Abstract
Four novel carotenoid derivatives, rehmannins A–D (1–4), were isolated from the fresh roots of the traditional Chinese medicinal plant Rehmannia glutinosa. The structures of these new compounds were determined by extensive spectroscopic methods (high-resolution mass spectrometry, NMR, and electronic circular dichroism) and X-ray diffraction analysis. Rehmannins B–D (2–4) possess a novel tricyclic ring system bearing five contiguous stereocenters on the heterocyclic core together with a unique amide group on the long chain, rendering them attractive targets for total synthesis. Furthermore, plausible biosynthetic routes for these metabolites are suggested. Compound 3 exhibited the inhibition of NO secretion in LPS-induced RAW264.7 cells.
Rehmannia glutinosa, a valuable medicinal herb in Traditional Chinese Medicine, is widely used in the treatment of anemia, liver yin deficiency, and renal failure. First documented more than two thousand years ago in Shennong’s Classic of Materia Medica, it is classified as a Top-Grade herb, reflecting its safety for long-term or dietary consumption. The fresh root of R. glutinosa (Fresh Rehmannia) is used in medicine for its traditional heat-clearing and blood-cooling effects, which might correspond to anti-inflammatory activities. The Chinese name, which means “yellow earth”, is derived from the distinct yellow hue of its roots. Histological examination reveals that yellow-orange pigments are predominantly localized in the epidermis and phloem tissues (Figure ), indicating the presence of specific chromogenic compounds. Although more than several hundred secondary metabolitessuch as iridoids, ionones, and phenylethanoidshave been isolated from this plant, the pigments responsible for its characteristic color have remained unidentified. This suggests the potential presence of other carotenoid-derived metabolites, such as C27/C30 intermediates, which have not yet been identified. To address this gap, we focused on the pigmented tissues of R. glutinosa roots and successfully isolated four novel pigmented carotenoid derivatives, designated rehmannins A–D (1–4, C30), along with an ionone derivative, 5,6-dihydroxy-β-ionone (5, C13) (Figure ). This work reports on their isolation, structural elucidation, proposed biogenetic pathway, and bioactive assessment of these new pigmental molecules.
1.
Histological structure of R. glutinosa roots and biosynthetic analysis of ionones.
2.
Rehmannins A–D (1–4) and an ionone (5).
The molecular formula of rehmannin A (1) was established as C32H45NO4 by HR-ESI-MS (m/z 508.3435 [M + H]+, calcd 508.3427), corresponding to 11 degrees of unsaturation. Analysis of the NMR data disclosed the presence of seven methyls (δC 24.6, 24.6, 30.0, 12.9, 12.8, 12.8, and 13.2), five methylenes (δC 39.9, 19.4, 44.0, 43.5, and 62.0), one quaternary carbon (δC 47.0), 16 olefinic carbons (four quaternary alkenes at δC 135.3, 137.7, 137.5, and 131.4), three carbonyls (δC 207.8, 203.3, and 169.7), and one amino group. Six proton spin systems, including C-2–C-3–C-4, C-7–C-8, C-10–C-11–C-12, C-14–C-15–C-16–C-17, and −NH–C-1′–C-2′, were identified from 1H–1H COSY correlations. The connectivity of these units was resolved using key HMBC correlations: from 27-Me to C-8/C-9/C-10; from 28-Me to C-12/C-13/C-14; from 29-Me to C-17/C-18/C-19; and from 30-Me to C-21/C-22/C-23, confirming the linkages around C-9, C-13, C-18, and C-22, respectively. Additional HMBC correlations from 24-Me/25-Me (δH 1.12) to C-1 (δC 47.0)/C-2 (δC 39.9)/C-6 (δC 203.3), from 26-Me (δH 2.05) to C-4 (δC 44.0)/C-5 (δC 207.8), from H-7 (δH 6.74) to C-1 (δC 47.0)/C-6 (δC 203.3), and from H-1′ (δH 3.39) to C-23 (δC 169.7) completed the planar assembly as shown in Figure . The geometry of all double bonds was assigned as the trans-configuration based on the X-ray crystallographic analysis of 1 (Figure ).
3.
Key HMBC and ROESY correlations of 1/2 and X-ray crystal structure of 1 with thermal ellipsoids set at the 50% probability level (Diamond Software 4.6).
Rehmannin B (2) was assigned the molecular formula C32H45NO5 based on HR-ESI-MS (m/z 524.3386 [M + H]+, calcd 524.3376), indicating 11 degrees of unsaturation, the same as in compound 1. The HMQC spectrum revealed 14 olefinic carbon signals and one carbonyl signal, suggesting the presence of three rings in the structure. NMR analysis (Table ) identified a structural fragment from C-9 to C-23-NH-C-1′-C-2′ identical to that in compound 1. Additional spin systems for C-2–C-3–C-4 and C-7–C-8 were established by 1H–1H COSY correlations. Key HMBC correlations from 24-Me/25-Me (δH 0.98/1.04) to C-1 (δC 37.1)/C-2 (δC 33.2)/C-6 (δC 83.0), from 26-Me (δH 1.23) to C-4 (δC 83.8)/C-5 (δC 83.8)/C-6 (δC 83.0), and from 6-OH (δH 5.02) to C-1 (δC 37.1)/C-5 (δC 83.8)/C-6 (δC 83.0)/C-7 (δC 80.7) supported the formation of a cyclohexane ring substituted with C-24/C-25 at C-1, C-26 at C-5, and a hydroxyl group at C-6 (Figure ). An ether linkage between C-4 and C-7 was indicated by the HMBC correlation from H-4 to C-7, while the connection between C-5 and C-8 via an oxygen atom was supported by the correlation from H-8 to C-5, which was further confirmed by a long-range HMBC correlation from Me-26 (δH 1.23) to C-8 (δC 82.8). Based on these observations, a planar structure was established, revealing a unique tricyclic heterocyclic system (Figure ).
1. 1H (500 MHz) and 13C NMR (125 MHz) Data of 1 (Acetone-d 6) and 2 (DMSO-d 6).
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1
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2
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| Pos. | δH, Mult. (J in Hz) | δC, Type | δH, Mult. (J in Hz) | δC, Type |
| 1 | 47.0, C | 37.1, C | ||
| 2 | 1.56, m | 39.9, CH2 | 1.19, m | 33.2, CH2 |
| 1.73, m | ||||
| 3 | 1.41, m | 19.4, CH2 | 1.54, m | 25.5, CH2 |
| 4 | 2.42, m | 44.0, CH2 | 3.93, d (5.0) | 83.8, CH |
| 5 | 207.8, C | 83.8, C | ||
| 6 | 203.3, C | 83.0, C | ||
| 7 | 6.74, d (15.0) | 120.6, CH | 4.07, s | 80.7, CH |
| 8 | 7.32, d (15.0) | 147.2, CH | 4.57, s | 82.8, CH |
| 9 | 135.3, C | 137.3, C | ||
| 10 | 6.65, d (11.5) | 140.8, CH | 6.33, d (11.5) | 124.3, CH |
| 11 | 6.80, dd (11.5, 15.0) | 125.8, CH | 6.59, dd (11.5, 15.0) | 125.2, CH |
| 12 | 6.64, d (15.0) | 142.6, CH | 6.32, d (15.0) | 135.6, CH |
| 13 | 137.7, C | 137.1, C | ||
| 14 | 6.47, d (11.5) | 135.7, CH | 6.30, d (11.5) | 131.4, CH |
| 15 | 6.83 | 132.2, CH | 6.78, dd (11.5, 15.0) | 131.6, CH |
| 16 | 6.84 | 132.2, CH | 6.69, dd (11.5, 15.0) | 129.5, CH |
| 17 | 6.46, d (11.5) | 135.3, CH | 6.43, d (11.5) | 134.5, CH |
| 18 | 137.5, C | 135.3, C | ||
| 19 | 6.61, d (15.0) | 142.4, CH | 6.59, d (15.0) | 141.5, CH |
| 20 | 6.68, dd (10.5, 15.0) | 125.0, CH | 6.56, d (15.0, 9.5) | 123.7, CH |
| 21 | 7.06, d (10.5) | 134.3, CH | 6.95, d (9.5) | 133.1, CH |
| 22 | 131.4, C | 130.2, C | ||
| 23 | 169.7, C | 168.2, C | ||
| 24 | 1.12, s | 24.6, CH3 | 0.98, s | 24.3, CH3 |
| 25 | 1.12, s | 24.6, CH3 | 1.04, s | 28.1, CH3 |
| 26 | 2.05, s | 30.0, CH3 | 1.23, s | 13.3, CH3 |
| 27 | 2.02, s | 12.9, CH3 | 1.73, s | 14.5, CH3 |
| 28 | 2.02, s | 12.8, CH3 | 1.94, s | 12.6, CH3 |
| 29 | 2.01, s | 12.8, CH3 | 1.94, s | 12.7, CH3 |
| 30 | 2.01, s | 13.2, CH3 | 1.93, s | 13.1, CH3 |
| 1′ | 3.39, m | 43.5, CH2 | 3.20, m | 42.1, CH2 |
| 2′ | 3.61, m | 62.0, CH2 | 3.43, m | 59.8, CH2 |
| 6-OH | 5.02, s | |||
| 2′-OH | 4.01, br s | 4.67, t (5.5) | ||
| -NH- | 7.25, br s | 7.77, t (5.5) | ||
Overlapped.
The relative configuration of 2 was deduced from the ROESY spectrum. The observed correlations of 24-Me (δH 0.98)/6-OH (δH 5.02)/H-8 (δH 4.57) and 24-Me (δH 0.98)/26-Me (δH 1.23)/H-4 (δH 3.93)/H-10 (δH 6.33) collectively suggested a β-orientation for H-4, 26-Me, 6-OH, and 24-Me. Meanwhile, the ROESY correlations between H-7 (δH 4.07) and 25-Me (δH 1.04)/27-Me (δH 1.73) indicated an α-orientation for these groups on the tricyclic core (Figure ). The absolute configuration of 2 was determined by the comparison of the ECD spectrum recorded in MeOH and the DT-DFT-calculated spectrum of 2 at the cam-B3LYP/6-311++G(2d,p) level. , The calculated ECD spectrum of 2 matched well with the experimental spectrum (Figure ), which indicated the absolute configuration to be 4R, 5R, 6S, 7R, and 8S with the same stereochemistry of 5,6-dihydroxy-β-ionone (5) isolated from the same plant. Although various methods were employed to grow X-ray crystals of compound (2), several attempts were unsuccessful due to its instability, which caused it to readily degrade into different metabolites.
4.
Experimental and calculated ECD spectra of 2.
Rehmannins C and D (3 and 4) were determined to share the same molecular formula as rehmannin B (2) based on HR-ESI-MS data. Their NMR spectra were nearly identical to those of 2, with the only significant differences observed in the chemical shifts of certain olefinic protons, suggesting that they were geometric isomers. The Z-configuration of the C-13/C-14 double bond in 3 was established based on the ROESY correlation between H-28 and H-14 (Figure S6 and Figure S26), supported by isomerization shifts (Δδ cis–trans ) (Figure S5 and Table S6) and the characteristic chemical shift of C-28 (δC 20.5). Similarly, the Z-geometry of the C-17/C-18 double bond in 4 was assigned from the ROESY correlations of H-29/H-17 and H-16/H-19 (Figure S6 and Figure S34), alongside analogous isomerization shifts (Δδ cis–trans ) and the chemical shift of C-29 (δC 20.2). The absolute configurations of 3 and 4 were determined as 4R, 5R, 6S, 7R, and 8S, identical to 2 by ECD spectra analysis (Figures S27 and S35). Compound 5 was identified as 5,6-dihydroxy-β-ionone by comparison of its MS and NMR spectra with published data (Figures S36–S38). Structural assignments were made with additional information from 1H–1H COSY, HSQC, ROESY, and HMBC experiments in the General Methods of the Experimental Section (Supporting Information).
Based on the structural features of 1–5 and colorful characteristics of rehmannins A–D (1–4), it suggested that these compounds might be originated from carotenoid biosynthesis. The possible biosynthetic pathway of rehmannins A–D (1–4) and ionone (5) was proposed (Scheme ). The initial substrate IPP/DMAPP first formed the β-carotene through the carotenoid biosynthetic pathway. Then, β-carotene oxidated through pathway A produced ionone with ten carbons (C10, such as rehmapicrogenin reported in fresh R. glutinosa) and carotenoid derivatives with 30 carbons (C30, rehmannins A–D, 1–4 in this report), which was further modified via a series of different postmodifications including oxidation, amination, dehydration, and cyclization to shape the pigmental molecules (1–4). Pathway B formed an ionone with 13 carbons (C13, such as 5 isolated from R. glutinosa) and carotenoid derivatives with 27 carbons (C27) though without isolation in this report but detected by UPLC-MS analysis (Figure S1), indicating that these new carotenoid analogues (C27) might exist in this medicinal plant.
1. Possible Biosynthetic Pathway of 1–5 .
The effects of compounds 1–5 on nitric oxide (NO) production were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. At 100 μM, rehmannin C (3) and 5,6-dihydroxy-β-ionone (5) showed substantial NO inhibition (66.8% and 60.5%, respectively), comparable to quercetin (65.6% inhibition) (Figure S3 and Table S2). Importantly, compounds 3 and 5 showed no cytotoxicity at 100 μM, underscoring their favorable safety profile and potential as a candidate for further drug development (Table S1). The observed structure–activity relationship suggests that the presence of a cyclo-ring and the trans/cis configuration of the double bonds in the long chain might influence anti-inflammatory efficacy (Figure S3 and Table S2).
Rehmannins A–D (1–4) represent a novel class of carotenoid derivatives characterized by the incorporation of an amide functional groupa structural feature reported here for the first time among all known carotenoids (comprising over 700 analogues). , More importantly, rehmannins B–D (2–4) contain a novel tricyclic ring system bearing five contiguous stereocenters on the heterocyclic core, rendering them attractive targets for total synthesis. Furthermore, the unusual oxygenation cleavage observed at the C-5/C-6 position in 1 never occurred in all carotenoids, indicating the presence of a dedicated oxygenase enzyme. This cleavage pattern offers a promising biosynthetic module for engineering oxygenated carotenoid pathways in synthetic biological systems. The first discovery of carotenoid derivatives not only validates the rationale of combining histochemical localization with biosynthetic pathway analysis for targeted pigmental molecule discovery but also suggests that this medicinal plant might harbor a largely untapped reservoir of structurally novel and biologically promising carotenoid-like molecules worthy of further exploration.
Supplementary Material
Acknowledgments
We acknowledge financial support from CAMS Innovation Fund for Medical Sciences (CIFMS) (grant no. 2023-I2M-2-006), the Key Project at the Central Government Level: Capacity Building for the Sustainable Utilization of Valuable Chinese Medicine Resources (2060302), the China Agriculture Research System of MOF and MARA (Grant No. CARS-21), Henan Province’s New Round Key Discipline (Pharmacy) (Jiao Yan [2023] No. 235, for L.C.), Zhengzhou Basic and Applied Basic Research (ZZSZX202421, for L.C.), and Key Discipline Construction Project of Traditional Chinese Medicine in Henan Province (No. 1 [2024] of Henan Health TCM Science and Education).
The data underlying this study are available in the published article and its Supporting Information.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.orglett.5c04732.
Detailed experimental procedures, NMR data, biological assay, MS/MS spectra, ECD, and NMR spectra (PDF)
Δ.
L.-W.Z. and P.S. have contributed equally. All authors have given approval to the final version of the manuscript.
The authors declare no competing financial interest.
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Supplementary Materials
Data Availability Statement
The data underlying this study are available in the published article and its Supporting Information.