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. 2026 Sep 30;11(40):61528–61536. doi: 10.1021/acsomega.6c06988

Homoisoflavanones, Alkaloid, and Norsesquiterpenoid from Polygonatum cyrtonema and Their Immunomodulatory Effects

Hongsu Zhao †, Caixia Lu †,§, Fucai Ren ‡, Xiaoxiao Wei †, Tongsheng Wang †, Wuxi Zhou †, Deling Wu †,∥,⊥,*, Fengqing Xu †,∥,⊥,*, Renzhong Wang †,∥,*
PMCID: PMC13670285  PMID: 42889847

Abstract

Polygonatum cyrtonema (Huang jing) is widely used in both the food and traditional Chinese medicine industries. A chemical investigation of the rhizome of cultivated P. cyrtonema yielded six previously undescribed compoundsone norsesquiterpenoid (1), one pyridine alkaloid (2), and four homoisoflavanones (3a, 4a, 5a, 6a)together with 9 known compounds (3b, 4b, 5b, 6b, 7–11). Their structures were established on the basis of extensive spectroscopic data and quantum chemical calculations, including NMR, HRESIMS, and calculated ECD. The enantiomers of the C-6-methylated homoisoflavanones (3a/3b–6a/6b) were successfully isolated via chiral HPLC. All compounds were evaluated for their immunomodulatory activity using RAW264.7 macrophages at concentrations ranging from 3.125 to 25 μM. Only compounds 8 and 11 enhanced the proliferation and phagocytosis of RAW264.7 macrophages and upregulated the secretion of NO, IL-6, and TNF-α.

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Introduction

Polygonatum cyrtonema Hua is a perennial herb belonging to the genus Polygonatum (Asparagaceae), which is mainly distributed in subtropical areas of China. Its dried rhizome, known “Huang Jing”, has been used in traditional Chinese medicine since the Han Dynasty for replenishing Qi, enriching Yin, moistening the lungs, strengthening the spleen, and invigorating the kidneys. Beyond its medicinal applications, the processed rhizome of P. cyrtonemais popular for its health benefits and is often made into herbal tea, medicinal wine, soup, congee, and other functional foods in daily life. − P. cyrtonemapossesses diverse biological properties, including antioxidant, anti-inflammatory, antifatigue, antidepressant, and immunomodulatory effects. Phytochemical studies have identified various constituents in P. cyrtonema, including flavonoids, , saponins, alkaloids, , polysaccharides, and lectins, all of which are implicated in the observed bioactivities.

Homoisoflavanones, a unique subclass of flavones, have a 3-benzylchromone skeleton that occurs mainly in plants of a few families, such as Liliaceae, Leguminosae, Portulacaceae, and Asparagaceae. − Since 1967, when homoisoflavanones were isolated from Eucomis bicolo, they have attracted the attention of synthetic biologists and chemists owing to their anti-inflammatory, antibacterial, antioxidant, antitumor, antiangiogenic, and cholinesterase-inhibitory activities. Currently, 54 homoisoflavanone compounds have been discovered in Polygonatum plants. However, the stereochemical configuration at the chiral C-3 position has been elucidated in only a few homoisodihydroflavonoids. , Our study aims to investigate the phytochemical profile of the raw material of P. cyrtonemaHua (from Chizhou, Anhui Province) to discover its bioactive constituents, thereby exploring its medicinal value and its potential for future development. Herein, we report the isolation and structural characterization of homoisoflavanones, an alkaloid, and a norsesquiterpenoid (Figure ). All isolates were tested for their immunomodulatory potential in RAW264.7 cells.

1.

1

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Structures of compounds 1–11.

Results and Discussion

The HRESIMS data of compound 1 (m/z 437.1606 [M + Cl]−, calcd. for C19H30O9Cl: 437.1578) established its molecular formula as C19H30O9. 1H NMR spectrum (Table ) exhibited two olefinic signals at δH 7.27 and 6.55 (1H each, d, J = 15.7), one oxygen-substituted methine signal at δH 4.26 (tt, J = 10.4, 7.0), three sets of methylene signals at δH 3.81 (dd, J = 7.6, 2.0)/ 3.78 (d, J = 7.6), 2.21 (ddd, J = 13.9, 6.9, 1.2)/1.81 (dd, J = 13.9, 10.8), and 2.04 (ddd, J = 13.8, 7.0, 1.2)/1.79 (dt, J = 13.8, 1.2), three singlet methyl signals at δH 2.33, 1.13, and 0.93, and an anomeric proton signal at δH 4.36 (d, J = 7.8). Nineteen carbon signals were observed in its 13C NMR spectrum, including 13 resonances assigned to a ionone skeleton, 6 carbon resonances from a glucose unit. Careful analysis of the NMR data showed that they were similar to 5,11-epoxy-3-hydroxymegastigman-7-en-9-one-3-O-β-d-glucopyranoside, except for an oxygenated quaternary carbon signal at δC 83.3 observed in 1. By integrating the correlations of 2D NMR, an α/β-unsaturated 2-butanone fragment was connected to C-6, which was supported by the spin system of H-7/H-8 in 1H–1H COSY spectrum, as well as long-range HMBC correlations of H-7 with C-1, C-5, C-6, and C-9, and of H-8 with C-6 and C-10. In addition, a furan ring fragment (C1–C6–C5–O–C11) was confirmed by HMBC correlations of H-11 with C-2, C-5, C-6, and C-12 (Figure ). A large coupling constant of 7.8 Hz for H-1’, together with correlations of H-1’ with C-3 in the HMBC spectrum, indicated that the glucopyranosyl unit was attached to the aglycone via a β- glycosidic linkage. Furthermore, the coupling constant J 7–8 = 15.7 Hz confirmed the E configuration of the double bond. Herein, the planar structure of 1 was determined. The ROESY spectrum exhibited key correlations of H-11 with H-3, as well as H-2α, suggesting that both H-3 and H-11 were α-oriented. Additionally, the correlations between H-7 and H-2β, H-4β, H3-12 and H3-13 suggested that all of these protons are β-oriented (Figure ). The absolute configuration of the glucose unit in 1 was assigned as D by HPLC of its thiocarbamoylthiazoline derivative (Figure S39), which was generated from the hydrolyzed glucose upon reaction with l-cysteine methyl ester hydrochloride and O-tolyl isothiocyanate. , The absolute configurations of C-1, C-3, C-5, and C-6 were determined as 1R,3S,5R,6S by comparing the experimental and time-dependent density functional theory (TD-DFT)-calculated ECD spectra (Figure ), and the calculations were performed at the B3LYP/6-311+G­(2d,p) level using Gaussian software. Therefore, compound 1 was identified as (1R,3S,5R,6S,7E)-6-hydroxy-5,11-epoxymegastigmane-7-en-9-one-3-O-β-d-glucopyranoside.

1. 1H and 13C NMR (600/150 MHz, CD3OD) Data of Compounds 1 and 2 .

  1
 2
NO δC δH (J in Hz) δC δH (J in Hz)
1 50.2 - - -
2 42.8 2.04 (1H, ddd, 13.8, 7.0, 1.2) 1.79 (1H, td, 13.8, 1.2) 156.1 -
3 73.5 4.26 (1H, tt, 10.4, 7.0) 123.5 7.53 (1H, d, 8.0)
4 42.8 2.21 (1H, ddd, 13.9, 6.9, 1.2) 1.81 (1H, dd, 13.9, 10.8) 139.1 7.68 (1H, dd, 8.0, 1.8)
5 87.8 - 138.7 -
6 83.3 - 149.4 8.32 (1H, d, 1.8)
7 145.8 7.27 (1H, d, 15.7) 33.2 2.65 (2H, t, 7.8)
8 134.0 6.55 (1H, d, 15.7) 34.5 1.60 (2H, m)
9 200.5 - 23.2 1.36 (2H, m)
10 27.8 2.33 (3H, s) 14.2 0.96 (3H, t, 7.4)
11 77.3 3.81 (1H, dd, 7.6, 2.0) 3.78 (1H, d, 7.6) 71.8 4.97 (1H, d, 13.0)
4.72 (1H, d, 13.0)
12 16.3 0.93 (3H, s)    
13 19.7 1.13 (3H, s)    
1’ 102.9 4.36 (1H, d, 7.8) 104.1 4.39 (1H, d, 7.2)
2’ 75.1 3.13 (1H, dd, 9.1, 7.8) 75.2 3.29 (1H, overlap)
3′ 78.1 3.34 (1H, m) 78.1 3.30 (1H, overlap)
4’ 71.7 3.27 (1H, m) 71.6 3.31 (1H, overlap)
5′ 78.0 3.24 (1H, m) 78.0 3.37 (1H, m)
6’ 62.8 3.86 (1H, dd, 12.0, 1.8) 62.8 3.86 (1H, dd, 11.9, 2.0)
3.64 (1H, dd, 12.0, 4.8) 3.67 (1H, dd, 11.9, 5.4)
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2.

2

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Key 2D-NMR correlations of compounds 1–6.

3.

3

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Key ROESY correlations of compound 1.

4.

4

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Experimental and calculated ECD spectra of compounds 1, 3–6.

The HRESIMS data of compound 2 confirmed its molecular formula to be C16H25NO6 (m/z: 328.1750 [M + H]+, calcd. for C16H26NO6: 328.1760). Its 1H NMR spectrum of 2 exhibited AMX-system signals at δH 8.32 (d, J = 1.8), 7.68 (dd, J = 8.0) and 7.53 (d, J = 8.0), a set of oxygen-substituted methylene signals at δH 4.97 and 4.72 (1H each, d, J = 13.0), and an anomeric proton signal at δH 4.39 (d, J = 7.2) (Table ). Its 1H–1H COSY spectrum confirmed that the presence of an n-butyl group in 2. Resonance signals at δC 156.1, 149.4, 139.1, 138.7, and 123.5 were assigned to the pyridine ring in its 13C NMR spectrum. The NMR data of 2 were similar to those of fusarinol, except for a glucose unit. The glucopyranosyl unit was assigned to C-11 in 2 via β-glycosidic linkage, which was established by HMBC correlation from H-1′ to δC 71.8 as well as the coupling constant of 7.2 Hz for H-1′. The n-butyl group was located at C-5, which was deduced from the correlations of H-7 with C-4, C-5, and C-9 and of H-11 with C-2 and C-3 in the HMBC spectrum (Figure ). D-Glucose was identified using the method described for compound 1. Thus, 2 was designated as fusarinol-11-O-β-d-glucopyranoside.

The HRESIMS of compound 3 showed an ion peak at m/z 301.1064 [M + H]+ (calcd. for C17H17O5: 301.1076), which confirmed its molecular formula as C17H16O5. An AA′XX′ spin system of aromatic protons at δH 7.07 and 6.74 (2H each, d, J = 8.5) and a singlet peak at δH 5.88) were observed in1H NMR spectrum. Twelve aromatic carbon signals (δC 166.2, 162.9, 162.2, 157.1, 131.2, 131.2, 130.3, 116.4, 116.4, 105.3, 102.6, 94.9) (Table ) were observed in 13C NMR spectrum. Analysis of the 1D NMR and HSQC spectra indicated the presence of a singlet methyl peak [δH 1.94/δC 7.0], two methylenes [δH 4.20 (dd, J = 11.3, 4.2) and 4.04 (dd, J = 11.3, 7.0)/δC 70.1; 3.07 (dd, J = 13.9, 4.6) and 2.62 (dd, J = 13.9, 10.4)/δC 33.2], a methine [δH 2.74 (m)/δC 48.3] and a carbonyl (δC 199.6). The NMR data of 3 were the same as those of 3-(4’-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-chroman-4-one. However, the absolute configuration of 3-(4’-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-chroman-4-one was not established. Compound 3 was separated by chiral HPLC using a CHIRALPAK AY-H column (isopropanol-ethanol, 65:35), yielding enantiomers 3a and 3b with an ee value of 78% for 3b. Their ECD experiments gave mirror image-like curves. The C-3 absolute configuration was verified based on comparing the experimental and calculated ECD spectra; the absolute configurations of 3a and 3b were assigned as 3S and 3R, respectively. In addition, the ECD spectrum of homoisoflavanones showed a negative Cotton effect at 287–295 nm, indicating the R configuration at C-3. Thus, compound 3a was elucidated as (3S)-5,7-dihydroxy-6-methyl-3-(4′-hydroxybenzyl)-chroman-4-one, which is a new compound. Compound 3b was elucidated as (3R)-5,7-dihydroxy-6-methyl-3-(4′-hydroxybenzyl)-chroman-4-one, which was reported in literature.

2. 1H and 13C NMR (600/150 MHz, CD3OD) Data of Compounds 3–6 .

  3
 4
 5
 6
NO δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz)
2 70.1 4.20 (1H, dd, 11.3, 4.2) 4.04 (1H, dd, 11.3, 7.0) 70.01 4.21 (1H, dd, 11.3, 4.2) 4.04 (1H, dd, 11.3, 7.1) 70.5 4.23 (1H, dd, 11.3, 5.5) 4.08 (1H, dd, 11.3, 3.5) 70.6 4.25 (1H, brd, 11.4) 4.11 (1H, brd, 11.4)
3 48.3 2.74 (1H, m) 48.1 2.72 (1H, m) 46.0 2.91 (1H, m) 45.7 2.92 (1H, m)
4 199.6 - 200.0 - 200.7 - 200.5 -
5 162.9 - 160.6 - 160.5 - 163.2 -
6 105.3 - 104.7 - 104.7 - 105.2 -
7 166.2 - 163.9 - 164.1 - 166.4 -
8 94.9 5.88 (1H, s) 103.7 - 103.6 - 96.0 5.88 (1H, s)
9 162.2 - 159.1 - 159.2 - 161.6 -
10 102.6 - 102.7 - 102.7 - 102.7 -
11 33.2 3.07 (1H, dd, 13.9, 4.6) 2.62 (1H, dd, 13.9, 10.4) 33.1 3.06 (1H, dd, 13.9, 4.6) 2.59 (1H, dd, 13.9, 10.4) 28.2 3.14 (1H, dd, 13.7, 5.0) 2.57 (1H, dd, 13.7, 9.9) 28.3 3.14 (1H, d, 13.7) 2.57 (1H, d, 13.7)
1’ 130.3 - 130.3 - 118.3 - 118.2 -
2’ 131.2 7.04 (1H, d, 8.5) 131.1 7.04 (1H, d, 8.4) 157.6 - 157.6 -
3′ 116.4 6.72 (1H, d, 8.5) 116.4 6.73 (1H, d, 8.4) 102.4 6.38 (1H, d, 2.5) 102.4 6.38 (1H, d, 2.4)
4’ 157.1 - 157.1 - 161.1 - 162.3 -
5′ 116.4 6.72 (1H, d, 8.5) 116.4 6.73 (1H, d, 8.4) 105.6 6.35 (1H, dd, 8.2, 2.5) 105.6 6.35 (1H, dd, 8.2, 2.4)
6’ 131.2 7.04 (1H, d, 8.5) 131.1 7.04 (1H, d, 8.4) 132.6 6.97 (1H, d, 8.2) 132.6 6.96 (1H, d, 8.2)
Me-6 7.0 1.94 (3H, s) 7.9 1.98 (3H, s) 7.4 1.99 (3H, s) 7.0 1.92 (3H, s)
Me-8 - - 7.4 1.97 (3H, s) 7.9 1.97 (3H, s) - -
OMe-2’ - - - - 55.6 3.73 (3H, s) 55.6 3.73 (3H, s)
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The HRESIMS of compound 4 showed an ion peak at m/z 315.1241 [M + H]+ (calcd. for C18H19O5: 315.1233), suggesting that its molecular formula is C18H18O5. A comparison of the 1D NMR data for compounds 4 and 3 demonstrated a similarity between them (Table ). In compound 4, an additional methyl group (δH 1.97, s) was observed, which was attached to the C-8 position (Figure ) based on HMBC correlations from δH 1.97 to C-7, C-8, and C-9. The 1H NMR and 13C NMR data were the same as 4’,5,7-trihydroxy-6,8-dimethyl-homoisoflavanone. However, the absolute configuration of 4’,5,7-trihydroxy-6,8-dimethyl-homoisoflavanone was also not established. Similar to compound 3, enantiomers 4a and 4b (76% ee for 4b) were obtained through chiral HPLC separation. Compounds 4a and 4b were determined to possess 3S and 3R configurations, respectively, through comparison of their experimental and calculated ECD spectra (Figure ). Thus, compound 4a was elucidated as (3S)-4’,5,7-trihydroxy-6,8-dimethyl-homoisoflavanone. Compound 4b was a known constituent.

The molecular formula of compound 5 was determined to be C19H20O6 by the HRESIMS ion at m/z 343.1173 [M – H]− (calcd. for C19H19O6: 343.1176). In its 1H NMR spectrum, an ABX system [δH 6.97 (d, J = 8.2), 6.38 (d, J = 2.5), and 6.35 (dd, J = 8.2, 2.5)], two methyl singlets (δH 1.99, 1.97), and a methoxy group (δH 3.73) were observed. Nineteen carbon signals appeared in 13C NMR spectrum, including 12 aromatic carbons (δC 164.1, 161.1, 160.5, 159.2, 157.6, 132.6, 118.3, 105.6, 104.7, 103.6, 102.7, 102.4), three methyl carbons (δC 55.6, 7.9, 7.4), two methylene carbons (δC 70.5, 28.2), a methine carbon (δC 46.0), and a carbonyl carbon (δC 200.7). The 1D NMR data were the same as those of 5,7-dihydroxy-6,8-dimethyl-3-(2′-hydroxy-4′-methoxybenzyl)­chroman-4-one. The planar structure of compound 5 was confirmed based on 1H–1H COSY as well as HMBC correlations (Figure ). Compound 5 was analyzed by a chiral HPLC column to afford 5a and 5b, a pair of enantiomers (approximately 50% ee for 5b); their absolute configurations were assigned based on the comparison of the experimental and calculated ECD spectra (Figure ), in conjunction with the observed Cotton effects in the 287–295 nm range. Compound 5a was elucidated as (3S)-5,7-dihydroxy-6,8-dimethyl-3-(2′-hydroxy-4′-methoxybenzyl)­chroman-4-one and compound 5b was identified as (3R)-5,7-dihydroxy-6,8-dimethyl-3-(2′-hydroxy-4′-methoxybenzyl)­chroman-4-one, which have been reported.

The HRESIMS data of compound 6 confirmed its molecular formula to be C18H18O6 (m/z 331.1137 [M + H]+, calcd. for C18H19O6: 331.1182). Compared with those of compound 5, the NMR data of compound 6 (Table ) revealed a singlet at δH 5.88, which was assigned to C-8. The 1H NMR and 13C NMR data of 6 were the same as those of (3R)-5,7-dihydroxy-6-methyl-3-(2’-hydroxy-4’-methoxybenzyl)-chroman-4-one. Compound 6 was further separated on a chiral HPLC column to give 6a and 6b, a pair of enantiomers, with approximately 50% ee for 6b. The absolute configuration of C-3 was determined by comparing the experimental and calculated ECD spectra (Figure ). The experimental ECD curve of 6a matched its calculated curve well, assigning compound 6a as (3S)-5,7-dihydroxy-6-methyl-3-(2’-hydroxy-4’-methoxybenzyl)-chroman-4-one.

Consistent with NMR and MS data from the literature , the remaining compounds were identified as: (3R)-5,7-dihydroxy-8-methyl-3-(4’-hydroxybenzyl)-chroman-4-one (7); (3R)-5,7-dihydroxy-3-(2′-hydroxy-4′-methoxybenzyl)-chroman-4-one (8); 3-(2’,4’-dihydroxybenzyl)-5,7-dihydroxychroman 4-one (9); (3R)-5,7-dihydroxy-8-methyl-3-(2’,4’-dihydroxybenzyl)-chroman-4-one (10); (E)-3-(3,4-dihydroxybenzylidene)-5,7-dihydroxy-6,8-dimethylchroman-4-one (11).

The immunomodulatory activity of all compounds was evaluated in RAW264.7 macrophages. To investigate the safe concentration range of the compounds, RAW264.7 macrophages were treated with them for 24 h. Cell viability, assessed by a CCK-8 assay, showed that only compounds 8 and 11 stimulated cell proliferation at concentrations of 3.125–25 μM (Figure ), while other compounds exhibited cytotoxicity toward RAW264.7 macrophages. The phagocytic activity of RAW264.7 macrophages was significantly enhanced by treatment with compounds 8 and 11 (Figure ). To further evaluate the immunomodulatory potential of the two isolates, the release of NO, IL-6, and TNF-α from RAW264.7 macrophages treated with these compounds was measured. As shown in Figure , compounds 8 and 11 at the indicated concentrations (3.125–25 μM) significantly promoted the release of NO, IL-6, and TNF-α. These results indicate that compounds 8 and 11 exhibit potential immunomodulatory activity.

5.

5

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Effect of compounds 8 (A) and 11 (B) with different concentrations on the viability and phagocytosis of RAW264.7 cells. Control: untreated cells. LPS: positive control group. *p < 0.05, **p < 0.01,***p < 0.001 versus the control group.

6.

6

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Effects of compounds 8 (A) and 11 (B) with different concentrations on NO, IL-6, and TNF-α release. Control: untreated cells. LPS: positive control group. *p < 0.05, **p < 0.01,***p < 0.001 versus the control group.

Conclusion

Six new compoundsone norsesquiterpenoid (1), one pyridine alkaloid (2), and four homoisoflavanones (3a–6a)together with 9 known compounds (3b–6b, 7–11) were obtained from Polygonatum cyrtonema Hua. Chiral HPLC successfully separated the enantiomers (3a/3b–6a/6b) of the homoisoflavanones with a methyl substitution at the C-6 position. Among these homoisoflavanones, some existed as partial racemates, such as compounds 3 and 4. However, compounds 7–10 showed single peaks on chiral HPLC. In the evaluation of immunomodulatory activity, only compounds 8 and 11 enhanced the proliferation and phagocytosis of RAW264.7 macrophages, as well as the secretion of NO, IL-6, and TNF-α.

Experimental Section

General Experimental Procedures

HRESIMS was recorded on an Agilent 6500 Q-TOF mass spectrometer. The NMR and ECD spectra were measured on Bruker AVANCE III-600 and JASCO P-1500 spectrometers, respectively. Column chromatography (CC) was performed using AB-8 macroporous resin, silica gel G, ODS, and Sephadex LH-20. Semipreparative HPLC was carried out using a Waters 1525 instrument equipped with a YMC-Pack-ODS-A (10.0 mm × 250 mm, 5 μm) column. TLC was performed on silica gel G precoated plates and visualized by spraying with 10% H2SO4 in ethanol, then heating.

Plant Material

The plant material (fresh rhizomes of P. cyrtonema) was obtained in October 2023 from the herbal medicine cultivation base of Jingtian Food Development Co., Ltd., located in Qingyang County, Anhui Province, China, and was authenticated by Professor Shoujin Liu (Anhui University of Chinese Medicine). The specimen was archived at the Herbarium of Anhui University of Chinese Medicine under the voucher number ACM2023101801. The fresh rhizomes of P. cyrtonema were processed into samples (original medicinal material) at Jinzhai Ryunyuan Biological Technology Co., Ltd.

Extraction and Isolation

50 kg of the sample was cut into pieces and extracted three times (2 h each) by refluxing with 75% ethanol (100 L per extraction). Following evaporation of the filtrates under reduced pressure, the residue was dissolved in deionized water and then passed through an AB-8 macroporous resin using gradient elution with ethanol–water solutions of varying concentrations (0%, 30%, 50%, 70%, 95%), yielding four fractions: A (30% ethanol, 230 g), B (50% ethanol, 46 g), C (70% ethanol, 17 g), and D (95% ethanol, 29 g). Separation of fraction A (230 g) used a silica gel column and afforded nine fractions (A1–A9) by eluting with a dichloromethane-methanol gradient (DCM-MeOH, 19:1 to 1:1). Fraction A4 was fractionated on Sephadex LH-20 (MeOH) to give fractions A4-1–A4-4. Further purification of A4-3 via MPLC afforded A4-3-1–A4-3-6. Purification of fraction A4-3-3 on Sephadex LH-20 (MeOH), followed by semipreparative HPLC, yielded compound 1 (2.1 mg). Separation of fraction B (46 g) on a Sephadex LH-20 column (MeOH-H2O, 4:1) afforded five fractions (B1–B5). Fraction B2 was chromatographed using MPLC to yield fractions B2-1–B2-4. Fraction B2-3 was further purified via semipreparative HPLC to afford compound 2 (1.5 mg). Fraction B4 was successively subjected to Sephadex LH-20, silica gel CC, and semipreparative HPLC to obtain compounds 9 (10.1 mg) and 10 (6.0 mg). Fraction D (29 g) was chromatographed using Sephadex LH-20 (DCM-MeOH, 1:1) to yield ten fractions (D1–D10). Fraction D6 was fractionated by silica gel CC using a petroleum ether-EtOAc gradient (9:1 to 1:1), yielding six fractions (D6-1–D6-6). Fraction D6-2 was further purified by semipreparative HPLC to obtain compounds 4 (18.0 mg), 3 (4.8 mg), and 7 (1.5 mg). Semipreparative HPLC purification of fraction D6-3 yielded compounds 6 (8.55 mg), 5 (4.35 mg), and 8 (2.05 mg). Compound 11 (1.5 mg) was isolated from fraction D8 using semipreparative HPLC.

(1R,3S,5R,6S,7E)-6-hydroxy-5,11-epoxymegastigmane-7-en-9-one-3-O-β-d-glucopyranoside (1): white powder; CD (MeOH) λmax (mdeg): 204 (+0.883), 235 (−8.41) nm; NMR data: see Table . HRESIMS m/z 437.1606 [M + Cl]− (calcd. for C19H30O9Cl, 437.1578).

Fusarinol-11-O-β-d-glucopyranoside (2): white powder; NMR data: see Table . HRESIMS m/z 328.1750 [M + H]+ (calcd. C16H26NO6, 328.1760).

5,7-Dihydroxy-6-methyl-3-(4′-hydroxybenzyl)-chroman-4-one (3): yellowish powder; NMR data: see Table . HRESIMS m/z 301.1064 [M + H]+ (calcd for C17H17O5, 301.1076). (3S)-5,7-dihydroxy-6-methyl-3-(4′-hydroxybenzyl)-chroman-4-one (3a): CD (MeOH) λmax (mdeg) 218 (9.73), 255 (4.36), and 291 (+10.86) nm; (3R)-5,7-dihydroxy-6-methyl-3-(4′-hydroxybenzyl)-chroman-4-one (3b): CD (MeOH) λmax (mdeg) 218 (−7.53), 255 (−5.07), and 291 (−8.34) nm.

4’,5,7-Trihydroxy-6,8-dimethyl-homoisoflavanone (4): yellowish powder; NMR data: see Table . HRESIMS m/z 315.1241 [M + H]+ (calcd for C18H19O5, 315.1233). (3S)-4’,5,7-trihydroxy-6,8-dimethyl-homoisoflavanone (4a): CD (MeOH) λmax (mdeg) 219.6 (2.39), 256.2 (−0.81) and 294.6 (+2.09) nm; (3R)-4’,5,7-trihydroxy-6,8-dimethyl-homoisoflavanone (4b): CD (MeOH) λmax (mdeg) 220 (−12.33), 256.6 (+5.33), and 294.4 (−8.91) nm.

5,7-Dihydroxy-6,8-dimethyl-3-(2′-hydroxy-4′-methoxybenzyl)­chroman-4-one (5): yellowish powder; NMR data: see Table . HRESIMS m/z 343.1173 [MH]− (calcd for C19H19O6, 343.1176). (3S)-5,7-dihydroxy-6,8-dimethyl-3-(2′-hydroxy-4′-methoxybenzyl)­chroman-4-one (5a): CD (MeOH) λmax (mdeg) 294.6 (+7.88) nm; (3R)-5,7-dihydroxy-6,8-dimethyl-3-(2′-hydroxy-4′-methoxybenzyl)­chroman-4-one (5b): CD (MeOH) λmax (mdeg) 295 (−7.76) nm.

5,7-Dihydroxy-6-methyl-3-(2′-hydroxy-4′-methoxybenzyl)-chroman-4-one (6): yellowish powder; NMR data: see Table . HRESIMS m/z 331.1137 [M + H]+ (calcd for C18H19O6, 331.1182). (3S)-5,7-dihydroxy-6-methyl-3-(2′-hydroxy-4′-methoxybenzyl)-chroman-4-one (6a): CD (MeOH) λmax (mdeg) 293.6 (+12.95) nm; (3R)-5,7-dihydroxy-6-methyl-3-(2′-hydroxy-4′-methoxybenzyl)-chroman-4-one (6b): CD (MeOH) λmax (mdeg) 294.4 (−13.30) nm.

Acid Hydrolysis

The compound was refluxed with 1.0 M HCl (3 mL, 1.5 h). After concentration, the residue was dissolved in water (3 mL) and extracted three times with ethyl acetate. The residue of the aqueous layer was dissolved in pyridine (0.1 mL) and reacted with l-cysteine methyl ester hydrochloride (0.5 mg, 60 °C, 1 h). To this mixture, O-tolyl isothiocyanate (10 μL) was added, and stirring was continued at 60 °C for 2 h. Reference samples (d-glucose and l-glucose) underwent the same derivatization procedure. HPLC analysis of the derivatives was performed using a YMC-Triart C18 column (250 mm × 4.6 mm, 5 μm). Analysis conditions were as follows: mobile phase, water–acetonitrile (75:25, v/v); column temperature: 30 °C; flow rate: 1.0 mL/min; detection wavelength: 250 nm.

ECD Calculation

Electronic circular dichroism (ECD) calculations were conducted by using Gaussian 16 software. Conformational searches were carried out with GMMX3, and the conformers were optimized using DFT at the B3LYP/6-31G­(d) level. The theoretical calculations of the ECD spectra were conducted in MeOH at the B3LYP/6-311+G­(d,p) level for the compounds. The final ECD spectra were obtained according to the Boltzmann distribution.

Immunomodulatory Effect on RAW 264.7 Cells

RAW264.7 murine macrophages were selected to screen for the immunomodulatory effects of the isolates. In brief, the cells were cultured in DMEM (Servicebio, Wuhan) containing 10% FBS (Kangyuan, Tianjin) at 37 °C under 5% CO2. The isolates were dissolved in DMSO and diluted to the required concentrations with culture medium (final DMSO concentration <0.1%). Lipopolysaccharide (LPS, 1 μg/mL) was employed as a positive control. Cell viability was determined using a CCK-8 assay kit (Sparkjade, China). The neutral red uptake assay was used to assess the phagocytic activity of RAW264.7 macrophages. Nitric oxide (NO) release levels were measured using the Griess reagent system (Beyotime, China). The concentrations of cytokines (IL-6 and TNF-α) were determined by the corresponding ELISA kits (Jianglai Biotechnology, China). The absorbance was measured using a SpectraMax i3x microplate reader (Molecular Devices, USA). All of the experiments were carried out in triplicate. Data were analyzed using one-way analysis of variance and LSD-t test by GraphPad Prism 10.4.1 software. A p-value less than 0.05 was considered statistically significant.

Supplementary Material

ao6c06988_si_001.pdf (3.4MB, pdf)

Acknowledgments

This work was partially supported by the University Collaborative Innovation Project of Anhui Province (GXXT-2022-066). We thank Anhui Jingtian Food Development Co., Ltd., and Anhui Jinzhai Runyuan Biological Technology Co., Ltd., for plant material collection and processing, respectively. We also thank the analytical group of the Experimental and Testing Center at Anhui University of Chinese Medicine for spectra test.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c06988.

  • ECD, HRESIMS, and NMR spectra of the new compounds; an HPLC chromatogram of sugar derivatives (PDF)

#.

H.Z. and C.L. contributed equally to this work. Writing – original draft, isolation, identification, and bioactivity evaluation. F.R.: Extraction and ECD calculations. X.W., T.W. and W. Z.: Methodology, investigation, and data curation. R.W.: Writing – review and editing, and validation. D.W. and F.X.: Review and editing, project administration, methodology, investigation, and funding acquisition.

The authors declare no competing financial interest.

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Supplementary Materials

ao6c06988_si_001.pdf (3.4MB, pdf)

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