Combination of 1H and 13C NMR for quantitative analysis of the orange pigments produced by Monascus kaoliang KB9

Abstract 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 1H NMR (qHNMR) and 13C NMR experiments were used to determine the amounts of the two orange pigments in a crude extract in which most of the 1H 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 13C 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. Graphical Abstract


Introduction
Fungi of the genus Monascus are widely used in the Asian food industry where rice is typically used as a carbohydrate source for fungal growth (Wang and Lin 2007). Fermentation with Monascus spp. usually imparts a red colour and the production is known as red yeast rice (RYR). Three pairs of major azaphilone pigments produced by Monascus spp. can be categorised into three colour groups, namely yellow (monascin and ankaflavin), orange (rubropunctatin and monascorubrin) and red (rubropunctamine and monascorubramine). All these pigments exhibit a broad spectrum of biological activities, especially the orange rubropunctatin, which displays potent anticancer activity (Zheng et al. 2016). Thus, quantitative analysis of the orange pigments from Monascus-fermented rice could be beneficial to the pharmaceutical and food industries. To the best of our knowledge, no quantitative analysis has been reported for this kind of application in the orange pigments by using NMR methods. Most analyses have been based on liquid chromatography or mass spectrometry or a combination of both techniques (Miyake et al. 2008;Huang et al. 2016;Li et al. 2019). By the other side, due to its quick data acquisition and sample preparation, quantitative NMR analysis has been used for quantification of bioactive compounds in plants (Al-Tamimi et al. 2019;Ongaro et al. 2020) and this could be an alternative method for quantifying the Monascus pigments.
Here, we report the high production of orange pigments from Monascus kaoliang KB9-fermented rice (MKR) and this is the first time that these orange pigments have been isolated from this Monascus species. The qHNMR and 13 C NMR methods were also performed to quantifying these orange pigments for a future use in the food industries.

Results and discussion
2.1. Isolation and structure determination of orange and yellow pigments MKR was successively extracted with hexane and dichloromethane using a Soxhlet extractor. HPLC analysis of the crude dichloromethane extract exhibited two compounds in large amounts ( Figure S1), which further NMR analysis confirmed those are the orange pigments, rubropunctatin (1) and monascorubrin (2) (Figure 1 and S2). Additionally, compounds 3 and 4 were identified as the yellow pigments monascin and ankaflavin, respectively (Figure 1 and S1-S2). These yellow pigments were also found when M. kaoliang KB9 was grown in submerged culture (manuscript under review).

Quantitative analysis of orange pigments by NMR and HPLC
The high content of orange pigments could make MKR a potential source of food supplements, so quantification of these bioactive components would be essential for quality control. The qHNMR method, using 2,3,5,6-tetrachloronitrobenzene (TCNB) as internal standard ( Figure S3), was selected due to its fast processes of sample preparation and data collection (Sakarat et al. 2021). However, the 1 H NMR spectrum alone was found unsuitable for separately quantifying the two compounds 1 and 2 as most signals were identical ( Figure S2). However, proton-decoupled 13 C NMR with short delays between pulses has been used to determine ratios for a range of compounds (Otte et al. 2014), and this technique was applied successfully to determine the mole ratio of compounds 1 and 2 ( Figure S4). The compound ratio obtained was further converted to the amounts of compounds 1 and 2. The amounts of orange pigments quantified by NMR are similar to those obtained from HPLC (Table 1 and Figure S5-6).

Experimental
Experimental information is provided in supplementary material.

Conclusions
Four Monascus pigments (compounds 1-4) were isolated and identified in MKR. Orange pigments (compounds 1-2) were the major components, and these were quantified by combination of 1 H and 13 C NMR experiments. Our ability to quantify Monascus orange pigments in this work could provide a platform for quantifying other Monascus pigments and invaluable natural products with indistinguishable 1 H NMR signals. This will be of benefit for both the pharmaceutical and food industries.