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Monascorubrin and rubropunctatin: Preparation and reaction characteristics with amines
Dyes and Pigments 170 (2019) 107629
Contents lists available at ScienceDirect
Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Monascorubrin and rubropunctatin: Preparation and reaction characteristics with amines
T
Lili Jiaa, Xuan Tua, Kun Hea, Chengtao Wangb, Sheng Yinb, Youxiang Zhouc, Wanping Chena,∗ a Hubei International Scientific and Technological Cooperation Base of Traditional Fermented Foods, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province, 430070, China b Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University, Beijing, 100048, China c Institute of Quality Standard and Testing Technology for Agro-Products, Hubei Academy of Agricultural Sciences, Wuhan, Hubei Province, 430064, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Monascorubrin Rubropunctatin Amination Monascus pigments
Monascorubrin and rubropunctatin are the two classical orange Monascus pigments, and serve as indispensable precursors in the formation of food colorants Monascus red pigments by amination with primary amines. However, this chemical transformation has been less investigated in vitro due to the hard preparation and preservation of monascorubrin and rubropunctatin. In this study, a way for the preparation of high-purity monascorubrin and rubropunctatin was developed. The in vitro reaction characteristics with various amino acids in different pH environments indicated that an alkaline environment could greatly promote the amination, while the amination was obviously inhibited in an acidic environment. The kinetic study of monascorubrin and rubropunctatin with ammonia suggested that their amination occurred via a second-order reaction. This study not only provides a better understanding of the chemical properties of monascorubrin and rubropunctatin, but also has significance in guiding the industrial applications of Monascus pigments.
1. Introduction Monascus pigments are a large class of secondary metabolites with a similar azaphilone skeleton produced by the filamentous fungi Monascus spp. via a polyketide pathway, and they have been widely used as food coloring agents for over two thousand years [1,2]. At present, it was estimated that more than one billion people may eat products containing Monascus pigments during their daily lives, and the annual output of Monascus pigments, usually in the form of red mold rice, is approximately 20, 000 tons in China alone [3–5]. Moreover, the demand for Monascus pigments as food additives or in other applications is growing rapidly due to their advantages, such as apparent heat and pH stability, diverse biological activities, efficient and economical production on cheap substrates, good solubility in water and ethanol, high safety, and vivid colors [4,6–9]. Monascus pigments are classified into red, orange, and yellow pigments [7,10], but in practice, they are commonly used as a mixture without further separation, although the main commercial interest lies in the red compounds [11]. The structural analysis of Monascus pigments dates back to 1932 [12,13]. Until 1970s, 6 compounds were clarified, namely, orange monascorubrin 1 [14] and rubropunctatin 2 [15], red monascorubramine 3 and rubropunctamine 4 [16], and
∗
yellow ankaflavin 5 [17] and monascin 6 [18], which are now well known as the fundamental compound types of Monascus pigments [2,4]. However, until recently, the biosynthetic pathway for these six classic Monasucs pigments have been fully clarified [7]. Briefly, as presented in Fig. 1, intermediates 7 and 8 are the last common node compounds in the biosynthetic pathway to generate the six classic Monascus pigments. In one branch of the pathway, the classical yellow ankaflavin 5 and monascin 6 are produced by the reduction of the C5(2’) double bonds of 7 and 8. The other branch yields the classical orange pigments monascorubrin 1 and rubropunctatin 2 by restoring the C6(7) double bonds of 7 and 8. Subsequently, amination of 1 and 2 leads to the classical red monascorubramine 3 and rubropunctamine 4. Currently, more than 111 Monascus pigment types have been reported, most of which are amino acid derivatives of monascorubrin and rubropunctatin [4]. Essentially, monascorubrin and rubropunctatin have a unique structure responsible for their high affinity for primary amines (so-called aminophiles), wherein the pyranyl oxygen is replaced with nitrogen by insertion of a primary amine, resulting in the formation of vinylogous γ-pyridones and a corresponding color change to red from orange [9,19,20]. The details mechanism of this aminophilic reaction was described in the comprehensive review [21]. Despite the indispensable role of these compounds in generating high-economic-
Corresponding author. E-mail address: [email protected] (W. Chen).
https://doi.org/10.1016/j.dyepig.2019.107629 Received 1 April 2019; Received in revised form 5 June 2019; Accepted 5 June 2019 Available online 06 June 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
Dyes and Pigments 170 (2019) 107629
L. Jia, et al.
Fig. 1. Biosynthetic relationship among six classic Monascus pigments. These pigments share a very close synthetic relationship. For the complete biosynthetic pathway, please refer to the recent report [7].
hexane, methanol solutions at different concentrations, and chloroform, combined with solid-phase and liquid-phase extraction methods, were used to compare the extraction ability for monascorubrin and rubropunctatin.
value red pigments, the chemical characteristic of pure monascorubrin and rubropunctatin have been less investigated in vitro due to their difficult preparation and preservation. Thus far, knowledge about these compounds has largely come from indirect observation via Monascus fermentation studies. Some key observations include the following: 1) Various derivatives could be produced by Monascus fermentation in the presence of different natural or non-natural primary amines in culture media [21–26]. 2) The production of pigments category was affected by the ambient pH. Generally, yellow and orange pigments predominate at lower pH values, while red pigments predominate at higher pH values [11,27–29]. For example, it was observed that by keeping the pH of the culture medium at a value in the range from 2 to 4, the transformation of orange into red pigments was inhibited, and hardly any red pigment, such as rubropunctatamine or monascorubramine, was formed under this condition [30]. Furthermore, the chemical reactions were also carried out with preliminary separation or solvent extraction of monascorubrin and rubropunctatin from Monascus fermentation [21,31,32]. In this study, a new reliable and effective way to prepare pure monascorubrin and rubropunctatin was developed. The preparation of a sufficient amount of pure monascorubrin and rubropunctatin enables the comprehensive investigation of their chemical characteristics, especially their reaction with amines, which has significance in guiding the industrial production of Monascus pigments.
2.3. Purification of rubropunctatin and monascorubrin Monascorubrin and rubropunctatin were further purified from the abovementioned optimized crude extract by a semi-preparative Waters HPLC system with a Waters 600 pump and controller, a Waters 717 autosampler, and a Waters 996 PDA, using an Intertsil ODS-3 column (250 mm × 10 mm, 5 μm), an isocratic elution with a mobile phase of 90% methanol, a flow rate of 3 ml/min, 35 min, detection at 470 nm with a PDA detector and an injection volume of 50 μL. Fractions containing monascorubrin and rubropunctatin were respectively collected, dried under nitrogen flow, and dissolved in methanol.
2.4. In vitro reaction of monascorubrin with amino acids The detailed experimental process is presented in Supporting Information section 2. Briefly, different types of amino acids were chosen as substrates to study their reactions with monascorubrin under different concentration gradients, while deionized water was used as a negative control. The reactions were performed at room temperature and followed by UPLC.
2. Materials and methods 2.1. Preparation of red mold rice M. ruber M7 was cultured on PDA plates at 28 °C for 10 days. Fresh conidia were harvested from the plate with distilled water, and filtered through lens wiping paper to remove hyphae. Then, the conidia were centrifuged at 6,000×g for 5 min and resuspended in distilled water to a final concentration of 1 × 105 spores/ml. A 1 ml spore suspension was inoculated into rice medium, prepared by steaming 15 g polished japonica rice, and incubated at 28 °C. After 13 days of fermentation, the red mold rice was dried at 40 °C and then ground into a powder. The powder was sealed and kept in a fridge at −20 °C.
2.5. Kinetic study of monascorubrin and rubropunctatin with ammonia Ammonia was used as a representative for the kinetic study of the amination of monascorubrin and rubropunctatin. Briefly, 25% ammonia was diluted with methanol to form working solutions with different concentration gradients. The reactions were placed in 96-well microtiter plates. In each well, 100 μL monascorubrin and rubropunctatin solutions were separately mixed with 100 μL ammonia working solutions at different concentrations, with 100 μL chromatographically pure methanol as a control. Three replicates were performed for each reaction. The reaction process was monitored by a microplate spectrophotometer (MK3, Thermo) at a detection wavelength of 450 nm via time interval measurements. The reactions were performed at room temperature.
2.2. Optimization of crude extraction condition of monascorubrin and rubropunctatin The detailed experimental process is presented in Supporting Information section 1. Briefly, the solvent acetone, deionized water, 2
Dyes and Pigments 170 (2019) 107629
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Fig. 2. Semi-preparative HPLC of monascorubrin 1 and rubropunctatin 2 recorded at 380 and 470 nm.
related products. In this connection, different concentrations of methanol (10–90%) were used to evaluate their ability to elute monascorubrin and rubropunctatin from solid-phase extraction columns. The results showed that the elution abilities of monascorubrin and rubropunctatin differ depending on the methanol concentration (Supporting Information section 1). The elution with 10–60% methanol contains many complex components and impurities, while elution with 90% methanol results in almost no monascorubrin and rubropunctatin. 70–80% methanol shows a good elution ability for monascorubrin and rubropunctatin, but also for monascin and ankaflavin, troubling the further purification. Therefore, methanol is not recommended for isolating monascorubrin and rubropunctatin. Next, the frequently used extractants deionized water, N-hexane, chloroform, and acetone were applied to determine their ability to extract monascorubrin and rubropunctatin (Supporting Information section 1). The results indicated that acetone and chloroform have a strong ability for monascorubrin and rubropunctatin extraction, while deionized water and N-hexane exhibited a poor extraction ability and dissolved many interfering substances. In view of their solubility properties, a mixture of deionized water and chloroform was considered an ideal two-phase liquid-liquid extraction system for crude extraction of monascorubrin and rubropunctatin. Collectively, the preparation and preservation of monascorubrin and rubropunctatin on the bench-scale are summarized as follows.
2.6. Effect of pH on the reaction of monascorubrin and rubropunctatin with amino acids The acidic amino acid Glu and the basic amino acid Arg were selected as representatives to study their reaction characteristics with monascorubrin and rubropunctatin at different pH values. The reaction concentration of monascorubrin and rubropunctatin was prepared at around 5 × 10−4 M, while that of Glu and Arg was prepared at 10−3 M. The different ambient pH values ranging from 2.0 to 10.0 were maintained by sodium hydrogen carbonate-sodium carbonate buffer and citric acid-disodium hydrogen phosphate buffer. The reactions were placed in 96-well microtiter plates. In each well, 100 μL monascorubrin and rubropunctatin solutions were separately mixed with 100 μL Glu and Arg solutions, with 100 μL deionized water as a control. Three replicates were performed for each reaction. The reaction process was monitored by a microplate spectrophotometer as described above. The reactions were performed at room temperature.
3. Results and discussion 3.1. A reliable and effective way to prepare monascorubrin and rubropunctatin Based on the author's knowledge, neither commercially available monascorubrin and rubropunctatin standards nor satisfactory methods for the high-yield and high-purity production of these components existed previously, which limited the study of monascorubrin and rubropunctatin. Therefore, at the beginning of this study, a rapid extraction and separation process for monascorubrin and rubropunctatin was developed. First, an effective extractant system for monascorubrin and rubropunctatin should be screened. Methanol is the most common extractant for recovering total azaphilone pigments from red mold rice or
Step 1: A deionized water and chloroform two-phase liquid-liquid extraction system is used to extract monascorubrin and rubropunctatin from red mold rice. After stratification, the chloroform layer is collected and evaporated by a rotary evaporator. Then, a crude solution of monascorubrin and rubropunctatin is prepared by redissolution of the evaporated extract with chromatographically pure methanol. Step 2: The crude solution is filtered through the membrane and 3
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(Supporting Information section 2), the amination reaction between the acidic amino acid Glu and monascorubrin and rubropunctatin was inhibited in aqueous solution, while the reactivity was much higher under alkaline conditions (Fig. 3). Similarly, the amination reaction between the basic amino acid Arg and monascorubrin and rubropunctatin was active in aqueous solution, while the reactivity was significantly inhibited under acidic conditions. Further experiments also clearly suggested that the lower pH values resulted in the stronger inhibition of the amination (Supporting Information section 3). However, a clear boundary was observed between ambient alkalinity and acidity. Previously, it was speculated that Monascus pigment production was closely related to the isoelectric point of amino acids based on observations of Monascus fermentation [27]. In this study, the in vitro reaction directly showed that the alkaline environment could greatly promote this amination conversion. However, for Monascus fermentation, a suitable pH environment for growth should be considered, and the difference between intracellular and extracellular pH environments should also be noted.
loaded on a semi-preparative HPLC under the optimized conditions suggested above for the purification of monascorubrin and rubropunctatin. The separation efficiency shown in Fig. 2 indicated a clear isolation of monascorubrin 1 and rubropunctatin 2 from ankaflavin 5 and monascin 6, which are usually hard to separate. Step 3: The collected monascorubrin and rubropunctatin fractions are dried under nitrogen flow, and redissolved in chromatographically pure methanol. The stock solutions are sealed and kept at −20 °C. It was observed that monascorubrin and rubropunctatin in the stock solutions remained stable for three days. However, freshly prepared monascorubrin and rubropunctatin are recommended for chemical analysis. 3.2. Reaction of monascorubrin with amino acids At present, based on the structure summary of red Monascus pigments [4], most of these pigments are amino acid derivatives. Our previous studies observed that the amination of monascorubrin and rubropunctatin with amino acids differed greatly by amino acid type [7]. In this study, the amino acids Arg, Asp, Glu, GABA, Lys and Thr were tested for their in vitro reactivity with monascorubrin, the derivatives of which are common types of Monascus red pigments. The details are presented in Supporting Information section 2. The results showed that only Arg and GABA led to obvious color changes from orange to red upon reacting with monascorubrin, and a positive correlation between color and concentration was also observed, but these results were not obviously observed for the other tested amino acids. The results were somewhat surprising, since other amino acid derivatives are commonly present in Monascus fermentation, and this amination is enzyme-independent. It was speculated that Arg and GABA create an alkaline environment in deionized water, which promotes the amination reactions. To support the speculation that the ambient pH rather than amino acid bias affects the reactions, further reactions were monitored as described below.
3.4. Kinetic study of monascorubrin and rubropunctatin with ammonia It is well known that the classical orange pigments monascorubrin 1 and rubropunctatin 2 can directly react with ammonia to form the classical red pigments monascorubramine 3 and rubropunctatamine 4 [4]. In vitro, a very obvious chemical reaction between ammonia and monascorubrin 1 and rubropunctatin 2 was observed, which was used as a positive control for the reactions with amino acids. Therefore, ammonia was chosen to analyze the kinetics of its reaction with monascorubrin and rubropunctatin. First, the reactions at different ammonia concentrations were compared (Fig. 4). As revealed, the reactions under different ammonia concentrations shared a similar trend. Generally, the higher the ammonia concentration is, the more intense the reaction is. Thus, reactions at an ammonia concentration of 2.67 × 10−4 M were selected for kinetics analysis. Based on the characteristics of the chemical reactions, and the abovementioned reaction trend (Fig. 4), the amination of monascorubrin and rubropunctatin may occur via a second-order reaction. That is, the reaction rate (r) is proportional to the substrate monascorubrin/ rubropunctatin, and ammonia concentrations, and is given by equation (1).
3.3. Effect of pH on the amination As indicated above, it was speculated that the pH could affect the amination. To support this speculation, the acidic amino acid Glu and the basic amino acid Arg were selected as representatives to study their reaction characteristics with monascorubrin and rubropunctatin at different pH values. As expected, the results showed that the alkaline environment greatly promoted amination, while the acidic environment significantly inhibited amination (Fig. 3). As indicated
r=
dx = k (a − x )(b − x ) dt
(1)
Fig. 3. The reaction characteristics of Arg and Glu with monascorubrin and rubropunctatin at different pH values. A and B correspond to the reactions with monascorubrin and rubropunctatin respectively. 4
Dyes and Pigments 170 (2019) 107629
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Fig. 4. The reactions of monascorubrin and rubropunctatin with different concentrations of ammonia. The reactions were performed in methanol at room temperature and monitored by absorbance changes. A. The reactions of monascorubrin with ammonia at different concentrations. B. The reactions of rubropunctatin with ammonia at different concentrations.
Fig. 5. Reaction kinetics analysis of monascorubrin and rubropunctatin with ammonia by second-order reaction. A and B correspond to the reactions of monascorubrin and rubropunctatin, respectively. The initial concentrations of monascorubrin and rubropunctatin were 2.82 × 10−4 M and 2.85 × 10−4 M rea−x spectively. The initial concentration of ammonia was 2.67 × 10−4 M. R2 is the square of the Pearson correlation coefficient between ln b − x and t.
second-order reaction. These observations will not only contribute to our understanding of the chemical properties of monascorubrin and rubropunctatin, but also have significance in guiding the industrial applications of Monascus pigments.
where a, b, k, and x stand for the initial monascorubrin/rubropunctatin concentration, the initial ammonia concentration, the rate constant, and the product monascorubramine/rubropunctamine concentration, respectively. Then, equation (2) is obtained by the integration of equation (1).
ln
a−x a = (a − b)⋅k⋅t + ln b−x b
Conflicts of interest
(2)
a−x ln b − x
and t showed a high linear relationship in both As revealed, monascorubrin and rubropunctatin amination reactions (Fig. 5), which suggests that these aminations are in line with second-order reactions.
The authors declare that they have no conflicts of interest.
Acknowledgements 4. Conclusions This work was supported by the fund of the Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU).
In this study, we developed a reliable and effective method for the preparation of two classical orange Monascus pigments monascorubrin and rubropunctatin on the laboratory scale. The characteristics of the reactions with various amino acids at different pH values indicated that an alkaline environment could greatly promote the amination of rubropunctatin and monascorubrin, while amination was obviously inhibited in an acidic environment. The kinetic study of monascorubrin and rubropunctatin with ammonia suggested that the amination was a
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107629. 5
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References
[17] Manchand PS, Whalley WB, Chen F-C. Isolation and structure of ankaflavin: a new pigment from Monascus anka. Phytochemistry 1973;12(10):2531–2. [18] Fielding B, Holker J, Jones D, Powell A, Richmond K, Robertson A, et al. 898. The chemistry of fungi. Part XXXIX. The structure of monascin. J Chem Soc 1961:4579–89. [19] Wei WG, Yao ZJ. Synthesis studies toward chloroazaphilone and vinylogous gamma-pyridones: two common natural product core structures. J Org Chem 2005;70(12):4585–90. [20] Gao J-M, Yang S-X, Qin J-C. Azaphilones: chemistry and biology. Chem Rev 2013;113(7):4755–811. [21] Liu L, Zhao J, Huang Y, Xin Q, Wang Z. Diversifying of chemical structure of native Monascus pigments. Front Microbiol 2018;9:3143. [22] Jung H, Kim C, Kim K, Shin CS. Color characteristics of Monascus pigments derived by fermentation with various amino acids. J Agric Food Chem 2003;51(5):1302–6. [23] Lin T, Yakushijin K, Büchi G, Demain A. Formation of water-soluble Monascus red pigments by biological and semi-synthetic processes. J Ind Microbiol 1992;9(3–4):173–9. [24] Martinkova L, Patakova-Juzlova P, Krent V, Kucerova Z, Havlicek V, Olsovsky P, et al. Biological activities of oligoketide pigments of Monascus purpureus. Food Addit Contam 1999;16(1):15–24. [25] Lian X, Wang C, Guo K. Identification of new red pigments produced by Monascus ruber. Dyes Pigment 2007;73(1):121–5. [26] Huang Y, Liu L, Zheng G, Zhang X, Wang Z. Efficient production of red Monascus pigments with single non-natural amine residue by in situ chemical modification. World J Microb Biot 2019;35(1):13. [27] Vendruscolo F, Schmidell W, Moritz DE, Bühler RMM, de Oliveira D, Ninow JL. Isoelectric point of amino acid: importance for Monascus pigment production. Biocatal Agric Biotechnol 2016;5:179–85. [28] Kang B, Zhang X, Wu Z, Qi H, Wang Z. Effect of pH and nonionic surfactant on profile of intracellular and extracellular Monascus pigments. Process Biochem 2013;48(5):759–67. [29] Lee B-K, Park N-H, Piao HY, Chung W-J. Production of red pigments by Monascus purpureus in submerged culture. Biotechnol Bioproc Eng 2001;6(5):341–6. [30] Shepherd D, Carels MS. Red pigment production. US Patents; 1979, US4145254A. [31] Choe D, Lee J, Woo S, Shin CS. Evaluation of the amine derivatives of Monascus pigment with anti-obesity activities. Food Chem 2012;134(1):315–23. [32] Xiong X, Zhang X, Wu Z, Wang Z. Coupled aminophilic reaction and directed metabolic channeling to red Monascus pigments by extractive fermentation in nonionic surfactant micelle aqueous solution. Process Biochem 2015;50(2):180–7.
[1] Feng Y, Shao Y, Chen F. Monascus pigments. Appl Microbiol Biot 2012;96(6):1421–40. [2] Chen W, He Y, Zhou Y, Shao Y, Feng Y, Li M, et al. Edible filamentous fungi from the species Monascus: early traditional fermentations, modern molecular biology, and future genomics. Compr Rev Food Sci Food Saf 2015;14(5):555–67. [3] Yang Y, Liu B, Du X, Li P, Liang B, Cheng X, et al. Complete genome sequence and transcriptomics analyses reveal pigment biosynthesis and regulatory mechanisms in an industrial strain, Monascus purpureus YY-1. Sci Rep 2015;5:8331. [4] Chen W, Feng Y, Molnar I, Chen F. Nature and nurture: confluence of pathway determinism and metabolic serendipity diversifies Monascus azaphilone pigments. Nat Prod Rep 2019;36:561–72. [5] Schweiggert RM. Perspective on the ongoing replacement of artificial and animalbased dyes with alternative natural pigments in foods and beverages. J Agric Food Chem 2018;66(12):3074–81. [6] Vendruscolo F, Meinicke Buhler RM, Cesar de Carvalho J, de Oliveira D, Moritz DE, Schmidell W, et al. Monascus: a reality on the production and application of microbial pigments. Appl Biochem Biotechnol 2016;178(2):211–23. [7] Chen W, Chen R, Liu Q, He Y, He K, Ding X, et al. Orange, red, yellow: biosynthesis of azaphilone pigments in Monascus fungi. Chem Sci 2017;8(7):4917–25. [8] Patakova P. Monascus secondary metabolites: production and biological activity. J Ind Microbiol Biotechnol 2013;40(2):169–81. [9] Mapari SA, Nielsen KF, Larsen TO, Frisvad JC, Meyer AS, Thrane U. Exploring fungal biodiversity for the production of water-soluble pigments as potential natural food colorants. Curr Opin Biotechnol 2005;16(2):231–8. [10] Zhong S, Zhang X, Wang Z. Preparation and characterization of yellow Monascus pigments. Sep Purif Technol 2015;150:139–44. [11] Carvalho JC, Pandey A, Babitha S, Soccol CR. Production of Monascus biopigments: an overview. Agro Food Ind Hi-Tech 2003;14(6):37–43. [12] Nishikawa H. Biochemistry of filamentous fungi. I: colouring matters of Monascus purpureus Went. Part I. J Agric Chem Soc Japan 1932;8(4–6):78–9. [13] Salomon H, Karrer P. Pflanzenfarbstoffe XXXVIII. Ein Farbstoff aus „rotem” Reis. Monascin. Helv Chim Acta 1932;15(1):18–22. [14] Kumasaki S, Nakanishi K, Nishikawa E, Ohashi M. Structure of monascorubrin. Tetrahedron 1962;18(10):1171–84. [15] Haws E, Holker J, Kelly A, Powell A, Robertson A. 722. The chemistry of fungi. Part XXXVII. The structure of rubropunctatin. J Chem Soc 1959:3598–610. [16] Whalley W. The sclerotiorin group of fungal metabolites: their structure and biosynthesis. Pure Appl Chem 1963;7(4):565–88.
6
Contents lists available at ScienceDirect
Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Monascorubrin and rubropunctatin: Preparation and reaction characteristics with amines
T
Lili Jiaa, Xuan Tua, Kun Hea, Chengtao Wangb, Sheng Yinb, Youxiang Zhouc, Wanping Chena,∗ a Hubei International Scientific and Technological Cooperation Base of Traditional Fermented Foods, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province, 430070, China b Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University, Beijing, 100048, China c Institute of Quality Standard and Testing Technology for Agro-Products, Hubei Academy of Agricultural Sciences, Wuhan, Hubei Province, 430064, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Monascorubrin Rubropunctatin Amination Monascus pigments
Monascorubrin and rubropunctatin are the two classical orange Monascus pigments, and serve as indispensable precursors in the formation of food colorants Monascus red pigments by amination with primary amines. However, this chemical transformation has been less investigated in vitro due to the hard preparation and preservation of monascorubrin and rubropunctatin. In this study, a way for the preparation of high-purity monascorubrin and rubropunctatin was developed. The in vitro reaction characteristics with various amino acids in different pH environments indicated that an alkaline environment could greatly promote the amination, while the amination was obviously inhibited in an acidic environment. The kinetic study of monascorubrin and rubropunctatin with ammonia suggested that their amination occurred via a second-order reaction. This study not only provides a better understanding of the chemical properties of monascorubrin and rubropunctatin, but also has significance in guiding the industrial applications of Monascus pigments.
1. Introduction Monascus pigments are a large class of secondary metabolites with a similar azaphilone skeleton produced by the filamentous fungi Monascus spp. via a polyketide pathway, and they have been widely used as food coloring agents for over two thousand years [1,2]. At present, it was estimated that more than one billion people may eat products containing Monascus pigments during their daily lives, and the annual output of Monascus pigments, usually in the form of red mold rice, is approximately 20, 000 tons in China alone [3–5]. Moreover, the demand for Monascus pigments as food additives or in other applications is growing rapidly due to their advantages, such as apparent heat and pH stability, diverse biological activities, efficient and economical production on cheap substrates, good solubility in water and ethanol, high safety, and vivid colors [4,6–9]. Monascus pigments are classified into red, orange, and yellow pigments [7,10], but in practice, they are commonly used as a mixture without further separation, although the main commercial interest lies in the red compounds [11]. The structural analysis of Monascus pigments dates back to 1932 [12,13]. Until 1970s, 6 compounds were clarified, namely, orange monascorubrin 1 [14] and rubropunctatin 2 [15], red monascorubramine 3 and rubropunctamine 4 [16], and
∗
yellow ankaflavin 5 [17] and monascin 6 [18], which are now well known as the fundamental compound types of Monascus pigments [2,4]. However, until recently, the biosynthetic pathway for these six classic Monasucs pigments have been fully clarified [7]. Briefly, as presented in Fig. 1, intermediates 7 and 8 are the last common node compounds in the biosynthetic pathway to generate the six classic Monascus pigments. In one branch of the pathway, the classical yellow ankaflavin 5 and monascin 6 are produced by the reduction of the C5(2’) double bonds of 7 and 8. The other branch yields the classical orange pigments monascorubrin 1 and rubropunctatin 2 by restoring the C6(7) double bonds of 7 and 8. Subsequently, amination of 1 and 2 leads to the classical red monascorubramine 3 and rubropunctamine 4. Currently, more than 111 Monascus pigment types have been reported, most of which are amino acid derivatives of monascorubrin and rubropunctatin [4]. Essentially, monascorubrin and rubropunctatin have a unique structure responsible for their high affinity for primary amines (so-called aminophiles), wherein the pyranyl oxygen is replaced with nitrogen by insertion of a primary amine, resulting in the formation of vinylogous γ-pyridones and a corresponding color change to red from orange [9,19,20]. The details mechanism of this aminophilic reaction was described in the comprehensive review [21]. Despite the indispensable role of these compounds in generating high-economic-
Corresponding author. E-mail address: [email protected] (W. Chen).
https://doi.org/10.1016/j.dyepig.2019.107629 Received 1 April 2019; Received in revised form 5 June 2019; Accepted 5 June 2019 Available online 06 June 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
Dyes and Pigments 170 (2019) 107629
L. Jia, et al.
Fig. 1. Biosynthetic relationship among six classic Monascus pigments. These pigments share a very close synthetic relationship. For the complete biosynthetic pathway, please refer to the recent report [7].
hexane, methanol solutions at different concentrations, and chloroform, combined with solid-phase and liquid-phase extraction methods, were used to compare the extraction ability for monascorubrin and rubropunctatin.
value red pigments, the chemical characteristic of pure monascorubrin and rubropunctatin have been less investigated in vitro due to their difficult preparation and preservation. Thus far, knowledge about these compounds has largely come from indirect observation via Monascus fermentation studies. Some key observations include the following: 1) Various derivatives could be produced by Monascus fermentation in the presence of different natural or non-natural primary amines in culture media [21–26]. 2) The production of pigments category was affected by the ambient pH. Generally, yellow and orange pigments predominate at lower pH values, while red pigments predominate at higher pH values [11,27–29]. For example, it was observed that by keeping the pH of the culture medium at a value in the range from 2 to 4, the transformation of orange into red pigments was inhibited, and hardly any red pigment, such as rubropunctatamine or monascorubramine, was formed under this condition [30]. Furthermore, the chemical reactions were also carried out with preliminary separation or solvent extraction of monascorubrin and rubropunctatin from Monascus fermentation [21,31,32]. In this study, a new reliable and effective way to prepare pure monascorubrin and rubropunctatin was developed. The preparation of a sufficient amount of pure monascorubrin and rubropunctatin enables the comprehensive investigation of their chemical characteristics, especially their reaction with amines, which has significance in guiding the industrial production of Monascus pigments.
2.3. Purification of rubropunctatin and monascorubrin Monascorubrin and rubropunctatin were further purified from the abovementioned optimized crude extract by a semi-preparative Waters HPLC system with a Waters 600 pump and controller, a Waters 717 autosampler, and a Waters 996 PDA, using an Intertsil ODS-3 column (250 mm × 10 mm, 5 μm), an isocratic elution with a mobile phase of 90% methanol, a flow rate of 3 ml/min, 35 min, detection at 470 nm with a PDA detector and an injection volume of 50 μL. Fractions containing monascorubrin and rubropunctatin were respectively collected, dried under nitrogen flow, and dissolved in methanol.
2.4. In vitro reaction of monascorubrin with amino acids The detailed experimental process is presented in Supporting Information section 2. Briefly, different types of amino acids were chosen as substrates to study their reactions with monascorubrin under different concentration gradients, while deionized water was used as a negative control. The reactions were performed at room temperature and followed by UPLC.
2. Materials and methods 2.1. Preparation of red mold rice M. ruber M7 was cultured on PDA plates at 28 °C for 10 days. Fresh conidia were harvested from the plate with distilled water, and filtered through lens wiping paper to remove hyphae. Then, the conidia were centrifuged at 6,000×g for 5 min and resuspended in distilled water to a final concentration of 1 × 105 spores/ml. A 1 ml spore suspension was inoculated into rice medium, prepared by steaming 15 g polished japonica rice, and incubated at 28 °C. After 13 days of fermentation, the red mold rice was dried at 40 °C and then ground into a powder. The powder was sealed and kept in a fridge at −20 °C.
2.5. Kinetic study of monascorubrin and rubropunctatin with ammonia Ammonia was used as a representative for the kinetic study of the amination of monascorubrin and rubropunctatin. Briefly, 25% ammonia was diluted with methanol to form working solutions with different concentration gradients. The reactions were placed in 96-well microtiter plates. In each well, 100 μL monascorubrin and rubropunctatin solutions were separately mixed with 100 μL ammonia working solutions at different concentrations, with 100 μL chromatographically pure methanol as a control. Three replicates were performed for each reaction. The reaction process was monitored by a microplate spectrophotometer (MK3, Thermo) at a detection wavelength of 450 nm via time interval measurements. The reactions were performed at room temperature.
2.2. Optimization of crude extraction condition of monascorubrin and rubropunctatin The detailed experimental process is presented in Supporting Information section 1. Briefly, the solvent acetone, deionized water, 2
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Fig. 2. Semi-preparative HPLC of monascorubrin 1 and rubropunctatin 2 recorded at 380 and 470 nm.
related products. In this connection, different concentrations of methanol (10–90%) were used to evaluate their ability to elute monascorubrin and rubropunctatin from solid-phase extraction columns. The results showed that the elution abilities of monascorubrin and rubropunctatin differ depending on the methanol concentration (Supporting Information section 1). The elution with 10–60% methanol contains many complex components and impurities, while elution with 90% methanol results in almost no monascorubrin and rubropunctatin. 70–80% methanol shows a good elution ability for monascorubrin and rubropunctatin, but also for monascin and ankaflavin, troubling the further purification. Therefore, methanol is not recommended for isolating monascorubrin and rubropunctatin. Next, the frequently used extractants deionized water, N-hexane, chloroform, and acetone were applied to determine their ability to extract monascorubrin and rubropunctatin (Supporting Information section 1). The results indicated that acetone and chloroform have a strong ability for monascorubrin and rubropunctatin extraction, while deionized water and N-hexane exhibited a poor extraction ability and dissolved many interfering substances. In view of their solubility properties, a mixture of deionized water and chloroform was considered an ideal two-phase liquid-liquid extraction system for crude extraction of monascorubrin and rubropunctatin. Collectively, the preparation and preservation of monascorubrin and rubropunctatin on the bench-scale are summarized as follows.
2.6. Effect of pH on the reaction of monascorubrin and rubropunctatin with amino acids The acidic amino acid Glu and the basic amino acid Arg were selected as representatives to study their reaction characteristics with monascorubrin and rubropunctatin at different pH values. The reaction concentration of monascorubrin and rubropunctatin was prepared at around 5 × 10−4 M, while that of Glu and Arg was prepared at 10−3 M. The different ambient pH values ranging from 2.0 to 10.0 were maintained by sodium hydrogen carbonate-sodium carbonate buffer and citric acid-disodium hydrogen phosphate buffer. The reactions were placed in 96-well microtiter plates. In each well, 100 μL monascorubrin and rubropunctatin solutions were separately mixed with 100 μL Glu and Arg solutions, with 100 μL deionized water as a control. Three replicates were performed for each reaction. The reaction process was monitored by a microplate spectrophotometer as described above. The reactions were performed at room temperature.
3. Results and discussion 3.1. A reliable and effective way to prepare monascorubrin and rubropunctatin Based on the author's knowledge, neither commercially available monascorubrin and rubropunctatin standards nor satisfactory methods for the high-yield and high-purity production of these components existed previously, which limited the study of monascorubrin and rubropunctatin. Therefore, at the beginning of this study, a rapid extraction and separation process for monascorubrin and rubropunctatin was developed. First, an effective extractant system for monascorubrin and rubropunctatin should be screened. Methanol is the most common extractant for recovering total azaphilone pigments from red mold rice or
Step 1: A deionized water and chloroform two-phase liquid-liquid extraction system is used to extract monascorubrin and rubropunctatin from red mold rice. After stratification, the chloroform layer is collected and evaporated by a rotary evaporator. Then, a crude solution of monascorubrin and rubropunctatin is prepared by redissolution of the evaporated extract with chromatographically pure methanol. Step 2: The crude solution is filtered through the membrane and 3
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(Supporting Information section 2), the amination reaction between the acidic amino acid Glu and monascorubrin and rubropunctatin was inhibited in aqueous solution, while the reactivity was much higher under alkaline conditions (Fig. 3). Similarly, the amination reaction between the basic amino acid Arg and monascorubrin and rubropunctatin was active in aqueous solution, while the reactivity was significantly inhibited under acidic conditions. Further experiments also clearly suggested that the lower pH values resulted in the stronger inhibition of the amination (Supporting Information section 3). However, a clear boundary was observed between ambient alkalinity and acidity. Previously, it was speculated that Monascus pigment production was closely related to the isoelectric point of amino acids based on observations of Monascus fermentation [27]. In this study, the in vitro reaction directly showed that the alkaline environment could greatly promote this amination conversion. However, for Monascus fermentation, a suitable pH environment for growth should be considered, and the difference between intracellular and extracellular pH environments should also be noted.
loaded on a semi-preparative HPLC under the optimized conditions suggested above for the purification of monascorubrin and rubropunctatin. The separation efficiency shown in Fig. 2 indicated a clear isolation of monascorubrin 1 and rubropunctatin 2 from ankaflavin 5 and monascin 6, which are usually hard to separate. Step 3: The collected monascorubrin and rubropunctatin fractions are dried under nitrogen flow, and redissolved in chromatographically pure methanol. The stock solutions are sealed and kept at −20 °C. It was observed that monascorubrin and rubropunctatin in the stock solutions remained stable for three days. However, freshly prepared monascorubrin and rubropunctatin are recommended for chemical analysis. 3.2. Reaction of monascorubrin with amino acids At present, based on the structure summary of red Monascus pigments [4], most of these pigments are amino acid derivatives. Our previous studies observed that the amination of monascorubrin and rubropunctatin with amino acids differed greatly by amino acid type [7]. In this study, the amino acids Arg, Asp, Glu, GABA, Lys and Thr were tested for their in vitro reactivity with monascorubrin, the derivatives of which are common types of Monascus red pigments. The details are presented in Supporting Information section 2. The results showed that only Arg and GABA led to obvious color changes from orange to red upon reacting with monascorubrin, and a positive correlation between color and concentration was also observed, but these results were not obviously observed for the other tested amino acids. The results were somewhat surprising, since other amino acid derivatives are commonly present in Monascus fermentation, and this amination is enzyme-independent. It was speculated that Arg and GABA create an alkaline environment in deionized water, which promotes the amination reactions. To support the speculation that the ambient pH rather than amino acid bias affects the reactions, further reactions were monitored as described below.
3.4. Kinetic study of monascorubrin and rubropunctatin with ammonia It is well known that the classical orange pigments monascorubrin 1 and rubropunctatin 2 can directly react with ammonia to form the classical red pigments monascorubramine 3 and rubropunctatamine 4 [4]. In vitro, a very obvious chemical reaction between ammonia and monascorubrin 1 and rubropunctatin 2 was observed, which was used as a positive control for the reactions with amino acids. Therefore, ammonia was chosen to analyze the kinetics of its reaction with monascorubrin and rubropunctatin. First, the reactions at different ammonia concentrations were compared (Fig. 4). As revealed, the reactions under different ammonia concentrations shared a similar trend. Generally, the higher the ammonia concentration is, the more intense the reaction is. Thus, reactions at an ammonia concentration of 2.67 × 10−4 M were selected for kinetics analysis. Based on the characteristics of the chemical reactions, and the abovementioned reaction trend (Fig. 4), the amination of monascorubrin and rubropunctatin may occur via a second-order reaction. That is, the reaction rate (r) is proportional to the substrate monascorubrin/ rubropunctatin, and ammonia concentrations, and is given by equation (1).
3.3. Effect of pH on the amination As indicated above, it was speculated that the pH could affect the amination. To support this speculation, the acidic amino acid Glu and the basic amino acid Arg were selected as representatives to study their reaction characteristics with monascorubrin and rubropunctatin at different pH values. As expected, the results showed that the alkaline environment greatly promoted amination, while the acidic environment significantly inhibited amination (Fig. 3). As indicated
r=
dx = k (a − x )(b − x ) dt
(1)
Fig. 3. The reaction characteristics of Arg and Glu with monascorubrin and rubropunctatin at different pH values. A and B correspond to the reactions with monascorubrin and rubropunctatin respectively. 4
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Fig. 4. The reactions of monascorubrin and rubropunctatin with different concentrations of ammonia. The reactions were performed in methanol at room temperature and monitored by absorbance changes. A. The reactions of monascorubrin with ammonia at different concentrations. B. The reactions of rubropunctatin with ammonia at different concentrations.
Fig. 5. Reaction kinetics analysis of monascorubrin and rubropunctatin with ammonia by second-order reaction. A and B correspond to the reactions of monascorubrin and rubropunctatin, respectively. The initial concentrations of monascorubrin and rubropunctatin were 2.82 × 10−4 M and 2.85 × 10−4 M rea−x spectively. The initial concentration of ammonia was 2.67 × 10−4 M. R2 is the square of the Pearson correlation coefficient between ln b − x and t.
second-order reaction. These observations will not only contribute to our understanding of the chemical properties of monascorubrin and rubropunctatin, but also have significance in guiding the industrial applications of Monascus pigments.
where a, b, k, and x stand for the initial monascorubrin/rubropunctatin concentration, the initial ammonia concentration, the rate constant, and the product monascorubramine/rubropunctamine concentration, respectively. Then, equation (2) is obtained by the integration of equation (1).
ln
a−x a = (a − b)⋅k⋅t + ln b−x b
Conflicts of interest
(2)
a−x ln b − x
and t showed a high linear relationship in both As revealed, monascorubrin and rubropunctatin amination reactions (Fig. 5), which suggests that these aminations are in line with second-order reactions.
The authors declare that they have no conflicts of interest.
Acknowledgements 4. Conclusions This work was supported by the fund of the Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU).
In this study, we developed a reliable and effective method for the preparation of two classical orange Monascus pigments monascorubrin and rubropunctatin on the laboratory scale. The characteristics of the reactions with various amino acids at different pH values indicated that an alkaline environment could greatly promote the amination of rubropunctatin and monascorubrin, while amination was obviously inhibited in an acidic environment. The kinetic study of monascorubrin and rubropunctatin with ammonia suggested that the amination was a
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107629. 5
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