Rubropunctatin

Mannitol improves Monascus pigment biosynthesis with rice bran as a substrate in Monascus purpureus.[Pubmed:38156009]

Front Microbiol. 2023 Dec 13;14:1300461.

To reduce the production cost of Monascus pigments (MPs), the utilization of rice bran (RB), an agricultural waste product, as a substrate in submerged fermentation was conducted in this study. To improve MP production, different nutritional ingredients including mannitol (Man), NH(4)NO(3) (AN), ZnSO(4) (Zn), and optimization (Opti), which was a synthesis of the three above ones, were added in rice bran (RB) medium. The yields of MPs, pigment constituents, and growth and development of Monascus purpureus M9 were investigated in this study. Man had the maximum color value of 3,532 U/g, which was 18.69 times more than that of RB and reached up to 76.65% of the value of rice (Rice) fermentation. Man significantly increased the production of two orange pigments, monascorubrin and Rubropunctatin, of which the yields were 69.49 and 95.36% of the counterpart of Rice. The biomass and colony diameter of Opti presented the maximum value among different groups. AN and RB induced more asexual spore formation, whereas Opti and Man promoted sexual spore production. Comparative transcriptomic analysis showed that different nutritional ingredients led to changes in pigment production, promoting the growth and development of M. purpureus M9 through the regulation of related gene expression. Man and Opti improved MP production by regulating the primary metabolism, including the Embden-Meyerhof pathway (EMP), the pentose phosphate (PP) pathway, the tricarboxylic (TCA) cycle, fatty acid degradation (FAD), fatty acid biosynthesis (FAB), amino acid metabolism (AAM), and fructose and mannose metabolism (FMM), to provide the precursors (acetyl-CoA and malonyl-CoA) for MP biosynthesis. This study presents a low-cost method for increasing MP production and explains the molecular mechanisms of different nutritional ingredients for enhancing MP biosynthesis.

Precursor-directed production of water-soluble red Monascus pigments with high thermal stability via azaphilic addition reaction-based semi-synthesis.[Pubmed:38144809]

Food Chem X. 2023 Oct 10;20:100940.

Red Monascus pigments (MPs) are a large group of polyketides from the fungus Monascus which have been widely used as food colorants. In this study, a variety of red MPs congeners were prepared to explore promising water-soluble candidates for application in liquid food formulations. The results showed that by combining the two-stage, low-pH fermentation strategy with a downstream purification step of fractional crystallization, precursors of red MPs, namely monascorubrin and Rubropunctatin, were obtained with a purity of 91.9%. Then, via the azaphilic addition reaction, 18 types of red MPs congeners carrying different amino acid moieties (MPs-aa) were semi-synthesized. Compared to rubropunctamine and monascorubramine, the water solubility, pH and thermal stability of MPs-aa were improved greatly. MPs-His, MPs-Phe, MPs-Tyr and MPs-Trp were identified to be the most resistant to pasteurization. These findings provide water-soluble red MPs candidates with high thermal stability and an attractive approach for their large scale production.

Rubropunctatin-silver composite nanoliposomes for eradicating Helicobacter pylori in vitro and in vivo.[Pubmed:38043750]

Int J Pharm. 2024 Jan 5;649:123655.

Helicobacter pylori (H. pylori) is a major factor in peptic ulcer disease and gastric cancer, and its infection rate is rising globally. The efficacy of traditional antibiotic treatment is less effective, mainly due to bacterial biofilms and the formation of antibiotic resistance. In addition, H. pylori colonizes the gastrointestinal epithelium covered by mucus layers, the drug must penetrate the double barrier of mucus layer and biofilm to reach the infection site and kill H. pylori. The ethanol injection method was used to synthesize nanoliposomes (EPI/R-AgNPs@RHL/PC) with a mixed lipid layer containing rhamnolipids (RHL) and phosphatidylcholine (PC) as a carrier, loaded with the urease inhibitor epiberberine (EPI) and the antimicrobial agent Rubropunctatin silver nanoparticles (R-AgNPs). EPI/R-AgNPs@RHL/PC had the appropriate size, negative charge, and acid sensitivity to penetrate mucin-rich mucus layers and achieve acid-responsive drug release. In vitro experiments demonstrated that EPI/R-AgNPs@RHL/PC exhibited good antibacterial activity, effectively inhibited urease activity, removed the mature H. pylori biofilm, and inhibited biofilm regeneration. In vivo antibacterial tests showed that EPI/R-AgNPs@RHL/PC exhibited excellent activity in eradicating H. pylori and protecting the mucosa compared to the traditional clinical triple therapy, providing a new idea for the treatment of H. pylori infection.

Lecithin/chitosan nanoparticle drug carrier improves anti-tumor efficacy of Monascus pigment rubropunctatin.[Pubmed:37236571]

Int J Biol Macromol. 2023 Jul 1;242(Pt 3):125058.

Rubropunctatin, a metabolite isolated from the fungi of the genus Monascus, is a natural lead compound applied for the suppression of tumors with good anti-cancer activity. However, its poor aqueous solubility has limited its further clinical development and utilization. Lecithin and chitosan are excellently biocompatible and biodegradable natural materials, which have been approved by the FDA as drug carrier. Here, we report for the first time the construction of a lecithin/chitosan nanoparticle drug carrier of the Monascus pigment Rubropunctatin by electrostatic self-assembly between lecithin and chitosan. The nanoparticles are near-spherical with a size 110-120 nm. They are soluble in water and possess excellent homogenization capacity and dispersibility. Our in vitro drug release assay showed a sustained release of Rubropunctatin. CCK-8 assays revealed that lecithin/chitosan nanoparticles loaded with Rubropunctatin (RCP-NPs) had significantly enhanced cytotoxicity against mouse mammary cancer 4T1 cells. The flow cytometry results revealed that RCP-NPs significantly boosted cellular uptake and apoptosis. The tumor-bearing mice models we developed indicated that RCP-NPs effectively inhibited tumor growth. Our present findings suggest that lecithin/chitosan nanoparticle drug carriers improve the anti-tumor effect of the Monascus pigment Rubropunctatin.

Eco-Friendly Green Synthesis of Rubropunctatin Functionalized Silver Nanoparticles and Evaluation of Antibacterial Activity.[Pubmed:36432337]

Nanomaterials (Basel). 2022 Nov 17;12(22):4052.

In order to solve the problems of Rubropunctatin insoluble in water and its low bioavailability, and explore the synthesis method of green silver nanoparticles, Rubropunctatin was used as reducing agent and blocking agent, Rubropunctatin-functionalized silver nanoparticles (R-AgNPs) were successfully synthesized. The distinctive absorption peak at 410 nm confirmed the formation of R-AgNPs. Zeta potential measurement showed excellent stability of R-AgNPs with negative values of -29.81 +/- 0.37 mV. The results of TEM and XRD showed that the prepared R-AgNPs were round, well dispersed and crystallized with average particle size of 13.54 +/- 0.42 nm. FT-IR and XPS studies show that functional groups are involved in R-AgNPs synthesis. The antibacterial activity of R-AgNPs was compared with commercial silver nanoparticles (AgNPs) by microdilution method. The results showed that R-AgNPs (MIC 7.81 mug/mL) has stronger antibacterial activity than commercial AgNPs. The bacteria morphology was observed by the live and dead (SYTO 9/PI) staining assay and SEM showed that the antibacterial effect of R-AgNPs was caused by the destruction of the bacterial cell membrane. Cytotoxicity of Rubropunctatin-functionalized silver nanoparticles and commercial silver nanoparticles on mouse fibroblast 3T3 cells was assessed by CCK-8 assay. The results showed that the toxicity of Rubropunctatin-functionalized silver nanoparticles to 3T3 cells was lower than that of commercial silver nanoparticles. In summary, synthesis of silver nanoparticles using Rubropunctatin is a green synthesis method, and R-AgNPs is a potential antibacterial agent.

A new endophyte Monascus ruber SRZ112 as an efficient production platform of natural pigments using agro-industrial wastes.[Pubmed:35871189]

Sci Rep. 2022 Jul 23;12(1):12611.

A number of biopigment applications in various industrial sectors are gaining importance due to the growing consumer interest in their natural origin. Thus, this work was conducted to valorize endophytic fungi as an efficient production platform for natural pigments. A promising strain isolated from leaves of Origanum majorana was identified as Monascus ruber SRZ112 produced several types of pigments. The nature of the pigments, mainly rubropunctamine, monascin, ankaflavin, Rubropunctatin, and monascorubrin in the fungal extract was studied by LC/ESI-MS/MS analyses. As a first step towards developing an efficient production of red pigments, the suitability of seven types of agro-industrial waste was evaluated. The highest yield of red pigments was obtained using potato peel moistened with mineral salt broth as a culture medium. To increase yield of red pigments, favourable culture conditions including incubation temperature, incubation period, pH of moistening agent, inoculum concentration, substrate weight and moisture level were evaluated. Additionally, yield of red pigments was intensified after the exposure of M. ruber SRZ112 spores to 1.00 KGy gamma rays. The final yield was improved by a 22.12-fold increase from 23.55 to 3351.87 AU g(-1). The anticancer and antioxidant properties of the pigment's extract from the fungal culture were also studied. The obtained data indicated activity of the extract against human breast cancer cell lines with no significant cytotoxicity against normal cell lines. The extract also showed a free radical scavenging potential. This is the first report, to our knowledge, on the isolation of the endophytic M. ruber SRZ112 strain with the successful production of natural pigments under solid-state fermentation using potato peel as a substrate.

Red yeast rice dietary intervention reduces oxidative stress-related inflammation and improves intestinal microbiota.[Pubmed:35621018]

Food Funct. 2022 Jun 20;13(12):6583-6595.

Inflammation and oxidative stress play key roles in the aging process, while red yeast rice (RYR), a traditional Chinese fermented food, has anti-oxidant and anti-inflammatory effects. To understand the anti-aging function of RYR in vivo, this study established a D-galactose-induced aging mouse model to verify the positive effects of RYR dietary intervention on aging and explore the related underlying mechanism. Eight weeks of RYR dietary intervention was shown to have a significant inhibitory effect on cognitive decline and hippocampal damage. The molecular mechanistic studies showed that the anti-aging effects of RYR were achieved by (i) improving the oxidative stress-related damage (increasing SOD, CAT, and GSH, and reducing MDA), (ii) regulating the NF-kappaB inflammation pathway induced by oxidative stress (decreasing the pro-inflammatory cytokines IL-6, TNF-alpha, IFN-gamma, iNOs, and IL-1beta, increasing the anti-inflammatory cytokine IL-10, and decreasing the expression of the NF-kappaB protein), (iii) slowing down apoptosis caused by oxidative stress (reducing the expression of P21 and P53), (iv) restoring the abundance of Lactobacillus, Lachnospiraceae and Rikenellaceae downregulated by D-galactose, and (v) reducing the abundance of Akkermansia and Helicobacter enriched by D-galactose. Mass spectrometry revealed orange pigments (Rubropunctatin and monascorubrin) as the main antioxidant components in RYR, which might play key roles in aging inhibition. This study provides theoretical support for the wide application of orange pigments as an antioxidant dietary supplement.

Solid-state co-culture fermentation of simulated food waste with filamentous fungi for production of bio-pigments.[Pubmed:35608668]

Appl Microbiol Biotechnol. 2022 Jun;106(11):4029-4039.

The use of waste stream residues as feedstock for material production simultaneously helps reduce dependence on fossil-based resources and to shift toward a circular economy. This study explores the conversion of food waste into valuable chemicals, namely, bio-pigments. Here, a simulated food waste feedstock was converted into pigments via solid-state fermentation with the filamentous fungus Talaromyces albobiverticillius (NRRL 2120). Pigments including monascorubrin, Rubropunctatin, and 7-(2-hydroxyethyl)-monascorubramine were identified as products of the fermentation via ultra-performance liquid chromatography coupled with quadrupole-time-of-flight electrospray ionization mass spectrometry. Pigments were obtained at concentrations of 32.5, 20.9, and 22.4 AU/gram dry substrate for pigments absorbing at 400, 475, and 500 nm, respectively. Pigment production was further enhanced by co-culturing T. albobiverticillius with Trichoderma reesei (NRRL 3652), and ultimately yielded 63.8, 35.6, and 43.6 AU/gds at the same respective wavelengths. This represents the highest reported production of pigments via solid-state fermentation of a non-supplemented waste stream feedstock. KEY POINTS: * Simulated food waste underwent solid-state fermentation via filamentous fungi. * Bio-pigments were obtained from fermentation of the simulated food waste. * Co-culturing multiple fungal species substantially improved pigment production.

Combination of (1)H and (13)C NMR for quantitative analysis of the orange pigments produced by Monascus kaoliang KB9.[Pubmed:34933631]

Nat Prod Res. 2023 Apr;37(8):1406-1409.

Two orange pigments, Rubropunctatin (1) and monascorubrin (2), along with the yellow pigments, monascin (3) and ankaflavin (4), were isolated from M. kaoliang KB9-fermented rice, also known as red yeast rice. The orange pigments exhibit a broad spectrum of biological activities and appeared to be the major components of this fermented rice. In this work, quantitative (1)H NMR (qHNMR) and (13)C NMR experiments were used to determine the amounts of the two orange pigments in a crude extract in which most of the (1)H NMR signals of the two compounds were indistinguishable. The quantitative values obtained by NMR techniques were found to be similar to those obtained by HPLC. Thus, the combined qHNMR with (13)C experiment described in this work could be further developed to quantifying Monascus pigments or other invaluable natural products when qHNMR alone is insufficient for quantitative analysis.

Effect of a Monascus sp. Red Yeast Rice Extract on Germination of Bacterial Spores.[Pubmed:34108955]

Front Microbiol. 2021 May 24;12:686100.

The pink-red color of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant color of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for coloring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, Rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2% v/v. For B. subtilis, total inhibition of spore germination was observed only after addition of 4% v/v RYR extract to the medium containing 1.3% w/w NaCl.

A facile macroporous resin-based method for separation of yellow and orange Monascus pigments.[Pubmed:33936846]

Food Sci Biotechnol. 2021 Mar 8;30(4):545-553.

The yellow Monascus pigments (YMPs) named monascin and ankaflavin and the orange Monascus pigments (OMPs) named Rubropunctatin and monascorubrin are two groups of bioactive components in a mixture state in the Monascus fermented products. In order to separate these two groups of bioactive pigments, a facile macroporous resin-based method was developed. The weak-polar resin CAD-40 was selected from the seven tested macroporous resins as it revealed better properties for the adsorption and desorption of the YMPs and OMPs. Then, CAD-40 resin was used for column-chromatographic separation. After eluted by 4 bed volumes of ethanol, the yellow group (monascin and ankaflavin) and the orange group (Rubropunctatin and monascorubrin) were successfully separated and purified, with an increased content from 49.3% and 44.2% in the crude pigment extract to 85.2% and 83.0% in the final products, respectively. This method would be helpful for the large-scale separation and purification of Monascus pigment products with specific bioactivity.

Ammonium nitrate regulated the color characteristic changes of pigments in Monascus purpureus M9.[Pubmed:33398480]

AMB Express. 2021 Jan 4;11(1):3.

Monascus pigments (MPs) with different color characteristics, produced by submerged fermentation of Monascus purpureus M9, have potential application in food industry. In the present study, the effects and regulatory mechanisms of ammonium nitrate (AN) on the color characteristics of MPs were investigated. The concentration of intracellular pigments was significantly decreased when subjected to AN. The hue and lightness value indicated AN altered the total pigments appearance from original red to orange. The HPLC analysis for six major components of MPs showed that the production of Rubropunctatin or monascorubrin, was significantly reduced to the undetectable level, whereas the yields of monascin, ankaflavin, rubropunctamine and monascorubramine, were apparently increased with AN supplement. To be noted, via real-time quantitative PCR strategy, the expression level of mppG, closely relative to orange pigments biosynthesis, was significantly down-regulated. However, the expression of mppE, involved in yellow pigments pathway, was up-regulated. Moreover, the broth pH value was dropped to 2.5-3.5 in the fermentation process resulted from AN treatment, along with the increased extracellular polysaccharide biosynthesis. Taken together, the change of MPs categories and amounts by AN might be the driving force for the color characteristics variation in M. purpureus M9. The present study provided useful data for producing MPs with different compositions and modified color characteristics.

Effects of mrpigG on Development and Secondary Metabolism of Monascus ruber M7.[Pubmed:32872515]

J Fungi (Basel). 2020 Aug 29;6(3):156.

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and are now used throughout the world via Asian catering. The MP biosynthetic pathway has been well-illustrated, but the functions of a few genes, including mrpigG, in the MP gene cluster are still unclear. In the current study, in order to investigate the function of mrpigG in M. ruber M7, gene deletion (DeltamrpigG), complementation (DeltamrpigG::mrpigG) and overexpression (M7::PtrpC-mrpigG) mutants were successfully obtained. The morphologies and biomasses, as well as the MP and citrinin production, of these mutants were analyzed. The results revealed that the disruption, complementation and overexpression of mrpigG showed no apparent defects in morphology, biomass or citrinin production (except MP production) in DeltamrpigG compared with M. ruber M7. Although the MP profiles of DeltamrpigG and M. ruber M7 were almost the same-with both having four yellow pigments, two orange pigments (OPs) and two red pigments (RPs)-their yields were decreased in DeltamrpigG to a certain extent. Particularly, the content of Rubropunctatin (an OP) and its derivative rubropunctamine (an RP) in DeltamrpigG, both of which have a five-carbon side chain, accounted for 57.7%, and 22.3% of those in M. ruber M7. On the other hand, monascorubrin (an OP) and its derivative monascorubramine (an RP), both of which have a seven-carbon side chain, were increased by 1.15 and 2.55 times, respectively, in DeltamrpigG compared with M. ruber M7. These results suggest that the MrPigG protein may preferentially catalyze the biosynthesis of MPs with a five-carbon side chain.

Preparation and evaluation of a Rubropunctatin-loaded liposome anticancer drug carrier.[Pubmed:35498569]

RSC Adv. 2020 Mar 11;10(17):10352-10360.

Rubropunctatin is a naturally occurring constituent of polyketide compounds that has great potential in the development of cancer-assisted chemotherapy. However, it has certain shortcomings such as water insolubility and photo instability that limit its clinical application. In this study, we constructed a Rubropunctatin-loaded liposome (R-Liposome) anticancer drug carrier for the first time. The results indicate that R-Liposome is water soluble, has spherical morphology, great homogeneity and dispersibility with high encapsulation efficiency (EE%, 90 +/- 3.5%) and loading rate (LR%, 5.60 +/- 2.5%) values. Moreover, the carrier improves the photostability, storage and pH stabilities of Rubropunctatin. The R-Liposome also prolongs the release of Rubropunctatin, enhances the anticancer activity of Rubropunctatin and encourages the mechanism of Rubropunctatin to promote apoptosis. Therefore, liposomal nanoparticles have great potential as drug delivery vehicles of Rubropunctatin for cancer treatment.

Transfigured Morphology and Ameliorated Production of Six Monascus Pigments by Acetate Species Supplementation in Monascus ruber M7.[Pubmed:31936171]

Microorganisms. 2020 Jan 7;8(1):81.

Monascus species have been used for the production of many industrially and medically important metabolites, most of which are polyketides produced by the action of polyketide synthases that use acetyl-CoA and malonyl-CoA as precursors, and some of them are derived from acetate. In this study the effects of acetic acid, and two kinds of acetates, sodium acetate and ammonium acetate at different concentrations (0.1%, 0.25% and 0.5%) on the morphologies, biomasses, and six major Monascus pigments (MPs) of M. ruber M7 were investigated when M7 strain was cultured on potato dextrose agar (PDA) at 28 degrees C for 4, 8, 12 days. The results showed that all of the added acetate species significantly affected eight above-mentioned parameters. In regard to morphologies, generally the colonies transformed from a big orange fleecy ones to a small compact reddish ones, or a tightly-packed orange ones without dispersed mycelia with the increase of additives concentration. About the biomass, addition of ammonium acetate at 0.1% increased the biomass of M. ruber M7. With respect to six MPs, all acetate species can enhance pigment production, and ammonium acetate has the most significant impacts. Production of monascin and ankaflavin had the highest increase of 11.7-fold and 14.2-fold in extracellular contents at the 8th day when 0.1% ammonium acetate was supplemented into PDA. Intracellular Rubropunctatin and monascorubrin contents gained 9.6 and 6.46-fold at the 8th day, when 0.1% ammonium acetate was added into PDA. And the extracellular contents of rubropunctamine and monascorubramine were raised by 1865 and 4100-fold at the 4th day when M7 grew on PDA with 0.5% ammonium acetate.