Keywords: Anthocyanins HPLC/DAD/MS Black rice Degradation kinetics

a b s t r a c t Black rice is rich in anthocyanins-plant pigments. The aim of this work was to identify anthocyanins in black rice using high-performance liquid chromatography (HPLC)-electrospray ionization — mass spectrometry with diode array detection. Four different anthocyanins (cyanidin-3-glucoside, peonidin-3-glucoside, cyanidin-3,5-diglucoside, cyanidin-3-rutinoside) were identified in black rice. Thermal stability of the four anthocyanins in black rice extract was studied at selected temperatures (80 °C, 90 °C and 100 °C) in the range of pH 1.0–pH 6.0. The results indicated that the thermal degradation of anthocyanins followed the first-order reaction kinetics. The temperature-dependent degradation was adequately modeled on the Arrhenius equation. The calculated values of activation energies (Ea), t1/2 and k were different for the four anthocyanins. The degradation rate of monomeric anthocyanin increased with increasing heating temperature and pH values. Especially, as heating temperature increasing to 100 °C and pH value to 5.0. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Anthocyanins are a group of plant pigments that are widely distributed in nature (Wu & Prior, 2005), which are responsible for the attractive colors of many flowers, fruits, grains and related products derived from them (Escribano-Bailón, Santos-Buelga, & Rivas-Gonzalo, 2004; Kong, Chia, Goh, Chia, & Brouillard, 2003). Anthocyanins are water-soluble glycosides and acylglycosides of anthocyanidins (Wu, Gu, Prior, & McKay, 2004), and they are found in the form of polyhydroxylated and or methoxylated heterosides which derive from the flavylium ion or 2-phenylbenzopyrilium in nature. Six anthocyanidins are widespread in fruits and vegetables (Table 1), which are pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin (De Pascual-Teresa, Santos-Buelga, & Rivas-Gonzalo, 2002; Di Paola-Naranjo, Sanchez-Sanchez, Gonzalez-Paramas, & Rivas-Gonzalo, 2004; Lohachoompol, Mulholland, Srzednicki, & Craske, 2008). These compounds are based on the same 2-phenylbenzopyrilium (flavylium) skeleton hydroxylated in 3, 5, and 7 positions, and different in the number and position of hydroxyl and methoxyl groups in the B-ring (Escribano-Bailón et al., 2004). Anthocyanins are valuable as kinds of important quality indicators in foods and chemotaxonomic indicators in plants. They attract much interest for its health function such as their potential antioxidant ability (Kong et al., 2003; Marco & Scarminio, 2007).

⁎ Corresponding author. Tel.: + 86 10 62 11 5596; fax: + 86 10 62 15 6596. E-mail address: [email protected] (G. Ren). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.07.037

Black rice (Oryza sativa L.) is a special cultivar of rice and widely consumed since ancient times in China and other Eastern Asia countries (Guo et al., 2007). It has been regarded as a health-promoting food for its abundant content of anthocyanins, but, anthocyanins are not stable and vulnerable to be destroyed by a number of factors such as: pH, light, oxygen, enzymes, ascorbic acid, thermal treatment, sulfur dioxide or sulfite salts, metal ions and copigments (Gradinaru, Biliaderis, Kallithraka, Kefalas, & Garcia-Viguera, 2003; Tiwari, O' Donnell, Muthukumarappan, & Cullen, 2009; Zhang et al., 2008). Preventing anthocyanin degradation is an important aspect that can benefit both processors and consumers. Thermal degradation of anthocyanins is a major problem for the food industry. Thermal degradation of anthocyanins has been studied for blackberry (Wang & Xu, 2007), sour cherry (Cemeroglu, Velioglu, & Isik, 1994), strawberry (Strede, Wrolstad, Lea, & Enersen, 1992) juices and concentrates. Cyanidin-3-glucoside was a major anthocyanin present in red raspberry, blackberry and black rice etc. The previous investigations were focused on the effect of temperature, time and pH etc. on cyanidin-3-glucoside (Zhang et al., 2008) and malvidin-3-glucoside (Tseng, Chang, & Wu, 2006) degradation. Rubinskiene, Viskelis, Jasutiene, Viskeliene, and Bobinas (2005) demonstrated that an average reduction of cyanidin-3-glucoside in blackcurrant extract was 53% at 95 °C for 150 min. Few studies have focused on the stability of anthocyanins in black rice extracts. It is difficult to determine each anthocyanin degradation, because black rice contains rich anthocyanins, such as cyanidin-3-glucoside and peonidin3-glucoside (Yawadio, Tanimori, & Morita, 2007). The composition and contents of anthocynins are different, and most studies are solely based

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Table 1 Common anthocyanidins present in nature. Anthocyanidins

Substitution pattern

MWa

Delphinidin Cyanidin Pelagonidin Petunidin Peonidin Malvidin

3,5,7,3′,4′,5′-OH 3,5,7,3′,4′-OH 3,5,7,4′-OH 3,5,7,3′,4′,5′-OH; 3′-OMe 3,5,7,3′,4′-OH; 3′-OMe 3,5,7,4′-OH; 3′,5′-OMe

303 287 271 317 301 333

a

Molecular weight.

on monitoring the absorption decrease at λmax (Sadilova, Stintzing, & Carle, 2006). It's hard to predict the loss of each anthocyanin during heat process. To obtain further understanding of the thermal degradation of anthocyanins in black rice, it is necessary to investigate the degradation kinetics of each major anthocyanin, respectively. The first objective of this work was to identify the pigment composition of black rice using HPLC coupled to diode array detection and mass spectrometer (MS) detector. Secondly, investigate the effect of pH/temperature/time on each anthocyanin stability by creating a kinetic reaction model. The kinetic studies reported here could be useful in establishing appropriate processing. 2. Materials and methods 2.1. Materials Black rice (Longjin No.1, Oryza sativa L. Japonica) was purchased from a local market in Jilin province, China. The kernels were dried at 40 °C, ground with a laboratory mill, and passed through a 60 mesh screen sieve. Trifluoroacetic acid (TFA, analytical grade) was purchased from Sigma-Aldrich (Shanghai, China). Acetonitrile and other solvents (HPLC grade) were purchased from Fisher Chemicals (Shanghai, China). Anthocyanins standards (Cyanidin-3-glucoside, Cyanidin-3,5diglucoside, Peonidin-3-glucoside, HPLC grade) were obtained from Polyphenols Laboratories (Sandnes, Norway). The mobile phase and the samples were all filtered through 0.45 μm membrane.

wavelength. The column temperature was set at 35 °C. The flow rate was 1 mL/min, and the injection volume was 20 μL. All experiments were performed in triplicate simultaneously. Peak identification was performed with retention time as compared with the standard and confirmed with characteristic MS spectra detection. 2.4. Mass spectrometry analysis Electrospray mass spectrometry detection was performed with an Esquire-LC mass spectrometer (MS) (Bruker Daltoniks, Billerica, MA) and an ion trap instrument equipment with an electrospray interface. The experimental conditions were described previously (De PascualTeresa et al., 2002; Wu et al., 2004). Spectra were recorded in positive ion mode between m/z 50 and 1500. Major MS parameters were as follows: capillary exit, 4000 V; capillary offset, 500 V; skim 1, 38.3 V; nebulizer, 40 psi; dry gas, 12 L/min; temperature, 300 °C. 2.5. Degradation studies The thermal stability of each anthocyanin from black rice was studied at 80, 90 and 100 °C. Two grams of AEBR were dissolved with 1000 mL 0.2 M citrate-phosphate buffer. Aliquots of 10 mL anthocyanins solution were put into each of six plastic tubes. The sample tubes covered with aluminum foil were well capped to avoid evaporation and placed in a thermostatic water bath (Memmert WB 14, Schwabach, Germany) preheated to a given temperature. At regular time intervals (0, 30, 60, 90, 120, 150, and 180 min), six tubes were randomly taken from the water bath and rapidly cooled by plunging into an ice water bath (Yue & Xu, 2008). The contents of cooled tubes were analyzed for monomeric anthocyanin content. The effect of pH on thermal stability was also studied at six different pHs (1.0, 2.0, 3.0, 4.0, 5.0 and 6.0) at each temperature. Citrate-phosphate buffers were prepared to provide the specified pH situation. 2.6. Statistical analysis Linear regression analysis was applied to obtain the degradation rate constants (k) for all compounds studied. Data was analyzed by Statistical Analysis System (SAS 6.0) program.

2.2. Preparation of anthocyanin-rich extract from black rice (AEBR)

3. Results and discussion

AEBR was prepared as previously described (Prior et al., 2008). Briefly, black rice powder was extracted twice with ethanol/water/ hydrochloric acid (50:50:0.5, v/v/v) of solid–liquid ratio (1:10) for 2 h at 50 °C. The filtrates were combined and subjected to vacuum evaporation (Rotavapor R 210; Büchi, Flawil, Sweden) to remove ethanol. The concentrated extracts were loaded onto AB-8 resin. The AB-8 resin was washed with distilled water, and subsequently the absorbed anthocyanins were recovered with 80% ethanol. The ethanol eluent was sprayed to yield anthocyanin-rich powders.

3.1. Identification of anthocyanins from black rice

2.3. Instrumentation and chromatographic conditions The HPLC system consisted of two Alltech 626 HPLC pumps, an auto sampler (AS3000), and an UV6000 detector (Spectra system thermo Finnigan, San Jose, CA, USA). Compounds separation was carried out on a VP-ODS column (5 μm, 250 mm ∗ 4.6 mm, Shimadzu, Japan). Mobile phases were composed of 0.1% trifluoroacetic acid (A) and acetonitrile (B) with the following gradients: isocratic 10% B for 5 min, and the linear increase to 15% B in the following 15 min, hold 15% B for 5 min, then increased from 15 to 18% B during next 5 min and from 18 to 35% B over 20 min. 520 nm was selected as the preferred

Identification and peak assignment of anthocyanins in all extracts was based on comparison of their retention times and mass spectral data with those of standards and published data. Anthocyanins which have been studied extensively, served as references for identification purposes. Four anthocyanins were identified as cyanidin-3,5-diglucoside (peak 1), cyanidin-3-glucoside (peak 2), cyanidin-3-rutinoside (peak 3), peonidin-3-glucoside (peak 4). Two of the predominant anthocyanins in black rice were cyanidin-3-glucoside (91.01%) and peonidin-3-glucoside (7.13%), followed by cyanidin-3,5-diglucoside (0.92%) and cyanidin-3-rutinoside (0.94%) (Figs. 1 and 2; Table 2). Fig. 1 shows the HPLC anthocyanin profile of black rice. Data (retention time, λuv, λvis, molecular ion and main fragment ion) obtained for the anthocyanin peak in the HPLC-DAD-MS analysis are presented in Table 2. Spectral properties are very useful for characterizing anthocyanins, especially for identifying the anthocyanidin type (Escribano-Bailón et al., 2004). Introducing and jointing diode array detection systems (DAD) with HPLC opened a new perspective for anthocyanin separation, identification and quantification conveniently. It is possible to acquire on-line UV–vis spectra during chromatographic

Z. Hou et al. / Food Research International 50 (2013) 691–697

mV

693

2

400 300 200 100 1

0 0

3

20

10

4

30

40

50

60

min

Fig. 1. Anthocyanin profile in black rice extract analyzed using HPLC. Peak 1, cyanidin-3,5-diglucoside; peak 2, cyanidin-3-glucoside; peak 3, cyanidin-3-rutinoside; peak 4, peonidin-3-glucoside.

ananlysis (Escribano-Bailón et al., 2004). Fig. 3 showed the UV–vis spectra of the detected four anthocyanins obtained with DAD. Anthocyanins are flavonoids that are characterized by a flavylium nucleus exhibiting maximum absorbance in green/blue spectrum at 510 nm (Cisse, Vaillant, Acosta, Dhuique-Mayer, & Dornier, 2009). Duenas, Pérez-Alonso, Santo-Buelga, and Escribano-Bailón (2008) have recently shown λuv, λvis at 276, 513 characteristic of cyanidin derivatives. Using peak spectral characteristics λvis and λuv and their corresponding absorptivities (Table 2), it was possible to identify the anthocyanins as either mono- or biosides (E440/Evis absorptivity ratio of 29–35% indicating a monoside and a ratio of 15–24% indicating a bioside) (Dyrby, Westergaard, & Stapelfeldt, 2001).

The identification of isolated anthocyanins was confirmed by LC-MS analysis, the MS data were summarized in Table 2. Fragmentation patterns and positively monocharged molecular ions of each anthocyanin were shown in Fig. 2. Peonidin with m/z at 301 and cyanidin with m/z at 287 were detected in their respective mass spectrogram and the difference of 14 between both values attested the presence of a methoxy group in compound A compared with a hydroxyl group in B. Mass spectral characteristics of isolated anthocyanins were consistent with those of standards. Comparing the MS data with those recently published (Aoki, Kuze, & Kato, 2002; Cao, Liu, Pan, Lu, & Xu, 2009; Wu et al., 2004; Yawadio et al., 2007), four different anthocyanins were identified. Peaks 2 and 4 were the major pigments identified in AEBR. MS data of peak 2 showed a molecular ion of 449 ([M]+m/z 435) and a

x106 1.50

x106

1.25

2.50

1.00

2.00

1.75

1.50

0.50

1.00

0.25

0.50

0.00 100

200

300

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700

800

m/z

0.00

Peak 1. Cyanidin-3,5-diglucoside

150 200 250 300 350 450 450 500 550 m/z

Peak 2. Cyanidin-3-glucoside

x105 1.50

x105 8

1.25 6

1.00 0.75

4

0.50 2 0.25 0.00 100

200

300

400

500

600

700

Peak 3. Cyanidin-3-rutinoside

800 m/z

0 150 200 250 300 350 450 450 500 550

m/z

Peak 4. Peonidin-3-glucoside

Fig. 2. MS fragmentation patterns of anthocyanins in black rice. Panels (1–4) show the ESI-MS of cyanidin-3,5-diglucoside, cyanidin-3-glucoside, cyanidin-3-rutinoside and peonidin-3-glucoside, respectively.

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Table 2 Chromatographic and spectral characteristics of peaks in Fig. 1. Peak no.

Compound

RTa (min)

λvisb (nm)

λuvb (nm)

E440/Evisb (%)

E440/Evisb indicating

[M + H]+ (m/z)

Fragments[M + H]+ (m/z)

1 2 3 4

Cyanidin-3,5-diglucoside Cyanidin-3-glucoside Cyanidin-3-rutinoside Peonidin-3-glucoside

20.27 23.08 24.69 29.48

516.52 516.37 517.76 514.20

280.15 276.31 279.15 277.95

37.71 33.99 32.84 34.50

Diside Monoside Monoside Monoside

611.4 449.3 595.4 463.0

287.2 287.2 287.2 301.0

Peak numbering refers to the peaks of Fig. 1. a Retention time. b The absorption maxima λvis and λuv are due to the presence of the anthocyanidin chromophore, Evis is the absorptivities at the corresponding maxima while E440 is the absorptivity at 440 nm.

fragment ion of 287 (m/z 287). The neutral loss of 162 (m/z) from their respective molecular ions indicated the existence of a glucose moiety in the structures of both identified anthocyanins. The fragment loss of 162 revealed this anthocyanin to be a cyanidin 3-glucoside (Aoki et al., 2002). Peak 4 showed a molecular ion at m/z 463. Its MS gave a fragment at m/z 301 ([M-162]+ loss of a glucoside moiety). The fragmentation pattern was characteristic of glucoside and the compound was identified as peonidin-3-glucoside. Two anthocyanins (cyanidin-3-glucoside and peonidin-3-glucoside) had been identified in black rice (Yawadio et al., 2007). Peak 1 had the MS data ([M]+m/z 611, m/z 287) as cyanidin-3, 5-diglucoside published previously (Lopes-Lutz et al., 2010). Peak 3 had the MS data ([M]+m/z 595, m/z 287). The fragmentation pattern was characteristic of rutinosides and the compound was identified as cyanidin-3-rutinoside published previously (Duenas et al., 2008).

The most principal anthocyanin in rice is cyanidin-3-glucoside, followed, in minor proportion by peonidin-3-glucoside (Hu, Zawistowski, Ling, & Kitts, 2003). Small quantities of other derivatives of cyanidin such as cyanidin-3-gentiobioside (Fossen, Slimestad, Ovstedal, & Andersen, 2002), cyanidin-3-rhamnoglucoside and cyanidin-3,5-diglucoside have also been found. Our results showed that black rice had a similar constitute with the previous reports. 3.2. Kinetics of anthocyanins degradation in heat treatment and different acidic atmosphere Thermal degradation of anthocyanins from black rice was studied at 80, 90 and 100 °C in the range of pH 1.0–pH 6.0. The anthcoyanin contents of black rice during heating were plotted with a regular

maU

maU

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0

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nm

Peak 1. Cyanidin-3,5-diglucoside

Peak 2. Cyanidin-3-glucoside

maU

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Peak 3. Cyanidin-3-rutinoside

Peak 4. Peonidin-3-glucoside

Fig. 3. HPLC-UV–vis spectra of major peaks recorded in chromatogram shown in Fig. 1.

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interval of 30 min (Fig. 4a). It is clear from Fig. 4a that the thermal degradation of monomeric anthocyanin of black rice followed first order reaction kinetics with respect to temperature. Our results were in agreement with those from the previous studies, reporting a first-order reaction model for the degradation of monomeric anthocyanins from various sources (Yang, Han, Gu, Fan, & Chen, 2008). The first-order reaction rate constants (k) and half-lives (t1/2), i.e. the time needed for 50% degradation of anthocyanins, were calculated by the following equations: ln ðCt =C0 Þ = −k × t

ð1Þ

t1=2 = −ln0:5=k

ð2Þ

Dependence of the degradation rate constant on temperature is represented by the Arrhenius equation: ln k = ln k0 −Ea =RT

ð3Þ

intermolecular association with anthocyanins. However, not all compounds enhance copigmentation; for example, sugars and their degradation products tend to accelerate the degradation of anthocyanins. The rate of anthocyanin degradation is associated with the rate at which the sugar is degraded to furfural-type compounds derive from the Maillard reaction (Cevallos-Casals & Cisneros-Zevallos, 2004). As the temperature is raised, Maillar reaction increased, pigment–copigment complexes become less stable, competition between hydration and copigmentation turns against copigmentation and this may explain the lack of any major effect of the copigment on anthocyanin stability during heating (Gradinaru et al., 2003). pH level had a strong influence on the stability of anthocyanins. As seen in Table 3, black rice anthocyanins showed two distinct stability profiles: (1) at lower pHs (pH 1.0, 2.0, 3.0 and 4.0), and (2) at higher pHs (pH 5.0 and 6.0). Increasing the pH from 1.0 to 6.0 hastened the degradation of anthocyanins, especially, cyanidin-3,5-diglucoside. Thermal stabilities of cyanidin-3-rutinoside and peonidin-3-rutinoside in citrate buffers (Tanchev & Joncheva, 1973) and cyanidin-3,5-diglucoside in model systems (Daravingas & Cain, 1968) were reported to decrease as the pH increased. Significant decrease in anthocyanin stability was observed at pHs above 5.0 indicated by low t1/2 values (Table 3). pH value higher than 3.0 were shown to considerably reduce anthocyanin stability (Cisse et al., 2009). Tuker and Erdogdu (2006) reported that anthocyanins were known to be the most stable state at pH= 2.0, an increase in pH may result in reduction of pigment stability. In contrast, Cevallos-Casals and Cisneros-Zevallos (2004) reported that anthocyanins from the extracts of red sweet potato, purple corn and commercial purple carrot colorant were more resistant to 98 °C at pH 3.0 than at pH 1.0 (Kırca, Özkan, & Cemeroğlu, 2007). In fact, Torskangerpoll and Andersen (2005) reported that color stability of anthocyanins depended highly on pH and anthocyanin structure. In relation to the stability, anthocyanins may suffer reactions that altered their structures due to the electronic deficiency of their flavylium nuclei. Anthocyanins stability increases with the number of methoxyls in the B ring and decreases as hydroxyls increase. In general, anthocyanins are more stable at an acid pH (Escribano-Bailón et al., 2004). These findings suggest that anthocyanins are unstable to high temperature, particularly for high pH conditions. In other words, anthocyanins would be more resistant to heat when anthocyanins are used in acidic atmosphere. 3.3. Temperature dependence The dependence of the degradation of black rice anthocyanins on temperature was determined by calculating the activation energy (Ea) values from the following equations: K = k0 e

1.2

−Ea = RT

ð4Þ

8

1 6

0.8

-Link

Anthocyanin content (C/CO)

where C0 is the initial monomeric anthocyanin content and Ct is the monomeric anthocyanin content after t minute heating at a given temperature. t1/2 is the half life time, k is the first order kinetic rate constant (min− 1), k0 is the frequency factor (min− 1), Ea is the activation energy (kJ/mol), R is the universal gas constant (8.314 J/mol K) and T is the absolute temperature (Kelvin). The kinetic parameters of anthocyanins (cyanidin-3-glucoside, peonidin-3-glucoside, cyanidin-3,5-diglucoside, cyanidin-3-rutinoside) degradation during heating are shown in Table 3. To determine the effect of temperature on the parameters studied, the constants obtained from Eq. (3) were fitted to an Arrhenius-type equation in each of the kinetic models studied (Fig. 4b). At different pH levels, there was an increase of each anthocyanin in the rate constant (k), and a corresponding decline in the half-life values (t1/2) with increasing heating temperature. The decrease of anthocyanins over time fitted a first-order equation with a good regression coefficient (0.8960b R2 b 0.9989). The k values showed that the thermal stability of the four anthocyanins decreased with increasing temperature, especially from 90 to 100 °C. The t1/2 values of cyanidin-3-glucoside ranged from 19.25 to 0.62 h. At the same temperature, the t1/2 values for cyanidin-3-glucoside were lower than that for peonidin-3-glucoside and cyanidin-3,5diglucoside, indicating that cyanidin-3-glucoside was less stable than peonidin-3-glucoside and cyanidin-3,5-diglucoside. At 80 °C and 90 °C, the t1/2 values for cyanidin-3-rutinoside were longer than other anthocyanins, however, at 100 °C, the t1/2 values decreased drastically. Stability of anthocyanins is related to structures and compigmentation. Four anthocyanin structures exist in equilibrium: flavylium cation, quinonoidal base, carbinol pseudobase and chalcone. Stability of anthocyanins can increase with inter molecular copigmentation. Aqueous fruit, vegetable, and grain extracts, with high anthocyanin content, contain mixtures of different compounds that may serve as copigments for

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0.6

4

0.4 2

0.2 0

0

50

100

150

200

0 0.0026

0.00265

0.0027

0.00275

times(min)

1/T (1/K)

(a)

(b)

◆

0.0028

0.00285

Fig. 4. Degradation o f cyanidin-3-glucoside in black rice during heating at 80, 90 and 100 °C. ( ,80; ■, 90; ▲, 100 °C) and its arrhenius pots for degradation during heating at 90 °C.

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Table 3 Effect of temperature and pH on the k, t1/2 values of anthocyanins degradation in black rice. pH

1.0

2.0

3.0

4.0

5.0

6.0

a b c d e f g

Cy3Ga

Temperature (°C)

80 90 100 80 90 100 80 90 100 80 90 100 80 90 100 80 90 100

Pn3Gb t1/2 (h)f

k × 103 (min− 1)

t1/2 (h)

k × 103 (min− 1)

t1/2 (h)

k × 103 (min− 1)

t1/2 (h)

0.6 (0.9414)g 1.3 (0.9913) 10.2 (0.9989) 0.9 (0.9163) 1.7 (0.9667) 5.6 (0.9487) 1.8 (0.9683) 2.6 (0.9633) 6.2 (0.9475) 2.2 (0.9630) 2.9 (0.9531) 7.7 (0.9886) 4.6 (0.9823) 6.1 (0.9777) 8.1 (0.9567) 9.9 (0.9661) 12.7 (0.9694) 18.6 (0.9355)

19.25 8.88 1.13 12.83 6.79 2.06 6.42 4.44 1.86 5.25 3.98 1.50 2.51 1.89 1.43 1.17 0.91 0.62

0.5 (0.8933) 1.2 (0.9439) 9.2 (0.9986) 1.0 (0.9690) 1.6 (0.9819) 5.6 (0.9486) 1.7 (0.9652) 2.5 (0.9784) 4.9 (0.9915) 2.1 (0.9686) 2.9 (0.9487) 6.0(0.9802) 4.1 (0.9811) 5.7 (0.9409) 7.8 (0.9509) 11.7 (0.9716) 15.3 (0.9925) 21.6 (0.8936)

23.10 9.63 1.26 11.55 7.22 2.06 6.79 4.62 2.36 5.50 3.98 1.93 2.82 2.03 1.48 0.98 0.75 0.53

0.5 (0.9124) 1.0 (0.9549) 6.7 (0.9414) 0.8 (0.9075) 1.2 (0.7681) 3.4 (0.9321) 1.7 (0.896) 2.6 (0.9113) 5.7 (0.9751) 2.0 (0.9797) 3.5(0.912) 4.7 (0.9821) 3.1 (0.9642) 4.6 (0.9287) 7.8 (0.8607)

23.10 11.55 1.72 14.44 9.63 3.39 6.79 4.44 2.03 5.78 3.04 2.46 3.73 2.51 1.48

0.4 (0.9299) 0.7 (0.9014) 15.7 (0.986) 1.1 (0.9180) 1.4 (0.9378) 5.0 (0.8567) 1.7 (0.9693) 2.8 (0.8697) 6.3 (0.9645) 1.8 (0.9200) 2.8 (0.9737) 4.0 (0.9558) 4.4 (0.9305) 6.2 (0.902) 8.2 (0.9511) 11.1 (0.9418) 14.0 (0.8972) 17.2 (0.9019)

28.88 16.50 0.74 10.50 8.25 2.31 6.79 4.13 1.83 6.42 4.13 2.89 2.63 1.86 1.41 1.04 0.83 0.67

Cyanidin-3-glucoside. Peonidin-3-glucoside. Cyanidin-3,5-diglucoside. Cyanidin-3-rutinoside. k = reaction rate constant. t1/2 = half-life of anthocyanins degradation. Numbers in parentheses are the determination coefficients.

Table 4 Effect of temperature and pH on the Ea values of anthocyanins degradation in black rice. pH

1.0 2.0 3.0 4.0 5.0 6.0

c d e

cyanidin-3-glucoside ranged from 99.75 to 67.38 kJ mol − 1 at pH 2.0– 4.0 and from 30.97 to 34.41 kJ mol − 1 at pH 5.0–6.0, respectively. It can be observed from the activation energies that anthocyanins are comparatively stable at lower temperatures and are more sensitive to higher temperature as indicated by higher activation energy. 4. Conclusions Four anthocyanins were detected in black rice using HPLC-DAD-MS. The four anthocyanins were identified as cyanidin-3-glucoside (91.01%), peonidin-3-glucoside (7.13%), cyanidin-3,5-diglucoside (0.92%), cyanidin-3-rutinoside (0.94%). The main anthocyanins were cyandin-3-glucoside and peonidin-3-glucoside. The present study provided detailed information regarding the changes in kinetic stability of the four anthocyanins in black rice at temperature (80 °C, 90 °C and 100 °C) at different pH levels (1.0–6.0). The present data showed that degradation of anthocyanins followed first-order reaction kinetics. The stability of anthocyanins depended on temperature and pH. The four anthocyanins degraded more quickly with increasing heating temperature and pH values, especially, as heating temperature increasing to 100 °C and pH value to 5.0. Thus higher stability of anthocyanins was achieved by using lower temperature, lower pH values and short-time. Further studies on the stability of black rice anthocyanins are needed considering that the great potential of black rice anthocyanins to develop functional foods or serve as functional colorant.

Ea (kJ/mol) Cy3Ga

b

Cy3Rd

k × 10 (min− 1)e

To determine the effect of temperature on the parameters studied, the constants obtained from Eq. (1) were fitted to an Arrhenius-type equation. Increasing pH resulted in lower Ea values of individual anthocyanins during heating at 80–100 °C (Table 4). The Ea values were obtained for black rice anthocyanins in citrate–phosphate buffers. The calculated Ea values of cyanidin-3-glucoside, peonidin-3-glucoside, cyanidin-3,5-diglucoside and cyanidin-3-rutinoside ranged from 154.43 to 30.97 kJ mol − 1, 158.76 to 33.51 kJ mol − 1, 141.39 to 46.92 kJ mol− 1 and 199.54 to 23.99 kJ mol− 1 during heating, respectively. Since high activation energy reactions are more sensitive to temperature changes, that is a substrate in the reaction with a higher activation energy tends to be degraded by a smaller temperature change, is to be less stable. So anthocyanins in black rice are more susceptible to temperature elevation during heating. There were differences among the Ea values for the thermal degradation of individual anthocyanins of black rice at different pHs (1.0–6.0). The Ea values decreased sharply when pH changed from 1.0 to 2.0 and with a continuous decrease as the increasing pH values. The Ea values at pH 1.0 were higher than those at other pHs. The Ea of cyanidin-3-rutinoside was highest, which indicated that it was the most stable at pH= 1.0. At higher pHs (5.0 and 6.0), the calculated Ea values of

a

Cy3,5Gc

154.43 (0.9278) 99.75 (0.9644) 67.38 (0.9406) 68.16 (0.8961) 30.97 (0.9997) 34.41 (0.9815)

e

Pn3Gb

Cy3,5Gc

Cy3Rd

158.76 (0.9426) 99.69 (0.9494) 57.75 (0.9711) 57.25 (0.9460) 35.19 (0.9721) 33.51 (0.9925)

141.39 (0.9243) 78.86 (0.9319) 66.03 (0.9658) 46.92 (0.9742) 50.39 (0.9901)

199.54 (0.8502) 82.33 (0.8549) 71.53 (0.9769) 43.71 (0.9980) 34.07 (0.9981) 23.99 (0.9970)

Cyanidin-3-glucoside. Peonidin-3-glucoside. Cyanidin-3,5-diglucoside. Cyanidin-3-rutinoside. Numbers in parentheses are the determination coefficients.

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