Ankaflavinime,an Unprecedented Polyketide From Red Yeast Rice

Abstract

Objective

The objective of this study was the isolation, structural characterization, and anti-tumor activity evaluation of a new compound isolated from Red Yeast Rice (RYR).

Method

The dichloromethane extract of RYR was separated by silica gel column chromatography using petroleum ether/acetone as eluent, affording four fractions. Fraction C was further purified by preparative high-performance liquid chromatography (HPLC) to afford ankaflavinime (1). The structure of 1 was elucidated by comprehensive spectral analysis, including 1D NMR, 2D NMR, and HRESIMS. Furthermore, the cytotoxicity of 1 was evaluated using the CCK-8 assay.

Results

A new polyketide compound, ankaflavinime (1), was isolated from RYR. The compound possessed an additional chiral center at C-15 compared with the known compound monasphilone A (2). Compound 1 exhibited potential anti-tumor activity against A549 and MDA-MB-231 cell lines.

Conclusion

In this study, a new polyketide compound, ankaflavinime (1), was isolated from Red Yeast Rice, and its relative configuration was characterized. The new compound showed promising anti-tumor activity against A549 and MDA-MB-231 cell lines, with IC₅₀ values of 2.5 μM and 5.0 μM, respectively.

Graphical Abstract

Keywords

ankaflavinime red yeast rice polyketide spectroscopic analysis

Introduction

Red Yeast Rice (RYR) is a traditional fermented product derived from rice with the yeast Monascus purpureus and other related molds.¹ It is characterized by a distinctive red color that develops during the fermentation process. For hundreds of years, RYR has been used as a food preservative, a nutraceutical in East Asian cuisines, and an ingredient in traditional Chinese medicine.^2-4

Modern pharmacological studies have demonstrated that RYR possesses diverse pharmacological properties, including improved lipid metabolism, reduced cholesterol levels, lowered blood pressure and blood glucose, as well as anti-cancer, anti-inflammatory, and neuroprotective effects against age-related cognitive decline.^5-9 Among its bioactive constituents, monacolin K has been the most extensively studied, as it is structurally and chemically identical to the clinically used cholesterol-lowering drug lovastatin.¹⁰ Nowadays, RYR is widely applied as a dietary supplement for statin-intolerant individuals pursuing lifestyle interventions.¹¹ However, other potentially active constituents in RYR, such as polyketides, flavonoids, lignans, and terpenoids, remain underexplored. Therefore, further investigation into the bioactive components of this traditionally used and widely available product is of great significance.

To date, more than ten polyketide compounds have been isolated from RYR, including monasphilone A (2), monasphilone B (3), monascin (4), ankaflavin (5), rubropunctatin (6), monascrubrin (7), rubropunctamine (8), monascrubramine (9), rubropunctin (10), monarubrin (11), monafluore A (12), and monafluore B (13)^5,12,13 (Figure 1). Nevertheless, many bioactive constituents, particularly trace components, remain to be fully explored. In addition, the absolute configurations of certain compounds, such as 10 and 11, have yet to be established. Given the current study of RYR’s active ingredients, numerous components exhibit considerable potential as lead compounds for drug development.

Figure 1.

The structures of representative compounds isolated from RYR

To further explore the bioactive constituents and expand the chemical diversity of RYR molecular library, this study aimed to isolate and structurally characterize novel compounds from RYR. Ankaflavinime (1), a novel polyketide, was isolated from the dichloromethane crude extract of RYR via column chromatography. Its structure was subsequently elucidated, and its anti-tumor activity against A549 and MDA-MB-231 cell lines was evaluated.

Materials and Methods

Plant Material

The RYR was purchased from Fujian Province, China, in February 2019. The specie was identified by Professor Y. Chen of Leshan Normal University. A voucher specimen (no. 201903-1) was deposited in the herbarium of Leshan Normal University.

General Experimental Procedures

Silica gel for column chromatography (CC) was purchased from Qingdao Marine Chemical, Inc. (Qingdao, China). TLC was performed on silica gel plates (Qingdao Marine Chemical, Inc.). IR Spectrum was recorded on a Nicolet FT-IR 200S spectrometer. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. 1D and 2D NMR spectra were acquired on a Varian Unity-INOVA-400 instrument. HR-ESI-MS data were obtained on a Waters Aqcquity UPLC/Q-TOF micro mass spectrometer.

Extraction and Isolation

RYR (50 kg) sourced from Fujian Province was pulverized and soaked three times with CH₂Cl₂ (80 L). Then reflux extracted with CH₂Cl₂ at 40 °C for three hours. After filtrating, the residue repeated the above operation two more times. The filtrates were combined and concentrated under reduced pressure to give the oily crude extract (1.22 kg).¹⁴ The crude extract was then subsequently to column chromatographed over silica gel, eluted with a gradient of petroleum ether-acetone (10:1 → 2:1), affording four fractions, including Fr. A (600 g, 10:1 and 9:1, v/v), Fr. B (319 g, 8:1 and 7:1, v/v), Fr. C (26 g, 6:1, 5:1, and 4:1, v/v) and Fr. D (28 g, 3:1 and 2:1, v/v). Fr. C was further purified by preparative high-performance liquid chromatography (HPLC) with a mobile phase of CH₃CN - H₂O (30 %:70 % → 90 %:10 %, v/v) to afford ankaflavinime (1.56 mg).

Cytotoxicity Assay

Cells viability assay was tested on human lung carcinoma cells A549 and human breast cancer cells MDA-MB-231 using CCK-8 kit.^15,16 The compound was screened at six concentrations (0.5, 1, 2, 4, 8 and 16 μM) against two cell lines. Camptothecin (CPT) was used as positive control. The A549 and MDA-MB-231 cell lines used in this study were purchased from Procell (China, Wuhan), that stored in CCL-185 ATCC (no. CL-0016) and CRL-12532 ATCC (no. CL-0150B), respectively.

Statistical Analysis

All bioassays were expressed as mean ± standard deviation. Statistical analysis was performed using Excel software.

Results

Ankaflavinime (1) was obtained as a yellow solid with

{[α]}_{D}^{25}

+ 210 (c = 1.0, CHCl₃). The IR (KBr) spectrum showed absorption bands corresponding to hydroxyl (3476 cm^-1), carbonyl (1682 cm^-1, 1708 cm^-1), and double bond (1646 cm^-1, 1536 cm^-1) functionalities. The molecular formula of 1 was determined as C₂₄H₃₄O₆ based on HRESIMS at m/z 441.2250 [M + Na] ⁺ (calcd for 441.2248) (Figure S1). The ¹H NMR and ¹³C NMR data of compound 1 (Table 1 and Figure S2 – S4) revealed the presence of one tertiary methyl group (δ_H 1.22, 3 H, s; δ_C 20.3), two ketone carbonyl groups (δ_C 204.8 and δ_C 197.2), an ester carbonyl group (δ_C 169.3), a quaternary carbon (δ_C 74.5), three pairs of carbon-carbon double bonds (δ_C 160.7, 152.4, 135.1, 124.7, 112.7, and 103.4; δ_H 6.54 – 6.40, 1H, m; 5.87, 1H, d, J = 15.2 Hz; 5.19, 1 H, s), and three olefinic protons, including a singlet and a trans-olefinic group (J = 15.2 Hz). Further analysis of ¹H-¹H COSY and 2D NMR spectra (Figure S5 – S7) indicated three key structural fragments: i) The octanoyl moiety: The octanoyl moiety was identified by a contiguous spin system from H₃-23 to H₂-17, supported by the characteristic proton signals at δ_H 0.88 (3H, t, J = 7.0 Hz), 1.33 – 1.23 (8H, m), 1.62 – 1.53 (2 H, m), and 2.62 – 2.54 (2 H, m). HMBC correlations from H-15 (δ_H 3.84) to C-16 (δ_C 204.8) and C-17 (δ_C 43.5) confirmed that the octanoyl group was connected to C-15 (δ_C 52.8). ii) The fused bicyclic core: The fused bicyclic core, featuring an α, β – unsaturated ketone and an enol ether motif, was established by NMR spectra (Figure 2 and S5 – S7). The protons H₂-1 (δ_H 4.74 and δ_H 5.00, each 1H) showed correlations to C-5 (δ_C 152.4), C-3 (δ_C 160.7), and C-9 (δ_C 197.2). Furthermore, the olefinic proton H-4 (δ_H 5.19) correlated to C-6 (δ_C 30.7), and H₂-6 (δ_H 2.54 – 2.43 and 2.31 – 2.20, each 1H) displayed a key correlation to the olefinic carbon C-10 (δ_C 112.7), confirming the ring connectivity. The connectivity between the fused bicyclic core and the octanoyl chain was further confirmed by HMBC correlations. The HMBC cross-peak from the H₂-6 to C-15 and C-16, and from H-15 (δ_H 3.84, d, J = 9.2 Hz, 1H) to both C-16 and C-17, verified the linkage between the bicyclic unit and the octanoyl moiety via C-15 to the ketone carbonyl C-16 of the octanoyl chain. iii) The allyl moiety: The propenyl substituent was identified by a ¹H-¹H COSY spin system spanning H₃-13, H-12, and H-11, with characteristic proton signals at δ_H 1.86 (3H, d, J = 6.8 Hz, H₃-13), 6.54 – 6.40 (1H, m, H-12), and 5.87 (1H, d, J = 15.2 Hz, H-11). The large coupling constant of H-11 (J = 15.2 Hz) indicated that it was a trans-propenyl moiety. The HMBC correlation from H-12 to C-3 (δ_C 160.7) suggested that the propenyl group was connected at C-3. Moreover, the NMR data indicated the compound 1 possessed the same carbon skeleton as the known compound monasphilone A (2) (Figure 2 and Table 1), except for an additional methoxycarbonyl group (δ_H 3.70, s, 3H; δ_C 169.3 and δ_C 59.2), which was attached to C-15 as confirmed by HMBC correlation from H-15 to the ester carbonyl carbon at δ_C 169.3. Therefore, the planar structure of compound 1 was elucidated as shown in Figure 2.

Table 1.

¹H and ¹³C NMR Data of Ankaflavinime (1) ^a

No.	1		2 ^{b, 12}
No.	δ_H (J in Hz)	δ _C	δ_H (J in Hz)	δ _C
1	5.00, d (12.4)	64.0	4.71, d (12.4)	64.3
1	4.75, d (12.4)	64.0	4.88, d (12.4)	64.3
3	–	160.7	–	160.3
4	5.19, s	103.4	5.41, s	104.3
5	–	152.4	–	152.4
6	2.54 – 2.43, m	30.7	2.21, m	32.9
6	2.31 – 2.20, m	30.7	2.49, m	32.9
7	3.04 – 2.91, m	43.8	2.54, m	40.3
8	–	74.5	–	74.8
9	–	197.2	–	198.6
10	–	112.7	–	114.2
11	5.87, d (15.2)	124.7	6.01, d (15.2)	125.7
12	6.54 – 6.40, m	135.1	6.39, dq (15.2, 7.2)	134.1
13	1.86, d (6.8)	18.6	1.82, d (7.2)	18.3
14	1.22, s	20.3	1.15, s	20.0
15	3.84, d (9.2)	52.8	2.47, m	42.4
15	3.84, d (9.2)	52.8	2.91, d (15.2)	42.4
16	–	204.8	–	209.8
17	2.62 – 2.54, m	43.5	2.49, m	43.4
18	1.62 – 1.53, m	23.6	1.54, m	24.4
19	1.33 – 1.23, m	29.2	1.27, m	29.9
20	1.33 – 1.23, m	29.0	1.27, m	29.9
21	1.33 – 1.23, m	31.8	1.27, m	32.4
22	1.33 – 1.23, m	22.7	1.27, m	23.2
23	0.88, t (6.4)	14.2	0.87, t (6.8)	14.2
1′	–	169.3	–	–
2′	3.70, s	59.2	–	–

^aCDCl₃; ¹H NMR (400 MHz); ¹³C NMR (100 MHz).

^bThe data sourced from ref. 12.

Figure 2.

(A) Structures of ankaflavinime (1) and monasphilone A (2). (B) The¹ H –¹ H correlations and HMBC correlations of 1

Monascin (4) and ankaflavin (5) are two of the most abundant constituents identified in RYR. A possible biosynthetic pathway of ankaflavinime (1) was proposed as shown in Figure 3. Compound 5 was proposed to serve as the biosynthetic precursor of compound 1. Initially, compound 5 undergoes hydrolysis, resulting in the opening of the lactone ring. Subsequently, compound 1 is formed through a methylation reaction. From a biosynthetic perspective, the stereochemistry configurations of the three chiral centers at C-7, C-8 and C-15 in 1 and 5 would remain unchanged throughout this process. The stereochemistry of 1 was established by NOE and coupling constant analysis. In the 1D NOE experiment, irradiation of H-7 resulted in no observable enhancement of H-14, indicating a trans configuration between H-7 and H-14 in compound 1. Furthermore, the doublet signal of H-15 at δ_H 3.84 (J = 9.2 Hz) showed a similar coupling constant comparable to that of the known compound monascin (4) (H-14 at δ_H 4.44, d, J = 10.2 Hz). The similar coupling constant suggests similar dihedral angles, supporting the assignment of the same relative configuration at C-15 in compound 1 and compound 4. Therefore, the relative configuration of 1 was determined as shown in Figure 2.

Figure 3.

Plausible biosynthetic pathway of 1

Moreover, the anti - tumor activity of compound 1 was evaluated using CCK-8 assay. Compound 1 exhibited potential activity against A549 and MDA-MB-231 cell lines, with IC₅₀ value of 2.5 μM and 5.0 μM, respectively (Table 2).

Table 2.

Anti - Tumor Activity of Compound 1 by CCK-8

Compound	IC₅₀ (μM) ^a
Compound	A549	MDA – MB - 231
1	2.5 ± 1.23	5.0 ± 2.19
Camptothecin ^b	0.1 ± 0.75	0.2 ± 0.38

^aThe data were repeated at three times in independent experiment, and presented as the mean ± SD (n = 3).

^bCamptothecin was used as the positive control.

Discussion

In this study, a new polyketide compound, ankaflavinime (1), was isolated from RYR. The structure of compound 1 was elucidated by comprehensive spectroscopic analyses, including 1D and 2D NMR techniques and HREMIMS. Although X-ray crystallographic data is lacking, a plausible biosynthetic pathway for ankaflavinime (1) is proposed, in which ankaflavin (5) serves as the biosynthetic precursor.^17,18 The stereochemical configurations at C-7, C-8, and C-15 would remain unchanged throughout the biosynthesis transformation. Moreover, the similar coupling constant of H-15 observed on both 1 and the known compound monascin (4) supported the relative configuration of C-15 in 1.^19,20 On the basis of the above evidences, the relative configuration of compound 1 was determined in the present study. However, the absolute configuration requires future confirmation by X-ray or ECD.²¹ The anti - tumor activity of compound 1 was evaluated using the CCK-8 assay. Compound 1 exhibited potential activity against A549 and MDA-MB-231 cell lines. Due to the trace amount of compound 1 obtained, only a preliminary bioactivity was conducted. Further isolation, in-depth mechanism study, and confirmation of absolute configuration are warranted.

Conclusion

In this study, a new polyketide compound, ankaflavinime (1), was identified from RYR. Its relative configuration was established by spectroscopic analysis (1D NMR, 2D NMR, and HREMIMS) and possible biosynthetic pathway, whereas its absolute configuration needs further confirmation. This work expands the chemical component library and enhances the molecular diversity of RYR. Notably, the new compound exhibited promising anti - tumor activity.

Supplemental Material

Supplemental Material - Ankaflavinime, an Unprecedented Polyketide From Red Yeast Rice

Supplemental Material for Ankaflavinime, an Unprecedented Polyketide From Red Yeast Rice by Fang-Tao Xiang, Yi Tian, Qiu-Shan Huang, Shu-Hua Li, Feng-Zheng Chen in Natural Product Communications

Footnotes

Acknowledgements

This research was supported by the Solid-state Fermentation Resource Utilization Key Laboratory of Sichuan Province(No. 2018GTJ011).

ORCID iD

Feng-Zheng Chen

Ethical Considerations

Ethical Approval is not applicable for this article.

Consent to Participate

There are no human subjects in this article and informed consent is not applicable.

Author contributions

CRediT: Fang-Tao Xiang: Data curation, investigation and writing-original draft;

Yi Tian: Methodology & investigation;

Qiu-Shan Huang: Writing-review & edition;

Shu-Hua Li: Writing-review & edition;

Feng-Zheng Chen: Conceptualization, Funding acquisition, Project administration.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Solid-state Fermentation Resource Utilization Key Laboratory of Sichuan Province(No. 2018GTJ011)

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Supplemental Material

Supplemental material for this article is available online.

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