Food and Chemical Toxicology 83 (2015) 125e132
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Preparation and toxicological evaluation of methyl pyranoanthocyanin Zhenzhou Zhu, Nao Wu, Minjie Kuang, Olusola Lamikanra, Gang Liu, Shuyi Li, Jingren He* College of Food Science and Engineering, Wuhan Polytechnic University, 430023 Wuhan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 March 2015 Received in revised form 24 April 2015 Accepted 5 May 2015 Available online 29 May 2015
Anthocyanins are increasingly valued in the food industry for their functional properties and as food colorants. The broadness of their applications has, however, been limited by the lack of stability of these natural pigment extracts in a number of food systems. The potential application of pyranoanthocyanins, anthocyanin derivatives with better stability conferred by the added pyran ring, as a food ingredient was determined. Methylpyranoanthocyanin (MPA) was prepared from reaction of acetone and anthocyanin extracts from red grapes. Reaction products were sequentially purified with polyamide resin, TSK gel resin and semi-preparative HPLC to a purity level >98%. Cytoprotective influence tests of the purified MPA indicated its significant protective effect against H2O2 induced MRC-5 cell damage. Results of evaluations of possible acute toxicity effects on MPA-fed mice, including macro and microscopic assessments, support the conclusion of a non-toxic effect of MPA, and its potential safe use as a food additive. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cytoprotective effect Methyl pyranoanthocyanin Acute toxicity HPLC-MS
1. Introduction Anthocyanins (Fig. 1(a)) are polyphenolic substances, and they are one of the most widespread pigments found inplants (Hwang rez et al., 2008). The presence of anthocyaet al., 2011; Rivero-Pe nins confers the red, blue and purple colors of a variety of fruits and flowers, such as cherries, strawberries, plums, blackberries, raspberries, grapes, red currants and black currants (Sui et al., 2014). Although anthocyanins were found with over 600 known molecular structures in the natural world, they mainly occur as glycosides of flavylium (2-phenylbenzopyrylium) salts, based on six anthocyanidins: pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin (Fernandes et al., 2014; Jampani et al., 2014). Interest in anthocyanins has considerably increased in recent years (Barba et al., 2015a, 2015b), mainly driven by their perceived functional properties. In the food industry, anthocyanins are widely used as natural color alternatives to synthetic colorants. The watersolubility of anthocyanin pigments facilitates their incorporation with numerous aqueous food systems, making them attractive natural colorants (Assous et al., 2014). Moreover, anthocyanins are regarded safer and healthier than synthetic colorants by consumers. An important attribute of anthocyanins is based on their
* Corresponding author. E-mail addresses: [email protected] (Z. Zhu), [email protected] (J. He). http://dx.doi.org/10.1016/j.fct.2015.05.004 0278-6915/© 2015 Elsevier Ltd. All rights reserved.
antioxidant properties that have been correlated to potential benefits in the reduction of risk of cardiovascular and oxidative related stress and diseases (Jiang et al., 2013; Saluk et al., 2012). The antioxidant property of anthocyanins is based on their ability to donate hydrogen to highly reactive radicals, thus retarding and/or preventing formation of undesirable oxidative products (Fukumoto and Mazza, 2000; Jiang et al., 2013). Applications of anthocyanins in food preparations and processing have, however, been limited by their instability, especially after isolation from plant cells. Isolated anthocyanins are highly unstable and very susceptible to degradation (Giusti and Wrolstad, 2003), due to their sensitivity to a number of factors such as pH, storage temperature, chemical structure, concentration, light, oxygen, solvents, the presence of enzymes, flavonoids, proteins and ~ eda-Ovando et al., 2009). These often alter metallic ionsas (Castan anthocyanin properties and are particularly detrimental to their ability to retain their color and nutritional properties. The development of stabilizing technologies and/or ability to isolate more stable anthocyanins and their derivatives will be beneficial to the food industry. Pyranoanthocyanins are one of the most important classes of anthocyanin-derived pigments occurring naturally in red wine (Blanco-Vega et al., 2011; Hayasaka and Asenstorfer, 2002; Mateus et al., 2001). Their stability is derived mainly by an additional cyclic ring (Fig. 1b), apyran ring located between the C4 position in the C ring and the hydroxyl group on the C5 position in the A ring of the anthocyanin molecule (He et al., 2012). Methyl pyranoanthocyanin
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2. Materials and methods 2.1. Preparation of MPA
Fig. 1. Chemical structure of anthocyanins (a) and pyranoanthocyanins (b).
(MPA) has a methyl group (-CH3) as R3 (Fig. 1b). They are typically yellow and have maximum UVeVis absorption at 478 nm (He et al., 2006). The formation of MPA has been reported to be derived from reactions of anthocyanins with acetone or acetoacetic acid (de Freitas and Mateus, 2011; Hayasaka and Asenstorfer, 2002; He n et al., 2004). Optimiet al., 2006; Lu and Foo, 2001; Pozo-Bayo zation of MPAs synthetic conditions from reactions between grape anthocyanin extracts and acetone was reported by Kuang et al. (2014). Their report included optimum ratios of anthocyanin extract to acetone and their concentrations, reaction temperature and pH. The chemical structure and mechanism of formation of synthesized MPAs were identified by HPLC-MS/MS. They proposed a reaction pathway in which malvidin 3-glucoside (Mv-3-G) and acetone under weak acidic conditions are combined by way of enolization of C atoms at a and b positions of acetone, followed by cycloaddition reaction of C atoms at the a and b positions. Subsequent dehydration, oxidation, and decarboxylation then lead to formation of the anthocyanin oxonium ion of a pyran ring (Fig. 2). Previous studies related to MPA have been mainly limited to their synthetic methods and structure. To our knowledge, their cytoprotective properties and potential toxicity have not been reported. These are important considerations for their potential applications in food. Thus, the objective of this study is to evaluate the cytoprotective effectiveness of MPA against H2O2 induced cell damage and its potential acute toxicity.
The red grape skin extracts containing anthocyanins were purchased from Yunnan Haitong Nature Product Co., Ltd. China. The pigments extract was consisting mainly of cyanidin-3-glucoside (Cy-3-G), peonidin-3-glucoside (Pn-3-G), delphinidin-3-glucoside (Dp-3-G), malvidin-3-glucoside (Mv-3-G), and petunidin-3glucoside (Pt-3-G), which were analyzed by HPLC-DAD-MS (Fig. 3a). It is obvious that Mv-3-G was the most important component of the extracts. Methyl pyranoanthocyanin (MPA) was synthesized by reaction of the above crude anthocyanins pigments extracts and acetone as described by Kuang et al. (2014). The ratio of red grape skin extract to acetone was 15:1 (mg:mL), resulting in grape skin pigments concentration of 2.5 mg/mL. The reaction temperature was set at 45 C and pH was 3.0. The reaction lasted 9 days, with oxygen gas being passed through the reaction mixture every 3 days. Separation and purification of MPA was carried out in three subsequent steps. Initial separation was done using polyamide resin. The resin was preconditioned by initial soaking in ethanol (95%) for 24 h after which it was rinsed with distilled water to remove ethanol and then soaked in 5% NaOH solution for an additional 1 h. The resin was rinsed with water until neutral, soaked in 5% HCl for 4 h, further rinsed until neutral, and then immersed in 50% ethanol until used. Excess acetone from the malvidin 3-glucoside and acetone reaction mixture was removed with a rotary evaporator after which the mixture (0.5 B V) was loaded onto a glass column (30 mm 600 mm I.D.) (wet-packed with polyamide resin) at a velocity of 1 B V/h. After total adsorption, the column was washed with water to remove non-reacted anthocyanins, then sample was eluted with eluted with 2e2.5 B V of 10% (v/v) aqueous ethanol solution containing HCl (10%) at a velocity of 1 B V/h, yielding the polyamide resin purified MPA. The collected sample was loaded onto a TSK Toyopearl gel HW-40(s) column (40 mm 600 mm I. D)
Fig. 2. The proposed formation mechanism of MPA.
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Fig. 3. HPLC chromatogram of red grape skin extracts (a) and MS fragmentation scheme of the anthocyanin-acetone adducts. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)
and eluted with 10e20% aqueous methanol solution containing HCl (10%), The yellow colored fractions based on the absorbance values at 478 nm were collected. TSK purified MPA was obtained by removing methanol from the collected sample using rotary evaporation. Further purification was carried out using semi-preparative HPLC (SSI Series 1500) equipped with a 250 10 mm i. d. 5 mm C18-reverse phase column (Allsphere ODS-2) and a UV detector. Eluent solvents were A: H2O/HCOOH (9:1), and B: HCOOH/H2O/ CH3CN (8:1). Samples (5 mL) were injected and eluted at a rate of 4 mL/min. The solvent mixture at the time of injection contained 20% of solvent B. The gradient program was set to increase B to 30% after 30 min and then to 100% after 33 min. Eluent solvent was held at 100% B until 43 min, and programed to decrease back to 20% at
45 min. Fractions eluted were detected at 478 nm and the eluent recovered between 16 and 21 min were collected Then MPA was purified by removing formic acid and freeze-dried to powders for further use.
2.2. Chemical structure identification The chemical structure of red grape skin extracts and reaction products was determined using a LTQ-XL mass spectrometer (Thermo, USA) with an ESI (ElectroSpray Ionisation) interface. The capillary voltage and temperature were 47 V and 350 C, respectively. Nitrogen gas was applied at 30 L/min. Spectra were recorded in positive ion mode between m/z 100 and 1500.
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2.3. Determination of MPA purity Progress of the successive purification steps were assessed using a SSI Series 1500 HPLC equipped with a 250 4.6 mm i. d. 5 mm C18-reverse phase column (LiChrospher) and a DAD detector. The solvents were A: H2O/HCOOH (9:1, v/v), and B: HCOOH/H2O/CH3CN (8:1:1, v/v/v). The injection volume was 20 mL at a velocity of 1 mL/ min. The solvent mixture at the time of injection contained 15% of solvent B. The gradient program was set to increase B to 24% after 30 min and then to 100% after 35 min. Eluent solvent was held at 100% B until 37 min, and programed to decrease back to 20% at 40 min. MPA retention time of 23 min was confirmed by comparing eluent retention time with that of a standard with 99%.purity provided by the University of Porto. Purity of eluted MPA was determined from the relationship:
Purity ¼ peak area of sample=peak area of standard
(1)
2.4. Cytoprotective effect investigations 2.4.1. Cell culture Human embryonic lung fibroblast (MRC-5) cells obtained from the China Center for Culture Type Collection (Wuhan University), were cultured in Modification of Eagle Medium-Earls Balanced Salt Solution (MEM-EBSS) (Hyclone) containing 10% (v/v) heatinactivated fetal bovine serum (FBS) (Gibico), 1% penicillinestreptomycin (Hangzhou Ginuo Biomedical Technology Co., Ltd. China), 5% non-essential amino acid and 5% L-Glutamine in an incubator (95% air and 5% CO2) at 37 C. A flask with a bottom area of 25 m2 was used for MRC-5 cell culture. The medium was replaced every 2 days and the cells were passaged at approximately 60e80% confluence using a trypsin solution (0.25%)with 0.02% EDTA (Hangzhou Ginuo Biomedical Technology Co., Ltd. China). Passages 2730 were used in this study. 2.4.2. Cytotoxicity assay The cytotoxicity of MPA to MRC-5 cells was evaluated by 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe nyl)-2H-tetrazolium, inner salt (MTS) assays (Beijing Promega Biotechnology Co., Ltd. China). Briefly, MRC-5 cells were grown in 96-well plates at a density of 104 cells/well. The cells were grown in an atmosphere with 5% CO2 and 95% relative humidity. After an incubation of 24 h, cells were washed twice with Phosphate Buffer Saline (PBS) solution and the culture medium was replaced by 100 mL fresh MEM medium which contains 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonicaci (HEPES) buffer. After 20 min's incubation, cells were re-washed and treated by100 mL MEM medium with different MPA concentrations (0, 20, 40, 60, 80, 200, 400, 600, 800 mg/mL), following an incubation of 24 h. After the incubation, the medium was removed and cells were washed with PBS trice, 120 mL solution consisting of 20 mL MTS and 100 mL MEM medium was added to each well. After an additional incubation of 4 h, the 96-well plate was placed in the microplate reader (iMark, BIO-RAD, USA) with gentle shaking for 1 min, and absorbance was determined at 490 nm, for the viability measurement of MRC-5 cells (Eq. (2)). Cells incubated without the test compounds were used as control. In each MTT assay, every sample was tested in at least three replicates.
cell viability ¼ As490 Ac490 100%
(2)
where As490 means absorbance of the sample at 490 nm and Ac490 means absorbance of the control at 490 nm.
The cytotoxicity of H2O2 to MRC-5 cells was measured using the same method as above, except that cells were treated by 100 mL MEM medium with different H2O2 (Sinopham Chemical Reagent Co, Ltd) concentrations (0, 100, 200, 300, 400, 500, 600, 700, 800, 900 mmol/L). 2.4.3. Protective effect of MPA on H2O2 induced MRC-5 cell damage MRC-5 cells were grown in 96-well plates at a density of 104 cells/well. The cells were grown in an atmosphere with 5% CO2 and 95% relative humidity. After an incubation of 24 h, cells were washed twice with Phosphate Buffer Saline (PBS) solution and the culture medium was replaced by 100 mL fresh MEM medium which contains 10 mmol/L 4-(2-hydroxyethyl)-1piperazineethanesulfonicaci (HEPES) buffer. After 20 min incubation, cells were re-washed and treated by 100 mL MEM medium with different MPA concentrations (0, 40, 60, 80 mg/mL), following an incubation of 24 h. The medium was then removed and cells were washed with PBS trice, after which they were incubated for 4 h in the MEM medium with an H2O2 concentration of 500 mmol/L. Thereafter, the culture medium was removed, cells were washed with PBS trice, 120 mL solution consisting of 20 mL MTS and 100 mL MEM medium was added to each well. After an additional incubation of 4 h, the 96-well plate was placed in the microplate reader with gentle shaking for 1 min, and absorbance was determined at 490 nm using iMark microplate reader (BIO-RAD, USA) for the viability measurement of MRC-5 cells. Cells incubated without the test compounds were used as control. In each MTT assay, every sample was tested at least three times. For the purpose of comparison, the same process was carried out to evaluate the protective effect of Mv-3-G on H2O2 induced MRC-5 cell damage. 2.5. Acute toxicity evaluation The acute oral toxicity of MPA was evaluated in mice (provided by Experimental Animals Research Centre, Hubei Province Centre for Disease Control and Prevention, China) using median lethal dose (LD50) method according to the procedures outlined by Procedure and methods of food safety toxicological assessment, GB15193.3-2003 (in Chinese) (China's Ministry of Health, 2003). After 3e5-day periods of quarantine and acclimatization, 6 healthy mice of each sex were used for pre-study. MPA was solved in distilled water (10 g/40 mL) and the sample was administered by oral gavage at a dose of 5000 mg/kg body weight (BW), and was observed during 24 h. The LD50 was estimated 5000 mg/kg since no death of the animal occurred during pre-study. Acute toxicity evaluation was carried out according to Horns method. A total of 50 mice, body weights ranging from 20 to 25 g, were used in this study. The animals were randomly divided into four dose groups and one control group (five males, five females per group). The dose groups were fed with MPA by oral gavage at doses of 1000, 2150, 4540 and 10,000 mg/kg BW (based on the LD50estimation from pre-study) respectively, before normal feeding. Observations were carried out once every 30 min in 2 h and every 4 h in 24 h after gavage, additional observation was done every morning and evening. The body weight of mice was measured once a week during two weeks. Animals were observed for signs of toxicity and mortality throughout the experimental period (14 days). Animals that died during the test were dissected for organ analysis. At the end of the test, the surviving animals were anesthetized and sacrificed. Organs such as liver, pancreas, kidneys were collected and weighed. Histopathological examinations were performed on liver. Liver slices were fixed with 10% formalin in phosphate buffered saline for 24 h and embedded in paraffin. Sections of 4 mm thickness were made using a microtome and
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stained with hematoxylin-eosin and observed under microscope to observe histopathological changes in the liver. 2.6. Statistical analysis All the tests were performed at least trice, data were analyzed with commercial statistical software and standard deviation (SD) was calculated. One-way ANOVA with Dun-can's test was used for inter-group comparison. P values less than 0.05 were considered statistically significant. 3. Results and discussion 3.1. Chemical structure identification and purity analysis of reaction products Mass spectrometry fragmentation patterns of the purified pyroanthocyanin is shown in Fig. 3(b). The presence of a [M]þ ion at m/z 531, and a major fragment ion [M162]þ at m/z 369 as observed for the purified compound is consistent with previously reported fragmentation for methyl pyroanthocyaninin (He et al., 2006; Kuang et al., 2014). The efficiency of each of the stepwise purification process was assessed from the relative GC-MS fraction peak areas of the extracts. The peak area of standard (with a recovery of 100%) was 7,353,143 mAU*min. Calculated recovery of polyamide resin purified sample, TSK gel resin purified sample and Semi-preparative HPLC purified sample were 76.16%, 91.60% and 98.31%, respectively, with peak area of 5,599,786, 6,735,479 and 7,228,875 mAU*min, respectively. Purity of recovered MPA after the semipreparative HPLC process indicated a recovery level of about 98%. 3.2. Cytoprotective effect of MPA The effects of MPA and H2O2 concentration on MRC-5 cell viability are presented in Fig. 4. For the purpose of comparison, the effect of malvidin-3-glucoside (Mv-3-G), the original anthocyanin for MPA synthesis, on MRC-5 cell viability is also presented in Fig. 4.
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MRC-5 cell viability remained at about 100% when MPA concentration was lower than 200 mg/mL, indicating its lack or minimal toxicity effect to MRC-5 at concentrations within this range. Increase in MPA concentrations above 200 mg/mL, however, resulted in reduced MRC-5 cell viability. For example, the MRC-5 cell viability decreased to 80% when MPA concentration was 760 mg/mL. Concentrations in the range of 40e80 mg/mL were thus selected to investigate the protective effect of MPA on H2O2 induced MRC-5 cell damage. The effect of Mv-3-G on MRC-5 cell viability shows the same tendency as that of MPA. H2O2 induced MRC-5 cell damage was more severe at higher concentrations. Significant loss in MRC-5 viability was observed at H2O2 concentrations higher than 200 mmol/L. MRC-5cell viability was 58% at a H2O2 concentration of 500 mmol/L, and was less than 50% at 600 mmol/L of H2O2. This may be explained by the rupture of MRC-5 cells at higher H2O2 concentration (600 mmol/L). Thus, a H2O2 concentration of 500 mmol/L, under which concentration cell growth was limited but without significant cell rupture, was selected forMRC-5 cell damage inducing to evaluate the protective function of MPA. Fig. 5 represents the protective effect of MPA and Mv-3-G on MRC-5 cell damage induced by H2O2 at 500 mmol/L. It is obvious that MPA does not have better protective effect than Mv-3-G. Cell viability was 75% after 24 h in the presence of MPA (40 mg/mL), while the value was 56% in the control without MPA. These results showed a significant protective effect (P < 0.05) of MPA on MRC-5 cell. However, this protective effect was dose dependent. For example, the protective effect was not significant (P > 0.05) when MPA concentration was increased to 80 mg/mL. The lack of effect at higher concentrations could be related to a competitive reaction between H2O2 and MPA, possibly similar to oxidative effects of H2O2 on anthocyanins (He et al., 2010), which will reduce the amount of effective MPA concentration on MRC-5 protective action. 3.3. Acute toxicity evaluation The number of mice that died after different gavage dosing levels with MPA was investigated for male and female separately.
Fig. 4. Influence of concentrations of Mv-3-G, MPA and H2O2 on MC-5 cell viability.
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Fig. 5. Protective effect of MPA and Mv-3-G on H2O2 induced MRC-5 cell. Columns followed by different letters within the same row represent significant differences at P < 0.05.
Fig. 6. The cumulative number of male and female mice deaths at different time after oral administration with MPA.
As observed, a dose of 4640 mg/kg BW did not cause the death of any animal. The mice, male and female, exhibited no behavioral changes during the treatment period (14 days). There were also no changes in water and food consumption, in relation to the control group. However, at a dose of 10,000 mg/kg BW, the mice became less active after oral dosing and death of some mice occurred after 8 h and 1.5 h for male and female, respectively (Fig. 6). After 24 h, the cumulative death number attained 3 for male and 5 for female. This suggests that the male mice might be more resistant to MPA than the female. LD50 value and 95% confidence limit was calculated according to procedure and methods of food safety
toxicological assessment, GB15193.3-2003 (in Chinese) (China's Ministry of Health, 2003). A LD50 value of 7945 mg/kg BW with a 95% confidence limit of 5716e11,043.3 mg/kg BW was presented in Table 1 for male. While for female, the LD50 value was 6816 mg/kg BW without 95% confidence limit. The high LD50 value for both male and female showed that MPA was practically non-toxic according to chemical-test method of acute oral toxicity for chemicals-acute toxic class method, GB/T 21757-2008 (in Chinese) (General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China). Changes in body and organ weights are a clear indications of damage caused by the substance test (Traesel et al., 2014). The increase of body weights of mice treated at different doses after two weeks were noted and presented in Fig. 7(a). There was no statistical difference in the weight increase between MPA administrated and control groups. Organ (liver, kidney, and pancreas) weights for both male and female mice were also measured and presented in Fig. 7(b) and (c). Results showed that organ weights were not statistically affected by MPA treatment over a period of 14 days. There, however, appears to be a decrease in liver weight of male mice at 1000 mg/kg BW which was not observed for all the other doses and did not occur in female mice at the same dosage. The reason for this observation is unknown, but need further investigation. Results of histopathological analysis suggest the deaths of some mice in this study is unrelated to toxic effects of MPA but could be attributed to the ability of the mice to adequately readjust to the metabolic changes related to the sudden change in diet. The assessment of pathological changes in the organs of treated animals, both macro and microscopically, is the basis of a safety assessment (Traesel et al., 2014). In this study, the histopathological analyses of survived mice's liver after 14 days' test showed no findings suggestive of toxic effects (Fig. 8). These results proved to be consistent with above analyses, confirming the safety of using
Table 1 LD50 and 95% confidence limits of male and female mice administered orally with MPA. Group
Dosage (mg/kg BW)
Logarithmic dose
Mortality number
Mortality rate (%)
LD50 and 95% confidence limit (CL
Male Female
10,000 10,000
4.0000 4.0000
3 5
60 100
LD50 ¼ 7945, CL95%: 5716e11043.3 LD50 ¼ 6816, No CL95%
95%)
(mg/kg)
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Fig. 7. Body weight of mice (a) and principal internal organ of male (b) and female (c) mice treated by gavage with methyl-pyranoanthocyanin. Columns followed by different letters within the same row represent significant differences at P < 0.05.
Fig. 8. Histopathological analysis of liver of surviving mice treated with MPA after 14 days.
the MPA with the dose range and administration period of this study. 4. Conclusions The results from this study are consistent with a non-toxic effect of MPA, The pyranoanthocyanin, when fed to mice showed no
evidence that would indicate toxicity based on their body and organ weights, and histopathological analysis of their livers. MPA, however, had significant protective effect on H2O2 damaged MRC-5 cells, increasing cell viability from 56% (control) to 75%. The nontoxic effect of MPA and its demonstrated cytoprotective effect on H2O2 induced MRC-5 cell damage suggest its potential use as a functional ingredient that could be safely incorporated in food and
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beverages. The stability of MPA relative to many anthocyanin compounds would also provide an added advantage over many other anthocyanin food additives. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment The authors appreciate the support of International Cooperation Project of Ministry of Science and Technology of China (2014DFG32310), National Natural Science Foundation of China (31371727) and Natural Science Foundation of Hubei Province of China (2014CFB891). References Assous, M.T.M., Abdel-Hady, M.M., Medany, G.M., 2014. Evaluation of red pigment extracted from purple carrots and its utilization as antioxidant and natural food colorants. Ann. Agric. Sci. 59, 1e7. Barba, F.J., Brianceau, S., Turk, M., Boussetta, N., Vorobiev, E., 2015a. Effect of alternative physical treatments (ultrasounds, pulsed electric fields, and highvoltage electrical discharges) on selective recovery of bio-compounds from fermented grape pomace. Food Bioprocess Technol. 8, 1139e1148. Barba, F.J., Galanakis, C.M., Esteve, M.J., Frigola, A., Vorobiev, E., 2015b. Potential use of pulsed electric technologies and ultrasounds to improve the recovery of high-added value compounds from blackberries. J. Food Eng. http://dx.doi.org/ 10.1016/j.jfoodeng.2015.02.001. Blanco-Vega, D., Lopez-Bellido, F.J., Alia-Robledo, J.M., Hermosin-Gutierrez, I., 2011. HPLC-DAD-ESI-MS/MS characterization of pyranoanthocyanins pigments formed in model wine. J. Agric. Food. Chem. 59, 9523e9531. ~ eda-Ovando, A., Pacheco-Herna ndez, M.d.L., P ndez, M.E., Castan aez-Herna Rodríguez, J.A., Gal an-Vidal, C.A., 2009. Chemical studies of anthocyanins: a review. Food Chem. 113, 859e871. China's Ministry of Health, 2003. Procedure and Methods of Food Safety Toxicological Assessment. GB15193-2003 (in Chinese). de Freitas, V., Mateus, N., 2011. Formation of pyranoanthocyanins in red wines: a new and diverse class of anthocyanin derivatives. Anal. Bioanal. Chem. 401, 1463e1473. Fernandes, I., Faria, A., Calhau, C., de Freitas, V., Mateus, N., 2014. Bioavailability of anthocyanins and derivatives. J. Funct. Foods 7, 54e66. Fukumoto, L.R., Mazza, G., 2000. Assessing antioxidant and prooxidant activities of phenolic compounds. J. Agric. Food Chem. 48, 3597e3604.
Giusti, M.M., Wrolstad, R.E., 2003. Acylated anthocyanins from edible sources and their applications in food systems. Biochem. Eng. J. 14, 217e225. Hayasaka, Y., Asenstorfer, R.E., 2002. Screening for potential pigments derived from anthocyanins in red wine using nanoelectrospray tandem mass spectrometry. J. Agric. Food. Chem. 50, 756e761. He, F., Mu, L., Yan, G.-L., Liang, N.-N., Pan, Q.-H., Wang, J., Reeves, M.J., Duan, C.-Q., 2010. Biosynthesis of anthocyanins and their regulation in colored grapes. Molecules 15, 9057e9091. He, F., Liang, N.N., Mu, L., Pan, Q.H., Wang, J., Reeves, M.J., Duan, C.Q., 2012. Anthocyanins and their variation in red wines. II. Anthocyanin derived pigments and their color evolution. Molecules 17, 1483e1519. He, J., Santos-Buelga, C., Silva, A.M., Mateus, N., De Freitas, V., 2006. Isolation and structural characterization of new anthocyanin-derived yellow pigments in aged red wines. J. Agric. Food. Chem. 54, 9598e9603. Hwang, Y.P., Choi, J.H., Yun, H.J., Han, E.H., Kim, H.G., Kim, J.Y., Park, B.H., Khanal, T., Choi, J.M., Chung, Y.C., Jeong, H.G., 2011. Anthocyanins from purple sweet potato attenuate dimethylnitrosamine-induced liver injury in rats by inducing Nrf2mediated antioxidant enzymes and reducing COX-2 and iNOS expression. Food Chem. Toxicol. 49, 93e99. Jampani, C., Naik, A., Raghavarao, K.S.M.S., 2014. Purification of anthocyanins from jamun (Syzygium cumini L.) employing adsorption. Sep. Purif. Technol. 125, 170e178. Jiang, D.Q., Guo, Y., Xu, D.H., Huang, Y.S., Yuan, K., Lv, Z.Q., 2013. Antioxidant and anti-fatigue effects of anthocyanins of mulberry juice purification (MJP) and mulberry marc purification (MMP) from different varieties mulberry fruit in China. Food Chem. Toxicol. 59, 1e7. Kuang, M., Wu, N., Qi, M., He, J., Li, S., Deng, L., Chen, X., 2014. Optimized method for preparation of methyl pyranoanthocyanins and analyzed by HPLC-MS/MS. Food Ind. Technol. 20, 061. Lu, Y., Foo, L.Y., 2001. Unusual anthocyanin reaction with acetone leading to pyranoanthocyanin formation. Tetrahedron Lett. 42, 1371e1373. Mateus, N., Silva, A.M., Vercauteren, J., de Freitas, V., 2001. Occurrence of anthocyanin-derived pigments in red wines. J. Agric. Food. Chem. 49, 4836e4840. Monagas, M., Polo, M.C., Go n, M.A., mez-Cordove s, C., 2004. Occurrence Pozo-Bayo of pyranoanthocyanins in sparkling wines manufactured with red grape varieties. J. Agric. Food. Chem. 52, 1300e1306. rez, M.D., Mun ~ iz, P., Gonz , M.L., 2008. Contribution of Rivero-Pe alez-Sanjose anthocyanin fraction to the antioxidant properties of wine. Food Chem. Toxicol. 46, 2815e2822. Saluk, J., Bijak, M., Kołodziejczyk-Czepas, J., Posmyk, M.M., Janas, K.M., Wachowicz, B., 2012. Anthocyanins from red cabbage extract d evidence of protective effects on blood platelets. Cent. Eur. J. Biol. 7, 655e663. Sui, X., Dong, X., Zhou, W., 2014. Combined effect of pH and high temperature on the stability and antioxidant capacity of two anthocyanins in aqueous solution. Food Chem. 163, 163e170. Traesel, G.K., de Souza, J.C., de Barros, A.L., Souza, M.A., Schmitz, W.O., Muzzi, R.M., Oesterreich, S.A., Arena, A.C., 2014. Acute and subacute (28 days) oral toxicity assessment of the oil extracted from Acrocomia aculeata pulp in rats. Food Chem. Toxicol. 74, 320e325.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Preparation and toxicological evaluation of methyl pyranoanthocyanin Zhenzhou Zhu, Nao Wu, Minjie Kuang, Olusola Lamikanra, Gang Liu, Shuyi Li, Jingren He* College of Food Science and Engineering, Wuhan Polytechnic University, 430023 Wuhan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 March 2015 Received in revised form 24 April 2015 Accepted 5 May 2015 Available online 29 May 2015
Anthocyanins are increasingly valued in the food industry for their functional properties and as food colorants. The broadness of their applications has, however, been limited by the lack of stability of these natural pigment extracts in a number of food systems. The potential application of pyranoanthocyanins, anthocyanin derivatives with better stability conferred by the added pyran ring, as a food ingredient was determined. Methylpyranoanthocyanin (MPA) was prepared from reaction of acetone and anthocyanin extracts from red grapes. Reaction products were sequentially purified with polyamide resin, TSK gel resin and semi-preparative HPLC to a purity level >98%. Cytoprotective influence tests of the purified MPA indicated its significant protective effect against H2O2 induced MRC-5 cell damage. Results of evaluations of possible acute toxicity effects on MPA-fed mice, including macro and microscopic assessments, support the conclusion of a non-toxic effect of MPA, and its potential safe use as a food additive. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cytoprotective effect Methyl pyranoanthocyanin Acute toxicity HPLC-MS
1. Introduction Anthocyanins (Fig. 1(a)) are polyphenolic substances, and they are one of the most widespread pigments found inplants (Hwang rez et al., 2008). The presence of anthocyaet al., 2011; Rivero-Pe nins confers the red, blue and purple colors of a variety of fruits and flowers, such as cherries, strawberries, plums, blackberries, raspberries, grapes, red currants and black currants (Sui et al., 2014). Although anthocyanins were found with over 600 known molecular structures in the natural world, they mainly occur as glycosides of flavylium (2-phenylbenzopyrylium) salts, based on six anthocyanidins: pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin (Fernandes et al., 2014; Jampani et al., 2014). Interest in anthocyanins has considerably increased in recent years (Barba et al., 2015a, 2015b), mainly driven by their perceived functional properties. In the food industry, anthocyanins are widely used as natural color alternatives to synthetic colorants. The watersolubility of anthocyanin pigments facilitates their incorporation with numerous aqueous food systems, making them attractive natural colorants (Assous et al., 2014). Moreover, anthocyanins are regarded safer and healthier than synthetic colorants by consumers. An important attribute of anthocyanins is based on their
* Corresponding author. E-mail addresses: [email protected] (Z. Zhu), [email protected] (J. He). http://dx.doi.org/10.1016/j.fct.2015.05.004 0278-6915/© 2015 Elsevier Ltd. All rights reserved.
antioxidant properties that have been correlated to potential benefits in the reduction of risk of cardiovascular and oxidative related stress and diseases (Jiang et al., 2013; Saluk et al., 2012). The antioxidant property of anthocyanins is based on their ability to donate hydrogen to highly reactive radicals, thus retarding and/or preventing formation of undesirable oxidative products (Fukumoto and Mazza, 2000; Jiang et al., 2013). Applications of anthocyanins in food preparations and processing have, however, been limited by their instability, especially after isolation from plant cells. Isolated anthocyanins are highly unstable and very susceptible to degradation (Giusti and Wrolstad, 2003), due to their sensitivity to a number of factors such as pH, storage temperature, chemical structure, concentration, light, oxygen, solvents, the presence of enzymes, flavonoids, proteins and ~ eda-Ovando et al., 2009). These often alter metallic ionsas (Castan anthocyanin properties and are particularly detrimental to their ability to retain their color and nutritional properties. The development of stabilizing technologies and/or ability to isolate more stable anthocyanins and their derivatives will be beneficial to the food industry. Pyranoanthocyanins are one of the most important classes of anthocyanin-derived pigments occurring naturally in red wine (Blanco-Vega et al., 2011; Hayasaka and Asenstorfer, 2002; Mateus et al., 2001). Their stability is derived mainly by an additional cyclic ring (Fig. 1b), apyran ring located between the C4 position in the C ring and the hydroxyl group on the C5 position in the A ring of the anthocyanin molecule (He et al., 2012). Methyl pyranoanthocyanin
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2. Materials and methods 2.1. Preparation of MPA
Fig. 1. Chemical structure of anthocyanins (a) and pyranoanthocyanins (b).
(MPA) has a methyl group (-CH3) as R3 (Fig. 1b). They are typically yellow and have maximum UVeVis absorption at 478 nm (He et al., 2006). The formation of MPA has been reported to be derived from reactions of anthocyanins with acetone or acetoacetic acid (de Freitas and Mateus, 2011; Hayasaka and Asenstorfer, 2002; He n et al., 2004). Optimiet al., 2006; Lu and Foo, 2001; Pozo-Bayo zation of MPAs synthetic conditions from reactions between grape anthocyanin extracts and acetone was reported by Kuang et al. (2014). Their report included optimum ratios of anthocyanin extract to acetone and their concentrations, reaction temperature and pH. The chemical structure and mechanism of formation of synthesized MPAs were identified by HPLC-MS/MS. They proposed a reaction pathway in which malvidin 3-glucoside (Mv-3-G) and acetone under weak acidic conditions are combined by way of enolization of C atoms at a and b positions of acetone, followed by cycloaddition reaction of C atoms at the a and b positions. Subsequent dehydration, oxidation, and decarboxylation then lead to formation of the anthocyanin oxonium ion of a pyran ring (Fig. 2). Previous studies related to MPA have been mainly limited to their synthetic methods and structure. To our knowledge, their cytoprotective properties and potential toxicity have not been reported. These are important considerations for their potential applications in food. Thus, the objective of this study is to evaluate the cytoprotective effectiveness of MPA against H2O2 induced cell damage and its potential acute toxicity.
The red grape skin extracts containing anthocyanins were purchased from Yunnan Haitong Nature Product Co., Ltd. China. The pigments extract was consisting mainly of cyanidin-3-glucoside (Cy-3-G), peonidin-3-glucoside (Pn-3-G), delphinidin-3-glucoside (Dp-3-G), malvidin-3-glucoside (Mv-3-G), and petunidin-3glucoside (Pt-3-G), which were analyzed by HPLC-DAD-MS (Fig. 3a). It is obvious that Mv-3-G was the most important component of the extracts. Methyl pyranoanthocyanin (MPA) was synthesized by reaction of the above crude anthocyanins pigments extracts and acetone as described by Kuang et al. (2014). The ratio of red grape skin extract to acetone was 15:1 (mg:mL), resulting in grape skin pigments concentration of 2.5 mg/mL. The reaction temperature was set at 45 C and pH was 3.0. The reaction lasted 9 days, with oxygen gas being passed through the reaction mixture every 3 days. Separation and purification of MPA was carried out in three subsequent steps. Initial separation was done using polyamide resin. The resin was preconditioned by initial soaking in ethanol (95%) for 24 h after which it was rinsed with distilled water to remove ethanol and then soaked in 5% NaOH solution for an additional 1 h. The resin was rinsed with water until neutral, soaked in 5% HCl for 4 h, further rinsed until neutral, and then immersed in 50% ethanol until used. Excess acetone from the malvidin 3-glucoside and acetone reaction mixture was removed with a rotary evaporator after which the mixture (0.5 B V) was loaded onto a glass column (30 mm 600 mm I.D.) (wet-packed with polyamide resin) at a velocity of 1 B V/h. After total adsorption, the column was washed with water to remove non-reacted anthocyanins, then sample was eluted with eluted with 2e2.5 B V of 10% (v/v) aqueous ethanol solution containing HCl (10%) at a velocity of 1 B V/h, yielding the polyamide resin purified MPA. The collected sample was loaded onto a TSK Toyopearl gel HW-40(s) column (40 mm 600 mm I. D)
Fig. 2. The proposed formation mechanism of MPA.
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Fig. 3. HPLC chromatogram of red grape skin extracts (a) and MS fragmentation scheme of the anthocyanin-acetone adducts. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)
and eluted with 10e20% aqueous methanol solution containing HCl (10%), The yellow colored fractions based on the absorbance values at 478 nm were collected. TSK purified MPA was obtained by removing methanol from the collected sample using rotary evaporation. Further purification was carried out using semi-preparative HPLC (SSI Series 1500) equipped with a 250 10 mm i. d. 5 mm C18-reverse phase column (Allsphere ODS-2) and a UV detector. Eluent solvents were A: H2O/HCOOH (9:1), and B: HCOOH/H2O/ CH3CN (8:1). Samples (5 mL) were injected and eluted at a rate of 4 mL/min. The solvent mixture at the time of injection contained 20% of solvent B. The gradient program was set to increase B to 30% after 30 min and then to 100% after 33 min. Eluent solvent was held at 100% B until 43 min, and programed to decrease back to 20% at
45 min. Fractions eluted were detected at 478 nm and the eluent recovered between 16 and 21 min were collected Then MPA was purified by removing formic acid and freeze-dried to powders for further use.
2.2. Chemical structure identification The chemical structure of red grape skin extracts and reaction products was determined using a LTQ-XL mass spectrometer (Thermo, USA) with an ESI (ElectroSpray Ionisation) interface. The capillary voltage and temperature were 47 V and 350 C, respectively. Nitrogen gas was applied at 30 L/min. Spectra were recorded in positive ion mode between m/z 100 and 1500.
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2.3. Determination of MPA purity Progress of the successive purification steps were assessed using a SSI Series 1500 HPLC equipped with a 250 4.6 mm i. d. 5 mm C18-reverse phase column (LiChrospher) and a DAD detector. The solvents were A: H2O/HCOOH (9:1, v/v), and B: HCOOH/H2O/CH3CN (8:1:1, v/v/v). The injection volume was 20 mL at a velocity of 1 mL/ min. The solvent mixture at the time of injection contained 15% of solvent B. The gradient program was set to increase B to 24% after 30 min and then to 100% after 35 min. Eluent solvent was held at 100% B until 37 min, and programed to decrease back to 20% at 40 min. MPA retention time of 23 min was confirmed by comparing eluent retention time with that of a standard with 99%.purity provided by the University of Porto. Purity of eluted MPA was determined from the relationship:
Purity ¼ peak area of sample=peak area of standard
(1)
2.4. Cytoprotective effect investigations 2.4.1. Cell culture Human embryonic lung fibroblast (MRC-5) cells obtained from the China Center for Culture Type Collection (Wuhan University), were cultured in Modification of Eagle Medium-Earls Balanced Salt Solution (MEM-EBSS) (Hyclone) containing 10% (v/v) heatinactivated fetal bovine serum (FBS) (Gibico), 1% penicillinestreptomycin (Hangzhou Ginuo Biomedical Technology Co., Ltd. China), 5% non-essential amino acid and 5% L-Glutamine in an incubator (95% air and 5% CO2) at 37 C. A flask with a bottom area of 25 m2 was used for MRC-5 cell culture. The medium was replaced every 2 days and the cells were passaged at approximately 60e80% confluence using a trypsin solution (0.25%)with 0.02% EDTA (Hangzhou Ginuo Biomedical Technology Co., Ltd. China). Passages 2730 were used in this study. 2.4.2. Cytotoxicity assay The cytotoxicity of MPA to MRC-5 cells was evaluated by 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe nyl)-2H-tetrazolium, inner salt (MTS) assays (Beijing Promega Biotechnology Co., Ltd. China). Briefly, MRC-5 cells were grown in 96-well plates at a density of 104 cells/well. The cells were grown in an atmosphere with 5% CO2 and 95% relative humidity. After an incubation of 24 h, cells were washed twice with Phosphate Buffer Saline (PBS) solution and the culture medium was replaced by 100 mL fresh MEM medium which contains 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonicaci (HEPES) buffer. After 20 min's incubation, cells were re-washed and treated by100 mL MEM medium with different MPA concentrations (0, 20, 40, 60, 80, 200, 400, 600, 800 mg/mL), following an incubation of 24 h. After the incubation, the medium was removed and cells were washed with PBS trice, 120 mL solution consisting of 20 mL MTS and 100 mL MEM medium was added to each well. After an additional incubation of 4 h, the 96-well plate was placed in the microplate reader (iMark, BIO-RAD, USA) with gentle shaking for 1 min, and absorbance was determined at 490 nm, for the viability measurement of MRC-5 cells (Eq. (2)). Cells incubated without the test compounds were used as control. In each MTT assay, every sample was tested in at least three replicates.
cell viability ¼ As490 Ac490 100%
(2)
where As490 means absorbance of the sample at 490 nm and Ac490 means absorbance of the control at 490 nm.
The cytotoxicity of H2O2 to MRC-5 cells was measured using the same method as above, except that cells were treated by 100 mL MEM medium with different H2O2 (Sinopham Chemical Reagent Co, Ltd) concentrations (0, 100, 200, 300, 400, 500, 600, 700, 800, 900 mmol/L). 2.4.3. Protective effect of MPA on H2O2 induced MRC-5 cell damage MRC-5 cells were grown in 96-well plates at a density of 104 cells/well. The cells were grown in an atmosphere with 5% CO2 and 95% relative humidity. After an incubation of 24 h, cells were washed twice with Phosphate Buffer Saline (PBS) solution and the culture medium was replaced by 100 mL fresh MEM medium which contains 10 mmol/L 4-(2-hydroxyethyl)-1piperazineethanesulfonicaci (HEPES) buffer. After 20 min incubation, cells were re-washed and treated by 100 mL MEM medium with different MPA concentrations (0, 40, 60, 80 mg/mL), following an incubation of 24 h. The medium was then removed and cells were washed with PBS trice, after which they were incubated for 4 h in the MEM medium with an H2O2 concentration of 500 mmol/L. Thereafter, the culture medium was removed, cells were washed with PBS trice, 120 mL solution consisting of 20 mL MTS and 100 mL MEM medium was added to each well. After an additional incubation of 4 h, the 96-well plate was placed in the microplate reader with gentle shaking for 1 min, and absorbance was determined at 490 nm using iMark microplate reader (BIO-RAD, USA) for the viability measurement of MRC-5 cells. Cells incubated without the test compounds were used as control. In each MTT assay, every sample was tested at least three times. For the purpose of comparison, the same process was carried out to evaluate the protective effect of Mv-3-G on H2O2 induced MRC-5 cell damage. 2.5. Acute toxicity evaluation The acute oral toxicity of MPA was evaluated in mice (provided by Experimental Animals Research Centre, Hubei Province Centre for Disease Control and Prevention, China) using median lethal dose (LD50) method according to the procedures outlined by Procedure and methods of food safety toxicological assessment, GB15193.3-2003 (in Chinese) (China's Ministry of Health, 2003). After 3e5-day periods of quarantine and acclimatization, 6 healthy mice of each sex were used for pre-study. MPA was solved in distilled water (10 g/40 mL) and the sample was administered by oral gavage at a dose of 5000 mg/kg body weight (BW), and was observed during 24 h. The LD50 was estimated 5000 mg/kg since no death of the animal occurred during pre-study. Acute toxicity evaluation was carried out according to Horns method. A total of 50 mice, body weights ranging from 20 to 25 g, were used in this study. The animals were randomly divided into four dose groups and one control group (five males, five females per group). The dose groups were fed with MPA by oral gavage at doses of 1000, 2150, 4540 and 10,000 mg/kg BW (based on the LD50estimation from pre-study) respectively, before normal feeding. Observations were carried out once every 30 min in 2 h and every 4 h in 24 h after gavage, additional observation was done every morning and evening. The body weight of mice was measured once a week during two weeks. Animals were observed for signs of toxicity and mortality throughout the experimental period (14 days). Animals that died during the test were dissected for organ analysis. At the end of the test, the surviving animals were anesthetized and sacrificed. Organs such as liver, pancreas, kidneys were collected and weighed. Histopathological examinations were performed on liver. Liver slices were fixed with 10% formalin in phosphate buffered saline for 24 h and embedded in paraffin. Sections of 4 mm thickness were made using a microtome and
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stained with hematoxylin-eosin and observed under microscope to observe histopathological changes in the liver. 2.6. Statistical analysis All the tests were performed at least trice, data were analyzed with commercial statistical software and standard deviation (SD) was calculated. One-way ANOVA with Dun-can's test was used for inter-group comparison. P values less than 0.05 were considered statistically significant. 3. Results and discussion 3.1. Chemical structure identification and purity analysis of reaction products Mass spectrometry fragmentation patterns of the purified pyroanthocyanin is shown in Fig. 3(b). The presence of a [M]þ ion at m/z 531, and a major fragment ion [M162]þ at m/z 369 as observed for the purified compound is consistent with previously reported fragmentation for methyl pyroanthocyaninin (He et al., 2006; Kuang et al., 2014). The efficiency of each of the stepwise purification process was assessed from the relative GC-MS fraction peak areas of the extracts. The peak area of standard (with a recovery of 100%) was 7,353,143 mAU*min. Calculated recovery of polyamide resin purified sample, TSK gel resin purified sample and Semi-preparative HPLC purified sample were 76.16%, 91.60% and 98.31%, respectively, with peak area of 5,599,786, 6,735,479 and 7,228,875 mAU*min, respectively. Purity of recovered MPA after the semipreparative HPLC process indicated a recovery level of about 98%. 3.2. Cytoprotective effect of MPA The effects of MPA and H2O2 concentration on MRC-5 cell viability are presented in Fig. 4. For the purpose of comparison, the effect of malvidin-3-glucoside (Mv-3-G), the original anthocyanin for MPA synthesis, on MRC-5 cell viability is also presented in Fig. 4.
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MRC-5 cell viability remained at about 100% when MPA concentration was lower than 200 mg/mL, indicating its lack or minimal toxicity effect to MRC-5 at concentrations within this range. Increase in MPA concentrations above 200 mg/mL, however, resulted in reduced MRC-5 cell viability. For example, the MRC-5 cell viability decreased to 80% when MPA concentration was 760 mg/mL. Concentrations in the range of 40e80 mg/mL were thus selected to investigate the protective effect of MPA on H2O2 induced MRC-5 cell damage. The effect of Mv-3-G on MRC-5 cell viability shows the same tendency as that of MPA. H2O2 induced MRC-5 cell damage was more severe at higher concentrations. Significant loss in MRC-5 viability was observed at H2O2 concentrations higher than 200 mmol/L. MRC-5cell viability was 58% at a H2O2 concentration of 500 mmol/L, and was less than 50% at 600 mmol/L of H2O2. This may be explained by the rupture of MRC-5 cells at higher H2O2 concentration (600 mmol/L). Thus, a H2O2 concentration of 500 mmol/L, under which concentration cell growth was limited but without significant cell rupture, was selected forMRC-5 cell damage inducing to evaluate the protective function of MPA. Fig. 5 represents the protective effect of MPA and Mv-3-G on MRC-5 cell damage induced by H2O2 at 500 mmol/L. It is obvious that MPA does not have better protective effect than Mv-3-G. Cell viability was 75% after 24 h in the presence of MPA (40 mg/mL), while the value was 56% in the control without MPA. These results showed a significant protective effect (P < 0.05) of MPA on MRC-5 cell. However, this protective effect was dose dependent. For example, the protective effect was not significant (P > 0.05) when MPA concentration was increased to 80 mg/mL. The lack of effect at higher concentrations could be related to a competitive reaction between H2O2 and MPA, possibly similar to oxidative effects of H2O2 on anthocyanins (He et al., 2010), which will reduce the amount of effective MPA concentration on MRC-5 protective action. 3.3. Acute toxicity evaluation The number of mice that died after different gavage dosing levels with MPA was investigated for male and female separately.
Fig. 4. Influence of concentrations of Mv-3-G, MPA and H2O2 on MC-5 cell viability.
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Fig. 5. Protective effect of MPA and Mv-3-G on H2O2 induced MRC-5 cell. Columns followed by different letters within the same row represent significant differences at P < 0.05.
Fig. 6. The cumulative number of male and female mice deaths at different time after oral administration with MPA.
As observed, a dose of 4640 mg/kg BW did not cause the death of any animal. The mice, male and female, exhibited no behavioral changes during the treatment period (14 days). There were also no changes in water and food consumption, in relation to the control group. However, at a dose of 10,000 mg/kg BW, the mice became less active after oral dosing and death of some mice occurred after 8 h and 1.5 h for male and female, respectively (Fig. 6). After 24 h, the cumulative death number attained 3 for male and 5 for female. This suggests that the male mice might be more resistant to MPA than the female. LD50 value and 95% confidence limit was calculated according to procedure and methods of food safety
toxicological assessment, GB15193.3-2003 (in Chinese) (China's Ministry of Health, 2003). A LD50 value of 7945 mg/kg BW with a 95% confidence limit of 5716e11,043.3 mg/kg BW was presented in Table 1 for male. While for female, the LD50 value was 6816 mg/kg BW without 95% confidence limit. The high LD50 value for both male and female showed that MPA was practically non-toxic according to chemical-test method of acute oral toxicity for chemicals-acute toxic class method, GB/T 21757-2008 (in Chinese) (General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China). Changes in body and organ weights are a clear indications of damage caused by the substance test (Traesel et al., 2014). The increase of body weights of mice treated at different doses after two weeks were noted and presented in Fig. 7(a). There was no statistical difference in the weight increase between MPA administrated and control groups. Organ (liver, kidney, and pancreas) weights for both male and female mice were also measured and presented in Fig. 7(b) and (c). Results showed that organ weights were not statistically affected by MPA treatment over a period of 14 days. There, however, appears to be a decrease in liver weight of male mice at 1000 mg/kg BW which was not observed for all the other doses and did not occur in female mice at the same dosage. The reason for this observation is unknown, but need further investigation. Results of histopathological analysis suggest the deaths of some mice in this study is unrelated to toxic effects of MPA but could be attributed to the ability of the mice to adequately readjust to the metabolic changes related to the sudden change in diet. The assessment of pathological changes in the organs of treated animals, both macro and microscopically, is the basis of a safety assessment (Traesel et al., 2014). In this study, the histopathological analyses of survived mice's liver after 14 days' test showed no findings suggestive of toxic effects (Fig. 8). These results proved to be consistent with above analyses, confirming the safety of using
Table 1 LD50 and 95% confidence limits of male and female mice administered orally with MPA. Group
Dosage (mg/kg BW)
Logarithmic dose
Mortality number
Mortality rate (%)
LD50 and 95% confidence limit (CL
Male Female
10,000 10,000
4.0000 4.0000
3 5
60 100
LD50 ¼ 7945, CL95%: 5716e11043.3 LD50 ¼ 6816, No CL95%
95%)
(mg/kg)
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Fig. 7. Body weight of mice (a) and principal internal organ of male (b) and female (c) mice treated by gavage with methyl-pyranoanthocyanin. Columns followed by different letters within the same row represent significant differences at P < 0.05.
Fig. 8. Histopathological analysis of liver of surviving mice treated with MPA after 14 days.
the MPA with the dose range and administration period of this study. 4. Conclusions The results from this study are consistent with a non-toxic effect of MPA, The pyranoanthocyanin, when fed to mice showed no
evidence that would indicate toxicity based on their body and organ weights, and histopathological analysis of their livers. MPA, however, had significant protective effect on H2O2 damaged MRC-5 cells, increasing cell viability from 56% (control) to 75%. The nontoxic effect of MPA and its demonstrated cytoprotective effect on H2O2 induced MRC-5 cell damage suggest its potential use as a functional ingredient that could be safely incorporated in food and
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